2018

USP 41 THE UNITED STATES PHARMACOPEIA

NF 36 THE NATIONAL FORMULARY

Volume 2 By authority of the United States Pharmacopeial Convention

Prepared by the Council of Experts and its Expert Committees

Official from May 1, 2018

The designation on the cover of this publication, “USP NF 2018,” is for ease of
identification only. The publication contains two separate compendia: The United
States Pharmacopeia, Forty-First Revision, and The National Formulary, Thirty-Sixth
Edition.

THE UNITED STATES PHARMACOPEIAL CONVENTION

12601 Twinbrook Parkway, Rockville, MD 20852
 SIX-MONTH IMPLEMENTATION GUIDELINE

The United States Fearne pd ene Formulary and its supplements become official six months after being released to
the public. The USP-NF, which is released on November 1 of each year, becomes official on May 1 of the following year. This
six-month implementation timing gives users more time to bring their methods and procedures into compliance with new
and revised USP-NF requirements.
The table below describes the official dates of the USP-NF and its supplements. The 2017 USP 40-NF 35, and its supple-
ments, Interim Revision Announcements (IRAs) and Revision Bulletins to that edition, will be official until May 1, 2018, at which
time the USP 41-NF 36 becomes official.

Publication Release Date Official Date Official Until

USP 41-NF 36 November 1, 2017 May 1, 2018 May 1, 2019 (except as superseded by supplements, /RAs, and
Revision Bulletins)
First Supplement to the February 1, 2018 August 1, 2018 May 1, 2019 (except as superseded by Second Supplement, IRAs,
USP 41-NF 36 and Revision Bulletins)
Second Supplement to the June 1, 2018 December 1, 2018 May 1, 2019 (except as superseded by /RAs and Revision Bulletins)
USP 41-NF 36
USP 42-NF 37 November 1, 2018 May 1, 2019 May 1, 2020 (except as superseded by supplements, /RAs, and
Revision Bulletins)

The table below gives the details of the /RAs that will apply to USP 41-NF 36.

1, 2018
1, 2018 18
ber
4, 2018
1 31, 2019 1

Revision Bulletins published on the USP website become official on the date specified in the Revision Bulletin.

NOTICE AND WARNING

Concerning U.S. Patent or Trademark Rights—The inclusion in The United States Pharmacopeia or in the National Formulary of a
monograph on any drug in respect to which patent or trademark rights may exist shall not be deemed, and is not intended
as, a grant of, or authority to exercise, any right or privilege protected by such patent or trademark. All such rights and
privileges are vested in the patent or trademark owner, and no other person may exercise the same without express
permission, authority, or license secured from such patent or trademark owner.

Concerning Use of USP or NF Text—Attention is called to the fact that USP and NF text is fully copyrighted. Authors and
others wishing to use portions of the text should request permission to do so from the Secretary of the USPC Board of
Trustees.
Copyright © 2017 The United States Pharmacopeial Convention 12601 Twinbrook Parkway, Rockville, MD 20852

All rights reserved.

ISSN: 0195-7996
ISBN: 978-1-936424-70-2

Printed in the United States by United Book Press, Inc., Baltimore, MD

USP 41—NF 36 Contents iii

Contents

VOLUME 1 Guide to General Chapters .......... 13

USP 41
Mission Statement and Preface...... vii

People 2015-2020 Revision Cycle ..... xi Monographs

COTTICONS 6 ok 5 G9 GE EAS SAi eee
Rete xi Official Monographs for USP 47, A-l......... 19
Board ot TTuSt@@S 5.siawewaneaneB
Wide bA > xi
Gounel OF EXpert: « sassewew.e
ccadines canene xi Index
Expert-CoOnumuees-« vaisisaenee
x8568 KAa i ne xii Combined Index to USP 41 and NF 36....... I-1
IncMemoriam c's b wesmersuyuepses
oogeaaeoa xviii

Members of the United States

Pharmacopeial Convention, VOLUME 2
as of May 31, 2017................ xix
2016 Recognition of Monograph
and Reference Material Donors ... xxvi Notices
General Notices and Requirements ........... ix
Articles of Incorporation ........... xxviii
USP Governance ................... xxix Guide to General Chapters .......... xix
Bylaws: eaceses 3 21ers
eedSeFeFEY xxix
Rules and Procedures. « «a swiwiselos
We Pad XXix
Monographs
USP PONS: aa vas au ewGoaidaladsnadiaw
yHyditale xxix
Official Monographs for USP 41, J-Z....... 2303

Admissions ..................00.00 xxxii

Index
Articles Admitted to USP 47 by
Supplement :s:22¢s45ssshwewmmagdgs
ies Xxxiii Combined Index to USP 41 and NF 36....... 1-1

New Articles Appearing in USP 47 That Were Not

VOLUME 3
Included in USP 40 Including
Supplements... 0.2.0.0... cee eeeeeeXXxiV
Articles Included in USP 40 But Not
Included in USP 41 . 2.5. S328 en8.248 XXxXiV
Annotated List ..... 20... eee ee eee XXXVi Notices
General Notices and Requirements ........... ix
Notices
General Notices and Requirements ........... 3 Guide to General Chapters .......... xix
 iv Contents
Front Matter
Global Health
Official Monographs .................4. 4415
Dietary Supplements
Official Monographs ................00. 4417
Admissions
Articles Admitted to NF 36 by Supplement .. 5167
New Articles Appearing in NF 36 That Were Not
Included in NF 35 Including
Supplements ........... 0000000 e eae 5167
New Articles Appearing in NF 36 ......... 5167
ADMOLATER LISts paces 050 aye rons 6 wee y « papemronsennines 5168
Excipients
USP and NF Excipients, Listed by
COCGOIY wees eeu ee 420 o¥ sds oo ewReRE 5169
Monographs
Official Monographs for NF 36 ........... S179
Index
Combined Index to USP 41 and NF 36....... I-1
VOLUME 4
Notices
General Notices and Requirements ........... ix
Guide to General Chapters .......... xix
Reagents, Indicators, and
Solutions ....................04. 5659
Reagent Specifications..............-24-. 5664
Indicators and Indicator Test Papers ....... 5745
USP 41-NF 36
SOLUUONS: 5 sso wwemnmeemerne
eyesEEREERSS 5748
Buffer Solutions ........ 0.0.00. 000 008 5748
Colorimetric Solutions ..............0. 5749
Test SOMGHOMS ccosnmamanowey
eewseeeEeERS 5750
Volumetric Solutions ..............00. 5761
Chromatographic Columns .............. 5774
Reference Tables
Containers for Dispensing Capsules and
Tablets... 0.6... cee eee eee 5781
Description and Relative Solubility of USP
and NP AMIGOS wiscacaeea cseeeseeexs 5791
Approximate Solubilities of USP and
NFATTICIES5. ounsnincateresisnanene
ef224 th, DY 5851
AtOMiG: WEIGHTS samsyeusana are Sy Yee eh S 5859
Half-Lives of Selected Radionuclides ....... 5860
Alcoholometric Table...............0005 5861
Intrinsic Viscosity fable joo s.< + ,09,853
0.4bonaetes3 5863
General Chapters
See page xix for detailed contents
General Tests and Assays.............-4- 5915
General Requirements for Tests and Assays .. 5915
Apparatus for Tests and Assays ........... 5954
Microbiological Tests... ......0....0
e005 5959
Biological Tests and Assays .............. 5991
Chemical Tests and Assays ............4- 6094
Physical Tests and Determinations......... 6327
Index
Combined Index to USP 41 and NF 36....... 1-1
VOLUME 5
Notices
General Notices and Requirements ........... ix
USP 41-NF 36 Contents v

Pesala)NieleCoyEy
Guide to General Chapters .......... xix Dietary Supplements ....0
500e seesus

General Chapters Index

See page 63 for detailed contents Combined Index to USP 41 and NF 36

General Information
USP 41 General Notices vii

General Notices and

Requirements

Ce NMLACIE LD)
repIb
Applying to Standards, Tests,
Assays, and Other Specifications
of the United States Pharmacopeia

1. Title and Revision 6.50. Preparation of Solutions .............0 0005 xiv

6.60. Units Necessary to Complete a Test ......... xiv
6.70... Reagent « <2 i ssnwass wee as 2s wD HERRE EG xiv
2. Official Status and Legal OBOLEQUIPMENE « nameenies 48seEESYFERERSEGA xiv
Recognition. .
2.10. Official Text... 2. eee ee
2.20. Official Articles 7. M@St RESUS wevsess « 5 soon 4 owe 2 5 eam YY DEH xv
7.10. Interpretation of Requirements ............. xv
7.20. Rounding Rules .........0. 0.00ceeeeeee xv
3. Conformance to Standards
3.10. Applicability of Standards................. ix 8. Terms and Definitions .................. xv
3.20. Indicating Conformance 8.10. Abbreviations... 0.0.0... eeeeeeeee
B:Z0.; ADOUE. « snecrmuceament
wauomoKHemeneetiUES4
8.30. Alcohol Content .
4. Monographs and General Chapters .... xi 8.40.. Atomic Weights: «<< 233s ¥ see eeawaieey 3228
4.10. Monographs .... 0... 0... cece eee eee xi 8.50. Blank Determinations .................-. xv
4.20. General Chapters... 0.0.0.0... 00eeeeee xi 8.60. Concomitantly ..... 2.0.0.0... 00.eeeee xvi
8.70. Desiccator . 6... eee ee xvi
5. Mono raph Components ............... xi 8:80. LOQaTItAMS paneer ae ene eo eo ane as oo xvi
5.10. Molecular Formula... ... 0... eee eee eee xi 8.90.. Microbial Strain’ 0.4.06 6ee xcommen ¥298 xvi
5.20. Added Substances -ocfvessescvcavevneaaes a xi 8.100; Negligibletvicccovs aa ss vee » a sceppemie
s$275 xvi
8.110; NELINMEsccooon 5 ao 8 5 € 2b EREETRGES
Ze& xvi
5.30. Description and Solubility................. xii
5:40.. Identification = sesso g&2¥5 8BBEEFeyeeecee-«0 xii 8.1.20) OGG? » scyerscsog 3 ooo FE EEL Keveomemee aan xvi
5.50 nASSay" «2 3 x % gexqumaiiie SE 6% £6 ¥.1 evannmremanceve » xii 8.130, Percent xvi
5.60. Impurities and Foreign Substances .......... xii 8.140. Percentage Concentrations xvi
5.70. Performance Tests ... 0.0...0.0eeeeeeee xili BalSO. PRESSUPES romances 2 0 0 8 eocreteromnere . xvi
5.80. USP Reference Standards ................. xiti 8.160, Reaction TIME ».2.600568 wansmimman yea y xvi
8.1.20, SpeciiierGravity: a0 sau 2s pccaweey ga892 xvi
8.180. Temperatures 33.35 sees cece eoeabee xvi
6. Testing Practices and Procedures ..... xiii S190, [IVE camwcommyues & a DEF HE BEER eee ae ae 4 xvi
6.10. Safe Laboratory Practices ........ 0... .0005 xili 8.200; Tanstetcv igs saa ee ve eee eee xvi
6.20, Automated Procedures... ...........000 00 xiii 8.210. Vacuum 2... eee eeeee xvi
6.30. Alternative and Harmonized Methods and 8.220. Vacuum Desiccator ...........-.0-00 0085 xvi
ProCODUTCS esse sees & 3 35 5 EE € & Svecmmemeree
ea 8 xiii 8.230. WALCP cess 9oo 6 nw oo eR oeaEERG xvi
6.40. Dried, Anhydrous, Ignited, or Solvent- 8.240. Weights and Measures ..............-0-- xvi
EPEC BASIS ppniesscmsis ¢ 4 ws 2 4 8 oc evominomenea
aa 3a xiii
 viii. General Notices USP 41

9. Prescribing and Dispensing............ xvi 10. Preservation, Packaging, Storage,

9.10 Use of Metric Units; .. sy eae eeweremesa
yee xvii and Labeling ........................
9.20 Changes in Volume... 1... ee eee eee xviii 10.10. Packaging and Storage. .
HO 2OCEADENNG sas oy ob kG tines we ene es

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USP 41
GENERAL NOTICES AND
REQUIREMENTS
The General Notices and Requirements section (the General
Notices) presents the basic assumptions, definitions, and de-
fault conditions for the interpretation and application of the
Nie States Pharmacopeia (USP) and the National Formulary
NF).
Requirements stated in these General Notices apply to all
articles recognized in the USP and NF (the “compendia”)
and to all general chapters unless specifically stated
otherwise.
1. TITLE AND REVISION
The full title of this publication (consisting of five volumes
and including its Supplements), is The Pharmacopeia of the
United States of America, Forty-First Revision and the Na-
tional Formulary, Thirty-Sixth Edition. These titles may be ab-
breviated to USP 41, to NF 36, and to USP 41-NF 36. The
United States Pharmacopeia, Forty-First Revision, and the Na-
tional Formulary, Thirty-Sixth Edition, supersede all earlier re-
visions. Where the terms “USP,” “NF,” or “USP—NF’ are used
without further qualification during the period in which
these compendia are official, they refer only to USP 41, NF
36, and any Supplement(s) thereto. The same titles, with no
further distinction, apply equally to print or electronic pres-
entation of these contents. Although USP and NF are pub-
lished under one cover and share these General Notices, they
are separate compendia.
This revision is official beginning May 1, 2018 unless oth-
erwise indicated in specific text.
Supplements to USP and NF are published periodically.
Accelerated Revisions, published periodically on the Offi-
cial Text section of USP’s website (http://www.usp.org/usp-
nf/official-text), are designed to make revisions cHicial more
quickly than through the routine process for publishing
standards in the USP-NF. Interim Revision Announcements are
Accelerated Revisions to USP and NF that contain official re-
visions and their effective dates.
Revision Bulletins are Accelerated Revisions to official text
or postponements that require expedited publication. They
generally are official immediately unless otherwise specified
in the Revision Bulletin.
Errata are Accelerated Revisions representing corrections
to items erroneously published. Announcements of the avail-
ability of new USP Reference Standards and announcements
of tests or procedures that are held in abeyance pending
availability of required USP Reference Standards are also
available on the “Official Text” tab of USP’s website.
2. OFFICIAL STATUS AND LEGAL RECOGNITION
2.10. Official Text
Official text of the USP and NF is published in the USP-NF
Online (www.uspnf.com) in the edition identified as “CUR-
RENTLY OFFICIAL” and in Accelerated Revisions that super-
sede the USP-NF Online as described below.
Routine revisions are published in the USP-NF Online and
become official on the date indicated, usually six months
after publication. Accelerated Revisions supersede the
USP-NF Online and become official on the date indicated.
Links to Accelerated Revisions on the USP website can be
found in any superseded monograph or general chapter in
the USP-NF Online.
Print and USB flash drive versions of the USP and NF also
are available. Routine revisions are provided with the same
timing as the USP-NF Online. Official text published in Sup-
plements supersedes that in the previously published print or
General Notices ix
a
USB flash drive versions of USP-NF. These versions also are i)
superseded by Accelerated Revisions as described above. R
®
=
In the event of any disparity between the print or USB
flash drive versions and the USP-NF Online, the USP-NF On- a
line will be deemed to apply. Ts
2.20. Official Articles °
=
An official article is an article that is recognized in USP or a
NF. An article is deemed to be recognized and included in a ©
ww
compendium when a monograph for the article is published
in the compendium and an official date is generally or spe-
cifically assigned to the monograph.
The title specified in a monograph is the official title for
such article. Other names considered to be synonyms of the
official titles may not be used as substitutesfat official titles.
Official articles include both official substances and official
products. An official substance is a drug substance, excipient,
dietary ingredient, other ingredient, or component ofa fin-
ished device for which the monograph title includes no indi-
cation of the nature of the finished form.
An official product is a drug product, dietary supplement,
compounded preparation, or finished device for which a
monograph is provided.
2.30. Legal Recognition
The USP and NF are recognized in the laws and regula-
tions of many countries throughout the world. Regulatory
authorities may enforce the standards presented in the USP
and NF, but because recognition of the USP and NF may
vary by country, users should understand applicable laws
and regulations. In the United States under the Federal
Food, Drug, and Cosmetic Act (FDCA), both USP and NF are
recognized as official compendia. A drug with a name rec-
ognized in USP-NF must comply with compendial identit
standards or be deemed adulterated, misbranded, or both.
See, e.g., FDCA § 501(b) and 502(e)(3)(b); also FDA regula-
tions, 21 CFR § 299.5(a&b). To avoid being deemed
adulterated, such drugs must also comply with compendial
standards for strength, quality, and purity, unless labeled to
show all respects in which the drug differs. See, e.g., FDCA
§ 501(b) and 21 CFR § 299.5(c). In addition, to avoid being
deemed misbranded, drugs recognized in USP-NF must also
be packaged and labeled in compliance with compendial
standards. See FDCA § 502(g).
A dietary supplement represented as conforming to speci-
fications in USP will be deemed a misbranded foodif it fails
to so conform. See FDCA § 403(s)(2)(D).
Enforcement of USP standards is the responsibility of FDA
and other government authorities in the U.S. and elsewhere.
USP has no role in enforcement.
Change to read:
3. CONFORMANCE TO STANDARDS
3.10, Applicability of Standards
Standards for an article recognized in the compendia
(USP-NF) are expressed in the article’s monograph, applica-
ble general chapters, and General Notices. The identity,
strength, quality, and purity of an article are determined by
the official tests, procedures, and acceptance criteria, and
other requirements incorporated in the monograph, in ap-
plicable general chapters, or in the General Notices. “Appli-
cable general chapters” means general chapters numbered
 x General Notices
below 1000 or above 2000 that are made applicable to an
article through reference in General Notices, a monograph,
or another applicable general chapter numbered below
1000. Where the requirements of a monograph differ from
the requirements specified in these General Notices or an
applicable general chapter, the monograph requirements
apply and supersede the requirements of the General Notices
or applicable general chapters, whether or not the mono-
graph explicitly states the difference.
General chapters numbered 1000 to 1999 are for infor-
mational purposes only. They contain no mandatory tests,
assays, or other requirements applicable to any official arti-
General Notices
cle, regardless of citation in a general chapter numbered
below 1000, a monograph, or these General Notices. Gen-
eral chapters numbered above 2000 apply only to articles
that are intended for use as dietary ingredients and dietary
spelen. General chapter citations in NF monographs
refer to USP general chapters.
Early adoption of revised standards in advance of the offi-
cial date is allowed by USP unless specified otherwise at the
time of publication. Where revised standards for anexisting
article have been published as final approved “official text”
(as approved in section 2.10 Official Text) but have not yet
reached the official date (six months after publication, un-
less otherwise specified; see “official date”, section 2.20. Of-
ficial Articles), compliance with the revised standard shall not
preclude a finding or indication of conformance with com-
pendial standards, unless USP specifies otherwise by prohib-
iting early adoption in a particular standard.
The standards in the relevant monograph, general chap-
ter(s), and General Notices apply at all times in the life of the
article from production to expiration. It is also noted that
the manufacturer's specifications, and manufacturing prac-
tices (e.g., Quality by Design, Process Analytical Technology,
and Real Time Release Testing initiatives), generally are fol-
lowed to ensure that the article will comply with com-
pendial standards until its expiration date, when stored as
directed. Every compendial article in commerce shall be so
constituted that when examined in accordance with these
assays and test procedures, it meets all applicable pharma-
copeial requirements (General Notices, monographs, and
general chapters). Thus, any official article is expected to
meet the compendial standards if tested, and any official
article actually tested as directed in the relevant monograph
must meet such standards to demonstrate compliance.
Some tests, such as those for Dissolution and Uniformity of
Dosage Units, require multiple dosage units in conjunction
with a decision scheme. These tests, albeit using a number
of dosage units, are in fact one determination. These proce-
dures should not be confused with statistical sampling
plans. The similarity to statistical procedures may seem to
suggest an intent to make inference to some larger group of
units, but in all cases, statements about whether the com-
pendial standard is met apply only to the units tested. Re-
peats, replicates, statistical rejection of outliers, or extrapola-
tions of results to larger populations, as well as the necessity
and appropriate regusney of batch testing, are neither
specified nor proscribed by the compendia; such decisions
are based on the objectives of the testing. Frequency of
testing and sampling are left to the preferences or direction
of those performing compliance testing, and other users of
USP-NF, including manufacturers, buyers, or regulatory
authorities.
Official products are prepared according to recognized
prneipies of good manufacturing practice and from ingredi-
ents that meet USP or NF standards, where standards for
such ingredients exist (for dietary supplements, see section
3.10.20 Applicability of Standards to Medical Devices, Dietary
Supplements, and Their Components and Ingredients).
Official substancesate peppared according to recognized
principles of good manufacturing practice and from ingredi-
ents complying with specifications designed to ensure that
the resultant substances meet the requirements of the com-
pendial monographs.
USP 41
3.10.10. Applicability of Standards to Drug Products,
Drug Substances, and Excipients
The applicable USP or NF standard applies to any article
marketed in the United States that (1) is recognized in the
compendium and (2) is intended or labeled for use as a
drug or as an ingredient in a drug. Such articles (drug prod-
ucts, rag substances, and excipients) include both human
drugs (whether dispensed by prescription, “over the
counter,” or otherwise), as well as animal drugs. The appli-
cable standard applies to such articles whether or not the
added designation “USP” or “NF” is used. The standards
apply equally to articles bearing the official titles or names
derived by transposition of the definitive words of official
titles or transposition in the order of the names of two or
more Adrug substancesauses: in official titles, or where there
is use of synonyms with the intent or effect of suggesting a
significant degree of identity with the official title or name.
3.10.20. Applicability of Standards to Medical Devices,
Dietary Supplements, and Their Components and
Ingredients
An article recognized in USP or NF shall comply with the
compendial standards if the article is a medicaldevice: com-
ponent intended for a medical device, dietary supplement,
dietary ingredient, or other ingredient that is intended for
incorporation into a dietary supplement, and is labeled as
conforming to the USP or NF.
Generally, dietary supplements are prepared from ingredi-
ents that meet USP, NF, or Food Chemicals Codex standards.
Where such standards do not exist, substances may be used
in dietary supplements if they have been shown to be of
acceptable food grade quality using other suitable
procedures.
3.10.30. Applicability of Standards to the Practice of
Compounding (New)
USP compounding practice standards, Pharmaceutical
Compounding—Nonsterile Preparations (795) and Pharmaceu-
tical Compounding—Sterile Preparations (797), as appropriate,
apply to compounding practice or activity regardless of
whether a monograph exists for the compounded prepara-
tion or these chapters are referenced in such a monograph.
In the United States, (795) and (797) are not applicable to
drugs compounded by entities registered with FDA as out-
sourcing facilities as defined by FDCA § 503B, because such
facilities are required to comply with FDA’s current good
manufacturing practice requirements. Compounded prepa-
rations, including drug products compounded by outsourc-
ing facilities, may also be subject to applicable monographs;
see section 2.20 Official Articles and section 4.10
Monographs.
3.20. Indicating Conformance
A drug product, drug substance, or excipient may use the
designation “USP” or “NF” in conjunction with its official
title or elsewhere on the label only when (1) a monograph
is provided in the specified compendium and (2) the article
complies with the identity prescribed in the specified
compendium.
Whena drug product, drug substance, compounded
preparation, or excipient differs from the relevant USP or NF
standard ofstrength, quality, or purity, as determined by
the application of the tests, procedures, and acceptance cri-
teria set forth in the relevant compendium, its difference
shall be plainly stated on its label.
When a drug product, drug substance, compounded
preparation, or excipient fails to comply with the identity
prescribed in USP or NF or contains an added substance that
interferes with the prescribed tests and procedures, the arti-
cle shall be designated by a name that is clearly distinguish-
ing and differentiating from any name recognized in USP or
NF.
A medical device, dietary supplement, or ingredient or
component of a medical levee or dietary supplement may
use the designation “USP” or “NF” in conjunction with its
official title or elsewhere on the label only when (1) a mon-
ograph is provided in the specified compendium and (2)
USP 41
the article complies with the monograph standards and
other applicable standards in that compendium.
The designation “USP” or “NF” on the label may not and
does not constitute an endorsement by USP and does not
represent assurance by USP that the article is known to
comply with the relevant standards. USP may seek legal re-
dress if an article purports to be or is represented as an
official article in one of USP’s compendia and such claim is
determined by USP not to be made in good faith.
The designation “USP-NF” may be used on the label of
an article provided that the label also bears a statement
such as “Meets NF standards as published by USP,” indicat-
ing the particular compendium to which the article purports
to apply.
When the letters “USP,” “NF,” or “USP—NF” are used on
the label of an article to indicate compliance with com-
pendial standards, the letters shall appear in conjunction
with the official title of the article. The letters are not to be
enclosed in any symbol such as acircle, square, etc., and
shall appear in capital letters.
If a dietary supplement does not comply with all applica-
ble compendial requirements but contains one or more die-
tary ingredients or other ingredients that are recognized in
USP or NF, the individual ingredient(s) may be designated as
complying with USP or NF standards or being of USP or NF
quality provided that the designation is limited to the indi-
vidual ingredient(s) and does not suggest that the dietary
supplement complies with USP standards.
4. MONOGRAPHS AND GENERAL CHAPTERS
4.10. Monographs
Monographs set forth the article’s name, definition, speci-
fication, and other requirements related to packaging, stor-
age, and labeling. The specification consists ofrests; ploce:
dures, and acceptance criteria that help ensure the identity,
strength, quality, and purity of the article. For general re-
quirements relating to specific monograph sections, see sec-
tion 5 Monograph Components.
Because monographs may not provide standards for all
relevant characteristics, some official substances may con-
form to the USP or NF standard but differ with regard to
nonstandardized properties that are relevant to their use in
specific preparations. To assure substitutability in such in-
stances, users may wish to ascertain functional equivalence
or determine such characteristics before use.
4.10.10. Applicability of Test Procedures
A single monograph may include more than one test,
proce and/or acceptance criterion for the same attri-
ute. Unless otherwise specified in the monograph, all tests
are requirements. In some cases, monograph instructions al-
low the selection of tests that reflect attributes of different
manufacturers’ articles, such as different polymorphic forms,
impurities, hydrates, and dissolution. Monograph instruc-
tions indicate the tests, procedures, and/or acceptance crite-
ria to be used and the required labeling.
The order in which the tests are listed in the monograph
is based on the order in which they are approved by the
relevant Expert Committee for inclusion in the monograph.
Test 1 is not necessarily the test for the innovator or for the
reference product. Depending on monograph instructions, a
labeling statement is not typically required if Test 1 is used.
4.10.20. Acceptance Criteria
The acceptance criteria allow for analytical error, for una-
voidable variations in manufacturing and compounding, and
for deterioration to an extent considered acceptable under
practical conditions. The existence of compendial accep-
tance criteria does not constitute a basis for a claim that an
official substance that more nearly approaches 100% purity
“exceeds” compendial quality. Similarly, the fact that an ar-
ticle has been prepared to tighter criteria than those speci-
fied in the monograph does not constitute a basis for a
claim that the article “exceeds” the compendial
requirements.
An official product shall be formulated with the intent to
provide 100% of the quantity of each ingredient declared
General Notices xi
on the label. Where the minimum amount of a substance
prea in a dietary supplement is required by law to be
igher than the lower acceptance criterion allowed for in
the monograph, the upper acceptance criterion contained
in the monograph may be increased by a corresponding
amount.
The acceptance criteria specified in individual monographs
and in the general chapters for compounded Peres
are based on such attributes of quality as might be ex-
ae to characterize an article compounded from suitable
ulk drug substances and ingredients, using the procedures
provided or recognized principles of good compounding (9)
practice, as described in these compendia. fe}
4,20. General Chapters
si
Each general chapter is assigned a number that appears in
1)
angle brackets adjacent to the chapter name (e-9. Chroma- =
tography (621)). General chapters may contain the Zz
following: °
a7
¢ Descriptions of tests and procedures for application a)
through individual monographs, ©
"
¢ Descriptions and specifications of conditions and prac-
tices for pharmaceutical compounding,
° General information for the interpretation of the com-
pendial requirements,
e Descriptions of general pharmaceutical storage, dispens-
ing, and packaging practices, or
e General guidance to manufacturers of official substances
or official products.
Whena general chapter is referenced in a monograph,
acceptance criteria may be presented after a colon.
Some chapters may serve as introductory overviews of a
test or of analytical techniques. They may reference other
general chapters that contain techniques, details of the pro-
cedures, and, at times, acceptance criteria.
Change to read:
5. MONOGRAPH COMPONENTS
5.10. Molecular Formula
The use of the molecular formula for the 4official sub-
stance(S)ausps; named in defining the required strength of a
compendial article is intended to designate the chemical en-
tity or entities, as given in the complete chemical name of
the article, having absolute (100%) purity.
5.20. Added Substances
Added substances are presumed to be unsuitable for in-
clusion in an official atid and therefore prohibited, if their
presence impairs the bioavailability, therapeutic efficacy, or
safety of the official article; or they interfere with the assays
and tests prescribed for determining compliance with the
compendial standards (see section 3.20 Indicating
Conformance).
The air in a container of an official article may, where
appropiate, be evacuated or be replaced by carbon diox-
ide, helium, argon, or nitrogen, or by a mixture of these
gases. The use of such gas need not be declared in the
labeling.
5.20.10. Added Substances in Official Substances
Official substances may contain only the specific added
substances that are permitted by the individual monograph.
Such added substances shall not exceed the quantity re-
quired for providing their intended effect. Where such addi-
tion is permitted, the label shall indicate the name(s) and
amount(s) of any added substance(s).
5.20.20. Added Substances (Excipients and Ingredients)
in Official Products
Suitable substances and excipients such as antimicrobial
agents, pharmaceutical bases, carriers, coatings, flavors, pre-
servatives, stabilizers, and vehicles may be added to an offi-
cial product to enhance its stability, usefulness, or elegance,
or to facilitate its preparation, unless otherwise specified in
the individual monograph.
 xii General Notices
Added substances and excipients employed solely to im-
part color may be incorporated into official products other
than those intended for parenteral or ophthalmic use, in
accordance with the regulations pertaining to the use of
colors issued by the U.S. Food and Drug Administration
(FDA), provided such added substances or excipients are
otherwise appropriate in all respects. (See also Injections
and Implanted Drugs Products (1), Product Quality Tests Com-
mon to Parenteral Dosage Forms, Specific Tests, Vehicles and
added substances, Added substances.)
The proportions of the substances constituting the base in
ointment and suppository products and preparations may
General Notices
be varied to maintain a suitable consistency under different
climatic conditions, provided that the concentrations of
Adrug substancesausex are not varied and provided that the
bioavailability, therapeutic efficacy, and safety of the prepa-
ration are not impaired.
5.20.20.1. In Compounded Preparations
Compounded preparations for which a complete compo-
sition is given shall contain only the ingredients named in
the formulas unlessSpec exempted herein or in the
individual monograph. Deviation from the specified
processes or methods of compounding, although not from
the ingredients or proportions thereof, may occur provided
that the finished preparation conforms to the relevant stan-
dards and to preparations produced by following the speci-
fied process.
Where a monograph for a compounded preparation calls
for an ingredient in an amount expressed on the dried ba-
sis, the ingredient need not be dried before use if due al-
lowance is made for the water or other volatile substances
present in the quantity taken.
Specially denatured alcohol formulas are available for use
in accordance with federal statutes and regulations of the
Internal Revenue Service. A suitable formula of specially de-
natured alcohol may be substituted for Alcohol in the manu-
facture of official preparations intended for internal or topi-
cal use, provided that the denaturant is volatile and does
not remain in the finished product. A preparation that is
intended for topical app eaen to the skin may contain spe-
cially denatured alcohol, provided that the denaturant is ei-
ther a usual ingredient in the preparation or a permissible
added substance; in either case the denaturant shall be
identified on the label of the topical preparation. Where a
process is given in the individual recede ae prepara-
tion compounded using denatured alcohol shall be identical
to that prepared by the monograph process.
5.20.20.2. In Dietary Supplements
Additional ingredients may be added to dietary supple-
ment products provided that the additional ingredients: (1)
comply with applicable regulatory requirements; and (2) do
not interfere with the assays and tests prescribed for deter-
mining compliance with compendial standards.
5.30. Description and Solubility
Only where a quantitative solubility test is given in a
monograph and is designated as such is it a test for purity.
A monograph may include information regarding the arti-
cle’s description. Information about an article’s “description
and solubility” also is provided in the reference table
Description and Relative Solubility of USP and NF Articles. The
reference table merely denotes the properties of articles that
comply with monograph standards. The reference table is
intended primarily for those who use, prepare, and dispense
drugs conor related articles. Although the information pro-
vided in monographs and the information in the reference
table may indirectly assist in the preliminary evaluation of an
article, it is not intended to serve as a standard or test for
purity.
The approximate solubility of a compendial substance is
indicated by one of the following descriptive terms:
USP 41
Parts of Solvent Required
Less than 1
From 1 to 1
From 1
to 100
100 to 1
‘om 1
Greater than or equal to
ble, or | 10,000
5.40. Aldentificationausrs;
A compendial test titled 4auses; Identification is provided as
an aid in verifying the identity of articles as they are pur-
ported to be, e.g., those taken from labeled containers, and
to establish whether it is the article named in USP-NF. The
Agusea Identification test for a particular article may consist of
one or more procedures. When a compendial 4auspa; Identifi-
cation Atestaysps; is undertaken, all requirements of all speci-
fied procedures in the test must be met to satisfy the re-
quirements of the test. Failure of an article to meet all the
requirements of a prescribed 4gusp) Identification test (i.e.,
failure to meet the requirements of all of the specified pro-
cedures that are components of that test) indicates that the
article is mislabeled and/or adulterated.
5.50. Assay
Lee) tests for compounded preparauions are not in-
tended for evaluating a compounded preparation before
dispensing, but instead are intended to serve as the official
test in the event of a question or dispute regarding the
preparation’s conformance to official standards.
5.50.10. Units of Potency (Biological)
For substances that cannot be completely characterized
by chemical or physical means or that need confirmation of
functionality or tertiary structure, it may be necessary to ex-
press quantities of biological activity in units of biological
potency, each defined by an authoritative, designated refer-
ence standard. In cases where international reference mater-
ials have been discontinued, international units of potency
may be defined in terms of molecular mass, such as in the
cases of vitamins A, D, and E.
Where available, World Health Organization (WHO) inter-
national biological standards define the International Units
(IU). USP monographs refer to the units assigned by USP
Reference Standaras either directly as International Units (IU)
or as “USP Units.” For some biological products, units of
potency are value assigned against a corresponding U.S.
Standard established by FDA, whether or not International
Units or USP Units have been defined (see Biologics (1041)).
Note that product-related labeling, e.g., on containers, need
not use the full phrase “USP [product name] Units” that
appears in many USP monograph labeling sections. The
term “USP Units” can be used on product labeling consis-
tent with USP compendial requirements, provided it is clear
from the context that the A4potencyauses; Is stated in terms
of USP [product name] Units. In such circumstances it
should be clear that “USP Units” and “USP [product name]
Units” share the same meaning.
5.60. Impurities and Foreign Substances
Tests for the presence of impurities and foreign substances
are ea to limit such substances to amounts that are
unobjectionable under conditions in which the article is cus-
tomarily employed (see also Impurities in Drug Substances
and Drug Products {1086)).
Nonmonograph tests and acceptance criteria suitable for
detecting and controlling impurities that may result from a
change in the processing methods or that may be intro-
duced from external sources should be employed in addi-
tion to the tests provided in the individual monograph,
where the presence of the impurity is inconsistent with ap-
plicable good manufacturing practices or good pharmaceu-
tical practices.
USP 41
5.60.10. Other Impurities in USP and NF Articles
If a USP or NF monograph includes an assay or organic
impurity test based on chromatography, other than a test
for residual solvents, and that monograph procedure does
not detect an impurity present in the substance, the amount
and identity of the impurity, where both are known, shall
be stated in the labeling (certificate of analysis) of the offi-
cial substance, under the heading Other Impurity(ies).
The presence of any unlabeled other impurity in an offi-
cial substance is a variance from the standard if the content
is 0.1% or greater. The sum of all Other Impurities combined
with the monograph-detected impurities may not exceed
2.0% (see Ordinary Impurities (466)), unless otherwise stated
in the monograph.
The following categories of drug substances are excluded
from Other Impurities requirements:
e Fermentation products and semi-synthetics derived
therefrom,
Radiopharmaceuticals,
Biologics,
Biotechnology-derived products,
Peptides,
Herbals, and
© Crude products of animal or plant origin.
Any substance known to be toxic shall not be listed under
Other Impurities.
5.60.20. Residual Solvents in USP and NF Articles
All USP and NF articles are subject to relevant control of
residual solvents, even when no test is specified in the indi-
vidual monograph. If solvents are used during production,
they must be of suitable quality. In addition, the toxicity
residual level of each solvent shall be taken into consid-
eration, and the solvents limited according to the principles
defined and the requirements specified in Residual Solvents
(467), using the general methods presented therein or other
suitable methods.
5.60.30. Elemental Impurities in USP Drug Products and
Dietary Supplements
A,guses1 Elemental impurities 4auses; in official drug prod-
ucts 4are controlledauses; according to the principles defined
and requirements specified in Elemental Impurities—Limits
(232). Sausear Elemental contaminants Aausps; in official die-
tary supplements 4are controlledavsps; according to the prin-
ciples defined and requirements specified in Elemental Con-
taminants in Dietary Supplements (2232). ®auseai
5.70. Performance Tests
Where content uniformity determinations have been
made using the same analytical methodology specified in
the Assay, with appropriate allowances made for differences
in sample preparation, the average of all of the individual
content uniformity determinations may be used as the Assay
value.
5.80. USP Reference Standards
USP Reference Standards are authentic specimens that
have been approved as suitable for use as comparison stan-
dards in USP or NF tests and assays. (See USP Reference Stan-
dards (11).) Where USP or NF tests or assays call for the use
of a USP Reference Standard, only those results obtained
using the specified USP Reference Standard are conclusive.
Where a procedure calls for the use of a compendial article
rather than for a USP Reference Standard as a material stan-
dard of reference, a substance meeting all of the com-
pendial monograph requirements for that article shall be
used. If any new USP or NF standard requires the use of a
new USP Reference Standard that is not yet available, that
eee of the standard containing the requirement shall not
e official until the specified USP reference material is
available.
Unless a Reference Standard label bears a specific potency
or content, assume the Reference Standard is 100.0% pure
in the official application. Unless otherwise directed in the
procedure in the individual monograph or in a general
chapter, USP Reference Standards are to be used in accor-
General Notices xiii
dance with the instructions on the label of the Reference
Standard.
Change to read:
6. TESTING PRACTICES AND PROCEDURES
6.10. Safe Laboratory Practices
In performing compendial procedures, safe laboratory
practices shall be followed, including precautionary meas-
ures, protective equipment, and work practices consistent
with the chemicals and procedures used. Before undertaking
Cebypte CCIEID)
any procedure descuived In the compendia, the analyst
should be aware of the hazards associated with the chemi-
cals and the techniques and means of protecting against
them. These compendia are not designed to describe such
hazards or protective measures.
6.20. Automated Procedures
Automated and manual procedures employing the same
basic chemistry are considered equivalent 4provided the au-
tomated system is properly qualified as being suitable to
execute the compendial manual method and the analytical
procedure is verified under the new equipment conditions.
AUSPAT
6.30. Alternative and Harmonized Methods and
Procedures
4An alternative method or procedure is defined as any
method or ete other than the compendial method or
procedure for the article in question. The alternative method
or procedure must be fully validated (see Validation of Com-
pendial Procedures (1225)) and must produce comparable re-
sults to the compendia! method or procedure within allowa-
ble limits established on a case-by-case basis. Alternative
methods or procedures can be developed for any one of a
number of reasons not limited to simplification of sample
preparation, enhanced precision and accuracy, improved
(shortened) run time, or being better suited to automation
than the compendial method or procedure.gu:-z, Only those
results obtained by the methods and procedures given in
the compendia are conclusive.
4For evaluation as a potential replacement or addition to
the standard, ausr4: alternative 4methods andauses: proce-
dures should be submitted to USP 4ausps; (see section 4.70.
Monographs).
Certain general chapters contain a statement that the text
in question is harmonized with the corresponding text of
the European Pharmacopoeia and/or the Japanese Pharmaco-
poeia and that these texts are interchangeable. Therefore, if
a substance or preparation is found to comply with a re-
quirement using an interchangeable method or procedure
from one of these pharmacopeias, it should comply with the
requirements of the USP-NF. When a difference appears, or
in the event of dispute, only the result obtained by the
method and/or procedure given in the USP-NF is conclusive.
6.40. Dried, Anhydrous, Ignited, or Solvent-Free Basis
All calculations in the compendia assume an “as-is” basis
unless otherwise specified.
Test procedures may be performed on the undried or
unignited substance and the results calculated on the dried,
anhydrous, or ignited basis, provided a test for Loss on Dry-
ing, or Water Determination, or Loss on Ignition, respectively,
is given in the Pe orate Where the presence of moisture
or other volatile material may interfere with the procedure,
previous ae of the substance is specified in the individ-
ualmonograp and is obligatory.
The term “solvent-free” signifies that the calculation shall
be corrected for the presence of known solvents as deter-
mined using the methods described in (467) unless a test
for limit of organic solvents is provided in the monograph.
The term “previously dried” without qualification signifies
that the substance shall be dried as directed under Loss on
Drying (731) or Water Determination (921) (gravimetric
determination).
 xiv General Notices
Where drying in vacuum over a desiccant is directed, a
vacuum desiccator, a vacuum drying pistol, or other suitable
vacuum drying apparatus shall be used.
6.40.10. Ignite to Constant Weight
“Ignite to constant weight” means that ignition shall be
continued at 800
+25°, unless otherwise indicated, until
two consecutive weighings, the second of which is taken
after an additional period appropriate to the nature and
quantity of the residue, do not differ by more than 0.50 mg
per g of substance taken.
6.40.20. Dried to Constant Weight
w
i “Dried to constant weight” means that drying shall be
a continued until two consecutive weighings, the second of
= weighed accurately. The portion of mixed Capsules contents
fo} which is taken after an additional drying period appropriate
vA to the nature and quantity of the residue, do not differ by
more than 0.50 mg per g of substance taken.
wo
io
ev
6.50. Preparation of Solutions
& 6.50.10. Filtration
v Where a procedure gives direction to “filter” without fur-
UO ther qualification, the liquid shall be passed through suitable
filter paper or equivalent device until the filtrate is clear.
Due to the possibility of filter effects, the initial volumes of a
filtrate may be discarded.
6.50.20. Solutions
Unless otherwise specified, all solutions shall be prepared
with Purified Water. Solutions for quantitative measures shall
be prepared using accurately weighed or accurately meas-
ured analytes (see section 8.20 About).
An expression such as “(1 in 10)” means that 1 part by
volume of a liquid shall be diluted with, or 1 part by weight
of a solid shall be dissolved in, a sufficient quantity of the
diluent or solvent to make the volume of the finished solu-
tion 10 parts by volume. 4For example, a 1 in 10 solution is
prepared by diluting 1 mL of a liquid or dissolving 1 g of a
solid in sufficient solvent to make 10 mL of the solution.
usps An expression such as “(20:5:2)” means that the re-
spective numbers of parts, by volume, of the designated
liquids shall be mixed, unless otherwise indicated.
6.50.20.1. Adjustments to Solutions
When a specified concentration is called for in a proce-
dure, a solution of other normality or molarity may be used,
provided that allowance is made for the difference in con-
centration and that the change does not increase the error
of measurement.
Proportionately larger or smaller quantities than the speci-
fied weights and volumes of assay or test substances and
Reference Standards may be taken, provided the measure-
ment is made with at least equivalent accuracy.
Unless otherwise indicated, analyte concentrations shall be
prepared to within ten percent (10%) of the indicated
value. In the case in which a procedure is adapted to the
working range of an instrument, solution concentrations
may differ from the indicated value by more than ten per-
cent (10%), with appropriate changes in associated calcula-
tions. Any changes shall fall within the validated range of
the instrument.
When adjustment of pH is indicated with either an acid or
base and the concentration is not indicated, appropriate
concentrations of that acid or base may be used.
6.50.20.2. Test Solutions
Information on Test Solutions (TS) is provided in the Test
Solutions portion of the Reagents, Indicators, and Solutions
section of the USP-NF. Use of an alternative Test Solution or
a change in the Test Solution used may require validation.
6.50.20.3. Indicator Solutions
Where a procedure specifies the use of an indicator TS,
approximately 0.2 mL, or 3 drops, of the solution shall be
added unless otherwise directed.
6.60. Units Necessary to Complete a Test
Unless otherwise specified, a sufficient number of units to
ensure a suitable analytical result shall be taken.
USP 41
6.60.10. Tablets
Where the procedure of a Tablet monograph directs to
weigh and finely powder not fewer than a given number of
Tablets, a counted number of Tablets shall be weighed and
reduced to a powder. The portion of the powdered Tablets
taken shall be representative of the whole Tablets and shall,
in turn, be weighed accurately.
6.60.20. Capsules
Where the procedure of a Capsule monograph gives di-
rection to remove, as completely as possible, the contents of
not fewer than a given number of the Capsules, a counted
number of Capsules shall be carefully opened and the con-
tents quantitatively removed, combined, mixed, and
taken shall be representative of the contents of the Capsules
and shall, in turn, be weighed accurately.
6.70. Reagents
The proper conduct of the compendial procedures and
the reliability of the results depend, in part, upon the quality
of the reagents used in the performance of the procedures.
Unless otherwise specified, reagents conforming to the spec-
ifications set forth in the current edition of Reagent Chemi-
cals published by the American Chemical Society (ACS) shall
be used. Where such ACS reagent specifications are not
available or where the required purity differs, compendial
specifications for reagents of acceptable quality are provided
(see the Reagents, Indicators, and Solutions section of the
USP-NF). Reagents not covered by any of these specifica-
tions should be of a grade suitable to the proper perfor-
mance of the method of assay or test involved.
Listing of these reagents, including the indicators and so-
lutions employed as reagents, in no way implies that they
have therapeutic utility; furthermore, any reference to USP
or NF in their labeling shall include also the term “reagent”
or “reagent grade.” USP may supply reagents if they other-
wise may not be generally commercially available.
6.80. Equipment
Unless otherwise specified, a specification for a definite
size or type of container or apparatus in a procedure is
given solely as a recommendation. Other dimensions or
types may be used if they are suitable for the intended use.
6.80.10. Apparatus for Measurement
Where volumetric flasks or other exact measuring, weigh-
ing, or sorting devices are specified, this or other equipment
of at least equivalent accuracy shall be employed.
6.80.10.1. Pipet/Pipette
Where a pipet/pipette is specified, a suitable buret may be
substituted. Where a “to contain” pipet/pipette is specified,
a suitable volumetric flask may be substituted.
6.80.10.2. Light Protection
Where low-actinic or light-resistant containers are speci-
fied, either containers specially treated to protect contents
from light or clear containers that have been rendered
opaque by application of a suitable coating or wrapping
may be used.
6.80.20. Instrumental Apparatus
An instrument may be substituted for the specified instru-
ment if the substitute uses the same fundamental principles
of operation and is of equivalent or greater sensitivity and
accuracy. These characteristics shall be qualified as appropri-
ate. Where a particular brand or source of a material, instru-
ment, or piece of equipment, or the name and address of a
manufacturer or distributor, is mentioned (ordinarily in a
footnote), this identification is furnished solely for informa-
tonal PUIBOSE as a matter of convenience, without implica-
tion of approval, endorsement, or certification.
6.80.20.1. Chromatographic Tubes and Columns
The term “diameter” refers to internal diameter (ID).
6.80.20.2. Tubing
The term “diameter” refers to outside diameter (OD).
USP 41 General Notices xv

6.80.20.3. Steam Bath 7.20. Rounding Rules

Where use of a steam bath is directed, use actively flow-
ing steam or another regulated heat source controlled at an
equivalent temperature.
6.80.20.4. Water Bath
A water bath requires vigorously boiling water unless oth-
erwise specified.
6.80.30. Temperature Reading Devices
Temperature reading devices suitable for pharmacopeial
tests conform to specifications that are traceable to a Na-
tional Institute of Standards and Technology (NIST) standard
or equivalent. Temperature reading devices may be of the
liquid-in-glass type or an analog or digital temperature indi-
cator type, such as a resistance temperature device, thermis-
tor, or thermocouple. Standardization of thermometers is
performed on an established testing frequency with a tem-
perature standard traceable to NIST. For example, refer to
the current issue of American Society of Testing and Materi-
als (ASTM) standards E1 for liquid-in-glass thermometers.
7. TEST RESULTS
7.10. Interpretation of Requirements
Analytical results observed in the laboratory (or calculated
from experimental measurements) are compared with stated
acceptance criteria to determine whether the article con-
forms to compendial requirements.
The reportable value, which often is a summary value for
several individual determinations, is compared with the ac-
ceptance criteria. The reportable value is the end result of a
completed measurement procedure, as documented.
Where acceptance criteria are expressed numerically
herein through specification of an upper and/or lower limit,
ermitted values include the specified values themselves,
ut no values outside the limit(s). Acceptance criteria are
considered significant to the last digit shown.
7.10.5. Nominal Concentrations in Equations
Where a “nominal concentration” is specified, calculate
the concentration based on the label claim. In assay proce-
dures, water correction is typically stated in the Definition
and on the label of the USP Reference Standard. For other
procedures, correction for assayed content, potency, or both
is made prior to using the concentration in the equation
provided in the monograph.
7.10.10. Equivalence Statements in Titrimetric
Procedures
The directions for titrimetric procedures conclude with a
statement of the weight of the analyte that is equivalent to
each mL of the standardized titrant. In such an equivalence
statement, the number of significant figures in the concen-
tration of the titrant should he understood to correspond to
the number of significant figures in the weight of the
analyte. Corrections to calculations based on the blank de-
termination are to be made for all titrimetric assays where
appropriate (see Titrimetry (541)).
The observed or calculated values shall be rounded off to
the number of decimal places that is in agreement with the
limit expression. Numbers should not be rounded until the
final calculations for the reportable value have been com-
pleted. Intermediate calculations (e.g., slope for linearity)
may be rounded for reporting purposes, but the original
(not rounded) value should be used for any additional re-
quired calculations. Acceptance criteria are fixed numbers
and are not rounded.
When rounding is required, consider only one digit in the
decimal place to the right of the last place in the limit ex- (a)
pression. If this digit is smaller than 5, it is eliminated and ©
the preceding digit is unchanged. If this digit is equal to or |]
©
greater than 5, it is eliminated and the preceding digit is be
ae
increased by 1.
8. TERMS AND DEFINITIONS rs
°
8.10. Abbreviations i
© RS refers to a USP Reference Standard. a
o
° CS refers to a Colorimetric Solution. “
e TS refers to a Test Solution.
VS refers to a Volumetric Solution that is standardized in
accordance with directions given in the individual mon-
ograph or in the Reagents, Indicators, and Solutions sec-
tion of USP-NF.
8.20. About
“About” indicates a quantity within 10%.
If the measurement is stated to be “accurately measured”
or “accurately weighed,” follow the statements in Volumetric
Apparatus (31) and Balances (41), respectively.
8.30. Alcohol Content
Percentages of alcohol, such as those under the heading
Alcohol Content, refer to percentage by volume of C,HsOH
at 15.56°. Where a formula, test, or assay calls for alcohol,
ethyl alcohol, or ethanol, the USP monograph article Alcohol
shall be used. Where reference is made to “C2HsOH,” abso-
lute (100%) ethanol is intended. Where a procedure calls for
dehydrated alcohol, alcohol absolute, or anhydrous alcohol,
nen monograph article Dehydrated Alcohol shall be
used.
8.40. Atomic Weights
Atomic weights used in computing molecular weights and
the factors in the assays and elsewhere are those established
by the IUPAC Commission on Isotopic Abundances and
Atomic Weights.
8.50. Blank Determinations
Where it is directed that “any necessary correction” be
made by a blank determination, the determination shall be
conducted using the same quantities of the same reagents
treated in the same manner as the solution or mixture con-
taining the portion of the substance under assay or test, but
with the substance itself omitted.

Illustration of Rounding Numerical Values

for Comparison with Requirements
Compendial Requirement Unrounded Value Rounded Result Conforms
Assay limit 298.0% 97.96% 98.0% Yes
97.92% 97.9% No
97.95% 98.0% Yes
Assay limit <101.5% 101.55% 101.6% No
101.46% 101.5% Yes
101.45% 101.5% Yes
Limit test <0.02% 0.025% 0.03% No
0.015% 0.02% Yes
0.027% 0.03% No
Limit test <3 ppm 3.5 ppm 4 ppm No
3.4 ppm 3 ppm Yes
2.5 ppm 3 ppm Yes
 xvi General Notices
8.60. Concomitantly
“Concomitantly” denotes that the determinations or
measurements are to be performed in immediate
succession.
8.70. Desiccator
The instruction “in a desiccator” indicates use of a tightly
closed container of suitable size and design that maintains
an atmosphere of low moisture content by means ofa suita-
ble desiccant such as anhydrous calcium chloride, magne-
sium perchlorate, phosphorus pentoxide, or silica gel. See
also section 8.220 Vacuum Desiccator.
4
vu 8.80. Logarithms
3y
Logarithms are to the base 10.
° 8.90. Microbial Strain
a A microbial strain cited and identified by its American
rc Type Culture Collection (ATCC) catalog number shall be
~~ used directly or, if subcultured, shall be used not more than
a
= five passages removed from the original strain.
7 8.100. Negligible
Oo “Negligible” indicates a quantity not exceeding 0.50 mg.
8.110. NLT/NMT
“NLT” means “not less than.” “NMT” means “not more
than.”
8.120. Odor
“Odorless,” “practically odorless,” “a faint characteristic
odor,” and variations thereof indicate evaluation of a suita-
ble quantity of freshly opened material after exposure to the
air for 15 minutes. An odor designation is descriptive only
and should not be regarded as a standard of purity for a
particular lot of an article.
8.130. Percent
“Percent” used without qualification means:
e For HSBC of solids and semisolids, percent weight in
weight;
° For acliaiens or suspensions of solids in liquids, percent
weight in volume;
¢ For solutions of liquids in liquids, percent volume in
volume;
° For solutions of gases in liquids, percent weight in
volume.
For example, a 1 percent solution is prepared by dissolv-
ing 1 g of a solid or semisolid, or 1 mL of a liquid, in suffi-
cient solvent to make 100 mL of the solution.
8.140. Percentage Concentrations
Percentage concentrations are expressed as follows:
© Percent Weight in Weight (w/w) is defined as the num-
ber of g of a solute in 100 g of solution.
© Percent Weight in Volume (w/v) is defined as the number
of g of a solute in 100 mL of solution.
© Percent Volume in Volume (v/v) is defined as the number
of mL of a solute in 100 mL of solution.
8.150. Pressure
Pressure is determined by use of a suitable manometer or
barometer calibrated in terms of the pressure exerted by a
column of mercury of the stated height.
8.160. Reaction Time
Reaction time is 5 minutes unless otherwise specified.
8.170. Specific Gravity
Specific gravity is the weight of a substance in air at 25°
divided by the weight of an equal volume of water at the
same temperature.
8.180. Temperatures
Temperatures are expressed in centigrade (Celsius) de-
grees, and all measurements are made at 25° unless other-
wise indicated. Where moderate heat is specified, any tem-
perature not higher than 45° (113° F) is indicated.
8.190. Time
Unless otherwise specified, rounding rules, as described in
section 7.20 Rounding Rules, apply to any time specified.
8.200. Transfer
“Transfer” indicates a quantitative manipulation.
USP 41
8.210. Vacuum
“Vacuum” denotes exposure to a pressure of less than
20 mm of mercury (2.67 kPas), unless otherwise indicated.
8.220. Vacuum Desiccator
“Vacuum desiccator” indicates a desiccator that maintains
a low-moisture atmosphere at a reduced pressure of not
more than 20 mm of mercury (2.67 kPas) or at the pressure
designated in the individual monograph.
8.230. Water
8.230.10. Water as an Ingredient in an Official Product
As an ingredient in an official product, water meets the
requirements of the appropriate water monograph in USP or
NF.
8.230.20. Water in the Manufacture of Official
Substances
When used in the manufacture of official substances,
water shall meet the requirements for drinking water as set
forth in the U.S. Environmental Protection Agency National
Primary Drinking Water Regulations or in the drinking water
regulations of the European Union or of Japan, or in the
World Health Organization’s Guidelines for Drinking Water
Quality. Additional specifications may be required in
monographs.
8.230.30. Water in a Compendial Procedure
When water is called for in a compendial procedure, the
USP monograph article Purified Water shall be used unless
otherwise specified. Definitions for other types of water are
provided in Reagents, Indicators, and Solutions and in Water
for Pharmaceutical Purposes (1231).
8.240. Weights and Measures
In general, weights and measures are expressed in the
International System of Units (SI) as established and revised
by the Conférence générale des poids et mesures. For com-
pendial purposes, the term “weight” is considered to be
synonymous with “mass.”
Molality is designated by the a m precededby a
number that represents the number of moles of the desig-
nated solute contained in 1 kilogram of the designated
solvent.
eae is designated by the ure M preceded by a
number that represents the number of moles of the desig-
nated solute contained in an amount of the designated sol-
vent that is sufficient to prepare1 liter of solution.
Normality is designated by the symbol N preceded by a
number that represents the number of equivalents of the
designated solute contained in an amount of the designated
solvent that is sufficient to prepare 1 liter of solution.
The symbol for degrees (°) without a qualifying unit of
measure represents degrees Celsius.
Chart of Symbols and Prefixes commonly employed for S|
metric units and other units:
Previously referred to
a mi
Previously the symbol
mu (for millimicron)
was
ual to im.
USP 41 General Notices xvii

Units Symbol Notes Units Symbol Notes

The symbol jg is used pounds per
in the USP and NF to square
represent micrograms, inch psi
but micrograms may millimeter
be represented as of mercu-
“meg” for labeling ry mmHg Equal to 133.322 Pa
and prescribing pur- Electrical
poses. The term units
“gamma,” symbolized
. ampere A
by y, frequently is
used to represent mi- volt V a
crograms in biochemi- millivolt, mv 2
microgram ug cal literature. hertz Hz Unit of frequency ro
nanogram ng kilohertz kHz Py
picogram pg megahertz MHz z
Also referred to as the electron °
unified atomic mass volt eV =a
unit and is equal to kilo-elec- Ay
1/12 times the mass tron volt keV a)
of the free carbon 12 mega-elec-
dalton Da atom. tron volt MeV
- kilodalton kDa Radiation
Time SI unit of activity for
second Ss becquerel Bq radionuclides
minute min kilobec-
hour h querel kBq
Volume megabec-
1 Lis equal to 1000 querel MBq
cm (cubic centime- gigabec-
liter L: ters) querel GBq
deciliter db Non-SI unit of activity
1 mL is equal to 1 cm3, curie Ci for radionuclides
sometimes referred to millicurie mCi
milliliter mL as CC microcurie uCi
microliter wh nanocurie ncr
Tempera- Other
ture acceleration
Celsius °C due to Used to express rate of
Amount of gravit g centrifugation
Substance revolutions
Historically referred to per min- Used to express rate of
as gram-molecular ute rpm centrifugation
weight or gram-atom-
mole mol ic weight
millimole mmol Selected SI Prefixes
micromole Lumol bol
femtomole fmol
Also referred to as
gram-equivalent
weight. It is used in
the calculation of sub-
stance concentration
in units of normality.
This unit is no longer micro
preferred for use in nano
analytical chemistry or
equivalent Eq metrology.
i . femto f
milli equiv-
oes mea 5 7 9. PRESCRIBING AND DISPENSING
“olution related to |
substance concentra:
9:10. Use of Metric Units | ,
Prescriptions for compendialarticles shall be written to
sericea Osiiol tion state the quantity and/or strength desired in metric units
5 * unless otherwise indicated in the individual monograph [see
mulliosmole mOsmol also section 5.50.70 Units of Potency (Biological) above]. If an
Pressure amount is prescribed by any other system of measurement,
pascal Pa only an amount that is the metric equivalent of the pre-
kilopascal kPa scribed amount shall be dispensed. Abbreviations for the
terms “Units” or “International Units” shall not be used for
 xviii General Notices USP 41

labeling or prescribing purposes. Apothecary unit designa- quirements (659), unless different requirements are provided
tions oa labels and labeling shall a be ued in an individual monograph.
9,20. Changes in Volume 10.20. Labeling
In the dispensing of prescription medications, slight All articles in USP or NF are subject to the labeling re-
changes in volume owing to variations in room tempera- quirements specified in Labeling (7), unless different require-
tures may be disregarded. ments are provided in an individual monograph.
10. PRESERVATION, PACKAGING, STORAGE, AND
LABELING
10.10. Packaging and Storage
All articles in USP or NF are subject to the packaging and
w storage requirements specified in Packaging and Storage Re-
oo)
m4
ver]
{o}
C4
ms
fo
7
c
o
oO
USP 41 Guide to General Chapters xix

Guide to General Chapters

(For complete alphabetical list of all general chapters in this Pharmacopeia, see under “General chapters” in the index.)

GENERAL TESTS AND ASSAYS (124) Erythropoietin Bioassays.............00. 6061

(126) Somatropin Bioidentity Tests ............ 6063
(127) Flow Cytometric Enumeration of CD34+
General Requirements Cells: oewsgotvieemes
aaoiElkHe VignetteasSe 6065
(129) Analytical Procedures for Recombinant
for Tests and Assays Therapeutic Monoclonal Antibodies ....... 6070
(130) Protein A Quality Attributes ............. 6076
(1) Injections and Implanted Drug Products (Parenter- CTS
1) PYTOGEN TOSt wince ie SUR Ns he tee.
als 6083
als)—Product Quality Tests .... 02. .....0.000, 5915 (161) Medical Devices—Bacterial Endotoxin and a
(2) Oral Drug Products—Product Quality Tests ... 5921 Pyrogen Tests sacksates « Pesan ltte led 6085 2)
(3) Topical and Transdermal Drug Products— =}
(162) Diphtheria Antitoxin Potency Testing for Human
Product Quality; Tests is. j, ance si enn ye.t eee ats 5926 Immune :Globulins: « «22 veh eek Se 6088 =
(4) Mucosal Drug Products—Product Quality (165) Prekallikrein Activator................-. 6070 o
“TOSES: & & 5 2 2 & 5 & Esisunldve surgetied ate temctonmlanhs 5933 (171) Vitamin Biz Activity Assay... 2... eee 6091 fa}
(5) Inhalation and Nasal Drug Products—General >
Information and Product Quality Tests ...... 5938 EY
(7) Labelling! 3 x + + 5 mesdvectonncact
enyanaysiedoaauctvours 5945 Chemical Tests and Assays mo)7
ol
(11) USP Reference Standards .............4-. 5951 rap’
Identification Tests my

Apparatus for Tests and Assays
(17) Prescription Container Labeling ........... 5954
(31) Volumetric Apparatus......2..2....0.000. 5957
Al) Balances. . . arohiomedanorsial ovindeala ed. Ot 5958
Microbiological Tests
(51) Antimicrobial Effectiveness Testing ......... 5959
(55) Biological Indicators—Resistance Performance
Tests Aer IN PIU Rooke, SMeDURSieMc) 5962
(61) Microbiological Examination of Nonsterile
Products: Microbial Enumeration Tests ..... . 5965
(62) Microbiological Examination of Nonsterile
Products: Tests for Specified Microorganisms. . 5971
(63) Mycoplasma Tests... 0.2... eeeeeeee 5978
(71) Sterility Tests 7 OEE dg ese 5984
Biological Tests and Assays
(81) Antibiotics—Microbial Assays .............% 5991
(85) Bacterial Endotoxins Test ................ 6011
(87) Biological Reactivity Tests, In Vitro ......... 6017
(88) Biological Reactivity Tests, In Vivo.......... 6020
(89) Enzymes Used as Ancillary Materials in
Pharmaceutical Manufacturing............ 6025
(89:1) Collagenase es i eiccneon sare §oeaRee 6029
(89.2) Collagenase IV) = ¢sss0-<5
s6 3.552FkRaGeoh 6033
(90) Fetal Bovine Serum—Quality Attributes and
Functionality Tests... 0.0... eee ee
(91) Calcium Pantothenate Assay..............
(92) Growth Factors and Cytokines Used in Cell
Therapy Manufacturing .............-005
(111) Design and Analysis of Biological Assays .
(115) Bexpanthenol ASSAY 2... eee ee ee ee
(121) Insulin Assays 2... eee eee eee
(121.1) Physicochemical Analytical Procedures
POR IMSUIINS cog a osgeeanuavas
Saoe2ow 8EERE
(181) Identification—Organic Nitrogenous Bases . . 6094
(191) Identification Tests—General............. 6094
(193) Identification—Tetracyclines ............. 6100
(197) Spectrophotometric Identification Tests..... 6101
(201)ThlecLaver Chromatographic Identification
TOSt ono gasses 6ey#2,92 pttatpiaate 6102
(202) Identification of Fixed Oils By Thin-Layer
Chromatography:jovi signin ath aoeatylmtn le 6103
(203) High-Performance Thin-Layer Chromatography
Procedure for Identification of Articles
of Botanical Origin ................000. 6105
Limit Tests
(206) Aluminum... 6. eee 6107
(207) Test for 1,6-Anhydro Derivative for Enoxaparin
SOUIUM: = este aoo9s¥eo5ReaEERSGS 6108
(208) Anti-Factor Xa and Anti-Factor lla Assays for
Unfractionated and Low Molecular Weight
Heparins.. 2... ee ee 6113
(209) Low Molecular Weight Heparin Molecular
Weight Determinations ................ 6117
(210) Monosaccharide Analysis .............2. 6118
OLA ASCNIC: Sy ean Alte Gee ey bas eee a es 6124
(212) Oligosaccharide Analysis .............25 6125
(221) Chloride and Sulfate... 6.0.0... eee 6139
(223) Dimethylaniline.. 2.0.0... 00... 6138
(226) 4-Epianhydrotetracycline ............004 6140
(227) 4-Aminophenol in Acetaminophen-Containing
Drug Productswiscwsci occa e ee ee peeteewad 6141
(228) Ethylene Oxide and Dioxane ............ 6142
231) Heavy Metals gsaa jaa os ov sealant a 6145
(232) Elemental Impurities—Limits............. 6147
(233) Elemental Impurities—Procedures ......... 6151
(241) ION oe eee eee
(251) Lead....
(261) Mercury
(267) Porosimetry by Mercury Intrusion......... 6160
 xx Guide to General Chapters USP 41

(268) Porosity by Nitrogen Adsorption— (661.2) Plastic Packaging Systems for

Desorption & osimngias.a&4 o4 228ROE 6163 Pharmaceutical US ieee rTree 6424
(271) Readily Carbonizable Substances Test . . . 6168 (670) Auxiliary Packaging Components ......... 6428
(281) Residue on Ignition ...............- 6168 (671) Containers—Performance Testing ......... 6436
(291), SCIONS Foci spades Lhe sie8 Se 0. 0 cyevstenar tina 6169 (691): COLON 2% as a az GES w thighe Receuaqed w iale' 6443
(695) Crystallinity... 6... eee 6445
(696) Characterization of Crystalline Solids by
Other Tests and Assays Microcalorimetry and Solution Calorimetry .. 6445
(697) Container Content for Injections.......... 6449
(301) Acid-Neutralizing Capacity.............. (698) Deliverable Volume ............00-00005 6450
(311) Alginates Assay ............. ‘ (699) Density of Solids ........... 00...0000. 6453
(341) Antimicrobial Agents—Content ¢701). Disintegration’ 35 ses bk umaetital eaOsos 6455
(345) Assay for Citric Acid/Citrate and Phosphate . . 6176 (705) Quality Attributes of Tablets Labeled
(351) Assay for Steroids ..........-..000000- 6177 as Having a Functional Score ............ 6457
(381) Elastomeric Closures for Injections - 6178 CATV DISSOIULION wen eo ooo oe erties BRS
(391) Epinephrine Assay ........... - 6183 (721) Distilling Range as
(401) Fats and Fixed Oils .... . . 6184 (724) DrugeRelease.sau% 0s booed eet’. 3 es
(411) Folic Acid Assay. ..........-.-. . 6197 (729) Globule Size Distribution in Lipid Injectable
(413) Impurities Testing in Medical Gases. . 6201 EUISIONSisrss 6 25 5 FE Ey onmeewamee aeeeeeH 6478
(415) Medical Gases Assay .......... .... 6202 (730) Plasma Spectrochemistry ...........- - - 6482
(425) lodometric Assay—Antibiotics............ 6205 (731) Loss ON. DIYING. .g,.00- «ceiereeavs « . . 6485
(429) Light Diffraction Measurement of Particle (733) Loss:on Ignition. “os Saalewccsa s . 6486
SIZE oo os cuenereemarayie eg waite ately alle afimenomngsed hy As 6206 (735) X-Ray Fluorescence Spectrometry .. . - - 6486
(431) Methoxy Determination................ 6212 (736) Mass Spectrometry ........... - . 6491
ad (441) Niacin or Niacinamide Assay ............ 6213 (741) Melting Range or Temperature . . . . 6497
-
a (451) Nitrite Titration... 6... eee eee 6218 (755) Minimum Fill oom eo oer tyereve ace #2 ee soe & 6499
Pd (461) Nitrogen Determination..............4. 6219
io (761) Nuclear Magnetic Resonance Spectroscopy . . 6500
S (466) Ordinary Impurities ................00- 6220 (771) Ophthalmic Products—Quality Tests
a (467) Residual Solvents..............2000-08 6222
1S) (776) Optical Microscopy ............. #4
(469) Ethylene Glycol, Diethylene Glycol, and <781)-Optical Rotation, « ssa aero
hg4.5.4a038,55
s
~
Triethylene Glycol in Ethoxylated (782) Vibrational Circular Dichroism
a SUbStANCES ers ee ae eR SSIS Baeo 6237 Spectroscopy ..........
i= (471) Oxygen Flask Combustion ............-. 6238 {785) Osmolality and Osmolarity
C7 (481) Riboflavin Assay... 2.2... 0.60.00. eee 6239
o (786) Particle Size Distribution Estimation by
(501) Salts of Organic Nitrogenous Bases........ 6245 Analytical Sieving .........-. eseee eee 6530
(503) AceticAcldin Peptides... «<< ccnusiown aaea 6246 (787) Subvisible Particulate Matter in Therapeutic
(503.1) Trifluoroacetic Acid (TFA) in Peptides ..... 6247 Protein Injections... «saqiwnwwossastreetws 6534
(507) Protein Determination Procedures......... 6248 (788) Particulate Matter in Injections ........... 6537
(511) Single-Steroid Assay. 2.206.602.0620 eeeae 6253 (789) Particulate Matter in Ophthalmic Solutions . . 6540
(S25)Sultur DiOkide se e's ee oe EM eles on‘aXe 6254 (790) Visible Particulates in Injections........... 6542
(531) Thiamine Assay 0). 2002 ciel nee s lee 6260 (791) th egog0 B42 EE & ww poemmprneue a 8 e8 Fo Hake 6543
(S47) TUIMOEIY oceie eee cae comer aes 6268 (795) Pharmaceutical Compounding—Nonsterile
(551) Vitamin E.Assay®: i. 2 22 0 be eentimedsaoes 6272 Preparations: «» ne 6 se ecamsausnsces
«aaa@8&wai 6546
(561) Articles of Botanical Origin. ............; 6279 (797) Pharmaceutical Compounding—Sterile
(563) Identification of Articles of Botanical Origin .. 6293 Preparations: + sc4-958 acsaeurtne dpeiscieile¢ 6554
(565) Botanical’ Extracts 22.5. 5. ee . (800) Hazardous Drugs—Handling In Healthcare
(571) Vitamin A Assay. .... 2.00600 005 SOLINGS) ay54s hare, dacereriederaOtalelar ed tend 6598
(580) Vitamin C Assay .....-..- 0.000 ee (801) Polarography .. 1.6... ceceeeeeeeeeee 6617
6581) Vitamin Di Assay ic). .o-0 o's o: wrenenenceunens (81 T): Powder FINENESS! 6:45: cor deepensia ial a erst tot 6621
(591) Zinc Determination (821) Radioactivity) icsie sis sseetoesesey?
sagsistssbos 6622
(823) Positron Emission Tomography
Physical Tests and Determinations Drugs for Compounding, Investigational,
and Research Uses... 2.0...
eeeeeeeens 6629
(601) Inhalation and Nasal Drug Products: Aerosols, (831) Refractive Index... 2.2...
eeeeeeeee 6639
Sprays, and Powders—Performance Quality (841) Specific Gravity... 26... ceeee eeeee 6639
TESESiccsserasuaseice ie «9 # wi vm wt or asisinetrncanesbiabebi
dlWARina 6327 (846) Specific Surface Area... 6... ee eee eee 6640
(602) Propellants ........-.. < (852) Atomic Absorption Spectroscopy ......... 6644
(603) Topical Aerosols $35 (853) Fluorescence Spectroscopy............-. 6648
(604)iLeak: Rate: sa.0 5 «02 ge gh chit eel: OY (854) Mid-Infrared Spectroscopy .............. 6654
(610) Alternative Microbiological Sampling Methods (855) Nephelometry, Turbidimetry, and Visual
for Nonsterile Inhaled and Nasal Products . . . 6356 COMPpanisOn: ec. fh sae sols BS Sok eae beter 6658
(611) Alcohol Determination...........-..-4- 6358 (857) Ultraviolet-Visible Spectroscopy........... 6660
(616) Bulk Density and Tapped Density of (861) Sutures—Diameter............00000005 6666
POWGESS eines oc 2 SF Heh a Gohabtindnie
WhiolaDeHH 6360 (871) Sutures—Needle Attachment ....... . 6667
(621) Chromatography. . 6363 (881) Tensile Strength ........... 2 . 6668
(631) Color and Achromicity . . . 6375 (891) Thermal Analysis ............. . . 6669
(641) Completeness of Solution............-.-- 6376 (905) Uniformity of Dosage Units ... . 6673
(643) Total Organic Carbon ............--2-05 6377 (911) Viscosity—Capillary Methods .. . 6677
(645) Water Conductivity... 2.0 eee eee 6378 (912) Viscosity—Rotational Methods ... . . 6679
(651) Congealing Temperature ....-.......-45 6382 (913) Viscosity—Rolling Ball Method ...... - . 6684
(659) Packaging and Storage Requirements ...... 6384 (914) Viscosity—Pressure Driven Methods ... 6686
(660) Containers—Glass ... 2.1...
0... eeeeee 6390 (921) Water Determination .............2.005 6687
(661) Plastic Packaging Systems and Their Materials (941) Characterization of Crystalline and Partially
Of CONStIUCHON . . 65 oS srenintsten
aivvtteEs 6396 Crystalline Solids by X-Ray Powder
(661.1) Plastic Materials of Construction ........ 6403 Diffraction: (XRPD) scsvoc x3 o's SF 4 Ee eee 6692
USP 41
GENERAL INFORMATION
(1004) Mucosal Drug Products—
PerformanceTests ),..h oie) a!a ee Fa ibe ot 6699
(1005) Acoustic Emission ............0 000005 6702
(1010) Analytical Data—Interpretation and
Treatment
(1024) Bovine’ Serulia si, iYoa
(1025) Pancreatin.....
(1027) Flow Cytometry
(1029) Good Documentation Guidelines ........ 6760
(1030) Biological Assay Chapters—Overview and
GIOSSAIY os oe e-auipeniuecesed
6<9SRASaigen 6764
(1031) The Biocompatibility of Materials Used
in Drug Containers, Medical Devices, and
Imiplantsiee: 2 -6Ols uit, iO) SOUP ON idly 6775
(1032) Design and Development of Biological
ASSAYSINET ROMS RE AN, Be SRE GG we 6785
(1033) Biological Assay Validation . . 6803
(1034) Analysis ofBiological Assays . . 6818
41039)!Ghemomietrics 3 0.5 lal! Ores . . 6831
(1041) Biologics:. . . 27 UNAC Ma ci eUT EE IU, ao 6849
(1043) Ancillary Materials for Cell, Gene, and Tissue-
Engineered Products, 052.4... Sebveces
(1044) Cryopreservation of Cells ........ a (1105) Immunological Test Methods—Surface
(1046) Cellular and Tissue-Based Products . is
(1047) Gene Therapy Products ...............
(1048) Quality of Biotechnological Products: Analysis
of the Expression Construct in Cells Used for
Production of r-DNA Derived Protein
Productss2{VfPs) gnCloa.Joga easze hey 6928
(1049) Quality of Biotechnological Products: Stability
Testing of Biotechnological/Biological
Productssue Wiebi1 pn Data tey alors, Ct Bey 6930
(1050) Viral Safety Evaluation of Biotechnology
Products Derived from Cell Lines of Human
or Animal Origin 2ivocacooas rears hy 6935
(1050.1) Design, Evaluation, And Characterization
of Viral Clearance Procedures
(1051) Cleaning Glass Apparatus. . . ne
(1052) Biotechnology-Derived Articles—Amino Acid
‘Analysisl pO Oey WS SLIN SIO gOS AON VG,
(1053) Capillary Electrophoresis..............
(1054) Biotechnology-Derived Articles—Isoelectric
Focusing’, (Aor cvitt, luau bsg laa. 6981
(1055) Biotechnology-Derived Articles—Peptide
Mapping 4": Schade “Gsie aessie(A 6984
(1056) Biotechnology-Derived Articles—Polyacrylamide
Gel Electrophoresis’! 2)/.s0.i Soak, (At 6991
(1057) Biotechnology-Derived Articles—Total Protein
ASSAY vecse ase x 8 2 a 2 paenpeyene
§SOE ia « «= 6998
(1058) arial Instrument Qualification . . . 7005
(1059) Excipient Performance ..........- - 7011
(1061) Color—Instrumental Measurement ....... 7040
(1062) Tablet Compression Characterization... ... 7042
(1063) Shear Cell Methodology for Powder
Flow? TESting: « a «0s 5s eagece titel
WES ooa 7054
(1064) Identification of Articles of Botanical Origin
by High-Performance Thin-Layer
Chromatography Procedure ............ 7065
(1065) lon Chromatography ...... -... 7075
(1066) Physical Environments That Promote Saft
Medication: Use). « «= geretdhtleid
oSEG«ee 7078
(1072) Disinfectants and Antiseptics ........... 7090
(1074) Excipient Biological Safety Evaluation
GUIDELINES. ss a5 0542 eemewmems awe ge 7095
(1078) Good Manufacturing Practices for Bulk
Pharmaceutical Excipients.............. 7100
(1079) Good Storage and Distribution Practices for
Drug Productsis «os 2» agecemmagews a2 6ax 6 7120
(1079.1) Storage and Transportation of Investigational
Drug Products’: «siya gucweamee sassy 7130
(1080) Bulk Pharmaceutical Excipients—Certificate of
Analysis... 6.0 cc eee eee eee 7133
Guide to General Chapters xxi
(1084) Glycoprotein and Glycan Analysis—General
CORSIGEFAUONS 2be a7. ites ny sists tielletabiale
tsbce wes 7141
(1086) Impurities in Drug Substances and Drug
PRODUCSin ringers § ale! cay ere gatesmie ys « a! AlS2
(1087) Apparent Intrinsic Dissolution—Dissolution
Testing Procedures for Rotating Disk and
Stationary DISK. sss 94s 2s ee ucsemme saeG 7155
(1088) In Vitro and In Vivo Evaluation of Dosage
FORMS. ecoseuonewurde ddelgl onalie tii 2 u: imienebetesieias
ceo 7159
(1090) Assessment of Drug Product
Performance—Bioavailability, Bioequivalence,
and: Dissolution x: sins!as«
ox SevSew aas 7170
(1091) Labeling of Inactive Ingredients ......... 7178
(1092) The Dissolution Procedure: Development
and Validation: .sulichs
edsolwhBobetoo 7178
(1094) Capsules—Dissolution Testing and Related
Quality-Attributes.ss). at. oc 6 atin hanhincton
GIs 7198
(1097) Bulk Powder Sampling Procedures ....... 7206
(1102) Immunological Test Methods—General
CONsideratiOns seve sy so ¥ 2s y enetiweewn 7219
(1103) Immunological Test Methods—Enzyme-
Linked Immunosorbent Assay (ELISA) ..... 7226
(1104) Immunological Test Methods—Immunoblot
ANALYSIS: 0. x. cennoeseice yp 9 a atetdlvdinye bestest benadsfe 7237
2)
Plasmon: Resonance: .3eiea eyeeens 7248 oO
=]
(1106) Immunogenicity Assays—Design and o
Validation of Immunoassays to Detect =
Sy
Anti-Drug Antibodies ................. 7264
(1106.1) Immunogenicity Assays—Design and fa)
Validation of Assays to Detect Anti-Drug sz
3
ey
Neutralizing Antibody
(1111) Microbiological Examination of Nonsterile i.
Products: Acceptance Criteria for Pharmaceutical St
7
Preparations and Substances for Pharmaceutical
USE: 5 5 ee EE ee aRPAVAN BIER STT USI Ss ener 7297
(1112) Application of Water Activity Determination to
Nonsterile Pharmaceutical Products. ...... 7298
(1113) Microbial Characterization, Identification, and
SAIN: TYPING aia todresiaesilsre
te eter)Mare Wienateebien 7301
(1115) Bioburden Control of Nonsterile Drug
Substances and Products .............. 7305
(1116) Microbiological Control and Monitoring of
Aseptic Processing Environments......... 7312
(1117) Microbiological Best Laboratory Practices... 7325
(1118) Monitoring Devices—Time, Temperature, and
Humidity: ti2co) a denciscton ath a aoeies sewn scone 7331
(1119) Near-Infrared Spectroscopy ............ 7337
(1120) Raman Spectroscopy .........00 005 7343
(1121) Nomenclature =» em.ieedice 6eeoe xOeRats 7351
(1125) Nucleic Acid-Based Techniques—General . . . 7353
(1126) Nucleic Acid-Based Techniques—Extraction,
Detection, and Sequencing ............ 7359
(1127) Nucleic Acid-Based Techniques—
Amplification: évcstesissig wasseus 62xeon 7369
(1128) Nucleic Acid-Based Techniques—
Mictoattay, <= 2 swavarsms yeae23eaBbBE 7379
(1129) Nucleic Acid-Based Techniques—
GENOLWPING + = cadebeopotiba ttlegeWeadLan 7385
(1130) Nucleic Acid-Based Techniques—Approaches for
Detecting Trace Nucleic Acids (Residual DNA
TOSti) a.3is is so eestraw ieee cee e oe a ae 7389
(1132) Residual Host Cell Protein Measurement in
Biophatmaceuticals sccrsewe gsee39 Rs 7393
(1136) Packaging and Repackaging—Single-Unit
Containers 2.2... 1 eee ee es
(1151) Pharmaceutical Dosage Forms .......
(1152) Animal Drugs for Use in Animal Feeds
(1160) Pharmaceutical Calculations in Pharmacy
PraCtie@s «as gus ony wwrermern aea eo8E 7451
(1163) Quality Assurance in Pharmaceutical
Compounding......... 0.06.0 e eee eee 7475
(1174) Powder Flow... 2... 0.0... eeeeeee ee 7481
(1176) Prescription Balances and Volumetric
Apparatus voces ess 2 weimanawa aweRes 7485
 xxii Guide to General Chapters USP 41

(1177) Good Packaging Practices ............. 7492 (1237) Virology Test Methods ................ 7812
(1178) Good Repackaging Practices............ 7495 (1238) Vaccines for Human Use—Bacterial
41780) Human Plasma «fee ¢ sae oa see et 7497 VAEGINES: . . » as dcnckae eae hfale, saayti Roehl 7833
(1181) Scanning Electron Microscopy .......... 7519 (1240) Virus Testing of Human Plasma for Further
(1184) Sensitization Testing... ........-.0000- 7529 Manitifacture: « « « x sarensorerga dycrysBOIS 7846
(1191) Stability Considerations in Dispensing (1241) Water-Solid Interactions in Pharmaceutical
PLAGUCE rs ow ss og a eotindionale aMay+&5 7540 SYSEOIMS oo ii. 5 oi ec ornpenneme e uliinie olen ene 7856
(1195) Significant Change Guide for Bulk (1251) Weighing on an Analytical Balance ....... 7860
Pharmaceutical Excipients.............. 7545 (1265) Written Prescription Drug Information—
(1197) Good Distribution Practices for Bulk Guidelines... «6 o: w.pegsuceiaizysrang
3 adFaas 7866
Pharmaceutical Excipients.............. 7556 (1285) Preparation of Biological Specimens for
(1207) Sterile Product Packaging—Integrity Histologic and Immunohistochemical
BVAlUARIOMys co's 1S Y ae oo ABhRhadllleondh
olW bawahs 7578 Analysis «sb eeaam gins fa eG OLH ose 7868
(1207.1) Package Integrity Testing in the Product (1285.1) Hematoxylin and Eosin Staining of Sectioned
Life Cycle—Test Method Selection Tissue for Microscopic Examination ....... 7872
and Validation 3's 2324. dames oale's & ad S 7585 (1601) Products for Nebulization—Characterization
(1207.2) Package Integrity Leak Test Technologies. . 7597 TeStSisiace ih its Ysera siigialtupels de wintitdark bdSees 7874
(1207.3) Package Seal Byalty Test Technologies. . . 7614 (1602) Spacers and Valved Holding Chambers
(1208) Sterility Testing—validation of Isolator Used With Inhalation Aerosols—
SYSTEMS nice one ain HAHEI ATo « 7617 Characterization Tests ..............4. 7878
(1210) Statistical Tools for Procedure Validation ... 7622 (1644) Theory and Practice of Electrical Conductivity
(1211) Sterilization and Sterility Assurance of Measurements of Solutions............. 7890
‘Gompendial Articles 32:5 s!sog'teeeS
e2623wit§ 7633 (1660) Evaluation of the Inner Surface Durability
(1216) Tablet Friability of Glass: Containers: aijistsil eneanigas a. 7897
al
— (1217) Tablet Breaking Force (1661) Evaluation of Plastic Packaging Systems and
23 (1222) Terminally Sterilized Pharmaceutical Products— Their Materials of Construction with Respect
Q Parametric Release’... ’s, os oh RRR rin 7638 to Their User Safety Impact ............ 7902
Ss (1223) Validation of Alternative Microbiological (1663) Assessment of Extractables Associated with
&
1) Methods:. «s5.0 foes
2Aae 7642 Pharmaceutical Packaging/Delivery
(1223.1) Validation of Alternative Methods to Antibiotic SYSCEMIS18)tsee7cL GAC. & ke REIS neBesos 7910
Si Microbial’Assays2 vc o's oie staal we 7656 (1664) Assessment of Drug Product Leachables
o (1224) Transfer of Analytical Procedures......... 7663 Associated with Pharmaceutical Packaging/
|
Cy (1225) Validation of Compendial Procedures ..... 7665 Delivery: Systems astorsiadsiasig warsoakee 7924
1) (1226) Verification of Compendial Procedures..... 7671 (1664.1) Orally Inhaled and Nasal Drug Products . . 7937
(1227) Validation of Microbial Recovery from (1724) Semisolid Drug Products—Performance
Pharmacopeial Articles ................ 7672 TSSts $c. tet Hod caged deie® otmbiaa ocx 7944
(1228) Depyrogenation’ :? sii) ci.tek eT ts ae 7676 (1730) Plasma Spectrochemistry—
(1228.1) Dry Heat Depyrogenation ............ 7681 Theory-and-Practice naiteid guide cy ered) fel 7956
(1228.3) Depyrogenation by Filtration .......... 7685 (1735) X-Ray Fluorescence Spectrometry—
(1228.5) Endotoxin Indicators For Theory'and Practic@sc:,’. sik nsiesw eed 7963
Depyrogenation”. 2. oi yee 7688 (1736) Applications of Mass Spectrometry ....... 7982
(1229) Sterilization of Compendial Articles... .... 7692 (1761) Applications of Nuclear Magnetic Resonance
(1229.1) Steam Sterilization by Direct Contact .... 7698 Spectroscopy’... .. sissyesigu)jael? soallac Ruse 8004
(1229.2) Moist Heat Sterilization of Aqueous (1771) Ophthalmic Products—Performance Tests . . 8024
Liquids ..cotiasde Ll2sd jeawa Cin NT, 7701 (1782) Vibrational Circular Dichroism Spectroscopy—
(1229.3) Monitoring of Bioburden............. 7706 Theoryiand) Practice yisatt-sronlorida-viuits
sore 8025
(1229.4) Sterilizing Filtration of Liquids ......... 7709 (1787) Measurement of Subvisible Particulate Matter in
(1229.5) Biological Indicators for Sterilization ..... 7716 Therapeutic Protein Injections........... 8038
(1229.6) Liquid-Phase Sterilization ............. 7719 (1788) Methods for the Determination of Particulate
(1229.7) Gaseous Sterilization .......00....4.. 7722 Matter in Injections and Ophthalmic
(1229.8) ‘Dry: Heat:Sterilization’62 20.6
2.2 es 7725 SOIUTONS's xe evngereg ancy © a 6 a 9 #\BhaGereea 8052
(1229.9) Physicochemical Integrators And (1790) Visual Inspection of Injections........... 8066
Indicators for Sterilization. ............. 7728 (1821) Radioactivity—Theory and Practice ....... 8084
(1229.10) Radiation Sterilization .............. 7728 (1823) Positron Emission Tomography Drugs—
(1229.11) Vapor Phase Sterilization ............ 7733 Informations: .ss0cd 2 aapeswene.: ¢sbdaieaGa 8098
(1229.12) New Sterilization Methods........... 7734 (1852) Atomic Absorption Spectroscopy—Theory
(1229.13) Sterilization-In-Place ............... 7735 AN Practice. i een e GESwidewae 8109
(1229.14) Sterilization Cycle Development ....... S137 (1853) Fluorescence Spectroscopy—Theory
(1229.15) Sterilizing Filtration Of Gases ......... 7740 and Practice.) rict.aaierstactietentiartesef
wo 8118
(1230) Water for Hemodialysis Applications ...... 7741 (1854) Mid-Infrared Spectroscopy—Theory
(1231) Water for Pharmaceutical Purposes ....... 7742 and Practice . csnackjgsonairouts nese 8127
(1234) Vaccines for Human Use—Polysaccharide (1857) Ultraviolet-Visible Spectroscopy—Theory
and Glycoconjugate Vaccines ........... 7778 and Practice « cscesjas cebtomd inning iiies. 8136
(1235) Vaccines for Human Use—General (1911) Rheometry
Considerations tr pesticide eeRTbalee 7795
USP 41 Guide to General Chapters xxiii

DIETARY SUPPLEMENTS (2040) Disintegration and Dissolution of Dietary

(2021) Microbial Enumeration Tests—Nutritional and
Dietary Supplements ...............0. 8153
(2022) Microbiological Procedures for Absence of
Specified Microorganisms—Nutritional and
Dietary Supplements ...............-. 8158
(2023) Microbiological Attributes of Nonsterile
Nutritional and Dietary Supplements... ... 8164
(2030) Supplemental Information for Articles of
Botanical Origin... 6.0... eee eee 8168
SUppleMenits « « 2 x v xem saa gece BRS
(2091) Weight Variation of Dietary Supplements .. .
(2232) Elemental Contaminants in Dietary
Supplements .. 0... 00.0... eeeeee
(2250) Detection of Irradiated Dietary
Supplements «<6 is eves a wascewne
(2251) Screening for Undeclared Drugs and
Drag Analogs «+ see eswumeres saeseeeRe
(2750) Manufacturing Practices for Dietary
Supplements ..... 0.2.0... 00eeeeee

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USP 41 Official Monographs / juniper 2303

e ROSIN OR ROSIN OILS

Juniper Tar Sample solution: Triturate 1 mL of Juniper Tar with
15 mL of solvent hexane, and filter.
DEFINITION Analysis: Mix equal volumes of Sample solution and cu-
Juniper Tar is the empyreumatic volatile oil obtained from pric acetate solution (10 mg/mL), shake vigorously, and
the woody portions of Juniperus oxycedrus L. (Fam. allow the liquids to separate. Decant the solvent hexane
Pinaceae). yer into a test tube, and add an equal volume of
ether.
IDENTIFICATION Acceptance criteria: The liquid does not become dark
°c A. green or blackish.
Sample solution: Shake 1 volume of Juniper Tar with
20 volumes of warm water, filter, and use the filtrate. ADDITIONAL REQUIREMENTS
Analysis: To 5 mL of the cold Sample solution add a few © PACKAGING AND STORAGE: Preserve in tight, light-resistant
drops of silver-ammonium nitrate TS. containers, and avoid exposure to excessive heat.
porceptence criteria: A black color is produced.
e .
Sample solution: Shake 1 volume of Juniper Tar with
20 volumes of warm water, filter, and use the filtrate.
Analysis: To 5 mL of the Sample solution add a few
drops of alkaline cupric tartrate TS, and heat the solu-
tion to boiling.
Acceptance criteria: A red precipitate is formed.
SPECIFIC TESTS
© SPECIFIC GRAVITY (841): 0.950-1.055
e REACTION: The filtrate prepared for Identification test A is
acid to litmus.

(<3
4)
ao}

kK
e
es
)
a=
i)
a}
=
w
 2304 Kanamycin / Official Monographs USP 41

Columns
Kanamycin Injection Guard: Packing L47
Analytical: 4-mm x 25-cm; packing L47
DEFINITION Flow rate: 0.5 mL/min
Kanamycin Injection contains an amount of kanamycin sul- Injection volume: 20 uL
fate equivalent to NLT 90.0% and NMT 115.0% of the System suitability
labeled amount of kanamycin (CisH36N4O13). It contains Samples: System suitability solution and Standard
suitable buffers and preservatives. solution
[Note—The relative retention times for kanamycin and
IDENTIFICATION amikacin are about 1.0 and 1.3, respectively.
© A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST Suitability requirements
(201) Resolution: NLT 3 between kanamycin and amikacin,
Sample solution: 1 mg/mL of kanamycin from Injection System suitability solution
in water Tailing factor: NMT 2, Standard solution
Chromatographic system Relative standard deviation: NMT 2.0%, Standard
Adsorbent: 0.25-mm layer of chromatographic silica solution
gel mixture, heated at 110° for 1 h and cooled imme- Analysis
diately before use Samples: Standard solution and Sample solution
Application volume: 10 uL Calculate the percentage of the labeled amount of
Developing solvent system: 150 mg/mL of monoba- kanamycin (CisH36N4O11) in the portion of Injection
sic potassium phosphate in water taken:
Spray reagent: 10 mg/mL of ninhydrin in butyl
alcohol Result = (ru/rs) x (Cs/Cy) x P x Fx 100
Analysis: Proceed as directed in the chapter. Allow the
ee to dry, and develop in a chamber previously equi- ry = peak area from the Sample solution
librated for 18 h with the Developing solvent system. Re- rs = peak area from the Standard solution
move the plate from the chamber, and air-dry. Spray Cs = concentration of USP Kanamycin Sulfate RS in
the plate with Spray reagent, and dry at 110° for 10 the Standard solution (tug/mL)
min. Cu = nominal concentration of kanamycin in the
Acceptance criteria: Meets the requirements Sample solution (ug/mL)
e B. The retention time of the kanamycin peak of the Sam- P = potency of kanamycin in USP Kanamycin
ple solution corresponds to that of the Standard solution, Sulfate RS (ug/mg)
as obtained in the Assay. F = conversion factor, 0.001 mg/yg
Acceptance criteria: 90.0%-115.0%
ASSAY
© PROCEDURE
SPECIFIC TESTS
Mobile phase: 0.115 N sodium hydroxide solution © PH (791): 3.5-5.0
System suitability solution: 20 g/mL of USP Amikacin © BACTERIAL ENDOTOXINS TEST (85): NMT 0.67 USP Endo-
&
“
toxin Unit/mg of kanamycin
es RS and 8 ug/mL of USP Kanamycin Sulfate RS in water
© STERILITY TESTS (71): It meets the requirements when
ii Standard solution: 8 t1g/mL of USP Kanamycin Sulfate
— tested as directed in Test for Sterility of the Product to Be
fo.) RS in water
° Sample solution: Nominally 6 g/mL of kanamycin Examined, Membrane Filtration.
S from Injection in water e PARTICULATE MATTER IN INJECTIONS (788): Meets the re-
5 Chromatographic system quirements for small-volume injections
= (See Chromatography (621), System Suitability.) e OTHER REQUIREMENTS: It meets the requirements in Injec-
a
Mode: LC tions and Implanted Drug Products (1).
al
=) Detector: Electrochemical ADDITIONAL REQUIREMENTS
Mode: Integrated amperometric ¢ PACKAGING AND STORAGE: Preserve in single-dose or mul-
Range: 300 nC tiple-dose containers, preferably of Type | or Type Ill
Output: 1Vfull-scale glass.
Electrodes
Indicator: Gold
Reference: pH silver-silver chloride Change to read:
Waveform: See Table 1.
e USP REFERENCE STANDARDS (11)
Table 1 YsP Amikacin RS
@ (CN 1-May-2018)
Potential USP Kanamycin Sulfate RS
USP 41 Official Monographs / Kanamycin 2305

Suitability requirements
Kanamycin Sulfate Resolution: NLT 3 between kanamycin and amikacin,
System suitability solution
NH
Tailing factor: NMT 2, Standard solution
Relative standard deviation: NMT 2.0%, Standard
* solution
Analysis
Samples: Standard solution and Sample solution
lS: we

HOM
4
Sa\ a 8 0 Ts80s Calculate the quantity, in ug/mg, of kanamycin
(CisH36N4O11) in the portion of Kanamycin Sulfate
taken:
te Ng
Result = (ru/rs) x (Cs/Cu) x P
CisH36N4Or1 - H2SO4 582.58 tu = peak area from the Sample solution
D-Streptamine, O-3-amino-3-deoxy-a-D-glucopyra- Is = peak area from the Standard solution
nosyl(1—-46)-O-[6-amino-6-deoxy-a-D-glucopyra- Cs = concentration of USP Kanamycin Sulfate RS in
nosyl(1—>4)]-2-deoxy-, sulfate (1:1) (salt); the Standard solution (g/mL)
Kanamycin sulfate (1:1) (salt) [25389-94-0]. CG = concentration of Kanamycin Sulfate in the
Sample solution (ug/mL)
DEFINITION P = potency of kanamycin in USP Kanamycin
Kanamycin Sulfate has a potency equivalent to NLT 750 pg/ Sulfate RS (ug/mg)
pee kanamycin (CisH36N4O11), calculated on the drie Acceptance criteria: NLT 750 ug/mg on the dried basis
asis.
IMPURITIES
IDENTIFICATION e RESIDUE ON IGNITION (281)
e A. INFRARED ABSORPTION (197K) Analysis: Moisten the charred residue with 2 mL of ni-
e B. IDENTIFICATION TESTS—GENERAL, Sulfate (191): Meets tric acid and 5 drops of sulfuric acid.
the requirements Acceptance criteria: NMT 1.0%
e C. The retention time of the major peak of the Sample © ORGANIC IMPURITIES
solution corresponds to that of the Standard solution, as Senter 0.25-mm layer of chromatographic silica
obtained in the Assay. gel
ASSAY Developing solvent system: 75 mg/mL of monobasic
© PROCEDURE potassium phosphate in water
Mobile phase: 0.115 N sodium hydroxide solution Spray reagent: 10 mg/mL of ninhydrin in buty! alcohol
System suitability solution: 20 g/mL of USP Amikacin Standard solution 1: 30 mg/mL of USP Kanamycin Sul-
RS and 8 g/mL of USP Kanamycin Sulfate RS in water fate RS in water
Standard solution: 8 g/mL of USP Kanamycin Sulfate Standard solution 2: 0.90 mg/mL of USP Kanamycin (=
RS in water Sulfate RS in water wn
Sample solution: 8 pg/mL of Kanamycin Sulfate in Sample solution: 30 mg/mL of Kanamycin Sulfate in ae)

water water K
Chromatographic system Application volume: 1 uL °
Analysis: Heat the plate at 110° for 1 _h immediately =
(See Chromatography (621), System Suitability.)
before use, and allow it to cool. Equilibrate for 90 min re)
Mode: LC
with the Developing solvent system.
ro}=
Detector: Pulsed amperometric electrochemical Ey
detector Apply all three solutions to the plate separately, allow bo}
Working electrode: Gold the spots to dry, and develop the chromatogram with =
Reference electrode: pH silver-silver chloride the Developing solvent system until the solvent front a)

Waveform: See Table 1. has moved three-fourths of the length of the plate.
Remove the plate from the chamber, and air-dry.
Spray the plate with Spray reagent. Dry the plate at
Table 1 110° for 10 min, and examine the chromatograms.
Potential Acceptance criteria: The chromatograms show princi-
pal spots at the same Rr value, and no secondary spot,
if present from the Sample solution, is more intense than
the principal spot of Standard solution 2.
SPECIFIC TESTS
e CRYSTALLINITY (695): Meets the requirements
e PH (791)
Sample solution: 10 mg/mL
Acceptance criteria: 6.5-8.5
80
e Loss ON DRYING (731)
Columns Analysis: Dry 100 mg in a vacuum ina capillary-stop-
Guard: 4-mm x 50-mm; 7.5-1m packing L47 pered bottle at a pressure not exceeding 5 mm of mer-
Analytical: 4-mm x 25-cm; 7.5-um packing L47 cury at 60° for 3 h.
Flow rate: 0.5 mL/min Acceptance criteria: NMT 4.0%
Injection volume: 20 pL © STERILITY Tests (71): Where the label states that
System suitability Kanamycin Sulfate is sterile, it meets the requirements
Note—tThe relative retention times for kanamycin and when tested as directed for membrane filtration in Test
amikacin are about 1.0 and 1.3, respectively. for Sterility of the Product to Be Examined.
Samples: System suitability solution and Standard e BACTERIAL ENDOTOXINS TEST (85): Where the label states
solution that Kanamycin Sulfate must be subjected to further pro-
cessing during the preparation of injectable dosage
 2306 Kanamycin / Official Monographs USP 41

forms, it contains NMT 0.67 USP Endotoxin Unit/mg of Mode: LC

Kanamycin. Detector: Electrochemical
Mode: Integrated amperometric
ADDITIONAL REQUIREMENTS Range: 300 nC
e PACKAGING AND STORAGE: Preserve in tight containers. Output: 1 Vfull-scale
e LABELING: Where it is intended for use in preparing in- Electrodes
jectable dosage forms, the label states that it is sterile or Indicator: Gold
must be subjected to further processing during the prep- Reference: pH silver-silver chloride
aration of injectable dosage forms. Waveform: See Table 1.

Change to read: Table 1

Potential
e USP REFERENCE STANDARDS (11)
ysP Amikacin RS
00
@ (CN 1-May-2018)
USP Kanamycin Sulfate RS
0.5!
+0.
0 +0.
0.71
Kanamycin Sulfate Capsules —0.80

DEFINITION
Kanamycin Sulfate Capsules contain the equivalent of NLT
90.0% and NMT 115.0% of the labeled amount of
kanamycin (CigH36N4On1).
IDENTIFICATION
e * oe CHROMATOGRAPHIC IDENTIFICATION TEST
2 solution
Sample solution: 1 mg/mL of kanamycin from Capsules
in water
Chromatographic system
Adsorbent: 0.25-mm layer of chromatographic silica
gel mixture, heated at 110° for 1 h and cooled imme-
diately before use
Application volume: 10 wL
os
nal
Developing solvent system: 150 mg/mL of monoba-
co sic potassium phosphate solution
i
— Spray reagent: 10 mg/mL of ninhydrin in butyl
aD
° alcohol
S Analysis: Proceed as directed in the chapter. Allow the
iS spots to dry, and develop in a suitable chamber, previ-
= ously equilibrated for 18 h with the Developing solvent
a system, Remove the plate from the chamber, and air-
” dry. Spray the plate with Spray reagent, and dry at 110°
=) for 10 min.
Acceptance criteria: Meet the requirements
e B. The retention time of the kanamycin peak of the Sam-
ple solution corresponds to that of the Standard solution,
as obtained in the Assay.
ASSAY
e PROCEDURE
Mobile phase: 0.115 N sodium hydroxide solution
System suitability solution: 20 g/mL of USP Amikacin
RS and 8 ug/mL of USP Kanamycin Sulfate RS
Standard solution: 8 tg/mL of USP Kanamycin Sulfate
RS
Sample stock solution: Nominally 0.32 mg/mL of
kanamycin, prepared as follows. Transfer a portion of
the mixed contents of Capsules (NLT 10) to a suitable
volumetric flask. Add water, using 20% of the final vol-
ume, and swirl to dissolve. Dilute with water to volume.
Sample solution: Nominally 6.4 g/mL of kanamycin
from the Sample stock solution in water
Chromatographic system
(See Chromatography (621), System Suitability.)
Columns
Guard: Packing L47
Analytical: 4-mm x 25-cm; packing L47
Flow rate: 0.5 mL/min
Injection volume: 20 uL
System suitability
Samples: System suitability solution and Standard
[Nott—The relative retention times for kanamycin and
amikacin are about 1.0 and 1.3, respectively.]
Suitability requirements
Resolution: NLT 3 between kanamycin and amikacin,
System suitability solution
Tailing factor: NMT 2, Standard solution
Relative standard deviation: NMT 2.0%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
kanamycin (CigH36N4O11) in the portion of Capsules
taken:
Result = (ru/rs) x (Cs/Cu) x P x Fx 100
ru = peak area from the Sample solution
rs = peak area from the Standard solution
Cs = concentration of USP Kanamycin Sulfate RS in
the Standard solution (ug/mL)
Cy = nominal concentration of kanamycin in the
Sample solution (ug/mL)
P = potency of kanamycin in USP Kanamycin
Sulfate RS (ug/mg)
F = conversion factor, 0.001 mg/ug
Acceptance criteria: 90.0%-115.0%
PERFORMANCE TESTS
e DISSOLUTION, Procedure for a Pooled Sample (711)
Medium: 0.01 N hydrochloric acid; 900 mL
Apparatus 1: 100 rpm
Time: 45 min
Standard solution: USP Kanamycin Sulfate RS in
Medium
Sample solution: Sample per Dissolution (711). Dilute
with Medium to a concentration that is similar to that of
the Standard solution.
Analysis: Determine the percentage of the labeled
amount of kanamycin (CisH36N4O1) dissolved by using
the procedure described in the Assay, making any nec-
essary modifications.
Tolerances: NLT 75% (Q) of the labeled amount of
kanamycin (CisH36N4O11) is dissolved.
USP 41
SPECIFIC TESTS
e Loss ON DRYING (731)
Sample: 100mg
Analysis: Dry the Sample in a capillary-stoppered bottle
under vacuum at 60° for 3 h.
Acceptance criteria: NMT 4.0%
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers.
e USP REFERENCE STANDARDS (11)
USP Amikacin RS
USP Kanamycin Sulfate RS
Kaolin
DEFINITION
Kaolin is a native hydrated aluminum silicate, powdered and
freed from gritty particles by elutriation.
IDENTIFICATION
¢ A. IDENTIFICATION TESTS—GENERAL, Aluminum (191)
Sample solution: Mix 1g of Kaolin with 10 mL of
water and 5 mL of sulfuric acid in a porcelain dish.
Evaporate the mixture until the excess water is re-
moved, and continue heating the residue until dense,
white fumes of sulfur trioxide se Cool, cautiously
add 20 mL of water, boil for a few min, and filter.
Acceptance criteria: A gray residue (impure silica) re-
mains on the filter, and the filtrate meets the require-
ments of the test.
IMPURITIES
e LOSS ON IGNITION (733)
Analysis: Ignite between 550° and 600°.
Acceptance criteria: NMT 15.0%
e LEAD 51)
Test preparation: Transfer 1.0 g of Kaolin to a centri-
fuge tube, add 10 mL of 1 N nitric acid, and digest for
1 hina boiling water bath. Centrifuge until the solids
are completely separated, and pour the supernatant
into a 100-mL volumetric flask. Add 5 mL of 1 N nitric
acid, and digest for 15 min in a boiling water bath.
Centrifuge, and add the supernatant to the previous ex-
tract in the volumetric flask. Dilute with water to
volume.
Analysis: Proceed as directed in the chapter, using a
50-mL portion of this solution, 3 mL of Ammonium Cit-
rate Solution, 500 L of Hydroxylamine Hydrochloride So-
lution, and 1 mL of Potassium Cyanide Solution.
sonore criteria: NMT 5 yg of lead (NMT 10 ppm)
e IRON
Sample: 2.0g
Analysis: Triturate the Sample in a mortar with 10 mL of
water, and add 0.50 g of sodium salicylate.
Acceptance criteria: The mixture does not acquire
more thanaslight reddish tint.
¢ ACID-SOLUBLE SUBSTANCES
Sample: 1.0g
Analysis: Digest the Sample with 20 mL of 3 N hydro-
chloric acid for 15 min, and filter.
Acceptance criteria: NMT 2.0%; 10 mL of the filtrate,
evaporated to dryness and ignited, leaves NMT 10 mg
of residue.
¢ CARBONATE
Sample: 1.0g
Analysis: Mix the Sample with 10 mL of water and 5 mL
of sulfuric acid.
Official Monographs / Ketamine 2307
Acceptance criteria: No effervescence occurs.
SPECIFIC TESTS
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): It meets the requirements of
the tests for absence of Escherichia coli.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed
containers.
Ketamine Hydrochloride
CH,
.NH HCL
°
cr
Ci3HieCINO - HCl 274.19
Cyclohexanone, 2-(2-chlorophenyl)-2-(methylamino)-,
hydrochloride.
(4)-2-(0-Chlorophenyl)-2-(methylamino)cyclohexanone hy-
drochloride [1867-66-9].
» Ketamine Hydrochloride contains not less than
98.0 percent and not more than 102.0 percent of
Ci3Hi6CINO - HCl.
Packaging and storage—Preserve in well-closed contain-
ers. Store at 25°, excursions permitted between 15° and
30°.
USP Reference standards (11)—
USP Ketamine Hydrochloride RS
USP Ketamine Related Compound A RS [=
1-[(2-Chlorophenyl)(methylimino)methy|]cyclopentanol. “
CisHigNOCI 237.73
ne}
Clarity and color of solution—Dissolve 1 g in 5 mL of =
°
water: the solution is clear and colorless. |
Identification— °
Ko}
A: Infrared Absorption (197K)—Do not dry specimens. =
i}
B: Acid solvent—The UV absorption spectrum of a solu- me}
tion in 0.1 N hydrochloric acid (1 in 3000) exhibits maxima >
a
and minima at the same wavelengths as that of a similar
solution of USP Ketamine Hydrochloride RS, concomitantly
measured, and the respective absorptivities, at the wave-
lengths of maximum absorbance at about 269 and 276 nm,
do not differ by more than 3.0%.
Basic solvent—The UV absorption spectrum of a solution
in 0.01 N sodium hydroxide (1 in 1250), in a mixture of
water and methanol (1 in 20), exhibits maxima and minima
at the same wavelengths as that of a similar solution of USP
Ketamine Hydrochloride RS, concomitantly measured, and
the respective absorptivities, at the wavelength of maximum
absorbance at about 302 nm, do not differ by more than
3.0%.
PH (791): between 3.5 and 4.1, in a solution (1 in 10).
Residue on ignition (281): not more than 0.1%.
Delete the following:
°Heavy metals, Method | (231): 0.002%. (official 1-Jan-2018)
Related compounds—
Mobile phase—Dissolve 0.95 g of sodium 1-hexanesul-
fonate in 1 L of a solution consisting of a mixture of water
and acetonitrile (3:1). Add 4 mL of acetic acid, and mix.
Standard solution—Dissolve accurately weighed quantities
of USP Ketamine Hydrochloride RS and USP Ketamine Re-
 2308 Ketamine / Official Monographs
lated CompoundA RS in Mobile phase (sonicate if necessary)
to prepare a solution containing about 0.005 mg per mL of
each compound. Prepare immediately before use.
Test solution—Transfer an accurately weighed quantity of
about 50.0 mg of Ketamine Hydrochloride to a 50-mL volu-
metric flask. Dissolve in and dilute with Mobile phase to vol-
ume, sonicating if necessary.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 215-nm detector
and a 4.0-mm x 4.0-mm guard column with a 4.0-mm x
12.5-cm analytical column that contains 5-um packing L1.
The flow rate is about 1.0 mL per minute. Chromatograph
the Standard solution, and record the peak responses as di-
rected for Procedure: the order of elution is ketamine hydro-
chloride followed by ketamine related compound A; the res-
olution, R, between these two peaks is not less than 2.0; the
retention time of ketamine hydrochloride is between 3.0
and 4.5 minutes (if necessary, adjust the concentration of
water and acetonitrile); and the tailing factor is not greater
than 1.5.
Procedure—Separately inject equal volumes (about 20 wL)
of the Standard solution and the Test solution into the chro-
matograph, record the chromatograms, identify the
ketamine hydrochloride and ketamine related compound A
peaks, and measure the areas of the major peaks. Calculate
the area percentage of each impurity, relative to ketamine
hydrochloride, in the portion of Ketamine Hydrochloride
taken by the formula:
5000(C/W)
(rr/ Fs)
in which C is the concentration, in mg per mL, of USP
Ketamine Hydrochloride RS in the Standard solution; W is
the weight, in mg, of Ketamine be ee taken to pre-
pare the Test solution; r,is the peak area of each individual
impurity peak in the Test solution; and rs is the response of
the ketamine hydrochloride peak obtained from the Stan-
a” dard solution. Not more than 0.1% of ketamine related com-
roa poundAis found; the response of no other unknown impu-
Kd rity is greater than 0.3% of the ketamine peak area; and the
a
) sum of the responses of all unknown impurity peaks is not
= greater than 1.0% of the ketamine peak response.
5 Assay—
P= Buffer—Dissolve 5.75 g of monobasic ammonium phos-
a
va) phate in 1000 mL of water. Add 6 mL of triethylamine, and
=) adjust with phosphoric acid to a pH of 3.0.
Mobile phase—Preparea filtered and degassed mixture of
Buffer and methanol (65:35). Make adjustments if necessary
(see System Suitability under Chromatography (621)).
System suitability solution—Transfer about 12.5 mg each,
of USP Ketamine Hydrochloride RS and USP Ketamine Re-
lated Compound A RS, both accurately weighed, to a 50-mL
volumetric flask, dissolve in Mobile phase with the aid of
sonification if necessary, dilute with Mobile phase to volume,
and mix. Transfer 10.0 mL of the solution so obtained to a
100-mL volumetric flask, dilute with Mobile phase to vol-
ume, and mix.
Standard preparation—Transfer about 10 mg of USP
Ketamine Hydrochloride RS, accurately weighed, to a 50-mL
volumetric flask, add about 20 mL of Mobile phase, and son-
icate to dissolve. Dilute with Mobile phase to volume, and
mix.
Assay preparation—Transfer about 20 mg of Ketamine Hy-
drochloride, accurately weighed, to a 100-mL volumetric
flask, add about 35 mL of Mobile phase, and sonicate to dis-
solve. Dilute with Mobile phase to volume, and mix.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 220-nm detector
and a 4.6-mm x 25-cm column that contains 5-1um packing
L1. The flow rate is about 1.0 mL per minute. Chromato-
graph the System suitability solution, and record the peak
responses as directed for Procedure: the order of elution is
ketamine followed by ketamine related compound A; the
USP 41
resolution, R, between ketamine and ketamine related com-
poundAis not less than 2.0; the column efficiency deter-
mined from the ketamine peak is not less than 9400 theo-
retical plates; and the tailing factor determined from the
ketamine peak is not more than 1.6. Chromatograph the
Standard preparation, and record the ketamine peak re-
sponse as directed for Procedure: the relative standard devia-
tion for replicate injections is not more than 0.6%.
Procedure—Separately inject equal volumes (about 20 ,L)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
ure the responses for the major peaks. Calculate the quan-
tity, in mg, of Ci3HieCINO » HCI in the portion of Ketamine
Hydrochloride taken by the formula:
100C(ru / rs)
in which C is the concentration, in mg per mL, of USP
Ketamine Hydrochloride RS in the Standard preparation; and
ry and rs are the ketamine peak responses obtained from the
Assay preparation and the Standard preparation, respectively.
Ketamine Hydrochloride Injection
» Ketamine Hydrochloride Injection is a sterile so-
lution of Ketamine Hydrochloride in Water for In-
jection. It contains an amount of ketamine hy-
drochloride (Ci3HieCINO - HCI) equivalent to not
less than 95.0 percent and not more than
105.0 percent of the labeled amount of ketamine
(CisHisCINO).
Packaging and storage—Preserve in single-dose or in
multiple-dose containers, preferably of Type | glass, pro-
tected from light and heat.
Change to read:
USP Reference standards (11)—
ba (CN i-May-2018) ¢
USP Ketamine Hydrochloride RS
Identification—
A: The UV absorption spectrum, measured in the region
between 250 and 350 nm, of a dilution of Injection in 0.01
N methanolic sodium hydroxide containing ketamine hydro-
chloride equivalent to about 800 yg of ketamine per mL,
exhibits maxima and minima at the same wavelengths as
that of a similar pee of USP Ketamine Hydrochloride
RS, concomitantly measured.
B: The UV apsOrpueD spectrum of the solution employed
for measurement of absorbance of the assay solution, pre-
pared as directed in the Assay, exhibits maxima and minima
at the same wavelengths as that of the Standard solution,
prepared as directed in the Assay.
Bacterial Endotoxins Test (85)—It contains not more
than 0.4 USP Endotoxin Unit per mg of ketamine hydro-
chloride.
PH (791): between 3.5 and 5.5.
Other requirements—It meets the requirements under Jn-
jections and Implanted Drug Products (1).
Assay—Transfer an accurately measured volume of Injec-
tion, equivalent to about 500 mg of ketamine hydrochlo-
ride, to a 200-mL volumetric flask, dilute with water to vol-
ume, and mix. Transfer 20.0 mL of this solution to a 125-mL
separator, add 3 mL of 0.1 N sodium hydroxide, and extract
with three 15-mL portions of chloroform. Collect the chloro-
form extracts in a second 125-mL separator, and extract
USP 41 Official Monographs / Ketoconazole 2309

with three 30-mL portions of 0.1 N sulfuric acid, collecting Table 1

the acid extracts in a 200-mL volumetric flask. Dilute with Solution A Solution B
0.1 N sulfuric acid (saturated with chloroform) to volume,
and mix. Concomitantly determine the absorbances of this
0
solution and a Standard solution of USP Ketamine Hydro- 71
chloride RS in the same medium having a known concentra-
tion of about 250 11g per mL, in 1-cm cells at the wave-
length of maximum absorbance at about 269 nm, with a
suitable spectrophotometer, using 0.1 N sulfuric acid (satu- 3
rated with chloroform) as the blank. Calculate the quantity,
in mg, of ketamine (Ci3HisCINO) in each mL of the Injec- Diluent: Methanol
tion taken by the formula: Standard solution: 0.1 mg/mL of USP Ketoconazole RS
in Diluent
(237.73 | 274.19)(2C
/ V)(Au/As) Sample solution: 0.1 mg/mL of Ketoconazole in Diluent
Chromatographic system
in which 237.73 and 274.19 are the molecular weights of (See Chromatography (621), System Suitability.)
ketamine and ketamine hydrochloride, respectively; C is the Mode: LC
concentration, in ug per mL, of USP Ketamine Hydrochlo- Detector: UV 225 nm
ride RS in the Standard solution; V is the volume, in mL, of Column: 4.6-mm x 10-cm; 3-um packing L1
Injection taken; and Ay and As are the absorbances of the Flow rate: 2.0 mL/min
solution from the Injection and the Standard solution, re- Injection volume: 10 pL
spectively. System suitability
Sample: Standard solution
Seite requirements
Tailing ‘a factor: NMT 2.0
Relative standard deviation: NMT 0.73%
Ketoconazole Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of ketoconazole
(CasH2sCl2N4Ox) in the portion of Ketoconazole taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response from the Sample solution
Is = peak response from the Standard solution
Cs = concentration of USP Ketoconazole RS in the
Standard solution (mg/mL)
Cu = concentration of Ketoconazole in the Sample (ox,
solution (mg/mL) Ra
Acceptance criteria: ~98.0%-102.0% on the dried basis PS
IMPURITIES °
e RESIDUE ON IGNITION (281) rs
CasH2gCl2NgO4 531.43 Sample: 2g at
Acceptance criteria: NMT 0.1% —
Piperazine, 1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1 H- By
imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-, Tc
cis-; =x
Delete the following: a)
(4)-cis-1-Acetyl-4-(p-[[2-(2,4-dichloropheny!)-2-(imidazol-1-
ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl)piperazine °e HEAVY METALS, Method II (231): NMT 20 ppme coral 1.
[65277-42-1]. Jan-2018)
© ORGANIC IMPURITIES
DEFINITION Buffer, Solution A, Solution B, Mobile phase, Diluent,
Ketoconazole contains NLT 98.0% and NMT 102.0% of and Chromatographic system: Proceed as directed in
petacanntale (CasH2eClzN4Ou), calculated on the dried the ee
asis. Standard solution: 0.01 mg/mL each of USP
IDENTIFICATION Ketoconazole RS and USP Terconazole RS in Diluent
e A, INFRARED ABSORPTION (197K) Sample solution 10.0 mg/mL of Ketoconazole in
e B. The retention time of the major peak of the Sample Diluent
solution corresponds to that of the Standard solution, as System suitability
obtained in the Assay. Sample: Standard solution
Suitability requirements
ASSAY Resolution: NLT 2.0 between ketoconazole and ter-
e PROCEDURE conazole peaks
Buffer: 3.4 mg/mL of tetrabutyl ammonium hydrogen Relative standard deviation: NMT 5.0% for the
sulfate in water ketoconazole peak
Solution A: Acetonitrile and Buffer (5:95) Analysis
Solution B: Acetonitrile and Buffer (50:50) Samples: Standard solution and Sample solution
Mobile phase: See Table 1. Calculate the percentage of any impurities in the por-
tion of Ketoconazole taken:
Result = (ru/rs) x (Cs/Cu) x 100

tu = peak response of any impurities from the

Sample solution
 2310 Ketoconazole / Official Monographs USP 41

rs = peak response of Ketoconazole from the ASSAY

Standard solution © PROCEDURE
Cs = concentration of USP Ketoconazole RS in the Mobile phase: Acetonitrile and 0.01 M tetrabutylam-
Standard solution (mg/mL) monium hydrogen sulfate (25:75). Pass throughafilter
G = concentration of Ketoconazole in the Sample of 5-um or finer pore size, and degas.
solution (mg/mL) Diluent: Methanol and water (50:50)
Acceptance criteria: Disregard any peak less than System suitability solution: Dissolve 4 mg of USP
0.05%. Ketoconazole RS in 1.0 mL of a solution of potassium
Any individual unspecified impurity: NMT 0.10% sorbate in water (1 in 5000). Dilute with Diluent to
Total impurities: NMT 2.0% 10.0 mL.
Standard solution: 0.4 mg/mL of USP Ketoconazole RS
SPECIFIC TESTS in Diluent
© OPTICAL ROTATION, Specific Rotation (781S) Sample solution: [NoTe—lf the Oral Suspension has
Sample solution: 40 mg/mL in methanol settled, invert the container 10-15 times, and sonicate
Acceptance criteria: —1° to +1° at 20° for 5 min, or stir on a magnetic stirrer until the Oral
e Loss ON DRYING (731) Suspension appears homogeneous. Examine the mix-
Analysis: Dry under vacuum at 80° for 4 h. ture for the presence of bubbles and unsuspended
Acceptance criteria: NMT 0.5% solids before sampling.] Transfer 5.0 mL of homogene-
ous Oral Suspension to a 250-mL volumetric flask, add
ADDITIONAL REQUIREMENTS 100 mL of water, and stir for 15 min to dissolve the
© PACKAGING AND STORAGE: Preserve in well-closed xanthan gum. Add 135 mL of methanol, and stir for an
containers. additional 15 min. Cool, and dilute with methanol to
e USP REFERENCE STANDARDS (11)
volume.
USP Ketoconazole RS Chromatographic system
USP Terconazole RS (See Chromatography (621), System Suitability.)
Piperazine, 1-[4-[[2-(2,4-dichlorophenyl)-2-(1H-1,2, Mode: LC
4-triazol-1-ylmethyl)-1,3-dioxolan-4-yl]methox- Detector: UV 223 nm
y]phenyl]-4-(1 mele cis-; Columns
cis-1-(p-{{2-(2,4-Dichlorop ne H-1,2,4-triazol- Guard: 5-um packing L1
1-ylmethyl)-1, 3-dioxolan-4-yl]methoxy}phenyl)- Analytical: 4.6-mm x 25-cm; 5-um packing L1
4-isopropylpiperazine. Column temperature: 30°
CosH3iClaNsO3 5532.46 Flow rate: 1 mL/min
Injection volume: 5 ul
System suitability
Samples: System suitability solution and Standard
solution
Ketoconazole Compounded Oral [Note—The relative retention times for ketoconazole
ie
“
Suspension and sorbate are 1.0 and 1.7, respectively.]
ry Suitability requirements
ct
- DEFINITION Resolution: NLT 2.0 between sorbate and
aD Ketoconazole Compounded Oral Suspension contains NLT ketoconazole, System suitability solution
i) 90.0% and NMT 110.0% of the labeled amount of Relative standard deviation: NMT 2.0% for replicate
=
GS ketoconazole (C26H2gCl2N4Ou). injections, Standard solution
Analysis
Ps Prepare Ketoconazole Compounded Oral Suspension 20 mg/
Samples: Standard solution and Sample solution
a
mL as follows (see Pharmaceutical Compounding—Nonster-
va) ile Preparations (795)). Calculate the percentage of the labeled amount of
=) ketoconazole (CasH2sClzN4O4) in the portion of Oral
Suspension taken:
Ketoconazole 2.0 q
Cetylpyridinium Chloride 10 mg Result = (ru/rs) x (Cs/Cu) x 100
Xanthan Gum 0.15 q
Purified Water 30 mL ry = peak response from the Sample solution
ig peak response from the Standard solution
Suspension Structured Vehicle or Sugar-Free
Cs concentration of USP Ketoconazole RS in the
Suspension Structured Vehicle, a sufficient quantity
to make 100 mL
Standard solution (mg/mL)
Cy = nominal concentration of ketoconazole in the
Place the required number of tablets in a glass mortar, and Sample solution (mg/mL)
comminute to a fine pest such that they pass through Acceptance criteria: 90.0%-110.0%
a 40-mesh or 45-mesh sieve, or add Ketoconazole powder ADDITIONAL REQUIREMENTS
to the mortar. Dissolve the Cetylpyridinium Chloride in Puri- ¢ PACKAGING AND STORAGE: Package in tight, light-resistant
fied Water, and dilute quantitatively, and stepwise if neces- containers. Store at controlled room temperature.
sary, with Purified Water to obtain 10 mL of a solution © BEYOND-USE DATE: NMT 14 days after the date on which
containing 10 mg of Cetylpyridinium Chloride. Transfer this it was compounded when stored at controlled room
solution, in divided portions, to the mortar containing the
temperature
powder, and mix to form a smooth paste. Place 20 mL of o LABELING: Label it to state that it is to be well shaken
Purified Water in a beaker. Using moderate heat, stir to
before use, and that it is to be protected from light. La-
form a vortex, and slowly sprinkle the Xanthan Gum into bel it to state the Beyond-Use Date.
the vortex to obtain a uniform dispersion. Add the disper- e USP REFERENCE STANDARDS (11)
sion to the wetted powder paste, and mix until smooth. USP Ketoconazole RS
Add a sufficient quantity of the Suspension Structured Vehi-
cle or Sugar-Free Suspension Structured Vehicle to bring to
final volume. Mix well.
USP 41 Official Monographs / Ketoprofen 2311

Relative standard deviation: NMT 2.0%

Ketoconazole Tablets
DEFINITION
Ketoconazole Tablets contain NLT 90.0% and NMT 110.0%
of the labeled amount of ketoconazole (C2sH2gCl2N4O4).
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
ketoconazole (C2sH2gClzN4Ox) in the portion of Tablets
taken:

IDENTIFICATION Result = (Ru/Rs) x (Cs/Cu) x 100

° A.
Standard solution: 1 mg/mL of USP Ketoconazole RS in Ry = peak response ratio of ketoconazole to
chloroform terconazole from the Sample solution
Sample solution: Nominally 1 mg/mL of ketoconazole Rs = peak response ratio of ketoconazole to
in chloroform prepared as follows. Transfer a quantity of terconazole from the Standard solution
finely powdered Tablets, equivalent to 50 mg of Gs = concentration of USP Ketoconazole RS in the
ketoconazole, to a suitable flask. Add 50 mL of chloro- Standard solution (mg/mL)
form, shake for 2 min, and filter. Cy = nominal concentration of ketoconazole in the
Chromatographic system Sample solution (mg/mL)
(See Chromatography (621), Thin-Layer Chromato- Acceptance criteria: 90.0%-110.0%
graphy.) PERFORMANCE TESTS
Mode: TLC. e DISSOLUTION (711)
Adsorbent: 0.25-mm layer of chromatographic silica Medium: 0.1 N hydrochloric acid; 900 mL
gel mixture Apparatus 2: 50 rpm
Application volume: 10 LL Time: 30 min
Developing solvent system: n-Hexane, ethyl acetate, Analytical wavelength: UV 270 nm
methanol, glacial acetic acid, and water (42:40:15:1:2) Standard solution: USP Ketoconazole RS in Medium
Analysis: Allow the spots to dry, and develop the chro- Sample solutions: Pass portions of the solution under
matogram in the Developing solvent system until the sol- test through a suitable filter of 0.45-um pore size, and
vent front has moved three-fourths of the length of the dilute with Medium to a concentration similar to that of
plate. Remove the plate from the chamber, air-dry, and the Standard solution.
view under short-wavelength UV light. Tolerances: NLT 80% (Q) of the labeled amount of
Ae criteria: The Rr value of the principal spot ketoconazole (C26H2sClzN4O,) is dissolved.
of the Sample solution corresponds to that of the Stan- e UNIFORMITY OF DOSAGE UNITS (905): Meet the
dard solution. requirements
ASSAY ADDITIONAL REQUIREMENTS
© PROCEDURE © PACKAGING AND STORAGE: Preserve in well-closed
Solution A: Diisopropylamine in methanol (1 in 500) containers.
Solution B: 5 mg/mL of ammonium acetate in water © USP REFERENCE STANDARDS (11) cS
Mobile phase: Solution A and Solution B (7:3) USP Ketoconazole RS ie]
Diluent: Methanol and methylene chloride (1:1) USP Terconazole RS Fd
Internal standard solution: 5 mg/mL of USP Ter-
conazole RS in Diluent °
Standard solution: 0.4 mg/mL of USP Ketoconazole RS 3
(-)
in Diluent prepared as follows. Transfer 20 mg of USP S|
Ketoconazole RS to a 50-mL volumetric flask. Add
5.0 mL of Internal standard solution, and dilute with Ketoprofen BI
Diluent. =A
Sample stock solution: Nominally 4 mg/mL of O° CHy ~

ketoconazole in Diluent prepared as follows. Weigh and a Sy OH

finely powder NLT 20 Tablets. Transfer the nominal
equivalent to 200 mg of ketoconazole to a suitable
screw-capped bottle. Add 50.0 mL of Diluent, shake by
mechanical means for 30 min, and centrifuge.
Sample solution: Nominally 0.4 mg/mL of
ketoconazole in Diluent prepared as follows. Transfer
5.0 mL of the clear supernatant so obtained from the
Sample stock solution to a 50-mL volumetric flask. Add
5.0 mL of Internal standard solution, and dilute with Dil-
uent to volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector; UV 225 nm
Column: 3.9-mm x 30-cm; packing L1
Flow rate: 3 mL/min
Injection volume: 20 uL
System suitability
Sample: Standard solution
[NoTte—The relative retention times for ketoconazole
and terconazole are 0.6 and 1.0, respectively.]
Suitability requirements
Resolution: NLT 2.0 between ketoconazole and
terconazole
lv lo oO
Cy6H1403 254.28
Benzeneacetic acid, 3-benzoyl-o.-methyl-, (+)-;
(+)-m-Benzoylhydratropic acid [22071-15-4].
DEFINITION
Ketoprofen contains NLT 98.5% and NMT 101.0% of
Ci6H1403, calculated on the dried basis.
IDENTIFICATION
© A. INFRARED ABSORPTION (197K)
e B. ULTRAVIOLET ABSORPTION (197U)
Analytical wavelength: 258 nm
Medium: Methanol and water (3:1)
Blank: Medium
Sample solution: 10 g/mL in Medium
Acceptance criteria: Absorptivities, calculated on the
dried basis, do not differ by more than 3.0%.
 2312 Ketoprofen / Official Monographs
ASSAY
© PROCEDURE
Sample: 450mg
Titrimetric system
(See Titrimetry (541).)
Mode: Direct titration
Titrant: 0.1 N sodium hydroxide VS
Endpoint detection: Visual
Analysis: Dissolve the Sample in 25 mL of alcohol. Add
25 mL of water and several drops of phenol red TS.
Perform a blank determination. Each mL of Titrant is
equivalent to 25.43 mg of Ci6Hi4O3, [NOTE—Standard-
ize the 0.1 N sodium hydroxide by a similar titration of
primary standard benzoic acid.]
Acceptance criteria: 98.5%-101.0% on the dried basis
IMPURITIES
© RESIDUE ON IGNITION (281): NMT 0.2%
Delete the following:
°e HEAVY METALS, Method |/ (231): NMT 0.002%e6 corteal 1.
jar-2018)
eORGANIC IMPURITIES
[NoTe—Protect the solutions from light.]
Buffer: 68 g/L of monobasic potassium phosphate in
pare and adjust with phosphoric acid to a pH of 3.5 +
.05
Mobile phase: Acetonitrile, water, and Buffer (43:55:2)
System suitability solution: 5 g/mL of USP
Ketoprofen RS and 1.5 g/mL of USP Ketoprofen Re-
lated Compound D RS in Mobile phase
Standard solution: 0.002 mg/mL of USP Ketoprofen RS
in Mobile phase
Sample solution: 1 mg/mL of Ketoprofen in Mobile
een
val hromatographic system
ca (See Chromatography (621), System Suitability.)
om Mode: LC
cS
i Detector: UV 233 nm
D Column: 4.6-mm x 15-cm; 5-um packing L1
ro)
= Flow rate: 1 mL/min
S Injection size: 20 uL
= Run time: Seven times the retention time for
i Ketoprofen
~“” System suitability
> Samples: System suitability solution and Standard
solution
[Note—The relative retention times for ketoprofen and
ketoprofen related compound D Saecobr lala
none) are about 1.0 and 1.6, respectively.]
Suitability requirements
Resolution: NLT 7.0 between ketoprofen related
compound D and ketoprofen, System suitability
solution
Column efficiency: NLT 2250 theoretical plates from
ketoprofen, System suitability solution
Tailing factor: NMT 2.0 for ketoprofen, System suita-
bility solution
Relative standard deviation: NMT 5%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the por-
tion of Ketoprofen taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response of impurity from the Sample
solution
rs = peak response of ketoprofen from the
Standard solution
Gs = concentration of USP Ketoprofen RS in the
Standard solution (mg/mL)
USP 41
Cy = concentration of the Sample solution (mg/mL)
Acceptance criteria
Any individual impurity: NMT 0.2%
Total impurities: NMT 1.0%
SPECIFIC TESTS
e MELTING RANGE OR TEMPERATURE, Procedure for Class |
(741): 92.0°-97.0°
¢ OPTICAL ROTATION, Specific Rotation (781S)
Sample solution: 10 mg/mL, in dehydrated alcohol
Acceptance criteria: +1° to -1°
e Loss ON DRYING (731): Dry a sample in a vacuum at 60°
for 4 h: it loses NMT 0.5% of its weight.
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers.
e USP REFERENCE STANDARDS (11)
USP Ketoprofen RS
USP Ketoprofen Related Compound D RS
3-Acetylbenzophenone.
Ketoprofen Capsules
DEFINITION
Ketoprofen Capsules contain NLT 90.0% and NMT 110.0%
of the labeled amount of ketoprofen (Ci6H1403).
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
Sample solution: Shake a quantity of the contents of
the Capsules containing 50 mg of ketoprofen with 5 mL
of chloroform for 5 min, filter, and evaporate to dryness
using a rotary evaporator.
Acceptance criteria: Meet the requirements
¢ B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
ASSAY
© PROCEDURE
The Standard solution and Sample solution must be pro-
tected from light.
Mobile phase: Acetonitrile, glacial acetic acid, and
water (90:1:110)
System suitability stock solution: 0.25 mg/mL of USP
Ketoprofen RS and 0.5 mg/mL of USP Ketoprofen Re-
lated Compound A RS in Mobile phase
System suitability solution: 0.02 a of USP
etoprofen RS and 0.04 mg/mL of USP Ketoprofen Re-
late Seopound ARS in Mobile phase from System suit-
ability stock solution
Standard stock solution: 0.24 mg/mL of USP
Ketoprofen RS in Mobile phase
Standard solution: 0.024 mg/mL of USP Ketoprofen RS
in Mobile phase from Standard stock solution
Sample solution: Nominally 0.024 mg/mL of
ketoprofen in Mobile phase fepared as follows. Remove
completely the contents of NLT 20 Capsules, and trans-
fer a quantity of the contents, equivalent to 200 mg of
ketoprofen, to a 250-mL volumetric flask. Add 150 mL
of Mobile phase, stir for 2 h, then dilute with Mobile
phase to volume. Centrifuge a portion of the prepara-
tion. Pipet 3.0 mL of clear supernatant into a 100-mL
volumetric flask. Dilute with Mobile phase to volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 250 nm
USP 41 Official Monographs / Ketoprofen 2313

Column: 4.6-mm x 25-cm; 5-um packing L1 Standard solution: 2 g/mL of USP Ketoprofen RS,
Flow rate: 1.2 mL/min 2ug/mi of USP Ketoprofen Related Compound C RS,
Injection volume: 20 uL and 3 ug/mL of USP Ketoprofen Related Compound D
System suitability RS in Diluent
Samples: System suitability solution and Standard Sample solution: Nominally 1 mg/mL of ketoprofen in
solution Mobile phase
Suitability requirements Chromatographic system
Resolution: NLT 3.0 between ketoprofen and (See Chromatography (621), System Suitability.)
ketoprofen related compound A, System suitability Mode: LC
solution Detector: UV 233 nm
Tailing factor: NMT 1.5 for the ketoprofen peak, Column: 4.6-mm x 15-cm; 5-um packing L1
System suitability solution Flow rate: 1 mL/min
Relative standard deviation: _NMT 2.0%, Standard Injection volume: 20 pL
solution Run time: 7 times the retention time of ketoprofen
Analysis System suitability
Samples: Standard solution and Sample solution Sample: System suitability solution
Calculate the percentage of the labeled amount of Suitability requirements
ketoprofen (CisH14O3) in the portion of Capsules taken: Resolution: NLT 7.0 between ketoprofen related
compoundDand ketoprofen
Result = (ru/rs) x (Cs/Cu) x 100 Relative standard deviation: NMT 10% for the
ketoprofen peak
ru = peak response from the Sample solution Analysis
Is = peak response from the Standard solution Samples: Standard solution and Sample solution
Cs = concentration of USP Ketoprofen RS in the Calculate the percentage of each impurity in the por-
Standard solution (mg/mL) tion of Capsules taken:
Cy = nominal concentration of ketoprofen in the
Sample solution (mg/mL) Result = (ru/rs) x (Cs/Cu) x 100
Acceptance criteria: 90.0%-110.0%
fu = peak response of each impurity from the
PERFORMANCE TESTS Sample solution
e DISSOLUTION (711) Is = peak response of the corresponding related
The Standard solution and Sample solution must be pro- compound from the Standard solution
tected from light. Gs = concentration of the corresponding USP
Medium: 0.05 M phosphate buffer, pH 7.4; 1000 mL Ketoprofen Related Compound RS in the
Apparatus 2: 50 rpm Standard solution (mg/mL); use the
Time: 30 min concentration of the USP Ketoprofen RS for
Standard solution: USP Ketoprofen RS in Medium unknown impurities
Sample solution: Pass a portion of the solution under Cu = nominal concentration of ketoprofen in the os
test through a suitable filter. Dilute with Medium, if nec- Sample solution (mg/mL) 7A)
essary, to a concentration similar to that of the Stan- Acceptance criteria: See Table 1. a]
dard solution. =
Instrumental conditions °
(See Ultraviolet-Visible Spectroscopy (857).) Table 1 |
Mode: UV
Analytical wavelength: 260 nm
Relative
Retention
Acceptance
Criteria,
re=
Cellpath length: 1 cm sy
me]
Blank: Medium >
Analysis al

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of
ketoprofen (Ci6H1403) dissolved: ivi

Result = (Au/As) x (Cs/L) x D x Vx 100 2 2-(3-Carboxyphenyl) propionic acid.

»3-Acetylbenzophenone,
Au = absorbance from the Sample solution
As = absorbance from the Standard solution ADDITIONAL REQUIREMENTS
Cs = concentration of the Standard solution © PACKAGING AND STORAGE: Preserve in tight containers,
(mg/mL) and store at controlled room temperature.
iE = label claim (mg/Capsule) e USP REFERENCE STANDARDS (11)
D = dilution factor of the Sample solution USP Ketoprofen RS
V = volume of Medium, 1000 mL (+)-m-Benzoylhydratropic acid.
Tolerances: NLT 80% (Q) of the labeled amount of CisHy4O3 254.28
ketoprofen (CisH14O3) is dissolved. USP Ketoprofen Related Compound A RS
a-Methyl-3-(4-methylbenzoyl) benzeneacetic acid.
IMPURITIES Ci7zHi6O3 268.31
© ORGANIC IMPURITIES USP Ketoprofen Related Compound C RS
The System suitability solution, Standard solution, and 2-(3-Carboxyphenyl) propionic acid.
Sample solution must be protected from light. CioH10O, 194.18
Buffer: 68.0 g/L of monobasic potassium phosphate in USP Ketoprofen Related Compound D RS
water. Adjust with phosphoric acid to a pH of 3.5 + 3-Acetylbenzophenone.
0.05. CisHi202 224.25
Mobile phase: Acetonitrile, water, and Buffer (43:55:2)
Diluent: Acetonitrile and water (2:3)
System suitability solution: 5 g/mL of USP
Ketoprofen RS and 1.5 g/mL of USP Ketoprofen Re-
lated CompoundD RS in Diluent
 2314 Ketoprofen / Official Monographs USP 41

Acceptance criteria: 90.0%-110.0%

Ketoprofen Extended-Release Capsules PERFORMANCE TESTS
e DISSOLUTION (711)
DEFINITION Medium: pH 6.8 phosphate buffer; 1000 mL
Ketoprofen Extended-Release Capsules contain NLT 90.0% Apparatus 2: 50 rpm
and NMT 110.0% of the labeled amount of ketoprofen Time: 1, 4, and 8h
(Ci6H140s). Detector: UV 258 nm
Standard solution: About 0.1 mg/mL of USP
IDENTIFICATION Ketoprofen RS in Medium
e A. The retention time of the major peak of the Sample Sample solution: Pass a portion of the solution under
solution corresponds to that of the Standard solution, as test through a suitable filter of 10-um pore size, then
obtained in the Assay. pass the filtrate througha suitable filter of 0.45-um
e B. ULTRAVIOLET ABSORPTION (197): The UV spectrum from pore size.
the Sample solution in the Analysis for the Dissolution sec- Capsules labeled to contain 200 mg: In a test tube,
tion corresponds to the spectrum from the Standard solu- dilute 5.0 mL of filtrate with 5.0 mL of Medium.
tion. Capsules labeled to contain 150 mg: In a test tube,
dilute 6.0 mL of filtrate with 3.0 mL of Medium.
ASSAY Capsules labeled to contain 100 mg: No dilution is
© PROCEDURE laceg
[Note—Protect the Standard solution and Sample solution
Capsule blank: Place 10 empty, clean Capsules of the
from light.] appropiate dosage into a 1000-mL volumetric flask.
Mobile phase: Acetonitrile, water, and glacial acetic
acid (90:110:1) Add about 800 mL of Medium at 37°. Stir until Capsule
Standard stock solution: 0.24 mg/mL of USP shells are disintegrated. After equilibration to room tem-
Ketoprofen RS in Mobile phase perature, dilute with Medium to volume. Transfer
Standard solution: 0.024 mg/mL of USP Ketoprofen RS 100.0 mL to a 1000-mL volumetric flask, and dilute
with Medium to volume. Pass through a suitable filter of
in Mobile phase, from the Standard stock solution
10-um pore size, then pass the filtrate through a suita-
System suitability solution: 0.25 mg/mL of USP ble filter of 0.45-1m pore size.
Ketoprofen RS and 0.5 mg/mL of USP Ketoprofen Re- Capsules labeled to contain 200 mg: Inaflask, dilute
lated Compound A RS in Mobile phase. Pipet 4.0 mL of 25.0 mL with 25.0 mL of Medium.
this solution into a 50-mL volumetric flask, and dilute Capsules labeled to contain 150 mg: Inaflask, dilute
with Mobile phase to volume. 30.0 mL with 15.0 mL of Medium.
Sample solution: Remove completely the contents of Capsules labeled to contain 100 mg: No dilution is
NLT 20 Capsules, and transfer a quantity of the beads,
equal to 200 mg of ketoprofen, to a 250-mL volumetric
necessary.
Analysis
flask. Add 150 mL of Mobile phase and mix; bring to Samples: Standard solution, Sample solution, and Cap-
Ps volume. Centrifuge, and pipet 3.0 mL of clear superna- sule blank, using Medium as the blank
<= tant that contains about 2.4 mg of ketoprofen into a Calculate the concentration, in mg/mL, of ketoprofen in
rs 100-mL volumetric flask. Dilute with Mobile phase to the sample withdrawn at each time point:
Ss volume.
—
D Chromatographic system
} (See Chromatography (621), System Suitability.)
Result = (Au — Ace) x (Cs/As)
iS
S Mode: LC Au = absorbance of the Sample solution
= Detector: UV 254 nm Acs = absorbance of the Capsule blank
io
Column: 4.6-mm x 25-cm; 5-um packing L1 Cs = concentration of USP Ketoprofen RS in the
al Flow rate: 1.2 mL/min Standard solution (mg/mL)
=) Injection size: 20 uL As = absorbance of the Standard solution
System suitability Calculate the percentage of ketoprofen dissolved at
Samples: Standard solution and System suitability each time point:
solution
Suitability requirements Result = (D + ZR) x 100/L
Resolution: NLT 3.0 between ketoprofen and
ketoprofen related compound A, System suitability D = [amount dissolved (mg)] = volume (mL)
solution remaining before draw x concentration
Tailing factor: NMT 1.5 for the ketoprofen peak, Sys- (mg/mL) of sample withdrawn at the
tem suitability solution sampling time point
Relative standard deviation: NMT 2.0%, Standard R = [amount removed (mg)] = volume (mL) of
solution sample withdrawn x concentration (mg/mL)
Analysis of sample withdrawn at each time point
Samples: Standard solution and Sample solution 100 =conversion factor for percentage
Calculate the percentage of Cis6H14O3 in the portion of L = Capsule label claim (mg)
Capsules taken: Tolerances: The percentage of the labeled amount of
ketoprofen released at the times specified conforms to
Result = (ru/ts) x (Cs/Cu) x 100 Acceptance Table 2.
tu = peak response from the Sample solution
Ts = peak response from the Standard solution Time
Cs = concentration of USP Ketoprofen RS in the
Standard solution (mg/mL)
Cy = concentration of ketoprofen in the Sample
solution (mg/mL)
e UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
USP 41
Procedure for content uniformity: [NoTE—Protect the
Standard solution and Sample solution from light.]
Mobile phase, Standard solution, System suitability
solution, and Chromatographic system: Proceed as
directed in the Assay.
Sample solution: Transfer the contents of 10 Capsules,
1 Capsule each, to each of 10 250-mL volumetric flasks,
add about 150 mL of Mobile phase to each flask, and
stir for 2 h. Dilute with Mobile phase to volume, and
mix. Centrifuge, and pipet a volume of clear superna-
tant that contains about 2.4 mg of ketoprofen into a
100-mL volumetric flask. Dilute with Mobile phase to
volume.
System suitability
Samples: Standard solution and System suitability
solution
Suitability requirements
Resolution: NLT 3.0 between ketoprofen and
ketoprofen related compound A, System suitability
solution
Tailing factor: NMT 1.5 for the ketoprofen peak, Sys-
tem suitability solution
Relative standard deviation: NMT 2.0%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of CisH14O3 in each Capsule:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response from the Sample solution
rs = peak response from the Standard solution
Cs = concentration of USP Ketoprofen RS in the
Standard solution (mg/mL)
Cy = concentration of ketoprofen in the Sample
solution (mg/mL)
SPECIFIC TESTS
e WATER DETERMINATION, Method | (921): NMT 3.0%
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers,
and store at controlled room temperature.
© USP REFERENCE STANDARDS (11)
USP Ketoprofen RS
USP Ketoprofen Related Compound A RS
a-Methyl-3-(4-methylbenzoyl) benzeneacetic acid.
Ketorolac Tromethamine
9 for identifying the peaks due to the ketorolac 1-keto
* “OH oH
~ § \ a 1-keto analog and ketorolac.]
lL l or
8 Tromethamine RS in Diluent
CisHisNO3 - Cai NO3 376.40
1H-Pyrrolizine-1-carboxylic acid, 5-benzoyl-2,3-dihydro, (+)-,
compound with 2-amino-2-(hydroxymethyl)-1,3-
propanediol (1:1);
(+)-5-Benzoyl-2,3-dihydro-1 H-pyrrolizine-1-carboxylic acid,
compound with 2-amino-2-(hydroxymethyl)-1,3-
propanediol (1:1) [74103-07-4].
DEFINITION
Ketorolac Tromethamine contains NLT 98.5% and NMT
101.5% of ketorolac tromethamine (CisHi3NO3 -
CaH1iNO3), calculated on the dried basis.
Official Monographs / Ketorolac 2315
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
e C. THIN-LAYER CHROMATOGRAPHY, Tromethamine Test
Diluent: Dichloromethane and methanol (2:1)
Standard solution: 5 mg/mL of USP Ketorolac
Tromethamine RS in Diluent
Sample solution: 5 mg/mL of Ketorolac Tromethamine
in Diluent
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
raphy.)
Mode.” Te
Adsorbent: 0.25-mm layer of chromatographic silica
gel mixture
Application volume: 40 pL
Developing solvent system: Dichloromethane, ace-
tone, and glacial acetic acid (95:5:2)
Spray reagent: Freshly prepared alcoholic solution
containing 30 mg/mL oFninhydrin
Analysis
Samples: Standard solution and Sample solution
Develop the chromatogram until the solvent front has
moved about three-fourths of the length of the plate.
Remove the plate from the chamber, and allow the
solvent to evaporate. Spray the plate with Spray rea-
gent, and heat the plate at about 150° for 2-5 min.
Acceptance criteria: Yellow spots with pink to purple
borders develop on the plate in the areas where the
Standard solution and the Sample solution were applied.
ASSAY
¢ PROCEDURE
Protect all the solutions from light.
Buffer: 5.75 g/L of monobasic ammonium phosphate.
Adjust with phosphoric acid to a pH of 3.0. =
Mobile phase: Tetrahydrofuran and Buffer (30:70) 4)
Diluent: Tetrahydrofuran and water (30:70) v
System suitability solution: In a 250-mL separator, mix <<
100 mL of water, 100 mL of dichloromethane, 30 mg of i}
USP Ketorolac Tromethamine RS, and 1 mL of 1 N hy- —]
}
drochloric acid. Insert the stopper, shake, and allow the ro}=
layers to separate. Transfer the lower dichloromethane
2
layer to a stoppered borosilicate glass flask, and discard a}
the upper layer. Expose the dichloromethane solution >
ww
to direct sunlight for 10-15 min. Transfer 1.0 mL of the
solution to a vial, evaporate in a current of air or in a
stream of nitrogen to dryness, add 1.0 mL of Diluent,
and swirl to dissolve. [NoTE—This solution may be
stored under refrigeration and used as long as the chro-
matogram obtained as directed for Analysis is suitable
analog and ketorolac 1-hydroxy analog, and for the
measurement of the resolution between the ketorolac
Standard solution: 0.4 mg/mL of USP Ketorolac
Sample solution: 0.4 mg/mL of Ketorolac
Tromethamine in Diluent
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 313 nm
Column: 4.6-mm x 25-cm; 5-4m packing L7
Column temperature: 40°
Flow rate: 1.5 mL/min
Injection volume: 10 uL
System suitability
Samples: System suitability solution and Standard
solution
[Note—The relative retention times for the ketorolac
1-hydroxy analog, the ketorolac 1-keto analog, and
ketorolac are about 0.63, 0.89, and 1.0, respectively.
 2316 Ketorolac / Official Monographs USP 41

Make adjustments, if necessary, to achieve a retention Table 1 (Continued)

time for ketorolac of about 8-12 min.] Relative Relative Acceptance
Suitability requirements Retention Response Criteria,
Resolution: NLT 1.5 between ketorolac 1-keto analog Name Time Factor NMT (%
and ketorolac, System suitability solution
Column efficiency: NLT 5500 theoretical plates, Impurity having a
Standard solution 0.66 relative
Relative standard deviation: NMT 1.5%, Standard retention time 0.66 0.91 0.5
solution Ketorolac 1-keto
Analysis analog 0.89 0.52 0.1
Samples: Standard solution and Sample solution Individual
Calculate the percentage of ketorolac tromethamine unspecified _
(CisHi3NO3 + C4HiiNOs) in the portion of Ketorolac impurity 1.0 0.5
Tromethamine taken: Ketorolac
tromethamine 1.0 1.0 a
Result = (ru/rs) x (Cs/Cu) x 100 Total impurities _— = 1.0
tu = peak area from the Sample solution
ls = peak area from the Standard solution SPECIFIC TESTS
Cs = concentration of USP Ketorolac Tromethamine e PH (791)
RS in the Standard solution (mg/mL) Sample solution: 10 mg/mL
Cu = concentration of Ketorolac Tromethamine in Acceptance criteria: 5./7-6.7
the Sample solution (mg/mL) e Loss ON DRYING (731)
Acceptance criteria: 98.5%-101.5% on the dried basis Analysis: Dry under vacuum at 60° for 3 h.
Acceptance criteria: NMT 0.5%
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.1% ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers. Store at 25°, excursions permitted between
Delete the following: 15° and 30°.
e USP REFERENCE STANDARDS (11)
°e HEAVY METALS, Method // (231): NMT 20 ppme coiraai1- USP Ketorolac Tromethamine RS
lan-2078)
e ORGANIC IMPURITIES
Mobile phase, Diluent, System suitability solution,
Standard solution, and Sample solution: Proceed as
directed in the Assay.
Chromatographic system Ketorolac Tromethamine Injection
cc
vw
(See Chromatography (621), System Suitability.)
a Mode: LC DEFINITION
sS
- Detector: UV 313 nm Ketorolac Tromethamine Injection is a sterile solution of
Dp Column: 4.6-mm x 25-cm; 5-1um packing L7 Ketorolac Tromethamine. It contains NLT 90.0% and NMT
o) Column temperature: 40° 110.0% of the labeled amount of ketorolac tromethamine
< (CisHi3NO3 - CaH11NOs).
GC) Flow rate: 1.5 mL/min
= Injection volume: 10 pL IDENTIFICATION
Qa
Run time: 3 times the retention time of ketorolac e A. The retention time of the major peak of the Sample
” Analysis solution corresponds to that of the Standard solution, as
=) Samples: Standard solution and Sample solution obtained in the Assay.
Calculate the percentage. of each individual impurity in e B. The UV spectrum of the ketorolac peak of the Sample
the portion of Ketorolac Tromethamine taken: solution corresponds to that of the Standard solution, as
Result = (ru/rr) x F x 100 obtained in the Assay.

tu = peak response of each individual impurity

ASSAY
e PROCEDURE
from the Sample solution [Note—Protect all solutions from light.]
tr = sum of all the peak responses from the Sample Mobile phase: Methanol, water, and glacial acetic acid
solution (55:44:1)
E = relative response factor (see Table 1) Diluent: Methanol and water (1:1)
Acceptance criteria: See Table 1. Standard solution: 0.05 mg/mL of USP Ketorolac
Tromethamine RS in Diluent
Table 1 Sample solution: Nominally equivalent to 0.05 mg/mL
Relative Relative Acceptance of ketorolac tromethamine inDiluent from Injection
Retention Response Criteria, Chromatographic system
Name Time Factor NMT (%) (See Chromatography (621), System Suitability.)
Impurity having a
Mode: LC
Detector: UV 254 nm. For Identification test B, use a
0.54 relative
retention time 0.54 2.2 0.5
diode array detector in the range of 200-600 nm.
Ketorolac 1-hydroxy
analog 0.63 0.67 0.1
USP 41 Official Monographs / Ketorolac 2317

Column: 4.6-mm x 25-cm; 5-um packing L1 fs = peak response of ketorolac related compound
Flow rate: 1.2 mL/min A, ketorolac related compound B, ketorolac
Injection volume: 100 pL related compound C, or ketorolac related
pee suitability compoundD from the Standard solution
ample: Standard solution Gs = concentration of the corresponding related
Suitability requirements compound in the Standard solution (mg/mL)
Tailing factor: NMT 1.5 for the ketorolac peak Cu = nominal concentration of ketorolac
Relative standard deviation: NMT 1.0% tromethamine in the Sample solution
Analysis (mg/mL)
Samples: Standard solution and Sample solution Calculate the percentage of any unspecified impurity in
Calculate the percentage of the labeled amount of the portion of Injection taken:
ketorolac tromethamine (CisHi3NO3 - C4H,NO3) in
each mL of Injection taken: Result = (ru/rs) x (Cs/Cu) x 100
Result = (ru/rs) x (Cs/Cu) x 100 tu = peak response of any unspecified impurity
from the Sample solution
tu = peak response of ketorolac from the Sample Is = peak response of ketorolac from the Standard
solution solution
Is = peak response of ketorolac from the Standard Gs = concentration of USP Ketorolac Tromethamine
solution RS in the Standard solution (mg/mL)
Cs = concentration of USP Ketorolac Tromethamine Cu = nominal concentration of ketorolac
RS in the Standard solution (mg/mL) tromethamine in the Sample solution
CG = nominal concentration of ketorolac (mg/mL) .
tromethamine in the Sample solution Acceptance criteria: See Table 1. Disregard any impu-
(mg/mL) rity peak less than 0.05%.
Acceptance criteria: 90.0%-110.0%
IMPURITIES Table 1
© ORGANIC IMPURITIES Relative Acceptance
[Note—Protect all solutions from light.] Retention Criteria,
Mobile phase, Diluent, and Chromatographic system: Name Time NMT (%)
Proceed as directed in the Assay. Ketorolac related
Standard stock solution: 0.10 mg/mL each of USP compound A 0.4 0.20
Ketorolac Tromethamine RS, USP Ketorolac Related Ketorolac related
Compound ARS, USP Ketorolac Related Compound B compound B 0.6 0.5
RS, USP Ketorolac Related Compound C RS, and USP
Ketorolac related
Ketorolac Related CompoundD RS in Diluent prepared compound C 0.8 0.5
as follows. Transfer USP Ketorolac Tromethamine RS,
USP Ketorolac Related Compound A RS, USP Ketorolac Ketorolac 1.0 &
Related Compound B RS, USP Ketorolac Related Com- Ketorolac related a]
pound C RS, and USP Ketorolac Related Compound D compound D 2.1 0.20 i
RS to a suitable volumetric flask. Add 4% of the volume Any unspecified Lit °
of the flask with methanol. Sonicate and dilute with impurity 0.20 =]
Diluent to volume. Total impurities = 1.50 a
Standard solution: 0.2 g/mL each of USP Ketorolac
Tromethamine RS, USP Ketorolac Related Compound A BY
RS, USP Ketorolac Related Compound B RS, USP SPECIFIC TESTS oe
Ketorolac Related Compound C RS, and USP Ketorolac © PH (791): 6.9-7.9 a)
Related CompoundD RS inDiluent from the Standard e BACTERIAL ENDOTOXINS TEST (85): It contains NMT 5.8
Stock solution USP Endotoxin Units/mg of ketorolac tromethamine.
Sample solution: Prepare nominally equivalent to © STERILITY TESTS (71): Meets the requirements when
0.2 mg/mL of ketorolac tromethamine in Diluent. tested as directed in Test for Sterility of the Product to Be
System suitability Examined, Membrane Filtration
Sample: Standard solution © PARTICULATE MATTER IN INJECTIONS (788): Meets the re-
[Note—See Table 7 for the relative retention times.] quirements for small-volume injections
Suitability requirements e OTHER REQUIREMENTS: Meets the requirements in Injec-
Resolution: NLT 2 between ketorolac related com- tions and Implanted Drug Products (1)
pound C and ketorolac
Relative standard deviation: NMT 2.8% for all the ADDITIONAL REQUIREMENTS
peaks e PACKAGING AND STORAGE: Preserve in single-dose contain-
Analysis ers, preferably of Type | glass, protected from light, and
Samples: Standard solution and Sample solution store at controlled room temperature.
Calculate the percentage of the labeled amounts of
ketorolac related compound A, ketorolac related com- Change to read:
ound B, ketorolac related compound C, and ketoro-
pereleed compoundDin the portion of Injection e USP REFERENCE STANDARDS (11)
taken: we «CN 1-May-2018)
USP Ketorolac Tromethamine RS
Result = (ru/rs) x (Cs/Cu) x 100 USP Ketorolac Related Compound A RS
5-Benzoyl-N-[1,3-dihydroxy-2-(hydroxymethyl)propan-
ru = peak response of ketorolac related compound 2-yl]-2,3-dihydro-1 H-pyrrolizine-1 -carboxamide.
A, ketorolac related compound B, ketorolac
related compound C, or ketorolac related
CisH22N20s 358.39
USP Ketorolac Related Compound B RS
compoundD from the Sample solution 5-Benzoyl-2,3-dihydro-1 H-pyrrolizin-1-ol.
CisHi3NO2 = 227.26
 2318 Ketorolac / Official Monographs
USP Ketorolac Related Compound C RS
5-Benzoyl-2,3-dihydro-1 H-pyrrolizin-1-one.
CigHiiNOz = 225.24
USP Ketorolac Related Compound D RS
5-Benzoyl-2,3-dihydro-1 H-pyrrolizine.
CiaHi3NO = 211.3
Ketorolac Tromethamine Tablets
DEFINITION
Ketorolac Tromethamine Tablets contain NLT 90.0% and
NMT 110.0% of the labeled amount of ketorolac
tromethamine (CisHi3NO3 - C4Hi7NO3).
IDENTIFICATION
e A. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
e B. The UV absorption spectra of the ketorolac peak of
the Sample solution and that of the Standard solution ex-
hibit maxima and minima at the same wavelengths, as
obtained in the Assay.
ASSAY
e PROCEDURE
Mobile phase: Methanol, water, and glacial acetic acid
(55:44:1)
Diluent: Methanol and water (1:1). [NoTE—Protect all
volumetric solutions from light.]
Standard stock solution: 0.24 mg/mL of USP Ketorolac
Tromethamine RS in methanol
Standard solution: 24 g/mL of USP Ketorolac
Tromethamine RS in Diluent from Standard stock solution
System suitability stock solution: 25 g/mL each of
” SP Ketorolac Related Compound A RS, USP Ketorolac
= Medium
is Related Compound B RS, USP Ketorolac Related Com-
i] pound C RS, and USP Ketorolac Related Compound D
— with Medium to a concentration that is similar to the
io RS in methanol
re) System suitability solution: 0.25 ug/mL each of USP
= Ketorolac Related Compound ARS, USP Ketorolac Re-
5 lated Compound B RS, USP Ketorolac Related Com-
= pound C RS, and USP Ketorolac Related Compound D
a RS in Standard solution from System suitability stock
2]
> solution
Sample stock solution: 0.2 mg/mL of ketorolac
tromethamine prepared as follows. Transfer 10 Tablets
to a suitable volumetric flask. Add a quantity of water
equivalent to about 10% of the volume of the flask,
and sonicate until the Tablets are disintegrated. Add a
quantity of methanol equivalent to 40% of the volume
of the flask, and sonicate for 10 min to dissolve the
ketorolac tromethamine. Coo! to ambient temperature,
dilute with methanol to volume, and mix. Centrifuge,
or allow to settle.
Sample solution: 0.02 mg/mL of ketorolac
tromethamine in Diluent from Sample stock solution
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector
Assay: UV 254 nm
Identification test B: Diode array, UV 200-400 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Flow rate: 1.2 mL/min
Injection volume: 100 pL
Run time: 3.8 times the retention time of the ketoro-
lac peak
USP 41
System suitability
Samples: Standard solution and System suitability
solution
[Note—The relative retention times are 0.8 for ketorolac
related compound B and 1.0 for the ketorolac peaks.]
Suitability requirements
Resolution: NLT 1.5 each between the ketorolac and
ketorolac related compound B, and ketorolac and
ketorolac related compound C peaks, System suitabil-
ity solution
Tailing factor: NMT 1.5, Standard solution
Relative standard deviation: NMT 1.5%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
ketorolac tromethamine (CisHi3NO3 - C4Hi,NOs) in the
portion of Tablets taken:
Result = (ru/rs) x (Cs/Cu) x 100
ru = response of the ketorolac peak from the
Sample solution
rs = response of the ketorolac peak from the
Standard solution
Cs = concentration of USP Ketorolac Tromethamine
RS in the Standard solution (mg/mL)
Cu = nominal concentration of ketorolac
tromethamine in the Sample solution
(mg/mL)
Acceptance criteria: 90.0%-110%
PERFORMANCE TESTS
e DISSOLUTION (711)
Medium: Water; 600 mL
Apparatus 2: 50 rpm
Time: 45 min
Standard solution: USP Ketorolac Tromethamine RS in
Sample solutions: Sample per Dissolution (711). Dilute
Standard solution.
Instrumental conditions
Mode: UV absorption spectroscopy
Analytical wavelength: 322 nm
Tolerances: NLT 75% (Q) of the labeled amount of
ketorolac tromethamine (CisHi3NOs3 - C4HiiNOs) is
dissolved.
e UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
IMPURITIES
e ORGANIC IMPURITIES
Mobile phase, Chromatographic system, and Diluent:
Proceed as directed in the Assay.
Standard solution: Use the System suitability solution,
prepared as directed in the Assay.
Sample solution: Proceed as directed for the Sample
solution in the Assay.
System suitability
Sample: Standard solution
Suitability requirements
Resolution: NLT 1.5 each between the ketorolac and
ketorolac related compound B, and ketorolac and
ketorolac related compound C peaks
Relative standard deviation: NMT 5.0% for ketoro-
lac related compound A, ketorolac related compound
B, ketorolac related compound C, and ketorolac re-
lated compound D
USP 41 Official Monographs / Krypton 2319

Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each known impurity in the Krypton Kr 81m
portion of Tablets taken: Kr 81m
Krypton, isotope of mass 81 (metastable).
Result = (ru/rs) x (Cs/Cu) x 100 Krypton, isotope of mass 81 (metastable) [15678-91-8].
tu = peak response of each known impurity in the » Krypton Kr 81m is a gas suitable only for in-
Sample solution
rs = peak response of each known impurity in the halation in diagnostic studies, and is obtained
Standard solution from a generator that contains rubidium 81 ad-
Cs = concentration of each impurity in the Standard sorbed on an immobilized suitable column sup-
Solution (mg/mL) port. Rubidium 81 decays with a half-life of
Cu = nominal concentration of ketorolac
tromethamine in the Sample solution 4.58 hours and forms its radioactive daughter
(mg/mL) 81mKr, which is eluted from the generator by pas-
Calculate the percentage of any other impurity in the sage of humidified oxygen or air through the col-
portion of Tablets taken: umn. Rubidium 81 is produced in an accelerator
Result = (ru/r7) x 100 by proton bombardment of Kr 82. Other radio-
isotopes of rubidium are produced and are pres-
Tu = response of each individual impurity peak in ent on the generator column. These other radio-
the Sample solution isotopes do not decay to §'™Kr. The column
tr = sum of responses for all the peaks in the
Sample solution contains not less than 90.0 percent and not more
Acceptance criteria: See Table 1. than 110.0 percent of the labeled amount of Rb
81 at the date and time indicated in the labeling,
Table 1 on elution yields not less than 80.0 percent
Relative Acceptance of 8'mKr.
Retention Criteria,
Packaging and storage—The generator column is en-
Name Time NMT (%)
closed in a lead container. The unit is stored at room tem-
Ketorolac related perature.
compound A 0.5 0.5
Labeling—The labeling indicates the name and address of
Ketorolac related the manufacturer, the name of the generator, the quantity
compound B 0.8 0.5 of ®'Rb at the date and time of calibration, and the state-
Ketorolac 1.0 ment, “Caution—Radioactive Material.” The labeling indi-
Ketorolac related cates that in making dosage calculations, correction is to be (23
compound C 1D 0.8 made for radioactive decay, and also indicates that the radi- nn
=")
Ketorolac related oactive half-life of 8'™Kr is 13.1 seconds.
compound D. 2.6 0.5 [NoTE—Perform the following tests and Assay quickly, be-
Total unspecified _ cause of the rapid decay of the 8'™Kr.] °
|
impurity 0.5 Radionuclide identification (see Radioactivity (821))— fo)
Total impurities = 1.0 The gamma-ray spectrum of eluted 8'™Kr exhibits a so}
=
monoenergetic gamma ray at a mean energy of 191 KeV. EY
me]
Radionuclidic Pa cceien a suitable counting assem- >
ADDITIONAL REQUIREMENTS bly, determine the radioactivity of each radionuclide present r
e PACKAGING AND STORAGE: Preserve in well-closed contain- in a specimen of Kr 81m gas obtained from eluting the
ers at controlled room temperature, protected from light generator by use of a calibrated system as directed under
and excessive humidity. Radioactivity (821). Not less than 99.9% of the radioactivity
e USP REFERENCE STANDARDS (11) in the specimen eluted from the generator is present as
USP Ketorolac Tromethamine RS 8imKr.
USP Ketorolac Related Compound A RS
5-Benzoyl-N-[1,3-dihydroxy-2-(hydroxymethyl)propan- Assay for radioactivity—Using a suitable counting assem-
2-yl]-2,3-dihydro-1H-pyrrolizine-1-carboxamide. bly (see Radioactivity (821)), determine the quantity, in MBq
CisH22N20s 358.39 (mCi), of Kr 81m contained in an elution of the generator.
USP Ketorolac Related Compound B RS Decay correct the result to the time of generator elution,
5-Benzoyl-2,3-dihydro-1 H-pyrrolizin-1-ol. and calculate the quantity of 8'Rb present in the column at
Cy4HisNO2 =227.26 the time of elution. The guantie of 8'mKr eluted is not less
USP Ketorolac Related Compound C RS than 80.0 percent of the labeled MBq (mCi) of §'Rb present
5-Benzoyl-2,3-dihydro-1 H-pyrrolizin-1-one. on the column at time of elution.
CyaHiiNOz = 225.24
USP Ketorolac Related Compound D RS
5-Benzoyl-2,3-dihydro-1 H-pyrrolizine.
CyaHi3NO 211.26
 2320 Labetalol / Official Monographs
Labetalol Hydrochloride
Ox UNH,
OH
cH,
° HCI
N size or intensity than the principal spot observed in the
H
oH
CisH24N203- HCl 364.87
Benzamide, 2-hydroxy-5-[1-hydroxy-2-[(1-methyl-
3-phenylpropyl)aminol]ethyl]-, monohydrochloride.
5-[1-Hydroxy-2-[(1-methyl-
3-phen Ipropyljaminofethyl|salicylamide monohydrochlo-
ride [32780-64-6].
» Labetalol Hydrochloride contains not less than
97.5 percent and not more than 101.0 percent of
Ci9H24N203 - HCI, calculated on the dried basis.
Packaging and storage—Preserve in tight, light-resistant
containers. Store at 25°, excursions permitted between 15°
and 30°.
USP Reference standards (11)—
USP Labetalol Hydrochloride RS
identification—
A: Infrared Absorption (197M).
B: It responds to the tests for Chloride (191).
pH (791): between 4.0 and 5.0, in a solution (1 in 100).
Loss on drying (731): Dry it in a vacuum at 105° for
4 hours: it loses not more than 1.0% of its weight.
Residue on ignition (281): not more than 0.1%.
Delete the following:
os
al
rm
Ss °Heavy metals, Method [I (231): 0.002%. (otticiat 1jan-2018)
a
J
Chromatographic purity—
ce}
= Detection reagent—Transfer 2.5 g of cadmium acetate to
5 a 500-mL volumetric flask, add 10 mL of glacial acetic acid,
= dilute with alcohol to volume, and mix. Just prior to use,
a. prepare a 0.2 in 100 solution of ninhydrin in the cadmium
” acetate solution for use as the Detection reagent.
= stepwise with 7-Butaneboronic acid solution to obtain a solu-
Solvent mixture—Prepare a solution of methanol and
water (4:1), and mix.
Ammonium chloride reference solution—Dissolve 60 mg of
ammonium chloride in 10.0 mL of water, and mix.
Standard stock solution—Dissolve USP Labetalol Hydro-
chloride RS in Solvent mixture, and mix to obtain a solution
having a known concentration of 40 mg per mL.
Standard solution 1—Quantitatively dilute a portion of the
Standard stock solution with Solvent mixture to obtain a solu-
tion having a known concentration of 0.2 mg per mL.
Standard solution 2—Quantitatively dilute a portion of the
Standard solution 1 with Solvent mixture to obtain a solution
having a known concentration of 0.1 mg per mL.
Test solution—Dissolve 200 mg of Labetalol Hydrochloride
in 5.0 mL of Solvent mixture, and mix.
Procedure I—Apply separately 5-uL portions of the Stan-
dard stock solution, Standard solution 1, Standard solution 2,
and the Test solution to a suitable thin-layer chromato-
graphic plate (see Chromatography (621)) coated with a
0.25-mm layer of chromatographic silica gel mixture. Allow
the spots to dry, and develop the chromatograms ina sol-
vent system consisting of a mixture of dichloromethane,
methanol, and ammonium hydroxide (15:5:1) until the sol-
vent front has moved about three-fourths of the length of
the plate: Remove the plate from the developing chamber,
mark the solvent front, and allow the solvent to evaporate.
Examine the plate under short-wavelength UV light: the Rr
USP 41
value of the principal spot from the Test solution corre-
sponds to that of the principal spot from the Standard stock
solution.
Spray the plate with Detection reagent, heat the plate at
105° for 15 minutes, cool to room temperature, and ex-
amine the chromatogram: no individual secondary spot ob-
served in the chromatogram of the Test solution is greater in
chromatogram of Standard solution 1 (0.5% each). [NOTE—
The spots appear as dark orange spots ona light orange to
yellow background. A “negative image” spot (white) near
the origin may be observed in the chromatogram of the
Test solution. This is due to the formation of ammonium
chloride during the chromatographic procedure and may be
ignored.]
Procedure II—Apply separately 10-uL portions of the Am-
monium chloride reference solution, the Standard stock solu-
tion, Standard solution 1, Standard solution 2, and the Test
solution to a suitable thin-layer chromatographic plate (see
Chromatography (621)), coated with a 0.25-mm layer of
chromatographic silica gel mixture. Allow the spots to dry,
and develop the chromatograms in a solvent system consist-
ing of a mixture of ethyl acetate, isopropyl alcohol, water,
and ammonium hydroxide (25:15:8:2) until the solvent
front has moved about three-fourths of the length of the
plate. Remove the plate from the developing chamber, mark
the solvent front, and allow the solvent to evaporate. Ex-
amine the plate under short-wavelength UV light: no indi-
vidual secondary spot (other than that due to ammonium
chloride) observed in the chromatogram of the Test solution
is greater in size or intensity than the punslal spot ob-
ane in the chromatogram of Standard solution 1 (0.5%
each).
Total impurities—The sum of the intensities of all second-
ary spots (other than those due to ammonium chloride) ob-
served in the chromatograms of the Test solution from both
Procedure | and Procedure II does not exceed 1.0%.
Diastereoisomer ratio—
1-Butaneboronic acid solution—Dissolve 1-butaneboronic
acid in pyridine, previously dried over a suitable molecular
sieve, and mix to obtain a solution having a known concen-
tration of 20 mg per mL.
System suitability solution—Dissolve an accurately weighed
quantity of USP Labetalol Hydrochloride RS in
1-Butaneboronic acid solution, and dilute quantitatively and
tion having a known concentration of about 1.4 mg of USP
Labetalo! Hydrochloride RS per mL. Allow the solution to
stand at room temperature for 20 minutes before using.
Test solution—Transfer about 1 mg of Labetalol Hydro-
chloride to a 1-mL reaction vial, add 0.7 mL of
1-Butaneboronic acid solution, and mix until the labetalol hy-
drochloride is completely dissolved. Allow the solution to
stand at room temperature for 20 minutes before using.
Chromatographic system (see Chromatography (621))—The
gas chromatograph is equipped with a flame-ionization de-
tector and a 2-mm x 1.8-m glass column packed with 10%
phase G3 on 100- to 120-mesh support STAB. The column
temperature is maintained at about 320°, and the injection
port and the detector block temperatures are maintained at
about 340°. Nitrogen is used as the carrier gas at the flow
rate of about 30 mL per minute. Chromatograph the System
suitability solution, and record the peak responses as directed
for Procedure: the relative retention times are about 0.8 for
the diastereoisomer B 1-butaneboronate derivative and 1.0
for the diastereoisomer A 1-butaneboronate derivative; the
resolution, R, between the diastereoisomer A
1-butaneboronate derivative and diastereoisomer B
1-butaneboronate derivative peaks is not less than 1.5; and
the relative standard deviation of the ratios of the peak ar-
eas of the diastereoisomers for replicate injections is not
more than 2.0%.
USP 41
Procedure—tnject about 2 uL of the Test solution into the
chromatograph, record the chromatograms, and measure
the responses for the major peaks. Calculate the diastereoi-
somer A content, in percentage, taken by the formula:
10064
/ (ra + Fa)
in which ra is the peak area of the diastereoisomer A
1-butaneboronate derivative peak; and rs is the peak area of
the diastereoisomer B 1-butaneboronate derivative peak.
The diastereoisomer A content is not less than 45.0% and
not more than 55.0%.
Assay—
Mobile Pe ee a suitable filtered and degassed
mixture of 0.1 M monobasic sodium phosphate and metha-
nol (65:35). Make adjustments if necessary (see System Suit-
ability under Chromatography (621)).
Standard preparation—Dissolve an accurately weighed
quantity of USP Labetalol Hydrochloride RS in Mobile phase
to obtain a solution having a known concentration of about
0.4 mg per mL.
Assay preparation—Transfer about 40 mg of Labetalol Hy-
drochloride, accurately weighed, to a 100-mL volumetric
flask, dilute with Mobile phase to volume, and mix.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 230-nm detector
and a 4.6-mm x 20-cm column that contains packing L1
and is maintained at 60 + 1°. The flow rate is about 1.5 mL
per minute. Chromatograph the Standard preparation, and
record the peak responses as directed for Procedure: the col-
umn efficiency determined from the analyte peak is not less
than 700 theoretical plates; the tailing factor for the analyte
peak is not more than 2.0; and the relative standard devia-
tion for replicate injections is not more than 1.5%.
Procedure—Separately inject equal volumes (about 5 wL)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
ure the area responses for the major peaks. Calculate the
quantity, in mg, of labetalol hydrochloride (Ci9H24N20Os -
an is the portion of Labetalol Hydrochloride taken by the
ormula:
100C(ru/ rs)
in which C is the concentration, in mg per mL, of USP
Labetalol Hydrochloride RS in the Standard preparation; and
ry and rs are the peak area responses obtained from the
Assay preparation and the Standard preparation, respectively.
Labetalol Hydrochloride Injection
» Labetalol Hydrochloride Injection is a sterile so-
lution of Labetalol Hydrochloride in Water for In-
jection. It contains not less than 90.0 percent and
not more than 110.0 percent of the labeled
wo of labetalol hydrochloride (Ci9H24N20s -
Packaging and storage—Preserve in single-dose contain-
ers, or in multiple-dose containers not exceeding 60 mL in
volume, preferably of lps | glass, at a temperature be-
tween 2° and 30°. Avoid freezing and exposure to light.
Change to read:
usP Reference standards (11)—
@ (CN 1-May-2018)
USP Labetalol Hydrochloride RS
Official Monographs / Labetalol 2321
Identification—The retention time of the major peak in
the chromatogram of the Assay preparation corresponds to
that of the Standard preparation, as obtained in the Assay.
Bacterial Endotoxins Test (85)—It contains not more
than 1.2 USP Endotoxin Units per mg of labetalol hydro-
chloride.
PH (791): between 3.0 and 4.5.
Other requirements—it meets the requirements under /n-
jections and Implanted Drug Products (1).
Assay—
Mobic poate —Fiepere a suitable filtered and degassed
mixture of 0.1 M monobasic sodium phosphate and metha-
nol (65:35). Make adjustments if necessary (see System Suit-
ability under Chromatography (621)).
Standard preparation—Dissolve an accurately weighed
quantity of USP Labetalol Hydrochloride RS in Mobile phase
to obtain a solution having a known concentration of about
0.5 mg per mL.
Resolution solution—Dissolve a quantity of methylparaben
in the Standard preparation to obtain a solution containing
about 0.08 mg per mL.
Assay preparation—Transfer an accurately measured vol-
ume of Injection, equivalent to about 50 mg of labetalol hy-
drochloride, to a 100-mL volumetric flask, dilute with Mobile
phase to volume, and mix.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 254-nm detector
and a 4.6-mm x 20-cm column that contains packing L1
and is maintained at 60+ 1°. The flow rate is about 71.5 mL
per minute. Chromatograph the Standard preparation, and
record the peak responses as directed for Procedure: the col-
umn efficiency determined from the analyte peak is not less
than 700 theoretical plates; the tailing factor for the analyte
peak is not more than 2.0; and the relative standard devia-
tion for replicate injections is not more than 1.5%. Chro-
matograph the Resolution solution, and record the peak re- [os
sponses as directed for Procedure: the relative retention a)
mo]
times are about 0.6 for methylparaben and 1.0 for labetalol;
and the resolution, R, between the methylparaben and =
labetalol is not less than 2.0. °
}
Procedure—Separately inject equal volumes (about 5 UL) °
of the Standard preparation and the Assay preparation into io}
a
the chromatograph, record the chromatograms, and meas- Cy
ure the area responses for the major peaks. Calculate the mo]
a
quantity, in mg, of labetalol hydrochloride (CysH24N2O3- a“
HCl) in each mL of the Injection taken by the formula:
100(C/ V)(ru/ rs)
in which C is the concentration, in mg per mL, of USP
Labetalol Hydrochloride RS in the Standard preparation; V is
the volume, in mL, of Injection taken; and ry and rs are the
peak area responses obtained from the Assay preparation
and the Standard preparation, respectively.
Labetalol Hydrochloride Compounded
Oral Suspension
DEFINITION
Labetalol Hydrochloride Compounded Oral Suspension con-
tains NLT 90.0% and NMT 110.0% of the labeled amount
of labetalol hydrochloride (CisH24N203 - HCl).
 2322 Labetalol / Official Monographs
Prepare Labetalol Hydrochloride Compounded Oral Suspen-
sion 40 mg/mL as follows (see Pharmaceutical Compound-
ing—Nonsterile Preparations {795)).
Labetalol Hydrochloride Aq
Vehicle: a 1:1 mixture of Vehicle for Oral Solution
(regular or sugar-free), NF, and Vehicle for Oral
Suspension, NF, a sufficient quantity to make 100 mL
Place the required number of tablets in a suitable mortar
and comminute to a fine powder, or use Labetalo! Hydro-
chloride powder. Add 20 mL of the Vehicle, and mix to
form a uniform paste. Add the Vehicle in small portions
almost to volume. Transfer the contents of the mortar,
stepwise and quantitatively, to a calibrated bottle. Add
the Vehicle in portions to rinse the mortar, then add suffi-
cient Vehicle to bring to final volume, and mix well.
ASSAY
© PROCEDURE
Mobile phase: Methanol and 0.1 M monobasic sodium
phosphate (35:65). Filter, and degas.
Standard solution: 400 g/mL of USP Labetalo!l Hydro-
chloride RS
Sample solution: Agitate the container of Oral Suspen-
sion for 30 min on a rotating mixer, remove a 5-mL
sample, and store in a clear glass vial at -70° until ana-
lyzed. At the time of analysis, remove the sample from
the freezer, allow it to reach room temperature, and
mix with a vortex mixer for 30 s. Pipet 1.0 mL of the
sample into a 100-mL volumetric flask, and dilute with
Mobile phase to volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 230 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
“vw
nod Flow rate: 1.3 mL/min
rm
iJ
Injection volume: 20 uL
System suitability
D
—
Sample: Standard solution
) [NoTE—The retention time for labetalol hydrochloride is
c
5 about 7.5 min.]
= Suitability requirements
Relative standard deviation: NMT 1.6% for replicate
a
” injections
> Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
labetalol hydrochloride (CysH24N2O3 - HCI) in the por-
tion of Oral Suspension taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response from the Sample solution
Is = peak response from the Standard solution
Cs = concentration of USP Labetalol Hydrochloride
RS in the Standard solution (g/mL)
Cu = nominal concentration of labetalol
hydrochloride in the Sample solution (ug/mL)
Acceptance criteria: 90.0%-110.0%
SPECIFIC TESTS
e PH (791): 4.0-5.0
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Package in tight, light-resistant
containers. Store at controlled room temperature, or in a
refrigerator.
© BEYOND-UsE DATE: NMT 60 days after the date on which
it was compounded when stored at controlled room
temperature, or in a refrigerator
USP 41
e LABELING: Label it to state that it is to be well shaken,
and to state the Beyond-Use Date.
e USP REFERENCE STANDARDS (11)
USP Labetalol Hydrochloride RS
Labetalol Hydrochloride Tablets
» Labetalol Hydrochloride Tablets contain not less
than 90.0 percent and not more than 110.0 per-
cent of the labeled amount of labetalol hydro-
chloride (CigH24N203 ‘ HCl).
Packaging and storage—Preserve in tight, light-resistant
containers, at a temperature between 2° and 30°.
USP Reference standards (11)—
USP Labetalol Hydrochloride RS
Identification—The retention time of the major peak in
the chromatogram of the Assay preparation corresponds to
that of the Standard preparation, as obtained in the Assay.
Dissolution (711)—
Medium: water; 900 mL.
Apparatus 2: 50 rpm.
Time: 45 minutes.
Procedure—Determine the amount of CisH24N2O3 - HCI
dissolved from UV absorbances at the wavelength of maxi-
mum absorbance at about 302 nm of filtered portions of
the solution under test, suitably diluted with water, if neces-
sary, in comparison with a Standard solution havin
known concentration of USP Labetalol Hydrochloride RS in
the same Medium.
Tolerances—Not less than 80% (Q) of the labeled amount
of CisH24N203 - HCl is dissolved in 45 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Assay—
Mobile phase, Standard preparation, and Chromatographic
system—Proceed as directed in the Assay under Labetalol Hy-
rochloride.
Assay piepaision Waste: an accurately counted num-
ber of Tablets, equivalent to about 2000 mg of labetalol hy-
drochloride, to a 500-mL volumetric flask, add 200 mL of
water, and shake by mechanical means for 60 minutes. Di-
lute with water to volume, and mix. Filter the solution
throughafilter of 0.5 um or finer porosity, discarding the
first few mL of the filtrate. Transfer 10.0 mL of the filtrate to
a 100-mL volumetric flask, dilute with Mobile phase to vol-
ume, and mix.
Procedure—Separately inject equal volumes (about 5 1L)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
ure the area responses for the major peaks. Calculate the
quantity, in mg, of labetalol hydrochloride (CisH24N2Os-
HCl) in each Tablet taken by the formula:
5000(C/ N)(ru/ rs)
in which C is the concentration, in mg per mL, of USP
Labetalol Hydrochloride RS in the Standard preparation; N is
the number of Tablets taken; and ry and rs are the peak area
responses obtained from the Assay preparation and the Stan-
dard preparation, respectively.
USP 41 Official Monographs / Lactic 2323

Acceptance criteria: NLT 30,000 USP Lactase Units/g

Lactase IMPURITIES
© ARSENIC (211): NMT 3 ug/g
DEFINITION
Lactase (B-D-galactoside galactohydrolase) is a hydrolytic en-
e LEAD (251): NMT 5ug/g
zyme derived from the mold Aspergillus oryzae. It contains
NLT 30,000 USP Lactase Units/g. Delete the following:
[Note—One USP Lactase Unit is the lactase activity con-
tained in the amount of enzyme that hydrolyzes one °o HEAVY METALS (231): NMT 30 1G/Ge (otiicial 1-jan-2018)
microequivalent a ee linkage per min at a pH of
4.5 and at 37°, as directed in the Assay for Lactase SPECIFIC TESTS
Activity.] e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): It meets the requirements of
ASSAY he tests for absence of Salmonella species and Escherichia
e LACTASE ACTIVITY coll.
Solution A: Dilute 57.5 mL of glacial acetic acid with e Loss ON DRYING (731)
sufficient water to make a 500-mL solution. Transfer Analysis: Dry under vacuum at 60? for 4 h.
50 mL of the glacial acetic acid solution into a 1000-mL Acceptance criteria: NMT 6.0%
volumetric flask, add 11.3 mL of 4 N sodium hydroxide,
and dilute with water to volume. If necessary, adjust ADDITIONAL REQUIREMENTS
with glacial acetic acid solution or 4 N sodium hydrox- © PACKAGING AND STORAGE: Preserve in tight containers at
ide to a pH of 4.50 + 0.05. room temperature.
Substrate solution: On the day of use, weigh e LABELING: Label it to indicate lactase activity in USP Units.
370.0 mg of o-nitrophenyl-B-D-galactopyranoside, and e USP REFERENCE STANDARDS (11)
place in a 100-mL volumetric flask. Add about 50 mL of USP Lactase RS
Solution A, swirl to dissolve, then dilute with Solution A
to volume.
Standard solution: Transfer about 0.4 g of USP Lactase
RS, accurately weighed, to a 1000-mL volumetric flask.
Add about 600 mL of water, allow to stand for 15 min, Lactic Acid
swirl gently, and dilute with water to volume. Pipet
3.0 mL of this solution into a 200-mL volumetric flask, Propanoic acid, 2-hydroxy-;
and dilute with water to volume. Lactic acid [50-21 Sh
Sample solution: Transfer an accurately weighed quan-
tity of about 0.4 g of Lactase to a 1000-mL volumetric DEFINITION
flask. Add about 600 mL of water, allow to stand for 15 Lactic Acid is a mixture of lactic acid (C3H6O3) and lactic
min, swirl gently, and dilute with water to volume. Pi- acid lactate (CsH10Os), equivalent to a total of NLT 88.0%
et 3.0 mL of this solution into a 200-mL volumetric and NMT 92.0%, by weight, of lactic acid (C3H.6Os). It is iS.
obtained by the lactic fermentation of sugars or is pre- wn
lask, and dilute with water to volume.
pared synthetically. Lactic Acid obtained by fermentation a)
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).) of sugars is levorotatory, whereas that prepared syntheti- cs
Mode: Vis cally is racemic. i}
[Note—Lactic Acid prepared by fermentation becomes dex- 3
Analytical wavelength: 420 nm )
Cell: 1.cm trorotatory on dilution, which hydrolyzes L-(-)-lactic acid ro}2
Analysis lactate to L-(+)-lactic acid.] oy
Samples: Standard solution and Sample solution 3
Pipet 2.0 mL of Substrate solution into three separate
IDENTIFICATION =e
e A. IDENTIFICATION TESTS—GENERAL, Lactate (191): Meets ww
test tubes labeled S, U, and B. Transfer the tubes to a the requirements
thermostated water bath maintained at 37.0 + 0.1°,
and incubate for 10 min. Following the incubation, ASSAY
rapidly add 0.5 mL of the Standard solution to tube S, © PROCEDURE
0.5 mL of the Sample solution to tube U, and 0.5 mL of Sample: 2.5 mL, accurately weighed
water to tube B (the reagent blank). Mix each tube on Titrimetric system
a vortex mixer for 1 s, and immediately return the (See Titrimetry (541).)
tubes to the water bath, which has been maintained Mode: Residual titration
at 37.0 + 0.1°. After 15 min of incubation, rapidly add Titrant: 1 .N sodium hydroxide VS
2.5 mL of a 10% sodium carbonate solution to each Back-titrant: 1N sulfuric acid VS
test tube to stop the enzyme reaction. Add 20.0 mL of Endpoint detection: Visual
water to each test tube, and mix. Concomitantly de- Analysis: Transfer the Sample to a tared 250-mL flask,
termine the absorbances of the three solutions. add 50.0 mL of Titrant, and boil the mixture for 20
Calculate the number of USP Lactase Units in the por- min. Add phenolphthalein TS, and titrate the excess al-
tion of Lactase taken: kali in the hot solution with Back-titrant. Perform a
blank determination. Each mL of Titrant is equivalent to
Result = [(Au — As)/(As — As)] x P x (Ws/Wu) 90.08 mg of lactic acid (C3H6Os).
Acceptance criteria: 88.0%-92.0% (w/w)
Au = absorbance of the Sample solution (tube U)
As = absorbance of the reagent blank (tube B) IMPURITIES
As = absorbance of the Standard solution (tube S) ¢ CHLORIDE
P = potency of USP Lactase RS (USP Units/g) Sample solution: 1 in 100
Ws = weight of USP Lactase RS in the Standard Analysis: To 10 mL of the Sample solution acidified with
solution (g) nitric acid add a few drops of silver nitrate TS.
Wy = weight of Lactase in the Sample solution (g) Acceptance criteria: No opalescence is produced
immediately.
 2324 Lactic / Official Monographs USP 41

© SULFATE Acceptance criteria: A red precipitate of cuprous oxide

Sample solution: 1 in 100 is formed.
Analysis: To 10 mL of the Sample solution add 2 drops
of hydrochloric acid and 1 mL of barium chloride TS. ASSAY
Acceptance criteria: No turbidity is produced. e PROCEDURE
Buffer: 1.15 g/L of monobasic sodium phosphate in
water
Delete the following: Mobile phase: Acetonitrile and Buffer (82:18). Ensure
that the concentration of acetonitrile in the Mobile
°e HEAVY MeTALs, Method |! (231): NMT 10 ug/ge comeia1- phase is between 78% and 85% to obtain appropriate
Jan-2018) retention times.
e RESIDUE ON IGNITION (281) Standard solution: 40 mg/mL of USP Lactulose RS,
Sample: 5 mL, accurately weighed 4.8 mg/mL of USP Anhydrous Lactose RS, and 3.2 mg/
Acceptance criteria: NMT 3 mg (0.05%) mL of USP Epilactose RS in a mixture of acetonitrile and
e LIMIT OF CITRIC, OXALIC, PHOSPHORIC, OR TARTARIC ACID water (1:1)
Sample solution: 1 in 10 Sample solution: Nominally equivalent to 40 mg/mL of
Analysis: To 10 mL of the Sample solution add 40 mL of lactulose prepared as follows. Transfer a quantity of
calcium hydroxide TS, and boil for 2 min. Concentrate containing 2.0 g of lactulose to a $0-mL
Acceptance criteria: No turbidity is produced. volumetric flask, and dissolve in 20 mL of water. Add
25.0 mL of acetonitrile, allow the solution to reach am-
SPECIFIC TESTS bient temperature, and dilute with water to volume.
e READILY CARBONIZABLE SUBSTANCES Chromatographic system
Sample: 5 mL (See Chromatography (621), System Suitability.)
Analysis: Rinse a test tube with sulfuric acid, and allow Mode: LC
to drain for 10 min. Add 5 mL of sulfuric acid to the Detector: Refractive index
test tube, carefully overlay it with the Sample, and Column: 4.6-mm x 15-cm; 3-11m packing L8
maintain the tube at 15°. Temperatures
Acceptance criteria: No dark color develops at the in- Column: 40+1°
terface of the two acids within 15 min. Detector: 40+1°
© OPTICAL ROTATION, Angular Rotation (781A): —0.05° to Flow rate: 1.3 mL/min
+0.05° for racemic Lactic Acid Injection volume: 20 pL
© SUGARS System suitability
Sample: 5 drops Sample: Standard solution
Analysis: To 10 mL of hot alkaline cupric tartrate TS {Note—The relative retention times are given in Table
add the Sample. is
Acceptance criteria: No red precipitate is formed. Suitability requirements
ADDITIONAL REQUIREMENTS Resolution: NLT 1.5 between lactulose and lactose;
v
© PACKAGING AND STORAGE: Preserve in tight containers. NLT 0.9 between lactulose and epilactose
£
5Ss
© LABELING: Label it to indicate whether it is levorotatory or Relative standard deviation: NMT 2.0% for the
main peak
racemic.
i=.)
_
Analysis
i}i= Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of lac-
5 tulose (Ci2H2201) in the portion of Concentrate taken:
=
5 Lactulose Concentrate Result = (ru/rs) x (Cs/Cu) x 100
a)
3) oH oH tu = peak response from the Sample solution
= rs
Gs
= peak response from the Standard solution
= concentration of USP Lactulose RS in the
mp Looe
Standard solution (mg/mL)
Ho ‘oH HO{on Cu = nominal concentration of lactulose in the
Sample solution (mg/mL)
Acceptance criteria: 95.0%-105.0%
Cy2H2O011 342.30
D-Fructose, 4-O-B-D-galactopyranosyl-; IMPURITIES
4-O-B-D-Galactopyranosyl-D-fructofuranose [4618-18-2]. © RESIDUE ON IGNITION (281): NMT 0.1%
e@ ORGANIC IMPURITIES
DEFINITION Buffer, Mobile phase, Sample solution, Chromato-
Lactulose Concentrate is a solution of sugars prepared from graphic system, and System suitability: Proceed as
Lactose. It consists principally of lactulose together with directed in the Assay. To evaluate the Suitability require-
minor quantities of lactose and galactose, and traces of ments, use the Standard solution prepared as directed in
other related sugars and water. It contains NLT 95.0% the Assay.
and NMT 105.0% of the labeled amount of lactulose Standard solution: 6.4 mg/mL of USP Galactose RS,
(Ci2H2201,). It contains no added substances. 4.8 mg/mL of USP Anhydrous Lactose RS, 3.2 mg/mL of
IDENTIFICATION USP Epilactose RS, 1.2 mg/mL of USP Tagatose RS, and
° A. The retention time of the major peak of the Sample 0.4 mg/mL of USP Fructose RS in a mixture of acetoni-
solution corresponds to that of the Standard solution, as trile and water (1:1)
obtained in the Assay. Analysis
° B. Samples: Standard solution and Sample solution
Sample solution: Dilute a portion of Concentrate with Calculate the percentages of galactose, lactose, epilac-
water (1 in 20). tose, tagatose, and fructose, if found, in the portion of
Analysis: Add a few drops of the Sample solution to Concentrate taken:
5 mL of hot alkaline cupric tartrate TS.
Result = (ru/rs) x (Cs/Cu) x 100
USP 41 Official Monographs / Lactulose 2325

ru = peak response of the relevant related lution containing 2.0 g of lactulose to a 50-mL volumet-
compound from the Sample solution tic flask, and dissolve in 20 mL of water. Add 25.0 mL
rs = peak response of the relevant related of acetonitrile, allow the solution to reach ambient tem-
compound from the Standard solution perature, and dilute with water to volume.
Gs = concentration of the relevant USP Reference Chromatographic system
Standard in the Standard solution (mg/mL) (See Chromatography (621), System Suitability.)
Cu = nominal concentration of lactulose in oe Mode: LC
Sample solution (mg/mL) Detector: Refractive index
Acceptance criteria: See Table 7. Column: 4.6-mm x 15-cm; 3-m packing L&
Temperatures
Table 1 Column: 40+1°
Detector: 4041°
Relative Acceptance Flow rate: 1.3 mL/min
Retention Criteria, Injection volume: 20 uL
%' System suitability
4 Sample: Standard solution
[Nott—The relative retention times are given in Table
1 Te
Suitability requirements
Resolution: NLT 1.5 between lactulose and lactose;
NLT 0.9 between lactulose and epilactose
Relative standard deviation: NMT 2.0% for the
main peak
ADDITIONAL REQUIREMENTS Analysis
e PACKAGING AND STORAGE: Preserve in tight containers, Samples: Standard solution and Sample solution
preferably at a temperature between 2° and 30°. Avoid Calculate the percentage of the labeled amount of lac-
subfreezing temperatures. tulose (Ci2H22011) in the portion of Solution taken:
e LABELING: The label states that this article is not intended
for direct administration to humans or animals. Result = (ru/rs) x (Cs/Cu) x 100
e USP REFERENCE STANDARDS (11) ry = peak response from the Sample solution
USP Epilactose RS rs = peak response from the Standard solution
USP Fructose RS Cs = concentration of USP Lactulose RS in the
USP Galactose RS Standard solution (mg/mL)
USP Anhydrous Lactose RS Cu = nominal concentration of lactulose in the
USP Lactulose RS Sample solution (mg/mL)
USP Tagatose RS Acceptance criteria: 90.0%-110.0%
cS
PERFORMANCE TESTS “vn
e UNIFORMITY OF DOSAGE UNITS (905) i)
Oral Solution packaged in lt Nada containers a
Lactulose Solution Acceptance criteria: Meets the requirements i}
=
DEFINITION IMPURITIES i}
Lactulose Solution is a solution in water prepared from Lac- © ORGANIC IMPURITIES @3
Buffer, Mobile phase, Sample solution, Chromato- 2
tulose Concentrate. It contains NLT 90.0% and NMT
graphic system, and System suitability: Proceed as 3
110.0% of the labeled amount of lactulose (C12H22011).
directed in the Assay. To evaluate the Suitability require-
=e
“”

IDENTIFICATION ments, use the Standard solution prepared as directed in

e A. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
° B.
Sample solution: Dilute a portion of Solution with
water (1 in 20).
Analysis: Add a few drops of the Sample solution to
5 mL of hot alkaline cupric tartrate TS.
Acceptance criteria: A red precipitate of cuprous oxide
is formed.
ASSAY
¢ PROCEDURE
Buffer: 1.15 g/L of monobasic sodium phosphate in
water
Mobile phase: Acetonitrile and Buffer (82:18). Ensure
that the concentration of acetonitrile in the Mobile
phase is between 78% and 85% to obtain appropriate
retention times.
Standard solution: 40 mg/mL of USP Lactulose RS,
4.8 mg/mL of USP Anhydrous Lactose RS, and 3.2 mg/
mL of USP Epilactose RS in a mixture of acetonitrile and
water (1:1)
Sample solution: Nominally equivalent to 40 mg/mL of
lactulose prepared as follows. Transfer a quantity of So-
the Assay.
Standard solution: 6.4 mg/mL of USP Galactose RS,
4.8 mg/mL of USP Anhydrous Lactose RS, 3.2 mg/mL of
USP Epilactose RS, 1.2 mg/mL of USP Tagatose RS, and
0.4 mg/mL of USP Fructose RS in a mixture of acetoni-
trile and water (1:1)
Analysis
Samples: Standard solution and Sample solution
Calculate the percentages of galactose, lactose, epilac-
tose, tagatose, and fructose, if found, in the portion of
Solution taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response of the relevant related
compound from the Sample solution
rs = peak response of the relevant related
compound from the Standard solution
Cs = concentration of the relevant USP Reference
Standard in the Standard solution (mg/mL)
Cu = nominal concentration of lactulose in the
Sample solution (mg/mL)
Acceptance criteria: See Table 1.
 2326 Lactulose / Official Monographs USP 41

Table 1 Standard solution: 0.25 mg/mL of USP Lamivudine RS

Relative Acceptance
in Mobile phase
Retention Criteria,
Sample solution: 0.25 mg/mL of Lamivudine in Mobile
NMT
phase
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 277 nm
Column: 4.6-mm x 25-cm; packing L1
Column temperature: 35°
Flow rate: 1 mL/min
Injection volume: 10 LL
System suitability
SPECIFIC TESTS Samples: System suitability solution and Standard
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI- solution
FIED MICROORGANISMS (62): The total bacterial count is [Note—The relative retention times for lamivudine dias-
NMT 102 cfu/g of lactulose, and the tests for Salmonella tereomer and lamivudine are 0.9 and 1.0,
species and Escherichia coli are negative. respectively.]
© PH (791): 2.5-6.5, after 15 min of contact with the Suitability requirements
electrodes Resolution: NLT 1.5 between lamivudine and
lamivudine diastereomer, System suitability solution
ADDITIONAL REQUIREMENTS Relative standard deviation: NMT 2.0% for
¢ PACKAGING AND STORAGE: Preserve in tight containers, lamivudine, Standard solution
preferably at a temperature between 2° and 30°. Avoid Analysis
subfreezing temperatures. Samples: Standard solution and Sample solution
e USP REFERENCE STANDARDS (11) Calculate the percentage of lamivudine (CgHi;N3035S) in
USP Epilactose RS the portion of Lamivudine taken:
USP Fructose RS
USP Galactose RS Result = (ru/rs) x (Cs/Cu) x 100
USP Anhydrous Lactose RS
USP Lactulose RS ru = peak response from the Sample solution
USP Tagatose RS rs = peak response from the Standard solution
Cs = concentration of USP Lamivudine RS in the
Standard solution (mg/mL)
Cu = concentration of Lamivudine in the Sample
solution (mg/mL)
Lamivudine Acceptance criteria: 98.0%-102.0% on the anhydrous
” and solvent-free basis
ao
% R N.
If labeled as a methanol solvate: 98.0%-102.0% on
ct \ the anhydrous, methanol-free, and solvent-free basis
J
D Ho” Cy Na
° OTHER COMPONENTS
iS © CONTENT OF METHANOL (if labeled as lamivudine metha-
iS CsHiiN3O3S 229.26 nol solvate)
= Diluent: N,N-Dimethylformamide and t-butanol (500:1)
fan CgHii1N303S - 0.2 CH30H 235.66 Standard solution: 0.625 mg/mL of methanol in dilu-
a) ent. Transfer 2.0 mL of this solution into a headspace
=) CgH11N303S - 0.2 H2O
2(1H)-Pyrimidinone, 4-amino-1-[2-(hydroxymethyl)-1,3-ox-
232.86
vial, and immediately seal the vial.
athiolan-5-yl]-, (2R-cis)-; Sample solution: Transfer 50 mg of Lamivudine to a
(-)-1-[(2R,5S)-2-(Hydroxymethyl)-1,3-oxathiolan- headspace vial, add 2.0 mL of Diluent, and immediately
5-yl]cytosine [134678-17-4]. seal the vial.
Chromatographic system
DEFINITION (See Chromatography (621), System Suitability.)
Lamivudine contains NLT 98.0% and NMT 102.0% of Mode: GC headspace
lamivudine (CgHi1N303S), calculated on the anhydrous Detector: Flame ionization
and solvent-free basis. If labeled as a methanol solvate, it Column: 0.53-mm x 75-m, coated with a 3-um film of
contains NLT 98.0% and NMT 102.0% of lamivudine qe G43
(CgHi1N3035), calculated on the anhydrous, methanol- emperatures
free, and solvent-free basis. Injector: 180°
Detector: 250°
IDENTIFICATION Column: See Table 7.
e A. INFRARED ABSORPTION (197M)
e B. The retention time of the major peak of the Sample
solution corresponds to that of the System suitability solu- Table 1
tion, as obtained in the test for Limit of Lamivudine Hold Time at|
Enantiomer. Initial Temperature Final Final
Temperature Ramp Temperature | Temperature
ASSAY © @/min) ©) (min)
© PROCEDURE
40 = 40 13
Buffer: Transfer about 1.9 g of ammonium acetate to a
1000-mL volumetric flask, dissolve in about 900 mL of 40 40 240 12
water, adit with acetic acid to a pH of 3.8 + 0.2, and
dilute with water to volume.
Mobile phase: Methanol and Buffer (5:95)
System suitability solution: 0.25 mg/mL of USP
amivudine Resolution Mixture B RS in Mobile phase
USP 41 Official Monographs / Lamivudine 2327

Injection volume: 1.0 mL Sample solution: 0.25 mg/mL of Lamivudine in Mobile

Injection type: Split ratio, 15:1 phase
Carrier gas: Helium Analysis
Flow rate: 6 mL/min Samples: Salicylic acid standard solution and Sample
Headspace samplers solution
Oven: 95° Calculate the percentage of salicylic acid in the portion
Loop: 175° of Lamivudine taken:
Transfer line: 175°
Equilibrium time: 10 min Result = (ru/rs) x (Cs/Cu) x 100
System suitability
Sample: Standard solution tu = peak response of salicylic acid from the Sample
[NoTE—The relative retention times for methanol and t- solution
butanol are 1.0 and 1.9, respectively.] rs = peak response of USP Salicylic Acid RS from
Suitability requirements the Salicylic acid standard solution
Tailing factor: NMT 2.0 for methanol Cs = concentration of salicylic acid in the Salicylic
Column efficiency: NLT 25,000 for methanol acid standard solution (mg/mL)
Relative standard deviation: NMT 5.0% for Cu = concentration of Lamivudine in the Sample
methanol solution (mg/mL)
Analysis Calculate the percentage of other individual impurities
Samples: Standard solution and Sample solution in the portion of Lamivudine taken:
Calculate the percentage of methanol in the portion of
lamivudine methanol solvate taken: Result = (ru/r7) x 100

Result = (Ru/Rs) x (Cs/Cu) x 100 tu = peak response of each impurity other than
salicylic acid from the Sample solution
Ry = peak response ratio of methanol to t-butanol Ir = sum of the responses of all the peaks
from the Sample solution Acceptance criteria: See Table 2.
Rs = peak response ratio of methanol to t-butanol
from the Standard solution Table 2
Cs = concentration of methanol in the Standard
solution (mg/mL) Relative Acceptance
Gu = concentration of Lamivudine (as methanol Retention Criteria,
Name Time NMT (%)
solvate) in the Sample solution (mg/mL)
Acceptance criteria: 2.0%-3.0% Lamivudine-carboxylic
acida 0.4 0.3
IMPURITIES Lamivudine-trans
e Limit OF LAMIVUDINE ENANTIOMER (lamivudine diastere-
Buffer: 7.7 g/L of ammonium acetate in water comer)» 0.9 0.2 (ax
Mobile phase: Methanol and Buffer (5:95) Lamivudine 1.0 = Ky
System suitability solution: 0.25 mg/mL of USP
Salicylic acid 27 0.1
Lamivudine Resolution Mixture A RS in water
Sample solution: 0.25 mg/mL of Lamivudine in water Any other individual im- a
Chromatographic system purit 0.1 5
(See Chromatography (621), System Suitability.) Total impurities — 0.6 fo}
Mode: LC 2 (2RS,5SR)-5-(Cytosine-1 -yl)-1,3-oxathiolane-2-carboxylic acid. =
Detector: UV 270 nm > 1-[(2RS,5RS)-2-(Hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine. 3
Column: 4.6-mm x 25-cm; packing L45
Column temperature: 15°-30° (constant temperature) e RESIDUAL SOLVENTS a
Flow rate: 1 mL/min Internal standard solution: Dilute 1 mL of 2-penta-
Injection volume: 10 pL none with dimethyl sulfoxide and water (1:1) to
System suitability 100.0 mL.
Sample: System suitability solution Standard solution: Transfer 10 mL of the Internal stan-
[Note—The relative retention times for lamivudine and dard solution to a 100-mL volumetric flask. Add 100 pL
the lamivudine enantiomer are 1.0 and 1.2, each of the following: dehydrated alcohol, isopropyl
respectively.] acetate, methanol, and triethylamine. Dilute with di-
Suitability requirements methyl sulfoxide and water (1:1) to volume.
Resolution: NLT 1.5 between lamivudine and the Sample solution: Transfer 5 g of Lamivudine to a
lamivudine enantiomer 100-mL volumetric flask, add 10 mL of the Internal stan-
Analysis dard solution, and dilute with dimethyl sulfoxide and
Sample: Sample solution water (1:1) to volume.
Calculate the percentage of the lamivudine enantiomer Chromatographic system
in the portion of Lamivudine taken: (See Chromatography (621), System Suitability.)
Mode: GC
Result = [ru/(ru + r)] x 100 Detector: Flame ionization
Column: 0.53-mm x 50-m, coated with a 5-um film of
Ty = peak response of the lamivudine enantiomer phase G1
rs = peak response of lamivudine Temperatures
Acceptance criteria: NMT 0.3% Injector: 150°
e OTHER RELATED COMPOUNDS Detector: 250°
Buffer, Mobile phase, System suitability solution, Column: See Table 3.
Standard solution, Chromatographic system, and
System suitability: Proceed as directed in the Assay.
Salicylic acid standard solution: 0.625 g/mL of USP
Salicylic Acid RS in Mobile phase
 2328 Lamivudine / Official Monographs USP 41

Table 3 IDENTIFICATION
Hold Time at! ° A. The retention
, time of the lamivudine Pp peak of the
Initial Temperature Final Final sump sie aeoe to that of the Standard solu-
Temperature Ramp Temperature | Temperature # INSEME Seay.
cy (¢/min) @) (min) ASSAY
70 = 70 3 ¢ PROCEDURE
70 30 200 6.5 Solution A: 2.0 g/L of sodium heptanesulfonate in
an water. Add 1.0 mL of hydrochloric acid and 1.0 mL of
Injection volume: 0.5 pL . triethylamine per L of the solution.
Injection type: Split flow rate, 320 mL/min Solution B: Acetonitrile and Solution A (50:50)
we gas: Hydrogen (at pressure 5 psig) Mobile phase: See Table 7.
nalysis
Samples: Standard solution and Sample solution Table 1
Calculate the percentage of each residual solvent in the ae
portion of Lamivudine taken: Solution A Solution B
Result = (Ru/Rs) x (Cs/Cu) x 100 0
Ru = peak response ratio of the respective analyte
to the internal standard from the Sample
solution
Rs = peak response ratio of the respective analyte
to the internal standard from the Standard 100
solution
Cs = concentration of the respective analyte in the Diluent: Acetonitrile and water (10:90)
Standard solution (mg/mL) System suitability solution: Dissolve the contents of 1
G = concentration of the Sample solution (mg/mL) vial of USP Lamivudine Resolution Mixture C RS in
Acceptance criteria: See Table 4. 2.5 mL of Diluent.
Standard solution: 0.2 mg/mL of USP Lamivudine RS
Table 4 in Diluent
Sample solution: Nominally 0.2 mg/mL of lamivudine
Acceptance in water
Criteria, Chromatographic system
% (See Chromatography (621), System Suitability.)
Alcohol Mode: LC
Detector: UV 277 nm
rr Column: 4.6-mm x 10-cm; 3-um packing L1
3 Ti Flow rate: 1 mL/mL
> : Injection volume: 10 wL
% Jotal solvents System suitability
= Samples: System suitability solution and Standard
fy) SPECIFIC TESTS solution .
I) © WATER DETERMINATION, Method Ic (921): NMT 2.0% Suitability requirements set .
= e LIGHT ABSORPTION Resolution: NLT 1.5 between lamivudine-S-sulfoxide
cS (See Ultraviolet-Visible Spectroscopy (857).) and lamivudine-R-sulfoxide, System suitability solution
” Mode: Vis Tailing factor: NMT 2.0 for the lamivudine peak, Sys-
= Sample solution: 50 mg/mL in water tem suitability solution _
Analytical wavelength: 440 nm Relative standard deviation: NMT 2% for the
Cell: 4cm peau peak, Standard solution
Acceptance criteria: Absorptivity NMT 0.0015 Analysis
if Pay! Samples: Standard solution and Sample solution
ADDITIONAL REQUIREMENTS Calculate the percentage of the labeled amount of
e PACKAGING AND STORAGE: Preserve in well-closed, light- lamivudine (CgHi;N303S) in the portion of Oral Solu-
resistant containers. Store at room temperature. tion taken:
e LABELING: Where it is a methanol solvate form, the label
so indicates. Result = (ru/rs) x (Cs/Cu) x 100
e USP REFERENCE STANDARDS (11) s 3
USP Lamivudine RS tu = peak response of lamivudine from the Sample
USP Lamivudine Resolution Mixture A RS solution Shey
USP Lamivudine Resolution Mixture B RS Is = peak response of lamivudine from the
USP Salicylic Acid RS Standard solution Var :
Gs = concentration of USP Lamivudine RS in the
Standard solution (mg/mL)
Cu = nominal concentration of lamivudine in the
Sample solution (mg/mL)
Lamivudine Oral Solution Acceptance criteria: 90.0%-110.0%
PERFORMANCE TESTS
DEFINITION . , ¢ DELIVERABLE VOLUME (698): Meets the requirements
Lamivudine Oral Solution contains NLT 90.0% and NMT
110.0% of the labeled amount of lamivudine IMPURITIES
(CgHii1N303S). It may contain a suitable preservative. © ORGANIC IMPURITIES
Solution A, Solution B, Mobile phase, Diluent, System
suitability solution, Sample solution, Chromato-
USP 41 Official Monographs / Lamivudine 2329

graphic system, and System suitability: Proceed as the filtrate. Evaporate the filtrate to dryness under a
directed in the Assay. gentle stream of nitrogen, and use the residue.
Analysis Standard: Dissolve a suitable amount of USP
Sample: Sample solution Lamivudine RS in a small amount of methanol, shaking
Calculate the percentage of any individual impurity in until completely dissolved. Evaporate to dryness under a
the portion of Oral Solution taken: gentle stream of nitrogen, and use the residue.
Acceptance criteria: Meet the requirements
Result = (ru/rs) x 100 e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
Tu = peak response of each individual impurity obtained in the Assay.
rs = sum of the responses of all of the peaks
excluding peaks due to added preservative(s) ASSAY
or excipients © PROCEDURE
Acceptance criteria: See Table 2. Buffer: 1.9 g/L of ammonium acetate in water. Adjust
with acetic acid to a pH of 3.8.
Table 2 Mobile phase: Methanol and Buffer (50:950)
System suitability solution: 0.2 mg/mL of USP
Relative Acceptance Lamivudine Resolution Mixture B RS in Mobile phase
Retention Criteria, Standard solution: 0.2 mg/mL of USP Lamivudine RS
Name Time NMT (%) in Mobile phase
Lamivudine-uracil derivatives 0.34 12 Sample stock solution: Nominally about 3-4 mg/mL of
Cytosine’ 0.52 0:3: lamivudine in water prepared as follows. Transfer the
Lamivudine-S-sulfoxides 0.61 03 required number of Tablets, based on the labeled
Lamivudine-R-sulfoxided 0.63 0.6 amount, to a suitable volumetric flask, and soak or
shake for at least 15 min in water to disperse the sam-
Lamivudine carboxylic acide! 0.89 = ple. Dilute with water to volume, mix, and pass
Lamivudine trans‘s 0.94 = throughasuitable filter or centrifuge.
Lamivudine 1.0 = Sample solution: Nominally 0.2 mg/mL of lamivudine
Salicylic acid! 1.38 = in Mobile phase from the Sample stock solution
Any other identified impurit = 0.3 Chromatographic system
Any individual unidentified (See Chromatography (621), System Suitability.)
impurit 0.2 Mode: LC
Detector: UV 277 nm
Total impurities = 2.0 Column: 4.6-mm x 25-cm; 5-um packing L1
@ 1-[(2R,55S)-2-(Hydroxymethyl)-1,3-oxathiolan-5-ylJuracil. Column temperature: 30+ 5°
» 4-Aminopyrimidin-2(1H)-one. Flow rate: 1 mL/min
¢1-[(2R,35,55)-2-(Hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine S-oxide. Injection volume: 20 uL
4 1-[(2R,3R,55)-2-(Hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine S-oxide. System suitability
© (2RS,5SR)-5-(Cytosine-1 -yl)-1,3-oxathiolane-2-carboxylic acid.
=
Samples: System suitability solution and Standard as
‘This impurity is controlled in the drug substance and is not to be includ- solution a)
ed in the total impurities Disregard any peak less than 0.01%.
9 1-[(25,55)-2-(Hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine.
[Note—The relative retention times for lamivudine dias- =
tereomer and lamivudine are 0.9 and 1.0, i}
SPECIFIC TESTS respectively.] Fi
}
© PH (791): 5.7-6.3 Suitability requirements eo}=
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI- Resolution: NLT 1.5 between lamivudine and i
FIED MICROORGANISMS (62): The total aerobic microbial lamivudine diastereomer, System suitability solution ne}
count does not exceed 10? cfu/mL. The total molds and Relative standard deviation: NMT 2.0%, Standard my
ww
yeasts count does not exceed 102 cfu/mL. It meets the solution
requirements of the test for absence of Escherichia coli. Analysis
Samples: Standard solution and Sample solution
ADDITIONAL REQUIREMENTS Calculate the percentage of the labeled amount of
e PACKAGING AND STORAGE: Preserve in light-resistant con- lamivudine (CsH1;N303S) in the portion of Tablets
tainers at controlled room temperature. taken:
e USP REFERENCE STANDARDS (11)
USP Lamivudine RS Result = (ru/rs) x (Cs/Cu) x 100
USP Lamivudine Resolution Mixture C RS
[Note—This reference standard contains lamivudine and ru = peak response from the Sample solution
several related impurities.] rs = peak response from the Standard solution
G = concentration of USP Lamivudine RS in the
Standard solution (mg/mL)
Cu = nominal concentration of lamivudine in the
Sample solution (mg/mL)
Lamivudine Tablets Acceptance criteria: 90.0%-110.0%
PERFORMANCE TESTS
DEFINITION e DISSOLUTION (711)
Lamivudine Tablets contain NLT 90.0% and NMT 110.0% of Test 1
the labeled amount of lamivudine (CgH1;N3O3S). Procedure for products labeled as Lamivudine Tab-
IDENTIFICATION lets 100-mg or 150-mg
© A. INFRARED ABSORPTION (197M) Medium: Water, degassed; 900 mL
Sample: Crush 1 Tablet and transfer it to a suitable Apparatus 2: 50 rom
container. Add 5 mL of methanol and shake for 15 min. Time: 30 min
Pass through a suitable filter, collecting about 2 mL of Standard solution: (1/900) mg/mL of USP
Lamivudine RS in Medium, where Lis the Tablet label
claim in mg
 2330 Lamivudine / Official Monographs USP 41

Sample solution: Pass a portion of the solution under Chromatographic system

test through a suitable filter to obtain a concentration (See Chromatography (621), System Suitability.)
similar to that of the Standard solution. Mode: LC
Instrumental conditions Detector: 285 nm
Mode: UV Column: 4.6-mm x 25-cm; 5-um packing L1
Analytical wavelength: 270 nm Flow rate: 1 mL/min
Cell: 1mm Injection volume: 5 ul
Blank: Medium System suitability
Analysis Sample: Standard solution
Samples: Standard solution and Sample solution Suitability requirements
Calculate the percentage of the labeled amount of Relative standard deviation: NMT 2.0%
lamivudine (CgHi1N303S) dissolved: Tailing factor: NMT 1.5
Analysis
Result = (Au/As) x Cs x Vx (1/L) x 100 Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
Au = absorbance of the Sample solution lamivudine (CsH11N303S) dissolved:
As = absorbance of the Standard solution
Cs = concentration of USP Lamivudine RS in the Result = (ru/rs) x Cs x Vx (1/L) x 100
Standard solution (mg/mL)
Vv = volume of Medium, 900 mL ru = peak response from the Sample solution
L = label claim (mg/Tablet) rs = peak response from the Standard solution
Tolerances: NLT 80% (Q) of the labeled amount of Gs = concentration of USP Lamivudine RS in the
lamivudine (CgH;1N3035S) is dissolved. Standard solution (mg/mL)
Procedure for products labeled as Lamivudine Tab- Vv = volume of Medium, 900 mL
lets 300-mg L = label claim (mg/Tablet)
Medium: 0.1 N hydrochloric acid; 900 mL Tolerances: NLT 80% (Q) of the labeled amount of
Apparatus 2: 75 rpm lamivudine (CgH1;N303S) is dissolved.
Time: 15 min e UNIFORMITY OF DOSAGE UNITS (905): Meet the
Standard solution: (1/900) mg/mL of USP requirements
Lamivudine RS in Medium, where L is the Tablet label
claim in mg IMPURITIES
Sample solution: Pass a portion of the solution under © ORGANIC IMPURITIES
test through a suitable filter. Buffer, Mobile phase, System suitability solution,
Instrumental conditions Standard solution, Sample solution, Chromato-
Mode: UV graphic system, and System suitability: Proceed as
Analytical wavelength: 280 nm directed in the Assay.
Cell: 0.5mm Analysis
Blank: Medium Sample: Sample solution
=
”

Analysis Calculate the percentage of each individual impurity in

rm Samples: Standard solution and Sample solution the portion of Tablets taken:
i
—
Dp Calculate the percentage of the labeled amount of
° lamivudine (CgH1;N303S) dissolved: Result = (ru/rr) x 100
=
5 Result = (Au/As) x Cs x Vx (1/L) x 100 Ty = peak response of each individual impurity
3 tr = sum of all the peak responses
a Au = absorbance of the Sample solution Acceptance criteria: See Table 7.
nn As = absorbance of the Standard solution
— G = concentration of USP Lamivudine RS in the Table 1
Standard solution (mg/mL)
Relative Acceptance
Vv = volume of Medium, 900 mL
L = label claim (mg/Tablet) Retention Criteria,
%o
Tolerances: NLT 80% (Q) of the labeled amount of
lamivudine (CgH1;N303S) is dissolved.
Test 2: If the product complies with this test, the label- Lamivudine-carboxylic
ing indicates that it meets USP Dissolution Test 2. db

Medium: 0.1 N hydrochloric acid; 900 mL ‘oxides

Apparatus 2: 50 rpm
Time: 15 min
Buffer: 1.93 g/L of ammonium acetate in water. Ad-
just with glacial acetic acid to a pH of 3.8.
Mobile phase: Methanol and Buffer (40:60)
Standard solution: (1/900) mg/mL of USP Lamivudine
RS in Medium, whereLis the Tablet label claim in mg
Sample solution: Pass a portion of the solution under
test through a suitable filter to obtain a concentration
similar to that of the Standard solution.
Lamivudine diastereomer
it 1
ic 2
a 4-Aminopyrimidin-2(1 H)-one.
» (2RS,5SR)-5-(Cytosine-1 -yl)-1,3-oxathiolane-2-carboxylic acid.
¢ Process impurity included in the table for identification only. Process im-
purities are controlled in the drug substance and are not to be reported
or included in the total impurities for the drug product.
4 1-[(2R,35,55S)-2-(Hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine S-oxide.
© 1-[(2R, 3R,55)-2-(Hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine S-oxide.
1 1-[(2RS,5RS)-2-(Hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine,
USP 41 Official Monographs / Lamivudine 2331

Table 1 (Continued) 750 mg of lamivudine, into a 500-mL volumetric flask.

Relative Acceptance
Add 250 mL of water, and disintegrate completely by
Retention Criteria, shaking for a minimum of 15 min. Dilute with water to
Name Time NMT (%) volume, and mix.
Sample solution: Pass a portion of the Sample stock
Any other individual aa solution througha filter of 0.45-um pore size, discarding
impurity 0.2
the first 2-3 mL. Further dilute the filtrate to obtain
Total impurities = 0.6 0.15 mg/mL of lamivudine and 0.30 mg/mL of
2 4-Aminopyrimidin-2(1 H)-one. zidovudine in Diluent.
» (2RS,5SR)-5-(Cytosine-1 -yl)-1,3-oxathiolane-2-carboxylic acid. Chromatographic system
© Process impurity included in the table for identification only. Process im- (See Chromatography (621), System Suitability.)
purities are controlled in the drug substance and are not to be reported Mode: LC
or included in the total impurities for the drug product.
4 1-[(2R,35,55)-2-(Hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine S-oxide.
Detector: UV 270 nm
© 1-[(2R,3R,55)-2-(Hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine S-oxide.
Column: 3-mm x 25-cm; packing L1
1 1-[(2RS,5R5)-2-(Hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine.
Flow rate: 0.5 mL/min
Injection volume: 10 uL
ADDITIONAL REQUIREMENTS System suitability
© PACKAGING AND STORAGE: Preserve in tight, light-resistant Samples: System suitability solution and Standard
containers. Store at room temperature. solution
e LABELING: When more than one Dissolution test is given, [Note—The relative retention times for lamivudine dias-
the labeling states the Dissolution test used only if Test 7 tereomer and lamivudine are 0.50 and 0.52,
is not used. respectively.]
e USP REFERENCE STANDARDS (11) Suitability requirements
USP Lamivudine RS Resolution: NLT 1.5 between lamivudine diastere-
USP Lamivudine Resolution Mixture B RS omer and lamivudine, System suitability solution
[NoTte—The resolution mixture contains lamivudine and Relative standard deviation: NMT 2.0% for
lamivudine diastereomer. Other impurities may also be zidovudine and lamivudine, Standard solution
present.] Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
lamivudine (CgH1;N303S) and zidovudine (CioHi3NsOa)
in the portion of Tablets taken:
Lamivudine and Zidovudine Tablets
Result = (ru/rs) x (Cs/Cu) x 100
DEFINITION tu = peak response of zidovudine or lamivudine
Lamivudine and Zidovudine Tablets contain NLT 90.0% and from the Sample solution
NMT 110.0% of the labeled amount of lamivudine ls = peak response of zidovudine or lamivudine e
(CsH1iN303S) and zidovudine (CioHi3NsO4). from the Standard solution “
IDENTIFICATION Cs = concentration of USP Zidovudine RS or USP ui:
e A. The retention times of the lamivudine and zidovudine Lamivudine RS in the Standard solution c<
peaks of the Sample solution correspond to those of the (mg/mL) 2
Standard solution, as obtained in the Assay. Cu = nominal concentration of zidovudine or 3
lamivudine in the Sample solution (mg/mL) a
ASSAY Acceptance criteria: 90.0%-110.0% Rj
© PROCEDURE
Solution A: 25 mM of ammonium acetate. Adjust with PERFORMANCE TESTS =
glacial acetic acid to a pH of 4.0. ¢ DISSOLUTION (711) wy
Solution B: Methanol Test 1
Solution C: Acetonitrile Medium: 0.1 N hydrochloric acid; 900 mL, deaerated
Mobile phase: See Table 7. Apparatus 2: 75 rpm
Time: 15 min
Lamivudine response factor solutions: 0.167 mg/mL
Table 1 of USP Lamivudine RS in Medium. [NoTe—Prepare in
Solution A Solution B Solution C duplicate.]
Zidovudine response factor solutions: 0.333 mg/mL
of USP Zidovudine RS in Medium. [NoTe—Prepare in
duplicate.]
Sample solution: Pass a portion of the solution under
70 test througha suitable filter (PTFE, PVDF, or equiva-
lent) of 0.45-um pore size.
1 0 Detector: UV 240-300 nm
45.0 Blank: Medium
45.1 95
Cell length: 0.02-cm flowcell
Analysis: The calculations of the percentages dissolved
95
are done using a multicomponent analysis software.
Diluent: Solution A and Solution B (19:1) Tolerances: NLT 85% (Q) of the labeled amount of
System suitability solution: 0.17 mg/mL of USP zidovudine and lamivudine is dissolved.
Lamivudine Resolution Mixture B RS in Diluent Test 2: If the product complies with this test, the label-
Standard solution: 0.15 mg/mL of USP Lamivudine RS ing indicates that it meets USP Dissolution Test 2.
and 0.30 mg/mL of USP Zidovudine RS in Diluent Medium: 0.1 N hydrochloric acid; 900 mL
Sample stuel solution: Transfer a counted number of Apparatus 2: 75 rpm
Tablets equivalent to 1500 mg of zidovudine and Time: 30 min
Buffer solution: 7.7 g/L of ammonium acetate in
water
 2332 Lamivudine / Official Monographs USP 41

Mobile phase: Acetonitrile and Buffer solution (1:9) nr = sum of the peak responses of zidovudine, all
Standard stock solution: 1.4 mg/mL of USP zidovudine related impurities, and
Lamivudine RS and 2.8 mg/mL of USP Zidovudine RS unspecified impurities from the Sample
in Medium. A small amount of methanol, NMT 20% of solution
the final volume, can be used to dissolve both F = relative response factor (see Table 2)
compounds. Acceptance criteria: See Table 2.
Standard solution: 0.168 mg/mL of lamivudine and
0.336 mg/mL of zidovudine in Medium from the Stan- Table 2
dard stock solution
Sample solution: Pass a portion of the solution under Relative Relative | Acceptance
test througha suitable filter. Retention | Response Criteria,
Chromatographic system Name Time Factor NMT (%)
(See Chromatography (621), System Suitability.) Lamivudine-(cytosine)? 0.11 1.0 —_
Mode: LC Lamivudine-(uracil)< 0.14 1.0 —_
Detector: UV 270 nm Lamivudine-(carboxylic
Column: 4.6-mm x 15-cm; packing L1 acid)? 0.17 1.0 0.3
Column temperature: 40° Lamivudine-(S-sulfox- np
Flow rate: 1.2 mL/min
ide)« 0.20 1.0
Injection volume: 10 pL
System suitability Lamivudine-(R-sulfox- 4
Sample: Standard solution ide) 0.22 1.0
Suitability requirements Zidovudine related
Column efficiency: NLT 1500 theoretical plates for compound C9 0.27 V7. i125
lamivudine and NLT 3000 theoretical plates for Lamivudine
zidovudine diastereomerh 0.50 1.0 0.2
Tailing factor: NMT 2.0 for lamivudine and Lamivudine 0.52 = =
zidovudine Zidovudine- ime
Relative standard deviation: NMT 2.0% for _(thymidine)i 0.60 1.0
zidovudine and lamivudine
Lamivudine-(uracil _»
Calculate the percentages of lamivudine (CsH11N303S) derivative) 0.70 1.0
and zidovudine (CioHi3NsO«) dissolved:
Lamivudine-(salicylic iy
Result = (ru/rs) x (Cs/L) x Vx 100 acid) 0.80 1.0
Zidovudine 1.00 = =
ru = peak response of lamivudine or zidovudine Zidovudine related orig
from the Sample solution compound B! 1.10 1.0
ig = peak response of lamivudine or zidovudine Any individual unspeci-
from the Standard solution fied impurity 1.0 0.1
cn
"

oe Cs = concentration of lamivudine or zidovudine in

the Standard solution (mg/mL) Total lamivudine
Ss
related impurities
—
Dp L = label claim for lamivudine or zidovudine (mg/ (the limit includes — —_
° Tablet)
= Vv = volume of Medium, 900 mL
all lamivudine related
5 Tolerances: NLT 80% (Q) of the labeled amount of impurities) 0.6
= zidovudine and lamivudine is dissolved. Total zidovudine
a
”
© UNIFORMITY OF DosaGE UNITS (905): Meet the require- related impurities
ments for zidovudine and lamivudine (the limit includes _— _
p=) individual unspecified
IMPURITIES impurities) 2.0
© ORGANIC IMPURITIES @ 4-Aminopyrimidin-2(1H)-one.
Solution A, Solution B, Solution C, Mobile phase, Dil- > The individual impurity limit is not included because these are process/
uent, System suitability solution, Sample solution, other impurities monitored individually in the drug substances.
Chromatographic system, and System suitability: ¢ Pyrimidine-2,4(1 H,3H)-dione.
Proceed as directed in the Assay. 9 (2R,5S)-5-(4-Amino-2-oxopyrimidin-1(2H)-yl)-1,3-oxathiolane-2-carboxyl-
ic acid (2R,55)-5-(cytosine-1-yl)-1,3-oxathiofane-2-carboxylic acid.
Analysis
© 1-[(2R,3S,5SS)-2-(Hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine S-oxide.
Sample: Sample solution
£1-[(2R,3R,55)-2-(Hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine S-oxide.
Calculate the percentage of each lamivudine related im-
purity in the portion of Tablets taken: 9 5-Methylpyrimidine-2,4(1 H,3H)-dione.
h 1 -[(25,55)-2-(Hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine.
Result = (ru/rr) x 100 i [1-(2-Deoxy-B-p-ribofuranosyl)]thymine.
i (2RS,5SR)1-[(2R,5S)-2-(Hydroxymethyl)-1,3-oxathiolan-5-ylJuracil.
ru = peak response of each individual impurity k 2-Hydroxybenzoic acid.
from the Sample solution '3’-Chloro-3'-deoxythymidine.
tr = sum of the peak responses of lamivudine and ADDITIONAL REQUIREMENTS
all lamivudine related impurities from the © PACKAGING AND STORAGE: Preserve in well-closed contain-
Sample solution ers, protected from light, and store between 2° and 30°.
Calculate the percentage of each zidovudine related e LABELING: When more than one Dissolution test is given,
impurity and unspecified impurity in the portion of the labeling states the Dissolution test used only if Test 7
Tablets taken: is not used.
Result = (ru/rr) x (1/F) x 100
tu = peak response of each individual impurity
from the Sample solution
USP 41 Official Monographs / Lamotrigine 2333

e USP REFERENCE STANDARDS (11) Chromatographic system

USP Lamivudine RS (See Chromatography (621), System Suitability.)
USP Lamivudine Resolution Mixture B RS Mode: LC
USP Zidovudine RS Detector: UV 270 nm
Column: 4.6-mm x 15-cm; 5-um packing L1
Column temperature: 35°
Flow rate: 1 mL/min
Injection size: 10 uL
Lamotrigine System suitability
Sample: Standard solution
Suitability requirements
ToL
wey ope
Tailing factor: NMT 1.5
Relative standard deviation: NMT 1.5%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of lamotrigine (CsH7Cl2Ns) in
CoH7Cla2Ns 256.09 the portion of Lamotrigine taken:
1,2,4-Triazine-3,5-diamine, 6-(2,3-dichlorophenyl);
3,5-Diamino-6-(2,3-dichlorophenyl)-as-triazine(84057-84-1 Is Result = (ru/rs) x (Cs/Cu) x 100
DEFINITION tu = peak response from the Sample solution
Lamotrigine contains NLT 98.0% and NMT 102.0% of rs = peak response from the Standard solution
CoH7Cl2Ns, calculated on the dried basis. Cs = concentration of USP Lamotrigine RS in the
Standard solution (mg/mL)
IDENTIFICATION Cu = concentration of Lamotrigine in the Sample
¢ A. INFRARED ABSORPTION (197K) solution (mg/mL)
e B. The retention time of the major peak of the Sample Acceptance criteria: 98.0%-102.0% on the dried basis
solution corresponds to that of the Standard solution, as
obtained in the Assay. IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.1%
ASSAY
¢ PROCEDURE
Diluent: Dilute 8.5 mL of hydrochloric acid with water Delete the following:
to 1 L (0.1 M hydrochloric acid).
Buffer: 2.7 g/L of monobasic potassium phosphate in °e HEAVY METALS, Method II (231): 10 ppmecotticis 1Jen-2018)
water ¢ LIMIT OF LAMOTRIGINE RELATED COMPOUND B
Solution A: Triethylamine and Buffer (1:150). Adjust Diluent, Solution A, and Sample solution: Prepare as
with phosphoric acid to a pH of 2.0. directed in the Assay.
Solution B: Acetonitrile Mobile phase: Acetonitrile and Solution A (35:65) (=
a)
Mobile phase: See Table 1. System suitability stock solution: 0.2 mg/mL of USP 7
Lamotrigine RS prepared as follows. Transfer the re-
quired amount of USP Lamotrigine RS to a suitable vol- =
Table 1 °
umetric flask, and add 5% of the final volume with |
Solution A Solution B methanol to facilitate dissolution, Dilute with Diluent to °
volume. 2
m
a
0 76. 2 Standard stock solution: 0.01 mg/mL of USP Lamo- Ey
trigine Related Compound B RS prepared as follows. mo)
4 Z as
1 Transfer the required amount of USP Lamotrigine Re- a
lated CompoundB RS to a volumetric flask. Add 80%
15
of the flask volume of methanol, and acidify with 1% of
1 the flask volume of hydrochloric acid. Allow to cool,
and dilute with methanol to volume. Dilute a portion of
Standard solution: 0.2ma/m. of USP Lamotrigine RS this solution with Diluent.
prepared as follows. Transfer the required amount of System suitability solution: 1 j1g/mL of lamotrigine re-
USP Lamotrigine RS to a suitable volumetric flask, and ated compound B from the Standard stock solution in
add 5% of the final volume with methanol to facilitate System suitability stock solution
dissolution. Dilute with Diluent to volume. Standard solution: 5 g/mL of lamotrigine related
Sample solution: 0.2 mg/mL of Lamotrigine prepared compoundB from the Standard stock solution in Diluent
as follows. Transfer the required amount of lamotrigine Chromatographic system
to a suitable volumetric flask, and add 5% of the final (See Chromatography (621), System Suitability.)
volume with methanol to facilitate dissolution. Dilute Mode: LC
with Diluent to volume. Detector: UV 210 nm
Column: 4.6-mm x 15-cm; 5-4m packing L1
Column temperature: 35°
Flow rate: 1 mL/min
Injection size: 10 wL
Run time: 2 times the retention time of lamotrigine
related compound B
System suitability
Sample: System suitability solution
[Note—Identify the peaks in the System suitability solu-
tion, taking into account that lamotrigine is un-
retained, eluting at or near the solvent front.]
 2334 Lamotrigine / Official Monographs USP 41

Suitability requirements Table 2

Tailing factor: NMT 2.0 for the lamotrigine related Relative Relative Acceptance
compound B peak Retention Response Criteria,
Relative standard deviation: NMT 5.0% for the Name Time Factor NMT (%)
lamotrigine related compound B peak
Analysis Lamotrigine 1.0 1.0 =
Samples: Standard solution and Sample solution Lamotrigine
Calculate the percentage of lamotrigine related com- related
poundBin the portion of Lamotrigine taken: compound Ca 1.5 1.0 0.1
Lamotrigine
Result = (ru/rs) x (Cs/Cu) x 100 related _ —
compound Bb« 522.
tu = peak response for lamotrigine related Lamotrigine
compoundB from the Sample solution related
rs = peak response for the lamotrigine related compound Dé 3.7 0.8 0.2
compound B from the Standard solution
Any individual,
Cs = concentration of USP Lamotrigine Related
unspecified _
Compound B RS in the Standard solution
(ug/ml) | ae impurity
Total impurities,
1.0 0.1
Cu = concentration of Lamotrigine in the Sample
solution (g/mL) excluding lamo-
Acceptance criteria: NMT 0.1% of lamotrigine related trigine related
compound B. [NoTte—Lamotrigine related compound D, compound B 0.2
if present will elute at a retention time of about 1.5 2 3-Amino-6-(2,3-dichlorophenyl)-1,2,4-triazin-5(4H)-one.
relative to lamotrigine related compound B. Disregard » 2,3-Dichlorobenzoic acid.
this peak as it is quantified in the test for Organic Included only for identification.
Impurities.] 9 N-[5-Amino-6-(2,3-dichlorophenyl)-1,2,4-triazin-3-yl]-2,3-
© ORGANIC IMPURITIES dichlorobenzamide.
Diluent, Buffer, Solution A, Solution B, Mobile phase, SPECIFIC TESTS
Sample solution, and Chromatographic system: Pro- e Loss ON DRYING (731): Dry a sample at 105° for 3 h: it
ceed as directed in the Assay. loses NMT 0.5% of its weight.
System suitability stock solution: 0.2 mg/mL of USP
Lamotrigine RS prepared as follows. Transfer the re- ADDITIONAL REQUIREMENTS
quired amount of USP Lamotrigine RS to a suitable vol- © PACKAGING AND STORAGE: Preserve in tight containers.
umetric flask, and add 5%of the final volume with Store at room temperature.
methanol to facilitate dissolution. Dilute with Diluent to e USP REFERENCE STANDARDS (11)
volume. USP Lamotrigine RS
al Impurities stock solution: 0.1 mg/mL of each of USP USP Lamotrigine Related Compound B RS
a Lamotrigine Related Compound C RS and USP Lamo-
5 trigine Related Compound D RS prepared as follows.
2,3-Dichlorobenzoic acid.
i CrHsClz2O2 191.01
7
aD Transfer suitable quantities of the Reference Standards USP Lamotrigine Related Compound C RS
i} to a suitable volumetric flask. Add an amount of meth- 3-Amino-6-(2,3-dichlorophenyl)-1,2,4-triazin-5(4H)-one.
= anol equal to 80% of the flask volume, and acidify with CoHeClaNgO 257.08
Sj 1% of the flask volume of hydrochloric acid. Allow to USP Lamotrigine Related Compound D RS
= cool. Dilute with methanol to volume. N-[5-Amino-6-(2,3-dichlorophenyl)-1,2,4-triazin-3-yl]-
rs System suitability solution: 0.5 g/mL each of lamo- 2,3-dichlorobenzamide.
a)
=) trigine related compound C and lamotrigine related CieHsClaNsO 429.09
compound D from Impurities stock solution in System
Suitability stock solution
System suitability
Sample: System suitability solution
[Note—Refer to Table 2 for relative retention times.] Lamotrigine Tablets
Suitability requirements
Resolution: NLT 2.0 between lamotrigine and lamo-
trigine related compound C peaks DEFINITION
Analysis Lamotrigine Tablets contain NLT 90.0% and NMT 110.0%
Samples: Diluent and Sample solution of the labeled amount of lamotrigine (CyH7Cl2Ns).
[Note—Disregard any peak that may be present in IDENTIFICATION
the chromatogram of the Diluent injection. Disre- e A. ULTRAVIOLET ABSORPTION (197U)
gard any peak due to lamotrigine related compound Standard solution: 0.02 mg/mL of USP Lamotrigine RS
B, because it is quantified in the test for Limit of in 0.01 N hydrochloric oad
Lamotrigine Related Compound B.] Sample solution: 0.02 mg/mL of lamotrigine from
Calculate thepercentage of each impurity in the por- crushed powdered Tablets in 0.01 N hydrochloric acid
tion of Lamotrigine taken: Acceptance criteria: The spectra of the Standard solu-
tion and Sample solution exhibit maxima and minima at
Result = (ru/rs) x (1/F) x 100 the same wavelengths.
ty = peak response for each impurity from the e B. The retention time of the lamotrigine peak of the
Sample solution Sample solution corresponds to that of the Standard solu-
tion, as obtained in the Assay.
rs = peak response for the lamotrigine peak from
the Sample solution
F = relative response factor for each impurity from
Table 2
Acceptance criteria: See Table 2.
USP 41 Official Monographs / Lamotrigine 2335

ASSAY Instrumental conditions

© PROCEDURE (See Ultraviolet-Visible Spectroscopy (857).)
Buffer: 0.8 g/L of ammonium acetate. Adjust with gla- Mode: UV
cial acetic acid to a pH of 4.5. ee wavelength: 267 nm
Mobile phase: Methanol and Buffer (60:40) Blank: Medium
Standard solution: 0.05 mg/mL of USP Lamotrigine RS Analysis
in Mobile phase Calculate the percentage of the labeled amount of
Sample solution: Transfer an amount equivalent to lamotrigine (CyH7Cl2Ns) dissolved:
100 mg of lamotrigine from a portion of crushed Tab-
lets (NLT 20) to a suitable volumetric flask to obtain a Result = (Au/As) x (Cs/L) x D xV
x 100
nominal concentration of lamotrigine of 1.0 mg/mL.
Dissolve in 70% of the flask volume of Mobile phase by Au = absorbance of the Sample solution
sonicating for 20 min. Dilute with Mobile phase to vol- As = absorbance of the Standard solution
ume. Centrifuge the solution. Quantitatively dilute a Cs = concentration of the Standard solution
suitable volume of centrifugate with Mobile phase to (ms/ ml)
obtain a nominal concentration of 0.05 mg/mL of L = label claim (mg/Tablet)
lamotrigine. D = dilution factor of the Sample solution
Chromatographic system Vv = volume of Medium, 900 mL
(See Chromatography (621), System Suitability.) Chromatographic method
Mode: LC Buffer and Mobile phase: Prepare as directed in the
Detector: UV 210 nm Assay.
Column: 4.6-mm x 15-cm; 5-1um packing L1 Standard stock solution: 0.5 mg/mL of USP Lamo-
Flow rate: 1 mL/min trigine RS in Medium, prepared as follows. Dissolve a
Injection size: 10 pL suitable quantity in 15% of the flask volume of meth-
System suitability anol, then dilute with Medium to volume.
Sample: Standard solution Standard solution: (1/1000) mg/mL of USP Lamo-
Suitability requirements trigine RS from the Standard stock solution in Medium,
Tailing factor: NMT 2.0 for lamotrigine where L is the label claim in mg/Tablet
Relative standard deviation: NMT 2.0% for Sample solution: Pass a portion of the solution under
lamotrigine test throughasuitable filter of 0.45-11m pore size.
Analysis Chromatographic system
Samples: Standard solution and Sample solution (See Chromatography (621), System Suitability.)
Calculate the percentage of the labeled amount of Mode: LC
lamotrigine (CoH7CI2Ns) in the portion of Tablets taken: Column: 4.6-mm x 15-cm; 5-um packing L1
Detector: UV 310 nm
Result = (ru/rs) x (Cs/Cu) x 100 Flow rate: 1 mL/min
Injection size: See Table 2.
ty = peak response from the Sample solution i
Is = peak response from the Standard solution Table 2 S
Cs = concentration of USP Lamotrigine RS in the
Standard solution (mg/mL) Label Claim Injection Size c<
Cu = nominal concentration of lamotrigine in the (mg/Tablet) (ul) fe)
Sample solution (mg/mL) 25 50 3
Acceptance criteria: 90.0%-110.0% 100, 150, 200 10 io}
PERFORMANCE TESTS System suitability
S
me}
¢ DISSOLUTION (711) Sample: Standard solution >s
Test 1 Peay requirements
uv

Medium: 0.1 N hydrochloric acid; 900 mL Tailing factor: NMT 2.0 for lamotrigine
Apparatus 2: 50 rpm Relative standard deviation: NMT 2.0%
Time: 30 min Analysis
Determine the amount of lamotrigine (CsH7Cl2Ns) dis- Samples: Standard solution and Sample solution
solved by using one of the following methods. Calculate the percentage of the labeled amount of
Spectrometric method lamotrigine (CsH7Cl2Ns) dissolved:
Standard stock solution: 0.15 mg/mL of USP Lamo-
trigine RS in Medium prepared as follows. Dissolve a Result = (ru/rs) x (Cs/L) x Vx 100
suitable quantity in 5% of the flask volume of metha-
nol, then dilute with Medium to volume. ty = peak response from the Sample solution
Standard solution: Dilute the Standard stock solution ls = peak response from the Standard solution
with Medium to obtain a final concentration of Gs = concentration of the Standard solution
0.028 mg/mL. (mg/mL)
Sample solution: Pass a portion of the solution under L = label claim (mg/Tablet)
test through a suitable filter of 0.45-1um pore size. Vv = volume of Medium, 900 mL
Dilute with Medium according to Table 1. Tolerances: NLT 80% (Q) of the labeled amount of
lamotrigine is dissolved.
Table 1 Test 2: If the product complies with this test, the label-
ing indicates that it meets USP Dissolution Test 2.
Tablet Volume of Volume of Final Medium, Apparatus, and Time: Proceed as directed
Label Claim Sample Volumetric | Concentration for Test 1.
(mg) (mL) Flask (mg/mL) Analysis: Determine the amount of lamotrigine dis-
25 = = 0.028 solved using either the Spectrometric method or Chro-
100 5.0 20 0.029 matographic method described in Test 1.
150 4.0 25 0.027 Tolerances: NLT 75% (Q) of the labeled amount of
200 3.0 25 0.027
lamotrigine is dissolved.
 2336 Lamotrigine / Official Monographs USP 41

Test 3: If the product complies with this test, the label- Analysis
ing indicates that it meets USP Dissolution Test 3. Samples: Standard solution and Sample solution
Medium: 0.1 N hydrochloric acid; 900 mL Calculate the percentage of any individual impurity in
Apparatus 2: 50 rpm the portion of Tablets taken:
Time: 15 min
Standard solution: (L/900) mg/mL of USP Lamo- Result = (ru/ts) x (Cs/Cu) x (1/F) x 100
trigine RS in Medium, where L is the Tablet label claim
Inm tu = peak response of each individual impurity
sample solution: Pass a portion of the solution under from the Sample solution
test through a suitable filter of 0.45-m pore size. rs = peak response of lamotrigine from the
Instrumental conditions Standard solution
(See Ultraviolet-Visible Spectroscopy (857).) Cs = concentration of USP Lamotrigine RS in the
Mode: UV Standard solution (mg/mL)
ae wavelength: 270 nm Cu = nominal concentration of lamotrigine in the
Cel Sample solution (mg/mL)
For Tablets labeled to contain 100, 150, or F = relative response factor for the corresponding
200 mg: 0.2-cm flow cell impurity (see Table 3)
For Tablets labeled to contain 25mg: 1 cm Acceptance criteria: See Table 3.
Blank: Medium
Analysis Table 3
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of Relative Relative Acceptance
Retention Response Criteria,
lamotrigine (CsH7Cl2Ns) dissolved:
Name Time Factor NMT (%)
Result = (Au/As) x (Gs/L) x Vx 100 Lamotrigine related
compound B+ 0.67 0.75 0.2
Au = absorbance of the Sample solution Lamotrigine 1.0 = —
As = absorbance of the Standard solution Lamotrigine related
Cs = concentration of the Standard solution compound Cb 5 1.0 0.5
(mg/ml) Any individual un-
E = label claim (mg/Tablet) specified degrada- —
Vv = volume of Medium, 900 mL
tion impurity 1.0 0.2
Tolerances: NLT 80% (Q) of the labeled amount of
lamotrigine is dissolved. Total impurities = — 0.75
e UNIFORMITY OF DOSAGE UNITS (905): Meet the 2 2,3-Dichlorobenzoic acid.
requirements » 3-Amino-6-(2,3-dichlorophenyl)-1,2,4-triazin-5(4H)-one.

IMPURITIES ADDITIONAL REQUIREMENTS

aa
al
@ ORGANIC IMPURITIES © PACKAGING AND STORAGE: Preserve in well-closed contain-
[a Buffer: Prepare as directed in the Assay. ers, and store at controlled room temperature.
i) e LABELING: When more than one Dissolution test is given,
— Mobile phase: Acetonitrile, methanol, and Buffer
Da the labeling states the Dissolution test used only if Test 1
o) (10:30:60)
is not used.
I Diluent: Methanol and Buffer (60:40)
G System suitability solution: 1 g/mL of Lamotrigine e USP REFERENCE STANDARDS (11)
= Related Compound B RS and 0.4 mg/mL of USP Lamo- USP Lamotrigine RS
rs trigine RS in Diluent USP Lamotrigine Related Compound B RS
al Standard solution: 1.0 4g/mL of USP Lamotrigine RS in 2,3-Dichlorobenzoic acid.
> Diluent C7H4Cl202 ~=—-191.01
Sample solution: Transfer an amount equivalent to
100 mg of lamotrigine from a portion of crushed Tab-
lets (NLT 20) to a suitable volumetric flask to obtain a
nominal concentration of lamotrigine of about 0.4 mg/
mL. Dissolve in 70% of the flask volume of Mobile phase Lamotrigine Extended-Release Tablets
by sonicating and shaking intermittently for 30 min. Di-
lute with Diluent to volume. Pass through a membrane DEFINITION
filter of 0.45-um pore size. Lamotrigine Extended-Release Tablets contain NLT 90.0%
Chromatographic system and NMT 110.0% of the labeled amount of lamotrigine
(See Chromatography (621), System Suitability.) (CoH7Cl2Ns).
Mode: LC
Detector: UV 210 nm IDENTIFICATION
Column: 4.6-mm x 25-cm; 5-um packing L1 e A. The retention time of the a of the Sample
Flow rate: 1 mL/min solution corresponds to that of the Standard solution, as
Injection size: 5 pL obtained in the Assay.
System suitability ASSAY
Samples: System suitability solution and Standard
solution
© PROCEDURE
Mobile phase: Acetonitrile, water, and trifluoroacetic
Suitability requirements acid (25: 75: 0.05)
Resolution: NLT 2.0 between lamotrigine related
Diluent: Acetonitrile, methanol, and water (10:20:70)
compoundB and lamotrigine, System suitability Standard solution: 0.25 mg/mL of USP Lamotrigine RS
solution in Diluent. Sonication may be used to aid dissolution.
Tailing factor: NMT 2.0 for lamotrigine, Standard Sample stock solution: 1.0-3.0 mg/mL of lamotrigine
solution prepared as follows. Transfer NLT 5 Tablets to a suitable
Relative standard deviation: NMT 10.0% for lamo-
volumetric flask containing 10% of the flask volume of
trigine, Standard solution
USP 41 Official Monographs / Lamotrigine 2337

acetonitrile. Allow the Tablets to disperse. Add 20% of Analysis .

the flask volume of methanol. Sonicate for 10 min. Add Samples: Standard solution and Sample solution
30% of the flask volume of 0.1 M hydrochloric acid. Calculate the percentage of the labeled amount (Q)) of
Sonicate for 25 min or until a fine, even dispersion is lamotrigine (CsH7Cl2Ns) dissolved at each time point
obtained. Allow to cool to room temperature. Dilute i,
with 0.1 M hydrochloric acid to volume. Pass a portion
of the solution through a nylon filter of 0.45-um pore Result = (Au/As) x Cs x Vx D x (1/L) x 100
size and use the filtrate.
Sample solution: Nominally 0.2-0.3 mg/mL of lamo- Au = absorbance of the Sample solution
trigine in 0.1 M hydrochloric acid from a suitable vol- As = absorbance of the Standard solution
ume of Sample stock solution Gs = concentration of the Standard solution
Chromatographic system (mg/mL)
(See Chromatography (621), System Suitability.) V = volume of Medium, 900 mL
Mode: LC D = dilution factor if needed
Detector: UV 270 nm L = label claim (mg/Tablet)
Column: 4.6-mm x 15-cm, 3-m packing L1 Tolerances
Column temperature: 40° For Tablets with 25- or 50-mg label claim: See Ta-
Flow rate: 1 mL/min ble 1.
Injection volume: 5 ul For Tablets with 100-, 200-, or 250-mg label claim:
Run time: NLT 8 times the retention time of See Table 2.
lamotrigine For Tablets with 300-mg label claim: See Table 3.
System suitability
Sample: Standard solution Table 1
malality requirements
Tailing factor: NMT 2.0 Time Point Time
Relative standard deviation: NMT 1.5%
Analysis 2
Samples: Standard solution and Sample solution 7
Calculate the percentage of the labeled amount of
lamotrigine (CsH7Cl2Ns) in the portion of Tablets taken:

Result = (ru/rs) x (Cs/Cu) x 100 Table 2

tu = peak response from the Sample solution
Is = peak response from the Standard solution Time Point
Cs = concentration of USP Lamotrigine RS in the
Standard solution (mg/mL)
Cu = nominal concentration of lamotrigine in the oa
Sample solution (mg/mL) a)
Acceptance criteria: 90.0%-110.0% i)
=
PERFORMANCE TESTS °
e DISSOLUTION (711) Table 3 =
Test 1 }
Medium 1: 0,01 M hydrochloric acid; 700 mL
Time Point eo}=
i
Medium 2: 7.8g of tribasic sodium phosphate, and no)
22.5 g of sodium dodecyl sulfate in 1 L of water. This
%
oe
solution has a pH of about 12. T
a]

ApRaatus 2: 50 rpm with sinkers (see Figure 2a in

Times
ps pene labeled to contain 25 or 50 mg: 2, 7,
For Tablets labeled to contain 100, 200, or
250mg: 2,5,12h
For Tablets labeled to contain 300 as 2,6,13h
Procedure: Run the test with Medium 7 for 2 h. Add
200 mL of Medium 2, preheated at 37°. Within 5 min
of the addition of Medium 2, withdraw the sample for
the 2 h time point. Continue the testing by drawin
samples at the time points specified in Table 1, rable
2, or Table 3, depending on the label claim.
Diluent: Medium 7 and Medium 2 (70:20)
Standard solution: (L/900) mg/mL of USP Lamo-
tri ine RS in Diluent, whereL is the label claim in mg/
Tablet
Sample solution: Pass a suitable portion of the solu-
tion under test through a suitable filter of 0.45-um
pore size. Dilute with Diluent if necessary.
Blank: Diluent
Instrumental conditions
Mode: UV
Analytical wavelength: 260 nm. [NoTt—Depending
on the label claim, cells with suitable path lengths
may be used.]
The percentages of the labeled amount of lamotrigine
(CsH7Cl2Ns) dissolved at the times specified conform
to Dissolution (711), Acceptance Table 2.
Test 2: If the product complies with this test, the label-
ing indicates that it meets USP Dissolution Test 2.
Acid stage medium: 0.01 M hydrochloric acid;
700 mL
Buffer stage stock medium: 2.83 g of sodium phos-
phate monobasic, 1.72 g of sodium hydroxide, and
22.5 g of sodium dodecyl sulfate in 1 L of water
Buffer stage medium: Acid stage medium and Buffer
stage stock medium (70:20); 900 mL. Adjust with solu-
tion A (phosphoric acid in water prepared by diluting
1 mL of phosphoric acid with water to 50 mL) or 0.17
N sodium hydroxide, if necessary, to a pH of 6.8. Re-
cord the required volume of solution A or 0.1 N so-
dium hydroxide for adjustment of pH to 6.8.
Apparatus 2: 50 rpm with stationary tablet basket.
ee Figures 1 and 2.
Times
For Tablets labeled to contain 25 or 50 mg: 2hin
Acid stage medium; 4, 7, 9, and 15h in Buffer stage
medium
 2338 Lamotrigine / Official Monographs USP 41

NOTES

1. Rod and Basket with a Tablet

cover placed in the horizontal
diagonal of the basket.
2. Basket and Tablet cover
material; stainless steel.
3. Basket gauze wire size:
8 mesh.

CKD
SSS
SKY
oS
100mm +2mm
a
w“

a I
i]
—
Dd <———— _ 354mm = ———+|
°
= £2mm
S
Ps Figure 1. Stationary tablet basket.
a
a)
> Buffer stage medium, whereLis the label claim in mg/
For Tablets labeled to contain 100, 200, or
300 mg: 2h in Acid stage medium; 3, 5, 7, and 12h
in Buffer stage medium
[Note—The times in the Buffer stage medium include
the time in the Acid stage medium.]
Procedure: Run the test with Acid stage medium for 2
h followed by collecting the Acid stage medium sample
and replacing it with the same volume of Acid stage
medium. Add 200 mL of Buffer stage stock medium to
the above solution. If necessary, add either solution A
or 0.1 N sodium hydroxide to the solution to reach a
pH of 6.8. Continue the resting by drawing samples at
the time points specified in Table 4 or Table 5, de-
pending on the label claim. Replace each of the
volumes withdrawn with an equal volume of Buffer
stage medium.
Buffer: Dissolve 2.76 g of sodium phosphate monoba-
sic in 1 L of water. Add 2 mL of triethylamine and ad-
just with solution A to a pH of 7.0.
Mobile phase: Methanol and Buffer (55:45)
Standard stock solution: 1.4 mg/mL of USP Lamo-
trigine RS in methanol
Acid stage standard solution: (1/900) mg/mL of USP
Lamotrigine RS from Standard stock solution, in Acid
stage medium, whereLis the label claim in mg/Tablet
Buffer stage standard solution: (1/900) mg/mL of
USP Lamotrigine RS from Standard stock solution, in
Tablet
Acid stage sample solution: Withdraw a 10.0-mL ali-
uot, and pass a portion of the solution under test
throughasuitable filter of 0.45-tum pore size. Replace
the 10.0-mL aliquot withdrawn for analysis with a
10.0-mL aliquot of Acid stage medium.
Buffer stage sample solution: Withdraw a 10.0-mL
aliquot, and pass a portion of the solution under test
through a suitable filter of 0.45-um pore size. Replace
the 10.0-mL aliquot withdrawn for analysis with a
10.0-mL aliquot of Buffer stage medium.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 270 nm
Column: 4.6-mm x 15-cm; 5-um packing L7
Flow rate: 1 mL/min
Injection volume
For 25-mg Tablets: 80 uL
For 50-mg Tablets: 40 uL
For 100-mg Tablets: 20 uL
For 200- or 300-mg Tablets: 10 ul
Run time: NLT 1.8 times the retention time of
lamotrigine
System suitability
Samples: Acid stage standard solution and Buffer stage
standard solution
USP 41 Official Monographs / Lamotrigine 2339

Calculate the percentage of the labeled amount of

lamotrigine (CyH7Cl2Ns) dissolved at each time point
(i) during the buffer stage:
Result; = [C; x Vg x (1/L) x 100] + (Q4 x Vs/Va)

Result, = {[(C2 x Vs) + (Cr x Vs)] x (1/L) x 100} + (Qa x

SIVA,

Results = ({(C3 x Ve) + [(C2 + C;) x Vs]} x (1/L) x 100) +

a X Vs/Va,

Results = ({(Cy x Va) + [(C3 + C2 + Ci) x Vs]} x (1/L) x

100) + (Qa x Vs/Va)
G = concentration of lamotrigine in the Buffer
stage sample solution withdrawn at time
point i (mg/mL)
Ve = volume of the Buffer stage medium, 900 mL
L = label claim (mg/Tablet)
Qa = the percentage of the labeled amount of
lamotrigine dissolved in the Acid stage
medium
Vs = volume of the Sample solution withdrawn at
each time point (/) during the acid stage or
buffer stage (mL)
Va = volume of the Acid stage medium, 700 mL
Figure 2. Drug release stationary tablet basket configuration Tolerances
diagram. For Tablets labeled to contain 25 or 50mg: See
Suitability requirements Table 4.
Tailing factor: NMT 2.0 For Tablets labeled to contain 100, 200, or
Relative standard deviation: NMT 2.0% 300 mg: See Table 5.
Analysis
Samples: Acid stage standard solution, Acid stage sam- Table 4
ple solution, Buffer stage standard solution, and Buffer Time Point Time =
stage sample solution a)
Calculate the percentage (Q,) of the labeled amount
2
a]
of lamotrigine (CsH7Cl2Ns) dissolved in the Acid stage 4
4
medium: re}
7 =
Result; = (ru/rs) x Cs x Va x (1/L) x 100 i}
to)=
15
tu = peak response from the Acid stage sample 2
solution a}
fs = peak response from the Acid stage standard
Py
wv
solution Table 5
Gs = concentration of USP Lamotrigine RS in the Time Point Time
Acid stage standard solution (mg/mL)
Va = volume of the Acid stage medium, 700 mL NMT1
L = label claim (mg/Tablet)
Calculate the concentration (C) of lamotrigine
%-5:
(CoH7Cl2Ns) in the sample withdrawn from the vessel
at each time point (/) during the buffer stage: 50%-7
12
Result; = (ru/rs) x Cs
The percentages of the labeled amount of lamotrigine
tu = peak response from the Buffer stage sample (CsH7Cl2Ns) dissolved at the times specified conform
solution at time point i to Dissolution (711), Acceptance Table 2.
fs = peak response from the Buffer stage standard e UNIFORMITY OF DOSAGE UNITS (905): Meet the
solution requirements
Cs = concentration of USP Lamotrigine RS in the
Buffer stage standard solution (mg/mL) IMPURITIES
¢ ORGANIC IMPURITIES
Mobile phase, Sample solution, and Chromatographic
system: Proceed as directed in the Assay.
Diluent 1: Acetonitrile, methanol, and 0.1 M hydro-
chloric acid (10:20:70)
Diluent 2: Acetonitrile, methanol, and water (10:20:70)
System suitability stock solution: 0.025 mg/mL of USP
Lamotrigine Related Compound C RS in Diluent 7
System suitability solution: 1.25 tug/mL of USP Lamo-
trigine Related Compound C RS and 0.25 mg/mL of
USP Lamotrigine RS in Diluent 2 prepared as follows.
 2340 Lamotrigine / Official Monographs USP 41

Transfer a suitable amount of USP Lamotrigine RS to a paper. Evaporate the solution to dryness. Add 250 mg
suitable volumetric flask. Transfer a suitable volume of of potassium bromide to the dried residue, and prepare
System suitability stock solution to the flask. Dissolve and the pellet.
dilute with Diluent 2 to volume. Acceptance criteria: Absorption bands at 1491 cm",
System suitability 1557 cm, 1621 cm, 3213 cm", 3320 cm", and
Sample: System suitability solution 3451 cm” are found in the Sample anda similarly pre-
Suitability requirements pared Standard.
Resolution: NLT 10 between the lamotrigine and e B. The retention time of the major peak of the Sample
lamotrigine related compound C peaks solution corresponds to that of the Standard solution, as
Signal-to-noise ratio: NLT 100 for lamotrigine re- obtained in the Assay.
lated compound C
Analysis ASSAY
Sample: Sample solution o PROCEDURE
Calculate the percentage of each degradation product Buffer: 0.77 g/L of ammonium acetate, adjusted with
in the portion of Tablets taken: glacial acetic acid to a pH of 4.5
Mobile phase: Acetonitrile, methanol, and Buffer
Result = (ru/rr) x 100 (30:10:60)
Diluent: Acetonitrile, methanol, and Buffer (30:30:40)
tu = response of each impurity from the Sample Standard solution: 0.05 mg/mL of USP Lamotrigine RS
solution in Diluent
tr = sum of all of the impurity peak responses and Sample solution: Transfer NLT 6 Tablets for Oral Sus-
the lamotrigine peak response from the pension to a suitable volumetric flask to obtain a nomi-
Sample solution nal concentration of about 0.05 mg/mL of lamotrigine.
Acceptance criteria: See Table 6. Disregard peaks less Sonicate in 70% of the flask volume of Diluent for 30
than 0.05%. min with intermittent shaking. Dilute with Diluent to fi-
nal volume, and pass a portion througha suitable
Table 6 membrane filter.
Chromatographic system
Relative Acceptance (See Chromatography (621), System Suitability.)
Retention Criteria, Mode: LC
Name Time NMT (%) Detector: 210 nm
Lamotrigine 10 — Column: 4.6-mm x 25-cm; 5-um packing L1
Lamotrigine related Flow rate: 1.5 mL/min
compound C LZ 0.3 Injection size: 10 pL
Lamotrigine dimer 6.0 0.2 System suitability
Any individual unspecified _
Sample: Standard solution
degradation product 0.2 Suitability requirements
a) Tailing factor: NMT 2.0, lamotrigine
a] Total impurities as 0.5
a @This is either lamotrigine o-dimer [N5,N%-methylenebis(6-(2,3-
Relative standard deviation: NMT 2.0%
6 Analysis
— dichlorophenyl)-1,2,4-triazine-3,5-diamine)] or famotrigine-dimer N3,N¥-
D methylenebis(6-(2,3-dichlorophenyl)-1,2,4-triazine-3,5-diamine). Samples: Standard solution and Sample solution
° Calculate the percentage of lamotrigine (CsH7Cl2Ns),
iS ADDITIONAL REQUIREMENTS based on the label claim, in the portion of Tablets for
iS ® PACKAGING AND STORAGE: Preserve in well-closed contain- Oral Suspension taken:
= ers. Store at controlled room temperature.
ion e LABELING: When more than one Dissolution test is given, Result = (ru/rs) x (Cs/Cu) x 100
va
=) the labeling states the Dissolution test used only if Test 7
is not used. ry = peak response from the Sample solution
e¢ USP REFERENCE STANDARDS (11) rs = peak response from the Standard solution
USP Lamotrigine RS Cs = concentration of USP Lamotrigine RS in the
USP Lamotrigine Related Compound C RS Standard solution (mg/mL)
3-Amino-6-(2,3-dichlorophenyl)-1,2,4-triazin-5(4H)-one. Cu = nominal concentration of lamotrigine in the
CoH6Cl,NsO = 257.08 Sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0%
PERFORMANCE TESTS
e DISSOLUTION (711)
Medium: 0.1 N hydrochloric acid; 900 mL, degassed
Lamotrigine Tablets for Oral Apparatus 2: 50 rpm
Suspension Time: 15 min
[Note—The Sample solution may be analyzed using either
DEFINITION HER UsegEpae procedure 1 or Chromatographic proce-
Lamotrigine Tablets for Oral Suspension contain NLT 90.0% lure 2.]
and NMT 110.0% of the labeled amount of lamotrigine Standard stock solution: 0.5 mg/mL of USP Lamo-
(CsH7Cl2Ns). trigine RS in methanol
Standard solution: (1/1000) mg/mL of USP Lamo-
IDENTIFICATION trigine RS in Medium from the Standard stock solution,
e A. INFRARED ABSORPTION (197K) whereLis the Tablet label claim in mg
Sample: Transfer crushed powder of the Tablets for Oral Sample solution: Pass a portion of the solution under
Suspension, equivalent to 30 mg of lamotrigine, into an test throughasuitable filter of 0.45-t1m pore size.
Erlenmeyer flask, and add 10 mL of chloroform. Soni- Determine the amount of lamotrigine dissolved by em-
cate for about 15 min. Shake the flask for another 2 ploying one of the following chromatographic
min, Pass the sample through a Whatman No. 1 filter procedures.
USP 41 Official Monographs / Lamotrigine 2341

Chromatographic procedure 1 Chromatographic system

Buffer: To 1 L of 0.77 g/L of ammonium acetate in (See Chromatography (621), System Suitability.)
water add 2 mL of triethylamine, and adjust with glacial Mode: LC
acetic acid to a pH of 7.5. Detector: UV 210 nm
Mobile phase: Acetonitrile, methanol, and Buffer Column: 4.6-mm x 25-cm column; 5-um packing L1
(20:15:65) Flow rate: 1 mL/min
Chromatographic system Injection size: 20 ul
(See Chromatography (621), System Suitability.) System suitability
Mode: LC Sample: — Standard solution
Detector: UV 310 nm [Note—Relative retention times are in Table 1.]
Column: 4.6-mm x 15-cm; 5-um packing L1 Suitability requirements
Flow rate: 1 mL/min Tailing factor: NMT 2.0
Injection size: 100 uL Relative standard deviation: NMT 10%
System suitability Analysis
Sample: Standard solution Samples: Standard solution and Sample solution
Suitability requirements Calculate the percentage of each impurity in the por-
Tailing factor; NMT 2.0 tion of Tablets for Oral Suspension taken:
Relative standard deviation: NMT 2.0%
Chromatographic procedure 2 Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
Mobile phase and Chromatographic system: Proceed
as directed in the test for Organic Impurities, Procedure tu = peak response of the impurity from the
23 Sample solution
System suitability rs = peak response of lamotrigine from the
Sample: Standard solution Standard solution
Suitability requirements Cs = concentration of USP Lamotrigine RS in the
Tailing factor: NMT 2.0 Standard solution (mg/mL)
Relative standard deviation: NMT 2.0% Cu = nominal concentration of lamotrigine in the
Analysis Sample solution (mg/mL)
Samples: Standard solution and Sample solution F = relative response factor for each impurity
Calculate the percentage of lamotrigine dissolved: listed in Table 1
Acceptance criteria: See Table 1.
Result = (ru/rs) x (Cs/L) x Vx 100
Table 1
tu = peak response from the Sample solution
ts = peak response from the Standard solution Relative Relative Acceptance
G = concentration of USP Lamotrigine RS in the Retention Response Criteria,
Standard solution (mg/mL) Name Time Factor NMT (%)
L = label claim (mg/Tablet, Lamotrigine 1.0 = = (om!
Vv = volume of Medium, 900 mL Lamotrigine related a
Tolerances: NLT 80% (Q) of the labeled amount of compound Ba 1.59 0.69 0.1
lamotrigine is dissolved. Any other individual =
¢ UNIFORMITY OF DOSAGE UNITS (905): Meet the degradation prod- — =
requirements uct 1.0 0.2 °
IMPURITIES Total impurities = = 0.4 =}
Organic Impurities a 2,3-Dichlorobenzoic acid, a
[Note—Procedure 7 is recommended if lamotrigine related
compoundBis a potential organic impurity. Procedure 2 is © PROCEDURE 2 a
recommended if lamotrigine related compound Cis a po- Mobile phase: Acetonitrile, water, glacial acetic acid,
tential organic mpurtyat and triethylamine (47:148:4:1). [NOTE—The Mobile
© PROCEDURE 1
phase is stable for 48 h at room temperature.]
Diluent: Methanol and water (40:60),
Buffer, Mobile phase, and Diluent: Prepare as di-
rected in the Assay. Standard solution: 0.2 mg/mL of USP Lamotrigine RS
Standard solution: 0.8 g/mL of USP Lamotrigine RS and 0.002 mg/mL of USP Lamotrigine Related Com-
in Diluent pound C RS prepared as follows. Transfer suitable
Sample solution: From NLT 20 Tablets for Oral Sus- amounts of USP Lamotrigine RS and USP Lamotrigine
pension ground to a fine powder, transfer an amount Related Compound C RS to a suitable volumetric flask.
Add 40% of the flask volume of methanol, and soni-
of powder toa suitable flask, to obtain a nominal con- cate until dissolved. Allow to cool to room tempera-
centration of 0.25 mg/mL of lamotrigine in Diluent. ture, and dilute with water to volume.
Sonicate for 15 min to dissolve the contents. Filter a
portion, and discard the first 1 mL of the filtrate. Sample solution: Nominally 0.2 mg/mL of lamotrigine.
Use 10 Tablets for Oral Suspension for a label claim of
25 mg or less and 5 Tablets for Oral Suspension for a
label claim of 50 mg or more prepared as follows.
Transfer the appropriate number of Tablets for Oral
Suspension to a suitable volumetric flask. Add 40% of
the flask volume of water. Swirl until the Tablets have
disintegrated. Allow the effervescence to stop, and
then add an additional 40% of the flask volume of
methanol. Sonicate the flask for 10 min, and cool to
room temperature. Dilute with water to volume.
[Note—For Tablets for Oral Suspension with a 50 mg
or higher label claim, a suitable intermediate concen-
tration may be chosen. The final dilution to arrive at
the nominal concentration is made using Diluent.]
 2342 Lamotrigine / Official Monographs USP 41

Chromatographic system e USP REFERENCE STANDARDS (11)

(See Chromatography (621), System Suitability.) USP Lamotrigine RS
Mode: LC 1,2,4-Triazine-3,5-diamine, 6-(2,3-dichlorophenyl).
Detector: UV 270 nm CoH7Cl2Ns 256.09
Column: 4.6-mm x 15-cm column; 5-um packing L1 USP Lamotrigine Related Compound C RS
Flow rate: 1 mL/min 3-Amino-6-(2,3-dichlorophenyl)-1,2,4-triazin-5(4H)-one.
Injection size: 10 pL CoHeClaNsO —-257.08
System suitability
Sample: Standard solution
[NoTe—Relative retention times are in Table 2.]
Suitability requirements
Resolution: NLT 2.0 between lamotrigine and lamo- Lamotrigine Compounded Oral
trigine related compound C
Tailing factor: NMT 2.0 for lamotrigine and lamo- Suspension
trigine related compound C
Relative standard deviation: NMT 5.0% for lamo- DEFINITION
trigine related compound C and NMT 1.5% for Lamotrigine Compounded Oral puspension contains NLT
lamotrigine 90.0% and NMT 110.0% of the labeled amount of lamo-
Analysis trigine (CsH7Cl2Ns).
Samples: Standard solution and Sample solution Prepare Lamotrigine Compounded Oral Suspension 1 mg/
Calculate the percentage of lamotrigine related com- mL as follows (see Pharmaceutical Compounding—Nonster-
poundCin the portion of Tablets for Oral Suspen- ile Preparations (795)).
sion taken:
Lamotrigine tablet? equivalent to 100 mg
Result = (ru/rs) x (Cs/Cu) x 100 Ora-Blend,® a sufficient quantity to make 100 mL
tu = peak response of lamotrigine related @Lamotrigine 100-mg tablet, Torrent Pharmaceuticals LTD, Kalamazoo, MI.
compound C from the Sample solution »Perrigo, Minneapolis, MN.
ig = peak response of lamotrigine related Place the required number of tablet(s) in a suitable mortar,
compound C from the Standard solution and comminute to a fine powder. Add the Ora-Blend in
Cs = concentration of USP Lamotrigine Related small portions, and triturate to make a smooth paste. Add
Compound C RS in the Standard solution increasing volumes of the Ora-Blend to make a lamo-
(mg/mL) trigine liquid that is pourable. Transfer the contents of the
Cy = nominal concentration of lamotrigine in the mortar, stepwise and quantitatively, to a calibrated bottle.
Sample solution (mg/mL) Add enough of the Ora-Blend to bring to final volume,
Calculate the percentage of any other individual and mix well.
unspecified degradation product in the portion of
Tablets for Oral Suspension taken: ASSAY
s
“”

a © PROCEDURE
fs) Result = (ru/rs) x (Cs/Cu) « 100 Solution A: Dissolve 2.7 g of monobasic potassium
—
Dd phosphate in 1000 mL of water. Add 6.5 mL of triethyl-
° tu = peak response of any other impurity from the amine, and adjust with phosphoric acid to a pH of 2.0.
< Sample solution Mobile phase: Acetonitrile and Solution A (20:80). Fil-
S ls = peak response of lamotrigine from the ter, and degas.
2 Standard solution Diluent: 0.1 M hydrochloric acid
5 Cs = concentration of USP Lamotrigine RS in the Standard solution: 0.4 mg/mL of USP Lamotrigine RS
” Standard solution (mg/mL)
=] in Diluent
= nominal concentration of lamotrigine in the
SoO

Sample solution: Shake thoroughly each bottle of Oral

j

Sample solution (mg/mL) Suspension. Transfer 4 mL of Oral Suspension into a

Acceptance criteria: See Table 2. 10 mL volumetric flask, dilute with Diluent to volume,
and mix well.
Table 2 Chromatographic system
(See Chromatography (621), System Suitability.)
Relative Acceptance Mode: LC
Retention Criteria,
Detector: UV 270 nm
Name Time NMT (%)
Column: 4.6-mm x 25-cm; 5-m packing L1
Lamotrigine 1.0 = Flow rate: 1.0 mL/min
Lamotrigine related com- Injection volume: 5 uL
pound Ca Ls 0.3 System suitability
Any other individual unspeci- mer Sample: Standard solution
fied degradation product 0.2 > ie retention time for lamotrigine is about 9.8
Total impurities — 0.5 min.
2 3-Amino-6-(2,3-dichlorophenyl)-1,2,4-triazin-5(4H)-one.
Suitability requirements
Tailing factor: NMT 2.0
ADDITIONAL REQUIREMENTS Relative standard deviation: NMT 2.0% for replicate
e PACKAGING AND STORAGE: Store in tight, light-resistant injections
containers, at controlled room temperature. Analysis
e LABELING: If a procedure for Organic Impurities other than Samples: Standard solution and Sample solution
Procedure 1 is used, then the labeling states with which Calculate the percentage of the labeled amount of
Organic Impurities procedure the article complies. The la- lamotrigine (CsH7CI2Ns) in the portion of Oral Suspen-
bel states that the Tablets for Oral Suspension may be sion taken:
swallowed whole, chewed, or dispersed in water or di-
luted fruit juice. Result = (ru/rs) x (Cs/Cu) x 100
USP 41
ty = peak response of lamotrigine from the Sample
solution
ls = peak response of lamotrigine from the
Standard solution
Cs concentration of lamotrigine in the Standard
solution (mg/mL)
Cu = nominal concentration of lamotrigine in the
Sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0%
SPECIFIC TESTS
© PH (791): 4.0-5.0
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Package in tight, light-resistant
contaligs. Store at controlled room temperature or
28°.
¢ LABELING: Label it to indicate that it is to be well-shaken
before use, and to state the Beyond-Use Date.
e BEYOND-UsE DATE: NMT 90 days after the date on which
it was compounded when stored at controlled room
temperature or 2°-8°
e USP REFERENCE STANDARDS (11)
USP Lamotrigine RS
Lanolin
DEFINITION
Lanolin is the purified, wax-like substance from the wool of
sheep, Ovis aries L. (Fam. Bovidae), that has been cleaned,
decolorized, and deodorized. It contains NMT 0.25% of
water. It may contain NMT 0.02% ofa suitable
antioxidant.
IMPURITIES
© RESIDUE ON IGNITION (281): NMT 0.1%
e CHLORIDE AND SULFATE, Chloride (221)
Sample solution: Boil 20 mL of alcohol with 1.0g of
Lanolin under a reflux condenser. Cool, add 1 mL of
2N nitric acid, and filter. To the filtrate add 5 drops of
a solution of 20 mg/mL of silver nitrate in alcohol.
Blank: Boil 20 mL of alcohol under a reflux condenser.
Cool, add 1 mL of 2 N nitric acid, and filter. To the
filtrate add 5 drops of a solution of 20 mg/mL of silver
mileate in alcohol. Add 0.50 mL of 0.020 N hydrochloric
acid.
Acceptance criteria: 0.035%; any turbidity produced
by the Sample solution does not exceed that produced
by the Blank.
© FOREIGN SUBSTANCES
Use pesticide-free grade reagents and solvents through-
out this test. [NoTE—Reference materials of pesticides
for use in the Standard solution may be obtained from
any commercial source."]
Standard stock solutions: Prepare stock solutions for
each reference pesticide containing 100 mg/L in hex-
ane.
[Note—Concentrated stock solutions may be stored in
glass-stoppered containers in a dark refrigerator at
2°-5° for up to 1 year. Most pesticides may be dis-
1 Suitable materials may be obtained from either Chem Service, 660 Tower
Lane, P.O. Box 3108, Westchester, PA 19381-3108 or Greyhound, 88 Grange
Road West, Birkenhead, Merseyside, L43 4XF, England U.K.
Official Monographs / Lanolin 2343
solved directly in hexane; however, the hexachloro-
cyclohexane isomers and the DDT group of pesticides
may require initial dissolution in the minimum volume
of acetone followed by dilution with hexane to the
specified concentration.]
Standard solution: Dilute volumes of the Standard
stock solutions quantitatively with hexane, and combine
to obtain a composite Standard solution having the con-
centrations indicated in Table 1. Store the composite
Standard solution in a glass-stoppered glass container in
the dark at 2°-5°, and replace it every 2 months.
[Note—Two or more separate composite Standard solu-
tions, each eae containing NMT8reference pesti-
cides, may be prepared if needed. Reference pesticides
should be selected for composite Standard solutions on
the basis that relative retention times (see Table 1) differ
sufficiently so that peaks in chromatograms will not be
expected to overlap, andthey should be selected and
combined appropriately for the chromatographic sys-
tem and detector used.]
Gel permeation chromatography cleanup system
Eluant: Methylene chloride and hexane (1:1)
Column: 25-mm x 50-cm; packed with a slurry of
35 g of styrene—divinylbenzene copolymer beads com-
pressed to a bed length of about 20 cm
Operating pressure: 8-11 psi
Flow rate: 5 mL/min
Set up the chromatograph, adjusting to discard the
fraction eluting from 0 to 12 min. Collect the fraction
eluting from 12 to 32 min, and rinse for 2 min, dis-
carding the rinse fraction.
System suitability
Elution of lanolin: Melt a suitable quantity of Lano-
lin, and pass throughafluted filter paper into a con-
tainer. Transfer 6.0 g to a 50-mL volumetric flask. Di-
lute with E/uant to volume, and filter. Transfer 5.0 mL
of this solution to the gel permeation chromato-
graphic column, and elute with Eluant. Collect <
100 mL of the column effluent in tared beakers in 4)
10-mL increments. Evaporate the solvent, cool, weigh
Ss)
the beakers and contents, and calculate the amount rd
of lanolin eluted in each 10-mL increment. The col- re}
=
umn is suitable if NLT 96% of the lanolin elutes in i}
the first 60 mL. ito)=
Elution of pesticide from lanolin: Dissolve suitable i)
quantities of diazinon, diclofenthion, bromophos i}
ethyl, lindane, and dieldrin in hexane to obtain a re
Standard solution having concentrations of 0.4, 0.4,
a
1.0, 0.1, and 0.6 tg/mL, respectively. Transfer 5.0 mL
of this solution to a 10-mL volumetric flask containing
1g of USP Lanolin RS. Dilute with methylene chloride
to volume. Transfer 5 mL of this solution to the gel
permeation chromatographic column, and elute with
160 mL of Eluant. Discard the first 60-mL fraction,
and collect the next 100-mL fraction (from 60 to
160 mL). Transfer this collection fraction to a concen-
trator fitted with a graduated collection flask, add
50 mL of hexane, and concentrate by evaporation to
5 mL. Inject this fraction into the chromatographs de-
scribed in Chromatographic system | and Chromato-
graphic system II. Record the chromatograms, and
measure the heights of the peaks obtained from the
five pesticides in the Standard solution. Calculate the
recoveries of each of the five pesticides used in the
fortified USP Lanolin RS solution.
 2344 Lanolin / Official Monographs USP 41

Table 1
Standard Solution Relative Retention Times
Cone:
Flame-
Electron- Photometric

Te loron| 0.05
a
beta-Hi

1,1’-Dichloro-2-(2-chlorophenyl)-2-(4-

1,1’-Dichloro-2-(4-chlorophenyl)-2-(4-

in
1,1’-Dichloro-2-(2-chlorophenyl)-2-(4-
TO.

c
”

rae Endrin
S
_
D 1,1-Dichloro-2,2-bis(4-chlorophenyl)ethane (p,pp-
°
c
Sj 1,1,1-Trichloro-2-(2-chloropheny!)-2-(4-
2
aq.
Al
-
1,1,1-Trichloro-2,2-bix(4-chlorophenyl)ethane (p,p’-
Di
Metho
ie 00 a 0

@ Suitable materials may be obtained from either Chem Service, 660 Tower Lane, P.O. Box 3108, Westchester, PA 19381-3108 or Greyhound, 88 Grange
Road West, Birkenhead, Merseyside, L43 4XF, England U.K.

Prepare a test solution by mixing hexane with the
Standard solution (1:1). Inject this into the chro-
matographs described in Chromatographic system |
and Chromatographic system II. Record the chromat-
ograms, and measure the peak heights of the five
pesticides in the chromatogram of the Sample solu-
tion. Compare the peak heights from the fraction of
the Standard solution to the peak heights of the cor-
responding pesticides from the Sample solution: NLT
85% of the added amounts of each of the five pesti-
cides is recovered.
Sample solution: Transfer 6 g of Lanolin, previously
melted to liquid form by heating on a hot water bath if
necessary, to a 50-mL volumetric flask. Dissolve in
25 mL of Eluant, dilute with Eluant to volume, and filter.
Transfer 5.0 mL of this solution to the column, and
elute with 160 mL of Eluant. Discard the first 60-mL
fraction, and collect the remaining fraction in a suitable
evaporator. Concentrate by evaporation on a steam
bath to 3 mL, add 50 mL of hexane, and evaporate
again to remove all traces of methylene chloride, ad-
justing the volume with hexane to 3.0 mL.
Chromatographic system |
(See Chromatography (621), System Suitability.)
Mode: GC
Detector: Electron capture
Column: 0.53-mm x 30-m fused silica capillary;
bonded with a 1.5-um layer of phase G1, and a 0.53-
mm x 6-m fused silica uncoated guard column con-
nected to a modified packed column-type injector
system
Column temperature: 200°. [NoTE—The initial tem-
perature of the column may be adjusted so that the
retention times of ethion and p,p’-DDT are 2.56 and
3.1, respectively, relative to chlorpyrifos.]
Carrier gas: Helium
Flow rate: 25 mL/min. Adjust so that the retention
time of chlorpyrifos is 4 min.
USP 41
Makeup gas: Nitrogen, 40 mL per minute
Injection volume: 5 uL . Sample solution: 10 mL of the solution from Water-Sol-
Chromatographic system II
Mode: GC
Detector: Flame photometric
Column: 0.53-mm x 30-m fused silica capillary;
bonded with a 1.0-1m layer of phase G3, and a 0.53-
mm x 6-m fused silica uncoated guard column con-
nected to a modified packed column-type injector
system
Column temperature: 200°. [NoTE—The initial tem-
perature of the column may be adjusted so that the
retention time of ethion is 3.36 relative to that of
chlorpyrifos.]
Carrier gas: Helium
Flow rate: 25 mL/min. Adjust so that the retention
time of chlorpyrifos is 4 min.
Makeup gas: Nitrogen, 40 mL/min
Injection volume: 5 uL
Analysis
The following procedure is to be followed for Chromato-
graphic systems | and Il.
Samples: Standard solution and Sample solution
Calculate the quantity of the individual specified residue
found in the sample taken:
Result = (ru/rs) x (C/W) x 30
ru = peak area of each residue from the Sample
solution
Is = peak area of each residue from the Standard
solution
C = concentration of the reference pesticide in the
Standard solution (mg/L)
w = weight of Lanolin taken (g)
Acceptance criteria
Individual specified residue: NMT 10 ppm
Total specified residue: NMT 40 ppm
SPECIFIC TESTS
© MELTING RANGE OR TEMPERATURE, Class // (741)
Analysis: Determine on a sample previously cooled to
Acceptance criteria: 38°-44°
© FATS AND FIXED OILS, Acid Value (Free Fatty Acids) (401)
Sample: 10.0g
Acceptance criteria: The free acids obtained from the
Sample require NMT 2.0 mL of 0.10 N sodium hydrox-
ide for neutralization.
e FATS AND FIXED OILS, /odine Value (401)
Sample: 780-820 mg
Acceptance criteria: 18-36
¢ ALKALINITY
Sample: 2.0g
Analysis: Dissolve the Sample in 10 mL of ether, and
add 2 drops of phenolphthalein TS.
Acceptance criteria: The liquid is not colored red.
¢ WATER-SOLUBLE ACIDS AND ALKALIES
Sample: 10.09
Analysis: Warm the Sample with 50 mL of water on a
steam bath, constantly stirring the mixture until the
Lanolin is melted.
Acceptance criteria: The fat separates completely on
cooling, leaving the water layer nearly clear and neutral
to litmus. Retain the water layer for the test for Water-
Soluble Oxidizable Substances and Ammonia.
© WATER-SOLUBLE OXIDIZABLE SUBSTANCES
Sample solution: 10 mL of the solution from Water-Sol-
uble Acids and Alkalies
Analysis: Add the Sample solution to 50 uL of 0.10 N
potassium permanganate.
Acceptance criteria: The resulting solution does not
completely decolorize within 10 min.
Official Monographs / Lanolin 2345
e AMMONIA
uble Acids and Alkalies
Analysis: Add 1 mL of 1 N sodium hydroxide to the
Sample solution, and boil.
Acceptance criteria: The vapors do not turn red litmus
to blue.
e@ WATER DETERMINATION, Method | (921)
Solution A: Chloroform and methanol (3:2)
Sample solution: 250 mg/mL of Lanolin in Solution A
Analysis: Determine the water content of a 10.0-mL
portion of the Sample solution. Perform a blank determi-
nation on 10.0 mL of Solution A, and make any neces-
sary correction.
Acceptance criteria: NMT 0.25%
e PETROLATUM
Sample: 3g
Analysis: Heat the Sample on a steam bath, with fre-
quent stirring, until its weight loss is NLT its water con-
tent. Boil 40 mL of dehydrated alcohol with 500 mg of
the dried lanolin so obtained.
Acceptance criteria: The solution is clear or NMT
opalescent.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, preferably at controlled room temperature.
e LABELING: The label states that it is not to be used
undiluted.
e USP REFERENCE STANDARDS (11)
USP Lanolin RS
Modified Lanolin
DEFINITION (=
wv
Modified Lanolin is the purified wax-like substance from the =)
wool of sheep, Ovis aries L. (Fam. Bovidae), that has been
processed to reduce the contents of free lanolin alcohols =
and residues of detergent and pesticide. It contains NMT i}
=]
0.25% of water. It may contain NMT 0.02% ofa suitable ro}
antioxidant. Re}=
i)
IMPURITIES ao}
rd3
e LIMIT OF FREE LANOLIN ALCOHOLS
Gel permeation chromatographic cleanup system
Eluant: Methylene chloride
Column: 25-mm x 100-cm; packed with a slurry of
styrene-divinylbenzene copolymer beads compressed
to a bed length of approximately 77 cm
Flow rate: 4 mL/min
Set up the chromatograph, adjusting to discard the
fraction eluting from 0 to 43 min. Collect the fraction
eluting from 43 to 60 min, and rinse for 20 min,
discarding the rinse fraction.
an suitabilit
lution of lanolin alcohols: Melt a suitable quantity
of USP Lanolin Alcohols RS, and pass through a fluted
filter paper into a container. Transfer 1.0 g to a
10-mL volumetric flask. Dilute with Eluant to volume.
Transfer 5 mL to the gel permeation snreraLogtabhte
column, and elute with Eluant. Collect 172-240 mL of
the column effluent in a suitable evaporator. Evapo-
rate the solvent, cool, weigh the evaporator, and cal-
culate the amount of lanolin alcohols eluted in the
evaporator. The column is suitable if NLT 99% of the
lanolin alcohols elute in the first 172-240 mL.
Standard solution: 0.5 mg/mL of USP Lanolin Alcohols
RS in hexane. Store this solution in a cold, dark place
for up to 4 weeks. Before using, warm just sufficiently
to dissolve any precipitate if necessary.
Sample solution: Transfer 1 g of Modified Lanolin, pre-
viously melted to liquid form by heating on a hot water
 2346 Lanolin / Official Monographs USP 41

bath if necessary, to a 10-mL volumetric flask. Dissolve [Note—Concentrated stock solutions may be stored in
in 7 mL of Eluant, dilute with Eluant to volume, and glass-stoppered containers in a dark refrigerator at
filter. Transfer 5.0 mL of this solution to the column, 2°-5° for up to 1 eae Most pesticides may be dis-
and elute with 320 mL of Eluant. Discard the first solved directly in hexane; however, the hexachloro-
172-mL fraction, and collect the next 68-mL fraction cyclohexane isomers and the DDT group of pesticides
(from 172 to 240 mL) in a suitable evaporator. Concen- may require initial dissolution in the minimum volume
trate by evaporation on a steam bath to 3 mL. Add of acetone followed by dilution with hexane to the
50 mL of hexane, and transfer this solution to a 100-mL specified concentration.]
volumetric flask, adjusting the volume with hexane to Standard solution: Dilute volumes of the Standard
100 mL. stock solutions quantitatively with hexane, and combine
Chromatographic system to obtain a composite Standard solution having the con-
(See Chromatography (621), System Suitability.) centrations indicated in Table 2. Store the composite
Mode: GC Standard solution in a glass-stoppered glass container in
Detector: Flame ionization the dark at 2°-5°, and replace it every 2 months.
Columns [NoTE—Two or more separate composite Standard solu-
Guard: 0.32-mm x 50-cm fused silica uncoated tions, each preterbly containing NMT8 reference pesti-
Analytical: 0.33-mm x 50-m fused silica capillary; cides, may be prepared if needed. Reference pesticides
bonded with a 0.50-um layer of phase G2 should be selected for composite Standard solutions on
Temperatures the basis that relative retention times (see Table 2) differ
Detector: 290° sufficiently so that peaks in chromatograms will not be
Column: See Table 1. expected to overlap, and they should be selected and
combined appropriately for the chromatographic sys-
Table 1 tem and detector seth]
Gel permeation chromato raphy cleanup system
Hold Time at Eluant: Methylene chloride and hexane (1:1)
Initial Temperature Final Final Column: 25-mm x 50-cm; packed with a slurry of
Temperature Ramp Temperature | Temperature 35 g of styrene-divinylbenzene copolymer beads com-
©) (¢/min) () (min) pressed to a bed length of about 20 cm
210 3 280 a Operating pressure: 8-11 psi
Flow rate: 5 mL/min. Set up the chromatograph, ad-
Flow rate: 7 mL/min justing to discard the fraction eluting from 0 to 12
Carrier gas: Nitrogen min. Collect the fraction eluting from 12 to 32 min,
Makeup gas: Nitrogen at 50 mL/min and rinse for 2 min, discarding the rinse fraction.
Injection volume: 1 uL System suitability
Analysis Elution of lanolin: Melt a suitable quantity of Lano-
Samples: Standard solution and Sample solution lin, and pass througha fluted filter paper into a con-
[NoTE—Allow both the Standard solution and the Sam- tainer. Transfer 6.0 g to a 50-mL volumetric flask. Di-
ple solution to elute for NLT 40 min.] lute with Eluant to volume, and filter. Transfer 5.0 mL
eS
a)

re Calculate the percentage of free lanolin alcohols in the of this solution to the gel permeation chromato-
i} portion of Modified Lanolin taken: graphic column, and elute with Eluant. Collect
io)
—
100 mL of the column effluent in tared beakers in
re} Result = (ru/rs) x [(C x K)/(I x W)] x 100 10-mL increments. Evaporate the solvent, cool, weigh
S the beakers and contents, and calculate the amount
S ty = total peak response from the Sample solution of lanolin eluted in each 10-mL increment. The col-
= rs = total peak response from the Standard solution umn is suitable if NLT 96% of the lanolin elutes in
3 Cc = concentration of USP Lanolin Alcohols RS in the first 60 mL.
a) the Standard solution (mg/mL) Elution of pesticide from lanolin: Dissolve suitable
> | = volume injected into the gel permeation quantities of diazinon, diclofenthion, bromophos
chromatography column (mL) ethyl, lindane, and dieldrin in hexane to obtain a
w = weight of Modified Lanolin taken (g) Standard solution having concentrations of 0.4, 0.4,
K = corrected fraction of free lanolin alcohols in 1.0, 0.1, and 0.6 g/mL, respectively. Transfer 5.0 mL
the USP Lanolin Alcohols RS in the Standard of this solution to a 10-mL volumetric flask containing
solution taken: 1g of USP Lanolin RS. Dilute with methylene chloride
to volume. Transfer 5 mL of this solution to the gel
K=1 + (0.0062A — 0.01195) permeation chromatographic column, and elute with
A = acid value of USP Lanolin Alcohols RS 160 mL of Eluant. Discard the first 60-mL fraction,
and collect the next 100-mL fraction (from 60 to
S = saponification value of USP Lanolin Alcohols
160 mL). Transfer this collection fraction to a concen-
RS
Acceptance criteria: NMT 6% trator fitted with a graduated collection flask, add
e FOREIGN SUBSTANCES
50 mL of hexane, and concentrate by evaporation to
Use pesticide-free grade reagents and solvents through- 5 mL. Inject this fraction into the chromatographs de-
out this test. [NoTE—Reference materials of pesticides scribed in Chromatographic system | and Chromato-
for use in the Standard solution may be obtained from graphic system II. Record the chromatograms, and
any commercial source."] measure the heights of the peaks obtained from the
Standard stock solutions: Prepare stock solutions for five pesticides in the Standard solution. Calculate the
each reference pesticide containing 100 mg/L in hex- recoveries of each of the five pesticides used in the
fortified USP Lanolin RS solution.
ane.
1 Suitable materials may be obtained from either Chem Service, 660 Tower
Lane, P O. Box 3108, Westchester, PA 19381-3108 or Greyhound, 88 Grange
Road West, Birkenhead, Merseyside, L43 4XF, England U.K.
USP 41 Official Monographs / Lanolin 2347

Table 2
Standard Solution Relative Retention Times
for
Flame-
Electron- Photometric

5.
0.0.

0.

lorf

1,1-Dichloro-2-(2-chlorophenyl)-2-(4-
loro,
1,1-Dichloro-2-(4-chlorophenyl)-2-(4-

1,1-Dichloro-2-(2-chlorophenyl)-2-(4-
I

Cc
“
~~

1 I E
1,1,1-Trichloro-2-(2-chlorophenyl)-2-(4-
°
J
°
Xe)
os
Sy
mo]
1,1,1-Trichloro-2,2-bis(4-chlorophenyl)ethane (p,p- s
7
1}
I

fe = 00
# Suitable materials may be obtained from either Chem Service, 660 Tower Lane, P.O, Box 3108, Westchester, PA 19381-3108 or Greyhound, 88 Grange
Road West, Birkenhead, Merseyside, L43 4XF, England U.K.

Prepare a test solution by mixing hexane with the
Standard solution (1:1). Inject into the chro-
matographs described in Chromatographic system |
and Chromatographic system Il. Record the chromat-
ograms, and measure the peak heights of the five
pesticides in the chromatogram of the Sample solu-
tion. Compare the peak heights from the fraction of
the Standard solution to the oa heights of the cor-
responding pesticides from the Sample solution: NLT
85% of the added amounts of each of the five pesti-
cides is recovered.
Sample solution: Transfer 6 g of Lanolin, previously
melted to liquid form by heating on a hot water bath if
necessary, to a 50-mL volumetric flask. Dissolve in
25 mL of Eluant, dilute with E/uant to volume, and filter.
Transfer 5.0 mL of this solution to the column, and
elute with 160 mL of Eluant. Discard the first 60-mL
fraction, and collect the remaining fraction in a suitable
evaporator. Concentrate by evaporation on a steam
bath to 3 mL, add 50 mL of hexane, and evaporate
again to remove all traces of methylene chloride, ad-
ausing the volume with hexane to 3.0 mL.
hromatographic system |
(See Chromatography (621), System Suitability.)
Mode: GC
Detector: Electron capture
Column: 0.53-mm x 30-m fused silica capillary;
bonded with a 1.5-um layer of phase G1, and a 0.53-
mm x 6-m fused silica uncoated guard column con-
nected to a modified packed column-type injector
system
Column temperature: 200°. [NoTe—The initial tem-
perature of the column may be adjusted so that the
retention times of ethion and p,p’-DDT are 2.56 and
3.1, respectively, relative to chlorpyrifos.]
Carrier gas: Helium
Flow rate: 25 mL/min. Adjust so that the retention
time of chlorpyrifos is 4 min.
 2348 Lanolin / Official Monographs USP 41

Makeup gas: Nitrogen, 40 mL/min Analysis: Add 1 mL of 1 N sodium hydroxide to the

Injection volume: 5 pL Sample solution, and boil.
Chromatographic system II Acceptance criteria: The vapors do not turn red litmus
Mode: GC to blue.
Detector: Flame photometric e WATER DETERMINATION, Method | (921)
Column: 0.53-mm x 30-m fused silica capillary; Solution A: Chloroform and methanol (3:2)
bonded with a 1.0-um layer of phase G3, and a 0.53- Sample solution: 250 mg/mL of Modified Lanolin in
mm x 6-m fused silica uncoated guard column con- Solution A
nected to a modified packed column-type injector Analysis: Determine the water content of a 10.0-mL
system portion of the Sample solution. Perform a blank determi-
Column temperature: 200°. [NoTE—The initial tem- nation on 10.0 mL of Solution A, and make any neces-
perature of the column may be adjusted so that the sary correction.
retention time of ethion is 3.36 relative to that of Acceptance criteria: NMT 0.25%
chlorpyrifos.] © PETROLATUM
Carrier gas: Helium Sample: 3g
Flow rate: 25 mL/min. Adjust so that the retention Analysis: Heat the Sample on a steam bath, with fre-
time of chlorpyrifos is 4 min. quent stirring, until it loses about 0.25% of its weight.
Makeup gas: Nitrogen, 40 mL/min Boil 40 mL of dehydrated alcohol with 500 mg of the
Injection volume: 5 ul dried lanolin so obtained.
Analysis Acceptance criteria: The solution is clear or NMT
The following procedure is to be followed for Chromato- Opalescent.
graphic systems | and Il.
Samples: Standard solution and Sample solution ADDITIONAL REQUIREMENTS
Inject the appropriate composite Standard solution and © PACKAGING AND STORAGE: Preserve in tight, preferably
the Sample solution into the gas chromatograph, re- rust-proof containers, preferably at controlled room
cord the chromatograms, and measure the areas of temperature.
all the peaks observed in the chromatograms. Com- e USP REFERENCE STANDARDS (11)
pare the peak areas of any of the pesticide residues in USP Lanolin RS
the Sample solution from each chromatographic sys- USP Lanolin Alcohols RS
tem with the peak areas that correspond to the re-
tention times in the appropriate composite Standard
solution from each corresponding chromatographic
system.
Calculate the quantity, in ppm, of the individual speci- Lansoprazole
fied residue found in the sample taken:
/>\
Result = (ru/rs) x (C/W) x 30 has
— Hs RF
\ eh A
”
N is
Ox, eke
<= tu = peak area of each residue from the Sample
oy solution ae
i]
-
fo) rs = peak area of each residue from the Standard
° solution Ci6Hi4F3N302S 369.36
= c = concentration of the reference pesticide in the 1H-Benzimidazole, 2-[[[3-methyl-4-(2,2,2-trifluoroethoxy)-
S Standard solution (mg/L)
3 2-pyridinyl|methy!|sulfinyl]-;
Ww = weight of Lanolin taken (g) 2-[[[3-Methyl-4-(2,2, 2-trifluoroethoxy)-2-pyridyl]-methyl-
a Acceptance criteria JsulfinylJbenzimidazole [103577-45-3].
va)
> Individual specified residue: NMT 1 ppm
Total specified residues: NMT 3 ppm DEFINITION
Lansoprazole contains NLT 98.0% and NMT 102.0% of
SPECIFIC TESTS CisHi4F3N302S.
© FATS AND FIXED OILS, Acid Value (Free Fatty Acids) (401)
Sample: 12.59 IDENTIFICATION
Acceptance criteria: The free acids obtained from the A. INFRARED ABSORPTION (197K)
Sample require NMT 2.0 mL of 0.10 N sodium hydrox- e B. ULTRAVIOLET ABSORPTION (197U)
ide for neutralization. Sample solution: 10par in methanol
e ALKALINITY Acceptance criteria: Meets the requirements
Sample: 2.5g
Anois Dissolve the Sample in 10 mL of ether, and ASSAY
add 2 drops of phenolphthalein TS. ¢ PROCEDURE
Acceptance criteria: No red color is produced. Mobile phase: Acetonitrile, water, and triethylamine
e WATER-SOLUBLE ACIDS AND ALKALIES (40:60:1). Adjust with phosphoric acid to a pH of 7.0.
Sample: 12.59 Diluent: Acetonitrile, water, and triethylamine
Analysis: Warm the Sample with 50 mL of water on a (40:60:1).pele with phosphoric acid to a pH of 10.0.
steam bath, constantly stirring the mixture until the System suitability solution: 0.1 mg/mL of USP Lan-
Lanolin is melted. soprazole RS and 0.1 mg/mL of USP Lansoprazole Re-
Acceptance criteria: The fat separates completely on lated Compound A RS in Diluent
cooling, leaving the water layer eel clear and neutral Standard solution: 0.1 mg/mL of USP Lansoprazole RS
to litmus. Retain the water layer for the test for in Diluent
Ammonia.
© AMMONIA
Sample solution: 10 mL of the solution from Water-Sol-
uble Acids and Alkalies
USP 41 Official Monographs / Lansoprazole 2349

Sample solution: 0.1 mg/mL of Lansoprazole in Diluent Blank: Methanol and Diluent (1:9)
Chromatographic system Chromatographic system
(See Chromatography (621), System Suitability.) (See Chromatography (621), System Suitability.)
Mode: LC Mode: LC
Detector: UV 285 nm Detector: UV 285 nm
Column: 4.6-mm x 25-cm; 5-um packing L1 Column: 4.6-mm x 15-cm; 5-um packing L1
Flow rate: 1 mL/min Flow rate: 0.8 mL/min
Injection size: 10 uL Injection size: 40 uL
System suitability System suitability
Samples: System suitability solution and Standard Sample: System suitability solution
solution Suitability requirements
Suitability requirements Resolution: NLT 6 between lansoprazole and lan-
Resolution: NLT 5 between lansoprazole and lan- soprazole related compound A
soprazole related compound A, System suitability Relative standard deviation: NMT 3%
solution Analysis
Relative standard deviation: NMT 1.0%, Standard anes Standard solution, Sample solution, and
solution Blan
Analysis Identify the lansoprazole peak and the peaks due to
Samples: Standard solution and Sample solution the impurities listed in Table 2. Measure the areas
Calculate the percentage of lansoprazole for the major peaks, excluding peaks obtained from
(CieHiaF3N302S) in the portion of Lansoprazole taken: the Blank.
Calculate the percentage of lansoprazole related com-
Result = (ru/rs) x (Cs/Cu) x 100 poundBin the portion of Lansoprazole taken:
tu = peak response from the Sample solution Result = (ru/rs) x (Cs/Cu) x 100
Is = peak response from the Standard solution
Cs = concentration of USP Lansoprazole RS in the ty = peak response for lansoprazole related
Standard solution (mg/mL) compound B from the Sample solution
Cu = concentration of Lansoprazole in the Sample rs = peak response for lansoprazole related
solution (mg/mL) compoundBfrom the Standard solution
Acceptance criteria: 98.0%-102.0% Cs = concentration of USP Lansoprazole Related
Compound B RS in the Standard solution
IMPURITIES
Inorganic Impurities (ug/ml) :
Cu = concentration of Lansoprazole in the Sample
e RESIDUE ON IGNITION (281): NMT 0.1% solution (ug/mL)
Organic Impurities Calculate the percentage of ae N-oxide,
© PROCEDURE lansoprazole sulfone, and any other individual
[NoTe—Store and inject the lansoprazole solutions at or impurity in the portion of Lansoprazole taken: =
below 5° using a cooled autosampler. The solutions are 4)
stable for about 24 h when stored at 5°.] Result = (ru/rs) x (Cs/Cu) x (1/F) x 100 z
Solution A: Water <
Solution B: Acetonitrile, water, and triethylamine tu = peak response for each impurity from the }
(160:40:1). Adjust with phosphoric acid to a pH of 7.0. Sample solution =:
Diluent: Methanol and 0.1 N sodium hydroxide (1:3) ls = peak response for lansoprazole from the i)
Mobile phase: See Table 7. Standard solution
eo}=
Sy
Gs = concentration of USP Lansoprazole RS in the 3
Table 1 Standard solution (ug/mL) =H
Cu = concentration of Lansoprazole in the Sample ww

Solution A Solution B solution (g/mL)

F = relative response factor for each impurity (see
90 1 Table 2)
Acceptance criteria
Individual impurities: See Table 2. Disregard any
peak below 0.05%.

Table 2
System suitability solution: Prepare a solution contain- Relative Relative Acceptance
og 25 g/mL of USP Lansoprazole RS and 25 ug/mL of Retention Response Criteria,
USP Lansoprazole Related Compound ARS in metha- Name Time Factor NMT (%)
nol. Transfer 1 mL of this solution into a 10-mL volu-
Lansoprazole
metric flask, and dilute with Diluent to volume.
N-oxide? 0.8 1.3 0.1
Standard solution: Prepare a solution containing
25 g/mL of USP Lansoprazole RS and 25 ug/mL of Lansoprazole 1.0 = =
USP Lansoprazole Related Compound B RS in metha- Lansoprazole related
nol. Transfer 1 mL of this solution into a 100-mL volu- compound A(lan-
metric flask, and dilute with Diluent to volume. soprazole sulfone)? 1.1 0.82 0.4
Sample solution: 2.5 mg/mL of Lansoprazole in meth- @ [[(1H-Benzimidazole-2-yl)sulfinyl]methyl]-3-methyl-4-(2,2,2-
anol. Transfer 1 mL of this solution into a 10-mL volu- trifluoroethoxy)-pyridine 1-oxide.
metric flask, and dilute with Diluent to volume. 7oilsMethy! 4 (22/2 tnllucroethoxy -2spyndylimethyt:
Jsulfonyl]benzimidazole.
< 2-[[[3-Methyl-4-(2,2,2-trifluoroethoxy)-pyridin-2-yl]methyl]sulfanyl]-14-
benzimidazole.
 2350 Lansoprazole / Official Monographs USP 41

Table 2 (Continued) Dissolution (711)—Proceed as directed for Procedure for

Relative Relative Acceptance Method A under Apparatus 1 and Apparatus 2, Delayed-Re-
Retention Response Criteria,
lease Dosage Forms.
Name Time Factor NMT (%) ACID STAGE—
Lansoprazole related Acid stage medium: 0.1 N hydrochloric acid; 500 mL.
compound B(lan- — Apparatus 2:75 rpm.
soprazole sulfide): 1.2 0.1 Time: 60 minutes.
Other individual Procedure—Withdraw a 25-mL aliquot and then proceed
impurit _ 1.00 0.1 immediately as directed for Test solution in the Buffer stage,
Total impurities — — 0.6 leaving the remaining 475 mL in the vessel for use in the
@ [[(1H-Benzimidazole-2-yl)sulfinyl]methy!]-3-methyl-4-(2,2,2- Buffer stage. Usinga filtered portion of the aliquot, deter-
trifluoroethoxy)-pyridine 1-oxide. mine the amount of CisHi4F3 N3O2S dissolved by employing
» 2-[[[3-Methyl-4-(2,2,2-trifluoroethoxy)-2-pyridyl]methyl- UV absorption at the wavelength of maximum absorbance
Jsulfonyl]benzimidazole.
at about 306 nm, using Acid stage medium as the blank.
¢ 2-[[[3-Methyl-4-(2,2,2-trifluoroethoxy)-pyridin-2-yl]methy!]sulfanyl]-1 H-
Concomitantly determine the absorbance of the Acid stage
benzimidazole.
test solution in comparison with a Standard solution of USP
SPECIFIC TESTS Lansoprazole RS having a known concentration equivalent
e WATER DETERMINATION, Method Ia (921) to about 8% of the labeled amount of lansoprazole dis-
Sample: 1.09 solved per 500 mL of Acid stage medium. [NOTE—A volume
[Note—Use 50 mL of a dehydrated mixture of pyridine of methanol not to exceed 0.5% of the total volume of the
and ethylene glycol (9:1 to 8:2) as thesolvent Standard solution may be used to dissolve USP Lansoprazole
Acceptance criteria: NMT 0.1% RS prior to dilution with Acid stage medium.]
Tolerances—Not more than 10% of the labeled amount
ADDITIONAL REQUIREMENTS of CigHi4F3N3O2S is dissolved in 60 minutes.
e PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers. Store at room temperature, and protect from
BUFFER STAGE—
excessive heat. Buffer concentrate—Transfer 65.4 g of monobasic sodium
e USP REFERENCE STANDARDS (11) phosphate, 28.2 g of sodium hydroxide, and 12 g of sodium
USP Lansoprazole RS dodecyl sulfate to a suitable container, and add enough
USP Lansoprazole Related Compound A RS water to dissolve. Dilute with water to 4.L, and mix well.
2-[[[3-Methyl-4-(2,2,2-trifluoroethoxy)- Blank solution—Prepare a mixture of Acid stage medium
2-pyridyl]methyl]sulfonyl]benzimidazole. and Buffer concentrate (19:17). Adjust, if necessary, with ei-
CicHiaF3N303S 385.36 ther phosphoric acid or sodium hydroxide to a pH of 6.8.
USP Lansoprazole Related Compound B RS Test solution—Add 425 mL of Buffer concentrate to the re-
2-[[[3-Methyl-4-(2,2,2-trifluoroethoxy)-pyridin-2-yl] maining 475 mL of solution in each vessel from the Acid
methyl]sulfanyl]-1 H-benzimidazole. stage. Adjust, if necessary, with either phosphoric acid or
es
a)
CisHiaF3N30S 353.36 sodium hydroxide to a pH of 6.8.
§ Apparatus 2: 75 rpm.
i]
— Time: 60 minutes.
Dd
° Procedure—Determine the amount of CysH;4F3N302S dis-
= solved in filtered portions of the Test solution, using the dif-
5 Lansoprazole Delayed-Release Capsules ference between the absorbances at the wavelengths of
= about 286 nm and 650 nm, with Blank solution as the
a
2)
» Lansoprazole Delayed-Release Capsules contain blank. Concomitantly determine the absorbances of the Test
> not less than 90.0 percent and not more than solution in comparison with a Standard solution of USP Lan-
soprazole RS having a known concentration equivalent to
110.0 percent of the labeled amount of lan- about 70% of the labeled amount of lansoprazole dissolved
soprazole (CisHi4F3N302S). in 900 mL of Blank solution. [NoTE—An amount of methanol
not to exceed 2% of the total volume of the Standard solu-
Packaging and storage—Preserve in tight containers, and tion may be used to dissolve USP Lansoprazole RS prior to
store at controlled room temperature. dilution with Blank solution.]
USP Reference standards (11)— Tolerances—Not less than 80% (Q) of the labeled amount
USP Lansoprazole RS of CisHi4F3N302S is dissolved in 60 minutes.
CisHiaF3N3028 369.36
USP Lansoprazole Related Compound A RS Uniformity of dosage units (905): meet the require-
2-[[[3-Methyl-4-(2,2,2-trifluoroethoxy)-2-pyridyl]- ments.
methyl]sulfonyl|benzimidazole. PROCEDURE FOR CONTENT UNIFORMITY—
CisHiaF3N303S 385.36 Test solution—Transfer the contents of 1 Capsule to a
Identification— 100-mL volumetric flask, add 30 mL of 0.1 M sodium hy-
A: Ultraviolet Absorption (197U)— droxide, and sonicate to disintegrate. Add 65 mL of acetoni-
trile, cool, and dilute with acetonitrile to volume. Centrifuge
Medium: methanol. a portion of the suspension and pass through a membrane
Procedure—Powder a portion of Capsule contents equiva- filter having a 0.5-um or finer porosity. Quantitatively dilute
lent to 5 mg of lansoprazole. Add 5 mL of methanol, shake a volume of the filtrate with a mixture of acetonitrile and
well, and centrifuge. To 0.1 mL of the supernatant, add 0.1 M sodium hydroxide (7:3) to obtain a solution contain-
10 mL of methanol. ing about 0.012 mg of lansoprazole per mL.
B: The retention time of the major peak in the chromato- Procedure—Concomitantly determine the absorbances of
gram of the Assay preparation corresponds to that in the the Test solution and a solution of USP Lansoprazole RS in
chromatogram of the Standard preparation, as obtained in the same medium and having a known concentration of
the Assay. about 0.012 mg of lansoprazole per mL, in 1-cm cells, at
the wavelength of maximum absorbance at about 294 nm,
with a suitable spectrophotometer, using a mixture of ace-
tonitrile and 0.1 M sodium hydroxide (7:3) as the blank.
USP 41
Calculate the quantity, in mg, of CisHi4F3N302S in the Cap-
sule taken by the formula:
(LC/D)(Au / As)
in which L is the labeled quantity of lansoprazole in the
Capsule; C is the concentration, in mg per mL, of USP Lan-
soprazole RS in the Standard solution; D is the concentra-
tion, in mg per mL, of lansoprazole in the Test solution,
based on the labeled quantity of lansoprazole per Capsule
and the extent of dilution; and Ay and As are the ab-
sorbances of the Test solution and the Standard solution, re-
spectively.
Loss on drying (731)—Dry about 1 g of the Capsule con-
tents in vacuum over phosphorus pentoxide at a pressure
not exceeding 5 mm of mercury at 60° for 5 hours: it loses
not more than 5.0% of its weight.
Assay—
Diluent, Mobile phase, and Resolution solution—Prepare as
directed in the Assay under Lansoprazole.
Internal standard solution—Dissolve an accurately
weighed quantity of 4’-ethoxyacetophenone in acetonitrile
to obtain a solution having a known concentration of about
7.5 mg per mL.
Standard preparation—Dissolve an accurately weighed
Suan of USP Lansoprazole RS in a mixture of 0.1 M so-
ium hydroxide and acetonitrile (3:2) to obtain a solution
having a known concentration of 3.0 mg per mL. Transfer
25.0 mL of this solution and 5.0 mL of Internal standard so-
lution to a 50-mL volumetric flask, dilute with Diluent to vol-
ume, and mix. Quantitatively dilute with Diluent to obtain a
solution having a known concentration of about 0.1 mg of
USP Lansoprazole RS per mL.
Assay preparation—Transfer the contents of not fewer
than 10 Capsules, equivalent to about 300 mg of lan-
soprazole, to a 300-mL conical flask containing 60.0 mL of
0.1 M sodium hydroxide, and sonicate until completely dis-
integrated. Add 20.0 mL of acetonitrile and 20.0 mL of Inter-
nal standard solution, shake well, and centrifuge a portion of
the suspension. Quantitatively dilute a volume of the super-
natant with Diluent to obtain a solution containing about
0.1 mg of lansoprazole per mL, and pass through a mem-
brane filter having a 0.5-1m or finer porosity.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 285-nm detector
and _a 4.6-mm x 25-cm column that contains 5-wm packin
L1. The flow rate is about 1 mL per minute. Chromatograp!
the Resolution solution, and record the peak responses as
directed for Procedure: the resolution, R, between the two
major peaks is not less than 5. Chromatograph the Standard
preparation, and record the peak responses as directed for
Procedure: the relative standard deviation for replicate injec-
tions is not more than 2.0%.
Procedure—Separately inject equal volumes (about 10 ,1L)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
ure the peak responses. Calculate the quantity, in mg, of
es (CisHi4F3N302S) in each Capsule taken by the
formula:
(LC/D)(Ru / Rs)
in whichLis the labeled quantity, in mg, of lansoprazole in
each Capsule taken; C is the concentration, in mg per mL,
of USP Lansoprazole RS in the Standard preparation; D is the
concentration, in mg per mL, of lansoprazole in the Assay
preparation, based on the labeled quantity of lansoprazole in
the Capsules taken and the extent of dilution; and Ry and Rs
are the peak response ratios obtained from the Assay prepa-
ration and the Standard preparation, respectively.
Official Monographs / Lansoprazole 2351
Lansoprazole Compounded Oral
Suspension
DEFINITION
Lansoprazole Compounded Oral Suspension contains NLT
90.0% and NMT 110.0% of the labeled amount of lan-
soprazole (CsHi4F3N302S). Prepare Lansoprazole Com-
pounded Oral Suspension 3 mg/mL as follows (see Phar-
maceutical Compounding—Nonsterile Preparations {795)).
Lansoprazole delayed-release capsule(s)*
equivalent to 300 mg
Vehicle: A mixture of Ora-Blend® and Sodium
Bicarbonate Injection (8.4%) (1:1), a suffi-
cient quantity to make 100 mL
Lansoprazole 30-mg delayed-release capsules, Dr. Reddy‘s Laboratory
Limited, Bridgewater, NJ.
>Perrigo Pharmaceuticals, Allegan, MI.
Empty the required number of delayed-release capsules, and
pour the contents into a mortar or other suitable con-
tainer. If necessary, crush the contents into a fine powder
by using a pestle or other mechanical means. Wet the
powder with a small amount of Vehicle, and triturate to
make a smooth paste. Add Vehicle to make the mortar
contents pourable. Transfer the contents stepwise and
uantitatively to a calibrated container using the remain-
ler of Vehicle. Add sufficient Vehicle to bring to final vol-
ume. Shake to mix well.
Alternatively, a compounded 8.4% sodium bicarbonate solu-
tion may be used instead of Sodium Bicarbonate Injection
(8.4%). Prepare an 8.4% sodium bicarbonate solution by
dissolving 8.4 g of Sodium Bicarbonate in sufficient Puri-
fied Water to make 100 mL.
ASSAY
¢ PROCEDURE
res
nn
i)
Solution A: 10 mM sodium phosphate adjusted with
sodium hydroxide to a pH of 7.5. Pass through a nylon
x
filter of 0.45-1m pore size, and degas. 5
Solution B: Acetonitrile and water (50:50) Ss
Solution C: Water adjusted with 1M sodium hydroxide )
to a pH of 6.5
a=
Mobile phase: Acetonitrile and Solution A (45:55) y
i}
Standard stock solution: 3 mg/mL of USP Lansoprazole a
RS in Solution B. Mix well, and sonicate for 3 min. Store a]
at 2°-8°.
Standard solution: Transfer 2.0 mL of the Standard
stock solution to a 500-mL volumetric flask, and dilute
with Solution C to volume. Centrifuge an aliquot of the
solution for 5 min at 14,000 rpm, and use the superna-
tant. Protect from light, and store at 2°-8°.
Sample solution: Shake each bottle of Oral Suspension
thoroughly. Transfer 2.0 mL of Oral Suspension to a
500-mL volumetric flask, and dilute with Solution C to
volume. Centrifuge an aliquot of the solution for 5 min
at 14,000 rpm, and use the supernatant. Protect from
light, and store at 2°-8°.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 285 nm
Column: 4.6-mm x 25-cm; 5-"um packing L1
Temperatures
Column: 35°
Autosampler: 5°
Flow rate: 1.0 mL/min
Injection volume: 20 UL
System suitability
Sample: Standard solution
[Note—The retention time for lansoprazole is about 5.2
min.]
 2352 Lansoprazole / Official Monographs USP 41

Suitability requirements ASSAY

Tailing factor: NMT 2.0 © PROCEDURE
Relative standard deviation: NMT 2.0% for replicate Mobile phase: Chromatographic hexane and dehy-
injections drated alcohol (94:6)
Analysis System suitability solution: 2.0 mg/mL of USP Lata-
Samples: Standard solution and Sample solution noprost RS and 20 g/mL of USP Latanoprost Related
Calculate the percentage of the labeled amount of lan- CompoundA RS prepared as follows. Transfer USP Lata-
soprazole (CisHi4FsN3O02S) in the portion of Oral Sus- noprost RS and USP Latanoprost Related Compound A
pension taken: RS into a suitable volumetric flask, dissolve in dehy-
drated alcohol equivalent to 20% of the final volume,
Result = (ru/rs) x (Cs/Cu) x 100 and dilute with chromatographic hexane to volume.
Standard solution: Transfer 2.0 mg/mL of USP Lata-
i = peak response of lansoprazole from the Sample noprost RS into a suitable volumetric flask, dissolve in
solution dehydrated alcohol equivalent to 20% of the final vol-
Is = peak response of lansoprazole from the ume, and dilute with chromatographic hexane to
Standard solution volume.
Cs = concentration of lansoprazole in the Standard Sample solution: Transfer 2.0 mg/mL of Latanoprost
solution (mg/mL) into a suitable volumetric flask, dissolve in dehydrated
Cc = nominal concentration of lansoprazole in the alcohol equivalent to 20% of the final volume, and di-
Sample solution (mg/mL) lute with chromatographic hexane to volume.
Acceptance criteria: 90.0%-110.0% Chromatographic system
(See Chromatography (621), System Suitability.)
SPECIFIC TESTS Mode: LC
e PH (791): 8.0-8.5 Detector: UV 210 nm
ADDITIONAL REQUIREMENTS Column: 4.0-mm x 25-cm; 5-um packing L3
e PACKAGING AND STORAGE: Package in tight, light-resistant Column temperature: 30°
containers. Store at 2°-8° or at controlled room Flow rate: 1 mL/min
temperature. Injection volume: 10 pL
e LABELING: Label Oral Suspension to indicate that it is to System suitability
be well shaken before use, and to state the Beyond-Use Samples: System suitability solution and Standard
Date. solution
© BEYOND-UsE DATE: NMT 90 days after the date on which [Note—The relative retention times for latanoprost and
it was compounded, when stored at 2°-8° or at con- latanoprost related compound A are 1.0 and 1.1,
trolled room temperature respectively.]
e USP REFERENCE STANDARDS (11) Suitability requirements
USP Lansoprazole RS Resolution: NLT 2.0 between latanoprost and lata-
noprost related compound A, System suitability
=
“
solution
a Relative standard deviation: NMT 1.0%, Standard
6S
—
solution
D Latanoprost
Analysis
° Samples: Standard solution and Sample solution
iS Calculate the percentage of latanoprost (C2sH4oOs) in
iS oh the portion of Latanoprost taken:
= Jk
a
al
Result = (ru/rs) x (Cs/Cu) x 100
= HO. ol
OE y tu = peak area from the Sample solution
Is = peak area from the Standard solution
Cs = concentration of USP Latanoprost RS in the
ns én Standard solution (mg/mL)
Cy = concentration of Latanoprost in the Sample
CogHaoOs 432.59 solution (mg/mL)
5-Heptenoic acid, 7-[3,5-dihydroxy-2-(3-hydroxy- Acceptance criteria: =94.0%-102.0% on the anhydrous
5-phenylpentyl)cyclopentyl]-1-methylethy! ester, [1R- and solvent-free basis
(102),2B(R*), 301,501]; IMPURITIES
Isopropyl (Z)-7-[(1 R,2R,3R,55S)-3,5-dihydroxy-2-[(3R)-3-hy-
droxy-5-phenylpentyl]cyclopentyl]-5-heptenoate e RESIDUE ON IGNITION (281):NMT 0.50%
[130209-82-4]. e ORGANIC IMPURITIES
Mobile phase, System suitability solution, Sample so-
DEFINITION lution, and Chromatographic system: Proceed as di-
Latanoprost contains NLT 94.0% and NMT 102.0% of lata- rected in the Assay.
noprost (C26H40Os), calculated on the anhydrous and sol- Standard solution: 0.04 mg/mL of USP Latanoprost RS
vent-free basis. in a mixture of chromatographic hexane and dehy-
[CauTION—Wear protective glasses and gloves while han- drated alcohol (80:20) prepared as follows. Transfer USP
dling the material. Avoid contact during pregnancy or Latanoprost RS into a suitable volumetric flask, dissolve
while nursing.] in dehydrated alcohol equivalent to 20% of the final
volume, and dilute with chromatographic hexane to
IDENTIFICATION volume.
e A. INFRARED ABSORPTION (197F) System suitability
e B. The retention time of the major peak of the Sample Samples: System suitability solution and Standard
solution corresponds to that of the Standard solution, as solution
obtained in the Assay.
USP 41 Official Monographs / Leflunomide 2353

Suitability requirements Sample solution: 1.0 mg/mL of Latanoprost in Diluent

Resolution: NLT 2.0 between latanoprost and lata- Chromatographic system
noprost related compound A, System suitability (See Chromatography (621), System Suitability.)
solution Mode: LC
Relative standard deviation: NMT 5.0%, Standard Detector: UV 200 nm
solution Column: 4.0-mm x 15-cm; 5-um packing L1
Analysis Column temperature: 60°
Samples: Standard solution and Sample solution Flow rate: 1.0 mL/min
Calculate the percentage of each impurity in the por- Injection volume: 50 LL
tion of Latanoprost taken: System suitability
Sample: Standard solution
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100 Suitability requirements
Relative standard deviation: NMT 5.0%
ty = peak area of each impurity from the Sample Analysis
solution Samples: Standard solution and Sample solution
rs = peak area of latanoprost from the Standard Calculate thepercentage of latanoprost related com-
solution poundEin the portion of Latanoprost taken:
Cs = concentration of USP Latanoprost RS in the
Standard solution (mg/mL) Result = (ru/rs) x (Cs/Cu) x 100
Cu = concentration of Latanoprost in the Sample
solution (mg/mL) tu = peak area of latanoprost related compound E
F = relative response factor for each individual from the Sample solution
impurity (see Table 1) rs = peak area of latanoprost related compound E
Acceptance criteria: See Table 1. Disregard any impu- from the Standard solution
rity peak less than 0.05%. Cs = concentration of USP Latanoprost Related
CompoundERS in the Standard solution
Table 1 (mg/mL)
Cy = concentration of Latanoprost in the Sample
Relative Relative Acceptance solution (mg/mL)
Retention | Response Criteria, Acceptance criteria: NMT 0.2%
Name Time Factor NMT (%)
Isopropyl! diphenylphos- SPECIFIC TESTS
phorylpentanoate? 0.79 2.4 0.1 e OPTICAL ROTATION, Specific Rotation (781S)
Latanoprost related Sample solution: 10 mg/mL of Latanoprost in
compound B> 0.89 1.0 0.5 acetonitrile
Acceptance criteria: +31° to +38°
Latanoprost 1.00 = e WATER DETERMINATION, Method Ic (921)
Latanoprost related Sample solution: 100 mg/mL of Latanoprost in ethyl
compound A‘ 1.10 1.0 3.5) =
acetate, [NoTE—Alternatively, Water Determination a)
Any unspecified may (921), Method la may be used.] z
impurity 1.0 0.1 Acceptance criteria: NMT 2.0% =
Total impurities¢ = = 0.5 i}
ADDITIONAL REQUIREMENTS 3
? Isopropyl 5-(diphenylphosphoryl)pentanoate.
© PACKAGING AND STORAGE: Preserve in well-closed, light- i)
» Isopropyl (Z)-7-[(1 R,2R,3R,55)-3,5-dihydroxy-2-[(35)-3-hydroxy-5-
phenylpentyl]cyclopenty!]-5-heptenoate. resistant containers, and store in a refrigerator or a To)S
freezer. ESS)
¢lsopropyl (F)-7-[(1R,2R,3R,55)-3,5-dihydroxy-2-[(3R)-3-hydroxy-5- 3
phenylpenty!}cyclopentyl]-5-heptenoate. e USP REFERENCE STANDARDS (11) =e
4Latanoprost related compound A and latanoprost related compound B USP Latanoprost RS “

are excluded. USP Latanoprost Related Compound A RS

Koppel HTC R,2R,3R,55S)-3,5-dihydroxy-2-[(3R)-
© Limit OF LATANOPROST RELATED COMPOUND E 3-hydroxy-5-phenylpentyl]cyclopentyl}-5-heptenoate.
Solution A: Acetonitrile, phosphoric acid, and water CasHaoOs 432.59
(300:1:700) USP Latanoprost Related Compound E RS
Solution B: Acetonitrile, phosphoric acid, and water (Z)-7-{(1 R,2R, 3R,5S)-3,5-Dihydroxy-2-[(3R)-3-hydroxy-
(800:1:200) 5-phenylpentyl]cyclopentyl}-5-heptenoic acid.
Mobile phase: See Table 2. C23H3405 390.51
Table 2
Solution A Solution B

100 0 Leflunomide
1

ort
0

100
/\,
FOF

Diluent: Acetonitrile and water (30:70) Cy2HoF3N202 270.21

Standard solution: 1.0 g/mL of USP Latanoprost Re- 4-lsoxazolecarboxamide, 5-methyl-N-[4-(trifluoromethyl)-
lated CompoundE RS in Diluent
phenyil-;
o,a,0-Trifluoro-5-methyl-4-isoxazolecarboxy-p-toluidide
[75706-12-6].
 2354 Leflunomide / Official Monographs USP 41

DEFINITION Standard solution: 0.5 g/mL of USP Leflunomide Re-

Leflunomide contains NLT 98.0% and NMT 102.0% of lated Compound ARS, from the Standard stock solution
Ci2HeF3N2O2, calculated on the dried basis. in Mobile phase
Sample solution: 2.5 mg/mL of Leflunomide, in aceto-
IDENTIFICATION nitrile and Mobile phase (1:9)
e A. INFRARED ABSORPTION (197K) Injection size: 20 uL
Sample: Dry the substance for 10 min at 130°. Analysis
e B, The retention time of the major peak of the Sample Samples: Standard solution and Sample solution
solution corresponds to that of the Standard solution, as Calculate the percentage of leflunomide related com-
obtained in the Assay. poundAin the portion of Leflunomide taken:
ASSAY Result = (ru/rs) x (Cs/Cu) x 100
© PROCEDURE
Mobile phase: Acetonitrile, triethylamine, and water tu = peak area of leflunomide related compound
(70:1:130). Adjust with phosphoric acid to a pH of 4. A from the Sample solution
Standard solution: 0.5 mg/mL of USP Leflunomide RS Is = peak area of leflunomide related compound
in acetonitrile and Mobile phase (1:9). [NoTE—First dis- A from the Standard solution
solve in acetonitrile. Protect solutions from light.] Gg = concentration of USP Leflunomide Related
System suitability solution: 0.5 maim. of USP Leflu- CompoundA RS in the Standard solution
nomide RS, 0.15 mg/mL of USP Leflunomide Related (mg/mL)
Compound B RS, and 0.05 mg/mL of USP Leflunomide Cu = concentration of Leflunomide in the Sample
Related CompoundC RS in Mobile phase. [NoTe—Dis- solution (mg/mL)
solve the Reference Standards in acetonitrile, and dilute Acceptance criteria: NMT 0.02 %
with Mobile phase.] © PROCEDURE 2
Sample solution: 0.5 mg/mL of Leflunomide in acetoni- Mobile phase, Sample solution, System suitability so-
trile and Mobile phase (1:9). [NoTE—First dissolve in ace- lution, and Chromatographic system: Proceed as di-
tonitrile. Protect solutions from light.] rected in the Assay.
Chromatographic system Standard solution: 0.5 g/mL of USP Leflunomide RS,
(See Chromatography (621), System Suitability.) from the Standard solution in Mobile phase
Mode: LC Sensitivity solution: 0.25 g/mL of Leflunomide, from
Detector: UV 210 nm the Standard solution in Mobile phase
Column: 4-mm x 12.5-cm; packing L1 System suitability
Flow rate: 1 mL/min Samples: System suitability solution and Sensitivity
Injection size: 20 uL solution
System suitability Resolution: NLT 1.0 between leflunomide and leflu-
Sample: System suitability solution nomide related compound C
[Note—The relative retention times for leflunomide re- Signal-to-noise ratio: NLT 10, Sensitivity solution
a lated compoundB and leflunomide related compound Analysis
= C are 0.2 and 0.9, respectively.] Samples: Standard solution and Sample solution
ra Suitability requirements [NoTr—Disregard any peak with an area less than the
J
_ Resolution: NLT 1.0 between the leflunomide and leflunomide peak from the Sensitivity solution. Con-
aD
i} leflunomide related compound C peaks tinue the elution for two times the retention time of
i= Analysis the leflunomide peak.]
GS Samples: Standard solution and Sample solution Calculate the percentage of each related compound
= Calculate the percentage of CizH»F3sN2Oz in the portion and any unknown impurity (see Impurity Table 1) in
ian of Leflunomide taken: the portion of Leflunomide taken:
a
=) Result = (ru/ts) x (Cs/Cu) x 100 Result = (ru/rs) x (Cs/Cu) x 100
ty = peak response from the Sample solution tu = peak area for each impurity from the Sample
Is = peak response from the Standard solution solution
Cs = concentration of USP Leflunomide RS in the Is = peak area of leflunomide from the Standard
Standard solution (mg/mL) solution
Cu = nominal concentration of Leflunomide in the Cs = concentration of USP Leflunomide RS in the
Sample solution (mg/mL) Standard solution (mg/mL)
Acceptance criteria: 98.0%-102.0% on the dried basis Cu = concentration of Leflunomide in the Sample
solution (mg/mL)
IMPURITIES
Inorganic Impurities
e RESIDUE ON IGNITION (281): NMT 0.1% Impurity Table 1
Relative Relative Acceptance
Delete the following: Retention Response Criteria,
Name Time Factor NMT (%)

°e Heavy METALS, Method If (231): NMT 20 ppme cortical

1- 5-Methylisoxazole-
Jan-2018) carboxylic acid 0.05 1.0 0.1
Organic Impurities Leflunomide related
e PROCEDURE 1: LIMIT OF LEFLUNOMIDE RELATED COMPOUND A compound B 0.22 1.0 0.3
Mobile phase, System suitability solution, and Chro- N-(2’-Trifluoromethyl-
matographic system: Proceed as directed in the phenyl)-5-
Assay. methylisoxazole-4-
Standard stock solution: 0.125 mg/mL of USP Leflu- Carboxamide 0.29 1.0 0.1
nomide Related Compound A RS, in acetonitrile and 2-Cyano-acetic acid-
Mobile phase (1:19) (4’-trifluoromethyl)-
anilide 0.36 1.0 0.1
USP 41 Official Monographs / Leflunomide 2355

Impurity Table 1 (Continued) Chromatographic system

Relative Relative Acceptance
(See Chromatography (621), System Suitability.)
Retention Response Criteria,
Mode: LC
Name Time Factor NMT (%) Detector: UV 210 nm
Column: 4.0-mm x 12.5-cm; packing L1
Leflunomide related Flow rate: 1 mL/min
compound C 0.94 1.0 0.1 Injection size: 10 pL
Any other individual System suitability
impurity ae tae 0.1 Samples: System suitability solution B and Standard
Total impurities, solution
excluding [Note—The relative retention times for leflunomide re-
leflunomide related lated compound B, leflunomide related compound A,
compound B and _ ~ leflunomide related compound C, and leflunomide are
leflunomide related 0.2, 0.4, 0.9, and 1.0, respectively.]
compound C 0.2 Suitability requirements
Total impurities = — 0.4 Resolution: NLT 1.5 between leflunomide related
compoundC and leflunomide, System suitability solu-
tion B
SPECIFIC TESTS Tailing factor: NMT 3.0 for leflunomide, Standard
© MELTING RANGE OR TEMPERATURE (741): 164°-168° solution
e Loss ON DRYING (731): Dry a sample in a vacuum over Relative standard deviation: NMT 2.0%, Standard
diphosphorus pentoxide at 60° for 4 h: it loses NWT solution
0.5% of its weight. Analysis
Samples: Standard solution and Sample solution
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in a well-closed con- Calculate the percentage of the labeled amount of leflu-
nomide (C;2HeF3N202) in the portion of Tablets taken:
tainer. Store at a temperature not exceeding 30°.
e USP REFERENCE STANDARDS (11) Result = (ru/rs) x (Cs/Cu) x 100
USP Leflunomide RS
USP Leflunomide Related Compound A RS tu = peak response from the Sample solution
USP Leflunomide Related Compound B RS ls = peak response from the Standard solution
USP Leflunomide Related Compound C RS Cs = concentration of USP Leflunomide RS in the
Standard solution (mg/mL)
Cu = nominal concentration of leflunomide in the
Sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0%
Leflunomide Tablets
PERFORMANCE TESTS (ox
DEFINITION e DISSOLUTION (711) a)
Leflunomide Tablets contain NLT 90.0% and NMT 110.0% Test 1 a]
of the labeled amount of leflunomide (Ci2HeF3N202). Medium =
For Tablets labeled to contain 10 or 20mg: Water, °
IDENTIFICATION 1000 mL, deaerated J
© A. ULTRAVIOLET ABSORPTION (197U) For Tablets labeled to contain 100 mg: Water con- i)
Wavelength range: 220-360 nm taining 0.6% of peyopetivicrie (23) lauryl ether;
ro}=
EY
Sample solution: 0.01 mg/mL in methanol 1000 mL, deaerate a}
e B. The retention time of the major peak of the Sample Apparatus 2: 100 rpm =
solution corresponds to that of the Standard solution, as Time: 30 min “

obtained in the Assay. Determine the amount of leflunomide (Ci2HeF3N202)

ASSAY
© PROCEDURE
Mobile phase: Acetonitrile, triethylamine, and water
(70:1:130). Adjust with phosphoric acid to a pH of 4.0.
System suitability solution A: 10 j1g/mL of USP Leflu-
nomide Related Compound A RS, 1 mg/mL of USP
Leflunomide Related Compound B RS, and 100 g/mL
of USP Leflunomide Related Compound C RS in a mini-
mum amount of acetonitrile, and diluted with Mobile
phase
System suitability solution B: Transfer 100.0 mg of
USP Leflunomide RS to a 100-mL volumetric flask. Dis-
solve in 2 mL of acetonitrile, add 1 mL of System suita-
bility solution A and 80 mL of Mobile phase, and shake
by mechanical means for 10 min. Dilute with Mobile
phase to volume.
Standard solution: 1 mg/mL of USP Leflunomide RS in
a minimum volume of acetonitrile, and diluted with
Mobile phase
Sample solution: Transfer equivalent to 100 mg
leflunomide, from finely powdered Tablets (NLT 20), to
a 100-mL volumetric flask. Add 20 mL of acetonitrile,
dilute with Mobile phase to volume, and shake by me-
chanical means for 10 min. Pass through a membrane
filter.
dissolved by using one of the following methods.
Spectrometric method
See Ultraviolet-Visible Spectroscopy (857).)
Mode: UV
Analytical wavelength: 262 nm
Standard solution: USP Leflunomide RS in Medium.
[NoTte—A volume of methanol not exceeding 2% of
the final volume of the Standard solution may be used
to dissolve leflunomide.]
Sample solution: Pass a portion of the solution under
test en a suitable filter of 0.45-1um pore size.
Dilute with Medium to a concentration that is similar
to that of the Standard solution, if necessary.
Chromatographic method
Mobile phases Acetonitrile and water (1:1)
Standard solution: Transfer 22 mg of USP Leflu-
nomide RS to a 100-mL volumetric flask. Add 40 mL
of acetonitrile, and sonicate until dissolved. Add
40 mL of water, and cool to room temperature. Di-
lute with water to volume. Transfer 10.0 mL of this
of solution to a 100-mL volumetric flask, and dilute with
water to volume.
Sample solution: Use portions of the solution under
test passed through a suitable filter of 0.45-11m pore
size.
 2356 Leflunomide / Official Monographs USP 41

Chromatographic system Acceptance criteria

(See Chromatography (621), System Suitability.) Leflunomide related compound A: NMT 0.1%
Mode: LC Leflunomide related compound B: NMT 3.5%
Detector: UV 260 nm Leflunomide related compound C: NMT 0.2%
Column: 4.6-mm x 15-cm; 5-m packing L1 Individual impurities: NMT 0.2%
Flow rate: 1.5 mL/min Total impurities; NMT 4.0%
Injection size: 40 wL
System suitability ADDITIONAL REQUIREMENTS
Sample: Standard solution e PACKAGING AND STORAGE: Preserve in tight, light-resistant,
Suitability requirements and humidity-resistant containers.
Tailing factor: NMT 2.0 e LABELING: When more than one Dissolution test is given,
Relative standard deviation: NMT 2.0% the labeling states the Dissolution test used only if Test 7
Analysis is not used.
Samples: Standard solution and Sample solution e USP REFERENCE STANDARDS (11)
Calculate the amount of leflunomide (Ci2H9F3N202) USP Leflunomide RS
dissolved: USP Leflunomide Related Compound A RS
USP Leflunomide Related Compound B RS
Result = (ru/ts) x (Cs/L) x Vx 100 USP Leflunomide Related Compound C RS
ru = peak response from the Sample solution
rs = peak response from the Standard solution
Gs = concentration of USP Leflunomide RS in the
Standard solution (mg/mL) Letrozole
L = label claim (mg/Tablet
Vv = volume of Medium, 1000 mL
Tolerances: NLT 80% (Q) of the labeled amount of
leflunomide (Ci2H9F3N20z) is dissolved.
Test 2: If the product complies with this test, the label-
ing indicates that the product meets USP Dissolution
Test 2.
Medium, Apparatus 2, Time, Spectrometric method,
and Chromatographic method: Proceed as directed
for Test 1. Cy7HiiNs 285.30
Tolerances: NLT 75% (Q) of the labeled amount of Benzonitrile, 4,4’-(1 H-1,2,4-triazol-1-ylmethylene)bis-;
leflunomide (Ci2H9F3N202) is dissolved. 4,4’-(1H-1,2,4-Triazol-1-ylmethylene)dibenzonitrile
e UNIFORMITY OF DOSAGE UNITS (905): Meet the [112809-51-5].
requirements DEFINITION
Procedure for content uniformity
fe
“
Letrozole contains NLT 98.0% and NMT 102.0% of
a Mobile phase, System suitability solution A, System CizHiiNs, calculated on the anhydrous basis.
Ss suitability solution B, Standard solution, Chromato-
J
Dp graphic system, and Analysis: Proceed as directed in IDENTIFICATION
° the Assay. e A. INFRARED ABSORPTION (197M)
rs Sample solution: Transfer 1 Tablet to a suitable volu- e B. The retention time of the major peak of the Sample
Sj metric flask, and prepare a solution having a concen- solution corresponds to that of the Standard solution, as
= tration of 1 mg/mL of leflunomide. Add Mobile phase obtained in the Assay.
a 50% by volume, and shake to disintegrate the Tablet.
Al
After the Tablet is completely disintegrated, add aceto- ASSAY
>
nitrile 20% by volume, dilute with Mobile phase to vol- © PROCEDURE
ume, and shake again. Pass through a membrane Solution A: Water
filter. Solution B: Acetonitrile
Mobile phase: See the gradient table below.
IMPURITIES
e PROCEDURE Time Solution A Solution B
Mobile phase, System suitability solution A, System
(min) (%) (%)
suitability solution B, Standard solution, Sample so-
lution, and Chromatographic system: Proceed as di- 0 70 30
rected in the Assay. 25 30 70
Analysis
Samples: Standard solution and Sample solution Diluent: Acetonitrile and water (3:7)
Calculate the percentage of each individual impurity in Standard solution: 10 g/mL of USP Letrozole RS in
the portion of Tablets taken: Diluent. [NoTE—Dissolve USP Letrozole RS in acetoni-
trile, then dilute with water.]
Result = (ru/rz) x 100 Sample solution: 10 g/mL of Letrozole in Diluent.
[Note—Dissolve Letrozole in acetonitrile, then dilute
fu = peak response of each individual impurity with water.]
from the Sample solution Chromatographic system
Ir = sum of all the peak responses of the related (See Chromatography (621), System Suitability.)
compounds and leflunomide from the Mode: LC
Sample solution Detector: UV 230 nm
Column: 4,6-mm x 12.5-cm; 5-1um packing L1
Flow rate: 1 mL/min
Injection size: 20 wL
USP 41 Official Monographs / Letrozole 2357

System suitability Impurity Table 1

Sample: Standard solution
Suitability requirements Relative Acceptance
Retention Criteria,
Tailing factor: 0.8-1.5
Name Time NMT (%)
Relative standard deviation: NMT 2.0%
Analysis Letrozole related compound
Samples: Standard solution and Sample solution he 0.67 0.3
Calculate the percentage of Cy7HiiNs in the portion of Letrozole 1.0 =
Letrozole taken: 4,4',4"-Methanetriyl-
tribenzonitrile 24 0.2
Result = (ru/rs) x (Cs/Cu) x 100 Any unspecified impurity _ 0.1
2 4,4’-(1H-1,3,4-triazol-1-ylmethylene)dibenzonitrile.
tu = peak response from the Sample solution
ts = peak response from the Standard solution [Not&—Disregard any impurity peaks less than 0.05%.]
Cs = concentration of USP Letrozole RS in the
Standard solution (mg/mL) SPECIFIC TESTS
Cu = nominal concentration of letrozole in the © WATER DETERMINATION, Method | (921): NMT 0.3%
Sample solution (mg/mL)
Acceptance criteria: 98.0%-102.0% on the anhydrous ADDITIONAL REQUIREMENTS
basis © PACKAGING AND STORAGE: Preserve in tight containers at
controlled room temperature.
IMPURITIES © USP REFERENCE STANDARDS (11)
Inorganic Impurities USP Letrozole RS
e RESIDUE ON IGNITION (281): NMT 0.1% USP Letrozole Related Compound A RS
4,4’-(1H-1,3,4-Triazol-1-ylmethylene)dibenzonitrile.
GizHuNs 285.31
Delete the following:

°e HEAVY METALS, Method II (231): 10 PPMee (official 1-Jon-2078)

Organic Impurities
e PROCEDURE
Solution A, Solution B, Mobile phase, Chromato-
Letrozole Tablets
graphic system, and Diluent: Proceed as directed in
the Assay. DEFINITION
System suitability solution: 2 g/mL of USP Letrozole Letrozole Tablets contain NLT 95.0% and NMT 105.0% of
Related Compound A RS and 10 g/mL of USP Le- the labeled amount of letrozole (C)7HiiNs).
trozole RS in Diluent. [NoTE—Dissolve Letrozole and IDENTIFICATION
USP Letrozole Related CompoundARS in acetonitrile, e A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
then dilute with water.] (201) (os
“
Standard solution: 1 g/mL of USP Letrozole RS in Dil- Sample solution: Equivalent to 2 mg/mL of letrozole i)
uent. [NoTtE—Dissolve USP Letrozole RS in acetonitrile, from powdered Tablets in methanol. [NoTe—Shake
then dilute with water.] s
Sample solution: Transfer 25 mg of Letrozole to a
thoroughly, sonicate for 10 min, and centrifuge.] i}
Application volume: 5 ul mS
250-mL volumetric flask. Dissolve in 75 mL of acetoni- Developing solvent system: Ethyl acetate and metha- i}
trile, and dilute with water to volume. nol (9:1)
ro}=
System suitability Acceptance criteria: Meet the requirements iy
Samples: System suitability solution and Standard a}
solution
e B. The retention time of the major peak of the Sample =
al
solution corresponds to that of the Standard solution, as
Suitability requirements obtained in the Assay.
Resolution: NLT 2.0 between letrozole related com-
pound A and letrozole, System suitability solution ASSAY
Relative standard deviation: NMT 10.0%, Standard ¢ PROCEDURE
solution Mobile phase: Acetonitrile and water (48:52)
Analysis Diluent: Acetonitrile and water (30:70)
Samples: Standard solution and Sample solution Standard stock solution: 0.2 mg/mL of USP Letrozole
Calculate the percentage of each impurity in the por- RS in Diluent. [NotE—Dissolve letrozole in acetonitrile,
tion of Letrozole taken: and then dilute with water.]
Standard solution: 10 g/mL of USP Letrozole RS in
Result = (ru/rs) x (Cs/Cu) x 100 Mobile phase from the Standard stock solution
Sample stock solution: Equivalent to 50 mg of le-
tu = peak response of each individual impurity trozole from Tablets in a 250-mL volumetric flask. Add
from the Sample solution 20 mL of water and shake for 5 min to dissolve the
Is = peak response of letrozole from the Standard Tablets. Add 75 mL of acetonitrile, shake for 30 min,
solution and dilute with water to volume. Centrifuge a portion
Cs = concentration of USP Letrozole RS in the of the solution.
Standard solution (mg/mL) Sample solution: 10 tg/mL of letrozole in Mobile phase
Cu = concentration of Letrozole in the Sample from the Sample stock solution
solution (mg/mL) Chromatographic system
Acceptance criteria (See Chromatography (621), System Suitability.)
Individual impurities: See Impurity Table 1. Mode: LC
Total unspecified impurities: NMT 0.3% Detector: UV 230 nm
Column: 4.6-mm x 12.5-cm; 5-um packing L1
Flow rate: 1 mL/min
Injection volume: 20 uL
 2358 Letrozole / Official Monographs USP 41

System suitability System suitability

Sample: Standard solution Sample: Standard solution
Suitability requirements Suitability requirements
Tailing factor: 0.8-1.5 Tailing factor: NMT 2
Relative standard deviation: NMT 2.0% Relative standard deviation: NMT 2.0%
Analysis Analysis
Samples: Standard solution and Sample solution Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of le- Calculate the percentage of the labeled amount of le-
trozole (Ci7HiiNs) in the portion of Tablets taken: trozole (CizHi1Ns) dissolved:
Result = (ru/rs) x (Cs/Cu) x 100 Result = (ru/rs) x (Cs/L) x Vx 100

Tu = peak response from the Sample solution tu = peak response from the Sample solution
Is = peak response from the Standard solution Is = peak response from the Standard solution
Cs = concentration of USP Letrozole RS in the Cs = concentration of USP Letrozole RS in the
Standard solution (g/mL) Standard solution (mg/mL)
Cu = nominal concentration of letrozole in the L = label claim (mg/Tablet)
Sample solution (yug/mL) Vv = volume of Medium, 900 mL
Acceptance criteria: 95.0%-105.0% Tolerances: NLT 80% (Q) of the labeled amount of
letrozole (Ci7H1iNs) is dissolved.
PERFORMANCE TESTS Test 3
e DISSOLUTION (711) Medium: 0.1 N hydrochloric acid; 500 mL
Test 1 Apparatus 2: 75 rpm
Medium: 0.1 N hydrochloric acid; 500 mL Time: 30 min
Apparatus 2: 100 rpm Mobile phase: Acetonitrile and water (48:52)
Time: 30 min Standard stock solution: 0.25 mg/mL of USP Le-
Standard solution: Transfer USP Letrozole RS to a suit- trozole RS in Mobile phase
able volumetric flask, dissolve in acetonitrile equivalent Standard solution: 0.005 mg/mL of USP Letrozole RS
to 10% of the final volume, and dilute with Medium to in Medium from the Standard stock solution
volume to obtain a solution of 0.05 mg/mL of le- Sample solution: Pass a portion of the solution under
trozole. Dilute this solution with Medium to obtain a test throughasuitable filter of 0.45-11m pore size and
solution of 0.005 mg/mL of letrozole. discard the first few mL of the filtrate.
Sample solution: Centrifuge a portion of the solution Chromatographic system
under test at 4000 rpm for 5 min. (See Chromatography (621), System Suitability.)
Mobile phase and Chromatographic system: Proceed Mode: LC
as directed in the Assay, except use an injection vol- Detector: UV 230 nm
ume of 200 pL. Column: 4.6-mm x 15-cm; 5-um packing L1
“ Analysis Flow rate: 1 mL/min
a Samples: Standard solution and Sample solution Injection volume: 50 uL
is Calculate the percentage of the labeled amount of le- System suitability
6
- trozole (Ci7H11Ns) dissolved: Sample: Standard solution
io.)
co) Suitability requirements
i= Result = (ru/rs) x (G/L) x Vx 100 Tailing factor: 0.8-1.5
iS Relative standard deviation: NMT 2.0%
= Tu = peak response from the Sample solution Analysis
3 Is = peak response from the Standard solution Samples: Standard solution and Sample solution
a) Cs = concentration of USP Letrozole RS in the Calculate the percentage of the labeled amount of le-
=) Standard solution (mg/mL) trozole (Ci7H1\Ns) dissolved:
L = label claim (mg/Tablet
Vv = volume of Medium, 500 mL Result = (ru/rs) x (Cs/L) x Vx 100
Tolerances: NLT 80% (Q) of the labeled amount of
letrozole (CizH11Ns) is dissolved. ty = peak response from the Sample solution
Test 2 Ts peak response from the Standard solution
Medium: 0.1 N hydrochloric acid solution adjusted Cs concentration of USP Letrozole RS in the
with 50% sodium hydroxide (NaOH) to a pH of 1.2; Standard solution (mg/mL)
900 mL, deaerated L = label claim (mg/Tablet,
Apparatus 2: 75 rpm V = volume of Medium, 500 mL
Time: 30 min Tolerances: NLT 80% (Q) of the labeled amount of
Mobile phase: Acetonitrile and water (45:55) letrozole (Ci7H1iNs) is dissolved.
Standard stock solution: 0.3 mg/mL of USP Letrozole e UNIFORMITY OF DosAGE UNITS (905): Meet the
RS in Mobile phase requirements
Standard solution: 3.0 tug/mL of USP Letrozole RS in
Medium from the Standard stock solution IMPURITIES
Sample solution: Pass a portion of the solution under © ORGANIC IMPURITIES
test through a suitable filter of 35-um pore size. Solution A: Water
Chromatographic system Solution B: Acetonitrile
(See Chromatography (621), System Suitability.) Mobile phase: See Table 7.
Mode: LC
Detector: UV 230 nm Table 1
Column: 4.6-mm x 15-cm; 5-um packing L1
Flow rate: 1 mL/min Time Solution A Solution B
Injection volume: 100 uL (min) (%) (%)
0 70 30
25 30 70
USP 41 Official Monographs / Leucine 2359

Diluent: Prepare as directed in the Assay.

System suitability solution: 10 1g/mL of USP Letrozole
RS and 2 g/mL of USP Letrozole Related Compound A
RS in Diluent. [NoTE—Dissolve letrozole and letrozole re- Leucine
lated compoundAin acetonitrile, then dilute with
water.]
Standard solution: 1 ug/mL of USP Letrozole RS in Dil- HyC. I on
uent. [NoTE—Dissolve letrozole in acetonitrile, then di-
lute with water.] ha be
Sample solution: Nominally 0.1 mg/mL of letrozole in
Diluent. Shake the whole Tablets (NLT 10) for about 15 CeHi3NO2 131.17
min in a portion of Diluent to aid in dissolution. Centri- L-Leucine [61-90-5].
fuge, and use the supernatant.
Chromatographic system DEFINITION
(See Chromatography (621), System Suitability.) Leucine contains NLT 98.5% and NMT 101.5% of L-leucine
Mode: LC (CsHi3NO2), calculated on the dried basis.
Detector: UV 230 nm
IDENTIFICATION
Column: 4.6-mm x 12.5-cm; 5-"um packing L1
Flow rate: 1 mL/min e A. INFRARED ABSORPTION (197K)
Injection volume: 50 uL ASSAY
System suitability ¢ PROCEDURE
Samples: System suitability solution and Standard Sear 130 mg of Leucine
solution Blank: Mix 3 mL of formic acid and 50 mL of glacial
Suitability requirements acetic acid.
Resolution: NLT 2.0 between letrozole and letrozole Titrimetric system
related compound A, System suitability solution (See Titrimetry (541).)
Relative standard deviation: NMT 10.0% for le- Mode: Direct titration
trozole, Standard solution Titrant: 0.1 N perchloric acid VS
Analysis Endpoint detection: Potentiometric
Samples: Standard solution and Sample solution Analysis: Dissolve the Sample in 3 mL of formic acid
Calculate the percentage of each impurity in the por- and 50 mL of glacial acetic acid. Titrate with the Titrant.
tion of Tablets taken: Perform the blank determination.
Calculate the percentage of leucine (CsH:3NO2z) in the
Result = (ru/rs) x (Cs/Cu) x 100 portion of the Sample taken:
ru = peak response of each individual impurity Result = {[(Vs — Vs) x Na x FI/W} x 100
from the Sample solution
rs = peak response of letrozole from the Standard Vs = Titrant volume consumed by the Sample (mL) [==
solution Ve = Titrant volume consumed by the Blank (mL) vn
Gs = concentration of USP Letrozole RS in the Na = actual normality of the Titrant (mEq/mL)
uv
Standard solution (mg/mL) F = equivalency factor, 131.2 mg/mEq =
Cy = nominal concentration of letrozole in the Ww = Sample weight (mg) iS
Sample solution men p=
Acceptance criteria: See Table 2. Disregard any impu-
Acceptance criteria: 98.5%-101.5% on the dried basis }
rity peaks less than 0.05%.
rr}=
IMPURITIES i
© RESIDUE ON IGNITION (281): NMT 0.4% a}
Table 2 © CHLORIDE AND SULFATE (221), Chloride Sy
“
Standard solution: 0.50 mL of 0.020 N hydrochloric
Relative Acceptance acid
Retention Criteria, Sample: 0.73 g of Leucine
Acceptance criteria: NMT 0.05%
compound _— © CHLORIDE AND SULFATE (221), Sulfate
Standard solution: 0.10 mL of 0.020 N sulfuric acid
benzonitri = Sample: 0.33 g of Leucine
An: if ed i - a1 Acceptance criteria: NMT 0.03%
@ IRON (241): NMT 30 ppm
I im i os
4,4'-(1H-1,3,4-Triazol-1-ylmethylene)dibenzonitrile.
(Note—Letrozole related compound A and 4,4’,4’”-Methanetriyltribenzoni- Delete the following:
trile are process impurities and are controlled in the drug substance mon-
ograph.] °e HEAVY METALS, Method I! (231): NMT 15 ppme comical 1-
Jan-2018)
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers at
controlled room temperature. Change to read:
e LABELING: When more than one Dissolution test is given,
the labeling states the Dissolution test used only if Test 7 e RELATED COMPOUNDS
is not used. © (8 1-Au9-2017)
e USP REFERENCE STANDARDS (11) System suitability solution: 0.4 mg/mL each of USP L-
USP Letrozole RS Leucine RS and USP L-Valine RS in 0.1 N hydrochloric
USP Letrozole Related Compound A RS acid
4,4’-(1H-1,3,4-Triazol-1 -ylmethylene)dibenzonitrile. Standard solution: 0.05 mg/mL of USP L-Leucine RS in
CiHuNs = 285.31 0.1 N hydrochloric acid. [NoTe—This solution has a
concentration equivalent to 0.5% of that of the Sample
solution.]
 2360 Leucine / Official Monographs USP 41

Sample solution: 10 mg/mL of Leucine in 0.1 N hydro- DEFINITION

chloric acid Leucovorin Calcium contains NLT 95.0% and NMT 105.0%
Chromatographic system of leucovorin calcium (C29H21CaN7O7), calculated on the
(See Chromatography (621), General Procedures, Thin- anhydrous basis.
Layer Chromatography.)
Mode: TLC IDENTIFICATION
Adsorbent: 0.25-mm layer of chromatographic silica e A. INFRARED ABSORPTION (197K)
gel mixture Do not dry specimens.
Application volume: 5 pL Acceptance criteria: Meets the requirements
Developing solvent system: Butyl alcohol, glacial ace-
tic acid, and water (3:1:1) ASSAY
Spray reagent: 2 mg/mL of ninhydrin in a mixture of © PROCEDURE
utyl alcohol and 2 N acetic acid (95:5) Use only freshly deionized water wherever water is speci-
System suitability fied throughout this Procedure. Use low-actinic glass-
Sample: System suitability solution ware for solutions containing leucovorin calcium, and
Suitability requirements: The chromatogram of the otherwise protect the solutions from unnecessary expo-
System suitability solution exhibits two clearly separated sure to light. Complete the assay without prolonge
spots. interruption.
Analysis Solution A: 250 mg/mL of tetrabutylammonium hy-
Samples: System suitability solution, Standard solution, droxide in methanol
and Sample solution Solution B: 276 mg/mL of monobasic sodium phos-
After air-drying the plate, spray with Spray reagent, and phate monohydrate in water
heat between 100° and 105° for 15 min. Examine the Mobile phase: Mix 15 mL of Solution A with 835 mL of
plate under white light. water. Add 125 mL of acetonitrile, adjust with Solution B
Acceptance criteria: Any secondary spot of the Sample to an apparent pH of 7.5 + 0.1, dilute with water to
solution is not larger or more intense than the principal 1000 mL, and filter. Adjust the concentration of aceto-
spot of the Standard solution. nitrile, if necessary.
Diluent: Mix 15 mL of Solution A with 900 mL of water,
Individual impurities: NMT 0.5%
Total impurities: NMT 2.0% and adjust with Solution B to a pH of 7.5 + 0.1. Dilute
with water to 1000 mL.
SPECIFIC TESTS Standard solution: 0.175 mg/mL of USP Leucovorin
© OPTICAL ROTATION (7815S), Procedures, Specific Rotation Calcium RS in Diluent
Sample solution: 40mg/mL in 6 N hydrochloric acid Sample solution: 0.2 mg/mL of Leucovorin Calcium in
Acceptance criteria: +14.9° to +17.3° Diluent
e PH (791) System suitability stock solution: 0.175 mg/mL of folic
Sample solution: 10 mg/mL in water acid in Diluent
Acceptance criteria: 5.5-7.0 System suitability solution: System suitability stock solu-
e Loss ON DRYING (731) tion and Standard solution (1:4)
te
”
Analysis: Dry at 105° for 3 h. Chromatographic system
oy Acceptance criteria: NMT 0.2% (See Chromatography (621), System Suitability.)
i
— Mode: LC
i) Detector: UV 254 nm
i} ADDITIONAL REQUIREMENTS
te © PACKAGING AND STORAGE: Preserve in well-closed Column: 4-mm x 30-cm; packing L1
5 containers. Flow rate: 1-2 mL/min
2 Injection volume: 15 uL
oe Change to read:
System suitability
wal Sample: System suitability solution
=) [Note—The relative retention times for leucovorin and
e USP REFERENCE STANDARDS (11) folic acid are 1.0 and about 1.6, respectively.]
% (RB 71-Aug-2017) Suitability requirements
USP L-Leucine RS Resolution: NLT 3.6 between leucovorin calcium and
USP L-Valine RS folic acid
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of leucovorin calcium
Leucovorin Calcium aa in the portion of Leucovorin Calcium
taken:
Han, A Result = (ru/rs) x (Cs/Cu) x 100
ry
tu = peak response from the Sample solution
°
a) rs = peak response from the Standard solution
Cs = concentration of USP Leucovorin Calcium RS
in the Standard solution (mg/mL)
Cu = concentration of Leucovorin Calcium in the
C20H21CaN7O7 511.50 Sample solution (mg/mL)
L-Glutamic acid, N-[4-[[(2-amino-5-formyl-1,4,5,6,7,8-hex- Acceptance criteria: 95.0%-105.0% on the anhydrous
ahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-, cal- basis
cium salt (1:1);
Calcium N-[p-[[[(6R5)-2-amino-5-formyl-5,6,7,8-tetrahydro- IMPURITIES
Fe ee cre rice enna pena eeegiutomate
Gs ) [1492-18-8]. Delete the following:

°e HEAVY METALS, Method // (231): 50 ppMe‘offs! 1Jan-2018)

USP 41 Official Monographs / Leucovorin 2361

SPECIFIC TESTS Column: 4-mm x 30-cm; packing L1

e WATER DETERMINATION, Method | (921): NMT 17.0% Flow rate: 1-2 mL/min
Injection volume: 15 uL
ADDITIONAL REQUIREMENTS System suitability
e PACKAGING AND STORAGE: Preserve in well-closed, light- Sample: System suitability solution
resistant containers. [Note—The relative retention times for leucovorin and
e USP REFERENCE STANDARDS (11) folic acid are 1.0 and about 1.6, respectively.]
USP Leucovorin Calcium RS Suitability requirements
Resolution: NLT 3.6 between leucovorin calcium and
folic acid
Relative standard deviation: NMT 2.0%
Analysis
Leucovorin Calcium Injection Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
DEFINITION eueovonn (Ca0H23N707) in the portion of Injection
Leucovorin Calcium Injection is a sterile solution of taken:
leucovorin calcium (Cz0H21CaN7O;) in Water for Injection.
It contains NLT 90.0% and NMT 120.0% of the labeled Result = (ru/rs) x (Cs/Cu) x (Mi/M,2) x 100
amount of leucovorin (C29H23N7O7).
ty = peak response from the Sample solution
IDENTIFICATION Is = peak response from the Standard solution
e A. INFRARED ABSORPTION (197K) Cs = concentration of USP Leucovorin Calcium RS
Analysis: Transfer a volume of Injection, equivalent to in the Standard solution (mg/mL)
6 mg of leucovorin calcium, to a glass-stoppered, Cy = nominal concentration of leucovorin in the
50-mL centrifuge tube. Add 40 mL of acetone, mix, Sample solution (mg/mL) .
centrifuge for a few min, and decant and discard the Ma — = molecular weight of leucovorin, 473.45
liquid phase. Repeat the washing process with an addi- M2 = molecular weight of leucovorin calcium,
tional 40 mL of acetone. Dry the precipitate so obtained 511.50
with a stream of dry nitrogen. Acceptance criteria: 90.0%-120.0%
Acceptance criteria: Meets the requirements
SPECIFIC TESTS
ASSAY e PH (791): 6.5-8.5
e PROCEDURE e BACTERIAL ENDOTOXINS TEST (85): NMT 1.95 USP Endo-
Use only freshly deionized water wherever water is speci- toxin Units/mg of leucovorin calcium
fied throughout this Procedure. Use low-actinic glass- © OTHER REQUIREMENTS: It meets the requirements in Injec-
ware for solutions containing leucovorin calcium, and tions and Implanted Drug Products (1).
otherwise protect the solutions from unnecessary expo-
sure to light. Complete the assay without prolonge ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in single-dose, light- =
interruption. “
Solution A: 250 mg/mL of tetrabutylammonium hy- resistant containers, preferably of Type | glass. a-]
droxide in methanol E
Solution B: 276 mg/mL of monobasic sodium phos- Change to read: fo)
phate monohydrate in water =]
Mobile phase: Mix 15 mL of Solution A with 835 mL of fo]
° usP REFERENCE STANDARDS (11) Q
water. Add 125 mL of acetonitrile, adjust with Solution B a
@ (CN J-May-2018) i
to an apparent pH of 7.5 + 0.1, dilute with water to USP Leucovorin Calcium RS mo)
1000 mL, and filter. Adjust the concentration of aceto- =
nitrile, if necessary. ry
Diluent: Mix 15 mL of Solution A with 900 mL of water,
and adjust with Solution B to a pH of 7.5 + 0.1. Dilute
with water to 1000 mL.
Standard solution: 0.175 mg/mL of USP Leucovorin Leucovorin Calcium Compounded Oral
Calcium RS in Diluent Suspension
Sample solution: Transfer a measured volume of Injec-
tion, equivalent to 9 mg of leucovorin, to a 50-mL volu- DEFINITION
metric flask, and dilute with Diluent to volume. Pipet Leucovorin Calcium Compounded Oral Suspension contains
25 mL of this solution into a 60-mL separator, add NLT 90.0% and NMT 110.0% of the labeled amount of
25 mL of methylene chloride, shake the mixture, allow leucovorin (CaoH23N7O7).
the layers to separate, and discard the methylene chlo- Prepare Leucovorin Calcium Compounded Oral Suspension
ride extract. Repeat the extraction with two more containing 5 mg/mL of leucovorin as follows (see Pharma-
25-mL portions of methylene chloride, discarding the ceutical Compounding—Nonsterile Preparations (795)).
methylene chloride extracts. Filter the aqueous layer,
discarding the first 5 mL of the filtrate, and collect the Leucovorin Calcium tablets? equivalent to 500 mg of leucovorin
remaining filtrate in a glass-stoppered conical flask.
Sodium Hydroxide (1_N) To adjust pH to 7.1-7.6
System suitabilitystock solution: 0.175 mg/mL of folic
acid in Diluent Syrup, a sufficient quantity to make 100 mL
System suitability solution: System suitability stock solu- ? Leucovorin calcium 25-mg tablets, Teva Pharmaceuticals, Sellersville, PA.
tion and Standard solution (1:4)
Chromatographic system Place the Leucovorin Calcium tablets into a suitable container.
(See Chromatography (621), System Suitability.) Wet the tablets with a small amount of Syrup and triturate
Mode: LC to make a smooth paste. Add the Syrup to make the con-
Detector: UV 254 nm tents pourable. Transfer contents stepwise and quantita-
tively to a calibrated container using the Syrup. Adjust
with Sodium Hydroxide (1 N) to a pH of 7.1-7.6. Add suffi-
cient Syrup to bring to final volume. Shake to mix well.
 2362 Leucovorin / Official Monographs USP 41

[NoTE—pH may decrease to 6.1 after bringing to final vol- e USP REFERENCE STANDARDS (11)
ume with Syrup without affecting the stability of the USP Leucovorin Calcium RS
preparation.]
ASSAY
e PROCEDURE
Solution A: Methanol and 5 mM tetrabutylammonium ; ‘
be Wy (20:80). Adjust with tetrabutylammonium Leucovorin Calcium Tablets
ydroxide to a pH of 6.6. [NoTE—Tetrabutylammonium
phosphate appearing wet should not be used as it may DEFINITION
coelute with leucovorin.] Leucovorin Calcium Tablets contain NLT 90.0% and NMT
Mobile phase: See Table 7. 110.0% of the labeled amount of leucovorin
(C2oH23N7O7).
Table 1 IDENTIFICATION
Methanol Solution A
oA
%
Sample: Equivalent to 200 mg of leucovorin calcium
from finely powdered Tablets
0 1
Analysis: Transfer the Sample to a conical flask. Add
10 mL of water, shake vigorously, sonicate for 10 min,
1 and filter. Transfer the filtrate to a stoppered centrifuge
0 1 tube, add 125 mg of ammonium oxalate, shake vigor-
ously, and centrifuge until a clear supernatant is ob-
Diluent: Methanol and water (20:80) tained. Transfer the supernatant to another stoppered
Standard solution: 0.05 mg/mL of leucovorin prepared centrifuge tube, add 1 mL of methanol and 3 drops of
from USP Leucovorin Calcium RS and Diluent. Vortex hydrochloric acid, and shake vigorously. If the prepara-
and sonicate until dissolved. tion is cloudy, add methanol until a clear solution is
Sample solution: Transfer 1.0 mL of Oral Suspension to obtained, and filter if necessary to remove any undis-
a 100-mL volumetric flask, and rinse the pipette with solved material. Cool the preparation at 0° until a pre-
about 2 mL of Diluent. Dilute with Diluent to volume. cipitate forms, and centrifuge for 1-2 min. [NoTE—The
Chromatographic system cooling and centrifuging steps may be repeated if nec-
(See Chromatography (621), System Suitability.) essary to increase the amount of precipitate collected.]
Mode: LC Decant the supernatant, add 2 mL of methanol to the
Detector: UV 290 nm tube, shake vigorously to dissolve the precipitate, and
Column: 4.6-mm x 15-cm; 2.7-'4m packing L7 transfer the contents to a beaker. Evaporate under a
Column temperature: 55° current of air to dryness, and dry the residue at 50° for
Flow rate: 0.75 mL/min 30 min.
Injection volume: 10 pL Acceptance criteria: The IR absarplion spectrum of a
fe
”
System suitability potassium bromide dispersion of the residue exhibits
a Sample: Standard solution maxima only at the same wavelengths as that of a simi-
Ss
—
eye retention time for leucovorin is about 20.3 lar preparation of USP Leucovorin Calcium RS.
Dd min. e B. The retention time of the major peak of the Sample
i} Suitability requirements solution corresponds to that of the Standard solution, as
©
S Tailing factor: NMT 2.0 obtained in the Assay.
= Relative standard deviation: NMT 2.0% for replicate
injections ASSAY
[3
” Analysis © PROCEDURE
= Samples: Standard solution and Sample solution Diluent: Methanol and water (20:80)
Calculate the percentage of the labeled amount of Mobile phase: 5 mM tetrabutylammonium phosphate
leucovorin (C2oH23N7O7) in the portion of Oral Suspen- in Diluent. Adjust with 50% (w/v) sodium hydroxide to
sion taken: a pH of 7.5.
Standard solution: 0.5 mg/mL of USP Leucovorin Cal-
Result = (ru/rs) x (Cs/Cu) x 100 cium RS and 10 ug/mL of USP 10-Formylfolic Acid RS in
water
tu = peak response from the Sample solution Sample solution: Transfer finely powdered Tablets (NLT
Is = peak response from the Standard solution 20), equivalent to 50 mg of leucovorin, to a 100-mL
Gs = concentration of leucovorin from USP volumetric flask. Add 50 mL of water, sonicate for 30
Leucovorin Calcium RS in the Standard min, dilute with water to volume, mix, and filter.
solution (mg/mL) Chromatographic system
GC = nominal concentration of leucovorin in the (See Chromatography (621), System Suitability.)
Sample solution (mg/mL) Mode: LC
Acceptance criteria: 90.0%-110.0% Detector: UV 254 nm
Column: 4.6-mm x 15-cm; packing L1
SPECIFIC TESTS Flow rate: 2.0 mL/min
© PH (791): 6.1-7.1 Injection volume: 20 uL
System suitability
ADDITIONAL REQUIREMENTS Sample: Standard solution
e PACKAGING AND STORAGE: Package in tight, light-resistant [Note—The relative retention times for leucovorin and
plastic containers. Store in a refrigerator or at controlled 10-formylfolic acid are about 1.0 and 2.3,
room temperature. respectively]
e BEYOND-UsE DATE: NMT 90 days after the date on which Suitability requirements
it was compounded when stored in a refrigerator; NMT Resolution: NLT 1.5 between leucovorin and
30 days after the date on which it was compounded 10-formylfolic acid
when stored at controlled room temperature Relative standard deviation: NMT 2.0% for
e LABELING: Label it to be well shaken before use, and to leucovorin
state the Beyond-Use Date.
USP 41 Official Monographs / Leuprolide 2363

Analysis Acceptance criteria: Meet the requirements

Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of IMPURITIES
puso (C2oH23N7O7) in the portion of Tablets © ORGANIC IMPURITIES
taken: Diluent, Mobile phase, Standard solution, Sample so-
lution, Chromatographic system, and System suitabil-
Result = (ru/rs) x (Cs/Cu) x (Ma/M,2) x 100 ity: Proceed as directed in the Assay.
Analysis
ru = peak area of the Sample solution Sample: Sample solution
ls = peak area of the Standard solution Calculate the percentage of each impurity in the por-
Gs = concentration of USP Leucovorin Calcium RS tion of Tablets taken:
in the Standard solution (mg/mL)
Cu = nominal concentration of leucovorin in the Result = (ru/r7) x 100
Sample solution (mg/mL)
My = molecular weight of leucovorin, 473.45 ty = response of each impurity peak
M2 = molecular weight of leucovorin calcium, rr = sum of the responses of all the peaks
511.50 Acceptance criteria
Acceptance criteria: 90.0%-110.0% Individual impurities: NMT 2.5%
Total impurities: NMT 4.0 %
PERFORMANCE TESTS
@ DISSOLUTION (711) ADDITIONAL REQUIREMENTS
Medium: Water; 900 mL ¢ PACKAGING AND STORAGE: Preserve in well-closed contain-
Apparatus 2: 50 rpm ers, protected from light, at controlled room
Time: 30 min temperature.
Detector: UV, at a maximum of about 284 nm e USP REFERENCE STANDARDS (11)
Standard solution: USP Leucovorin Calcium RS in USP 10-Formylfolic Acid RS
Medium USP Leucovorin Calcium RS
Sample solution: Use filtered portion of solution under
test, and dilute with water if necessary to a concentra-
tion similar to that of the Standard solution.
Calculate the percentage of the labeled amount of
leucovorin (C20H23N7O7) dissolved: Leuprolide Acetate
Result = (Au/As) x Cs x Vx Dx (Mi/Myz) x (1/L) x 100 tte
Au = absorbance of the Sample solution we ae (q
As = absorbance of the Standard solution
Cs = concentration of the Standard solution ° ee 9 me ° OK ° fe ‘a
(mg/mL) AS atA Swet OC et SO =
V = volume of Medium, 900 mL ¢\-NH i Ke
3 ee oePK
Tt Baty ae a]
é
° 8 Yom
D =
Mn, — =
dilution factor
molecular weight of leucovorin, 473.45 ee Ales =" ~on
Le TB
°o
M2 = molecular weight of leucovorin calcium, =|
511.50
L = label claim (mg/Tablet) ; | °i a°
nc” ~oul, 3
Tolerances: NLT 75% (Q) of the labeled amount of
leucovorin (C29H23N7O7) is dissolved. ao}
e UNIFORMITY OF DOSAGE UNITS (905)
=
CsgHeaNi6O12 - (C2H4O2)n, 1 = 1-2 1209.41 (as free base) v
Analysis for content uniformity Luteinizing hormone-releasing factor, 6-D-leucine-9-(N-ethyl-
Standard solution: 10 g/mL of USP Leucovorin Cal- L-prolinamide)-10-desglycinamide acetate (salt);
cium RS 5-Oxo-L-prolyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-leucyl-
Sample solution: 10 g/mL of leucovorin calcium, use L-leucyl-L-arginyl-N-ethyl-L-prolinamide acetate (salt);
individual intact Tablets. Free base [53714-56-0].
Blank: Water Acetate salt [74381-53-6].
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).) DEFINITION
Mode: UV Leuprolide Acetate is a synthetic nonapeptide agonist ana-
Cell: 1m log of luteinizing hormone-releasing factor. It contains
Analytical wavelength: UV, at maxima about 284 NLT 97.0% and NMT 103.0% of leuprolide
nm (CsoHgaNi6O12), calculated on the anhydrous, acetic acid-
Analysis free basis.
Samples: Standard solution and Sample solution [Note—Due to the hi Gioscapic nature of this material, anal-
Calculate the percentage of the labeled amount of yses are performed immediately after opening the con-
leucovorin (C2oH23N7O7) in each Tablet taken: tainer in a glove box under dry nitrogen purge.]
[CauTion—Leuprolide Acetate is a potent hormonal manipu-
Result = (Au/As) x (Cs/Cu) X (Mr/M,2) x 100 lator. Avoid skin contact and inhalation of dusts and
mists.]
Au = absorbance of the Sample solution
As = absorbance of the Standard solution IDENTIFICATION
Cs = concentration of USP Leucovorin Calcium RS e A. HPLC
in the Standard solution (g/mL) Solution A, Solution B, Mobilephase, Standard solu-
Cu = nominal concentration of leucovorin in the tion, Degradation standard solution, Sample solu-
Sample solution (g/mL) tion, Chromatographic system, and System suitabil-
M, = molecular weight of leucovorin, 473.45 ity: Proceed as directed in theAssay.
Mz = molecular weight of leucovorin calcium, Identity sample solution: Mix equal volumes of the
511.50 Standard solution and the Sample solution.
 2364 Leuprolide / Official Monographs
Analysis
Samples: Standard solution, Sample solution, and Iden-
tity sample solution
Examine the chromatograms of the Standard solution,
the Sample solution, and the Identity sample solution.
Acceptance criteria: The retention time of the major
peak of the Sample solution corresponds to that of the
Standard solution, and the major peak of the /dentity
sample solution elutes as a single peak.
e B. AMINO ACID ANALYSIS
[Note—The following method is given for informational
urposes; any validated amino acid analysis method can
e used.]
Standard solutions: Prepare a solution having known
equimolar amounts of L-alanine, L-arginine, L-aspartic
acid, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-
leucine, L-lysine, L-methionine, L-phenylalanine, L-pro-
line, L-serine, L-threonine, L-tyrosine, and L-valine with
half the equimolar amount of L-cystine. Prepare a sepa-
rate, equimolar solution of L-tryptophan.
Sample solution: Transfer about 6.4 mg of Leuprolide
Acetate to a suitable vacuum hydrolysis tube. Add
2.0 mL of 6 N hydrochloric acid to the tube, evacuate,
and seal. Heat at 120° for 16 h. Allow to cool. Remove
the solvent under vacuum. Dissolve in, and dilute to a
suitable volume in, a buffer solution suitable for amino
acid analysis.
Analysis: Standardize the instrument with the Standard
solutions. Inject suitable volumes of the Standard solu-
tions and the Sample solution into the amino acid ana-
lyzer. Record and measure the responses for each
amino acid peak. Express the content of each amino
acid in nmol.
Calculate the mean nmol of each of the amino acids
taken:
Result = (nmol found in the Analysis for Glu, Pro, Tyr,
His, Arg, Leu)/7
aa
Sal
ro Divide the nmol of each amino acid by the Result to
i]
7
Dp determine the amino acid ratios that must meet the
° Acceptance criteria.
4 Acceptance criteria
) Glutamic acid, proline, tyrosine, histidine, and argi-
= nine: 0.85-1.1
a Leucine: 1.8-2.2
a)
| Serine and tryptophan: Present
ASSAY
© PROCEDURE
Solution A: 15.2 mg/mL of triethylamine in water. Ad-
just with phosphoric acid to a pH of 3.0.
Solution B: Acetonitrile and n-propyl alcohol (3:2)
Mobile phase: Solution A and Solution B (17:3)
Standard stock solution: 1 mg/mL of USP Leuprolide
Acetate RS in Mobile phase
Standard solution: 50 g/mL of USP Leuprolide Ace-
tate RS in Mobile phase prepared from the Standard
stock solution
Degradation standard solution: Dilute the Standard
stock solution with water to 0.1 mg/mL. Transfer 5 mL of
the solution into a scintillation vial. Add 100 uL of 1N
sodium hydroxide solution, cap tightly, and shake vig-
orously. Place in an oven at 100° for 60 min. Remove,
allow to cool, add 50 ul of 1 M phosphoric acid, recap,
and shake vigorously to mix.
Sample solution: 50 ug/mL of Leuprolide Acetate in
Mobile phase
Chromatographic system
(See Chromatography (621), System Suitability.)
USP 41
Mode: LC
Detector: UV 220 nm
Column: 4.6-mm x 10-cm; 3-1m packing L1
Flow rate: 1-1.5 mL/min
Injection volume: 20 uL
System ee
Samples: Mobile phase, Standard solution, and Degra-
dation standard solution
[Note—Chromatograph the Mobile phase and verify
that no extraneous peaks are present.]
[NoTtr—The relative retention times for the degradation
product and leuprolide are about 0.90 and 1.0,
respectively.]
Suitability requirements
Resolution: NLT 1.5 between leuprolide and the deg-
radation product, Degradation standard solution
Tailing factor: 0.8-1.5, Standard solution
Retention time: 41-49 min for leuprolide, Degrada-
tion standard solution
Relative standard deviation: NMT 1.5% for
leuprolide acetate, Standard solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of leuprolide (CssHg4N16O12) in
the portion of Leuprolide Acetate taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak area from the Sample solution
rs = peak area from the Standard solution
Cs = concentration of USP Leuprolide Acetate RS in
the Standard solution (ug/mL)
Cy = concentration of Leuprolide Acetate in the
Sample solution (ug/mL) on the anhydrous
and acetic acid-free basis
Acceptance criteria: 97.0%-103.0% on the anhydrous
and acetic acid-free basis
OTHER COMPONENTS
e ACETIC ACID IN PEPTIDES (503): 4.7%-9.0%
PRODUCT-RELATED SUBSTANCES AND IMPURITIES
e LEUPROLIDE-RELATED IMPURITIES
Solution A, Solution B, Mobile phase, Standard stock
solution, Degradation standard solution, and Chro-
matographic system: Proceed as directed in the
Assay.
Standard solution: 0.01 mg/mL of USP Leuprolide Ace-
tate RS in Mobile phase prepared by diluting the Stan-
dard stock solution
Sample solution: 1mg/mL of Leuprolide Acetate in
Mobile phase
System mais
Samples: Mobile phase, Degradation standard solution,
and Standard solution
[Note—Chromatograph the Mobile phase and verify
that no extraneous peaks are present.]
[NoTte—The relative retention times for the degradation
product and leuprolide are about 0.90 and 1.0,
respectively.]
Suitability requirements
Resolution, Tailing factor, and Retention time: Pro-
ceed as directed in the Assay.
Relative standard deviation: NMT 1.5% for
leuprolide acetate, Standard solution
Analysis
Samples: Standard solution and Sample solution
[Note—Record the chromatograms for 90 min.]
Calculate the percentage of each impurity in the por-
tion of Leuprolide Acetate taken:
Result = (ru/rs) * (Cs/Cu) x 100
tu = peak response of each impurity from the
Sample solution
USP 41 Official Monographs / Levalbuterol 2365

ls = peak response of leuprolide from the Standard ASSAY

solution e PROCEDURE
Cs = concentration of USP Leuprolide Acetate RS in Solution A: Phosphoric acid in water (1 in 1000)
the Standard solution (mg/mL) Solution B: Acetonitrile, methanol, water, and phos-
Cu = concentration of Leuprolide Acetate in the phoric acid (350:350:300:1)
Sample solution (mg/mL) Mobile phase: See Table 1.
Acceptance criteria: See Table 1.
Table 1
Table 1 Solution A Solution B
Relative Acceptance
Retention Criteria,

0.9
1.0 —
12 0.5 30 8.5
1 1.0
ual i — Diluent: Dissolve 9.0 g of sodium chloride in 950 mL of
= 2.5 water. Adjust with dilute sulfuric acid to a pH of 4.0,
and dilute with water to 1000 mL. Mix, and pass
2 5-Oxo-L-prolyl-L-histidyl-L-tryptophyl-p-seryl-L-tyrosyl-D-leucyl-L-leucyl-L-
arginy|l-N-ethyl-L-prolinamide. througha filter of 0.45-m pore size.
» 5-Oxo-L-prolyl-D-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-leucyl-L-leucyl-L- Standard solution: 0.1 mg/mL of USP Levalbuterol Hy-
arginyl-N-ethyl-t-prolinamide. drochloride RS in Diluent
¢5-Oxo-L-prolyl-t-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-t-leucyl-L-leucyl-L- apne solution: Nominally 0.1 mg/mL of levalbutero!
arginyl-N-ethyl-L-prolinamide. hydrochloride (equivalent to 0.087 mg/mL of
a Se KOT prOW TCT SUCH Ctyptop hela Crarcty sepia atu eueyet levalbuterol free base) in Diluent from an appropriately
leucyl-L-arginyl-N-ethyl-L-prolinamide. diluted volume of Inhalation Solution
PROCESS-RELATED IMPURITIES Chromatographic system
© TRIFLUOROACETIC ACID (TFA) IN PEPTIDES (503.1): NMT (See Chromatography (621), System Suitability.)
0.25%. [NoTe—Perform this test if trifluoroacetic acid is Mode: LC
used in the manufacturing process.] Detector: UV 220 nm
Column: 4.6-mm x 15-cm; 5-m packing L1
SPECIFIC TESTS Column temperature: 35°
¢ WATER DETERMINATION (921), Method |, Method Ic) NMT Flow rate: 1 mL/min
8.0% Injection volume: 10 pL
e BACTERIAL ENDOTOXINS TEST (85): It contains NMT 166.7 System suitability Cc
USP Endotoxin Units/mg of leuprolide acetate. Sample: Standard solution “v
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI- Suitability requirements i)
FIED MICROORGANISMS (62): The total aerobic microbial Column efficiency: NLT 5500 theoretical plates =
count does not exceed 10? cfu/g. The total yeast and Tailing factor: NMT 2.3 }
mold count does not exceed 10? cfu/g. Relative standard deviation: NMT 2.0% =
re}
ADDITIONAL REQUIREMENTS
Analysis e=
Samples: Standard solution and Sample solution
© PACKAGING AND STORAGE: Preserve in tight containers. i)
Calculate the percentage of the labeled amount of a)
Store at a temperature not higher than 30°. levalbuterol (Ci3H2:1NOs) in the portion of Inhalation me
Solution taken: “v

Change to read:
Result = (ru/rs) x (Cs/Cu) x (Ma/M,2) x 100
e USP REFERENCE STANDARDS (11) ty = peak response of levalbuterol hydrochloride
© (CN I-May-2018) from the Sample solution
USP Leuprolide Acetate RS Is = peak response of levalbuterol hydrochloride
from the Standard solution
Cs = concentration of USP Levalbuterol
Hydrochloride RS in the Standard solution
Levalbuterol Inhalation Solution
(mg/ml)
nominal concentration of levalbuterol in the
©

Sample solution (mg/mL)

DEFINITION Ma = molecular weight of levalbuterol (free base),
Levalbuterol Inhalation Solution is a sterile, aqueous solution 239,31
of Levalbuterol Hydrochloride, prepared with Sodium Mz = molecular weight of levalbuterol
Chloride. It contains NLT 90.0% and NMT 110.0% of the hydrochloride, 275.77
labeled amount of levalbuterol (C:3H2iNOs). Acceptance criteria: 90.0%-110.0%
IDENTIFICATION PERFORMANCE TESTS
e A. The retention time of the major peak of the Sample e UNIFORMITY OF DosAGE UNITS (905): Meets the
Solution corresponds to that of the Standard solution, as requirements
obtained in the Assay.
IMPURITIES
© ORGANIC IMPURITIES
Solution A, Solution B, Diluent, and Sample solution:
Prepare as directed in the Assay.
 2366 Levalbuterol / Official Monographs USP 41

Mobile phase: See Table 2. Table 3 (Continued)

Relative Relative Acceptance
Table 2 Retention | Response Criteria,
Time Solution A Solution B Name Time Factor NMIT (%)
(min) (%) (%) Levalbuterol related
0 100 0 compound Be 15 at _
30 70 30 Levalbuterol related
50 28 72
compound C» 1.6 _ _
50.01 oO 100
Levalbuterol related
compound D 17 3.0 0.08
55 0 100
Levalbuterol related
55.01 100 0
compound E> 2.1
70 100 0
Levalbuterol related
compound F> 305
System suitability solution: Prepare a solution contain-
ing the following in Diluent. Any individual
USP Levalbuterol Hydrochloride RS, 100 ug/mL unspecified = =
USP Levalbuterol Related Compound A RS, 0.05 g/mL degradation product 0.10
USP Levalbuterol Related CompoundBRS, 0.05 jug/mL Total impurities = = 0.70
USP Levalbuterol Related Compound C RS, 0.05 pg/mL 2 2-(fere Buby laming) 4 -hydroxyethyl]-3-(hydroxymethyl)benzene-1,2-di-
USP Levalbuterol Related CompoundD RS, 0.05 yg/ ol.

mL Process impurity, included for identification purposes only. Not to be

included in the Total impurities.
USP Levalbuterol Related Compound E RS, 0.05 g/mL
USP Levalbuterol Related Compound F RS, 0.05 g/mL ¢4-[2-(tert-Butylamino)-1-methoxyethyl]-2-(hydroxymethyl)phenol.
Chromatographic system e ENANTIOMERIC PURITY
(See Chromatography (621), System Suitability.) Mobile phase: Acetonitrile, methanol, acetic acid, and
Mode: LC triethylamine (500:500:3:1)
Detector: UV 220 nm Diluent: Mobile phase
Column: 4.6-mm x 15-cm; 5-um packing L1 System suitability solution: 0.10 mg/mL of USP
Column temperature: 45° Levalbuterol Hydrochloride RS and 0.04 mg/mL of USP
Flow rate: 1 mL/min Albuterol RS in Diluent
Injection volume: 50 wL Sample solution: Inhalation Solution
System suitability Chromatographic system
Sample: System suitability solution (See Chromatography (621), System Suitability.)
Suitability requirements Mode: LC
Resolution: NLT 4.9 between levalbuterol and Detector: UV 225 nm
levalbuterol related compound A; NLT 1.5 between Column: 4.6-mm x 25-cm; 5-4um packing L63
te
ww

3 levalbuterol related compound Band levalbuterol re- Flow rate: 1 mL/min

Ss lated compound C Injection volume: 10 UL of the System suitability solu-
—
a Tailing factor: NMT 4.0 for the levalbutero! peak tion anda suitable volume of Sample solution to obtain
° Analysis 4.2 ug of levalbuterol injected on the column
= Sample: Sample solution Run time: NLT 30 min
9 [NoTe—Integrate all peaks with an area greater than System suitability
P 0.05%of the area corresponding to the levalbuterol Sample: System suitability solution
a eak.] [Note—The relative retention times of levalbutero! and
w
r=) Calculate the percentage of each impurity in the por- (S)-albuterol are 1.0 and 1.16, respectively.]
tion of Inhalation Solution taken: Suitability requirements
Resolution: NLT 3.0 between levalbuterol and (S)-
Result = (ru/rz) x (1/F) x 100 albuterol
Tailing factor: NMT 1.6 for levalbuterol and NMT
ru = peak response of each impurity from the 2.0 for (S)-albuterol
Sample solution Relative standard deviation: NMT 20% for (S)-al-
rr = sum of the responses of all the peaks buterol, for three injections
F = relative response factor for each impurity (see Analysis
Table 3) Sample: Sample solution
Acceptance criteria: See Table 3. Calculate the percentage of (5)-albuterol in the portion
of Inhalation Solution taken:
Table 3
Relative Relative Acceptance
Result = (ru/r7) x 100
Retention | Response Criteria,
ty = peak response of (5)-albuterol
Name Time Factor NMT (%)
rr = sum of the peak responses of levalbuterol and
5-Hydroxyalbuterol 0.90 1.0 0.10 (S)-albuterol
Levalbuterol — _ — Acceptance criteria: NMT 2.50% of (5)-albuterol in the
Levalbuterol related Sample solution
compound A® 1.2: 7 ae
Levalbuterol related
SPECIFIC TESTS
compound H»« 1.3 _ ~~ © STERILITY TESTS (71): Meets the requirements
© PH (791): 3.3-4.5
25-[2-(tert-Butylamino)-1-hydroxyethyl]-3-(hydroxymethyl)benzene-1,2-di- © PARTICULATE MATTER IN INJECTIONS (788): See Table 4.
ol.
» Process impurity, included for identification purposes only. Not to be
included in the Total impurities.
¢4-[2-(tert-Butylamino)-1-methoxyethyl]-2-(hydroxymethyl)phenol
USP 41 Official Monographs / Levalbuterol 2367

Table 4 ASSAY
Particle Size
e PROCEDURE
Solution A: Phosphoric acid in water (1 in 1000)
Solution B: Acetonitrile, methanol, phosphoric acid,
and water (350:350:1:300)
Mobile phase: See Table 7.
1
Table 1
© OSMOLALITY AND OSMOLARITY,Osmolality (785): 280-320 Solution A Solution B
mOsmol/kg
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in low-density poly-
ethylene single-use ampuls, with a multilayer foil over-
wrap. Store at controlled room temperature.
e LABELING: The outer label indicates the dose and that the 1.5
ampuls should be discarded if the solution is not 91
colorless.
e USP REFERENCE STANDARDS (11) Diluent: Solution A
USP Albuterol RS Standard solution: 100 g/mL of USP Levalbuterol Hy-
USP Levalbuterol Hydrochloride RS drochloride RS in Diluent
USP Levalbuterol Related Compound A RS Sample solution: 100 ug/mL of Levalbutero! Hydro-
4-(2-tert-Butylamino-ethyl)-2-hydroxymethyl-phenol. chloride in Diluent
Ci3H2NO2 223:31 Chromatographic system
USP Levalbuterol Related Compound B RS (See Chromatography (621), System Suitability.)
a[{(1,1-Dimethylethyl)amino}methyl]-4-hydroxy- Mode: LC
3-methyl-benzenemethanol. Detector: UV 220 nm
CisH2iNO2 223.31 Column: 4.6-mm x 15-cm; 5-um packing L1
USP Levalbuterol Related Compound C RS Column temperature: 35°
a[{(1,1-Dimethylethyl)amino}methyl]-4-hydroxy- Flow rate: 1 mL/min
3-(methoxymethyl)-benzenemethanol. Injection volume: 10 pL
Cy4H23NO3 253.34 System suitability
USP Levalbuterol Related Compound D RS Sample: Standard solution
5-[2-{(1,1-Dimethylethyl)amino}-1-hydroxyethyl]-2-hy- Suitability requirements
droxy-benzaldehyde; Column efficiency: NLT 5500 theoretical plates
Also known as 5-[2-{(1,1-Dimethylethyl)amino}methyl]- Tailing factor: NMT 2.3
4-hydroxy-3-(methoxymethyl)-benzenemethanol. Relative standard deviation: NMT 2.0% S
CysHigNO3 237.29 Analysis wn
[Note—This Reference Standard is available as the Samples: Standard solution and Sample solution z
benzenesulfonic acid salt.] Calculate thepercentage of levalbutero! hydrochloride =
USP Levalbuterol Related Compound E RS (Ci3H21NO3 - HCl) in the portion of Levalbuterol Hydro- i}
a[{(1,1-Dimethylethyl)amino}methyl]-3-(ethoxymethyl)- chloride taken: =
i}
4-hydroxy-benzenemethanol.
Result = (ru/rs) x (Cs/Cy) x 100
a2
CisH2sNO3 267.36 Ey
USP Levalbuterol Related Compound F RS so}
af{{(1,1-Dimethylethyl)amino}methyl]- ry = peak response from the Sample solution =
J ee ,3-benzenedimethanol. rs = peak response from the Standard solution “

C20H27NO3 = 329.43 Gs = concentration of USP Levalbuterol

Hydrochloride RS in the Standard solution
(ug/ml)_ ’
Cu = concentration of the Sample solution (g/mL)
ae criteria: 98.0%-102.0% on the anhydrous
asis
Levalbuterol Hydrochloride
a
apie
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.1%
» + Hel
HC CH, Delete the following:
Hom

®e HEAVY METALS, Method | (231): NMT 10 ppme coffcat 1.

Cy3H2iNO3 - HCl 275.77
(R)-c'-[(tert-Butylamino)methyl]-4-hydroxy-m-xylene-o,,o’- Jan-2018)
diol hydrochloride [50293-90-8]. e ORGANIC IMPURITIES
Solution A, Solution B, Diluent, and Sample solution:
DEFINITION Proceed as directed in the Assay.
Levalbuterol Hydrochloride contains NLT 98.0% and NMT Mobile phase: See Table 2.
102.0% of levalbuterol hydrochloride (Ci3H2i1NOs - HCl),
calculated on the anhydrous basis. Table 2
IDENTIFICATION Solution A Solution B
e A. INFRARED ABSORPTION (197K) %’
e B. The retention time of the major peak of the Diluted 100
samp solution corresponds to that of the levalbuterol
peak of the System suitability solution, as obtained in the 28
test for Enantiomeric Purity.
 2368 Levalbuterol / Official Monographs USP 41

Table 2 (Continued) Table 3 (Continued)

Time Solution A Solution B Relative Relative Acceptance
(min) (%) (%) Retention Response Criteria,
50.01 0 100 Name Time Factor NMT (%)
55 0 100 Any individual
55.01 100 0 unspecified impurity _ _ 0.10
70 100 0 Total impurities = — 0.5

System suitability solution: Prepare a solution contain- e ENANTIOMERIC PURITY

ing the following in Diluent: Mobile phase: Acetonitrile, methanol, acetic acid, and
USP Levalbuterol Hydrochloride RS, 100 pg/mL triethylamine (500:500:3:1)
USP Levalbuterol Related Compound A RS, 0.05 g/mL Diluent: Mobile phase
USP Levalbutero! Related CompoundB RS, 0.05 g/mL System suitability solution: 0.10 mg/mL of USP
USP Levalbuterol Related Compound C RS, 0.05 g/mL Levalbuterol Hydrochloride RS and 0.04 mg/mL of USP
USP Levalbuterol Related CompoundD RS, 0.05 .g/ Albuterol RS in Diluent
mL Sample solution: 0.8 mg/mL of Levalbuterol Hydro-
USP Levalbuterol Related CompoundE RS, 0.05 pg/mL chloride in Diluent
USP Levalbuterol Related Compound F RS, 0.05 g/mL Diluted sample solution: 0.1 mg/mL from the Sample
USP Levalbuterol Related Compound H RS, 0.05 pg/mL solution
Chromatographic system Chromatographic system
(See Chromatography (621), System Suitability.) (See Chromatography (621), System Suitability.)
Mode: LC Mode: LC
Detector: UV 220 nm Detector: UV 225 nm
Column: 4.6-mm x 15-cm; 5-um packing L1 Column: 4.6-mm x 25-cm; 5-um packing L63
Column temperature: 45° Flow rate: 1 mL/min
Flow rate: 1 mL/min Injection volume: 10 LL
Injection volume: 50 uL System suitability
System suitability Sample: System suitability solution
Sample: System suitability solution [Note—The relative retention times of levalbuterol and
Suitability requirements (S)-albuterol are 1.0 and 1.16, respectively.]
Resolution: NLT 4.9 between levalbuterol and Suitability requirements
levalbuterol related compound A; NLT 1.5 between Resolution: NLT 2.0 between levalbuterol and (5)-
levalbuterol related compound B and levalbuterol re- albuterol
lated compound C Tailing factor: NMT 2.2 for levalbuterol and (5)-
Tailing factor: NMT 4.0 for levalbuterol albuterol
Analysis Relative standard deviation: NMT 20% for (5)-al-
a Sample: Sample solution buterol for three injections
= Analysis
5 [Nore isa all peaks with an area greater than
Samples: Sample solution and Diluted sample solution
i 0.05% of the area corresponding to the levalbuterol
— Calculate the percentage of (5)-albuterol in the portion
a eak.]
ic} Calculate the percentage of each impurity in the por- of Levalbuterol Hydrochloride taken:
= tion of Levalbuterol Hydrochloride taken:
iS Result = (ru/rr) x 100
= Result = (ru/rr) x (1/FA) x 100
a ty = peak response of (5)-albuterol from the Sample
ww tu = peak response of each impurity from the solution
= Sample solution rr = sum of the peak responses for levalbuterol and
tr = sum of all the peak responses from the Sample (S)-albuterol from the Sample solution
solution ; ; Acceptance criteria: NMT 0.2% of (5)-albuterol
F = relative response factor for each impurity (see SPECIFIC TESTS
Table 3) e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
Acceptance criteria: See Table 3. FIED MICROORGANISMS (62): The total aerobic bacterial
count is less than 101 cfu/g. The total combined molds
Table 3 and yeasts count is less than 10' cfu/g. It meets the re-
Relative Relative Acceptance quirements of the tests for the absence of Salmonella spe-
Retention Response Criteria, cies, Staphylococcus aureus, Escherichia coli, and Pseudo-
Name Time Factor NMT (%) monas aeruginosa.
e PH (791): 4.5-5.5, in a 10-mg/mL solution
Levalbuterol 1.0 — a
e WATER DETERMINATION, Method Ic (921): NMT 0.3%
Levalbuterol related
compound A 12 1.0 0.1 ADDITIONAL REQUIREMENTS
Levalbuterol related ¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant
compound H 13 10 0.15 containers, and store at controlled room temperature.
Levalbuterol related e USP REFERENCE STANDARDS (11)
compound B 15 1.0 0.10 USP Albuterol RS
Levalbuterol related USP Levalbuterol Hydrochloride RS
compound C 1.6 1.0 0.15 USP Levalbuterol Related Compound A RS
4-(2-tert-Butylamino-ethyl)-2-hydroxymethyl-phenol.
Levalbuterol related Ci3H2NOz 223.31
compound D 1 3.0 0.05
USP Levalbuterol Related Compound B RS
Levalbuterol related a-{[(1,1-Dimethylethyl)amino]methy|}-4-hydroxy-
compound E 2h 1.0 0.1 3-methyl-benzenemethanol.
Levalbuterol related Ci3H2NO2 223.31
compound F 3:5 1.2. 0.10
USP 41
USP Levalbuterol Related Compound C RS
a-{[(1,1-Dimethylethyl)amino]methyl}-4-hydroxy-
3-(methoxymethyl)-benzenemethanol.
CyaH23NO3 253.34
USP Levalbuterol Related Compound D RS
5-{2-[(1,1-Dimethylethyl)amino]-1-hydroxyethyl}-2-hy-
droxy-benzaldehyde.
CisHigNO3 237.29
USP Levalbuterol Related Compound E RS
a-{[(1,1-Dimethylethyl)amino]methyl}-3-(ethoxymethyl)-
4-hydroxy-benzenemethanol.
CisH2sNO3 267.36
USP Levalbuterol Related Compound F RS
a-{[(1,1-Dimethylethyl)amino]methyl}-
4-(phenylmethoxy)-1,3-benzenedimethanol.
C29H27NO3 =3329.43
USP Levalbuterol Related Compound H RS
4-[2-(tert-Butylamino)-1-methoxyethyl]-2-(hydrox-
Pains rai acetate.
14H23NO3-CoHsO2 3313.39
Levamisole Hydrochloride
ge ° HCI
CiiHizN2S + HCl 240.75
Imidazo[2,1-b]thiazole, 2,3,5,6-tetrahydro-6-phenyl-,
monohydrochloride, (5)-.
(-)-2,3,5,6-Tetrahydro-6-phenylimidazo[2,1-b]thiazole mono-
hydrochloride © [16595-80-5].
» Levamisole Hydrochloride contains not less
than 98.5 percent and not more than 101.0 per-
cent of CiiHizN2S - HCl, calculated on the dried
asis.
Packaging and storage—Preserve in well-closed contain-
ers, protected from light.
USP Reference standards (11)—
USP Levamisole Hydrochloride RS
Completeness of solution (641)—A test solution of
500 mg of Levamisole Hydrochloride dissolved in 10 mL of
water meets the requirements.
Color of solution—The test solution prepared for the test
for Completeness of solution is colorless or not more intensely
colored than a color matching fluid prepared by ae
2.5 mL of Matching Fluid F (see Color and Achromicity (631))
with 97.5 mL of 0.12 N hydrochloric acid.
Identification—
A: The IR absorption spectrum of a potassium bromide
dispersion of it, previously dried, exhibits maxima only at
the same wavelengths as that of a similar preparation of
USP Levamisole Hydrochloride RS.
B: The color, size, and Ry value of the principal spot in
the chromatogram of Test solution B obtained in the test for
Chromatographic purity, when examined under short-wave-
length UV light, correspond to the respective characteristics
of the principal spot in the chromatogram of Reference solu-
tion A obtained in the test for Chromatographic purity.
a 9 A solution of it responds to the tests for Chloride
Melting range (741): between 226° and 231°.
Light absorption—its absorbance (see Ultraviolet-Visible
Spectroscopy (857)) at 310 nm, determined in a 0.2 N meth-
anolic hydrochloric acid solution containing 1 mg per mL
using a 1-cm cell, is not more than 0.20.
Official Monographs / Levamisole 2369
Specific rotation (7815S): between —121.5° and -128.0°.
Test solution: 50mg per mL, in water.
PH (791): between 3.0 and 4.5, in a solution (1 in 20).
Loss on drying (731): Dry it at 105° for 4 hours: it loses
not more than 0.5% of its weight.
Residue on ignition (281): not more than 0.1%.
Delete the following:
°Heavy metals, Method | (231): 0.001%.¢(otficiat 14jan-2018)
Chromatographic purity—Prepare a solution of it in
methanol containing 50 mg per mL (Test solution A). Dilute
1.0 mL of Test solution A to 10 mL with methanol, and mix
(Test solution B). Prepare a solution of USP Levamisole Hy-
drochloride RS in methanol having a concentration of 5 mg
per mL (Reference solution A). Dilute 1.0 mL of Test solution B
to 20 mL with methanol, and mix (Reference solution B). Ap-
ply separate 10-L portions of the four solutions on the
ae line to a suitable thin-layer chromatographic plate
(see Cromatooraply (621)), coated with a 0.25-mm layer of
chromatographic silica gel mixture. Allow the spots to dry,
and develop the chromatogram in a solvent system consist-
ing of a mixture of toluene, acetone, and ammonium hy-
droxide (60:40:1) until the solvent front has moved about
three-fourths of the length of the plate. Remove the plate
from the developing chamber, and dry it at 105° for
15 minutes. Locate the spots on the plate by examination
under short-wavelength UV light: any spot obtained from
Test solution A, other than the one corresponding to
levamisole, does not exceed, in size or intensity, the princi-
pal spot obtained from Reference solution B, corresponding
to not more than 0.5% of any individual impurity. Expose
the plate to iodine vapor in a closed chamber for 15 min-
utes, and locate the spots on the plate: any spot obtained
from Test solution A, other than the one corresponding to
levamisole, does not exceed, in size or intensity, the princi- iS
4)
pal spot obtained from Reference solution B, corresponding a)
to not more than 0.5% of any individual im urity, and the
total of all impurities found does not exceed 1.0%. K
)
Assay—Dissolve about 200 mg of Levamisole Hydrochlo- =
ride, accurately weighed, in 30 mL of alcohol. Add 5.0 mL ro)
of 0.01 N hydrochloric acid, and titrate with 0.1 N sodium reo)=
hydroxide VS, determining the two inflection points potenti- i
ometrically. Determine the volume, in mL, of 0.1 N sodium as)
hydroxide consumed between the two inflection points.
oy
7)
Each mL of 0.1 N sodium hydroxide consumed is equivalent
to 24.08 mg of CyHi2N2S - HCI.
Levamisole Hydrochloride Tablets
» Levamisole Hydrochloride Tablets contain an
amount of Levamisole Hydrochloride equivalent
to not less than 90.0 panei and not more than
110.0 percent of the labeled amount of levamis-
ole (Ci1Hi2N2S).
Packaging and storage—Preserve in well-closed contain-
ers,
Labeling—Label it to state both the content of the active
ae and the content of the salt used in formulating the
article.
USP Reference standards (11)—
USP Levamisole Hydrochloride RS
Identification—
A: The retention time of the major peak for levamisole in
the chromatogram of the Assay preparation corresponds to
 2370 Levamisole / Official Monographs
that in the chromatogram of the Standard preparation, as
obtained in the Assay.
B: The R; value of the principal spot obtained from Test
solution B in the Chromatographic purity test corresponds to
that from Standard solution A.
Dissolution (711)—
Medium: 0.01 N hydrochloric acid; 900 mL.
Apparatus 2: 50 rpm.
Time: 45 minutes.
Procedure—Determine the amount of levamisole
(CiiHi2N2S) dissolved by employing UV absorption at the
wavelength of maximum absorbance at about 214 nm on
filtered portions of the solution under test, suitably diluted
with Dissolution Medium, if necessary, in comparison with a
Standard solution having a known concentration of USP
Levamisole Hydrochloride RS in the same Medium.
Tolerances—Not less than 80% (Q) of the labeled amount
of Ci,Hi2N2S is dissolved in 45 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Chromatographic purity—
Test solution A—Transfer an amount of powdered Tablets,
equivalent to 100 mg of levamisole, to a glass test tube.
Add 5.0 mL of methanol, shake for 2 minutes, and filter.
Test solution B—Dilute 1.0 mL of Test solution A to 10 mL
with methanol, and mix.
Standard solution A—Prepare a solution of USP Levamisole
Hydrochloride RS in methanol having a concentration of
2.4 mg per mL (equivalent to 2.0 mg of levamisole per mL).
Standard solution B—Dilute 1.0 mL of Standard solution A
to 20 mL with methanol, and mix.
Procedure—Apply separate 10-uL portions of Test solutions
A and B and Standard solutions A and B to the starting line
of a suitable thin-layer chromatographic plate (see Chroma-
fe tography (621)) coated with a 0.25-mm layer of chromato-
ww
a graphic silica gel mixture. Allow the spots to dry, and de-
Ss velop the chromatogram in a solvent system conseting ofa
—
2) mixture of toluene, acetone, and ammonium hydroxide
i} (60:40:1) until the solvent front has moved about three-
=
5 fourths of the length of the plate. Remove the plate from
Ps the developing chamber, and dry the plate at 105° for
15 minutes. Locate the spots on the plate by examination
os under short-wavelength UV light: any spot obtained from
A)
=) Test solution A, other than that of levamisole, does not ex-
ceed, in size or intensity, the principal spot obtained from
Standard solution B, corresponding to not more than 0.5%
of any individual impurity. Expose the plate to iodine vapor
in a closed chamber for 15 minutes, and locate the spots on
the plate: any spot obtained from Test solution A, other than
that of levamisole, does not exceed, in size or intensity, the
principal spot obtained from Standard solution B, corre-
sponding to not more than 0.5% of any individual impurity.
Assay—
Solution A—Prepare a 0.75% solution of monobasic am-
monium phosphate in water, and adjust with diisopropyla-
mine to a pH of 7.
Solution B—Use acetonitrile.
Mobile phase—Use variable mixtures of Solution A and So-
lution B as directed for Chromatographicsystem. Make ad-
justments if necessary (see System Suitability under Chroma-
tography (621)).
Standard preparation—Transfer about 20 mg of USP
Levamisole Hydrochloride RS, accurately weighed, to a
100-mL volumetric flask, add 10 mL of water, and swirl to
dissolve. Dilute with methanol to volume, and mix to obtain
a solution having a known concentration of about 0.2 mg of
USP Levamisole Hydrochloride RS per mL.
Resolution solution—Dissolve 20 mg of Levamisole Hydro-
chloride in 5 mL of 0.1 N sodium hydroxide, and heat at
USP 41
100° in a closed vial for 5 hours. Allow to cool, and dilute
1 mL of the solution to 25 mL with methanol.
Assay persaiie case an accurately counted num-
ber of Tablets, equivalent to about 150 mg of levamisole
(CiiHi2N2S), to a 100-mL volumetric flask. Add 25 mL of
water, and shake by mechanical means for 30 minutes. Di-
lute with water to volume, and mix. Transfer 10.0 mL of this
solution to a second 100-mL volumetric flask, dilute with
methanol to volume, and mix.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 215-nm detector
and a 4.6-mm x 10-cm column that contains 3-um packing
L1. The flow rate is about 2 mL per minute. The chromato-
graph is programmed as follows.
Time Solution A Solution B
(minutes) (%) (%) Elution
0-5 8020 20-80 linear gradient
5-7 20 80 isocratic
7-8 20-80 80-20 linear gradient
8-12 80 20 isocratic
Chromatograph the Resolution solution, and record the peak
responses as directed for Procedure: the relative retention
times are 1.0 for levamisole and about 1.3 for the major
degradation product; and the resolution, R, between
levamisole and the major degradation product is not less
than 6.0. Chromatograph the Standard preparation, and re-
cord the peak responses as directed for Procedure: the ca-
pacity factor, k’, is not less than 3.0; the tailing factor is not
more than 1.8; and the relative standard deviation for repli-
cate injections is not more than 2.0%.
Procedure—Separately inject equal volumes (about 10 uL)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
ure the areas for the major peaks. Calculate the quantity, in
mg, of levamisole (C;,Hi2N2S) in the Tablets taken by the
formula:
(204.29 / 240.75)(10000(ru
/rs)
in which 204.29 and 240.75 are the molecular weights of
levamisole and levamisole hydrochloride, respectively; C is
the concentration, in mg per mL, of USP Levamisole Hydro-
chloride RS in the Standard preparation; and ry and rs are
the levamisole peak responses obtained from the Assay prep-
aration and the Standard preparation, respectively.
Levetiracetam
CgHi4N202 170.21
1-Pyrrolidineacetamide, a-ethyl-2-oxo-, («5)-;
(-)-(S)-a-Ethyl-2-oxo-1-pyrrolidineacetamide [102767-28-2].
DEFINITION
Levetiracetam contains NLT 98.0% and NMT 102.0% of
levetiracetam (CgH14N202), calculated on the anhydrous
and solvent-free basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
e B. The retention time of the major peak of the Identifica-
tion solution corresponds to that of the levetiracetam S-
USP 41 Official Monographs / Levetiracetam 2371

enantiomer from the System suitability solution, as ob- IMPURITIES

tained in the test for Limit of Levetiracetam R-Enantiomer. e RESIDUE ON IGNITION (281): NMT 0.1%
ASSAY
© PROCEDURE Delete the following:
Buffer: 2.7 g/L of monobasic potassium phosphate in
water. Adjust with 2% aqueous potassium hydroxide ®o HEAVY METALS, Method iI (231): 20 ppme‘otic 1-4an-2018)
(w/v) to a pH of 5.5. e LIMIT OF LEVETIRACETAM R-ENANTIOMER
Solution A: Acetonitrile and Buffer (1:19) Mobile phase: n-Hexane and dehydrated alcohol
Solution B: Acetonitrile (80:20)
Mobile phase: See Table 1. System suitability solution: 0.1 mg/mL of USP Leve-
tiracetam Racemic Mixture RS in Mobile phase
Table 1 Standard solution: 0.05 mg/mL of USP Levetiracetam
RS in Mobile phase
Solution A Solution B Sample solution: 10 mg/mL of Levetiracetam in Mobile
ase
IGentification solution: 0.05 mg/mL of Levetiracetam
100 from Sample solution in Mobile phase
Chromatographic system
(See Chromatography (621), System Suitability.)
System suitability solution: 0.2 mg/mL of USP Leve- Mode: LC
tiracetam RS and 0.08 mg/mL of USP Levetiracetam Re- Detector: UV 215 nm
lated Compound A RS in Solution A. Prepare by first Column: 4.6-mm x 25-cm; 10-4um packing L51
dissolving the required amount of USP Levetiracetam RS Flow rate: 1.0 mL/min
in a suitable volumetric flask. Add 10% of the flask vol- Injection volume: 20 uL
ume of 0.1 N potassium hydroxide. Let the mixture re- System suitability
act at room temperature for about 15 min, and then Samples: System suitability solution and Identification
neutralize by adding 0.1 N hydrochloric acid at 10% of solution
the flask volurhe, Add the required amount of USP {[Note—The relative retention times for levetiracetam R-
Levetiracetam Related CompoundARS, sonicate to dis- enantiomer and levetiracetam S-enantiomer are 0.55
solve, dilute with Solution A to volume, and mix. and 1.0, respectively. Use the chromatogram from the
[Nott—Levetiracetam related compoundAis included Identification solution for Identification test B.]
for peak identification purposes.] Suitability requirements
Standard solution: 0.1 mg/mL of USP Levetiracetam RS Resolution: NLT 4.0 between the R- and S-enanti-
in Solution A omers, System suitability solution. [NoTE—If a loss of
Sample solution: 0.1 mg/mL of Levetiracetam in Solu- resolution (less than 4.0) is observed, it is recom-
tion A mended that the column temperature be maintained
Chromatographic system at 25° to stabilize the system.
(See Chromatography (621), System Suitability.) Analysis a
Mode: LC Samples: Standard solution and Sample solution v
Detector: UV 205 nm Calculate the percentage of levetiracetam R-enantiomer |<
Column: 4.6-mm x 15-cm; packing L1 in the portion of Levetiracetam taken: g
Flow rate: 0.9 mL/min
Injection volume: 10 LL Result = (ru/rs) x (Cs/Cu) x 100 AS
System suitability
|
Sample: System suitability solution ru = peak response of levetiracetam R-enantiomer a
[Note—See Table 2 for relative retention times.] from the Sample solution =
Suitability requirements ls = peak response of levetiracetam from the oa
Relative standard deviation: NMT 1.0%, for the Standard solution
levetiracetam peak Cs = concentration of USP Levetiracetam RS in the
[Note—lf system suitability criteria cannot be met, it is Standard solution (mg/mL)
recommended that the column temperature be main- Cu = concentration of Levetiracetam in the Sample
tained at 20° to stabilize the system.] solution (mg/mL)
Analysis Acceptance criteria: NMT 0.8%
Samples: Standard solution and Sample solution e LIMIT OF LEVETIRACETAM RELATED COMPOUND B
Calculate the percentage of levetiracetam (CsH14N202) [Notr—Perform this test only if levetiracetam related
in the portion of Levetiracetam taken: compoundBis a known process impurity.]
Buffer: 1.22 g of sodium 1-decanesulfonate in 1 L of
Result = [(ru/rs) x (Cs/Cu) x 100] - F water containing about 1.3 mL of phosphoric acid. Ad-
ie with 20% (w/v) potassium hydroxide to a pH of
ru = peak response of levetiracetam from the
Sample solution Mobile phase: Acetonitrile and Buffer (3:17)
rs = peak response of levetiracetam from the System suitability solution: 2 mg/mL of USP Leve-
Standard solution tiracetam Related CompoundB RS in Mobile phase
Gs = concentration of USP Levetiracetam RS in the Standard solution: 0.002 mg/mL of USP Levetiracetam
Standard solution (mg/mL) Related Compound B RS in Mobile phase
Cy = concentration of Levetiracetam in the Sample sample solution: 2.0 mg/mL of Levetiracetam in Mobile
solution (mg/mL) phase
F = percentage of levetiracetam R-enantiomer Chromatographic system
from the test for Limit of Levetiracetam R- (See Chromatography (621), System Suitability.)
Enantiomer Mode: LC
Acceptance criteria: 98.0%-102.0% on the anhydrous Detector: UV 200 nm
and solvent-free basis Column: 4.6-mm x 25-cm; packing L1
 2372 Levetiracetam / Official Monographs USP 41

Flow rate: 1.0 mL/min Table 2

Injection volumes Relative Relative Acceptance
System suitability: 10 wL Retention Response Criteria,
Analysis: 50 ub Name Time Factor NMT (%)
System suitability
Sample: System suitability solution Pyridin-2-ole 0.37 1,0) 0.025
[Note—The retention time for levetiracetam related Levetiracetam acide 0.62 1.2 0.3
compoundB is 9 min.] Levetiracetam 1.00 — =
Suitability requirements Levetiracetam related
Tailing factor: NMT 3.0. [NoTeE—If a significant tail- compound As 4.25 0.35 0.05
ing of the levetiracetam related compound B peak is Any individual _
observed (greater than 3.0), it is recommended that unspecified impurity 1.0 0.05
the column temperature be maintained at 27° to sta- Total impurities = = 0.4
bilize the system.]
@ Not included in the total impurities limit.
Relative standard deviation: NMT 2.0%
Hee eee alain -yl)butanoic acid. Included in the total impurities
Analysis limit,
Samples: Standard solution and Sample solution ¢ (S)-N-(1-Amino-1-oxobutan-2-yl)-4-chlorobutanamide. Included in the to-
Calculate theperceniage of levetiracetam related com- tal impurities limit only if levetiracetam related compound B is a known
poundBin the portion of Levetiracetam taken: process impurity.

Result = (ru/rs) x (Cs/Cu) X (Ma/Mjz) x 100 SPECIFIC TESTS

tu = peak response of levetiracetam related
compound B from the Sample solution
rs = peak response of levetiracetam related
compoundB from the Standard solution
G = concentration of USP Levetiracetam Related
Compound BRS in the Standard solution
(mg/mL)
cy = concentration of Levetiracetam in the Sample
solution (mg/mL)
My = molecular weight of levetiracetam related
compoundBfree base, 102.1
Mr = molecular weight of levetiracetam related
compound B, 138.6
Acceptance criteria: NMT 0.10%
[Note—The amount of levetiracetam related compound
B measured is to be included in the total impurities in
fe
“
© WATER DETERMINATION (921), Method la: NMT 0.5%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, and store at room temperature.
e USP REFERENCE STANDARDS (11)
USP Levetiracetam RS
USP Levetiracetam Racemic Mixture RS
A 1:1 mixture of:
Levetiracetam S-enantiomer-(25)-2-(2-oxopyrrolidin-
1-yl)butanamide;
Levetiracetam R-enantiomer (2R)-2-(2-oxopyrrolidin-
1-yl)butanamide.
USP Levetiracetam Related Compound A RS
(S)-N-(1-Amino-1 -oxobutan-2-yl)-4-chlorobutanamide.
CsHisCIN202 206.67
USP Levetiracetam Related Compound B RS

the test for Organic Impurities. ] (S)-2-Aminobutanamide hydrochloride.

ro © ORGANIC IMPURITIES CaHioN20 HCl 138.6
S
aD
ond
Buffer, Solution A, Solution B, Mobile phase, System
S} suitability solution, Chromatographic system, and
fe System suitability: Proceed as directed in the Assay.
5 Standard solution: 0.005 mg/mL of USP Levetiracetam
= RS in Solution A Levetiracetam Injection
a Sample solution: 5 mg/mL of Levetiracetam in Solution
al
=) A DEFINITION
Analysis Levetiracetam Injection is a sterile solution of levetiracetam
Samples: Standard solution and Sample solution in Water for Injection and contains NLT 90.0% and NMT
Calculate the percentage of each impurity in the por- 110.0% of the labeled amount of levetiracetam
tion of Levetiracetam taken: (CgHi4N20z). Levetiracetam Injection may contain buffer-
ing and isotonicity agents. Levetiracetam Injection con-
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100 tains no antimicrobial agent.
tu = peak response of each impurity from the IDENTIFICATION
Sample solution e A. The retention time of theinaios peak of the Sample
Is = peak response of levetiracetam from the solution corresponds to that of the Standard solution, as
Standard solution obtained in the Assay.
Cs = concentration of USP Levetiracetam RS in the
Standard solution (mg/mL) ASSAY
Cy = concentration of Levetiracetam in the Sample © PROCEDURE
solution (mg/mL) Buffer: 1.0 g/L of anhydrous dibasic potassium phos-
F = relative response factor (see Table 2) phate in water. Adjust with phosphoric acid to a pH of
[Note—Disregard any peak witha relative retention
time of 0.19 or less.] Mobile phase: Acetonitrile and Buffer (6:94)
Acceptance criteria: See Table 2. Diluent: Acetonitrile and water (6:94)
System suitability solution: Solution containing leve-
tiracetam and levetiracetam acid prepared from a solu-
tion of 0.2 mg/mL of USP Levetiracetam RS as follows.
Dissolve the required amount of USP Levetiracetam RS
in 10% of the final volume of 0.1 N potassium hydrox-
ide. Let the mixture react at room temperature for
about 15 min, then neutralize by adding 10% of the
USP 41 Official Monographs / Levetiracetam 2373

flask volume of 0.1 N hydrochloric acid. Dilute with Dil- Cs = concentration of USP Levetiracetam RS in the
uent to volume. Standard solution (g/mL)
Standard solution: 100 t1g/mL of USP Levetiracetam RS Cu = nominal concentration of levetiracetam in the
in Diluent. Sonication may be used to aid in dissolution Sample solution (ug/mL)
if necessary. Acceptance criteria: See Table 1.
Sample solution: Nominally 100 g/mL of leve-
tiracetam from NLT 2 mL of Injection in Diluent Table 1
Chromatographic system
(See Chromatography (621), System Suitability.) Relative Acceptance
Mode: LC Retention Criteria,
Detector: UV 205 nm Name Time NMT (%)
Column: 3.9-mm x 30-cm; 10-m packing L1 Levetiracetam acid® 0.4 0.3
Flow rate: 1 mL/min Levetiracetam 1.0 =
Injection volume: 20 uL Any individual unspecified _
Run time: NLT 1.5 times the retention time of degradation product 0.10
levetiracetam Total impurities = 1.00
System suitability
@ (S)-2-(2-Oxopyrrolidin-1-yl)butanoic acid
Samples: System suitability solution and Standard
solution SPECIFIC TESTS
[Note—Identify the peaks using the relative retention e PH (791): 5.0-6.0
times given in Table 7.] e BACTERIAL ENDOTOXINS TEST (85): Contains NMT 0.175
Sultability requirements USP Endotoxin Units/mg of levetiracetam
Tailing factor: NMT 2.0 for the levetiracetam peak, e STERILITY TESTS (71): Meets the requirements when
System suitability solution tested as directed for Aqueous Solutions under Test for Ste-
Relative standard deviation: NMT 1.5%, Standard rility of the Product to Be Examined, Membrane Filtration
solution © PARTICULATE MATTER IN INJECTIONS (788): Meets the re-
Analysis quirements for small-volume injections
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of leve- ADDITIONAL REQUIREMENTS
Breccia (CsHi4N202) in the portion of Injection © PACKAGING AND STORAGE: Preserve in well-closed Type |
taken: glass vials. Store at controlled room temperature.
¢ LABELING: Label the article to indicate that the Injection is
Result = (ru/rs) x (Cs/Cu) x 100 to be diluted prior to administration.
ty = peak response of levetiracetam from the
Sample solution Change to read:
rs = peak response of levetiracetam from the
Standard solution © USP REFERENCE STANDARDS (11) =
Cs = concentration of USP Levetiracetam RS in the “Ss (CN 1-May-2018) 4)
Standard solution (\ug/mL) USP Levetiracetam RS a]
Cu = nominal concentration of levetiracetam in the =
Sample solution (g/mL) °
Acceptance criteria: 90.0%-110.0% |
°
io}
IMPURITIES Levetiracetam Oral Solution a)
»
¢ ORGANIC IMPURITIES mo]
Buffer, Mobile phase, Diluent, System suitability solu- DEFINITION _
tion, Sample solution, and Chromatographic sys- a
Levetiracetam Oral Solution contains NLT 90.0% and NMT
tem: Proceed as directed in the Assay. 110.0% of the labeled amount of levetiracetam
Standard solution: 0.1 g/mL of USP Levetiracetam RS
in Diluent (CsHi4N20z).
System suitability IDENTIFICATION
Samples: System suitability solution and Standard e A. The retention time of the gion peat in the Sample
solution solution corresponds to that of the Standard solution, as
[Note—Identify the peaks using the relative retention obtained in the Assay.
times in Table 7.]
Suitability requirements ASSAY
Tailing factor: NMT 2.0 for the levetiracetam peak, © PROCEDURE
System suitability solution Solution A: Dilute 1 mL of phosphoric acid with water
Relative standard deviation: NMT 10.0%, Standard toll.
solution Solution B: Acetonitrile
Signal-to-noise ratio: NLT 10, Standard solution Mobile phase: See Table 1.
Analysis
Samples: Standard solution and Sample solution Table 1
Calculate the percentage of levetiracetam acid and any
other unspecified degradation product in the portion Solution A Solution B
of Injection taken:
Result = (ru/rs) x (Cs/Cu) x 100

tu = peak response of levetiracetam acid or any

individual unspecified degradation product
from the Sample solution
ls = peak response of levetiracetam from the
Standard solution
 2374 Levetiracetam / Official Monographs USP 41

Standard solution: 1.0 mg/mL of USP Levetiracetam RS ent to volume. [NOTE—This solution contains leve-
in Solution A tiracetam, levetiracetam acid, and levetiracetam related
Sample solution: Nominally 1.0 mg/mL of leve- compound A.]
tiracetam prepared as follows. Transfer a suitable vol- Standard solution: 3 g/mL of USP Levetiracetam RS in
ume of the Oral Solution to a suitable volumetric flask Solution A
to obtain 1.0 mg/mL final concentration of leve- Sample solution: Nominally 2 mg/mL of levetiracetam
tiracetam. Add 60% of the flask volume of Solution A, prepared as follows. Transfer a suitable volume of the
and sonicate at room temperature for 5 min with inter- Oral Solution to a suitable volumetric flask. Add 60% of
mittent shaking. Allow the solution to cool, and dilute the flask volume of Solution A, and sonicate at room
with Solution A to volume. Pass a portion of the solution temperature for 5 min with intermittent shaking. Allow
under test through a suitable filter. the solution to cool, and dilute with Solution A to vol-
Chromatographic system Aras: Pass a portion of the solution through a suitable
(See Chromatography (621), System Suitability.) ilter.
Mode: LC Chromatographic system
Detector: UV 230 nm (See Chromatography (621), System Suitability.)
Column: 4.6-mm x 15-cm; 5-um packing L1 Mode: LC
Flow rate: 1.5 mL/min Detector: UV 210 nm
Injection volume: 20 uL Column: 4.6-mm x 15-cm; 5-um packing L1
System suitability Column temperature: 45°
Sample: Standard solution Flow rate: 71 mL/min
Suitability requirements Injection volume: 20 pL
Tailing factor: NMT 2.0 System suitability
Relative standard deviation: NMT 2.0% Samples: System suitability solution and Standard
Analysis solution
Samples: Standard solution and Sample solution Suitability requirements
Calculate the percentage of the labeled amount of leve- Resolution: NLT 2.0 between levetiracetam related
tiracetam (CgH;4N2Oz) in the portion of Oral Solution compoundA and levetiracetam acid, System suitabil-
taken: ity solution
Tailing factor: NMT 2.0, Standard solution
Result = (ru/rs) x (Cs/Cu) x 100 Relative standard deviation: NMT 5.0%, Standard
solution
ty = peak response of levetiracetam from the Analysis
Sample solution Samples: Standard solution and Sample solution
rs = peak response of levetiracetam from the Calculate the percentage of each impurity in the por-
Standard solution tion of Oral Solution taken:
Cs = concentration of USP Levetiracetam RS in the
Standard solution (mg/mL) Result = (ru/rs) x (Cs/Cu) x (1/7) x 100
G = nominal concentration of levetiracetam in the
oe
wal

a Sample solution (mg/mL) ru = peak response of the impurity from the

ci Acceptance criteria: 90.0%-110.0% Sample solution
J
=) rs = peak response of levetiracetam from the
3 IMPURITIES Standard solution
iS © ORGANIC IMPURITIES Cs = concentration of USP Levetiracetam RS in the
3 Solution A: Dilute 2 mL of phosphoric acid with water Standard solution (mg/mL)
= to 1L. Cu = nominal concentration of levetiracetam in the
os Solution B: Acetonitrile Sample solution (mg/mL)
a) Diluent: Acetonitrile and Solution A (5:95) é = relative response factor for each impurity (see
=) Mobile phase: See Table 2. Table 3)
Acceptance criteria: See Table 3.
Table 2
Solution A Solution B Table 3
%' Relative Relative Acceptance!
Retention Response Criteria,
e 5 Name Time Factor NMT (%)
Levetiracetam 1.00 = i
0. 25 Levetiracetam related
50 compound Aa» 1.38 “a a
40 50 Levetiracetam acid¢ 1.46 0.92 0.3
41 0 Any individual unspeci-
fied degradation _—
50 0
product 1.0 0.10
System suitability solution: 0.2 mg/mL of USP Leve- Total impurities = = 1.0;
tiracetam RS and 0.1 mg/mL of USP Levetiracetam Re- 2(S)-N-(1-Amino-1-oxobutan-2-yl)-4-chlorobutanamide.
lated Compound ARS in Diluent prepared as follows. aps is a process impurity and included for peak identification purposes
Dissolve the required amount of USP Levetiracetam RS only.
in 10% of the final volume of 0.1 N potassium hydrox- ¢(S)-2-(2-Oxopyrrolidin-1-yl)butanoic acid.
ide. Let the mixture react at room temperature for SPECIFIC TESTS
about 15 min, and then neutralize by adding 0.1 N © PH (791): 4.8-6.3
hydrochloric acid at 10% of the flask volume. Add the e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
required amount of USP Levetiracetam Related Com- FIED MICROORGANISMS (62): The total aerobic microbial
poundA RS, sonicate to dissolve, and dilute with Dilu- count does not exceed 10? cfu/mL. The total yeasts and
USP 41 Official Monographs / Levetiracetam 2375

molds count does not exceed 10! cfu/mL. It meets the System suitability
requirement of the test for absence of Escherichia coli. Sample: Standard solution
Suitability requirements
ADDITIONAL REQUIREMENTS Tailing factor: NMT 2.0
© PACKAGING AND STORAGE: Preserve in light-resistant con- Relative standard deviation: NMT 2.0%
tainers. Store at controlled room temperature. Analysis
e USP REFERENCE STANDARDS (11) Samples: Standard solution and Sample solution
USP Levetiracetam RS Calculate the percentage of the labeled amount of leve-
USP Levetiracetam Related Compound A RS tiracetam (CsHi4N2Oz) in the portion of Tablets taken:
(S)-N-(1-Amino-1 -oxobutan-2-yl)-4-chlorobutanamide.
CsHisCIN202 206.67 Result = (ru/rs) x (Cs/Cu) x 100
ry = peak response from the Sample solution
rs = peak response from the Standard solution
Cs = concentration of USP Levetiracetam RS in the
Levetiracetam Tablets Standard solution (mg/mL)
Cy = nominal concentration of levetiracetam in the
DEFINITION Sample solution (mg/mL)
Levetiracetam Tablets contain NLT 90.0% and NMT 110.0% Acceptance criteria: 90.0%-110.0%
of the labeled amount of levetiracetam (CgHi4N202). PERFORMANCE TESTS
IDENTIFICATION e DISSOLUTION (711)
¢ A. INFRARED ABSORPTION (197K), (197A) Test 1
Standard solution: 1 mg/mL solution of USP Leve- Medium: Water; 900 mL
tiracetam RS in solution prepared as follows. Transfer a Apparatus 2: 50 rpm
suitable quantity of USP Levetiracetam RS to a suitable Time: See Table 1.
volumetric flask. Add 70% of the flask volume of ace-
tone. Sonicate for 15 min. Dilute with acetone to Table 1
volume. Tablet Strength
Standard: Pass 10 mL of the Standard solution through
a membrane filter of 0.45-11m pore size. Evaporate ace-
tone from the filtrate completely to form crystals.
Scratch the crystals. Weigh 2-4 mg of the residue and
200 mg of KBr in a mortar and pestle. Mix and grind
well, and prepare the KBr pellet.
Sample solution: Transfer an amount of finely pow-
dered Tablets (NLT 20) equivalent to 250 mg of leve- Buffer: 6.8git of monobasic potassium phosphate,
tiracetam to a 50-mL volumetric flask. Add35 mL of adjusted with dilute potassium hydroxide to a pH of =
acetone. Sonicate for 15 min. Dilute with acetone to 5.6 wn
a)
volume. Mobile phase: Acetonitrile and Buffer (15:85)
Sample: Pass 10 mL of the Sample solution through a Standard solution: (1/1000) mg/mL in Medium, where =
membrane filter of 0.45-t1m pore size. Evaporate ace- Lis the Tablet label claim, in mg }
Sample solution: Pass a portion of the solution under oo]
tone from the filtrate completely to form crystals. }
Scratch the crystals. Weigh 2-4'mg of the residue and test though a suitable filter of 0.45-um pore size. eo}=)
200 mg of KBr in a mortar and pestle. Mix and grind Chromatographic system i)
well, and prepare the KBr pellet. (See Chromatography (621), System Suitability.) a}
Analysis: Record the spectra of the Standard and Sam- Mode: LC Ee
aad
ple between 4000 cm: and 650 cm". Detector: UV 220 nm
Acceptance criteria: The spectrum of the Sample corre- Column: 4.6-mm x 15-cm; 5-um packing L1
sponds to that of the Standard. Flow rate: 1.2 mL/min
° B. The retention time of the major peak of the Sample Injection volume: 10 pL
solution corresponds to that of the Standard solution, as System suitability
obtained in the Assay. Sample: Standard solution
Suitability requirements
ASSAY Tailing factor: NMT 2.0
© PROCEDURE Relative standard deviation: NMT 2.0%
Buffer: 1.4 g/L of monobasic potassium phosphate and Analysis
0.6 g/L of sodium 1-heptanesulfonate, adjusted with Samples: Standard solution and Sample solution
phosphoric acid to a pH of 2.8 Calculate the percentage of the labeled amount of
Mobile phase: Acetonitrile and Buffer (8:92) levetiracetam (CgH:4N2O2) dissolved:
Diluent: Acetonitrile and water (20:80)
Standard solution: 0.35 mg/mL of USP Levetiracetam Result = (ru/rs) x (Cs/L) x V x 100
RS in Diluent. Sonication may be used to aid dissolution.
Sample solution: Bemba 0.4 mg/mL of leve- ru = peak response from the Sample solution
tiracetam from NLT 20 Tablets, finely crushed, in Dilu- ls = peak response from the Standard solution
ent. Sonication may be used to aid dissolution. Cs = concentration of USP Levetiracetam RS in the
Chromatographic system Standard solution (mg/mL)
(See Chromatography (621), System Suitability.) L = label claim (mg/Tablet)
Mode: LC Vv = volume of Medium, 900 mL
Detector: UV 220 nm Tolerances
Column: 4.6-mm x 25-cm; 4-1um packing L1 NLT 70% (Q) of the labeled amount of levetiracetam
Flow rate: 2 mL/min (CgHi4N2O2) in 15 min for Tablets labeled to contain
Injection volume: 10 LL 250, 500, or 750 mg; NLT 80% (Q) of the labeled
amount of levetiracetam (CsH:4N202) in 30 min for
Tablets labeled to contain 1000 mg.
 2376 Levetiracetam / Official Monographs USP 41

Test 2: If the product complies with this test, the label- phase. [NoTE—Sonicate if necessary, and centrifuge the
ing indicates that the product meets USP Dissolution solution before passing through a suitable filter.]
Test 2. Chromatographic system
Medium: Water; 900 mL, deaerate, if necessary (See Chromatography (621), System Suitability.)
Apparatus 2: 50 rpm Mode: LC
Time: 15 min Detector: UV 200 nm
Buffer: 1.36 g/L of monobasic potassium phosphate, Column: 4.6-mm x 25-cm; 4-m packing L1
adjusted with 10% potassium hydroxide to a pH of Flow rate: 1 mL/min
5.0 Injection volume: 10 uL
Mobile phase: Acetonitrile and Buffer (10:90) System suitability
Standard solution: 54 g/mL of USP Levetiracetam RS Samples: System suitability solution and Standard
in Medium solution
Sample solution: Pass a portion of the solution under Suitability requirements
test throughasuitable filter. Dilute an aliquot with Resolution: NLT 2.0 between levetiracetam related
Medium to obtain a concentration similar to that of compound B and levetiracetam, System suitability
the Standard solution. solution
Chromatographic system Tailing factor: NMT 2.0, Standard solution
(See Chromatography (621), System Suitability.) Relative standard deviation: NMT 10.0%, Standard
Mode: LC solution
Detector: UV 210 nm Analysis
Column: 4.6-mm x 15-cm; 5-um packing L1 Samples: Standard solution and Sample solution
Column temperature: 30° Calculate the percentage of each impurity in the por-
Flow rate: 1.5 mL/min tion of Tablets taken:
Injection volume: 20 LL
System suitability Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
Sample: Standard solution
Suitability requirements tu = peak response of each impurity from the
Tailing factor: NMT 1.5 Sample solution
Relative standard deviation: NMT 1.0% rs = peak response of levetiracetam from the
Analysis Standard solution
Samples: Standard solution and Sample solution Gs = concentration of USP Levetiracetam RS in the
Calculate the percentage of the labeled amount of Standard solution (mg/mL)
levetiracetam (CsHi4N202) dissolved: Cu = nominal concentration of levetiracetam in the
Sample solution (mg/mL)
Result = (ru/rs) x (Cs/L) x D x Vx 100 F = relative response factor (see Table 2)
Acceptance criteria: See Table 2.
tu = peak response from the Sample solution
fs = peak response from the Standard solution
Rr
“
Table 2
is Cs = concentration of USP Levetiracetam RS in the
ys} Standard solution (mg/mL) Relative Relative Acceptance
h
D b = label claim (mg/Tablet Retentlon | Response Criterla,
S) D = dilution factor of the Sample solution Name Time Factor NMT (%)
= Vv = volume of Medium, 900 mL Levetiracetam related uy fic
5 Tolerances: NLT 80% (Q) of the labeled amount of compound B= 0.54
= levetiracetam (CsHi4N202) is dissolved. Levetiracetam 1.0 = =
is Test 3: If the product complies with this test, the label- Levetiracetam related a ei
w
=) ing indicates that the product meets USP Dissolution compound Az» 17
Test 3. Levetiracetam acid< Zi 0.79 0.3
Medium: Water; 900 mL
Apparatus 2: 50 rpm Any individual
Time: 30 min unspecified degrada- -
Buffer, Mobile phase, Standard solution, Sample so- tion product 1.0 0.1
lution, Chromatographic system, System suitability, Total impurities — = 0.6
and Analysis: Proceed as directed for Test 7. These impurities are listed for information only; they are process impuri-
Tolerances: NLT 80% (Q) of the labeled amount of ties, which are controlled in the drug substance.
levetiracetam (CgHi4N202) is dissolved. » (5)-N-(1-Amino-1 -oxobutan-2-yl)-4-chlorobutanamide.
e UNIFORMITY OF DOSAGE UNITS (905): Meet the € (5)-2-(2-Oxopyrrolidine-1-yl)butanoic acid.
requirements ADDITIONAL REQUIREMENTS
IMPURITIES © PACKAGING AND STORAGE: Preserve in tight containers.
© ORGANIC IMPURITIES Store at controlled room temperature.
Buffer: 6.8 g/L of monobasic potassium phosphate and e LABELING: When more than one Dissolution test is given,
0.85 g/L of sodium 1-heptanesulfonate, adjusted with the jabeling states the Dissolution test used only if Test 7
phosphoric acid to a pH of 2.8 is not used.
Mobile phase: Acetonitrile and Buffer (5:95) © USP REFERENCE STANDARDS (11)
System suitability solution: 3.6 ug/mL of USP Leve- USP Levetiracetam RS
tiracetam RS and 3.6 g/mL of USP Levetiracetam Re- USP Levetiracetam Related Compound B RS
lated Compound BRS in Mobile eee (S)-2-Aminobutanamide hydrochloride.
Standard solution: 3.6 g/mL of USP Levetiracetam RS C4HioN20- HCI 138.60
in Mobile phase
Sample solution: Equivalent to 1.2 mg/mL of leve-
tiracetam from NLT 20 Tablets, finely crushed, in Mobile
USP 41 Official Monographs / Levetiracetam 2377

Analysis
Samples: Standard solution and Sample solution
Levetiracetam Extended-Release Tablets Calculate the percentage of the labeled amount of leve-
tiracetam (CgHi4N2O2) in the portion of Tablets taken:
DEFINITION
Levetiracetam Extended-Release Tablets contain NLT 90.0% Result = (ry/rs) x (Cs/Cu) x 100
and NMT 110.0% of the labeled amount of levetiracetam
(CgHi4N202). ry = peak response of levetiracetam from the
Sample solution
IDENTIFICATION ls = peak response of levetiracetam from the
e A. The retention time of the malo peat of the Sample Standard solution
solution corresponds to that of the Standard solution, as Cs = concentration of USP Levetiracetam RS in the
obtained in the Assay. Standard solution (mg/mL)
ASSAY Cu = nominal concentration of levetiracetam in the
Sample solution (mg/mL)
© PROCEDURE Acceptance criteria: 90.0%-110.0%
Buffer: 1.4 g/L of anhydrous dibasic sodium pis bate
in water. Adjust with phosphoric acid to a pH of 3.5. PERFORMANCE TESTS
Mobile phase: Acetonitrile and Buffer (10:90)
Standard stock solution: 1.0 mg/mL of USP Leve-
tiracetam RS prepared as follows. Weigh a suitable Change to read:
quanuty of the Reference Standard into a volumetric
flask. Add Mobile phase to fill 60% of flask volume and e DISSOLUTION (711)
tetrahydrofuran to fill 4% of flask volume. Sonicate in Test 1
cool water to dissolve. Equilibrate to room temperature. Buffer A: Dissolve 6.8 g of potassium dihydrogen
Dilute with Mobile phase to volume. phosphate and 0.2 g of sodium hydroxide in 1 L of
Standard solution: 0.08 mg/mL of USP Levetiracetam water. If necessary, adjust with 1 N sodium hydroxide
RS in Mobile phase from Standard stock solution. Pass a to a pH of 6.0.
portion of the solution through a suitable filter of 0.45- Medium: Buffer A; 900 mL
Lum pore size. Apparatus 1: 100 rpm
Sample stock solution: Nominally (L/100) mg/mL of Times: 1, 2,4, and 8h
levetiracetam from NLT 5 Tablets prepared as follows, Buffer B: 1.4 g/L of anhydrous dibasic sodium phos-
where L is the label claim in mg/Tablet. Transfer the phate in water. Adjust with phosphoric acid to a pH of
Tablets to a volumetric flask containing tetrahydrofuran 3:5
to fill about 5% of flask volume. Stir for 30 min, and Mobile phase: Acetonitrile and Buffer B (10:90)
allow to stand for 5 min. Sonicate for 20 min with in- Standard stock solution: 1.7 mg/mL of USP Leve-
termittent shaking. Add Mobile phase to fill 80% of final tiracetam RS in water. Sonication may be used to aid
volume, and sonicate in cold water for 20 min with in dissolution,
intermittent shaking. Add methanol to fill 10% of flask Standard solution: (1/900) mg/mL of USP Leve- [=
wr
volume. Dilute with Mobile phase to volume. une tiracetam RS in Medium from Standard stock solution, a=]
for 15 min, and pass a portion of the solution throug where L is the label claim in mg/Tablet. Pass a portion
a suitable filter of 0.2-um pore size. through a suitable filter of 0.45-uum pore size. =
fo)
Alternatively, the Sample stock solution, having a nomi- Sample solution: Pass a portion of the solution under S
nal concentration of 3 mg/mL of levetiracetam, may test throughasuitable filter of 0.45-um pore size. fe]
be prepared as follows. Finely grind NLT 10 Tablets, Chromatographic system Ko}
a
and transfer an amount equivalent to 750 mg of leve- (See Chromatography (621), System Suitability.) iy
tiracetam to a suitable volumetric flask. Add 18% of Mode: LC me]
a
the flask volume of acetonitrile. Sonicate for 10 min Detector: UV 205 nm my
followed by shaking using a mechanical shaker for 10 Column: 4.6-mm x 25-cm; 5-14m packing L7
min. Add 18% of the flask volume of water, and shake Temperatures
for 15 min using a mechanical shaker. Allow the sam- Column: 30°
ple to equilibrate to room temperature, and dilute Autosampler: 10°
with a mixture of acetonitrile and water (50:50) to vol- Flow rate: 1.5 mL/min
ume. Pass a portion of the solution through a suitable Injection volume: 5 uL
filter of 0.45-~um pore size. Run time: 2 times the retention time of levetiracetam
Sample solution: Nominally 0.08 mg/mL of leve- System suitability
tiracetam in Mobile phase from Sample stock solution Sample: Standard solution
Chromatographic system Suitability requirements
(See Chromatography (621), System Suitability.) Tailing factor: NMT 2.0
Mode: LC Relative standard deviation: NMT 2.0%
Detector: UV 205 nm Analysis
Column: 4.6-mm x 25-cm; 5-m packing L7 Samples: Standard solution and Sample solution
Temperatures Calculate the concentration, CG, of levetiracetam
Column: 30° (CsHi4N202) in Medium (mg/mL) after time point i:
Autosampler: 10°
Flow rate: 1.5 mL/min Result; = (ru/rs) x Cs
Injection volume: 10 uL
Run time: 3 times the retention time of levetiracetam tu = peak response from the Sample solution
System suitability Is = peak response from the Standard solution
Sample: Standard solution Cs = concentration of the Standard solution
Suitability requirements (mg/mL)
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0%
 2378 Levetiracetam / Official Monographs USP 41

Calculate the percentage of the labeled amount of Analysis

levetiracetam (CgH:4N2O2) dissolved at each time Samples: Standard solution and Sample solution
point (i): Calculate the concentration, C, of levetiracetam
(CgHi4N202) in Medium (mg/mL) after time point i:
Result; = C; x Vx (1/L) x 100
Result, = (ru/rs) x Cs

Results = [(C2 x V) + (Ci x Vs)] x (1/L) x 100 Tu = peak response from the Sample solution
rs = peak response from the Standard solution
Gs = concentration of the Standard solution
Results = {(C3 x V) + [(C2 + Ci) x VsJ} x (1/L) x 100 (mg/mL)
Calculate the percentage of the labeled amount of
levetiracetam (CgHi4N2O2) dissolved at each time
Results = {(C4 x V) + Kas C, + Ci) x Vs} x (/L) x point ():
00
Result; = CG) x Vx (1/L) x 100
G = concentration of levetiracetam in the portion
of sample withdrawn at the specified time
point (mg/mL) Resultz = {[C. x (V— Vs)] + (Gi x Vs)} x (1/L) x 100
Vv = volume of Medium, 900 mL
L = label claim (mg/Tablet)
Vs = volume of the Sample solution withdrawn at Results = ({C3 x [V— (2 x Vs)]} + [(C2 + Gi) x Vs]) x (1/
each time point and replaced with Medium 1) x 100
mL
Tolerances See Table 1.
Results = ({Ca x [V— (3 x Vs)]} + [(C3 + Co + Gy) x Vs]) x
(1/L) x 100
Table 1
Amount Dissolved G = concentration of levetiracetam in Medium in
500 mg/ 750 mg/ the portion of sample withdrawn at time
Time Point Time Tablet Tablet point i (mg/mL)
i) (h) (%) (%) Vv = volume of Medium, 900 mL
1 1 25-45 33-53
L = label claim (mg/Tablet)
Vs = volume of the Sample solution withdrawn from
2 2 45-65 45-65
the Medium (mL)
3 4 60-80 65-85 Tolerances: See Table 2.
4 8 NLT 80 NLT 80

The percentages of the labeled amount of leve- Table 2

vn
oa tiracetam (CsHi4N2O2), dissolved at the times speci- Amount Dissolved
rs
i] fied, conform to Dissolution (711), Acceptance Table 2. 500 mg/ 750 mg/
Test 2: If the product complies with this procedure, the
i) Time Point Time Tablet Tablet
—

} labeling indicates that it meets USP Dissolution Test 2.

Buffer A: Dissolve 6.8 g of potassium dihydrogen
(i) (h) (%) (%)
c
5 phosphate and 0.2 g of sodium hydroxide in 1 L of
fl 1 22-42 16-36

3 water. If necessary, adjust with 1 N sodium hydroxide Z

3
2
4
39-59
62-82
30-50.
50-70
a to a pH of 6.0.
a) Medium: Buffer A; 900 mL 4 8 NLT 80 NLT 80
=) Apparatus 1: 100 rpm
Times: 1, 2, 4, and 8h The percentages of the labeled amount of leve-
Buffer B: 2.82 g/L of potassium dihydrogen phosphate tiracetam (CgHi4N202), dissolved at the times speci-
in water fied, conform to Dissolution (711), Acceptance Table 2.
Mobile phase: Acetonitrile and Buffer B (5:95). Adjust Test 3: If the product complies with this procedure, the
with phosphoric acid to a pH of 2.0. labeling indicates that it meets USP Dissolution Test 3.
Standard solution: (1/900) mg/mL of USP Leve- Buffer A: Dissolve 6.8 g of potassium dihydrogen
tiracetam RS in Medium, where L is the label claim in phosphate and 0.5 g o ocilies hydroxide in 1 L of
mg/Tablet water. Adjust to a pH of 6.0.
Sample solution: Pass a portion of the solution under Medium: Buffer A; 900 mL
test through a suitable filter of 0.45-um pore size. Apparatus 1: 100 rpm
Chromatographic system Times: 1, 2,4, and 8h
(See Chromatography (621), System Suitability.) Buffer B: 7.8 g/L of monobasic sodium phosphate di-
Mode: LC hydrate in water. Adjust with sodium hydroxide to a
Detector: UV 235 nm H of 5.6.
Columns Mobile phase: Acetonitrile and Buffer B (15:85)
Guard: 4.6-mm x 1-cm, 4.6-mm x 2-cm, or 4.0-mm Standard solution: (1/900) mg/mL of USP Leve-
x 2-cm; 5-tum packing L1 tiracetam RS in Medium, where L is the label claim in
Analytical: 4.6-mm x 5-cm; 5-um packing L1 mg/Tablet
Flow rate: 0.8 mL/min Sample solution: Centrifuge a portion of the solution
Injection volume: 10 uL naer test.
Run time: 2 times the retention time of levetiracetam Chromatographic system
System suitability (See Chromatography (621), System Suitability.)
Sample: Standard solution Mode: LC
Suitability requirements Detector: UV 220 nm
Tailing factor: NMT 2.0 Column: 4.6-mm x 15-cm; 5-um packing L1
Relative standard deviation: NMT 1.5% for five Column temperature: 30°
replicate injections
USP 41 Official Monographs / Levetiracetam 2379

Flow rate: 1.5 mL/min Blank: Medium

Injection volume: 10 uL Instrumental conditions
Run time: 2 times the retention time of levetiracetam Mode: UV
System suitability Analytical wavelength: 210 nm
Sample: Standard solution Analysis
Suitability requirements Samples: Standard solution and Sample solution
Column efficiency: NLT 1500 theoretical plates Calculate the concentration, C, of levetiracetam
Relative standard deviation: NMT 2.0% for six rep- (CeHi4N2O2) in Medium (mg/mL) after time point i:
licate injections
Analysis Result, = (Au/As) x Cs
Samples: Standard solution and Sample solution
Calculate the concentration, G, of levetiracetam Au = absorbance of the Sample solution
(CgHi4N2Oz) in Medium (mg/mL) after time point i: As = absorbance of the Standard solution
Cs = concentration of the Standard solution
Result; = (ru/rs) x Cs (mg/mL)
Calculate the percentage of the labeled amount of
tu = peak response from the Sample solution levetiracetam (CsHi4N202) dissolved at each time
Is = peak response from the Standard solution point (/):
Gs = concentration of USP Levetiracetam RS in the
Standard solution (mg/mL) Result; = C; x Vx (1/L) x 100
Calculate the percentage of the labeled amount of
levetiracetam (CsH14N202) dissolved at each time
point (/): Resultz = [(C2 x V) + (CG, x Vs] x (1/L) x 100

Result; = C; x Vx (1/L) x 100

Results = {(C3 x V) + [(C2 + Cy) x Vs]} x (1/L) x 100

Results = {[Co x (V— Vs)] + (Ci x V9} x (1/L) x 100 Results = {(C, x V) + Lh + C1) x Vs}} x (1/L) x

Results = ({Cz x [V— (2 x Vs)]} + [(Go + G) x Vs}) x (1/

1) x 100 G = concentration of levetiracetam in the portion
of sample withdrawn at the specified time
point (mg/mL)
Result, = ({C; x [V— (3 x Vs)]} + [(G3 + Co + Gi) x Vs]) x Vv = volume of Medium, 900 mL
(1/L) x 100 L = label claim (mg/Tablet)
Vs = volume of the Sample solution withdrawn at
G = concentration of levetiracetam in Medium in each time point and replaced with Medium
the portion of sample withdrawn at time (mL) fans
point / (mg/mL) Tolerances: See Table 4. “
a}
V = volume of Medium, 900 mL
L = label claim (mg/Tablet) Table 4 os
Vs = volume of the Sample solution withdrawn from °
the Medium (mL) |
°
Tolerances: See Table 3. 500 mg/ 750 mg/ io}
Time Point Tablet Tablet PY
Table 3 mo]
42 1 7
1
Amount Dissolved
2
Time 500 mg/ | 750 mg/ 1000 mg/
82
Point Time Tablet Tablet Tablet
(i) (h) (%) (%) (%) 4 LT
1 1 42-62 35-55, 35-55 The percentages of the labeled amount of leve-
2 Z 59-79 50-70 50-70 tiracetam (CgHi4N2O2), dissolved at the times speci-
3 4 78-98 70-90 70-90 fied, conform to Dissolution (711), Acceptance Table 2.
4 8 NLT 80 NLT 80 NLT 80 Test 5: If the product complies with this procedure, the
labeling indicates that it meets USP Dissolution Test 5.
The percentages of the labeled amount of leve- Medium: pH 6.0 phosphate buffer (6.8 g/L of mono-
tiracetam (CgHi4N202), dissolved at the times speci- basic re phosphate in water. Adjust with so-
fied, conform to Dissolution (711), Acceptance Table 2. dium hydroxide to a pH of 6.0.); 900 mL
Test 4: If the product complies with this procedure, the Apparatus 1: 100 rpm
labeling indicates that it meets USP Dissolution Test 4. Times: 1, 4, 8,and 12h
Buffer: 6.8 g/L of monobasic potassium phosphate in Buffer: 2.7 g/L of monobasic potassium phosphate in
water. Adjust with sodium hydroxide to a pH of 6.0. water
Medium: Buffer; 900 mL Mobile phase: Acetonitrile and Buffer (10:90)
Apparatus 1: 100 rpm Standard stock solution: 2.8 mg/mL of USP Leve-
Times: 1, 2, 4, and 8h tiracetam RS in Medium prepared as follows. Transfer a
Standard solution: (L/900) mg/mL of USP Leve- suitable quantity of USP Levetiracetam RS to a suitable
tiracetam RS in Medium, where L is the label claim in volumetric flask. Dissolve in 20% of the flask volume of
mg/Tablet methanol. Dilute with Medium to volume.
Sample solution: Pass a suitable portion of the solu- Standard solution: (1/900) mg/mL of USP Leve-
tion under test through a suitable filter of 0.45-4m tiracetam RS in Medium from Standard stock solution,
pee size. Discard the first 3 mL of the filtrate. Dilute a where Lis the label claim in mg/Tablet
nown volume of the remaining filtrate quantitatively
with Medium.
 2380 Levetiracetam / Official Monographs USP 41

Sample solution: At each time point withdraw 1 mL Mode: LC

of the solution under test, and pass it through a suita- Detector: UV 230 nm
ble filter of 0.45-um pore size. Column: 4.6-mm x 5-cm; 5-um packing L1
Chromatographic system Column temperature: 30°
(See Chromatography (621), System Suitability.) Flow rate: 0.9 mL/min
Mode: LC Injection volume: 10 uL
Detector: UV 220 nm Run time: 2 times the retention time of levetiracetam
Column: 4.6-mm x 15-cm; 5-um packing L11
Flow rate: 1 mL/min System suitability
Injection volume: 10 uL Sample: Standard solution
Run time: 2 times the retention time of levetiracetam Suitability requirements
System suitability Tailing factor: NMT 2.0
Sample: Standard solution Relative standard deviation: NMT 2.0%
Suitability requirements Analysis
Column efficiency: NLT 4000 theoretical plates Samples: Standard solution and Sample solution
Tailing factor: NMT 1.5 Calculate the concentration, C, of levetiracetam
Relative standard deviation: NMT 2.0% for five (CgH14N2O2) in Medium (mg/mL) after time point i:
replicate injections
Analysis Result; = (ru/rs) x Cs
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of tu = peak response from the Sample solution
levetiracetam (CgHi4N2O2) dissolved in Medium rs = peak response from the Standard solution
(mg/mL) after time point i: Gs = concentration of the Standard solution
(mg/mL)
Result, = (ru/rs) x Cs x Vx (1/L) x 100 Calculate the percentage of the labeled amount of
levetiracetam (CgHi4N202) dissolved at each time
tu = peak response from the Sample solution point (i):
rs = peak response from the Standard solution
Cs = concentration of USP Levetiracetam RS in the Result; = C; x Vx (1/L) x 100
Standard solution (mg/mL)
Vv = volume of Medium, 900 mL
L = label claim (mg/Tablet) Resultz = {[C2 x (V— Vs)] + (Ci x Vs)} x (1/L) x 100
Tolerances: See Table 5.
Results = ({Ca x [V— (2 Vs)]} + [(Co + C1) x Vs]) x C1/
Table 5 L) x 100
Time Point Time Amount Dissolved
(i) (h) (%)
an
ww
Results = ({C, x [V— (3 x Vs)]} + [(C3 + Co + Gi) x Vs]) x
iy 1 1 NMT 40
(1/L) x 100
i} 2 4 55-80
_
Dd 3 8 NLT 75 Gi = concentration of levetiracetam in Medium in
i) the portion of sample withdrawn at time
< 4 12 NLT 85
S point i (mg/mL)
= The percentages of the labeled amount of leve- Vv = volume of Medium, 900 mL
tiracetam (CgH;4N202), dissolved at the times speci- L = label claim (mg/Tablet)
[5
a) fied, conform to Dissolution (711), Acceptance Table 2. Vs = volume of the Sample solution withdrawn from
= Test 6: If the product complies with this procedure, the the solution under test (mL)
labeling indicates that it meets USP Dissolution Test 6. Tolerances: See Table 6.
Medium: pH 6.0 phosphate buffer (6.9 g of monoba-
sic sodium phosphate, and 0.23 g of sodium hydrox- Table 6
ide in 1 L of water. Adjust with sodium hydroxide or
phosphoric acid to a pH of 6.0.); 900 mL Time Point Time Amount Dissolved
Apparatus 1: 100 rpm (i) (h) (%)
Times: 1, 2, 4, and 8h 1 1 25-45
Mobile phase: Acetonitrile and water (10:90) 2 2 45-65
Standard solution: 0.5 mg/mL of USP Levetiracetam 3 4 60-80
RS in Medium prepared as follows. Transfer a suitable
4 8 NLT 80
quantity of USP Levetiracetam RS to a suitable volu-
metric flask. Add 4% of the flask volume of methanol The percentages of the labeled amount of leve-
and 60% of the flask volume of the Medium. Sonicate tiracetam (CsHi4N202), dissolved at the times speci-
for NLT 5 min. Dilute with Medium to volume. fied, conform to Dissolution (711), Acceptance Table 2.
Sample solution: At the end of specified time interval, Test 7: If the product complies with this procedure, the
withdraw a known volume of the solution from the labeling indicates that it meets USP Dissolution Test 7.
dissolution vessel. Pass a suitable portion of the solu- Medium: Acetate buffer, pH 4.5, prepared as follows.
tion under test through a suitable filter of 0.45-um Dissolve 3.0 g of sodium acetate in 1 L of water and
pore size. add 1.4 mL of glacial acetic acid. Adjust with 5 N so-
Chromatographic can dium hydroxide or glacial acetic acid to a pH of 4.5;
(See Chromatography (621), System Suitability.) 230 mL.
USP 41 Official Monographs / Levetiracetam 2381

Apparatus 3: 15 dips per min, with suitable screens The percentages of the labeled amount of leve-
Times tiracetam (CsHi4N202), dissolved at the times speci-
For 500-mg Tablets: 1, 2,4, and 8h fied, conform to Dissolution (711), Acceptance Table 2.
For 750-mg Tablets: 1, 2,4, and 10h °Test 8: If the product complies with this procedure,
Buffer: 13.6 g/L of monobasic re phosphate in Hie labeling indicates that it meets USP Dissolution Test
yee Adjust with 5 N sodium hydroxide to a pH of
Medium: Phosphate buffer, pH 6.0, prepared as fol-
Mobile phase: Methanol and Buffer (15:85) lows. Dissolve 6.8 g of monobasic potassium phos-
Standard solution: 0.55 mg/mL of USP Levetiracetam phate in 1 L of water. Adjust with 10 N sodium hy-
RS in Medium. Sonication may be used to aid in droxide solution to a pH of 6.0; 900 mL.
dissolution. Apparatus 1: 100 rpm
Sample solution: Pass a suitable portion of the solu- mes: 1,2,4,and12h
tion under test through a suitable filter of 0.45-um
pore size. Discard the first 5 mL. Dilute a suitable vol- Buffer: 0.26 g/L of monobasic potassium phosphate in
ume of the filtrate with Medium, as needed. water. Adjust with 20 g/L aqueous potassium hydrox-
Chromatographic system ide to a pH of 5.5.
(See Chromatography (621), System Suitability.) Solution A: Acetonitrile and Buffer (5:95)
Mode: LC Mobile phase: Acetonitrile and Solution A (10:90)
Detector: UV 210 nm Standard solution: (1/900) mg/mL of USP Leve-
Column: 4.6-mm x 10-cm; 3-um packing L1 tiracetam RS in Medium, where L is the label claim in
Column temperature: 30° mg/Tablet. Sonicate to dissolve as needed.
Flow rate: 1 mL/min Sample solution: Pass a portion of the solution under
Injection volume: 10 pL test through a suitable filter of 0.45-um pore size.
Run time: 2 times the retention time of levetiracetam Chromatographic system
System suitability (See Chromatography (621), System Suitability.)
Sample: Standard solution Mode: LC
Suitability requirements Detector: UV 220 nm
Tailing factor: NMT 2.0 Column: 4.6-mm x 15-cm; 5-um packing 11
Relative standard deviation: NMT 2.0% Column temperature: 20°
Analysis Flow rate: 1 mL/min
Samples: Standard solution and Sample solution Injection volume: 5 ul
Calculate the concentration, C, of levetiracetam Run time: NLT 1.6 times the retention time of
(CgHi4N2O2) in Medium (mg/mL) after time point i: levetiracetam
System suitability
Result; = (ru/rs) x D x Cs Sample: Standard solution
Tu = peak response from the Sample solution Suitably requirements
rs = peak response from the Standard solution Tailing factor: NMT 1.5
Relative standard deviation: NMT 1.8% (=
D = dilution factor, as needed wn
Gs = concentration of the Standard solution Analysis a)
Samples: Standard solution and Sample solution
(mg/mL) Calculate the concentration, C, of levetiracetam ms
Calculate the percentage of the labeled amount of CS)
levetiracetam (CgHi4N202) dissolved at each time (CaHi4N2O2) in Medium (mg/mL) after time point /: be
°
point (i):
Result; = (ru/rs) x Cs eo}2
st)
Result; = C; x Vx (1/L) x 100 ty = peak response from the Sample solution ae
ts = peak response from the Standard solution FSy
Resultz = C2 x Vx (1/L) x 100 + Result, G = concentration of the Standard solution
(mg/mL) ;
Calculate the percentage of the labeled amount of
Result; = C3 x Vx (1/L) x 100 + Resultz levetiracetam (CsH;4N202) dissolved at each time
point (/):
Result, = C, x Vx (1/L) x 100 + Results Result; = C; x Vx (1/K x 100
G = concentration of levetiracetam in the portion
of sample withdrawn at the specified time Result, = {[C2 x (V— V3] + (Ci x Vs)} x (/D x 100
point (mg/mL)
V = volume of Medium, 230 mL
L = label claim (mg/Tablet) Results = (GX [V= (2X Vel + (Ge + G) X Va) XC
Tolerances: See Table 7. 1) x 100
Table 7 Results = ({C, x [V— (3 x Vs)]} + [((Cs + C2 + Gi) x Vs]) x
(1/L) x 100
500 mg/ 750 mg/ G = concentration of levetiracetam in the portion
Time Point Tablet Tablet of sample withdrawn at time point i
(mg/mL)
V = volume of Medium, 900 mL
L = label claim (mg/Tablet)
Vs = volume of the Sample solution withdrawn from
the Medium (mL)
Tolerances: See Table 8.
 2382 Levetiracetam / Official Monographs USP 41

Table 8 Suitability requirements

Time Point Time Amountbiscived Resolution: NLT 1.5 betweenlevetiracetam and leve-
tiracetam acid peaks, System suitability solution
Tailing factor: NMT 2.0, Standard solution
1 25-4. Relative standard deviation: NMT 5.0%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of any unspecified degrada-
The percentages of the labeled amount of leve- tion product in the portion of Tablets taken:
tiracetam (C3HisN2O2), dissolved at the times speci-
fied, conform to Dissolution (711), Acceptance Table 2. Result = (ru/rs) * (Cs/Cu) x 100
© (RB 17-Jun-2017) ej is
e UNIFORMITY OFDosAcE UNITS (905): Meet the fu = peak response of each impurity from the
requirements Sample solution
rs = peak response of USP Levetiracetam RS from
IMPURITIES the Standard solution
© ORGANIC IMPURITIES Cs = concentration of USP Levetiracetam RS in the
Solution A: Dilute 2 mL of phosphoric acid with water Standard solution (mg/mL)
to1L. Cu = nominal concentration of levetiracetam in the
Diluent: Acetonitrile and Solution A (5:95) Sample solution (mg/mL)
Buffer: oe of anhydrous dibasic sodium phosphate Acceptance criteria: See Table 9.
in water. Adjust with phosphoric acid to a pH of 3.5.
Mobile phase: Acetonitrile and Buffer (5:95). To each L Table 9
of the mixture, add 1 g of sodium 1-hexanesulfonate
monohydrate. Relative Acceptance
System suitability solution: 0.3 mg/mL of USP Leve- Retention Criteria,
tiracetam RS in Diluent prepared as follows. Dissolve the Name Time NMT (%)
required amount of USP Levetiracetam RS in 10% of Levetiracetam related
the final volume of 0.1 N potassium hydroxide. Let the compound Bab 0.40 F
mixture react at room temperature for about 15 min, Levetiracetam 1.0 me
and then neutralize by adding 0.1 N hydrochloric acid Levetizacetarencile 13 0.30
at 10% of the flask volume. Dilute with Diluent to vol- Lévetinacetary related
ume. [Note—This solution contains levetiracetam and =
levetiracetam acid.] compound Ate 19
Standard solution: 12.5 ug/mL of USP Levetiracetam Any individual
RS in water. Sonication may be used to aid in dissolu- unspecified =
my tion. Pass a portion of the solution through a suitable degradation product 0.10
Ce filter of 0.2-um pore size. Total impurities = 1.0
ry Sample solution: Nominally equivalent to 2.5 mg/mL 4(5)-2-Aminobutanamide.
a of levetiracetam in water, from a portion of crushed » Process impurities controlled in the drug substance. Included for identifi-
ro} Tablets (NLT 20) prepared as follows. Transfer the cation purposes only. Not reported for the drug product, and not includ-
7 weighed amount of crushed Tablet powder to a volu- a I) total ABURUES. ;
= metric flask containing water to fill 80% of final vol- <(9-2-(2-Oxopyrrolidin-1-y))butanoic acid. /
= ume. Sonicate in cold water for 10 min. Equilibrate to (3)-N-(l-Amino-1-oxobutan-2-y})-4-chlorobutanamide.
S room temperature. Dilute with water to volume. Pass a ADDITIONAL REQUIREMENTS
| portion through a suitable filter of 0.2-4m pore size. e PACKAGING AND STORAGE: Preserve in well-closed contain-
Alternatively, the Sample solution having a nominal con- ers. Store at controlled room temperature.
centration of 2-3 mg/mL of levetiracetam may be pre- e LABELING: When more than one Dissolution test is given,
pared as follows. Finely grind NLT 10 Tablets, and the labeling states the Dissolution test used only if Test 1
transfer an amount equivalent to one Tablet to a suita- is not used.
ble volumetric flask. Add NLT 30 mL of acetonitrile. e USP REFERENCE STANDARDS (11)
Sonicate for 10 min, and shake using a mechanical USP Levetiracetam RS
shaker for 10 min. Add NLT 30 mL of water, and shake
for 15 min using a mechanical shaker. Allow the result-
ing mixture to equilibrate to room temperature. Add
NMT 25% of the final flask volume of acetonitrile. Di-
lute with water to volume. Centrifuge for 15 min, and
pass a portion througha suitable filter of 0.45-um Levmetamfetamine
pore size. am
Chromatographic system “Sp an
(See Chromatography (621), System Suitability.) Lo) fem
Mode: LC
Detector: UV 205 nm : CioHisN 149.23
Column: 4.6-mm x 25-cm; 5-um packing L1 Benzeneethanamine, N, o-dimethyl-, (R)-.
Temperatures (-)-(R)-N, a-Dimethylphenethylamine —[33817-09-3].
Column: 30° Q .
Autosampler: 10° » Levmetamfetamine contains not less than
Flow rate: 2 mL/min 98.0 percent and not more than 100.5 percent of
Injection volume: 20 uL GrakicsN
Run time: 5 times the retention time of levetiracetam HOMME
System suitabilit in ti ight-resi
Samples: systenn suitability solution and Standard Packaging and storage—Preserve in tight, light-resistant
solution
USP 41 Official Monographs / Levobunolol 2383

USP Reference standards (11)—
USP Levmetamfetamine RS
USP Methamphetamine Hydrochloride RS
Identification—
A: Infrared Absorption (197F).
B: The retention time of the major peak in the chromato-
gram of the Test solution corresponds to that in the chro-
matogram of the Sen suitability solution, as obtained in
the test for Limit of methamphetamine.
Specific rotation (7815S): between -18.5° and -21.5°.
fest solution: 16mg per mL, in 1.2 N hydrochloric
acid.
Limit of methamphetamine—
Mobile phase—Prepareafiltered and degassed mixture of
hexane, isopropyl alcohol, and acetonitrile (98:1.5:0.5).
Make adjustments if necessary (see System Suitability under
Chromatography (621)).
Resolution solution—Mix suitable quantities of a solution
of USP Methamphetamine Hydrochloride RS in chloroform
and USP Levmetamfetamine RS in chloroform to obtain a
solution containing about 0.025 mg per mL and 2.5 mg per
mL of methamphetamine hydrochloride and
levmetamfetamine, respectively. Transfer 2.0 mL of this solu-
tion to a suitable container, add 10 mg of 2-naphthyl
chloroformate and 2.0 mL of chloroform, mix with a vortex
mixer, and allow to stand for 5 minutes. To this solution,
levmetamfetamine obtained from the Test solution: not more
than 0.1% is found.
Limit of nonvolatile residue—Heat about 1.0 g, accu-
rately weighed, at 150° to constant weight: the limit is not
more than 0.5%.
Ordinary impurities (466)—
Test solution: chloroform.
Standard solution: chloroform.
Eluant: a mixture of chloroform, cyclohexane, and di-
ethylamine (5:4:1).
Visualization: 1.
Limits—No impurity exceeds 0.1%, and the total does
not exceed 0.5%.
Assay—Transfer about 400 mg of Levmetamfetamine to a
suitable container, add 50.0 mL of glacial acetic acid, and
mix. Add two drops of crystal violet TS, and titrate with 0.1
N perchloric acid VS. Perform a blank determination, and
make any necessary correction. Each mL of 0.1 N perchloric
acid is equivalent to 14.92 mg of CioHisN.
Levobunolol Hydrochloride

Ay Le hoe
add 2 mL of 1 N sodium hydroxide, mix with a vortex
mixer, allow to stand for 5 minutes, and discard the aque-
ous layer. Wash the organic layer twice with 2 mL of 1N HC CHy
sodium hydroxide, discarding the aqueous layer. To the or-
ganic layer add 2 mL of 1 N hydrochloric acid, mix with a
vortex mixer, and discard the aqueous layer. Wash the or- Ci7H2sNO3 - HCl 327.85
ganic layer twice with 2 mL of 1 N hydrochloric acid, dis- 1(2H)-Naphthalenone, 5-[3-[(1,1-dimethylethyl)amino]-
carding the aqueous layer. To the organic layer add 2 mL of 2-hydroxypropoxy]-3,4-dihydro-, hydrochloride, (-)-(5);
water, mix with a vortex mixer, and discard the aqueous (-)-5-[3-(tert-Butylamino)-2-hydroxypropoxy]-3,4-dihydro-
layer. Wash the organic layer twice with 2 mL of water, dis- 1(2H)-naphthalenone hydrochloride [27912-14-7].
[os
carding the aqueous layer. To the organic layer add about DEFINITION a)
1.0 g of anhydrous sodium sulfate, and mix with a vortex a]
Levobunolol Hydrochloride contains NLT 98.0% and NMT
mixer. Transfer 1.0 mL of this solution to a 10-mL volumet- 102.0% of levobunolol hydrochloride (C;7H2sNO3 - HCl), c=
ric flask, dilute with Mobile phase to volume, mix, and filter. calculated on the dried basis. i}
J
Test solution—Transfer about 62.5 mg of }
Levmetamfetamine, accurately weighed, to a 25-mL volu- IDENTIFICATION a=
metric flask, dissolve in and dilute with chloroform to vol- © A. INFRARED ABSORPTION (197M) 2
ume, and mix. Transfer 2.0 mL of this solution to a suitable e B. The retention time of the major peak of the Sample bo}
container, and proceed as directed in Resolution solution be- solution corresponds to that of the Standard solution, as 7
w
grey with “add 10 mg of 2-naphthyl chloroformate and obtained in the Assay.
mL of chloroform.” © C. IDENTIFICATION TESTS—GENERAL, Chloride (191): Meets
Chromatographic system (see Chromatography (621))—The the requirements
liquid chromatograph is equipped with a 274-nm detector ASSAY
and a 4.6-mm x 25-cm column that contains packing L36. ¢ PROCEDURE
The flow rate is about 1.5 mL per minute. Chromatograph
the Resolution solution, and record the peak responses as
Solution A: 5 mM sodium 1-heptanesulfonate in
methanol
directed for Procedure; the relative retention times are about Solution B: 5 mM sodium 1-heptanesulfonate in water
0.9 for methamphetamine and 1.0 for levmetamfetamine;
Mobile phase: Solution A, 0.5 M sulfuric acid, and Solu-
and the resolution, R, between methamphetamine and
levmetamfetamine is not less than 1.4. Chromatograph the tion B (53:1:47)
Standard solution: 100 g/mL of USP Levobunolol Hy-
Test solution, and record the peak responses as directed for drochloride RS in Mobile phase
Procedure: the relative standard deviation for replicate injec-
tions is not more than 2.0%. Sample solution: 100 g/mL of Levobunolol Hydro-
chloride in Mobile phase
Procedure—tnject a volume (about 50 uL) of the Test solu- Chromatographic system
tion into the chromatograph, record the chromatogram, (See Chromatography (621), System Suitability.)
and measure the responses for the major peaks. Calculate
the percentage of methamphetamine in the portion of
Levmetamfetamine taken by the formula:
100ru / (rw + 1)

in which ry is the methamphetamine peak response ob-

tained from the Test solution, and r, is the peak response of
 2384 Levobunolol / Official Monographs USP 41

Mode: LC Acceptance criteria

Detector: UV 223 nm Individual impurity: NMT 0.5%
Column: 3.9mm x 25 cm; 10-um packing L7 Total impurities: NMT 1%
Flow rate: 1 mL/min
Injection volume: 20 uL SPECIFIC TESTS
System suitability e OPTICAL ROTATION, Specific Rotation (781S)
Sample: Standard solution Sample solution: 30 mg/mL in methanol
Suitability requirements Acceptance criteria: —19° to -20°
Tailing factor: NMT 1.5 © PH (791)
Relative standard deviation: NMT 0.73% Sample: 50 mg/mL
Analysis Acceptance criteria: 4.5-6.5
Samples: Standard solution and Sample solution e Loss ON DRYING (731)
Calculate the percentage of levobunolol hydrochloride Sample: 1g
(CizH2sNO3+ HCl) in the portion of Levobunolol Hydro- Analysis: Dry under vacuum over phosphorus pentox-
chloride taken: ide at 110° for 4 h.
Acceptance criteria: NMT 0.5%
Result = (ru/rs) x (Cs/Cu) x 100
ADDITIONAL REQUIREMENTS
tu = peak response from the Sample solution ° PACKAGING AND STORAGE: Preserve in well-closed contain-
rs = peak response from the Standard solution ers. Protect from light. Store at room temperature.
Cs = concentration of USP Levobunolol e USP REFERENCE STANDARDS (11)
Hydrochloride RS in the Standard solution USP Atenolol RS /
(g/mL) USP Levobunolol Hydrochloride RS
CG = concentration of Levobunolol Hydrochloride in
the Sample solution (g/mL)
Acceptance criteria: 98.0%-102.0% on the dried basis
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.1%
Levobunolol
*
Hydrochloride Ophthalmic
¢ ORGANIC IMPURITIES . Solution
Solution A, Solution B, and Mobile phase: Proceed as DEFINITION
directed in the Assay.
System suitability solution: 50 j1g/mL each of USP Levobunolol Hydrochloride Ophthalmic Solution contains
Levobunolol Hydrochloride RS and USP Atenolol RS in NLT 90.0% and NMT 110.0% of the labeled amount of
Mobile phase levobunolol hydrochloride (Ci7H2sNO3 - HCl).
Standard solution: 5 g/mL of USP Levobunolol Hydro- IDENTIFICATION
chloride RS in Mobile phase e A. The retention time of the major peak of the Sample
Sample solution: 1 mg/mL of Levobunolol Hydrochlo- solution corresponds to that of the Standard solution, as
ca
al
tide in Mobile phase
a obtained in the Assay.
ig Chromatographic system: Proceed as directed in the
_ Assay using Mobile phase. ASSAY
a
i} Run time: 3 times the retention time of levobunolol © PROCEDURE
c System suitability Solution A: 5 mM sodium 1-heptanesulfonate in water
5 Sample: System suitability solution Mobile phase: Methanol, glacial acetic acid, and Solu-
= Suitability requirements tion A (550:5:450)
a Resolution: NLT 8 between levobunolol and atenolol Standard solution: 50 g/mL of USP Levobunolol Hy-
a) peaks
po drochloride RS in Mobile phase
Analysis Sample solution: Nominally equivalent to 50 ug/mL of
Samples: Standard solution and Sample solution levobunolol hydrochloride in Mobile phase from
Calculate the percentage of each impurity in the por- Ophthalmic Solution
tion of Levobunolol Hydrochloride taken: Chromatographic system
(See Chromatography (621), System Suitability.)
Result = (ru/rs) x (Cs/Cu) x 100 Mode: LC
Detector: UV 254 nm
tu = peak response of each impurity from the Column: 3.9 mm x 30 cm; 10-um packing L1
Sample solution Flow rata’ 1S AL g
Is = peak response of levobunolol from the Injection-vélumie: ©26:AL
Standard 3solution 5 stem
L AO UmE:
suitability HY
Cs = concentration of USP Levobunolol Sample: Standard solution
Hydrochloride RS in the Standard solution Suitability requirements
(ug/ml) et Tailing factor: NMT 1.2
Cy = concentration of Levobunolol Hydrochloride in Relative standard deviation: NMT 1.5%
the Sample solution (\ug/mL) Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
levobunolol hydrochloride (Ci7H2sNO3+ HCl) in the
portion of Ophthalmic Solution taken:
Result = (ru/rs) x (Cs/Cy) x 100
Tu peak response from the Sample solution
Is peak response from the Standard solution
Cs = concentration of USP Levobunolol
Hydrochloride RS in the Standard solution
(mg/mL)
USP 41 Official Monographs / Levocabastine 2385

Cu = nominal concentration of levobunolol e USP REFERENCE STANDARDS (11)

hydrochloride in the Sample solution USP Edetate Disodium RS
(mg/mL) USP Levobunolol Hydrochloride RS
Acceptance criteria: 90.0%-110.0%
IMPURITIES
@ ORGANIC IMPURITIES
Mobile phase: Proceed as directed in the Assay.
Standard solution: 10 ttg/mL each of USP Levobunolol Levocabastine Hydrochloride
Hydrochloride RS and USP Edetate Disodium RS in Mo-
bile phase
Sample solution: Nominally equivalent to 1 mg/mL of
levobunolol hydrochloride in Mobile phase from
Ophthalmic Solution HCI
Chromatographic system and System suitability: Pro-
ceed as directed in the Assay using wavelengths of UV
254 nm and 400 nm.
[Note—The relative retention times for levobunolol and CasH29FN2O2 - HCI 456.98
edetate disodium are 1.0 and 0.46, respectively.] 4-Piperidinecarboxylic acid, 1-[4-cyano-4-(4-fluorophen-
Analysis yl)cyclohexyl]-3-methyl-4-phenyl-, monohydrochloride,
Samples: Standard solution and Sample solution ©-I1 (cis), 30,,4B)-;
Calculate the percentage of any individual impurity at (S-trans-1 aise Gyan (priueropheny ycoheair
254 nm in the portion of Ophthalmic Solution taken: 3-methyl-4-phenylisonipecotic acid monohydrochloride
[79547-78-7].
Result = (ru/rs) x (Cs/Cu) x 100
DEFINITION
i = peak response of each impurity from the Levocabastine Hydrochloride contains NLT 98.5% and NMT
Sample solution 101.5% of C2gH2sFN2O2- HCI, calculated on the dried
rs = peak response of levobunolol from the basis.
Standard solution
Cs = concentration of USP Levobunolol IDENTIFICATION
Hydrochloride RS in the Standard solution e A. INFRARED ABSORPTION (197K)
© B. IDENTIFICATION TESTS—GENERAL, Chloride (191): Meets
(g/mL) p the requirements
Cu = nominal concentration of levobunolol
evenlon in the Sample solution (ug/mL) e C. OPTICAL ROTATION, Specific Rotation (781S): Meets the
Calculate the percentage of any impurity at the requirements
retention time of levobunolol (obtained using the ASSAY
detector at 254 nm) using the detector at 400 nm in © PROCEDURE c
the portion of Ophthalmic Solution taken: Sample solution: Dissolve 175 mg of Levocabastine Hy- a)
uu
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100 drochloride in 50 mL of alcohol, and add 5.0 mL of
0.01 N hydrochloric acid. =
ty = peak response of the impurity from the Titrimetric system i}
Ss
Sample solution at 400 nm (See Titrimetry (541).) i)
Us = peak response of levobunolol from the Mode: Direct titration
Titrant: 0.1 N sodium hydroxide VS
=oy
Standard solution at 254 nm 2
Cs = concentration of USP Levobunolol Endpoint detection: Potentiometric i}
Hydrochloride RS in the Standard solution Analysis a]
as

U
(ug/ml)
= nominal concentration of levobunolol
Sample: Sample solution
The volume of titrant required to titrate Levocabastine
hydrochloride in the Sample solution (g/mL) Hydrochloride is the difference between the first and
EB = relative response factor for the impurity, 0.2 third endpoints. Perform a blank determination and
Acceptance criteria make any necessary correction. Each mL of 0.1 N so-
Individual impurity: NMT 1% dium hydroxide VS is equivalent to 22.85 mg of
Total impurities: NMT 2.5%. Disregard any peak ob- CasH2sFN2O2 - HCI.
tained with the detector at 254 nm with the retention Acceptance criteria: 98.5%-101.5% on the dried basis
time of edetate disodium. IMPURITIES
SPECIFIC TESTS Inorganic Impurities
¢ ANTIMICROBIAL EFFECTIVENESS TESTING (51): Meets the e RESIDUE ON IGNITION (281): NMT 0.1%, based on a sam-
requirements ple weight of about 1.000 g
e STERILITY TESTS (71): Meets the requirements when Organic Impurities
tested as directed for Test for Sterility of the Product to Be e PROCEDURE
Examined, Membrane Filtration. [NotE—Prepare solutions immediately before use.]
© PH (791): 5.5-7.5 Diluent: 2 mg/mL of sodium hydroxide in water
Solution A: Dissolve 1.39 g of boric acid in water, and
ADDITIONAL REQUIREMENTS adjust with 1 N sodium hydroxide to a pH of 9.0. Di-
e PACKAGING AND STORAGE: Preserve in tight containers. lute with water to 100 mL.
Protect from light. Store at controlled room temperature. Run buffer: Dissolve 1.08 g of sodium dodecyl! sulfate
and 650mg of hydroxypropyl-B-cyclodextrin in 5 mL of
isopropyl alcohol, then dilute with Solution A to 50 mL.
System suitability solution: 12.5 g/mL of USP Levo-
cabastine Hydrochloride RS and 12.5 ug/mL of USP
Levocabastine Related CompoundARS in Diluent
Standard solution: Dilute 5.0 mL of the Sample solu-
tion with Diluent to 100 mL. Dilute 1.0 mL of this solu-
 2386 Levocabastine / Official Monographs USP 41

tion with Diluent to 10 mL to obtain a solution contain- ¢ USP REFERENCE STANDARDS (11)
ing 12.5 ug/mL of Levocabastine Hydrochloride. USP Levocabastine Hydrochloride RS
Sample solution: 2.5 mg/mL of Levocabastine Hydro- USP Levocabastine Related Compound A RS
chloride in Diluent
Capillary electrophoresis system
Detector: UV 214 nm
Column: 75-4m x 50-cm uncoated fused-silica capil-
lary column Levocarnitine
Column temperature: 50°
Current: See the gradient table below. 3G, CH, GH O
eee
Bye eS
Time Current

0 0
75
1
60 200
[Notte—Before performing the System suitability, equi-
librate the capillary column with Diluent for 2 min,
then equilibrate with Run buffer for at least 5 min.]
System suitability
Sample: System suitability solution
{[Notr—The relative migration times for levocabastine
and levocabastine related compound A are approxi-
mately 1.0 and 1.07, respectively.]
Suitability requirements
Resolution: NLT 4 between levocabastine and levo-
cabastine related compound A
[NoTe—If necessary, adjust the current gradient to
achieve the required resolution.]
Analysis
Samples: Diluent (blank), Standard solution, and Sam-
ple solution
Separately inject equal volumes (pressure of 3450 Pa
“
for 5 s) of the Samples, and record the peak
<=
5 responses.
cd [Note—Disregard any peak originating from the Dilu-
— Analysis: Dissolve the Sample in a mixture of 3 mL of
io) ent. Disregard any peak with an area of less than 0.1
° times the major peak area of the Standard solution
iS (0.05%).]
S to an emerald green endpoint. Perform the Blank
= Acceptance criteria: The area for any peak in the Sam-
ple solution, other than the major peak, is not greater
1.0
than the major peak area of the Standard solution
A)
=) (0.5%); and the sum of all peak areas in the Sample
solution, except for the major peak, is not greater than
twice the major peak area of the Standard solution
(1.0%).
SPECIFIC TESTS
© OPTICAL ROTATION, Specific Rotation (781S): -102° to
—106° at 20°
Sample solution: 10 mg/mL in methanol
e Loss ON DRYING (731): Dry about 1.000 g of the sample
at 105° to constant weight: it loses NMT 0.5% of its
weight.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. Protect from light.
C7HisNO3 161.20
(R)-3-Carboxy-2-hydroxy-N,
N,N-trimethyl-1-propanaminium,
inner salt;
(R)-(3-Carboxy-2-hydroxypropyl)trimethylammonium, inner
salt [541-15-1]. ”
DEFINITION
Levocarnitine contains NLT 97.0% and NMT 103.0% of
eae (C7HisNOs), calculated on the anhydrous
asis.
IDENTIFICATION
© A. INFRARED ABSORPTION (197K)
Analysis: Dry the sample and the USP Levocarnitine RS
under vacuum at 50° for 5 h.
Acceptance criteria: Meets the requirements
ASSAY
e PROCEDURE
bample: 100 mg of Levocarnitine
Blank: A mixture of 3 mL of formic acid and 50 mL of
glacial acetic acid
Titrimetric system
(See Titrimetry (541).)
Mode: Direct titration
Titrant: 0.1 N perchloric acid VS
Endpoint detection: Visual
formic acid and 50 mL of glacial acetic acid. Add
2 drops of crystal violet TS, and titrate with the Titrant
determination.
Calculate the percentage of levocarnitine (C7H;sNOs) in
the portion of Levocarnitine taken:
Result = {[(Vs — Vs) x N x F ]/W} x 100
Vs = Titrant volume consumed by the Sample (mL)
Ve = Titrant volume consumed by the Blank (mL)
N = actual normalilty of the Titrant (mEq/mL)
F = equivalency factor, 161.2 mg/mEq
w = Sample weight (mg)
Acceptance criteria: 97.0%-—103.0% on the anhydrous
basis
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.5%
e CHLORIDE AND SULFATE, Chloride (221)
Standard: 0.50 mL of 0.020 N hydrochloric acid
Sample: 0.090 g of Levocarnitine
Acceptance criteria: NMT 0.4%

Delete the following:

°e HEAVY METALS (231): NMT 20 ppme ‘offical 1jan-2018)

e Limit oF POTASSIUM
[Note—The Standard solution and the Sample solutions
may be modified, if necessary, to obtain solutions of
suitable concentrations adaptable to the linear or work-
ing range of the instrument.]
USP 41 Official Monographs / Levocarnitine 2387

Standard solution: 31.25 g/mL of potassium in water, ume. This solution contains 32 mg/mL of Levocarnitine
prepared from potassium chloride, previously dried at and 40 g/mL of added sodium from the Standard
105° for 2h solution.
Sample stock solution: 0.625 mg/mL of Levocarnitine Blank: Water
in water Instrumental conditions
Sample solution A: Transfer 20.0 mL of the Sample (See Atomic Absorption Spectroscopy (852).)
stock solution to a 25-mL volumetric flask, and dilute Mode: Atomic absorption spectrophotometry
with water to volume. This solution contains 500 g/mL Analytical wavelen th: 589.0 nm
of Levocarnitine and 0 g/mL of added potassium from Lamp: Sodium hollow-cathode
the Standard solution. Flame: Air—acetylene
Sample solution B: Transfer 20.0 mL of the Sample Analysis
stock solution to a 25-mL volumetric flask, add 2.0 mL Samples: ele solution A, Sample solution B, Sample
of the Standard solution, and dilute with water to vol- solution C, and Blank
ume. This solution contains 500 g/mL of Levocarnitine Determine the absorbances of the solutions against the
and 2.5 ug/mL of added potassium from the Standard Blank. Plot the absorbances of the three Sample solu-
solution. tions versus their added sodium concentrations, in
Sample solution C: Transfer 20.0 mL of the Sample g/mL. Draw the straight line best fitting the three
stock solution to a 25-mL volumetric flask, add 4.0 mL points, and extrapolate the line until it intercepts the
of the Standard solution, and dilute with water to vol- concentration axis. From the intercept determine the
ume. This solution contains 500 jug/mL of Levocarnitine concentration, in g/mL, of sodium in Sample solution
and 5.0 ug/mL of added potassium from the Standard A.
solution. , Calculate the percentage of sodium in the portion of
Blank: Water Levocarnitine taken:
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).) Result = (Cya/Cu) x 100
Mode: Atomic ee spectrophotometry
Analytical wavelength: 766.7 nm Cva | = concentration of sodium in Sample solution A
Lamp: Potassium hollow-cathode (ug/mL), determined from the intercept of
Flame: Air-acetylene the linear regression line
Analysis Cu = concentration of Levocarnitine in Sample
Samples: Sample solution A, Sample solution B, Sample solution A (g/mL)
solution C, and Blank Acceptance criteria: NMT 0.1%
Determine the absorbances of the solutions against the
Blank. Plot the absorbances of the three Sample solu- SPECIFIC TESTS
tions versus their added potassium concentrations, in ¢ OPTICAL ROTATION, Specific Rotation (781S)
ug/mL. Draw the straight line best fitting the three Sample solution: 100 mg/mL in water
points, and extrapolate the line until it intercepts the pean criteria: —29° to —32°
concentration axis. From the intercept determine the © PH (791) =
concentration, in g/mL, of potassium in Sample solu- Sample solution: 50 mg/mL solution wv
tion A. Acceptance criteria: 5.5-9.5 z
Calculate the percentage of potassium in the portion of e@ WATER DETERMINATION (921): NMT 4.0% E
Levocarnitine taken: i)
ADDITIONAL REQUIREMENTS =
© PACKAGING AND STORAGE: Preserve in tight containers. )
Result = (Ck/Cu) x 100 e USP REFERENCE STANDARDS (11) to}=
USP Levocarnitine RS i}
Cx = concentration of potassium in Sample solution so)
A (g/mL), determined from the intercept of ee
”
the linear regression line
Cu = concentration of Levocarnitine in Sample
solution A (tug/mL)
Acceptance criteria: NMT 0.2% Levocarnitine Injection
e Limit OF SODIUM
[Nott—The Standard solution and the Sample solutions DEFINITION
may be modified, if necessary, to obtain solutions of Levocarnitine Injection is a sterile solution of Levocarnitine in
suitable concentrations adaptable to the linear or work- Water for Injection. It contains NLT 90.0% and NMT
ing range of the instrument.] 110.0% of the labeled amount of levocarnitine
Standard solution: 250 g/mL of sodium in water, pre- (C7HisNO3).
pared from sodium chloride, previously dried at 105°
for 2h IDENTIFICATION
Sample stock solution: 40.0 mg/mL of Levocarnitine in e A. The retention time of the major peak of the Sample
water solution corresponds to that of the Standard solution, as
Sample solution A: Transfer 20.0 mL of the Sample obtained in the Assay.
stock solution to a 25-mL volumetric flask, and dilute e B. COLOR REACTION
with water to volume. This solution contains 32 mg/mL Analysis: Transfer 2 mL of Injection to a test tube, add
of Levocarnitine and 0 ug/mL of added sodium from 5 mL of 1 N hydrochloric acid and a few drops of am-
the Standard solution. monium reineckate TS.
Sample solution B: Transfer 20.0 mL of the Sample Acceptance criteria: A red-violet precipitate is
stock solution to a 25-mL volumetric flask, add 2.0 mL produced.
of the Standard solution, and dilute with water to vol- ASSAY
ume. This solution contains 32 mg/mL of Levocarnitine e PROCEDURE
and 20 g/mL of added sodium from the Standard Buffer: 0.05 M phosphate buffer, prepared by dissolv-
solution. ing 6.805 g of monobasic potassium phosphate in 1 L
Sample solution C: Transfer 20.0 mL of the Sample of water
stock solution to a 25-mL volumetric flask, add 4.0 mL
of the Standard solution, and dilute with water to vol-
 2388 Levocarnitine / Official Monographs USP 41

Mobile phase: Acetonitrile and Buffer (65:35). Adjust

with phosphoric acid to a pH of 4.7, and mix.
System suitability solution: 5 mg/mL of USP Levocarni- Levocarnitine Oral Solution
tine RS and 0.024 mg/mL of USP Levocarnitine Related
Compound A RS in water DEFINITION
Standard solution: 10 mg/mL of USP Levocarnitine RS Levocarnitine Oral Solution is a solution of levocarnitine in
in water water, and it contains suitable antimicrobial agents. It
Sample solution: Pool the contents of 10 containers, may contain a suitable flavor. It contains NLT 90.0% and
and dilute an accurately measured volume of Injection NMT 110.0% of the labeled amount of levocarnitine
quantitatively with water to obtain a solution having a (C7HisNO3).
nominal concentration of about 10 mg/mL of IDENTIFICATION
levocarnitine. e A. The retention time of the major peak of the Sample
Chromatographic system solution corresponds to that of the Standard solution,
(See Chromatography (621), System Suitability.) both relative to the internal standard, as obtained in the
Mode: LC Assay.
Detector: UV 205 nm
Column: 3.9-mm x 30-cm; 10-14m packing L8 ASSAY
Flow rate: 1 mL/min © PROCEDURE
Injection volume: 5 pL Buffer: 0.05 M phosphate buffer, pH 2.4, prepared by
System suitability van 11.5 mL of phosphoric acid, 1900 mL of water,
Samples: System suitability solution and Standard and about 100 mL of 1 N sodium hydroxide
solution Mobile phase: Dissolve 555 mg of sodium 1-heptane-
Suitability requirements sulfonate in 980 mL of Buffer with stirring. Add 20 mL
Resolution: NLT 1.0 between levocarnitine related of methanol, and mix.
compound A(crotonoylbetaine) and levocarnitine, Internal standard solution: 0.02 mg/mL of p-ami-
System suitability solution nobenzoic acid in water
Relative standard deviation: NMT 2.0% for levo- Standard solution: Transfer about 10 mg of USP Levo-
carnitine, Standard solution carnitine RS to a 5-mL volumetric flask, add 1.0 mL of
Analysis the Internal standard solution, and dilute with water to
Samples: Standard solution and Sample solution volume.
Calculate the percentage of the labeled amount of levo- Sample stock solution: Equivalent to 10 mg/mL of
carnitine (C7HisNO3) in the portion of Injection taken: levocarnitine in water from an accurately measured vol-
ume of Oral Solution
Result = (ru/rs) x (Cs/Cu) x 100 Sample solution: Wash a 10-mm x 4-cm disposable
column containing 500 mg of packing L1, in order,
ty = peak area of levocarnitine from the Sample with two column volumes of methylene chloride, two
solution column volumes of methanol, and three column
ls = peak area of levocarnitine from the Standard
i
”
volumes of water. Pipet 5.0 mL of the Sample stock solu-
Q solution tion into the washed disposable column, and rinse the
3— Gs = concentration of USP Levocarnitine RS in the column twice with 6.0-mL portions of water. Collect
1o2) Standard solution (mg/mL) the filtrate and washings in a 25-mL volumetric flask,
° nominal concentration of levocarnitine in the
©

c add 5.0 mL of the Internal standard solution, and dilute

I

Sample solution (mg/mL) with water to volume.

° Acceptance criteria: 90.0%-110.0%
2 Chromatographic system
oe SPECIFIC TESTS (See Chromatography (621), System Suitability.)
a e BACTERIAL ENDOTOXINS TEST (85): It contains NMT 0.1 Mode: ne
= USP Endotoxin Units/mg of levocarnitine. Detector: UV 225 nm
¢ PH (791): 6.0-6.5 Column: 3.9-mm x 30-cm; 10-4um packing L1
e PARTICULATE MATTER IN INJECTIONS (788): Meets the re- Flow rate: 1.5 mL/min
quirements for small-volume injections Injection size: 40 uL
e INJECTIONS AND IMPLANTED DRUG PRODUCTS (1): Meets the System suitability
requirements Sample: Standard solution
[Note—The relative retention times for levocarnitine
ADDITIONAL REQUIREMENTS and p-aminobenzoic acid are 0.56 and 1.0,
e PACKAGING AND STORAGE: Preserve in single-dose contain- respectively.]
ers, preferably of Type | glass. Store below 25°. Do not Suitability requirements
freeze. Resolution: NLT 1.5 between the levocarnitine and
internal standard peaks
Relative standard deviation: NMT 2.0% for
Change to read: levocarnitine
Analysis
e USP REFERENCE STANDARDS (11) Samples: Standard solution and Sample solution
°o {CN T-May-2018) Calculate the percentage of the labeled amount of levo-
USP Levocarnitine RS ae (C7HisNOs) in the portion of Oral Solution
USP Levocarnitine Related Compound A RS taken:
2-Propen-1-aminium, 3-carboxy-N,N,N-trimethyl-,
chloride. Result = (Ru/Rs) x (Cs/Cu) x 100
CyHisCINOz =:179.65
Ru = peak area ratio of levocarnitine to p-
aminobenzoic acid from the Sample solution
Rs = peak area ratio of levocarnitine to p-
aminobenzoic acid from the Standard
solution
Cs = concentration of USP Levocarnitine RS in the
Standard solution (mg/mL)
USP 41 Official Monographs / Levocetirizine 2389

Cu = nominal concentration of levocarnitine in the Tu = peak area of levocarnitine from the Sample
Sample solution (mg/mL) solution
Acceptance criteria: 90.0%-110.0% rs = peak area of levocarnitine from the Standard
solution
SPECIFIC TESTS Cs = concentration of USP Levocarnitine RS in the
© PH (791): 4.0-6.0 Standard solution (mg/mL)
ADDITIONAL REQUIREMENTS Gu = nominal concentration of levocarnitine in the
e PACKAGING AND STORAGE: Preserve in tight containers. Sample solution (mg/mL)
e USP REFERENCE STANDARDS (11) Acceptance criteria: 90.0%-110.0%
USP Levocarnitine RS PERFORMANCE TESTS
e DISSOLUTION (711)
Medium: Water; 900 mL
Apparatus 2: 75 rpm
Time: 30 min
Levocarnitine Tablets Standard solution: Known concentration of USP Levo-
carnitine RS in Medium
DEFINITION Sample solution: Filtered portion of the solution under
Levocarnitine Tablets contain NLT 90.0% and NMT 110.0% test, suitably diluted with Medium if necessary
of the labeled amount of levocarnitine (C7HisNOs). Analysis
Samples: Standard solution and Sample solution
IDENTIFICATION Proceed as directed in the Assay, making any necessary
e A. The retention time of the major peak of the Sample modifications.
solution corresponds to that of the Standard solution, as Determine the percentage of the labeled amount of
obtained in the Assay. levocarnitine (C7HisNO3) dissolved:
e B. COLOR REACTION
Analysis: Dissolve 1 Tablet in 5 mL of water, filter, and Result = (ru/rs) x (Cs x D x V/L) x 100
add 5 mL of 1 N hydrochloric acid. Place 2 mL of the
filtrate in a test tube, and add a few drops of ammo- ru = peak area of levocarnitine in the Sample
nium reineckate TS. solution
Acceptance criteria: A red-violet precipitate is rs = peak area of levocarnitine in the Standard
produced. solution
Cs = concentration of USP Levocarnitine RS in the
ASSAY Standard solution (mg/mL)
© PROCEDURE D = dilution factor for the Sample solution
Buffer: 0.05 M phosphate buffer, pH 4.5, prepared by Vv = volume of Medium, 900 mL
dissolving 6.805 g of monobasic potassium phosphate L = label claim (mg/Tablet)
in 1 L of water
Mobile phase: Acetonitrile and Buffer (65:35). Adjust
Tolerances: NLT 75% (Q) of the labeled amount of
levocarnitine (C7HisNOs) is dissolved.
cs
wn
with phosphoric acid to a pH of 4.7, and mix. © UNIFORMITY OF DOSAGE UNITS (905): Meet the require- uv
System suitability solution: 1.5 mg/mL of USP Levo- ments for Weight Variation
carnitine RS and 7 g/mL of USP Levocarnitine Related =
CompoundA RS in water ADDITIONAL REQUIREMENTS
2}
p=
Standard solution: 3 mg/mL of USP Levocarnitine RS in e PACKAGING AND STORAGE: Preserve in tight containers. }
water e USP REFERENCE STANDARDS (11) ro}=
Sample solution: Transfer 10 Tablets, accurately USP Levocarnitine RS 2
weighed, to a 500-mL volumetric flask, and add water USP Levocarnitine Related Compound A RS 3
7
to volume. Shake until the Tablets have disintegrated 2-Propen-1-aminium, 3-carboxy-N,N,N-trimethyl-, a

completely, and pass throughafilter of 0.45-1m pore chloride.

size. Dilute a portion of the filtrate quantitatively with C7Hy4CINO2 = 179.65
water to a nominal concentration of about 3 mg/mL of
levocarnitine.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC Levocetirizine Dihydrochloride
Detector: UV 205 nm
Column: 3.9-mm x 30-cm; 10-um packing L8
Flow rate: 1 mL/min
Injection volume: 20 uL O
System suitability + 2Her
Samples: System suitability solution and Standard
solution a
Suitability requirements
Resolution: NLT 1.0 between levocarnitine related
compound A (crotonoylbetaine) and levocarnitine, Cai H2sCIN2O3 + ZHCI 461.81
System suitability solution Acetic acid, [2-[4-[(R)-(4-chlorophenyl)phenylmethyl]-
Relative standard deviation: NMT 2.0% for levo- 1-piperazinylJethoxy]-, Sone:
carnitine, Standard solution 21(8,(4-Chlorophenyp enylmethyl]piperazin-
Analysis
1-yl}ethoxy)acetic acid dihydrochloride [130018-87-0].
Samples: Standard solution and Sample solution Levocetirizine free base
Calculate the percentage of the labeled amount of levo-
carnitine (C7HisNOs) in the portion of Tablets taken: Ca HasCIN2O3 388.89
[130018-77-8].
Result = (ru/rs) x (Cs/Cu) x 100
 2390 Levocetirizine / Official Monographs USP 41

DEFINITION Mode: LC
Levocetirizine Dihydrochloride contains NLT 98.0% and Detector: UV 230 nm
NMT 102.0% of levocetirizine dihydrochloride Column: 4.6-mm x 25-cm; 5-um packing L3
(CaiHasCIN2O3 - 2HCI), calculated on the dried basis. Column temperature: 30°
Flow rate: 1 mL/min
IDENTIFICATION Injection volume: 20 pL
e A. INFRARED ABSORPTION (197K) Run time: 3 times the retention time of levocetirizine
e B. The retention time of the major peak of the Sample System suitability
solution corresponds to that of the levocetirizine peak of Samples: System suitability solution and Standard
the System suitability solution, as obtained in the test for solution
Enantiomeric Purity. [Note—See Table 1 for the relative retention times.]
e C, IDENTIFICATION TESTS—GENERAL (191), Chloride: Meets Suitability requirements
the requirements Resolution: NLT 3.0 between levocetirizine and
chlorobenzhydryl piperazine, System suitability solution
ASSAY Tailing factor: NMT 2.0 for levocetirizine, System
¢ PROCEDURE suitability solution
Mobile phase: Acetonitrile, water, and 1 M sulfuric acid Relative standard deviation: NMT 5.0% for levoce-
(93: 6.6: 0.4) tirizine, Standard solution
Standard solution: 0.05 mg/mL of USP Levocetirizine Analysis
Dihydrochloride RS in Mobile phase Samples: Standard solution and Sample solution
Sample solution: 0.05 mg/mL of Levocetirizine Dihy- Calculate the percentage of levocetirizine amide or
drochloride in Mobile phase chlorobenzhydryl piperazine in the portion of Levoce-
Chromatographic system tirizine Dihydrochloride taken:
(See Chromatography (621), System Suitability.)
Mode: LC Result = (ru/rs) x (Cs/Cu) x 100
Detector: UV 230 nm
Column: 4.6-mm x 25-cm; 5-um packing L3 ru = peak response of levocetirizine amide or
Column temperature: 30° chlorobenzhydryl piperazine from the Sample
Flow rate: 1 mL/min solution
Injection volume: 20 pL I
= peak response of levocetirizine amide or
System suitability chlorobenzhydryl piperazine from the
Sample: Standard solution Standard solution
Suitability requirements Cs = concentration of USP Levocetirizine Amide RS
Tailing factor: NMT 2.0 or USP Chlorobenzhydryl Piperazine RS in
Relative standard deviation: NMT 1.0% the Standard solution (ug/ml)
Analysis Cu = concentration of Levocetirizine
Samples: Standard solution and Sample solution Dihydrochloride in the Sample solution
Calculate the percentage of levocetirizine dihydrochlo- (ug/ml) costaralienA
<<
”
ride (C2H2sCIN2O3 - 2HCI) in the portion of Levoce- Calculate the percentage of any unspecified impurity in
% tirizine Dihydrochloride taken: the portion of Levocetirizine Dihydrochloride taken:
hs]
Dp
—

i} Result = (ru/rs) x (Cs/Cu) x 100 Result = (ru/rs) x (Cs/Cu) x 100

=
C ty = peak response of levocetirizine from the ty = peak response of any unspecified impurity
Pi Sample solution from the Sample solution
a rs = peak response of levocetirizine from the rs = peak response of levocetirizine from the
4) Standard solution Standard solution
he Cs = concentration of USP Levocetirizine Gs = concentration of USP Levocetirizine
Dihydrochloride RS in the Standard solution Dihydrochloride RS in the Standard solution
Cu
(mg/mL)
= concentration of Levocetirizine
(ug/ml) | :
Cu = concentration of Levocetirizine
Dihydrochloride in the Sample solution Dihydrochloride in the Sample solution
(mg/mL) (ug/ml)
Acceptance criteria: 98.0%-102.0% on the dried basis Acceptance criteria: See Table 1.
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.2% Table 1
© ORGANIC IMPURITIES Relative Acceptance
Mobile phase: Acetonitrile, water, and 1 M sulfuric acid Retention i Criteria,
(93: 6.6: 0.4) Name Time NMT (%)
System suitability solution: 0.2 mg/mL of USP Levoce- Levocetirizine
tirizine Dihydrochloride RS and 0.2 g/mL each of USP dihydrochloride 1.0 A
Levocetirizine Amide RS and USP Chlorobenzhydryl Pi-
perezine RS in Mobile phase. Use the solution within 16 Chlorobenzhydryl
piperazine 13! 0.2%
Standard solution: 0.2 g/mL each of USP Levoce- Levocetirizine amide 255) 0.2%
tirizine Dihydrochloride RS, USP Levocetirizine Amide Any individual
RS, and USP Chlorobenzhydryl Piperazine RS in Mobile unspecified —
phase. Use the solution within 16 h. impurity 0.1%
Sample solution: 200 g/mL of Levocetirizine Dihydro- Total impurities _— 0.5%
chloride in Mobile phase. Use the solution within 16 h.
Chromatographic system © ENANTIOMERIC PURITY
(See Chromatography (621), System Suitability.) Analyze the solutions within 24 h of preparation.
Mobile phase: Prepare a mixture of chromatographic
solvent hexane and absolute alcohol (95:5). Add 2 mL
of trifluoroacetic acid per L of mixture.
USP 41 Official Monographs / Levocetirizine 2391

System suitability solution: 5 mg/mL of USP Cetirizine IDENTIFICATION

Hydrochloride RS in absolute alcohol e A. ULTRAVIOLET ABSORPTION (197U)
Sensitivity solution: 0.05 mg/mL of USP Levocetirizine Medium: Water
Dihydrochloride RS in absolute alcohol Sample solution: Nominally 0.1 mg/mL of levoce-
Sample solution: 5 mg/mL of Levocetirizine Dihydro- tirizine dihydrochloride from Tablets in water prepared
chloride in absolute alcohol as follows. Transfer 1 Tablet to a suitable volumetric
Chromatographic system flask, and add 40% of the flask volume of water. Shake
(See Chromatography (621), System Suitability.) for NLT 5 min to promote the disintegration of the Tab-
Mode: LC let. Dilute with water to volume. Pass a 10-mL portion
Detector: UV 230 nm of the resulting solution through a suitable filter, and
Column: 4.6-mm x 25-cm; 5-um packing L40 discard the first mL. Use the filtrate.
Column temperature: 30° Acceptance criteria: Meet the requirements
Flow rate: 1.5 mL/min e B. The retention time of the major peak of the Sample
Injection volume: 10 uL solution corresponds to that of the Standard solution, as
System suitability obtained in the Assay.
Samples: System suitability solution and Sensitivity
solution ASSAY
[Note—The relative retention times for the S-enanti- © PROCEDURE
omer (of cetirizine) and levocetirizine, which is the R- Solution A: 1M sulfuric acid and water (5.7: 94.3)
enantiomer of cetirizine, are about 0.7 and 1.0, Mobile aed Acetonitrile, water, and 1 M sulfuric acid
respectively.] (93: 6.6: 0.4)
Suitability requirements System suitability solution: 0.2 mg/mL of USP Levoce-
Resolution: NLT 1.5 between the S-enantiomer and tirizine Dihydrochloride RS and 0.2 ug/mL of USP
levocetirizine, System suitability solution Chlorobenzhydry! Piperazine RS in Mobile phase
Signal-to-noise ratio: NLT 10 for levocetirizine, Sensi- Standard solution: 0.2 mg/mL of USP Levocetirizine Di-
tivity solution hydrochloride RS in Mobile phase
Analysis Sample solution: Nominally 0.2 mg/mL of levoce-
Sample: Sample solution tirizine dihydrochloride prepared as follows. Transfer a
Calculate the percentage of the S-enantiomer in the number of Tablets (NLT 10), equivalent to 50 mg of
portion of Levocetirizine Dihydrochloride taken: levocetirizine dihydrochloride, to a 250-mL volumetric
flask. Add 20 mL of Solution A, and put the flask on a
Result = (ru/rr) x 100 mechanical shaker for 15 min. Add 150 mL of acetoni-
trile, and place the flask in an ultrasonic bath for 10
ty = peak response for the S-enantiomer from the min. Allow the contents to cool to room temperature, if
Sample solution necessary, and dilute with acetonitrile to final volume.
tr = sum of all the peak responses for the S- Homogenize the solution, and centrifuge a 10-mL por-
enantiomer and levocetirizine from the tion for 5 min. Use the supernatant solution.
Sample solution Chromatographic system Cc
Acceptance criteria: NMT 2.0% of the S-enantiomer (See Chromatography (621), System Suitability.) 4)
Mode: LC a]
SPECIFIC TESTS Detector: UV 230 nm ss
e Loss ON DRYING (731) Columns °
Analysis: Dry at 105° to constant weight. Guard: 4-mm x 0.3-cm; 5-um packing L3 =]
Acceptance criteria: NMT 1.0% fo)
Analytical: 4.6-mm x 25-cm; 5-m packing L3 a
e PH (791) Column temperature: 30° ma
Sample solution: 50 mg/mL of Levocetirizine Dihydro- Ey
Flow rate: 1 mL/min mol
chloride in water Injection volume: 20 uL a
Acceptance criteria: 1,.2-1.8 7
System suitability
Sample: System suitability solution
ADDITIONAL REQUIREMENTS [Note—See Table 7 for relative retention times.]
© PACKAGING AND STORAGE: Preserve in well-closed contain- Suitability requirements
ers, protected from light at controlled room temperature. Resolution: NLT 3.0 between levocetirizine and
e USP REFERENCE STANDARDS (11)
chlorobenzhydryl piperazine
USP Cetirizine Hydrochloride RS Tailing factor: NMT 1.5 for levocetirizine
USP Chlorobenzhydry! Piperazine RS Relative standard deviation: NMT 1.0% for
(R)-1 olf enlorapne wupnenyimetiylpiperazines levocetirizine
Ci7zHigCIN2 286.80 Analysis
USP Levocetirizine Amide RS Samples: Standard solution and Sample solution
(R)-2-(2-{4-[(4-Chlorophenyl)phenylmethyl]piperazin- Calculate the percentage of the labeled amount of
1-yl}ethoxy)acetamide. levocetirizine dihydrochloride (C2H2sCIN2O3 - 2HCI) in
CaiH26CIN302 387.90 the portion of Tablets taken:
USP Levocetirizine Dihydrochloride RS
Result = (ru/rs) x (Cs/Cu) x 100
ty = peak response of levocetirizine from the
Sample solution
Levocetirizine Dihydrochloride Tablets ls = peak response of levocetirizine from the
Standard solution
DEFINITION Cs = concentration of USP Levocetirizine
Levocetirizine Dihydrochloride Tablets contain NLT 90% and Dihydrochloride RS in the Standard solution
NMT 110% of the labeled amount of levocetirizine dihy- (mg/mL)
drochloride (C2i1H2sCIN2O3 - 2HCI). Cu = nominal concentration of levocetirizine
dihydrochloride in the Sample solution
(mg/mL)
 2392 Levocetirizine / Official Monographs USP 41

Acceptance criteria: 90%-110% Mn = molecular weight of levocetirizine (free base),

388.89
PERFORMANCE TESTS Mz = molecular weight of levocetirizine
e DISSOLUTION (711) dihydrochloride, 461.81
Medium: Water; 900 mL Acceptance criteria: See Table 1. Disregard peaks less
Apparatus 2: 50 rpm than 0.1%.
Time: 30 min
Standard solution: (1/900) mg/mL of USP Levoce-
tirizine Dihydrochloride RS in Medium, where Lis the Table 1
label claim in mg/Tablet Relative Acceptance
Sample solution: Pass a portion of the solution under Retention Criteria,
test through a suitable filter.
Instrumental conditions 1.0 =
(See Ultraviolet-Visible Spectroscopy (857).)
Mode: UV-Vis
Analytical wavelength: 230 or 231 nm; use a suitable
wavelength for background correction Any individual unspecified
Cell: 1 or 2cm
Blank: Medium
Analysis @This is a process impurity that is included in this table for identification
Samples: Standard solution, Sample solution, and Blank only. This impurity is controlled in the drug substance. This impurity is
not to be reported for the drug product and is not to be included in the
Calculate the percentage of the labeled amount of total impurities.
levocetirizine dihydrochloride (C2:H2sCIN2O3 - 2HCI) » (R)-2-(2-{4-[(4-Chlorophenyl)phenylmethyl]piperazin-1 -yl}ethoxy)
dissolved: acetamide.

Result = (Au/As) x Cs x V x (1/L) x 100 ADDITIONAL REQUIREMENTS

© PACKAGING AND STORAGE: Preserve in well-closed contain-
Au = absorbance of the Sample solution ers, and store at controlled room temperature.
As = absorbance of the Standard solution e USP REFERENCE STANDARDS (11)
Cs = concentration of USP Levocetirizine USP Chlorobenzhydryl Piperazine RS
Dihydrochloride RS in the Standard solution (R)-1-[(4-Chlorophenyl)phenylmethyl]piperazine.
(mg/mL) Cy7HisCIN2 286.80
Vv = volume of Medium, 900 mL USP Levocetirizine Dihydrochloride RS
L = label claim (mg/Tablet)
Tolerances: NLT 80% (Q) of levocetirizine dihydrochlo-
ride (C2H2sCIN2O3 - 2HCI) is dissolved.
e UNIFORMITY OF DOSAGE UNITS (905): Meet the
" requirements Levodopa
ee
r= IMPURITIES

SS
— © ORGANIC IMPURITIES
aD Solution A, Mobile phase, System suitability solution,
°
= and Sample solution: Prepare as directed in the Assay.
5 Standard solution: 0.002 mg/mL of USP Levocetirizine
= Dihydrochloride RS in Mobile phase
a Chromatographic system: Proceed as directed in the
al Assay, except for the Run time.
—) Run time: 2.3 times the retention time of
levocetirizine
System suitability
Sample: System suitability solution
[Note—See Table 1 for relative retention times.]
Suitability requirements
Resolution: NLT 3.0 between levocetirizine and
chlorobenzhydryl piperazine
Tailing factor: NMT 1.5 for levocetirizine
Relative standard deviation: NMT 1.0% for levoce-
tirizine; NMT 5.0% for chlorobenzhydryl piperazine
Analysis
Samples: Sample solution and Standard solution
Calculate the percentage of each impurity in the por-
tion of Tablets taken:
Result = (u/s) x (Cs/Cu) x (Mr/Mi2z) x 100
tu = peak response of each impurity from the
Sample solution
ig = peak response of levocetirizine from the
Standard solution
G = concentration of USP Levocetirizine
Dihydrochloride RS in the Standard solution
(mg/mL)
Cu = nominal concentration of levocetirizine
dihydrochloride in the Sample solution
(mg/mL)
so
HO.
C5H1iNO4 197019.
L-Tyrosine, 3-hydroxy-;
(-)-3-(3,4-Dihydroxyphenyl)-L-alanine [59-92-7].
DEFINITION
Levodopa contains NLT 98.0% and NMT 102.0% of
levodopa (CsHi;NOa), calculated on the dried basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197M)
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
ASSAY
© PROCEDURE
Protect all solutions from light, and maintain them at
10° until they are injected into the chromatograph.
Diluent: 0.1% trifluoroacetic acid in water
Mobile phase: Tetrahydrofuran and Diluent (3:97)
System suitability solution: 10 g/mL each of USP
Levodopa RS, USP Levodopa Related Compound BRS,
and USP L-Tyrosine RS in Diluent
Standard solution: 0.4 mg/mL of USP Levodopa RS in
Diluent
Sample solution: 0.4 mg/mL of Levodopa in Diluent
Chromatographic system
(See Chromatography (621), System Suitability.)
USP 41 Official Monographs / Levodopa 2393

Mode: LC Table 1 (Continued)

Detector: UV 280 nm Relative Relative Acceptance
Column: 4.6-mm x 25-cm; 5-~um L1 packing Retention Response Criteria,
Flow rate: 1 mL/min Name Time Factor NMT (%)
Injection volume: 20 wL
System suitability L-Tyrosine 1.3 0.44 0.1
Sample: System suitability solution Levodopa related
[Note—Refer to Table 1 in the test for Organic Impurities compound B 1.6 0.83 0.5
for the relative retention times.] L-Veratrylglycine> ra 0.76 0.1
Suitability requirements Individual un-
Resolution: NLT 3.0 between levodopa and L-tyrosine known impurity _ 1.0 0.1
Tailing factor: NMT 2.0 for levodopa Total impurities = ed 11
Relative standard deviation: NMT 2.0% for 2 3-(3,4,6-Trihydroxyphenyl)alanine.
levodopa » 3-(3,4-Dimethoxyphenyl)-L-alanine.
Analysis
Samples: Standard solution and Sample solution SPECIFIC TESTS
Calculate the percentage of levodopa (CsH1;NO,) in the © OPTICAL ROTATION, Specific Rotation (7815S)
portion of the sample taken: Sample solution: 500 mg of Levodopa in a 25-mL vol-
umetric flask. Add 10 mL of 1 N hydrochloric acid to
Result = (ru/rs) x (Cs/Cu) x 100 dissolve the solid, add 5 g of methenamine, swirl the
contents to dissolve the methenamine, and add 1 N hy-
ru = peak response of the Sample solution drochloric acid to volume.
Is = peak response of the Standard solution Analysis: Allow the Sample solution to stand in the dark
G = concentration of USP Levodopa RS in the at 25° for 3 h, and measure the rotation.
Standard solution (mg/mL) Acceptance criteria: —160° to -167°
Cy = concentration of Levodopa in the Sample e Loss ON DRYING (731)
solution (mg/mL) Analysis: Dry at 105° for 4 h.
Acceptance criteria: 98.0%-102.0% on the dried basis Acceptance criteria: NMT 0.5%
IMPURITIES ADDITIONAL REQUIREMENTS
e RESIDUE ON IGNITION (281): NMT 0.1% © PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, store in a dry place, and prevent exposure to
Delete the following: excessive heat.
e USP REFERENCE STANDARDS (11)
°e HEAVY METALS, Method Il (231): NMT 20 ppme cortciat1- USP Levodopa RS
Jan-2018) USP Levodopa Related Compound B RS
© ORGANIC IMPURITIES 3-Methoxytyrosine.
Protect all solutions from light, and maintain them at CioHisNO4 = -.211.22 =
USP L-Tyrosine RS 2)
10° until they are injected into the chromatograph. z
Diluent, Mobile phase, System suitability solution,
Standard solution, Sample solution, Chromato- =
re}
graphic system, and System suitability: Proceed as S
directed in the Assay. i}
Analysis Levodopa Capsules a=
Samples: Standard solution and Sample solution ES)
Calculate the percentage of each impurity in the por- DEFINITION so)
tion of Levodopa taken: Levodopa Capsules contain NLT 90.0% and NMT 110.0% of Gp
as
the labeled amount of levodopa (CsH1iNO,).
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
IDENTIFICATION
ty = peak area for any impurity in the Sample e A. INFRARED ABSORPTION (197M)
solution Sample: Shake a quantity of the contents of the Cap-
Is = peak area for Levodopa in the Standard sules, equivalent to about 500 mg of levodopa, with
solution 25 mL of 3N pecineca lore acid, and filter. Adjust the
Cs = concentration of USP Levodopa RS in the acidity of the filtrate with 6 N ammonium hydroxide to
Standard solution (mg/mL) a pH of 3, added dropwise with stirring, and allow to
Cu = concentration of Levodopa in the Sample stand protected from light for several h. Filter, wash the
solution (mg/mL) precipitate with water, and dry at 105°.
FE = relative response factor of each impurity (see Acceptance criteria: The residue meets the
Table 1) requirements.
Acceptance criteria: See Table 1.
ASSAY
¢ PROCEDURE
Table 1
Standard solution: 35 g/mL of USP Levodopa RS in
Relative Relative Acceptance 0.1 N hydrochloric acid
Retention Response Criteria, Sample stock solution: 1.75 mg/mL of levodopa in 0.1
Name Time Factor NMT (%) N hydrochloric acid, from the contents of NLT 20 Cap-
Levodopa related sules. Shake the mixture by mechanical means for 5
compound Aa 0.9 0.41 0.1 min, and filter discarding the first 20 mL of the filtrate.
Levodopa 1.0 = Sample solution: 35 jug/mL of levodopa in 0.1 N hy-
drochloric acid from the Sample stock solution
2 3-(3,4,6-Trihydroxyphenyl)alanine.
» 3-(3,4-Dimethoxyphenyl)-L-alanine.
 2394 Levodopa / Official Monographs USP 41

Blank: 0.1 N hydrochloric acid Analysis

Instrumental conditions Samples: Standard solution A, Standard solution B, and
Mode: UV-Vis Sample solution
Cell: 1cm Apply the Samples at separate points about 3 cm from
Analytical wavelength: 280 nm the bottom of the ie Dry the spots in a stream of
Analysis nitrogen, and develop the chromatogram in a suitable
Samples: Standard solution and Sample solution low-actinic chamber equilibrated for 5 min with a
Calculate the percentage of the labeled amount of freshly mixed portion of Developing solvent until the
levodopa (CsH;;NOs4) in the portion of Capsule con- solvent front has moved about 15 cm from the line of
tents taken: application. Remove the plate from the chamber, mark
the solvent front, and dry in a current of air for about
Result = (Au/As) x (Cs/Cu) x 100 10 min. Spray the plate with Spray reagent. Levodopa
related compound A produces a spot at an R- of about
Au = absorbance of the Sample solution 0.25, and levodopa related compoundB at an R; of
As = absorbance of the Standard solution about 0.5.
Cs = concentration of USP Levodopa RS in the Acceptance criteria: No spot at R; 0.25 from the Sam-
Standard solution (g/mL) ple solution is greater in size or intensity than the corre-
Cu = nominal concentration of levodopa in the sponding spot from Standard solution A corresponding
Sample solution (g/mL) to NMT 0.1% levodopa related compound A. No spot
Acceptance criteria: 90.0%-110.0% at Rr 0.5 from the Sample solution is greater in size or
intensity than the corresponding spot from Standard so-
PERFORMANCE TESTS lution B corresponding to NMT 0.5% of levodopa re-
e DISSOLUTION (711) lated compound B. [NoTte—Disregard the bleached
Medium: 0.01 N hydrochloric acid; 900 mL spot, which is an artifact resulting from the develop-
Apparatus 1: 100 rpm ment of sodium metabisulfate from Diluent. It may ap-
Time: 30 min pear at an R; value of about 0.6.]
Instrumental conditions
Mode: UV ADDITIONAL REQUIREMENTS
Analytical wavelength: Maximum at about 280 nm @ PACKAGING AND STORAGE: Preserve in tight, light-resistant
Standard solution: USP Levodopa RS in Medium containers, in a dry place, and prevent exposure to ex-
Sample solution: Sample per Dissolution (711). Dilute cessive heat.
with Medium to a concentration that is similar to that of e USP REFERENCE STANDARDS (11)
the Standard solution. USP Levodopa RS
Tolerances: NLT 75% (Q) of the labeled amount of USP Levodopa Related Compound A RS
levodopa (CsH;;NOs,) is dissolved. 3-(3,4,6-Trihydroxyphenyl)alanine.
e UNIFORMITY OF DOSAGE UNITS (905): Meet the CsHiiNOs 213.19
requirements USP Levodopa Related Compound B RS
3-Methoxytyrosine.
fn
al
IMPURITIES CioHi3NO, = =—.211.22
at ¢ ORGANIC IMPURITIES
i]
— [Note—Use low-actinic glassware for all volumetric
a solutions.]
)
= Diluent: Dissolve 100 mg of sodium metabisulfite in
5 10 mL of 1.2 N hydrochloric acid, and dilute with ace-
Levodopa Tablets
= tone to 100 mL.
Pe Standard solution A: 10 g/mL of USP Levodopa Re-
al lated Compound A RS and 10 mg/mL of USP Levodopa DEFINITION
p>) RS in Diluent Levodopa Tablets contain NLT 90.0% and NMT 110.0% of
Standard solution B: 50 g/mL of USP Levodopa Re- the labeled amount of levodopa (CoH1;NOx,).
lated Compound B RS in Diluent
Sample solution: Just prior to application, dissolve IDENTIFICATION
100 mg of the residue obtained in the /dentification test e A. INFRARED ABSORPTION (197M)
in 10.0 mL of Diluent. Sample: Shake a quant of powdered Tablets equiva-
Chromatographic system lent to 500 mg of oe with 25 mL of 3 N hydro-
(See Chromatography (621), Thin-Layer Chromato- chloric acid, and filter. Adjust the acidity of the filtrate
with 6 N ammonium hydroxide, added dropwise with
graphy.)
stirring, and allow to stand, protected from light, for
Mode: TLC several h. Filter, wash the precipitate with water, and
Adsorbent: Thin-layer chromatographic plate, coated
with a 0.25-mm layer of microcrystalline cellulose dry at 105°.
Predevelop a plate in Developing solvent until the sol- Acceptance criteria: The residue meets the
vent front has moved NLT 18 cm from the origin. requirements.
Remove the plate from the chamber, and dry in a ASSAY
current of air for about 10 min. e@ PROCEDURE
[Note—The plate may be developed overnight: solvent Protect all solutions from light, and maintain them at
overflow during predevelopment is of no 10° until they are injected into the chromatograph.
consequence.] Mobile phase: 0.01 M monobasic potassium phos-
Application volume: 10 pL phate; adjust with phosphoric acid and acetonitrile
Developing solvent: Butyl alcohol, methanol, glacial (97:3) to a pH of 2.0.
acetic acid, and water (150:15:75:75) Standard solution: 0.4 mg/mL of USP Levodopa RS in
Spray reagent: Just before use, mix 2 volumes of fer- Mobile phase
tic chloride solution (100 mg/mL) with 1 volume of Sample solution: 0.4 mg/mL of levodopa in Mobile
potassium ferricyanide solution (50 mg/mL) to obtain phase, from finely powdered Tablets (NLT 20). Filter.
about 100 mL of solution. Sonicate for 5 min.
System suitability solution: 10 j1g/mL each of USP
Levodopa RS, USP Levodopa Related Compound B RS,
USP 41 Official Monographs / Levofloxacin 2395

and USP Levodopa Related Compound A RS in Mobile Acceptance criteria: See Table 1.
hase
hromatographic system Table 1
(See Chromatography (621), System Suitability.)
Mode: LC Relative Relative Acceptance
Detector: UV 280 nm Retention Response Criteria,
Column: 3.0-mm x 25-cm; packing L1 Name Time Factor NMT (%)
Flow rate: 1 mL/min Levodopa related
Injection volume: 20 uL compound A 0.9 0.83 0.1
System suitability Levodopa 1.0 =_ =
Sample: System suitability solution Levodopa related
[NotE—The relative retention times for levodopa related compound B 2.8 0.83 0.5
compound A, levodopa, and levodopa related com-
5,6-Dihydroxy-in-
poundBare 0.7, 1.0, and 2.8, respectively.]
dole-2-carboxylic
Suitability requirements
Resolution: NLT 3.5 between levodopa related com- acid 6.0 25 0.1
pound A and levodopa 0.1
Relative standard deviation: NMT 2.0% for individual
levodopa i 0.3 total
Analysis Unknown impurities 1.0 unknown
Samples: Standard solution and Sample solution Total impurities = = 11
Calculate the percentage of the labeled amount of
levodopa (CsHi:NOx) in the portion of Tablets taken: ADDITIONAL REQUIREMENTS
Result = (ru/rs) x (Cs/Cu) x 100 e PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, in a dry place, and prevent exposure to ex-
ry = peak response from the Sample solution cessive heat.
rs = peak response from the Standard solution e USP REFERENCE STANDARDS (11)
Cs = concentration of USP Levodopa RS in the USP Levodopa RS
Standard solution (mg/mL) USP Levodopa Related Compound A RS
Cc = nominal concentration of levodopa in the ae Moye eepbenyjalanine,
Sample solution (mg/mL) CoHNOs = 213.
Acceptance criteria: 90.0%-110.0% USP Levodopa Related Compound B RS
3-Methoxytyrosine.
PERFORMANCE TESTS CioHisNO, = =—-211.22
© DISSOLUTION (711)
Medium: 0.01 N hydrochloric acid; 900 mL
Apparatus 1: 100 rpm
Time: 30 min c
al
Detector: UV maximum at about 280 nm Levofloxacin a]
Standard solution: USP Levodopa RS in Medium
Sample solution: Sample per Dissolution (711). Dilute =
°
with Medium to a concentration that is similar to that of
the Standard solution. bade =]
°
Tolerances: NLT 75% (Q) of the labeled amount of
levodopa (CsH1;NOx) is dissolved.
LT ivom Ko}
y
a
mo)
© UNIFORMITY OF DOSAGE UNITS (905): Meet the a
requirements 7)
CisHaoFN3Oq - 1/2H20 370.38
IMPURITIES 7H-Pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid,
© ORGANIC IMPURITIES 9-fluoro-2,3-dihydro-3-methyl-10-(4-methyI-1-piperazinyl)-
Protect all solutions from light, and maintain them at 7-oxo-hydrate (2:1), (5)-;
10° until they are injected into the chromatograph. (-)-(S)-9-Fluoro-2,3-dihydro-3-methy|-10-(4-methyl-1 -piper-
Mobile phase, System suitability solution, Standard Ser reel ee ee -Car-
solution, Sample solution, Chromatographic system, boxylic acid, hemihydrate [138199-71-0].
and System suitability: Prepare as directed in the Anhydrous [100986-85-41].
Assay.
Analysis DEFINITION
Samples: Standard solution and Sample solution Levofloxacin contains NLT 98.0% and NMT 102.0% of
Calculate the percentage of each impurity in the por- CisH20FN3Ox4, calculated on the anhydrous basis.
tion of Tablets taken:
IDENTIFICATION
Result = (ru/rs) x (Cs/Cu) x (1/P) x 100 e A. INFRARED ABSORPTION (197K)
e B. The retention time of the major peak of the Sample
ty = peak area for any impurity from the Sample solution corresponds to that of the Standard solution, as
solution obtained in the Assay.
Is = peak area for levodopa from the Standard
solution ASSAY
Cs = concentration of USP Levodopa RS in the ¢ PROCEDURE
Standard solution (mg/mL) Buffer: 8.5 g/L of ammonium acetate, 1.25 g/L of cu-
Cu = nominal concentration of levodopa in the pric sulfate, pentahydrate, and 1.3 g/L of L-isoleucine in
Sample solution (mg/mL) water
F = relative response factor of the impurity (see Mobile phase: Methanol and Buffer (3:7)
Table 1) Standard solution: 1 mg/mL of USP Levofloxacin RS in
Mobile phase
 2396 Levofloxacin / Official Monographs USP 41

Sample solution: 1 mg/mL of Levofloxacin in Mobile Table 1

dus Relative Relative Acceptance
hromatographic system
Retention Response Criteria,
(See Chromatography (621), System Suitability.) Name Time Factor NMT (%)
Mode: LC
Detector: UV 360 nm N-Desmethyl
Column: 4.6-mm x 25-cm; 5-1um packing L1 levofloxacin 0.47 1.0 0.3
Column temperature: 45° Diamine derivative> 0.52 0.9 0.3
Flow rate: 0.8 mL/min Levofloxacin
Injection size: 25 uL N-oxidec 0.63 11 0.3
System suitability 9-Desfluoro levoflox-
Sample: Standard solution acin¢ 0.73 1.0 0.3
Suitability requirements Levofloxacin 1.0 = =
Tailing factor: 0.5-1.5 D-Isomer® 1.23 1.0 0.8
Relative standard deviation: NMT 1.0%
Analysis Any unknown _
Samples: Standard solution and Sample solution impurity 1.0 0.1
Calculate the percentage of levofloxacin (CisH2oFN3O.) Total Impurities =— = 0.5*
in the portion of Levofloxacin taken: 2 (5)-9-Fluoro-2,3-dihydro-3-methyl-1 0-(piperazin-1-yl)-7-oxo-7H-pyrido[1,
2,3-de)[1,4]benzoxazine-6-carboxylic acid.
Result = (ru/rs) x (Cs/Cu) x 100 » (5)-9-Fluoro-2,3-dihydro-3-methyl-10-[2-(methylamino)ethylamino]-7-
oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid.
tu = peak response of levofloxacin from the Sample ¢ (S)-4-(6-Carboxy-9-fluoro-2, 3-dihydro-3-methyl-7-0xo-7H-pyrido-[1,2,3-
de}{1,4]benzoxazine-10-yl)-1-methyl-piperazine-1-oxide.
solution 4 (S)-2,3-Dihydro-3-methyl-10-(4-methyI-1-piperazinyl)-7-oxo-7H-pyrido[1,
rs = peak response of levofloxacin from the 2,3-de)[1,4]benzoxazine-6-carboxylic acid.
Standard solution s Cy} Fluora- 23 cinare.Smetiyt| 0-(4-methyl-1 -piperazinyl)-7-oxo-7H-
Cs = concentration of USP Levofloxacin RS in the pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid.
Standard solution (mg/ml) * Do not include the D-isomer in the calculation for total impurities.
Cu = concentration of Levofloxacin in the Sample
solution (mg/mL) © ORGANIC IMPURITIES, PROCEDURE 2
Acceptance criteria: 98.0%-102.0% on the anhydrous [NoTte—Solutions of levofloxacin are not stable in light;
basis use amber bottles.]
Buffer: Dissolve 3.08 g/L of ammonium acetate and
IMPURITIES 8.43 g/L of sodium perchlorate monohydrate in water.
e RESIDUE ON IGNITION (281): NMT 0.2%. Use a platinum Adjust with phosphoric acid to a pH of 2.2.
crucible. Solution A: Acetonitrile and Buffer (16:84)
Solution B: Acetonitrile, methanol, and Buffer
(30:20:50)
f= Delete the following:
”
Solution C: 0.4 mg/mL of USP Levofloxacin RS by dis-
a solving in acetonitrile at about 8% of volume and dilut-
ij
®e HEAVY METALS, Method /! (231): NMT 10 pome corrcial1. ing with water to volume
aD
—
Solution D: 0.05 mg/mL of USP Levofloxacin Related
° Jan-2018)
€ e ORGANIC IMPURITIES, PROCEDURE 1 CompoundA RS in 0.2% ammonium hydroxide in
5 [Note—Procedure 1 is recommended if levofloxacin N-ox- methanol
= ide is a potential organic impurity. Procedure 2 and Pro- Mobile phase: See Table 2.
re cedure 3 are recommended if levofloxacin related com-
2) poundBis a potential organic impurity.]
Table 2
=) Solution A, Mobile phase, Sample solution, and Chro-
matographic system: Proceed as directed in the Solution A Solution B
Assay.
System suitability solution: 1 mg/mL of USP Levoflox- 1
acin RS in Mobile phase
Sensitivity solution: 0.3 g/mL of USP Levofloxacin RS
in Mobile phase
System suitability
Samples: System suitability solution and Sensitivity
solution
Suitability requirements
Relative standard deviation: NMT 1.0%, System suit-
ability solution System suitability solution: 0.1 mg/mL of USP
Signal-to-noise ratio: NLT 10, Sensitivity solution levofloxacin RS and 5 g/mL of USP Levofloxacin Re-
Analysis lated CompoundA RS in water from Solution C and
Sample: Sample solution Solution D
Calculate the percentage of each individual impurity in Levofloxacin stock solution: 0.4 mg/mL of USP
the portion of Levofloxacin taken: Levofloxacin RS. Dissolve USP Levofloxacin RS in aceto-
nitrile at about 8% of final volume, sonicate, and dilute
Result = (ru/rs) x (1/F) x 100 with water to volume.
Levofloxacin standard solution: 0.02 mg/mL of USP
ru = peak response of each impurity Levofloxacin RS in acetonitrile and water (1:10) from
rs = peak response of levofloxacin Levofloxacin stock solution
F = relative response factor (see Table 1) Levofloxacin related compoundBstock solution:
Acceptance criteria: See Table 1. 0.2 maior USP Levofloxacin Related Compound B RS
in methanol. [NoTe—Sonicate if necessary.
Levofloxacin related compoundBstandard solution:
0.04 mg/mL USP Levofloxacin Related Compound B RS
USP 41 Official Monographs / Levofloxacin 2397

in methanol from Levofloxacin related compound B stock Mobile phase: Methanol and Buffer (15:85)
solution System suitability solution: 0.01 mg/mL of USP Oflox-
Standard solution: 0.4 g/mL of levofloxacin and acin RS and 0.01 mg/mL of USP Levofloxacin RS in
0.8 g/mL of levofloxacin related compoundBin aceto- water
nitrile and water (1:10) from Levofloxacin standard solu- Sample solution: 0.08 mg/mL in water
tion and Levofloxacin related compound B standard Chromatographic ye
solution (See Chromatography (621), System Suitability.)
Sample solution: 0.4 mg/mL by dissolving the sample Mode: LC
in acetonitrile at about 8% of final volume and diluting Detector: 294 nm
with water to volume. [NoTE—Sonicate if necessary.] Column: 4.6-mm x 15-cm; 3.5-1m packing L1
Chromatographic system Column temperature: 40°
(See Chromatography (621), System Suitability.) Flow rate: 0.7 mL/min
Mode: LC Injection size: 10 uL
Detector: 280 nm System suitability
Column: 4.0-mm x 15-cm; 3.0-um packing L1 Sample: System suitability solution
Column temperature: 38° [Note—The relative retention times for D-ofloxacin and
Flow rate: 1.0 mL/min levofloxacin are 0.91 and 1.0, respectively.]
Injection size: 10 uL Suitability requirements
System suitability Resolution: NLT 2.0 between D-ofloxacin (D-isomer)
Sample: System suitability solution and levofloxacin
Suitability requirements Analysis
Relative standard deviation: NMT 2.0% for Sample: Sample solution
levofloxacin Calculate the percentage of D-ofloxacin in the portion
Analysis of Levofloxacin taken:
Samples: Standard solution and Sample solution
Calculate the Cer of levofloxacin related com- Result = (ru/r7) x 100
poundBin the portion of Levofloxacin taken:
tu = peak response for D-ofloxacin
Result = (ru/rs) x (Cs/Cu) x 100 rr = sum of responses of all peaks
Acceptance criteria: NMT 1.0%
ty = peak response for levofloxacin related
compound B from the Sample solution SPECIFIC TESTS
Is = peak response for levofloxacin related e OPTICAL ROTATION, Specific Rotation (781S)
compound B from the Standard solution Solvent: Methanol
G = concentration of USP Levofloxacin Related Sample solution: 5 mg/mL in Solvent
CompoundBRS in the Standard solution Acceptance criteria: —92° to —106°, at 20°
(mg/mL) © WATER DETERMINATION, Method Ia (921): 2.0%-3.0%
Cu = concentration of Levofloxacin in the Sample
ADDITIONAL REQUIREMENTS [=
solution (mg/mL) “
Calculate the percentage of other impurities in the @ PACKAGING AND STORAGE: Preserve in tight, light-resistant a~}
containers. Store at room temperature.
portion of Levofloxacin taken:
e LABELING: If a procedure for Organic Impurities other than ms
i}
Result = (ru/rs) x (Cs/Cu) x 100 Procedure 1 is used, then the labeling states with which =
Organic Impurities procedure the article complies. }
tu = peak response of any other impurity from the © USP REFERENCE STANDARDS (11) eo}=
Sample solution USP Levofloxacin RS ES)
a}
Is = peak response of levofloxacin from the USP Levofloxacin Related Compound A RS 7
Standard solution (S)-9-Fluoro-3-methyl-10-(piperazin-1-yl)-7-oxo-2,3- "“

G = concentration of USP Levofloxacin RS in the Spee eae 1,2,3-de][1,4]benzoxazine-6-carbox-

standard solution (mg/mL) ic acid.
Cy = concentration of Levofloxacin in the Sample CithaEN;Os 347.34
solution (mg/mL) USP Levofloxacin Related Compound B RS
Acceptance criteria: See Table 3. (5)-9,10-Difluoro-3-methyl-7-oxo-2,3-dihydro-7 H-pyrido
[1,2,3-de][1,4]benzoxazine-6-carboxylic acid.
CisHoF2NOg 281.21
Table 3
USP Ofloxacin RS
Relative Acceptance
Retention Criteria,

Levofloxacin related compound A

N- loxacii
1.0 Levofloxacin Oral Solution
im, =_ 10 DEFINITION
Te ay Levofloxacin Oral Solution contains NLT 90.0% and NMT
2 (S)-9-Fluoro-3-methyl-10-(piperazin-1-yl)-7-oxo-2,3-dihydro-7H-pyrido[1, 110.0% of the labeled amount of levofloxacin
2,3-de][1,4-benzoxazine-6-carbocylic acid. (CisH2oFN30).
» (S)-9,10-Difluoro-3-methyl-7-0xo-2,3-dihydro-7H-pyrido[1 ,2,3-de][1,4-
benzoxazine-6-carbocylic acid. IDENTIFICATION
e A. The retention time of the major peak of the Sample
© ORGANIC IMPURITIES (ENANTIOMERIC PURITY), PROCEDURE 3 solution corresponds to that of the Standard solution, as
Buffer: hoagit of D-phenylalanine and 0.75 g/L of obtained in the Assay.
copper(Il)sulfate pentahydrate in water
 2398 Levofloxacin / Official Monographs USP 41

ASSAY Acceptance criteria

© PROCEDURE Individual impurities: See /mpurity Table 1.
[Note—Protect the solutions of levofloxacin from light.]
Diluent: Acetonitrile and water (18:82) Impurity Table 1
Mobile phase: Diluent that contains 1 mL of trifluoroa-
cetic acid in each 1000 mL of solution Relative Relative Acceptance
Standard solution: 102.5 ug/mL of USP Levofloxacin Retention Response Criteria,
RS in Diluent Name Time Factor NMT (%)
System suitability solution: 102.5 g/mL each of USP 9-Desfluoro iSa
Levofloxacin RS and USP Levofloxacin Related Com- levofloxacine 0.64 1.0
pound A RS in Diluent Diamine oe
Sample solution: 102.5 ug/mL of levofloxacin in Dilu- derivative> 0.75 1.0
ent based on the label claim. [NoTE—Mix the solution Levofloxacin
well after equilibrating the solution for 4 h at room related
temperature while protected from light.] compound Ac 0.91 0.81 0.5
Chromatographic system Levofloxacin 1.0 = =
(See Chromatography (621), System Suitability.)
Mode: LC Levofloxacin
Detector: UV 294 nm N-oxided 1.55 0.93 0.5
Column: 4.6-mm x 15-cm; 3.5-m packing L11 Any other individual us
Column temperature: 30° impurity 1.0 0.2
Flow rate: 0.7 mL/min Total impurities = = 1.0
Run time: 2.5 times the retention time of the levoflox- 4 ($)-2,3-Dihydro-3-methy!-1 0-(4-methyI-1 -piperazinyl)-7-oxo-7H-pyrido[1,
acin peak 2)3-del[1,4]benzoxazine-6-carboxylic acid. ” nt
Injection size: 20 pL » (S)-9-Fluoro-2, 3-dihydro-3-methyI-10-[2-(methylamino)ethylamino]-7-
System suitability oxo-7H-pyrido[1 ,2,3-de][1,4]benzoxazine-6-carboxylic acid.
Samples: Standard solution and System suitability ¢ (S)-9-Fluoro-2,3-dihydro-3-methyl-10-(piperazin-1-yl)-7-oxo-7H-pyrido[1,
2,3-de)[1,4]benzoxazine-6-carbocylic acid.
solution 9 (S)-4-(6-Carboxy-9-fluoro-2,3-dihydro-3-methy|-7-oxo-7H-pyrido-[1,2,3-
Suitability requirements de][1,4]benzoxazine-10-yl)-1-methylpiperazine 1-oxide.
Resolution: NLT 1.9 between levofloxacin related * Disregard this peak because this is a process impurity controlled for the
compound A and levofloxacin, System suitability drug substance.
solution
Relative standard deviation: NMT 2.0%, Standard SPECIFIC TESTS
solution e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
Analysis FIED MICROORGANISMS (62): The total aerobic microbial
Samples: Standard solution and Sample solution count does not exceed 10? cfu/mL, and the total com-
Calculate the percentage of the labeled amount of bined molds and yeast count does not exceed 10! cfu/
"
levofloxacin (CisHaoFN3O,) in the portion of Oral Solu- mL. It also meets the requirement for absence of Escher-
oo ichia coli.
3 tion taken:
© DELIVERABLE VOLUME (698): Meets the requirements
i
—
e PH (791): 5.0-6.0
Dp Result = (ru/rs) x (Cs/Cu) x 100
°
is Tu = peak response from the Sample solution ADDITIONAL REQUIREMENTS
S peak response from the Standard solution e PACKAGING AND STORAGE: Store at controlled room tem-
= Gs concentration of USP Levofloxacin RS in the perature, and protect from light.
a Standard solution (mg/mL) e USP REFERENCE STANDARDS (11)
a)
Cu = nominal concentration of levofloxacin in the USP Levofloxacin RS
= Sample solution (mg/mL) 7H-Pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid,
Acceptance criteria: 90.0%-110.0% 9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1 -piper-
azinyl)-7-oxo-hydrate (2:1), (5)-;
IMPURITIES (-)-($)-9-Fluoro-2,3-dihydro-3-methyl-10-(4-methyl-
Organic Impurities ea roe ,2,3-de]-1,4-benzox-
© PROCEDURE azine-6-carboxylic acid, hemihydrate.
{Note—Protect the solutions of levofloxacin from light.] Anhydrous.
Diluent, Mobile phase, Standard solution, System CisH2oFN3Oz - /2H20 370.38
suitability solution, Sample solution, Chromato- USP Levofloxacin Related Compound A RS
graphic system, and System suitability: Proceed as Beeae 0-(piperazin-1-yl)-
directed in the Assay. 7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carbo-
Analysis cylic acid.
Samples: Standard solution and Sample solution Ci7HisFN304 347.34
Calculate the percentage of each individual impurity in
the portion of Oral Solution taken:
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
Levofloxacin Tablets
tu = peak response of each individual impurity
from the Sample solution DEFINITION
rs = peak response of levofloxacin from the Levofloxacin Tablets contain NLT 90.0% and NMT 110.0%
Standard solution of the labeled amount of levofloxacin (C;gsH20FN3O,).
Cs = concentration of USP Levofloxacin RS in the
Standard solution (mg/mL) IDENTIFICATION
Cy = nominal concentration of levofloxacin in the e A. The retention time of the major peak of the Sample
Sample solution (mg/mL) solution corresponds to that of the Standard solution, as
F = relative response factor for each impurity (See obtained in the Assay.
Impurity Table 1)
USP 41 Official Monographs / Levofloxacin 2399

ASSAY Au = absorbance of the Sample solution

e PROCEDURE As = absorbance of the Standard solution
Diluent: Acetonitrile and water (20:80) Cs = concentration of the Standard solution
Mobile phase: Transfer 874 mg of cupric sulfate, (mg/mL)
918 mg of L-isoleucine, and 5.94 g of ammonium ace- Vv = volume of Medium, 900 mL
tate to a suitable container. Add 700 mL of water, and D = dilution factor of the Sample solution
mix until dissolved. Add 300 mL of methanol. L = label claim (mg/Tablet)
Standard stock solution: 2 mg/mL of USP Levofloxacin Tolerances: NLT 80% (Q) of the labeled amount of
RS in Diluent levofloxacin (CisH2oFN3O,) is dissolved.
Standard solution: 0.2 mg/mL of USP Levofloxacin RS Test 2
in Mobile phase from the Standard stock solution Medium: 0.1 N hydrochloric acid; 900 mL
Sample stock solution: Nominally 5 mg/mL of levoflox- Apparatus 1: 100 rpm
acin prepared as follows. Transfer intact Tablets (NLT 5) Time: 30 min
to a volumetric flask, add 75% of the final volume of Standard solution: 1/900 mg/mL of USP Levofloxacin
Diluent, and allow to stand for 15 min. Shake for 30 RS in Medium, and mix to obtain solutions with known
min, and dilute with Diluent to volume. Pass a portion concentrations as indicated in Table 1, where Lis the
of the solution through a suitable filter of 0.45-um pore label claim in mg/Tablet.
size, discarding the first 1-2 mL of the filtrate. Sample solution: Pass a portion of the solution under
Sample solution: Nominally 0.2 mg/mL of levofloxacin test having a concentration similar to that of the Stan-
in Mobile phase from the Sample stock solution dard solution througha suitable filter of 0.45-4m pore
Chromatographic system size.
(See Chromatography (621), System Suitability.)
Mode: LC Table 1
Detector: UV 360 nm
Column: 4.6-mm x 25-cm; 5-m packing L1 Tablet Label Claim Final Concentration
Column temperature: 45° (mq) (mg/mL)
Flow rate: 0.8 mL/min 250 0.27
Injection volume: 25 pL 500 0.55
Run time: 2 times the retention time of levofloxacin 750 0.83
System suitability
Sample: Standard solution Instrumental conditions
Suitability requirements (See Ultraviolet-Visible Spectroscopy (857).)
Tailing factor: NMT 1.8 Mode: UV
Relative standard deviation: NMT 2.0% Analytical wavelength: 293 nm
Analysis Cell length: 0.1 mm
Samples: Standard solution and Sample solution Blank: Medium
Calculate the percentage of the labeled amount of Analysis
Hevetioxecin CisH20FN3Ox) in the portion of Tablets Samples: Standard solution and Sample solution four
taken: Calculate the percentage (Q) of the labeled amount of rv)
ne
levofloxacin(CHEN.O;) dissolved:
Result = (ru/rs) x (Cs/Cu) x 100 =
Result = (Au/As) x Cs x Vx D x (1/L) x 100 °
ty = peak response of levofloxacin from the Sample 3
°
solution Au = absorbance of the Sample solution a
Is peak response of levofloxacin from the As = absorbance of the Standard solution =
i
Standard solution Cs = concentration of the Standard solution tT
Gs concentration of USP Levofloxacin RS in the (mg/mL) =
a
"

Standard solution (mg/mL) Vv = volume of Medium, 900 mL

CG = nominal concentration of levofloxacin in the D = dilution factor of the Sample solution
Sample solution (mg/mL) L = label claim (mg/Tablet)
Acceptance criteria: 90.0%-110.0% Tolerances: NLT 80% (Q) of the labeled amount of
levofloxacin (CigH2oFN3Os) is dissolved.
PERFORMANCE TESTS Test 3
e DISSOLUTION (711) Medium: 0.1 N hydrochloric acid; 900 mL
Test 1 Apparatus 1: 100 rpm
Medium: 0.1 N hydrochloric acid; 900 mL Time: 30 min
Apparatus 2: 75 rpm Standard solution: 1/900 mg/mL of USP Levofloxacin
Time: 30 min RS in Medium, and mix to obtain solutions with known
Standard solution: 0.56 mg/mL of USP Levofloxacin concentrations as indicated in Table 7, where L is the
RS in Medium label claim in mg/Tablet.
Sample solution: Pass a portion of the solution under Sample solution: Pass a portion of the solution under
test through a suitable filter of 0.45-tum pore size. test having the same concentration as that of the Stan-
Instrumental conditions dard solution through a suitable filter of 0.45-um pore
(See Ultraviolet-Visible Spectroscopy (857).) size.
Mode: UV Instrumental conditions
Analytical wavelength: 294 nm (See Ultraviolet-Visible Spectroscopy (857).)
Cell length: 0.1 mm Mode: UV
Blank: Medium Analytical wavelength: 326 nm
Analysis Cell length: 1mm for a 250-mg Tablet, 0.5 mm for a
Samples: Standard solution and Sample solution 500-mg Tablet, and 0.2 mm for a 750-mg Tablet
Calculate the percentage (Q) of the labeled amount of
levofloxacin (CigH2oFN30,) dissolved:

Result = (Au/As) x Cs x Vx Dx (1/L) x 100

 2400 Levofloxacin / Official Monographs USP 41

Blank: Medium tu = peak response of levofloxacin related

Analysis compound A from the Sample solution
Samples: Standard solution and Sample solution rs = peak response of levofloxacin related
Calculate the percentage (Q) of the labeled amount of compound A from the Standard solution
levofloxacin (CisH2oFN304) dissolved: Cs = concentration of USP Levofloxacin Related
CompoundA RS in the Standard solution
Result = (Au/As) x Cs x Vx Dx (1/L) x 100 (mg/mL)
Cu = nominal concentration of levofloxacin in the
Au = absorbance of the Sample solution Sample solution (mg/mL)
As = absorbance of the Standard solution Calculate the percentage of any other impurities in the
G = concentration of the Standard solution portion of Tablets taken:
(mg/mL)
Vv = volume of Medium, 900 mL Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
D = dilution factor of the Sample solution
ii = label claim (mg/Tablet) tu = peak response of any impurity from the
Tolerances: NLT 80% (Q) of the labeled amount of Sample solution
levofloxacin (CisH2oFN3O,) is dissolved. fs = peak response of levofloxacin from the
Test 4 Standard solution
Medium: 0.1 N hydrochloric acid; 900 mL Cs = concentration of USP Levofloxacin RS in the
Apparatus 1: 100 rpm Standard solution (mg/mL)
Time: 30 min Cu = nominal concentration of levofloxacin in the
Standard solution: 16 g/mL of USP Levofloxacin RS Sample solution (mg/mL)
in Medium F = relative response factor (see Table 2)
Sample solution: Pass a portion of the solution under Acceptance criteria: See Table 2.
test having the same concentration as that of the Stan-
dard soluiton throughasuitable filter of 0.45-um pore Table 2
size.
Instrumental conditions Relative Relative Acceptance
(See Ultraviolet-Visible Spectroscopy (857).) Retention Response Criteria,
Mode: UV Name Time Factor NMT (%)
Analytical wavelength: 332 nm Decarboxy
Cell length: 1cm levofloxacine 0.38 0.60 0:3:
Blank: Medium Levofloxacin related 2
Analysis compound A> 0.47 0.7
Samples: Standard solution and Sample solution Diamine derivatives 0.52 0.83 0.3
Calculate the percentage (Q) of the labeled amount of
Levofloxacin
levofloxacin (CigH2oFN304) dissolved: N-oxided 0.63 0.68 0.7
“
= Result = (Au/As) x Cs x Vx D x (1/L) x 100 9-Desfluoro _ beiy
levofloxacine 0.73
5 Au = absorbance of the Sample solution Levofloxacin 1.00 = =
} As = absorbance of the Standard solution Dextrofloxacins 1.23 — —
= Cs = concentration of the Standard solution Levofloxacin
c (mg/mL) 9-piperazino — a
= V = volume of Medium, 900 mL isomer? 1.69
a. D = dilution factor of the Sample solution Any unspecified ag
4 L = label claim (mg/Tablet) impurity 1.0 0.2
Tolerances: NLT 80% (Q) of the labeled amount of
levofloxacin (CigHzoFN3O,) is dissolved. Total impurities = = 1
e UNIFORMITY OF DOSAGE UNITS (905): Meet the ®{5):9:Fluoro:d, sdllydro:3 meaty 0-(4-methyl-1-piperazinyl)-7-oxo-7H-
pyrido[1,2,3-de][1,4]benzoxazine.
requirements
»(S)-9-Fluoro-3-methyl-10-(piperazin-1-yl)-7-oxo-2,3-dihydro-7H-pyrido[1,
2,3-del[1 vAlbenzoxazine-6-carboxylic acid,
IMPURITIES
£(5)-9-Fluoro-2,3-dihydro-3-methyl-1 0-[2-(methylamino)ethylamino]-7-
© ORGANIC IMPURITIES oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid.
Diluent, Mobile phase, Standard stock solution, Sam- 4(S)-4-(6-Carboxy-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido-[1,2,3-
ple solution, and Chromatographic system: Proceed de][1,4]benzoxazine-10-yl)-1-methylpiperazine 1-oxide.
as directed in the Assay. © (S)-2,3-Dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-ox0-7H-pyrido[1,
Standard solution: 0.2 mg/mL of USP Levofloxacin RS 2,3-dél[1,4]penzoxazine.6-carboxylicacid. ® i
from the Standard stock solution and 1 wg/mL of USP ‘Process impurity, for information only.
Levofloxacin Related Compound A RS in Mobile phase a2 Hluare:2 S-dlbyate-samethy | 0-(4-methyl-1-piperazinyl)-7-oxo-7H-
System suitability pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid.
Sample: Standard solution h(5)-10-fluoro-3-methyl-9-(4-methylpiperazin-1-yl)-7-oxo-3,7-dihydro-7H-
Suitability requirements pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid.
Tailing factor: NMT 1.8 for the levofloxacin peak ADDITIONAL REQUIREMENTS
Relative standard deviation: NMT 2.0% for the © PACKAGING AND STORAGE: Preserve in tight containers.
levofloxacin peak Store at controlled room temperature.
Analysis e LABELING: When more than one Dissolution test is given,
Samples: Standard solution and Sample solution the labeling states the Dissolution test used only if Test 7
Calculate the peresntage of levofloxacin related com- is not used.
poundAin the portion of Tablets taken:
Result = (ru/rs) x (Cs/Cu) x 100
USP 41 Official Monographs / Levonorgestrel 2401

e USP REFERENCE STANDARDS (11) (70:20:10) until the solvent front has moved about three-
USP Levofloxacin RS fourths of the length of the plate. Remove the plate from
USP Levofloxacin Related Compound A RS the developing chamber, mark the solvent front, and allow
(5)-9-Fluoro-3-methyl-10-(piperazin-1-yl)-7-oxo-2,3- the solvent to evaporate in warm, circulating air. Examine
dihydro-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carbox- the plate under short-wavelength UV light. Expose the plate
lic acid. to iodine vapors, and examine again. Compare the intensi-
a7HisFN304 347.34 ties, observed by both visualizations, of any secondary spots
observed in the chromatogram of the Test solution with
those of the principal spots in the chromatograms of the
Standard solutions: the sum of the intensities of secondary
spots obtained from the Test solution corresponds to not
more than 1.0% of related compounds, with no single im-
Levonordefrin purity corresponding to more than 0.5%.
HOH Assay—Transfer about 350 mg of Levonordefrin, previously
HO, ee dried and accurately weighed, to a small flask, dissolve in
S | HN OH
50 mL of glacial acetic acid, heating, if necessary, add 1
HO drop of crystal violet TS, and titrate with 0.1 N perchloric
acid VS to a green endpoint. Perform a blank determination,
CsHi3NO3 183.20 and make any necessary correction. Each mL of 0.1 N per-
1,2-Benzenediol, 4-(2-amino-1-hydroxypropyl)-, [R-(R*,5*)]-. chloric acid is equivalent to 18.32 mg of CoHi3NO3.
(-)-a-(1-Aminoethyl)-3,4-dihydroxybenzyl alcohol
[18829-78-2; 829-74-3].
» Levonordefrin, dried in vacuum at 60° for
15 hours, contains not less than 98.0 percent and Levonorgestrel
not more than 102.0 percent of C9H:3NO3.
Packaging and storage—Preserve in well-closed contain-
ers.
USP Reference standards (11)—
USP Levonordefrin RS
Identification—
A: Infrared Absorption (197K). CxH2s02 312.45
B: Ultraviolet Absorption (197U)— 18,19-Dinorpregn-4-en-20-yn-3-one, 13-ethyl-17-hydroxy-,
Solution: 25 wg per mL. (170)-C)-.
(-)-13-Ethyl-17-hydroxy-18,19-dinor-1 7a-pregn-4-en-20-yn-
Medium: 0.1 N hydrochloric acid. 3-one [797-63-7]. c
a)
Specific rotation (781S): between —28° and -31°. vu
Test solution: 50mg, previously dried, per mL, in 0.3 N » Levonorgestrel contains not less than 98.0 per-
hydrochloric acid. cent and not more than 102.0 percent of ce
3
Loss on drying (731)—Dry it in vacuum at 60° for Cx1H2gO2, calculated on the dried basis. ]
15 hours: it loses not more than 1.0% of its weight. )
Packaging and storage—Preserve in well-closed, light-re- ro}=
Residue on ignition (281): not more than 0.2%. sistant containers. ESS}
Chromatographic purity— 3
Standard solutions—Dissolve an accurately weighed quan-
USP Reference standards (11)— y
USP Levonorgestrel RS a]
tity of USP Levonordefrin RS in a mixture of methanol and
glacial acetic acid (96:4) to obtain a Standard stock solution Identification—
having a known concentration of 5 mg per mL. Dilute this A: Infrared Absorption (197K).
solution quantitatively with a mixture of methanol and gla- B: Meeting the requirements of the tests for Specific rota-
cial acetic acid (96:4) to obtain Standard solutions, desig- tion and Melting range provides identification distinguishing
nated below by letter, having the following compositions: it from norgestrel.
Melting range (741): between 232° and 239°, but the
Concentra- Percentage (%, range between beginning and end of melting does not ex-
tion for comparison ceed 4°.
Standard (ug RS per with test speci- Specific rotation (7815S): between —30° and —35°.
solution Dilution mL) men) Test solution: 20mg per mL, in chloroform.
A (1 in 10) 500 1.0 Loss on drying (731)—Dry it at 105° for 5 hours: it loses
B (1 in 20) 250 0.5 not more than 0.5% of its weight.
c (1 in 50) 100 0.2 Residue on ignition (281): not more than 0.3%.
D (in 100) 50 0.1 Limit of athyny! group—Dissolve 200 mg in about 40 mL
of tetrahydrofuran. Add 10 mL of silver nitrate solution (1 in
Test solution—Dissolve an accurately ajae quantity of 10), and titrate with 0.1 N sodium hydroxide VS, using ei-
Levonordefrin in a mixture of methanol and glacial acetic ther a glass-calomel ora silver-silver chloride electrode sys-
acid (96:4) to obtain a solution containing 50 mg per mL. tem with potassium nitrate filling solution. Perform a blank
Procedure— Apply separately 5 uL of the Test solution and determination, and make any necessary correction. Each mL
5 wl of each Standard solution to a suitable thin-layer chro- of 0.1 N sodium hydroxide is equivalent to 2.503 mg of
matographic plate (see Chromatography (621)) coated with ethynyl group (-C=CH). Not less than 7.81% and not more
0.25-mm layer of chromatographic silica gel mixture. Posi- than 8.18% of ethynyl group is found.
tion the plate in a chromatographic chamber, and develop Chromatographic purity—Proceed as directed in the test
the chromatograms in a solvent system consisting of a mix- for Chromatographic purity under Norgestrel, using USP Levo-
ture of n-butyl alcohol, water, and glacial acetic acid norgestrel RS in place of USP Norgestrel RS. The require-
 2402 Levonorgestrel / Official Monographs
ments of the test are met if the sum of the impurities in the
Test preparation does not exceed 2.0% and no single impu-
rity is greater than 0.5%.
Assay—Using USP Levonorgestrel RS, proceed as directed
in the Assay under Norgestrel, except to read “Levonorges-
trel” in place of “Norgestrel.”
Levonorgestrel and Ethinyl Estradiol
Tablets
DEFINITION
Levonorgestrel and Ethinyl Estradiol Tablets contain NLT
90.0% and NMT 110.0% of the labeled amount of levo-
norgestrel (C2H28O2) and NLT 90.0% and NMT 110.0%
of the labeled amount of ethinyl estradiol (C2oH2402).
IDENTIFICATION
e A. The retention times of the two major peaks of the
Sample solution correspond to those of levonorgestrel and
ethinyl estradiol in the Standard solution, as obtained in
the Assay.
e B. Finely powder 20 Tablets and transfer a portion of the
powder, equivalent to 4 mg of levonorgestrel, to a suita-
le container. Add 250 mL of a solvent mixture consist-
ing of isooctane and chloroform (3:1). Sonicate the mix-
ture for 3 min, and then stir it by mechanical means for
30 min. Filter the mixture and evaporate the filtrate to
dryness in a rotating vacuum evaporator. Dissolve the
residue in 3 mL of chloroform, and transfer with a pipet
to a 60-mL separator containing 18 mL of isooctane.
Rinse the evaporator flask with an additional 3-mL por-
tion of chloroform, and add the rinsing to the separator.
Add 10 mL of 1 N sodium hydroxide, shake vigorously,
a and allow the layers to separate. Discard the lower aque-
<= ous phase, and filter the organic phase through 3 g of
a
i]
aatiyarous sodium sulfate on filter paper into a 50-mL
beaker. Rinse the filter with several small portions of the
Dp
—
i) mixture of isooctane and chloroform (3:1), adding the
filtered rinsings to the filtrate, and evaporate under nitro-
—
S gen ona steam bath to dryness. Dissolve the residue in
= 1-2 mL of hot toluene, and transfer with a pipet to a
a small glass vial. Reduce the volume of the solution to
w 0.1 mL under nitrogen with warming. [NoTe—During this
=) step, any crystals that deposit on the vial wall should be
transferred to the bottom, and allowed to redissolve.]
Store the vial containing the clear toluene solution at 4°
renin to allow crystallization to occur. Remove and
discard the mother liquor with a pipet, rinse the crystals
with two 0.5-mL portions of anhydrous ether, and dis-
card the rinsings. Dry the vial containing the rinsed
crystals in a vacuum desiccator at 60° for 4 h.
Acceptance criteria: The melting point of the dried
crystals of levonorgestrel so obtained is not lower than
220°, using the procedure described under Melting
Range or Temperature (741), Class |.
ASSAY
e PROCEDURE
Mobile phase: Acetonitrile, methanol, and water
(35:15:45)
Standard solution: 15 ug/mL of USP Levonorgestrel RS
and 3 ug/mL of USP Ethinyl Estradiol RS in Mobile phase
Sample solution: Transfer a number of Tablets, equiva-
lent to 3 mg of levonorgestrel, to a 200-mL volumetric
flask. Dilute with Mobile phase to volume, sonicate to
disintegrate the Tablets, then shake by mechanical
means for 20 min. Centrifuge, and use the clear
supernatant.
Chromatographic system
(See Chromatography (621), System Suitability.)
USP 41
Mode: LC
Detector: UV 215 nm
Column: 4.6-mm x 15-cm; 5- to 7-um packing L7
Flow rate: 1 mL/min
Injection size: 50 uL
System suitability
Sample: Standard solution
[Note—The relative retention times for ethinyl estradiol
and levonorgestrel are about 0.7 and 1.0,
reapectivehi|
Suitability requirements
Resolution: NLT 2.5 between the two major peaks
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of C21H2sO2 and C29H24Q2 in
the portion of Tablets taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response of the corresponding analyte
from the Sample solution
Is = peak response of the corresponding analyte
from the Standard solution
Cs = concentration of the appropriate USP
Reference Standard in the Standard solution
(ug/ml) i
Cu = nominal concentration of the corresponding
analyte in the Sample solution (ug/mL)
Acceptance criteria: 90.0%-110.0% of the labeled
amount of C21H2sO2, 90.0%-110.0% of the labeled
amount of C29H2402
PERFORMANCE TESTS
e DISSOLUTION (711): Determine the amount of C2:H2sO2
and C29H24O02 dissolved by employing the following
method.
Medium: Polysorbate 80 (5 g/g) in water; 500 mL
Apparatus 2: 75 rpm
Time: 60 min
Mobile phase: Acetonitrile and water (6:4)
Standard solution: Prepare a solution of USP Levo-
norgestrel RS and USP Ethinyl Estradiol RS in Medium
having known concentrations corresponding approxi-
mately to the concentrations that would be obtained by
dissolving 1 Tablet in 500 mL of Medium.
[Note—A volume of alcohol not exceeding 2% of the
final total volume of solution may be used to aid in
dissolving the Reference Standards.]
Sample solution: Withdraw 15-mL portions of liquid
from each vessel, and pass through a polyvinylidene fil-
ter, discarding the first 10 mL of the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 247 nm (for levonorgestrel analysis); a
spectrofluorometric detector (for ethinyl estradiol anal-
ysis), with an excitation wavelength of 285 nm, and
an emission wavelength of 310 nm
Column: 4-mm x 15-cm; packing L7
Flow rate: 1 mL/min
Injection size: 100 wL
System suitability
Sample: Standard solution
[NotE—The relative retention times for ethinyl estradiol
and levonorgestrel are about 0.7 and 1.0,
respectvehit
Suitability requirements
Relative standard deviation: NMT 3.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of C2H2sO2 and C2H24O2
dissolved:
Result = (ru/ts) x (Cs/Cu) x 100
USP 41 Official Monographs / Levorphanol 2403

tu = peak response of the corresponding analyte B: Ultraviolet Absorption (197U)—

from the Sample solution
ts = peak response of the corresponding analyte
from the Standard solution
Cs = concentration of the appropriate USP
Reference Standard in the Standard solution
(ug/mL) j )
Cu = nominal concentration of the corresponding
analyte in the Sample solution (g/mL)
Tolerances
Uncoated Tablets: NLT 80% (Q) of the labeled
amount of C21H2s02, and 75% (Q) of the labeled
amount of Cz9H24O2 is dissolved.
Coated Tablets: NLT 60% (Q) of the labeled amount
of C21H2gO2 and CzoH24Oz is dissolved.
e UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
Sample solution: Place 1 Tablet in a 40-mL centrifuge
tube, add 10.0 mL of Mobile phase, and proceed as di-
rected in the Assay.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed
containers.
e USP REFERENCE STANDARDS (11)
USP Ethiny! Estradiol RS
USP Levonorgestrel RS
Solution: 130 wg per mL.
Medium: 0.1 N hydrochloric acid.
Absorptivities at 279 nm, calculated on the anhydrous ba-
sis, do not differ by more than 3.0%.
Specific rotation (7815): between -14.7° and -16.3°.
Test solution: 30 mg per mL, in water. Heat on a water
bath or sonicate to dissolve 750 mg in 20 mL of water in a
25-mL volumetric flask, dilute with water to volume, and
mix.
Water Determination, Method | (921): between 7.0%
and 9.0%.
Residue on ignition (281): not more than 0.1%.
Ordinary impurities (466)—
Test solution: water.
Standard solution: water.
Fluant: a mixture of hexanes, dehydrated alcohol, and
ammonium hydroxide (80:25:1).
Visualization: 17; then view immediately under short-
wavelength UV light.
meray Peres about 900 mg ofSarpy accurately
weighed, in 85 mL of glacial acetic acid, warming slightly if
necessary. Titrate with 0.1 N perchloric acid VS and deter-
mine the endpoint potentiometrically. Perform a blank de-
termination and make any nececssary corrections. Each mL
of 0.1 N perchloric acid consumed by the sample is equiva-
lent to 40.75 mg of CizH23NO - C4HeOc.

Levorphanol Tartrate
H

CSB) «AY sm
NCH,
( 9 HOH Levorphanol Tartrate Injection
» Levorphanol Tartrate Injection is a sterile solu-
HO
f HOH oO
tion of Levorphanol Tartrate in Water for Injec- (=
A)
tion. It contains not less than 93.0 percent and ae}
CizH23NO - C4HpO6- 2H20 443.49 not more than 107.0 percent of the labeled ms
Morphinan-3-ol, 17-methyl-, [R-(R*,R*)]-2,3-dihydroxy- amount of Ci7H23NO.C4HeOc + 2H20.
°
|]
butanedioate (1:1) (sald, dihydrate. fe)
17-Methylmorphinan-3-ol, tartrate (1:1) (salt) dihydrate Packaging and storage—Preserve in single-dose or in a}=
[5985-38-6]. multiple-dose containers, preferably of Type | glass. 2
Anhydrous 407.47 [125-72-4]. ao}
a
» Levorphanol Tartrate contains not less than Delete the following:
a]

99.0 percent and not more than 101.0 percent of

pasa » C4HeOo, calculated on the anhydrous
asis.
Packaging and storage—Preserve in well-closed contain-
ers. Store at 25°, excursions permitted between 15° and
30°.
USP Reference standards (11)—
USP Levorphanol Tartrate RS
identification—
A: Infrared Absorption (197K)—Obtain the test specimen
as follows. Dissolve 50 mg in 25 mL of water in a 125-mL
separator. Add 2 mL of 6N ammonium hydroxide, extract
with 25 mL of chloroform, and filter the chloroform extract
throughalayer of 4g of granular anhydrous sodium sulfate
supported on glass wool into a 125-mL conical flask. Evapo-
rate the chloroform extract on a steam bath with the aid of
a stream of nitrogen to dryness. Dissolve the residue in 1 mL
of acetone, and evaporate to dryness. Dry in vacuum at 90°
for 1 hour. Proceed as directed with the dried levorphanol
so obtained and a similar preparation of USP Levorphanol
Tartrate RS.
°USP Reference standards (11)—
USP Endotoxin RS
@ (CN 1-May-2018)
Identification—
A: To 1 mL of meron add 1 drop of 3 N hydrochloric
acid and 2 drops of ferric chloride TS. Heat to boiling, and
add 1 mL of potassium ferricyanide solution (1 in 200): a
blue-green color develops.
B: The angular rotation of the Injection is levorotatory
(see Optical Rotation (781)).
Bacterial Endotoxins Test (85)—It contains not more
than 125.0 USP Endotoxin Units per mg of levorphanol tar-
trate.
PH (791): between 4.1 and 4.5.
Other requirements—It meets the requirements under /n-
jections and Implanted Drug Products (1).
Assay—Transfer an accurately measured volume of Injec-
tion, equivalent to about 40 mg of levorphanol tartrate, to a
125-mL separator. Add 5 g of sodium chloride and sufficient
sodium bicarbonate to render the solution alkaline to litmus,
add an additional 100 mg of sodium bicarbonate, and ex-
tract the levorphanol wath five 20-mL portions of a mixture
of 3 volumes of ether and 1 volume of chloroform. Pass the
combined extracts through a layer of about 10 g of granular
 2404 Levorphanol / Official Monographs
anhydrous sodium sulfate into a 500-mL conical flask, and
evaporate to a volume of about 30 mL. Add about 50 mL of
chloroform and 1 drop of methanolic methyl red TS, and
titrate with 0.01 N perchloric acid in dioxane VS to a red
endpoint. Perform a blank determination, and make an
necessary correction. Each mL of 0.01 N perchloric aci
equivalent to 4.435 mg of Ci7H23NO - C4HeO¢ - 2H20.
Levorphanol Tartrate Tablets
» Levorphanol Tartrate Tablets contain not less
than 93.0 percent and not more than 107.0 per-
cent of the labeled amount of levorphanol tar-
trate (C17H23NO . C4HeO6 . 2H20).
Packaging and storage—Preserve in well-closed contain-
ers,
USP Reference standards (11)—
USP Levorphanol Tartrate RS
identification—
A: Powder finely a number of Tablets. To a portion of the
powder, equivalent to about 1 mg of eve anol tartrate,
add 1 mL of water, 1 drop of 3 N hydrochloric acid, and
2 drops of ferric chloride TS, and heat to boiling. To the hot
solution add 1 mL of potassium ferricyanide solution (1 in
200): a bluish color develops.
B: Powder a number of Tablets, equivalent to about
60 mg of levorphanol tartrate, and transfer the mixture to a
small separator. Add 10 mL of water, dissolve as much of
the powder as possible, add about 400 mg of sodium bicar-
bonate, and extract with a 50-mL portion of chloroform.
Evaporate the filtered chloroform extract on a steam bath to
al
<= a small volume, dilute with chloroform to 10 mL, and deter-
a mine the angular rotation: the solution is levorotatory (see
ii Optical Rotation (781)).
aD
a
Dissolution (711)—
i}
im Medium: water; 500 mL.
S Apparatus 2: 50 rpm.
= Time: 30 minutes.
a
al Procedure—Determine the amount of Ci7H23NO - CaHeOc +
= 2H20 dissolved from UV absorbances at the wavelength of
maximum absorbance at about 279 nm on filtered portions
of the solution under test, suitably diluted with water, in
comparison with a Standard solution having a known con-
centration of USP Levorphanol Tartrate RS in the same me-
dium.
Tolerances—Not less than 75% (Q) of the labeled amount
of Cy7H23NO - C4HoOc - 2H20 is dissolved in 30 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Procedure for content uniformity—Transfer 1 Tablet to a
glass-stoppered flask, add 25.0 mL of 0.1 N hydrochloric
acid, and allow the Tablet to disintegrate. Shake well, and
filter through a small filter paper, discarding the first portion
of the filtrate. Dilute a portion of the filtrate quantitatively
and stepwise, if necessary, to provide a solution containing
about 80 tg of levorphanol tartrate per mL. Concomitantly
determine the absorbances of this solution and of a solution
of USP Levorphanol Tartrate RS in the same medium having
a known concentration of about 80 ug of anhydrous
levorphanol tartrate per mL, in 1-cm cells at the wavelength
of maximum absorbance at about 279 nm, witha suitable
spectrophotometer, using 0.1 N hydrochloric acid as the
USP 41
blank. Calculate the quantity, in mg, of Ci7H2sNO - CaHoOc
-
2H,0O in the Tablet taken by the formula:
(443.49 / 407.47)(TC / D)(Au / As)
in which 443.49 and 407.47 are the molecular weights of
the hydrated and anhydrous forms of levorphanol tartrate,
respectively; T is the labeled quantity, in mg, of levorphanol
tartrate in the Tablet; C is the concentration, in ug per mL,
of USP Levorphanol Tartrate RS, on the anhydrous basis, in
the Standard solution; D is the concentration, in ug per mL,
of levorphanol tartrate in the solution from the Tablet,
based on the labeled quantity per Tablet and the extent of
dilution; and Ay and As are the absorbances of the solution
from the Tablet and the Standard solution, respectively.
pasay We and finely powder not less than 20 Tablets.
Weigh accurately a portion of the powder, equivalent to
about 40 mg of levorphanol tartrate, transfer to a 125-mL
separator, add 20 mL of water and sufficient sodium bicar-
bonate to render the suspension alkaline to litmus, and pro-
ceed as directed in the Assay under Levorphanol Tartrate In-
jection, beginning with “add an additional 100 mg of
sodium bicarbonate.”
Levothyroxine Sodium
Ho. |
ae
rank.
Set
i
Nev at i leuk wo xi
' UNA © Za NH
'
CisHiolaNNaOg - xH20 (anhydrous) 798.85
L-Tyrosine, O-(4-hydroxy-3,5-diiodophenyl)-3,5-diiodo-,
monosodium salt, hydrate;
Monosodium L-thyroxine hydrate [25416-65-3].
Anhydrous [55-03-8].
DEFINITION
Levothyroxine Sodium is the sodium salt of L-3,3’,5,5’-te-
traiodothyronine. It contains NLT 97.0% and NMT
103.0% of levothyroxine sodium (C;sHiolaNNaOs), calcu-
lated on the anhydrous basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197): [NoTE—Methods de-
ane in Infrared Absorption (197K) or (197A) may be
used.
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
Change to read:
© C. IDENTIFICATION TESTS—GENERAL (191), Sodium
Sample solution: To 200 mg add 2 mL of 2 N sulfuric
acid. Heat on a water bath and then carefully heat over
an open flame, increasing the temperature gradually up
to about 600°. [NOTE—Al eecae for ignit-
ing the material could also be used.] Continue the igni-
tion until most of the particles have disappeared. Dis-
solve the residue in 2 mL of water.
Acceptance criteria: The Sample solution meets the re-
quirements of ®test A.e civ1-may-z018)
ASSAY
e PROCEDURE
Mobile phase: Acetonitrile and water (4:6) that con-
tains 0.5 mL of phosphoric acid in each 1000 mL
Solution A: 400 mg of sodium hydroxide in 500 mL of
water. Cool and add 500 mL of methanol.
USP 41 Official Monographs / Levothyroxine 2405

Levothyroxine stock solution: 0.4 mg/mL of USP Levo- Standard solution: Pipet 4.0 mL of the System suitabil-
thyroxine RS in Solution A ity solution into a 100-mL volumetric flask. Add 7 mL of
Liothyronine stock solution: 0.4 mg/mL of liothyronine Solution A, and dilute with Diluent to volume.
from USP Liothyronine RS in Solution A. Make a 1:100 Sample solution: Transfer 25 mg of Levothyroxine So-
dilution of this solution using Mobile phase. dium to a 100-mL volumetric flask. Add 50 mL of Dilu-
Standard solution: 10 g/mL of levothyroxine from Le- ent, and sonicate until dissolved. Add 7 mL of Solution
vothyroxine stock solution and 0.2 ug/mL of liothyronine A, and dilute with Diluent to volume.
from Liothyronine stock solution in Mobile phase Blank solution: Transfer 7 mL of Solution A to a 100-mL
Sample solution: 10 g/mL of Levothyroxine Sodium in volumetric flask, and dilute with Diluent to volume.
Mobile phase. [NoTE—A small amount of 0.01 M metha- Chromatographic system
nolic sodium hydroxide can be used to facilitate the (See Chromatography (621), System Suitability.)
dissolution of the sample.] Mode: LC
Chromatographic system Detector: UV 225 nm
(See Chromatography (621), System Suitability.) Column: 4.6-mm x 15-cm; 5-um packing L7
Mode: LC Column temperature: 35°
Detector: UV 225 nm Flow rate: 1.5 mL/min
Column: 4.6-mm x 25-cm; packing L10 Injection volume: 15 uL
Flow rate: 1.5 mL/min System suitability
Injection volume: 100 uL Samples: System suitability solution and Standard
System suitability solution
Sample: Standard solution Suitability requirements
Suitability requirements Resolution: NLT 5.0 between levothyroxine and
Resolution: NLT 5.0 between liothyronine and liothyronine, System suitability solution
levothyroxine Relative standard deviation: NMT 2.0% for the le-
Relative standard deviation: NMT 2.0% for vothyroxine peak, Standard solution
levothyroxine Analysis
Analysis Samples: Standard solution, Sample solution, and Blank
Samples: Standard solution and Sample solution solution
Calculate the percentage of levet tyrone sodium [Note—Record the chromatograms for at least six times
(CisHiolaNNaO,) in the portion of Levothyroxine So- the retention time of the levothyroxine peak. Verify
dium taken: that no peaks elute in the Blank solution at the ex-
pected retention times for levothyroxine and related
Result = (ru/ts) x (Cs/Cu) x (Mr/Mi2) x 100 compounds.]
Calculate the area percentage of each related com-
tu = peak response of levothyroxine from the pound in the portion of Levothyroxine Sodium taken:
Sample solution
fs = peak response of levothyroxine from the Result = (ru/rs) x (Cs/Cu) x (Mri/M,2) x 100
Standard solution (a
Cs = concentration of USP Levothyroxine RS in the tu = peak response of each impurity from the un
Standard solution (\ug/mL) Sample solution 2
Cu = concentration of Levothyroxine Sodium in the fs = peak response of levothyroxine from the <=
Sample solution (agin ) Standard solution °
My = molecular weight of levothyroxine sodium, Cs = concentration of USP Levothyroxine RS in the =
798,85 Standard solution (mg/mL) tot
Ma = molecular weight of levothyroxine, 776.87 Cu = concentration of Levothyroxine Sodium in the a
Agoayranicl criteria: 97.0%-103.0% on the anhydrous Sample solution (mgiml) os
asis Mn = molecular weight of levothyroxine sodium, zz
798,85
IMPURITIES Mz = molecular weight of levothyroxine, 776.87
[NotrE—On the basis of the synthetic route, perform either [Note—The relative response factor for the impurities
Organic Impurities, Procedure 1 or Organic Impurities, Proce- listed in Table 1 is 1,00. Any unspecified impurity
dure 2, Procedure 2 is recommended when related com- ae should be assigned a relative response factor of
pounds listed in Table 3 may be present.]
© ORGANIC IMPURITIES, PROCEDURE 1 Disregard peaks corresponding to those of the Blank so-
Diluent: Acetonitrile and water (1:1) lution, and disregard peaks corresponding to less than
Solution A: Dilute 5 mL of phosphoric acid with Diluent 0.03%,
to 100,0 mL. Acceptance criteria: See Table 1.
Mobile phase: Dissolve 1.0 g of sodium 1-heptanesul-
fonate In 200 mL of water. Add 200 mL of acetonitrile,
400 mL of methanol, and 1.0 mL of phosphoric acid. Table 1
Dilute with water to 1 L. Relative Acceptance
Standard stock solution 1: Transfer 25 mg of USP Le- Retention Criteria,
vothyroxine RS to a 100-mL volumetric flask. Add Name Time NMT (%)
50 mL of Diluent and 1 drop of 10 N sodium hydroxide, Liothyronine 0.65-0.70 1.0
and sonicate until dissolved. Add 7 mL of Solution A, B-Hydroxy-T4@ 0.71-0.76 0.15
and dilute with Diluent to volume.
Standard stock solution 2: Transfer 25 mg of USP 2 0-(4-Hydroxy-3,5-diiodophenyl)-3,5-diiodo-B-hydroxy-L-tyrosine.
Liothyronine RS to a 100-mL volumetric flask. Add b 2-Hiydrony-2e14-Crhydroxy-3,-dlledephienoxy):3,2-dliodophenyilacetle
acid,
50 mL of Diluent and 1 drop of 10 N sodium hydroxide, ¢ N-Formyl-O-(4-hydroxy-3,5-diiodopheny!)-3,5-diiodo--tyrosine.
and sonicate until dissolved. Add 7 mL of Solution A, 4 2-[4-(4-Hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyllacetamide.
and dilute with Diluent to volume. © N-Acetyl-O-(4-hydroxy-3,5-diiodophenyl)-3,5-diiodo-L-tyrosine.
System suitability solution: Transfer 5.0 mL of Standard t2-[4-(4-Hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyljacetic acid.
stock solution 1 and 5.0 mL of Standard stock solution 2 9 4-(4-Hydroxy-3,5-diiodophenoxy)-3,5-diiodobenzaldehyde.
to a 100-mL volumetric flask. Add 7 mL of Solution A,
) 4-(4-Hydroxy-3,5-diiodophenoxy)-3,5-diiodobenzoic acid.
and dilute with Diluent to volume.
 2406 Levothyroxine / Official Monographs USP 41

Table 1 (Continued) Blank solution: Use Diluent 2.

Relative Acceptance
Chromatographic system
Retention Criteria,
(See Chromatography (621), System Suitability.)
Name Time NMT (%) Mode: LC
Detector: UV 225 nm
Levothyroxine 1.0 — Column: 4.0-mm x 15-cm; 3-um packing L1
T4-Hydroxyacetic acid’ 1.13-1.28 0.15 Flow rate: 1.0 mL/min
N-Formyl-T4¢ and Injection volume: 25 pL
T4-acetamidet 1.47-1.53 0.15 System suitability
N-Acetyl-T4¢ 1.50-1.86 0.20 Samples: Standard solution and Sensitivity solution
T4-Acetic acidt 2.42-2.51 0.30 Suitability requirements
T4-Aldehydes 3.17-3.45 0.15
Resolution: NLT 5 between levothyroxine and
liothyronine, Standard solution
T4-Benzoic acid’ 3.46-3.70 0.15
Signal-to-noise ratio: NLT 5 for each peak from the
Individual unspecified _ Sensitivity solution, calculated as follows:
impurit 0.10
Total impurities — 2.0 Result = (2H)/h
@ Q-(4-Hydroxy-3,5-diiodophenyl)-3,5-ditodo-B-hydroxy-L-tyrosine.
6 2-Hydroxy-2-[4-(4-hydroxy-3,5-dliodophenoxy)-3,5-diodophenyllacetic H = measured height of the peak
acid. A = amplitude of the average measured baseline
¢ N-Formyl-O-(4-hydroxy-3,5-diiodophenyl)-3,5-diiodo-L-tyrosine noise
4 2-[4-(4-Hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyljacetamide. Analysis
¢ N-Acetyl-O-(4-hydroxy-3,5-diiodopheny!)-3,5-diiodo-L-tyrosine. Samples: Blank solution, Standard solution, Identification
2-[4-(4-Hydroxy-3,5-dilodophenoxy)-3,5-diiodophenylJacetic acid. solution, and Sample solution
94-(4-Hydroxy-3,5-diiodophenoxy)-3,5-diiodobenzaldehyde. [Note—Identify the components on the basis of their
h 4-(4-Hydroxy-3,5-diiodophenoxy)-3,5-diiodobenzoic acid. relative retention times as listed in Table 3.]
Calculate the percentage of liothyronine sodium in the
© ORGANIC IMPURITIES, PROCEDURE 2 portion of Levothyroxine Sodium taken:
Solution A: Dissolve 9.7 g of sulfamic acid in 2000 mL
of water. Add 1.5 g of sodium hydroxide, mix to dis- Result = (ru/rs) x (Cs/Cu) x (Mn/Mj2) x 100
solve, and adjust with 2 N sodium hydroxide to a pH of
2.0 ru = peak response of liothyronine from the Sample
Solution B: Acetonitrile solution
Diluent 1: Methanol and Solution A (90:10) rs = peak response of liothyronine from the
Diluent 2: Acetonitrile and Solution A (30:70); mix with Standard solution
Diluent 1 (1:1). Cs = concentration of USP Liothyronine RS in the
Mobile phase: See Table 2. Standard solution (mg/mL)
Cu = concentration of Levothyroxine Sodium in the
fs
w“
Sample solution (mg/mL)
a Table 2
Ma — = molecular weight of liothyronine sodium,
Ss
7 Solution A Solution B 672.96
D
3 Ma = molecular weight of liothyronine, 650.98
= Calculate the percentage of any other impurity in the
iS 70 30 portion of Levothyroxine Sodium taken:
= 2 80
a Result = (ru/rs) x (Cs/Cu) x (Mr/M,2) x 100
va)
=) 70 30 ty = peak response of any impurity from the
30 Sample solution
rs = peak response of levothyroxine from the
Identification solution: Dissolve 5.0 mg of USP Levo- Standard solution
thyroxine for Peak Identification RS in 4.5 mL of metha- Cs = concentration of USP Levothyroxine RS in the
nol. Add 0.5 mL of Solution A. Further dilute a portion Standard solution (mg/mL)
of this solution with Diluent 2 to obtain a solution con- Cu = concentration of Levothyroxine Sodium in the
taining about 0.2 mg/mL. Sample solution (mg/mL)
Standard stock solution: 0.1 mg/mL each of USP Levo- Ma = molecular weight of levothyroxine sodium,
thyroxine RS and USP Liothyronine RS in Diluent 1 798.85
Standard solution: 0.002 mg/mL each of USP Levothy- Mz = molecular weight of levothyroxine, 776.87
roxine RS and USP Liothyronine RS, prepared using the [Note—The relative response factor for the impurities
Standard stock solution in Diluent 2 listed in Table 3 is 1.00. Any unspecified impurity
Sensitivity solution: 0.0002 mg/mL each of USP Levo- ere should be assigned a relative response factor of
thyroxine RS and USP Liothyronine RS, prepared using 1.00.
the Standard solution in Diluent 2 Disregard peaks corresponding to those of the Blank so-
Sample solution: Dissolve an amount of Levothyroxine lution, and disregard peaks corresponding to less than
Sodium in Diluent 1 to obtain a solution with a known 0.03%.
concentration of about 1.0 mg/mL. Further dilute a por- Acceptance criteria: See Table 3.
tion of this solution with Diluent 2 to obtain a solution
with a known concentration of about 0.2 mg/mL.
USP 41 Official Monographs / Levothyroxine 2407

Table 3 Identification—The retention time of the major peak in

the chromatogram of the Assay preparation corresponds to
Relative Acceptance
Retention Criteria,
that in the chromatogramof the Standard preparation, as
Time
obtained in the Assay.
a
Loss on drying (731)—Dry it in vacuum at 60° for 3 hours:
it loses not more than 2.0% of its weight.
eo 0.1
97
Assay—
Mobile phase—Prepare a filtered and degassed mixture of
Le
water and acetonitrile (65:35) that contains 1 mL of phos-
Triiodothyroacetic acid, or phoric acid in each 1000 mL. Make adjustments if necessary
(see System Suitability under Chromatography (621)).
O-(4-Hydroxy-3,5-
0.01 M Methanolic sodium hydroxide—Dissolve 400 mg of
Té¢
sodium hydroxide in 500 mL of water. Cool, add 500 mL of
O-Methyl-tetraiodothyroeth- methanol, and mix.
ine,
Standard preparation—Dissolve an accurately weighed
79 Suan of USP Levothyroxine RS in 0.07 M Methanolic so-
Ini ual Im — lium hydroxide, and dilute quantitatively and stepwise with
0.01 M Methanolic sodium hydroxide to obtain a solution
2 (S)-2-Amino-3-[3-chloro-4-(4-hydroxy-3,5-diiodophenoxy)-5-iodophenyl- having a known concentration of about 4 ug per mL.
Jpropanoic acid. Assay preparation—Transfer an accurately weighed por-
» (S)-2-Amino-3-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]-N- tion of Oral Powder, equivalent to about 5 mg of levothy-
methylpropanamide.
roxine sodium, to a 2sb-mL volumetric flask. Dilute with
¢ [4-(4-Hydroxy-3-iodophenoxy)-3,5-diiodophenylJacetic acid.
0.01 M Methanolic sodium hydroxide to volume, mix, and
4 ($)-2-Amino-3-[4-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenoxy]-
3,5-diiodophenyl]propanoic acid. allow to stand for 4 hours, with occasional mixing. Pass a
© 2-[4-(3,5-Diiodo-4-methoxyphenoxy)-3,5-diiodophenylJethanamine. portion of this mixture throughafilter that does not absorb
f 2-(4-(4-Hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl)acetic acid. levothyroxine. Transfer 10.0 mL of the filtrate to a 50-mL
volumetric flask, dilute with 0.07 M Methanolic sodium hy-
SPECIFIC TESTS droxide to volume, and mix.
e OPTICAL ROTATION, Specific Rotation (781S) Chromatographic system (see Chromatography (621))—The
Sample solution: Equivalent to 30 mg/mL of anhydrous liquid chromatograph is equipped with a 225-nm detector
Levothyroxine Sodium in alcohol and 1 N sodium hy- and a 4.6-mm x 25-cm column that contains packing L10.
droxide (2:1) The flow rate is about 1 mL per minute. Chromatograph the
Acceptance criteria: —5° to —6° Standard preparation, and record the peak responses as di-
© WATER DETERMINATION, Method | (921): NMT 11.0% rected for Procedure: the tailing factor is not more than 1.8;
ADDITIONAL REQUIREMENTS and the relative standard deviation for replicate injections is
© PACKAGING AND STORAGE: Preserve in tight containers, not more than 2.0%. S
protected from light. Store as stated in the labeling Procedure—Separately inject equal volumes (about 50 pL) a
of the Standard preparation and the Assay preparation into a]
instructions.
e LABELING: If a test for Organic Impurities other than Proce- the chromatograph, record the chromatograms, and meas- F<
dure 1 is used, the labeling states the test with which the ure the responses for the major peaks. Calculate the quan- °
tity, in mg, of ee sodium (CisHiglaNNaO,) in the =}
article complies. fo)
e USP REFERENCE STANDARDS (11) portion of Oral Powder taken by the formula: Ko}
=
USP Levothyroxine RS i
O-(4-Hydroxy-3,5-diiodophenyl)-3,5-diiodo-L-tyrosine. (798.85 / 776.87)(1.250(ru/ rs) Tv
CysHiil4NOg 76.87 a
a
USP Levothyroxine for Peak Identification RS in which 798.85 and 776.87 are the molecular weights of
Levothyroxine sodium spiked with liothyronine, levothyroxine sodium and levothyroxine, respectively; C is
triiodothyroacetic acid, and tetraiodothyroacetic acid. the concentration, in ug per mL, of USP Levothyroxine RS in
USP Levothyroxine Sodium RS the Standard preparation; and ry and rs are the peak re-
USP Liothyronine RS sponses obtained from the Assay preparation and the Stan-
O-(4-Hydroxy-3-iodophenyl)-3,5-diiodo-L-tyrosine. lard preparation, respectively.
CisHialsNO4 650.98

Levothyroxine Sodium Tablets

Levothyroxine Sodium Oral Powder DEFINITION
Levothyroxine Sodium Tablets contain NLT 95.0% and NMT
» Levothyroxine Sodium Oral Powder contains 105.0% of the labeled amount of levothyroxine sodium
not less than 90.0 percent and not more than (CisHiolaNNaO,).
110.0 percent of the labeled amount of levothy- IDENTIFICATION
roxine sodium (CisHiolaNNaQO,). e A. The retention time of the major peak of the Sample
solution corresponds to the ee peak of the
Packaging and storage—Preserve in tight, light-resistant Standard solution, as obtained in the Assay.
containers.
Labeling—Label it to indicate that it is for veterinary use ASSAY
only. e PROCEDURE
[Note—Use Sample solution 2 for Tablets labeled to meet
USP Reference standards (11)— the requirements of Dissolution Test 3. For all other
USP Levothyroxine RS products, use the Sample solution.]
 2408 Levothyroxine / Official Monographs
Mobile phase: Acetonitrile and water (4:6) containing
0.5 mL of phosphoric acid per liter of mixture
Solution A: Dissolve 400 mg of sodium hydroxide in
500 mL of water. Cool, and add 500 mL of methanol.
Diluent: Methanol and water (6:4) containing 0.5 mL
of phosphoric acid per liter of mixture
Levothyroxine stock solution: 0.4 mg/mL of USP Levo-
thyroxine RS in Solution A
Liothyronine stock solution: 0.4 mg/mL of USP
Liothyronine RS in Solution A. Make a 1:100 dilution of
this solution using Mobile phase.
Standard solution: 10 g/mL of levothyroxine from Le-
vothyroxine stock solution and 0.2 g/mL of liothyronine
from Liothyronine stock solution, in Mobile phase
Sample solution: Transfer an equivalent to about
100 yg of levothyroxine sodium, from finely powdered
Tablets (NLT 20), to a centrifuge tube, add two glass
beads, pipet 10 mL of Mobile phase into the tube, and
mix on a vortex mixer for 3 min. Centrifuge to obtain a
clear supernatant, filtering if necessary.
Sample solution 2 (for Tablets labeled to meet the
requirements of Dissolution Test 3): Place the appro-
priate number of Tablets (see Table 7) into a suitable
container, add 100.0 mL of Diluent, and shake by me-
chanical means for at least 30 min, or until the Tablets
are fully disintegrated. Pass through a PTFE filter of
0.45-um pore size.
Table 1
Tablet Strength
Chromatographic system
a
al
(See Chromatography (621), System Suitability.)
rm Mode: LC
iin]
a Detector: UV 225 nm
Dp Column: 4.6-mm x 25-cm; packing L10
S) Flow rate: 1.5 mL/min
S Samples: Standard solution and Sample solution
) Injection volume: 100 pL
= System suitability
Sample: Standard solution
a Suitability requirements
va)
=) Resolution: NLT 5.0 between liothyronine and
levothyroxine
Relative standard deviation: NMT 2.0% for the le-
vothyroxine peak
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of levo-
thyroxine sodium (CisHiol4aNNaQO,) in the portion of
Tablets taken:
Result = (ru/rs) x (Cs/Cu) x (Mir/M,z) x 100
tu = peak response from the Sample solution
rs = peak response from the Standard solution
Cs = concentration of USP Levothyroxine RS in the
Standard solution (ug/mL)
Cu = nominal concentration of levothyroxine
sodium in the Sample solution (g/mL)
Ma = molecular weight of levothyroxine sodium,
798.85
Mz = molecular weight of levothyroxine, 776.87
Acceptance criteria: 95.0%-105.0%
PERFORMANCE TESTS
e DISSOLUTION (711) . Calculate the percentage of the labeled amount of le-
[Note—All containers that are in contact with solutions
containing levothyroxine sodium are to be made of
glass.]
USP 41
Test 1
Medium: 0.01 N hydrochloric acid containing 0.2%
sodium lauryl sulfate; 500 mL
Apparatus 2: 50 rpm
Time: 45 min
vera) phase: Methanol and 0.1% phosphoric acid
4)
Standard stock solution: 0.1 mg/mL of USP Levothy-
roxine RS in methanol
Standard solution: Dilute the Standard stock solution
with Medium to obtain a solution having a concentra-
tion similar to that expected in the Sample solution.
Sample solution: Pass a portion of the solution under
test through a suitable filter. [NoTE—Before use, check
the filters for absorptive loss of drug.]
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 225 nm
Column: 4.6-mm x 25-cm; packing L1
Flow rate: 2 mL/min
Injection volume: 800 pL
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 1.5
Relative standard deviation: NMT 4.0% for
levothyroxine
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of le-
vothyroxine sodium (CisHiolaNNaQO,) dissolved.
Tolerances: NLT 70% (Q) of the labeled amount of
levothyroxine sodium (C;sHiol4NNaOa) is dissolved.
Test 2: If the product complies with this test, the label-
ing indicates that it meets USP Dissolution Test 2.
Medium, Apparatus 2, Mobile phase, Standard solu-
tion, Sample solution, Chromatographic system,
and System suitability: Proceed as directed for Test
ds
Time: 15 min
Analysis
Calculate the percentage of the labeled amount of le-
vothyroxine sodium (CisHiol4NNaO,) dissolved.
Tolerances: NLT 80% (Q) of the labeled amount of
levothyroxine sodium (CisHiolaNNaOa) is dissolved.
Test 3: If the product complies with this test, the label-
ing indicates that it meets USP Dissolution Test 3.
Medium, Apparatus 2, Time, Standard solution, and
Sample solution: Proceed as directed for Test 7.
[Note—Filter the Standard solution in a manner identi-
cal to that used for the Sample solution.]
Mobile phase: Acetonitrile and water (35:65) that
contains 0.5 mL of phosphoric acid per liter of mixture
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 225 nm
Column: 4.6-mm x 25-cm; 5-um packing L10
Column temperature: 30°
Flow rate: 1.5 mL/min
Injection volume: 100 pL
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 1.5
Relative standard deviation: NMT 4.0%
Analysis
Samples: Standard solution and Sample solution
vothyroxine sodium (CisHiolaNNaO,) dissolved.
Tolerances: NLT 80% (Q) of the labeled amount of
levothyroxine sodium (CisHiolaNNaOs) is dissolved.
USP 41 Official Monographs / Lidocaine 2409

Test 4: If the product complies with this test, the label- Mn = molecular weight of liothyronine sodium,
ing indicates that it meets USP Dissolution Test 4. 672.96
[Note—Do not use paddle stirrers with synthetic M, = molecular weight of liothyronine, 650.98
coating.] Acceptance criteria: NMT 2.0% of liothyronine sodium
Medium: 0.01 N hydrochloric acid; 500 mL for Tablets
labeled to contain between 25 and 175 wg of levothy- ADDITIONAL REQUIREMENTS
roxine sodium; and 900 mL for Tablets labeled to con- ° PACKAGING AND STORAGE: Preserve in tight, light-resistant
tain 200 or 300 Wg of levothyroxine sodium containers.
Apparatus 2: 75 rpm e LABELING: When more than one Dissolution test is given,
Time: 45 min the labeling states the Dissolution test used only if Test 7
Mobile phase: Acetonitrile, water, and phosphoric is not used.
acid (500:700:2) e USP REFERENCE STANDARDS (11)
Standard stock solution: Transfer about 100 mg of USP Levothyroxine RS
USP Levothyroxine RS to a 100-mL volumetric flask. USP Liothyronine RS
Add 80 mL of alcohol and 1 mL of 1 N hydrochloric
acid, sonicate for 2 min, dilute with alcohol to volume,
and mix.
Standard solution: Dilute the Standard stock solution
with a mixture of alcohol and water (1:1) to obtain a Lidocaine
concentration of 0.01 mg/mL of levothyroxine. Dilute
the resulting solution with Medium to obtaina final CH,
concentration similar to that expected in the Sample
ocr ncn,
solution.
Sample solution: Sample per Dissolution (711). Centri-
fuge the solution under analysis.
Chromatographic system
(See Chromatography (621), System Suitability.) Cy4H22N20 234.34
Mode: LC Acetamide, 2 eypoiaa ee ay rere
Detector: UV 225 nm 2-(Diethylamino)-2’,6’-acetoxylidide [137-58-6].
Column: 4.0-mm x 12.5-cm; packing L7
Flow rate: 1.5 mL/min DEFINITION
Injection volume: 500 pL Lidocaine contains NLT 97.5% and NMT 102.5% of
System suitability Cy4H22N20.
Sample: Standard solution IDENTIFICATION
Suitability requirements e A. INFRARED ABSORPTION (197K): Previously dried in vac-
Tailing factor: NMT 1.5 uum over silica gel for 24 h
Relative standard deviation: NMT 4.0% of e B. The retention time of the major peak of the Sample
levothyroxine solution corresponds to that of the Standard solution, as —
Analysis obtained in the Assay.
nw
a
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of le- ASSAY E<
vothyroxine sodium (CisHiolaNNaO,) dissolved. e PROCEDURE }
=:
Tolerances: NLT 80% (Q) of the labeled amount of Solution A: Water and glacial acetic acid (930:50). Ad- re}
levothyroxine sodium (CisHiola4NNaQO,) is dissolved. just with 1 N sodium hydroxide to a pH of 3.40. eo}2
¢ UNIFORMITY OF DOSAGE UNITS (905): Meet the Mobile phase: Acetonitrile and Solution A (1:4), so that ES)
requirements the retention time of lidocaine is 4-6 min i}
Standard solution: Dissolve 85 mg of USP Lidocaine =e
“
IMPURITIES RS, with warming if necessary, in 0.5 mL of 1 N hydro-
© LIMIT OF LIOTHYRONINE SODIUM chloric acid in a 50-mL volumetric flask. Dilute with Mo-
[Note—Use Sample solution 2 for Tablets labeled to meet bile phase to volume.
the requirements of Dissolution Test 3. For all other System suitability stock solution: 220 g/mL of meth-
products, use the Sample solution.] ylparaben in Mobile phase
Mobile phase, Liothyronine stock solution, Standard system suitability solution: Mix 2 mL of System suita-
solution, Sample solution, Chromatographic system, ility stock solution and 20 mL of Standard solution.
i System suitability: Proceed as directed in the Sample solution: Dissolve 85 mg of Lidocaine, with
say. warming if necessary, in 0.5 mL of 1 N hydrochloric
Liothyronine standard solution: 0.2 g/mL of acid in a 50-mL volumetric flask. Dilute with Mobile
Pau weenie from Liothyronine stock solution, in Mobile pe to volume.
phase hromatographic system
Analysis (See Chromatography (621), System Suitability.)
Samples: Sample solution and Liothyronine standard Mode: LC
solution Detector: UV 254 nm
Calculate the percentage of levothyroxine sodium Column: 3.9-mm x 30-cm; packing L1
(CisHiilsNNaQOx) in the portion of Tablets taken: Flow rate: 1.5 mL/min
Result = (ru/rs) x (Cs/Cu) x (Ma/M2) x 100 Injection size: 20 uL
System suitability
ry = peak response of liothyronine from the Sample Samples: Standard solution and System suitability
solution solution
rs = peak response of liothyronine from the Suitability requirements
Liothyronine standard solution Resolution: NLT 3.0 between lidocaine and methyl-
Cs = concentration of USP Liothyronine RS in the paraben, System suitability solution
Liothyronine standard solution (tug/mL) Relative standard deviation: NMT 1.5%, Standard
Cy = nominal concentration of levothyroxine solution
sodium in the Sample solution (ug/mL)
 2410 Lidocaine / Official Monographs
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of Ci4H22N20 in the portion
of Lidocaine taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response from the Sample solution
rs = peak response from the Standard solution
Cs = concentration of USP Lidocaine RS in the
Standard solution (mg/mL)
Cu = concentration of Lidocaine in the Sample
solution (mg/mL)
Acceptance criteria: 97.5%-102.5%
IMPURITIES
Inorganic Impurities
© RESIDUE ON IGNITION (281): NMT 0.1%
© CHLORIDE AND SULFATE, Chloride (221): Dissolve 1.0g ina
mixture of 3 mL of 2.N nitric acid and 12 mL of water,
and add 1 mL of silver nitrate TS: the turbidity does not
exceed that produced by 50 ul of 0.020 N hydrochloric
acid (0.0035%).
© CHLORIDE AND SULFATE, Sulfate (221): Dissolve 100 mg in
a mixture of 1 mL of 2 N nitric acid and 10 mL of water.
Filter if necessary, and add 1 mL of barium chloride TS.
The turbidity does not exceed that produced by 0.10 mL
of 0.020 N sulfuric acid (NMT 0.1%).
Delete the following:
°e HEAVY METALS, Method | (231)
Test preparation: 1.0g
Analysis: Dissolve the jest preparation in a mixture of
2 mL of 3 N hydrochloric acid and 10 mL of water.
Evaporate on a steam bath to dryness, and dissolve the
residue in 25 mL of water.
ve
nal
Acceptance criteria: 20 ppMe (official 1-jan-2018)
rs
Ss SPECIFIC TESTS
_ PERFORMANCE TESTS
fo.) © MELTING RANGE OR TEMPERATURE (741): 66°-69°
)
= ADDITIONAL REQUIREMENTS
5 e PACKAGING AND STORAGE: Preserve in well-closed contain-
3 ers. Store at room temperature.
a e USP REFERENCE STANDARDS (11)
va)
=) USP Lidocaine RS
Lidocaine Topical Aerosol
DEFINITION
Lidocaine Topical Aerosol is a solution of Lidocaine in a suit-
able flavored vehicle with suitable propellants in a pres-
surized container equipped with a metering valve. It con-
tains NLT 90.0% and NMT 110.0% of the labeled amount
of lidocaine (Ci4H22N20), and it delivers NLT 85.0% and
NMT 115.0% of the labeled amount of lidocaine
(Ci4H22N20) per actuation.
IDENTIFICATION
oA.
Sample solution: To 5 mL of Aerosol spray, collected in
a separator, add 10 mL of water and 3 mL of dilute
hydrochloric acid (1 in 2), wash with two 15-mL por-
tions of chloroform, and discard the chloroform wash-
ings. Render the solution in the separator alkaline with
5-6 mL of ammonium hydroxide, and extract with
three 20-mL portions of chloroform, filtering the chloro-
form extracts through a pledget of cotton previously
moistened with chloroform. Evaporate the combined
chloroform extracts with the aid of gentle heat to dry-
USP 41
ness, and dry the residue under vacuum over silica gel
for 24 h.
Acceptance criteria: A potassium bromide dispersion of
the lidocaine exhibits maxima only at the same wave-
lenges as that of a similar preparation of USP Lidocaine
° B.
Analysis: To 2 mL of Aerosol spray, collected in a test
tube, add 10-15 drops of cobaltous chloride TS, and
shake for 2 min.
Acceptance criteria: A bright green color develops, and
a fine precipitate is formed (lidocaine).
e *
Analysis: To 2 mL of Aerosol spray, collected in a test
tube, add 5 mL of water, 1 mLof) N nitric acid, and
3 mL of mercuric nitrate TS.
Acceptance criteria: A light yellow color develops
(lidocaine).
ASSAY
© PROCEDURE
Sample solution: Weigh 1 Aerosol container and actua-
tor. Transfer a counted number of NLT 10 doses to a
125-mL conical flask by carefully discharging the doses
in such a manner as to avoid loss of material, and take
precautions to protect the sample from absorption of
atmospheric moisture. Weigh the container and actua-
tor to obtain the sample weight. To the specimen, add
20 mL of chloroform, mix, and add 10 mL of dioxane
and 2 drops of crystal violet TS.
Analysis: Titrate with 0.1 N perchloric acid in dioxane
VS to a blue endpoint. Perform a blank determination,
and make any necessary correction. Each mL of 0.1 N
perchloric acid is equivalent to 23.43 mg of lidocaine
(Ci4H2N20).
Acceptance criteria: It contains 90.0%-110.0% of the
labeled amount of lidocaine (C:4H22N20), and it delivers
85.0%-115.0% of the labeled amount of lidocaine
(Ci4H22N20) per actuation.
e INHALATION AND NASAL DRUG PRODUCTS: AEROSOLS,
SPRAYS, AND POWDERS—PERFORMANCE QUALITY TESTS
(601) andToPICcAL AEROSOLS (603)
For Delivered-Dose Uniformity and Number of Dis-
charges per Container
Acceptance criteria: Meets the requirements
SPECIFIC TESTS
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): It meets the requirements of
the tests for absence of Staphylococcus aureus and Pseu-
domonas aeruginosa.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in nonreactive aerosol
containers equipped with metered-dose valves.
e USP REFERENCE STANDARDS (11)
USP Lidocaine RS
Lidocaine Ointment
DEFINITION
Lidocaine Ointment is Lidocaine in a suitable hydrophilic
ointment base. It contains NLT 95.0% and NMT 105.0%
of the labeled amount of lidocaine (Ci4H22N20).
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
Sample: Stir a quantity of Ointment, equivalent to
300 mg of lidocaine, with 20 mL of water, transfer to a
separator, and extract with two 30-mL portions of sol-
USP 41 Official Monographs / Lidocaine 2411

vent hexane. Wash the combined hexane extracts with Acceptance criteria: 95.0%-105.0%
10 mL of water, evaporate with the aid of a current of
warm air, and dry the residue in vacuum over silica gel PERFORMANCE TESTS
for 24 h. e Minimum FILL (755): Meets the requirements
Acceptance criteria: The crystalline precipitate so ob-
SPECIFIC TESTS
tained meets the requirements.
e B. The retention time of the major peak of the Sample e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): It meets the requirements of
solution corresponds to that of the Standard solution, as
obtained in the Assay. the tests for absence of Staphylococcus aureus and Pseu-
domonas aeruginosa.
ASSAY ADDITIONAL REQUIREMENTS
¢ PROCEDURE
Solution A: 0.1% phosphoric acid, piepated by adding e PACKAGING AND STORAGE: Preserve in tight containers,
1.0 mL of 85% phosphoric acid to 1 L of water and store at controlled room temperature.
Solution B: Acetonitrile e USP REFERENCE STANDARDS (11)
Mobile phase: See Table 7. USP Lidocaine RS
USP Ropivacaine Related Compound A RS
2,6-Dimethylaniline hydrochloride.
Table 1 CsHi2CIN 157.64
Solution A Solution B

Lidocaine Oral Topical Solution

15 10
» Lidocaine Oral Topical Solution contains not
Diluent: Acetonitrile and Solution A (1:1) less than 95.0 percent and not more than
System suitability solution: 0.1 mg/mL of USP Lido-
caine RS and 0.04 mg/mL of USP Ropivacaine Related 105.0 percent of the labeled amount of lidocaine
Compound ARS in Diluent (Ci4H22N20). It contains a suitable flavor.
[Note—USP Ropivacaine Related CompoundA RS is
2,6-dimethylaniline hydrochloride.] Packaging and storage—Preserve in tight containers.
Standard solution: 0.1 mg/mL of USP Lidocaine RS in USP Reference standards (11)—
Diluent USP Lidocaine RS
Sample solution: Nominally 0.1 mg/mL of lidocaine in Identification—Transfer a quantity of Oral Topical Solu-
Diluent from a portion of Ointment. Sonicate the solu- tion, equivalent to about 250 mg of lidocaine, to a
tion for about 5 min. separator with 20 mL of water, and extract with 20 mL of
Chromatographic system chloroform. Wash the chloroform extract with 20 mL of Cc
a)
(See Chromatography (621), System Suitability.) water, and evaporate the chloroform extract with the aid of uv
Mode: LC a current of warm air. Dissolve the residue in hexane, evap-
Detector: UV 210 nm orate with the aid of a current of warm air, and dry the a
Column: 4.6-mm x 15-cm; 5-m packing L1 residue in vacuum over silica gel for 24 hours: the crystalline °
I
Flow rate: 0.8 mL/min precipitate so obtained responds to /dentification test A °
Injection volume: 5 uL under Lidocaine. so}
=
System suitability Assay—Transfer an accurately measured volume of Oral Ey
Samples: System suitability solution and Standard mo)
Topical Solution, equivalent to about 150 mg of lidocaine, a
solution to a 125-mL conical flask, and protect from atmospheric 7)
[Note—The relative retention times for 2,6-dimethy- moisture with a stopper fitted with a tube containing silica
laniline and lidocaine are about 0.93 and 1.0, gel. Add 20 mL of glacial acetic acid and 2 drops of crystal
respectively.] violet TS. Titrate immediately with 0.1 N perchloric acid VS
Suitability requirements to a blue endpoint. Perform a blank determination, and
Tailing factor: NMT 1.5 for the lidocaine peak, Sys- make any necessary correction. Each mL of 0.1 N perchloric
tem suitability solution acid is equivalent to 23.43 mg of C,4H22N20.
Relative standard deviation: NMT 2.0%, Standard
solution
Resolution: NLT 1.8 between lidocaine and 2,6-
dimethylaniline, System suitability solution
Analysis Lidocaine Hydrochloride
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of lido-
caine (Ci4H22N20) in the portion of Ointment taken: Ci4H22N20 - HCl - H2O 288.81
Acetamide, 2-(diethylamino)-N-(2,6-dimethylpheny))-,
Result = (ru/rs) x (Cs/Cu) x 100 monohydrochloride, monohydrate;
2-(Diethylamino)-2’,6’-acetoxylidide monohydrochloride
tu = peak response from the Sample solution monohydrate [6108-05-0].
Is = peak response from the Standard solution Anhydrous 270.80
Cs = concentration of USP Lidocaine RS in the [73-78-9].
Standard solution (mg/mL)
Cy = nominal concentration of lidocaine in the DEFINITION
Sample solution (mg/mL) Lidocaine Hydrochloride contains NLT 97.5% and NMT
102.5% of lidocaine hydrochloride (C14H22N20 - HCl), cal-
culated on the anhydrous basis.
 2412 Lidocaine / Official Monographs USP 41

IDENTIFICATION caine Related Compound H RS and USP Lidocaine Hy-

© A. INFRARED ABSORPTION (197) drochloride RS in Mobile phase
¢ B. The retention time of the major peak of the Sample Sample solution: 5 mg/mL of Lidocaine Hydrochloride
solution corresponds to that of the Standard solution, as in Mobile phase
obtained in the Assay. Chromatographic system
e C. IDENTIFICATION TESTS—GENERAL, Chloride (191): Meets (See Chromatography (621), System Suitability.)
the requirements Mode: LC
Detector: UV 230 nm
ASSAY Column: 3.9-mm x 15-cm; 5-um packing L1
© PROCEDURE Column temperature: 30°
Solution A: Water and glacial acetic acid (930:50). Ad- Flow rate: 1 mL/min
just with 1 N sodium hydroxide to a pH of 3.40. Injection volume: 20 uL
Mobile phase: Acetonitrile and Solution A (1:4) Run time: NLT 3.5 times the retention time for
Standard solution: 2.0 mg/mL of USP Lidocaine Hydro- lidocaine
chloride RS in Mobile phase System suitability
System suitability stock solution: 220 g/mL of meth- Sample: Standard solution
ylparaben in Mobile phase [Note—See Table 7 for relative retention times.]
System suitability solution: Mix 2 mL of System suita- Suitability requirements
bility stock solution and 20 mL of Standard solution. Resolution: NLT 1.5 between lidocaine related com-
Sample solution: 2 mg/mL of Lidocaine Hydrochloride pound H and ropivacaine related compound A; NLT
in Mobile phase 2.0 between ropivacaine related compound A and
Chromatographic system lidocaine
(See Chromatography (621), System Suitability.) Relative standard deviation: NMT 10.0% for ropiva-
Mode: LC caine related compound A
Detector: UV 254 nm Analysis
Column: 3.9-mm x 30-cm; packing L1 Samples: Standard solution and Sample solution
Flow rate: 1.5 mL/min Calculate the percentage of lidocaine related compound
Injection volume: 20 wL H or ropivacaine related compoundA in the portion of
System suitability Lidocaine Hydrochloride taken:
Samples: Standard solution and System suitability
solution Result = (ru/rs) x (Cs/Cu) x 100
Suitability requirements
Resolution: NLT 3.0 between lidocaine and methyl- tu = peak response for lidocaine related compound
paraben, System suitability solution H or ropivacaine related compound A from
Relative standard deviation: NMT 1.5%, Standard the Sample solution
solution rs = peak response of lidocaine related compound
Analysis H or ropivacaine related compound A from
“ Samples: Standard solution and Sample solution the Standard solution
<= Calculate the percentage of lidocaine hydrochloride Cs = concentration of USP Lidocaine Related
a (Ci4H22N20 - HCl) in the portion of Lidocaine Hydro- Compound H RS or USP Ropivacaine Related
i
—
fo.) chloride taken: CompoundARS in the Standard solution
}
< Result = (ru/rs) x (Cs/Cu) x 100
(ug/mL) ‘ Hul>.
Cu = concentration of Lidocaine Hydrochloride in
S the Sample solution (g/mL)
= tu = peak response from the Sample solution Calculate the percentage of any individual unspecified
os Is = peak response from the Standard solution {rari in the portion of Lidocaine Hydrochloride
a) Gs = concentration of USP Lidocaine Hydrochloride
= RS in the Standard solution (mg/mL)
taken:

Cu = concentration of Lidocaine Hydrochloride in Result = (ru/rs) x (Cs/Cu) x 100

the Sample solution (mg/mL)
Acceptance criteria: 97.5%-102.5% on the anhydrous ty = peak response for each unspecified impurity
basis from the Sample solution
rs = peak response of lidocaine from the Standard
IMPURITIES solution
e RESIDUE ON IGNITION (281): NMT 0.1% Gs = concentration of USP Lidocaine Hydrochloride
e CHLORIDE AND SULFATE, Sulfate (221) RS in the Standard solution (\ug/mL)
Sample: 100mg Cy = concentration of Lidocaine Hydrochloride in
Analysis: Dissolve Sample in 10 mL of water, and add the Sample solution (g/mL)
1 mL of 3.N hydrochloric acid. Mix, and add 1 mL of Acceptance criteria: See Table 7.
barium éhletine TS:
Acceptance criteria: The turbidity does not exceed that
produced by 0.10 mL of 0.020 N sulfuric acid (NMT Table 1
0.1%). Relative Acceptance
Retention Criteria,
Name Time NMT (%)
Delete the following:
Lidocaine related
compound H 0.38 0.10
°e HEAVY METALS, Method | (231): NMT 20 ppme crtaa1-
jan-2018)
Ropivacaine related
° ORGANIC IMPURITIES compound A 0.42 0.01
Buffer: 4.85 g/L of monobasic potassium phosphate in Lidocaine 1.0 =
water. Adjust with sodium hydroxide solution to a pH Any individual
of 8.0. unspecified a
Mobile phase: Acetonitrile and Buffer (30:70) impurity 0.10
Standard solution: 0.5 g/mL of USP Ropivacaine Re- Total impurities = 0.5
lated Compound A RS and 5 g/mL each of USP Lido-
USP 41 Official Monographs / Lidocaine 2413

SPECIFIC TESTS Bacterial Endotoxins Test (85)—It contains not more

© WATER DETERMINATION, Method | (921): 5.0%-7.0%
e STERILITY TESTS (71): Where the label states that Lido-
caine Hydrochloride is sterile, it meets the requirements.
e BACTERIAL ENDOTOXINS TEST (85): Where the label states
that Lidocaine Hydrochloride is sterile or must be sub-
jected to further processing during the preparation of in-
jectable dosage forms, it contains NMT 1.1 USP Endo-
toxin Units/mg of lidocaine hydrochloride.
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. Store at controlled room temperature.
e LABELING: Where it is intended for use in preparing in-
jectable dosage forms, the label states that it is sterile or
must be subjected to further processing during the prep-
aration of injectable dosage forms.
than 1.1 USP Endotoxin Units per mg of lidocaine hydro-
chloride.
pH (791): between 5.0 and 7.0.
Particulate Matter in Injections (788): meets the re-
quirements for small-volume injections.
Other requirements—It meets the requirements under /n-
jections and Implanted Drug Products (1).
Assay—Proceed with Injection as directed in the Assay for
lidocaine hydrochloride under Lidocaine Hydrochloride and Epi-
nephrine Injection.
Lidocaine Hydrochloride Jelly

Change to read: DEFINITION

Lidocaine Hydrochloride Jelly is Lidocaine Hydrochloride in a
° usP REFERENCE STANDARDS (11) suitable, water-soluble, sterile, viscous base. It contains
NLT 95.0% and NMT 105.0% of the labeled amount of
@ (CN 1-May-2018)
USP Lidocaine Hydrochloride RS lidocaine hydrochloride (C;4H22N20 - HCl).
USP Lidocaine Related Compound H RS IDENTIFICATION
2-Chloro-N-(2,6-dimethylphenyl)acetamide. e A. INFRARED ABSORPTION (197K)
CioHiz2CINO 197.66 Standard: Prepare as directed in Infrared Absorption
USP Ropivacaine Related Compound A RS (197K), using USP Lidocaine RS.
2a ene ane hydrochloride. Sample: Place in a separator containing 10-15 mL of
CsHiN-HCl 157.64 water a quantity of Jelly, equivalent to 300 mg of lido-
caine hydrochloride. Mix to assure thorough dilution of
the Jelly, and add 4 mL of 6 N ammonium hydroxide.
Extract with four 15-mL portions of chloroform. Com-
bine the chloroform extracts, and evaporate with the
Lidocaine Hydrochloride Injection aid of a current of warm air to dryness. Redissolve the
crystals in solvent hexane, evaporate with the aid of
warm air, and dry the residue in vacuum over silica gel
» Lidocaine Hydrochloride Injection is a sterile so-
lution of Lidocaine Hydrochloride in Water for In-
for 24 h. fo
Acceptance criteria: The residue so obtained meets the wn
jection, ora sterile solution prepared from Lido- equirements. vu

caine with the aid of Hydrochloric Acid in Water e B. The retention time of the major peak of the Sample =
for Injection. It contains not less than 95.0 per- solution corresponds to that of the Standard solution, as i}
obtained in the Assay. J
cent and not more than 105.0 percent of the la- i}
beled amount of lidocaine hydrochloride ASSAY
a=
2
(Cy4H22N20 - HCl). © PROCEDURE ae}
Solution A: 0.1% phosphoric acid, prepared by adding =%
Packaging and storage—Preserve in single-dose or in 1.0 mL of 85% phosphoric acid to 1 L of water ”

multiple-dose containers, preferably of Type | glass. Injection Solution B: Acetonitrile

may be packaged in 50-mL multiple-dose containers. Mobile phase: See Table 1.
Labeling—Injections that are of such concentration that
they are not intended for direct injection into tissues are Table 1
labeled to indicate that they are to be diluted prior to ad-
Solution A Solution B
ministration.
Yo

Change to read: 10 10
10.1
USP Reference standards (11)— 15
@ (CN T-May-2018)
USP Lidocaine RS Diluent: Acetonitrile and Solution A (1:1)
Identification—Place in a separator a volume of Injection System suitability solution: 0.1 mg/mL of USP Lido-
equivalent to about 300 mg of lidocaine hydrochloride, and caine RS and 0.04 mg/mL of USP Ropivacaine Related
extract with four 15-mL portions of chloroform, discarding Compound A RS in Diluent
the chloroform extracts. Add 2 mL of 2 N sodium hydroxide [NoTE—USP Ropivacaine Related Compound A RS is
to the aqueous solution remaining in the separator, and ex- 2,6-dimethylaniline hydrochloride.]
tract with four 15-mL portions of chloroform. Combine the Standard solution: 0.1 mg/mL of USP Lidocaine RS in
chloroform extracts, and evaporate with the aid of a current Diluent
of warm air to dryness. Dissolve the crystals so obtained in Sample solution: Nominally 0.12 mg/mL of lidocaine
solvent hexane, evaporate with the aid of warm air, and dry hydrochloride in Diluent from a portion of Jelly. Soni-
the residue in vacuum over silica gel for 24 hours: the resi- cate the solution for about 10 min.
due so obtained responds to /dentification test A under Lido-
caine.
 2414 Lidocaine / Official Monographs USP 41

Chromatographic system ing in the separator, and extract with four 15-mL por-
(See Chromatography (621), System Suitability.) tions of chloroform. Combine the chloroform extracts,
Mode: LC and evaporate with the aid of a current of warm air to
Detector: UV 210 nm dryness. Dissolve the crystals in solvent hexane, evapo-
Column: 4.6-mm x 15-cm; 5-um packing L1 rate with the aid of warm air, and dry the residue
Flow rate: 0.8 mL/min under vacuum over silica gel for 24 h.
Injection volume: 5 uL Acceptance criteria: Residue obtained from the Sample
System suitability meets the requirements.
Samples: System suitability solution and Standard e B. The retention time of the major peak of the Sample
solution solution corresponds to that of the Standard solution, as
[Note—The relative retention times of 2,6-dimethy- obtained in the Assay.
laniline and lidocaine are about 0.93 and 1.0,
respectively.] ASSAY
Suitability requirements © PROCEDURE
Tailing factor: NMT 1.5 for the lidocaine peak, Sys- Solution A: 4.85 g/L of monobasic potassium phos-
tem suitability solution phate. Adjust with 10 N sodium hydroxide solution to a
Relative standard deviation: NMT 2.0%, Standard pH of 8.00.
solution Mobile phase: Acetonitrile and Solution A (30:70)
Resolution: NLT 1.8 between lidocaine and 2,6- System suitability stock solution: 0.043 mg/mL of USP
dimethylaniline, System suitability solution Lidocaine RS (equivalent to 0.05 mg of lidocaine hydro-
Analysis chloride), 0.05 mg/mL of USP Lidocaine Related Com-
Samples: Standard solution and Sample solution pound H RS, and 0.0065 mg/mL of USP Ropivacaine
Calculate the perceptige of the labeled amount of lido- Related CompoundA RS in Mobile phase prepared as
caine hydrochloride (Ci4H22N2O - HCl) in the portion of follows. Transfer a weighed quantity of USP Lidocaine
Jelly taken: RS, USP Lidocaine Related Compound H RS, and USP
Ropivacaine Related Compound A RS to a suitable volu-
Result = (ru/rs) x (Cs/Cu) x (Mi1/Mi2z) x 100 metric flask, and add a small amount of acetonitrile.
Swirl to dissolve, and dilute with Mobile phase to
ty = peak response from the Sample solution volume.
fs = peak response from the Standard solution System suitability solution: Transfer 2.0 mL of System
Gs = concentration of USP Lidocaine RS in the suitability stock solution to a 20-mL volumetric flask, and
Standard solution (mg/mL) dilute with Mobile phase to volume.
Cu = nominal concentration of lidocaine Standard solution: 0.85 mg/mL of USP Lidocaine RS
hydrochloride in the Sample solution (equivalent to 1 mg/mL of lidocaine hydrochloride) in

M,
(mg/mL)
= molecular weight of lidocaine hydrochloride,
Mobile phase
Sample solution: Nominally equivalent to 1 mg/mL of
270.80 lidocaine hydrochloride from Oral Topical Solution in
Mr = molecular weight of lidocaine, 234.34 Mobile phase
fe
”

Qa Acceptance criteria: 95.0%-105.0% Chromatographic system

J (See Chromatography (621), System Suitability.)
D
- PERFORMANCE TESTS Mode: LC
° e MINIMUM FILL (755): Meets the requirements Detector: UV 230 nm
4 Column: 4.6-mm x 15-cm; 3.5-1um packing L1
5 SPECIFIC TESTS Column temperature: 45°
= e PH (791): 6.0-7.0 Flow rate: 1 mL/min
oe © STERILITY TESTS (71): Meets the requirements Injection volume: 20 ul
a)
p>) ADDITIONAL REQUIREMENTS System suitability
e PACKAGING AND STORAGE: Preserve in tight containers, Samples: System suitability solution and Standard
and store at controlled room temperature. solution
e USP REFERENCE STANDARDS (11) [Note—See Table 7 for the relative retention times.]
USP Lidocaine RS Suitability requirements
USP Ropivacaine Related Compound A RS Resolution: NLT 1.5 between lidocaine related com-
2,6-Dimethylaniline hydrochloride. pound H and ropivacaine related compound A, Sys-
CsHi2CIN = 157.64 tem suitability solution
Tailing factor: NMT 2.0, Standard solution
Relative standard deviation: NMT 2.0%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Lidocaine Hydrochloride Oral Topical Calculate thepercentage of the labeled amount of lido-
Solution caine hydrochloride (C,4H22N20 - HCl) in the portion of
Oral Topical Solution taken:
DEFINITION
Lidocaine Hydrochloride Oral Topical Solution contains NLT Result = (ru/rs) x (Cs/Cu) X (Mi/M,2) x 100
95.0% and NMT 105.0% of the labeled amount of lido-
caine hydrochloride (Cy4H22Nz2O
- HCl). It contains a suita- tu = peak response of lidocaine from the Sample
ble flavor and/or sweetening agent. solution
Is = peak response of lidocaine from the Standard
IDENTIFICATION solution
e A. INFRARED ABSORPTION (197K) Cs = concentration of USP Lidocaine RS in the
Sample: Place in a separator a volume of Oral Topical Standard solution (mg/mL)
Solution, equivalent to 300 mg of lidocaine hydrochlo- Cu = nominal concentration of lidocaine
ride, and extract with four 15-mL portions of chloro- hydrochloride in the Sample solution
form, scaring the chloroform extracts. Add 2 mL of (mg/mL)
2.N sodium hydroxide to the aqueous solution remain-
USP 41 Official Monographs / Lidocaine 2415

Ma = molecular weight of lidocaine hydrochloride, Acceptance criteria: See Table 1. Disregard any impu-
270.80 rity peak less than 0.05%.
M2 = molecular weight of lidocaine, 234.34
Acceptance criteria: 95.0%-105.0% Table 1
IMPURITIES Relative Acceptance
© ORGANIC IMPURITIES Retention Criteria,
Solution A, Mobile phase, System suitability solution, Name Time NMT (%)
and Chromatographic system: Proceed as directed in Lidocaine related
the ue compound H 0.33 0.1
Standard solution: 0.0043 mg/mL of USP Lidocaine RS, Dimethylaniline 0.37 0.01
0.005 mg/mL of USP Lidocaine Related Compound H
Lidocaine 1.0 =
RS, and 0.00065 mg/mL of USP Ropivacaine Related
Compound A RS in Mobile phase Any other individual,
Sample solution: Nominally equivalent to 5 mg/mL of unspecified —
lidocaine hydrochloride in Mobile phase impurity 0.10
System suitability Total impurities = =
Samples: System suitability solution and Standard
solution
Suitability requirements SPECIFIC TESTS
e PH (791): 5.0-7.0
Resolution: NLT 1.5 between lidocaine related com-
pound H and ropivacaine related compound A, Sys- ADDITIONAL REQUIREMENTS
tem suitability solution © PACKAGING AND STORAGE: Preserve in tight containers.
Relative standard deviation: NMT 2.0% for lido- Store at controlled room temperature.
caine, Standard solution ¢ USP REFERENCE STANDARDS (11
Analysis USP Lidocaine RS
Samples: Standard solution and Sample solution USP Lidocaine Related Compound H RS
Calculate the percentage of lidocaine related compound N-(Chloroacetyl)-2,6-xylidide.
H in the portion of Oral Topical Solution taken: CioHi2CINO "197.66
USP Ropivacaine Related Compound A RS
Result = (ru/rs) x (Cs/Cu) x 100 EG bess aolline hydrochloride.
CgsHiN+HCl 157.64
ry = peak response of lidocaine related compound
H from the Sample solution
Is = peak response of lidocaine related compound
H from the Standard solution
Gs = concentration of USP Lidocaine Related
Compound H RS in the Standard solution Lidocaine Hydrochloride Topical
Solution S
(mg/mL) 4)
cu = nominal concentration of lidocaine ao]
hydrochloride in the Sample solution DEFINITION ms
(mg/mL) Lidocaine Hydrochloride Topical Solution contains NLT 5
Calculate the percentage of dimethylaniline in the 95.0% and NMT 105.0% of the labeled amount of lido- BS
portion of Oral Topical Solution taken: i)
caine hydrochloride (C4H22N20 - HCl). to}<
Result = (ru/rs) x (Cs/Cu) x (Mr/Mi2) X 100 IDENTIFICATION i
e A. INFRARED ABSORPTION (197K)
i}
>
i = peak response of dimethylaniline from the Sample: Place in a separator a volume of Topical Solu- w”
Sample solution tion, equivalent to 200 mg of lidocaine hydrochloride,
Is = peak response of dimethylaniline from the and extract with four 15-mL portions of chloroform,
Standard solution discardin the chloroform extracts. Add 2 mL of 2 N so-
Cs = concentration of USP Ropivacaine Related dium hydroxide to the aqueous solution remaining in
CompoundA RS in the Standard solution the separator, and extract with four 15-mL portions of
(mg/mL) chloroform. Combine the chloroform extracts, and
Cu = nominal concentration of lidocaine in the evaporate with the aid of a current of warm air to dry-
Sample solution (mg/mL) ness. Dissolve the crystals in solvent hexane, evaporate
M, — = molecular weight of ropivacaine related with the aid of warm air, and dry the residue under
compound A, 157.64 vacuum over silica gel for 24 h.
Mz = molecular weight of dimethylaniline, 121.18 Acceptance criteria: The residue obtained from the
Calculate the percentage of any other individual Sample meets the requirements.
impurity in the portion of Oral Topical Solution taken: e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
Result = (ru/rs) x (Cs/Cu) x (Mn/Mi2z) x 100 obtained in the Assay.
ry = peak response of each impurity from the ASSAY
Sample solution © PROCEDURE
rs = peak response of lidocaine from the Standard Solution A: Glacial acetic acid and water (5:93). Adjust
solution with 1 N sodium hydroxide to a pH of 3.40.
Cs = concentration of USP Lidocaine RS in the Mobile phase: Acetonitrile and Solution A (1:4)
Standard solution (mg/mL) Standard solution: 1.7 mg/mL of USP Lidocaine RS
Cu nominal concentration of lidocaine (equivalent to 2 mg/mL of lidocaine hydrochloride) in
hydrochloride in the Sample solution Mobile phase prepared as follows. Transfer a weighed
(mg/mL) quanity of USP Lidocaine RS to a suitable volumetric
Mn = molecular weight of lidocaine hydrochloride, flask, and add 1 N hydrochloric acid to fill 1% of the
270.80
molecular weight of lidocaine, 234.34
=
I
s
 2416 Lidocaine / Official Monographs USP 41

final volume. Warm if necessary, and dilute with Mobile [NoTe—See Table 1 for the relative retention times.]
phase to volume. Suitability requirements
System suitability stock solution: 220 g/mL of USP Resolution: NLT 2.0 between lidocaine related com-
Methylparaben RS in Mobile phase pound H and ropivacaine related compound A, Sys-
System suitability solution: Mix 2 mL of the System tem suitability solution
suitability stock solution with 20 mL of the Standard Relative standard deviation: NMT 2.0% for lido-
solution. caine, Standard solution
Sample solution: Nominally equivalent to 2 mg/mL of Analysis
iis hydrochloride from Topical Solution in Mobile Samples: Standard solution and Sample solution
phase Calculate the percentage of lidocaine related compound
Chromatographic system H in the portion of Topical Solution taken:
(See Chromatography (621), System Suitability.)
Mode: LC Result = (ru/rs) x (Cs/Cu) x 100
Detector: UV 254 nm
Column: 3.9-mm x 30-cm; packing L1 tu = peak response of lidocaine related compound
Flow rate: 1.5 mL/min H from the Sample solution
Injection volume: 20 UL ls = peak response of lidocaine related compound
System suitability H from the Standard solution
Samples: Standard solution and System suitability Cs = concentration of USP Lidocaine Related
solution Compound H RS in the Standard solution
Suitability requirements (mg/mL)
Resolution: NLT 3.0 between lidocaine and methyl- Cu = nominal concentration of lidocaine
paraben, System suitability solution hydrochloride in the Sample solution
Relative standard deviation: NMT 1.5%, Standard (mg/mL)
solution Calculate the percentage of dimethylaniline in the
Analysis portion of Topical Solution taken:
Samples: Standard solution and Sample solution
Calculate thepercentage of the labeled amount of lido- Result = (ru/rs) x (Cs/Cu) x (Mrr/Mj2) x 100
caine hydrochloride (Cy4H22N2O - HCl) in the portion of
Topical Solution taken: ry = peak response of dimethylaniline from the
Sample solution
Result = (ru/rs) x (Cs/Cu) (Mr/Miz) x 100 Is peak response of dimethylaniline from the
Standard solution
ry = peak response of lidocaine from the Sample Cs = concentration of USP Ropivacaine Related
solution Compound A RS in the Standard solution
Is = peak response of lidocaine from the Standard (mg/mL)
solution Cy nominal concentration of lidocaine
il

G = concentration of USP Lidocaine RS in the hydrochloride in the Sample solution

us
al

ry Standard solution (mg/mL) (mg/mL)

Cc = nominal concentration of lidocaine Ma — = molecular weight of ropivacaine related
- hydrochloride in the Sample solution compound A, 157.64
D
° (mg/mL) Me = molecular weight of dimethylaniline, 121.84
= Ma = molecular weight of lidocaine hydrochloride, Calculate the percentage of any impurity in the portion
5 270.80 of Topical Solution taken:
= M2 = molecular weight of lidocaine, 234.34
5 Acceptance criteria: 95.0%-105.0% Result = (ru/rs) x (Cs/Cu) x (Ma/M,2) x 100
2)
= IMPURITIES ru = peak response of each impurity from the
© ORGANIC IMPURITIES Sample solution
Solution A and Mobile phase: Proceed as directed in rs = peak response of lidocaine from the Standard
the Assay. solution
System suitability solution: 2.6 j1g/mL of USP Lido- Cs = concentration of USP Lidocaine RS in the
caine RS, 3.9 1g/mL of USP Ropivacaine Related Com- Standard solution (mg/mL)
pound A RS, and 3 g/mL of USP Lidocaine Related G = nominal concentration of lidocaine
Compound H RS in Mobile phase hydrochloride in the Sample solution
Standard solution: 0.0017 mg/mL of USP Lidocaine RS, (mg/mL)
0.0026 mg/mL of USP Ropivacaine Related Compound Mn = molecular weight of lidocaine hydrochloride,
A RS (equivalent to 0.002 mg/mL of gedmetnye 270.80
laniline), and 0.002 mg/mL of USP Lidocaine Related Mz = molecular weight of lidocaine, 234.34
CompoundH RS in Mobile phase Acceptance criteria: See Table 1. Disregard any impu-
Sample solution: Nominally equivalent to 2 mg/mL of tity peak less than 0.05%.
lidocaine hydrochloride in Mobile phase
Chromatographic system Table 1
(See Chromatography (621), System Suitability.)
Mode: LC Relative Acceptance
Retention Criteria,
Detector: UV 254 nm
Name Time NMT (%)
Column: 4.6-mm x 25-cm; 5-ym packing L1
Flow rate: 1.5 mL/min Lidocaine 1.0 =
Injection volume: 50 LL Dimethylaniline 3.2 0.1
System suitability Lidocaine related
Samples: System suitability solution and Standard compound H 3.8 0.1
solution
USP 41 Official Monographs / Lidocaine 2417

Table 1 (Continued) Assay for dextrose—Determine the angular rotation of In-

jection in a suitable polarimeter tube (see Optical Rotation
Relative Acceptance
(781)). Calculate the percentage (g per 100 mL) of dextrose
Retention Criteria,
(C6Hi206¢- H2O) in the portion of Injection taken by the
Name Time NMT (%)
formula:
Any unspecified deck
impurity 0.10 (100/52.9)(198.17/180.16)AR
Total impurities = 2.0
in which 100 is the percentage; 52.9 is the midpoint of the
specific rotation range for anhydrous dextrose, in degrees;
SPECIFIC TESTS 198.17 and 180.16 are the molecular weights for dextrose
© PH (791): 5.0-7.0 monohydrate and anhydrous dextrose, respectively; A is
ADDITIONAL REQUIREMENTS 100 mm divided by the length of the polarimeter tube, in
mm; and Ris the observed rotation, in degrees.
© PACKAGING AND STORAGE: Preserve in tight containers.
Store at controlled room temperature.
e USP REFERENCE STANDARDS (1 1b
USP Lidocaine RS
USP Lidocaine Related Compound H RS
N-(Chloroacetyl)-2,6-xylidide. Lidocaine Hydrochloride and
CioHi2CINO = 197.66 Epinephrine Injection
USP Methylparaben RS
USP Ropivacaine Related Compound A RS » Lidocaine Hydrochloride and Epinephrine Injec-
2,6-Dimethylaniline hydrochloride.
CeHuN-HCl 157.64 tion is a sterile solution prepared from Lidocaine
Hydrochloride and Epinephrine with the aid of
Hydrochloric Acid in Water for Injection, or a
sterile solution prepared from Lidocaine and Epi-
nephrine with the aid of Hydrochloric Acid in
Lidocaine Hydrochloride and Dextrose Water for Injection, or a sterile solution of Lido-
Injection caine Hydrochloride and Epinephrine Bitartrate in
Water for Injection. The content of epinephrine
» Lidocaine Hydrochloride and Dextrose Injection does not exceed 0.002 percent (1 in 50,000).
is a sterile solution of Lidocaine Hydrochloride Lidocaine Hydrochloride and Epinephrine Injec-
and Dextrose in Water for Injection. It contains tion contains the equivalent of not less than
not less than 95.0 percent and not more than 95.0 percent and not more than 105.0 percent of
105.0 percent of the labeled amounts of lido- (=
the labeled amount of lidocaine hydrochloride a)
caine hydrochloride (Ci4H22N2O - HCl) and dex- (CisH22N20 - HCl) and the equivalent of not less a}
trose (CsHi206 - H20). than 90.0 percent and not more than 115.0 per- E<
cent of the labeled amount of epinephrine 2
Packaging and storage—Preserve in single-dose glass or =
plastic containers. Glass containers are preferably of Type | (CoH13NOs). )
or Type Il glass.
(ro)af
Packaging and storage—Preserve in single-dose or multi- 2
Bo)
ple-dose light-resistant containers, preferably of Type | glass. sI
Change to read: Labeling—The label indicates that the Injection is not to be
used if its color is pinkish or darker than slightly yellow or if
usP Reference standards (11)— it contains a precipitate.
@ (CN \-May-2018)
USP Lidocaine RS
Identification— Change to read:
A: Place in a separator a volume of Injection equivalent
to about 300 mg of lidocaine hydrochloride, add 2 mL of usP Reference standards (11)—
@ (CN i-May-2018)
2.N sodium hydroxide, and extract with four 15-mL por-
tions of chloroform. Combine the chloroform extracts, and USP Epinephrine Bitartrate RS
evaporate with the aid of a current of warm air to dryness. USP Lidocaine RS
Dissolve the crystals so obtained in solvent hexane, evapo- Color and clarity—Using the Injection as the Test solution,
rate with the aid of warm air, and dry the residue in vac- proceed as directed for Color and clarity under Epinephrine
uum over silica gel for 24 hours: the residue so obtained Injection.
responds to Identification test A under Lidocaine. Bacterial Endotoxins Test (85)—It contains not more
B: It responds to the /dentification test under Dextrose. aan 0.7 USP Endotoxin Unit per mg of lidocaine hydrochlo-
Bacterial Endotoxins Test (85)—It contains not more ride.
than 1.1 USP Endotoxin Units per mg of lidocaine hydro- pH (791): between 3.3 and 5.5.
chloride. Other requirements—lt responds to the /dentification test
pH (791): between 3.0 and 7.0. under Lidocaine Hydrochloride Injection. It meets also the re-
Other requirements—It meets the requirements under /n- quirements under Injections and Implanted Drug Products (1).
jections and Implanted Drug Products (1). Assay for lidocaine hydrochloride—
Assay for lidocaine hydrochloride—Proceed with Injec- Mobile phase—Mix 50 mL of glacial acetic acid and
tion as directed in the Assay for lidocaine hydrochloride under 930 mL of water, and adjust with 1 N sodium hydroxide to
Lidocaine and Epinephrine Injection. a pH of 3.40. Mix about 4 volumes of this solution with 1
volume of acetonitrile, so that the retention time of lido-
caine is about 4 to 6 minutes. Pass through a membrane
 2418 Lidocaine / Official Monographs
filter having a 1-um or finer porosity, and degas. Make ad-
justments if necessary (see System Suitability under Chroma-
tography (621)).
Standard preparation—Dissolve about 85 mg of USP Lido-
caine RS, accurately weighed, with warming if necessary, in
0.5 mL of 1 N hydrochloric acid in a 50-mL volumetric flask,
dilute with Mobile phase to volume, and mix to obtain a
Standard preparation having a known concentration of about
1.7 mg of lidocaine per mL.
Assay preparation—Transfer an accurately measured vol-
ume of Injection, equivalent to about 100 mg of lidocaine
hydrochloride, to a 50-mL volumetric flask, dilute with Mo-
bile phase to volume, and mix.
Resolution preparation—Prepare a solution of methylpara-
ben in Mobile phase containing about 220 ug per mL. Mix
2 mL of this solution and 20 mL of the Standard preparation.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 254-nm detector
and a 3.9-mm x 30-cm column that contains packing L1.
The flow rate is about 1.5 mL per minute. Chromatograph
about 20 wL of the Resolution preparation, and record the
peas responses as directed for Procedure: the resolution, R,
tween lidocaine and methylparaben is not less than 3.0.
Chromatograph the Standard preparation, and record the
peak responses as directed for Procedure: the relative stan-
dard deviation for replicate injections is not more than
1.5%
Procedure—Separately inject equal volumes (about 20 jL)
of the Assay preparation and the Standard preparation into
the chromatograph. Record the chromatograms, and meas-
ure the responses for the major peaks. Calculate the quan-
tity, in mg, of lidocaine hydrochloride (Cy4H22N20- HCl) in
each mL of the Injection taken by the formula:
(270.80/234.34)(50)(C/V)(ru/ rs)
<a
ww
in which 270.80 and 234,34 are the molecular weights of
a lidocaine hydrochloride and lidocaine, respectively; C is the
oS
—
concentration, in mg per mL, of USP Lidocaine RS in the
2} Standard preparation; V is the volume, in mL, of Injection
9 taken; and ry and rs are the lidocaine peak responses ob-
= tained from the Assay preparation and the Standard prepara-
iS tion, respectively.
= USP Prilocaine Hydrochloride RS
a
Assay for epinephrine—
” Mobile phase—Mix 50 mL of glacial acetic acid and
=} 930 mL of water, and adjust with 1 N sodium hydroxide to
a pH of 3.40. Dissolve 1.1 g of sodium 1-heptanesulfonate
in this solution, add 1.0 mL of 0.1 M edetate disodium, and
mix. Mix about 9 volumes of this solution with 1 volume of
methanol, so that the retention time of epinephrine is about
4 to 6 minutes. Pass through a membrane filter having a
1-1um or finer porosity, and degas.
Standard preparation—Dissolve an accurately weighed
quantity of USP Epinephrine Bitartrate RS in Mobile phase to
obtain a solution having a known concentration of about
9 wg of epinephrine bitartrate per mL. Pipet 10 mL of this
solution into a 50-mL volumetric flask, dilute with Mobile
phase to volume, and mix to obtain a Standard preparation
having a known concentration of about 1.8 11g of epineph-
rine bitartrate per mL.
Assay preparation—Transfer an accurately measured vol-
ume of Injection, equivalent to about 50 yg of epinephrine,
to a 50-mL volumetric flask, dilute with Mobile phase to vol-
ume, and mix.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is fitted with a 3.9-mm x 30-cm stain-
USP 41
less steel column that contains packing L1 and is equipped
with an electrochemical detector held at a potential of +650
mV, a controller capable of regulating the background cur-
rent, and a suitable recorder. The flow rate is about 1 mL
per minute. Chromatograph the Standard preparation as di-
rected for Procedure: the relative standard deviation of the
peak responses of successive injections of the Standard prep-
aration is not more than 1.5%.
Procedure—Separately inject equal volumes (about 20 wL)
of the Assay preparation and the Standard preparation into
the chromatograph by means of a suitable microsyringe or
sampling valve, adjusting the specimen size and other oper-
ating parameters so that satisfactory chromatography and
peak responses are obtained. Record the chromatograms,
and measure the responses for the major peaks. Calculate
the quantity, in jug, of epinephrine (CysH;3NO3) in each mL
of the Injection taken by the formula:
(183.20/333.29)(50)(C/V)(ru/ rs)
in which 183.20 and 333.29 are the molecular weights of
epinephrine and epinephrine bitartrate, respectively; C is the
concentration, in ug per mL, of USP Epinephrine Bitartrate
RS in the Standard preparation; V is the volume, in mL, of
Injection taken; and ry and rs are the peak responses ob-
tained from the Assay preparation and the Standard prepara-
tion, respectively.
Lidocaine and Prilocaine Cream
» Lidocaine and Prilocaine Cream contains not
less than 90.0 percent and not more than
110.0 percent of the labeled amounts of lido-
caine (Ci4H22N2O) and prilocaine (Ci3H20N20).
Packaging and storage—Preserve in collapsible tubes or
in tight containers. Do not store above 30°. Do not freeze.
USP Reference standards (11)—
USP Lidocaine RS
USP Prilocaine Related Compound B RS
(RS)-N-(4-Methylphenyl)-2-(propylamino)propanamide.
CisH2N20 220.31
Identification—The retention times of the major peaks in
the chromatogram of the Assay preparation correspond to
those in the chromatogram of the Standard preparation, as
obtained in the Assay.
Microbial enumeration tests (61) and Tests for speci-
fied microorganisms (62)—It meets the requirements of
the tests for absence of Siapliy orcas aureus and Pseudo-
monas aeruginosa. The total aerobic microbial count does
not exceed 100 cfu per g, and the total combined molds
and yeasts count does not exceed 50 cfu per g.
Minimum fill (755): meets the requirements.
pH (791): between 8.7 and 9.7, determined in a solution
(1 in 10) or in the undiluted Cream.
Related compounds—
Solution A, Solution B, Mobile phase, System suitability solu-
tion, and Chromatographic system—Proceed as directed i
the Assay.
USP 41 Official Monographs / Lidocaine 2419

Table 1
Relative Relative
Retention Response
Related Compound Time? Factor (A) Limit
o-Toluidine 0.38 2.3 (Pye not more than 2.0%
n-Chloroacetyl-2,6-xylidine 0.54 1.0 (Le not more than 0.1%
2,6-Dimethylaniline 0.67 3.3 (Ls not more than 0.1%
Prilocaine 1.00 _ —
2-Diethylaminoaceto-2,4-xylidine 1.33 0.8 (L)< not more than 0.1%
Lidocaine 2.14 — _—
n-Dichloroacetyl-2,6-xylidine 2.98 2.2 (Le not more than 0.1%
Any other individual related compounds _ 1.0 (Pe not more than 0.2%
Total related compounds, excluding — - not more than 1.0%
o-toluidine
? Relative to the prilocaine peak.
©P designates a prilocaine related compound.
L designatesa lidocaine related compound.

sodium hydroxide to a pH of 7.20 + 0.02. Dilute with aceto-

Standard solution—Dissolve accurately weighed quantities
of USP Lidocaine RS and USP Prilocaine Hydrochloride RS in
Solution A, and dilute quantitatively, and stepwise if neces-
sary, with Solution A to obtain a solution having a known
concentration of about 0.002 mg per mL of each com-
pound. Immediately store this solution at or below 10°.
Test solution—Use the Assay preparation, prepared as di-
rected in the Assay.
Chromatographic system (see Chromatography (621))—
Proceed as rected in the ASSey: Chromatograph the System
suitability solution, and record the peak responses as directed
for Procedure: the relative retention times are listed in Table
1; and the resolution, R, between prilocaine and prilocaine
related compoundBis not less than 1.4. Chromatograph
the Standard solution a minimum of six times, and record
the peak responses as directed for Procedure: the relative
standard deviation for replicate injections is not more than
5.0%.
Procedure—Separately inject equal volumes (about 50 pL)
of the Standard solution and the Test solution into the chro-
matograph, record the chromatograms, and measure the
peak responses. Calculate the percentage of each related
gompause in the portion of the Cream taken by the
formula:
100C(ru/ r5)(V/W)(100/L)(1/F)\(220.31/256.77)
nitrile to 1 L.
Solution B—Dissolve about 2.73 g of monobasic potas-
sium phosphate in 900 mL of water, and adjust with 5 N
sodium hydroxide to a pH of 7.20 + 0.02. Dilute with aceto-
nitrile to 1 L.
Mobile phase—Use variable mixtures of filtered and
degassed Solution A and Solution B as directed for Chromato-
graphic system. Make adjustments if necessary (see System
Suitability under Chromatography (621)).
Standard preparation—Dissolve accurately weaned quan-
tities of USP Lidocaine RS and USP Prilocaine Hydrochloride
RS in Solution A, and dilute quantitatively, and stepwise if
necessary, with Solution A to obtain a solution having a
known concentration of about 0.2 mg per mL of each com-
pound. Immediately store this solution at or below 10°.
System suitability solution—Dissolve an accurately weighed cS
a
quantity of USP Prilocaine Related CompoundBRS in the as]
Standard preparation, and dilute quantitatively, and stepwise
if necessary, with the Standard preparation, to obtain a solu- c
i}
tion having a known concentration of about 0.08 mg per 5.
mL of prilocaine related compound B. )
Assay preparation—Transfer a portion of the Cream,
©4
i)
equivalent to about 20 mg each of lidocaine and prilocaine, Gs}
accurately weighed, to a 100-mL volumetric flask. Add 5 mL a
of 5 N sodium hydroxide to disperse the Cream, and mix. a)

Add 5 mL of 5 N hydrochloric acid, and dilute with Solution

in whichCis the individual concentration, in mg per mL, of A to volume, and mix. Pass a portion through a nylon filter
either USP Lidocaine RS or USP Prilocaine Hydrochloride RS having a 0.2-1m or finer porosity, discarding the first 1 mL,
in the Standard solution; ry is the individual peak response of and use the filtrate. Immediately store this solution at or
the impurities obtained from the Test solution; rs is the indi- below 10°.
vidual peak response for either lidocaine or prilocaine ob- Chromatographic system (see Chromatography (621))—The
tained from the Standard solution; V is the volume, in mL, of liquid chromatograph is equipped with a 232-nm detector
the Test solution; W is the weight, in m9 of the Cream and _a 4.6-mm x 10-cm column that contains 3-4m packing
taken to prepare the Test solution; L is the individual label L1. The flow rate is about 1.5 mL per minute. The column
claim, in percent, for either lidocaine or prilocaine; F is the temperature is maintained at 40°. The samples are main-
relative response factor for each related compound as listed tained at or below 10°. The chromatograph is programmed
in Table 1; and 220.31 and 256.77 are the molecular as follows.
weign’s of prilocaine and prilocaine hydrochloride, respec-
tively (these are used only for calculation involving prilo-
Time Solution A Solution B
caine related compounds). The percentages of lidocaine re-
(minutes) (%) (%) Elution
lated compounds and prilocaine related compounds are
calculated using the concentration and peak response from 0 67 33 equilibration
USP Lidocaine RS and USP Prilocaine Hydrochloride RS, re- 0-11.0 67 33 isocratic
spectively. The designation of whether an impurity is a lido- 11.0-22.0 67-100 330 linear gradient
caine related compound or prilocaine related compound is 22.0-32.0 100 0 isocratic
specified in Table 1. The percentage of any individual un-
known related compound is determined using the concen- Chromatograph the System suitability solution, and record
tration and peak response from USP Prilocaine Hydrochlo- the peak responses as directed for Procedure: the relative
ride RS in the Standard solution. retention times are 1.00 for prilocaine, 1.09 for prilocaine
Assay— related compound B, and 2.14 for lidocaine; and the resolu-
Solution A—Dissolve about 2.73 g of monobasic potas- tion, R, between prilocaine and prilocaine related compound
sium phosphate in 630 mL of water, and adjust with 5 N B is not less than 1.4. Chromatograph the Standard prepara-
 2420 Lidocaine / Official Monographs
tion a minimum of five times, and record the peak re-
sponses as directed for Procedure: the column efficiency is
not less than 5000 theoretical plates, based on the prilo-
caine peak; the tailing factor is not more than 1.5, based on
the prilocaine peak; and the relative standard deviation for
replicate injections is not more than 2.0%.
Procedure—Separately inject equal volumes (about 50 uL)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
ure theresponses for the lidocaine and prilocaine peaks.
Calculate the percentage of the label claim of lidocaine
(Cy4H22N20) and pilocaing (Ci3H20N20) in the portion of
Cream taken by the formula:
100C(ry
/ rs)(V/W)(100/L)(220.31/256.77)
in whichC is the individual concentration, in mg per mL, of
either USP Lidocaine RS or USP Prilocaine Hydrochloride RS
in the Standard preparation; ry and rs are either the individ-
ual peak responses of lidocaine or prilocaine obtained from
the Assay preparation and the Standard preparation, respec-
tively; Vis the volume, in mL, of the Assay preparation; W is
the weight, in mg, of the Cream taken to prepare the Assay
preparation; L is the individual label claim, in percent, for
either lidocaine or prilocaine; and 220.31 and 256.77 are
the molecular weights of prilocaine and prilocaine hydro-
chloride, respectively (these are used only for calculating the
percentage of prilocaine in the Cream).
Lime
caO 56.08
Calcium oxide [1305-78-8].
i
ww
DEFINITION
5 Lime, when freshly ignited to constant weight, contains NLT
ic]
— 95.0% of lime (CaO).
D Lincomycin Hydrochloride in Water for Injection
°
iS IDENTIFICATION
S oA.
= Analysis: Moisten a suitable quantity of Lime with
[om water: heat is generated, and a white powder is ob-
”n tained (calcium hydroxide or slaked lime). Mix the pow-
=) der with 3 or 4 times its weight of water.
Acceptance criteria: A smooth magma of lime forms
that is alkaline to litmus.
e B. IDENTIFICATION TESTS—GENERAL, Calcium (191)
Sample solution: Slake 1 g with 20 mL of water, and
add 6N acetic acid until the lime is dissolved.
Acceptance criteria: Meets the requirements
ASSAY
© PROCEDURE
Sample solution: Ignite 1g of Lime in a muffle furnace
to constant weight. Cool, weigh accurately, and dis-
solve in 20 mL of 3 N hydrochloric acid. Cool the solu-
tion, transfer to a 500-mL volumetric flask with the aid
of water, and dilute with water to volume.
Analysis: Transfer 50.0 mL to a suitable container, add
100 mL of water, 15 mL of 1 N sodium hydroxide, and
300 mg of hydroxy naphthol blue. Titrate with 0.05 M
edetate disodium VS until the solution is deep blue in
color. Each mL of 0.05 M edetate disodium is equiva-
lent to 2.804 mg of lime (CaO).
Acceptance criteria: NLT 95.0%
IMPURITIES
© INSOLUBLE SUBSTANCES
Sample: 5.0g
Analysis: Slake the Sample, then mix with 100 mL of
water, followed By hydrochloric acid, dropwise, with
agitation, until solution takes place: the resulting solu-
USP 41
tion after boiling and cooling is acid. Filter the solution
through a tared crucible, wash with water until free of
chlorides, and dry at 105° for 1 h.
Acceptance criteria: NMT 50 mg (1.0%) of insoluble
substances
© MAGNESIUM AND ALKALI SALTS
Sample solution: Dissolve 500 mg in 30 mL of water
and 15 mL of 3 N hydrochloric acid. Neutralize the so-
lution with 6 N ammonium hydroxide, heat to boiling,
and add ammonium oxalate TS to precipitate the cal-
cium completely. Heat the mixture on a steam bath for
1h. Cool, dilute with water to 100 mL, mix, and filter.
Analysis: To 50 mL of the filtrate add 0.5 mL of sulfuric
acid, evaporate to dryness, and ignite in a tared plati-
num crucible to constant weight.
Acceptance criteria: The weight of the residue does
not exceed 9 mg.
e CARBONATE
Sample: 1g
Analysis: Slake the Sample, mix with 50 mL of water,
and decant the greater portion of the milky liquid.
Acceptance criteria: The addition of an excess of 3N
hydrochloric acid to the residue does not cause more
thanaslight effervescence.
SPECIFIC TESTS
e LOSS ON IGNITION (733)
Analysis: Ignite a portion to constant weight in a tared
platinum crucible at 1100 + 50°.
Acceptance criteria: NMT 10.0%
ADDITIONAL REQUIREMENTS
° PACKAGING AND STORAGE: Preserve in tight containers.
Lincomycin Injection
» Lincomycin Injection contains an amount of
equivalent to not less than 90.0 percent and not
more than 120.0 percent of the labeled amount
of lincomycin (CisH34Nz O65). It contains benzyl
alcohol as a preservative.
Packaging and storage—Preserve in single-dose or in
multiple-dose containers, preferably of Type | glass.
Change to read:
USP Reference standards (1 1)—
@ (CN 1-May-2078)
USP Lincomycin Hydrochloride RS
Bacterial Endotoxins Test (85)—It contains not more
than 0.5 USP Endotoxin Unit per mg of lincomycin.
Sterility Tests (71)—It meets the requirements when
tested as directed for Membrane Filtration under Test for Ste-
rility of the Product to be Examined.
pH (791): between 3.0 and 5.5.
Particulate Matter in Injections (788): meets the re-
quirements for small-volume injections.
Other requirements—It meets the requirements under In-
jections and Implanted Drug Products (1).
Assay—
Mobile phase, Standard preparation, and Chromatographic
system—Proceed as directed in the Assay under Lincomycin
Hydrochloride.
Assay preparation—Transfer an accurately measured vol-
ume of Injection, equivalent to about 600 mg of lincomycin,
to a 50-mL volumetric flask, dilute with Mobile phase to vol-
USP 41 Official Monographs / Lincomycin 2421

ume, and mix. Transfer 2.0 mL of this solution to a 25-mL

volumetric flask, dilute with Mobile phase to volume, and
mix. Lincomycin Hydrochloride
Procedure—Proceed as directed for Procedure in the Assay ie
CH,
under Lincomycin Hydrochloride. Calculate the quantity, in Hc,\ Neng HOH
mg, of lincomycin (CigH34N2O6S) in each mL of the Injection ED iT CN CH, @ HCl © HO
taken by the formula: ie
H 8 HO} 9! SH

‘OH )
0.625(CP/V)(ru/ rs)
‘SCH,
OH
in which V is the volume, in mL, of Injection taken, and the
other terms are as defined therein.
CisH34N206S - HCl: H20 461.01
D-erythro-o.-D-galacto-Octopyranoside, methyl 6,8-dideoxy-6-
{a ceed Eee prreudiny econ Marine ts
thio-, monohydrochloride, monohydrate, (25-trans)-.
Methyl 6,8-dideoxy-6-(1-methy!-trans-4-propyl-L-
Lincomycin Oral Solution 2-pyrrolidinecarboxamido)-1-thio-D-erythro-o.-D-galacto-
eae monohydrochloride monohydrate
» Lincomycin Oral Solution contains an amount [7179-49-9].
of lincomycin hydrochloride (CigH34N20¢S - HCI- Anhydrous 443.01 [859-18-7].
H20) equivalent to not less than 90.0 percent » Lincomycin Hydrochloride has a potency equiv-
and not more than 120.0 percent of the labeled alent to not less than 790 yg of lincomycin
amount of lincomycin (CigH34N20¢S), and one or
(CisH34N206S) per mg.
more suitable colors, flavors, preservatives, and
sweeteners in water. Packaging and storage—Preserve in tight containers.
Labeling—Where it is intended for use in preparing inject-
Packaging and storage—Preserve in tight containers. able dosage forms, the label states that it is sterile or must
USP Reference standards (11)— be subjected to further processing during the preparation of
USP Lincomycin Hydrochloride RS injectable dosage forms.
Uniformity of dosage units (905)—
FOR ORAL SOLUTION PACKAGED IN SINGLE-UNIT CONTAINERS: Change to read:
meets the requirements.
Deliverable volume (698): meets the requirements. USP Reference standards (11)—
pH (791): between 3 and 5.5. @ (CN 1-May-2018)
Assay— USP Lincomycin Hydrochloride RS (ss
a)
Mobile phase, Standard preparation, and Chromatographic Identification, Infrared Absorption (197M). a]
system—Proceed as directed in the Assay under Lincomycin Specific rotation (7815S): between +135° and +150°. E
Hydrochloride. Test solution: 20mg per mL, in water. 3
Assay preparation—Transfer an accurately measured vol- 2
Crystallinity (695): |meets the requirements. )
ume of Oral Solution, freshly mixed and free of air bubbles,
PH (791): between 3.0 and 5.5, in a solution (1 in 10). to)
=
equivalent to about 100 mg of lincomycin, to a suitable i}
container. Add 0.5 mL of sodium carbonate solution (3 in Water Determination, Method | (921): between 3.0% no)
10), and swirl for 30 seconds, noting that a precipitate and 6.0%. =
a
forms. Add 1.0 mL of 0.2 N sodium hydroxide, swirl for Limit of lincomycin B—Use the chromatogram obtained
30 seconds, add 10.0 mL of chloroform, and shake by me- from the Assay preparation in the Assay: the area of the
chanical means for 10 minutes. Centrifuge, and remove the lincomycin B peak is not greater than 5.0% of the sum of
upper aqueous layer by suction, Transfer 1.0 mL of the clear the areas of the lincomycin B peak and the lincomycin peak.
chloroform layer to a suitable container, and evaporate Other requirements—Where the label states that Linco-
under a stream of nitrogen to dryness. Add 10.0 mL of Mo- mycin Hydrochloride is sterile, it meets the requirements for
bile phase to the residue, and dissolve by swirling, sonicating Sterility and Bacterial endotoxins under Lincomycin Injection.
if necessary. Where the label states that Lincomycin Hydrochloride must
Procedure—Proceed as directed in the Assay under Linco- be subjected to further processing during the preparation of
mycin Hydrochloride, recording the chromatogram over a pe- injectable dosage forms, it meets the requirements for Bacte-
riod seven times the retention time of lincomycin. Calculate rial endotoxins under Lincomycin Injection.
the quantity, in mg, of lincomycin (CisH34N206S) in each Assay—
mL of the Oral Solution taken by the formula:
Mobile phase—Add 13.5 mL of phosphoric acid to
1000 mL of water, and adjust with ammonium hydroxide to
(CP/ 10V)(ru / rs) a pH of 6.0. Prepareafiltered and degassed mixture of this
in which V is the volume, in mL, of Oral Solution taken; and solution, acetonitrile, and methanol (780:150:150). Make
the other terms are as defined therein. adjustments if necessary (see System Suitability under Chro-
matography (621)).
Standard preparation—Dissolve an accurately weighed
quantity of USP Lincomycin Hydrochloride RS in Mobile
phase to obtain a solution having a known concentration of
about 1.2 mg per mL, using sonication if necessary to effect
solution.
Assay preparation—To about 12 mg of Lincomycin Hydro-
chloride, accurately weighed, add 10.0 mL of Mobile phase.
Shake by mechanical means for 5 minutes, and sonicate if
necessary to effect solution.
 2422 Lincomycin / Official Monographs
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 210-nm detector
and a 4.6-mm x 25-cm column that contains 5-um packing
L7 and is maintained at a temperature of 46°. The flow rate
is about 1 mL per minute. Chromatograph the Standard
preparation, and record the responses as directed for Proce-
dure: the tailing factor for the main lincomycin peak is not
more than 1.3; the column efficiency determined from the
main lincomycin peak is not less than 4000 theoretical
plates; and the relative standard deviation for replicate injec-
tions is not more than 2.0%.
Procedure—Separately inject equal volumes (about 20 uL)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
ure the areas for the major peaks. The relative retention
times are about 0.5 for lincomycin B and 1.0 for lincomycin.
Calculate the quantity, in jg, of lincomycin (CisH3aN2O6S) in
pack mg of the Lincomycin Hydrochloride taken by the
ormula:
10(CP
/ W)(ru/ rs)
in which C is the concentration, in mg per mL, of USP
Lincomycin Hydrochloride RS in the Standard preparation; P
is the designated potency, in tg of lincomycin per mg, of
USP Lincomycin Hydrochloride RS; W is the weight, in mg,
of the portion of Lincomycin Hydrochloride taken to prepare
the Assay preparation; and ry and rs are the lincomycin peak
responses obtained from the Assay preparation and the Stan-
dard preparation, respectively.
Lincomycin Hydrochloride Capsules
” » Lincomycin Hydrochloride Capsules contain an
= amount of CisH34N206S - HCI - HzO equivalent to
a Labeling—Label it to indicate that it is for veterinary use
i
- not less than 90.0 percent and not more than
Dd 120.0 percent of the labeled amount of linco-
i}
= mycin (CigH34N20¢S).
Sj
= Packaging and storage—Preserve in tight containers.
ian USP Reference standards (11)—
al
=) USP Lincomycin Hydrochloride RS
Dissolution (711)—
Medium: water; 500 mL.
Apparatus 1: 100 rpm.
Time: 45 minutes.
Procedure—Filter a portion of about 20 mL of the solution
under test. Transfer about 5 mL of the eluant into a small
test tube, and add 250 uL of 0.01 M sodium sulfate internal
standard solution. Evaporate until dry using a vacuum cen-
trifuge. Add 10.0 uL of water to the precipitate and place
on a vortex mixer until all solid material is dissolved. Trans-
fer this solution to a capillary tube, place it in a Raman
spectrometer, and obtain the Raman spectrum using suita-
ble instrumental conditions . Integrate the Raman intensity,
applying baseline corrections, between 660 cm"! and
720 cm". Divide this result by the integrated intensity be-
tween 966 cm and 994 cm-'. Determine the amount of
CisH34N20¢S dissolved in comparison with an aqueous Stan-
dard solution having a known concentration of USP Linco-
mycin Hydrochloride RS.
Tolerances—Not less than 75% (Q) of the labeled amount
of CigH34N20¢S is dissolved in 45 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Water Determination, Method | (921): not more than
7.0%.
USP 41
Assay—
Mobile phase, Standard preparation, and Chromatographic
system—Proceed as directed in the Assay under Lincomycin
Hydrochloride.
Assay preparation—Remove, as completely as possible,
the contents of not less than 10 Capsules, taking care to
prevent capsule shell fragments from being combined with
the eapsile contents and to remove any shell fragments
from the contents. Weigh and mix the combined contents,
and transfer an accurately weighed portion of the powder,
equivalent to about 50 mg of lincomycin, to a suitable con-
tainer. Add 50.0 mL of Mobile pes. and shake by mechani-
cal means for 5 minutes. Use the solution thus obtained as
the Assay preparation.
Procedure—Proceed as directed for Procedure in the Assay
under Lincomycin Hydrochloride. Calculate the quantity, in
mg, of lincomycin (GrattauNeO,5) in the portion of Capsule
contents taken by the formula:
(CP
/ 20)(ru/ rs)
in which the terms are as defined therein.
Lincomycin Hydrochloride Soluble
Powder
» Lincomycin Hydrochloride Soluble Powder con-
tains an amount of Lincomycin Hydrochloride
equivalent to not less than 90.0 percent and not
more than 110.0 percent of the labeled amount
of lincomycin (CisH34N206S).
Packaging and storage—Preserve in tight containers.
only.
USP Reference standards (11)—
USP Lincomycin Hydrochloride RS
Identification—The retention time of the major peak in
the chromatogram of the Assay preparation corresponds to
that in the chromatogram of the Standard preparation, as
obtained in the Assay.
Minimum fill (755): |meets the requirements.
Water Determination, Method | (921): not more than
6.0%.
Assay—
Mobile phase and Chromatographic system—Proceed as di-
rected in the Assay under Lincomycin Hydrochloride.
Standard preparation—Dissolve an accurately weighed
quantity of USP Lincomycin Hydrochloride RS in Mobile
phase to obtain a solution having a known concentration of
about 1.2 mg per mL.
Assay preparation—Remove as completely as possible the
contents of not fewer than 5 containers. Weigh and mix the
combined contents, and transfer an accurately weighed por-
tion of the Soluble Powder, equivalent to about 400 mg of
lincomycin (CigH34N20¢S), to a 100-mL volumetric flask. Add
about 80 mL of Mobile phase, and swirl to dissolve. Dilute
with Mobile phase to volume, and mix. Transfer 25.0 mL of
this solution to a second 100-mL volumetric flask, dilute
with Mobile phase to volume, and mix.
Procedure—Proceed as directed in the Assay under Linco-
mycin Hydrochloride. Calculate the quantity, in mg, of linco-
USP 41
mycin (CisH3gN2O0¢S) in the portion of Soluble Powder taken
by the formula:
0.4CP(ru / rs)
in which the terms are as defined therein.
Lindane
a of the Standard preparation and the Assay preparation into
Cl AL -el
ek
a
CeHeCle 290.83
Cyclohexane, 1,2,3,4,5,6-hexachloro-, (10,201, 3B,401,50,6B)-.
y-1,2,3,4,5,6-Hexachlorocyclohexane [58-89-9].
» Lindane is the gamma isomer of hexachloro-
cyclohexane. It contains not less than 99.0 per-
cent and not more than 101.0 percent of lindane
(y-CeHeCle).
Packaging and storage—Preserve in well-closed contain-
ers.
USP Reference standards (11)—
USP Lindane RS
Identification, Infrared Absorption (197K).
Congealing temperature (651): not less than 112.0°.
Water Determination, Method | (921): not more than
0.5%.
Chloride ion—Place about 100 mg ina test tube with
10 mL of water, shake, and filter. Add 1 mL of nitric acid
and 3 mL of silver nitrate TS to the filtrate: no turbidity de-
velops.
Assay—
Internal standard solution—Dissolve n-octadecane in
methylene chloride to obtain a solution having a concentra-
tion of about 0.5 mg per mL.
Standard preparation—Dissolve an accurately weighed
quantity of USP Lindane RS in Internal standard solution to
obtain a solution having a known concentration of about
2™mg per mL.
System suitability solution—Prepare solutions of a-benzene
hexachlorides (BHC) at 1000 yg per mL of methanol, B-BHC
at 1000 ug per mL of acetone, and 8-BHC at 1000 1g per
mL of methanol. Transfer 100 iL each of o-BHC, B-BHC and
5-BHC solutions to a 4-mL conical vial, and evaporate under
a stream of nitrogen to dryness. Add to the vial a 100-uL
aliquot of the Standard preparation. Insert the stopper, and
shake vigorously to dissolve the residue.
Assay preparation—Transfer about 10 mg of Lindane, ac-
curately weighed, to a 5-mL volumetric flask. Dissolve in
and dilute with Internal standard solution to volume.
Chromatographic system (see Chromatography (621))—The
gas chromatograph is equipped with a flame-ionization de-
tector and a 0.32-mmx30-m fused-silica column coated
with a 1-um phase G46. The chromatograph is pro-
grammed as follows. The initial column temperature is
maintained at 120° for 1 minute. Then, the temperature is
increased at a rate of 20° per minute to 150°, and then
ramped at a rate of 10° per minute to 280° and maintained
at that temperature for 4 minutes. The injectionport and
detector temperatures are maintained at 300°. The injection
split ratio is 50:1. Chromatograph the Standard preparation
and the System suitability solution, and record the peak re-
sponses as directed for Procedure: the relative retention
times for n-octadecane and lindane are about 0.85 and 1.0,
Official Monographs / Lindane 2423
respectively. [NOTE—Typical retention times for a-BHC, B-
BHC, y-BHC, 5-BHC, and n-octadecane are 15.7, 17.8, 16.5,
18.8, and 13.9 minutes, respectively.] The resolution, R, be-
tween n-octadecane and «-BHC is not less than 21, be-
tween lindane (y-BHC) and a-BHC is not less than 9, be-
tween B-BHC and lindane is not less than 14, and between
8-BHC and B-BHC is not less than 8; the tailing factors for n-
octadecane and lindane are less than 1.5 and 1.2, respec-
tively; and the relative standard deviation of the ratios of
peak area responses of lindane to n-octadecane for replicate
injections of Standard preparation is not more than 1.5%.
Procedure—Separately inject equal volumes (about 1 pL)
the chromatograph, record the chromatograms, and meas-
ure the responses for the major peaks. Calculate the quan-
tity, in mg, of y-CsHeCle in the portion of Lindane taken by
the formula:
5C(Ru/ Rs)
in which C is the concentration, in mg per mL, of USP
Lindane RS in the Standard preparation; and Ru and Rs are
the ratios of the peak responses of lindane to n-octadecane,
obtained from the Assay preparation and the Standard prepa-
ration, respectively.
Lindane Cream
DEFINITION
Lindane Cream is Lindane in a suitable cream base. It con-
tains NLT 90.0% and NMT 110.0% of the labeled amount
of lindane (y-CeHeCle).
IDENTIFICATION
eA. (=
Analysis: Winda strip of 20-mesh copper gauze 1.5 cm vA)
~~
wide and 5 cm long around the end of a copper wire.
Heat the gauze in the nonluminous flame of a Bunsen =
burner until it glows without coloring the flame green. °
po]
Allow the gauze to cool, and repeat the heating and °
cooling step several times until a thorough coating of sco)
oxide is formed. Apply a small amount of Cream to the =
2
cooled gauze, ignite, and allow to burn freely in the air. mo}
Hold the gauze in the outer edge of the burner flame a
vy
at a height of 4 cm.
Acceptance criteria: A bright green color is imparted
to the flame.
ASSAY
© PROCEDURE
Mobile phase: Mix 18 mL of anhydrous ethyl ether
with 280 mL of chromatographic hexane.
Internal standard solution: 1 mg/mL of n-docosane in
methylene chloride
Standard stock solution: 2mg/mL of USP Lindane RS
in methylene chloride
Standard solution: Transfer 5.0 mL of Standard stock
solution to a graduated centrifuge tube, add 5.0 mL of
Internal standard solution, and evaporate with the aid of
gentle heat anda current of dry air to 3 mL. Avoid
svaporating to dryness. If the mixture is inadvertently
evaporated to dryness, discard it, and begin another
Standard solution.
Solid support: 60- to 100-mesh magnesium silicate
that has been heated previously at 300° for 2h
Sample stock solution: Nominally 2 mg/mL of lindane
from a quantity of Cream, equivalent to 10 mg of
lindane, prepared as follows. Place a pledget of cotton
on a removable porous plate at the base of a 25-mm x
200-mm chromatographic tube fitted with a polytef
stopcock. Add 50 mL of Mobile phase and 10 g of Solid
support, and stir the mixture to expel air bubbles. Add
 2424 Lindane / Official Monographs
1.5 g of anhydrous sodium sulfate to the column, and
elute until the surface of the liquid is 4 cm above the
Solid support, discarding the eluate. Transfer a portion of
Cream to a 150-mL beaker, and add 10g of Solid sup-
port. Mix with a spatula, adding chromatographic hex-
ane as necessary to produce a homogeneous mixture,
and continue stirring until a free-flowing powder is pro-
duced. Transfer this mixture to the chromatographic
column with the aid of three 5-mL portions of Mobile
phase, and elute the column with 225 mL of the Mobile
phase at a flow rate of 2-3 mL/min, collecting the elu-
ate in a 250-mL beaker. Remove the chromatographic
column, add 5.0 mL of Internal standard solution to the
eluate, and evaporate with the aid of gentle heat and a
current of dry air to 5 mL.
Sample solution: Transfer the Sample stock solution to a
graduated centrifuge tube with the aid of 1 mL of
methylene chloride, and evaporate with the aid of gen-
tle heat and a current of dry air to 3 mL. Avoid evapo-
rating to dryness. If the mixture is inadvertently evapo-
rated to dryness, discard it, and begin another Sample
solution.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC
Detector: Flame ionization
Column: 1.8-m x 2-mm glass; packed with 3% liquid
phase G3 on support S1A
Temperatures
Column: 195°
Injection port: 250°
Detector: 250°
Flow rate: 40 mL/min
Injection volume: 1 wl
Carrier gas: Dry nitrogen
System suitability
Sample: Standard solution (6-10 replicate injections)
as
al
Suitability requirements
a Resolution: NLT 5 between lindane and n-docosane
i]
—
Tailing factor: NMT 2.0
D Relative standard deviation: NMT 3.0%
i} Analysis
iJ
S Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
= lindane (y-CsH6Cle) in the portion of Cream taken:
3 Analysis: Wind a 1.5-cm x 5-cm strip of 20-mesh cop-
al
= Result = (Ru/Rs) x (Cs/Cy) x 100
Ry = peak response ratio of lindane to n-docosane
in the Sample solution
Rs = peak response ratio of lindane to n-docosane
in the Standard solution
Cs concentration of USP Lindane RS in the
I
Standard stock solution (mg/mL)
Cu = nominal concentration of lindane in the
Sample stock solution (mg/mL)
USP 41
Acceptance criteria: 90.0%-110.0%
SPECIFIC TESTS
PH (791)
Sample solution: 1-in-5 dilution
Acceptance criteria: 8.0-9.0
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers.
e USP REFERENCE STANDARDS (11)
USP Lindane RS
Lindane Lotion
» Lindane Lotion is Lindane in a suitable aqueous
vehicle. It contains not less than 90.0 percent
and not more than 110.0 percent of the labeled
amount of lindane (y-CéHeCle).
Packaging and storage—Preserve in tight containers.
USP Reference standards (11)—
USP Lindane RS
Identification—It responds to the Identification test under
Lindane Cream.
pH (791): between 6.5 and 8.5.
Assay—Proceed as directed in the Assay under Lindane
Cream, substituting “Lotion” for “Cream” throughout.
Lindane Shampoo
DEFINITION
Lindane Shampoo is Lindane in a suitable vehicle. It con-
tains NLT 90.0% and NMT 110.0% of the labeled amount
of lindane (y-CeH6Cle).
IDENTIFICATION
oA
per gauze around the end of a copper wire. Heat the
gauze in the nonluminous flame of a Bunsen burner
until it glows, without coloring the flame green. Allow
the gauze to cool, and repeat the heating and cooling
step several times until a thorough coating of oxide is
formed. Apply a small amount of Shampoo to the
cooled gauze, ignite, and allow to burn freely in the air.
Hold the gauze in the outer edge of the burner flame
at a height of 4.cm.
Acceptance criteria: A bright green color is imparted
to the flame.
ASSAY
© PROCEDURE
Mobile phase: Anhydrous ethyl ether and chromato-
graphic solvent hexane (18:280)
Internal standard solution: 1 mg/mL of n-docosane in
methylene chloride
Standard stock solution: 2 mg/mL of USP Lindane RS
in methylene chloride
Standard solution: Transfer 5.0 mL of Standard stock
solution to a graduated centrifuge tube, add 5.0 mL of
Internal standard solution, and evaporate with the aid of
gentle heat and a current of dry air to 3 mL. Avoid
evaporating to dryness. If the mixture is inadvertently
evaporated to dryness, discard it, and begin another
Standard solution.
Solid support: Use 60- to 100-mesh magnesium silicate
that has been previously heated at 300° for 2 h.
USP 41 Official Monographs / Linezolid 2425

Sample stock solution: Nominally 2 mg/mL of lindane Acceptance criteria: 90.0%-110.0%

from a quantity of Shampoo, equivalent to 10 mg of
lindane, prepared as follows. Place a pledget of cotton SPECIFIC TESTS
on a removable porous plate at the base of a 25-mm x e PH (791): 6.2-7.0
200-mm chromatographic column that is fitted with a
polytef stopcock. Add 50 mL of Mobile phase and 10g ADDITIONAL REQUIREMENTS
of Solid support, and stir the mixture to expel air bub- © PACKAGING AND STORAGE: Preserve in tight containers.
bles. Add 1.5 g of anhydrous sodium sulfate to the col- © USP REFERENCE STANDARDS (11)
umn, and elute until the surface of the liquid is 4 cm USP Lindane RS
above Solid support, discarding the eluate. Transfer a
weighed portion of Shampoo, corresponding to 10 mg
of lindane, to a 150-mL beaker, and add 10g of Solid
support. Mix with a spatula, adding chromatographic
solvent hexane as necessary to produce a homogeneous Linezolid
mixture, and continue stirring until a free-flowing pow-
der is produced. Transfer this mixture to the chromato-
graphic column with the aid of three 5-mL portions of
Mobile phase. Elute the column with 225 mL of Mobile
phase at a flow rate of 2-3 mL/min, collecting the elu-
ate in a 250-mL beaker. Remove the chromatographic
column, add 5.0 mL of Internal standard solution to the
eluate, and evaporate with the aid of gentle heat and a
current of dry air to 5 mL. Ci6H20FN3O4 337.35
Sample solution: Transfer the Sample stock solution to a Acetamide, N-[[3-[3-fluoro-4-(4-morpholinyl)phenyl]-2-oxo-
graduated centrifuge tube with the aid of 1 mL of 5-oxazolidinyl]methyl]-, (5S)-;
methylene chloride, and evaporate with the aid of gen- N-[[(S)-3-(3-Fluoro-4-morpholinophenyl)-2-oxo-5-ox-
tle heat and a current of dry air to 3 mL. Avoid evapo- azolidinyl]methyl]acetamide [165800-03-3].
rating to dryness. If the mixture is inadvertently evapo-
rated to dryness, discard it, and begin another Sample DEFINITION
solution. Linezolid contains NLT 98.0% and NMT 102.0% of linezolid
Chromatographic system (Ci6H20FN3O,), calculated on the anhydrous, solvent-free
(See Chromatography (621), System Suitability.) basis.
Mode: GC
Detector: Flame ionization IDENTIFICATION
Column: 1.8-m x 2-mm glass; packed with 3% liquid e A. INFRARED ABSORPTION (197K): If a difference appears
phase G3 on support S1A in the IR spectra of the analyte and the Standard, dis-
Temperatures solve equal portions of the test specimen and the USP
Injector: 250° Reference Standard in equal volumes of methanol. Evap- ¢
Detector: 250° orate the solutions to dryness and perform the test on 4)
the residues. ~~
Column: 195°
Carrier gas: Dry nitrogen e B. The retention time of the major peak of the Sample x
Flow rate: 40 mL/min solution corresponds to that of the Standard solution, as °
obtained in the Assay. =]
Injection volume: 1 uL °
System suitability io}
Sample: Standard solution ASSAY =
oy
Suitability requirements © PROCEDURE me)
Resolution: NLT 5 between lindane and n-docosane Buffer: 1.4 g/L of monobasic potassium phosphate a
Solution A: Methanol, acetonitrile, and Buffer (15:5:80) 7)
Tailing factor: NMT 2.0
Relative standard deviation: NMT 3.0% for 6-10 Solution B: Methanol and Buffer (50:50)
replicate injections Mobile phase: See Table 7.
Analysis
Samples: Standard solution and Sample solution Table 1
Calculate the percentage of the labeled amount of Solution A Solution B
lindane (y-CeHeCle) in the portion of Shampoo taken:
Result = (Ru/Rs) x (Cs/Cu) x 100

Ru = peak response ratio of lindane to n-docosane

from the Sample solution
Rs = peak response ratio of lindane to n-docosane
from the Standard solution Return to original conditions, and equilibrate the system
Gs = concentration of USP Lindane RS in the for 5 min.
Standard stock solution (mg/mL) Diluent: Acetonitrile and water (35:65)
Cu = nominal concentration of lindane in the System suitability solution: 50 g/mL each of USP
Sample stock solution (mg/mL) Linezolid RS and USPLinezolidRelated Compound D RS
in Diluent
Standard solution: 0.08 mg/mL of USP Linezolid RS in
Diluent
Sample solution: 0.08 mg/mL of Linezolid in Diluent
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
 2426 Linezolid / Official Monographs USP 41

Column: 4.6-mm x 7.5-cm; 3-4m packing L1 Table 2

Temperatures Relative Acceptance
Autosampler: 15° Retention Criteria,
Column: 30° Name Time NMT (%)
Flow rate: 1.5 mL/min
Injection volume: 10 uL Linezolid N-oxide« 0.20 0.3
System suitability Linezolid related compound C
Samples: System suitability solution and Standard (linezolid amine)» 0.31 0.2
solution Desfluoro linezolid (if present)«4 0.63 0.3
[Note—The relative retention times for linezolid and Linezolid 1.0 =
linezolid related compound D are about 1.0 and 1.4, Any individual unspecified a.
respectively.] impurity 0.1
Suitability requirements a(S)-4-{4-[5-(Acetamidomethyl)-2-oxooxazolidin-3-yl]-2-
Resolution: NLT 3.0 between linezolid and linezolid fluorophenyl}morpholine 4-oxide.
related compound D, System suitability solution ’(S)-5-(Aminomethyl)-3-(3-fluoro-4-morpholinophenyl)oxazolidin-2-one.
Tailing factor: NMT 1.5, Standard solution ©(5)-N-{[3-(4-Morpholinophenyl)-2-oxooxazolidin-5-yl]methyl}acetamide.
Relative standard deviation: NMT 1.0%, Standard 4If possible from the manufacturing process.
solution
Analysis © ENANTIOMERIC PURITY
Samples: Standard solution and Sample solution Mobile phase: Hexane, absolute alcohol, and trifluoroa-
Calculate the percentage of linezolid (CisHaoFN3O«) in cetic acid (650:350:1)
the portion of Linezolid taken: System suitability solution: 25 g/mL each of USP
Linezolid RS and USP Linezolid R-Isomer RS in absolute
Result = (ru/rs) x (Cs/Cu) x 100 alcohol
Sample solution: 0.5 mg/mL of Linezolid in absolute
tu = peak response from the Sample solution alcohol. Sonicate as needed to dissolve.
rs peak response from the Standard solution Chromatographic system
oul

Gs concentration of USP Linezolid RS in the (See Chromatography (621), System Suitability.)

Standard solution (mg/mL) Mode: LC
Cu = concentration of Linezolid in the Sample Detector: UV 254 nm
solution (mg/mL) Column: 4.6-mm x 15-cm; 5-wm packing L51
Acceptance criteria: 98.0%-102.0% on the anhydrous, Flow rate: 1.5 mL/min
solvent-free basis Injection volume: 20 pL
Run time: NLT 2.8 times the retention time of
IMPURITIES linezolid
e RESIDUE ON IGNITION (281): NMT 0.2% System suitability
e ORGANIC IMPURITIES Sample: System suitability solution
Buffer, Solution A, Solution B, Mobile phase, Diluent, [NoTte—The relative retention times for linezolid R-iso-
i
”

is System suitability solution, and Chromatographic mer and linezolid are 0.84 and 1.0, respectively.]
3 system: Proceed as directed in the Assay. _ : Suitability requirements
—
Dp Standard solution: 0.0008 mg/mL of USP Linezolid RS Resolution: NLT 1.5 between linezolid R-isomer and
i) in Diluent linezolid
< Sample solution: 0.8 mg/mL of Linezolid in Diluent Analysis
Sj System suitability Sample: Sample solution
= Samples: System suitability solution and Standard Calculate the percentage of linezolid R-isomer in the
[3 solution portion of Linezolid taken:
” [Note—The relative retention times for linezolid and
=) linezolid related compound D are about 1.0 and 1.4, Result = (ru/r7) x 100
respectively.]
Suitability requirements ty = peak response of linezolid R-isomer from the
Resolution: NLT 3.0 between linezolid and linezolid Sample solution
related compound D, System suitability solution la = sum of the peak responses of both
Tailing factor: NMT 1.5, Standard solution enantiomers from the Sample solution
Relative standard deviation: NMT 5.0%, Standard Acceptance criteria: NMT 0.3%
solution
Analysis SPECIFIC TESTS
Samples: Standard solution and Sample solution e@ WATER DETERMINATION (921), Method |, Method Ic) NMT
Calculate the percentage of each impurity in the por- 0.5%
tion of Linezolid taken:
ADDITIONAL REQUIREMENTS
Result = (ru/rs) x (Cs/Cu) x 100 © PACKAGING AND STORAGE: Preserve in tight containers.
Store at controlled room abe
tu = peak response of each impurity from the ¢ USP REFERENCE STANDARDS (11
Sample solution USP Linezolid RS
Is = peak response of linezolid from the Standard USP Linezolid Related Fceeeung DRS
solution (R)-[3-(3-Fluoro-4-morpholinopheny!)-2-oxooxazolidin-
Cs = concentration of USP Linezolid RS in the 5-yl]methyl methanesulfonate.
Standard solution (mg/mL) CisHisFN2O06S_ 374.38
Cy = concentration of Linezolid in the Sample USP Linezolid R-lsomer RS
solution (mg/mL) N-{[(R)-3-(3-Fluoro-4-morpholinophenyl)-2-oxo-
Acceptance criteria: See Table 2. The reporting thresh- 5-oxazolidinyl]methyl}acetamide.
old is 0.05%. Ci6HaoFN304, 337.35
USP 41
Liothyronine Sodium
He ee,a WR
;ee
AALS th aR So” Nat
| Suitability requirements
CisHiilsNNaOg 672.96
L-Tyrosine, O-(4-hydroxy-3-iodophenyl)-3,5-diiodo-,
monosodium salt;
Monosodium L-3-[4-(4-hydroxy-3-iodophenoxy)-3,5-
diiodophenylJalanine [55-06-1].
DEFINITION
Liothyronine Sodium is the sodium salt of L-3,3’,5-triiodothy-
ronine. It contains NLT 95.0% and NMT 101.0% of
liothyronine sodium (CisHiilsNNaO,), calculated on the
dried basis.
IDENTIFICATION
eA.
Diluent: Solution of hydrochloric acid in 80% alcohol
(1 in 50)
Sample solution: 0.1-mg/mL solution in Diluent
Acceptance criteria: The UV absorption spectrum of
the Sample solution exhibits maxima at the same wave-
lengths as those of a similar solution of USP
Liothyronine RS, concomitantly measured; and the re-
spective absorptivities, both calculated on the dried ba-
sis in terms of the acid at the wavelength of maximum
absorbance at about 297 nm, do not differ by more
than 5.0%.
° B.
Analysis: Heat 50 mg with a few drops of sulfuric acid
in a porcelain crucible.
Acceptance criteria: Violet vapors of iodine are
evolved.
© C. IDENTIFICATION TESTS—GENERAL (191), Sodium: The res-
idue from the ignition of Liothyronine Sodium meets the
requirements.
e D. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
ASSAY
¢ PROCEDURE
Mobile phase: Mixture of acetonitrile and water (4:6)
that contains 0.5 mL of phosphoric acid in each liter of
the mixture
Solution A: Dissolve 400 mg of sodium hydroxide in
500 mL of water. Cool, and add 500 mL of methanol.
Levothyroxine stock solution: 0.4 mg/mL of USP Levo-
thyroxine RS in Solution A. Make a 1:100 dilution of this
solution using Mobile phase.
Liothyronine stock solution: 0.4 mg/mL of USP
Liothyronine RS in Solution A
Standard solution: 10 g/mL of liothyronine from
Liothyronine stock solution and 0.5 g/mL of levothyrox-
ine from Levothyroxine stock solution, in Mobile phase
Sample solution: 10 g/mL of Liothyronine Sodium in
Mobile phase
[Note—A small amount of 0.01 M methanolic sodium
hydroxide can be used to facilitate the dissolution of
the sample.]
Chromatographic system
(See Chromatography (621), System Suitability.)
Official Monographs / Liothyronine 2427
Mode: LC
Detector: UV 225 nm
Column: 4.6-mm x 25-cm; packing L10
Flow rate: 1.5 mL/min
Injection volume: 100 nL
System suitability
Sample: Standard solution
Resolution: NLT 5.0 between levothyroxine and
liothyronine
Relative standard deviation: NMT 2.0% for
liothyronine
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of liothyronine sodium
ae in the portion of Liothyronine Sodium
taken:
Result = (ru/rs) x (Cs/Cu) X (Mr1/Mr2) x 100
tu = peak response of liothyronine from the Sample
solution
rs = peak response of liothyronine from the
Standard solution
Cs = concentration of USP Liothyronine RS in the
Standard solution (g/mL)
Cu = concentration of Liothyronine Sodium in the
Sample solution ual mL)
M, = molecular weight of liothyronine sodium,
672.96
Mz = molecular weight of liothyronine, 650.97
Acceptance criteria: 95.0%-101.0% on the dried basis
IMPURITIES
e CHLORIDE CONTENT
Sample solution: Transfer 100 mg of ae So-
lun preslasly dried, into a platinum dish. Ignite over
a low flame, protecting the dish from air currents dur- Cc
ing the ignition. When carbonization is complete, cool “
the dish, add 2 drops of water, and break up the char- co]
red mass thoroughly witha stirring rod. Add 10 mL of =
water and 5 mL of ammonium hydroxide, and mix. °
Transfer the slurry to a glass-stoppered, 50-mL flask, |
and wash the platinum dish and the stirring rod with °
io}
water, adding the washings to the flask, until the vol- =
i)
ume of the solution is about 25 mL. Add 10 mL of silver me]
nitrate solution (1 in 20), shake thoroughly, and filter a
through a retentive paper into a 50-mL color-compari- al
son tube. Wash the flask and the filter paper with
10 mL of water, and add the washings to the tube.
Acidify the combined filtrate and washings to litmus
with nitric acid, and dilute with water to 50 mL.
Control solution: Mix 5 mL of ammonium hydroxide,
20 mL of water, and 10 mL of silver nitrate solution (1
in 20). Filter the mixture through a retentive paper into
a 50-mL color-comparison tube, and then wash the fil-
ter paper with 10 mL of water into the tube. Acidify the
contents of the tube to litmus with nitric acid, and di-
lute with water to 50 mL.
Sodium chloride solution: 1-mg/mL solution of sodium
chloride in water
Analysis: Add the Sodium chloride solution in 0.1-mL in-
crements to the Contro/ solution until the turbidity of
the Control solution matches that of the Sample solution.
Acceptance criteria: NMT 2.0 mL of Sodium chloride so-
lution is required (1.2%).
¢ LIMIT OF LEVOTHYROXINE SODIUM
Mobile phase, Levothyroxine stock solution, Standard
solution, Sample solution, Chromatographic system,
and System suitability: Proceed as directed in the
Assay.
Levothyroxine standard solution: 0.5 g/mL of levo-
thyroxine from Levothyroxine stock solution, in Mobile
phase
 2428 Liothyronine / Official Monographs
Analysis
Samples: Levothyroxine standard solution and Sample
solution
Calculate the percentage of levothyroxine sodium
fee elsralanle) in the portion of Liothyronine Sodium
taken:
Result = (ru/rs) x (Cs/Cu) x (Mr/M,2) X 100
tu = peak response of levothyroxine from the
Sample solution
Is = peak response of levothyroxine from the
Levothyroxine standard solution
Cs = concentration of USP Levothyroxine RS in the
Levothyroxine standard solution (g/mL)
Cu = concentration of Liothyronine Sodium in the
Sample solution (ug/ml)
Ma = molecular weight of levothyroxine sodium,
798.85
Mz = molecular weight of levothyroxine, 776.87
Acceptance criteria: NMT 5.0% of levothyroxine
sodium
SPECIFIC TESTS
e SODIUM CONTENT
Analysis: Transfer 100 mg, previously dried, into a plati-
num dish. Add 8-10 drops of sulfuric acid, and ignite to
constant weight, taking care to avoid spattering. Each
ne residue is equivalent to 0.324 mg of sodium
a).
Correct the result for the quantity of sodium equivalent
to the sodium chloride (NaCl) found in the test for
Chloride Content.
Acceptance criteria: 2.9%-4.0%
© OPTICAL ROTATION (7815S), Procedures, Specific Rotation
Diluent: A mixture of alcohol and 1.2 N hydrochloric
acid (4:1)
2 Sample solution: 20 mg/mL in Diluent
ed Acceptance criteria: +18° to +22°
a e Loss ON DRYING (731)
J
— Analysis: Dry at 105° for 2 h.
im)
f-) Acceptance criteria: NMT 4.0%
~ and levothyroxine is not less than 3.0. Chromatograph the
3 ADDITIONAL REQUIREMENTS
= e PACKAGING AND STORAGE: Preserve in tight containers.
a e USP REFERENCE STANDARDS (11)
a) USP Levothyroxine RS
= USP Liothyronine RS
Liothyronine Sodium Tablets
» Liothyronine Sodium Tablets contain an
amount of CisHiilsNNaOsz equivalent to not less
than 90.0 percent and not more than 110.0 per-
cent of the labeled amount of liothyronine
(CisHi2lsNOs).
Packaging and storage—Preserve in tight containers.
USP Reference standards (11)—
USP Levothyroxine RS
USP Liothyronine RS
Identification—The retention time of the major peak in
the chromatogram of the Assay preparation corresponds to
the liothyronine peak in the chromatogram of the Standard
preparation, as obtained in the Assay.
Dissolution (711)—[NOTE—AIl containers that are in con-
tact with solutions containing liothyronine sodium are to be
made of glass.]
USP 41
Medium: pH 10.0 + 0.05 alkaline borate buffer (see
Buffer Solutions in the section Reagents, Indicators, and Solu-
tions); 250 mL.
Apparatus 3: 30calig per minute, using 20-mesh screen
on the top and 40-mesh screen on the bottom of the glass
reciprocating cylinder.
Time: _ 45 minutes.
Determine the amount of liothyronine sodium
(CisHi2lsNO«) dissolved by employing the following method.
Ammoniated solution—Add 0.05 mL of ammonium hy-
droxide to 200 mL of water.
Mobile phase—Prepare a filtered and degassed mixture of
water and acetonitrile (55:45) that contains 1 mL of phos-
phoric acid in each 1000 mL of solution. Make adjustments
‘6 vo (see System Suitability under Chromatography
Standard solution—Dissolve an accurately weighed quan-
tity of USP Liothyronine RS in Ammoniated solution, and di-
lute quantitatively, and stepwise if necesorl) with Ammoni-
ated solution to obtain a solution having a known
concentration of about 10 wg of USP Liothyronine RS per
mL. Dilute a portion of this solution quantitatively, and step-
wise if necessary, with water to obtain a solution having a
known concentration of about 0.5 ug of USP Liothyronine
RS per mL.
Test solution—Transfer 20 mL of the solution under test to
a centrifuge tube, and centrifuge until a clear supernatant is
obtained.
Resolution solution—Prepare a solution of USP
Liothyronine RS and USP Levothyroxine RS in Ammoniated
solution having known concentrations of about 10 pg of
each USP Reference Standard per mL. Dilute with water to
obtain a concentration of about 0.5 1g of each USP Refer-
ence Standard per mL.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 225-nm detector
and a 4.6-mm x 25-cm column that contains packing L10.
The flow rate is about 2 mL per minute. Chromatograph the
Resolution solution, and record the peak responses as di-
rected for Procedure: the resolution, R, between liothyronine
Standard solution, and record the peak responses as directed
for Procedure: the relativestands’ deviation for replicate
injections is not more than 4.0%.
Procedure—Separately inject equal volumes (about
200 uL) of the Standard solution and the Test solution into
the chromatograph, record the chromatograms, and meas-
ure the responses for the major peaks. Calculate the amount
of CisHi2lsNOs dissolved.
Tolerances—Not less than 70% (Q) of the labeled amount
of CisHialsNOz is dissolved in 45 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Assay—
Mobile phase, Standard preparation, and Chromatographic
system—Proceed as directed in the Assay under Liothyronine
odium.
Assay preparation—Weigh and finely powder not fewer
than 20 Tablets. Transfer an accurately weighed portion of
the powder, equivalent to about 100 ug of liothyronine so-
dium, to a centrifuge tube, add 2 glass beads, pipet 10 mL
of Mobile phase into the tube, and mix using a vortex mixer
for 3 minutes. Centrifuge to obtain a clear supernatant, fil-
tering if necessary.
Procedure—Proceed as directed in the Assay under
Liothyronine Sodium. Calculate the quantity, in ug, of
USP 41
liothyronine (C;sHi2l3 NOx) in the portion of Tablets taken
by the formula:
10C(ru/ rs)
in which C is the concentration, in ug per mL, of USP
Liothyronine RS in the Standard preparation; and ry and rs
are the liothyronine peak responses obtained from the Assay
preparation and the Standard preparation, respectively.
Liotrix Tablets
» Liotrix Tablets contain not less than 90.0 per-
cent and not more than 110.0 percent of the la-
beled amounts of Levothyroxine Sodium
(CisHiolaNNaO.) and Liothyronine Sodium
(CisHiilsNNaQO,z) in a ratio by weight of 4 to 1,
respectively.
Packaging and storage—Preserve in tight containers.
USP Reference standards (11)—
USP Levothyroxine RS
USP Liothyronine RS
Identification—The retention time of the two major peaks
in the chromatogram of the Assay preparation corresponds
to the levothyroxine and liothyronine peaks in the chromat-
ogram of the Standard preparation, as obtained in the Assay.
Disintegration (701): 30 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Assay—
Mobile phase—Prepare a filtered and degassed mixture of
water and acetonitrile (65:35) that contains 2 mL of trifluor-
oacetic acid in each 1000 mL of solution. Make adjustments
if necessary (see System Suitability under Chromatography
(621)).
0.01 M Methanolic sodium hydroxide—Dissolve 400 mg of
sodium hydroxide in 500 mL of water. Cool, add 500 mL of
methanol, and mix.
Levothyroxine stock solution—Dissolve an accurately
weighed quantity of USP Levothyroxine RS in 0.07 M Meth-
anolic sodium hydroxide to obtain a solution having a known
concentration of about 0.4 mg of levothyroxine per mL.
Liothyronine stock solution—Dissolve an accurately
weighed quantity of USP Liothyronine RS in 0.07 M Metha-
nolic sodium hydroxide to obtain a solution having a known
concentration of about 0.4 mg of eis A ae per mL. Make
a 1:10 dilution of this solution using Mobile phase.
Standard preparation—Transfer appropriate volumes of Le-
vothyroxine stock solution and Liothyronine stock solution to a
suitable container, and dilute quantitatively and stepwise, if
necessary, with Mobile phase to obtain a solution having
known concentrations of about 10 jg of levothyroxine per
mL and 2.5 wg of liothyronine per mL.
Assay preparation—Transfer 20 Tablets to a 200-mL volu-
metric flask, add 180 mL of Mobile phase, and sonicate for
15 minutes, occasionally swirling the flask to accelerate the
disintegration of the Tablets. Cool to room temperature,
and dilute with Mobile phase to volume. Transfer a portion
of the solution to a centrifuge tube, and centrifuge for
10 minutes at 5000 rpm. Quantitatively dilute a portion of
the clear supernatant with Mobile phase to obtain concen-
trations of about 10.0 ug of levothyroxine sodium per mL
and 2.5 yg of liothyronine sodium per mL.
Chromatographic system—Proceed as directed in the As-
say under Levothyroxine Sodium.
Official Monographs / Lipid 2429
Procedure—Proceed as directed for Procedure in the Assay
under Levothyroxine Sodium. Calculate the quantity, in yg, of
levothyroxine sodium (CisHiolaNNaQOa) in the portion of Tab-
lets taken by the formula:
(798.86/776.87)(10O(ru
/ rs)
in which 798.86 and 776.87 are the molecular weights of
levothyroxine sodium and levothyroxine, respectively; C is
the concentration, in ug per mL, of USP Levothyroxine RS in
the Standard preparation; and ry and rs are the levothyroxine
peak responses obtained from the Assay preparation and the
Standard preparation, respectively.
Calculate the quantity, in ug of liothyronine sodium
(CisHijlsNNaO,) in the portion of Tablets taken by the
formula:
(672.96/650.98)(10O(ru/ rs)
in which 672.96 and 650.98 are the molecular weights of
liothyronine sodium and liothyronine, respectively; C is the
concentration, in ug per mL, of USP Liothyronine RS in the
Standard preparation; and ry and rs are the liothyronine peak
responses obtained from the Assay preparation and the Stan-
dard preparation, respectively.
Lipid Injectable Emulsion
» Lipid Injectable Emulsion used in total paren-
teral nutrition is a sterile 10 (0.10 g per mL), 20
(0.20 g per mL), or 30 (0.30g per mL) percent
w/v emulsion in an aqueous vehicle. The aque-
ous phase contains 0.6 percent to 1.8 percent
w/v parenteral Egg Phospholipids in Water for In- [=
4)
jection and contains, if necessary, an osmotic uv
agent, such as glycerin in amounts of 1.7 percent =
to 2.5 percent w/v, or a suitable stabilizer, such i}
=]
as a fatty acid salt. The most frequently used oil i}
present is Soybean Oil, which provides an ample
ro}=
i
supply of the essential fatty acids: linoleic aci be}
2
and linolenic acid. Other oils, such as Safflower a)
Oil, Medium-Chain Triglycerides, Olive Oil, Fish
Oil, or other suitable oils, can be mixed with
Soybean Oil. Hence, Soybean Oil can be the only
oil or be part of a mixture of these other oils. It
contains not less than 90.0 percent and not more
than 110.0 percent of the labeled amount of the
total oil(s). It contains no antimicrobial agents.
The final products are terminally sterilized.
Packaging and storage—Preserve in an appropriate con-
tainer (see Packaging and Storage Requirements (659), Injec-
tion Packaging). Use elastomeric closures that are compatible
with both the oil and water phases of the Emulsion. Store at
a temperature not below 4° (protect from freezing) or
above 30° (protect from excessive heat).
Labeling—The label states the identity and the quantities
of the specific oils in the Emulsion. The label states the total
osmolar concentration (or osmolarity) in mOsm per L. The
labeling provides the following information: do not use if
there is evidence of excessive creaming or aggregation, if
excessive free oil droplets are visible, or if there are other
indications of compromised integrity, such as microbial
growth, present in the product.
 2430 Lipid / Official Monographs
Delete the following:
°USP Reference standards (11)—
USP Endotoxin RS
@ (CN 1-May-2018)
Fatty acid composition—Transfer a volume of the Emul-
sion, equivalent to about 200 mg of lipids, to a stoppered
extraction vessel, add 10 mL of ether, and mix. Add 5 g of
anhydrous sodium sulfate, mix, and allow the mixture to
stand until separation of the layers is complete. Wet the
packing of a chromatographic silica cartridge with a few mL
of ether, transfer about 5 mL of the ether layer from the
extraction vessel to the column reservoir, and elute at a rate
of between 5 and 10 drops per minute into a suitable ves-
sel. Evaporate the ether from the eluant, and dissolve the
residue in 5.0 mL of toluene. Transfer 1.0 mL of the toluene
solution to a reaction vial, and add 0.4 mL of (m-trifluoro-
methylphenyl) trimethylammonium hydroxide in methanol.
Cover, mix, and allow to stand for 30 minutes. Inject about
1 uL of this solution into a gas chromatograph equipped
with a 0.53-mm x 50-m wide-bore, fused-silica capillary col-
umn coated with a 2.0-um thickness of liquid phase G16
and maintained at a temperature of 200°. The column is
connected to a flame-ionization detector. Helium is used as
the carrier gas at a flow rate of about 10 mL per minute.
Measure the main peak areas of the methyl esters of the
fatty acids. The relative peak areas expressed as a percent-
age of the main peaks are in the known ranges for the oil
(e.g., Soybean Oil, USP; Safflower Oil, USP) as specified on
the label. For oil mixtures, analysis of each oil should be
performed to identify known peaks prior to emulsification as
specified on the label.
Bacterial Endotoxins Test (85)—It contains not more
than 0.5 USP Endotoxin Unit per mL.
pH (791): between 6.0 and 9.0.
Globule size limits—The Injectable Emulsion meets the re-
ee
”
quirements of the limits specified in both Method | and
a Method II as directed under Globule Size Distribution in Lipid
i]
— Injectable Emulsions (729).
Dd
° Limit of oil droplet mean diameters (See Method I—Light
= Scattering Method under Globule Size Distribution in Lipid In-
S jectable Emulsions (729))—Using the method of light scatter-
= ing, determine the mean droplet diameter (MDD): the sam-
rs ple meets the requirements. The intensity-weighted mean
a2) droplet diameter (MDD) for the Injectable Emulsion must be
=) <500 nm, or 0.5 jum, irrespective of the concentration of
the dispersed lipid phase.
Limit of large globule volume-diameter (See Method |i—
Light Obscuration or Extinction Method under Globule Size
Distribution in Lipid Injectable Emulsions (729))—Using the
method of light obscuration, determine the size distribution
of globules in the large-diameter tail of the dispersion (de-
tection threshold 22.0 um). Calculate the volume-weighted
mass of lipid in the form of globules with diameters in ex-
cess of 5.0 um per 100 mL of the Injectable Emulsion. The
volume-weighted, large-diameter fat globule limits of the
dispersed phase, expressed as the percentage of fat residing
in globules larger than 5 um (PFATS) for a given Injectable
Emulsion, is not to exceed 0.05%.
Limit of free fatty acid—
Solvent—Prepare a mixture of heptane, isopropanol, and
water (400:400:200) in a separatory funnel. Allow the
phases to separate, and discard the lower phase. Filter the
upper phase (heptane solution) through 40 g of anhydrous
sodium sulfate. Store in a tightly capped glass container,
and use within 1 week.
Chromatographic column—Prepare a slurry of heptane and
chromatographic silica gel having an average pore size of 6
nm, and activate at a temperature of 110° for not less than
USP 41
1 hour prior to use. Transfer the slurry to a 2.3-cm chromat-
ographic tube (see Column Chromatography under Chroma-
tography (621)), and pack to a bed height of between 5 cm
and 6 cm. Wash the column with about 40 mL of heptane,
and drain the heptane through the column to a level of
about 0.5 cm above the silica gel bed.
Procedure—Transfer 20.0 mL of the Injectable Emulsion to
a flask, freeze, and lyophilize. Dissolve the residue in 30 mL
of Solvent, and transfer the solution to the column. Rinse
the flask with three 30-mL portions of Solvent, and transfer
the washings to the column, allowing each rinsing to drain
to the 4 of the column bed before applying the next
rinse. Collect a total of 120 mL of effluent. Add 10 drops of
phenolphthalein TS to the effluent, bubble nitrogen through
the solution, and titrate with 0.02 N alcoholic potassium
hydroxide VS until the solution remains pale pink after mix-
ing for 10 seconds. Titrate a blank using 120 mL of Solvent.
Calculate the quantity, in mEq, of free fatty acids per g of
oil in the Injectable Emulsion using the formula:
(Vu — Va)N
/20C
in which Vy is the volume, in mL, of 0.02 N alcoholic potas-
sium hydroxide consumed by the eluant; Vs is the volume,
in mL, of 0.02 N alcoholic potassium hydroxide consumed
by the blank; N is the normality of the 0.02 N alcoholic
potassium hydroxide; andCis the labeled concentration, in
g per mL, of the total oil(s) in the Injectable Emulsion: not
more than 0.07 mEq of free fatty acids per g of oil is found.
Other requirements—It meets the requirements under /n-
jections and Implanted Drug Products (1).
Assay—
Mobile phase—Preparea filtered and degassed mixture of
isopropanol, ethyl acetate, and glacial acetic acid
(179:20:1).
Standard preparation—Dissolve an accurately weighed
portion of Soybean Oil (or other relevant oils used in the
Emulsion) in Mobile phase to obtain a solution having a
known concentration of about 8 mg per mL.
AssayPicparaner ,Nausicr an accurately measured por-
tion of Emulsion, equivalent to about 800 mg of oil, to a
100-mL valaretie ask with the aid of additional portions
of Mobile phase. Dilute with Mobile phase to volume, and
mix to obtain a solution containing about 8 mg of oil per
mL.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a refractive index
detector and a 4.1-mm x 25-cm column that contains pack-
ing L21. The flow rate is about 1 mL per minute, adjusted
so that the peak due to oil elutes at about 6.5 minutes.
Chromatograph the Standard preparation, and record the
peak responses as directed for Procedure: the capacity factor,
k’, is not less than 1.0; the tailing factor for the oil peak is
not more than 2.5; and the relative standard deviation for
replicate injections is not more than 2.0%.
Procedure—Separately inject equal volumes (about 50 j1L)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
ure the peak responses. Calculate the quantity, in mg, of oil
in the portion of Emulsion taken by the formula:
100C(ru/ rs)
in whichCis the concentration, in mg per mL, of Soybean
Oil or other relevant oils used in the Emulsion in the Stan-
dard preparation; and ry and rs are the peak responses ob-
tained from the Assay preparation and the Standard prepara-
tion, respectively.
USP 41 Official Monographs / Lisinopril 2431

Cu = concentration of Lisinopril in the Sample

Lisinopril solution (mg/mL)
Acceptance criteria: 98.0%-102.0% on the anhydrous
basis
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.1%
+ 2H,0
Delete the following:

°e HEAVY METALS, Method I! (231): 0.001%e ‘omic tjan-2018)

C2 H3iN3Os - 2H20 441.52 © ORGANIC IMPURITIES
L-Proline, 1-[N2-(1-carboxy-3-phenylpropyl)-L-lysyl]-, dihy- Buffer: 3.53 g/L of monobasic sodium phosphate dihy-
drate, (S)-; drate in water adjusted with phosphoric acid to a pH of
1-[N2-[(S)-1-Carboxy-3-phenylpropyl]-t-lysyl]-L-proline dihy- 4.1
drate [83915-83-7]. Solution A: Acetonitrile and Buffer (7:193)
Solution B: Acetonitrile and Buffer (20:80)
DEFINITION Mobile phase: See Table 1.
Lisinopril contains NLT 98.0% and NMT 102.0% of lisinopril
(CaH3iN30s), calculated on the anhydrous basis. Table 1
IDENTIFICATION Solution A Solution B
e A. INFRARED ABSORPTION (197M)
e B. The retention time of the major peak of the Sample 1 0
solution corresponds to that of the Standard solution, as
obtained in the Assay. 40
ASSAY
© PROCEDURE
Solution A: 2.76 g/L of monobasic sodium phosphate Standard solution: 0.006 mg/mL of USP Lisinopril RS in
in water prepared as follows. Dissolve 2.76g of mono- Solution A
basic sodium phosphate in about 900 mL of water in a Sensitivity solution: 1.0 t1g/mL of USP Lisinopril RS in
1000-mL volumetric flask. Adjust with 1 N sodium hy- Solution A from Standard solution
droxide to a pH of 5.0 and dilute with water to volume. Sample solution: 2 mg/mL of Lisinopril in Solution A
Mobile phase: Acetonitrile and Solution A (4:96) Chromatographic system
Standard solution: 0.3 mg/mL of USP Lisinopril RS in (See Chromatography (621), System Suitability.)
water Mode: LC
Sample solution: 0.3 mg/mL of Lisinopril in water Detector: UV 210 nm (ox
Column: 4.6-mm x 25-cm; 5-um packing L7 ry
Column temperature: 45° i)
Flow rate: 1.8 mL/min E
Injection volume: 20 uL °
Chromatographic system System suitability =
°
(See Chromatography (621), System Suitability.) Samples: Standard solution and Sensitivity solution to}S
Mode: LC Suitability requirements Py
Detector: UV 210 nm Relative standard deviation: NMT 10.0%, Standard mo]
Column: 4.6-mm x 25-cm; 5-um packing L7 solution = vw
Column temperature: 50° Signal-to-noise ratio: NLT 10, Sensitivity solution
Flow rate: 1 mL/min Analysis
Injection volume: 20 uL Samples: Standard solution and Sample solution
System suitability Calculate the percentage of each impurity in the por-
Sample: Standard solution tion of Lisinopril taken:
Suitability requirements
Tailing factor: NMT 1.7 Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
Relative standard deviation: NMT 0.73% ru = peak response of each impurity from the
Analysis Sample solution
Samples: Standard solution and Sample solution peak response of lisinopril from the Standard
Calculate the percentage of lisinopril (C2H3iN3Os) in ls
solution
the portion of Lisinopril taken: Gs = concentration of USP Lisinopril RS in the
Result = (ru/rs) x (Cs/Cu) x 100 Standard solution (mg/mL)
Cu = concentration of Lisinopril in the Sample
tu = peak response of lisinopril from the Sample solution (mg/mL)
solution F = relative response factor (see Table 2)
ls = peak response of lisinopril from the Standard Acceptance criteria: See Table 2. Disregard any peak
solution less than 0.05%.
Gs = concentration of USP Lisinopril RS in the
Standard solution (mg/mL)
 2432 Lisinopril / Official Monographs USP 41

Table 2 Vehicle: a 1:1 mixture of Ora-Sweet® and Ora-

Relative Relative Acceptance Plus,» a sufficient quantity to make 100 mL
Retention | Response Criteria, 4Prinivil 10-mg tablets, Merck & Co., West Point, PA.
Name Time Factor NMT (%) >Paddock Laboratories, Minneapolis, MN.

N-Alkyl-L-lysinea 0.57 0.35 0.3 Calculate the required quantity of each ingredient for the
DlL-Homopheny- total amount to be prepared. Place the required number
lalanine> 0.72 1.08 0.30 of Lisinopril tablets in a suitable mortar, and comminute to
Lisinopril 1.00 1.00 — a fine powder. Add the Vehicle in small portions, and tritu-
Lisinopril epimer< 1233 0.76 0.3 rate to make a smooth paste. Add increasing volumes of
Lisinopril cyclohexyl
the Vehicle to makea lisinopril liquid that is pourable.
analog 2.93 0.39 0.30
Transfer the contents of the mortar, stepwise and quan-
titatively, to a calibrated bottle. Add enough of the Vehicle
R,S,5-Diketopiperazinee 3.88 079 0.3 to bring to final volume, and mix well.
5,S,S-Diketopiperazine
(lisinopril related ASSAY
compound A)f 4.04 0.76 0.3 ¢ PROCEDURE
N-Alkyl lisinoprils 4.60 0.86 0.15 Solution A: 4.1 g/L of monobasic potassium phosphate.
Any individual _ Adjust with phosphoric acid to a pH of 2.0.
unspecified impurity 1.00 0.1
Mobile phase: 1.0 g/L of sodium 1-hexanesulfonate in
acetonitrile and Solution A (18:82). Filter and degas.
Total impurities* = = 0.5 Diluent: Methanol and water (20:80)
a[(S)-1-Carboxy-3-phenylpropyl]-t-lysine. Standard solution: 0.2 mg/mL of USP Lisinopril RS in
> 2-Amino-4-phenylbutanoic acid. Diluent
©[(R)-1-Carboxy-3-phenylpropyl]-t-lysyl-L-proline. Sample solution: Shake thoroughly by hand each bot-
4[(S)-1-Carboxy-3-cyclohexylpropyl]-L-lysyl-L-proline. tle of Oral Suspension. Mix 1.0 mL of Oral Suspension
©(5)-2-[(35,8aR)-3-(4-Aminobutyl)-1,4-dioxohexahydropyrrolo[1,2- with 4.0 mL of Diluent to obtain a solution having a
a]pyrazin-2(1 H)-yl]-4-phenylbutanoic acid.
nominal concentration of 0.2 mg/mL of lisinopril.
£(S)-2-[(35,8a5)-3-(4-Aminobutyl)-1,4-dioxohexahydropyrrolo[1 ,2-
al|pyrazin-2(1 H)-yl]-4-phenylbutanoic acid. Chromatographic system
s N2,N6-Bis[(5)-1-Carboxy-3-phenylpropyl]-L-lysyl-L-proline. (See Chromatography (621), System Suitability.)
Total impurities does not include lisinopril epimer. Mode: LC
Detector: UV 215 nm
SPECIFIC TESTS Column: 4.6-mm x 25-cm; 5-um packing L7
© OPTICAL ROTATION (7815S), Specific Rotation Column temperature: 40°
Diluent: 0.25 M zinc acetate solution prepared as fol- Flow rate: 1.0 mL/min
lows. Mix 600 mL of water with 150 mL of glacial acetic Injection volume: 20 uL
acid and 54.9 g of zinc acetate, and stir to dissolve the System suitability
zinc acetate. While stirring, add 150 mL of ammonium Sample: Standard solution
ro
al
hydroxide, cool to room temperature, and adjust with [Note—The retention time for lisinopril is about 12.9
iy ammonium hydroxide to a pH of 6.4. Transfer the solu- min.]
iJ
— tion to a 1000-mL volumetric flask, and dilute with Suitability requirements
Dd water to volume. Column efficiency: NLT 1800 theoretical plates
} Sample solution: 10 mg/mL of Lisinopril in Diluent Tailing factor: NMT 2.0
5 Acceptance criteria: —115.3° to -122.5° (4 = 405 nm) Relative standard deviation: NMT 2.0% for replicate
Ps e WATER DETERMINATION (921), Method |: 8.0%-9.5% injections
a ADDITIONAL REQUIREMENTS
Analysis
a) Samples: Standard solution and Sample solution
=) e PACKAGING AND STORAGE: Preserve in well-closed Calculate the percentage of the labeled amount of lisi-
containers. nopril (C2iH3:N3Os) in the portion of Oral Suspension
e USP REFERENCE STANDARDS (11) taken:
USP Lisinopril RS
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response from the Sample solution
ig = peak response from the Standard solution
Cs = concentration of USP Lisinopril RS in the
Lisinopril Compounded Oral Suspension Standard solution (mg/mL)
Cu = nominal concentration of lisinopril in the
Sample solution (mg/mL)
DEFINITION \ Acceptance criteria: 90.0%-110.0%
Lisinopril Compounded Oral Suspension contains NLT
90.0% and NMT 110.0% of the labeled amount of lisi- SPECIFIC TESTS
nopril (C2i1H3:N3Os). e PH (791): 4.3-5.3
Prepare Lisinopril Compounded Oral Suspension 1 mg/mL
as follows (see Pharmaceutical Compounding—Nonsterile ADDITIONAL REQUIREMENTS
Preparations (795)). © PACKAGING AND STORAGE: Package in tight, light-resistant
containers. Store in a refrigerator or at controlled room
100 mg of temperature.
Lisinopril tablets? equivalent to lisinopril e BEYOND-UsE DATE: NMT 90 days after the date on which
it was compounded when stored in a refrigerator or at
aPrinivil 10-mg tablets, Merck & Co., West Point, PA. controlled room temperature
» Paddock Laboratories, Minneapolis, MN e LABELING: Label it to indicate that it is to be well shaken
before use, and to state the Beyond-Use Date.
USP 41
e USP REFERENCE STANDARDS (11)
USP Lisinopril RS
Lisinopril Tablets
» Lisinopril Tablets contain not less than 90.0 per-
cent and not more than 110.0 percent of the la-
beled amount of C2H3iN30s.
Packaging and storage—Preserve in tight containers.
USP Reference standards (11)—
USP Lisinopril RS
Identification—The retention time of the major peak in
the chromatogram of the Assay preparation corresponds to
that of the Standard preparation as obtained in the Assay.
Dissolution (711)—
Medium: 0.1 N hydrochloric acid; 900 mL.
Apparatus 2: 50 rpm.
Time: 30 minutes.
Determine the amount of lisinopril dissolved using the fol-
lowing method.
Mobile phase and Chromatographic system—Prepare as di-
rected in the Assay.
Determine the amount of lisinopril dissolved by one of
the following procedures.
PROCEDURE FOR POOLED SAMPLE—Proceed as directed for Pro-
cedure in Apparatus 1 and Apparatus 2, Immediate-Release
Dosage Forms under Dissolution (711). Combine evel
volumes of the filtered solutions of the 6 or 12 individual
specimens withdrawn, and use the pooled sample as the
test solution. Inject a volume of the pooled sample into the
chromatograph, record the chromatogram, and measure
the response for the major peak. Calculate the quantity of
C2H3iN3Os dissolved in comparison with a Standard solu-
tion having a known concentration of USP Lisinopril RS in
the same Medium and similarly chromatographed.
Tolerances—Not less than 80% (Q) of the labeled amount
of Cz1H3iN3Os in the Tablets is dissolved in 30 minutes: the
requirements are met if the quantities of active ingredient
dissolved from the pooled sample conform to the accompa-
nying Acceptance Table for a Pooled Sample. Continue testing
through the three stages unless the results conform at either
S; or So. The quantity, Q, is the amount of dissolved active
ingredient specified, expressed as a percentage of the la-
beled content.
Acceptance Table for a Pooled Sample
Number
Stage Tested Acceptance Criteria
Sy 6 Average amount dissolved is not less than Q
+ 10%.
Sz 6 Average amount dissolved (S; + S2) is equal
to or greater than Q + 5%.
S3 12 Average amount dissolved (S; + Sz + Ss) is
equal to or greater than Q.
PROCEDURE FOR UNIT SAMPLE—Proceed as directed for Proce-
dure in Apparatus 1 and Apparatus 2, Immediate-Release Dos-
age Forms under Dissolution (711). Inject a volume of a
filtered portion of the solution under test into the chromato-
raph, record the chromatogram, and measure the response
or the major peak. Calculate the amount of C21H3iN3Os dis-
solved in comparison with a Standard solution having a
known concentration of USP Lisinopril RS in the Medium and
similarly chromatographed.
Official Monographs / Lisinopril 2433
Tolerances—Not less than 80% (Q) of the labeled amount
of C2H31N3QOs is dissolved in 30 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Procedure for content uniformity—
Phosphate solution, Mobile phase, and Chromatographic
system—Prepare as directed in the Assay.
Diluent—Dissolve 2.72 g of monobasic potassium phos-
phate in 800 mL of water, adjust with phosphoric acid to a
pH of 4.0, dilute with water to 1000 mL, and mix.
Standard preparation—Dissolve an accurately weighed
quantity of USP Lisinopril RS in Diluent to obtain a solution
having a known concentration of about 0.2 mg per mL.
Test preparation—Place one Tablet in a volumetric flask of
appropriate size, based on the labeled quantity, in mg, of
lisinopril in the Tablet, to obtain a solutioncontaining
0.2 mg of lisinopril per mL. Fill the flask to about 50% vol-
ume with Diluent, sonicate for 5 minutes, and shake by me-
chanical means for 20 minutes. Dilute with Diluent to vol-
ume, mix, and filter.
Procedure—[NOTE—Use peak areas where peak responses
are indicated.] Separately inject equal volumes (about 20 pL)
of the Test preparation and the Standard preparation into the
chromatograph, record the chromatograms, and measure
the responses for the major peaks. Calculate the quantity, in
mg, of CoiH3iN3Os in the Tablet taken by the formula:
(TC/ D)(ru/ 15)
in whichTis the labeled quantity, in mg, of lisinopril in the
Tablet; C is the concentration, in mg per mL, calculated on
the anhydrous basis, of USP Lisinopril RS in the Standard
preparation; D is the concentration, in mg per mL, of lisi-
nopril in the Test preparation, based upon the labeled quan-
tity per Tablet and the extent of dilution; and ry and rs are
the lisinopril peak responses obtained from the Test prepara-
tion and the Standard preparation, respectively. tm
al
ao]
Related compounds—
Phosphate solution, Mobile phase, Diluent, and Chromato- “<
graphic system—Prepare as directed in the Assay. °
=)
Standard solution—Dilute the Standard preparation, pre- °
pared as directed in the Assay, with Diluent to obtain a solu- Ko}
BI
tion having a known concentration of about 20 ug per mL. Eo)
Test solution—Use the Assay preparation. mo]
>
Procedure—Proceed as directed in the Assay. Measure the 7
responses of the lisinopril peak obtained from the Standard
solution, and of all peaks other than that of lisinopril ob-
tained from the Test solution. Calculate the percentage of
related compounds in each Tablet taken by the formula:
100(V/ 10)(C/ L)(ru/rs)
in which V is the volume, in mL, of the Test solution; C is the
concentration, in mg pet mL, calculated on the anhydrous
basis, of USP Lisinopril RS in the Standard solution; L is the
quantity, in mg, of lisinopril in each Tablet, taken as deter-
mined in the Assay; ru is the sum of all peak responses other
than that of lisinopril obtained from the Test solution; and rs
is the lisinopril peak response obtained from the Standard
solution: not more than 2.0%, calculated on the basis of the
quantity, in mg, of lisinopril in the portion of Tablets taken,
as determined in the Assay, is found.
Assay—
Phosphate solution—Dissolve 4.1 g of monobasic potas-
sium phosphate in about 900 mL of water in a 1000-mL
volumetric flask, and adjust with phosphoric acid to a pH of
2.0. Dilute with water to volume, and mix.
Mobile phase—Dissolve 1.0 g of sodium 1-hexanesul-
fonate in 820 mL of Phosphate solution. Add 180 mL of ace-
tonitrile, mix, filter, and degas. Make adjustments if neces-
sary (see System Suitability under Chromatography (621)).
 2434 Lisinopril / Official Monographs USP 41

Diluent—Prepare a mixture of water and methanol (4:1).
Standard preparation—Dissolve an accurately weighed
quantity of USP Lisinopril RS in Diluent to obtain a solution
having a known concentration of about 0.2 mg per mL.
Assay preparation—Transfer to a suitable size volumetric
flask 10 Tablets, which when diluted with Diluent will yield a
solution having a concentration of about 0.2 mg per mL.
Add Diluent, and sonicate for 5 minutes. Shake the flask by
mechanical means for 20 minutes, dilute with Diluent to vol-
ume, mix, and filter.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 215-nm detector
and a 4.6-mm x 20-cm column that contains packing L7
and is maintained at a temperature of 40°. The flow rate is
about 1 mL per minute. Chromatograph the Standard prepa-
ration, and record the peak responses as directed for Proce-
dure: the column efficiency determined from the analyte
peak is not less than 700 theoretical plates; the tailing factor
for the analyte peak is not more than 2.0; the capacity fac-
tor, k’, for the analyte peak is greater than 1.5; and the
relative standard deviation for replicate injections is not
more than 2%.
Procedure—Separately inject equal volumes (about 20 mL)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
ure the area responses for the major peaks. Calculate the
sare in mg, of Cz:H3:N3Os in each Tablet taken by the
ormula:
(L/D)C(tu / rs)
in which L is the labeled quantity, in mg, of lisinopril in
each Tablet, D is the concentration, in mg per mL, of lisi-
nopril in the Assay preparation based on the labeled quantity
per Tablet and the extent of dilution; C is the concentration,
in mg per mL, calculated on the anhydrous basis, of USP
Lisinopril RS in the Standard preparation; and ry and rs are
c
“
Benzothiadiazine related compound A stock solu-
tion: 0.016 mg/mL of USP Benzothiadiazine Related
Compound A RS in methanol
Standard solution: 0.1 mg/mL of USP Lisinopril RS,
0.125 mg/mL of USP Hydrochlorothiazide RS, 2 g/mL
of USP Lisinopril Related Compound A RS, and 1.3 ug/
mL of USP Benzothiadiazine Related Compound A RS in
Buffer from Lisinopril standard stock solution, Hydrochloro-
thiazide standard stock solution, Lisinopril related com-
pound A stock solution, and Benzothiadiazine related com-
ound A stock solution. For Tablet strengths 20/12.5 of
isinopril/hydrochlorothiazide, the concentration of USP
Lisinopril RS and USP Lisinopril Related Compound A RS
in the Standard solution is 0.2 mg/mL and 4 ug/mL,
respectively.
Sample stock solution: Transfer 10 Tablets to a suitable
volumetric flask. Add Buffer (0.25 mL/mg of total lisi-
nopril), sonicate for 5 min, and then add methanol
(0.5 mL/mg of total lisinopril). Sonicate for an addi-
tional 10 min. Add more Buffer (0.75 mL/mg of total
lisinopril), and mix by mechanical means for 20 min.
Dilute with water to volume to prepare solutions as de-
scribed in Table 1.
Table 1
Tablet Strength of Nominal Concentration of
Lisinopril/ Lisinopril/
Hydrochlorothiazide Hydrochlorothiazide
(mg/Tablet) (mg/mL)
10/12.5 0.4/0.5
20/12.5 0.4/0.25
20/25 0.4/0.5
Sample solution: Dilute the Sample stock solution with
Buffer to prepare solutions as described in Table 2. Pass
a portion through a suitable filter of 0.45-1m pore size.

a the lisinopril peak area responses obtained from the Assay

ic preparation and the Standard preparation, respectively. Table 2
—
io) Tablet Strength of Nominal Concentration of
i) Lisinopril/ Lisinopril/
=
iS Hydrochlorothiazide Hydrochlorothiazide
P ° e
Lisinopril and Hydrochlorothiazide
° e (mg/Tablet)
TOADS
(mg/mL)
01/0.13
[5
”“ Tablets 20/12.5 0.2/0.125
=) 20/25 0.1/0.12
DEFINITION
Lisinopril and Hydrochlorothiazide Tablets contain NLT Chromatographic system
90.0% and NMT 110.0% of the labeled amount of lisi- (See Chromatography (621), System Suitability.)
nopril (C2H3:N30s) and hydrochlorothiazide Mode: LC
(C7HsCIN304Sz2). Detector: UV 215 nm
Column: 4.6-mm x 15-cm; 5-41m packing L7
IDENTIFICATION Flow rate: 1.5 mL/min
e A. The retention times of the major peaks of the Sample Injection volume: 10 uL
solution correspond to those of the Standard solution, as Run time: NLT 10 min
obtained in the Assay. System suitability
ASSAY Sample: Standard solution
Suitability requirements
© PROCEDURE
Resolution: NLT 3.0 between lisinopril and
The Tablets must be assayed within 24 h of the time benzothiadiazine related compound A and NLT 4.0
they are put in the solution. between hydrochlorothiazide and benzothiadiazine
Buffer: Dissolve 4.08 g of monobasic potassium phos- related compound A
phate in 800 mL of water. Adjust with phosphoric acid
to a pH of 2.5, and dilute with water to 1 L. Tailing factor: NMT 2 for both the lisinopril and hy-
Mobile phase: Acetonitrile, triethylamine, water, and
drochlorothiazide peaks
phosphoric acid (280:3:1480:15) Relative standard deviation: NMT 2.0% for both
the lisinopril and hydrochlorothiazide peaks
Lisinopril standard stock solution: 0.5 mg/mL of USP Analysis
Lisinopril RS in Buffer Samples: Standard solution and Sample solution
Hydrochlorothiazide standard stock solution: Calculate the percentage of the labeled amount of lisi-
3.12 mg/mL of USP Hydrochlorothiazide RS in methanol nopril (C2H3;N30s) and hydrochlorothiazide
Lisinopril related compoundA stock solution: (C7HeCIN3O,S2) in the portion of Tablets taken:
0.05 mg/mL of USP Lisinopril Related Compound A RS
in methanol Result = (ru/rs) x (Cs/Cu) x 100
USP 41 Official Monographs / Lisinopril 2435

ty = peakresponse of lisinopril or Vv = volume of Medium, 900 mL

hydrochlorothiazide from the Sample solution Tolerances: NLT 80% (Q) of the labeled amounts of
ls = peak response of lisinopril or lisinopril (C21H3iN3Os) and hydrochlorothiazide
hydrochlorothiazide from the Standard (C;HsCIN304Sz2) is dissolved.
solution Test 2: If the product complies with this test, the label-
Gs = concentration of USP Lisinopril RS or USP ing indicates that the product meets USP Dissolution
Hydrochlorothiazide RS in the Standard Test 2.
solution (mg/mL) Medium: 0.1 N hydrochloric acid; 900 mL
Cu = nominal concentration of lisinopril or Apparatus 2: 50 rpm
hydrochlorothiazide in the Sample solution Times: 30 min for both lisinopril and
(mg/mL) hydrochlorothiazide
Acceptance criteria: 90.0%-110.0% Buffer: 2.76 g/L of monobasic sodium phosphate in
water. Adjust with phosphoric acid to a pH of 3.0.
PERFORMANCE TESTS Mobile phase: Acetonitrile and Buffer (6:94)
e DISSOLUTION (711) Standard stock solution 1 (for Tablets labeled to con-
Test 1 tain 10 mg/12.5 mg or 20 mg/25 mg of lisinopril/hy-
Medium: 0.1 N hydrochloric acid; 900 mL drochlorothiazide): 0.11 mg/mL of USP Lisinopril RS
Apparatus 2: 50 rpm and 0.14 mg/mL of USP Hydrochlorothiazide RS, pre-
Time: 45 min for hydrochlorothiazide; 30 min for pared as follows. Add acetonitrile to fill 1.5% of the
lisinopril total volume, sonicate until dissolved, and dilute with
Buffer and Mobile phase: Prepare as directed in the Medium to volume.
Assay. Standard stock solution 2 (for Tablets labeled to con-
Lisinopril standard stock solution: 0.5 mg/mL of USP tain 20 mg/12.5 mg of lisinopril/hydrochlorothiazide):
Lisinopril RS in Buffer 0.22 mg/mL of USP Lisinopril RS and 0.14 mg/mL of
Hydrochlorothiazide standard stock solution: USP Hydrochlorothiazide RS, prepared as follows. Add
0.44 mg/mL of USP Hydrochlorothiazide RS in acetonitrile to fill 1.5% of the total volume, sonicate
methanol until dissolved, and dilute with Medium to volume.
Standard solution: Prepare solutions in Medium as de- Standard solution: Prepare solutions, in Medium, of
scribed in Table 3 from the Lisinopril standard stock so- lisinopril and hydrochlorothiazide as described in Table
lution and Hydrochlorothiazide standard stock solution. 4 from either Standard stock solution 1 or Standard
stock solution 2.
Table 3
Tablet Strength of Nominal Concentration of Table 4
Lisinopril/ Lisinopril/ Tablet Strength of Nominal Concentration of
Hydrochlorothiazide Hydrochlorothiazide Lisinopril/ Lisinopril/
(mg/Tablet)
_ (mg/mL) Hydrochlorothiazide Hydrochlorothiazide
10/12.5 0.01/0.013 (mg/Tablet) (mg/mL) é
20/12.5 0.02/0.013 10/12.5 0.011/0.014 a
uv
20/25 0.02/0.026 20/12.5 0.022/0.014
20/25 0.022/0.028 E
Sample solution: Pass a portion of the solution under 3
3
test throughasuitable filter of 0.45-um pore size, and Sample solution: Pass a portion of the solution under )
discard the first few mL of the filtrate. test through a suitable filter of 0.45-m pore size. a=.
Chromatographic system: Proceed as directed in the Chromatographic system ES)
Assay, except use an injection volume of 20 pL. (See Chromatography (621), System Suitability.) 3
System suitability -
Mode: LC ay
Sample: Standard solution Detector: UV 210 nm
Suitability requirements Column: 4.6-mm x 20-cm; 5-um packing L7
Resolution: NLT 4.0 between the lisinopril and hy- Column temperature: 50°
drochlorothiazide peaks Flow rate: 1.0 mL/min
Tailing factor: NMT 2 for both the lisinopril and Injection volume: 50 uL
hydrochlorothiazide peaks System suitability
Column efficiency: NLT 6000 theoretical plates for Sample: Standard solution
the hydrochlorothiazide peak Suitability requirements
Relative standard deviation: NMT 2.0% for both Tailing factor: NMT 1.8 for the lisinopril ee and
the lisinopril and hydrochlorothiazide peaks NMT 1.5 for the hydrochlorothiazide peal
Analysis Resolution: NLT 3.5 between the lisinopril and hy-
Samples: Standard solution and Sample solution drochlorothiazide peaks
Calculate the percentage of the labeled amounts of Relative standard deviation: NMT 2.0% for both
lisinopril (C21H3i1N30s) and hydrochlorothiazide peaks
(C;HsCIN304S2) dissolved: Analysis
Samples: Standard solution and Sample solution
Result = (ru/rs) x (CGs/L) x V x 100 Calculate the percentage of the labeled amounts of
lisinopril (C2i1H3i1N30s) and hydrochlorothiazide
ty = peak response of lisinopril or (CzHsCIN3O4S2) dissolved:
hydrochlorothiazide from the Sample solution
ls = peak sets of lisinopril or Result = (ru/rs) x (Cs/L) x V x 100
hydrochlorothiazide from the Standard
solution tu = peakresponse of lisinopril or
Gs = concentration of lisinopril and hydrochlorothiazide from the Sample solution
hydrochlorothiazide in the Standard solution rs = peak Sa of lisinopril or
from Table 3 (mgimt) hydrochlorothiazide from the Standard
b = labeled amount of lisinopril and solution
hydrochlorothiazide (mg/Tablet)
 2436 Lisinopril / Official Monographs
Cs = concentration of lisinopril or
hydrochlorothiazide in the Standard solution
(mg/mL)
iL = labeled amounts of lisinopril or
hydrochlorothiazide (mg/Tablet)
Vv = volume of Medium, 900 mL
Tolerances: NLT 80% (Q) of the labeled amount of
lisinopril (C21H3i1N3Os) and NLT 75% (Q) of the labeled
amount of hydrochlorothiazide (C7HsCIN30.S2) are
dissolved.
¢ UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
IMPURITIES
© ORGANIC IMPURITIES
Buffer, Mobile phase, Benzothiadiazine related com-
pound A stock solution, Sample solution, Chromato-
graphic system, and System suitability: Proceed as
directed in the Assay.
Standard solution 1: Use the Standard solution, pre-
pared as directed in the Assay.
Standard solution 2: 1.28 g/mL of USP
Benzothiadiazine Related Compound ARS in Buffer from
the Benzothiadiazine related compound A stock solution
Analysis
Samples: Standard solution 1, Standard solution 2, and
Sample solution
Calculate the percentage of lisinopril related compound
A and benzothiadiazine related compoundAin the
portion of Tablets taken:
Result = (ru/rs) x (Cs/Cu) x (Ma/M,2) x 100
ry = peak response of lisinopril related compound
A or benzothiadiazine related compound A
from the Sample solution
rs = peak response of lisinopril related compound
“ A or benzothiadiazine related compound A
= from Standard solution 1 or Standard solution
is 2
i Transfer a suitable volume of the Standard stock solution
— Cs = concentration of USP Lisinopril Related
ro Compound A RS in Standard solution 1 or
i}
iS USP Benzothiadiazine Related Compound A
S RS in Standard solution 2 (mg/mL)
Ps Cu = nominal concentration of lisinopril or
qj hydrochlorothiazide in the Sample solution
a)
=] (mg/mL) o Oral Solution equivalent to NLT 60 mg of lithium to a
Mr = molecular weight of lisinopril, 405.5, for
calculating the percentage of lisinopril
related compound A; or molecular weight of
hydrochlorothiazide, 297.74, for calculating
the percentage of benzothiadiazine related
compound A
M2 = molecular weight of lisinopril related
compound A, 387.47; or benzothiadiazine
related compound A, 285.73
Acceptance criteria
Individual impurities: NMT 2% of lisinopril related
compound A; NMT 1% of benzothiadiazine related
compound A
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, Store at controlled room temperature.
e LABELING: When more than one Dissolution test is given,
the labeling states the test used only if Test 7 is not used.
e USP REFERENCE STANDARDS (11)
USP Benzothiadiazine Related Compound A RS
4-Amino-6-chloro-1 ,3-benzenedisulfonamide.
CoHsCIN3O4S2 285.73
USP 41
USP Hydrochlorothiazide RS
USP Lisinopril RS
USP Lisinopril Related Compound A RS
(S)-2-{(35,8a5)-3-(4-Aminobutyl)-1 ,4-dioxohex-
ahydropyrrolo[1,2-a]pyrazin-2(1 H)-yl}-4-phenylbutanoic
acid.
CaH2N304 387.47
Lithium Oral Solution
DEFINITION
Lithium Oral Solution is prepared from Lithium Citrate or
Lithium Hydroxide to which an excess of Citric Acid has
been added. It contains NLT 90.0% and NMT 110.0% of
the labeled amount of lithium (Li).
IDENTIFICATION
e A. The emission intensity at 671 nm of the Sample solu-
tion corresponds to that of the Standard solution, as ob-
tained in the Assay.
e B. IDENTIFICATION TESTS—GENERAL, Citrate (191): Meets
the requirements
ASSAY
¢ PROCEDURE
Surfactant solution: 1%-2% solution of nonionic
surfactant, such as t-dodecyl mercaptan ethoxylate or
polyoxyethylene (20) sorbitan monolaurate, in water
Standard stock solution: 0.3 mg/mL of USP Lithium
Carbonate RS prepared as follows. Transfer the required
quantity of USP Lithium Carbonate RS toa suitable vol-
umetric flask, and add 20% of the flask volume of
water and 0.5% of the flask volume of hydrochloric
acid. Shake until dissolved.
Standard solution: 6 g/mL of USP Lithium Carbonate
RS from the Standard stock solution prepared as follows.
to a suitable volumetric flask. Add 80% of the flask vol-
ume of water and 2% of the flask volume of Surfactant
solution, and dilute with water to volume. Determine
the pH of the solution.
Sample stock solution: Nominally 0.06 mg/mL of lith-
ium in water prepared as follows. Transfer a volume of
suitable volumetric flask. Dilute with water to volume.
Sample solution: Nominally 1.2 ug/mL of lithium from
the Sample stock solution prepared as follows. Transfer a
suitable volume of the Sample stock solution to a suita-
ble volumetric flask. Add 95% of the flask volume of
water, 0.2% of the flask volume of 1 N hydrochloric
acid, and 2% of the flask volume of the Surfactant solu-
tion, Adjust with 1 N hydrochloric acid or 1 N sodium
hydroxide to the same pH (+0.1 pH unit) as that of the
Standard solution, and dilute with water to volume.
Blank: Surfactant solution
Instrumental conditions
Mode: Flame photometry
Analytical wavelength: About 671 nm
Analysis
Samples: Standard solution, Sample solution, and Blank
Use the Blank to zero the instrument. Measure the
emission responses for the Standard solution and Sam-
ple solution.
Calculate the percentage of the labeled amount of lith-
ium (Li) in the portion of Oral Solution taken:
Result = (ru/rs) x (Cs/Cu) x (Ar/M,) x F x 100
tu = photometer reading of the Sample solution
rs = photometer reading of the Standard solution
Cs = concentration of USP Lithium Carbonate RS in
the Standard solution (g/mL)
USP 41 Official Monographs / Lithium 2437

Cu = nominal concentration of lithium in the Analysis: Boil the Sample solution, then cool it. To 5 mL
Sample solution (g/mL) of the solution add 6 N ammonium hydroxide until the
A = atomic weight of lithium, 6.94 reaction is alkaline.
M, = molecular weight of lithium carbonate, 73.89 Acceptance criteria: No turbidity or precipitate is
F = number of lithium ions in one mole of lithium observed.
carbonate, 2 e CALCIUM
Acceptance criteria: 90.0%-110.0% Sample solution: Suspend 5.0g of Lithium Carbonate
in 50 mL of water, and add a one excess of 3 N hy-
PERFORMANCE TESTS drochloric acid. Boil the clear solution to expel carbon
© UNIFORMITY OF DOSAGE UNITS (905): Meets the require- dioxide, add 5 mL of ammonium oxalate TS, render al-
ments for oral solution packaged in single-unit containers kaline with 6 N ammonium hydroxide, and allow to
stand for 4 h. Pass througha filtering crucible, and
SPECIFIC TESTS wash with warm water until the last washing yields no
© PH (791): 4.0-5.0
turbidity with calcium chloride TS. Place the crucible in
ADDITIONAL REQUIREMENTS a beaker, cover the crucible with water, add 3 mL of
¢ PACKAGING AND STORAGE: Preserve in tight containers. sulfuric acid, and heat to 70°.
Store at controlled room temperature. Analysis: Titrate the Sample solution with 0.10 N potas-
e USP REFERENCE STANDARDS (11 sium permanganate to a pale pink color that persists for
USP Lithium Carbonate RS 30s.
Acceptance criteria: NMT 3.8 mL of 0.10 N potassium
permanganate is consumed (0.15%).
e SODIUM
Standard stock solution: 500 j1g/mL of sodium pre-
pared as follows. Dissolve 1.271 g of sodium chloride,
Lithium Carbonate previously dried at 130° to constant weight, in water in
a 1000-mL volumetric flask. Dilute with water to
LizCO3 73.89 volume.
Carbonic acid, dilithium salt; Sample stock solution: 100 mg/mL of Lithium Carbon-
Dilithium carbonate [554-13-2]. ate prepared as follows. Suspend 20.0 g of Lithium Car-
bonate in 100 mL of water, cautiously add 50.0 mL of
DEFINITION hydrochloric acid, transfer to a 200-mL volumetric flask,
Lithium Carbonate contains NLT 99.0% of lithium carbonate and dilute with water to volume.
(LizCO3), calculated on the dried basis. Standard solution: Transfer 1 mL of Standard stock solu-
IDENTIFICATION tion and 5 mL of Sample stock solution to a 100-mL vol-
e A. It effervesces upon the addition of an acid, yielding a umetric flask, and dilute with water.
colorless gas that, when passed into calcium hydroxide Sample solution: 5 mg/mL of Lithium Carbonate from
TS, immediately forms a white precipitate. Sample stock solution diluted with water
e B. When moistened with hydrochloric acid, it imparts an Instrumental conditions c
al
intense crimson color to a nonluminous flame. Mode: Flame photometry a
Analytical wavelengths: 580 and 589 nm
ASSAY Analysis =
© PROCEDURE Samples: Standard solution and Sample solution iS}
=
Sample solution: Dissolve 0.5 g of Lithium Carbonate Set the flame photometer for maximum emission at )
in 25.0 mL of 1 N hydrochloric acid VS. 589 nm, using the Standard solution. Measure the to}~
Blank: 25.0 mL of 1 N hydrochloric acid VS emission intensities of the Sample solution at 580 and 2
Titrimetric system 589 nm. Bo]
(See Titrimetry (541).) Acceptance criteria: The difference between the inten- Sr
“
Mode: Residual titration sities observed at 580 and 589 nm for the Sample solu-
Titrant: 1N sodium hydroxide VS tion does not exceed the difference between the inten-
Endpoint detection: Visual sities observed at 589 nm for the Sample solution and
Indicator: Methyl orange TS the Standard solution, respectively (0.1%).
Analysis
Samples: Sample solution and Blank Delete the following:
Titrate the excess acid in the Sample solution with
Titrant.
Calculate the percentage of lithium carbonate (LixCO3) °e HEAVY METALS (231)
in the portion of Lithium Carbonate taken: Sample solution: Dissolve 1g of Lithium Carbonate in
10 mL of 3 N hydrochloric acid, and dilute with water
Result = (Vs
— Vs) x Nx Fx (1/W) x 100 to 25 mL.
Acceptance criteria: NMT 20 ppme (official 1-jan-2018)
Ve = Titrant volume consumed by the Blank (mL) e CHLORIDE AND SULFATE, Sulfate (221)
Vs = Titrant volume consumed by the Sample Standard solution: Transfer 1 mL of 0.020N sulfuric
solution (mL) acid and 1 mL of 3 N hydrochloric acid to a suitable
N = normality of Titrant (mEq/mL) container. Dilute with water to 40 mL.
F = equivalent weight of Lithium Carbonate, Sample solution: Transfer 1.0 g of Lithium Carbonate
36.95 mg/mEq to a suitable container. Dissolve 10 mL of 3 N hydro-
Ww = weight of Lithium Carbonate in the Sample chloric acid. Dilute with water to 40 mL.
solution (mg) Analysis: To the Standard solution and the Sample solu-
Acceptance criteria: NLT 99.0% on the dried basis tion, separately, add 1 mL of barium chloride TS.
Acceptance criteria: The turbidity produced in the
IMPURITIES Sample solution, after 3 min, is NMT that produced in
¢ ALUMINUM AND IRON the Standard solution (0.1%).
Sample solution: Dissolve 500 mg of Lithium Carbon-
ate in 10 mL of water by the dropwise addition, with
agitation, of hydrochloric acid.
 2438 Lithium / Official Monographs USP 41

© CHLORIDE AND SULFATE, Chloride (221) Instrumental conditions

Standard solution: Transfer 0.5 mL of 0.02 N hydro-
chloric acid and 1.2 mL of nitric acid to a suitable con-
tainer. Dilute with water to 50 mL.
Sample solution: Transfer 500 mg of Lithium Carbon-
ate to a suitable container. Add 1.2 mL of nitric acid.
Dilute with water to 50 mL.
Analysis: To the Standard solution and the Sample solu-
tion, separately, add 1 mL of silver nitrate TS.
Acceptance criteria: The turbidity produced in the
Sample solution is NMT that produced in the Standard
solution (0.07%).
SPECIFIC TESTS
e Loss ON DRYING (731)
Analysis: Dry at 200° for 4 h.
Acceptance criteria: NMT 1.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE:
containers.
Preserve in well-closed
Lithium Carbonate Capsules
DEFINITION
Lithium Carbonate Capsules contain NLT 95.0% and NMT
105.0% of the labeled amount of lithium carbonate
(LizCO3).
IDENTIFICATION
e A. A portion of the Capsule contents effervesces upon
the addition of an acid, yielding a colorless gas that,
when passed into calcium hydroxide TS, immediately
forms a white precipitate.
al e B. The emission intensity at 671 nm of the Sample solu-
£ tion corresponds to that of the Standard solution as ob-
is tained in the Assay.
J
— carbonate (LizCO3) dissolved:
22) ASSAY
° e PROCEDURE
¢
3 Surfactant solution: 1%-2% (v/v) solution of nonionic
= surfactant such as t-dodecyl mercaptan ethoxylate or
[a polyoxyethylene (20) sorbitan monolaurate in water
al Blank: Surfactant solution and water (1:50)
= Standard stock solution: 0.3 mg/mL of USP Lithium
Carbonate RS prepared as follows. Transfer the required
quantity of USP Lithium Carbonate RS to a suitable vol-
umetric flask, and add water to 20% of the flask vol-
ume and hydrochloric acid to 0.5% of the flask volume.
Shake until dissolved, and dilute with water to volume.
Standard solution: 0.006 mg/mL of USP Lithium Car-
bonate RS from Standard stock solution prepared as fol-
lows. Pipet a suitable volume of Standard stock solution
into a suitable volumetric flask. Add water to 80% of
the flask volume and Surfactant solution to 2% of the
flask volume, and dilute with water to volume.
Sample stock solution: Nominally 0.6 mg/mL of lith-
ium carbonate from the contents of NLT 20 Capsules
prepared as follows. Transfer a portion of powder,
equivalent to NLT 600 mg of lithium carbonate, into a
suitable volumetric flask. Add water to 4% of the flask
volume and hydrochloric acid to 0.5% of the flask vol-
ume, shake until the solid is well disintegrated, and di-
lute with water to volume.
Sample solution: Nominally 0.006 mg/mL of lithium
carbonate from Sample stock solution prepared as fol-
lows. Pipet a suitable volume of Sample stock solution
into a suitable volumetric flask. Add water to 80% of
the flask volume and Surfactant solution to 2% of the
flask volume, and dilute with water to volume.
Mode: Flame photometry
Analytical wavelength: About 671 nm
Analysis
Samples: Blank, Standard solution, and Sample solution
Use the Blank to zero the instrument. Measure the
emission responses for the Standard solution and Sam-
ple solution.
Calculate the percentage of the labeled amount of lith-
ae carbonate (LizCO3) in the portion of Capsules
taken:
Result = (ru/rs) x (Cs/Cy) x 100
ru = emission response from the Sample solution
rs = emission response from the Standard solution
Cs = concentration of USP Lithium Carbonate RS in
the Standard solution (mg/mL)
Cu = nominal concentration of the Sample solution
(mg/ml)
Acceptance criteria: 95.0%-105.0%
PERFORMANCE TESTS
e DISSOLUTION (711)
Medium: Water; 900 mL
Apparatus 1: 100 rpm
Time: 30 min
Surfactant solution, Blank, and Standard solution:
Prepare as directed in the Assay.
Sample solution: Pass the solution under test through
a suitable filter. Transfer 20.0 mL of the filtrate to a
1000-mL volumetric flask. Add 500 mL of water, 1 drop
of hydrochloric acid, and 20 mL of Surfactant solution.
Dilute with water to volume.
Analysis
Samples: Blank, Standard solution, and Sample solution
Use the Blank to zero the instrument. Measure the
emission responses for the Standard solution and Sam-
ple solution.
Calculate percentage of the labeled amount of lithium
Result = (ru/rs) x Cs x Vx Dx (1/L) x 100
tu = emission response from the Sample solution
Is = emission response from the Standard solution
Gs = concentration of USP Lithium Carbonate RS in
the Standard solution (mg/mL)
Vv = volume of the Medium, 900 mL
D = dilution factor
L = label claim (mg/Capsule)
Tolerances: NLT 80% (Q) of the labeled amount of lith-
ium carbonate (LizCO3) is dissolved.
e UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. Store at controlled room temperature.
e USP REFERENCE STANDARDS (11)
USP Lithium Carbonate RS
Lithium Carbonate Tablets
DEFINITION
Lithium Carbonate Tablets contain NLT 95.0% and NMT
105.0% of the labeled amount of lithium carbonate
(LizCO3).
IDENTIFICATION
A portion of the powdered Tablets meets the requirements
of the following tests.
USP 41
e A. It effervesces upon the addition of an acid, yielding a
colorless gas that, when passed into calcium hydroxide
TS, immediately forms a white precipitate.
e B. The emission intensity at 671 nm of the Sample solu-
tion corresponds to that of the Standard solution as ob-
tained in the Assay.
ASSAY
© PROCEDURE
Surfactant solution: 1%-2% solution of nonionic
surfactant such as t-dodecyl mercaptan ethoxylate or
polyoxyethylene (20) sorbitan monolaurate in water
Blank: ‘Surfactant solution and water (1:50)
Standard solution: Transfer 30 mg of USP Lithium Car-
bonate RS to a 100-mL volumetric flask, and add 20 mL
of water and 0.5 mL of hydrochloric acid. Shake until
dissolved, and dilute with water to volume. Pipet 20 mL
of the resulting solution into a 1000-mL volumetric
flask, add 800 mL of water and 20 mL of a suitable
surfactant solution, and dilute with water to volume.
Sample solution: Powder NLT 20 Tablets. Transfer a
ortion of powder, nominally equivalent to 600 mg of
ithium carbonate, into a 1000-mL volumetric flask. Add
40 mL of water and 5 mL of hydrochloric acid, shake
until the solid is well disintegrated, and dilute with
water to volume. Pipet 10 mL of the resulting solution
into a 1000-mL volumetric flask, add 800 mL of water
and 20 mL of the surfactant solution, and dilute with
water to volume.
Instrumental conditions
Mode: Flame photometer
Analytical wavelength: About 671 nm
[NoTe—Adjust the instrument with the Surfactant
solution.]
Analysis
Samples: Blank, Standard solution, and Sample solution
Calculate the percentage of the labeled amount of lith-
ium carbonate (LizCO3) in the portion of Tablets taken:
Result = (ru/rs) x (Cs/Cu) x 100
ty = peak response from the Sample solution
rs = peak response from the Standard solution
Gs = concentration of the Standard solution
(mg/mL)
Cu = nominal concentration of lithium carbonate in
the Sample solution (mg/mL)
Acceptance criteria: 95.0%-105.0%
PERFORMANCE TESTS
¢ DISSOLUTION (711)
Medium: Water; 900 mL
Apparatus 1: 100 rpm
Time: 30 min
Surfactant solution, Blank, and Standard solution:
Proceed as directed in the Assay.
Sample solution: Dilute 900 mL of the solution under
test with Medium to 1000 mL. Pass through a suitable
filter. Transfer 20.0 mL of the filtrate to a 1000-mL volu-
metric flask. Add 500 mL of water, 1 drop of hydrochlo-
ric acid, and 20 mL of Surfactant solution. Dilute with
water to volume.
Instrumental conditions
Mode: Flame photometer
Analytical wavelength: About 671 nm
Analysis
Samples: Blank, Standard solution, and Sample solution
Calculate the percentage of the labeled amount of lith-
ium carbonate (LizCO3) dissolved:
Result = (ru/rs) x Cs x Vx (1/L) x 100
ty = peak response from the Sample solution
Is = peak response from the Standard solution
Gs = concentration of USP Lithium Carbonate RS in
the Standard solution (mg/mL)
Official Monographs / Lithium 2439
Vv = volume of the Medium, 900 mL
L = label claim (mg/Tablet)
Tolerances: NLT 80% (Q) of the labeled amount of lith-
ium carbonate (LizCO3) is dissolved.
e UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers.
Protect from moisture. Store at controlled room
temperature.
e USP REFERENCE STANDARDS (11)
USP Lithium Carbonate RS
Lithium Carbonate Extended-Release
Tablets
DEFINITION
Lithium Carbonate Extended-Release Tablets contain NLT
90.0% and NMT 110.0% of the labeled amount of lith-
ium carbonate (LizCOs).
IDENTIFICATION
e A. A portion of powdered Tablets effervesces upon the
addition of an acid, yielding a colorless gas that, when
passed into calcium hydroxide TS, immediately forms a
white precipitate.
¢ B. The emission intensity at 671 nm of the Sample solu-
tion corresponds to that of the Standard solution as ob-
tained in the Assay.
ASSAY
© PROCEDURE
Diluent: Dilute 5 mL of hydrochloric acid with water to
dbs os
Standard solution: 0.3 mg/mL of USP Lithium Carbon- vw
ate RS in Diluent prepared as follows. Transfer a suitable
i)
quantity of USP Lithium Carbonate RS to an pie etiate =
volumetric flask. Add water to 20% of the flask volume, i}
=
and hydrochloric acid to 0.5% of the flask volume. i}
Shake until dissolved, and dilute with water to volume. ro)
=
Sample stock solution: Nominally 12 mg/mL of lithium i)
carbonate from a number of Tablets, nominally equiva- 3)
lent to NLT 1200 mg of lithium carbonate prepared as =
follows. Transfer the required number of Tablets to a
a)
suitable container. Add the required amount of Diluent.
Shake until completely dissolved. Filter, and discard the
first 25 mL. Use the remaining filtrate.
Sample solution: Nominally 0.3 mg/mL from Sample
stock solution in Diluent
[NotE—The Standard solution and Sample solution may
be diluted quantitatively with Diluent, if necessary, to
yield solutions of suitable concentrations adaptable to
the linear or working range of the instrument.]
Instrumental conditions
Mode: Flame photometry
Analytical wavelength: About 671 nm
Analysis
Samples: Standard solution and Sample solution
Use the Diluent to zero the instrument. Measure the
emission responses for the Standard solution and the
Sample solution.
Calculate the percentage of labeled amount of lithium
carbonate (LizCOs) in the portion of Tablets:
Result = (ru/rs) x (Cs/Cu) x D x 100
ru = emission response from the Sample solution
fs = emission response from the Standard solution
Cs = concentration of USP Lithium Carbonate RS in
the Standard solution (mg/mL)
 2440 Lithium / Official Monographs USP 41

Cy = nominal concentration of lithium carbonate in Calculate the percentage of the labeled amount of lith-
the Sample solution (agin) ium carbonate (LizCO3) dissolved at each time point as
D = dilution factor, if neede described in Test 7.
Acceptance criteria: 90.0%-110.0% Tolerances: See Table 2.
PERFORMANCE TESTS
e DISSOLUTION (711) Table 2
Test 1 Time Point Time Amount Dissolved
Medium: Dilute hydrochloric acid (7 in 1000); 800 mL (i) (h) (%)
Apparatus 1: 100 rpm 1 1 NMT 40
Time: 15, 45, 90, and 120 min zZ 3: 45-75
Standard solution: USP Lithium Carbonate RS at a 3 i: NLT 70
known concentration in Medium
Sample solution: At each time point j, withdraw The percentages of the labeled amount of lithium car-
8.0 mL of the Sample solution, and pass througha filter bonate (LizCO3) dissolved at the times specified con-
of 35-um or finer pore size. Use the filtrate as the Sam- form to Acceptance Table 2 in Dissolution (711).
ple solution, suitably diluted with Medium if necessary. Test 3: If the product complies with this test, the label-
Instrumental conditions ing indicates that it meets USP Dissolution Test 3.
Mode: Flame photometry Medium: Water; 250 mL
Analytical wavelength: About 671 nm Apparatus 3: 6 dips/min, 20-mesh top screen and
Analysis 100-mesh bottom screen
Samples: Standard solution and Sample solution Time: 1, 2, and 6h
Use the Medium to zero the instrument. Measure the Standard solution, Sample solution, Instrumental
emission responses for the Standard solution and the conditions, and Analysis: Proceed as directed in Test
Sample solution. 1
Calculate the concentration, C, of lithium carbonate Calculate the percentage of the labeled amount of lith-
(LixCO3) in Medium (mg/mL) at each time point i: ium carbonate (LizCO3) dissolved at each time point as
described in the Test 7.
CG = (tufts) x Cs Tolerances: See Table 3.
ty = emission response from the Sample solution
ls = emission response from the Standard solution Table 3
Gs = concentration of USP Lithium Carbonate RS in Time Point Time Amount Dissolved
the Standard solution (mg/mL) (i) (h) (%)
Calculate the percentage of the labeled amount (Q) of 1 1 10-45
lithium carbonate (LizCO3) dissolved at each time
2 2 25-75
point i:
3 6 NLT 70
r= Result; = C;) x Vx (1/L) x 100 The percentages of the labeled amount of lithium car-
S
— bonate (LizCO3) dissolved at the times specified con-
aD Result, = ({C, x [V— (i- 1) x Vs]} + [(G-1 + G-2.. + Cr) form to Acceptance Table 2 in Dissolution (711).
Sl x Vs}) x (1/L) x 100 Test 4: If the product complies with this procedure, the
fo} labeling indicates that it meets USP Dissolution Test 4.
>= G = concentration of lithium carbonate in Medium Medium: Dilute hydrochloric acid (7 in 1000); 800 mL
Ps in the portion of sample withdrawn at time Apparatus 1: 100 rpm
rs point i (mg/mL) Time: 15, 45, 90, and 120 min
b=| Vv = volume of Medium, 900 mL Standard solution, Sample solution, Instrumental
L = label claim (mg/Capsule) conditions, and Analysis: Proceed as directed in Test
Vs = volume of the Sample solution withdrawn from Ts
the Medium (mL) Calculate the percentage of the labeled amount of lith-
Tolerances: See Table 1. ium carbonate (LizCO3) dissolved at each time point as
described in the Test 7.
Table 1 Tolerances: See Table 4.
Time Point Time Amount Dissolved
Table 4
1 2-16 Time Point Time Amount Dissolved

3.
4 1

The percentages of the labeled amount of lithium car- 4 NLT

bonate (LizCO3) dissolved at the times specified con-
form to Acceptance Table 2 in Dissolution (711).
Test 2: If the product complies with this procedure, the
labeling indicates that it meets USP Dissolution Test 2.
Medium: Water; 900 mL
Apparatus 1: 100 rpm
Time: 1, 3, and7h
Standard solution, Sample solution, Instrumental
conditions, and Analysis: Proceed as directed in Test
he
The percentages of the labeled amount of lithium car-
bonate (LizCO3) dissolved at the times specified con-
form to Acceptance Table 2 in Dissolution (711).
Test 5: If the product complies with this procedure, the
labeling indicates that it meets USP Dissolution Test 5.
Medium: Water; 900 mL
Apparatus 1: 100 rpm
Time: 30, 90, and 150 min
Standard solution, Sample solution, Instrumental
conditions, and Analysis: Proceed as directed in Test
1,
USP 41 Official Monographs / Lithium 2441

Calculate the percentage of the labeled amount of lith- water and 0.5% of the flask volume of hydrochloric
ium carbonate (LizCO3) dissolved at each time point as acid. Shake until dissolved, and dilute with water to
described in the Test 7. volume.
Tolerances: See Table 5. Standard solution: 0.006 mg/mL of USP Lithium Car-
bonate RS from Standard stock solution prepared as fol-
Table 5 lows. Pipet a suitable volume of Standard stock solution
into a suitable volumetric flask. Add 80% of the flask
Time Point Time Amount Dissolved volume of water and 2% of the flask volume of the
(i) (min) (%) Surfactant solution, and dilute with water to volume.
1 30 10-30 Sample stock solution: 0.8 mg/mL of Lithium Citrate
2 90 55-75 prepared as follows. Transfer a suitable amount of Lith-
3 150 NLT 85% ium Citrate to a suitable volumetric flask. Dissolve in
water. Add 0.05% of the flask volume of hydrochloric
The percentages of the labeled amount of lithium car- acid. Dilute with water to volume.
bonate (LizCO3) dissolved at the times specified con- Sample solution: Nominally 0.006 mg/mL of Lithium
form to Acceptance Table 2 in Dissolution (711). Citrate from Sample stock solution prepared as follows.
e UNIFORMITY OF DOSAGE UNITS (905): Meet the Pipet a suitable volume of Sample stock solution into a
requirements suitable volumetric flask. Add 80% of the flask volume
of water and 2% of the flask volume of the Surfactant
ADDITIONAL REQUIREMENTS solution, and dilute with water to volume.
e PACKAGING AND STORAGE: Preserve in well-closed contain- Instrumental conditions
ers. Protect from moisture. Store at controlled room Mode: Flame photometry
temperature. Analytical wavelength: 671 nm
e LABELING: When more than one Dissolution test is given, Analysis
the fabeling states the Dissolution test used only if Test 7 Samples: Blank, Standard solution, and Sample solution
is not used. Use the Blank to zero the instrument. Measure the
e USP REFERENCE STANDARDS (11) emission responses for the Standard solution and the
USP Lithium Carbonate RS Sample solution.
Calculate the percentage of lithium citrate (CsHsLisO7)
in the portion of Lithium Citrate taken:
Result = (ru/rs) x (Cs/Cu) x (Mr/M,2) x F x 100
Lithium Citrate tu = emission response of the Sample solution
ls = emission response of the Standard solution
Cs = concentration of USP Lithium Carbonate RS in
the Standard solution (mg/mL)
om

f Ho) + ano Cu = concentration of Lithium Citrate in the Sample {ow

solution (mg/mL) wm)
Mn, = molecular weight of anhydrous lithium citrate, bi
209.93
CeHsLis3O7 - 4H20 281.98 M2 = molecular weight of lithium carbonate, 73.89 °
F = ratio of the number of moles of lithium in 1 s
Ce6HsLisO7 209.93
mol of lithium carbonate to that of the a
1,2,3-Propanetricarboxylic acid, 2-hydroxy-trilithium salt number of moles of lithium in 1 mol of Fy
tetrahydrate;
lithium citrate, 0.66 co}
Trilithium citrate tetrahydrate [6080-58-6]. Acceptance criteria: 98.0%-102.0% on the anhydrous 2
Anhydrous [919-16-4].
basis
DEFINITION
Lithium Citrate contains NLT 98.0% and NMT 102.0% of IMPURITIES
qa citrate (CsHsLi307), calculated on the anhydrous
asIS. Delete the following:
IDENTIFICATION
e A. The emission intensity at 671 nm of the Sample solu- °o HEAVY METALS (231)
Test Penenton Dissolve 2.0g of Lithium Citrate in
tion corresponds to that of the Standard solution, as ob- 2 mL of 0.1 N hydrochloric acid, and dilute with water
tained in the Assay. to 25 mL.
e B. IDENTIFICATION TESTS—GENERAL, Citrate (191): Meets Acceptance criteria: NMT 10 ppMe cotfciai 1420-2018)
the requirement
SPECIFIC TESTS
ASSAY © CARBONATE
° PROCEDURE Analysis: Add 0.5 g of Lithium Citrate to 5 mL of 6N
Surfactant solution: 1%-2% solution of nonionic acetic acid.
surfactant such as t-dodecyl mercaptan ethoxylate or Acceptance criteria: NMTaslight effervescence is
Bisyis (20) sorbitan monolaurate in water produced.
Blank: ‘Surfactant solution and water (1:50) PH (791)
Standard stock solution: 0.3 mg/mL of USP Lithium Sample solution: 50-mg/mL solution of Lithium Citrate
Carbonate RS prepared as follows. Transfer the required in water
quantity of USP Lithium Carbonate RS to a suitable vol-
umetric flask, and add 20% of the flask volume of
 2442 Lithium / Official Monographs
Acceptance criteria: 7.0-10.0
© WATER DETERMINATION, Method II] (921)
Analysis: Dry a sample at 150° for 3 h.
Acceptance criteria: 24.0%-28.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers.
e USP REFERENCE STANDARDS (11)
USP Lithium Carbonate RS
Lithium Hydroxide
LiOH - H20 41.96
LiOH 23.95
Lithium hydroxide monohydrate [1310-66-3].
Anhydrous [1310-65-2].
DEFINITION
Lithium Hydroxide contains NLT 98.0% and NMT 102.0%
of lithium hydroxide (LiOH), calculated on the anhydrous
basis.
[CauTion—Exercise great care in handling Lithium Hydrox-
ide, as it rapidly destroys tissues.]
IDENTIFICATION
e A. When moistened with hydrochloric acid, it imparts an
intense crimson color to a nonluminous flame.
ASSAY
e PROCEDURE
Sample solution: Nominally equivalent to 10 mg/mL of
anhydrous lithium hydroxide from Lithium Hydroxide in
carbon dioxide-free water
Analysis
Preliminary titration: Pipet 50 mL of the Sample solu-
ate
”
tion into a 250-mL conical flask. Start the titration by
a adding 35 mL of 0.5 N hydrochloric acid VS with con-
J
h tinuous vigorousSting: Add 20 mL of 1.N barium
Dp chloride and 3 drops of phenolphthalein TS, and allow
° Acceptance criteria: NMT 20 ppme coticial i-Jan-2018)
rs to stand for 2 min. Continue the titration with 0.5 N
3 hydrochloric acid VS. At the discharge of the pink
= color of the indicator, record the volume of acid solu-
a tion consumed.
Al Final titration: Pipet 50 mL of the Sample solution into
= a 250-mL conical flask. While pipeting and during the
subsequent titrations, keep the contents of the flask
blanketed with a stream of carbon dioxide-free air.
Start the titration by adding with continuous vigorous
swirling a volume of 0.5 N hydrochloric acid VS that
is 0.50 mL less than that consumed in the preliminary
titration. Add 20 mL of 1 N barium chloride and
3 drops of phenolphthalein TS, and allow to stand for
2 min. Rinse the sides of the flask with carbon diox-
ide-free water, and continue the titration with 0.1 N
hydrochloric acid VS. At the discharge of the pink
color of the indicator, record the volume of acid solu-
tion consumed. Each mL of 0.5 N hydrochloric acid
VS and 0.1 N hydrochloric acid VS is equivalent to
11.975 and 2.395 mg of total alkali, respectively, cal-
culated as lithium hydroxide (LiOH).
Receptanes criteria: 98.0%-102.0% on the anhydrous
asis
IMPURITIES
e CHLORIDE AND SULFATE, Sulfate (221)
Sample: 2.0g
Acceptance criteria: |t shows no more sulfate than cor-
responds to 1.0 mL of 0.020 N sulfuric acid (0.05%).
e CALCIUM
Sample solution: Dissolve 3.33.9 in 50 mL of 3 N hy-
drochloric acid. Boil the clear solution to expel carbon
dioxide, add 5 mL of ammonium oxalate TS, render al-
USP 41
kaline with 6 N ammonium hydroxide, and allow to
stand for 4 h. Pass through afiltering crucible, and
wash with warm water until the last washing yields no
turbidity with calcium chloride TS. Place the crucible in
a beaker, cover it with water, add 3 mL of sulfuric acid,
and heat to 70°.
Analysis: Titrate the Sample solution with 0.10 N potas-
sium permanganate to a pale pink color that persists for
30 s.
Acceptance criteria: NMT 3.34 mL of 0.10 N potas-
sium permanganate is consumed (0.20%).
e CARBONATE
[NotE—While pipeting and li the subsequent titra-
tions, keep the contents of the flasks blanketed with a
stream of carbon dioxide-free air.]
Analysis: To the flask containing the completed Final
titration obtained in the Assay, add 1 drop of methyl
orange TS. Titrate with 0.1 N hydrochloric acid VS until
a persistent orange color is produced and no undis-
solved barium carbonate remains. Perform a blank titra-
tion to determine the volume of 0.1 N hydrochloric
acid consumed in going from the phenolphthalein
endpoint to the methyl orange endpoint. To 100 mL of
carbon dioxide-free water in a 250-mL conical flask,
add 3 drops of the Sample solution from the Assay,
20 mL of 1N barium chloride, and 3 drops of phenol-
phthalein TS. Allow to stand for 2 min. Titrate this solu-
tion with 0.1 N hydrochloric acid. At the discharge of
the pink color of the indicator, add 1 drop of methyl
orange TS, and titrate with 0.1 N hydrochloric acid VS
until a persistent orange color is produced.
Acceptance criteria: The titration shows no more car-
bon dioxide than corresponds to 1.5 mL of 0.10 N hy-
drochloric acid (0.7%).
Delete the following:
®e HEAVY METALS, Method | (231)
Sample solution: Dissolve 1.0 g in 15 mL of 3 N hydro-
chloric acid, and dilute with water to 25 mL.
SPECIFIC TESTS
e LITHIUM CONTENT
Standard stock solution: 0.3 mg/mL of USP Lithium
Carbonate RS prepared as follows. Dissolve first in water
using 20% final volume and hydrochloric acid using
0.5% of final volume. Dilute with water to volume.
Standard solution: 6.0 g/mL of USP Lithium Carbon-
ate RS from the Standard stock solution prepared as fol-
lows. Pipet a volume of the Standard stock solution into
a suitable volumetric flask, add water to fill 80% of final
volume, and a suitable surfactant solution to fill 2% of
final volume. Dilute with water to volume. Measure the
pH.
Sample stock solution: 0.4 mg/mL of Lithium Hydrox-
ide in water
Sample solution: Pipet 20 mL of the Sample stock solu-
tion into a 1000-mL volumetric flask. Add 950 mL of
water, 2mL of 1 N hydrochloric acid, and 20 mL of a
surfactant solution, and mix. Adjust with 1 N hydro-
chloric acid or 1 N sodium hydroxide to the same pH
(40.1 pH unit) as that of the Standard solution, and di-
lute with water to volume.
Instrumental conditions
Mode: Flame photometry
Analytical wavelength: 671 nm. Adjust the instru-
ment with the surfactant solution.
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of lithium (Li) in the portion
of Lithium Hydroxide taken:
Result = (ru/rs) x (Cs/Cu) x (Ar/M,) x F x 100
USP 41 Official Monographs / Lomustine 2443

ru = photometric reading of the Sample solution Mode: LC

Is = photometric reading of the Standard solution Detector: UV 230 nm
Gs = concentration of USP Lithium Carbonate RS in Column: 4.6-mm x 75-mm; 3-m packing L1
the Standard solution (mg/mL) Flow rate: 1.5 mL/min
Cy = concentration of Lithium Hydroxide in the Injection volume: 10 pL
Sample solution (mg/mL) System suitability
Ar = atomic weight of lithium, 6.94 Sample: Standard solution
M, = molecular weight of lithium carbonate, 73.89 Suitability requirements
E = number of lithium ions per mole of lithium Relative standard deviation: NMT 1.0%
carbonate, 2 Tailing factor: NMT 1.3
Acceptance criteria: 28.1%-29.9% on the anhydrous Analysis
asis Samples: Standard solution and Sample solution
e WATER DETERMINATION, Method III (921) Calculate the percentage of lomustine (CoHisCIN3Oz) in
Analysis: Dry at 135° at a pressure of NMT 5 mm of the portion of Lomustine taken:
mercury for 1h.
Acceptance criteria: 41.0%-43.5% Result = (ru/rs) x (Cs/Cu) x 100
ADDITIONAL REQUIREMENTS Ty = peak response from the Sample solution
© PACKAGING AND STORAGE: Preserve in tight containers. ls = peak response from the Standard solution
e USP REFERENCE STANDARDS (11) Cs = concentration of USP Lomustine RS in the
USP Lithium Carbonate RS Standard solution (mg/mL)
cu = concentration of Lomustine in the Sample
solution (mg/mL)
Acceptance criteria: 97%-103% on the as-is basis

Lomustine IMPURITIES

Delete the following:

or °e HEAVY METALS
Magnesium sulfate solution: 250 mg/ml of magne-
sium sulfate in 2 N sulfuric acid
CyHi6CIN302 233.70 Lead nitrate stock solution and Standard lead solu-
Urea, N-(2-chloroethyl)-N’-cyclohexyl-N-nitroso-; tion: Prepare as directed in Heavy Metals (231), Special
1-(2-Chloroethyl)-3-cyclohexyl-1-nitrosourea [13010-47-4]. Reagents.
Standard solution: Transfer 4.0 mL of Standard lead so-
DEFINITION lution inte a 50-mL color-comparison tube, and dilute
Lomustine contains NLT 97% and NMT 103% of lomustine with water to 25 mL. c=4
(CoHisCIN3O2), calculated on the as-is basis. 72)
Sample solution: Transfer 2.0 g of Lomustine to a ao)
[CautTion—Great care should be taken to prevent inhalation 50-mL silica crucible. Add 4 mL of Magnesium sulfate
ofparticles of lomustine and to prevent exposure to the solution, and mix using a thin glass rod. Place the cruci- Es
skin. ble on a steam bath, and heat cautiously until charring °
J
begins. Then piace the crucible on a hot plate, and °
IDENTIFICATION continue the charring. When the charring is complete, ©
e A. INFRARED ABSORPTION (197K) =
carry out the ignition at a temperature not exceeding Sy
e B. The retention time of the major peak of the Sample 800° until an almost white or a mostly grayish residue mo]
solution corresponds to that of the Standard solution, as mH
is obtained. Allow to cool, and moisten the residue with “
obtained in the Assay. 0.5 mL of 2 N hydrochloric acid, evaporate, ignite
ASSAY again, and allow to cool. The total period of ignition
© PROCEDURE must not exceed 2 h. Dissolve the residue in two por-
[Note—Protect the solutions from light. Use freshly pre- tions, 5 mL each, of 2 N hydrochloric acid. Add 0.1 mL
pared solutions.] of phenolphthalein TS, followed by ammonium hydrox-
Mobile phase: Acetonitrile and water (35:65) ide, until a pink color is obtained. Cool, add glacial ace-
Standard solution: 0.2 mg/mL of USP Lomustine RS in tic acid until the solution is decolorized, then add an
acetonitrile additional 0.5 mL of glacial acetic acid. Filter if neces-
Sample solution: 0.2 mg/mL of Lomustine in sary, dilute with water to 20 mL, and quantitatively
acetonitrile age this solution into a 50-mL color-comparison
Chromatographic system tube.
(See Chromatography (621), System Suitability.) Analysis
Samples: Standard solution and Sample solution
Adjust with either 1 N acetic acid or 6 N ammonium
hydroxide to a pH of between 3.0 and 4.0 using lit-
mus paper. Dilute with water to 40 mL. Add 1.2 mL
of thioacetamide—glycerin base TS, and dilute with
water to volume. Allow to stand for 2 min, and view
downward over a white surface.
Acceptance criteria: NMT 20 g/g; any brown color in
the Sample solution is not more intense than that in the
Standard solution.e cottica: 1-jan-2018)
© ORGANIC IMPURITIES
[Note—Protect the solutions from light. Use freshly pre-
pared solutions.]
 2444 Lomustine / Official Monographs USP 41

Solution A: Water Detect at 230 nm, and compare the peak area of
Solution B: Acetonitrile lomustine related compoundD in the Sample solution
Mobile phase: See Table 1. with the peak area of lomustine related compound D
in Standard solution A. The peak area of lomustine
Table 1 related compoundD in the Sample solution is NMT the
peak area of lomustine related compoundD in
Solution A Solution B Standard solution A (0.5%).
% Calculate the percentage of any unspecified impurity in
90. the portion of Lomustine taken:
10
5
Result = (ru/rs) x (Cs/Cu) x 100
5 tu = peak area of any unspecified impurity from
the Sample solution, determined at 195 nm
or 230 nm. If the impurity is detected at
5. both wavelengths, use the higher peak area
60 in the formula.
10 rs = peak area of lomustine from Standard solution
B, determined at 230 nm
6: 90 Cs = concentration of USP Lomustine RS in
« 4 Standard solution B (mg/mL)
Standard solution A: 0.032 mg/mL each of USP Cu = concentration of Lomustine in the Sample
Carmustine Related Compound A RS, USP Lomustine solution (mg/mL)
Related Compound B RS, and USP Lomustine Related Acceptance criteria: See Table 2, Disregard any impu-
Compound C RS, and 0.04 mg/mL of USP Lomustine tity peak less than 0.05%.
Related Compound D RS, and 2 mg/mL of USP Lomus-
tine RS in acetonitrile
Standard solution B: 8 g/mL of USP Lomustine RS in Table 2
acetonitrile ‘ ee a Relative Acceptance
Sample solution: 8 mg/mL of Lomustine in acetonitrile Retention Criteria,
Chromarcgeipie s Stet é a Name Time NMT (%)
(eee etnategrap y (621), System Suitability.) Carmustine related
Detector: UV 195 and 230 nm compound a q oh 0.4
Column: 4.6-mm x 10-cm; 2.6-um packing L43 Lomustine telate
Temperatures compound Be 0.39 0.4
Column: 35° Lomustine related
Sample: 15° compound C4 0.73 0.4
4 Flow rate: 0.8 mL/min Lomustine 1.0 a
2S Injection volume: 4 uL Lomustine related
pd System suitability . compound De 1.02 0.5
m Samples: Standard solution A and Standard solution B Any individual
= Suitability requirements : unspecified impurit _ 0.2
° Resolution: NLT 1.2 between the lomustine and Total impuritiest _ 1
= lomustine related compound D peaks determined at is(2-chi hl
4 220 bine standard POLO A malts aa Drees urity (carmustine related compound A,
3 Relative standard deviation: NMT 10% for carmus- lomustine related compound ; oy lomustine related compound C) 1s
tine related compound A and lomustine related com- greater than 0.2%.
pounds B and C determined at 195 nm, Standard © 1-(2-Chloroethyl)-3-cyclohexylurea.
solution A; NMT 10% for lomustine related com- 91,3-Dicyclohexylurea.
pound D determined at 230 nm, Standard solution A; 3-(2-Chloroethyl)-1 -cyclohexyl-1-nitrosourea.
NMT 10% for lomustine determined at 230 nm, f Lomustine related compoundD 1s not included in the Total impurities.
Standard solution B
Tailing factor: Between 0.7 and 1.3 for the lomustine SPECIFIC TESTS
peak determined at 230 nm, Standard solution A e WATER DETERMINATION, Method | (921): NMT 0.5%
Analysis ADDITIONAL REQUIREMENTS
Samples: Standard solution A, Standard solution B, and e PACKAGING AND STORAGE: Preserve in well-closed contain-
Sample solution ers, and store between 2° and 8°.
Calculate the percentage of carmustine related com- e USP REFERENCE STANDARDS (11)
pound A and lomustine related compounds B andC in USP Carmustine Related Compound A RS
the portion of Lomustine taken: 1,3-Bis(2-chloroethyl)urea.
Result = (ru/rs) x (Cs/Cu) x 100 CsHioClaN20 185.05
USP Lomustine RS
tu = peak area of each impurity from the Sample USP Lomustine Related Compound B RS
OAlution, determined at 195 nm 1-(2-Chloroethyl)-3-cyclohexylurea.
ts = peak area of the corresponding related CsHizCIN2Z0 204.70
compound from Standard solution A, USP Lomustine Related Compound C RS
determined at 195 nm 1,3-Dicyclohexylurea.
Cs = concentration of the corresponding USP CisHaaN20 224.34
Carmustine Related Compound A RS, USP USP Lomustine Related Compound D RS
Lomustine Related Compound B RS, or USP 3-(2-Chloroethyl)-1-cyclohexyl-1-nitrosourea.
Lomustine Related CompoundC RS in CsHisCIN302 ~— 233.70
Standard solution A (mg/mL)
Cu = concentration of Lomustine in the Sample
solution (mg/mL)
USP 41 Official Monographs / Lomustine 2445

Table 1
Lomustine Capsules Solution A Solution B
Yo
DEFINITION
Lomustine Capsules contain NLT 90.0% and NMT 110.0% 10
of the labeled amount of lomustine (CsHigCIN3O2).
[CAuTION—Great care should be taken to prevent inhalation 5
st ee of lomustine and to prevent exposure to the
skin.
0
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
Sample: Transfer the contents of 2 Capsules to a stop-
pered Erlenmeyer flask containing 25 mL of methylene
1
chloride. Shake vigorously, and filter, Evaporate the
methylene chloride from the filtrate under a stream of Standard solution A: 0.032 mg/mL each of USP
dry nitrogen. Prepare a potassium bromide pellet of the Carmustine Related Compound A RS, USP Lomustine
residue. Related Compound B RS, and USP Lomustine Related
Acceptance criteria: Meet the requirements CompoundC RS, and 0.04 mg/mL of USP Lomustine
° B. The retention time of the major peak of the Sample Related Compound D RS and 2 mg/mL of USP Lomus-
solution corresponds to that of the Standard solution, as tine RS in acetonitrile
obtained in the Assay. Standard solution B: 8 g/mL of USP Lomustine RS in
ASSAY acetonitrile
e PROCEDURE Sample solution: 8 mg/mL of lomustine in acetonitrile
[NoTe—Protect the solutions from light. Use freshly pre- prepared as follows. Transfer a portion of the Capsule
pared solutions.] fill (from NLT 20 Capsules) to a suitable volumetric
Mobile phase: Acetonitrile and water (7:13) flask, and dilute with acetonitrile to volume. Sonicate
Standard solution: 0.2 mg/mL of USP Lomustine RS in for 30 min, and then stir for 30 min. Pass a portion of
acetonitrile the solution through asuitable filter of 0.2-um pore
Sample solution: 0.2 mg/mL of lomustine in acetoni- size.
trile prepared as follows. Transfer a portion of the Cap- Chromatographic system
sule fill (from NLT 20 Capsules) to a suitable volumetric (See Chromatography (621), System Suitability.)
flask, and add acetonitrile equivalent to 75% of the vol- Mode: LC
ume. Shake for 15 min, and dilute with acetonitrile to Detector: UV 195 and 230 nm
volume. Pass through a suitable filter, and discard the Column: 4.6-mm x 10-cm; 2.6-um packing L43
first few mL of filtrate. Temperatures
Chromatographic system Column: 35° a
Sample: 15° Al
(See Chromatography (621), System Suitability.) ao]
Mode: LC Flow rate: 0.8 mL/min
Detector: UV 230 nm Injection volume: 4 uL cs
System suitability °
Column: 4.6-mm x 7.5-cm; 3-4m packing L1 S
Flow rate: 1.5 mL/min Samples: Standard solution A and Standard solution B fo)
Injection volume: 10 LL Suitability requirements Ko}
Resolution: NLT 1.2 between the lomustine and ra
System suitability 2
Sample: Standard solution lomustine related compound D peaks determined at me}
230 nm, Standard solution A =a
Suitability requirements 7
Relative standard deviation: NMT 2.0% Relative standard deviation: NMT 10% for carmus-
Tailing factor: NMT 1.3 tine related compound A, lomustine related com-
Analysis pounds B and C determined at 195 nm, Standard
Samples: Standard solution and Sample solution solution A; NMT 10% for lomustine determined at
Calculate the percentage of the labeled amount of 230 nm, Standard solution B
fepacesnines (CsHisCIN3O2) in the portion of Capsules Tailing factor: Between 0.7 and 1.3 for the lomustine
taken: peak determined at 230 nm, Standard solution A
Analysis
Result = (ru/rs) x (Cs/Cu) x 100 Samples: Standard solution A, Standard solution B, and
Sample solution
ty = peak response from the Sample solution Calculate the percentage of carmustine related com-
rs = peak response from the Standard solution pound A and lomustine related compounds B and C in
Cs = concentration of lomustine in the Standard the portion of Capsules taken:
solution (mg/mL)
Cy = nominal concentration of lomustine in the Result = (ru/rs) x (Cs/Cu) x 100
Sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0% tu = peak area of each impurity from the Sample
solution, determined at 195 nm
IMPURITIES Is = peak area of the corresponding related
© ORGANIC IMPURITIES compound from Standard solution A,
[NoTtE—Protect the solutions from light. Use freshly pre- determined at 195 nm
pared solutions.] Cs = concentration of the corresponding USP
Solution A: Water Carmustine Related Compound A RS, USP.
Solution B: Acetonitrile Lomustine Related CompoundBRS, or USP
Mobile phase: See Table 7. Lomustine Related Compound C RS in
Standard solution A (mg/mL)
Cu = nominal concentration of lomustine in the
Sample solution (mg/mL)
 2446 Lomustine / Official Monographs USP 41

Calculate the percentage of any unspecified impurity in Au = absorbance from the Sample solution
the portion of Capsules taken: As = absorbance from the Standard solution
Cs = concentration of USP Lomustine RS in the
Result = (ru/rs) x (Cs/Cu) x 100 Standard solution (mg/mL)
Cu = nominal concentration of lomustine in the
tu = peak area of any unspecified impurity from Sample solution (mg/mL)
the Sample solution, determined at 195 nm Acceptance criteria: Meet the requirements
or 230 nm. If the impurity is detected at
both wavelengths, use the higher peak area ADDITIONAL REQUIREMENTS
in the formula. e PACKAGING AND STORAGE: Preserve in well-closed contain-
rs = peak area of lomustine from Standard solution ers, and store at controlled room temperature.
B, determined at 230 nm e USP REFERENCE STANDARDS (11)
Cs = concentration of USP Lomustine RS in the USP Carmustine Related Compound A RS
Standard solution B (mg/mL) 1,3-Bis(2-chloroethyl)urea.
Cu = nominal concentration of lomustine in the CsHioClaNzO0 185.05
Sample solution (mg/mL) USP Lomustine RS
Acceptance criteria: See Table 2. Disregard any impu- USP Lomustine Related Compound B RS
rity peak less than 0.05%. 1-(2-Chloroethyl)-3-cyclohexylurea.
CoHi7zCIN20 204.70
Table 2 USP Lomustine Related Compound C RS
1,3-Dicyclohexylurea.
Relative Acceptance Cy3HasN20 224.34
Retention Criteria, USP Lomustine Related Compound D RS
Name Time NMT (%) 3-(2-Chloroethyl)-1-cyclohexyl-1-nitrosourea.
Carmustine related CsHisCIN3O2 — 233.70
compound Aa 0.11 0.4
Lomustine related
compound Be 0.39 0.4
Lomustine related
compound C4 0.73 0.45 Loperamide Hydrochloride
Lomustine 1.0 =
Lomustine related ta
compound De 1.02
Any individual un- ee
specified impurity 0.2
cr
Total impurities a 2
a 1,3-Bis(2-chloroethyl)urea.
ea
”

is ’ No more than one such impurity (carmustine related compound A,

S
“4
lomustine related compound B, or lomustine related compound C) 1s
greater than 0.2%.
Ca9H33CIN2O> + HCl 513.50
Dd 1-Piperidinebutanamide, 4-(4-chlorophenyl)-4-hydroxy-N,N-
i) ¢ 1-(2-Chloroethy!)-3-cyclohexylurea.
dimesnyy gecepees monohydrochloride.
e 4 1,3-Dicyclohexylurea.
)
4-(p-Chlorophenyl)-4-hydroxy-N, N-dimethyl-c,,a-dipheny|-
S © This process impurity is included in the table for identification only, and
1-piperidinebutyramide monohydrochloride
P= it is not to be reported or included in the Total impurities.
[34552-83-5].
a PERFORMANCE TESTS
A)
=) e DISINTEGRATION (701): 20 min » Loperamide Hydrochloride contains not less
e UNIFORMITY OF DosaGE UNITS (905) than 98.0 percent and not more than 102.0 per-
Procedure for content uniformity cent of Co9H33CIN2O2
- HCI, calculated on the
Complete the analysis within 1 h after the preparation dried basis.
of Sample solution.
Standard solution: 0.02 mg/mL of USP Lomustine RS Packaging and storage—Preserve in well-closed contain-
in methanol ers.
Sample solution: Nominally equivalent to 0.02 mg/mL
of lomustine in methanol prepared as follows. Transfer USP Reference standards (11)—
the content of a Capsule into a 100-mL volumetric USP Loperamide Hydrochloride RS
flask, and dilute with methanol to volume. Allow the identification—
excipients to settle for at least 15 min. Transfer a A: Infrared Absorption (197K).
measured volume of the clear supernatant to a suita- B: Transfer about 40 mg, accurately weighed, to a
ble volumetric flask, and dilute to volume with 100-mL volumetric flask, dissolve in about 50 mL of isopro-
methanol. pyl alcohol, add 10 mL of 0.1 N hydrochloric acid, dilute
Instrumental conditions with isopropyl alcohol to volume, and mix: the UV absorp-
(See Ultraviolet-Visible Spectroscopy (857).) tion spectrum between 250 and 300 nm of this solution
Mode: UV exhibits maxima and minima at the same wavelengths as
Analytical wavelength: 230 nm that of a similar solution of USP Loperamide Hydrochloride
Cell length: 1cm RS, concomitantly measured.
Analysis Loss on drying (731)—Dry it in vacuum at 80° for 4 hours:
Samples: Standard solution and Sample solution it loses not more than 0.5% of its weight.
Calculate the percentage of the labeled amount of
lomustine (CsHisCIN302) in the Capsule taken:
Result = (Au/As) x (Cs/Cu) x 100
USP 41
Residue on ignition (281): not more than 0.2%.
Delete the following:
°Heavy metals, Method !/ (231): 0.002%. core 1an-2018)
Chromatographic purity—Prepare a test solution in chlo-
roform containing 10 mg per mL. Apply 10 uL of this solu-
tion and 10 wL of a Standard solution of USP Loperamide
Hydrochloride RS in chloroform containing 10 mg per mL to
a thin-layer chromatographic plate (see Chromatography
(621)) coated with a 0.25-mm layer of chromatographic sil-
ica gel mixture. Allow the spots to dry, and develop the
chromatogram in a solvent system consisting of a mixture of
chloroform, methanol, and formic acid (85:10:5) until the
solvent front has moved about three-fourths of the length of
the ree Remove the plate from the developing chamber,
mark the solvent front, and allow the plate to air-dry. Locate
the spots on the plate by exposing it to fumes of iodine: the
spot obtained from the test solution corresponds in R, value,
color, and intensity to that obtained from the Standard solu-
tion, and no secondary spots are observed.
Chloride content—Using about 13 mg, accurately
weighed, proceed as directed under Oxygen Flask Combus-
tion (471), using a mixture of 10 mL of 0.02 N sodium hy-
droxide and 2 drops of 30 percent hydrogen peroxide as the
absorbing liquid. When combustion is complete and the
combustion gases absorbed, rinse the stopper, sample
holder, and inner walls of the flask with 50 mL of isopropyl
alcohol. Add 4 mL of 0.1 N nitric acid, and titrate with 0.01
N mercuric nitrate VS, using diphenylcarbazone TS as the
indicator. Each mL of 0.01 N mercuric nitrate is equivalent
to 0.3545 mg of chlorine: between 13.52% and 14.20% is
found.
Assay—
Neutralized acetic acid—Dissolve 10 mg of a-naphthol-
benzein in 100 mL of glacial acetic acid, and titrate with 0.1
N perchloric acid to a green endpoint, disregarding the
amount of titrant consumed.
Procedure—Dissolve about 375 mg, accurately weighed,
of Loperamide Hydrochloride in 25 mL of Neutralized acetic
acid. Add 10 mL of mercuric acetate solution (prepared b'
dissolving 1 g of mercuric acetate in 33 mL of Neutralize
acetic acid) and titrate with 0.1 N perchloric acid VS to the
original green color of the Neutralized acetic acid. Each mL
of 0.1 N perchloric acid is equivalent to 51.35 mg of
C29H33CIN2O2 - HCI.
Loperamide Hydrochloride Capsules
» Loperamide Hydrochloride Capsules contain
not less than 90.0 percent and not more than
110.0 percent of the labeled amount of loper-
amide hydrochloride (C29H33CIN2O2 - HCI).
Packaging and storage—Preserve in well-closed contain-
ers.
USP Reference standards (11)—
USP Loperamide Hydrochloride RS
Identification—
A: Thin-Layer Chromatographic Identification Test (201)—
Test solution—Transfer a quantity of the contents of the
Capsules equivalent to about 10 mg of loperamide hydro-
chloride, to a 37-mL stoppered vial, add 10 mL of methanol,
shake for 5 minutes, and filter.
Standard solution: a solution of USP Loperamide Hydro-
chloride RS in methanol containing about 10 mg per mL.
Official Monographs / Loperamide 2447
Application volume: 10 uL of the Test solution and 1 wl
of the Standard solution.
Developing solvent system: a mixture of chloroform,
methanol, and formic acid (85:10:5).
Procedure—Proceed as directed in the chapter. Visualize
the spots by exposing to iodine vapors.
B: The retention time of the major peak in the chromato-
gram of the Assay preparation corresponds to that in the
chromatogram of the Standard preparation, as obtained in
the Assay.
Dissolution (711)—
Medium: pH 4.7 acetate buffer, prepared by mixing
200 mL of 1 N acetic acid with 600 mL of water, adjusting
with 1 N sodium hydroxide to a pH of 4.70 + 0.05, diluting
with water to 1000 mL, and mixing; 500 mL.
Apparatus 1: 100 rpm.
Time: 30 minutes.
Determine the amount of loperamide hydrochloride dis-
solved using the following method.
Mobile phase and Chromatographic system—Proceed as di-
rected in the Assay.
Procedure—nject a volume (about 50 pL) of a filtered
portion of the solution under test into the chromatograph,
record the chromatogram, and measure the response for
the major peak. Calculate the quantity of C2sH3sCIN2Oz - HCI
dissolved in comparison with a Standard solution having a
known concentration of USP Loperamide Hydrochloride RS
in the same medium and similarly chromatographed.
Tolerances—Not less than 80% (Q) of the labeled amount
of C29H33CIN2O2 « HCI is dissolved in 30 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Assay—
Mobile phase—Transfer 500 mL of acetonitrile to a
1000-mL volumetric flask. Dilute with water to volume, add Cc
20 drops of phosphoric acid, mix, and filter. Make adjust- “nn
uv
ments if necessary (see System Suitability under Chromatog-
raphy (621)). c=
Standard preparation—Dissolve an accurately weighed °
|
quantity of USP Loperamide Hydrochloride RS in a mixture °
of acetonitrile and 0.5 N hydrochloric acid (1:1) to obtain a ©
x
solution having a known concentration of about 0.2 mg per i
mL. Transfer 5.0 mL of this solution to a 100-mL volumetric mo}
ae
flask, dilute with a mixture of acetonitrile and water (1:1) to a
volume, and mix to obtain a solution having a known con-
centration of about 10 ug per mL.
Assay preparation—Transfer, as completely as possible,
the contents of not less than 20 Capsules to a suitable tared
container, and determine the average weight per capsule.
Mix the combined contents, and transfer an accurately
weighed portion of the powder, equivalent to about 20 mg
of loperamide hydrochloride, to a 100-mL volumetric flask.
Add about 35 mL of 0.5 N hydrochloric acid and sonicate
for 15 minutes. Add 35 mL of acetonitrile and sonicate for
an additional 15 minutes. Dilute with a mixture of acetoni-
trile and 0.5 N hydrochloric acid (1:1) to volume, mix, and
filter. Transfer 5.0 mL of this solution to a 100-mL volumet-
tic flask, dilute with a mixture of acetonitrile and water (1:1)
to volume, and mix.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 220-nm detector
and a 4-mm x 25-cm column that contains 10-um packing
L10. The flow rate is about 2 mL per minute. Chromato-
graph the Standard preparation, and record the peak re-
sponses as directed under Procedure: the column efficiency,
N, determined from the analyte peak is not less than 1900
theoretical plates, the capacity factor, kK’, is not less than
3.5, and the relative standard deviation for replicate injec-
tions is not more than 2.0%.
Procedure—Separately inject equal volumes (about 50 wL)
of the Standard preparation and the Assay preparation into
 2448 Loperamide / Official Monographs
the chromatograph, record the chrematpara’ and meas-
ure the responses for the major peaks. Calculate the quan-
yy in mg, of C2sH33CIN2O2 - HCI in the portion of Capsules
taken by the formula:
2000C(ry / rs)
in which C is the concentration, in mg per mL, of USP
Loperamide Hydrochloride RS in the Standard preparation;
and ry and fs are the peak responses obtained from the
Assay preparation and the Standard preparation, respectively.
Loperamide Hydrochloride Oral
Solution
DEFINITION
Loperamide Hydrochloride Oral Solution contains NLT
90.0% and NMT 110.0% of the labeled amount of loper-
amide hydrochloride (C29H33CIN2O2 - HCl).
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
Sample: Transfer a volume of Oral Solution containing
a suitable amount of loperamide hydrochloride (typi-
cally 12-24 mg) to a separator containing about
100 mL of water and 1 mL of 50% sodium hydroxide
solution, and gently swirl the contents. Add 50 mL of
methylene chloride, shake gently by hand, releasing
pressure often, and then shake by mechanical means
for 20 min. Allow the layers to separate. Transfer the
lower methylene chloride layer to a separator contain-
ing 100 mL of water. Shake gently by hand, releasing
pressure often, and then shake by mechanical means
for 10 min. Allow the layers to separate. Transfer the
a]
a lower methylene chloride layer to a 250-mL beaker, and
a
i}
evaporate to dryness on a steam bath with the aid of a
— current of air. Add 10 mL of methanol and 500 mg of
=) potassium bromide to the beaker. Evaporate to dryness
° on a steam bath with the aid of a current of air, and
S SPECIFIC TESTS
iS) use the residue.
= Acceptance criteria: The spectrum obtained from the
Sample shows bands at approximately 3400 cm,
[a
a) 2929 cm, 1624 cm-!, and 1493 cm“, similar to the
=) spectrum from a Standard similarly obtained.
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
ASSAY
© PROCEDURE
Buffer: 3.0 g/L of monobasic potassium phosphate in
water
Mobile phase: Acetonitrile and Buffer (37:63), adjusted
with 5% phosphoric acid to a pH of 3.0
Standard stock solution: Prepare 2 mg/mL of USP
Loperamide Hydrochloride RS in methanol. Dilute this
solution with water to obtain a 0.1-mg/mL solution.
Standard solution: 0.01 mg/mL of USP Loperamide
Hydrochloride RS in Mobile phase from the Standard
stock solution
Sample solution: Nominally 0.01 mg/mL of loperamide
Rvarochlotlde from Oral Solution, prepared as follows.
Transfer a volume of Oral Solution, equivalent to about
1.0 mg of loperamide hydrochloride, to a 100-mL volu-
metric flask. Dilute with Mobile phase to volume, mix,
and filter.
Chromatographic system
(See Chromatography (621), System Suitability.)
USP 41
Mode: LC
Detector: UV 214 nm
Column: 4.0-mm x 8.0-cm; 5-14m packing L7, 4.6-mm
x 7.5-cm; 3.5-um packing L7, or 4.6-mm x 12.5-cm;
5-"um packing L7
Flow rate: 1.5 mL/min
Injection volume: 10 uL
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
loperamide hydrochloride (C29H33CIN2O2 - HCl) in the
portion of Oral Solution taken:
Result = (ru/rs) x (Cs/Cu) x 100
ty = peak area from the Sample solution
Is = peak area from the Standard solution
Cs = concentration of USP Loperamide
Hydrochloride RS in the Standard solution
(mg/mL)
Cy = nominal concentration of loperamide
hydrochloride in the Sample solution
(mg/mL)
Acceptance criteria: 90.0%-110.0%
OTHER COMPONENTS
e ALCOHOL DETERMINATION, Method I/ (611) (if present):
90.0%-110.0% of the labeled amount of alcohol
(C2HsOH)
PERFORMANCE TESTS
e UNIFORMITY OF DOSAGE UNITS (905)
For single-unit containers
Acceptance criteria: Meets the requirements
e DELIVERABLE VOLUME (698)
For multiple-unit containers
Acceptance criteria: Meets the requirements
e PH (791): 2.7-6.5
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers. Store below 40°, preferably between 15° and
30°, unless otherwise specified by the manufacturer.
e USP REFERENCE STANDARDS (11)
USP Loperamide Hydrochloride RS
Loperamide Hydrochloride Tablets
DEFINITION
Loperamide Hydrochloride Tablets contain NLT 90.0% and
NMT 110.0% of the labeled amount of loperamide hydro-
chloride (C29H33CIN2O>2 - HCl).
IDENTIFICATION
eA.
ULTRAVIOLET ABSORPTION (197U)
[Note—This procedure is not applicable for Tablets la-
beled as chewable.]
Wavelength range: 250-300 nm
Seandard solutions About 0.4 mg/mL of USP Loper-
amide Hydrochloride RS, prepared as follows. Transfer
an amount of USP Loperamide Hydrochloride RS to a
suitable volumetric flask, dissolve first in fonteey alco-
hol, using 50% of the final volume. Add 0.1 N hydro-
chloric acid equivalent to 10% of the final volume,
and dilute with isopropyl alcohol to volume.
USP 41 Official Monographs / Loperamide 2449

Sample solution: Transfer a quantity of fey pow- rs = peak response from the Standard solution
dered Tablets equivalent to about 10 mg of loper- Gs = concentration of USP Loperamide
amide hydrochloride to a test tube. Add 20.0 mL of Hydrochloride RS in the Standard solution
isopropyl alcohol, shake by mechanical means for 1 (mg/mL) . J
min, and allow to settle. Pipet 9.0 mL of the superna- Cu = nominal concentration of loperamide
tant into a 10-mL volumetric flask, and dilute with 0.1 hydrochloride in the Sample solution
N hydrochloric acid to volume.
Acceptance criteria: The spectrum of the Sample solu-
(mg/mL)
Acceptance criteria: 90.0%-110.0%
tion exhibits maxima and minima at the same wave-
lengths as those of the Standard solution, concomi- PERFORMANCE TESTS
tantly measured. e DISSOLUTION (711)
THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST (201) Test 1
[Note—For Tablets labeled as chewable, use the follow- Medium: 0.01 N hydrochloric acid; 900 mL
ing procedure.] Apparatus 2: 50 rpm
Standard solution: 1.0 mg/mL of USP Loperamide Hy- Time: 30 min
drochloride RS in methanol Standard solution: USP Loperamide Hydrochloride RS
Sample solution: Grind a number of Tablets, equiva- at a known concentration in Medium. [NoTe—lIf neces-
lent to 10 mg of loperamide hycrochionde, with sary, dissolve USP Loperamide Hydrochloride RS in a
10 mL of methanol for about 2 min. Centrifuge the minimal amount of methanol, and then dilute with
mixture, and use the supernatant. Medium to final concentration.]
Application volume: 10 pL Sample solution: Filtered solution under test
Developing solvent system: Chloroform, methanol, Buffer: Transfer 3.0 g of triethylamine hydrochloride
and formic acid (75:25:1) and 1.0 mL of phosphoric acid to a 1-L flask, and add
Analysis: Visualize the spots by using Dragendorff’s TS. 550 mL of water.
Acceptance criteria: Meet the requirements Mobile phase: Acetonitrile and Buffer (45:55)
e B. The retention time of the major peak of the Sample Chromatographic system
solution corresponds to that of the Standard solution, as (See Chromatography (621), System Suitability.)
obtained in the Assay. Mode: LC
Detector: UV 214 nm
ASSAY Column: 4.6-mm x 7.5-cm; 3.5-'um packing L7 or
¢ PROCEDURE 4.6-mm x 12.5-cm; 5-um packing L7
Solvent mixture: Methanol and acetonitrile (3:1) Flow rate: 1.5 mL/min
lon pairing solution: Solution containing 2.35 g/L of Injection volume: 50 LL
sodium 1-hexanesulfonate and 2.88 g/L of monobasic System suitability
ammonium phosphate in water, adjusted with phos- Sample: Standard solution
phoric acid to a pH of 3.2 Suitability requirements
Mobile phase: Solvent mixture and lon pairing solution Tailing factor: NMT 2.0
(55:45) Relative standard deviation: NMT 2.0% S
System suitability solution: 0.2 mg/mL of USP Loper- Analysis wn
amide Hydrochloride RS and 0.002 mg/mL of USP Samples: Standard solution and Sample solution z
Loperamide Related CompoundF RS in Mobile phase Calculate the percentage of the labeled amount of S
Standard solution: 0.2 mg/mL of USP Loperamide Hy- loperamide hydrochloride (C2sH33CIN2O2« HCl) i)
drochloride RS in Mobile nae dissolved: S
i)
Sample solution: Fill a 100-mL volumetric flask with ro)=
Mobile phase. Immediately transfer a number of Tablets Result = (ru/rs) x (Gs/L) x Vx 100 2
equivalent to 20 mg of loperamide hydrochloride to the 3
flask, and cap tightly. Sonicate for 15-30 min with in- tu = peak response from the Sample solution =
termittent shaking. Allow the contents to settle, and use ts = peak response from the Standard solution w

a clear supernatant. Gs = concentration of the Standard solution

Chromatographic system (mg/mL)
(See Chromatography (621), System Suitability.) L = label claim (mg/Tablet)
Mode: LC Vv = volume of Medium, 900 mL
Detector: UV 219 nm Tolerances: NLT 80% (Q) of the labeled amount of
Column: 3.9-mm x 15-cm; 5-um or 10-um packing L1 loperamide hydrochloride (C29H33CIN2Oz - HCl) is
Flow rate: 2 mL/min dissolved.
Injection volume: 50 uL Test 2: If the product complies with this test, the label-
System suitability ing indicates that the product meets USP Dissolution
Samples: System suitability solution and Standard Test 2.
solution Medium: 0.01 N hydrochloric acid; 900 mL
Suitability requirements Apparatus 2: 50 rpm
Resolution: NLT 3.0 between loperamide and loper- Time: 10 min
amide related compound F, System suitability solution Standard stock solution: 0.44 mg/mL of USP Loper-
Tailing factor: NMT 2.0 for both peaks, System suita- amide Hydrochloride RS in methanol. Use sonication
bility solution as necessary to dissolve.
Relative standard deviation: NMT 2.0%, Standard Standard solution: 0.0022 mg/mL of USP Loperamide
solution Hydrochloride RS in Medium, from the Standard stock
Analysis solution
Samples: Standard solution and Sample solution Sample solution: Pass a portion of the solution under
Calculate the percentage of the labeled amount of test through a suitable membrane filter of 0.45-4m
loperamide hydrochloride (C29H33CIN2O2 - HCl) in the pore size, discarding first few milliliters of the filtrate.
portion of Tablets taken: Buffer: Transfer 3.0 g of triethylamine hydrochloride
and 1.0 mL of phosphoric acid to a 1-L flask, and add
Result = (ru/rs) x (Cs/Cy) x 100 550 mL of water.
tu = peak response from the Sample solution
 2450 Loperamide / Official Monographs USP 41

Mobile phase: Acetonitrile and Buffer (40:60) Table 1 (Continued)

Chromatographic system Relative Accent
(See Chromatography (621), System Suitability.) . Sess,
. Retention Criteria,
Mode: LG UV 214 nm
Detector: - Nami = Time NMT (%)
2
Column: 4.6-mm x 15-cm; 5-uum packing L7 Loperamide trans-N-oxide
Flow rate: 2.0 mL/min (loperamide related compound F) NES}
Injection volume: 50 uL Loperamide cis-N-oxide> 187. 2.08
System suitability For the sum of trans and cis isomers.
Sample: Standard solution ba §40/4-(4-Chlorophenyl)-1 -[4(dimethylamino)-4-oxo-3,3-
Suitability requirements diphenylbutyl]-4-hydroxypiperidine 1-oxide.
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0%
Analysis . , ADDITIONAL REQUIREMENTS
Samples: Standard solution and Sample solution é ; ;
Calculate the percentage of the labeled amount of ° PACKAGINGANDSTORAGE: Presenvelin wellsclosed, light:
loperamide hydrochloride (CasHssCINzO2 - HCl) e LABELING: Label chewable Tablets to indicate that they
. are to be chewed before swallowing. When more than
_ wayne one Dissolution test is given, the labeling states the Disso-
Result = (rulrs) x (Cs/L) x Vx 100 lution test used only if Test 7 is not used.
tu = peak response from the Sample solution eo USP REFERENCE STANDARDS (11) :
Is = peak response from the Standard solution USP Loperamide Hydrochloride RS
Cs = concentration of the Standard solution USP Loperamide Related Compound F RS
(mg/mL) Loperamide trans-N-oxide; ; ;
L = label claim (mg/Tablet) (11,4s)-4-(4-Chlorophenyl)-1-[4-(dimethylamino)-4-oxo-
V = volume of Medium, 900 mL 3,3-diphenylbutyl|-4-hydroxypiperidine 1-oxide.
Tolerances: NLT 80% (Q) of the labeled amount of Ca9H33CIN203 493.04
loperamide hydrochloride (Cz9H33CIN2O2 + HCl) is
dissolved.
e UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
Lopinavir
IMPURITIES
© ORGANIC IMPURITIES a
Solvent mixture, lon pairing solution, Mobile phase, ( \
System suitability solution, Sample solution, and a NES Ht
“ Chromatographic system: Proceed as directed in the J Ao As JLo |
= Assay. eid AP an I 5]
3S Standard solution: 0.002 mg/mL of USP Loperamide yo ° a ne
a Related Compound F RS in Mobile phase [ 3
} System suitability Y
i= Sample: System suitability solution
i} Suitability requirements C37HagN4Os 628.80
= Resolution: NLT 3.0 between loperamide and loper- [15-[1R*(R%),3R*,4R*]]-N-[4][(2,6-Dimethylphenoxy)
o amide related compound F acetyl]Jamino]-3-hydroxy-5-phenyl-
a
SS Tailing
i factor: NMT 2.0 for both peaks a (atieaylimetiw pent)
wee -tetrahydro-c-(1-methylethyl)-
Analysis . . 2-0xo-1(2H)-pyrimidineacetamide;
Samples: Sample solution and Standard solution . (aS)-Tetrahydro-N-[(0.5)-c.-[(25, 35)-2-hydroxy-4-phenyl-3-
Calculate the percentage of loperamide N-oxide in the [2-(2,6-xylyloxy)acetamido]butyl]phenethyl]-c-isopropyl-
portion of Tablets taken: 2-0xo-1(2H)-pyrimidineacetamide [192725-17-0].
Result = (r7/rs) « (Cs/Cu) x 100 DEFINITION
4 Lopinavir contains NLT 98.0% and NMT 102.0% of
tr = sum of the peak responses of the cis and trans lopinavir (C37H4gN4Os), calculated on the anhydrous basis.
isomers of N-oxide from the Sample solution
Is = peak response of loperamide related IDENTIFICATION
compoundF from the Standard solution e A. INFRARED ABSORPTION (197A)
Cs = concentration of USP Loperamide Related e B. The retention time of the lopinavir peak of the Sample
Compound F RS in the Standard solution solution corresponds to that of the Standard solution, as
(mg/mL) obtained in the Assay.
cy = nominal concentration of loperamide
hydrochloride in the Sample solution ASSAY
(mg/mL) © PROCEDURE
Acceptance criteria: See Table 1. Buffer: 2.7 g/L of monobasic potassium phosphate and
0.9 g/L of dibasic potassium phosphate in water. Adjust
with phosphoric acid to a pH of 6.0. Pass the solution
Table 1 througha suitable filter of 0.45-um pore size.
Relative Acceptance Diluent: Acetonitrile and water (1:1)
Retention Criteria, Solution A: Acetonitrile and Buffer (9:11)
Name Time NMT (%) Mobile phase: Solution A
Loperamide 1.0 = Azanigars) solutions: 0.025 mg/mL of USP Lopinavir RS
?For the sum of trans and cis isomers.
bd SAO a Chloropheny!)-1 -[4-(dimethylamino)-4-0xo-3,
3-
diphenylbuty!]-4-hydroxypiperidine 1-oxide.
USP 41 Official Monographs / Lopinavir 2451

Sample solution: 0.025 mg/mL of Lopinavir in Diluent System suitability solution: 0.5 mg/mL of USP
Chromatographic system Lopinavir System Suitability Mixture RS in Diluent
(See Chromatography (621), System Suitability.) Standard solution: 0.005 mg/mL of USP Lopinavir RS
Mode: LC in Diluent
Detector: UV 215 nm Sample solution: 0.5 mg/mL of Lopinavir in Diluent
Column: 4.6-mm x 25-cm; 4-'4m packing L1 Chromatographic system
Column temperature: 50° (See Chromatography (621), System Suitability.)
Flow rate: 1 mL/min Mode: LC
Injection volume: 20 uL Detector: UV 215 nm
Run time: 60 min Column: 4.6-mm x 25-cm; 4-m packing L1
System suitability Column temperature: 50°
Sample: Standard solution Flow rate: 1 mL/min
Suitability requirements Injection volume: 20 pL
Column efficiency: NLT 8000 theoretical plates Run time: 100 min
Capacity factor: NLT 15 [Note—Data collection is only for the first 60 min. The
Tailing factor: 0.8-1.5 remaining godien steps wash out the late-eluting im-
Relative standard deviation: NMT 2.0% purities and re-equilibrate the column.]
Analysis System suitability
Samples: Standard solution and Sample solution Samples: System suitability solution and Standard
Calculate the percentage of lopinavir (C37H4gsN4Os) in solution
the portion of Lopinavir taken: [Note—The relative retention times are listed in
Table 2.]
Result = (ru/rs) x (Cs/Cu) x 100 Suitability requirements
Resolution: NLT 1.2 between lopinavir N-formylphe-
ty = peak response from the Sample solution noxyacetamide and lopinavir N-acetylphenoxy-
ls = peak response from the Standard solution acetamide, System suitability solution
Gs = concentration of USP Lopinavir RS in the Capacity factor: NLT 15, Standard solution
Standard solution (mg/mL) Column efficiency: NLT 8000, Standard solution
Cy = concentration of Lopinavir in the Sample Tailing factor: 0.8-1.5, Standard solution
solution (mg/mL) Relative standard deviation: NMT 3.0%, Standard
Acceptance criteria: 98.0%-102.0% on the anhydrous solution
basis Analysis
IMPURITIES Samples: Diluent, System suitability solution, Standard
e RESIDUE ON IGNITION (281): NMT 0.2%
solution, and Sample solution
Calculate the percentage of each lopinavir related impu-
rity and unidentified impurity in the portion of
Delete the following: Lopinavir taken:
(=
°e HEAVY METALS, Method {1 (231): NMT 20 tig/ge corrcia. Result = (ru/rs) x (Cs/Cu) x (1/F) x 100 3
Jan-2018)
e@ ORGANIC IMPURITIES: PROCEDURE 1
ty = peak response of each impurity from the <
[Note—For early-eluting impurities.] Sample solution °
Buffer, Diluent, and Solution A: Prepare as directed in rs = peak response of lopinavir from the Standard SS
the Assay. solution iol
Solution B: Acetonitrile and Buffer (3:1) Gs = concentration of USP Lopinavir RS in the Ss
Mobile phase: See Table 7. Standard solution (mg/mL) cs
Cu = concentration of Lopinavir in the Sample ra
solution (mg/mL)
Table 1 F = relative response factor (see Table 2)
Solution A Solution B
 2452 Lopinavir / Official Monographs USP 41

Table 2 Mode: LC
Relative Detector: UV 215 nm
Relative | Response Criteria, Column: 4.6-mm x 25-cm; 4-4m packing L1
%’
Column temperature: 50°
Flow rate: 1 mL/min
amine? 0.1
Injection volume: 20 uL
Lopinavir N-formylami- Run time: 50 min
System suitability
ites Sample: Standard solution
If inavird [Note—The relative retention times are listed in
Lopinavir phenoxy- Table 3.]
Suitability requirements
Lopinavir N-formylphe- oo factor: NLT 1.5
Column efficiency: NLT 3000
Tailing factor: 0.8-1.5
Lopinavir N-acetylphe- Relative standard deviation: NMT 3.0%
ides Analysis
Samples: Diluent, System suitability solution, Standard
solution, and Sample solution
Calculate the percentage of each lopinavir related impu-
Lopinavir 2,4-phenoxy rity and unidentified impurity in the portion of
Lopinavir taken:
Lopinavir D-valine dias-
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
tu = peak response of each impurity from the
Lopinavir (2R,4R) diaster- Sample solution
rm rs = peak response of lopinavir from the Standard
ir (4 solution
Any other individual im- Gs = concentration of USP Lopinavir RS in the
1 Standard solution (mg/mL)
2 (S)-N-[(25,45,55)-5-Amino-4-hydroxy-1 ,6-diphenylhexan-2-yl]-3-methyl-2-
Cy = concentration of Lopinavir in the Sample
[2-oxotetrahydropyrimidin-1 (2#)-yl]butanamide. solution (mg/mL)
» (S)-N-[(25,45,55)-5-Formamido-4-hydroxy-1,6-diphenylhexan-2-yl]-3- F = relative response factor (see Table 3)
methyl-2-[2-oxotetrahydropyrimidin-1 (2H)-yl]butanamide.
© (25,2'S)-N,N’-[(25,35,55)-3-Hydroxy-1,6-diphenylhexane-2,5-diyl]bis{3- Table 3
methyl-2-[2-oxotetrahydropyrimidin-1 (2H)-yl]butanamide}.
es 4 (25,35,55)-2-[2-(2,6-Dimethylphenoxy)acetamido]-5-{(5)-3-methyl-2-[2- Relative
i oxotetrahydropyrimidin-1(2H)-yl]butanamido}-1,6-diphenylhexan-3-yl hy- Relative Response Criteria,
a drogen sulfate.
Retention F.
i] © N-[(25,35,55)-5-Amino-3-hydroxy-1,6-diphenylhexan-2-yl]-2-(2,6-
7
i) dimethylphenoxy)acetamide.
° f 2-(2,6-Dimethylphenoxy)-N-[(25,35,55)-5-formamido-3-hydroxy-1,6- Lopinavir 1.49
¢ diphenylhexan-2-yllacetamide.
S 9 N-[(25,35,55)-5-Acetamido-3-hydroxy-1,6-diphenylhexan-2-yl]-2-(2,6-
Lopinavir (2R)
= dimethylphenoxy)acetamide.
1.91
» N-{(5)-1-[(45,65)-4-Benzyl-2-oxo-1,3-oxazinan-6-yl]-2-phenylethyl}-2-(2,6- Li 4.39
r.0
” dimethylphenoxy)acetamide.
=) (S)-N-{(25,35,55)-5-[2-(2,6-Dimethylphenoxy)acetamido]-3-hydroxy-1,6- Lopinavir O-phenox-
diphenylhexan-2-yl}-3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)-
ylbutanamide.
1(S)-N-{(25,45,55)-5-[2-(2,4-Dimethylphenoxy)acetamido]-4-hydroxy-1,6- Lopinavir amino-
diphenylhexan-2-yl}-3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)- alcohol 8.46 1
ylbutanamide.
k (R)-N-{(25,45,55)-5-[2-(2,6-Dimethylphenoxy)acetamido]-4-hydroxy-1,6- a(5)-{(25,35,55)-2-[2-(2,6-Dimethylphenoxy)acetamido]-5-[(5)-3-methyl-2-
diphenylhexan-2-yl}-3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)- (2-oxotetrahydropyrimidin-1(2H)-y!)butanamido]-1,6-diphenylhexan-3-yl}
yllbutanamide. 3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)-yl]outanoate.
'(Z)-N,N'-(Ethene-1,2-diyl)bis[2-(2,6-dimethylphenoxy)acetamide]. » (SPN 2BA555) os 2-t2.6-Dimethy pnenexyjaceamides tyatony:] /6-
diphenylhexan-2-yl}-3-methyl-2-[2-oxotetrahydropyrimidin-1 (2H)-
m(SN ZRARS) S22 6 Dimmetiyipnene sy acetamido]-4-hydroxy-1,6- yllbutanamide
diphenylhexan-2-yl}-3-methyl-2-[2-oxotetrahydropyrimidin-1 (2H)- ¢ N,N’-[(2S,35,55)-3-Hydroxy-1,6-diphenylhexane-2,5-diy|]bis[2-(2,6-
yl]butanamide.
dimethylphenoxy)acetamide].
9 (S)-N-{(25,4R,55)-5-[2-(2,6-Dimethylphenoxy)acetamido]-4-hydroxy-1,6-
diphenylhexan-2-yl}-3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)- 4 (S)-N-{(25,45,55)-5-[2-(2,6-Dimethylphenoxy)acetamido]-4-hydroxy-1,6-
diphenylhexan-2-yl}-2-{3-[2-(2,6-dimethylphenoxy)acetyl]-2-oxote-
yl]butanamide. trahydropyrimidin-1 (2H)-yl}-3-methylbutanamide.
© ORGANIC IMPURITIES: PROCEDURE 2 © (25,35,55)-2-[2-(2,6-Dimethylphenoxy)acetamido]-5-{(5)-3-methyl-2-[2-
oxotetrahydropyrimidin-1 (2H)-yl]butanamido}-1,6-diphenylhexan-3-yl 2-
[Note—For late-eluting impurities.] (2,6-dimethylphenoxy)acetate.
Buffer, Diluent, and Solution A: Prepare as directed in f N,N’-(2S,2’S,35,3’S,55,5’S)-5,5’-Carbonylbis(azanediyl)bis(3-hydroxy-1,6-
the Assay. diphenylhexane-5,2-diyl)bis[2-(2,6-dimethylphenoxy)acetamide].
Solution B: Acetonitrile and Buffer (3:1) 3 Exclude from Organic Impurities, Procedure 2, \opinavir (4R) epimer and
Mobile phase: Solution A and Solution B (3:7) any other peak eluting prior to this peak because these are already moni-
tored in Procedure 1.
System suitability solution: 0.5 mg/mL of USP
Lopinavir System Suitability Mixture RS in Diluent
Standard solution: 0.005 mg/mL of USP Lopinavir RS
in Diluent
Sample solution: 0.5 mg/mL in Diluent
Chromatographic system
(See Chromatography (621), System Suitability.)
USP 41 Official Monographs / Lopinavir 2453

Table 3 (Continued) Standard stock solution: 0.1 mg/mL each of USP

Relative Acceptance} Lopinavir RS and USP Ritonavir RS in Solution B
Relative Response Criteria,
Standard solution: 0.025 mg/mL each of USP Lopinavir
Name Retention Factor NMT (%)
RS and USP Ritonavir RS in Solution B from the Standard
Stock solution
Any other individual Sample stock solution: Nominally 4 mg/mL of lopinavir
impurity — 1.0 0.1 and 1 mg/mL of ritonavir in Solution A prepared as fol-
Total impurities from lows. Transfer a volume of Oral Solution equivalent to
Procedure 1 and =. 400 mg of lopinavir and 100 mg of ritonavir to a
Procedure 2 1.0 0.79 100-mL volumetric flask with the aid of several small
# (S)-{(25,35,55)-2-[2-(2,6-Dimethylphenoxy)acetamido]-5-[(5)-3-methyl-2- portions of Solution A, and then dilute with Solution A
(2-oxotetrah dropyrimidin-1(2H)-ybutanamido]-1,6-diphenyihexan-3-yi} to volume.
3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)-yl]butanoate.
6(SNC 455 pieate,b-Dirnctny pheno pacetamidgh hyarony-,6-
Sample solution: Nominally 0.05 mg/mL of lopinavir
diphenylhexan-2-yl}-3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)- and 0.0125 mg/mL of ritonavir in Solution B from the
yljbutanamide. Sample stock solution
© N,N’-[(25,35,55)-3-Hydroxy-1 ,6-diphenylhexane-2,5-diyl]bis[2-(2,6- Chromatographic system
dimethylphenoxy)acetamide]. (See Chromatography (621), System Suitability.)
4 (S)-N-{(25,45,55)-5-[2-(2,6-Dimethylphenoxy)acetamido]-4-hydroxy-1,6- Mode: LC
diphenylhexan-2-yl}-2-{3-[2-(2,6-dimethylphenoxy)acetyl]-2-oxote-
trahydropyrimidin-1(2H)-yl}-3-methylbutanamide. Detector: UV 215 nm
€ (25,35,55)-2-[2-(2,6-Dimethylphenoxy)acetamido]-5-{(S)-3-methyl-2-[2- Column: 4.6-mm x 15-cm; 5-um packing L7
oxotetrahydropyrimidin-1 (2H)-yl]butanamido}-1,6-diphenylhexan-3-yl 2- Column temperature: 40°
(2,6-dimethylphenoxy)acetate. Flow rate: 1.5 mL/min
1 N,N’-(25,2’S,35,3’S,55,5’S)-5,5’-Carbonylbis(azanediyl)bis(3-hydroxy-1,6- Injection volume: 50 uL
diphenylhexane-5,2-diyl)bis[2-(2,6-dimethylphenoxy: acetamide], System suitability
9 Exclude from Organic Impurities, Procedure 2, lopinavir (4R) epimer and
any other peak eluting prior to this peak because these are already moni-
Sample: Standard solution
tored in Procedure 1. Suitability requirements
Tailing factor: 0.8-1.2 for the ritonavir peak
Acceptance criteria: See Table 2 and Table 3. Relative standard deviation: NMT 2.0% each for
ritonavir and lopinavir
SPECIFIC TESTS Analysis
© WATER DETERMINATION, Method | (921): NMT 4.4% Samples: Standard solution and Sample solution
ADDITIONAL REQUIREMENTS Calculate the percentages of the labeled amounts of
© PACKAGING AND STORAGE: Preserve in tight containers. lopinavir (C37H4sN4Os) and ritonavir (C37H4sNeOsSz) in
Store at room temperature. the portion of Oral Solution taken:
e USP REFERENCE STANDARDS (11)
Result = (ru/rs) x (Cs/Cu) x 100
USP Lopinavir RS
USP Lopinavir System Suitability Mixture RS
Lopinavir System Suitability Mixture contains lopinavir (omy
N-formylphenoxyacetamide, lopinavir N-acetylphenoxy- ty = peak response of the corresponding analyte a]
acetamide, and several other minor components. from the Sample solution
Lopinavir N-formylphenoxyacetamide is (2-(2,6- rs = peak response of the corresponding analyte E
dimethylphenoxy)-N-[(25,35,55)-5-formamido-3-hy- from the Standard solution =
droxy-1,6-diphenylhexan-2-yl]acetamide. Cs = concentration of USP Lopinavir RS or USP ro)
CosH34N204 474.59 Ritonavir RS in the Standard solution @
Lopinavir N-acetylphenoxyacetamide is (N-[(25,35,55)- (mg/mL) Ey
5-acetamido-3-hydroxy-1,6-diphenylhexan-2-yl]-2-(2,6- Gu = nominal concentration of lopinavir or ritonavir es
dimethylphenoxy)acetamide. in the Sample solution (mg/mL) a
C3oH36N20, 488.62 Acceptance criteria: 90.0%-110.0% of the labeled
amounts of Sia (C37HagN4Os) and ritonavir
(C37HagNeOsS2

Lopinavir and Ritonavir Oral Solution
DEFINITION
Lopinavir and Ritonavir Oral Solution contains NLT 90.0%
and NMT 110.0% of the labeled amounts of lopinavir
(C37HasN4Os) and ritonavir (C37HasNeOsSz).
IDENTIFICATION
e A. The retention times of the lopinavir and ritonavir
peaks of the Sample solution correspond to those of the
Standard solution, as obtained in the Assay.
ASSAY
© LOPINAVIR AND RITONAVIR
Buffer: 4.1 g/L of monobasic potassium phosphate in
water
Solution A: Acetonitrile and Buffer (65:35)
Solution B: Acetonitrile and Buffer (50:50)
Mobile phase: Acetonitrile, methanol, tetrahydrofuran,
and Buffer (175:100:100:625). Separately filter the Buffer
and the premixed solvents before combining them to
make the Mobile phase.
PERFORMANCE TESTS
e DELIVERABLE VOLUME (698)
For multiple-unit containers
Acceptance criteria: Meets the requirements
IMPURITIES
@ ORGANIC IMPURITIES
Buffer A: 4.1 g/L of monobasic potassium phosphate in
water
Buffer B: 3.8 g/L of monobasic potassium phosphate
and 0.25 g/L of dibasic potassium phosphate in water
Solution A: Acetonitrile and BufferA (50:50)
Solution B: Acetonitrile, butyl alcohol, and Buffer A
(15:5:80)
Solution C: Acetonitrile and Buffer A (65:35)
Mobile phase: Acetonitrile, tetrahydrofuran, butyl alco-
hol, and Buffer B (18:8:5:69). Adjust with 1M phos-
phoric acid or 1 M potassium hydroxide, if necessary, to
a pH of 6.3.
Standard stock solution: 0.1 mg/mL each of USP
Lopinavir RS and USP Ritonavir RS in Solution A
Standard solution: 0.01 mg/mL each of USP Lopinavir
RS and USP Ritonavir RS from Standard stock solution in
Solution B
 2454 Lopinavir / Official Monographs
Peak identification solution: Transfer a weighed por-
tion of Oral Solution to a crimp-top container. Add an
amount of citric acid equivalent to 1% by weight of the
Oral Solution taken and mix until dissolved. Seal the
container, and heat at 50° for approximately 4 days.
Use this degradation solution and follow the procedure
described below in the Sample stock solution and Sample
solution sections to prepare the Peak identification
solution.
Sample stock solution: Transfer 5 mL of Oral Solution
with the aid of several small portions of Solution C to a
100-mL volumetric flask, and dilute with Solution C to
volume.
Sample solution: Dilute 25.0 mL of Sample stock solu-
tion with Solution B to 50.0 mL. Transfer 15.0 mL of this
solution to a 50-mL connhe tube that has been pre-
viously rinsed with methanol and dried. Add 20.0 mL of
n-heptane, and shake vigorously until a uniform emul-
sion is formed. Vent the tube periodically while shaking.
Centrifuge the emulsion for about 5 min. Carefully re-
move the top heptane layer by aspiration, leaving the
clear Sample solution layer. The middle viscous, cloudy
layer should be considered part of the heptane layer for
removal by aspiration. Precondition a strong anion-ex-
change cartridge (quaternary ammonium functionality
ona styrene/divinylbenzene base) with a sorbent mass
of 600 mg by rinsing the cartridge with 3 mL of metha-
nol, then 3 mL of Solution C, and repeating these rinse
steps. Dry the cartridge for about 10 min with the aid
of a low vacuum. Transfer 5.0 mL of the clear Sample
solution to the preconditioned cartridge. With the aid of
a vacuum, slowly pass the silline solution completely
through the cartridge, collect the extract in a 5-mL vol-
umetric flask, and then dilute with Solution C to
volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
=
al
(oe Detector: UV 215 nm and 240 nm
As) Column: 4.6-mm x 15-cm; 3-um packing L26
—
a Column temperature: 60°
° Flow rate: 1 mL/min
c Injection volume: 50 pL
5 Run time: 2 times the retention time of lopinavir
= System suitability
a Sample: Standard solution
a)
Suitability requirements
=,
Resolution: NLT 2.5 between the ritonavir and
lopinavir peaks at 215 nm
Tailing factor: 0.8—-1.2 for the ritonavir peak at 240
nm
Relative standard deviation: NMT 3.0% for the
lopinavir peak at 215 nm; NMT 3.0% for the ritonavir
peak at 240 nm
USP 41
Analysis
Samples: Standard solution, Peak identification solution,
and Sample solution
[Note—Determine the relative retention values (r) for
the components listed in Table 1 and Table 2, using
the time measured at the first baseline deflection of
the Standard solution chromatogram as the void vol-
ume (tw).]
To identify the ritonavir impurities, determine the rela-
tive retention value from the 240-nm chromatogram
relative to ritonavir (see Table 1). The Peak identifica-
tion solution may also be used to identify ritonavir
degradants. Unspecified ritonavir impurities are as-
signee according to the algorithm outlined in Table 3.
Calculate the percentage of each ritonavir impurity in
the portion of Oral Solution taken:
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
ru = peak response of each individual impurity
from the Sample solution
Is = peak response of ritonavir from the Standard
solution
Cs = concentration of USP Ritonavir RS in the
Standard solution (mg/mL)
Cu = nominal concentration of ritonavir in the
Sample solution (mg/mL)
F = relative response factor (see Table 7)
Acceptance criteria: See Table 1.
To identify the lopinavir impurities, determine the rela-
tive retention value from the 215-nm chromatogram
relative to ritonavir (see Table 2). Compare the
215-nm chromatogram to the 240-nm chromatogram.
Any impurity assigned as a ritonavir impurity at 240
nm that is also observed at 215 nm is discounted. Un-
spectied ritonavir impurities are assigned according to
the algorithm outlined in Table 3.
Calculate the percentage of each unspecified lopinavir
impurity at 215 nm in the portion of Oral Solution
taken:
Result = (ru/rs) x (Cs/Cy) x 100
Tu = peak response of each individual impurity
from the Sample solution
rs = peak response of lopinavir from the Standard
solution
Cs = concentration of USP Lopinavir RS in the
Standard solution (mg/mL)
Cu = nominal concentration of lopinavir in the
Sample solution (mg/mL)
Acceptance criteria: See Table 2.
USP 41 Official Monographs / Lopinavir 2455

Table 1
Relative
Retention Relative Acceptance
Value Response Criteria,

03 —
ine 11 is 0.
mate 14
midoalcohole

2,5-Thi

Ritonavir
mate anal
antoi

isomere

Isobut

‘ohol ui
5R-Epimer
SR oy
valine ureaz
un.
Total ritonavir impurities, specified and

2 [N-Methyl[(2-isopropyl-4-thiazolyl)methyl]amino]carbonyl-t-valine. =
4)
> These are process impurities which are included in this table for identification only. These impurities are controlled in the drug substance. They are not to be
reported for the drug product and are not included in the total impurities. a)
¢ Thiazol-5-ylmethyl (25,35,55)-5-[(S)-2-amino-3-methylbutanamido]-3-hydroxy-1,6-diphenylhexan-2-ylcarbamate. =
4 Thiazol-5-ylmethyl (25,35,55)-5-[(25)-2-(2,3-dihydroxypropoxycarbonylamino)-3-methylbutanamido]-3-hydroxy-1,6-diphenylhexan-2-ylcarbamate. °
=|
© Thiazol-5-ylmethyl (25,35,55)-5-[(25)-2-(2-hydroxypropoxycarbonylamino)-3-methylbutanamido]-3-hydroxy-1,6-diphenylhexan-2-ylcarbamate.
}
{Thiazol-5-ylmethyl (25,35,55)-5-acetamido-3-hydroxy-1,6-diphenylhexan-2-ylcarbamate. to}=
9 If two peaks appear witha relative retention value of 0.24, the second peak will be identified as 2,5-thiazolylmethyldicarbamate.
Cy
h 2,5-Thiazolylmethyldicarbamate. a]
'A sna peak witha relative retention value of 0.44 should be reported as the ethyl carbamate analog due to possible coelution with ritonavir hydroperoxide mg
a)
Impurity.
i Thiazol-5-ylmethyl (25,35,55)-3-hydroxy-5-[2-(3-{[2-(2-hydroxypropan-2-yl)thiazol-4-yl]methyl}-3-methylureido)acetamido]-1,6-diphenylhexan-2-ylcarbamate.
k Thiazol-5-ylmethyl (25,35,55)-3-hydroxy-5-[(S)-4-isopropyl-2,5-dioxoimidazolidin-1 -yl]-1,6-diphenylhexan-2-yicarbamate.
' Thiazol-5-ylmethyl (25,35,55)-5-[(S)-2-ethoxycarbonylamino-3-methylbutanamido]-3-hydroxy-1,6-diphenylhexan-2-ylicarbamate.
™ Thiazol-5-ylmethyl (25,35,55)-5-[(S)-2-{3-[(2-ethylthiazol-4-yl)methyl]-3-methylureido}-3-methylbutanamido]-3-hydroxy-1,6-diphenylhexan-2-yicarbamate.
” (48,55)-Thiazol-5-ylmethy! 4-benzyl-5-{(5)-2-[(5S)-4-isopropyl-2,5-dioxoimidazolidin-1 -yl]-3-phenylpropyl}-2-oxooxazolidine-3-carboxylate.
° Thiazol-5-ylmethyl (25, 35,55)-5-[(5)-2-{3-[(2-ethylthiazol-4-yl)methyl]-3-methylureido}-3-methylbutanamido]-3-hydroxy-1,6-diphenylhexan-2-ylcarbamate.
P (S)-{(25,35,55)-5-Amino-1,6-diphenyI-2-[(thiazol-5-ylmethoxy)carbonylamino]hexan-3-yl} 2-{3-[(2-isopropylthiazol-4-yl)methyl]-3-methylureido}-3-methylbuta-
noate.
4 Thiazol-5-ylmethy! (25,35,55)-(5-t-butoxycarbonylamino)-3-hydroxy-1,6-diphenylhexan-2-ylcarbamate.
‘ Thiazol-S-ylmethyl (25,35,55)-(5-isobutoxycarbonylamino)-3-hydroxy-1,6-diphenylhexan-2-ylcarbamate.
5 (5)-N-[(5)-1-[(45,55)-4-Benzyl-2-oxooxazolidin-5-yl]-3-phenylpropan-2-yl]-2-{3-[(2-isopropylthiazol-4-yl)methyl]-3-methylureido}-3-methyloutanamide.
t(5)-lsobutyl 2-{3-[(2-isopropylthiazol-4-yl)methy]-3-methylureido}-3-methylbutanoate.
¥ Thiazol-5-ylmethyl (25,45,55)-4-hydroxy-5-[(5)-2-{3-[(2-isopropylthiazol-4-yl)methyl]-3-methylureido}-3-methylbutanamido]-1,6-diphenylhexan-2-ylcarbamate.
* Thiazol-5-ylmethyl (25,3R,55)-3-hydroxy-5-[(S)-2-{3-[(2-isopropylthiazol-4-yl)methy|]-3-methylureido}-3-methylbutanamido]-1,6-diphenylhexan-2-ylcarbamate.
Bis(thiazol-5-ylmethyl) (25,2’S,35,3’S,55,5’S)-5,5’-carbonylbis(azanediyl)bis(3-hydroxy-1 ,6-diphenythexane-5,2-diyl)dicarbamate.
*Thiazol-5-ylmethy! (25,3R,5)-3-hydroxy-5-[(S)-2-{3-[(2-isopropylthiazol-4-yl)methyl]-3-methylureido}-3-methylbutanamido]-1 ,6-diphenylhexan-2-ylcarbamate.
’ Thiazol-5-ylmethyl (25,35,5R)-3-hydroxy-5-[(S)-2-{3-[(2-isopropylthiazol-4-yl)methyl]-3-methylureido}-3-methylbutanamido]-1,6-diphenylhexan-2-ylcarbamate.
z BETS 455 5)->.{ I hlazols-yvimethoxyearbony amino) nyarony:t ,6-diphenylhexan-2-yl]-2-{3-[(25,45,55)-5-(thiazol-5-ylmethoxycarbonylamino)-4-hydroxy-
1,6-diphenylhexan-2-ylJureido}-3-methylbutanamide.
a Disregard any peak less than 0.01% in the calculation of total impurities.
 2456 Lopinavir / Official Monographs USP 41

Table 2 Table 3
Relative Acceptance Unspecified
Retention Value Criteria, Wavelength Impurity
Name (A) NMT (%) nm
Lopinavir aminoalcohol 0.06 — 2.
Lopinavir N-formylami- _s
noalcohol« 0.12
Lopinavir divalinate? 0.21 =
Lopinavir phenoxy- ab
acetamidee 0.35
SPECIFIC TESTS
Lopinavir N-formylphe- _5
e ALCOHOL DETERMINATION (611)
noxyacetamide! 0.67
Internal standard solution: Transfer 10.0 mL of butyl
Lopinavir N-acetylphe- _6 alcohol to a 200-mL volumetric flask and dilute with
noxyacetamides 0.69 methanol to volume.
Lopinavir oxazineh 0.77 — Internal standard identification solution: Dilute
Z-Diacylethenediamine! 0.92 = 5.0 mL of Internal standard solution with methanol to
Ritonavir 1.0 —_ 100 mL.
lsolopinaviri 1.18 — Standard stock solution: 4.0% (v/v) of dehydrated al-
cohol in methanol
Lopinavir 2,4-
Standard solution: 0.4% (v/v) of dehydrated alcohol
dimethylphenoxy — prepared as follows. Transfer 10.0 mL of Standard stock
isomer* 1.21
solution and 5.0 mL of the Internal standard solution to a
Lopinavir 4-epimer'! 1.26 — 100-mL volumetric flask, and dilute with methanol to
Lopinavir D-valine 6 volume.
diastereomer™ 1.33) Sample stock solution: Transfer 5.0 mL of Oral Solution
Lopinavir (2R,4R) ag to a 50-mL volumetric flask with the aid of several por-
diastereomer? 1.42 tions of methanol, and dilute with methanol to volume.
Lopinavir 2-epimere 1.79 —> Sample solution: Transfer 10.0 mL of Sample stock solu-
Any unspecified lopinavir _ tion and 5.0 mL of the Internal standard solution to a
impurit 0.2 100-mL volumetric flask, and dilute with methanol to
volume.
Total unspecified _ Chromatographic system
lopinavir impurities 0.5P (See Chromatography (621), System Suitability.)
4 (5S)-N-[(25,45,55)-5-Amino-4-hydroxy-1,6-diphenylhexan-2-yl]-3-methyl-2- Mode: GC
[2-oxotetrahydropyrimidin-1(2H)-yl]butanamide.
Detector: Flame ionization
> These are process impurities which are included in this table for identifi-
Column: 0.53-mm x 30-m fused silica capillary; coated
a3
”
cation only. These impurities are controlled in the drug substance. They
with a 1-uum film of liquid phase G16
rm are not to be reported for the drug product and are not included in the
3— total impurities. Temperatures
Dp € (S)-N-[(25,45,55)-5-Formamido-4-hydroxy-1,6-diphenylhexan-2-yl]-3- Injection port: 185°
methyl-2-[2-oxotetrahydropyrimidin-1(2H)-yl]butanamide.
-) Detector: 220°
iS 4 (25,2'S)-N,N’-[(25,35,55)-3-Hydroxy-1,6-diphenylhexane-2, 5-diyl]bis{3- Column: See Table 4.
C) methyl-2-[2-oxotetrahydropyrimidin-1 (2H)-yl]butanamide}.
= © N-[(25,35,55)-5-Amino-3-hydroxy-1,6-diphenylhexan-2-yl]-2-(2,6-
dimethylphenoxy)acetamide. Table 4
a t 2-(2,6-Dimethylphenoxy)-N-[(25,35,55)-5-formamido-3-hydroxy-1,6-
al diphenylhexan-2-yllacetamide. Hold Time
=) 9 N-[(25,35,55)-5-Acetamido-3-hydroxy-1,6-diphenylhexan-2-yl]-2-(2,6- Initial Temperature Final at Final
dimethylphenoxy)acetamide. Temperature Ramp Temperature | Temperature
» N-{(S)-1-[(45,65)-4-Benzyl-2-oxo-1 ,3-oxazinan-6-yl]-2-phenylethy]}-2-(2,6- ©) (¢/min) ©) (min)
dimethylphenoxy)acetamide. 40 O 40 5
i (2-N,N’-(Ethene-1,2-diyl)bis[2-(2,6-dimethylphenoxy)acetamide].
40 10 145 6
i (S)-N-{(25,35,55)-5-[2-(2,6-Dimethylphenoxy)acetamido]-3-hydroxy-1,6-
diphenylhexan-2-yl}-3-methy|-2-[2-oxotetrahydropyrimidin-1(2H)- 145 20 200 9.75
ylloutaramide.
k (5)-N-{(25,45,55)-5-[2-(2,4-Dimethylphenoxy)acetamido]-4-hydroxy-1,6- Carrier gas: Helium
diphenylhexan-2-yl}-3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)- Flow rate: 4.5 mL/min
ylloutaramide.
Makeup gas flow: 30 mL/min
1 (S)-N-{(25,4R, 5S)-5-[2-(2,6-Dimethylphenoxy)acetamido]-4-hydroxy-1,6-
diphenylhexan-2-yl}-3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)- Injection volume: 1 wL
yl]butanamide. Injection type: Split ratio 4:1
™ (R)-N-{(25,45,55)-5-[2-(2,6-Dimethylphenoxy)acetamido]-4-hydroxy-1,6- System suitability
diphenylhexan-2-yl}-3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)- Sample: Standard solution
yllbutanamide. Suitabi requirements
(S)-N-{(2R,4R, 5S)-5-[2-(2,6-Dimethylphenoxy)acetamido]-4-hydroxy-1,6- Tailing factor: 0.8-1.2 for the alcohol peak
diphenylhexan-2-yl}-3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)-
ylbutatamide, Relative standard deviation: NMT 3.0% for the peak
© (S)-N-{(2R,45,55)-5-[2-(2,6-Dimethylphenoxy)acetamido]-4-hydroxy-1,6- area ratio of alcohol to the internal standard
diphenylhexan-2-yl}-3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)- Analysis
yllbutaramide. Samples: Internal standard identification solution, Stan-
® Disregard any peak less than 0.01%. dard solution, and Sample solution
For calculating and reporting impurities, follow the al- Calculate the percentage of the labeled amount of alco-
gorithm outlined in Table 3. hol in the portion of Oral Solution taken:
Result = (Ru/Rs) X (Cs/Cu) x D x 100

Ru = peak response ratio of alcohol to buty! alcohol

from the Sample solution
USP 41 Official Monographs / Lopinavir 2457

Rs = peak response ratio of alcohol to butyl alcohol Relative standard deviation: NMT 2.0% for the
from the Standard solution ritonavir and lopinavir peaks
Gs = concentration of dehydrated alcohol in the Analysis
Standard solution (% v/v) Samples: Standard solution and Sample solution
Cu = nominal concentration of alcohol in the Oral Calculate the percentage of the labeled amount of
Solution (% v/v) lopinavir (C37H4sN4Os) and ritonavir (C37H4sNeOsSz) in
D = dilution factor used to prepare the Sample the portion of Tablets taken:
solution
Acceptance criteria: 85.0%-115.0% of the labeled Result = (ru/rs) * (Cs/Cu) x 100
amount of alcohol (C2H6O)
¢ MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI- ru = peak response of lopinavir or ritonavir from
FIED MICROORGANISMS (62): The total aerobic microbial the Sample solution
count does not exceed 102 cfu/mL. rs = peak response of lopinavir or ritonavir from
the Standard solution
ADDITIONAL REQUIREMENTS Cs = concentration of lopinavir or ritonavir in the
© PACKAGING AND STORAGE: Preserve in well-closed contain- Standard solution (g/mL)
ers, protected from light. Store at 2°-8°. Cy = nominal concentration of lopinavir or ritonavir
e USP REFERENCE STANDARDS (11) in the Sample solution (ug/mL)
USP Lopinavir RS Acceptance criteria: 90.0%-110.0% of the labeled
USP Ritonavir RS amounts of lopinavir (C37H4sN4Os) and ritonavir
(C37HasNeOsSz
PERFORMANCE TESTS
e DISSOLUTION (711)
Lopinavir and Ritonavir Tablets Medium: 60 mM polyoxyethylene 10 lauryl ether
(37.56 g/L) in water; 900 mL
DEFINITION Apparatus 2: 75 rpm
Time: 90 min
Lopinavir and Ritonavir Tablets contain NLT 90.0% and Mobile phase: Acetonitrile and 4.1 g/L potsssuln phos-
NMT 110.0% of the labeled amounts of lopinavir
(C37H4gN4Os) and ritonavir (C37H4gNe6OsSz). phate monobasic (55:45). Adjust with phosphoric acid
to an apparent pH of 4.0 + 0.05.
IDENTIFICATION Standard solution: Dissolve USP Lopinavir RS in metha-
e A. The retention times of the major peaks of the Sample nol to obtain a solution containing 2.6 mg/mL. Dissolve
solution correspond to those of the Standard solution, as USP Ritonavir RS in methanol to obtain a solution con-
obtained in the Assay. taining 1.3 mg/mL. Combine portions of these solutions
to make a solution containing approximately 0.104 mg/
ASSAY mL of lopinavir and 0.026 mg/mL of ritonavir in
¢ LOPINAVIR AND RITONAVIR Medium. (=
Buffer 1: 4.1 g/L of monobasic potassium phosphate in Sample solutions: Pass a portion of the solution under 2)
water test through a suitable filter. If necessary, dilute the so- mo]
Solution A: Acetonitrile and Buffer 1 (50:50) lution with Medium to obtain a final sample solution —
Buffer 2: 2.1 g/L of monobasic potassium phosphate in containing approximately 0.104 mg/mL of lopinavir and i}
water 0.026 mg/mL of ritonavir. =)
Solution B: Acetonitrile and 1-butanol (13:3) Chromatographic system re}
Solution C: Acetonitrile, 1-butanol, Buffer 1, and water (See Chromatography (621), System Suitability.)
re}=
Mode: LC Cy
(65:15:10:10) a}
Standard solution: 6.25 g/mL of USP Ritonavir RS and Detector: UV 215 nm >
25 g/mL of USP Lopinavir RS in Solution A Column: 4.6-mm x 15-cm; 5-1um packing L1 ww

Sample solution: Place a number of Tablets equivalent Flow rate: 1.5 mL/min
to 1000 mg of lopinavir and 250 mg of ritonavir in a Injection volume: 25 uL
250-mL volumetric flask, add 25 mL of Buffer 2, and System suitability
agitate to dissolve the Tablet coating, if necessary. Add Sample: Standard solution
100 mL of Solution B, and shake mechanically until the Suitability requirements
Tablets are dissolved. Dilute with Solution C to volume. Resolution: NLT 2.0 between lopinavir and ritonavir
Centrifuge a portion of this solution, and then further Tailing factor: 0.9-1.5 for the lopinavir and ritonavir
dilute with Solution A to a nominal concentration of eaks
6.25 g/mL of ritonavir and 25 pg/mL of lopinavir. Relative standard deviation: NMT 2.0% for the
Mobile phase: Acetonitrile, methanol, feaanerea ial lopinavir and ritonavir peaks
and Buffer 7 (175:100:100:625) Analysis
Chromatographic system Samples: Standard solution and Sample solution
(See Chromatography (621), System Suitability.) Calculate the percentage of lopinavir (C37H4sN4Os) and
Mode: LC ritonavir (C37H4gsNeOsS2) dissolved:
Detector: UV 215 nm
Column: 4.6-mm x 15-cm; 5-{1m packing L7 Result = (ru/rs) x (Cs/L) x D x Vx 100
Column temperature: 40°
Flow rate: 1.5 mL/min tu = peak response of lopinavir or ritonavir from
Injection volume: 50 pL the Sample solution
System suitability rs = peak response of lopinavir or ritonavir from
Sample: Standard solution the Standard solution
[Note—The elution order is ritonavir, then lopinavir.] G = concentration of USP Lopinavir RS or USP
Suitability requirements Ritonavir RS in the Standard solution
Capacity factor: 15-24 for the ritonavir peak
L
(mg/mL)
= re label claim for lopinavir or ritonavir
Tailing factor: 0.8-1.2 for the ritonavir peak
Thearetieal plates: More than 5000 for the ritonavir mg)
pea D =alunes factor of the Sample solution
 2458 Lopinavir / Official Monographs
Vv = volume of Medium, 900 mL
Tolerances: NLT 80% (Q) of the labeled amounts of
lopinavir (C37H4gN4Os) and ritonavir (C37H4gN¢OsS2) are
dissolved.
e UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
IMPURITIES
© ORGANIC IMPURITIES
Buffer 1: 4.1 g/L of monobasic potassium phosphate in
water
Solution A: Buffer 1 and acetonitrile (50:50)
Buffer 2: 2.1 g/L of monobasic potassium phosphate in
water
Solution B: Acetonitrile, 1-butanol, and Buffer 7
(15:5:80)
Solution C: Acetonitrile, 1-butanol, Buffer 1, and water
(65:15:10:10)
Solution D: Acetonitrile and 1-butanol (13:3)
Buffer solution: 3.8 g/L of monobasic potassium phos-
phate and 0.25 g/L of dibasic potassium phosphate in
water
Mobile phase: Acetonitrile, tetrahydrofuran,1-butanol,
and Buffer solution (18:8:5:69). Adjust with 1M phos-
phoric acid or 1 M potassium hydroxide, if necessary, to
a pH of 6.3 + 0.1.
Standard stock solution: 0.025 mg/mL of USP
Ritonavir RS in Solution A
Standard solution: 2.5 j1g/mL of USP Ritonavir RS in
Solution B from Standard stock solution
Ritonavir degradant identification solution: Transfer
two 5.0 mL portions of a 1 mg/mL solution of USP
Ritonavir RS in Solution A to separate 50-mL volumetric
flasks. Add 1 g of citric acid to one flask, and shake
until dissolved. Heat both flasks at 80° for approxi-
mately 24 h. Cool the flasks, and add 13 mL of 1N
sodium hydroxide to the flask containing the citric acid.
Dilute both flasks with Solution B to volume. Combine
s
“
ra equal volumes of both solutions. This solution contains
S—_ ritonavir and the ritonavir degradation products (N-dea-
Dp cylvaline ritonavir, hydantoin aminoalcohol, O-acyl iso-
io) mer, and oxazolidinone derivative).
r= Ritonavir related compounds identification solution:
5 1 mg/mL of USP Ritonavir Related Compounds Mixture
3 RS decked in Solution C and further diluted with Solu-
a tion B to 0.5 mg/mL.
v2)
= Sample solution: Place a number of Tablets equivalent
to 1000 mg of lopinavir and 250 mg of ritonavir into a
250-mL volumetric flask. Add 25 mL of Buffer 2, and
USP 41
agitate to dissolve the Tablet coating, if necessary. Add
100 mL of Solution D, and shake mechanically until the
Tablets are dissolved. Dilute with Solution C to volume.
Centrifuge a portion of this solution, and further dilute
with Solution B to a concentration of 2 mg/mL of
lopinavir and 0.5 mg/mL of ritonavir.
Chromatographic system
(See Chromatography (621), System Suitability.)
Column: 4.6-mm x 15-cm; 3-um packing L26
Column temperature: 60°
Detector: UV 240 nm
Injection volume: 50 uL
Flow rate: 1.0 mL/min
System suitability
Samples: Ritonavir degradant identification solution,
Ritonavir related compounds identification solution, and
Standard solution
Suitability requirements
Resolution: NLT 1.0 between the peaks for O-acyl
isomer and oxazolidinone derivative, Ritonavir degra-
dant identification solution. NLT 0.7 between the
peaks for hydroxyritonavir and hydantoin ami-
noalcohol, Ritonavir related compounds identification
solution
Capacity factor: NLT 10.8, Standard solution
Tailing factor: 0.8-1.2, Standard solution
Column efficiency: NLT 5000, Standard solution
Relative standard deviation: NMT 5.0%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each ritonavir degradation
product in the Sample solution:
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
tu = peak area of individual degradation product
from the Sample solution
rs peak response of ritonavir from the Standard
solution
Cs = concentration of USP Ritonavir RS in the
Standard solution (mg/mL)
Cu = nominal concentration of ritonavir in the
Sample solution (mg/mL)
F = relative response factor
Acceptance criteria: See Table 7. [NoTE—Disregard all
peaks eluting before the retention time of the N-dea-
cylvaline ritonavir peak from the Ritonavir degradant
identification solution.]
USP 41 Official Monographs / Loracarbef 2459

Table 1
Relative Acceptance
Relative Response Criteria,
Factor
N- itonavire
Acetam| b
2,5-Thiazolylmethyl-

cohole
xidet
inone

ro

inoalco

imert a
1
= 1.0
T i a =
4 Thiazol-5-ylmethyl (25,35,55)-5-[(S)-2-amino-3-methylbutanamido]-3-hydroxy-1,6-diphenylhexan-2-ylcarbamate.
» Thiazol-5-ylmethyl (25,35,55)-5-acetamido-3-hydroxy-1,6-diphenylhexan-2-ylcarbamate.
© Bis(thiazol-5-ylmethyl) (25, 35,55)-3-hydroxy-1,6-diphenylhexane-2,5-diyldicarbamate.
Seep oyimetnyl(25,35,55)-3-hydroxy-5-[(S)-2-(3-{[2-(2-hydroxypropan-2-yl)thiazol-4-yl]methy]}-3-methylureido)-3-methylbutanamido]-1,6-diphenylhexan-
-yicarbamate.
© Thiazol-S-ylmethyl (25,35,55)-3-hydroxy-5-[(S)-4-isopropyl-2,5-dioxoimidazolidin-1-yl]-1,6-diphenylhexan-2-ylcarbamate.
‘ Thiazol-5-ylmethy! (25,35,55)-5-[(S)-2-(3-{[2-(2-hydroperoxypropan-2-yl)thiazol-4-yl]methyl}-3-methylureido)-3-methylbutanamido]-3-hydroxy-1,6- (=
diphenylhexan-2-ylcarbamate. “
9 (45,55)-Thiazol-5-ylmethyl 4-benzyl-5-{(S)-2-[(S)-4-isopropyl-2,5-dioxoimidazolidin-1-yl]-3-phenylpropy|}-2-oxooxazolidine-3-carboxylate.
z
» Thiazol-5-ylmethyl (25,35,55)-5-[(S)-2-{3-[(2-ethylthiazol-4-yl)methy|]-3-methylureido}-3-methylbutanamido]-3-hydroxy-1,6-diphenylhexan-2-ylcarbamate. E
'(S)-{(25,35,55)-5-Amino-1,6-diphenyl-2-[(thiazol-5-ylmethoxy)carbonylamino]hexan-3-yl} 2-{3-[(2-isopropylthiazol-4-yl)methy|]-3-methylureido}-3-methylbuta- iS
noate. =
2}
i Thiazol-5-ylmethy! (25,35,55)-(5-t-butoxycarbonylamino)-3-hydroxy-1,6-diphenylhexan-2-ylcarbamate.
k Thiazol-5-ylmethyl (25,35,55)-(5-isobutoxycarbonylamino)-3-hydroxy-1,6-diphenylhexan-2-ylcarbamate.
re}=
i)
' (S)-N-[(S)-1-[(45,55)-4-Benzyl-2-oxooxazolidin-5-yl]-3-phenylpropan-2-y!]-2-{3-[(2-isopropylthiazol-4-yl)methy!]-3-methylureido}-3-methylbutanamide. ie}
™ (5S)-lsobuty! 2-{3-[(2-isopropylthiazol-4-yl)methyl]-3-methylureido}-3-methylbutanoate. oa
a
" Thiazol-5S-ylmethyl (25,45,55)-4-hydroxy-5-[(5)-2-{3-[(2-isopropylthiazol-4-yl)methy!]-3-methylureido}-3-methylbutanamido]-1,6-diphenylhexan-2-ylcarbamate.
© Thiazol-5-ylmethyl (25,3R,55)-3-hydroxy-5-[(S)-2-{3-[(2-isopropylthiazol-4-yl)methyl]-3-methylureido}-3-methylbutanamido]-1,6-diphenylhexan-2-ylcarbamate.
P Bis(thiazol-5-ylmethyl) (25,2’S,35,3'S,55,5’5)-5,5’-carbonylbis(azanediy)bis(3-hydroxy-1, 6-diphenylhexane-5,2-diyl)dicarbamate.
4Thiazol-5-ylmethyl (25,3R,5R)-3-hydroxy-5-[(5)-2-{3-[(2-isopropylthiazol-4-yl)methyl]-3-methylureido}-3-methylbutanamido]-1,6-diphenylhexan-2-ylcarbamate.
' Thiazol-5-ylmethyl (25,35,5R)-3-hydroxy-5-[(S)-2-{3-[(2-isopropylthiazol-4-yl)methyl]-3-methylureido}-3-methylbutanamido]-1,6-diphenylhexan-2-ylcarbamate.
5 (35,45,65,105S,135,155,165)-Bis(thiazol-5-ylmethyl)-4,15-dihydroxy-10-isopropyl-8,1 1 -dioxo-3,6,13,16-tetrabenzyl-2,7,9,12,17-pentaazaoctadecanedioate.
* Process impurities; for information only.
** Disregard any peak less than 0.05%.

ADDITIONAL REQUIREMENTS 1-Azabicyclo[4.2.0][oct-2-ene-2-carboxylic acid, 7-

e USP REFERENCE STANDARDS (11) [{(aminophenylacetyl)amino]-3-chloro-8-oxo-,
USP Lopinavir RS monohydrate, [6R-[60,7B(R*)]]-.
USP Ritonavir RS (6R,75)-7-[(R)-2-Amino-2-phenylacetamido]-3-chloro-8-oxo-I-
USP Ritonavir Related Compounds Mixture RS azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, monohy-
drate [121961-22-6].
Anhydrous 349.78
» Loracarbef contains not less than 960 ug and
not more than 1020yg of anhydrous loracarbef
Loracarbef (Ci6HisCIN304) per acalculated on the anhy-
Ho. 0 drous basis.
OL VonN™ oH Packaging and storage—Preserve in tight containers.
agi rT t USP Reference standards (11)—
USP Cefaclor RS
CigHigCIN3O4-H2O 367.78 USP Loracarbef RS
USP Loracarbef L-lsomer RS
 2460 Loracarbef / Official Monographs
Identification—
A: Infrared Absorption (197K).
B: The retention time of the loracarbef peak in the chro-
matogram of the Assay preparation corresponds to that in
the chromatogram of the Standard preparation, as obtained
in the Assay.
Specific rotation (7815S): between +27° and +33°, cal-
culated on the anhydrous basis, determined in a solution in
0.1 N hydrochloric acid containing 10 mg in each mL.
Crystallinity (695): meets the requirements.
PH (791): between 3.0 and 5.5, in a suspension (1 in 10).
Water Determination, Method | (921): between 3.5%
and 6.0%.
Related compounds—
Solution A—Dissolve 6.9 g of monobasic ammonium
phosphate in 1960 mL of water. Adjust with phosphoric acid
to a pH of 2.5, add 40 mL of acetonitrile, and mix. Filter, if
necessary, to obtain a clear solution, and degas.
Solution B—Dissolve 6.9 g of monobasic ammonium
phosphate in 600 mL of water. Adjust with phosphoric acid
to a pH of 2.5, add 1400 mL of acetonitrile, and mix. Filter,
if necessary, to obtain a clear solution, and degas.
Mobile phase—Use variable mixtures of Solution A and So-
lution B as directed for Chromatographic system.
[NoTE—When preparing the System suitability solution, the
Standard solution, and the Test solution, if necessary, sonicate
and mix on a vortex mixer to aid in dissolution. Use the
solutions immediately after preparation or refrigerate and
use within 24 hours.)
Phenylglycine solution—Dissolve an accurately weighed
quantity of phenylglycine in Solution A to obtain a solution
having a known concentration of about 0.0075 mg per mL.
System suitability solution—Dissolve accurately weighed
quantities of USP Loracarbef RS and USP Cefaclor RS in Solu-
tion A to obtain a solution having known concentrations of
en
val
oe about 0.01 mg of each per mL.
i}
—_
Standard solution—Dissolve an accurately weighed quan-
Dp tity of USP Loracarbef RS in Solution A to obtain a solution
° having a known concentration of about 0.01 mg per mL.
=
G Test solution—Transfer about 50 mg of Loracarbef, accu-
Pe rately weighed, to a 10-mL volumetric flask, add about 8 mL
of Solution A, and dissolve. Dilute with Solution A to volume,
ie and mix. Filter, if necessary, to obtain a clear solution.
a)
=) Chromatographic system (seeChrometograpty (621))—The
liquid chromatograph is equipped with a 220-nm detector
and a 4.6-mm x 15-cm column that contains 5-11m packing
L1. The flow rate is about 2 mL per minute, and is main-
tained at a constant temperature of about 40°. The chro-
matograph is programmed as follows. Initially it is equili-
brated with Solution A, then the proportion of Solution B is
increased linearly from 0% to 14.5% over 9.5 minutes, then
increased from 14.5% to 100% over 7.5 minutes, and held
at 100% for an additional 1.5 minutes. Finally, the composi-
tion of the Mobile phase is changed to 100% Solution A, and
is allowed to re-equilibrate for about 4 minutes or until a
stable baseline is obtained. Chromatograph the System suita-
bility solution, and record the peak responses as directed for
Procedure: the relative retention times are about 0.9 for
cefaclor and 1.0 for loracarbef; the resolution, R, between
the cefaclor peak and the loracarbef peak is between 4.0
and 8.0; and the tailing factor for the loracarbef peak is not
more than 1.3. Calculate the recovery of loracarbef from the
System suitability solution by the formula:
100(C/ L)(n/ rs)
in which C is the concentration, in mg per mL, of USP Lora-
carbef RS in the Standard solution; L is the concentration, in
mg per mL, of USP Loracarbef RS in the System suitability
solution; and r, and rs are the loracarbef responses in the
chromatograms obtained from the System suitability solution
USP 41
and the Standard solution, respectively: the recovery is be-
tween 95% and 105%.
Procedure—Separately inject equal volumes (about 20 pL)
of Solution A, the Phenylglycine solution, the Standard solu-
tion, and the Test solution into the chromatograph, record
the chromatograms, and measure the peak responses. Disre-
gard any peak responses in the chromatograms that corre-
spond to those in the chromatogram obtained from Solution
A, and identify any peak in the chromatogram of the Test
solution that corresponds to the peak forPhenvlglycine in
the chromatogram obtained from the Phenylglycine solution.
Separately calculate the percentage of each related com-
pound in the portion of Loracarbef taken by the formula:
100(C/ Y)(rv/ rs)
in which Y is the concentration, in mg per mL, of Loracarbef
in the Test solution; ry is the response of any related com-
pound in the chromatogram obtained from the Test solu-
tion; and C and rs are as defined above: not more than
0.15% of phenylglycine is found, not more than 0.5% of
any other related compound is found, and the sum of all
other related compounds is not more than 2.0%.
Assay—
Mobile phase—Dissolve 2.0 g of sodium 1-pentanesulfo-
nate in 1560 mL of water, add 20 mL of triethylamine, and
adjust with phosphoric acid to a pH of 2.5. Add 440 mL of
methanol, mix, and pass throughafilter having aporosity
of 0.5 um or finer, and degas. Make adjustments if neces-
sary (see System Suitability under Chromatography (621)).
Standard preparation—Transfer about 10 mg of USP Lora-
carbef RS, accurately weighed, to a 50-mL volumetric flask,
dissolve in Mobile phase, using sonication, if necessary, to
achieve dissolution, dilute with Mobile phase to volume, and
mix.
Assay preparation—Transfer about 10 mg of Loracarbef,
accurately weighed, to a 50-mL volumetric flask, dissolve in
Mobile phase, using sonication, if necessary, to achieve dis-
solution, dilute with Mobile phase to volume, and mix.
Resolution solution—Prepare a solution in Mobile phase
containing about 0.2 mg each of USP Loracarbef RS and of
USP Loracarbef L-Isomer RS in each mL.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 265-nm detector
and a 4.6-mm x 25-cm column that contains 5-um packing
L1. The flow rate is about 1.5 mL per minute. Chromato-
graph the Resolution solution, and record the responses as
directed for Procedure: the relative retention times are about
0.6 for loracarbef L-isomer and 1.0 for loracarbef; and the
resolution, R, between the loracarbef L-isomer peak and the
loracarbef peak is not less than 6.0. Chromatograph the
Standard preparation, and record the responses as directed
for Procedure: the capacity factor for the loracarbef peak is
not less than 5 and not more than 8; the tailing factor is
not less than 0.8 and not more than 1.3; the column effi-
ciency, determined from the loracarbef peak, is not less than
2500 theoretical plates; and the relative standard deviation
for replicate injections is not more than 2.0%.
Procedure—{NOTE—Use peak areas where peak responses
are indicated.] Separately inject equal volumes (about 20 pL)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
ure the responses for the major peaks. Calculate the quan-
tity, in ug, of anhydrous loracarbef (Ci6HisCIN3Ox) in each
mg of the Loracarbef taken by the formula:
(WP
/ w)(ru / ts)
in whichWis the quantity, in mg, of USP Loracarbef RS
taken to prepare the Standard preparation; P is the assigned
potency, in ug of anhydrous loracarbef (Ci6HisCIN3O,) in
each mg of USP Loracarbef RS; w is the quantity, in mg, of
Loracarbef taken to prepare the Assay preparation; and ru
USP 41
and rs are the loracarbef peak responses obtained from the
Assay preparation and the Standard preparation, respectively.
Loracarbef Capsules
» Loracarbef Capsules contain not less than
90.0 percent and not more than 110.0 percent of
the labeled amount of anhydrous loracarbef
(CisHisCIN3Ox).
Packaging and storage—Preserve in well-closed contain-
ers.
USP Reference standards (11)—
USP Cefaclor RS
USP Loracarbef RS
USP Loracarbef L-Isomer RS
Identification—The retention time of the loracarbef peak
in the chromatogram of the Assay preparation, corresponds
to that in the chromatogram of the Standard preparation, as
obtained in the Assay.
Dissolution (711)—
Medium: water; 900 mL.
Apparatus 2: 50 rpm.
Time: 30 minutes.
Procedure—Determine the amount of anhydrous lora-
carbef (CisHisCIN30.4) dissolved from UV absorbances at the
wavelength of maximum absorption at about 260 nm of
filtered portions of the solution under test, suitably diluted
with Dissolution Medium, if necessary, in comparison with a
Standard solution having a known concentration of USP
Loracarbef RS in the same medium.
Tolerances—Not less than 75% (Q) of the labeled amount
of anhydrous loracarbef (CisHisCIN3O4) is dissolved in
30 minutes.
Uniformity of dosage units (905)—meet the require-
ments.
Water Determination, Method | (921): not more than
8.5%.
Related compounds—
Solution A, Solution B, Mobile phase, System suitability solu-
tion, Standard solution, and Chromatographic system—Pro-
ceed as directed in the test for Related compounds under
Loracarbef.
Test solution—Remove, as completely as possible, the
contents of not less than 5 Capsules. Weigh the contents,
and determine the average weight of the content of each
Capsule. Transfer an accurately weighed peer of the pow-
der, equivalent to 125 mg of loracarbef, based on the la-
beled amount per Capsule, to a 25-mL volumetric flask. Add
about 20 mL of Solution A to the flask, mix, sonicate, and
mix on a vortex mixer to aid in dissolution. Dilute with Solu-
tion A to volume, and mix. Filter, and use the filtrate as the
Test solution immediately, or refrigerate and use within
24 hours.
Procedure—Proceed as directed for Procedure in the test
for Related compounds under Loracarbef, except to omit the
injection of Phenylglycine solution. Calculate the percentage
of each related compound in the portion of Capsule con-
tents taken by the formula:
100(C/Y)(ni / rs)
in which C is the concentration, in mg per mL, of USP Lora-
carbef RS in the Standard solution; Y is the concentration, in
mg per mL, of loracarbef in the Test solution; r; is the re-
sponse of any related compound obtained from the Test
solution; and rs is the loracarbef response obtained from the
Official Monographs / Loracarbef 2461
Standard solution: not more than 1.0% of any individual re-
lated compound is found, and the sum of all related com-
pounds is not more than 3.0%.
Assay—
Mobile phase, Standard preparation, Resolution solution,
and Chromatographic system—Proceed as directed in the
Assay under Loracarbef.
Assay preparation—Remove, as completely as possible,
the contents of not less than 20 Capsules. Transfer an accu-
rately weighed portion of the powder, equivalent to about
10 mg of loracarbef, to a 50-mL volumetric flask. Add about
40 mL of Mobile phase, and dissolve with the aid of swirling
and sonication. Dilute with Mobile phase to volume, and
mix. Pass 2 portion of this solution through a filter having a
porosity of 0.5 um or finer, and use the filtrate as the Assay
preparation.
Procedure—Proceed as directed for Procedure in the Assay
under Loracarbef. Calculate the quantity, in mg, of lora-
carbef (Ci6HisCIN3O.) in the portion of Capsules taken by
the formula:
(CP/20)(ru / rs)
in which C is the concentration, in mg per mL, of USP Lora-
carbef RS in the Standard preparation; P is the specified po-
tency, in ug of anhydrous loracarbef (CisHiesCIN3O4) per mag,
of USP Loracarbef RS; and ry and rs are the loracarbef peak
responses obtained from the Assay preparation and the Stan-
dard preparation, respectively.
Loracarbef for Oral Suspension
» Loracarbef for Oral Suspension is a dry mixture (ex,
of Loracarbef and one or more suitable sus- 4)
uv
pending agents, preservatives, coloring agents,
antifoaming agents, flavorings, and sweeteners, It a
fo)
contains not less than 90.0 percent and not more S
than 115.0 percent of the labeled amount of an- °
©
hydrous loracarbef (CisHi6CIN3O4). =
Sy
Tv
Packaging and storage—Preserve in tight containers. a
7
USP Reference standards (11)—
USP Cefaclor RS
USP Loracarbef RS
USP Loracarbef L-lsomer RS
Identification—The retention time of the loracarbef peak
in the chromatogram of the Assay preparation corresponds
to that in the chromatogram of the Standard preparation, as
obtained in the Assay.
Uniformity of dosage units (905)—
FOR SOLIDS PACKAGED IN SINGLE-UNIT CONTAINERS: meets the
requirements.
Deliverable volume (698): meets the requirements.
PH (791): between 3.0 and 5.5, in the Loracarbef for Oral
Suspension constituted as directed in the labeling.
Water Determination, Method | (921): not more than
2.0%.
Related compounds—
Solution A, Solution B, Mobile phase, System suitability solu-
tion, Standard solution, and Chromatographic system—Pro-
ceed as directed in the test for Related compounds under
Loracarbef.
Test solution—Constitute a container of Loracarbef for
Oral Suspension as directed in the labeling. Transfer an ac-
cuaey measured portion of the Suspension thus obtained,
equivalent to 100 mg of loracarbef, based on the labeled
amount per mL of the Suspension, to a 25-mL volumetric
 2462 Loracarbef / Official Monographs USP 41

flask. Add about 20 mL of Solution A to the flask, mix, soni- DEFINITION

cate, and mix on a vortex mixer to effect dissolution. Dilute Loratadine contains NLT 98.5% and NMT 101.0% of
with Solution A to volume, and mix. Filter, and use the fil- loratadine (C22H23CIN2O2), calculated on the dried basis.
trate as the Test solution immediately, or refrigerate and use
within 24 hours. IDENTIFICATION
Procedure—Proceed as directed for Procedure in the test © A. INFRARED ABSORPTION (197M)
for Related compounds under Loracarbef, except to omit the e B. The retention time of the major peak of the Sample
injection of the Phenylglycine solution. Calculate the percent- solution corresponds to that of the Standard solution, as
age of each related compound in the Suspension taken by obtained in the Assay.
the formula: ASSAY
e PROCEDURE
100(C/Y)(ri/ rs)
Buffer A (0.01 M dibasic po phosphate):
1.74 g/L of anhydrous dibasic potassium phosphate in
in which C is the concentration, in mg per mL, of USP Lora- water
carbef RS in the Standard solution; Y is the concentration, in Buffer B (0.6 M dibasic potassium phosphate): 105 g/L
mg per mL, of loracarbef in the Test solution; r, is the re- of anhydrous dibasic potassium phosphate in water
sponse of any related compound obtained from the Test 0.05 N hydrochloric acid: Transfer 500 mL of water to
solution; and rs is the loracarbef response obtained from the a 1000-mL volumetric flask, add 83 mL of hydrochloric
Standard solution: not more than 1.0% of a individual re- acid, and dilute with water to volume. Transfer 50 mL
lated compound is found, and the sum of all related com- of this solution into a 1000-mL volumetric flask, and
pounds is not more than 4.0%. dilute with water to volume.
Assay— Mobile phase: Acetonitrile, methanol, and Buffer
A
Mobile phase, Standard preparation, Resolution solution, (60:60:70). Adjust with 10% phosphoric acid to an ap-
and Chromatographic system—Proceed as directed in the parent pH of 7.2.
Assay under Loracarbef. Diluent: Transfer 400 mL of 0.05 N hydrochloric acid
fk oe ae 1 container of Loracarbef and 80 mL of Buffer B to a 1-L volumetric flask. Dilute
for Oral Suspension as directed in the labeling. Transfer an with a mixture of acetonitrile and methanol (1:1) to
accurately measured volume of Loracarbef for Oral Suspen- volume.
sion, freshly mixed and free from air bubbles, equivalent to Standard solution: 0.4 mg/mL of USP Loratadine RS in
about 200 mg of Loracarbef, to a 100-mL volumetric flask, Diluent
dilute with Mobile phase to volume, and mix. Transfer Sample solution: 0.4 mg/mL of Loratadine in Diluent
10.0 mL of this solution to a second 100-mL volumetric Chromatographic system
flask, dilute with Mobile phase to volume, and mix. Pass a (See Chromatography (621), System Suitability.)
portion of this solution Bough a filter having a porosity of Mode: LC
0.5 um or finer, and use the filtrate as the Assay preparation. Detector: UV 254 nm
Column: 4.6-mm x 15-cm; 5-1m packing L7
Procedure—Proceed as directed for Procedure in the Assay
Column temperature: 25°-35°
fe under Loracarbef. Calculate the quantity, in mg, of anhy-
al
Flow rate: 1 mL/min
rm drous loracarbef (CisHi6CIN3O4) in each mL of the Lora-
Injection volume: 15 ul
&
i carbef for Oral Suspension taken by the formula:
System suitability
Dp Sample: Standard solution
° (CP/V)(ru/ r3)
s Suitability requirements
iS in which C is the concentration, in mg per mL, of USP Lora- Relative standard deviation: NMT 2.0%
= carbef RS in the Standard preparation; P is the specified po- Analysis
a tency, in 4g of anhydrous loracarbef (CisHisCIN3O4) per mg, Samples: Standard solution and Sample solution
va) Calculate the percentage of loratadine (C22H23CIN2O2) in
oS of USP Loracarbef RS; V is the volume, in mL, of Loracarbe'
the portion of Loratadine taken:
for Oral Suspension taken to prepare the Assay preparation;
and ry and rs are the loracarbef peak responses obtained
from the Assay preparation and the Standard preparation, re- Result = (ru/rs) x (Cs/Cu) x 100
spectively. ty = peak response from the Sample solution
rs = peak response from the Standard solution
Cs = concentration of USP Loratadine RS in the
Standard solution (mg/mL)
Cu = concentration of the Sample solution (mg/mL)
Loratadine Acceptance criteria: 98.5%-101.0% on the dried basis
Ox OA IMPURITIES
| e RESIDUE ON IGNITION (281): NMT 0.1%
Y
Delete the following:
AX J °e HEAVY METALS, Method Ii (231): 10 ppMecotiicia 1400-2018)
© ORGANIC IMPURITIES, PROCEDURE 1
[NoTte—On the basis of the synthetic route, perform ei-
CopHa3CIN2O2 382.88 ther Procedure 1 or Procedure 2. Procedure 2 is recom-
1-Piperidinecarboxylic acid, 4-(8-chloro-5,6-dihydro-11H- mended if 4,8-dichloro-5,6-dihydro-11H-benzo
benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-, ethy| [5,6]cyclohepta[1,2-b]pyridin-11-one is a potential re-
ester; lated compound.]
Ethyl 4-(8-chloro-5,6-dihydro-11 H-benzo[5,6]cyclohepta[1,2- Mobile phase and Diluent: Proceed as directed in the
b]pyridin-11-ylidene)-1-piperidinecarboxylate Assay.
[79794-75-5]. Standard solution: 0.8 g/mL of USP Loratadine RS in
Diluent
USP 41 Official Monographs / Loratadine 2463

Sample solution: 0.4 mg/mL of Loratadine in Diluent Loratadine Related Compound BRS prepared as fol-
Chromatographic system lows. Transfer 1.0 mL of the Standard stock solution to a
(See Chromatography (621), System Suitability.) 10-mL volumetric flask, add 2 mL of Solution A, and di-
Mode: LC lute with methanol to volume.
Detector: UV 254 nm Sample solution: 10 mg/mL of Loratadine prepared as
Column: 4.6-mm x 15-cm; 5-'um packing L7 follows. Transfer 100 mg of Loratadine to a 10-mL volu-
Column temperature: 25°-35° metric flask, and dissolve in 2 mL of methanol. Add
Flow rate: 1 mL/min 2 mL of Solution A, and then dilute with methanol to
Injection volume: 50 pL volume.
System suitability ChromatographicSystem
Sample: Standard solution (See Chromatography (621), System Suitability.)
Suitability requirements Mode: LC
Relative standard deviation: NMT 4.0% Detector: UV 254 nm
Analysis Column: 4.6-mm x 25-cm; 5-um packing L1
Samples: Standard solution and Sample solution Flow rate: 1.2 mL/min
Calculate the percentage of each impurity in the por- Injection volume: 20 LL
tion of Loratadine taken: System suitability
Sample: Standard solution
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100 Suitability requirements
Resolution: NLT 1.5 between loratadine related com-
tu = peak area of each impurity from the Sample pound A and loratadine related compound B
solution Relative standard deviation: NMT 10% for the
ls = peak area of loratadine from the Standard loratadine peak
solution Analysis
Cs = concentration of USP Loratadine RS in the Sample: Sample solution
Standard solution (mg/mL) Calculate the percentage of each impurity in the por-
Cy ° = concentration of Loratadine in the Sample tion of Loratadine taken:
solution (mg/mL)
F = relative response factor as listed in Table 7 Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
Acceptance criteria: See Table 7.
tu = peak area of each individual impurity from the
Table 1 Sample solution
rs = peak area of loratadine from the Standard
Relative Relative Acceptance solution
Retention Response Criteria, Cs = concentration of USP Loratadine RS in the
Name Time Factor NMT (%) Standard solution (mg/mL)
Fluoroloratadine# 0.79 0.25 0.2 Cy = concentration of Loratadine in the Sample
Loratadine 1.0 — = solution (mg/mL) (aq
Any other individual _ F = relative response factor as listed in Table 3 “
impurity 1.0 0.1 Acceptance criteria: See Table 3. a
Total impurities — = 0.3 “=<
2 Ethyl 4-(8-chloro-1 1-fluoro-5,6-dihydro-1 1 H-benzo[5,6]cyclohepta[1,2- Table 3 =
b)pyridin-11-yl) piperidin-1-carboxylate.
Relative Relative Acceptance ft
@ ORGANIC IMPURITIES, PROCEDURE 2 Retention Response Criteria, Ss
Solution A: Dissolve 0.96 g of 1-pentanesulfonic acid Name Time Factor NMT (%) so
sodium salt in 900 mL of water, Adjust with phosphoric Loratadine related 2
acid solution (1 in 10) to a pH of 3.00 + 0.05, and compound A 0.50 1.00 0.1
dilute with water to 1 L. Loratadine related
Solution B: Acetonitrile compound B 0.53 0.89 0.1
Mobile phase: See Table 2. Loratadine related
compound C* 0.70 0.60 0.1
Table 2 Hydroxy deacyl
Solution A Solution B analog? 0.75 0.46 0.1
Loratadine 1.00 =—
75, Dichlorobenzo-
cycloheptapyridi-
none 1.23 0.92 0.1
Hydroxyloratadine? 1.60 0.42 0.1
3.
4-Chloroloratadines 1.83 1.08 0.1

5 75 8-Chloro-5,6-dihydro-1 1 H-benzo[5,6]cyclohepta[1 ,2-b]pyridin-11-one.

Standard stock solution: 0.1 mg/mL each of USP
Loratadine RS, USP Loratadine Related CompoundA RS,
and USP Loratadine Related Compound BRS in
methanol
Standard solution: 0.01 mg/mL each of USP Loratadine
RS, USP Loratadine Related Compound A RS, and USP
> 8-Chloro-5,6-dihydro-1 1-hydroxy-11-(1-methylpiperidin-4-yl)-11H-benzo
[5,6]cyclohepta[1,2-b]pyridine.
¢ 4,8-Dichloro-5,6-dihydro-11 H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-one.
4 Ethyl 4-(8-chloro-11-hydroxy-5,6-dihydro-1 1 H-benzo[5,6]cyclohepta[1,2-
b)pyridin-11-yl) piperidin-1-carboxylate.
© Ethyl 4-(4,8-dichloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-
b]pyridin-11-ylidene)piperidin-1 -carboxylate.
 2464 Loratadine / Official Monographs USP 41

Table 3 (Continued) 50-mL volumetric flask. Pipet 5.0 mL of Internal stan-

Relative Relative Acceptance dard solution into the flask, and dilute with Diluent to
Retention Response Criteria,
volume.
Name Time Factor NMT (%)
Chromatographic system
(See Chromatography (621), System Suitability.)
Any individual Mode: LC
unknown impurit _ 1.0 0.10 Detector: UV 254 nm. For /dentification B, use a diode
Total impurities — = 0.3 array detector in the range of 200-400 nm.
2 8-Chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta{1,2-b]pyridin-11-one. Column: 3.9-mm x 30-cm; 10-um packing L11
» 8-Chloro-5,6-dihydro-11-hydroxy-11-(1-methylpiperidin-4-yl)-1 1 H-benzo Column temperature: 20°-30°
[5,6]cyclohepta[1,2-b]pyridine. Flow rate: 2 mL/min
¢ 4,8-Dichloro-5,6-dihydro-1 1 H-benzo[5,6]cyclohepta[1 ,2-b]pyridin-11-one. Injection volume: 10 LL
4 Ethyl 4-(8-chloro-11-hydroxy-5,6-dihydro-11 H-benzo[5,6]cyclohepta[1 ,2- System suitability
b\pyridin-11-yl) piperidin-1 -carboxylate.
© Ethyl 4-(4,8-dichloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-
Sample: Standard solution
b]pyridin-11-ylidene)piperidin-1 -carboxylate. [Note—The relative retention times for butylparaben
and loratadine are about 0.78 and 1.0, respectively.]
SPECIFIC TESTS Suitability requirements
e Loss ON DRYING (731) Resolution: NLT 1.9 between loratadine and
Analysis: Dry a sample at 100° to constant weight. butylparaben
Acceptance criteria: NMT 0.5% Tailing factor: NMT 1.6 for the loratadine and
butylparaben peaks
ADDITIONAL REQUIREMENTS Relative standard deviation: NMT 2%
© PACKAGING AND STORAGE: Preserve in well-closed contain- Analysis
ers, and store between 2° and 30°. Samples: Standard solution and Sample solution
e LABELING: If a test for Organic Impurities other than Proce- Calculate the percentage of the labeled amount of
dure 7 is used, then the labeling states with which Or- loratadine (C22H23CIN2O2) in the portion of Oral Solu-
ganic Impurities test the article complies. tion taken:
e USP REFERENCE STANDARDS (11)
USP Loratadine RS Result = (Ru/Rs) x (Cs/Cu) x 100
USP Loratadine Related Compound A RS
8-Chloro-5,6-dihydro-1 1-(piperidin-4-ylidene)-11H- Ry = peak response ratio of loratadine to the
benzo[5,6]cyclohepta[1,2-b]pyridine. internal standard from the Sample solution
CisHisCIN, ~ 310.82 Rs = peak response ratio of loratadine to the
USP Loratadine Related Compound B RS internal standard from the Standard solution
8-Chloro-5,6-dihydro-11-(N-methylpiperidin-4-ylidene)- G = concentration of USP Loratadine RS in the
11H-benzo[5,6]cyclohepta[1,2-b]pyridine. Standard solution (mg/mL)
CoHaCIN2 324.85 Cu = nominal concentration of loratadine in the
as Sample solution (mg/mL)
ao Acceptance criteria: 94.0%-105.0%
%
s
— PERFORMANCE TESTS
2)
9 Loratadine Oral Solution © DELIVERABLE VOLUME (698): Meets the requirements
<
iS DEFINITION
IMPURITIES
= Loratadine Oral Solution contains NLT 94.0% and NMT
@ ORGANIC IMPURITIES
2 Mobile phase: 4.3 g/L of sodium dodecyl sulfate in a
al 105.0% of the labeled amount of loratadine mixture of acetonitrile and water (1:1). Adjust with
=) (C22H23CIN202). phosphoric acid to a pH of 2.6 + 0.1.
IDENTIFICATION Diluent: Mobile phase and water (2:1)
e A. The retention time of the major peak of the Sample System suitability solution 1: 2 g/mL of USP
solution corresponds to that of the Standard solution, as Loratadine RS in Diluent
obtained in the Assay. System suitability solution 2: 0.2 g/mL of USP
e B. The UV spectrum of the major peak of the Sample Loratadine RS in Diluent from System suitability
solution corresponds to that of the Standard solution, as
solution 1
obtained in the Assay. System suitability solution 3: Transfer an amount of
Oral Solution, equivalent to 20 mg of loratadine, into a
ASSAY screw-cap glass container. Add 1 mL of 3% aqueous hy-
e PROCEDURE drogen peroxide and mix. Cap and heat at 65° for
Buffer: 6.8 g/L of monobasic potassium phosphate in 18-24 h. Allow to cool to room temperature and then
water, Adjust with phosphoric acid to a pH of 3.0 + dilute 5 mL of the resulting solution with Diluent to
0.1. 25 mL.
Mobile phase: Acetonitrile and Buffer (3:7) Sample solution: Nominally 0.2 mg/mL of loratadine
Diluent: Acetonitrile and water (3:7) from a volume of Oral Solution in Diluent
Internal standard solution: 0.3 mg/mL of USP Chromatographic system
Butylparaben RS in Diluent (See Chromatography (621), System Suitability.)
Standard stock solution: 1.0 mg/mL of USP Loratadine Mode: LC
RS in acetonitrile Detector: UV 254 nm
Standard solution: Transfer 5.0 mL of Internal standard Column: 4.6-mm x 25-cm; 5-um packing L7
solution, 5.0 mL of Standard stock solution, and 12 mL of Column temperature: 30°-40°
water into a 50-mL volumetric flask. Dilute with Diluent Flow rate: 2 mL/min
to volume. Injection volume: 50 uL
Sample solution: Transfer a portion of Oral Solution, System suitability
nominally equivalent to 5 mg of loratadine, into a Samples: System suitability solution 1, System suitability
solution 2, and System suitability solution 3
USP 41 Official Monographs / Loratadine 2465

[Note—See Table1 for relative retention times.] ASSAY

Suitability requirements ¢ PROCEDURE
Resolution: NLT 3.0 between loratadine and 2-hy- Buffer A: 0.01 M dibasic potassium phosphate (1.74 g/
droxymethyl loratadine, System suitability solution 3 L of anhydrous dibasic potassium phosphate in water)
Tailing factor: 0.7-1.1, System suitability solution 1 Buffer B: 0.6 M dibasic potassium phosphate (105 g/L
Relative standard deviation: NMT 10%, System suit- of anhydrous dibasic potassium phosphate in water)
ability solution 2 0.05 N hydrochloric acid: Transfer 500 mL of water
Analysis into a 1000-mL volumetric flask, add 83 mL of hydro-
Sample: Sample solution chloric acid, and dilute with water to volume. Transfer
Calculate the percentage of each individual impurity in 50 mL of this solution into a 1000-mL volumetric flask
the portion of Oral Solution taken: and dilute with water to volume.
Mobile phase: Acetonitrile, methanol, and Buffer A
Result = (ru/r7) x 100 (60:60:70). Adjust with 10% phosphoric acid to a pH of
Ld,
fu = peak response of each individual impurity in Diluent: Transfer 400 mL of 0.05 N hydrochloric acid
the Sample solution and 80 mL of Buffer B into a 1-L volumetric flask. Dilute
tr = sum of all the peak responses in the Sample with a mixture of acetonitrile and methanol (1:1) to
solution, excluding excipient peaks volume.
Acceptance criteria: See Table 7. Standard solution: 0.4 mg/mL of USP Loratadine RS in
Diluent
Table 1 Sample solution: Transfer 10 Tablets to a 250-mL volu-
metric flask, add 100 mL of 0.05 N hydrochloric acid,
Relative Acceptance and shake for 40 min or until the Tablets are com-
Retention Criteria,
pletely disintegrated. Add 75 mL of a mixture of aceto-
Name Time NMT (%)
nitrile and methanol (1:1), and 20 mL of Buffer B, and
4-Hydroxymethyl mix for 5 min. Dilute with a mixture of acetonitrile and
loratadine 0.70 0.3 methanol (1:1) to volume.
2-Hydroxymethyl Chromatographic system
loratadine” 0.84 0.3 (See Chromatography (621), System Suitability.)
Loratadine 1.0 = Mode: LC
Any other individual _ Detector: UV 254 nm. For Identification B, use a diode
impurity 0.2 array detector in the range of 200-400 nm.
Total impurities = 0.5 Column: 4.6-mm x 15-cm; 5-"um packing L7
Column temperature: 25°-35°
2 Ethyl 4-[8-chloro-5,6-dihydro-4-(hydroxymethyl)-11H-benzo[5,
6]cyclohepta[1 ,2-b]pyridin-1 1-ylidene]-1 -piperidinecarboxylate. Flow rate: 1 mL/min
» Ethyl 4-[8-chloro-5,6-dihydro-2-(hydroxymethyl)-11H-benzo[5, Injection volume: 15 pL
6]cyclohepta[1,2-b]pyridin-11-ylidene]-1-piperidinecarboxylate. System suitability
Sample: Standard solution (=
SPECIFIC TESTS Suitability requirements a)
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI- Capacity factor: NLT 3.5
i)
FIED MICROORGANISMS (62): The total aerobic microbial Tailing factor: NMT 1.7 F<
count is NMT 102 cfu/mL, and the total combined molds Relative standard deviation: NMT 2.0% i}
and yeasts count is NMT 5 x 10! cfu/mL. It meets the =
Analysis )
requirements for the absence of Salmonella species and Samples: Standard solution and Sample solution a=
Escherichia coli. Calculate the percentage of the labeled amount of i
© PH (791): 2.2-3.1 loratadine (C22H23CIN2O2) in the portion of Tablets ao)
taken: =
ADDITIONAL REQUIREMENTS “

¢ PACKAGING AND STORAGE: Preserve in tight containers, Result = (ru/rs) x (Cs/Cu) x 100
and store between 2° and 25°.
e USP REFERENCE STANDARDS (11) ry = peak response from the Sample solution
USP Butylparaben RS Is = peak response from the Standard solution
USP Loratadine RS Cs = concentration of USP Loratadine RS in the
Standard solution (mg/mL)
Cu = nominal concentration of loratadine in the
Sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0%
Loratadine Tablets
PERFORMANCE TESTS
DEFINITION e DISSOLUTION (711)
Loratadine Tablets contain NLT 90.0% and NMT 110.0% of Medium: 0.1 N hydrochloric acid; 900 mL
the labeled amount of loratadine (C22H23CIN2O2). Apparatus 2: 50 rom
ime: 60 min
IDENTIFICATION Standard solution: USP Loratadine RS at a known con-
e A. The retention time of the major peak of the Sample centration in Medium
solution corresponds to that of the Standard solution, as Sample solution: Afiltered portion of the solution
obtained in the Assay. under test, suitably diluted with Medium, if necessary
e B. The UV spectrum of the major peak of the Sample Instrumental conditions
solution corresponds to that of the Standard solution, as Mode: UV-Vis
obtained in the Assay. Analytical wavelength: Maximum absorbance at
about 280 nm
 2468 Loratadine / Official Monographs USP 41

Table 1 (Continued) Mode: LC

Relative Relative Acceptance Detector: UV 254 nm
Retention Response Criteria,
Column: 4.6-mm x 15-cm; 5-um packing L1
Name Time Factor NMT (%)
Flow rate: 1.0 mL/min
Injection size: 20 uL
Any other individual System suitability
impurity ati 1.0 0.10 Sample: Standard solution
Total impurities — = 0.5 Suitability requirements
This is a process impurity and is included in the table for identification Column efficiency: NLT 3000 theoretical plates
only This impurity is controlled in the drug substance. It is not to be Tailing factor: NMT 2.0
reported for the drug product and should not be included in the total
impurities.
Relative standard deviation: NMT 2.0%
Analysis
ADDITIONAL REQUIREMENTS Samples: Standard solution and Sample solution
© PACKAGING AND STORAGE: Preserve in tight containers, Calculate the percentage of the labeled amount of
and store between 20° and 25°. loratadine in the portion of Tablets taken:
e LABELING: Label it to indicate that the Chewable Tablets
are to be chewed before swallowing. Result = (ru/rs) x (Cs/Cu) x 100
e USP REFERENCE STANDARDS (11)
USP Loratadine RS tu = peak response of loratadine from the Sample
USP Loratadine Related Compound A RS solution
8-Chloro-5,6-dihydro-11 -(piperidin-4-ylidene)-11H- rs = peak response of loratadine from the Standard
benzo[5,6]cyclohepta[1,2-b]pyridine. solution
CisHisCIN2 — 310.82 Gs = concentration of USP Loratadine RS in the
USP Loratadine Related Compound B RS Standard solution (mg/mL)
8-Chloro-5,6-dihydro-1 1-(N-methylpiperidin-4-ylidene)- Cu = nominal concentration of loratadine in the
11H-benzo[5,6]cyclohepta[1 2: bipynditie Sample solution (mg/mL)
CooHaiCIN2 324.85 Acceptance criteria: 95.0%-105.0%
USP Loratadine Related Compound C RS © PROCEDURE 2
8-Chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2- Buffer: 2.28 g/L of dibasic potassium phosphate
b]pyridin-11-one. trihydrate
CysHioCINO =.243.69 Mobile phase: Methanol, acetonitrile, and Buffer
(6:6:7), adjusted with 10% phosphoric acid to an ap-
parent pH of 7.2
Diluent: Methanol and water (1:1)
System suitability solution: 0.8 g/mL each of USP
Loratadine Related Compound A RS, USP Loratadine Re-
Loratadine Orally Disintegrating lated Compound B RS, and USP Loratadine Related
Tablets Compound C RS in Diluent
<
”
Standard solution: 0.4 mg/mL of USP Loratadine RS in
oe DEFINITION Diluent. [NoTE—The solution may be sonicated for 5
s
—
D Loratadine Orally Disintegrating Tablets contain NLT 95.0% min to aid in dissolving.]
} and NMT 105.0% of the labeled amount of loratadine Sample solution: Transfer a number of Tablets into a
& (C22H23CIN202). 250-mL volumetric flask so that the final concentration
Sj is 0.4 mg/mL, based on the label claim. Add 50 mL of
P= IDENTIFICATION water, and sonicate, if necessary, to disperse the Tab-
a e A. The retention time of the major peak of the Sample lets. Add 50 mL of methanol, and shake to dissolve. Di-
w solution corresponds to that of the Standard solution, as lute with Diluent to volume.
a obtained in the Assay. Chromatographic yo
(See Chromatography (621), System Suitability.)
ASSAY Mode: LC
[Note—Matrix effects have been observed that affect the Detector: UV 254 nm
extraction of loratadine. Depending on the composition of Column: 4.6-mm x 15-cm; 5-um packing L7
the Tablet, use Assay, Procedure 1 or Procedure 2.] Flow rate: 1.0 mL/min
e PROCEDURE 1 Injection size: 15 ul for the Standard solution and
Buffer: 2.72 g/L of monobasic potassium phosphate in Sample solution; 50 wL for the System suitability solution
water. Adjust with 5 N sodium hydroxide solution to a System suitability
pH of 6.50 + 0.05, and filter. Samples: System suitability solution and Standard
Mobile phase: Acetonitrile and Buffer (70:30) solution
Diluent: Acetonitrile and Buffer (40:60) [Note—tThe relative retention times for loratadine re-
Standard solution: 0.1 mg/mL of USP Loratadine RS in lated compound A, loratadine related compound C,
Mobile phase loratadine related compound B, and loratadine are
Sample solution: Transfer 10 Tablets into a 500-mL vol- about 0.26, 0.31, 0.42, and 1.0, respectively.]
umetric flask, add 400 mL of acetonitrile, and stir for 10 Suitability requirements
min. Sonicate the solution for 10 min, and stir for an- Resolution: NLT 1.2 between loratadine related com-
other 10 min. Dilute with acetonitrile to volume, and pound A and loratadine related compound C and
mix. Dilute an aliquot of the resulting solution with Dil- NLT 1.2 between loratadine related compound C and
uent to obtain a solution having a concentration of loratadine related compound B, System suitability
about 0.1 mg/mL, based on the label claim. Pass a por- solution
tion of this solution through a PVDF filter of 0.45-1m Tailing factor: NMT 2.0, Standard solution
pore size, and discard the first 5 mL of filtrate. Relative standard deviation: NMT 2.0%, Standard
Chromatographic system solution
(See Chromatography (621), System Suitability.)
USP 41 Official Monographs / Loratadine 2469

Analysis stir for another 10 min. Dilute with acetonitrile to vol-

Samples: Standard solution and Sample solution ume, and mix. Dilute an aliquot of the resulting solu-
Calculate the percentage of the labeled amount of tion with Diluent to obtain a solution having a concen-
loratadine in the portion of Tablets taken: tration of about 0.1 mg/mL, based on the label claim.
Centrifuge the solution for about 10 min, and use the
Result = (ru/rs) x (Cs/Cu) x 100 supernatant.
Chromatographic system
ru = peak response of loratadine from the Sample (See Chromatography (621), System Suitability.)
solution Mode: LC
Ts peak response of loratadine from the Standard Detector: UV 254 nm
solution Column: 4.6-mm x 15-cm; 5-uwm packing L1
Gs = concentration of USP Loratadine RS in the Flow rate: 1.0 mL/min
Standard solution (mg/mL) Injection size: 50 uL
Cy = nominal concentration of loratadine in the System suitability
Sample solution (mg/mL) Samples: System sensitivity solution and Standard
Acceptance criteria: 95.0%-105.0% solution
Suitability requirements
PERFORMANCE TESTS Signal-to-noise ratio: NLT 10, System sensitivity
e DISINTEGRATION (701) solution
Test 1 Column efficiency: NLT 3000 theoretical plates,
Stage 1: All 6 Tablets completely disintegrate in 1 Standard solution
min. Tailing factor: NMT 2.0, Standard solution
Stage 2: NLT 16 of 18 Tablets completely disintegrate Relative standard deviation: NMT 2.0%, Standard
in 1 min. solution
Test 2: If the product complies with this test, the label- Analysis
ing indicates that it meets USP Disintegration Test 2. Samples: Standard solution and Sample solution
Analysis: Place a stainless steel wire clip on each Tablet Calculate the percentage of each impurity in the por-
to prevent the Tablet from floating. tion of Tablets taken:
Acceptance criteria: NMT 30s
¢ DISSOLUTION (711) Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
Medium: Simulated gastric fluid without enzymes;
900 mL, deaerated ry = peak response of each impurity from the
Apparatus 1; 50 rpm Sample solution
Time: 6 min ls = peak response of loratadine from the Standard
Standard solution: Prepare a solution of USP solution
Loratadine RS in Medium at a concentration similar to Cs = concentration of USP Loratadine RS in the
that expected in the Sample solution. Standard solution (mg/mL)
Sample solution: Pass a portion of the solution under Cu = nominal concentration of loratadine in the (on
test through a suitable filter. Sample solution (mg/mL) “
Analysis F = relative response factor (see Table 1) ay
Detector: UV 278 nm Acceptance criteria: See Table 1. i=
Cell length: 1m °
Blank: Medium a
Table 1 fe]
Calculate the percentage of the labeled amount of
loratadine (C22H23CIN2O2) dissolved: Relative Relative Acceptance =
Retention Response Criteria, a
Result = (Au/As) x Cs x (V/L) x 100 Name Time Factor NMT (%’ =
Loratadine related i
Au = absorbance from the Sample solution compound C 0.5 0.64 0.2
As = absorbance from the Standard solution
Loratadine 1.0 =
Gs = concentration of the Standard solution
Individual unspeci- _—
(mg/mL)
L = label claim (mg/Tablet) fied impurity 1.0 0.1
Vv = volume of Medium, 900 mL Total impurities = — 0.3
Tolerances: NLT 80% (Q) of the labeled amount of
loratadine is dissolved. e PROCEDURE 2
© UNIFORMITY OF DOSAGE UNITS (905): Meet the [Note—Use Organic Impurities, Procedure 2 if Assay, Proce-
requirements dure 2 is used.]
Buffer, Mobile phase, Diluent, Sample solution, and
IMPURITIES System suitability solution: Proceed as directed in As-
Organic Impurities Say, Procedure 2.
¢ PROCEDURE 1 System sensitivity solution: 0.04 ug/mL of USP
[Nott—Use Organic Impurities, Procedure 1 if Assay, Proce- Loratadine RS in Diluent
dure1 is used.] Standard solution: 0.8 g/mL of USP Loratadine RS in
Buffer, Mobile phase, and Diluent: Proceed as di- Diluent
rected in Assay, Procedure 1. Chromatographic system
System sensitivity solution: 0.05 ug/mL of USP (See Chromatography (621), System Suitability.)
Loratadine RS in Mobile phase Mode: LC
Standard solution: 0.5 ug/ml of USP Loratadine RS in Detector: UV 254 nm
Mobile phase Column: 4.6-mm x 15-cm; 5-um packing L7
Sample solution: Transfer 10 Tablets into a 500-mL Flow rate: 1.0 mL/min
volumetric flask, add 400 mL of acetonitrile, and stir for Injection size: 50 wL
about 10 min. Sonicate the solution for 10 min, and
 2470 Loratadine / Official Monographs USP 41

System suitability USP Loratadine Related Compound B RS

Samples: System suitability solution, Standard solution, 8-Chloro-5,6-dihydro-11-(N-methylpiperidin-4-ylidene)-
and System sensitivity solution 11H-benzo[5,6]cyclohepta[1,2-b]pyridine.
[Note—The relative retention times for loratadine re- CaoHaiCIN2 324.85
lated compound A, loratadine related compound C, USP Loratadine Related Compound C RS
loratadine related compound B, and loratadine are 8-Chloro-5,6-dihydro-1 1 H-benzo[5,6]cyclohepta[1,2-
about 0.26, 0.31, 0.42, and 1.0, respectively.] b]pyridin-11-one.
Suitability requirements Ci4HioCINO =.243.69
Resolution: NLT 1.2 between loratadine related
compoundA and loratadine related compound C
and NLT 1.2 between loratadine related compound
C and loratadine related compound B, System suita-
bility solution Lorazepam
Tailing factor: NMT 2.0, Standard solution
Relative standard deviation: NMT 4.0%, Standard i
solution (ON NN
Signal-to-noise ratio: NLT 3.0, System sensitivity
solution =n
Analysis =
Samples: Standard solution and Sample solution \
Calculate the percentage of each impurity in the por-
tion of Tablets taken:
CysHioClzN202 321.16
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100 2H-1,4-Benzodiazepin-2-one, 7-chloro-5-(2-chlorophenyl)-
1,3-dihydro-3-hydroxy-, (+)-;
ru = peak response of each impurity from the (4)-7-Chloro-5-(o-chlorophenyl)-1,3-dihydro-3-hydroxy-2H-
Sample solution 1,4-benzodiazepin-2-one [846-49-1].
rs = peak response of loratadine from the
DEFINITION
= Standard,
tratisolution i in th Lorazepam contains NLT 98.0% and NMT 102.0%: of .
‘s oranda Corwen RS the lorazepam (CisHi0ClzN202), calculated on the dried basis.
Cu = nominal concentration of loratadine in the IDENTIFICATION
Sample solution (mg/mL) e A. INFRARED ABSORPTION (197): [NOTE—Procedures de-
F = relative response factor (see Table 2) scribed in (197K) or (197A) may be used.]
Acceptance criteria: See Table 2. e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
rs Table 2 obtained in the Assay.
Ta Relative Relative Acceptance ASSAY
] Retention Response Criteria, e PROCEDURE
= Name Time Factor NMT (%) Mobile phase: Acetonitrile, glacial acetic acid, and
° Loratadine related water (50: 1.2: 50)
= compound A 0.26 0.9 0.1 Diluent: Methanol and water (75:25)
= Loratadine related _ _ Standard solution: 0.1 mg/mL of USP Lorazepam RS in
compound Ba 0.42 Diluent
= Unspecified = = Sample solution: 0.1 mg/mL of Lorazepam in Diluent
i impurity 0.76 Chromatographic system oo
Loratadine 1.0 _ ae (See Chromatography (621), System Suitability.)
UI ified = — Mode: LC
SRE Detector: UV 230 nm
impurity? 1.5 Column: 4.6-mm x 25-cm; 5-1m packing L1
Individual unspeci- = Temperatures
fied impurity 1.0 0.1 Column: 5°
Total impurities = — 0.1 Sample chamber: 4°
4These impurities are controlled in the drug substance and are listed here Flow rate: 1 mL/min
for information only. These impurities are not included when determining Injection volume: 5 wl
total impurities. System suitability
ADDITIONAL REQUIREMENTS Sample: Standard solution
e PACKAGING AND STORAGE: Preserve in tight containers. Collect data for at least 50 min.
Store between 20° and 25°. Suitability requirements
e LABELING: The labeling states with which Organic Impuri- Tailing factor: NMT 2.0
ties and Assay procedure the article complies, if other Relative standard deviation: NMT 2.0%
than Procedure 1. When more than one Disintegration test Analysis
is BN the labeling states the Disintegration test used Samples: Standard solution and Sample solution
only if Test 7 is not used. Calculate the percentage of lorazepam (CisHioClaN2O2)
e USP REFERENCE STANDARDS (11) in the portion of Lorazepam taken:
USP Loratadine RS
USP Loratadine Related Compound A RS Result = (ru/rs) x (Cs/Cu) x 100
8-Chloro-5,6-dihydro-11-(piperidin-4-ylidene)-11H-
benzols.tlcyeldneptall Pp eyridine: ) ru =Pea ee of lorazepam from the Sample
solution
CrelsGiN, 10182 Is = peak response of lorazepam from the Standard
solution
Cs = concentration of USP Lorazepam RS in the
Standard solution (mg/mL)
USP 41 Official Monographs / Lorazepam 2471

Gu = concentration of Lorazepam in the Sample Table 1 (Continued)

solution (mg/mL) Relative Relative Acceptance
Acceptance criteria: 98.0%-102.0% on the dried basis Retention Response Criteria,
IMPURITIES Name Time Factor NMT (%)
e RESIDUE ON IGNITION (281): NMT 0.3% Lorazepam related
compound A 7 1.0 0.10
Lorazepam related
Delete the following: compound E iD 1.3 0.15
Lorazepam related
°e HEAVY METALS, Method I! (231): NMT 20 ppme coma s-
_compound C__ nee] 1.0 0.30
Jan-2018)
© ORGANIC IMPURITIES Lorazepam related
Mobile phase and Diluent: Proceed as directed in the compound B 535. 1.0 0.01
Assay. Any individual
System suitability solution: 3.2 mg/mL of USP unspecified =
Lorazepam RS and 32 g/mL each of USP Lorazepam _impurity_ 1.0 0.10
Related Compound A RS, USP Lorazepam Related Com- Total impurities = = 0.75
pound B RS, USP Lorazepam Related Compound C RS,
USP Lorazepam Related CompoundD RS, and USP
Lorazepam Related Compound E RS in Diluent SPECIFIC TESTS
Standard solution: 32 1g/mL of USP Lorazepam RS in e Loss ON DRYING (731)
Diluent Analysis: Dry under vacuum at 105° for 3 h.
Sample solution: 3.2 mg/mL of Lorazepam in Diluent Acceptance criteria: NMT 0.5%
Chromatographic system ADDITIONAL REQUIREMENTS
(See Chromatography (621), System Suitability.) © PACKAGING AND STORAGE: Preserve in tight, light-resistant
Mode: LC containers.
Detector: UV 230 nm e USP REFERENCE STANDARDS (11)
Column: 4.6-mm x 25-cm; 5-um packing L1 USP Lorazepam RS
Temperatures USP Lorazepam Related Compound A RS
Column: 5° 7-Chloro-5-(o-chlorophenyl)-1,3-dihydro-3-acetoxy-2H-
Sample chamber: 4° 1,4-benzodiazepin-2-one.
Flow rate: 1 mL/min Cy7Hi2ClzN203 363.20
Injection volume: 100 pL USP Lorazepam Related Compound B RS
System suitability 2-Amino-2’,5-dichlorobenzophenone.
Samples: System suitability solution and Standard CisHaCl2NO 266.12
solution USP Lorazepam Related Compound C RS
[Note—See Table 7 for the relative retention times.]
Collect data for at least 50 min.
Se c
2-quinazolinecarboxaldehyde. a
Suitability requirements CisHsClzN20 303.14 a)
Resolution: NLT 1.2 between lorazepam related com-
pound A and lorazepam related compound E, System
USP Lorazepam Related Compound D RS =
og ater"teach expen)-2-Gallsae Neca So
suitability solution acid. 3
Tailing factor: NMT 2.0 for lorazepam, Standard }
solution
CisHsClaN2O2 319.14 i}=
USP Lorazepam Related Compound E RS ES)
Relative standard deviation: NMT 5% for
lorazepam, Standard solution
6-Chloro-4-(0-chlorophenyl)-2-quinazoline methanol. 5}
CisHioClzaN20 305.16 =>
Analysis “

Samples: Standard solution and Sample solution

Calculate the percentage of each impurity in the por-
tion of Lorazepam taken:
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100 Lorazepam Injection
tu = peak response of each impurity from the DEFINITION
Sample solution Lorazepam Injection is a sterile solution of Lorazepam in a
rs = peak response of lorazepam from the Standard suitable medium. It contains NLT 90.0% and NMT
solution 110.0% of the labeled amount of lorazepam
Cs = concentration of USP Lorazepam RS in the (CisHioClaN2O2).
Standard solution (mg/mL)
Cy concentration of Lorazepam in the Sample IDENTIFICATION
solution (mg/mL) e A. The retention time of the major peak of the Sample
F = relative response factor for any given impurity solution corresponds to that of the Standard solution, as
(see Table 1) obtained in the Assay.
Acceptance criteria: See Table 1.
Delete the following:
Table 1
Relative Relative Acceptance 4e B. THIN-LAYER CHROMATOGRAPHY
Retention Response Criteria, Standard stock solution: 1 mg/mL of USP Lorazepam
Name Time Factor NMT (%) RS in alcohol
Standard solution: Transfer 10 mL of Standard stock so-
Lorazepam 1.0 1.0 =
lution to a suitable container. Add 5 mL of hydrochloric
Lorazepam related acid, heat the solution on a steam bath for 20 min, and
compound D 1.4 1.0 0.15 cool. Transfer the solution to a separator, and add 8 mL
of 10 N sodium hydroxide. Extract the solution with
 2472 Lorazepam / Official Monographs
two 25-mL portions of ether, filtering the ether extracts
through cotton plugs. Evaporate the ether extract to
2mL, and add § mL of methanol.
Sample solution: Transfer a volume of Injection, equiv-
alent to 10 mg of lorazepam, to a container. Add 5 mL
of hydrochloric acid, heat on a steam bath for 20 min,
and cool. Transfer the solution to a separator, and add
8 mL of 10 N sodium hydroxide. Extract with two
25-mL portions of ether, filtering the ether extracts
through cotton flocs. Evaporate the ether extract to
2 mL, and add 8 mL of methanol.
Chromatographic system
(See Chromatography {621}, Thin-Layer Chromato-
raphy.)
lode: TLC
Adsorbent: Thin-layer chromatographic plate, coated
with a 0.25-mm layer of chromatographic silica gel
Application volume: 10 iL
Developing solvent system: Toluene
ee reagent 1: 12.5 mg/mL of sodium nitrite in 0.5
hydrochloric acid, oy prepared
Spray reagent 2: 1 mg/mL of N-(1-naphthylethylene-
diamine dihydrochloride in alcohol
Analysis
Samples: Standard solution and Sample solution
Allow the spots to dry, and develop the chromatograms
until the solvent front has moved 15 cm. Remove the
late from the developing chamber, mark the solvent
Font and allow the solvent to evaporate. Spray the
pie with Spray reagent 1. Heat the plate at 100° for
min, allow to cool, and spray with Spray reagent 2.
Asceplance criteria: The Rr value of the principal spot
of the Sample solution corresponds to that of the Stan-
dard solution. usps:
Add the following:
2
ia 4e B. The UV spectrum of the major peak of the Sample
rm solution corresponds to that of the Standard solution, as
Ss
—
toa) obtained in the Assay.ausea
-)
to ASSAY
So
= Change to read:
os
a)
= © PROCEDURE
Buffer: 0.05 M monobasic ammonium phosphate
Mobile phase: Methanol and Buffer (50:50). Adjust
with ammonium hydroxide to a pH of 6.5.
System suitability solution: 0.04 mg/mL of lorazepam
and 32 g/mL each of USP Lorazepam Related Com-
pound CRS and USP Lorazepam Related Compound D
RS in Mobile phase
Standard stock solution: 1mg/mL of USP Lorazepam
RS in methanol
Standard solution: 0.16 mg/mL of USP Lorazepam RS
in Mobile phase from the Standard stock solution
Sample solution: Nominally 0.16 maim of lorazepam
from Injection, diluted with Mobile phase
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 240 nm. 4For Identification B, use a di-
ode array detector in the range of 210-400 nm.auseas
Column: 4.6-mm x 10 to 15-cm; 45-LMauseat packing
L1
Flow rate: 2 mL/min
Injection volume: 20 uL
aRun time: NLT 3 times the retention time of
lorazepamausear
System suitability
Samples: System suitability solution and Standard
solution
USP 41
[NoTte—The relative retention times for lorazepam re-
lated compound D, lorazepam, and lorazepam related
compoundC are 0.7, 1.0, and 2.7, respectively.]
Suitability requirements
Resolution: NLT 1.2 between lorazepam related com-
pound D and lorazepam; NLT 1.2 between lorazepam
and lorazepam related compound C, System suitability
solution
Relative standard deviation: NMT 2.0%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
peean (CisHioClzN2O2) in the portion of Injection
taken:
Result = (ru/rs) x (Cs/Cu) x 100
ru = peak response of lorazepam from the Sample
solution
rs = peak response of lorazepam from the Standard
solution
Cs = concentration of USP Lorazepam RS in the
Standard solution (mg/mL)
Cu = nominal concentration of lorazepam in the
Sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0%
IMPURITIES
Change to read:
© ORGANIC IMPURITIES
Mobile phase, System suitability solution, Sample so-
lution, and Chromatographic system: Proceed as di-
rected in the Assay.
Standard solution 1: Prepare as directed for the Stan-
dard solution in the Assay.
Standard solution 2: 3.2 g/mL each of USP
Lorazepam Related Compound C RS and USP
Lorazepam Related CompoundD RS in Mobile phase
System suitability
Samples: System suitability solution and Standard solu-
tion 1
[Note—The relative retention times for lorazepam re-
lated compound D, lorazepam, and lorazepam related
compoundC are 0.7, 1.0, and 2.7, respectively.]
Suitability requirements
Resolution: NLT 1.2 between lorazepam related com-
pound D and lorazepam; NLT 1.2 between lorazepam
and lorazepam related compound C, System suitability
solution
Relative standard deviation: NMT 2.0%, Standard
solution 1
Analysis
Samples: Sample solution and Standard solution 2. Do
not include as an impurity any peak from the Sample
solution that has a retention time shorter than that of
the lorazepam related compound D peak from Stan-
dard solution 2.
Calculate ee peeps of lorazepam related com-
pound C and lorazepam related compoundD in the
portion of Injection taken:
Result = (ru/rs) * (Cs/Cu) x 100
ru = peak response of lorazepam related
compoundCor lorazepam related
compoundD from the Sample solution
rs = peak response of the corresponding related
compound from Standard solution 2auses
Gs = concentration of the corresponding related
compound in 4Standard solution 2ausesi
(ug/ml)
USP 41 Official Monographs / Lorazepam 2473

Cu = nominal concentration of lorazepam in the Change to read:

Sample solution (g/mL)
Calculate the percentage of any other impurity in the e USP REFERENCE STANDARDS (11)
portion of Injection taken: °e (CN 1-May-2018)
USP Lorazepam RS
Result = (ru/rr) x 100 USP Lorazepam Related Compound B RS
tu = peak response of the individual impurity from 2-Amino-2’,5-dichlorobenzophenone.
the Sample solution CisHeChNO 4266.12ausrsi
rr = peak response of lorazepam from the Sample USP Lorazepam Related Compound C RS
solution 6-Chloro-4-(o-chlorophenyl)-
Acceptance criteria: NMT 4.0% of all impurities 2-quinazolinecarboxaldehyde.
CisHsClaN20 4303.14aisesi
USP Lorazepam Related Compound D RS
Change to read: 6-Chloro-4-(o-chlorophenyl)-2-quinazolinecarboxylic
acid.
© Limit OF LORAZEPAM RELATED COMPOUND B CisHsClzN202 4319.14ausear
4Buffer: 0.05 M monobasic ammonium phosphate. Ad-
just with ammmonium hydroxide to a pH of 6.5.
Mobile phase: Methanol and Buffer (55:45)
Diluent: Methanol and Buffer (50:50)
Standard solution: 0.16 g/mL of USP Lorazepam Re- Lorazepam Oral Concentrate
lated Compound B RS in Diluent. Sonicate to dissolve if
necessary. DEFINITION
Sample solution: Nominally 0.16 mg/mL of lorazepam Lorazepam Oral Concentrate contains NLT 90.0% and NMT
from Injection, diluted with Diluent. Sonicate to dissolve 110.0% of the labeled amount of lorazepam
if necessary. (CisHioClaN20z).
Chromatographic system
(See Chromatography (621), System Suitability.) IDENTIFICATION
Mode: LC e A. The retention time of the major peak of the Sample
Detector: UV 240 nm solution corresponds to that of the Standard solution, as
Column: 4.6-mm x 10-cm; 5-jum packing L1 obtained in the Assay.
Flow rate: 2 mL/min e B. The UV spectrum of the major peak of the Sample
Injection volume: 50 uL solution corresponds to that of the Standard solution, as
Run time: 1.5 times the retention time of lorazepam obtained in the Assay.
related compound B
System suitability ASSAY
ample: Standard solution © PROCEDURE
Suitability requirements Mobile phase: Acetonitrile, glacial acetic acid, and a
a)
Relative standard deviation: NMT 5.0% water (45: 0.2: 55) a)
Signal-to-noise ratio: NLT 10 System suitability solution: 0.1 mg/mL each of USP
Lorazepam RS and USP Lorazepam Related Compound os
Analysis >)
Samples: Standard solution and Sample solution E RS in methanol 3
Calculate the Foceoe of lorazepam related com- Standard solution: 0.05 mg/mL of USP Lorazepam RS i)
pound B in the portion of Injection taken: in methanol e=
Sample solution: Nominally 0.05 mg/mL of lorazepam 2
prepared as follows. Transfer a suitable volume of Oral 5S.
Result = (ru/rs) x (Cs/Cu) x 100
Concentrate to a volumetric flask, and dilute with meth-
=p
Sal
fu = peak response of lorazepam related anol to volume.
compound 8 from the Sample solution Chromatographic system
Ks = peak response of lorazepam related (See Chromatography (621), System Suitability.)
compound B from the Standard solution Mode: LC
CG; = concentration of USP Lorazepam Related Detector: UV 254 nm. For /dentification B, use a diode-
Compound 8 RS in the Standard solution array detector in the range of 220-400 nm.
(mg/mL) Column: 4-mm x 30-cm; packing L1
Cu = nominal concentration of lorazepam in the Flow rate: 2 mL/min
Sample solution (mg/mL) Injection volume: 20 pL
Acceptance criteria: NMT 0.1%ausesr System suitability
Samples: System suitability solution and Standard
SPECIFIC TESTS solution
e BACTERIAL ENDOTOXINS TEST (85): Contains NMT 102 USP [NoTe—The relative retention times for lorazepam and
Endotoxin Units/mg of lorazepam lorazepam related compoundE are 0.6 and 1.0,
© OTHER REQUIREMENTS: It meets the requirements in Injec- respectively.]
tions and Implanted Drug Products (1). Suitability requirements
ADDITIONAL REQUIREMENTS Resolution: NLT 2.0 between lorazepam and
lorazepam related compound E, System suitability
solution
Change to read: Relative standard deviation: NMT 2.0%, Standard
solution
¢ PACKAGING AND STORAGE: Preserve in single-dose or mul- Analysis
tiple-dose containers, preferably of Type | glass, protected Samples: Standard solution and Sample solution
from light. 4Store in arefrigerator.ausear Calculate the percentage of the labeled amount of
lorazepam (CisHioClzN2O02) in the portion of Oral Con-
centrate taken:
Result = (ru/rs) x (Cs/Cu) x 100
 2474 Lorazepam / Official Monographs USP 41

ru = peak response from the Sample solution Table 1

rs = peak response from the Standard solution Relative Acceptance
Cs = concentration of USP Lorazepam RS in the Retention Criteria,
Standard solution (mg/mL) Name Time NMT (%)
Cu = nominal concentration of lorazepam in the
Lorazepam related
Sample solution (mg/mL)
compound D 0.8 4.02
Acceptance criteria: 90.0%-110.0%
Lorazepam 10 =
IMPURITIES Lorazepam related
e@ ORGANIC IMPURITIES compound C 2:3 4.08
Mobile phase: Methanol and 0.05 M monobasic am- Lorazepam related
monium phosphate (64:36) compound B 2.9 0.1
Diluent: Methanol and 0.05 M monobasic ammonium @ Includes the sum of lorazepam related compound C and lorazepam re-
phosphate (50:50). Adjust with ammonium hydroxide lated compound D.
to a pH of 6.5.
System suitability solution: 0.04 mg/mL of USP ADDITIONAL REQUIREMENTS
Lorazepam RS, and 0.032 mg/mL each of USP e PACKAGING AND STORAGE: Preserve in well-closed, light-
Lorazepam Related Compound C RS and USP resistant containers, and store in a refrigerator.
Lorazepam Related Compound DRS in Diluent e USP REFERENCE STANDARDS (11)
Standard stock solution: 1.0 mg/mL of USP Lorazepam USP Lorazepam RS
RS in methanol USP Lorazepam Related Compound B RS
Standard solution 1: 0.16 g/mL of lorazepam from 2-Amino-2’,5-dichlorobenzophenone.
the Standard stock solution in Diluent Cy3HsClANO =.266.12
Standard solution 2: 0.16 hg/mi of USP Lorazepam USP Lorazepam Related Compound C RS
Related Compound B RS, and 3.2 ug/mL each of USP 6-Chloro-4-(o-chlorophenyl)-
Lorazepam Related Compound C RS and USP 2-quinazolinecarboxaldehyde.
Lorazepam Related Compound DRS in Mobile phase CysHgClaN20 303.14
Sample solution: Nominally 0.16 mg/mL of lorazepam USP Lorazepam Related Compound D RS
prepared as follows. Transfer a suitable volume of Oral 6-C pare ae enlerophest ee ane Zeln aaa
Concentrate to a volumetric flask, and dilute with Mo- acid.
bile phase to volume. CisHsClaN2O2 _ 319.14
Chromatographic system USP Lorazepam Related Compound E RS
(See Chromatography (621), System Suitability.) 6-Chloro-4-(o-chlorophenyl)-2-quinazoline methanol.
Mode: LC CisHioClaN20 =:3305.16
Detector: UV 240 nm
Column: 4.6-mm x 10- to 15-cm; packing L1
Flow rate: 0.7 mL/min
”“ Injection volume: 20 pL
= System suitability Lorazepam Tablets
(oy Samples: System suitability solution and Standard solu-
S
7
Dp tion 1 DEFINITION
3 Suitability requirements Lorazepam Tablets contain NLT 90.0% and NMT 110.0% of
i= Resolution: NLT 1.2 between lorazepam related com- the labeled amount of lorazepam (CisHioCl2zN202).
5 poundD and lorazepam; NLT 1.2 between lorazepam
= and lorazepam related compound C, System suitability IDENTIFICATION
2 solution e A. INFRARED ABSORPTION (197M)
”
=) Relative standard deviation: NMT 2.0% for Sample: Stir a portion of finely powdered Tablets,
lorazepam, Standard solution 1 equivalent to 15 mg of lorazepam, with 40 mL of ace-
Analysis tone for 5 min. Pass through very retentive filter ied
Samples: Standard solution 2 and Sample solution pre-washed with acetone. Evaporate the filtrate to dry-
[NoTE—Disregard peaks eluting prior to lorazepam re- ness on a steam bath with the aid of a current of air.
lated compound D.] Dissolve the residue in 1 mL of acetone, and add 20 mL
Calculate the percentage of lorazepam related com- of Sp let penta es Heat the solution on a hot
ound B, lorazepam related compound C, and plate to a gentle boil, and evaporate to a volume of
lorazepam related compoundDin the portion of Oral about 10 mL. Remove the solution from the hot plate,
Concentrate taken: and evaporate to dryness with the aid of a current of
air. Dry the residue under vacuum at 60° for 1h.
Result = (ru/rs) x (Cs/Cu) x 100 Acceptance criteria: Meet the requirements
e B. The retention time of the major peak of the Sample
tu = peak response of lorazepam related solution corresponds to that of the Standard solution, as
compound B, lorazepam related compound obtained in the Assay.
C, or lorazepam related compound D from
the Sample solution ASSAY
is peak response of the corresponding related e PROCEDURE
compound from Standard solution 2 Diluent: Methanol and water (85:15)
Gs = concentration of the corresponding related Mobile phase: Acetonitrile, glacial acetic acid, and
compound in Standard solution 2 (mg/mL) water (40: 0.4: 60)
Cu = nominal concentration of lorazepam in the Standard solution: 0.1 mg/mL of USP Lorazepam RS in
Sample solution (mg/mL) Diluent
Acceptance criteria: See Table 1. Sample solution: Nominally 0.1 mg/mL of lorazepam
prepared as follows. Transfer 20 Tablets to a 100-mL
volumetric flask, add 50 mL of Diluent, sonicate for 10
min, and shake by mechanical means for 20 min. Dilute
USP 41 Official Monographs / Lorazepam 2475

with Diluent to volume, mix, and centrifuge a portion of ls = peak response from the Standard solution
the solution at 2000 rpm for 10 min. Dilute a portion Cs = concentration of USP Lorazepam RS in the
of the clear supernatant with Diluent. Standard solution (mg/mL)
Chromatographic system Gu = nominal concentration of lorazepam in the
(See Chromatography (621), System Suitability.) Sample solution uid
Mode: LC Acceptance criteria: Meet the requirements
Detector: UV 230 nm
Column: 4.6-mm x 25-cm; 5-um packing L1 IMPURITIES
Flow rate: 1 mL/min © ORGANIC IMPURITIES
Injection volume: 20 uL Buffer: 67.7 g/L of sodium acetate trihydrate in water.
System suitability Adjust with glacial acetic acid to a pH of 5.0 + 0.05.
Sample: Standard solution Mobile phase: Acetonitrile, glacial acetic acid, and
Suitability requirements water (50: 1.2: 50)
Tailing factor: NMT 2.0 Diluent: Methanol and Buffer (75:25)
Relative standard deviation: NMT 2.0% Standard solution: 1.6 g/mL of USP Lorazepam RS in
Analysis Diluent
Samples: Standard solution and Sample solution Peak identification solution: 0.16 mg/mL of USP
Calculate the percentage of the labeled amount of Lorazepam RS, 1.6 g/mL each of USP Lorazepam Re-
aera (CisHioClzN2O2) in the portion of Tablets lated Compound A RS, USP Lorazepam Related Com-
taken: pound B RS, USP Lorazepam Related CompoundC RS,
USP Lorazepam Related Compound D RS, and USP
Result = (ru/rs) x (Cs/Cu) x 100 Lorazepam Related Compound ERS in Diluent
Sample solution: Nominally 0.16 mg/mL of lorazepam
ru = peak response from the Sample solution prepared as follows. Transfer a weighed amount of
rs = peak response from the Standard solution jorazepam, equivalent to 21.3 mg from powdered Tab-
Gs = concentration of USP Lorazepam RS in the lets, to a 25-mL volumetric flask. Add 20 mL of Diluent,
Standard solution (mg/mL) and stir for 15 min. Do not dilute to volume. Centrifuge
Cu = nominal concentration of lorazepam in the at 2000 rpm for 15 min. Pass the supernatant through
Sample solution (mg/mL) a polyethersulfone membrane of 0.45-1m pore size. Di-
Acceptance criteria: 90.0%-110.0% lute a portion of the filtrate with Diluent.
Chromatographic system
PERFORMANCE TESTS (See Chromatography (621), System Suitability.)
© DISSOLUTION (711) Mode: LC. Use an instrument equipped with a sample
Medium: Water; 500 mL compartment chiller maintained at 4°.
Apparatus 1: 100 rpm Detector: UV 230 nm
Times: 30 and 60 min Column: 4.6-mm x 25-cm; 5-"um packing L1
Mobile phase and Chromatographic system: Prepare Column temperature: 5°
as directed in the Assay, except use an Injection volume Flow rate: 1 mL/min (=
of 50 uL. Injection volume: 20 uL a)
Standard solution: USP Lorazepam RS at a known con- Run time: At least 50 min uv
centration in Medium. Initially, use a volume of alcohol System suitability 4
not exceeding 10% of the final volume of the Standard Samples: Standard solution and Peak identification i}
solution to dissolve the Reference Standard. solution J
Sample solution: Sample per Dissolution (711). }
Analysis
[Nott—See Table 7 for the approximate relative reten- @=
tion times.] ES)
Samples: Standard solution and Sample solution Suitability requirements so}
Tolerances: NLT 60% (Q) of the labeled amount of Resolution: NLT 1.2 between lorazepam related com- =>
lorazepam (CysHioClzN202) is dissolved in 30 min. NLT pound A and lorazepam related compound E, Peak
“”

80% (Q) of the labeled amount of lorazepam identification solution

(GisHioClzN2Oz) is dissolved in 60 min.
e UNIFORMITY OF DOSAGE UNITS (905)
Procedure for content uniformity
Diluent, Mobile phase, Standard solution, and Chro-
matographic system: Proceed as directed in the
Assay.
Sample solution: Nominally, 0.1 mg/mL of lorazepam
prepared as follows. Place 1 Tablet in a volumetric flask
of appropriate size, based on the labeled quantity, in
mg, of lorazepam in the Tablet. Add a volume of Dilu-
ent equal to about 50% of the volume of the flask, tu
sonicate for 10 min, and shake by mechanical means
for 20 min. Dilute with Diluent to volume, mix, and
centrifuge a portion of the solution for 10 min at 2000
rpm. Cs
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
lorazepam (CisHioClzN202) in the portion of the Tablet
taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response from the Sample solution
Tailing factor: NMT 2.0, Standard solution
Relative standard deviation: NMT 5%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the por-
tion of Tablets taken:
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
= peak response for each impurity from the
Sample solution
rs = peak response for lorazepam from the
Standard solution
= concentration of lorazepam in the Standard
solution (mg/mL)
Cu = nominal concentration of lorazepam in the
Sample solution (mg/mL)
E = relative response factor for any given impurity
(see Table 1)
Acceptance criteria: See Table 1.
 2476 Lorazepam / Official Monographs USP 41

Table 1
Relative Relative Acceptance Losartan Potassium
Retention Response Criteria,
Name Time Factor NMT (%)
Lorazepam 1.0 1.0 =
Lorazepam relat-
ed compound
De 1.4 1.0 0.5
Lorazepam relat-
ed compound _ —
Abe 17.
Lorazepam relat- C22H22CIKNeO 461.00
ed compound 1H-Imidazole-5-methanol, 2-butyl-4-chloro-1-[[2’-(1 H-tetra-
Ed 19 i} 0.5
zol-5-yl)[1,1’-biphenyl]-4-yl]methyl]-, monopotassium salt;
Lorazepam relat- 2Butyl chives! ee H-tetrazol-
ed compound 5-ylphenyl)benzyljimidazole-5-methanol, monopotassium
Ce 21 1.0 3.0 salt [124750-99-8].
Lorazepam relat-
ed compound DEFINITION
Br 535 1.0 0.1 Losartan Potassium contains NLT 98.5% and NMT 101.0%
Any individual of losartan potassium (C22H22CIKNeO), calculated on the
unspecified deg- anhydrous, solvent-free basis.
radation prod- IDENTIFICATION
uct 1.0 0.2 © A. INFRARED ABSORPTION (197M) or (197K): Meets the
Total impurities = = 4.0 requirements
@ 6-Chloro-4-(o-chlorophenyl)-2-quinazolinecarboxylic acid. e B. ULTRAVIOLET ABSORPTION (197U)
» 7-Chloro-5-(o-chlorophenyl)-1,3-dihydro-3-acetoxy-2H-1,4- Sample solution: 10 g/mL in methanol
benzodiazepin-2-one. Acceptance criteria: Meets the requirements
Lorazepam related compoundAis included only for peak identification e C. IDENTIFICATION TESTS—GENERAL, Potassium (191):
urposes. It is not quantified and should not be included in the total
impurities calculation. Meets the requirements
4 6-Chloro-4-(o-chlorophenyl)-2-quinazoline methanol.
© 6-Chloro-4-(o-chlorophenyl)-2-quinazolinecarboxaldehyde.
ASSAY
f 2-Amino-2’,5-dichlorobenzophenone.
¢ PROCEDURE
Solution A: 0.1% solution of phosphoric acid in water
ADDITIONAL REQUIREMENTS Solution B: Acetonitrile
al © PACKAGING AND STORAGE: Preserve in tight, light-resistant Mobile phase: Solution B and Solution A (2:3)
aa Standard solution: 0.25 mg/mL of USP Losartan Potas-
% containers.
e USP REFERENCE STANDARDS (11) sium RS in methanol
i]
-
Dd USP Lorazepam RS Sample solution: 0.25 mg/mL of Losartan Potassium in
} USP Lorazepam Related Compound A RS methanol
iJ 7-Chloro-5-(o-chlorophenyl)-1,3-dihydro-3-acetoxy-2H- Chromatographic system
S 1,4-benzodiazepin-2-one. (See Chromatography (621), System Suitability.)
= Cy7Hi2ClzN2O3 _ 363.20 Mode: LC
rs USP Lorazepam Related Compound B RS Detector: UV 254 nm
2)
=) 2-Amino-2’,5-dichlorobenzophenone. Column: 4.0-mm x 25-cm; packing L1
Ci3HsClANO =.266.13 Column temperature: 35°
USP Lorazepam Related Compound C RS Flow rate: 1 mL/min
6-Chloro-4-(o-chloropheny!)- Injection volume: 10 pL
2-quinazolinecarboxaldehyde. System suitability
CisHgClzN2O 303.15 Sample: Standard solution
USP Lorazepam Related Compound D RS Suitability requirements
6-Chloro-4-(o-chloropheny!)-2-quinazolinecarboxylic Column efficiency: NLT 5600 theoretical plates
acid. Tailing factor: NMT 1.4
CisHsClaN202 _ 319.15 Relative standard deviation: NMT 0.5%
USP Lorazepam Related Compound E RS Analysis
6-Chloro-4-(o-chlorophenyl)-2-quinazoline methanol. Samples: Standard solution and Sample solution
CisHioClzN20 305.16 Calculate the percentage of losartan potassium
(C22H22CIKNeO) in the portion of Losartan Potassium
taken:
Result = (ry/rs) x (Cs/Cu) x 100
i = peak area from the Sample solution
Is = peak area from the Standard solution
Cs = concentration of USP Losartan Potassium RS in
the Standard solution (mg/mL)
Cu = concentration of the Sample solution (mg/mL)
Acceptance criteria: 98.5%-101.0% on the anhydrous,
solvent-free basis
USP 41 Official Monographs / Losartan 2477

IMPURITIES e USP REFERENCE STANDARDS (11)

USP Losartan Potassium RS
Delete the following:

°o Heavy METALS, Method Ii (231): NMT 10 ppMe cofteiai

1.
Jan-2018)
© ORGANIC IMPURITIES Losartan Potassium Tablets
Solution A: 0.1% solution of phosphoric acid in water
Solution B: Acetonitrile DEFINITION
System suitability solution: 0.3 mg/mL of USP Losartan Potassium Tablets contain NLT 95.0% and NMT
osartan Potassium RS and 2 g/mL of triphenylmetha- 105.0% of the labeled amount of losartan potassium
nol in methanol (Ca2H22ClIKNeO).
Sample solution: 0.3 mg/mL of Losartan Potassium in IDENTIFICATION
methanol e A. The retention time of the major peak of the Sample
Mobile phase: See Table 7. solution corresponds to that of the Standard solution, as
obtained in the Assay.
Table 1
ASSAY
Solution A Solution B e PROCEDURE
Buffer: 1.25 mg/mL of monobasic potassium phosphate
7 and 1.5 mg/mL of dibasic sodium phosphate in water.
The resulting pH is approximately 7.0. Pass the solution
through a PTFE or equivalent filter of 0.45-14m pore
size, and degas before use.
Solution A: Acetonitrile and Buffer (15:85)
Solution B: Use acetonitrile.
Chromatographic system Mobile phase: See Table 1.
(See Chromatography (621), System Suitability.)
Mode: LC Table 1
Detector: UV 220 nm
Solution A Solution B
Column: 4.0-mm x 25-cm; packing L1
Flow rate: 1 mL/min
Injection volume: 10 LL
System suitability
Sample: System suitability solution
[Notte—The relative retention times for losartan and
triphenylmethanol are 1.0 and 1.9, respectively. The (<4
al
pal retention time for triphenylmethanol is 20 System suitability stock solution: Dissolve 12 mg of a]
min. USP Losartan Potassium RS in a 50-mL volumetric flask,
Suitability requirements first using 5 mL of water, followed by 5 mL of 0.1 N 4
Tailing factor: NMT 1.6 hydrochloric acid. Place the flask in a 105° oven for 1-2 °
=]
Analysis h, and allow to cool to room temperature. Pipet 5 mL 5°
Sample: Sample solution of 0.1 N sodium hydroxide into the flask, and dilute co}
a
Calculate the percentage of each impurity in the por- with water to volume. Adjust with either 0.1 N hydro- a
tion of losartan potassium (C22H22CIKN6O) taken: chloric acid or 0.1 N sodium hydroxide to a pH of 6.0. mo]
>
[NoTe—The resulting solution contains the 1H-dimer 7
Result = (ru/rz) x 100 and 2H-dimer, and the resulting solution may be
cloudy.]
ru = peak response for each impurity System suitability solution: Add 3 mL of acetonitrile to
nr = sum of the responses for all peaks 7 mL of System suitability stock solution to clear the
Acceptance criteria cloudy solution, and mix well.
Individual impurities: NMT 0.2% Standard solution: 0.25 mg/mL of USP Losartan Potas-
Total impurities: NMT 0.5% sium RS in Solution A. Pass through a PTFE or equivalent
SPECIFIC TESTS filter of 0.45-11m pore size.
Sample stock solution: Transfer 10 Tablets to a 500-mL
e@ WATER DETERMINATION, Method | (921): NMT 0.5%. If
volumetric flask, add SolutionA to fill the flask to about
labeled as an amorphous form, NMT 5.0%.
50% of the final volume, and sonicate with intermittent
ADDITIONAL REQUIREMENTS shaking for 15 min. Sonicate for an additional 10 min.
e LABELING: Where it is an amorphous form, the label so Dilute with Solution A to volume, and mix well.
indicates. Sample solution: 0.25 mg/mL of losartan potassium in
© PACKAGING AND STORAGE: Preserve in well-closed contain- Solution A from the Sample stock solution. Mix well. Pass
ers, Store at controlled room temperature. an aliquot of the solution through a PTFE filter of 0.45-
im pore size, and use the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 250 nm
Column: 3.9-mm x 15-cm; 5-1um packing L7
Flow rate: 1.0 mL/min
Injection volume: 10 uL
System suitability
Samples: System suitability solution and Standard
solution
 2478 Losartan / Official Monographs USP 41

Suitability requirements Mode: LC

Tailing factor: NMT 2.0 for the losartan, 1H-dimer, Detector: UV 254 nm
and 2H-dimer peaks; System suitability solution Column: 4.0-mm x 25-cm; 5-um packing L1
Resolution: NLT 2.0 between the 1H-dimer and 2H- Column temperature: 35°
dimer, System suitability solution Flow rate: 1 mL/min
Column efficiency: NLT 3000 theoretical plates, Injection volume: 20 wL
Standard solution Run time: NLT 1.5 times the retention time of
Tailing factor: NMT 2.0, Standard solution losartan
Relative standard deviation: NMT 2.0%, Standard System suitability
solution Sample: Standard solution
Analysis Suitability requirements
Samples: Standard solution and Sample solution Tailing factor: NMT 2.0
Calculate the percentage of the labeled amount of Relative standard deviation: NMT 2.0%
losartan potassium (C22H22CIKNeO) in the portion of Calculate the percentage of the labeled amount of
Tablets taken: losartan potassium (C22H22CIKNeO) dissolved:
Result = (ru/rs) x (Cs/Cu) x 100 Result = (ru/rs) x Cs x Vx (1/L) x 100

ty = peak response of losartan from the Sample ty = peak response from the Sample solution
solution rs = peak response from the Standard solution
rs = peak response of losartan from the Standard Cs = concentration of USP Losartan Potassium RS in
solution the Standard solution (mg/mL)
Cs = concentration of USP Losartan Potassium RS in V = volume of Medium, 900 mL
the Standard solution (mg/mL) L = label claim (mg/Tablet)
Cy = nominal concentration of losartan potassium Tolerances: NLT 75% (Q) of the labeled amount of
in the Sample solution (mg/mL) losartan potassium (C22H22CIKNeO) is dissolved.
Acceptance criteria: 95.0%-105.0% Test 2: If the product complies with this test, the label-
ing indicates that the product meets USP Dissolution
PERFORMANCE TESTS Test 2.
e DISSOLUTION (711) Medium: Water; 900 mL
Test 1 Apparatus 2: 75 rpm
Medium: Water; 900 mL, deaerated Time: 30 min
Apparatus 2: 50 rpm Buffer: 1.4 g/L of anhydrous monobasic potassium
Time: 30 min phosphate in water. Adjust with phosphoric acid to a
Standard solution: (1/1000) mg/mL of USP Losartan pH of 3.3 + 0.1.
Potassium RS in Medium, where L is the Tablet label Mobile phase: Methanol, acetonitrile, and Buffer
claim, in mg (20:20:60)
” Sample solution: Pass a portion of the solution under Standard solution: 0.028 mg/mL of USP Losartan Po-
= test through a suitable filter of 0.45-11m pore size. tassium RS in Medium
% Analysis: Determine the amount of losartan potassium Sample solution
i]
_ (C22H22CIKNeO) dissolved by using one of the follow- For Tablets labeled to contain 25 mg: Pass a por-
aD ing procedures:
) tion of the solution under test through a suitable fil-
i Instrumental conditions ter of 0.45-um fae size.
S Analytical wavelength: Maximum absorbance at For Tablets labeled to contain 50 and 100 mg: Pass
= about 256 nm a portion of the solution under test through a suita-
[3 Path length: See Table 2 or make the appropriate ble filter of 0.45-4um pore size. Further dilute the fil-
2) dilution of the solutions with Medium to be within trate with Medium to prepare a 0.028-mg/mL
=) the linearity range of the spectrophotometer. solution.
Chromatographic system
Table 2 (See Chromatography (621), System Suitability.)
Mode: LC
Tablet Strength Cell Size Detector: UV 265 nm
Column: 4.6-mm x 15-cm; 5-um packing L10
1 Column temperature: 45°
Flow rate: 1.5 mL/min
100 Injection volume: 10 uL
System suitability
Blank: Medium Sample: Standard solution
Calculate the percentage of the labeled amount of Suitability requirements
losartan potassium (C22H22CIKN.O) dissolved: Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0%
Result = (Au/As) x (Cs/L) x Vx 100 Analysis
Samples: Standard solution and Sample solution
Au = absorbance of the Sample solution Calculate the percentage of the labeled amount of
As = absorbance of the Standard solution losartan potassium (C22H22CIKNeO) dissolved:
Cs = concentration of USP Losartan Potassium RS in
the Standard solution (mg/mL) Result = (ru/rs) x (Cs/L) x Vx 100
L = label claim (mg/Tablet)
Vv = volume of Medium, 900 mL ru = peak response from the Sample solution
Chromatographic procedure rs = peak response from the Standard solution
Solution A: 0.1% v/v phosphoric acid in water Gs = concentration of USP Losartan Potassium RS in
Mobile phase: Acetonitrile and Solution A (40:60) the Standard solution (mg/mL)
Chromatographic system L = label claim (mg/Tablet)
(See Chromatography (621), System Suitability.) Vv = volume of Medium, 900 mL
USP 41
Tolerances: NLT 85% (Q) of the labeled amount of
losartan potassium (C22H22CIKNeO) is dissolved.
Test 3: If the product complies with this test, the label-
ing indicates that the product meets USP Dissolution
Test 3.
Medium: Water; 900 mL, deaerated
Apparatus 2: 50 rom
Time: 30 min for 25-mg and 50-mg Tablet strengths,
and 45 min for 100-mg Tablet strength
Buffer: 0.025 M phosphoric acid. Adjust with 1 N so-
dium hydroxide to a pH of 2.15.
Mobile phase: Acetonitrile and Buffer (400:600)
Standard stock solution: 0.27 mg/mL of USP Losartan
Potassium RS prepared as follows. Add methanol to
USP Losartan Potassium RS to fill about 10% of the
volume of the flask, and add Medium to fill about 50%
of the volume of the flask. Sonicate for NLT 15 min.
Cool to room temperature, and dilute with Medium to
volume.
Standard solution: Prepare as directed in Table 3 from
the Standard stock solution.
Table 3
Tablet Strength Concentration
7 Calculate the percentage of the labeled amount of
1 1
Sample solution: Pass a portion of the solution under
test through a suitable polyethylene filter of 10-m
pore size. tu
Chromatographic system
(See Chromatography (621), System Suitability.) rs
Mode: LC
Detector: UV 220 nm Gs
Column: 4.6-mm x 10-cm; 3.5-14m packing L7
Column temperature: 40°
Flow rate: 1.5 mL/min
Injection volume: 10 uL
System suitability
IMPURITIES
Sample: Standard solution © ORGANIC IMPURITIES
Suleepiity requirements
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
losartan potassium (C22H22CIKNeO) dissolved:
Result = (ru/rs) x (G/L) x V x 100
ty = peak response of losartan from the Sample
solution
rs = peak response of losartan from the Standard
solution
Gs = concentration of USP Losartan Potassium RS in
the Standard solution (mg/mL)
L = label claim (mg/Tablet)
Vv = volume of Medium, 900 mL
Tolerances: NLT 75% (Q) of the labeled amount of
losartan potassium (C22H22CIKNeO) is dissolved for
25-mg and 50-mg Tablet strengths. NLT 80% (Q) of
the labeled amount of losartan potassium
(C22H22CIKNeO) is dissolved for 100-mg Tablet
strength.
e UNIFORMITY OF DOSAGE UNITS (905)
Procedure for content uniformity
Buffer: Dissolve 1.36 mg/mL of monobasic potassium
phosphate in water. Adjust with phosphoric acid to a
PH of 2.5.
Diluent: Dissolve 17.42 g of dibasic potassium phos-
phate in 900 mL of water. Adjust with phosphoric acid
to a pH of 8.0. Dilute with water to a volume of
Official Monographs / Losartan 2479
1000 mL, and mix well. Further dilute with water (1 in
10), and mix well.
Mobile phase: Acetonitrile and Buffer (60:40)
Standard solution: 0.05 mg/mL of USP Losartan Po-
tassium RS in Diluent
Sample stock solution: Transfer 1 Tablet to a 100-mL
volumetric flask, add about 65 mL of Diluent, and
shake mechanically for 30 min. Dilute with Diluent to
volume, and mix well.
Sample solution: 0.05 mg/mL of losartan potassium in
Diluent from the Sample stock solution. Filter an aliquot
of the solution, and use the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 230 nm
Column: 4.6-mm x 25-cm; 10-um packing L7
Flow rate: 1.4 mL/min
Injection volume: 20 uL
System suitability
Sample: Standard solution
Suitability requirements
Column efficiency: NLT 3000 theoretical plates
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
losartan potassium (C22H22CIKNcO) in the portion of
the Tablet taken:
Result = (ru/rs) x (Cs/Cu) x 100
= peak response of losartan from the Sample
solution
= peak response of losartan from the Standard
solution
= concentration of USP Losartan Potassium RS in
the Standard solution (mg/mL) e
Cu = nominal concentration of losartan potassium “
in the Sample solution (mg/mL) ao)
Acceptance criteria: Meet the requirements E
°
3
}
Solution A, Solution B, Mobile phase, System suitabil-
ro}~
ity solution, Sample solution, and Chromatographic Ly
system: Prepare as directed in the Assay.
a}
s
Standard stock solution: Use the Standard solution, a)
prepared as directed in the Assay.
Standard solution: 2.5 g/mL of USP Losartan Potas-
sium RS in Solution A from the Standard stock solution
Sensitivity solution: Dilute 1 mL of the Standard solu-
tion to 10 mL in Solution A.
System suitability
Samples: System suitability solution, Standard solution,
and Sensitivity solution
Suitability requirements
Tailing factor: NMT 2.0 for the losartan, 1H-dimer,
and 2H-dimer peaks; System suitability solution
Resolution: NLT 2.0 between the 1H-dimer and 2H-
dimer, System suitability solution
Column efficiency: NLT 3000 theoretical plates,
Standard solution
Tailing factor: NMT 2.0, Standard solution
Relative standard deviation: NMT 5.0%, Standard
solution
Signal-to-noise ratio: NLT 10 for the losartan peak
from the first injection. If this is not met, then the
Signal-to-noise ratio must be greater than 3 with a
relative standard deviation of area counts less than
25% for three replicate injections, Sensitivity solution.
Analysis
Samples: Standard solution and Sample solution
[Notr—Identify the peaks using the relative retention
times provided in Table 4.]
 2480 Losartan / Official Monographs USP 41

Calculate the percentage of each impurity in the por- Table 1

tion of Tablets taken: Solution A Solution B
%'
Result = (ru/rs) x (Cs/Cu) x 100
100
ru = peak response of each individual impurity
from the Sample solution
rs = peak response of losartan from the Standard
solution 1
Cs = concentration of USP Losartan Potassium RS in
the Standard solution (nairal) Standard solution: Transfer USP Losartan Potassium RS
Cy = nominal concentration of losartan potassium and USP Hydrochlorothiazide RS into a suitable volu-
in the Sample solution (mg/mL) metric flask, and dissolve in Diluent (50% of the volume
Acceptance criteria: See Table 4. of the flask). Dilute with Buffer A to volume to obtain a
solution having concentrations as directed in Table 2.
Table 4 Pass a portion of the solution through a PTFE or equiva-
Relative Acceptance
lent filter of 0.45-um pore size.
Retention Criteria,
Name Time NMT (%) Table 2
Losartan 1.0 = Tablet Strength
1H-Dimer@ 2.4 05 Losartan Potas- Concentration of Concentration of
2H-Dimer® 29 0.5 sium/Hydrochlo- USP Losartan USP Hydrochlo-
Total impurities: = 1.0 rothiazide Potassium RS rothiazide RS
2 5-[4’-({2-Butyl-5-[(5-{4’-[(2-butyl-4-chloro-S-hydroxymethyl-1 H-imidazol-
(mg) (mg/mL) (mg/mL)
1-yl)methyl]bipheny|-2-yl}-1 H-tetrazol-1-yl)methyl]-4-chloro-1 H-imidazol-1- 50/12.5 0.4 0.1
yl}methyl)biphenyl-2-iItetrazol, potassium salt. 100/12.5 04 0.05
5 3 Fe Butyl (G14 1G Baty (4 -chlorb-5-hydronymethyi) H-imidazol-
1-yl)methyl]bipheny|-2-yl}-2H-tetrazol-2-yl)methyl]-4-chloro-1 H-imidazol-1- 100/25 0.4 0.1
yiimethylpbiphenyi-2-yiftetrazol, potassium salt.
¢ The total impurities include the sum of all the specified impurities and Sample stock solution: Transfer 10 Tablets into a suita-
the sum of all the unspecified impurities. Disregard peaks less than 0.1%. ble volumetric flask and add Diluent as directed in Table
3. Mix well and mechanicall shake or stir until the
ADDITIONAL REQUIREMENTS solid is dispersed. Dilute wit! Buffer A to volume, and
e PACKAGING AND STORAGE: Store in tightly closed contain- sonicate.
ers, protected from light, at controlled room
temperature.
e LABELING: When more than one Dissolution test is given, Table 3
Py the labeling states the test used only if Test 7 is not used. Tablet Strength
a
a e USP REFERENCE STANDARDS (11) Losartan Potassium/ Volume of
it
—
USP Losartan Potassium RS Hydrochlorothiazide Flask Size Diluent
i) (mg) (mL) (mL)
° 50/12.5 250 210
=
S 100/12.5 500 420
= Losartan Potassium and
100/25 500 420
rs
”“ Hydrochlorothiazide Tablets Sample solution: Dilute a ortion of the Sample stock
=) solution first with acetonitrile (20% of the volume of the
DEFINITION flask) and then with Buffer A, to obtain a solution hav-
Losartan Potassium and Hydrochlorothiazide Tablets contain ing nominal concentrations as directed in Table 4. Pass
NLT 95.0% and NMT 105.0% of the labeled amounts of a portion of this solution through a PTFE or equivalent
losartan potassium (C22H22CIKNeO) and hydrochlorothia- filter of 0.45-11m pore size, and use the filtrate.
zide (C7HsCIN3O.Sz2).
Table 4
IDENTIFICATION
e A. The retention times of the major peaks of the Sample Tablet Strength
solution correspond to those of the Standard solution, as Losartan Potas- Concentration of Concentration of
obtained in the Assay. sium/Hydrochlo- USP Losartan USP Hydrochlo-
rothiazide Potassium RS rothiazide RS
ASSAY (mg) (mg/mL) (mg/mL)
e PROCEDURE 50/12.5 0.4 0.1
Buffer A: 2.76 g/L of monobasic sodium phosphate in 100/12.5 0.4 0.05
water. Adjust with phosphoric acid to a pH of 2.5. 100/25 0.4 0.1
Buffer B: 1.25 g/L of monobasic potassium phosphate
and 1.5 g/L of dibasic sodium phosphate in water. The Chromatographic system
pH of the resulting solution is about 7.0-7.5. (See Chromatography (621), System Suitability.)
Diluent: Acetonitrile and Buffer A (3:2) Mode: LC
Solution A: Acetonitrile and Buffer B (7:93) Detector: UV 280 nm
Solution B: Use acetonitrile. Column: 3.9-mm x 15-cm; 5-um packing L7
Mobile phase: See Table 7. Column temperature: 35°
Flow rate: 1 mL/min
Injection volume: 20 uL
USP 41 Official Monographs / Losartan 2481

System suitability Mode: LC

Sample: Standard solution Detector: UV 230 nm
Suitability requirements Column: 4.6-mm x 25-cm; 10-um packing L7
Tailing factor: Less than 2.5 for the losartan peak Column temperature: 35°
Relative standard deviation: Less than 2.0% for Flow rate: 2.3 mL/min
both hydrochlorothiazide and losartan peaks Injection volume: 20 uL
Analysis System suitability
Samples: Standard solution and Sample solution Sample: Standard solution
Calculate the percentage of the labeled amount of Suitability requirements
losartan potassium (C22H22CIKN6O) or hydrochlorothia- Resolution: NLT 2 between the hydrochlorothiazide
zide (C7HsCIN304S2) in the portion of Tablets taken: and losartan peaks
Relative standard deviation: NMT 2.0% for both
Result = (ru/rs) x (Cs/Cu) x 100 the hydrochlorothiazide and losartan peaks
Analysis
tu = peak response of losartan or Samples: Standard solution and Sample solution
hydrochlorothiazide from the Sample solution Calculate the percentage of the labeled amount of
ts = peak response of losartan or losartan potassium (C22H22CIKN6O) or hydrochlorothi-
hydrochlorothiazide from the Standard azide (C7HgCIN30,S2) dissolved:
solution
Cs = concentration of USP Losartan Potassium RS or Result = (ru/rs) x (Cs/L) x Vx 100
USP Hydrochlorothiazide RS in the Standard
solution (mg/mL) ty = peak response of losartan or
Cy = nominal concentration of losartan potassium hydrochlorothiazide from the Sample solution
or hydrochlorothiazide in the Sample solution rs = peak response of losartan or
(mg/ml) hydrochlorothiazide from the Standard
Acceptance criteria: 95.0%-105.0% solution
Cs = concentration of USP Losartan Potassium RS or
PERFORMANCE TESTS USP Hydrochlorothiazide RS in the Standard
e DISSOLUTION (711) solution (mg/mL)
Test 1 L = label claim (mg/Tablet)
Medium: Water; 900 mL, deaerated Vv = volume of Medium, 900 mL
Apparatus 1: 100 rpm Tolerances: NLT 85% (Q) of the labeled amount of
Time: 30 min for both losartan and losartan en (C22H22CIKNeO) and NLT 75% (Q)
hydrochlorothiazide of the labeled amount of hydrochlorothiazide
Buffer: Dissolve 1.36 g/L of monobasic potassium (C7HsCIN304S2) is dissolved.
picspiiate in water. Adjust with phosphoric acid to a Test 2: If the product complies with this test, the label-
H of 2.5. ing indicates that the product meets USP Dissolution
Mobile phase: Acetonitrile and Buffer (2:3) Test 2. ss
Losartan potassium stock solution: 0.44 mg/mL of Medium, Apparatus 1, and Time: Proceed as directed Al
USP Losartan Potassium RS in Medium in Test 1. a)
Hydrochlorothiazide stock solution: 0.14 mg/mL of Buffer: Dissolve 1.78 g of dibasic sodium phosphate K
USP Hydrochlorothiazide RS prepared by dissolving in dihydrate in 1 L of water. Adjust with phosphoric acid i}
methanol (10% of the volume of the flask). Dilute with to a pH of 6.5. 3
Medium to volume. )
Standard solution: Transfer the appropriate volumes
Mobile phase: Acetonitrile and Buffer (32:68) to}=
Diluent: Acetonitrile and water (40:60) 2
of Losartan potassium stock solution and Hydrochlorothi- Standard stock solution 1: 1.1 mg/mL of USP 3
azide stock solution to a 100-mL volumetric flask ac- Losartan Potassium RS in Diluent. Sonication may be =
cording to the dilution schemes in Table 5. Dilute with necessary for complete dissolution.
“

Medium to volume. Standard stock solution 2: 0.28 mg/mL of USP Hy-

drochlorothiazide RS in Diluent. Sonication may be
Table 5 peeeenaly tae a be asaititionk z
7 tandard solution: Transfer appropriate volumes o
eeSurenat ae oars Standard stock solution 1 and Standard stock solution 2
sium/Hydrochlo- patariatesesek auacideStock to a 100-mL volumetricflask according to the dilution
pothiazidé Soliittor Solution schemes in Table 6. Dilute with Medium to volume.
(mg) (mL) (mL)
50/12.5 12.5 10.0 Table 6
100/12.5 25.0 10.0 Tablet Strength
100/25 25.0 20.0 Losartan Potas- Aliquot of Aliquot of
sium/Hydrochlo- Standard Standard
Sample solution: Pass a portion of the solution under rothiazide Stock Solution 1 Stock Solution 2
test through a suitable filter of 0.45-um pore size. (mg) (mL) (mL)
Chromatographic system 50/12.5 5 5
(See Chromatography (621), System Suitability.) 100/12.5 10 5
100/25 10 10
Sample solution: Pass a portion of the solution under
test through a suitable filter of 0.45-um pore size.
 2482 Losartan / Official Monographs USP 41

Chromatographic system Sample stock solution: Transfer 1 Tablet into a suita-

(See Chromatography (621), System Suitability.) ble volumetric flask, and add Diluent as directed in Ta-
Mode: LC ble 8. Mix well, and mechanically shake for 30 min or
Detector: UV 225 nm until the solid is finely dispersed. Dilute with Buffer A
Column: 4.6-mm x 25-cm; 5-uum packing L1 to volume, and mix well.
Autosampler temperature: 8°
Flow rate: 1.2 mL/min Table 8
Injection volume: 10 uL
System suitability Tablet Strength
Sample: Standard solution Losartan Potassium/ Volume of
Suitability requirements Hydrochlorothiazide Flask Size Diluent
Relative standard deviation: NMT 2.0% for both (mg) (mL) (mL)
the hydrochlorothiazide and losartan peaks 50/12.5 100 50
Analysis 100/12.5 200 100
Samples: Standard solution and Sample solution 100/25 200 100
Calculate the percentage of the labeled amount of
losartan potassium (C22H22CIKNeO) and hydrochloro- Sample solution: Dilute 10 mL of the Sample stock so-
thiazide (C7HsCIN304S2) dissolved: lution in a 100-mL volumetric flask, with 45 mL of Dilu-
ent, and then dilute with Buffer A to volume. Pass an
Result = (ru/rs) x Cs x (1/L) x Vx 100 aliquot of the solution through a PTFE or equivalent
filter of 0.45-4m pore size, and use the filtrate.
ru = peak response of losartan or Chromatographic system
hydrochlorothiazide from the Sample solution (See Chromatography (621), System Suitability.)
Is = peak response of losartan or Mode: LC
hydrochlorothiazide from the Standard Detector: UV 230 nm
solution Column: 4.6-mm x 25-cm; 10-m packing L7
Cs = concentration of USP Losartan Potassium RS or Column temperature: 35°
USP Hydrochlorothiazide RS in the Standard Flow rate: 2.3 mL/min
solution (mg/mL) Injection volume: 20 UL
L = label claim (mg/Tablet) System suitability
Vv = volume of Medium, 900 mL Sample: Standard solution
Tolerances: NLT 85% (Q) of the labeled amount of Suitability requirements
losartan potassium (C22H22CIKNeO) and NLT 80% (Q) Resolution: NLT 2 between the hydrochlorothiazide
of the labeled amount of hydrochlorothiazide and losartan peaks
(C7HaCIN3O4Sz2) is dissolved. Relative standard deviation: NMT 2.0% for both
e UNIFORMITY OF Dosace UNITS (905) the hydrochlorothiazide and losartan peaks
Procedure for content uniformity Analysis
Py Buffer A: Prepare as directed in the Assay. Samples: Standard solution and Sample solution
ca Buffer B: Dissolve 1.36 g/L of monobasic potassium
is phosphate in water. Adjust with phosphoric acid to a
Calculate the percentage of the labeled amount of
i] losartan potassium (C22H22CIKNeO) or hydrochlorothi-
i) pH of 2.5.
—
azide (C7;HsCIN304Sz2) in the Tablet taken:
i} Diluent: Prepare a mixture of acetonitrile and Buffer
A
r= (3:2) Result = (ru/rs) x (Cs/Cu) x 100
So Mobile phase: Acetonitrile and Buffer B (2:3)
= Standard stock solution 1: 0.46 mg/mL of USP Tu = peak tesporise of losartan or
a. Losartan Potassium RS prepared by dissolving in Dilu- hydrochlorothiazide from the Sample solution
a)
=) ent (50% of the total volume of the flask). Mechani- Is = peak response of losartan or
cally shake for 15 min or until dissolved. Dilute with hydrochlorothiazide from the Standard
Buffer A to volume. solution
Standard stock solution 2: 0.35 mg/mL of USP Hy- Cs = concentration of USP Losartan Potassium RS or
drochlorothiazide RS prepared by dissolving in Diluent USP Hydrochlorothiazide RS in the Standard
(50% of the total volume of the flask). Mechanically solution (mg/mL)
shake for 15 min or until dissolved. Dilute with Buffer A Cu = nominal concentration of losartan potassium
to volume. or hydrochlorothiazide in the Sample solution
Standard solution: Transfer aliquots of Standard stock (mg/ml)
solution 1 and Standard stock solution 2 into a suitable Acceptance criteria: Meet the requirements
volumetric flask, and add Diluent, up to 42% of the
total volume of the flask. Dilute with Buffer A to vol- IMPURITIES
ume, mix well, and sonicate for 2 min to obtain a e ORGANIC IMPURITIES
solution having concentrations based on Tablet Buffer A, Buffer B, Diluent, Solution A, Solution B,
strength as listed in Table 7. Pass a portion of the solu- Mobile phase, Standard solution, Sample solution,
tion through a PTFE or equivalent filter of 0.45-um and Chromatographic system: Proceed as directed in
pore size, and use the filtrate. the Assay.
Chlorothiazide standard solution: 0.1 mg/mL of USP
Table 7
Chlorothiazide RS prepared by dissolving in Diluent
(50% of the volume of the flask). Dilute with Buffer A to
Tablet Strength volume, and sonicate.
Losartan Potas- Concentration of Concentration of Benzothiadiazine related compoundAstandard solu-
sium/Hydrochlo- USP Losartan USP Hydrochlo- tion: 0.1 mg/mL of USP Benzothiadiazine Related
rothiazide Potassium RS rothiazide RS Compound A RS prepared by dissolving in Diluent (50%
(mg) (mg/mL) (mg/mL) of the volume of the flask). Dilute with Buffer A to vol-
50/12.5 0.06 0.014 ume, and sonicate.
100/12.5 0.06 0.007 Stressed losartan solution: [Note—This solution con-
100/25 0.06 0.014
tains the degradates 1-H-dimer and 2-H-dimer and
losartan potassium.] Weigh 12 mg of the USP Losartan
USP 41 Official Monographs / Losartan 2483

Potassium RS in a 50-mL flask. Dissolve in 5 mL of Analysis

water. Pipet 5.0 mL of 0.1 N hydrochloric acid into this Samples: Sample solution and Diluted standard solution
solution, and place it in an oven at 105° for 1-2 h. [Note—The run time is about 1.6 times the retention
Remove from the oven and allow to cool to room tem- time of the losartan peak. Identify the peaks using the
perature, Pipet 5.0 mL of 0.1 N sodium hydroxide into relative retention times provided in Table 10.]
the flask, and dilute with water to volume. Calculate the percentage of benzothiadiazine related
Diluted standard solution: Dilute portions of the Stan- compoundA (expressed as hydrochlorothiazide equiv-
dard solution and Benzothiadiazine related compound A alent) in the portion of Tablets taken:
standard solution first with acetonitrile (30% of the vol-
ume of the flask), then with Buffer A to obtain a solu- Result = (ru/rs) x (Cs/Cu) x (Mr1/Miz) x 100
tion having nominal concentrations based on Tablet
strength as listed in Table 9. ry = peak response of benzothiadiazine related
compound A from the Sample solution
rs = peak response of benzothiadiazine related
Table 9
compoundA from the Diluted standard
Concentra- solution
Tablet Concentra- tion of USP Cs = concentration of USP Benzothiadiazine Related
Strength tion of Concentra- Benzothia- CompoundA RS in the Diluted standard
Losartan usP tion of USP diazine solution (mg/mL)
Potassium/ Losartan Hydrochlo- Related Cu = nominal concentration of hydrochlorothiazide
Hydrochlo- Potassium rothiazide Compound in the Sample solution (mg/mL)
rothiazide RS RS ARS Mi = molecular weight of hydrochlorothiazide, 298
(mg) (g/mL) (ug/ml) (ug/mL) M2 = molecular weight of benzothiadiazine related
50/12.5 4 1 1 compound A, 286
100/12.5 4 0.5 1 Calculate the percentage of each specified impurity in
100/25 4 1 1
the portion of Tablets taken:

System suitability solution: Dissolve weighed quanti-
ties of USP Losartan Potassium RS and USP Hydrochlo- ty
rothiazide RS in a suitable volumetric flask in Diluent
(50% of the volume of the flask). Add the Stressed rs
losartan solution, about 25% of the volume of the flask,
into the same flask. Transfer appropriate amounts of Cs
Chlorothiazide standard solution and Benzothiadiazine re-
lated compound A standard solution into the same flask, Cu
and dilute with Buffer A to volume to obtain a solution
having a known concentration of about 0.4 mg/mL of
losartan, 0.1 mg/mL of hydrochlorothiazide, and
0.001 mg/mL each of benzothiadiazine related com-
pound A and chlorothiazide. Adjust with phosphoric
acid to a pH of 2.5, and mix well. Pass an aliquot of the
solution through a PTFE or equivalent filter of 0.45-um
pore size, and use the filtrate.
Limit of quantitation solution: Pipet 5.0 mL of the Di- ty
luted standard solution into a 50-mL volumetric flask.
Add 15 mL of acetonitrile, dilute with Buffer A to vol- Is
ume, and mix well.
System suitability Cs
Samples: Standard solution, Diluted standard solution,
System suitability solution, and Limit of quantitation Cu
solution
Suitability requirements For a Tablet strength of 100/12.5 for losartan
Resolution: Greater than 1.5 between chlorothiazide
and benzothiadiazine related compound A; greater
than 1.5 between the benzothiadiazine related com-
pound A and hydrochlorothiazide peak, System suita-
bility solution
Tailing factor: Less than 2.5 for the losartan peak,
Standard solution ty
Relative standard deviation: Less than 2.0% for
both the hydrochlorothiazide and losartan peaks, ls
Standard solution; less than 10.0% for both the hy-
drochlorothiazide and losartan peaks, Diluted standard Cs
solution
Signal-to-noise ratio: NLT 10 for each component Cu
from the first injection. If this is not met, then the
signal-to-noise ratio must be greater than 3 with a Acceptance criteria See Table 10.
relative standard deviation of area counts less than
25% for three replicate injections, Limit of quantita-
tion solution
Result = (ru/rs) x (Cs/Cy) x 100
= peak response of each individual impurity
from the Sample solution
= peak response of losartan from the Diluted
standard solution
= concentration of USP Losartan Potassium RS in
the Diluted standard solution (mg/mL)
= nominal concentration of losartan potassium
in the Sample solution (mg/mL)
For Tablet strengths of 50/12.5 and 100/25 for losartan c
potassium/hydrochlorothiazide, respectively, calculate vy
the percentage of any other impurity in the portion of a)
Tablets taken: 4
Result = (ru/rs) x (Cs/Cy) x 100 5
= peak response of each individual impurity a
from the Sample solution S
= peak response of losartan from the Diluted 1
standard solution a
= concentration of USP Losartan Potassium RS in
the Diluted standard solution (mg/mL)
= nominal concentration of losartan potassium
in the Sample solution (mg/mL)
potassium/hydrochlorothiazide, calculate the
dae of any other impurity in the portion of
ablets taken:
Result = (ru/rs) x (Cs/Cu) x 100
= peak response of each individual impurity
from the Sample solution
= peak response of hydrochlorothiazide from the
Diluted standard solution
= concentration of USP Hydrochlorothiazide RS
in the Diluted standard solution (mg/mL)
= nominal concentration of hydrochlorothiazide
in the Sample solution (mg/mL)
 2484 Losartan / Official Monographs USP 41

Table 10 (15,35S,4aR,75,85,8aS)-8-{2-[(2R,4R)-4-hydroxy-6-oxote-
Relative Acceptance trahydro-2H-pyran-2-yllethyl}-3,7-dimethyl-1,2,3,4,4a,
Retention Criteria,
7,8,8a-octahydronaphthalen-1-yl (S)-
Name Time NMT (%) 2-methylbutanoate.
Co4H3s05 406.56
Chlorothiazides 0.57 =
Benzothiadiazine related
Identification—
compound A 0.69 1.0 A: Infrared Absorption (197M).
Hydrochlorothiazide 1.0 = B: Ultraviolet Absorption (197U)—
Losartan 27 = Solution: 10 ug per mL.
1-H-Dimer> 3.3 0.5 Medium: acetonitrile.
2-H-Dimer< 3.5. 0.5 Specific rotation (7815S): between +324° and +338°.
Total impurities¢ = 2.0 Test solution: 5 mg per mL, in acetonitrile.
This process impurity (not a degradation product) is related to hydro- Loss on drying (731)—Dry it in vacuum at a pressure not
chlorothiazide and is controlled in the drug g substance. exceeding 5 mm of mercury at 60° for 3 hours: it loses not
» Related to Se apo SILA eee more than 0.3% of its weight.
droxymethyl-1 H-imidazol-1-yl)met! iphenyl-2-yl]-1 H-tetrazol-1-
Ylmethyll-sehlores|H-imidazol-1 “ybmethyl]biphenyl-2-yiltetrazol,potassi- Residue on ignition (281): not more than 0.2%.
um salt.
¢ Related to aide yi OMitbpher Loui oieoscce
droxymethyl-1 H-imidazol-1-yl)met! iphenyl-2-yl]-2H-tetrazol-2- Delete the following:
yllmethyi--chioro H-imidazol-1“y)methyl|biphenyl-2-ylltetrazol, potassi-
um salt.
Total impurities include the sum of all the specified impurities and the °Heavy metals, Method I! (231): 0.002%.« (oiniat 1Jan-2018)
unspecified impurities that are equal to or greater than 0.1%. Limit of lovastatin related compound A—
ADDITIONAL REQUIREMENTS Mobile phase—Prepare a filtered and degassed mixture of
e LABELING: When more than one Dissolution test is given, acetonitrile and 0.01 M phosphoric acid (13:7). Make ad-
the labeling states the test used only if Test 7 is not used. justments if necessary (see System Suitability under Chroma-
© PACKAGING AND STORAGE: Preserve in tightly closed con- tography (621)).
tainers protected from light, and store at controlled room System suitability solution—Dissolve accurately weighed
temperature. quantities of USP Lovastatin RS and USP Lovastatin Related
e USP REFERENCE STANDARDS (11) CompoundARS in acetonitrile, and dilute quantitatively,
USP Benzothiadiazine Related Compound A RS and stepwise if necessary, to obtain a solution containing
4-Amino-6-chloro-1,3-benzenedisulfonamide. 2.0 ug of each per mL.
CeHsCIN3O4S2 285.73 Standard solution—Dissolve an accurately weighed quan-
USP Chlorothiazide RS tity of USP Lovastatin RS in acetonitrile, and dilute quantita-
USP Hydrochlorothiazide RS tively, and stepwise if necessary, to obtain a solution having
USP Losartan Potassium RS a known concentration of about 2.0 ug per mL.
=
“

a Test solution—transfer about 25 mg of Lovastatin, accu-

< rately weighed, to a 25-mL volumetric flask, dissolve in and
a dilute with acetonitrile to volume, and mix.
° Chromatographic system (see Chromatography {621))—The
¢
Sj Lovastatin liquid chromatograph is equipped with a 200-nm detector
= and a 4.6-mm x 25-cm column that contains 5-um packing
Pm L7. The column temperature is maintained at 40°. The flow
”“ HC rate is about 1.5 mL per minute. Chromatograph the System
=} suitability solution, and record the peak responses as directed
wy
é
for Procedure: the relative retention times are about 1.0 for
lovastatin and 1.3 for lovastatin related compound A; and
H,C- the resolution, R, between lovastatin and lovastatin related
if compoundAis not less than 6.0. Chromatograph the Stan-
dard solution, and record the peak responses as directed for
Co4H36Os 404.54 Procedure: the relative standard deviation for replicate injec-
Butanoic acid, 2-methyl-, 1,2,3,7,8,8a-hexahydro-3,7- tions is not more than 5.0%.
dimethyl-8-[2-(tetra pate ee Ge ae ran-2-yl)- Procedure—Separately inject equal volumes (about 10 wL)
aoe ester, [1 S-[1a(R*),30,78,8B(25*,45*), of the Standard solution and the Test solution into the chro-
8aB]I-. matograph, record the chromatograms, and measure all the
(S)-2-Methylbutyric acid, 8-ester with (4R,6R)-6-[2-[(15,25, eak responses. Calculate the percentage of lovastatin re-
6R,85,8aR)-1,2,6,7,8,8a-hexahydro-8-hydroxy-2,6- ated compound Ain the portion of Lovastatin taken by the
dimethyl-1-naphthyljethyl]tetrahydro-4-hydroxy-2H-pyran- formula:
2-one — [75330-75-5].
2.5F(C/W)(ru
/ rs)
» Lovastatin contains not less than 98.5 percent
and not more than 101.0 percent of C24H36Os, in which F is the response factor for lovastatin related com-
calculated on the dried basis. poundA and is equal to 1.6; C is the concentration, in jg
per mL, of USP Lovastatin RS in the Standard solution; W is
Packaging and atorage: {ister in tight containers the weight, in mg, of Lovastatin in the Test solution; ry is the
under nitrogen in a cold place. peak response for lovastatin related compound A obtained
USP Reference standards (11)— from the Test solution; and rs is the peak response for lovas-
USP Lovastatin RS tatin obtained from the Standard solution: not more than
USP Lovastatin Related Compound A RS 0.5% of lovastatin related compoundAis found.
Chromatographic purity—
Solution A—Prepare a 0.001 M phosphoric acid solution,
adjusted with 1 M sodium hydroxide to a pH of 4.0.
USP 41
Solution B—Use acetonitrile.
Mobile phase—Use variable mixtures of Solution A and So-
lution B as directed for Chromatographic system. Make ad-
justments if necessary (see System Suitability under Chroma-
tography (621)).
System suitability solution—Dissolve accurately weighed
quantities of USP Lovastatin RS and compactin in acetoni-
trile, and dilute quantitatively, and stepwise if necessary,
with acetonitrile to obtain a solution containing 2.0 ug of
each per mL.
Standard solution—Dissolve an accurately weighed quan-
tity of USP Lovastatin RS in acetonitrile, and dilute quantita-
tively, and stepwise if necessary, to obtain a solution having
a known concentration of about 2.0 ug per mL.
Test solution—Transfer about 25 mg of Lovastatin, accu-
rately weighed, to a 25-mL volumetric flask, dissolve in and
dilute with acetonitrile to volume, and mix.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 238-nm detector
and a 4.0-mm x 12.5-cm column that contains 4-\um pack-
ing L1. The column temperature is maintained at 40°. The
flow rate is about 1.5 mL per minute. The chromatograph is
programmed as follows.
Time Solution A Solution B
(minutes) (%) (%) Elution
0-2 60 40 isocratic
2-5 6045 40-55 linear gradient
5-8 45 55 isocratic
8-16 4510 5590 linear gradient
16-25 10 90 isocratic
25-27 10-60 9040 linear gradient
27-35 60 40 isocratic
Chromatograph the System suitability solution and the Stan-
dard solution, and record the peak responses as directed for
Procedure: the relative retention times are about 1.00 for
lovastatin and 0.85 for compactin; the resolution, R, be-
tween lovastatin and compactin is not less than 3.5; and the
relative standard deviation for replicate injections is not
more than 5%.
Procedure—Separately inject equal volumes (about 10 LL)
of the Standard solution and the Test solution into the chro-
matograph, record the chromatograms, and measure all the
peak responses. Calculate the percentage of each impurity
in the portion of Lovastatin taken by the formula:
2.5(C/W)(ni | rs)F
in which C is the concentration, in ug per mL, of USP Lovas-
tatin RS in the Standard solution; W is the weight, in mg, of
Lovastatin in the Test solution; r, is the peak response for
each impurity obtained from the Test solution; rs is the peak
response for lovastatin obtained from the Standard solution;
and Fis the response factor for each impurity and is equal
to 1.4 for the impurity with a relative retention time o’
about 0.73 and 1.0 for all other impurities. Disregard any
peak with less than 0.04%: not more than 0.2% of any
individual impurity is found; and not more than 1.0% of
total impurities is found.
Assay—
Dilute phosphoric acid—Transfer 1 mL of phosphoric acid
to a 1-L volumetric flask, and dilute with water to volume.
Mobile phase—Prepare a filtered and degassed mixture of
acetonitrile and Dilute phosphoric acid (65:35). Make adjust-
ments if necessary (see System Suitability under Chromatog-
raphy (621)).
Standard preparation—Dissolve an accurately weighed
quantity of USP Lovastatin RS in acetonitrile to obtain a so-
lution having a known concentration of about 0.3 mg per
mL.
Official Monographs / Lovastatin 2485
Assay preparation—Transfer about 30 mg of Lovastatin,
accurately weighed, to a 100-mL volumetric flask, dissolve in
and dilute with acetonitrile to volume, and mix.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is Pauipped with a 238-nm detector
and a 4.6-mm x 25-cm column that contains 5-11m packing
L7. The flow rate is about 1.5 mL per minute. Chromato-
graph the Standard preparation, and record the peak re-
sponses as directed for Procedure: the column efficiency is
not less than 3000 theoretical plates, the tailing factor is not
more than 1.4, and the relative standard deviation for repli-
cate injections is not more than 1.0%.
Procedure—Separately inject equal volumes (about 10 LiL)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
ure the responses for the major peaks.Calculate the quan-
tity, in mg, of C24H36Os in the portion of Lovastatin taken by
the formula:
100C(ru/ rs)
in which C is the concentration, in mg per mL, of USP Lova-
statin RS in the Standard preparation; and ry and rs are the
peak responses obtained from the Assay preparation and the
Standard preparation, respectively.
Lovastatin Tablets
» Lovastatin Tablets contain not less than
90.0 percent and not more than 110.0 percent of
the labeled amount of lovastatin (C24H36Os).
Packaging and storage—Preserve in well-closed, light-re-
sistant containers. Protect from light and store either in a =
a)
cool place or at controlled room temperature. i)
USP Reference standards (11)—
E
USP Lovastatin RS °
Identification— =
i)
A: Thin-Layer Chromatographic Identification Test (201)— ea2
Test solution: Ey
ao]
FOR TABLETS CONTAINING 10 MG OF LOVASTATIN— Transfer 1 ms
Tablet to a centrifuge tube, add 0.4 mL of water and 1.6 mL ww
of acetonitrile. Mix on a vortex mixer until the tablet disin-
tegrates, and sonicate for 4 minutes. Centrifuge for 4 min-
utes, and use the clear supernatant.
FOR TABLETS CONTAINING 20 MG OR 40 MG OF LOVASTATIN—
Transfer 1 Tablet to a centrifuge tube, add 1 mL of water
and 4.0 mL of acetonitrile. Mix on a vortex mixer until the
tablet disintegrates, and sonicate for 4 minutes. Centrifuge
for 4 minutes, and use the clear supernatant.
Standard solution—Prepare a solution of USP Lovastatin
RS in acetonitrile containing 8 mg per mL.
Application volume: 5 wL of the Test solution, 3 wl of the
Standard solution for 10-mg and 20-mg Tablets, 5 wL of the
Standard solution for 40-mg Tablets.
Developing solvent solution: _a mixture of cyclohexane,
chloroform, and isopropyl alcohol (5:2:1).
B: The retention time of the major peak in the chromato-
gram of the Assay preparation corresponds to that in the
chromatogram of the Standard preparation, as obtained in
the Assay.
Dissolution (711)—
Medium—Dissolve 1.38g of monobasic sodium phos-
phate and 20 g of sodium ar sulfate in 900 mL of water.
Adjust with 1 N sodium hydroxide to a pH of 7.0, dilute
with water to 1000 mL, and mix; 900 mL.
 2486 Lovastatin / Official Monographs
Apparatus 2: 50 rpm.
Time: 30 minutes.
Mobile phase—Proceed as directed in the Assay.
Standard solution—Accurately weigh approximately
44 mg of USP Lovastatin RS into a 500-mL volumetric flask,
and dissolve in no more than 20 mL of methanol. Dilute
with Medium to volume, and mix well. Further dilute this
solution with Medium to obtain a solution containing
1/900 mg per mL, with L being the Tablet label claim, in
mg.
Test solution—Pass the solution under test through a suit-
able 0.45-um filter.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 230-nm detector
and a 4.6-mm x 5-cm column that contains 5-11m packing
L1. The flow rate is about 2.0 mL per minute. Chromato-
graph the Standard solution, and record the peak responses
as directed for Procedure: the capacity factor, k’, is greater
than 2.0; the tailing factor is less than 2.0; and the relative
standard deviation for replicate injections is not more than
2.0%.
Procedure—Separately inject equal volumes (about 10 uL)
of the Standard solution and the Test solution into the chro-
matograph, record the chromatograms, and measure the
peak responses. Calculate the percentage of lovastatin dis-
solved by the formula:
7 X Cy ¥ 900 x 100
ro XL
in which ry and rs are the peak responses obtained from the
Test solution and the Standard solution, respectively; Cs is the
concentration, in mg per mL, of the Standard solution; 900
is the volume, in mL, of Medium; and L is the Tablet label
claim, in mg.
i
a)
Tolerances—Not less than 80% (Q) of the labeled amount
iy of C24H36Os is dissolved in 30 minutes.
g Butanedioic acid, compd. with 2-chloro-11-(4-methyl-
io.) Uniformity of dosage units (905): meet the require-
) ments.
= 2-Chloro-11-(4-methyl-1-piperazinyl)dibenz[b, fl[1,4] ox-
6 Assay—
P= Buffer solution—Dissolve 3.45 g of monobasic sodium
a phosphate in 900 mL of water, adjust with phosphoric acid
~” to a pH of 4.0, dilute with water to 1000 mL, and mix.
=) Dissolving solvent—Add 3.0 mL of glacial acetic acid to
900 mL of water contained in a 1 L beaker, adjust to a pH
of 4.0, determined electrometrically, by the addition of a
solution of sodium hydroxide (20%), and mix. Transfer the
contents of the beaker to a 1000-mL volumetric flask, dilute
with water to volume, and mix. Prepare a mixture of aceto-
nitrile and the resultant solution (80:20).
Mobile phase—Prepare a filtered and degassed mixture of
acetonitrile, Buffer solution, and methanol (5:3:1). Make ad-
justments if necessary (see System Suitability under Chroma-
tography (621)).
Standard preparation—Dissolve an accurately weighed
quantity of USP Lovastatin RS in Dissolving solvent to obtain
a solution having a known concentration of about 40 ug per
mL.
Assay preparation—Weigh and finely powder not fewer
than 20 Tablets. Transfer an accurately weighed portion of
the powder, equivalent to about 40 mg of lovastatin, to a
200-mL volumetric flask. Add about 150 mL of Dissolving
solvent, and sonicate for 20 minutes. Cool to room tempera-
ture, and allow the solution to stand for 30 minutes. Dilute
with Dissolving solvent to volume, and mix. Centrifuge a
portion of this solution, transfer 5.0 mL of the clear superna-
tant to a 25-mL volumetric flask, dilute with Dissolving sol-
vent to volume, and mix.
USP 41
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 230-nm detector
and a 4.6-mm x 25-cm column that contains packing L1
and is maintained at 45°. The flow rate is about 1.5 mL per
minute. Chromatograph the Standard preparation, and re-
cord the peak responses as directed for Procedure: the col-
umn efficiency is greater than 3000 theoretical plates, the
tailing factor is less than 2.0, and the relative standard
deviation for replicate injections is not more than 2.0%.
Procedure—Separately inject equal volumes (about 50 1L)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chremateg ars, and meas-
ure the responses for the major peaks. Calculate the per-
centage of the labeled amount of C24H36Os in the portion of
Tablets taken by the formula:
100(Cs
/ Cu)(ru/ rs)
in which Cs is the concentration, in ug per mL, of USP Lova-
statin RS in the Standard preparation; Cy is the concentration
of lovastatin, in ug per mL, in the Assay preparation, based
on the label claim; and ry and rs are the peak responses
obtained from the Assay preparation and the Standard prepa-
ration, respectively.
Loxapine Succinate
2,
CysHisCIN3O + C4HeOx 445.90
mK : HO LN.
“~on
om
\Ryley
Hye
1-piperazinyl)dibenz[b,f][1,4]oxazepine (1:1);
azepine succinate (1:1) [27833-64-3].
DEFINITION
Loxapine Succinate contains NLT 98.5% and NMT 101.0%
of CigHigCIN3O - C4HeQOu, calculated on the dried basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
e B. ULTRAVIOLET ABSORPTION (197U)
Sample solution: 20 g/mL in 0.01 N hydrochloric acid
ASSAY
© PROCEDURE
Sample: 400mg of Loxapine Succinate
Analysis: Dissolve in 80 mL of glacial acetic acid, and
titrate with 0.1 N perchloric acid VS, determining the
endpoint potentiometrically. Perform a blank determi-
nation, and make any necessary correction (see Titrime-
try (541)).
Calculate the percentage of CigHisCIN3O - CaHoOx in the
portion of Loxapine Succinate taken:
Result = [(V - B) x N x Fx 100]/W
sample titrant volume (mL)
STZ0<
tl
blank titrant volume (mL)
ie
normality of titrant fea
uw
equivalence factor, 22.29 mg/mEq
tou
sample weight (mg)
USP 41 Official Monographs / Loxapine 2487

Acceptance criteria: 98.5%-101.0% on the dried basis Impurity Table 1 (Continued)

IMPURITIES Relative
Inorganic Impurities Reten- Relative | Acceptance
© RESIDUE ON IGNITION (281): NMT 0.1% tion Response Criteria,
Name Time Factor NMT (%)
Loxapine succinate 1.0 — =
Delete the following:
Loxapine related com-
pound Ac 1.2 1.2 0.15
°e HEAVY METALS, Method I! (231): NMT 20 ppme coticat 1-
Any unspecified individu-
Jan-2018)
al impurity _ 1.0 0.10
Organic Impurities
© PROCEDURE 2 2-Chloro-11-(piperazin-1 -yl)dibenzo[b, f[1,4]oxazepine,
Buffer: 3.9 g/L of ammonium acetate in water. Adjust » 2-Chlorodibenzo[b, f|-1,4-oxazepin-11-one.,
with 20% acetic acid in water or 6 N ammonium hy- < 3-Chloro-11-(4-methylpiperazin-1-yl)dibenzo[b,f][1 ,4Joxazepine.
droxide to a pH of 7.3. SPECIFIC TESTS
Mobile phase: Acetonitrile and Buffer (3:7) e Loss ON DRYING (731): Dry a sample at 105° for 4 h; it
Diluent: Acetonitrile and Buffer (7:3) loses NMT 0.5% of its weight.
Standard solution: 10 g/mL of USP Loxapine Succi-
nate RS and 1.5 g/mL each of USP Amoxapine RS and ADDITIONAL REQUIREMENTS
USP Loxapine Related CompoundA RS in Diluent. © PACKAGING AND STORAGE: Preserve in tight containers.
[NotE—Amoxapine has been included in the Standard e USP REFERENCE STANDARDS (11)
solution for peak identification purposes only.] USP Amoxapine RS
Sample solution: 1 mg/mL of Loxapine Succinate in USP Loxapine Related Compound A RS
Diluent 3-Chloro-11-(4-methylpiperazin-1 -yl)dibenzo[b,f]
Chromatographic system [1,4]oxazepine.
(See Chromatography (621), System Suitability.) CisHisCIN30 3:27.81
Mode: LC USP Loxapine Succinate RS
Detector: UV 254 nm
Column: 4.6-mm x 5-cm; 2.7-um packing L1
Column temperature: 35°
Flow rate: 1.5 mL/min
Injection size: 10 uL Loxapine Capsules
Run time: 2 times the retention time of loxapine
succinate DEFINITION
System suitability Loxapine Capsules contain an amount of loxapine succinate
Sample: Standard solution (CigHigCIN3O - C4HeO4) equivalent to NLT 90.0% and
Suitability requirements
Resolution: NLT 2.0 between loxapine succinate
NMT 110.0% of the labeled amount of loxapine S
and loxapine related compound A
(CisHigCIN30). 4)
a)
Relative standard deviation: NMT 5.0% IDENTIFICATION
Analysis S
° A. The retention time of the major peak of the Sample i}
Samples: Standard solution and Sample solution solution corresponds to that of the Standard solution, as =
Calculate the percentage of any individual impurity in obtained in the Assay. }
the portion of Loxapine Succinate taken: i}=
ASSAY i)
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100 © PROCEDURE
a}
a
Mobile phase: Dissolve 4 g of tetramethylammonium “”
tu = peak response of each impurity from the chloride in 800 mL of water, and add 200 mL of aceto-
Sample solution nitrile and 1 mL of phosphoric acid.
Ts = peak response of loxapine succinate from the Standard solution: 0.136 mg/mL of USP Loxapine Suc-
Standard solution cinate RS prepared as follows. Dissolve a suitable quan-
Cs = concentration of USP Loxapine Succinate RS tity of USP Loxapine Succinate RS in 0.01 N hydrochlo-
in the Standard solution (mg/mL) ric acid in a suitable volumetric flask, and dilute with
Cu = concentration of Loxapine Succinate in the Mobile phase to volume.
Sample solution (mg/mL) Sample solution: Nominally 0.1 mg/mL of loxapine
Acceptance criteria prepared as follows. Weigh the contents of NLT 20
Individual impurities: See Impurity Table 1. Capsules. Transfer a portion of the contents,nominally
Total impurities: NMT 0.5% equivalent to NLT 50 mg of loxapine, to a suitable volu-
metric flask. Add 10% of the flask volume of 0.1 N
Impurity Table 1 hydrochloric acid. Shake, and sonicate for 5 min. Dilute
with Mobile phase to volume, and filter. Discard the first
Relative
8 mL of the filtrate.
Reten- Relative | Acceptance Chromatographic system
tion Response Criteria, (See Chromatography (621), System Suitability.)
Name Time Factor NMT (%) Mode: LC
Amoxapine? 0.19 13 0.15 Detector: UV 254 nm
Chlorodibenzoxazepi- Column: 4.6-mm x 15-cm; 5-um packing L10
none 0.45 0.70 0.15 Flow rate: 1.6 mL/min
a 2-Chloro-11-(piperazin-1-yl)dibenzo[b,fl[1 ,4]oxazepine. Injection volume: 20 pL
» 2-Chlorodibenzo[b,f]-1,4-oxazepin-1 1-one.
© 3-Chloro-11 -(4-methylpiperazin-1-yl)dibenzo[b,f][1,4]oxazepine.
 2488 Loxapine / Official Monographs USP 41

System suitability ASSAY

Sample: Standard solution © PROCEDURE
Suitability requirements Solution A: 0.1 mL of phosphoric acid diluted with
Column efficiency: NLT 1500 theoretical plates water to 1L
Tailing factor: NMT 2.0 Solution B: Acetonitrile
Relative standard deviation: NMT 2.0% Mobile phase: See Table 7. Return to original condi-
Analysis tions, and equilibrate the system.
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of lox- Table 1
apine (CisHigCIN3O) in the portion of Capsules taken:
Solution A Solution B
Result = (ru/rs) x (Cs/Cu) X (Ma/M,2z) x 100
ry = peak area from the Sample solution
rs = peak area from the Standard solution
15
Gs = concentration of USP Loxapine Succinate RS in
the Standard solution (mg/mL) 17
Cu = nominal concentration of loxapine in the
Sample solution (mg/mL) Diluent: Acetonitrile and water (70:30)
Mr = molecular weight of loxapine, 327.81
System suitability solution: 0.4 mg/mL of USP
Lufenuron RS and 0.14 mg/mL of USP Lufenuron Re-
Mz = molecular weight of loxapine succinate, lated CompoundG RS in Diluent. Sonicate if necessary
445.90
Acceptance criteria: 90.0%-110.0% to facilitate dissolution.
Standard stock solution: 0.4 mg/mL of USP Lufenuron
PERFORMANCE TESTS RS in Diluent. Sonicate if necessary to facilitate
e DISSOLUTION (711) dissolution.
Medium: Water; 900 mL Standard solution: 0.04 mg/mL of USP Lufenuron RS
Apparatus 1: 100 rpm in Diluent from Standard stock solution
Time: 45 min Sample stock solution: 0.4 mg/mL of Lufenuron in Dil-
Standard solution: USP Loxapine Succinate RS at a uent. Sonicate if necessary to facilitate dissolution.
known concentration in Medium Sample solution: 0.04 mg/mL of Lufenuron in Diluent
Sample solution: Dilute with water as needed. from Sample stock solution
Instrumental conditions Chromatographic system
Mode: UV (See Chromatography (621), System Suitability.)
Analytical wavelength: 254 nm Mode: LC
Analysis Detector: UV 255 nm
Samples: Standard solution and Sample solution Column: 4-mm x 25-cm; 5-m packing L1
Tolerances: NLT 75% (Q) of the labeled amount of lox- Flow rate: 1 mL/min
4
"
apine (CisHisCIN30) is dissolved. Injection volume: 20 pL
is e UNIFORMITY OF DOSAGE UNITS (905): Meet the System suitability
i]
— requirements Samples: System suitability solution and Standard
m2) solution
° ADDITIONAL REQUIREMENTS [Note—The relative retention times for lufenuron re-
<I
5 © PACKAGING AND STORAGE: Preserve in tight containers. lated compound G and lufenuron are 0.9 and 1.0,
3 e USP REFERENCE STANDARDS (11) respectively.]
a USP Loxapine Succinate RS Suitability requirements
al Resolution: NLT 3.0 between lufenuron related com-
=) pound G and lufenuron, System suitability solution
Tailing factor: NMT 2.0, Standard solution
Relative standard deviation: NMT 0.73%, Standard
Lufenuron solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of lufenuron (Ci7HgClaFgN2O3)
in the portion of Lufenuron taken:
Result = (ru/rs) x (Cs/Cu) x 100
ru = peak response from the Sample solution
Cy7HsClaFgN2O3, S11015 rs = peak response from the Standard solution
Benzamide, N-[[[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropro- Cs = concentration of USP Lufenuron RS in the
oxy)pheny!Jamino]carbonyl]-2,6-difluoro-; Standard solution (mg/mL)
1-[2,5-Dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]- Cu = concentration of Lufenuron in the Sample
3-(2,6-difluorobenzoyl)urea [1 0305-07-81. solution (mg/mL)
Acceptance criteria: 98.0%-102.0% on the dried basis
DEFINITION
Lufenuron contains NLT 98.0% and NMT 102.0% of IMPURITIES
lufenuron (Ci7HgClaFgN2O3), calculated on the dried basis. e RESIDUE ON IGNITION (281): NMT 0.1%
@ ORGANIC IMPURITIES
IDENTIFICATION Mobile phase, Diluent, Standard solution, Chromato-
e A. INFRARED ABSORPTION (197K) graphic system, and System suitability: Proceed as
e B. The retention times of the et ea of the Sample directed in the Assay.
solution correspond to those of the Standard solution, as Identification solution: 1.2 g/mL of USP Lufenuron
obtained in the Assay. Related Compound B RS and 1.6 ug/mL of USP
Lufenuron Related CompoundC RS in Diluent
USP 41 Official Monographs / Lumefantrine 2489

Diluted standard solution: 0.4 ug/mL of USP

Lufenuron RS in Diluent from Standard solution
Sample solution: 0.4 mg/mL of Lufenuron in Diluent. Lumefantrine
Sonicate if necessary to facilitate dissolution.
Analysis i
Nass
Samples: /dentification solution, Diluted standard solu- ¢
tion, and Sample solution
Chromatograph the /dentification solution, and identify
Yow J
the components on the basis of their relative retention = LOCA at
times, given in Table 2.
Calculate the percentage of each impurity in the por- cd cl
tion of Lufenuron taken: el
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
C3oH32Cl;NO 528.94
tu = peak response of each impurity from the (4)-2,7-Dichloro-9-[(Z)-p-chlorobenzylidine]-a[(dibutylami-
Sample solution no)methyl]-fluorene-4-methanol [82186-77-4].
rs = peak response of lufenuron from the Diluted
standard solution DEFINITION
Cs = concentration of USP Lufenuron RS in the Lumefantrine contains NLT 98.0% and NMT 102.0% of
Diluted standard solution (\ug/mL) lumefantrine (C30H32Cl3NO).
Cu = concentration of Lufenuron in the Sample IDENTIFICATION
solution (ug/mL) © A. INFRARED ABSORPTION (197K)
F = relative response factor (see Table 2) e B. The retention time of the lumefantrine peak of the
Acceptance criteria: See Table 2. The reporting level Sample solution corresponds to that of the Standard solu-
for impurities is 0.1%. tion, as obtained in the Assay.

Table 2 ASSAY
© PROCEDURE
Relative Relative Acceptance Buffer: Dissolve 5.65 g of sodium 1-hexanesulfonate
Retention Response Criteria, and 2.75 g of monobasic sodium phosphate in 900 mL
Name Time Factor NMT (%) of water. Adjust with phosphoric acid to a pH of 2.3
Lufenuron before dilution with water to a final volume of
related 1000 mL.
compound B 0.3 0.77 0.3 Solution A: Acetonitrile and Buffer (300:700)
Lufenuron Solution B: Acetonitrile and 2-propanol (540:460)
related Mobile phase: See Table 7.
compound C 0.7 0.77 0.4 (=
Lufenuron 1.0 = = “
Table 1 uv
Any other
individual —
Solution A Solution B ms
%' Yo °
impurity 1.0 0.20 |]
Total °
impurities _ ue 1.0 ve}
=e
50 50 oy
me}
SPECIFIC TESTS 3
25 75 7)
e Loss ON DRYING (731)
Analysis: Dry at 105° to constant weight.
Acceptance criteria: NMT 0.5% 15.1 65 5
2 65 35
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain- System suitability stock solution: 10 g/mL of USP
ers. Store at room temperature. Lumefantrine Related CompoundA RS prepared as fol-
°PADALING! Label it to indicate that it is for veterinary use lows. Transfer a suitable quantity of USP Lumefantrine
only. Related Compound A RS to a volumetric flask, dissolve
e USP REFERENCE STANDARDS (11) in 10% volume dichloromethane, and dilute with aceto-
USP Lufenuron RS nitrile to volume.
USP Lufenuron Related Compound B RS System suitability solution: 1 mg/mL of USP Lumefan-
N-[(2,5-Dichloro-4-hydroxyphenyl)carbamoyl]-2,6- trine RS and 1 pg/mL of USP Lumefantrine Related
difluorobenzamide. CompoundA RS prepared as follows. Transfer 10 mg of
CisHsClaF2N203 361.13 USP Lumefantrine RS to a 10-mL volumetric flask, and
USP Lufenuron Related Compound C RS dissolve in 1 mL of dichloromethane. Add 1.0 mL of the
N-[3-Chloro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl- System suitability stock solution, and dilute with acetoni-
carbamoyl]-2,6-difluorobenzamide. trile to volume.
Cy7HsCIFsN203_ 476.71 Standard solution: 1mg/mL of USP Lumefantrine RS
USP Lufenuron Related Compound G RS prepared as follows. Transfer a suitable quantity of USP
2,5-Dichloro-4-[3-(2,6-difluorobenzoyl)ureido]phenyl Lumefantrine RS to a volumetric flask, dissolve in 10%
phenyl carbonate. volume of dichloromethane, and dilute with acetonitrile
CarHi2ClaF2N20s 481.23 to volume.
Sample solution: 1 mg/mL of Lumefantrine prepared as
follows. Transfer a suitable quantity of Lumefantrine to a
volumetric flask, dissolve in 10% volume of dichloro-
methane, and dilute with acetonitrile to volume.
 2490 Lumefantrine / Official Monographs USP 41

Chromatographic system Table 2

(See Chromatography (621), System Suitability.)
Relative Relative Acceptance
Mode: LC
Detector: UV 265 nm Retention Response Criteria,
Column: 4.6-mm x 50-mm; 1.8-11m packing L1 Name Time Factor NMT (%)
Column temperature: Beginning of column, 50°; end Desbuty! lumefan-
of column, 35° trinea 0.68 dad 0.05
Flow rate: 2.5 mL/min Lumefantrine 1.0 = =
Injection volume: 2.5 uL Any other individual ea
Run time: 20 min impurity 1.0 0.10
System suitability Total impurities = — 0.3
Samples: System suitability solution and Standard # (Z)-2-(Butylamino)-1-(2,7-dichloro-9-(4-chlorobenzylidene)-9H-fluoren-4-
solution ylethanol.
[Note—The relative retention times for lumefantrine re-
lated compound A and lumefantrine are 0.9 and 1.0, SPECIFIC TESTS
respectively.] e CLARITY OF SOLUTION
Suitability requirements Hydrazine sulfate solution: Transfer 1.0 g of hydrazine
Resolution: NLT 1.3 between lumefantrine and lume- sulfate to a 100-mL volumetric flask, and dissolve in and
fantrine related compound A, System suitability dilute with water to volume. Allow to stand for 4-6 h
solution before use.
Tailing factor: NMT 2.1, Standard solution Methenamine solution: Transfer 2.5 g of methenamine
Relative standard deviation: NMT 1.0%, Standard to a 100-mL glass-stoppered flask, add 25 mL of water,
solution insert the glass stopper, and mix to dissolve.
Analysis Primary opalescent suspension: Transfer 25.0 mL of
Samples: Standard solution and Sample solution Hydrazine sulfate solution to the Methanamine solution in
Calculate the percentage of lumefantrine (C3oH32ClsNO) the 100-mL glass-stoppered flask. Allow to stand for 24
in the portion of Lumefantrine taken: h. This suspension is stable for 2 months, provided it is
stored in a glass container free from surface defects.
Result = (ru/rs) x (Cs/Cu) x 100 The suspension must not adhere to the glass and must
be well mixed before use.
ru = peak response from the Sample solution Stock opalescence suspension: Transfer 15.0 mL of the
rs = peak response from the Standard solution Primary opalescent suspension to a 1000-mL volumetric
G = concentration of the Standard solution flask, and dilute with water to volume. This suspension
(mg/mL) should not be used beyond 24h after preparation.
Cu = concentration of the Sample solution (mg/mL) Sample solution: Dissolve 1.0 g of Lumefantrine in di-
Acceptance criteria: 98.0%-102.0% chloromethane, and dilute with dichloromethane to
10.0 mL.
” IMPURITIES Standard suspension: Transfer 5.0 mL of Stock opales-
<= e RESIDUE ON IGNITION (281): NMT 0.1%
re cence suspension to a 100-mL volumetric flask, and di-
cS
—
lute with water to volume. Prepare only if the Sample
D Delete the following: solution is not as clear as water or dichloromethane.
° Analysis
= Samples: Sample solution, Standard suspension, water,
S °e HEAVY METALS, Method Il 231): NMT 10 ppme coificial 1-
P= Jon-2018) and dichloromethane
© ORGANIC IMPURITIES Transfer a sufficient portion of the Sample solution to a
5 test tube of colorless, transparent, neutral glass with a
~“” Buffer, Solution A, Solution B, Mobile phase, System
=) suitability stock solution, System suitability solution, flat base and an internal diameter of 15-25 mm to ob-
Sample solution, Chromatographic system, and Sys- tain a depth of 40 mm. Similarly transfer portions of
tem suitability: Proceed as directed in the Assay. Standard suspension and dichloromethane to separate
Standard stock solution: 50 j1g/mL of USP Lumefan- matching test tubes. Compare the Sample solution,
trine RS prepared as follows. Transfer a suitable quantity Standard suspension, water, and dichloromethane in
of USP Lumefantrine RS into a volumetric flask, dissolve diffused daylight, viewing vertically against a black
in 10% volume of dichloromethane, and dilute with ac- background. The diffusion of light must be such that
etonitrile to volume. the Standard suspension can readily be distinguished
Standard solution: 1 j1g/mL of USP Lumefantrine RS in from dichloromethane. If the Sample solution is as clear
acetonitrile from the Standard stock solution as water or dichloromethane, it is not necessary to
Analysis prepare the Standard suspension.
Samples: Sample solution and Standard solution Acceptance criteria: The Sample solution shows the
Calculate the percentage of desbutyl lumefantrine or same or more clarity than water, dichloromethane, or
any other impurity in the portion of Lumefantrine the Standard suspension.
taken:
ADDITIONAL REQUIREMENTS
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100 e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. Store at room temperature.
ru = peak response of desbutyl lumefantrine or any e USP REFERENCE STANDARDS (11)
other impurity from the Sample solution USP Lumefantrine RS
Is peak response from the Standard solution USP Lumefantrine Related Compound A RS
"ou

Cs concentration of the Standard solution (RS,Z)-2-(Dibutylamino)-2-(2, 7-dichloro-

(mg/mL) 9-(4-chlorobenzylidene)-9H-fluoren-4-yl)ethanol.
Cu = concentration of the Sample solution (mg/mL) C3oH32Ch35NO 528.94
F = relative response factor
Acceptance criteria: See Table 2. Disregard any peak
less than 0.05%.
USP 41 Official Monographs / Lysine 2491

Application volume: 5 ul
Developing solvent system: Isopropyl alcohol and
Lysine Acetate ammonium hydroxide (7:3)
Spray reagent: 0.2 of ninhydrin in a mixture of bu-
tyl alcohol and 2N acetic acid (95:5)
tNBoA al oh
f
System suitability
Suitability requirements: The chromatogram of the
System suitability solution exhibits two clearly sepa-
rated spots.
CeH14N2O2 - CzH402 206.24 Analysis
L-Lysine monoacetate [57282-49-2]. Samples: Standard solution, System suitability solution,
DEFINITION and Sample solution
Lysine Acetate contains NLT 98.0% and NMT 102.0% of Dry the plate between 100° and 105° until the am-
L-lysine acetate (C6H14N2O2 - C2H4O2), calculated on the monia completely disappears. Spray with Spray rea-
dried basis. ent, and heat between 100° and 105° for 15 min.
xamine the plate under white light.
IDENTIFICATION Acceptance criteria: Any secondary spot of the Sample
e A. INFRARED ABSORPTION (197K) Solution is not larger or more intense than the principal
spot of the Standard solution.
ASSAY Individual impurities: NMT 0.5%
© PROCEDURE Total impurities: NMT 2.0%
Sample: 100 mg of Lysine Acetate
Blank: Mix 3 mL of formic acid and 50 mL of glacial SPECIFIC TESTS
acetic acid. © OPTICAL ROTATION, Specific Rotation (781S)
Titrimetric system Sample solution: 100 mg/mL in water
(See Titrimetry (541).) Acceptance criteria: +8.4° to +9.9°
Mode: Direct titration e Loss ON DRYING (731): Dry a sample at 80° for 3 h: it
Titrant: 0.1 N perchloric acid VS loses NMT 0.2% of its weight.
Endpoint detection: Potentiometric
Analysis: Dissolve the Sample in 3 mL of formic acid ADDITIONAL REQUIREMENTS
and 50 mL of glacial acetic acid. Titrate with the Titrant. © PACKAGING AND STORAGE: Preserve in well-closed
Perform the Blank determination. containers.
Calculate the percentage of lysine acetate (CéH14N20z - e@ USP REFERENCE STANDARDS (11)
CzH4Oz2) in the Sample taken: USP Arginine Hydrochloride RS
USP L-Lysine Acetate RS
Result = {[(Vs — Ve) x N x F]/W} x 100
Vs = Titrant volume consumed by the Sample (mL) Cc
Ve = Titrant volume consumed by the Blank (mL) 4)
N = actual normality of the Titrant (mEq/mL) Lysine Hydrochloride uv
F = equivalency factor, 103.1 mg/mEq =
w = Sample weight (mg) i}
Acceptance criteria: 98.0%-102.0% on the dried basis HANS i ‘OH + Hel }
}

IMPURITIES NH. ro}=

e RESIDUE ON IGNITION (281): NMT 0.4% i
© CHLORIDE AND SULFATE, Chloride (221)
bo}
CeHi4N2O2 - HCl 182.65 >
—— solution: 0.50 mL of 0.020 N hydrochloric L-Lysine hydrochloride [657-27-2].
al

aci
Sample: 0.73 g of Lysine Acetate DEFINITION
Acceptance criteria: NMT 0.05% Lysine Hydrochloride contains NLT 98.5% and NMT 101.5%
e CHLORIDE AND SULFATE, Sulfate (221) of L-lysine hydrochloride (CeéHisN2O2
- HCl), calculated on
Standard solution: 0.10 mL of 0.020 N sulfuric acid the dried basis.
Sample: 0.33g of Lysine Acetate
Acceptance criteria: NMT 0.03% IDENTIFICATION
© IRON (241): NMT 30 ppm e A. INFRARED ABSORPTION (197K)
ASSAY
Delete the following: © PROCEDURE
Sample: 90 mg of Lysine Hydrochloride
°e HEAVY METALS, Method | (231): NMT 15 ppme coricial1- Blank: Mix 3 mL of formic acid and 50 mL of glacial
Jan-2018) acetic acid.
e RELATED ComPpouNDs Titrimetric system
Standard solution: 0.05 mg/mL of USP L-Lysine Acetate (See Titrimetry (541).)
RS in water Mode: Direct titration
[NoTte—This solution has a concentration equivalent to Titrant: 0.1 N perchloric acid VS
0.5% of that of the Sample solution.] Endpoint detection: Potentiometric
Sample solution: 10 mg/mL of Lysine Acetate in water Analysis: Dissolve the Sample in 3 mL of formic acid
System suitability solution: 0.4 mg/mL each of USP and 50 mL of glacial acetic acid. Add 10 mL of mercuric
L-Lysine Acetate RS and USP Arginine Hydrochloride RS acetate TS, and titrate with theTitrant. Perform the
Chromatographic system Blank determination.
(See Chromatography (621), Thin-Layer Chromato- Calculate the percentage of lysine hydrochloride
graphy.) (CeHi4N202 - HCl) in the Sample taken:
Mode: TLC
Adsorbent: 0.25-mm layer of chromatographic silica Result = {[(Vs — Vs) x N x F]/W} x 100
gel mixture
 2492 Lysine / Official Monographs USP 41

Vs = Titrant volume consumed by the Sample (mL) Sample solution: 10 mg/mL of Lysine Hydrochloride in
Ve = Titrant volume consumed by the Blank (mL) water
N = actual normality of the Titrant (mEq/mL) System suitability solution: 0.4 mg/mL each of USP
F = equivalency factor, 91.33 mg/mEq Lae Hydrochloride RS and USP Arginine Hydrochlo-
w = Sample weight (mg) ride RS
Acceptance criteria: 98.5%-101.5% on the dried basis Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
OTHER COMPONENTS
© CONTENT OF CHLORIDE
graphy.)
Mode: TLC
Saniple: 350 mg of Lysine Hydrochloride Adsorbent: 0.25-mm layer of chromatographic silica
Blank: 140 mL of water gel mixture
Titrimetric system Application volume: 5 wL
(See Titrimetry (541).) Developing solvent system: Isopropyl alcohol and
Mode: Direct titration ammonium hydroxide (7:3)
Titrant: 0.1 N silver nitrate VS Spray reagent: 0.2g of ninhydrin in a mixture of bu-
Endpoint detection: Visual tyl alcohol and 2N acetic acid (95:5)
Analysis: Transfer the Sample to a porcelain casserole, System suitability
and add 140 mL of water and 1 mL of dichlorofluores- Suitability requirements: The chromatogram of the
cein TS. Titrate with the Titrant until the silver chloride System suitability solution exhibits two clearly sepa-
flocculates and the mixture acquires a faint pink color. rated spots.
Perform the Blank determination. Analysis
Calculate the percentage of chloride (Cl) in the Sample Samples: Standard solution, System suitability solution,
taken: and Sample solution
Dry the plate between 100° and 105° until the am-
Result = {[(Vs — Va) x N x FI/W} x 100 monia completely disappears. Spray with Spray rea-
gent, and heat between 100° and 105° for 15 min.
Titrant volume consumed by the Sample (mL) Examine the plate under white light.
Vp = Titrant volume consumed by the Blank (mL) Acceptance criteria: Any secondary spot of the Sample
N = actual normality of the Titrant (mEq/mL) solution is not larger or more intense than the principal
F = equivalency factor, 35.45 mg/mEq spot of the Standard solution.
w = Sample weight (mg) Individual impurities: NMT 0.5%
Acceptance criteria: 19.0%-19.6% Total impurities: NMT 2.0%
IMPURITIES SPECIFIC TESTS
¢ RESIDUE ON IGNITION (281): NMT 0.1% © OPTICAL ROTATION, Specific Rotation (781S)
© CHLORIDE AND SULFATE, Sulfate (221) Sample solution: 80 mg/mL in 6 N hydrochloric acid
Standard solution: 0.10 mL of 0.020 N sulfuric acid Acceptance criteria: +20.4° to +21.4°
ww
Sample: 0.33 g of Lysine Hydrochloride e Loss ON DRYING (731): Dry a sample at 105° for 3 h: it
<= Acceptance criteria: NMT 0.03% loses NMT 0.4% of its weight.
a e IRON (241): NMT 30 ppm
i]
a ADDITIONAL REQUIREMENTS
a e PACKAGING AND STORAGE: Preserve in well-closed
2} Delete the following:
containers.
<7
iS °o HEAVY METALS, Method | (231): NMT 15 ppme wiricia1-
e USP REFERENCE STANDARDS (11)
= USP Arginine Hydrochloride RS
2
Jan-2018) USP L-Lysine Hydrochloride RS
al e RELATED COMPOUNDS
=) Standard solution: 0.05 mg/mL of USP L-Lysine Hydro-
chloride RS in water. [NoTE—This solution has a con-
centration equivalent to 0.5% of that of the Sample so-
lution.]
USP 41 Official Monographs / Mafenide 2493

nol to obtain Standard solution E and Standard solution

Mafenide Acetate F. The compositions are shown in Table 7.

a9 Table 1
re SE
WF Oo

oS J “NH * J!
HyC “OH
Percentage
(%, for
comparison
Standard Concentration with test
C7HioN202S - CoH4O2 246.28 Solution Dilution (ug/ml) specimen)
Benzenesulfonamide, 4-(aminomethyl)-, monoacetate;
a-Amino-p-toluenesulfonamide monoacetate [13009-99-9]. A (undiluted) 500 1.0
B Sin 10 250 0.5
DEFINITION Cc lin $ 100 0.2
Mafenide Acetate contains NLT 98.0% and NMT 102.0% of D (undiluted) 500 1.0
mafenide acetate (C7Hio0N202S - C2H4O2), calculated on the
E Sin 10 250 0.5
anhydrous basis.
Fi 1ins 100 0.2
IDENTIFICATION
e A. INFRARED ABSORPTION (197K) Sample solution: 50 mg/mL of Mafenide Acetate in
e B. The R; value of the principal spot of the /dentification methanol
solution corresponds to that of Standard solution A, as ob- Identification solution: 500 g/mL from the Sample so-
tained in the test for Organic Impurities. lution in methanol
Ninhydrin solution: 300 mg of ninhydrin in 100 mL of
ASSAY butyl alcohol. Add 3 mL of glacial acetic acid.
© PROCEDURE Chromatographic system
Standard solution: 200 t1g/mL of USP Mafenide Ace- (See Chromatography (621), Thin-Layer Chromato-
tate RS in 0.01 N hydrochloric acid graphy.)
Sample stock solution: 2mg/mL of Mafenide Acetate Mode: TLC
Sample solution: 200 g/mL of Mafenide Acetate pre- Adsorbent: 0.25-mm layer of chromatographic silica
pared as follows. Pipet 10 mL of the Sample stock solu- gel mixture
tion into a 100-mL volumetric flask containing 1 mL of Application volume: 5 ul
1N hydrochloric acid, and dilute with water to volume. Developing solvent system: Ethyl acetate, methanol,
Instrumental conditions and isopropylamine (77:20:3)
Mode: UV Spray reagent: Ninhydrin solution
Analytical wavelength: 267 nm Analysis
Cell: 1.cm Samples: Standard solutions, Sample solution, and Iden-
Blank: 0.01 N hydrochloric acid tification solution
Analysis Apply the Samples separately to the chromatographic =
Samples: Standard solution, Sample solution, and Blank late. Position the plate in a chromatographic cham- 4)
Calculate the percentage of mafenide acetate er, and develop the chromatograms in the Developing v
(C7HioN202S - C2zH4Oz) in the portion of Mafenide Ace- solvent system until the solvent front has moved about E
tate taken: three-fourths of the length of the plate. Remove the )
Eat from the developing chamber, mark the solvent rs]
Result = (Au/As) x (Cs/Cu) x 100. ry
ront, and allow the solvent to evaporate in warm, cir-
culating air. Examine the plate under short-wavelength
to)=
Au = absorbance of the Sample solution 2
UV light, and compare the intensities of any secondary a)
As = absorbance of the Standard solution spots observed in the chromatogram of the Sample so- oe
G = concentration of USP Mafenide Acetate RS in lution at the Rr value corresponding to those of the al

the Standard solution (\1g/mL) principal spots in the chromatograms of Standard solu-
Cu = concentration of Mafenide Acetate in the tions D, E, and F. Spray the plate with Spray reagent,
Sample solution (ug/mL) heat the plate at 105° for 5 min, and examine the
eceparice criteria: 98.0%-102.0% on the anhydrous plate. Compare the intensities of any secondary spots
aSIS observed in the chromatogram of the Sample solution
to those of the principal spots in the chromatograms
IMPURITIES
of Standard solutions A, B, and C.
e RESIDUE ON IGNITION (281): NMT 0.2% Acceptance criteria: No secondary spot, observed by
¢ SELENIUM (291) both visualizations, from the chromatogram of the Sam-
Sample: 200mg ple solution is larger or more intense than the principal
Acceptance criteria: NMT 30 ppm spots obtained from Standard solution B (0.5%) and
Standard solution E (0.5%). The sum of the intensities of
Delete the following: all secondary spots obtained from the Sample solution
corresponds to NMT 1.0%.
°e HEAVY METALS, Method I! (231): NMT 20 ppme coicia-
SPECIFIC TESTS
lan-2018)
e ORGANIC IMPURITIES © MELTING RANGE OR TEMPERATURE (741): Between 162°
Standard solution A: 500 g/mL of USP Mafenide Ace- and 171°, but the range between the beginning and end
tate RS in methanol of melting does not exceed 4°.
Standard solution D: 500 g/mL of USP Mafenide Re- e PH (791)
lated Compound A RS in methanol Sample solution: 100 mg/mL
[Note—USP Mafenide Related Compound ARS is Acceptance criteria: 6.4-6.8
Baer ae acl e@ WATER DETERMINATION, Method | (921): NMT 1.0%
Standard solutions: Quantitatively dilute portions of ADDITIONAL REQUIREMENTS
Standard solution A with methanol to obtain Standard e PACKAGING AND STORAGE: Preserve in tight, light-resistant
solution B and Standard solution C. Similarly, quantita- containers.
tively dilute portions of Standard solution D with metha-
 2494 Mafenide / Official Monographs
e USP REFERENCE STANDARDS (11)
USP Mafenide Acetate RS
USP Mafenide Related Compound A RS
4-Formylbenzenesulfonamide.
C;H7NO3S ~—-185.20
Mafenide Acetate Cream
DEFINITION
Mafenide Acetate Cream is Mafenide Acetate in a water-
miscible, oil-in-water cream base, containing suitable pre-
servatives. It contains NLT 90.0% and NMT 110.0% of
mafenide acetate (C7HioN202S « C2H4Oz), in terms of the
labeled amount of mafenide (C7Hi9N202S).
IDENTIFICATION
e A. ULTRAVIOLET ABSORPTION (197U)
Sample solution: Proceed as directed in the Assay.
Acceptance criteria: Meets the requirements
Change to read:
e B. IDENTIFICATION TESTS—GENERAL, Acetate (191)
Sample solution: Place about 1g in a 60-mL separa-
tory funnel, and add 20 mL of chloroform to dissolve it.
Add 20 mL of water, shake for 2 min, allow the layers
to separate completely, and discard the lower chloro-
form layer. Repeat this washing with another 20-mL
portion of chloroform, and discard the chloroform
washing. Centrifuge the aqueous layer. Use a 1-mL ali-
uot of the supernatant with 2 mL of water for °test
© (GN t-May-2018) and a 3-mL aliquot of the supernatant
for “test Bie cen 1-May-2018)
Acceptance criteria: Meets the requirements
cs
vw
a tate prepared as follows. Constitute the Topical Solution
S ASSAY
—
Db © PROCEDURE
} Standard solution: 200 g/mL of USP Mafenide Ace-
iS tate RS in 0.01 N hydrochloric acid
S Sample solution: Nominally 200 ug/mL of mafenide
= acetate prepared as follows. Transfer a portion of Cream
es containing 100 mg of mafenide acetate to a 60-mL
a) separator, and add 20 mL of chloroform to dissolve it.
= Mode: LC
Add 20 mL of water, shake for 2 min, allow the layers
to separate completely, and discard the lower chloro-
form layer. Repeat this washing with two separate
20-mL portions of chloroform, and discard the chloro-
form washings. Pass the aqueous phase through a dry
filter into a 100-mL volumetric flask. Rinse the separator
and the filter with water, passing all rinses through the
filter, and add water to volume. Centrifuge 30 mL, then
pipet 20 mL of the clear supernatant into a 100-mL vol-
umetric flask. Add 1 mL of 1 N hydrochloric acid, and
add water to volume.
Instrumental conditions
Mode: UV
Analytical wavelength: 267 nm
Cell: 1. cm
Blank: 0.01 N hydrochloric acid
Analysis
Samples: Standard solution, Sample solution, and Blank
Calculate the percentage of the labeled amount of
mafenide acetate (C7HioN2O2S - CzH4Oz) in the portion
of Cream taken:
Result = (Au/As) x (Cs/Cu) x 100
Au = absorbance of the Sample solution
As = absorbance of the Standard solution
USP 41
Cs = concentration of USP Mafenide Acetate RS in
the Standard solution (g/mL)
CG = nominal concentration of mafenide acetate in
the Sample solution (g/mL)
Acceptance criteria: 90.0%-110.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and avoid exposure to excessive heat.
e USP REFERENCE STANDARDS (11)
USP Mafenide Acetate RS
Mafenide Acetate for Topical Solution
DEFINITION
Mafenide Acetate for Topical Solution contains NLT 98.0%
and NMT 102.0% of mafenide acetate (C7HioN202S
-
C2H4O2), calculated on the anhydrous basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
ASSAY
© PROCEDURE
Solution A: Dissolve 6.8 g of monobasic potassium
phosphate and 1.0g of sodium 1-hexanesulfonate in
800 mL of water. Adjust with phosphoric acid to a pH
of 2.5, and dilute to 1000 mL.
Mobile phase: Acetonitrile and Solution A (1:9)
Standard solution: 1 mg/mL of USP Mafenide Acetate
RS in Mobile phase. Sonicate to dissolve if necessary.
Sample solution: Nominally 1 mg/mL of mafenide ace-
as directed in the labeling. Transfer a volume of the
constituted Topical Solution, equivalent to 25 mg of
mafenide acetate, to a 25-mL volumetric flask. Using
sonication, dissolve in 12 mL of Mobile phase. Dilute
with Mobile phase to volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Detector: UV 267 nm
Column: 4.6-mm x 15-cm; 5-um packing L1
Flow rate: 1 mL/min
Injection volume: 20 pL
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
mafenide acetate (C7HioN202S »C2H4O2) in the portion
of the constituted Topical Solution taken:
Result = (ru/rs) x (Cs/Cu) x 100
ry = peak response from the Sample solution
rs = peak response from the Standard solution
Cs = concentration of USP Mafenide Acetate RS in
the Standard solution (mg/mL)
Cu = nominal concentration of mafenide acetate in
the Sample solution (mg/mL)
Acceptance criteria: 98.0%-102.0% on the anhydrous
basis
USP 41 Official Monographs / Mafenide 2495

OTHER COMPONENTS Acceptance criteria: 22.0%-26.8%

¢ CONTENT OF ACETIC ACID
Internal standard solution: 0.5% (v/v) of propionic IMPURITIES
acid in water © ORGANIC IMPURITIES
Standard stock solution: Transfer 50 mL of water to a Solution A: Dissolve 6.8 g of monobasic potassium
100-mL volumetric flask, insert a stopper, and weigh. phosphate and \.c9 of sodium 1-hexanesulfonate in
Add 0.5 mL of glacial acetic acid to the flask, insert the 800 mL of water. Adjust with phosphoric acid to a pH
stopper, weigh, and calculate, by difference, the of 2.5, and dilute to 1000 mL.
amount of acetic acid added. Dilute with water to Mobile phase: Acetonitrile and Solution A (1:9)
volume. Standard stock solution: 25 g/mL of USP Mafenide
Standard solution: 530 g/mL of acetic acid prepared Related Compound A RS in Mobile phase
as follows. Add 10.0 mL of Standard stock solution and [Note—USP Mafenide Related Compound A RS is
10.0 mL of Internal standard solution to a 100-mL volu- 4-formylbenzenesulfonamide.]
metric flask containing 200 mg of oxalic acid. Dilute System suitability solution: 1.0 mg/mL of USP
with water to volume. jafenide Acetate RS and 10 g/mL of USP Mafenide
Sample solution: Nominally 2 mg/mL of mafenide ace- Related Compound A RS from Standard stock solution in
tate prepared as follows. Constitute the Topical Solution Mobile phase. Initially dissolve the USP Mafenide Acetate
as directed in the labeling. Transfer a volume of the RS using 20% of the final volume by sonication in 2 mL
constituted Topical Solution, equivalent to 200 mg of of Mobile phase, add the appropriate volume of USP
mafenide acetate, to a 100-mL volumetric flask contain- Mafenide Related Compound ARS, and dilute to final
ing 200 mg of oxalic acid. Pipet 10.0 mL of Internal volume.
standard solution into the flask, and dilute with water to Standard solution A: 5 g/mL of USP Mafenide Related
volume. soe A RS from Standard stock solution in Mobile
Chromatographic system jase
(See Chromatography (621), System Suitability.) Stanciaid solution B: 1 g/mL of USP Mafenide Related
Mode: GC eau A RS from Standard solution A in Mobile
Detector: Flame ionization jase
Column: 0.25-mm x 60-m fused-silica capillary col- Sample solution: Proceed as directed in the Assay.
umn coated with a 0.5-um layer of acid-deactivated Chromatographic system
phase G35 (See Chromatography (621), System Suitability.)
Temperatures Mode: LC
Injection: 250° Detector: UV 267 nm
Detector: 250° Column: 4.6-mm x 15-cm; 5-um packing L1
Column: See Table 7. Flow rate: 1 mL/min
Injection volume: 20 pL
Run time: 3 times the retention time of mafenide
Table 1 System suitability
Hold Time Samples: System suitability solution, Standard solution fos
Initial Temperature Final at Final A, and Standard solution B “
Temperature Ramp Temperature | Temperature Suitability requirements z
©) (¢/min) () (min) Resolution: NLT 3.0 between mafenide acetate and i
150 0 150 ala) mafenide related compound A, System suitability i}
solution |
150 25 240 10 )
240 25 150 1
Tailing factor: NMT 2.0, System suitability solution ro}=
Relative standard deviation: NMT 2.0%, Standard iy
Carrier gas: Helium solution A no}
Flow rate: 40 cm/s Analysis a
al
Injection volume: 1 uL Samples: Mobile phase, Standard solution A, and Sam-
System suitability le solution
Sample: Standard solution hromatograph the Standard solution and adjust the in-
Suitability requirements tegration parameters so that the response is 5%-15%
Resolution: NLT 3.0 between acetic acid and propi- of full-scale deflection. Disregard the peaks corre-
onic acid sponding to those obtained from the Mobile phase.
Relative standard deviation: NMT 6.0% for peak re- Calculate the percentage of each impurity in the por-
sponse ratios tion of the constituted Topical Solution taken:
Analysis
Samples: Standard solution and Sample solution Result = (ru/rs) x (Cs/Cu) x 100
Calculate the percentage of acetic acid in the portion of ru = peak response for each impurity from the
the constituted Topical Solution taken: Sample solution
Result = (Ru/Rs) x (Cs/Cu) x 100 rs = peak response of mafenide related compound
A from Standard solution A
Ru = peak response ratio of acetic acid to propionic Cs = concentration of USP Mafenide Related
acid from the Sample solution CompoundA RS in Standard solution A
Rs = peak response ratio of acetic acid to propionic (ug/ml) / : :
acid from the Standard solution Cu = nominal concentration of mafenide acetate in
Cs = concentration of acetic acid in the Standard the Sample solution (g/mL)
solution (mg/mL)
Cu = nominal concentration of mafenide acetate in
the Sample solution (mg/mL)
 2496 Mafenide / Official Monographs
Acceptance criteria
Individual impurity: NMT 0.5%
Total impurities: NMT 1.0%
SPECIFIC TESTS
e PH (791)
Sample solution: Nominally 100 mg/mL
Acceptance criteria: 6.4-6.8
© WATER DETERMINATION, Method | (921): NMT 1.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, at controlled room temperature. For prepared
solutions, use within 48 h of preparation.
e USP REFERENCE STANDARDS (11)
USP Mafenide Acetate RS
USP Mafenide Related Compound A RS
4-Formylbenzenesulfonamide.
C7H7NO3S_ 185.20
Magaldrate
Aluminum magnesium hydroxide sulfate
(AlsMg10(OH)s3:(SO4)2 - xH20).
Aluminum magnesium hydroxide sulfate, hydrate
[74978-16-8].
Anhydrous 1097.38
» Magaldrate is a chemical combination of alumi-
num and magnesium hydroxides and sulfate,
corresponding approximately to the formula:
AlsMg10(OH)31(SO4)2 i xH20.
“
od It contains the equivalent of not less than
rm 90.0 percent and not more than 105.0 percent of
£ AlsMgio(OH)s31(SO4)2, calculated on the dried
=)
3 basis.
2 rately weighed, in 3 mL of dilute hydrochloric acid (1 in 10),
S Packaging and storage—Preserve in well-closed contain-
Ps ers.
i for 5 minutes. Then add 20 mL of acetic acid-ammonium
va) USP Reference standards (11)—
=) USP Magaldrate RS
Identification—
A: Dissolve about 600 mg in 20 mL of 3 N hydrochloric
acid, add 3 drops of methyl red TS and about 30 mL of
water, and heat to boiling. Add 6 N ammonium hydroxide
until the color just changes to yellow, continue boiling for
2 minutes, and filter: the filtrate responds to the tests for
Magnesium (191).
B: Wash the precipitate obtained in Identification test A
with 50 mL of hot ammonium chloride solution (1 in 50),
then dissolve the precipitate in 15 mL of 3 N hydrochloric
acid: the solution responds to the tests for Aluminum (191).
C: Its X-ray diffraction pattern (see X-ray Diffraction (941))
in the d-spacings region below 0.257 nm (2.57 angstrom
units) conforms to that of USP Magaldrate RS.
Microbial enumeration tests (61) and Tests for speci-
fied microorganisms (62)—It meets the requirements of
the test for absence of Escherichia coli.
Loss on drying (731)—Dry it at 200° for 4 hours: it loses
between 10.0% and 20.0% of its weight.
Soluble chloride—Boil 1 g of it, accurately weighed, with
50.0 mL of water for 5 minutes, cool, add water to restore
the original volume, mix, and filter. To 25.0 mL of the fil-
trate add 0.1 mL of potassium chromate TS, and titrate with
USP 41
0.10Nsilver nitrate until a persistent pink color is obtained:
not more than 5.0 mL of 0.10Nsilver nitrate is required
(3.5%).
Soluble sulfate (221)—A 2.5-mL portion of the filtrate ob-
tained in the test for Soluble chloride shows no more sulfate
than corresponds to 1.0 mL of 0.020 N sulfuric acid (1.9%).
Sodium—transfer 2 g of it, accurately weighed, to a
100-mL volumetric flask, place in an ice bath, add 5 mL of
nitric acid, and swirl to dissolve. Allow to warm to room
temperature, dilute with water to volume, and mix. Filter, if
necessary, to obtain a clear solution. Dilute 10.0 mL of the
filtrate with water to 100.0 mL: the emission intensity of this
solution, determined with a suitable flame photometer at
589 nm and corrected for background transmission at 580
nm, is not greater than that produced by a standard con-
taining 2.2 ug of Na per mL, similarly measured (0.11%).
Arsenic, Method | (211): 8 ppm.
Delete the following:
Hea metals {231)—Dissolve 330 mg in 10 mL of 3N
hydrochloric acid, filter if necessary to obtain a clear solu-
tion, and dilute with water to 25 mL: the limit is 0.006%.
@ (Official 1-fan-2018)
Magnesium hydroxide content—Dissolve about 100 mg,
accurately weighed, in 3 mL of dilute hydrochloric acid (1 in
10), and dilute with water to about 200 mL. Add, with stir-
ring, 1 g of ammonium chloride, 20 mL of triethanolamine,
10 mL of ammonia-ammonium chloride buffer TS, and
0.1 mL of eriochrome black TS, and titrate with 0.05 M ede-
tate disodium VS to a blue color. Perform a blank determi-
nation, and make any necessary correction. Each mL of 0.05
M edetate disodium Is equivalent to 2.916 mg of Mg(Oh)z:
between 49.2% and 66.6% of Mg(OH), is found, calculated
on the dried basis.
Aluminum hydroxide content—
Edetate disodium titrant—Prepare and standardize as di-
rected in the Assay under Ammonium Alum.
Procedure—Dissolve about 100 mg of Magaldrate, accu-
and dilute with water to about 30 mL. Add, with stirring,
25.0 mL of Edetate disodium titrant, mix, and allow to stand
acetate buffer TS, 60 mL of alcohol, and 2 mL of dithizone
TS, and titrate with 0.05 M zinc sulfate to a bright rose-pink
color. Perform a blank determination, and make any neces-
sary correction. Each mL of 0.05 M Edetate disodium titrant
is equivalent to 3.900 mg of Al(OH): between 32.1% and
45.9% of Al(OH) is found, calculated on the dried basis.
Sulfate content—
Chromatographic column—transfer 15 mL of strongly
acidic 50- to 100-mesh styrene-divinylbenzene cation-ex-
change resin to a 1-cm inside diameter glass column. Wash
the resin with 30 mL of water.
Indicator solution—Prepare a solution in water containing
2 mg of sodium alizarinsulfonate per mL.
Magnesium acetate solution—Dissolve 26.8 g of magne-
sium acetate in 500 mL of water.
0.05 M Barium chloride—Dissolve 12.2 g of barium chlo-
ride in about 900 mL of water, adjust with 1 N hydrochloric
acid to a pH of 3.0, dilute with water to 1000 mL, and mix.
Standardize this solution as follows: Transfer 10.0 mL of 0.1
N sulfuric acid VS to a 125-mL conical flask. Adjust by add-
ing Magnesium acetate solution to a pH of 3.0. Add 25 mL
of methanol and 3 or 4 drops of Indicator solution. Add from
a buret an accurately measured volume of 8 to 9 mL of 0.05
M barium chloride. Add an additional 4 drops of Indicator
solution, and titrate slowly until the yellow color disappears
USP 41
anda pink tinge is visible. Calculate the molarity of the
barium chloride titrant taken by the formula:
5(N/V)
in which N is the normality of the sulfuric acid; and V is the
volume, in mL, of titrant consumed.
Test preparation—Transfer about 875 mg of Magaldrate,
accurately weighed, to a 25-mL volumetric flask. Dissolve in
10 mL of water and 5 mL of glacial acetic acid, dilute with
water to volume, and mix. Transfer 5.0 mL of this solution
to the chromatographic column and wash the column with
15 mL of water, collecting the eluate in a 125-mL conical
flask (Test preparation).
Procedure—Add to the Test preparation 5 mL of Magne-
sium acetate solution, 32 mL of methanol, and 3 or 4 drops
of Indicator solution. Add from a buret an accurately meas-
ured volume of 5.0 to 5.5 mL of 0.05 M barium chloride.
Add an additional 3 drops of Indicator solution, and titrate
slowly until the yellow color disappears and a pink tinge is
visible. Each mL of 0.05 M barium chloride is equivalent to
4.803 mg of sulfate (SO,): between 16.0% and 21.0% of
SOx, is found, calculated on the dried basis.
enc: psoas! about 3 g of Magaldrate, accurately
weighed, to a 250-mL beaker, add 100.0 mL of 1 N hydro-
chloric acid VS, and stir until the solution becomes clear.
Titrate the excess acid with 1 N sodium hydroxide VS to a
pH of 3.0, determined potentiometrically. Perform a blank
determination (see Residual Titrations under Titrimetry (541)).
Each mL of 1 N hydrochloric acid is equivalent to 35.40 mg
ofAlsMgio(OH)s1 (SOx)2.
Magaldrate Oral Suspension
» Magaldrate Oral Suspension contains not less
than 90.0 percent and not more than 110.0 per-
cent of the labeled amount of magaldrate
[AlsMg10(OH)31(SOa)2].
Packaging and storage—Preserve in tight containers.
USP Reference standards (11)—
USP Magaldrate RS
Identification—
A: Dissolve an amount of Oral Suspension, equivalent to
about 800 mg of magaldrate, in 20 mL of 3 N hydrochloric
acid, dilute with water to about 50 mL, add 3 drops of
methyl red TS, and proceed as directed in Identification test
A under Magaldrate, beginning with “and heat to boiling.”
B: It responds to /dentification test B under Magaldrate.
C: Transfer an amount of Oral Suspension, equivalent to
about 1 g of magaldrate, to a 100-mL centrifuge tube. Add
about 60 mL of water, cap, and shake for 3 minutes. Centri-
fuge the suspension, and discard the supernatant. Repeat
the washing of the residue with three 60-mL portions of
water. Transfer the residue to a 250-mL beaker, and heat on
a steam bath to dryness: the X-ray diffraction pattern (see
X-ray Diffraction (941)), in the d-spacings region below 2.57
angstrom units, of the residue so obtained conforms to that
of USP Magaldrate RS.
Microbial enumeration tests (61) and Tests for speci-
fied microorganisms (62)—Its total aerobic microbial
count does not exceed 100 cfu per mL, and it meets the
requirements of the test for absence of Escherichia coli.
Acid-neutralizing capacity (301)—The acid consumed by
the minimum single dose recommended in the labeling is
Official Monographs / Magaldrate 2497
not less than 5 mEq, and not less than the number of mEq
calculated by the formula:
0.8(0.0282M)
in which 0.0282 is the theoretical acid-neutralizing capacity,
in mEq per mg, of magaldrate, and M is the quantity, in
mg, of the labeled amount of magaldrate.
Magnesium hydroxide content—
Test preparation—Transfer an accurately measured quan-
tity of Oral Suspension, equivalent to about 1 g of magal-
drate, to a 100-mL volumetric flask, add 30 mL of dilute
hydrochloric acid (1 in 10), shake to dissolve, dilute with
water to volume, and mix.
Procedure—Transfer 10.0 mL of Test preparation to a
400-mL beaker, and proceed as directed in the test for Mag-
nesium hydroxide content under Magaldrate, beginning with
“and dilute with water to about 200 mL.” Not less than
492 mg and not more than 666 mg of magnesium hydrox-
ie IMg(OH)2] per g of the labeled amount of magaldrate is
ound.
Aluminum hydroxide content—
Edetate disodium titrant—Prepare and standardize as di-
rected in the Assay under Ammonium Alum.
Test preparation—Prepare as directed in the test for Mag-
nesium hydroxide content.
Procedure—Transfer 10.0 mL of Test preparation and
20 mL of water to a 250-mL beaker, and proceed as di-
rected for Procedure in the test for Aluminum hydroxide con-
tent under Magaldrate, beginning with “Add, with stirring,
25.0 mL of Edetate disodium titrant.” Not less than 321 mg
and not more than 459 mg of aluminum hydroxide
IAWOH)s} per g of the labeled amount of magaldrate is
ound.
Change to read: (=a
al
a]
Other requirements—Evaporate a volume of Oral Suspen-
E
sion, equivalent to about 5 g of magaldrate, on a steam °
bath to dryness: the residue so obtained meets the require- 3
ments of the tests for Arsenic®© (oficial 1-jan-2018) Under Magal- °
drate. Ko}
s
Cy
Assay—Transfer an accurately measured quantity of Oral me]
Suspension, equivalent to about 3 g of magaldrate, to a a
beaker. Add 100.0 mL of 1 N hydrochloric acid VS, and mix, a
using a magnetic stirrer to achieve dissolution. Titrate the
excess acid with 1 N sodium hydroxide VS to a pH of 3.0,
determined potentiometrically. Perform a blank determina-
tion (see Residual Titrations under Titrimetry (541)). Each mL
of 1. N hydrochloric acid is equivalent to 35.40 mg of
AlsMgio(OH)s1(SOa)2.
Magaldrate Tablets
» Magaldrate Tablets contain not less than
90.0 percent and not more than 110.0 percent of
the labeled amount of magaldrate
[AlsMg10(OH)31(SOx)a].
Packaging and storage—Preserve in well-closed contain-
ers.
Labeling—Label the Tablets to indicate whether they are to
be swallowed or to be chewed.
USP Reference standards (11)—
USP Magaldrate RS
Identification—Transfer a quantity of powdered Tablets,
equivalent to about 2 g of magaldrate, to a 100-mL centri-
 2498 Magaldrate / Official Monographs
fuge tube. Add about 60 mL of water, cap, and shake for
3 minutes. Centrifuge the suspension, and discard the super-
natant. Repeat the washing with three more 60-mL portions
of water. Transfer the residue to a 250-mL beaker, and heat
on a steam bath to dryness: the residue so obtained meets
the requirements of the /dentification tests under Magaldrate.
Microbial enumeration tests (61) and Tests for speci-
fied microorganisms (62)—Tablets meet the requirements
of the test for absence of Escherichia coli.
Disintegration (701): 2 minutes, for Tablets labeled to
be swallowed.
Uniformity of dosage units (905): meet the require-
ments for Weight Variation.
Acid-neutralizing capacity—Proceed as directed under
Acid-Neutralizing Capacity (301). The acid consumed by the
minimum single dose recommended in the labeling is not
less than 5 mEq, and not less than the number of mEq
calculated by the formula:
0.8(0.0282M)
in which 0.0282 is the theoretical acid-neutralizing capacity,
in mEq per mg, of magaldrate; and M is the quantity, in
mg, of the labeled amount of magaldrate.
Magnesium hydroxide content—
Test preparation—Weigh and finely powder not fewer
than 20 Tablets. Transfer an accurately weighed portion of
the powder, equivalent to about 1 g of magaldrate, to a
100-mL volumetric flask, add 30 mL of dilute hydrochloric
acid (1 in 10), shake for 15 minutes, dilute with water to
volume, and mix.
Procedure—transfer 10.0 mL of Test preparation to a
400-mL beaker, and proceed as directed in the test for Mag-
nesium hydroxide content under Magaldrate, beginning with
“and dilute with water to about 200 mL.” Not less than
“ 492 mg and not more than 666 mg of magnesium hydrox-
i ide [Mg(OH)2] per g of the labeled amount of magaldrate is
rs found.
FS Aluminum hydroxide content—
=)
) Edetate disodium titrant—Prepare and standardize as di-
ts rected in the Assay under Ammonium Alum.
Gj
= Test preparation—Prepare as directed in the test for Mag-
nesium hydroxide content.
a
al Procedure—Transfer 10.0 mL of Test preparation and
=} 20 mL of water to a 250-mL beaker, and proceed as di-
rected for Procedure in the test for Aluminum hydroxide con-
tent under Magaldrate, beginning with “Add, with stirring,
25.0 mL of Edetate disodium titrant.” Not less than 321 mg
and not more than 459 mg of aluminum hydroxide
[Al(OH)3] per g of the labeled amount of magaldrate is
found.
Assay—Weigh and finely powder not fewer than 20 Tablets.
Transfer an accurately weighed portion of the powder,
equivalent to about 6 g of magaldrate, to a 200-mL volu-
metric flask. Add 100.0 mL of 2 N hydrochloric acid VS, and
swirl by mechanical means for 30 minutes. Dilute with water
to volume, mix, and filter. Transfer 100.0 mL of the filtrate
to a beaker. Titrate the excess acid with 1 N sodium hydrox-
ide VS to a pH of 3.0, determined potentiometrically. Per-
forma blank determifation (see Residual Titrations under Ti-
trimetry (541)). Each mL of 2 N hydrochloric acid is
equivalent to 70.80 mg of AlsMgio(OH)31(SOa)2.
Magaldrate and Simethicone Oral
Suspension
» Magaldrate and Simethicone Oral Suspension
contains not less than 90.0 percent and not more
USP 41
than 110.0 percent of the labeled amount of
magaldrate [AlsMgio(OH)3:(SO4)2], and an
amount of polydimethylsiloxane [-(CH3)2SiO-],
that is not less than 85.0 percent and not more
than 115.0 percent of the labeled amount of
simethicone.
Packaging and storage—Preserve in tight containers, and
keep from freezing.
USP Reference standards (11)—
USP Magaldrate RS
USP Polydimethylsiloxane RS
Identification—
A: Dissolve an amount of Oral Suspension, equivalent to
about 800 mg of magaldrate, in 20 mL of 3 N hydrochloric
acid, dilute with water to about 50 mL, add 3 drops of
methyl red TS, and proceed as directed in Identification test
A under Magaldrate, beginning with “and heat to boiling.”
B: Wash the precipitate obtained in Identification test A
with hot ammonium chloride solution (1 in 50), and dis-
solve the precipitate in hydrochloric acid. Divide this solu-
tion into two portions: the dropwise addition of 6 N ammo-
nium hydroxide to one portion yields a gelatinous white
precipitate, which does not dissolve in an excess of 6 N am-
monium hydroxide. The dropwise addition of 1 N sodium
hydroxide to the other portion yields a gelatinous white pre-
cipitate, which dissolves in an excess of 1 N sodium hydrox-
ide, leaving some turbidity.
C: Transfer an amount of Oral Suspension, equivalent to
about 1 g of magaldrate, to a 100-mL centrifuge tube. Add
about 60 mL of water, insert the cap, and shake for 3 min-
utes. Coe the suspension, and discard the superna-
tant. Repeat the washing of the residue with three 60-mL
portions of water. Transfer the residue to a 250-mL beaker,
and heat on a steam bath to dryness: the X-ray diffraction
pattern (see X-ray Diffraction (941)), in the d-spacings region
elow 2.57 angstrom units, of the residue so obtained con-
forms to that of USP Magaldrate RS.
D: The IR absorption spectrum, in the 7- to 15-um re-
gion, determined in a 0.1-mm cell, of the Assay preparation
repared as directed in the Assay for polydimethylsiloxane ex-
Fiblts maxima only at the same wavelengths as that of the
Standard preparation prepared as directed in the Assay for
polydimethylsiloxane.
Microbial enumeration tests (61) and Tests for speci-
fied microorganisms (62)—Its total aerobic microbial
count does not exceed 100 cfu per mL, and it meets the
requirements of the test for absence of Escherichia coli.
Acid-neutralizing capacity (301)—The acid consumed by
the minimum single dose recommended in the labeling is
not less than 5 mEq, and not less than the number of mEq
calculated by the formula:
0.8(0.0282M)
in which 0.0282 is the theoretical acid-neutralizing capacity,
in mEq per mg, of magaldrate; and M is the quantity, in
mg, of the labeled amount of magaldrate.
Magnesium hydroxide content—
Test Blepatgtion.anstey an accurately measured quan-
tity of Oral Suspension, equivalent to about 1 g of magal-
drate, to a 100-mL volumetric flask, add 30 mL of dilute
hydrochloric acid (1 in 10), shake to dissolve, dilute with
water to volume, and mix.
Procedure—Transfer 10.0 mL of Test preparation to a
400-mL beaker, and proceed as directed in the test for Mag-
nesium hydroxide content under Magaldrate, beginning with
“and dilute with water to about 200 mL.” Not less than
492 mg and not more than 666 mg of magnesium hydrox-
ide [Mg(OH)2] per g of the labeled amount of magaldrate is
found.
USP 41
Aluminum hydroxide content—
Edetate disodium titrant—Prepare and standardize as di-
rected in the Assay under Ammonium Alum.
Test preparation—Prepare as directed in the test for Mag-
nesium hydroxide content.
Procedure—tTransfer 10.0 mL of Test preparation and
20 mL of water to a 250-mL beaker, and proceed as di-
rected for Procedure in the test for Aluminum hydroxide con-
tent under Magaldrate, beginning with “Add, with stirring,
25.0 mL of Edetate disodium titrant.” Not less than 321 mg
and not more than 459 mg of aluminum hydroxide
[Al(OH)3] per g of the labeled amount of magaldrate is
found.
Change to read:
Other requirements—Evaporate a volume of Oral Suspen-
sion, equivalent to about 5 g of magaldrate, on a steam
bath to dryness: the residue so obtained meets the require-
ments of the tests for Arsenic? comical 1-jan-2018) Under Magal-
drate.
Assay for magaldrate—Transfer an accurately measured
ae of Oral Suspension, equivalent to about 3 g of
magaldrate, to a beaker. Add 100.0 mL of 1 N hydrochloric
acid VS, and mix, using a magnetic stirrer to achieve disso-
lution. Titrate the excess acid with 1 N sodium hydroxide VS
to a pH of 3.0, determined potentiometrically. Perform a
blank determination (see Residual Titrations under Titrimetry
(541)). Each mL of 1 N hydrochloric acid is equivalent to
35.40 mg of magaldrate YalsMgro(OH)si(SO-)al.
Assay for polydimethylsiloxane—Transfer an accurately
measured quantity of Oral Suspension, equivalent to about
250 mg of simethicone, to a 200-mL centrifuge bottle. Add
an equal volume of hydrochloric acid, swirl to dissolve the
Oral Suspension, add 25.0 mL of hexanes, and immediately
close the bottle securely with a cap having an inert liner.
Shake the bottle for 30 minutes, and centrifuge the mixture
until a clear supernatant layer is obtained (Assay prepara-
tion). Prepare a Standard preparation of USP Polydimethyl-
siloxane RS in hexanes having a known concentration of
about 10 mg per mL. Concomitantly determine the ab-
sorbances of the Assay preparation and the Standard prepa-
ration in 0.1-mm cells at the wavelength of maximum ab-
sorbance at about 7.9 um and at the wavelengths of
minimum absorbance at about 7.5 um and 8.3 um, with a
suitable IR spectrophotometer, using hexanes as the blank.
Drawa linear baseline between the two minima, and deter-
mine the absorbances for the Standard preparation and the
Assay preparation with respect to the baseline, making any
necessary correction for the blank. Calculate the quantity, in
mg, of [-(CH3)2SiO-], in the portion of Oral Suspension
taken by the formula:
25C(Au | As)
in which C is the concentration, in mg per mL, of USP
Polydimethylsiloxane RS in the Standard preparation; and Ay
and As are the absorbances of the Assay preparation and the
Standard preparation, respectively.
Magaldrate and Simethicone Chewable
Tablets
Former Title: Magaldrate and Simethicone Tablets
» Magaldrate and Simethicone Chewable Tablets
contain not less than 90.0 percent and not more
than 110.0 percent of the labeled amount of
magaldrate [AlsMgio(OH)s31(SO4)2], and an
Official Monographs / Magaldrate 2499
amount of polydimethylsiloxane [—(CH3)2SiO-],
that is not less than 85.0 percent and not more
than 115.0 percent of the labeled amount of
simethicone.
Packaging and storage—Preserve in well-closed contain-
ers.
Labeling—Label the Chewable Tablets to indicate that they
are to be chewed before being swallowed.
USP Reference standards (11)—
USP Magaldrate RS
USP Polydimethylsiloxane RS
identification—
A: Transfer a quantity of powdered Chewable Tablets,
equivalent to about 2 g of magaldrate, to a 100-mL centri-
fuge tube. Add about 60 mL of water, cap, and shake for
3 minutes. Centrifuge the suspension, and discard the super-
natant. Repeat the washing with three more 60-mL portions
of water. Transfer the residue to a 250-mL beaker, and heat
on a steam bath to dryness: the residue so obtained meets
the requirements of the /dentification tests under Magaildrate.
B: The IR absorption spectrum, in the 7- to 11-m re-
gion, determined in a 0.5-mm cell, of the Assay preparation
prepared as directed in the Assay for polydimethylsiloxane,
exhibits maxima only at the same wavelengths as that of
the Standard preparation containing about 2 mg of USP
Polydimethylsiloxane RS per mL prepared as directed in the
Assay for polydimethylsiloxane.
Microbial enumeration tests (61) and Tests for speci-
fied microorganisms (62)—Chewable Tablets meet the
requirements of the test for absence of Escherichia coli.
Uniformity of dosage units (905): meet the require-
ments for Weight Variation with respect to magaldrate.
Acid-neutralizing capacity—Proceed as directed under
Acid-neutralizing Capacity (301). The acid consumed by the =
minimum single dose recommended in the labeling is not “
less than 5 mEq, and not less than the number of mEq uv
calculated by the formula:
=
°
0.8(0.0282 M) ]
°
in which 0.0282 is the theoretical acid-neutralizing capacity, Ke]
=
in mEq per mg, of magaldrate, and Mis the quantity, in i}
mo}
mg, of the labeled amount of magaldrate. a
a
Magnesium hydroxide content—
Test pore vege and finely powder not fewer
than 20 Chewable Tablets. Transfer an accurately weighed
portion of the powder, equivalent to about 1 g of magal-
drate, to a 100-mL volumetric flask, add 30 mL of dilute
hydrochloric acid (1 in 10), shake for 15 minutes, dilute
with water to volume, and mix.
Procedure—Transfer 10.0 mL of the Test preparation to a
400-mL beaker, and proceed as directed in the test for Mag-
nesium hydroxide content under Magaldrate, beginning with
“and dilute with water to about 200 mL.” Not less than
492 mg and not more than 666 mg of magnesium hydrox-
ide [Mg(OH)2] per g of the labeled amount of magaldrate is
found.
Aluminum hydroxide content—
Edetate disodium titrant—Prepare and standardize as di-
rected in the Assay under Ammonium Alum.
Test preparation—Prepare as directed in the test for Mag-
nesium hydroxide content.
Procedure—Transfer 10.0 mL of Test preparation and
20 mL of water to a 250-mL beaker, and proceed as di-
rected for Procedure in the test for Aluminum hydroxide con-
tent under Magaldrate, beginning with “Add, with stirring,
25.0 mL of Edetate disodium.” Not less than 321 mg and not
more than 459 mg of aluminum hydroxide [AI(OH)3] per g
of the labeled amount of magaldrate is found.
 2500 Magaldrate / Official Monographs
Assay for magaldrate—Weigh and finely powder not
fewer than 20 Chewable Tablets. Transfer an accurately
weighed portion of the powder, equivalent to about 6 g of
magaldrate, to a 200-mL volumetric flask. Add 100.0 mL of
2.N hydrochloric acid VS, and swirl by mechanical means
for 30 minutes. Dilute with water to volume, mix, and filter.
Transfer 100.0 mL of the filtrate to a beaker. Titrate the ex-
cess acid with 1 N sodium hydroxide VS to a pH of 3.0,
determined potentiometrically. Perform a blank determina-
tion (see Residual Titrations under Titrimetry (541)). Each mL
of 2 N hydrochloric acid is equivalent to 70.80 mg of
AlsMgio(OH)31(SOa)2-
Assay for polydimethylsiloxane—Weigh and finely pow-
der not fewer than 20 Chewable Tablets. Transfer an accu-
rately weighed portion of the powder, equivalent to about
20 mg of simethicone, to a 60-mL separator. Add 10.0 mL
of hexanes and 25 mL of 6 N hydrochloric acid, cap the
separator, and shake by mechanical means for not less than
2 hours. Allow to stand for about 10 minutes, and drain off
as much of the lower, aqueous layer as possible without
removing any of the unseparated interphase. Add 25 mL of
4N sodium hydroxide to the separator, cap it, and shake by
mechanical means for 1 hour. Transfer the mixture from the
separator to a 50-mL centrifuge tube, cap, and centrifuge to
obtain clear layers. Transfer not less than 5 mL of the clear
upper hexanes layer to a test tube containing about 0.5 g of
anhydrous sodium sulfate. Cap the tube, shake vigorously,
and allow to stand to obtain a clear supernatant (Assay
reparation). Prepare three Standard preparations in hexanes
aving known concentrations of about 1.6, 2.0, and 2.4 mg
of USP Polydimethylsiloxane RS per mL, respectively. Con-
comitantly determine the absorbances of the Assay prepara-
tion and the Standard preparations in a 0.5-mm cell at the
wavelength of maximum absorbance at about 1260 cm"
with an IR spectrophotometer, using hexanes as the blank.
[NoTE—Between each measurement, rinse the cell with hep-
tane, empty, and dry it.] Plot the absorbances for the Stan-
zo
“
dard preparations versus concentration, in mg per mL, of
a USP Fae a RS, and draw the straight line best
S fitting the three plotted points. From the graph so obtained,
7
aD determine the concentration, C, in mg per mL, of
iS} polydimethylsiloxane in the Assay preparation. Calculate the
¢ edetate disodium VS to a blue endpoint. Each mL of 0.05 M
iS quantity, in mg, of [-(CH3)2SiO-], in the portion of Chew-
= able Tablets taken by multiplying C by 10.
(a
a)
=)
Milk of Magnesia
Mg(Oh)2 58.32
Magnesium hydroxide.
Magnesium hydroxide [1309-42-8].
» Milk of Magnesia is a suspension of Magnesium
Hydroxide. Milk of Magnesia, Double-Strength
Milk of Magnesia, and Triple-Strength Milk of
Magnesia contain not less than 90.0 percent and
not more than 115.0 percent of the labeled
amount of Mg(Oh), the labeled amount being
80, 160, and 240 mg of Mg(OH)2 per mL, re-
spectively. It may contain not more than
0.05 percent of a volatile oil or a blend of volatile
oils, suitable for flavoring purposes.
Packaging and storage—Preserve in tight containers,
preferably at a temperature not exceeding 35°. Avoid freez-
ing.
Labeling—Double- or Triple-Strength Milk of Magnesia is
so labeled, or may be labeled as 2x or 3x Concentrated
Milk of Magnesia, respectively.
Identification—A solution of the equivalent of 1 g of regu-
lar-strength Milk of Magnesia in 2 mL of 3 N hydrochloric
USP 41
acid meets the requirements of the tests for Magnesium
(191).
Microbial enumeration tests (61) and Tests for speci-
fied microorganisms (62)—lts total aerobic microbial
count does not exceed 100 cfu per mL, and it meets the
requirements of the test for absence of Escherichia coli.
Acid-neutralizing capacity (301)—Not less than 5 mEq
of acid is consumed by the minimum single dose recom-
mended in the labeling, and not less than the number of
mEq calculated by the formula:
0.8(0.0343M)
in which 0.0343 is the theoretical acid-neutralizing capacity,
in mEq, of Mg(OH)2; and M is the quantity, in mg, of
Mg(OR), in the specimen tested, based on the labeled
quantity.
Soluble alkalies—Centrifuge about 50 mL of Milk of Mag-
nesia. Dilute 5.0 mL of the clear supernatant with 40 mL of
water. Add 1 drop of methyl red TS, and titrate the solution
with 0.10 N sulfuric acid to the production of a persistent
pink color: not more than 1.0 mL of the acid is required.
Where the specimen is Double- or Triple-Strength Milk of
Magnesia, not more than 2.0 or 3.0 mL of the acid is re-
quired, respectively.
Carbonate and acid-insoluble matter—To the equiva-
lent of 1 g of regular-strength Milk of Magnesia add 2 mL of
3.N hydrochloric acid: not more than a slight effervescence
occurs, and the solution is not more than slightly turbid.
Assay—Transfer an accurately measured quantity of Milk of
Magnesia, previously shaken in its original container, equiva-
lent to about 800 mg of magnesium hydroxide, to a
250-mL volumetric flask. Dissolve in 30 mL of 3 N hydro-
chloric acid, dilute with water to volume, and mix. Filter, if
necessary, and transfer 25.0 mL of the filtrate to a beaker
containing 75 mL of water, and mix. Adjust the reaction of
the solution with 1 N sodium hydroxide to a pH of 7 (using
pH indicator paper; see Indicator and Test Papers under Re-
agents, in the section Reagents, Indicators, and Solutions),
add 5 mL of ammonia-ammonium chloride buffer TS and
0.15 mL of eriochrome black TS, and titrate with 0.05 M
edetate disodium is equivalent to 2.916 mg of Mg(Oh)2.
Magnesia Tablets
DEFINITION
Magnesia Tablets contain NLT 93.0% and NMT 107.0% of
the labeled amount of magnesium hydroxide [Mg(OH))].
IDENTIFICATION
e A. IDENTIFICATION TESTS—GENERAL, Magnesium (191)
Sample solution: Crush several Tablets, and dissolve
1g of the powder in 20 mL of 3 N hydrochloric acid.
Acceptance criteria: Meet the requirements
ASSAY
e PROCEDURE
Sample solution: Finely powder NLT 20 Tablets. Trans-
fer a portion of the powder, equivalent to 250 mg of
magnesium hydroxide, to a 100-mL volumetric flask.
Dissolve in 10 mL of 3 N hydrochloric acid, and dilute
with water to volume. Filter, if necessary, and transfer
25.0 mL of the filtrate to a beaker containing 75 mL of
water.
Analysis: Adjust the reaction of the solution with 1N
sodium hydroxide to a pH of 7 (using pH indicator pa-
per; see Reagents, Indicators, and Solutions—Iindicator
and Test Papers), and add 5 mL of ammonia-am-
monium chloride buffer TS and 0.15 mL of eriochrome
black TS. Titrate with 0.05 M edetate disodium VS to a
USP 41
blue endpoint. Each mL of 0.05 M edetate disodium is
equivalent to 2.916 mg of Mg(Oh)2.
Acceptance criteria: 93.0%-107.0%
PERFORMANCE TESTS
e DISINTEGRATION (701)
Time: NMT 10 min, simulated gastric fluid TS being
substituted for water in the test
e UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
SPECIFIC TESTS
e ACID-NEUTRALIZING CAPACITY (301)
Analysis: NLT 5 mEq of acid is consumed by the mini-
mum single dose recommended in the labeling, and
NLT the number of mEq calculated by the formula:
Result = (Fu x M) x 0.8
Fu = theoretical acid-neutralizing capacity of
Mg(Oh).2, 0.0343 mEq
M = quantity of Mg(OH), in the sample tested,
based on the labeled quantity (mg)
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed
containers.
Magnesium Carbonate
Carbonic acid, magnesium salt, basic; or, Carbonic acid,
magnesium salt (1:1), hydrate;
Magnes carbonate, basic; or, Magnesium carbonate
(1:1) hydrate [23389-33-5].
Anhydrous 84.31
[546-93-0].
DEFINITION
Magnesium Carbonate is a basic hydrated magnesium car-
bonate or a normal hydrated magnesium carbonate. It
contains the equivalent of NLT 40.0% and NMT 43.5% of
magnesium oxide (MgO).
IDENTIFICATION
© A. IDENTIFICATION TESTS—GENERAL, Magnesium (191):
When treated with 3 N hydrochloric acid, it dissolves
with effervescence, and the resulting solution meets the
requirements.
ASSAY
¢ PROCEDURE
Sample: 1g
Analysis: Dissolve the Sample in 30.0 mL of 1 N sulfuric
acid VS, add methyl orange TS, and titrate the excess
acid with 1 N sodium hyeronid VS. Perform the blank
determination. Calculate the volume, Vs, of 1 N sulfuric
acid, in mL, consumed by the Sample:
Result = (Vs — Va) X Nwoow
Ve = volume of 1 N sodium hydroxide consumed
by the blank determination (mL)
Va = volume of 1 N sodium hydroxide consumed
by the Sample (mL)
Nwaow = exact normality of the sodium hydroxide
solution
Calculate the volume of 1 N sulfuric acid, Veo, in mL,
consumed by calcium, which is present in the portion
of Magnesium Carbonate taken for the Assay:
Result = (W x Lca)/(Fea x 100)
w = weight of Magnesium Carbonate taken (mg)
Official Monographs / Magnesium 2501
Lea = content of calcium as determined in the test
for Limit of Calcium (%)
Fea = weight of Ca that is equivalent to each mL of
1N sulfuric acid, 20.04 mg
Calculate the percentage of magnesium oxide (MgO) in
the portion of Magnesium Carbonate taken:
Result = (Vs — Vea) X Figo/W x 100
Vs = volume of 1 N sulfuric acid consumed by the
Sample, as calculated above (mL)
Vea = volume of 1 N sulfuric acid consumed by
calcium, as calculated above (mL)
Fugo = weight of MgO that is equivalent to each mL
of 1N sulfuric acid, 20.15 mg
Ww = weight of Magnesium Carbonate taken (mg)
Acceptance criteria: 40.0%-43.5% of MgO
IMPURITIES
e SOLUBLE SALTS
Sample: 2.0g
Analysis: Mix the Sample with 100 mL of a mixture of
equal volumes of n-propyl alcohol and water. Heat the
mixture to the boiling point with constant stirring, cool
to room temperature, dilute with water to 100 mL, and
filter. Evaporate 50 mL of the filtrate on a steam bath to
dryness, and dry at 105° for 1 h.
Acceptance criteria: The weight of the residue does
not exceed 10 mg (NMT 1.0%).
© ACID-INSOLUBLE SUBSTANCES
Sample: 5.0g
Analysis: Mix the Sample with 75 mL of water, add hy-
drochloric acid in small portions, with agitation, until
no more of the magnesium carbonate dissolves, and
boil for 5 min. If an insoluble residue remains, filter,
wash well with water until the last washing is free from
chloride, and ignite.
Acceptance criteria: The weight of the ignited residue
does not exceed 2.5 mg (NMT 0.05%).
(=
“
e ARSENIC, Method | (211) uv
Tees prepatacearts 750 mg in 25 mL of 3 N hydrochloric <<
aci °
Acceptance criteria: NMT 4 ppm =]
e Limit oF CALCIUM fo]
Ko}
[NoTe—A commercially available atomic absorption stan- a
2
dard solution for calcium may be used where prepara- mo}
tion of a calcium standard stock solution is described a
below. Concentrations of the Standard solutions and the 3)
Sample solution may be modified to fit the linear or
working range of the instrument.]
Dilute hydrochloric acid: Dilute 100 mL of hydrochlo-
ric acid with water to 1000 mL.
Lanthanum solution: To 58.65 g of lanthanum oxide
add 400 mL of water, and add, gradually with stirring,
250 mL of hydrochloric acid. Stir until dissolved, and
dilute with water to 1000 mL.
Standard solutions: Transfer 249.7 mg of calcium car-
bonate, previously dried at 300° for 3h and cooled in a
desiccator for 2 h, to a 100-mL volumetric flask. Dis-
solve in a minimum amount of hydrochloric acid, and
dilute with water to volume. Transfer 1.0, 5.0, 10.0,
and 15.0 mL of this stock solution to separate 1000-mL
volumetric flasks, each containing 20 mL of Lanthanum
solution and 40 mL of Dilute hydrochloric acid. Dilute
with water to volume. These Standard solutions contain
1.0, 5.0, 10.0, and 15.0 g/mL of calcium, respectively.
Blank solution: Transfer 4 mL of Lanthanum solution
and 10 mL of Dilute hydrochloric acid to a 200-mL volu-
metric flask, and dilute with water to volume.
Sample solution: Transfer 250 mg of Magnesium Car-
bonate to a beaker, add 30 mL of Dilute hydrochloric
acid, and stir until dissolved, heating if necessary. Trans-
fer the solution so obtained to a 200-mL volumetric
flask containing 4 mL of Lanthanum solution, and dilute
with water to volume.
 2502 Magnesium / Official Monographs USP 41

Instrumental conditions Acid that when constituted as directed in the labeling

(See Atomic Absorption Spectroscopy (852).) yields a solution that contains NLT 90.0% and NMT
Mode: Atomic absorption spectrophotometry 110.0% of the labeled amount of magnesium citrate
Lamp: Calcium hollow-cathode (Ci2HioMg3O14).
Flame: Nitrous oxide-acetylene
Analytical wavelength: Calcium emission line at IDENTIFICATION
422.7 nm © A. IDENTIFICATION TESTS—GENERAL, Magnesium (191)
Analysis Sample solution: Constitute as directed in the labeling.
Samples: Standard solutions, Blank solution, and Sample Acceptance criteria: Meets the requirements
solution e B. The retention time of the citrate peak of the Sample
Using the Blank solution as blank, determine the con- solution corresponds to that of Standard solution 1, as ob-
centration, C, in g/mL, of calcium in the Sample solu- tained in the test for Content of Anhydrous Citric Acid.
tion using the calibration graph.
Calculate the percentage of calcium in the portion of ASSAY
© PROCEDURE
Magnesium Carbonate taken:
Sample solution: Transfer a volume of constituted oral
Result
=(V/Wx C x F) x 100 solution, equivalent to 18.7 g of magnesium citrate
(Ci2HioMg30i4), to a 1000-mL volumetric flask. Add
volume of the Sample solution (mL) 200 mL of 1 N hydrochloric acid, swirl, and allow to
NOSS

wu

weight of Magnesium Carbonate taken (mg) stand for 10 min. Dilute with water to volume. Stir by
as defined above mechanical means for 30 min.
ou

conversion factor from jg/mL to mg/mL, Analysis: Transfer 10.0 mL of the Sample solution to a
0.001 250-mL beaker. Add 10 mL of ammonia-ammonium
Acceptance criteria: NMT 0.45% chloride buffer TS, 5 mL of triethanolamine, and 0.3 mL
of eriochrome black TS. Titrate with 0.05 M edetate
disodium VS until the last hint of violet disappears (blue
Delete the following: endpoint). Each mL of 0.05 M edetate disodium is
equivalent to 7.520 mg of magnesium citrate
°e HEAVY METALS, Method | (231) (Ci2HioMg3O14).
Test preparation: Dissolve 0.67 gin 10 mL of 3 N hy- Acceptance criteria: 90.0%-110.0%
drochloric acid in a suitable crucible, and evaporate the
solution on a steam bath to dryness. Ignite at 550 + 25° OTHER COMPONENTS
until all carbonaceous material is consumed. Dissolve e CONTENT OF ANHYDROUS CITRIC ACID
the residue in 15 mL of water and 5 mL of hydrochloric Mobile phase, Standard solution 1, Chromatographic
acid, and evaporate to dryness. Toward the end of the system, and System suitabi ty: Proceed as directed in
evaporation, stir frequently to disintegrate the residue Assay for Citric Acid/Citrate and Phosphate (345).
so that finally a dry powder is obtained. Dissolve the Sample solution: Transfer an appropriate volume of the
residue in 20 mL of water, and evaporate in the same constituted oral solution into a suitable volumetric flask,
=
as
manner as before to dryness. Redissolve the residue in and proceed as directed for the Sample solution in Assay
is 20 mL of water, filter, if necessary, and add to the fil- for Citric Acid/Citrate and Phosphate (345), Assay Prepa-
s ration for Citric Acid/Citrate Assay.
trate 2 mL of 1 N acetic acid and water to make 25 mL.
a
—
Analysis
° Acceptance criteria: NMT 30 ppMe (ficial 1-jan-2018)
Samples: Standard solution 1 and Sample solution
fs e IRON (241)
iS Test preparation: Boil 50 mg with 5 mL of 2 N nitric Proceed as directed for Assay for Citric Acid/Citrate and
= acid for 1 min. Cool, dilute with water to 45 mL, add Phosphate (345), Procedure.
= 2 mL of hydrochloric acid, and mix. Calculate the percentage of anhydrous citric acid
a) Acceptance criteria: NMT 200 ppm (CsHgO7) in relation to the labeled amount of magne-
=) sium citrate in the volume of constituted oral solution
SPECIFIC TESTS taken:
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): It meets the requirements of Result = (ru/rs) x (Cs/Cu) x (Ma/M,2) x 100
the test for absence of Escherichia coli.
tu = peak area of citrate from the Sample solution
ADDITIONAL REQUIREMENTS rs = peak area of citrate from Standard solution 1
e PACKAGING AND STORAGE: Preserve in well-closed Cs = concentration of citrate in Standard solution 1
containers. (mg/mL)
Cy = nominal concentration of magnesium citrate
in the Sample solution (mg/mL)
Mr = molecular weight of anhydrous citric acid,
192.12
Magnesium Carbonate and Citric Acid Mz = molecular weight of citrate (CsHsO7), 189.10
Acceptance criteria: 76.6%-107.8% of the labeled
for Oral Solution amount of magnesium citrate
DEFINITION
Magnesium Carbonate and Citric Acid for Oral Solution con-
tains a dry mixture of Magnesium Carbonate and Citric
USP 41
PERFORMANCE TESTS
e UNIFORMITY OF DOSAGE UNITS (905): Meets the
requirements
IMPURITIES
e CHLORIDE AND SULFATE, Chloride (221)
Sample solution: Constitute as directed in the labeling.
Acceptance criteria: A 2.0-mL portion of Sample solu-
tion shows no more chloride than corresponds to
0.30 mL of 0.020 N hydrochloric acid (NMT 0.01%).
© CHLORIDE AND SULFATE, Sulfate (221)
Sample solution: Constitute as directed in the labeling.
Acceptance criteria: A 2.0-mL portion of Sample solu-
tion shows no more sulfate than corresponds to
0.30 mL of 0.020 N sulfuric acid (NMT 0.015%).
e TARTARIC ACID
Sample solution: Constitute as directed in the labeling.
Analysis: To 10 mL of Sample solution in a test tube,
add 1 mL of glacial acetic acid and 3 mL of a solution
of potassium acetate (1 in 2). Shake the mixture vigor-
ously, then gently rub the inner wall of the test tube
with a glass rod for a few min, and allow to stand for 1
A,
Acceptance criteria: No white, crystalline precipitate
soluble in 6 N ammonium hydroxide is formed.
SPECIFIC TESTS
© [MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): It meets the requirements of
the test for absence of Escherichia coli and Salmonella
species.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers.
e LABELING: The label contains directions for constitution of
the powder and states the equivalent amount of magne-
sium citrate (C;2Hio0Mg3014) in a given volume of the oral
solution obtained after constitution.
Delete the following:
°e USP REFERENCE STANDARDS (11)
USP Citric Acid RS
@ (CN 1-May-2018)
Magnesium Carbonate, Citric Acid, and
Potassium Citrate for Oral Solution
DEFINITION
Magnesium Carbonate, Citric Acid, and Potassium Citrate
for Oral Solution contains a dry mixture of Magnesium
Carbonate, Citric Acid, and Potassium Citrate that when
constituted as directed in the labeling yields a solution
that contains NLT 90.0% and NMT 710.0% of the labeled
amount of magnesium citrate (Ci2HioMg3O14).
IDENTIFICATION
¢ A, IDENTIFICATION TESTS—GENERAL, Magnesium (191)
Sample solution: Constitute as directed in the labeling.
Acceptance criteria: Meets the requirements
¢ B. The retention time of the citrate peak of the Sample
solution corresponds to that of Standard solution 1, as ob-
tained in the test for Content of Anhydrous Citric Acid.
ASSAY
© PROCEDURE
Sample solution: Transfer a volume of constituted oral
solution, equivalent to 1.9 g of magnesium citrate to a
100-mL volumetric flask and dilute with water to
volume.
Analysis: Transfer 10.0 mL of the Sample solution to a
Official Monographs / Magnesium 2503
beaker. While stirring, add 10 mL of ammonia—am-
monium chloride buffer TS, 5 mL of triethanolamine,
and 0.3 mL of eriochrome black TS. Titrate with 0.05 M
edetate disodium VS until the last hint of violet disap-
pears (blue endpoint). Each mL of 0.05 M edetate diso-
dium is equivalent to 7.520 mg of magnesium citrate
(Ci2HioMg3On,).
Acceptance criteria: 90.0%-110.0%
OTHER COMPONENTS
© CONTENT OF ANHYDROUS CiTRIC ACID
Mobile phase, Chromatographic system, and System
suitability: Proceed as directed in Assay for Citric Acid/
Citrate and Phosphate (345).
Standard solution: 0.02 mg/mL of anhydrous citric
acid, from USP Citric Acid RS in a freshly prepared 1
mM sodium hydroxide
Sample solution: Transfer a volume of constituted oral
solution equivalent to 500 mg of magnesium citrate, to
a suitable volumetric flask, and dilute quantitatively,
and stepwise if necessary, with a freshly prepared 1 mM
sodium hydroxide to obtain a solution having a concen-
tration of 0.02 mg/mL of magnesium citrate, based on
the label claim. Pass throughafilter of 0.5-11m or finer
pore size, and use the filtrate.
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of anhydrous citric acid
(CsHgO7) in relation to the labeled amount of magne-
aud citrate in the volume of constituted oral solution
taken:
Result = (ru/rs) X (Cs/Cu) x 100
ty = peak area of citrate from the Sample solution
Is = peak area of citrate from the Standard solution
Cs = concentration of citrate in the Standard
solution (mg/mL) S
Cy = nominal concentration of magnesium citrate nn
in the Sample solution (mg/mL) S)
Acceptance criteria: 126.1%-154.4% of the labeled “=
amount of magnesium citrate °
ej
PERFORMANCE TESTS re}
e UNIFORMITY OF DOSAGE UNITS (905): Meets the @s
requirements i)
so}
IMPURITIES =
ww“
© CHLORIDE AND SULFATE, Chloride (221)
Sample solution: Constitute as directed in the labeling.
Acceptance criteria: A 2.0-mL portion of Sample solu-
tion shows no more chloride than corresponds to
0.30 mL of 0.020 N hydrochloric acid (NMT 0.01%).
e CHLORIDE AND SULFATE,Sulfate (221)
Sample solution: Constitute as directed in the labeling.
Acceptance criteria: A 2.0-mL portion of Sample solu-
tion shows no more sulfate than corresponds to
0.30 mL of 0.020 N sulfuric acid (NMT 0.015%).
© TARTARIC ACID
Sample solution: Constitute as directed in the labeling.
Analysis: To 10 mL of Sample solution in a test tube,
add 1 mL of glacial acetic acid and 3 mL of a solution
of potassium acetate (1 in 2). Shake the mixture vigor-
ously, then gently rub the inner wall of the test tube
with a glass rod for a few min, and allow to stand for 1
h.
Acceptance criteria: No white, crystalline precipitate
soluble in 6 N ammonium hydroxide is formed.
SPECIFIC TESTS
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
count does not exceed 1000 cfu/g, and the total com-
bined molds and yeasts count does not exceed 100 cfu/
g. It meets the requirements of the test for absence of
Escherichia coli.
 2504 Magnesium / Official Monographs USP 41

© PH (791) containing 6.0, 12.0, and 18.0 1g of sodium chloride per

Sample solution: Constitute as directed in the labeling. mL, respectively.
Acceptance criteria: 3.3-4.3 Assay preparation—Transfer an accurately measured vol-
ume of the stock solution remaining from the Assay for mag-
ADDITIONAL REQUIREMENTS
nesium carbonate, equivalent to about 180 mg of NaHCOs,
© PACKAGING AND STORAGE: Preserve in tight, single-dose to a 100-mL volumetric flask, dilute with water to volume,
containers. Store at controlled room temperature. and mix. Transfer 10.0 mL of the resulting solution to a
e LABELING: The label specifies the directions for the consti- 1000-mL volumetric flask, dilute with water to volume, and
tution of the powder and states the equivalent amount mix.
of magnesium citrate (Ci2zHioMg3O1,).
Procedure—Concomitantly determine the absorbances of
the Standard preparations and the Assay preparation at the
Delete the following: sodium emission line at about 589.0 nm, with a suitable
atomic absorption spectrophotometer (see Atomic Absorption
eo USP REFERENCE STANDARDS (11) Spectroscopy (852)) equipped with a sodium hollow-cathode
USP Citric Acid RS lamp and an air—acetylene flame, using water as the blank.
© (CN 1-May-2018) Plot the absorbances of the Standard preparations versus
concentration, in j4g of sodium chloride per mL, and draw
the straight line best fitting the three plotted points. From
the graph so obtained, determine the concentration, in ug
per mL, of sodium chloride equivalent in the Assay prepara-
tion. Calculate the quantity, in g, of NaHCO; in the portion
Magnesium Carbonate and Sodium of Magnesium Carbonate and Sodium Bicarbonate for Oral
Bicarbonate for Oral Suspension Suspension taken by the formula:

» Magnesium Carbonate and Sodium Bicarbo- (84.01 / 58.44)(5C/ V)

nate for Oral Suspension contains not less than in which 84.01 and 58.44 are the molecular weights of so-
90.0 percent and not more than 110.0 percent of dium bicarbonate and sodium chloride, respectively; C is the
the labeled amounts of MgCO3 and NaHCOs. It concentration, in ug per mL, of sodium chloride equivalent
may contain suitable flavors. in the Assay preparation; and V is the volume, in mL, of the
stock solution remaining from the Assay for magnesium car-
Packaging and storage—Preserve in tight containers. bonate taken.
Identification—
A: Place about 1g ina flask equipped with a stopper and
glass tubing, the tip of which is immersed in calcium hy-
droxide TS in a test tube. Add 5 mL of 3 N hydrochloric
val acid to the flask, and immediately insert the stopper: gas Magnesium Chloride
= evolves in the flask and a precipitate is formed in the test
a tube. MgCl - 6H20 203.30
6
- Magnesium chloride, hexahydrate [7791-18-6].
Dp B: The solution remaining in the flask responds to the
° tests for Magnesium (191) and for Sodium (191). Anhydrous 95.21
e [7786-30-3].
Co Minimum fill (755): |meets the requirements.
es Acid-neutralizing capacity (301)—Not less than 5 mEq DEFINITION
Qa of acid is consumed by the minimum single dose recom- Magnesium Chloride contains NLT 98.0% and NMT 101.0%
” mended in the labeling, and not less than the number of of MgCl - 6H20.
=) mEq calculated by the formula:
IDENTIFICATION
0.8(0.024M) + 0.8(0.01195) e A. IDENTIFICATION TESTS—GENERAL, Magnesium (191)
Sample solution: 50 mg/mL
in which 0.024 and 0.0119 are the theoretical acid-neutral- e B. IDENTIFICATION TESTS—GENERAL, Chloride (191)
izing capacities, in mEq, of MgCO3 and NaHCOs, respec- Sample solution: 50 mg/mL
tively; and M and S are the quantities, in mg, ofMg O3 [Note—Acidify the Sample solution with diluted nitric
and NaHCO; in the specimen tested, based on the labeled acid before adding 6 N ammonium hydroxide.]
quantities.
ASSAY
Assay for magnesium carbonate—Transfer an accurately ¢ PROCEDURE
weighed portion of Magnesium Carbonate and Sodium Bi- Sample: 450mg
carbonate for Oral Suspension, equivalent to about 4.2 g of Analysis: Dissolve the Sample in 25 mL of water, add
MgCO3, to a 500-mL volumetric flask, Add 200 mL of 1N 5 mL of ammonia~ammonium chloride buffer TS and
hydrochloric acid, and mix. When dissolved, dilute with 0.1 mL of eriochrome black TS, and titrate with 0.05 M
water to volume, and mix. Transfer 10.0 mL of this stock edetate disodium VS to a blue endpoint. Each mL of
solution to a suitable container, dilute with water to 0.05 M disodium edetate is equivalent to 10.17 mg of
100 mL, add 10 mL of ammonia—ammonium chloride buffer MgCl - 6H20.
TS, 5 mL of triethanolamine, and 0.3 mL of eriochrome Acceptance criteria: 98.0%-101.0%
black TS, and titrate with 0.05 M edetate disodium VS to a
blue endpoint. Each mL of 0.05 M edetate disodium con- IMPURITIES
sumed is equivalent to 4.216 mg of MgCOs. © INSOLUBLE MATTER
Assay for sodium bicarbonate— Sample: 20g
Standard preparations—Dissolve a suitable quantity of so- Analysis: Dissolve the Sample in 200 mL of water, heat
dium chloride, Previcsy dried at 125° for 30 minutes and to boiling, and digest in a covered beaker on a steam
accurately weighed, in water, and dilute quantitatively with bath for 1 h. Filter through a tared filtering crucible,
water to obtain a solution having a known concentration of wash thoroughly, dry at 115°, and determine the
about 600 ug per mL. On the day of use, further dilute this weight of the residue.
solution quantitatively with water to obtain three solutions Acceptance criteria: NMT 0.005%
USP 41 Official Monographs / Magnesium 2505

© CHLORIDE AND SULFATE, Sulfate (221) Delete the following:

Sample: 10g
Acceptance criteria: It shows no more sulfate than cor- ®e HEAVY METALS (231)
responds to 0.50 mL of 0.020N sulfuric acid (0.005%). Test preparation: Dissolve 2 g in water, and dilute with
e BARIUM water to 25 mL.
Sample: 1 Acceptance criteria; NMT 10 ppme coffciat 1-1an-2018)
Analysis: Dissolve the Sample in 10 mL of water, and
add 1 mL of 2 N sulfuric acid. SPECIFIC TESTS
Mpoepteriee criteria: No turbidity is produced within 2 @ PH (791)
Sample solution: 50 mg/mL in carbon dioxide-free
e Limit oF CALCIUM water
[NoTteE—A commercially available atomic absorption stan- Acceptance criteria: 4.5-7.0
dard solution for calcium may be used where prepara-
tion of a calcium standard stock solution is described ADDITIONAL REQUIREMENTS
below. Concentrations of the Standard solutions and the © PACKAGING AND STORAGE: Preserve in tight containers.
Sample solution may be modified to fit the linear or e LABELING: Where Magnesium Chloride is intended for use
working range of the instrument.] in hemodialysis, it is so labeled.
Dilute hydrochloric acid: Dilute 100 mL of hydrochlo-
ric acid with water to 1000 mL.
Lanthanum solution: To 58.65 g of lanthanum oxide
add 400 mL of water, and add, gradually with stirring,
250 mL of hydrochloric acid. Stir until dissolved, and Magnesium Citrate
dilute with water to 1000 mL.
Standard solutions: Transfer 249.7 mg of calcium car-
bonate, previously dried at 300° for 3 h and cooled in a
desiccator for 2 h, to a 100-mL volumetric flask. Dis-
solve in a minimum amount of hydrochloric acid, and
dilute with water to volume. Transfer 1.0, 5.0, 10.0,
and 15.0 mL of this stock solution to separate 1000-mL
volumetric flasks, each containing 20 mL of Lanthanum Cy2HioMg3Or4 -, 451.11
solution and 40 mL of Dilute hydrochloric acid. Dilute 1,2,3-Propanetricarboxylic acid, hydroxy-, magnesium salt
with water to volume. These Standard solutions contain
1.0, 5.0, 10.0, and 15.0 ug/mL of calcium, respectively. Magnesium citrate (3:2) [3344-18-1].
Blank solution: Transfer 4 mL of Lanthanum solution
and 10 mL of Dilute hydrochloric acid to a 200-mL volu- DEFINITION
metric flask, and dilute with water to volume. Magnesium Citrate contains NLT 14.5% and NMT 16.4% of
Sample solution: Transfer 10.0 9 of Magnesium Chlo- magnesium (Mg), calculated on the dried basis.
ride to a 200-mL volumetric flask, and add water to cS
dissolve. Add 4 mL of Lanthanum solution, and dilute IDENTIFICATION 4)

with water to volume. e A. IDENTIFICATION TESTS—GENERAL, Magnesium (191) z

Instrumental conditions Sample: 10 mg/mL =
(See Atomic Absorption Spectroscopy (852).) Acceptance criteria: Meets the requirements re)
e B. IDENTIFICATION TESTS—GENERAL, Citrate (191) S,
Mode: Atomic absorption spectrophotometry )
Lamp: Calcium hollow-cathode Sample: 80 mg/mL a=
Flame: Nitrous oxide-acetylene Acceptance criteria: Meets the requirements 2
Analytical wavelength: Calcium emission line at 3
422.7 nm ASSAY Ke
Analysis © PROCEDURE a

Samples: Standard solutions, Blank solution, and Sample Sample: 400mg

solution. Analysis: Dissolve the Sample in 50 mL of water. Add
Determine the concentration, C, in ug/mL, of calcium 20 mL of ammonia-ammonium chloride buffer TS and
in the Sample solution using the calibration graph. 0.1 mL of eriochrome black TS. Titrate with 0.05 M
Calculate the percentage of calcium in the portion of edetate disodium VS to a blue endpoint. Perform a
Magnesium Chloride taken: blank determination (see Titrimetry (541)), and make
any necessary correction. From the volume of 0.05 M
Result
=(V/Wx Cx F)
x 100 edetate disodium consumed, deduct the volume of
0.05 M edetate disodium corresponding to the amount
Vv = volume of the Sample solution (mL) of calcium in the portion of Magnesium Citrate taken,
Ww = weight of Magnesium Chloride taken (mg) based on the amount of calcium found in the test for
G = as defined above Limit of Calcium. Each mg of Ca is equivalent to
F = cormersion factor from ug/mL to mg/mL, 0.25 mL of 0.05 M edetate disodium. The difference is
0.0 the volume of 0.05 M edetate disodium consumed by
Acceptance criteria: NMT 0.01% the magnesium. Each mL of 0.05 M edetate disodium is
¢ POTASSIUM equivalent to 1.215 mg of Mg.
Sample solution: 5g Acceptance criteria: 14.5%-16.4% on the dried basis
Analysis: Dissolve the Sample in 5 mL of water, and add
0.2 mL of sodium bitartrate TS. IMPURITIES
Acceptance criteria: No turbidity is produced within 5 e CHLORIDE AND SULFATE, Chloride (221)
min. Sample: 300mg
e ALUMINUM (206) (where it is labeled as intended for use in Acceptance criteria: It shows no more chloride than
hemodialysis) corresponds to 0.20 mL of 0.020 N hydrochloric acid
Test preparation: Prepare as directed in the chapter, (0.05%).
using 2.0 g. e CHLORIDE AND SULFATE, Sulfate (221)
Acceptance criteria: NMT 1 ppm Sample: 100mg
Acceptance criteria: It shows no more sulfate than cor-
 2506 Magnesium / Official Monographs
responds to 0.20 mL of 0.020 N sulfuric acid (0.2%).
© ARSENIC, Method | (211): NMT 3 ppm
Delete the following:
°e HEAVY METALS, Method | (231)
Test ht aoa Dissolve 0.4 g in 25 mL of water, and
proceed as directed in the chapter, except use glacial
acetic acid to adjust ope
Acceptance criteria: NMT 50 ppme official 1-Jan-2018)
e IRON (241)
Test preparation: Boil 50 mg with 5 mL of 2N nitric
acid for 1 min. Cool, dilute with water to 45 mL, and
add 2 mL of hydrochloric acid.
Acceptance criteria: NMT 200 ppm
e Limit OF CALCIUM
[NoTE—A commercially available atomic absorption stan-
dard solution for calcium may be used where prepara-
tion of a calcium standard stock solution is described
below. Concentrations of the Standard solutions and the
Sample solutionrma be modified to fit the linear or
working range of the instrument.]
Dilute hydrochloric acid: Dilute 100 mL of hydrochlo-
ric acid with water to 1000 mL.
Lanthanum solution: To 58.65g of lanthanum oxide
add 400 mL of water, and add,gradually with stirring,
250 mL of hydrochloric acid. Stir until dissolved, and
dilute with water to 1000 mL.
Standard solutions: Transfer 249.7 mg of calcium car-
bonate, previously dried at 300° for 3h and cooled in a
desiccator for 2 h, to a 100-mL volumetric flask. Dis-
solve in a minimum amount of hydrochloric acid, and
dilute with water to volume. Transfer 1.0, 5.0, 10.0,
and 15.0 mL of this stock solution to separate 1000-mL
volumetric flasks, each containing 20 mL of Lanthanum
solution and 40 mL of Dilute hydrochloric acid. Dilute
with water to volume. These Standard solutions contain
te
“
1.0, 5.0, 10.0, and 15.0 g/mL of calcium, respectively.
3
cS Sample solution: Transfer 250 mg of Magnesium Cit-
—~
rate to a beaker, add 30 mL of Dilute hydrochloric acid,
Dp
3 and stir until dissolved. Transfer the solution to a
iJ 200-mL volumetric flask containing 4 mL of Lanthanum
Sj solution, and dilute with water to volume.
= Blank solution: Transfer 4 mL of Lanthanum solution
a and 10 mL of Dilute hydrochloric acid to a 200-mL volu-
a) metric flask, and dilute with water to volume.
> Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic penne spectrophotometry
Analytical wavelength: Calcium emission line at
422.7 nm
Lamp: Calcium hollow-cathode
Flame: Nitrous oxide-acetylene
Analysis
Samples: Standard solutions, Sample solution, and Blank
solution
Determine the concentration, C, in ug/mL, of calcium
in the Sample solution using the calibration graph.
Calculate the percentage of calcium in the portion of
Magnesium Citrate taken:
Result
=(V/Wx C xF) x 100
= volume of the Sample solution (mL)
a=~<
= weight of Magnesium Citrate taken (mg)
= concentration of calcium in the Sample
solution (4ug/mL)
F = conversion from g/mL to mgimt. 0.001
Acceptance criteria: NMT 1.0% on the dried basis
SPECIFIC TESTS
e PH (791): 5.0-9.0, in a suspension (50 mg/mL)
e Loss ON DRYING (731) Dry 1g in a mechanical convec-
tion oven at 135° for 16 h, then to constant weight: it
USP 41
loses NMT 29% of its weight, except that where it is
labeled as anhydrous, it loses NMT 2.0% of its weight.
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers.
e LABELING: Magnesium Citrate that loses NMT 2.0% of its
weight in the test for Loss on Drying may be labeled as
Anhydrous Magnesium Citrate.
Magnesium Citrate Oral Solution
DEFINITION
Magnesium Citrate Oral Solution is a sterilized or pasteur-
ized solution containing NLT 7.59 g of anhydrous citric
acid (CsHgQ7) and an amount of magnesium citrate equiv-
alent to NLT 1.55 g and NMT 1.9 g of magnesium oxide
(MgO) in each 100 mL of Oral Solution.
Prepare Magnesium Citrate Oral Solution as follows.
m_Carbonate
mb
ate
Pi icien L
Dissolve the Anhydrous Citric Acid in 150 mL of hot Purified
Water in a suitable dish, slowly add the Magnesium Car-
bonate previously mixed with 100 mL of Purified Water,
and stir until it is dissolved. Add the Syrup, heat the
mixed liquids to the boiling point, and immediately add
the Lemon Oil previously triturated with Talc. Filter the
mixture, while hot, into a strong bottle of suitable capac-
ity previously rinsed with boiling Purified Water. Add
boiled Purified Water to bring the preparation to final vol-
ume. Use Purified Cotton as a stopper for the bottle, and
allow to cool. Add the Potassium Bicarbonate, and imme-
diately insert the stopper in the bottle securely. Shake the
solution occasionally until the Potassium Bicarbonate is dis-
solved, cap the bottle, and sterilize or pasteurize the solu-
tion.
Alternatively, 30 g of citric acid containing 1 molecule of
water of hydration, equivalent to 2749 of Anhydrous Cit-
ric Acid, may be used in the foregoing formula. In this
process, replace the 2.5 g of Potassium Bicarbonate with
2.1 g of sodium bicarbonate, preferably in tablet form.
The Oral Solution may be further carbonated by the use
of carbon dioxide under pressure.
IDENTIFICATION
¢ A. IDENTIFICATION TESTS—GENERAL, Magnesium (191):
Meets the requirements
° B.
Sample: 5 mL
Analysis: To the Sample add 1 mL of potassium per-
manganate TS and 5 mL of mercuric sulfate TS, and
heat the solution.
Acceptance criteria: A white precipitate is formed.
ASSAY
e Citric AciD
Mobile phase, Standard preparation 1, and Chromat-
ographic system: Proceed as directed in Assay for Cit-
ric Acid/Citrate and Phosphate (345).
Assay preparation: Transfer 10 mL of Oral Solution that
has been previously freed from excessive carbon dioxide
by repeated pouring to a suitable volumetric flask, and
proceed as directed in Assay for Citric Acid/Citrate and
Phosphate (345), Assay Preparation for Citric Acid/Citrate
Assay.
USP 41
Analysis: Proceed as directed in Assay for Citric Acid/
Citrate and Phosphate (345), Procedure.
Calculate the quantity, g, of anhydrous citric acid
(CséHgO7) in 100 mL of Oral Solution:
Result = (ru/rs) x Cs x VX D x (Ma/M,2) x F
ru = peak area of citrate from the Assay preparation
rs = peak area of citrate from Standard peparation
1
Cs = concentration of citrate in Standard
preparation 1 (ug/mL)
Vv = final volume of Oral Solution, 100 mL
D = dilution factor
M, = molecular weight of citric acid (CsHsO7),
192.12
M2 = molecular weight of citrate (CsHsO7), 189.10
F = conversion factor, 10-* g/ug
Acceptance criteria: NLT 7.59 g in 100 mL of Oral
Solution
© MAGNESIUM OXIDE
Sample: 50.0 mL of Oral Solution that has been previ-
ously freed from excessive carbon dioxide by repeated
pouring
Analysis: Transfer the Sample to a 100-mL volumetric
flask, and dilute with water to volume. Transfer 5.0 mL
of the resulting solution to a beaker containing 150 mL
of water heated to 70°-80°, and add 1 mL of ammo-
nium chloride TS and 3 mL of ammonium hydroxide.
Mix, and slowly add 8 mL of Blvatoe unos US
with stirring. After standing for 30 min, filter through a
sintered-glass crucible, previously dried and weighed,
and wash the precipitate with ten 10-mL portions of
water. Dry the crucible and contents at 105° for 3 h,
cool, and weigh. Determine the equivalent of magne-
sium oxide (MgO) in 100 mL of Oral Solution by multi-
plying the weight of CisHizMgNzO2 - 2H20 so obtained
y 4.624.
Acceptance criteria: 1.55-1.9 g in 100 mL of Oral
Solution
IMPURITIES
e CHLORIDE AND SULFATE, Chloride (221)
Sample: 2.0 mL
Acceptance criteria: 0.01%; it shows no more chloride
a corresponds to 0.30 mL of 0.020 N hydrochloric
acid.
© CHLORIDE AND SULFATE, Sulfate (221)
Sample: 2.0 mL
Acceptance criteria: 0.015%; it shows no more sulfate
than corresponds to 0.30 mL of 0.020 N sulfuric acid.
e TARTARIC ACID
Sample: 10 mL
Analysis: Place the Sample in a test tube, add 1 mL of
glacial acetic acid and 3 mL ofa solution of potassium
acetate (1 in 2), shake the mixture vigorously, then
gently rub the inner wall of the test tube with a glass
rod for a few min, and allow to stand for 1 h.
Acceptance criteria: No white, crystalline precipitate
soluble in 6 N ammonium hydroxide is formed.
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Package in bottles NLT 200 mL
in capacity. Store at controlled room temperature or in a
cool place.
Delete the following:
®o USP REFERENCE STANDARDS (11)
USP Citric Acid RS
© (CN 1-May-2018)
Official Monographs / Magnesium 2507
Magnesium Citrate for Oral Solution
DEFINITION
Magnesium Citrate for Oral Solution, when constituted as
directed in the labeling, yields a solution that contains
NLT 90.0% and NMT 110.0% of the labeled amount of
magnesium citrate (C12HioMg3O14).
IDENTIFICATION
© A, IDENTIFICATION TESTS—GENERAL, Magnesium (191)
Sample solution: Constitute as directed in the labeling.
Acceptance criteria: Meets the requirements
e B. The retention time of the citrate peak of the Sample
solution corresponds to that of Standard solution 1, as ob-
tained in the test for Content of Anhydrous Citric Acid.
ASSAY
¢ PROCEDURE
Sample solution: Transfer a volume of constituted oral
solution, equivalent to 18.7 g of magnesium citrate
(Ci2HioMg3O14), to a 1000-mL volumetric flask. Add
200 mL of 1 N hydrochloric acid, swirl, and allow to
stand for 10 min. Dilute with water to volume. Stir by
mechanical means for about 30 min.
Analysis: Transfer 10.0 mL of the Sample solution to a
250-mL beaker. Add 10 mL of ammonia~-ammonium
chloride buffer TS, 5 mL of triethanolamine, and 0.3 mL
of eriochrome black TS. Titrate with 0.05 M edetate
disodium VS until the last hint of violet disappears (blue
endpoint). Each mL of 0.05 M edetate disodium is
equivalent to 7.520 mg of magnesium citrate
(Ci2HioMg30i,).
Acceptance criteria: 90.0%-110.0%
OTHER COMPONENTS
(a
Change to read: vw
a]
© CONTENT OF ANHYDROUS CITRIC ACID i
Mobile phase, Standard solution 1, Chromatographic °
system, and System suitability: Proceed as directed in =)
Assay for Citric Acid/Citrate and Phosphate (345). °
©
Sample solution: Transfer an appropriate volume of the =
)
constituted oral solution into a suitable volumetric flask, me]
and proceed as directed for the Sample solution in >
°Assay for Citric Acid/Citrate and Phosphate (345), Sam- a
ple solution (for the assay of citric acid/citrate)e cen i-xay.
2018)-
Analysis
Samples: Standard solution 1 and Sample solution
Proceed as directed for Assay for Citric Acid/Citrate and
prosorate (345), Procedure.
Calculate the percentage of anhydrous citric acid
(CeHsO7) in relation to the labeled amount of magne-
sium citrate in the volume of constituted oral solution
taken:
Result = (ru/rs) x (Cs/Cu) x (Mu/Mi2z) x 100
tu = peak area of citrate from the Sample solution
ls = peak area of citrate from Standard solution 7
Cs = concentration of citrate in Standard solution 1
(mg/mL)
Cu = nominal concentration of magnesium citrate
in the Sample solution (mg/mL)
My = molecular weight of anhydrous citric acid,
192.12
Mz = molecular weight of citrate (CsHsO;), 189.10
Acceptance criteria: 76.6%-93.7% of the labeled
amount of magnesium citrate
 2508 Magnesium / Official Monographs
PERFORMANCE TESTS
e UNIFORMITY OF DOSAGE UNITS (905): Meets the
requirements
IMPURITIES
e CHLORIDE AND SULFATE, Chloride (221)
Sample solution: Constitute as directed in the labeling.
Acceptance criteria: A 2.0-mL portion of Sample solu-
tion shows no more chloride than corresponds to
0.30 mL of 0.020 N hydrochloric acid (NMT 0.01%).
e CHLORIDE AND SULFATE, Sulfate (221)
Sample solution: Constitute as directed in the labeling.
Acceptance criteria: A 2.0-mL portion of Sample solu-
tion shows no more sulfate than corresponds to
0.30 mL of 0.020 N sulfuric acid (NMT 0.015%).
¢ TARTARIC ACID
Sample solution: Constitute as directed in the labeling.
Analysis: To 10 mL of Sample solution in a test tube,
add 1 mL of glacial acetic acid and 3 mL of a solution
of potassium acetate (1 in 2). Shake the mixture vigor-
ously, then gently rub the inner wall of the test tube
pan a glass rod for a few min, and allow to stand for 1
Acceptance criteria: No white, crystalline precipitate
soluble in 6 N ammonium hydroxide is formed.
SPECIFIC TESTS
¢ MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): It meets the requirements of
the test for absence of Escherichia coli and Salmonella
species.
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers.
e LABELING: The label contains directions for constitution of
the powder and states the equivalent amount of magne-
sium citrate (Ci2Hio0Mg3014) in a given volume of the oral
solution obtained after constitution.
ke
”
is
i] Delete the following:
—
D (Ci2H22MgOv4) in the Sample taken:
° ®e USP REFERENCE STANDARDS (11)
= Result = {[(Vs — Vs) x M x F ]/W} x 100]
5 USP Citric Acid RS
3 @ (CN 1-May-2018)
[5
a]
=)
Magnesium Gluconate
QH OH 9
2
Ci2H22MgOr4 414.60
450.64
Ci2H2MgOnr4 - 2H20
D-Gluconic acid, magnesium salt (2:1), hydrate;
Magnesium D-gluconate (1:2) hydrate;
Magnesium D-gluconate (1:2) dihydrate [59625-89-7].
Anhydrous [3632-91-5].
DEFINITION
Magnesium Gluconate contains NLT 98.0% and NMT
102.0% of anhydrous magnesium gluconate
(Ci2H22MgOu4), calculated on the anhydrous basis.
IDENTIFICATION
e A. IDENTIFICATION TESTS—GENERAL, Magnesium (191): A
100-mg/mL solution meets the requirements.
e B. THIN-LAYER CHROMATOGRAPHY
Standard solution: 10 mg/mL of USP Potassium Gluco-
nate RS
USP 41
Sample solution: 10 mg/mL of Magnesium Gluconate,
heating in a water bath at 60°, if necessary, to dissolve
Chromatographic system
(See promatograp y (621), Thin-Layer Chromato-
graphy.
Mode: TLC
ae 0.25-mm layer of chromatographic silica
ge
Application volume: 5 uL
Developing solvent system: Alcohol, ethyl acetate,
ammonium hydroxide, and water (50:10:10:30)
Spray reagent: Dissolve 2.5 g of ammonium molyb-
date in 50 mL of 2N sulfuric acid in a 100-mL volu-
metric flask, add 1.0 g of ceric sulfate, swirl to dissolve,
and dilute with 2 N sulfuric acid to volume.
Analysis
Samples: Standard solution and Sample solution
Develop the chromatogram until the solvent front has
moved about three-fourths of the length of the plate.
Remove the plate from the chamber, and dry at 110°
for 20 min. Allow to cool, and spray with the Spray
reagent. Heat the plate at 110° for about 10 min.
Acceptance criteria: The pea spot of the Sample
solution corresponds in color, size, and R; value to that
of the Standard solution.
ASSAY
e PROCEDURE
Sample: 800 mg of Magnesium Gluconate
Blank: 20 mL of water
Titrimetric system
(See Titrimetry (541).)
Mode: Direct titration
Titrant: 0.05 M edetate disodium VS
Endpoint detection: Visual
Analysis: Dissolve the Sample in 20 mL of water. Add
5 mL of ammonia—ammonium chloride buffer TS and
0.1 mL of eriochrome black TS. Titrate with Titrant to a
blue endpoint. Perform the Blank determination.
Calculate the percentage of magnesium gluconate
Vs = Titrant volume consumed by the Sample (mL)
Ve = Titrant volume consumed by the Blank (mL)
M Titrant molarity (mM/mL)
Wout
F
equivalency factor, 414.6 mg/mM
Ww = Sample weight (ng)
Acceptance criteria: 98.0%-102.0% on the anhydrous
basis
IMPURITIES
e CHLORIDE AND SULFATE, Chloride (221)
rae solution: 0.7 mL of 0.020 N hydrochloric
aci
Sample: 1.0g
Acceptance criteria: NMT 0.05%
e CHLORIDE AND SULFATE, Sulfate (221)
Standard solution: 1.0 mL of 0.020N sulfuric acid
Sample: 2.0g
Acceptance criteria: NMT 0.05%
Delete the following:
°e HEAVY METALS (231)
Test preparation: 1.09 in 10 mL of water. Add 6 mL of
3.N hydrochloric acid, and dilute with water to 25 mL.
USP 41 Official Monographs / Magnesium 2509

Acceptance criteria: NMT 20 ppme ‘orriciat 1ja0-2018) IDENTIFICATION

e REDUCING SUBSTANCES e A. IDENTIFICATION TESTS—GENERAL, Magnesium (191)
lt 1.0 g of Magnesium Gluconate Sample solution: Afiltered solution in water from pow-
Blank: 10 mL of water dered Tablets, equivalent to 100 mg/mL of magnesium
Titrimetric system gluconate
(See Titrimetry (541).) Acceptance criteria: Meet the requirements
Mode: Residual titration e B. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Titrant: 0.1 N iodine VS Standard solution: 10 mg/mL of USP Potassium Gluco-
Back titrant: 0.1 N sodium thiosulfate VS nate RS
Endpoint detection: Visual Sample solution: Equivalent to 10 mg/mL of magne-
Analysis: Transfer the Sample to a 250-mL conical flask, sium gluconate from a dilution of the Sample solution
dissolve in 10 mL of water, and add 25 mL of alkaline obtained for the Identification test A
cupric citrate TS. Cover the flask, boil gently for 5 min, Chromatographic system
accurately timed, and cool rapidly to room tempera- (See Chromatography (621), Thin-Layer Chromato-
ture. Add 25 mL of 0.6 N acetic acid, 10.0 mL of Ti- graphy.)
trant, and 10 mL of 3 N hydrochloric acid, and titrate Mode: TLC
with Back titrant, adding 3 mL of starch TS as the Adsorbent: 0.25-mm layer of chromatographic silica
endpoint is approached. Perform the Blank determina- gel
tion. Application volume: 5 wl
Calculate the percentage of reducing substances (as dex- Developing solvent system: Alcohol, ethyl acetate,
trose) in the Sample taken: ammonium hydroxide, and water (50:10:10:30)
Spray reagent: Dissolve 2.5 g of ammonium molyb-
Result = {[(Vs — Vs) x N x FJ/W} x 100 date in 50 mL of 2Nsulfuric acid in a 100-mL volu-
metric flask, add 1.0g of ceric sulfate, swirl to dissolve,
Vp = Back titrant volume consumed by the Blank and dilute with 2 N sulfuric acid to volume.
(mL) Analysis
Vs = Back titrant volume consumed by the Sample Samples: Standard solution and Sample solution
(ml) Develop the chromatogram until the solvent front has
N = Back titrant normality (mEq/mL) moved about three-fourths of the length of the plate.
F = equivalency factor, 27 mg/mEq Remove the plate from the chamber, and dry at 110°
w = Sample weight (mg) for 20 min. Allow to cool, and spray with Spray rea-
Acceptance criteria: NMT 1.0% gent. Heat the plate at 110° for about 10 min.
Acceptance criteria: The principal spot of the Sample
SPECIFIC TESTS solution corresponds in color, size, and R; value to that
© PH (791) of the Standard solution.
Sample solution: 50 mg/mL
Acceptance criteria: 6.0-7.8 ASSAY
e WATER DETERMINATION, Method Ib (921) © PROCEDURE (ox
Test preparation: Proceed as directed in the chapter. Sample: A portion of the powder from NLT 20 finely 4)
Allow 30 min for solubilization of Magnesium Gluco- powdered Tablets, equivalent to 800 mg of magnesium )
nate and for the reaction to reach completion. luconate =
Blank: Use the same volume of Reagent as that of the Blank: Proceed as directed in the Analysis without the i}
Test preparation but without the specimen. Sample. =
Analysis fo)
samp Test preparation and Blank
Titrimetric system a!
(See Titrimetry (541).) S
Calculate the water content, in mg, of the Magnesium Mode: Direct titration 3
Gluconate taken: Titrant: 0.05 M edetate disodium VS Bi
“
Endpoint detection: Visual
Result = F x (Xs— X) xR Analysis: Transfer the Sample to a crucible, and ignite,
gently at first, until free from carbon. Cool the crucible,
Xe = volume of standardized Water—Methanol add 25 mL of water and 5 mL of hydrochloric acid, and
Solution required to neutralize the stir. Heat on a steam bath for 5 min. Filter, rinsing the
unconsumed Reagent in the Blank filter with several portions of water. Dilute the com-
determination (mL) bined filtrate and washings with water to 150 mL. Add
The other terms are as defined in Water Determination, ammonia—ammonium chloride buffer TS until the solu-
Method Ib (921). tion is neutral to litmus. Add an excess of 5 mL of am-
Acceptance criteria: 3.0%-12.0% monia—ammonium chloride buffer TS and 0.1 mL of eri-
ADDITIONAL REQUIREMENTS ochrome black TS, and titrate with Titrant to a blue
© PACKAGING AND STORAGE: Preserve in well-closed endpoint. Perform a Blank determination.
containers. Calculate the percentage of the labeled amount of
e USP REFERENCE STANDARDS (11) magnesium gluconate (Ci2H22MgQ;,) in the portion of
USP Potassium Gluconate RS Tablets taken:
Result = {[(Vs — Vs) x M x FI/W} x 100
Vs = Titrant volume consumed by the Sample (mL)
Ve = Titrant volume consumed by the Blank (mL)
Magnesium Gluconate Tablets M = actual molarity of the Titrant (mM/mL)
F = equivalency factor, 414.6 mg/mM
DEFINITION Ww = nominal amount of magnesium gluconate in
Magnesium Gluconate Tablets contain NLT 95.0% and NMT the Sample taken (mg)
105.0% of the labeled amount of magnesium gluconate
(Ci2H22MgOu4).
 2510 Magnesium / Official Monographs
Acceptance criteria: 95.0%-105.0%
PERFORMANCE TESTS
e DISSOLUTION (711)
Medium: Water; 900 mL
Apparatus 2: 50 rpm
Time: 30 min
Standard solution: Solution having a known concentra-
tion of magnesium in the Medium
Sample solution: Filtered portion of the solution under
test, suitably diluted with the Medium if necessary
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic a sorpuen spectrophotometry
Analytical wavelength: 285.2 nm
Lamp: Magnesium hollow-cathode
Flame: Air—acetylene
Analysis
Samples: Standard solution and Sample solution
Determine the concentration of magnesium (Mg) in the
Sample solution in comparison with a Standard solution.
Calculate the percentage of the labeled amount of
magnesium gluconate (C;2H22MgOx4) dissolved:
Result = (C x D x V/L) x (M/A) x 100
iG = determined concentration of magnesium in
the Sample solution (mg/mL)
D = dilution factor for the Sample solution
Vv = volume of Medium, 900 mL
E = label amount of magnesium gluconate
(mg/Tablet)
M, = molecular weight of magnesium gluconate,
414.60
A = atomic weight of magnesium, 24.31
Tolerances: NLT 80.0% (Q) of the labeled amount of
magnesium gluconate (CizH22MgOv,) is dissolved.
e UNIFORMITY OF DosaGeE UNITS (905): Meet the
te
“w
requirements
ro
cS
— ADDITIONAL REQUIREMENTS
io e PACKAGING AND STORAGE: Preserve in well-closed
° mercially prepared thallium ICP standard solution
= containers.
So e USP REFERENCE STANDARDS (11)
3 USP Potassium Gluconate RS
5 Standard stock solution 100 ppb—Prepare this solution
a)
=)
Magnesium Hydroxide
Mg(OH)2 58.32
Magnesium hydroxide.
Magnesium hydroxide [1309-42-8].
» Magnesium Hydroxide, dried at 105° for
2 hours, contains not less than 95.0 percent and
not more than 100.5 percent of Mg(OH)2.
Packaging and storage—Preserve in tight containers.
Identification—A 1 in 20 solution in 3 N hydrochloric acid
responds to the tests for Magnesium (191).
Microbial enumeration tests (61) and Tests for speci-
fied microorganisms (62)—It meets the requirements of
the test for absence of Escherichia coli.
Loss on drying (731)—Dry it at 105° for 2 hours: it loses
not more than 2.0% of its weight.
Loss on ignition (733)—lgnite it at 800°, increasing the
heat gradually, to constant weight: it loses between 30.0%
and 33.0% of its weight.
Soluble salts—Boil 2.0 g with 100 mL of water for 5 min-
utes in a covered beaker, filter while hot, cool, and dilute
the filtrate with water to 100 mL. Titrate 50 mL of the di-
luted filtrate with 0.10 N sulfuric acid, using methyl red TS
USP 41
as the indicator: not more than 2.0 mL of the acid is con-
sumed. Evaporate 25 mL of the diluted filtrate to dryness,
and dry at 105° for 3 hours: not more than 10 mg of resi-
due remains.
Carbonate—Boil a mixture of 0.10 g with 5 mL of water,
cool, and add 5 mL of 6N acetic acid: not more than a
slight effervescence is observed.
Limit of calcium—
Dilute hydrochloric acid, Lanthanum solution, Standard
preparations, and Blank solution—Prepare as directed in the
test for Limit of calcium under Magnesium Carbonate.
Test preparation—Transfer 250 mg of Magnesium Hydrox-
ide, previously dried, to a beaker, add 30 mL of Dilute hydro-
chloric acid, and stir until dissolved, heating if necessary.
Transfer the solution so obtained to a 200-mL volumetric
flask containing 4 mL of Lanthanum solution, dilute with
water to volume, and mix.
Procedure—Proceed as directed in the test for Limit of cal-
cium under Magnesium Carbonate: the limit is 1.5%.
Delete the following:
Heed metals, Method | (231)—Dissolve 1.0 g in 15 mL
of 3 N hydrochloric acid, and evaporate the solution on a
steam bath to dryness. Toward the end of the evaporation,
stir the residue ogee: disintegrate it so that finally a dry
powder is obtained, dissolve the residue in 20 mL of water,
and filter. To the filtrate, which should be neutral to litmus,
add 2 mL of 1 N acetic acid, and dilute with water to
25 mL: the limit is 20 jug per 9-¢ <otficial 14an-2013)
Limit of lead—[NoTE—When water is specified as a diluent,
use deionized ultra-filtered water.]
Blank solution—Transfer 3.0 mL of nitric acid to a SO-mL
volumetric flask, and dilute with water to volume.
Thallium internal standard 20 ppb—{NoTE—Use this solu-
tion only if an ICP-MS instrument is used. This internal stan-
dard is added in-line via a mixing block between the sample
probe and the spray chamber.] Dilute 20.0 mL of a com-
(1000 ppb) with water to 1 L.
Dilute nitric acid—Dilute 2.0 mL of nitric acid with water
to 100 mL.
fresh every two months. Quantitatively dilute an accurately
measured volume of a commercially prepared lead ICP stan-
dard (1000 ppm) with Dilute nitric acid to obtain a solution
containing 10 ppm of lead. Further dilute this solution with
Dilute nitric acid to obtain a solution containing 1000 ppb of
lead. Transfer 10.0 mL of this solution to a separate 100-mL
volumetric flask, add 2.0 mL of nitric acid, and dilute with
water to volume.
Standard solutions—Prepare these solutions fresh weekly.
[NoTte—The concentrations specified below are recom-
mended if an ICP-MS instrument is used. If an ICP-AES in-
strument is used, the concentrations of the Standard solu-
tions may be modified to adapt to the working range of the
instrument.] Transfer 5.0 mL of the Standard stock solution
100 ppb to a 50-mL volumetric flask, add 3.0 mL of nitric
acid, and dilute with water to volume (Standard lead solu-
tion 10 ppb). Transfer 5.0 mL of Standard lead solution
10 ppb to a 50-mL volumetric flask, add 3.0 mL of nitric
acid, and dilute with water to volume (Standard lead solu-
tion 1 ppb).
Test solution—{NoTeE—The concentration specified below
is recommended if an ICP-MS instrument is used. If an
ICP-AES instrument is used, the concentration of the Test
solution may be modified to adapt to the working range of
the instrument.]
Accurately weigh about 0.25 g of Magnesium Hydroxide.
Cautiously add 3.0 mL of nitric acid, and mix until the sam-
USP 41
ple is dissolved. Accurately transfer this solution to a 50-mL
volumetric flask, and dilute with water to volume.
Procedure (see Plasma Spectrochemistry (730))—The induc-
tively coupled plasma—mass spectrometer (ICP-MS) is
equipped with a quadrupole mass spectrometer and an ion
detector maintained under vacuum. The instrument should
read all isotopes for lead (206, 207, and 208 amu) and the
thallium internal standard (205 amu), and should report the
total lead content using the most naturally abundant iso-
tope at 208 amu. Alternatively, lead Rodbe determined
using an inductively coupled plasma—atomic emission spec-
trometer (ICP-AES) by measuring the emission at 220.353
nm, with the settings optimized as directed by the manufac-
turer. [NOTE—To minimize matrix interference when using an
ICP-AES instrument, it is recommended that the method of
standard additions be used.]
Instrument performance must be verified to conform to
the manufacturer’s specifications for resolution and sensitiv-
ity. Before analyzing samples, the instrument must pass a
suitable performance check. Generate the calibration curve
using the Blank solution, Standard lead solution 1 ppb, and
Standard lead solution 10ppb: a linear regression coefficient
is not less than 0.999.
Aspirate the Test solution, at least in duplicate, and calcu-
late the amount of lead using the calibration curve. Report
the average reading as the lead content of the sample. Cal-
culate the content of lead in the portion of Magnesium Hy-
aroxide taken: not more than 0.00015% (1.5 ppm) is
‘ound.
Assay—Transfer about 75 mg of Magnesium Hydroxide,
previously dried and accurately weighed, to a conical flask.
Add 2 mL of 3 N hydrochloric acid, and swirl to dissolve.
Add 100 mL of water, adjust the reaction of the solution to
a pH of 7 (using pH indicator paper; see Indicator and Test
Papers under Reagents in the section Reagents, Indicators,
and Solutions) with 1 N sodium hydroxide, add 5 mL of am-
monia-ammonium chloride buffer TS and 0.15 mL of eri-
ochrome black TS, and titrate with 0.05 M edetate diso-
dium VS to a blue endpoint. Each mL of 0.05 M edetate
disodium is equivalent to 2.916 mg of Mg(OH)2.
Magnesium Hydroxide Paste
» Magnesium Hydroxide Paste is an aqueous
aste of Magnesium Hydroxide. It contains not
ess than 93.0 percent and not more than
107.0 percent of the labeled amount of magne-
sium hydroxide [Mg(OH),], the labeled amount
being not less than 28.0 percent and not more
than 70.0 percent of magnesium hydroxide.
Packaging and storage—Preserve in tight containers.
Identification—One g of Paste dissolved in 10 mL of 3N
hydrochloric acid responds to the tests for Magnesium (191).
Microbial enumeration tests (61) and Tests for speci-
fied microorganisms (62)—Its total aerobic microbial
count does not exceed 400 cfu per g, and it meets the
requirements of the test for absence of Escherichia coli.
Change to read:
Soluble alkalies—Accurately weigh a portion of Paste,
equivalent to about 7.75 g of magnesium hydroxide, and
mix with 75.0 mL of water. Transfer about 25 mL of this
diluted Paste toafilter, and reject the first 5 mL of the fil-
trate. [NOTE—Retain the remaining diluted Paste for the test
for Carbonate and acid-insoluble matter.®@ (oficial 1Jan-201@)] Di-
Official Monographs / Magnesium 2511
lute 5 mL of the clear filtrate with 40 mL of water. Add 1
drop of methyl red TS, and titrate the solution with 0.10 N
sulfuric acid to the production of a persistent pink color: not
more than 1.0 mL of the acid is required.
Soluble salts—To 5.0 mL of the clear filtrate obtained in
the test for Soluble alkalies add 3 drops of sulfuric acid,
evaporate on a steam bath to dryness, and ignite gently to
constant weight: the residue weighs not more than 12 mg.
Carbonate and acid-insoluble matter—To 1 mL of the
diluted Paste obtained in the test for Soluble alkalies add
2 mL of 3 N hydrochloric acid: not more thanaslight effer-
vescence occurs, and the solution is not more than slightly
turbid.
Limit of calcium—
Dilute hydrochloric acid, Lanthanum solution, Standard
preparations, and Blank solution—Prepare as directed in the
test for Limit of calcium under Magnesium Carbonate.
Test preparation—Transfer a portion of the Paste, equiva-
lent to 250 mg of Mg(OH)2, to a beaker, add 30 mL of Di-
lute hydrochloric acid, and stir until dissolved, heating if nec-
essary. Transfer the solution so obtained to a 200-mL
volumetric flask containing 4 mL of Lanthanum solution, di-
lute with water to volume, and mix.
Procedure—Proceed as directed in the test for Limit of cal-
cium under Magnesium Carbonate: the limit is 1.5%.
Delete the following:
“Heavy metals, Method / (231)—To 4.0 mL of the diluted
Paste obtained in the test for Soluble alkalies add 6 mL of
3 N hydrochloric acid, and evaporate the solution on a
steam bath to dryness, with frequent stirring. Dissolve the
residue in 20 mL of water, and evaporate to dryness in the
same manner as before. Redissalve in 20 mL of water, filter
if necessary, and dilute with water to 25 mL: the limit is
5 ppm, based on the amount of diluted Paste taken.e (otiica 1- c
al
Jan-2018) a]
Limit of lead—
Blank solution, Thallium internal standard 20 ppb, Dilute ni-
EK
°
tric acid, Standard stock solution 100 ppb, and Standard solu- |
tions—Proceed as directed in the test for Limit of lead under °
io}
Magnesium Hydroxide. a]
a
Test solution—Accurately weigh an amount of Paste me]
oe to 0.25 g of magnesium hydroxide. Cautiously a
a
add 3.0 mL of nitric acid, and mix until the sample is dis-
solved. Accurately transfer this solution to a 50-mL volumet-
tic flask, and dilute with water to volume. [NOTE—This con-
centration is recommended if an ICP-MS instrument is used.
If an ICP-AES instrument is used, the concentration of the
Test solution may be modified to adapt to the working range
of the instrument.]
Procedure—Proceed as directed in the test for Limit of
lead under Magnesium Hydroxide. Calculate the content of
lead in the portion of Paste taken based on the content of
magnesium hydroxide in the Paste, as determined in the
Assay: not more than 0.00015% (1.5 ppm) is found.
Assay—Transfer an accurately weighed portion of Paste,
equivalent to about 250 mg of magnesium hydroxide, to a
100-mL volumetric flask. Dissolve in 10 mL of 3 N hydro-
chloric acid, dilute with water to volume, and mix. Filter, if
necessary, and transfer 25.0 mL of the filtrate to a beaker
containing 75 mL of water, and mix. Adjust the reaction of
the solution to a pH of 7 (using pH indicator paper; see
Indicator and Test Papers under Reagents in the section Re-
agents, Indicators, and Solutions) with 1 N sodium hydroxide,
add 5 mL of ammonia-ammonium chloride buffer TS and
0.15 mL of eriochrome black TS, and titrate with 0.05 M
edetate disodium VS to a blue endpoint. Each mL of 0.05 M
edetate disodium is equivalent to 2.916 mg of magnesium
hydroxide [Mg(OH)z].
 2512 Magnesium / Official Monographs
Magnesium Oxide
MgO 40.30
Magnesium oxide [1309-48-4].
DEFINITION
Magnesium Oxide, after ignition, contains NLT 96.0% and
NMT 100.5% of magnesium oxide (MgO).
IDENTIFICATION
© A. IDENTIFICATION TESTS—GENERAL, Magnesium (191): A
solution in diluted hydrochloric acid meets the
requirements.
ASSAY
© PROCEDURE
Diluted ammonium hydroxide: Dilute 67 g of ammo-
nium hydroxide (about 75 mL) with water to 100 mL.
Buffer: Prepare ammonium chloride buffer pH 10 as
follows. In a 100-mL volumetric flask, dissolve 5.4 g of
ammonium chloride in 20 mL of water, add 35 mL of
Diluted ammonium hydroxide, and dilute with water to
volume.
Sample stock solution: Ignite a sample of Magnesium
Oxide to a constant weight in the temperature range of
(800°-900°) + 25°. Weigh 320 mg of the ignited sam-
ple into a 100-mL volumetric flask, dissolve in 20 mL of
2.N hydrochloric acid, and dilute with water to volume.
Sample solution: Transfer 20.0 mL of the Sample stock
solution to a 500-mL flask, and dilute with water to
300 mL. The Sample solution is equivalent to 64 mg of
ignited Magnesium Oxide.
Analysis: To the Sample solution, add 10 mL of Buffer
and 50 mg of eriochrome black T-sodium chloride indi-
cator. Heat the sample to 40°, and titrate to a blue
endpoint with 0.1 M edetate disodium VS. Perform a
blank determination, and make any necessary correc-
iS
a)
o tion to determine the volume of 0.1 M edetate diso-
S
—
dium consumed (Vs).
i=.) Calculate the volume of 0.1 M edetate disodium, Vea, in
° mL, consumed by calcium, which is present in the
S portion of Magnesium Oxide taken:
iS
= Vea = (W x Lea)/(Fea x 100)
a and dilute with water to volume.
2) w = amount of ignited Magnesium Oxide in the
= Sample solution (mg)
Lea = content of calcium as determined in the test
for Limit of Calcium (%)
Fea = weight of calcium equivalent to each mL of
0.1 M edetate disodium, 4.008 mg
Calculate the percentage of magnesium oxide (MgO) in
the portion of Magnesium Oxide taken:
Result = (Vs — Vea) x (Fivgo/W) x 100
Vs = volume of 0.1 M edetate disodium consumed
by the Sample solution (mL)
Vea = volume of 0.1 M edetate disodium consumed
by calcium (mL)
Fugo = Weight of Magnesium Oxide equivalent to
each mL of 0.1 M edetate disodium,
4.030 mg
w = amount of the ignited Magnesium Oxide in
the Sample solution (mg)
Acceptance criteria: 96.0%-100.5% after ignition
IMPURITIES
e FREE ALKALI AND SOLUBLE SALTS
Sample solution: Boil 2.0 g with 100 mL of water for 5
min in a covered beaker, and filter while hot. Allow to
cool, and dilute with water to 100 mL.
Analysis 1: To 50 mL of the Sample solution add methyl
red TS, and titrate with 0.10 N sulfuric acid.
USP 41
Acceptance criteria 1: NMT 2.0 mL of the acid is
consumed.
Analysis 2: Evaporate 25 mL of the remaining Sample
solution to dryness, and dry at 105° for 1 h.
Acceptance criteria 2: NMT 10 mg of residue remains
(NMT 2.0%).
e ACID-INSOLUBLE SUBSTANCES
Sample: 2g
Analysis: Mix the Sample with 75 mL of water, add hy-
drochloric acid in small portions with agitation until no
more dissolves, and boil for 5 min. If an insoluble resi-
due remains, filter, wash well with water until the last
washing is free from chloride, and ignite.
Acceptance criteria: The weight of the ignited residue
is NMT 2 mg (NMT 0.1%).
e Limit OF CALCIUM
[NoTE—A commercially available atomic absorption stan-
dard solution for calcium may be used where prepara-
tion of a calcium standard stock solution is described
below. Concentrations of the Standard solutions and the
Sample solution ay be modified to fit the linear or
working range of the instrument.]
Dilute hydrochloric acid: Dilute 100 mL of hydrochlo-
ric acid with water to 1000 mL.
Lanthanum solution: To 58.65 g of lanthanum oxide,
add 400 mL of water, and add, gradually with stirring,
250 mL of hydrochloric acid. Stir until dissolved, and
dilute with water to 1000 mL.
Standard solutions: Transfer 249.7 mg of calcium car-
bonate, previously dried at 300° for 3 h and cooled in a
desiccator for 2 h, to a 100-mL volumetric flask. Dis-
solve in a minimum amount of hydrochloric acid, and
dilute with water to volume. Transfer 1.0, 5.0, 10.0,
and 15.0 mL of this stock solution to separate 1000-mL
volumetric flasks, each containing 20 mL of the Lantha-
num solution and 40 mL of Dilute hydrochloric acid, and
dilute with water to volume. These Standard solutions
contain 1.0, 5.0, 10.0, and 15.0 g/mL of calcium,
respectively.
Sample solution: Transfer 250 mg of Magnesium Ox-
ide, freshly ignited for 1 h in the temperature range of
(800°-900°) + 25°, to a beaker. Add 30 mL of Dilute
hydrochloric acid, and stir until dissolved, heating if nec-
essary. Transfer the solution so obtained to a 200-mL
volumetric flask containing 4 mL of Lanthanum solution,
Blank solution: Transfer 4 mL of the Lanthanum solution
and 10 mL of Dilute hydrochloric acid to a 200-mL volu-
metric flask, and dilute with water to volume.
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic al sorpuon spectrophotometry
Analytical wavelength: Calcium emission line at
422.7 nm
Lamp: Calcium hollow-cathode
Flame: Nitrous oxide-acetylene
Analysis
Samples: Standard solutions, Sample solution, and Blank
solution
Using the calibration graph, determine the concentra-
tion, CG, in ug/mL, of calcium in the Sample solution.
Calculate the percentage of calcium in the portion of
Magnesium Oxide taken:
Result
= [(V/W) x C x F] x 100
= volume of the Sample solution (mL)
moss
= weight of Magnesium Oxide (mg)
= concentration of calcium in the Sample
solution (g/mL)
= conversion from g/mL to mg/mL, 0.001
USP 41
Acceptance criteria: NMT 1.1%
Delete the following:
°e HEAVY METALS (231)
Test preparation: Dissolve 2.0 g in 35 mL of 3 N hy-
drochloric acid, and evaporate the solution on a steam
bath to dryness. Toward the end of the evaporation, stir
frequently to disintegrate the residue so that finally a
dry powder is obtained. Dissolve the residue in 20 mL
of water, and evaporate to dryness in the same manner
as before. Redissolve the residue in 20 mL of water, fil-
ter if necessary, and dilute with water to 40 mL. To
20 mL, add water to make 25 mL.
Acceptance criteria: NMT 20 ppmee coitciat ).jan-2018)
© IRON (241)
Test preparation: Boil 40 mg with 5 mL of 2Nnitric
acid for 1 min. Cool, dilute with water to 50 mL, and
mix. Dilute 25 mL of this solution with water to 45 mL,
and add 2 mL of hydrochloric acid.
Acceptance criteria: NMT 0.05%
SPECIFIC TESTS
e LOSS ON IGNITION (733)
Sample: 500-1000 mg
Analysis: Transfer the Sample to a tared platinum cruci-
ble, and ignite in the temperature range of (800°-900°)
+ 25° to constant weight.
Acceptance criteria: NMT 10.0%
e BULK DENSITY AND TAPPED DENSITY OF POWDERS, Bulk Den-
sity, Method | (616): Using the procedure specified in
He eps determine the bulk density of Magnesium
Oxide.
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers.
e LABELING: Label it to indicate its bulk density. The indi-
cated density may be in the form of a range.
Magnesium Oxide Capsules
DEFINITION
Magnesium Oxide Capsules contain NLT 90.0% and NMT
110.0% of the labeled amount of magnesium oxide
(MgO).
IDENTIFICATION
© A, IDENTIFICATION TESTS—GENERAL, Magnesium (191)
Sample solution: Transfer the contents of 1 Capsule to
a beaker. Add 10 mL of 3 N hydrochloric acid and
5 drops of methyl red TS, and heat to boiling. Add 6 N
ammonium hydroxide until the color of the solution
changes to deep yellow, then continue boiling for 2
min, and filter. Use the filtrate.
Acceptance criteria: Meet the requirements
ASSAY
© PROCEDURE
Eriochrome black indicator solution: Dissolve 200 mg
of eriochrome black T in a mixture of 15 mL of trietha-
nolamine and 5 mL of dehydrated alcohol.
Sample solution: Mix the contents of NLT 20 Capsules.
Transfer a portion of the powder, equivalent to 500 mg
of magnesium oxide, to a beaker. Add 20 mL of water,
and slowly add 40 mL of 3 N hydrochloric acid with
mixing. Heat the mixture to boiling, cool, and filter into
a 200-mL volumetric flask. Wash the beaker with water,
adding the washings to the filter. Add water to volume.
Analysis: Transfer 20.0 mL of the Sample solution to a
400-mL beaker. Add 180 mL of water and 20 mL of tri-
ethanolamine, and stir. Add 10 mL of ammonia—am-
monium chloride buffer TS and 3 drops of Eriochrome
Official Monographs / Magnesium 2513
black indicator solution. Cool the solution to 3°-4° by
immersion of the beaker in an ice bath. Remove, and
titrate with 0.05 M edetate disodium VS to a blue
endpoint. Perform a blank determination, substituting
20 mL of water for the Sample solution, and make any
necessary correction (see Titrimetry (541)). Each mL of
0.05 M edetate disodium consumed is equivalent to
2.015 mg of MgO.
Acceptance criteria: 90.0%-110.0%
PERFORMANCE TESTS
e DISSOLUTION (711)
Medium: 0.1 N hydrochloric acid; 900 mL
Apparatus 1: 100 rpm
Time: 45 min
Analysis: ppsing atomic absorption one
at a wavelength of 285.2 nm, determine the amount of
MgO dissolved, using filtered portions of the solution
under test, suitably diluted with Medium if necessary, in
comparison with a standard solution having a known
concentration of magnesium in the same Medium.
Tolerances: NLT 75% (Q) of the labeled amount of
MgO is dissolved.
e UNIFORMITY OF DosaceE UNITS (905): Meet the
requirements
SPECIFIC TESTS
e ACID-NEUTRALIZING CAPACITY (301): NLT 5 mEq of acid is
consumed by the minimum single dose recommended in
the labeling, and NLT 85.0% of the expected mEq value
calculated from the results of the Assay is obtained. Each
m9 of MgO has an expected acid-neutralizing capacity
value of 0.0492 mEq.
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed
containers.
=
“
a]
Magnesium Oxide Tablets
=
2°
Ss
°
DEFINITION ©
Magnesium Oxide Tablets contain NLT 90.0% and NMT ma
)
110.0% of the labeled amount of magnesium oxide me]
(MgO). a
a
IDENTIFICATION
e A. IDENTIFICATION TESTS—GENERAL, Magnesium (191)
Sample solution: Finely powder 1 Tablet. Transfer the
powder to a beaker, add 10 mL of 3 N hydrochloric
acid and 5 drops of methyl red TS, and heat to boiling.
Add 6 N ammonium hydroxide until the color of the
solution stones to deep yellow, then continue boiling
for 2 min, and filter. Use the filtrate.
Acceptance criteria: Meet the requirements
ASSAY
e PROCEDURE
Eriochrome black indicator solution: Dissolve 200 mg
of eriochrome black T in a mixture of 15 mL of trietha-
nolamine and 5 mL of dehydrated alcohol.
Sample solution: Finely powder NLT 20 Tablets. Trans-
fer a portion of the powder, equivalent to 500 mg of
a oxide, to a beaker. Add 20 mL of water,
and slowly add 40 mL of 3 N hydrochloric acid, with
mixing. Heat the mixture to boiling, cool, and filter into
a 200-mL volumetric flask. Wash the beaker with water,
adding the washings to the filter. Add water to volume.
Analysis: Transfer 20.0 mL of the Sample solution to a
400-mL beaker. Add 180 mL of water and 20 mL of tri-
ethanolamine, and stir. Add 10 mL of ammonia—am-
monium chloride buffer TS and 3 drops of Eriochrome
black indicator solution. Cool the solution to 3°-4° by
 2514 Magnesium / Official Monographs
immersion of the beaker in an ice bath. Remove, and
titrate with 0.05 M edetate disodium VS to a blue
endpoint. Perform a blank determination, substituting
20 mL of water for the Sample solution, and make any
necessary correction (see Titrimetry (541)). Each mL of
0.05 M edetate disodium consumed is equivalent to
2.015 mg of MgO.
Acceptance criteria: 90.0%-110.0%
PERFORMANCE TESTS
e DISSOLUTION (711)
Medium: 0.1 N hydrochloric acid; 900 mL
Apparatus 2: 75 rpm
Time: 45 min
Analysis: Using atomic absorption spectrophotometry
at a wavelength of 285.2 nm, determine the amount of
MgO dissolved, using filtered portions of the solution
under test, suitably diluted with Medium if necessary, in
comparison with a standard solution having a known
concentration of magnesium in the same Medium.
Tolerances: NLT 75% (Q) of the labeled amount of
MgO is dissolved.
e UNIFORMITY OF DosaGE UNITS (905): Meet the
requirements
SPECIFIC TESTS
e ACID-NEUTRALIZING CAPACITY (301) (where Tablets are la-
beled as intended for antacid use): NLT 5 mEq of acid
is consumed by the minimum single dose recommended
in the labeling, and NLT 85.0% of the expected mEq
value calculated from the results of the Assay is obtained.
Each mg of MgO has an expected acid-neutralizing ca-
pacity value of 0.0492 mEq.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed
containers.
<
v
is
Ss
—
fo.)
i) Magnesium Phosphate
r=
S Mgs(POa4)2 » SH20 352.93
= Phosphoric acid, magnesium salt (2:3), pentahydrate;
i.e Magnesium phosphate (3:2) pentahydrate [10233-87-1].
va)
=) Mga3(POs)2 262.86
[7757-87-1].
DEFINITION
Magnesium Phosphate, ignited at 425° to constant weight,
contains NLT 98.0% and NMT 101.5% of Mg3(PO,)2.
IDENTIFICATION
cA.
Sample: 200mg
Analysis: Dissolve the Sample in 10 mL of 2 N nitric
acid, and add, dropwise, ammonium molybdate TS.
Acceptance criteria: A greenish-yellow precipitate of
ammonium phosphomolybdate is formed, and it is sol-
uble in 6 N ammonium hydroxide.
e B. IDENTIFICATION TESTS—GENERAL, Magnesium (191)
Sample solution: Dissolve 0.1 g in 0.7 mL of 1 N acetic
acid and 20 mL of water. Add 1 mL of ferric chloride
TS, allow to stand for 5 min, and filter. Use 5 mL of the
filtrate.
Acceptance criteria: Meets the requirements
ASSAY
e PROCEDURE
Sample: 200 mg, previously ignited at 425° to constant
weight
Analysis: Dissolve the Sample in a mixture of 25 mL of
water and 10 mL of 2N nitric acid. Filter, if necessary.
Wash any precipitate, add sufficient 6 N ammonium hy-
droxide to the filtrate to producea slight precipitate,
USP 41
and then dissolve the precipitate by the addition of
1 mL of 2N nitric acid. Adjust the temperature to 50°,
add 75 mL of ammonium molybdate TS, and maintain
the temperature at 50° for 30 min, stirring occasionally.
Wash the precipitate once or twice with water by de-
cantation, using 30-40 mL each time and passing the
washings throughafilter. Transfer the precipitate to the
filter, and wash with potassium nitrate solution (1 in
100) until the last washing is not acid to litmus. Trans-
fer the precipitate and filter to the precipitation vessel.
Add 50 mL of water and 40.0 mL of 1 N sodium hy-
droxide VS, and agitate until the precipitate is dissolved.
Add phenolphthalein TS, and then titrate the excess al-
kali with 1 N sulfuric acid VS. Each mL of 1 N sodium
hydroxide is equivalent to 5.716 mg of Mg3(PO,)2.
Acceptance criteria: 98.0%-101.5% on the previously
ignited basis
IMPURITIES
© ACID-INSOLUBLE SUBSTANCES
[Note—Perform if an insoluble residue remains in the
test for Carbonate.]
Analysis: Filter the solution, wash well with hot water
until the last washing is free from chloride, and ignite
the residue.
Acceptance criteria: The weight of the residue does
not exceed 4 mg (NMT 0.2%).
e SOLUBLE SUBSTANCES
Sample: 2.0g
Analysis: Digest the Sample with 100 mL of water on a
steam bath for 30 min. Cool, add sufficient water to
restore the original volume, mix, and filter. Evaporate
50 mL of the filtrate to dryness, and ignite gently to
constant weight.
Acceptance criteria: The weight of the residue does
not exceed 15 mg (NMT 1.5%).
e CARBONATE
Sample: 2.0g
Analysis: Mix the Sample with 20 mL of water, and add
hydrochloric acid, dropwise, to dissolve.
Acceptance criteria: No effervescence occurs when the
acid is added.
e CHLORIDE AND SULFATE, Chloride (221)
Sample: 0.50g
Analysis: Dissolve the Sample in 50 mL of 2 N nitric
acid, and add 1 mL of silver nitrate TS.
Acceptance criteria: The turbidity does not exceed that
produced by 1.0 mL of 0.020 N hydrochloric acid
(NMT 0.14%).
e CHLORIDE AND SULFATE, Sulfate (221)
Sample: 0.50g
Analysis: Dissolve the Sample in the smallest possible
amount of 3 N hydrochloric acid, dilute with water to
48 mL, and add 2 mL of barium chloride TS.
Acceptance criteria: The turbidity does not exceed that
produced by 3.0 mL of 0.020N sulfuric acid (NMT
0.6%).
© LIMIT OF NITRATE
Sample: 0.209
Analysis: Mix the Sample with 5 mL of water, and add
just sufficient hydrochloric acid to dissolve. Dilute with
water to 10 mL, add 0.1 mL of indigo carmine TS, then
add, with stirring, 10 mL of sulfuric acid.
Acceptance criteria: The blue color persists for NLT 5
min.
e ARSENIC, Method | (211)
Test preparation: Prepare as directed in the chapter,
using 1309 and dissolving it first in just a sufficient
amount of 3 N hydrochloric acid (about 9 mL).
Acceptance criteria: NMT 3 ppm
e BARIUM
Sample: 2.0g
Analysis: Mix the Sample with 40 mL of water. Heat,
add hydrochloric acid, dropwise, to dissolve, and then
add 1 mL of acid in excess. Cool, dilute with water to
USP 41 Official Monographs / Magnesium 2515

50 mL, and filter. To 5 mL of the filtrate add 1 mL of IDENTIFICATION

potassium sulfate TS. e A. INFRARED ABSORPTION (197K)
Acceptance criteria: No turbidity is produced within 15 e B. The retention time of the major peak of the Sample
min. solution corresponds to that of the Standard solution, as
© CALCIUM obtained in the Assay.
Sample: 0.50g © C. IDENTIFICATION TESTS—GENERAL, Magnesium (191):
Analysis: Mix the Sample with 15 mL of water. Heat, Meets the requirements
and add sufficient hydrochloric acid, in small portions,
to dissolve. Cool, add 6 N ammonium hydroxide, in ASSAY
small portions, to produce a slight permanent precipi- © PROCEDURE
tate, then add 2 mL of 6 N acetic acid. Dilute with Mobile phase: Methanol, phosphoric acid, and water
water to 25 mL, and filter. To 10 mL of the filtrate add (40: 0.1: 60), prepared by adding 1 mL of phosphoric
2 mL of ammonium oxalate TS. acid to a solution containing 400 mL of methanol and
Acceptance criteria: NMTaslight turbidity is produced 600 mL of water
within 5 min. Standard solution: 0.5 mg/mL of USP Magnesium Sa-
© DIBASIC SALT AND MAGNESIUM OXIDE licylate RS, in Mobile phase
Sample: Ignite about 2.5 g to constant weight and use Sample solution: 0.5 mg/mL of Magnesium Salicylate,
2g of the ignited salt. in Mobile phase
Analysis: Dissolve the Sample by warming with 50.0 mL Chromatographic system
of 1 N hydrochloric acid VS. Cool, add 1 or 2 drops of (See Chromatography (621), System Suitability.)
methyl orange TS, and slowly titrate the excess 1 N hy- Mode: LC
drochloric acid VS with 1 N sodium hydroxide VS to a Detector: UV 236 nm
yellow color, vigorously shaking the mixture during the Column: 4.6-mm x 10-cm; 5-4m packing L1
titration. Flow rate: 1 mL/min
Acceptance criteria: 14.8-15.4 mL of 1 N hydrochloric Injection volume: 10 uL
acid is consumed for each g of the ignited salt. System suitability
e LEAD (251) Sample: Standard solution
Test preparation: Dissolve 1.0 g in 20 mL of 3 N hy- Suitability requirements
drochloric acid, evaporate on a steam bath to 10 mL, Tailing factor: NMT 1.5
dilute with water to 20 mL, and cool. Relative standard deviation: NMT 1.10%
Analysis: Proceed as directed in the chapter, using Analysis
5 mL of Diluted Standard Lead Solution (5 ug of Pb). Samples: Standard solution and Sample solution
Acceptance criteria: NMT 5 ppm Calculate the percentage of magnesium salicylate
(Cis4HioMgQOc - 4H20) in the portion of Magnesium Sa-
licylate taken:
Delete the following:
Result = (ru/rs) x (Cs/Cy) x 100
°e HEAVY METALS, Method | (231) ec
Test preparation: Dissolve 0.67 g in 4.5 mL of 3 N hy- ru = peak response from the Sample solution “
drochloric acid, and dilute with water to 25 mL. Is peak response from the Standard solution z
oi

Acceptance criteria: NMT 30 ppme (otcial 1-4an-2018) Cs concentration of USP Magnesium Salicylate RS re
in the Standard solution (mg/mL) i)
SPECIFIC TESTS Cy = concentration of Magnesium Salicylate in the Ss)
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI- Sample solution (mg/mL) -)
FIED MICROORGANISMS (62): It meets the requirements of Acceptance criteria: 98.0%-103.0%
a5
the test for the absence of Escherichia coli. i}
IMPURITIES ao]
e LOSs ON IGNITION (733): Ignite a sample at 425° to con- <r
stant weight; it loses 20.0%-27.0% of its weight. ”

ADDITIONAL REQUIREMENTS Delete the following:

© PACKAGING AND STORAGE: Preserve in well-closed
containers. °e HEAVY METALS, Method | (231): 40 ppMe cortical 14an-2018)
© ORGANIC IMPURITIES
Mobile phase: Methanol, phosphoric acid, and water
(40: 0.1: 60), prepared by adding 1 mL of phosphoric
acid to a solution containing 400 mL of methanol and
Magnesium Salicylate 600 mL of water
Standard stock solution: 0.25 mg/mL of USP Magne-
sium Salicylate RS, 0.25 mg/mL of USP Salicylic Acid Re-
lated Compound A RS, 0.125 mg/mL of USP Salicylic
ag? ft ° + 44,0 Acid Related Compound B RS, and 0.05 mg/mL of USP
Phenol RS, in Mobile phase
Standard solution: 0.005 mg/mL of USP Magnesium
Salicylate RS, 0.005 mg/mL of USP Salicylic Acid Related
Cra4HioMgOc - 4H20 370.59 Compound A RS, 0.0025 mg/mL of USP Salicylic Acid
CysHioMgOc 298.54 Related Compound B RS, and 0.001 mg/mL of USP
Magnesium, bis(2-hydroxybenzoato-0',02)-, tetrahydrate;
Phenol RS, in Mobile phase prepared from Standard
Magnesium salicylate (1:2), tetrahydrate [18917-95-8]. stock solution
Anhydrous [18917-89-0]. Sample solution: 5 mg/mL of Magnesium Salicylate, in
Mobile phase
DEFINITION
Magnesium Salicylate contains NLT 98.0% and NMT
103.0% of magnesium salicylate (Ci4HioMgOs - 4H20).
 2516 Magnesium / Official Monographs USP 41

Chromatographic system Titrimetric system

(See Chromatography (621), System Suitability.) Mode: Direct titration
Mode: LC Titrant: 0.05 M edetate disodium VS
Detector: UV 212 nm Endpoint detection: Visual
Column: 4.6-mm x 10-cm; 5-um packing L1 Analysis: Transfer 50.0 mL of the Sample solution to a
Flow rate: 1 mL/min 250-mL conical flask. Add 50 mL of water, 5 mL of am-
Injection volume: 10 LL monia—ammonium chloride buffer TS, and 0.15 mL of
System suitability eriochrome black TS. Titrate with Titrant to a blue
Sample: Standard solution endpoint. Each mL of Titrant is equivalent to 1.215 mg
Suitability requirements of magnesium.
Resolution: NLT 2.0 between any two peaks Acceptance criteria: 6.3%-6.7% of magnesium
Relative standard deviation: NMT 3% for each peak e WATER DETERMINATION, Method | (921): 17.5%-21.0%
Analysis
Samples: Standard solution and Sample solution ADDITIONAL REQUIREMENTS
Calculate the percentage of salicylic acid related com- e PACKAGING AND STORAGE: Store in tight containers at
pound A, salicylic acid related compound B, and phe- controlled room temperature.
nol in the portion of Magnesium Salicylate taken: © USP REFERENCE STANDARDS (11)
USP Magnesium Salicylate RS
Result = (ru/rs) x (Cs/Cu) x 100 USP Phenol RS
USP Salicylic Acid Related Compound A RS
ru = peak response of salicylic acid related 4-Hydroxybenzoic acid.
compound A, salicylic acid related C7HsO3 138.12
compound B, or phenol from the Sample USP Salicylic Acid Related Compound B RS
solution 4-Hydroxyisophthalic acid.
ig = peak response of salicylic acid related CsHeOs 182.13
compound A, salicylic acid related
compound B, or phenol from the Standard
solution
Cs = concentration of USP Salicylic Acid Related
CompoundA RS, USP Salicylic Acid Related Magnesium Salicylate Tablets
Compound B RS, or USP Phenol RS in the
Standard solution (mg/mL) DEFINITION
Cu = concentration of Magnesium Salicylate in the Magnesium Salicylate Tablets contain an amount of magne-
Sample solution (mg/mL) sium salicylate (C14HioMgO¢ - 4H20) equivalent to NLT
Calculate the percentage of any other individual 95.0% and NMT 105.0% of the labeled amount of anhy-
impurity in the portion of Magnesium Salicylate taken: drous magnesium salicylate (CigHioMgQOs).
Result = (ru/rs) x (Cs/Cu) x 100 IDENTIFICATION
=
vv

% ru = peak response of any other individual impurity

e A. The IR absorption spectrum of a potassium bromide
Ss dispersion of a quantity of finely powdered Tablets exhib-
— from the Sample solution
a2) its maxima at the same wavelengths as those of a similar
° rs = peak response of salicylic acid related preparation of USP Magnesium Salicylate RS.
c compound B from the Standard solution © B. IDENTIFICATION TESTS—GENERAL, Magnesium (191)
Sj Cs = concentration of USP Salicylic Acid Related Sample solution: Prepare a 50-mg/mL magnesium sa-
= CompoundBRS in the Standard solution licylate solution from Tablets, and filter.
ta (mg/mL) Acceptance criteria: Meet the requirements
a) Cu = concentration of Magnesium Salicylate in the
= Sample solution (mg/mL)
e C. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
Acceptance criteria: See Table 7. obtained in the Assay.
Table 1 ASSAY
© PROCEDURE
Relative Acceptance
Mobile phase: Methanol, trifluoroacetic acid, and water
Retention Criteria, (40: 0.1: 60), prepared by adding 1 mL of trifluoroacetic
Name Time NMT (%) acid to a solution containing 400 mL of methanol and
Salicylic acid related 600 mL of water
compound A 03 0.1 Standard solution: 0.05 mg/mL of anhydrous USP
Phenol 0.4 0.02 Magnesium Salicylate RS in Mobile phase
Salicylic acid related Sample stock solution: Nominally 0.5 mg/mL of anhy-
compound B 0.6 0.05 drous magnesium salicylate from NLT 20 finely pow-
Salicylic acid 1.0 — dered Tablets in Mobile phase prepared as follows.
Any other individual fe
Transfer a suitable amount of the powder toa suitable
impurit 0.05
volumetric flask. Add 75% of the flask volume of Mobile
phase, and sonicate for 10 min. Allow the solution to
Total impurities = 0.2 cool to room temperature and then dilute with Mobile
phase to volume.
SPECIFIC TESTS Sample solution: Nominally 0.05 mg/mL of anhydrous
o MAGNESIUM CONTENT magnesium salicylate in Mobile phase, from the Sample
Sample solution: Transfer 800 mg of Magnesium Salic- stock solution. Pass through a suitable filter of 0.20-m
late to a 200-mL volumetric flask. Dissolve in and di- pore size, discarding the first 2-3 mL of the filtrate.
ute with water to volume. Stir the solution continu-
ously for 15 min, and filter, discarding the first 10 mL of
the filtrate, into a flask.
USP 41 Official Monographs / Magnesium 2517

Chromatographic system acid to a solution containing 400 mL of methanol and

(See Chromatography (621), System Suitability.) 600 mL of water
Mode: LC System suitability solution: 0.5 mg/mL of USP Magne-
Detector: UV 212 nm sium Salicylate RS, 0.5 g/mL of USP Salicylic Acid Re-
Column: 2.1-mm x 5-cm; 1.7-um packing L1 lated Compound A RS, 0.5 g/mL of USP Salicylic Acid
Column temperature: 30° Related CompoundB RS, aa 0.5 ug/mL of USP Phenol
Flow rate: 0.2 mL/min RS, in Mobile phase
Injection volume: 2 UL Standard solution: 2.5 g/mL of anhydrous USP Mag-
System suitability nesium Salicylate RS in Mobile phase
Sample: Standard solution Sample solution: Nominally 2.5 mg/mL of anhydrous
Suitability requirements magnesium salicylate from NLT 20 finely powdered
Tailing factor: 0.8-1.8 Tablets in Mobile phase prepared as follows. Transfer a
Relative standard deviation: NMT 1.0% suitable amount of the powder to a suitable volumetric
Analysis flask. Add 80% of the flask volume of Mobile phase, and
Samples: Standard solution and Sample solution sonicate for 15 min. Allow the solution to cool to room
Calculate the percentage of the labeled amount of an- temperature and then dilute with Mobile phase to vol-
hydrous magnesium salicylate (Ci4HioMgQO¢) in the ume. Centrifuge the solution, and use the supernatant.
portion of Tablets taken: Chromatographic oe
(See Chromatography (621), System Suitability.)
Result = (ru/rs) x (Cs/Cu) x 100 Mode: LC
Detector: UV 212 nm
ru = peak response from the Sample solution Column: 2.1-mm x 5-cm; 1.7-44m packing L1
ls = peak response from the Standard solution Column temperature: 30°
Cs = concentration of anhydrous USP Magnesium Flow rate: 0.2 mL/min
Salicylate RS in the Standard solution Injection volume: 2 uL
(mg/mL) System suitability
Cu = nominal concentration of anhydrous Samples: System suitability solution and Standard
magnesium salicylate in the Sample solution solution
(mg/mL) Suitability requirements
Acceptance criteria: 95.0%-105.0% Resolution: NLT 2.0 between any two peaks, System
suitability solution
PERFORMANCE TESTS Relative standard deviation: NMT 3%, Standard
© DISSOLUTION (711) solution
Medium: Water; 900 mL Analysis
Apparatus 2: 50 rpm Samples: Standard solution and Sample solution
Time: 120 min Calculate the percentage of any individual unspecified
Standard solution: USP Salicylic Acid RS in Medium impurity in the portion of Tablets taken:
Sample solution: Pass a portion of the solution under (ox
test througha suitable filter, and dilute with Medium if Result = (ru/rs) x (Cs/Cu) x 100 4)
necessary. SS)
Instrumental conditions tu = peak response of any individual unspecified =
Mode: UV impurity from the Sample solution i}
Analytical wavelength: Maximum wavelength at rs = peak response of magnesium salicylate from =|
about 296 nm )
Blank: Water
the Standard solution reo}|
Gs = concentration of anhydrous USP Magnesium i}
Analysis Salicylate RS in the Standard solution a}
Samples: Standard solution and Sample solution (mg/mL) =)
Calculate the percentage of the labeled amount of an- Cu = nominal concentration of anhydrous
a)

hydrous magnesium salicylate (Ci4HioMgQO6c) dissolved: magnesium salicylate in the Sample solution
(mg/mL)
Result = (Au/As) x (1/L) x (Mi/Miz) x Cs x Vx 100 Acceptance criteria: See Table 1.
Au = absorbance from the Sample solution
As = absorbance from the Standard solution Table 1
L = nominal concentration of anhydrous Relative Acceptance
magnesium salicylate in the Sample solution Retention Criteria,
(mg/mL) Name Time NMT (%)
Mn, — = molecular weight of anhydrous magnesium Salicylic acid related
salicylate, 298.54 compound A 0.3 =
M2 = twice the molecular weight of salicylic acid, 0.4 —
Phenol
276.24
Gs = concentration of USP Salicylic Acid RS in the Salicylic acid related
Standard solution (mg/mL) compound B 0.6 =
Vv = volume of Medium, 900 mL Salicylic acid 1.0 =
Tolerances: NLT 80% (Q) of the labeled amount of an- These are process impurities, which are included in the table for identifi-
hydrous magnesium salicylate (Ci4HioMgOs) is cation only. These impurities are controlled in the drug substance. They
dissolved. are not to be reported for the drug product andshould not be included
in the total impurities.
e UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
IMPURITIES
© ORGANIC IMPURITIES
Mobile phase: Methanol, trifluoroacetic acid, and water
(40: 0.1: 60), prepared by adding 1 mL of trifluoroacetic
 2518 Magnesium / Official Monographs USP 41

Table 1 (Continued) N = titrant molarity (mmol/mL)

Relative Acceptance F = equivalency factor, 120.36 mg/mmol
Retention Criteria, w = weight of Sample (mg)
Name Time NMT (%) Acceptance criteria: 99.0%-100.5% on the anhydrous
by ignition basis
Any individual unspecified
impurit _ 0.10 IMPURITIES
Total impurities = 1.0 o LIMIT OF CHLORIDE (221)
These are process impurities, which are included in the table for identifi- Sample: 1.0g
cation only. These impurities are controlled in the drug substance. They Acceptance criteria: The Sample shows no more chlo-
are not to be reported for the drug product andshould not be included
in the total impurities. ride than corresponds to 0.20 mL of 0.020 N hydro-
chloric acid (0.014%).
ADDITIONAL REQUIREMENTS e Limit OF IRON (241)
¢ PACKAGING AND STORAGE: Preserve in tight containers. Magnesium Sulfate intended for use in preparing
e USP REFERENCE STANDARDS (11) nonparenteral dosage forms
USP Magnesium Salicylate RS Sample solution: Dissolve 0.50 g in 40 mL of water.
USP Phenol RS Analysis: Proceed as directed in the test for /ron (241).
USP Salicylic Acid RS Acceptance criteria: NMT 20 jig/g
USP Salicylic Acid Related Compound A RS Magnesium Sulfate intended for use in preparing par-
4-Hydroxybenzoic acid. enteral dosage forms
C7HeOz 138.12 [NoTte—Rinse all glassware used in this test with Dilute
USP Salicylic Acid Related Compound B RS hydrochloric acid.]
4-Hydroxyisophthalic acid. Dilute hydrochloric acid: 1 mL of hydrochloric acid
CsHeOs ~~ 182.13 diluted with water to 1000 mL
Solution A: 500 mg/mL of ammonium acetate in
water
Solution B: 13.4 mg/mL of ascorbic acid in water.
[NoTte—Use this solution on the day prepared.]
Magnesium Sulfate Color reagent: 3.8 mg/mL of 3-(2-pyridyl)-5,6-di-
(2-furyl)-1,2,4-triazine-5’,5”-disulfonic acid, disodium
MgSOg - xH20
salt in Solution A. Shake by mechanical means if neces-
sary. Use this solution on the day prepared.
Sulfuric acid magnesium salt (1:1), hydrate;
Standard stock solution: 1.0 g/mL of iron, from
Magnesium sulfate (1:1) monohydrate 138.36 Standard Iron Solution in Dilute Aydisthiere acid
{14168-73-1]. Standard solutions: To three separate 50-mL volumet-
Magnesium sulfate (1:1) heptahydrate 246.47 ric flasks transfer 2.0, 5.0, and 10.0 mL of Standard
[10034.99-8]. stock solution, and dilute each with Dilute hydrochloric
”
Anhydrous 120.37 acid to 35 mL. These solutions contain 2.0, 5.0, and
Es [7487-88-9]. 10.0 tg of iron, respectively.
a Sample solution: Transfer 10.0 g of Magnesium Sul-
Ss
4 DEFINITION
=) Magnesium Sulfate, rendered anhydrous by ignition, con- fate to a 50-mL volumetric flask, add Dilute hydrochlo-
} tains NLT 99.0% and NMT 100.5% of MgSO.. ric acid to 35 mL, and sonicate, if necessary, to
= dissolve.
S IDENTIFICATION Blank: Transfer 35 mL of Dilute hydrochloric acid to a
3 oe A. IDENTIFICATION TESTS—GENERAL, Magnesium (191) and- 50-mL volumetric flask.
a Sulfate (191) Instrumental conditions
a)
=} Sample solution: 50 mg/mL (See Ultraviolet-Visible Spectroscopy (857).)
Acceptance criteria: Meets the requirements Mode: UV-Vis
Analytical wavelength: 594 nm
ASSAY Analysis
© PROCEDURE Samples: Standard solutions, Blank, and Sample
Sample: 250 mg of the ignited Magnesium Sulfate ob- solution
tained in the test for Loss on Ignition To each of the flasks containing the Standard solu-
Titrimetric system tions, the Sample solution, and the Blank add 5 mL of
Mode: Direct titration Solution B and 5 mL of Color reagent. Dilute each
Titrant: 0.05M edetate sodium VS solution with Dilute hydrochloric acid to volume, mix,
Endpoint detection: Colorimetric and allow to stand for 10 min.
Analysis: Dissolve the Sample in 100 mL of water and Plot the absorbance values of the Standard solutions
the minimum amount of 3 N hydrochloric acid required versus their iron contents in ug and draw the
for a clear solution. Adjust the reaction of the solution straight line best fitting the three plotted points.
(using pH indicator paper; see Reagents, Indicators, and From the graph, determine the iron content, C, in
Solutions—Reagents—Indicator and Test Papers) with 1 N ug, of the Sample solution.
sodium hydroxide to a pH of 7, add 5 mL of am- Calculate the content, in g/g, of iron in the portion
monia—ammonium chloride buffer TS and 0.15 mL of of Magnesium Sulfate taken:
eriochrome black TS, and titrate with the Titrant to a
blue endpoint. Caclulate the perecentage of MgSO, in Result = C/W
the portion of the ignited Magnesium Sulfate taken:
Cc = content of iron in the Sample solution in ug,
Result = [Vx Nx Fx 100]/W determined from the graph
Ww = weight of Magnesium Sulfate in the Sample
Vv = Sample titrant volume (mL) solution (g)
USP 41
Acceptance criteria: NMT 0.5 ug/g
e SELENIUM (291)
Test solution: 200 mg in 50 mL of 0.25 N nitric acid
Acceptance criteria: NMT 30 ug/g
Delete the following:
°e HEAVY METALS (231)
Sample solution: 2g in 25 mL of water
Acceptance criteria: NMT 10 ppmMecoricial 14Jan-2018)
SPECIFIC TESTS
© PH (791)
Sample solution: 50 mg/mL
Acceptance criteria: 5.0-9.2
e Loss ON DRYING (731): Dry a sample at 105° for 2 h: the
anhydrous form loses NMT 2% of its weight.
e Loss ON IGNITION (733)
Sample: 1g
Analysis: Weigh the Sample in a crucible, heat at 105°
for 2 h, then ignite in a muffle furnace at 450 + 25° to
constant weight.
Acceptance criteria
Monohydrate: Loses 13.0%-16.0% of its weight
Dried form: Loses 22.0%-28.0% of its weight
Heptahydrate: Loses 40.0%-52.0% of its weight
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed
containers.
e LABELING: The label states whether it is the monohydrate,
the dried form, or the heptahydrate. Magnesium Sulfate
intended for use in preparing parenteral dosage forms is
so labeled. Magnesium Sulfate not intended for use in
preparing parenteral dosage forms is so labeled. In addi-
tion, it may be labeled also as intended for use in prepar-
ing nonparenteral dosage forms.
Magnesium Sulfate Injection
DEFINITION
Magnesium Sulfate Injection is a sterile solution of Magne-
sium Sulfate in Water for Injection. It contains magnesium
sulfate (MgSO,) equivalent to NLT 93.0% and NMT
107.0% ofthe labeled amount of magnesium sulfate
heptahydrate (MgSO, - 7H20).
IDENTIFICATION
© A. IDENTIFICATION TESTS—GENERAL, Magnesium (191) and-
Sulfate (191): Meets the requirements
ASSAY
© PROCEDURE
Sample: A known volume of Injection equivalent to
250 mg of anhydrous magnesium sulfate
Titrimetric system
Mode: Direct titration
Titrant: 0.05 M edetate disodium VS
Endpoint detection: Visual
Analysis
Sample: Sample
Transfer the Sample to a beaker, and dilute with water
to 100 mL. Adjust with 1 N sodium hydroxide to a pH
of 7 (using pH indicator paper; see Reagents, Indica-
tors, and Solutions—indicators and Indicator Test Pa-
pers). Add 5 mL of ammonia~-ammonium chloride
buffer TS and 0.15 mL of eriochrome black TS. Titrate
with Titrant to a blue endpoint. Each mL of Titrant
consumed is equivalent to 12.32 mg of magnesium
sulfate heptahydrate (MgSO,- 7H20).
Official Monographs / Magnesium 2519
Acceptance criteria: 93.0%-107.0%
SPECIFIC TESTS
e BACTERIAL ENDOTOXINS TEST (85): It contains NMT 0.09
USP Endotoxin Unit/mg of magnesium sulfate.
© PH (791)
Sample solution: 50 mg/mL
Acceptance criteria: 5.57.0
© PARTICULATE MATTER IN INJECTIONS (788): Meets the re-
quirements for small-volume injections
e OTHER REQUIREMENTS: It meets the requirements in Injec-
tions and Implanted Drug Products (1).
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in single-dose or mul-
tiple-dose containers, preferably of Type | glass.
e LABELING: The label states the total osmolar concentra-
tion in mOsmol/L. Where the contents are less than
100 mL, or where the label states that the Injection is not
for direct injection but is to be diluted before use, the
label alternatively may state the total osmolar concentra-
tion in mOsmol/mL.
Delete the following:
®o USP REFERENCE STANDARDS (11)
USP Endotoxin RS
© (CN 1-May-2018)
Magnesium Sulfate in Dextrose
Injection
DEFINITION
Magnesium Sulfate in Dextrose Injection is a sterile solution fe
of Magnesium Sulfate and Dextrose in Water for Injection. 4)
It contains NLT 93.0% and NMT 107.0% of the labeled a]
amount of magnesium sulfate (MgSO, - 7H2O) and NLT =
90.0% and NMT 110.0% of the labeled amount of dex- )I
. trose (CsH120¢6
- H20). )
IDENTIFICATION
eo}4
ES)
eA.
Sample solution 1: A suitable volume of Injection
co]>
equivalent to 50 mg/mL of dextrose a)
Analysis 1: Add a few drops of the Sample solution to
5 mL of hot alkaline cupric tartrate TS.
Acceptance criteria 1: A copious red precipitate of cu-
prous oxide is formed.
Analysis 2: Proceed as directed in Identification Tests—
General (191), Magnesium.
Acceptance criteria 2: Meets the requirements
ASSAY
© MAGNESIUM SULFATE
Sample: A known volume of Injection equivalent to
250 mg of anhydrous magnesium sulfate
Titrimetric system
Mode: Direct titration
Titrant: 0.05 M edetate disodium VS
Endpoint detection: Visual
Analysis: Transfer the Sample to a beaker, and dilute
with water to 100 mL. Adjust with 1 N sodium hydrox-
ide to a pH of 7 using pH indicator paper (see Reagents,
Indicators, and Solutions—Indicator and Test Papers), and
add 5 mL of ammonia-ammonium chloride buffer TS
and 0.15 mL of eriochrome black TS. Titrate with Titrant
to a blue endpoint. Each mL of Titrant is equivalent to
12.32 mg of magnesium sulfate (MgSO4- 7H20).
Acceptance criteria: 93.0%-107.0% of the labeled
amount of magnesium sulfate (MgSO. - 7H2O)
 2520 Magnesium / Official Monographs USP 41

¢ DEXTROSE 20.0 percent of magnesium oxide (MgO) and

Sample solution: Nominally 2 g/100 mL of dextrose
prepared as follows. Transfer a suitable volume of Injec-
tion to a 100-mL volumetric flask. Add 0.2 mL of 6N
ammonium hydroxide, and dilute with water to
volume.
Analysis: Determine the angular rotation in a suitable
polarimeter tube (see Optical Rotation (781)).
Calculate the percentage of the labeled amount of dex-
trose (CsHi206+H2O) in the portion of Injection taken:
Result = [(100 x a)/(I x a] x (1/Cu) x (Ma/M,2) x 100
a = observed angular rotation of the Sample
solution (°)
I length of the polarimeter tube, decimeter
wou
(S05 than 45.0 percent of silicon dioxide
IO).
Packaging and storage—Preserve in well-closed contain-
ers.
Identification—
A: Mix about 500 mg with 10 mL of 3 N hydrochloric
acid, filter, and neutralize the filtrate to litmus paper with
6 N ammonium hydroxide: the neutralized filtrate responds
to the tests for Magnesium (191).
B: Prepare a bead by fusing a few crystals of sodium
ammonium phosphate ona platinum loop in the flame of a
Bunsen burner. Place the hot, transparent bead in contact
with Magnesium Trisilicate, and again fuse: silica floats

a mi pein’ of the specific rotation range for about in the bead, producing, upon cooling, an opaque
anhydrous dextrose, 52.9° bead with a web-like structure.
Cu = nominal concentration of dextrose in the
Sample solution, g/100 mL Water Determination, Method I] (921)—Weigh accu-
M, = molecular weight of dextrose monohydrate, rately about 1 g in a tared platinum crucible provided with
198.17 a cover. Gradually apply heat to the crucible at first, then
Mz = molecular weight of anhydrous dextrose, strongly ignite to constant weight: it loses between 17.0%
180.16 and 34.0% of its weight.
Acceptance criteria: 90.0%-110.0% of the labeled Soluble salts—Boil 10.0 g with 150 mL of water for
amount of dextrose (CsHi20¢ - H2O) 15 minutes. Cool to room temperature, allow the mixture to
stand for 15 minutes, filter with the aid of suction, transfer
IMPURITIES the filtrate to a 200-mL volumetric flask, dilute with water to
e Limit OF 5-HYDROXYMETHYLFURFURAL AND RELATED volume, and mix. Evaporate 50.0 mL of this solution, repre-
SUBSTANCES senting 259 of the Trisilicate, in a tared platinum dish to
Sample solution: Nominally 2 mg/mL of dextrose dryness, and ignite gently to constant went the weight of
(CsH120¢ - H2O) in water from a suitable volume of In- the residue does not exceed 38.0 mg (1.5%),
jection containing 1.0 g of dextrose in water Chloride (221)—A 20-mL portion of the diluted filtrate pre-
Instrumental conditions pared in the test for Soluble salts, representing 1 g of Mag-
Mode: UV nesium Trisilicate, shows no more chloride than corresponds
Analytical wavelength: 284 nm to 0.75 mL of 0.020 N hydrochloric acid (0.055%).
Cell: 1.cm Sulfate—Treat the residue obtained in the test for Soluble
Blank: Water salts with 2 mL of hydrofluoric acid, and evaporate on a
se
”
Analysis
a Samples: Sample solution and Blank
steam bath to dryness. Mix the residue with water, transfer
i] to a filter, and wash, using approximately 50 mL of water
— Acceptance criteria: Absorbance of the Sample solution
io) for the complete procedure. Heat the filtrate to boiling, and
° is NMT 0.25. add 0.1 mL of hydrochloric acid and 5 mL of barium chlo-
iS ride TS. Maintain the mixture near its ae point for
Sj SPECIFIC TESTS
1 hour, filter, wash the precipitate thorough y with water,
= e BACTERIAL ENDOTOXINS TEST (85): NMT 0.039 USP Endo-
dry, and ignite to constant weight: the weight of the resi-
a toxin Unit/mg of magnesium sulfate
due does not exceed 30 mg (0.5%).
“” e PH (791): 3.5-6.5
> e OTHER REQUIREMENTS: |t meets the requirements in /njec- Free alkali—Add 2 dropsof phenolpitialeln TS to 20 mL
tions and Implanted Drug Products (1). of the diluted filtrate prepared in the test for Soluble salts,
representing 1 g of the Trisilicate: if a pink color is pro-
ADDITIONAL REQUIREMENTS duced, not more than 1.0 mL of 0.10 N hydrochloric acid is
e PACKAGING AND STORAGE: Preserve in single-dose glass or required to discharge it.
plastic containers. Glass containers are preferably of Type Arsenic, Method | (211): 8 ppm.
1 or Type Il glass.

Delete the following: Delete the following:

°o USP REFERENCE STANDARDS (11) °Heavy metals (231)—Boil 2.67 g with a mixture of 50 mL
USP Endotoxin RS of water and 5 mL of hydrochloric acid for 20 minutes, add-
ing water to maintain the volume during the boiling. Add
@ (CN 14-May-2018)
ammonium hydroxide until the mixture ts only slightly acid
to litmus paper. Filter with the aid of suction, and wash
with 15 to 20 mL of water, combining the washing with the
original filtrate. Add 2 drops of phenolphthalein TS, then
add aslight excess of 6 N ammonium hydroxide. Discharge
Magnesium Trisilicate the pink color with dilute eens acid (1 in 100), then
2MgO - 3SiO2 - xH2O(anhydrous) 260.86 add 8 mL of dilute hydrochloric acid (1 in 100). Dilute with
Silicic acid (H4Si3Os), magnesium salt (1:2), hydrate. water to 100 mL, and use 25 mL of the solution for the test:
Magnesium silicate hydrate (Mg2Si3Os - xH20) the limit is 0.003%.e soficiat 1-Jan-2018)
39365-87-2]. Acid-consuming capacity—Weigh accurately about
Anhydrous —[14987-04-3]. 200 mg into a glass-stoppered, 125-mL conical flask. Add
30.0 mL of 0.1 N hydrochloric acid VS and 20.0 mL of
» Magnesium Trisilicate is a compound of Mag- water. Place the flask in a bath maintained at 37°, and
nesium Oxide and silicon dioxide with varying shake the mixture occasionally during a period of 4 hours
but leave the mixture undisturbed during the last 15 min-
proportions of water. It contains not less than
USP 41 Official Monographs / Malathion 2521

utes of the heating period. Cool to room temperature. To
25.0 mL of the supernatant add methyl red TS, and titrate
the excess acid with 0.1 N sodium hydroxide VS. Oneg of
Magnesium Trisilicate, calculated on the anhydrous basis,
consumes not less than 140 mL and not more than 160 mL
of 0.10 N hydrochloric acid.
Assay for magnesium oxide—Weigh accurately about
1.5 g, and transfer to a 250-mL conical flask. Add 50.0 mL
of 1 N sulfuric acid VS, and digest on a steam bath for
1 hour. Cool to room temperature, add methyl orange TS,
and titrate the excess acid with 1 N sodium hydroxide VS.
Each mL of 1 N sulfuric acid is equivalent to 20.15 mg of
MgO.
Assay for silicon dioxide—Transfer about 700 mg of
Magnesium Trisilicate, accurately weighed, to a small plati-
num dish. Add 10 mL of 1 N sulfuric acid, and heat on a
steam bath to dryness, leaving the dish uncovered. Treat the
residue with 25 mL of water, and digest on a steam bath for
15 minutes. Decant the supernatant through an ashless filter
paper, with the aid of suction, and wash the residue, by
decantation, three times with hot water, passing the wash-
ings through the filter paper. pay transfer the residue to
the filter, and wash thoroughly with hot water. Transfer the
filter paper and its contents to the platinum dish previously
used. Heat to dryness, incinerate, ignite strongly for 30 min-
utes, cool, and weigh. Moisten the residue with water, and
add 6 mL of hydrofluoric acid and 3 drops of sulfuric acid.
Evaporate to dryness, ignite for 5 minutes, cool, and weigh:
the loss in weight represents the weight of SiOz.
Ratio of SiO, to MgO—Divide the percentage of SiO2 ob-
tained in the Assay for silicon dioxide by the percentage of
MgO obtained in the Assay for magnesium oxide: the quo-
tient obtained is between 2.10 and 2.37.
Magnesium Trisilicate Tablets
hydrochloric acid, with mixing. Heat the mixture to boiling,
cool, and filter into a 200-mL volumetric flask. Wash the
beaker with water, adding the washings to the filter. Add
water to volume, and mix. Transfer 20.0 mL of this solution
to a 400-mL beaker, add 180 mL of water and 20 mL of
triethanolamine, and stir. Add 10 mL of ammonia—am-
monium chloride buffer TS and 3 drops of an eriochrome
black indicator solution prepared by dissolving 200 mg of
eriochrome black T in a mixture of 15 mL of triethanolamine
and 5 mL of dehydrated alcohol, and mix. Cool the solution
to between 3° and 4° by immersion of the beaker in an ice
bath, then remove, and titrate with 0.05 M edetate diso-
dium VS to a blue endpoint. Perform a blank determination,
substituting 20 mL of water for the assay solution, and make
any necessary correction. Each mL of 0.05 M edetate diso-
dium consumed is equivalent to 6.521 mg of Mg2SizOs.
Malathion
a
_ ON
140 . j] i
ae i H a
CioHisO6PS2 330.36
Butanedioic acid, [(dimethoxyphosphinothioyl)-thio]-,
diethyl ester, (+)-;
Diethyl (£)-mercaptosuccinate, S-ester with O,O-dimethyl
phosphorodithioate [121-75-5].
DEFINITION
Malathion contains NLT 98.0% and NMT 102.0% of mala-
thion (CioHi9O¢PSz).
IDENTIFICATION
=
e A. INFRARED ABSORPTION (197F)
4)
x]

» Magnesium Trisilicate Tablets contain not less ASSAY =

¢ PROCEDURE
re}
than 90.0 percent and not more than 110.0 per- p=
cent of the labeled amount of Mg2Si3Os. Mobile phase: Methanol and water (50:30) i}
Internal standard solution: 0.6 mg/mL of propylpara- eo}=
Packaging and storage—Preserve in well-closed contain- ben in Mobile phase ES}

ers. Standard solution: 10 mg/mL of USP Malathion RS and be}

s
Identification— 0.06 mg/mL of propylparaben from Internal standard “

solution in methanol
A: Powder 1 Tablet, add 10 mL of 3 N hydrochloric acid Sample solution: 10 mg/mL of Malathion and
and 5 drops of methyl red TS, heat to boiling, add 6 N am- 0.06 mg/mL of propylparaben from Internal standard
monium hydroxide until the color of the solution changes solution in methanol
to deep yellow, then continue boiling for 2 minutes, and Chromatographic system
filter: the filtrate so obtained responds to the tests for Mag- (See Chromatography (621), System Suitability.)
nesium (191). Mode: LC
B: Wash the solids on the filter obtained in Identification Detector: UV 254 nm
test A with hot ammonium chloride solution (1 in 50), add Column: 4-mm x 30-cm; 10-4m packing L1
10 mL of 3 N hydrochloric acid, and filter. Transfer the filter Flow rate: 1 mL/min
paper and contents to a small platinum dish, ignite, cool in Injection volume: 20 uL
a desiccator, and weigh. Moisten the residue with water, System suitability
and add 6 mL of hydrofluoric acid. Evaporate to dryness, Sample: Standard solution
ignite for 5 minutes, cool in a desiccator, and weigh: a loss [Note—The relative retention times for propylparaben
of more than 10% in relation to the weight of the residue and malathion are about 0.6 and 1.0, respectively.]
from the initial ignition indicates SiO2. Suitability requirements
Disintegration (701): 10 minutes, simulated gastric fluid Resolution: NLT 4 for propylparaben and malathion
TS being substituted for water in the test. Relative standard deviation: NMT 2.0%
Uniformity of dosage units (905): meet the require- Analysis
ments. Samples: Standard solution and Sample solution
Calculate the percentage of malathion (CioHi9O¢PSz2) in
Acid-neutralizing capacity (301)—Not less than 5 mEq
the portion of Malathion taken:
of acid is consumed by the minimum single dose recom-
mended in the labeling. Result = (Ru/Rs) x (Cs/Cy) x 100
Assay—Weigh and finely powder not fewer than 20 Tablets.
Transfer an accurately weighed portion of the powder, Ru = peak response ratio of malathion to the
equivalent to about 1 g of magnesium trisilicate, to a internal standard from the Sample solution
beaker, add 20 mL of water, and slowly add 40 mL of 3N
 2522 Malathion / Official Monographs USP 41

Rs = peak response ratio of malathion to the ASSAY

internal standard from the Standard solution ¢ PROCEDURE
Gs = concentration of USP Malathion RS in the Solution A: Methyl ethyl ketone and n-hexane (4:1)
Standard solution (mg/mL) Internal standard solution: 2 mg/mL of parathion in
Cy = concentration of Malathion in the Sample Solution A
solution (mg/mL) Standard stock solution: 2mg/mL of USP Malathion
Acceptance criteria: 98.0%-102.0% RS in Solution A
Standard solution: 0.4 mg/mL of USP Malathion RS
IMPURITIES from the Standard stock solution and 0.4 mg/mL of
e LIMIT OF ISOMALATHION parathion from the Internal standard solution in Solution
Mobile phase: Methanol and water (50:30) A
Standard solution: 0.1 mg/mL of USP Isomalathion RS Sample solution: Nominally 0.4 mg/mL of malathion
in methanol and 0.4 mg/mL of parathion in Solution A, prepared as
Sample solution: 20 mg/mL of Malathion in methanol follows. Transfer a volume of Lotion, equivalent to
Chromatographic system 10 mg of malathion, to a 25-mL volumetric flask. Add
(See Chromatography (621), System Suitability.) 5.0 mL of Internal standard solution, and dilute with So-
Mode: LC lution A to volume.
Detector: UV 210 nm Chromatographic system
Column: 4-mm x 30-cm; 10-~4m packing L1 (See Chromatography (621), System Suitability.)
Flow rate: 1 mL/min Mode: GC
Injection volume: 20 pL Detector: Flame ionization
System suitability Column: 2-mm x 1.8-m glass; packed with 5% G6
Sample: Standard solution liquid phase on 110- to 120-mesh support S1A
[Note—The relative retention times for isomalathion Temperatures
and malathion are about 0.5 and 1.0, respectively.] Injector: 230°
Suitability requirements Detector: 250°
Relative standard deviation: NMT 2.0% Column: 190°
Analysis Carrier gas: Dry nitrogen
Samples: Standard solution and Sample solution Flow rate: 15 mL/min
Calculate the percentage of isomalathion in the portion Injection volume: 1 uL
of Malathion taken: System suitability
Sample: Standard solution
Result = (ru/rs) x (Cs/Cu) x 100 [Note—The relative retention times for malathion and
parathion are 1.0 and about 1.3, respectively.]
tu = peak response of isomalathion from the Suitability requirements
Sample solution Resolution: NLT 3.0 between malathion and
rs = peak response of isomalathion from the parathion
Standard solution Relative standard deviation: NMT 2.0%
fred
al
Cs = concentration of USP Isomalathion RS in the
a Standard solution (mg/mL)
Analysis
J— Gu = concentration of Malathion in the Sample
Samples: Standard solution and Sample solution
aD Calculate the percentage of the labeled amount of
re} solution (mg/mL) malathion (CjoHi9O6PS2) in the portion of Lotion taken:
- Acceptance criteria: NMT 0.3%
3 Result = (Ru/Rs) x (Cs/Cu) x 100
= SPECIFIC TESTS
a © SPECIFIC GRAVITY (841): Between 1.220 and 1.240 Ru = peak response ratio of malathion to the
al) e WATER DETERMINATION, Method | (921): NMT 0.1% internal standard from the Sample solution
=) Rs = peak response ratio of malathion to the
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight, light-resistant internal standard from the Standard solution
containers. Gs = concentration of USP Malathion RS in the
e USP REFERENCE STANDARDS (11) Standard solution (mg/mL)
USP Isomalathion RS Cu = nominal concentration of malathion in the
Butanedioic acid, [[methoxy(methylthio)phosphinyl] Sample solution (mg/mL)
thio]-, diethyl ester. Acceptance criteria: 90.0%-110.0%
CroHisO6PS2 330.36 OTHER COMPONENTS
USP Malathion RS e CONTENT OF ISOPROPYL ALCOHOL
Internal standard solution: Ethyl acetate and dehy-
drated alcohol (4:1)
Standard solution: Transfer 2.0 mL of isopropyl! alcohol
and 5.0 mL of Internal standard solution to a 200-mL
Malathion Lotion volumetric flask, and dilute with ethyl acetate to
volume.
DEFINITION Sample solution: Transfer a volume of Lotion, equiva-
Malathion Lotion is Malathion in a suitable isopropyl! alcohol lent to 2.0 mL of isopropy! alcohol, to a 200-mL volu-
vehicle. It contains NLT 90.0% and NMT 110.0% of the metric flask. Add 5.0 mL of Internal standard solution,
labeled amount of malathion (CioHi906PSz2). and dilute with ethyl acetate to volume.
Chromatographic system
IDENTIFICATION (See Chromatography (621), System Suitability.)
e A. The retention time of the major peak for malathion of Mode: GC
the Sample solution corresponds to that of the Standard Detector: Flame ionization
solution, both relative to the internal standard, as ob- Column: 2-mm x 1.8-m glass; packed with 110- to
tained in the Assay. 120-mesh support S2
 USP 41 Official Monographs / Manganese 2523

Temperatures and stir the solution, preferably using a magnetic stirrer.

Injector: 200° Begin the titration by adding 25 mL of 0.05 M edetate
Detector: 220° disodium VS, then add 10 mL of ammonia-ammonium
Column: 130° chloride buffer TS, and 1 mL of eriochrome black TS.
Carrier gas: Dry nitrogen Continue to titrate with 0.05 M edetate disodium VS to
Flow rate: 7 mL/min a blue endpoint. Each mL of 0.05 M edetate disodium
Injection volume: 1 pL is equivalent to 6.292 mg of MnCl.
System suitability Acceptance criteria: 98.0%-101.0% of MnCl, on the
Sample: Standard solution dried basis
Suitability requirements
Relative standard deviation: NMT 2.0% for the peak IMPURITIES
response ratio of isopropyl alcohol to the internal © CHLORIDE AND SULFATE, Sulfate (221)
standard Sample: 2.0g
Analysis Acceptance criteria: Shows no more sulfate than corre-
Samples: Standard solution and Sample solution sponds to 0.10 mL of 0.020N sulfuric acid (0.005%).
Calculate the percentage of the labeled amount of iso- e IRON (241)
propyl alcohol (C3HgO) in the portion of Lotion taken: Sample solution: 2.0g in 40 mL of water
Acceptance criteria: NMT 5 ppm
Result = (Ru/Rs) x (Cs/Cy) x 100 e ZINC
Sample solution: Dissolve 1 g in a mixture of 48 mL of
Ry = peak a ratio of isopropyl alcohol to the water and 2 mL of sulfuric acid.
internal standard from the Sample solution Analysis: To the Sample solution, add, slowly and with
Rs = peakresponse ratio of isopropyl alcohol to the constant agitation, 1 mL of potassium ferrocyanide solu-
internal standard from the Standard solution tion (1 in 100).
Gs = concentration of isopropyl alcohol in the Acceptance criteria: No turbidity is produced within 5
Standard solution (mg/mL) min.
Cu = nominal concentration of isopropyl alcohol in
the Sample solution (mg/mL)
Acceptance criteria: 90%-110% Delete the following:

ADDITIONAL REQUIREMENTS °° HEAVY METALS, Method | (231)

e PACKAGING AND STORAGE: Preserve in tight glass
containers.
© LABELING: The labeling states the percentage (v/v) of iso-
propyl alcohol in the Lotion.
e USP REFERENCE STANDARDS (11)
USP Malathion RS
Manganese Chloride
MnCl - 4H20 T9791
Manganese chloride (MnCl2) tetrahydrate;
Manganese (2+) chloride tetrahydrate [13446-34-9].
Anhydrous 125.84
[7773-01-5].
DEFINITION
Manganese Chloride contains NLT 98.0% and NMT 101.0%
of MnClz, calculated on the dried basis.
IDENTIFICATION
© A. IDENTIFICATION TESTS—GENERAL, Chloride (191): Yields
white, curdy precipitate of silver chloride with silver ni-
trate TS, which is insoluble in nitric acid. After being
washed with water, this precipitate is soluble in a slight
excess of 6 N ammonium hydroxide.
© B. IDENTIFICATION TESTS—GENERAL, Manganese (191):
Meets the requirements
ASSAY
e PROCEDURE
Sample: 425mg
Analysis: Transfer the Sample to a 400-mL beaker, dis-
solve in 25 mL of water, add 300 mg of ammonium
chloride and 0.5 g of hydroxylamine hydrochloride, and
swirl to dissolve. Warm slightly on a hot plate, and di-
lute with water to 100 mL. Add 3 mL of triethanolamine
Sample solution: Dissolve 6.0g in 30 mL of water.
[NotE—Use 25 mL of this solution in the Test Prepara-
tion and use the remaining 5.0 mL in preparing the
Standard Preparation.]
Acceptance criteria: NMT 5 ppme ‘official 1-jan-2018)
SPECIFIC TESTS
e INSOLUBLE MATTER a
wn
Sample solution: Transfer 10 g to a 250-mL beaker, i)
add 150 mL of water, cover the beaker, and heat to
boiling. <
}
Analysis: Digest the hot Sample solution on a steam =|
bath for 1 h, and pass through atared filtering crucible -)
of fine pore size. Rinse the beaker with hot water, pass- eo}3
ing the rinsings through the filter, and finally wash the By
filter with additional hot water. Dry the filter at 105°. a}
Acceptance criteria: The residue weighs NMT 0.5 mg
=
“
(0.005%).
e SUBSTANCES NOT PRECIPITATED BY AMMONIUM SULFIDE
Sample solution: Dissolve 2.0 g in 90 mL of water, add
5 mL of ammonium hydroxide, and warm the solution
to 80°. Pass a stream of hydrogen sulfide through the
solution for 30 min. Dilute with water to 100 mL, mix,
and allow the precipitate to settle. Decant the superna-
tant through afilter of fine pore size, and transfer
50.0 mL to an evaporating dish that previously has
been ignited and tared.
Analysis: Evaporate the filtrate to dryness, cool, add
0.5 mL of sulfuric acid, heat gently to remove the ex-
cess acid, and ignite at 800 + 25° for 15 min.
Acceptance criteria: The weight of the residue is NMT
2.0 mg (NMT 0.2% as sulfate).
PH (791)
Sample solution: 10g in 200 mL of carbon dioxide-
and ammonia-free water
Acceptance criteria: 3.5-6.0
e Loss ON DRYING (731): Dry a sample at 50° for 2 h, then
raise the temperature to 150° for 24 h: it loses
36.0%-38.5% of its weight.
 2524 Manganese / Official Monographs
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers.
Manganese Chloride Injection
» Manganese Chloride Injection is a sterile solu-
tion of Manganese Chloride in Water for Injec-
tion. It contains not less than 90.0 percent and
not more than 110.0 percent of the labeled
amount of manganese (Mn).
Packaging and storage—Preserve in single-dose or in
multiple-dose containers, preferably of Type | or Type Il
glass.
Labeling—Label the Injection to indicate that it is to be
diluted to the appropriate strength with Sterile Water for
Injection or other suitable fluid prior to administration.
Delete the following:
°USP Reference standards (11)—
USP Endotoxin RS
@ (CN I-May-2018)
Identification—The Assay preparation, prepared as di-
rected in the Assay, exhibits an absorption maximum at
about 279 nm when tested as directed for Procedure in the
Assay.
Bacterial Endotoxins Test (85)—It contains not more
than 0.45 USP Endotoxin Unit per ug of manganese.
pH (791): between 1.5 and 2.5.
Particulate Matter in Injections (788): meets the re-
i
”
quirements for small-volume injections.
rs
i] Other requirements—It meets the requirements under /n-
— requirements.
a) jections and Implanted Drug Products (1).
i}
is Assay—
S Sodium chloride solution—Dissolve 1.8 g of sodium chlo-
= ride in water, dilute with water to 2000 mL, and mix.
ie Manganese stock solution—Transfer 1.000 g of manganese
va) to a 1000-mL volumetric flask, dissolve in 20 mL of nitric
p=) acid, dilute with 0.1 N hydrochloric acid to volume, and
mix. This solution contains 1000 yg of manganese per mL.
Store in a polyethylene bottle.
Standard preparations—Pipet 10 mL of the Manganese
stock solution into a 500-mL volumetric flask, dilute with
water to volume, and mix. Transfer 4.0, 5.0, and 6.0 mL,
respectively, of this solution to separate 50-mL volumetric
flasks, containing 10 mL of Sodium chloride solution, dilute
the contents of each flask with water to volume, and mix.
These Standard preparations contain, respectively, 1.6, 2.0,
and 2.4 ug of manganese per mL.
Assay preparation—Transfer an accurately measured vol-
ume of Injection, equivalent to about 1 mg of manganese,
to a 100-mL volumetric flask, dilute with water to volume,
and mix. Pipet 10 mL of this solution into a 50-mL volumet-
ric flask. From the labeled amount of sodium chloride, if
any, in the Injection, calculate the amount, in mg, of so-
dium chloride in the initial dilution, and add sufficient So-
dium chloride solution to bring the total sodium chloride
content in this flask to 9 mg. Dilute with water to volume,
and mix.
Procedure—Concomitantly determine the absorbances of
the Standard preparations and the Assay preparation at the
manganese emission line of 279 nm, with a suitable atomic
absorption spectrophotometer (see Atomic Absorption Spec-
USP 41
troscopy (852)) equipped with a manganese hollow-cathode
lamp and an air—acetylene flame, using a dilution of Sodium
chloride solution (1:5) as the blank. Plot the absorbances of
the Standard preparations versus concentration, in [4g per
mL, of manganese, and draw the straight line best fitting
the three plotted points. From the graph so obtained, deter-
mine the concentration, in ug per mL, of manganese in the
Assay preparation. Calculate the quantity, in mg, of manga-
nese in each mL of the Injection taken Cthe Pomaalas
0.5C/V
in which C is the concentration, in jug per mL, of manga-
nese in the Assay preparation, and V is the volume, in mL, of
Injection taken.
Manganese Chloride for Oral Solution
» Manganese Chloride for Oral Solution contains
not less than 90.0 percent and not more than
110.0 percent of the labeled amount of manga-
nese (Mn). It may contain one or more suitable
flavors, sweetening agents, thickening agents,
and stabilizers.
Packaging and storage—Preserve in tight, light-resistant,
single-dose containers.
Labeling—tThe label contains directions for constitution of
the powder and states the amount of manganese in a given
volume of the Oral Solution obtained after constitution.
Identification—It meets the requirements of the tests for
Chloride (191) and for Manganese (191).
Uniformity of dosage units (905)—
FOR POWDER PACKAGED IN SINGLE-UNIT CONTAINERS: meets the
Deliverable volume (698)—
FOR POWDER PACKAGED IN MULTIPLE-UNIT CONTAINERS: meets
the requirements.
pH (791): between 6.0 and 8.0, when constituted to
300 mL with water.
Osmolality and Osmolarity (785): 230 mOsmol pH 6.0
to 8.0.
Assay—
Sodium chloride solution, Manganese stock solution, and
Standard preparations—Prepare as directed in the Assay
under Manganese Chloride Injection.
fssay preparation—Constitute the Manganese Chloride
for Oral Solution as directed in the labeling. Transfer about
25 mL, accurately measured, of the constituted Manganese
Chloride for Oral Solution to a 100-mL volumetric flask, di-
lute with water to volume, and mix. Proceed as directed for
Assay preparation in the Assay under Manganese Chloride In-
jection, beginning with “Pipet 10 mL of this solution.”
Procedure—Proceed as directed in the Assay under Man-
ganese Chloride Injection. Calculate the quantity, in Lg, of
manganese in each mL of the constituted for Oral Solution
taken by the formula:
500C/V
in which C is the concentration, in ug per mL, of the man-
ganese in the Assay preparation; and V is the volume, in mL,
of the constituted Manganese Chloride for Oral Solution
taken.
USP 41
Manganese Gluconate
oH OH oo
~ AM
oh on 2
Ci2H22MnOr4
Ci2H22MnOx4 - 2H20
Bis(D-gluconato-O',
02) manganese;
Manganese D-gluconate (1:2).
Anhydrous [6485-39-8].
DEFINITION
Manganese Gluconate is dried or contains two molecules of
water of hydration. It contains NLT 98.0% and NMT
102.0% of manganese gluconate (Ci2H22MnOy,,4), calcu-
lated on the anhydrous basis.
IDENTIFICATION
e A. IDENTIFICATION TESTS—GENERAL, Manganese (191): A
50-mg/mL solution meets the requirements.
e B. THIN-LAYER CHROMATOGRAPHY
Standard solution: 10 mg/mL of USP Potassium Gluco-
nate RS
Sample solution: 10 mg/mL of Manganese Gluconate,
heating in a water bath at 60°, if necessary, to dissolve
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.)
Mode: TLC
Adore: 0.25-mm layer of chromatographic silica
ge
Application volume: 5 uL
Developing solvent system: Alcohol, ethyl acetate,
ammonium hydroxide, and water (50:10:10:30)
Spray reagent: Dissolve 2.5 g of ammonium molyb-
date in 50 mL of 2 N sulfuric acid in a 100-mL volu-
metric flask. Add 1.0 g of ceric sulfate, swirl to dis-
solve, and dilute with 2 N sulfuric acid to volume.
Analysis
Samples: Standard solution and Sample solution
Develop the chromatogram until the solvent front has
moved about three-fourths of the length of the plate.
Remove the plate from the chamber, and dry at 110°
for 20 min. Allow to cool, and spray with Spray rea-
gent. Heat the plate at 110° for about 10 min.
Acceptance criteria: The principal spot of the Sample
solution corresponds in color, size, and R- value to that
of the Standard solution.
ASSAY
© PROCEDURE
pample: 700 mg of Manganese Gluconate
Blank: 50mL of water
Titrimetric system
(See Titrimetry (541).)
Mode: Direct titration
Titrant: 0.05 M edetate disodium VS
Endpoint detection: Visual
Analysis: Dissolve the Sample in 50 mL of water. Add
1g of ascorbic acid and 10 mL of ammonia-ammonium
chloride buffer TS and 0.1 mL of eriochrome black TS.
Titrate with the Titrant until the solution is deep blue in
color. Perform the blank determination.
Calculate the percentage of manganese gluconate
(Ci2H22MnQy,) in the Sample taken:
Result = {[(Vs — Vs) x M x F]/W} x 100
Vs = Titrant volume consumed by the Sample (mL)
Official Monographs / Manganese 2525
Ve = Titrant volume consumed by the Blank (mL)
M = actual molarity of the Titrant (mmol/mL)
E = equivalency factor, 445.2 mg/mmol
Ww = Sample weight (mg)
Acceptance criteria: 98.0%-102.0% on the anhydrous
basis
IMPURITIES
445.23 ¢ CHLORIDE AND SULFATE, Chloride (221)
481.26 Standard: 0.70 mL of 0.020 N hydrochloric acid
Sample: 1.0 g of Manganese Gluconate
Acceptance criteria: NMT 0.05%
© CHLORIDE AND SULFATE, Sulfate (221)
Standard: 4.0 mL of 0.020 N sulfuric acid
Sample: 2.0 g of Manganese Gluconate
Acceptance criteria: NMT 0.2%
Delete the following:
°e HEAVY METALS (231)
Test preparation: Dissolve 1 g of Manganese Gluconate
in 10 mL of water, add 6 mL of 3 N hydrochloric acid,
and dilute with water to 25 mL.
Acceptance criteria: NMT 20 ppMe (oticial 1jan-2018)
e LEAD
[Note—For the preparation of all aqueous solutions and
for the rinsing of glassware before use, use water that
has been passed through a strong-acid, strong-base,
mixed-bed ion-exchange resin. Select all reagents to
have as low a content of lead as practicable, and store
all reagent solutions in containers of borosilicate glass.
Cleanse glassware before use by soaking in warm 8 N
nitric acid for 30 min and by rinsing with deionized
water.]
Ascorbic acid-sodium iodide solution: 100 mg/mL of
ascorbic acid and 192.5 mg/mL of sodium iodide
Trioctylphosphine oxide solution: 50 mg/mL of trioc-
tylphosphine oxide in 4-methyl-2-pentanone. (=
“n
[CAUTION—This solution causes irritation. Avoid contact a)
with eyes, skin, and clothing. Take special precautions
in disposing of unused portions of solutions to which ES
i}
this reagent is added.] s
Standard solution: Transfer 5.0 mL of lead nitrate stock °
solution TS, to a 100-mL volumetric flask. Dilute with to)
=
water to volume. Transfer 2.0 mL of the resulting solu- i
ao}
tion to a 50-mL volumetric flask. Add 10 mL of 9 N hy- =
drochloric acid and 10 mL of water. Add 20 mL of As- as
corbic acid-sodium iodide solution and 5.0 mL of
Trioctylphosphine oxide solution. Shake for 30 s, and al-
low to separate. Add water to bring the organic solvent
layer into the neck of the flask, shake again, and allow
to separate. The organic layer is the Standard solution,
and it contains 2.0 ug/mL of lead.
Sample solution: To a 50-mL volumetric flask add 1.0 g
of Manganese Gluconate, 10 mL of 9 N hydrochloric
acid, 10 mL of water, 20 mL of Ascorbic acid-sodium io-
dide solution, and 5.0 mL of Trioctylphosphine oxide solu-
tion. Shake for 30 s, and allow to separate. Add water
to bring the organic solvent layer into the neck of the
flask, shake again, and allow to separate. The organic
layer is the Sample solution.
Blank: To a 50-mL volumetric flask add 10 mL of 9N
hydrochloric acid, 10 mL of water, 20 mL of Ascorbic
acid-sodium iodide solution, and 5.0 mL of Trioctylphos-
phine oxide solution. Shake for 30 s, and allow to sepa-
rate. Add water to bring the organic solvent layer into
the neck of the flask, shake again, and allow to sepa-
rate. The organic layer is the Blank, and it contains
0 yg/mL of lead.
 2526 Manganese / Official Monographs
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometry
Analytical waveleretns 283.3 nm
Lamp: Lead hollow-cathode
Flame: Air-acetylene
System suitability
Samples: Standard solution and Blank
Suitability requirements: The absorbance of the Stan-
dard solution and the absorbance of the Blank are sig-
nificantly different.
Analysis
Samples: Standard solution, Sample solution, and Blank
Concomitantly determine the absorbances of the Blank,
Standard solution, and the Sample solution. Use the
Blank to set the instrument to zero.
Acceptance criteria: The absorbance of the Sample so-
lution does not exceed that of the Standard solution
(NMT 10 ppm).
e REDUCING SUBSTANCES
Semple: 1.0 g of Manganese Gluconate
Blank: Proceed as directed in the Analysis, omitting the
Sample.
Titrimetric system
(See Titrimetry (541).)
Mode: Residual titration
Titrant: 0.1 N iodine VS
Back-titrant: 0.1 N sodium thiosulfate VS
Endpoint detection: Visual
Analysis: Transfer the Sample to a 250-mL conical flask,
dissolve in 10 mL of water, and add 25 mL of alkaline
cupric citrate TS. Cover the flask, boil gently for 5 min,
accurately timed, and cool rapidly to room tempera-
ture. Add 25 mL of 0.6 N acetic acid, 10.0 mL of Ti-
trant, and 10 mL of 3 N hydrochloric acid, and titrate
with Back-titrant, adding 3 mL of starch TS as the
endpoint is approached. Perform the blank
a)
determination.
3 Calculate the percentage of reducing substances (as dex-
Q
S
—
trose) in the Sample taken:
=)
io} Result = {[(Vs — Vs) x N x FJ/W} x 100
S e SUBSTANCES NOT PRECIPITATED BY AMMONIUM SULFIDE
) Ve = Back-titrant volume consumed by the Blank
= (ml)
re Vs = Back-titrant volume consumed by the Sample
a pass hydrogen sulfide through the solution for about 30
—) (ml)
N = Back-titrant normality (mEq/mL)
Fe = equivalency factor, 27 mg/mEq
Ww = Sample weight (mg)
Acceptance criteria: NMT 1.0%
SPECIFIC TESTS
e WATER DETERMINATION, Method | (921)
Analysis: Proceed as directed in the chapter. Maintain
the mixture containing the Test preparation at 50°, and
stir for 30 min before titrating with the Reagent.
Acceptance criteria
Where labeled as the dried form: 3.0%-9.0%
Where labeled as the dihydrate: 6.0%-9.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed
containers.
USP 41
e LABELING: The label indicates whether it is the dried or
the dihydrate form.
e USP REFERENCE STANDARDS (11)
USP Potassium Gluconate RS
Manganese Sulfate
MnSO, - H20 169.02
Sulfuric acid, manganese(2+) salt (1:1) monohydrate;
Manganese(2+) sulfate (1:1) monohydrate [10034-96-5].
Anhydrous 151.00
[7785-87-7].
DEFINITION
Manganese Sulfate contains NLT 98.0% and NMT 102.0%
of MnSOq - H20.
IDENTIFICATION
e A. IDENTIFICATION TESTS—GENERAL, Manganese (191)
Sample solution: 100 mg/mL
Acceptance criteria: Meets the requirements
e B. IDENTIFICATION TESTS—GENERAL, Sulfate (191)
Sample solution: 100 mg/mL
Acceptance criteria: Meets the requirements
ASSAY
e PROCEDURE
Sample: 350mg
Analysis: Dissolve the Sample in 200 mL of water. Add
10 mg of ascorbic acid. Begin the titration by adding
25 mL of 0.05 M edetate disodium VS using a suitable
buret, then add 10 mL of ammonia—ammonium chlo-
ride buffer TS and 0.15 mL of eriochrome black TS.
Complete the titration with 0.05 M edetate disodium
VS to a blue endpoint. Each mL of 0.05 M edetate diso-
dium is equivalent to 8.451 mg of MnSO,- H20.
Acceptance criteria: 98.0%-102.0%
IMPURITIES
Sample: 2.0g
Analysis: Dissolve the Sample in 90 mL of water, add
5 mL of ammonium hydroxide, warm the solution, and
min. Dilute with water to 100 mL, mix, and allow the
Pee to settle. Decant the supernatant through a
ilter, transfer 50 mL of the clear filtrate to a tared dish,
evaporate to dryness, and ignite, gently at first and fi-
nally at 800 + 25°.
Acceptance criteria: The weight of the residue does
not exceed 5 mg (NMT 0.5%).
SPECIFIC TESTS
e Loss ON IGNITION (733)
Analysis: Ignite a sample at 450° to constant weight: it
loses 10.0%-13.0% of its weight.
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers.
Store at controlled room temperature.
Manganese Sulfate Injection
» Manganese Sulfate Injection is a sterile solution
of Manganese Sulfate in Water for Injection. It
contains not less than 90.0 percent and not more
than 110.0 percent of the labeled amount of
manganese (Mn).
USP 41 Official Monographs / Mannitol 2527

Packaging and storage—Preserve in single-dose or in a dry residue is obtained. Non-sticky, white, or slightly
multiple-dose containers, preferably of Type | or Type Il yellowish powders are obtained.
glass. Analysis: Record new spectra using the residues from
Labeling—Label the Injection to indicate that it is to be the Standard solution and the Sample solution.
diluted to the appropriate strength with Sterile Water for
Injection or other suitable fluid prior to administration. ASSAY
¢ PROCEDURE
Mobile phase: Degassed water
Delete the following: iey! suitability solution A: 25.0 mg/mL each of sor-
ito! and USP Mannitol RS
°USP Reference standards (11)— System suitability solution B: 1.0 mg/mL each of mal-
USP Endotoxin RS titol and isomalt
Standard solution A: 50.0maiml, of USP Mannitol RS
@ (CN 1-May-2018)
Standard solution B: Dilute 2.0 mL of the Sample solu-
Identification—The Assay preparation, prepared as di- tion with water to 100.0 mL.
rected in the Assay, exhibits an absorption maximum at Standard solution C: Dilute 0.5 mL of Standard solution
about 279 nm when tested as directed for Procedure in the B with water to 20.0 mL.
Assay. Sample solution: 50.0 mg/mL of Mannitol
Bacterial Endotoxins Test (85)—It contains not more Chromatographic system
than 0.45 USP Endotoxin Unit per ug of manganese. (See Chromatography (621), System Suitability.)
pH (791): between 2.0 and 3.5. Mode: LC
Particulate Matter in Injections (788): meets the re- Detector: Refractive index
quirements for small-volume injections. Column: 7.8-mm x 30-cm; packing L19
Temperatures
Other requirements—\t meets the requirements under /n- Detector: 40° (maintain at a constant temperature)
jections and Implanted Drug Products (1). Column: 85 + 2°
Assay— Flow rate: 0.5 mL/min
Sodium chloride solution, Manganese stock solution, and Injection volume: 20 uL
Standard preparations—Prepare as directed in the Assay Run time: NLT 1.5 times the retention time of the
under Manganese Chloride Injection. mannitol peak. [NoTE—The retention time for mannitol
Assay preparation—Transfer an accurately measured vol- is about 20 min.]
ume of Injection, equivalent to about 1 mg of manganese, System suitability
to a 100-mL volumetric flask, dilute with water to volume, Samples: System suitability solution A, System suitability
and mix. Pipet 10 mL of this solution into a S0-mL volumet- solution B, Standard solution B, and Standard solution C
ric flask, dilute with water to volume, and mix. Suitability requirements
Procedure—Proceed as directed for Procedure in the Assay Resolution: NLT 2.0 between sorbitol and mannitol,
under Manganese Chloride Injection. System suitability solution A
Analysis Joy
Samples: Standard solution A and Sample solution wn
Calculate the percentage of mannitol (CsHi40¢) in the uv
portion of Mannitol taken: E
Mannitol °
Result = (ru/rs) x (Cs/Cu) x 100 |
Portions of the monograph text that are national USP text, °
io}
and are not part of the harmonized text, are marked with tu peak response from the Sample solution bl
symbols (*¢) to specify this fact. Ey
Is peak response from Standard solution A me]
Cs concentration of USP Mannitol RS in Standard 7
OH OH solution A (mg/mL) a
,
HOt Bye
4 i.
~
OH
Cy = concentration of the Sample solution (mg/mL)
Acceptance criteria: 97.0%-102.0% on the dried basis
IMPURITIES
CoH1406 182.17 © RELATED SUBSTANCES
D-Mannitol [69-65-8]. Mobile phase, System suitability solution A, System
suitability solution B, Standard solution B, Standard
DEFINITION solution C, Sample solution, Chromatographic sys-
Mannitol contains NLT 97.0% and NMT 102.0% of manni- tem, and System suitability: Proceed as directed in
tol (CeéH14O¢), calculated on the dried basis. the Assay.
IDENTIFICATION Analysis
e A. INFRARED ABSORPTION (197K) Samples: Standard solution B, Standard solution C, and
If the spectra shows differences, proceed as directed. Sample solution
Standard solution: Dissolve 25 mg of USP Mannitol RS Acceptance criteria: See Table 1 for the relative reten-
in a glass vial with 0.25 mL of distilled water without tion times.
heating. The solution is clear. Evaporate to dryness by
one of the following methods. Heat in a microwave Table 1
oven with a power range of 600-700 W for 20 min, or Relative
heat in an oven at 100° for 1 h, then gradually apply Retention
vacuum until a dry residue is obtained. Non-sticky,
white, or slightly yellowish powders are obtained.
Sample solution: Dissolve 25 mg of Mannitol in a glass
vial with 0.25 mL of distilled water without heating. The
solution is clear. Evaporate to dryness by one of the
following methods. Heat in a microwave oven with a
power range of 600-700 W for 20 min, or heat in an
oven at 100° for 1 h, then gradually apply vacuum until
 2528 Mannitol / Official Monographs
[Nott—Impurity A—Sorbitol; Impurity B—Maltitol; Im-
purity C—Isomalt.]
[Note—lIsomalt elutes in two peaks.]
[NoTt—Coelution of impurity B and the second peak of
impurity C may be observed.]
Disregard limit: NMT 0.05%; any peak NMT the area
of the principal peak obtained with Standard solution C
Sorbitol: NMT 2.0%; NMT the area of the principal
peak obtained with Standard solution B
Sum of isomalt and maltitol: NMT 2.0%; NMT the
area of the principal peak obtained with Standard solu-
tion B
Unspecified impurities: NMT 0.10% for each impu-
rity; NMT twice the area of the principal peak ob-
tained with Standard solution C
Total impurities: NMT 2.0%; NMT the area of the
principal peak obtained with Standard solution B
e REDUCING SUGARS
Ferric sulfate solution: Dissolve 50 g of ferric sulfate in
an excess of water, add 200 mL of sulfuric acid, and
dilute with water to 1000 mL.
Copper sulfate solution: 69.2 mg/mL of copper sulfate
(CuSO, - 5H20) in water
Sodium potassium tartrate solution: Dissolve 173 g of
sodium potassium tartrate (C4H4aKNaOg - 4H20) and
50 g of sodium hydroxide in 400 mL of water. Heat to
boiling, allow to cool, and dilute with carbon dioxide-
free water to 500 mL.
Cupri-tartaric solution: Mix equal volumes of Copper
sulfate solution and Sodium potassium tartrate solution
immediately before use.
Sample: 7.0g
Analysis: To the Sample add 13 mL of water. Ror gently
with 40 mL of Cupri-tartaric solution for 3 min, and al-
low to stand for about 2 min. A precipitate is formed.
Pass through a sintered-glass filter (10-16 1m) coated
with diatomaceous earth or a sintered-glass filter (5-10
" Lum). Wash the precipitate with hot water (at about
<=
a 50°-60°) until the washing is no longer alkaline, and
£ pass the washings through the filter described above.
io)
3
Discard all the filtrate at this step. Immediately dissolve
the precipitate in 20 mL of Ferric sulfate solution, pass
< Standard solution: Pipet 3.0 mL of ferric chloride CS,
iS through the filter described above in a clean flask, and
wash the filter with 15-20 mL of water. Combine the
Ps washings and the filtrate, heat to 80°, and titrate with
a 0.02 M potassium permanganate VS.
va)
ma Acceptance criteria: NMT 0.1%, expressed as glucose;
NMT 3.2 mL of 0.02 M potassium permanganate VS is
required to change the color of the solution. The green
color turns to pink, and the color persists at least 10 s.
e NICKEL
Sample solution: Suspend 10.0 g of Mannitol in 30 mL
of dilute acetic acid [115-125 g/L of acetic acid
(C2H4O2)], add water, and shake to dissolve. Dilute with
water to 100.0 mL. Add 2.0 mL of a saturated solution
of ammonium pyrrolidinedithiocarbamate (CsHi2N2S2)
(about 10 g/L) and 10.0 mL of water-saturated methyl
isobutyl ketone (CeHi20, 4-methyl-2-pentanone), and
then shake for 30 s protected from bright light. Allow
the layers to separate, and use the methyl isobutyl ke-
tone layer.
Blank solution: Treat water-saturated methyl isobutyl
ketone as described for preparation of the Sample solu-
tion, omitting the mannitol.
Standard solutions: Prepare three reference solutions in
the same manner as the Sample solution but adding
0.5, 1.0, and 1.5 mL, respectively, of nickel standard
solution TS [10 ppm nickel (Ni)] in addition to the
10.0 g of the substance to be examined.
USP 41
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometry
Analytical wavelengttt 232.0 nm
Lamp: Nickel hollow-cathode
Flame: Air-acetylene
Analysis
Samples: Sample solution and Standard solutions
Set the zero of the instrument using the blank. Record
the average of the steady readings for each of the
Standard solutions and the Sample solution. Between
each measurement rinse with water, and ascertain that
the reading returns to zero with the blank. Plot the
absorbances of the Standard solutions and the Sample
solution versus the added quantity of nickel. Extrapo-
late the line joining the points on the graph until it
meets the concentration axis. The distance between
this point and the intersection of the axes represents
the concentration of nickel in the Sample solution.
Acceptance criteria: NMT 1 ug/g
SPECIFIC TESTS
e MELTING RANGE OR TEMPERATURE (741), Class |
Melting point: 165°-170°
© APPEARANCE OF SOLUTION
Hydrazine sulfate solution: 10.0 mg/mL of hydrazine
sulfate. Allow to stand for 4-6 h.
Methenamine solution: 2.5 g of methenamine in
25 mL of water, in a ground-glass-stoppered flask
Primary opalescent suspension: To the Methenamine
solution, add 25.0 mL of the Hydrazine sulfate solution.
Mix, and allow to stand for 24 h. This suspension is
stable for 2 months, provided it is stored in a glass
container free from surface defects. The suspension
must not adhere to the glass and must be well mixed
before use.
Opalescence standard: Dilute 15.0 mL of the Primary
opalescent suspension with water to 1000.0 mL. This sus-
pension is freshly prepared and may be stored for up to
24h.
Reference suspension: To 5.0 mL of Opalescence stan-
dard add 95.0 mL of water. Mix, and shake before use.
3.0 mL of cobaltous chloride CS, and 2.4 mL of cupric
sulfate CS into a 1-L volumetric flask. Dilute with 1%
(w/v) hydrochloric acid to volume.
Sample solution:
Analysis:
100.0 mg/mL of Mannitol
Compare the color, clarity, and opalescence
of equal volumes of the Reference suspension, Standard
solution, and Sample solution.
Acceptance criteria: The Sample solution is clear and
colorless; its clarity is the same as that of water, or its
opalescence is not more pronounced than that of the
Reference suspension, and it is not more intensely
colored than the Standard solution.
e Loss ON DRYING (731)
Sample: 1.000g
Analysis: Dry the Sample at 105° for 4 h.
Acceptance criteria: NMT 0.5%
© CONDUCTIVITY
Sample: 20.0g
Analysis: Dissolve the Sample in carbon dioxide-free
water prepared from distilled water by heating to
40°-50°, and dilute with the same solvent to 100 mL.
After cooling, measure the conductivity of the solution
while gently stirring with a magnetic stirrer.
Acceptance criteria: NMT 20 pS/cm at 25°
¢ MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
count (TAMC) is NMT 103 cfu/g, and the total combined
molds and yeasts count is NMT 102 cfu/g. It meets the
requirements of the test for absence of Escherichia coli.
If intended for use in the manufacture of parenteral dos-
age forms, the TAMC is NMT 102 cfu/g.
USP 41
¢ BACTERIAL ENDOTOXINS TEST (85): {f intended for use in
the manufacture of parenteral dosage forms without a
further appropriate procedure for the removal of bacterial
endotoxins, less than 4 IU/g for parenteral dosage forms
with a concentration of 100 g/L or less of mannitol, and
less than 2.5 |U/g for parenteral dosage forms with a
concentration of more than 100 g/L of mannitol
ADDITIONAL REQUIREMENTS
© *PACKAGING AND STORAGE: Preserve in well-closed con-
tainers.¢
e LABELING
The label states, where applicable, the maximum con-
centration of bacterial endotoxins.
The label states, where applicable, that the substance is
plteioe for use in the manufacture of parenteral dosage
orms.
Change to read:
° USP REFERENCE STANDARDS (11)
@ (CN }-May-2018)
USP Mannitol RS
Mannitol Injection
» Mannitol Injection is a sterile solution, which
may be supersaturated, of Mannitol in Water for
Injection. It may require warming or autoclaving
before use if crystallization has occurred. It con-
tains not less than 95.0 percent and not more
than 105.0 percent of the labeled amount of
mannitol (CsH1406¢). It contains no antimicrobial
agents.
Packaging and storage—Preserve in single-dose glass or
plastic containers. Glass containers are preferably of Type |
or Type Il glass.
Labeling—tThe label states the total osmolar concentration
in mOsmol per L. Where the contents are less than 100 mL,
or where the label states that the Injection is not for direct
injection but is to be diluted before use, the label alterna-
tively may state the total osmolar concentration in mOsmol
er mL. The label also states that it should be warmed
efore use to dissolve any crystals that may have formed.
Change to read:
UsP Reference standards (11)—
@ (CN 13-May-2018)
USP Mannitol RS
Identification—
A: Evaporate a portion of Injection on a steam bath to
dryness, and od the residue at 105° for 4 hours. To 3 mL of
freshly prepared solution of catechol in water (1 in 10) add
6 mL of sulfuric acid with cooling. Place 3 mL of this solu-
tion in each of two separate test tubes. To one tube add
0.3 mL of water (reagent blank) and to the other add
0.3 mL of a solution of it in water (1 in 10). Heat the tubes
over an open flame for about 30 seconds: the solution in
the tube containing mannitol is dark pink or wine red, and
Beesclution in the tube containing the reagent blank is light
pink.
B: The retention time for the major peak in the chromat-
ogram of the Assay preparation corresponds to that in the
chromatogram of the Standard preparation, as obtained in
the Assay.
Official Monographs / Mannitol 2529
Specific rotation (781)—+137° to +145°. Transfer an ac-
curately measured volume of Injection, equivalent to about
1g of mannitol as determined by the Assay, to a 100-mL
aalimetie flask. Add 40 mL of a 1-in-10 ammonium molyb-
date solution, previously filtered if necessary. Add 20 mL of
1N sulfuric acid, and dilute with water to volume.
Bacterial Endotoxins Test (85)—It contains not more
than 0.04 USP Endotoxin Unit per mg of mannitol where
the labeled amount of mannitol in the Injection is 10% or
less, and not more than 2.5 USP Endotoxin Units per g of
mannitol where the labeled amount of mannitol in the In-
jection is greater than 10%.
pH (791): between 4.5 and 7.0, determined potentiomet-
rically, on a portion to which 0.30 mL of saturated potas-
sium chloride solution has been added for each 100 mL,
and which previously has been diluted with water, if neces-
sary, to a concentration of not more than 5% of mannitol.
Particulate Matter in Injections (788): meets the re-
quirements for small-volume injections.
Other requirements—It meets the requirements under In-
jections and Implanted Drug Products (1).
Assay—
Mobile phase—Use degassed water.
Resolution solution—Dissolve sorbitol and USP Mannitol
RS in water to obtain a solution having concentrations of
about 4.8 mg per mL of each.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is Pe with a refractive index
detector that is maintained at a constant temperature and a
4-mm x 25-cm column that contains packing L19. The col-
umn temperature is maintained at a temperature between
30° and 85° controlled within +2° of the selected tempera-
ture, and the flow rate is about 0.5 mL per minute. Chro-
matograph the Standard preparation, and record the peak
responses as directed for Procedure: the relative standard
deviation for replicate injections is not more than 2.0%. Ina (=
similar manner, chromatograph the Resolution solution: the “
resolution, R, between the sorbitol and mannitol peaks is a)
not less than 2.0. =
Standard preparation—Dissolve an accurately weighed i}
=
quantity of USP Mannitol RS in water, and dilute quantita- i}
tively with water to obtain a solution having a known con- a=
centration of about 5 mg per mL. »
Assay preparation—Transfer an accurately measured vol- a}
ume of Injection, equivalent to about 500 mg of mannitol,
my
ww
to a 100-mL volumetric flask, dilute with water to volume,
and mix.
Procedure—Separately inject equal volumes (about 20 pL)
of the Assay preparation and the Standard preparation into
the chromatograph, record the chromatograms, and meas-
ure the responses for the major peaks. Calculate the quan-
tity, in mg, of mannitol (CeH14O¢) in each mL of the Injec-
tion taken by the formula:
100(C/V)(ru / 15)
in which V is the volume, in mL, of Injection taken; and the
other terms are as defined therein.
Mannitol in Sodium Chloride Injection
» Mannitol in Sodium Chloride Injection is a ster-
ile solution of Mannitol and Sodium Chloride in
Water for Injection. It contains not less than
95.0 percent and not more than 105.0 percent of
the labeled amounts of C6Hi40¢ and NaCl. It
contains no antimicrobial agents.
 2530 Mannitol / Official Monographs USP 41

Labeling—The label states the total osmolar concentration ASSAY

in mOsmol per L. Where the contents are less than 100 mL, e PROCEDURE
or where the label states that the Injection is not for direct Sample solution: 600 mg of Maprotiline Hydrochloride
injection but is to be diluted before use, the label alterna- in 25 mL of mercuric acetate TS
tively may state the total osmolar concentration in mOsmol Blank: Mercuric acetate TS
per mL. Titrimetric system
(See Titrimetry (541).)
Mode: Potentiometric
Change to read: Titrant: 0.1 N perchloric acid VS
Analysis: Titrate the Sample solution with Titrant using a
USP Reference standards (11)— glass electrode and a calomel electrode containing satu-
@ (CN 1-May-2018) rated lithium chloride in glacial acetic acid. Perform a
USP Mannitol RS blank determination. Each mL of 0.1 N perchloric acid
Identification— is equivalent to 31.39 mg of maprotiline hydrochloride
A: Evaporate a portion of Injection on a steam bath to (C2oHa3N + HCl).
dryness, and dry the residue at 105° for 4 hours: the residue Acceptance criteria: 99.0%-101.0% on the dried basis
responds to the Identification test A under Mannitol Injection. IMPURITIES
B: It responds to the tests for Sodium (191) and for Chio- © RESIDUE ON IGNITION (281); NMT 0.1%
ride (191).
Bacterial Endotoxins Test (85)—It contains not more
than 0.04 USP Endotoxin Unit per mg of mannitol. Delete the following:
pH (791): between 4.5 and 7.0. °e HEAVY METALS, Method II (231): NMT 10 ppme circ.
Other requirements—It meets the requirements for Pack- Jan-2018)
aging and storage under Mannitol Injection. It meets also the ¢@ ORGANIC IMPURITIES
requirements under Injections and Implanted Drug Products Mobile phase: Dissolve 0.6 g of ammonium acetate in
Ts 200 mL of water, and add 2'mL of a 70-g/L ammonia
Assay for mannitol—Proceed with Injection as directed in solution, 150 mL of 2-propanol, and 650 mL of metha-
the Assay under Mannitol Injection. nol. The resulting apparent pH value is 8.2-8.4.
Assay for sodium chloride—Proceed with Injection as di- Standard solution: 2 g/mL of USP Maprotiline Hydro-
rected in the Assay under Sodium Chloride Injection. chloride RS in Mobile phase
System suitability solution: 1. mg/mL of USP Mapro-
tiline Hydrochloride RS and 0.1 mg/mL of USP Mapro-
tiline Related CompoundD RS in Mobile phase
Sample solution: 1 mg/mL of Maprotiline Hydrochlo-
Maprotiline Hydrochloride tide in Mobile phase
Chromatographic system
ea
”
(See Chromatography (621), System Suitability.)
s Mode: LC
i
— Detector: UV 272 nm
aD
i) Column: 4.6-mm x 25-cm; 5-4um packing L1
iS Flow rate: 1 mL/min
S Injection volume: 20 uL
= Run time: 1.5 times the retention time of maprotiline
a System suitability
a) CooH23N - HCI 313.86 Sample: System suitability solution
= 9,10-Ethanoanthracene-9(10H)-propanamine, N-methyl-, Suitability requirements
hydrochloride; [Note—See Table 7 for relative retention times.]
N-Methyl-9, 10-ethanoanthracene-9(10H)-propylamine hy- Resolution: 1.8-3.2 between the maprotiline related
drochloride [10347-81-6]. compound D and maprotiline peaks
[NoTe—lf necessary, adjust the pH of the Mobile phase,
DEFINITION in steps of 0.1 pH unit, by adding a 50% v/v solution
Maprotiline Hydrochloride contains NLT 99.0% and NMT
101.0% of the labeled amount of maprotiline hydrochlo- of acetic acid if the resolution is ee than 1.8, or by
ride (C2oH23N - HCI), calculated on the dried basis. adding a 70-g/L solution of ammonia if the resolution
is greater than 3.2.]
IDENTIFICATION Analysis
© A. INFRARED ABSORPTION (197K) Samples: Standard solution and Sample solution
e B. ULTRAVIOLET ABSORPTION (197U) Calculate the percentage of each impurity in the por-
Sample solution: 100 ug/mL in methanol tion of Maprotiline Hydrochloride taken:
Acceptance criteria: Absorptivities at 266 nm and 272
nm, calculated on the dried basis, do not differ by Result = (ru/rs) x (Cs/Cu) x 100
more than 3.0%.
ry = peak area of any impurity from the Sample
solution
Change to read: rs = peak area of maprotiline from the Standard
solution
© C. IDENTIFICATION TESTS—GENERAL (191), Chloride Gs = concentration of USP Maprotiline
Sample solution: 5 mg/mL Hydrochloride RS in the Standard solution
Acceptance criteria: Responds to °tests A and Be «ni. (ng/mL)
May-2018) Cu = concentration of Maprotiline Hydrochloride in
the Sample solution (mg/mL)
Acceptance criteria: See Table 7. Disregard any peak
representing less than 0.05% of the area of the main
peak.
USP 41 Official Monographs / Maprotiline 2531

Table 1 Analysis
Samples: Standard solution and Sample solution
Relative Acceptance
In a suitable chromatographic chamber, place a vol-
Retention Criteria,
ume of the Developing solvent system sufficient to de-
Name Time NMT (%) velop a chromatogram. Place a beaker containin
Maprotiline acry- 25 mL of ammonium hydroxide in the bottom of the
laldehyde analog! 0.3 0.2 chamber, and allow it to equilibrate for 1 h. Apply
Maprotiline dimer? 0.5 0.2 Samples and allow the spots to mi Develop the
Desmethylmapro- chromatograms until the solvent front has moved
tiline? 0.7 0.2 three-fourths of the length of the plate, remove the
Maprotiline related plate from the developing chamber, mark the solvent
compound D¢ 0.8 0.2 front, and allow the solvent to evaporate. Expose the
Maprotiline 1.0 = plate to hydrogen chloride vapor for 30 min, and ex-
N-Methylmaprotilines 13 0.2
pose it to a high-intensity UV light irradiator (1000 to
1600 watts) for 5 min. [CAUTION—UV irradiators emit
Any individual un- _ UV radiation that is harmful to eyes and skin.] Com-
known impurit 0.10 pare the chromatograms under long-wavelength UV
Total impurities = 1.0 ight.
1 ep OOityrone 1 Oethancahdiacen 2 pacyacenyee (EP Impurity Acceptance criteria: The R; value of the principal spot
A). of the Sample solution corresponds to that of the Stan-
2 N-Methyl-N, N-bis[3-(9,1 0-dihydro-9,10-ethanoanthracen-9-yl)propyl]- dard solution.
amine (EP Impurity B).
e B. The retention time of the major peak in the Sample
3 3-(9,10-Dihydro-9,10-ethanoanthracen-9-yl)propan-1-amine (EP Impurity
solution corresponds to that of the Standard solution, as
°
4 3-(9,10-Dihydro-9, 10-ethanoanthracen-9-yl)-N-methylprop-2-en-1-amine obtained in the Assay.
(EP Impurity D).
$ 3-(9,10-Dihydro-9,10-ethanoanthracen-9-yl)-N, N-dimethylpropan-1- ASSAY
amine (EP Impurity E). ¢ PROCEDURE
Mobile phase: Dissolve 4 g of tetramethylammonium
SPECIFIC TESTS chloride in 495 mL of water, and add 500 mL of aceto-
e Loss ON DRYING (731) nitrile and 1 mL of phosphoric acid.
Sample: Dry a sample under vacuum at 80° to constant Standard solution: 0.75 mg/mL of USP Maprotiline Hy-
weight. drochloride RS prepared as follows. Transfer a suitable
Acceptance criteria: NMT 1.0% quantity of the USP Maprotiline Hydrochloride RS to a
suitable volumetric flask. Add 10% of the flask volume
ADDITIONAL REQUIREMENTS each of methanol and 0.1 N hydrochloric acid. Sonicate
© PACKAGING AND STORAGE: Preserve in tight containers. for 5 min, and then dilute to volume with water.
e USP REFERENCE STANDARDS (11) Sample stock solution: Transfer NLT 15 Tablets to a
USP Maprotiline Hydrochloride RS 500-mL volumetric flask, add 100 mL of 0.1 N hydro- a
USP Maprotiline Related Compound D RS chloric acid, sonicate, and shake occasionally for 5 min “
3-(9,10-Dihydro-9,10-ethanoanthracen-9-yl)-N-methyl- to disintegrate the Tablets. Add 100 mL of methanol, uv
prop-2-en-1-amine. shake, and sonicate for 5 min. Dilute with water to vol- =
CooH22N 276.4 ume, and centrifuge. Use the supernatant. io)
Sample solution: Nominally 0.75 mg/mL of maprotiline 3
°
hydrochloride from Sample stock solution and water. ro}
Chromatographic system ba
i
(See Chromatography (621), System Suitability.) me}
Maprotiline Hydrochloride Tablets Mode: LC 1
ww
Detector: UV 272 nm
DEFINITION Column: 8-mm x 10-cm; 10-um packing L10
Maprotiline Hydrochloride Tablets contain NLT 90.0% and Flow rate: 2 mL/min
NMT 110.0% of the labeled amount of maprotiline hy- Injection volume: 20 uL
drochloride (C2oH23N « HCl). System suitability
Sample: Standard solution
IDENTIFICATION Suitability requirements
e A. THIN-LAYER CHROMATOGRAPHY Column efficiency: NLT 1500 theoretical plates
Standard solution: 20 mg/mL of USP Maprotiline Hy- Tailing factor: NMT 2.0
drochloride RS in methanol Relative standard deviation: NMT 2.0%
Sample solution: Transfer a portion of powdered Tab- Analysis
lets, equivalent to 100 mg of maprotiline hydrochloride, Samples: Standard solution and Sample solution
to a glass-stoppered centrifuge tube. Add 5.0 mL of Calculate the percentage of the labeled amount of
methanol to the tube, sonicate for 10 min, shake by maprotiline hydrochloride (CaoH23N - HCI) in the por-
mechanical means for 10 min, and centrifuge. tion of Tablets taken:
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato- Result = (ru/rs) x (Cs/Cu) x 100
graphy.)
Adsorbent: 0.25-mm layer of chromatographic silica tu = peak response of maprotiline from the Sample
gel that has been prewashed with chloroform by al- solution
owing chloroform to travel the full length of the plate, ts peak response of maprotiline from the
and dried at 100° for 30 min Standard solution
Application volume: 5 uL Cs = concentration of USP Maprotiline
Developing solvent system: Secondary butyl alcohol, Hydrochloride RS in the Standard solution
ethyl acetate, and 2 N ammonium hydroxide (6:3:1) (mg/mL)
Cu = nominal concentration of maprotiline
hydrochloride in the Sample solution
(mg/mL)
 2532 Maprotiline / Official Monographs USP 41

Acceptance criteria: 90.0%-110.0% Table 1

PERFORMANCE TESTS Solution A Solution B
e DISSOLUTION (711) % %'
Medium: Dilute hydrochloric acid (7 in 1000); 900 mL
Apparatus 2: 50 rpm 10
Time: 60 min
Standard solution: USP Maprotiline Hydrochloride RS
in Medium
Sample solution: Sample per Dissolution (711).
Instrumental conditions
Mode: UV
Analytical wavelengths: About 268 nm absorbance
minimum; about 272 nm absorbance maximum
Analysis Standard solution: 0.4 mg/mL of marbofloxacin in So-
Samples: Standard solution and Sample solution lution A
Determine the percentage of the labeled amount of Sample solution: Transfer 1.6 mL of Oral Suspension,
maprotiline hydrochloride (C2oH23N « HCl) dissolved Veterinary to a 100-mL volumetric flask, dilute with So-
from UV absorbances, using the difference between lution A to volume, and mix well. Pass through a PVDF
the absorbance maximum and the absorbance mini- filter of 0.2-'um pore size.
mum of filtered portions of the Sample solution, suita- Chromatographic system
bly diluted with Medium, in comparison with the (See Chromatography (621), System Suitability.)
Standard solution having a known concentration of Mode: LC
USP Maprotiline Hydrochloride RS. Detector: UV-Vis 327 nm
Tolerances: NLT 75% (Q) of the labeled amount of Columns
maprotiline hydrochloride (C2oH23N - HCl) is dissolved. Guard: Packing L1
e UNIFORMITY OF DOSAGE UNITS (905): Meet the Analytical: 4.6-mm x 15-cm; 5-um packing L1
requirements Column temperature: 50°
Flow rate: 1.5 mL/min
ADDITIONAL REQUIREMENTS Injection volume: 2 wL
© PACKAGING AND STORAGE: Preserve in well-closed System suitability
containers. Sample: Standard solution
e USP REFERENCE STANDARDS (11) [NotE—The retention time for marbofloxacin is about
USP Maprotiline Hydrochloride RS 6.2 min.]
Suitability requirements
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0% for replicate
w“ injections
a
ey Marbofloxacin Compounded Oral Analysis
Samples: Standard solution and Sample solution
i
— Suspension, Veterinary Calculate the percentage of the labeled amount of
io)
i} DEFINITION
marbofloxacin (Ci7Hi9FN4Ox) in the portion of Oral
is Suspension, Veterinary taken:
So Marbofloxacin Compounded Oral Suspension, Veterinar
= contains NLT 90.0% and NMT 110.0% of the labele Result = (ru/rs) x (Cs/Cu) x 100
[3
amount of marbofloxacin (CizHi9FN4O4).
Ww Prepare Marbofloxacin Compounded Oral Suspension, Vet- tu = peak response of marbofloxacin from the
= erinary 25 mg/mL as follows (see Pharmaceutical Com- Sample solution
pounding—Nonsterile Preparations (795)). rs = peak response of marbofloxacin from the
Standard solution
Marbofloxacin powder 259 Cs = concentration of marbofloxacin in the
Purified Water A small amount Standard solution (mg/mL)
Cu = nominal concentration of marbofloxacin in the
Vehicle: 1:1 mixture of Ora-Plus#
Sample solution (mg/mL)
and Ora-Sweet; or Ora-Blenda,
Acceptance criteria: 90.0%-110.0%
a sufficient quantity to make 100 mL
@Perrigo Pharmaceuticals, Allegan, MI. SPECIFIC TESTS
© PH (791): 6.2-7.2
Wet the Marbofloxacin powder with a small amount of Puri-
fied Water and triturate to make a smooth paste. Add the ADDITIONAL REQUIREMENTS
Vehicle to make the mortar contents pourable. Transfer @ PACKAGING AND STORAGE: Package in tight, light-resistant,
the contents of the mortar stepwise and quantitatively to plastic containers. Store in a refrigerator (2°-8°) or at
a calibrated container using the Vehicle. Add sufficient Ve- controlled room temperature.
hicle to bring to final volume, and mix well. e BEYOND-UsE DATE: NMT 90 days after the date on which
it was compounded, when stored in a refrigerator (2°-8°)
ASSAY or at controlled room temperature. [NOTE—A slight dark-
e PROCEDURE
ening in yellow color may occur in the suspension with
Solution A: 10 mM ammonium formate containing 1:1 Ora-Plus and Ora-Sweet that does not affect the
0.1% (v/v) formic acid
strength of the preparation.]
Solution B: Methanol containing 0.1% formic acid e LABELING: Label it to indicate that it is for veterinary use
Mobile phase: See Table 7. only. Label it to indicate that it is to be well shaken
before use, and to state the Beyond-Use Date.
USP 41
Mazindol
N 0.50%, and 0.25% of impurities, respectively. No individual
it
aan 7,
\
Y \ + cl
OH
CisHisCIN20 284.74
3H-Imidazo[2,1-alisoindol-5-ol, 5-(4-chlorophenyl)-2,5-
dihydro-, (+)-.
(£)-5-(p-Chlorophenyl)-2,5-dihydro-3H-imidazo[2, 1-
ajisoindol-5-ol —[22232-71-9].
» Mazindol contains not less than 98.0 percent
and not more than 102.0 percent of
CisHisCIN2O, calculated on the dried basis.
Packaging and storage—Preserve in tight containers.
USP Reference standards (11)—
USP Mazindol RS
Clarity and color of solution—A 1 in 100 solution of
Mazindol in a mixture of chloroform and methanol (9:1) is
clear and not darker in color than a solution prepared by
mixing equal volumes of Matching Fluid C (see Color and
Achromicity (631)) and water.
Identification—
A: Infrared Absorption (197K).
B: Ultraviolet Absorption (197U)—
Solution: 10g per mL.
Medium: 0.6 N hydrochloric acid.
Absorptivities at 272 nm, calculated on the dried basis, do
not differ by more than 3.0%.
Loss on drying (731)—Dry it in vacuum at 60° for 2 hours:
it loses not more than 0.5% of its weight.
Residue on ignition (281): not more than 0.1%.
Delete the following:
°Heavy metals, Method // (231): 0.002%. ‘official 1-jan-2018)
Sulfate (221)—Triturate a 500-mg portion with 10 mL of
water in a mortar. Filter the suspension through a water-
washed filter, and rinse the mortar and filter with 30 mL of
water, collecting the combined filtrate and washings in a
50-mL color-comparison tube. The filtrate shows no more
sulfate than corresponds to 0.20 mL of 0.020N sulfuric acid
(0.04%).
Chromatographic purity—Dissolve 10 mg in 2.0 mL of a
mixture of chloroform and methanol (9:1) to obtain the test
solution. Dissolve a suitable quantity of USP Mazindol RS in
a mixture of chloroform and methanol (9:1) to obtain a
Standard solution having a concentration of 5.0 mg per mL.
Dilute portions of this solution quantitatively and stepwise
with the mixture of chloroform and methanol (9:1) to ob-
tain a series of diluted standard solutions having concentra-
tions of 0.100, 0.050, 0.025, and 0.0125 mg per mL, re-
spectively. Separately apply a 20-uL portion of the test
solution and 20-uL portions of the Standard solution and
each diluted standard solution to a suitable thin-layer chro-
matographic plate (see Chromatography (621)) coated with
a 0.25-mm layer of chromatographic silica gel mixture. Al-
low the spots to dry, and develop the chromatogram in a
solvent system consisting of a mixture of chloroform, alco-
hol, and ammonium hydroxide (80:20:1) until the solvent
front has moved about three-fourths of the length of the
plate. Remove the plate from the developing chamber, mark
the solvent front, and allow the solvent to evaporate. Locate
the spots on the ne by examination under short-wave-
length UV light: the chromatograms show principal spots at
about the same R; value. Estimate the concentration of any
Official Monographs / Mazindol 2533
secondary spots present in the chromatogram from the test
solution by comparison with the diluted standard solutions:
the principal spots from the 0.100, 0.050, 0.025, and
0.0125 mg per ml dilutions are equivalent to 2.0%, 1.0%,
impurity is greater than 1.0%, and the sum of the impurities
is not greater than 2.0%.
Aasoy=liansiet about 230 mg of Mazindol, accurately
weighed, to a suitable flask, dissolve in 40 mL of glacial ace-
tic acid, add 3 drops of crystal violet TS, and titrate with 0.1
N perchloric acid VS to an emerald-green endpoint. Perform
a blank determination, and make any necessary correction.
Each mL of 0.1 N perchloric acid is equivalent to 28.47 mg
of CyeHisCIN2O.
Mazindol Tablets
» Mazindol Tablets contain not less than
90.0 percent and not more than 110.0 percent of
the labeled amount of mazindol (Cis6Hi3CIN2O).
Packaging and storage—Preserve in tight containers, at a
temperature not exceeding 25°.
USP Reference standards (11)—
USP Mazindol RS
Identification—Place a portion of powdered Tablets,
equivalent to about 1 mg of mazindol, in a suitable flask.
Add 40 mL of methanol, shake by mechanical means for not
less than 5 minutes, and heat for several minutes on a steam
bath to boiling. Cool, dilute with methanol to about
100 mL, and filter. Separate the filtrate into two approxi-
mately equal portions, add 2 drops of hydrochloric acid to
one portion, and mix: the UV absorption spectra of the so-
=
lutions so obtained exhibit maxima and minima at the same wv
wavelengths as those of similar solutions prepared from USP 7
Mazindol RS, concomitantly measured. ra
Dissolution (711)— i}
=
Medium: 0.01 N hydrochloric acid; 500 mL. re}
Apparatus 2: 50 rpm. =ts}oy
Time: _ 120 minutes. iS
Determine the amount of CisHi3CIN2O dissolved by em- =
ploying the following method. “
Mobile phase—Mix 11.50 g of monobasic ammonium
phosphate and 1.32 g of dibasic ammonium phosphate
with water to obtain 1000 mL of an ammonium phosphate
buffer. The Mobile phase is a suitably filtered and degassed
mixture of the ammonium phosphate buffer and acetonitrile
(55:45). Make adjustments if necessary (see System Suitabil-
ity under Chromatography (621)).
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 271-nm detector
and a 4-mm x 30-cm column that contains packing L7. The
flow rate is about 2 mL per minute. Chromatograph three
replicate injections of the Standard solution, and record the
peak responses as directed for Procedure: the relative stan-
dard deviation is not more than 3.0%.
Procedure—inject an appropriate volume (50 pL to
500 uL) of a filtered portion of the solution under test into
the chromatograph, record the chromatogram, and meas-
ure the response for the major peak. Calculate the quantity
of CisHi3CIN2O dissolved in comparison with a Standard so-
lution having a known concentration of USP Mazindol RS in
the same Medium and similarly chromatographed.
Tolerances—Not less than 80% (Q) of the labeled amount
of CisHi3CIN2O is dissolved in 120 minutes.
Uniformity of dosage units (905): meet the require-
ments.
 2534 Mazindol / Official Monographs USP 41

PROCEDURE FOR CONTENT UNIFORMITY— (CisHi3CIN2©) in the portion of Tablets taken by the
Dye solution—Dissolve 100 mg of bromocresol purple in formula:
1000 mL of 0.33 N acetic acid, and mix.
Standard solution—Dissolve an accurately weighed quan- 25C(Ru/ Rs)
tity of USP Mazindol RS in 0.33 N acetic acid, and dilute in which C is the concentration, in mg per mL, of USP
quantitatively and stepwise with 0.33 N acetic acid to ob- Mazindol RS in the Standard preparation; and Ry and Rs are
tain a solution having a known concentration of about the peak response ratios of mazindol to amitriptyline hydro-
20 ug per mL. chloride obtained from the Assay preparation and the Stan-
Test solution—Mix 1 finely powdered Tablet with an accu- dard preparation, respectively.
rately measured volume of 0.33 N acetic acid, sufficient to
provide a solution having a concentration of about 20 jg of
mazindol per mL, shake by mechanical means for 30 min-
utes, and filter, discarding the first few mL of the filtrate.
Procedure—Transfer 25.0 mL each of the Standard solu- Mebendazole
tion, the Test solution, and 0.33 N acetic acid to provide the
blank, to individual 125-mL separators. Add 30 mL of Dye i
solution and 50.0 mL of chloroform to each, and shake by cot
mechanical means for 15 minutes. Allow the layers to sepa-
rate, and filter the chloroform layers. Concomitantly deter- Croce &
mine the absorbances of the filtered solutions obtained from
the Test solution and the Standard solution at the wavelength
of maximum absorbance at about 420 nm, using the blank CieHi3N303 295.29
to set the instrument. Calculate the quantity, in mg, of Carbamic acid, (5-benzoyl-1H-benzimidazol-2-yl), methyl
mazindol (CisHi3CIN2O) in the Tablet by the foul: ester;
Methyl 5-benzoyl-2-benzimidazolecarbamate [31431-39-7].
(TC/ D)(Au/ As) DEFINITION
Mebendazole contains NLT 98.0% and NMT 102.0% of me-
in whichTis the labeled quantity, in mg, of mazindol in the bendazole (Cis6Hi3N303), calculated on the dried basis.
Tablet; C is the concentration, in ug per mL, of USP
Mazindol RS in the Standard solution; D is the concentration, IDENTIFICATION
in ug per mL, of mazindol in the Test solution, based on the e A. INFRARED ABSORPTION (197K)
labeled quantity per Tablet and the extent of dilution; and ° B. The retention time of the major peak of the Sample
Au and As are the absorbances of the solutions from the Test solution corresponds to that of the Standard solution, as
solution and the Standard solution, respectively. obtained in the Assay.
Assay—
Internal standard solution—Dissolve 50 mg of amitriptyline ASSAY
e PROCEDURE
=
“
hydrochloride in 250 mL of methanol, and mix.
ry Standard preparation—Transfer about 32 mg of USP.
Solution A: 7.5 g/L of ammonium acetate
Solution B: Acetonitrile
i]
Mazindol RS, accurately weighed, to a 100-mL volumetric
aD Mobile phase: See Table 7.
-

3 flask, add about 50 mL of Internal standard solution, and

iS shake by mechanical means for 30 minutes. Dilute with /n-
Cj ternal standard solution to volume, and mix. Table 1
= Assay preparation—Weigh and finely powder not fewer Time Solution A Solution B
a than 20 Tablets. Transfer an accurately weighed portion of (min) (%) (%)
va) the powder, equivalent to about 8 mg of mazindol, to a
| suitable flask, add 25.0 mL of Internal standard solution, and
0 80 20
15 70 30
shake by mechanical means for 30 minutes. Pass through a 20 10 90
fine-porosity, sintered-glass filter, discarding the first few mL
25 10 90
of the filtrate.
26 80 20
Mobile phase—Transfer 200 mL of aqueous 0.01 M diba-
sic ammonium phespuc to a 1000-mL volumetric flask, 30 80 20
dilute with methanol to volume, and mix. Pass through a
0.5-1um porosity polytef filter, and degas under vacuum. Diluent: Acetonitrile and water (50:50)
Protect this solution from light. System suitability solution: 50 ug/mL of USP Meben-
dazole RS and 2.5 g/mL of USP Mebendazole Related
Chromatographic system (see Chromatography (621))—The Compound D RS in Diluent. Sonicate in Diluent using
liquid chromatograph is equipped with a 254-nm detector 80% of the final volume at 45°-50° for 10 min. Dilute
and a 4-mm x 30-cm column that contains packing L10. with Diluent to final volume.
Inject three replicate portions of the Standard preparation, Standard solution: 0.05 mg/mL of USP Mebendazole
and record the peak responses as directed for Procedure: the RS in Diluent. Sonicate in Diluent using 80% of the final
resolution, R, is not less than 2.0: and the relative standard volume at 45°-50° for 10 min. Dilute with Diluent to
deviation is not more than 3.0%. final volume.
Procedure—Separately inject equal volumes (about 20 LL) Sample solution: 0.05 mg/mL of Mebendazole in Dilu-
of the Standard preparation and the Assay preparation into ent. Sonicate in Diluent using 80% of the final volume
the chromatograph, record the chromatograms, and meas- at 45°-50° for 10 min. Dilute with Diluent to final
ure the peak responses for mazindol and amitriptyline hy- volume.
drochloride. Calculate the quantity, in mg, of mazindol
USP 41 Official Monographs / Mebendazole 2535

Chromatographic system F = relative response factor (see Table 2)

(See Chromatography (621), System Suitability.) Acceptance criteria: See Table 2. Disregard peaks less
Mode: LC than 0.05%.
Detector: UV 250 nm
Column: 4.6-mm x 10-cm; 3-m packing L1 Table 2
Column temperature: 40°
Flow rate: 1.2 mL/min Relative Relative Acceptance
Injection volume: 10 pL Retention Response Criteria,
System suitability Ti
Samples: System suitability solution and Standard 2-Amino
solution
[Note—The relative retention times of mebendazole 2-Hydroxy
and mebendazole related compoundD are 1.0 and
1.2, respectively.] 2-Amino-1-methyl
Suitability requirements
Resolution: NLT 5.0 between mebendazole and me-
bendazole related compound D, System suitability
solution Mebendazole relat-
Tailing factor: NMT 2.0, Standard solution c
Relative standard deviation: NMT 0.73%, Standard
solution Toluoyl
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of mebendazole (CisH13N3O3) Any other unspeci-
in the portion of Mebendazole taken: i
Total im| i =
Result = (ru/rs) x (Cs/Cu) x 100
2 2-Amino-5-benzoylbenzimidazole.
tu = peak response of mebendazole from the » 5-Benzoyl-2-hydroxybenzimidazole.
Sample solution ¢ 2-Amino-5-benzoyl-1-methylbenzimidazole.
rs = peak response of mebendazole from the ¢ Ethyl 5-benzoyl-1-methylbenzimidazol-2-ylcarbamate.
Standard solution © Methyl 5-(4-toluoyl)-1-methylbenzimidazol-2-ylcarbamate.
Cs concentration of USP Mebendazole RS in the f 1,3-Bis(5-benzoylbenzimidazol-2-yl)urea.
Standard solution (mg/mL)
SPECIFIC TESTS
Cu = concentration of Mebendazole in the Sample e Loss ON DRYING (731)
solution (mg/mL) Analysis: Dry at 105° for 4 h.
Acceptance criteria: 98.0%-102.0% on the dried basis Acceptance criteria: NMT 0.5%
(ss
IMPURITIES yn
© RESIDUE ON IGNITION (281): NMT 0.1%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed
Z
containers. =<
Delete the following: e USP REFERENCE STANDARDS (11) i}
3
USP Mebendazole RS )
°e HEAVY METALS, Method I/ 231): NMT 20 ppme corrui1- USP Mebendazole Related Compound D RS ito}2
Jan-2078) Methyl 5-benzoyl-1-methylbenzimidazol-2-ylcarbamate. i}
¢ ORGANIC IMPURITIES Ci7HisN303 = 309.32 Bo}
Solution A, Solution B, Mobile Pas Diluent, System
a
a)
suitability solution, Sample solution, and Chromato-
graphic system: Prepare as directed in the Assay.
Standard solution: 2.5 g/mL of USP Mebendazole RS
in Diluent Mebendazole Oral Suspension
System suitability
Sample: System suitability solution
Suitability requirements » Mebendazole Oral Suspension is Mebendazole
Resolution: NLT 5.0 between the mebendazole and in an aqueous vehicle. It contains not less than
mebendazole related compound D peaks 90.0 percent and not more than 110.0 percent of
Relative standard deviation: NMT 1.0% for the me- the labeled amount of mebendazole
bendazole peak and NMT 5.0% for the mebendazole
related compound D peak (Ci6Hi3N30s).
Analysis ree and storage—Preserve in tight containers at
Samples: Sample solution and Standard solution controlled room temperature.
Calculate the percentage of each impurity in the por-
tion of Mebendazole taken: Labeling—Label it to indicate that it is for veterinary use
only.
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100 USP Reference standards (11)—
USP Mebendazole RS
tu = peak response of each impurity from the Identification—Mix a quantity of Oral Suspension, equiva-
Sample solution lent to about 200 mg of mebendazole, with 20 mL of a mix-
ls = peak response of mebendazole from the ture of chloroform and 96 percent formic acid (19:1). Pro-
Standard solution ceed as directed for Identification under Mebendazole Tablets,
Gs = concentration of USP Mebendazole RS in the beginning with “Warm the suspension on a water bath for a
Standard solution (mg/mL) few minutes.” The specified result is obtained.
Cu = concentration of Mebendazole in the Sample
solution (mg/mL)
 2536 Mebendazole / Official Monographs
pH (791): between 6.0 and 7.0.
Assay—
Standard preparation—Transfer about 10 mg of USP Me-
bendazole RS, accurately weighed, to a 100-mL volumetric
flask, and add 90 mL of chloroform, 7 mL of isopropyl alco-
hol, and 2 mL of 96 percent formic acid. Agitate until the
solid has dissolved, add isopropyl alcohol to volume, and
mix. Transfer 5.0 mL of this solution to a second 100-mL
volumetric flask, dilute with isopropyl alcohol to volume,
and mix to obtain a solution having a known concentration
of about 5 ug per mL.
Assay preparation 1—Transfer an accurately measured
quantity of Oral Suspension, equivalent to about 1000 mg
of mebendazole, to a 100-mL volumetric flask, dilute with
96 percent formic acid to volume, and mix. Transfer
10.0 mL of this mixture to a second 100-mL volumetric
flask, add 40 mL of 96 percent formic acid, and heat in a
water bath at a temperature of 50° for 15 minutes. Cool,
add water to volume, mix, and pass through a medium-
porosity, sintered-glass filter. Transfer 10.0 mL of the filtrate
to a 250-mL separator, and add 50 mL of water and 50 mL
of chloroform. Shake for about 2 minutes, allow the phases
to separate, and transfer the chloroform layer to a second
250-mL separator. Wash the aqueous layer with two 10-mL
portions of chloroform, add the chloroform washings to the
second separator, and discard the aqueous layer. Wash the
combined chloroform solutions with a mixture of 4 mL of
1N hydrochloric acid and 50 mL of a 1 in 10 solution of
96 percent formic acid in water, and transfer the chloroform
layer to a 100-mL volumetric flask. Extract the aqueous
washing with two 10-mL portions of chloroform, add these
chloroform extracts to the chloroform solution in the volu-
metric flask, add 2 mL of 96 percent formic acid and 7 mL
of isopropyl alcohol, dilute with chloroform to volume, and
mix. Transfer 5.0 mL of this solution to another 100-mL vol-
umetric flask, dilute with isopropyl alcohol to volume, and
mix.
=
”
Assay preparation 2 (where the Oral Suspension is pack-
is
S aged in syringes calibrated to deliver stated increments of
— Apply the Samples to the plate, and allow the spots to
a) mebendazole)—Express an increment of Oral Suspension to
2) a volumetric flask of an appropriate nominal volume so that
< when diluted with 96 percent formic acid to volume a mix-
S ture containing about 10 mg of mebendazole per mL is ob-
= tained. Transfer 10.0 mL of this mixture to a 100-mL volu-
[5 metric flask, add 40 mL of 96 percent formic acid, and heat
”
in a water bath at a temperature of 50° for 15 minutes.
=) Proceed as directed for Assay preparation 7 beginning with
“Cool, add water to volume.”
Procedure—Mix 90 mL of chloroform with 2 mL of
96 percent formic acid in a 100-mL volumetric flask, add
isopropyl alcohol to volume, and mix. Transfer 5.0 mL of
this solution to a second 100-mL volumetric flask, dilute
with isopropyl alcohol to volume, and mix to obtain a rea-
gent blank. Concomitantly determine the absorbances of
the relevant mers preparation and the Standard preparation
at the wavelength of maximum absorbance at qbaut 247
nm with a spectrophotometer, using the reagent blank to
set the instrument. Calculate the quantity, in mg, of meben-
dazole (CisHi3N3Os) in the portion of Oral Suspension taken
to prepare Assay preparation 1 by the formula:
200C(Au / As)
in which C is the concentration, in ug per mL, of USP Me-
bendazole RS in the Standard preparation; and Ay and As are
the absorbances of Assay preparation 1 and the Standard
preparation, respectively. Where appropriate, calculate the
quantity, in mg, of mebendazole (CisHi3N3Os) in the incre-
USP 41
ment of Oral Suspension taken to prepare Assay preparation
2 by the formula:
20,000(C
/ V)(Au
/ As)
in which V is the volume, in mL, of the volumetric flask into
which the increment of Oral Suspension was expressed; Au
is the absorbance of Assay preparation 2; and the other
terms are as defined above.
Mebendazole Tablets
DEFINITION
Mebendazole Tablets contain NLT 90.0% and NMT 110.0%
of the labeled amount of mebendazole (Ci6Hi3N303).
IDENTIFICATION
eA.
Standard solution: 10 mg/mL of USP Mebendazole RS
in chloroform and 96% formic acid (19:1)
Sample solution: Finely powder a quantity of Tablets,
equivalent to 200 mg of mebendazole, and mix the
powder with 20 mL of a mixture of chloroform and
96% formic acid (19:1). Warm the suspension on a
water bath for a few min. Cool, and pass through a
medium-porosity, sintered-glass filter.
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.)
Mode: TLC
Adsorbent: 0.25-mm layer of chromatographic silica
gel mixture
Application volume: 10 ul
Developing solvent system: Chloroform, methanol,
and 96% formic acid (90:5:5)
Analysis
Samples: Standard solution and Sample solution
dry. Develop the chromatogram in the Developing sol-
vent system until the solvent front has moved about
three-fourths of the length of the plate. Remove the
late from the developing chamber, mark the solvent
ront, allow the solvent to evaporate, and examine the
plate under short-wavelength UV light.
Acceptance criteria: The R; value of the principal spot
of the Sample solution corresponds to that of the Stan-
dard solution.
ASSAY
e PROCEDURE
Solution A: 0.05 M monobasic potassium phosphate
Mobile phase: Methanol and Solution A (60:40). Adjust
with 0.1 M phosphoric acid or 1 N sodium hydroxide
to a pH of 5.5, and filter.
Standard stock solution: 0.25 mg/mL of USP Meben-
dazole RS prepared as follows. Transfer 25 mg of USP
Mebendazole RS into a 100-mL volumetric flask. Add
10 mL of formic acid, and heat in a water bath at 50°
for 15 min. Shake by mechanical means for 5 min, add
90 mL of methanol, and allow to cool. Dilute with
methanol to volume.
Standard solution: 0.05 mg/mL of USP Mebendazole
RS in Mobile phase from the Standard stock solution
Sample stock solution: Nominally 0.25 mg/mL of me-
bendazole prepared as follows. Transfer an equivalent to
500 mg of mevendazele, from finely powdered Tablets
(NLT 20), to a 100-mL volumetric flask. Add 50 mL of
formic acid, and heat in a water bath at 50° for 15
min. Shake by mechanical means for 1 h, dilute with
water to volume, mix, and filter. Transfer 5.0 mL of the
filtrate to a 100-mL volumetric flask, and dilute with a
solution of formic acid in methanol (1:9) to volume.
USP 41 Official Monographs / Mebrofenin 2537

Sample solution: Neminally 0.05 mg/mL of mebenda- of 96% formic acid, and mix to dissolve. Add isopropyl
zole in Mobile phase from the Sample stock solution. Pass alcohol to volume.
the solution througha suitable filter of 0.5-um pore Standard solution: 0.01 mg/mL of USP Mebendazole
size. RS in isopropyl alcohol, from the Standard stock
Chromatographic system solution
(See Chromatography (621), System Suitability.) Sample solution: Mix 1 Tablet with 20 mL of 96%
Mode: LC formic acid in a 100-mL volumetric flask, and heat on
Detector: UV 247 nm a steam bath for 15 min. Cool, add isopropyl alcohol
Columns to volume, mix, and pass through a medium pore size,
Precolumn: Contains packing L1 sintered-glass filter. Transfer an equivalent to 1 mg of
Analytical column: 3.9-mm x 30-cm; packing L1 mebendazole from the filtrate to a 100-mL volumetric
Column temperature: 30° flask, and dilute with isopropyl alcohol to volume.
Flow rate: 1.5 mL/min Instrumental conditions
Injection volume: 15 pL (See Ultraviolet-Visible Spectroscopy (857).)
System suitability Mode: UV
Sample: Standard solution Analytical wavelength: Absorption maximum at
ey requirements about 310 nm
Tailing factor: NMT 2.0 Cell length: 1c¢m
Column efficiency: NLT 2500 theoretical plates Blank: 1-in-500 solution of 96% formic acid in iso-
Relative standard deviation: NMT 1% propyl alcohol
Analysis Analysis
Samples: Standard solution and Sample solution Samples: Standard solution and Sample solution
Calculate the percentage of mebendazole (Cis6H13N3O3) Calculate the quantity, in mg, of mebendazole
in the portion of Tablets taken: (CisH13N3O3) in the Tablet taken:

Result = (ru/rs) x (Cs/Cu) x 100 Result = (Au/As) x (Cs/Gu) x Lx 100

ty = peak response of mebendazole from the Au = absorbance of the Sample solution
Sample solution As = absorbance of the Standard solution
rs = peak response of mebendazole from the Cs = concentration of USP Mebendazole RS in the
Standard solution Standard solution (mg/mL)
Cs = concentration of USP Mebendazole RS in the Cy = nominal concentration of mebendazole in the
Standard solution (mg/mL) Sample solution (mg/mL)
Cu = nominal concentration of mebendazole in the L = label claim (mg/Tablet)
Sample solution (mg/mL) Acceptance criteria: Meet the requirements
Acceptance criteria: 90.0%-110.0%
ADDITIONAL REQUIREMENTS
PERFORMANCE TESTS © PACKAGING AND STORAGE: Preserve in well-closed cS
e DISSOLUTION (711) containers. 4)
Medium: 0.1 N hydrochloric acid containing 1.0% so- e USP REFERENCE STANDARDS (11) i)
dium lauryl sulfate; 900 mL USP Mebendazole RS =
Apparatus 2: 75 rpm i}
Time: 120 min =
Solution A: Dissolve 8.0 g of sodium hydroxide in 2 L }
of water. Add 3.0 g of sodium lauryl sulfate, and mix.
a=)
2
Add 20 mL of phosphoric acid, and adjust with phos- Mebrofenin i}
phoric acid to a pH of 2.5. Se
Mobile phase: Acetonitrile and Solution A (3:7) al

Standard solution: 0.5 mg/mL of USP Mebendazole RS

prepared as follows. Transfer the appropriate amount of
USP Mebendazole RS to a volumetric flask. Add 20% of
the final volume of formic acid, and dissolve. Dilute
with methanol to volume. Dilute a portion of this solu-
tion with Medium to obtain a solution having a known
concentration similar to the expected concentration in
the solution under test.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 3-cm; packing L7
Flow rate: 1 mL/min
Injection volume: 10 uL
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 2.0%
Analysis: Determine the percentage of mebendazole
(Ci6H13N3O3) dissolved.
Tolerances: NLT 75% (Q) of the labeled amount of me-
bendazole (CieéHi3N303) is dissolved.
e UNIFORMITY OF DOSAGE UNITS (905)
Procedure for content uenrpeniy
Standard stock solution: Transfer 20 mg of USP Me-
bendazole RS into a 10-mL volumetric flask. Add 4 mL
CisHigBrN2Os 387.23
Glycine, N-[2-[(3-bromo-2,4,6,-trimethylphenyl)amino]-2-
oxoethyl]-N-(carboxymethyl)-;
([[(3-Bromomesityl)carbamoyl]methylJimino]diacetic acid
[78266-06-5].
DEFINITION
Mebrofenin contains NLT 97.0% and NMT 101.0% of
mebrofenin (CisHigBrN2Os), calculated on the dried basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
ASSAY
© PROCEDURE
Sample solution: Dissolve 100 mg of Mebrofenin in
40 mL of dimethylformamide in a conical flask with the
aid of sonication if necessary. Add 3 drops of thymol
blue TS.
Analysis: Titrate the Sample solution with 0.1 N sodium
methoxide (in toluene) VS to a blue endpoint while
flushing the flask with a gentle stream of nitrogen. Per-
 2538 Mebrofenin / Official Monographs USP 41

form a blank determination, and make any necessary Chromatographic system

correction. Each mL of 0.1 N sodium methoxide is (See Chromatography (621), System Suitability.)
equivalent to 19.36 mg of mebrofenin (CisHi9BrN2Os). Mode: LC
Acceptance criteria: 97.0%-101.0% on the dried basis Detector: UV 220 nm
Column: 4.6-mm x 25-cm; packing L1
IMPURITIES Flow rate: 1 mL/min
e RESIDUE ON IGNITION (281): NMT 0.1% Injection volume: 20 uL
Run time: Twice the retention time of mebrofenin
Delete the following: System suitabilit
Sample: Sample solution
Suitability requirements
°e HEAVY METALS, Method II (231): NMT 30 ppme cotta.
Capacity factor, K: NLT 1.2
Jan-2018)
e LIMIT OF NITRILOTRIACETIC ACID
Column efficiency: NLT 200 plates
Mobile phase: Add 10 mL of a solution (1 in 4) of tet- Tailing factor: NMT 4.0
Relative standard deviation: NMT 2.0% for the
rabutylammonium hydroxide in methanol to 200 mL of
water, and adjust with 1M phosphoric acid to a pH of mebrofenin peak
7.5 + 0.1. Transfer this solution to a 1000-mL volumet- Analysis
ric flask, add 90 mL of methanol, and dilute with water Sample: Sample solution
Calculate the percentage of impurities in the portion of
to volume.
Diluent: 10 mg/mL of cupric nitrate solution Mebrofenin taken:
Standard stock solution: 0.5 mg/mL of nitrilotriacetic Result = (ru/rz) x 100
acid in dilute ammonium hydroxide (1 in 20)
Standard solution: 0.05 mg/mL of nitrilotriacetic acid ru = sum of the peak responses of the individual
from a suitable volume of Standard stock solution in Dil- impurities
uent. [NoTE—Prepare fresh on the day of use.] fe = total of all of the peak responses in the
Sample solution: 10 mg/mL of Mebrofenin in Diluent. chromatogram
Sonicate, if necessary, to dissolve. [NoTE—Prepare fresh Acceptance criteria: NMT 3%
on the day of use.]
Chromatographic system SPECIFIC TESTS
(See Chromatography (621), System Suitability.) e MELTING RANGE OR TEMPERATURE, Class | (741):
Mode: LC 185°-200°, but the range between the beginning and
Detector: UV 254 nm end of melting does not exceed 4°.
Column: 4.6-mm x 25-cm; packing L7 ¢ Loss ON DRYING (731)
Flow rate: 0.8 mL/min Analysis: Dry under vacuum at 100° for 3 h.
Injection volume: 20 uL Acceptance criteria: NMT 0.3%
System suitability
Sample: Standard solution ADDITIONAL REQUIREMENTS
5
al
[NoTe—Peaks containing copper may be present.] ¢ PACKAGING AND STORAGE: Preserve in tight containers.
a Suitability requirements Store at 25°, excursions permitted between 15° and 30°.
J e USP REFERENCE STANDARDS (11)
— Resolution: NLT 1.7 between the major peak and
Dp any other peak USP Mebrofenin RS
i} Relative standard deviation: NMT 2.0%
i=]
5 Analysis
3 Samples: Standard solution and Sample solution
i Calculate the percentage of nitrilotriacetic acid in the
2) portion of Mebrofenin taken: Mecamylamine Hydrochloride
=
Result = (ru/rs) x (Cs/Cu) x 100

ty = peak response of copper nitrilotriacetic acid (Cre + a

from the Sample solution ~ ee
Is = peak response of copper nitrilotriacetic acid
from the Standard solution CiHaN-+HCl 203.75
Cs = concentration of the Standard solution Bicyclo[2.2.1]heptan-2-amine, N,2,3,3-tetramethyl-,
(mg/mL) hydrochloride.
Cu = concentration of the Sample solution (mg/mL) N,2,3,3-Tetramethyl-2-norbornanamine hydrochloride
Acceptance criteria: NMT 0.1% [826-39-1].
© ORGANIC IMPURITIES
Buffer: Prepare a mixture of equal volumes of 0.025 M » Mecamylamine Hydrochloride contains not less
monobasic potassium phosphate and 0.025 M dibasic than 98.0 percent and not more than 100.5 per-
sodium phosphate. cent of CiiH2iN - HCI, calculated on the dried
Mobile phase: Mix 400 mL of Buffer with 600 mL of
methanol in a 1-L volumetric flask. Dilute with water to basis.
volume. Adjust with 1 N hydrochloric acid to a pH of Packaging and storage—Preserve in tight containers.
5.0 + 0.1.
Sample solution: 0.5 mg/mL of Mebrofenin in Mobile USP Reference standards (11)—
phase USP Mecamylamine Hydrochloride RS
USP Mecamylamine Related Compound A RS
N,1,7,7-Tetramethyl bicyclo [2.2.1]heptan-2-amine.
CiHaN 167.29
USP 41
Identification—
A: Infrared Absorption (197K).
B: It responds to the tests for Chloride (191).
Acidity—Dissolve 5.0 g in 100 mL of methanol, and titrate
potentiometrically with 0.10 N alcoholic potassium hydrox-
ide to an apparent pH of 5.5, using a calomel-glass elec-
trode system and a potentiometer previously standardized
with pH 5.0 neutralized phthalate buffer (see Solutions in
the section Reagents, Indicators, and Solutions): after correc-
tion for the volume of alkali consumed by 100 mL of metha-
nol, not more than 0.55 mL of 0.10 N alcoholic potassium
hydroxide is required.
Loss on drying (731)—Dry it at a pressure not exceeding
5 mm of mercury at 105° for 1 hour: it loses not more than
1.0% of its weight.
Residue on ignition (281): not more than 0.5%.
Delete the following:
°Heavy metals, Method | (231)—Dissolve 400 mg in
20 mL of water, add 2 mL of 1 N acetic acid, and dilute
with water to 25 mL: the limit is not more than 50 ppm.
© (Official 1-Jan-2018)
Limit of residual solvents—
Diluent—Prepare a mixture of dimethyl sulfoxide and
water (2:1).
Internal standard solution—Prepare a solution of absolute
alcohol in Diluent having a known concentration of about
15 wL per mL.
Standard stock solution—Transfer 50 mL of the Diluent to
a 100-mL volumetric flask, add 0.64 mL of isopropyl alcohol,
dilute with Diluent to volume, and mix.
Standard solution—Pipet 1.0 mL of the Standard stock so-
lution into a 25-mL volumetric flask, dilute with Diluent to
volume, and mix (Solution 1). Transfer about 500 mg of so-
dium chloride, accurately weighed, to a headspace vial, add
1.5 mL of Solution 1 and 1.5 mL of the Internal standard
Solution, and mix.
Test solution—Transfer about 150 mg of Mecamylamine
Hydrochloride, accurately weighed, to a headspace vial, add
about 500 mg of sodium chloride, 1.5 mL of Diluent, and
1.5 mL of the Internal standard solution, and mix.
Chromatographic system (see Chromatography (621))—The
gas chromatograph is equipped with a flame-ionization de-
tector and a 0.53-mm x 30-m capillary column whose inter-
nal wall is coated with a 1.0-um film of liquid phase G16.
This column is joined with a 0.53-mm x 25-m capillary col-
umn whose internal wall is coated with a 5.0-um film of
liquid phase G1. The G16 column is connected to the de-
tector, and the G1 column is connected to the injector. The
injection port temperature is maintained at about 100°; the
detector temperature is maintained at about 210°; and the
column temperature is maintained at 50° for 10 minutes,
then increased at a rate of 5° per minute to 110°, then
increased at a rate of 30° per minute to 210°, and main-
tained for 5 minutes at 210°. Nitrogen is used as the carrier
gas, flowing at a rate of about 6.5 mL per minute. The split
low is 15 mL per minute.
Procedure—Allow the Standard solution, the Internal stan-
dard solution, and the Test solution to stand for 20 minutes
at 90°. Separately inject equal volumes (about 1 mL) of the
headspace of the Standard solution, the Internal standard so-
lution, and the Test solution into the gas chromatograph,
record the chromatograms, and measure the peak responses
of the internal standard and isopropyl alcohol. Calculate the
Official Monographs / Mecamylamine 2539
quantity, in ppm, of isopropyl alcohol in the portion of Me-
camylamine Hydrochloride taken by the formula:
150(Ru)(Ws) / (Rs)(Wu)
in which Ws is the amount, in ppm, of isopropyl! alcohol in
the Standard solution; Wy is the weight, in mg, of Mecamy-
lamine Hydrochloride taken to prepare the Test solution; and
Ru and Rs are the peak response ratios of isopropyl alcohol
to the internal standard obtained from the Test solution and
the Standard solution, respectively: not more than 2000 ppm
of isopropyl alcohol is found.
Related compounds—
Internal standard solution—Proceed as directed in the As-
say.
Solution 1—Prepare a solution of d/-camphene and USP
Mecamylamine Related Compound ARS in the Internal stan-
dard solution containing 625 wg of each per mL.
System suitability solution—Transfer about 125 mg of USP
Mecamylamine Hydrochloride RS, accurately weighed toa
50-mL volumetric flask, add 1 mL of Solution 1, dilute with
Internal standard solution to volume, and mix.
Test solution—Use the Assay preparation.
Chromatographic system—Prepare as directed in the As-
say. Chromatograph the System suitability solution, and re-
cord peak responses as directed for Procedure: the resolu-
tion, R, between the mecamylamine and mecamylamine
related compoundAis not less than 5; the column effi-
ciency is not less than 4000 theoretical plates; the tailing
factor is not more than 1.5; and the relative standard devia-
tion for replicate injections is not more than 2.0%.
Procedure—tinject a volume (about 1 yL) of the Test solu-
tion into the chromatograph, record the chromatogram,
and measure all the peak responses. Calculate the percent-
age of each impurity in the portion of Mecamylamine Hy-
drochloride taken by the formula:
(=
4)
100(ni/ 3) uv
in which r; is the peak response for each impurity; and r, is E
i}
the sum of the responses for all the peaks: not more than =
0.5% of mecamylamine related compoundAis found; not i}
more than 0.5% of di-camphene is found; and not more aat
than 1.0% of total impurities is found. i)
Chloride content—Dissolve about 500 mg, accurately
i}
>
weighed, in 5 mL of water. Add 5 mL of glacial acetic acid, ”
50 mL of methanol, and 1 drop of eosin Y TS, and titrate
with 0.1 N silver nitrate VS. Each mL of 0.1 N silver nitrate
is equivalent to 3.545 mg of Cl: the content is between
17.0% and 17.8%.
Assay—
Internal standard solution—Transfer about 600 mg of so-
dium hydroxide pellets to a 1 L volumetric flask, dissolve in
about 800 mL of methanol. Add an accurately weighed
quantity of about 1.7 g of biphenyl to the flask, and dilute
with methanol to volume.
Standard preparation—Dissolve an accurately weighed
quantity of USP Mecamylamine Hydrochloride RS in Internal
standard solution, and dilute with Internal standard solution,
quantitatively and stepwise if necessary, to obtain a solution
having a known concentration of about 2.5 mg per mL.
Assay preparation—Transfer about 125 mg of Mecamy-
lamine Hydrochloride, accurately weighed, to a 50-mL volu-
metric flask, dissolve in and dilute with Internal standard so-
lution to volume, and mix.
Chromatographic system (see Chromatography (621))—The
gas chromatograph is equipped with a flame-ionization de-
tector connected to a 0.53-mm x 30-m capillary column,
coated with a 1.5-um film of liquid phase G27. The injec-
tion port temperature is maintained at about 200°, the de-
tector temperature is maintained at about 280°, and the
column temperature is at 120° for 15 minutes then in-
 2540 Mecamylamine / Official Monographs
creased at 25° per minute to 250° and maintained for
7 minutes at 250°. Nitrogen is used as the carrier gas at
7.4mL per minute. Chromatograph the Standard prepara-
tion, and record the peak responses as directed for Proce-
dure: the column efficiency is not less than 4000 theoretical
plates; the tailing factor is not more than 1.5; and the rela-
tive standard deviation for replicate injections is not more
than 2.0%.
Procedure—Inject equal volumes (about 1 uL) of the Assay
preparation and the Standard preparation into the gas chro-
matograph, record the chromatogram, and measure the re-
sponses for the major peaks. Calculate the quantity, in mg,
of CiH2aiN - HCI in the portion of Mecamylamine Hydro-
chloride taken by the formula:
SOC(Ru/ Rs)
in which C is the concentration of USP Mecamylamine Hy-
drochloride RS, in mg per mL, in the Standard preparation;
and Ry and Rs are the peak response ratios of mecamy-
lamine hydrochloride to the internal standard biphenyl ob-
tained from the Assay preparation and the Standard prepara-
tion, respectively.
Mecamylamine Hydrochloride Tablets
» Mecamylamine Hydrochloride Tablets contain
not less than 90.0 percent and not more than
110.0 percent of the labeled amount of mecamy-
lamine hydrochloride (Cy;H2iN - HCl).
Packaging and storage—Preserve in well-closed contain-
ers.
i
vw
USP Reference standards (11)—
oe USP Mecamylamine Hydrochloride RS
i]
—
Dd Identification—
° A: To a quantity of powdered Tablets, equivalent to
=
5 about 75 mg of mecamylamine hydrochloride, add 50 mL of
chloroform, and triturate the mixture for 5 minutes. Filter,
= to a glass-stoppered, 125-mL conical flask. Add about 25 mL
and evaporate the filtrate on a steam bath with the aid of a
[5 current of air to dryness: the IR absorption spectrum of a
a)
= potassium bromide dispersion of a portion of the residue so
obtained exhibits maxima only at the same wavelengths as
that of a similar preparation of USP Mecamylamine Hydro-
chloride RS.
B: A portion of the residue obtained in Identification test
A responds to the tests for Chloride (191).
Dissolution (711)—
Medium: water; 750 mL.
Apparatus 2: 50 rpm.
Time: 30 minutes.
Determine the amount of Cy;H2iN - HCI dissolved using
the following procedure.
Diluent—Prepare a solution of triethylamine in alcohol
(1:100).
Internal standard solution—Prepare a solution of biphenyl
in Diluent having a concentration of 82.5 ug per mL.
Standard solution—Prepare a solution of USP Mecamy-
lamine Hydrochloride RS and biphenyl in Diluent having
concentrations of 8.25 ug per mL of each.
Test solution—{NoTE—Condition the solid-phase extraction
column specified in this procedure in the following manner.
Wash the column with 5 mL of water, then with 5 mL of
Diluent, and finally with two 5-mL portions of water.] Trans-
fer by pipetting 25.0 mL of the solution under test through
a freshly conditioned solid-phase extraction column contain-
ing L1 packing with a sorbent-mass to column volume ratio
of 360 mg per 5 mL, or equivalent. Wash the pipet and the
USP 41
solid-phase extraction column with two 5-mL portions of
water. Discard the filtrate. Elute the solid-phase extraction
column with two 4-mL portions of Diluent, and collect the
eluate in a 10-mL volumetric flask containing 1.0 mL of In-
ternal standard solution. Dilute with Diluent to volume, and
mix.
Chromatographic system (see Chromatography (621))—The
gas chromatograph is equipped with a flame-ionization de-
tector, a splitless injection system, and a 0.53-mm x 30-m
analytical column coated with a 1-5-um layer of phase G27.
The carrier gas is helium at a flow rate of 5.2 mL per min-
ute. The detector and column temperatures are maintained
at 250° and 150°, respectively. Chromatograph replicate in-
jections of the Standard solution, and record the peak re-
sponses as directed for Procedure: the column efficiency is
not less than 4000 theoretical plates, the tailing factor is not
more than 2, and the relative standard deviation is not
more than 2.0%.
Procedure—Separately inject equal volumes (about 2 jL)
of the Standard solution and the Test solution into the chro-
matograph, record the chromatograms, and measure the re-
sponses for the al eaks. Calculate the amount in mg, of
CiiHaiN « HCI dissolved by the formula:
0.3C(Ru / Rs)
in which C is the concentration, in ug per mL, of USP Me-
camylamine Hydrochloride RS in the Standard solution, and
Ru and Rs are the peak response ratios of the mecamylamine
hydrochloride peak to the internal standard peak obtained
from the Test solution and Standard solution, respectively.
Tolerances—Not less than 75% (Q) of the labeled amount
of CyH2iN - HCl is dissolved in 30 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Procedure for content uniformity—Place 1 Tablet in the di-
gestion flask, and proceed as directed under Nitrogen Deter-
mination, Method II (461). Each mL of 0.01 N sulfuric acid is
equivalent to 2.038 mg of mecamylamine hydrochloride.
Assay—Weigh and finely powder not fewer than 30 Tablets.
Transfer an accurately weighed portion of the powder,
equivalent to about 50 mg of mecamylamine hydrochloride,
of water, insert the stopper in the flask, and shake by me-
chanical means for 20 minutes. Transfer the contents of the
flask to a 250-mL separator with the aid of small portions of
water. Add 1 mL of 1 N sodium hydroxide and 36 of so-
dium chloride, and extract the mixture successively with
two 50-mL and three 25-mL portions of ether. Wash the
combined ether extracts with three 10-mL portions of
water, and wash, in turn, the combined water washes with
a 10-mL portion of ether, adding it to the washed com-
bined ether extracts. Transfer the ether phase to a 250-mL
conical flask containing 25.0 mL of 0.02N sulfuric acid VS,
and evaporate the ether on a steam bath. Cool the solution,
add methyl red TS, and titrate the excess acid with 0.02 N
sodium hydroxide VS. Each mL of 0.02 N sulfuric acid is
equivalent to 4.075 mg of mecamylamine hydrochloride
(CiHoiN + HCI).
Mechlorethamine Hydrochloride
cl
HyC—N. © HCI
YY
b
CsHiChN
+HCl 192.51
Ethanamine, 2-chloro-N-(2-chloroethyl)-N-methyl-, hydro-
chloride.
USP 41 Official Monographs / Meclizine 2541

2,2’-Dichloro-N-methyldiethylamine hydrochloride Change to read:

[55-86-7].
» Mechlorethamine Hydrochloride contains not
less than 97.5 percent and not more than
100.5 Fereane of CsHiiClN - HCI, calculated on
the anhydrous basis.
Packaging and storage—Preserve in tight, light-resistant
containers.
Labeling—The label bears a warning that great care should
be taken to prevent inhaling particles of Mechlorethamine
Hydrochloride and exposing the skin to it.
USP Reference standards (11)—
USP Mechlorethamine Hydrochloride RS
Identification—
A: Infrared Absorption (197K).
B: Transfer 100 mg to a test tube pane 1 mL of
sodium thiosulfate solution (prepared by dissolving 1 g of
sodium thiosulfate and 100 mg of sodium carbonate in
40 mL of water), shake, allow to stand for 2 hours, then add
1 drop of iodine TS: the color of free iodine remains.
Melting range (741): between 108° and 111°.
pH (791): between 3.0 and 5.0, in a solution (1 in 500).
Water Determination, Method | (921): not more than
0.4%.
lonic chloride content—Dissolve about 30 mg, accurately
weighed, in 30 mL of water contained in a beaker. Add
5 mL of nitric acid and stir. Titrate with 0.02 N silver nitrate
VS to a potentiometric endpoint, using a silver combination
electrode. Perform a blank determination (see Titrimetry
(541)), and make any necessary correction. Each mL of 0.02
N silver nitrate is equivalent to 0.709 mg of ionic chloride:
not less than 18.0% and not more than 19.3% of ionic
chloride is found.
Assay—Transfer about 100 mg of Mechlorethamine Hydro-
chloride, accurately weighed, to a 125-mL conical flask. Add
100 mg of sodium bicarbonate and 20.0 mL of 0.1 N so-
dium thiosulfate VS. Allow to stand for 2'/2 hours, add 3 mL
of starch TS, and titrate the excess sodium thiosulfate with
0.1 N iodine VS. Each mL of 0.1 N sodium thiosulfate is
equivalent to 9.626 mg of CsH1iCl2N - HCI.
USP Reference standards (11)—
"a (CN 1-May-2018)
USP Mechlorethamine Hydrochloride RS
Completeness of solution (641)—A 0.10-g portion dis-
solves in 10 mL of carbon dioxide-free water to yield a clear
solution.
Constituted solution—At the time of use, it meets the
requirements for Injections and Implanted Drug Products (1),
Specific Tests, Completeness and clarity of solutions.
Identification—It meets the requirements of the /dentifica-
tion tests under Mechlorethamine Hydrochloride.
Bacterial Endotoxins Test (85)—It contains not more
than 12.5 USP Endotoxin Units per mg of mechlorethamine
hydrochloride.
pH (791): between 3.0 and 5.0, in a solution (1 in 50).
Water Determination, Method | (921): not more than
1.0%.
Particulate Matter in Injections (788): meets the re-
quirements for small-volume injections.
Other requirements—It meets the requirements for Steril-
ity Tests (71) and Uniformity of Dosage Units (905).
Assay—
Assay preparation—Select a counted number of not fewer
than 10 containers of Mechlorethamine Hydrochloride for
Injection, equivalent to about 100 mg of mechlorethamine
hydrochloride. Dissolve the contents of each container in
water, and transfer the resulting solutions to a 250-mL coni-
cal flask.
Procedure—Immediately proceed as directed in the Assa
under Mechlorethamine Hydrochloride, beginning with “Ad
100 mg of sodium bicarbonate.” Calculate the average con-
tent, in mg, of mechlorethamine hydrochloride (CsHiiCloN -
HCl) per container of Mechlorethamine Hydrochloride for (<=
Injection taken by the formula: 4)
Z
9.626(V/N) m=
}
in which V is the volume, in mL, of 0.1 N sodium thiosulfate 3
°
consumed; andN is the number of containers selected to reo}=
prepare the Assay preparation. By
me}
rey’
wv

Mechlorethamine Hydrochloride for Meclizine Hydrochloride

Injection
» Mechlorethamine Hydrochloride for Injection is
a sterile mixture of Mechlorethamine Hydrochlo- seh C
+ 2HCl + HO
\4

ride with Sodium Chloride or other suitable dilu- oo™to NINO ‘CH
ent. It contains not less than 90.0 percent and
not more than 110.0 percent of the labeled
C2sH27CIN2 + ZHCI - HzO 481.89
amount of mechlorethamine hydrochloride
(CsHiiCl2N - HCI). CosH27ClNo - 2HCI 463.88
Piperazine, Pie clorey rey ery eet
Packaging and storage—Preserve as described in Packag- [(3-methylphenyl)methyl]-, dihydrochloride, monohydrate;
ing and Storage Requirements (659), Injection Packaging, 1-(p-Chloro-o-phenylbenzyl)-4-(m-methylbenzy!) piperazine
Packaging for constitution. dihydrochloride monohydrate [31884-77-2].
Labeling—It meets the requirements for Labeling (7), Labels Anhydrous [1104-22-9].
and Labeling for Injectable Products. The label bears a warn- DEFINITION
ing that great care should be taken to prevent inhaling par- Meclizine Hydrochloride contains NLT 97.0% and NMT
ticles of Mechlorethamine Hydrochloride for Injection and 102.0% of C2sH27CIN2 - ZHCI, calculated on the anhydrous
exposing the skin to it. basis.
 2542 Meclizine / Official Monographs USP 41

IDENTIFICATION [Note—The elution order is meclizine, followed by

e A. INFRARED ABSORPTION (197K) 4-chlorobenzophenone.]
e B. IDENTIFICATION TESTS—GENERAL, Choride (191) Suitability requirements
Sample solution: Dissolve 25 mg in a mixture of 3 mL Resolution: NLT 2.0 between meclizine hydrochloride
of 2.N nitric acid and 5 mL of alcohol. and 4-chlorobenzophenone, System suitability solution
Acceptance criteria: Meets the requirements Column efficiency: NLT 1800 theoretical plates, de-
termined from the analyte peak, Standard solution
ASSAY Tailing factor: NMT 1.5 for the analyte peak, Stan-
e PROCEDURE dard solution
Mobile phase: Dissolve 1.5 g of sodium 1-heptanesul- Relative standard deviation: NMT 1.5%, Standard
fonate in 300 mL of water, and mix this solution with solution
700 mL of acetonitrile. Adjust with 0.1 N sulfuric acid to Analysis
a pH of 4. Samples: Standard solution and Sample solution
Standard solution: 0.1 mg/mL of USP Meclizine Hydro- Allow the Sample solution to elute for NLT three times
chloride RS in Mobile phase the retention time of meclizine hydrochloride.
Sample solution: 0.1 mg/mL of Meclizine Hydrochlo- Calculate the percentage of each impurity in the por-
ride in Mobile phase tion of Meclizine Hydrochloride taken:
Chromatographic system
(See Chromatography (621), System Suitability.) Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
Mode: LC
Detector: UV 230 nm tu = peak response of each impurity from the
Column: 4.6-mm x 25-cm; 5-um packing L1 Sample solution
Flow rate: 1.3 mL/min rs = peak response of meclizine from the Standard
Injection size: 20 uL solution
System suitability Cs = concentration of USP Meclizine Hydrochloride
Sample: Standard solution RS in the Standard solution (mg/mL)
Suitability requirements Cu = concentration of Meclizine Hydrochloride in
Relative standard deviation: NMT 1.0% the Sample solution (mg/mL)
Analysis F = relative response factor, 0.72 for the
Samples: Standard solution and Sample solution 4-chlorobenzophenone peak and 1.0 for all
Calculate the percentage of meclizine hydrochloride other peaks
(CosHa7CIN2 - ZHCI) in the portion of Meclizine Hydro- Acceptance criteria
chloride taken: Any individual impurity: NMT 0.5%
Total impurities: NMT 1.0%
Result = (ru/rs) x (Cs/Cu) x 100 oe ORGANIC IMPURITIES, PROCEDURE 2
Mobile phase: Dissolve 5 g of sodium 1-heptanesulfon-
tu = peak response of meclizine from the Sample ate in 1000 mL of water, and mix 600 mL of this solu-
solution tion with 400 mL of acetonitrile. Adjust with 0.1 N sul-
us
“
rs = peak response of meclizine from the Standard furic acid to a pH of 4.0 + 0.1.
a solution System suitability solution: 2.5 g/mL each of USP
Ss
- Cs = concentration of USP Meclizine Hydrochloride Meclizine Hydrochloride RS, USP Meclizine Related
Dp RS in the Standard solution (mg/mL)
ro) Compound A RS, and USP Meclizine Related Com-
= Cu = concentration of Meclizine Hydrochloride in pound B RS in Mobile phase
So the Sample solution (mg/mL) Standard solution: 2.5 g/mL of USP Meclizine Hydro-
= Acceptance criteria: 97.0%-102.0% on the anhydrous chloride RS in Mobile phase
Cs basis Sample solution: 0.5 mg/mL of Meclizine Hydrochlo-
a) ride in Mobile phase. [NoTE—Store this solution no
ta IMPURITIES longer than 24 h.]
o RESIDUE ON IGNITION (281): NMT 0.1% Chromatographic yam
e ORGANIC IMPURITIES, PROCEDURE 1 (See Chromatography (621), System Suitability.)
[NoTe—On the basis of the synthetic route, perform ei- Mode: LC
ther Procedure 1 or Procedure 2. Procedure 2 is recom- Detector: UV 210 nm
mended when the isomeclizine impurity may be Column: 4.6-mm x 25-cm; 5-1m packing L1
present.] Column temperature: 50°
Mobile phase: Dissolve 1.5 g of sodium 1-heptanesul- Flow rate: 2.0 mL/min
fonate in 300 mL of water, and mix this solution with Injection size: 30 pL
700 mL of acetonitrile. Adjust with 0.1 N sulfuric acid to System suitability
a pH of 4. Samples: System suitability solution and Standard
System suitability solution: 0.01 mg/mL each of USP solution
Meclizine Hydrochloride RS and 4-chlorobenzophenone Suitability requirements
in Mobile phase Resolution: NLT 2.0 between meclizine related com-
Standard solution: 2.5 g/mL of USP Meclizine Hydro- pound B and meclizine, System suitability solution
chloride RS in Mobile phase Tailing factor: NMT 2.0, Standard solution
Sample solution: 0.5 mg/mL of Meclizine Hydrochlo- Relative standard deviation: NMT 6.0%, Standard
tide in Mobile phase solution
Chromatographic system Analysis
(See Chromatography (621), System Suitability.) Samples: Standard solution and Sample solution
Mode: LC Calculate the percentage of each impurity in the por-
Detector: UV 230 nm tion of Meclizine Hydrochloride taken:
Column: 4.6-mm x 25-cm; 5-\um packing L1
Flow rate: 1.3 mL/min Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
Injection size: 20 pL
System suitability tu = peak response of each impurity from the
Samples: System suitability solution and Standard Sample solution
solution
USP 41 Official Monographs / Meclizine 2543

fs = peak response of meclizine from the Standard Identification—

solution A: The retention time of the major peak in the chromato-
Gs = concentration of USP Meclizine Hydrochloride gram of the Assay preparation corresponds to that in the
RS in the Standard solution (mg/mL) chromatogram of the Standard preparation, as obtained in
Cu = concentration of Meclizine Hydrochloride in the Assay.
the Sample solution (mg/mL) B: Thin-Layer Chromatographic Identification Test (201)—
F = relative response factor (see Table 7)
Acceptance criteria: See Table 1. Disregard any peak Adsorbent: 0.5-mm layer of chromatographic silica gel
eluting before 1.75 min. mixture.
Test solution—Extract a quantity of finely powdered Tab-
Table 1
lets, equivalent to about 125 mg of meclizine hydrochloride,
by shaking for 15 minutes with 50 mL of methanol.
Relative Relative Acceptance Standard solution—Prepare a solution of USP Meclizine
Retention Response Criteria, Hydrochloride RS in methanol, containing 2.5 mg per mL.
Name Time Factor NMT (%)
Application volume: 50 uL.
3-Methylbenzyl
alcohol 0.11 1.0 0.10 Developing solvent system: _a mixture of cyclohexane,
toluene, and diethylamine (15:3:2).
1,4-Bis(3-
methylbenzyl) Procedure—Proceed as directed in the chapter, except to
piperazine 0.22 0.73 0.10 place the plate in a developing chamber that contains and
has been equilibrated with Developing solvent system.
4-Chlorobenzhydrole 0.53 13! 0.15
Meclizine o-chloro
Dissolution, Procedure for a Pooled Sample (711)—
isomer? 0.81 1.0 0.10 Medium: 0.01 N hydrochloric acid; 900 mL.
Isomeclizine Apparatus 1: 100 rpm.
(meclizine o-methyl Time: 45 minutes.
isomer): 0.90 A 0.15 Determine the amount of C2sH27CIN2 - 2HCI dissolved by
Meclizine 1.0 = = employing the following method.
Any individual un- _ Mobile ee a suitable degassed and filtered
specified impurity 1.0 0.10 mixture of water and methanol (55:45) that contains 0.69 g
Total impurities _ =_ 1.0 of monobasic sodium phosphate in each 100 mL and is ad-
* USP Meclizine Related Compound A.
justed with phosphoric acid, if necessary, to a pH of 4.0.
» 1-[2-Chlorophenyl)(phenyl)methyl]-4-(3-methylbenzyl) piperazine. Chromatographic system (see Chromatography (621))—The
© USP Meclizine Related Compound B. liquid chromatograph is equipped with a 230-nm detector
and a 4.6-mm x 25-cm analytical column that contains
SPECIFIC TESTS packing L9. The flow rate is about 2 mL per minute. Chro-
e@ WATER DETERMINATION, Method | (921): NMT 5.0% matograph replicate injections of the Standard solution, and
record the peak responses as directed for Procedure: the rela- NS
ADDITIONAL REQUIREMENTS tive standard deviation is not more than 2.0%. al
© PACKAGING AND STORAGE: Preserve in tight containers. ao]
Procedure—Inject about 100 uL of a filtered portion of the
Store at room temperature.
solution under test, suitably diluted with Mobile phase, if =
¢ LABELING: If a test for Organic Impurities other than Proce- °
dure 1 is used, the labeling states the test with which the necessary, into the chromatograph, record the chromato- =]
article complies. gram, and measure the response for the major peak. Deter- °
mine the amount of C2sH27CIN2 - 2HCI dissolved from the Ko}
e USP REFERENCE STANDARDS (11) oa
USP Meclizine Hydrochloride RS peak response obtained in comparison with the peak re- Ey
sponse obtained from a Standard solution having a known mo]
USP Meclizine Related Compound A RS >
4-Chlorobenzhydrol. concentration of USP Meclizine Hydrochloride RS in a mix- 7
Ci3HnClo 18.68 ture of Medium and Mobile phase (1:1), similarly chromato-
USP Meclizine Related Compound B RS graphed. An amount of alcohol not to exceed 1% of the
lsomeclizine total volume of the Standard solution may be used to dis-
1-[(4-Chlorophenyl)(phenyl)methyl]- solve USP Meclizine Hydrochloride RS prior to dilution.
Peametiyibencybeiperaene dihydrochloride Tolerances—Not less than 75% (Q) of the labeled amount
monohydrate. of C2sH27CIN2 - 2HCI is dissolved in 45 minutes.
CosH27CIN2-2HCl-H20 481.88 Uniformity of dosage units (905): meet the require-
ments.
Procedure for content uniformity—Place 1 Tablet in a
100-mL volumetric flask, add 50 mL of dilute hydrochloric
acid (1 in 100), shake by mechanical means for 30 minutes,
Meclizine Hydrochloride Tablets add the dilute acid to volume, and filter, discarding the first
20 mL of the filtrate. Dilute quanta and stepwise with
the same acid to obtain a solution having a concentration of
» Meclizine Hydrochloride Tablets contain not about 15 ug of meclizine hydrochloride per mL. Similarly,
less than 95.0 percent and not more than prepare a Standard solution of USP Meclizine Hydrochloride
110.0 percent of the labeled amount of meclizine RS in dilute hydrochloric acid (1 in 100) having a known
hydrochloride (C2sH27CIN2 - 2HCI). concentration of about 15 ug per mL. Concomitantly deter-
mine the absorbances of the solution from the Tablet and
Packaging and storage—Preserve in well-closed contain- the Standard solution in 1-cm cells at the wavelength of
ers. maximum absorbance at about 232 nm, with a suitable
USP Reference standards (11)— spectrophotometer, using dilute hydrochloric acid (1 in 100)
USP Meclizine Hydrochloride RS as the blank. Calculate the quantity, in mg, of meclizine
 2544 Meclizine / Official Monographs
hydrochloride (C2sH27CIN2 - 2HCI) in the Tablet taken by the
formula:
(T/D)CAu/ As)
in which T is the quantity, in mg, of meclizine hydrochloride
in the Tablet; D is the concentration, in ug per mL, of
meclizine hydrochloride in the solution from the Tablet, on
the basis of the labeled quantity per Tablet and the extent
of dilution; C is the concentration, in jg per mL, of USP
Meclizine Hydrochloride RS in the Standard solution; and Ay
and As are the absorbances of the solution from the Tablet
and the Standard solution, respectively.
Related compounds—
Mobile phase and Buffer pH 7.5—Prepare as directed in
the Assay.
Standard solution—Dissolve an accurately weighed quan-
tity of USP Meclizine Hydrochloride RS in Mobile phase, soni-
cating for about 5 minutes or until the material is dissolved,
and dilute quantitatively, and stepwise if necessary, with
Mobile phase to obtain a solution having a known concen-
tration of about 0.025 mg per mL. [NOTE—This solution is
stable for 72 hours when stored at controlled room temper-
ature protected from light.]
Sensitivity solution—Dilute an aliquot of the Standard solu-
tion with Diluent to obtain a solution containing about
1.25 ug per mL. [NoTE—Prepare this solution fresh daily.]
Test solution—Weigh and finely powder not fewer than
20 Tablets. Transfer an accurately weighed portion of the
powder, equivalent to about 250 mg of meclizine hydro-
chloride based on the label claim, to a 100-mL volumetric
flask. Add about 50 mL of Mobile phase and shake by me-
chanical means for not less than 30 minutes. Dilute with
Mobile phase to volume, mix, allow to settle for about
15 minutes, and pass through a 0.45-m nylon filter, dis-
carding the first 5 mL of the filtrate.
al
<< Chromatographic system (see Chromatography (621))—
o Prepare as directed in the Assay. Chromatograph the Stan-
s dard solution, and record the peak responses as directed for
i)
—
Procedure: the column efficiency, N, is not less than 1200
° theoretical plates; and the relative standard deviation for
iS
GS replicate injections is not more than 2.0%. Chromatograph
= the Sensitivity solution, and record the peak responses as di-
is rected for Procedure: the signal-to-noise ratio, S/N, is not
less than 10.
”
=) Procedure—Separately inject equal volumes (about 20 uL)
of the Standard solution and the Test solution into the chro-
matograph. Allow the Test solution to elute for not less than
two times the retention time of meclizine hydrochloride. Re-
cord the chromatograms and measure all of the peak areas.
Calculate the percentage of each impurity relative to the
labeled content of meclizine hydrochloride in the portion of
the Tablets taken by the formula:
10001 / F\(Cs/ Cr)(ri/ rs)
in whichFis the relative response factor, which is equal to
0.72 for the 4-chlorobenzophenone peak eluting at a rela-
tive retention time of about 0.23 and equal to 1.0 for all
other peaks; Cr is the concentration, in mg per mL, of
meclizine hydrochloride in the Test solution, based on the
label claim; Cs is the concentration, in mg per mL, of
meclizine hydrochloride in the Standard solution; r, is the
peakfespolse for each impurity obtained from the Test solu-
tion; and rs is the response of the meclizine peak obtained
from the Standard solution: not more than 0.5% of any indi-
vidual impurity is found; and not more than 1.0% of total
impurities is found. Reporting level for impurities is 0.1%.
Assay—
Buffer pH 7.5—Dissolve 1.32 g of dibasic ammonium
phosphate in 1000 mL of water. Adjust with phosphoric
acid to a pH of 7.5 + 0.05.
USP 41
Mobile phase—Prepare a mixture of Buffer pH 7.5, metha-
nol, and acetonitrile (350:325:325). Make adjustments if
ey (see System Suitability under Chromatography
Standard preparation—Dissolve an accurately weighed
quantity of USP Meclizine Hydrochloride RS in Mobile phase,
sonicating for about 5 minutes or until the material is dis-
solved, and dilute quantitatively, and stepwise if necessary,
with Mobile phase to obtain a solution having a known con-
centration of about 0.125 mg per mL. [NOTE—This solution
is stable for 72 hours when stored at controlled room tem-
perature protected from light.]
Assay preparation—Weigh and finely powder not fewer
than 20 Tablets. Transfer an accurately weighed portion of
the powder, equivalent to about 12.5 mg of meclizine hy-
drochloride based on the label claim, to a 100-mL volumet-
ric flask. Add about 50 mL of Mobile phase, and shake by
mechanical means for not less than 30 minutes. Dilute with
Mobile phase to volume, mix, and filter through a 0.45-um
nylon filter, discarding the first 5 mL of the filtrate.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 232-nm detector
and a 4.6-mm x 25-cm column that contains 5-~um packing
L11. The column temperature is maintained at 30°, and the
flow rate is about 2.0 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as di-
rected for Procedure: the relative standard deviation for repli-
cate injections is not more than 2.0%.
Procedure—Separately inject equal volumes (about 20 pL)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
ure the responses for the major peaks. Calculate the per-
centage of the labeled amount of meclizine cya eee
(C2sH27CINz - 2HCI) in each Tablet taken by the formula:
100(CV
/ W)(ru/ Fs)
in which C is the concentration, in mg per mL, of meclizine
hydrochloride in the Standard preparation; V is the volume,
in mL, of the Assay preparation; W is the quantity, in mg, of
meclizine hydrochloride based on the label claim, taken to
prepare the Assay preparation; and ry and rs are the peak
responses obtained from the Assay preparation and the Stan-
dard preparation, respectively.
Meclocycline Sulfosalicylate
s IL O. LOH
Cr”
HAH
‘OH
i
Cl HAC HOH H N(CH),
Co2H2iCIN2Og - C7H6O6S 695.05
2-Naphthacenecarboxamide, 7-chloro-4-(dimethylamino)-
1,4,4a,5,5a,6,11,12a-octahydro-3,5,10,12,12a-
pentahydroxy-6-methylene-1,11-dioxo-, [45-(4a,,4aa,5a,
Sac,12aa)]-, mane sydrauySault be pate) (salt).
(45,4aR,55,5aR, 12a5S)-7-Chloro-4-(dimethylamino)-1,4,4a,
5,5a,6,11,12a-octahydro-3,5,10,12,12a-pentahydroxy-
6-methylene-1,11-dioxo-2-naphthacene carboxamide
mono(5-sulfosalicylate) (salt) [73816-42-9].
» Meclocycline Sulfosalicylate has a potency
equivalent to not less than 620 ug of meclo-
cycline (C22H21CIN2Og) per mg.
are and storage—Preserve in tight containers,
protected from light.
USP 41
USP Reference standards (11)—
USP Meclocycline Sulfosalicylate RS
Identification, Infrared Absorption (197K).
Crystallinity (695): meets the requirements.
pH (791): between 2.5 and 3.5, in a solution containing
10 mg per mL.
Water Determination, Method | (921): not more than
4.0%.
Assay—
0.001 M Ammonium edetate—Transfer 293 mg of edetic
acid, accurately weighed, to a 1000-mL volumetric flask,
add 1 mL of methanol and 7 mL of ammonium hydroxide,
and shake to dissolve the edetic acid. Add 900 mL of water,
adjust with glacial acetic acid to a pH of 6.6, dilute with
water to volume, and mix.
Mobile phase—Prepare a mixture of 0.007 M Ammonium
edetate and tetrahydrofuran (85 : 15). Filter and degas the
solution before use.
Standard stock preparation—Dissolve an accurately
weighed quantity of USP Meclocycline Sulfosalicylate RS in
methanol to obtain a solution having a known concentra-
tion of about 0.5 mg of meclocycline per mL.
Standard preparation—immediately prior to injection, di-
lute the Standard stock ie itt quantitatively, and step-
wise if necessary, with Mobile phase to obtain a solution
having a known concentration of about 60 11g of meclo-
cycline per mL.
Assay stock preparation—Transfer 36 mg of Meclocycline
Sulfosalicylate, accurately weighed, to a 50-mL volumetric
flask, dilute with methanol to volume, and mix.
Assay preparation—immediately prior to injection, transfer
3.0 mL of the Assay stock pebatuor to a 25-mL volumetric
flask, dilute with Mobile phase to volume, and mix to obtain
a solution having a nominal concentration of about 60 1g of
meclocycline per mL.
Chromatographic system—The liquid chromatograph is
equipped with a 340-nm detector and a 4-mm x 25-cm
column that contains packing L1. The flow rate is about
0.8 mL per minute. Chromatograph the Standard prepara-
tion, and record the peak responses as directed for Proce-
dure: the relative standard deviation of the meclocyline peak
for replicate injections is not more than 3.0%.
Procedure—Separately inject equal volumes (about 10 w1L)
of the Standard preparation and the Assay preparation into
the chromatograph, and measure the responses for the
meclocycline peak. Calculate the quantity in jg of
C22H2iCIN2Og in each mg of Meclocycline Sulfosalicylate
taken by the formula:
(Cs/ Cu)(ru/ rs)
in which Cs is the concentration, in ug per mL, of meclo-
cycline in the Standard preparation; Cy is the concentration,
in mg per mL, of Meclocycline Sulfosalicylate in the Assay
preparation; and ry and rs are the peak responses obtained
from the Assay preparation and the Standard preparation, re-
spectively.
Meclocycline Sulfosalicylate Cream
» Meclocycline Sulfosalicylate Cream contains the
equivalent of not less than 90.0 percent and not
more than 125.0 percent of the labeled amount
of meclocycline (C22H2iCIN2Og).
Packaging and storage—Preserve in tight containers,
protected from light.
Official Monographs / Meclofenamate 2545
USP Reference standards (11)—
USP Meclocycline Sulfosalicylate RS
Minimum fill (755): |meets the requirements.
Assay—
0.001 M Ammonium edetate—Transfer 293 mg of edetic
acid, accurately weighed, to a 1000-mL volumetric flask,
add 1 mL of methanol and 7 mL of ammonium hydroxide,
and shake to dissolve the edetic acid. Add 900 mL of water,
adjust with glacial acetic acid to a pH of 6.6, dilute with
water to volume, and mix.
Mobile phase—Prepare a mixture of 0.007 M Ammonium
edetate and tetrahydrofuran (85 : 15). Filter and degas the
solution before use.
Standard stock preparation—Dissolve an accurately
weighed quantity of USP Meclocycline Sulfosalicylate RS in
methanol to obtain a solution having a known concentra-
tion of about 0.5 mg of meclocycline per mL.
Standard preparation—Immediately prior to injection, di-
lute the Standard stock preparation quantitatively, and step-
wise if necessary, with Mobile phase, to obtain a solution
having a known concentration of about 10 Ug of meclo-
cycline per mL.
Assay stock preparation—Transfer an accurately weighed
quantity of Cream, equivalent to about 5 mg of meclo-
cycline, to a glass-stoppered, 50-mL centrifuge tube. Add
20 mL of methanol and 20 mL of 0.025 N sulfuric acid, and
shake vigorously for 15 minutes. Transfer the solution to a
50-mL volumetric flask, rinse the centrifuge tube with two
5-mL portions of methanol, and add the rinsings to the
flask. Dilute with methanol to volume, and mix.
AssayPian oh Cenriige a portion of the Assay stock
preparation for 5 minutes. Immediately prior to injection,
transfer 5 mL of the supernatant to a 50-mL volumetric
flask, dilute with Mobile phase to volume, mix, and filter to
obtain a solution having a nominal concentration of about
10 ug of meclocycline per mL. co
Chromatographic system—tThe liquid chromatograph is a)
equipped with a 340-nm detector and a 4-mm x 25-cm
i)
column that contains packing L1. The flow rate is about i
0.8 mL per minute. Chromatograph the Standard prepara- }
=)
tion, and record the peak responses as directed for Proce- )
dure: the relative standard deviation of the meclocyline peak to)=
for replicate injections is not more than 3.0%. ES)
Procedure—Separately inject equal volumes (about 10 yL) 3
of the Standard preparation and the Assay preparation into
=
“”
the chromatograph, record the chromatograms, and meas-
ure the responses for the meclocycline peak. Calculate the
percent label claim of C22H21CIN2Os in the portion of Cream
taken by the formula:
(Cs/ Cu(ru
/ rs)(100)
in which Cs is the concentration, in ug per mL, of meclo-
cycline in the Standard preparation; Cy is the nominal con-
centration, in ug per mL, of meclocycline in the Assay
preparation; and ry and rs are the peak responses obtained
from the Assay preparation and the Standard preparation, re-
spectively.
Meclofenamate Sodium
Ox 0Na
a
HN CH,
° H,0
on
Ci4HioClaNNaOz-H20 336.15
Benzoic acid, 2-[(2,6-dichloro-3-methylphenyl)amino]-,
monosodium salt, monohydrate.
 2546 Meclofenamate / Official Monographs
Monosodium N-(2,6-dichloro-m-tolyl)anthranilate monohy-
drate [6385-02-0].
Anhydrous 318.13
» Meclofenamate Sodium contains not less than
97.0 percent and not more than 103.0 percent of
Cy4HioClzNNaOz, calculated on the anhydrous
basis.
Packaging and storage—Preserve in tight, light-resistant
containers.
USP Reference standards (11)—
USP Meclofenamate Sodium RS
Identification—
A: Infrared Absorption (197K).
B: Ultraviolet Absorption 1 (197U)—
Solution: 25 ug per mL.
Medium: 0.01 N hydrochloric acid in methanol.
Absorptivities at 242 nm, 279 nm, and 336 nm, calcu-
lated on the anhydrous basis, do not differ by more than
3.0%.
C: Ultraviolet Absorption 2 (197U)—
Solution: 1 in 40,000.
Medium: 0.1 N sodium hydroxide.
Absorptivities at 279 nm and 317 nm, calculated on the
anhydrous basis, do not differ by more than 3.0%.
Water Determination, Method | (921): between 4.8%
and 5.8%.
Copper—
Standard copper solution—Dissolve 1000 mg of copper
wire in 6 mL of nitric acid in a 1 L volumetric flask. Add
8 mL of hydrochloric acid, dilute with water to volume, and
mix. Dilute this solution ae and stepwise with
water to obtain a Standard copper solution having a known
al
oe concentration of 0.6 ug per mL.
a Test solution—Transfer 2 g of Meclofenamate Sodium, ac-
Ss
_ curately weighed, to a 100-mL volumetric flask, and add 1
a drop of ammonium hydroxide. Dissolve in water, dilute with
i)
a water to volume, and mix.
B Procedure—Concomitantly determine the absorbances of
= the Standard copper solution and the Test solution at the cop-
a. per emission line at about 325 nm, with a suitable atomic
A) absorption spectrophotometer (see Atomic Absorption Spec-
=) troscopy (852)) equipped with a copper hollow-cathode
lamp, using water as the blank. Adjust the operating condi-
tions to obtain about 70% full-scale detector response with
the Standard copper solution. The detector response ob-
tained with the Test solution is not greater than that ob-
tained with the Standard copper solution (0.003%).
Chromatographic purity—
Standard solutions—Dissolve an accurately weighed quan-
tity of USP Meclofenamate Sodium RS in methanol to obtain
a solution containing 20 mg per mL (Standard solution A).
Dilute 1.0 mL of Standard solution A with sufficient methanol
to obtain 200 mL of solution (Standard solution B).
Test solution—Dissolve 200 mg of Meclofenamate Sodium
in 10.0 mL of methanol.
Procedure—Apply 10-uL portions of Standard solution A,
Standard solution B, and the Test solution to a suitable thin-
layer chromatographic plate (see Chromatography (621))
coated with a 0.25-mm layer of chromatographic silica gel
mixture. Allow the spots to dry, and develop the chromato-
gram in a solvent system consisting of a mixture of methyl-
ene chloride, methyl ethyl ketone, and glacial acetic acid
(50:48:2) until the solvent front has moved about eight-
tenths of the length of the plate. Remove the plate from the
developing chamber, mark the solvent front, and allow the
solvent to evaporate. Examine the plate under short-wave-
length UV light: the chromatograms show a principal spot
at about the same R; value, and any secondary spot, if pres-
USP 41
ent in the chromatogram from the Test solution is not more
intense than the principal spot obtained from Standard solu-
tion B (0.5%).
Assay—Transfer about 350 mg of Meclofenamate Sodium,
accurately weighed, to a 125-mL separator, add 10 mL of
water, and mix to dissolve. To this solution add 3 mL of 3N
hydrochloric acid, shake, and extract with three 30-mL por-
tions of chloroform, collecting the chloroform extracts in an
evaporating flask. Evaporate the chloroform extracts to dry-
ness. Dissolve the residue in 5 mL of dimethyl sulfoxide and
25 mL of methanol. Mix, add 5 drops of phenolphthalein
TS, and titrate the mixture with 0.1 N sodium hydroxide VS.
Each mL of 0.1 N sodium hydroxide is equivalent to
31.81 mg of CigHioClANNaOo.
Meclofenamate Sodium Capsules
» Meclofenamate Sodium Capsules contain an
amount of Ci4HioClaNNaQOz equivalent to not less
than 90.0 percent and not more than 110.0 per-
cent of the labeled amount of meclofenamic acid
(CyaH11ClzNO2).
Packaging and storage—Preserve in tight, light-resistant
containers.
USP Reference standards (11)—
USP Meclofenamate Sodium RS
Identification—Prepare a solution of Capsule contents in
methanol containing 20 mg per mL, and filter. The clear
filtrate so obtained meets the requirements of the Thin-Layer
Chromatographic Identification Test (201), the solvent mixture
consisting of methylene chloride, methyl ethyl ketone, and
glacial acetic acid (50:48:2).
Dissolution (711)—
Medium: 0.05 M pH 7.5 phosphate buffer (see under
Buffer Solutions in the section Reagents, Indicators, and Solu-
tions); 900 mL.
Apparatus 2: 50 rpm.
Time: 45 minutes.
Procedure—Determine the amount of meclofenamic acid
(Ci4H11Cl2NOz2) dissolved from UV absorbances at the wave-
length of maximum absorbance at about 279 nm of filtered
portions of the solution under test, suitably diluted with Me-
dium, if necessary, in comparison with a Standard solution
having a known concentration of USP Meclofenamate So-
dium RS in the same Medium.
Tolerances—Not less than 75% (Q) of the labeled amount
of Ci4HiiCl2NOz is dissolved in 45 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Assay—Remove, as completely as possible, the contents of
not fewer than 20 Capsules, and weigh accurately. Mix the
combined contents, and transfer an accurately weighed
quantity of the powder, equivalent to about 50 mg of
meclofenamic acid, to a 200-mL volumetric flask. Add 0.01
N hydrochloric acid in methanol to volume, and mix. Filter,
discarding the first 20 mL of the filtrate. Transfer 10.0 mL of
the filtrate to a 100-mL volumetric flask, add 0.01 N hydro-
chloric acid in methanol to volume, and mix. Dissolve an
accurately weighed quantity of USP Meclofenamate Sodium
RS in 0.01 N hydrochloric acid in methanol to obtain a
solution having a known concentration of about 27 ug per
mL. Concomitantly determine the absorbances of both solu-
tions in 1-cm cells at the wavelength of maximum absorb-
ance at about 336 nm, with a suitable spectrophotometer,
using 0.01 N hydrochloric acid in methanol as the blank.
Calculate the quantity, in mg, of meclfenamate acid
USP 41 Official Monographs / Medroxyprogesterone 2547

(CiaHi1ClaNOz) in the portion of Capsule contents taken by Cu = concentration of Medroxyprogesterone

the formula:
2€(296.15 / 318.13)(Au/As)
in whichCis the concentration, in ug per mL, of USP
Meclofenamate Sodium RS in the Standard solution; 296.15
and 318.13 are the molecular weights of meclofenamic acid
and meclofenamate sodium, respectively; and Ay and As are
the absorbances of the solution from the Capsule contents
and the Standard solution, respectively.
Medroxyprogesterone Acetate
Acetate in the Sample solution (mg/mL)
Acceptance criteria: 97.0%-103.0% on the dried basis
IMPURITIES
© ORGANIC IMPURITIES
Mobile phase: Acetonitrile and water (60:40)
System suitability solution: 40 g/mL each of meges-
trol acetate and USP Medroxyprogesterone Acetate RS
in Mobile phase
Standard solution: 50 g/mL of USP Medroxyproges-
terone Acetate RS in Mobile phase
Sample solution: 2.5 mg/mL of Medroxyprogesterone
Acetate in Mobile phase
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; packing L1
Flow rate: 1 mL/min

Jt
Injection volume: 20 uL
System suitability
Samples: System suitability solution and Standard
Cry solution
Suitability requirements
CoaHa404 386.52 Resolution: NLT 1.5 between megestrol acetate and
Pregn-4-ene-3,20-dione, 17-(acetyloxy)-6-methyl-, (6a)-; medroxyprogesterone acetate, System suitability
1 Tee ee are ae acetate solution
[71-58-9]. Relative standard deviation: NMT 3.0%, Standard
solution
DEFINITION Analysis
Medroxyprogesterone Acetate contains NLT 97.0% and Samples: Standard solution and Sample solution
NMT 103.0% of medroxyprogesterone acetate (C24H34O.), Calculate the percentage of each impurity in the por-
calculated on the dried basis. tion of Medroxyprogesterone Acetate taken:
IDENTIFICATION Result = (ru/rs) x (Cs/Cu) x 100
e A. INFRARED ABSORPTION (197K)
e B. ULTRAVIOLET ABSORPTION (197U) tu = peak response for each impurity from the iS
Analytical wavelength: 241 nm Sample solution 4)
Sample solution: 10 {g/mL in alcohol Is = peak response of medroxyprogesterone i)
Acceptance criteria: Absorptivities, calculated on the acetate from the Standard solution
E
dried basis, do not differ by more than 2.0%. Gs = concentration of USP Medroxyprogesterone i)
Acetate RS in the Standard solution (mg/mL) =|
ASSAY Cu = concentration of Medroxyprogesterone )
© PROCEDURE Acetate in the Sample solution (mg/mL) a=
Mobile phase: Acetonitrile and water (40:60) Acceptance criteria i)
Standard solution: 1 mg/mL of USP Medroxyprogester- 3
one Acetate RS in acetonitrile
Individual impurity: NMT 1.0% =
Total impurities: NMT 1.5% a)
Sample solution: 1 mg/mL of Medroxyprogesterone e LIMIT OF MEDROXYPROGESTERONE ACETATE RELATED COM-
Acetate in acetonitrile POUND A
Chromatographic system Standard solution: 20 mg/mL of USP Medroxyproges-
(See Chromatography (621), System Suitability.) terone Acetate RS and 0.1 mg/mL of USP Medroxypro-
Mode: LC gesterone Acetate Related CompoundA RS in methyl-
Detector: UV 254 nm ene chloride
Column: 4-mm x 30-cm; packing L1 Sample solution: 20 mg/mL of Medroxyprogesterone
Flow rate: 2 mL/min Acetate in methylene chloride
Injection volume: 10 uL Chromatographic system
System suitability (See Chromatography (621), Thin-Layer Chromato-
Sample: Standard solution graphy.)
eae requirements Mode: TLC
Tailing factor: NMT 2 Adsorbent: 0.25-mm layer of chromatographic silica
Relative standard deviation: NMT 2.0% gel mixture
Analysis Application volume: 10 pL
Samples: Standard solution and Sample solution Developing solvent system: Hexanes, tert-butyl
Calculate the percentage of medroxyprogesterone ace- methyl ether, and tetrahydrofuran (45:45:10)
tate (C2sH34Ox) in the portion of Medroxyprogesterone Spray reagent: 200 mg/mL of p-toluenesulfonic acid
Acetate taken: in alcoho
Analysis
Result = (ru/rs) x (CGs/Cu) x 100 Samples: Standard solution and Sample solution
Develop the chromatogram until the solvent front has
ty = peak response from the Sample solution moved about 10 cm. Allow the plate to air-dry, and
rs = peak response from the Standard solution develop the chromatogram again until the solvent
Gs = concentration of USP Medroxyprogesterone front has moved about 10 cm. Allow the plate to dry
Acetate RS in the Standard solution (mg/mL) at 120° for 10 min. Spray the plate with Spray reagent.
 2548 Medroxyprogesterone / Official Monographs
Heat the plate for 10 min at 120°, and examine the
plate under UV light at 365 nm.
Acceptance criteria: NMT 0.5%; any blue fluorescent
spot with an R; value higher than that of the principal
spot due to medroxyprogesterone acetate of the Sample
solution is not more intense than the corresponding
blue fluorescent spot of the Standard solution.
SPECIFIC TESTS
© OPTICAL ROTATION, Specific Rotation (781S)
Sample solution: 10 mg/mL in dioxane
Acceptance criteria: +45° to +51°
e Loss ON DRYING (731)
Analysis: Dry a sample at 105° for 3 h.
Acceptance criteria: NMT 1.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers. Store at 25°, excursions pernitied between
15° and 30°.
e USP REFERENCE STANDARDS (11)
USP Medroxyprogesterone Acetate RS
USP Medroxyprogesterone Acetate Related
Compound A RS
4,5B-Dihydromedroxyprogesterone acetate.
Cr4H36O4 388.54
Medroxyprogesterone Acetate
Injectable Suspension
DEFINITION
Medroxyprogesterone Acetate Injectable Suspension is a
sterile suspension of Medroxyprogesterone Acetate in a
a)
suitable aqueous medium. It contains NLT 90.0% and
a NMT 110.0% of the labeled amount of medroxyproges-
a terone acetate (Co4H34Ox).
ci
=
Dp IDENTIFICATION
) e A. INFRARED ABSORPTION (197K)
iS
S Sample: Transfer a volume of Injectable Suspension,
= equivalent to 50 mg of medroxyprogesterone acetate,
to a centrifuge tube, centrifuge, decant the superna-
a
a) tant, and wash the solids with two 15-mL portions of
=) water, discarding the water washings. Dissolve the
solids in 10 mL of chloroform, transfer to a small
beaker, evaporate the chloroform on a steam bath, and
dry the residue at 105° for 3 h.
Acceptance criteria: Meets the requirements
ASSAY
e PROCEDURE
Mobile phase: 700 mL of butyl chloride, 300 mL of
hexane, both previously saturated with water, and
80 mL of acetonitrile. The acetonitrile concentration
may be varied to meet System suitability requirements
and to provide elution times of about 12 and 15 min
for progesterone and medroxyprogesterone acetate, re-
spectively. Pass the solution through a membrane filter
of 1 um or less pore size.
Internal standard solution: 0.25 mg/mL of progester-
one in Mobile phase
Standard solution: 0.4 mg/mL of USP Medroxyproges-
terone Acetate RS in Internal standard solution
Sample solution: Nominally 0.4 mg/mL of medroxy-
progesterone acetate in Internal standard solution, pre-
pared as follows. Transfer a volume of Injectable Sus-
pension, equivalent to 50 mg of medroxyprogesterone
acetate, to a suitable container. Transfer 25 mL of chlo-
roform into the container, shake for 20 min, and centri-
fuge. Transfer 4 mL of the chloroform layer into a suita-
ble container, and evaporate to dryness. Dissolve the
residue in 20 mL of Internal standard solution.
USP 41
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 2-mm x 25-cm; 5-4um packing L3
Flow rate: The Mobile phase is maintained at a flow
rate capable of giving the required resolution and suit-
able elution times.
Injection volume: 10 ul
System suitability
Sample: Standard solution
Suitability requirements
Resolution: NLT 5.0 between progesterone and med-
roxyprogesterone acetate
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
medroxyprogesterone acetate (C24H3404) in the por-
tion of Injectable Suspension taken:
Result = (Ru/Rs) x (Cs/Cu) x 100
Ru = peak area ratio of medroxyprogesterone
acetate to the internal standard from the
Sample solution
Rs = peak area ratio of medroxyprogesterone
acetate to the internal standard from the
Standard solution
Cs = concentration of USP Medroxyprogesterone
Acetate RS in the Standard solution (mg/mL)
Cu = nominal concentration of
medroxyprogesterone acetate in the Sample
solution Cage)
Acceptance criteria: 90.0%-110.0%
SPECIFIC TESTS
e PH (791): 3.0-7.0
© OTHER REQUIREMENTS: |t meets the requirements in Injec-
tions and Implanted Drug Products (1).
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in single-dose or mul-
tiple-dose containers, preferably of Type | glass.
e USP REFERENCE STANDARDS (11)
USP Medroxyprogesterone Acetate RS
Medroxyprogesterone Acetate Tablets
DEFINITION
Medroxyprogesterone Acetate Tablets contain NLT 93.0%
and NMT 107.0% of the labeled amount of medroxypro-
gesterone acetate (C24H34Ox).
IDENTIFICATION
© A. INFRARED ABSORPTION (197K)
Sample: Triturate a number of Tablets, equivalent to
about 25 mg of medroxyprogesterone acetate, with
15 mL of chloroform. Filter, evaporate the chloroform
on a steam bath, and dry the residue at 105° for 3 h.
Acceptance criteria: Meet the requirements
ASSAY
© PROCEDURE
Mobile phase: Acetonitrile and water (40:60)
Standard solution: 1 mg/mL of USP Medroxyprogester-
one Acetate RS in acetonitrile
Sample solution: Finely powder NLT 20 Tablets. Weigh
a portion of the powder, equivalent to 25 mg of med-
roxyprogesterone acetate, into a 50-mL glass centrifuge
tube. Transfer 25 mL of acetonitrile into the tube, shake
USP 41 Official Monographs / Mefenamic 2549

to wet the powder Sisiovably, sonicate for NLT 10 ing the responses from the Sample solution and Stan-
min, and centrifuge. Use the clear supernatant. dard solution.
Chromatographic system Tolerances: NLT 50% (Q) of the labeled amount of
(See Chromatography (621), System Suitability.) medroxyprogesterone acetate (C24H34O.) is dissolved.
Mode: LC e UNIFORMITY OF DOSAGE UNITS (905)
Detector: UV 254 nm Procedure for content uniformity
Column: 4-mm x 30-cm; packing L1 Diluent: Alcohol and water (3:1)
Flow rate: 2 mL/min Standard solution: 15 g/mL of USP Medroxyproges-
Injection volume: 10 LL terone Acetate RS in Diluent
System suitability Sample solution: Nominally 15 g/mL of medroxypro-
Sample: Standard solution esterone acetate in Diluent prepared as follows. Trans-
Suitability requirements er 1 Tablet to a volumetric flask, dilute with Diluent to
Tailing factor: NMT 2 volume, and shake for 15 min. Filter, and quantita-
Relative standard deviation: NMT 2.0% tively dilute a portion of the filtrate as needed.
Analysis Instrumental conditions
Samples: Standard solution and Sample solution Mode: UV-Vis
Calculate the percentage of the labeled amount of Analytical wavelength: Maximum at about 242 nm
precio pasireie acetate (C24H340,) in the por- Cell; 1.cm
tion of Tablets taken: Analysis
Samples: Standard solution and Sample solution
Result = (ru/rs) x (Cs/Cu) x 100 Calculate the percentage of the labeled amount of
faesiraseyprogestenone acetate (C24H34Ox) in the Tablet
tu = peak response from the Sample solution taken:
ls = peak response from the Standard solution
Cs = concentration of USP Medroxyprogesterone Result = (Au/As) x (Cs/Cy) x 100
Acetate RS in the Standard solution (mg/mL)
Cu = nominal concentration of Au = absorbance of the Sample solution
medroxyprogesterone acetate in the Sample As = absorbance of the Standard solution
solution Cann Cs = concentration of USP Medroxyprogesterone
Acceptance criteria: 93.0%-107.0% Acetate RS in the Standard solution (g/mL)
Cu = nominal concentration of
PERFORMANCE TESTS medroxyprogesterone acetate in the Sample
e DISSOLUTION (711) solution (4ug/mL)
Medium: 0.5% sodium lauryl sulfate; 900 mL Acceptance criteria: Meet the requirements
Apparatus 2: 50 rpm
Time: 45 min ADDITIONAL REQUIREMENTS
Mobile phase: Acetonitrile and water (60:40) © PACKAGING AND STORAGE: Preserve in well-closed
Sodium lauryl sulfate stock solution: Transfer 180.0 g containers. =
of sodium lauryl sulfate to a 2000-mL volumetric flask. e USP REFERENCE STANDARDS (11) “
Add 1500 mL of water, and stir until dissolved. [NoTE— USP Medroxyprogesterone Acetate RS i}
Several hours of stirring are required.] Dilute with water =
to volume. fo)
Standard stock solution: 70mg of USP Medroxypro- Po]
gesterone Acetate RS in 140 mL of Sodium laury! sulfate °
iro}
stock solution. Dilute with water to 250 mL. [NoTE—It Mefenamic Acid iad
2
may be necessary to sonicate the solution to bring the Tv
Reference Standard into solution before dilution with Om fa a >
water.] Prepare the Standard stock solution fresh daily. 7
Standard solution: Transfer a 20-mL aliquot of Stan-
dard stock solution into a 1-L volumetric flask. Add
40 mL of Sodium lauryl sulfate stock solution, and dilute
be water to volume. This solution is stable for up to 7
lays. CisHisNO2 241.29
Sara le solution: Withdraw 15 mL of the solution Benzoic acid, 2-(2,3-dimethylphenyl)amino-;
under test and filter, discarding the first 5 mL of the N-2,3-Xylylanthranilic acid [61-68-7].
filtrate.
Chromatographic system DEFINITION
(See Chromatography (621), System Suitability.) Mefenamic Acid contains NLT 98.0% and NMT 102.0% of
Mode: LG mefenamic acid (CisHisNOz), calculated on the dried
Detector: UV 254 nm basis.
Column: 4-mm x 8-cm; packing L7 IDENTIFICATION
Flow rate: 1.5 mL/min
Injection volume: 20 uL
System suitability Change to read:
Sample: Standard solution
Suitability requirements e A. INFRARED ABSORPTION 4(197): [NoTE—Methods de-
Tailing factor: NMT 1.2 scribed in (197K) or (197A) may beused.]ausea1
Relative standard deviation: NMT 2.0% e B. The retention time of the major peak of the Sample
Analysis solution corresponds to that of the Standard solution, as
Samples: Standard solution and Sample solution obtained in the Assay.
Calculate the percentage of the labeled amount of
medroxyprogesterone acetate (C4H34O4) dissolved us-
 2550 Mefenamic / Official Monographs USP 41

ASSAY Signal-to-noise ratio: NLT 10, Sensitivity solutionausess

Analysis
Change to read: Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the por-
tion of Mefenamic Acid taken:
© PROCEDURE
Buffer: 50 mM monobasic ammonium phosphate. Ad- Result = (ru/rs) x (Cs/Cu) x 100
just with 3 M ammonium hydroxide to a pH of 5.0.
Mobile phase: Acetonitrile, tetrahydrofuran, and Buffer tu = peak response of each impurity from the
(23:7:20) Sample solution
Standard solution: 0.2 mg/mL of USP Mefenamic Acid rs = peak response of mefenamic acid from the
RS in Mobile phase Standard solution
Sample solution: 0.2 mg/mL of Mefenamic Acid in Mo- Gs = concentration of USP Mefenamic Acid RS in
bile phase the Standard solution (mg/mL)
Chromatographic system Cu = concentration of Mefenamic Acid in the
(See Chromatography (621), System Suitability.) Sample solution (mg/mL)
Mode: LC Acceptance criteria: 4The reporting threshold is
Detector: UV 254 nm 0.03%.auspat
Column: 4.6-mm x 25-cm; 45-tUMauser: packing L1 Any impurity: NMT 40.07%ausrs1
Flow rate: 1 mL/min Total impurities: NMT 0.5%
Injection volume: 10 pL
System suitability SPECIFIC TESTS
Sample: Standard solution © Loss ON DRYING (731)
Suitability requirements Analysis: Dry at 105° for 4 h.
Column efficiency: NLT 8200 theoretical plates Acceptance criteria: NMT 1.0%
Tailing factor: NMT 1.6
Relative standard deviation: NMT 40.73%avspai ADDITIONAL REQUIREMENTS
Analysis e PACKAGING AND STORAGE: Preserve in tight, light-resistant
Samples: Standard solution and Sample solution containers.
Calculate the percentage of mefenamic acid e USP REFERENCE STANDARDS (11)
(CisHisNOz) in the portion of Mefenamic Acid taken: USP Mefenamic Acid RS

Result = (ru/rs) x (Cs/Cy) x 100

ru = peak response of mefenamic acid from the
Sample solution
rs = peak response of mefenamic acid from the Mefenamic Acid Capsules
Standard solution
is
“
Gs = concentration of USP Mefenamic Acid RS in » Mefenamic Acid Capsules contain not less than
roy the Standard solution (mg/mL) 90.0 percent and not more than 110.0 percent of
it
— Gu = concentration of Mefenamic Acid in the the labeled amount of mefenamic acid
io) Sample solution (mg/mL)
° (CisHisNOz).
S Acceptance criteria: 98.0%-102.0% on the dried basis
S Packaging and storage—Preserve in tight containers.
= IMPURITIES
USP Reference standards (11)—
a © RESIDUE ON IGNITION (281): NMT 0.1%
al USP Mefenamic Acid RS
E>} Identification—
Delete the following:
A: Place a portion of Capsule contents, equivalent to
°e HEAVY METALS, Method I! (231): NMT 20 ppme coricia1 about 250 mg of mefenamic acid, in a 250-mL volumetric
Jan-2018) flask, add about 100 mL of a mixture of chloroform and
methanol (3:1), and shake vigorously. Dilute with a mixture
of chloroform and methanol (3:1) to volume, mix, and filter:
Change to read: the filtrate so obtained responds to the Thin-layer Chromato-
graphic Identification Test (201), a solvent system consisting
© ORGANIC IMPURITIES of a mixture of chloroform, ethyl acetate, and glacial acetic
Buffer, Mobile phase, and Chromatographic system: acid (75:25:1) and the Ordinary Impurities (466) visualization
Proceed as directed in the Assay. technique 17 being used.
Standard solution: 0.01 mg/mL of USP Mefenamic B: The retention time of the major peak in the chromato-
Acid RS in Mobile phase gram of the Assay preparation corresponds to that of the
Sensitivity solution: 0.3 g/mL of USP Mefenamic Standard preparation, obtained as directed in the Assay.
Acid RS in Mobile phase from the Standard solutionguses: Dissolution (711)—
Sample solution: 1 mg/mL of Mefenamic Acid in Mo-
bile phase 0.05 M Tris buffer—Dissolve 60.5 g of tris(hydroxymeth-
System suitability yl)aminomethane in 6 L of water, and dilute with water to
4Samples: Standard solution and Sensitivity solution 10 L. Adjust with phosphoric acid to a pH of 9.0 + 0.05. To
es requirements a second container, transfer about6liters of this solution,
Tailing factor: NMT 1.6, Standard solution add 100 g of sodium lauryl sulfate, and mix to dissolve the
oe standard deviation: NMT 5.0%, Standard solid material. Transfer this solution back into the first con-
solution tainer, and mix.
USP 41 Official Monographs / Mefloquine 2551

Medium: 0.05 M Tris buffer; 900 mL. Sample solution: 0.2 mg/mL of Mefloquine Hydrochlo-
Apparatus 1: 100 rpm. ride in Mobile phase
Time: 45 minutes. Chromatographic system
(See Chromatography (621), System Suitability.)
Procedure—Determine the amount of CisHisNOz dis- Mode: LC
solved, employing the procedure set forth in the Assay, Detector: UV 280 nm
making any necessary volumetric adjustments. Guard column: 4-mm x 3-cm; C18 (recommended)
Tolerances—Not less than 75% (Q) of the labeled amount Column: 4.0-mm x 25-cm; 5-um packing L1
of CisHisNOz is dissolved in 45 minutes. Column temperature: 25°
Uniformity of dosage units (905): meet the require- Flow rate: 0.8 mL/min
ments. Injection size: 20 uL
Assay— System suitability
Mobile phase, Standard preparation, and Chromatographic Samples: System suitability solution and Standard
solution
system—Proceed as directed in the Assay under Mefenamic
Acid. [Note—The relative retention times for mefloquine re-
lated compound A and mefloquine are about 0.7 and
Assay preparation—Remove, as completely as possible, 1.0, respectively.]
the contents of not fewer than 20 Capsules. Weigh the con- Suitability requirements
tents, and determine the average weight per capsule. Mix Resolution: NLT 2.0 between mefloquine related
the combined contents, and transfer an accurately weighed compound A and mefloquine, System suitability
quantity of the powder, equivalent to about 100 mg o solution
mefenamic acid, to a 500-mL volumetric flask. Add 10.0 mL Tailing factor: NMT 2.0, Standard solution
of tetrahydrofuran, and sonicate for about 5 minutes with Relative standard deviation: NMT 1.0%, Standard
occasional mixing. Dilute with Mobile phase to volume, mix, solution
and filter. Analysis
Procedure—Proceed as directed for Procedure in the Assay Samples: Standard solution and Sample solution
under Mefenamic Acid. Calculate the quantity, in mg, of Calculate the percentage of mefloquine hydrochloride
CisHisNOz in the portion of Capsules taken by the formula: (CizHisFeN2O - HCI) in the portion of Mefloquine Hy-
drochloride taken:
500C(ru / rs)
Result = (ru/rs) x (Cs/Cy) x 100
in which the terms are as defined therein.
tu = peak response of mefloquine from the Sample
solution
rs = peak response of mefloquine from the
Standard solution
Mefloquine Hydrochloride Cs = concentration of USP Mefloquine
Hydrochloride RS in the Standard solution (=
(mg/mL) v
Cu = concentration of Mefloquine Hydrochloride in ea
the Sample solution (mg/mL) =
Acceptance criteria: 98.0%-102.0% on the anhydrous °
basis 2
ito}
IMPURITIES By
© RESIDUE ON IGNITION (281): NMT 0.1% mo]
=>
ww“
CizHieFsN2O - HCl 414.77 Delete the following:
4-Quinolinemethanol, «-2-piperidinyl-2,8-bis(trifluoro-
methyl)-, monohydrochloride, (R*,S*)- (+)-; °e HEAVY METALS, Method I! (231): NMT 20 ppme crical1
DL-erythro-a-2-Piperidyl-2,8-bis(trifluorometh pels uin- Jan-2018)
olinemethanol monohydrochloride [1773- 234. © ORGANIC IMPURITIES
DEFINITION Mobile phase: Dissolve 1 g of tetraheptylammonium
Mefloquine Hydrochloride contains NLT 98.0% and NMT bromide in a 1-L mixture of a 1.5-g/L solution of so-
102.0% of CizHisFeN2O - HCI, calculated on the anhy- dium hydrogen sulfate, acetonitrile, and methanol
drous basis. (2:2:1).
System suitability solution: 4 ug/mL each of USP
IDENTIFICATION Mefloquine Hydrochloride RS and USP Mefloquine Re-
© A. INFRARED ABSORPTION (197K) lated Compound A RS in Mobile phase. [NoTe—Meflo-
e B. IDENTIFICATION TESTS—GENERAL, Chloride (191) quine related compoundAis threo-mefloquine.]
Sample stock solution: 4 mg/mL of Mefloquine Hydro-
ASSAY chloride in Mobile phase
© PROCEDURE Sample solution: 4 g/mL from the Sample stock solu-
Solution A: 1.5 g/L of sodium hydrogen sulfate in tion in Mobile phase
water Chromatographic system
Mobile phase: Dissolve 1 g of tetraheptylammonium (See Chromatography (621), System Suitability.)
bromide in a 1000-mL mixture of acetonitrile, metha- Mode: LC
nol, and Solution A (2:1:2). Detector: UV 280 nm
System suitability solution: 4 j1g/mL each of USP Guard column: 4-mm x 2.5-cm; 5-uum packing L1
Mefloquine Hydrochloride RS and USP Mefloquine Re- Column: 4.0-mm x 25-cm; 5-um packing L1
lated Compound ARS in Mobile phase Flow rate: 0.8 mL/min
Standard solution: 0.2 mg/mL of USP Mefloquine Hy- Injection size: 20 uL. [NoTE—Equilibrate the column
drochloride RS in Mobile phase win Monte phase at a flow rate of 0.8 mL/min for 30
min.
 2552 Mefloquine / Official Monographs USP 41

System suitability volume to obtain a solution having a concentration of

Sample: System suitability solution 2.5 mg/mL of mefloquine hydrochloride.
[NoT&—The relative retention times for mefloquine re- Sample solution: Nominally 0.05 mg/mL of mefloquine
lated compound A and mefloquine are about 0.7 and hydrochloride in Diluent from the Sample stock solution
1.0, respectively.] Chromatographic system
Suitability requirements (See Chromatography (621), System Suitability.)
Resolution: NLT 2.0 between mefloquine related Mode: LC
compound A and mefloquine Detector: UV 222 nm
Relative standard deviation: NMT 2.0% Column: 4.6-mm x 15-cm; 5-4um packing L68
Analysis Flow rate: 1 mL/min
Samples: Sample stock solution and Sample solution Injection size: 10 uL
Record the chromatogram for a time that is 10 times System suitability
the retention time of the main peak. Samples: Standard solution and Sensitivity solution
Acceptance criteria: The response of the mefloquine Suitability requirements
related compound A peak in the Sample stock solution is Column efficiency: NLT 4000 theoretical plates,
NMT twice the area of the main peak of the Sample Standard solution
solution (0.2%). The response of any other individual Tailing factor: NMT 1.5, Standard solution
peak, other than the main peak of the Sample stock Signal-to-noise ratio: NLT 5, Sensitivity solution
solution, is NMT that of the main peak of the Sample Relative standard deviation: NMT 2.0%, Standard
solution (0.1%); and the sum of the responses of any solution
such peaks of the Sample stock solution is NMT five Analysis
times the response of the main peak of the Sample solu- Samples: Standard solution and Sample solution
tion (0.5%). [NoTE—Exclude the main peak and any Calculate the percentage of mefloquine hydrochloride
other peak producing a response of less than 0.2 times (Ci7zHi6FeN2O - HCl) in the portion of Tablets taken:
(0.02%) the main peak of the Sample solution.]
Result = (ru/rs) x (Cs/Cu) x 100
SPECIFIC TESTS
© OPTICAL ROTATION, Specific Rotation (781S) ty = peak response from the Sample solution
Sample solution: 50 mg/mL in methanol ls = peak response from the Standard solution
Acceptance criteria: —0.2° to +0,2° G = concentration of USP Mefloquine
e WATER DETERMINATION, Method | (921): NMT 3.0% Hydrochloride RS in the Standard solution
(mg/mL)
ADDITIONAL REQUIREMENTS WG = renin concentration of mefloquine
¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant hydrochloride in the Sample solution
containers. Store between 15° and 30°. (mg/mL)
e USP REFERENCE STANDARDS (11) Acceptance criteria: 90.0%-110.0%
USP Mefloquine Hydrochloride RS
ry USP Mefloquine Related Compound A RS PERFORMANCE TESTS
= threo-Mefloquine. e DISSOLUTION (711)
a
Ln CizHisF6N20- HCI 414.78 Test 1
— Medium: 0.1 N hydrochloric acid; 900 mL
=) Apparatus 2: 50 rpm
° Time: 30 min
i=]
5 Standard stock solution: 0.2 mg/mL of USP Meflo-
= Mefloquine Hydrochloride Tablets quine Hydrochloride RS in Medium. A small amount of
[2 methanol, not exceeding 5% of the final volume, may
al
DEFINITION be used to help solubilize mefloquine.
= Standard solution: 0.04 mg/mL of USP Mefloquine
Mefloquine Hydrochloride Tablets contain NLT 90.0% and
NMT 110.0% of the labeled amount of mefloquine hydro- Hydrochloride RS in Medium from the Standard stock
chloride (Ci7HieFeN2O - HCl). solution
Sample solution: Dilute a portion of the solution
IDENTIFICATION under test with Medium (1:5), and pass a portion
e A. The retention time of the major peak of the Sample throughasuitable filter of 0.8-4m pore size.
solution corresponds to that of the Standard solution, as Instrumental conditions
obtained in the Assay. (See Ultraviolet-Visible Spectroscopy (857).)
e B. ULTRAVIOLET ABSORPTION (197U) Mode: UV
Diluent, Standard solution, and Sample solution: Pro- Analytical wavelength: 285 nm
ceed as directed in the Assay. Cell length: 1m
Blank: Diluent Blank: Medium
Analysis
ASSAY Samples: Standard solution and Sample solution
e PROCEDURE Calculate the percentage of mefloquine hydrochloride
Buffer: 2.7 g/L of monobasic potassium phosphate. Ad- (CizHieFsN2O - HCl) dissolved:
just with phosphoric acid to a pH of 3.0 + 0.1.
Diluent: Methanol and water (23:27) Result = (Au/As) x (Cs/L) x D x Vx 100
Mobile phase: Methanol, acetonitrile, and Buffer
(13:10:27) Av = absorbance from the Sample solution
Standard solution: 0.05 mg/mL of USP Mefloquine Hy- As = absorbance from the Standard solution
drochloride RS in Diluent Cs = concentration of USP Mefloquine
Sensitivity solution: 0.025 ug/mL of USP Mefloquine Hydrochloride RS in the Standard solution
Hydrochloride RS in Diluent (mg/ml)
Sample stock solution: Transfer a suitable number of L = label claim (mg/Tablet)
Tablets to a volumetric flask, dilute with methanol (ap- D = dilution factor of the Sample solution
proximately 80% of the total volume), shake for 30 Vv = volume of Medium, 900 mL
min, allow to sit for 1 h, and dilute with methanol to
USP 41 Official Monographs / Megestrol 2553

Tolerances: NLT 80% (Q) of the labeled amount of Table 1

mefloquine hydrochloride (Ci7HieFeN2O - HCl) is Relative Acceptance
dissolved. Retention Criteria,
Test 2: If the product complies with this test, the label- Name Time NMT (%)
ing indicates that it meets USP Dissolution Test 2.
Medium: 0.01 N hydrochloric acid; 900 mL Specified (unidentified) 0.67 0.15
Apparatus 2: 50 rpm Specified (unidentified) 0.70 0.15
Time: 30 min threo-Mefloquine (DL-threo-a-2-piper-
Standard solution: 0.278 mg/mL of USP Mefloquine idyl-2,8-bis(trifluoromethyl)-4- _—
Hydrochloride RS in Medium. A small amount of meth- quinolinemethanol) 0.75
anol, not exceeding 2.5% of the final volume, may be Specified (unidentified) 0.84 0.25
used to helpsolubihize mefloquine. Mefloquine hydrochloride 1.0 =
Sample solution: Pass a portion of the solution under
Any other unknown individual _
test throughasuitable filter.
impurity 0.15
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).) Total impurities — 0.50
Mode: UV
Analytical wavelength: 284 nm ADDITIONAL REQUIREMENTS
Cell length: 0.2 cm ° PACKAGING AND STORAGE: Preserve in tight, light-resistant
Blank: ~“Medium containers. Store at controlled room temperature.
Analysis o LABELING: When more than one Dissolution test is given,
Samples: Standard solution and Sample solution the labeling states the Dissolution test used only if Test 7
Calculate the percentage of mefloquine hydrochloride is not used.
(Ci7HieFeN2O - HCl) dissolved: e USP REFERENCE STANDARDS (11)
USP Mefloquine Hydrochloride RS
Result = (Ay/As) x (Cs/L) x Vx 100

Au = absorbance from the Sample solution

As = absorbance from the Standard solution
Cs = concentration of USP Mefloquine
Hydrochloride RS in the Standard solution Megestrol Acetate
(mg/mL)
L = label claim (mg/Tablet) Ox CH,
Vv = volume of Medium, 900 mL FHS] 20. 1
Tolerances: NLT 75% (Q) of the labeled amount of ce] °
mefloquine hydrochloride (Ci7HieFeN2O + HCl) is
dissolved. @
SoDNs :
e@ UNIFORMITY OF DosAGE UNITS (905): Meet the =
requirements cH rv
a}
IMPURITIES Co4H3204 384.51 =
e ORGANIC IMPURITIES Pregna-4,6-diene-3,20-dione, 17-(acetyloxy)-6-methyI-; fe)
Buffer, Diluent, Mobile phase, Standard solution, Sen- 17-Hydroxy-6-methylpregna-4,6-diene-3,20-dione acetate a
sitivity solution, Sample stock solution, Sample solu- [595-33-5]. a4
tion, Chromatographic system, and System suitabil- 4
ity: Proceed as directed in the Assay. DEFINITION a
Analysis Megestrol Acetate contains NLT 97.0% and NMT 103.0% of [iy
Samples: Standard solution and Sample solution megestrol acetate (C24H320.), calculated on the anhydrous |
Calculate the percentage of each impurity in the por- basis.
tion of Tablets taken:
IDENTIFICATION
Result = (ru/rs) x (Cs/Cu) x 100 e A. INFRARED ABSORPTION (197K)
tu = peak response of each impurity from the ASSAY
Sample solution e PROCEDURE
fs = peak response of mefloquine hydrochloride Mobile phase: Acetonitrile and water (55:45)
from the Standard solution Diluent: Acetonitrile and water (40:60)
Cs = concentration of USP Mefloquine Internal standard solution: 0.8 mg/mL of propylpara-
Hydrochloride RS in the Standard solution ben in acetonitrile
(mg/mL) Standard stock solution: 1 mg/mL of USP Megestrol
Cu = nominal concentration of mefloquine Acetate RS in acetonitrile
hydrochloride in the Sample solution Standard solution: 80 g/mL each of USP Megestrol
(mg/mL)
Acceptance criteria: See Table 1.
Acetate RS and propylparaben in Diluent from the Stan-
dard stock solution and Internal standard solution,
[Note—Do not include the threo isomer, a process im- respectively
purity monitored in the drug substance, in the calcula- Sample stock solution: 1 mg/mL of Megestrol Acetate
tion of total impurities. Disregard any peak less than in acetonitrile
0.05%.] Sample solution: 80 g/mL each of Megestrol Acetate
and propylparaben in Diluent from the Sample stock so-
lution and Internal standard solution, respectively
 2554 Megestrol / Official Monographs USP 41

Chromatographic system IDENTIFICATION

(See Chromatography (621), System Suitability.) e THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST (201)
Mode: LC Standard solution: 4.0 mg/mL of USP Megestrol Ace-
Detector: UV 280 nm tate RS in chloroform
Column: 3.9-mm x 30-cm; packing L1 Sample solution: Transfer Oral Suspension, equivalent
Flow rate: 1 mL/min to 160 mg of megestrol acetate, to a separatory funnel,
Injection volume: 25 uL add 50 mL of water and 40 mL of chloroform, and
System suitability shake. Allow the phases to separate, and discard the
Sample: Standard solution aqueous layer.
[Note—The relative retention times for propylparaben Developing solvent system: Chloroform and ethyl ace-
and megestrol acetate are about 0.4 and 1.0, tate (4:1)
respectively.]
Suitability requirements ASSAY
Resolution: NLT 8.0 between propylparaben and © PROCEDURE
megestrol acetate Mobile phase: Acetonitrile and water (11:9)
Relative standard deviation: NMT 2.0% for the peak Standard solution: 80 j1g/mL of USP Megestrol Acetate
response ratio of megestrol acetate to propylparaben RS in Mobile phase
Analysis Sample solution: A volume of Oral Suspension diluted
Samples: Standard solution and Sample solution with Mobile phase to obtain nominally 80 ng/mL of
Calculate the percentage of megestrol acetate megestrol acetate
(C24H320.) in the portion of Megestrol Acetate taken: Chromatographic system
(See Chromatography (621), System Suitability.)
Result = (Ru/Rs) x (Cs/Cu) x 100 Mode: LC
Detector: UV 280 nm
Ru = peak response ratio of megestrol acetate to Column: 3.9-mm x 30-cm; packing L1
propylparaben from the Sample solution Flow rate: 1.5 mL/min
Rs = peak response ratio of megestrol acetate to Injection size: 25 wL
propylparaben from the Standard solution System suitability
Cs = concentration of USP Megestrol Acetate RS in Sample: Standard solution
the Standard solution (mg/mL) Suitability requirements
Gu = concentration of Megestrol Acetate in the Column efficiency: NLT 2500 theoretical plates
Sample solution (mg/mL) Tailing factor: NMT 1.4
Acceptance criteria: 97.0%-103.0% on the anhydrous Relative standard deviation: NMT 2.0%
basis Analysis
Samples: Standard solution and Sample solution
IMPURITIES Calculate the percentage of C24H32O, in the portion of
© RESIDUE ON IGNITION (281): NMT 0.2%, with a platinum Oral Suspension taken:
dish being used and ignition at 600 + 25°
Ato
“
Result = (ru/ts) x (Cs/Cu) x 100
oy
ig— Delete the following: tu = peak response from the Sample solution
aD
° Is = peak response from the Standard solution
= °o HEAVY METALS, Method I! (231): NMT 20 ppme coral
1- Cs = concentration of USP Megestrol Acetate RS in
5 Jan-2018) the Standard solution (g/mL)
= SPECIFIC TESTS Cu = nominal concentration of the Sample solution
Ps e COMPLETENESS OF SOLUTION (641) (ug/mL)
Al
Sample: 500mg in 10 mL of acetone Acceptance criteria: 90.0%-110.0%
=)
Acceptance criteria: Meets the requirements PERFORMANCE TESTS
e MELTING RANGE OR TEMPERATURE (741): 213°-220°, but e DISSOLUTION (711)
the range between the beginning and the end of melting Test 1
does not exceed 3°. Medium: 0.5% sodium lauryl sulfate in water; 900 mL
© OPTICAL ROTATION, Specific Rotation (781S) Apparatus 2: 25 rpm
Sample solution: 20 mg/mL in chloroform Time: 30 min
Acceptance criteria: +8.8° to +12.0° (t = 20°) Detector: UV 292 nm
e WATER DETERMINATION, Method | (921): NMT 0.5% Standard solution: 45 mg of USP Megestrol Acetate
ADDITIONAL REQUIREMENTS RS in a 250-mL volumetric flask. Add 12 mL of metha-
© PACKAGING AND STORAGE: Preserve in well-closed contain- nol, and place the flask in a warm water bath until the
ers, protected from light. solid is dissolved. Dilute with Medium to volume. The
e USP REFERENCE STANDARDS (11) final concentration is 180 tug/mL of megestrol acetate.
USP Megestrol Acetate RS Dilute with Medium, if necessary.
Sample solution: Transfer to the surface of the Me-
dium in the dissolution vessel an accurately measured
volume of Oral Suspension, freshly mixed and free
from air bubbles, equivalent to 160 mg of megestrol
acetate. At the sampling time, withdraw a volume of
Megestrol Acetate Oral Suspension the solution under test and pass through a suitable
filter with 0.45-um pore size. Dilute with Medium, if
DEFINITION necessary.
Megestrol Acetate Oral Suspension contains NLT 90.0% and Analysis
NMT 110.0% of the labeled amount of megestro! acetate Samples: Standard solution and Sample solution
(C24H3204). Calculate the percentage of C24H32O, released:
Result = (Au/As) x (Cs/V) X Vo x (100/L)
Au = absorbance of the Sample solution
USP 41 Official Monographs / Megestrol 2555

As = absorbance of the Standard solution Injection size: 10 pL

Cs = concentration of the Standard solution Analysis
Vv
(mg/mL)
= volume of Oral Suspension taken
Samples: Standard solution and Sample solution
Calculate the percentage of C24H32O, released:
Vo = volume of Medium, 900 mL
L = label claim (mg/mL) Result = (ru/ts) x (Cs/W) x Vo x d x (100/L)
Tolerances: NLT 80% (Q) of the labeled amount of
C24H320, is dissolved. tu = peak response from the Sample solution
Test 2: If the product complies with this test, the label- ts = peak response from the Standard solution
ing indicates that it meets USP Dissolution Test 2. Cs = concentration of the Standard solution
Medium: 0.5% sodium lauryl sulfate in water; 900 mL (mg/ml) i
Apparatus 2: 25 rpm Ww = weight of the Oral Suspension taken (mg)
Time: 30 min Vo = volume of the Medium in the dissolution
Detector: UV 292 nm, using 0.5-cm pathlength vessel, 900 mL
cuvettes d = density of the Oral Suspension (mg/mL),
Standard solution: 45 mg of USP Megestrol Acetate obtained by dividing the weight of Oral
RS ina 250-mL volumetric flask. Add 5 mL of metha- Suspension taken by 10 mL
nol. Dilute with Medium to volume. Transfer 10 mL of L = label claim (mg/mL)
this solution to a 100-mL volumetric flask, and dilute Tolerances: NLT 80% (Q) of the labeled amount of
with Medium to volume. The final concentration is C24H320s is dissolved.
18 ua/me © DELIVERABLE VOLUME (698): Meets the requirements
Sample solution: [NoT&—Use a separate syringe for
each vessel.] Withdraw more than 10 mL of the Oral SPECIFIC TESTS
Suspension, using a 10-mL syringe with a long can- ° PH (791): 3.0-4.7
nula. Remove air bubbles from the syringe. Adjust the ADDITIONAL REQUIREMENTS
volume to the 10-mL mark on the syringe, and re- e PACKAGING AND STORAGE: Preserve in well-closed, light-
move the needle. Wipe the tip of the syringe, and resistant containers.
weigh (gross weight). Operate the apparatus, and rap- e LABELING: When more than one test for Dissolution is
idly dispense the Oral Suspension to the side of the given, the labeling states the test used only if Test 7 is
vessel at about halfway from the bottom. Similarly dis- not used.
pense the Oral Suspension into other vessels. Weigh o USP REFERENCE STANDARDS (11)
each syringe after dispensing the sample (tare weight). USP Megestrol Acetate RS
Record sample weights. After completion of the disso-
lution, pass an aliquot through a suitable nylon filter
with 0.45-um pore size, and dilute 2.0 mL of the fil-
trate with Medium to 50.0 mL to obtain a solution hav-
ing a theoretical concentration of about 18 g/mL.
Analysis Megestrol Acetate Tablets (=
Samples: Standard solution and Sample solution 4)
Calculate the percentage of C24H32O0,4 released: DEFINITION a)
Megestrol Acetate Tablets contain NLT 93.0% and NMT E
Result = (Au/As) x (Cs/W) x Vo x d x (100/L) 107.0% of the labeled amount of megestrol acetate i)
mS
(Co4H320,).
[NoTt—Megestrol Acetate Tablets labeled solely for veteri- ry
Au = absorbance of the Sample solution
nary use are exempt from the requirements of the Dissolu-
to)=
As = absorbance of the Standard solution i}
Cs = concentration of the Standard solution tion test.] a]
(mg/mL) IDENTIFICATION
Ke
“
WwW = weight of the Oral Suspension taken (mg)
Vo = volume of the Medium in the dissolution oA.
vessel, 900 mL Sample solution: Grind a suitable number of Tablets in
d = density of the Oral Suspension (mg/mL), a known volume of chloroform, NLT 10 mL, to obtain a
obtained by dividing the weight of Oral solution containing 4 mg/mL of megestrol acetate.
Suspension taken by 10 mL Analysis: Filter the Sample solution into a beaker. Pipet
L = label claim (mg/mL) 0.6 mL of the filtrate into a stainless steel grinding vial
Tolerances: NLT 80% (Q) of the labeled amount of containing 500 mg of potassium bromide, dry with a
C24H320, is dissolved. current of air, grind, pellet, and record the IR spectrum.
Test 3: If the product complies with this test, the label- Acceptance criteria: The IR absorption spectrum of the
ing indicates that it meets USP Dissolution Test 3. potassium bromide dispersion so obtained exhibits
Medium: 0.5% sodium lauryl sulfate in degassed maxima only at the same wavelengths as that of a simi-
water; 900 mL. [NoTE—Use ultrapure sodium lauryl sul- lar preparation of USP Megestrol Acetate RS.
fate with an Assay content of NLT 99.0%.]
ASSAY
Apparatus 2: 50 rpm
Time: 30 min e PROCEDURE
Mobile phase: Acetonitrile and water (55:45)
Determine the amount of C24H32Ox dissolved by using
the following method. Diluent: Acetonitrile and water (40:60)
Mobile phase: Proceed as directed in the Assay. Internal standard solution: 0.8 mg/mL of propylpara-
Standard solution: 0.46 mg/mL of USP Megestrol
ben in acetonitrile
Acetate RS in Mobile phase Standard stock solution: 1 mg/mL of USP Megestrol
Sample solution: Proceed as directed for Test 2, intro- Acetate RS in acetonitrile
Standard solution: 80 g/mL each of USP Megestrol
ducing the sample into the vessel over a 10- to 15-s Acetate RS and propylparaben in Diluent from the Stan-
period (about 1 mL/s).
dard stock solution and Internal standard solution,
Chromatographic system: Proceed as directed in the
respectively
Assay.
Sample solution: Nominally 80 ug/mL of megestrol
acetate prepared as follows. Transfer the equivalent of
 2556 Megestrol / Official Monographs
80 mg of megestrol acetate from powdered Tablets
(NLT 20 Tablets) to a 100-mL volumetric flask. Add
10 mL of water, and shake for 10 min. Add 75 mL of
acetonitrile, shake for 30 min, then dilute with acetoni-
trile to volume. Place a 25-mL aliquot in a glass-stop-
pered 35-mL centrifuge tube, insert the stopper, and
centrifuge for 10 min. Transfer 5.0 mL of the superna-
tant and 5.0 mL of Internal standard solution to a 50-mL
volumetric flask, and dilute with Diluent to volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 280 nm
Column: 3.9-mm x 30-cm; packing L1
Flow rate: 1 mL/min
Injection volume: 25 uL
System suitability
Sample: Standard solution
[NoTte—The relative retention times for propylparaben
and megestrol acetate are about 0.4 and 1.0,
respectively.]
Suitability requirements
Resolution: NLT 8.0 between propylparaben and
megestrol acetate
Relative standard deviation: NMT 2.0% for the peak
response ratio of megestrol acetate to propylparaben
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
megestrol acetate (C24H32O.) in the portion of Tablets
taken:
Result = (Ru/Rs) x (Cs/Cu) x 100
Ru = peak response ratio of megestrol acetate to
propylparaben from the Sample solution
Rs = peak response ratio of megestrol acetate to
val propylparaben from the Standard solution
a Gs = concentration of USP Megestrol Acetate RS in
5 the Standard solution (mg/mL)
S
—
Cu = nominal concentration of megestrol acetate in
Dd
° the Sample solution (mg/mL)
i<j Acceptance criteria: 93.0%-107.0%
5
= PERFORMANCE TESTS
[5 ¢ DISINTEGRATION (701)
Al
a)
Sample: Tablets labeled solely for veterinary use; pro-
ceed as directed for plain-coated Tablets, but use film-
coated Tablets instead.
Time: 30 min
Acceptance criteria: Meet the requirements
e DISSOLUTION (711)
Medium: 1% sodium lauryl sulfate; 900 mL
Apparatus 2: 75 rpm
Time: 60 min
Standard solution: USP Megestrol Acetate RS in
Medium
Sample solution: Afiltered portion of the solution
under test, suitably diluted with Medium, if necessary,
to a concentration that is similar to that of the Standard
solution.
Instrumental conditions
Analytical wavelength: UV 292 nm
Analysis
Samples: Standard solution and Sample solution
Determine the amount of megestrol acetate (C24H3204)
dissolved.
Tolerances: NLT 75% (Q) of the labeled amount of
megestrol acetate (C24H320.) is dissolved.
e UNIFORMITY OF DosaGE UNITS (905)
Procedure for content uniformity
Standard solution: 10 g/mL of USP Megestrol Ace-
tate RS in methanol
Sample solution: Nominally 10 g/mL of megestrol
acetate prepared as follows. Place 1 Tablet in a volu-
USP 41
metric flask of suitable size so that the final expected
solution concentration is between 0.2 and 1.0 mg of
megestrol acetate per mL. Add 1 mL of water, and
gen shake until the Tablet has disintegrated. Fill the
lask to three-quarters of its nominal capacity with
methanol, and shake by mechanical means for 20 min.
Dilute with methanol to volume, mix, and filter, dis-
carding the first 15 mL of the filtrate. Dilute 5.0 mL of
the subsequent filtrate with methanol.
Instrumental conditions
Mode: UV-Vis
Wavelength range: 260-350 nm
Analytical wavelength: Absorption maximum at
about 288 nm
Cell: 1.cm
Blank: Methanol
Analysis
oe Standard solution, Sample solution, and
Blank
Record the absorbances of the Standard solution and
the Sample solution against the Blank, scanning from
260 to 350 nm.
Calculate the percentage of megestrol acetate
(C24H3204) in the Tablet taken:
Result = (Au/As) x (Cs/Cu) x 100
Au = absorbance of the Sample solution
As = absorbance of the Standard solution
Gs = concentration of USP Megestrol Acetate RS in
the Standard solution (ug/mL)
Cu = nominal concentration of megestro! acetate in
the Sample solution (ug/mL)
Acceptance criteria: Meet the requirements
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed
containers.
e LABELING: Tablets intended solely for veterinary use are so
labeled.
e USP REFERENCE STANDARDS (11)
USP Megestrol Acetate RS
Meglumine
woLP
Hon
by oon
C7HizyNOs 195.21
D-Glucitol, 1-deoxy-1-(methylamino)-;
1-Deoxy-1-(methylamino)-D-glucitol [6284-40-8].
DEFINITION
Meglumine contains NLT 99.0% and NMT 100.5% of
meglumine (C7Hi7NOs), calculated on the dried basis.
IDENTIFICATION
cA.
Sample solution: Transfer 250 mg to a dry, 50-mL cen-
trifuge tube, add 500 mg of sodium metaperiodate,
then add 5 mL of water rapidly in one portion. Allow to
stand undisturbed.
Analysis: The solution instantly turns yellow, and heat is
produced. The color then changes from deep yellow to
orange-brown (rust), and after 20 min, the rust-colored
solution is cloudy. Then add 2 mL of 2.5 N sodium
hydroxide.
Acceptance criteria: The mixture turns bright yellow
and becomes clear.
USP 41 Official Monographs / Melengestrol 2557

ASSAY » Melengestrol Acetate contains not less than

© PROCEDURE 97.0 percent and not more than 103.0 percent of
Sample: 500mg
Titrimetric system C25H320,4, calculated on the dried basis.
Mode: Direct titration Packaging and storage—Preserve in tight, light-resistant
Titrant: 0.1 N hydrochloric acid containers, and store at controlled room temperature.
Endpoint detection: Visual
Analysis: Transfer the Sample into a conical flask, dis- Labeling—Label it to indicate that it is for veterinary use
solve in 40 mL of water, and add 2 drops of methyl red only.
TS. Titrate with Titrant. Each mL of Titrant is equivalent USP Reference standards (11)—
to 19.52 mg of meglumine (C7Hi7NOs). USP Melengestro! Acetate RS
Acceptance criteria: 99.0%-100.5% on the dried basis USP Melengestrol Acetate Related Compound A RS
16-Methylene-17a-hydroxy-4-pregnene-3,20-dione
IMPURITIES 17-acetate.
USP Melengestrol Acetate Related Compound B RS
Delete the following: 17a-Hydroxy-6, 16-dimethyleneprogna-4-ene-3,20-dione
17-acetate.
°e HEAVY METALS (231) Identification—
Test preparation: Dissolve 1 g in 20 mL of water, add A: Infrared Absorption (197K).
phenolphthalein TS, neutralize with 3 N hydrochloric B: Ultraviolet Absorption (197U)—
acid, and dilute with water to 25 mL. Solution: 10 ug per mL.
Acceptance criteria: NMT 20 Ha/ge (Oificial (-Jan-2018)
e RESIDUE ON IGNITION (281): NMT 0.17% Medium: alcohol.
C: The retention time of the melengestrol acetate peak in
SPECIFIC TESTS the chromatogram of the Assay preparation corresponds to
© ABSENCE OF REDUCING SUBSTANCES that in the chromatogram of the Standard preparation, as
Sample solution: 50 mg/mL obtained in the Assay.
Analysis: To 5 mL of Sample solution add 5 mL of alka- Melting temperature (741): between 219° and 226°.
line cupric tartrate TS, and heat to boiling.
Acceptance criteria: The color of the solution does not Specific rotation (7815S): between -132.0° and -122.0°,
change. at 20°.
COMPLETENESS AND COLOR OF SOLUTION Test solution: 10.0 mg per mL, in chloroform.
Sample solution: 200 mg/mL Loss on drying (731)—Dry it at 105° for 3 hours: it loses
Instrumental conditions not more than 0.5% of its weight.
(See Ultraviolet-Visible Spectroscopy (857).)
Mode: Vis
Analytical wavelength: 420 nm Delete the following:
Cell: 1.cm (oa
Blank: Water °Heavy metals, Method |! (231): not more than 0.001%. al
~~
Analysis © (Official }-Jan-2018)
Sample: Sample solution Related compounds— =
Acceptance criteria: Adsorbance is NMT 0.030. i}
Mobile phase—Prepare a mixture of acetonitrile and water PJ
Loss ON DRYING (731) (50:50). re}
Sample: 1g a=
Analysis: Dry the Sample at 105° to constant weight. Standard solution—Dissolve an accurately weighed quan- i
Acceptance criteria: NMT 1.0% tity of USP Melengestrol Acetate RS, USP Melengestrol Ace- so}
MELTING RANGE OR TEMPERATURE (741): 128°-132° tate Related Compound A RS, and USP Melengestrol Acetate =>
al
OPTICAL ROTATION, Specific Rotation (781S) Related Compound BRS in methanol to obtain a solution
Sample solution: 100 mg/mL, undried, in water having known concentrations of about 0.005 mg of each
Acceptance criteria: —15.7° to -17,3° per mL.
Test solution—Use the Assay preparation.
ADDITIONAL REQUIREMENTS Chromatographic system (see Chromatography (621))—The
e PACKAGING AND STORAGE: Preserve in well-closed liquid chromatograph is equipped with a multiwavelength
containers. detector set at 240 and 262 nm and a 4.6-mm x 25-cm
column that contains 5-um packing L7. The flow rate is
about 1.0 mL per minute. Chromatograph the Standard so-
lution, and record the peak responses as directed for Proce-
dure [NoTE—Melengestrol acetate and melengestrol related
Melengestrol Acetate compound B will generate larger peak areas at 262 nm than
at 240 nm; melengestrol acetate related compound A will
generate a larger peak area at 240 nm than at 262 nm]: the
relative retention times are about 0.78, 1.0, and 1.05 for
melengestrol acetate related compound A, melengestrol
acetate, and melengestrol acetate related compound B, re-
spectively; the resolution, R, between melengestrol acetate
related compound A and melengestrol acetate related com-
poundBis not less than 5.0; the column efficiency for the
melengestrol acetate related compound A peak is greater
than 1500 theoretical plates; the tailing factor is less than
C2sH3204 396,52 2.0; and the relative standard deviation for replicate injec-
Pregna-4,6-diene-3,20-dione, 17-(acetyloxy)-6-methyl- tions is not more than 5.0%.
16-methylene-. Procedure—Separately inject equal volumes (about 20 LL)
17-Hydroxy-6-methyl-1 6-methylenepregna-4,6-diene-3,20- of the Standard solution and the Test solution into the chro-
dione acetate [2919-66-6]. matograph, record the chromatograms, identify the peaks,
 2558 Melengestrol / Official Monographs USP 41

and determine which detector wavelength generates the

larger peak area for each impurity. Using the larger peak
area, calculate the percentage of each impurity in the por-
tion of Melengestrol Acetate taken by the formula:
100(Cs/ Cu)(ri/ rs)
in which Cs is the concentration, in mg per mL, of either
melengestrol related compound A or melengestrol related
compoundBin the Standard solution [NoTE—If using the
impurity peak area generated at 240 nm, Cs is the concen-
tration of melengestrol related compound A; if using the
impurity peak area generated at 262 nm, Cs is the concen-
tration of melengestrol related compound B]; Cy is the con-
centration, in mgper mL, of melengestrol acetate in the
Test solution; ris the peak area of each impurity obtained
from the Test solution; and rs is the peak area of either
melengestrol related compound A or melengestrol related
compound B obtained from the Standard solution [NoTe—lf
using the impurity peak area generated at 240 nm, rs is the
peak area of melengestrol related compound A; if using the
impurity peak area generated at 262 nm, Cs is the peak area
of melengestro! related compound BJ: not more than 0.5%
of any identified impurity is found; not more than 0.2% of
any unidentified impurity is found; and not more than 1.0%
of total impurities is found.
Assay—
Mobile phase—Prepare a mixture of acetonitrile and water
(50:50).
Standard preparation—Dissolve an accurately weighed
quantity of USP Melengestrol Acetate RS in methanol to ob-
tain a solution having a known concentration of about
0.5 mg per mL.
Assay preparation—Transfer about 50 mg of Mice al
Acetate, accurately weighed, to a 100-mL volumetric flask,
and dissolve in and dilute with methanol to volume.
" Chromatographic system (see Chromatography (621))—The
J prepared as follows. Dissolve USP Meloxicam RS in 50%
a liquid chromatograph is equipped with a 287-nm detector
i] and a 4.6-mm x 25-cm column that contains 5-11m packing
- volume.
D L7. The flow rate is about 1.0 mL per minute. Chromato-
° graph the Standard preparation as directed for Procedure: the
iJ column efficiency is not less than 1500 theoretical plates;
Sj the tailing factor is not more than 2; and the relative stan-
= dard deviation for replicate injections is not more than
is 2.0%.
a)
=) Procedure—Separately inject equal volumes (about 20 LL)
of the Standard preparation and the Assay preparation in du-
plicate into the chromatograph, record the chromatograms,
and measure the areas for the major peaks. Calculate the
quantity, in mg, of C2sH320. in the portion of Melengestrol
Acetate taken by the formula:
2CWru / rs)
in which C is the concentration, in mg per mL, of the Stan-
dard preparation; W is the weight, in mg, of Melengestrol
Acetate used to prepare the Assay preparation; ry is the aver-
age peak area of the melengestrol acetate peak obtained
from the Assay preparation; and rs is the average peak area
of the melengestrol acetate peak obtained from the Stan-
dard preparation.
Meloxicam
CO
LITW
CHy
Crath3NsO.S2 351.40
aeee eee 1)-2H-1,2-
benzothiazine-3-carboxamide 1,1-dioxide [71125-38-7].
DEFINITION
Meloxicam contains NLT 98.0% and NMT 102.0% of
meloxicam (C4Hi3N304S2), calculated on the dried basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
e B. The retention time of the meloxicam peak of the Sam-
ple solution corresponds to that of the Standard solution,
as obtained in the Assay.
ASSAY
¢ PROCEDURE
Solution A: Mixture of a 0.1% (w/v) solution of ammo-
nium acetate adjusted with 10% ammonia solution to a
pH of 9.1
Mobile phase: Methanol and Solution A (21:29)
Diluent: Methanol and 1 N sodium hydroxide (250:1)
System suitability solution: 0.08 mg/mL each of USP
Meloxicam RS and USP Meloxicam Related Compound
A RS prepared as follows. Dissolve USP Meloxicam RS
and USP Meloxicam Related Compound A RS in 50% of
the flask volume of Diluent, and dilute with water to
volume.
Standard solution: 0.2 mg/mL of USP Meloxicam RS
of the flask volume of Diluent, and dilute with water to
Sample solution: 0.2 mg/mL of Meloxicam prepared as
follows. Dissolve Meloxicam in 50% of the flask volume
of Diluent, and dilute with water to volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 360 nm
Column: 4.6-mm x 15-cm; 5-41m packing L1
Column temperature: 45°
Flow rate: 1 mL/min
Injection volume: 10 uL
System suitability
Samples: System suitability solution and Standard
solution
[Note—The relative retention times for meloxicam re-
lated compound A and meloxicam are 0.7 and 1.0,
respectively.]
Suitability requirements
Resolution: NLT 3.0 between meloxicam related
compound A and meloxicam, System suitability
solution
Tailing factor: NMT 2.0 for the meloxicam peak, Sys-
tem suitability solution
Relative standard deviation: NMT 0.73%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of meloxicam (Ci4Hi3N304S2)
in the portion of Meloxicam taken:
Result = (ru/rs) x (Cs/Cu) x 100
ry = peak response of meloxicam from the Sample
solution
USP 41 Official Monographs / Meloxicam 2559

rs = peak response of meloxicam from the Chromatographic system

Standard solution (See Chromatography (621), System Suitability.)
Gs = concentration of USP Meloxicam RS in the Mode: LC
Standard solution (mg/mL) Detector: UV 260 and 350 nm (variable wavelength
Cu = concentration of the Sample solution (mg/mL) or multi-wavelength detector)
Acceptance criteria: 98.0%-102.0% on the dried basis Column: 4.6-mm x 15-cm; 5-ym packing L1
Column temperature: 45°
IMPURITIES Flow rate: 1 mL/min
e RESIDUE ON IGNITION (281): NMT 0.1% Injection volume: 5 uL
System suitability
Delete the following: Samples: System suitability solution and Standard
solution
°e HEAVY METALS, Method I! (231): NMT 10 ppme coitica 1- [Note—The relative retention times are listed in Table
Jan-2078)
ws
© ORGANIC IMPURITIES, PROCEDURE 1 suitability requirements
Perform either Procedure 1 or Procedure 2, depending on Resolution: NLT 3.0 between meloxicam related
the manufacturing process used. compound A and meloxicam at 350 nm; NLT 3.0
Solution A: 0.1% (w/v) solution of monobasic potas- between meloxicam related compound B and meloxi-
sium phosphate adjusted with 1 N sodium hydroxide to cam at 260 nm, System suitability solution
a pH of 6.0 Relative standard deviation: NMT 10%, Standard
Solution B: Methanol solution
Diluent: Methanol and 1 N sodium hydroxide (50:3) Analysis
Mobile phase: See Table 7. Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the por-
tion of Meloxicam taken:
Table 1
Solution A Solution B Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
%
ty = peak response of each impurity from the
60
Sample solution
rs = peak response of meloxicam at 350 nm from
the Standard solution
Cs = concentration of USP Meloxicam RS in the
60 Standard solution (mg/mL)
Cu = concentration of the Sample solution (mg/mL)
F = relative response factor (see Table 2)
System suitability solution: 0.08 mg/mL each of USP [Note—For the specified impurities, calculate the per-
Meloxicam RS, USP Meloxicam Related Compound A centage content of each impurity, using the peak re- =
RS, and USP Meloxicam Related Compound B RS pre- sponses from the Sample solution recorded at the de- “
tection wavelength given in Table 2. For an unknown vu
pared as follows. Dissolve USP Meloxicam RS, USP
Meloxicam Related Compound A RS, and USP Meloxi- impurity, calculate the percentage content, using peak =
cam Related Compound B RS in 10% of the flask vol- responses recorded at the wavelength that gives the °
greater response.] =|
ume of Diluent, and dilute with water to volume. °
Standard stock solution: 0.6 mg/mL of USP Meloxicam Acceptance criteria: See Table 2. Ko}
© ORGANIC IMPURITIES, PROCEDURE 2 =
RS prepared as follows. Dissolve USP Meloxicam RS in 2
25% of the flask volume of Diluent, and dilute with If an article complies with this test, the labeling indicates me]
methanol to volume. that it meets the requirements under Organic Impurities, a
7
Standard solution: 0.012 mg/mL of USP Meloxicam RS Procedure 2.
in methanol from the Standard stock solution Solution A and Solution B: Proceed as directed in Pro-
Sample solution: 4 mg/mL of Meloxicam prepared as cedure 1.
follows. Dissolve Meloxicam in 25% of the flask volume
of Diluent, and dilute with methanol to volume.

Table 2
Relative Relative Acceptance
Retention Wavelength Response Criteria,
Ti
0.4 1

Meloxicam 1.4
xical 1
Ei 1.9
i I =
otal rities =
2 5-Methylthiazol-2-amine.
» Ethyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide.
¢ N-[3,5-Dimethylthiazol-2(3H]-ylidene)-4-hydroxy-2-methyl-2H-benzo[e][1,2]thiazine-3-carboxamide 1,1-dioxide.
4 N-[3-Ethyl-5-methylthiazol-2(3H)-ylidene]-4-hydroxy-2-methyl-2H-benzo[e][1,2]thiazine-3-carboxamide 1,1-dioxide.
 2560 Meloxicam / Official Monographs USP 41

Mobile phase: See Table 3. Cu = concentration of the Sample solution (mg/mL)

[Note—Use the peak response and concentration of
Table 3 USP Meloxicam RS for unknown impurities; for the
specified impurities, calculate the percentage content
Solution A Solution B of each impurity using the Sample solution peak re-
sponses recorded at the detection wavelength given in
0 45 55 Table 4. For an unknown impurity, calculate the per-
25 45 centage content using peak responses recorded at the
70 wavelength that gives the gad response.]
40 Acceptance criteria: See Table 4.

50 45 5
Table 4
Relative Wave- Acceptance
ONO) A: Diluent B and 0.4 N sodium hydroxide Retention length Criteria,
50:3 Name Time (mm) NMT (%)
Diluent B: Methanol and water (2:3) Meloxicam 1.0 350 =
Standard stock solution A: 0.01 mg/mL of USP Melox-
Meloxicam related
icam RS prepared as follows. Dilute a solution of
compound Ba 0.8 260 0.1
0.05 mg/mL of USP Meloxicam RS in Diluent A with Dil-
uent B. Meloxicam related
Standard stock solution B: 0.05 mg/mL each of USP compound Ce 3:2 350 0.1
Meloxicam Related Compound B RS and USP Meloxi- Individual unknown _
cam Related Compound C RS prepared as follows. impurity 260/350 0.1
Transfer suitable amounts of USP Meloxicam Related Total impurities — a 0.3
Compound B RS and USP Meloxicam Related Com- a 5-Methylthiazol-2-amine.
pound C RS to an adequate volumetric flask. Add 0.4 N b: Eopropyttchydroxy-2emethyl:2t1 ,2-benzothiazine-3-carboxylate-1,1-di-
sodium hydroxide to 6% of the flask volume, and soni- oxide,
cate for 2 min. Add an additional 40% of the flask vol-
ume of methanol, sonicate for 2 min, and dilute with SPECIFIC TESTS
water to volume. e Loss ON DRYING (731)
Standard solution: 0.001 mg/mL of USP Meloxicam RS Analysis: Dry at 105° for 4 h.
and 0.0015 mg/mL each of USP Meloxicam Related Acceptance criteria: NMT 0.5%
Compound B RS and USP Meloxicam Related Com- ADDITIONAL REQUIREMENTS
pound C RS prepared as follows. Transfer suitable ¢ PACKAGING AND STORAGE: Preserve in well-closed contain-
volumes of Standard stock solution A and Standard stock ers. Store at room temperature.
solution B to an adequate volumetric flask, and dilute e LABELING: The labeling states with which Procedure under
al with Diluent B to volume. Organic Impurities the article complies if a test other than
<= Sample solution: 1 mg/mL of Meloxicam prepared as
ry follows. Dissolve a suitable amount of Meloxicam with
Procedure 1 is used.
S e USP REFERENCE STANDARDS (11)
_
50% of the flask volume of Diluent A, and dilute with
im) USP Meloxicam RS
i} Diluent B to volume. USP Meloxicam Related Compound A RS
te Chromatographic system Ethyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-car-
Gj (See Chromatography (621), System Suitability.) boxylate 1,1-dioxide.
= Mode: LC Ci2HizNOsS 283.30
[5 Detector: UV variable wavelength or multi-wavelength USP Meloxicam Related Compound B RS
va) detector at 260 and 350 nm
= Column: 4.6-mm x 25-cm; 5-um packing L1
5-Methylthiazol-2-amine.
C4aHeN2S 114.175
Column temperature: 45° USP Meloxicam Related Compound C RS
Flow rate: 1 mL/min ee ee roxy-2-methyl-2H-1,2-benzothiazine-
Injection volume: 20 uL 3-carboxylate-1,1-dioxide.
System suitability CisHisNOsS = 297.33
Sample: Standard solution
[Note—The relative retention times are listed in Table
4]
Suitability requirements
Relative standard deviation: NMT 5.0% for meloxi-
cam, meloxicam related compound B, and meloxi- Meloxicam Oral Suspension
cam related compound C
Analysis » Meloxicam Oral Suspension contains not less
Samples: Standard solution and Sample solution than 90.0 percent and not more than 110.0 per-
Calculate the percentage of each impurity in the por-
tion of Meloxicam taken: cent of the labeled amount of meloxicam
(Ci4H13N304S2).
Result = (ru/rs) x (Cs/Cu) x 100
Packaging and storage—Preserve in well-closed contain-
ry = peak response of each impurity from the os Store at 25°, excursions permitted between 15° and
Sample solution
rs = peak response of the corresponding related USP Reference standards (11)—
compound from the Standard solution USP Meloxicam RS
G = concentration of the corresponding USP USP Meloxicam Related a BRS
Related Compound RS in the Standard 2-Amino-5-methyl-thiazole.
solution (mg/mL). [NoTe—Use the concentra-
tion of USP Meloxicam RS for unknown im-
purities.]
USP 41
Identification—
A: Thin-Layer Chromatographic Identification Test (201)—
Test solution—Transfer a volume of Oral Suspension,
equivalent to about 2.5 mg of meloxicam, to a 10-mL volu-
metric flask. Dilute with acetone to volume, and mix for
10 minutes. If necessary, pass through fluted filter paper.
Standard solution: 0.25 mg per mL, prepared by dis-
solving USP Meloxicam RS in T mL of water and diluting
with acetone to volume.
Developing solvent solution: a mixture of chloroform,
methanol, and ammonium hydroxide (80:20:1)
Procedure—Proceed as directed in the chapter. After re-
moving the plate from the chamber and drying, examine
the chromatograms under UV light at 254-nm: the Ry value
(approximately 0.45) of the principal dark spot obtained
from the Test solution corresponds to that obtained from the
Standard solution.
B: The retention time of the major peak in the chromato-
gram of the Assay preparation corresponds to that in the
chromatogram of the Standard preparation, as obtained in
the Assay.
PH (791): between 3.5 and 4.5.
Viscosity—Rotational Methods (912)—Determine using
a shear rate programmable rotational viscometer: between
40 and 100 centipoises, determined at 20°.
Dissolution (711)—
Medium: pH 7.5 phosphate buffer; 900 mL.
Apparatus 2: 25 rpm.
Time: 15 minutes.
Determine the amount of C14H13N304S2 dissolved by em-
ploying the following method.
Standard solution—Transfer about 20.83 mg of USP
Meloxicam RS, accurately weighed, into a 100-mL volumet-
ric flask. Dissolve in 5 mL of methanol and 1 mL of 0.1 M
sodium hydroxide, and dilute with Medium to volume. Di-
lute with Medium to a final concentration of about 8.3 ug
per mL of meloxicam.
Test solution—Shake each sample for 15 minutes. Weigh
six portions, equivalent to 7.5 mg of the Oral Suspension,
into separate tared 10-mL beakers, and record each weight.
Introduce each of the samples to the middle of the dissolu-
tion vessels, and rinse each beaker with about 20 mL of the
Medium withdrawn from the vessel. Carefully lower the pad-
dle to the appropriate height and start the rotation. After
completion of the dissolution, pass a 20-mL aac through
a nylon filter having a 0.45-11m porosity, discarding the first
3 mL of the filtrate.
Procedure—Determine the amount of Cy4Hi3N30,S> dis-
solved by employing UV absorption at the wavelength of
maximum absorbance at about 362 nm on the Test solution
in comparison with the Standard solution, using Medium as
the blank. Calculate the percentage of Ci4Hi3N3O4S2 re-
leased by the formula:
Ay xC, x900xd x100
A; xW,,xLC
in which Ay and As are the absorbances obtained from the
Test solution and the Standard solution, ey Cs is the
concentration, in mg per mL, of the Standard solution; d is
the density, in ope mL, of the Oral Suspension; Wy is the
weight, in mg, of the Oral Suspension taken; 900 is the
volume, in mL of the Medium; 100 is the conversion factor
to percentage; and LC is the label claim, in mg per mL.
Tolerances—Not less than 75% (Q) of the labeled amount
of Ci4Hi3N3O4S2 is dissolved in 15 minutes.
Microbial enumeration tests (61) and Tests for speci-
fied microorganisms (62)—The total aerobic microbial
count does not exceed 100 cfu per g or 100 cfu per mL.
The total yeasts and molds count does not exceed 50 cfu
Official Monographs / Meloxicam 2561
perg or 50 cfu per mL. It meets the requirements of the
test for the absence of Escherichia coli.
Chromatographic purity—
Buffer, Mobile phase, and Diluent—Proceed as directed in
the Assay.
Related compound standard stock solution—Proceed as di-
rected for Related compound standard stock preparation in
the Assay.
Sensitivity solution—Dilute the Related compound standard
stock solution with Diluent to a final concentration of about
0.08 tg per mL.
Related compound standard solution—Dilute Related com-
pound standard stock preparation with Diluent to a final con-
centration of about 0.5 1g per mL.
Test solution—Proceed as directed for Assay preparation in
the Assay.
Chromatographic system (see Chromatography (621))—
Proceed as directed in the Assay. Chromatograph the Sensi-
uae solution (about 10 wL), and record the peak responses
as directed for Procedure at 260 nm: the relative standard
deviation of three replicate injections is not more than 10%
for meloxicam related compound B. Chromatograph the Re-
lated compound standard solution (about 10 wL), and record
the peak responses as directed for Procedure at 260 nm: the
tailing factor for meloxicam related compoundBis not
more than 2.0.
Procedure—Separately inject equal volumes (about 10 pL)
of the Related compound standard solution and the Test solu-
tion into the chromatograph, record the chromatograms,
and record the peak areas at 260 nm and 360 nm. The run
time is about 20 minutes or two times the retention time of
meloxicam. Calculate the percentage of meloxicam related
compoundBin the portion of Oral Suspension taken by the
formula:
(5000/L)(C/V)(ru/ rs) e
wn
in whichL is the label claim, in mg per mL;C is the concen- uv
tration, in mg per mL, of USP Meloxicam Related Com- E
pound B RS in the Related compound standard solution; V is °
the volume, in mL, of Oral Suspension taken to prepare the =)
Test solution; ry is the peak area obtained for meloxicam i)
to}=
related compoundB in the Test solution at 260 nm; and rs is
2
the peak area for meloxicam related compoundBin the 3
Related compound standard solution at 260 nm. Calculate the 1
percentage of each unknown degradation product in the a)
portion of Oral Suspension taken by the formula:
100(r,
/ r5)
in which 1; is the area of any unknown degradant at 360
nm; Fr; is the sum of areas of meloxicam and all impurities in
the Test solution at 360 nm. Not more than 0.15% of
meloxicam related compoundB is found; not more than
0.2% of any individual unknown degradation product is
found; and not more than 0.5% of total degradation prod-
ucts is found.
Assay—
Buffer—Dissolve 2 g of monohydrate citric acid and 2 g of
boric acid in 1000 mL of water, and adjust with dihydrate
trisodium citrate to a pH of 2.9.
Mobile phase—Mix 565 mL of Buffer, 260 mL of metha-
nol, and 200 mL of acetonitrile. Degas the solution, and
then dissolve 200 mg of sodium dodecyl sulfate in 1000 mL
of the resulting solution.
Diluent—Dissolve 3 g of boric acid and 1.5 g of dihydrate
trisodium citrate in 1000 mL of water, and adjust with 2M
sodium hydroxide to a pH of 8.3. Mix 420 mL of the result-
ing buffer with 420 mL of methanol and 160 mL of acetoni-
trile.
Standard stock preparation—Transfer about 67 mg of USP
Meloxicam RS, accurately weighed, into a 100-mL volumet-
 2562 Meloxicam / Official Monographs
tic flask. Add 3.0 mL of dimethylformamide. Swirl the flask,
and allow to stand for about 5 minutes. Add 15 mL of
methanol. Dilute with Diluent to just below volume. Soni-
cate for 30 minutes, and mix until dissolved. Cool to room
temperature. Dilute with Diluent to volume.
Standard preparation—Dilute Standard stock preparation
with Diluent to a final concentration of about 0.27 mg per
mL.
Related compound standard stock preparation—Transfer
about 21 mg of USP Meloxicam Related CompoundB RS,
accurately weighed, into a 100-mL volumetric flask. Add
3.0 mL of dimethylformamide, 15 mL of methanol, and
about 60 mL of Diluent. Sonicate, and mix until dissolved.
Cool to room temperature. Dilute with Diluent to volume.
Dilute further with Diluent to a concentration of about
8.4 ug per mL.
System suitability solution—Transfer a volume of Oral Sus-
pension, equivalent to about 15 mg of meloxicam, accu-
rately weighed, to a 50-mL volumetric flask. Add 3.0 mL of
Related compound standard stock preparation. Add 3.0 mL of
dimethylformamide. Swirl the flask, and allow to stand for
about 5 minutes. Add 15 mL of methanol. Dilute with Dilu-
ent to just below volume. Sonicate for 30 minutes, mixing
the flask vigorously about every 5 minutes. Cool to room
temperature. Dilute with Diluent to volume. Mix, and allow
particulates to settle. Pass through a 0.45-um membrane
filter with a fiberglass prefilter.
Assay preparation—Transfer an accurately meaured vol-
ume of Oral Suspension, equivalent to about 15 mg of
meloxicam, to a 50-mL volumetric flask. Add 3.0 mL of di-
methylformamide. Swirl the flask, and allow to stand for
about 5 minutes. Add 15 mL of methanol. Dilute with Dilu-
ent to just below volume. Sonicate for 30 minutes, mixing
the flask vigorously about every 5 minutes. Cool to room
temperature. Dilute with Diluent to volume. Mix, and allow
particulates to settle. Pass through a 0.45-lum membrane
filter with a fiberglass prefilter.
£
al
5 Chromatographic system (see Chromatography (621))—The
i
7 liquid chromatograph is equipped with a programmable
ia) dual wavelength detector, a single wavelength detector in
2) series, or a photodiode array detector capable of detecting
=
C wavelengths from 190 nm to 400 nm, or equivalent, and a
3 4-mm x 12.5-cm analytical column that contains 5-um
packing L1. The column temperature is maintained at 40°.
rs The flow rate is about 1.0 mL per minute. The run time is
al
=) about 20 minutes or two times the retention time of meloxi-
cam. Chromatograph the System suitability solution (about
10 uL), and record the peak responses as directed for Proce-
dure at 360 nm and 260 nm: at 360 nm the resolution, R,
between meloxicam and any other adjacent peak is not less
than 1.5. The tailing factor for the meloxicam peak is not
more than 2.0. Chromatograph the Standard preparation,
and record the peak responses as directed for Procedure at
360 nm: the relative standard deviation for replicate injec-
tions of the Standard preparation is not more than 1.5%.
Procedure—Separately inject equal volumes (about 10 uL)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and record
the peak areas at 360 nm. Calculate the amount of meloxi-
cam (Cy4Hi3N30,S2), in mg per mL, in the portion of Oral
Suspension taken by the formula:
50(C/V)(ru
/ rs)
in which C is the concentration, in mg per mL, of USP
Meloxicam RS in the Standard preparation; V is the volume,
in mL, of Oral Suspension taken to prepare the Assay pepe
ration; ru is the peak area obtained for meloxicam in the
Assay preparation at 360 nm; and rs is the peak area for
meloxicam in the Standard solution at 360 nm.
USP 41
Meloxicam Tablets
» Meloxicam Tablets contain not less than
90.0 percent and not more than 110.0 percent of
the labeled amount of meloxicam
(Ci4Hi3N30,Sz).
Packaging and storage—Preserve in well-closed contain-
ers. Store at 25°, excursions permitted between 15° and
30°.
USP Reference standards (11)—
USP Meloxicam RS
Identification—
A: Thin-Layer Chromatographic Identification Test (201)—
0.1 N Methanolic sodium hydroxide—Dilute 100 mL of 1 N
sodium hydroxide with methanol to 1000 mL.
Test solution—Transfer a portion of finely powdered Tab-
lets, equivalent to about 50 mg of meloxicam, to a suitable
flask. Add 5 mL of 0.1 N Methanolic sodium hydroxide, and
mix. Add 20 mL of methanol, and stir for about 15 minutes.
Filter the mixture to remove insoluble material, and use the
filtrate.
Standard solution—Transfer about 20 mg of USP Meloxi-
cam RS, accurately weighed, to a 10-mL volumetric flask,
dissolve in 2 mL of 0.1 N Methanolic sodium hydroxide, dilute
with methanol to volume, and mix.
Developing solvent system—Prepare a mixture of chloro-
form, methanol, and ammonia water (25%) (80:20:1).
Procedure—Proceed as directed in the chapter.
B: The retention time of the major peak in the chromato-
gram of the Assay preparation corresponds to that in the
chromatogram of the Standard preparation, as obtained in
the Assay.
Dissolution (711)—
Medium: pH 7.5 phosphate buffer (prepared by dissolv-
ing 6.81 g of potassium dihydrogen phosphate in 800 mL of
water, adjusting the pH to 7.5 with 0.5 N sodium hydrox-
ide, and diluting with water to 1 L); 900 mL.
Apparatus 2: 75 rpm.
Time: 30 minutes.
Determine the amount of meloxicam dissolved by em-
ploying the following method.
Standard solution—
FOR TABLETS LABELED TO CONTAIN 7 5 MG—Transfer about
33.3 mg of USP Meloxicam RS, accurately weighed, to a
100-mL volumetric flask. Add 5.0 mL of methanol, 1.0 mL of
0.1 N sodium hydroxide, dilute with Medium to volume,
and mix. Transfer 5.0 mL to a 100-mL volumetric flask, di-
lute with Medium to volume, and mix. Transfer 25.0 mL of
the resulting solution to a 50-mL volumetric flask, dilute
with Medium to volume, and mix.
FOR TABLETS LABELED TO CONTAIN 15 MG—Transfer about
33.3 mg of USP Meloxicam RS, accurately weighed, to a
100-mL volumetric flask. Add 5.0 mL of methanol, 1.0 mL of
0.1 N sodium hydroxide, dilute with Medium to volume,
and mix. Transfer 5.0 mL to a 100-mL volumetric flask, di-
lute with Medium to volume, and mix.
Test solution—Use portions of the solution under test
passed througha suitable 10-um filter, discarding the first
few mL.
Procedure—Determine the percentage of the labeled
amount of meloxicam dissolved by employing UV absorp-
tion, using a suitable spectrophotometer, at the wavelength
of maximum absorbance at about 362 nm, using 1-cm cu-
vettes, on the Test solution in comparison with the Standard
USP 41
solution usin Medium as blank. Calculate the percentage of
meloxicam issolved by the formula:
A, xC,x900x100
Ax LC
in which Ay and As are the absorbances obtained from the
Test solution and the Standard solution, respectively; Cs is the
concentration, in mg per mL, of the Standard solution; 900
is the volume, in mL, of Medium; 100 is the conversion fac-
tor to percentage; and LC is the Tablet label claim, in mg.
Tolerances—Not less than 70% (Q) of the labeled amount
of meloxicam is dissolved in 30 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Related compounds—
Solution A, Solution B, and Mobile phase—Proceed as di-
rected in the Assay.
Standard solution—Use the Standard preparation from the
Assay.
System sensitivity solution—Transfer 4 mL of the Standard
solution to a 100-mL volumetric flask, dilute with methanol
to volume, and mix. Transfer 5 mL of the resulting solution
to a 50-mL volumetric flask, add 5 mL of 1 N sodium hy-
droxide, and dilute with methanol to volume.
Test solution—Use the Assay preparation.
Chromatographie system (see Chromatography (621))—
Proceed as directed in the Assay, except to chromatograph
the Standard solution and the System sensitivity solution: the
tailing factor for the meloxicam peak is not more than 2.0;
the relative standard deviation for replicate injections of the
Standard solution is not more than 2.0%; and the signal-to-
noise ratio of the meloxicam peak in the chromatogram of
the System sensitivity solution is not less than 10.
Procedure—Separately inject equal volumes (about 25 iL)
of the Standard solution and the Test solution into the chro-
matograph, record the pnrenaealatrs, and measure the
peak responses. Determine the relative retention times for
the impurity peaks relative to that of the meloxicam peak.
Calculate the percentage of each impurity in the portion of
Tablets taken by the formula:
(5000/3)(1/F\(C/W)(A/L)(ni / rs)
in whichFis the relative response factor for each impurit
and is equal to 2.7 for the impurity with a relative retention
time of about 0.5 (meloxicam related compound B
[2-amino-5-methylthiazole]) and 1.0 for all other impurities;
Cis the concentration, in mg per mL, of USP Meloxicam RS
in the Standard solution; W is the weight, in mg, of pow-
dered Tablets taken to prepare the Test solution; A is the
average weight of aTablet Lis the labeled amount, in mg,
of meloxicam in each Tablet; 1, is the peak response ob-
tained for each impurity in the Test solution; and rs is the
peak response for meloxicam in the Standard solution: not
more than 0.15% of meloxicam related compoundBis
found; not more than 0.2% of any individual unknown im-
purity is found; and not more than 0.5% of total impurities
is found.
Assay—
Solution A—Dissolve 2.0 g of dibasic ammonium phos-
phate in 1 L of water, and adjust with phosphoric acid to a
pH of 7.0 + 0.1.
Solution B—Mix 650 mL of methanol and 100 mL of iso-
propyl alcohol.
Mobile phase—Prepare a filtered and degassed mixture of
Solution A and Solution B (63:37). Make adjustments if nec-
essary (see System Suitability under Chromatography (621)).
Standard stock preparation—{NoTe—The Standard stock
preparation is prepared so that the final concentration of
Official Monographs / Melphalan 2563
meloxicam, in mg per mL, is approximately equivalent to
the concentration of the Assay stock preparation.] Transfer a
suitable quantity of USP Meloxicam RS, accurately weighed,
to a 50-mL volumetric flask, dissolve in 1 mL of T N sodium
hydroxide and 30 mL of methanol, and dilute with metha-
nol to volume. Transfer 10 mL of the resulting solution to a
100-mL volumetric flask, add 10 mL of 1 N sodium hydrox-
ide, and dilute with methanol to volume.
Standard preparation—Transfer 15 mL of the Standard
stock preparation to a 25-mL volumetric flask, and dilute
with water to volume.
Assay stock Pee et 10 Tablets to a 1000-mL
volumetric flask, add about 100 mL of 1 N sodium hydrox-
ide, shake to disperse the Tablets, and add 800 mL of meth-
anol. Sonicate the solution for about 15 minutes, then stir
for 30 minutes. Dilute with methanol to volume, and mix.
Filter the resulting solution, and use the filtrate.
Assay preparation—Transfer 15 mL of the Assay stock prep-
aration to a 25-mL volumetric flask, and dilute with water to
volume.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 254-nm detector,
a guard column that contains packing L1, and a 4-mm
x10-cm column that contains packing L1. The flow rate is
about 0.8 mL per minute. The column temperature is main-
tained at 40°. Chromatograph the Standard preparation, and
record the peak responses as directed for Procedure: the tail-
ing factor for the meloxicam peak is not more than 2.0; and
the relative standard deviation for replicate injections is not
more than 2.0%.
Procedure—Separately inject equal volumes (about 25 yL)
of the Standard preparation and the Assay preparation to the
chromatograph, record the chromatograms, and measure
the responses for the meloxicam peak. Calculate the quan-
tity, in mg, of meloxicam (Ci4Hi3N30,4S2) in the portion of
Tablets taken by the formula:
c
“
5000(C/3)(ru/ rs) mo]
in which C is the concentration, in mg per mL, of USP =
Meloxicam RS in the Standard preparation; and ry and rs are °
=]
the peak responses obtained from the Assay preparation and °
the Standard preparation, respectively. ro}
a
2
mo)
=a
7)
Melphalan
°
CLAY HONH,,
oJ
Ci3HigClaN202 305.20
L-Phenylalanine, 4-bis(2-chloroethyl)amino]-.
L-3-[p (Bis(2-chloroethyhamino}phenyllalanine [148-82-3].
» Melphalan contains not less than 93.0 percent
and not more than 100.5 percent of
Ci3HisClaN202, calculated on the dried and ioniz-
able chlorine-free basis.
[Caution—Handle Melphalan with exceptional care
because it is a highly potent agent.]
Packaging and storage—Preserve in tight, light-resistant,
glass containers.
USP Reference standards (11)—
USP Melphalan Hydrochloride RS
 2564 Melphalan / Official Monographs
identification—
A: Ultraviolet Absorption (197U)—
Solution: 5 ug per mL.
Medium: — methanol.
B: To 1 mL of 1 in 10,000 solution in alcohol in a glass-
stoppered test tube add 1 mL of pH 4.0 acid phthalate
buffer (see under Solutions in the section Reagents, Indica-
tors, and Solutions), 1 mL of a 1 in 20 solution of 4-(p-ni-
trobenzyl)pyridine in acetone, and 1 mL of saline TS. Heat
on a water bath at 80° for 20 minutes, and cool quickly.
Add 10 mL of alcohol and 1 mL of 1 N potassium hydrox-
ide: a violet to red-violet color is produced.
C: Heat 100 mg with 10 mL of 0.1 N sodium hydroxide
on a water bath for 10 minutes: the resulting solution, after
acidification with 2 N nitric acid, responds to the tests for
Chloride (191).
Specific rotation (7815S): between —30° and —36°.
Test solution: 7 mg per mL, in methanol, prepared with
the aid of gentle heating.
Loss on drying (731): Dry it in vacuum at 105° to con-
stant weight: it loses not more than 7.0% of its weight.
Residue on ignition (281): not more than 0.3%.
lonizable chlorine—Dissolve about 500 mg of Melphalan,
accurately weighed, in a mixture of 75 mL of water and
2 mL of nitric acid, allow to stand for 2 minutes, and titrate
with 0.1 N silver nitrate VS, determining the endpoint po-
tentiometrically: not more than 1.0 mL of 0.1 N silver ni-
trate is required for each 500 mg of test specimen.
Nitrogen Determination (461)—Determine the nitrogen
content as directed under Method II, using about 325 mg of
Melphalan, accurately weighed, and 0.1 N sulfuric acid VS
for the titration: not less than 8.90% and not more than
9.45% of N is found, calculated on the dried basis.
Assay—Transfer to a beaker about 200 mg of Melphalan,
accurately weighed, and dissolve in 20 mL of 0.5 N sodium
ae
“
hydroxide. Cover the beaker with a watch glass, and boil
a the solution for 30 minutes, adding water as necessary to
ii
—
maintain the volume. Cool, neutralize to phenolphthalein TS
Dp
° with acetic acid, and add 1 mL of acetic acid in excess. Ti-
e trate with 0.1 Nsilver nitrate VS, determining the endpoint
5 otentiometrically, using silver and calomel electrodes, the
Ps
atter modified to contain saturated potassium sulfate solu-
rs tion. From the results obtained in the test for lonizable chlo-
va) rine, calculate the volume, in mL, of 0.1 N silver nitrate that
> is equivalent to the ionizable chlorine in the quantity of
Melphalan taken for the Assay, and subtract it from the As-
say titration volume. Each mL of 0.1 Nsilver nitrate is equiv-
alent to 15.26 mg of Ci3HisCl2N2O2.
Melphalan Tablets
» Melphalan Tablets contain not less than
90.0 percent and not more than 110.0 percent of
the labeled amount of melphalan
(Ci3HigCl2aN202).
Packaging and storage—Preserve in well-closed, light-re-
sistant, glass containers.
USP Reference standards (11)—
USP Melphalan Hydrochloride RS
Identification—
A: The retention time of the major peak in the chromato-
gram of the Assay preparation corresponds to that in the
chromatogram of the Standard preparation, as obtained in
the Assay.
B: Shake a portion of finely powdered Tablets, equivalent
to about 2 mg of melphalan, with 20 mL of alcohol, and
USP 41
filter: a 1-mL portion of the solution so obtained responds
to Identification test B under Melphalan.
Dissolution (711)—
Medium: 0.1 N hydrochloric acid; 900 mL.
Apparatus 2: 50 rpm.
Time: 30 minutes.
Determine the amount of C;3HigClzN2O2 dissolved by em-
ploying the following method.
Mobile phase—Transfer 2 grams of ammonium acetate,
2 mL of glacial acetic acid, and 0.4 mL of triethylamine to a
suitable flask containing 1500 mL of water and 500 mL of
acetonitrile. Stir until all solids are dissolved and well mixed,
then filter and degas.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 254-nm detector
and a 4.6-mm x 5-cm column that contains packing L7.
The flow rate is about 1.5 mL per minute. Chromatograph
replicate injections of the Standard solution, and record the
peak responses as directed for Procedure: the relative stan-
dard deviation is not more than 3.0%.
Procedure—inject a volume (about 50 pL) ofa filtered
portion of the solution under test into the chromatograph,
record the chromatogram, and measure the response for
the major peak. Calculate the quantity of CisHisClzN2Oz dis-
solved in comparison with a Standard solution havin
known concentration of USP Melphalan Hydrochloride RS in
the same Medium and similarly chromatographed.
Tolerances—Not less than 80% (Q) of the labeled amount
of Ci3HisCl2N2Oz is dissolved in 30 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Procedure for content uniformity—Place 1 Tablet in a
200-mL volumetric flask, add 10 mL of water and 10 mL of
alcohol, sonicate to dissolve the soluble components in the
mixture, dilute with alcohol to volume, mix, and filter to
obtain a clear solution. Dissolve an accurately weighed
quantity of USP Melphalan Hydrochloride RS in alcohol to
obtain a Standard solution having a known concentration of
about 10 ug per mL. Concomitantly determine the ab-
sorbances of the solution from the Tablet and the Standard
solution in 1-cm cells at the wavelength of maximum ab-
sorbance at about 260 nm, witha suitable spectrophotome-
ter, using alcohol as the blank. Calculate the quantity, in
mg, a melphalan (Ci3HisCl2N2Oz) in the Tablet taken by the
ormula:
(305.20/341.66)(T/D)C(Au / As)
in which 305.20 and 341.66 are the molecular weights of
melphalan and melphalan hydrochloride, respectively, T is
the labeled quantity, in mg, of melphalan in the Tablet; D is
the concentration, in ug per mL, of melphalan in the solu-
tion from the Tablet, on the basis of the labeled quantity
per Tablet and the extent of dilution; C is the concentration,
in ug per mL, of USP Melphalan Hydrochloride RS in the
Standard solution; and Ay and As are the absorbances of the
solution from the Tablet and the Standard solution, respec-
tively.
Assay—
Mobile phase—Prepare a solution of 0.025 M diethyl-
amine in a mixture of methanol and water (1:1), adjust with
3.5 N hydrochloric acid to a pH of 5.5, filter, and degas.
Make adjustments if necessary (see System Suitability under
Chromatography (621)).
Standard preparation—Dissolve an accurately weighed
quantity of USP Melphalan Hydrochloride RS in alcohol, and
quantitatively dilute with alcohol to obtain a solution having
a known concentration of about 0.9 mg of melphalan hy-
drochloride per mL. Pipet 10 mL of this solution into a
100-mL volumetric flask containing 75 mL of alcohol and
2.0 mL of glacial acetic acid, dilute with alcohol to volume,
and mix to obtain a Standard preparation having a known
USP 41 Official Monographs / Memantine 2565

concentration of about 90 ug of USP Melphalan Hydrochlo- separate, and filter a portion of the top hexane layer
ns RS per mL (equivalent to about 80 ug of melphalan per through anhydrous sodium sulfate. Use the clear filtrate.
ml). Sample solution: 4.0 mg/mL of Memantine Hydrochlo-
Ay preparation—Weigh and finely powder not fewer tide in Internal standard solution prepared as follows.
than 20 Tablets. Transfer an accurately weighed portion of Transfer 100 mg of Memantine Hydrochloride to a
the powder, equivalent to 8 mg of anhydrous melphalan, to 50-mL centrifuge tube. Add 15 mL of 1 N sodium hy-
a 100-mL volumetric flask. Add about 75 mL of alcohol and droxide, and mix. Add 25 mL of Internal standard solu-
2.0 mL of glacial acetic acid to the flask, and sonicate for tion, and shake for 15 min. Allow the layers to separate,
15 minutes. Cool, dilute with alcohol to volume, and mix. and filter a portion of the top hexane layer through
Filter through a medium-porosity, sintered-glass funnel, dis- anhydrous sodium sulfate. Use the clear filtrate.
carding the first few mL of the filtrate, and use the remain- Chromatographic system
der of the filtrate as the Assay preparation. (See Chromatography (621), System Suitability.)
Chromatographic system (see Chromatography (621))—The Mode: GC
liquid chromatograph is equipped with a 254-nm detector Detector: Flame ionization
and a 4.2-mm x 25-cm column that contains packing L7. Column: 50-m x 0.32-mm; 0.52-um packing G27
The flow rate is about 1 mL per minute. Chromatograph the Temperatures
Standard preparation, and record the peak responses as di- Injection port: 220°
rected for Procedure: the tailing factor for the analyte peak is Detector: 300°
not more than 2.0; and the relative standard deviation for Column: See Table 1.
replicate injections is not more than 2.0%.
Procedure—Separately inject equal volumes (between 10 Table 1
and 20 uL) of the Standard preparation and the Assay prepa- Hold Time at’
ration into the chromatograph, record the chromatograms, Initial Temperature Final Final
and measure the responses for the major peaks. Calculate Temperature Ramp Temperature | Temperature
the quantity, in mg, of melphalan (Ci3HisClzN2O2) in the ©) (¢/min) () _(min)___|
portion of Tablets taken by the formula: 50 5 145 0
145 10 250 20
(305.20/341.67)(0.1Q(ru/ rs)
Carrier gas: Helium
in which 305.20 and 341.67 are the molecular weights of Flow rate: 4.0 + 0.4 mL/min
melphalan and melphalan hydrochloride, respectively; C is Injection volume: 17 uL
the concentration, in ug per mL, of melphalan hydrochlo- Injection es Split ratio, 1:50
tide in the Standard preparation; and ry and rs are the peak System suitability
responses obtained from the Assay preparation and the Stan- Sample: Standard solution
dard preparation, respectively. Suitability requirements
Tailing factor: NMT 2.0 each for memantine and
adamantane co
Relative standard deviation: NMT 2.0% for the ratio a)
mo]
of the peak areas of adamantane and memantine
Memantine Hydrochloride Analysis =
Samples: Standard solution and Sample solution °
Calculate the percentage of memantine hydrochloride =]
fe)
(Cy2H2iN - HCl) in the portion of Memantine Hydro- io]
chloride taken: =
HAN’ Et}
me]
Result = (Ru/Rs) x (Cs/Cu) x 100 7
Cy2HaiN - HCI 215.76 7)
Tricyclo[3.3.1.137]decan-1-amine, 3,5-dimethyl-, Ru = peak response ratio of memantine to the
hydrochloride; internal standard from the Sample solution
1-Amino-3,5-dimethyladamantane hydrochloride Rs = peak response ratio of memantine to the
[41100-52-1]. internal standard from the Standard solution
Cs = concentration of USP Memantine
DEFINITION Hydrochloride RS in the Standard solution
Memantine Hydrochloride contains NLT 98.0% and NMT (mg/mL)
102.0% of memantine hydrochloride (C;2H2iN - HCI), cal- Cu = concentration of Memantine Hydrochloride in
culated on the anhydrous basis. the Sample solution (mg/mL)
IDENTIFICATION erates criteria: 98.0%-102.0% on the anhydrous
e A. INFRARED ABSORPTION (197K)
asis
e B. The retention time of the major peak of the Sample IMPURITIES
solution corresponds to that of the Standard solution, as
obtained in the Assay.
© C. IDENTIFICATION TESTS—GENERAL, Chloride (191): Meets Delete the following:
the requirements
®e HEAVY MeTALs, Method Ii (231): NMT 10 ppme (oil
1-
ASSAY jan-2018)
© PROCEDURE e RESIDUE ON IGNITION (281): NMT 0.1%
Internal standard solution: 4.0 mg/mL of adamantane @ ORGANIC IMPURITIES
in n-hexane Standard stock solution A: 2.5 mg/mL each of USP
Standard solution: 4.0 mg/mL of USP Memantine Hy- Memantine Related Compound A RS, USP Memantine
drochloride RS in Internal standard solution prepared as Related Compound B RS, USP Memantine Related Com-
follows. Transfer 100 mg of USP Memantine Hydrochlo- pound C RS, USP Memantine Related CompoundD RS,
ride RS to a 50-mL centrifuge tube. Add 15 mL of 1N and USP Memantine Related CompoundE RS in n-
sodium hydroxide, and mix. Add 25 mL of Internal stan- hexane
dard solution, and shake for 15 min. Allow the layers to
 2566 Memantine / Official Monographs USP 41

Standard stock solution B: 2.5 mg/mL of USP Meman- Calculate the percentage of any other impurity in the
tine Hydrochloride RS prepared as follows. To the flask portion of Memantine Hydrochloride taken:
containing a weighed amount of USP Memantine Hy-
drochloride RS, add 5.0 N sodium hydroxide to fill 20% Result = (ru/rs) x (Cs/Cy) x 100
of the final volume and n-hexane to fill 20% of the final
volume. Shake for 10 min, and transfer the contents to tu = peak response of any other impurity from the
a separator. Allow the layers to separate, filter a portion Sample solution
of the top hexane layer, dry the organic layer by rs = peak response of memantine hydrochloride
ae with anhydrous sodium sulfate, and allow to from the Standard solution
stand for a few min to ensure all the remaining water Cs = concentration of USP Memantine
has been removed. Use the clear filtrate. Hydrochloride RS in the Standard solution
System suitability solution: 25 g/mL each of USP (mg/mL)
Memantine Related Compound A RS, USP Memantine Cu = concentration of Memantine Hydrochloride in
Related Compound B RS, USP Memantine Related Com- the Sample solution (mg/mL)
pound C RS, USP Memantine Related Compound D RS, Acceptance criteria: See Table 2.
and USP Memantine Related CompoundE RS, from
Standard stock solution A in Standard stock solution B. Table 2
The concentration of USP Memantine Hydrochloride RS
is 2.5 mg/mL. Relative Acceptance
Retention Criteria,
Standard solution: 25 g/mL each of USP Memantine
Name Time NMT (%)
Related Compound A RS, USP Memantine Related Com-
pound B RS, USP Memantine Related CompoundC RS, Memantine related
USP Memantine Related Compound D RS, USP Meman- compound A ORE. 0.15
tine Related Compound E RS, and USP Memantine Hy- Memantine 1.0 —
drochloride RS, from Standard stock solution A and Stan- Memantine related
dard stock solution B, respectively, in n-hexane compound B 1.03 0.15
Sample solution: 25 mg/mL of Memantine Hydrochlo- Memantine related
ride prepared as follows. Transfer the weighed amount compound C 1.07 0.15
of Memantine Hydrochloride to a suitable volumetric Memantine related
flask. Add 5.0 N sodium hydroxide to fill 30% of the compound D 1.19 0.15
final volume and n-hexane to fill 40% of the final vol-
ume. Shake for 10 min, and transfer the contents to a Memantine related
compound E 1.44 0.15
separator. Allow the layers to separate, filter a portion
of the top hexane layer, dry the organic layer by Any individual a
swirling with anhydrous sodium sulfate, and allow to unspecified impurity 0.10
stand for a few min to ensure all the remaining water Total impurities — 0.50
has been removed. Use the clear filtrate.
es
ww
Chromatographic system: Proceed as directed in the SPECIFIC TESTS
iy Assay. © WATER DETERMINATION, Method | (921): NMT 1.0%
c
-_
System suitability
=) Samples: Syste suitability solution and Standard ADDITIONAL REQUIREMENTS
° solution e PACKAGING AND STORAGE: Preserve in well-closed contain-
= [Note—See Table 2 for the relative retention times.]
iS Suitability requirements
ers. Store at controlled room temperature.
Ps Resolution: NLT 6.0 between memantine and mem-
e USP REFERENCE STANDARDS (11)
Qa
USP Memantine Hydrochloride RS
”
antine related compound B; NLT 2.0 between mem- USP Memantine Related Compound A RS
=} antine related compound B and memantine related 1,3-Dimethyladamantane.
compound C, System suitability solution CiaH20 =:164.29
Tailing factor: NMT 2.0 for memantine, Standard USP Memantine Related Compound B RS
solution 3,5-Dimethyladamantane-1-ol.
Relative standard deviation: NMT 10.0% for mem- Ci2H200 =:180.29
antine, Standard solution USP Memantine Related Compound C RS
Analysis 1-Chloro-3,5-dimethyladamantane.
Samples: Standard solution and Sample solution CyaHisCl = 198.73
[NoTte—Ignore the peaks at the relative retention times USP Memantine Related Compound D RS
0.11, 0.12, 0.13, 0.18, and 0.26 with respect to the 1-Bromo-3,5-dimethyladamantane.
memantine peak, as they correspond to residual Ci2HigBr = 243.18
solvents.] USP Memantine Related Compound E RS
Calculate the percentage of each of memantine related N-3,5-Dimethyladamantan-1-yl formamide.
compounds A, B, C, D, and E in the portion of Mem- Ci3H2NO 207.31
antine Hydrochloride taken:
Result = (ru/rs) x (Cs/Cu) x 100
Tu = peak response of memantine related
compounds A, B, C, D, or E from the Sample Memantine Hydrochloride Tablets
solution
rs = peak response of the corresponding USP DEFINITION
Memantine Related Compound RS from the Memantine Hydrochloride Tablets contain an amount of
Standard solution memantine hydrochloride equivalent to NLT 90.0% and
Cs = concentration of the corresponding USP NMT 110.0% of the labeled amount of memantine hy-
Memantine Related Compound RS in the drochloride (Ci2H2N - HCl).
Standard solution (mg/mL)
Cu = concentration of Memantine Hydrochloride in
the Sample solution (mg/mL)
USP 41 Official Monographs / Memantine 2567

IDENTIFICATION Table 1
e A. INFRARED ABSORPTION (197K) Hold Time at
Analytical range: 4000-400 cm Initial Temperature Final Final
Standard: 6.7 mg/mL of USP Memantine Hydrochloride Temperature Ramp Temperature | Temperature
RS in dichloromethane. Shake for 10 min, and pass
©) (¢/min) () (min)
throughasuitable filter. Evaporate the solvent at room
temperature. Collect the residue powder, and dry at 50 o 50 2
60° for 15 min. Prepare an approximate 1% (w/w) dis- 50 20 140 0
persion of the sample in potassium bromide. 140 30 200 5:
Sample: 6.7 mg/mL of memantine hydrochloride in di-
chloromethane from NLT 20 crushed Tablets. Shake for Carrier gas: Helium
10 min, and omg for 10 min. Pass the superna- Flow rate: 34.8 psi
tant through a suitable filter. Evaporate the solvent at Injection volume: 4 wL
room temperature. Collect the residue powder, and dry Injection type: Split ratio, 1:10
at 60° for 15 min. Prepare an approximate 1% (w/w) System suitability
dispersion of the sample in potassium bromide. Sample: Standard solution
Acceptance criteria: Fingerprint region of the Standard [Note—The relative retention times for amantadine and
and Sample spectrum exhibit maxima at the same wave memantine are 0.97 and 1.0, respectively.]
numbers. Suitability requirements
e B. The retention time of the memantine peak of the Resolution: NLT 2.0 between amantadine and
Sample solution corresponds to that of the memantine memantine
peak of the Standard solution, as obtained in the Assay. Tailing factor: NMT 2.5 for amantadine; NMT 2.0 for
memantine
ASSAY Relative standard deviation: NMT 2.0% for the ratio
© PROCEDURE of the peak areas of amantadine and memantine
Solution A: 200 mg/mL of sodium hydroxide in water Analysis
Internal standard solution: 25 g/mL of USP Samples: Standard solution, Sample solution, and Blank
Amantadine Hydrochloride RS in water Calculate the percentage of the labeled amount of
Standard stock solution: 25 «g/mL of USP Memantine memantine hydrochloride (Ci2H2iN - HCl) in the por-
Hydrochloride RS prepared as follows. Weigh a suitable tion of Tablets taken:
quantity of the Standard into a volumetric flask. Add
methanol to fill 40% of the final flask volume, and soni- Result = (Ru/Rs) x (Cs/Cy) x 100
cate. Dilute with water to volume.
Standard solution: Pipet 4.0 mL each of the Internal Ru = peak area ratio of memantine to amantadine
standard solution and the Standard stock solution into a from the Sample solution
test tube. Add 2 mL of Solution A, and mix on a vortex Rs = peak area ratio of memantine to amantadine
mixer for 1 min. Add 4.0 mL of toluene, and mix on a from the Standard solution
vortex mixer for 3 min. Allow the two layers to sepa- Cs = concentration of USP Memantine (8s
rate. Inject the toluene layer. Hydrochloride RS in the Standard solution 4)
Sample stock solution: Nominally 20 ug/mL of mem- (ug/mL) | . Ss)
antine hydrochloride prepared as follows. Transfer a Cy = nominal concentration of memantine <
suitable number of Tablets to a volumetric flask to ob- hydrochloride in the Sample solution (g/mL) i)
tain a 0.1 mg/mL memantine hydrochloride solution. Acceptance criteria: 90.0%-110.0% =)
é
Add methanol to fill 40% of the final flask volume, and
PERFORMANCE TESTS iro)=
sonicate for 30 min with intermittent shaking. Add
e DISSOLUTION (711) i)
water to fill 40% of the final flask volume, and sonicate
Medium: 0.1 N hydrochloric acid with sodium chloride i}
for 30 min with intermittent shaking. Dilute with water
(2 g/L of sodium chloride in water), adjusted with hy-
=P
“
to volume, and centrifuge a portion for 10 min. Pipet a
suitable volume of the clear centrifugate into a volu- drochloric acid to a pH of 1.2; 900 mL
metric flask, and dilute with water to volume. Apparatus 1: 100 rpm
Sample solution: Pipet 5.0 mL of the Sample stock solu- Time: 30 min
tion, 4.0 mL of the Internal standard solution, and 2 mL Standard stock solution: (L/900) mg/mL of USP Mem-
of Solution A into a test tube, and mix on a vortex antine Hydrochloride RS in Medium, whereLis the label
mixer for 1 min. Add 4.0 mL of toluene, and mix on a claim in mg/Tablet
vortex mixer for 5 min. Allow the two layers to sepa- Internal standard solution: 28 g/mL of USP
rate. Inject the toluene layer. Amantadine Hydrochloride RS in Medium
Blank: To 5.0 mL of 80 uL/mL of methanol in water add Standard solution
2 mL of Solution A, and mix on a vortex mixer for 1 For Tablets labeled to contain 5 mg: Transfer 5 mL of
min. Add 4.0 mL of toluene, and mix on a vortex mixer the Standard stock solution to a test tube, add 1 mL of
for 5 min. Allow the two layers to separate. Inject the the Internal standard solution and 2 mL of 5 N sodium
toluene layer. hydroxide, and mix for 1 min. Add 3 mL of toluene,
Chromatographic system and mix for 2 min. Use the toluene layer.
(See Chromatography (621), System Suitability.) For Tablets labeled to contain 10 mg: Transfer 5 mL
Mode: GC of the Standard stock solution to a test tube, add 2 mL
Detector: Flame ionization of the Internal standard solution and 2 mL of 5 N so-
Column: 30-m x 0.32-mm; 0.25-um packing G27 dium hydroxide, and mix for 1 min. Add 3 mL of tolu-
Temperatures ene, and mix for 2 min. Use the toluene layer.
Injection port: 210° Sample solution: Pass a portion of the solution under
Detector: 300° test through a suitable filter.
Oven: See Table 1. For Tablets labeled to contain 5 mg: Transfer 5 mL of
the filtrate to a test tube, add 1 mL of the /nternal
standard solution and 2 mL of 5 N sodium hydroxide,
and mix for 1 min. Add 3 mL of toluene, and mix for
2 min. Use the toluene layer.
 2568 Memantine / Official Monographs USP 41

For Tablets labeled to contain 10 mg: Transfer 5 mL for 30 min. Centrifuge, andpass a portion of the cen-
of the filtrate to a test tube, add 2 mL of the /nternal trifugate through a suitable filter of 0.45-um pore size.
standard solution and 2 mL of 5 N sodium hydroxide, Chromatographic system
and mix for 1 min. Add 3 mL of toluene, and mix for (See Chromatography (621), System Suitability.)
2 min. Use the toluene layer. Mode: LC
Chromatographic system Detector: Refractive index
(See Chromatography (621), System Suitability.) Column: 4.6-mm x 15-cm; 5-um packing L1
Mode: GC, splitless Temperatures
Detector: Flame ionization Column: 40°
Column: 30-m x 0,32-mm,; 0.25-m packing G27 Detector: 35°
Flow rate: 34.8 psi Flow rate: 1.3 mL/min
Temperatures Injection volume: 50 uL
Injection port: 210° Run time: 1.3 times the retention time of the meman-
Detector: 300° tine peak
Oven: See Table 2. System suitability
Sample: Standard solution
Table 2 Suitability requirements
Tailing factor: NMT 3.5
Hold Time at! Relative standard deviation: NMT 10.0%
Initial Temperature Final Final Analysis
Temperature Ramp Temperature | Temperature Samples: Standard solution and Sample solution
@) (¢/min) () (min) Calculate the percentage of the memantine-lactose ad-
50 0 50 2 duct in the portion of Tablets taken:
50 20 140 0
140 30 200 5
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100

Carrier gas: Helium ru = peak response of the memantine-lactose

Injection volume: 4 pL adduct from the Sample solution
System suitability ig = peak response of memantine from the
Sample: Standard solution Standard solution
Suitability requirements Cs = concentration of USP Memantine
Resolution: NLT 2.0 between amantadine and Hydrochloride RS in the Standard solution
memantine (mg/mL)
Tailing factor: NMT 2.0 each for amantadine and Cy = nominal concentration of memantine
memantine hydrochloride in the Sample solution
Relative standard deviation: NMT 2.0% for the ratio (mg/mL)
of memantine to amantadine peaks F = relative response factor of the
al
Analysis memantine-lactose adduct (see Table 3)
a] Acceptance criteria: See Table 3.
ra Samples: Standard solution and Sample solution
Disregard all peaks other than the memantine-lactose
£Ss Calculate the percentage of the labeled amount of
adduct peak.
Dp memantine hydrochloride (C2H2iN - HCl) dissolved:
2)
= Result = (Ru/Rs) x (G/L) x V x 100
iS Table 3
= Ru = peak area ratio of memantine to amantadine Relative Relative Acceptance
5 Retention Response Criteria,
from the Sample solution
” Name Time Factor NMT (%
=) Rs = peak area ratio of memantine to amantadine
Memantine-
from the Standard solution
Cs = concentration of USP Memantine lactose adduct 0.41 0.53 14
Hydrochloride RS in the Standard solution Memantine 1.0 1.0 =
(ug/mL) © ORGANIC IMPURITIES
L = label claim (mg/Tablet)
Vv = volume of Medium, 900 mL Solution A: 1 .N sodium hydroxide
Tolerances: NLT 80% (Q) of the labeled amount of System suitability stock solution A: 0.5 mg/mL each
memantine hydrochloride (Ci2H2:N - HCl) is dissolved. of USP Memantine Related Compound A RS, USP Mem-
e UNIFORMITY OF DOSAGE UNITS (905): Meet the antine Related Compound B RS, USP Memantine Re-
requirements lated Compound C RS, USP Memantine Related Com-
pound D RS, and USP Memantine Related Compound E
IMPURITIES RS in n-hexane
e LIMIT OF MEMANTINE-LACTOSE ADDUCT System suitability stock solution B: Transfer 75 mg of
[Note—Perform this test if lactose is present in the USP Memantine Hydrochloride RS into a suitable con-
formulation.] tainer, add 9 mL of 1.0 N sodium hydroxide and 6 mL
Solution A: 40 mg/mL of sodium hydroxide in water of n-hexane, and mix for 10 min.
Buffer: Dissolve 3.3 g of monobasic potassium phos- System suitability solution: Pipet 4.0 mL of the n-hex-
phate and 2.3 g of sodium 1-octane sulfonate in 1 L of ane layer from System suitability stock solution B into a
water, Adjust with Solution A to a pH of 6.1. 10-mL volumetric flask. Add 0.5 mL of System suitability
Mobile phase: Acetonitrile, methanol, and Buffer Stock solution A, and dilute with n-hexane to volume.
(26:4:70) Standard stock solution: 1.3 mg/mL of USP Meman-
Standard solution: 0.2 mg/mL of USP Memantine Hy- tine Hydrochloride RS in n-hexane prepared as follows.
drochloride RS in Mobile phase Weigh a suitable quantity of the Standard into a volu-
Sample solution: Nominally 10 mg/mL of memantine metric flask. Add Solution A to fill 30% of the final flask
hydrochloride from NLT 25 crushed Tablets, prepared volume, and mix for 5 min. Add n-hexane to fill 40% of
as follows. Transfer an amount of powder equivalent to the final flask volume, and shake for 10 min. Transfer
100 mg of memantine hydrochloride to a 20-mL volu- the contents of the flask into a separator. Allow the
metric flask. Add 10 mL of Mobile phase, and sonicate layers to separate, and filter a portion of the top n-
USP 41 Official Monographs / Menadiol 2569

hexane layer through anhydrous sodium sulfate. Use Acceptance criteria: See Table 5.
the clear solution.
Standard solution: Pipet 2.0 mL of the clear solution Table 5
from the Standard stock solution into a 100-mL volumet-
tic flask, and dilute with n-hexane to volume. Relative Acceptance
Sample solution: Nominally 5 mg/mL of memantine Retention Criteria,
hydrochloride in n-hexane from NLT 20 crushed Tab- Name Time NMT (%)
lets, prepared as follows. Transfer a weighed amount of Memantine related
powder equivalent to 100 mg of memantine hydrochlo- compound A? 0.77 _
ride to a suitable volumetricflask. Add Solution A to fill Memantine 1.0 =
15% of the final flask volume. Shake to disperse the Memantine related
material, and then shake for 5 min. Sonicate for 5 min compound B? 1.03 Ee
with intermittent shaking. Add n-hexane to fill 20% of Memantine related
the final flask volume, and shake for 10 min. Transfer
compound C# 11 att
the contents into a separator. Allow the layers to sepa-
rate, and filter a portion of the top hexane layer Memantine related
through anhydrous sodium sulfate. Use the clear compound De 1.2 a
solution. Memantine related
Chromatographic system compound E 1.4 0.3
(See Chromatography (621), System Suitability.) Any individual
Mode: GC unspecified
Detector: Flame ionization degradation a
Column: 50-m x 0.32-mm; 0.52-um packing G27 product 0.20
Temperatures Total impurities? — 0.5
Injection port: 220° @Process impurities controlled in the drug substance and are included for
Detector: 300° identification only. Not reported for the drug product and not included in
Oven: See Table 4. the total impurities.
Excludes memantine-lactose adduct monitored in the test for Limit of
Memantine-Lactose Adduct.
Table 4
ADDITIONAL REQUIREMENTS
Hold Time at
© PACKAGING AND STORAGE: Preserve in tight containers.
Initial Temperature Final Final
Temperature Ramp Temperature | Temperature
Store at controlled room temperature.
© USP REFERENCE STANDARDS (1 1S
C) C/min) «) (min) USP Amantadine Hydrochloride RS
50 0 50 2 USP Memantine Hydrochloride RS
50 5 145 0 USP Memantine Related Compound A RS
145 10 250 20 1,3-Dimethyladamantane.
CizH20 ~=—-164.29 i=
al
Carrier gas: Helium USP Memantine Related Compound B RS a4
Flow rate: 4.0 + 0.2 mL/min 3,5-Dimethyladamantane-1-ol.
Injection volume: 3 uL CizH2O 180.29 =
Injection type: Split ratio, 1:20 USP Memantine Related Compound C RS i}
]
System suitability 1-Chloro-3,5-dimethyladamantane. )
Samples: System suitability solution and Standard CyaHisCl = 198.73 ro}=
solution USP Memantine Related Compound D RS Et)
[Note—See Table 5 for the relative retention times.] 1-Bromo-3,5-dimethyladamantane. i}
Suitability requirements CyaHiBr =—.243.18
=
a
Resolution: NLT 2.0 between memantine and mem- USP Memantine Related Compound E RS
antine related compound B; NLT 2.0 between mem- N-3,5-Dimethyladamantan-1-yl formamide.
antine related compound B and memantine related Ci3H2NO = 207.31
compound C, System suitability solution
Tailing factor: NMT 2.0, Standard solution
Relative standard deviation: NMT 10.0%, Standard
solution
Analysis
Samples: Standard solution and Sample solution Menadiol Sodium Diphosphate
Calculate the percentage of memantine related com- OPO,Na,
pound E orany individual degradation product in the CH,
portion of Tablets taken: CO © aH,
Result = (ru/rs) x (Cs/Cu) x 100 OPO,Na
ty = peak response of memantine related
an E or any individual degradation CiHgNagOgP2-6H20 530.17
product from the Sample solution 1,4-Naphthalenediol, 2-methyl-, bis(dihydrogen phosphate),
Is = peak response of memantine hydrochloride tetrasodium salt, hexahydrate.
from the Standard solution 2-Methyl-1,4-naphthalenediol bisidlbyellogen hosphate)
Cs = concentration of USP Memantine tetrasodium salt, hexahydrate [6 00-42-11
Hydrochloride RS in the Standard solution Anhydrous 422.09 [131-13-5].
(mg/mL)
Gu nominal concentration of memantine » Menadiol Sodium Diphosphate contains not
hydrochloride in the Sample solution less than 97.5 percent and not more than
(mg/mL)
 2570 Menadiol / Official Monographs
102.0 percent of Cii;HsNasOgP2, calculated on the
anhydrous basis.
Packaging and storage—Preserve in tight, light-resistant
containers, and store in a cold place.
Identification—
A: Dissolve about 200 mg of Menadiol Sodium
Diphosphate in 10 mL of water, add 10 mL of 2 N sulfuric
acid, 10 mL of 0.1 N ceric sulfate, and 1 mL of 30 percent
hydrogen peroxide previously diluted with 5 mL of water,
and extract the solution with two 10-mL portions of chloro-
form. Gently evaporate the clear chloroform solution on a
steam bath to dryness, and dry the residue at 80° for
1 teak the menadione so obtained melts between 104° and
7 Bacterial Endotoxins Test (85)—It contains not more
B: To 50 mg of the dried residue obtained in Identification
test A add 5 mL of water, then add 75 mg of sodium bisul-
fite, and heat on a steam bath, shaking vigorously until the
substance is dissolved and the solution is practically color-
less. Dilute with water to 50 mL, and mix. To 2 mL of the
solution add 2 mL of alcoholic ammonia (prepared by mix-
ing equal volumes of alcohol and ammonium hydroxide),
shake, and add 3 drops of ethyl cyanoacetate: a deep pur-
plish blue color is produced, and on the addition of 1 mL of
sodium hydroxide solution (1 in 3), it changes to green and
then to yellow.
C: To about 20 mg contained in a small beaker add 1 mL
of water, 2 drops of nitric acid, and 1 mL of sulfuric acid,
and heat slowly to the evolution of white fumes. Cool, cau-
tiously dilute with water to about 10 mL, and filter if not
clear. Render the filtrate slightly alkaline to litmus with 6 N
ammonium hydroxide, then render it acid with nitric acid,
and add to the warm solution 3 mL of ammonium molyb-
date TS: a yellow precipitate is formed within a few min-
utes.
Water Determination, Method | (921): between 19.0%
i
”
and 21.5%.
ay
S— Assay—Dissolve about 100 mg of Menadiol Sodium
Dp Diphosphate, accurately weighed, in 25 mL of water, and
} add 25 mL of glacial acetic acid and 25 mL of 3 N hydro-
4 chloric acid. Titrate the solution with 0.02N ceric sulfate
5 VS, determining the endpoint potentiometrically using a cal-
= omel-platinum electrode system. Each mL of 0.02 N ceric
a sulfate is equivalent to 4.221 mg of CisHsNasOgP2.
”
>]
Menadiol Sodium Diphosphate
Injection
» Menadiol Sodium Diphosphate Injection is a
sterile solution of Menadiol Sodium Diphosphate
in Water for Injection. It contains not less than
95.0 percent and not more than 110.0 percent of
the labeled amount of Ci;3HgNa4OgP2 - 6H20.
Packaging and storage—Preserve in single-dose, light-re-
sistant containers, preferably of Type | glass.
Change to read:
USP Reference standards (11)—
°. (CN 1-May-2018)
USP Menadione RS
Identification—
A: Transfer a volume of Injection, equivalent to about
100 mg of menadiol sodium diphosphate, to a separator,
add 10 mL of 2.N sulfuric acid, and extract with six 25-mL
portions of ether, discarding the ether extracts. To the aque-
USP 41
ous solution add 1 mL of 0.5N ceric sulfate and 1 mL of
30 percent hydrogen peroxide, and extract with two 10-mL
portions of chloroform. Evaporate the combined chloroform
extracts on a steam bath just to dryness, then dry at 80° for
1 hour: the IR absorption spectrum of a potassium bromide
dispersion of the menadione so obtained exhibits maxima at
the same alot as that of a similar preparation of
USP Menadione RS. The solid also Espen to Identification
test B under Menadiol Sodium Diphosphate.
B: Adjust, if necessary, a volume of Injection, equivalent
to about 20 mg of menadiol sodium diphosphate, b evap -
ration or dilution with water, as required, to 2 mL: the solu-
tion responds to Identification test C under Menadiol Sodium
Diphosphate.
than 25.0 USP Endotoxin Units per mg of menadiol sodium
diphosphate.
pH (791): between 7.5 and 8.5.
Other requirements—It meets the requirements under In-
jections and Implanted Drug Products (1).
Assay—Transfer an accurately measured volume of Injec-
tion, equivalent to about 50 mg of menadiol sodium
diphosphate, to a 125-mL separator, and extract with three
25-mL portions of chloroform, discarding the chloroform ex-
tracts. Transfer the aqueous solution to a 250-mL beaker,
add 25 mL of glacial acetic acid and 25 mL of 3 N hydro-
chloric acid, vigorously bubble nitrogen through this solu-
tion for not less than 15 minutes, and titrate with 0.01 N
ceric sulfate VS, determining the endpoint potentiometri-
cally using a calomel-platinum electrode system. Each mL of
0.01 N ceric sulfate is equivalent to 2.651 mg of
CriHsNagOgP2 - 6H20.
Menadiol Sodium Diphosphate Tablets
» Menadiol Sodium Diphosphate Tablets contain
not less than 95.0 percent and not more than
110.0 percent of the labeled amount of
CiHsNasOgP2 e 6H20.
Packaging and storage—Preserve in well-closed, light-re-
sistant containers.
USP Reference standards (11)—
USP Menadione RS
Identification—
A: Triturate a quantity of powdered Tablets, equivalent to
about 100 mg of menadiol sodium diphosphate, with a mix-
ture of 10 mL of water and 10 mL of 2N sulfuric acid, cen-
trifuge the mixture, and filter the supernatant. To the filtrate
add 1 mL of 0.5 N ceric sulfate, mix, extract with 10 mL of
chloroform, and centrifuge. Evaporate the chloroform ex-
tract on a steam bath just to dryness, then dry at 80° for
1 hour: the IR absorption spectrum of a potassium bromide
dispersion of the menadione so obtained exhibits maxima at
the same wavelengths as that of a similar preparation of
USP Menadione RS.
B: To 50 mg of the menadione obtained in /dentification
test A add 5 mL of water, then add 75 mg of sodium bisul-
fite, and heat on a steam bath, shaking vigorously until the
substance is dissolved and the solution is almost colorless.
Add water to make 50 mL, and mix. To 2 mL of the solution
add 2 mL of alcoholic ammonia (prepared by mixing equal
volumes of alcohol and ammonium hydroxide), shake, and
add 3 drops of ethyl cyanoacetate: a deep purplish blue
color is produced, and, on the addition of 1 mL of sodium
hydroxide solution (1 in 3), it changes to green and then to
yellow.
USP 41 Official Monographs / Menadione 2571

C: Triturate a quantity of powdered Tablets, equivalent to metrically using a calomel-platinum electrode system. Each
about 20 mg of menadiol sodium diphosphate, with 10 mL mL of 0.01 N ceric sulfate is equivalent to 2.651 mg of
of water, centrifuge the mixture, filter the supernatant, and CiiHgNagOgP2 - 6H20.
evaporate to a volume of about 2 mL. Add 2 drops of nitric
acid and 1 mL of sulfuric acid, and heat slowly to the evolu-
tion of white fumes. Cool, cautiously dilute with water to
about 10 mL, and filter if not clear. Render the filtrate
slightly alkaline to litmus with 6 N ammonium hydroxide, Menadione
then render it acid with nitric acid, and add to the warm
solution 3 mL of ammonium molybdate TS: a yellow precipi-
tate is formed within a few minutes.
Dissolution (711)—
Medium: 0.1 N hydrochloric acid; 900 mL.
Apparatus 1: 100 rpm.
Time: 30 minutes. 172.18
CiHsO2
Procedure—Determine the amount of Cy,HsNaqOgP2 - 1,4-Naphthalenedione, 2-methyl-;
6H20 dissolved from UV absorbances at the wavelength of 2-Methyl-1,4-naphthoquinone [58-27-5].
maximum absorbance at about 227 nm on filtered portions
of the solution under test, suitably diluted with Medium, in DEFINITION
comparison with a standard solution prepared by dissolving Menadione contains NLT 98.5% and NMT 101.0% of men-
in the same Medium an accurately weighed quantity of adione (C;;:HsOz), calculated on the dried basis.
Menadiol Sodium Diphosphate, previously dried in vacuum [CauTlon—Menadione powder is irritating to the respiratory
over phosphorus pentoxide for 4 hours, the dried sample tract and to the skin, and a solution of it in alcohol is a
having a known concentration determined by titration with vesicant.]
0.01 N ceric sulfate VS as directed in the Assay.
Tolerances—Not less than 75% (Q) of the labeled amount IDENTIFICATION
of CisHgNasOgP2
- 6H20 is dissolved in 30 minutes. e A. INFRARED ABSORPTION (197K)
e B, ULTRAVIOLET ABSORPTION (197U)
Uniformity of dosage units (905): meet the require- Standard solution: 5 \1g/mL of USP Menadione RS in
ments. alcohol
Procedure for content uniformity—{NoTE—Use low-actinic Sample solution: 5 g/mL in alcohol
glassware.] Transfer 1 Hosly powdered Tablet to a glass-stop- Analytical wavelength: 250 nm
pred centrifuge tube, add 25 mL of pH 8.0 phosphate Acceptance criteria: Absorptivities, calculated on the
uffer (see under Solutions in the section Reagents, Indica- dried basis, do not differ by more than 3.0%.
tors, and Solutions), and shake vigorously for several min-
utes. Filter into a 50-mL volumetric flask, rinse the centri- ASSAY
fuge tube, and filter with three 5-mL portions of pH © PROCEDURE S
8.0 phosphate buffer, adding the rinsings to the volumetric Sample solution: Transfer about 150 mg of Menadione ww
flask, dilute with pH 8.0 phosphate buffer to volume, and into a 150-mL volumetric flask. Add 15 mL each of gla- uv
mix. Dilute a portion of this solution quantitatively and step- cial acetic acid and 3 N hydrochloric acid, and rotate cs
wise, if necessary, with pH 8.0 phosphate buffer to provide the flask until Menadione is dissolved. Add about 3 g of i}
a solution containing approximately 40 1g of menadiol so- zinc dust, and close the flask with a stopper bearing a S
Bunsen valve. Shake, and allow to stand in the dark for °
dium diphosphate per mL. Concomitantly determine the ab-
1 h, with frequent shaking. Rapidly decant the solution
ro}2
sorbances of this solution and of a solution of Menadiol So-
dium Diphosphate, previously dried in vacuum over through a pledget of cotton into another flask, immedi- so}
phosphorus pentoxide for 4 hours, in the same Medium hav- ately wash the reduction flask with three 10-mL por- mF
ing a known concentration of about 40 yg per mL, at the tions of freshly boiled and cooled water, and add a)

wavelength of maximum absorbance at about 297 nm, with
a suitable spectrophotometer, using pH 8.0 phosphate
buffer as the blank. Calculate the quantity, in mg, of
CiiNeNa4gOgP2 - 6H20 in the Tablet taken by the Forman
(TC/ D)(Au/ As)
in whichTis the labeledquantity. in mg, of menadiol so-
dium diphosphate in the Tablet, C is the concentration, in
ug per mL, of Ci;HsNasOgP2 - 6H20 in the Standard solution,
D is the concentration, in ug per mL, of menadiol sodium
diphosphate in the test solution, based upon the labeled
quantityper Tablet and the extent of dilution, and Ay and As
are the absorbances of the solution from the Tablet and the
standard solution, respectively.
Assay—Weigh and finely powder not less than 20 Tablets.
Transfer an accurately weighed portion of the powder,
equivalent to about 50 mg of menadiol sodium
diphosphate, to a 250-mL beaker. Moisten the powder with
a few mL of glacial acetic acid, and then add sufficient
guantity of the acid to make 25 mL. Add 25 mL of 3 N hy-
rochloric acid and 25 mL of water, mix, and titrate with
0.01 N ceric sulfate VS, determining the endpoint potentio-
0.1 mL of orthophenanthroline TS.
Titrimetric system
Mode: Direct titration
Titrant: 0.1 N ceric sulfate VS
Endpoint detection: Potentiometric
Analysis: Immediately titrate the combined filtrate and
washings with Titrant. Perform a blank determination,
and make any necessary correction. Each mL of 0.1 N
ceric sulfate is equivalent to 8.609 mg of menadione
(CiHgO2z).
Acceptance criteria: 98.5%-101.0% on the dried basis
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.1%
© ORDINARY IMPURITIES (466)
Standard solution and Sample solution: Methanol
Eluant: Chloroform
Visualization: 1
Acceptance criteria: Meets the requirements
SPECIFIC TESTS
© MELTING RANGE OR TEMPERATURE, Class | (741):
105°-107°
 2572 Menadione / Official Monographs USP 41

e Loss ON DRYING (731) the solutions from the Assay preparation and the Standard
Analysis: Dry over silica gel for 4 h. preparation, respectively.
Acceptance criteria: NMT 0.3%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed, light-
resistant containers. Store at 25°, excursions permitted Menthol
between 15° and 30°.

Ok
eo USP REFERENCE STANDARDS (11) cH.
USP Menadione RS

HyC~ CH,

Menadione Injection CioH200 156.27

» Menadione Injection is a sterile solution of
Menadione in oil. It contains not less than
90.0 percent and not more than 120.0 percent of
the labeled amount of C1i1HgOo.
Packaging and storage—Preserve in single-dose or in
multiple-dose containers, preferably of Type | glass.
Change to read:
USP Reference standards (11)—
Ss (CN 1-May-2018)
USP Menadione RS
Bacterial Endotoxins Test (85)—It contains not more
than 58.3 USP Endotoxin Units per mg of menadione.
Other requirements—It meets the requirements under /n-
jections and Implanted Drug Products (1).
Assay—[NoTE—Avoid exposing Menadione and its solutions
to light throughout the Tsay
ES
“
Cyclohexanol, 5-methyl-2-(1-methylethyl)- [1490-04-6].
DEFINITION
Menthol is an alcohol obtained from oils derived from a
variety of mints or prepared synthetically. Menthol may
be levorotatory (/-menthol) from natural or synthetic
sources, or racemic (d/-menthol). It contains NLT 98.0%
and NMT 102.0% of menthol (CioH200).
IDENTIFICATION
e A. The retention time of the major peak of the Sample
solution corresponds to that of the menthol peak of the
Standard solution, as obtained in the Assay.
e B. It meets the requirements in Specific Tests for Optical
Rotation (7815S), Procedures, Specific Rotation.
ASSAY
Change to read:
e PROCEDURE
4Standard solution: 10 mg/mL of USP Menthol RS in

ro Standard preparation—Transfer about 25 mg of USP Men- hexanes

i] adione RS, accurately weighed, to a 100-mL volumetric Sample solution: 10 mg/mL of Menthol in hexanes
=
Dp flask, dissolve in a mixture of equal volumes of alcohol and Chromatographic system
° ether, dilute with the same mixture to volume, and mix. (See Chromatography (621), System Suitability.)
= Keep the solution tightly closed in a dark, cool place, and Mode: GC
5 use it within 7 days. Detector: Flame ionization
2 Column: 0.18-mm x 20-m fused silica; coated with a
Assay preparation—Transfer an accurately measured vol-
[3
ume of Injection, equivalent to about 25 mg of menadione, 0.18-4m layer of G16 stationary phase
2)
=) to a 100-mL volumetric flask, dilute with a mixture of equal Temperatures
volumes of ether and alcohol to volume, and mix. Injection port: 250°
Detector: 260°
Procedure—transfer 1.0 mL each of the Standard prepara- Column: See Table 7.
tion and the Assay preparation to separate 50-mL volumetric
flasks, add to each 4 mL of alcohol, and mix. Then to each
flask add 1.0 mL of a solution prepared by dissolving 50 mg Table 1
of 2,4-dinitrophenylhydrazine in 20 mL of a mixture of 2 Hold Time
volumes of 3 N hydrochloric acid and 1 volume of water. Initial Temperature Final at Final
Place the flasks in a bath maintained at 70° to 75° for Temperature Ramp Temperature | Temperature’
15 minutes, shaking vigorously every 2 to 3 minutes. Imme- ©) (/min) ©) (min)
diately after the heating, cool the flasks to about 25°; then 60 20 0 10
add to each 5 mL of alcoholic ammonia, prepared by mix-
ing equal volumes of alcohol and ammonium hydroxide. Carrier gas: Hydrogen
Shake the flasks thoroughly, add alcohol to make 50.0 mL, Flow ae 0.9’ml/min
mix, allow to stand for 15 minutes, and decant from any injection volume: 0.5 ul
separated oil. Determine the absorbances of the solutions, in Injection type: Split ratio, 50:1
1-cm cells at the wavelength of maximum absorbance at System suitability
about 635 nm, with a suitable spectrophotometer, using a Sample: Standard solution
reagent blank to set the instrument. Calculate the quantity, Suitability requirements
K mg, of Cy;HgO2 in each mL of the Injection taken by the Relative standard deviation: NMT 2.0% for the
ormula: menthol peak in replicate injections
Analysis
(0.1C/ V)(Au /As) Samples: Standard solution and Sample solution
Calculate the percentage of menthol (CjoH200) in the
in which C is the concentration, in ug per mL, of USP Men- portion of Menthol taken:
adione RS in the Standard preparation, V is the volume, in
mL, of Injection taken, and Ay and As are the absorbances of Result = (ru/rs) x (Cs/Cu) x 100
USP 41 Official Monographs / Menthol 2573

fe = peak area of menthol from the Sample solution Acceptance criteria: 41°-44°
fs = peak area of menthol from the Standard ¢ OPTICAL ROTATION (7815S), Procedures, Specific Rotation
solution Sample solution: 100 mg/mL in alcohol
G = concentration of USP Menthol RS in the Acceptance criteria
Standard solution a Menthol: —45° to —51°
Gs = concentration of Menthol in the Sample di-Menthol: —2° to +2°
Solution (mg/mL)auseai
Acceptance criteria: 98.0%-102.0% ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers,
IMPURITIES preferably at controlled room temperature.
e LIMIT OF NONVOLATILE RESIDUE e LABELING: Label it to indicate whether it is levorotatory or
Analysis: Evaporate 2 g, accurately weighed, in a tared racemic.
open porcelain dish on a steam bath, and dry the resi- e USP REFERENCE STANDARDS (11)
due at 105° for 1 h. USP Menthol RS
Acceptance criteria: NMT 0.05%

Change to read:

e RELATED COMPOUNDS Menthol Lozenges

Aguspa? Standard solution, 4Sample solution,ause,; Chro-
matographic spereliy and System suitability: Proceed DEFINITION
as directed in the Assay. Menthol Lozenges contain NLT 90.0% and NMT 125.0% of
Aauspat the labeled amount of menthol (C;oH200), in a suitable
Analysis molded base.
Sample: Sample solution IDENTIFICATION
Calculate the percentage of each individual impurity in e A. The retention time of the menthol peak of the Sample
the portion of Menthol taken: solution corresponds to that of the Standard solution, as
Result = (ru/r) x 100 obtained in the Assay.
ASSAY
ty = peak area of each impurity from the Sample © PROCEDURE
solution Solution A: 250 mg/mL of sodium chloride in water
lea = sum of the peak areas from the Sample Internal standard solution: 2 mg/mL of anethole in
solution hexanes
Acceptance criteria Standard solution: 0.201 mg/mL of USP Menthol RS in
Individual impurities: 4ANMT 0.5% for isomenthol (rel- Internal standard solution, where L is the labeled quan-
ative retention time is 1.08) from synthetic racemic
menthol and 0.3% for all other impurities from natural
tity, in mg, of menthol in each Lozenge S
Sample solution: Transfer 20 Lozenges to a 1-L screw- A)
and synthetic mentholausps1 =P ed conical flask. [NoTE—Use caps with inert white vu
Total impurities: NMT 2.0%
© READILY OXIDIZABLE SUBSTANCES IN d/-IMENTHOL
ru ber liners.] Add 200 mL of water, 260 mL of Solution =
Sample solution: Place 500 mg of di-menthol in a
A, and 100.0 mL of the Internal standard solution, and °
shake by mechanical means for 30 min. Allow the J
clean, dry test tube, and add 10 mL of a solution of i}
potassium permanganate, prepared by diluting 3 mL of
phases to separate, and transfer a portion of the hex- i}=
anes phase to a suitable container.
0.1 N potassium permanganate with water to 100 mL. i)
Analysis: Place the test tube in a beaker with water at a
Chromatographic system i}
temperature between 45° and 50°. Remove the tube
(See Chromatography (621), System Suitability.) =
a]
Mode: GC
ae the bath at intervals of 30 s, and mix quickly by Detector: Flame ionization
shaking. Column: 0.53-mm x 30-m fused silica; coated with a
Acceptance criteria: The purple color of potassium per- 1-um layer of G16 stationary phase
manganate is still apparent after 5 min. Temperatures
SPECIFIC TESTS Column: 125° (isothermally)
© CONGEALING RANGE OF d/-MENTHOL Injection port: 250°
(See Congealing Temperature (651).) Detector: 250°
[Note—Perform this test preferably in a room having a Carrier gas: Helium
temperature below 30° and a relative humidity below Flow rate: 10 mL/min
50%.] Injection volume: 1 uL
Sample: 10g of d/-menthol, previously dried in a desic- Injection type: Split ratio of 10:1
cator over silica gel for 24 h System suitability
Analysis: Place the Sample in a dry test tube having an Sample: Standard solution
internal diameter of 18-20 mm, and melt the contents [Note—The relative retention times for menthol and
at a temperature of about 40°. Suspend the test tube in anethole are about 0.5 and 1.0, respectively.]
water having a temperature of 23°-25°, and stir the Suitability requirements
contents of the tube continually with a thermometer, Resolution: NLT 15 between menthol and anethole
rene the bulb of the thermometer immersed in the Tailing factor: NMT 2.0 for menthol and anethole
iquid. Relative standard deviation: NMT 2.0% for replicate
Acceptance criteria: d/-Menthol congeals at a tempera- injections
ture between 27° and 28°. Shortly after the tempera- Analysis
ture has stabilized at the congealing point, add a few Samples: Standard solution and Sample solution
milligrams of dried d/-menthol to the congealed mass, Calculate the percentage of the labeled amount of
and continue stirring. After a few minutes, the tempera- menthol (CioH200) in the portion of Lozenges taken:
ture of the mass quickly rises to 30.5°-32.0°.
¢ MELTING RANGE OF J-IMENTHOL Result = (Ru/Rs) x (Cs/Cu) x 100
(See Melting Range or Temperature (741).)
 2574 Menthol / Official Monographs USP 41

Ru = peak response ratio of the menthol to the Chromatographic system

anethole from the Sample solution (See Chromatography (621), System Suitability.)
Rs = peak response ratio of the menthol to the Mode: LC
anethole from the Standard solution Detector: UV 230 nm
Cs = concentration of USP Menthol RS in the Column: 3.9-mm x 30-cm; packing L1
Standard solution (mg/mL) Flow rate: 1 mL/min
Cy = nominal concentration of menthol in the Injection volume: 20 uL
hexanes phase of the Sample solution System suitability
(mg/mL) Sample: Standard solution
Acceptance criteria: 90.0%-125.0% Suitability requirements
Column efficiency: NLT 2000 theoretical plates
ADDITIONAL REQUIREMENTS Tailing factor: NMT 2 for the meperidine peak
© PACKAGING AND STORAGE: Preserve in well-closed Relative standard deviation: NMT 2%
containers. Analysis
e USP REFERENCE STANDARDS (11) Samples: Standard solution and Sample solution
USP Menthol RS Calculate the percentage of meperidine hydrochloride
(CsH21NOz - HCl) in the portion of Meperidine Hydro-
chloride taken:
Result = (ru/rs) x (Cs/Cu) x 100
Meperidine Hydrochloride
ty = peak response of meperidine from the Sample
solution
ls = peak response of meperidine from the
+ HCI Standard solution
G = concentration of USP Meperidine
Hydrochloride RS in the Standard solution
(mg/mL)
CisH2iNOz - HCl 283.79 Cu = concentration of Meperidine Hydrochloride in
4-Piperidinecarboxylic acid, 1-methyl-4-phenyl-, ethyl ester, the Sample solution (mg/mL)
hydrochloride; Acceptance criteria: 98.0%-102.0% on the dried basis
Ethyl 1-methyl-4-phenylisonipecotate hydrochloride OTHER COMPONENTS
[50-13-5]. @ CONTENT OF CHLORIDE
DEFINITION Sample solution: Transfer about 500 mg of Meperidine
Meperidine Hydrochloride contains NLT 98.0% and NMT Hydrochloride, previously dried, to a 250-mL conical
102.0% of meperidine hydrochloride (CisH2i1NO> - HCl), flask. Add 15 mL of water, 5 mL of glacial acetic acid,
calculated on the dried basis. 50 mL of methanol, and 0.2 mL of eosin Y TS.
ie
”
Analysis: Titrate the Sample solution with 0.1 N silver
a IDENTIFICATION nitrate VS to a rose-colored endpoint. Each mL of 0.1 N
J
—
e A. IDENTIFICATION—ORGANIC NITROGENOUS BASES (181): silver nitrate is equivalent to 3.545 mg of chloride.
D Meets the requirements Acceptance criteria: 12.2%-12.7% of chloride is
°
< © B. IDENTIFICATION TESTS—GENERAL, Chloride (191) ound.
iS Sample solution: 10 mg/mL
= Acceptance criteria: Meets the requirements IMPURITIES
i e C. The retention time of the major peak of the Sample e RESIDUE ON IGNITION (281):NMT 0.1%
A) solution corresponds to that of the Standard solution, as ¢ ORGANIC IMPURITIES
=) obtained in the Assay. Sample solution: 10 mg/mL in water
Chromatographic system
ASSAY Mode: GC
e PROCEDURE Detector: Flame ionization
Solution A: Transfer about 6.8 g of monobasic potas- Column: 2-mm x 2-m glass; 10% phase G3 on sup-
sium phosphate to a 1000-mL volumetric flask. Dissolve port SIA
in and dilute with water to volume. Add 10 mL of tri- Temperatures
ethylamine, and mix. Adjust with phosphoric acid to a Column: 190°
pH of 7.0, and filter. Injection port: 255°
Mobile phase: Acetonitrile and Solution A (550:450), Detector: 280°
filtered and degassed Carrier gas: Helium
Standard stock solution: 0.6 mg/mL of USP Meper- Flow rate: 28 mL/min
idine Hydrochloride RS in water Injection volume: 2.0 pL
Standard solution: 0.12 mg/mL of USP Meperidine Hy- Analysis
drochloride RS from the Standard stock solution in Mo- Sample: Sample solution
bile phase Calculate the area percentage of each peak.
Sample stock solution: 0.6 mg/mL of Meperidine Hy- Acceptance criteria: No peak other than the principal
drochloride in water peak (except for the solvent peak) constitutes more
Sample solution: 0.12 mg/mL of Meperidine Hydro- than 1.0% of the total area.
chloride from the Sample stock solution in Mobile phase
SPECIFIC TESTS
e Loss ON DRYING (731)
Analysis: Dry under vacuum at a pressure between 20
and 40 mm of mercury at 80° for 4 h.
USP 41
Acceptance criteria: NMT 1.0%
e MELTING RANGE OR TEMPERATURE (741)
Sample: Dried under vacuum at 80° for 4h
Acceptance criteria: 186°-189°
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in well-closed, light-
resistant containers, and store at room temperature.
e USP REFERENCE STANDARDS (11)
USP Meperidine Hydrochloride RS
Meperidine Hydrochloride Injection
DEFINITION
Meperidine Hydrochloride Injection is a sterile solution of
Meperidine Hydrochloride in Water for Injection. It con-
tains NLT 95.0% and NMT 105.0% of the labeled amount
of meperidine hydrochloride (CisH21NOz2 - HCl).
IDENTIFICATION
e A. IDENTIFICATION—ORGANIC NITROGENOUS BASES (181):
Meets the requirements
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
ASSAY
¢ PROCEDURE
Buffer: Transfer about 6.8 g of monobasic potassium
phosphate to a 1000-mL volumetric flask. Dissolve in
and dilute with water to volume. Add 10 mL of triethyl-
amine, and mix. Adjust with phosphoric acid to a pH of
7.0, and filter.
Mobile phase: Acetonitrile and Buffer (550:450), filtered
and degassed
Standard stock solution: 0.6 mg/mL of USP Meper-
idine Hydrochloride RS in water
Standard solution: 0.12 mg/mL of USP Meperidine Hy-
drochloride RS from the Standard stock solution in Mo-
bile phase
Sample stock solution: Transfer a measured volume of
Injection equivalent to about 300 mg to a 100-mL volu-
metric flask, and dilute with water to volume.
Sample solution: Transfer 1.0 mL of the Sample stock
Solution to a 25-mL volumetric flask, dilute with Mobile
phase to volume, and mix.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 230 nm
Column: 3.9-mm x 30-cm; packing L1
Flow rate: 1 mL/min
Injection volume: 20 pL
System suitability
Sample: Standard solution
Suitability requirements
Column efficiency: NLT 2000 theoretical plates
Tailing factor: NMT 2
Relative standard deviation: NMT 2%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
meperidine hydrochloride (CisH21NOz - HCl) in the
portion of Injection taken:
Result = (ru/rs) x (Cs/Cy) x 100
tu = peak response from the Sample solution
Is = peak response from the Standard solution
Gs = concentration of USP Meperidine
Hydrochloride RS in the Standard solution
(mg/mL)
Official Monographs / Meperidine 2575
Cu = nominal concentration of meperidine
hydrochloride in the Sample solution
(mg/ml)
Acceptance criteria: 95.0%-105.0%
SPECIFIC TESTS
° PH (791): 3.5-6.0
e BACTERIAL ENDOTOXINS TEST (85): It contains NMT 2.4
USP Endotoxin Units/mg of meperidine hydrochloride.
¢ OTHER REQUIREMENTS: It meets the requirements in Injec-
tions and Implanted Drug Products (1).
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in single-dose or mul-
tiple-dose containers, preferably of Type | glass.
Change to read:
° USP REFERENCE STANDARDS (11)
@ (CN 1-May-2018)
USP Meperidine Hydrochloride RS
Meperidine Hydrochloride Oral Solution
DEFINITION
Meperidine Hydrochloride Oral Solution contains NLT
95.0% and NMT 105.0% of the labeled amount of
meperidine hydrochloride (CisH21NOz - HCl).
IDENTIFICATION
eA.
Sample solution: Transfer a volume of Oral Solution
nominally equivalent to about 100 mg of meperidine
hydrochloride to a 125-mL separator. Add 40 mL of
water and 3 mL of 1 N sodium hydroxide, and extract my
“
with three 25-mL portions of n-hexane. Wash the com- a]
bined extracts with two 20-mL portions of water, dis-
card the water, and then extract with three 25-mL por- K
°
tions of 0.1 N hydrochloric acid. Transfer the extracts to =]
a 100-mL volumetric flask, dilute with 0.1 N hydrochlo- °
ric acid to volume, and mix. ico}
=
Standard solution: Prepare in a way similar to that for ES}
the Sample solution, using USP Meperidine Hydrochlo- mo}
a
ride RS. 7)
Acceptance criteria: The UV absorption spectrum of
the Sample solution exhibits maxima and minima at the
same wavelengths as those of the Standard solution.
ASSAY
e PROCEDURE
Sample solution: Transfer a suitable volume of Oral So-
lution nominally equivalent to about 250 mg of meper-
idine hydrochloride to a separator, and add 3 mL of 1N
sodium hydroxide. Extract with five 20-mL portions of
chloroform, and filter the extracts through a pledget of
cotton into a 250-mL conical flask. Wash the cotton
with 5 mL of chloroform, and add the washing to the
combined filtrates. Add 10 mL of glacial acetic acid and
2 drops of crystal violet TS.
Analysis: Titrate with 0.1 N perchloric acid VS to a blue
endpoint. Perform a blank determination, and make
any necessary correction. Each mL of 0.1 N perchloric
acid is equivalent to 28.38 mg of meperidine hydro-
chloride (C1sH21NOz2 - HCl).
Acceptance criteria; 95.0%-105.0%
PERFORMANCE TESTS
e DELIVERABLE VOLUME (698): Meets the requirements for
oral solution packaged in multiple-unit containers
© UNIFORMITY OF DOSAGE UNITS (905): Meets the require-
ments for oral solution packaged in single-unit containers
 2576 Meperidine / Official Monographs USP 41

SPECIFIC TESTS Chromatographic system

© PH (791): 3.5-4.1 (See Chromatography (621), System Suitability.)
Mode: LC
ADDITIONAL REQUIREMENTS Detector: UV 230 nm
e PACKAGING AND STORAGE: Preserve in tight, light-resistant Column: 3.9-mm x 30-cm; packing L1
containers. Flow rate: 1 mL/min
e USP REFERENCE STANDARDS (11) Injection volume: 20 uL
USP Meperidine Hydrochloride RS System suitability
Sample: Standard solution
Suitability requirements
Column efficiency: NLT 2000 theoretical plates
Tailing factor: NMT 2 for the meperidine peak
Meperidine Hydrochloride Tablets Relative standard deviation: NMT 2%
Analysis
DEFINITION Samples: Standard solution and Sample solution
Meperidine Hydrochloride Tablets contain NLT 95.0% and Calculate the percentage of meperidine hydrochloride
NMT 105.0% of the labeled amount of meperidine hy- (CisH21NOz - HCl) in the portion of Tablets taken:
drochloride (C)sH21NOz2 + HCI).
Result = (ru/rs) x (Cs/Cu) x 100
IDENTIFICATION
e A. IDENTIFICATION—ORGANIC NITROGENOUS BASES (181) fu = peak response of meperidine from the Sample
Sample solution: Transfer an amount nominally equiva- solution
lent to about 50 mg of meperidine hydrochloride from rs = peak response of meperidine from the
powdered Tablets to a separator, add 10 mL of water, Standard solution
and shake. Add 5 mL of saturated sodium chloride solu- Cs = concentration of USP Meperidine
tion and 1 mL of sodium hydroxide solution (1 in 25). Hydrochloride RS in the Standard solution
Extract with three 20-mL portions of chloroform, filter- (mg/mL)
ing the extracts through cotton overlaid with anhydrous Cy = nominal concentration of meperidine
sodium sulfate. Evaporate the chloroform on a steam hydrochloride in the Sample solution
bath, and dissolve the residue in 4 mL of carbon (mg/mL)
disulfide. Acceptance criteria: 95.0%-105.0%
Standard solution: In a second separator, proceed as
directed in the Sample solution, using 50 mg of USP PERFORMANCE TESTS
Meperidine Hydrochloride RS. e DISSOLUTION (711)
Analysis: Proceed as directed in the chapter, beginning Medium: Water; 500 mL
with “Determine the absorption spectra”. Apparatus 1: 100 rpm
Acceptance criteria: Meet the requirements Time: 45 min
e B. The retention time of the major peak of the Sample Standard solution: A known concentration of USP
ia
ww
Meperidine Hydrochloride RS in Medium
(oH solution corresponds to that of the Standard solution, as
Sample solution: Filter portions of the solution under
ii obtained in the Assay.
— test, andsuitably dilute with Medium, if necessary.
io)
2} ASSAY Chromatographic system and System suitability: Pro-
c © PROCEDURE ceed as directed in the Assay.
iS Solution A: Transfer about 6.8 g of monobasic potas- Analysis: Determine the labeled amount of meperidine
= sium phosphate to a 1000-mL volumetric flask. Dissolve hydrochloride (CisH2i1NOz - HCl) dissolved by comparing
rs in and dilute with water to volume. Add 10 mL of tri- the peak response of meperidine from the Sample solu-
a) ethylamine, and mix. Adjust with phosphoric acid to a tion with that from the Standard solution.
=: pH of 7.0, and filter. Tolerances: NLT 75% (Q) of the labeled amount of
Mobile phase: Acetonitrile and Solution A (550:450), meperidine hydrochloride (CisH2i1NOz2 - HCl) is dissolved.
filtered and degassed e UNIFORMITY OF DOSAGE UNITS (905): Meet the
Standard stock solution: 0.6 mg/mL of USP Meper- requirements
idine Hydrochloride RS in water
Standard solution: 0.12 mg/mL of USP Meperidine Hy- ADDITIONAL REQUIREMENTS
drochloride RS from the Standard stock solution in Mo- e PACKAGING AND STORAGE: Preserve in well-closed, light-
bile phase resistant containers.
Sample stock solution: Transfer an amount nominally e USP REFERENCE STANDARDS (11)
equivalent to about 60 mg of meperidine hydrochloride USP Meperidine Hydrochloride RS
from NLT 20 finely powdered Tablets to a 100-mL volu-
metric flask. Add about 70 mL of Mobile phase, and
sonicate for 10 min with occasional shaking. Shake by
mechanical means for about 30 min, dilute with Mobile
phase to volume, mix, and filter. Mephenytoin
Sample solution: Nominally equivalent to 0.12 mg/mL
of meperidine hydrochloride in Mobile phase from the °
Sample stock solution

ot Za CH,

Ci2Hi4N202 218.25
2,4-Imidazolidinedione, 5-ethyl-3-methyl-5-phenyl-, (+)-;
(+)-5-Ethyl-3-methyl-5-phenylhydantoin [50-12-4].
USP 41 Official Monographs / Mephenytoin 2577

DEFINITION Chromatographic system

Mephenytoin contains NLT 98.0% and NMT 102.0% of (See Chromatography (621), System Suitability.)
mephenytoin (Ci2Hi4N202), calculated on the dried basis. Mode: LC
Detector: UV 225 nm
IDENTIFICATION Column: 3.9-mm x 15-cm; packing L7
e A. INFRARED ABSORPTION (197K) Flow rate: 1 mL/min
e B. The retention time of the major peak of the Sample Injection volume: 10 uL
solution corresponds to that of the Standard solution as System suitability
obtained in the Assay. Sample: System suitability solution
[Note—See Table 7 for relative retention times.]
ASSAY Suitability requirements
© PROCEDURE Column efficiency: NLT 4000 theoretical plates for
Mobile phase: Acetonitrile, methanol, and water the mephenytoin peak
(10:38:52) Relative standard deviation: NMT 2.0% for the
System suitability solution: 0.015 mg/mL of propi- mephenytoin peak
ophenone and 1.5 mg/mL of USP Mephenytoin RS in Analysis
Mobile phase. Sonicate if necessary. Sample: Sample solution
Standard solution: 5.0 mg/mL of USP Mephenytoin RS Calculate the percentage of each impurity in the por-
in Mobile phase. Sonicateif necessary. tion of Mephenytoin taken:
Sample solution: 5.0 mg/mL of Mephenytoin in Mobile
phase Result = (ru/rz) x (1/F) x 100
Chromatographic system
(See Chromatography (621), System Suitability.) ru = peak response for each ey
Mode: LC rr = sum of the responses of all of the peaks
Detector: UV 257 nm F = relative response factor for the corresponding
Column: 3.9-mm x 15-cm; packing L7 impurity (see Table 1)
Flow rate: 1 mL/min Acceptance criteria: See Table 1.
Injection volume: 10 pL
System suitability
Table 1
Sample: System suitability solution
[NoTte—See Table 1 for relative retention times.] Relative Relative Acceptance
Suitability requirements Retention Response Criteria,
Column efficiency: NLT 4000 theoretical plates for Name Time Factor NMT (%)
the mephenytoin peak Desmethyl
Relative standard deviation: NMT 2.0% for the phenytoin? 0.66 0.86 1.0
mephenytoin peak Mephenytoin 1.0 = os
Analysis
Methyl
Samples: Standard solution and Sample solution mephenytoin® 112 1.0 1.0 S
Calculate the percentage of mephenytoin (C;2Hi4N2O2)
in the portion of Mephenytoin taken: Propiophe- uv
none 15 27 1.0 z
Result = (ru/rs) x (Cs/Cu) x 100 Any individual }
unspecified — =]
tu = peak response from the Sample solution impurity 1.0 0.10 °
Is = peak response from the Standard solution vo}
Total = — 15 a
Cs = concentration of USP Mephenytoin RS in the 2 5-Ethyl-5-phenylimidazolidine-2,4-dione. Ts
Standard solution (mg/mL) » 5-Ethyl-1,3-dimethyl-5-phenylimidazolidine-2,4-dione. ra
Cu = concentration of the Sample solution (mg/mL)
Acceptance criteria: 98.0%-102.0% on the dried basis SPECIFIC TESTS
e LOSS ON DRYING (731)
IMPURITIES Analysis: Dry at 105° for 4 h.
e RESIDUE ON IGNITION (281): NMT 0.1% Acceptance criteria: NMT 1.0%
ADDITIONAL REQUIREMENTS
Delete the following: © PACKAGING AND STORAGE: Preserve in tight containers be-
low 30°.
°e HEAVY METALS, Method I! (231): NMT 20 ppme cofical 1. e USP REFERENCE STANDARDS (11)
Jan-20183 USP Mephenytoin RS
© ORGANIC IMPURITIES
Mobile phase: Acetonitrile, methanol, and water
(10:38:52)
System suitability solution: 0.015 mg/mL of propi-
ophenone and 1.5 mg/mL of USP Mephenytoin RS in
Mobile phase Mephenytoin Tablets
Sample solution: 5.0 mg/mL of Mephenytoin in Mobile
phase DEFINITION
Mephenytoin Tablets contain NLT 90.0% and NMT 110.0%
of the labeled amount of mephenytoin (Ci2Hi4N202).
IDENTIFICATION
e A. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
 2578 Mephenytoin / Official Monographs USP 41

ASSAY Au = absorbance of the Sample solution

Noid
e PROCEDURE As absorbance of the Standard solution
Mobile phase: Acetonitrile, methanol, and water Cs concentration of USP Mephenytoin RS in the
(10:38:52) Standard solution (mg/mL)
System suitability solution: 0.015 mg/mL of propi- D = dilution factor, if used
ophenone and 1.5 mg/mL of USP Mephenytoin RS in V = volume of Medium, 500 mL
Mobile phase. Sonicate if necessar L = label claim (mg/Tablet)
Standard solution: 5.0 mg/mL of USP Mephenytoin RS Tolerances: NLT 70% (Q) of the labeled amount of
in Mobile phase. Sonicate if necessary. mephenytoin (Cy2Hi4N202) is dissolved.
Sample solution: Nominally 5.0 mg/mL of e UNIFORMITY OF DOSAGE UNITS (905): Meet the
mephenytoin prepared with NLT 500 mg from NLT requirements
20 powdered Tablets as follows. Transfer the powder to
a suitable volumetric flask. Add 60% of the flask volume IMPURITIES
of Mobile phase, sonicate for 10 min, and shake by me- e ORGANIC IMPURITIES
chanical means for 30 min. Dilute with Mobile phase to Mobile phase: Acetonitrile, methanol, and water
volume, and filter, discarding a suitable portion of the (10:38:52)
filtrate. System suitability solution: 0.015 mg/mL of propi-
Chromatographic system ophenone and 1.5 mg/mL of USP Mephenytoin RS in
(See Chromatography (621), System Suitability.) Mobile phase. Sonicate if necessary.
Mode: LC Sample solution: Nominally 5.0 mg/mL of
Detector: UV 257 nm mephenytoin prepared with NLT 500 mg from NLT
Column: 3.9-mm x 15-cm; packing L7 20 powdered Tablets as follows. Transfer the powder to
Flow rate: 1 mL/min a suitable volumetric flask. Add 60% of the flask volume
Injection volume: 10 uL of Mobile phase, sonicate for 10 min, and shake by me-
System suitability chanical means for 30 min. Dilute with Mobile phase to
Sample: System suitability solution volume, and filter, discarding a suitable portion of the
[Note—See Table 7 for relative retention times.] filtrate.
Suitability requirements Chromatographic system
Column efficiency: NLT 4000 theoretical plates for (See Chromatography (621), System Suitability.)
the mephenytoin peak Mode: LC
Relative standard deviation: NMT 2.0% for the Detector: UV 225 nm
mephenytoin peak Column: 3.9-mm x 15-cm; packing L7
Analysis Flow rate: 1 mL/min
Samples: Standard solution and Sample solution Injection volume: 10 uL
Calculate the percentage of the labeled amount of System suitability
mephenytoin (C2H;4N202) in the portion of Tablets Sample: System suitability solution
ken: [Note—See Table 1 for relative retention times.]
Pa} ‘ais Suitability requirements
as Result = (ru/rs) x (Cs/Cu) x 100 Column efficiency: NLT 4000 theoretical plates for
c the mephenytoin peak
a ty = peak response from the Sample solution Relative standard deviation: NMT 2.0% for the
fo} rs = peak response from the Standard solution mephenytoin peak
= Cs = concentration of USP Mephenytoin RS in the Analysis
eS Standard solution (mg/mL) Sample: Sample solution
Cu = nominal concentration of mephenytoin in the Calculate the percentage of each impurity in the por-
c Sample solution (mg/mL) tion of Tablets taken:
cs Acceptance criteria: 90.0%-110.0%
Result = (ru/rr) x (1/F) x 100
PERFORMANCE TESTS
© DISSOLUTION (711) tu = peak response of each impurity
Medium: Water; 500 mL tr = sum of the responses of all of the peaks
Apparatus 2: 75 rpm F = relative response factor (see Table 1)
Time: 60 min Acceptance criteria: See Table 7.
Instrumental conditions
Mode: UV . Table 1
Analytical wavelength: Wavelength of maximum ab- a
sorbance at about 257 nm Relative Relative Acceptance
Standard solution: 0.2 mg/mL of USP Mephenytoin RS Retention | Response Criteria,
in Medium Name Time Factor NMT (%)
Sample solution: Filter a portion of the solution under Desmethyl phenytoin 0.66 0.86 1.0
test. Dilute the filtrate, if necessary, with Medium. Mephenytoin 1.0 tt pay
Analysis Methyl mephenytoin® 1 1.0 1.0
Samples: Standard solution and Sample solution Propiophenone 15 22 1.0
Calculate the percentage of the labeled amount of
mephenytoin (Ci2Hi4N2Oz) dissolved: 2 5-Ethyl-5-phenylimidazolidine-2,4-dione
© 5-Ethyl-1,3-dimethyl-5-phenylimidazolidine-2,4-dione.
Result= (Au/As) x Cs x D x Vx (1/L) x 100
USP 41 Official Monographs / Mephobarbital 2579

Table 1 (Continued) Acceptance criteria: 98.0%-100.5% on the dried basis

Relative Relative Acceptance IMPURITIES
Retention Response Criteria, e RESIDUE ON IGNITION (281): NMT 0.1%
Name Time Factor NMT (%)
Any individual SPECIFIC TESTS
unspecified degrada- - e Loss ON DRYING (731)
tion product 1.0 0.2 Analysis: Dry a sample at 105° for 4 h.
Total impurities = = 2.0 Acceptance criteria: NMT 1.0%
25-Ethyl-5-phenylimidazolidine-2,4-dione. e MELTING RANGE OR TEMPERATURE, Class | (741):
»5-Ethyl-1,3-dimethyl-5-phenylimidazolidine-2,4-dione.
176°-181°

ADDITIONAL REQUIREMENTS ADDITIONAL REQUIREMENTS

e PACKAGING AND STORAGE: Preserve in tight containers, © PACKAGING AND STORAGE: Preserve in well-closed
and store below 30°. containers.
e USP REFERENCE STANDARDS (11) e USP REFERENCE STANDARDS (11)
USP Mephenytoin RS USP Mephobarbital RS

Mephobarbital Mephobarbital Tablets

Fe DEFINITION
Mephobarbital Tablets contain NLT 95.0% and NMT
110.0% of the labeled amount of mephobarbital
= (CisHiaN20s).
% IDENTIFICATION
e A. INFRARED ABSORPTION—GENERAL (197M)
Sample: Residue obtained in the Assay
Ci3HiaN203 246.26 Acceptance criteria: The infrared absorption spectrum
2,4,6(1H,3H,5H)-Pyrimidinetrione, 5-ethyl-1-methyl- of Sample is consistent with that of USP Mephobarbital
5-phenyl-; RS.
5-Ethyl-1-methyl-5-phenylbarbituric acid [115-38-8]. e B. MELTING RANGE OR TEMPERATURE (741)
DEFINITION Sample: Residue obtained in the Assay
Mephobarbital contains NLT 98.0% and NMT 100.5% of Acceptance criteria: 174°-181°
mephobarbital (C13H;4N2O3), calculated on the dried ASSAY (==
basis. © PROCEDURE
al
a]
IDENTIFICATION Lae Weigh a suitable number of powdered Tablets
e A. INFRARED ABSORPTION (197M) (NLT 20), and transfer an accurately weighed portion of Es
°
e B. powder, equivalent to 300 mg of mephobarbital, to a |
Sample: 200 mg of Mephobarbital suitable extraction thimble. Extract with 15 mL of hex- °
ane, allow the thimble to drain, transfer to a continu- vo}
Analysis: Boil the Sample with 10 mL of 1 N sodium mf
hydroxide. ous-extraction apparatus provided with a tared flask, 2
and extract the mephobarbital with chloroform for 2 h. no]
Acceptance criteria: Ammonia is evolved. a
°C. Evaporate the chloroform on a steam bath with the aid 7
Diluent: Sodium hydroxide solution (1 in 500) of a current of air, and cool.
Sample solution: 12 mg/mL of Mephobarbital prepared Analysis: Dissolve the residue in 10 mL of alcohol, and
as follows. Shake about 60 mg of Mephobarbital with evaporate. Dry the residue at 105°; for 1 h, cool, and
5 mL of Diluent, and filter. Use the filtrate. weigh. The weight of the residue represents the noe
Analysis 1: Add 3 drops of mercuric nitrate TS to 1 mL ue (Ci3Hi4N2O3) in the portion of Tablets
of the Sample solution. taken.
Acceptance criteria 1: A white precipitate is formed, Acceptance criteria: 95.0%-110.0%
and it is soluble in 6 N ammonium hydroxide. PERFORMANCE TESTS
Analysis 2: Add silver nitrate TS to 1 mL of the Sample e DISSOLUTION (711)
solution. Buffer: 1.1L of 1% of 3-(dodecyldimethylammonio)
Acceptance criteria 2: A white precipitate is formed, propanesulfonate in pH 8.0 noel buffer prepared
and it dissolves readily in 6 N ammonium hydroxide. as follows. Transfer 10.0
g of 3-(dodecyldimethylam-
ASSAY roe pcp aa in 400 mL of warm water,
¢ PROCEDURE and add 250 mL of 0.2 M monobasic potassium phos-
Sample solution: 10 mg/mL of Mephobarbital in phate and about 220 mL of 0.2 M sodium hydroxide.
dimethylformamide Cool to room temperature, adjust with 0.2 M sodium
Analysis: Transfer 50 mL of Sample solution to a 200-mL hydroxide to a pH of 8.0, dilute with water to 1000 mL,
flask. Add 4 drops of thymolphthalein TS. Titrate with mix, and degas.
0.1 N lithium methoxide in toluene VS using a mag- Medium: Buffer, 900 mL
netic stirrer and a cover for the flask to protect against Apparatus 2: 75 rpm
atmospheric carbon dioxide. Perform a blank determi- Time: 75 min
nation. Each mL of 0.1 N lithium methoxide is equiva- Standard solution: Known concentration of USP
lent to 24.63 mg of mephobarbital (Ci3Hi4N203). Mephobarbital RS in Medium
Sample solution: Pass a portion of the solution through
a nylon filter of 0.45-11m pore size, and, if necessary,
suitably dilute with Medium.
 2580 Mephobarbital / Official Monographs USP 41

Instrumental conditions IDENTIFICATION

Mode: UV
Analytical wavelength: 244 nm
Analysis
Samples: Standard solution and Sample solution
Tolerances: NLT 70% (Q) of the labeled amount of
mephobarbital (Ci3Hi4N20s) is dissolved.
© UNIFORMITY OF DosAGE UNITS, Content Uniformity (905)
Prepare all of the solutions concomitantly.
Diluent: 6.2g of boric acid and 7.45 g of potassium
chloride in 500 mL of water. Add 210 mL of sodium
hydroxide solution (1 in 25). Add water to make
2000 mL of Diluent.
Standard stock solution: 10 mg/mL of USP
Mephobarbital RS in Diluent
Standard solution: 1.5 mg/mL of USP Mephobarbital
RS in water from Standard stock solution
Sample solution: Nominally 1.5 mg/mL of
mephobarbital prepared as follows. Transfer 1 Tablet to
a glass-stoppered centrifuge tube, crush the Tablet, and
add 25.0 mL of Diluent. Insert the stopper, shake for 10
min, and, if necessary, centrifuge until clear, filtering
the supernatant. Dilute a portion of the subsequent liq-
uid with water.
Instrumental conditions
Mode: UV
Analytical wavelength: 245 nm
Cell: 71cm
Blank: Diluent and water (1:3)
Analysis
Samples: Standard solution and Sample solution
Transfer 3.0 mL each of the Standard solution and the
Sample solution to separate 200-mL volumetric flasks,
and dilute each with Blank to volume.
Determine thepercentade of the labeled amount of
mephobarbital (Ci3H14N203) in the Tablet taken:
Result = (Au/As) x (Cs/Cu) x 100
US
“
e A. INFRARED ABSORPTION (197K)
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
© C. IDENTIFICATION TESTS—GENERAL, Chloride (191)
Sample solution: 10 mg/mL
Acceptance criteria: Meets the requirements
ASSAY
© PROCEDURE
Buffer: 2.25 git solution of phosphoric acid, adjusted
with 50% sodium hydroxide to a pH of 7.6
Mobile phase: Acetonitrile and Buffer (35:65)
System suitability solution: 2 g/mL of USP Mepiva-
caine Hydrochloride RS and 3 ug/mL of USP Bupiva-
caine Related Compound BRS in Mobile phase
Standard solution: 0.2 mg/mL of USP Mepivacaine Hy-
drochloride RS in Mobile phase
Sample solution: 0.2 mg/mL of Mepivacaine Hydro-
chloride in Mobile phase
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: Lc
Detector: UV 220 nm
Column: 4.6-mm x 15-cm; 5-um packing L1
Flow rate: 1 mL/min
Injection volume: 20 pL
System suitability
Samples: System suitability solution and Standard
solution
Suitability requirements
Resolution: NLT 2.5 between bupivacaine related
compound B and mepivacaine, System suitability
solution
Tailing factor: NMT 1.5, Standard solution
Relative standard deviation: NMT 1.0%, Standard
solution

oy Analysis
i Au = absorbance of the Sample solution Samples: Standard solution and Sample solution
—
=) As = absorbance of the Standard solution Calculate the percentage of mepivacaine hydrochloride
° Cs = concentration of the USP Mephobarbital RS in (CisH22N20 - HCl) in the portion of Mepivacaine Hy-
= the Standard solution (mg/mL) drochloride taken:
5 Cu = nominal concentration of mephobarbital in
= the Sample solution (ngimLy Result = (ru/rs) x (Cs/Cy) x 100
os Acceptance criteria: Meet the requirements
a) Tu = peak response from the Sample solution
> ADDITIONAL REQUIREMENTS ts = peak response from the Standard solution
© PACKAGING AND STORAGE: Preserve in well-closed Cs = concentration of USP Mepivacaine
containers. Hydrochloride RS in the Standard solution
e USP REFERENCE STANDARDS (11) (mg/mL)
USP Mephobarbital RS Gu = concentration of Mepivacaine Hydrochloride
in the Sample solution (mg/mL)
Acceptance criteria: 98.0%-102.0% on the dried basis
IMPURITIES
Mepivacaine Hydrochloride e RESIDUE ON IGNITION (281): NMT 0.1%
© ORGANIC IMPURITIES
Mobile phase, System suitability solution, and Chro-
matggtaphic system: Proceed as directed in the As-
say. The run time is three times the retention time of
the mepivacaine peak.
Standard solution: 2 g/mL of USP Mepivacaine Hy-
drochloride RS in Mobile phase
CisH22N20 - HCl 282.81 Sample solution: 2 mg/mL of Mepivacaine Hydrochlo-
2-Piperidinecarboxamide, N-(2,6-dimethylphenyl)-1-methyl-, ride in Mobile phase
monohydrochloride, (+)-; System suitability
(4)-1-Methyl-2’,6’-pipecoloxylidide monohydrochloride Samples: System suitability solution and Standard
[1722-62-9]. solution
Suitability requirements
DEFINITION Resolution: NLT 2.5 between bupivacaine related
Mepivacaine Hydrochloride contains NLT 98.0% and NMT compound B and mepivacaine, System suitability
102.0% of mepivacaine hydrochloride (CisH22N2O- HCl), solution
calculated on the dried basis.
USP 41 Official Monographs / Mepivacaine 2581

Tailing factor: NMT 1.5, Standard solution Carrier gas: Helium

Relative standard deviation: NMT 2.0%, Standard Flow rate: 4 mL/min
solution Injection type: Split ratio, 1:1
Analysis Headspace sampler
Samples: Standard solution and Sample solution Equilibration time: 15 min
Calculate the percentage of any impurity in the portion Equilibration temperature: 90°
of Mepivacaine Hydrochloride taken: Loop temperature: 215°
Transfer line temperature: 220°
Result = (ru/rs) x (Cs/Cu) x 100 Vial pressure: About 15 psi
Loop size: 3 mL
tu = peak response of any individual impurity from System suitability
the Sample solution Sample: Standard solution
Is = peak response of mepivacaine from the Suitability requirements
Standard solution Tailing factor: NMT 2.0
Cs = concentration of USP Mepivacaine Relative standard deviation: NMT 15%
Hydrochloride RS in the Standard solution Analysis
(mg/mL) Samples: Standard solution and Sample solution
Cu = concentration of Mepivacaine Hydrochloride Calculate the quantity, in ppm, of 2,6-dimethylaniline
in the Sample solution (alin). in the portion of Mepivacaine Hydrochloride taken:
Acceptance criteria: See Table 7.
Result = (ru/rs) x (Cs/Cu) x (Mn/M,2) x 106
Table 1
ty = peak response of 2,6-dimethylaniline from the
Relative Acceptance Sample solution
Retention Criteria, ls = peak response of 2,6-dimethylaniline from the
Name Time NMT (%) Standard solution
Bupivacaine related compound B G = concentration of USP Ropivacaine Related
(desmethyl mepivacaine) 0.4 0.15 CompoundA RS in the Standard solution
Mepivacaine 1.0 = (ug/ml) _ whe :
Picolinamide analog? 2A. 0.15 Cu = concentration of Mepivacaine Hydrochloride
Any individual unspecified impurity — 0.10 in the Sample solution (ug/mL)
Mn = eae weight of 2,6-dimethylaniline,
Total impurities pe 0.4 121.
4 N-(2,6-Dimethylpheny|)piperidine-2-carboxamide. Mz = molecular weight of 2,6-dimethylaniline
» N-(2,6-Dimethylphenyl)picolinamide. hydrochloride (ropivacaine related
© 2,6-DIMETHYLANILINE compound A), 157.64
Prepare the Standard solution and Sample solution fresh, Acceptance criteria: NMT 20 ppm
just before use. iS
SPECIFIC TESTS wn“
Standard stock solution: 0.6 g/mL of USP Ropiva- © Loss ON DRYING (731) i)
caine Related Compound A RS in 1 N hydrochloric acid. Analysis: Dry at 105° for 4 h.
[Note—Ropivacaine related compoundAis 2,6- =
dimethylaniline hydrochloride.]
Acceptance criteria: NMT 1.0% }
=}
Standard solution: Transfer 2.0 mL of the Standard ADDITIONAL REQUIREMENTS ey
stock solution and 1.0 mL of 3 N sodium hydroxide to a ¢ PACKAGING AND STORAGE: Preserve in well-closed @|
20-mL headspace vial, and immediately close the vial containers. »
with a cap. e USP REFERENCE STANDARDS (11) 3
Sample stock solution: 30 mg/mL of Mepivacaine Hy- USP Bupivacaine Related Compound B RS
SE
w
drochloride in 1 N hydrochloric acid PEGS OPENS Ertl eriainiese catmoxanicle,
Sample solution: Transfer 2.0 mL of the Sample stock Cy4H20N20 32.32
solution and 1.0 mL of 3 N sodium hydroxide to a USP Mepivacaine Hydrochloride RS
20-mL headspace vial, and immediately close the vial USP Ropivacaine Related Compound A RS
with a cap. ee hydrochloride.
Chromatographic system CsHiN-HCl 157.64
(See Chromatography (621), System Suitability.)
Mode: GC
Detector: Flame ionization
Column: 0.53-mm x 30-m capillary; coated with 3-um
film of G43
Temperatures
Mepivacaine Hydrochloride Injection
Injection port: 225°
Detector: 250° DEFINITION
Column: See Table 2. Mepivacaine Hydrochloride Injection is a sterile solution of
Mepivacaine Hydrochloride in Water for Injection. It con-
tains NLT 95.0% and NMT 105.0% of the labeled amount
Table 2 of mepivacaine hydrochloride (CisH22N20
- HCl).
Hold Time
IDENTIFICATION
Initial Temperature Final at Final
Temperature Ramp Temperature | Temperature
© A. IDENTIFICATION—ORGANIC NITROGENOUS BASES (181):
Meets the requirements
() (¢/min) (°) (min)
130 10 230 5
 2582 Mepivacaine / Official Monographs USP 41

° B. ADDITIONAL REQUIREMENTS
Analysis: Extract a volume of Injection, equivalent to e PACKAGING AND STORAGE: Preserve in single-dose or mul-
200 mg of mepivacaine, with two 10-mL portions of tiple-dose containers, preferably of Type | glass. Injection
ether, and discard the ether extracts. Render the re- labeled to contain 2% or less of mepivacaine hydrochlo-
maining solution slightly alkaline with sodium carbonate ride may be packaged in 50-mL multiple-dose containers.
TS, and extract the precipitate with ether. Evaporate
the ether extract on a steam bath to snes and dry
the residue under vacuum at 60° for 1 h. Change to read:
Acceptance criteria: The mepivacaine obtained melts
between 149° and 153°. ° USP REFERENCE STANDARDS (11)
@ (CN 1-May.2018)
ASSAY USP Mepivacaine Hydrochloride RS
¢ PROCEDURE
Buffer: 3.40 g/L of monobasic potassium phosphate
and 4.35 g/L of dibasic potassium phosphate in water.
Adjust with potassium hydroxide or phosphoric acid to
a pH of 6.3. Mepivacaine Hydrochloride and
Mobile phase: Acetonitrile and Buffer (35:65) Levonordefrin Injection
System suitability solution: 0.05 mg/mL ofsoe lba~
aben and 1.0 mg/mL of USP Mepivacaine Hydrochlo-
tide RS in Mobile phase DEFINITION
Standard solution: 1.0 mg/mL of USP Mepivacaine Hy- Mepivacaine Hydrochloride and Levonordefrin Injection is a
drochloride RS in Mobile phase sterile solution of Mepivacaine Hydrochloride and Levo-
Sample solution: Nominally 1 mg/mL of mepivacaine nordefrin in Water for Injection. It contains NLT 95.0%
hydrochloride from Injection in Mobile phase and NMT 105.0% of the labeled amount of mepivacaine
Chromatographic system hydrochloride (CisH22N2O - HCl) and NLT 90.0% and
(See Chromatography (621), System Suitability.) NMT 110.0% of the labeled amount of levonordefrin
Mode: LC (CoHi3NOs).
Detector: UV 263 nm IDENTIFICATION
Column: 4.6-mm x 25-cm; 5-um packing L1! cA.
Column temperature: 40° Analysis: Extract a volume of Injection, equivalent to
Flow rate: 1 mL/min 200 mg of mepivacaine, with two 10-mL portions of
Injection volume: 10 uL ether, and discard the ether extracts. Render slightly al-
System suitability kaline with sodium carbonate TS, extract the precipitate
Samples: System suitability solution and Standard with ether, evaporate the ether extract on a steam bath
solution ie anes and dry the residue under vacuum at 60°
[NoTte—The relative retention times for mepivacaine or 1h.
al and methylparaben are 1.0 and 1.4, respectively.] Acceptance criteria: The mepivacaine obtained melts
25 Suitability requirements
a Resolution: NLT 2.0 between methylparaben and
between 149° and 153°.
ic e B. IDENTIFICATION TESTS—GENERAL, Chloride (191): Meets
—
i) mepivacaine, System suitability solution the requirements
2} Capacity factor: NLT 1.0 for the mepivacaine peak,
i= System suitability solution ASSAY
Sj Tailing factor: NMT 2.0 for the mepivacaine peak, e MEPIVACAINE HYDROCHLORIDE
= System suitability solution Buffer: 3.40 g/L of monobasic potassium phosphate
[a5 Relative standard deviation: NMT 2.0%, Standard and 4.35 g/L of dibasic potassium phosphate in water.
“wn
> solution Adjust with potassium hydroxide or phosphoric acid to
Analysis a pH of 6.3.
Samples: Standard solution and Sample solution Mobile phase: Acetonitrile and Buffer (35:65)
Calculate the percentage of the labeled amount of System suitability solution: 0.05 mg/mL of methyspar:
mepivacaine hydrochloride (CisH22N2O - HCl) in the aben and 1.0 mg/mL of USP Mepivacaine Hydrochlo-
volume of Injection taken: ride RS in Mobile phase
Standard solution: 1.0 mg/mL of USP Mepivacaine Hy-
Result = (ru/rs) x (Cs/Cu) x 100 drochloride RS in Mobile phase
Sample solution: Nominally 1 mg/mL of mepivacaine
ru = peak response from the Sample solution nydrocitonide from Injection in Mobile phase
ls = peak response from the Standard solution Chromatographic system
Cs = concentration of USP Mepivacaine (See Chromatography (621), System Suitability.)
Hydrochloride RS in the Standard solution Mode: LC
(mg/mL) : Detector: UV 263 nm
Cu = nominal concentration of the Sample solution Column: 4.6-mm x 25-cm; 5-"m packing L1!
(mg/mL) Column temperature: 40°
Acceptance criteria: 95.0%-105.0% Flow rate: 1 mL/min
SPECIFIC TESTS
Injection volume: 10 uL
© PH (791): 4.5-6.8 System suitability
e BACTERIAL ENDOTOXINS TEST (85): It contains NMT 0.8 Samples: System suitability solution and Standard
USP Endotoxin Unit/mg of mepivacaine hydrochloride. solution
e OTHER REQUIREMENTS: It meets the requirements in Injec- [Note—The relative retention times for mepivacaine
tions and Implanted Drug Products (1).
and methylparaben are 1.0 and 1.4, respectively.]
Suitability requirements
1A Whatman Partisphere RTF C18 brand of L1 column has been shown to be Resolution: NLT 2.0 between methylparaben and
an appropriate column. mepivacaine, System suitability solution
1A Whatman Partisphere RTF C18 brand of L1 column has been shown to be
an appropriate column.
USP 41 Official Monographs / Meprednisone 2583

Capacity factor: NLT 1.0 for the mepivacaine peak,
System suitability solution
Tailing factor: NMT 2.0 for the mepivacaine peak,
System suitability solution
Relative standard deviation: NMT 2.0%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
mepivacaine hydrochloride (C;sH22N20 - HCI) in the
volume of Injection taken:
Result = (ru/rs) x (Cs/Cu) x 100
ty = peak response from the Sample solution
rs = peak response from the Standard solution
Cs = concentration of USP Mepivacaine
Hydrochloride RS in the Standard solution
© PH (791): 3.3-5.5
e BACTERIAL ENDOTOXINS TEST (85): It contains NMT 0.8
USP Endotoxin Unit/mg of mepivacaine hydrochloride.
© OTHER REQUIREMENTS: It meets the requirements in Injec-
tions and Implanted Drug Products (1).
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in single-dose or mul-
tiple-dose containers, preferably of Type | glass.
© LABELING: The label indicates that the Injection is not to
be used if its color is pinkish or darker than slightly yel-
low or if it contains a precipitate.
Change to read:
e UsP REFERENCE STANDARDS (11)
@ (CN I-May-2018)

(mg/mL) USP Levonordefrin RS

Cu = nominal concentration of mepivacaine USP Mepivacaine Hydrochloride RS
hydrochloride in the Sample solution
(mg/mL)
Acceptance criteria: 95.0%-105.0%
o LEVONORDEFRIN
Ferro-citrate solution and Buffer solution: Prepare as Meprednisone
directed in Epinephrine Assay (391).
Standard stock solution: With the aid of 20 mL of so-
dium bisulfite solution (1 in 50), transfer 25 mg of USP
Levonordefrin RS to a 50-mL volumetric flask, and dilute
with water to volume.
Standard solution: 50 tg/mL of USP Levonordefrin RS
in sodium bisulfite solution (1 in 500) from the Stan-
dard stock solution. Make the final dilution at the time
the Assay is to be carried out. Co2H2gOs 372.45
Sample solution: Nominally 50 g/mL of levonordefrin Pregna-1,4-diene-3,11,20-trione, 17,21-dihydroxy-
from Injection, diluting if necessary 16-methyl-, (168)-;
Analysis: Proceed as directed in Epinephrine Assay (391), 17,21-Dihydroxy-16B-methylpregna-1,4-diene-3,11,20-trione
except use levonordefrin wherever epinephrine [base] is
called for. When the Ferro-citrate solution and the Buffer
[1247-42-3]. e
4)
solution are mixed with the Sample solution, a fine pre- DEFINITION i>]
cipitate may be formed. Remove this precipitate by cen- Meprednisone contains NLT 97.5% and NMT 102.5% of cs
trifugation or by passing through dry filter paper before meprednisone (C22H2gOs), calculated on the dried basis. °
the colorimetric measurements are taken. 2
Calculate the percentage of the labeled amount of levo- IDENTIFICATION re}
nordefrin (CsH;3NO3) in the volume of Injection taken: e A. INFRARED ABSORPTION (197M) =
e B. ULTRAVIOLET ABSORPTION (197U) Ey)
Result = (Au/As) x (Cs/Cy) x 100 Analytical wavelength: 238 nm Bs}a
Standard solution: 10 g/mL of USP Meprednisone RS “

Au = absorbance of the Sample solution in methanol

As = absorbance of the Standard solution
Gs = concentration of USP Levonordefrin RS in the
Standard solution (ug/mL)
Cu = nominal concentration of levonordefrin in the
Sample solution (g/mL)
Acceptance criteria: 90.0%-110.0%
SPECIFIC TESTS
e COLOR AND CLARITY
Standard solution: Transfer 2.0 mL of 0.100 N iodine
VS to a 500-mL volumetric flask, and dilute with water
to volume.
Analysis
Samples: Standard solution and Sample solution
Visually examine a portion of the Injection (Sample so-
lution) in a suitable clear glass test tube against a
white background: it is not pinkish, and it contains
no precipitate. If any yellow color is observed in the
Sample solution, concomitantly determine the ab-
sorbances of the Sample solution and Standard solu-
tion in 1-cm cells with a suitable spectrophotometer
set at 460 nm.
Acceptance criteria: The absorbance of the Sample so-
lution does not exceed that of the Standard solution.
Sample solution: 10 g/mL in methanol
Acceptance criteria: Absorptivities, calculated on the
dried basis, do not differ by more than 3.0%.
° 6 + lala CHROMATOGRAPHIC IDENTIFICATION TEST
201
Diluent: Toluene and alcohol (1:1)
Sample solution: 20 mg/mL in Diluent
Chromatographic system
Spray reagent: 10% (v/v) sulfuric acid in alcohol
Analysis: Proceed as directed in the chapter. Locate the
spots on the plate by spraying with Spray reagent and
heating at 105° for 10 min.
Acceptance criteria: Meets the requirements
ASSAY
© PROCEDURE
Diluent: Alcohol and chloroform (1:1)
Standard solution: Prepare as directed in Single-Steroid
Assay (511), Standard Preparation using USP
Meprednisone RS.
Sample solution: 2 mg/mL of previously dried
Meprednisone in Diluent
Instrumental conditions
Mode: UV
Analytical wavelength: Maximum at about 238 nm
Cell: 11cm
 2584 Meprednisone / Official Monographs USP 41

Analysis
Samples: Standard solution and Sample solution
Proceed as directed in Single-Steroid Assay (511), Proce-
dureusing a solvent system consisting of chloroform,
methanol, and water (180:15:1), through the fourth
sentence of the second paragraph. Then centrifuge the
tubes for 5 min. Determine the absorbances of the
supernatants against a blank.
Calculate the perceniage of meprednisone (C22H2¢Os) in
the portion of Meprednisone taken:
Result = (Au/As) x (Cs/Cu) x 100
Au = absorbance of the Sample solution
As = absorbance of the Standard solution
G = concentration of USP Meprednisone RS in the
Standard solution (mg/mL)
Cy = concentration of the Sample solution (mg/mL)
Acceptance criteria: 97.5%-102.5% on the dried basis
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.1%
SPECIFIC TESTS
e Loss ON DRYING (731)
Analysis: Dry at 105° for 3 h.
Acceptance criteria: NMT 1.0%
e OPTICAL ROTATION, Specific Rotation (7815S)
Sample solution: 10mg/mL in dioxane
Acceptance criteria: +180° to +188°
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and avoid exposure to excessive heat.
e USP REFERENCE STANDARDS (11)
USP Meprednisone RS
ASSAY
e PROCEDURE
Mobile phase: Acetonitrile and water (30:70)
Standard solution: 5 mg/mL of USP Meprobamate RS
prepared as follows. Dissolve the Standard first in aceto-
nitrile using 30% of final volume. Sonicate if necessa\
to dissolve, and cool to room temperature. Dilute witl
water to volume.
Sample solution: 5 mg/mL of Meprobamate prepared
as follows. Dissolve the sample first in acetonitrile using
30% of final volume. Sonicate if necessary to dissolve,
and cool to room temperature. Dilute with water to
volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 200 nm
Column: 4.6-mm x 25-cm; 4-4m packing L1
Flow rate: 1 mL/min
Injection volume: 20 pL
Run time: 2 times the retention time of meprobamate
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of meprobamate (CsHisN2O.)
in the portion of Meprobamate taken:
Result = (ru/rs) x (Cs/Cy) x 100
tu = peak response from the Sample solution
Is peak response from the Standard solution
ou

Gs concentration of USP Meprobamate RS in the

Standard solution (mg/mL)
Cu = concentration of Meprobamate in the Sample
iS
”

a solution (mg/mL)
i] Acceptance criteria: 97.0%-101.0% on the dried basis
a Meprobamate
oa)
° IMPURITIES
i ¢ ORGANIC IMPURITIES: PROCEDURE 1
6 Standard solutions: Dissolve USP Meprobamate RS in
= alcohol, and mix to obtain Standard solution A with a
om known concentration of 1.0 mg/mL. Dilute quantita-
a) tively with alcohol to the obtain the Standard solutions
=| with the compositions given in Table 1.
CoHigN204 218.25
1,3-Propanediol, 2-methyl-2-propyl-, dicarbamate; Table 1
2-Methyl-2-propyl-1,3-propanediol dicarbamate [57-53-4]. Percentage
DEFINITION (%, for
Meprobamate contains NLT 97.0% and NMT 101.0% of Concentra- Comparison
meprobamate (CsHisN2O,), calculated on the dried basis. Standard tion with Sam-
Solution Dilution (mg RS/mL) ple)
IDENTIFICATION A (Undiluted) 1.0 1.0
e A. INFRARED ABSORPTION (197K) B (4 in 5) 0.8 0.8
Sample: 1 mg in 200 mg c (3 in 5) 0.6 0.6
Acceptance criteria: The IR absorption spectrum of a
potassium bromide dispersion of the Sample, previously D (2 in 5) 0.4 0.4
dried, exhibits maxima only at the same wavelengths as E (in 5) 0.2 0.2
that of a similar preparation of USP Meprobamate RS. If
a difference appears, dissolve portions of both the Sam- Sample solution: 100 mg/mL of Meprobamate in
ple and the Reference Standard in acetone at a concen- alcohol
tration of 8 mg/mL. Dilute 0.1-mL portions of the ace- Chromatographic system
tone solutions with 1 mL of n-heptane, and remove the (See Chromatography (621), Thin-Layer Chromato-
solvents by evaporation under nitrogen at a tempera- graphy.)
ture of 30°. Dry the residues under vacuum at room Mode: TLC
temperature for 30 min, and repeat the test on the Adsorbent: Thin-layer chromatographic plate coated
with a 0.25-mm layer of chromatographic silica gel
residues. Application volume: 2 uL
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as Developing solvent system: Hexane, acetone, and
pyridine (70:30:10)
obtained in the Assay. Spray reagent: 5 mg/mL of vanillin in a cooled mix-
ture of sulfuric acid and alcohol (80:20)
USP 41
Analysis
Samples: Standard solutions and Sample solution
Position the plate in a chromatographic chamber, and
develop the chromatograms in the Developing solvent
system until the solvent front has moved about three-
fourths of the length of the plate. Remove the plate
from the developing chamber, mark the solvent front,
and air-dry the plate for 15 min. Heat the plate at
100° for 15 min, cool, and spray with Spray reagent.
Heat the plate at 110° for 15-20 min, cool, and allow
the plate to develop blue-purple spots at room tem-
perature. [NoTE—Color development requires about
30-60 min.] Examine the plate, and compare the in-
tensities of any secondary spots of the Sample solution
with those of the principal spots of the Standard
solutions.
Acceptance criteria: No secondary spot of the Sample
solution is larger or more intense than the principal spot
of Standard solution A (1.0%), and the sum of the in-
tensities of all secondary spots of the Sample solution
corresponds to NMT 2.0%.
© ORGANIC IMPURITIES, PROCEDURE 2: LIMIT OF METHYL
CARBAMATE
Standard solution: 1.0 mg/mL of methyl carbamate
Sample solution: Transfer 1.0g of finely powdered Me-
probamate to a beaker, add 5.0 mL of water, and stir to
wet the powder completely. Filter the slurry through a
small plug of glass wool in the stem of a glass funnel.
Use the clear filtrate.
Mobile phase: Water
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 200 nm
Column: 3.9-4.6-mm x 25-30-cm; packing L1
Flow rate: 1 mL/min
Injection volume: 50 uL
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Acceptance criteria: The peak response of the Sample
solution is not greater than that of the Standard solution,
corresponding to NMT 0.5% of methyl carbamate.
SPECIFIC TESTS
e Loss ON DRYING (731)
Analysis: Dry a sample under vacuum at 60° for 3 h.
Acceptance criteria: NMT 0.5%
© MELTING RANGE OR TEMPERATURE (741): 103°-107°, but
ue fanige between the beginning and end of melting is
MT 2°.
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers.
e USP REFERENCE STANDARDS (11)
USP Meprobamate RS
Meprobamate Oral Suspension
DEFINITION
Meprobamate Oral Suspension contains NLT 95.0% and
NMT 110.0% of the labeled amount of meprobamate
(CoHigN20,).
Official Monographs / Meprobamate 2585
IDENTIFICATION
cA.
Sample solution: 2 mL of Oral Suspension
Analysis: Mix the Sample solution with 2 mL of acetone
and 2 mL of furfural in glacial acetic acid (1 in 100),
add 5 mL of hydrochloric acid, and shake.
Acceptance criteria: A purer color is produced, and,
on standing, it changes to blue, then to blue-black, and
finally to black-brown.
ASSAY
© PROCEDURE
Sample solution: Transfer an equivalent to 400 mg of
meprobamate from Oral Suspension to a separator, and
completely extract the meprobamate with 20-mL por-
tions of chloroform, filtering the extracts through a
pee of cotton enclosed in glass wool that previously
as been moistened with chloroform. Collect the filtrate
in a conical flask, add several glass beads to the flask,
and evaporate on a steam bath to dryness. To the resi-
due add 20 mL of water, heat on a steam bath for sev-
eral min, then add 40 mL of hydrochloric acid, and re-
flux for 90 min. Remove the condenser, and continue
boiling until the volume is reduced to about 20 mL.
Cool to room temperature, add 50 mL of water, and
cool in an ice bath. Add 1 drop of methyl red TS, and,
while cooling continuously, cautiously neutralize with
sodium hydroxide solution (4 in 10) until the indicator
begins to change color. Add hydrochloric acid, if neces-
sary, to restore the pink color, then carefully neutralize
with 0.1 N sodium hydroxide VS. Add 30 mL of neutral
formaldehyde solution (18% w/w).
Analysis: Titrate with 0.1 N sodium hydroxide VS until
the solution becomes yellow. Add 0.2 mL of phenol-
phthalein TS, and continue the titration with 0.1 N so-
dium hydroxide VS to a distinct pink color. Perform a
blank determination. Each mL of the total volume of
0.1 N sodium hydroxide consumed after the addition of =
the formaldehyde solution is equivalent to 10.91 mg of nn
meprobamate (CoHigN2O,). a)
Acceptance criteria: 95.0%-110.0% =
fo}
PERFORMANCE TESTS =
e UNIFORMITY OF DosaGeE UNITS (905): Meets the require- °
ments for oral suspension packaged in single-unit =}
containers iy
mo}
e DELIVERABLE VOLUME (698): Meets the requirements for Pz.
oral suspension packaged in multiple-unit containers “
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers.
Meprobamate Tablets
DEFINITION
Meprobamate Tablets contain NLT 90.0% and NMT 110.0%
of the labeled amount of meprobamate (CsHisN20,).
IDENTIFICATION
o A. INFRARED ABSORPTION (197K)
Sample: A portion of finely powdered Tablets, equiva-
lent to 800 mg of meprobamate
Analysis: To the Sample add 5 mL of dehydrated alco-
hol, and heat to just below boiling for about 5 min,
with occasional swirling. Cool, and filter into 15 mL of
solvent hexane. With the aid of suction, filter the crys-
tals that form, and dry at 60°.
Acceptance criteria: The IR absorption spectrum of a
potassium bromide dispersion (about 1 mg in 200 mg)
from a portion of crystals obtained from the Sample ex-
hibits maxima only at the same wavelengths as that of
a similar preparation of USP Meprobamate RS. If a dif-
 2586 Meprobamate / Official Monographs USP 41

ference appears, dissolve portions of both the Sample Acceptance criteria: 90.0%-110.0%
and the Reference Standard in acetone at a concentra-
tion of 8 mg/mL. Dilute 0.1-mL portions of the acetone PERFORMANCE TESTS
solutions with 1 mL of n-heptane, and remove the sol- ¢ DISSOLUTION (711)
vents by evaporation under nitrogen at a temperature Procedure for a pooled sample
of about 30°. Dry the residues under vacuum at room Medium: Deaerated water; 900 mL
temperature for 30 min, and repeat the test on the Apparatus 1: 100 rpm
residues. Time: 30 min
e B. The retention time of the major peak of the Sample Standard solution, System suitability solution, Chro-
solution corresponds to that of the Standard solution, as matographic system, and System suitability: Pro-
obtained in the Assay. ceed as directed in the Assay.
Analysis
ASSAY Calculate the percentage of the labeled amount of me-
e PROCEDURE probamate (C9HigN2O.) dissolved:
Mobile phase: Acetonitrile and water (30:70)
Phenacetin stock solution: 125 t1g/mL of phenacetin in Result = (ru/rs) x Cs x Vx (1/L) x 100
acetonitrile
Phenacetin solution: 25 g/mL of phenacetin prepared tu = peak response from the Sample solution
as follows from the Phenacetin stock solution. Pipet a rs = peak response from the Standard solution
suitable volume of Phenacetin stock solution into a volu- Cs = concentration of USP Meprobamate RS in the
metric flask. Add acetonitrile to fill 30% of the final flask Standard solution (mg/mL)
volume, and dilute with water to volume. Vv = volume of the Medium, 900 mL
Standard solution: 5 mg/mL of USP Meprobamate RS L = label claim (mg/Tablet)
prepared as follows. Transfer a suitable amount of the Acceptance criteria: NLT 75% (Q) of the labeled
Reference Standard to a suitable volumetric flask. Dis- amount of meprobamate (CsHigN2O,) is dissolved.
solve in 30% of the final flask volume of acetonitrile, e UNIFORMITY OF DOSAGE UNITS (905): Meet the
and dilute with water to volume. requirements
System suitability solution: 5 mg/mL of USP Meproba-
mate RS and 5 g/mL of phenacetin prepared as fol- ADDITIONAL REQUIREMENTS
lows. Dissolve a weighed amount of USP Meprobamate © PACKAGING AND STORAGE: Preserve in well-closed
RS, first in acetonitrile, using 20% final volume. Shake containers.
to dissolve. Add a suitable volume of Phenacetin solu- e USP REFERENCE STANDARDS (11)
tion, and dilute with water to volume. USP Meprobamate RS
Sample solution: Nominally equivalent to 5 mg/mL of
meprobamate prepared as follows. Transfer an amount
of meprobamate from a portion of finely powdered
Tablets (NLT 20) to a suitable volumetric flask. Add ace-
ww tonitrile to fill 30% of final volume, and shake to dis- Meradimate
<=
Se solve. Dilute with water to volume, and filter, discarding
the first 10 mL of the filtrate.
i did
—
i=) Chromatographic system
}
=
(See Chromatography (621), System Suitability.) LO
Sj Mode: LC
( .
> °o |

Detector: UV 200 nm
= Column: 3.9-4.6-mm x 25-30-cm; 5-um packing L1
~~ NH, HC” CH,

Pw Flow rate: 1 mL/min

”“
Injection volume: 20 uL Ci7H2sNOz 275;39
2 Cyclohexanol, 5-methyl-2-(1-methylethyl)-,
System suitability
Samples: System suitability solution and Standard 2-aminobenzoate;
solution Anthranilic acid, p-menth-3-yl ester [134-09-8].
[Note—The relative retention times for meprobamate
and phenacetin are about 0.7 and 1.0, respectively.] DEFINITION
Suitability requirements Meradimate contains NLT 95.0% and NMT 105.0% of mer-
adimate (Ci7H2sNOz).
Resolution: NLT 2.0 between the meprobamate and
the phenacetin peaks, System suitability solution IDENTIFICATION
Relative standard deviation: NMT 2.0%, Standard e A. INFRARED ABSORPTION (197F)
solution e B. ULTRAVIOLET ABSORPTION (197U)
Analysis Sample solution: 5 tug/mL in alcohol
Samples: Standard solution and Sample solution Acceptance criteria: Meets the requirements
Calculate the percentage of the labeled amount of me- eC, The retention time of the major peak of the Sample
probamate (CyHisN20,) in the portion of Tablets solution corresponds to that of the Standard solution, as
taken: obtained in the Assay.
Result = (ru/rs) * (Cs/Cu) x 100 ASSAY
e PROCEDURE
ru = peak response of meprobamate from the Standard solution: 20.0 mg/mL of USP Meradimate RS
Sample solution in tert-butyl methyl ether
Is = peak response of meprobamate from the Sample solution: 20 mg/mL of Meradimate in tert-bu-
Standard solution tyl methyl ether
Cs = concentration of USP Meprobamate RS in the Ehromateeraphte system
Standard solution (mg/mL) (See Chromatography (621), System Suitability.)
Cu = nominal concentration of meprobamate in the Mode: GC
Sample solution (mg/mL) Detector: Flame ionization
Column: 0.32-mm x 25-m; coated with a 0.1-um film
of G1
USP 41 Official Monographs / Mercaptopurine 2587

Temperatures © OPTICAL ROTATION, Specific Rotation (781S): —4° to +4°

Injector: 240° Sample solution: 10 mg/mL in alcohol
Detector: 260° e REFRACTIVE INDEX (831): 1.540-1.544 at 20°
Column: See Table 1.
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers.
Table 1 e USP REFERENCE STANDARDS (11)
Tempera- Hold Time at USP Meradimate RS
Initial ture Final Final
Temperature Ramp Temperature | Temperature
(@) (/min)_ «) (min)
60. 8 240 10
Mercaptopurine
Carrier gas: Helium
Flow rate: 6 mL/min
Injection type: Split ratio, 30:1 J.
[Nott—The split ratio can be modified to optimize
LP
HN Y + HO.
performance.]
Injection volume: 1 wL
System suitability
Sample: Standard solution CsHaNaS - H20 170.19
Suitability requirements CsH4NaS 152.18
Relative standard deviation: NMT 2.0% 6H-Purine-6-thione, 1,7-dihydro-, monohydrate;
Analysis Purine-6-thiol monohydrate [6112-76-1].
Samples: Standard solution and Sample solution Anhydrous [50-44-2].
Calculate the percentage of meradimate (Ci7H2sNOz) in
the portion of Meradimate taken: DEFINITION
Mercaptopurine contains NLT 97.0% and NMT 102.0% of
Result = (ru/rs) x (Cs/Cu) x 100 pie eapagulis (CsH4N,S), calculated on the anhydrous
asis.
ru = peak response from the Sample solution
rs = peak response from the Standard solution IDENTIFICATION
Gs = concentration of USP Meradimate RS in the © A. INFRARED ABSORPTION (197K)
Standard solution (mg/mL) e B. The retention time of the major peak of the Sample
Cu = concentration of the Sample solution (mg/mL) solution corresponds to that of the Standard solution, as
Acceptance criteria: 95.0%-105.0% obtained in the Assay.
IMPURITIES ASSAY
© ORGANIC IMPURITIES e PROCEDURE =
4)
Standard solution, Sample solution, Chromatographic Solution A: 0.77 g/L of ammonium acetate in water i)
system, and System suitability: Proceed as directed in Mobile phase: Methanol and Solution A (25:75)
the Assay. Ee
Standard stock solution: 0.2 mg/mL of USP Mer- re}
Analysis captopurine RS in a mixture of methanol and water 3
Sample: Sample solution (1:1). Transfer USP Mercaptopurine RS into a suitable o)
Calculate the percentage of each impurity in the por- volumetric flask, and add methanol equivalent to 50% to)=
tion of Meradimate taken: of the final volume. Shake mechanically to dissolve, and ey
dilute with water to volume. 3
Result = (ru/r7) x 100
my
Standard solution: 0.02 mg/mL of USP Mercaptopurine “

RS in Mobile phase from the Standard stock solution

tu = peak response of each impurity Sample stock solution: Transfer 25 mg of Mer-
I = sum of all the peak responses captopurine into a 50-mL volumetric flask. Add 25 mL
Acceptance criteria of methanol, shake mechanically for at least 45 min,
Any individual impurity: NMT 0.5% and dilute with water to volume. Transfer 20 mL of this
Total impurities: NMT 2.0% solution into a 25-mL volumetric flask, and dilute with
Mobile phase to volume.
SPECIFIC TESTS Sample solution: 0.02 mg/mL of Mercaptopurine in
e ACIDITY Mobile phase from the Sample stock solution
Sample: 5.0 mL of Meradimate Chromatographic system
Titrimetric system
(See Chromatography (621), System Suitability.)
Mode: Direct titration Mode: LC
Titrant: 0.1 N sodium hydroxide VS Detector: UV 325 nm
Endpoint detection: Visual Column: 4.6-mm x 15-cm; 5-um packing L68
Analysis: Transfer 50 mL of alcohol to a suitable con- Flow rate: 1.0 mL/min
tainer, add 1 mL of phenolphthalein TS, and add suffi- Injection volume: 20 uL
cient volume of Titrant to obtain a persistent pink color. System suitability
Transfer 50 mL of this solution to a suitable container,
Sample: Standard solution
add the Sample, and titrate with Titrant. Suitability requirements
Acceptance criteria: NMT 0.2 mL of Titrant per mL of Tailing factor: NMT 2.0
Meradimate is necessary. Relative standard deviation: NMT 0.73%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of mercaptopurine (CsH4N,S)
in the portion of Mercaptopurine taken:
Result = (ru/rs) x (Cs/Cu) x 100
 2588 Mercaptopurine / Official Monographs USP 41

ru = peak response from the Sample solution Cu = concentration of Mercaptopurine in the

Is = peak response from the Standard solution Sample solution (mg/mL)
Cs = concentration of USP Mercaptopurine RS in F = relative response factor for each individual
the Standard solution (mg/mL) impurity (see Table 2)
Cu = concentration of Mercaptopurine in the Acceptance criteria: See Table 2. Disregard any impu-
Sample solution (mg/mL) rity peak less than 0.05%.
Acceptance criteria: 97,0%-102.0% on the anhydrous
basis Table 2
IMPURITIES Relative Relative | Acceptance
e RESIDUE ON IGNITION (281): NMT 0.1% Retention | Response Criteria,
© ORGANIC IMPURITIES Name Time Factor NMT (%)
Solution A: 0.1% (v/v) Formic acid in water Didanosine related
poeon B: yt ae ie A (2:98) compound A= 0.54 63 0.15
Solution C: Methanol and Solution A (1:1) i ial f
Mobile phase: See Table 1. Mercaptopurine 1.09
Mercaptopurine
disulfide> 2.90 4.4 0.15
Table 1 Any unspecified
Solution B Solution C impurity _ 1.0 0.10
Total impurities = = 0.5
1 2 Hypoxanthine.
8 1 »1,2-Di(9H-purin-6-yl)disulfane.

a SPECIFIC TESTS
25 e PHOSPHORUS
27: 100 Standard phosphate solution: 43.96 ug/ml of dried
1 monobasic potassium phosphate (equivalent to 10 ug
of phosphorus)
Standard stock solution: 0.06 mg/mL of USP Mer- Standard solution: Transfer 2 mL of Standard phosphate
captopurine RS in Solution A. [NoTE—Use methanol solution to a 25-mL volumetric flask. Add 1 mL of 15 N
equivalent to 2.5% of the final volume to help dissolve.] sulfuric acid, 0.5 mL of nitric acid, 0.75 mL of ammo-
Standard solution: 1.2 g/mL of USP Mercaptopurine nium molybdate TS, and 1 mL of aminonaphtholsul-
RS in Solution B from the Standard stock solution fonic acid TS, then dilute with water to volume, and
Sensitivity solution: 0.06 g/mL of USP Mer- mix. Allow to stand for 5 min.
captopurine RS in Solution B from the Standard solution Sample solution: Digest 200 mg with 2 mL of 15 N sul-
Sample solution: 0.12 mg/mL of Mercaptopurine in So- furic acid in a large test tube, periodically adding nitric
lution A. [NoTe—Inject the Sample solution within 1 h of acid, dropwise and with caution. Continue heating until
aS
”"
preparation.] practically all of the liquid has evaporated and the resi-
a Chromatographic system due is colorless. Transfer the residue, with the aid of
4
i]
(See Chromatography (621), System Suitability.) small portions of water, to a 25-mL volumetric flask.
oa) Mode: LC Add 1 mL of 15N sulfuric acid, 0.5 mL of nitric acid,
o)
= Detector: UV 260 nm 0.75 mL of ammonium molybdate TS, and 1 mL of ami-
C) Column: 4.6-mm x 10-cm; 3-um packing L1 nonaphtholsulfonic acid TS, then dilute with water to
> Temperatures volume. Allow to stand for 5 min.
a Column: 30° Blank: Transfer 2 mL of 15 N sulfuric acid to a large test
2] Sample: 4° tube, periodically adding nitric acid, pres and with
=) Flow rate: 1.0 mL/min caution. Continue heating unti! practically all of the liq-
Injection volume: 50 pL uid has evaporated and the residue is colorless, Transfer
System suitability the residue, with the aid of small portions of water, to a
Samples: Standard solution and Sensitivity solution 25-mL volumetric flask. Add 1 mL of 15.N sulfuric acid,
Suitability requirements 0.5 mL of nitric acid, 0.75 mL of ammonium molybdate
Tailing factor: NMT 2.0, Standard solution TS, and 1 mL of aminonaphtholsulfonic acid TS, then
Signal-to-noise ratio: NLT 10, Sensitivity solution dilute with water to volume. Allow to stand for 5 min.
Relative standard deviation: NMT 2.0%, Standard Instrumental conditions
solution (See Ultraviolet-Visible Spectroscopy (857).)
Analysis Mode: UV-Vis
Samples: Standard solution and Sample solution Analytical wavelength: 750 nm
Calculate the percentage of each impurity in the por- Analysis
tion of Mercaptopurine taken: Samples: Standard solution, Sample solution, and Blank
Acceptance criteria: The absorbance of the Sample so-
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100 lution is NMT that of the Standard solution (NMT
100 BPI).
ty = peak response of each impurity from the © WATER TERMINATION, Method | (921)
Sample solution Medium: 30 mL of methanol and 5g of salicylic acid in
rs = peak response of mercaptopurine from the the titration vessel
Standard solution
Cs = concentration of USP Mercaptopurine RS in
the Standard solution (mg/mL)
USP 41 Official Monographs / Mercaptopurine 2589

Sample: 0.3 g of Mercaptopurine Cs = concentration of USP Mercaptopurine RS in

Acceptance criteria: NMT 12.0% the Standard solution (mg/mL)
Gu = nominal concentration of mercaptopurine in
ADDITIONAL REQUIREMENTS the Sample solution (mg/mL)
e PACKAGING AND STORAGE: Preserve in tight containers, Mn = molecular weight of mercaptopurine, 170.19
protected from light. Store at room temperature. Mz = molecular weight of anhydrous
e USP REFERENCE STANDARDS (11) mercaptopurine, 152.18
USP Mercaptopurine RS Acceptance criteria: 93.0%-110.0%
PERFORMANCE TESTS
e DISSOLUTION (711)
Test 1
Mercaptopurine Tablets Medium: 0.1 N hydrochloric acid; 900 mL
Apparatus 2: 50 rpm
DEFINITION Time: 60 min
Mercaptopurine Tablets contain NLT 93.0% and NMT Mobile phase: 0.1% acetic acid in water
110.0% of the labeled amount of mercaptopurine Standard solution: USP Mercaptopurine RS in Medium
(CsH4NaS + H20). Sample solution: Pass a portion of the solution under
test through a suitable filter. Dilute with Medium to a
IDENTIFICATION concentration that is similar to the Standard solution, if
e A. The UV absorption spectrum exhibits a maximum at necessary.
325 +2 nm, and the ratio Azss/A3zs does not exceed Chromatographic system
0.09. (See Chromatography (621), System Suitability.)
Sample: 5 g/mL of mercaptopurine in a mixture of Mode: LC
methanol and water (1:1), from the Sample solution in Detector: UV 230 nm
the Assay Column: 3.9-mm x 15-cm; packing L1
e B. The retention time of the major peak of the Sample Flow rate: 2.5 mL/min
solution corresponds to that of the Standard solution, as Injection size: 10 uL
obtained in the Assay. System suitability
Sample: Standard solution
ASSAY ee retention time for mercaptopurine is NLT 4
© PROCEDURE min.
Solution A: 0.77 g/L of ammonium acetate in water Suitability requirements
Solution B: Methanol and Solution A (5:95) Relative standard deviation: NMT 2.0%
Solution C: Methanol and Solution A (30:70) Analysis
Mobile phase: Solution B and Solution C (80:20) Samples: Standard solution and Sample solution
Diluent: Methanol and water (1:1) Tolerances: NLT 80% (Q) of the labeled amount of
Standard solution: 0.25 mg/mL of USP Mercaptopurine CsH4NgS - H20 is dissolved. im
RS in a mixture of methanol and water (1:1). Transfer Test 2: If the product complies with this test, the label- wv
USP Mercaptopurine RS into a suitable volumetric flask, ing indicates that it meets USP Dissolution Test 2. i]
and add methanol equivalent to 50% of the final vol- Medium, Apparatus 2, Chromatographic system, and =
ume. Shake mechanically to dissolve, and dilute with Analysis: Proceed as directed for Test 7. °
water to volume. Time: 120 min p=}
Sample stock solution: 0.5 mg/mL of mercaptopurine fe)
Tolerances NLT 80% (Q) of the labeled amount of
in Diluent from NLT 5 Tablets. Place the Tablets into a CsHa4NaS - H20 is dissolved. =
Ne}
a
suitable volumetric flask, add methanol equivalent to e UNIFORMITY OF DOSAGE UNITS (905): Meets the se}
50% of the final volume, and shake mechanically for a requirements Zs
minimum of 30 min. Dilute with water to volume. Pass as

through a PVDF filter of 0.45-um pore size, and discard IMPURITIES

the first 3 mL of filtrate. Organic Impurities
Sample solution: 0.25 mg/mL of mercaptopurine in © PROCEDURE
Diluent from the Sample stock solution Solution A: 0.1% (v/v) formic acid in water
Chromatographic system Solution B: Methanol and Solution A (2:98)
(See Chromatography (621), System Suitability.) Solution C: Methanol and Solution A (1:1)
Mode: LC Mobile phase: See Table 7.
Detector: UV 260 nm
Column: 4.6-mm x 10-cm; 3-um packing L1 Table 1
Column temperature: 30°
Flow rate: 1.0 mL/min Solution B Solution C
Injection size: 10 uL %
System suitability 1
Sample: Standard solution
Suitability requirements 0
Tailing factor: NMT 2.0 0
Relative standard deviation: NMT 2.0%
Analysis 27 1
Samples: Standard solution and Sample solution 100
Calculate the percentage of mercaptopurine (CsH4NaS
-
H20) in the portion of Tablets taken: Standard stock solution: 0.06 mg/mL of USP Mer-
captopurine RS in Solution A. [NoTE—Use methanol
Result = (ru/rs) x (Cs/Cu) x (Mn/M,2) x 100 equivalent to 2.5% of the final volume to help
dissolve.]
tu = peak response from the Sample solution Standard solution: 1.2 j1g/mL of USP Mercaptopurine
rs = peak response from the Standard solution RS in Solution B from the Standard stock solution
 2590 Mercaptopurine / Official Monographs USP 41

Sensitivity solution: 0.06 g/mL of USP Mer- e LABELING: When more than one Dissolution test is given,
captopurine RS in Solution B from the Standard solution the labeling states the Dissolution test used only if Test 7
Sample stock solution: 0,5 mg/mL of mercaptopurine is not used.
in a mixture of methanol and Solution A (1:9) from NLT e USP REFERENCE STANDARDS (11)
5 Tablets. Place the Tablets into a suitable volumetric USP Mercaptopurine RS
flask, add methanol equivalent to 10% of the final vol-
ume, and shake mechanically for a minimum of 30
min. Dilute with Solution A to volume. Pass through a
PVDF filter of 0.45-1m pore size, and discard the first
3 mL of filtrate. . Ammoniated Mercury
Sample solution: 0.12 men of mercaptopurine in
Solution A. Transfer 6.0 mL of the Sample stock solution
into a 25-ml volumetric flask, and dilute with solutin — NONHICIae gn 4-ap.8) aneh
A to volume. Pass through a PVDF filter of 0.45-um y .
pore size, and discard the first 5 mL of filtrate. [NoTE— DEFINITION
Inject the aan solution within 1 h of preparation.] Ammoniated Mercury contains NLT 98.0% and NMT
Chromatographic system Sen 57 100.5% of ammoniated mercury [Hg(NH2)CI].
(See Chromatography (621), System Suitability.)
Mode: LC IDENTIFICATION
Detector: UV 260 nm oA.
Column: 4.6-mm x 10-cm; 3-um packing L1 Sample: 0.1g
Temperature Analysis: Place the Sample in a cold solution of 1 g of
Column: 30° sodium thiosulfate in 2 mL of water.
Sample: 4° Acceptance criteria: The Sample is soluble, with the
Flow rate: 1.0 mL/min evolution of ammonia. When this solution is heated
Injection size: 50 uL gently, a rust-colored mixture is formed, from which a
System suitability red precipitate is obtained on centrifugation. If the solu-
Samples: Standard solution and Sensitivity solution tion is strongly heated, a black mixture forms.
Suitability requirements ° B.
Tailing factor: NMT 2.0, Standard solution Sample: A suitable quantity
Signal-to-noise ratio: NLT 10, Sensitivity solution Analysis: Heat the Sample with 1 N sodium hydroxide.
Relative standard deviation: NMT 2.0%, Standard Acceptance criteria: The solution becomes yellow, and
solution ammonia is evolved.
Analysis eC.
Samples: Standard solution and Sample solution says solution: A suitable quantity in warm acetic
Calculate the percentage of each impurity in the por- aci
tion of Tablets taken: Analysis: Sample solution with potassium iodide TS
4 Acceptance criteria: The solution yields a red precipi-
om Result = (ru/'s) x (Cs/Cu) x (1/F) x 100 tate that is soluble in an excess of the reagent. The
A . . solution yields a white precipitate with silver nitrate TS.
D> i = peak response of each impurity from the
fo} Sample solution ASSAY
£ rs = peak response of mercaptopurine from the e PROCEDURE
eS Standard solution Sample solution: Mix 0.25 g of Ammoniated Mercury
Gs = concentration of USP Mercaptopurine RS in with 10 mL of water. Add 3g of potassium iodide, mix
aj the Standard solution (mg/mL) occasionally until dissolved, add about 40 mL of water,
S Cu = nominal concentration of mercaptopurine in and add methyl red TS.
the Sample solution (mg/mL) Analysis: Titrate with 0.1 N hydrochloric acid VS. Per-
F = relative response factor for each individual form a blank determination, and make any necessary
impurity (see Table 2) correction. Each mL of 0.1 N hydrochloric acid is equiv-
Acceptance criteria alent to 12.60 mg of ammoniated mercury
Individual impurities: See Table 2. [NoTe—Disregard [Hg(NHz2)Cl].
any impurity peak less than 0.05%.] Acceptance criteria: 98.0%-100.5%
Table 2 IMPURITIES
— e RESIDUE ON IGNITION (281): NMT 0.2%
Relative Relative Acceptance e MEeRCUROUS COMPOUNDS
Retention Response Criteria, Sample: 2.5g
Name Time Factor NMT (%) Analysis: Dissolve the Sample in 25 mL of warm hydro-
Didanosine related chloric acid. Pass througha tared filtering crucible,
compound Ae 0.54 63 0.3 wash withwater, and dry at 60° to constant weight. _
Mercaptopurine 1.00 _ _ Acceptance criteria: NMT 0.2%; the weight of the resi-
M : due does not exceed 5 mg.
jercaptopurine
disulfide? 2.90 44 0.4 ADDITIONAL REQUIREMENTS
Any unspecified a e PACKAGING AND STORAGE: Preserve in well-closed, light-
impurity 1.0 0.2 resistant containers.
Total impurities a = 0.6
? Hypoxanthine.
» 1,2-Di(9H-purin-6-yl)disulfane.

ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed
containers.
USP 41
Meropenem
Naya N-CH,,
us 3°.
ae 4 ye
HO oH Hf “cH,
Ci7H2sN30sS - 3H20 437.51
1-Azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, 3-[[5-
[(dimethylamino)carbonyl]-3-pyrrolidinyl]thio]-
6-(1-hydroxyethyl)-4-methyl-7-oxo, trihydrate, [4R-[3(35*,
55*) 401,58, 6B(R*)]I-
(4R,5S,6S)-3-[[(35,5S)-5-(Dimethylcarbamoyl)-
3-pyrrolidin Ithiol-¢-[(1 fl hydroxyen 1]-4-methyl-
7-0xo-1-azabicyclo[3.2.0]hept-2-ené-carboxylic acid, trihy-
drate [119478-56-7].
Anhydrous 383.47 [96036-03-2].
» Meropenem contains not less than 98.0 per-
cent and not more than 101.0 percent of
Ci7H2sN30sS, calculated on the anhydrous basis.
Packaging and storage—Preserve in tight containers.
Store the dry powder at controlled room temperature.
Labeling—Where it is intended for use in preparing inject-
able dosage forms, the label states that it is sterile or must
besubjected to further processing during the preparation of
injectable dosage forms.
Change to read:
UsP Reference standards (11)—
@ (CN 1-Meay-2018)
USP Meropenem RS
Identification—
A: Infrared Absorption (197K).
B: Ultraviolet Absorption (197U)—
Solution: 30g per mL.
Medium: water.
Specific rotation (781): between -17° and —21°, meas-
ured at 20°.
Test solution: 5 mg per mL, in water.
PH (791): between 4.0 and 6.0, in a solution (1 in 100).
Water Determination, Method Ic (921): between 11.4%
and 13.4%.
Residue on ignition (281): not more than 0.1%, igniting
at 500 + 50°, instead of at 800 + 25°. Use a desiccator con-
taining silica gel.
Delete the following:
°Heavy metals—
Sodium sulfide reagent—Dissolve 5 g of sodium sulfide in
a mixture of 10 mL of water and 30 mL of glycerin. Preserve
in well-filled, light-resistant bottles, and use within 3
months.
Test solutien—Transfer 1.0 g of Meropenem to a quartz or
porcelain crucible, cover loosely with a lid, and carbonize by
gentle ignition. After cooling, add 2 mL of nitric acid and
5 drops of sulfuric acid, heat cautiously until white fumes
evolve, and incinerate by ignition at 500° to 600°. Cool,
add 2 mL of hydrochloric acid, and evaporate on a water
bath to dryness. Moisten the residue with 3 drops of hydro-
chloric acid, add 10 mL of hot water, and warm for 2 min-
utes. Add 1 drop of phenolphthalein TS, add ammonia TS,
dropwise, until the solution develops a pale red color, and
add 2 mL of 1 N acetic acid. Filter, if necessary, to obtain a
Official Monographs / Meropenem 2591
clear solution, washing the filter with 10 mL of water. Trans-
fer the filtrate and the Wasi to a 50-mL color-compari-
son tube, and add water to obtain a volume of 50 mL.
Standard solution—tvaporate a mixture of 2 mL of nitric
acid, 5 drops of sulfuric acid, and 2 mL of hydrochloric acid
on a water bath, further evaporate to dryness on a hot sand
bath, and moisten the residue with 3 drops of hydrochloric
acid. Proceed as directed for Test solution, beginning with
“add 10 mL of hot water,” except add water to obtain a
volume of 49 mL. Add 1.0 mLof Standard Lead Solution (see
Heavy Metals (231)).
Procedure—To the tubes containing the Test solution and
the Standard solution, add 1 drop of Sodium sulfide reagent,
mix, and allow to stand for 5 minutes. The color in the tube
containing the Test solution is not darker than the color in
the tube containing the Standard solution (0.001%). cotfical1-
Jan-2018)
Limit of acetone—
Internal standard solution—Prepare a solution in dimethyl-
formamide containing 0.05 Ll of ethy! acetate per mL.
Standard solution—Transfer about 50 mg of acetone, ac-
curately weighed, to a 100-mL volumetric flask, dilute with
dimethylformamide to volume, and mix. To 1.0 mL of this
solution, add 10.0 mL of the /nternal standard solution, and
mix.
Test solution—Dissolve 100 mg of Meropenem, accurately
weighed, in 0.2 mL of dimethylformamide and 2.0 mL of
Internal standard solution.
Chromatographic system (see Chromatography (621))—The
gas chromatograph is equipped with a flame-ionization de-
tector and a 3-mm x 2-m column that contains support $2
and is maintained at a constant temperature of about 150°.
The injection port temperature is maintained at about 170°.
Nitrogen is the carrier gas, with the flow rate adjusted so
that the retention time for acetone is about 3 minutes.
Procedure—Separately inject equal volumes (about 2 uL) jo
of the Standard solution and the Test solution into the chro- a)
matograph, record the chromatograms, and measure the me]
peak responses for the acetone peak and the internal stan- x
dard peak. Calculate the percentage of acetone in the por- °
tion of Meropenem taken by the formula: |
fo)
Xe}
(Wal/5Wu)(Ru / Rs) a
ES}
mo]
in which W, is the weight, in mg, of acetone in the Stan- 7
dard solution; Wy is the quantity, in mg, of Meropenem in a
the Test solution; and Ry and Rs are the peak area ratios of
acetone to the internal standard obtained from the Test So-
lution and the Standard solution, respectively. Not more than
0.05% is found.
Chromatographic purity—
Diluted phosphoric acid—Dilute 10 mL of phosphoric acid
with water to make 100 mL of solution.
Solvent—Transfer 1.0 mL of triethylamine to a 1000-mL
volumetric flask containing 900 mL of water. Adjust with Di-
luted phosphoric acid to a pH of 5.0 + 0.1, dilute with water
to volume, and mix.
Mobile phase—Transfer 1.0 mL of triethylamine to a
1000-mL volumetric flask containing 900 mL of water. Ad-
just with Diluted phosphoric acid to a pH of 5.0 + 0.1, dilute
with water to volume, and mix. Mix this solution with
70 mL of acetonitrile. Make adjustments if necessary (see
System Suitability under Chromatography (621)).
Standard solution—Prepare a solution of USP Meropenem
RS in Solvent having a known concentration of about
0.025 mg of USP Meropenem RS per mL. [NoTE—Immedi-
ately after preparation, store this solution in a refrigerator
use within 24 hours.]
Test solution—Dissolve an accurately weighed quantity of
Meropenem quantitatively in Solvent to obtain a solution
having a known concentration of about 5 mg per mL. Use
this Test solution immediately.
 2592 Meropenem / Official Monographs
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 220-nm detector
and a 4.6-mm x 25-cm column that contains 5-um packing
L1 and is maintained at a constant temperature of about
40°. The flow rate is about 1.6 mL per minute, and is ad-
justed so that the retention time of meropenem is between
5 and 7 minutes. Chromatograph the Standard solution, and
record the peak responses as directed for Procedure: the col-
umn efficiency is not less than 2500 theoretical plates; the
tailing factor is not more than 1.5; and the relative standard
deviation for replicate injections is not more than 2.0%.
Procedure—Separately inject equal volumes (about 10 uL)
of the Standard solution and the Test solution into the chro-
matograph, record the chromatograms, using a period of
chromatography for the Test solution that is about 3 times
the retention time of meropenem, and measure the peak
responses. Major impurity peaks may be observed at reten-
tion times of about 0.45 and 1.9 in relation to the retention
time of meropenem. Calculate the percentage of each im-
pu in the chromatogram obtained from the Test solution
y the formula:
(Cs / Cu)(P)(r/ rs)
in which Cs is the concentration, in mg per mL, of USP
Meropenem RS in the Standard solution; Cy is the concentra-
tion, in mg per mL, of Meropenem in the Test solution; P is
the stated percentage, calculated on the anhydrous basis, of
meropenem in USP Meropenem RS; 1 is the peak response
of any individual impurity obtained from the Test solution;
and rs is the peak response of meropenem obtained from
the Standard solution. Not more than 0.3% of any of two
major impurities is found, calculated on the anhydrous ba-
sis; not more than 0.1% of any other impurity is found,
calculated on the anhydrous basis; and the sum of all such
other impurities is not more 0.3%.
Other requirements—Where the label states that Mer-
ee
a)
openem is sterile, it meets the requirements for Sterility Tests
a (71) and for Bacterial endotoxins under Meropenem for Injec-
i]
— tion. Where the label states that Meropenem must be sub-
aD jected to further processing during the preparation of inject-
} solution (25% in water) with water to 750 mL. Adjust
a able dosage forms, it meets the requirements for Bacterial
5 endotoxins under Meropenem for Injection.
= Assay—
a Diluted phosphoric acid—Dilute 10 mL of phosphoric acid
al
=) with water to make 100 mL of solution.
Solvent—tTransfer 1.0 mL of triethylamine to a 1000-mL
volumetric flask containing 900 mL of water. Adjust with Di-
luted phosphoric acid to a pH of 5.0 + 0.1, dilute with water
to volume, and mix.
Mobile phase—Prepare a mixture of Solvent and methanol
(5:1). Make adjustments if necessary (see System Suitability
under Chromatography (621)).
Standard preparation—Transfer about 25 mg of USP Mer-
openem RS, accurately weighed, to a 50-mL volumetric
flask, add Solvent, swirl to dissolve, dilute with Solvent to
volume, and mix. [NoTE—Immediately after preparation,
store this solution in a refrigerator. It may be used for
24 hours.]
Assay preparation—Transfer about 25 mg of Meropenem,
accurately weighed, to a 50-mL volumetric flask, add Sol-
vent, swirl to dissolve, dilute with Solvent to volume, and
mix. Use this solution immediately after preparation.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 300-nm detector
and a 4.6-mm x 25-cm column that contains 5-m packing
L1. Adjust the flow rate so that the retention time for mer-
openem is about 6 to 8 minutes. The flow rate is about
1.5 mL per minute. Chromatograph the Standard prepara-
tion, and record the peak responses as directed for Proce-
dure: the column efficiency is not less than 2500 theoretical
USP 41
plates; the tailing factor is not more than 1.5; and the rela-
tive standard deviation for replicate injections is not more
than 2.0%.
Procedure—Separately inject equal volumes (about 5 wL)
of Standard preparation and Assay preparation into the chro-
matograph, record the chromatograms, and measure the ar-
eas for the major peaks. Calculate the quantity, in mg, of
prea in the portion of Meropenem taken by the
‘ormula:
(Ws/Wu)(P)(ru
/ 5)
in which Ws is the weight, in mg, of USP Meropenem RS
taken to prepare the Standard preparation, calculated on the
anhydrous basis; Wy is the weight, in mg, of Meropenem
taken to prepare the Assay preparation; P is the stated per-
centage, calculated on the anhydrous basis, of meropenem
in USP Meropenem RS; and ry and rs are the meropenem
peak responses obtained from the Assay preparation and the
Standard preparation, respectively.
Meropenem for Injection
DEFINITION
Meropenem for Injection is a sterile dry mixture of Mer-
openem and Sodium Carbonate. It contains NLT 90.0%
and NMT 120.0% of the labeled amount of meropenem
(CizH2sN3O5S).
IDENTIFICATION
° A. The retention time of the meropenem peak of the
Sample solution corresponds to that of the Standard solu-
tion, as obtained in the Assay.
ASSAY
© PROCEDURE
Solution A: 1:10 solution of phosphoric acid and water
Buffer: Dilute 15 mL of tetrabutylammonium hydroxide
with Solution A to a pH of 7.5 +0.1.
Mobile phase: Acetonitrile, methanol, and Buffer
(150:100:750)
Standard solution: 0.11 mg/mL of USP Meropenem RS
in Mobile phase. Immediately after preparation, store
this solution in a refrigerator, and use within 24 h.
Sample stock solution 1 (where it is represented as be-
ing a single-dose container): Nominally 1 mg/mL of
meropenem, prepared as follows. Constitute a container
of Meropenem for Injection with a volume of water,
corresponding to the quantity of solvent specified in
the labeling. Withdraw all of the withdrawable con-
tents, using a suitable hypodermic needle and syringe,
and transfer to a suitable volumetric flask. Dilute with
water to volume, and mix.
Sample solution 1: _Nominally 0.1 mg/mL of mer-
openem in Mobile phase from Sample stock solution 17.
Hold this Sample solution 1 for 2 h at 25+ 1° before
testing.
Sample stock solution 2 (where the label states the
quantity of meropenem in a given volume of consti-
tuted solution): Nominally 1 mg/mL of meropenem,
prepared as follows. Constitute a container of Mer-
openem for Injection with a volume of water corre-
sponding to the quantity of solvent specified in the la-
pela) and dilute with water.
Sample solution 2: Nominally 0.1 mg/mL of mer-
openem in Mobile phase from Sample stock solution 2.
Hold this Sample solution 2 for 2 h at 25 + 1° before
testing.
USP 41 Official Monographs / Meropenem 2593

Chromatographic system Burner: Single-slot

(See Chromatography (621), System Suitability) Flame: Air—-acetylene
Mode: LC Lamp: Sodium hollow-cathode
Detector: UV 300 nm Analysis
Column: 4.6-mm x 25-cm; 5-um packing L1 Samples: Standard solution, Sample solution, and Blank
Flow rate: 1.5 mL/min. [NoTE—Adjust the flow rate to Calculate the percentage of sodium (Na) in the portion
obtain a retention time for meropenem of about 6-8 of Meropenem for Injection taken:
min.
Injection volume: 20 pL Result = (Au/As) x (Cs/Cu) x (Mu/M,2) x 100
System suitability
Sample: Standard solution Au = absorbance of the Sample solution
Suitability requirements As = absorbance of the Standard solution
Relative standard deviation: NMT 2.0% Cs = concentration of sodium chloride in the
Tailing factor: NMT 1.5 Standard solution (g/mL)
Analysis Cu = nominal concentration of meropenem in the
Samples: Standard solution and Sample solution 1 or Sample solution (g/mL)
Sample solution 2 Ma = atomic weight of sodium, 22.99
Calculate the percentage of the labeled amount of Mr = molecular weight of sodium chloride, 58.44
meropenem (Ci7H2sN3OsS) in the portion of Mer- Acceptance criteria: 80%-120% of the labeled amount
openem for Injection taken: of sodium

Result = (ru/rs) x (Cs/Cu) x P x 100 PERFORMANCE TESTS

e UNIFORMITY OF DOSAGE UNITS (905): Meets the
tu = peak response of meropenem from Sample requirements
solution 1 or Sample solution 2
rs = peak response of meropenem from the IMPURITIES
Standard solution © ORGANIC IMPURITIES
Cs = concentration of USP Meropenem RS in the Solution A: 1:10 solution of phosphoric acid and water
Standard solution (mg/mL) Buffer: Mix 1 mL of triethylamine and 900 mL of water.
Cu = nominal concentration of meropenem in Adjust with Solution A to a pH of 5.0 + 0.1, and dilute
Sample solution 1 or Sample solution 2 with water to 1000 mL.
Mobile phase: Acetonitrile and Buffer (70:1000)
(mg/mL) Standard solution: 0.029 mg/mL of USP Meropenem
P = potency of meropenem in USP Meropenem RS
RS in Buffer. Store this solution in a refrigerator immedi-
(mg/mg) ately after preparation, and use within 24 h.
Acceptance criteria: 90.0%-120.0%
Sample solution: Nominally prepare 5 mg/mL of mer-
OTHER COMPONENTS openem in Buffer from Meropenem for Injection. This
¢ CONTENT OF SODIUM solution has to be prepared fresh and used
immediately. (ss
Solution A: 38.1 g/L of potassium chloride in water .4)
Standard stock solution: 25.42 \1g/mL of sodium chlo- Chromatographic system i)
ride (previously dried at 105° for 2 h) in water (See Chromatography (621), System Suitability.)
Mode: LC =
Standard solution: 2.5 ug/mL of sodium chloride from re}
the Standard stock solution mixed first with Solution A to Detector: UV 220 nm =
10% of the final volume and diluted with water to Column: 4.6-mm x 25-cm; 5-um packing L1 }
volume Temperature: 40° eo}=
Flow rate: 1.6 mL/min. [NoTE—Adjust to obtain a re- »
Sample stock solution 1 (where it is represented as be- a}
ing a single-dose container): Nominally 0.125 mg/mL tention time for meropenem of 5-7 min.] =r
of meropenem prepared as follows. Constitute a con- Injection volume: 10 uL “

tainer of Meropenem for Injection with a volume of Run time: 3 times the retention time of meropenem
water corresponding to the quantity of solvent specified System suitability
in the labeling. Withdraw all of the withdrawable con- Sample: Standard solution
tents, using a suitable hypodermic needle andsyringe, Suitability requirements
and transfer to a suitable volumetric flask. Dilute wit Relative standard deviation: NMT 2.0%
water to volume. Tailing factor: NMT 1.5
Sample stock solution 2 (where the label states the Analysis
quantity of meropenem in a given volume of consti- Samples: Standard solution and Sample solution
tuted solution): Nominally 0.125 mg/mL of mer- Calculate the percentage of each impurity in the por-
openem prepared as follows. Constitute a container of tion of Meropenem for Injection taken:
Meropenem for Injection with a volume of water, corre-
sponding to the quantity of solvent specified in the la- Result = (ru/rs) x (Cs/Cu) x P x 100
beling. Transfer the constituted solution to a suitable
volumetric flask, and dilute with water to volume. ty = peak response of each individual impurity
Sample solution: Nominally 0.0125 mg/mL of mer- from the Sample solution
openem from Sample stock solution 1 or Sample stock rs = peak response of meropenem from the
solution 2 mixed first with Solution A to 10% of the final Standard solution
volume, and dilute with water to volume Cs = concentration of USP Meropenem RS in the
Blank: 1:10 mixture of Solution A and water Standard solution (mg/mL)
Instrumental conditions Cu = nominal concentration of meropenem in the
(See Atomic Absorption Spectroscopy (852).) Sample solution (mg/mL)
Mode: Atomic ai sorption spectrophotometry P = potency of meropenem in USP Meropenem RS
a wavelength: 589.6 nm sodium emission (mg/mg)
ine Acceptance criteria: See Table 1.
 2594 Meropenem / Official Monographs USP 41

Table 1 to a 1000-mL volumetric flask, add 500 mL of water,

Relative Acceptance and swirl to dissolve. Add 7.5 mL of a solution of tetra-
Retention Criteria,
butylammonium hydroxide 30-hydrate in methanol (1
Name Time NMT (%)
in 4), dilute with water to volume, and mix.
Mobile phase: Methanol and Buffer (15:85)
Meropenem
System suitability solution: 0.25 mg/mL of 4-ami-
impurity l 0.45 0.8
nosalicylic acid and 0.4 mg/mL of USP Mesalamine RS
Meropenem in Mobile phase
impurity Ile 1.9 0.6 Standard stock solution: 1 mg/mL of USP Mesalamine
2 Specified, unidentified impurities. RS in Mobile phase
Standard solution: 0.4 mg/mL of USP Mesalamine RS
SPECIFIC TESTS
e BACTERIAL ENDOTOXINS TEST (85): NMT 0.125 USP Endo-
in Mobile phase from the Standard stock solution
Sample stock solution: 1 mg/mL of Mesalamine in Mo-
toxin Unit/mg of meropenem bile phase
© CONSTITUTED SOLUTION: At the time of use, it meets the
requirements for Injections and Implanted Drug Products Sample solution: 0.4 mg/mL of Mesalamine in Mobile
phase from the Sample stock solution
(1), Specific Tests, Completeness and clarity of solutions. Chromatographic system
Loss ON DrvING (731)
Analysis: Dry in a vacuum at 65° for 6 h. (See Chromatography (621), System Suitability.)
Mode: LC
Acceptance criteria: 9.0%-12.0% Detector: UV 254 nm
PARTICULATE MATTER IN INJECTIONS (788): Meets the re-
Column: 4-mm x 30-cm; 10-um packing L1
quirements for small-volume injections Flow rate: 2 mL/min
e PH (791) Injection volume: 15 pL
Sample solution: 50 mg/mL System suitability
Acceptance criteria: 7.3-8.3 Samples: System suitability solution and Standard
e STERILITY TESTS (71): Meets the requirements when
solution
tested as directed for Test for Sterility of the Product to Be Suitability requirements
Examined, Membrane Filtration
Resolution: NLT 2.0 between 4-aminosalicylic acid
ADDITIONAL REQUIREMENTS and mesalamine, System suitability solution
© PACKAGING AND STORAGE: Preserve in tight containers as Tailing factor: NMT 2.5, Standard solution
described in Packaging and Storage Requirements (659), Relative standard deviation: NMT 0.73%, Standard
Injection Packaging, Packaging for constitution. Store at solution
controlled room temperature. Analysis
e LABELING: Meets the requirements for Labeling (7), Labels Samples: Standard solution and Sample solution
and Labeling for Injectable Products. Label it to state the Calculate the percentage of mesalamine (C7H7NOs) in
quantity, in mg, of sodium (Na) in a given dosage of the portion of Mesalamine taken:
meropenem.
Result = (ru/rs) x (Cs/Cu) x 100
oa
al

ry ru = peak response of mesalamine from the Sample

CS
-
Change to read:
D solution
9 ° UsP REFERENCE STANDARDS (11) rs = peak response of mesalamine from the
is Standard solution
iS @ (CN }-May-2018)
USP Meropenem RS Cs = concentration of USP Mesalamine RS in the
= Standard solution (mg/mL)
a G = concentration of Mesalamine in the Sample
Ww
> solution (mg/mL)
Acceptance criteria: 98.5%-101.5% on the dried basis
Mesalamine IMPURITIES
e CHLORIDE AND SULFATE, Chloride (221)
Sample solution: Disperse 500 mg in 40 mL of water,
sonicate for 5 min, and filter the dispersion. Use the
filtrate.
Analysis: Add 1 mL of nitric acid to the Sample solution.
Acceptance criteria: No more chloride than corre-
C)H7NO3 153.14 sponds to 0.7 mL of 0.020 N hydrochloric acid (0.1%)
Benzoic acid, 5-amino-2-hydroxy-; e CHLORIDE AND SULFATE, Sulfate (221)
5-Aminosalicylic acid [89-57-6]. Sample solution: Dissolve 500 mg in water. Filter if
necessary. Use the filtrate.
DEFINITION Acceptance criteria: The Sample solution shows no
Mesalamine contains NLT 98.5% and NMT 101.5% of more sulfate than corresponds to 1.0 mL of 0.02 N sul-
mesalamine (C7H7NOs), calculated on the dried basis. furic acid (0.2%).
IDENTIFICATION
e A. INFRARED ABSORPTION (197K) Delete the following:
e B. The retention time of the mesalamine peak of the
Sample solution corresponds to that of the Standard solu- °e HEAVY METALS, Method II (231): NMT 0.002%6 cortea1-
tion, as obtained in the Assay. jan-2018)
° RESIDUE ON IGNITION (281): NMT 0.2%
ASSAY e HYDROGEN SULFIDE AND SULFUR DIOXIDE
o PROCEDURE Analysis: Dissolve about 500 mg in 5 mL of 1 N sodium
Buffer: Transfer 7.1 g of anhydrous dibasic sodium hydroxide, add 6 mL of 3 N hydrochloric acid, and stir
phosphate and 6.9 g of monobasic sodium phosphate vigorously. Hold a piece of moistened lead acetate test
paper over the mixture.
USP 41 Official Monographs / Mesalamine 2595

Acceptance criteria: The test paper so obtained does ¢ CONTENT OF ANILINE, 2-AMINOPHENOL, AND 4-AMINOPHENOL
not become discolored. Standard stock solution: 0.05 mg/mL of aniline, 2 mg/
© CONTENT OF 3-AMINOSALICYLIC ACID AND OTHER RELATED mL of 2-aminophenol, and 2 mg/mL of USP 4-Ami-
IMPURITIES nophenol RS in methanol
[NoTe—Use this test to measure 3-aminosalicylic acid and Standard solution: 0.5 ug/mL of aniline, 20 ug/mL of
other related impurities not measured in the test for 2-aminophenol, and 20 j1g/mL of USP 4-Aminophenol
Content of Aniline, 2-Aminophenol, and 4-Aminophenol.] RS from the Standard stock solution in methylene
Mobile phase: Dissolve 1.36 g of monobasic potassium chloride
phosphate and 2.2 g of sodium 1-octanesulfonate in Sample solution: 100 mg/mL of Mesalamine in enact
890 mL of water, and adjust with phosphoric acid to a ene chloride. Allow to settle, and use the clear methyl-
pH of 2.2. Pass througha filter of 0.5-11m or finer pore ene chloride solution.
size. To the filtrate add 80 mL of methanol and 30 mL Chromatographic system
of acetonitrile. (See Chromatography (621), System Suitability.)
Standard solution: 1 g/mL each of USP Mesalamine Mode: GC
RS and 3-aminosalicylic acid in Mobile phase Detector: Flame ionization
Sample solution: 0.5 mg/mL of Mesalamine in Mobile Column: 0.53-mm ~ 10-m fused-silica capillary; 2.65-
phase. Initially add about 75% of the final volume of um film of G27
Mobile phase, and sonicate briefly to dissolve. Dilute Temperatures
with Mobile phase to volume, and mix. Injection port: 280°
Chromatographic system Detector: 300°
(See Chromatography (621), System Suitability.) Column: See Table 2.
Mode: LC
Detector: UV 220 nm Table 2
Column: 4.6-mm x 15-cm; 5-um packing L7
Flow rate: 1.2 mL/min Hold Time at)
Injection volume: 20 pL Initial Temperature Final Final
Run time: 3 times the retention time of mesalamine Temperature Ramp Temperature | Temperature
System suitability ©) (¢/min) «°) (min)
Sample: Standard solution 70. = 70 2
[NotE—See Table 1 for the relative retention times.] 70 30 150 1
Suitability requirements
Resolution: NLT 2 between mesalamine and 3-ami- Carrier gas: Helium
nosalicylic acid Flow rate: 15 mL/min
Relative standard deviation: NMT 5.0% for both Injection volume: 2 uL
mesalamine and 3-aminosalicylic acid System suitability
Analysis Sample: Standard solution
Samples: Standard solution and Sample solution {Note—See Table 3 for the relative retention times.]
Calculate the percentage of 3-aminosalicylic acid: Suitability requirements e
Resolution: NLT 2.0 between aniline and 2-ami- “
Result = (ru/rs) x (Cs/Cu) x 100 nophenol; NLT 2.0 between 2-aminophenol and
a)
4-aminophenol =
tu = peak response of 3-aminosalicylic acid from Relative standard deviation: NMT 10.0% for aniline, i}
the Sample solution ej
2-aminophenol, and 4-aminophenol }
rs = peak response of 3-aminosalicylic acid from Analysis ro}=
the Standard solution Samples: Standard solution and Sample solution i
Gs = concentration of 3-aminosalicylic acid in the Calculate the percentage of aniline, 2-aminophenol, 3
Standard solution (g/mL) ane 4-aminophenol in the portion of Mesalamine =
a)
Cu = concentration of Mesalamine in the Sample taken:
solution (ug/mL)
Calculate the percentage of any other impurity: Result = (ru/rs) x (Cs/Cu) x 100
Result = (ru/rs) x (Cs/Cu) x 100 i = peak response of aniline, 2-aminophenol, or
4-aminophenol from the Sample solution
tu = peak response of any individual impurity from Is = peak response of aniline, 2-aminophenol, or
the Sample solution 4-aminophenol from the Standard solution
rs = peak response of mesalamine from the Cs = concentration of aniline, 2-aminophenol, or
Standard solution USP 4-Aminophenol RS in the Standard
Cs = concentration of USP Mesalamine RS in the solution (ug/mL)
Standard solution (g/mL) Cu = concentration of Mesalamine in the Sample
Gu = concentration of Mesalamine in the Sample solution (ug/mL)
solution (\ug/mL) Acceptance criteria: See Table 3.
Acceptance criteria: See Table 1.
Table 3
Table 1
Relative Acceptance
Accep- Retention Criteria,
Relative tance Name Time NMT (%)
Retention Criteria, Aniline 0.5 0.0005
2-Aminophenol 0.9 0.02
lai 1
4-Aminophenol 1.0 0.02
i i 1
oO i a
Total impuriti
 2596 Mesalamine / Official Monographs USP 41

SPECIFIC TESTS 4.0 mL of the Internal standard solution, dilute with

e CLARITY OF SOLUTION Buffer to volume, and mix. Dilute 5.0 mL of this solution
Sample solution: Freshly prepare a solution of 0.25 g with Buffer to 25 mL.
of Mesalamine in 10 mL of 1 N hydrochloric acid. Sample solution: Transfer, as completely as possible,
Acceptance criteria: The Sample solution is clear. the contents of NLT 20 Capsules to a suitable tared
e Loss ON DRYING (731) container, and determine the average weight of the
Analysis: Dry under vacuum at 105° for 3 h. contents of a Capsule. Finely powder the Capsule con-
Acceptance criteria: NMT 0.5% tents so that the powder thus obtained passes through
e PH (791) a No. 40 sieve (see Powder Fineness (811)). Transfer a
Sample: A suspension (1 in 40) portion of the powder, nominally equivalent to about
Acceptance criteria: 3.5-4.5 250 mg of mesalamine, to a 500-mL volumetric flask.
Add 20.0 mL of the Internal standard solution and about
ADDITIONAL REQUIREMENTS yn , . 300 mL of Buffer, and shake by mechanical means for 1
e PACKAGING AND STORAGE: Preserve in tight, light-resistant h. Dilute with Buffer to volume, and mix. Transfer
containers. 5.0 mL of this solution to a 25-mL volumetric flask. Di-
¢ USP REFERENCE STANDARDS (11) lute with Buffer to volume, mix, and pass about 10 mL
USP 4-Aminophenol RS of this solution througha filter of 0.5-4um or finer pore
4-Aminophenol. size. Use the filtrate as the Sample solution.
CsH7NO 109.13 Chromatographic system
USP Mesalamine RS (See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 240 nm
Column: 4.6-mm x 25-cm; 5-4um packing L1
Flow rate: 1.5 mL/min
Mesalamine Extended-Release Capsules Injection volume: 10 uL
System suitability
DEFINITION Sample: Standard solution
Mesalamine Extended-Release Capsules contain NLT 90.0% [NoTe—The relative retention times for mesalamine and
and NMT 110.0% of the labeled amount of mesalamine sodium benzoate are about 0.6 and 1.0, respectively.]
(C;H7NO3). Suitability requirements
Resolution: NLT 2.5 between mesalamine and so-
IDENTIFICATION dium benzoate
o A. INFRARED ABSORPTION (197K) Relative standard deviation: NMT 2.0%
Sample: Use the powdered, undried Capsule contents. Analysis
Analysis: Record the spectra in the range between Samples: Standard solution and Sample solution
2000 cm- and 1240 cm. Calculate the percentage of the labeled amount of
Acceptance criteria: Meet the requirements mesalamine (C;H7NOs) in the portion of Capsule con-
2 tents taken:
roe ASSAY
rs] ¢ PROCEDURE 7 . . Result = (Ru/Rs) x (Cs/Cu) x 100
ey Buffer: Dissolve 6.8 g of monobasic potassium phos-
fo} phate and 1.65 g of sodium hydroxide in 800 mL of Ry = peak response ratio of mesalamine to sodium
¢ water. Adjust with 1 N sodium hydroxide to a pH of benzoate from the Sample solution
© 7.5, dilute with water to 1000 mL, and mix. Rs = peak response ratio of mesalamine to sodium
Ps Solution A: Dissolve 3.4 g of tetrabutylammonium hy- benzoate from the Standard solution
[= drogen sulfate and 1.4 g of sodium acetate trihydrate in Cs = concentration of USP Mesalamine RS in the
3 1000 mL of water. Adjust with 1 N sodium hydroxide to Standard solution (mg/mL)
a pH of 6.6. Add 200 mL of acetonitrile, mix, and pass Cy = nominal concentration of mesalamine in the
throughafilter of 0.5-um or finer pore size. [NOTE— Sample solution (mg/mL)
Increasing the proportion of acetonitrile decreases the Acceptance criteria: 90.0%-110.0%
retention times. Prepare fresh daily.]
Solution B: Dissolve 4.6 g of tetrabutylammonium hy- PERFORMANCE TESTS
drogen sulfate and 1.9 g of sodium acetate trihydrate in e DISSOLUTION (711)
1000 mL of water, and adjust with 1 N sodium hydrox- Buffer: 0.05 M pH 7.5 phosphate buffer prepared as
ide to a pH of 6.6. Add 650 mL of acetonitrile, mix, and follows. Dissolve 6.8 g of monobasic potassium phos-
ass throughafilter of 0.5-1m or finer pore size. phate and 1 gof sodium hydroxide in water to make
Note—Prepare fresh daily.] 1000 mL of solution, and adjust with 10 N sodium hy-
Mobile phase: See Table 1. droxide to a pH of 7.50 + 0.05.
Medium: Buffer, 900 mL
Table 1 Apparatus 2: 100 rpm
Times: 1, 2, 4, and 8h
Solution A Solution B Standard solution: A known concentration of USP
Mesalamine RS in Medium
Sample solution: Filter portions of the solution under
test suitably diluted with Medium, if necessary.
Analysis: Calculate the percentages of the labeled
amount of mesalamine (C7;H;NOs3) dissolved at the
wavelength of maximum absorbance at about 330 nm
by comparing the UV absorbances of the Sample solu-
20 1 tion wile that of the Standard solution.
Tolerances: See Table 2.
Internal standard solution: 35 mg/mL of sodium ben-
Zoate in Buffer
Standard solution: Transfer about 50 mg of USP
Mesalamine RS to a 100-mL volumetric flask. Add
USP 41 Official Monographs / Mesalamine 2597

Table 2 Suitability requirements

Resolution: NLT 2.0 between 4-aminosalicylic acid
and mesalamine, System suitability solution
Tailing factor: NMT 2.5, Standard solution
Relative standard deviation: NMT 2.0%, Standard
solution
4 Analysis
8 Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
The percentages of the labeled amount of mesalamine mesalamine (C7H7NOs) in the portion of Rectal Sus-
(C7H7NO3) dissolved at the times specified conform to pension taken:
Acceptance Table 2 in (711).
e UNIFORMITY OF DOSAGE UNITS (905): Meet the Result = (ru/rs) x (Cs/Cu) x 100
requirements
tu = peak response of mesalamine from the Sample
ADDITIONAL REQUIREMENTS solution
© PACKAGING AND STORAGE: Preserve in tight, light-resistant fs = peak response of mesalamine from the
containers. Standard solution
e USP REFERENCE STANDARDS (11) Cs = concentration of USP Mesalamine RS in the
USP Mesalamine RS Standard solution (mg/mL)
Cu = nominal concentration of mesalamine in the
Sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0%

Mesalamine Rectal Suspension OTHER COMPONENTS

© CONTENT OF SODIUM BENZOATE (if present)
Mobile phase: Transfer 390 mg of ammonium acetate
DEFINITION to a 1000-mL volumetric flask, add 100 mL of water,
Mesalamine Rectal Suspension is a suspension of
Mesalamine in a suitable aqueous vehicle. It contains NLT and dissolve by swirling. Add 6 mL of glacial acetic acid
and 300 mL of methanol, dilute with water to volume,
90.0% and NMT 110.0% of the labeled amount of and mix. Pass this solution throughafilter of 0.5-m or
mesalamine (C7H7NOs). It contains one or more suitable
preservatives. finer pore size.
Standard solution: 1 mg/mL of sodium benzoate in
IDENTIFICATION water. To 5.0 mL of this solution add 40 mL of metha-
e A. The retention time of the et of the Sample nol, and dilute with water to 100 mL. Pass this solution
solution corresponds to that of the Standard solution, as througha filter of 0.5-um or finer pore size.
obtained in the Assay. Sample solution: Transfer about 5 g of well-shaken Rec-
tal Suspension to a 100-mL volumetric flask. Add 40 mL c
ASSAY of methanol, dilute with water to volume, and mix. wv
¢ PROCEDURE Pass the solution throughafilter of 0.5-um or finer u
Buffer: Transfer 7.1 g of anhydrous dibasic sodium pore size. B=
phosphate and 6.9 g of monobasic sodium phosphate Chromatographic system i)
to a 1000-mL volumetric flask, add 500 mL of water, Mode: LC =
and swirl to dissolve. Add 7.5 mL of a solution of tetra- Detector: UV 254 nm )
butylammonium hydroxide in methanol (1 in 4), dilute Column: 4.6-mm x 25.0-cm; packing L7
(eo)|
2
with water to volume, and mix. Flow rate: 1.5 mL/min a]
Mobile phase: Methanol and Buffer (15:85) Injection volume: 15 pL y
System suitability solution: 0.25 mg/mL of 4-ami- System suitability “

nosalicylic acid and 0.4 mg/mL of USP Mesalamine RS Sample: Standard solution
in Mobile phase Suitability requirements
Standard stock solution: 1 mg/mL of USP Mesalamine Tailing factor: NMT 2.5
RS in Mobile phase Relative standard deviation: NMT 2.0%
Standard solution: 0.4 mg/mL of USP Mesalamine RS Analysis
in Mobile phase from the Standard stock solution Samples: Standard solution and Sample solution
Sample solution: Transfer an accurately measured, well- Calculate the percentage (w/w) of sodium benzoate in
shaken quantity of Rectal Suspension, nominally equiva- the Rectal Suspension taken:
lent to about 100 mg of mesalamine, to a 100-mL volu-
metric flask. Add 55 mL of Mobile phase, and dissolve Result = (ru/rs) x Cs x (10/W)
by shaking for about 10 min. Dilute with Mobile phase
to volume, and mix. Transfer 10.0 mL of this solution to ru = peak response of sodium benzoate from the
a 25-mL volumetric flask, dilute with Mobile phase to Sample solution
volume, and mix. Pass this solution through a suitable Is = peak response of sodium benzoate from the
filter of 0.5-14m or finer pore size, and use the filtrate as Standard solution
the Sample solution. Cs = concentration of sodium benzoate in the
Chromatographic system Standard solution (mg/mL)
(See Chromatography (621), System Suitability.) Ww = weight of Rectal Suspension taken (g)
Mode: LC Acceptance criteria: 0.05%-0.125%
Detector: UV 254 nm
Column: 4-mm x 30-cm; packing L1 PERFORMANCE TESTS
Flow rate: 2 mL/min ¢ UNIFORMITY OF DOSAGE UNITS (905)
Injection volume: 15 uL Procedure for content uniformity
System suitability Buffer: Transfer 7.1 g of anhydrous dibasic sodium
Samples: System suitability solution and Standard phosphate and 6.9 g of monobasic sodium phosphate
solution to a 1000-mL volumetric flask, add 500 mL of water,
and swirl to dissolve. Add 7.5 mL of a solution of tetra-
 2598 Mesalamine / Official Monographs USP 41

butylammonium hydroxide in methanol (1 in 4), dilute a volume of about 80 mL, and arise with phosphoric
with water to volume, and mix. acid to a pH of 2.0. Sonicate briefly to dissolve, transfer
Mobile phase: Methanol and Buffer (15:85) to a 100-mL volumetric flask, dilute with water to vol-
System suitability solution: 0.25 mg/mL of 4-ami- ume, and mix.
nosalicylic acid and 0.4 mg/mL of USP Mesalamine RS Chromatographic system
in Mobile phase (See Chromatography (621), System Suitability.)
Standard stock solution: Transfer about 100 mg of Mode: LC
USP Mesalamine RS to a 50-mL volumetric flask, add Detector: UV 220 nm
15 mL of 2.N hydrochloric acid, and dissolve by Column: 4.6-mm x 15-cm; 5-um packing L7
swirling. Dilute with 2 N hydrochloric acid to volume, Flow rate: 1.2 mL/min
and mix. Injection volume: 20 uL
Standard solution: Add 5 mL of 2 N sodium hydrox- Run time: 3 times the retention time of mesalamine
ide to 5.0 mL of the Standard stock solution, and dilute System suitability
with Mobile phase to 25 mL. Pass this solution through Sample: Standard solution
a filter of 0.5-um or finer pore size. [Note—The relative retention times for mesalamine and
Sample stock solution: Transfer, with the aid of 2N 3-aminosalicylic acid are about 1.0 and 1.3,
hydrochloric acid, the contents of a container of Rectal respectively.
Suspension to a 200-mL volumetric flask. Add 2 N hy- Suitability requirements
drochloric acid to obtain about 160 mL of solution, Resolution: NLT 2 between mesalamine and 3-ami-
and shake for 10 min. Dilute with 2 N hydrochloric nosalicylic acid
acid to volume, and mix. Analysis
Sample solution: Transfer a suitable volume of the Samples: Standard solution and Sample solution
Sample stock solution, nominally equivalent to 40 mg Calculate the Ferenge of each impurity in the por-
of mesalamine, to a 100-mL volumetric flask. Add a tion of Rectal Suspension taken:
volume of 2 N hydrochloric acid, equal to the added
volume of the Sample stock solution, dilute with Mobile Result = (ru/rs) x (Cs/Cu) x 100
phase to volume, and mix. Pass this solution through a
suitable filter of 0.5-4m or finer pore size. tu = peak response of any individual impurity from
Chromatographic system the Sample solution
(See Chromatography (621), System Suitability.) rs = peak response of mesalamine from the
Mode: LC Standard solution
Detector: UV 254 nm Cs = concentration of USP Mesalamine RS in the
Column: 4-mm x 30-cm; packing L1 Standard solution (ug/mL)
Flow rate: 2 mL/min Cy = nominal concentration of mesalamine in the
Injection volume: 15 ul Sample solution (g/mL)
System suitability Acceptance criteria
Samples: System suitability solution and Standard Individual impurities: NMT 0.2%
£ solution Total impurities: NMT 1.0%
5 Suitability requirements
Ss Resolution: NLT 2.0 between 4-aminosalicylic acid SPECIFIC TESTS
fo) and mesalamine, System suitability solution ° PH (791)
° Tailing factor: NMT 2.5, Standard solution Sample solution: Dilute the Rectal Suspension 1 to 10
5 Relative standard deviation: NMT 2.0%, Standard
with water.
Acceptance criteria: 3.5-5.5
S solution
ei Analysis ADDITIONAL REQUIREMENTS
“ Samples: Standard solution and Sample solution e PACKAGING AND STORAGE: Preserve in tight, light-resistant
=) Calculate the percentage of the labeled amount of containers.
mesalamine (C7H7NO3) in the container of Rectal Sus- e USP REFERENCE STANDARDS (11)
pension taken: USP Mesalamine RS
Result = (ru/rs) x (Cs/Cu) x 100
ru = peak response of mesalamine from the Sample
solution
Is = peak response of mesalamine from the Mesalamine Delayed-Release Tablets
Standard solution
Cs = concentration of USP Mesalamine RS in the DEFINITION
Standard solution (mg/mL) Mesalamine Delayed-Release Tablets contain NLT 90.0% and
Cy = nominal concentration of mesalamine in the NMT 110.0% of the labeled amount of mesalamine
Sample solution (mg/mL) (C7H7NO3).
Acceptance criteria: Meets the requirements
IDENTIFICATION
IMPURITIES e A. INFRARED ABSORPTION (197K)
© ORGANIC IMPURITIES Sample solution: To about 50 mL of water add a quan-
Mobile phase: Dissolve 1.36 g of monobasic potassium tity of finely powdered Tablets, nominally equivalent to
phosphate and 2.2 g of sodium 1-octanesulfonate in about 800 mg of mesalamine. Boil the mixture for
890 mL of water, and adjust with phosphoric acid to a about 5 min, with constant stirring. Filter the hot solu-
pH of 2.2. Pass throughafilter of 0.5-um or finer pore tion, and allow the filtrate to cool. Collect the precipi-
size. To the filtrate add 80 mL of methanol and 30 mL tated crystals, and dry at about 110°.
of acetonitrile. Acceptance criteria: Meet the requirements
Standard solution: 1 g/mL each of USP Mesalamine
RS and 3-aminosalicylic acid in Mobile phase ASSAY
Sample solution: Transfer a volume of Rectal Suspen- e PROCEDURE
sion, previously well shaken, nominally equivalent to Mobile phase: Dissolve 4.3 g of sodium 1-octanesulfon-
100 mg of mesalamine, to a beaker. Add water to give ate in 1 L of water. Adjust with phosphoric acid to a pH
USP 41
of 2.15, pass througha filter of 0.45-4m or finer pore
size, and degas.
System suitability stock solution: Transfer about
20 mg each of 3-aminosalicylic acid and USP Salicylic
Acid RS to a 200-mL volumetric flask. Dissolve in 50 mL
of 1. N hydrochloric acid, sonicate to dissolve, dilute
with water to volume, and mix.
System suitability solution: 0.01 mg/mL each of
3-aminosalicylic acid and salicylic acid in water from the
System suitability stock solution
Standard stock solution: Transfer about 25 mg of USP
Mesalamine RS to a 25-mL volumetric flask. Dissolve in
5 mL of 0.25 N hydrochloric acid, sonicate to dissolve,
dilute with water to volume, and mix.
Standard solution: Transfer 10.0 mL of the Standard
stock solution and 5.0 mL of the System suitability solu-
tion to a 50-mL volumetric flask. Dilute with water to
volume, mix, and pass throughafilter of 0.5-1m or
finer pore size.
Sample solution: Pipet a 25.0-mL aliquot of the Sample
solution, obtained as directed in Organic Impurities, into
a 100-mL volumetric flask. Dilute with water to volume,
and pass throughafilter of 0.5-m or finer pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 230 nm
Columns
Precolumns: Two 4.6-mm x 3.0-cm; each containing
10-"um packing L1 and located between the pump
and the injector
Analytical: 4.6-mm x 3.3-cm; 3-m base-deactivated
packing L1
Flow rate: 2 mL/min
Injection volume: 20 uL
System suitability
Sample: Standard solution
Suitability requirements
Resolution: NLT 2 between mesalamine and salicylic
acid or 3-aminosalicylic acid
Tailing factor: NMT 2
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
mesalamine (C7H7NO3) in the portion of Tablets taken:
Result = (ru/rs) x (Cs/Cu) x 100
ru = peak response of mesalamine from the Sample
solution
rs = peak response of mesalamine from the
Standard solution
Cs = concentration of USP Mesalamine RS in the
Standard solution (mg/mL)
Cu = nominal concentration of mesalamine in the
Sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0%
PERFORMANCE TESTS
e DISSOLUTION (711)
Solution A: Transfer about ae of monobasic potas-
sium phosphate and 1.65 g of sodium hydroxide to a
2-L volumetric flask. Dissolve in and dilute with water to
volume, and mix. Adjust with 1 N sodium hydroxide or
phosphoric acid to a pH of 6.0, and mix.
Solution B: Transfer 133.6 g of sodium hydroxide to a
2-L volumetric flask, dissolve in and dilute with water to
volume, and mix.
Official Monographs / Mesalamine 2599
Medium
Acid stage: 500 mL of 0.1 N hydrochloric acid
Buffer stages: 900 mL of Solution A
Apparatus 2
Acid stage: 100 rpm
Buffer stage 1: 100 rpm
Buffer stage 2: 50 rpm
Times
Acid stage: 2h
Buffer stage 1: 1h
Buffer stage 2: 90 min
Acid stage
After 2 h of operation, withdraw an aliquot of the fluid,
discard the remaining solution, and retain the Tablets
in proper order so that each will be returned later to
its respective vessel. Blot the Tablets with a paper
towel to dry, and proceed immediately as directed in
Buffer stage 1.
Standard solution: A known concentration of USP
Mesalamine RS in Medium, equivalent to about 1% of
the labeled amount of mesalamine (C7H7NO3)
Sample solution: Filter portions of the solution under
test, and suitably dilute with Medium, if necessary.
Analysis: Calculate the percentage of the labeled
amount of mesalamine (C7H7NO3) dissolved by com-
paring the UV maximum absorbance at about 302 nm
of the Sample solution with that of the Standard
solution.
Tolerances: See Table 1. Continue testing through all
levels unless the results conform at an earlier level.
Buffer stage 1
[NoTe—Use Solution A that has been equilibrated to a
temperature of 37 + 0.5°.]
Transfer Solution A to each of the dissolution vessels,
and place each Tablet from the Acid stage into its re-
spective vessel. After 1 h, remove a 50-mL aliquot, and
proceed immediately as directed in Buffer stage 2.
Standard solution: A known concentration of USP (=
Mesalamine RS in Medium, equivalent to about 1% of Ww
me]
the labeled amount of mesalamine (C7H7NO3)
Sample solution: Filter portions of the solution under cE
test, and suitably dilute with Medium, if necessary. i}
J
Analysis: Calculate the percentage of the labele °
amount of mesalamine (C7H7NO3) dissolved by com- eo}=
paring the UV maximum absorbance at about 330 nm 2
of the Sample solution with that of the Standard 3
solution. >
al
Tolerances: See Table 1. Continue testing through all
levels unless the results conform at an earlier level.
Table 1
Number
Level Tested Acceptance Criteria
U1 6 No individual value exceeds 1% dissolved.
Average of the 12 units (Li + Lz) is NMT
1% dissolved, and no individual unit is
L2 6 greater than 10% dissolved.
Average of the 24 units (Li + L2 + Ls) is
NMT 1% dissolved, and NMT one individ-
L3 12 ual unit is greater than 10% dissolved.
Buffer stage 2
Add 50 mL of Solution B to each dissolution vessel to
adjust to a pH of 7.2, and continue the run.
Standard solution: A known concentration of USP
Mesalamine RS in Medium
Sample solution: Filter portions of the solution under
test, and suitably dilute with Medium, if necessary.
Analysis: Calculate the percentage of the labele
amount of mesalamine (C7H7NOs) dissolved by com-
paring the UV maximum absorbance at about 332 nm
of the Sample solution with that of the Standard
solution.
 2600 Mesalamine / Official Monographs USP 41

Tolerances: NLT 80% (Q) of the labeled amount of 0.1 N sodium thiosulfate VS, adding 1 mL of starch TS
mesalamine (C7H7NOs) is dissolved. The requirements near the endpoint. Perform a blank determination, and
are met if the quantities dissolved from the product make any necessary corrections (see Titrimetry (541)).
conform to Acceptance Table 4 in (711). Continue test- Each mL of sodium thiosulfate is equivalent to
ing through all levels unless the results conform at an 16.42 mg of mesna (C2HsNaO3Sz).
earlier level. Acceptance criteria: 96.0%-102.0% on the dried basis
© UNIFORMITY OF DOSAGE UNITS (905): Meet the require-
ments for Weight Variation IMPURITIES
e LIMIT OF CHLORIDE
IMPURITIES Chloride standard solution: 8.24 g/mL of sodium
© ORGANIC IMPURITIES chloride in water
Mobile phase, System suitability stock solution, Sys- Sample solution: 200 mg/mL of Mesna in carbon diox-
tem suitability solution, Standard stock solution, ide-free water
Standard solution, Chromatographic system, and Analysis: To 1 mL of the Sample solution and 15 mL of
System suitability: Proceed as directed in the Assay. water add 1 mL of 2M nitric acid. Add the resulting
Sample solution: Transfer a portion nominally equiva- solution to 1 mL of silver nitrate solution (17 g in
lent to about 400 mg of mesalamine, from NLT 20 1000 mL), and allow to stand for 5 min, protected from
finely powdered Tablets, to a 500-mL volumetric flask. light. To 10 mL of the Chloride standard solution add
Add 50 mL of 1 N hydrochloric acid, and sonicate to 5 mL of water and 1 mL of 2M nitric acid. To this solu-
dissolve. Shake by mechanical means for 10 min, dilute tion add 1 mL of silver nitrate solution (17 g in
with water to volume, mix, and pass throughafilter of 1000 mL) and allow to stand for 5 min, protected from
0.5-um or finer pore size. light. When viewed against a dark background, the
[Note—Use an aliquot of this solution for the preparation Sample solution is not more turbid than the Chloride
of the Sample solution in the Assay.] standard solution.
Analysis Acceptance criteria: NMT 250 ppm
Sample: Sample solution o LIMIT OF SULFATE
Calculate the percentage of each impurity in the por- Diluent: 30% (v/v) ethanol in water
tion of Tablets taken: Sulfate standard stock solution: 1.81 mg/mL of potas-
sium sulfate in Diluent
Result = (ru/rz) x 100 Sulfate standard solution: 0.0181 mg/mL of potassium
sulfate in Diluent, prepared immediately before use from
ru = peak response for each impurity Sulfate standard stock solution
rr = sum of all the peak responses Sample solution: Add 5.0 mL of the Sample solution
Acceptance criteria prepared as directed in the test for Limit of Chloride to a
Individual impurity: The largest secondary peak is 30-mL volumetric flask, and dilute with water to
NMT 1.0% of the total area. volume.
Any other individual impurity: NMT 0.5% Analysis: Add 3 mL of a 250-g/L solution of barium
” Total impurities: NMT 2.0% chloride to 4.5 mL of Sulfate standard solution. Shake
<= and allow to stand for 1 min. To 2.5 mL of this solution
(or ADDITIONAL REQUIREMENTS add 15 mL of the Sample solution and 0.5 mL of acetic
i}
J e PACKAGING AND STORAGE: Preserve in tight containers. acid. Use 15 mL of this mixture for comparison with
Dp e USP REFERENCE STANDARDS (11)
fo) 15 mL of the Sulfate standard solution, prepared in the
S USP Mesalamine RS same manner, but using the Sulfate standard solution
C) USP Salicylic Acid RS instead of the Sample solution. After 5 min, any opales-
PE cence in the Sample solution is not more intense than
a that in the Sulfate standard solution.
al
Acceptance criteria: NMT 300 ppm
=)
Delete the following:
ae
yi °e HEAVY METALS, Method | (231): 10 PPMe (Official 1-42-2018)
Ks7 “ONa
¢ ORGANIC IMPURITIES
Mobile phase: In a 1000-mL volumetric flask dissolve
CoHsNaO3S2 / 164.18 2.94 g of potassium dihydrogen phosphate, 2.94 g of
Ethanesulfonic acid, 2-mercapto-, monosodium salt; dipatassium hydrogen PRespnate, and 2.6g of tetrabu-
Sodium 2-mercaptoethanesulfonate [19767-45-4]. tylammonium hydrogen sulfate in about 600 mL of
water. paalush with phosphoric acid to a pH of 2.3, add
DEFINITION 335 mL of methanol, and dilute with water to volume.
Mesna contains NLT 96.0% and NMT 102.0% of mesna System suitability solution: 0.18 mg/mL and
(CzHsNaO3Sz2), calculated on the dried basis. 0.004 mg/mL of USP Mesna RS and USP Mesna Related
IDENTIFICATION
Compound A RS, respectively, in Mobile phase
e A. INFRARED ABSORPTION (197K)
Standard solution 1: 8 g/mL and 120 Baym of USP
e B. IDENTIFICATION TESTS—GENERAL (191), Sodium
Mesna Related Compound A RS and USP Mesna Related
Acceptance criteria: Meet the requirements CompoundBRS, respectively, in Mobile phase
Standard solution 2: 12 g/mL of USP Mesna RS in
ASSAY Mobile phase
¢ PROCEDURE
Sample solution: 120mg of Mesna in 10 mL of water
Analysis: To the Sample solution add 10 mL of 1 M sul-
furic acid and 10 mL of 0.1 N iodine VS. Titrate with
USP 41 Official Monographs / Mesoridazine 2601

Sample solution: 4 mg/mL of Mesna in Mobile phase Table 1

Chromatographic system Relative Relative Acceptance
(See Chromatography (621), System Suitability.) Retention Response Criteria,
Mode: LC Name Time Factor NMT (%)
Detector: UV 235 nm
Thiouronium
Column: 4.6-mm x 25-cm; 10-um packing L1
Flow rate: 1 mL/min ethanesulfonic acide 0.6 100 0.3
Run time: Four times the elution time for mesna Guanidinethiouronium
Injection volume: 20 WL ethanesulfonic acide 0.6 100 0.3
System suitability Mesna triazine analogs 0.8 100 0.3
Sample: System suitability solution Mesna 1.0 — =
[NotE—The relative retention times for mesna and Mesna related
mesna related compoundAare about 1.0 and 1.4, compound A 1.4 _ 0.2
respectively.] Mesna related _
Suitability requirements compound B 2.3 3.0
Resolution: NLT 3.0 between mesna and mesna re-
lated compound A Individual unspecified _ 1.0
impurities 0.1
Analysis
Samples: Standard solution 1, Standard solution 2, and Total unspecified _ _
Sample solution impurities 0.3
{Notr—Identify the peaks using the relative retention 22-(Carbamimidoylthio)ethane-1-sulfonic acid.
times provided in Table 1.] » 2-[(N-Carbamimidoylcarbamimidoyl)thioJethane-1 -sulfonic acid.
Calculate the percentage of mesna related compound A © 2-((4,6-Diamino-1,3,5-triazin-2-yl)thio)ethane-1 -sulfonic acid.
in the portion of Mesna taken:
SPECIFIC TESTS
Result = (ru/rs) x (Cs/Cu) x 100 e Loss ON DRYING
Sample: 1g
ty = peak response of mesna related compound A Analysis: Dry the Sample under vacuum at a pressure
from the Sample solution not exceeding 1 mm of mercury at 60° over phos-
rs = peak response of mesna related compound A phorus pentoxide for 2 h.
from Standard solution 1 Acceptance criteria: NMT 1.0%
G = concentration of USP Mesna Related © PH (791)
Compound A RS in Standard solution 1 Sample solution: 100 mg/mL of Mesna in carbon diox-
(mg/mL) ide-free water
Cu = concentration of Mesna in the Sample solution Acceptance criteria: 4.5—6.0
(mg/mL) ADDITIONAL REQUIREMENTS
Calculate the percentage of mesna related compound B
¢ PACKAGING AND STORAGE: Preserve in a tight container,
in the portion of Mesna taken: =
and store at room temperature. a)
Result = (ru/rs) x (Cs/Cu) x 100 e USP REFERENCE STANDARDS (11) uv
USP Mesna RS
tu = peak response of mesna related compound B USP Mesna Related Compound A RS =
fe)
from the Sample solution 2-(Acetylthio)ethane-1-sulfonic acid. S
ls peak response of mesna related compound B C4HgO4S2 184.22 °
©
i

from Standard solution 1 USP Mesna Related Compound B RS =

Cs = concentration of USP Mesna Related 2,2'-Disulfanediylbis(ethane-1-sulfonic acid). J
C4Hip06S4 282.36 mo]
CompoundB RS in Standard solution 1 >
(mg/mL) my
Cu = concentration of Mesna in the Sample solution
(mg/mL)
Calculate the percentage of anyepectied impurities
(thiouronium ethanesulfonic acia, Mesoridazine Besylate
guanidinethiouronium ethanesulfonic acid, and mesna
triazine analog) and any unspecified impurities in the
portion of Mesna taken:
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100

ru = peak response of any specified or unspecified

individual impurity from the Sample solution
ls = peak response of mesna from Standard
solution 2 Ca H26N2OS2 - CeHeO3S 544.75
Gs = concentration of USP Mesna RS in Standard 10H-Phenothiazine, 10-[2-(1-methyl-2-piperidinyl)ethyl]-
solution 2 (mg/mL) 2-(methylsulfinyl)-, (£)-, monobenzenesulfonate.
Cu = concentration of Mesna in the Sample solution (4)-10-[2-(1 -Methyl-2-piperidyl)ethy!]-
(mg/mL) 2-(methylsulfinyl)phenothiazine monobenzenesulfonate
F = relative response factors (see Table 1) [32672-69-8].
Acceptance criteria: See Table 1.
» Mesoridazine Besylate contains not less than
98.0 percent and not more than 102.0 percent of
arena » CeHeO3S, calculated on the dried
asis.
 2602 Mesoridazine / Official Monographs
Packaging and storage—Preserve in tight, light-resistant
containers.
USP Reference standards (11)—
USP Mesoridazine Besylate RS
[NoTE—Throughout the following procedures, protect test
or assay specimens, the USP Reference Standard, and solu-
tions containing them, by conducting the procedures with-
out delay, under subdued light, or using low-actinic
glassware.]
Identification—
A: Infrared Absorption (197M).
B: Ultraviolet Absorption (197U)—
Solution: 10 ug per mL.
Medium: methanol.
Absorptivities at 263 nm, calculated on the dried basis, do
not differ by more than 3.0%.
PH (791): between 4.2 and 5.7, in a freshly prepared solu-
tion (1 in 100).
Loss on drying (731)—Dry it at 105° for 4 hours: it loses
not more than 0.5% of its weight.
Residue on ignition (281): not more than 0.2%.
Delete the following:
°Heavy metals, Method I! (231): 0.002%.
e ‘oiticia 1-Jan-2018)
Selenium (291)—The absorbance of the solution from the
Test Solution, prepared with 100 mg of Mesoridazine Besy-
late and 100 mg of magnesium oxide, is not greater than
one-half that from the Standard Solution (0.003%).
Ordinary impurities (466)—
Test solution: a solution in methanol having a known con-
centration of 14.1 mg per mL equivalent to 10 mg of
mesoridazine per mL.
Standard solution: methanol.
ie
“
a Eluant: a mixture of chloroform, isopropyl alcohol, and
rg— ammonium hydroxide (87:12:1).
Dp Visualization: 3, followed by spraying with 3% (v/v) aque-
° © PACKAGING AND STORAGE: Preserve in single-dose contain-
r= ous hydrogen peroxide.
5 Application volume: 10 wL.
= Limit: 3.0%.
a Assay—Dissolve about 150 mg of Mesoridazine Besylate,
~”
pe accurately weighed, in 70 mL of acetic anhydride, and ti-
trate with 0.1 N perchloric acid VS, determining the
endpoint potentiometrically. Perform a blank determination,
and make any necessary correction. Each mL of 0.1 N per-
chloric acid is equivalent to 27.24 mg of C2iH26N20S2-
CsH6O3S.
Mesoridazine Besylate Injection
DEFINITION
Mesoridazine Besylate Injection is a sterile solution of
Mesoridazine Besylate in Water for Injection. It contains
mesoridazine besylate (C21H26N2OS2 - CeHeO3S) equivalent
to NLT 90.0% and NMT 110.0% of the labeled amount
of mesoridazine (C21H26N20Sz).
Throughout the following procedures, protect samples, the
Reference Standard, and solutions containing them by
conducting the procedures without delay, under subdued
light, or using low-actinic glassware.
IDENTIFICATION
e A, IDENTIFICATION—ORGANIC NITROGENOUS BASES (181)
Sample solution: Dilute a volume of Injection, equiva-
lent to 50 mg of mesoridazine besylate, with 0.01 N
hydrochloric acid to 25 mL.
USP 41
Analysis: Proceed as directed in the chapter, beginning
with “Transfer the liquid to a separator”.
Acceptance criteria: Meets the requirements
ASSAY
© PROCEDURE
Conduct this procedure with minimum exposure to
light.
Standard solution and Sample solution: Proceed with
Injection as directed in Salts of Organic Nitrogenous
Bases (501), except use 1.0 mL each of the Standard
Preparation and the Assay Preparation in the Procedure.
Instrumental conditions
Mode: UV-Vis
Analytical wavelength: Maximum at about 262 nm
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
mesoridazine (C2iH2sN20Sz) in the portion of Injection
taken:
Result = (Au/As) x (Cs/Cu) x (Mn/Mr2) x 100
Au = absorbance of the Sample solution
As = absorbance of the Standard solution
Cs = concentration of USP Mesoridazine Besylate RS
in the Standard solution (g/mL)
Cu = nominal concentration of mesoridazine
besylate in the Sample solution (g/mL)
Ma = molecular weight of mesoridazine, 386.59
M2 = meu weight of mesoridazine besylate,
44.75
Acceptance criteria: 90.0%-110.0%
SPECIFIC TESTS
e BACTERIAL ENDOTOXINS TEST (85): It contains NMT 7.0
USP Endotoxin Units/mg of mesoridazine besylate.
e PH (791): 4.0-5.0
¢ OTHER REQUIREMENTS: It meets the requirements in Injec-
tions and Implanted Drug Products (1).
ADDITIONAL REQUIREMENTS
ers, preferably of Type | glass, protected from light.
Change to read:
° USP REFERENCE STANDARDS (11)
@ (CN 1-Alay-2018)
USP Mesoridazine Besylate RS
Mesoridazine Besylate Oral Solution
DEFINITION
Mesoridazine Besylate Oral Solution contains NLT 90.0%
and NMT 110.0% of the labeled amount of mesoridazine
(Ca HasN20S2).
Throughout the following procedures, protect samples, the
Reference Standard, and solutions containing them by
conducting the procedures without delay, under subdued
light, or using low-actinic glassware.
IDENTIFICATION
° A.
Conduct this test without exposure to daylight and with
the minimum necessary exposure to artificial light.
Standard solution: 14 mg/mL of USP Mesoridazine
Besylate RS in methanol
Sample solution: Transfer 4.0 mL of Oral Solution into
a separator, add 6 mL of 1 N sodium hydroxide and
10 mL of chloroform, shake for 2 min, and filter the
USP 41 Official Monographs / Mesoridazine 2603

chloroform layer through anhydrous sodium sulfate into Mn = molecular weight of mesoridazine, 386.59
a small, glass-stoppered conical flask. Mz = molecular weight of mesoridazine besylate,
Chromatographic system 544.75
Adsorbent: 0.25-mm layer of chromatographic silica Acceptance criteria: 90.0%-110.0%
gel mixture
Application volume: 10 uL PERFORMANCE TESTS
Developing solvent system: To a separator add ben- e DELIVERABLE VOLUME (698)
zene, alcohol, and ammonium hydroxide (10:2:1). For multiple-unit containers
sakes and allow the layers to separate. Use the upper Acceptance criteria: Meets the requirements
ayer. e UNIFORMITY OF DosAGE UNITS (905)
Spray reagent: Perchloric acid and water (15:85) For single-unit containers
Analysis Acceptance criteria: Meets the requirements
Samples: Standard solution and Sample solution
Allow the spots to dry and develop the chromatogram SPECIFIC TESTS
until the solvent front has moved about three-fourths © ALCOHOL DETERMINATION, Method | (611): 0.25%-1.0% of
of the length of the plate. Remove the plate from the the labeled amount of alcohol (C2HsOH) is found.
developing chamber, mark the solvent front, and allow ADDITIONAL REQUIREMENTS
the solvent to evaporate in a fume hood, Spray the © PACKAGING AND STORAGE: Preserve in tight, light-resistant
plate with Spray reagent, and heat at 80° for 2 min. containers, and store at a temperature not exceeding
Acceptance criteria: The principal spot of the Sample 25°.
solution corresponds in Rr value and color to that of the e LABELING: Label it to indicate that it is to be diluted to
Standard solution. the apenas strength with water or other suitable
ASSAY fluid before administration.
© PROCEDURE e USP REFERENCE STANDARDS (11)
Conduct this peocedure with the minimum necessary ex- USP Mesoridazine Besylate RS
posure to light.
Standard stock solution: 0.14 mg/mL of USP
Mesoridazine Besylate RS in chloroform prepared as fol-
lows. Transfer 14mg of USP Mesoridazine Besylate RS
to a 125-mL separator containing 30 mL of water. Mesoridazine Besylate Tablets
Render the solution alkaline with 10 mL of 1 N sodium
hydroxides and extract with three 30-mL periahsof DEFINITION
chloroform. Filter the extracts through anhydrous so- Mesoridazine Besylate Tablets contain mesoridazine besylate
dium sulfate into a 100-mL volumetric flask. Rinse the (Ca HasN20S2 - CeH6O3S) equivalent to NLT 90.0% and
filter with small portions of chloroform, collecting the NMT 110.0% of the labeled amount of mesoridazine
rinsings in the volumetric flask, and dilute with chloro- (C2iH26N20Sz).
form to volume. Throughout the following procedures, protect samples, the 4
Standard solution: 0.014 mg/mL of USP Mesoridazine Reference Standard, and solutions containing them by wn
Besylate RS from a suitable volume of Standard stock conducting the procedures without delay, under subdued Ss)
solution and chloroform light, or using low-actinic glassware. =
Sample stock solution: Nominally 1 mg/mL of re}
mesoridazine in chloroform prepared as follows. Pipet a IDENTIFICATION =
© A, IDENTIFICATION—ORGANIC NITROGENOUS BASES (181): ey
volume of Oral Solution, equivalent to 100 mg of
Meet the requirements eo}|
mesoridazine, into a separator containing 30 mL of 2
water. Render the solution alkaline with 10 mL of 1N ASSAY Be)
sodium bydeoide, and extract with three 30-mL por- © PROCEDURE
ay
“
tions of chloroform. Filter the extracts through anhy- Mobile phase: Acetonitrile, triethylamine, and water
drous sodium sulfate into a 100-mL volumetric flask. (850:1:150)
Rinse the filter with small portions of chloroform, col- Standard solution: 0.35 mg/mL of USP Mesoridazine
lecting the rinsings in the volumetric flask, and dilute Besylate RS in methanol
with chloroform to volume. System suitability solution: 0.025 mg/mL of thi-
Sample solution: Nominally 0.1 mg/mL of oridazine hydrochloride in Standard solution
mesoridazine from a suitable volume of Sample stock Sample solution: Nominally 0.25 mg/mL of
solution and chloroform mesoridazine prepared as follows. Transfer an amount
Instrumental conditions of powder equivalent to NLT 50 mg of mesoridazine,
Mode: UV-Vis from NLT 20 powdered Tablets, to a suitable volumetric
Analytical wavelength: About 267 nm flask. Add 75% of the flask volume of methanol, shake
Cell: 1.cm by mechanical means for 15 min, and dilute with meth-
Blank: Chloroform
anol to volume. Sonicate for 30 min, and allow dis-
Analysis persed material to settle. Filter through a 0.25-um disk,
Samples: Standard solution, Sample solution, and Blank discarding the first 20 mL of the filtrate.
Calculate the percentage of labeled amount of Chromatographic system
mesoridazine (C21H26N2OS2) in the portion of Oral So- (See Chromatography (621), System Suitability.)
lution taken: Mode: LC
Result = (Au/As) x (Cs/Cu) x (Mnl/Mj2) x 100 Detector: UV 265 nm
Column: 4.6-mm x 25-cm; packing L1
Au = absorbance of the Sample solution Flow rate: 2.5 mL/min
As = absorbance of the Standard solution Injection volume: 10 uL
Gs = concentration of USP Mesoridazine Besylate RS System suitability
in the Standard solution (g/mL) Samples: Standard solution and System suitability
Cy = nominal concentration of mesoridazine in the solution
Sample solution (ug/mL)
 2604 Mesoridazine / Official Monographs
Suitability requirements
Resolution: NLT 1.0 between mesoridazine besylate
and thioridazine hydrochloride, System suitability
solution
Column efficiency: NLT 750 theoretical plates for the
mesoridazine peak, System suitability solution
Relative standard deviation: NMT 2.0%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
mesoridazine (C21H26N2OSz) in the portion of Tablets
taken:
Result = (ru/rs) x (Cs/Cu) x (Mn/M,2) x 100
ru = peak response from the Sample solution
Is peak response from the Standard solution
Cs concentration of USP Mesoridazine Besylate RS
in the Standard solution (mg/mL)
Cu = nominal concentration of mesoridazine in the
Sample solution (mg/mL)
Mn = molecular weight of mesoridazine, 386.59
M2 = molecular weight of mesoridazine besylate,
544.75
Acceptance criteria: 90.0%-110.0%
PERFORMANCE TESTS
e DISSOLUTION (711)
Medium: 0.01 N hydrochloric acid; 1000 mL
Apparatus 2: 100 rpm
Time: 60 min
Standard solution: USP Mesoridazine Besylate RS in
Medium. [NoTe—A volume of methanol not exceeding
1% of the final total volume may be used to prepare
the Standard solution.]
Sample solution: Filter the solution under test, suitably
” diluted with Medium, if necessary, to a concentration
£ that is similar to the Standard solution.
Q.
3 Instrumental conditions
- Mode: UV
D Analytical wavelength: Maximum at about 261 nm
fo}
e Tolerances: NLT 80% (Q) of the labeled amount of
fo} mesoridazine (C2:H26N2OSz) is dissolved.
= e UNIFORMITY OF DOSAGE UNITS (905): Meet the
a requirements
7)
=) ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed, light-
resistant containers. Preserve Tablets with an opaque
coating in well-closed containers.
e USP REFERENCE STANDARDS (11)
USP Mesoridazine Besylate RS
Mestranol
CrH26O2
19-Norpregna-1,3,5(10)-trien-20-yn-17-ol, 3-methoxy-,
(17a)-; . Au
3-Methoxy-19-nor-17a-pregna-1 ,3,5(10)-trien-20-yn-17-ol
[72-33-3].
DEFINITION
Mestranol contains NLT 97.0% and NMT 102.0% of mestra-
nol (CzH26O2), calculated on the dried basis.
USP 41
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
e B, ULTRAVIOLET ABSORPTION (197U)
Sample solution: 100 tg/mL of Mestranol in methanol
Acceptance criteria: Meets the requirements
ec
Standard solution: 1 mg/mL of USP Mestranol RS in
chloroform
Sample solution: 1 mg/mL of Mestranol in chloroform
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.)
Mode: TLC
Adsorbent: 0.25-mm layer of chromatographic silica
gel mixture
Application volume: 10 uL
Developing solvent system: Chloroform and dehy-
drated alcohol (29:1)
Spray reagent: Prepare as directed in Solution A in the
Mee a .
Analysis
Samples: Standard solution and Sample solution
Apply both solutions toa line parallel to and 2.5 cm
rom the bottom edge of a thin-layer chromatographic
plate. Place the plate in the Developing solvent system.
Develop the chromatogram until the solvent front has
moved about 15 cm from the origin. Remove the plate
from the chamber, allow the solvent to evaporate, and
spray with Spray reagent. Heat the plate in an oven at
105° for 5 min, and observe under long-wavelength
UV light.
Weare criteria: The R; value of the principal spot
of the Sample solution corresponds to that of the Stan-
dard solution.
ASSAY
¢ PROCEDURE
Solution A: Pipet 30 mL of methanol into a 100-mL
volumetric flask contained in an ice bath. Add slowly
and cautiously and with continuous stirring about
65 mL of sulfuric acid, taking care that the temperature
remains below 15°. Allow the solution to warm to room
temperature, and dilute with sulfuric acid to volume.
Standard solution: 5 g/mL of USP Mestranol RS in
chloroform
Sample solution: 5 g/mL of Mestranol in chloroform
Analysis: Pipet 4 mL each of the Standard solution and
the Sample solution into separate glass-stoppered,
25-mL conical flasks. Evaporate the solutions under a
slow current of air, without the aid of heat, to dryness.
Dissolve the residue in 0.3 mL of methanol. Place the
flasks in a water bath maintained at a temperature of
25°, and pipet into each, with constant swirling, 10 mL
of Solution A. Insert the me in the flasks. At 6 min,
accurately timed after the addition of Solution A, con-
comitantly determine the absorbances of the solutions.
Instrumental conditions
Mode: Vis
Analytical wavelength: Maximum at about 545 nm
Cell: 1.cm
Blank: Solution A
Analysis
Samples: Standard solution, Sample solution, and Blank
Calculate the percentage of mestranol (C2:H26O2) in the
portion of Mestranol taken:
310.43 # -
Result= (Au/As) x (Cs/Cu) * 100
= absorbance of the Sample solution
As = absorbance of the Standard solution
Cs = concentration of USP Mestranol RS in the
Standard solution (ug/mL)
Cu = concentration of Mestranol in the Sample
solution (g/mL)
USP 41 Official Monographs / Metacresol 2605

Acceptance criteria: 97.0%-102.0% on the dried basis Carrier gas: Helium

Flow rate: 1.8 mL/min
SPECIFIC TESTS Injection type: Split ratio of 1:40
e Loss ON DRYING (731) Injection volume: 1 uL
Analysis: Dry at 105° for 3 h. System suitability
Acceptance criteria: NMT 1.0% Samples: System suitability solution and Standard
© MELTING RANGE OR TEMPERATURE (741); 146°-154°, but solution
the range between beginning and end of melting does [Note—The relative retention times for phenol,
not exceed 4°. orthocresol, paracresol, and metacresol are about 0.77,
e OPTICAL ROTATION, Specific Rotation (781S) 0.85, 0.99, and 1.00, respectively.]
Sample solution: 20mg, previously dried, per mL, in Suitability requirements
dioxane Resolution: NLT 1.4 between the paracreso! and
Acceptance criteria: +2° to +8° metacresol peaks, System suitability solution
Relative standard deviation: NMT 1.0% for the peak
ADDITIONAL REQUIREMENTS ratio of metacresol to phenol, Standard solution
e PACKAGING AND STORAGE: Preserve in well-closed, light- Analysis
resistant containers. Samples: Standard solution and Sample solution
e USP REFERENCE STANDARDS (11) Calculate the percentage of metacresol (C7HgO) in the
USP Mestranol RS portion of Metacresol taken:
Result = (Ru/Rs) x (Cs/Cu) x 100
Ru = ratio of the metacresol peak response to the
Metacresol phenol peak response from the Sample
solution
Rs = ratio of the metacresol peak response to the
phenol peak response from the Standard
solution
Gs = concentration of USP Metacresol RS in the
CHO 108.14 Standard solution (mg/mL)
3-Methylphenol; Cy = concentration of Metacresol in the Sample
3-Hydroxytoluene [108-39-4]. solution (mg/mL)
Acceptance criteria: 98.0%-102.0%
DEFINITION
Metacresol contains NLT 98.0% and NMT 102.0% of IMPURITIES
metacresol (C7Hg0). © ORGANIC IMPURITIES
System suitability solution: 5 mg/mL each of
IDENTIFICATION metacresol, orthocresol, and paracresol in methanol c
e A. INFRARED ABSORPTION (197F) Sensitivity solution: 5 g/mL of USP Metacresol RS in 4)
e B. The retention time of the major peak of the Sample methanol an]
solution corresponds to that of the Standard solution, as Sample solution: 10 mg/mL of Metacresol in methanol =
obtained in the Assay. Chromatographic system i}
(See Chromatography (621), System Suitability.) =
ASSAY Mode: GC }
e PROCEDURE Detector: Flame-ionization
ro}=
Internal standard solution: 1 mg/mL of USP Phenol RS 2
Column: 0.25-mm x 30-m; 0.25-um coating of G7 i}
in methanol Temperatures =
System suitability solution: 5 mg/mL each of Injection port: 200° as

metacresol, orthocresol, and paracresol in methanol Detector: 200°

Standard solution: 1mg/mL of USP Metacresol RS in Column: See Table 2.
the Internal standard solution
Sample solution: 1 mg/mL of Metacresol in the Internal
Table 2
standard solution
Chromatographic system Hold Time
(See Chromatography (621), System Suitability.) Initial Temperature Final at Final
Mode: GC Temperature Ramp Temperature | Temperature
Detector: Flame-ionization () (¢/min) () (min)
Column: 0.25-mm x 30-m; 0.25-jum coating of G7 90. 0 90. 10
Temperatures 90 2 150 15
Injection port: 200°
Detector: 200° Carrier gas: Helium
Column: See Table 1. Flow rate: 1.8 mL/min
Injection type: Split ratio of 1:40
Table 1 Injection volume: 1 pL
System suitability
Hold Time Samples: System suitability solution and Sensitivity
Initial Temperature Final at Final solution
Temperature Ramp Temperature | Temperature
Suitability requirements
Cy (¢/min) C) (min) Resolution: NLT 1.4 between the paracresol and
90 o 90 10 metacresol peaks, System suitability solution
90 2 120 0
120 10 150 3
 2606 Metacresol / Official Monographs USP 41

Signal-to-noise ratio: NLT 10, Sensitivity solution USP Reference standards(1 1)—
Analysis USP Metaproterenol Sulfate RS
Sample: Sample solution Identification—
Calculate the percentage of each individual impurity in A: Infrared Absorption (197K).
the portion of Metacresol taken:
B: To a solution of 10 mg in 1 mL of water add 1 drop of
Result = (ru/rz) x 100 ferric chloride TS: a violet color is produced.
C: It responds to the tests for Sulfate (191).
tu = peak area of each individual impurity from the D: The chromatogram of the Assay preparation obtained
Sample solution as directed in the Assay exhibits a major peak for
rr = sum of all the peak areas from the Sample metaproterenol, the retention time of which corresponds
solution with that exhibited in the chromatogram of the Standard
Acceptance criteria: See Table 3. Disregard any peak preparation obtained as directed in the Assay.
less than 0.05%.
pH (791): between 4.0 and 5.5, in a solution containing
100 mg per mL.
Table 3
Water Determination, Method | (921): not more than
Relative Acceptance 2.0%.
Retention Criteria, Residue on ignition (281): not more than 0.1%.

35:
0.5 Delete the following:

°Heavy metals, Method [1 (231): 0.001%. cortical 1-jan-2018)

0.1
fron (241)—Dissolve 2.0 g in 45 mL of water, add 2 mL of
1.0
hydrochloric acid, and mix: the limit is 5 ppm.
Limit of metaproterenone sulfate—lts absorptivity (see
SPECIFIC TESTS Ultraviolet-Visible Spectroscopy {857)) at 328 nm, determined
© CLARITY OF SOLUTION in an aqueous solution containing 9.0 mg per mL, is not
A. more than 0.009 (0.1%).
Analysis: Add 10 mL of Metacresol to 10 mL of hex- Isopropyl alcohol and methanol—
ane, and mix.
Isopropyl alcohol standard solution—Transfer about 0.3
Acceptance criteria: A clear solution is obtained. of isopropyl alcohol, accurately weighed, to a 100-mL volu-
B. metric flask containing about 10 mL of water, dilute with
Analysis: Add 1.0 mL of Metacresol to 20 mL of 1 N water to volume, and mix. Pipet 10 mL of the resulting solu-
sodium hydroxide, and mix. tion into a 100-mL volumetric flask, add about 85 mL of
Acceptance criteria: A clear solution is obtained. pyridine, mix, and allow to stand for 1 hour. Dilute with
py
vw
pyridine to volume, and mix. Pipet 5 mL of this solution to a
% ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant 50-mL volumetric flask, dilute with pyridine to volume, and
i
mix. The solution so obtained contains about 30 jg of iso-
D
-
containers.
i} e USP REFERENCE STANDARDS (11) propyl alcohol per mL.
rs USP Metacresol RS Methanol standard solution—Prepare as directed for /so-
Sj USP Phenol RS propyl alcohol standard solution, using about 0.1 g of metha-
2 Phenol. nol, accurately weighed. The resulting solution contains
s CeHoO = 94.11 about 10 jig of methanol per mL.
”
= Test preparation—Transfer about 1 g of Metaproterenol
Sulfate, accurately weighed, to a 100-mL volumetric flask,
dissolve in about 2 mL of water, dilute with pyridine to vol-
ume, and mix.
Metaproterenol Sulfate Chromatographic system—The gas chromatograph is
equipped with a flame-ionization detector and contains a
OH
H
2-m x 2-mm column packed with 0.1% liquid phase G25
HO. Nv CH, on 80- to 100-mesh support $7. The injection port is main-
J © H,S0, tained at a temperature of about 150°; the column is,pra-
SS chy
grammed for 2 minutes at 40°, to increase at a rate o
OH 2 about 15° per minute to 200°, and for 10 minutes at 200°;
the detector is maintained at about 250°; and helium is
(CinHi7NO3)2 + H2SO, 520.59 used as the carrier gas at a flow rate of about 15 mL per
1,3-Benzenediol, 5-[1-hydroxy-2-(1-methylethyl)amino]- minute.
ethyl-, (+)-, sulfate (2:1) (salt). Procedure—inject equal volumes (about 2 uL) of the Test
(+)-3,5-Dihydroxy-o-[(isopropylamino)methyl]benzyl alcohol preparation, the Isopropyl alcoho! standard solution, and the
sulfate (2:1) [5874-97-5]. Methanol standard solution successively into the gas chro-
matograph. Measure the responses of the isopropyl alcohol
» Metaproterenol Sulfate contains not less than peak and the methanol peak in each Se Deter-
98.0 percent and not more than 102.0 percent of mine the quantities, in mg, of isopropyl alcohol and metha-
(Ci1Hi7NO3)2 - H2SOx, calculated on the anhy- nek in the portion of Metaproterenol Sulfate taken by the
ormula:
drous, isopropyl alcohol-free, and methanol-free
basis. 0.1C(ru/ rs)
Packaging and storage—Preserve in tight, light-resistant in which C is the concentration, in ug per mL, of isopropyl
containers. alcohol or methanol in the /sopropy! alcohol standard solution
or the Methanol standard solution; and ry and rs are the re-
USP 41
sponses of the respective analytes in the Test preparation
and of the corresponding Isopropyl alcoho! standard solution
or Methanol standard solution: not more than 0.3% of iso-
propyl alcohol and not more than 0.1% of methanol are
found.
Assay—
Mobile phase—Dissolve 11.9 g of anhydrous dibasic so-
dium phosphate in water to make 1000 mL of solution, and
mix (Solution A). Dissolve 9.1 g of monobasic potassium
phosphate in water to make 1000 mL of solution, and mix
(Solution B). Mix 735 mL of Solution A and 140 mL of Solu-
tion B, add 125 mL of methanol, and mix. Filter and degas
this solution before use.
Standard preparation—Dissolve an accutately weighed
quantity of USP Metaproterenol Sulfate RS in 0.01 N hydro-
chloric acid to obtain a solution having a known concentra-
tion of about 2 mg per mL.
Assay preparation—Transfer about 100 mg of Metaproter-
enol Sulfate, accurately weighed, to a 50-mL volumetric
flask, dilute with 0.01 N hydrochloric acid to volume, and
mix.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 278-nm detector
and a 4.6-mm x 5-cm guard column that contains packing
L7 and a 4.6-mm x 25-cm analytical column that contains
10-uum packing L7. The flow rate is about 2 mL per minute.
Chromatograph the Standard preparation, and record the
peak responses as directed for Procedure: the column effi-
ciency determined from the analyte peak is not less than
500 theoretical plates, the tailing factor for the analyte peak
is not more than 3.0, and the relative standard deviation for
replicate injections is not more than 2.0%.
Procedure—Separately inject equal volumes (about 10 pL)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
ure the responses for the major peaks. Calculate the quan-
tity, in mg, of (CiiHi7NO3)2- H2SO, in the portion of
Metaproterenol Sulfate taken by the formula:
50C(ru/ rs)
in which C is the concentration, in mg per mL, of USP
Metaproterenol Sulfate RS in the Standard preparation, and
ry and rs are the peak responses from the Assay preparation
and the Standard preparation, respectively.
Metaproterenol Sulfate Inhalation
Aerosol
» Metaproterenol Sulfate Inhalation Aerosol is a
suspension of microfine Metaproterenol Sulfate in
fluorochlorohydrocarbon propellants in a pres-
surized container. It contains not less than
90.0 percent and not more than 110.0 percent of
the labeled amount of metaproterenol sulfate
[(C1iHizNO3)2 + H2SOa].
Packaging and storage—Preserve in small, nonreactive,
light-resistant aerosol containers equipped with metered-
dose valves and provided with oral inhalation actuators.
USP Reference standards (11)—
USP Metaproterenol Sulfate RS
Identification—The UV absorption spectrum of the solu-
tion from the Assay preparation, obtained as directed in the
Assay, exhibits maxima and minima at the same wave-
lengths as that of the Standard preparation prepared as di-
rected in the Assay.
Official Monographs / Metaproterenol 2607
Delivered dose uniformity over the entire contents:
meets the requirements for Metered-Dose Inhalers under
Aerosols, Nasal Sprays, Metered-Dose Inhalers, and Dry Powder
Inhalers (601).
PROCEDURE FOR DOSE UNIFORMITY—
Standard preparation—Using a suitable quantity of USP
Metaproterenol Sulfate RS, accurately weighed, prepare a
solution in 0.01 N hydrochloric acid to obtain a solution
having a known concentration of 0.05 mg per mL.
Test preparation—Discharge the minimum recommended
dose into the sampling apparatus and detach the inhaler as
directed. Rinse the apparatus (filter and interior) with four
5.0-mL portions of 0.01 N hydrochloric acid, and quantita-
tively transfer the rinsings to a 25-mL volumetric flask. Di-
lute with 0.01 N hydrochloric acid to volume, and mix.
Procedure—Transfer 20.0 mL portions of the Standard
preparation, the Test preparation, and 0.01 N hydrochloric
acid to serve as a blank to separate centrifuge tubes. Add
10.0 mL of chloroform to each, shake by mechanical means
for 5 minutes, and separate the layers by centrifuging for
5 minutes. Determine the absorbances of the respective
aqueous layers in 1-cm cells, at the wavelength of maxi-
mum absorbance at about 276 nm, with a suitable spectro-
photometer, against the blank. Calculate the quantity, in
mg, of metaproterenol sulfate [(Ci1Hi7NOs)2 - H2SO,] con-
tained in the minimum dose taken by the formula:
12.5CN(Au /As)
in which C is the concentration, in mg per mL, of USP
Metaprotereno! Sulfate RS in the Standard preparation; N is
the number of sprays discharged to obtain the minimum
dose; and Ay and As are the absorbances of the solutions
from the Test preparation and the Standard preparation, re-
spectively.
Particle size—Prime the valve of an Inhalation Aerosol
container by alternately shaking and firing it several times, Ce
and then actuate one measured spray onto a clean, dry mi- wa
uv
croscope slide held 5 cm from the end of the oral inhalation
actuator, perpendicular to the direction of the spray. Care- <<
fully rinse the slide with about 2 mL of chloroform, and al- i}
|
low to dry. Examine the slide under a microscope equipped i}
with a calibrated ocular micrometer, using 450x magnifica- eo}=
tion. Focus on the particles of 25 fields of view near the 2
center of the test specimen pattern, and note the size of the ib}
great majority of individual patties: they are less than 5 a
"
uum along the longest axis. Record the number and size of
all individual crystalline particles (not agglomerates) more
than 10 um in length measured along the longest axis: not
more than 10 such particles are observed.
Water—Transfer the contents of a weighed container to the
titration vessel by attaching the valve stem to an inlet tube.
Weigh the ie container and determine the weight of
the specimen taken. The water content, determined by
Method | under Water Determination (921), is not more than
0.075%.
Assay—Cool an accurately weighed Inhalation Aerosol con-
tainer for 10 minutes in a bath consisting of a mixture of
acetone and solid carbon dioxide. Cut the valve from the
aerosol container and allow the container to warm to room
temperature. When most of the propellants have evapo-
rated, transfer the residue in the container to a 250-mL
separator with the aid of 30 mL of chloroform and 50 mL of
0.01 N hydrochloric acid. Reserve the valve and the empty
container. Shake the separator for 1 minute and allow the
phases to separate. Transfer the chloroform phase to a sec-
ond 250-mL separator and the aqueous phase to a 250-mL
volumetric flask. Wash the chloroform phase with two
50-mL portions of 0.01 N hydrochloric acid, add the wash-
ings to the 250-mL volumetric flask, dilute with 0.01 N hy-
drochloric acid to volume, and mix. Transfer an accurately
measured volume of this stock solution, equivalent to about
10 mg of metaproterenol sulfate, to a 100-mL volumetric
 2608 Metaproterenol / Official Monographs
flask, dilute with 0.01 N hydrochloric acid to volume, and
mix. Dissolve an accurately weighed quantity of USP
Metaproterenol Sulfate RS in 0.01 N hydrochloric acid, and
dilute quantitatively and stepwise with the same solvent to
obtain a Standard solution having a known concentration of
about 100 tg per mL. Concomitantly determine the ab-
sorbances of both solutions at the wavelength of maximum
absorbance at about 276 nm, witha suitable spectropho-
tometer, using 0.01 N hydrochloric acid as the blank. Rinse
the empty aerosol container and the valve with water and
dry them at 105° for 10 minutes, allow to cool, and weigh.
Subtract the weight thus obtained from the original weight
of the Inhalation Aerosol container to obtain the weight of
the Inhalation Aerosol taken. Calculate the quantity, in mg,
of metaproterenol sulfate [(Ci1Hi7NO3)2 - H2SOx] in each mL
of the Inhalation Aerosol taken by the formula:
25(C/ V)(d/ W)(Au/As)
in which C is the concentration, in ug per mL, of USP
Metaproterenol Sulfate RS in the Standard solution, V is the
volume, in mL, of stock solution taken, W is the weight, in
g, of the Inhalation Aerosol taken, and Ay and As are the
absorbances of the solution from the Inhalation Aerosol and
the Standard solution, respectively. [The density, d, is deter-
mined as follows: Weigh a known volume (v) of the Inhala-
tion Aerosol in a suitable 5-mL gas-tight syringe equipped
witha linear valve. Calibrate the volume of the syringe by
filling to the 5-mL mark with dichlorotetrafluoroethane with-
drawn from a plastic-coated glass vial sealed with a neo-
prene multiple-dose rubber stopper and an aluminum seal,
using 1.456g per ml as the density of the calibrating liq-
uid. Maintain the dichlorotetrafluoroethane, the Inhalation
Aerosol sample, and the syringe (protected from becoming
wet) at 25° in a water bath. Obtain the sample, equivalent
to the same volume as that obtained during the sampling
procedure, from the Inhalation Aerosol by means of a sam-
pling device consisting of a replaceable rubber septum en-
os
nal
gaged in the plate threads at one end of a threaded fitting,
iy the opposite end of which contains a sharpened tube capa-
i]
_
D ble of puncturing the aerosol container, and a rubber gasket
re) areuneare tube to prevent leakage of the container con-
iS tents after puncture.* Calculate the density taken by the
5 formula:
= Assay preparation—Transfer an accurately measured vol-
a wiv
~ metaproterenol sulfate, to a 100-mL volumetric flask, dilute
=) in which w is the weight of the volume, v, of the Inhalation
Aerosol taken.]
Metaproterenol Sulfate Inhalation
Solution
» Metaproterenol Sulfate Inhalation Solution is a
sterile solution of Metaproterenol Sulfate in Puri-
fied Water. It may contain Sodium Chloride. It
contains not less than 90.0 percent and not more
than 110.0 percent of the labeled amount of
metaproterenol sulfate [(C11Hiz7NO3)2 - H2SO,].
Packaging and storage—Store in small, tight containers
that are well-filled or otherwise protected from oxidation.
Protect from light.
Labeling—Label it to indicate that the Inhalation Solution
is not to be used if its color is pinkish or darker than slightly
yellow or if it contains a precipitate.
USP Reference standards (11)—
* A suitable sampling system is available from Alltek Associates, P. O. Box
498, Arlington Heights, IL 60006.
USP 41
USP Metaproterenol Sulfate RS
Color and clarity—
Standard solution—Transfer 2.0 mL of 0.100 N iodine VS
to a 500-mL volumetric flask, dilute with water to volume,
and mix.
Procedure—Visually examine a portion of the Inhalation
Solution (Test solution) in a suitable clear glass test tube
against a white background: it is not pinkish, and it contains
no precipitate. If any yellow color is observed in the Test
solution, concomitantly determine the absorbances of the
Test solution and the Standard solution in 1-cm cells with a
suitable spectrophotometer set at 460 nm: the absorbance
of the Test solution does not exceed that of the Standard
solution.
Identification—
A: Apply 4 wl of the Inhalation Solution and 4 uL of an
aqueous solution of USP Metaproterenol Sulfate RS contain-
ing about 50 mg per mL toa suitable thin-layer chromato-
graphic plate (see Chromatography (621)) coated with a
0.25-mm layer of chromatographic silica gel mixture. Allow
the spots to dry, and develop the chromatogram in a sol-
vent system consisting of the upper layer of a freshly pre-
pared mixture of butyl alcohol, water, and formic acid
(50:25:7) until the solvent front has moved about three-
fourths of the length of the plate. Remove the plate from
the developing chamber, mark the solvent front, and allow
the solvent to evaporate. Locate the spots on the plate by
examination under short-wavelength UV light: the Rr value
of the principal spot obtained from the Inhalation Solution
corresponds to that obtained from the Standard solution.
B: The chromatogram of the Assay preparation obtained
as directed in the Assay exhibits a major peak for
metaproterenol, the retention time of which corresponds to
that exhibited in the chromatogram of the Standard prepa-
ration obtained as directed in the Assay.
Sterility Tests (71): meets the requirements.
pH (791): between 2.8 and 4.0.
Assay—
Mobile phase, Standard preparation, and Chromatographic
system—Prepare as directed in the Assay under Metaprotere-
nol Sulfate.
ume of Inhalation Solution, equivalent to about 200 mg of
with 0.01 N hydrochloric acid to volume, and mix.
Procedure—Proceed as directed for Procedure in the Assay
under Metaproterenol Sulfate. Calculate the quantity, in mg,
of metaproterenol sulfate [(C11H17NOs)2 + H2SOx] in each mL
of the Inhalation Solution taken by the formula:
100(C/V)(ru/ rs)
in which V is the volume, in mL, of Inhalation Solution
taken; and C, ru, and rs are as defined therein.
Metaproterenol Sulfate Oral Solution
» Metaproterenol Sulfate Oral Solution contains
not less than 90.0 percent and not more than
110.0 percent of the labeled amount of
metaproterenol sulfate [(C11Hi7NO3)2 - H2SOa].
Packaging and storage—Preserve in tight, light-resistant
containers.
USP Reference standards (11)—
USP Metaproterenol Sulfate RS
USP 41
Identification—
A: Transfer a portion of Oral Solution, equivalent to
about 10 mg of metaproterenol sulfate, to a separator, and
extract with four 30-mL portions of ether, discarding the
ether extracts. Apply 10 uL of the extracted portion of Oral
Solution to the lower right corner of a suitable thin-layer
chromatographic plate (see Chromatography (621)) coated
with a 0.25-mm layer of chromatographic silica gel mixture,
and allow to dry. gyclop the chromatogram in a solvent
system consisting of the lower layer of a well-shaken mix-
ture of dioxane, methylene chloride, alcohol, and ammo-
nium hydroxide (4:4:1:1). Allow the solvent front to move
about three-fourths of the length of the plate. Remove the
plate from the developing chamber, mark the solvent front,
and dry in vacuum at 35° to 40° for 30 minutes. Rotate the
plate 90°. At a point about four-fifths of the distance be-
tween the initial application of the Oral Solution extract and
the solvent front, apply 10 uL of a Standard solution of USP
Metaproterenol Sulfate RS in water containing about 2 mg
per mL. Proceed as directed in Identification test A under
Metaproterenol Sulfate Inhalation Solution, beginning with
“Allow the spots to dry”: the R- value of the principal spot
obtained from the Oral Solution corresponds to that ob-
tained from the Standard solution.
B: The retention time of the major peak for metaprotere-
nol in the chromatogram of the Assay preparation corre-
sponds to that in the chromatogram of the Standard prepa-
ration, as obtained in the Assay.
pH (791): between 2.5 and 4.0, in a solution obtained by
mixing 1 volume of Oral Solution and 4 volumes of water.
Assay—
Mobile phase—Mix 10 mL of formic acid and water to
make 1000 mL of solution. Filter and degas this solution
before use. Make adjustments if necessary (see System Suita-
bility under Chromatography (621)).
Standard preparation—Dissolve an accurately weighed
quantity of USP Metaproterenol Sulfate RS in water to ob-
tain a solution having a known concentration of about
0.2 mg per mL.
Assay preparation—Transfer an accurately measured vol-
ume of Oral Solution, equivalent to about 20 mg of
metaproterenol sulfate, to a 100-mL volumetric flask, dilute
with water to volume, and mix.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 278-nm detector,
a 4.6-mm x 5-cm guard column that contains packing L2,
and a 3.9-mm x 30-cm analytical column that contains
packing L1. [NoTe—After use, rinse the analytical column
with water and store with water in it.] The flow rate is
about 2 mL per minute. Chromatograph the Standard prepa-
ration, and record the peak responses as directed for Proce-
dure: the tailing factor for the analyte peak is not more than
3.0; and the relative standard deviation for replicate injec-
tions is not more than 2.0%.
Procedure—Separately inject equal volumes (about
100 uL) of the Standar pa porien and the Assay prepara-
tion into the chromatograph, record the chromatograms,
and measure the responses for the major peaks. Calculate
the quantity, in mg, of metaproterenol sulfate
[(Ci1HizNO3)2 - H2SOx] in each mL of the Oral Solution taken
by the formula:
100(C/ V)(ru
/rs)
in which C is the concentration, in mg per mL, of USP
Metaproterenol Sulfate RS in the Standard preparation;V is
the volume, in mL, of Oral Solution taken; and ry and rs are
oe responses from the Assay preparation and the Stan-
dard preparation, respectively.
Official Monographs / Metaproterenol 2609
Metaproterenol Sulfate Tablets
» Metaproterenol Sulfate Tablets contain not less
than 92.0 percent and not more than 108.0 per-
cent of the labeled amount of metaproterenol
sulfate [(Ci1Hi7NO3)2 . H2SOa,].
Packaging and storage—Preserve in well-closed, light-re-
sistant containers.
USP Reference standards (11)—
USP Metaproterenol Sulfate RS
Identification—
A: Powder a number of Tablets, equivalent to about
100 mg of metaproterenol sulfate, add 10 mL of water, stir
for about 3 minutes, and centrifuge. Use the clear solution
so obtained as the Test solution. Dissolve a suitable quantity
of USP Metaprotereno! Sulfate RS in water to obtain a Stan-
dard solution having a concentration of 10 mg per mL. Ap-
ply separate 10-uL portions of the Test solution and the
Standard solution to a thin-layer chromatographic plate (see
Chromatography (621)) coated with a 0.25-mm layer of
chromatographic silica gel mixture. Proceed as directed in
Identification test A under Metaproterenol Sulfate Inhalation
Solution, beginning with “Allow the spots to dry”: the Rr
value of the principal spot obtained from the Test solution
corresponds to that obtained from the Standard solution.
B: Mix a quantity of powdered Tablets, equivalent to
about 20 mg of metaproterenol sulfate, with 5 mL of water,
and filter: the filtrate responds to the tests for Sulfate (191).
C: The chromatogram of the Assay preparation obtained
as directed in the Assay exhibits a major peak for
metaproterenol, the retention time of which corresponds to
that exhibited in the chromatogram of the Standard prepa-
ration obtained as directed in the Assay.
cS
Dissolution (711)— al
uv
Medium: — water; 500 mL.
Apparatus 2: 50 rpm. =
fo}
Time: 30 minutes. |
°
Procedure—Determine the amount of (C1,Hi7NO3)2 - io}
H2SO, dissolved from UV absorbances at the wavelength of me
2
maximum absorbance at about 276 nm of filtered portions mo]
of the solution under test, suitably diluted with Dissolution a
a
Medium, if necessary, in comparison with a Standard solu-
tion having a known concentration of USP Metaproterenol
Sulfate RS in the same Medium.
Tolerances—Not less than 70% (Q) of the labeled amount
of (CiHiz7NO3)2 - H2SOx is dissolved in 30 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Assay—
Mobile phase, Standard preparation, and Chromatographic
system—Prepare as directed in the Assay under Metaprotere-
nol Sulfate.
Assay preparation—Transfer 20 Tablets to a 500-mL coni-
cal flask. Add an accurately measured volume of 0.01 N
yngtots acid sufficient to yield a solution containing
about 2 mg of metaproterenol sulfate per mL, shake by me-
chanical means for 30 minutes, and filter. Use the filtrate so
obtained as the Assay preparation.
Procedure—Proceed as directed for Procedure in the Assay
under Metaprotereno! Sulfate. Calculate the quantity, in mg,
of metaproterenol sulfate [(Ci;Hi7NO3)2- H2SOx] in each
Tablet taken by the formula:
(CV
/ 20)(ru/ rs)
in which Vis the volume, in mL, of 0.01 N hydrochloric
acid added; and C, ry, and rs are as defined therein.
 2610 Metaraminol / Official Monographs
Metaraminol Bitartrate
HOH OHO H
HO. \ UCHs 9 Oe
rs Ho
HONS HOH 6
CoHi3NO2 + C4HeOg 317.29
Benzenemethanol, a-(1-aminoethyl)-3-hydroxy-, [R-(R*, 5*)]-,
[R-(R*, R*)]-2,3-dihydroxybutanedioate (1:1) (salt).
(-)-a-(1-Aminoethyl)-m-hydroxybenzyl alcohol tartrate (1:1)
(salt) [33402-03-8].
» Metaraminol Bitartrate contains not less than
99.0 percent and not more than 100.5 percent of
CsHi3NO2 + C4HeO¢, calculated on the dried basis.
Packaging and storage—Preserve in well-closed contain-
ers. Store at 25°, excursions permitted between 15° and
30°:
USP Reference standards (11)—
USP Metaraminol Bitartrate RS
Identification—
A: Infrared Absorption (197K).
B: To 0.5 mL of a solution (1 in 2000) add 1 mL of Folin-
Ciocalteu phenol TS, then add 5 mL of sodium carbonate
solution (1 in 10), mix, and allow to stand for 5 minutes: an
intense blue color appears (presence of a phenol).
C: To 4 mL of a solution (1 in 2000) add 5 mL of pH 9.6
alkaline borate buffer (see Buffer Solutions in the section Re-
agents, Indicators, and Solutions), then add about 5 mg of B-
naphthoquinone-4-sodium sulfonate, mix until dissolved,
and allow to stand for 5 minutes. Add 0.2 mL of benzalko-
nium chloride solution (1 in 100), mix, add 5 mL of toluene,
al
and shake: the toluene layer turns purple immediately (dis-
fs tinction from phenylephrine).
os Melting range (741): between 171° and 175°.
ic]
—
D Specific rotation (7815S): between —31.5° and —33.5° (A=
° 405 nm).
= Test solution: 100 mg per mL, in 0.5 N hydrochloric acid.
C}
3 PH (791): between 3.2 and 3.5, in a solution (1 in 20).
Q Loss on drying (731)—Dry it at 105° for 2 hours: it loses
wv not more than 1.0% of its weight.
> Residue on ignition (281): not more than 0.1%.
Delete the following:
°Heavy metals, Method | (231): 0.002%. covticial 1-Jan-2018)
Assay—Dissolve about 600 mg of Metaraminol Bitartrate,
accurately weighed, in 20 mL of glacial acetic acid, warming
slightly to effect solution. Cool the solution to room temper-
ature, add 2 drops of crystal violet TS, and titrate with 0.1 N
erchloric acid VS to an emerald-green color. Perform a
Elan determination, and make any necessary correction.
Each mL of 0.1 N perchloric acid is equivalent to 31.73 mg
of CoHi3NOz - CxHoOc.
Metaraminol Bitartrate Injection
» Metaraminol Bitartrate Injection is a sterile solu-
tion of Metaraminol Bitartrate in Water for Injec-
tion. It contains, in each mL, an amount of
metaraminol bitartrate equivalent to not less than
9.0 mg and not more than 11.0 mg of metarami-
nol (CoH13NO2).
USP 41
Packaging and storage—Preserve in single-dose or in
multiple-dose containers, preferably of Type | glass, pro-
tected from light.
Change to read:
usP Reference standards (11)—
@ (CN 1-May-2018)
USP Metaraminol Bitartrate RS
Identification—
A: Evaporate a 1-mL portion to dryness: the residue so
obtained meets the requirements for Identification test A
under Metaraminol Bitartrate.
B: It meets the requirements for Identification tests B and
C under Metaraminol Bitartrate.
Bacterial Endotoxins Test (85)—It contains not more
than 3.5 USP Endotoxin Units per mg of metaraminol.
pH (791): between 3.2 and 4.5.
Particulate Matter in Injections (788): meets the re-
quirements for small-volume injections.
Other requirements—it meets the requirements under /n-
jections and Implanted Drug Products (1).
Assay—
0.0032 M Hexanesulfonate buffer—Mix 600 mg of sodium
1-hexanesulfonate with water to obtain 1000 mL of solution,
adjust with phosphoric acid to a pH of 3.0 + 0.05, and
ilter.
Mobile phase—Prepare a suitable degassed and filtered
mixture of methanol and 0.0032 M Hexanesulfonate buffer
(7:3). Make adjustments if necessary (see System Suitability
under Chromatography (621)).
Standard preparation—Dissolve an accurately weighed
quantity of USP Metaraminol Bitartrate RS in water to obtain
a solution having a known concentration of about 0.2 mg of
metaraminol per mL.
Assay preparation—Transfer an accurately measured vol-
ume of Injection, equivalent to about 20 mg of metarami-
nol, to a 100-mL volumetric flask, dilute with water to vol-
ume, and mix.
System suitability preparation—Prepare a solution of pro-
pylparaben in alcohol containing 0.4 mg per mL. Mix 1 vol-
ume of this solution with 99 volumes of the Standard prepa-
ration.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 264-nm detector
and a 4-mm x 25-cm column that contains packing L7. The
flow rate is about 1 mL per minute. Chromatograph the Sys-
tem suitability preparation and the Standard preparation, and
record the peak responses as directed for Procedure: the col-
umn efficiency is not less than 2600 theoretical plates, the
resolution, R, between the metaraminol bitartrate and pro-
pylparaben peaks is not less than 3.0 with propylparaben
eluting first, and the relative standard deviation for replicate
injections is not more than 2.0%.
Procedure—Separately inject equal volumes (about 10 uL)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
ure the responses for the major peaks. Calculate the quan-
tity, in mg, of metaraminol (CsH:3NOz2) in each mL of the
Injection taken by the formula:
100(C/ V)(ru / 15)
in which C is the concentration, in mg per mL, of metarami-
nol represented by the USP Metaraminol Bitartrate RS in the
Standard preparation; V is the volume, in mL, of Injection
taken; and ry and rs are the peak responses obtained from
Hie. assay preparation and the Standard preparation, respec-
tively.
USP 41 Official Monographs / Metaxalone 2611

Cu = concentration of Metaxalone in the Sample

Metaxalone solution (mg/mL)
Acceptance criteria: 98.0%-102.0% on the dried basis
Na IMPURITIES
Hey oa Ly © RESIDUE ON IGNITION (281): NMT 0.30%
W © ORGANIC IMPURITIES, PROCEDURE 1
If metaxalone related compound B, metaxalone related
|
Oy compound C, or N-benzyl metaxalone is a known pro-
cess impurity, Organic Impurities, Procedure 2 is
Cy2HisNO3 224.25, recommended.
2-Oxazolidinone, 5-[(3,5-dimethylphenoxy)methyl]-; Solution A: 0.1% Trifluoroacetic acid in water
5-[(3,5-Xylyloxy)methy|]-2-oxazolidinone; Solution B: 0.1% Trifluoroacetic acid in acetonitrile
5-[(3,5-Dimethylphenoxy)methyl]-1,3-oxazolidin-2-one Mobile phase: See Table 1.
[1665-48-1].
Table 1
DEFINITION
Metaxalone contains NLT 98.0% and NMT 102.0% of Solution A Solution B
metaxalone (C;2H:sNO3), calculated on dried basis.
IDENTIFICATION 1
e A. INFRARED ABSORPTION (197A) or (197K)
eh
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Sample solution, as 15.
obtained in the Assay.
Diluent: Acetonitrile and water (75:25)
ASSAY System suitability solution: 4mg/mL of USP Metax-
© PROCEDURE alone RS and 0.01 mg/mL of 3,5-dimethylphenol in
Buffer: 0.68 g/L of monobasic potassium phosphate in Diluent
water. Adjust with phosphoric acid to a pH of 4.5. Sensitivity solution: 0.002 mg/mL of USP Metaxalone
Mobile phase: Methanol and Buffer (50:50) RS in Diluent
Standard stock solution: 0.5 mg/mL of USP Metax- Sample solution: 4 mg/mL of Metaxalone in Diluent
alone RS prepared as follows. Transfer a suitable quan- Chromatographic system
tity of USP Metaxalone RS to a suitable volumetric flask. (See Chromatography (621), System Suitability.)
Add 50% of the flask volume of methanol. Sonicate for Mode: LC
5 min to dissolve. Add 40% of the flask volume of Detector: UV 273 nm
Buffer, and mix. Cool to room temperature. Dilute with Column: 4.6-mm x 25-cm; 5-um packing L68
Buffer to volume. Flow rate: 2.0 mL/min
Standard solution: 0.05 mg/mL of USP Metaxalone RS Injection volume: 10 uL =
Ww
from the Standard stock solution in Mobile phase System suitability xe]
Sample stock solution: 0.5 mg/mL of Metaxalone pre- Samples: System suitability solution and Sensitivity
pared as follows. Transfer a suitable quantity of Metax- solution Ex
alone to a suitable volumetric flask. Add 50% of the [Note—See Table 2 for the relative retention times.] i}
PJ
flask volume of methanol. Sonicate for 5 min to dis- Suitability requirements }
solve. Add 40% of the flask volume of Buffer, and mix. Resolution: NLT 2.0 between metaxalone and 3,5- ro}=
Cool to room temperature. Dilute with Buffer to dimethylphenol, System suitability solution
volume. Signal-to-noise ratio: NLT 10, Sensitivity solution 3
Analysis 7
Sample solution: 0.05 mg/mL of Metaxalone from the a)
Sample stock solution in Mobile phase Sample: Sample solution
Chromatographic system Calculate the percentage of each impurity in the por-
(See Chromatography (621), System Suitability.) tion of Metaxalone taken:
Mode: LC
Detector: UV 226 nm Result = (ru/r7) x 100
Column: 4.6-mm x 15-cm; 5-um packing L1
Column temperature: 50° tu = peak response of each impurity from the
Flow rate: 1 mL/min Sample solution
Injection volume: 20 uL nr = sum of the peak responses of metaxalone and
Run time: NLT 2 times the retention time of impurities from the Sample solution
metaxalone Acceptance criteria: See Table 2. Disregard any impu-
System suitability rity peaks less than 0.03%.
Sample: Standard solution
ee requirements Table 2
Tailing ‘a factor: NMT 2.0 Relative Acceptance
Relative standard deviation: NMT 0.73% Retention Criteria,
Analysis Name Time NMT (%)
Samples: Standard solution and Sample solution
Calculate the percentage of metaxalone (Ci2HisNOs) in Metaxalone 1.0 —
the portion of Metaxalone taken: 3,5-Dimethylphenol 11 0.05
Any individual a
Result = (ru/rs) x (Cs/Cy) x 100 unspecified impurity 0.05
Total impurities = 0.50
ty = peak response from the Sample solution
ls = peak response from the Standard solution © ORGANIC IMPURITIES, PROCEDURE 2
G = concentration of USP Metaxalone RS in the If metaxalone related compound B, metaxalone related
Standard solution (mg/mL) compound C, or N-benzy! metaxalone is a known pro-
 2612 Metaxalone / Official Monographs USP 41

cess impurity, Organic Impurities, Procedure 2 is recom- Table 3 (Continued)

mended. If the article complies with Procedure 2, the
Relative Relative Acceptance
labeling indicates that it meets Organic Impurities, Proce-
Retention | Response Criteria,
dure 2.
Name Time Factor NMT (%)
Buffer, Mobile phase, and Standard stock solution:
Proceed as directed in the Assay. Metaxalone related
Standard solution: 0.001 mg/mL of USP Metaxalone compound C 3.6 1.0 0.05
RS from the Standard stock solution in Mobile phase N-Benzylmetaxalone? 6.9 0.64 0.05
Impurity stock solution: 0.2 mg/mL each of USP Any individual _
Metaxalone Related Compound B RS and USP Metax- unspecified impurity 1.0 0.05
alone Related Compound C RS in methanol. Sonicate to Total impurities — => 0.3
dissolve if necessary. 2 3-Benzyl-5-[(3,5-dimethylphenoxy)methyl]oxazolidin-2-one.
Peak identification solution: 1 mg/mL of USP Metax-
alone RS and 0.02 mg/mL each of USP Metaxalone Re- SPECIFIC TESTS
lated Compound B RS and USP Metaxalone Related e Loss ON DRYING (731)
Compound C RS prepared as follows. Transfer a suitable Analysis: Dry at 90° for 2 h.
qua of USP Metaxalone RS to a suitable volumetric Acceptance criteria: NMT 0.5%
flask. Add 50% of the flask volume of methanol, and
sonicate to dissolve. Transfer suitable volumes of /mpu- ADDITIONAL REQUIREMENTS
rity stock solution to the flask. Dilute with Buffer to © PACKAGING AND STORAGE: Preserve in tight containers.
volume. e LABELING: The label states with which Organic Impurities
Sample solution: 2.0 mg/mL of Metaxalone prepared procedure the article complies if Organic Impurities, Proce-
as follows. Transfer a suitable quantity of Metaxalone to dure 7 is not used.
a suitable volumetric flask. Add 50% of the flask volume e USP REFERENCE STANDARDS (11)
of methanol. Sonicate for 5 min to dissolve. Add 40% USP Metaxalone RS
of the flask volume of Buffer, and mix. Cool to room USP Metaxalone Related Compound B RS
temperature. Dilute with Buffer to volume. 1-Amino-3-(3,5-dimethylphenoxy)propan-2-ol.
Chromatographic system CiHiyNO2 = 195.26
(See Chromatography (621), System Suitability.) USP Metaxalone Related Compound C RS
Mode: LC Bis[2-hydroxy-3-(3,5-dimethylphenoxy)propyl]amine.
Detector: UV 226 nm CoHsiNOs = 373.49
Column: 4.6-mm x 15-cm; 5-um packing L1
Column temperature: 50°
Flow rate: 1 mL/min
Injection volume: 20 uL
Run time: NLT 8 times the retention time of Metaxalone Tablets
metaxalone
ee
”
System suitability DEFINITION
a Sample: Standard solution Metaxalone Tablets contain NLT 90.0% and NMT 110.0%
—
J Suitability requirements of the labeled amount of metaxalone (Ci2HisNOs).
Dd Tailing factor: NMT 2.0
° Relative standard deviation: NMT 10.0% IDENTIFICATION
= Signal-to-noise ratio: NLT 25 e A. The retention time of the major peak of the Sample
iS Analysis solution corresponds to that of the Standard solution, as
= Samples: Standard solution and Sample solution obtained in the Assay.
2 Use the Peak identification solution to identify the peaks.
a)
Calculate the percentage of each impurity in the por- ASSAY
=} © PROCEDURE
tion of Metaxalone taken:
Buffer: 0.68 g/L of monobasic potassium phosphate.
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100 Adjust with phosphoric acid to a pH of 4.5.
Mobile phase: Methanol and Buffer (50:50)
ru = peak response of each impurity from the Standard stock solution: 0.5 mg/mL of USP Metax-
Sample solution alone RS prepared as follows. Transfer a suitable
rs = peak response of metaxalone from the amount of USP Metaxalone RS to a suitable volumetric
Standard solution flask. Add 50% of the flask volume of methanol and
Gs = concentration of USP Metaxalone RS in the sonicate to dissolve. Dilute with Buffer to volume.
Standard solution (mg/mL) Standard solution: 0.05 mg/mL of USP Metaxalone RS
Cu = concentration of Metaxalone in the Sample from Standard stock solution in Mobile phase
solution (mg/mL) Sample stock solution: Nominally 1.0 mg/mL of
F = relative response factor for the corresponding metaxalone from NLT 20 Tablets prepared as follows.
impurity (see Table 3) Transfer a portion of finely powdered Tablets equivalent
Acceptance criteria: See Table 3. Disregard any impu- to NLT 500 mg of metaxalone to a suitable volumetric
rity peaks less than 0.03%. flask. Add 50% of the flask volume of methanol and
sonicate for 10 min with occasional swirling. Shake on a
mechanical shaker for 15 min. Add 40% of the flask
Table 3
volume of Buffer and allow the solution to cool to room
Relative Relative Acceptance temperature. Dilute with Buffer to volume. Pass a por-
Retention | Response Criteria, tion of the solution through a PVDF filter of 0.45-m
Name Time Factor NMT (%) pore size. Discard the first 5 mL. Use the filtrate.
Metaxalone related Sample solution: Nominally 0.05 mg/mL of metax-
compound B 0.35: 1.0 0.05 alone from Sample stock solution and Mobile phase
Metaxalone 1.0 = =
2 3-Benzyl-5-[(3,5-dimethylphenoxy)methyl]oxazolidin-2-one.
USP 41 Official Monographs / Metaxalone 2613

Chromatographic system Impurity stock solution: 0.2 mg/mL each of USP

(See Chromatography (621), System Suitability.) Metaxalone Related Compound B RS and USP Metax-
Mode: LC alone Related CompoundC RS in methanol. Sonicate to
Detector: UV 226 nm dissolve if necessary.
Column: 4.6-mm x 15-cm; 5-"um packing L1 Peak identification solution: 1 mg/mL of USP Metax-
Column temperature: 50° alone RS and 0.02 mg/mL each of USP Metaxalone Re-
Flow rate: 1 mL/min lated Compound B RS and USP Metaxalone Related
Injection volume: 20 uL CompoundC RS prepared as follows. Transfer a suitable
Run time: NLT 2 times the retention time of uantity of USP Metaxalone RS to a suitable volumetric
metaxalone flask. Add 50% of the flask volume of methanol and
System suitability sonicate to dissolve. Transfer suitable volumes of Impu-
Sample: Standard solution rity stock solution to the flask. Dilute with Buffer to
Suitability requirements volume.
Tailing factor: NMT 2.0 Sensitivity solution: 0.5 g/mL of USP Metaxalone RS
Relative standard deviation: NMT 0.73% from Standard solution and Mobile phase
Analysis Sample solution: Nominally 1.0 mg/mL of metaxalone
Samples: Standard solution and Sample solution prepared from NLT 20 Tablets as follows. Transfer a por-
Calculate the percentage of the labeled amount of tion of NLT 20 finely powdered Tablets equivalent to
metaxalone (Ci2HisNOs) in the portion of Tablets NLT 500 mg of metaxalone to a suitable volumetric
taken: flask. Add 50% of the flask volume of methanol and
sonicate for 10 min with occasional swirling. Shake on a
Result = (ru/rs) x (Cs/Cy) x 100 mechanical shaker for 15 min. Add 40% of the flask
volume of Buffer and cool to room temperature. Dilute
tu = peak response of metaxalone from the Sample with Buffer to volume. Pass a portion of the solution
solution through a PVDF filter of 0.45-1m pore size. Discard the
rs = peak response of metaxalone from the first 5 mL.
Standard solution System suitability
Cs = concentration of USP Metaxalone RS in the Samples: Peak identification solution and Sensitivity
Standard solution (mg/mL) Solution
Cu = nominal concentration of metaxalone in the [Note—See Table 1 for relative retention times.]
Sample solution (mg/mL) Suitability requirements
Acceptance criteria: 90.0%-110.0% Tailing factor: NMT 2.0, Sensitivity solution
Relative standard deviation: NMT 10.0% for the
PERFORMANCE TESTS metaxalone peak, Sensitivity solution
e DISSOLUTION (711) Signal-to-noise ratio: NLT 25 for the metaxalone
Medium: 0.5% sodium lauryl sulfate; 900 mL peak, Sensitivity solution
Apparatus 2: 100 rpm Analysis
Time: 60 min Samples: Standard solution, Peak identification solution, fons
Buffer, Mobile phase, Chromatographic system, and and Sample solution a)
System suitability: Proceed as directed in the Assay, Use the Peak identification solution to identify the peaks.
Z
cca use 270 nm for analysis. Calculate the percentage of each degradation product m4
Standard solution: (1/900) mg/mL of USP Metaxalone in the portion of Tablets taken: i)
RS, where L is the label claim of metaxalone, in mg/ =
Tablet, prepared as follows. Transfer a suitable quantity }
of USP Metaxalone RS to a suitable volumetric flask.
Result = (ru/rs) x (Cs/Cu) x 100 (to)|
iy
Add 4% of the flask volume of methanol, sonicate to ru = peak response of each degradation product 3
dissolve, and dilute with Medium to volume. from the Sample solution mr
Sample solution: Pass a portion of the solution under Is = peak response of metaxalone from the
a

test through a suitable PVDF membrane filter of 0.45- Standard solution

um pore size. Discard the first 5 mL of the filtrate and Cs = concentration of USP Metaxalone RS in the
use the remaining amount for analysis. Standard solution (mg/mL)
Analysis Cy = nominal concentration of metaxalone in the
Samples: Standard solution and Sample solution Sample solution (mg/mL)
Calculate the percentage of the labeled amount of Acceptance criteria: See Table 1.
metaxalone (C;2HisNO3) dissolved:
Result = (ru/rs) x Cs x Vx (1/L) x 100 Table 1
. Relative Acceptance
ty = peak response from the Sample solution Retention Cilteria,
rs = peak response from the Standard solution Name Time NMT (%)
Cs = concentration of: USP Metaxalone RS in the Metaxalone related
Standard solution (mg/mL) compounds 0.35 0.15
Vv = volume of the Medium, 900 mL = -
L = label claim of metaxalone (mg/Tablet) Metaxalone 1.0 =
Tolerances: NLT 60% (Q) of the labeled amount of Metaxalone related _
metaxalone (Ci2HisNOs) is dissolved. compound Ce 3.6
e UNIFORMITY OF DOSAGE UNITS (905): Meet the N-Benzylmetaxalone? 6.9 =
* Gocaineteenrrics = roces$ IipuLy, included for peak identification only; monitored in the
F . rug substance.
Buffer, Mobile phase, Standard solution, and Chro- »3-Benzyl-5-[(3,5-dimethylphenoxy)methylJoxazolidin-2-one.
matographic system: Proceed as directed in the
Assay.
 2614 Metaxalone / Official Monographs USP 41

Table 1 (Continued) Acceptance criteria: 98.5%-101.0% on the dried basis

Relative Acceptance
IMPURITIES
Retention Criteria, e RESIDUE ON IGNITION (281): NMT 0.1%
Name Time NMT (%)
Any individual unspecified _
degradation product 0.10 Delete the following:
Total degradation
roducts a 0.5 °e HEAVY METALS, Method | (231): NMT 10 ppme comes.
Process impurity, included for peak identification only; monitored in the fari-2018)
drug substance. ¢ ORGANIC IMPURITIES
» 3-Benzyl-5-[(3,5-dimethylphenoxy)methyl]oxazolidin-2-one Mobile phase: 17 g/L of monobasic ammonium phos-
phate in water, adjusted with phosphoric acid to a pH
ADDITIONAL REQUIREMENTS of 3.
e PACKAGING AND STORAGE: Preserve in well-closed, light- System suitability stock solution: 0.25 mg/mL of
resistant containers. Store at controlled room metformin hydrochloride and 0.1 mg/mL of melamine
temperature. in water
e USP REFERENCE STANDARDS (11) System suitability solution: Transfer 1.0 mL of the Sys-
USP Metaxalone RS tem suitability stock solution to a 50-mL volumetric flask,
USP Metaxalone Related Compound B RS and dilute with Mobile phase to volume.
1-Amino-3-(3,5-dimethylphenoxy)propan-2-ol. Standard stock solution: 0.2 mg/mL of USP Metformin
CyHizNOz 195.26 Related Compound A RS in water
USP Metaxalone Related Compound C RS Standard solution: 0.001 mg/mL of USP Metformin Re-
Bis[2-hydroxy-3-(3,5-dimethylphenoxy)propyl]amine. lated Compound A RS in Mobile phase from the Stan-
Co2H3iNO4g =373.49 dard stock solution
Sample solution: 5 mg/mL of Metformin Hydrochloride
in Mobile phase
Diluted sample solution: 0.005 mg/mL of Metformin
Hydrochloride in Mobile phase from the Sample solution
Metformin Hydrochloride Chromatographic system
(See Chromatography (621), System Suitability.)
NH NH Mode: LC
wc NSN
J NH, Hel
Detector: UV 218 nm
1 48 Column: 4.6-mm x 25-cm; packing L9
CHy Flow rate: 1.0-1.7 mL/min
Run time: NLT twice the retention time of metformin
C4HNs - HCl 165.62 Injection volume: 20 wL
Imidodicarbonimidic diamide, N,N-dimethyl-, System suitability
i
a)
monohydrochloride; Sample: System suitability solution
5 1,1-Dimethylbiguanide monohydrochloride [1115-70-4]. Suitability requirements
i]
J Resolution: NLT 10 between melamine and
a DEFINITION metformin
°
= Metformin Hydrochloride contains NLT 98.5% and NMT Analysis
S 101.0% of metformin hydrochloride (C4HiiNs + HCI), cal- Samples: Standard solution, Sample solution, and Di-
3 culated on the dried basis. luted sample solution
3 Calculate the percontage of metformin related com-
2) IDENTIFICATION pound A in the portion of Metformin Hydrochloride
> e A. INFRARED ABSORPTION (197K) taken:
e B. IDENTIFICATION TESTS—GENERAL, Chloride (191): Meets
the requirements Result = (ru/rs) x (Cs/Cu) x 100
ASSAY ty = peak response of metformin related
e PROCEDURE compound A from the Sample solution
Sample: 60mg of Metformin Hydrochloride ls = peak response of metformin related
Analysis compound A from the Standard solution
[NoTE—To avoid overheating of the reaction medium, G = concentration of USP Metformin Related
mix thoroughly throughout the titration, and stop the Compound A RS in the Standard solution
titration immediately after the endpoint has been
reached.] (mg/mL)
Cy = concentration of Metformin Hydrochloride in
Dissolve the Sample in 4 mL of anhydrous formic acid, the Sample solution (mg/mL)
and add 50 mL of acetic anhydride. Titrate with 0.1 N Calculate the percentage of any other impurity in the
perchloric acid VS, determining the endpoint potentio- portion of Metformin Hydrochloride taken:
metrically. Perform a blank determination, and make
any necessary correction (see Titrimetry (541)). Each Result = (ru/rs) x D x 100
mL of 0.1 N perchloric acid is equivalent to 8.28 mg
of metformin hydrochloride (C4H11Ns + HCl). ty = peak response of an individual impurity from
the Sample solution
rs = peak response of metformin from the Diluted
sample solution
D = dilution factor for the preparation of the
Diluted sample solution, 0.001
Acceptance criteria
Individual impurities: NMT 0.02% for metformin re-
lated compound A; NMT 0.1% for any other impurity
USP 41
Total impurities: NMT 0.5%
SPECIFIC TESTS
e Loss ON DRYING (731)
Analysis: Dry a sample at 105° for 5 h.
Acceptance criteria: NMT 0.5%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. Store at room temperature.
e¢ USP REFERENCE STANDARDS (11)
USP Metformin Hydrochloride RS
USP Metformin Related Compound A RS
1-Cyanoguanidine.
CoH«Ng = 84.08
Metformin Hydrochloride Tablets
DEFINITION
Metformin Hydrochloride Tablets contain NLT 95.0% and
NMT 105.0% of the labeled amount of metformin hydro-
chloride (C4HiiNs - HCl).
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
Sample: Transfer an amount of powdered Tablets,
equivalent to 20 mg of metformin hydrochloride, to a
suitable flask. Add 20 mL of dehydrated alcohol, and
shake. Filter, evaporate the filtrate on a water bath to
dryness, and dry the residue at 105° for 2 h.
Acceptance criteria: Meet the requirements
° B.
Solution A: Dissolve 1 g of 1-naphthol in a solution
containing 6 g of sodium hydroxide and 16g of anhy-
drous sodium carbonate in 100 mL of water.
Sample solution: Triturate an amount of powdered
Tablets, equivalent of 50 mg of metformin hydrochlo-
ride, with 10 mL of water, filter, and use the filtrate.
Analysis: To 5 mL of the Sample solution add 1.5 mL of
5 N sodium hydroxide solution and 1 mL of Solution A.
Add 0.5 mL of sodium hypochlorite TS, dropwise, and
with shaking.
Acceptance criteria: An orange-red color is produced
that darkens on standing.
© C. IDENTIFICATION TESTS—GENERAL, Chloride (191)
Sample solution: Prepare as directed for the Sample so-
lution in Identification test B.
Acceptance criteria: Meet the requirements
ASSAY
e PROCEDURE
Standard solution: 10 ug/mL of USP Metformin Hydro-
chloride RS in water
ee solution: Weigh and finely powder NLT 20
Tablets. Transfer the amount of powder, equivalent to
100 mg of metformin hydrochloride, to a 100-mL volu-
metric flask. Add 70 mL of water, shake by mechanical
means for 15 min, dilute with water to volume, and
filter, discarding the first 20 mL of the filtrate. Dilute
10.0 mL of the filtrate with water to 100.0 mL, and di-
lute 10.0 mL of the resulting solution with water to
100.0 mL. The nominal concentration of this solution is
10 pg/mL.
Penal corel eons
See Ultraviolet-Visible Spectroscopy (857).
Mode: UV : py e Buffer: Dissolve 1.38 g of monobasic sodium phos-
Analytical wavelength: Wavelength of maximum ab-
sorbance at about 232 nm
Official Monographs / Metformin 2615
Cell: 1m
Blank: Water
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
metformin hydrochloride (C4Hi1Ns - HCI) in the por-
tion of the Tablets taken:
Result = (Au/As) « (Cs/Cu) x 100
Au = absorbance of the Sample solution
As = absorbance of the Standard solution
Cs = concentration of USP Metformin
Hydrochloride RS in the Standard solution
(g/mL) : }
Cu = nominal concentration of metformin
hydrochloride in the Sample solution (\ug/mL)
Acceptance criteria: 95.0%-105.0%
PERFORMANCE TESTS
e DISSOLUTION (711)
Test 1
Medium: pH 6.8 phosphate buffer; 1000 mL
Apparatus 1: 100 rpm
Time: 45 min
Standard solution: USP Metformin Hydrochloride RS
in Medium
Sample solution: Pass a portion of the solution under
test through a suitable filter.
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).)
Mode: UV
Analytical wavelength: Wavelength of maximum ab-
sorbance at about 233 nm
Analysis: Determine the amount of metformin hydro-
chloride (C4HiiNs - HCI) dissolved by using UV absorp-
tion of filtered portions of the Sample solution, suitably
diluted with Medium, if necessary, in comparison with Cc
the Standard solution. 4)
Tolerances: NLT 70% (Q) of the labeled amount of a)
metformin hydrochloride (C4H1iNs - HCl) is dissolved.
=
Test 2: If the product complies with this test, the label- i}
ing indicates that it meets USP Dissolution Test 2. i
For products labeled to contain 500 mg of i}
metformin hydrochloride ro}BI
Medium: pH 6.8 phosphate buffer; 1000 mL By
Be}
Apparatus 2: 50 rpm s
Time: 30 min a
Standard solution, Sample solution, Instrumental
conditions, and Analysis: Proceed as directed in
Test 1.
Tolerances: NLT 80% (Q) of the labeled amount of
metformin hydrochloride (C4HiiNs- HCl) is dissolved.
For products labeled to contain 850 or 1000 mg of
metformin hydrochloride
Medium: pH 6.8 phosphate buffer; 1000 mL
Apparatus 2: 75 rpm
Time: 30 min
Standard solution, Sample solution, Instrumental
conditions, and Analysis: Proceed as directed in
Test 1.
Tolerances: NLT 75% (Q) of the labeled amount of
metformin hydrochloride (C4H1iNs - HCl) is dissolved.
Test 3: If the product complies with this test, the label-
ing indicates that it meets USP Dissolution Test 3.
Medium: pH 6.8 phosphate buffer; 1000 mL
Apparatus 1: 100 rpm
Time: 60 min
phate in about 1800 mL of water. Add 3.484g of
1-pentanesulfonic acid sodium salt. Adjust with diluted
phosphoric acid to a pH of 3.00 + 0.05. Dilute with
water to 2000 mL.
 2616 Metformin / Official Monographs
Mobile phase: Acetonitrile and Buffer (1:19)
Standard stock solution: 0.25 mg/mL of USP
Metformin Hydrochloride RS in Medium. Use sonication
to dissolve.
Standard solution: 0.05 mg/mL of USP Metformin Hy-
drochloride RS in Medium from the Standard stock
solution
Sample solution: Pass a portion of the solution under
test through a nylon filter of 0.45-1m pore size. Dilute
with Medium, if necessary, to obtain a solution with a
concentration similar to that of the Standard solution.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 230 nm
Column: 4.6-mm x 25-cm; 5-4um packing L1
Flow rate: 1.0 mL/min
Injection volume: 40 pL
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 2.0
Column efficiency: NLT 1500 theoretical plates
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
metformin hydrochloride (C4H1iNs - HCl) dissolved:
Result = (ru/rs) x (Gs/L) x (V/D) x 100
tu = peak response from the Sample solution
rs = peak response from the Standard solution
Gs = concentration of the Standard solution
(mg/mL)
L = label claim (mg/Tablet)
Vv = volume of Medium, 1000 mL
D = dilution factor of the Sample solution
in
"
% Tolerances: NLT 70% (Q) of the labeled amount of
metformin hydrochloride (C4HiiNs - HCl) is dissolved.
=
i]
e UNIFORMITY OF DosAGE UNITS (905): Meet the
ro.)
° requirements
= ASSAY
5 IMPURITIES
= ¢ ORGANIC IMPURITIES
rs Mobile phase: 17 g/L of monobasic ammonium phos-
al phate in water, adjusted with phosphoric acid to a pH
=) of 3.0
System suitability stock solution: 0.25 mg/mL of
metformin hydrochloride and 0.1 mg/mL of melamine
in water
System suitability solution: Transfer 1.0 mL of the Sys-
tem suitability stock solution to a 50-mL volumetric flask,
and dilute with Mobile phase to volume.
Sample solution: Weigh and finely powder NLT 20
Tablets. Transfer the amount of powder, equivalent to
500 mg of metformin hydrochloride, to a 100-mL volu-
metric flask. Dissolve in Mobile phase with shaking, di-
lute with Mobile phase to volume, and filter.
Diluted sample solution: Nominally 0.005 mg/mL of
metformin hydrochloride in Mobile phase from the Sam-
pe solution
hromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 218 nm
Column: 4.6-mm x 25-cm; packing L9
Flow rate: 1.0-1.7 mL/min
Run time: NLT twice the retention time of metformin
Injection volume: 20 pL
System suitability
Sample: System suitability solution
Suitability requirements
Resolution: NLT 10 between melamine and
metformin
USP 41
Analysis
Samples: Sample solution and Diluted sample solution
Calculate the percentage of any individual impurity in
the portion of the Tablets taken:
Result = (ru/rs) x D x 100
tu = peak response of any individual impurity from
the Sample solution
rs = peak response of metformin from the Diluted
sample solution
D = dilution factor for the preparation of the
Diluted sample solution, 0.001
Acceptance criteria
Any individual impurity: NMT 0.1%
Total impurities: NMT 0.6%
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers.
Store at controlled room temperature.
e LABELING: When more than one Dissolution test is given,
the labeling states the Dissolution test used only if Test 7
is not used.
e USP REFERENCE STANDARDS (11)
USP Metformin Hydrochloride RS
Metformin Hydrochloride Extended-
Release Tablets
DEFINITION
Metformin Hydrochloride Extended-Release Tablets contain
NLT 90.0% and NMT 110.0% of the labeled amount of
metformin hydrochloride (C4H1;Ns - HCl).
IDENTIFICATION
e A. The retention time of the major peak from the Sample
solution corresponds to that from the Standard solution,
as obtained in the Assay.
e PROCEDURE
Buffer solution: 0.5 g/L of sodium 1-heptanesulfonate
and 0.5 g/L of sodium chloride in water. Before final
dilution, adjust with 0.06 M phosphoric acid to a pH of
3.85.
Mobile phase: Acetonitrile and Buffer solution (1:9).
[Note—To improve the separation, the composition of
acetonitrile and Buffer solution may be changed to 1:19,
if necessary.]
Diluent: 1.25% solution of acetonitrile in water
Standard solution: (1/4000) mg/mL of USP Metformin
Hydrochloride RS in Diluent, where L is the labeled
quantity, in mg, of metformin hydrochloride in each
Tablet
System suitability stock solution: 12.5 g/mL each of
USP Metformin Related Compound B RS and USP
Metformin Related Compound C RS in Diluent
System suitability solution: Dilute 0.5 mL of the Sys-
tem suitability stock solution with the Standard solution
to 50 mL.
Sample stock solution: Finely powder NLT 10 Tablets.
Transfer powder, equivalent to the average Tablet
weight, to a homogenization vessel, and add 500 mL of
a 10% acetonitrile solution. Alternately, homogenize
and allow to soak until the sample is fully homogen-
ized. [NoTE—A suggested homogenization sequence is
as follows. Homogenize the sample using five pulses,
each of 5 s, at about 20,000 rpm, and allow to soak for
2 min. Repeat these steps two additional times.]
Sample solution: Pass a portion of the Sample stock
solution throughasuitable filter of 0.45-11m pore size,
discarding the first 3 mL of filtrate. Transfer 25 mL of
USP 41 Official Monographs / Metformin 2617

the filtrate to a 200-mL volumetric flask, and dilute with Cs = concentration of the Standard solution
water to volume. (mg/mL)
Chromatographic system Vv = initial volume of Medium in the vessel (mL)
(See Chromatography (621), System Suitability.) Vs = volume withdrawn from the vessel for
Mode: LC previous samplings (mL)
Detector: UV 218 nm Ceo | = concentration of metformin hydrochloride in
Column: 3.9-mm x 30-cm; 10-m packing L1 Medium determined at 1 h (mg/mL)
Column temperature: 30° Cigo += concentration of metformin hydrochloride in
Flow rate: 1 mL/min Medium determined at 3 h (mg/mL)
Injection volume: 10 uL L label claim (mg/Tablet)

I
Run time: Until after the elution locus of metformin Tolerances: See Table 7.
related compound C
System suitability Table 1
Sample: System suitability solution
[Nott—The relative retention times for metformin re- Amount Dissolved, Amount Dissolved,
lated compound B, metformin, and metformin related Time 500-mg Tablet 750-mg Tablet
compoundCare 0.86, 1.0, and 2.1-2.3, respectively. (h) (%) (%)
Metformin related compound C can have a variable 1 20-40 22-42
retention time. The composition of the Mobile phase 3 45-65 49-69
may be changed to 1:19, if it elutes at a relative reten- 10 NLT 85 NLT 85
tion time of less than 2.1.]
Suitability requirements The percentages of the labeled amount of metformin
Resolution: NLT 1.5 between the peaks due to hydrochloride (C4H1iNs - HCl) dissolved at the times
metformin related compound B and metformin species conform to Dissolution (711), Acceptance Ta-
Tailing factor: NLT 0.8 and NMT 2.0 for the ble 2.
metformin peak Test 2: If the product complies with this test, the label-
Relative standard deviation: NMT 1.5% for the ing indicates that it meets USP Dissolution Test 2.
metformin peak and NMT 10% for each of the peaks Medium: Prepare as directed for Test 71; 1000 mL.
due to metformin related compound B and Apparatus 2: 100 rpm
metformin related compound C Times: 1, 2,6, and 10h
Analysis Detector: UV 232 nm
Samples: Standard solution and Sample solution Standard solution: USP Metformin Hydrochloride RS
Calculate the percentage of the labeled amount of in Medium
metformin hydrochloride (C4H1Ns - HCl) in the portion Sample solution: Pass a portion of the solution under
of Tablets taken: test through a suitable polyethylene filter of 0.45-um
pore size. Dilute, if necessary, with Medium to a con-
Result = (ru/rs) x (Cs/Cu) x 100 centration that is similar to that of the Standard
solution. i
ru = peak response from the Sample solution Analysis: Calculate, in mg/mL, the content of Kd
rs = peak response from the Standard solution metformin hydrochloride (CsHiiNs - HCl), G, in Me-
Cs = concentration of USP Metformin dium at each time point, t: ES
Hydrochloride RS in the Standard solution

Cu
(mg/mL)
= nominal concentration of metformin
Result = (Ay x Cs x Du)/As FS
Ko}
hydrochloride in the Sample solution Au = absorbance of the Sample solution Py
Acceptance criteria: 90.0%-110.0% Cs = concentration of metformin hydrochloride in me}
PERFORMANCE TESTS
the Standard solution (mg/mL) a
Du = dilution factor of the solution under test
e DISSOLUTION (711) As = absorbance of the Standard solution
Test 1 Calculate the percentage of the labeled amount of
Medium: pH 6.8 phosphate buffer solution; 1000 mL metformin hydrochloride (C4HiiNs - HCI) dissolved at
Apparatus 1: 100 rpm for Tablets labeled to contain each time point by the following formulas.
750mg Percentage dissolved at the first time point (1 h):
Apparatus 2: 100 rpm for Tablets labeled to contain
500 mg Result = (C; x V x 100)/L
Times: 1, 3, and 10h
Detector: UV 232 nm Gq = content of metformin hydrochloride in
Standard solution: USP Metformin Hydrochloride RS Medium at the first time interval (mg/mL)
in Medium Vv = volume of Medium, 1000 mL
Sample solution: Pass a portion of the solution under EL = label claim (mg/Tablet)
test through a suitable hydrophilic polyethylene filter Percentage dissolved at the second time point (2 h):
of 0.45-11m pore size. Dilute, if necessary, with Medium
to a concentration similar to that of the Standard Result = [C, x (V—- SV;) + C, x SV;] x (100/L)
solution.
Analysis: Calculate the percentage of the labeled G = content of metformin hydrochloride in
amount of metformin hydrochloride (C4HiiNs«HCl) re- Medium at the second time interval (mg/mL)
leased at each time point: Vv = volume of Medium, 1000 mL
SV; = volume of the sample withdrawn at 1 h (mL)
Result = [(Au/As) x Cs x (V— Vs) + (Coo x Vs) + (Ciao X G = content of metformin hydrochloride in
Vs)] x (100/L) Medium at 1 h (mg/mL)
L = label claim (ng/tabiew
Au = absorbance of the Sample solution
As = absorbance of the Standard solution
 2618 Metformin / Official Monographs USP 41

Percentage dissolved at the nth time point: L = label claim (mg/Tablet)

Tolerances: See Tables 3 and 4.
Result = {C, x [V—(n-1)Vs] + (Ci + Gat... + Gi) x
Vs} x (100/L)
Table 3. For Tablets Labeled to Contain 500 mg
Ch = content of metformin hydrochloride in Amount
Medium at the nth time interval (mg/mL) Time Dissolved
Vv = volume of Medium, 1000 mL
n = time interval of interest 1
Vs = volume of sample withdrawn at each time
interval (mL)
€ = as Cy, Cz, C3, ... Car, the content of
metformin hydrochloride in Medium at each
time interval (mg/mL)
L = label claim (mg/Tablet)
Tolerances: See Table 2. Table 4. For Tablets Labeled to Contain 750 mg
Amount
Table 2 Time Dissolved
%
Amount
Dissolved 22-42

8.

The percentages of the labeled amount of metformin

1 Berge conform to Dissolution (711), Acceptance Ta-
The percentages of the labeled amount of metformin
hydrochloride (CsHiiNs - HCl) dissolved at the times
specified conform to Dissolution (711), Acceptance Ta-
ble 2.
Test 3: If the product complies with this test, the label-
ing indicates that it meets USP Dissolution Test 3.
Medium, Apparatus 1, and Apparatus 2: Proceed as
directed in Test 7.
Times: 1, 2, 5, and 12 h for Tablets labeled to contain
500 mg; and 1, 3, and 10 h for Tablets labeled to
”
aod contain 750 mg
a Detector: UV 232 nm
cS
— Standard solution: USP Metformin Hydrochloride RS
Dd in Medium
° Sample solution: Pass a portion of the solution under
fod
Sj test through a suitable eee polyethylene filter
= of 0.45-1m pore size. Dilute, if necessary, with Medium
to a concentration similar to that of the Standard
a
al solution.
=) Analysis: Calculate the percentage of the labeled
amount of metformin hydrochloride (C4H1iNs
- HCl) re-
leased at each time point:
Result = {[(Au/As) x Cs x (V— Vs) + (Ceo x Vs) + (Cr20 X
Vs) + (C300 x Vs) + (Crz0 x Vs)] x 100}/L
Au = absorbance of the Sample solution
As = absorbance of the Standard solution
Cs = concentration of the Standard solution
(mg/mL)
Vv = initial volume of Medium in the vessel (mL)
Vs = volume withdrawn from the vessel for
previous samplings (mL)
Coo = concentration of metformin hydrochloride in
Medium determined at 1 h (mg/mL)
Ciz0 +== concentration of metformin hydrochloride in
Medium determined at 2 h (mg/mL)
C300 +== Concentration of metformin hydrochloride in
Medium determined at 5 h (mg/mL)
C20 += concentration of metformin hydrochloride in
Medium determined at 12 h (mg/mL)
hydrochloride (C4H11Ns - HCI) dissolved at the times
(ara
Test 4: If the product complies with this test, the label-
ing indicates that it meets USP Dissolution Test 4.
Medium: Prepare as directed for Test 1; 1000 mL.
Apparatus 2: 100 rpm
Times: 1, 3, 6,and 10h
Detector: UV 250 nm (shoulder)
Standard solution: USP Metformin Hydrochloride RS
in Medium
Sample solution: Pass a portion of the solution under
test through a suitable filter of 0.45-1um pore size. Di-
lute, if necessary, with Medium to a concentration sim-
ilar to that of the Standard solution.
Analysis: Calculate, in mg/mL, the content of
metformin hydrochloride (C4HiiNs « HCl), CG, in Me-
a each time point, t, by the formulas specified in
Test 2.
Tolerances: See Table 5.
Table 5
Amount
Dissolved
The percentages of the labeled amount of metformin
hydrochloride (C4HiiNs - HCI) dissolved at the times
speciied conform to Dissolution (711), Acceptance Ta-
2:
Test 5: If the product complies with this test, the label-
ing indicates that it meets USP Dissolution Test 5.
Medium: pH 6.8 phosphate buffer solution; 900 mL,
deaerated
USP 41 Official Monographs / Metformin 2619

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J RAD TYP

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1. MATERIAL: 316SS OR EQUIVALENT .017 WIRE VERTICAL MEAS

SQUARE WEAVE WITH .039 SQUARE OPENINGS.
2. ALL DIMENSIONS ARE IN INCHES. TOLERANCES TO BE +/-.010

Figure 1

Apparatus 1: 100 rpm, with the vertical holder de- Calculate, in mg/mL, the content of metformin hydro-
scribed in Figure 1 and Figure 2 chloride (C4HiiNs - HCI), G, in Medium at each time
Times: 2, 8, and 16h point, t, by the formulas specified in Test 2.
Detector: UV 250 nm Tolerances: See Table 6.
Standard solution: USP Metformin Hydrochloride RS
in Medium Table 6
Sample solution: Pass a portion of the solution under
test through a suitable filter of 0.45-tm pore size. Di- Amount Dissolved, Amount Dissolved,
lute, if necessary, with Medium to a concentration sim- Time 500-mg Tablet 1000-mg Tablet
ilar to that of the Standard solution. (h) (%) (%)
Analysis: Place a vertical sample holder into each bas- 2 NMT 30 NMT 30
ket (see Figures 1 and 2). Place 1 Tablet inside the 8 60-85 65-90
sample holder, making sure that the Tablets are verti- 16 NLT 90 NLT 90
cal at the bottom of the baskets.
 2620 Metformin / Official Monographs USP 41

017 NOM
J RAD TYP

404 ID

—{ .555 ID L | 438

7404
E Hd
17 Egeltaadt
1 pesgeeear
mitt ni
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NOTES:
1. MATERIAL: 3168S OR EQUIVALENT .017 WIRE VERTICAL MEAS
SQUARE WEAVE WITH .039 SQUARE OPENINGS.
2. ALL DIMENSIONS ARE IN INCHES. TOLERANCES TO BE +/-.010
Figure 2

The percentages of the labeled amount of metformin to a concentration similar to that of the Standard
hydrochloride (C4HiNs - HCl) dissolved at the times solution.
specified conform to Dissolution (711), Acceptance Ta- Analysis: Calculate the percentage of the labeled
ble 2. amount of metformin hydrochloride (C4HiiNs - HCl) re-
leased at each time point:
Test 6: If the product complies with this test, the label- Result = {[(Au/As) x Cs x (V— Vs) + (Coo X Vs) + (Cigo X
ing indicates that it meets USP Dissolution Test 6. au! » + vanesVat sett ee
Medium: pH 6.8 phosphate buffer solution; 1000 mL,
deaerated . . . Au = absorbance of the Sample solution
Apparatus 2: 100 rpm, with USP sinker, if necessary As = absorbance of the Standard solution
Detector: UV 233 nm Cs = concentration of the Standard solution
Standard solution: USP Metformin Hydrochloride RS (mg/mL)
in Medium V = initial volume of Medium in the vessel (mL)
Sample solution: Pass a portion of the solution under Vs = volume withdrawn from the vessel for
test through a suitable pyaropplte polyethylene filter previous samplings (mL)
of 0.45-um pore size. Dilute, if necessary, with Medium Céo | = concentration of metformin hydrochloride in
Medium determined at 1 h (mg/mL)
USP 41 Official Monographs / Metformin 2621

Ciso = concentration of metformin hydrochloride in Medium: Prepare as directed in Test 1; 1000 mL.
Medium determined at 3 h (mg/mL) Apparatus 1: 100 rpm for Tablets labeled to contain
Ceoo = concentration of metformin hydrochloride in 750mg
Medium determined at 10 h (mg/mL) Apparatus 2: 100 rpm, with sinker, for Tablets labeled
L = label claim (mg/Tablet) to contain 500 mg
Tolerances: See Table 7. Times: 1, 2, 6, and 10h
Detector: UV 232 nm
Table 7 Standard solution: USP Metformin Hydrochloride RS
in Medium
Amount Dissolved, Amount Dissolved, Sample solution: Pass a portion of the solution under
Time 500-mg Tablet 750-mg Tablet test through a suitable filter of 0.45-um pore size. Di-
(h) (%) (%) lute, if necessary, with Medium to a concentration sim-
1 20-40 20-40 ilar to that of the Standard solution.
3 45-65 45-65 Analysis: Calculate the percentage of the labeled
10 NLT 85 NLT 85 amount of metformin hydrochloride (C4HiiNs - HCI) re-
leased at each time point:
The percentages of the labeled amount of metformin
hydrochloride (C4H11Ns- HCI) dissolved at the times Result = {[(Au/As) x Cs x (V
— Vs) + (Ceo x Vs) + (Ci20 X
ened conform to Dissolution (711), Acceptance Ta- Vs) + (C360 x Vs) + (Ce00 x Vs)] x 100}/L
ex2,
Test 7: If the product complies with this test, the label- Au = absorbance of the Sample solution
ing indicates that it meets USP Dissolution Test 7. As = absorbance of the Standard solution
Medium: Prepare as directed in Test 1; 1000 mL. Cs = concentration of the Standard solution
Apparatus 1: 100 rpm for Tablets labeled to contain (mg/mL)
750 mg Vv = initial volume of Medium in the vessel (mL)
Apparatus 2: 50 rpm, with USP sinker, for Tablets la- Vs = volume withdrawn from the vessel for
beled to contain 500 mg previous samplings (mL)
Times: 1, 3,and 10h Ceo = concentration of metformin hydrochloride in
Detector: UV 232 nm Medium determined at 1 h (mg/mL)
Standard solution: USP Metformin Hydrochloride RS Ciz0 +== concentration of metformin hydrochloride in
in Medium Medium determined at 2 h (mg/mL)
Sample solution: Pass a portion of the solution under C360 = Concentration of metformin hydrochloride in
test through a suitable filter of 0.45-1m pore size. Di- Medium determined at 6 h (mg/mL)
lute, if necessary, with Medium to a concentration sim- Céoo + = Concentration of metformin hydrochloride in
ilar to that of the Standard solution. Medium determined at 10 h (mg/mL)
Analysis: Calculate the percentage of the labeled L = label claim (mg/Tablet)
amount of metformin hydrochloride (C4H1iNs - HCl) re- Tolerances: See Table 9.
leased at each time point: fx
4)
Table 9 uv
— Vs) + (Ceo x Vs) + (Ciao x
Result = {[(Au/As) x Cs x (V
Vs) + (Ceoo x Vs)] x 100}/L
Amount Dissolved, Amount Dissolved,
cs
Time 500-mg Tablet 750-mg Tablet ro}
(h) _(%) (%) =
Au = absorbance of the Sample solution i}
As = absorbance of the Standard solution 1 20-40 20-40 to}=|
Gs = concentration of the Standard solution 2 30-50 35-55 iy
(mg/mL) 6 65-85 75-95 a}
Vv = initial volume of Medium in the vessel (mL) 10 NLT 85 NLT 85
s”“
Vs = volume withdrawn from the vessel for
previous samplings (mL) The percentages of the labeled amount of metformin
Ceo = concentration of metformin hydrochloride in hydrochloride (C4H11Ns - HCI) dissolved at the times
Medium determined at 1 h (mg/mL) pened conform to Dissolution (711), Acceptance Ta-
Cigo = Concentration of metformin hydrochloride in le 2.
Medium determined at 3 h (mg/mL) Test 9: If the product complies with this test, the label-
Ceoo = Concentration of metformin hydrochloride in ing indicates that it meets USP Dissolution Test 9.
Medium determined at 10 h (mg/mL) Medium: 0.05 M phosphate buffer, pH 6.8; 1000 mL
L = label claim (mg/Tablet) Aaa 1: 100 rpm, for Tablets labeled to contain
Tolerances: See Table 8. Omg
Apparatiss 2: 100 rpm, for Tablets labeled to contain
Table 8 10 mg
Times: 1, 5, 12, and 20 h for Tablets labeled to con-
Amount Dissolved, Amount Dissolved,
tain 500 mg; and 1, 4, 10, and 24h for Tablets la-
Time 500-mg Tablet 750-mg Tablet beled to contain 750 mg
(h) (%) (%) Standard solution: 0.5 mg/mL of USP Metformin Hy-
1 20-40 20-40 drochloride RS in Medium
3 45-65 40-60 Sample solution: Pass a portion of the solution under
10 NLT 85 NLT 80 test through a suitable filter of 0.45-um pore size.
Detector: UV 232 nm
The percentages of the labeled amount of metformin Path length: 0.01 cm, flow cell
hydrochloride (C4Hi1Ns - HCl) dissolved at the times Blank: Medium
specified conform to Dissolution (711), Acceptance Ta- Analysis: Calculate the percentage of the labeled
ble 2. amount of metformin hydrochloride (C4H1iNs + HCl) re-
Test 8: If the product complies with this test, the label- leased at each time point:
ing indicates that it meets USP Dissolution Test 8.
Result = {[(Au/As) x Cs x (V— Vs) + (Ci x Vs) + (C2 x Vs)
+ (C3 x Vs) + (Cy x Vs)] x 100}/L
 2622 Metformin / Official Monographs USP 41

Au = absorbance of the Sample solution Detector: UV 233 nm

As = absorbance of the Standard solution Path length: 1 cm
Cs = concentration of the Standard solution Blank: Medium
(mg/mL) Analysis: Calculate the concentration (mg/mL) of
Vv = initial volume of Medium in the vessel (mL) metformin hydrochloride (C) at each time point:
Vs = volume withdrawn from the vessel for
previous samplings (mL) G = (Au/As) x Cs
C = concentration of metformin hydrochloride in
Medium determined at the first time point Au = absorbance of the Sample solution
(mg/mL) As = absorbance of the Standard solution
G = concentration of metformin hydrochloride in Cs = concentration of the Standard solution
Medium determined at the second time point (mg/mL)
(mg/mL) Calculate the cumulative percentage of the labeled
G = concentration of metformin hydrochloride in amount of metformin hydrochloride (CsHiiNs » HCl)
Medium determined at the third time point dissolved (Q)) at each time point (i):
(mg/mL) Ati=1:
G = concentration of metformin hydrochloride in
Medium determined at the fourth time point Q, =(G x V/L) x 100
(mg/mL)
L = label claim (mg/Tablet) At i = 3:
Tolerances: See Tables 10 and 11.
Qs = [CV
— Vs) + (GC; x V9] x 100/L
Table 10. For Tablets Labeled to Contain 500 mg At i= 10:
Amount
Time Dissolved Quo = [Cro(V
— 2Vs) + (Cy + C3)Vs] x 100/L
Vv = initial volume of Medium, 1000 mL
Vs =sampling volume, 10 mL
L = label claim (mg/Tablet)
Tolerances: See Table 12.

Table 11. For Tablets Labeled to Contain 750 mg
Amount
Time Dissolved
vv
a h
is
cS
—
Da
3 1
c 4.
Sj
3 Te es of the labeled amount of metformin
a hydrochloride (C4HiiNs - HCl) dissolved at the times
“ specified conform to Dissolution (711), Acceptance Ta-
Ea ble 2.
Test 10: If the product complies with this test, the la-
beling indicates that it meets USP Dissolution Test 10.
Medium: 0.05 M phosphate buffer (prepared by dis-
solving 6.8 g of monobasic potassium phosphate in
250 mL of water, adding 77 mL of 0.2 N sodium hy-
droxide and 500 mL of water, adjusting with 2 N so-
dium hydroxide or 2 N hydrochloric acid to a pH 6.8,
and diluting with water to 1000 mL)
Apparatus 1: 100 rpm for Tablets labeled to contain
750 mg
Apparatus 2: 100 rpm for Tablets labeled to contain
500 mg
Times: 1, 3, and 10h
Standard solution: (L/100,000) mg/mL of USP
Metformin Hydrochloride RS in Medium, whereLis the
label claim, in mg/Tablet. This solution is stable for 72
h at room temperature.
Sample solution: At the times specified, withdraw
10 mL of the solution under test and replace with
10 mL of Medium previously equilibrated at 37.0 +
0.5°. Centrifuge at 2500 rpm for 10 min. Dilute a por-
tion of the supernatant with Medium to obtain a theo-
retical concentration of (L/100,000) mg/mL, whereLis
the label claim, in mg/Tablet.
Table 12
Amount
Dissolved
%
1
The percentages of the labeled amount of metformin
hydrochloride (C4HiiNs - HCl) dissolved at the times
pein conform to Dissolution (711), Acceptance Ta-
le 2.
Test 11: If the product complies with this test, the la-
beling indicates that it meets USP Dissolution Test 11.
Medium: pH 6.8 phosphate buffer solution; 1000 mL
Apparatus 1: 100 rpm for Tablets labeled to contain
750 mg
ont 2: 100 rpm for Tablets labeled to contain
500 mg
Times: 1, 3, and 10h
Standard solution: 7.5 g/mL of USP Metformin Hy-
drochloride RS in Medium
Sample solution: At the times specified, withdraw
10 mL of the solution under test, and pass it through a
suitable filter of 0.45-11m pore size, discarding the first
3 mL of filtrate. Dilute 3.0 mL of the filtrate with Me-
dium to 200 mL. For Tablets labeled to contain
750 mg, dilute 2.0 mL of the filtrate with Medium to
200 mL. Replace the volume of Medium taken with the
same volume of Medium preheated at 37.0 + 0.5°.
Detector: UV 232 nm
Path length: 1 cm
Blank: Medium
Analysis: Calculate the percentage of the labeled
amount of metformin hydrochloride (C4HiiNs - HCl)
dissolved at each time point:
Q = (Au/As) x (Gs/L) x Vx D x 100
At 1h:
Result = Q;
USP 41 Official Monographs / Metformin 2623

At 3 h: G = concentration of metformin hydrochloride in

the portion of sample withdrawn at time
Result = Qs + [(Q: x 10)/V] point i (mg/mL)
Vv initial volume of Medium, 1000 mL
At 10h: iL label claim (mg/Tablet)
Vs = volume of the Sample solution withdrawn,
Result = Qro + {{(Qi x 10)/V] + [(Qs x 10)/V]} 10 mL
Tolerances: See Table 14.
Au = absorbance of the Sample solution
As = absorbance of the Standard solution
a = concentration of the Standard solution Table 14
(mg/mL) Amount
L = label claim (mg/Tablet) Time point Time Dissolved
Vv = volume of Medium, 1000 mL
D = dilution factor of the Sample solution 1 1
Tolerances: See Table 13.
3 T
Table 13
Amount The percentages of the labeled amount of metformin
Dissolved hydrochloride (C4HiiNs + HCl) dissolved at the times
weuiee conform to Dissolution (711), Acceptance Ta-
le 2.
Test 13: If the product complies with this test, the la-
50-7
beling indicates that it meets USP Dissolution Test 13.
NLT Medium: pH 6.8 phosphate buffer solution; 1000 mL
Apparatus 1: 100 rpm
The percentages of the labeled amount of metformin Times: 1, 4, 6, and 14h
hydrochloride (C4H1;Ns - HCl) dissolved at the times Standard stock solution: 0.2 mg/mL of USP
pages conform to Dissolution (711), Acceptance Ta- Metformin Hydrochloride RS prepared as follows.
ble 2. Transfer a suitable amount of USP Metformin Hydro-
Test 12: If the product complies with this test, the la- chloride RS into an appropriate volumetric flask. Dis-
beling indicates that it meets USP Dissolution Test 12. solve by adding Medium to fill 50% of the flask vol-
Medium: pH 6.8 phosphate buffer solution; 1000 mL ume and dilute with Medium to volume.
Apparatus 1: 100 rpm Standard solution: 0.01 mg/mL of USP Metformin Hy-
Times: 1,4, and 12h drochloride RS from Standard stock solution in water
Standard stock solution: 0.2 mg/mL of USP Sample stock solution: At the times specified, with-
Metformin Hydrochloride RS in Medium draw 10 mL of the solution under test, and replace
Standard solution: 0.01 mg/mL of USP Metformin Hy- with the same volume of Medium preheated at 37.0 + =
drochloride RS in water, from the Standard stock 4)
solution
0.5°. Pass a portion of the solution under test through i)
a suitable filter of 0.45-um pore size, discard the first
Sample solution: At the times specified, withdraw few mL, and use the filtrate. =
10 mL of the solution under test, and replace with Sample solution i)
10 mL of Medium previously equilibrated at 37.0 + 3
For Tablets labeled to contain 500 mg: Dilute 2 mL }
0.5°. Pass it through a suitable filter, discarding the of Sample stock solution with water to 100 mL. Ro}=
first few mL of the filtrate. For Tablets labeled to contain 1000 mg: Dilute ES)
For Tablets labeled to contain 500 mg: Dilute 1 mL of Sample stock solution with water to 100 mL. a}
2.0 mL of the filtrate with water to 100 mL. Instrumental conditions ay
a)
For Tablets labeled to contain 1000 mg: Dilute (See Ultraviolet-Visible Spectroscopy (857).)
1.0 mL of the filtrate with water to 100 mL. Mode: UV
Detector: UV 232 nm Analytical wavelength: 232 nm
Blank: Dilute 1 mL of Medium with water to 100 mL. Blan
Analysis: Calculate the concentration, CG, in mg/mL of For Tablets labeled to contain 500 mg: Dilute 2 mL
metformin hydrochloride (C4Hi1Ns - HCI) in the sample of Medium with water to 100 mL.
withdrawn at each time point (): For Tablets labeled to contain 1000 mg: Dilute
Result; = (Au/As) X Cs x D 1 mL of Medium with water to 100 mL.
System suitability
Au = absorbance of the Sample solution Sample: Standard solution
As = absorbance of the Standard solution Suitability requirements
G = concentration of the Standard solution Relative standard deviation: NMT 2.0%
(mg/mL) Analysis
D = dilution factor of the Sample solution Samples: Standard solution, Sample solution, and
Calculate the percentage of the labeled amount of Blank
metformin hydrochloride (C4Hi1Ns - HCl) dissolved (Q)) Calculate the concentration (C), in mg/mL, of
at each time point (i): metformin hydrochloride (C4HiiNs - HCI) in the sam-
ple withdrawn from the vessel at each time point (():
Result; = C; x Vx (1/L) x 100
Result; = (Ay/As) x Cs x D

Resultz = {[C2 x V] + [C; x Vs]} x (1/L) x 100 Au = absorbance of the Sample solution
As = absorbance of the Standard solution
Cs = concentration of the Standard solution
Results = {[C3 x V] + [(C2 + GC) x Vs]} x (1/L) x 100 (mg/mL)
D = dilution factor of the Sample solution
 2624 Metformin / Official Monographs USP 41

Calculate the percentage of the labeled amount of

metformin hydrochloride (C4H1iNs + HCl) dissolved at
each time point (/): Methacholine Chloride
Result; = C; x Vx (1/L) x 100
AA an
Results = [(C2 x V) + (Gi x Vs)] x (1/2) x 100

Results = {[C3 x V] + [(C2 + Cr) x Vs]} x (1/L) x 100 CsHisCINO2 195.69

1-Propanaminium, 2-(acetyloxy)-N,N,N-trimethyl-, chloride,
)-;
Results = {[C; x V] + ae + Cj) x Vs]} x (1/L) x (4)-(2- ie a i ii chloride acetate
[62-51-1].
G = concentration of metformin hydrochloride in DEFINITION
the portion of sample withdrawn at the Methacholine Chloride contains NLT 98.0% and NMT
specified time point (mg/mL) 101.0% of methacholine chloride (CsHisCINO2), calcu-
Vv = volume of Medium, 1000 mL lated on the dried basis.
L = label claim (mg/Tablet)
Vs = volume of the Sample solution withdrawn at IDENTIFICATION
each time point and replaced with Medium e A. INFRARED ABSORPTION (197M)
mL) e B. IDENTIFICATION TESTS—GENERAL, Chloride (191)
Sample solution: 20 mg/mL
Tolerances: See Table 15.
Acceptance criteria: Meets the requirements
e C. The retention time of themaior peak of the Sample
Table 15 solution corresponds to that of the Standard solution, as
Time point Time Amount Dissolved obtained in the Assay.
() (h) (%)
1 1 NMT 20
ASSAY
e PROCEDURE
2 4 45-65
Mobile phase: 0.5 g/L of methanesulfonic acid in water
3 6 65-85 Standard solution: 50 g/mL of USP Methacholine
4 14 NLT 85 Chloride RS in water
System suitability solution: 10 jug/mL of USP Acetyl-
The percentages of the labeled amount of metformin choline Chloride RS in Standard solution
hydrochloride (C4Hi;Ns
- HCI) dissolved at the times Sample solution: 50 g/mL of Methacholine Chloride
specified conform to Dissolution (711), Acceptance Ta- in water
p=
w”
ble 2. Chromatographic system
rm e UNIFORMITY OF DOSAGE UNITS (905): Meet the (See Chromatography (621), System Suitability.)
i]
— requirements Mode: IC
Dd
i) IMPURITIES Detector: Conductivity
= Columns
Sj © ORGANIC IMPURITIES
Guard: 4.0-mm x 50-mm; L77 packing
3 Mobile phase, Sample solution, and Chromatographic Analytical: 4.0-mm x 25-cm; L77 packing
Q.
system: Proceed as directedin the Assay. Suppressor: lon-exchange membrane autosuppressor’
Ww Analysis: From the Sema of the Sample solu- or a suitable chemical suppression system
=) tion obtained in the Assay, calculate the percentage of Suppressant: Autosuppression
each impurity in the portion of Tablets taken: Flow rate: 1 mL/min
Result = (ru/rz) x 100 Injection volume: 20 uL
System suitability
tu =peak response for each impurity Samples: System suitability solution and Standard
tr = sum of all the peak responses solution
Acceptance criteria [Note—See Table 7 for the relative retention times.]
Individual impurities: NMT 0.1% Suitability requirements
Total impurities: NMT 0.6% Resolution: NLT 2 between acetylcholine and
[Note—Disregard any peak less than 0.05%, and disre- methacholine, System suitability solution
gard any peak observed in the blank.] Relative standard deviation: NMT 1.0%, Standard
solution
ADDITIONAL REQUIREMENTS Analysis
© PACKAGING AND STORAGE: Preserve in well-closed, light- Samples: Standard solution and Sample solution
resistant containers, and store at controlled room Calculate the percentage of methacholine chloride
temperature. (CgHigCINOz) in the portion of Methacholine Chloride
e LABELING: When more than one dissolution test is given, taken:
the labeling states the Dissolution test used only if Test 7
is not used. Result = (ru/rs) x (Cs/Cu) x 100
e USP REFERENCE STANDARDS (11)
USP Metformin Hydrochloride RS ty = peak response from the Sample solution
USP Metformin Related Compound B RS Is =peak response from the Standard solution
1-Methylbiguanide hydrochloride. Cs = concentration of USP Methacholine Chloride
C3HyNsHCl = 151.60 RS in the Standard solution (g/mL)
USP Metformin Related Compound C RS Gu = concentration of Methacholine Chloride in the
N,N-Dimethyl-[1,3,5]triazine-2,4,6-triamine. Sample solution (g/mL)
CsHioNe = 154.17 1 Available as Cation Self-Regenerating Suppressor (CSRS) from Dionex Inc., or
equivalent.
USP 41 Official Monographs / Methacycline 2625

Acceptance criteria: 98.0%-101.0% on the dried basis Table 1

IMPURITIES Relative Acceptance

e RESIDUE ON IGNITION (281): NMT 0.1% Retention Criteria,

1
Delete the following:
M 0
°e HEAVY METALS, Method I (231): NMT 20 ppme cofficiai 1.
@ 2-Hydroxy-N,N,N-trimethylpropan-1-aminium chloride.
Jan-2078)
¢ ORGANIC IMPURITIES SPECIFIC TESTS
Mobile phase: 0.5 g/L of methanesulfonic acid in water e Loss ON DRYING (731)
System suitability solution: 1 mg/mL of USP Analysis: Dry a sample at 105° for 4 h.
Methacholine Chloride RS and 1 jug/mL of USP Acetyl- Acceptance criteria: NMT 1.5%
choline Chloride RS in water
Standard solution: 1 tug/mL of USP Methacholine Chlo- ADDITIONAL REQUIREMENTS
ride RS in water e PACKAGING AND STORAGE: Preserve in tight containers.
Sample solution: 1 mg/mL of Methacholine Chloride in e USP REFERENCE STANDARDS (11)
water USP Acetylcholine Chloride RS
Chromatographic system USP Methacholine Chloride RS
(See Chromatography (621), System Suitability.)
Mode: IC
Detector: Conductivity
Columns
Guard: 4.0-mm x 50-mm; L77! packing
Analytical: 4.0-mm x 25-cm; L77! packing Methacycline Hydrochloride
Suppressor: lon-exchange membrane autosuppressor! HO 9 HOO
HO
°
or a suitable chemical suppression system
Suppressant: Autosuppression
es iret
NH,

Flow rate: 1 mL/min

Injection volume: 20 wL
SS
HAR?
H,C
‘OH
"HOH H N(CH),
Run time: Two times the retention time of
methacholine C22H22N20g-HCl 478.88
System suitability 2-Naphthacenecarboxamide, 4-(dimethylamino)-1,4,4a,5,5a,
Sample: System suitability solution 6,11,lum1 2a-octahydro-3,5,10,12,12a-pentahydroxy-
[Note—See Table 1 for the relative retention times.] 6-methylene-1,11-dioxo-, monohydrochloride,(45-40,
Suitability requirements 4aa,50,,5aa,12aa)]-.
Resolution: NLT 2 between acetylcholine and 4-(Dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,5, 35
methacholine 10,12,12a-pentahydroxy-6-methylene-1,11-dioxo- 4)
Signal-to-noise ratio: NLT 2 for acetylcholine 2-naphthacenecarboxamide monohydrochloride z
Analysis =
[3963-95-9].
Samples: Standard solution and Sample solution i)
Calculate the percentage of beta-methylcholine or ace- » Methacycline Hydrochloride has a potency =i
tylcholine in the portion of Methacholine Chloride }
taken: equivalent to not less than 832 ug and not more eo}=
than 970 ug of methacycline (C22H22N2Ox) per Sy

Result = (ru/rs) x (Cs/Cu) x 100
Tu = peak response of beta-methylcholine or
acetylcholine from the Sample solution
Is = peak response of methacholine chloride from
the Standard solution
G = concentration of USP Methacholine Chloride
RS in the Standard solution (\ug/mL)
Cu = concentration of Methacholine Chloride in the
Sample solution (g/mL)
Acceptance criteria: See [able 1.
mg. be}
=~
al
Packaging and storage—Preserve in tight, light-resistant
containers.
USP Reference standards (11)—
USP Doxyeveline Hyclate RS
USP Methacycline Hydrochloride RS
Identification, Ultraviolet Absorption (197U)—
Solution: 20 ug per mL.
Medium: hydrochloric acid in methanol (1 in 1200).
Absorptivity at 345 nm, calculated on the dried basis, is
between 88.4% and 96.4% of the USP Methacycline Hydro-
chloride RS, the potency of the Reference Standard being
taken into account.
Crystallinity (695): | meets the requirements.
PH (791): between 2.0 and 3.0, in a solution containing
10 mg of methacycline per mL.
Water Determination, Method | (921): not more than
2.0%.
Assay—
Mobile phase—Prepare a mixture of 0.2 M ammonium
oxalate, dimethylformamide, and 0.1 M edetate disodium
(11:5:4), adjust with tetrabutylammonium hydroxide,
40 percent in water, to a pH of 7.0, and filter. Make adjust-
ments, if necessary (see System Suitability under Chromatog-
raphy (621)).
 2626 Methacycline / Official Monographs
System suitability preparation—Prepare a solution of USP
Methacycline Hydrochloride RS and USP Doxycycline
Hyclate RS in Mobile phase containing about 0.5 mg of each
per mL.
Standard preparation—Quantitatively dissolve an accu-
rately weighed quantity of USP Methacycline Hydrochloride
RS in Mobile phase to obtain a solution having a known
concentration of about 0.5 mg per mL.
ssa preparation—Transfer about 50 mg of Methacycline
Hydrochloride, accurately weighed, to a 100-mL volumetric
flask, dilute with Mobile phase to volume, and mix.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 354-nm detector
and a 4.6-mm x 15-cm column that contains 3.5-um pack-
ing L1. The flow rate is about 1 mL per minute. Chromato-
graph the System suitability preparation, and record the peak
responses as directed for Procedure: the relative retention
times are about 0.75 for methacycline and 1.0 for doxycy-
cline; and the resolution, R, between methacycline an
doxycycline is not less than 1.5. Chromatograph the Stan-
dard preparation, and record the peak responses as directed
for Procedure: the tailing factor is not more than 1.5; and
the relative standard deviation for replicate injections is not
more than 1.0%. : 2.5 mg of methacycline (Cz2H22N2Ox) per mL. Filter, transfer
Procedure—Separately inject equal volumes (about 20 pL)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
ure the areas for the major peaks. Calculate the quantity, in
Lig, of methacycline (C22H22N20s) in each mg of Metha-
cycline Hydrochloride taken by the formula:
100(CE
/ W)(ru/ rs)
in which C is the concentration, in mg per mL, of USP
Methacycline Hydrochloride RS in the Standard preparation;
Eis theinethacyelne content, in ig per mg, of USP Metha-
cycline Hydrochloride RS; W is the quantity, in mg, of Metha-
a
ww
is cycline Hydrochloride taken to prepare the Assay prepara-
S tion; and ry and rs are the methacycline peak areas obtained
—
D from the Assay preparation and the Standard preparation, re-
° spectively.
=
iS
= Methacycline Hydrochloride Oral
oo
va)
=) Methacycline Hydrochloride Capsules
» Methacycline Hydrochloride Capsules contain
the equivalent of not less than 90.0 percent and
not more than 120.0 percent of the labeled
amount of methacycline (C22H22N2Os).
Packaging and storage—Preserve in tight, light-resistant
containers.
USP Reference standards (11)—
USP Doxycycline Hyclate RS
USP Methacycline Hydrochloride RS
Identification—Shake a suitable quantity of Capsule con-
tents with methanol to obtain a solution containing the
equivalent of about 1 mg of methacycline per mL, and filter.
Using the filtrate as the Test Solution, proceed as directed for
Method II under Identification—Tetracyclines (193).
Dissolution (711)—
Medium: water; 900 mL.
Apparatus 1: 100 rpm.
Time: 60 minutes.
Procedure—Determine the amount of C22H22N2Os - HCI
dissolved from UV absorbances at the wavelength of maxi-
mum absorbance at about 345 nm of filtered portions of
USP 41
the solution under test, suitably diluted with water, in com-
parison with a Standard solution having a known concentra-
tion of USP Methacycline Hydrochloride RS in the same Me-
dium.
Tolerances—Not less than 70% (Q) of the labeled amount
of Co2H22N2Os+ HCI is dissolved in 60 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Water Determination, Method | (921): not more than
7.5%.
Assay—
Mobile phase, System suitability preparation, and Chromat-
ographic system—Proceed as directed in the Assay under
Methacycline Hydrochloride.
Standard preparation—Transfer about 28 mg of USP
Methacycline Hydrochloride RS, accurately weighed, to a
50-mL volumetric flask, add 10 mL of water, dilute with Mo-
bile phase to volume, and mix.
Assay preparation—Place no fewer than 5 Capsules in a
high-speed, glass blender jar containing an accurately meas-
ured volume of water, and blend for 3 to 5 minutes to ob-
tain a stock solution having a concentration of about
10.0 mL of the filtrate to a 50-mL volumetric flask, add
10 mL of water, dilute with Mobile phase to volume, and
mix.
Procedure—Proceed as directed in the Assay under Metha-
cycline Hydrochloride. Calculate the quantity, in mg, of
inethagycline (C22H22N20s) in each Capsule taken by the
‘ormula:
5(CE/ 1000)(V
/NY(ru/r5)
in which V is the volume, in mL, of water used to prepare
the stock solution for the Assay preparation; N is the number
of Capsules taken to prepare the stock solution for the Assay
preparation; and the other terms are as defined therein.
Suspension
» Methacycline Hydrochloride Oral Suspension
contains the equivalent of not less than 90.0 per-
cent and not more than 125.0 percent of the la-
beled amount of methacycline (C22H22N2Os). It
contains one or more suitable and harmless buff-
ers, colors, diluents, dispersants, flavors, and
preservatives.
Packaging and storage—Preserve in tight, light-resistant
containers.
USP Reference standards (11)—
USP Doxpayaiine Hyclate RS
USP Methacycline Hydrochloride RS
Identification—To an accurately measured volume of Oral
Suspension, equivalent to about 50 mg of methacycline,
add 50 mL of methanol, shake, and allow the mixture to
settle. Using the clear supernatant as the Test Solution, pro-
ceed as directed for Method Ii under Identification—Tetracy-
clines (193).
Uniformity of dosage units (905)—
FOR SUSPENSION PACKAGED IN SINGLE-UNIT CONTAINERS: meets
the requirements.
USP 41 Official Monographs / Methadone 2627

Deliverable volume (698): meets the requirements. Acceptance criteria: 98.5%-100.5% on the dried basis
pH (791): between 6.5 and 8.0. IMPURITIES
Assay— e RESIDUE ON IGNITION (281): NMT 0.1%
Mobile phase, System suitability preparation, and Chromat- © ORDINARY IMPURITIES (466)
ee system—Proceed as directed in the Assay under Standard solution: Alcohol
Methacycline Hydrochloride. Sample solution: Alcohol
Standard preparation—Transfer about 28 mg of USP Eluant: Methanol and ammonium hydroxide (100: 1.5)
Methacycline Hydrochloride RS, accurately weighed, to a Visualization: 3
50-mL volumetric flask, add 10 mL of water, dilute with Mo- Acceptance criteria: The sum of the intensities of all
bile phase to volume, and mix. secondary spots from the Sample solution corresponds
Assay preparation—tTransfer an accurately measured quan- to NMT 1.0%.
tity of Oral Suspension, freshly mixed and free from air bub- SPECIFIC TESTS
bles, equivalent to about 50 mg of methacycline ¢ PH (791)
(C22H22N20z), to a 100-mL volumetric flask, dilute with Mo- Sample solution: 10 mg/mL
bile phase to volume, mix, and filter. Acceptance criteria: 4.5-6.5
Procedure—Proceed as directed in the Assay under Metha- e Loss ON DRYING (731)
cycline Hydrochloride. Calculate the quantity, in mg, of Sample: 500 mg
methacycline (C22H22N2Og) in each mL of the Oral Suspen- Analysis: Dry the Sample at 105° for 1 h.
sion taken by the formula: Acceptance criteria: NMT 0.3%
100(CE /1000V)(ru / rs) ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight, light-resistant
in which V is the volume, in mL, of Oral Suspension taken containers. Store at 25°, excursions permitted between
to prepare the Assay preparation; and the other terms are as 15° and 30°.
defined therein. e USP REFERENCE STANDARDS (11)
USP Methadone Hydrochloride RS

Methadone Hydrochloride
Methadone Hydrochloride Oral
HC, Nyc,
Concentrate
et
VY CH;
DEFINITION
C oY cu, Methadone Hydrochloride Oral Concentrate contains, in
each mL, NLT 9.0 mg and NMT 11.0 mg of methadone =
hydrochloride (C2:H27NO- HCl). It contains a suitable pre- $
CaH27NO - HCI 345.91 servative and may contain suitable coloring, flavoring, and
3-Heptanone, 6-(dimethylamino)-4,4-diphenyl-, surface-active agents. FS
hydrochloride;
IDENTIFICATION Z
6-(Dimethylamino)-4,4-diphenyl-3-heptanone hydrochloride
[1095-90-5]. ¢ A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST eo}
(201) a
DEFINITION Sample solution: A volume of Oral Concentrate equiva- me]
Methadone Hydrochloride contains NLT 98.5% and NMT lent to 5 mg of methadone hydrochloride =
“
100.5% of methadone hydrochloride (C2:H27NO - HCl), Chromatographic system
calculated on the dried basis. Developing solvent system: Alcohol, glacial acetic
acid, and water (5:3:2)
IDENTIFICATION Analysis: Shake the Sample solution with 5 mL of so-
e A. INFRARED ABSORPTION (197K) dium carbonate TS, and extract with 5 mL of chloro-
e B, IDENTIFICATION TESTS—GENERAL, Chloride (191): A solu- form. Proceed as directed, using iodoplatinate TS to vis-
tion meets the requirements of the tests. ualize the spots.
Acceptance criteria: Meets the requirements
ASSAY e B. IDENTIFICATION TESTS—GENERAL, Chloride (191): Meets
¢ PROCEDURE the requirements
Sample: 500mg
Mode: Direct titration ASSAY
Titrimetric system ¢ PROCEDURE
Titrant: 0.1 N perchloric acid VS Mobile phase: Acetonitrile and 0.033 M monobasic po-
Endpoint detection: Visual tassium phosphate (40:60). Adjust with phosphoric acid
Analysis: Dissolve the Sample in a mixture of 10 mL of to a pH of 4.0, filter, and degas.
glacial acetic acid and 10 mL of mercuric acetate TS, Standard solution: 0.4 mg/mL of USP Methadone Hy-
warming slightly if necessary to dissolve. Cool the solu- drochloride RS in Mobile phase
tion to room temperature, add 10 mL of dioxane, then Sample stock solution: Nominally 1 mg/mL of metha-
add crystal violet TS, and titrate rapidly with Titrant. Gone hydrochloride from Oral Concentrate in Mobile
Perform a blank determination, and make any necessary phase
correction (see Titrimetry (541)). Each mL of Titrant is Sample solution: 0.4 mg/mL of methadone hydrochlo-
equivalent to 34.59 mg of methadone hydrochloride tide in Mobile phase from Sample stock solution
(Cai H27NO - HCl).
 2628 Methadone / Official Monographs USP 41

Chromatographic system bining the extracts in a vessel containing about 3 g of

(See Chromatography (621), System Suitability.) anhydrous sodium sulfate. Transfer 2.0 mL of Internal
Mode: LC standard solution to the vessel containing the extracts,
Detector: UV 254 nm insert the stopper, and mix. Decant 15 mL of the meth-
Column: 3.9-mm x 30-cm; packing L11 ylene chloride solution to a test tube, and evaporate to
Flow rate: 2 mL/min a volume of 2-3 mL using vacuum or a stream of
Injection volume: 10 pL nitrogen.
System suitability Sample solution: Transfer 1.0 mL of Injection, equiva-
Sample: Standard solution lent to 10 mg of methadone hydrochloride, to a 60-mL
Suitability requirements separator. Add 2 mL of 0.5 N sodium hydroxide, and
Column efficiency: NLT 1500 theoretical plates extract with three 10-mL portions of methylene chlo-
Tailing factor: NMT 2.0 tide, combining the extracts in a vessel containing
Relative standard deviation: NMT 2.0% for replicate about 3 g of anhydrous sodium sulfate. Transfer 2.0 mL
injections of Internal standard solution to the vessel containing the
Analysis extracts, insert the stopper, and mix. Decant 15 mL of
Samples: Standard solution and Sample solution the methylene chloride solution to a test tube, and
Calculate the labeled amount of methadone hydrochlo- evaporate to a volume of 2-3 mL using vacuum or a
ride (CzH27NO - HCl) in the portion of Oral Concen- stream of nitrogen.
trate taken: Chromatographic system
(See Chromatography (621), System Suitability.)
Result = (ru/rs) x (Cs/Cu) x L Mode: GC
Detector: Flame ionization
Tu = peak response from the Sample solution Column: Glass column, 1.2-m long and 4-mm in di-
ts = peak response from the Standard solution ameter; packed with 3% phase G2 on 100- to
Cs = concentration of USP Methadone 200-mesh support S1A
Hydrochloride RS in the Standard solution Temperatures
(mg/mL) 5 Column: 170°
Cu = nominal concentration of the Sample solution Injection port: 225°
(mg/mL) Detector: 240°
L = label claim (mg/mL) Carrier gas: Dry helium
Acceptance criteria: 9.0-11.0 mg/mL Flow rate: 55 mL/min
Injection volume: Containing 5 ug of methadone
SPECIFIC TESTS System suitability
© PH (791): 1.0-6.0 Sample: Standard solution (six replicate injections)
ADDITIONAL REQUIREMENTS Suitability requirements
e PACKAGING AND STORAGE: Preserve in tight containers, Resolution: NLT 5.0 between methadone and
protected from light, and store at controlled room procaine
a
a)
temperature. Coefficient of variation: NMT 1% in the ratios of
iss e LABELING: Label it to indicate that it is to be diluted with the peak areas of methadone to the peak area of
_
Ss
water or other liquid to 30 mL or more before procaine
D administration. Analysis
i) Samples: Standard solution and Sample solution
rs e USP REFERENCE STANDARDS (11)
Calculate the quantlh in mg, of methadone hydrochlo-
o USP Methadone Hydrochloride RS
tide (C2H27NO - HCl) in each mL of Injection taken:
=
a Result = (Ru/Rs) x W x 100
”
pe
Ru = peak area ratio of methadone to procaine
Methadone Hydrochloride Injection from the Sample solution
Rs = peak area ratio of methadone to procaine
DEFINITION from the Standard solution
Methadone Hydrochloride Injection is a sterile solution of Ww = weight, in mg, of USP Methadone
Methadone Hydrochloride in Water for Injection. It con- Hydrochloride RS in the Standard solution
tains, in each mL, NLT 9.5 mg and NMT 10.5 mg of Acceptance criteria: 9.5-10.5 mg/mL
methadone hydrochloride (C2;H27NO - HCl).
SPECIFIC TESTS
IDENTIFICATION © PH (791): 3.0-6.5
© A, IDENTIFICATION—ORGANIC NITROGENOUS BASES (181): e BACTERIAL ENDOTOXINS TEST (85): NMT 8.8 USP Endo-
Meets the requirements toxin Units/mg of methadone hydrochloride
e OTHER REQUIREMENTS: It meets the requirements in Injec-
ASSAY tions and Implanted Drug Products (1).
© PROCEDURE
Internal standard solution: 5 mg/mL of procaine in ADDITIONAL REQUIREMENTS
methylene chloride ¢ PACKAGING AND STORAGE: Preserve in single-dose or mul-
Standard solution: Transfer 10 mg of USP Methadone tiple-dose, light-resistant containers, preferably of Type |
Hydrochloride RS to a 60-mL separator. Add 1 mL of glass.
water and 2 mL of 0.5 N sodium hydroxide, and extract
with three 10-mL portions of methylene chloride, com-
USP 41
Change to read:
° UsP REFERENCE STANDARDS (11)
@ (CN 1-May-2018)
USP Methadone Hydrochloride RS
Methadone Hydrochloride Oral Solution
DEFINITION
Methadone Hydrochloride Oral Solution contains NLT
90.0% and NMT 110.0% of the labeled amount of meth-
adone hydrochloride (C2H27NO - HCl).
IDENTIFICATION
e * oe CHROMATOGRAPHIC IDENTIFICATION TEST
201
Sample solution: A volume of Oral Solution equivalent
to 5 mg of methadone hydrochloride
Developing solvent system: Alcohol, glacial acetic
acid, and water (5:3:2)
Analysis: Shake the Sample solution with 5 mL of so-
dium carbonate TS, and extract with 5 mL of chloro-
form. Proceed as directed using iodoplatinate TS to vis-
ualize the spots.
Acceptance criteria: Meets the requirements
e B. IDENTIFICATION TESTS—GENERAL, Chloride (191): Meets
the requirements
ASSAY
¢ PROCEDURE
Mobile phase: Acetonitrile and 0.033 M monobasic po-
tassium phosphate (40:60). Adjust dropwise with phos-
phoric acid to oie of 4.0.
Internal standard solution: 250 g/mL of pyrilamine
maleate
Standard solution: Transfer 20 mg of USP Methadone
Hydrochloride RS to a 25-mL volumetric flask. Add
2.0 mL of Internal standard solution, and dilute with
water to volume.
Sample solution: Transfer a volume of Oral Solution
equivalent to 20 mg of methadone hydrochloride to a
125-mL separator. Extract the specimen with two
50-mL portions of ether, collecting the ether extracts in
a second separator. Wash the combined ether extracts
with 2 mL of water, and discard the ether extract.
Transfer the aqueous wash and the aqueous specimen
to a 25-mL volumetric flask. Add 2.0 mL of Internal
standard solution, and dilute with water to volume. Pass
the solution throughafilter of 5-l1m pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 3.9-mm x 30-cm; packing L11
Flow rate: 1.3 mL/min
Injection volume: 10 uL
System suitability
Sample: Standard solution
The retention times for the internal standard and
methadone hydrochloride are about 5.5 and 9 min,
respectively.
Suitability requirements
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of methadone hydrochloride
(CaH27NO - HCl) in the Oral Solution taken:
Result = (Ru/Rs) x (Cs/Cu) x 100
Official Monographs / Methadone 2629
Ru = peak response ratio of methadone
hydrochloride to the internal standard from
the Sample solution
Rs = peak response ratio of methadone
hydrochloride to the internal standard from
the Standard solution
Cs = concentration of USP Methadone
Hydrochloride RS in the Standard solution
(mg/mL)
Cy = nominal concentration of methadone
hydrochloride in the Sample solution
(mg/mL)
Acceptance criteria: 90.0%-110.0%
OTHER COMPONENTS
e ALCOHOL DETERMINATION, Method II (611) (if present):
90.0%-115.0% of the labeled amount of C2HsOH, deter-
mined by the gas Ghpamategraphi procedure using ace-
tone as the internal standar
PERFORMANCE TESTS
e UNIFORMITY OF DOSAGE UNITS (905): Meets the require-
ments for Oral Solution packaged in single-unit
containers
e DELIVERABLE VOLUME (698): Meets the requirements for
Oral Solution packaged in multiple-unit containers
SPECIFIC TESTS
° PH (791): 1.0-4.0
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers,
protected from light, and store at controlled room
temperature.
e USP REFERENCE STANDARDS (11)
USP Methadone Hydrochloride RS
=
4)
a)
Methadone Hydrochloride Tablets <
)
DEFINITION Fe}
Methadone Hydrochloride Tablets contain NLT 93.0% and °
NMT 107.0% of the labeled amount of methadone hy- to)=
drochloride (C21H27NO - HCl). Q
a]
IDENTIFICATION
=F
“
cA. oo CHROMATOGRAPHIC IDENTIFICATION TEST
(201
Sample: Equivalent to 5 mg of methadone hydrochlo-
ride from a quantity of finely powered Tablets
Chromatographic system
Developing solvent system: Alcohol, glacial acetic
acid, and water (5:3:2)
Analysis: Shake the Sample with 5 mL of sodium car-
bonate TS, and extract with 5 mL of chloroform. Pro-
ceed as directed using iodoplatinate TS to visualize the
spots.
Acceptance criteria: Meet the requirements
ASSAY
© PROCEDURE
Mobile phase: Acetonitrile and 0.03 M monobasic po-
tassium phosphate (40:60). Adjust with phosphoric acid
to a pH of 3.2.
Standard solution: 0.4 mg/mL of USP Methadone Hy-
drochloride RS in Mobile phase
Sample solution: 0.4. mg/mL of methadone hydrochlo-
ride in Mobile phase. Prepare by transferring an amount
of finely powdered Tablets (NLT 20) equivalent to
10 mg of methadone hydrochloride to a 25-mL volu-
metric flask, add 10 mL of Mobile phase, and sonicate
briefly. Shake by mechanical means for 15 min, dilute
with Mobile phase to volume, and filter.
 2630 Methadone / Official Monographs USP 41

Chromatographic system e USP REFERENCE STANDARDS (11)

(See Chromatography (621), System Suitability.) USP Methadone Hydrochloride RS
Mode: LC
Detector: UV 254 nm
Column: 3,.9-mm x 30-cm; packing L11
Flow rate: 1.5 mL/min
Injection volume: 10 uL Methadone Hydrochloride Tablets for
System suitability
Sample: Standard solution Oral Suspension
Suitability requirements
Tailing factor: NMT 2.0 DEFINITION
Relative standard deviation: NMT 2.0% Methadone Hydrochloride Tablets for Oral Suspension con-
Analysis tain NLT 93.0% and NMT 107.0% of the labeled amount
Samples: Standard solution and Sample solution of methadone hydrochloride (C2iH27NO - HCl).
Calculate the percentage of the labeled amount of IDENTIFICATION
methadone hydrochloride (C21H27NO - HCl) in the por-
e * eR CHROMATOGRAPHIC IDENTIFICATION TEST
tion of Tablets taken: 20
Result = (ru/rs) x (Cs/Cu) x 100 Sample solution: Shake a quantity of powdered Tablets
for Oral Suspension equivalent to 5 mg of methadone
ty = peak response from the Sample solution hydrochloride with 5 mL of sodium carbonate TS, and
rs = peak response from the Standard solution extract with 5 mL of chloroform.
Cs = concentration of USP Methadone Developing solvent system: Alcohol, glacial acetic
Hydrochloride RS in the Standard solution acid, and water (5:3:2)
(mg/mL) Analysis: Proceed as directed, using iodoplatinate TS to
Cy = nominal concentration of methadone visualize the spots.
hydrochloride in the Sample solution Acceptance criteria: Meet the requirements
(mg/mL)
Acceptance criteria: 93.0%-107.0%
ASSAY
© PROCEDURE
PERFORMANCE TESTS Mobile phase: Acetonitrile and 0.03 M monobasic po-
e DISSOLUTION (711) tassium phosphate (40:60). Adjust with phosphoric acid
Medium: Water; 500 mL to a pH of 3.2.
Apparatus 1: 100 rpm Standard solution: 0.4 mg/mL of USP Methadone Hy-
Time: 45 min drochloride RS in Mobile phase
Standard solution: USP Methadone Hydrochloride RS Sample solution: 0.4 mg/mL of methadone hydrochlo-
in Medium tide in Mobile phase. Prepare by transferring an amount
Sample solution: Sample per Dissolution (711). of ine powdered Tablets for Oral Suspension (NLT 20)
fe
“”
Instrumental conditions equivalent to 10 mg of methadone hydrochloride to a
ie Mode: Vis 25-mL volumetric flask, adding 10 mL of Mobile phase,
Ss and sonicating briefly. Shake by mechanical means for
Analytical wavelength: Maximum at about 405 nm
D
J
15 min, dilute with Mobile phase to volume, and filter.
iS) Analysis: Filter a portion of the Sample solution, and
Chromatographic system
= pipet a volume of the filtrate equivalent to about
S 400 ug of methadone hydrochloride into a suitable (See Chromatography (621), System Suitability.)
= separator. Add 1 mL of glacial acetic acid and 20 mL of Mode: Lc
i a solution of bromocresol purple prepared by dissolving Detector: UV 254 nm
“ 200 mg of bromocresol purple in 1000 mL of dilute gla- Column: 3.9-mm x 30-cm; packing L11
> cial acetic acid (1 in 50). Mix, and extract with 20.0 mL Flow rate: 1.5 mL/min
of chloroform. Injection volume: 10 pL
Determine the amount of methadone hydrochloride System suitability
(C21H27NO - HCl) dissolved from visible absorbances at Sample: Standard solution
the wavelength of maximum absorbance of the chlo- Suitability requirements
roform extract so obtained in comparison with the Tailing factor: NMT 2.0
chloroform extract similarly prepared from the Stan- Relative standard deviation: NMT 2.0%
dard solution. Analysis
Tolerances: NLT 75% (Q) of the labeled amount of Samples: Standard solution and Sample solution
methadone hydrochloride (CzH27NO - HCl) is dissolved. Calculate the percentage of the labeled amount of
e UNIFORMITY OF DOSAGE UNITS (905): Meet the methadone hydrochloride (C2:H27NO - HCl) in the por-
requirements tion of Tablets for Oral Suspension taken:

ADDITIONAL REQUIREMENTS Result = (ru/rs) x (Cs/Cu) x 100

e PACKAGING AND STORAGE:
containers.
Preserve in well-closed
ru = peak response from the Sample solution
ts = peak response from the Standard solution
Cs = concentration of USP Methadone
Hydrochloride RS in the Standard solution
(mg/mL)
Gu = nominal concentration of methadone
hydrochloride in the Sample solution
(mg/mL)
USP 41
Acceptance criteria: 93.0%-107.0%
PERFORMANCE TESTS
e DISINTEGRATION (701)
Time: 15 min
Acceptance criteria: Meet the requirements
e UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed
containers.
e LABELING: Label the Tablets for Oral Suspension to indi-
cate that they are intended for dispersion in a liquid
before oral administration of the prescribed dose.
e USP REFERENCE STANDARDS (11)
USP Methadone Hydrochloride RS
Methamphetamine Hydrochloride
CroHisN - HCI 185.69
Benzeneethanamine, N,o-dimethyl-, hydrochloride, (S)-;
Caan methyiphenethylamine hydrochloride
51-57-0].
DEFINITION
Methamphetamine Hydrochloride contains NLT 98.5% and
NMT 100.5% of methamphetamine hydrochloride
(CioHisN - HCI), calculated on the dried basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
e B. IDENTIFICATION TESTS—GENERAL, Chloride (191)
ASSAY
e PROCEDURE
Mobile phase: Prepare a degassed solution of 1.1 g of
sodium 1-heptanesulfonate in a mixture of water, meth-
anol, and diluted glacial acetic acid (7 in 50)
(575:400:25). Adjust with acetic acid to a pH of 3.3 +
0.1, if necessary. Filter through a 0.5-um disk.
Standard solution: 0.2 mg/mL of USP
Methamphetamine Hydrochloride RS in 0.12 M phos-
phoric acid. Sonicate if necessary.
Sample solution: 0.2 mg/mL of Methamphetamine Hy-
drochloride in 0.12 M phosphoric acid
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 257 nm
Column: 3.9-mm x 30.0-cm; packing L1
Flow rate: 2 mL/min
Injection volume: 20 pL
System suitability
Sample: Standard solution
Suitability requirements
Column efficiency: NLT 1000 theoretical plates
Tailing factor: NMT 1.5
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of methamphetamine hydro-
chloride (CioHisN - HCl) in the portion of
Methamphetamine Hydrochloride taken:
Result = (ru/rs) x (Cs/Cu) x 100
Official Monographs / Methamphetamine 2631
ru = peak response from the Sample solution
Is = peak response from the Standard solution
Cs = concentration of USP Methamphetamine
Hydrochloride RS in the Standard solution
(mg/mL)
Cu = concentration of Methamphetamine
Hydrochloride in the Sample solution
(mg/ml)
Acceptance criteria: 98.5%-100.5% on the dried basis
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.1%
© ORDINARY IMPURITIES (466)
Standard solution and Sample solution: Chloroform
Eluant: Chloroform, cyclohexane, and diethylamine
(5:4:1)
Visualization: 1
Acceptance criteria: Meets the requirements
SPECIFIC TESTS
e MELTING RANGE OR TEMPERATURE (741): 171°-175°
e OPTICAL ROTATION, Specific Rotation (7815S)
Sample solution: 20 mg/mL in water
Acceptance criteria: Between +16° and +19°
e Loss ON DRYING (731)
Analysis: Dry at 105° for 2 h.
Acceptance criteria: NMT 0.5%
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers.
e USP REFERENCE STANDARDS (11)
USP Methamphetamine Hydrochloride RS
Methamphetamine Hydrochloride (<4
Tablets nw
i)
DEFINITION c=
Methamphetamine Hydrochloride Tablets contain NLT i}
3
90.0% and NMT 110.0% of the labeled amount of ry
methamphetamine hydrochloride (CioHisN - HCl). eet
ES)
IDENTIFICATION so}
e A. The UV absorption spectrum of the Sample solution, a
“
prepared as described in Procedure for content uniformity
in Uniformity of Dosage Units, exhibits maxima and min-
ima at the same wavelengths as those of the Standard
solution, concomitantly measured.
ASSAY
e PROCEDURE
Mobile phase: Prepare a degassed solution of 1.1 g of
sodium 1-heptanesulfonate in a mixture of water, meth-
anol, and diluted glacial acetic acid (7 in 50)
(575:400:25). Adjust with acetic acid to apts of 3.3.4
0.1, if necessary. Filter through a 0.5-um disk.
Standard solution: 0.2 mg/mL of USP
Methamphetamine Hydrochloride RS in 0.12 M phos-
phoric acid. Sonicate if necessary.
Sample solution: Transfer a portion of fine powder
from NLT 20 Tablets, nominally equivalent to about
10 mg of methamphetamine hydrochloride, to a 50-mL
volumetric flask. Add 20 mL of 0.12 M phosphoric acid,
and sonicate for 5 min. Dilute with 0.12 M phosphoric
acid to volume, and filter.
Chromatographic system
(See Chromatography (621), System Suitability.)
 2632 Methamphetamine / Official Monographs
Mode: LC
Detector: UV 257 nm
Column: 3.9-mm x 30.0-cm; packing L1
Flow rate: 2 mL/min
Injection volume: 20 pL
System suitability
Sample: Standard solution
Suitability requirements
Column efficiency: NLT 1000 theoretical plates
Tailing factor: NMT 1.5
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
methamphetamine hydrochloride (CjoHisN - HCI) in
the portion of Tablets taken:
Result = (ru/rs) x (Cs/Cu) x 100
i = peak response from the Sample solution
ls = peak response from the Standard solution
Cs = concentration of USP Methamphetamine
Hydrochloride RS in the Standard solution
(mg/mL)
Cu = nominal concentration of methamphetamine
hydrochloride in the Sample solution
(mg/mL)
Acceptance criteria: 90.0%-110.0%
PERFORMANCE TESTS
e DISSOLUTION (711)
Medium: Water; 900 mL
Apparatus 2: 50 rpm
Time: 45 min
Mobile phase: Acetonitrile and dilute perchloric acid (1
in 20) (300:700). Filter, and degas.
Sample solution: Filter aliquots of the solution in the
al test, and dilute 2:1 with 0.15 M perchloric acid.
aod Standard solution: Dissolve USP Methamphetamine
oy Hydrochloride RS in water to obtain a concentration
=J similar to the one expected in the Sample solution. Di-
i)
° lute 2:1 with 0.15 M perchloric acid.
<7 Chromatographic system
Sj Mode:
3 Detector: UV 211 nm
a Column: 3.9-mm x 30-cm; packing L1
a) Flow rate: 2.5 mL/min
=) Injection volume: 100 LL
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 1.5
Relative standard deviation: NMT 3.0%
Analysis
Samples: Sample solution and Standard solution
Calculate the percentage of the labeled amount of
methamphetamine hydrochloride (CioHisN - HCI) dis-
solved by comparing the major peak response of the
Sample solution with that of the Standard solution.
Tolerances: NLT 75% (Q) of the labeled amount of
Ges eee tetera hydrochloride (CicHisN + HCl) is
dissolved.
e UNIFORMITY OF DosAGE UNITS (905)
Procedure for content uniformity
Solution A: Shake 250 mL of 0.1 N sulfuric acid with
25 mL of chloroform for 10 min. Allow to stand for 1
h with occasional shaking. Drain off the chloroform,
and retain the chloroform-saturated sulfuric acid in a
eorpeteeflask.
Standard solution: 0.5 mg/mL of USP
Methamphetamine Hydrochloride RS in Solution A
Sample solution: Place 1 Tablet in a 125-mL
separator. Add 15 mL of water, and shake by mechani-
al surons for 15 min to dissolve. Add 2.5 mL of 1N
sodium hydroxide, and shake. Extract the liberated
USP 41
nefhampbelamie with four 10-mL portions of chloro-
form, collecting the chloroform extracts in a second
125-mL separator. Transfer 10.0 mL of Solution A to
the second separator, and shake by mechanical means
for 10 min. Allow the layers to separate, and collect
the aqueous layer.
Instrumental conditions
Mode: UV
Analytical wavelength: Maximum at about 257 nm
Cell: 1.cm
Blank: Solution A
Analysis
ne Standard solution, Sample solution, and
lan
Calculate the percentage of the labeled amount of
methamphetamine hydrochloride (CjoHisN « HCl) in
the portion of Tablets taken:
Result = (Au/As) x (Cs/Cu) x 100
Au = absorbance of the Sample solution
As = absorbance of the Standard solution
Cs = concentration of USP Methamphetamine
Hydrochloride RS in the Standard solution
(mg/mL)
Cy = nominal concentration of methamphetamine
hydrochloride in the Sample solution
(mg/mL)
Acceptance criteria: Meet the requirements
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers.
e USP REFERENCE STANDARDS (11)
USP Methamphetamine Hydrochloride RS
Methazolamide
CsHgN4O3S2 236,27
Acetamide, N-[5-(aminosulfonyl)-3-methyl-1,3,4-thiadiazol-
2(3H)-ylidene]-.
N-(4-Methyl-2-sulfamoyl-A2-1,3,4-thiadiazolin-5-ylidene)-
acetamide [554-57-4].
» Methazolamide contains not less than 98.0 per-
cent and not more than 102.0 percent of
CsHgN4O3S2, calculated on the dried basis.
Packaging and storage—Preserve in well-closed, light-re-
sistant containers.
USP Reference standards (11)—
USP Methazolamide RS
Identification—
A: Infrared Absorption (197K).
B: Ultraviolet Absorption (197U)—
Solution: 10g per mL.
ee sodium hydroxide solution in water (1 in
250).
Loss on drying (731)—Dry it at 105° for 2 hours: it loses
not more than 0.5% of its weight.
Residue on ignition (281): not more than 0.1%.
Selenium (291): 0.003%, a 200-mg specimen being
used.
USP 41
Delete the following:
°Heavy metals, Method // (231): 0.002%. (official 1-Jan-2018)
Assay—
Buffer solution—Dissolve 1.80 g of anhydrous sodium ace-
tate in 1 L of water. Adjust, if necessary, with glacial acetic
acid to a pH of 4.5 + 0.2.
Mobile phase—Preparea filtered and degassed mixture of
Buffer solution and acetonitrile (86:14). Make adjustments if
Peed (see System Suitability under Chromatography
621)).
Standard preparation—Dissolve about 20 mg of USP
Methazolamide RS, accurately weighed, in 20 mL of acetoni-
trile contained in a 200-mL volumetric flask. Dilute with
Buffer solution to volume, and mix. Quantitatively dilute an
accurately measured volume of this solutionwith Buffer solu-
tion to obtain a solution having a known concentration of
about 50 ug of USP Methazolamide RS per mL.
Resolution solution—Prepare a solution of acetaminophen
and USP Methazolamide RS in acetonitrile containing
0.3 mg of acSapepe and 0.5 mg of methazolamide
per mL. Quantitatively dilute an accurately measured vol-
ume of this solution with Buffer solution to obtain a solution
containing 30 yg of acetaminophen and 50 ug of methazo-
lamide per mL. : 4.5; 900 mL.
Assay preparation—tTransfer about 100 mg of Methazo-
lamide, accurately weighed, to a 200-mL volumetric flask,
dissolve in 20 mL of acetonitrile, dilute with Buffer solution to
volume, and mix. Quantitatively dilute an accurately meas-
ured volume of this solution with Buffer solution to obtain a
solution having a known concentration of about 50 1g per
mL.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 265-nm detector
and a 3.9-mm x 15.0-cm column containing packing L1.
The flow rate is about 1.5 mL per minute. Chromatograph
the Resolution solution, and record the peak responses as
directed for Procedure: the relative retention times are about
0.6 for acetaminophen and 1.0 for methazolamide, the res-
olution, R, between the acetaminophen peak and the
methazolamide peak is not less than 4.0, and the tailing
factor is not more than 2,0. Chromatograph the Standard
preparation, and record the peak responses as directed for
Procedure: the relative standard deviation for replicate injec-
tions is not more than 2%.
Procedure—Separately inject equal volumes (about 10 yL)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
ure the areas of the responses for the major peaks. Calculate
the quantity, in mg, of CsHsN4O3S2 in the portion of
Methazolamide taken by the formula:
2C(ru/ rs)
in which C is the concentration, in ug per mL, of USP
Methazolamide RS in the Standard preparation; and ry and rs
are the peak responses obtained from the Assay preparation
and the Standard preparation, respectively.
Methazolamide Tablets
» Methazolamide Tablets contain not less than
90.0 percent and not more than 110.0 percent of
the labeled amount of methazolamide
(CsHgNzO3Sz).
Packaging and storage—Preserve in well-closed contain-
ers.
Official Monographs / Methazolamide 2633
USP Reference standards (11)—
USP Methazolamide RS
Identification—
A: Extract a quantity of finely powdered Tablets, equiva-
lent to about 250 mg of methazolamide, with about 50 mL
of acetone. Filter, and add solvent hexane until a heavy
white precipitate is formed. Collect the solid onafilter, and
dry: the IR absorption sao of a potassium bromide dis-
persion of the methazolamide so obtained exhibits maxima
only at the same wavelengths as that of a similar prepara-
tion of USP Methazolamide RS.
B: Dissolve about 100 mg of the dried solid obtained in
Identification test A in 5 mL of 1 N sodium hydroxide, and
add 5 mL of a mixture of 1 g of hydroxylamine hydrochlo-
ride and 500 mg of cupric sulfate in 100 mL of water. Heat
the solution on a steam bath for 15 minutes: the solution
turns dark amber, then a black precipitate is formed.
C: The retention time of the major peak in the chromato-
gram of the Assay preparation corresponds to the chromato-
gram of the Standard preparation, as obtained in the Assay.
Dissolution (711)—
Medium: _ pH 4.5 acetate buffer, prepared by mixing
2.99 g of sodium acetate and 1.66 mL of glacial acetic acid
with water to obtain 1000 mL of a solution having a pH of
Apparatus 2: 75 rpm.
Time: 45 minutes.
Procedure—Determine the amount of CsHgN4O3S2 dis-
solved from UV absorbances at the wavelength of maximum
absorbance at about 252 nm of filtered portions of the solu-
tion under test, suitably diluted with pH 4.5 acetate buffer,
in comparison with a Standard solution having a known
: ae of USP Methazolamide RS in the same Me-
ium.
Tolerances—Not less than 75% (Q) of the labeled amount
of CsHgN4O3S2 is dissolved in 45 minutes. (=
al
Uniformity of dosage units (905): meet the require- a)
ments.
=
Assay— °
pH 2.5 Buffer—Transfer 16.8 mL of dibutylamine to a bj
°
beaker containing 70 mL of water. Adjust with phosphoric Ke}
acid to a pH of 2.5, dilute with water to 100 mL, and mix. =
Ey
Mobile phase—Prepare a mixture of water, methanol, and mo]
pH 2.5 Buffer (375:15:6). Make adjustments if necessary (see a
“
System Suitability under Chromatography (621)).
pH 4.5 Acetate buffer—Dissolve 2.99 g of sodium acetate
and 1.66 mL of glacial acetic acid in water, dilute with
water to 1000 mL, and mix. Adjust, if necessary, with glacial
acetic acid or sodium hydoxide to a pH of 4.5.
Standard preparation—Dissolve an accurately weighed
quantity of USP Methazolamide RS in methanol to obtain a
solution having a concentration of about 0.5 mg per mL.
Dilute this solution quantitatively, and stepwise if necessary,
with pH 4.5 Acetate buffer to obtain a solution having a
known concentration of about 50 ug per mL.
Assay preparation—Weigh and finely powder not fewer
than 20 Tablets. Transfer an accurately weighed portion of
the powder, equivalent to about 50 mg of methazolamide,
to a 250-mL volumetric flask, add 65 mL of pH 4.5 Acetate
buffer, and sonicate to dissolve. Add 65 mL of methanol,
and sonicate again until dissolved. Dilute with pH 4.5 Ace-
tate buffer to volume, mix, and filter. Dilute an ape
measured volume of the filtrate with pH 4.5 Acetate buffer to
obtain a solution having a concentration of about 50 ug of
methazolamide per mL.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 252-nm detector
and an 8-mm x 10-cm column that contains packing L10.
The flow rate is about 2 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as di-
rected for Procedure: the tailing factor is not more than 1.5;
 2634 Methazolamide / Official Monographs
and the relative standard deviation for replicate injections is
not more than 3.0%.
Procedure—Separately inject equal volumes (about 25 uL)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
ure the areas for the major peaks. Calculate the quantity, in
mg, of methazolamide (CsHgN4O3S2) in the portion of Tab-
lets taken by the formula:
Cru
/ 15)
in which C is the concentration, in ug per mL, of USP
Methazolamide RS in the Standard preparation; and ry and rs
are the peak responses obtained from the Assay preparation
and the Standard preparation, respectively.
Methdilazine Hydrochloride
CigH2oN2S + HCl 332.89
10H-Phenothiazine, 10-[(1-methyl-3-pyrrolidinyl)methyl]-,
monohydrochloride.
10-[(1 cue yrrolidinyl)methyl]phenothiazine monohy-
drochloride 1299-35-91,
» Methdilazine Hydrochloride contains not less
than 97.0 percent and not more than 103.0 per-
cent of CigH2oN2S - HCI, calculated on the dried
basis.
Packaging and storage—Preserve in tight, light-resistant
containers.
USP Reference standards (11)—
USP Methdilazine Hydrochloride RS
[NoTE—Throughout the following procedures, protect test
is
“
or assay specimens, the Reference Standard, and solutions
5 containing them, by conducting the procedures without de-
S
— lay, under subdued light, or using low-actinic glassware.]
Dd
° Identification—
=)
3 A: Infrared Absorption (197K).
= B: Ultraviolet Absorption (197U)—
Solution: 5 wg per mL.
a
al
Medium: water.
=)
agi A solution of it responds to the tests for Chloride
Melting range, Class | (741): between 184° and 190°.
PH (791): between 4.8 and 6.0, in a solution (1 in 100).
Loss on drying (731)—Dry it in vacuum at 65° for
16 hours: it loses not more than 1.0% of its weight.
Residue on ignition (281): not more than 0.5%.
Selenium (291)—The absorbance of the solution from the
Test Solution, prepared with 100 mg of Methdilazine Hydro-
chloride andfoo mg of magnesium oxide, is not greater
than one-half that from the Standard Solution (0.003%).
Delete the following:
°Heavy metals, Method II (231): 0.002%. cotiicial 1-Jan-2018)
Ordinary impurities (466)—
Test solution: methanol.
Standard solution: methanol.
Application volume: 10 pL.
Eluant: a mixture of toluene, isopropyl alcohol, and
ammonium hydroxide (70:29:1), in a nonequilibrated cham-
ber.
USP 41
Visualization: 5.
Assay—Transfer about 100 mg of Methdilazine Hydrochlo-
ride, accurately weighed, to a 1000-mL volumetric flask, add
water to volume, and mix. Transfer 5.0 mL of this solution
to a 100-mL volumetric flask, dilute with water to volume,
and mix. Concomitantly determine the absorbances of this
solution and of a Standard solution of USP Methdilazine Hy-
drochloride RS in the same medium having a known con-
centration of about 5 ug per mL, in 1-cm cells at 252 and
275 nm, with a suitable spectrophotometer, using water as
the blank. Calculate the quantity, in mg, of CisH2oN2S- HCl
n ue pete of Methdilazine Hydrochloride taken by the
ormula:
20C(Azs2 — Azzs)u / (Azs2 — Azzs)s
in which C is the concentration, in ug per mL, of USP
Methdilazine Hydrochloride RS in the Standard solution; and
the parenthetic expressions are the differences in the ab-
sorbances of the two solutions at the wavelengths indicated
it the subscripts, for the solution of Methdilazine Hydro-
chloride (U) and the Standard solution (5), respectively.
Methdilazine Hydrochloride Oral
Solution
» Methdilazine Hydrochloride Oral Solution con-
tains not less than 93.0 percent and not more
than 107.0 percent of the labeled amount of
methdilazine hydrochloride (CigH2oN2S - HCl).
Packaging and storage—Preserve in tight, light-resistant
containers.
USP Reference standards (11)—
USP Methdilazine Hydrochloride RS
[NoTE—Throughout the following procedures, protect test
or assay specimens, the USP Reference Standard, and solu-
tions containing them, by conducting the procedures with-
out delay, under subdued light, or using low-actinic
glassware.]
Identification—Transfer a volume of Oral Solution, equiva-
lent to about 4 mg of methdilazine hydrochloride, to a
60-mL separator, add 5 mL of 0.1 N hydrochloric acid, and
extract with 10 mL of ether, discarding the extract. Add
10 mL of sodium bicarbonate solution (1 in 10) to the
separator, and extract with 3 mL of chloroform. Filter the
extract through a pledget of cotton. Evaporate the chloro-
form, carefully removing the last trace of solvent in a small
vacuum flask: the IR absorption spectrum of a potassium
bromide dispersion of the methdilazine so obtained exhibits
maxima only at the same wavelengths as that of a similar
pleparaiien of USP Methdilazine Hydrochloride RS that has
een treated in the same manner.
PH (791): between 3.3 and 4.1.
Alcohol Determination, Method |! (611): between
6.5% and 7.5% of C2HsOH.
Assay—
Standard preparation—Dissolve a suitable quantity of USP
Methdilazine Hydrochloride, accurately weighed, in chloro-
form, and quantitatively dilute with chloroform to obtain a
solution having a known concentration of about 400 yg per
mL.
Assay preparation—Transfer a volume of Oral Solution,
equivalent to about 4 mg of methdilazine hydrochloride, to
a 60-mL separator, add 10 mL of a saturated solution of
sodium chloride, and extract with three 10-mL portions of
chloroform, transferring the extracts to a 100-mL volumetric
flask.
USP 41
Procedure—Transfer 10.0 mL of Standard preparation to a
100-mL volumetric flask, and add 20 mL of chloroform. To
this flask and to the flask containing the Assay preparation
add 4.0 mL of buffered palladium chloride TS, dilute with
alcohol to volume, and mix. Concomitantly determine the
absorbances of the solutions in 1-cm cells at the wavelength
of maximum absorbance at about 460 nm, with a suitable
spectrophotometer, using a mixture of 30 mL of chloroform,
4 mL of palladium chloride TS, and 66 mL of alcohol as the
blank. Calculate the quantity, in mg, of methdilazine hydro-
chloride (CisH2oN2S - HCI ) in each mL of the Oral Solution
taken by the formula:
(0.01C/ V)(Au/ As)
in which C is the concentration, in wg per mL, of USP
Methdilazine Hydrochloride RS in the Standard preparation;
Vis the volume, in mL, of Oral Solution taken; and Ay and
As are the absorbances of the solutions from the Assay prep-
aration and the Standard preparation, respectively.
Methdilazine Hydrochloride Tablets
» Methdilazine Hydrochloride Tablets contain not
less than 93.0 percent and not more than
107.0 percent of the labeled amount of methdi-
lazine hydrochloride (CisH2oN2S - HCl).
Packaging and storage—Preserve in tight, light-resistant
containers.
USP Reference standards (11)—
USP Methdilazine Hydrochloride RS
[NoTE—Throughout the following procedures, protect test
or assay specimens, the Reference Standard, and solutions
containing them, by conducting the procedures without de-
lay, under subdued light, or using low-actinic glassware.]
Identification—Transfer a portion of finely powdered Tab-
lets, equivalent to about 8 mg of methdilazine hydrochlo-
ride, to a 60-mL separator, add 10 mL of sodium bicarbo-
nate solution (1 in 10), and extract with 3 mL of
chloroform. Filter the extract through a pledget of cotton.
Evaporate the chloroform, carefully removing the last trace
of solvent in a small vacuum flask: the IR absorption spec-
trum of a potassium bromide dispersion of the methdilazine
so obtained exhibits maxima only at the same wavelengths
as that of a similar preparation of USP Methdilazine Hydro-
chloride RS, similarly treated and measured.
Dissolution (711)—
Medium: water; 900 mL.
Apparatus 1: 100 rpm.
Time: 45 minutes.
Procedure—Determine the amount of CisHzoN2S - HCI dis-
solved from UV absorbances at the wavelength of maximum
absorbance at about 252 nm of filtered portions of the solu-
tion under test, suitably diluted with Dissolution Medium, if
necessary, in comparison with a Standard solution having a
known concentration of USP Methdilazine Hydrochloride RS
in the same Medium.
Tolerances—Not less than 75% (Q) of the labeled amount
of CisH2oN2S - HCI is dissolved in 45 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Assay—
Standard preparation—Dissolve a suitable quantity of USP
Methdilazine Hydrochloride RS, accurately weighed, in chlo-
roform, and dilute queritsartively with chloroform to obtain
a solution having a known concentration of about 400 pg
per mL.
Official Monographs / Methenamine 2635
Assay preparation—Weigh and finely powder not fewer
than 20 Tablets, and transfer an accurately weighed portion
of the powder, equivalent to about 80 mg of methdilazine
hydrochloride, to a 200-mL volumetric flask. Add 60 mL of
chlorororti, shake for 20 minutes, dilute with chloroform to
volume, and mix. Filter, discarding the first 15 mL of the
filtrate. Use the subsequent filtrate as directed in the Proce-
dure.
Procedure—into three separate 100-mL volumetric flasks
transfer 10.0 mL each of the Standard preparation, the Assay
preparation, and chloroform to provide the blank. To each
flask add 20 mL of chloroform and 4.0 mL of buffered palla-
dium chloride TS, dilute with alcohol to volume, and mix.
Concomitantly determine the absorbances of the solutions
in 1-cm cells at the wavelength of maximum absorbance at
about 460 nm, with a suitable spectrophotometer, using the
blank to set the instrument. Calculate the quantity, in mg,
of methdilazine hydrochloride (CisHz0N2S - HCI) in the por-
tion of Tablets taken by the formula:
0.2C(Au/ As)
in which C is the concentration, in ug per mL, of USP
Methdilazine Hydrochloride RS in the Standard preparation;
and Ay and As are the absorbances of the solutions from the
Assay preparation and the Standard preparation, respectively.
Methenamine
CeHi2Na 140.19 [ie
1,3,5,7-Tetraazatricyclo[3.3.1.137]decane; vy
Hexamethylenetetramine [100-97-0]. oe
DEFINITION =
Methenamine, dried over phosphorus pentoxide for 4 h, s
contains NLT 99.0% and NMT 100.5% of methenamine &
(CéHi2Na). =
IDENTIFICATION 2
e A. INFRARED ABSORPTION (197K) ry
e B.
Analysis: Heat a solution (1 in 10) with 2 N sulfuric
acid.
Acceptance criteria: Formaldehyde is liberated, recog-
nizable by its odor and by its darkening of paper moist-
ened with silver ammonium nitrate TS. On the subse-
uent addition of an excess of 1 N sodium hydroxide to
the solution, ammonia is evolved.
ASSAY
e PROCEDURE
Chromotropic acid spot test solution: Suspend
100 mg of chromotropic acid in 2 mL of water, and
cautiously add 3 mL of sulfuric acid. Allow to cool, and
add 25 mL of sulfuric acid. If excessive heat generated
during mixing causes a violet color to appear in the
solution, discard the solution and prepare another, tak-
ing precautions to avoid excessive heat.
Sample solution: Transfer \9 of Methenamine, previ-
ously dried, to a beaker. Add 40.0 mL of 1 N sulfuric
acid VS, and heat to a gentle boil, adding water from
time to time if necessary, until the formaldehyde has
been expelled. Test for the absence of formaldehyde by
adding a drop of the assay solution to a glass fiber filter
disk, on a watch glass, on which has previously been
placed 3 or 4 drops of Chromotropic acid spot test solu-
tion. Formaldehyde produces a violet color with this re-
agent. Repeat the test until no violet color is obtained
 2636 Methenamine / Official Monographs USP 41

on the warmed test filter disk upon comparison with a ened with silver ammonium nitrate TS. On the subse-
blank filter disk to which no assay specimen is added. uent addition of an excess of 1 N sodium hydroxide to
Cool, add 20 mL of water, then add methyl red TS. the solution, ammonia is evolved.
Titrimetric system
Mode: Residual titration ASSAY
Titrant: 1 N sulfuric acid VS © PROCEDURE
Back-titrant: 1.N sodium hydroxide VS Chromotropic acid solution: Mix 100 mg of chromo-
Endpoint detection: Visual tropic acid with 50 mL of water in a 100-mL volumetric
Analysis: Titrate the excess acid in the Sample solution flask. Cool in an ice bath and, while cooling, cautiously
with 1 N sodium hydroxide VS. Perform a blank deter- and slowly add 50 mL of sulfuric acid. Allow the solu-
mination (see Titrimetry (541), Residual Titrations). Each tion to reach room temperature, and add dilute sulfuric
mL of 1 N sulfuric acid is equivalent to 35.05 mg of acid (1 in 2) to volume. If excessive heat generated
methenamine (Ce6Hi2Na). during mixing causes a violet color to appear in the
Acceptance criteria: 99.0%-100.5% solution, discard the solution and prepare another, tak-
ing precautions to avoid excessive heat.
IMPURITIES Standard stock solution: 0.05 mg/mL of USP Methena-
e RESIDUE ON IGNITION (281): NMT 0.1% mine RS
e CHLORIDE AND SULFATE, Chloride (221) Standard solution: 1 g/mL of USP Methenamine RS
Sample: 1.0g prepared as follows. Transfer 2.0 mL of Standard stock
Acceptance criteria: 0.014%; shows no more chloride Solution to a 100-mL volumetric flask, then add 25 mL
aa. corresponds to 0.20 mL of 0.020 N hydrochloric of Chromotropic acid solution and 50 mL of dilute sulfu-
acid. ric acid (1 in 2). Place the flask in a boiling water bath
© SULFATE for 30 min, accurately timed, then remove it from the
Sample solution: 20 mg/mL bath. Cool immediately to room temperature, then add
Analysis: 10 mL of the Sample solution, acidified with dilute sulfuric acid (1 in 2) to volume.
5 drops of hydrochloric acid. Add 5 drops of barium Standard blank: Transfer 2.0 mL of Standard stock solu-
chloride TS. tion to a 100-mL volumetric flask, then add 75 mL of
Acceptance criteria: No turbidity is produced within 1 dilute sulfuric acid (1 in 2). Place the flask in a boiling
min. water bath for 30 min, accurately timed, then remove it
from the bath. Cool immediately to room temperature,
then add dilute sulfuric acid (1 in 2) to volume.
Delete the following: Sample stock solution: Nominally 60 ug/mL of methe-
namine from Oral Solution
®e HEAVY METALS, Method / (231) Sample solution: Nominally 1.2 ug/mL of methena-
Test preparation: 2g in 10 mL of water mine prepared as follows. Transfer 2.0-mL of Sample
Analysis: Add 2 mL of 3 N hydrochloric acid, and dilute Stock solution to a 100-mL volumetric flask, then add
with water to 25 mL. Proceed as directed, except use 25 mL of Chromotropic acid solution and 50 mL of dilute
glacial acetic acid to ae the pH. sulfuric acid (1 in 2). Place the flask in a boiling water
rs
al
Acceptance criteria: NMT 10 ppme cofficat 1-jan-2018)
(os bath for 30 min, accurately timed, then remove it from
the bath. Cool immediately to room temperature, then
i] SPECIFIC TESTS
—
D e Loss ON DRYING (731) add dilute sulfuric acid (1 in 2) to volume.
° Analysis: Dry over phosphorus pentoxide for 4 h. Sample blank: Transfer 2.0 mL of Sample stock solution
¢ to a 100-mL volumetric flask, then 75 mL of dilute sul-
S Acceptance criteria: NMT 2.0%
furic acid (1 in 2). Place the flask in a boiling water
3 e AMMONIUM SALTS
Sample solution: 50 mg/mL bath for 30 min, accurately timed, then remove it from
a
Analysis: Add to 10 mL of the Sample solution 1 mL of the bath. Cool immediately to room temperature, and
”
=) alkaline mercuric—potassium iodide TS. add dilute sulfuric acid (1 in 2) to volume.
Acceptance criteria: The mixture is not darker in color Instrumental conditions
than a mixture of 1 mL of the reagent and 10 mL of Mode: Vis
water. Analytical wavelength: Maxima at about 570 nm
Cell: 71cm
ADDITIONAL REQUIREMENTS Blank: Dilute sulfuric acid (1 in 2)
e PACKAGING AND STORAGE: Preserve in well-closed Analysis
containers. Samples: Standard solution, Standard blank, Sample so-
¢ USP REFERENCE STANDARDS (11) lution, Sample blank, and Blank
USP Methenamine RS Calculate the percentage of the labeled amount of me-
thenarning (CeHi2Na) in each mL of Oral Solution
taken:
Result = [(Au — Bu)/(As — Bs)] x (Cs/Cu) x 100
Methenamine Oral Solution Au = absorbance of the Sample solution
By = absorbance of the Sample blank
DEFINITION As = absorbance of the Standard solution
Methenamine Oral Solution contains NLT 90.0% and NMT Bs = absorbance of the Standard blank
110.0% of the labeled amount of methenamine Cs = concentration of USP Methenamine RS in the
(CeHi2Na). Standard solution (g/mL)
Cu = nominal concentration of methenamine in the
IDENTIFICATION Sample solution (j1g/mL)
eA. Acceptance criteria: 90.0%-110.0%
Sample solution: Heat a volume of Oral Solution,
equivalent to 1 g of methenamine, with 10 mL of 2N OTHER COMPONENTS
sulfuric acid. e ALCOHOL DETERMINATION, Method | (611): 90.0%-110.0%
Acceptance criteria: Formaldehyde is liberated, recog- of the labeled amount of C2zHsOH
nizable by its odor and by its darkening of paper moist-
USP 41 Official Monographs / Methenamine 2637

PERFORMANCE TESTS bath. Cool immediately to room temperature, then add

e UNIFORMITY OF DOSAGE UNITS (905): Meets the require- dilute sulfuric acid (1 in 2) to volume.
ments for Oral Solution packaged in single-unit Sample blank: Transfer 2.0 mL of Sample stock solution
containers to a 100-mL volumetric flask, then add 75 mL of dilute
e DELIVERABLE VOLUME (698): Meets the requirements for sulfuric acid (1 in 2). Place the flask in a boiling water
Oral Solution packaged in multiple-unit containers bath for 30 min, accurately timed, then remove it from
the bath. Cool immediately to room temperature, then
ADDITIONAL REQUIREMENTS add dilute sulfuric acid (1 in 2) to volume.
e PACKAGING AND STORAGE: Preserve in tight containers. Instrumental conditions
e USP REFERENCE STANDARDS (11) Mode: Vis
USP Methenamine RS Analytical wavelength: Maxima at about 570 nm
Cell: 1m
Blank: Dilute sulfuric acid (1 in 2)
Analysis
Samples: Standard solution, Standard blank, Sample so-
Methenamine Tablets lution, Sample blank, and Blank
Calculate the percentage of the labeled amount of me-
DEFINITION thenamine (CeHi2N,) in the portion of Tablets taken:
Methenamine Tablets contain NLT 95.0% and NMT 105.0%
of the labeled amount of methenamine (Ce6H:2N,). Result = [(Au — Bu)/(As — Bs)] x (Cs/Cu) x 100
IDENTIFICATION Au =
absorbance of the Sample solution
oA. By =
absorbance of the Sample blank
Sample: 500 mg of powdered Tablets As =
absorbance of the Standard solution
Analysis: Dissolve the Sample in 10 mL of water, add Bs =
absorbance of the Standard Blank
10 mL of 2.N sulfuric acid, and heat. Cs =
concentration of USP Methenamine RS in the
Acceptance criteria: Formaldehyde is liberated, recog- Standard solution (ug/mL)
nizable by its odor and by its darkening of paper moist- Cu = nominal concentration of methenamine in the
ened with silver ammonium nitrate TS. On the subse- Sample solution (g/mL)
uent addition of an excess of 1 N sodium hydroxide to Acceptance criteria: 95.0%-105.0%
the solution, ammonia is evolved.
PERFORMANCE TESTS
ASSAY e DISSOLUTION (711)
¢ PROCEDURE Procedure for a pooled sample
Chromotropic acid solution: Mix 100 mg of chromo- Medium: Water; 900 mL
tropic acid with 50 mL of water in a 100-mL volumetric Apparatus 1: 100 rpm
flask. Cool in an ice bath and, while cooling, cautiously ime: 45 min
and slowly add 50 mL of sulfuric acid. Allow the solu- Analysis: Determine the amount of methenamine (3
tion to reach room temperature, and add dilute sulfuric (CéHi2Na) dissolved by using the procedure set forth in wn
acid (1 in 2) to volume. If excessive heat generated the Assay, making any necessary modifications. a)
during mixing causes a violet color to appear in the Tolerances: NLT 75% (Q) of the labeled amount of c
solution, discard the solution and prepare another, tak- methenamine (CeHi2N,) is dissolved. re}
ing precautions to avoid excessive heat. © UNIFORMITY OF DOSAGE UNITS (905): Meet the A
Standard stock solution: 50 j1g/mL of USP Methena- requirements i}
mine RS
a=
ADDITIONAL REQUIREMENTS 2
Standard solution: 1 g/mL of USP Methenamine RS so}
prepared as follows. Transfer 2.0 mL of Standard stock © PACKAGING AND STORAGE: Preserve in well-closed oy
solution to a 100-mL volumetric flask, add 25 mL of containers. a)

Chromotropic acid solution and 50 mL of dilute sulfuric e USP REFERENCE STANDARDS (11)
acid (1 in 2). Place the flask in a boiling water bath for USP Methenamine RS
30 min, accurately timed, then remove it from the
bath. Cool immediately to room temperature, then add
dilute sulfuric acid (1 in 2) to volume.
Standard blank: Transfer 2.0 mL of Standard stock solu-
tion to a 100-mL volumetric flask, then add 75 mL of Methenamine Hippurate
dilute sulfuric acid (1 in 2). Place the flask in a boiling
water bath for 30 min, accurately timed, then remove it
from the bath. Cool immediately to room temperature,
then add dilute sulfuric acid (1 in 2) to volume.
Sample stock solution: Nominally 50 ug/mL of methe-
namine prepared as follows. Transfer an equivalent to
500 mg of methenamine from powdered Tablets (NLT C6Hi2N4 « CoHsNO3 319.36
20) to a 250-mL volumetric flask. Dilute with water to Glycine, N-benzoyl, compd. with 1,3,5,7-tetraazatricyclo
volume, mix, and filter, discarding the first 20 mL of the 3.3.1.137]decane (1:1);
filtrate. Transfer 25.0 mL of the subsequent filtrate to a Hexamethylenetetramine monohippurate [5714-73-8].
1000-mL volumetric flask. Dilute with water to volume.
Sample solution: Nominally 1 g/mL of methenamine DEFINITION
prepared as follows. Transfer 2.0-mL of Sample stock so- Methenamine Hippurate, dried under vacuum at 60° for 1
lution to a 100-mL volumetric flask, then add 25 mL of h, contains NLT 95.5% and NMT 102.0% of methena-
Chromotropic acid solution and 50 mL of dilute sulfuric mine hippurate (CsHi2N4 - CoHsNO3), and contains NLT
acid (1 in 2). Place the flask in a boiling water bath for 54.0% and NMT 58.0% of hippuric acid (CsHgNO3).
30 min, accurately timed, then remove it from the
 2638 Methenamine / Official Monographs USP 41

IDENTIFICATION (NLT 20) to a 250-mL conical flask. Add 50 mL of alco-

e A. INFRARED ABSORPTION (197M) hol, then add thymolphthalein TS.
Titrimetric system
ASSAY Mode: Direct titration
e PROCEDURE Titrant: 0.1 N sodium hydroxide VS
Sample solution: Dissolve 700 mg of Methenamine Endpoint detection: Visual
Hippurate in 50 mL of glacial acetic acid. Analysis: Titrate the Sample solution with Titrant. Per-
Titrimetric system form a blank determination on a mixture of 50 mL of
Mode: Direct titration alcohol and 20 mL of water, and make any necessary
Titrant: 0.1 N perchloric acid VS correction. Each mL of 0.1 N sodium hydroxide is
Endpoint detection: Potentiometric equivalent to 31.94 mg of methenamine hippurate
Analysis: Titrate with Titrant. Perform a blank determi- (CoHi2Na + CoHgNOs).
nation, and make any necessary correction. Each mL of Acceptance criteria: 95.0%-105.0%
0.1 N perchloric acid is equivalent to 31.94 mg of me-
thenamine hippurate (CsHi2N4 - CoHoNO3). PERFORMANCE TESTS
Acceptance criteria: 95.5%-102.0% on the previously e DISSOLUTION (711)
dried basis Test 1
Medium: Water; 900 mL
OTHER COMPONENTS Apparatus 2: 100 rpm
e CONTENT OF HipPuRIC ACID Time: 30 min
Sample solution: Transfer 1 g to a 250-mL conical flask, Standard solution: 22 g/mL of USP Methenamine
and add 50 mL of water. When the solution is com- Hippurate RS
plete, add phenolphthalein TS. Instrumental conditions
Titrimetric system Mode: UV
Mode: Direct titration Analytical wavelength: Maxima at about 227 nm
Titrant: 0.1 N sodium hydroxide VS Analysis: Determine the quantity of methenamine hip-
Endpoint detection: Visual purate (CsHi2N4 - CoHsNOs) dissolved of filtered por-
Analysis: Titrate with Titrant. Each mL of 0.1 N sodium tions of the solution under test, suitably diluted with
hydroxide is equivalent to 17.92 mg of hippuric acid water if necessary, in comparison with the Standard
(CsHsNOs). solution.
Acceptance criteria: 54.0%-58.0% Tolerances: NLT 80% (Q) of the labeled amount of
methenamine hippurate (CsHi2N4 - CsH9NOs) is
IMPURITIES dissolved.
e RESIDUE ON IGNITION (281): NMT 0.1%
Test 2
© SULFATE [NoTte—If the product complies with this test, the label-
Sample solution: 200 mg in 10 mL of water ing indicates that it meets USP Dissolution Test 2.]
Analysis: Add 5 a of 3 N hydrochloric acid and Medium: Water; 900 mL
5 drops of barium chloride TS. Apparatus 2: 50 rpm
‘e
”
Acceptance criteria: No turbidity appears within 1 min. Time: 60 min
iy Standard solution: 22 j1g/mL of USP Methenamine
i]
—
io.) Delete the following: Hippurate RS
° Instrumental conditions
iS Mode: UV
} °e HEAVY METALS, Method |i (231): NMT 15 ppme cortical 1-
Analytical wavelength: Maxima at about 227 nm
= Jan-2018)
Analysis: Determine the quantity of methenamine hip-
a SPECIFIC TESTS purate (CsHi2N4 - CoHeNO3) dissolved of filtered por-
”
=) e Loss ON DRYING (731) tions of the solution under test, suitably diluted with
Analysis: Dry under vacuum at 60° for 1 h. water if necessary, in comparison with the Standard
Acceptance criteria: NMT 1.0% solution.
Tolerances: NLT 80% (Q) of the labeled amount of
ADDITIONAL REQUIREMENTS methenamine hippurate (CsHi2N4 - CoH9NOs) is
e PACKAGING AND STORAGE: Preserve in well-closed dissolved.
containers. e UNIFORMITY OF DosAGE UNITS (905): Meet the
e USP REFERENCE STANDARDS (11) requirements
USP Methenamine Hippurate RS
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed
containers.
© LABELING: When more than one Dissolution test is given,
Methenamine Hippurate Tablets the labeling states the Dissolution test used only if Test 7
is not used.
DEFINITION e USP REFERENCE STANDARDS (11)
MethenamineHippurate Tablets contain NLT 95.0% and USP Methenamine Hippurate RS
NMT 105.0% of the labeled amount of methenamine hip-
purate (CeHi2Nq - CsH9NOs).
IDENTIFICATION
e A. INFRARED ABSORPTION (197M) Methenamine Mandelate
Sample: A portion of finely powdered Tablets
Acceptance criteria: Meet the requirements
ASSAY
¢ PROCEDURE
oO Oo
Sample solution: Transfer an equivalent to 700 mg of
methenamine hippurate from finely powdered Tablets CoHi2Na - CsHgOs 292.33
USP 41
Benzeneacetic acid, a-hydroxy-, (+)-, compd. with 1,3,5,7-
tetraazatricyclo[3.3.1.137]decane (1:1);
Hexamethylenetetramine mono-(+)-mandelate [587-23-5].
DEFINITION
Methenamine Mandelate contains NLT 95.5% and NMT
102.0% of methenamine mandelate (CsHi2N4 - CsHgOs),
and contains NLT 50.0% and NMT 53.0% of mandelic
acid (CgHgO3), calculated on the dried basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
ASSAY
© PROCEDURE
Sample solution: Transfer 60 mg of Methenamine
Mandelate to a 250-mL conical flask. Add 15 mL of de-
hydrated alcohol, stir to dissolve, and add 40 mL of
chloroform.
Titrimetric system
Mode: Direct titration
Titrant: 0.05 N silver nitrate in dehydrated alcohol
prepared as follows. Dissolve by stirring 8.5 g of silver
nitrate in 1000 mL of dehydrated alcohol. Transfer
100 mg of sodium chloride, previously dried at 110°
for 2 h, to a 100-mL beaker, and dissolve in 50 mL of
water. Titrate with the silver nitrate solution to the
potentiometric endpoint, usinga silver billet indicator
electrode anda silver-silver chloride double-junction
reference electrode containing a potassium nitrate salt
bridge. Calculate the normality of the titrant.
Endpoint detection: Potentiometric
Analysis: Titrate the Sample solution with Titrant, deter-
mining the endpoint potentiometrically, using a silver
billet indicator electrode andasilver-silver chloride
double-junction reference electrode containing a potas-
sium nitrate salt bridge. Each mL of 0.05 N silver nitrate
is equivalent to 7.308 mg of methenamine mandelate
(CeHi2Nq - CgHgOs).
Acceptance criteria: 95.5%-102.0% on the dried basis
OTHER COMPONENTS
© CONTENT OF IMANDELIC ACID
Sample solution: Transfer 90 mg to a 250-mL conical
flask containing 50 mL of water. When the solution is
complete, titrate the magnetically stirred solution.
Titrimetric system
Mode: Direct titration
Titrant: 0.05 N ceric ammonium nitrate VS
Endpoint detection: Potentiometric
Analysis: Titrate the Sample solution with Titrant, deter-
mining the endpoint potentiometrically. Each mL of
0.05 N ceric ammonium nitrate is equivalent to
3.804 mg of mandelic acid (CgHgO3).
Acceptance criteria: 50.0%-53.0% on the dried basis
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.1%
© CHLORIDE AND SULFATE, Chloride (221)
Sample: 1.0g
Analysis: Dissolve the Sample in 10 mL of water, and
add gradually 500 mg of anhydrous sodium carbonate.
Evaporate to dryness, and ignite the residue at a dull-
id heat. Add 20 mL of 2 N nitric acid, stir gently, and
ilter.
Acceptance criteria: 0.01%; the filtrate shows no more
chloride than corresponds to 0.15 mL of 0.020 N hy-
drochloric acid.
© SULFATE
Sample: 0.20g
Analysis: Dissolve the Sample in 10 mL of water. Add
5 drops of 3 N hydrochloric acid and 5 drops of barium
chloride TS.
Official Monographs / Methenamine 2639
Acceptance criteria: No turbidity appears within 1 min.
Delete the following:
°o HEAVY METALS (231)
Test Baier Dissolve 1.3 g in 10 mL of water, add
2 mL of 3.N hydrochloric acid, and dilute with water to
25 mL.
Acceptance criteria: NMT 15 ppme cotica 1-jan-2018)
SPECIFIC TESTS
e Loss ON DRYING (731)
Analysis: Dry over silica gel for 18 h.
Acceptance criteria: NMT 1.5%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed
containers.
e USP REFERENCE STANDARDS (11)
USP Methenamine Mandelate RS
Methenamine Mandelate for Oral
Solution
DEFINITION
Methenamine Mandelate for Oral Solution contains NLT
90.0% and NMT 110.0% of the labeled amount of me-
thenamine mandelate (CsHi2Nq - CgHgOs).
IDENTIFICATION
e A. INFRARED ABSORPTION (197)
Sample: A finely powdered portion, equivalent to
100 mg of methenamine mandelate
Acceptance criteria: The IR absorption spectrum of a
=
potassium bromide dispersion of the residue so ob- wn
tained exhibits maxima only at the same wavelenths as se)
that of a potassium bromide dispersion of USP Methe- =
namine Mandelate RS. i}
3
ASSAY re)
© PROCEDURE @=
Sample solution: Accurately weigh the contents of NLT i)
i}
10 containers of Methenamine Mandelate for Oral Solu- =,
tion, and reduce to a fine powder. Transfer an equiva- “
lent to 60 mg of methenamine mandelate from the
powder to a 150-mL beaker, Add 15 mL of dehydrated
alcohol, stir to dissolve, and add 40 mL of chloroform.
Titrimetric system
Mode: Direct titration
Titrant: 0.05 N silver nitrate in dehydrated alcohol
prepared as follows. Dissolve by stirring 8.5 g of silver
nitrate in 1000 mL of dehydrated alcohol. Transfer
100 mg of sodium chloride, previously dried at 110°
for 2 h, to a 100-mL beaker, and dissolve in 50 mL of
water. Titrate with the silver nitrate solution to the
potentiometric endpoint, usinga silver billet indicator
electrode andasilver-silver chloride double-junction
reference electrode containing a potassium nitrate salt
bridge. Calculate the normality of the titrant.
Endpoint detection: Potentiometric
Analysis: Titrate the Sample solution with Titrant, deter-
mining the endpoint potentiometrically, using a silver
billet indicator electrode anda silver-silver chloride
double-junction reference electrode containing a potas-
sium nitrate salt bridge. Each mL of 0.05Nsilver nitrate
is equivalent to 7.308 mg of methenamine mandelate
(CeHi2Nq + CgHgOs).
 2640 Methenamine / Official Monographs
Acceptance criteria: 90.0%-110.0%
PERFORMANCE TESTS
e UNIFORMITY OF DOSAGE UNITS (905): Meets the require-
ments for powder packaged in single-unit containers
e DELIVERABLE VOLUME (698%: Meets the requirements for
powder packaged in multiple-unit containers
SPECIFIC TESTS
e PH (791)
Sample solution: 1g in 30 mL of water
Acceptance criteria: 4.0-4.5
¢ WATER DETERMINATION, Method | (921): NMT 0.5%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed
containers.
e LABELING: Label Methenamine Mandelate for Oral Solu-
tion that contains insoluble ingredients to indicate that
the aqueous constituted Oral Solution contains dissolved
methenamine mandelate but may remain turbid because
of the presence of added substances.
e USP REFERENCE STANDARDS (11)
USP Methenamine Mandelate RS
Methenamine Mandelate Oral
Suspension
DEFINITION
Methenamine Mandelate Oral Suspension is Methenamine
Mandelate suspended in vegetable oil. It contains NLT
90.0% and NMT 110.0% of the labeled amount of me-
thenamine mandelate (CsHi2N4 - CgHgO3).
a) IDENTIFICATION
a= e A. INFRARED ABSORPTION (197)
a Sample solution: Triturate the equivalent to 100 mg of
i]
methenamine mandelate, with 10 mL of chloroform,
a)
4
° and pass through a 0.45-1m pore size membrane filter.
fad Analysis: Evaporate the solvent in the Sample solution,
iS wash the residue with five small portions of ether, and
= ASSAY
allow it to air-dry.
a Acceptance criteria: The IR absorption spectrum of a
2)
> potassium bromide dispersion of the residue so ob-
tained exhibits maxima only at the same wavelenths as
that of a potassium bromide dispersion of USP Methe-
namine Mandelate RS.
ASSAY
e PROCEDURE
Sample solution: Shake the Oral Suspension, then pi-
pet, using a “to contain” pipet, an amount equivalent
to 1g of methenamine mandelate, into a 100-mL volu-
metric flask. Add 5.0 mL of dehydrated alcohol, mix,
and add methylene chloride to volume. Pipet 5 mL of
this solution into a 250-mL conical flask. Add 15 mL of
dehydrated alcohol, and add 40 mL of chloroform.
Titrimetric system
Mode: Direct titration
Titrant: 0.05Nsilver nitrate in dehydrated alcohol
prepared as follows. Dissolve by stirring 8.5 g of silver
nitrate in 1000 mL of dehydrated alcohol. Transfer
100 mg of sodium chloride, previously dried at 110°
for 2 h, to a 100-mL beaker, and dissolve in 50 mL of
water. Titrate with the silver nitrate solution to the
potentiometric endpoint, using asilver billet indicator
electrode and a silver-silver chloride double-junction
reference electrode containing a potassium nitrate salt
bridge. Calculate the normality of the titrant.
USP 41
Endpoint detection: Potentiometric
Analysis: Titrate the Sample solution with Titrant, deter-
mining the endpoint potentiometrically, using a silver
billet indicator electrode and a silver-silver chloride
double-junction reference electrode containing a potas-
sium nitrate salt bridge. Each mL of 0.05Nsilver nitrate
is equivalent to 7.308 mg of methenamine mandelate
(CeHi2Nq - CgHgOs).
Acceptance criteria: 90.0%-110.0%
PERFORMANCE TESTS
© UNIFORMITY OF DOSAGE UNITS (905): Meets the require-
ments for Oral Suspension packaged in single-unit
containers
© DELIVERABLE VOLUME (698): Meets the requirements for
Oral Suspension packaged in multiple-unit containers
SPECIFIC TESTS
e WATER DETERMINATION, Method | (921): NMT 0.1%
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers.
e USP REFERENCE STANDARDS (11)
USP Methenamine Mandelate RS
Methenamine Mandelate Tablets
DEFINITION
Methenamine Mandelate Tablets contain NLT 95.0% and
NMT 105.0% of the labeled amount of methenamine
mandelate (C6Hi2N4 - CgHsgOs).
IDENTIFICATION
e A. INFRARED ABSORPTION (197)
Sample: Triturate a portion of finely powdered Tablets,
equivalent to 5.0 mg of methenamine mandelate, with
5 mL of chloroform, and pass through a 0.45-um mem-
brane filter. Evaporate the solvent, and allow the resi-
due to air-dry.
Acceptance criteria: Meet the requirements
e PROCEDURE
Sample solution: Transfer an equivalent to 60 mg of
methenamine mandelate, from NLT 20 finely powdered
Tablets, to a 250-mL conical flask. Add 15 mL of dehy-
drated alcohol, stir to dissolve, and add 40 mL of
chloroform.
Titrimetric system
Mode: Direct titration
Titrant: 0.05N silver nitrate in dehydrated alcohol
prepared as follows. Dissolve by stirring 8.5 g of silver
nitrate in 1000 mL of dehydrated alcohol. Transfer
100 mg of sodium chloride, previously dried at 110°
for 2 h, to a 100-mL beaker, and dissolve in 50 mL of
water. Titrate with the silver nitrate solution to the
potentiometric endpoint, usinga silver billet indicator
electrode anda silver-silver chloride double-junction
reference electrode containing a potassium nitrate salt
bridge. Calculate the normality of the titrant.
Endpoint detection: Potentiometric
Analysis: Titrate the Sample solution with Titrant, deter-
mining the endpoint potentiometrically, using a silver
billet indicator electrode anda silver-silver chloride
double-junction reference electrode containing a potas-
sium nitrate salt bridge. Each mL of 0.05N silver nitrate
is equivalent to 7.308 mg of methenamine mandelate
(CeHi2Na - CgHgQOs).
USP 41 Official Monographs / Methimazole 2641

Acceptance criteria: 95.0%-105.0% billet indicator electrode and a silver-silver chloride

double-junction reference electrode containing a potas-
PERFORMANCE TESTS sium nitrate salt bridge. Each mL of 0.05Nsilver nitrate
e DISSOLUTION (711) is equivalent to 7.308 mg of methenamine mandelate
For uncoated or plain coated Tablets (CeHi2N4 - CgHeO3).
Medium: Water; 900 mL Acceptance criteria: 95.0%-105.0%
Apparatus 1: 100 rpm
Time: 45 min PERFORMANCE TESTS
Standard solution: USP Methenamine Mandelate RS © DISINTEGRATION (701): 2.5 h, determined as directed in
in Medium Delayed-Release (Enteric-Coated) Tablets
Instrumental conditions e UNIFORMITY OF DOSAGE UNITS (905): Meet the
Mode: UV requirements
Analytical wavelength: UV maximum at about 257
nm ADDITIONAL REQUIREMENTS
Analysis: Determine the amount of methenamine e PACKAGING AND STORAGE: Preserve in well-closed
mandelate (CsHi2N4 - CsHgO3) dissolved in the portion containers.
of the solution under test, passed throughafilter of e USP REFERENCE STANDARDS (11)
0.45-1um pore size and suitably diluted with Medium, if USP Methenamine Mandelate RS
necessary, in comparison with a Standard solution hav-
ing a known concentration of USP Methenamine
Mandelate RS in the same Medium.
Tolerances: NLT 75% (Q) of the labeled amount of me-
thenamine mandelate (CsHi2N4 - CsHgOs3) is dissolved. Methimazole
e UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
ADDITIONAL REQUIREMENTS CF8
e PACKAGING AND STORAGE: Preserve in well-closed CNH
containers.
© USP REFERENCE STANDARDS (11) CaHoN2S 114.17
USP Methenamine Mandelate RS 2H-Imidazole-2-thione, 1,3-dihydro-1-methyl-;
1-Methylimidazole-2-thiol [60-56-0].
DEFINITION
Methimazole contains NLT 98.0% and NMT 101.0% of
Methenamine Mandelate Delayed- methimazole (C4HgN2S), calculated on the dried basis.
Release Tablets IDENTIFICATION
e A. INFRARED ABSORPTION (197K) Cc
“nn
DEFINITION a]
Methenamine Mandelate Delayed-Release Tablets contain ASSAY
NLT 95.0% and NMT 105.0% of the labeled amount of © PROCEDURE Ec
methenamine mandelate (CsHi2Nq - CsHsO3). Sample: 250mg of Methimazole i}
]
Analysis: Dissolve the Sample in 75 mL of water. Add i}
IDENTIFICATION 15.0 mL of 0.1 N sodium hydroxide VS, mix, and add, i=
e A. INFRARED ABSORPTION (197K) with stirring, 30 mL of 0.1 Nsilver nitrate. Continue the ey
Sample: Triturate an equivalent to 5.0 mg of methena- titration with the 0.1 N sodium hydroxide VS, deter- be}
mining the end-point potentiometrically. Each mL of zs
mine mandelate from finely powdered Tablets with ww
5 mL of chloroform, and pass throughafilter of 0.45- 0.1 N sodium hydroxide is equivalent to 11.42 mg of
um pore size. Evaporate the solvent, and allow the resi- methimazole (C4H6N2S).
due to air-dry. Acceptance criteria: 98.0%-101.0% on the dried basis
Acceptance criteria: Meet the requirements
IMPURITIES
ASSAY © RESIDUE ON IGNITION (281): NMT 0.1%
© PROCEDURE e SELENIUM (291)
Sample solution: Transfer an Se to 60 mg of Sample: 200 mg of Methimazole
methenamine mandelate, from finely powdered Tablets Acceptance criteria: NMT 30 ppm
(NLT 20), to a 250-mL conical flask. Add 15 mL of de- © ORGANIC IMPURITIES
hydrated alcohol, stir to dissolve, and add 40 mL of Standard solution: 0.01 mg/mL each of USP
chloroform. Methimazole RS, USP Methimazole Related Compound
Titrimetric system A RS, 1-methylimidazole, and USP Methimazole Related
Mode: Direct titration CompoundC RS in chloroform
Titrant: 0.05 N silver nitrate in dehydrated alcohol Sample solution: 10 mg/mL of Methimazole in
prepared as follows. Dissolve by stirring 8.5 g of silver chloroform
nitrate in 1000 mL of dehydrated alcohol. Transfer Chromatographic system
100 mg of sodium chloride, previously dried at 110° (See Chromatography (621), System Suitability.)
for 2 h, to a 100-mL beaker, and dissolve in 50 mL of Mode: GC
water. Titrate with the silver nitrate solution to the Detector: Flame ionization
potentiometric endpoint, using a silver billet indicator Column: 30-m x 0.25-mm; 0.5-um coating of G27
electrode anda silver-silver chloride double-junction Temperatures
reference electrode containing a potassium nitrate salt Injection port: 150°
bridge. Calculate the normality of the titrant. Detector: 250°
Endpoint detection: Potentiometric Column: See Table 7.
Analysis: Titrate the Sample solution with Titrant, deter-
mining the endpoint potentiometrically, using a silver
 2642 Methimazole / Official Monographs USP 41

Table 1 e USP REFERENCE STANDARDS (11)

Hold Time at USP Methimazole RS
USP Methimazole Related Compound A RS
Initial Temperature Final Final
Temperature Ramp Temperature | Temperature
2,2-Dimethoxy-N-methylethanamine.
CsHi3NO2 119.16
Cy (¢/min) ©) (min) USP Methimazole Related Compound C RS
100 = 100 2 1-Methyl-2-(methylthio)-1 H-imidazole.
100 30 250 15 CsHsN2S 128.20
Carrier gas: Helium
Flow rate: 1.5 mL/min
Injection volume: 1 wL
Injection type: Split ratio, 3:20
System suitability Methimazole Tablets
Sample: Standard solution
Suitability requirements DEFINITION
Resolution: NLT 1.5 between methimazole related Methimazole Tablets contain NLT 94.0% and NMT 106.0%
compound A and 1-methylimidazole of the labeled amount of methimazole (C4HeN2S).
Analysis IDENTIFICATION
Samples: Standard solution and Sample solution e A. INFRARED ABSORPTION (197K)
Calculate the percentage of methimazole related com- Sample: Digest a quantity of powdered Tablets, equiva-
pound A, 1-methylimidazole, and methimazole related lent to 10 mg of methimazole, with 10 mL of warm
compoundCin the portion of Methimazole taken: chloroform for 20 min, filter, and evaporate the filtrate
on a steam bath to dryness.
Result = (ru/rs) x (Cs/Cu) x 100 Acceptance criteria: Meet the requirements
ru = peak response of each related compound from ASSAY
the Sample solution e PROCEDURE
ts peak response of the corresponding related Sample solution: Finely powder NLT 20 Tablets. Trans-
compound from the Standard solution fer a portion of the powder, equivalent to 120 mg of
Gs concentration of the corresponding related methimazole, to a 100-mL volumetric flask. Add about
compound in the Standard solution (mg/mL) 80 mL of water, insert the stopper, and shake by me-
cy = concentration of Methimazole in the Sample chanical means or occasionally by hand for 30 min. Di-
solution (mg/mL) lute with water to volume, and filter.
Calculate the percentage of any other individual Analysis: Add 3.5 mL of 0.1 N sodium hydroxide VS to
impurity in the portion of Methimazole taken: 50.0 mL of Sample solution, mix, and add, with stirring,
Result = (ru/rs) x (Cs/Cu) x 100 7 mL of 0.1 N silver nitrate. Continue the titration wit
the 0.1 N sodium hydroxide VS, determining the end-
=s
”

[= ry = peak response of each impurity from the oily pebniann Healy, Each mL of 0.1 N sodium hy-
s Sample solution droxide is equivalent to 11.42 mg of methimazole
—
Dp Is = peak response of methimazole from the (C4HeN2S).
° Standard solution Acceptance criteria: 94.0%-106.0%
S
S Cs = concentration of USP Methimazole RS in the PERFORMANCE TESTS
= Standard solution (mg/mL) e DISSOLUTION (711)
Gy = concentration of Methimazole in the Sample Medium: Water; 500 mL
a
a) solution (mg/mL) Apparatus 1: 100 rpm
=) Acceptance criteria: See Table 2. Disregard any peak Time: 30 min
below 0.02%. Standard solution: USP Methimazole RS at a known
concentration in Medium
Table 2 Sample solutions: Filtered solution under test, suitably
Accep- diluted with Medium
Relative tance Instrumental conditions
Retention Criteria, Mode: UV
Name Time NMT (%) Analytical wavelength: Maximum absorbance at
about 252 nm
Methimazole related compound A 0.3 0.1 Tolerances: NLT 80% (Q) of the labeled amount of
1-Methylimidazole 0.4 0.1 methimazole (C4HeN2S) is dissolved.
Methimazole related compound C 0.7 0.1 e UNIFORMITY OF DOSAGE UNITS (905): Meet the
Methimazole 1.0 —_ requirements
Any other individual impurity — 0.1 Procedure for content uniformity
Total impurities eo 0.5 Standard solution: 5 g/mL of USP Methimazole RS in
water
Sample stock solution: Place 1 Tablet, previously
SPECIFIC TESTS crushed or finely powdered, in a 100-mL volumetric
e Loss ON DRYING (731) flask. Add 50 mL of water, and shake by mechanical
Analysis: Dry at 105° for 2 h. means for 30 min. Dilute with water to volume, mix,
Acceptance criteria: NMT 0.5% and filter, discarding the first 20 mL of filtrate.
Sample solution: Nominally 5 ug/mL of methimazole
ADDITIONAL REQUIREMENTS in water from Sample stock solution
e PACKAGING AND STORAGE: Preserve in well-closed, light- Instrumental conditions
resistant containers. Mode: UV
Analytical wavelength: Maximum absorbance at
about 252 nm
USP 41 Official Monographs / Methionine 2643

Cell: 1m Sample: 0.73 g of Methionine

Blank: Water Acceptance criteria: NMT 0.05%
Analysis e CHLORIDE AND SULFATE (221), Sulfate
Samples: Standard solution, Sample solution, and Standard solution: 0.10 mL of 0.020 N sulfuric acid
Blank Sample: 0.33 g of Methionine
Calculate the percentage of the labeled amount of Acceptance criteria: NMT 0.03%
methimazole (C4HeN2S) in the Tablet taken: e IRON (241): NMT 30 ppm
Result = (Au/As) x (Cs/Cu) x 100
Delete the following:
Au = absorbance of the Sample solution
As = absorbance of the Standard solution °e HEAVY METALS (231), Method Ik NMT 15 ppme cortical 1.
Cs = concentration of USP Methimazole RS in the Jai-2018)
Standard solution (g/mL)
Cy = nominal concentration of methimazole in the Change to read:
Sample solution (g/mL)
ADDITIONAL REQUIREMENTS e RELATED COMPOUNDS
¢ PACKAGING AND STORAGE: Preserve in well-closed, light- Phosphoric acid solution: 23gil of phosphoric acid
resistant containers. Solution A: Acetonitrile, Phosphoric acid solution, and
e USP REFERENCE STANDARDS (11) water (0.5: 12.5: 87)
USP Methimazole RS Solution B: Acetonitrile, Phosphoric acid solution, and
water (47.5: 12.5: 40)
Mobile phase: Gradient elution. See Table 7.

Table 1
Methionine Solution A Solution B
° 100
BC" [ OH 100
Ne

60
CsHiiNO2S 149.21 70 00
t-Methionine [63-68-3].
System suitability solution: Transfer 5 mg each of USP
DEFINITION L-Methionine RS and L-methionine sulfoxide to a 50-mL
Methionine contains NLT 98.5% and NMT 101.5% of L-me- volumetric flask, and dissolve in and dilute with Solution c
thionine (CsHi;NO2S), calculated on the dried basis. A to volume. 4)
Standard solution: 30 g/mL of USP L-Methionine RS v
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
in Solution A =
N-Acetyl-D,L-methionine standard solution: 0.06 mg/ 3
mL of USP N-Acetyl-D,L-methionine RS in Solution A =]
ASSAY )
© PROCEDURE Sample solution: 30 mg/mL of Methionine in Solution re)=
Sample: 140 mg of Methionine A 2
Blank: Mix 3 mL of formic acid and 50 mL of glacial Chromatographic system i)
acetic acid. (See Chromatography (621), System Suitability.) Sy
a
Titrimetric system Mode: LC
(See Titrimetry (541).) Detector: UV 205 nm
Mode: Direct titration Column: 4.6-mm x 25-cm; 5-4um packing L1
Titrant: 0.1 N perchloric acid VS Column temperature: 30°
Endpoint detection: Potentiometric Flow rate: 1.0 mL/min
Analysis: Dissolve the Sample in 3 mL of formic acid Injection volume: 50 LL
and 50 mL of glacial acetic acid, and titrate with the System suitability
Titrant. Sample: System suitability solution
Calculate the percentage of L-methionine (CsH;;NO2S) [Note—The relative retention times for L-methionine
in the portion of the Sample taken: and L-methionine sulfoxide are 1.0 and 0.5,
respectively.]
Result = {[(Vs — Va) x N
x FJ/W} x 100 Suitability requirements
Resolution: NLT 5.0 between L-methionine and L-me-
Vs = Titrant volume consumed by the Sample (mL) thionine sulfoxide peaks
Ve = Titrant volume consumed by the Blank (mL) Analysis
N = actual normality of the Titrant (mEq/mL) Samples: Standard solution, N-Acetyl-D,L-methionine
F = equivalency factor, 149.2 mg/mEq standard solution, and Sample solution
w = Sample weight (mg) Calculate the percentage of N-acetyl-D,L-methionine in
Acceptance criteria: 98.5%-101.5% on the dried basis the portion of Methionine taken:
IMPURITIES Result = (ru/rs) x (Cs/Cy) x 100
e RESIDUE ON IGNITION (281): NMT 0.4%
© CHLORIDE AND SULFATE (221), Chloride ry = peak response of N-acetyl-D,L-methionine from
aema solution: 0.50 mL of 0.020 N hydrochloric the Sample solution
aci rs = peak response of N-acetyl-D,l-methionine from
the N-Acetyl-D,l-methionine standard solution
 2644 Methionine / Official Monographs USP 41

Cs = concentration of USP N-Acetyl-D,L-methionine DEFINITION

RS in the N-Acetyl-D,L-methionine standard
solution (mg/mL,
Cu — = concentration of Methionine in the Sample
solution (mg/mL)
Separately calculate the percentage of methionine
sulfoxide and any unspecified impurity in the portion
of Methionine taken:
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
Methocarbamol contains NLT 98.5% and NMT 101.5% of
methocarbamol (Ci;HisNOs), calculated on the dried
basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.

ty = peak response of methionine sulfoxide or any ASSAY

unspecified impurity from the Sarmple solution © PROCEDURE
rs = peak response of methionine from the Buffer: 6.8 g/L of monobasic potassium phosphate in
Standard solution water. Adjust with phosphoric acid or sodium hydroxide
Cs = concentration of USP L-Methionine RS in the to a pH of 4.5.
Standard solution (mg/mL) Mobile phase: Methanol and Buffer (30:70)
Cu = concentration of Methionine in the Sample System suitability solution: 1.0 mg/mL of USP Metho-
solution (mg/mL) carbamol RS and 0.005 mg/mL of USP Guaifenesin RS
F = relative response factor (see Table 2) in Mobile phase
Acceptance criteria: See Table 2. Disregard any impuri- Standard solution: 0.1 mg/mL of USP Methocarbamol
ties less than 0.05%. RS in Mobile phase
Sample solution: 0.1 mg/mL of Methocarbamol in Mo-
bile phase
Table 2
Chromatographic system
Relative Relative Acceptance (See Chromatography (621), System Suitability.)
Retention Response Criteria, Mode: LC
Name Time Factor NMT (%) Detector: UV 274 nm
Methionine sulfoxide 0.5 0.53 0.1 Column: 4.6-mm x 15-cm; 3-um packing L1
Methionine 1.00 = = Column temperature: 30°
Flow rate: 0.8 mL/min
N-Acetyl-D,L- _
Injection volume: 20 pL
methionine 3.4 0.2 Run time: 1.5 times the retention time of
Any unspecified "el methocarbamol
impurity — 2017) 0.1 System suitability
Total unspecified a _ Samples: System suitability solution and Standard
impurities 0.30 solution
[Note—See Table 7 for relative retention times.]
=
”
Suitability requirements
os SPECIFIC TESTS Resolution: NLT 3.5 between methocarbamol and
ic} e OPTICAL ROTATION (7815S), Procedures, Specific Rotation
— guaifenesin, System suitability solution
Dp Sample solution: 20 mg/mL in 6 N hydrochloric acid Tailing factor: NMT 2.0, Standard solution
° Acceptance criteria: +22.4° to +24.7°
= © PH (791)
Relative standard deviation: NMT 0.73%, Standard
5 Sample solution: 10 mg/mL of solution
solution
Ps Acceptance criteria: 5.6-6.1
Analysis
a Samples: Standard solution and Sample solution
va) e Loss ON DRYING (731) Calculate the percentage of methocarbamol
f=) Analysis: Dry at 105° for 3 h. (CyiHisNOs) In the portion of Methocarbamol taken:
Acceptance criteria: NMT 0.3%
ADDITIONAL REQUIREMENTS Result = (ru/rs) x (Cs/Cu) x 100
e PACKAGING AND STORAGE: Preserve in well-closed ru = peak response of methocarbamol from the
containers. Sample solution
e USP REFERENCE STANDARDS (11) fs = peak response of methocarbamol from the
USP N-Acetyl-D,L-methionine RS Standard solution
USP L-Methionine RS Cs = concentration of USP Methocarbamol RS in
the Standard solution (mg/mL)
Cu = concentration of Methocarbamol in the
Sample solution (mg/mL)
Acceptance criteria: 98.5%—101.5% on the dried basis
Methocarbamol
IMPURITIES
OH e RESIDUE ON IGNITION (281): NMT 0.1%
apa
l “OCH: J Delete the following:

°o HEAVY METALS (231), Method |

CirHisNOs 241.24 Sample solution: 1.0g in a 10-mL mixture of methanol
1,2-Propanediol, 3-(2-methoxyphenoxy)-, 1-carbamate, (+)-; and 1 N acetic acid (7:3), diluted with water to 25 mL
(4)-3-(o-Methoxyphenoxy)-1,2-propanediol 1-carbamate Acceptance criterias NMT 20 ppme (otical 1-jen-2013)
[532-03-6]. e ORGANIC IMPURITIES
Mobile phase, System suitability solution, and Chro-
matographic system: Proceed as directed in the
Assay.
USP 41 Official Monographs / Methocarbamol 2645

Standard solution: 0.005 mg/mL of USP Metho- IDENTIFICATION

carbamol RS in Mobile phase e A. INFRARED ABSORPTION (197K)
pis solution: 1mg/mL of Methocarbamol in Mobile Sample: Mix a volume with 40 mL of water equivalent
phase to 500 mg of methocarbamol from Injection in a small
System suitability separator. Extract with 10 mL of ethyl acetate, and dry
Samples: System suitability solution and Standard the ethyl acetate layer over anhydrous sodium sulfate.
solution Evaporate the ethyl acetate with the use of a water
[Note—See Table 1 for the relative retention times.] bath maintained at 40° under a stream of nitrogen to
Suitability requirements dryness.
Resolution: NLT 3.5 between methocarbamol and e B. The retention time of the major peak of the Sample
guaifenesin, System suitability solution solution corresponds to that of the Standard solution, as
Tailing factor: NMT 2.0, Standard solution obtained in the Assay.
Relative standard deviation: NMT 5.0%, Standard
solution ASSAY
Analysis © PROCEDURE
Samples: Standard solution and Sample solution Buffer: 6.8 g/L of monobasic potassium phosphate in
Calculate the percentage of each impurity in the por- water. Adjust with phosphoric acid or potassium hy-
tion of Methocarbamol taken: droxide to a pH of 4.5.
Mobile phase: Methanol and Buffer (30:70)
Result = (ru/rs) x (Cs/Cu) x (1/FA) x 100 Standard solution: 1 mg/mL of USP Methocarbamol RS
in Mobile phase
tu = peak response of each impurity from the Sample solution: Nominally 1 mg/mL of metho-
Sample solution carbamol from a suitable volume of Injection containing
fs = peak response of methocarbamol from the NLT 100 mg of methocarbamol in Mobile phase
Standard solution Chromatographic system
G = concentration of USP Methocarbamol RS in (See Chromatography (621), System Suitability.)
the Standard solution (mg/mL) Mode: LC
Cy = concentration of Methocarbamol in the Detector: UV 274 nm
Sample solution (mg/mL) Column: 4.6-mm x 10.0-cm; 3-uum or 5-~4m packing
F = relative response factor (see Table 1) u1
Acceptance criteria: See Table 7. Column temperature: 30°
Flow rate: 1 mL/min
Table 1 Injection volume: 20 pL
System suitability
Relative Relative Acceptance Sample: Standard solution
Retention Response Criteria, Suitability requirements
Name Time Factor NMT (%) Relative standard deviation: NMT 2.0%
Guaifenesin 0.84 AD 0.15 Analysis (el
Methocarbamol Samples: Standard solution and Sample solution “
isomer@ 0.90 1.0 0.05 Calculate the percentage of the labeled amount of ny
Methocarbamol 1.0 =— — methocarbamol (Ci;HisNOs) in the portion of Injection [E¢
Methocarbamol
taken: =
dioxolone> is 1.0 0.05 Result = (ru/rs) x (Cs/Cu) x 100 a3J
Any individual
unspecified — _ tu = peak response from the Sample solution a
impurity 0.05 Is = peak response from the Standard solution md
Total impurities = — 1.0 Cs = concentration of USP Methocarbamol RS in "6
@1-Hydroxy-3-(2-methoxyphenoxy)propan-2-yl carbamate. the Standard solution (mg/mL)
»4-[(2-Methoxyphenoxy)methyl]-1,3-dioxolan-2-one. Cu = nominal concentration of methocarbamol in
the Sample solution reg
SPECIFIC TESTS Acceptance criteria: 95.0%-105.0%
e Loss ON DRYING (731)
Analysis: Dry at 60° for 2 h. IMPURITIES
Acceptance criteria: NMT 0.5% © LIMIT OF ALDEHYDES
Diluent: Alcohol and water (20:80)
ADDITIONAL REQUIREMENTS Solution A: 10 mg/mL of phenylhydrazine hydrochlo-
© PACKAGING AND STORAGE: Preserve in tight containers. ride in Diluent
Store at controlled room temperature. Solution B: 10 mg/mL of potassium ferricyanide in
¢ USP REFERENCE STANDARDS (11 water
USP Guaifenesin RS Solution C: 10 g/mL of formaldehyde in water pre-
USP Methocarbamol RS pared as follows. Dissolve 1.37 g of formaldehyde solu-
tion in 1 L of water. Dilute 10 mL of the resulting solu-
tion with water to 500 mL.
Standard solution: Transfer 4 mL of Solution C to a
25-mL volumetric flask. Add 2.0 mL of filtered Solution
Methocarbamol Injection A. Allow to stand for 10 min. Add 1 mL of Solution B,
and allow to stand for 5 min. Add 4 mL of hydrochloric
DEFINITION acid, and dilute with alcohol to volume.
Methocarbamol Injection is a sterile solution of Metho- Sample solution: Empty the contents of NLT 10 vials of
carbamol in an aqueous solution of Polyethylene Glycol Injection to a suitable container. Transfer 4.0 mL of the
300. It contains NLT 95.0% and NMT 105.0% of the la- composite sample of Injection to a 25-mL volumetric
beled amount of methocarbamol (CiiHisNOs). flask. Add 2.0 mL of filtered Solution A, and allow to
stand for 10 min. Add 1 mL of Solution B, and allow to
 2646 Methocarbamol / Official Monographs
stand for 5 min. Add 4 mL of hydrochloric acid, and
dilute with alcohol to volume.
Blank: Transfer 4 mL of water to a 25-mL volumetric
flask. Add 2.0 mL of filtered Solution A, and allow to
stand for 10 min. Add 1 mL of Solution B, and allow to
stand for 5 min. Add 4 mL of hydrochloric acid, and
dilute with alcohol to volume.
Instrumental conditions
Mode: Vis
Analytical wavelength: 515 nm
Cell: 1.cm
Analysis
Samples: Standard solution, Sample solution, and Blank
Determine the absorbances of the Samples.
Acceptance criteria: The absorbance of the Sample so-
lution is NMT the absorbance of the Standard solu-
tion(NMT 10 ug of formaldehyde in each mL of
Injection).
SPECIFIC TESTS
© PH (791): 3.5-6.0
e BACTERIAL ENDOTOXINS TEST (85): NMT 0.2 USP Endo-
toxin Units/mg of methocarbamol
e PARTICULATE MATTER IN INJECTIONS (788): Meets the re-
quirements for small-volume injections
© OTHER REQUIREMENTS: Meets the requirements in Injec-
tions and Implanted Drug Products (1)
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in single-dose contain-
ers. Store at controlled room temperature.
Change to read:
e USP REFERENCE STANDARDS (11)
*. (CN 1-May-2078)
USP Methocarbamol RS
iS
aa}
oh
i]
2)
—
° Methocarbamol Tablets
3 Acceptance criteria: 95.0%-105.0%
S
P= DEFINITION
o Methocarbamol Tablets contain NLT 95.0% and NMT
Ww 105.0% of the labeled amount of methocarbamol
p>} (CiiHisNOs).
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
Sample: Mix a portion of finely powdered Tablets
equivalent to 1 g of methocarbamol with 25 mL of
water in a separator, and extract with 25 mL of chloro-
form. Filter the extract, and evaporate to dryness.
Acceptance criteria: Meet the requirements
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
ASSAY
© PROCEDURE
Buffer: 6.8 g/L of monobasic potassium phosphate in
water. Adjust with phosphoric acid or sodium hydroxide
to a pH of 4.5.
Mobile phase: Methanol and Buffer (30:70)
System suitability solution: 1.0 mg/mL of USP Metho-
carbamol RS and 0.005 mg/mL of USP Guaifenesin RS
in Mobile phase
Standard solution: 0.1 mg/mL of USP Methocarbamol
RS in Mobile phase
Sample stock solution: Nominally 1 mg/mL of metho-
carbamol solution prepared as follows. Transfer a por-
tion of finely powdered Tablets (NLT 10) to a volumet-
ric flask of suitable size. Add 60% of the volume of the
USP 41
flask with Mobile phase. Sonicate for 30 min with inter-
mittent shaking. Dilute with Mobile phase to volume.
Pass a portion of the solution throughasuitable filter of
0.45-um pore size.
Sample solution: Nominally 0.1 mg/mL of metho-
carbamol from the Sample stock solution in Mobile phase
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 274 nm
Column: 4.6-mm x 15-cm; 3-um packing L1
Column temperature: 30°
Flow rate: 0.8 mL/min
Injection volume: 20 uL
Run time: 1.5 times the retention time of
methocarbamol
System suitability
Samples: System suitability solution and Standard
solution
{[Note—See Table 7 for relative retention times.]
Suitability requirements
Resolution: NLT 3.5 between methocarbamol and
guaifenesin, System suitability solution
Tailing factor: NMT 2.0, Standard solution
Relative standard deviation: NMT 2.0%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
methocarbamol (CiHisNOs) in the portion of Tablets
taken:
Result = (ru/rs) x (Cs/Cu) x 100
fu = peak response of methocarbamol from the
Sample solution
rs = peak response of methocarbamol from the
Standard solution
Cs = concentration of USP Methocarbamol RS in
the Standard solution (mg/mL)
Cu = nominal concentration of methocarbamol in
the Sample solution (mg/mL)
PERFORMANCE TESTS
e DISSOLUTION (711)
Medium: Water; 900 mL
Apparatus 2: 50 rpm
Time: 45 min
Mobile phase, Chromatographic system, and System
suitability: Proceed as directed in the Assay.
Standard solution: USP Methocarbamol RS in Medium
Sample solution: Filtered portion of the solution under
test, diluted with Medium if necessary
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
methocarbamol (CiiHisNOs) dissolved:
Result = (ru/rs) x Cs x Vx (1/L) x 100
tu = peak response of methocarbamol from the
Sample solution
rs = peak response of methocarbamol from the
Standard solution
Cs = concentration of USP Methocarbamol RS in
the Standard solution (mg/mL)
Vv = volume of Medium, 900 mL
L = label claim for methocarbamol (mg/Tablet)
Tolerances: NLT 75% (Q) of the labeled amount of
methocarbamol (Cy;HisNOs) is dissolved.
e UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
USP 41 Official Monographs / Methohexital 2647

IMPURITIES e USP REFERENCE STANDARDS (11)

© ORGANIC IMPURITIES USP Guaifenesin RS
Mobile phase, System suitability solution, and Chro- USP Methocarbamol RS
matographic system: Proceed as directed in the
Assay.
Standard solution: 0.005 mg/mL of USP Metho-
carbamol RS in Mobile phase
Sample solution: Use the Sample stock solution from
the Assay. Methohexital
System suitability
Samples: System suitability solution and Standard
solution
[Note—See Table 1 for relative retention times.]
Suitability requirements Het °
Resolution: NLT 3.5 between methocarbamol and
guaifenesin, System suitability solution CisHisN203 262.30
Tailing factor: NMT 2.0, Standard solution 2,4,6(1H,3H,5H)-Pyrimidinetrione, 1-methyl-5-(1-methyl-
Relative standard deviation: NMT 5.0%, Standard 2-pentynyl)-5-(2-propenyl)-, (+)-.
solution (4)-5-Allyl-T-methy|-5-(1-methyl-2-pentynyl)barbituric acid
Analysis [151-83-7].
Samples: Standard solution and Sample solution
Calculate the percentage of each degradation product » Methohexital contains not less than 98.0 per-
in the portion of Tablets taken: cent and not more than 101.0 percent of
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100 Ci4HigN2O3, calculated on the anhydrous basis.
ru = peak response of each degradation product Packaging and storage—Preserve in well-closed contain-
from the Sample solution ers.
Is = peak response of methocarbamol from the USP Reference standards (11)—
Standard solution USP Methohexital RS
Cs = concentration of USP Methocarbamol RS in Identification, Infrared Absorption (197S)—
the Standard solution (mg/mL) Solution: 1 in 100.
Cu = nominal concentration of methocarbamol in
the Sample solution (mg/mL) Medium: — chloroform.
F = relative response factor (see Table 1) Melting range (741): between 92° and 96°, but the
Acceptance criteria: See Table 1. range between beginning and end of melting does not ex-
ceed 3°.
Table 1 Water Determination, Method | (921): not more than (ox
2.0%. “
Relative Relative Acceptance a]
Chloride (221)—Dissolve 200 mg in a mixture of 75 mL of
Name
Retention
Time
Response
Factor
Criteria,
NMT (%)
ether and 25 mL of water, agitate, and allow to separate: K
the water solution shows no more chloride than corre- °
Guaifenesin 0.84 1.2 0.15 |
sponds to 0.17 mL of 0.010 N hydrochloric acid (0.03%). fo]
Methocarbamol vo]
isomer@ 0.90 1.0 0.05 a}
i)
Methocarbamol 1.0 = eae Delete the following: me}
a
Methocarbamol vy
dioxolone® 13. 1.0 0.05 °Heavy metals, Method I! (231): 0.001%.¢ (ificia! 14an-2018)
Any individual Ordinary impurities (466)—
unspecified em ga Test solution: methanol.
degradation Standard solution: methanol.
product. 0.10
Eluant: a mixture of chloroform and acetone (7:3).
Total impurities _ = 1.0
Visualization—Expose the plate to chlorine gas for 1 min-
2 1-Hydroxy-3-(2-methoxyphenoxy)propan-2-yl carbamate.
ute, and air-dry the plate at room temperature for 2 min-
» 4-[(2-Methoxyphenoxy)methyl]-1,3-dioxolan-2-one.
utes. ate a solution of 0.5 g of potassium iodide in
ADDITIONAL REQUIREMENTS 50 mL of water, and prepare a solution of 1.5 g of soluble
© PACKAGING AND STORAGE: Preserve in tight containers. starch in 50 mL of hot water. Mix 10 mL of each solution
Store at controlled room temperature. with 4 mL of alcohol to obtain the Detection reagent.
[NoTE—The Detection reagent so obtained may be used for
up to 3 or 4 days.] Spray the plate with the Detection rea-
gent.
Ae ce about 100 mg of Methohexital, accurately
weighed, in chloroform, and dilute quantitatively and step-
wise with chloroform to obtain a solution having a concen-
tration of about 10 mg per mL. Dissolve an accurately
weighed quantity of USP Methohexital RS in chloroform,
and dilute quantitatively and stepwise with chloroform to
obtain a Standard solution having a known concentration of
about 10 mg per mL. Concomitantly determine the ab-
sorbances of both solutions in 0.1-mm cells at the wave-
length of maximum absorbance at about 5.93 4m, with a
suitable spectrophotometer, using chloroform as the blank.
 2648 Methohexital / Official Monographs
Calculate the quantity, in mg, of Ci4HisN2O3 in the portion
of Methohexital taken by the formula:
10C(Au
/ As)
in which C is the concentration, in mg per mL, of USP
Methohexital RS in the Standard solution; and Au and As are
the absorbances of the solution of Methohexital and the
Standard solution, respectively.
Methohexital Sodium for Injection
Cy4HizN2NaO3 284.29
2,4,6(1H,3H,5H)-Pyrimidinetrione, 1-methyl-5-(1-methyl-
2-pentynyl)-5-(2-propenyl)-, (£)-, monosodium salt.
Sodium 5-allyl-1-methyl-5-(1 -methyl-2-pentynyl)bar-
biturate [309-36-4; 22151-68-4].
» Methohexital Sodium for Injection is a freeze-
dried, sterile mixture of methohexital sodium and
anhydrous Sodium Carbonate as a buffer, pre-
pared from an aqueous solution of Methohexital,
Sodium Hydroxide, and Sodium Carbonate. It
contains not less than 90.0 percent and not more
than 110.0 percent of the labeled amount of
methohexital sodium (Ci4Hi7N2NaQOs).
Packaging and storage—Preserve as described in Packag-
ing and Storage Requirements (659), Injection Packaging,
Store at controlled room temperature. Injection may be
packaged in 50-mL multiple-dose containers.
Change to read:
re
”“
oy
J usP Reference standards (11)—
—
Dp @ (CN 1-May-2018)
° USP Methohexital RS
re
Sj Completeness of solution—Mix 1 g with 20 mL of car-
= bon dioxide-free water: after 1 minute, the solution is clear
and free from undissolved solid.
~ Internal standard solution—Dissolve aprobarbital in chloro-
“ Constituted solution—At the time of use, it meets the
2) requirements for Injections and Implanted Drug Products
(Parenterals)—Product Quality Tests (1), Specific Tests, Com-
pleteness and clarity of solutions.
Identification—
A: Dissolve about 500 mg in 10 mL of water in a
separator, add 10 mL of 3 N hydrochloric acid, and extract
the liberated methohexital with two 25-mL portions of chlo-
roform. Evaporate the combined chloroform extracts to dry-
ness, add 10 mL of ether, evaporate again, and dry the resi-
due in vacuum at 80° for 4 hours. Dissolve 50 mg of the
residue so obtained in 5 mL of chloroform: the solution ex-
hibits IR absorption maxima at the same wavelengths as
that of a similar preparation of USP Methohexital RS.
B: The methohexital obtained and dried as directed for
Identification test A melts between 92° and 96°.
Bacterial Endotoxins Test (85)—It contains not more
than 0.5 USP Endotoxin Unit per mg of methohexital so-
dium.
Uniformity of dosage units (905): meets the require-
ments.
Procedure for content uniformity—
Standard solution—Transfer about 23 mg of USP Metho-
hexital RS, accurately weighed, to a 250-mL volumetric
flask, add 50 mL of water, 0.5 mL of sodium hydroxide solu-
tion (1 in 10), and 1.5 mL of sodium carbonate solution (1
in 1000), and mix. Dilute with water to volume, and mix.
USP 41
Transfer 20.0 mL of this solution to a 100-mL volumetric
flask, dilute with water to volume, and mix.
Test solution—Transfer the contents of 1 vial of Metho-
hexital Sodium for Injection with the aid of water to a
1000-mL volumetric flask, dilute with water to volume, and
mix. Transfer an accurately measured volume of this solu-
tion, equivalent to about 100 mg of methohexital sodium,
to a 1000-mL volumetric flask, add about 200 mL of water
and 2.0 mL of sodium hydroxide solution (1 in 10), mix,
dilute with water to volume, and again mix. Transfer
20.0 mL of the resulting solution to a 100-mL volumetric
flask, dilute with water to volume, and mix.
Procedure—Concomitantly determine the absorbances of
the Standard solution and the Test solution in 1-cm cells at
the wavelength of maximum absorbance at about 247 nm,
with a suitable spectrophotometer, using water as the blank.
Calculate the quantity, in mg, of Ci4HizN2NaQ3 in the
Methohexital Sodium for Injection taken by the formula:
(284,29/262.30)(TC/D)(Av / As)
in which 284.29 and 262.30 are the molecular weights of
methohexital sodium and methohexital, respectively; T is
the labeled quantity; in mg, of methohexital sodium in the
Methohexital Sodium for Injection; C is the concentration, in
lug per mL, of USP Methohexital RS in the Standard solution;
D is the concentration, in ug per mL, of methohexital so-
dium in the Test solution based on the labeled quantity per
container and the extent of dilution; and Ay and As are the
absorbances of the Test solution and the Standard solution,
respectively.
pH (791): between 10.6 and 11.6 in the solution prepared
in the test for Completeness of solution.
Loss on drying (731)—Dry it at 105° for 4 hours: it loses
not more than 2.0% of its weight.
Delete the following:
°Heavy metals, Method I! (231): 0.001%. (oiricial 1-jan-2018)
Other requirements—it meets the requirements under /n-
jections and Implanted Drug Products (1).
Assay—
form to obtain a solution having a concentration of about
1.35 mg per mL.
Standard preparation—Dissolve an accurately weighed
quantity of USP Methohexital RS in chloroform to obtain a
solution having a known concentration of about 0.46 mg
per mL. Transfer 5.0 mL of the resulting solution to a 10-mL
volumetric flask, add 2.0 mL of Internal standard solution, di-
lute with chloroform to volume, and mix to obtain a Stan-
dard preparation having a known concentration of about
230 Wg per mL.
Assay preparation—Combine and mix the constituted so-
lutions prepared from the contents of 5 vials of Methohexi-
tal Sodium for Injection. Transfer an accurately measured
volume of the resulting solution, equivalent to about 50 mg
of methohexital sodium, to a 125-mL separator containing
25 mL of water, and mix. Add 0.2 mL of dilute hydrochloric
acid (1 in 2), and mix. Extract with three 25-mL portions of
chloroform, shaking each extraction for 2 minutes and filter-
ing the extracts through about 15 g of anhydrous sodium
sulfate, that previously has been washed with about 5 mL of
chloroform, into a 100-mL volumetric flask. Wash the so-
dium sulfate with several small portions of chloroform, col-
lecting the washings in the 100-mL volumetric flask. Dilute
with chloroform to volume, and mix. Transfer 5.0 mL of this
solution to a 10-mL volumetric flask, add 2.0 mL of Internal
standard solution, dilute with chloroform to volume, and
mix,
Chromatographic system (see Chromatography (621))—The
gas chromatograph is equipped with a flame-ionization de-
USP 41 Official Monographs / Methotrexate 2649

tector and contains a 1.2-m x 4-mm column packed with Solution A Solution B
3% phase G10 on support S1AB. The column is maintained
at about 230°, the injection port at about 265°, and the 0
detector block at about 265°. Dry helium is used as the
carrier gas at a flow rate of about 60 mL per minute. Chro-
matograph replicate injections of the Standard preparation,
and record the peak responses as directed for Procedure; the
resolution, R, between methohexital and aprobarbital is not
less than 4.0, and the relative standard deviation is not
more than 2.0%. Standard stock solution: 1.0 mg/mL of USP Metho-
trexate RS prepared as follows. Transfer a known
Procedure—Separately inject equal volumes (about 2 uL) amount of USP Methotrexate RS to a suitable volumet-
of the Assay preparation and the Standard preparation into ric flask, dissolve in dimethyl! sulfoxide equivalent to 5%
thegas chromatograph, and measure the peak responses
of the final volume, and dilute with Solution A to
for the major peak. The relative retention times are about
0.6 for methohexital and 1.0 for aprobarbital. Calculate the volume.
Standard solution: 0.2 mg/mL of USP Methotrexate RS
uantity, in mg, of methohexital sodium (Ci4Hi7N2NaQs) in in Solution A, from the Standard stock solution
the ee of Methohexital Sodium for Injection taken by
the formula: Sample stock solution: Transfer 200 mg of Methotrex-
ate to a 200-mL volumetric flask, and dissolve in 10 mL
(284.29/262.30)(0.2©(Ru/ Rs) of dimethyl sulfoxide with sonication for 5 min. Add
150 mL of Solution A, and sonicate again for 5 min.
in which 284.29 and 262.30 are the molecular weights of Dilute with Solution A to volume to obtain 1.0 mg/mL
methohexital sodium and methohexital, respectively; C is of Methotrexate. [NoTE—Sonicate as needed.]
the concentration, in ug per mL, of USP Methohexital RS in Sample solution: 0.2 mg/mL of Methotrexate in Solu-
the Standard preparation; and Ry and Rs are the peak re- tion A, from the Sample stock solution
sponse ratios obtained from the Assay preparation and the Chromatographic system
Standard preparation, respectively. (See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 280 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Flow rate: 1.0 mL/min
Methotrexate Injection size: 20 uL
System suitability
HNC UNO UN,
Sample: Standard solution
YT 3 Hy Suitability requirements
NaUL
% N’
a N,
as 9. Tailing factor: NMT 1.6
| H 0H Relative standard deviation: NMT 2.0%
NH AA,
7 OH Analysis c
oO wy < Samples: Standard solution and Sample solution vy
Calculate the percentage of C2oH22NgQOs in the portion s
of Methotrexate taken: c<
C20H22NgOs 454.44 Result = (u/s) x (Cs/Cu) x 100
°
2
L-Glutamic acid, N-[4-[[(2,4-diamino-6-pter-
idinyl)methyl]methylamino]benzoyl]-; ty = peak response from the Sample solution =
ROTEL Gd Diathin eG piendinymethy Imetaylamt- rs = peak response from the Standard solution s
no]benzoyl] glutamic acid; Cs = concentration of USP Methotrexate RS in the a
(S)-2-(4-{[(2,4-Diaminopteridin-6-yl)methyl](methyl)ami- Standard solution (mg/mL) Cs
no}benzamido)pentanedioic acid [59-05-2]. Cu = concentration of Methotrexate in the Sample
DEFINITION solution (mg/mL)
Methotrexate is a mixture of 4-amino-10-methylfolic acid Acceptance criteria: 98.0%-102.0% on the anhydrous
and closely related compounds. It contains NLT 98.0% basis
and NMT 102.0% of C2oH22NgOs, calculated on the anhy- IMPURITIES
drous basis. Inorganic Impurities
[CauTioN—Great care should be taken to prevent inhaling e RESIDUE ON IGNITION (281): NMT 0.1%
particles of Methotrexate and exposing the skin to it.]
IDENTIFICATION Delete the following:
© A, INFRARED ABSORPTION (197K): Do not dry specimens.
e B. ULTRAVIOLET ABSORPTION (197U) °e Heavy METALs, Method II (231): NMT 20 ppme cofticett-
Sample solution: 10 g/mL in 0.1 N hydrochloric acid jan-2018)
Organic Impurities
ASSAY ¢ PROCEDURE 1: RELATED COMPOUNDS
e PROCEDURE
Solution A, Solution B, Mobile phase, Sample solu-
Buffer: 3.4 mg/mL of anhydrous monobasic sodium tion, and Chromatographic system: Proceed as di-
phosphate in water. Adjust with 1 N sodium hydroxide rected in the Assay.
to a pH of 6.0. Standard stocksolution A: Use the Standard solution
Solution A: Acetonitrile and Buffer (1:19) from the Assay.
Solution B: Acetonitrile and Buffer (1:1) Standard solution A: 0.1beime of USP Methotrexate
Mobile phase: See the gradient table below. RS in Solution A, from Standard stock solution A
Standard stock solution B: Transfer known quantities
of USP Methotrexate Related Compound C RS, USP
Methotrexate Related Compound B RS, and USP Meth-
otrexate Related CompoundERS to a suitable volu-
metric flask, dissolve in dimethyl sulfoxide equivalent to
 2650 Methotrexate / Official Monographs USP 41

1% of the final volume, and dilute with Solution A to Tu = peak response from the Sample solution
volume to obtain 0.1 mg/mL of USP Methotrexate Re- Ts = peak response of methotrexate from Standard
lated Compound BRS, 0.2 mg/mL of USP Methotrex- solution A
ate Related Compound C RS, and 0.1 mg/mL of USP Cs = concentration of USP Methotrexate RS in
Methotrexate Related CompoundE RS. Standard solution A (mg/mL)
Standard solution B: 0.4 g/mL of USP Methotrexate Cu = concentration of Methotrexate in the Sample
Related Compound C RS, 0.2 g/mL of USP Methotrex- solution (mg/mL)
ate Related CompoundB RS, and 0.2 g/mL of USP F = relative response factor for each individual
Methotrexate Related CompoundE RS in Solution A, impurity (see Impurity Table 1)
from Standard stock solution B Acceptance criteria
System suitability solution: Transfer a known quantity Individual impurities: See Impurity Table 1. [NoTE—
of USP Methotrexate System Suitability Mixture RS to a Disregard any impurity peak less than 0.05%.]
suitable volumetricflask, and dissolve in dimethyl sulf- Total impurities: NMT 1.0%
oxide equivalent to about 1% of the final volume. Add
Standard stock solution B equivalent to 0.2% of the final Impurity Table 1
volume, and dilute with Solution A to volume to pre-
pare 0.1 mg/mL of USP Methotrexate System Suitabil- Relative Relative Acceptance
ity Mixture RS, 0.2 g/mL of USP Methotrexate Related Retention Response Criteria,
CompoundB RS, 0.4 ug/mL of USP Methotrexate Re- Name Time Factor NMT (%)
lated Compound C RS, and 0.2 g/mL of USP Metho- Methotrexate related
trexate Related Compound E RS. compound Be 0.71 = 0.3
System suitability Methotrexate related
Samples: Standard solution A and System suitability compound C 0.75 — 0.5
solution Methotrexate 1.00 — —
Suitability requirements Methotrexate related
Resolution: NLT 1.7 between methotrexate related compound E free
compound B and methotrexate related compound basec 1.39 — 0.3
C, NLT 10.0 between methotrexate related com-
pound C and methotrexate, and NLT 5.0 between Methotrexate
methotrexate related compound | and methotrexate dimethylamide? and
related compound H; System suitability solution Methotrexate related
Relative standard deviation: NMT 5.0%, Standard compound Ie 1.55 0.71 0.2!
solution A Methotrexate related
Analysis compound Hy 1.68 1.0 0.2
Samples: Standard solution A, Standard solution B, and Any unspecified impu-
Sample solution rity =— 1.0 0.10
Calculate the percentage of methotrexate related com- a Gy ACE /4-Diaminoptendin:6-y)metnylarning penzamida)pentanedioie
rs pound B and methotrexate related compound Cin acid.
ro the portion of Methotrexate taken: » (S)-2-(4-{[(2-Amino-4-oxo-1,4-dihydropteridin-6-yl)methy!](methyl)ami-
S no}benzamido)pentanedioic acid.
ey Result = (ru/ts) x (Cs/Cu) x 100 ¢ 4-{[(2,4-Diaminopteridin-6-yl)methy!](methyl)amino}benzoic acid.
4 2-(4-{[(2,4-Diaminopteridin-6-yl)methy|](methyl)amino}benzamido)-5-(di-
methylamino)-5-oxopentanoic acid.
S tu = peak response from the Sample solution
© (5)-4-(4-{[(2,4-Diaminopteridin-6-yl)methyl](methyl)amino}benzamido)-5-
Ss Is = peak response from Standard solution B methoxy-5-oxopentanoic acid.
Cs = concentration of the corresponding ‘If present, methotrexate dimethylamide and Methotrexate related com-
ra methotrexate related compound in Standard
solution B (mg/mL)
jound | may not be completely resolved by the method. These peaks are
integrated together to determine conformance.
>) Cu = concentration of Methotrexate in the Sample 9 (S)-2-(4-{[(2,4-Diaminopteridin-6-yl)methyl](methyl)amino}benzamido)-5-
solution (mg/mL) methoxy-5-oxopentanoic acid.
Calculate the percentage of methotrexate related
compoundEfree base in the portion of Methotrexate © PROCEDURE 2: ENANTIOMERIC PURITY
taken: Solution A: 7.1 g/L of anhydrous dibasic sodium phos-
phate in water
Result = (ru/rs) x (Cs/Cu) x (Mn/My2) x 100 Solution B: 6.9 g/L of monobasic sodium phosphate in
water
tu = peak response from the Sample solution Solution C: Solution A and Solution B (5:6). Adjust with
Ts = peak response from Standard solution B 2.N sodium hydroxide to a pH of 6.9.
Cs = concentration of USP Methotrexate Related Mobile phase: n-Propanol and Solution C (2:23)
CompoundERS in Standard solution B System suitability solution: 0.02 mg/mL each of USP
(mg/mL) \ as aa es RS and USP R-Methotrexate RS in Mobile
Cu = concentration of Methotrexate in the Sample phase
solution (mg/mL) Sample solution: 0.2 mg/mL of Methotrexate in Mo-
Mn = molecular weight of methotrexate related bile phase
compoundEfree base, 325.33 Diluted sample solution: 2 j1g/mL of Methotrexate in
M,2 = molecular weight of USP Methotrexate Mobile phase, from the Sample solution
Related CompoundE RS, 343.56 Chromatographic system
[Note—USP Methotrexate Related Compound E RS is (See Chromatography (621), System Suitability.)
4-{[(2,4-diaminopteridin-6-yl)methy!](methyl)amino}- Mode: LC
benzoic acid, hemihydrochloride.] Detector: UV 302 nm
Calculate the percentage of methotrexate related com-
pound H, methotrexate related compound |, and any
unspecified impurity in the portion of Methotrexate
taken:

Result = (ru/ts) x (Cs/Cu) x (1/F) x 100

USP 41 Official Monographs / Methotrexate 2651

Column: 4.0-mm x 15-cm; 7-~m packing L75 to obtain a solution having a concentration of about
Flow rate: 1.5 mL/min 2.5 mg/mL. Adjust with 0.1 N hydrochloric acid to a
Injection size: 20 uL pH of 4.0. Place the slurry in a 50-mL centrifuge tube,
System suitability and centrifuge. Decant the supernatant, add 25 mL of
Sample: System suitability solution. [NoTE—The rela- acetone, shake, and filter through a solvent-resistant
tive retention times for methotrexate and R-metho- membrane filter of 0.45-um pore size. Air-dry the
trexate are 1.0 and 1.95, respectively.] filtered precipitate.
Suitability requirements Acceptance criteria: Meets the requirements
Resolution: NLT 1.3 between methotrexate and
R-methotrexate ASSAY
Relative standard deviation: NMT 5.0% for the e PROCEDURE
methotrexate peak Buffer: 0.2 M dibasic sodium phosphate and 0.1 M
Analysis citric acid (63:37), adjusted if necessary with 0.1 M cit-
Samples: Sample solution and Diluted sample solution eae or 0.2 M dibasic sodium phosphate to a pH of
Calculate the percentage of R-methotrexate in the por- 6.
tion of Methotrexate taken: Mobile phase: Acetonitrile and Buffer (10:90)
System suitability solution: 0.1 mg/mL each of USP
Result = [ru/(rs x 100)] x 100 Methotrexate RS and folic acid in Mobile phase
Standard solution: 100 g/mL of USP Methotrexate RS
tu = peak area of R-methotrexate from the Sample in Mobile phase
solution Sample solution: Equivalent to 100 ug/mL of metho-
Is = peak area of Methotrexate from the Diluted trexate from Injection in Mobile phase
sample solution Chromatographic system
Acceptance criteria: NMT 3.0% (See Chromatography (621), System Suitability.)
Mode: LC
SPECIFIC TESTS Detector: UV 302 nm
@ WATER DETERMINATION, Method | (921): NMT 12.0% Column: 4.6-mm x 25-cm; packing L1
Flow rate: 1.2 mL/min
ADDITIONAL REQUIREMENTS Injection volume: 10 uL
© PACKAGING AND STORAGE: Preserve in tight, light-resistant System suitability
containers. Sample: System suitability solution
© USP REFERENCE STANDARDS (11) [Note—The relative retention times for folic acid and
USP Methotrexate RS methotrexate are 0.35 and 1.0, respectively.]
USP Methotrexate Related Compound B RS Suitability requirements
USP Methotrexate Related Compound C RS Resolution: NLT 8.0 between the folic acid and
USP Methotrexate Related Compound E RS methotrexate peaks
USP Methotrexate System Suitability Mixture RS Relative standard deviation: NMT 2.5% for the
It contains Methotrexate, Methotrexate Dimethylester methotrexate peak
Hydrochloride (oss
Analysis 2)
Gepinetnyired ac 4-diaminapteneln Syamelie Samples: Standard solution and Sample solution uv
viGneth amino}benzamido) pentanedioate
ydrochloride.
Calculate the percentage of the labeled amount metho- ms
trexate (C20H22NsOs) in the portion of Injection taken: io)
CazH2sNsOs-HCl 518.95 |
and a small amount of Methotrexate related compound Result = (ru/rs) x (Cs/Cu) x 100 °
| so}
om
(S)-4-(4-{[(2,4-Diaminopteridin-6-yl)meth- tu peak response from the Sample solution Ly
mo]
wt

Pile eaiine beneaentdeyee Methexioeexepenter Ts peak response from the Standard solution a
Won

noic acid. concentration of USP Methotrexate RS in the 7

Cs
CaH2sNgOs 468.47 Standard solution (ug/mL)
and Methotrexate related compound H Cy = nominal concentration of methotrexate in the
(5)-2-(4-{[(2,4-Diaminopteridin-6-yl)meth- Sample solution (ug/mL)
PeerYael Detar NO) SAE ONS ACRENa Acceptance criteria: 90.0%-110.0%
noic acid.
CrH2NsOs 468.47 SPECIFIC TESTS
USP R-Methotrexate RS e PH (791): 7.0-9.0
(R)-2-(4-{[(2,4-Diaminopteridin-6-yl)meth- e BACTERIAL ENDOTOXINS TEST (85): NMT 0.4 USP Endo-
yl](methyl)amino}benzamido)pentanedioic acid. toxin Unit/mg of methotrexate sodium
CaoH22NsOs 454.44 © OTHER REQUIREMENTS: Meets the requirements in Injec-
tions (1)
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in single-dose or in
Methotrexate Injection multiple-dose containers, preferably of Type | glass, pro-
tected from light.
DEFINITION
Methotrexate Injection is a sterile solution of Methotrexate Change to read:
in Water for Injection prepared with the aid of Sodium
Hydroxide. It contains NLT 90.0% and NMT 110.0% of ° USP REFERENCE STANDARDS (11)
the labeled amount of methotrexate (C2oH22NgOs). @ (CN 1-May-2018)
USP Methotrexate RS
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
Sample: Dilute, if necessary, a volume of Injection,
equivalent to about 25 mg of methotrexate, with water
 2652 Methotrexate / Official Monographs
Methotrexate for Injection
DEFINITION
Methotrexate for Injection is a sterile, freeze-dried prepara-
tion of methotrexate sodium with or without suitable
added substances, buffers, and/or diluents. It contains
NLT 95.0% and NMT 115.0% of the labeled amount of
methotrexate (C2oH22NgOs).
[CAuTION—Great care should be taken to prevent inhalin
particles of methotrexate sodium and exposure to skin.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
Sample: 2.5 mg/mL of methotrexate in water from
Methotrexate for Injection. Adjust with 0.1 N hydro-
chloric acid to a pH of 4.0. Place the slurry in a 50-mL
centrifuge tube, and centrifuge. Decant the superna-
tant, add 25 mL of acetone, shake, and filter through a
solvent-resistant membrane filter of 0.45-14m pore size.
Air-dry the filtered precipitate.
Acceptance criteria: Meets the requirements
ASSAY
© PROCEDURE
Buffer: 0.2 M dibasic sodium phosphate and 0.1 M
citric acid (63:37), adjusted if necessary with 0.1 M cit-
ric acid or 0.2 M dibasic sodium phosphate to a pH of
6.0
Mobile phase: Acetonitrile and Buffer (10:90)
System suitability solution: 0.1 mg/mL each of USP
Methotrexate RS and folic acid in Mobile phase
Standard solution: 100 ug/mL of USP Methotrexate RS
in Mobile phase
Sample solution: Equivalent to 100 ug/mL of metho-
trexate from 1 container of Methotrexate for Injection
in Mobile phase
Chromatographic system
=
”
a (See Chromatography (621), System Suitability.)
i] Mode: Lc
—
D Detector: UV 302 nm
S Column: 4.6-mm x 25-cm; packing L1
S
S Flow rate: 1.2 mL/min
= Injection volume: 10 LL
System suitability
a
A) Sample: System suitability solution
= {[Note—The relative retention times for folic acid and
methotrexate are 0.35 and 1.0, respectively.]
Suitability requirements
Resolution: NLT 8.0 between the folic acid and
methotrexate peaks
Relative standard deviation: NMT 2.5% for the
methotrexate peak
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
methotrexate (CzoH22NgOs) in the portion of Metho-
trexate for Injection taken:
Result = (ru/rs) x (Cs/Cu) x 100
ry = peak response from the Sample solution
rs = peak response from the Standard solution
Cs = concentration of USP Methotrexate RS in the
Standard solution (g/mL)
Cy = nominal concentration of methotrexate in the
Sample solution (4ug/mL)
Acceptance criteria: 95,0%-115.0%
PERFORMANCE TESTS
e UNIFORMITY OF DosAGE UNITS (905): Meets the
requirements
SPECIFIC TESTS
e PH (791)
USP 41
Sample: Constituted as directed in the labeling, except
that water is used as the diluent
Acceptance criteria: 7.0-9.0
CONSTITUTED SOLUTION: At the time of use, it meets the
requirements in Injections and Implanted Drug Products
(1), Specific Tests, eopiten ess and clarity of solutions.
BACTERIAL ENDOTOXINS TEST (85): NMT 0.4 USP Endo-
toxin Unit/mg of methotrexate sodium
STERILITY TESTS (71): Meets the requirements
OTHER REQUIREMENTS: Meets the requirements in Labeling
(7), Labels and Labeling for Injectable Products
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve as described in Pack-
aging and Storage Requirements (659), Injection Packaging,
protected from light.
Change to read:
e UsP REFERENCE STANDARDS (11)
@ (CN 1-May-2018)
USP Methotrexate RS
Methotrexate Tablets
DEFINITION
Methotrexate Tablets contain NLT 90.0% and NMT 110.0%
of the labeled amount of methotrexate (C29H22NgOs).
IDENTIFICATION
© A. ULTRAVIOLET ABSORPTION (197U)
Standard solution: 25 «g/mL of USP Methotrexate RS
in dilute hydrochloric acid (1 in 100)
Sample solution: Dissolve 1 Tablet in 100 mL of dilute
hydrochloric acid (1 in 100), and filter the solution.
Acceptance criteria: The UV absorption spectrum of
the Sample solution exhibits maxima and minima at the
same wavelengths as that of the Standard solution.
ASSAY
© PROCEDURE
Buffer: 0.2 M dibasic sodium phosphate and 0.1 M
citric acid (63:37), adjusted if necessary with 0.1 M cit-
ric acid or 0.2 M dibasic sodium phosphate to a pH of
6.0
Mobile phase: Acetonitrile and Buffer (10:90)
System suitability solution: 0.1 mg/mL each of USP
Methotrexate RS and folic acid in Mobile phase
Standard solution: 100 g/mL of USP Methotrexate RS
in Mobile phase
Sample solution: Equivalent to 100 g/mL of metho-
trexate from powdered Tablets (NLT 20 Tablets) in Mo-
bile phase. Dissolve the methotrexate using a mechani-
cal shaker or ultrasonic bath.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: Ka
Detector: UV 302 nm
Column: 4.6-mm x 25-cm; packing L1
Flow rate: 1.2 mL/min
Injection volume: 10 pL
System suitability
Sample: System suitability solution
[NoTe—The relative retention times for folic acid and
methotrexate are 0.35 and 1.0, respectively.]
Suitability requirements
Resolution: NLT 8.0 between the folic acid and
methotrexate peaks
Relative standard deviation: NMT 2.5% for the
methotrexate peak
USP 41 Official Monographs / Methotrimeprazine 2653

Analysis IDENTIFICATION
Samples: Standard solution and Sample solution e A. INFRARED ABSORPTION (197K)
Calculate the percentage of the labeled amount of e B. ULTRAVIOLET ABSORPTION (197U)
methotrexate (C2oH22NgOs) in the portion of Tablets Solution: 7 ug/mL
taken: Medium: Alcohol
Acceptance criteria: Absorptivities at 255 nm, calcu-
Result = (ru/rs) x (Cs/Cu) x 100 ie on the dried basis, do not differ by more than
3.0%.
ty = peak response from the Sample solution
ls = peak response from the Standard solution ASSAY
Gs = concentration of USP Methotrexate RS in the e PROCEDURE
Standard solution (uug/mL) Sample solution: Dissolve about 700 mg of Metho-
Cy nominal concentration of methotrexate in the trimeprazine in 100 mL of chloroform. Add 1 drop of a
ul

Sample solution (\ug/mL) solution (1 in 500) of crystal violet in chloroform.

Acceptance criteria: 90.0%-110.0% Analysis: Titrate the Sample solution with 0.1 N perchlo-
ric acid VS to the first disappearance of the violet tinge.
PERFORMANCE TESTS Perform a blank determination, and make any necessary
e DISSOLUTION (711) correction. Each mL of 0.1 N perchloric acid is equiva-
Medium: 0.1 N hydrochloric acid; 900 mL lent to 32.85 mg of methotrimeprazine (Ci9H24N20S).
Apparatus 2: 50 rpm Acceptance criteria: 98.0%-101.0% on the dried basis
Time: 45 min
Standard solution: USP Methotrexate RS in Medium IMPURITIES
Detector: UV 306 nm (maximum absorbance) e SELENIUM (291)
Analysis: Determine the amount of C2oH22NgOs dis- Test solution: Prepare with 100 mg of Metho-
solved from UV absorbances on filtered portions of the trimeprazine and 100 mg of magnesium oxide.
solution under test, suitably diluted with Medium, in Acceptance criteria: The absorbance of the Test solu-
comparison with a Standard solution. tion is NMT one-half that of the Standard Solution
Tolerances: NLT 75% (Q) of the labeled amount of (0.003%).
methotrexate (C2oH22NeOs) is dissolved.
e UNIFORMITY OF DosAGE UNITS (905): Meet the SPECIFIC TESTS
requirements © OPTICAL ROTATION, Specific Rotation (781S)
Sample solution: 50 mg/mL in chloroform
ADDITIONAL REQUIREMENTS Acceptance criteria: —15° to -18°
e PACKAGING AND STORAGE: Preserve in well-closed contain- e Loss ON DRYING (731)
ers. A unit-of-use container contains a quantity of Tablets Analysis: Dry at 100° for 3 h.
sufficient to provide one week's therapy as indicated in Acceptance criteria: NMT 0.5%
the labeling.
e LABELING: When packaged in a unit-of-use container, the ADDITIONAL REQUIREMENTS
label indicates the total amount of methotrexate present © PACKAGING AND STORAGE: Preserve in well-closed, light- jog
a)
as one week’s supply. resistant containers. Store at 25°, excursions permitted me)
© USP REFERENCE STANDARDS (11) between 15° and 30°.
USP Methotrexate RS e USP REFERENCE STANDARDS (11) =
USP Methotrimeprazine RS }
=
}
eo}=
i)
Methotrimeprazine ie}
be
Methotrimeprazine Injection a

‘oer
DEFINITION
Methotrimeprazine Injection is a sterile solution of Metho-
CH CHy trimeprazine in Water for Injection, prepared with the aid
Teg son of hydrochloric acid. It contains NLT 90.0% and NMT
110.0% of the labeled amount of methotrimeprazine
(CisH24N20S), as the hydrochloride.
CigH24N20S 328.47 [Nott—Throughout the following procedures, protect test
10H-Phenothiazine-10-propanamine, 2-methoxy-N,N,B- or assay specimens, the Reference Standard, and solutions
trimethyl-, (-)-; containing them by conducting the procedures without
(-)-1 Fre a pe ean nop aemnethoxyplies delay under subdued light or by using low-actinic
nothiazine [60-99-1]. glassware.]

DEFINITION
Methotrimeprazine contains NLT 98.0% and NMT 101.0%
of methotrimeprazine (CisH24N20S), calculated on the
dried basis.
[Note—Throughout the following procedures, protect test
or assay specimens, the USP Reference Standard, and the
solutions containing them by conducting the procedures
without delay under subdued light or by using low-actinic
glassware.]
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
Sample: Place 1 mL of Injection in a 125-mL separator,
and add 1 N sodium hydroxide dropwise until the solu-
tion becomes opaque white. Extract with 50 mL of
ether, wash the ether extract with 25 mL of water, and
discard the washing. Filter the ether extract through a
layer of anhydrous sodium sulfate into a beaker, and
evaporate the filtrate by means of a stream of nitrogen
to complete dryness. Dry at 100° for 3 h.
 2654 Methotrimeprazine / Official Monographs USP 41

Acceptance criteria: Meets the requirements Change to read:

ASSAY
© PROCEDURE e USP REFERENCE STANDARDS (11)
Solution A: Transfer 23.5 mL of 85% phosphoric acid °o (CN 1-May-2018)

into a 100-mL volumetric flask containing water, and USP Methotrimeprazine RS

dilute with water to volume.
Mobile phase: Add 20 mL of Solution A to 450 mL of
water. To this solution add 5 mL of triethylamine, and
adjust with 1 N sodium hydroxide to a pH of 3.0. Add
500 mL of acetonitrile, and dilute with water to Methoxsalen
1000 mL. Filter, and degas.
System suitability solution: 2.0 mg/mL of benzyl alco-
hol, using appropriate amounts of 1% benzyl alcohol,
a 0.1 mg/mL of USP Methotrimeprazine RS in Mobile
phase
Standard solution: 0.1 mg/mL of USP Metho-
trimeprazine RS in Mobile phase CizHgO4 216.19
Sample solution: Nominally equivalent to 0.1 mg/mL 7H-Furo[3,2-g][1 ]benzopyran-7-one, 9-methoxy-.
of methotrimeprazine in Mobile phase from an appropri- 9-Methoxy-7H-furo[3,2-g][1]benzopyran-7-one [298-81-7].
ate amount of Injection
Chromatographic system » Methoxsalen contains not less than 98.0 per-
(See Chromatography (621), System Suitability.) cent and not more than 102.0 percent of
Mode: LC Ci2HsOx, calculated on the anhydrous basis.
Detector: UV 254 nm [Caution—Avoid contact with the skin.]
Column: 4.6-mm x 25-cm; packing L7
Flow rate: 1 mL/min Packaging and storage—Preserve in well-closed, light-re-
Injection volume: 20 pL sistant containers.
System suitability
USP Reference standards (11)—
Samples: System suitability solution and Standard USP Methoxsalen RS
solution
Suitability requirements Identification, Infrared Absorption (197K).
Resolution: NLT 4.0 between benzyl alcohol and Melting range, Class | (741): between 143° and 148°.
methotrimeprazine, System suitability solution Water Determination, Method | (921): not more than
Tailing factor: NMT 1.2, System suitability solution 0.5%.
Relative standard deviation: NMT 1.5%, Standard Residue on ignition (281): not more than 0.1%, a 1-g
solution specimen being used.
al Analysis
Es Samples: Standard solution and Sample solution
a Calculate the percentage of the labeled amount of
Ss Delete the following:
—
D methotrimeprazine (CigH24N2OS) in the portion of In-
° jection taken: °Heavy metals, Method !/ (231): 0.002%. cotficiai 1jan-2018)
=
S Result = (ru/rs) x (Cs/Cu) x 100 Chromatographic impurities—Prepare a solution of it in
2 chloroform containing about 20 mg per mL (Solution A). Di-
a ru = peak response from the Sample solution lute 1.0 mL of it with chloroform to 100.0 mL (Solution B).
w rs = peak response from the Standard solution Apply 5-uL portions of both solutions at points along a line
=) Cs = concentration of USP Methotrimeprazine RS in about 2.5 cm from one edge of a thin-layer chromato-
the Standard solution (mg/mL) graphic plate coated with a 0.25-mm layer of chromato-
Cu = nominal concentration of methotrimeprazine graphic silica gel mixture and previously dried at 105° for
in the Sample solution (mg/mL) 30 minutes. Develop the plate in a suitable chamber, with-
Acceptance criteria: 90.0%-110.0% out previous equilibration, using a mixture of 9 volumes of
benzene and 1 volume of ethyl acetate, until the solvent
SPECIFIC TESTS front has moved to about 15 cm above the line of applica-
© PH (791): 3.0-5.0 tion. Remove the plate from the chamber, air-dry, and ob-
e BACTERIAL ENDOTOXINS TEST (85): NMT 17.9 USP Endo- serve under long-wavelength UV light: any spot in the chro-
toxin Units/mg of methotrimeprazine matogram from Solution A, other than the principal spot, is
© OTHER REQUIREMENTS: It meets the requirements in /njec- not more intense than the spot from Solution B (1.0%).
tions and Implanted Drug Products (1). Assay—
ADDITIONAL REQUIREMENTS Mobile phase—Prepare a solution of acetonitrile in water
¢ PACKAGING AND STORAGE: Preserve in single-dose or mul- (35 in 100). Make adjustments if necessary (see System Suit-
tiple-dose containers, preferably of Type | glass, protected ability under Chromatography (621)).
from light. Internal standard preparation—Dissolve trioxsalen in alco-
hol to obtain a solution containing about 0.2 mg per mL.
Standard preparation. sing an accurately weighed quan-
tity of USP Methoxsalen RS, prepare a solution in alcohol
having a known concentration of about 0.2 mg per mL.
Transfer 2.0 mL of this solution to a 100-mL volumetric flask,
add 2.0 mL of Internal standard preparation, dilute with Mo-
bile phase to volume, and mix to obtain a Standard prepara-
tion having a known concentration of about 4 ug of USP
Methoxsalen RS per mL. Pass through a 0.45-um disk before
using.
USP 41
Assay preparation—Using 20 mg of Methoxsalen, accu-
rately weighed, proceed as directed for Standard prepara-
tion.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 254-nm detector
and a 4-mm x 30-cm column that contains packing L1. The
flow rate is about 1.5 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as di-
rected for Procedure: the resolution, R, between the analyte
and internal standard peaks is not less than 4.0, and the
relative standard deviation for replicate injections is not
more than 2.0%.
Procedure—Separately inject equal volumes (about 20 uL)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
ure the responses for the major peaks. The relative retention
times are about 2.1 for trioxsalen and 1.0 for methoxsalen.
Calculate the quantity, in mg, of C:2HsOu, in the portion of
Methoxsalen taken by the formula:
5C(Ru / Rs)
in whichCis the concentration, in ug per mL, of USP
Methoxsalen RS in the Standard preparation; and Ry and Rs
are the ratios of the peak responses of methoxsalen to the
internal standard obtained from the Assay preparation and
the Standard preparation, respectively.
Methoxsalen Capsules
» Methoxsalen Capsules contain not less than
90.0 percent and not more than 110.0 percent of
the labeled amount of methoxsalen (C;2HgO,).
Packaging and storage—Preserve in tight, light-resistant
containers.
rime ai the Capsules to state that Methoxsalen
Hard Gelatin Capsules may not be interchangeable with
Methoxsalen Soft Gelatin Capsules without retitration of the
patient.
USP Reference standards (11)—
USP Methoxsalen RS
Identification—
A: The retention time exhibited by methoxsalen in the
chromatogram of the Assay preparation corresponds to that
of methoxsalen in the chromatogram of the Standard prepa-
ration as obtained in the Assay.
B: Place one Capsule in 50 mL of alcohol contained in a
high-speed glass blender jar and blend thoroughly until the
shell is capt dispersed. Dilute a portion quantitatively
with alcohol to obtain a solution having a concentration of
about 4 ug per mL: the UV absorption spectrum of the solu-
tion so obtained exhibits maxima and minima at the same
wavelengths as that of a similar solution of USP Methox-
salen RS, concomitantly measured.
Dissolution (711)—
FOR SOFT GELATIN CAPSULES—
Medium: water; 900 mL.
Apparatus 2: 50 rpm.
Time: 45 minutes.
Procedure—Determine the amount of C;2HsO. dissolved
from UV absorbances at the wavelength of maximum ab-
sorbance at about 300 nm using filtered portions of the
solution under test, suitably diluted with water, if necessary,
in comparison with a Standard solution having a known
concentration of USP Methoxsalen RS in the same Medium.
[NoTE—An amount of alcohol not to exceed 1% of the total
volume of the Standard solution may be used to bring the
Official Monographs / Methoxsalen 2655
Reference Standard into solution prior to dilution with
Medium.]
Tolerances—Not less than 75% (Q) of the labeled amount
of Ci2HgOs is dissolved in 45 minutes.
FOR HARD GELATIN CAPSULES—
Medium: water; 900 mL.
Apparatus 1: 150 rpm.
Time: 90 minutes.
Procedure—Determine the amount of Ci2HgO, dissolved
from UV absorbances at the wavelength of maximum ab-
sorbances at about 252 nm of filtered portions of the solu-
tion under test in comparison with a Standard solution hav-
ing a known concentration of USP Methoxsalen RS prepared
in alcohol and diluted with water.
Tolerances—Not less than 75% (Q) of the labeled amount
of Ci2HsO, is dissolved in 90 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Assay—
Mobile phase—Prepare a filtered and degassed mixture of
acetonitrile and water (65:35). Make adjustments if neces-
sary (see System Suitability under Chromatography (621)).
Standard preparation—Prepare a solution in alcohol hav-
ing an accurately known concentration of 0.2 mg of USP
Methoxsalen RS per mL. Pipet 2.0 mL of this solution into a
100-mL volumetric flask, dilute with Mobile phase to vol-
ume, and mix.
Assay preparation—
FOR HARD GELATIN CAPSULES—Place not less than 10 Cap-
sules in a high-speed glass blender jar containing 100.0 mL
of alcohol, and blend thoroughly. Transfer an accurately
measured volume of the aliquot from the blender jar, equiv-
alent to about 2 mg of Methoxsalen, to a 50-mL volumetric
flask, dilute with alcohol to volume, mix, and filter. Transfer
5.0 mL of this solution to a S0-mL volumetric flask, dilute foe
with Mobile phase to volume, mix, and filter. 4)
z
FOR SOFT GELATIN CAPSULES—Place the end of a long-stem
glass funnel on a 250-mL volumetric flask, punch a hole at =
each end of a Capsule with a syringe containing 15 mL of i)
3
alcohol, and rinse the contents into the flask. Cut the Cap- i)
sule shell with a scalpel, and wash the inside of the shell to)Bt
with 15 mL of alcohol into the same flask. Repeat these 2
steps for not less than 4 additional Capsules, and collect the 3
rinse. Wash the funnel, and collect the rinse in the same =
”
flask. Dilute with alcohol to volume, and mix. Transfer an
accurately measured volume of this solution, equivalent to
about 2 mg of methoxsalen, to a 50-mL volumetric flask,
dilute with alcohol to volume, mix, and filter. Transfer
5.0 mL of this solution to a 50-mL volumetric flask, dilute
with Mobile phase to volume, mix, and filter.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 254-nm detector
and a 4-mm x 30-cm column that contains packing L1. The
flow rate is about 1.5 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as di-
rected for Procedure: the relative standard deviation for rep-
licate injections is not more than 2.0%.
Procedure—Separately inject equal volumes (about 20 uL)
of the Standard preparation and the Assay preparation into
the chromatograph, record the Sea and meas-
ure the responses for the major peaks. Calculate the quan-
tity, in percentage of the label claim, of methoxsalen
(Ci2HgO«) in the portion of the Capsule taken by the
formula:
100(Cs / Cu)(ru/ rs)
in which Cs is the concentration, in mg per mL, of USP
Methoxsalen RS in the Standard preparation; Cy is the nomi-
nal concentration, in mg per mL, of methoxsalen in the
Assay preparation, Based on the label claim; and ry and rs
 2656 Methoxsalen / Official Monographs USP 41

are the peak responses obtained from the Assay preparation Sample solution: Methoxyflurane (1 in 20) in
and the Standard preparation, respectively. chloroform
Acceptance criteria: The IR absorption spectrum of the
Sample solution exhibits maxima only at the same wave-
lengths as those of the Standard solution.
e s

Methoxsalen Topical Solution Sample: Methoxyflurane

Analysis: To 1 mL of the Sample in a test tube add 1 mL
of sulfuric acid.
» Methoxsalen Topical Solution is a solution of Acceptance criteria: The Sample forms a layer over the
Methoxsalen in a suitable vehicle. It contains not acid (distinction from halothane).
less than 9.2 mg and not more than 10.8 mg of °C
methoxsalen (Ci2HgO4) per mL. Analysis: Cautiously heat the contents of the test tube
from Identification test B with agitation.
Packaging and storage—Preserve in tight, light-resistant Acceptance criteria: The interface disappears, and hy-
containers. drofluoric acid is evolved (distinction from chloroform,
USP Reference standards (11)— trichloroethylene, and halothane).
USP Methoxsalen RS ASSAY
Identification—Transfer a volume of Topical Solution to a e PROCEDURE
100-mL volumetric flask, and dilute quantitatively and step- Sample: Methoxyflurane
wise with alcohol to obtain a concentration of about 8 uw Chromatographic system
per mL: the UV absorption spectrum of this solution exhibits (See Chromatography (621), System Suitability.)
maxima and minima at the same wavelengths as that of a Mode: GC
similar solution of USP Methoxsalen RS, concomitantly Detector: Thermal conductivity
measured. Column: 4-mm x 3-m; stainless steel packed with liq-
Alcohol Determination (611) (if present): between uid phase G11 on support S1A
66.5% and 77.0% of C2HsOH. Temperatures
Assay— Injection port: 150°
Column: 100°-110°
Mobile phase, Internal standard preparation, Standard Carrier gas: Dry helium
preparation, and Chromatographic system—Prepare as di- Flow rate: 60 mL/min
rected in the Assay under Methoxsalen. Injection volume: NMT 30 pL
Assay preparation—Transfer an accurately measured vol- Analysis
ume of Topical Solution, equivalent to about 20 mg of Sample: Sample
methoxsalen, to a 100-mL volumetric flask. Dilute with alco- Calculate the percentage of methoxyflurane
hol to volume, and mix. Transfer 2.0 mL of this solution and (C3H4Cl2F20) in the portion of Methoxyflurane taken:
2.0 mL of Internal standard preparation to a 100-mL volu-
=
”
metric flask. Dilute with Mobile phase to volume, and mix. Result = (ru/r7) x 100
is Pass through a 0.45-um disk before using.
i
Procedure—Proceed as directed for Procedure in the Assay ry = peak area of methoxyflurane
D rr = sum of all the peak areas
i) under Methoxsalen. Calculate the quantity, in mg, of
Acceptance criteria: 99.9%-100.0%
c methoxsalen (Ci2HgO4) in each mL of the Topical Solution
S taken by the formula:
= SPECIFIC TESTS
e SPECIFIC GRAVITY (841): 1.420-1.425
a 5(C/V)(Ru /Rs) e ACIDITY
al
= in which V is the volume, in mL, of Topical Solution taken, Sample: 25 mL of Methoxyflurane
and the other terms are as defined therein. Titrimetric system
Mode: Direct titration
Titrant: 0.010 N sodium hydroxide
Endpoint detection: Visua
Analysis: Shake the Sample with 25 mL of carbon diox-
Methoxyflurane ide-free water for 2 min, and allow the layers to sepa-
rate. Add 1 drop of methyl red TS to the water extract,
boil for 1 min, and titrate with Titrant.
cl
Acceptance criteria: NMT 0.50 mL of Titrant is required
2On
cn ‘CH, to produce a distinct yellow color.
FoF e@ WATER DETERMINATION, Method | (921): NMT 0.1%
e Limit OF NONVOLATILE RESIDUE
C3H4ClaF20 164.97 Sample: 50 mL of Methoxyflurane
Ethane, 2,2-dichloro-1,1-difluoro-1-methoxy-; Analysis: Evaporate the Sample at room temperature in
2,2-Dichloro-1,1-difluoroethyl methyl ether [76-38-0]. awae evaporating dish, and dry the residue at 105°
or 1h.
DEFINITION Acceptance criteria: The weight of the residue does
Methoxyflurane contains NLT 99.9% and NMT 100.0% of not exceed 1 mg.
methoxyflurane (C3H4Cl2F20). It may contain a suitable
stabilizer. ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight, light-resistant
IDENTIFICATION containers, and avoid exposure to excessive heat.
© A. INFRARED ABSORPTION
Standard solution: USP Methoxyflurane RS (1 in 20) in
chloroform
USP 41 Official Monograph / Methscopolamine 2657

e USP REFERENCE STANDARDS (11) area less than that of the methscopolamine peak in the
USP Methoxyflurane RS chromatogram obtained from the Diluted standard solution,
and disregard any peak that is due to Solution A. Calculate
the percentage of each impurity in the portion of Methsco-
polamine Bromide taken by the formula:
100F(ri/ rs)
Methscopolamine Bromide
Hac,N CHy in which F is the relative response factor for the methsco-
polamine bromide impurities (see Table 1); r; is the peak
area of any impurity obtained from the Test solution; and rs
is the peak area of methscopolamine obtained from the
chromatogram of the Test solution: not more than 0.1% of
any individual impurity is found; and not more than 0.5%
Sox of total impurities is found.
CisHa4BrNO, 398,29
3-Oxa-9-azoniatricyclo[3.3.1.024]nonane, pele drei Table 1

(1,28,48,50, 76]
1-oxo-2-phenylpropoxy)-9,9-dimethyl-, bromide, [7(5)-
Relative Relative
68,7B-Epoxy-30.-hydroxy-8-methyI-1 oH, 50H-tropanium Retention Response
Name Time Factor (fF)
bromide (-)-tropate ~ [155-41-9].
Tropic acid 0.4 0.4
» Methscopolamine Bromide contains not less Scopolamine 0.9 1.0
than 97.0 percent and not more than 103.0 per- hydrobromide
cent of CigH24BrNO., calculated on the dried Methylatropine bromide TZ 1.0
basis. Apomethscopolamine 3.5 0.6
bromide
Packaging and storage—Preserve in tight, light-resistant Any other impurity =— 1.0
containers, and store at room temperature.
USP Reference standards (11)— Assay—
USP Methscopolamine Bromide RS Buffer solution—Prepare a solution containing 5.16 g of
USP Scopolamine Hydrobromide RS sodium 1-hexanesulfonate monohydrate and 3.40 g of mon-
Identification— obasic potassium phosphate in 1000 mL of water, adjust
A: Infrared Absorption (197K). with 1 M phosphoric acid to a pH of 2.8, and mix.
B: A solution (1 in 20) meets the requirements of the Solution A—Mix 850 mL of Buffer solution and 150 mL of
tests for Bromide (191). acetonitrile, filter, and degas.
wo
Specific rotation (781): between -21° and -25°, deter- Solution B—Mix 500 mL of Buffer solution and 500 mL of wn
mined in a solution containing 500 mg in each 10 mL. acetonitrile, filter, and degas. uv
Loss on drying (731)—Dry it at 105° for 2 hours: it loses Mobile phase—Use variable mixtures of Solution A and So- m5
not more than 2.0% of its weight. lution B as directed for Chromatographic sree Make ad- °
justments if necessary (see System Suitability under Chroma- =]
Residue on ignition (281): not more than 0.1%. °
Chromatographic purity— tography (621)). Ko}
me
Buffer solution, Solution A, Solution B, and Mobile phase— Standard preparation—Dissolve an accurately wees )
quantity of USP Methscopolamine Bromide RS in Solution A mo}
Proceed as directed in the Assay. =>
to obtain a solution having a known concentration of about 7
Standard solution—Prepare as directed for the Standard 1.0 mg per mL.
preparation in the Assay.
Assay preparation—Transfer about 50 mg of Methscopo-
Diluted standard solution—Dilute 5 wL of the Standard so- lamine Bromide, accurately weighed, to a 50-mL volumetric
lution with Solution A to 10.0 mL. flask, dissolve in and dilute with Solution A to volume, and
_ Test solution—Prepare as directed for the Assay prepara- mix.
tion. Chromatographic system (see Chromatography (621))—The
Scopolamine hydrobromide solution—Dissolve an accu- liquid chromatograph is equipped with a 210-nm detector
rately weighed quantity of USP Scopolamine Hydrobromide and a 4.6-mm x 10-cm column that contains packing L1.
RS in Solution A to obtain a solution having a known con- The flow rate is about 3 mL per minute. The column tem-
centration of about 0.05 mg per mL. perature is maintained at 50°. The chromatograph is pro-
System suitability solution—Dissolve about 50 mg of USP grammed as follows.
Methscopolamine Bromide RS in Solution A, add 1.0 mL of
Scopolamine hydrobromide solution, and dilute with Solution A Time Solution A Solution B
to 50.0 mL. This solution contains about 0.1% of scopol- (minutes) (%) (%) Elution
amine hydrobromide. 0-3 100 0 isocratic
Chromatographic system (see Crremarogmaphy (621))— 3-10 100-85 0315 linear gradient
Proceed as directed in the Assay. In addition, chromato-
10-10.1 85-100 1530 linear gradient
graph the System suitability solution, and record the peak
responses as directed for Procedure; the resolution, R, be- 10.1-13 100 0 re-equilibration
tween methscopolamine and scopolamine is not less than
1.5; and the tailing factor for the methscopolamine peak is Chromatograph the Standard preparation, and record the
not more than 2.0. peak responses as directed for Procedure: the relative stan-
dard deviation for six replicate injections is not greater than
Procedure—Separately inject equal volumes (about 5 wL) 1%.
of the Diluted standard solution and the Test solution into the
chromatograph, record the chromatogram for four times Procedure—Separately inject equal volumes (about 5 uL)
the retention time of methscopolamine, and measure the of the Standard preparation and the Assay preparation into
responses for the major peaks. Disregard any peak with an the chromatograph, record the chromatograms, and meas-
 2658 Methscopolamine / Official Monograph
ure the peak area responses. Calculate the quantity, in mg,
of CisH24BrNOg in the portion of Methscopolamine Bromide
taken by the formula:
50Cru/ rs)
in which C is the concentration, in mg per mL, of USP
Methscopolamine Bromide RS in the Standard preparation;
and ry and rs are the peak area responses of methscopo-
lamine obtained from the Assay preparation and the Stan-
dard preparation, respectively.
Methscopolamine Bromide Tablets
» Methscopolamine Bromide Tablets contain not
less than 93.0 percent and not more than
107.0 percent of the labeled amount of methsco-
polamine bromide (CigH24BrNO,).
Packaging and storage—Preserve in tight containers, and
store at controlled room temperature.
USP Reference standards (11)—
USP Methscopolamine Bromide RS
Identification—
A: Thin-Layer Chromatographic Identification Test (201 )—
pH 7.3 Dye-buffer solution—Prepare a solution containing,
in each 500 mL, 200 mg of bromothymol blue, 3.2 mL of
0.1 N sodium hydroxide, 577.5 mg of citric acid monohy-
drate, and 6.3 mg of anhydrous dibasic sodium phosphate.
Test solution—Finely powder 1 Tablet, and transfer an
amount, equivalent to about 0.5 mg of methscopolamine
bromide, to a suitable container. Add 20 mL of water, heat
ad
Amy for 5 minutes on a steam bath with frequent agitation, and
rm
i}
centrifuge to obtain a clear supernatant. Transfer 10 mL of
the supernatant to a vessel containing 10 mL of chloroform
=)
-
and 10 mL of pH 7.3 Dye-buffer solution. Shake vigorously
i)
tm5 for 3 minutes, centrifuge, and transfer 8 mL of the chloro-
form layer to a suitable container. Evaporate to dryness, and
= dissolve the residue in 1 mL of chloroform.
3 Standard solution—Prepare a solution in water containing
a) about 0.025 mg of USP Methscopolamine Bromide RS per
=) mL, and treat as directed above, beginning with “Transfer
10 mL of the supernatant.”
Application volume: 50 wL.
Developing solvent system—tn a suitable container, mix
water, butyl alcohol, and glacial acetic acid (5:4:1), then
transfer a measured volume of the upper organic layer to a
suitable container, and mix with a volume of alcohol equiva-
lent to 20% of the volume of the organic layer.
Procedure—Allow the solvent front to move about three-
fourths of the length of the plate, remove the plate from
the developing chamber, mark the solvent front, and dry
the plate under a current of air for 30 minutes. Spray the
plate evenly with potassium-bismuth iodide TS: the chro-
matogram of the Test solution shows a bright orange spot
on a yellow background corresponding in Rr value (about
0.25) to that in the chromatogram obtained from the Stan-
dard solution. [Note—Bromothymol blue produces a dark
yellow spot at an Ry value of about 0.8.]
B: Powder a number of Tablets, equivalent to about
5 mg of methscopolamine bromide, digest with 5 mL of
water for 10 minutes, and filter: a portion of the clear solu-
tion so obtained responds to the test for Bromide (191).
USP 41
Dissolution (711)—
Medium: 0.1 N hydrochloric acid; 500 mL.
Apparatus 2: 50 rpm.
Time: 30 minutes.
Determine the percentage of the labeled amount of
methscopolamine bromide dissolved using the following
method.
pH 3.0 Phosphate buffer—Dissolve 5.44 g of monobasic
potassium phosphate in 1 L of water. Adjust with 1 N phos-
phoric acid to a pH of 3.0.
Mobile phase—Preparea filtered and degassed mixture of
pH 3.0 Phosphate buffer and methanol (3:1). Make adjust-
ments if necessary (see System Suitability under Chromatog-
raphy (621)).
Standard solution—Dissolve an accurately weighed quan-
tity of USP Methscopolamine Bromide RS in Medium, and
dilute quantitatively, and stepwise if necessary, with Medium
to obtain a solution having a known concentration similar to
the one expected in the Test solution.
Test solution—Use portions of the solution under test that
have been passed through a 0.45-um PTFE filter.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 204-nm detector
and a 4.6-mm x 15-cm column that contains packing L1.
The flow rate is about 1.0 mL per minute. The column tem-
perature is maintained at 30°. Chromatograph the Standard
solution, and record the peak responses as directed for Pro-
cedure: the tailing factor is not more than 2.0; and the rela-
tive standard deviation for replicate injections is not more
than 2.0%.
Procedure—Separately inject equal volumes (about 25 wL)
of the Standard solution and the Test solution into the chro-
matograph, record the chromatograms, and measure the re-
sponses for the major peaks. Calculate the percentage of
methscopolamine bromide dissolved by the formula:
ty x Cy x 500 x 100
Roe hG
in which ry and rs are the peak responses obtained from the
Test solution and the Standard solution, respectively; Cs is the
concentration, in mg per mL, of USP Methscopolamine Bro-
mide RS in the Standard solution; 500 is the volume, in mL,
of Medium; 100 is the factor for conversion to percentage;
and LC is the tablet label claim, in mg.
Tolerances—Not less than 80% (Q) of the labeled amount
of CigH24BrNO, is dissolved in 30 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Assay—
Mobile phase—Prepare a solution containing 2.6 g of de-
cyl sodium sulfate in 450 mL of water. Add 550 mL of meth-
anol, adjust with 1 N sulfuric acid to a pH of 3.5, mix, filter,
and degas.
Standard preparation—Transfer about 25 mg of USP
Methscopolamine Bromide RS, accurately weighed, to a
100-mL volumetric flask, dissolve in and dilute with Mobile
phase to volume, and mix.
Assay preparation—
FOR TABLETS THAT CONTAIN 2.5 MG OF METHSCOPOLAMINE BRO-
MIDE—Place 10 Tablets in a 100-mL volumetric flask, add
about 50 mL of Mobile phase, and sonicate for 30 minutes.
Shake by mechanical means for 30 minutes, dilute with Mo-
bile phase to volume, and mix. Pass a portion through a
0.45-1um PTFE filter, discarding the first 2 to 3 mL of the
filtrate.
FOR TABLETS THAT CONTAIN 5 MG OF METHSCOPOLAMINE BRO-
MIDE—Place 10 Tablets in a 200-mL volumetric flask, add
about 100 mL of Mobile phase, and sonicate for 30 minutes.
Shake by mechanical means for 30 minutes, dilute with Mo-
USP 41 Official Monographs / Methsuximide 2659

bile phase to volume, and mix. Pass a portion through a Mode: LC

0.45-um PTFE filter, discarding the first 2 to 3 mL of the Detector: UV 254 nm
filtrate. Column: 3.9-mm x 30-cm; 10-4m packing L1
Chromatographic system (see Chromatography (621))—The Flow rate: 1 mL/min
liquid chromatograph is equipped with a 254-nm detector Injection volume: 20 nL
and a 4.6-mm x 25-cm column that contains packing L1. System suitability
Chromatograph the Standard preparation, and record the Sample: Standard solution
peak responses as directed for Procedure: the relative stan- Suitability requirements
dard deviation for replicate injections is not more than Column efficiency: NLT 5800 theoretical plates
2.0%. Tailing factor: NMT 1.3
Relative standard deviation: NMT 0.6%
Procedure—Separately inject a volume (about 50 LL) of
the Standard preparation and the Assay preparation into the Analysis
Samples: Standard solution and Sample solution
chromatograph, record the chromatogram, and measure Calculate the percentage of methsuximide (Ci2Hi3NO2)
the peak responses. Calculate the quantity, in mg, of meth- in the portion of Methsuximide taken:
scopolamine bromide (CisH24BrNOxz) in the portion of Tab-
lets taken by the formula: Result = (ru/rs) x (Cs/Cu) x 100
100C(ru
/ rs) tu = peak response from the Sample solution
rs = peak response from the Standard solution
in which C is the concentration, in mg per mL, of USP Cs = concentration of USP Methsuximide RS in the
Methscopolamine Bromide RS in the Standard preparation; Standard solution (mg/mL)
and ry and rs are the peak responses of the methscopo- Cu = concentration of Methsuximide in the Sample
lamine bromide obtained from the Assay preparation and solution (mg/mL)
the Standard preparation, respectively. Acceptance criteria: 97.0%-103.0% on the dried basis
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.2%
© ORGANIC IMPURITIES
Methsuximide Mobile phase and Chromatographic system: Proceed
as directed in the Assay.
System suitability solution: 0.6 mg/mL of Methsux-
imide in Mobile phase
Standard solution: 0.006 mg/mL of USP Methsuximide
a

RS in Mobile phase
Sample solution: 6.0 mg/mL of Methsuximide in Mo-
bile phase
Ci2Hi3NO2 203.24 System suitability
2,5-Pyrrolidinedione, 1,3-dimethyl-3-phenyl-, (+)-; Sample: System suitability solution =
(+)-N,2-Dimethyl-2-phenylsuccinimide [77-41-8]. Suitability requirements a)
Column efficiency: NTL 5800 theoretical plates <<
DEFINITION Tailing factor: NMT 1.3 ro)
Methsuximide contains NLT 97.0% and NMT 103.0% of Relative standard deviation: NMT 0.6% |
methsuximide (C;2Hi3NO2), calculated on the dried basis. Analysis S
IDENTIFICATION Samples: Standard solution and Sample solution =
e A. INFRARED ABSORPTION (197K) Calculate the percentage of each impurity in the por- a
e B. The retention time of the major peak of the Sample tion of Methsuximide taken: >
solution corresponds to that of the Standard solution, as Result = (ru/rs) x (Cs/Cu) x 100
obtained in the Assay.
ASSAY ru = peak response for each impurity from the
e PROCEDURE Sample solution
Mobile phase: Acetonitrile and water (9:11) rs = peak response for methsuximide from the
Standard solution: 0.6 mg/mL of USP Methsuximide Standard solution
RS in Mobile phase Cs = concentration of USP Methsuximide RS in the
Sample solution: 0.6 mg/mL of Methsuximide in Mo- Standard solution (mg/mL)
bile phase Cu = concentration of Methsuximide in the Sample
Chromatographic system solution (mg/mL)
(See Chromatography (621), System Suitability.) Acceptance criteria
Any individual impurity: NMT 0.1%
Total impurities: NMT 2.0%
SPECIFIC TESTS
e Loss ON DRYING (731)
Avalysis: Dry a sample over phosphorus pentoxide for
16h.
 2660 Methsuximide / Official Monographs USP 41

Acceptance criteria: NMT 0.5% Acceptance criteria: 92.0%-108.0%

ADDITIONAL REQUIREMENTS PERFORMANCE TESTS
¢ PACKAGING AND STORAGE: Preserve in tight containers. e DISSOLUTION (711)
e USP REFERENCE STANDARDS (11) Medium: Water; 900 mL
USP Methsuximide RS Apparatus 1: 100 rpm
Time: 120 min
Analysis: Determine the percentage of the labeled
amount of methsuximide (Ci2H13NOz) dissolved by us-
ing the procedure set forth in the Assay, making any
Methsuximide Capsules necessary adjustments.
Tolerances: NLT 75% (Q) of the labeled amount of
DEFINITION methsuximide (C12H;3NOz) is dissolved.
Methsuximide Capsules contain NLT 92.0% and NMT ¢ UNIFORMITY OF DosAGE UNITS (905): Meet the
108.0% of the labeled amount of methsuximide requirements
(Cy2Hi3NOz). ADDITIONAL REQUIREMENTS :
IDENTIFICATION e PACKAGING AND STORAGE: Preserve in tight containers,
e A. INFRARED ABSORPTION (197K) and avoid exposure to excessive heat.
Sample: Mix a portion of the content of Capsules, e USP REFERENCE STANDARDS (11)
equivalent to 200 mg of methsuximide with 25 mL of USP Methsuximide RS
water in a separator, extract with 50 mL of ether, and
discard the aqueous layer. Wash the ether extract with
25 mL of water, and discard the water. Filter the ex-
tract, evaporate with the aid of a current of warm air to
dryness, and dry the methsuximide over phosphorus Methyclothiazide
pentoxide for 16 h.
Acceptance criteria: Meet the requirements °. o a0
we Ww
e B. The retention time of the major peak of the Sample eC
solution corresponds to that of the Standard solution, as
obtained in the Assay. cr yon

ASSAY CoH11ClaN30482 360.24

¢ PROCEDURE 2H-1,2,4-Benzothiadiazine-7-sulfonamide, 6-chloro-
Mobile phase: Acetonitrile and water (45:55). Filter, 3-(chloromethyl)-3,4-dihydro-2-methyl-, 1,1-dioxide, (+)-.
and degas. (+)-6-Chloro-3-(chloromethyl)-3,4-dihydro-2-methyl-2H-1,2,
Standard solution: 0.6 mg/mL of USP Methsuximide 4-benzothiadiazine-7-sulfonamide 1,1-dioxide
RS in Mobile phase
aS
“
Sample stock solution: Place 10 Capsules in a 500-mL [135-07-9].
% volumetric flask, and add 280 mL of water. Sonicate in
i] » Methyclothiazide contains not less than
-
a water bath at 40°-50°, with occasional shaking, until
aD 97.0 percent and not more than 102.0 percent of
° the Capsules have broken, and cool to room tempera-
CoH11Cl2N3O4S2, calculated on the dried basis.
i—j ture. Dilute with acetonitrile to volume, mix, and filter.
iS Sample solution: Nominally 0.6 mg/mL of methsux-
= innigie prepared from Sample stock solution in Mobile Packaging and storage—Preserve in well-closed contain-
3 ase ers.
a) hromatographic system USP Reference standards (11)—
= (See Chromatography (621), System Suitability.) USP Methyclothiazide RS
Mode: LC USP Methyclothiazide Related Compound A RS
Detector: UV 254 nm 4-Amino-6-chloro-N3-methyl-m-benzenedisulfonamide.
Column: 3.9-mm x 30-cm; packing L1 C7Hi0CIN3O4S2 299.76
Flow rate: 1 mL/min Identification—
Injection volume: 20 pL A: Infrared Absorption (197K).
System suitability
Sample: Standard solution B: Ultraviolet Absorption (197U)—
Suitability requirements Solution: 20g per mL.
Column efficien NLT 2100 theoretical plates Medium: methanol.
Relative standard deviation: NMT 1.5% C: Fuse about 100 mg of it with a pellet of sodium hy-
Analysis droxide: the ammonia fumes produced turn moistened red
Samples: Standard solution and Sample solution litmus paper blue. The fused mixture responds to the test
Calculate the percentage of the labeled amount of for Sulfite (191).
methsuximide (Ci2Hi3NOz) in the portion of Capsules Loss on drying (731)—Dry it at 105° for 4 hours: it loses
taken: not more than 0.5% of its weight.
Result = (ru/rs) x (Cs/Cu) x 100 Residue on ignition (281): not more than 0.2%.
Chloride (221)—Shake 750 mg with 25 mL of water for
tu = peak response of methsuximide from the 2 minutes, filter through a suitable membrane filter, and
Sample solution add 4 or 5 drops of 2N nitric acid: the acidified filtrate
rs = peak response of methsuximide from the shows no more chloride than corresponds to 0.20 mL of
Standard solution 0.020 N hydrochloric acid (0.02%).
Cs = concentration of USP Methsuximide RS in the
Standard solution (mg/mL)
Cu = nominal concentration of methsuximide in the
Sample solution (mg/mL)
USP 41
Selenium (291): 0.003%.
Delete the following:
°Heavy metals, Method I! (231): 0.002%.
© coiticint 1-jan-2018)
Diazotizable substances—
Standard preparation—Transfer about 10 mg of USP
Methyclothiazide Related CompoundA RS, accurately
weighed, to a 50-mL volumetric flask, dilute with acetoni-
trile to volume, and mix. Pipet 25 mL of the solution into a
100-mL volumetric flask, dilute with acetonitrile to volume,
and mix. Each mL of Standard preparation contains about
50 ug of the Reference Standard.
Test preparation—Transfer about 500 mg of Methyclothia-
zide, accurately weighed, to a 100-mL volumetric flask, dis-
solve in and dilute with acetonitrile to volume, and mix.
Procedure—Pipet 2 mL each of the Standard preparation
and the Test preparation into separate 50-mL volumetric
flasks. Pipet 2 mL of acetonitrile into a third 50-mL flask to
provide the blank. To each flask add 4 mL of 0.1 N hydro-
chloric acid, and mix. Add 3.0 mL of sodium nitrite solution
(1 in 200) to each flask, mix, and place the flasks in an ice
bath for 5 minutes, shaking occasionally. Add to each flask
3.0 mL of ammonium sulfamate solution (1 in 50), mix, and
allow the flasks to remain in the ice bath for 1 additional
minute. Remove the flasks from the ice bath, add 1.0 mL of
N-(1-naphthyl)ethylenediamine dihydrochloride solution (1
in 1000), and mix. Allow the flasks to stand at room tem-
perature for 1 minute, then dilute with water to volume,
and mix. Concomitantly determine the absorbances of the
solutions obtained from the Standard preparation and the
Test preparation in 1-cm cells at 525 nm, with a suitable
spectrophotometer, using the reagent blank to set the in-
strument. The absorbance of the solution from the Test
preparation does not exceed that of the solution from the
Standard preparation, corresponding to not more than 1.0%
of diazotizable substances.
Assay—Transfer about 350 mg of ieee accu-
rately weighed, to a 250-mL conical flask, add 40 mL of a 1
in 20 solution of potassium hydroxide in methanol, and re-
flux at full boil for 1 hour. Cool, rinse the inner walls of the
condenser with 20 mL of water and two 20-mL portions of
methanol, add 10 mL of glacial acetic acid and 2 drops of
eosin Y TS, and titrate with 0.1 N silver nitrate VS to the
first appearance of a definite pink color. Each mL of 0.1 N
silver nitrate is equivalent to 36.02 mg of CoHi1Cl2N304S2.
Methyclothiazide Tablets
» Methyclothiazide Tablets contain not less than
90.0 percent and not more than 110.0 percent of
the labeled amount of methyclothiazide
(CoH11Cl2N304S2).
Packaging and storage—Preserve in well-closed contain-
ers.
USP Reference standards (11)—
USP Methyclothiazide RS
Identification, Ultraviolet Absorption (197U)—
Solution—Powder a number of Tablets, equivalent to
about 50 mg of methyclothiazide, and transfer to a 100-mL
volumetric flask with the aid of methanol. Add about 60 mL
of methanol, and shake the flask for 1 hour. Dilute with
methanol to volume, mix, and centrifuge a portion of the
solution. Pipet 2 mL of the clear supernatant into a second
100-mL volumetric flask, dilute with methanol to volume,
and mix: the UV absorption spectrum of this solution exhib-
Official Monographs / Methyclothiazide 2661
its maxima and minima only at the same wavelengths as
that of a similar solution of USP Methyclothiazide RS.
Dissolution (711)—
Medium: 0.01 N hydrochloric acid; 900 mL.
Apparatus 2: 50 rpm.
Time: 60 minutes.
Procedure—Determine the amount of C9H1;Cl2N3O4S2 dis-
solved by enbleying UV absorption at the wavelength of
maximum absorbance at about 270 nm on filtered portions
of the solution under test, suitably diluted with Dissolution
Medium, if necessary, in comparison with a Standard solu-
tion having a known concentration of USP Methyclothiazide
RS in the same Medium. An amount of alcohol not to ex-
ceed 1% of the total volume of the Standard solution may
be used to dissolve USP Methyclothiazide RS prior to dilu-
tion with Dissolution Medium.
Tolerances—Not less than 70% (Q) of the labeled amount
of CoH11ClzN30.S2 is dissolved in 60 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Procedure for content uniformity—Transfer 1 finely pow-
dered Tablet to a 50-mL volumetric flask, add about 30 mL
of methanol, and shake by mechanical means for 1 hour.
Dilute with methanol to volume, mix, and centrifuge a por-
tion of the mixture. Dilute quantitatively with methanol to
obtain a solution containing approximately 10 ug per mL of
methyclothiazide. Concomitantly determine the absorbances
of this solution and a Standard solution of USP
Methyclothiazide RS in the same medium, having a known
concentration of about 10 wg per mL, in 1-cm cells at the
wavelength of maximum absorbance at about 267 nm, with
a suitable spectrophotometer, using methanol as the blank.
Calculate the quantity, in mg, of CsHi1Cl2N3O4S2 in the Tab-
let taken by the formula:
(TC/ D)(Au/ As) oy
4)
in which Tis the labeled quantity, in mg, of methyclothia- z
zide in the Tablet; C is the concentration, in ug per mL, of c
USP Methyclothiazide RS in the Standard solution; D is the °
concentration, in ug per mL, of methyclothiazide in the so- =
re)
lution from the Tablet, based upon the labeled quantity per @I
Tablet and the extent of dilution; and Ay and As are the
i
absorbances of the solution from the Tablet and the Stan- a}
dard solution, respectively. =
al
Assay—
Standard preparation—Transfer about 20 mg of USP
Methyclothiazide RS, accurately weighed, to a 100-mL volu-
metric flask, add methanol to volume, and mix. Transfer
10.0 mL of this solution to a 200-mL volumetric flask, add
chloroform to volume, and mix.
Assay preparation—Weigh and finely powder not fewer
than 20 Tablets. Transfer an accurately weighed portion of
the powder, equivalent to about 2 mg of methyclothiazide,
to a 150-mL beaker, add 2.0 mL of methanol, mix, allow
the mixture to stand for 30 minutes while taking precau-
tions against loss of solvent, add 2.0 mL of 0.1 M sodium
bicarbonate, and mix.
Procedure—{NOTE—Use water-saturated solvents through-
out this procedure.] Mix about 3 g of chromatographic sili-
ceous earth with 2.0 mL of 0.1 M sodium bicarbonate in a
150-mL beaker. Pack the mixture into a 25- x 200-mm
chromatographic column. Add 4 g of chromatographic sili-
ceous earth to the Assay preparation, mix, transfer the mix-
ture to the column, and pack. Dry-wash the beaker with 1 g
of the siliceous earth mixed with 3 drops of water, and
transfer to the column. Place a small pad of glass wool
above the column packing, pass 75 mL of a mixture of iso-
octane and ether (9:1) through the column, and discard the
eluate. Using a 200-mL volumetric flask as a receiver, pass
100 mL of chloroform through the column, wash the tip of
column with ether, add 10.0 mL of methanol, dilute with
 2662 Methyclothiazide / Official Monographs USP 41

chloroform to volume, and mix. Concomitantly determine System suitability

the absorbances of this solution and the Standard prepara- Sample: System suitability solution
tion at the wavelength of maximum absorbance at about [Note—The relative retention times for benzethonium
268 nm, with a suitable spectrophotometer, using chloro- and methylbenzethonium are 0.7 and 1.0,
form as the blank. Calculate the quantity, in mg, of respectively]
methyclothiazide (CyH1:ClzN3O.S2) in the portion of Tablets Suitability requirements
taken by the formula: Resolution: NLT 5.0 between the benzethonium and
methylbenzethonium peaks
0.2C(Au /As) Taling factor: NMT 1.8 for the methylbenzethonium
ea
in which C is the concentration, in ug per mL, of USP Relative standard deviation: NMT 0.5% for the
Methyclothiazide RS in the Standard preparation; and Ay and methylbenzethonium peak
As are the absorbances of the solution from the Assay prepa- Analysis
ration and the Standard preparation, respectively. Samples: Standard solution and Sample solution
Calculate the percentage of methylbenzethonium chlo-
ride (C2gH44CINOz- H2O) in the portion of
Methylbenzethonium Chloride taken:
Methylbenzethonium Chloride Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response of methylbenzethonium from
Hac, y the Sample solution
Ct |
Hee
Ay e ca + H:O
rs = peak response of methylbenzethonium from
the Standard solution
x CH, Cs = concentration of USP Methylbenzethonium
Chloride RS in the Standard solution (mg/mL)
Cu = concentration of Methylbenzethonium
CogHaaCINOz - H20 480.12 Chloride in the Sample solution (mg/mL)
CosH4aCINO2 462.12 Acceptance criteria: 97.0%-103.0% on the dried basis
Benzenemethanaminium, N,N-dimethyl-N-[2-[2-[methyl- IMPURITIES
4-(1,1,3,3-tetramethylbutyl)phenoxy]ethoxy]ethyl]-, chlo- © RESIDUE ON IGNITION (281): NMT 0.1%
ride, acaey
Benzyldimethyl[2-[2-[[4-(1,1,3, 3-tetramethylbutyl)tolyl]- SPECIFIC TESTS
oxy]ethoxyjethyllammonium chloride monohydrate e MELTING RANGE OR TEMPERATURE (741): 159°-163°, the
[1320-44-1]. specimen having been previously dried
Anhydrous [25155-18-4]. e Loss ON DRYING 2731): Dry a sample at 105° for 4 h: it
loses NMT 5.0% of its weight.
DEFINITION
<
”

rs Methylbenzethonium Chloride contains NLT 97.0% and ADDITIONAL REQUIREMENTS

ic] NMT 103.0% of C2gH44CINOz, calculated on the dried e PACKAGING AND STORAGE: Preserve in tight containers.
ia) basis.
—
e USP REFERENCE STANDARDS (11)
) IDENTIFICATION
USP Benzethonium Chloride RS
c
GC e A. PROCEDURE
USP Methylbenzethonium Chloride RS
= Analysis: To 1 mL of solution (1 in 100) add 2 mL of
a alcohol, 0.5 mL of 2.N nitric acid, and 1 mL of silver
” nitrate TS.
=) Acceptance criteria: A white precipitate, which is insol-
uble in 2.N nitric acid and soluble in 6 N ammonium Methylbenzethonium Chloride Lotion
hydroxide, is formed.
e B. INFRARED ABSORPTION (197K) DEFINITION
Methylbenzethonium Chloride Lotion is an emulsion con-
ASSAY taining NLT 90.0% and NMT 110.0% of the labeled
e PROCEDURE amount of methylbenzethonium chloride (C2sH44CINOz -
Buffer: Dilute 20 mL of triethylamine with water to H20).
1000 mL, and adjust the pH to 3.0 with phosphoric
acid. IDENTIFICATION
Mobile phase: Acetonitrile and Buffer (45:55) ° A.
Standard solution: 0.15 mg/mL of USP Sample: Suspend 0.5 mL of methylbenzethonium chlo-
Methylbenzethonium Chloride RS in Mobile phase ride lotion in 20 mL of water.
System suitability solution: 0.15 mg/mL each of USP Analysis: Add 0.1 g of sodium carbonate, 1 mL of bro-
Benzethonium Chloride RS and USP mophenol blue TS, and 10 mL of chloroform to the
Methylbenzethonium Chloride RS in Mobile phase Sample, and shake the mixture.
Sample solution: 0.15 mg/mL of methylbenzethonium Acceptance criteria: The chloroform layer is blue.
chloride in Mobile phase
Chromatographic system ASSAY
(See Chromatography (621), System Suitability.) e PROCEDURE
Mode: LC Standard stock solution: 4.446 mg/mL of USP Docu-
Detector: UV 225 nm sate Sodium RS in isopropyl alcohol (equivalent to 0.01
Column: 4.6-mm x 15-cm; 5-um packing L7 M of USP Docusate Sodium RS). Store this solution in a
Column temperature: 40° tightly-stoppered glass container. Prepare the Standard
Flow rate: 1 mL/min solution on the day of use.
Injection size: 10 uL Standard solution 44.46 i1g/mL of USP Docusate So-
Run time: 1.5 times the retention time of the dium RS in water from the Standard stock solution
methylbenzethonium peak (equivalent to 0.1 mM of USP Docusate Sodium RS)
USP 41
Sample solution: Transfer an amount of Lotion, equiva-
lent to 0.5 mg of methylbenzethonium chloride, to a
glass-stoppered, 50-mL cylinder. Add 5 mL of chloro-
form (fres| by purified by shaking 100 mL with 10 g of
silica gel, allowing to settle, and withdrawing the super-
natant), 5 mL of phosphoric acid solution (1 in 10), and
1 mL of 0.05 mg/mL of safranin O solution.
Analysis: Titrate the Sample solution with the Standard
Solution until 1 mL from the endpoint, then shake the
stoppered tube vigorously for about 2 min, and con-
tinue the titration in 0.1-mL increments, shaking vigor-
ously after each addition, until a pink color appears in
the chloroform layer. Perform a blank determination,
and make any necessary correction (see Titrimetry
(541)). Each mL of Standard solution is equivalent to
48.01 wg of methylbenzethonium chloride
(CasH44CINOz - H20).
Acceptance criteria: 90.0%-110.0%
SPECIFIC TESTS
° PH (791): 5.2-6.0
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers.
e USP REFERENCE STANDARDS (11)
USP Docusate Sodium RS
Methylbenzethonium Chlorid
Ointment : Acceptance criteria: The chloroform layer is blue.
DEFINITION
Methylbenzethonium Chloride Ointment contains NLT
90.0% and NMT 110.0% of the labeled amount of
methylbenzethonium chloride (C2sH44CINOz - H20).
IDENTIFICATION
oA.
Sample: Suspend 0.5 g of methylbenzethonium chlo-
ride ointment in 10 mL of water.
Analysis: Add 0.1 g of sodium carbonate, 1 mL of bro-
mophenol blue TS, and 10 mL of chloroform to the
Sample, and shake the mixture.
Acceptance criteria: The chloroform layer is blue.
ASSAY
© PROCEDURE
Standard stock solution: 4.446 mg/mL of USP Docu-
sate Sodium RS in isopropyl alcohol (equivalent to 0.01
M of USP Docusate Sodium RS). Store this solution in a
tightly-stoppered glass container. Prepare the Standard
Solution on the day of use.
Standard solution 44.46 g/mL of USP Docusate So-
dium RS in water from the Standard stock solution
(equivalent to 0.1 mM of USP Docusate Sodium RS)
Sample solution: Transfer an amount of Ointment,
equivalent to 0.5 mg of methylbenzethonium chloride,
to a glass-stoppered, 50-mL cylinder. Add 5 mL of chlo-
roform (freshly purified by shaking 100 mL with 10 g of
silica gel, allowing to settle, and withdrawing the super-
natant), 5 mL of phosphoric acid solution (1 in 10), and
1 mL of 0.05 mg/mL of safranin O solution.
Analysis: Titrate the Sample solution with the Standard
solution until 1 mL from the endpoint, then shake the
stoppered tube vigorously for about 2 min, and con-
tinue the titration in 0.1-mL increments, shaking vigor-
ously after each addition, until a pink color appears in
the chloroform layer. Perform a blank determination,
and make any necessary correction (see Titrimetry
(541)). Each mL of Standard solution is equivalent to
48.01 ug of methylbenzethonium chloride
(CagH4aCINO2+ H20).
Official Monographs / Methylbenzethonium 2663
Acceptance criteria: 90.0%-110.0%
SPECIFIC TESTS
e PH (791): 5.0-7.0, in a dispersion of 10 mg/mL of the
sample in carbon dioxide-free water
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in collapsible tubes or
in tight containers.
e USP REFERENCE STANDARDS (11)
USP Docusate Sodium RS
Methylbenzethonium Chloride Topical
Powder
DEFINITION
Methylbenzethonium Chloride Topical Powder contains NLT
85.0% and NMT 115.0% of the labeled amount of
methylbenzethonium chloride (C2sH44CINOz - H2O) in a
suitable fine powder base, free from grittiness.
IDENTIFICATION
° A.
Sample: Suspend 0.1 g of methylbenzethonium chlo-
ride topical powder in 10 mL of water.
Analysis: Add 0.1 g of sodium carbonate, 1 mL of bro-
mophenol blue TS, and 10 mL of chloroform to the
Sample, and shake the mixture.
ASSAY
© PROCEDURE
Standard stock solution: 4.446 mg/mL of USP Docu-
sate Sodium RS in isopropyl alcohol (equivalent to 0.01
M of USP Docusate Sodium RS). Store this solution in a SS
tightly-stoppered glass container. Prepare the Standard A
solution on the day of use. uv
Standard solution: 44.46 t1g/mL of USP Docusate So- =
dium RS in water from the Standard stock solution °
(equivalent to 0.1 mM of USP Docusate Sodium RS) J
fo)
Sample solution: Transfer an amount of Topical Pow- Ko}
der, equivalent to 0.5 mg of methylbenzethonium chlo- Ba]
s
ride, to a glass-stoppered, 50-mL cylinder. Add 5 mL of mo]
chloroform (freshly purified by shaking 100 mL with =>
a
10g of silica gel, allowing to settle, and withdrawini
the supernatant), 5 mL of phosphoric acid solution cl in
10), and 1 mL of 0.05 mg/mL of safranin O solution.
Analysis: Titrate the Sample solution with the Standard
solution until 1 mL from the endpoint, then shake the
stoppered tube vigorously for about 2 min, and con-
tinue the titration in 0.1-mL increments, shaking vigor-
ously after each addition, until a pink color appears in
the chloroform layer. Perform a blank determination,
and make any necessary correction (see Titrimetry
(541)). Each mL of Standard solution is equivalent to
48.01 ug of methylbenzethonium chloride
(CasHaaCINOz - H20).
Acceptance criteria: 85.0%-115.0%
SPECIFIC TESTS
e PH (791): 9.0-10.5, in a dispersion of 10 mg/mL of the
sample in carbon dioxide-free water
© POWDER FINENESS (811): NLT 99% of it passes through a
No. 200 sieve.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed
containers.
 2664 Methylbenzethonium / Official Monographs
e USP REFERENCE STANDARDS (11)
USP Docusate Sodium RS
Methylcellulose
Portions of the monograph text that are national USP text,
and are not part of the harmonized text, are marked with
symbols (*¢) to specify this fact.
Cellulose, methyl ether;
Cellulose methyl ether [9004-67-5].
DEFINITION
Methylcellulose is a methyl ether of cellulose. When dried at
105° for 1 h, it contains NLT 26.0% and NMT 33.0% of
methoxy (-OCHs3) groups.
IDENTIFICATION
eA.
Sample: 1g
Analysis: Evenly distribute the Sample onto the surface
of 100 mL of water in a beaker, tapping the top of the
beaker gently, if necessary, to ensure a uniform layer on
the surface, and allow to stand for 1-2 min.
Acceptance criteria: The powdered material aggregates
on the surface.
° B.
Sample: 1g
Analysis: Evenly distribute the Sample into 100 mL of
boiling water and stir the mixture using a magnetic stir-
rer with a 25-mm long bar: a slurry is formed and the
particles do not dissolve. Allow the slurry to cool to 5°
and stir using a magnetic stirrer.
Acceptance criteria: A clear or slightly turbid solution
occurs with its thickness dependent on the viscosity
grade.
fe
”
°C
ry Solution A: Sulfuric acid and water (9 in 10). [NoTE—
i]
= Carefully add the sulfuric acid to the water.]
D contents of the vial continuously for 60 min while heat-
° Sample solution: 0.1 mL of the solution prepared for
c Identification test B
iS Analysis: To the Sample solution add 9 mL of Solution A
= shake. Heat in a water bath for exactly 3 min, im-
[5 mediately cool in an ice bath, and add carefully 0.6 mL
” of ninhydrin TS. Shake and allow to stand at 25°.
=) Acceptance criteria: A red color develops immediately
and it does not change to purple within 100 min.
e D.
Sample solution: 2-3 mL of the solution prepared for
Identification test B
Analysis: Pour the Sample solution onto a glass slide as
a thin film and allow the water to evaporate.
Acceptance criteria: A coherent, clear film forms on
the glass slide.
o E.
Sample solution: 50 mL of the solution prepared in
Identification test B
Analysis: Add the Sample solution to exactly 50 mL of
water in a beaker. Insert a thermometer into the solu-
tion. Stir the solution on a magnetic stirrer/hot plate,
and begin heating at a rate of 2°-5°/min. Determine
the temperature at which a turbidity increase begins to
occur and designate this temperature as the flocculation
temperature.
Acceptance criteria: The flocculation temperature is
higher than 50°.
ASSAY
e PROCEDURE
[CauTion—Perform all steps involving Hydriodic acid care-
fully, in a well-ventilated hood. Use goggles, acid-resistant
gloves, and other appropriate safety equipment. Be ex-
ceedingly careful when handling the hot vials because
USP 41
they are under pressure. In the event of hydriodic expo-
sure, wash with copious amounts of water, and seek med-
ical attention at once.]
Apparatus
Reaction vial: A 5-mL pressure-tight serum vial,
20 mm in outside diameter, 50 mm in height, and
20 mm in outside diameter and 13 mm in inside diam-
eter at the mouth, equipped with a pressure-tight sep-
tum having a polytetrafluoroethylene-faced butyl rub-
ber and an airtight seal using an aluminum crimp or
any sealing system that provides sufficient airtightness
Heater: A heating module with a square-shaped alu-
minum block having holes 20 mm in diameter and
32 mm in depth, so that the reaction vial fits. The
heating module is also equipped with a magnetic stir-
rer capable of mixing the contents of the vial, or a
reciprocal shaker that performs a reciprocating motion
approximately 100 times/min can be used.
Hydriodic acid: Use a reagent having aspa tie gravity
of at least 1.69, equivalent to 55%-57% hydrogen io-
dide (HI).
Internal standard solution: 30 mg/mL of n-octane in
o-xylene
Standard solution: Into a suitable serum vial weigh
60-100 mg of adipic acid, add 2.0 mL of Hydriodic acid,
and then pipet 2.0 mL of the Internal standard solution
into the vial. Close the vial securely with a suitable sep-
tum stopper. Weigh the vial and contents, add 45 uL of
methyl iodide with a syringe through the septum,
weigh again, and calculate the weight of methyl iodide
added, by difference. Shake, and allow the layers to
separate. Use the upper layer as the Standard solution.
Sample solution: Transfer 0.065 g of Methylcellulose to
a 5-mL thick-walled reaction vial equipped with a pres-
sure-tight septum closure, add 60-100 mg of adipic
acid, and pipet 2.0 mL of the Internal standard solution
into the vial. Cautiously pipet 2.0 mL of Hydriodic acid
into the mixture, immediately secure the closure, and
weigh accurately. Using the magnetic stirrer from the
heating module, or using a reciprocal shaker, mix the
ing the block so that the temperature of the contents is
maintained at 130 + 2°. If a reciprocal shaker or mag-
netic stirrer cannot be used, shake the vial well by hand
at 5-min intervals during the initial 30 min of the heat-
ing time. Allow the vial to cool, and weigh again. If the
weight loss is less than 0.50% of the contents and there
is no evidence of a leak, use the upper layer of the
mixture as the Sample solution.
Chromatographic system
Mode: GC
Detector: Thermal conductivity or hydrogen flame
ionization
Column: 3- to 4-mm x 1.8- to 3-m; packed with
10%-20% liquid phase G1, 125-150 um in diameter
on 100- to 120-mesh support S1A. [NoTE—Use a col-
umn giving well-resolved peaks of methyl iodide and
the internal standard, in that order.]
Column temperature: 100°
Carrier gas: Helium for the thermal conductivity de-
tector, and helium or nitrogen for the hydrogen flame
ionization detector
Flow rate: Adjust so that the retention time of the
internal standard is about 10 min.
Injection volume: 1 or 2 uL
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of methoxy in the portion of
Methylcellulose taken:
Result = X x (Ru/Rs) x (Ws/W)
Xx = ratio of the formula weights of methoxy to
methyl iodide times 100%, 21.864
USP 41 Official Monographs / Methylcellulose 2665

Ry = ratio of the peak area of methyl iodide to that Apparatus: Brookfield type LV model or equivalent.
of the internal standard from the Sample For rotor no., revolution, and calculation multiplier,
solution apply the conditions specified in Table 1.
Rs = ratio of the peak area of methyl iodide to that
of the internal standard from the Standard Table 1
solution
Ws = weight of methyl iodide in the Standard Labeled
solution (mg) Viscosity? Rotor Revolution Calculation
w = weight of Methylcellulose, calculated on the (mPa - s) No. (rpm) Multiplier
dried basis, taken for the Assay (mg) 600 or more and
Acceptance criteria: 26.0%-33.0% calculated on the less than 1400 3 60 20
dried basis 1400 or more and
less than 3500 3 12 100
IMPURITIES
3500 or more and
e RESIDUE ON IGNITION (281): NMT 1.5%
less than 9500 4 60 100
9500 or more and
Delete the following: less than 99,500 4 6 1000
99,500 or more 4 3 2000
°e HEAVY METALS (231), Method III @ The Labeled Viscosity 1s based on the manufacturer's specifications.
Analysis: For the Standard Preparation, add the Stan-
dard Lead Solution before digestion. Omit the Monitor Operation of apparatus: Allow the spindle to rotate
Preparation. for 2 min before taking the measurement. Allow a
Acceptance criteria: NMT 20 ppm; the color of the test rest period of at least 2 min between subsequent
solution is not darker than that of the control solution. measurements. Repeat the operation to rotate the
@ (Official 1-4an-2018) spindle specified above twice, and average the three
readings.
SPECIFIC TESTS Acceptance criteria: 80.0%-120.0% of that stated on
e Loss ON DRYING (731) the label for viscosity types less than 600 mPa -s, and
Analysis: Dry at 105° for 1 h. 75.0%-140.0% of that stated on the label for viscosity
Acceptance criteria: NMT 5.0% types 600 mPa -s or higher
e Viscosity—CAPILLARY METHODS (911) and Viscosity—Ro- e PH (791)
TATIONAL METHODS (912) Analysis: Measure the pH of the solution aS in
[Note—The density is 1.00 g/mL, so there is no necessity the test for Viscosity. Read the indicated pH value after
for determining the density at every measurement in the probe has been immersed for 5+ 0.5 min.
the case of having the confirmation data.] Acceptance criteria: 5.0-8.0
Method 1: This method is applied to samples with a
viscosity of less than 600 mPa-s. Weigh a quantity of ADDITIONAL REQUIREMENTS
Methylcellulose, equivalent to 4.000 g, calculated on e *PACKAGING AND STORAGE: Preserve in well-closed con- =
w
the dried basis, transfer into a wide-mouth bottle, and tainers.» z
add hot water (90°-99°) to obtain the total weight of e LABELING: Label it to indicate its nominal viscosity type
the sample and water of 200.0 g. Cap the bottle and eo, of a solution (1 in 50)] in milli-Pascal seconds =
}
stir by mechanical means at 400 + 50 rpm for 10-20 (mPa -s). =
min until particles are thoroughly dispersed and wetted i}
out. Scrape down the walls of the bottle with a spatula, to)2.
if necessary, to ensure that there is no undissolved ma- ES)
terial on the sides of the bottle. Continue the stirring in i}
a cooling water bath equilibrated at a temperature be-
roe
Methylcellulose Ophthalmic Solution
“
low 5° for another 20-40 min. Adjust the solution
weight, if necessary, to 200.0 g using cold water. Cen-
trifuge the solution, if necessary, to expel any en- » Methylcellulose Ophthalmic Solution is a sterile
trapped air bubbles. Using a spatula, remove any foam, solution of Methylcellulose. It contains not less
if present. Perform the test with this solution at 20 +0. than 85.0 percent and not more than 115.0 per-
1° to obtain the kinematic viscosity (v). Separately, de-
termine the density (p) of the solution, and calculate cent of the labeled amount of methylcellulose. It
the viscosity (n) as n = pv. may contain suitable antimicrobial, buffering,
Method 2: This method is applied to samples with a and stabilizing agents.
viscosity of 600 mPa-s or ight. Weigh a quantity of
Methylcellulose, equivalent to 10.00 g, calculated on Packaging and storage—Preserve in tight containers.
the dried basis, transfer into a wide-mouth bottle, and Identification—
add hot water (90°-99°) to obtain the total weight of A: Heat a few mL of Ophthalmic Solution: the solution
the sample and water of 500.0 g. Capping the bottle, becomes cloudy and a flaky precipitate, which redissolves as
stir by mechanical means at 400 + 50 rpm for 10-20 the solution cools, appears.
min until particles are thoroughly dispersed and wetted B: Pour a few mL of Ophthalmic Solution onto a glass
out. Scrape down the walls of the bottle with a spatula, plate, and allow the water to evaporate: a thin, self-sus-
if necessary, to ensure that there is no undissolved ma- taining film results.
terial on the sides of the bottle. Continue the stirring in
a cooling water bath equilibrated at a temperature be- Sterility Tests (71): meets the requirements.
low 5° for another 20-40 min. Adjust the solution PH (791): between 6.0 and 7.8.
weight, if necessary, to 500.0 g using cold water. Cen- Assay—To boiling flask A, as described under Methoxy De-
trifuge the solution, if necessary, to expel any en- termination (431), pipet a quantity of Ophthalmic Solution,
trapped air bubbles. Using a spatula, remove any foam, equivalent to 50 mg of methylcellulose. Evaporate on a
if present. Determine the viscosity of this solution at steam bath to dryness, cool the flask in an ice bath, add the
20+0.1° using a single-cylinder type rotational specified amount of hydriodic acid, and proceed as directed
viscometer. under Methoxy Determination (431). Each mL of 0.1 N so-
 2666 Methylcellulose / Official Monographs
dium thiosulfate is equivalent to 1.753 mg of methylcel-
lulose.
Methylcellulose Oral Solution
» Methylcellulose Oral Solution is a flavored solu-
tion of Methylcellulose. It contains not less than
85.0 percent and not more than 115.0 percent of
the labeled amount of methylcellulose.
Packaging and storage—Preserve in tight, light-resistant
containers, and avoid exposure to direct sunlight and to ex-
cessive heat. Avoid freezing.
identification—
A: Heat a few mL of Oral Solution: the solution becomes
cloudy anda flaky precipitate, which redissolves as the solu-
tion cools, appears.
B: Pour a few mL of Oral Solution onto a glass plate, and
allow the water to evaporate:a thin, self-sustaining film re-
sults.
Microbial enumeration tests (61) and Tests for speci-
fied microorganisms (62)—Its total aerobic microbial
count does not exceed 100 cfu per mL, and it meets the
requirements of the test for the absence of Escherichia coli.
Alcohol Determination, Method |/ (611): between
3.5% and 6.5% of C2HsOH.
Assay—To boiling flask A, as described under Methoxy De-
termination (431), transfer an accurately measured volume
of Oral Solution, equivalent to 50 mg of methylcellulose.
Evaporate on a steam bath to dryness, cool the flask in an
iceBath, add the specified amount of hydriodic acid, and
proceed as directed under Methoxy Determination (431).
2S
nal
Each mL of 0.1 N sodium thiosulfate is equivalent to
roy 1.753 mg of methylcellulose.
J
— Methyldopa contains NLT 98.0% and NMT 102.0% of
i)
°
3 basis.
Sj
3 Methylcellulose Tablets
re
Al
= » Methylcellulose Tablets contain not less than
90.0 percent and not more than 110.0 percent of
the labeled amount of methylcellulose.
Packaging and storage—Preserve in well-closed contain-
ers.
identification—
A: Gently add about 250 mg of the residue obtained in
the Assay to the top of 25 mL of water in a beaker, and
allow to disperse over the surface, tapping the top of the
container to ensure an even dispersion of the test specimen.
Allow the beaker to stand until the specimen becomes
transparent and mucilaginous (about 5 hours), swirl the
beaker to wet the remaining substance, addastirring bar,
and stir until dissolved: the mixture remains stable when an
equal volume of 1 N sodium hydroxide or 1 N hydrochloric
acid is added.
B: Heat a few mL of the solution prepared for /Identifica-
tion test A: the solution becomes cloudy, anda flaky precipi-
tate, which redissolves as the solution cools, appears.
C: Pour a few mL of the solution prepared for Identifica-
tion test A onto a glass plate, and allow the water to evapo-
rate: a thin, self-sustaining film results.
Disintegration (701): 30 minutes.
Uniformity of dosage units (905): meet the require-
ments.
USP 41
fissay—Weigh and finely powder not less than 20 Tablets.
Weigh accurately a portion of the powder, equivalent to
about 500 mg of methylcellulose, and transfer to a tared,
fine fritted-glass, low-form, 30-mL crucible having a fitted
crucible lid. Add 20 mL of alcohol, and macerate the solid
for about 5 minutes, mixing intermittently with a glass stir-
ring rod. Repeat the extraction with ten consecutive 10-mL
portions of alcohol. Test for completeness of extraction b
evaporating the last alcohol extract on a steam bath to dry-
ness, taking up the residue in about 1 mL of water, and
adding this to 5 mL of hot alkaline cupric tartrate TS (no red
precipitate of cuprous oxide is formed within 5 minutes). If
a precipitate is formed, continue with the alcohol extrac-
tions until the test is negative. Wash the completely ex-
tracted residue with a 10-mL portion of ether, using suction
to drain off the liquid. Dry the residue in the crucible in a
drying oven at 105° to constant weight. Weigh the crucible
with the crucible lid in place. The weight of residue is the
weight of methylcellulose present in the portion of pow-
dered Tablets taken.
Methyldopa
CK.
“OH + THO
Ho SF
CroHi3NOz + 11/2H20 238.24
CioHi3NO4 211.22
L-Tyrosine, 3-hydroxy-a-methyl-, sequilyarale:
L-3-(3,4-Dihydroxyphenyl)-2-methylalanine sesquihydrate
[41372-08-1].
Anhydrous [555-30-6].
DEFINITION
methyldopa (CioH13NO,), calculated on the anhydrous
IDENTIFICATION
o A. INFRARED ABSORPTION (197): [NoTE—Methods de-
scribed in (197K) or (197A) may be used.]
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
ASSAY
Change to read:
e PROCEDURE
[Note—Freshly prepare the Standard solution and Sample
solution before use.]
Buffer: 0.1 M monobasic sodium phosphate. Adjust
with phosphoric acid to a pH of 3.0.
Mobile phase: Buffer and methanol (850:150)
Diluent: 0.1 N hydrochloric acid
Standard solution: 0.4 mg/mL of USP Methyldopa RS
in Diluent
Sample solution: 0.4 mg/mL of Methyldopa in Diluent
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 280 nm
USP 41 Official Monographs / Methyldopa 2667

Column: 4.6-mm x 25-cm; 5-14m packing L1 Acceptance criteria: See Table 1. Disregard any peaks
Flow rate: 1.0 mL/min below 0.03%.
Injection volume: °20 pile cre t-jun-2017)
System suitability Table 1
Sample: Standard solution
Suitability requirements Relative Relative Acceptance
Tailing factor: 0.9-1.5 Retention Response Criteria,
Relative standard deviation: NMT 0.73% Name Time Factor NMT (%)
Analysis Methyldopa 1.0 1.0 _—
Samples: Standard solution and Sample solution 3-0-
Calculate the percentage of methyldopa (CioHi3NO«) in Methylmethyldopae 9 1.0 0.15
the portion of Methyldopa taken: Methyldopa related
compound Be 4.3 0.38 0.15
Result = (ru/rs) x (Cs/Cu) x 100
Methyldopa related
ty = peak response of methyldopa from the Sample compound C« 4.9 0.77 0.15
solution Any individual i.
Is = peak response of methyldopa from the unspecified impurity 1.0 0.05
Standard solution Total impurities _ = 0.5
Cs = concentration of USP Methyldopa RS in the 2 (S)-2-Amino-3-(4-hydroxy-3-methoxypheny!)-2-methylpropanoic acid.
Standard solution (mg/mL) » (S)-2-Amino-3-(4-methoxyphenyl)-2-methylpropanoic acid.
Cu = concentration of Methyldopa in the Sample © (S)-2-Amino-3-(3,4-dimethoxyphenyl)-2-methylpropanoic acid.
solution (mg/mL)
i criteria: 98.0%-102.0% on the anhydrous SPECIFIC TESTS
aSIS © OPTICAL ROTATION (781S), Procedures, Specific Rotation
Sample solution: 44 mg/mL, in a solvent that is a solu-
IMPURITIES tion of aluminum chloride in water (2 in 3) that previ-
© RESIDUE ON IGNITION (281): NMT 0.1% ously has been treated with activated charcoal, filtered,
and adjusted with 0.25 N sodium hydroxide to a pH of
155
Delete the following:
Acceptance criteria: —25° to -28°
e ACIDITY
°o HEAVY METALS, Method !/ (231): NMT 10 ppme corte. Sample solution: Dissolve 1.0 g in carbon dioxide-free
Jan-2012) ee with the aid of heat, and add 1 drop of methyl
© ORGANIC IMPURITIES red TS.
{Note—Freshly prepare the Standard solution and Sample Analysis: Titrate the Sample solution with 0.10 N so-
solution before use.] dium hydroxide to a yellow endpoint.
Buffer, Mobile phase, and Diluent: Prepare as directed Acceptance criteria: NMT 0.50 mL is required.
in the Assay. © WATER DETERMINATION (921), Method |: 10.0%-13.0%
fo
nw
System suitability solution: 4 ug/mL of USP mo]
ethyldopa RS and 6 g/mL each of USP 3-O- ADDITIONAL REQUIREMENTS
Methylmethyldopa RS, USP eee Related Com- e PACKAGING AND STORAGE: Preserve in well-closed, light- c=
pound B RS, and USP Methyldopa Related Compound i}
resistant containers. oS
C RS in Diluent e USP REFERENCE STANDARDS (11) iS
Standard solution: 4 jig/mL of USP Methyldopa RS in USP Methyldopa RS eo}=
Diluent USP Methyldopa Related Compound B RS i)
Sample solution: 4 mg/mL of Methyldopa in Diluent (S)-2-Amino-3-(4-methoxyphenyl)-2-methylpropanoic is}
-
Chromatographic system: Proceed as directed in the acid hydrochloride. Sal
Assay, except for the Run time. CiHisNO3 245.70
Run time: NLT 6 times the retention time of USP Methyldopa Related Compound C RS
methyldopa (S)-2-Amino-3-(3,4-dimethoxyphenyl)-2-methylpropa-
System suitability noic acid hydrochloride.
Samples: System suitability solution and Standard CyaHizNO, = 275.73
solution USP 3-O-Methylmethyldopa RS
Suitability requirements (S)-2-Amino-3-(4-hydroxy-3-methoxyphenyl)-2-methyl-
Resolution: NLT 2.0 between methyldopa related propanoic acid.
compound B and methyldopa related compound C, CutisNO4, 225.24
System suitability solution
Relative standard deviation: NMT 5%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the por- Methyldopa Oral Suspension
tion of Methyldopa taken:
» Methyldopa Oral Suspension is an aqueous sus-
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100 pension of Methyldopa. It contains one or more
tu = peak response of each impurity from the suitable flavors, wetting agents, and preserva-
Sample solution tives, and it may contain Sucrose. It contains not
ls = peak response of methyldopa from the less than 90.0 percent and not more than
Standard solution 110.0 percent of the labeled amount of
Cs = concentration of USP Methyldopa RS in the
Standard solution (mg/mL) CioHi3NOx.
Cu = concentration of Methyldopa in the Sample
solution (mg/mL)
F = relative response factor (see Table 1)
 2668 Methyldopa / Official Monographs
Packaging and storage—Preserve in tight, light-resistant
containers, and store at a temperature not exceeding 26°.
USP Reference standards (11)—
USP Methyldopa RS
Identification—Apply 10-uL portions of the Assay prepara-
tion and the Standard preparation prspated as directed in
the Assay to a suitable thin-layer chromatographic plate (see
Chromatography (621)) coated with a 0.25-mm layer of
chromatographic silica gel mixture. Allow to dry, develop
the chromatogram in a solvent system consisting of equal
volumes of acetone, butyl alcohol, glacial acetic acid, tolu-
ene, and water until the solvent front has moved about
three-fourths of the length of the plate. Remove the plate
from the developing chamber, mark the solvent front, and
allow the solvent to evaporate. Locate the spots by staining
the plate with iodine vapor for about 50 minutes, then view
the plate under short-wavelength UV light: the Rr value of
the principal spot obtained from the Assay preparation corre-
sponds to that obtained from the Standard preparation.
Uniformity of dosage units (905)—
FOR ORAL SUSPENSION PACKAGED IN SINGLE-UNIT CONTAINERS:
meets the requirements.
Deliverable volume (698)—
FOR ORAL SUSPENSION PACKAGED IN MULTIPLE-UNIT CONTAINERS:
meets the requirements.
pH (791): between 3.0 and 5.0; between 3.2 and 3.8 if
sucrose is present.
Assay—
Mobile phase—To 6.8 g of monobasic potassium phos-
phate add 750 mL of water, and stir until solution is com-
lete. Adjust with 1M phosphoric acid to a pH of 3.5, di-
ute with water to 1000 mL, mix, and pass irugh a filter
having a 10-um or finer porosity.
Standard preparation—Dissolve an accurately weighed
quantity of USP Methyldopa RS in 0.1 N sulfuric acid to
ro
”
obtain a solution having a known concentration of about
ah 1 mg of anhydrous methyldopa per mL.
i solution, and reject the first 20 mL of the filtrate.
— Assay preparation—Transfer an accurately measured vol-
aD ume of Oral Suspension, freshly mixed, equivalent to about
° Instrumental conditions
iS 250 mg of methyldopa, to a 250-mL volumetric flask, dilute
iS with 0.1 N sulfuric acid to volume, and mix to dissolve the
= methyldopa. Pass the solution through a 0.45-1zm mem-
rs brane filter before using.
va) Chromatographic system (see Chromatography (621))—The
> liquid chromatograph is equipped with a 280-mm detector
and a 3.9-mm x 30-cm column that contains packing L1.
The flow rate is about 1 mLpet minute. Chromatograph
three replicate injections of the Standard preparation, and
record the peak responses as directed for Procedure: the rela-
tive standard deviation is not more than 2.0%.
Procedure—Separately inject equal volumes (about 50 LL)
of the Standard preparation and the Assay preparation into
the chromatograph by means of a suitable microsyringe or
sampling valve, record the chromatograms, and measure
the responses for themajor peaks. Calculate the quantity, in
mg, of CioHi3NOx in each mL of the Oral Suspension taken
by the formula:
250(C/V)(ru / Fs)
in which C is the concentration, in mg per mL, of USP
Methyldopa RS in the Standard preparation; V is the volume,
in mL, of Oral Suspension taken; and ry and rs are the peak
responses obtained from the Assay preparation and the Stan-
dard preparation, respectively.
USP 41
Methyldopa Tablets
DEFINITION
Methyldopa Tablets contain NLT 90.0% and NMT 110.0%
of the labeled amount of methyldopa (CioHi3NOa).
IDENTIFICATION
oA
Analysis: To about 10 mg of finely ground Tablets add
ao of a solution of ninhydrin in sulfuric acid (1 in
Acceptance criteria: A dark purple color is produced
within 5-10 min that changes to pale brownish yellow
on addition of 3 drops of water.
e B.
Analysis: To about 10 mg of finely ground Tablets add
2 mL of 0.1 N sulfuric acid and 2 mL of Solution A, pre-
pared as directed in the Assay, followed by 0.25 mL of
6 N ammonium hydroxide.
Acceptance criteria: A dark purple color is produced
immediately.
ASSAY
¢ PROCEDURE
Solution A: Freshly dissolve 1 9 of ferrous sulfate, 2 g of
potassium sodium tartrate, and 100 mg of sodium bi-
sulfite in water to make a 100-mL solution.
Solution B: 50 g/L of ammonium acetate in 20% alco-
hol. Adjust with 6 N ammonium hydroxide to a pH of
8.5.
Standard solution: 1 mg/mL of anhydrous methyldopa
from USP Methyldopa RS in 0.1 N sulfuric acid
Sample solution: Nominally 1 mg/mL of methyldopa in
0.1 N sulfuric acid from NLT 20 powdered Tablets pre-
pared as follows. To a 100-mL volumetric flask add 0.1
N sulfuric acid to fill about 50% of the volume of the
flask, agitate by mechanical means for 15 min, and
then dilute with 0.1 N sulfuric acid to volume. Filter the
Blank: Water
Mode: UV-Vis
Analytical wavelength: 520 nm
Cell: 1.cm
Analysis
Samples: Standard solution, Sample solution, and Blank
Pipet 5 mL each of Standard solution, Sample solution,
and Blank into separate 100-mL volumetric flasks. Add
to each flask 5 mL of Solution A, and dilute with Solu-
tion B to volume.
Calculate the percentage of the labeled amount of
methyldopa (CioHi3NO,) in the portion of Tablets
taken:
Result = (Au/As) x (Cs/Cu) x 100
Au = absorbance of the Sample solution
As = absorbance of the Standard solution
Cs = concentration of USP Methyldopa RS in the
Standard solution (Gigirald.
Cy = nominal concentration of methyldopa in the
Sample solution (mg/mL)
USP 41
Acceptance criteria: 90.0%-110.0%
PERFORMANCE TESTS
© DISSOLUTION (711)
Medium: 0.1 N hydrochloric acid; 900 mL
Apparatus 2: 50 rpm
Time: 20 min
Instrumental conditions
Mode: UV
one wavelength: 280 nm
Standard solution: USP Methyldopa RS in Medium
Sample solution: Sample per Dissolution (711). Dilute
with Medium to a concentration similar to that of the
Standard solution.
Analysis: Determine the amount of methyldopa
(CioH13NOx) dissolved.
Tolerances: NLT 80% (Q) of the labeled amount of
methyldopa (CioH13NO,) is dissolved.
e UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in well-closed
containers.
e USP REFERENCE STANDARDS (11)
USP Methyldopa RS
Methyldopa and Chlorothiazide Tablets
» Methyldopa and Chlorothiazide Tablets contain
not less than 90.0 percent and not more than
110.0 pean of the labeled amounts of
methyldopa (CioH:3NOxz) and chlorothiazide
(C7H6CIN304S2).
Packaging and storage—Preserve in well-closed contain-
ers.
USP Reference standards (11)—
USP Chlorothiazide RS
USP Methyldopa RS
Identification—Transfer the finely ground contents of 1
Tablet to a test tube, add 10 mL of dilute alcohol (1 in 2),
shake for 5 minutes, and centrifuge. Use the clear superna-
tant as the Test solution. Prepare a solution of alcohol and
0.1 N sodium hydroxide (1:1) containing in each mL about
10 mg of USP Methyldopa RS and 10 mg of USP Chlorothia-
zide RS. Apply 20 uL of the Test solution ona line parallel to
and about 2 cm from the bottom edge of a 20-cm x 10-cm
thin-layer chromatographic plate (see Chromatography
(621)) coated with chromatographic silica gel mixture, and
apply 20 wL of the Standard solution separately on the start-
ing line. Allow the spots to dry, develop the chromatogram
in a solvent system consisting of equal volumes of glacial
acetic acid, acetone, butyl alcohol, toluene, and water until
the solvent front has moved about three-fourths of the
length of the plate. Remove the plate from the developing
tank, and allow the solvent to evaporate. View the plate
under short-wavelength UV light: the solution under test ex-
hibits two major spots having R; values corresponding to
those of the two major spots obtained with the Standard
solution.
Dissolution (711)—
PROCEDURE FOR METHYLDOPA—
Medium: 0.1 N hydrochloric acid; 900 mL.
Apparatus 2: 75 rpm.
Time: 30 minutes.
Standard preparation—Dissolve an accurately weighed
quantity of USP Methyldopa RS in Medium, and dilute quan-
titatively with the same solvent to obtain a solution having a
Official Monographs / Methyldopa 2669
known concentration of about 275 ug of anhydrous
methyldopa per mL.
Ferrous tartrate solution—Dissolve 1 g of ferrous sulfate,
2g of potassium sodium tartrate, and 100 mg of sodium
bisulfite in water to make 100 mL, and mix. Use a freshly
prepared solution.
Buffer solution—Dissolve 50 g of ammonium acetate in
1000 mL of dilute alcohol (1 in 5). Adjust with 6 N ammo-
nium hydroxide to a pH of 8.5.
Procedure—Filter 35 mL of the solution under test, and
transfer an aliquot estimated to contain between 2 mg and
3 mg of methyldopa to a 100-mL volumetric flask. Adjust
the final volume, if necessary, with Medium to 10 mL. To a
second 100-mL volumetric flask add 10.0 mL of Standard
preparation, and to a third 100-mL volumetric flask add
10.0 mL of Medium to provide a blank. Pipet 5.0 mL of Fer-
rous tartrate solution into each flask, dilute with Buffer solu-
tion to volume, and mix. Concomitantly determine the ab-
sorbances of the treated Standard preparation and test
solution at the wavelength of maximum absorbance at
about 520 nm, with a suitable spectrophotometer, against
the reagent blank. Calculate the amount of CioHi3NOs dis-
solved, in mg, taken by the formula:
9(C/ V)(Au/ As)
in whichCis the concentration, in ug of anhydrous
methyldopa per mL, of USP Methyldopa RS in the Standard
preparation; V is the volume, in mL, of the aliquot of test
solution used; and Ay and As are the absorbances of the
solutions from the test solution and the Standard prepara-
tion, respectively.
Tolerances—Not less than 80% (Q) of the labeled amount
of CioHi3NO, is dissolved in 30 minutes.
PROCEDURE FOR CHLOROTHIAZIDE—
Medium: 0.05 M, pH 8.0 phosphate buffer (see Buffer
Solutions in the section Reagents, Indicators, and Solutions) c
ww
containing sodium sulfite (1 in 5000); 900 mL. vu
Apparatus 2: 75 rpm. =
Time: 60 minutes. i}
3
Procedure—Determine the amount of C7HeCIN3O.S2 dis- )
solved from UV absorbances of the solution under test, suit- a2
ably diluted with Medium, if necessary, at the wavelength 2
of maximum absorbance at about 317 nm in comparison a}
with a Standard solution having a known concentration of =e
”
USP Chlorothiazide RS in the same Medium.
Tolerances—Not less than 75% (Q) of the labeled amount
of C7H6CIN30.S2 is dissolved in 60 minutes.
Uniformity of dosage units (905): meet the require-
ments with respect to methyldopa and to chlorothiazide.
Assay—
Mobile phase—Preparea filtered and degassed mixture of
0.08 M monobasic sodium phosphate and methanol (95:5).
Adjustby the addition of phosphoric acid to a pH of 2.8.
Make adjustments if necessary (see System Suitability under
Chromatography (621)).
Standard preparation—Transfer to a 100-mL volumetric
flask accurately weighed quantities of USP Methyldopa RS
and USP Chlorothiazide RS, equivalent to one-fifth of their
labeled amounts, in mg, per Tablet. Add 15 mL of water
and 5 mL of 1 N hydrochloric acid, and sonicate for about
3 minutes. Add 10 mL of acetonitrile, and sonicate for
2 minutes. Dilute with water to volume, and mix.
Assay preparation—Weigh and finely powder not less
than 10 Tablets. Transfer an accurately weighed portion of
the powder, equivalent to the weight of 1 Tablet, to a
500-mL volumetric flask. Add 75 mL of water and 25 mL of
1N hydrochloric acid, and sonicate for about 5 minutes.
Add 50 mL of acetonitrile, and sonicate for 10 minutes. Di-
lute with water to volume, and mix. Filter through a 0.45-
to 2.0-um membrane filter, discarding the first TO mL.
 2670 Methyldopa / Official Monographs USP 41

Chromatographic system (see Chromatography (621))—The Chromatographic system

liquid chromatograph is equipped with a 280-nm detector (See Chromatography (621), System Suitability.)
and a 3.9-mm x 30-cm column that contains packing L1. Mode: LC
The flow rate is about 2 mL per minute. Chromatograph the Detector: UV 270 nm
Standard preparation, and record the peak responses as di- Column: 3.9-mm x 30.0-cm; packing L1
rected under Procedure: the column efficiency determined Flow rate: 2 mL/min
from the chlorothiazide peak is not less than 1300 theoreti- Injection volume: 10 uL
cal plates, the tailing factor for chlorothiazide peak is not System suitability
more than 2, the resolution, R, between the chlorothiazide Sample: Standard solution
and methyldopa peaks is not less than 7, and the relative [Note—Adjust the flow rate to obtain relative retention
standard deviation for replicate injections is not more than times of about 0.38 and 1.0 for methyldopa and hy-
2.0%. drochlorothiazide, respectively.]
Procedure—Separately inject equal volumes (about 10 pL) Suitability requirements
of the Standard preparation and the Assay preparation into Resolution: NLT 6.0 between methyldopa and
the chromatograph, record the chromatograms, and meas- hydrochlorothiazide
ure the responses for the major peaks. The relative retention Relative standard deviation: NMT 2.0%
times are about 1.0 for methyldopa and 2.5 for chlorothia- Analysis
zide. Calculate the quantity, in mg, of chlorothiazide Samples: Standard solution and Sample solution
eee) in the portion of Tablets taken by the Calculate the percentage of the labeled amount of
formula: enw eepe (CioHi3NO«) in the portion of Tablets
taken:
500C(ru/ rs)
Result = (ru/rs) x (Cs/Cu) x 100
in which C is the concentration, in mg per mL, of USP
Chlorothiazide RS in the Standard Beepagton, and ry and rs Ty = peak response of methyldopa from the Sample
are the peak responses of the chlorothiazide peak obtained solution
from the a preparation and the Standard preparation, re- rs = peak response of methyldopa from the
spectively. Calculate the quantity, in mg, of methyldopa Standard solution
(CioHi3NO«) taken by the same formula, reading Cs = concentration of USP Methyldopa RS in the
“methyldopa” instead of “chlorothiazide.” Standard solution (mg/mL)
Cy = nominal concentration of methyldopa in the
Sample solution (mg/mL)
Calculate the percettage of the labeled amount of
hydrochlorothiazide (C7HgCIN30.S2) in the portion of
Methyldopa and Hydrochlorothiazide Tablets taken:
Tablets Result = (ru/rs) x (Cs/Cu) x 100

te
vy
DEFINITION
a Methyldopa and Hydrochlorothiazide Tablets contain NLT
iy
7 90.0% and NMT 110.0% of the labeled amounts of
Dd methyldopa (CioHi3NO4) and hydrochlorothiazide
°
2 (C7HsCIN3O.Sz).
S in the Standard solution (mg/mL)
2 IDENTIFICATION
3 e A. The retention times of the two major peaks of the
va) Sample solution correspond to those of the Standard solu-
=) tion, as obtained in the Assay.
° B. PERFORMANCE TESTS
Analysis: To 10 mg of methyldopa from a portion of e DISSOLUTION (711)
crushed Tablets add 0.15 mL of a solution of ninhydrin
in sulfuric acid (1 in 250).
Acceptance criteria: A dark purple color is produced
within 5-10 min. The color changes to pale brownish
yellow upon adding 0.15 mL of water.
ASSAY
e PROCEDURE
Buffer: 11.04 g/L of monobasic sodium phosphate. Ini-
tially add 950 mL of water, adjust with phosphoric acid
to a pH of 2.8, and then dilute with water to volume.
Mobile phase: Methanol and Buffer (5:95)
Standard solution: Transfer a suitable quantity of USP
Methyldopa RS to a suitable volumetric flask to pipes
a 1-mg/mL solution of anhydrous methyldopa. Add a
quantity of USP Hydrochlorothiazide RS that corre-
sponds to the ratio of hydrochlorothiazide to
methyldopa in the Tablets. Dissolve in 25% of the total
volume a mixture of water, acetonitrile, and 1 N hydro-
chloric acid (1: 1: 0.5). Dilute with water to volume.
Sample solution: Transfer an equivalent to 250 mg of
methyldopa (NLT 20 Tablets) to a 250-mL volumetric
flask, and add 50 mL of water, 25 mL of acetonitrile,
and 13 mL of 1 N hydrochloric acid. Shake the flask for
5 min, and dilute with water to volume.
ty = peak response of hydrochlorothiazide from the
Sample solution
rs = peak response of hydrochlorothiazide from the
Standard solution
Cs = concentration of USP Hydrochlorothiazide RS
Cu = nominal concentration of hydrochlorothiazide
in the Sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0%
Methyldopa
Medium: 0.1 N hydrochloric acid; 900 mL
Apparatus 2: 50 rpm
Time: 30 min
Solution A: 10 mg/mL of ferrous sulfate, 20 mg/mL of
potassium sodium tartrate, and 1 mg/mL of sodium bi-
sulfite in water. [NoTE—Use a freshly prepared
solution.]
Solution B: 50 mg/mL of ammonium acetate in dilute
alcohol (1 in 5). Adjust with 6 N ammonium hydrox-
ide to a pH of 8.5.
Standard solution: 0.275 mg/mL of anhydrous
methyldopa from USP Methyldopa RS in Medium
Sample solution: Filter 35 mL of the solution under
test through paper.
Instrumental conditions
Mode: Vis
Analytical wavelength: 520 nm
Cell: 1.cm
Analysis
Samples: Standard solution and Sample solution
Transfer an aliquot of the Sample solution estimated to
contain 2-3 mg of methyldopa to a 100-mL volumet-
ric flask. Adjust the final volume, if necessary, with
Medium to 10 mL. To a second 100-mL volumetric
USP 41
flask add 10.0 mL of Standard solution, and to a third
100-mL volumetric flask add 10.0 mL of Medium to
provide a blank. Pipet 5.0 mL of Solution A into each
flask, dilute with Solution B to volume, and mix. De-
termine the absorbances of the Standard solution and
the Sample solution at the wavelength of maximum
absorbance, using the blank in the reference cell.
Calculate the percentage of the labeled amount of
methyldopa (CioHi3NO.) dissolved:
Result = (Au/As) x (Cs/L) x V x 100
Au = absorbance of the Sample solution
As = absorbance of the Standard solution
Cs = concentration of anhydrous methyldopa in the
Standard solution (g/mL)
L = label claim (mg/Tablet)
Vv = volume of the medium, 900 mL
Tolerances: NLT 80% (Q) of the labeled amount of
methyldopa (CioHi3NOx) is dissolved.
Hydrochlorothiazide
Medium: 0.1 N hydrochloric acid; 900 mL
Apparatus 2: 50 rpm
Time: 60 min
Instrumental conditions
Mode: Vis
Analytical wavelength: 317 nm
Cell: 1. cm
Standard solution: USP Hydrochlorothiazide RS in
Medium
Sample solution: Pass a portion of the solution under
test throughasuitable filter. Dilute with Medium to a
concentration that is similar to the Standard solution.
Tolerances: NLT 80% (Q) of the labeled amount of
hydrochlorothiazide (C7HgCIN3O4Sz) is dissolved.
e UNIFORMITY OF DOSAGE UNITS (905)
Procedure for content uniformity: Proceed as directed
in the Assay, except use the following Sample solution.
Sample solution: Transfer 1 Tablet to a 250-mL volu-
metric flask, add 50 mL of water, and shake gently, if
necessary, to disintegrate the Tablet. Do not sonicate.
After the Tablet has completely disintegrated, add
25 mL of acetonitrile, and shake by mechanical means
for 30 min. Add 13 mL of 1 N hydrochloric acid, and
shake by mechanical means for an additional 5 min.
Dilute with water to volume.
Acceptance criteria: Meet the requirements with re-
spect to methyldopa and hydrochlorothiazide
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed
containers.
e USP REFERENCE STANDARDS (11)
USP Hydrochlorothiazide RS
USP Methyldopa RS
Methyldopate Hydrochloride
°
WZ x Yo“ cH,
wos Yad Sty © Her
Cy2zHiyNO4- HCl 275.73
L-Tyrosine, 3-hydroxy-a-methyl-, ethyl ester, hydrochloride.
L-3-(3,4-Dihydroxyphenyl)-2-methylalanine ethyl ester hy-
drochloride [5123-53-5; 2508-79-4].
» Methyldopate Hydrochloride contains not less
than 98.0 percent and not more than 101.0 per-
Official Monographs / Methyldopate 2671
cent of Ci2HizNO, - HCI, calculated on the dried
basis.
Packaging and storage—Preserve in well-closed contain-
as Store at 25°, excursions permitted between 15° and
USP Reference standards (11)—
USP Methyldopate Hydrochloride RS
identification—
A: Infrared Absorption (197M).
B: Ultraviolet Absorption (197U)—
Solution: 50 1g per mL.
Medium: 0.1 N hydrochloric acid.
Absorptivities at 280 nm, calculated on the dried basis, do
not differ by more than 3.0%.
C: It responds to Identification test C under Methyldopa.
D: It responds to the tests for Chloride (191), except that
on the addition of the slight excess of 6 N ammonium hy-
droxide a brown precipitate is formed.
Specific rotation (7815S): between —13.5° and -14.9°
(A = 405 nm).
Test solution: 40 mg per mL, in 0.1 N hydrochloric acid.
PH (791): between 3.0 and 5.0, in a solution (1 in 100).
Loss on drying (731)—Dry it at 100° and at a pressure not
exceeding 5 mm of mercury for 2 hours: it loses not more
than 0.5% of its weight.
Residue on ignition (281): not more than 0.1%.
Delete the following:
°Heavy metals, Method II (231): 0.001%.‘oficial 1-jan-2018)
Assay—
Mobile solvent—Prepare a suitable solution of 0.02 M =
monobasic sodium phosphate and 0.015 M phosphoric acid a)
in a water and methanol solution (approximately 15.5:4.5) ao]
such that the retention time of methyldopate hydrochloride =
is approximately 6.5 minutes. i)
Standard preparation—Dissolve an accurately weighed =
i)
quantity of USP Methyldopate Hydrochloride RS in the Mo- ro}=
bile solvent to obtain a solution containing about 1 mg per ESS)
mL. so)
Assay preparation—Transfer about 50 mg of Methyldo- a
a
pate Hydrochloride, accurately weighed, to a 50-mL volu-
metric flask, dissolve in and dilute with Mobile solvent to
volume.
Procedure—Introduce separately 20-u1L portions of the As-
say preparation and the Standard preparation into a high-
pressure liquid chromatograph (see Chromatography (621))
operated at 25°, by means of a suitable microsyringe or
sampling valve, adjusting the operating parameters such
that the peak obtained with the Standard preparation is
100% full-scale. Typically, the apparatus is fitted with a
4-mm x 30-cm column that contains packing L1, is
equipped with an UV detector capable of monitoring ab-
sorption at 280 nm andasuitable recorder, and is capable
of operatg at a column pressure between 700 and
1700 psi. In a suitable chromatogram, three replicate injec-
tions of the Standard preparation showarelative standard
deviation of not more than 1.5%. Determine the peak areas,
at equivalent retention times, obtained with the Assay prepa-
ration and the Standard preparation, and calculate the quan-
tity, in mg, of Ci2Hi7NO, - HCI in the portion of Methyldo-
pate Hydrochloride taken by the formula:
S0C(Au / As)
in which C is the concentration, in mg per mL, of USP
Methyldopate Hydrochloride RS in the Standard preparation;
and Ay and As are the peak areas obtained from the Assay
preparation and the Standard preparation, respectively.
 2672 Methyldopate / Official Monographs
Methyldopate Hydrochloride Injection
» Methyldopate Hydrochloride Injection is a ster-
ile solution of Methyldopate Hydrochloride in
Water for Injection. It contains not less than
90.0 percent and not more than 110.0 percent of
the labeled amount of methyldopate hydrochlo-
ride (Cy2Hi7NO« * HCl).
Packaging and storage—Preserve in single-dose contain-
ers, preferably of Type | glass.
Change to read:
USP Reference standards (11)—
%o (CN i-May-2018)
USP Methyldopate Hydrochloride RS
Identification—
A: Dilute a volume of Injection with a mixture of chloro-
form and methanol (1:1), i necessary, to obtain a solution
containing about 5 mg of methyldopate hydrochloridePe
mL. Apply separately 10 uL of this solution and 10 wL of a
Standard solution of USP Methyldopate Hydrochloride RS in
a solvent mixture of chloroform and methanol (1:1) contain-
ing 5 mg per mL to a suitable thin-layer chromatographic
late (see Chromatography (621)) coated with a 0.25-mm
ayer of chromatographic silica gel. Allow the spots to dry,
and develop the chromatogram in a saturated chamber
with a solvent system consisting of a mixture of butyl alco-
hol, water, and formic acid (7:2:1), until the solvent front
has moved about three-fourths of the length of the plate.
Remove the plate from the developing chamber, mark the
a
solvent front, and allow the solvent to evaporate. Locate the
ao spots on the plate by lightly spraying with Folin-Ciocalteu
a henol TS followed by spraying with sodium carbonate so-
i]
— ution (1 in 10): the R; value arthe principal spot obtained
Dp from the test solution corresponds to that obtained from
°
c the Standard solution.
Sj B: It responds to the tests for Chloride (191), with the
= exception that the 6 N ammonium hydroxide is omitted.
fam Bacterial Endotoxins Test (85)—It contains not more
“A
than 0.5 USP Endotoxin Unit per mg of methyldopate hy-
> drochloride.
pH (791): between 3.0 and 4.2.
Particulate Matter in Injections (788): meets the re-
quirements under small-volume injections.
Other requirements—\t meets the requirements under /n-
jections and Implanted Drug Products <1).
Assay—
Buffer solution—To 214 g of monobasic potassium phos-
phate add 700 mL of water, and stir. Cautiously add 75 mL
of sodium hydroxide solution (1 in 2), and stir until solution
is complete. Adjust with sodium hydroxide solution (1 in 2)
to a pH of 8.0, and dilute with water to 1000.0 mL.
Water-saturated tributy! phosphate—Shake 800 mL of
tributyl phosphate with 100 mL of water, and discard the
lower, aqueous phase. Filter the upper phase.
Standard preparation—Transfer about 25 mg of USP
Methyldopate Hydrochloride RS, accurately weighed, to a
25-mL volumetric flask, add water to volume, and mix.
Transfer 5 mL of this solution to a 100-mL volumetric flask,
add 0.1 N sulfuric acid to volume, and mix. Use a freshly
prepared solution. The Standard preparation contains about
50 yg per mL.
Assay preparation—Transfer to a 50-mL volumetric flask
an accurately measured volume of Injection, equivalent to
about 50 mg of methyldopate hydrochloride, add water to
volume, and mix. Transfer a 5.0-mL aliquot of the solution
USP 41
to a 60-mL separator, add 15 mL of Buffer solution and
10 mL of Water-saturated tributyl phosphate, and shake for
about 1 minute. Allow the phases to separate, and transfer
the lower, aqueous phase to a second 60-mL separator. To
this separator add a second 10-mL portion of Water-satu-
rated tributyl phosphate, shake for SeoL 1 minute, allow the
phases to separate, discard the lower, aqueous phase, and
add the upper tributyl phosphate phase to the phase re-
tained in the first separator. Rinse the second separator with
about 2 mL of Water-saturated tributy! phosphate, and add
the rinsing to the first separator. Extract the phase con-
tained in the first separator with two 25-mL portions of 0.1
N sulfuric acid. Collect the acid extracts in a 100-mL volu-
metric flask, add 0.1 N sulfuric acid to volume, and mix.
Filter, if necessary, to obtain a clear solution.
Procedure—Concomitantly determine the absorbances of
the Assay preparation and the Standard preparation in 1-cm
cells at the wavelength of maximum absorbance at about
283 nm, with a suitable spectrophotometer, using 0.1 N
sulfuric acid as the blank. Calculate the quantity, in mg, of
methyldopate hydrochloride (C12Hi7NO,- HCl) in each mL
of the Injection taken by the formula:
(C/ V)(Au/ As)
in which C is the concentration, in ug per mL, of USP
Methyldopate Hydrochloride RS in the Standard preparation;
Vis the volume, in mL, of Injection taken; and Ay and As are
the absorbances of the Assay preparation and the Standard
preparation, respectively.
Methylene Blue
; 8 2
Hyc ~ re cu, ct xH,O
a iOS
ON LO
Ci6HisCINsS - xH20
Phenothiazin-5-ium, 3,7-bis(dimethylamino)-, chloride;
3,7-Bis(dimethylamino)phenothiazin-5-ium chloride;
Pentahydrate 409.93
[32680-41-4].
Trihydrate 373.90
7220-79-3].
Monohydrate 337.90
[122965-43-9].
Anhydrous 319.85
[61-73-4].
DEFINITION
Methylene Blue contains NLT 97.0% and NMT 103.0% of
methylene blue (CisHisCIN3S), calculated on the dried
basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
° B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
e C. IDENTIFICATION TESTS—GENERAL (191), Chloride
Sample solution: Ignite 50 mg of Methylene Blue with
0.5 g of anhydrous sodium carbonate. Cool and dis-
solve the residue in 10 mL of 2 N nitric acid. Filter and
use 2 mL of the filtrate for performing the test.
Acceptance criteria: Meets the requirements
ASSAY
© PROCEDURE
[Note—All solutions containing methylene blue are rec-
ommended to be prepared fresh before analysis.]
USP 41 Official Monographs / Methylene 2673

Solution A: 0.1% trifluoroacetic acid in water System suitability a

Solution B: Acetonitrile
Diluent: Solution A and Solution B (70:30)
Mobile phase: See Table 7.
Table 1
Solution A
Samples: System suitability solution and Sensitivity
solution
Suitability requirements
Resolution: NLT 3.5 between methylene blue and az-
ure B peaks, System suitability solution
Signal-to-noise ratio: NLT 10, Sensitivity solution
Solution B Analysis
Sample: Sample solution
20 Calculate the percentage of azure B or any unspecified
impurity in the portion of Methylene Blue taken:
Result = (ru/r7) x 100
i peak response of azure B or any unspecified

i}
impurity from the Sample solution
Iv sum of all the peak responses from the Sample

ut
Standard solution: 1 mg/mL of USP Methylene Blue RS solution
in Diluent. Stirring and sonication may be necessary for Acceptance criteria: See Table 2. Disregard any peak
complete dissolution. less than 0.05%.
Sample solution: 1 mg/mL of Methylene Blue in Dilu-
ent. Stirring and sonication may be necessary for com-
plete dissolution. Table 2
Chromatographic system Relative Acceptance
(See Chromatography (621), System Suitability.) Retention Criteria,
Mode: LC Name Time NMT (%)
Detector: UV 246 nm Azure Ba 0.8 2.5;
Column: 4.6-mm x 10-cm; 3.5-um packing L11 Methylene blue 1.0 —
Column temperature: 30° Any unspecified _
Flow rate: 1 mL/min impurity 0.10
Injection volume: 5 uL
System suitability Total impurities? = 0.5
Sample: Standard solution @3-(Dimethylamino)-7-(methylamino)-phenothiazine-5-ium chloride.
Suitability requirements ’Total impurities does not include azure B.
Tailing factor: NMT 3.0 SPECIFIC TESTS
Relative standard deviation: NMT 1.10% e Loss ON DRYING (731)
Analysis Analysis: Dry at 105° for 5 h.
Samples: Standard solution and Sample solution Acceptance criteria: 8.0%-22.0% rex
Calculate the percentage of methylene blue e BACTERIAL ENDOTOXINS TEST (85): NMT 2.5 USP Endo- nn
(CisHigCINsS) in the portion of Methylene Blue taken: toxin Units/mL
a]

Result = (ru/rs) x (Cs/Cu) x 100 e MICROBIAL ENUMERATION TESTS (61): The total aerobic mi- os
crobial count is NMT 102 cfu/g. °
|
tu = peak response of methylene blue from the °
Sample solution
ADDITIONAL REQUIREMENTS te}~
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ls = peak response of methylene blue from the Ay
Standard solution
ers and protect from light. Store below 30°. so}
e USP REFERENCE STANDARDS (11) a
Cs = concentration of USP Methylene Blue RS in USP Azure B RS
al

the Standard solution (mg/mL) 3-(Dimethylamino)-7-(methylamino)-phenothiazine-

Cu = concentration of Methylene Blue in the Sample 5-ium chloride.
solution (mg/mL) CisHisCIN3S 305.82
Acceptance criteria: 97.0%-103.0% on the dried basis USP Endotoxin RS
IMPURITIES USP Methylene Blue RS
© RESIDUE ON IGNITION (281): NMT 0.15%
e ARSENIC
Analysis: Proceed as directed in Elemental Impurities—
Procedures (233), Procedure 2: ICP-MS.
Acceptance criteria: 8 ppm Methylene Blue Injection
© COPPER OR ZINC
Analysis: Proceed as directed in Flemental Impurities— DEFINITION
Procedures (233), Procedure 2: ICP-MS. Methylene Blue Injection is a sterile solution of Methylene
Acceptance criteria: NMT 100 ppm of zinc and NMT Blue in Water for Injection. It contains NLT 95.0% and
200 ppm of copper NMT 105.0% of the labeled amount of methylene blue
© ORGANIC IMPURITIES (CisHisCINsS » 3H20).
Diluent, Mobile phase, Standard solution, Sample so-
lution, and Chromatographic system: Proceed as di- IDENTIFICATION
rected in the Assay. e A, The UV absorption spectra of the major peak of the
System suitability solution: 1 mg/mL of USP Methyl- Sample solution exhibit maxima and minima at the same
ene Blue RS and 0.025 mg/mL of USP Azure B RS in ae as those of the corresponding peak of the
Diluent Standard solution, as obtained in the Assay.
Sensitivity solution: 0.5 j1g/mL of USP Methylene Blue e B. The retention time of the major peak of the Sample
RS in Diluent from the Standard solution solution corresponds to that of the Standard solution, as
obtained in the Assay.
 2674 Methylene / Official Monographs USP 41

ASSAY System suitability

© PROCEDURE Samples: System suitability solution, Standard solution,
All solutions containing methylene blue are recom- and Sensitivity solution
mended to be freshly prepared prior to analysis. Suitability requirements
Solution A: 0.1% trifluoroacetic acid in water Resolution: NLT 3.5 between the methylene blue
Solution B: Acetonitrile and azure B peaks, System suitability solution
Diluent: Solution A and Solution B (70:30) Relative standard deviation: NMT 5.0%, Standard
Mobile phase: Solution A and Solution B (75:25) solution
Standard solution: 100 g/mL of USP Methylene Blue Signal-to-noise ratio: NLT 10, Sensitivity solution
RS in Diluent Analysis
Sample solution: Nominally 100 g/mL of methylene Samples: Standard solution and Sample solution
blue in Diluent from Injection Calculate the percentage of each specified impurity in
Chromatographic system the portion of Injection taken:
(See Chromatography (621), System Suitability.)
Mode: LC Result = (ru/rs) x (Cs/Cy) x 100
Detector: UV 246 nm. For Identification A, use a diode
array detector in the range of 200-400 nm. tu = peak response of each impurity from the
Column: 4.6-mm x 10-cm; 3.5-um packing L11 Sample solution
Temperatures rs = peak response of methylene blue from the
Autosampler: 5° Standard solution
Column: 30° Cs = concentration of USP Methylene Blue RS in
Flow rate: 1 mL/min the Standard solution (g/mL)
Injection volume: 5 uL Cu = nominal concentration of methylene blue in
System suitability the Sample solution (ug/mL)
Sample: Standard solution Calculate the percentage of any unspecified impurity in
Suitability requirements the portion of Injection taken:
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0% Result = (ru/rz) x 100
Analysis
Samples: Standard solution and Sample solution fu =Peak Fite Saat a, wispeciirsl impurity
Calculate the percentage of the labeled amount of _ Cae Ilthe beak res 1GH from the Sampl
methylene blue (CisHisCIN3S - 3H20) in the portion of & = lation enpeak responsessifom Ane: Sample
Injection taken:
Acceptance criteria: See Table 2. Disregard any peak
Result = (ru/rs) x (Cs/Cu) x (Ma/Mr2) x 100 less than 0.05%.

tu = peak response of methylene blue from the Table 2

ra} Sample solution late Z
S Is = peak response of methylene blue from the Relative Ce reance
a Standard solution Retention Rebate
oy Cs = concentration of USP Methylene Blue RS in Name: Time T (%)
3} the Standard solution (ug/mL) Azure C 0.32 0.20
¢ Cu = nominal concentration of methylene blue in Azure A 0.52 0.20
eS the Sample solution (1g/mL) Azure Ba 0.82 30
a Ma = =rig ettet weight of methylene blue Methylene blue 1.00 _
trihydrate, 373.90
Es Mrz = molecular weight of methylene blue Ay unspectied = 0.20
anhydrous, 319.85 Total j ae :
Acceptance criteria: 95.0%-105.0% otal impurities = 5.0
Azure B is quantiated using the test for Limit of Azure B and is included
IMPURITIES in the table for identification purposes.
© ORGANIC IMPURITIES e LIMIT OF AZURE B
Solution A, Solution B, Diluent, and Chromatographic Diluent, Mobile phase, Sample solution, and Chromat-
system: Proceed as directed in the Assay. ographic system: Proceed as directed in the Assay.
Mobile phase: For analyzing azure A, azure C, and un- Standard solution: 3 g/mL of USP Methylene Blue RS
specified impurities, see Table 1. in Diluent
System suitability: Proceed as directed in the Assay us-
Table 1 ing the Standard solution from the Assay.
S 5 [Note—The relative retention times for azure B and
Time
(min)
Solution
(%)
A Solution
(%)
8 Analysis
methylene blue are 0.70 and 1.00, respectively.]

o 80 20 Samples: Sample solution and Standard solution

5 80 20 Calculate the percentage of azure B in the portion of
2S: 30 70 Injection taken:
32 30 70
Result = (ru/rs) x (Cs/Cu) x 100
System suitability solution: 1 mg/mL of USP Methyl-
ene Blue RS and 0.03 mg/mL of USP AzureB RS in ry = peak response of azure B from the Sample
Diluent solution
Standard solution: 1 g/mL of USP Methylene Blue RS Is = peak response of methylene blue from the
in Diluent Standard solution .
Sensitivity solution: 0.25 11g/mL of USP Methylene Blue Cs = concentration of USP Methylene Blue RS in
RS in Diluent from the Standard solution the Standard solution
Sample solution: Nominally equivalent to 500 g/mL
of methylene blue (anhydrous) in Diluent from Injection
USP 41 Official Monographs / Methylene 2675

Cu = nominal concentration of methylene blue in tion. Remove the plate from the chamber, and allow
the Sample solution the solvent to evaporate.
Acceptance criteria: NMT 3.0% Acceptance criteria: The Rr value of the principal spot
from the Sample solution corresponds to that from the
SPECIFIC TESTS Standard solution.
© PH (791): 3.0-4.5
e BACTERIAL ENDOTOXINS TEST (85): NMT 2.5 USP Endo- ASSAY
toxin Units/mL © PROCEDURE
e OTHER REQUIREMENTS: It meets the requirements in /njec- Standard solution: 2 g/mL of USP Methylene Blue RS
tions and Implanted Drug Products (1). in diluted alcohol
Sample solution: Dilute a volume of Injection with
ADDITIONAL REQUIREMENTS diluted alcohol to obtain a solution with a nominal con-
e PACKAGING AND STORAGE: Preserve in single-dose contain- centration of about 2.4 ug of methylene blue trihydrate
ers, preferably of Type | glass. Store at room tempera- (2 ug of anhydrous methylene blue)/mL.
ture, protected from light. Do not refrigerate or freeze. Spectrometric conditions
(See Ultraviolet-Visible Spectroscopy (857).)
Change to read: Mode: UV-Vis
Detector: 663 nm
e USP REFERENCE STANDARDS (11) Cell: 1.cm
USP Azure B RS Analysis
3-(Dimethylamino)-7-(methylamino)phenothiazin-5-ium Samples: Standard solution and Sample solution. Con-
chloride. comitantly determine the absorbance of both solu-
eCisHisCINas 305.82 tions, using diluted alcohol as the blank.
@ (CN 1-May-2018)
Calculate the quantity, in mg, of CisHigCIN3S - 3H2O in
USP Methylene Blue RS each mL of Injection taken:
Result = (Au/As) x (Cs/Cu) x L x (Mn/My2)

Au = absorbance of the Sample solution

As = absorbance of the Standard solution
Methylene Blue Injection, Veterinary Cs = concentration of methylene blue trihydrate in
the Standard solution (mg/mL)
DEFINITION Cu = nominal concentration of methylene blue
Methylene Blue Injection, Veterinary is a sterile solution of trihydrate in the Sample solution (mg/mL)
Methylene Blue in Water for Injection. It contains NLT Mn = molecular weight of methylene blue trihydrate
9.5 mg/mL and NMT 10.5 mg/mL of methylene blue tri- (373.90)
hydrate (CisHisCIN3S- 3H2O). Prepare Methylene Blue In- Mz = molecular weight of methylene blue
jection, Veterinary as follows (see Pharmaceutical Com- anhydrous (319.86) c
pounding—Sterile Preparations {797)): L; = labeled amount of methylene blue in the a
Injection (10.0 mg/mL) v
Methylene Blue 5g Acceptance criteria: 9.5-10.5 mg/mL of CisHisCINsS - z
Sterile Water for Injection or Sodium Chloride 3H20 °
=)
Injection (0.9%), a sufficient quantity to SPECIFIC TESTS 9
make 500 mL © PH (791): 3.0-4.5 ©
e BACTERIAL ENDOTOXINS TEST (85): NMT 0.17 USP Endo- By
Dissolve an accurately weighed quantity of Methylene Blue toxin Unit/mg of methylene blue mo]
in Sterile Water for Injection or Sodium Chloride Injection a
e STERILITY TESTS (71): Meets the requirements a
(0.9%), and dilute to volume, with mixing. Sterilize by a © OTHER REQUIREMENTS: It meets the requirements under
suitable means, such as sterile filtration or autoclaving. Labeling (7), Labels and Labeling for Injectable Products.
IDENTIFICATION ADDITIONAL REQUIREMENTS
e A. The visible absorption spectrum of the Sample solu- ¢ PACKAGING AND STORAGE: Preserve in single-dose contain-
tion exhibits maxima and minima at the same wave- ers, preferably of Type | amber glass. Store at room tem-
lengths as that of the Standard solution, concomitantly perature, protected from light.
measured as directed in the Assay. e LABELING: Label it to indicate that it is to be discarded
° eo van CHROMATOGRAPHIC IDENTIFICATION TEST after its beyond use date, to state that it is to be kept
out of the reach of children, and to indicate the nominal
Diluent: Methanol and water (1:1) content of methylene blue in the Injection and whether
Standard solution: 5 mg/mL of USP Methylene Blue RS it was prepared in Sterile Water for Injection or in So-
in Diluent dium Chloride Injection (0.9%). Label it to indicate that
Sample solution: Dilute a portion of the Injection with the dose is not to exceed 30 mg of methylene blue/kg of
an equal volume of methanol. body weight/h. Label it to indicate that it is for veterinary
Application volume: 1 ul use only and to state the Beyond-Use Date.
Developing solvent system: Alcohol, acetic acid, and e BEYOND-UsE DATE: NMT 365 days after the date on
water (3:3:4) which it was compounded
Analysis
Samples: Standard solution and Sample solution
Allow the spots to dry, and develop until the solvent
front has moved about 10 cm above the line of applica-
 2676 Methylene / Official Monographs
Change to read:
° USP REFERENCE STANDARDS (11)
@ (CN i-May-2018)
USP Methylene Blue RS
Methylergonovine Maleate
LOH
C2oH25N3O2 + CaHgOu 455.50
Ergoline-8-carboxamide, 9,10-didehydro-N-[1-(hydroxy-
methyl)propyl]-6-methyl-, [8B(5)]-, (Z)-2-butenedioate
(1:1) (salt);
9,10-Didehydro-N-[(5S)-1-(hydroxymethyl)propyl]-
6-methylergoline-8B-carboxamide maleate (1:1) (salt)
[57432-61-8].
DEFINITION
Methylergonovine Maleate contains NLT 97.0% and NMT
103.0% of methylergonovine maleate (C2oH2sN3O2 -
C4H4Ox), calculated on the dried basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
e B. The R; values of the principal fluorescent spot and the
principal blue spot of the Sample solution correspond to
those of the Standard stock solution, as obtained in the
test for Related Alkaloids.
Ea
“
a ASSAY
ii © PROCEDURE
— Diluent
io.) Seances this procedure with a minimum exposure to
} ight.
S Mobile phase: Acetonitrile and 2.0 g/L of monobasic
6 potassium phosphate (1:4)
= Diluent: 2.5 mg/mL of tartaric acid prepared as follows.
re Transfer a Satable amount of tartaric acid to a suitable
a)
volumetric flask, add 50% of the flask volume of water,
p=]
and mix with shaking. Dilute with methanol to volume.
Allow the mixture to cool before use.
Standard stock solution: 0.1 mg/mL of USP
Methylergonovine Maleate RS in Diluent. Shake by me-
chanical means for 15 min.
Standard solution: 4 g/mL of USP Methylergonovine
Maleate RS from the Standard stock solution in Diluent
Sample stock solution: 0.2 gira of Methylergo-
novine Maleate in Diluent. Shake by mechanical means
for 15 min or until completely dissolved.
Sample solution: 4 g/mL of Methylergonovine
Maleate from the Sample stock solution in Diluent
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: Fluorescence with excitation at 315 nm
and emission at 423 nm
USP 41
Column: 4.6-mm x 25-cm; packing L7
Temperature: 30°
Flow rate: 2 mL/min
Injection volume: 20 pL
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of methylergonovine maleate
(C2oH2sN3O2 - C4HgOx) in the portion of Methylergo-
novine Maleate taken:
Result = (ru/rs) x (Cs/Cu) x 100
ru = peak response from the Sample solution
Is = peak response from the Standard solution
Cs = concentration of USP Methylergonovine
Maleate RS in the Standard solution (\ug/mL)
Cu = concentration of Methylergonovine Maleate in
the Sample solution (\1g/mL)
Acceptance criteria: 97.0%-103.0% on the dried basis
IMPURITIES
© RESIDUE ON IGNITION (281): NMT 0.1%
e RELATED ALKALOIDS
Conduct this test promptly, without exposure to daylight
and with minimum exposure to artificial light. Solutions
containing methylergonovine maleate should be pre-
pared immediately before use.
Diluent: Alcohol and ammonium hydroxide (9:1)
Standard stock solution: 10 mg/mL of USP
Methylergonovine Maleate RS in Diluent
Standard solution A: 0.20 mg/mL of USP Methylergo-
novine Maleate RS from the Standard stock solution in
Diluent
Standard solution B: 0.10 mg/mL of USP Methylergo-
novine Maleate RS from the Standard stock solution in
Standard solution C: 0.05 mg/mL of USP Methylergo-
novine Maleate RS from the Standard stock solution in
Diluent
Sample solution: 10 mg/mL of Methylergonovine
Maleate in Diluent
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.)
Mode: TLC
Adsorbent: 0.25-mm layer of chromatographic silica
gel mixture
Application volume: 5 uL
Developing solvent system: Chloroform, methanol,
and water (75:25:3), equilibrated for 30 min
Spray reagent: 10 mg/mL of p-dimethylaminobenzal-
dehyde in a cooled mixture of alcohol and hydrochlo-
ric acid (1:1)
Analysis
Samples: Standard stock solution, Standard solution A,
Standard solution B, Standard solution C, and Sample
solution
Proceed as directed in the chapter. Locate the spots on
the plate by spraying thoroughly and evenly with
Spray reagent. Immediately dry in a stream of nitrogen
for 2 min.
Acceptance criteria: The R; value of the principal spot
of the Sample solution corresponds to that of the Stan-
dard stock solution. Estimate the concentration of any
other spots observed from the Sample solution by com-
parison with Standard solution A, Standard solution B,
and Standard solution C. The spots from Standard solu-
tion A, Standard solution B, and Standard solution C are
equivalent to 2.0%, 1.0%, and 0.50% of impurities, re-
spectively. The sum of the impurities is NMT 2.0%.
USP 41 Official Monographs / Methylergonovine 2677

SPECIFIC TESTS Mode: LC

© OPTICAL ROTATION (7815), Specific Rotation Detector: UV 240 nm
Sample solution: 5 mg/mL of Methylergonovine Column: 4-mm x 25-cm; packing L7
Maleate in water Column temperature: 30°
Acceptance criteria: +44° to +50° Flow rate: 2 mL/min
© PH (791) Injection size: 20 uL
Sample solution: 0.2 mg/mL of Methylergonovine System suitability
Maleate in water Sample: Standard solution
Acceptance criteria: 4.4-5.2 Suitability requirements
e Loss ON DRYING (731) Column efficiency: NLT 1000 theoretical plates
Analysis: Dry under vacuum at 80° to constant weight. Tailing factor: NMT 2.0
Acceptance criteria: NMT 2.0% Relative standard deviation: NMT 2.0%
Analysis
ADDITIONAL REQUIREMENTS Samples: Standard solution and Sample solution
© PACKAGING AND STORAGE: Preserve in tight, light-resistant Calculate the percentage of CzoH2sN3O2 - C4H4Ox in
containers, and store in a cold place. each mL of Injection taken:
e USP REFERENCE STANDARDS (11)
USP Methylergonovine Maleate RS Result = (ru/ts) x (Cs/Cu) x 100
tu = peak response from the Sample solution
Ts = peak response from the Standard solution
Cs = concentration of USP Methylergonovine
Methylergonovine Maleate Injection Maleate RS in the Standard solution (\ug/mL)
Cu = nominal concentration of the Sample solution
DEFINITION (g/mL)
Methylergonovine Maleate Injection is a sterile solution of Acceptance criteria: 90.0%-110.0%
Methylergonovine Maleate in Water for Injection. Each mL
IMPURITIES
contains NLT 90.0% and NMT 110.0% of the labeled
amount of methylergonovine maleate (C2oH2sN3O2 - Organic Impurities
C4H4O,). ¢ PROCEDURE: RELATED ALKALOIDS
[Note—Conduct this test promptly, without exposure to
IDENTIFICATION daylight and with minimum exposure to artificial light.]
e A. The R; values of the principal fluorescent spot and the Diluent: Alcohol and ammonium hydroxide (9:1)
principal blue spot of the Sample solution correspond to [Note—All solutions should be prepared immediately
those of the Standard stock solution, as obtained in the before use.]
procedure for Organic Impurities, Related Alkaloids. Standard stock solution: 10 mg/mL of USP
© B. PROCEDURE Methylergonovine Maleate RS in Diluent
Sample solution: 0.67 mg/mL of methylergonovine Standard solution A: 0.50 mg/mL of USP Methylergo- i
maleate from Injection in water novine Maleate RS from the Standard stock solution in a)
Analysis: The Sample solution exhibits a bluish fluores- Diluent ae]
cence under UV light. To this solution add 2 mL of a Standard solution B: 0.20 mg/mL of USP Methylergo- x
solution of glacial acetic acid in ethyl acetate (1:2), and novine Maleate RS from the Standard stock solution in °
stratify 2 mL of sulfuric acid, by pipetting, under the Diluent =]
Standard solution C: 0.10 mg/mL of USP Methylergo- °
solution. vo}
Acceptance criteria: A bluish-purple ring appears at the novine Maleate RS from the Standard stock solution in =
Diluent Ey
interface of the two liquids. mo)
Standard solution D: 0.05 mg/mL of USP Methylergo- >
ASSAY novine Maleate RS from the Standard stock solution in a
© PROCEDURE Diluent
[Note—Conduct this procedure with minimum exposure Sample solution: Transfer the equivalent of 5 mg of
to a methylergonovine maleate from Injection to a
Mobile phase: Acetonitrile and 0.015 M monobasic po- separator, and extract with three 5-mL portions of
tassium phosphate solution (1:4) chloroform. Discard the chloroform extracts. Render al-
Diluent: 5 mg/mL of tartaric acid in water and metha- kaline to litmus with 6 N ammonium hydroxide, and
nol (1:1). Allow the mixture to cool before use. [NOTE— extract with three 5-mL portions of chloroform. Evapo-
Dissolve tartaric acid with water, then add an equal vol- rate the combined extracts with the aid of a current of
ume of methanol.] air, but without heat, to dryness. Dissolve the residue
Standard solution: 100 g/mL of USP Methylergo- so obtained in 0.5 mL of Diluent.
novine Maleate RS in Diluent. [NoTE—Shakeby. mechan- Chromatographic system
ical means for 15 min or until completely dissolved.] (Gee aeaiilitled (621), Thin-Layer Chromatogra-
Sample solution: 100 g/mL of methylergonovine phy.
maleate from Injection in Diluent Mode: TLC
Chromatographic system Adsorbent: 0.25-mm layer of chromatographic silica
(See Chromatography (621), System Suitability.) gel mixture
Application volume: 5 ul
Developing solvent system: Chloroform, methanol,
and water (75:25:3), equilibrated for 30 min
Spray reagent: 10 mg/mL of p-dimethylamino-
enzaldehyde in a cooled mixture of alcohol and hy-
drochloric acid (1:1)
Analysis
Samples: Standard stock solution, Standard solution A,
Standard solution B, Standard solution C, Standard so-
lution D, and Sample solution
 2678 Methylergonovine / Official! Monographs
Proceed as directed in the chapter. Locate the spots
on the plate by spraying thoroughly and evenly with
Spray reagent. Immediately dry in a stream of nitro-
gen for 2 min.
Acceptance criteria: The R- value of the principal spot
from the Sample solution corresponds to that from the
Standard stock solution. Estimate the concentration of
any other spots observed in the lane for the Sample
solution by comparison with Standard solution A, Stan-
dard solution B, Standard solution C, and Standard solu-
tion D: the spots from the 0.50-, 0.20-, 0.10-, and
0.05-mg/mL dilutions are equivalent to 5.0%, 2.0%,
1.0%, and 0.50% of impurities, respectively. The sum
of the impurities is NMT 5.0%.
SPECIFIC TESTS
e PH (791): 2.7-3.5
© BACTERIAL ENDOTOXINS TEST (85): NMT 1.7 USP Endo-
toxin Units/ug of methylergonovine maleate
© OTHER REQUIREMENTS: Meets the requirements under /n-
jections and Implanted Drug Products (1)
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in single-dose, light-
resistant containers, preferably of Type | glass. Store in a
refrigerator.
Change to read:
° usP REFERENCE STANDARDS (11)
@ (CN 1-May-2018)
USP Methylergonovine Maleate RS
Methylergonovine Maleate Tablets
fe
”
a
S7 » Methylergonovine Maleate Tablets contain not
aD
°
less than 90.0 percent and not more than
= 110.0 percent of the labeled amount of
° methy’ ergonovinemaleate (CooH25N302 . C4H4Ox).
= tions, designated below by letter, having the following con-
a Packaging and storage—Preserve in tight, light-resistant
ai containers.
=
USP Reference standards (11)—
USP Methylergonovine Maleate RS
Identification—
A: The Ry values of the principal fluorescent spot and the
principal blue spot in the chromatogram of the Test prepara-
tion correspond to those in the chromatogram of the Stan-
dard preparation, as obtained in the test for Related alkaloids
under Ergonovine Maleate, using the Tablets instead of Ergo-
novine Maleate.
B: Transfer a quantity of powdered Tablets, equivalent to
about 4 mg of methylergonovine maleate, to a separator,
add 20 mL of water, and render alkaline to litmus with so-
dium carbonate solution (1 in 10). Extract with three 20-mL
portions of chloroform, filter the combined chloroform ex-
tracts into a small evaporating dish, and evaporate on a
steam bath to dryness. Dissolve the residue in a mixture of
6 mL of water and 0.3 mL of hydrochloric acid, and filter, if
necessary: the solution so obtained exhibits a bluish fluores-
cence under UV light. To this solution, add 2 mL of a solu-
tion of glacial acetic acid in ethyl acetate (1 in 2), and strat-
ify 2 mL of sulfuric acid, by pipetting, under the solution: a
bluish purple ring appears at the interface of the two liq-
uids.
USP 41
Dissolution (711)—
Medium: _ tartaric acid solution (1 in 200); 900 mL.
Apparatus 2: 75 rpm.
Time: 30 minutes.
Procedure—Filter a portion of the solution under test into
a flask. Concomitantly determine the fluorescence intensity
of this solution in comparison with a Standard solution of
USP Methylergonovine Maleate RS in the same medium
having a known concentration of about 0.22 tg per mL ina
fluorometer at an excitation wavelength of about 327 nm
and an emission wavelength of about 428 nm, using tartaric
acid solution (1 in 200) as the blank.
Tolerances—Not less than 70% (Q) of the labeled amount
of CroH2sN3Oz2- C4H4Os is dissolved in 30 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Related alkaloids—[NoTE—Conduct this test without ex-
posure to daylight and with the minimum exposure to artifi-
cial light.]
Solvent mixture—Mix 75 volumes of chloroform, 25
aoe of methanol, and 1 volume of ammonium hydrox-
ide.
Detecting reagent—Cautiously dissolve 800 mg of p-di-
methylaminobenzaldehyde in a mixture of alcohol and sul-
furic acid (101:11).
Test preparation—Transfer a quantity of finely powdered
Tablets, equivalent to 5.0 mg of methylergonovine maleate,
to a suitable container, add 50 mL of Solvent mixture, and
stir with the aid of a magnetic stirrer for 40 minutes. Filter,
rinsing the container with two 10-mL portions of Solvent
mixture. Evaporate the combined filtrates in vacuum at 25°
to 30°, and dissolve the residue in 2.0 mL of Solvent mixture.
Standard stock solution—Transfer 25 mg of USP
Methylergonovine Maleate RS to a 10-mL volumetric flask,
add Solvent mixture to volume, and mix to obtain a solution
having a known concentration of 2.5 mg per mL.
Standard preparations A, B, C, and D—Dilute accurately
measured volumes of Standard stock solution quantitatively
with Solvent mixture (designated below as parts by volume
of Standard stock solution in total parts by volume of the
finished Standard preparation) to obtain Standard prepara-
centrations and percentage assignments:
A: (1 in 20); 125 ug per mL (5.0%).
B: (1 in 33); 75 ug per mL (3.0%).
C: (1 in 100); 25 wg per mL (1.0%).
D: (1 in 200); 12.5 wg per mL (0.5%).
Procedure—Apply separately 20 uL of the Test preparation
and 20 uL of each Standard preparation to a suitable thin-
layer chromatographic plate (see Chromatography (621))
coated with a 0.25-mm aye! of chromatographic silica gel
mixture. Dry the plate with the aid of a stream of cool air.
Position the plate in a chromatographic chamber, and de-
velop the chromatograms in Solvent mixture until the solvent
front has moved about three-fourths of the length of the
plate. Remove the plate from the developing chamber, mark
the solvent front, and allow the solvent to evaporate in a
stream of cool air. Examine the plate under long-wavelength
UV light. Mark the principal and any secondary fluorescent
spots. Spray the plate with Detecting reagent, and mark the
principal and secondary blue spots. Compare the intensities
of any secondary spots observed in the chromatogram of
the Test preparation with those of the principal spots in the
chromatograms of the Standard preparations: the sum of the
intensities of secondary spots obtained from the Test prepa-
ration corresponds to not more than 5.0% of related com-
pounds.
Assay—[NOTE—Conduct this procedure with a minimum ex-
posure to light.]
USP 41 Official Monographs / Methylnaltrexone 2679

Mobile phase, Solvent mixture, Standard preparation, and Table 1

Chromatographic system—Proceed as directed in the Assay Time Solution A Solution B
under Methylergonovine Maleate.
(min) (%) (%)
Assay preparation—Place 10 Tablets in 1 500-mL volumet- 0 90 10
ric flask, add 400 mL of Solvent mixture, and shake by me-
15 30 70
chanical means for 15 minutes or until completely disinte-
grated. Dilute with Solvent mixture to volume, and mix. 50 30. 70
Allow the solution to settle for not less than 30 minutes
before use, and then filter to obtain the Assay preparation. Diluent: Solution A
Standard solution: 3.0 mg/mL of USP Methylnaltrex-
Procedure—Separately inject equal volumes (about 10 wL) one Bromide RS in Diluent
of the Standard preparation and the Assay preparation into Sample solution: 3.0 mg/mL of Methylnaltrexone Bro-
the chromatograph, record the chromatograms, and meas- mide in Diluent
ure the responses for the major peaks. Calculate the quan- Chromatographic system
tity, in mg, of methylergonovine maleate (C2oH2sN302 - (See Chromatography (621), System Suitability.)
C4H,O,) in the portion of Tablets taken by the formula: Mode: LC
Detector: UV 280 nm
(L/ D)(O(ru/ 5) Column: 4.6-mm x 25-cm; 3-4um packing L1
in whichL is the labeled quartiy. in mg, of methylergo- Column temperature: 50°
Flow rate: 1.0 mL/min
novine maleate in each Tablet, D is the concentration, in ug
per mL, of methylergonovine maleate in the Assay prepara- Injection volume: 50 uL
tion, based on the labeled quantity per Tablet and the ex- System suitability
tent of dilution, C is the concentration, in ug per mL, of Sample: Standard solution
USP Methylergonovine Maleate RS in the Standard prepara- Suitability requirements
tion, and ry and rs are the responses obtained from the As-
Tailing factor: NMT 2.0
say preparation and the Standard preparation, respectively. Relative standard deviation: NMT 0.73%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of methylnaltrexone bromide
(C2iH2sBrNOs) in the portion of Methylnaltrexone Bro-
mide taken:
Methylnaltrexone Bromide
Result = (ru/rs) x (Cs/Cu) x 100

ru = peak response from the Sample solution

ls = peak response from the Standard solution
ANTS oF Cs = concentration of USP MethyInaltrexone
Bromide RS in the Standard solution (mg/mL) c
Gy = concentration of Methylnaltrexone Bromide in a)
the Sample solution (mg/mL) 7
Co H26BrNO, 436.34 Acceptance criteria: 98.0%-102.0% on the anhydrous nx
Morphinanium, 17-(cyclopropylmethyl)-4,5-epoxy-3,14-
dihydroxy-17-methyl-6-oxo-, bromide, (5a,17R)-;
and solvent-free basis i}]
(17R)-17-(Cyclopropylmethyl)-4,50.-epoxy-3, 14-dihydroxy- IMPURITIES ey
17-methyl-6-oxomorphinanium bromide [916055-92-0]. e RESIDUE ON IGNITION (281): NMT 0.1% =
© ORGANIC IMPURITIES 2
DEFINITION Protect solutions containing methylnaltrexone bromide 1)=a
MethyInaltrexone Bromide contains NLT 98.0% and NMT from light. “
102.0% of methylnaltrexone bromide (C2H2sBrNO,), cal- Solution A, Solution B, Mobile phase, Diluent, Sample
culated on the anhydrous and solvent-free basis. solution, and Chromatographic system: Proceed as
directed in the Assay.
IDENTIFICATION System suitability solution: 3.0 mg/mL of USP
e A. INFRARED ABSORPTION (197): [NoTE—Methods de- MethyInaltrexone Bromide RS and 4.5 jug/mL of USP
scribed in (197K) or (197A) may be used.] Naltrexone RS in Diluent
e B. The retention time of the major peak of the Sample Sensitivity solution: 1.5 ug/mL of USP Methylnaltrex-
solution corresponds to that of the Standard solution, as one Bromide RS in Diluent
obtained in the Assay. Standard solution: 4.5 tug/mL of USP Methylnaltrexone
© C. IDENTIFICATION TESTS—GENERAL (191), Chemical Identifi- Bromide RS in Diluent
cation Tests, Bromide: Meets the requirements of the sil- System suitability
ver nitrate precipitation test Samples: System suitability solution, Sensitivity solution,
ASSAY and Standard solution
© PROCEDURE
Suitability requirements
Protect solutions containing methylnaltrexone bromide Resolution: NLT 1.5 between methylnaltrexone bro-
from light. mide and naltrexone, System suitability solution
Buffer: Dilute 3.0 mL of heptafluorobutyric acid with Relative standard deviation: NMT 5.0%, Standard
solution
water to 1000 mL. Adjust with ammonium hydroxide to
a pH of 2.4. Signal-to-noise ratio: NLT 10, Sensitivity solution
Solution A: Buffer and methanol (85:15) Analysis
Solution B: Buffer, methanol, and tetrahydrofuran Samples: Sample solution and Standard solution
(85:5:10) Calculate the percentage of each impurity in the por-
Mobile phase: See Table 1. Return to original condi- tion of Methylnaltrexone Bromide taken:
tions and re-equilibrate the system for about 15 min. Result = (ru/rs) x (Cs/Cu) x 100

ru = peak response of each impurity from the

Sample solution
 2680 Methylnaltrexone / Official Monographs USP 41

ls = peak response of methylnaltrexone from the Mode: LC

Standard solution Detector: UV 225 nm
Cs = concentration of USP Methylnaltrexone Column: 3.0-mm x 15-cm; 3.5-14m packing L1
Bromide RS in the Standard solution (mg/mL) Column temperature: 15°
Cu = concentration of Methylnaltrexone Bromide in Flow rate: 0.4 mL/min
the Sample solution (mg/mL) Injection volume: 20 LL
Acceptance criteria: See Table 2. The reporting thresh- System suitability
old is 0.05%. Sample: Standard solution
Suitability requirements
Table 2 Relative standard deviation: NMT 10.0%
Analysis
Relative Acceptance Samples: Peak identification solution, Standard solution,
Retention Criteria, and Sample solution
%’ Identify the peak due to methylnaltrexone related com-
pound A from the Peak identification solution. [NoTE—
Typical relative retention times for methylnaltrexone
and methylnaltrexone related compound A are 1.0
and 1.2, respectively.]
Calculate the amount, in ppm, of methylnaltrexone re-
lated compoundAin the portion of Methylnaltrexone
Bromide taken:
Itrexone®
Any individual unspecified Result = (ru/rs) x (Cs/Cu) « (1/F) x 1000
tu = peak response of methylnaltrexone related
tal_im, =
compound A from the Sample solution
4 (17S)-17-(Cyclopropylmethyl)-4,50-epoxy-3, 14-dihydroxy-1 7-methyl-6- rs = peak response of methylnaltrexone from the
oxomorphinanium bromide.
© (17RS)-17-(But-3-en-1-yl)-4,50-epoxy-3, 14-dihydroxy-1 7-methyl-6-ox- Standard solution
omorphinanium bromide. Cs = concentration of USP Methylnaltrexone
¢ 3-Acetyloxy-17-cyclopropylmethyl-4,50-epoxy-1 4-hydroxy-1 7-methyl-6- Bromide RS in the Standard solution (\ug/mL)
oxomorphinanium bromide. Cy = concentration of Methylnaltrexone Bromide in
417-(Cyclopropylmethyl)-4,5c.-epoxy-14-hydroxy-6-oxomorphinan-3-yl the Sample solution eee
acetate. P = relative response factor for methylnaltrexone
© (17RS)-17-(Cyclopropylmethyl)-4,5a-epoxy-14-hydroxy-17-methyl-3- related compound A, 1.3
methoxy-6-oxomorphinanium bromide.
Acceptance criteria: NMT 100 ppm
o LIMIT OF METHYLNALTREXONE RELATED COMPOUND A.
Buffer: 1.1 g/L solution of 1-octanesulfonate sodium SPECIFIC TESTS
Py salt in water (5 mM). Adjust with phosphoric acid to a PH (791)
ts Sample solution: 75 mg/mL in water
oy pH of 2.00 + 0.05.
Acceptance criteria: 4,3-5.3
S Solution A: Tetrahydrofuran and Buffer (50:950)
— e@ WATER DETERMINATION (921), Method |, Method la: NMT
Dp Solution B: Acetonitrile, tetrahydrofuran, and Buffer
fe} (600:50:350) . . 1.0%
a Mobile phase: See Table 3. Return to original condi- OPTICAL ROTATION (7815S), Procedures, Specific Rotation
5 tions and re-equilibrate the system for about 8 min. Sample solution: 10 mg/mL in water
= Acceptance criteria: —160° to -166°
re Table 3
MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
al FIED MICROORGANISMS (62): The total aerobic microbial
=) count is NMT 103 cfu/g, and the total combined yeasts
Solution A Solution B
and molds count is NMT 102 cfu/g.
0 o5' 5 BACTERIAL ENDOTOXINS TEST (85): Tine level of bacterial
endotoxins is such that the requirement under the rele-
36 6 14
vant dosage form enegraphrs) in which Methylnaltrex-
40 one Bromide is used can be met. Where the label states
that Methylnaltrexone Bromide must be subjected to fur-
ther processing during the preparation of injectable dos-
Diluent: Acetonitrile, water, and phosphoric acid age forms, the level of bacterial endotoxins is such that
(100: 900: 1.2). [NoTE—This is v:v:v ratio equivalent to the requirement under the relevant dosage form mono-
(100:900:2, v:v:w).] raph(s) in which Methylnaltrexone Bromide is used can
Peak identification solution: 5 mg/mL of USP e met.
Methylnaltrexone Peak Identification Mixture RS in
Diluent ADDITIONAL REQUIREMENTS
Standard solution: 0.5 j1g/mL of USP Methylnaltrexone © PACKAGING AND STORAGE: Preserve in well-closed contain-
Bromide RS in Diluent ers and store at room temperature.
Sample solution: 5 mg/mL of Methylnaltrexone Bro- e LABELING: Where it must be subjected to further process-
mide in Diluent ing during the preparation of injectable dosage forms to
Chromatographic system ensure acceptable levels of bacterial endotoxins,
(See Chromatography (621), System Suitability.) MethyInaltrexone Bromide is so labeled. Where it is ster-
ile, it is so labeled.
e USP REFERENCE STANDARDS (11)
USP Endotoxin RS
USP Methylnaltrexone Bromide RS
USP Methylnaltrexone Peak Identification Mixture RS
It contains Methylnaltrexone Bromide and a small
amount of methylnaltrexone related compound A:
USP 41 Official Monographs / Methylphenidate 2681

7,8-Didehydromethylnaltrexone bromide, or ABUK- Relative standard deviation: NMT 2.0% for the
Methylnaltrexone. methylphenidate peak
(17RS)-1 A ee atapy Analysis
dihydroxy-17-methyl-6-oxomorphinan-7-enium bro- Samples: Standard solution and Sample solution
mide. Calculate the percentage of methylphenidate hydro-
CaiHoaBrNOg 434.32 chloride (C)4HigNOz - HCl) in the portion of the sample
USP Naltrexone RS taken:
Result = (ru/rs) x (Cs/Cu) x 100
ru = peak response from the Sample solution
Methylphenidate Hydrochloride Is = peak response from the Standard solution
Cs = concentration of USP Methylphenidate
Hydrochloride RS in the Standard solution
(4 Cu
(mg/mL)
= concentration of Methylphenidate
ere a Hydrochloride in the Sample solution
[ole (mg/mL)
Acceptance criteria: 98.0%-102.0% on the dried basis

CS IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.1%

et Delete the following:

CisHieNO2 - HCl 269.77 °e HEAvY Metals, Method II (231): NMT 10 ppme coricatt-
2-Piperidineacetic acid, o-phenyl-, methyl ester, hydrochlo- Jan-2018)
ride, (R*,R*)-(4)-; © ORGANIC IMPURITIES, PROCEDURE 1
Buffer, Mobile phase, System suitability solution,
Methyl «-phenyl-2-piperidineacetate hydrochloride;
(RS)-Methyl-2-phenyl-2-[(R5)-piperidin-2-yl] acetate, hydro- Sample solution, Chromatographic system, and Sys-
chloride [23655-65-4]. tem suitability: Proceed as directed in the Assay.
Analysis
DEFINITION Sample: Sample solution
Methylphenidate Hydrochloride contains NLT 98.0% and Identify each impurity using the relative retention times
NMT 102.0% of methylphenidate hydrochloride in Table 7. Calculate the percentage of each impurity
(Cy4HigNOz2 - HCl), calculated on the dried basis. in the portion of Methylphenidate Hydrochloride
taken:
IDENTIFICATION Se
e A. INFRARED ABSORPTION (197M) Result = (ru/r) x 100 a)

tu = peak response for each impurity in the Sample =

Change to read: solution Ss
rr = sum of the responses for all impurity peaks ra)
o B. IDENTIFICATION TESTS—GENERAL (191), Chloride: Meets including the methylphenidate peak in the te]
the requirement °of test Ae cen 1-ay-2018) Sample solution S
ASSAY Acceptance criteria: See Table 1. me}
© PROCEDURE a
Buffer: 2.7 g/L of monobasic potassium phosphate Table 1
Mobile phase: Methanol and Buffer (1:2). Adjust with Relative Acceptance
phosphoric acid to a pH of 4.6 + 0.1. Retention Criteria,
System suitability solution: 0.005 mg/mL of USP Name Time NMT (%)
Methylphenidate Related Compound A RS and 0.5 mg/ Erythro isomera 0.58 0.15
mL of USP Methylphenidate Hydrochloride RS in the
Mobile phase Methylphenidate related compound
Standard solution: 0.5 mg/mL of USP Methylphenidate A 085 0.5
Hydrochloride RS in Mobile phase Methylphenidate 1.0 =
Sample solution: 0.5 mg/mL of Methylphenidate Hy- Any individual, unspecified impurity = 0.10
drochloride in Mobile phase Total impurities = 1.0
Chromatographic system 2 (RS)-Methyl-2-phenyl-2-[(SR)-piperidin-2-yl] acetate.
(See Chromatography (621), System Suitability.)
Mode: LC ¢ ORGANIC IMPURITIES, PROCEDURE 2
Detector: UV 209 nm [Note—Perform this test only if ethylphenidate or bis-
Column: 4.6-mm x 25-cm; 5-uum packing L1 1,2-(-carboxymethylbenzyl) piperidine is a known pro-
Flow rate: 1.0 mL/min cess impurity.]
Injection volume: 10 pL Buffer A: 5.7 g of monobasic ammonium phosphate
Run time: 2 times the retention time of and 1.6 g of 1-octanesulfonate sodium in 1 L of water
methylphenidate Buffer B: Add 4 mL of triethylamine to 1 L of Buffer A.
System suitability Adjust with phosphoric acid to a pH of 2.9.
Sample: System suitability solution Solution A: Acetonitrile and Buffer B (7:43)
Suitability requirements Solution B: Acetonitrile and BufferA (4:1)
Tailing actor: NMT 3.0 for the methylphenidate System suitability solution: 0.5 mg/mL of USP
peal Methylphenidate Hydrochloride RS; and 3 g/mL each
Resolution: NLT 2.5 between methylphenidate re- of USP Methylphenidate Related Compound A RS,
lated compound A and rriethwiphentaate
 2682 Methylphenidate / Official Monographs USP 41

phenylacetic acid, and USP Methylphenidate Hydrochlo- Table 3

tide Erythro Isomer Solution RS in Solution A Relative Relative Acceptance
Standard solution: 0.5 t1g/mL of USP Methylphenidate Retention Response Criteria,
Hydrochloride RS in Solution A Name Time Factor NMT (%)
Sample solution: 0.5 mg/mL of Methylphenidate Hy-
drochloride in Solution A. [NoTE—Allow the solution to Methylphenidate
stand for at least 2 h.] related compound A 0.55 45, 0.2
Mobile phase: See Table 2. (See also Chromatography Phenylacetic acid 0.67 1.0 0.1
(621), System Suitability). Erythro isomer= 0.80 1.0 0.2
Methylphenidate 1.0 = a=
Table 2 Ethylphenidate> 1.22 0.9 0.1
Solution A Solution B Bis-methylphenidates 1.80 2.6 0.1
‘mi %' %o’ Any individual, _
0 unspecified impurity 1.0 0.1
Total impurities — _— 0.5
Z 65
2 (RS)-Methyl-2-phenyl-2-[(SR)-piperidin-2-yl] acetate.
» Ethyl -2-phenyl-2-(piperidin-2-yl)acetate.
12 50
© 1,2-Bis(carboxymethylbenzyl)piperidine. [NoTE—Also known as 1,2-(a-
z carboxymethylbenzyl}piperiaine.)
16
SPECIFIC TESTS
[NoTte—Equilibration of the chromatographic system at e Loss ON DRYING (731)
the initial conditions for a minimum of 30 min is rec- Analysis: Dry a sample in a vacuum at 60° for 4 h.
ommended before the first injection.] Acceptance criteria: NMT 0.5%
Chromatographic system ADDITIONAL REQUIREMENTS
(See Chromatography (621), System Suitability.) © PACKAGING AND STORAGE: Preserve in well-closed
Mode: LC containers.
Detector: UV 220 nm e LABELING: If a test for Organic Impurities other than Proce-
Column: 3.9-mm x 15-cm; 5-um packing L7 dure 1 is used, then the labeling states the procedure
Column temperature: 40° with which the article complies.
Flow rate: 2.8 mL/min e USP REFERENCE STANDARDS (11)
Injection volume: 10 uL USP Methylphenidate Hydrochloride RS
System suitability USP Methylphenidate Hydrochloride Erythro Isomer Solu-
sample: System suitability solution. ore ey the tion RS
peaks using the relative retention times in Table 3.] This solution contains 0.5 mg of methylphenidate hy-
Suitability requirements drochloride erythro isomer per mL in methanol.
Resolution: NLT 2.7 between methylphenidate re- USP Methylphenidate Related Compound A RS
aos
”“
lated compound A and phenylacetic acid; NLT than
is 3.6 between phenylacetic acid and erythro isomer
a-Phenyl-2-piperidineacetic acid hydrochloride.
Ss CisHiyNOz2-HCl 255.74
— Tailing factor: NMT 2.0 for the methylphenidate
Dd eal
co}
c Relative standard deviation: NMT 2.0% for the
S methylphenidate peak; NMT 5.0% forcaer eile
= date related compound A, phenylacetic acid, and
[3 methylphenidate hydrochloride erythro isomer Methylphenidate Hydrochloride Tablets
a) Analysis
=) Samples: Standard solution and Sample solution DEFINITION
Calculate the percentage of any individual impurity in Methylphenidate Hydrochloride Tablets contain NLT 93.0%
the portion of Methylphenidate Hydrochloride taken: and NMT 107.0% of the labeled amount of methylpheni-
date hydrochloride (Ci4HisNOz - HCl).
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
IDENTIFICATION
ru = peak response for each impurity peak from the e A. INFRARED ABSORPTION (197M)
Sample solution Sample: Equivalent to 50 mg of methylphenidate hy-
rs = peak response for the methylphenidate peak drochloride from a portion of powdered Tablets in a
from the Standard solution 40-mL centrifuge tube. Add 10 mL of chloroform,
Gs = concentration of USP Methylphenidate shake, and centrifuge. Filter the clear extract through a
Hydrochloride RS in the Standard solution medium-sized sintered-glass funnel into a beaker, and
(mg/mL) repeat the extraction with an additional 10-mL portion
of chloroform. Evaporate the combined chloroform ex-
Cu = concentration of Methylphenidate
Hydrochloride in the Sample solution tracts on a steam bath to dryness. Agitate the dried
(mg/mL) residue with 2 mL of acetonitrile, and filter the mixture
F = relative response factor (see Table 3) through a small sintered-glass funnel. Wash the crystals
Acceptance criteria: See Table 3. with an additional 2 mL of acetonitrile, and dry them
with the aid of suction.
Acceptance criteria: Meet the requirements
ASSAY
¢ PROCEDURE
Buffer: Dissolve 1.6 g of anhydrous sodium acetate in
900 mL of water. Adjust with acetic acid to a pH of 4.0.
Dilute with water to 1 L.
USP 41
Mobile phase: Methanol, acetonitrile, and Buffer (4:3:3)
Internal standard solution: 0.4 mg/mL of phenyl-
ephrine hydrochloride in Mobile phase
Standard stock solution: 0.2 mg/mL of USP
Methylphenidate Hydrochloride RS in Mobile phase
Standard solution: Mix 10.0 mL of the Standard stock
solution with 5.0 mL of the Internal standard solution
Sample stock solution: 0.2 mg/mL of methylphenidate
hydrochloride from finely powdered Tablets (NLT 20
Tablets) prepared as follows. Dissolve in Mobile phase
using 70% of the final volume. Sonicate for 15 min,
and cool to room temperature. Dilute with Mobile phase
to volume. Pass a portion of this solution through a
suitable membrane filter, discarding the first portion of
the filtrate. Avoid the use of glass filters. Polypropylene
filters are suitable for use.
Sample solution: Mix 10.0 mL of the clear filtrate from
the Sample stock solution with 5.0 mL of the Internal
standard solution.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 210 nm
Column: 4.6-mm x 25-cm; packing L10
Flow rate: 1.5 mL/min
Injection size: 50 uL
System suitability
Sample: Standard solution
[Note—The relative retention times for phenylephrine
hydrochloride and methylphenidateteldioeblotide are
0.8 and 1.0, respectively.]
Suitability requirements
Resolution: NLT 2.0 between the analyte and the in-
ternal standard peaks
Relative standard deviation: NMT 2.0% from the
peak pApale ratios of the analyte to the internal
standar
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of methylphenidate hydro-
chloride (Cy4H1gNO2- HCI ) in the portion of Tablets
taken:
Result = (Ru/Rs) x (Cs/Cu) x 100
Ry = peakresponse ratio of the analyte to the
internal standard from the Sample solution
Rs = peak esponse ratio of the analyte to the
internal standard from the Standard solution
G = concentration of USP Methylphenidate
Hydrochloride RS in the Standard solution
(mg/mL)
Cu = nominal concentration of Methylphenidate
Hydrochloride in the Sample solution
(mg/mL)
Acceptance criteria: 93.0%-107.0%
PERFORMANCE TESTS
¢ DISSOLUTION (711), Procedure for a Pooled Sample
Medium: Water; 900 mL
Apparatus 1: 100 rpm
Time: 45 min
Analysis: Determine the amount of methylphenidate
hydrochloride (CisHisNOz - HCl) dissolved by using the
procedure in the Assay, making any necessary volumet-
tic adjustments.
Tolerances: NLT 75% (Q) of the labeled amount of
CisHigNO> - HCI is dissolved.
© UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
Official Monographs / Methylphenidate 2683
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers.
e USP REFERENCE STANDARDS (11)
USP Methylphenidate Hydrochloride RS
Methylphenidate Hydrochloride
Extended-Release Tablets
DEFINITION
Methylphenidate Hydrochloride Extended-Release Tablets
contain NLT 90.0% and NMT 110.0% of the labeled
amount of methylphenidate hydrochloride (Ci4Hi9NOz -
HCl).
IDENTIFICATION
e A. INFRARED ABSORPTION
Sample: Place a portion of powdered Tablets, equiva-
lent to 100 mg of methylphenidate hydrochloride, in a
100-mL beaker. Add 20 mL of chloroform, stir for 5
min, and filter, collecting the filtrate. Evaporate the fil-
trate to about 5 mL. Add ethyl ether slowly, with stir-
ring, until crystals form. Filter the crystals, wash with
ethyl ether, and dry at 80° for 30 min.
Acceptance criteria: The IR absorption spectrum of a
mineral oil oan of the crystals so obtained exhib-
its maxima only at the same wavelengths as those of a
ry preparation of USP Methylphenidate Hydrochlo-
ride RS.
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
ASSAY
(oe
Change to read: 4)
i]
© PROCEDURE cs
Mobile phase: Dissolve 2 g of octanesulfonic acid so- i}
dium salt in 730 mL of water. Adjust with phosphoric =
}
acid to a pH of 2.7. Mix with 270 mL of acetonitrile. ir}=
Solution A: Acidified water; adjusted with phosphoric 2
acid to a pH of 3 a}
Diluent A: Acetonitrile and Solution A (25:75) a
a]
Diluent B: Acetonitrile and methanol (50:50)
System suitability solution: 80 g/mL of USP
Methylphenidate Hydrochloride RS, 1 ug/mL of
methylphenidate hydrochloride erythro isomer from
USP Methylphenidate Hydrochloride Erythro lsomer So-
lution RS, and 2 g/mL of USP Methylphenidate Related
Compound A RS in Diluent A
Standard solution: 0.1 mg/mL of USP Methylphenidate
Hydrochloride RS in Diluent A
Sample stock solution: Nominally 1 mg/mL of
methylphenidate hydrochloride prepared as follows.
Dissolve NLT 10 Tablets in a suitable volumetric flask
with 20% of the total flask volume of Diluent B. [NOTE—
Alternatively, a portion of powder from NLT 10 Tablets
may be transferred to a suitable volumetric flask and
suspended in 20% of the total flask volume of Diluent
B.] Stir for 4 h. Dilute with Solution A to volume.
Sample solution: Nominally 0.1 mg/mL of
methylphenidate hydrochloride in Solution A from the
Sample stock solution. [NotE—Centrifuge before chro-
matographic analysis.]
 2684 Methylphenidate / Official Monographs USP 41

Chromatographic system Chromatographic system

(See Chromatography (621), System Suitability.) (See Chromatography (621), System Suitability.)
Mode: LC Mode: LC
Detector: UV 210 nm Detector: UV 210 nm
Column: 3.9-mm x 15-cm; 5-um packing L1 Column: 4.6-mm x 25-cm; packing L10
Column temperature: 30° Flow rate: 1.5 mL/min
Flow rate: 1 mL/min Injection volume: 50 LL
Injection volume: 25 ul System suitability
Run time: 2 times the retention time of Sample: Standard solution
methylphenidate [Note—The relative retention times for phenylephrine
System suitability hydrochloride and methylphenidate hydrochloride are
Samples: System suitability solution and Standard 0.8 and 1.0, respectively.]
solution Suitability requirements
[Note—See Table "80 (8 1-Apr-2017) for relative retention Resolution: NLT 2.0 between the analyte and inter-
times.] nal standard peaks
Suitability requirements Relative standard deviation: NMT 2.0% for the
Resolution: NLT 4.0 between ene ae re- peak papenie ratios of the analyte to the internal
lated compound A and methylphenidate hydrochlo- standar
ride erythro isomer; NLT 6.0 between the Analysis
methylphenidate and erythro isomer peaks, System Samples: Standard solution and Sample solution
suitability solution Calculate the percentage of the labeled amount of
Tailing factor: NMT 2.0 for the methylphenidate eyeredcas hydrochloride (Ci4HigNOz - HCl) dis-
peak, Standard solution solved by using the procedure in the Assay, making
Relative standard deviation: NMT 2.0% for the any necessary volumetric adjustments.
methylphenidate peak, Standard solution Tolerances: See Table 1.
Analysis
Samples: Standard solution and Sample solution Table 1
Calculate the percentage of the labeled amount of
methylphenidate hydrochloride (Ci4HigNOz - HCl) in Amount Dissolved
the portion of Tablets taken:
Result = (ru/rs) x (Cs/Cu) x 100

ty = peak response from the Sample solution

Is = peak response from the Standard solution
Cs = concentration of USP Methylphenidate Z,
Hydrochloride RS in the Standard solution
“” (mg/mL) The percentages of the labeled amount of
&
Gu = nominal concentration of methylphenidate methylphenidate hydrochloride (Ci4His3NOz - HCl) dis-
iy hydrochloride in the Sample solution solved at the times specified conform to Dissolution
A]
—
io.) (mg/mL) (711), Acceptance Table 2.
For products labeled for dosing every 24h
° Acceptance criteria: 90.0%-110.0%
= Test 2: If the product complies with this test, the label-
5 PERFORMANCE TESTS ing indicates that it meets USP Dissolution Test 2.
= Medium: Acidified water; adjusted with phosphoric
ie Change to read:
acid to a pH of 3; 50 mL at 37 +0.5°
Al Apparatus 7: 30 cycles/min; 2-3 cm amplitude. Follow
= Drug Release (724), General Drug Release Standards, Ap-
e DISSOLUTION (711) paratus 7, Sample preparation A ng a metal spring
Test 1 sample holder (Drug Release (724), Figure 5d). Place
Medium: Water; 500 mL one Tablet in the holder with the Tablet orifice facing
Apparatus 2: 50 rpm down, and cover the top of the holder with Parafilm™.
Times: 1, 2, 3.5, 5, and 7h At the end of each specified test interval, the systems
Buffer: Dissolve 1.6 g of anhydrous sodium acetate in are transferred to the next row of new test tubes con-
900 mL of water. Adjust with acetic acid to a pH of taining 50 mL of fresh Medium.
4.0 and dilute with water to 1000 mL. Times: 1-h intervals for a duration of 10 h
Mobile phase: Methanol, acetonitrile, and Buffer Calculate the percentage of the labeled amount of
(40:30:30) insee phen ate hydrochloride (Ci4HigNOz - HCl) dis-
Internal standard solution: 0.4 mg/mL of phenyl- solved by using the following method.
ephrine hydrochloride in Mobile phase Solution A: Dissolve 2.0 g of sodium 1-octanesulfonate
Standard stock solution: (1.5 x [L/500]) mg/mL of in 700 mL of water, mix well, and adjust with phos-
USP Methylphenidate Hydrochloride RS in Mobile phoric acid to a pH of 3.0.
hase whereL is the label claim of methylphenidate Mobile phase: Acetonitrile and Solution A (30:70)
hydrochloride in mg/Tablet Diluent: Acetonitrile and Medium (25:75)
Standard solution: Transfer 10.0 mL of the Standard Standard stock solution: 0.3 mg/mL of USP
stock solution to a glass-stoppered, 25-mL conical flask, Methylphenidate Hydrochloride RS in Diluent
add 5.0 mL of the Internal standard solution, and mix. Standard solutions: Prepare at least six solutions by
Sample stock solution: Use portions of the solution making serial dilutions of the Standard stock solution in
under test passed through a suitable filter of 0.45-um Diluent to bracket the expected drug concentration
pore size. Do not use glass fiber filters. range.
Sample solution: Transfer 10.0 mL of the Sample stock
solution to a ee 25-mL conical flask, add
5.0 mL of the /nternal standard solution, and mix.
USP 41 Official Monographs / Methylphenidate 2685

Chromatographic system Table 3

(See Chromatography (621), System Suitability.) Flow Rate
Mode: LC
Detector: UV 220 nm
Column: 3.2-mm x 5-cm; 5-um packing L1
Column temperature: 30°
Flow rate: 1 mL/min
Injection volume: 25 wL
System suitability 6.5
Sample: Middle range concentration of the Standard 7.
solutions
Suitability requirements Injection volume: 10 pL
Tailing factor: NMT 2 System suitability
Relative standard deviation: NMT 2% for the peak Sample: Standard solution
response of the analyte; NMT 2% for the retention [NoTe—The relative retention times for methylphenidate
time of the analyte related compound A, the erythro isomer, an
Analysis ces enigets are 0.47, 0.65, and 1.0, respectively.]
Samples: Standard solutions and the solution under Suitability requirements
test Relative standard deviation: NMT 2.0%
Construct a calibration curve by plotting the peak re- Analysis
sponse versus the concentration of the Standard solu- Samples: Standard solution and Sample solution
tions. Determine the amount of methylphenidate hy- Calculate the concentration (C) of methylphenidate
drochloride (Ci4HigNOz - HCl) in each interval by hydrochloride (C;4HigNO2- HCl) in the sample with-
linear regression analysis of the standard curve. aan from the vessel at each time point (/) shown in
Tolerances: See Table 2. Table 4:

Table 2 Result; = (ru/rs) x Cs

Amount Dissolved tu = sum of the peak responses of methylphenidate
and methylphenidate related compound A
1 12-32 from the Sample solution
rs = peak response of methylphenidate from the
10 T85 Standard solution
9 Cs = concentration of USP Methylphenidate
Hydrochloride RS in the Standard solution
The percentages of the labeled amount of Calculate the percentage of the labeled amount of
methylphenidate hydrochloride (Ci4HigNO2 + HCl) dis- methylphenidate hydrochloride (Ci4HisNOz - HCl)
solved at the times specified conform to Dissolution dissolved at each time point ()) shown in Table 4: c
“n
(711), Acceptance Table 2. 7v
Calculate the average percentage released from 3-6 h: Result; = C; x Vx (1/L) x 100
=
i}
Result = (Y
— X)/3
Resultz = {[C2 x (V— Vs)] + [Ci x Vs]} x (1/L) x 100 =|
}
% = cumulative drug released from 0-6 h a=
x = cumulative drug released from 0-3 h 2
Results = ({C3 x [V— (2 x Vs)]} + [((Go + Ci) x Vs}) x (1/
For products labeled for dosing every 24h
L) x'100 so}
Test 3: If the product complies with this test, the label- =e
“
ing indicates that it meets USP Dissolution Test 3. G = concentration of methylphenidate
Medium: pH 6.8 phosphate buffer (6.8 g/L of mono- hydrochloride in the portion of sample
basic poe in water; adjusted with 2 N withdrawn at time point (ij) (mg/mL)
sodium hydroxide or 10% phosphoric acid to a pH of Vv = volume of Medium, 900 mL
6.80); 900 mL E = label claim (mg/Tablet)
Apparatus 1: 100 rpm Vs = volume of the Sample solution withdrawn from
Times: 0.75, 4, and 10h
the Medium (mL)
Buffer: pH 4.0 nestle buffer (2.72 g/L of monoba- Tolerances: See Table 4.
sic potassium phosphate in water; adjusted with 2 N
con hydroxide or 10% phosphoric acid to a pH of
.00 Table 4
Mobile phase: Acetonitrile and Buffer (17.5: 82.5) Amount
Standard solution: 0.06 mg/mL of USP Methylpheni- Time Point Time Dissolved
date Hydrochloride RS in 0.1 N hydrochloric acid (i) (h) (%)
Sample solution: Pass a portion of the solution under 1 0.75 12-30
test througha suitable polytetrafluoroethylene (PTFE)
2 4 55-80
filter of 0.45-1um pore size.
Chromatographic system 3 10 NLT 80
(See Chromatography (621), System Suitability.)
Mode: LC The percentages of the labeled amount of
Detector: UV 210 nm methylphenidate hydrochloride (Cy4HigNOz2 « HCl) dis-
Column: 3.0-mm x 5-cm; 2.5-1m packing L1 solved at the times specified conform to Dissolution
Column temperature: 50° (711), Acceptance Table 2.
Flow rate: See Table 3. Test 4: If the product complies with this test, the label-
ing indicates that it meets USP Dissolution Test 4.
 2688 Methylphenidate / Official Monographs USP 41

lution RS, and 2 ug/mL of USP Methylphenidate Related Cy = nominal concentration of methylphenidate
CompoundA RS in Diluent A hydrochloride in the Sample solution
Standard solution: 0.2 g/mL of USP eee ae (mg/mL)
Hydrochloride RS, 0.5 g/m of methylphenidate hydro- Acceptance criteria: See Table °8.e (aa t-apr-2017)
chloride erythro isomer from USP Methylphenidate Hy-
drochloride Erythro Isomer Solution RS, and 1.5 ug/mL
Table °8e {RB 1-Apr-2017)
of bet Methylphenidate Related Compound A RS in Dil-
uent Relative Acceptance
Sample stock solution: Nominally 1 mg/mL of Retention Criteria,
methylphenidate hydrochloride prepared as follows. Name Time NMT (%)
Dissolve NLT 10 Tablets in a suitable volumetric flask Methylphenidate related
with 20% of the total flask volume of Diluent B. [NoTE— compound A 0.47 5
Alternatively, a portion of powder from NLT 10 Tablets Erythro isomer@ 0.65 0.5
may be transferred to a suitable volumetric flask and Methylphenidate 1.0 —
suspended in 20% of the total flask volume of Diluent Any unspecified on
B,] Stir for 4 h. Dilute with Solution A to volume.
degradation product 0.2
Sample solution: 0.1 mg/mL of methylphenidate hy-
drochloride in Solution A from the Sample stock solution. Total degradation ee
[Note—Centrifuge before chromatographic analysis.] products 26.
Chromatographic system 4Methyl (RS, SR)-2-phenyl-2-(piperidin-2-yl)acetate.
(See Chromatography (621), System Suitability.) ADDITIONAL REQUIREMENTS
Mode: LC © PACKAGING AND STORAGE: Preserve in tight containers.
Detector: UV 210 nm Store at controlled room temperature.
Column: 3.9-mm x 15-cm; 5-um packing L1 e LABELING: The labeling states the Dissolution test with
Column temperature: 30° which the product complies if other than Test 7.
Flow rate: 1 mL/min e USP REFERENCE STANDARDS (11)
Injection volume: 25 uL USP Methylphenidate Hydrochloride RS
Run time: 2 times the retention time of USP Methylphenidate Hydrochloride Erythro lsomer Solu-
methylphenidate tion RS
System suitability USP Methylphenidate Related Compound A RS
Sample: System suitability solution a-Phenyl-2-piperidineacetic acid hydrochloride.
Suitability requirements CisHiyNO2-HCl = 255.74
Resolution: NLT 6.0 between the methylphenidate
and erythro isomer peaks
Tailing factor: NMT 2.0 for the methylphenidate
eak
Relative standard deviation: NMT 2.0% for the
es
”“
methylphenidate peak; NMT 4.0% each for the Methylprednisolone
a methylphenidate related compound A and erythro
i
—
isomer peaks
Dp Analysis
iS) Samples: Standard solution and Sample solution
S Calculate the percentage of methylphenidate related
} compound Aor erythro isomer in the portion of Tab-
= lets taken:
a
a)
> Result = (ru/rs) x (Cs/Cu) x 100
Cx2H300s 374.47
ru = peak response of methylphenidate related Pregna-1,4-diene-3,20-dione, 11,17,21-trihydroxy-6-methyl-,
compound Aor erythro isomer from the (60,118)-.
Sample solution 11B,17,21-Trihydroxy-6a-methylpregna-1,4-diene-3,20-di-
Is peak response of methylphenidate related one [83-43-2].
compound A or erythro isomer from the
Standard solution » Methylprednisolone contains not less than
Cs = concentration of USP Methylphenidate Related 97.0 percent and not more than 103.0 percent of
CompoundA RS or methylphenidate C22H30Os, calculated on the dried basis.
hydrochloride erythro isomer in the Standard
solution (mg/mL) Packaging and storage—Preserve in tight, light-resistant
Cu = nominal concentration of methylphenidate containers.
hydrochloride in the Sample solution USP Reference standards (11)—
(mg/mL) USP Methylprednisolone RS
Calculate the percentage of any unspecified Identification—
degradation product in the portion of Tablets taken:
A: Infrared Absorption (197K).
Result = (ru/rs) x (Cs/Cu) x 100 B: Ultraviolet Absorption (197U)—
Solution: 10 ug per mL.
ru = peak response of each unspecified degradation
Medium: — alcohol.
product from the Sample solution Absorptivities at 243 nm, calculated on the dried basis, do
rs = peak response of USP Methylphenidate
Hydrochloride RS from the Standard solution not differ by more than 3.0%.
Cs = concentration of USP Methylphenidate C: Dissolve about 5 mg in 2 mL of sulfuric acid: a red
Hydrochloride RS in the Standard solution color is produced.
(mg/mL)
USP 41
Specific rotation (781S): between +79° and +86°.
Test solution: —5 mg per mL, in dioxane.
Loss on drying (731)—Dry it at 105° for 3 hours: it loses
not more than 1.0% of its weight.
Residue on ignition (281): not more than 0.2%.
Chromatographic purity—
Mobile phase—Preparea filtered and degassed mixture of
water, tetrahydrofuran, dimethyl sulfoxide, and butanol
(149:40:10:1). Make adjustments if necessary (see System
Suitability in Chromatography (621)).
Diluting solution—Prepare a filtered mixture of water, tet-
rahydrofuran, and glacial acetic acid (72:25:3).
Standard solution—Dissolve an accurately weighed quan-
tity of USP Methylprednisolone RS in Diluting soar. Dilute
quantitatively, and stepwise if necessary, with Diluting solu-
tion to obtain a solution having a known concentration of
about 0.01 mg per mL.
Test solution—Transfer about 25 mg of Methyl-
prednisolone, accurately weighed, to a 25-mL volumetric
flask, dissolve in and dilute with Diluting solution to volume,
and mix.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 254-nm detector
and a 4.6-mm x 20-cm column that contains packing L1.
The flow rate is about 1 mL per minute. Chromatograph the
Standard solution, and record:the peak responses as directed
for Procedure: the column efficiency is not less than 800 the-
oretical plates; and the relative standard deviation for repli-
cate injections is not more than 5.0%.
Procedure—Separately inject equal volumes (about 10 j1L)
of the Standard solution and the Test solution into the chro-
matograph, record the chromatograms, and measure the
peak responses. Calculate the percentage of each impurit
in the portion of Methylprednisolone taken by the formula:
100(Cs
/ Cu)(r
/ rs)
in which Cs and Cy are the concentrations, in mg per mL, of
Methylprednisolone in the Standard solution and the Test so-
lution, respectively; r; is the peak response for each impurity
obtained from the Test solution; and rs is the peak response
formethylprednisolone in the Standard solution: not more
than 1.0% of any individual impurity is found, and not
more than 2.0% of total impurities is found.
Assay—
Mobile phase—Prepare a solution containing a mixture of
butyl chloride, water-saturated butyl chloride, tetrahydro-
furan, methanol, and glacial acetic acid (475:475:70:35:30).
Internal standard solution—Dissolve prednisone in a 3 in
100 solution of glacial acetic acid in chloroform to obtain a
solution having a concentration of about 0.2 mg per mL.
Standard preparation—Dissolve an accurately weighed
quantity of USP Methylprednisolone RS in Internal standard
solution to obtain a solution having a known concentration
of about 0.2 mg per mL.
Assay preparation—Using about 10 mg of Methyl-
prednisolone, accurately weighed, proceed as directed for
Standard preparation.
Chromatographic system (see Chromatography)—The liq-
uid chromatograph is equipped with a 254-nm detector and
a 4-mm x 25-cm column that contains packing L3. The flow
rate is about 1 mL per minute. Chromatograph the Standard
preparation, and record the peak responses as directed for
Procedure: the resolution, R, between the methyl-
prednisolone and internal standard peaks is not less than
4.0; and the relative standard deviation for replicate injec-
tions is not more than 2.0%.
Procedure—Separately inject equal volumes (about 10 pL)
of the Standard preparation and the Assay preparation into
Official Monographs / Methylprednisolone 2689
the chromatograph, record the chromatograms, and meas-
ure the responses for the major peaks: the relative retention
times are about 0.7 for prednisone and 1.0 for methyl-
prednisolone. Calculate the quantity, in percent, of C22H300s
in the portion of Methylprednisolone taken by the formula:
100(Cs
/ Cu)(Ru
/ Rs)
in which Cs is the concentration of methylprednisolone, in
mg per mL, in the Standard preparation; Cy is the nominal
concentration, in mg per mL, of Methylprednisolone in the
Assay preparation; and Ry and Rs are the ratios of the peak
responses for the imethyiprecpiolonc peak and the internal
standard peak obtained from the Assay preparation and the
Standard preparation, respectively.
Methylprednisolone Tablets
» Methylprednisolone Tablets contain not less
than 92.5 percent and not more than 107.5 per-
cent of the labeled amount of methyl-
prednisolone (C22H30Os).
Packaging and storage—Preserve in tight containers.
USP Reference standards (11)—
USP Methylprednisolone RS
Identification—Powder a number of Tablets, equivalent to
about 40 mg of methylprednisolone, and digest with 25 mL
of solvent hexane for 15 minutes. Filter, and discard the fil-
trate. Digest the residue with 25 mL of chloroform for
15 minutes. Filter, evaporate the filtrate to dryness, and dry
at 105° for 2 hours: the residue so obtained responds to
Identification tests A and C under Methylprednisolone. =
Dissolution (711)— vA)
Medium: water; 900 mL.
i)
Apparatus 2: 50 rpm. cK
i}
Time: 30 minutes. 3
Procedure—Measure the UV absorption of filtered aliquots }
removed from the Dissolution Medium and suitably diluted, if
eo}=
Ey
necessary, in 1-cm cells at 246 nm, with a suitable spectro- 3
photometer, using water as the blank and utilizing a stan- my
dard curve, representing the absorbance versus concentra- nw
tion of USP Methylprednisolone RS. [NoTE—Dissolve about
20 mg of USP Methylprednisolone RS, accurately weighed,
in 1 mL of alcohol, dilute in a 1000-mL volumetric flask with
water to volume, and mix. Prepare quantitative dilutions of
this solution for the development of a standard curve.]
Tolerances—Not less than 70% (Q) of the labeled amount
of C22H30Os is dissolved in 30 minutes.
Uniformity of dosage units (905): meet the require-
ments.
PROCEDURE FOR CONTENT UNIFORMITY—
Mobile phase, Internal standard solution, Standard prepara-
tion, and See system—Proceed as directed in
the Assay under Methylprednisolone.
Test preparation—Place 1 Tablet in a suitable container.
For tablet labeled strengths of 10 mg or less, add 0.5 mL of
water. For tablet labeled strengths greater than 10 mg, add
1.0 mL of water. Allow the tablet to stand for about 2 min-
utes, then swirl the container to disperse the tablet. Add
5.0 mL of Internal standard solution for each mg of labeled
tabletstrenigu shake for 15 minutes, and filter or centrifuge
a portion of the test specimen. Analyze the clear solution as
directed under Procedure.
 2690 Methylprednisolone / Official Monographs USP 41

Procedure—Proceed as directed for Procedure in the Assay
under Methylprednisolone. Calculate the quantity, in mg, of
Co2H300s in the Tablet taken by the formula:
(FWs)(Ru / Rs)
in whichF is the ratio of the volume of Internal standard
preparation, in mL, in the Test preparation to the volume, in
mL, of the Internal standard preparation in the Standard
preparation, Ws is the weight, in mg, of USP Methyl-
prednisolone RS taken for the Standard preparation; and the
other terms are as defined for Procedure in the Assay under
Methylprednisolone.
Assay—
Mobile phase, Internal standard solution, Standard prepara-
tion, and Chromatographic system—Proceed as directed in
the Assay under Methylprednisolone.
Assay preparation—Accurately weigh 20 Tablets, and
grind to a fine powder in a mortar and pestle. Accurately
weigh a portion of the powder, equivalent to about 10 mg
of methylprednisolone, and transfer to a suitable container.
Add 2.5 mL of water to the ground tablet material and swirl
to form a fine slurry. Add 50.0 mL of Internal standard solu-
tion, and shake for 15 minutes. Filter or centrifuge a portion
of the liquid so obtained, if necessary, and analyze the clear
solution as directed under Procedure.
Procedure—Proceed as directed for Procedure in the Assay
underBea i pingone, Calculate the quantity, in mg, of
C22H30Os in the portion of Tablets taken by the formula:
SOC(Ru
/ Rs)
in which the terms are as defined therein.
Rs
2
cal
Methylprednisolone Acetate
a acetate to prednisone from the Standard
i
a CoaH3206 416.51 Cs
)= Pregna-1,4-diene-3,20-dione, 21-(acetyloxy)-11,17-
dihydroxy-6-methyl-, (6a,11B)-;
S 11B,17,21-Trihydroxy-6a-methylpregna-1,4-diene-3,20-di-
= one 21-acetate [53-36-1].
a”“
DEFINITION IMPURITIES
=) Methylprednisolone Acetate contains NLT 97.0% and NMT e RESIDUE ON IGNITION (281): NMT 0.2%
103.0% of methylprednisolone acetate (C24H320¢), calcu- © ORGANIC IMPURITIES
lated on the dried basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
e B. ULTRAVIOLET ABSORPTION (197U)
Analytical wavelength: 243 nm
Standard solution: 10 g/mL of USP Methyl-
prednisolone Acetate RS in alcohol
Sample solution: 10 ug/mL of methylprednisolone ace-
tate in alcohol
Acceptance criteria: Absorptivities, calculated on the
dried basis, do not differ by more than 3.0%.
ASSAY
¢ PROCEDURE
Mobile phase: n-Butyl chloride, water-saturated n-butyl
chloride, tetrahydrofuran, methanol, and glacial acetic
acid (95:95:14:7:6)
Internal standard solution: 6 mg/mL of prednisone
prepared as follows. Transfer an appropriate amount of
prednisone to a suitable volumetric flask. Add 3% of
the flask volume of glacial acetic acid, and sonicate. Di-
lute with chloroform to volume, slowly adding the chlo-
roform. Sonicate, and shake to dissolve.
Standard solution: 0.2 mg/mL of USP Methyl-
prednisolone Acetate RS prepared as follows. Transfer
an appropriate amount of USP Methylprednisolone Ace-
tate RS to a suitable volumetric flask, and add 5% of
the flask volume of the /nternal standard solution. Dilute
with chloroform to volume, and shake to dissolve.
Sample solution: 0.2 mg/mL of Methylprednisolone
Acetate prepared as follows. Transfer an appropriate
amount of methylprednisolone acetate to a suitable vol-
umetric flask, and add 5% of the flask volume of the
Internal standard solution. Dilute with chloroform to vol-
ume, and shake to dissolve.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4-mm x 25-cm; packing L3
Flow rate: 1 mL/min
Injection volume: 10 pL
System suitability
Sample: Standard solution
[Note—The relative retention times for methyl-
prednisolone acetate and prednisone are about 1.0
and 1.3, respectively.]
Suitability requirements
Resolution: NLT 2.5 between methylprednisolone
acetate and prednisone
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of methylprednisolone acetate
(C24H320¢) in the portion of Metis lnredrisolone Ace-
tate taken:
Result = (Ru/Rs) x (Cs/Cu) x 100
Ru = peak height ratio of methylprednisolone
acetate to prednisone from the Sample
solution
= peak height ratio of methylprednisolone
solution
= concentration of USP Methylprednisolone
Acetate RS in the Standard solution (mg/mL)
Cu = concentration of Methylprednisolone Acetate
in the Sample solution (mg/mL)
Acceptance criteria: 97.0%-103.0% on the dried basis
Mobile phase: Tetrahydrofuran and water (51:149)
Diluent: Tetrahydrofuran, acetonitrile, glacial acetic
acid, and water (250:250:1:499)
Standard solution: 20 g/mL of USP Methyl-
prednisolone Acetate RS in Diluent. Sonicate, if neces-
sary, to dissolve.
Sample solution: 1 mg/mL of Methylprednisolone Ace-
tate in Diluent. Sonicate, if necessary, to dissolve.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; packing L1
Flow rate: 1 mL/min
Injection volume: 20 pL
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 5.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the por-
tion of Methylprednisolone Acetate taken:
Result = (ru/rs) * (Cs/Cu) x 100
USP 41
tu = peak response for each impurity from the
Sample solution
rs = peak response for methylprednisolone acetate
from the Standard solution
Gs = concentration of USP Methylprednisolone
Acetate RS in the Standard solution (mg/mL)
Cu = concentration of Methylprednisolone Acetate
in the Sample solution (mg/mL)
Acceptance criteria
Any individual impurity: NMT 1.0%
Total impurities: NMT 2.0%
SPECIFIC TESTS
© OPTICAL ROTATION, Specific Rotation (781S)
Sample solution: 10 mg/mL in dioxane
Acceptance criteria: +97° to +105°
e Loss ON DRYING (731)
Analysis: Dry at 105° for 3 h.
Acceptance criteria: NMT 1.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers. Store at 25°, excursions permitted between
15° and 30°.
e USP REFERENCE STANDARDS (11)
USP Methylprednisolone Acetate RS
Methylprednisolone Acetate Cream
DEFINITION
Methylprednisolone Acetate Cream contains NLT 90.0% and
NMT 110.0% of the labeled amount of methyl-
prednisolone acetate (C24H3206.).
IDENTIFICATION
oA.
Analysis: Use the thin-layer chromatogram, prepared as
directed in Analysis 71 in the Assay.
Acceptance criteria: The R, value of the principal spot
from the Sample stock solution corresponds to that from
the Standard stock solution.
ASSAY
© PROCEDURE
(See scromatography (621).)
Solution A: Alcohol and chloroform (1:1)
Solution B: Alcohol and tetramethylammonium hydrox-
ide TS (9:1)
Standard stock solution: 500 tg/mL of USP Methyl-
prednisolone Acetate RS in Solution A
Sample stock solution: Transfer the equivalent of 5 mg
of methylprednisolone acetate from Cream to a 125-mL
separator, and add 50 mL of solvent hexane. Extract
with three 10-mL portions of acetonitrile, and evaporate
the combined extracts on a steam bath with the aid of
a current of air nearly to dryness. Transfer the residue to
a 10-mL volumetric flask with the aid of one 5-mL por-
tion and two 2-mL portions of Solution A, and dilute
with Solution A to volume.
Adsorbent: 0.5-mm layer of chromatographic silica gel
mixture
Application volume: 250 uL
Developing solvent system: Ethyl acetate and chloro-
form (7:5)
Analysis 1
Samples: Standard stock solution and Sample stock
solution
Divide the plate into three equal sections, with the left
and right sections to be used for the Sample stock solu-
tion and Standard stock solution, respectively, and the
center section for the blank. Apply the solutions as
streaks 2.5 cm from the bottom of the designated sec-
Official Monographs / Methylprednisolone 2691
tion of the plate, and dry the streaks with the aid of a
current of air. Develop the chromatogram in the De-
veloping solvent system until the solvent front has
moved about three-fourths of the length of the plate.
Remove the plate from the chamber, mark the solvent
front, and allow the solvent to evaporate. Locate the
principal bands from the Standard stock solution and
the Sample stock solution (see also Identification test A)
by viewing under short-wavelength UV light.
Standard solution, Sample solution, and Blank: Mark
the Standard stock solution and Sample stock solution
bands and the corresponding band section for the
Blank on the TLC plate from Analysis 1. Quantitatively
remove the silica gel containing these bands, and trans-
fer to separate glass-stoppered, 50-mL centrifuge tubes.
Add 25.0 mL of alcohol to each tube, shake for 2 min,
and centrifuge for 5 min. Transfer 20.0 mL of each su-
ernatant to separate glass-stoppered, 50-mL conical
lasks. Add 2.0 mL of blue tetrazolium TS to each solu-
tion, mix, and add 2.0 mL of Solution B to each flask.
Mix, and allow the solutions to stand in the dark for 90
min.
Instrumental conditions
Mode: Vis
Analytical wavelength: Maximum absorbance at
about 525 nm
Cell: 1.cm
Analysis 2
Samples: Standard solution, Sample solution, and Blank
Determine the absorbances of the Standard solution and
the Sample solution at the wavelength of maximum
absorbance against the Blank.
Calculate the percentage of the labeled amount of
methylprednisolone acetate (C24H3206¢) in the portion
of Cream taken:
Result = (Ay/As) x (Cs/Cu) x 100
(<a
Au = absorbance of the Sample solution y
As = absorbance of the Standard solution Me
Cs = concentration of USP Methylprednisolone =
Acetate RS in the Standard stock solution °
(ug/mL) % . 3
Cu = nominal concentration of methylprednisolone ol
in the Sample stock solution (g/mL) Ps
Acceptance criteria: 90.0%-110.0% no}
2
PERFORMANCE TESTS od
e Minimum FILL (755): Meets the requirements
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in collapsible tubes or
in rae containers, protected from light.
e USP REFERENCE STANDARDS (11)
USP Methylprednisolone Acetate RS
Methylprednisolone Acetate Injectable
Suspension
DEFINITION
Methylprednisolone Acetate Injectable Suspension is a sterile
suspension of Methylprednisolone Acetate in a suitable
aqueous medium. It contains NLT 90.0% and NMT
110.0% of the labeled amount of methylprednisolone
acetate (C24H3206).
IDENTIFICATION
¢ A. INFRARED ABSORPTION (197K)
Sample: Nominally 100 mg of methylprednisolone ace-
tate from taecople Suspension
 2692 Methylprednisolone / Official Monographs
Analysis: Filter the Sample through paper. Wash the res-
idue with several 5-mL portions of water, and dry at
105° for 3h.
Acceptance criteria: Meets the requirements
ASSAY
© PROCEDURE
Mobile phase: n-Butyl chloride, water-saturated n-butyl
chloride, tetrahydrofuran, methanol, and glacial acetic
acid (95:95:14:7:6)
Internal standard solution: 6 mg/mL of prednisone
prepared as follows. Transfer an appropriate amount of
prednisone to a suitable volumetric flask. Add 3% of
the flask volume of glacial acetic acid, and sonicate. Di-
lute with chloroform to volume, slowly adding the chlo-
roform. Sonicate, and shake to dissolve.
Standard solution: 0.2 mg/mL of USP Methyl-
prednisolone Acetate RS prepared as follows. Transfer
an appropriate amount of USP Methylprednisolone Ace-
tate RS to a suitable volumetric flask, and add 5% of
the flask volume of the Internal standard solution. Dilute
with chloroform to volume, and shake to dissolve.
Sample solution: Swirl Injectable Suspension to ensure
uniformity before analysis. Transfer a suitable guar
of Injectable Suspension equivalent to 40 mg of methyl-
prednisolone acetate to a 25-mL volumetric flask, ad
10.0 mL of the /nternal standard solution, dilute with
chloroform to volume, and shake for 15 min or until
the aqueous layer is clear. Transfer 4.0 mL of the chloro-
form layer to a suitable vial, add 30 mL of chloroform
and a small quantity (about 400 mg) of anhydrous so-
dium sulfate, shake for 5 min. Use the clear solution.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4-mm x 25-cm; packing L3
Flow rate: 1 mL/min
fm
a)
a Injection volume: 10 uL
i System suitability
—
oy Sample: Standard solution
-) [Note—The relative retention times for methyl-
S prednisolone acetate and prednisone are 1.0 and 1.3,
3 respectively.]
Ps Suitability requirements
is Resolution: NLT 2.5 between methylprednisolone
al
=) acetate and prednisone
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
methylprednisolone acetate (C24H32O¢) in the portion
of Injectable Suspension taken:
Result = (Ru/Rs) x (Cs/Cu) x 100
Ru = peak height ratio of methylprednisolone
acetate to prednisone from the Sample
solution
Rs = peak height ratio of methylprednisolone
acetate to prednisone from the Standard
solution
Cs = concentration of USP Methylprednisolone
Acetate RS in the Standard solution (mg/mL)
Cu = nominal concentration of methylprednisolone
acetate in the Sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0%
PERFORMANCE TESTS
e UNIFORMITY OF DOSAGE UNITS (905): Meets the
requirements
USP 41
SPECIFIC TESTS
° PH (791): 3.0-7.0
© PARTICLE SIZE
Analysis: Transfer 1 drop to a microscope slide, and
spread it evenly, diluting with water if necessary, to de-
crease the density of the field. Examine the slide under
a microscope equipped with a calibrated ocular mi-
crometer, using 400x magnification. Scan the entire
slide, and note the size of the individual particles.
Acceptance criteria: NLT 99% of the particles are less
than 20 um in length when measured along the long-
est axis, and NLT 75% of the particles are less than 10
um.
© OTHER REQUIREMENTS: It meets the requirements in /njec-
tions and Implanted Drug Products (1).
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in single-dose or mul-
tiple-dose containers, preferably of Type | glass.
e USP REFERENCE STANDARDS (11)
USP Methylprednisolone Acetate RS
Methylprednisolone Hemisuccinate
CasH34Os 474.54
Pregna-1,4-diene-3,20-dione, 21-(3-carboxy-1-oxopropoxy)-
11,17-dihydroxy-6-methyl-, (6a,118)-;
11B,17,21-Trihydroxy-6a-methylpregna-1,4-diene-3,20-di-
one 21-(hydrogen succinate) [2921-57-5].
DEFINITION
Methylprednisolone Hemisuccinate contains NLT 97.0% and
NMT 103.0% of methylprednisolone hemisuccinate
(C26H34Os), calculated on the dried basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197M)
e B. ULTRAVIOLET ABSORPTION (197U)
Standard solution: 20 g/mL of USP Methyl-
prednisolone Hemisuccinate RS in alcohol
Sample solution: 20 g/mL of methylprednisolone
hemisuccinate in alcohol
Analytical wavelength: 243 nm
Acceptance criteria: Absorptivities, calculated on the
dried basis, do not differ by more than 3.0%.
ASSAY
e PROCEDURE
Solution A: Chloroform and glacial acetic acid (97:3)
Mobile phase: Butyl chloride, water-saturated butyl
chloride, tetrahydrofuran, methanol, and glacial acetic
acid (95:95:14:7:6)
Internal standard solution: 6 mg/mL of USP
Fluorometholone RS in tetrahydrofuran
Standard solution: 0.4 mg/mL of USP Methyl-
prednisolone Hemisuccinate RS prepared as follows.
Transfer a suitable quantity of USP Methylprednisolone
Hemisuccinate RS to a suitable volumetric flask, and
add 5.0% of the flask volume of Internal standard solu-
tion. Dilute with Solution A to volume.
Sample solution: 0.4 mg/mL of Methylprednisolone
Hemisuccinate prepared as follows. Transfer a suitable
quantity of Methylprednisolone Hemisuccinate to a suit-
able volumetric flask, and add 5.0% of the flask volume
of intemal standard solution. Dilute with Solution A to
volume.
USP 41
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4-mm x 30-cm; packing L3
Injection volume: 4-8 pL
System suitability
Sample: Standard solution
Suitability requirements
Resolution: NLT 2.0 between methylprednisolone
hemisuccinate and fluorometholone
Relative standard deviation: NMT 2.0% for six repli-
cate injections
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of methylprednisolone hemi-
succinate (C26H340g) in the portion of Methyl-
prednisolone Hemisuccinate taken:
Result = (Ru/Rs) x (Cs/Cu) x 100
Ry = peak area ratio of methylprednisolone
hemisuccinate to fluorometholone from the
Sample solution
Rs = peak area ratio of methylprednisolone
hemisuccinate to fluorometholone from the
Standard solution
Cs = concentration of USP Methylprednisolone
Hemisuccinate RS in the Standard solution
(mg/mL)
Cu = concentration of Methylprednisolone
Hemisuccinate in the Sample solution
(mg/mL)
Acceptance criteria: 97.0%-103.0% on the dried basis
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.2%
© ORGANIC IMPURITIES
Mobile phase: Tetrahydrofuran, formic acid, and water
(255:1:745)
Diluent: Tetrahydrofuran, acetonitrile, acetic acid, and
water (25:25:3:47)
Standard solution: 0.02 mg/mL of USP Methyl-
prednisolone Hemisuccinate RS in Diluent
Sample solution: 1 mg/mL of Methylprednisolone
Hemisuccinate in Diluent. Shake or sonicate to aid in
solubilization.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 20-cm; 5-um packing L1
Flow rate: 1 mL/min
Injection volume: 20 pL
System suitability
Sample: Standard solution
Suitability requirements
Column efficiency: NLT 5000 theoretical plates
Relative standard deviation: NMT 5.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the por-
tion of Methylprednisolone Hemisuccinate taken:
Result = (ru/rs) x (Cs/Cu) x 100
ty = peak area of each impurity from the Sample
solution
ts = peak area of methylprednisolone
hemisuccinate from the Standard solution
Cs = concentration of USP Methylprednisolone
Hemisuccinate RS in the Standard solution
(mg/mL)
Official Monographs / Methylprednisolone 2693
Cu = concentration of Methylprednisolone
Hemisuccinate in the Sample solution
(mg/mL)
Acceptance criteria
Any individual impurity: NMT 1.0%
Total impurities: NMT 2.0%
SPECIFIC TESTS
e OPTICAL ROTATION, Specific Rotation (781S)
Sample solution: 10 mg/mL of Methylprednisolone
Hemisuccinate in dioxane
Acceptance criteria: +87° to +95°
e Loss ON DRYING (731)
Analysis: Dry at 105° for 3 h.
Acceptance criteria: NMT 1.0%
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers.
e USP REFERENCE STANDARDS (11)
USP Fluorometholone RS
USP Methylprednisolone Hemisuccinate RS
Methylprednisolone Sodium Succinate
Co6H33NaOg 496.53
Pregna-1,4-diene-3,20-dione, 21-(3-carboxy-1-oxopropoxy)-
11,17-dihydroxy-6-methyl-, monosodium salt, (60,11B)-;
11B,17,21-Trihydroxy-6a-methylpregna-1,4-diene-3,20-di-
one 21-(sodium succinate) [2375-03-3].
DEFINITION
Methylprednisolone Sodium Succinate contains NLT 97.0%
and NMT 103.0% of methylprednisolone sodium succi-
nate (C2sH33NaQOg), calculated on the dried basis.
(al
IDENTIFICATION A)
e A. INFRARED ABSORPTION x]
Sample: 100 mg of Methylprednisolone Sodium =
Succinate °
Analysis: Transfer the Sample to a separator, dissolve in 3
10 mL of water, add 1 mL of 3 N hydrochloric acid, and fo]
Xo}
extract immediately with 50 mL of chloroform. Filter the +
2
chloroform extract through cotton, evaporate on a me]
steam bath to dryness, and dry under vacuum at 60° >
or 3h. a
Acceptance criteria: The IR absorption spectrum of a
mineral oil dispersion of the residue so obtained exhib-
its maxima only at the same wavelengths as those of a
similar preparation of USP Methylprednisolone Hemisuc-
cinate RS.
e B. ULTRAVIOLET ABSORPTION (197U)
Standard solution: 20 g/mL of USP Methyl-
prednisolone Hemisuccinate RS in methanol
Sample solution: 20boimt, of Methylprednisolone So-
dium Succinate in methanol
Analytical wavelength: 243 nm
Acceptance criteria: Absorptivities, calculated on the
dried basis, do not differ by more than 3.0%.
e C. The sample imparts an intense yellow color to a
nonluminous flame.
ASSAY
e PROCEDURE
Solution A: 5 mg/mL of blue tetrazolium in alcohol
Solution B: Alcohol and tetramethylammonium hydrox-
ide TS (9:1)
Standard solution: Proceed as directed for Assay for
Steroids (351), Standard Preparation, preparing 12.5 wg/
mL of USP Methylprednisolone Hemisuccinate RS in
alcohol.
Sample solution: 12.5 ug/mL of Methylprednisolone
Sodium Succinate in alcohol
 2694 Methylprednisolone / Official Monographs
Blank: Alcohol
Instrumental conditions
Mode: Vis
Analytical wavelength: 525 nm
Analysis
Samples: Standard solution, Sample solution, and Blank
Pipet 20.0 mL of the Blank, Standard solution, and Sam-
ple solution into three different glass-stoppered, 50-mL
conical flasks. Add 2.0 mL of Solution A, and mix. To
each flask add 4.0 mL of Solution B. Mix, and allow to
stand in the dark for 90 min. Add 1.0 mL of glacial
acetic acid, and mix. Without delay, determine the ab-
sorbances of the Standard solution and the Sample so-
lution against the Blank.
Calculate the percentage of methylprednisolone sodium
succinate (CzsH33NaQOg) in the portion of Methyl-
prednisolone Sodium Succinate taken:
Result = (Au/As) x (Cs/Cu) x (Ma/M,2) x 100
Au = absorbance of the Sample solution
As = absorbance of the Standard solution
Cs = concentration of USP Methylprednisolone
Hemisuccinate RS in the Standard solution
Cy
(ug/ml)
= concentration of Methylprednisolone Sodium
Succinate in the Sample solution (ug/mL)
Ma = molecular weight of methylprednisolone
sodium succinate, 496.53
Mz = molecular weight of methylprednisolone
hemisuccinate, 474.54
Acceptance criteria: 97.0%-103.0% on the dried basis
OTHER COMPONENTS
e SODIUM CONTENT
Sample solution: Dissolve, with gentle heating, about
1g of Methylprednisolone Sodium Succinate in 75 mL
of glacial acetic acid. Add 20 mL of dioxane, then add
fe
“a
1 drop of crystal violet TS.
a Titrimetric system
i]
— Mode: Direct
a tuted solutions (mix the constituted solutions prepared
° Titrant: 0.1 N perchloric acid VS
= Endpoint detection: Visual
S Analysis: Titrate with Titrant to a blue-green endpoint.
= Perform a blank determination, and make any necessary
a correction. Each mL of Titrant is equivalent to 2.299 mg
a) of sodium (Na).
=) Acceptance criteria: 4.49%-4.77% on the dried basis
SPECIFIC TESTS
e OPTICAL ROTATION (7815S), Specific Rotation
Sample solution: 10 mg/mL of Methylprednisolone So-
dium Succinate in alcohol
Acceptance criteria: +96° to +104°
e Loss ON DRYING (731)
Analysis: Dry at 105° for 3 h.
Acceptance criteria: NMT 3.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers.
e USP REFERENCE STANDARDS (11)
USP Methylprednisolone Hemisuccinate RS
Methylprednisolone Sodium Succinate
for Injection
DEFINITION
Methylprednisolone Sodium Succinate for Injection is a ster-
ile mixture of Methylprednisolone Sodium Succinate with
suitable buffers. It may be prepared from Methyl-
prednisolone Sodium Succinate or from Methyl-
USP 41
prednisolone Hemisuccinate with the aid of Sodium Hy-
droxide or Sodium Carbonate. It contains the equivalent
of NLT 90.0% and NMT 110.0% of the labeled amount
of methylprednisolone (C22H300s) in the volume of consti-
tuted solution designated on the label.
IDENTIFICATION
© A. INFRARED ABSORPTION
Sample: Nominally 100 mg of methylprednisolone so-
dium succinate from Methylprednisolone Sodium Succi-
nate for Injection
Analysis: Transfer the Sample to a separator, dissolve in
10 mL of water, add 1 mL of 3 N hydrochloric acid, and
extract immediately with 50 mL of chloroform. Filter the
chloroform extract through cotton, evaporate on a
seat pe to dryness, and dry under vacuum at 60°
or 3h.
Acceptance criteria: The IR absorption spectrum of a
mineral oil dispersion of the residue so obtained exhib-
its maxima only at the same wavelengths as those of a
similar preparation of USP Methylprednisolone Hemisuc-
cinate RS.
ASSAY
* PROCEDURE si Bae Le
Diluent: Chloroform and glacial acetic acid (97:3)
Mobile phase: Butyl chloride, water-saturated butyl _
chloride, tetrahydrofuran, methanol, and glacial acetic
acid (95:95:14:7:6) _
Standard stock solution: 0.30 mg/mL of USP Methyl-
prednisolone RS in Diluent
Internal standard solution: 3 mg/mL of USP
Fluorometholone RS in tetrahydrofuran
Standard solution: 0.65 mg/mL of USP Methyl-
prednisolone Hemisuccinate RS prepared as follows.
‘Transfer an appropriate amount of USP Methyl-
prednisolone Hemisuccinate RS to a suitable volumetric
flask. Pipet 10% of the flask volume of the Internal stan-
dard solution and 10% of the flask volume of the Stan-
dard stock solution. Dilute with Diluent to volume.
Sample solution: Transfer a suitable quantity of consti-
from the contents of 10 vials of Methylprednisolone So-
dium Succinate for Injection) equivalent to 50 mg of
methylprednisolone to a suitable flask eonbainiag
10.0 mL of the Internal standard solution, and dilute
with Diluent to 100.0 mL. Shake thoroughly for 5 min,
fey allow the phases to separate, discarding the upper
phase.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 3.9-mm x 30-cm; packing L3
Flow rate: 1 mL/min
Injection volume: 6 pL
[NoTe—The order of elution of peaks in the Standard so-
lution is as follows. Internal standard peak, methyl-
prednisolone hemisuccinate peak, and successive
smaller peaks of free methylprednisolone and methyl-
prednisolone 17-hemisuccinate.]
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
methyprednisolons (C22H30Os) in the portion of con-
stituted Methylprednisolone Sodium Succinate for In-
jection taken:
Result = (Ru/Rs) x (Cs/Cu) x (Mn/M2) x 100
Ry = ratio of the sum of the peak areas for
methylprednisolone hemisuccinate and
methylprednisolone 17-hemisuccinate to the
peak area of the internal standard from the
Sample solution
USP 41 Official Monographs / Methyltestosterone 2695

Rs = ratio of the sum of the peak areas for Change to read:

methylprednisolone hemisuccinate and
methylprednisolone 17-hemisuccinate to the e USP REFERENCE STANDARDS (11)
peak area of the internal standard from the * KEN T-May-2018)
Standard solution USP Fluorometholone RS
Cs = concentration of USP Methylprednisolone USP Methylprednisolone RS
Hemisuccinate RS in the Standard solution USP Methylprednisolone Hemisuccinate RS
(mg/mL)
Cu = nominal concentration of methylprednisolone
in the Sample solution (rain)
Mn = molecular weight of methylprednisolone,
374.47
Mz = molecular weight of methylprednisolone Methyltestosterone
hemisuccinate, 474.54
To this calculated amount, add the percentage of free
methylprednisolone found in the test for Free
Methylprednisolone.
Acceptance criteria: 90.0%-110.0%
aes
pl
_ OPH,

oF Za

OTHER COMPONENTS
¢ FREE METHYLPREDNISOLONE
Analysis C20H3002 302.45
Samples: Standard solution and Sample solution Androst-4-en-3-one, 17-hydroxy-17-methyl-, (178)-;
Using the chromatograms obtained in the Assay, meas- 17B-Hydroxy-17-methylandrost-4-en-3-one [58-18-4].
ure the areas of the peaks from the internal standard DEFINITION
and free methylprednisolone. Methyltestosterone contains NLT 97.0% and NMT 103.0%
Calculate the percentage of free methylprednisolone in of methyltestosterone (C20H3002), calculated on the dried
the portion of Sample solution taken: basis.
Result = (Ru/Rs) x (Cs/Cu) x 100 IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
Ru = peak area ratio of the free methylprednisolone e B. The retention time of the major peak of the Sample
to the internal standard from the Sample solution corresponds to that of the Standard solution, as
solution obtained in the Assay.
Rs = peak area ratio of the free methylprednisolone
to the internal standard from the Standard ASSAY
solution ¢ PROCEDURE
G = concentration of USP Methylprednisolone RS Mobile phase: Acetonitrile and water (55:45)
in the Standard solution (mg/mL) iS
Standard stock solution: 0.25 mg/mL of USP wn
Cu = nominal concentration of methylprednisolone Methyltestosterone RS in methanol a)
in the Sample solution (mg/mL) Standard solution: 20 g/mL of USP Methyltestoster- =
Acceptance criteria: The amount of free methyl- one RS in Mobile phase from the Standard stock solution i}
prednisolone is NMT 6.6% of the labeled amount of System suitability stock solution: 250 g/mL of USP PJ
methylprednisolone (C22H300s). estosterone RS in methanol i}
System suitability solution: Dilute 4 mL of the System
Re}=
PERFORMANCE TESTS 2
© UNIFORMITY OF DOSAGE UNITS (905): Meets the
suitability stock solution with the Standard solution to ko}
requirements
50 mL. me
Pd
Sample stock solution: 0.50 mg/mL of Methyltestoster-
SPECIFIC TESTS one in methanol
e PH (791) Sample solution: 20 1g/mL of Methyltestosterone in
Sample solution: 50 mg/mL of methylprednisolone so- Mobile phase from the Sample stock solution
dium succinate Chromatographic aoe
Acceptance criteria: 7.0-8.0 (See Chromatography (621), System Suitability.)
eo STERILITY TESTS (71): Meets the requirements Mode: LC
¢ BACTERIAL ENDOTOXINS TEST (85): NMT 0.17 USP Endo- Detector: UV 241 nm
toxin Units/mg of methylprednisolone Column: 4-mm x 25-cm; packing L1
© Loss ON DRYING (731) Flow rate: 1 mL/min
Analysis: Dry at 105° for 3 h. Injection volume: 50 uL
Acceptance criteria: NMT 2.0% System suitability
e CONSTITUTED SOLUTION: At the time of use, it meets the Samples: Standard solution and System suitability
requirements for Injections and Implanted Drug Products solution
(1), Specific Tests, Completeness and clarity of solutions. [Note—The relative retention times for testosterone and
© PARTICULATE IMIATTER IN INJECTIONS (788): Meets the re- methyltestosterone are about 0.8 and 1.0,
quirements for small-volume injections respectively.]
¢ OTHER REQUIREMENTS: Meets the requirements in Labeling Suitability requirements
(7), Labels and Labeling for Injectable Products Resolution: NLT 2.0 between testosterone and
methyltestosterone, System suitability solution
ADDITIONAL REQUIREMENTS Column efficiency: NLT 2000 theoretical plates,
e PACKAGING AND STORAGE: Preserve as described in Pack- Standard solution
aging and Storage Requirements (659), Injection Packaging, Tailing factor: NMT 2.7, Standard solution
Packaging for constitution. Relative standard deviation: NMT 2.0%, Standard
solution
 2696 Methyltestosterone / Official Monographs USP 41

Analysis Acceptance criteria: +79° to +85°

Samples: Standard solution and Sample solution e Loss ON DRYING (731)
Calculate the percentage of eed Analysis: Dry at 105° for 4 h.
(C20H30O2) in the portion of Methyltestosterone taken: Acceptance criteria: NMT 2.0%
Result = (ru/rs) x (Cs/Cu) x 100 ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed, light-
ry = peak response of methyltestosterone from the resistant containers.
Sample solution e USP REFERENCE STANDARDS (11)
Is = peak response of methyltestosterone from the USP Methyltestosterone RS
Standard solution USP Testosterone RS
Gs = concentration of USP Methyltestosterone RS in
the Standard solution (mg/mL)
Cu = concentration of Methyltestosterone in the
Sample solution (mg/mL)
Acceptance criteria: 97.0%-103.0% on the dried basis Methyltestosterone Capsules
IMPURITIES
e ORGANIC IMPURITIES DEFINITION
Solution A: Methanol and water (55:45) Methyltestosterone Capsules contain NLT 90.0% and NMT
Solution B: Methanol 110.0% of the labeled amount of methyltestosterone
Mobile phase: See Table 7. (C20H3002).
IDENTIFICATION
Table 1 e A. INFRARED ABSORPTION
Solution A Solution B Sample: Evaporate to dryness 25 mL of the Sample
stock solution from the Assay.
Acceptance criteria: The IR absorption spectrum of a
1 0
potassium bromide dispersion of the residue so ob-
tained exhibits maxima at the same wavelengths as
yee of a similar preparation of USP Methyltestosterone
100 RS.
1
ASSAY
Sample solution: 0.5 mg/mL of Methyltestosterone in e PROCEDURE
methanol Standard solution: 10 g/mL of USP Methyltestoster-
System suitability solution: 0.005 mg/mL of one RS in alcohol
Methyltestosterone in methanol from the Sample Sample stock solution: Nominally 0.2 mg/mL of
solution methyltestosterone prepared as follows. Transfer the
red
a)
equivalent to 10 mg of methyltestosterone from the
a Chromatographic system
contents of NLT 20 Capsules to a 125-mL separator
S (See Chromatography (621), System Suitability.)
Dp
I
Mode: LC with the aid of about 5 mL of water. Extract with four
° Detector: UV 254 nm 20-mL portions of chloroform, filtering each through
iS Column: 4.6-mm x 25-cm; 5-um packing L1 chloroform-washed cotton. Evaporate the combined ex-
G) Flow rate: 1 mL/min tracts on a steam bath, with the aid of a current of air,
2 Injection volume: 5 wL to dryness. Dissolve the residue in alcohol, transfer to a
a System suitability 50-mL volumetric flask, and dilute with alcohol to vol-
”
m=) Samples: Sample solution and System suitability solution ume.
Suitability requirements Sample solution: Nominally 10 ug/mL of methyltestos-
Relative standard deviation: NMT 2.0%, Sample terone in alcohol from the Sample stock solution
solution Instrumental conditions
Signal-to-noise ratio: NLT 100, System suitability Mode: UV
solution Analytical wavelength: Maximum absorbance at
Analysis about 241 nm
Sample: Sample solution Cell: 1m
Calculate the percentage of each impurity in the por- Blank: Alcohol
tion of Methyltestosterone taken: Analysis
Samples: Standard solution and Sample solution
Result = (ru/rz) x 100 Calculate the percentage of the labeled amount of
methyltestosterone (C2oH3002) in the portion of Cap-
ru = peak response of each impurity from the sules taken:
Sample solution
Ir = sum of all the peak responses from the Sample Result = (Au/As) x (Cs/Cu) x 100
solution
[Note—Disregard any impurity peak less than 0.05%.] Au = absorbance of the Sample solution
Acceptance criteria As = absorbance of the Standard solution
Any individual impurity: NMT 0.5% Cs = concentration of USP Methyltestosterone RS in
Total impurities: NMT 1.0% the Standard solution (g/mL)
Cu = nominal concentration of methyltestosterone
SPECIFIC TESTS in the Sample solution (ug/mL,
© OPTICAL ROTATION, Specific Rotation (781S)
Sample solution: 10 mg/mL of Methyltestosterone in
alcohol
USP 41 Official Monographs / Methyltestosterone 2697

Acceptance criteria: 90.0%-110.0% IDENTIFICATION

e A, INFRARED ABSORPTION
PERFORMANCE TESTS Sample: Evaporate to dryness 25 mL of the Sample
e DISSOLUTION (711) stock solution from the Assay.
Medium: Water; 900 mL Acceptance criteria: The IR absorption spectrum of a
Apparatus 1: 100 rpm potassium bromide dispersion of the residue so ob-
Time: 45 min tained exhibits maxima at the same wavelengths as
Standard solution: 10 g/mL of USP Methyltestoster- those of a similar preparation of USP Methyltestosterone
one RS in Medium RS.
Sample solution: Filter a portion of the solution under
test. Dilute with Medium to obtain a solution containing ASSAY
about 10 g/mL of methyltestosterone. ¢ PROCEDURE
Instrumental conditions Standard solution: 10 g/mL of USP Methyltestoster-
Mode: UV one RS in alcohol
Analytical wavelength: Maximum absorbance at Sample stock solution: Nominally 0.2 mg/mL of
about 248 nm methyltestosterone prepared as follows. Transfer the
Blank: Medium equivalent to 10 mg of methyltestosterone from NLT
Analysis 20 powdered Tablets to a 125-mL separator with the
Samples: Standard solution and Sample solution aid of about 5 mL of water. Extract with four 25-mL
Tolerances: NLT 70% (Q) of the labeled amount of ortions of chloroform, filtering each through chloro-
methyltestosterone (C20H3002) is dissolved. ‘orm-washed cotton. Evaporate the combined extracts
e UNIFORMITY OF DOSAGE UNITS (905) on a steam bath, with the aid of a current of air, to
Procedure for content uniformity dryness. Dissolve the residue in alcohol, transfer to a
Standard solution: 0.010 mg/mL of USP Methyltestos- 50-mL volumetric flask, and dilute with alcohol to vol-
terone RS in methanol ume.
Sample stock solution: Transfer the contents of 1 Sample solution: Nominally 10 ug/mL of methyltestos-
Capsule to a 100-mL volumetric flask. Add 50 mL of terone in alcohol from the Sample stock solution
methanol, and shake by mechanical means for 60 min. Instrumental conditions
Dilute with methanol to volume, and filter, discarding Mode: UV
the first 20 mL of the filtrate. Analytical wavelength: Maximum absorbance at
Sample solution: Dilute a suitable volume of the Sam- about 241 nm
ple stock solution with methanol to obtain 0.010 mg/ Cell: 1.cm
mL of methyltestosterone. Blank: Alcohol
Instrumental conditions Analysis
Mode: UV Samples: Standard solution and Sample solution
Analytical wavelength: Maximum absorbance at Calculate the percentage of the labeled amount of
about 241 nm methyltestosterone (C20H300z) in the portion of Tablets
Cell: 1cm taken: (ex
Blank: Methanol a
Analysis Result = (Au/As) x (Cs/Cu) x 100 v
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of Au = absorbance of the Sample solution =
methyltestosterone (C20H30O2) in the Capsule taken: As = absorbance of the Standard solution =}
Cs = concentration of USP Methyltestosterone RS in iS
Result = (Au/As) x (Cs/L) x Vx D x 100 the Standard solution (ug/mL) al
Cu = nominal concentration of methyltestosterone a
Au = absorbance of methyltestosterone from the in the Sample solution (g/mL =
Sample solution Acceptance criteria: 90.0%-110.0%
As = absorbance of methyltestosterone from the
Standard solution PERFORMANCE TESTS
Cs = concentration of USP Methyltestosterone RS in © DISINTEGRATION (701)
the Standard solution (mg/mL) Time: 30 min
i = label claim (mg/Capsule) Acceptance criteria: Tablets intended for buccal admin-
Vv = volume of the Sample solution (mL) istration meet the requirements for Buccal Tablets.
D = dilution factor of the Sample solution e UNIFORMITY OF DosAGE UNITS (905)
Acceptance criteria: Meet the requirements Procedure for content uniformity
Standard solution: 0.010 mg/mL of USP Methyltestos-
ADDITIONAL REQUIREMENTS terone RS in methanol
© PACKAGING AND STORAGE: Preserve in well-closed Sample stock solution: Transfer 1 finely powdered
containers. Tablet to a 100-mL volumetric flask. Add 50 mL of
e USP REFERENCE STANDARDS (11) methanol, and shake by mechanical means for 60 min.
USP Methyltestosterone RS Dilute with methanol to volume, and filter, discarding
the first 20 mL of the filtrate.
Sample solution: Dilute a suitable volume of the Sam-
ple stock solution with methanol to obtain 0.010 mg/
mL of methyltestosterone.
Methyltestosterone Tablets Instrumental conditions
Mode: UV
DEFINITION Analytical wavelength: Maximum absorbance at
Methyltestosterone Tablets contain NLT 90.0% and NMT about 241 nm
110.0% of the labeled amount of methyltestosterone
(C20H3002).
 2698 Methyltestosterone / Official Monographs USP 41

Cell: 1.cm monium hydroxide in the chamber, cover, and allow

Blank: Methanol to equilibrate for 30 min. Apply the Samples, and de-
Analysis velop the chromatogram until the solvent front has
Samples: Standard solution and Sample solution moved about three-fourths of the length of the plate.
Calculate the percentage of the labeled amount of Remove the plate from the developing chamber,
methyltestosterone (CzoH30O2) in the Tablet taken: mark the solvent front, and allow the solvent to evap-
orate. Locate the spots on the plate by lightly spray-
Result = (Au/As) x (Cs/L) x Vx D x 100 ing with Spray reagent. Allow the plate to dry, then
expose it briefly to fumes of a mixture of nitric and
Au = absorbance of methyltestosterone from the hydrochloric acids.
Sample solution Acceptance criteria: The R; value of the principal spot
As = absorbance of methyltestosterone from the of the Sample solution corresponds to that of the Stan-
Standard solution dard solution.
Gs = concentration of USP Methyltestosterone RS in
the Standard solution (mg/mL) ASSAY
E = label claim (mg/Tablet) e PROCEDURE
Vv = volume of the Sample solution (mL) Sample solution: 200 mg of Methysergide Maleate in
D = dilution factor of the Sample solution 30 mL of glacial acetic acid. Add 1 drop of crystal violet
Acceptance criteria: Meet the requirements S
Analysis: Titrate with 0.1 N perchloric acid VS to a blue
ADDITIONAL REQUIREMENTS endpoint. Perform a blank determination, and make
e PACKAGING AND STORAGE: Preserve in well-closed any necessary correction. Each mL of 0.1 N perchloric
containers. acid is equivalent to 46.95 mg of methysergide maleate
e USP REFERENCE STANDARDS (11) (Co1H27N3O2 - C4HaOa).
USP Methyltestosterone RS Acceptance criteria: 97.0%-103.0% on the dried basis
IMPURITIES
e ORDINARY IMPURITIES (466)
Standard solution and Sample solution: Methanol
Methysergide Maleate Eluant: Use the Developing solvent from Identification
test B.
OH
Visualization: 1
SDre ioe Acceptance criteria: Meets the requirements
°

woo + a ~te c oi
| i Gi SPECIFIC TESTS
e OPTICAL ROTATION, Specific Rotation (781S)
H |
CHs °o Sample solution: 2.5 mg/mL in water
” a criteria: +35° to +45°
ao CaH27N3Qz - CaHgOa 469.53 e PH (791)
os
i Ergoline-8-carboxamide, 9,10-didehydro-N-[1 -(hydroxy-
Sample solution: 1 in 500 in carbon dioxide-free water
-
methy!)propy!]-1,6-dimethyl-, (8B, (Z)-2-butenedioate Acceptance criteria: 3.7-4.7
Dp e Loss ON DRYING (731)
i) (1:1) (salt); Analysis: Dry a sample under vacuum at 120° for 2 h.
= 9,10-Didehydro-N-[1-(hydroxymethyl)propyl]-1,6-
S dimethylergoline-8B-carboxamide maleate (1:1) (salt)
Acceptance criteria: NMT 7.0%
= [129-49-7]. ADDITIONAL REQUIREMENTS
[any
” ¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant
DEFINITION
=) Methysergide Maleate contains NLT 97.0% and NMT containers, in a cold place.
e USP REFERENCE STANDARDS (11)
103.0% of methysergide maleate (C2i1H27N3O2 - CsH4Ox),
USP Methysergide Maleate RS
calculated on the dried basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
e B. THIN-LAYER CHROMATOGRAPHY
Conduct this test without exposure to daylight and with Methysergide Maleate Tablets
minimum exposure to artificial light.
Standard solution: 5 mg/mL of USP Methysergide DEFINITION
Maleate RS in methanol Methysergide Maleate Tablets contain NLT 90.0% and NMT
Sample solution: 5 mg/mL of Methysergide Maleate in 110.0% of the labeled amount of methysergide maleate
methanol (CaiH27zN3Oz2 +» CaHaOa).
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato- IDENTIFICATION
raphy.) e A. The retention time of the major peak of the Sample
Adsorbent: 0.25-mm layer of chromatographic silica solution corresponds to that of the Standard solution, as
gel obtained in the Assay.
Application volume: 25 uL
Developing solvent: Chloroform and methanol (20:1) ASSAY
Spray reagent: 8 mg/mL of p-dimethylaminobenzalde- © PROCEDURE
de in a cooled mixture of alcohol and sulfuric acid oneuce this procedure with a minimum exposure to
ight.
(8:2) Mobile phase: Dissolve 6.8 g of monobasic potassium
Analysis
Samples: Standard solution and Sample solution phosphate in 700 mL of water, add 300 mL of acetoni-
In the chromatographic chamber, place a volume of trile, and mix.
the Developing solvent sufficient to develop the chro- Diluent: Methanol and 10 g/L of tartaric acid (50:50)
matogram. Place a beaker containing 25 mL of am- Standard solution: 0.1 mg/mL of USP Methysergide
Maleate RS in Diluent
USP 41 Official Monographs / Metoclopramide 2699

[Note—Sonication may be used.] e USP REFERENCE STANDARDS (11)

Sample solution: Nominally 0.1 mg/mL of USP Methysergide Maleate RS
methysergide maleate prepared as follows. Transfer a
portion of NLT 20 finely powdered Tablets equivalent to
NLT 10 mg of methysergide maleate to a suitable volu-
metric flask. Add 75% of the flask volume with Diluent.
Shake by mechanical means for 60 min. Dilute with Dil- Metoclopramide Hydrochloride
uent to volume. Filter, and discard the first 20 mL of the
filtrate. a CH, Ib
Chromatographic system
(See Chromatography (621), System Suitability.) a Oe
Mode: LC H
Detector: UV 318 nm HANz ‘OCH,
Column: 4.6-mm x 15-cm; 5-1m packing L7
Flow rate: 2 mL/min
Injection volume: 20 uL Cy4H22ClN3O02 » HCI - H2O 354.27
System suitability Benzamide, 4-amino-5-chloro-N-[2-(diethylamino)ethyl]-
Sample: Standard solution 2-methoxy-, monohydrochloride, monohydrate;
Suitability requirements 4-Amino-5-chloro-N-[2-(diethylamino)ethy!]-o-anisamide
Resolution: NLT 1.0 between the analyte and the monohydrochloride monohydrate [54143-57-6].
closest adjacent peak DEFINITION
Tailing factor: NMT 2.5 Metoclopramide Hydrochloride contains NLT 98.0% and
Relative standard deviation: NMT 2.0% NMT 101.0% of metoclopramide hydrochloride
Analysis (Ci4H22CIN3O2
- HCI), calculated on the anhydrous basis.
Samples: Standard solution and Sample solution
Calculate the percentage of methysergide maleate IDENTIFICATION
(Ca1H27N3Oz - C4H4O,) in the portion of Tablets taken: e A. INFRARED ABSORPTION (197)
[NoteE—Methods described in Infrared Absorption (197K),
Result = (ru/rs) x (Cs/Cu) x 100 (197M), or (197A) may be used.]
© B. IDENTIFICATION TESTS—GENERAL, Chloride (191)
ty = peak response from the Sample solution Sample solution: Dissolve 100 mg in 2 mL of water,
rs = peak response from the Standard solution and acidify the solution with dilute nitric acid.
Cs = concentration of USP Methysergide Maleate Acceptance criteria: Meets the requirements
RS in the Standard solution (mg/mL)
Cu = nominal concentration of methysergide ASSAY
maleate in the Sample solution (mg/mL) © PROCEDURE
Acceptance criteria: 90.0%-110.0% Sample: 250 mg
PERFORMANCE TESTS
Analysis: Dissolve the Sample in a mixture of 5.0 mL of c
0.01 N hydrochloric acid and 50 mL of alcohol. Titrate wn
e DISSOLUTION (711) with 0.1 N sodium hydroxide VS (see Titrimetry (541)), uv
Medium: Tartaric acid solution (1 in 200); 900 mL determining the endpoint potentiometrically. Read the c=
Apparatus 2: 100 rom volume of 0.1 N sodium hydroxide added between the re}
ime: 30 min two points of inflection. Each mL of 0.1 N sodium hy- s
Standard solution: 0.002 mg/mL of USP Methysergide i)
Maleate RS in Medium
droxide is equivalent to 33.63 mg of the anhydrous Re}=
metoclopramide hydrochloride (C,4H22CIN3O2 - HCl). a
Sample solution: Portions of the solution under test Acceptance criteria: 98.0%-101.0% on the anhydrous i}
suitably diluted with Medium basis =
Instrumental conditions al

Mode: Fluorescence IMPURITIES

Excitation wavelength: 327 nm
Emission wavelength: 428 nm
Blank: Tartaric acid solution (1 in 200)
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
methysergide maleate (C21H27N302 - C4H4Ox) dissolved:
Result = (Au/As) x Cs x Vx (1/L) x 100
Au = absorbance from the Sample solution
As = absorbance from the Standard solution
Cs = concentration of USP Methysergide Maleate
RS in the Standard solution (mg/mL)
Vv = volume of Medium, 900 mL
L = label claim of methysergide maleate (mg/
Tablet)
Tolerances: NLT 70% (Q) of the labeled amount of
methysergide maleate (C2i1H27N3O2 - C4H4Oa) is
dissolved.
© UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers.
Store below 30°.
e RESIDUE ON IGNITION (281): NMT 0.1%
¢ ORGANIC IMPURITIES
Buffer: Dissolve 6.8 g of monobasic potassium phos-
phate in 700 mL of water. Add 0.2 mL of N,N-
dimethyloctylamine, and adjust with dilute phosphoric
acid to a pH of 4.0. Dilute with water to 1000 mL.
Mobile phase: Acetonitrile and Buffer (250:1000)
Standard stock solution: 0.1 mg/mL each of USP
Metoclopramide Hydrochloride RS, USP
Metoclopramide Related Compound A RS, USP
Metoclopramide Related CompoundB RS, and USP
pi sinclgpramie Related CompoundD RS in Mobile
jase
standard solution: 2.0 g/mL each of USP
Metoclopramide Hydrochloride RS, USP
Metoclopramide Related Compound A RS, USP
Metoclopramide Related Compound B RS, and USP
Metoclopramide Related Compound D RS in Mobile
phase from the Standard stock solution
Sample solution: 1.0 mg/mL of Metoclopramide Hy-
drochloride in Mobile phase
 2700 Metoclopramide / Official Monographs USP 41

Chromatographic system Table 1 (Continued)

(See Chromatography (621), System Suitability.) Relative Acceptance
Mode: LC Retention Criteria,
Detector: UV 240 nm Name Time NMT (%)
Column: 4.6-mm x 25-cm; 5-uum packing L7
Flow rate: 1.5 mL/min Any other individual impurity == 0.10
Injection volume: 10 uL Total impurities = 0.50
Run time: At least 8 times the retention time of the # 4-Acetamido-5-chloro-N-[2-(diethylamino)ethyl]-2-methoxybenzamide.
metoclopramide peak > Methyl 4-acetamido-5-chloro-2-methoxybenzoate.
System suitability
Sample: Standard solution SPECIFIC TESTS
Suitability requirements e@ WATER DETERMINATION, Method | (921): 4.5%-6.0%
Resolution: NLT 3.0 for metoclopramide related ADDITIONAL REQUIREMENTS
compound A and metoclopramide e PACKAGING AND STORAGE: Preserve in tight, light-resistant
Analysis containers. Store at room temperature.
Samples: Standard solution and Sample solution e USP REFERENCE STANDARDS (11)
Calculate the percentage of each of metoclopramide re- USP Metoclopramide Hydrochloride RS
lated compounds A, B, andD in the portion of USP Metoclopramide Related Compound A RS
Metoclopramide Hydrochloride taken: N-Acetylmetoclopramide.
4-Acetamido-5-chloro-N-[2-(diethylamino)ethyl]-
Result = (ru/rs) x (Cs/Cu) x 100 2-methoxybenzamide.
tu = peak response of the relevant metoclopramide CisH2sCIN303 341.83
related compound from the Sample solution USP Metoclopramide Related Compound B RS
ig = peak response of the relevant metoclopramide N-Acetyl methyl ester analog.
related compound from the Standard solution Methyl 4-acetamido-5-chloro-2-methoxybenzoate.
Cs = concentration of the relevant USP CyHi2CINO, 257.67
Metoclopramide Related Compound RS in USP Metoclopramide Related Compound D RS
the Standard solution (mg/mL) Methyl 4-acetamido-2-methoxybenzoate.
Cu = concentration of Metoclopramide CuHi3NO, 223.23
Hydrochloride in the Sample solution
(mg/mL)
Calculate the percentage of any other individual
impurity in the portion of Metoclopramide
Hydrochloride taken: Metoclopramide Injection
Result = (ru/rs) x (Cs/Cu) x 100 DEFINITION
Metoclopramide Injection is a sterile solution of
ts
al
ru = peak response of any other individual impurity Metoclopramide Hydrochloride in Water for Injection. It
rs from the Sample solution contains the equivalent of NLT 90.0% and NMT 110.0%
J
— rs = peak response of metoclopramide from the of the labeled amount of metoclopramide (C14H22CIN3O2).
Dd Standard solution
i)
= Gs = concentration of USP Metoclopramide IDENTIFICATION
S Hydrochloride RS in the Standard solution e A. The retention time of the major peak of the Sample
3
Cu
(mg/ml)
= concentration of Metoclopramide
solution corresponds to that of the Standard solution, as
obtained in the Assay.
a
a) Hydrochloride in the Sample solution e B. The UV spectrum of the major peak of the Sample
= (mg/mL) solution corresponds to that of the Standard solution, as
Acceptance criteria: See Table 1. obtained in the Assay.
ASSAY
Table 1 e PROCEDURE
Relative Acceptance Mobile phase: Dissolve 2.7 g of sodium acetate in
Retention Criteria, 500 mL of water. Add 500 mL of acetonitrile and 2 mL
Name Time NMT (%) of tetramethylammonium hydroxide solution in metha-
N-Acetylmetoclopramide nol (1 in 5), and mix. Adjust with glacial acetic acid to
(metoclopramide related a pH of 6.5, filter, and degas.
compound A)? 0.8 0.15 System suitability stock solution: Transfer 12.5 mg of
Metoclopramide 1.0 —
benzenesuitonaivide to a 25-mL volumetric flask. Add
15 mL of methanol, and shake to dissolve. Dilute with
Methyl 4-acetamido-2-methox- 0.01 del evade acid to volume.
ybenzoate (metoclopramide Standard stock solution: 0.9 mg/mL of USP
related compound D) 2.4-2.7 0.15 Metoclopramide Hydrochloride RS_ in 0.01 M phos-
N-Acetyl methyl ester analog phoric acid
(metoclopramide related System suitability solution: Transfer 5 mL of System
compound B)> 5.2-6.7 0.15 suitability stock solution and 5 mL of Standard stock solu-
2 4-Acetamido-5-chloro-N-[2-(diethylamino)ethyl]-2-methoxybenzamide. tion into a 100-mL volumetric flask, and dilute with
> Methyl 4-acetamido-5-chloro-2-methoxybenzoate. 0.01 M phosphoric acid to volume.
Standard solution: 45 tg/mL of USP Metoclopramide
Hydrochloride RS (equivalent to 40 g/mL of
metoclopramide) from Standard stock solution. Dilute
with 0.01 M phosphoric acid.
Sample solution: Nominally 40 g/mL of
metoclopramide, prepared as follows. Transfer a volume
USP 41 Official Monographs / Metoclopramide 2701

of Injection, ee to about 40 mg of Mode: LC

metoclopramide, to a 100-mL volumetric flask, and di- Detector: UV 265 nm
lute with 0.01 M phosphoric acid to volume. Transfer Column: 4.6-mm x 25-cm; 5-um packing L1
10.0 mL of this solution to a 100-mL volumetric flask, Flow rate: 2 mL/min
and dilute with 0.01 M phosphoric acid to volume. Injection volume: 20 LL
Chromatographic system System suitability
(See Chromatography (621), System Suitability.) Sample: Standard solution
Mode: LC saree requirements
Detector: UV 215 nm or diode array. [NOTE—Use the Tailing a factor: NMT 1.8
diode array detector to perform /dentification test B.] Relative standard deviation: NMT 5.0%
Column: 4.6-mm x 25-cm; packing L1 Analysis
Flow rate: 1.5 mL/min Samples: Standard solution and Sample solution
Injection volume: 20 pL Calculate the percentage of any individual impurity in
System suitability the portion of Injection taken:
Samples: System suitability solution and Standard
solution Result = (ru/ts) x (Cs/Cu) X (Mn/M2z) x 100
Suitability requirements
[Note—The relative retention times for ru = peak response of each impurity from the
benzenesulfonamide and metoclopramide are 0.7 and Sample solution
1.0, respectively.] rs = peak response of metoclopramide from the
Resolution: NLT 1.5 between the Standard solution
benzenesulfonamide and metoclopramide peaks, Sys- Cs = concentration of USP Metoclopramide
tem suitability solution Hydrochloride RS in the Standard solution
Tailing factor: NMT 2.0 for the metoclopramide (ug/ml) j eeepe
peak, Standard solution Cu = nominal concentration of metoclopramide in
Relative standard deviation: NMT 2.0%, Standard the Sample solution (ug/mL)
solution Mn = molecular weight of metoclopramide, 299.80
Analysis Mz = molecular weight of anhydrous
Samples: Standard solution and Sample solution metoclopramide hydrochloride, 336.26
Calculate the percentage of the labeled amount of Acceptance criteria: NMT 0.5% of any individual im-
metoclopramide (Cy4H22CIN3Oz) in the portion of In- purity is found.
jection taken:
SPECIFIC TESTS
Result = (ru/rs) x (Cs/Cu) x (Ma/M,2) x 100 © PH (791): 2.5-6.5
e BACTERIAL ENDOTOXINS TEST (85): NMT 2.5 USP Endo-
ty = peak response from the Sample solution toxin Units/mg of metoclopramide
Is = peak response from the Standard solution e PARTICULATE MATTER IN INJECTIONS (788): It meets the re-
Cs = concentration of USP Metoclopramide quirements for small-volume injections. [=
Hydrochloride RS in the Standard solution e OTHER REQUIREMENTS: It meets the requirements in Injec- a
(ug/ml) . a. tions and Implanted Drug Products (1). a)
Cu = nominal concentration of metoclopramide in
ADDITIONAL REQUIREMENTS =
the Sample solution (ug/mL) °
My = molecular weight of metoclopramide, 299.80 e PACKAGING AND STORAGE: Preserve in single-dose or mul- =}
M,2 = molecular weight of anhydrous tiple-dose containers, preferably of Type | glass, protected °
from light. [NoTE—Injection containing an antioxidant to)=
metoclopramide hydrochloride, 336.26
Acceptance criteria: 90.0%-110.0% agent does not require protection from light.] Store at 2
mo]
controlled room temperature. z
IMPURITIES “

© ORGANIC IMPURITIES
Mobile phase: Prepare a 1.88 g/L solution of sodium
1-hexanesulfonate solution (0.01 M solution) in a mix-
ture of acetonitrile and water (60:40), and adjust with
glacial acetic acid to a pH of 4.0.
Standard solution: 5.5 g/mL of USP Metoclopramide
Hydrochloride RS in Mobile phase
Sample solution: Dilute a volume of Injection with Mo-
bile phase to obtain a solution containing about
1.0 mg/mL of metoclopramide.
Chromatographic system
(See Chromatography (621), System Suitability.)
Change to read:
¢ USP REFERENCE STANDARDS (11)
*. (EN 1-May-2018)
USP Metoclopramide Hydrochloride RS
Metoclopramide Oral Solution
DEFINITION
Metoclopramide Oral Solution contains an amount of
metoclopramide hydrochloride (Ci4H22CIN3O2 - HCI - H2O)
equivalent to NLT 90.0% and NMT 110.0% of the labeled
amount of metoclopramide (C;4H22CIN3O2).
IDENTIFICATION
e A. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
e B. The UV spectrum of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
 2702 Metoclopramide / Official Monographs USP 41

ASSAY e DELIVERABLE VOLUME (698)

¢@ PROCEDURE
Mobile phase: Dissolve 2.7 g of sodium acetate in
600 mL of water, and add 400 mL of acetonitrile and
4 mL of tetramethylammonium hydroxide solution in
methanol (25%). Adjust with glacial acetic acid to a pH
of 6.5, filter, and degas.
System suitability stock solution: Transfer 125 mg of
peiren ditopariide to a 25-mL volumetric flask. Add
15 mL of methanol, and shake to dissolve. Dilute with
0.01 M phosphoric acid to volume.
Standard stock solution: 9 mg/mL of USP
Metoclopramide Hydrochloride RS in 0.01 M phos-
phoric acid
System suitability solution: Transfer 15 mL of System
suitability stock solution and 5 mL of Standard stock solu-
tion into a 250-mL volumetric flask, and dilute with
0.01 M phosphoric acid to volume.
Standard solution: 180 paral of USP Metoclopramide
Hydrochloride RS (equivalent to 160 g/mL of
metoclopramide) from Standard stock solution. Dilute
with 0.01 M phosphoric acid.
Sample solution: Nominally 160 g/mL of
metoclopramide, prepared as follows. Transfer a volume
of Oral Solution, equivalent to about 4 mg of
metoclopramide, to a 25-mL volumetric flask, and di-
lute with 0.01 M phosphoric acid to volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 215 nm or diode array. [NOTE—Use the
diode array detector to perform /dentification test B.]
Column: 4.6-mm x 25-cm; packing L1
Jnjectionvoli a
For oral solution packaged in multiple-unit contain-
ers: Meets the requirements
IMPURITIES
© ORGANIC IMPURITIES
Mobile phase: Prepare a 1.88 g/L solution of sodium
1-hexanesulfonate solution (0.01 M solution) in a mix-
ture of acetonitrile and water (60:40), and adjust with
glacial acetic acid to a pH of 4.0.
Standard solution: 5.5 g/mL of USP Metoclopramide
Hydrochloride RS in Mobile phase
Sample solution: Dilute a volume of Ora! Solution with
Mobile phase to obtain a solution containing about
1.0 mg/mL of metoclopramide.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 265 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Flow rate: 2 mL/min
Injection volume: 20 uL
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 1.8
Relative standard deviation: NMT 5.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of any individual impurity in
the portion of Oral Solution taken:
Result = (ru/rs) x (Cs/Cu) x (Mn/M,2) x 100
ty = Pe nieeution each impurity from the

i Samples: Gen suitability solution and Standard soit Peatiesrence efgnctoctonharnide from the
a4 cheer i Cs = concentration of USP Metoclopramide
es TNORE ‘ areative: remeron times for eee RS in the Standard solution

ee oo and metoclopramide are Ow ang Cu = nominal concentration of metoclopramide in

= n: 1.5 between ‘the
ResolutioNLT the Sample solution (ug/ml)
ee 299.80
= benzenesulfonamide and metoclopramide peaks, Sys- Me = moan vent

StandardNMT2.0
for the metoclopramide metoclopramide hydrochloride, 336.26
Ga
S peak, factor.
Talling solution Acceptance ene i 0.5% a aneee ims
Relative standard deviation: NMT 2.0%, Standard pa,AineOF 0 ie peak with arelative re
:
solution
Analysis SPECIFIC TESTS
Samples: Standard solution and Sample solution e PH (791): 2.0-5.5
Calculate the percentage of the labeled amount of
metoclopramide (C4H22CIN3Oz) in the portion of Oral ADDITIONAL REQUIREMENTS
Solution taken: e PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and store at controlled room temperature.
Result = (ru/rs) x (Cs/Cu) x (Mri/M,2) x 100 Protect from freezing.
e USP REFERENCE STANDARDS
; (11)hloride RS
tu = peak response from the Sample solution USP Met
rs = peak response from the Standard solution letoclopramide Hydrochloride
Cs = concentration from USP Metoclopramide
Hydrochloride RS in the Standard solution

Cuv oa
= nominal concentration metocl ide n
trati of f metoclopramidei i
the Sample solution (ug/ml) i Metoclopramide Tablets
Ma = molecular weight of metoclopramide, 299.80
M2 = molecular weight of anhydrous DEFINITION
metoclopramide hydrochloride, 336.26 Metoclopramide Tablets contain an amount of
Acceptance criteria: 90.0%-110.0% metoclopramide hydrochloride (Ci4H22CIN3O2- HCI - H20)
equivalent to NLT 90.0% and NMT 110.0% of the labeled
PERFORMANCE TESTS amount of metoclopramide (Ci4H22CIN30O2).
o UNIFORMITY OF DosaGe UNITS (905)
For oral solution packaged in single-unit containers: IDENTIFICATION
Meets the requirements e A. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
USP 41 Official Monographs / Metoclopramide 2703

e B. The UV spectrum of the major peak of the Sample Ma molecular weight of metoclopramide, 299.80
solution corresponds to that of the Standard solution, as M2 molecular weight of anhydrous
obtained in the Assay. metoclopramide hydrochloride, 336.26
Acceptance criteria: 90.0%-110.0%
ASSAY
¢ PROCEDURE PERFORMANCE TESTS
Mobile phase: Dissolve 2.7 g of sodium acetate in e DISSOLUTION (711)
500 mL of water. Add 500 mL of acetonitrile and 2 mL Medium: Water; 900 mL
of tetramethylammonium hydroxide solution in metha- Apparatus 1: 50 rpm
nol (1 in 5), and mix. Adjust with glacial acetic acid to Time: 30 min
a pH of 6.5, filter, and degas. Standard solution: USP Metoclopramide Hydrochloride
Seren suitability stock solution: Transfer 12.5 mg of RS at a known concentration in Medium
enzenesulfonamide to a 25-mL volumetric flask. Add Sample solution: Filtered portion of the solution under
15 mL of methanol, and shake to dissolve. Dilute with test, suitably diluted with Medium
0.01 M phosphoric acid to volume. Instrumental conditions
Standard stock solution: 0.9 mg/mL of USP Mode: UV-Vis
Metoclopramide Hydrochloride RS in 0.01 M phos- Analytical wavelength: Wavelength of maximum ab-
phoric acid sorbance at about 309 nm
System suitability solution: Transfer 5 mL of System Tolerances: NLT 75% (Q) of the labeled amount of
Suitability stock solution and 5 mL of Standard stock solu- metoclopramide (C;4H22CIN3O2) is dissolved.
tion into a 100-mL volumetric flask, and dilute with e UNIFORMITY OF DOSAGE UNITS (905): Meet the
0.01 M phosphoric acid to volume. requirements
Standard solution: 45 t1g/mL of USP Metoclopramide
Hydrochloride RS (equivalent to 40 ng/mL of IMPURITIES
metoclopramide) from Standard stock solution diluted © ORGANIC IMPURITIES
with 0.01 M phosphoric acid Mobile phase: Prepare a 1.88 g/L solution of sodium
Sample solution: Nominally 40 g/mL of 1-hexanesulfonate solution (0.01 M solution) in a mix-
metoclopramide, prepared as follows. Weigh and finely ture of acetonitrile and water (60:40), and adjust with
powder NLT 20 Tablets. Transfer an accurately weighed glacial acetic acid to a pH of 4.0.
portion of the powder, equivalent to about 40 mg of Standard solution: sou /mL of USP Metoclopramide
metoclo| raraide to a 100-mL volumetric flask, add Hydrochloride RS in Mobile phase
about 70 mL of 0.01 M phosphoric acid, and sonicate Sample solution: Shake a quantity of the powdered
for 5 min. Cool to room temperature, dilute with 0.01 Tablets containing the equivalent of 100 mg of
M phosphoric acid to volume, and mix. Pass the solu- metoclopramide with 20 mL of methanol for 5 min,
tion throughafilter of 0.45-um pore size, sliscarcing and pass through a suitable filter, discarding the first
the first portion of the filtrate. Transfer 10.0 mL of this few mL of the filtrate. Dilute a portion of the filtrate
solution to a 100-mL volumetric flask, and dilute with with Mobile phase to obtain a solution containing about
0.01 M phosphoric acid to volume. 1.0 mg/mL of metoclopramide. cS
Chromatographic system Chromatographic system “n
(See Chromatography (621), System Suitability.) (See Chromatography (621), System Suitability.) a]
Mode: LC Mode: LC =
Detector: UV 215 nm or diode array. [NOTE—Use the Detector: UV 265 nm i}
diode array detector to perform /dentification test B.] Column: 4.6-mm x 25-cm; 5-um packing L1 =]
Flow rate: 2 mL/min )
Column: 4.6-mm x 25-cm; packing L1
Injection volume: 20 uL
re}
2
Flow rate: 1.5 mL/min 2
Injection volume: 20 pL System suitability io}
System suitability Sample: Standard solution =
Samples: System suitability solution and Standard Suitability requirements a)

solution Tailing factor: NMT 1.8

Suitability requirements Relative standard deviation: NMT 5.0%
[Note—The relative retention times for Analysis
benzenesulfonamide and metoclopramide are 0.7 and Samples: Standard solution and Sample solution
1.0, respectively.] Calculate the percentage of any individual impurity in
Resolution: NLT 1.5 between the the portion of Tablets taken:
benzenesulfonamide and metoclopramide peaks, Sys-
tem suitability solution Result = (ru/rs) x (Cs/Cu) X (Mn/M2) x 100
Tailing factor: NMT 2.0 for the metoclopramide
peak, Standard solution ty = peak response of each impurity from the
Relative standard deviation: NMT 2.0%, Standard Sample solution
solution ls = peak response of metoclopramide from the
Analysis Standard solution
Samples: Standard solution and Sample solution Gs = concentration of USP Metoclopramide
Calculate the percentage of the labeled amount of Hydrochloride RS in the Standard solution
metoclopramide (Cy4H22CIN3O2) in the portion of Tab- (ug/ml) . .
lets taken: Cu = nominal concentration of metoclopramide in
the Sample solution (ug/mL)
Result = (ru/rs) x (Cs/Cu) x (Ma/M,2) x 100 Ma = molecular weight of metoclopramide, 299.80
M2 = molecular weight of anhydrous
ry = peak response from the Sample solution metoclopramide hydrochloride, 336.26
ls = peak response from the Standard solution Acceptance criteria: NMT 0.5% of any individual im-
Cs = concentration of USP Metoclopramide purity is found.
Hydrochloride RS in the Standard solution
ADDITIONAL REQUIREMENTS
(ug/mL) e PACKAGING AND STORAGE: Preserve in tight, light-resistant
Cu = nominal concentration of metoclopramide in
the Sample solution (ug/mL) containers. Store at controlled room temperature.
 2704 Metoclopramide / Official Monographs USP 41

e USP REFERENCE STANDARDS (11) Cu = concentration of Metolazone in the Sample

USP Metoclopramide Hydrochloride RS solution (g/mL)
Acceptance criteria: 97.0%-102.0% on the dried basis
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.1%
Metolazone
Delete the following:

°e HEAVY Metals, Method I! (231): 15 ppme coificiai r4an-2018)

© ORGANIC IMPURITIES
Buffer and Mobile phase: Proceed as directed in the
BSS
Standard stock solution: 0.48 mg/mL of USP Meto-
CrsHisCIN3035 365.83 lazone RS in tetrahydrofuran
6-Quinazolinesulfonamide, 7-chloro-1,2,3,4-tetrahydro- Standard solution: 6 j1g/mL of USP Metolazone RS in
2 neyoe eye alcohol from the Standard stock solution
7-Chloro-1,2,3,4-tetrahydro-2-methyl-4-oxo-3-o0-tolyl- Sample solution: 0.6 mg/mL of Metolazone, prepared
6-quinazolinesulfonamide [17560-51-9]. as follows. Dissolve a suitable amount of Metolazone
with tetrahydrafuran in 50% of the total volume, and
DEFINITION dilute with alcohol to volume.
Metolazone contains NLT 97.0% and NMT 102.0% of Chromatographic system: Proceed as directed in the
metolazone (CisHisCIN303S), calculated on the dried Assay, except for the following:
basis. Column: 4.6-mm x 25-cm; 5-um packing 1
Run time: NLT 3.5 times the retention time of
IDENTIFICATION metolazone
© A. INFRARED ABSORPTION (197K) System suitability
e B. ULTRAVIOLET ABSORPTION (197U) Sample: Standard solution
Sample solution: 5 jug/mL in methanol Suitability requirements
Acceptance criteria: Meets the requirements Tailing factor: NMT 2.4
e C. The retention time of the major peak of the Sample Relative standard deviation: NMT 5.0%
solution corresponds to that of the Standard solution, as
Analysis
obtained in the Assay. Samples: Standard solution and Sample solution
ASSAY Calculate the percentage of each individual impurity in
e PROCEDURE
the portion of sample taken:
Protect all solutions of Metolazone from light.
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
2 Buffer: 5.4 g/L of monobasic potassium phosphate. Ad-
os just with phosphoric acid to a pH of 3.0.
a Mobile phase: Acetonitrile, methanol, and Buffer
Tu = peak response of each individual impurity
Ss from the Sample solution
—
a (10:25:65) rs = peak response of metolazone from the
-) Standard stock solution: 0.5 mg/mL of USP Meto-
Standard solution
fe lazone RS in tetrahydrofuran
C Standard solution: 0.05 mg/mL of USP Metolazone RS
Cs = concentration of USP Metolazone RS in the
2 Standard solution (mg/mL)
in alcohol from the Standard stock solution Cy = concentration of Metolazone in the Sample
a Sample stock solution: 1 mg/mL of Metolazone in solution (mg/mL)
al tetrahydrofuran E = relative response factor of each individual
= Sample solution: 0.05 mg/mL of Metolazone in alcohol impurity (see Table 1)
from the Sample stock solution Acceptance criteria: See Table 7.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC Table 1
Detector: UV 230 nm Relative Relative Acceptance
Column: 4.6-mm x 25-cm; 10-um packing L1 Retention Response Criteria,
Flow rate: 1.5 mL/min Name Time Factor NMT (%)
Injection volume: 15 pL Desmethyl meto-
System suitability lazone? 0.7 1.0 0.5
Sample: Standard solution
Suitability requirements Metolazone
Tailing factor: NMT 2.4 benzamide analog® 0.8 0.83 0.5
Relative standard deviation: NMT 1.0% Metolazone 1.0 1.0 =
Analysis meta-Metolazone* 1.3. 0.91 0.5
Samples: Standard solution and Sample solution para-Metolazone? 1.4 0.91 0.5
Calculate the percentage of metolazone Didehydrometo-
(CisHisCIN3O3S) in the portion of sample taken: lazonee 1.5 0.83 0.5
Result = (ru/rs) x (Cs/Cy) x 100 2 7-Chloro-2-methyl-4-oxo-3-phenyl-1,2,3,4-tetrahydroquinazoline-6-sul-
fonamide.
» 2-Amino-4-chloro-5-sulfamoyl-N-(o-tolyl)benzamide.
ru = peak response of metolazone from the Sample
¢ 7-Chloro-2-methyl-4-0xo-3-(m-tolyl)-1,2,3,4-tetrahydroquinazoline-6-sul-
solution fonamide.
fs = peak response of metolazone from the 4 7-Chloro-2-methyl-4-oxo-3-(p-tolyl)-1,2,3,4-tetrahydroquinazoline-6-sul-
Standard solution fonamide.
Cs = concentration of USP Metolazone RS in the FeChloto-2-Methy A-OxO.S:(oly) 3 cLaihydrequinazoline-6-sultonans:
Standard solution (g/mL) ide.
Sum of all individual impurities. Disregard any peaks less than 0 05%.
USP 41 Official Monographs / Metolazone 2705

Table 1 (Continued) Chromatographic system

Relative Relative Acceptance (See Chromatography (621), System Suitability.)
Retention Response Criteria, Mode: LC
Name Time Factor NMT (%) Detector: UV 254 nm
Column: 4.6-mm x 20-cm; 5-14m packing L3
Any other individual Flow rate: 1.0 mL/min
impurity _ a0 0.10 Injection volume: 20 LL
Total impurities! = = 1.0 System suitability
2 7-Chloro-2-methyl-4-oxo-3-phenyl-1,2,3,4-tetrahydroquinazoline-6-sul- Sample: Standard solution
fonamide. >a retention time for metolazone is about 6.0
» 2-Amino-4-chloro-5-sulfamoyl-N-(0-tolyl)benzamide. min.
¢ 7-Chloro-2-methyl-4-o0xo-3-(m-tolyl)-1,2,3,4-tetrahydroquinazoline-6-sul- Suitability requirements
fonamide.
Relative standard deviation: NMT 2.2% for replicate
¢ 7-Chloro-2-methyl-4-oxo-3-(p-tolyl)-1,2,3,4-tetrahydroquinazoline-6-sul-
fonamide. injections
¢ 7-Chloro-2-methyl-4-oxo-3-(0-toly!)-3,4-dihydroquinazoline-6-sulfonam- Analysis
ide. Samples: Standard solution and Sample solution
Sum of all individual impurities. Disregard any peaks less than 0.05%. Calculate the percentage of the labeled amount of
metolazone (Ci6HisCIN3O3S) in the portion of Oral
SPECIFIC TESTS Suspension taken:
e Loss ON DRYING (731)
Analysis: Dry a sample at 105° for 2 h. Result = (ru/rs) x (Cs/Cu) x 100
Acceptance criteria: NMT 1.0%
tu = peak response from the Sample solution
ADDITIONAL REQUIREMENTS Is = peak response from the Standard solution
© PACKAGING AND STORAGE: Preserve in tight, light-resistant Cs = concentration of USP Metolazone RS in the
containers. Standard solution (g/mL)
e USP REFERENCE STANDARDS (11) Cu = nominal concentration of metolazone in the
USP Metolazone RS Sample solution (ug/ml)
Acceptance criteria: 90.0%-110.0%
SPECIFIC TESTS
° PH (791): 3.6-4.6
Metolazone Compounded Oral ADDITIONAL REQUIREMENTS
Suspension e PACKAGING AND STORAGE: Package in tight, light-resistant
containers. Store at controlled room temperature, or in a
DEFINITION refrigerator.
Metolazone Compounded Oral Suspension contains NLT ¢ BEYOND-UsE DATE: NMT 60 days after the date on which
90.0% and NMT 110.0% of the labeled amount of meto- it was compounded when stored at controlled room (=x
al
lazone (CigHisCIN303S). temperature, or in a refrigerator a]
Prepare Metolazone Compounded Oral Suspension 1 mg/ e LABELING: Label it to state that it is to be well shaken,
mL as follows (see Pharmaceutical Compounding—Nonster- and to state the Beyond-Use Date. ce
ile Preparations {795)). fo}
e USP REFERENCE STANDARDS (11) 3
USP Metolazone RS fo]
Ke}
Metolazone 100 mg st
i
Vehicle: a 1:1 mixture of Vehicle ao]
for Oral Solution, (regular or a
sugar-free), NF, and Vehicle for
a
Oral Suspension, NF, a suffi- Metolazone Tablets
cient quantity to make 100 mL
DEFINITION
Place the required number of tablets in a suitable mortar Metolazone Tablets contain NLT 90.0% and NMT 110.0%
and comminute to a fine powder, or use Metolazone pow- of the labeled amount of metolazone (Ci6HisCIN303S).
der. Add 20 mL of Vehicle, and mix to a uniform paste.
Add the Vehicle in small portions, and transfer the con- IDENTIFICATION
tents of the mortar, stepwise and quantitatively, to a cali- ¢ A. ULTRAVIOLET ABSORPTION (197U)
brated bottle. Add Vehicle in portions to rinse the mortar, Sample solution: Dilute 3 mL of the Sample solution in
then ane sufficient Vehicle to bring to final volume, and the Assay with methanol to 25 mL.
mix well. Acceptance criteria: Meet the requirements
ASSAY ASSAY
e PROCEDURE © PROCEDURE
Mobile phase: Methanol and water (70:30) containing [Note—Use low-actinic glassware throughout the Assay.]
1.5 g/L of ammonium acetate and 1 mL/L of diisoprop- Buffer: 1.38 g of monobasic potassium phosphate
ylamine. Filter, and degas. monohydrate in 900 mL of water. Adjust with phos-
Standard solution: 1.0 g/mL of USP Metolazone RS phoric acid to a pH of 3.0, and dilute with water to
Sample solution: Agitate the container of Oral Suspen- 1000 mL.
sion for 30 min on a rotating mixer, remove a 5-mL Mobile phase: Methanol, acetonitrile, and Buffer
sample, and store in a clear glass vial at —70° until ana- (28:7:65)
lyzed. At the time of analysis, remove the sample from Standard stock solution: 0.25 mg/mL of USP Meto-
the freezer, allow it to reach room temperature, and lazone RS in methanol
mix on a vortex mixer for 30 s. Pipet 1.0 mL of the Standard solution: 5 g/mL of USP Metolazone RS in
sample to a 1000-mL volumetric flask, and dilute with Mobile phase from Standard stock solution
Mobile phase to volume. Sample stock solution: Transfer 10 Tablets to a 200-mL
volumetric flask. Add 3 mL of water and 100 mL of
 2706 Metolazone / Official Monographs USP 41

methanol, and sonicate for 30 min. If disintegration is Mode: LC

not complete, sonicate for an additional 30 min. Shake Detector: UV 254 nm
by mechanical means for 30 min. Dilute with methanol Column: 4.6-mm x 15-cm; 5-um packing L7
to volume. Column temperature: 30°
Sample solution: Nominally equivalent to 5 ug/mL of Flow rate: 1.2 mL/min
metolazone in Mobile phase from the Sample stock Injection volume: 50 uL
solution System suitability
Chromatographic system Sample: Standard solution
(See Chromatography (621), System Suitability.) Suitability requirements
Mode: LC Tailing factor: NMT 2.0
Detector: UV 235 nm Column efficiency: NLT 2000 theoretical plates
Column: 3.9-mm x 15-cm; packing L1 Relative standard deviation: NMT 2.0%
Flow rate: 1.1 mL/min Analysis
Injection volume: 100 pL Samples: Standard solution and Sample solution
System suitability Calculate the percentage of the labeled amount of
Sample: Standard solution metolazone (CisHisCIN3O3S) dissolved:
Suitability requirements
Relative standard deviation: NMT 2.0% Result = (ru/rs) x (Cs/L) x V x 100
Analysis
Samples: Standard solution and Sample solution ru = peak response from the Sample solution
Calculate the percentage of the labeled amount of rs = peak response from the Standard solution
metolazone (CisHisCIN3O35) in the portion of Tablets G = concentration of the Standard solution
taken: (mg/mL)
L = label claim (mg/Tablet)
Result = (ru/rs) x (Cs/Cu) x 100 Vv = volume of Medium, 900 mL
Tolerances: NLT 75% (Q) of the labeled amount of
ty = peak response from the Sample solution metolazone (CisHi6CIN3O3S) is dissolved.
rs = peak response from the Standard solution Test 2: If the product complies with this test, the label-
Cs = concentration of USP Metolazone RS in the ing indicates that the product meets USP Dissolution
Standard solution (ug/ml) Test 2.
Cu = nominal concentration of the Sample solution Medium: Prepare a solution of 0.05 M dibasic sodium
(g/mL) phosphate ina suitable flask, and adjust with phos-
Acceptance criteria: 90.0%-110.0% phoric acid to a pH of 7.5. Dissolve a suitable amount
of sodium lauryl sulfate to obtain a 20-g/L solution;
PERFORMANCE TESTS 900 mL
¢ DISSOLUTION (711) Apparatus 2: 75 rpm
[NoTe—Protect all solutions from light.] Time: 120 min
Test 1 Standard stock solution: 0.275 mg/mL of USP Meto-
=
wal
Medium: 2% w/v sodium lauryl sulfate in 0.05 M lazone RS. Initially add methanol to 10% of the vol-
5 monobasic sodium phosphate. Heat the mixture to ume of the flask. Sonicate to dissolve, and dilute with
i]
J about 37° to dissolve the sodium lauryl sulfate, and Medium to volume.
Dp adjust with 10 N sodium hydroxide to a pH of 7.5;
° 900 mL, deaerated
Standard solution: (1/900) mg/mL in Medium from
i= the Standard stock solution, where Lis the label claim
S Apparatus 2: 75 rpm in mg/Tablet
= Time: 120 min Sample solution: Pass a portion of the solution under
fo0 Buffer: 0.05 M monobasic potassium phosphate. Ad- test through a suitable filter of 0.45-um pore size.
a) just with phosphoric acid to a pH of 3.00. Detector: UV 238 nm
=) Mobile phase: Acetonitrile, methanol, and Buffer Path length: 1 cm
(270:50:680) Analysis
Standard stock solution: 0.28 mg/mL of USP Meto- Samples: Standard solution and Sample solution
lazone RS. Initially add methanol to 2% of the volume Calculate the percentage of the labeled amount of
of the flask. Sonicate to dissolve, and dilute with Me- metalozone (Ci6HisCIN303S) dissolved:
dium to volume.
Standard solution: (L/900) mg/mL in Medium from Result = (Au/As) x (Cs/L) x Vx 100
the Standard stock solution, where L is the label claim
in mg/Tablet Au = absorbance of the Sample solution
Sample solution: Pass a portion of the solution under As = absorbance of the Standard solution
test through a suitable filter of 0.45-um pore size. Cs = concentration of the Standard solution
Chromatographic system (mg/mL)
(See Chromatography (621), System Suitability.) L = label claim (mg/Tablet)
Vv = volume of Medium, 900 mL
Tolerances: NLT 75% (Q) of the labeled amount of
metolazone (CisHisCIN3O3$) is dissolved.
© UNIFORMITY OF DosAGE UNITS (905): Meet the
requirements
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and store below 30°.
© LABELING When more than one test for Dissolution is
given, the Labeling section states the test for Dissolution
used only if Test 7 is not used.
USP 41 Official Monographs / Metoprolol 2707

e USP REFERENCE STANDARDS (11) Acceptance criteria: 98.0%-102.0% on the dried basis
USP Metolazone RS
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.1%

Delete the following:

Metoprolol Fumarate
°e HEAVY MeTALs, Method | (231): NMT 10 ppme coriiai-
on Jan-2018)
ee oy A MeHy © ORGANIC IMPURITIES
Solution A: 1.3 g/L of sodium dodecyl sulfate in 0.1%
Ho” ese Cy bs (w/v) phosphoric acid
Solution B: Acetonitrile
Mobile phase: See Table 1.
(CisH2sNOs)2 - CaHsOu 650.80
2-Propanol, 1-[4-(2-methoxyethyl)phenoxy]-3-
cemeeiysaa amine, ()-, (£)-2-butanedioate (2:1) Table 1
(salt); Solution A Solution B
(4)-1-(lsopropylamino)-3-[p-(2-methoxyethyl)phenoxy]-
2-propanol fumarate (2:1) (salt) [119637-66-0]. 40
DEFINITION
Metoprolol Fumarate contains NLT 98.0% and NMT
102.0% of metoprolol fumarate [(CisH2sNOs)2 - CaHaOu],
calculated on the dried basis. 40
IDENTIFICATION Diluent: Solution A and Solution B (60:40)
e A. INFRARED ABSORPTION (197K) System suitability solution: 5 g/mL each of USP
e B. The retention time of the major peak of the Sample Metoprolol Fumarate RS, USP Metoprolol Related Com-
solution corresponds to that of the Standard solution, as pound A RS, USP Metoprolol Related CompoundB RS,
obtained in the Assay. and USP Metoprolol Related Compound CRS in Diluent
ASSAY Standard solution: 2.5 g/mL each of USP Metoprolol
Fumarate RS, USP Metoprolol Related Compound A RS,
© PROCEDURE
Solution A: 1.3 g/L of sodium dodecyl sulfate in 0.1% USP Metoprolol Related Compound B RS, USP
(w/v) phosphoric acid Metoprolol Compound C RS, and USP Metoprolol Re-
Solution B: Acetonitrile lated Compoun D RS in Diluent
Mobile phase: Solution A and Solution B (60:40) Sample solution: 1 mg/mL of Metoprolol Fumarate in
Standard solution: 1 mg/mL of USP Metoprolol Diluent ce
Chromatographic system wn
Fumarate RS in Mobile se
(See Chromatography (621), System Suitability.) v
Sample solution: 1 mg/mL of Metoprolol Fumarate in
Mobile phase Mode: LC s
Detector: UV 223 nm i}
Chromatographic system
Column: 4.6-mm x 15-cm; 5-um packing L7 =
(See Chromatography (621), System Suitability.) i}
Mode: LC Column temperature: 30° a=
Detector: UV 223 nm Flow rate: 1 mL/min i)
Column: 4.6-mm x 15-cm; 5-1m packing L7 Injection volume: 10 pL a}
Column temperature: 30° System suitability oH
vw
Flow rate: 1 mL/min Samples: System suitability solution and Standard
Injection volume: 10 uL solution
Run time: 10 min Suitability requirements
System suitability Resolution: NLT 1.5 between metoprolol related
Sample: Standard solution compound A and metoprolol related compound B;
Suitability requirements NLT 2.5 between metoprolol related compound B
Tailing factor: NMT 2 and metoprolol related compound C, System suitabil-
Relative standard deviation: NMT 0.73% ity solution
Analysis Relative standard deviation: NMT 2.0% for
Samples: Standard solution and Sample solution metoprolol, metoprolol related compound A,
Calculate the percentage of metoprolol fumarate metoprolol related compound B, metoprolol related
[(CisH2sNO3)2 + CaHaOa] in the portion of Metoprolol compound C, and metoprolol related compound D,
Fumarate taken: Standard solution
Analysis
Result = (ru/rs) x (Cs/Cy) x 100 Samples: Standard solution and Sample solution
Calculate the percentage of the corresponding
tu = peak response of metoprolol from the Sample metoprolol related compound in the portion of
solution Metoprolol Fumarate taken:
rs = peak response of metoprolol from the
Standard solution Result = (ru/rs) x (Cs/Cu) x 100
Cs = concentration of USP Metoprolol Fumarate RS
in the Standard solution (mg/mL) = peak response of the corresponding
Cu = concentration of Metoprolol Fumarate in the metoprolol related compound in the Sample
Sample solution (mg/mL) solution
ls = peak response of the corresponding
metoprolol related compound in the
Standard solution
 2708 Metoprolol / Official Monographs USP 41

Gs = concentration of the corresponding USP

Metoprolol Related Compound RS in the
Standard solution (g/mL) Metoprolol Succinate
Cu = concentration of Metoprolol Fumarate in the
Sample solution (ug/ml) H,CO. OL e oH
Calculate the percentage of any unspecified impurity in
the portion of Metoprolol Fumarate taken:
hen] + why
H
OH

OH 2
fe}

Result = (ru/rs) x (Cs/Cu) x 100

ty = peak response of each unspecified impurity in (CisH2sNO3)2 + CaHoO4 652.82

the Sample solution 2-Propanol, 1-[4-(2-methoxyethyl)phenoxy]-3-
rs = peak response of metoprolol in the Standard [(1-methylethyl)amino]-, @)-, butanedioate (2:1) (salt).
solution (4)-1-(Isopropylamino)-3-[p-(2-methoxyethyl)phenoxy]-
Cs = concentration of USP Metoprolol Fumarate RS 2-propanol succinate (2:1) (salt) [98418-47-4].
in the Standard solution (ug/mL)
Cy = concentration of Metoprolol Fumarate in the » Metoprolol Succinate contains not less than
Sample solution (ug/mL) 98.0 percent and not more than 102.0 percent of
Acceptance criteria: See Table 2. Disregard peaks be- (CisHasNO3)2 - C4HeOu, calculated on the dried
low 0.03%. basis.
Table 2 Packaging and storage—Preserve in tight containers at
controlled room temperature.
Relative Acceptance
Retention Criteria, USP Reference standards (11)—
Name Time NMT (%) USP Metoprolol Related Compound A RS
Fumaric acid 0:2 = (4)1-Ethylamino)-3-[4-(2-methoxyethyl)phenoxy]-
propan-2-ol.
Metoprolol related CisH23NO3 253.34
compound C 0.6 0.10 USP Metoprolol Related Compound B RS
Metoprolol related (+)1-Chloro-2-hydroxy-3-[4-(2-methoxyethyl)phenoxy]-
compound B> 0.7 0.10 - propane.
Metoprolol related CyaHi7zClO3 244.71
compound Ac 0.8 0.10 USP Metoprolol Related Compound C RS
Metoprolol 1.0 — (+)4-[2-Hydroxy-3-(1-methylethyl)aminopropoxy]
Metoprolol related benzaldehyde.
compound D¢ 15 0.10 Ci3HigNO3 237.29
Any individual _ USP Metoprolol Related Compound D RS
es
“
unspecified impurity 0.10 (+) Beis ae Nero 4-(2-methoxyethyl)phenoxy]
a propyl](1-methylethyl)amine.
J Total impurities — 1.0
- Co7HaiNOe 475.62
D @4-[2-Hydroxy-3-(isopropylamino)propoxy]benzaldehyde hydrochloride. USP Metoprolol Succinate RS
° ’1-Chloro-3-[4-(2-methoxyethyl)phenoxy]propan-2-ol.
Clarity and color of solution—A solution of Metoprolol
Cc
S © 1-Ethylamino-3-[4-(2-methoxyethyl)phenoxy]propan-2-ol.
Succinate having a concentration of 20 mg per mL is not
= 9 N,N-Bis{2-hydroxy-3-[4-(2-methoxyethyl)phenoxy]propyl}isopropylamine
hydrochloride. less clear than an equal volume of water in a test tube of
cs similar size. The absorbance of the solution determined at
“vw SPECIFIC TESTS 440 nm in a 5-cm cell, using water as the blank, is not
> e PH (791): 5.5-6.5, in a solution (1 in 10) more than 0.1.
e Loss ON DRYING (731) identification, Infrared Absorption (197K).
Analysis: Dry under vacuum at 60° for 4 h.
Acceptance criteria: NMT 0.5% pH (791): between 7.0 and 7.6, in a solution containing
65 mg per mL.
ADDITIONAL REQUIREMENTS Loss on drying (731)—Dry it in vacuum at 60° for 4 hours:
e PACKAGING AND STORAGE: Preserve in tight, light-resistant it loses not more than 0.2% of its weight.
containers. Store at controlled room temperature. Residue on ignition (281): not more than 0.1%.
e USP REFERENCE STANDARDS (11)
USP Metoprolol Fumarate RS
USP Metoprolol Related Compound A RS Delete the following:
1 eu arming ee netioryeany Uenenpraletopan-
2-ol. °Heavy metals, Method | (231): 0.001%.¢ cotticia 1-4an-2018)
CiaH23NO3 253.34 Related compounds—
USP Metoprolol Related Compound B RS
TEST 1—
1-Chloro-3-[4-(2-methoxyethyl)phenoxy]propan-2-ol.
CiaHizClO3 244.71 Adsorbent: 0.25-mm layer of chromatographic silica gel
USP Metoprolol Related Compound C RS mixture.
4-[2-Hydroxy-3-(isopropylamino)propoxy]benzaldehyde Test solution—Dissolve an accurately weighed quantity of
hydrochloride. Metoprolol Succinate in methanol to obtain a solution con-
Gi3HigNO3-HCl 273.76 taining 50 mg per mL.
USP Metoprolol Related Compound D RS Standard solution—Dilute the Test solution quantitatively,
N,N-Bis{2-hydroxy-3-[4-(2-methoxyethyl)phenox- and stepwise if necessary, with methanol to obtain a solu-
y]propyl}isopropylamine hydrochloride. tion having a concentration of 0.1 mg per mL.
Co7HaiNOe- HCl © 512.08 Application volume: 10 |tL.
Developing solvent system: a mixture of ethyl acetate and
methanol (80:20).
USP 41
Procedure—Proceed as directed for Thin-Layer Chromatog-
raphy under Chromatography (621). Place two 50-mL beak-
ers, each containing 30 mL of ammonium hydroxide, on the
bottom of a chromatographic chamber that is lined with
filter paper and contains the Developing solvent system, and
allow to equilibrate for 1 hour. Position the plate in the
chromatographic chamber, and develop the chromatogram
until the solvent front has moved about two-thirds of the
length of the plate. Remove the plate from the chamber,
mark the solvent front, and dry the plate for 3 hours in a
current of warm air. Place the plate in a chamber containing
iodine vapor, and allow to react for at least 15 hours. Com-
pare the intensities of the brown spots appearing on the
chromatogram: any secondary spot obtained from the Test
solution is not more intense than the corresponding spot
obtained from the Standard solution. Not more than 0.2% is
found.
TEST 2—
Sodium dodecy! sulfate solution, Mobile phase, and Resolu-
tion solution—Prepare as directed in the Assay.
Standard solution—Dissolve an eae weighed quan-
tity of USP Metoprolol Succinate RS in Mobile phase, and
dilute quantitatively, and stepwise if necessary, with Mobile
phase to obtain a solution having a known concentration of
about 1.0 ug per mL.
Test solution—Transfer about 50 mg of Metoprolol Succi-
nate, accurately weighed, to a 50-mL volumetric flask, dis-
solve in and dilute with Mobile phase to volume, and mix.
Chromatographic system (see Chromatography (621))—
Prepare as directed in the Assay. Chromatograph the Resolu-
tion solution, and record the peak responses as directed for
Procedure: the resolution, R, between metoprolol related
compound A and metoprolol related compoundBis not less
than 2.5; and the resolution, R, between metoprolol related
compound B and metoprolol related compoundCis not
less than 1.5. [NoTE—The relative retention times are about
0.6 for metoprolol related compound C, 0.7 for metoprolol
related compound B, 0.8 for metoprolol related compound
A, 1.0 for metoprolol, and 5.0 and 5.2 for the two diastere-
omers of metoprolol related compound D.] Chromatograph
the Standard solution, and record the peak responses as di-
rected for Procedure: the relative standard deviation for rep-
licate injections is not more than 5.0%.
Procedure—nject equal volumes (about 10 wL) of the
Standard solution and the Test solution into the chromato-
graph, record the chromatograms, and measure the peak
responses. Calculate the percentage of each impurity in the
portion of Metoprolol Succinate taken by the formula:
100(Cs/ Cr)(n/ rs)
in which Cs is the concentration, in mg per mL, of USP
Metoprolol Succinate RS in the Standara solution; Cr is the
concentration of metoprolol succinate in the Test solution; 1,
is the individual peak response of related impurities; and rs is
the peak response obtained from the Standard solution: not
more than 0.1% of any single impurity is found, and not
more than 0.5% of total impurities is found. [NoTE—The
sum of the peak responses for the two diastereomers of
metoprolol related compoundDis used in the above calcu-
lation to report the amount of metoprolol related com-
pound D.]
Assay—
Sodium dodecyl sulfate solution—Add 1.3 g of sodium
dodecyl sulfate to 1 L of aqueous phosphoric acid, 0.1%
(w/v).
Mobile phase—Preparea filtered and degassed mixture of
Sodium dodecyl sulfate solution and acetonitrile (60:40).
Make adjustments if necessary (see System Suitability under
Chromatography (621)).
Resolution solution—Prepare a solution in Mobile phase
containing about 5 jig each of USP Metoprolol Succinate RS,
USP Metoprolol Related Compound A RS, USP Metoprolol
Official Monographs / Metoprolol 2709
Related Compound B RS, USP Metoprolol Related Com-
pound C RS, and USP Metoprolol Related Compound D RS
per mL.
Standard preparation—Dissolve an accurately weighed
quantity of USP Metoprolol Succinate RS in Mobile phase,
and dilute quantitatively, and stepwise if necessary, with
Mobile phase to obtain a solution having a known concen-
tration of about 0.08 mg per mL.
Test preparation—Transfer about 80 mg of Metoprolol
Succinate, accurately weighed, to a 100-mL volumetric flask,
dissolve in and dilute with Mobile phase to volume, and mix.
Transfer 5.0 mL of this solution to a 50-mL volumetric flask,
dilute with Mobile phase to volume, and mix.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 223-nm detector
and a 4-mm x 12.5-cm column that contains 4-m packing
L7. The column temperature is maintained at 30°. The flow
rate is about 0.9 mL per minute. Chromatograph the Resolu-
tion solution, and record the peak responses as directed for
Procedure: the resolution, R, between metoprolol related
compound A and metoprolol related compoundBis not less
than 2.5; and the resolution, R, between metoprolol related
compound B and metoprolol related compoundCis not
less than 1.5. [NoTE—The relative retention times are about
0.6 for metoprolol related compound C, 0.7 for metoprolol
related compound B, 0.8 for metoprolol related compound
A, 1.0 for metoprolol, and 5.0 and 5.2 for the two diastere-
omers of metoprolol related compound D.] Chromatograph
the Standard preparation, and record the peak responses as
directed for Procedure: the relative standard deviation for
replicate injections is not more than 2.0%.
Procedure—Inject equal volumes (about 10 pL) of the
Standard preparation and the Test preparation into the chro-
matograph, record the chromatograms for at least 1.5 times
the retention of the metoprolol peak, and measure the peak
responses. Calculate the quantity, in mg, of (CrsHysNO3),
es in the portion of Metoprolol Succinate taken by the 5
‘ormula: wn
ae}
1000C(ru / rs) =
°
in which C is the concentration, in mg per mL, of USP |
°
Metoprolol Succinate RS in the Standard preparation; and ry Ke}
and rs are the peak responses obtained from the Test prepa- Ge)
ration and the Standard preparation, respectively.
2
mo}
=
nw
Metoprolol Succinate Extended-Release
Tablets
DEFINITION
Metoprolol Succinate Extended-Release Tablets contain NLT
90.0% and NMT 110.0% of the labeled amount of
metoprolol succinate [(CisH2sNOs3)2 - CaHeOa].
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
Sample solution: Equivalent to 200 mg of metoprolol
succinate from NLT 1 Tablet in a stoppered centrifuge
tube. Add 40 mL of pH 6.8 phosphate buffer (see Re-
agents, Indicators, and Solutions—Buffer Solutions) and
40 mL of methylene chloride, and shake for 5 min.
Centrifuge, filter, and use the aqueous phase as the
Sample solution.
Sample: Transfer 3 mL of the Sample solution to a
separator. Add 2 mL of ammonium hydroxide, and ex-
tract with 20 mL of methylene chloride. Filter the meth-
ylene chloride phase. Grind 1 mL of the filtrate with
300 mg of potassium bromide, dry in a current of
warm air, and prepare a disk.
Acceptance criteria: The IR spectrum of the Sample ex-
hibits maxima only at the same wavelengths as those
 2710 Metoprolol / Official Monographs USP 41

obtained from a similar preparation of USP Metoprolol Acceptance criteria: 90.0%-110.0%

Succinate RS (presence of metoprolol).
e B. INFRARED ABSORPTION (197K)
Sample: Transfer 5 mL of the Sample solution prepared
in Identification A to a glass-stoppered test tube. Add
2 mL of 5 N hydrochloric acid, and extract with 5 mL of
ether. Filter the ether phase. Grind 2 mL of the filtrate
with 300 mg of potassium bromide, dry in a current of
warm air, and prepare a disk.
Acceptance criteria: The IR spectrum of the Sample ex-
hibits maxima only at the same wavelengths as those
obtained from a similar preparation of succinic acid
(presence of succinate).
Add the following:
4e C. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.auspsy
ASSAY
PERFORMANCE TESTS
Change to read:
e DISSOLUTION (711)
Test 1
Medium: pH 6.8 phosphate buffer (see Reagents, In-
dicators, and Solutions—Buffer Solutions); 500 mL
Apparatus 2: 50 rpm
Times: 1, 4, 8, and 20h
Buffer, Mobile phase, and Standard solution: Pre-
pare as directed in the 4Assay. ausps:
Analysis: Proceed as directed in the AAssay,ausra) X-
cept use 5.0 mL of a filtered portion of the solution
under test as the Sample solution, and use Medium as
the blank, in comparison with a Standard solution with
a known concentration of USP Metoprolol Succinate
RS in the same Medium.
Acceptance criteria: See Table 7.

Change to read: Table 1

Time Amount Dissolved
© PROCEDURE (h) (%)
ABuffer: Mix 50 mL of 1 M monobasic sodium phos- 1 NMT 25
phate and 8.0 mL of 1 M phosphoric acid, and dilute
4 20-40
with water to 1000 mL. If necessary, adjust with 1M
monobasic potassium phosphate or 1 M phosphoric 8 40-60
acid to a pH of 3.0. 20 NLT 80
Mobile phase: Acetonitrile and Buffer (250:750)
Standard solution: 0.05 mg/mL of USP Metoprolol The percentages of the labeled amount of metoprolol
Succinate RS in Mobile phase succinate [(CysH2sNO3)2 + C4HeO,] dissolved atthe
Sample stock solution: Nominally 1 mg/mL of times specified conform to Dissolution (711), Accep-
metoprolol succinate pena as follows. Transfer a tance Table 2.
suitable number of Tablets to a suitable volumetric flask, Test 2: If the product complies with this test, the label-
ing indicates that the product meets USP Dissolution
fe
”
add about 5 mL of water, and allow the Tablets to dis-
a integrate. Add a volume of alcohol to fill 30% of the Test 2.
Medium: Simulated gastric fluid without enzyme, pH
Hd flask volume, and shake for 30 min. Add a portion of
1.2; 500 mL
i) 0.1 N hydrochloric acid to fill 50% of the flask volume,
° and shake for an additional 30 min. Dilute with 0.1 N Apparatus 2: 75 rpm
iS Times: 1, 4, 8, and 20h
S hydrochloric acid to volume. Filter, and discard the first
Buffer: 1M monobasic sodium phosphate, 1M phos-
= 10 mL of the filtrate.
Sample solution: Nominally 0.05 mg/mL of metoprolol phoric acid, and water (50:8:942). If necessary, adjust
a
succinate from the Sample stock solution in Mobile phase with 1M monobasic sodium phosphate or 1 M phos-
”
2 Chromatographic system phoric acid to a pH of 3.0.
(See Chromatography (621), System Suitability.) Mobile phase: Acetonitrile and Buffer (250:750)
Mode: LC Standard solution: Prepare a solution of USP
Detector: UV 280 nm Metoprolol Succinate RS in Medium as directed in Ta-
Column: 4-mm x 12.5-cm; 5-um packing L7 ble 2.
Flow rate: 1 mL/min
Injection volume: 40 pL Table 2
System suitability Tablet Strength Concentration
Sample: Standard solution mL
ha requirements
03
Tailing factor: NMT 2.0
Relative standard deviation; NMT 2.0% 0.190
Analysis 50
Samples: Standard solution and Sample solution 25
Calculate the percentage of the labeled amount of
metoprolol succinate [(CisH2sNO3)2 - CsHeO.] in the Sample solution: Pass the solution under test through
portion of Tablets taken: a suitable filter.
Chromatographic system
Result = (ru/rs) x (Cs/Cu) x 100 (See Chromatography (621), System Suitability.)
Mode: LC
tu = peak response of metoprolo! from the Sample Detector: UV 280 nm
solution Column: 4.0-mm x 12.5-cm; 4-um packing L7
rs = peak response of metoprolol from the Flow rate: 1 mL/min
Standard solution Injection volume: See Table 3.
G = concentration of USP Metoprolol Succinate RS
in the Standard solution (mg/mL)
Cu = nominal concentration of metoprolol succinate
in the Sample solution (mg/mL)auses:
USP 41 Official Monographs / Metoprolol 2711

Table 3 IMPURITIES
Tablet Strength Volume
Add the following:

4e ORGANIC IMPURITIES
Buffer; 1.15 mL of phosphoric acid in 2 L of water. Add
2.6g of sodium dodecyl sulfate. Sonicate to dissolve.
Solution A: Methanol and Buffer (30:70)
System suitability Solution B: Acetonitrile and Buffer (75:25)
Sample: Standard solution Mobile phase: See Table 5.
Suitability requirements
Column efficiency: NLT 1500 theoretical plates Table 5
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0% Solution A Solution B
Analysis
Samples: Standard solution and Sample solution
Calculate the concentration (Cj) of metoprolol succi-
nate dissolved in Medium at each time point (/):

Result = (ru/rs) x Cs
tu = peak response of metoprolol from the Sample
solution
ts = peak response of metoprolol from the
Standard solution Diluent: Acetonitrile and Buffer (40:60)
G = concentration of USP Metoprolol Succinate RS System suitability solution: 3 ug/ml of USP
in the Standard solution (mg/mL) letoprolol Related Compound A RS and 1 mg/mL of
Calculate the percentage of the labeled amount of USP Metoprolol Succinate RS in Diluent
metoprolol succinate UCisHosNOs)s - CaHeOx] Standard solution: 3 g/ml of USP Metoprolol Succi-
dissolved (Q), at each time point (i): nate RS in Diluent
SemOuty solution: 0.5 ig/ml of USP Metoprolol Suc-
Result; = C; x Vx (1/L) x 100 cinate RS from Standard solution in Diluent
Sample solution: Nominally 1 mg/mL of metoprolol
succinate from Tablets prepared as follows. Transfer a
Resultz = {[C2 « (V— Vs)] + (C; x Vs)} x (1/L) x 100 portion of finely powdered Tablets (NLT 20), equivalent
to 50 mg of metoprolol succinate, to a 50-mL volumet-
ric flask. Add Diluent to fill 60% of the flask volume and
Results = ({Cs x [V— (2 « Vs)]} + [(Co + G) x Vs]) x (1/ sonicate for 30 min with intermittent shaking. Dilute =
L x 100 with Diluent to volume. Pass the solution through a
“
~v
suitable filter of 0.45-um pore size.
=
Results = ({C, x [V— (3 x Vs)]} + [(C3 + Co + G) x Vs]) x Chromatographic system i}
(1/L) x 100 (See Chromatography (621), System Suitability.) |
Mode: LC re}
Detector: UV 223 nm a=
G = concentration of metoprolol succinate in the 2
Column: 4.6-mm x 15-cm; 5-14m packing L1
portion of sample withdrawn at time point no)
() (mg/mL) Column temperature: 30° Re
Vv volume of Medium, 500 mL Flow rate: 1 mL/min “

L label claim (mg/Tablet) Injection volume: 10 ul

Vs = volume of the Sample solution withdrawn from System suitability
the Medium (mL) Samples: System suitability solution, Standard solution,
Tolerances: See Table 4. and Sensitivity solution
Suitability requirements
Resolution: NLT 2.0 between metoprolol related
Table 4 compound A and metoprolol, System suitability
Amount solution
Time Point Time Dissolved Relative standard deviation: NMT 5.0%, Standard
(/) (h) (%) solution
1 1 NMT 20 Signal-to-noise ratio: NLT 10, Sensitivity solution
2 4 20-40
Analysis
Samples: Standard solution and Sample solution
3 8 55-85 Calculate the percentage of each unspecified degrada-
4 20 NLT 80 tion product in the portion of Tablets taken:
The percentages of the labeled amount of metoprolol Result = (ru/ts) x (Cs/Cu)} x 100
succinate [(CisH2sNOs3)2 - C4HeOa] dissolved at the
times specified conform to Dissolution (711), Accep- tr = peak response of each unspecified degradation
tance Table 2. product from the Sample solution
Is = peak response of metoprolol from the
Change to read: Standard solution
Gs = concentration of USP Metoprolol Succinate RS
¢ UNIFORMITY OF DOSAGE UNITS (905): Meet the require- in the Standard solution (g/mL)
ments
Cu = nominal concentration of metoprolol succinate
in the Sample solution (g/mL)
Agusrst
 2712 Metoprolol / Official Monographs USP 41

Age pane criteria: See Table 6. Reporting threshold: Mobile phase: Acetonitrile and Buffer (400:600)
. 0. Standard solution: 1 mg/mL of USP Metoprolol Tar-
trate RS in Mobile phase
Table 6 Sample solution: 1 mg/mL of Metoprolol Tartrate in
Mobile phase
Relative Chromatographic system
Retention (See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 223 nm
Metoprolol related Column: 4.6-mm x 15-cm; 5-um packing L7
Column temperature: 30°
Flow rate: 1 mL/min
Any unspecified Injection volume: 10 uL
System suitability
Sample: Standard solution
Suitability requirements
*Counter ion included for identification only. Tailing factor: NMT 2
Relative standard deviation: NMT 0.73%
AUSPAT
Analysis
ADDITIONAL REQUIREMENTS Samples: Standard solution and Sample solution
© PACKAGING AND STORAGE: Preserve in tight containers, Calculate the percentage of metoprolol tartrate
and store at controlled room temperature. [(CisH2sNO3)2 - C4HeOc] in the portion of sample taken:
e LABELING: Label it to indicate the content of metoprolol
succinate and its equivalent, expressed as metoprolol suc- Result = (ru/rs) x (Cs/Cy) x 100
cinate [(CisH2sNO3)2 - C4HeO¢]. When more than one Dis-
solution test is given, the labeling states the Dissolution ty = peak response of metoprolol from the Sample
test used only if Test 7 is not used. solution
Is = peak response of metoprolol from the
Standard solution
Change to read: Cs = concentration of USP Metoprolol Tartrate RS in
the Standard solution (mg/mL)
e USP REFERENCE STANDARDS (11) Cu = concentration of Metoprolol Tartrate in the
4USP Metoprolol Related Compound A RS Sample solution (mg/mL)
FE SS TERE ERONesy pene Ieper Acceptance criteria: 98.0%-102.0% on the dried basis
-0
CisHas3NOs 253.34,ausess IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.1%
USP Metoprolol Succinate RS
ra
i
is Delete the following:
i]
—
Dp °e HEAVY METALS, Method | (231): NMT 10 ppme cofiicai1-
2) Metoprolol Tartrate
te Jan-2018)
S © ORGANIC IMPURITIES
P= [Note—Use all solutions within 48 hrs.]
ii H
Buffer, Mobile phase, Sample solution, and Chromato-
is
2)
= H.co sipfey chats Me™
#2
2 Oe OH °
graphic system: Proceed as directed in the Assay.
System suitability solution: 5 g/mL each of USP
etoprolol Tartrate RS, USP Metoprolol Related Com-
pound A RS, USP Metoprolol Related CompoundB RS,
(CisHasNO3)2 + CaHoOo 684.81 a USP Metoprolol Related Compound C RS in Mobile
2-Propanol, 1-[4-(2-methoxyethyl)phenoxy]-3- jase
((1-methylethyl)amino]-, (+)-, [R-(R*,R*)]-2,3-dihydroxy- standard solution: 1 1g/mL each of USP eer ee
butanedioate (2:1) (salt); Tartrate RS, USP Metoprolol Related Compound A RS,
(4)-1 ee ee USP Metoprolol Related Compound B RS, USP
2-propanol |-(+)-tartrate (2:1) (salt); Metoprolol Related Compound C RS, and USP
1-(Isopropylamino)-3-[p-(2-methoxyethyl)phenoxy]-2-propa- Metoprolol Related CompoundD RS in Mobile phase
nol (2:1) dextro-tartrate salt [56392-17-7]. System suitability
DEFINITION Samples: System suitability solution and Standard
Metoprolol Tartrate contains NLT 98.0% and NMT 102.0% solution
of metoprolol tartrate [(CisH2sNOs3)2 - CsHeOc], calculated Suitability requirements
on the dried basis. Resolution: NLT 1.5 between metoprolol related
compound A and metoprolol related compound B;
IDENTIFICATION NLT 2.5 between metoprolol related compound B
e A. INFRARED ABSORPTION (197M) and metoprolol related compound C, System suitabil-
e B. The retention time of the major peak of the Sample ity solution
solution corresponds to that of the Standard solution, as Relative standard deviation: NMT 5.0% for the
obtained in the Assay. metoprolol peak, Standard solution
Analysis
ASSAY Samples: Standard solution and Sample solution
e PROCEDURE Calculate the percentage of each impurity in the por-
[Note—Use all solutions within 48 h.] tion of sample taken:
Buffer: 1.3 g/L of sodium dodecyl sulfate in 0.1% (w/v)
phosphoric acid Result = (ru/rs) x (Cs/Cu) x 100
USP 41 Official Monographs / Metoprolol 2713

tu = peak response of each individual impurity in USP Metoprolol Related Compound D RS

the Sample solution (4)-N,N-bis-[2-Hydroxy-3-[4-(2-methoxyethyl)phenox-
ts = peak response of the corresponding y]propy!](1-methylethyl)amine hydrochloride.
metoprolol related compound or metoprolol Caz7HaiNOg 512.08
(for calculating any individual unspecified
impurity) in the Standard solution
Cs = concentration of the corresponding USP
Metoprolol Related Compound RS or USP
Metoprolol Tartrate RS (for calculating any
individual unspecified impurity) in the Metoprolol Tartrate Injection
Standard solution (mg/mL)
Cu = concentration of Metoprolol Tartrate in the » Metoprolol Tartrate Injection is a sterile solution
Sample solution (mg/mL) of Metoprolol Tartrate in Water for Injection. It
Acceptance criteria: See Table 7. contains Sodium Chloride as a tonicity-adjusting
agent. It contains not less than 90.0 percent and
Table 1 not more than 110.0 Pesca of the labeled
Relative Acceptance amount of metoprolol tartrate [(CisH2sNOs3)2 -
Retention Criteria,
Name Time NMT (%)
C4HoOe].
Metoprolol related Packaging and storage—Preserve in single-dose, light-re-
compound Ca 0.65 0.10 sistant containers, preferably of Type | or Type Ill glass.
Metoprolol related
compound B> 0.72 0.10
Metoprolol related
Change to read:
compound A< 0.83 0.10
Metoprolol 1.00 —
USP Reference standards (11)—
%e (CN 1-May-2018)
Metoprolol related USP Metoprolol Tartrate RS
compound D¢« 4.78/5.00 0.10
USP Oxprenolol Hydrochloride RS
Any individual _ Identification—Place a volume of Injection, equivalent to
unspecified impurity 0.10 about 40 mg of metoprolol tartrate, in a separator, add
Total impurities' = 0.50 4 mL of dilute ammonium hydroxide (1 in 3), and extract
8(+)-4-[2-Hydroxy-3-(1 -isopropyl)aminopropoxy]benzaldehyde). with 20 mL of chloroform, filtering the chloroform extract
»(+)-1-Chloro-2-hydroxy-3-[4-(2-methoxyethyl)phenoxy]-propane. through chloroform-pre-rinsed anhistrous sodium sulfate.
¢(4)-1-(Ethylamino)-3-[4-(2-methoxyethyl)phenoxy]-propan-2-ol Evaporate the chloroform to dryness, and place in a freezer
4 (+)-N,N-bis-[2-Hydroxy-3-[4-(2-methoxyethyl)phenoxy]propyl](1- to congeal the residue: the IR absorption spectrum of a po-
methylethyl)amine hydrochloride. tassium bromide dispersion of the residue so obtained ex- ss
The sum of the peak responses of the two diastereomers is used to vw
calculate the amount of metoprolol related compound D.
hibits maxima only at the same wavelengths as that of a a>]
similar preparation of USP Metoprolol Tartrate RS.
‘Disregard any peak due to tartaric acid at about RRT 0.17.
Bacterial Endotoxins Test (85)—It contains not more =S
bo]
SPECIFIC TESTS than 25.0 USP Endotoxin Units per mg of metoprolol tar- |
© OPTICAL ROTATION, Specific Rotation (781S) trate. °
Sample solution: 20 mg/mL in water Ko}
Sterility Tests (71)—It meets the requirements when Sm
Acceptance criteria: +6.5° to +10.5° (t = 20°) tested as directed for Membrane Filtration under Test for
Cy
° PH (791) me}
Sterility of the Product to be Examined. >
Sample solution: 100 mg/mL of Metoprolol Tartrate in 7
water PH (791): between 5.0 and 8.0.
Acceptance criteria: 6.0-7.0 Other requirements—It meets the requirements under /n-
e Loss ON DRYING (731) jections and Implanted Drug Products (1).
Sample solution: Dry a sample in a vacuum at 60° for Assay—
yo re eer a degassed solution by dissolving
Acceptance criteria: NMT 0.5% 961 mg of 1-pentanesulfonic acid sodium salt (monohy-
ADDITIONAL REQUIREMENTS drate) and 82 mg of anhydrous sodium acetate in a mixture
e PACKAGING AND STORAGE: Preserve in tight, light-resistant of 550 mL of methanol and 470 mL of water and adding
containers. Store at controlled room temperature. 0.57 mL of glacial acetic acid.
© USP REFERENCE STANDARDS (11) Internal standard solution—Dissolve USP Oxprenolol Hy-
USP Metoprolol Tartrate RS drochloride RS in freshly prepared Mobile phase to obtain a
USP Metoprolol Related Compound A RS solution containing about 720 ug per mL.
(4)-1-(Ethylamino)-3-[4-(2-methoxyethyl)phenoxy]- Sodium chloride solution—Dissolve 9.0 g of sodium chlo-
propan-2-ol. ride in water to make 1000 mL.
CiaH23NO3 253.34 Standard preparation—Dissolve an accurately weighed
USP Metoprolol Related Compound B RS quantity of USP Metoprolol Tartrate RS in Sodium chloride
(4)-1-Chloro-2-hydroxy-3-[4-(2-methoxyethyl)phenoxy]- solution to obtain a stock solution having a known concen-
propane. tration of about 1000 11g per mL. Mix equal volumes, accu-
CyHi7ClO3 244.71 rately measured, of this stock solution and of Internal stan-
USP Metoprolol Related Compound C RS dard solution.
(+)-4-[2-Hydroxy- Assay preparation—Dilute an accurately measured volume
3-(1-isopropyl)aminopropoxy]benzaldehyde. of Injection, if necessary, quantitatively with Sodium chloride
CisHigNO3 | 237.29 solution to obtain a stock solution having a concentration of
about 1000 pg per mL. Mix equal volumes, accurately
oa of this stock solution and of Internal standard so-
lution.
 2714 Metoprolol / Official Monographs USP 41

Chromatographic system (see Chromatography (621))—The Chromatographicayer

liquid chromatograph is equipped with a 254-nm detector (See Chromatography (621), System Suitability.)
and a 3.9-mm x 30-cm column that contains packing L1. Mode: LC
The flow rate is about 1 mL per minute. Chromatograph Detector: UV 254 nm
three Fe peats injections of the Standard preparation, and Column: 4.6-mm x 25-cm; 5-um packing L1
record the peak responses as directed under Procedure: the Flow rate: 1.0 mL/min
relative standard deviation is not more than 2.0%, and the Injection volume: 20 uL
resolution factor between metoprolol tartrate and oxpre- System suitability
nolol hydrochloride is not less than 2.0. Sample: Standard solution
Procedure—Separately inject equal volumes (about 10 uL) [Note—The retention time for metoprolol tartrate is
of the Standard preparation and the Assay preparation into about 7.3 min.]
the chromatograph, record the chromatograms, and meas- Suitability requirements
ure the responses for the major peaks. The relative retention Relative standard deviation: NMT 1.3% for replicate
times are about 0.8 for metoprolol tartrate and 1.0 for ox- injections
prenolol hydrochloride. Calculate the quantity, in mg, of Analysis
metoprolol tartrate [(CisH2sNO3)2 - C4H6Q¢] in each mL of Samples: Standard solution and Sample solution
the Injection taken by the formula: Calculate the percentage of the labeled amount of
metoprolol tartrate [(C;sH2sNO3)2 - C4HeOc] in the por-
(£7 DC(Ru/ Rs) tion of Oral Solution taken:

in whichLis the labeled quantity, in mg, of metoprolol
tartrate in the Injection; D is the concentration, in ug per
mL, of metoprolol tartrate in the Assay preparation, on the
basis of thelabeled quantity in each mL of Injection taken
and the extent of dilution; C is the concentration, in ug per
mL, of USP Metoprolol Tartrate RS in the Standard prepara-
tion; and Ru and Rs are the peak response ratios of
metoprolol tartrate to oxprenolol hydrochloride obtained
from the Assay preparation and the Standard preparation, re-
spectively.
Metoprolol Tartrate Compounded Oral
Solution
Fs
”
DEFINITION
roe Metoprolol Tartrate Compounded Oral Solution contains
i]
— NLT 90.0% and NMT 110.0% of the labeled amount of
a e USP REFERENCE STANDARDS (11)
3 metoprolol tartrate [(CisH2sNO3)2 - C4HeOc].
= Prepare Metoprolol Tartrate Compounded Oral Solution
6 10 mg/mL as follows (see Pharmaceutical Compounding—
= Nonsterile Preparations (795)).
is
a)
Metoprolol Tartrate powder lq
—_
Vehicle for Oral Solution (regular
or sugar-free), NF, a sufficient
quantity to make 100 mL
Add Metoprolol Tartrate powder and 20 mL of Vehicle to a
mortar, and mix, Add the Vehicle in small portions almost
to volume, and mix thoroughly after each addition. Trans-
fer the contents of the mortar, stepwise and quantita-
tively, to a calibrated bottle. Add enough Vehicle to bring
to final volume, and mix well.
ASSAY
¢ PROCEDURE
Mobile phase: 961 mg of seu acid so-
dium salt (monohydrate) and 82 mg of anhydrous so-
dium acetate in a mixture of 550 mL of methanol and
470 mL of water. Add 0.57 mL of glacial acetic acid.
Filter, and degas.
Standard solution: 100 t1g/mL of USP Metoprolol Tar-
trate RS
Sample solution: Agitate the container of Oral Solution
for 30 min on a rotating mixer, remove a 5-mL sample,
and store in a clear glass vial at —70° until analyzed. At
the time of analysis, remove the sample from the
freezer, allow it to reach room temperature, and mix on
a vortex mixer for 30 s. Pipet 1.0 mL of the sample to a
100-mL volumetric flask, and dilute with Mobile phase
to volume.
Result = (ru/rs) x (Cs/Cu) x 100
ru = peak response from the Sample solution
rs = peak response from the Standard solution
Cs = concentration of USP Metoprolol Tartrate RS in
the Standard solution (\ug/mL)
Cu = nominal concentration of metoprolol tartrate
in the Sample solution (ug/' mL).
Acceptance criteria: 90.0%-110.0%
SPECIFIC TESTS
© PH (791): 3.6-4.6
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Package in tight, light-resistant
containers. Store at controlled room temperature or in a
refrigerator.
e BEYOND-UsE DATE: NMT 60 days after the date on which
it was compounded when stored at controlled room
temperature or in a refrigerator
e LABELING: Label it to state the Beyond-Use Date.
USP Metoprolol Tartrate RS
Metoprolol Tartrate Compounded Oral
Suspension
DEFINITION
Metoprolol Tartrate Compounded Oral Suspension contains
NLT 90.0% and NMT 110.0% of the labeled amount of
metoprolol tartrate [(CisH2sNO3)2 - CaHeOc].
Prepare Metoprolol Tartrate Compounded Oral Suspension
10 mg/mL as follows (see Pharmaceutical Compounding—
Nonsterile Preparations {795)).
Metoprolol Tartrate lg
Vehicle: a 1:1 mixture of Vehicle
for Oral Solution, (regular or
sugar-free), NF, and Vehicle for
Oral Suspension, NF, a suffi-
cient quantity to make 100 mL
Place the required number of tablets in a suitable mortar,
and comminute to a sine pode, or use Metoprolol Tar-
trate powder. Add the Vehicle in small portions, and mix
well. Transfer the contents of the mortar, stepwise and
quantitatively, to a calibrated bottle. Add the Vehicle in
jortions to rinse the mortar. Add sufficient Vehicle to
ting to final volume, and mix well.
USP 41
ASSAY
¢ PROCEDURE
Mobile phase: 961 mg of 1-pentanesulfonic acid so-
dium salt (monohydrate) and 82 mg of anhydrous so-
dium acetate in a mixture of 550 mL of methanol and
470 mL of water. Add 0.57 mL of glacial acetic acid.
Filter, and degas.
Standard solution: 100 g/mL of USP Metoprolol Tar-
trate RS
Sample solution: Agitate the container of Oral Suspen-
sion for 30 min ona rotating mixer, remove a 5-mL
sample, and store in a clear glass vial at —70° until ana-
lyzed. At the time of analysis, remove the sample from
the freezer, allow it to reach room temperature, and
mix with a vortex mixer for 30 s. Pipet 1.0 mL of the
sample to a 100-mL volumetric flask, and dilute with
Mobile phase to volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Flow rate: 1.0 mL/min
Injection volume: 20 pL
System suitability
Sample: Standard solution
[Note—The retention time for metoprolol tartrate is
about 7.3 min.]
Suitability requirements
Relative standard deviation: NMT 1.3% for replicate
injections
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
metoprolol tartrate [(CisH2sNO3)2 - C4HeOo] in the por-
tion of Oral Suspension taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response from the Sample solution
Is = peak response from the Standard solution
Cs = concentration of USP Metoprolol Tartrate RS in
the Standard solution (g/mL)
Cu = nominal concentration of metoprolol tartrate
in the Sample solution (ug/mL)
Acceptance criteria: 90.0%-110.0%
SPECIFIC TESTS
© PH (791): 3.6-4.6
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Package in tight, light-resistant
containers. Store at controlled room temperature, or in a
refrigerator.
e BEYOND-UsE DATE: NMT 60 days after the date on which
it was compounded when stored at controlled room
temperature, or in a refrigerator
¢ LABELING: Label it to state that it is to be well shaken,
and to state the Beyond-Use Date.
e USP REFERENCE STANDARDS (11)
USP Metoprolol Tartrate RS
Metoprolol Tartrate Tablets
DEFINITION
Metoprolol Tartrate Tablets contain NLT 90.0% and NMT
110.0% of the labeled amount of metoprolol tartrate
[(CisHasNOs)2 - CaHeOo].
Official Monographs / Metoprolol 2715
IDENTIFICATION
eA.
Standard solution: 0.1 mg/mL of USP Metoprolol Tar-
trate RS in water
Sample solution: Transfer an amount equivalent to
50 mg of metoprolol tartrate from a quantity of finely
powdered Tablets to a 500-mL volumetric flask, dilute
with water to volume, and mix. Pass a portion of the
solution througha filter of 1-1um or finer Pore size.
Acceptance criteria: The UV spectrum of the Sample
solution exhibits maxima and minima at the same wave-
lengths as those of the Standard solution.
e B. The retention time of the metoprolol peak of the Sam-
ple solution corresponds to that of the Standard solution,
as obtained in the Assay.
ASSAY
e PROCEDURE
Mobile phase: 961 mg of sodium 1-pentanesulfonate
and 82 mg of anhydrous sodium acetate in a mixture of
550 mL of methanol and 470 mL of water. Add
0.57 mL of glacial acetic acid.
Diluent: Methanol and 0.1 N hydrochloric acid (1:1)
System suitability stock solution: 0.72 mg/mL of USP
Oxprenolol Hydrochloride RS in Diluent
Standard stock solution: 1 mg/mL of USP Metoprolol
Tartrate RS in Diluent
System suitability solution: System suitability stock solu-
tion and Standard stock solution (1:1)
Standard solution: 0.5 mg/mL of USP Metoprolol Tar-
trate RS from theivanaand stock solution in Mobile phase
Sample stock solution: Nominally 1 mg/mL of
metoprolol tartrate from Tablets prepared as follows.
Transfer a portion of finely powdered Tablets (NLT 20),
equivalent to about 50 mg of metoprolol tartrate, to a
50-mL volumetric flask, add 30 mL of Diluent, shake by
mechanical means for 30 min, sonicate for 15 min, and
heat on a steam bath for 10 min. Allow the solution to &
cool to room temperature, dilute with Diluent to vol- A)
ume, and centrifuge a portion of the solution. Use the Z
supernatant. =
Sample solution: Nominally 0.5 mg/mL of metoprolol }
tartrate prepared from the Sample stock solution in Mo- =
}
bile phase. Pass a portion of the solution througha filter e=
of 0.5-um or finer pore size. Discard the first few millili- »
ters of the filtrate. i}
Chromatographic system =z
(See Chromatography (621), System Suitability.)
a
Mode: LC
Detector: UV 254 nm
Column: 3.9-mm x 30-cm; 10-um packing L1
Flow rate: 1 mL/min
Injection volume: 30 uL
System suitability
Samples: Standard solution and System suitability
solution
[Note—The relative retention times for metoprolol and
oxprenolol are 0.8 and 1.0, respectively]
Suitability requirements
Resolution: NLT 2.0 between metoprolol and oxpre-
nolol, System suitability solution
Relative standard deviation: NMT 2.0%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
metoprolol tartrate [(CisH2sNO3)2 - C4H6Oc] in the por-
tion of Tablets taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response of metoprolol from the Sample
solution
rs = peak response of metoprolol from the
Standard solution
 2716 Metoprolol / Official Monographs USP 41

Cs = concentration of USP Metoprolol Tartrate RS in Suitability requirements

the Standard solution (mg/mL) Resolution: NLT 1.5 between tyrosol and metoprolol
Cu = nominal concentration of metoprolol tartrate related compound C, System suitability solution
in the Sample solution (mg/mL) Relative standard deviation: NMT 5.0%, Standard
Acceptance criteria: 90.0%-110.0% solution
Signal-to-noise ratio: NLT 10, Sensitivity solution
PERFORMANCE TESTS Analysis ay
e DISSOLUTION (711) Samples: Standard solution and Sample solution
Medium: Simulated gastric fluid TS (without enzyme); Calculate the percentage of each unspecified degrada-
900 mL tion product in the portion of Tablets taken:
Apparatus 1: 100 rpm
Time: 30 min Result = (ru/s) x (Cs/Cu) x 100
Standard solution: USP Metoprolol Tartrate RS with a
known concentration in Medium tu = peak response of each unspecified degradation
Sample solution: Sample per the chapter. Dilute with product from the Sample solution
Medium as needed. rs = peak response of metoprolol from the
Instrumental conditions Standard solution
Mode: UV Cs = concentration of USP Metoprolol Tartrate RS in
Analytical wavelength: 275 nm the Standard solution (mg/mL)
Analysis Cu = nominal concentration of metoprolol tartrate
Samples: Standard solution and Sample solution in the Sample solution (mg/mL)
Tolerances: NLT 75% (Q) of the labeled amount of Acceptance criteria: See Table 2. Disregard peaks be-
metoprolol tartrate [(CisH2sNO3)2 - C4HeOc] is dissolved. low 0.1%.
¢ UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements Table 2
IMPURITIES Relative Acceptance
e ORGANIC IMPURITIES Retention Criteria,
Solution A: 3.9 g of ammonium acetate in 810 mL of Name Time NMT (%)
water. Add ein of Cee 10.0 rtof glacial Tyrosole 0.41 ak
acetic acid, and 3.0 mL of phosphoric acid. [NoTE—Ad-
just the pH of the solution tS 3.7 if needed.] —— e 0.43 ov
Solution B: Acetonitrile and Solution A (70:30) Metoprolol related ;
Mobile phase: See Table 1. eaapalin ie ng an
Metoprolol 1.00 —
Hable’ Metoprolol related
- Solution A Solution B compound Db 1.57 _
& Metoprolol related
2 1 compound Db 1.58 _
a Metoprolo! related _
fo} compound Bb 1.68
= Any individual
S “4 unspecified degradation _
0 product 0.2
a : Total degradation ~~
=) Diluent: Acetonitrile and Solution A (17:83) products 1.0
System suitability solution: 0.01 mg/mL each of #2-(4-Hydroxyphenyl)ethanol. For resolution measurement only.
tyrosol and USP Metoprolo! Related CompoundC RS in Specified impurities controlled in the drug substance.
Diluent ¢(4)-1-(Ethylamino)-3-[4-(2-methoxyethyl)phenoxy]-propan-2-ol.
Standard solution: 0.01 mg/mL of USP Metoprolol Tar- 4(+)-N,N-Bis-[2-hydroxy-3-[4-(2-methoxyethyl)phenoxy]propyl](1-
trate RS in Diluent methylethyl)amine hydrochloride. It has two diastereomers.
Sensitivity solution: 0.7 g/mL of USP Metoprolol Tar- © (+)-1-Chloro-2-hydroxy-3-[4-(2-methoxyethyl)phenoxy]-propane.
trate RS from the Standard solution in Diluent ADDITIONAL REQUIREMENTS
Sample solution: Nominally 1 mg/mL of metoprolol e PACKAGING AND STORAGE: Preserve in tight, light-resistant
tartrate from Tablets prepared as follows. Transfer containers. Store at controlled room temperature.
100 mg of metoprolol tartrate from a quantity of finely e USP REFERENCE STANDARDS (11)
Pei Tablets (NLT 20) to a 100-mL volumetric USP Metoprolol Related Compound C RS
lask, and add 50 mL of Diluent. Sonication and stirring 4-[2-Hydroxy-3-(isopropylamino)propoxy]benzaldehyde
may be necessary for complete dissolution. Dilute with hydrochloride.
Diluent to volume. Centrifuge a portion of the solution. Ci3HisNO3-HCl 273.76
Use the supernatant. USP Metoprolol Tartrate RS
Chromatographic system USP Oxprenolo! Hydrochloride RS
(See Chromatography (621), System Suitability.)
Mode: LC -
Detector: UV 275 nm
Column: 4.6-mm x 15-cm; 5-m packing L1
Flow rate: 1 mL/min
Injection volume: 20 pL
System suitability .
Samples: System suitability solution, Standard solution,
and Sensitivity solution
USP 41 Official Monographs / Metoprolol 2717

Tu = peak Fespanse of metoprolol or

hydrochlorothiazide from the Sample solution
Metoprolol Tartrate and Is peak Ueda of metoprolol or
Hydrochlorothiazide Tablets hydrochlorothiazide from the Standard
solution
DEFINITION Cs concentration of USP Metoprolol Tartrate RS
Metoprolol Tartrate and Hydrochlorothiazide Tablets contain or USP Hydrochlorothiazide RS in the
NLT 90.0% and NMT 110.0% of the labeled amount of Standard solution (mg/mL)
metoprolol tartrate [(CisH2sNO3)2 + CaHeOc] and hydro- Cy = nominal concentration of the corresponding
chlorothiazide (C7HgCIN30.S2). analyte in the Sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0% of the labeled
IDENTIFICATION amount of metoprolol tartrate [(CisH2sNO3)2 - C4H6Oc]
e A. The retention times of the metoprolol and hydrochlo- and hydrochlorothiazide (C7HgCIN3O.4S2)
rothiazide peaks of the Sample solution correspond to
those of the Standard solution, as obtained in the Assay. PERFORMANCE TESTS
e B. The UV spectra of the metoprolol and hydrochlorothi- e DISSOLUTION (711)
azide peaks of the Sample solution correspond to those of Medium: Simulated gastric fluid TS (without enzyme);
the Standard solution, as obtained in the Assay. 900 mL
Apparatus 1: 100 rpm
ASSAY Time: 30 min
¢ PROCEDURE Determination of dissolved metoprolol tartrate
Solution A: 4.1 g/L of monobasic sodium phosphate in Standard solution: 0.05 mg/mL of USP Metoprolol
water. Adjust with phosphoric acid to a pH of 3.0. Tartrate RS in Medium
Solution B: Acetonitrile Sample solution: Remove 125 mL of the solution
Mobile phase: See Table 7. under test, allow to cool to room temperature, and
filter, discarding the first 25 mL of the filtrate. [NoTE—
Table 1 Retain 30 mL of the remaining filtrate of the solution
under test for the Determination of dissolved hydrochlo-
Solution A Solution B rothiazide.] If necessary, prepare 0.05 mg/mL of
% Yo’
metoprolol! tartrate from the filtrate in fresh Medium.
8. Instrumental conditions
85 15 Mode: UV
10. Analytical wavelength: 276 nm
1 85 1 Cell: 2cm
13 Blank: Medium
Analysis
Diluent: Acetonitrile and Solution A (15:85) ieee Standard solution, Sample solution, and
Standard solution: 0.1 mg/mL of USP Metoprolol Tar- Blan c
trate RS and 0.05 mg/mL of USP Hydrochlorothiazide Transfer to separate separators 50.0 mL each of the wn
Standard solution, Sample solution, and Blank. Add a)
RS in Diluent
Sample solution: Nominally 0.1 pom.ofmetoprolol 10 mL of 2.5 N sodium hydroxide to each separator, =
tartrate and 0.05 mg/mL of hydrochlorothiazide in Dilu- and extract each with three 15-mL portions of chlo- ro}
roform, filtering the chloroform extracts through J
ent prepared as follows. Transfer a portion of the pow- re}
der from NLT 20 finely powdered Tablets to a suitable pledgets of chloroform-prerinsed glass wool into indi- ro}=
volumetric flask and dissolve in a suitable amount of vidual 50-mL volumetric flasks. Dilute the contents of Ey
Diluent. Sonication may be necessary for complete dis- each flask with chloroform to volume, and mix. De- so}
solution. Dilute with Diluent to volume. termine the absorbances of the solutions obtained. Es
“"
Chromatographic system Calculate the percentage of the labeled amount of
(See Chromatography (621), System Suitability.) metoprolol tartrate [(CisH2sNO3)2 - C4HeOc] dissolved:
Mode: LC
Detector: UV 223 nm. For Identification B, use a diode Result = (Au/As) x Cs x Vx D x (100/L)
array detector in the range of 200-400 nm.
Column: 4.6-mm x 10-cm; 3.5-1um packing L1 Au = absorbance of the Sample solution
Autosampler temperature: 4° As = absorbance of the Standard solution
Flow rate: 1 mL/min G = concentration of USP Metoprolol Tartrate RS in
Injection volume: 10 uL the Standard solution (mg/mL)
System suitability Vv = volume of Medium, 900 mL
Sample: Standard solution D = dilution factor of the Sample solution
[Note—The relative retention times for hydrochlorothia- L = label claim (mg/Tablet)
zide and metoprolol are 0.56 and 1.0, respectively.] Determination of dissolved hydrochlorothiazide
Suitability requirements Standard solution: 0.03 mg/mL of USP Hydrochloro-
Tailing factor: NMT 2.0 for metoprolol and thiazide RS in Medium
hydrochlorothiazide Sample solution: Pass a portion of the filtrate retained
Relative standard deviation: NMT 1.0% for from the Determination of dissolved metoprolol tartrate
metoprolol and hydrochlorothiazide through a filter of 0.8-um or finer pore size, and dis-
Analysis card the first 5 mL of the filtrate. If necessary, prepare
Samples: Standard solution and Sample solution 0.03 mg/mL of hydrochlorothiazide in fresh Medium.
Calculate the percentage of the labeled amount of Instrumental conditions
metoprolol tartrate [(CisH2sNO3)2 - C4HsOc] and hydro- Mode: UV
Guaculaace (C7HsCIN304Sz2) in the portion of Tablets Analytical wavelength: 316 nm
taken: Cell: 2cm
Blank: Medium
Result = (ru/rs) x (Cs/Cu) x 100 Analysis
Samples: Standard solution, Sample solution, and
Blank
 2718 Metoprolol / Official Monographs USP 41

Determine the absorbances of the Standard solution Column: 4.6-mm x 15-cm; 5-um packing L1
and the Sample solution at the wavelength of maxi- Flow rate: 1 mL/min
mum absorbance. Injection volume: 20 uL
Calculate the percentage of hydrochlorothiazide System suitability
(C7HsCIN304S2) dissolved: Samples: System suitability solution, Standard solution,
and Sensitivity solution
Result = (Au/As) x Cs x Vx D x (100/L) [Note—See Table 3 for relative retention times.]
Suitability requirements
Au = absorbance of the Sample solution Resolution: NLT 1.5 between maltol and
As = absorbance of the Standard solution benzothiadiazine related compound A; NLT 3.0 be-
Gs = concentration of USP Hydrochlorothiazide RS tween benzothiadiazine related compound A and hy-
in the Standard solution (mg/mL) drochlorothiazide, System suitability solution
Vv = volume of Medium, 900 mL Relative standard deviation: NMT 5.0% for
D = dilution factor of the Sample solution metoprolol and hydrochlorothiazide, Standard solution
L = label claim (mg/Tablet) Signal-to-noise ratio: NLT 10 for metoprolol and hy-
Tolerances: NLT 80% (Q) of the labeled amount of drochlorothiazide, Sensitivity solution
metoprolol tartrate [(CisH2sNO3)2 - C4HsO6] and NLT Analysis
80% (Q) of the labeled amount of hydrochlorothiazide Samples: Standard solution and Sample solution
(C7HgCIN304S2) is dissolved. For impurities detected at UV 275 nm
e UNIFORMITY OF DOSAGE UNITS (905), Content Uniformity: Calculate the percentage of maltol and any unspecified
Meet the requirements with respect to metoprolol tar- degradation product (excluding the peaks that ap-
trate and hydrochlorothiazide pear at 320 nm) in the portion of Tablets taken:
IMPURITIES Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
© ORGANIC IMPURITIES
Solution A: Dissolve 3.9 g of ammonium acetate in tu = peak response of maltol or any unspecified
810 mL of water. Add 2.0 mL of triethylamine, 10.0 mL degradation product from the Sample
of glacial acetic acid, and 3.0 mL of phosphoric acid. solution
[Note—Adjust the solution to a pH of 3.7 if needed.] rs = peak response of metoprolol from the
Solution B: Acetonitrile and Solution A (70:30) Standard solution
Mobile phase: See Table 2. Gs = concentration of USP Metoprolol Tartrate RS in
the Standard solution (ug/mL)
Table 2 Cy = nominal concentration of metoprolol tartrate
in the Sample solution (g/mL)
Solution A Solution B
F = relative response factor (autive to metoprolol)
%
For impurities detected at UV 320 nm
Calculate the percentage of benzothiadiazine related
20 compound A and any unspecified degradation
pel
”

¥ 60 product (excluding the peaks that appear at 275 nm)

in the portion of Tablets taken:
A]
—
D
° Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
€ 9 10
Sj ty = peak response of benzothiadiazine related
P= Diluent: Acetonitrile and Solution A (17:83) compound A or any unspecified degradation
a
system suitability solution: 0.7 g/mL of USP Maltol product from the Sample solution
wn“ S, 5 ug/mL of USP Benzothiadiazine Related Com- Is = peak response of hydrochlorothiazide from the
m, pound A RS, and 0.5 mg/mL of USP Hydrochlorothia- Standard solution
zide RS in Diluent Cs = concentration of USP Hydrochlorothiazide RS
Standard solution: 10.0 g/mL of USP Metoprolol Tar- in the Standard solution (ug/mL)
trate RS and 2.5 g/mL of USP Hydrochlorothiazide RS Cu = nominal concentration of hydrochlorothiazide
in Diluent in the Sample solution (g/mL)
Sensitivity solution: 1.0 41g/mL of USP Metoprolol Tar- F = relative response factor (relative to
trate RS and 0.25 pg/mL of USP Hydrochlorothiazide RS hydrochlorothiazide)
in Diluent from the Standard solution Acceptance criteria: See Table 3. Disregard peaks be-
Sample solution: Nominally 1 mg/mL of metoprolol low 0.1%.
tartrate in Diluent prepared as follows. Transfer a suita-
ble portion of NLT 20 finely powdered Tablets, equiva-
lent to 200 mg of metoprolol tartrate, into a suitable Table 3
volumetric flask and add Diluent to about 50% of the Relative Relative Acceptance
flask volume. Sonicate for 20 min with occasional Retention | Response Criteria,
swirling. Stir for 15 min. Dilute with Diluent to volume. Name Time Factor NMT (%)
Centrifuge a portion of the solution. Use the Maltol 0.29 7 0.2
supernatant.
Benzothiadiazine
Chromatographic system
related compound A? 0.31 1.0 1.0
(See Chromatography (621), System Suitability.)
Mode: LC Hydrochlorothiazide 0.46 —
Detectors Metoprolol 1.00 —- —
Metoprolol and related impurities: UV 275 nm 4Not to be included in the total degradation products.
Hydrochlorothiazide and related impurities: UV >Based on the sum of unspecified degradation products determined at
320 nm 275 nm and at 320 nm.
Excluding benzothiadiazine related compound A.
USP 41 Official Monographs / Metrifonate 2719

Table 3 (Continued) mediately examine the plate: the principal spot in the chro-
Relative Relative Acceptance
matogram obtained from the Test solution corresponds in Rr
Retention Response Criteria,
value, size, and blue color to that in the chromatogram ob-
Name Time Factor NMT (%) tained from the Standard solution.
Any unspecified C: Dissolve 20 mg of Metrifonate in 1 mL of 2 N sodium
degradation product? _ 1.0 0.2 hydroxide, add 1 mL of pyridine, shake, and heat on a
water bath for 2 minutes: a red color develops in the pyri-
Total degradation
dine layer.
products« _ a 1.0
Not to be included in the total degradation products. D: To 100 mg of Metrifonate add 0.5 mL of nitric acid,
bBased on the sum of unspecified degradation products determined at
0.5 mL of a 50% solution of ammonium nitrate, and 0.1 mL
275 nm and at 320 nm. of 30 percent hydrogen peroxide, and heat on a water bath
Excluding benzothiadiazine related compound A. for 10 minutes. Heat to boiling, and add 1 mL of ammo-
nium molybdate TS: a yellow color precipitate is formed.
ADDITIONAL REQUIREMENTS Acidity—Dissolve 2.5 g of it in carbon dioxide-free water,
¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant dilute with carbon dioxide-free water to 50 mL, and add
containers. Store at controlled room temperature. 0.1 mL of methyl red TS. Not more than 1.0 mL of 0.1 N
e¢ USP REFERENCE STANDARDS (11) sodium hydroxide is required to change the color of the
USP Benzothiadiazine Related Compound A RS indicator.
4-Amino-6-chloro-1,3-benzenedisulfonamide.
CeHsCIN3O482 285.73 Water Determination, Method | (921): not more than
USP Hydrochlorothiazide RS 0.3%.
USP Maltol RS
3-Hydroxy-2-methyl-4-pyrone. Delete the following:
CeHeO3 = 126.11
USP Metoprolol Tartrate RS
°Heavy metals (231): 0.001%. (oficiat Jan-2018)
Limit of free chloride—Dissolve 5.0g of Metrifonate in
30 mL of alcohol, and add a mixture of 100 mL of water
and 15 mL of nitric acid. Titrate with 0.1 N silver nitrate VS,
determining the endpoint potentiometrically using a silver
Metrifonate electrode. Not more than 0.7 mL of 0.1 Nsilver nitrate is
consumed (0.05%).
Chromatographic purity—
Solution A—Dissolve 1.36 g of monobasic potassium
phosphate in water, and dilute with water to 1000 mL. Ad-
just with phosphoric acid to a pH of 3.0.
C4HgCl304P 257.44 Solution B—Use acetonitrile. S
Phosphonic acid, (2,2,2-trichloro-1-hydroxyethyl)-, dimethyl Mobile phase—Use variable mixtures of Solution A and So- a)
ester.
lution B as directed for Chromatographic See Make ad- a]
Dimethyl! (2,2,2-trichloro-1-hydroxyethyl)phosphonate
[52-68-6]. justments if necessary (see System Suitability under Chroma- =
tography (621)). °
=]
» Metrifonate contains not less than 98.0 percent Diluent—Prepare a mixture of acetonitrile and water °
(1:1) ©
and not more than 100.5 percent of C4HgClsO,P, ad
calculated on the anhydrous basis. Standard preparation—Prepare a solution of USP Me- Ey
trifonate RS in Diluent containing 20 mg per mL. mo]
>
Packaging and storage—Preserve in well-closed contain- Test solution—Transfer 500 mg of Metrifonate, accurately a
ers at a temperature not exceeding 25°. weighed, to a 25-mL volumetric flask, dissolve in and dilute
Labeling lapel it to indicate that it is for veterinary use with Diluent to volume, and mix.
only. Chromatographic system (see Chromatography (621))—The
USP Reference standards (11)— liquid chromatograph is equipped with a 210-nm detector
USP Metrifonate RS and a 4-mm x 25-cm column that contains 5-um packing
Trichlorfon. L7. The column is maintained at a constant temperature of
about 40°. The flow rate is about 1.5 mL per minute. The
Completeness of solution (641): meets the require- chromatograph is programmed as follows.
ments, 0.5 g of it being dissolved in methanol.
Color of solution (631)—The solution obtained in the test Time Solution A Solution B
for Completeness of solution has no more color than Match-
ing Fluid F. (minutes) (%) (%) Elution
0 90 10 equilibration (10 min-
Identification— utes)
A: Infrared Absorption (197K). 0-5 90 10 isocratic
B: Thin-Layer Chromatographic Identification Test (201)— 5 90-85 10315 step gradient
Test solution: — Dissolve 10 mg of Metrifonate in metha- 5-25 85 15 isocratic
nol, and dilute with methanol to 10.0 mL. 25 85-45 1555 step gradient
Developing solvent sytstem: a mixture of toluene, diox- 25-end* 45 55 isocratic
ane, and glacial acetic acid (70:25:5)
*The elution concludes at 3 times the retention time of metrifonate.
Procedure—Proceed as directed in the chapter. After al-
lowing the plate to air-dry, spray the plate with a 5% solu-
tion of =e ine in acetone, and heat at Procedure—Separately inject equal volumes (about 50 uL)
120° for 15 minutes. Before the plate cools, spray it with a of the Standard solution and the Test solution into the chro-
10% solution of tetraethylenepentamine in acetone, and im- matograph, record the chromatograms, and measure the
 2720 Metrifonate / Official Monographs USP 41

fe areas. Calculate the percentage of each impurity taken Mode: LC

y the formula: Detector: UV 319 nm
Column: 4.6-mm x 15-cm; 5-um packing L7
100F(r, / rs) Column temperature: 30°
Flow rate: 1 mL/min
in which F is a response factor, being 0.38 for the Injection volume: 30 uL
desmethylmetrifonate peak, if present at a retention time of Run time: Twice the retention time of metronidazole
0.5 relative to that of Metrifonate, 0.03 for the dichlorvos System suitability
peak, if present, at a retention time of 1.9 relative to that of Sample: Standard solution
Metrifonate, and 1.0 for any other impurity; r, is the peak Suitability requirements
area for the individual impurity obtained from the Test solu- Tailing factor: NMT 2.0
tion; and rs is the peak area for Metrifonate obtained from Relative standard deviation: NMT 2.0%
the Standard solution: not more than 1.0% of desmethylme- Analysis
trifonate, 0.2% of dichlorvos, and 0.5% of any other impu- Samples: Standard solution and Sample solution
rity are found; anda total of not more than 1.0% of impuri- Calculate the percentage of metronidazole (CsHsN303)
ties other than desmethylmetrifonate and dichlorvos is in the portion of Metronidazole taken:
found.
Assay—Dissolve about 300 mg of Metrifonate, accurately Result = (ru/rs) x (Cs/Cu) x 100
weighed, in 30 mL of alcohol. Add 10 mL of monoethano-
lamine, and allow to stand for 1 hour at 21 +1°. Cool while Tu = peak response from the Sample solution
adding a mixture of 100 mL of water and 15 mL of nitric Is = peak response from the Standard solution
acid. While maintaining the temperature at 21 41°, titrate Cs = concentration of USP Metronidazole RS in the
with 0.1 Nsilver nitrate VS, determining the endpoint po- Standard solution (mg/mL)
tentiometrically using a silver electrode. Each mL of 0.1 N Cy = concentration of Metronidazole in the Sample
silver nitrate is equivalent to 25.74 mg of C4HsCl3O4P. solution (mg/mL)
Acceptance criteria: 99.0%-101.0% on the dried basis
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.1%
Metronidazole
Delete the following:

°o HEAVY METALS, Method Il (231): NMT 50 ppme coral 1.

Jan-2018)
@ ORGANIC IMPURITIES
Mobile phase and Chromatographic system: Proceed
as directed in the Assay. The run time is 30 min.
CeH9N303 171.15
=
Aa)
Standard solution: 1 j1g/mL of metronidazole from USP
1H-lmidazole-1-ethanol, 2-methyl-5-nitro-;
iy 2-Methyl-5-nitroimidazole-1-ethanol [443-48-1].
Metronidazole RS and 2 ug/mL of tinidazole related
Ss compound A from USP Tinidazole Related Compound A
-
i) DEFINITION RS in Mobile phase
i} Sample solution: 1.0 mg/mL of Metronidazole in Mo-
t= Metronidazole contains NLT 99.0% and NMT 101.0% of
6 metronidazole (CsHgN303), calculated on the dried basis. bile phase
Ps System suitability
IDENTIFICATION Sample: Standard solution
a e A. INFRARED ABSORPTION (197K): Meets the requirements [Note—See Table 7 for the relative retention times.]
al
=) e B. The retention time of the major peak of the Sample Suitability requirements
solution corresponds to that of the Standard solution, as Resolution: NLT 2.0 between tinidazole related com-
obtained in the Assay. pound A and metronidazole
Tailing factor: NMT 2.0 for the metronidazole peak
ASSAY Relative standard deviation: NMT 6.0% for both
e@ PROCEDURE tinidazole related compound A and metronidazole;
Mobile phase: Methanol and water (1:4) six replicate injections
Standard solution: 0.03 mg/mL of USP Metronidazole Analysis
RS in Mobile phase Samples: Standard solution and Sample solution
Sample solution: 0.03 mg/mL of Metronidazole in Mo- Calculate the percentage of tinidazole related com-
bile phase poundAin the portion of Metronidazole taken:
Chromatographic system
(See Chromatography (621), System Suitability.) Result = (ru/rs) x (Cs/Cy) x 100

ry = peak response of tinidazole related compound

A from the Sample solution
rs = peak response of tinidazole related compound
A from the Standard solution
Cs = concentration of USP Tinidazole Related
Compound A RS in the Standard solution
(mg/mL)
Cu = concentration of Metronidazole in the Sample
solution (mg/mL)
Calculate the Tenens of any single unspecified
impurity in the portion of Metronidazole taken:
Result = (ru/rs) x (Cs/Cu) x 100
USP 41 Official Monographs / Metronidazole 2721

ty = peak response of any single unspecified correction. Each mL of 0.1 N perchloric acid is equiva-
impurity from the Sample solution lent to 27.53 mg of metronidazole benzoate
ls = peak response of metronidazole from the (Ci3Hi3N30,). ; ;
Standard solution Acceptance criteria: 98.5%-101.0% on the dried basis
Cs = concentration of USP Metronidazole RS in the
Standard solution (mg/mL) IMPURITIES
Cu = concentration of Metronidazole in the Sample © RESIDUE ON IGNITION (281): NMT 0.1%
Solution (mg/mL)
Acceptance criteria: See Table 7. Delete the following:
Table 1 °e HEAVY METALS, Method If (231): NMT 20 ppme coir 1-
Relative Acceptance jan-2018)
Retention Criteria, ¢ ORGANIC IMPURITIES
Name Time NMT (%) Standard solution A: 0.1 mg/mL of USP Metronidazole
Tinidazole related com-
Benzoate RS in acetone
Standard solution B: 0.04 mg/mL of USP Me-
pound A 0.75 0.1
tronidazole Benzoate RS in acetone from Standard solu-
Metronidazole 1.00 — tion A
Any single unspecified —. Standard solution C: 0.2 mg/ml each of USP Me-
impurity 0.1 tronidazole RS and USP Tinidazole Related Compound A
Total impurities = 02 RS in acetone
Sample solution: 20 mg/mL of Metronidazole Benzoate
in acetone
SPECIFIC TESTS Chromatographic system
e Loss ON DRYING (731) (See Chromatography (621), Thin-Layer Chromato-
Analysis: Dry at 105° for 2 h.
Acceptance criteria: NMT 0.5% graphy.)
Mode: TLC
ADDITIONAL REQUIREMENTS Adsorbent: 0.2-mm layer of chromatographic silica
© PACKAGING AND STORAGE: Preserve in well-closed, light- gel mixture
resistant containers, and store at controlled room Application volume: 10 uL
temperature. Developing solvent system: Ethyl acetate
© USP REFERENCE STANDARDS (11) System suitability
USP Metronidazole RS Sample: Standard solution C
USP Tinidazole Related Compound A RS Suitability requirements: The test is valid only if the
2-MethyI-5-nitroimidazole. metronidazole and tinidazole related compound A
C4HsN302 127.10 spots are clearly separated.
Analysis
Samples: Standard solution A, Standard solution B, c
ww
Standard solution C, and Sample solution ae]
Proceed as directed in the chapter. Examine the plate
E
Metronidazole Benzoate under short-wavelength UV light. S
Acceptance criteria: No seem spot of the Sample =|
solution is larger or more intense than the principal spot i)
On re)=
° of Standard solution A (0.5%); and NMT three spots,

eae
excluding the principal spot, of the Sample solution are 2
I ng" bo}
larger or more intense than the principal spot of Stan- 3
dard solution B (0.2%). vw

SPECIFIC TESTS
Ci3HisN3O4 275.26 © ACIDITY
2-(2-Methyl-5-nitroimidazol-1-yl)ethyl benzoate Sample: 2.0g
[13182-89-3]. Analysis: Neutralize 40 mL of a mixture of dimethyl-
DEFINITION
formamide and water (1:1) with hydrochloric acid or
Metronidazole Benzoate contains NLT 98.5% and NMT 0.02 M sodium hydroxide. Add 0.2 mL of methyl red
TS and the Sample, mix to dissolve, and titrate with
101.0% of metronidazole benzoate (C13H13N3O,), calcu-
lated on the dried basis. 0.02 M sodium hydroxide.
Acceptance criteria: NMT 0.25 mL is required to pro-
IDENTIFICATION duce a color change.
© A. INFRARED ABSORPTION (197K) e Loss ON DRYING (731)
¢ B. The principal spot of the Sample solution corresponds Analysis: Dry at 80° for 3 h.
to that of Standard solution A, as obtained in the test for Acceptance criteria: NMT 0.5%
Organic Impurities.
ADDITIONAL REQUIREMENTS
ASSAY e PACKAGING AND STORAGE: Preserve in well-closed, light-
© PROCEDURE resistant containers. Store at 25°, excursions permitted
Sample solution: Dissolve with stirring 250 mg of Me- between 15° and 30°.
tronidazole Benzoate in 50.0 mL of glacial acetic acid. e USP REFERENCE STANDARDS (11)
Titrimetric system USP Metronidazole RS
(See Titrimetry (541).) USP Metronidazole Benzoate RS
Mode: Direct titration USP Tinidazole Related Compound A RS
Titrant: 0.1 N perchloric acid 2-Methyl-5-nitroimidazole.
Endpoint detection: Potentiometric C4HsN302 127.10
Analysis: Titrate the Sample solution with Titrant. Per-
form a blank determination, and make any necessary
 2722 Metronidazole / Official Monographs USP 41

SPECIFIC TESTS
Metronidazole Benzoate Compounded e PH (791): 3.6-4.6
Oral Suspension ADDITIONAL REQUIREMENTS
° PACKAGING AND STORAGE: Package in tight, light-resistant
DEFINITION containers. Store at 2°-8° or at controlled room
Metronidazole Benzoate Compounded Oral Suspension con- temperature.
tains NLT 90.0% and NMT 110.0% of the labeled amount e BEYOND-UsE DATE: NMT 90 days after the date on which
of metronidazole (CsH»N3O3). it was compounded when stored at 2°-8° or controlled
Prepare Metronidazole Benzoate Compounded Oral Suspen- room temperature.
sion containing 50 mg/mL of metronidazole as follows © LABELING: Label it to indicate that it is to be well-shaken
pinmaceutical Compounding—Nonsterile Preparations before use, and to state the Beyond-Use Date.
795)). eo USP REFERENCE STANDARDS (11)
USP Metronidazole Benzoate RS
Metronidazole (as the Benzoate)
owder 5q(8q)
Ora-Blend?, a sufficient quantity
to make 100 mL
Metronidazole Capsules
4Perrigo, Minneapolis, MN.

Place the Metronidazole Benzoate powder into a suitable mor- DEFINITION

tar. Wet the powder with a small amount of Ora-Blend,
and triturate to make a smooth paste. Add the Ora-Blend
in small oes almost to volume, and mix thoroughly
after each addition. Transfer the contents of the mortar,
stepwise and quantitatively, to a calibrated container. Add
sufficient Ora-Blend to bring the preparation to final vol-
ume. Shake to mix well.
ASSAY
e PROCEDURE
Solution A: 0.1% (v/v) glacial acetic acid in water
Mobile phase: Acetonitrile and Solution A (40:60). Fil-
ter, and degas.
Standard solution: 0.4 mg/mL of metronidazole pre-
pared from USP Metronidazole Benzoate RS in Mobile
phase. Mix well until dissolved.
al
Sample solution: Shake erent each bottle of Oral
pln
a Suspension. Transfer 0.8 mL of the Oral peperaien into
i a 100-mL volumetric flask, dilute with Mobile phase to
a volume, and mix well.
) Chromatographic system
S Capsules (NLT 20). Transfer an amount equivalent to
° (See Chromatography (621), System Suitability.)
= Mode: LC
Detector: UV 316 nm
a Column: 4.6-mm x 15-cm; 5-um packing L1
a)
=) Column temperature: 30°
Flow rate: 1.0 mL/min
Injection volume: 5 pL
System suitability
Sample: Standard solution
[Note—The retention time for metronidazole is about
7.7 min.]
Suitability requirements
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0% for replicate
injections
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of me-
tronidazole (CsHgN3O3) in the portion of Oral Suspen-
sion taken:
Result = (ru/rs) x (Cs/Cu) x 100
Tu = peak response from the Sample solution
rs = peak response from the Standard solution
Cs = concentration of metronidazole in the
Standard solution (mg/mL)
Cy = nominal concentration of metronidazole in the
Sample solution (mg/mL)
Metronidazole Capsules contain NLT 90.0% and NMT
110.0% of the labeled amount of metronidazole
(CéHsN303).
IDENTIFICATION
© A. INFRARED ABSORPTION (197K)
Wavelength range: Between 1600 and 1000 cm
Acceptance criteria: Capsule contents show maxima
only at the same wavelengths as those of similarly pre-
pared USP Metronidazole RS.
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
ASSAY
e PROCEDURE
Mobile phase: Methanol and water (1:4)
Standard solution: 0.03 mg/mL of USP Metronidazole
RS in Mobile phase
Sample stock solution: Nominally 1 mg/mL of me-
tronidazole prepared as follows. Mix the contents of
100 mg of metronidazole to a 100-mL volumetric flask,
add 80 mL of Mobile phase, and sonicate with intermit-
tent shaking for 10 min. Shake for 30 min, and dilute
with Mobile phase to volume. Centrifuge a portion of
the solution.
Sample solution: 0.03 mg/mL of metronidazole in Mo-
bile phase, from the Sample stock solution. Pass a portion
of the solution through a nylon membrane filter of
0.45-um or finer pore size. Discard the first 10 mL of
the filtrate, and use the remainder.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 319 nm
Column: 4.6-mm x 15-cm; 5-um packing L7
Column temperature: 30°
Flow rate: 1 mL/min
Injection volume: 30 uL
Run time: 2 times the retention time of the me-
tronidazole peak
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0% for five rep-
licate injections
USP 41 Official Monographs / Metronidazole 2723

Analysis tu = peak response of tinidazole related compound

Samples: Standard solution and Sample solution A from the Sample solution
Calculate the percentage of the labeled amount of me- rs = peak response of tinidazole related compound
tronidazole (CsHeN3O3) in the portion of Capsules A from the Standard solution
taken: Gs = concentration of USP Tinidazole Related
CompoundA RS in the Standard solution
Result = (ru/rs) x (Cs/Cu) x 100 (mg/mL)
Cu = nominal concentration of metronidazole in the
tu = peak response from the Sample solution Sample solution (mg/mL)
rs = peak response from the Standard solution Calculate the percentage of any unspecified
Cs = concentration of USP Metronidazole RS in the degradation product in the portion of Capsules taken:
Standard solution (mg/mL)
Cu = nominal concentration of metronidazole in the Result = (ru/rs) x (Cs/Cu) x 100
Sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0% tu = peak response of any unspecified degradation
product from the Sample solution
PERFORMANCE TESTS rs = peak response of metronidazole from the
e DISSOLUTION (711) Standard solution
Medium: 0.1 N hydrochloric acid; 900 mL Cs = concentration of USP Metronidazole RS in the
Apparatus 1: 100 rpm Standard solution (mg/mL)
Time: 30 min Cu = nominal concentration of metronidazole in the
Standard solution: 0.4 mg/mL of USP Metronidazole Sample solution imgim()
RS in Medium. Sonicate to dissolve if necessary. Acceptance criteria: See Table 1.
Sample solution: Pass a portion of the solution under
test throughasuitable filter of 0.45-m pore size, and
Table 1
discard the first few mL.
Instrumental conditions Relative Acceptance
Mode: UV Retention Criteria,
Analytical wavelength: Maximum at about 278 nm Name Time NMT (%)
Cell: 0.05 cm Tinidazole related compound A 0.75 0.1
Blank: Medium Metronidazole 1.0 =
Analysis
Each unspecified degradation pits
Samples: Standard solution and Sample solution
product 0.1
Calculate the percentage of the labeled amount of me-
tronidazole (CsH»N303) dissolved: Total impurities = 0.5

Result = (Au/As) x Cs x (1/L) x Vx 100 ADDITIONAL REQUIREMENTS

Au = absorbance of the Sample solution © PACKAGING AND STORAGE: Preserve in well-closed, light- lo
As = absorbance of the Standard solution resistant containers, and store at controlled room nn
a)
Gs = concentration of USP Metronidazole RS in the temperature.
Standard solution (mg/mL) e USP REFERENCE STANDARDS (11) KS
L = label claim (mg/Capsule) USP Metronidazole RS 2}
USP Tinidazole Related Compound A RS =]
Vv = volume of Medium, 900 mL }
Tolerances: NLT 85% (Q) of the labeled amount of me- 2-Methyl-5-nitroimidazole. a=
tronidazole (CsH9N303) is dissolved. C4HsN302 =127.10 i)
e UNIFORMITY OF DOSAGE UNITS (905): Meet the i}
requirements
2
a]

IMPURITIES
¢ ORGANIC IMPURITIES Metronidazole Gel
Mobile phase, Sample solution, and Chromatographic
system: Proceed as directed in the Assay. DEFINITION
Standard solution: 1 g/mL of metronidazole from USP Metronidazole Gel contains NLT 90.0% and NMT 110.0%
Metronidazole RS and 2 g/mL of tinidazole related of the labeled amount of metronidazole (CeHgN3Os3).
compound A from USP Tinidazole Related Compound A
RS in Mobile phase IDENTIFICATION
System suitability e A. The UV spectrum of the metronidazole peak of the
Sample: Standard solution Sample solution corresponds to that of the Standard solu-
[NoTte—The relative retention times for tinidazole re- tion, as obtained in the Assay.
lated compound A and metronidazole are 0.75 and e B. The retention time of the major peak of the Sample
1.0, respectively.] solution corresponds to that of the Standard solution, as
Suitability requirements obtained in the Assay.
Resolution: NLT 2.0 between tinidazole related com-
pound A and metronidazole ASSAY
Tailing factor: NMT 2.0 for metronidazole © PROCEDURE
Relative standard deviation: NMT 6.0% for both Solution A: Methanol and water (20:80)
tinidazole related compound A and metronidazole Solution B: Methanol
Analysis Mobile phase: See Table 7.
Samples: Standard solution and Sample solution
Calculate the perceiage of tinidazole related com- Table 1
poundAin the portion of Capsules taken:
Time Solution A Solution B
Result = (ru/rs) x (Cs/Cu) x 100 (min) (%) (%)
0 100 0
10.0 100 0
 2724 Metronidazole / Official Monographs USP 41

Table 1 (Continued) System suitability

Time Solution A Solution B Sample: Standard solution
[Note—See Table 2 for the relative retention times.]
(min) (%) (%)
Suitability requirements
15.0 10 90 Resolution: NLT 2.0 between metronidazole and
15.1 100 0 tinidazole related compound A
20.0 100 0 Relative standard deviation: NMT 2.0%
Analysis
System suitability solution: 0.6 g/mL of USP Me- Samples: Standard solution and Sample solution
tronidazole RS and 0.6 g/mL of USP Tinidazole Related Calculate the peeenisds of tinidazole related com-
CompoundA RS in Solution A poundAin the portion of Gel taken:
Standard solution: 30 g/mL of USP Metronidazole RS
in Solution A Result = (ru/ts) x (Cs/Cu) x 100
Sample stock solution: Nominally 300 tg/mL of me-
tronidazole in Solution A prepared as follows. Transfer a ru = peak response of tinidazole related compound
portion of Gel to a suitable volumetric flask. Add Solu- A from the Sample solution
tion A equivalent to 50% of the flask volume and soni- rs = peak response of tinidazole related compound
cate or vortex until dissolved. Dilute with Solution A to A from the Standard solution
volume. [NOTE—On the basis of formulation, if neces- Cs = concentration of USP Tinidazole Related
sary, centrifuge a portion of the solution at 3000 rpm Compound A RS in the Standard solution
for 10 min and pass a portion of the supernatant (ug/mL) . : ;
throughafilter of 0.45-um pore size. Use the filtrate.] Cy = nominal concentration of metronidazole in the
Sample solution: Nominally 30 ug/mL of me- Sample solution (ug/ml)
tronidazole in Solution A prepared from the Sample stock Calculate the percentage of each individual unspecified
solution impurity in the portion of Gel taken:
Chromatographic system
(See Chromatography (621), System Suitability.) Result = (ru/rs) x (Cs/Cu) x 100
Mode: LC
Detector: UV 319 nm. For Identification test A, use a ty = peak response of each unspecified impurity
diode-array detector in the range of 210-500 nm. from the Sample solution
Column: 4.6-mm x 15-cm; 5-um packing L7 rs = peak response of metronidazole from the
Column temperature: 30° Standard solution
Flow rate: 1 mL/min Cs = concentration of USP Metronidazole RS in the
Injection volume: 30 pL Standard solution (g/mL)
System suitability CG = nominal concentration of metronidazole in the
Samples: System suitability solution and Standard Sample solution (g/mL)
solution Acceptance criteria: See able 2.
“ [Note—See Table 2 for the relative retention times.]
= Suitability requirements
5 Resolution: NLT 2.0 between metronidazole and
Table 2
i] Relative Acceptance
—
fo.) tinidazole related compound A, System suitability Retention Criteria,
fe) solution
is Tailing factor: NMT 2.0, Standard solution Name Time NMT (%)
} Relative standard deviation: NMT 2.0%, Standard Tinidazole related compound A 0.76 0.2
Ps solution Metronidazole 1.0 =_
a Analysis Any individual unspecified 294
”
=) Samples: Standard solution and Sample solution impurity 0.3
Calculate the percentage of the labeled amount of me- Total impurities = 1.0
tronidazole (CeHgN3O3) in the portion of Gel taken:
Result = (ru/rs) x (Cs/Cu) x 100 PERFORMANCE TESTS
e MINIMUM FILL (755): Meets the requirements
fu = peak response of metronidazole from the
Sample solution SPECIFIC TESTS
rs = peak response of metronidazole from the e PH (791): The apparent pH determined potentiometri-
Standard solution cally is between 4.0 and 6.5.
Cs = concentration of USP Metronidazole RS in the
Standard solution (g/mL) ADDITIONAL REQUIREMENTS
Cu = nominal concentration of metronidazole in the © PACKAGING AND STORAGE: Preserve in laminated collapsi-
Sample solution (ug/ml) ble tubes at controlled room temperature.
Acceptance criteria: 90.0%-110.0% e USP REFERENCE STANDARDS (11)
USP Metronidazole RS
IMPURITIES USP Tinidazole Related Compound A RS
e ORGANIC IMPURITIES 2-Methyl-5-nitroimidazole.
Solution A, Solution B, Mobile phase, and Chromato- C4HsN302 127.10
graphic system: Proceed as directed in the Assay.
Standard solution: Use the System suitability solution
from the Assay.
Sample solution: Use the Sample stock solution from
the Assay. Metronidazole Injection
DEFINITION
Metronidazole Injection is a sterile, isotonic, buffered solu-
tion of Metronidazole in Water for Injection. It contains
USP 41 Official Monographs / Metronidazole 2725

NLT 90.0% and NMT 110.0% of the labeled amount of [Note—See Table 1 for the relative retention times.]
metronidazole (CsH9N303). Suitability requirements
Resolution: NLT 4.0 between metronidazole and
IDENTIFICATION tinidazole related compound A, System suitability
e A. The UV (UV-Vis) spectrum of the metronidazole peak solution
of the Sample solution corresponds to that of the Stan- Relative standard deviation: NMT 1.0%, Standard
dard solution, as obtained in the Assay. solution
e B. The retention time of the major peak of the Sample Analysis
solution corresponds to that of the Standard solution, as Samples: Standard solution and Sample solution
obtained in the Assay. Calculate the percentage of tinidazole related com-
poundAin the portion of Injection taken:
ASSAY
© PROCEDURE Result = (ru/rs) x (Cs/Cu) x 100
Mobile phase: Methanol and water (20:80)
System suitability solution: 1 t1g/mL of USP Me- Ta = peak response of tinidazole related compound
tronidazole RS and 2 g/mL of USP Tinidazole Related A from the Sample solution
Compound A RS in Mobile phase Is = peak response of tinidazole related compound
Standard solution: 0.03 mg/mL of USP Metronidazole A from the Standard solution
RS in Mobile phase Cs = concentration of USP Tinidazole Related
Sample solution: Nominally 0.03 mg/mL of me- CompoundA RS in the Standard solution
tronidazole in Mobile phase prepared as follows. Transfer (ug/ml)
jortion of Injection to a suitable volumetric flask, and Cu = nominal concentration of metronidazole in the
dilute with Mobile phase to volume. Sample solution (ug/ml)
Chromatographic system Calculate the percentage of any individual unspecified
(See Chromatography (621), System Suitability.) impurity in the portion of Injection taken:
Mode: LC
Detector: UV 319 nm. For Identification test A, use a Result = (ru/rs) x (Cs/Cu) x 100
diode array detector in the range of 210-800 nm.
Column: 4.6-mm x 15-cm; 5-um packing L7 ry = peak response for each unspecified impurity
Column temperature: 30° from the Sample solution
Flow rate: 1 mL/min Is = peak response of metronidazole from the
Injection volume: 30 ul Standard solution
System suitability Cs = concentration of USP Metronidazole RS in the
Samples: System suitability solution and Standard Standard solution (ug/mL)
solution Cu = nominal concentration of metronidazole in the
[Note—See Table 7 for the relative retention times.] Sample solution (g/mL)
Suitability requirements Acceptance criteria: See [able 1.
Resolution: NLT 4.0 between metronidazole and
tinidazole related compound A, System suitability Table 1 o
solution : v
Tailing factor: NMT 2.0, Standard solution Relative Acceptance z
Relative standard deviation: NMT 1.0%, Standard Retention Criteria, fo)
solution Name Time NMT (%) i
Analysis Tinidazole related iS
Samples: Standard solution and Sample solution compound A 0.7 0.15 =
Calculate the percentage of the labeled amount of me- Metronidazole 1.0 a a
tronidazole (CsHsN303) in the portion of Injection Any individual mz
taken: unspecified — ne
_ . degradation product 0.15
Result = (rufrs) x (Cs/Cu) x 100 Total impurities = 2.0
ty = peak response from the Sample solution
Is = peak response from the Standard solution SPECIFIC TESTS
Cs = concentration of USP Metronidazole RS in the © PH (791): 4.5-7.0
Cu
Standard solution (mg/ml)
= nominal concentration of metronidazole in the
: e BACTERIAL ENDOTOXINS TEST (85): NMT 0.35 USP Endo-
toxin Units/mg of metronidazole
Sample solution (mg/mL) PARTICULATE MATTER IN INJECTIONS (788): It meets the re-
Acceptance criteria: 90.0%-110.0% quirements for small-volume injections.
IMPURITIES OTHER REQUIREMENTS: It meets the requirements in Injec-
¢ ORGANIC IMPURITIES tions and Implanted Drug Products (1).
Mobile phase, Chromatographic system, and System ADDITIONAL REQUIREMENTS
suitability solution: Proceed as directed in the Assay. © PACKAGING AND STORAGE: Preserve in single-dose contain-
Standard solution: 0.75 g/mL each of USP Me- ers of Type | or ee Il glass, or in suitable plastic con-
tronidazole RS and USP Tinidazole Related Compound A tainers, protected from light. Store at controlled room
RS in Mobile phase temperature.
Sample solution: Nominally 500 g/mL of me-
tronidazole prepared as follows. Transfer a portion of
Injection to a suitable volumetric flask. Add Mobile Change to read:
phase equivalent to 50% of the flask size. Sonicate for 2
min. Dilute with Mobile phase to volume, and pass a e USP REFERENCE STANDARDS (11)
portion of the solution througha filter of 0.45-1um pore Se {CN 1-May-2018)
size. Use the filtrate. USP Metronidazole RS
System suitability USP Tinidazole Related Compound A RS
Samples: System suitability solution and Standard 2-Methyl-5-nitroimidazole.
solution C4HsN302 127.10
 2726 Metronidazole / Official Monographs
Metronidazole Tablets
DEFINITION
Metronidazole Tablets contain NLT 90.0% and NMT
110.0% of the labeled amount of metronidazole
(CsHgN30s3).
IDENTIFICATION
e A. ULTRAVIOLET ABSORPTION (197U)
Sample stock solution: Equivalent to 15 mg/mL of me-
tronidazole from powdered Tablets in dilute hydrochlo-
ric acid (1 in 100). Shake for several min, and filter.
Medium: Sulfuric acid in methanol (1 in 350)
Sample solution: 20 j1g/mL of metronidazole in Me-
dium from the Sample stock solution
Acceptance criteria: Meet the requirements
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
ASSAY
e PROCEDURE
Mobile phase: Methanol and water (20:80)
Standard solution: 0.5 mg/mL of USP Metronidazole
RS in Mobile phase
Sample stock solution: Nominally 10 mg/mL of me-
tronidazole in methanol from Tablets prepared as fol-
lows. Transfer 10 Tablets, whole or ground, to a suitable
size volumetric flask. Add methanol, and shake by me-
chanical means for 30 min or until the Tablets are disin-
tegrated. Dilute with methanol to volume, and allow
the solution to stand until the insoluble material has
settled.
Sample solution: Nominally 0.5 mg/mL of me-
tronidazole in Mobile phase prepared from the clear su-
pernatant of the Sample stock solution. Filter.
al
ie Chromatographic system
re (See Chromatography (621), System Suitability.)
is Mode: LC
Dp Detector: UV 254 nm
3 Column: 4.6-mm x 15-cm; packing L7
is
iS Flow rate: 1 mL/min
= Injection volume: 10 uL
a System suitability
“ Sample: Standard solution
=) Suitability requirements
Tailing factor: NMT 2
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of me-
tronidazole (CsHgN3O3) in the portion of Tablets taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response of metronidazole from the
Sample solution
Is = peak response of metronidazole from the
Standard solution
Cs = concentration of USP Metronidazole RS in the
Standard solution (mg/mL)
Cu = nominal concentration of metronidazole in the
Sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0%
PERFORMANCE TESTS
e DISSOLUTION (711)
Medium: 0.1 N hydrochloric acid; 900 mL
Apparatus 1: 100 rpm
Time: 60 min
Standard solution: USP Metronidazole RS in Medium
Sample solution: Filter a portion of the solution under
test, and dilute with Medium to a concentration similar
to that of the Standard solution.
USP 41
Instrumental conditions
Mode: UV
Analytical wavelength: 278 nm
Blank: Medium
Analysis
Samples: Standard solution, Sample solution, and Blank
Calculate the percentage of the labeled amount of me-
tronidazole (CeHsN3O3) dissolved.
Result = (Au/As) x Cs x Vx (1/L) x Dx 100
Au = absorbance of the Sample solution
As = absorbance of the Standard solution
Cs = concentration of USP Metronidazole RS in the
Standard solution (mg/mL)
Vv = volume of Medium, 900 mL
L = label claim (mg/Tablet)
D = dilution factor to prepare the Sample solution
Tolerances: NLT 85% (Q) of the labeled amount of me-
tronidazole (CsHsN3QO3) is dissolved.
e UNIFORMITY OF DosaGE UNITS (905)
Procedure for content uniformity
Diluent: Diluted hydrochloric acid (1 in 100)
Standard solution: 20 g/mL of USP Metronidazole
RS in Diluent
Sample stock solution: Transfer 1 Tablet to a 250-mL
volumetric flask. Add about 100 mL of Diluent, and
shake for 30 min. Dilute with Diluent to volume. Filter,
discarding the first 15 mL of the filtrate. Nominall
200 1g/mL of metronidazole is prepared by transfer-
ring the filtrate quantitatively with the Diluent.
Sample solution: 20 g/mL of metronidazole in Dilu-
ent from Sample stock solution
Instrumental conditions
Mode: UV
Analytical wavelength: 278 nm
Cell: 1.cm
Blank: Diluent
Analysis
Samples: Standard solution, Sample solution, and
Blank
Calculate the quantity, in mg, of metronidazole
(CéHsN303) in each Tablet taken:
Result = (Au/As) x (Cs/Cu) x L
Au = absorbance of the Sample solution
As = absorbance of the Standard solution
Cs = concentration of USP Metronidazole RS in the
Standard solution (g/mL)
Cu = nominal concentration of metronidazole in the
Sample solution (g/mL)
L = label claim (mg/Tablet)
Acceptance criteria: Meet the requirements
IMPURITIES
e@ ORGANIC IMPURITIES
Mobile phase: Methanol and water (20:80)
Standard solution: 0.5 g/mL of USP Metronidazole RS
and 2.5 g/mL of USP Tinidazole Related Compound A
RS in Mobile phase
Sample solution: Nominally 500 ug/mL of me-
tronidazole prepared as follows. Transfer a suitable
amount of powdered Tablets (NLT 20) to a suitable vol-
umetric flask. Add Mobile phase equivalent to 80% of
the flask size. Sonicate for 10 min. Dilute with Mobile
phase to volume, and pass a portion of the solution
through a suitable filter. Use the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
USP 41 Official Monographs / Metronidazole 2727

Mode: LC
Detector: UV 319 nm
Column: 4.6-mm x 15-cm; 5-14m packing L7 Metronidazole Extended-Release
Column temperature: 30° Tablets
Flow rate: 1 mL/min
Injection volume: 30 wL DEFINITION
System suitability Metronidazole Extended-Release Tablets contain NLT 90.0%
Sample: Standard solution and NMT 110.0% of the labeled amount of me-
[Note—See Table 7 for relative retention times.] tronidazole (CsHsN3O3).
Suitability requirements
Resolution: NLT 4.0 between metronidazole and IDENTIFICATION
tinidazole related compound A e A, ULTRAVIOLET ABSORPTION (197U)
Relative standard deviation: NMT 3.0% Diluent: Methanol and sulfuric acid (350:1)
Analysis Standard stock solution: 15 mg/ml. of USP Me-
Samples: Standard solution and Sample solution tronidazole RS in dilute hydrochloric acid (1 in 100).
Calculate the percentage of tinidazole related com- Sonicate to dissolve and pass through a suitable filter.
poundAin the portion of Tablets taken: Standard solution: 18.8 g/mL of USP Metronidazole
RS in Diluent from Standard stock solution
Result = (ru/rs) x (Cs/Cu) x 100 Sample stock solution: Nominally 15 mg/mL of me-
tronidazole prepared as follows. Finely powder NLT 5
tu = peak response of tinidazole related compound Tablets and transfer an amount equivalent to 300 mg of
A from the Sample solution metronidazole into a 20-mL volumetric flask. Add about
Is = peak response of tinidazole related compound 15 mL of dilute hydrochloric acid (1 in 100) and shake
A from the Standard solution mechanically for 30 min. Dilute with dilute hydrochloric
Cs = concentration of USP Tinidazole Related acid (1 in 100) to volume and shake well. Pass through
Compound A RS in the Standard solution a suitable filter.

Cy
(ug/ml)
= nominal concentrationof metronidazole in the
Sample solution: Nominally equivalent to 18.8 ug/mL
of metronidazole in Diluent from Sample stock solution
Sample solution (g/mL) Wavelength range: 200-400 nm
Calculate the percentage of each impurity in the Acceptance criteria: Meet the requirements
portion of Tablets taken: e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
Result = (ru/rs) x (Cs/Cu) x 100 obtained in the Assay.
tu = peak response for each impurity from the ASSAY
Sample solution © PROCEDURE
rs = peak response of metronidazole from the Buffer: 1.4 g/L of monobasic potassium phosphate in
Standard solution water (s
Gs = concentration of USP Metronidazole RS in the Mobile phase: Methanol and Buffer (30:70) 4)
Standard solution (g/mL) Standard solution: 0.1 mg/mL of USP Metronidazole ae]
Cy = nominal concentration of metronidazole in the RS in Mobile phase x
Sample solution (ug/mL) Sample stock solution: Nominally 2.0 mg/mL of me- °
Acceptance criteria: See Jable 1. tronidazole from NLT 20 finely powdered Tablets in Mo- |
bile phase, prepared as follows. Transfer a suitable )
amount of the powder to a suitable volumetric flask.
2=
Table 1 i}
Add 60% of the flask volume with Mobile phase, and ie)
Relative
Retention
Acceptance
Criteria,
shake by mechanical means for 30 min. Dilute with Mo- toa
bile phase to volume. Allow the solution to stand until a
Name Time NMT (%) the insoluble material settles.
Tinidazole related Sample solution: Nominally 0.1 mg/mL of me-
compound A 0.7 0.5 tronidazole in Mobile phase from the Sample stock solu-
Metronidazole 1.0 = tion supernatant. Pass the solution through a suitable
Any individual unspecified _ filter of 0.45-um pore size.
degradation product 0.10 Chromatographic system
Total impurities = 2.0 (See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 315 nm
ADDITIONAL REQUIREMENTS Column: 4.6-mm x 25-cm; 5-um packing L1
e PACKAGING AND STORAGE: Preserve in well-closed, light- Temperatures
resistant containers. Store at controlled room Column: 30°
temperature. Autosampler: 15°
e USP REFERENCE STANDARDS (11) Flow rate: 1 mL/min
USP Metronidazole RS Injection volume: 10 uL
USP Tinidazole Related Compound A RS Run time: 15 min
2-Methyl-5-nitroimidazole. System suitability
C4HsN302 127.10 Sample: Standard solution
Suitability requirements
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of me-
tronidazole (CsH»N303) in the portion of Tablets taken:

Result = (ru/rs) x (Cs/Cu) x 100

 2728 Metronidazole / Official Monographs USP 41

tu = peak response of metronidazole from the The percentages (Q) of the labeled amount of me-
Sample solution tronidazole (CsHsN3Os) released at the times specified
rs = peak response of metronidazole from the conform to Dissolution (711), Acceptance Table 2.
Standard solution e UNIFORMITY OF DOSAGE UNITS (905): Meet the
G = concentration of USP Metronidazole RS in the requirements
Standard solution (mg/mL)
Cu = nominal concentration of metronidazole in the IMPURITIES
Sample solution (mg/mL) © ORGANIC IMPURITIES
Acceptance criteria: 90.0%-110.0% Buffer: Dissolve 1.5 g of monobasicpotassium phos-
phate in 900 mL of water, adjust with phosphoric acid
PERFORMANCE TESTS to a pH of 3.2, and dilute with water to 1000 mL.
e DISSOLUTION (711) Diluent: Acetonitrile and Buffer (45:55)
Medium: Water; 900 mL Mobile phase: See Table 2.
Apparatus 2: 50 rpm
Times: 2, 6, 10, and 16h Table 2
Standard solution: 16.65 j1g/mL of USP Metronidazole
RS in Medium Buffer Acetonitrile
Sample solution: At the times specified, withdraw %o
10 mL of the solution under test and pass through a 5
suitable filter of 0.45-4um pore size. Replace the aliquots 5
withdrawn for analysis with equal volumes of fresh por- 50
tions of Medium maintained at 37°. Dilute with Medium 95
to a concentration similar to that of the Standard
3 5
solution.
Blank: Medium System suitability solution: 0.5 mg/mL of USP Me-
Instrumental conditions
tronidazole RS and 2.5 t1g/mL of USP Tinidazole Related
Mode: UV CompoundA RS in Diluent. Sonicate, if necessary, to
Analytical wavelength: 320 nm dissolve.
Cell: 1.cm Standard solution: 0.75 g/mL of USP Metronidazole
Analysis RS in Diluent
Samples: Standard solution and Sample solution Sample solution: Nominally 0.5 mg/mL of me-
Calculate the concentration (Cj) of metronidazole tronidazole from NLT 20 finely powdered Tablets in Dil-
(CsHsN3O3) in the sample withdrawn from the vessel uent, prepared as follows. Transfer a suitable amount of
at each time point (i). the powder to a suitable volumetric flask. Add Diluent
Result = (Au/As) x Cs x D equivalent to 70% of the flask volume, sonicate for 15
min with intermittent shaking, and dilute with Diluent
“ Au = absorbance of the Sample solution to volume. Allow the solution to stand until the insolu-
a As = absorbance of the Standard solution ble material settles, and pass the supernatant through a
os Cs = concentration of USP Metronidazole RS in the suitable filter of 0.45-um pore size.
ii Chromatographic system
— Standard solution (mg/mL)
D (See Chromatography (621), System Suitability.)
° D = dilution factor, if needed
Mode: LC
= Calculate the percentage of the labeled amount (Q) of
Sj metronidazole (CsH»N303) dissolved at each time point Detector: UV 315 nm
P= (). Column: 4.6-mm x 25-cm; 5-1um packing L1
[3
Autosampler temperature: 20°
a) Result; = CG; x Vx (1/L) x 100 Flow rate: 1 mL/min
= Injection volume: 10 uL
System suitability
Resultz = [(Cz x V) + (C; x Vs)] x (1/L) x 100 Samples: System suitability solution and Standard
solution
Suitability requirements
Results = {[C3 x V] + [(C2 + Ci) x Vs]} x (1/2) x 100 Resolution: NLT 2.0 between tinidazole related com-
pound A and metronidazole, System suitability solution
Relative standard deviation: NMT 5.0% for me-
Results = {(Cy x V) + [(C3 + Co + Cy) x Vs]} x (1/2) x 100 tronidazole, Standard solution
G = concentration of metronidazole in the portion Analysis
of sample withdrawn at the specified time Samples: Standard solution and Sample solution
point (mg/mL) Calculate the percentage of each individual degradation
Vv = volume of the Medium, 900 mL product in the portion of Tablets taken:
L = label claim (mg/Tablet) Result = (ru/rs) x (Cs/Cu) x 100
Vs = volume of the Sample solution withdrawn at
each time point and replaced with Medium tu = peak response of each individual degradation
(ml) product from the Sample solution
Tolerances: See Table 1. rs = peak response of metronidazole from the
Standard solution
Table 1 Gs = concentration of USP Metronidazole RS in the
Time Point Time Amount Dissolved Standard solution (mg/mL)
Cu = nominal concentration of metronidazole in the
Sample solution (mg/mL)
Acceptance criteria: See Table 3. Disregard any impu-
rity peaks less than 0.05%.
USP 41 Official Monographs / Metyrapone 2729

Table 3 Acceptance criteria: 98.0%-102.0% on the dried basis

Relative Acceptance IMPURITIES
Retention Criteria, e RESIDUE ON IGNITION (281): NMT 0.1%

c 9
0
Delete the following:
Any individual unspecified
°e HEAVY METALS, Method !! (231): NMT 10 ppme cota’ i.
Jan-2018)
Total ra — © ORGANIC IMPURITIES
Standard stock solution: 0.2 mg/mL of USP
ADDITIONAL REQUIREMENTS Metyrapone RS in methanol
© PACKAGING AND STORAGE: Preserve in well-closed contain- Standard solution A: 40 g/mL of USP Metyrapone RS
ers. Store at controlled room temperature. in methanol
e USP REFERENCE STANDARDS (11) Standard solution B: 20 g/mL of USP Metyrapone RS
USP Metronidazole RS in methanol
USP Tinidazole Related Compound A RS Sample solution: 20 mg/mL of Metyrapone in
2-Methyl-5-nitroimidazole. methanol
CaHsN302 127.10 Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.)
Mode: TLC
Adsorbent: 0.25-mm layer of chromatographic silica
Metyrapone gel mixture
Application volume: 5 uL
Developing solvent system: Chloroform and metha-
nol (48:3)
a Jl Analysis
Samples: Standard stock solution, Standard solution A,
Wd vial ie
Standard solution B, and Sample solution
Apply each of the Samples separately to the TLC plate.
Ci4HiaN20 226.27 Position the plate in a chromatographic chamber,
1-Propanone, 2-methyl-1,2-di-3-pyridinyI-; and develop the chromatograms in the Developing
2-MethyI-1,2-di-3-pyridyl-1-propanone [54-36-4]. solvent system until the solvent front has moved
about three-fourths of the length of the plate. Re-
DEFINITION move the plate from the developing chamber, mark
Metyrapone contains NLT 98.0% and NMT 102.0% of the solvent front, and dry under a stream of nitrogen
metyrapone (Ci4Hi4N20), calculated on the dried basis. for about 10 min. Position the dried plate once again (=
a]
in the same chromatographic chamber, and again me)
IDENTIFICATION develop the chromatograms until the solvent front
e A. INFRARED ABSORPTION (197M) c
¢ B. ULTRAVIOLET ABSORPTION (197U)
has moved about three-fourths of the length of the }
plate. Remove the plate from the developing cham- =
Sample solution: 10 g/mL in 1 N sulfuric acid er, mark the solvent front, and dry under a current i}
Acceptance criteria: Meets the requirements of warm air for about 15 min. Examine the plate =}
under short-wavelength UV light, and compare the i)
ASSAY
intensities of any secondary spots observed in the
so}
© PROCEDURE a
Diluent: 1 .N sulfuric acid chromatogram of the Sample solution with those of ww

Standard solution: 10 g/mL of USP Metyrapone RS in the principal spots in the chromatograms of the Stan-
Diluent dard solutions.
Sample solution: 10 g/mL of Metyrapone in Diluent Acceptance criteria: The intensity of any secondary
Instrumental conditions spot from the Sample solution is NMT the principal spot
Mode: UV from Standard solution A (0.2%), and the sum of the
Analytical wavelength: 260 nm intensities of the secondary spots from the Sample solu-
Cell: 1cm tion corresponds to NMT 1.0%.
Blank: Diluent SPECIFIC TESTS
Analysis e Loss ON DRYING (731)
Samples: Standard solution and Sample solution Analysis: Dry a sample under vacuum at room temper-
Calculate the percentage of metyrapone (Ci4Hi4N20) in ature for 6 h.
the portion of Metyrapone taken: Acceptance criteria: NMT 0.5%
Result = (Au/As) x (Cs/Cu) x 100 ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers,
Au = absorbance of the Sample solution protected from heat and light.
As = absorbance of the Standard solution e@ USP REFERENCE STANDARDS (11)
Gs = concentration of USP Metyrapone RS in the USP Metyrapone RS
Standard solution (\ug/mL)
Cu = concentration of Metyrapone in the Sample
solution (g/mL)
 2730 Metyrapone / Official Monographs USP 41

Calculate the percentage of the labeled amount of

metyrapone (Ci4Hi4N20) in the portion of Tablets
Metyrapone Tablets taken:
DEFINITION Result = (Au/As) x (Cs/Cu) x 100
Metyrapone Tablets contain NLT 95.0% and NMT 105.0%
of the labeled amount of metyrapone (Ci4Hi4N20). Au = absorbance of the Sample solution
As = absorbance of the Standard solution
IDENTIFICATION Cs = concentration of USP Metyrapone RS in the
e A. INFRARED ABSORPTION Standard solution (mg/mL)
Sample solution: Transfer 500 mg of metyrapone from Cu = nominal concentration of metyrapone in the
powdered Tablets into a centrifuge tube. Add 10 mL of Sample solution (mg/mL)
1.N sodium hydroxide, and mix. Extract with 10 mL of Acceptance criteria: 95.0%-105.0%
chloroform, centrifuge, and filter.
Acceptance criteria: The IR absorption spectrum of the PERFORMANCE TESTS
Sample solution, determined in a 0.5-mm cell against e DISSOLUTION (711)
chloroform, exhibits maxima only at the same wave- Medium: 0.1 N hydrochloric acid; 900 mL
lengths as that of a similar solution of USP Metyrapone Apparatus 1: 100 rpm
RS. Time: 45 min
e B. UV ABSORPTION Standard solution: Known concentration of USP
Sample solution: Transfer 1 mL of the filtrate obtained Metyrapone RS in Medium
in Identification test A to a centrifuge tube. Add 20 mL Sample solution: Portions of the solution under test
of chloroform, and extract with 30 mL of 1 N sulfuric suitably diluted with Medium, and filtered
acid, centrifuging and filtering the sulfuric acid layer Instrumental conditions
through a pledget of cotton. Mix 1 mL of this solution Mode: UV
with 99 mL of 7 N sulfuric acid. Analytical wavelength: 259 nm
Acceptance criteria: The UV absorption spectrum of Analysis
the Sample solution exhibits maxima and minima at the Samples: Standard solution and Sample solution
same wavelengths as that of a similar solution of USP Tolerances: NLT 60% (Q) of the labeled amount of
Metyrapone RS, concomitantly measured. metyrapone (Cy4Hi4N20) is dissolved.
e UNIFORMITY OF DosaGeE UNITS (905): Meet the
ASSAY requirements
© PROCEDURE
Solution A: 13.3 mg/mL of 2,4-dinitrophenylhydrazine ADDITIONAL REQUIREMENTS
in methanol. Shake by mechanical means for about 15 ¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant
min, and filter. Prepare fresh daily. containers, and avoid exposure to excessive heat.
Solution B: Solution A and hydrochloric acid (23:2) © USP REFERENCE STANDARDS (11)
Solution C: 50 mg/mL of potassium hydroxide in meth- USP Metyrapone RS
te
“vw
anol, and filter
ie Diluent: Chloroform and methanol (1:1)
i] Standard solution: 0.1 mg/mL of USP Metyrapone RS
—
Dd in chloroform
° Sample stock solution: Nominally equivalent to 25 mg
c
S of metyrapone from powdered Tablets (NLT 20) pre- Metyrosine
= pared as follows. Transfer a suitable amount of pow- °
a
dered Tablets to a centrifuge tube with the aid of
10 mL of 1 N sodium hydroxide. Shake gently, and ex-
al
2 tract with three 15-mL portions of chloroform. Centri- Ho
ad Yo
fuge each extract, filtering through a pledget of cotton,
previously washed with chloroform, into a 50-mL volu- CioHi3NO3 195.22
metric flask. Add chloroform to volume, and mix. L-Tyrosine, o-methyl-, (—)-
Sample solution: Nominally 0.1 mg/mL from the Sam- (-o-Methyl-t-tyrosine © [672-87-7].
ple stock solution in chloroform
Instrumental conditions » Metyrosine contains not less than 98.6 percent
Mode: Vis and not more than 101.0 percent of CioHi3NOs,
Analytical wavelength: 450 nm calculated on the dried basis.
Cell: 1cm
Analysis Packaging and storage—Preserve in well-closed contain-
Samples: Standard solution, Sample solution, and chlo- ers.
roform (blank) USP Reference standards (11)—
Pipet 3 mL each of the Standard solution, the Sample USP Metyrosine RS
solution, and the blank into separate 50-mL volumetric
flasks. To each flask add 1 mL of Solution B, and shake Identification—
lightly. Evaporate the solutions on a steam bath to A: Infrared Absorption (197M).
near dryness. Wash down the sides of the flask with B: Ultraviolet Absorption (197U)—
1 mL of Diluent, and again evaporate the solutions to Solution: 15g per mL.
near dryness. Heat the flasks in an oven maintained at
110°-120° for 30 min. Remove the flasks, pipet 10 mL
Medium: 0.1 N hydrochloric acid.
Absorptivities at 224 nm, calculated on the dried basis, do
of Solution C into each flask, and heat again in the
boiling water bath for 1 min. Allow to cool to room not differ by more than 3.0%.
temperature, insert the stoppers, and shake by me- Specific rotation (781S): between +185° and +195° (t=
chanical means for 5 min. Add methanol to volume, 30° 4 = 546 nm; | = 0.5 dm).
and mix. Measure the absorbance of the Standard so- Test solution: 5 mg per mL, in Diluent, with the aid of
lution and the Sample solution after the extraction sonication if necessary. Prepare the Diluent as follows.
against the blank.
USP 41
Solution A—Dissolve 20.0 g of anhydrous sodium acetate
in about 150 mL of water in a 250-mL volumetric flask. Add
50.0 mL of glacial acetic acid, dilute with water to volume,
and mix.
Solution B—Dissolve 62.5 g of cupric sulfate in water in a
200-mL volumetric flask, dilute with water to volume, and
mix.
Diluent—Mix Solution A and Solution B in a 1000-mL volu-
metric flask, dilute with water to volume, and mix.
Loss on drying (731)—Dry it at a pressure not exceeding
5mm of mercury at 100° for two hours: it loses not more
than 1.0% of its weight.
Residue on ignition (281): not more than 0.1%.
Delete the following:
°Heavy metals, Method // (231): 0.003%. oificiat 1-Jan-2018)
Chromatographic purity—
Standard solutions—Dissolve USP Metyrosine RS in a sol-
vent mixture of methanol and ammonium hydroxide (7:3)
to obtain a solution having a concentration of 10 mg per
mL (Standard solution A). Pipet 1 mL of Standard solution A
into a 100-mL volumetric flask, dilute with the same solvent
mixture to volume, and mix (Standard solution B). Pipet
5 mL of Standard solution B into a 10-mL volumetric flask,
dilute with the same solvent mixture to volume, and mix
(Standard solution ©). Pipet 5 mL of Standard solution C into
a 10-mL volumetric flask, dilute with the same solvent mix-
ture to volume, and mix (Standard solution D).
Test solution—Dissolve Metyrosine in the solvent mixture
of methanol and ammonium hydroxide (7:3) to obtain a
solution having a concentration of 10 mg per mL.
Procedure—Apply 10-uL portions of Standard solutions A,
B, CG, and D and the Test solution to a suitable thin-layer
chromatographic plate (see Chromatography (621)) coated
with a 0.25-mm layer of chromatographic silica gel mixture
and previously washed with methanol. Allow the spots to
dry, and develop the chromatogram in a solvent system
consisting of a mixture of n-propyl alcohol and ammonium
hydroxide (7:3) until the solvent front has moved about
three-fourths of the length of the plate. Remove the plate
from the developing chamber, mark the solvent front, and
dry the plate. Expose the plate to iodine vapors, and ex-
amine under short-wavelength UV light: the chromatogram
shows principal spots at about the same R; value. Estimate
the levels of any additional spots observed in the chromato-
gram of the Test solution by comparison with the spots in
the chromatograms of Standard solutions B, C, and D: the
sum of the intensities of any spots observed is not greater
than that of the principal spot obtained from standard solu-
tion B, corresponding to not more than 1%.
eee about 300 mg of Metyrosine, accurately
weighed, in about 100 mL of glacial acetic acid, sonicate for
about 5 minutes, and titrate with 0.1 N perchloric acid VS,
determining the endpoint potentiometrically, using a plati-
num ring electrode and a sleeve-type calomel electrode con-
taining 0.1 N lithium perchlorate in glacial acetic acid (see
Titrimetry (541)). Perform a blank determination, and make
any necessary correction. Each mL of 0.1 N perchloric acid
is equivalent to 19.52 mg of CioHi3NO3.
Metyrosine Capsules
» Metyrosine Capsules contain not less than
90.0 percent and not more than 110.0 percent of
the labeled amount of metyrosine (CioHi3NOs3).
Official Monographs / Mexiletine 2731
Packaging and storage—Preserve in well-closed contain-
ers.
USP Reference standards (11)—
USP Metyrosine RS
Identification—The UV absorption spectrum of a 1 in
10,000 solution of the Capsule contents in dilute hydrochlo-
ric acid (1 in 100) exhibits maxima and minima at the same
wavelengths as that of a similar solution of USP Metyrosine
RS, concomitantly measured.
Dissolution (711)—
Medium: 0.1 N hydrochloric acid; 750 mL.
Apparatus 1: 100 rpm.
Time: 60 minutes.
Procedure—Determine the amount of CioHi3NOs3 dis-
solved from UV absorbances at the wavelength of maximum
absorbance at about 274 nm of filtered portions of the solu-
tion under test, suitably diluted with Medium, if necessary,
in comparison with a Standard solution having a known
concentration of USP Metyrosine RS in the same medium.
Tolerances—Not less than 75% (Q) of the labeled amount
of CioH13NO3 is dissolved in 60 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Assay—
Standard preparation—Dissolve a suitable quantity of USP
Metyrosine RS, accurately weighed, in dilute hydrochloric
acid (1 in 100) to obtain a solution having a known concen-
tration of about 100 wg per mL.
Assay preparation—Combine the contents of not less than
20 Capsules, and transfer an accurately weighed portion of
the combined contents, equivalent to about 100 mg of
metyrosine, to a 100-mL volumetric flask. Add 50 mL of di-
lute hydrochloric acid (1 in 100), shake by mechanical
means for 45 minutes, dilute with dilute hydrochloric acid (1
in 100) to volume, mix, and filter. Transfer 10.0 mL of the Cc
filtrate to a 100-mL volumetric flask, dilute with dilute hy- 4)
drochloric acid solution (1 in 100) to volume, and mix. ao]
Concomitantly determine the absorbances of this solution my
and the Standard preparation at the wavelength of maxi- i}
mum absorbance at about 274 nm, witha suitable spectro- J
2}
photometer, using dilute hydrochloric acid solution (1 in ro}eI
100) as the blank. Calculate the quantity, in mg, of i)
metyrosine (CioHi3NO3) in the portion of Capsules taken by a}
the formula: os
al
C(Au / As)
in which C is the concentration, in 4g per mL, of USP
Metyrosine RS in the Standard preparation, and Ay and As
are the absorbances of the solutions obtained from the As-
say preparation and the Standard preparation, respectively.
Mexiletine Hydrochloride
oH, oH.
(> Ox -" SNH + HEL
Z
CiHizNO - HCl 215.72
2-Propanamine, 1-(2,6-dimethylphenoxy)-, hydrochloride,
(4)-;
(4)-1-Methyl-2-(2,6-xylyloxy)ethylamine hydrochloride
[5370-01-04].
DEFINITION
Mexiletine Hydrochloride contains NLT 98.0% and NMT
102.0% of mexiletine hydrochloride (CiiHizNO - HC)), cal-
culated on the dried basis.
 2732 Mexiletine / Official Monographs USP 41

IDENTIFICATION Standard solution: 0.2 mg/mL of USP Mexiletine Hy-

e A. INFRARED ABSORPTION (197M) drochloride RS in Mobile phase, from the Standard solu-
e B. The retention time of the major peak of the Sample tion in the Assay
solution corresponds to that of the Standard solution, as Sample solution: 20 mg/mL of Mexiletine Hydrochlo-
obtained in the Assay. ride in Mobile phase
°C System suitability
Sample solution: 3 mL of a solution (1 in 60) Samples: System suitability solution and Standard
Analysis: Add 1 mL of 6 N ammonium hydroxide to solution
the Sample solution, filter, and acidify the filtrate with [Note—The relative retention times for 2-phenylethy-
2 mL of nitric acid. Then add 1 mL of silver nitrate TS. lamine and mexiletine are 0.7 and 1.0, respectively.]
Acceptance criteria: A curdy, white precipitate is Suitability requirements
formed, and it is soluble in an excess of 6 N ammonium Resolution: NLT 3.0 between the 2-phenylethylamine
hydroxide (presence of chloride). and mexiletine peaks, System suitability solution
Relative standard deviation: NMT 3.0%, Standard
ASSAY solution
© PROCEDURE Analysis
Buffer: Dissolve 11.5 g of anhydrous sodium acetate in Samples: Standard solution and Sample solution
500 mL of water. Add 3.2 mL of glacial acetic acid, mix, Calculate the percentage of each impurity in the por-
and allow to cool. Adjust with hydrochloric acid to a tion of Mexiletine Hydrochloride taken:
pH of 4.8 + 0.1, and dilute with water to 1000 mL.
Mobile phase: Methanol and Buffer (600:400) Result = (ru/rs) x (Cs/Cu) x 100
Standard solution: 2 mg/mL of USP Mexiletine Hydro-
chloride RS in Mobile phase tu = peak area of each impurity from the Sample
System suitability solution: 1 mg/mL of 2-phenylethy- solution
amine hydrochloride in Standard solution ls = peak area of mexiletine from the Standard
Sample solution: 2 mg/mL of Mexiletine Hydrochloride solution
in Mobile phase Cs = concentration of USP Mexiletine Hydrochloride
Chromatographic system RS in the Standard solution (mg/mL)
(See Chromatography (621), System Suitability.) Cu = concentration of Mexiletine Hydrochloride in
Mode: LC the Sample solution (mg/mL)
Detector: UV 254 nm Acceptance criteria
Columns Any individual impurity: NMT 1%
Guard: Packing L1 Total impurities: NMT 1.5%
Analytical: 3.9-mm x 30-cm; 10-m packing L1
Flow rate: 1 mL/min SPECIFIC TESTS
Injection volume: 20 uL e PH (791)
System suitability Sample solution: 100 mg/mL
Samples: Standard solution and System suitability Acceptance criteria: 3.5-5.5
te
”

solution e Loss ON DRYING (731)

io [Note—The relative retention times for 2-phenylethy- Analysis: Dry at 105° for 2 h.
s
lamine and mexiletine are 0.7 and 1.0, respectively.] Acceptance criteria: NMT 0.5%
Da
J

° Suitability requirements ADDITIONAL REQUIREMENTS

c Resolution: NLT 3.0 between the 2-phenylethylamine
GC and mexiletine peaks, System suitability solution
e PACKAGING AND STORAGE: Preserve in tight containers.
= Relative standard deviation: NMT 2.0%, Standard e USP REFERENCE STANDARDS (11)
USP Mexiletine Hydrochloride RS
a solution
a)
2 Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of mexiletine hydrochloride
(CiHi7NO - HCl) in the portion of Mexiletine Hydro-
chloride taken: Mexiletine Hydrochloride Capsules
Result = (ru/rs) x (Cs/Cu) x 100 » Mexiletine Hydrochloride Capsules contain not
tu = peak area of mexiletine from the Sample less than 90.0 percent and not more than
solution 110.0 percent of the labeled amount of mexile-
Is = peak area of mexiletine from the Standard tine hydrochloride (C11HizNO - HCl).
solution
Cs = concentration of USP Mexiletine Hydrochloride Packaging and storage—Preserve in tight containers.
RS in the Standard solution (mg/mL) USP Reference standards (11)—
Cy = concentration of Mexiletine Hydrochloride in USP Mexiletine Hydrochloride RS
the Sample solution (mg/mL)
Acceptance criteria: 98.0%-102.0% on the dried basis Identification—
A: Transfer a quantity of Capsule contents, equivalent to
IMPURITIES about 250 mg of mexiletine hydrochloride, to a suitable test
e RESIDUE ON IGNITION (281); NMT 0.1% tube, add 10 mL of methanol, and mix on a vortex mixer
for 1 minute. Filter the mixture, evaporate the filtrate under
a stream of nitrogen to dryness, and dry the residue in vac-
Delete the following: uum at 60° for 1 hour: the IR absorption spectrum of a min-
eral oil dispersion of the dried residue so obtained exhibits
°e HEAVY METALS, Method II (231): NMT 10 ppme coticiai :- maxima only at the same wavelengths as that of a similar
Jen-2018) preparation of USP Mexiletine Hydrochloride RS.
¢ ORGANIC IMPURITIES
Mobile phase, System suitability solution, and Chro- B: The retention time of the major peak in the chromato-
matographic system: Proceed as directed in the gram of the Assay preparation corresponds to that in the
Assay.
USP 41 Official Monographs / Mezlocillin 2733

chromatogram of the Standard preparation, as obtained in tube. Add 25.0 mL of Mobile phase, insert the stopper, and
the Assay. shake by mechanical means for 15 minutes. Centrifuge, and
Dissolution (711)— use the clear supernatant as the Assay preparation. [NOTE—
Medium: water; 900 mL.
Reserve a portion of this solution for use as the Test solution
in the test for Chromatographic purity.]
Apparatus 2: 50 rpm. Procedure—Proceed as directed for Procedure in the Assay
Time: 30 minutes. under Mexiletine Deni, Calculate the quantity, in
Procedure—Determine the amount of Ci;HizNO - HCI dis- mg, of mexiletine hydrochloride (C;;Hi7NO - HCI) in the
solved from the difference between first derivative values at portion of Capsule contents taken by the formula:
the wavelengths of maximum and minimum first derivative
absorbance in the wavelength range from 230 to 290 nm 25C(ru/ rs)
on filtered portions of the solution under test, suitably di-
luted with Dissolution Medium, if necessary, in comparison in which C is the concentration, in mg per mL, of USP Mex-
with a Standard solution having a known concentration of iletine Hydrochloride RS in the Standard preparation; and ru
USP Mexiletine Hydrochloride RS in the same Medium. and rs are the mexiletine peak responses obtained from the
Tolerances—Not less than 80% (Q) of the labeled amount Assay preparation and the Standard preparation, respectively.
of CiHizNO - HCI is dissolved in 30 minutes.
Uniformity of dosage units (905): meet the require-
ments.
Chromatographic purity— Mezlocillin Sodium
Mobile phase, Standard preparation, and Resolution solu-
tion—Prepare as directed in the Assay under Mexiletine Hy-
drochloride.
Standard solution—Transfer 10.0 mL of the Standard prep- CH
aration prepared as directed in the Assay under Mexiletine
Hydrochloride to a 1000-mL volumetric flask, dilute with Mo-
bile phase to volume, and mix. This solution contains about
20 ug of USP Mexiletine Hydrochloride RS per mL.
Test solution—Use the Assay preparation prepared as di-
rected in the Assay. CaiH24NaNsOsS2 561.56
Chromatographic system (see Chromatography (621))— 4-Thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, 3,3-di-
Prepare as directed in the Assay under Mexiletine Hydrochlo- methyl-6-[[[[(3-(methylsul‘onyl)-2-0xo-
ride, except that the relative standard deviation of replicate 1-imidazolidinyl]carbonyl]amino]phenylacetyl] amino]-
injections of the Standard solution is not more than 3.0%. 7-oxo-, monosodium salt, [25-[20,50,6B (S*)}]-;
Procedure—Separately inject equal volumes (about 20 iL) Sodium (25,5R,6R)-3,3-dimethy|-6-[(R)-2-[3-(methylsulfonyl)-
of the Standard solution and the Test solution into the chro- Done: -lmidavolldinecarboxainidol-2!phenyiseet mide =
matograph; record the ey an ee al using a high sensi- 7-0xo-4-thia-1 -azabicyclo[3.2.0]heptane-2-carboxylate yv
[42057-22-7]. me]
tivity setting for the recorder; and measure the areas for the
pea 's. Calculate the percentage of each impurity observed Monohydrate 579.58 =
y the formula: °
DEFINITION =}
Mezlocillin Sodium contains the equivalent of NLT 838 j1g/ °
100(C/L)(ru / 15) me and NMT 978 ug/g of mezlocillin (C21H2sNsO8Sz),
Ko}
=
2
in which C is the concentration, in mg per mL, of USP Mex- calculated on the anhydrous basis. mo}
iletine Hydrochloride RS in the Standard solution; L is the ye
IDENTIFICATION a
uantity, in mg, of mexiletine hydrochloride in each mL of
the Test solution, based on the labeled amount in the por- © A. INFRARED ABSORPTION (197K)
tion of Capsule contents used to prepare the Assay prepara- e B. The retention time of the major peak of the Sample
tion and the extent of dilution; ry is the peak area obtained solution corresponds to that of the Standard solution, as
from an individual impurity observed in the chromatogram obtained in the Assay.
of the Test solution; and rs is the mexiletine peak area ob- e C. IDENTIFICATION TESTS—GENERAL, Sodium (191): Meets
tained from the Standard solution: not more than 1% of any the requirements
individual impurity is found; and the total of all observed ASSAY
impurities is not more than 1.5%. © PROCEDURE
Assay— Buffer: 4.9 g/L of monobasic potassium phosphate and
Mobile phase, Standard preparation, Resolution solution, 0.54 g/L of dibasic potassium phosphate in water
and Chromatographic system—Prepare as directed in the As- Mobile phase: Acetonitrile and Buffer (145:855)
say under Mexiletine Hydrochloride. Standard solution: 0.5 mg/mL of mezlocillin from USP
Assay preparation—Weigh the contents of not fewer than Mezlocillin Sodium RS in water
20 Capsules, and calculate the average weight per Capsule. Sample solution: 0.55 mg/mL of Mezlocillin Sodium in
Mix the combined contents of the Capsules, and transfer an water
accurately weighed portion, equivalent to about 50 mg of Chromatographic system
mexiletine hydrochloride, to a stoppered, 50-mL centrifuge (See Chromatography (621), System Suitability.)
 2734 Mezlocillin / Official Monographs USP 41

Mode: LC Packaging and storage—Preserve as described in Packag-

Detector: UV 210 nm ing and Storage Requirements (659), Injection Packaging,
Column: 4-mm x 12.5-cm; 5-m packing L1 Packaging for constitution.
Temperature: 40°
Flow rate: 2 mL/min
Injection volume: 20 uL Change to read:
System suitability
Sample: Standard solution USP Reference standards (11)—
Suitability requirements bs {CN 1-May-2018)
Relative standard deviation: NMT 1.5% USP Mezlocillin Sodium RS
Tailing factor: NMT 1.5 Constituted solution—At the time of use, it meets the
Analysis requirements for Injections and Implanted Drug Products (1),
Samples: Standard solution and Sample solution Specific Tests, Completeness and clarity of solutions.
Calculate the quantity, in ug/mg, of mezlocillin Bacterial Endotoxins Test (85)—It contains not more
pealiedisklee in each mg of Mezlocillin Sodium than 0.06 USP Endotoxin Unit per mg of mezlocillin.
taken:
Sterility Tests (71)—It meets the requirements when
Result = (ru/rs) x (Cs/Cu) x P tested as directed for Membrane Filtration under Test for Ste-
rility of the Product to be Examined.
ru = peak response from the Sample solution Particulate Matter in Injections (788): meets the re-
ls = peak response from the Standard solution quirements for small-volume injections.
Cs = concentration of USP Mezlocillin Sodium RS in Other requirements—It responds to the /dentification tests
the Standard solution (mg/mL) and meets the requirements for Specific rotation, pH, and
Gy = concentration of the Sample solution (mg/mL) Water under Mezlocillin Sodium. \t meets also the require-
P = potency of mezlocillin in USP Mezlocillin ments for Uniformity of Dosage Units (905) and Labeling (7),
Sodium RS (ugima) Labels and Labeling for Injectable Products.
Recepeariee criteria: 838-978 g/mg on the anhydrous
asis Assay—
Mobile phase, Standard preparation, Resolution solution,
SPECIFIC TESTS and Chromatographic system—Prepare as directed for the As-
e OPTICAL ROTATION, Specific Rotation (781S) say under Mezlocillin Sodium.
Sample solution: 10 mg/mL in water Assay preparation 1 (where it is represented as being in a
Acceptance criteria: +175° to +195° single-dose container)—Constitute Mezlocillin for Injection
PH (791) in a volume of water, accurately measured, corresponding
Sample solution: 100 mg/mL in water to the volume of solvent specified in the labeling. Withdraw
Acceptance criteria: 4.5-8.0 all of the withdrawable contents, using a suitable hypoder-
WATER DETERMINATION, Method | (921): NMT 6.0% mic needle and syringe, and dilute spar utatvely with water
"“ STERILITY TESTS (71): Where the label states that Mezlocil- to obtain a solution containing about 0.5 mg of mezlocillin
<j lin Sodium is sterile, it meets the requirements when per mL.
oe tested as directed for Test for Sterility of the Product to Be
i] Assay preparation 2 (where the label states the quantity
J Examined, Membrane Filtration.
D of mezlocillin in a aven volume of constituted solution)—
3 BACTERIAL ENDOTOXINS TEST (85): Where the label states Constitute Mezlocillin for Injection in a volume of water,
is that Mezlocillin Sodium is sterile or must be subjected to accurately measured, corresponding to the volume of sol-
5 further processing during the preparation of injectable vent specified in the labeling. Dilute an accurately measured
= dosage forms, it contains NMT 0.06 USP Endotoxin portion of the constituted solution Seen with water
a Units/mg of mezlocillin. to obtain a solution containing about 0.5 mg of mezlocillin
2)
= ADDITIONAL REQUIREMENTS per mL.
© PACKAGING AND STORAGE: Preserve in tight containers. Procedure—[NOTE—Use peak areas where peak responses
e LABELING: Where it is intended for use in preparing in- are indicated.] Separately inject equal volumes (about 20 pL)
jectable dosage forms, the label states that it is sterile or of the Standard preparation and Assay preparation 1 into the
must be subjected to further processing during the prep- chromatograph, record the chromatograms, and measure
aration of injectable dosage forms. the responses for the major peaks. Calculate the quantity, in
mg, of mezlocillin in the container, or in the portion of
constituted solution taken by the formula:
Change to read:
(L/ D(C /1000)(ru
/rs)
e USP REFERENCE STANDARDS (11)
@ (CN 1-May-2018) in whichLis the labeled quantity, in mg, of mezlocillin in
USP Mezlocillin Sodium RS the container, or in the volume of constituted solution
taken, D is the concentration, in mg per mL, of mezlocillin
in oy reparation 1 or in Assay preparation 2, on the basis
of the labeled quantity in the container, or in the portion of
constituted solution taken, respectively, and the extent of
dilution, C is the concentration, in ug per mL, of mezlocillin
Mezlocillin for Injection (C2H2sNsOgSz) in the Standard preparation, and ry and rs are
the mezlocillin peak responses obtained from the Standard
» Mezlocillin for Injection contains an amount of preparation and from Assay preparation 1 or Assay prepara-
Mezlocillin Sodium equivalent to not less than tion 2, as appropriate.
90.0 percent and not more than 115.0 percent of
the labeled amount of mezlocillin (C2iH2sNsOgSz).
USP 41
Mibolerone
Use foes,
f ie:
Ce i the labeled amount of mibolerone (C20H300z2).
a A ty
re
CooH3oO2 302.45
Estr-4-en-3-one, Ua 7-dimethyl-, (7a,178)-.
17B-Hydroxy-7a,17-dimethylestr-4-en-3-one [3704-09-4].
» Mibolerone contains not less than 96.0 percent
and not more than 106.0 percent of C29H3002,
calculated on the dried basis.
Packaging and storage—Preserve in well-closed contain-
ers.
Labeling—Label it to indicate that it is for veterinary use
only.
USP Reference standards (11)—
USP Mibolerone RS
Identification, Infrared Absorption (197M).
Specific rotation (781S): between +34° and +40°.
Test solution: 10 mg per mL, in chloroform.
Loss on drying (731)—Dry about 1 g, accurately weighed,
in a capillary-stoppered bottle in vacuum at a pressure not
exceeding 5 mm of mercury at 60° for 3 hours: it loses not
more than 0.5% of its weight.
Residue on ignition (281): not more than 0.5%.
Assay—
Mobile phase—Preparea filtered and degassed mixture of
water, tetrahydrofuran, and methanol (60:25:15). Make ad-
justments if necessary (see System Suitability under Chroma-
tography (621)).
Internal standard solution—Prepare a solution of proges-
terone in methanol containing 0.6 mg per mL.
Standard preparation—Prepare a solution of USP Miboler-
one RS in Internal standard solution having a known concen-
tration of about 0.4 mg per mL. Mix, and sonicate if neces-
sary to achieve complete solution.
Assay preparation—Transfer about 10 mg of Mibolerone,
accurately weighed, to a 25-mL volumetric flask, dilute with
Internal standard solution to volume, and mix. Sonicate if
necessary to achieve complete solution.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 254-nm detector
and a 3.9-mm x 30-cm column that contains packing L1.
The flow rate is about 2 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as di-
rected for Procedure: the relative retention times are about
0.6 for mibolerone and 1.0 for progesterone; and the rela-
tive standard deviation for replicate injections is not more
than 2.0%.
Procedure—Separately inject equal volumes (about 5 pL)
of the Standard preparation and the Assay preparation into
the chromatograph, record the Supmaiaea Ds and meas-
ure the responses for the major peaks. Calculate the quan-
tity, in mg, of CzoH30O2 in the portion of Mibolerone taken
by the formula:
25 (Ru / Rs)
in which C is the concentration, in mg per mL, of USP
Mibolerone RS in the Standard preparation; and Ry and Rs
are the ratios of the peak responses of the mibolerone peak
and the progesterone peak obtained from the Assay prepara-
tion and the Standard preparation, respectively.
Official Monographs / Mibolerone 2735
Mibolerone Oral Solution
» Mibolerone Oral Solution contains not less than
90.0 percent and not more than 115.0 percent of
shxvenalt | and storage—Preserve in tight containers,
protected from light.
Labeling—Label it to indicate that it is for veterinary use
only.
USP Reference standards (11)—
USP Mibolerone RS
Identification—The chromatogram of the Assay prepara-
tion exhibits a major peak for mibolerone, the retention
time of which corresponds to that in the chromatogram of
the Standard preparation, as obtained in the Assay.
Specific gravity (841): between 1.030 and 1.045.
Assay—
Internal standard solution—Prepare a solution of 1,3,
5-triphenylbenzene in chloroform containing about 0.25 mg
per mL.
Standard preparation—Prepare a solution of USP Miboler-
one RS in Internal standard solution having a known concen-
tration of about 0.5 mg per mL.
Assay preparation—Transfer an accurately weighed por-
tion of Oral Solution, equivalent to about 1000 yg of
mibolerone, to a 125-mL separator containing 60 mL of
water, and swirl to disperse. Add 30 mL of methylene chlo-
tide, shake gently for about 5 minutes, and allow the phases
to separate. Drain the lower oe chloride layer
through a pledget of methylene chloride-washed cotton
into a 50-mL conical flask. Evaporate to dryness under a
current of air. Re-extract the aqueous layer remaining in the
separator with an additional 30-mL portion of methylene (es
chloride, draining the filtered methylene chloride extract 4)
into the 50-mL conical flask, and evaporating it to dryness.
a)
Add 2.0 mL of Internal standard solution, and swirl to dis- Ss
solve. 5}
=
Chromatographic system (see Chromatography (621))—The }
gas chromatograph is equipped with a flame-ionization de- eo}e
tector and a 3-mm x 61-cm column packed with 1% liquid i)
phase G6 on support S1AB. The column is maintained at a}
about 175° and the detector at 195° to 225°. Helium is
a
nn
used as the carrier gas at a flow rate of about 60 mL per
minute. Chromatograph the Standard preparation, and re-
cord the peak responses as directed for Procedure: the rela-
tive retention times are about 0.6 for the internal standard
and 1.0 for mibolerone; and the relative standard deviation
for replicate injections is not more than 2.0%.
Procedure—Separately inject equal volumes (about 2 uL)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
ure the responses for the major peaks.Calculate the quan-
tity, in ug, of mibolerone (C20H3002) in each mL of the Oral
Solution taken by the formula:
2000(C/ Wy)(D)(Ru
/ Rs)
in whichCis the concentration, in mg per mL, of USP
Mibolerone RS in the Standard preparation; Wy is the
weight, in g, of Oral Solution taken to prepare the Assay
preparation; D is the specific gravity of the Oral Solution;
and Ry and Rs are the ratios of the rae height response of
the mibolerone peak to the internal standard peak obtained
from the Assay preparation and the Standard preparation, re-
spectively.
 2736 Miconazole / Official Monographs USP 41

ber with freshly prepared Developing solvent system

Miconazole until the solvent front has moved about three-fourths

es
of the length of the plate. Remove the plate from the
i chamber, and allow the solvent to evaporate. Expose
the plate to iodine vapors in a closed chamber for 30
min, and locate the spots.
rey Acceptance criteria: The R; value of the principal spot
from the Sample solution corresponds to that from Stan-
A dard solution A, and any other spot from the Sample
solution does not exceed, in size or intensity, the princi-
pal spot from Standard solution B (1.0%).
a
SPECIFIC TESTS
CisHi4ClaN2O 416.13 e Loss ON DRYING (731)
1H-Imidazole, 1-2-[(2,4-dichlorophenyl)-2-[(2,4-
Analysis: Dry a sample under vacuum at 60° for 4 h.
dichlorophenyl)|methoxy]ethyl]-, (4)-; Acceptance criteria: NMT 0.5%
(4)-1-[2,4-Dichloro-B-[(2,4-dichlorobenzyl)oxy] ADDITIONAL REQUIREMENTS
phenethyl]imidazole [2291 6-47-8]. e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light. Store at 25°, excursions permit-
DEFINITION ted between 15° and 30°.
Miconazole contains NLT 98.0% and NMT 102.0% of
e USP REFERENCE STANDARDS (11)
miconazole (CigHi4Cl4aN2O), calculated on the dried basis.
USP Miconazole RS
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
° B.
Sample solution: Dissolve 40 mg in 50 mL of isopropyl
alcohol in a 100-mL volumetric flask, and add 10 mL of Miconazole Compounded Ophthalmic
0.1 N hydrochloric acid. Dilute with isopropyl alcohol Solution
to volume.
Acceptance criteria: The UV absorption spectrum of DEFINITION
the Sample solution exhibits maxima and minima at the Miconazole Compounded Ophthalmic Solution contains
same wavelengths as that of a similar solution of USP NLT 90.0% and NMT 110.0% of the labeled amount of
Miconazole RS, concomitantly measured. miconazole (CigH;4Cl4aN20).
ASSAY Prepare Miconazole Compounded Ophthalmic Solution 1%
e PROCEDURE
(10 mg/mL) as follows (see Pharmaceutical Compounding—
Sterile Preparations (797)).
4 Sample solution: 300 mg of Miconazole in 40 mL of
a= glacial acetic acid. Add 4 drops of p-naphtholbenzein
rok TS
i]
— Titrimetric system Po! i
Dp
2} Mode: Direct titration ution (88%!
is Titrant: 0.1 N perchloric acid VS
iS Endpoint detection: Visual
Sterile Water for Injection, a sufficient amount
make 1
= Analysis: Titrate the Sample solution with Titrant to a
rs green endpoint. Perform a blank determination, and Add the Miconazole to a sterile container and gradually add
a) make any necessary correction. Each mL of 0.1 N per- the Polyoxyl 40 Hydrogenated Castor Oil. Mix into a
= chloric acid is equivalent to 41.61 mg of miconazole smooth viscous mixture. Add the Lactic Acid Solution
(CisHi4ClaN2O). (88%) and mix thoroughly. Add 80 mL of the Sterile Water
Acceptance criteria: 98.0%-102.0% on the dried basis for Injection and stir vigorously until the Miconazole is
completely dissolved. Transfer the contents stepwise and
IMPURITIES quantitatively to a sterile calibrated container and bring to
e RESIDUE ON IGNITION (281): NMT 0.2%
final volume with Sterile Water for Injection. Pass through a
© ORGANIC IMPURITIES sterile filter of 0.22-1m pore size into an empty sterile
Standard solution A: 10 mg/mL of USP Miconazole RS dropper bottle. [NoTE—Room temperature Sterile Water
in chloroform for Injection should be used to assist in solubilization.]
Standard solution B: 100 ug/mL of USP Miconazole RS
from Standard solution A in chloroform ASSAY
Sample solution: 10 mg/mL in chloroform © PROCEDURE
Chromatographic system Mobile phase: Dissolve 5.7 g of ammonium acetate in
(See Chromatography (621), Thin-Layer Chromato- 380 mL of water, and add 320 mL of methanol and
graphy.)
Mode: TLC
300 mL of acetonitrile. Mix well.
Standard solution: 0.05 mg/mL of miconazole pre-
Adsorbent: 0.25-mm layer of chromatographic silica pared from USP Miconazole RS in methanol
gel mixture Sample solution: Transfer 0.5 mL of Ophthalmic Solu-
Application volume: 5 pL tion to a 100-mL volumetric flask, dilute with methanol
Developing solvent system: n-Hexane, chloroform, to volume, and vortex to mix.
methanol, and ammonium hydroxide (60:30:10:1) Chromatographic system
Analysis (See Chromatography (621), System Suitability.)
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the Samples separately to the starting line of the
plate. Develop the chromatogram in a suitable cham-
USP 41 Official Monographs / Miconazole 2737

Mode: LC Solution B: 400 mg/mL of potassium iodide in water

Detector: UV 230 nm Standard solution: 5 mg/mL of USP Miconazole RS in
Column: 2.0-mm x 10-cm; 2.5-"um packing L1 methanol
Column temperature: 55° Sample solution: Nominally 5 mg/mL of miconazole in
Flow rate: 0.35 mL/min methanol
Injection volume: 10 uL Chromatographic system
System suitability (See Chromatography (621), Thin-Layer Chromato-
Sample: Standard solution graphy.)
a retention time for miconazole is about 18.0 Mode: TLC
min. Adsorbent: 0.25-mm layer of chromatographic silica
Suitability requirements gel mixture
Tailing factor: NMT 2.0 Application volume: 5 wl
Relative standard deviation: NMT 2.0% for replicate Developing solvent system: n-Hexane, chloroform,
injections methanol, and ammonium hydroxide (60:30:10:1)
Analysis Spray reagent (Dragendorff’s TS): Solution A, Solu-
Samples: Standard solution and Sample solution tion B, glacial acetic acid, and water (1:1:4:14)
Calculate the percentage of the labeled amount of Analysis
miconazole (CigH:4Cl4N2O) in the portion of Samples: Standard solution and Sample solution
Ophthalmic Solution taken: Apply the Samples separately to the starting line of the
ate. Develop the chromatogram in a suitable cham-
Result = (ru/rs) x (Cs/Cu) x 100 er with freshly prepared Developing solvent system un-
til the solventfront has moved about three-fourths of
ru = peak response of miconazole from the Sample the length of the plate. Remove the plate from the
solution chamber, and allow the solvent to evaporate. Locate
rs peak response of miconazole from the the spots on the plate by spraying with Spray reagent.
i

Standard solution Acceptance criteria: The R- value of one of the princi-

Cs = concentration of USP Miconazole RS in the pal spots from the Sample solution corresponds to that
Standard solution (mg/mL) from the Standard solution.
Cu = nominal concentration of miconazole in the
Sample solution (mg/mL) ASSAY
Acceptance criteria: 90.0%-110.0% e PROCEDURE
Solution A: 25 mg/mL of ammonium acetate in water
SPECIFIC TESTS Mobile phase: Acetonitrile, methanol, and Solution A
e PH (791): 2.9-3.9 (30:50:20)
e STERILITY TESTS (71), Test for Sterility of the Product to Be system suitability solution: 50 g/mL each of USP
Examined, Membrane Filtration: Meets the requirements iconazole RS and dibutyl phthalate in Mobile phase
¢ PARTICULATE MATTER IN OPHTHALMIC SOLUTIONS (789): Standard solution: 50 g/mL of USP Miconazole RS in
Meets the requirements Mobile phase =
Sample solution: Nominally 50 g/mL of miconazole in al
ADDITIONAL REQUIREMENTS Mobile phase i)
© PACKAGING AND STORAGE: Package in sterile plastic
ophthalmic dropper bottles for single-use in one patient
Chromatographic system E<
(See Chromatography (621), System Suitability.) i)
only. Store in a refrigerator (2°-8°) or at controlled room
Mode: LC =
temperature. )
e BEYOND-USE DATE: I|n the absence of performing and
Detector: UV 230 nm ro)=:
Column: 4.6-mm x 30-cm; packing L7
completingasterility test, the storage conditions for iy
high-risk level CSPs apply (see Pharmaceutical Compound-
Flow rate: 2 mL/min rc
Injection volume: 20 uL cy
ing—Sterile Preparations (797), CSP Microbial Contamina- System suitability
vw
tion Risk Levels, High-Risk Level CSPs). After successful Samples: System suitability solution and Standard
completion of sterility testing, NMT 30 days after the solution
date on which it was compounded when stored in a re- [Note—The relative retention times for dibutyl phthal-
frigerator (2°-8°); NMT 21 days after the date on which ate and miconazole are 0.7 and 1.0, respectively.]
it was compounded when stored at controlled room Suitability requirements
temperature Resolution: NLT 5.0 between the dibutyl phthalate
e LABELING: Label it to indicate that it is for ophthalmic use
only and to state the Beyond-Use Date. and miconazole peaks, System suitability solution
Tailing factor: NMT 1.3 for the miconazole peak,
© USP REFERENCE STANDARDS (11)
USP Miconazole RS System suitability solution
Relative standard deviation: NMT 2.0%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Allow the chromatograph to run for at least 16-18 min
Miconazole Injection between injections to allow for elution of all compo-
nents associated with the Injection vehicle.
DEFINITION Calculate the percentage of the labeled amount of
Miconazole Injection is a sterile solution of Miconazole in miconazole (CigH;4Cl4N20) in the portion of Injection
Water for Injection. It contains NLT 90.0% and NMT taken:
110.0% of the labeled amount of miconazole
(CigHi4Cl4N2O). Result = (ru/rs) x (Cs/Cu) x 100
IDENTIFICATION ru = peak response from the Sample solution
cA. rs = peak response from the Standard solution
Solution A: 17 mg/mL of bismuth subnitrate in a mix- Cs = concentration of USP Miconazole RS in the
ture of glacial acetic acid and water (1:4) Standard solution (ug/mL)
 2738 Miconazole / Official Monographs USP 41

Cy = nominal concentration of miconazole in the Acceptance criteria: 98.0%-102.0% on the dried basis
Sample solution (g/mL)
Acceptance criteria: 90.0%-110.0% IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.2%
SPECIFIC TESTS © ORGANIC IMPURITIES
© PH (791): 3.7-5.7 Mobile phase: Methanol, acetonitrile, and 0.2 M am-
e BACTERIAL ENDOTOXINS TEST (85): NMT 0.10 USP Endo- monium acetate (32:30:38)
toxin Unit/mg of miconazole System suitability solution: 25 j1g/mL each of USP
© PARTICULATE MATTER IN INJECTIONS (788): Meets the re- Miconazole Nitrate RS and USP Econazole Nitrate RS in
quirements for small-volume injections Mobile phase
e INJECTIONS AND IMPLANTED DRUG PRODUCTS (1): Meets the Sample stock solution: 10 mg/mL of Miconazole Ni-
requirements trate in Mobile phase
Sample solution: 25 tg/mL of Miconazole Nitrate,
ADDITIONAL REQUIREMENTS from Sample stock solution, in Mobile phase
e PACKAGING AND STORAGE: Preserve in single-dose contain- Chromatographic system
ers, preferably of Type | glass, at controlled room (See Chromatography (621), System Suitability.)
temperature. Mode: LC
Detector: UV 235 nm
Change to read: Column: 4.6-mm x 10-cm; 3-11m packing L1
Flow rate: 2 mL/min
e USP REFERENCE STANDARDS (11)
Injection volume: 10 UL
Run time: 1.2 times the retention time of the main
@ (CN 1-May-2018)
USP Miconazole RS
peak
System suitability
Sample: System suitability solution
[Note—The relative retention times for econazole and
miconazole are 0.5 and 1.0, respectively.]
Suitability requirements
Miconazole Nitrate Resolution: NLT 10 between econazole and
miconazole
Relative standard deviation: NMT 2.0%
scellet Analysis
Lt Samples: Sample stock solution and Sample solution
Measure the responses of all peaks, excluding the peak
representing nitrate ion and any peak producing a
response less than 0.2 times the response of the main
te 5 eak.
Acte tance criteria: The response of any individual
=
al a
peak, other than the main peak of the Sample stock
a solution, is NMT that of the main peak of the Sample
s CisHi4gCl4aN2O-HNO3 479.14
D
3=| 1H-Imidazole, 1-[2-(2,4-dichlorophenyl)-2-[(2,4-
solution (0.25%), and the sum of the responses of all
peaks, other than the main peak of the Sample stock
dichlorophenyl)methoxy]ethyl]-, mononitrate; solution, is NMT twice the response of the main peak of
5 1-[2,4-Dichloro-B-[(2,4-dichlorobenzyl)oxy] the Sample solution (0.5%).
= phenethyl]imidazole mononitrate [22832-87-7].
[5 SPECIFIC TESTS
v2) DEFINITION e Loss ON DRYING (731)
2 Miconazole Nitrate contains NLT 98.0% and NMT 102.0% Analysis: Dry a sample at 105° for 2 h.
of miconazole nitrate (CigHi4ClaN2O-HNO3), calculated Acceptance criteria: NMT 0.5%
on the dried basis.
ADDITIONAL REQUIREMENTS
IDENTIFICATION e PACKAGING AND STORAGE: Preserve in well-closed contain-
e A. INFRARED ABSORPTION (197K) ers, protected from light.
e B. ULTRAVIOLET ABSORPTION (197U) e USP REFERENCE STANDARDS (11)
Sample solution: 400 g/mL in a mixture of 0.1 N hy- USP Econazole Nitrate RS
drochloric acid in isopropyl alcohol (1 in 10) USP Miconazole Nitrate RS
Acceptance criteria: Meets the requirements
ASSAY
e PROCEDURE
Sample solution: 350 mg of Miconazole Nitrate in
50 mL of glacial acetic acid Miconazole Nitrate Cream
Titrimetric system
Mode: Direct titration DEFINITION
Titrant: 0.1 N perchloric acid VS Miconazole Nitrate Cream contains NLT 90.0% and NMT
Endpoint detection: Potentiometric 110.0% of the labeled amount of miconazole nitrate
Analysis: Titrate the Sample solution with Titrant using a (CigHi4Cl4N20 - HNO3).
glass-calomel electrode system. Perform a blank deter-
mination, and make any necessary correction. Each mL IDENTIFICATION
of 0.1 N perchloric acid is equivalent to 47.92 mg of e A. The retention time of the major peak of the Sample
miconazole nitrate (CisHi4Cl4N2O - HNOs). solution corresponds to that of the Standard solution, as
obtained in the Assay.
USP 41
ASSAY
e PROCEDURE
Buffer: Triethylamine and water (10:1000). Adjust with
phosphoric acid to a pH of 2.5.
Mobile phase: Methanol, acetonitrile, tetrahydrofuran,
and Buffer (5:4:3:8)
Standard solution: 0.28maim of USP Miconazole Ni-
Bale RS and 0.02 mg/mL of benzoic acid in Mobile
jase
sample solution: Nominally 0.28 mg/mL of miconazole
nitrate in Mobile phase prepared as follows. Dissolve a
weighed quantity of Cream in Mobile phase, and soni-
cate in a water bath at 40°-45° until the sample is
completely dispersed. Cool the solution to below room
temperature, and pass through a 0.45-um Teflon filter
into an HPLC vial.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 225 nm
Column: 4.6-mm x 25-cm; packing L11
Column temperature: 45°
Flow rate: 1 mL/min
Injection volume: 10 uL
System suitability
Sample: Standard solution
Suitability requirements
Resolution: NLT 13 between miconazole nitrate and
benzoic acid
Column efficiency: NLT 7500 theoretical plates for
the miconazole nitrate peak
Tailing factor: NMT 2.0 for the miconazole nitrate
eak
Relative standard deviation: NMT 2.0% from the
miconazole nitrate peak
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
miconazole nitrate (CigHi4Cl4N2O - HNO3) in the por-
tion of Cream taken:
Result = (ru/rs) x (Cs/Cu) x 100
ru = peak response from the Sample solution
Ts = peak response from the Standard solution
Cs = concentration of USP Miconazole Nitrate RS in
the Standard solution (mg/mL)
Cy = nominal concentration of miconazole nitrate
in the Sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0%
PERFORMANCE TESTS
e MINIMUM FiLL (755): Meets the requirements
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in collapsible tubes or
tight containers, and store at controlled room
temperature.
e LABELING: Cream that is packaged and labeled for use as
a vaginal preparation shall be labeled Miconazole Nitrate
Vaginal Cream.
e USP REFERENCE STANDARDS (11)
USP Miconazole Nitrate RS
Miconazole Nitrate Topical Powder
DEFINITION
Miconazole Nitrate Topical Powder contains NLT 90.0% and
NMT 110.0% of the labeled amount of miconazole nitrate
(CigHi4ClaN20 - HNO3).
Official Monographs / Miconazole 2739
IDENTIFICATION
oA.
Sample: Transfer nominally 100 mg of miconazole ni-
trate from Topical Powder to a 50-mL beaker, disperse
in 40 mL of methanol, and mix for a minimum of 5
min. Allow to settle for 5-10 min, and filter into a
100-mL beaker. Evaporate on a steam bath to dryness.
Dry the residue at 105° for 10 min.
Acceptance criteria: The IR absorption spectrum of a
potassium bromide dispersion of the residue obtained
from the Sample exhibits maxima only at the same
wavelengths as that of a similar preparation of USP
Miconazole Nitrate RS.
ASSAY
e PROCEDURE
Internal standard solution: 0.5 mg/mL of cholestane
in chloroform
Standard solution: 2 mg/mL of USP Miconazole Nitrate
RS prepared as follows. Transfer 5.0 mL of 0.8 mg/mL of
USP Miconazole Nitrate RS in a mixture of chloroform
and methanol (1:1) to a test tube, and add 2.0 mL of
Internal standard solution. Evaporate to dryness at a
temperature not higher than 40° with the aid of a cur-
rent of nitrogen. Dissolve the residue in 2.0 mL of a
mixture of chloroform and methanol (1:1).
Sample solution: Nominally 2 mg/mL of miconazole ni-
trate prepared as follows. Transfer an equivalent to
20 mg of miconazole nitrate from Topical Powder to a
50-mL centrifuge tube. Add 25.0 mL of methanol, and
shake by mechanical means for 30 min to dissolve the
miconazole nitrate. Centrifuge to obtain a clear super-
natant. Transfer 5.0 mL of this solution to a test tube,
add 2.0 mL of Internal standard solution, and evaporate
to dryness at a temperature not higher than 40° with
the aid of a current of nitrogen. Dissolve the residue in
2.0 mL of a mixture of chloroform and methanol (1:1).
Chromatographic system =
(See Chromatography (621), System Suitability.) a)
Mode: GC 7
Detector: Flame ionization c
Column: 2-mm x 1.2-m glass; packed with 3% phase i}
G32 on support S1A po}
}
Temperatures @=
Column: 250° i)
Injection port: 250° a}
Detector: 300° Pa
“
Carrier gas: Helium
Flow rate: 50 mL/min
Injection volume: 5 wl
System suitability
Sample: Standard solution
[NoTt—The relative retention times for cholestane and
miconazole nitrate are 0.5 and 1.0, respectively.]
Suitability requirements
Resolution: NLT 2.0 between the cholestane and
miconazole nitrate peaks
Relative standard deviation: NMT 3.0% for replicate
injections
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
miconazole nitrate (CigHi4Cl4N2O - HNOs) in the por-
tion of Topical Powder taken:
Result = (Ru/Rs) x (Cs/Cu) x 100
Ru = peak response ratio of miconazole nitrate to
cholestane from the Sample solution
Rs = peak response ratio of miconazole nitrate to
cholestane from the Standard solution
Cs = concentration of USP Miconazole Nitrate RS in
the Standard solution (mg/mL)
Cu = nominal concentration of miconazole nitrate
in the Sample solution (mg/mL)
 2740 Miconazole / Official Monographs USP 41

Acceptance criteria: 90.0%-110.0% Mode: GC

Detector: Flame ionization
PERFORMANCE TESTS Column: 2-mm x 1.2-m glass; packed with 3% phase
e MINIMUM FILL (755): Meets the requirements G32 on support S1A
Temperatures
SPECIFIC TESTS Column: 250°
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI- Injection port: 250°
FIED MICROORGANISMS (62): The total count does not ex- Detector: 300°
ceed 102 cfu/g. It meets the requirements of the tests for Carrier gas: Helium
the absence of Staphylococcus aureus and Pseudomonas Flow rate: 50 mL/min
aeruginosa. Injection volume: 1 pL
ADDITIONAL REQUIREMENTS System suitability
e PACKAGING AND STORAGE: Preserve in well-closed Sample: Standard solution
containers. [NoTtE—The relative retention times for cholestane and
e USP REFERENCE STANDARDS (11) miconazole nitrate are 0.44 and 1.0, respectively.]
USP Miconazole Nitrate RS Suitability requirements
Resolution: NLT 2.0 between the cholestane and
miconazole nitrate peaks
Relative standard deviation: NMT 3.0% for replicate
injections
Analysis
Miconazole Nitrate Vaginal Samples: Standard solution and Sample solution
Suppositories Calculate the percentage of the labeled amount of
miconazole nitrate (CisHi4Cla4N2O - HNOs) in the por-
DEFINITION tion of Suppository taken:
Miconazole Nitrate Vaginal Suppositories contain NLT 90.0%
and NMT 110.0% of the labeled amount of miconazole Result = (Ru/Rs) x (Cs/Cu) x 100
nitrate (CigHi4Cl4N2O - HNOs).
Ry = peak response ratio of miconazole nitrate to
IDENTIFICATION cholestane from the Sample solution
eA. Rs = peak response ratio of miconazole nitrate to
Sample: Place a portion of the Sample stock solution cholestane from the Standard solution
prepared as directed in the Assay, containing about Cs = concentration of USP Miconazole Nitrate RS in
25 mg of miconazole nitrate, in a 50-mL beaker. Evapo- the Standard solution (mg/mL)
rate on a steam bath to dryness with the aid of a cur- G = nominal concentration of miconazole nitrate
rent of filtered air. Dry the residue at 105° for 10 min. in the Sample solution (mg/mL)
Acceptance criteria: The IR oben en spectrum of a Acceptance criteria: 90.0%-110.0%
ww potassium bromide dispersion of the residue obtained
£ from the Sample exhibits maxima only at the same ADDITIONAL REQUIREMENTS
Se wavelengths as that of a similar preparation of USP © PACKAGING AND STORAGE: Preserve in tight containers at
3
Miconazole Nitrate RS. controlled room temperature.
io.)
—

° e USP REFERENCE STANDARDS (11)

USP Miconazole Nitrate RS
fy ASSAY
S © PROCEDURE
2 Internal standard solution: 1 mg/mL of cholestane in a
[a mixture of chloroform and methanol (1:1)
” Standard solution: 2.5 mg/mL of USP Miconazole Ni-
=} trate RS prepared as follows. Transfer a 10.0-mL aliquot Midazolam
of a solution containing 500 g/mL of USP Miconazole
Nitrate RS in methanol to a test tube, and evaporate on HO
a steam bath to dryness with the aid of a current of
filtered air. Dissolve the residue in 2.0 mL of Internal NA NG
standard solution.
Sample stock solution: Nominally 2.5 mg/mL of
Ay
miconazole nitrate prepared as follows. Transfer 1 Sup- mA eae
pository to a stoppered, 50-mL centrifuge tube. Add
30 mL of pentane, and shake by mechanical means for Ne
20 min to dissolve the suppository base and to disperse
the miconazole nitrate. Centrifuge to obtain a clear su- CigHisClFN3 325.77
pernatant. Aspirate, and discard the clear liquid. Wash 4-H-Imidazo[1 ,5-a][1,4]benzodiazepine, 8-chloro-
the residue with three 20-mL portions of pentane, shak- 6-(2-fluorophenyl)-1-methyl;
ing, centrifuging, and aspiating in the same manner. 8-Chloro-6-(0-fluorophenyl)-1-methyl-4H-imidazo[1,5-a]
Discard the pentane washings. Evaporate the residual [1,4]benzodiazepine [59467-70-8].
pentane from the residue with the aid of a current of
filtered air. Using small portions of methanol, transfer DEFINITION
the residue to a 100-mL volumetric flask. Dissolve in Midazolam contains NLT 98.5% and NMT 101.5% of
and dilute with methanol to volume. CigHi3CIFN3, calculated on the dried basis.
Sample solution: Transfer an aliquot containing nomi-
nally the equivalent to 5 mg of miconazole nitrate from IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
the Sample stock solution to a suitable container, and e B. The retention time of the major peak of the Sample
evaporate on a steam bath to dryness with the aid of a
current of filtered air. Dissolve the residue in 2.0 mL of solution corresponds to that of the Standard solution, as
obtained in the Assay.
Internal standard solution.
Chromatographic system
(See Chromatography (621), System Suitability.)
USP 41 Official Monographs / Midazolam 2741

ASSAY Acceptance criteria: See Impurity Table 1.

© PROCEDURE
Buffer: 7.7 g/L of ammonium acetate in water. Adjust Impurity Table 1
with glacial acetic acid to a pH of 5.5 + 0.1.
Mobile phase: Acetonitrile and Buffer (1:2) Relative Relative Acceptance
Standard solution: 0.04 mg/mL of USP Midazolam RS Retention Response Criteria,
in Mobile phase Name Time Factor NMT (%'
Sample solution: 0.04 mg/mL of Midazolam in Mobile Reduced midazo- 0.20 1.0 0.1
Pi lame
hromatographic system Reduced reduced 0.24 1.0 0.1
(See Chromatography (621), System Suitability.) midazolam?
Mode: LC Amino compound« 0.25 0.5 0.1
Detector: UV 254 nm Oxide midazolam¢ 0.46 1.3 0.1
Column: 4.6-mm x 25-cm; 5-11m packing L60
Flow rate: 1.5 mL/min Nitromethylene 0.76 1.0 0.1
Injection size: 25 uL compound:
System suitability Dihydromidazolam! 0.83 0.5 0.1
Sample: Standard solution Midazolam 1.0 — =
Suitability requirements Desfluoromidazo- 1.14 1.0 0.2
Column efficiency: NLT 10,000 theoretical plates lams
Tailing factor: NMT 2.0 6H-isomerh 2.48 0.7 0.1
Relative standard deviation: NMT 2.0% Unknown impurity = 1.0 0.1
Analysis
Total impurities — =— 05 |
Samples: Standard solution and Sample solution
Calculate the percentage of CisHi3CIFN3 in the portion 2 8-Chloro-3a,4-dihydro-6-(2-fluorophenyl)-1 -methyl-3H-imidazo[1,5-a][1,
4)-benzodiazepine.
of Midazolam taken:
» 8-Chloro-6-(2-fluorophenyl)-3a,4,5,6-tetrahydro-1-methyl-3H-imidazo[1,
5-a][1,4]-benzodiazepine.
Result = (ru/rs) x (Cs/Cy) x 100 ¢ 2-Aminomethyl-7-chloro-2, 3-dihydro-5-(2-fluorophenyl)-1 H-1,4-benzodi-
azepine.
tu = peak response from the Sample solution 4 8-Chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]-benzodiaze-
rs peak response from the Standard solution pine-S-oxide.
Wot

Cs concentration of USP Midazolam RS in the ¢ 7-Chloro-1,3-dihydro-2-nitromethylene-5-(2-fluorophenyl)-2H-1,4-benzo-

Standard solution (mg/mL) diazepine-4-oxide.
Cu = concentration of Midazolam in the Sample f 8-Chloro-6-(2-fluorophenyl)-5,6-dihydro-1-methy|-4H-imidazo[1,5-a][1,4]-
solution (mg/mL) benzodiazepine.
Acceptance criteria: 98.5%-101.5% on the dried basis 3 8-Chloro-6-phenyl-1-methyl-4H-imidazo-[1,5-a][1,4]-benzodiazepine.
» 8-Chloro-6-(2-fluorophenyl)-1 -methyl-6H-imidazo[1,5-a][1,4]-benzodiaze-
pine.
IMPURITIES
Inorganic Impurities
fos
SPECIFIC TESTS . 4)
e RESIDUE ON IGNITION (281): NMT 0.1% e Loss ON DryING (731): Dry a sample at 105° for 2 h: it a)
Organic Impurities loses NMT 0.5% of its weight. K
© PROCEDURE i}
Buffer, Mobile phase, Standard solution, and Chro- ADDITIONAL REQUIREMENTS =
matographic system: Proceed as directed in the ¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant 3
Assay. containers.
ro}-
»
Sensitivity check solution: Dilute the Standard solution © USP REFERENCE STANDARDS (11) a}
with Mobile phase to obtain a 0.2-11g/mL solution. USP Midazolam RS a
aap solution: 0.2 mg/mL of Midazolam in Mobile as

phase
System suitability
Samples: Standard solution and Sensitivity check
solution Midazolam Injection
Suitability requirements
Column efficiency: NLT 10,000 theoretical plates, DEFINITION
Standard solution Midazolam Injection is a sterile solution of Midazolam Hy-
Tailing factor: NMT 2.0, Standard solution drochloride in Water for Injection or of Midazolam in
Relative standard deviation: NMT 2.0%, Standard Water for Injection prepared with the aid of Hydrochloric
solution Acid. It contains the equivalent of NLT 90.0% and NMT
Peak ratio: The ratio of the area of the midazolam 110.0% of the labeled amount of midazolam
peak of the Standard solution to the area of the (CisHi3ClFNs). It may contain Sodium Chloride, Benzyl Al-
midazolam peak of the Sensitivity check solution cohol, and/or a chelating agent.
should be within 160-240.
Analysis IDENTIFICATION
Sample: Sample solution e The retention time of the major peak of the Sample solu-
Calculate the percentage of each impurity in the por- tion goesponds to that of the Standard solution, as ob-
tion of Midazolam taken: tained in the Assay.
Result = (ru/F)/[2(ru/F) + rr] x 100 ASSAY
[NoTe—Protect all prepared Standard and sample solutions
tu = peak response of each individual impurity from light.]
from the Sample solution e PROCEDURE
tr = peak response of Midazolam from the Sample Buffer: 6.7 g/L of dibasic sodium phosphate
solution heptahydrate in water. Adjust with phosphoric acid to a
F = relative response factor (see Impurity Table 1) pH of 5.0 + 0.1.
 2742 Midazolam / Official Monographs USP 41

Solution A: Preparea filtered and degassed mixture of System suitability

acetonitrile, methanol and Buffer (8:3:9). Samples: Standard solution and Control solution
Solution B: Acetonitrile and Buffer (3:1) Suitability requirements
Mobile phase: See the gradient table below. Tailing factor: NMT 2.5 for midazolam peak, Stan-
dard solution
Time Solution A Solution B Column efficiency: NLT 5500 theoretical plates,
(min) (%) (%) Standard solution
Signal-to-noise ratio: NLT 10, Control solution
0 100 0
Relative standard deviation: NMT 8.0%, Standard
15 100 0 solution
20 0 100 Analysis
35 0 100 Samples: Standard solution and Sample solution
37 100 0 Calculate the percentage of each impurity in the por-
45 100 0 tion of Injection taken:

Standard solution: Dissolve USP Midazolam RS in
about 2 mL of methanol, and dilute quantitatively, and
stepwise if necessary, with Solution A to obtain a 0.2-
mg/mL solution.
Sample solution: [NotE—The midazolam present in the
Injection converts from the open-ring form to the
closed-ring form when diluted with Solution A. The
midazolam potency is determined based on the peak
area of the closed-ring form. It takes approximately 60
min at 40° or 2-3 h at room temperature to complete
the conversion. The Standard solution is not subject to
this conversion process.] Transfer a volume of Injection
to a suitable volumetric flask, and dilute with Solution A
to obtain a solution containing about 0.2 mg/mL of
midazolam. Transfer the resulting solution into suitable
crimp top vials, seal tightly, and heat at about 40° for
60 min. Allow this solution to cool to room tempera-
ture before injection.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
fm
“
Column: 4.6-mm x 25-cm; packing L1
os Flow rate: 1.0 mL/min
it Injection size: 50 uL
4 Mobile phase: Acetonitrile and Buffer (7:13)
a System suitability
° Sample: Standard solution
a Midazolam RS and 0.5 mg/mL of USP Benzyl Alcohol RS
5 Suitability requirements
= Column efficiency: NLT 5500 theoretical plates
qa
Tailing factor: NMT 2.5
a) Relative standard deviation: NMT 2.0%
= Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of labeled amount of
CisHi3CIFN3 in the portion of Injection taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response from the Sample solution
rs = peak response from the Standard solution
Cs = concentration of USP Midazolam RS in the
Standard solution (mg/mL)
Cu = nominal concentration of Midazolam in the
Sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0%
IMPURITIES
Organic impurities
[Note—Protect all prepared Standard and sample solutions
from light.]
e PROCEDURE
Buffer, Solution A, Solution B, Mobile phase, Sample
solution, and Chromatographic system: Proceed as
directed in the Assay.
Standard stock solution: Use Standard solution in the
Assay.
Standatd solution: 0.5 g/mL USP Midazolam RS in
Solution A from Standard stock solution
Control solution: 0.1 ug/mL USP Midazolam RS in So-
lution A from Standard solution
Result = (ru/rs) * (Cs/Cu) x (1/F) x 100
Ty = peak response of the individual impurity from
the Sample solution
Ts = peak response of midazolam from the
Standard solution
Cs = concentration of USP Midazolam RS in the
Standard solution (mg/mL)
Cu = nominal concentration of Midazolam in the
Sample solution (mg/mL)
F = relative response factor; 0.51 for the peak
eluting at a relative retention between 0.79
and 0.97 with respect to midazolam; 1.0 for
all other peaks
Acceptance criteria
Individual known impurity: NMT 0.5%
Individual unknown impurity: NMT 0.1%
Total impurities: NMT 1.0%
[Note—Disregard all solvent- and excipient-related
peaks.]
SPECIFIC TESTS
e BENZYL ALCOHOL CONTENT (if present)
Buffer: 3.4 g/L of monobasic sodium phosphate in
water. Adjust with phosphoric acid to a pH of 3.5.
System suitability solution: 0.05 mg/mL of USP
in Mobile phase
Standard solution: 0.5 mg/mL of USP Benzyl Alcohol
RS in Mobile phase
Sample solution: Transfer a measured volume of Injec-
tion to a suitable volumetric flask. Dilute with Mobile
phase to obtain a concentration of about 0.5 mg/mL of
enzyl alcohol, based on the labeled content of benzyl
alcohol in the Injection.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; L1 packing
Flow rate: 1.0 mL/min
Injection size: 50 uL
System suitability
Sample: System suitability solution
Suitability requirements
Resolution: NLT 6.0 between benzyl alcohol and
midazolam
Tailing factor: NMT 2.0 for benzyl alcohol
Relative standard deviation: NMT 2.0% for benzyl
alcohol
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
benzyl alcohol in the volume of Injection taken:
Result = (ru/rs) x (Cs/Cu) x 100
Tu = peak response of benzyl alcohol from the
Sample solution
USP 41 Official Monographs / Midodrine 2743

Is = peak response of benzyl alcohol from the Chromatographic system

Standard solution (See Chromatography (621), System Suitability.)
Cs = concentration of USP Benzyl Alcohol RS in the Mode: LC
Standard solution (mg/mL) Detector: UV 290 nm
Cu = nominal concentration of benzyl alcohol in the Column: 4.6-mm x 15-cm; 5-uum packing L1
Sample solution (mg/mL) Flow rate: 1 mL/min
Acceptance criteria: The content of benzyl alcohol Injection size: 20 uL
meets the requirements in Injections and Implanted Drug System suitability
Products (1), Specific Tests, Vehicles and added Sample: Standard solution
substances. Suitability requirements
PARTICULATE MATTER IN INJECTIONS (788): Meets the re- Column efficiency: NLT 3000 theoretical plates
quirements for small-volume injections Tailing factor: NMT 2.0
BACTERIAL ENDOTOXINS TEST (85): It contains NMT 8.33 Relative standard deviation: NMT 2.0%
USP Endotoxin Units/mg of midazolam. Analysis
PH (791): 2.5-3.7 Samples: Standard solution and Sample solution
STERILITY TESTS (71): Meets the requirements Calculate the perce rage of Ci2HigN2O, - HCI in the por-
OTHER REQUIREMENTS: It meets the requirements for /njec- tion of Midodrine Hydrochloride taken:
tions and Implanted Drug Products (1).
Result = (ru/rs) x (Cs/Cu) x 100
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in single-dose contain- Tu = peak response of midodrine from the Sample
ers, preferably of Type 1 glass. Store between 15° and solution
30°. Is = peak response of midodrine from the Standard
e LABELING: Label to indicate the vehicle used and the solution
names and concentrations of any added preservatives. In- Cs = concentration of USP Midodrine Hydrochloride
dicate if the product is preservative free. RS in the Standard solution (mg/mL)
Cu = concentration of Midodrine Hydrochloride in
the Sample solution (mg/mL)
Change to read: Acceptance criteria: 98.0%-102.0% on the anhydrous
basis
e USP REFERENCE STANDARDS (11)
YsP Benzyl Alcohol RS IMPURITIES
@ (CN 1-May-2018) Inorganic Impurities
USP Midazolam RS e RESIDUE ON IGNITION (281): NMT 0.2%. A 1-g sample is
used.

Delete the following:

=
Midodrine Hydrochloride ®e HEAVY METALS, Method I! (231): NMT 10 ppme coffcial1-
a)
v
on Jan-2018)
E
4 Organic Impurities
i}
© PROCEDURE
wu A.och, 8J Buffer and Mobile phase: Proceed as directed in the
=]
}
i}=
Assay.
Standard solution: 1.0 g/mL of USP Midodrine Hy- i}
Ci2HigN20, - HCI 290.74 3
Acetamide, 2-amino-N-[2-(2,5-dimethoxyphenyl)-2-hydroxy-
drochloride RS and 2.0 g/mL of USP Midodrine Re-
lated Compound A RS in Mobile phase
=z
“
ethyl]-, monohydrochloride, (+)-; Sample solution: 1.0 mg/mL of Midodrine Hydrochlo-
(4)-2-Amino-N-(B-hydroxy-2,5-dimethoxyphenethyl)acet- tide in Mobile phase
amide monohydrochloride [3092-17-9]. Chromatographic system: Proceed as directed in the
Assay except for the following:
DEFINITION Injection size: 50 uL
Midodrine Hydrochloride contains NLT 98.0% and NMT System suey
102.0% of midodrine hydrochloride (Ci2HisN2O, - HCl), Sample: Standard solution
calculated on the aeiiriaen basis. Suitability requirements
IDENTIFICATION Resolution: NLT 2.0 between midodrine hydrochlo-
© A. INFRARED ABSORPTION (197K) tide and midodrine hydrochloride related compound
e B. The retention time of the major peak of the Sample A
solution corresponds to that of the Standard solution, as Tailing factor: NMT 2.0 for midodrine
obtained in the Assay. hydrochloride
© C. IDENTIFICATION TESTS—GENERAL, Chloride (191): A Relative standard deviation: NMT 2.0% for both
10 mg/mL solution of Midodrine Hydrochloride in water midodrine hydrochloride and midodrine related
meets the requirements. compound A
Analysis
ASSAY Samples: Standard solution and Sample solution
© PROCEDURE Calculate thepercentage of midodrine related com-
Buffer: 13.6 g/L of monobasic potassium phosphate. peutd A in the portion of Midodrine Hydrochloride
Adjust with phosphoric acid to a pH of 4.00 + 0.05. taken:
Mobile phase: Acetonitrile and Buffer (3:22)
Standard solution: 0.05 mg/mL of USP Midodrine Hy- Result = (ru/rs) x (Cs/Cu) x 100
drochloride RS in Mobile phase
Sample solution: 0.05 mg/mL of Midodrine Hydrochlo- tu = peak response of midodrine related
ride in Mobile phase compound A from the Sample solution
ts = peak response of midodrine related
compound A from the Standard solution
 2744 Midodrine / Official Monographs USP 41

Cs = concentration of USP Midodrine Related ASSAY

Compound A RS in the Standard solution e PROCEDURE
(mg/mL) Buffer: 13.6 g/L of monobasic potassium phosphate.
Cy = concentration of Midodrine Hydrochloride in Adjust with phosphoric acid to a pH of 4.00 + 0.05.
the Sample solution (mg/mL) Mobile phase: Acetonitrile and Buffer (3:22)
Calculate the percentage of any individual impurity in Standard solution: 0.05 mg/mL of USP Midodrine Hy-
the portion of Midodrine Hydrochloride taken: drochloride RS in Mobile phase
Sample solution: 0.05 mg/mL of midodrine hydrochlo-
Result = (ru/ts) x (Cs/Cu) x 100 ride in Mobile phase from NLT 5 Tablets (for 10-mg Tab-
let strength) or NLT 10 Tablets (for 5-mg and 2.5-mg
tu = peak response of each impurity from the Tablet strength). Initially add Mobile phase up to 80%
Sample solution of the volume of the flask. Sonicate for 10 min, stir for
Is = peak response of midodrine from the 15 min, and then dilute to volume, mix, and let stand
Standard solution for 10 min. Pass through a suitable PVDF filter of 0.45-
Cs = concentration of USP Midodrine lum pore size, and discard the first 5 mL.
Hydrochloride RS in the Standard solution Chromatographic system
(mg/mL) (See Chromatography (621), System Suitability.)
Cy = concentration of Midodrine Hydrochloride in Mode: LC
the Sample solution (mg/mL) Detector: UV 290 nm
Acceptance criteria: See Impurity Table 1. Column: 4.6-mm x 15-cm; 5-um packing L1
Flow rate: 1.0 mL/min
Impurity Table 1 Injection size: 20 uL
; System suitability
wees Ascpeance Sample: Standard solution
Name arene on Nea(se Suitability requirements
- me (°%) Column efficiency: NLT 3000 theoretical plates
Midodrine related compound 08 0.2% Tailing factor: NMT 2.0
A Relative standard deviation: NMT 2.0%
Midodrine hydrochloride 1 = Analysis
Individual unspecified impurity - 0.1% Samples: Standard solution and Sample solution
Total impurities = 0.5% Calculate the percentage of the labeled amount of
@ 1-(2,5 Dimethoxypheny!)-2-aminoethanol. eee eee vores (CizHiaN20s - HCl) in the por-

SPECIFIC TESTS
o WATER DETERMINATION, Method | (921): NMT 0.5% Result = (ru/ts) x (Cs/Cu) x 100
e PH (791): 4.0-5.0. Use 50 mg/mL of the midodrine hy-
drochloride sample. Tu = peak response from the Sample solution
% rs = peak response from the Standard solution
Wot ADDITIONAL REQUIREMENTS Cs = concentration of the Standard solution
J e PACKAGING AND STORAGE: Preserve in tight containers and (mg/mL)
a store at room temperature. Cu = nominal concentration of the Sample solution
° e USP REFERENCE STANDARDS (11) (mg/mL)
= USP Midodrine Hysieshlotice RS 4 Acceptance criteria: 90.0%-105.0%
USP Midodrine Related Compound A RS
2 1-(2,5Dinethoxyphenyie2 aniioethandl PERFORMANCE TESTS
a CioHisNO3 197.23 e DISSOLUTION (711)
5 Medium: 0.1 N HCl; 900 mL, deaerated
Apparatus 2: 50 rpm
Time: 15 min
Buffer: Proceed as directed in the Assay.
Mobile phase: Acetonitrile and Buffer (3:17)
Midodrine Hydrochloride Tablets Standard solution: L/900 mg/mL of USP Midodrine Hy-
drochloride RS in Medium, whereLis the label claim in
DEFINITION mg/Tablet
Midodrine Hydrochloride Tablets contain NLT 90.0% and Sample solution: Pass a portion of the solution under
NMT 105.0% of the labeled amount of Midodrine Hydro- test through a suitable filter of 45-um pore size.
chloride (Ci2H1gN2O4 - HCl). Chromatographic system
IDENTIFICATION (See Chromatography (621), System Suitability.)
e A. INFRARED ABSORPTION (197K) Mode: LC
Sample specimen: Weigh a quantity, from finely pow- Detector: UV 290 nm
dered Tablets (NLT 20), equivalent to 15 mg of Column: 4.6-mm x 15-cm; 5-um packing L1
midodrine hydrochloride, into a 50-mL disposable cen- Flow rate: 1.0 mL/min
trifuge tube. Add 20 mL of water, and stir for 2 min Injection size: 50 pL
using a vortex mixer. Pass the mixture through filter System suitability
paper into a 50-mL beaker, and boil it until about 2 mL Sample: Standard solution
of the solution is left. Evaporate the final solution in an Suitability requirements
oven at 105° for 1 h. Column efficiency: NLT 2000 theoretical plates
e B. The retention time of the major peak of the Sample Tailing factor: NMT 2.0
solution corresponds to that of the Standard solution, as Relative standard deviation: NMT 2.0%
obtained in the Assay. Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of midodrine hydrochloride
dissolved:
Result = (ru/ts) x (Cs/L) x V x 100
USP 41 Official Monographs / Milbemycin 2745

tu = peak area from the Sample solution Ts = peak response of midodrine from the
Ts = peak area from the Standard solution Standard solution
Cs = concentration of the Standard solution Cs = concentration of USP Midodrine
(mg/mL) Hydrochloride RS in the Standard solution
L = label claim (mg/Tablet) (ug/ml) * the
Vv = volume of Medium, 900 mL Cu = nominal concentration of midodrine
Tolerances: NLT 80% (Q) of the labeled amount of hydrochloride in the Sample solution (ug/mL)
midodrine hydrochloride is dissolved. Acceptance criteria
e UNIFORMITY OF DOSAGE UNITS (905): Meet the Individual impurities: NMT 0.5% of midodrine re-
requirements lated compound A; NMT 0.2% of any other individ-
ual impurity
IMPURITIES Total impurities: NMT 1.0%
Organic Impurities
¢ PROCEDURE ADDITIONAL REQUIREMENTS
Buffer and Mobile phase: Proceed as directed in the © PACKAGING AND STORAGE: Preserve in well-closed
Assay. containers.
Standard stock solution 1: 25 g/mL of USP e USP REFERENCE STANDARDS (11)
Midodrine Hydrochloride RS in Mobile phase USP Midodrine Hydrochloride RS
Standard stock solution 2: 25 g/mL of USP USP Midodrine Related Compound A RS
Midodrine Related Compound A RS in Mobile phase 1-(2,5 Dimethoxyphenyl)-2-aminoethanol.
Standard solution: 1.25 g/mL each of USP Midodrine CioHisNO3 197.23
Hydrochloride RS and USP Midodrine Related Com-
pound A RS in Mobile phase from Standard stock solu-
tion 1 and Standard stock solution 2
Sample solution: 0.25 mg/mL in Mobile phase from
NLT 5 Tablets (for 10-mg Tablet strength) and NLT 10 Milbemycin Oxime
Tablets (for 5-mg and 2.5-mg Tablet strength). Initially
add Mobile phase to about 80% of the volume of the
flask. Sonicate for 10 min, stir for 15 min, and then
iE“we 7
Yar
dilute to volume. Pass through a suitable PVDF filter of
0.45-um pore size, and discard the first 5 mL. yc | Y
Chromatographic system OeSy AS Re oH
(See Chromatography (621), System Suitability.) oy
Proceed as directed in the Assay except for the re
} ia ] A, Re CMe
following:
Injection volume: 40 uL Om; 1 “city
System sultan N
HO
Sample: Standard solution jess
Suitability requirements Mixture of milbemycin A3 oxime and milbemycin A, oxime 4)
{Nott—The relative retention times for midodrine re- [129496-10-2]. z
lated compound A and midrodrine hydrochloride Milbemycin Az Oxime =
are 0.83 and 1, respectively.] i}
Resolution: NLT 2.0 between midodrine hydrochlo- C3) Ha3NO7 541.68 =
Milbemycin B, 5-O-demethyl-28-deoxy-25-methyl-6,28-ep- }
tide and midodrine related compound A
oxy-23-hydroxyimino-, [6R,235,255()|-; (2a,4E,5’S,6R, to}2
Column efficiency: NLT 2000 theoretical plates for
68861 1R,13R,15S,17aR,200R, 20B5)-3’,4’,5’,6,6',7,10, 2
the midodrine peak
11,14,15,170,20,200,20B-tetradecahydro-20B-hydroxy-5’, a]
Tailing factor: NMT 2.0 for the midodrine peak >
Relative standard deviation: NMT 2.0% for the 6’,6,8,19-pentamethylspiro[11,15-methano-2H,13H,17H- a)

midodrine peak furo[4, 3,2-pq][2,6]benzodioxacyclooctadecin-1 3,2’-

Analysis
Samples: Standard solution and Sample solution
Calculate the peerage of midodrine related com-
poundAin the portion of Tablets taken:
Result = (ru/ts) x (Cs/Cu) x 100
tu = peak response of midodrine related
compound A from the Sample solution
Is = peak response of midodrine related
compound A from the Standard solution
Cs = concentration of USP Midodrine Related
Compound A RS in the Standard solution
Cu
(ug/ml) , aris
= nominal concentration of midodrine
hydrochloride in the Sample solution (ug/mL)
Calculate the percentage of any other unknown
impurity in the portion of Tablets taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response of any other unknown impurity
from the Sample solution
[2H]pyran]-17-one 20-oxime.
Milbemycin A, Oxime
C32HasNO7 555.70
Milbemycin B, 5-O-demethyl-28-deoxy-25-ethyl-6,28-epoxy-
23-hydroxyimino-, [6R,235,255S(E)]-; (20£,4E,5’S,6R,6'S,8E,
11R,13R,155S,17a0R,200R,20BS)-6’-ethyl-3’,4’,5’,6,6’,7,10,
11,14,15,17c,20,20a,20B-tetradecahydro-20B-hydroxy-5’,
6,8,19-tetramethylspiro[11,15-methano-2H,13H,17H-furo
[4,3,2-pq][2,6]benzodioxacyclooctadecin-1 3,2’-[2H]pyran]-
17-one 20-oxime.
DEFINITION
Milbemycin Oxime is a mixture of milbemycin A, oxime and
milbemycin A3 oxime. It contains NLT 95.0% and NMT
102.0% of the sum of milbemycin A, oxime and
milbemycin A3 oxime, calculated on the anhydrous basis.
The ratio of milbemycin A, oxime is NLT 0.75.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
e B. The retention times of the major peaks of the Sample
solution correspond to those of the Standard solution, as
obtained in the Assay.
 2746 Milbemycin / Official Monographs USP 41

ASSAY ru = peak response of milbemycin As from the

e PROCEDURE Sample solution
Solution A: Water rs = peak response of milbemycin A, from the
Solution B: Methanol Standard solution
Mobile phase: See Table 1. Cs = concentration of USP Milbemycin Oxime RS in
the Standard solution (mg/mL)
Table 1 Fag = potency of milbemycin A, oxime in USP
Milbemycin Oxime RS (mg/mg)
Solution A Solution B Cu = concentration of Milbemycin Oxime in the
Sample solution (mg/mL)
26 Calculate the percentage of the sum of milbemycin A3
26 oxime and milbemycin A, oxime in the portion of
24 Milbemycin Oxime taken:
47
Result = Pa3 + Pas
47.1 4
26 74 Pas = percentage of milbemycin A3 oxime
Pag = percentage of milbemycin A, oxime
Diluted phosphoric acid solution: 0.3-0.5 mL/L of Calculate the ratio of milbemycin A3 oxime in the
phosphoric acid in water portion of Milbemycin Oxime taken:
Diluent: Acetonitrile and Diluted phosphoric acid solution
(75:25) Result = Pa3/(Pas + Paa)
Standard solution: 0.2 mg/mL of USP Milbemycin Ox-
ime RS in Diluent. Sonicate if necessary to facilitate Pas = percentage of milbemycin A3 oxime
dissolution. Pas = percentage of milbemycin A, oxime
Sample solution: 0.2 mg/mL of Milbemycin Oxime in Calculate the ratio of milbemycin A, oxime in the
Diluent. Sonicate if necessary to facilitate dissolution. portion of Milbemycin Oxime taken:
This solution must be stored at 5° protected from light.
Chromatographic system Result = Pas/(Paz + Pas)
(See Chromatography (621), System Suitability.)
Mode: LC Psa = percentage of milbemycin A, oxime
Detector: UV 240 nm Ps3 = percentage of milbemycin A3 oxime
Column: 3.0-mm x 10-cm; 3-um packing L1 Acceptance criteria
Column temperature: 35° Sum of milbemycin A, oxime and milbemycin A3
Flow rate: 0.5 mL/min oxime: 95.0%-102.0% on the anhydrous basis
Injection volume: 10 uL Ratio of milbemycin A, oxime: NLT 0.75
System suitability
Sample: Standard solution IMPURITIES
ca
nw
[Note—The elution order is milbemycin A3 oxime fol-
© RESIDUE ON IGNITION (281): NMT 0.5%
rs lowed by milbemycin Ay oxime. (See Table 2 for the © ORGANIC IMPURITIES
Kd relative retention times.)] Mobile phase, Diluent, Standard solution, Sample so-
io) lution, Chromatographic system, and System suitabil-
i} Suitability requirements
ity: Proceed as directed in the Assay.
f= Resolution: NLT 9 between milbemycin A3 oxime
5 and milbemycin A, oxime
Diluted standard solution: 0.002 mg/mL of USP
= Tailing factor: NMT 2.0 each for milbemycin A3 ox- Milbemycin Oxime RS in Diluent from Standard solution
Ps ime and milbemycin Ay oxime Analysis
a) Relative standard deviation: NMT 0.73% each for Samples: Sample solution and Diluted standard solution
= milbemycin A3 oxime and milbemycin A, oxime Calculate the percentage of each impurity in the por-
Analysis tion of Milbemycin Oxime taken:
Samples: Standard solution and Sample solution Result = (ru/r7) x (Cs/Cu) x 100
Calculate the percentage of milbemycin A3 oxime
(CsiH43NO7) in the portion of Milbemycin Oxime tu = peak response of each impurity from the
taken: Sample solution
rr = sum of the peak responses of milbemycin A3
Result = (ru/rs) x [(Cs x Faz)/Cu] x 100 oxime and milbemycin A, oxime from the
tu = peak response of milbemycin A3 from the Diluted standard solution
Sample solution Cs = concentration of USP Milbemycin Oxime RS in
rs = peak response of milbemycin A3 from the the Diluted standard solution (mg/mL)
Standard solution Cu = concentration of Milbemycin Oxime in the
Cs = concentration of USP Milbemycin Oxime RS in Sample solution (mg/mL)
the Standard solution (mg/mL) Acceptance criteria: See Table 2. The reporting level
Fa3 = potency of milbemycin A3 oxime in USP
for impurities is 0.10%.
Milbemycin Oxime RS (mg/mg)
G = concentration of Milbemycin Oxime in the
Sample solution (mg/mL)
Calculate the percentage of milbemycin Ay oxime
(C32H4sNO7) in the portion of Milbemycin Oxime
taken:
Result = (ru/rs) < [(Cs x Faa)/Cu] x 100
USP 41 Official Monographs / Milrinone 2747

Table 2 The solution should become nearly transparent after ad-

Relative Acceptance
justment. Dilute with water to 1 L.
Mobile phase: Methanol, Buffer, and water
Retention Criteria,
Name Time NMT (%)
(320:40:640)
Diluent: Methanol, water, and lactic acid
Milbemycin Az oxime 1.00 =—
(320: 679: 1.2)
11’-Desmethylmilbemycin As Standard*solution: 0.1 mg/mL of USP Milrinone RS in
oxime? 1.17 0.7 Diluent. Sonicate until dissolved.
(20’R)-Hydroxymilbemycin Ay Sample solution: 0.1 mg/mL of Milrinone in Diluent.
keto form? 1:33) 0.5 Sonicate until dissolved.
Milbemycin A oxime 1.43 = Chromatographic system
Milbemycin As keto forme 1.51 0.7 (See Chromatography (621), System Suitability.)
Milbemycin D oxime? 2.18 3.0
Mode: LC
Detector: UV 268 nm
Any other individual impurity = 0.5 Column: 4.6-mm x 25-cm; 5-4um packing L1
Total impurities (excluding 4 Flow rate: 1 mL/min
milbemycin D oxime) 3:5 Injection volume: 20 LL
4(1/R,2R,4’S,5S,6R,8'R,10’E,1 3’R,14’E,16’E,20’R,21'Z,24’S)-6-Ethyl-24’-hy- System suitability
droxy-21’-(hydroxyimino)-5,13’,22’-trimethyl-3,4,5,6-tetrahydrospiro[py- Sample: Standard solution
ran-2,6'-[3,7,19]trioxatetracyclo[15.6.1.148.0224]pentacosa[10,14,16,
22]tetraene]-2’-one. Suitability requirements
©(1’R,2R,4’S,5S,6R,8'R,1 0°E,13’R,14’E, 16’E,20°R,21'Z,24’S)-6-Ethy|-20’,24’- Tailing factor: NMT 2.0
dihydroxy-21’-(hydroxyimino)-5,11’,13’,22’-tetramethyl-3,4,5,6-te- Relative standard deviation: NMT 0.73%
bahydrospirelpyran:s.6-13.7;) 9]trioxatetracyclo[15.6.1 148.020,24] Analysis
pentacosa[10,14,16,22]tetraene]-2’-one.
Samples: Standard solution and Sample solution
£(1'R,2R,4’S,55,6R,8'R, 10°E,13'R,14'E,1 6°E,20°S,24’S)-6-Ethyl-24’- ‘droxy-5, Calculate the percentage of milrinone (Ci2H9N30) in the
11’,13’,22’-tetramethyl-3,4,5,6-tetrahydrospiro[pyran-2,6' 3,7, 194trioxate
tracyclo[15.6.1.148.02°24]pentacosa[10,14,16,22]tetraene]-2’,21’-dione. portion of Milrinone taken:
4 (1’R,2R,4’S,5S,6R,8'R,10°E,1 3’R, 14’E, 16E,20'R,21’Z,24’S)-24’-Hydroxy-21’-
(hydroxyimino)-6-(propan-2-yl)-5,11’,1 3’,22’-tetramethyl-3,4,5,6-te- Result = (ru/rs) x (Cs/Cu) x 100
trahydrospiro[pyran-2,6’-[3,7,19]trioxatetracyclo[15.6.1.148.020.24]
pentacosa[10,14,16,22]tetraene]-2’-one. ru = peak response of milrinone from the Sample
SPECIFIC TESTS solution
© WATER DETERMINATION (921), Method |: NMT 3.0% rs = peak response of milrinone from the Standard
solution
ADDITIONAL REQUIREMENTS Cs = concentration of USP Milrinone RS in the
© PACKAGING AND STORAGE: Preserve in tight containers. Standard solution (mg/mL)
Store at room temperature, protected from light. Cu = concentration of Milrinone in the Sample
° ae Label it to indicate that it is for veterinary use solution (mg/mL)
only. Acceptance criteria: 98.0%-102.0% on the anhydrous ce
e USP REFERENCE STANDARDS (11) basis a)
uv
USP Milbemycin Oxime RS
IMPURITIES cs
© RESIDUE ON IGNITION (281): NMT 0.2% iS}
|
}
Delete the following: re}<j
Milrinone 2
°e HEAVY METALS, Method If (231): NMT 20 ppme (orciat- ne}
4N.-0
x
a)
Jan-2018)
© ORGANIC IMPURITIES
ao ee Buffer: To 2.7 g of dibasic potassium phosphate in
800 mL of water add 2.4 mL of triethylamine, and ad-
just with phosphoric acid to a pH of 7.5.
Mobile phase: Acetonitrile and Buffer (200:800)
Ci2HsN30 211.22 System suitability stock solution: 0.2 mg/mL of USP
[3,4’-Bipyridine]-5-carbonitrile, 1,6-dihydro-2-methyl-6-oxo-; Milrinone Related Compound A RS in Mobile phase.
1,6-Dihydro-2-methyl-6-oxo[3,4’-bipyridine]-5-carbonitrile Heat in a water bath at approximately 80°, and/or soni-
[78415-72-2]. cate if necessary to dissolve.
Standard stock solution: 2 mg/mL of USP Milrinone RS
DEFINITION in Mobile phase. Heat in a water bath at approximately
Milrinone contains NLT 98.0% and NMT 102.0% of milri- 80°, and/or sonicate if necessary to dissolve.
none (C;2H»9N30), calculated on the anhydrous basis. System suitability solution: 10.0 mL of System suitabil-
[CauTION—Milrinone is a cardiotonic agent.] ity stock solution and 1.0 mL of Standard stock solution
IDENTIFICATION in a 100-mL volumetric flask. Dilute with Mobile phase
to volume.
e A. INFRARED ABSORPTION (197K)
Standard solution: 0.006 mg/mL of USP Milrinone RS,
e B. The retention time of the major peak of the Sample from the Standard stock solution, in Mobile phase
solution corresponds to that of the Standard solution, as Sample solution: 2 mg/mL of Milrinone in Mobile
obtained in the Assay. phase. Heat in a water bath at approximately 80°, if
ASSAY necessary to dissolve.
¢ PROCEDURE Chromatographic system
Buffer: To 72.44 g of sodium tetraborate add 900 mL (See Chromatography (621), System Suitability.)
of water. Adjust with hydrochloric acid to a pH of 6.5.
 2748 Milrinone / Official Monographs USP 41

Mode: LC ASSAY
Detector: UV 220 nm © PROCEDURE
Column: 4.6-mm x 25-cm; packing L7 Buffer: Dissolve 3.3 g of dibasic potassium phosphate
Flow rate: 1 mL/min in 1 L of water and add 3 mL of triethylamine. Adjust
Injection volume: 20 uL with phosphoric acid to a pH of 7.5.
System suitability Mobile phase: Acetonitrile and Buffer (20:80)
Sample: System suitability solution - Standard solution: 0.05 mg/mL of USP Milrinone RS in
[Note—The relative retention times for milrinone related Mobile phase. Sonication may be necessary for complete
compound A and milrinone are 0.6 and 1.0, dissolution.
respectively.] Sample solution: Nominally equivalent to 0.05 mg/mL
Suitability requirements of milrinone prepared from a volume of Injection suita-
Resolution: NLT 4.0 between milrinone related com- bly diluted with Mobile phase
pound A and milrinone Chromatographic system
Relative standard deviation: NMT 5.0% from the (See Chromatography (621), System Suitability.)
milrinone peak Mode: LC
Analysis Detector: UV 220 nm. For /dentification B, use a diode
Samples: Standard solution and Sample solution array detector in the range of 200-400 nm.
Calculate the percentage of each impurity in the por- Column: 4.6-mm x 25-cm; 5-um packing L7
tion of Milrinone taken: Flow rate: 1 mL/min
Injection volume: 20 pL
Result = (ru/rs) x (Cs/Cu) x 100 Run time: NLT 1.7 times the retention time of
milrinone
ru = peak response of each impurity from the sea suitability
Sample solution ample: Standard solution
rs = peak response of milrinone from the Standard Suitability requirements
solution Tailing factor: NMT 2.0
Cs = concentration of USP Milrinone RS in the Relative standard deviation: NMT 2.0%
Standard solution (mg/mL) Analysis
CG = concentration of Milrinone in the Sample Samples: Standard solution and Sample solution
solution (mg/mL) Calculate the percentage of the labeled amount of
Acceptance criteria milrinone (Ci2HsN3O) in the portion of Injection taken:
Any individual impurity: NMT 0.3%
Total impurities: NMT 1.0% Result = (ru/ts) x (CGs/Gy) x 100
SPECIFIC TESTS re = peak response of milrinone from the Sample
e WATER DETERMINATION, Method | (921): NMT 2.0% solution
fs = peak response of milrinone from the Standard
ADDITIONAL REQUIREMENTS solution
fe
“
© PACKAGING AND STORAGE: Preserve in tight containers,
rs Gs = concentration of USP Milrinone RS in the
and store at controlled room temperature. Standard solution (mg/mL)
S
— e USP REFERENCE STANDARDS (11)
Dd USP Milrinone RS
Cu = nominal concentration of milrinone in the
° USP Milrinone Related Compound A RS
Sample solution (mg/mL)
¢ Acceptance criteria: 90.0%-110.0%
3 1,6-Dihydro-2-methyl-6-oxo-(3,4’-bipyridine)-
= 5-carboxamide. UMPURITIES
a Ci2HiiN3O2 229.23 © ORGANIC IMPURITIES
ww Buffer and Mobile phase: Prepare as directed in the
= Assay.
System suitability solution: 0.5 ug/ml each of USP
ilrinone RS and USP Milrinone Related Compound A
Add the following: RS in Mobile phase
Standard solution: 0.5 g/mL of USP Milrinone RS in
Mobile phase
Sensitivity solution: 0.1 ug/ml of USP Milrinone RS in
4Milrinone Lactate Injection Mobile phase from Standard solution
Sample solution: Nominally en of milrinone
DEFINITION from a volume of Injection in Mobile phase
Milrinone Lactate Injection is a sterile eee solution of Chromatographic system: Proceed as directed in the
Milrinone and a suitable osmolality-adjusting substance in Assay, except for the Run times.
Water for Injection, prepared with the aid of Lactic Acid. Run times
It contains NLT 90.0% and NMT 110.0% of the labeled Standard solution: NLT 1.7 times the retention time
amount of milrinone (C;2HeN30). of milrinone
Sample solution: NLT 4 times the retention time of
IDENTIFICATION milrinone
e A. The retention time of the milrinone peak of the Sam- System suitability
ple solution corresponds to that of the Standard solution, Samples: System suitability solution, Standard solution,
as obtained in the Assay. and Sensitivity solution
e B. The UV absorption spectrum of the major peak of the Suitability requirements
Sample solution exhibits maxima and minima at the same Resolution: NLT 5.0 between milrinone and milri-
wavelengths as those of the corresponding peak of the none related compound A, System suitability solution
Standard solution, as obtained in the Assay. te standard deviation: NMT 5.0%, Standard
solution
USP 41 Official Monographs / Mineral 2749

Signal-to-noise ratio: NLT 20, Sensitivity solution ty = peak response of lactic acid from the Sample
Analysis solution
Samples: Standard solution and Sample solution ts = peak response of lactic acid from the Standard
Calculate the percentage of each impurity in the por- solution
tion of Injection taken: G = concentration of USP Sodium Lactate RS in
the Standard solution (mg/mL)
Result = (ru/rs) x (Cs/Cu) x (Ma/Mr2) x 100 Cu = nominal concentration of milrinone in the
Sample solution friaimt)
ty = peak response of each impurity from the Mn = molecular weight of lactic acid, 90.08
Sample solution Mz = molecular weight of sodium lactate, 112.06
ts = peak response of milrinone from the Standard Acceptance criteria; 95.0%-129.0%
solution ¢ BACTERIAL ENDOTOXINS TEST (85): NMT 25 USP Endotoxin
G = concentration of USP Milrinone RS in the Seemilrinone
Standard solution (g/mL) © STERILITY TESTS (71): Meets the requirements
Cy — = nominal concentration of milrinone in the © PH (791): 3.2-4.0
Sample solution (ug/mL) @ PARTICULATE MATTER IN INJECTIONS (788): Meets the re-
M, — = molecular weight of milrinone free base, quirements for small-volume injections
211.22 © OTHER REQUIREMENTS: Meets the requirements in /njec-
M2 = molecular weight of milrinone lactate, 151.16 tions and Implanted Drug Products (1)
Acceptance criteria: See Table 1. Disregard peaks be-
low 0.01%. ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in single-dose contain-
Table 1 ers. Store at controlled room temperature.
e LABELING: Label it to indicate that it is to be suitably
Relative Acceptance diluted prior to administration.
Retention Criteria, e USP REFERENCE STANDARDS (11)
Name Time NMT_(%) USP Milrinone RS
Milrinone related USP Milrinone Related Compound A RS
compound As 0.6 = 1,6-Dihydro-2-methyl-6-0x0(3,4’-bipyridine)-
Milrinone 1.0 = 5-carboxamide.
Any unspecified GizHiwN302 229.24
degradation product = 0.20 Vy thas RS
Total impurities? = 0.5 odium 2-hydroxypropanoate.
CsHsNaO3 1 12.06
2Process-related impurity; 1,6-Dihydro-2-methy!-6-ox0(3,4’-bipyridine)-5-
carboxamide. AUSPST
»Total impurities include both process-related and degradation products.

SPECIFIC TESTS Cc
¢ CONTENT OF LACTIC ACID 4)
a]
Mobile phase: Water adjusted with phosphoric acid to Mineral Oil
a pH of 2.1 =
Standard solution: 0.2 mg/mL of USP Sodium Lactate DEFINITION iS
RS in Mobile phase =
Mineral Oil is a purified mixture of liquid hydrocarbons ob- }
Sample solution: Nominally equivalent to 0.2 mg/mL tained from petroleum. It may contain a suitable a=
of milrinone prepared as follows. Transfer a suitable vol- stabilizer. ES)
ume of Injection into a suitable volumetric flask and a}
add about 8% of the flask volume of 1.0 N sodium IDENTIFICATION =e
al
hydroxide solution. Shake well and keep for 10 min. e A. INFRARED ABSORPTION (197F)
Neutralize with an equal amount of 1.0 N sulfuric acid e B. It meets the requirements in Specific Tests for Viscos-
and dilute with Mobile phase to volume. ity—Capillary Methods (911).
Chromatographic system
(See Chromatography (621), System Suitability.) IMPURITIES
Mode: LC ¢ LIMIT OF POLYCYCLIC AROMATIC HYDROCARBONS
Detector UV 210 nm Dimethyl sulfoxide: Use spectrophotometric grade di-
Column: 4.6-mm x 25-cm; 5-1um packing L1 methyl sulfoxide.
Flow rate: 1 mL/min n-Hexane: Use n-hexane that has been washed by be-
Injection volume: 20 uL ing shaken previously twice with one-fifth its volume of
Run times Dimethy! sulfoxide.
Standard solution: NLT 2.4 times the retention time Standard solution: 7.0 g/mL of USP Naphthalene RS
of lactic acid in isooctane (2,2,4-trimethylpentane)
Sample solution: NLT 4 times the retention time of Standard blank: 2,2,4-Trimethylpentane
lactic acid Sample solution: Transfer 25.0 mL of Mineral Oil and
System suitability 25 mL of n-Hexane to a 125-mL separator, and mix.
Sample: Standard solution [Not&—Use no lubricants other than water on the stop-
Suitability requirements cock, or use a separator equipped with a suitable poly-
Tailing factor; NMT 2.0 meric stopcock.]
Relative standard deviation: NMT 2.0% Add 5.0 mL of Dimethyl sulfoxide, and shake the mixture
Analysis vigorously for 1 min. Allow to stand until the lower
Samples: Standard solution and Sample solution layer is clear, transfer the lower layer to another
Calculate the percentage of lactic acid in the portion of 125-mL separator, add 2 mL of n-Hexane, and shake
Injection taken: vigorously. Use the lower layer.
Sample blank: Dimethy/ sulfoxide that has been shaken
Result = (ru/rs) x (Cs/Cu) x (Me/M.2) x 100 previously vigorously for 1 min with n-Hexane in the
ratio of 5 mL of Dimethyl! sulfoxide to 25 mL of n-Hexane
 2750 Mineral / Official Monographs USP 41

Instrumental conditions Acceptance criteria: No dark brown color develops.

Mode: UV
Analytical wavelengths ADDITIONAL REQUIREMENTS
Standard solution: 275 nm ¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant
Sample solution: 260-350 nm containers. No storage requirements specified.
Cell: 1cm e LABELING: Label it to indicate the name and quantity of
Analysis any substance added asa stabilizer.
Samples: Standard solution, Standard blank, Sample so- e USP REFERENCE STANDARDS (11)
lution, and Sample blank USP Mineral Oil RS
Acceptance criteria: The absorbance at any wavelength USP Naphthalene RS
in the specified range of the Sample solution is NMT
one-third of the absorbance of the Standard solution.
SPECIFIC TESTS
e SPECIFIC GRAVITY (841): 0.845-0.905 Mineral Oil Emulsion
© ViscosiTy—CAPILLARY METHODS (911): 34.5-150.0 mm? -
s"| for kinematic viscosity, measured with a capillary vis- DEFINITION
cometer at 40 +0.1° Prepare Mineral Oil Emulsion as follows.
° ACIDITY
Sample: 10 mL
Analysis: Add 20 mL of boiling water to the Sample, Mineral Oil
and shake vigorously for about 1 min. Allow to cool, Acacia, in
and draw off the separated water. To 10 mL of the S$
piere aqueous layer add 0.1 mL of phenolphthalein m:
Alcohol 60 mL
Acceptance criteria: The solution does not produce a
pink color. NMT 1.0 mL of 0.01 N sodium hydroxide is Puri Wai (o make 1000 mL
required to to produce a pink color. Mix the Mineral Oil with the Acacia in a dry mortar. Add
e READILY CARBONIZABLE SUBSTANCES TEST (271)
250 mL of Purified Water all at once, and emulsify the mix-
Sample: 5 mL ture. Add, in divided portions, triturating after each addi-
Standard solution: In a glass-stoppered test tube that tion, a mixture of the Syrup, 50 mL of Purified Water, and
previously has been rinsed with hot nitric acid (see Vanillin dissolved in Alcohol. Add Purified Water to bring
Cleaning Glass Apparatus (1051)), mix 3 mL of ferric the preparation to final volume, and mix.
chloride CS, 1.5 mL of cobaltous chloride CS, and The Vanillin may be replaced by NMT 1% of any other offi-
0.5 mL of cupric sulfate CS then overlaid with 5 mL of cial flavoring substance or any mixture of official flavoring
Mineral Oil. substances. Alcohol may be replaced with 60 mL of sweet
Analysis: Place the Sample in a glass-stoppered test orange peel tincture or 2 g of benzoic acid as a
tube that previously has been rinsed with hot nitric acid
fe
as
preservative.
5 (see Cleaning Glass Apparatus (1051)), then rinsed with
Ss water, and dried. Add 5 mL of sulfuric acid containing SPECIFIC TESTS
—
boa) 94.5%-94.9% of H2SOx4, and heat in a boiling water e ALCOHOL DETERMINATION, Method | (611): 4.0%-6.0% of
° bath for 10 min. After the test tube has been in the C2HsOH
S bath for 30 s, remove it quickly, and while holding the
Gj stopper in place, give three vigorous, vertical shakes ADDITIONAL REQUIREMENTS
> over an amplitude of about 5 in. Repeat every 30 s. Do e PACKAGING AND STORAGE: Package in tight containers.
a not keep the test tube out of the bath longer than 3 s
na
=) for each shaking pees, At the end of 10 min from the
time when first placed in the water bath, remove the
test tube.
Acceptance criteria: The oil portion of the Sample may Mineral Oil, Rectal
turn hazy, but it remains colorless or shows a slight
pink or yellow color, and the acid portion of the Sample DEFINITION
does not become darker than the Standard solution. Mineral Oil, Rectal, is Mineral Oil that has been suitably
¢ SOLID PARAFFIN packaged.
Sample: Mineral Oil that has been dried previously in a
beaker at 105° for 2 h and cooled to room temperature IDENTIFICATION
in a desiccator over silica gel e A. INFRARED ABSORPTION (197F)
Analysis: Fill a tall, cylindrical, standard oil-sample bot- e B. It meets the requirements in Specific Tests for Viscos-
tle of colorless glass of 120-mL capacity with the Sam- ity—Capillary Methods (911).
ples insert the stopper, and immerse in an ice bath for 4
SPECIFIC TESTS
Acceptance criteria: The Sample is sufficiently clear that e SPECIFIC GRAVITY (841): 0.845-0.905
a black line 0.5 mm in width, on a white background, e ViscosiTy—CAPILLARY METHODS (911): 34.5-150.0 mm2-
held vertically behind the bottle, is clearly visible. s" for kinematic viscosity, measured with a capillary vis-
© LimiT OF SULFUR COMPOUNDS cometer at 40+ 0.1°
Solution A: Saturated solution of lead(Il) oxide in so- e ACIDITY
dium hydroxide (200 mg/mL) Sample: 10 mL
Sample: 4.0 mL Analysis: Add 20 mL of boiling water to the Sample,
Analysis: Combine the Sample, 2 mL of dehydrated al- shake vigorously for about 1 min. Allow to cool,
cohol, and 2 drops of Solution A, heat at 70° for 10 min and draw off the separated water. To 10 mL of the
with frequent shaking, and cool. filtered aqueous layer add 0.1 mL of phenolphthalein
TS
Acceptance criteria: The solution does not produce a
pink color. NMT 1.0 mL of 0.01 N sodium hydroxide is
required to produce a pink color.
USP 41
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight, light-resistant,
single-unit containers. No storage requirements specified.
e USP REFERENCE STANDARDS (11)
USP Mineral Oil RS
Topical Light Mineral Oil
DEFINITION
Topical Light Mineral Oil is Light Mineral Oil that has been
suitably packaged.
IDENTIFICATION
e A. INFRARED ABSORPTION (197F)
e B. It meets the requirements in Specific Tests for Viscos-
ity—Capillary Methods (911).
SPECIFIC TESTS
© SPECIFIC GRAVITY (841): 0.818-0.880
© Viscosity—CAPILLARY METHODS (911): 3.0-34.4 mmz2- s“1
for kinematic viscosity, measured with a capillary viscom-
eter at 40+0.1°
e ACIDITY
Sample: 10 mL
Analysis: Add 20 mL of boiling water to the Sample,
and shake vigorously for about 1 min. Allow to cool,
and draw off the separated water. To 10 mL of the
filtered aqueous layer add 0.1 mL of phenolphthalein
TS:
Acceptance criteria: The solution does not produce a
pink color. NMT 1.0 mL of 0.01 N sodium hydroxide is
required to produce a pink color.
© SOLID PARAFFIN
Sample: Topical Light Mineral Oil that has been dried
previously in a beaker at 105° for 2 h and cooled to
room temperature in a desiccator over silica gel
Analysis: Fill a tall, cylindrical, standard oil-sample bot-
tle of colorless glass of 120-mL capacity with the Sam-
ple. Insert the stopper, and immerse the bottle in a mix-
ture of ice and water for 4 h.
Acceptance criteria: The Sample solution is sufficiently
clear that a black line 0.5 mm in width, on a white
background, held vertically behind the bottle, is clearly
visible.
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers. No storage requirements specified.
© LABELING: Label it to indicate the name and quantity of
any substance added as a stabilizer, and label packages
intended for direct use by the public to indicate that it is
not intended for internal use.
e USP REFERENCE STANDARDS (11)
USP Mineral Oil RS
Minocycline for Injection
DEFINITION
Minocycline for Injection is sterile, freeze-dried Minocycline
Hydrochloride suitable for parenteral use. It contains the
equivalent of NLT 90.0% and NMT 120.0% of the labeled
amount of minocycline (C23H27N3O7).
IDENTIFICATION
e A. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
Official Monographs / Minocycline 2751
ASSAY
© PROCEDURE
Mobile phase: Dimethylformamide, tetrahydrofuran,
0.2 M ammonium oxalate, and 0.01 M edetate diso-
dium (120:80:600:180). Adjust with ammonium hy-
droxide to a pH of 7.2.
System suitability solution: Dissolve 10 mg of USP Mi-
nocycline Hydrochloride RS in 20 mL of 0.2 M ammo-
nium oxalate. Heat on a water bath at 60° for 3 h,
allow to cool, and dilute with water to 25.0 mL.
Standard solution: 0.5 mg/mL of minocycline from
USP Minocycline Hydrochloride RS in water. Use this
solution within 3 h.
Sample solution 1 (where it is represented as being in a
single-dose container): Nominally 0.5 mg/mL of mino-
cycline, prepared as follows. Constitute Minocycline for
Injection in a volume of water, corresponding to the
volume of solvent specified in the labeling. Withdraw all
of the withdrawable contents, using a hypodermic nee-
dle and syringe, and dilute with water.
Sample solution 2 (where the label states the quantity
of minocycline in a given volume of constituted solu-
tion): Nominally 0.5 mg/mL of minocycline, prepared
as follows. Constitute Minocycline for Injection in a vol-
ume of water, corresponding to the volume of solvent
specified in the labeling. Dilute a portion of constituted
solution with water.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 280 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 40°
Flow rate: 1.5 mL/min
Injection volume: 20 LL
System suitability
Samples: System suitability solution and Standard
solution (<3
[Note—The relative retention times for epiminocycline “
ae]
and minocycline are 0.7 and 1.0, respectively.]
Suitability requirements Es
Capacity factor: 5.0-11.5, Standard solution °
|
Resolution: NLT 4.6 between epiminocycline and mi- °
nocycline, System suitability solution io}
Tailing factor: 0.9-2.0 for minocycline, Standard Fi
ES)
solution mo]
Relative standard deviation: NMT 2.0%, Standard si
7)
solution
Analysis
Samples: Standard solution, and Sample solution 1 or
Sample solution 2
Calculate the percentage of the labeled amount of mi-
nocycline (C23H27N3O7) in the container, or in the por-
tion of constituted solution taken:
Result = (ru/rs) x (Cs/Cu) x Px F x 100
ty = peak response from Sample solution 1 or
Sample solution 2
rs = peak response from the Standard solution
Cs = concentration of USP Minocycline
Hydrochloride RS in the Standard solution
(mg/mL)
Gu = nominal concentration of Sample solution 1 or
Sample solution 2 (mg/mL)
P = potency of minocycline in USP Minocycline
Hydrochloride RS (ug/g)
F = conversion factor, 0.001 mg/ug
Acceptance criteria: 90.0%-120.0%
PERFORMANCE TESTS
e UNIFORMITY OF DOSAGE UNITS (905): Meets the
requirements
 2752 Minocycline / Official Monographs USP 41

IMPURITIES IDENTIFICATION
© LIMIT OF EPIMINOCYCLINE © INFRARED ABSORPTION (197K): Dry the Standard and Sam-
Mobile phase, System suitability solution, Standard ple at 100° for 2 h before use.
solution, Sample solution 1 or Sample solution 2,
Chromatographic system, and System suitability: ASSAY
Proceed as directed in the Assay. © PROCEDURE
[Note—The relative retention times for epiminocycline [Note—Protect the Standard solution and Sample solution
and minocycline are 0.7 and 1.0, respectively.] from light, store in a refrigerator, and use within 3 h.]
Analysis: Calculate the percentage of epiminocycline in Mobile phase: Dimethylformamide, tetrahydrofuran,
the portion of Minocycline for Injection taken: 0.2 M ammonium oxalate, and 0.01 M edetate diso-
dium (120:80:600:180). Adjust with ammonium hy-
Result = (ru/r7) x 100 droxide to a pH of 7.2.
System suitability solution: Dissolve 10 mg of USP Mi-
ru = peak area of epiminocycline from Sample nocycline Hydrochloride RS in 20 mL of 0.2 M ammo-
solution 1 or Sample solution 2 nium oxalate. Heat on a water bath at 60° for 3 h,
tr = total area of all the peaks from Sample solution allow to cool, and dilute with water to 25.0 mL.
1 or Sample solution 2 Standard solution: Equivalent to 500 g/mL of mino-
Acceptance criteria: NMT 6.0% cycline (C23H27N307) from USP Minocycline Hydrochlo-
ride RS in water
SPECIFIC TESTS Sample solution: Equivalent to 500 wg/mL of mino-
e PH (791) cycline (C23H27N307) from Minocycline Hydrochloride in
Sample solution: Nominally 10 mg/mL of minocycline water
Acceptance criteria: 2.0-3.5 Chromatographic system
e© WATER DETERMINATION, Method | (921) (See Chromatography (621), System Suitability.)
Test preparation: Prepare as directed for a hygroscopic Mode: LC
specimen. Detector: UV 280 nm
Acceptance criteria: NMT 3.0% Column: 4.6-mm x 25-cm; 5-um packing L1
e PARTICULATE MATTER IN INJECTIONS (788): Meets the re- Column temperature: 40°
quirements for small-volume injections Flow rate: 1.5 mL/min
e STERILITY TESTS (71): Meets the requirements Injection size: 20 uL
e BACTERIAL ENDOTOXINS TEST (85): It contains NMT 1.25 System suitability
USP Endotoxin Units/mg of minocycline. Samples: System suitability solution and Standard
© CONSTITUTED SOLUTION: At the time of use, it meets the solution
requirements for Injections and Implanted Drug Products [Note—The relative retention times for epiminocycline
(1), Specific Tests, Completeness and clarity of solutions. and minocycline are 0.7 and 1.0, respectively.]
e OTHER REQUIREMENTS: It meets the requirements for La- Suitability requirements
beling (7), Labels and Labeling for Injectable Products. Resolution: NLT 4.6 between epiminocycline and mi-
“ nocycline, System suitability solution
= ADDITIONAL REQUIREMENTS pits 7 -
re ¢ PACKAGING AND STORAGE: Preserve as described in Pack- ieee 0.9-2.0 for the analyte peak, Standard
s
Dd
J
aging and Storage Requirements (659), Injection Packaging, Relative
solution standard deviation: NMT 2.0%, ‘ Standard
Packaging for constitution, protected from light.
9
S Analysis
iS Change to read: Samples: Standard solution and Sample solution
= Calculate the quantity, in g/mg, of minocycline
(a ° USP REFERENCE STANDARDS (11) (C23H27N307) in the portion of Minocycline Hydrochlo-
a) ride taken:
=) @ (CN 1-May-2018)
USP Minocycline Hydrochloride RS
Result = (ru/ts) x (Cs/Cu) x P
tu = peak response from the Sample solution
Ts peak response from the Standard solution
° ° © trati f mi line in the Standard
Minocycline Hydrochloride . Spoturion(ag/mnis 2) Haentts sel babes
Cu = concentration of the Sample solution (ug/mL)
GG OH i PB = potency of USP Minocycline Hydrochloride RS
we ne (ug/mg)
| 3 I + et Acceptance criteria: 890-950 g/mg on the anhydrous
~~ Ty oem basis
UN. UM
os Sie IMPURITIES
Inorganic Impurities
C23H27N307 + HCl 493,94 e RESIDUE ON IGNITION (281): NMT 0.15%
2-Naphthacenecarboxamide, 4,7-bis(dimethylamino)-1,4,4a,
5,5a,6,11,1 acta ai 0,12,12a-tetrahydroxy-1,1 1di-
oxo-, monohydrochloride, [45-(40,4a0,5aa,1 Baer) Delete the following:
4,7-Bis(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-
3,10,12,12a-tetrahydroxy-1,11-dioxo-2-naphthacene- °e HEAVY METALS, Method |/ (231): NMT 50 ppme cofteai1-
carboxamide monohydrochloride [13614-98-7]. Jan-2018)
Organic Impurities
DEFINITION e PROCEDURE
Minocycline Hydrochloride contains the equivalent of NLT Mobile phase, Standard solution, System suitability
890 11g and NMT 950 ug of minocycline (C23H27N307) per solution, Chromatographic system, and System suit-
mg, calculated on the anhydrous basis. ability: Proceed as directed in the Assay
USP 41 Official Monographs / Minocycline 2753

[Note—Protect the Standard solution and the Sample Change to read:

solutions from light, store in a refrigerator, and use
within 3 h.] ° USP REFERENCE STANDARDS (11)
Sample solution 1: 0.25 mg/mL of Minocycline
@ (CN 1-May-2018)
Hydrochloride USP Minocycline Hydrochloride RS
Sample solution 2: 5 ug/mL of Minocycline Hydrochlo-
tide in water
Sample solution 3: 3 g/mL of Minocycline Hydrochlo-
ride in water
Run time: 2.6 times the retention time of minocycline,
Sample solution 1 Minocycline Hydrochloride Capsules
Analysis
Samples: Sample solution 1, Sample solution 2, and DEFINITION
Sample solution 3 Minocycline Hydrochloride Capsules contain the equivalent
Calculate the percentage of epiminiecyeline in the por- of NLT 90.0% and NMT 115.0% of the labeled amount
tion of Minocycline Hydrochloride taken: of minocycline (C23H27N307).

Result = (rei/tm3) X Dy x 100 IDENTIFICATION

e A. The retention time of the major peak of the Sample
Tet = peak response of epiminocycline from Sample solution corresponds to that of the Standard solution, as
solution 1 obtained in the Assay.
Tu = peak response of minocycline from Sample
ASSAY
solution 3 ¢ PROCEDURE
Dy = dilution factor for Sample solution 3 Mobile phase: linet nora tetrahydrofuran,
Calculate the total percentage of impurities other than 0.2 M ammonium oxalate, and 0.01 M edetate diso-
epiminocycline in the portion of Minocycline dium (120:80:600:180). Adjust with ammonium hy-
Hydrochloride taken: droxide to a pH of 7.2.
Result = (rt/tw2) x D2 x 100 System suitability solution: Dissolve 10 mg of USP Mi-
nocycline Hydrochloride RS in 20 mL of 0.2 M ammo-
tr = sum of peak responses of all impurities other nium oxalate. Heat on a water bath at 60° for 3 h,
than epiminocycline from Sample solution 1 allow to cool, and dilute with water to 25.0 mL.
2 = peak response of minocycline from Sample Standard solution: 0.5 mg/mL of minocycline from
solution 2 USP Minocycline Hydrochloride RS in water. Use this
D2 = dilution factor for Sample solution 2 solution within 3 h.
Acceptance criteria Sample solution: Nominally 0.5 mg/mL of minocycline
Individual impurities: NMT 1.2% epiminocycline in water from combined contents of NLT 20 Capsules.
Total impurities (excluding epiminocycline): NMT Pass throughasuitable filter.
Chromatographic system =
2.0% 4)
(See Chromatography (621), System Suitability.) a)
SPECIFIC TESTS Mode: LC
e CRYSTALLINITY (695): Meets the requirements Detector: UV 280 nm E
i}
e PH (791): 3.5-4.5, in a solution equivalent to 10 mg/mL Column: 4.6-mm x 25-cm; 5-um packing L1 3
of minocycline Column temperature: 40° )
e@ WATER DETERMINATION, Method | (921): 4.3%-8.0% Flow rate: 1.5 mL/min @2
© STERILITY TESTS (71): Where the label states that Mino- Injection volume: 20 pL 2
cycline Hydrochloride is sterile, it meets the System suitability 3
requirements. Samples: System suitability solution and Standard
Se
a)
e BACTERIAL ENDOTOXINS TEST (85): Where the label states solution
that Minocycline Hydrochloride is sterile or must be sub- [Note—The relative retention times for epiminocycline
jected to further processing during the preparation of in- and minocycline are 0.7 and 1.0, respectively.]
jectable dosage forms, it contains NMT 1.25 USP Endo- Suitability requirements
toxin Units/mg of minocycline. Capacity factor: 5.0-11.5, Standard solution
Resolution: NLT 4.6 between epiminocycline and mi-
ADDITIONAL REQUIREMENTS nocycline, System suitability solution
© PACKAGING AND STORAGE: Preserve in tight containers, Tailing factor: 0.9-2.0 for minocycline, Standard
protected from light. solution
© LABELING: Where it is intended for use in preparing in- Relative standard deviation: NMT 2.0%, Standard
jectable dosage forms, the label states that it is sterile or solution
must be subjected to further processing during the prep- Analysis
aration of injectable dosage forms. Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of mi-
eens (C23H27N307) in the portion of Capsules
taken:

Result = (ru/rs) x (Cs/Cu) x Px Fx 100

tu peak response from the Sample solution

Woueu

ls peak response from the Standard solution

Cs concentration of USP Minocycline
Hydrochloride RS in the Standard solution
(mg/mL)
Cu = nominal concentration of the Sample solution
(mg/mL)
P = potency of minocycline in USP Minocycline
Hydrochloride RS (1ug/mg)
 2754 Minocycline / Official Monographs USP 41

F = conversion factor, 0.001 mg/yg {Notte—The relative retention times for epiminocycline
Acceptance criteria: 90.0%-115.0% and minocycline are 0.7 and 1.0, respectively.]
Suitability requirements
PERFORMANCE TESTS Capacity factor: 5.0-11.5, Standard solution
e DISSOLUTION (711) Resolution: NLT 4.6 between epiminocycline and mi-
Medium: Water; 900 mL nocycline, System suitability solution
Apparatus 2: 50 rpm Tailing factor: 0.9-2.0 for minocycline, Standard
Time: 45 min solution
Detector: UV maximum at about 348 nm Relative standard deviation: NMT 2.0%, Standard
Standard solution: USP Minocycline Hydrochloride RS solution
in Medium Analysis
Sample solution: Sample per Dissolution (711). Dilute Samples: Standard solution and Sample solution
with Medium to a concentration that is similar to that of Calculate the percentage of the labeled amount of mi-
the Standard solution. nocycline (C23H27N307) in the portion of Oral Suspen-
Tolerances: NLT 75% (Q) of the labeled amount of mi- sion taken:
nocycline (C23H27N307) is dissolved.
e UNIFORMITY OF DOSAGE UNITS (905): Meet the Result = (ru/rs) x (Cs/Cu) x Px Fx 100
requirements
ru = peak response from the Sample solution
SPECIFIC TESTS Is = peak response from the Standard solution
e WATER DETERMINATION, Method | (921): NMT 12.0% G = concentration of USP Minocycline
ADDITIONAL REQUIREMENTS ee RS in the Standard solution
© PACKAGING AND STORAGE: Preserve in tight, light-resistant G, _ nominal Concentration of minocycline in the
containers 5 ~ Sample solution
i (mg/mL)
° erable <11) P = potency of minocycline in USP Minocycline
inocycline Hydrochloride RS Hydrochloride RS (ug/mg)
E = conversion factor, 0.001 mg/pg
Acceptance criteria: 90.0%-130.0%
PERFORMANCE TESTS
Minocycline Hydrochloride Oral e UNIFORMITY OF DOSAGE UNITS (905)
nsion For single-unit containers
Suspe 3 Acceptance criteria: Meets the requirements
DEFINITION e DELIVERABLE VOLUME (698): Meets the requirements
Minocycline Hydrochloride Oral Suspension contains the SPECIFIC TESTS
equivalent of NLT 90.0% and NMT 130.0% of the labeled e PH (791): 7.0-9.0
al
B=]
amount of minocycline (C2sH27N307) and one or more
a suitable diluents, flavors, preservatives, and wetting agents ADDITIONAL REQUIREMENTS
J
J
in an aqueous vehicle. © PACKAGING AND STORAGE: Preserve in tight, light-resistant
aD containers.
° IDENTIFICATION e USP REFERENCE STANDARDS (11)
= e A. The retention time of the major peak of the Sample USP Minocycline Hydrochloride RS
Sj solution corresponds to that of the Standard solution, as
Ps obtained in the Assay.
rc
“ ASSAY
> e PROCEDURE
Mobile phase: Dimethylformamide, tetrahydrofuran, Minocycline Hydrochloride Tablets
0.2 M ammonium oxalate, and 0.01 M edetate diso-
dium (120:80:600:180). Adjust with ammonium hy- DEFINITION
droxide to a pH of 7.2. Minocycline Hydrochloride Tablets contain the equivalent of
System lpr solution: 2 mg/mL of USP Mino- NLT 90.0% and NMT 115.0% of the labeled amount of
cycline Hydrochloride RS in water. Transfer 5 mL of this minocycline (C23H27N30;).
solution to a small beaker, and heat on a steam bath
for 60 min. Evaporate to dryness, and dissolve the resi- IDENTIFICATION
due in 25 mL of Mobile phase. Pass throughafilter. e A. The retention time of the major peak of the Sample
Standard solution: 0.5 mg/mL of minocycline from solution corresponds to that of the Standard solution, as
USP Minocycline Hydrochloride RS in Mobile phase. Use obtained in the Assay.
the solution within 1 h. ASSAY
Sample solution: Nominally 0.5 mg/mL of minocycline e PROCEDURE
from Oral Suspension, freshly mixed and free from air Mobile phase: Dimethylformamide, tetrahydrofuran,
bubbles, in Mobile phase. Use the solution within 1 h. 0.2 M ammonium oxalate, and 0.01 M edetate diso-
Chromatographic a dium (120:80:600:180). Adjust with ammonium hy-
(See Chromatography (621), System Suitability.) droxide to a pH of 7.2.
Mode: LC System suitability solution: Dissolve 10 mg of USP Mi-
Detector: UV 280 nm nocycline Hydrochloride RS in 20 mL of 0.2 M ammo-
Column: 4.6-mm x 25-cm; 5-m packing L1 nium oxalate. Heat on a water bath at 60° for 3 h,
Column temperature: 40° allow to cool, and dilute with water to 25.0 mL.
Flow rate: 1.5 mL/min Standard solution: 0.5 mg/mL of minocycline from
Injection volume: 20 pL USP Minocycline Hydrochloride RS in water. Use this
System suitability solution within 3 h.
Samples: System suitability solution and Standard Sample solution: Nominally 0.5 mg/mL of minocycline
solution from NLT 20 finely powdered Tablets in water. Shake
for 1 min.
USP 41 Official Monographs / Minocycline 2755

Chromatographic system IDENTIFICATION

(See Chromatography (621), System Suitability.) e A. The retention time of the major peak of the Sample
Mode: LC solution corresponds to that of the Standard solution, as
Detector: UV 280 nm obtained in the Assay.
Column: 4.6-mm x 25-cm; 5-um packing L1 e B. The UV absorption spectrum of the major peak of the
Column temperature: 40° Sample solution and that of the Standard solution exhibit
Flow rate: 1.5 mL/min maxima and minima at the same wavelengths, as ob-
Injection volume: 20 uL tained in the Assay.
System suitability
Samples: System suitability solution and Standard ASSAY
solution © PROCEDURE
[Note—The relative retention times for epiminocycline Protect solutions containing minocycline from light.
and minocycline are 0.7 and 1.0, respectively.] Buffer: 3.5 g/L of tetrabutylammonium hydrogen sul-
Suitability requirements fate, 2. g/L of anhydrous citric acid, and 6.8 g/L of mon-
Capacity factor: 5.0-11.5, Standard solution obasic potassium phosphate. Adjust with 10 N sodium
Resolution: NLT 4.6 between epiminocycline and mi- hydroxide to a pH of 7.0.
nocycline, System suitability solution Mobile phase: Acetonitrile and Buffer (24:76)
Tailing factor: 0.9-2.0 for minocycline, Standard Diluent: Acetonitrile and water (20:80)
solution Standard solution: 0.045 mg/mL of minocycline from
Relative standard deviation: NMT 2.0%, Standard USP Minocycline Hydrochloride RS in Diluent. Store at
solution 4° and use within 24 h.
Analysis Sample stock solution: Nominally about 0.9 mg/mL of
Samples: Standard solution and Sample solution minocycline from Tablets prepared as follows. Transfer a
Calculate the percentage of the labeled amount of mi- suitable portion of finely powdered Tablets (NLT 10) to
nocycline (C23H27N307) in the portion of Tablets taken: a suitable volumetric flask. Add acetonitrile, using 20%
of the final volume, and mix vigorously for 15 min. Add
Result = (ru/ts) x (Cs/Cu) x P x Fx 100 water, using 65% of the final volume, and mix vigor-
ously for 30 min. Dilute with water to volume and mix.
tu = peak response from the Sample solution Sample solution: Nominally 0.045 mg/mL of mino-
rs = peak response from the Standard solution cycline from Sample stock solution in Diluent. Centrifuge
Cs = concentration of USP Minocycline and use the clear supernatant. Store at 4° and use
Hydrochloride RS in the Standard solution within 24 h.
(mg/mL) Chromatographic system
Cu = nominal concentration of the Sample solution (See Chromatography (621), System Suitability.)
(mg/mL) Mode: LC
P = potency of minocycline in USP Minocycline Detector: UV 277 nm. When this procedure is used
Hydrochloride RS (1ug/mg) for Identification test B, use a diode-array detector set
F = conversion factor, 0.001 mg/ug at 200-400 nm. =
Acceptance criteria: 90.0%-115.0% Column: 4.6-mm x 15-cm; 5-um packing L1 2)
ao)
Temperatures
PERFORMANCE TESTS Column: 35° E<
© DISSOLUTION (711) Autosampler: 4° 5
Medium: Water; 900 mL Flow rate: 1.3 mL/min J
Apparatus 2: 50 rom }
Time: 45 min
Injection volume: 10 uL to}=
System suitability iy
Detector: UV maximum at about 348 nm Sample: Standard solution bo}
Standard solution: USP Minocycline Hydrochloride RS Suitability requirements Be
in Medium Tailing factor: NMT 1.5
w

Sample solution: Sample per Dissolution (711). Dilute Relative standard deviation: NMT 1.0%
with Medium to a concentration that is similar to that of Analysis
the Standard solution. Samples: Standard solution and Sample solution
Tolerances: NLT 75% (Q) of the labeled amount of mi- Calculate the percentage of the labeled amount of mi-
nocycline (C23H27N307) is dissolved. nocycline (C23H27N3O7) in the portion of Tablets taken:
© UNIFORMITY OF DosAGE UNITS (905): Meet the
requirements Result = (ru/rs) x (Cs/Cu) x P x Fx 100
SPECIFIC TESTS tu = peak response from the Sample solution
© WATER DETERMINATION, Method
| (921): NMT 12.0% rs = peak response from the Standard solution
Gs = concentration of USP Minocycline
ADDITIONAL REQUIREMENTS Hydrochloride RS in the Standard solution
e PACKAGING AND STORAGE: Preserve in tight, light-resistant (mg/mL)
containers. Cu = nominal concentration of minocycline in the
e USP REFERENCE STANDARDS (11) Sample solution (mg/mL)
USP Minocycline Hydrochloride RS P = potency of minocycline in USP Minocycline
Hydrochloride RS (4g/mg)
F = conversion factor, 0.001 mg/ug
Acceptance criteria: 90.0%-110.0%
Minocycline Hydrochloride Extended- PERFORMANCE TESTS
Release Tablets e DISSOLUTION (711)
Test 1
DEFINITION Protect solutions containing minocycline from light.
Minocycline Hydrochloride Extended-Release Tablets contain
NLT 90.0% and NMT 110.0% of the labeled amount of
minocycline (C23H27N307).
 2756 Minocycline / Official Monographs USP 41

Medium: pH 6.8 phosphate buffer; 900 mL Test 2

Apparatus 2: 50 rpm If the product complies with this test, the labeling indi-
Times: 1, 2, and 5h cates that it meets USP Dissolution Test 2.
Standard stock solution: 0.5 mg/mL of minocycline Protect solutions containing minocycline from light.
from USP Minocycline Hydrochloride RS in Medium Medium: 0.1 N hydrochloric acid: 900 mL
Standard solution: (1/900) mg/mL of minocycline Apparatus 1: 100 rom
from Standard stock solution in Medium, where Lis the Times: 1, 2, and 4
label claim of minocycline in mg/Tablet Standard solution: 0.0225 mgiml of minocycline
Sample solution: Pass a portion of the solution under from USP Minocycline Hydrochloride RS in Medium
test through a suitable filter. Sample solution: At the times specified, withdraw
Instrumental conditions 10 mL of the solution under test and replace with
Mode: UV 10 mL of Medium. Pass throughasuitable filter. Dilute
Analytical wavelength: 348 nm with Medium to a concentration that is similar to that
Cell: See Table 1. of the Standard solution.
Instrumental conditions
Table 1 Mode: UV
Analytical wavelength: 348 nm
Tablet Strength Cell Path Length Cell: 1.cm
(mg) (cm) Blank: Medium
45 05 Analysis
90 0.2 Samples: Standard solution and Sample solution
135 0.2 Autozero the instrument using the Blank.
Calculate the concentration (C) of minocycline
Blank: Medium (Cz3H27N307) in the sample withdrawn from the ves-
Analysis sel at each time point (/):
Samples: Standard solution, Sample solution, and
Blank Result = (Au/As) x Cs x Dx Px F
Autozero the instrument using the Blank.
Calculate the concentration (C) of minocycline Au = absorbance of the Sample solution at time
(C23H27N307) in the sample withdrawn from the ves- point /
sel at each time point (/): As = absorbance of the Standard solution
Cs = concentration of the Standard solution
Result = (Au/As) x Cs x Px F (mg/mL)
D = dilution factor (mL/mL)
Au = absorbance of the Sample solution at time P = potency of minocycline in USP Minocycline
point i Hydrochloride RS (4g/mg)
As = absorbance of the Standard solution = conversion factor, 0.001 mg/ug
ww Cs = concentration of the Standard solution Calculate the percentage of the labeled amount (Q) of
no
i
Q
P
(mg/mL)
= potency of minocycline in USP Minocycline
minocycline (C23H27N3O7) dissolved at each time
point (/):
Dd
-_
Hydrochloride RS (tug/mg)
° = conversion factor, 0.001 malig Result; = C; x Vx (1/L) x 100
Cc Calculate the percentage of the labeled amount (Q) of
Sj minocycline (C23H27N3O7) dissolved at each time
Ps point (i):
Results = [(C2 x V) + (CG, x Vs)] x (1/2) x 100
a
WY
=| Result; = C; x Vx (1/L) x 100 Result; = {(C3 x V) + [(C2 + Ci) x Vs]} x (1/L) x 100
G = concentration of minocycline in the portion of
Resultz = {[C2 x (V— Vs)] + (CG; x Vs} x (1/1) x 100 sample withdrawn at the specified time point
(mg/mL)
Results = ({C3 x [V— (2 x Vs)]} + [(C2 + Gi) x Vs}) x C1/ volume of Medium, 900 mL
1) x 100 L label claim (mg/Tablet)
volume of the Sample solution withdrawn at
Vs
G = concentration of minocycline in the portion of each time point and replaced with Medium
sample withdrawn at the specified time point (ml)
(mg/mL) Tolerances: See Table 3.
Vv = volume of Medium, 900 mL
L = label claim (mg/Tablet) Table 3
Vs = volume of the Sample solution withdrawn at
each time point (mL) Amount Dissolved
Tolerances: See Table 2. (%)
Time 90 mg/Tablet
Point Time and 135 mg/
Table 2
(i) (h) 45 mg/Tablet Tablet
Time Point Time Amount Dissolved 1 1 40-60 40-60
{/) (h) (%) 2 2 70-95, 70-90.
1 1 20-45 3 4 NLT 85 NLT 85.
2 2 40-70
3 5 NLT 85 The percentages of the labeled amounts of mino-
cycline (C23H27N307) dissolved at the times specified
The percentages of the labeled amounts of mino- conform to Dissolution (711), Acceptance Table 2.
cycline (C23H27N307) dissolved at the times specified
conform to Dissolution (711), Acceptance Table 2.
USP 41 Official Monographs / Minocycline 2757

Test 3 Medium: 0.1 N hydrochloric acid; 900 mL

If the product complies with this test, the labeling indi- Apparatus 1: 100 rpm
cates that it meets USP Dissolution Test 3. Times: 1, 2, and 4
Protect solutions containing minocycline from light. Standard solution: (1/900) mg/mL of minocycline
Medium: 0.1 N hydrochloric acid, 900 mL from USP Minocycline Hydrochloride RS in Medium,
Apparatus 1: 100 rpm where L is the label claim of minocycline inmig ianet
Times: 0.5, 1.5,and4h Sample solution: At the times specified, withdraw
Standard solution: 0.021 mg/mL of minocycline from 5 mL of the solution under test and replace with 5 mL
USP Minocycline Hydrochloride RS in Medium of Medium. Pass throughasuitable filter, Dilute with
Sample solution: At the times specified, withdraw Medium to a concentration that is similar to that of the
10 mL of the solution under test and replace with Standard solution.
10 mL of Medium. Pass throughasuitable filter. Dilute Instrumental conditions
with Medium to a concentration that is similar to that Mode: UV
of the Standard solution. Analytical wavelength: 353 nm
Instrumental conditions Cell: 1cm
Mode: UV Blank: Medium
Analytical wavelength: 265 nm Analysis
Cell: 1.cm Samples: Standard solution and Sample solution
Blank: Medium Autozero the instrument using the Blank.
Analysis Calculate the concentration (C) of minocycline
Samples: Standard solution and Sample solution (C23H27N307) in the sample withdrawn from the ves-
Autozero the instrument using the Blank. sel at each time point (/):
Calculate the concentration (C)) of minocycline
(C23H27N307) in the sample withdrawn from the ves- Result = (Au/As) x Csx Dx Px F
sel at each time point (i):
Au = absorbance of the Sample solution at time
Result = (Au/As) x Cs x Dx PX F point i
As = absorbance of the Standard solution
Au = absorbance of the Sample solution at time Cs = concentration of the Standard solution
point j (mg/mL)
As = absorbance of the Standard solution D = dilution factor (mL/mL)
G = concentration of the Standard solution Pp = potency of minocycline in USP Minocycline
(mg/mL) Hydrochloride RS (1ug/mg)
D = dilution factor (mL/mL) = conversion factor, 0.001 mg/ug
Pp = potency of minocycline in USP Minocycline Calculate the percentage of the labeled amount (Q) of
Hydrochloride RS (tug/mg) minocycline (C23H27N307) dissolved at each time
= conversion factor, 0.001 mg/g point (i):
Calculate the percentage of the labeled amount (Q)) of
minocycline (C23H27N307) dissolved at each time Result; = C; x Vx (1/L) x 100 g
point (/):
Result; = C; x Vx (1/L) x 100 Result, = [(C2 x V) + (C; x Vs)] x (1/L) x 100 ES
=]
fo)
Resultz = [(Cz x V) + (CG; x Vs)] x (1/L) x 100 Results = {(C3 x V) + [(C2 + Cr) x Vs]} x (1/L) x 100 2
RY
G = concentration of minocycline in the portion of —
Results = {(C3 x V) + [(C2 + C1) x Vs]} x (1/L) x 100 sample withdrawn at the specified time point a
(mg/mL)
G = concentration of minocycline in the portion of volume of Medium, 900 mL
noua

sample withdrawn at the specified time point L label claim (mg/Tablet)

(mg/mL) Vs volume of the Sample solution withdrawn at
Vv = volume of Medium, 900 mL each time point and replaced with Medium
L = label claim (mg/Tablet) (mL)
Vs = volume of the Sample solution withdrawn at Tolerances: See Table 5.
each time point and replaced with Medium
(mL) Table 5
Tolerances: See Table 4.
Amount Dissolved
(%)
Table 4
45/Tablet
Time Point Time Amount Dissolved Time Point Time and 90 mg/ 135 mg/
(i) (h) Tablet Tablet
1 1 35-50 35-50
2 Z 63-78 67-82
NLT 3 4 NLT 90 NLT 90
The percentages of the labeled amounts of mino- The percentages of the labeled amounts of mino-
cycline (C23H27N3O7) dissolved at the times specified cycline (C23H27N3O7) dissolved at the times specified
conform to Dissolution (711), Acceptance Table 2. conform to Dissolution (711), Acceptance Table 2.
Test 4 e UNIFORMITY OF DOSAGE UNITS (905): Meet the
If the product complies with this test, the labeling indi- requirements
cates that it meets USP Dissolution Test 4.
Protect solutions containing minocycline from light.
 2758 Minocycline / Official Monographs USP 41

IMPURITIES Table 6
Relative Acceptance
Change to read: Retention Criteria,
Name Time NMT (%)
© ORGANIC IMPURITIES 4-Epiminocycline 0.38 4.0
Protect solutions containing minocycline from light. Desmethyl minocycline®< 0.46 =
°Buffer, Mobile phase, and Diluentie ‘erg s-apr2017) Pre- Sancycline®.4 0.68 —
pare as directed in the Assay. 5a,6-Anhydrominocycline’e 0.81 =—
Standard stock solution: Use the Standard solution as
Hydroxymethylminocycline®t 0.92 —
directed in the Assay.
Standard solution: 0.009 mg/mL of minocycline from Minocycline 1.0 =
Standard stock solution in Diluent. Store at 4° and use Any individual unspecified =
within 24 h. degradation product 0.2
°Sample solution: Use the Sample stock solution as di- Total degradation productss —: 2.0.
rected in the Assay. ‘err 1-spr-2017) @(4R,4aS,5aR, 12a5)-4,7-Bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-
Sensitivity solution: 0.9 g/mL of minocycline from dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide.
Standard solution in Diluent. Store at 4° and use within Process impurities are controlled in the drug substance and are not to be
24h. reported here. They are not included in total degradation products
System suitability solution: Heat a portion of the Stan- ©(45,4aS,5aR,12a5)-4-Dimethylamino-3,10,12,12a-tetrahydroxy-7-methyl-
amino-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carbox-
dard stock solution at 60° for about 2 h and cool. This amide.
solution contains a mixture of Saal ee sins and mi- 46-Demethyl-6-deoxytetracycline, (45,4a5,5aR, 12aS)-4-Dimethylamino-3,
nocycline. Store at 4° and use within 24 h. 10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrote-
Chromatographic system: Proceed as directed in the tracene-2-carboxamide.
Assay, except use a flow rate of 1 mL/min. ©(45,4aS,12aS)-4,7-Bis(dimethylamino)-3,10,11,12a-tetrahydroxy-1,12-di-
oxo-1,4,4a,5,12,12a-hexahydrotetracene-2-carboxamide.
System suitability
Samples: Standard solution, Sensitivity solution, and 5(45,4aS,5aR,12a5)-4,7-Bis(dimethylamino)-3,10,12,12a-tetrahydroxy-N-
(hydroxymethyl)-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-
System suitability solution carboxamide.
Suitability requirements 9Total degradation products does not include 4-epiminocycline.
Resolution: NLT 4.6 between minocycline and
4-epiminocycline, System suitability solution ADDITIONAL REQUIREMENTS
Tailing factor: NMT 1.5, Standard solution e PACKAGING AND STORAGE: Store in tightly closed contain-
Relative standard deviation: NMT 2.0%, Standard ers at controlled room temperature
solution e LABELING: When more than one Dissolution test is given,
Signal-to-noise ratio: NLT 10, Sensitivity solution the labeling states the Dissolution test used only if Test 7
Analysis is not used.
Samples: Standard solution and Sample solution e USP REFERENCE STANDARDS (11)
al Calculate the percentage of each impurity in the por- USP Minocycline Hydrochloride RS
c= tion of Tablets taken:
er
io
Result = (ru/rs) x (Cs/Cu) x P x Fx 100
a)
—

°i=} ty = peak response of each impurity from the Minocycline Periodontal System
° Sample solution
3 rs = peak response of minocycline from the DEFINITION
a Standard solution Minocycline Periodontal System is an extended-release for-
Ww Cs = concentration of USP Minocycline mulation of Minocycline Hydrochloride containing the
=) Hydrochloride RS in the Standard solution equivalent of NLT 90.0% and NMT 120.0% of the labeled
(mg/mL) amount of minocycline (C23H27N307).
nominal concentration of minocycline in the
Scrma

Sample solution (mg/mL) IDENTIFICATION

potency of minocycline in USP Minocycline e A. ULTRAVIOLET ABSORPTION (197U)
I

Hydrochloride RS (ug/mg) Wavelength range: 250-450 nm

F = conversion factor, 0.001 mg/ug Standard stock solution: Transfer USP Minocycline Hy-
Acceptance criteria: See Table 6. The reporting thresh- drochloride RS to a suitable volumetric flask. Dissolve
old is 0.1%. first in dimethylformamide, using about 20% of the fi-
nal volume, then dilute with water to volume, and mix
to obtain a solution having a known concentration of
about 0.48 mg/mL of minocycline.
Standard solution: 0.024 mg/mL minocycline hydro-
chloride from Standard stock solution in water
Sample stock solution: Transfer Minocycline Periodon-
tal System equivalent to 12 mg of minocycline hydro-
chloride to a 25-mL volumetric flask. Add 5.0 mL of di-
methylformamide, and mix to dissolve. Dilute with
water to volume and filter.
Sample solution: 0.024 mg/mL minocycline hydrochlo-
ride from Sample stock solution in water
Acceptance criteria: The Sample solution exhibits max-
ima at the same wavelengths as the Standard solution,
concomitantly measured.
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
USP 41
ASSAY
© PROCEDURE
Mobile phase: Dimethylformamide, 0.2 M ammonium
oxalate, and 0.1 M edetate disodium (25:55:20), ad-
justed with 0.4 M aqueous tetrabutylammonium hy-
droxide solution to a pH of 6.3 + 0.2
Diluent: Dimethylformamide and methanol (1:1)
System suitability solution: Prepare a solution in water
containing 2 mg/mL USP Minocycline Hydrochloride RS.
Heat over a steam bath for 60 min. To one part of this
solution add four parts of Mobile phase and mix. Refrig-
erate the solution immediately after preparation and
during analysis, using a refrigerated autosampler.
Standard solution: 0.4 mg/mL of minocycline from
USP Minocycline Hydrochloride RS in Diluent. Refriger-
ate the solution immediately after preparation and dur-
ing analysis, using a refrigerated autosampler. [NOTE—
Use low-actinic glassware.]
Sample solution: Mix the contents of NLT 10 dispens-
ing units of Minocycline Periodontal System. Transfer a
portion of the mixture, equivalent to 10 mg of mino-
cycline, into a 25-mL volumetric flask. Add Diluent and
sonicate for 2-5 min, or until the sample is dissolved.
Dilute with Diluent to volume, and mix to obtain a solu-
tion having a nominal concentration of 0.4 mg/mL of
minocycline, based on the label claim. Refrigerate the
solution immediately after preparation and during anal-
ysis, using a refrigerated autosampler. [NoTE—Use low-
actinic aecwvatet
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 280 nm
Guard column: 4.6-mm x 3-cm; 10-1um packing L7
Column: 4.6-mm x 15-cm; 5-um packing L7
Flow rate: 2 mL/min
Autosampler temperature: 5°
Injection size: 10 uL
System suitability
Samples: System suitability solution and Standard
solution
[Note—The relative retention times of epiminocycline
and minocycline are 0.81 and 1, respectively.]
Suitability requirements
Resolution: NLT 2.0 between epiminocycline and mi-
nocycline, System suitability solution
Tailing factor: NMT 2.0 for the minocycline peak,
System suitability solution
Relative standard deviation: NMT 2.0% for the mi-
nocycline peak, Standard solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of minocycline (C23H27N307) in
the portion of the Minocycline Periodontal System
taken:
Result = (ru/rs) * (Cs/Cy) x P x F x 100
Ty = peak response of minocycline from the Sample
solution
rs = peak response of minocycline from the
Standard solution
Official Monographs / Minocycline 2759
Cs = concentration of USP Minocycline
Hydrochloride RS in the Standard solution
(mg/mL)
U nominal concentration of minocycline in the
Sample solution (mg/mL)
P = potency of minocycline in USP Minocycline
Hydrochloride RS (4ug/mg)
F = conversion factor, 0.001 mg/ug
Acceptance criteria: 90.0%-120.0%
PERFORMANCE TESTS
© DISSOLUTION
Medium: 6.9 g/L of monobasic sodium phosphate
monohydrate in water, adjusted with phos horic acid
to a pH of 4.2. This solution is stable for 10 days.
Apparatus: Tube rotator. [NoTE—Suitable equipment is
available as Labquake® tube rotator, catalog number
400110.]
0.1 M Edetate disodium: 37.2 g/L of edetate disodium
in water
0.2 M Ammonium oxalate: 28.4 g/L of ammonium ox-
alate in water
Mobile phase: Mix 310 mL of 0.7 M Edetate disodium
and 500 mL of 0.2 M Ammonium oxalate, adjust with
0.4 M aqueous tetrabutylammonium hydroxide to a pH
of 6.2, and add 175 mL of dimethylformamide. The in-
jector wash solution is a mixture of dimethylformamide
and water (25:75).
Standard stock solution: 0.11 mg/mL of USP Mino-
cycline Hydrochloride RS in Medium
Standard solutions: Dilute the Standard stock solution
with Medium to obtain solutions with final concentra-
tions of 0.088 mg/mL, 0.0528 mg/mL, 0.0352 mg/mL,
0.022 mg/mL, and 0.0176 mg/mL.
System suitability solution: Transfer 10 mg of USP Mi-
nocycline Hydrochloride RS to a 50-mL beaker. Add
5 mL of water and heat on a steam bath for 60 min.
Add 20 mL of Medium or Mobile phase, and mix well. Cc
Store at 5°. Al
Sample solution: Use borosilicate glass tubes, 25 mm a)
outside diameter and 15 cm long. Close the tubes with =
a snap type cell with a Teflon prong consisting of a °
Teflon closure and holder that snap together, two 3
°
25-4um stainless steel screens, two silicone gaskets, and Re}=
a Teflon spacer (see figure 1). Prepare six tubes as fol- Ry
lows: partially assemble a release tube and tare its me]
weight; dispense one dose of Minocycline Periodontal 3
a)
System into a partially assembled release cell (see Figure
1); record the sample weight in mg; assemble the cell
so that the sample is enclosed between the two 25-11m
screens; close the cells and place each one of them into
separate glass tubes containing 10 mL of Medium previ-
ously equilibrated at 37°; add the Teflon prong, and
cap the tube with Teflon faced rubber-lined caps; seal
with Teflon tape. Place the tubes in the tube rotator.
Place the tube rotator in a convection incubator that is
maintained at 37°. Allow the tubes to rotate for 4 h.
Remove the solution under test, and add 10 mL of Me-
dium previously equilibrated at 37°. Replace the tubes
in the apparatus and rotate for 20 h (24 htotal). Re-
peat the sampling procedure after 24 h (48htotal),
and after another 24 h (72h total).
 2760 Minocycline / Official Monographs USP 41

Teflon Release Container e UNIFORMITY OF DOSAGE UNITS (905): Meet the

requirements

IMPURITIES
Organic Impurities
© PROCEDURE
CTI Teton Clone Mobile phase, Diluent, System suitability solution,
Standard solution, Sample solution, Chromato-
graphic system, and System suitability: Proceed as
cH Teflon Spacer directed in the Assay.
Analysis
Sample: Sample solution
— Silicone Gasket
Calculate the percentage of each related compound in
the portion of Minocycline Periodontal System taken:
25 Micron Screen Result = (ru/rr) x 100
Sample
Tu = peak response of each impurity from the
25 Micron Screen Sample solution
tr = sum of the peak responses from the Sample
solution. [NoTE—Exclude peaks eluting in the
Scone} Silicone Gasket solvent front.]
Acceptance criteria
Individual impurities: NMT 6.0% of epiminocycline
Total impurities (excluding epiminocycline): NMT

Figure 1. Sample Extraction Configuration

SPECIFIC TESTS
Chromatographic system e@ WATER DETERMINATION, Method | (921): NMT 5.0%
(See Chromatography (621), System Suitability.) © MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
Mode: LC FIED ORGANISMS (62): The total aerobic microbial count
Detector: UV 280 nm does not exceed 1000 cfu/g; the total combined molds
Guard column: 4.6-mm x 3-cm; 10-4um packing L7 and yee count does not exceed 100 cfu/g; and the
Column: 4.6-mm x 3.3-cm; 5-um packing L1 product meets the requirements of the test for the ab-
Flow rate: 1.5 mL/min sence of Escherichia coli.
Autosampler temperature: 5°
Injection size: 20 ul for the 4 and 24h time points; ADDITIONAL REQUIREMENTS
50 ul for the 48 and 72 h time points © PACKAGING AND STORAGE: Preserve in a tight, light-resis-
a
”
Suitability requirements tant container. Store at controlled room temperature.
rm Samples: System suitability solution and Standard e USP REFERENCE STANDARDS (11)
sS USP Minocycline Hydrochloride RS
- solutions
Dp Resolution: NLT 2.0 between epiminocycline and mi-
re}
= nocycline. Inject 20 uL of the System suitability solution.
3 Tailing factor: NMT 2.0. Inject 20 uL of the System
= suitability solution.
oe Relative standard deviation: NMT 2.0% for the mi- Minoxidil
4) nocycline peak, any of the Standard solutions
=) Analysis: Construct a calibration curve for each sam-
pling interval by plotting the concentration of the Stan-
dard solutions versus peak area. Calculate the slopes and
y-intercepts using linear regression analysis.
Calculate the release rate of minocycline:
Result) = [(ru — y)/Si] x 10/( i x W x A)
CsHisNsO 209.25
i = sampling time, 4, 24, 48, 72h 2,4-Pyrimidinediamine, 6-(1-piperidinyl)-, 3-oxide;
Tui = peak response from each of the Standard 2,4-Diamino-6-piperidinopyrimidine 3-oxide [38304-91-5].
solutions at time i
yi = y-intercept of the calibration curve at DEFINITION
sampling time i Minoxidil contains NLT 97.0% and NMT 103.0% of minox-
Si = slope of the calibration curve at sampling time idil (CsHisNsO), calculated on the dried basis.
i
Ww = weight of the sample (mg) IDENTIFICATION
A = amount of minocycline in the sample (mg/mg ¢ A. INFRARED ABSORPTION (197M): Do not dry specimens.
of sample) as determined in the Assay ASSAY
Tolerances © PROCEDURE
Mobile phase: Methanol, glacial acetic acid, and water
Time Release Rate (11g/h) (700:10:300). Add 3.0 g/L of docusate sodium. Adjust
with perchloric acid to a pH of 3.0.
Internal standard solution: 0.2 mg/mL of medroxypro-
gesterone acetate in Mobile phase
Standard solution: 0.25 mg/mL of USP Minoxidil RS in
Internal standard solution
Sample solution: 0.25 mg/mL of Minoxidil in Internal
standard solution
USP 41 Official Monographs / Minoxidil 2761

Chromatographic system e USP REFERENCE STANDARDS (11)

(See Chromatography (621), System Suitability.) USP Minoxidil RS
Mode: LC
Detector: UV 254 nm
Column: 4-mm x 25-cm; packing L1
Flow rate: 1 mL/min
Injection volume: 10 uL Minoxidil Tablets
System suitability
Sample: Standard solution DEFINITION
[Note—The relative retention times for the internal Minoxidil Tablets contain NLT 90.0% and NMT 110.0% of
standard and minoxidil are 0.8 and 1.0, respectively.] the labeled amount of minoxidil (CsH;sNsO).
Suitability requirements
Resolution: NLT 2.0 between the internal standard IDENTIFICATION
and minoxidil © INFRARED ABSORPTION
Relative standard deviation: NMT 2.0% from NLT Sample: Transfer a portion of finely powdered Tablets,
four replicate injections equivalent to 10 mg of minoxidil, to a separator. Add
Analysis 25 mL of water, and extract with three 15-mL portions
Samples: Standard solution and Sample solution of chloroform. Combine the chloroform extracts, and
Calculate the percentage of minoxidil (CoHisNsO) in evaporate with the aid of a stream of nitrogen. Wash
the portion of Minoxidil taken: the inside of the container with 5 mL of alcohol, add
300 mg of potassium bromide, and evaporate under
Result = (Ru/Rs) x (Cs/Cu) x 100 vacuum at 50° until dry.
Acceptance criteria: The IR absorption spectrum of the
Ru = peak response ratio of minoxidil to the potassium bromide dispersion prepared from the Sam-
internal standard from the Sample solution ple exhibits maxima at the same wavelengths as that of
Rs = peak response ratio of minoxidil to the a similar preparation of USP Minoxidil RS.
internal standard from the Standard solution
Gs = concentration of the Standard solution ASSAY
(mg/mL) © PROCEDURE
Cy = concentration of the Sample solution (mg/mL) Mobile phase: Methanol, glacial acetic acid, and water
Acceptance criteria: 97.0%-103.0% on the dried basis (70:1:30). Add 3.0 g/L of docusate sodium, and adjust
with perchloric acid to a pH of 3.0.
IMPURITIES Internal standard solution: 0.2 mg/mL of medroxypro-
e RESIDUE ON IGNITION (281): NMT 0.5% gesterone acetate in Mobile phase
Standard solution: 0.25 mg/mL of USP Minoxidil RS in
Delete the following: Internal standard solution
Sample solution: Nominally 0.25 mg/mL of minoxidil
°o HEAVY METALS, Method I! (231); NMT 20 ppMe ‘otis - in Internal standard solution, prepared as follows. Dis- fo
Jan-2018) solve the equivalent to 5 mg of minoxidil, from pow- 4)
a]
© ORGANIC IMPURITIES deredTablets (NLT 10), in 20.0 mL of Internal standard
Mobile phase and Chromatographic system: Proceed solution, and shake for 5 min. =
as directed in the Assay. Chromatographic system }
(See Chromatography (621), System Suitability.) =]
Sample solution: 0.25 mg/mL of Minoxidil in Mobile )
phase Mode: LC i}=
Analysis Detector: UV 254 nm i)
Sample: Sample solution Column: 4-mm x 25-cm; packing L1 a}
Flow rate: 1 mL/min >
Calculate the total percentage of impurities in the por- ww
tion of Minoxidil taken: Injection volume: 10 uL
System suitability
Result = (ru/rr) x 100 Sample: Standard solution
[Note—The relative retention times for the internal
tu = sum of the peak responses of all impurities standard and minoxidil are 0.8 and 1.0, respectively.]
from the Sample solution Suitability requirements
tr = sum of all the peak responses from the Sample Resolution: NLT 2.0 between the internal standard
solution and minoxidil peaks
Acceptance criteria: NMT 1.5% Relative standard deviation: NMT 2.0% from NLT
four replicate injections
SPECIFIC TESTS Analysis
e LOss ON DRYING (731) Samples: Standard solution and Sample solution
Analysis: Dry a sample at 50° and at a pressure not Calculate the percentage of the labeled amount of mi-
exceeding 5 mm of mercury for 3 h. noxidil (CsHisNsO) in the portion of Tablets taken:
Acceptance criteria: NMT 0.5%
Result = (Ru/Rs) x (Cs/Cu) x 100
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed Ru = peak response ratio of minoxidil to the
containers. internal standard from the Sample solution
Rs = peak response ratio of minoxidil to the
internal standard from the Standard solution
Cs = concentration of the Standard solution
(mg/mL)
Cu = nominal concentration of the Sample solution
(mg/mL)
 2762 Minoxidil / Official Monographs USP 41

Acceptance criteria: 90.0%-110.0% Table 1

Relative Acceptance
PERFORMANCE TESTS
Retention Criteria,
e DISSOLUTION (711)
Medium: pH 7.2 Phosphate Buffer (see Reagents, Indica-
tors, and Solutions—Buffer Solutions); 900 mL 0.1
Apparatus 1: 75 rom 1.00
Time: 15 min imidi
Spectrometric conditions 1.45
ode: UV Any other unknown im-
Detector: UV 231 nm for Tablets containing up to
10 mg of minoxidil, and UV 287 nm for Tablets con-
Tc ie _—
taining more than 10 mg of minoxidil
Standard solution: USP Minoxidil RS in Medium 2 2,4,-Diamin-6-chloro-pyrimidine 3 oxide.
Sample solution: Sample per Dissolution (711). Dilute » 2,4,-Diamin-6-chloro-pyrimidine.
with Medium to a concentration that is similar to that of ¢ 2,4,-Diamin-6-pipridino-pyrimidine.
the Standard solution. * Total impurities is the sum of all the impurities, including process-related
impurities. Disregard peaks less than 0.05%.
Tolerances: NLT 75% (Q) of the labeled amount of mi-
** A process-related impurity that is controlled in the drug substance.
noxidil (CyHisNsO) is dissolved.
o UNIFORMITY OF DOSAGE UNITs (905): Meet the ADDITIONAL REQUIREMENTS
requirements e PACKAGING AND STORAGE: Preserve in tight containers.
e USP REFERENCE STANDARDS (11)
IMPURITIES USP Minoxidil RS
e ORGANIC IMPURITIES
Diluent: Methanol and water (50:50)
Mobile phase: Dissolve 2.0 g of sodium lauryl sulfate in
a mixture of methanol, glacial acetic acid, and water
(60:1:40). Adjust with perchloric acid to a pH of 3.0 +
0.1, and pass throughasuitable filter of 0.45-um pore Minoxidil Topical Solution
size.
Standard solution: 5 g/mL of USP Minoxidil RS in DEFINITION
Diluent Minoxidil Topical Solution contains NLT 90.0% and NMT
Sample solution: Nominally equivalent to 0.25 mg/mL 110.0% of the labeled amount of minoxidil (CoHisNsO).
of minoxidil in Diluent from powdered tablets (NLT 20). IDENTIFICATION
Initially add Diluent to about 60% of the volume of the e A. INFRARED ABSORPTION (197M)
flask, shake on a mechanical shaker for 20 min, and Sample: Evaporate 1 mL of the Topical Solution under a
then dilute with Diluent to volume. Pass through a suit- stream of nitrogen while heating at 50°.
able filter of 0.45-11m pore size. Acceptance criteria: Meets the requirements
od
“”
Chromatographic system
a (See Chromatography (621), System Suitability.)
e B. The retention time of the major peak of the Sample
ne
i] solution corresponds to that of the Standard solution, as
Mode: LC
Dp obtained in the Assay.
° Detector: UV 280 nm
te Column: 3.9-mm x 15-cm; 3-10-wm packing L1 ASSAY
6 Flow rate: 0.5 mL/min e PROCEDURE
= Run time: NLT 3 times the retention time of the main Solution A: Add 0.65 mL of heptafluorobutyric acid to
os eak a 1000-mL volumetric flask. Dilute with water to
“ iiestian volume: 40uL volume.
— System suitability Solution B: Acetonitrile
Sample: Standard solution Mobile phase: See Table 1.
Suitability requirements
Tailing factor: NMT 2.0
Table 1
Relative standard deviation: NMT 2.0%
Analysis Solution A Solution B
Samples: Sample solution and Standard solution %
Calculate the percentage of each individual impurity in
the portion of the Tablets taken:
Result = (ru/rs) x (Cu/Cs) x 100
1
ru = peak response of each impurity from the
Sample solution Diluent: Methanol and water (50:50)
ls = peak response of minoxidil from the Standard System suitability solution: 0.4 mg/mL of USP Minox-
solution idil RS and 0.001 mg/mL of USP Minoxidil Related
Cs = concentration of USP Minoxidil RS in the CompoundE RS in Diluent
Standard solution (mg/mL) Standard solution: 0.05 mg/mL of USP Minoxidil RS in
Cy = nominal concentration of minoxidil in the Diluent
Sample solution (mg/mL) Sample solution: Nominally 0.05 mg/mL of minoxidil
Acceptance criteria: See Table 1. in Diluent
Chromatographic system
(See Chromatography (621), System Suitability.)
USP 41 Official Monographs / Mirtazapine 2763

Mode: LC Table 2
Detector: UV 280 nm Relative Acceptance
Column: 2.1-mm x 10-cm; 1.7-4m packing L1 Retention Criteria,
Column temperature: 35° Name Time NMT (%)
Flow rate: 0.4 mL/min
Injection volume: 1 ul Minoxidil related
System suitability compound A
Samples: System suitability solution and Standard (pyrimidine oxide i
analog)?»_ 0.36
solution
Suitability requirements Pyrimidine analog? 0.51 =
Resolution: NLT 1.5 between minoxidil and minoxidil Minoxidil 1.00 —
related compound E, System suitability solution Minoxidil related
Relative standard deviation: NMT 1.0%, Standard compound E —
solution (deoxyminoxidil)®< 1.03
Analysis Individual
Samples: Standard solution and Sample solution unspecified iis
Calculate the percentage of the labeled amount of mi- impurity 0.2
ee (CsHisNsO) in the portion of Topical Solution Total impurities = 2.0
taken:
2 2,6-Diamino-4-chloropyrimidine 1-oxide
Result = (ru/rs) x (Cs/Cu) x 100 » Process impurity included in the table for identification only. Process
impurities are controlled in the drug substance, and are not to be report-
ed or included in the total impurities for the drug product.
tu = peak response from the Sample solution ¢ 6-Chloropyrimidine-2,4-diamine.
ts = peak response from the Standard solution 4 6-(Piperidin-1-yl)pyrimidine-2,4-diamine.
Cs = concentration of USP Minoxidil RS in the
Standard solution (mg/mL) ADDITIONAL REQUIREMENTS
G = nominal concentration of minoxidil in the © PACKAGING AND STORAGE: Preserve in tight containers.
Sample solution (mg/mL) e USP REFERENCE STANDARDS (11)
Acceptance criteria: 90.0%-110.0% USP Minoxidil RS
USP Minoxidil Related Compound E RS
IMPURITIES 6-(Piperidin-1-yl)pyrimidine-2,4-diamine.
© ORGANIC IMPURITIES CoHisNs = 193.25
Solution A, Solution B, Mobile phase, Diluent, System
suitability solution, and Chromatographic system:
Proceed as directed in the Assay.
Standard solution: 0.4 g/mL of USP Minoxidil RS in
Diluent Mirtazapine
Sample solution: Nominally 0.4 mg/mL of minoxidil in =
Diluent. Pass througha suitable filter of 0.2-um pore
size. 7 o S
System suitability
Samples: System suitability solution and Standard
solution
Suitability requirements
C
Nt
Ho
Ee
Xo}
Resolution: NLT 1.5 between minoxidil and minoxidil =}
related compound E, System suitability solution ey
Relative standard deviation: NMT 2.8%, Standard CizHigNs3 265.35 cS
solution Pyrazino[2, 1-a]pyrido[2,3-c][2]benzazepine,1,2,3,4,10,14b- 7)
Analysis ew aepieceaiae
Samples: Standard solution and Sample solution 1,2,3,4,10,14b-Hexahydro-2-methylpyrazino[2,1-a]pyrido
Calculate the percentage of each unspecified impurity [2,3-c][2]-benzazepine [85650-52-8].
in the portion of Topical Solution taken:
DEFINITION
Result = (ru/rs) x (Cs/Cu) x 100 Mirtazapine contains NLT 98.0% and NMT 102.0% of
mirtazapine (C;7HisN3), calculated on the anhydrous basis.
tu = peak response of each unspecified impurity
from the Sample solution IDENTIFICATION
Is = peak response of minoxidil from the Standard e A. INFRARED ABSORPTION (197K)
solution e B. The retention time of the major peak of the Sample
Gs = concentration of USP Minoxidil RS in the solution corresponds to that of the Standard solution, as
Standard solution (mg/mL) obtained in the Assay.
Cu = nominal concentration of minoxidil in the ASSAY
Sample solution (mg/mL) ¢ PROCEDURE
Acceptance criteria: See Table 2. Disregard any impu- Diluent: Acetonitrile and water (1:1)
rity peaks less than 0.05%. Buffer: Dissolve 18 g of tetramethylammonium hydrox-
ide pentahydrate in 950 mL of water. Adjust with phos-
phoric acid to a pH of 7.4. Dilute with water to 1 L.
Mobile phase: Acetonitrile, methanol, tetrahydrofuran,
and Buffer (15: 12.5: 7.5: 65)
Standard solution: 0.3 mg/mL of USP Mirtazapine RS
in Diluent
Sample solution: 0.3 mg/mL of Mirtazapine in Diluent
Chromatographic system
(See Chromatography (621), System Suitability.)
 2764 Mirtazapine / Official Monographs USP 41

Mode: LC rs = peak response of mirtazapine from the

Detector: UV 290 nm Standard solution
Column: 4.6-mm x 25-cm; packing L1 Gs = concentration of USP Mirtazapine RS in the
Column temperature: 40° Standard solution (mg/mL)
Flow rate: 1.5 mL/min Cy = concentration of Mirtazapine in the Sample
Injection volume: 10 pL solution (mg/mL)
System suitability F = relative response factor (see Table 1)
Sample: Standard solution [Not&—Disregard any peak with a result of 0.05% or
Suitability requirements less, as calculated using the formula given above.]
Column efficiency: NLT 7000 theoretical plates Acceptance criteria: See Table 1.
Tailing factor: NMT 2.0
Relative standard deviation: NMT 1.0% Table 1
Analysis
Samples: Standard solution and Sample solution Relative Relative | Acceptance;
Calculate the percentage of mirtazapine (C;7Hi9N3) in Retention Response Criteria,
the portion of Mirtazapine taken: impurity Name Time Factor NMT (%)
Mirtazapine N-oxide 0.2 0.8 0.1
Result = (ru/rs) x (Cs/Cu) x 100 Acyclomirtazapine al-
cohol? 0.3 0.8 0.1
ty = peak response from the Sample solution
1-Ketomirtazapinec 0.35 1.0 0.1
rs = peak response from the Standard solution
G = concentration of USP Mirtazapine RS in the Desmethylmirtazapine? 0.4 1.0 0.1
Standard solution (mg/mL) Mirtazapine 1.0 = =
Cu = concentration of Mirtazapine in the Sample Acyclomirtazapine
solution (mg/mL) methyl derivatives 1.3, 1.0 0.1
Acceptance criteria: 98.0%-102.0% on the anhydrous 10-Ketomirtazapinet 1.35 5.0 0.1
basis Any individual unspeci- _
IMPURITIES fied impurity 1.0 0.10
e RESIDUE ON IGNITION (281): NMT 0.1% Total impurities = = 0.5
[Note—Disregard any peak representing less than 0.05% of the main
peak and any peak that is due to the Diluent.]
Delete the following: 2 1,2,3,4,10,14b-Hexahydro-2-methylpyrazino[2, 1-a]pyrido[2,3-c]
[2]benzazepine 2-oxide (Impurity A).
°e HEAVY METALS, Method II (231): NMT 10 ug/ge cortices 1- »(2-(4-Methyl-2-phenylpiperazin-1-yl)pyridin-3-yl)methanol (Impurity B).
Jan-2018) ¢(2-Methyl-3,4,10,14b-tetrahydrobenzo[c]pyrazino[1,2-a]pyrido[3,2-
© ORGANIC IMPURITIES flazepin-1(2H)-one (Impurity C).
Diluent, Buffer, and Mobile phase: Prepare as directed 41,2,3,4,10,14b-Hexahydropyrazino[2, 1-a]pyrido[2,3-c][2]benzazepine
(Impurity D).
£
vw
in the Assay.
oy System suitability solution: 1.5 mg/mL of USP ¢4-Methyl-1-(3-methylpyridin-2-yl)-2-phenylpiperazine (Impurity E).
‘2-Methyl-1,2,3,4-tetrahydrobenzo[c]pyrazino[1,2-a]pyrido[3,2-flazepin-
i] Mirtazapine Resolution Mixture RS in Diluent
=
Dd Standard solution: 0.0015 mg/mL of USP Mirtazapine 10(14bH)-one (Impurity F).
i) RS in Diluent
= SPECIFIC TESTS
iS Sample solution: 1.5 mg/mL of Mirtazapine in Diluent e@ WATER DETERMINATION, Method | (921): NMT 3.5%
= Chromatographic system © OPTICAL ROTATION, Specific Rotation (781S)
3
(See Chromatography (621), System Suitability.) Sample solution: ‘10 mg/mL, in denatured alcohol
a) Mode: LC Acceptance criteria: +2° to -2°
= Detector: UV 240 nm
Column: 4.6-mm x 25-cm; packing L1 ADDITIONAL REQUIREMENTS
Column temperature: 40° © PACKAGING AND STORAGE: Preserve in tight containers,
Flow rate: 1.5 mL/min and store at controlled room temperature.
Injection volume: 10 uL © LABELING: Label it to indicate whether it is anhydrous or
Run time: Twice the retention time of mirtazapine hemihydrate.
System suitability e¢ USP REFERENCE STANDARDS (11)
Samples: System suitability solution and Standard USP Mirtazapine RS
solution USP Mirtazapine Resolution Mixture RS
[Note—The relative retention times are listed in Table 7. This resolution mixture contains approximately 0.1% w/
w each of the following:
Suitability requirements Impurity A: 1,2,3,4,10,14b-Hexahydro-2-methylpyrazino
Resolution: NLT 1.5 between acyclomirtazapine [2,1-a]pyrido[2,3-c][2]benzazepine 2-oxide.
methyl derivative (impurity E) and 10-ketomirtazapine Impurity B: (2-(4-Methyl-2-phenylpiperazin-1-yl)pyridin-
(impurity F), System suitability solution 3-yl)methanol.
Tailing factor: NMT 2.0, Standard solution Impurity C: (2-Methyl-3,4,10,14b-tetrahydrobenzo
Relative standard deviation: NMT 10.0%, Standard [apyrazinolt,2-alpyridels.2-flazepipel 2H)-one.
solution Impurity D: 1,2,3,4,10,14b-Hexahydropyrazino[2,1-
Analysis dpycdola.4¢clgibencazepine,
Samples: Standard solution and Sample solution Impurity E: 4-Methyl-1-(3-methylpyridin-2-yl)-
Calculate the percentage of each impurity in the por- 2-phenylpiperazine.
tion of Mirtazapine taken: Impurity F: 2-Methyl-1,2,3,4-tetrahydrobenzo[c]pyrazino
[1,2-alpyrido[3,2-Hazepine| 0(14bH)-one.
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
tu = peak response of any impurity from the
Sample solution
USP 41 Official Monographs / Mirtazapine 2765

Acceptance criteria: 90.0%-110.0%

Mirtazapine Tablets PERFORMANCE TESTS
e DISSOLUTION (711)
DEFINITION Medium: 0.1 N hydrochloric acid; 900 mL
Mirtazapine Tablets contain NLT 90.0% and NMT 110.0% Apparatus 2: 50 rpm
of the labeled amount of mirtazapine (Ci7Hi9Ns). Time: 15 min
Sample solution: Pass a portion of the solution under
IDENTIFICATION test through a suitable filter. Dilute with Medium, if
e A. INFRARED ABSORPTION (197K) necessary.
Extraction mixture: n-Hexane and water (1:1) Standard solution: USP Mirtazapine RS in Medium in a
Sample: Transfer an amount equivalent to 30 mg of concentration similar to the one expected in the Sample
mirtazapine from finely powdered Tablets to a suitable solution
centrifuge tube. Add Extraction mixture to obtain a solu- Instrumental conditions
tion of T mg/mL of mirtazapine in n-hexane. Shake for (See Ultraviolet-Visible Spectroscopy (857).)
5 min, and centrifuge. Decant, and evaporate the Mode: UV
supernatant. Analytical wavelength: 315 nm
Standard: Dissolve USP Mirtazapine RS in Extraction Analysis
mixture to obtain a solution having a concentration of Samples: Sample solution and Standard solution
about 1 mg/mL of mirtazapine in n-hexane. Shake for 5 Calculate the percentage of the labeled amount of
min, and centrifuge. Decant, and evaporate the mirtazapine (Ci7Hi9N3) dissolved:
supernatant.
e B. The retention time of the major peak of the Sample Result = (Au/As) x Cs x Vx (1/L) x 100
solution corresponds to that of the Standard solution, as
obtained in the Assay. Au = absorbance of the Sample solution
As = absorbance of the Standard solution
ASSAY Cs = concentration of USP Mirtazapine RS in the
¢ PROCEDURE Standard solution (mg/mL)
Diluent: Acetonitrile and water (1:1) Vv = volume of the Medium, 900 mL
Buffer: Dissolve 18.0 g of tetramethylammonium hy- L = label claim (mg/Tablet)
droxide pentahydrate in 950 mL of water. Adjust with Tolerances: NLT 80% (Q) of the labeled amount of
Pigsprcre acid to a pH of 7.4, and dilute with water mirtazapine (Ci7Hi9Ns) is dissolved.
to 1L. e UNIFORMITY OF DOSAGE UNITS (905): Meet the
Mobile phase: Acetonitrile, methanol, tetrahydrofuran, requirements
and Buffer (15: 12.5: 7.5: 65)
Standard solution: 0.3 mg/mL of USP Mirtazapine RS IMPURITIES
in Diluent © ORGANIC IMPURITIES
Sample solution: Nominally 0.3 mg/mL of mirtazapine Diluent, Buffer, and Mobile phase: Proceed as di-
(from an amount equivalent to the weight of 1 Tablet rected in the Assay. (a
~“
from NLT 20 finely powdered Tablets) in Diluent. Shake System suitability solution: 1.5 mg/mL of USP a)
vigorously for 10 min, centrifuge an aliquot, and use Mirtazapine Resolution Mixture RS in Diluent
the clear supernatant. Standard solution: 0.015 mg/mL of USP Mirtazapine E
re}
Chromatographic cago RS in Diluent Cs
(See Chromatography (621), System Suitability.) Sample solution: 1.5 mg/mL of mirtazapine (from an i}
Mode: LC amount equivalent to the weight of 1 Tablet from NLT =oy
Detector: UV 290 nm 20 finely powdered Tablets) in Diluent. Shake vigorously By
Column: 4.6-mm x 25-cm; packing L1 ie}
for 10 min, centrifuge an aliquot, and use the clear me
Column temperature: 40° supernatant. “

Flow rate: 1.5 mL/min Chromatographic system

Injection volume: 10 uL (See Chromatography (621), System Suitability.)
System suitability Mode: LC
Sample: Standard solution Detector: UV 240 nm
Suitability requirements Column: 4.6-mm x 25-cm; packing L1
Column efficiency: NLT 7000 theoretical plates Column temperature: 40°
Tailing factor: NMT 2.0 Flow rate: 1.5 mL/min
Relative standard deviation: NMT 1.5% Injection volume: 10 uL
Analysis Run time: 2 times the retention time of mirtazapine
Samples: Standard solution and Sample solution System suitability
Calculate the percentage of the labeled amount of Samples: System suitability solution and Standard
mirtazapine (Ci7Hi9N3) in the portion of Tablets taken: solution
eo relative retention times are listed in Table
Result = (ru/rs) x (Cs/Cu) x 100 Ts
Suitability requirements
ty = peak response from the Sample solution Resolution: NLT 1.5 between acyclomirtazapine
ls = peak response from the Standard solution methyl derivative (impurity E) and 10-ketomirtazpine
Cs = concentration of USP Mirtazapine RS in the (impurity F), System suitability solution
Standard solution (mg/mL) Tailing factor: NMT 2.0, Standard solution
Cu = nominal concentration of the Sample solution Relative standard deviation: NMT 10.0%, Standard
(mg/mL) solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the por-
tion of Tablets taken:
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100
 2766 Mirtazapine / Official Monographs USP 41

ry = peak response of any impurity from the Impurity F: 2-Methyl-1,2,3,4-tetrahydrobenzo[c]pyrazino

Sample solution [1,2-a]pyrido[3,2-flazepin-10(14bH)-one.
rs = mirtazapine peak response from the Standard
solution
Cs = concentration of USP Mirtazapine RS in the
Standard solution (mg/mL)
Cu = nominal concentration of the Sample solution Mirtazapine Orally Disintegrating
(mg/mL) Tablets
F = relative response factor for the corresponding
impurity (see Table 1)
Acceptance criteria: See Table 1. DEFINITION
Mirtazapine OrallyDisintegrating Tablets contain NLT 90.0%
and NMT 110.0% of the labeled amount of mirtazapine
Table 1
(Ci7HigNs).
Relative Relative Acceptance
Retention Response Criteria, IDENTIFICATION
Name Time Factor NMT (%)
e A. INFRARED ABSORPTION (197K)
Standard solution: Dissolve 30 mg of USP Mirtazapine
Mirtazapine N-oxide? 0.2 0.8 0.2 RS in a separatory funnel containing 30 mL of water,
Acyclomirtazapine al- _ = and add 30 mL of n-hexane. Shake vigorously for 5
coholbs 0.3 min. Allow the solution to separate into two layers. Fil-
1-Ketomirtazpines 0.35 1.0 0.2 ter the n-hexane layer through glass wool, and evapo-
Desmethylmirtaza- — os rate to dryness.
piness 0.4 Sample solution: Transfer a quantity of finely powdered
Mirtazapine 1.0 = = Tablets, equivalent to 30 mg of mirtazapine, to a sepa-
Acyclomirtazapine _ _ ratory funnel. Add 30 mL of water and 30 mL of n-hex-
methyl derivativees 13 ane. Shake vigorously for 5 min. Allow the solution to
separate into two layers. Filter the n-hexane layer
10-Ketomirtazapine! 1.35 5.0 0.2 through glass wool, and evaporate to dryness.
Any individual unspeci- e B. The retention time of the major peak of the Sample
fied degradation _- solution corresponds to that of the Standard solution, as
product 1.0 0.2 obtained in the Assay.
Total impurities — = 2.0
[Note—Disregard any peak representing less than 0.05% of the main ASSAY
peak and any peak that is due to the Diluent.] © PROCEDURE
21,2,3,4,10,1 4o-Hexahydro 2 methylpyrazinalé,1 -alpyrido[2,3-c] Diluent: Acetonitrile and water (50:50)
[2]benzazepine 2-oxide. (Impurity A) Diluted phosphoric acid: Water and phosphoric acid
» (2-(4-Methyl-2-phenylpiperazin-1-yl)pyridin-3-yl)methanol. (Impurity B) (1000:3)
ze
“
¢ (2-Methyl-3,4,10,14b-tetrahydrobenzo[c]pyrazino[1,2-a]pyrido[3,2- Buffer: Dissolve 1 g of monobasic potassium phosphate
a flazepin-1(2H)-one. (Impurity C) and 1.7 g of pentanesulfonic acid sodium salt in 1 L of
ig- 4 1,2,3,4,10,14b-Hexahydropyrazino[2,
1-a]pyrido[2,3-c][2]benzazepine. water. Adjust with Diluted phosphoric acid to a pH of 4.7
aD (Impurity D) + 0.1, and filter.
3° © 4-Methyl-1-(3-methylpyridin-2-yl)-2-phenylpiperazine. (Impurity E) Mobile phase: Acetonitrile and Buffer (25:75)
fo ‘ 2-Methyl-1,2,3,4-tetrahydrobenzo[c]pyrazino[1,2-a]pyrido[3,2-flazepin- Standard stock solution: 0.3 mg/mL of USP
° 10(14b#H)-one. (Impurity F)
= 9 Process impunt /. Included for identification purposes only. Not to be
Mirtazapine RS in Diluent
Standard solution: 0.036 mg/mL of USP Mirtazapine
re included in Total impurities.
vo ADDITIONAL REQUIREMENTS
RS in Mobile phase from the Standard stock solution
> Sample stock solution: 0.3 mg/mL of mirtazapine in
¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant Diluent (from NLT 20 Tablets, finely powdered). Soni-
containers, and store at controlled room temperature. cate for 15 min with occasional swirling, and shake for
e USP REFERENCE STANDARDS (11) 30 min. [NoTE—Alternatively, dissolve 10 Tablets in a
USP Mirtazapine RS volume of a mixture of acetonitrile and water (90:10)
USP Mirtazapine Resolution Mixture RS to obtain a 0.3 mg/mL solution of mirtazapine. Shake
This resolution mixture contains approximately 0.1% or stir until the mixture is free from lumps.]
w/w of each of the following: Sample solution: Nominally, 0.036 mg/mL of
Impurity A: 1,2,3,4,10,14b-Hexahydro-2-methylpyrazino mirtazapine in Mobile phase obtained as follows: transfer
[2,1-a]pyrido[2,3-c][2]benzazepine 2-oxide. 40 mL of the Sample stock solution into a centrifuge
Impurity B: (2-(4-Methyl-2-phenylpiperazin-1 -yl)pyridin- tube, and centrifuge at 3000 rpm for 10 min. Transfer
3-yl)methanol. 6.0 mL of the supernatant into a 50-mL volumetric
Impurity C: (2-Methyl-3,4,10,14b-tetrahydrobenzo flask, and dilute with Mobile phase to volume. Pass the
[apyrazino[1,2-alpyridol3,2-flazepin-1 2H)-one. portion through a polypropylene membrane filter of
Impurity D: 1,2,3,4,10,14b-Hexahydropyrazino[2, 1- 0.45-um pore size. Discard at least the first 5 mL of
alpyrido[2,3-c][2]benzazepine. filtrate.
Impurity E: 4-Methyl-1-(3-methylpyridin-2-yl)- Chromatographic system
2-phenylpiperazine. (See Chromatography (621), System Suitability.)
USP 41 Official Monographs / Mirtazapine 2767

Mode: LC Table 1 (Continued)

Detector: UV 290 nm Time Solution A Solution B
Column: 4.6-mm x 15-cm; 5-m packing L1 (min) (%) (%)
Flow rate: 1 mL/min 18.5 61 39
Injection size: 20 uL
System suitability 22.0 61 39
Sample: Standard solution
Suitability requirements System suitability solution: 0.3 mg/mL of USP
Tailing factor: NMT 2.0 Mirtazapine RS in Diluent
Relative standard deviation: NMT 2.0% Standard solution: 0.015 mg/mL each of USP
Analysis Mirtazapine RS, USP Mirtazapine Related Compound A
Samples: Standard solution and Sample solution RS, USP Mirtazapine Related CompoundB RS, USP
Calculate the percentage of the labeled amount of Mirtazapine Related CompoundCRS, and USP
mirtazapine (C;7HieNs) in the portion of Tablets taken: Mirtazapine Related CompoundD RS in Diluent
Sample solution: Nominally, 1.5 mg/mL of mirtazapine
Result = (ru/rs) x (Cs/Cu) x 100 in Diluent from NLT 5 Tablets
Chromatographic system
ty = peak response from the Sample solution (See Chromatography (621), System Suitability.)
Is = peak response from the Standard solution Mode: LC
Cs = concentration of USP Mirtazapine RS (mg/mL) Detector: UV 240 nm
Cu = nominal concentration of mirtazapine in the Column: 4.6-mm x 25-cm; 5-um packing L1
Sample solution (mg/mL) Column temperature: 40°
Acceptance criteria: 90.0%-110.0% Flow rate: 1.2 mL/min
Injection size: 10 pL
PERFORMANCE TESTS System suitability
e DISINTEGRATION (701): NMT 60 s Samples: System suitability solution and Standard
e DISSOLUTION (711) solution
Medium: 0.1 N hydrochloric acid; 900 mL Suitability requirements
Apparatus 2: 50 rpm Tailing factor: NMT 2.0, System suitability solution
Time: 15 min Relative standard deviation: NMT 2.0%, System suit-
Sample solution: Sample per Dissolution (711). Pass ability solution
throughafilter of 0.45-t1m pore size, and discard the Resolution: NLT 4.0 between the mirtazapine and
first 5 mL of the filtrate. mirtazapine related compound D peaks, Standard
Standard solution: 33 j1g/mL of USP Mirtazapine RS in solution
Medium Analysis
Instrumental conditions Samples: Standard solution and Sample solution
(See Ultraviolet-Visible Spectroscopy (857).) Calculate the percentage of each individual specified
Mode: UV impurity in the portion of Tablets taken: S
phe wavelength: 316 nm “
Blank: Medium Result = (ru/rs) x (Cs/Cu) x 100 a)
Cell: 0.5m =
Analysis: Determine the percentage of mirtazapine ru = peak response of each individual specified S
(Ci7HigN3) dissolved: impurity from the Sample solution =
peak response of the corresponding related )
Result = (Au/As) x Cs x Vx (1/L) x 100
ls
compound from the Standard solution (re)“
Gs concentration of each individual impurity in i
3
Au = absorbance of the Sample solution the Standard solution (mg/mL) =
As = absorbance of the Standard solution CG = nominal concentration of mirtazapine in the “

Cs = concentration of USP Mirtazapine RS in the Sample solution (mg/mL)

Standard solution (mg/mL) Calculate the percentage of each individual unspecified
Vv = volume, 900 mL impurity in the portion of Tablets taken:
L = label claim of mirtazapine (mg/Tablet)
Tolerances: NLT 80% (Q) of the labeled amount of Result = (ru/rs) x (Cs/Cu) x 100
mirtazapine (Ci7HieNs) is dissolved.
© UNIFORMITY OF DOSAGE UNITS (905): Meet the tu = peak response of each individual impurity
requirements from the Sample solution
rs = peak response of mirtazapine from the
IMPURITIES Standard solution
© ORGANIC IMPURITIES Gs = concentration of mirtazapine in the Standard
Solution A: Dissolve 7.2 g of tetramethylammonium hy- solution (mg/mL)
droxide pentahydrate in 4 L of water. Add 1 mL of tri- Cu = nominal concentration of mirtazapine in the
ethylamine. Adjust with phosphoric acid to a pH of 7.4. Sample solution (mg/mL)
Solution B: Acetonitrile, methanol, and tetrahydrofuran Acceptance criteria: See Table 2. [NoTe—Disregard
(170:145:85) any peak less than 0.05%.]
Diluent: Acetonitrile and water (50:50)
Mobile phase: See Table 7. Table 2
Relative Acceptance
Table 1
Retention Criteria,
Time Solution A Solution B Name Time NMT (%)
in % Mirtazapine related
compound B 0.23 0.5
61 Mirtazapine related
46 compound C 0.51 0.5
 2768 Mirtazapine / Official Monographs USP 41

Table 2 (Continued) Standard solution: 5.0 mg/mL of USP Misoprostol RS

[ Relative Acceptance
in Mobile phase
Retention Criteria,
Sample solution: 5.0 mg/mL of Misoprostol in Mobile
Name Time NMT (%)
phase
Chromatographic system
Mirtazapine related (See Chromatography (621), System Suitability.)
compound A 0.62 0.5 Mode: LC
Mirtazapine 1.0 = Detector: UV 210 nm
Mirtazapine related Column: 4.6-mm x 25-cm; 5-um packing L3
compound D 1.3 0.5 Flow rate: 2 mL/min
Any individual un- Injection size: 20 pL
specified degrada- — System suitability
tion product 0.5 Sample: Standard solution
Total impurities — 3.0 [Note—Identify the impurities based on the retention
times shown in Impurity Table 1.]
Suitability requirements
ADDITIONAL REQUIREMENTS Resolution: NLT 1.2, between the second diastere-
e PACKAGING AND STORAGE: Store at controlled room tem- ame, peak for 12-epimisoprostol and the Misoprostol
perature. Protect from light and moisture. peal
e USP REFERENCE STANDARDS (11) Relative standard deviation: NMT 1.0%, for three
USP Mirtazapine RS replicate injections
USP Mirtazapine Related Compound A RS Analysis
1,2,3,4,10, 14b-Hexahydropyrazino[2,1-a]pyrido[2, 3-c] Samples: Standard solution and Sample solution
[2]benzazepine. Calculate the percentage of C22H3gOs in the portion of
CisHizN3 251.33 Misoprostol taken:
USP Mirtazapine Related Compound B RS
1,2,3,4,10,14b-Hexahydro-2-methylpyrazino[2,1- Result = (ru/ts) x (Cs/Cu) x 100
a]pyrido[2,3-c][2]benzazepine 2-oxide monohydrate.
GizHigN30-H2O0 299,36 tu = peak response of the Sample solution
USP Mirtazapine Related Compound C RS rs = peak response of the Standard solution
2-Methyl-3,4,10,14b-tetrahydrobenzo[c]pyrazinof[1 ,2- Cs = concentration of the Standard solution
alpyrido[3,2-flazepin-1(2H)-one. (mg/mL)
Ci7Hi7N30 279.34 Cu = concentration of the Sample solution (mg/mL)
USP Mirtazapine Related Compound D RS Acceptance criteria: 97.0%-102.0% on the anhydrous
2-Methyl-1,2,3,4-tetrahydrobenzo[c]pyrazino[1,2- basis
alpyrido[3,2-flazepin-10(14bH)-one.
Ci7Hi7N30 279.34 IMPURITIES
a Organic Impurities
<= © PROCEDURE 1
a Mobile phase, Standard solution, Sample solution,
Ss
J Chromatographic system, and System suitability:
fo) Proceed as directed in the Assay.
° Misoprostol
iS Analysis
S Samples: Standard solution and Sample solution
=
A
Record the chromatogram for at least 3 times the
é retention time of the Misoprostol peak, and measure
Cem os
2
a) the peak responses. Identify the impurities based on
=) the retention times shown in Impurity Table 1.
eo NR Calculate the percentage of each impurity in the por-
tion of Misoprostol taken:
CooHagOs 382.53 Result = (ru/ts) x (Cs/Cu) x (1/F) x 100
Prost-13-en-1-oic acid, 11,16-dihydroxy-16-methyl-9-oxo-,
methyl ester, (1R*,2R*,3R*,E)-; tu = peak response of each impurity from the
(+)-Methyl (12,2R,3R)-3-hydroxy-2-[(E)-(4RS)-4-hydroxy- Sample solution
4-methyl-1-octenyl]-5-oxocyclopentaneheptanoate Ts = peak response of the Standard solution
[59122-46-2]. Cs = concentration of USP Misoprostol RS in the
Standard solution (mg/mL)
DEFINITION Cu = concentration of Misoprostol in the Sample
Misoprostol contains NLT 97.0% and NMT 102.0% of
C22H32Os, calculated on the anhydrous basis. solution (mg/mL)
F = relative response factor (see Impurity Table 1)
IDENTIFICATION Acceptance criteria
© A. INFRARED ABSORPTION (1975) Individual impurities: See Impurity Table 1.
Sample solution: 30 mg/mL Total impurities: NMT 1.5%
Medium: Chloroform
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
ASSAY
e PROCEDURE
Mobile phase: 2,2,4-Trimethylpentane, dioxane, and
acetonitrile (78:21.5:0.5)
USP 41 Official Monographs / Misoprostol 2769

Impurity Table 1 e USP REFERENCE STANDARDS (11)

Relative Relative Acceptance USP Misoprostol RS
Retention Response Criteria,
Name Time Factor NMT (%)
A-Type misoprostol® 0.22 7.8 0.1
B-Type misoprostol? 0.33 0.80 0.1 Misoprostol Dispersion
Norprostole O:51 8.4 0.1
8-Epimisoprostol¢ 0.71 1.05 0.3 DEFINITION
0.86 and Misoprostol Dispersion is a mixture of Misoprostol and Hy-
12-Epimisoprostole 0.92! 1.08 1.0! promellose. It contains NLT 95.0% and NMT 104.0% of
Misoprostol 1.0 im = the labeled amount of misoprostol (C22H33Os).
Autre dict ste ta IDENTIFICATION
SnpUEY 7 : : e A. ULTRAVIOLET ABSORPTION
@ Methyl 7-{(1R*,2.5*)-2-[(E)-4-hydroxy-4-methyloct-1-enyl]-5-oxocyclopent-
3-enyl}heptanoate.
Perform both Procedure 1 and Procedure 2.
» (E)-Methyl 7-[2-(4-hydroxy-4-methyloct-1-enyl)-5-oxocyclopent-1-
Procedure 1
enyl]heptanoate. Medium: Methanol and water (4:1)
¢ Methyl 7-(3-hydroxy-5-oxocyclopent-1-enyl)heptanoate. Sample solution: Nominally 16 g/mL of misoprostol
4 Methyl (1 5*,2R*,3R*)-3-hydroxy-2-[(£)-4-hydroxy-4-methyl-1-octenyl]-5- in Mediumpreparect as follows. Dissolve the amount of
oxocyclopentaneheptanoate. Misoprosto! Dispersion, equivalent to 400 1g of mis-
© Methyl! (1 S*,2R*,35*)-3-hydroxy-2-[(£)-4-hydroxy-4-methyl-1-octenyl]-5- oprostol, in 25 mL of Medium.
oxocyclopentaneheptanoate. Blank: Prepare a solution of hypromellose in Medium
112-Epimisoprostol consists of two diastereomers that are separated under having the same concentration as in the Sample
these conditions; integrate both peaks together for the impurity calcula-
tions.
solution.
Analysis: Determine UV absorption spectrum of Sam-
© PROCEDURE 2: CONTENT OF DIASTEREOMERS ple solution against the Blank from 330-230 nm.
Mobile phase: Hexane, ethanol, and isopropyl alcohol Acceptance criteria 1: It exhibits no maximum near
(94:4:2 280 nm.
Sample solution: 1.0 mg/mL of Misoprostol in Mobile Procedure 2
phase von Methanol and 1 N potassium hydroxide
Chromatographic system (4:1
(See Chromatography (621), System Suitability.) Sample solution: Add 10 mL of Medium to 10 mL of
Mode: LC the Sample solution prepared from Procedure 1. Allow
Detector: UV 205 nm to stand for 30 min at room temperature.
Column: 4.6-mm x 25-cm; 5-11m packing L3 Blank: Add 10 mL of Medium to 10 mL of the Blank
Column temperature: 40° prepared from Procedure 1. Allow to stand for 30 min
Flow rate: 1 mL/min at room temperature. c
4)
Injection size: 20 uL Analysis: Determine UV absorption spectrum of Sam- i}
System suitability ple solution against the Blank from 330-230 nm.
Sample: Sample solution Acceptance criteria 2: It exhibits a maximum near K¢
[Note—Identify the components based on their rela- i)
280 nm. =
tive retention times which are about 0.92 for the e B. The retention time of the major peak of the Sample i)
first diastereomer peak and 1.0 for the second dias- solution corresponds to that of the Standard solution, as a=
tereomer peak.] obtained in the Assay. »
Suitability requirements ao)
Reselustore NLT 2.0, between the two diastereomer ASSAY PeI
ea © PROCEDURE
Relative standard deviation: NMT 2.0% from the [Note—During addition of water to a solution of mis-
area of the first diastereomer peak oprostol in isopropyl alcohol, an exothermic reaction
Analysis takes place. After each addition of water, invert the flask
Sample: Sample solution to mix isopropyl alcohol and water. Allow the solution
Calculate the fraction of the first diastereomer in the to cool to room temperature before the final dilution.]
portion of Misoprostol taken: Buffer: 1.36 g/L of monobasic potassium phosphate in
water, adjusted with phosphoric acid to a pH of 3.0 +
Result = r/(ri+ r2) 1
Mobile phase: Isopropyl alcohol and Buffer (27:73)
n = peak response for the first diastereomer Standard stock solution: 0.5 mg/mL of USP Misopros-
rt = peak response for the second diastereomer tol RS in isopropyl alcohol. [NoTE—This solution is sta-
Acceptance criteria ble up to 28 days when stored at 5 + 3°.]
Fraction of the first diastereomer: 0.51-0.56 Standard solution: 0.1 mg/mL of USP Misoprostol RS
in water from Standard stock solution. [NoTE—This solu-
SPECIFIC TESTS tion is stable up to 7 days when stored at 5 + 3°.]
e WATER DETERMINATION, Method Ic (921): NMT 0.5% Sample solution: Nominally 0.1 mg/mL of misoprostol
prepared as follows. Place an amount of Misoprostol
ADDITIONAL REQUIREMENTS Dispersion, equivalent to about 10 mg of misoprostol,
© PACKAGING AND STORAGE: Preserve in tight containers, into a 100-mL volumetric flask, and add 25 mL of iso-
and store in a freezer.
propyl alcohol. Shake to disperse the solid, place the
solution in an ice bath, Si, and allow to cool for 10
min. Carefully add about 70 mL of water, previously
chilled in a refrigerator, remove from the ice bath, and
shake. Sonicate as necessary at a temperature not ex-
ceeding 20°. Equilibrate to room temperature, dilute
with water to volume, and immediately cool the solu-
 2770 Misoprostol / Official Monographs USP 41

tion to 5°. [NoTE—This solution is stable up to 7 days Cs = concentration of USP Misoprostol RS in the
when stored at 5 + 3°.] Diluted standard solution (g/mL)
Chromatographic system Cu = nominal concentration of misoprostol in the
(See Chromatography (621), System Suitability.) Sample solution (ug/mL)
Mode: LC F = relative response factor (see Table 1)
Detector: UV 205 nm Acceptance criteria: See Table 1. Disregard a peak from
Column: 4.6-mm x 15-cm; 5-um packing L7 the Diluent, eluting at about 4 min, and any other peak
Temperatures observed in the Blank.
Column: 50+ 2°; the Mobile phase must be
preheated prior to introduction on the column. Table 1
Autosampler: 5+ 3°
Flow rate: 1.5 mL/min Relative Relative Acceptance
Injection volume: 100 uL Retention Response Criteria,
System suitability Name Time Factor NMT (%)
Sample: Standard solution 12-Epimisoprostol 0.84 = =
[Note—USP Misoprostol RS contains 12-epimisoprostol 8-Epimisoprostol? 0.90 0.90 0.50
as a minor component. The relative retention times for Misoprostol 1.0 —_— —
12-epimisoprostol and misoprostol are 0.84 and 1.0, B-Type misoprostol« 1.6 0.78 0.30
respectively.]
Suitability requirements A-Type misoprostol¢ 1.9 2.6 0.50
Resolution: NLT 2.7 between 12-epimisoprostol and Any other individual
misoprostol impurity 1.0 0.1
Relative standard deviation: NMT 1.0% Total impurities = = 1.8
Analysis This 1s a process impurity in the manufacturing of misoprostol It is con-
Samples: Standard solution and Sample solution trolled in the misoprostol, which is the starting material for the Misopros-
tol Dispersion. This impurity is included in the table for identification only,
Calculate the percentage of the labeled amount of mis- and it is not to be reported or included in the total impurities for the
oprostol (C22H3gOs) in the portion of Misoprostol Dis- Misoprostol Dispersion.
persion taken: » Methyl (1 S*,2R*,3R*)-3-hydroxy-2-[(£)-4-hydroxy-4-methyl-1-octenyl]-5-
oxocyclopentaneheptanoate.
Result = (ru/rs) x (Cs/Cu) x 100 ¢(£)-Methy! 7-[2-(4-hydroxy-4-methyloct-1-enyl)-5-oxocyclopent-1-
enylJheptanoate
ty = peak response from the Sample solution 4 Methyl 7-[(1 R*,25S*)-2-[(B)-4-hydroxy-4-methyloct-1-enyl]-5-oxocyclopent-
Is = peak response from the Standard solution 3-enyl]heptanoate
Cs = concentration of USP Misoprostol RS in the
Standard solution (mg/mL) SPECIFIC TESTS
e WATER DETERMINATION, Method Ic (921)
Cy = nominal concentration of misoprostol in the
Sample solution (mg/mL) Sample: 300mg
al Acceptance criteria: 95.0%-104.0% Analysis: Perform the test immediately after opening
ao the sample container. Use the evaporation technique in
a IMPURITIES which water is released and evaporated from the Sam-
ie ple by heating it in an external oven at 105° and trans-
i)
°
© ORGANIC IMPURITIES
[Note—During the addition of water to a solution of ferring it to the reaction cell with the aid of an inert
¢ misoprostol in isopropyl alcohol, an exothermic reaction as.
Sj takes place. After each addition of water, invert the flask Arceataned criteria: NMT 1.0%
= to mix isopropyl alcohol and water. Allow the solution
ADDITIONAL REQUIREMENTS
a to cool to room temperature before the final dilution.]
~”“ e PACKAGING AND STORAGE: Preserve in tight containers,
Buffer, Mobile phase, Standard solution, Sample solu-
> tion, and Chromatographic system: Proceed as di-
protected from light, and store in a refrigerator.
e LABELING: The label states that this article is not intended
rected in the AS for direct administration to humans or animals. Label it
Diluent: Isopropyl alcohol and water (27:73)
Blank: Prepare a solution of hypromellose in the Diluent to indicate the nominal concentration or percentage of
having the same concentration as in the Sample misoprostol in the Misoprostol Dispersion.
e USP REFERENCE STANDARDS (11)
solution.
Diluted standard solution: 0.5 j1g/mL of USP Mis- USP Misoprostol RS
oprostol RS in Diluent from Standard solution
Sensitivity solution: 0.1 g/mL of USP Misoprostol RS
in Diluent from Diluted standard solution
System suitability
Samples: Standard solution and Sensitivity solution Mitomycin
Suitability requirements
Resolution: NLT 2.7 between 12-epimisoprostol and
misoprostol, Standard solution the rt 5 r
Signal-to-noise ratio: NLT 10, Sensitivity solution )
Analysis ee
nf oct
Samples: Sample solution, Blank, and Diluted standard C="
solution
Calculate the percentage of any individual impurity in 4 oN
the portion of Misoprostol Dispersion taken:
Result = (ru/rs) * (Cs/Cu) x (1/F) x 100 CisHisNsOs 334.33
Azirino[2’,3’:3,4]pyrrolo[1,2-a]indole-4,7-dione, 6-amino-8-
tu = peak response of any individual impurity from [[(aminocarbonyl)oxy]methyl]-1,1a,2,8,8a,8b-hexahydro-
the Sample solution 8a-methoxy-5-methyl-, [1a5-(1aa,,8B,8aa,8ba)]-;
rs = peak response of misoprostol from the Diluted
standard solution
USP 41 Official Monographs / Mitomycin 2771

(1aS,85,8aR,8bS)-(6-Amino-8a-methoxy-5-methyl-4,7-dioxo- Table 1
1,1a,2,4,7,8,8a,8b-octahydroazirino[2’,3’:3,4]pyrrolo[1 ,2- Solution B Solution C
ajindol-8-yl)methy! carbamate; %o'
Mitomycin C [50-07-7].
0
DEFINITION 0
Mitomycin has a potency of NLT 970 ug/mg of mitomycin
(CisHigN4Os). o 1
IDENTIFICATION 1 0
e A. INFRARED ABSORPTION (197M)
Analysis: Do not dry the sample and standard. Standard solution: 0.025 mg/mL of USP Mitomycin RS
Acceptance criteria: Meets the requirements in methanol
° B. The retention time of the major peak of the Sample Sensitivity solution: 2.5 g/mL of USP Mitomycin RS in
solution corresponds to that of the Standard solution, as methanol
obtained in the Assay. Sample solution: 5 mg/mL of Mitomycin in methanol
Chromatographic system
ASSAY (See Chromatography (621), System Suitability.)
© PROCEDURE Mode: LC
Mobile phase: Dissolve 1.54 g of ammonium acetate in Detector: UV 254 nm
250 mL of methanol. Add 5.0 mL of 0.83 N acetic acid Column: 4.6-mm x 25-cm; 5-um packing L1
and water to make 1000 mL. Column temperature: 30°
System suitability solution: 0.5 mg/mL of USP Mito- Flow rate: 1 mL/min
mycin RS and 7.5 mg/mL of 3-ethoxy-4-hydrox- Injection volume: 10 pL
ybenzaldehyde in N,N-dimethylacetamide System suitability
Standard solution: 0.5 mg/mL of USP Mitomycin RS in Samples: Sensitivity solution and Standard solution
N,N-dimethylacetamide Suitability requirements
Sample solution: 0.5 mg/mL of Mitomycin in N,N- Signal-to-noise ratio: NLT 10, Sensitivity solution
dimethylacetamide Tailing factor: NMT 2.0, Standard solution
Chromatographic system Relative standard deviation: NMT 5.0%, Standard
(See Chromatography (621), System Suitability.) solution
Mode: LC Analysis
Detector: UV 365 nm Samples: Standard solution and Sample solution
Column: 3.9-mm x 30-cm; 10-41m packing L11 Calculate the percentage of each impurity in the por-
Flow rate: 2 mL/min tion of Mitomycin taken:
Injection volume: 10 uL
System suitability Result = (ru/rs) x (Cs/Cu) x P x (Fi/F2) x 100
Samples: System suitability solution and Standard
solution ty = peak response of each impurity from the ce
[Nott—The relative retention times for mitomycin and Sample solution a
3-ethoxy-4-hydroxybenzaldehyde are 1.0 and 1.4, ls = peak response of mitomycin from the
respectively.] Standard solution =
Suitability requirements Cs = concentration of USP Mitomycin RS in the °
Resolution: NLT 1.8 between mitomycin and Standard solution (mg/mL) 3
3-ethoxy-4-hydroxybenzaldehyde, System suitability Cu = concentration of the Sample solution (mg/mL) a
solution P = potency of mitomycin in USP Mitomycin RS y
Tailing factor: NMT 1.3, Standard solution (ug/mg) Zz
Relative standard deviation: NMT 2.0%, Standard Fy = conversion factor, 0.001 mg/uug 2
solution Fa = relative response factor (see Table 2)
Analysis Acceptance criteria: See Table 2. The reporting thresh-
Samples: Standard solution and Sample solution old is 0.05%.
Calculate the quantity, in ug/mg, of mitomycin
(CisHigN4Os) in the portion of Mitomycin taken: Table 2
Relative Relative Acceptance
Result = (ru/rs) x (Cs/Cu) x P Retention Response Criteria,
tu = peak area from the Sample solution Name Time Factor NMT (%)
Is = peak area from the Standard solution Albomitomycin C+ 0.6 1.0 0.5
Gs = concentration of USP Mitomycin RS in the Mitomycin 1.0 =_ =
Standard solution (mg/mL) Mitomycin Be 1.2 1.0 OS
Cy = concentration of the Sample solution (mg/mL) Cinnamamide 13) 2.9 0.5
P = potency of mitomycin in USP Mitomycin RS Mitomycin Ac 1.6 1.0 0.5
(ug/mg)
Acceptance criteria: NLT 970 ug/mg 9{(1S,25,4aS,8aR,95,9aR)-7-Amino-9a-methoxy-6-methyl-5,8-dioxo-1,2,3,5,
8,8a,9,9a-octahydro-1,2,4a-metheno-(epinitrilo)pyrrolo[1,2-a]indol-9-
yl}methy! carbamate.
IMPURITIES
»{(1a5,85,8aR, 8bS)-8a-Hydroxy-6-methoxy-1,5-dimethyl-4, 7-dioxo-1,1a,2,
© ORGANIC IMPURITIES 4,7,8,8a,8b-octahydroazirino[2’,
3’:3,4]pyrrolo[1,2-a]indol-8-yl}methyl car-
Solution A: 0.77 g/L of ammonium acetate bamate.
Solution B: Methanol and Solution A (20:80) ¢{(1aS,85,8aR,8b5)-6,8a-Dimethoxy-5-methyl-4,7-dioxo-1,1a,2,4,7,8,8a,
Solution C: Methanol and Solution A (50:50) 8b-octahydroazirino[2’,3’:3,4]pyrrolo[1,2-a]indol-8-yl}methy! carbamate.
Mobile phase: See Table 1.
 2772 Mitomycin / Official Monographs USP 41

Table 2 (Continued) Sample solution: Add an accurately measured volume

Relative Relative Acceptance
of N,N-dimethylacetamide
wat :
to : 1 container
4
of Mitomycin
:
Retention | Response Criteria, pt ee i obtain a solution that is nominally
Name
individual
Time Factor NMT (%) -o mg/m or mitomycin.
Chromatographic system
Any individual (See Chromatography (621), System Suitability.)
unspecified impuri- — Mode: LC
ty _____ 1.0 05 Detector: UV 365 nm
Total impurities = 2.0 Column: 3.9-mm x 30-cm; 10-4m packing L11
@{(15,25,4aS,8aR,95,9aR)-7-Amino-9a-methoxy-6-methyl-5,8-dioxo-1,2,3,5, Flow rate: 2 mL/min
8,8a,9,9a-octahydro-1 ,2,4a-metheno-(epinitrilo)pyrrolo[1,2-a]indol-9-
yl}methyl carbamate.
Injection volume: 10 pL
» ((1aS,85,8aR,8bS)-8a-Hydroxy-6-methoxy-1,5-dimethyl-4,7-dioxo-1,1a,2,
System suitability
4,7,8,8a,8b-octahydroazirino[2’,3’°3,4]pyrrolo[1,2-ajindol-8-yl}methyl car- Samples: System suitability solution and Standard
bamate. solution
¢{(1a5,85,8aR,8b5)-6,8a-Dimethoxy-5-methyl-4,7-dioxo-1,1a,2,4,7,8,8a, [Note—The relative retention times for mitomycin and
8b-octahydroazirino[2’,3’:3,4]pyrrolo[1 ,2-a]indol-8-yl}methyl carbamate. 3-ethoxy-4-hydroxybenzaldehyde are 1.0 and 1.4,
respectively.]
SPECIFIC TESTS Suitability requirements
© CRYSTALLINITY (695): Meets the requirements Resolution: NLT 1.8 between mitomycin and
© PH (791) 3-ethoxy-4-hydroxybenzaldehyde, System suitability
Sample: 5-mg/mL suspension in water solution
Acceptance criteria: 6.0-7.5 Tailing factor: NMT 1.3, Standard solution
e WATER DETERMINATION, Method | (921): NMT 2.5% Relative standard deviation: NMT 2.0%, Standard
e STERILITY TESTS (71): Where the label states that Mito- solution
mycin is sterile, it meets the requirements when tested as Analysis
directed for Test for Sterility of the Product to Be Examined, Samples: Standard solution and Sample solution
Membrane Filtration. Calculate the percentage of mitomycin (CisHigN4Os) in
e BACTERIAL ENDOTOXINS TEST (85): Where the label states the container of Mitomycin for Injection taken:
that Mitomycin is sterile or must be subjected to further
processing during the preparation of injectable dosage Result = (ru/rs) x (Cs/Cu) x P x Fx 100
forms, it contains NMT 10.0 USP Endotoxin Units/mg of
mitomycin. tu = peak area from the Sample solution
ls = peak area from the Standard solution
ADDITIONAL REQUIREMENTS rea ; . Gs = concentration of USP Mitomycin RS in the
© PACKAGING AND STORAGE: Preserve in tight,lighieresistant Standard solution (mg/ml)
containers. Store at 25°, excursions permitted between Gi = nominal concentration of mitomycin in the
15° and 30°. I j L
e LABELING: Where it is intended for use in preparing in- Pp ee sebteyor niteadiyers ia ge Mitomycin RS
ce
”
jectable dosage forms, the label states that it is sterile or (ug/mg)
5 must be subjected to further processing during the prep- = conversion factor, 0.001 mg/g
Ss
aration of injectable dosage forms. Acceptance criteria: 90.0%-120.0%
=)
—

°
¢ PERFORMANCE TESTS
S Change to read:
e UNIFORMITY OF DOSAGE UNITS (905): Meets the
= ° USP REFERENCE STANDARDS (11)
requirements
PS
”“ @ (CN 11-May-2018) SPECIFIC TESTS
b=) USP Mitomycin RS © PH (791)
Sample solution: Constitute as directed in the labeling.
Acceptance criteria: 6.0-8.0 where it contains manni-
tol, and 5.5-8.5 where it contains hydroxypropyl
betadex
Mitomycin for Injection WATER DETERMINATION, Method Ia (921)
Sample solution: Prepare as directed for a hygroscopic
DEFINITION specimen, using the pooled contents of five containers.
Mitomycin for Injection contains NLT 90.0% and NMT Acceptance criteria: NMT 5.0%
120.0% of the labeled amount of mitomycin BACTERIAL ENDOTOXINS TEST (85): Contains NMT 10.0
(CisHigNaOs).
USP Endotoxin Units/mg of mitomycin
STERILITY TESTS (71): Meets the requirements when
IDENTIFICATION tested as directed for Test for Sterility of the Product to Be
e A. The retention time of the major peak from the Sample Examined, Membrane Filtration
solution corresponds to that from the Standard solution, CONSTITUTED SOLUTION: At the time of use, it meets the
as obtained in the Assay. requirements for Injections and Implanted Drug Products
(1), Specific Tests, Completeness and clarity of solutions.
ASSAY ¢ OTHER REQUIREMENTS: Meets the requirements in /njec-
© PROCEDURE tions and Implanted Drug Products (1)
Mobile phase: Dissolve 1.54 g of ammonium acetate in
250 mL of methanol. Add 5.0 mL of 0.83 N acetic acid ADDITIONAL REQUIREMENTS
and water to make 1000 mL. © PACKAGING AND STORAGE: Preserve as described in Pack-
System suitability solution: 0.5 mg/mL of USP Mito- aging and Storage Requirements (659), Injection Packaging,
mycin RS and 7.5 mg/mL of 3-ethoxy-4-hydroxy- Packaging for constitution, pulsed from light. Store at
benzaldehyde in N,N-dimethylacetamide 25°, excursions permitted between 15° and 30°.
Standard solution: 0.5 mg/mL of USP Mitomycin RS in
N,N-dimethylacetamide
USP 41
Change to read:
e USP REFERENCE STANDARDS (11)
*% (CN 1-May-2018)
USP Mitomycin RS
Mitotane
CygHioCla
Benzene, 1-chloro-2-[2,2-dichloro-1-(4-chlorophenyl)ethyl]-,
(RS)-;
(4)-1,1-Dichloro-2-(0-chlorophenyl)-2-(p-
chlorophenyl)ethane [53-19-0)
DEFINITION
Mitotane contains NLT 97.0% and NMT 103.0% of
mitotane (Ci4HioCl4), calculated on the anhydrous basis.
[CAUTION—Handle Mitotane with exceptional care, because
it is a highly potent agent.]
IDENTIFICATION
© A. INFRARED ABSORPTION (197M)
° B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
ASSAY
© PROCEDURE
Buffer: 1.38 g/L of monobasic potassium phosphate in
water. Adjust with phosphoric acid to a pH of 2.5.
Mobile phase: Acetonitrile and Buffer (75:25)
System suitability solution: 0.2 mg/mL of USP
Mitotane RS and 0.2 mg/mL of the p,p’-isomer of
mitotane in Mobile phase, [NotE—The p,p’-isomer of
mitotane is 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane
known as p,p’-DDD.]
Standard solution: 0.2 mg/mL of USP Mitotane RS in
Mobile phase
Sample solution: 0.2 mg/mL of Mitotane in Mobile
phase, [NoTE—Inject within 48 h of preparation.]
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 230 nm
Column: 4.6-mm x 25-cm; 5-14m packing L1
Flow rate: 1 mL/min
Injection volume: 10 pL
System suitability
Samples: System suitability solution and Standard
solution
[Nott—The relative retention times for the p,p’-isomer
of mitotane and mitotane are about 0.92 and 1.0,
respectively.]
Suitability requirements
Resolution: NLT 1.5 between the p,p’-isomer of
mitotane and mitotane, System suitability solution
Tailing factor: NMT 1.5, Standard solution
Relative standard deviation: NMT 1.0%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of mitotane (CisHioCla) in the
portion of Mitotane taken:
Result = (ru/rs) x (Cs/Cu) x 100
Official Monographs / Mitotane 2773
ry = peak response from the Sample solution
rs = peak response from the Standard solution
Cs = concentration of USP Mitotane RS in the
Standard solution (mg/mL)
Cy = concentration of Mitotane in the Sample
solution (mg/mL)
Acceptance criteria: 97.0%-103.0% on the anhydrous
basis
IMPURITIES
© RESIDUE ON IGNITION (281): NMT 0.5%
SPECIFIC TESTS
e WATER DETERMINATION, Method Ia (921): NMT 0.5%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers.
320.04 e USP REFERENCE STANDARDS (11)
USP Mitotane RS
Mitotane Tablets
» Mitotane Tablets contain not less than
90.0 percent and not more than 110.0 percent of
the labeled amount of mitotane (Cy4HioCla).
Packaging and storage—Preserve in tight, light-resistant
containers.
USP Reference standards (11)—
USP Mitotane RS
Identification—Triturate a quantity of finely powdered
Tablets, equivalent to about 500 mg of mitotane, with
10 mL of water, filter on a sintered-glass filter funnel, and (=
wash the residue with two 5-mL portions of water. Transfer 4)
the residue to a small beaker, add 4 mL of alcohol, heat to
=)
boiling, and filter immediately. Allow the filtrate to cool, fil- m=
ter the crystals of mitotane, wash once with 2 mL of alco- i)
=|
hol, and dry in vacuum at 60° for 2 hours: the IR absorption )
spectrum of a mineral oil dispersion of the mitotane so ob- @a
tained exhibits maxima only at the same wavelengths as 2
that of a similar preparation of USP Mitotane RS. ao}
Disintegration (701): 15 minutes, the use of disks being
=P
”
omitted.
Uniformity of dosage units (905): meet the require-
ments.
Assay—
Standard preparation—Dissolve about 50 mg of USP
Mitotane RS, accurately weighed, in methanol, and dilute
quantitatively and stepwise with methanol to obtain a solu-
tion having a known concentration of about 200 ug per mL.
Assay preparation—Weigh and finely powder not less
than 10 Tablets. Transfer an accurately weighed portion of
the powder, equivalent to about 100 mg of mitotane, to a
250-mL volumetric flask, add 100 mL of methanol, and
shake occasionally for 5 minutes, then dilute with methanol
to volume, and mix. Filter, rejecting the first portion of the
filtrate, transfer 25.0 mL of the filtrate to a 50-mL volumetric
flask, dilute with methanol to volume, and mix.
Procedure—Concomitantly determine the absorbances of
the Assay preparation and the Standard preparation in 1-cm
cells at the wavelength of maximum absorbance at about
268 nm, with a suitable spectrophotometer, using methanol
as the blank.
 2774 Mitotane / Official Monographs
Calculate the quantity, in mg, of Ci4HioCls in the portion
of Tablets taken by the formula:
0.5C(Au / As)
in which C is the concentration, in mg/mL, of USP Mitotane
RS in the Standard preparation, and Ay and As are the ab-
sorbances of the Assay preparation and the Standard prepa-
ration, respectively.
Mitoxantrone Injection
» Mitoxantrone Injection is a sterile solution of
Mitoxantrone Hydrochloride in Water for Injec-
tion. It contains the equivalent of not less than
90.0 percent and not more than 105.0 percent of
the labeled amount of mitoxantrone
(C22H28NaOo).
Packaging and storage—Preserve in single-dose or multi-
ple-dose containers, preferably of Type | glass.
Labeling—Label Injection to state both the content of the
active moiety and the name of the salt used in formulating
the article. Label Mitoxantrone Injection to indicate that it is
to be diluted to appropriate strength with water or other
suitable fluid prior to administration.
USP Reference standards (11)—
USP Mitoxantrone Hydrochloride RS
USP Mitoxantrone System Suitability Mixture RS
A mixture of 9,10-anthracenedione, 8-amino-1,4-
dihydroxy-5[[2-[(2-hydroxyethyl)amino]ethyl]amino]-,
hydrochloride (CigHigN3Os - HCI 393.83) and USP
a
al
Mitoxantrone Hydrochloride RS.
iy Identification—Transfer a volume of Injection, equivalent
ic to about 2 mg of mitoxantrone, to a 200-mL volumetric
—
aD flask, add 100 mL of water and 20 mL of 1 N hydrochloric
°
iS acid, dilute with water to volume, and mix: the UV absorp-
S tion spectrum of this solution exhibits maxima and minima
= at the same wavelengths as that of a similar solution of USP
a Mitoxantrone Hydrochloride RS.
a) Bacterial Endotoxins Test (85)—It contains not more
= Packaging and storage—Preserve in tight containers.
than 5 Endotoxin Units per mg of mitoxantrone.
Sterility Tests (71)—It meets the requirements when
tested as directed for Membrane Filtration under Test for Ste-
rility of the Product to be Examined, the entire contents of
each container being used.
pH (791): between 3.0 and 4.5.
Chromatographic purity—Using the chromatogram of
the Assay preparation obtained as directed in the Assay, cal-
culate the percentage of each impurity in the Injection
taken by the formula:
100(r, / rs)
in which r, is the response of any individual peak, other than
the main mitoxantrone peak; and rs is the sum of the re-
sponses of all the peaks in the chromatogram, including
that of the main mitoxantrone peak: not more than 1.5% of
any individual impurity and not more than 3.0% of the total
impurities is found.
Other requirements—lt meets the requirements under In-
jections and Implanted Drug Products (1).
Assay—
Sodium 1-heptanesulfonate solution, Mobile phase, System
suitability solution, and Chromatographic system—Proceed as
directed in the Assay under Mitoxantrone Hydrochloride.
Standard preparation—Transfer about 23 mg of USP Mito-
xantrone Hydrochloride RS, accurately weighed, to a 50-mL
USP 41
volumetric flask, add 40 mL of Mobile phase, and dissolve by
sonicating for about 5 minutes. Cool to room temperature,
dilute with Mobile phase to volume, and mix. This solution
contains the equivalent of about 0.4 mg of mitoxantrone
(C22H2gN4Oc) per mL.
Assay preparation—Transfer an accurately measured vol-
ume of Injection, equivalent to about 4 mg of mitoxantrone
(Ca2H2gN40o), to a 10-mL volumetric flask, dilute with Mobile
phase to volume, and mix.
Procedure—Proceed as directed for Procedure in the Assay
under Mitoxantrone Hydrochloride. Calculate the quantity, in
mg, of mitoxantrone (Cz2H2gN4Q¢) in each mL of the Injec-
tion taken by the formula:
(444.49 / 517.40)(10C/ V)(ru/ rs)
in which 444.49 and 517.40 are the molecular weights of
mitoxantrone and mitoxantrone hydrochloride, respectively;
Vis the volume, in mL, of the portion of Injection taken;
and the other terms are as defined therein.
Mitoxantrone Hydrochloride
CoaH2gN4O6- 2HCl 517.40
9,10-Anthracenedione, 1,4-dihydroxy-5,8-bis[[2-
[(2-hydroxyethyl)amino]ethyl]amino]-, dihydrochloride.
1,4-Dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]
ein laine lent nrsauinere difydrochioride.
[70476-82-3].
» Mitoxantrone Hydrochloride contains not less
than 97.0 percent and not more than 102.0 per-
cent of Co2H2sN4O¢ - 2HCI, calculated on the an-
hydrous basis.
USP Reference standards (11)—
USP Mitoxantrone Hydrochloride RS
USP Mitoxantrone System Suitability Mixture RS
A mixture of 9,10-anthracenedione, 8-amino-1,4-
dihydroxy-5[[2-[(2-hydroxyethyl)amino]ethyl]Jamino]-,
hydrochloride (CigHisN3Os » HCI 393.83) and USP
Mitoxantrone Hydrochloride RS.
identification, Infrared Absorption (197K).
weer Determination, Method | (921): not more than
0%.
Alcohol—
Standard solution—Transfer 5.0 mL of dehydrated alcohol
to a 250-mL volumetric flask, dilute with water to volume,
and mix. Transfer 5.0 mL of this solution to a 500-mL volu-
metric flask, dilute with water to volume, and mix.
Internal standard solution—Transfer 5.0 mL of n-propyl al-
cohol to a 250-mL volumetric flask, dilute with water to
volume, and mix. Transfer 5.0 mL of this solution to a
500-mL volumetric flask, dilute with water to volume, and
mix.
Standard preparation—Transfer 10.0 mL of the Standard
Solution to a 25-mL volumetric flask, add 10.0 mL of the
Internal standard solution, dilute with water to volume, and
mix. This solution contains 0.063 mg of alcohol (C2HsOH)
per mL.
USP 41
Test pepe er about 100 mg of Mitoxantrone
Hydrochloride, accurately weighed, to a 5-mL volumetric
flask, add 2.0 mL of the Internal standard solution, dilute
with water to volume, and mix. Sonicate for 2 minutes and
shake for 2 minutes, repeating these actions until the speci-
men is completely dissolved.
Chromatographic system (see Chromatography (621))—The
gas chromatograph is equipped with a flame-ionization de-
tector and a 2-mm x 3-m column that contains 20% phase
G1 and 0.1% phase G39 on silanized support S1A. Maintain
the column at 50° for 5 minutes, then increase the tempera-
ture at a rate of 30° per minute. When 140° is reached,
maintain that temperature for 20 minutes. Maintain the in-
jection port at 200° and the detection block at 250°. Use
lium as the carrier gas at a flow rate of about 15 mL per
minute. Make adjustments if necessary (see System Suitability
under Chromatography (621)). Chromatograph the Standard
preparation, and record the peak responses as directed for
Procedure: the relative retention times are about 0.5 for alco-
hol and 1.0 for n-propyl alcohol, the resolution, R, between
the alcohol and the n-propyl alcohol peaks is not less than
6.0, and the tailing factors for the two peaks are not more
than 2.0.
Procedure—{NOTE—Use peak areas where peak responses
are indicated.] Separately inject equal volumes (about 1 LL)
of the Standard preparation and the Test preparation into the
chromatograph, record the chromatograms, and measure
the responses for the major peaks. Calculate the percentage
of alcohol (C2HsOH) in the portion of Mitoxantrone Hydro-
chloride taken by the formula:
500(C/W)(Ru
/ Rs)
in which C is the concentration, in mg per mL, of alcohol
(C2HsOH) in the Standard preparation; W is the weight, in
mg, of Mitoxantrone Hydrochloride taken; and Ry and Rs are
the ratios of the response of the alcohol peak to that of the
Co alcohol peak obtained from the Test preparation
and the Standard preparation, respectively: not more than
1.5% is found.
Delete the following:
°Heavy metals (231)—Proceed as directed under Method
|, except in the Procedure to filter the final solutions
through a suitable acid-resistant membrane filter of 0.22 um
or finér porosity, instead of viewing them over a dark sur-
face: the precipitate on the filter obtained from the Test
Preparation is not darker than that obtained from the Stan-
dard Preparation. The limit is 0.002%. (ofticiat 1-jan-2018)
Chromatographic purity—Using the chromatogram of
the Assay preparation obtained as directed in the Assay, cal-
culate the percentage of each impurity in the Mitoxantrone
Hydrochloride taken by the formula:
100(n
/ rs)
in which 4; is the response of any individual peak, other than
the main mitoxantrone peak, and r; is the sum of the re-
spots of all the peaks in the chromatogram, including
that of the main mitoxantrone peak: not more than 1.0% of
any individual impurity and not more than 2.0% of total
impurities is found.
Assay—
Sodium 1-heptanesulfonate solution—Dissolve 22.0 g of so-
dium 1-heptanesulfonate in about 150 mL of water, pass
through asuitable filter having a 0.5-1m or finer porosity,
and transfer the filtrate to a 250-mL volumetric flask. Wash
the filter with about 50 mL of water, adding the filtrate to
the 250-mL volumetric flask. Add 32.0 mL of glacial acetic
an to the volumetric flask, dilute with water to volume,
and mix.
Official Monographs / Modafinil 2775
Mobile phase—Prepare a suitable degassed mixture of
water, acetonitrile, and Sodium 1-heptanesulfonate solution
(750:250:25). Make adjustments if necessary (see System
Suitability under Chromatography (621)).
System suitability solution—Prepare a solution of USP
Mitoxantrone System Suitability Mixture RS in a suitable vol-
ume of Mobile phase to obtain a solution containing about
0.2 mg of 8-amino-1,4-dihydroxy-5[[2-[(2-hydroxyethyl)ami-
nojethylJamino}-9,10-anthracenedione hydrochloride (mito-
xantrone related compound A) and 0.1 mg of mitoxantrone
hydrochloride per mL.
Standard preparation—Transfer about 20 mg of USP Mito-
xantrone Hydrochloride RS, accurately weighed, to a 50-mL
volumetric flask, add 40 mL of Mobile phase, and dissolve by
sonicating for about 5 minutes. Cool to room temperature,
dilute with Mobile phase to volume, and mix.
Assay preparation—Transfer about 20 mg of Mitoxantrone
Hydrochloride, accurately weighed, to a 50-mL volumetric
flask, add 40 mL of Mobile phase, and dissolve by sonicating
for about 5 minutes. Cool to room temperature, dilute with
Mobile phase to volume, and mix.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 254-nm detector
and a 3.9-mm x 30-cm column that contains packing L11.
The flow rate is about 3 mL per minute. Chromatograph the
System suitability solution, and record the peak responses as
directed for Procedure: the relative retention times are about
0.7 for mitoxantrone and 1.0 for mitoxantrone related com-
pound A; the resolution, R, between mitoxantrone and
mitoxantrone related compoundAis not less than 3.0; and
the tailing factor for the mitoxantrone peak is not more
than 2.0. Chromatograph the Standard preparation, and re-
cord the peak responses as directed for Procedure: the ca-
pacity factor, k’, for mitoxantrone is not less than 3.5; and
the relative standard deviation for replicate injections is not
more than 2.0%.
Procedure—Separately inject equal volumes (about 50 wL) Cc
nw
of the Standard preparation and the Assay preparation into A)
the chromatograph, record the chromatograms, and meas-
ure the areas for the major peaks. [NoTE—After use, wash =
the column with a mixture of acetonitrile and water (50:50), 2}
Ei
and store in this mixture.] Calculate the quantity, in mg, of }
Ca2H2gN4Oc - 2HCI in the portion of Mitoxantrone Hydro- a=
chloride taken by the formula: 2
i}
50C(ru/ rs) >
w
in which C is the concentration, in mg per mL, of anhy-
drous mitoxantrone hydrochloride in the Standard prepara-
tion, as determined from the content of USP Mitoxantrone
Hydrochloride RS corrected for the water content deter-
mined byatitrimetric water determination; and ry and rs
are the mitoxantrone peak areas obtained from the Assay
preparation and the Standard preparation, respectively.
Modafinil
CLL
“oO
CisHisNO2S 273.35
Acetamide, feltciphenyimeriy eur ls
2-[(Diphenylmethy!)sulfinyl]-acetamide [68693-1 1-8].
DEFINITION
Modafinil contains NLT 98.0% and NMT 101.5% of
CisHisNO2S, calculated on the anhydrous basis.
 2776 Modafinil / Official Monographs USP 41

IDENTIFICATION tr = sum of the responses of all the peaks

e A. INFRARED ABSORPTION (197K) F = relative response factor (see Table 1)
Acceptance criteria: See Table 1.
ASSAY
e PROCEDURE
Buffer: 6.8 g/L of potassium dihydrogen phosphate in Table 1
water. Adjust with phosphoric acid to a pH of 2.3. Relative Relative Acceptance
Mobile phase: Acetonitrile and Buffer (35:65) Retention Response Criteria,
Diluent: Acetonitrile and water (35:65) Name Time Factor NMT (%)
Syaem suitability solution: 5 g/mL of USP Modafinil Modafinil 1.0 =_ =—
S and 10 g/mL of USP Salicylic Acid RS in Diluent Salicylic acid? 1.1 — =
Standard solution: 0.1 mg/mL of USP Modafinil RS in
Modafinil acid? 1.4 1.0 0.5
Diluent
Sample solution: 0.1 mg/mL of Modafinil in Diluent Modafinil sulfones 17 0.9 0.5
Chromatographic system Modafinil esters 3.0 1.0 0.5
(See Chromatography (621), System Suitability.) Any other individual
Mode: LC unspecified impuri- —
Detector: UV 220 nm ty 1.0 0.05
Column: 4.6-mm x 15-cm; 5-m packing L1 Total impurities = = 1.0
Column temperature: 40° ®Salicylic acid is used for calculating resolution and is not a potential
Flow rate: 1 mL/min impurity.
Injection size: 20 ul » 2-[(Diphenylmethyl)sulfinylJacetic acid.
System suitability ¢ 2-[(Diphenylmethy|)sulfonyl]acetamide.
Sample: System suitability solution 4 2-[(Diphenylmethyl)sulfinyl]acetic acid methyl ester.
[Note—The relative retention times for modafinil and
salicylic acid are about 1.0 and 1.1, respectively.] SPECIFIC TESTS
Suitability requirements e@ WATER DETERMINATION, Method | (921): NMT 0.2%
aes NLT 1.3 between modafinil and salicylic
ADDITIONAL REQUIREMENTS
aci e PACKAGING AND STORAGE: Preserve in well-closed contain-
Tailing factor: NMT 1.5 for the modafinil peak
ers. Store at controlled room temperature.
Relative standard deviation: NMT 2.0% for the e USP REFERENCE STANDARDS (11)
modafinil peak USP Modafinil RS
Analysis USP Salicylic Acid RS
Samples: Standard solution and Sample solution
Calculate the percentage of modafinil (CisHisNO2S) in
the portion of Modafinil taken:
a Result = (ru/rs) x (Cs/Cu) x 100
Modafinil Tablets
a ru = peak response from the Sample solution
a rs = peak response from the Standard solution DEFINITION
ro) Cs = concentration of USP Modafinil RS in the Modafinil Tablets contain NLT 90.0% and NMT 110.0% of
= Standard solution (mg/mL) the labeled amount of modafinil (CisHisNO2S).
iS Cy = concentration of Modafinil in the Sample
= solution (mg/mL) IDENTIFICATION
- Acceptance criteria: =98.0%-101.5% on the anhydrous e A. INFRARED ABSORPTION (197K)
es basis Standard specimen: Transfer a quantity, in mg, of USP
= Modafinil RS, equivalent to the labeled amount of
IMPURITIES modafinil, to a suitable container. Add 50 mL each of
© RESIDUE ON IGNITION (281): NMT 0.1% dichloromethane and water. Shake the mixture, and al-
low the layers to separate. Filter a portion of the lower
(dichloromethane) layer, and evaporate to dryness, us-
Delete the following: ing a stream of nitrogen if necessary. Prepare a potas-
sium bromide pellet of the residue.
®o HEAVY METALS, Method I (231): NMT 20 ppme cottcat i- Sample specimen: Grind 1 Tablet, and add 50 mL each
Jan-2018) of dichloromethane and water. Shake the mixture, and
¢@ ORGANIC IMPURITIES allow the layers to separate. Filter a portion of the lower
Buffer, Mobile phase, Sample solution, System suita- (dichloromethane) layer, and evaporate to dryness, us-
bility solution, and Chromatographic system: Pre- ing a stream of nitrogen if necessary. Prepare a potas-
pare as directed in the Assay. sium bromide pellet of the residue.
System suitability Acceptance criteria: Meet the requirements
Sample: System suitability solution
Suitability requirements ASSAY
Resolution: NLT 1.3 between modafinil and salicylic e PROCEDURE
acid Buffer: 6.8 g/L of potassium dihydrogen phosphate in
Tailing factor: NMT 1.5 for the modafinil peak water. Adjust with phosphoric acid to a pH of 2.3.
Relative standard deviation: NMT 2.0% for the Mobile phase: Acetonitrile and Buffer (35:65)
modafinil peak Diluent A: Acetonitrile and water (35:65)
Analysis Diluent B: Acetonitrile, water, and acetic acid (35:65:1)
Sample: Sample solution System suitability solution: 5 g/mL of USP Modafinil
Calculate the percentage of each impurity in the por- RS and 10 pg/mL of USP Salicylic Acid RS in Diluent A
tion of Modafinil taken: Standard solution: 0.4 mg/mL of USP Modafinil RS in
Diluent B
Result = (ru/rz) x (1/F) x 100
fu = peak response of each individual impurity
USP 41 Official Monographs / Modafinil 2777

Sample solution: Weigh and finely powder NLT 20 Instrumental conditions

Tablets. Transfer a portion of the powder, equivalent to (See Ultraviolet-Visible Spectroscopy (857).)
100 mg of modafinil, to a 250-mL volumetric flask, add Mode: UV
200 mL of Diluent B, and sonicate for about 5 min with Analytical wavelength: Absorption maximum at
intermittent manual shaking. Dilute with Diluent B to about 225 nm
volume, and mix. Pass through a suitable filter of 0.45- Cell: 0.1 cm
um or finer pore size, and use the filtrate. Blank: Medium
Chromatographic system Analysis
(See Chromatography (621), System Suitability.) Samples: Standard solution and Sample solution
Mode: LC Calculate the percentage of modafinil (CisHisNO2S)
Detector: UV 220 nm dissolved:
Column: 4.6-mm x 15-cm; 5-m packing L1
Column temperature: 40° Result = (Au/As) x Cs x Vx (1/L) x 100
Flow rate: 1.0 mL/min
Injection volume: 5 pL Au = absorbance of the Sample solution
System suitability As = absorbance of the Standard solution
Sample: System suitability solution Gs = concentration of the Standard solution
[Note—The relative retention times for modafinil and (mg/mL)
salicylic acid are 1.0 and 1.1, respectively.] Vv = volume of Medium, 900 mL
Suitability requirements L = label claim (mg/Tablet)
fe aa NLT 1.3 between modafinil and salicylic Tolerances: NLT 75% (Q) of the labeled amount of
aci modafinil (CisHisNO2S) is dissolved.
Tailing factor: NMT 1.5 for the modafinil peak Test 3: If the product complies with this test, the label-
Relative standard deviation: NMT 2.0% for the ing potenti that the product meets USP Dissolution
modafinil peak Test 3.
Analysis Medium: 0.1 N hydrochloric acid; 900 mL, deaerated
Samples: Standard solution and Sample solution Apparatus 2: 75 rpm
Calculate the percentage of the labeled amount of Time: 30 min
modafinil (CisHisNO2S) in the portion of Tablets taken: Standard solution: (1/900) mg/mL of USP Modafinil
RS, whereLis the label claim (mg/Tablet). Prepare by
Result = (ru/rs) x (Cs/Cu) x 100 dissolving the standard in a volume of methanol
Sana to 5%-10% of the final volume and then
ru = peak response from the Sample solution diluting with Medium to volume.
rs = peak response from the Standard solution Sample solution: Pass a portion of the solution under
Cs = concentration of USP Modafinil RS in the test throughasuitable filter.
Standard solution (mg/mL) Instrumental conditions
Cu = nominal concentration of modafinil in the (See Ultraviolet-Visible Spectroscopy (857).)
Sample solution (mg/mL) Mode: UV c
Acceptance criteria: 90.0%-110.0% Analytical wavelength: Absorption maximum at 4)
about 220 nm uv
PERFORMANCE TESTS
© DISSOLUTION (711)
Cell: 0.1 cm 4
Blank: Medium fo)
Test 1 Analysis ]
Medium: 0.1 N hydrochloric acid; 900 mL °
Samples: Standard solution and Sample solution io]
Apparatus 2: 50 rom Calculate the percentage of modafinil (CisHisNO2S) =
Time: 30 min 2
dissolved: mo]
Standard solution: Prepare a solution having a known a
concentration of USP Modafinil RS in Medium. Result = (Au/As) x Cs x V x (1/L) x 100 7
Sample solution: A filtered portion of the solution
under test, suitably diluted with Medium if necessary Au = absorbance of the Sample solution
Instrumental conditions As = absorbance of the Standard solution
(See Ultraviolet-Visible Spectroscopy {857).) Cs = concentration of the Standard solution
Mode: UV
Analytical wavelength: Absorption maximum at
(mg/mL)
Vv = volume of Medium, 900 mL
about 222 nm L = label claim (mg/Tablet)
Analysis Tolerances: NLT 80% (Q) of the labeled amount of
Samples: Standard solution and Sample solution modafinil (CisHisNO2S) is dissolved.
Determine the amount of modafinil (CisHisNO2S) e UNIFORMITY OF DOSAGE UNITS (905): Meet the
dissolved. requirements
Tolerances: NLT 75% (Q) of the labeled amount of
modafinil (CisHisNO2S) is dissolved. IMPURITIES
Test 2: If the product complies with this test, the label- © ORGANIC IMPURITIES
ing eiieaies that the product meets USP Dissolution Buffer, Mobile phase, Systemsuitability solution,
est 2. Sample solution, and Chromatographic system: Pro-
Medium: 0.1 N hydrochloric acid; 900 mL ceed as directed in the Assay.
Apparatus 2: 50 rpm System suitability
Time: 45 min Sample: System suitability solution
Standard solution: (1/900) mg/mL of USP Modafinil Suitability requirements
RS, where L is the label claim (mg/Tablet). Prepare by een NLT 1.3 between modafinil and salicylic
dissolving the standard in a volume of methanol aci
Souivyatest to 5%-10% of the final volume and then Relative standard deviation: NMT 2.0% for the
diluting with Medium to volume. modafinil peak
Sample solution: Pass a portion of the solution under
test throughasuitable filter of 0.45-m pore size.
 2778 Modafinil / Official Monographs USP 41

Analysis ASSAY
Sample: Sample solution ¢ PROCEDURE
Calculate the percentage of each impurity in the por- Buffer: 1.32 g/L of dibasic ammonium phosphate. Ad-
tion of Tablets taken: just with diluted phosphoric acid to a pH of 7.5.
Solution A: Acetonitrile and tetrahydrofuran (95:5)
Result = (ru/rr) x (1/F) x 100 Mobile phase: Solution A and Buffer (30:70)
Standard solution: 0.1 mg/mL of USP Moexipril Hydro-
ry = peak fesponse of each individual impurity chloride RS in Mobile phase. [Note—Sonication may be
tr = sum of the responses of all the peaks necessary for complete dissolution.]
F = relative response factor (see Table 1) Sample solution: 0.1 mg/mL of Moexipril Hydrochlo-
Acceptance criteria: See Table 7. ride in Mobile phase. [NOTE—Sonication may be neces-
sary for complete dissolution.]
Table 1 Chromatographic system ore
malailve Ralative! |. Acceptance ifaespetagiap y (621), System Suitability.)
Retention Response Criteria, Detector: UV 215 nm
aD Time Factor: NMT (%) Column: 4.6-mm x 25-cm; 5-um packing L1
Modafinil 1.0 my = Column temperature: 35°
Salicylic acid 11 = — Flow rate: 1 mL/min
Modafinil acide 1.4 1.0 0.5 Injection volume: 10 wL
Modafinil sulfonec 137, 0.90 0.5 Runall NLT 3.2 times the retention time of
Any individual MOCXIDM ase
unspecified impurity = 1.0 0.2 “Sonate Gere sohutioni
Total impurities = = Ls Suitability requirements
2Salicylic acid is used for calculating resolution and is not a potential Relative standard deviation: NMT 2.0%
IMPLY:
» 2-[(Diphenylmethy)sulfinylJacetic acid.
Analysis
« 2-[(Diphenyimethy))sulfonyllacetamide. Samples: Standard solution and Sample solution
Calculate the percentage of moexipril hydrochloride
ADDITIONAL REQUIREMENTS (C27H34N207 - HCl) in the portion of Moexipril Hydro-
e PACKAGING AND STORAGE: Preserve in tight containers. chloride taken:
Store at controlled room temperature.
e LABELING: When more than one Dissolution test is given, Result = (ru/rs) x (Cs/Cu) x 100
the labeling states the test used only if Test 7 is not used. .
e USP REFERENCE STANDARDS (11) tu = peak response from the Sample solution
USP Modafinil RS rs = peak response from the Standard solution _
USP Salicylic Acid RS G = concentration of USP Moexipril Hydrochloride
a} RS in the Standard solution (mg/mL)
rt Cu = concentration of Moexipril Hydrochloride in
J the Sample solution (mg/mL)
a Acceptance criteria: 98.0%-102.0% on the anhydrous
<5) Moexipril Hydrochloride basis
ic) IMPURITIES
2
bn Delete the following:
=)
aco : + Hel °e HEAVY METALS, Method f/ (231): NMT 10 ppme cofficial 1-
Serene Jan-2018)
eT oe e RESIDUE ON IGNITION (281): NMT 0.20%
= © ORGANIC IMPURITIES
[NoTte—Use freshly prepared samples for analysis.]
Solution A and Chromatographic system: Proceed as
C27H34N2O07
- HCI . . 535.03 directed in the Assay.
3 oquinelinecarboxvlie acid, 2-[2-[[1-(ethoxycarbonyl)- Solution B: Proceed as directed for the Buffer in the
3-phenylpropyljamino]-1 een ,2,3,4-tetrahydro- Assay.
6,/-dimethoxy-, monohydrochloride, Senet /3R*])-; Diluent: Solution A and Solution B (20:80)
(35S)-2-[(25)-N-[ ee oyalanyl]-1,2, Mobile phase: See Table 1.
3,4-tetrah' Eto a inecarboxylic
acid, 2-ethyl ester, monohydrochloride [82586-52-5]. Table-7
DEFINITION 4 ’ Solution A Solution B
Moexipril Hydrochloride contains NLT 98.0% and NMT
102.0% of moexipril hydrochloride (C27H34N207 - HCl),
calculated on the anhydrous basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
e B. The relative retention time of the major peak of the 20
Sample solution corresponds to that of the Standard solu- 20
tion, as obtained in the Assay. .
© C. IDENTIFICATION TESTS—GENERAL (191), Chloride: Meets Standard solution 1: 4 g/mL of USP Moexipril Hydro-
the requirements chloride RS in Diluent. [NOTE—Sonication may be neces-
sary for complete dissolution.]
USP 41 Official Monographs / Moexipril 2779

Standard solution 2: 2 mg/mL of USP Moexipril Hydro- Table 2

chloride RS and 3 ug/mL each of USP Moexipril Related Relative Acceptance
Compound A RS, Use Moexipril Related Compound B Retention Criteria,
RS, USP Moexipril Related Compound C RS, USP Moex- Name Time NMT (%)
ipril Related Compound D RS, USP Moexipril Related
Compound E RS, USP Moexipril Related Compound F Moexipril related
RS, and USP Moexipril Related Compound G RS in Dilu- compound E* 0.14 0.15
ent. [Note—Sonication may be necessary for complete Moexipril related
dissolution.] compound Ae 0.28 0.2
Sample solution: 2 mg/mL of Moexipril Hydrochloride Moexipril related
in Diluent. [NoTeE—Sonication may be necessary for compound F« 0.62 0.15
complete dissolution.] Moexipril related
System suitability compound G¢ 0.90 0.15
Samples: Standard solution 1 and Standard solution 2 Moexipril 1.00 =
Suitability requirements Moexipril related
Resolution: NLT 3.5 between moexipril related com- compound De 1.28 0.15
pane A and moexipril related compound E; NLT 2.5
tween moexipril and moexipril related compound Moexipril related
G, Standard solution 2 compound Bt 1.62 0.2
Relative standard deviation: NMT 5.0% for moex- Moexipril related
ipril, Standard solution 7 compound C9 2.26 0.15
Analysis Any other individual _
Samples: Standard solution 1, Standard solution 2, and unspecified impurity 0.10
Sample solution Total impurities’ = 1.0
Calculate the percentage of each specified impurity in 2 (S)-6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.
the portion of Moexipril Hydrochloride taken: » (35)-2-{(25)-N-[(1 5)-1-Carboxy-3-phenylpropyllalanyl}-1,2,3,4-tetrahydro-
6,7-dimethoxy-3-isoquinolinecarboxylic acid.
Result = (ru/rs) x (Cs/Cu) x 100 © (5)-2-[(S)-1-Ethoxy-1-oxo-4-phenylbutan-2-ylamino]propanoic acid.
4 (5)-6,7-Dimethoxy-2-{(S)-2-[(5)-1-methoxy-1-0xo-4-phenylbutan-2-ylami-
tu = peak response of each specified impurity from no]propanoy\}-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.
the Sample solution © (S)-2-{(5)-2-[(5)-4-Cyclohexyl-1-ethoxy-1-oxobutan-2-ylamino]propanoyl}-
ls peak response of the corresponding Reference 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.
Standard from Standard solution *(5)-Ethyl 2-{(35,11a5)-8,9-dimethoxy-3-methyl-1,4-dioxo-3,4-dihydro-1 H-
Cs = concentration of each specified impurity in pyrazino[1,2-b]isoquinolin-2(6H,11H,11aH)-yl}-4-phenylbutanoate.
9 (S)-tert-Butyl 2-{(5)-2-[(S)-1-ethoxy-1-oxo-4-phenylbutan-2-ylami-
Standard solution 2 (mg/mL) no]propanoyl}-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxyl-
Cu = concentration of Moexipril Hydrochloride in ate.
the Sample solution (mg/mL) + Sum of all specified and unspecified impurities.
Calculate the percentage of any other individual Cc
unspecified impurity in the portion of Moexipril © CONTENT OF IMIDAZOLE “
Hydrochloride taken: Mobile phase: Hexane, isopropyl alcohol, and diethyl ss )
amine (52: 48: 0.025) =
Result = (ru/rs) x (Cs/Cu) x 100 Standard solution: 0.01 mg/mL of USP Imidazole RS in °
Mobile phase. [NOTE—Sonication may be necessary for |
Tu = peak response of each individual unspecified complete dissolution.] °
io}
impurity from the Sample solution Sample solution: 2mg/mL of Moexipril Hydrochloride =
rs = peak response of moexipril from Standard in Mobile phase. [NoTE—Sonication may be neessay i)
mo}
solution 1 for complete dissolution. Use freshly prepared Sample >
Cs = concentration of USP Moexipril Hydrochloride solution for analysis.] vy
RS in Standard solution 1 (mg/mL) Chromatographic system
Cu = concentration of Moexipril Hydrochloride in (See Chromatography (621), System Suitability.)
the Sample solution (mg/mL) Mode: LC
Acceptance criteria: See Table 2. Disregard peaks less Detector: UV 215 nm
than 0.05%. Column: 4.6-mm x 25-cm; 5-'um packing L3
Flow rate: 1 mL/min
Injection volume: 20 uL
Run time: NLT 3.3 times the retention time of
imidazole
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 5.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of imidazole in the portion of
Moexipril Hydrochloride taken:
Result = (ru/rs) x (Cs/Cu) x 100
ru = peak response of imidazole from the Sample
solution
rs = peak response of imidazole from the Standard
solution
G = concentration of USP Imidazole RS in the
Standard solution (mg/mL)
 2780 Moexipril / Official Monographs
Cu = concentration of Moexipril Hydrochloride in
the Sample solution (mg/mL)
Acceptance criteria: NMT 0.03%
SPECIFIC TESTS
© WATER DETERMINATION (921), Method |, Method la: NMT
1.5%
e OPTICAL ROTATION (7815S), Procedures, Specific Rotation
Sample solution: 0.011 g/mL of Moexipril Hydrochlo-
ride in alcohol. Sonicate to dissolve the sample.
Acceptance criteria: +30.0° to +38.0°
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers,
protected from moisture. Store at room temperature.
© USP REFERENCE STANDARDS (11)
USP Imidazole RS
USP Moexipril Hydrochloride RS
USP Moexipril Related Compound A RS
(35S)-2-{(25)-N-[(1 5)-1-Carboxy-3-phenylpropylJalanyl}-
1,2,3,4-tetrahydro-6,7-dimethoxy-3-isoquinoline-
carboxylic acid.
CosH30N207 470.51
USP Moexipril Related Compound B RS
(S)-Ethyl 2-{(35,11a5)-8,9-dimethoxy-3-methyl-1,4-di-
oxo-3,4-dihydro-1 H-pyrazino[1,2-b]isoquinolin-2(6H,
11H,11aH)-yl}-4-phenylbutanoate.
Co7H32N206 480.55
USP Moexipril Related Compound C RS
(S)-tert-Butyl 2-{(S)-2-[(S)-1-ethoxy-1-oxo-
4-phenylbutan-2-ylamino]propanoyl}-6, 7-dimethoxy-
1,2,3,4-tetrahydroisoquinoline-3-carboxylate.
C3iH42N207 554.67
USP Moexipril Related Compound D RS
(S)-2-{(S)-2-[(S)-4-Cyclohexyl-1-ethoxy-1-oxobutan-
2-ylamino]propanoy!}-6,7-dimethoxy-1,2,3,4-te-
trahydroisoquinoline-3-carboxylic acid.
Ca7HaoN2O7 504.62
ra)
ww
Q
ig_
USP Moexipril Related Compound E RS
(S)-6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-3-car-
boxylic acid.
Dy
3° Ci2HisNO4 = 237.25
S USP Moexipril Related Compound F RS
5 (S)-2-[(S)-1-Ethoxy-1 -oxo-4-phenylbutan-2-ylami-
= no]propanoic acid.
rs CisHaiNO, 279.33
a) USP Moexipril Related Compound G RS
=) (S)-6,7-Dimethoxy-2-{(5)-2-[(5)-1-methoxy-1-oxo-
4-phenylbutan-2-ylamino]propanoy}}-1,2,3,4-te-
trahydroisoquinoline-3-carboxylic acid.
CasH32N207 484.54
Moexipril Hydrochloride Tablets
DEFINITION
Moexipril Hydrochloride Tablets contain NLT 90.0% and
NMT 110.0% of the labeled amount of moexipril hydro-
chloride (C27H34N20;7 - HCl).
IDENTIFICATION
e A. ULTRAVIOLET ABSORPTION (197U)
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
ASSAY
© PROCEDURE
Buffer: 0.01 M potassium dihydrogen phosphate
Diluent: Acetonitrile and water (30:70)
Mobile phase: Acetonitrile and Buffer (350:650)
Standard solution: 0.075 mg/mL of USP Moexipril Hy-
drochloride RS in Diluent. Initially fill with Diluent to
USP 41
about 70% of the total volume, and sonicate. Further
dilute with Diluent to volume.
Sample solution: Nominally 0.075 mg/mL of moexipril
hydrochloride in Diluent, prepared from a sufficient
number of crushed Tablets as follows. Add Diluent to
about 75% of the total volume, and sonicate for 30
min with intermittent shaking. Dilute with Diluent to
volume, and pass throughasuitable filter of 0.45-14m
pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 210 nm
Column: 4.6-mm x 15-cm; 5-4m packing L7
Column temperature: 45°
Flow rate: 1.5 mL/min
Injection volume: 20 uL
Run times 4 times the retention time of the moexipril
pea
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
moexipril hydrochloride (C27H34N2O7 - HCl) in the por-
tion of Tablets taken:
Result = (ru/rs) x (Cs/Cu) x 100
ru = peak response from the Sample solution
ts = peak response from the Standard solution
Cs = concentration of USP Moexipril Hydrochloride
RS in the Standard solution (mg/mL)
Gu = nominal concentration of moexipril
hydrochloride in the Sample solution
(mg/mL)
Acceptance criteria: 90.0%-110.0%
PERFORMANCE TESTS
e DISSOLUTION (711)
Test 1
Buffer, Diluent, Mobile phase, Chromatographic sys-
tem, and System suitability: Proceed as directed in
the Assay.
Medium: Water; 900 mL
Apparatus 2: 50 rpm
Time: 15 min
Standard stock solution: 0.16 mg/mL of USP Moex-
ipril Hydrochloride RS in Diluent. [NoTE—Sonication
may be necessary for complete dissolution.]
Standard solution: 0.016 mg/mL of USP Moexipril
Hydrochloride RS in Medium from the Standard stock
solution for 15-mg Tablet strength and 0.008 mg/mL
of USP Moexipti Hydrochloride RS in Medium from the
Standard stock solution for 7.5-mg Tablet strength
Sample solution: Pass 10 mL of the solution under
test throughasuitable filter of 0.45-um pore size, dis-
carding the first 2-3 mL.
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
moexipril hydrochloride (C27H34N2O7 - HCl) dissolved:
Result = (ru/rs) x (Cs/L) x Vx 100
tu = peak response of moexipril from the Sample
solution
rs = peak response of moexipril from the Standard
solution
Cs = concentration of USP Moexipril Hydrochloride
RS in the Standard solution
L = label claim (mg/Tablet)
USP 41 Official Monographs / Moexipril 2781

Vv = volume of Medium, 900 mL Relative standard deviation: NMT 5.0%, Standard

Tolerances: NLT 80% (Q) of the labeled amount of solution
moexipril hydrochloride (C27H34N2O7 - HCl) is dissolved. Analysis .
Test 2: If the product complies with this test, the label- Samples: System suitability solution, Standard solution,
ing indicates that it meets USP Dissolution Test 2. and Sample solution
Buffer, Diluent, Mobile phase, Chromatographic sys- Calculate ite phe of moexipril related com-
tem, and System suitability: Proceed as directed in pound A and moexipril related compound Bin the
the Assay. portion of Tablets taken:
Medium, Apparatus 2, Standard stock solution, Stan-
dard solution, Sample solution, and Analysis: Pro- Result = (ru/rs) x (Cs/Cu) x 100
ceed as directed in Jest 1.
Time: 30 min ru = peak response of moexipril related compound
Tolerances: NLT 80% (Q) of the labeled amount of A and moexipril related compound B from
moexipril hydrochloride (C27H34N2O7 - HCl) is dissolved. the Sample solution
© UNIFORMITY OF DosAGE UNITS (905): Meet the require- rs = peak response of moexipril related compound
ments for Content Uniformity A and moexipril related compound B from
the Standard solution
IMPURITIES Cs = concentration of USP Moexipril Related
© ORGANIC IMPURITIES Compound A RS and USP Moexipril Related
Solution A: 0.025% trifluoroacetic acid in water Compound B RS in the Standard solution
Solution B: Acetonitrile and tetrahydrofuran (90:10) (mg/mL)
Diluent: Acetonitrile and water (30:70) Cy = nominal concentration of moexipril
Mobile phase: See Table 1. hydrochloride in the Sample solution
(mg/mL)
Table 1 Calculate the percentage of any other individual
unspecifieddeqradation product in the portion of
Solution A Solution B Tablets taken:
5, Result = (ru/rs) x (Cs/Cu) x 100
7
ty = peak response of any other individual
unspecified degradation product from the
0 5 Sample solution
ls = peak response of moexipril from the Standard
Impurity stock solution: 0.12 mg/mL of USP Moexipril
solution
Related CompoundG RS in Diluent. [NoTE—Sonication Gs = concentration of USP Moexipril Hydrochloride
may be necessary for complete dissolution.] RS in the Standard solution (mg/mL)
System suitability solution: 1.2 mg/mL of USP Moex- Cu = nominal concentration of moexipril fod
ipril Hydrochloride RS and 2.4 g/mL of USP Moexipril
Related Compound G RS from the Impurity stock solu- hydrochloride in the Sample solution a
tion in Diluent. [NoTE—Sonication may be necessary for (mg/mL)
Acceptance criteria: See Table 2. Disregard peaks less
z
z
complete dissolution.] than 0.1%. fo}
Standard stock solution: 1.2 mg/mL of USP Moexipril
Hydrochloride RS in Diluent. Initially add Diluent to
about 60% of the volume of the flask, and sonicate Table 2
F
wo
—
with intermittent shaking for complete dissolution. Relative Acceptance a
Standard solution: 6 jig/mL each of USP Moexipril Re- Retention Criteria, >
lated Compound A RS and USP Moexipril Related Com- Name Time NMT (%) ns
pound B RS, and 1.2 4g/mL of USP Moexipril Hydro- Moexipril related
chloride RS in Diluent from the Standard stock solution. compound E@ 0.31 =
[Note—Sonication may be necessary for complete
dissolution.] Moexipril related
Sample solution: Nominally 1.2 mg/mL of moexipril compound F# 0.77 Xe
hydrochloride in Diluent, prepared fromasufficient Moexipril related
number of crushed Tablets. Initially add Diluent to compound A> 0.85 2.0
about 60% of the volume of theflask, and sonicate for Moexipril related
20 min with intermittent shaking in ice cold water. Di- compound G: 0.94 _
lute with Diluent to volume. Pass through a suitable fil- Moexipril 1.00 =
ter of 0.45-um pore size. Moexipril related
Chromatographic system compound Da 1aA7 “te
(See Chromatography (621), System Suitability.) Moexipril related
Mode: LC
compound C* 1.27 a
Detector: UV 210 nm
Column: 4.6-mm x 25-cm; 5-um packing L1 Moexipril related
Column temperature: 30° compound B¢ 1.43 1S
Flow rate: 1 mL/min Process-related impurities controlled in the drug substance.
Injection volume: 10 LL »(3$)-2-{(25)-N-[(15)-1-Carboxy-3-phenylpropyl]alanyl}-1,2,3,4-tetrahydro-
System suitability 6,7-dimethoxy-3-isoquinolinecarboxylic acid.
Samples: System suitability solution and Standard ¢(5S)-Ethyl 2-{(35,11a5)-8,9-dimethoxy-3-methyl-1,4-dioxo-3,4-dihydro-1 H-
pyrazino[1,2-b]isoquinolin-2(6H,11H,11aH)-yl}-4-phenylbutanoate.
solution
$Total impurities do not include moexipril related compound A.
Suitability requirements
Resolution: NLT 2.5 between moexipril and moex-
ipril related compound G, System suitability solution
Tailing factor: NMT 2.0 for the moexipril peak, Sys-
tem suitability solution
 2782 Moexipril / Official Monographs USP 41

Table 2 (Continued) Table 1

Relative Acceptance Tablet Strength
Retention Criteria, Moexipril
Name Time NMT (%) Hydrochloride/ Concentration of Concentration of
Any unspecified Hydrochlorothia- Moexipril Hydrochlorothia-
degradation product _ 0.2 zide Hydrochloride zide
Total impurities¢ = 2.0 (mg/mq) (mg/mL) (mg/mL)
@Process-related impurities controlled in the drug substance. 75/125. 0.06 0.1
» (35)-2-{(25)-N-[(1 5)-1-Carboxy-3-phenylpropylJalanyl}-1,2,3,4-tetrahydro- 15/12.5 0.06 0.05
6,7-dimethoxy-3-isoquinolinecarboxylic acid. 15/25 0.06 0.1
(5)-Ethyl 2-{(35,11a5)-8,9-dimethoxy-3-methyl-1,4-dioxo-3,4-dihydro-1 H-
pyrazino[1,2-b]isoquinolin-2(6H,11H,11aH)-yl}-4-phenylbutanoate. Sample solution: Prepare the Sample solutions of nomi-
4Total impurities do not include moexipril related compound A. nal concentrations given in Table 7 from NLT 20 pow-
ADDITIONAL REQUIREMENTS dered Tablets as follows. Initially add Diluent to about
e PACKAGING AND STORAGE: Store at controlled room tem- 60% of the total volume, sonicate for 45 min with in-
erature in tight, well-closed containers, and protect termittent shaking, and then dilute with Diluent to vol-
rom moisture. ume. Pass through asuitable filter of 0.45-14m pore size.
e LABELING: When more than one Dissolution test is given, Chromatographic system
the labeling states the test used only if Test 7 is not used. (See Chromatography (621), System Suitability.)
e USP REFERENCE STANDARDS (11) Mode: LC
USP Moexipril Hydrochloride RS Detector: UV 210 nm
USP Moexipril Related Compound A RS Column: 4.6-mm x 25-cm; 5-14m packing L7
(35)-2-{(25)-N-[(1 5)-1-Carboxy-3-phenylpropy!Jalanyl}- Column temperature: 30°
1,2,3,4-tetrahydro-6,7-dimethoxy-3-isoquinoline- Flow rate: 1 mL/min
carboxylic acid. Injection volume: 20 wL
C2sH30N207_ 470.51 Run time: 2.2 times the retention time of the moex-
USP Moexipril Related Compound B RS ipril peak
(5)-Ethyl 2-{(35,11a5)-8,9-dimethoxy-3-methyl-1,4-di- System suitability
oxo-3,4-dihydro-1 H-pyrazino[1,2-b]isoquinolin-2(6H, Sample: Standard solution
11H,11aH)-yl}-4-phenylbutanoate. Suitability requirements
C27H32N206 480.55 Column efficiency: NLT 2500 theoretical plates for
USP Moexipril Related Compound G RS moexipril and NLT 4000 theoretical plates for the hy-
oes ee ches ce Sara pera drochlorothiazide peaks
4-phenylbutan- rcp es, teaae Tailing factor: NMT 2.0 for both moexipril and hy-
trahydroisoquinoline-3-carboxylic acid. drochlorothiazide peaks
C26H32N207 484.54 Relative standard deviation: NMT 2.0% for both
moexipril and hydrochlorothiazide peaks
3
"
Analysis
5 Samples: Standard solution and Sample solution
sS
—
Calculate the percentage of the labeled amounts of
Dp
i) Moexipril Hydrochloride and
moexipril hydrochloride (C27H34N2O7 - HCl) and hydro-
tS pipettes (C;HgCIN30,Sz2) in the portion of Tablets
iS Hydrochlorothiazide Tablets taken:
=
a DEFINITION Result = (ru/rs) x (Gs/Cu) x 100
val
=) Moexipril Hydrochloride and Hydrochlorothiazide Tablets
contain NLT 90.0% and NMT 110.0% each of the labeled ty = peakresponse of moexipril or
amounts of moexipril hydrochloride (C27H34N2O7 - HCl) hydrochlorothiazide from the Sample solution
and hydrochlorothiazide (C7HsCIN304S2). rs = peakresponse of moexipril or
hydrochlorothiazide from the Standard
IDENTIFICATION solution
e A. The retention times of the major peaks of the Sample Cs = concentration of USP Moexipril Hydrochloride
solution correspond to those of the Standard solution, as RS or USP Hydrochlorothiazide RS in the
obtained in the Assay. Standard solution (mg/mL)
Cy = nominal concentration of moexipril
ASSAY hydrochloride or hydrochlorothiazide in the
© PROCEDURE Sample solution (mg/mL)
Buffer: 0.01 M potassium dihydrogen phosphate Acceptance criteria: 90.0%-110.0% each of the la-
Mobile phase: Acetonitrile and Buffer (350:650) beled amounts of moexipril hydrochloride and
Diluent: Acetonitrile and water (30:70) hydrochlorothiazide
Standard solution: Prepare solutions of USP Moexipril
Hydrochloride RS and USP Hydrochlorothiazide RS in PERFORMANCE TESTS
Diluent, of concentrations stated in Table 1. Initially add e DISSOLUTION (711)
Diluent to 70% of the total volume, sonicate to dissolve, Buffer, Mobile phase, Diluent, Chromatographic sys-
and then dilute with Diluent to volume. tem, and System suitability: Proceed as directed in
the Assay.
Medium: 0.1 N hydrochloric acid; 900 mL
Apparatus 2: 50 rpm
Time: 15 min
Standard solution: Prepare solutions of USP Moexipril
Hydrochloride RS and USP Hydrochlorothiazide RS in
Medium of concentrations stated in Table 2.
USP 41 Official Monographs / Moexipril 2783

Table 2 volume, and pass through a suitable filter of 0.45-um

Tablet Strength
pore size.
Moexipril
Hydrochloride/ Concentration of Concentration of Table 4
Hydrochlorothia- USP Moexipril USP Hydrochloro- Tablet
zide Hydrochloride RS thiazide RS Strength Nominal Nominal
(mg/mg) (ug/mL) (ug/ml) Moexipril Concentra- Concentra-
7.5/12.5 8 14 Hydrochlo- tion tion
15/12.5 16 14 ride/ of Moexipril of Hydro-
15/25 16 28 Hydrochlo- Number of Hydrochlo- chlorothia-
rothiazide Tablets ride zide
Sample solution: Pass a portion of the solution under (mg/mq) (NLT) (mg/mL) (mg/mL
test through a suitable filter of 0.45-l1m pore size, dis- 7.5/12.5 20 1.2 2
carding the first 2-3 mL. 15/12.5 10 1.8 125.
Analysis 15/25 10 a 2
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amounts of Chromatographic system
moexipril hydrochloride (C27H34N2O7 - HCI) and hydro- (See Chromatography (621), System Suitability.)
chlorothiazide (C7HsCIN304S2) dissolved: Mode: LC
Detector: UV 210 nm
Result = (ru/rs) x (Cs/L) x Vx 100 Column: 4.6-mm x 25-cm; 5-4um packing L1
ty = peakresponse of moexipril or Column temperature: 30°
hydrochlorothiazide from the Sample solution Flow rate: 1 mL/min
rs = peak response of moexipril or Injection volume: 10 LL
hydrochlorothiazide from the Standard System suitability
solution Samples: System suitability solution and Standard
Gs = concentration of USP Moexipril Hydrochloride solution
RS or USP Hydrochlorothiazide RS in the Suitability requirements
Standard solution (mg/mL) Resolution: NLT 2.5 between the moexipril and
L = label claim for moexipril hydrochloride or moexipril related compoundG peaks, System suitabil-
hydrochlorothiazide (mg/Tablet) ity solution
Vv = volume of Medium, 900 mL Tailing factor: NMT 2.0 for both moexipril and hy-
Tolerances: NLT 70% (Q) of the labeled amounts each drochlorothiazide peaks, Standard solution
of moexipril hydrochloride (C27H34N2O7 - HCl) and hy- Relative standard deviation: NMT 5.0% for both
drochlorothiazide (C7HsCIN30.S2) are dissolved. moexipril and hydrochlorothiazide peaks, Standard
e UNIFORMITY OF DOSAGE UNITS (905): Meet the solution
requirements Analysis ce
Samples: Standard solution and Sample solution A
ao)
IMPURITIES Calculate the percentage of moexipril related com-
© ORGANIC IMPURITIES pound A and moexipril related compoundB in the ms
portion of Tablets taken: °
Solution A: Add 1 mL of trifluoroacetic acid to 4L of =
water. }
Solution B: Acetonitrile and tetrahydrofuran (90:10) Result = (ru/rs) x (Cs/Cu) x 100 re}
=
Mobile phase: See Table 3. EY)
ru = peak response of moexipril related compound a}
A or moexipril related compound B from the 2B
a
Table 3 Sample solution
Solution A Solution B rs = peak response of USP Moexipril Related
Compound A RS or USP Moexipril Related
CompoundB RS from the Standard solution
Cs = concentration of USP Moexipril Related
3
Compound A RS and USP Moexipril Related
95 CompoundB RS in the Standard solution
0 2 (mg/mL)
Cy = nominal concentration of moexipril
Diluent: Proceed as directed in the Assay. hydrochloride in the Sample solution
System suitability solution: 1.2 mg/mL of USP Moex- (mg/mL)
ipril Hydrochloride RS, 2 mg/mL of USP Hydrochlorothi- Calculate the percentage of benzothiadiazine related
azide RS, and 2.4 g/mL of USP Moexipril Related Com- compound A or chlorothiazide in the portion of
pound G RS in Diluent. Initially add Diluent to 70% of Tablets taken:
the total volume, sonicate to dissolve, and then dilute
with Diluent to volume. Result = (ru/rs) x (Cs/Cu) x 100
Standard solution: 1.2 ug/mL of USPNicexpal Hydro-
chloride RS, 12 g/mL each of USP Moexipril Related tu = peak response of benzothiadiazine related
Compound A RS and USP Moexipril Related Compound compound Aor chlorothiazide from the
B RS, 2 ug/mL of USP Hydrochlorothiazide RS, and Sample solution
40 g/mL each of USP Benzothiadiazine Related Com- Is = peak response of benzothiadiazine related
pound A RS and USP Chlorothiazide RS in Diluent. Ini- compound Aor chlorothiazide from the
tially add Diluent to 70% of the total volume, sonicate Standard solution
to dissolve, and then dilute with Diluent to volume. Cs = concentration of USP Benzothiadiazine Related
Sample solution: Prepare solutions of nominal concen- Compound A RS or USP Chlorothiazide RS in
tration given in Table 4. Initially add Diluent to 70% of the Standard solution me
the total volume, and sonicate for 15 min with intermit- Cu = nominal concentration of hydrochlorothiazide
tent shaking in ice cold water. Dilute with Diluent to in the Sample solution (mg/mL)
 2784 Moexipril / Official Monographs USP 41

Calculate thepercentage of any other individual USP Hydrochlorothiazide RS

impurity in the portion of Tablets taken: USP Moexipril Hydrochloride RS
USP Moexipril Related Compound A RS
Result = (ru/rs) x (Cs/Cu) x 100 (35)-2-{(25)-N-[(1 S)-1-Carboxy-3-phenylpropylJalanyl}-
1,2,3,4-tetrahydro-6,7-dimethoxy-3-isoquinoline-
Tu = peak response of any other individual impurity carboxylicacid.
from the Sample solution CosH30N207_ 470.51
rs = peak response of moexipril from the Standard USP Moexipril Related Compound B RS
solution (5)-Ethy! 2-{(35,11a5S)-8,9-dimethoxy-3-methyl-1,4-di-
Gs = concentration of USP Moexipril Hydrochloride oxo-3,4-dihydro-1 H- pyrazino[1, 2-bjisoquinolin-.2(6H,
RSin the Standard solution (mg/mL) 11H,1 4 ah)-ie-4--phenylbutanoate.
Cy = nominal concentration of moexipril CorHa2NoOs 480.55
hydrochloridein the Sample solution USP Moexipril Related Compound G RS
(mg/mL) (5)-6,7-Dimethoxy-2-{(5S)-2-[(S)-1-methoxy-1-oxo-
Acceptance chteriat See Table 5. 4- phenylbutan--2-ylarmina jpropancy' ls1,2,3,4-te-
ST ea ee 3-carboxylic acid.
Table 5 CasH32N207 484.54
Relative Acceptance
Retention Criteria,
Name Time NMT (%)
Moexipril related compound Ea* 0.31 _—
Benzothiadiazine related Molindone Hydrochloride
compound A 0.47 1.0
Chlorothiazides 0.53 0.5
Hydrochlorothiazide 0.57 = ort cb +H
5-Chlorohydrochlorothiazides’ 0.82 —
Moexipril related compound Ft» 0.77 =
Moexipril related compound A 0.85 1.0 CisHa4N2O2- HCl 312.83
Moexipril related compound Gh» 0.94 = 4H-Indol-4-one, 3-ethyl-1,5,6,7-tetrahydro-2-methyl-
Moexipril 100 —
5-(4-morpholinylmethy/)-, monohydrochloride.
3-Ethyl-6,7-dihydro-2-methyl--5-(morpholinomethyl)indol-
Moexipril related compound D> 1:12 =
4(SH)-one monohydrochloride [15622-65-8].
Moexipril related compound Ci. 1.27 =
Moexipril related compound Bk 1.43 1S » Molindone Hydrochloride contains not less
Any other individual unspecified _ than 98.0 percent and not more than 101.5 per-
ee
” impurity 0.2 cent of CisH24N2O2 - HCI, calculated on the anhy-
is Total impurities = 4.0! drous basis.
i 2 (S)-6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.
—
D » Process-related impurity controlled in the drug substance. Packaging and storage—Preserve in tight, light-resistant
ie) containers.
= ¢ 4-Amino-6-chloro-1,3-benzenedisulfonamide.
iS 4 2H-1,2,4-Benzothiadiazine-7-sulfonamide, 6-chloro-, 1,1-dioxide. USP Reference standards (11)—
= © 5-Chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide, 6- chloro- 3,4-di-
hydro-, 1,1-dioxide.
USP Molindone Hydrochloride RS
- f($)-2-[(5)-1-Ethoxy-1-oxo-4-phenylbutan-2-ylamino]propanoic acid. Identification—
a)
=} 9 (35)-2-{(25)-N-[(1 S)-1-Carboxy-3-sphenylpropyljalany|}- 1,2,3,4-tetrahydro- A: Infrared Absorption (197K). Do not dry specimens.
6,7-dimethoxy-3-isoquinolinecarboxylic acid.
B: Prepare a solution in methanol containing 10 mg of
4 (S)-6,7-Dimethoxy-2-{(5S)-2-[(S)-1-methoxy-AM-oxo-4-phenylbutan-2-ylami-
no]propanoy)}-1,2,3,4-tetrahydroisoquinoline--3-carboxylic acid.
molindone hydrochloride per mL. Separately apply 1 LL of
1 (S)-2-{(S)-2-[(5)-4-Cyclohexyl-1-ethoxy-1-oxobutan-2-ylamino]propanoyl}-
this solution and 1 uL of a Standard solution containing
6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. 10 mg per mL of USP Molindone Hydrochloride RS in meth-
J (S)-tert-Butyl 2-{(5)-2-[(S)-1-ethoxy-1-oxo-4-phenylbutan-2-ylami- anol to a thin-layer chromatographic plate (see Chromatog-
no]propanoyl}-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxyl- oy (621)) coated with a 0.25-mm layer of chromato-
ate. graphic silica gel mixture, and allow thespats to dry.
« (S)-Ethyl 2-{(35,11a5)-8,9-dimethoxy-3-methyl-1,4-dioxo-3,4-dihydro-1 H- Protect the chromatogram from light, and develop ina sol-
pyrazino[1,2-b]isoquinolin-2(6H, 11H, 11aH)-yl}-4-phenylbutanoate.
‘Total impurities is a sum total of all specified and unspecified impurities
vent system consisting of a mixture of alcohol, methanol,
and 1N hydrochloric acid (90:5:5) until the solvent front
ADDITIONAL REQUIREMENTS has moved about three-fourths of the length of the plate.
© PACKAGING AND STORAGE: Preserve in well-closed contain- Remove the plate from the developing chamber, mark the
ers, and protect from light. Store at controlled room solvent front, and allow the solvent to evaporate. Spray the
temperature. plate with a freshly prepared solution containing 100 mg of
e USP REFERENCE STANDARDS (11) potassium ferricyanide dissolved in 20 mL of 10% ferric
USP Benzothiadiazine Related Compound A RS chloride solution: the principal spot obtained from the test
4-Amino-6-chloro-1,3-benzenedisulfonamide. solution corresponds in R; value and intensity to that ob-
CeHgCIN30482 285.73 tained from the Standard solution.
USP Chlorothiazide RS C: It responds to the tests for Chloride (191).
2H-1,2,4-Benzothiadiazine-7-sulfonamide, 6-chloro-,1,1- pH (791): between 4.0 and 5.0, in a solution (1 in 100).
dioxide. Water Determination, Method | (921): not more than
C7HeCIN3048S2_ 295.73
0.5%.
USP 41
Residue on ignition (281): not more than 0.25%.
Delete the following:
°Heavy metals, Method [/ (231): not more than 0.003%.
© (Official 1-Jan-2018)
Chromatographic purity—
Mobile phase—Dissolve 1.1 g of sodium octanesulfonate
in 600 mL of water, add 400 mL of methanol, 1 mL of gla-
cial acetic acid, and 0.5 mL of triethylamine. Mix, filter
through afilter having a porosity of 0.45 jum or less, and
degas. Make adjustments if necessary (see System Suitability
under Chromatography (621)).
Solvent mixture—Proceed as directed in the Assay.
Standard solution—Prepare a solution of USP Molindone
Hydrochloride RS in Solvent mixture having a known concen-
tration of about 0.01 mg per mL.
Test solution—Transfer about 100 mg of Molindone Hy-
drochloride, accurately weighed, to a 50-mL volumetric
flask, dissolve in and dilute with Solvent mixture to volume.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 254-nm detector
and a 4.6-mm x 25-cm column that contains packing L11.
The column temperature is maintained at 35°. The flow rate
is about 1.5 mL per minute. Chromatograph the Standard
solution, and record the peak responses as directed under
Procedure: the relative standard deviation for replicate injec-
tions is not more than 5.0%.
Procedure—Separately inject equal volumes (about 20 j1L)
of the Standard solution and the Test solution into the chro-
matograph, record the chromatograms, and measure the re-
sponte of all peaks: no peak from the Test solution, other
than the molindone peak, is greater than the molindone
peak from the Standard preparation (0.5%), and the sum of
all the impurity peaks is not greater than 2.0%.
Assay—
Mobile phase—Dissolve 1.1 g of sodium octanesulfonate
in 480 mL of water, add 520 mL of methanol, 2 mL of gla-
cial acetic acid, and 0.4 mL of triethylamine. Mix, filter
through a 0.45-um filter, and degas. Make adjustments if
ony (see System Suitability under Chromatography
621)).
Solvent mixture—Prepare a mixture of 0.01 N hydrochlo-
ric acid and methanol (60:40).
Internal standard solution—Dissolve 200 mg of
butylparaben in 40 mL of methanol in a 100-mL volumetric
flask, dilute with water to volume, and mix.
Standard preparation—Transfer about 25 mg of USP
Molindone Hydrochloride RS, accurately weighed, to a
50-mL volumetric flask, add 5.0 mL of Internal standard solu-
tion, dilute with Solvent mixture to volume, and mix.
Assay preparation—Transfer about 50 mg of Molindone
Hydrochloride, accurately weighed, to a 100-mL volumetric
flask. Add 10.0 mL of Internal standard solution, dilute with
Solvent mixture to volume, and mix.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 254-nm detector
and a 4.6-mm x 25-cm column that contains packing L11.
The column temperature is maintained at 35°. The flow rate
is 1.5 mL per minute. Chromatograph the Standard prepara-
tion, and record the peak responses as directed under Proce-
dure: the resolution, R, between the molindone and
butylparaben peaks is not less than 2, and the relative stan-
dard deviation for replicate injections is not more than
2.0%.
Procedure—Separately inject equal volumes (about 10 pL)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
ure the responses for the major peaks. The relative retention
times are about 0.7 for molindone and 1.0 for
Official Monographs / Molindone 2785
butylparaben. Calculate the quantity, in mg, of CiséH24N2O2
-
HC] in the portion of Molindone Hydrochloride taken by
the formula:
100C(Ru / Rs)
in which C is the concentration, in mg per mL, of USP
Molindone Hydrochloride RS in the Standard preparation,
and Ry and Rs are the ratios of the peak response of
molindone to that of butylparaben obtained from the Assay
preparation and the Standard preparation, respectively.
Molindone Hydrochloride Tablets
» Molindone Hydrochloride Tablets contain not
less than 90.0 percent and not more than
110.0 percent of the labeled amount of
molindone hydrochloride (CisH24N2O2 - HCl).
Packaging and storage—Preserve in tight, light-resistant
containers.
USP Reference standards (11)—
USP Molindone Hydrochloride RS
Identification—Dissolve a portion of finely powdered Tab-
lets in methanol to obtain a test solution containing about
2.5 mg of molindone hydrochloride per mL. Separately ap-
ply 5 uL of the test solution and 5 wl of a Standard solution
of USP Molindone Hydrochloride RS in methanol containing
2.5 mg per mL to a thin-layer chromatographicplate (see
Chromatography (621)) coated with a 0.25-mm layer of
oer oe silica gel mixture. Allow the spots to dry,
protect the chromatogram from light, and develop ina sol-
vent system consisting of a mixture of alcohol and 1 N hy- (os
drochloric acid (95:5). Remove the plate from the develop- 2)
ing chamber, mark the solvent front, and allow the solvent uv
to evaporate. Locate the spots on the plate by spraying with =
Dragendorff’s reagent, prepared as directed for Visualization fo}
Technique 3 under Ordinary Impurities (466): the Rr value of S
the principal spot obtained from the test solution corre- fo]
Ko}
sponds to that obtained from the Standard solution. =
i)
Uniformity of dosage units (905): meet the require- me]
ments. a
my)
Dissolution (711)—
Medium: 0.1 N hydrochloric acid; 900 mL.
Apparatus 1: 100 rpm.
Time: 30 minutes.
Solvent A—Mix 300 mL of methanol and 700 mL of 0.1
N hydrochloric acid.
Solvent B—Mix 75 mL of methanol and 25 mL of 0.1 N
hydrochloric acid.
Standard solution—Transfer about 100 mg of USP
Molindone Hydrochloride RS, accurately weighed, to a
250-mL volumetric flask, and dissolve in and dilute with Sol-
vent A to volume. Pipet 5.0 mL of this stock solution into a
250-mL volumetric flask, and dilute with Solvent A to vol-
ume. Pipet 15.0 mL of the diluted stock solution into a
50-mL volumetric flask, and dilute with Solvent A to volume.
Test solution—Withdraw a portion of the solution under
test, and filter, discarding the first 3 mL of filtrate. Pipet
15.0 mL of this solution into a 25-mL volumetric flask, and
dilute with Solvent B to volume.
Mobile phase—Dissolve 1.08 g of sodium 1-octanesulfon-
ate in 480 mL of water. Add 520 mL of methanol, 2.0 mL of
acetic acid, and 0.4 mL of triethylamine, and mix. Make ad-
justments if necessary (see System Suitability under Chroma-
tography {621)).
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 254-nm UV detec-
 2786 Molindone / Official Monographs
tor and a 4.6-mm x 25-cm column that contains packing
L11. The flow rate is about 1.5 mL per minute.
Procedure—Separately inject equal volumes (about
100 uL) of the Standard solution and the Test solution into
the chromatograph, record the chromatograms, and meas-
ure the peak heights. Determine the amount of molindone
hydrochloride (CisHz4N2Oz
- HCl) dissolved.
Tolerances—Not less than 80% (Q) of the labeled amount
of CigH24N2O>
- HCI is dissolved in 30 minutes.
Assay—
Mobile phase, Solvent mixture, Internal standard solution,
Standard preparation, and Chromatographic system—Proceed
as directed in the Assay under Molindone Hydrochloride.
Assay preparation—Accurately weigh not less than 20
Tablets, grind the Tablets to a homogeneous mixture, and
transfer an accurately weighed portion, equivalent to about
50 mg of molindone hydrochloride, to a 250-mL conical
flask. Add 10.0 mL of Internal standard solution and 90.0 mL
of Solvent mixture, shake for 30 minutes, and filter.
Procedure—Proceed as directed for Procedure in the Assay
under Molindone Hydrochloride. Calculate the quantity, in
mg, of molindone hydrochloride (C;sH24N202
- HCl) in the
portion of Tablets taken by the formula:
100C(Ru / Rs)
in which C is the concentration, in mg per mL, of USP
Molindone Hydrochloride RS in the Standard preparation,
and Ry and Rs are the ratios of the peak response of
molindone to that of butylparaben obtained from the Assay
preparation and the Standard preparation, respectively.
Mometasone Furoate
is
a“
iy
i]
_
Dp
i)
is
iS
P=
a the internal standard from the Standard
A)
2 solution
Co7H39Cl2O6 521.43
Pregna-1,4-diene-3,20-dione, 9,21-dichloro-1 7-[(2-
furanylcarbonyl)oxy]-11-hydroxy-16-methyl-, (118, 16«)-;
9,21-Dichloro-11B,17-dihydroxy-16a-methylpregna-1,4-
diene-3,20-dione 17-(2-furoate) [83919-23-7].
DEFINITION
Mometasone Furoate contains NLT 97.0% and NMT
102.0% of mometasone furoate (C27H30Cl20¢), calculated
on the dried basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197M)
e B. The retention time of the Sample solution corresponds
to that of the Standard solution, both relative to the inter-
nal standard, as obtained in the Assay.
ASSAY
° PROCEDURE
Mobile phase: Methanol and water (65:35)
Diluent: Methanol, acetic acid, and water (65: 0.2: 35)
Internal standard solution: 0.4 mg/mL of
beclomethasone dipropionate in Diluent
Standard stock solution: 0.1 mg/mL of USP
Mometasone Furoate RS, preparedby dissolving USP
Mometasone Furoate RS in methanol and diluting
quantitatively and stepwise, if necessary, with Diluent
USP 41
Standard solution: 0.02 mg/mL of USP Mometasone
Furoate RS and 0.08 mg/mL of beclomethasone dipro-
pionate, pera by pipetting equal volumes of Stan-
dard stock solution and Internal standard solution into a
suitable volumetric flask and diluting with Diluent to
volume, if necessary
Sample stock solution: 0.1 mg/mL of mometasone
furoate, prepared by dissolving Mometasone Furoate in
methanol and diluting quantitatively and stepwise, if
necessary, with Diluent
Sample solution: 0.02 mg/mL of mometasone furoate
and 0.08 mg/mL of beclomethasone dipropionate, pre-
pared by pipetting 10 mL each of Sample stock solution
and Internal standard solution into a 50-mL volumetric
flask and diluting with Diluent to volume
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; packing L7
Flow rate: 1.7 mL/min
Injection size: 20 uL
System suitability
Sample: Standard solution
[NotE—The relative retention times for mometasone
furoate and beclomethasone dipropionate are about
1.0 and 1.6, respectively.]
Suitability requirements
Resolution: NLT 4.0 between the mometasone
furoate and beclomethasone dipropionate peaks
Taling factor: NMT 1.8 for the mometasone furoate
pea
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of mometasone furoate
eee) in the portion of Mometasone Furoate
taken:
Result = (Ru/Rs) X (Cs/Cu) x 100
Ru = peak response ratio of mometasone furoate to
the internal standard from the Sample
solution
Rs = peak response ratio of mometasone furoate to
Cs = concentration of USP Mometasone Furoate RS
in the Standard solution (mg/mL)
Cu = concentration of Mometasone Furoate in the
Sample solution (mg/mL)
Acceptance criteria: 97.0%-102.0% on the dried basis
IMPURITIES
© RESIDUE ON IGNITION (281): NMT 0.1%
Delete the following:
°e HEAVY METALS, Method I (231): NMT 30 Ug/ge cite 1-
Jan-2018)
e@ ORGANIC IMPURITIES
Standard stock solution: 10mg/mL of USP
Mometasone Furoate RS in dichloromethane
Standard solution A (5%): 0.5 mg/mL of USP
Mometasone Furoate RS in dichloromethane from the
Standard stock solution
Standard solution B (2%): 0.2 mg/mL of USP
Mometasone Furoate RS in dichloromethane, from the
Standard stock solution
Standard solution C (1%): 0.1 mg/mL of USP
Mometasone Furoate RS in dichloromethane, from the
Standard stock solution
USP 41 Official Monographs / Mometasone 2787

Standard solution D (0.2%): 0.02 mg/mL of USP ASSAY

Mometasone Furoate RS in dichloromethane, from the © PROCEDURE
Standard stock solution [Note—Protect from light.]
Standard solution E (0.1%) 0.01 mg/mL of USP Diluent A: Tetrahydrofuran and glacial acetic acid
Mometasone Furoate RS in dichloromethane, from the (100:1)
Standard stock solution Diluent B: Acetonitrile, water, and glacial acetic acid
Sample solution: 10 mg/mL of Mometasone Furoate in (50:50:1)
dichloromethane Solution A: Water
Chromatographic system Solution B: Acetonitrile
(See Chromatography (621), Thin-Layer Chromato- Mobile phase: See Table 1.
graphy.)
Mode: TLC Table 1
‘Adsorbent 0.25-mm layer of chromatographic silica
ge Solution A Solution B
Application volume: 40 pL
Developing solvent system: Chloroform and ethyl 70
acetate (3:1) A
Analysis
Samples: Standard solutions and Sample solution
Proceed as directed in the chapter. Examine the plate
under short-wavelength UVlight. Compare the inten-
sities of any secondary spots from the Sample solution
with those of the principal spots from the Standard Internal standard solution: 1.4 mg/mL of diethyl
phthalate in acetonitrile
solutions. Standard stock solution: 0.2 mg/mL of USP
Acceptance criteria: No secondary spot from the Sam- Mometasone Furoate RS in Diluent A
ple solution is larger or more intense than the principal
Standard solution: 0.05 mg/mL of mometasone
spot from Standard solution C; and the sum of the in- furoate and 0.35 mg/mL of diethyl phthalate from
tensities of the secondary spots from the Sample solu- equal quantities of the Standard stock solution and the
tion is NMT 2.0%. Internal standard solution, in Diluent B
SPECIFIC TESTS Sample solution: Transfer a portion of Cream, equiva-
¢ OPTICAL ROTATION, Specific Rotation (781S) lent to 1.0 mg of mometasone furoate, to a 50-mL,
Sample solution: 5 mg/mL in dioxane screw-capped centrifuge tube. Add 5.0 mL of Diluent A
Acceptance criteria: +56° to +62° and a few glass beads, and mix on a vortex mixer. Add
e Loss ON DRYING (731) 5.0 mL of Internal standard solution, and mix. Add
Analysis: Dry a sample at 105° for 3 h. 10.0 mL of Diluent B, mix on a vortex mixer for 1 min,
Acceptance criteria: NMT 0.5% and centrifuge for 10 min. Pass the aqueous phase
through a polypropylene filter of 0.2-um pore size, dis-
ADDITIONAL REQUIREMENTS carding the first 1-2 mL of filtrate. i
“
© PACKAGING AND STORAGE: Preserve in well-closed Chromatographic system vu
containers. (See Chromatography (621), System Suitability.)
Mode: LC c=
e USP REFERENCE STANDARDS (11) i}
USP Mometasone Furoate RS Detector: UV 254 nm =
Column: 4.6-mm x 25-cm; 5-um packing L60 )
Flow rate: 2 mL/min @=
Injection size: 20 uL iy
Bo}
System suitability Ge
Mometasone Furoate Cream Sample: Standard solution a]

[Note—The relative retention times for diethyl phthalate

DEFINITION
Mometasone Furoate Cream is Mometasone Furoate in a
suitable cream base. It contains NLT 90.0% and NMT
110.0% of the labeled amount of mometasone furoate
(Ca7H30Cl206).
IDENTIFICATION
e A. The retention time of the major peak of the Sample
solution corresponds to that of the Sander solution,
both relative to the internal standard, as obtained in the
Assay.
° *: THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
201)
Standard solution: 0.2 mg/mL of USP Mometasone
Furoate RS in acetonitrile
Sample solution: 0.2 mg/mL of mometasone furoate
from Cream in acetonitrile
Developing solvent system: Chloroform and ethyl ace-
tate (3:1)
Acceptance criteria: The R; value of the principal spot
of the Sample solution corresponds to that of the Stan-
dard solution.
and mometasone furoate are 0.4 and 1.0,
respectively]
Suitability requirements
Tall actor: NMT 1.5 for the mometasone furoate
ea
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of mometasone furoate
(C27H30Cl20¢) in the portion of Cream taken:
Result = (Ru/Rs) x (Cs/Cu) x 100
Ry = ratio of the mometasone furoate peak
response to the diethyl phthalate peak
response from the Sample solution
Rs = ratio of the mometasone furoate peak
response to the diethyl phthalate peak
response from the Standard solution
Gs = concentration of USP Mometasone Furoate RS
in the Standard solution (mg/mL)
Cu = nominal concentration of mometasone furoate
in the Sample solution (mg/mL)
 2788 Mometasone / Official Monographs USP 41

Acceptance criteria: 90.0%-110.0% Table 2 (Continued)

PERFORMANCE TESTS Relative Acceptance

e MINIMUM FILL (755): Meets the requirements Retention Criteria,
Name Time NMT (%)
IMPURITIES 21-Chloro-9B,11B-epoxy-17-
© ORGANIC IMPURITIES hydroxy-160-methylpregna-1,
[NoTe—Protect from light.] 4-diene-3,20-dione 17-(2-
Diluent A, Solution A, Solution B, Mobile phase, and furoate) 0.94 1.0
Standard stock solution: Prepare as directed in the Mometasone furoate 1.0 —
Assay. Unspecified individual impurit = 0.2
Diluent C: Acetonitrile, water, and glacial acetic acid
(30:70:1) Total specified and unspecified
impurities 1.0
System suitability solution: 0.1 ug/mL of USP
Mometasone Furoate RS from Standard stock solution in
Diluent C SPECIFIC TESTS
Blank solution: Diluent C and Diluent A (3:1) e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
Sample solution: Transfer a portion of Cream, equiva- FIED MICROORGANISMS (62): It meets the requirements of
lent to 2.0 mg of mometasone furoate, to a 50-mL, the tests for absence of Staphylococcus aureus, Pseudomo-
screw-capped centrifuge tube. Add 5.0 mL of Diluent A nas aeruginosa, Escherichia coli, and Salmonella species.
and a few glass beads, and mix on a vortex mixer. Add
15.0 mL of Diluent C, and mix. Centrifuge for 10 min. ADDITIONAL REQUIREMENTS
Pass the aqueous phase through a 0.2-11m polypropy- © PACKAGING AND STORAGE: Preserve in well-closed contain-
lene filter, discarding the first 1-2 mL of filtrate. ers, and store at controlled room temperature.
Chromatographic system e USP REFERENCE STANDARDS (11)
(See Chromatography (621), System Suitability.) USP Mometasone Furoate RS
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; 5-um packing L60
Column temperature: 25 + 5°
Flow rate: 2 mL/min Mometasone Furoate Ointment
Injection size: 50 uL
System suitability DEFINITION
Sample: System suitability solution Mometasone Furoate Ointment is Mometasone Furoate in a
Suitability requirements suitable ointment base. It contains NLT 90.0% and NMT
Relative standard deviation: NMT 10% 110.0% of the labeled amount of mometasone furoate
Analysis (Ca7H30Cl206).
” Samples: System suitability solution, Blank solution, and
<= Sample solution IDENTIFICATION
is [NotE—Exclude any peak areas less than those from the e A. The retention time of the major peak of the Sample
ii
- System suitability solution. Also exclude any peaks with solution corresponds to that of the Standard solution,
f=) both relative to the internal standard, as obtained in the
i} the same retention time as that observed in the Blank
= solution. Any peaks having a relative retention time of Assay.
5 about 1.04 or 1.13 are controlled in the Mometasone e B. THIN-LAYER CHROMATOGRAPHY
= Furoate monograph and, therefore, are not included in (See Chromatography (621), Thin-Layer Chromatography.)
a the total specified and unspecified impurities limit.] Standard solution: 0.6 mg/mL of USP Mometasone
a) Calculate the percentage of each impurity in the por- Furoate RS in methanol
= tion of Cream taken: Sample solution: Transfer the equivalent to 3 mg of
mometasone furoate from Ointment to a 50-mL screw-
Result = (ru/r7) x 100 capped centrifuge tube. Pipet 5.0 mL of methanol into
the tube, and attach the cap. Heat in a steam bath
ru = peak response of each impurity from the until the Ointment completely melts, and shake vigor-
Sample solution ously until the Ointment resolidifies. Place in an ice-
tr = sum of all the peak responses from the Sample water bath for 10 min. Centrifuge, and filter a portion
solution of the supernatant. Extract 1 mL of the filtrate with
Acceptance criteria: See Table 2. 1 mL of hexane, and use the lower phase.
Adsorbent: 0.25-mm layer of chromatographic silica
Table 2 gel mixture
Application volume: 10 pL
Relative Acceptance Developing solvent system A: Methanol
Retention Criteria,
Developing solvent system B: Chloroform and ethyl
Name Time NMT (%) acetate (3:1)
9a-Chloro-118,17,21-trihy- Analysis
droxy-16a-methylpregna-1,4- Samples: Standard solution and Sample solution
diene-3,20-dione 17-(2- Allow the spots to dry, and develop the chromato-
furoate) 0.56 0.1 ram in Developing solvent system A until the solvent
9a,21-Dichloro-11B,17- ront has moved 2 cm from the origin. Remove the
dihydroxy-1 6a-methylpregna- plate from the developing chamber, and air-dry. De-
1,4-diene-3,20-dione 0.73 0.1 velop the chromatogram in Developing solvent system
21-Chloro-17-hydroxy-1 6a- B until the solvent front has moved three-fourths of
methylpregna-1,4-diene-3,11, the length of the plate. Remove the plate from the
20-trione 17-(2-furoate) 0.88 0.1 developing chamber, mark the solvent front, and al-
low the spots to air-dry. Examine the plate under
short-wavelength UV light.
USP 41 Official Monographs / Mometasone 2789

Acceptance criteria: The R value of the principal spot Acceptance criteria: 90.0%-110.0%
of the Sample solution corresponds to that of the Stan-
dard solution. IMPURITIES
e ORGANIC IMPURITIES
ASSAY [Note—Protect from light.]
¢ PROCEDURE Diluent A, Solution A, Solution B, Mobile phase, and
{Note—Protect from light.] Standard stock solution: Prepare as directed in the
noo Tetrahydrofuran and glacial acetic acid Assay.
100: Diluent C: Acetonitrile, water, and glacial acetic acid
Diluent B: Acetonitrile, water, and glacial acetic acid (30:70:1)
(50:50:1) System suitability solution: 0.1 ug/mL of USP
Solution A: Water lometasone Furoate RS from Standard stock solution in
Solution B: Acetonitrile Diluent C
Mobile phase: See Table 1. Sample solution: Transfer a portion of Ointment,
equivalent to 2.0 mg of mometasone furoate, to a
Table 1 50-mL screw-capped centrifuge tube. Add 5.0 mL of
Diluent A and a few glass beads, and mix on a vortex
Solution A Solution B mixer. Add 15.0 mL of Diluent C, and mix. Centrifuge
for 10 min. Pass the aqueous phase through a polypro-
7 30 pylene filter of 0.2-um pore size, discarding the first
30 1-2 mL of filtrate.
Blank solution: Diluent C and Diluent A (3:1)
7
Chromatographic system
(See Chromatography (621), System Suitability.)
7 30
Mode: LC
Internal standard solution: 1.4 mg/mL of diethyl Detector: UV 254 nm
phthalate in acetonitrile Column: 4.6-mm x 25-cm; 5-1um packing L60
Standard stock solution: 0.2 mg/mL of USP
Column temperature: 25+ 5°
Mometasone Furoate RS in Diluent A Flow rate: 2 mL/min
Standard solution: 0.05 mg/mL of mometasone Injection size: 50 uL
furoate and 0.35 mg/mL of diethyl phthalate from System suitability
equal quantities of the Standard stock solution and the Sample: System suitability solution
Internal standard solution, in Diluent B Suitability requirements -
Sample solution: Transfer a portion of Ointment, Relative standard deviation: NMT 10%
equivalent to 1.0mg of mometasone furoate, to a
Analysis
50-mL screw-capped centrifuge tube. Add 5.0 mL of Samples: System suitability solution, Sample solution,
Diluent A and a few glass beads, and mix on a vortex and Blank solution
mixer. Add 5.0 mL of Internal standard solution, and [NoTte—Exclude any peak areas less than those from the 9
chromatogram of the System suitability solution. Also a)
mix. Add 10.0 mL of Diluent B, mix on a vortex mixer
exclude any peaks with the same retention time as z
for 1 min, and centrifuge for 10 min. Pass the aqueous
phase through a palypraay ene filter of 0.2-um pore that observed in the chromatogram of the Blank solu- =
tion. Any peaks havingarelative retention time of i)
size, discarding the first 1-2 mL of filtrate.
about 1.04 or 1.13 are controlled in the monograph =
Chromatographic system r)
(See Chromatography (621), System Suitability.) for Mometasone Furoate, and therefore are not in- a2
Mode: LC ice in the total specified and unspecified impurities iS)
Detector: UV 254 nm imit. i}
Column: 4.6-mm x 25-cm; 5-"um packing L60 Calculate the percentage of each impurity in the por- =y
a)
Flow rate: 2 mL/min tion of Ointment taken:
Injection size: 20 wl Result = (ru/r7) x 100
System suitability
Sample: Standard solution tu = peak response of each impurity from the
[NoTt—The relative retention times for diethyl phthalate Sample solution
and mometasone furoate are 0.4 and 1.0, tr = sum of all the peak responses from the Sample
respectively.] solution
een requirements Acceptance criteria: See Table 2.
Tailing a factor: NMT 1.5 for the mometasone furoate
peak
Relative standard deviation: NMT 2.0% Table 2
Analysis Relative Acceptance
Samples: Standard solution and Sample solution Retention Criteria,
Calculate the percentage of mometasone fuorate Name Time NMT (%)
(C27H30Cl2O¢) in the portion of Ointment taken: 9a-Chloro-11,17,21-trihy-
droxy-16a-methylpregna-1,
Result = (Ru/Rs) x (Cs/Cu) x 100 4-diene-3,20-dione 17-(2-
ratio of the mometasone furoate peak furoate) 0.56 0.2
Ru
response to the diethyl phthalate peak 9a,,21-Dichloro-11B,17-
response from the Sample solution dihydroxy-16a-methyl-
Rs = ratio of the mometasone furoate peak pregna-1,4-diene-3,20-dio-
response to the diethyl phthalate peak ne 0.73 0.2
response from the Standard solution 21-Chloro-17-hydroxy-160-
Cs = concentration of USP Mometasone Furoate RS methylpregna-1,4-diene-3,
in the Standard solution (mg/mL) 11,20-trione 17-(2-furoate) 0.88 0.2
nominal concentration of mometasone furoate
©
W

in the Sample solution (mg/mL)

 2790 Mometasone / Official Monographs USP 41

Table 2 (Continued) ASSAY

e PROCEDURE
Relative Acceptance
Retention Criteria,
[NoTe—Protect from light.]
Name Time NMT (%)
Diluent: Acetonitrile, water, and glacial acetic acid
(50:50:1)
21-Chloro-98,11B-epoxy-17- Solution A: Water
hydroxy-160-methylpregna- Solution B: Acetonitrile
1,4-diene-3,20-dione 17-(2-
Mobile phase: See Table 7.
furoate) 0.94 1.0
Mometasone furoate 1.0 =
Table 1
Unspecified individual _
impurit) 0.2 Solution A Solution B
Total specified and _ %
unspecified impurities 1.0 0 70 30
2 70 30

PERFORMANCE TESTS
e MinimuM FILL (755): Meets the requirements 46 70 30

SPECIFIC TESTS
© MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): It meets the requirements of
the tests for absence of Staphylococcus aureus, Pseudomo-
nas aeruginosa, Escherichia coli, and Salmonella species.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, and store at controlled room temperature.
e USP REFERENCE STANDARDS (11)
USP Mometasone Furoate RS
Mometasone Furoate Topical Solution
DEFINITION
Mometasone Furoate Topical Solution is Mometasone
hc
a“
Furoate in a suitable aqueous vehicle. It contains NLT
fe 90.0% and NMT 110.0% of the labeled amount of
g mometasone furoate (C27H30Cl20¢).
2)
3
= IDENTIFICATION
5 e A. The retention time of the major peak of the Sample
Ps solution corresponds to that of the Standard solution,
om both relative to the internal standard, as obtained in the
2) Assay.
=} ° B. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
(201)
Standard solution: 1 mg/mL of USP Mometasone Is
Furoate RS in a mixture of chloroform and methanol Cs
(4:1)
Sample solution: Transfer the equivalent of 2 mg of
mometasone furoate from Topical Solution to a 50-mL
centrifuge tube. Add 10 mL of water. Extract the aque-
ous solution with 20 mL of chloroform. Remove the
chloroform layer, dry over anhydrous sodium sulfate, IMPURITIES
and filter through a cotton pledget. Repeat the chloro- © ORGANIC IMPURITIES
form extraction, and combine the dried extracts. Evapo-
rate the chloroform solution to dryness on a steam bath
under a stream of nitrogen. Allow the sample specimen
to cool to room temperature. Dissolve the residue in a
mixture of chloroform and methanol (4:1) to obtain
1 mg/mL of Sample solution.
Application volume: 20 uL
Developing solvent system: Chloroform and ethyl ace-
tate (3:1)
Acceptance criteria: The R; value of the principal spot
of the Sample solution corresponds to the that of the
Standard solution.
Standard solution: 0.1 mg/mL of USP Mometasone
Furoate RS in Solution B
Sample solution: Transfer a portion of Topical Solution,
equivalent to about 2.5 mg of mometasone furoate, to
a 25-mL flask. Dilute with Diluent to volume, and mix.
Pass 4 ponion of the solution through a polypropylene
filter of 0.2-um pore size, discarding the first 1-2 mL of
filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; 5-uum packing L60
Flow rate: 2 mL/min
Injection size: 50 uL
System suitability
Sample: Standard solution
Suitability requirements:
Tailing factor: NMT 1.5 for the mometasone furoate
peak
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of mometasone furoate
(C27H30Cl2O¢) in the portion of Topical Solution taken:
Result = (ru/rs) x (Cs/Cu) x 100
ru = peak response from the Sample solution
= peak response from the Standard solution
= concentration of USP Mometasone Furoate RS
in the Standard solution (mg/mL)
Cu = nominal concentration of mometasone furoate
in the Sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0%
[Note—Protect from light.]
Diluent, Solution A, Solution B, Mobile phase, Stan-
dard solution, and Sample solution: Prepare as di-
rected in the Assay.
System suitability solution: 0.1 g/mL of USP
Mometaeone Furoate RS from Standard solution in
Diluent
USP 41 Official Monographs / Monensin 2791

Chromatographic system e USP REFERENCE STANDARDS (11)

(See Chromatography (621), System Suitability.) USP Mometasone Furoate RS
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; 5-1um packing L60
Column temperature: 25+ 5°
Flow rate: 2 mL/min
Injection size: 50 pL Monensin
System suitability
Sample: System suitability solution ot CH, CH,
6 9. OH
Suitability requirements ON OA. oS
Relative standard deviation: NMT 10% ? | 9 ~ OCH, 0
CH,
Analysis H.C! CH, CH,
3 oH
Samples: Diluent, System suitability solution, and Sam-
ple solution
[Note—Exclude any peak areas less than that of the Sys- C36H62011(monensin A) 670.87
tem suitability solution. Also, exclude any peaks with C3sHeoO11(monensin B) 656.84
the same retention times as those observed in the Dil- C37He4O11(monensin C) 684.90
uent. Any peaks having a relative retention time of Monensin.
1.04 or 1.13 are controlled in the Mometasone Furoate Stereoisomer of 2-[2-ethyloctahydro-3’-methyl-5’-
monograph, and therefore are not included in the to- [tetrahydro-6-hydroxy-6-(hydroxymethyl)-3,5-dimethyl-
tal specified and unspecified impurities limit.] 2H-pyran-2-y!][2,2’-bifuran-5-yl]]-9-hydroxy-B-methoxy-a,
Calculate the percentage of each impurity in the por- y,2,8-tetramethyl-1,6-dioxaspiro[4.5]decan-7-butanoic acid
tion of Topical Solution taken: [17090-79-8].

Result = (ru/r7) x 100 » Monensin is a mixture of antibiotic substances

produced by the growth of Streptomyces cin-
tu = peak response of each impurity from the namonensis. |t has a potency of not less than
Sample solution 110 ug of monensin per mg.
rr = sum of all the peak responses from the Sample
solution Packaging and storage—Preserve in well-closed contain-
Acceptance criteria: See Table 2. ers. Avoid moisture and excessive heat.
Labeling—Label it to indicate that it is for veterinary use
Table 2 only. Label it also to state that it is for manufacturing, pro-
Relative Acceptance cessing, or repackaging.
Retention Criteria, USP Reference standards (11)—
Name Time NMT (%) USP Monensin Sodium RS
USP Narasin RS (ex
9a-Chloro-11,17,21-trihy- a)
droxy-160-methylpregna-1,4- Identification—The chromatogram of the Assay prepara- ae]
diene-3,20-dione 17-(2- tion obtained as directed in the Assay exhibits a major peak c=
furoate) 0.56 0.3 for monensin A and a minor peak for monensin B, the re- °
9a,21-Dichloro-11B,17- tention times of which correspond to those exhibited in the =]
chromatogram of the Standard preparation, obtained as di- °
dihydroxy-1 6a-methylpregna- Ko}
1,4-diene-3,20-dione 0.73 0.1 rected in the Assay. =
=)
21-Chloro-17-hydroxy-160- Loss on drying (731)—Dry it in vacuum at 60° for 2 hours: me}
methylpregna-1,4-diene-3,11, it loses not more than 10% of its weight. >
7
20-trione 17-(2-furoate) 0.88 0.1 Content of monensin A andBactivity—Using the re-
21-Chloro-9B,11B-epoxy-17- sults of the calculations in the Assay, calculate the percent-
hydroxy-160-methylpregna- age of monensin A activity in the Monensin under test by
1,4-diene-3,20-dione 17-(2- the formula:
furoate)_ 0.94 1.0
Mometasone furoate 1.0 = 100A
/P
Unspecified individual impurity = 0.5
in which A is the potency, in ug per mg, of monensin A in
Total specified and unspecified _ the Monensin under test, as determined in the Assay, and P
impurities 2.0 is the potency, in ug of monensin, in each mg of the
Monensin under test, as determined in the Assay: not less
SPECIFIC TESTS than 90% is found. Calculate the percentage of monensin A
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI- activity plus monensin B activity in the Monensin under test
FIED MICROORGANISMS (62): It meets the requirements of by the formula:
the tests for absence of Staphylococcus aureus, Pseudomo-
nas aeruginosa, Escherichia coli, and Salmonella species. 100(A
+ B)/P
© PH (791): 4.0-5.0
in which B is the potency, in ug per mg, of monensin B in
ADDITIONAL REQUIREMENTS the Monensin under test, as determined in the Assay, and
e PACKAGING AND STORAGE: Preserve in well-closed contain- the other terms are as defined above: not less than 95% is
ers, and store at controlled room temperature. found.
Assay—
Mobile phase—Prepare a filtered and degassed mixture of
methanol, water, and glacial acetic acid (94:6:0.1). Make
adjustments if necessary (see System Suitability under Chro-
matography (621)).
 2792 Monensin / Official Monographs
Neutralized methanol—Add 1 g of sodium bicarbonate to
4 liters of methanol, mix, and filter.
Diluent—Prepare a mixture of methanol and water (9:1).
Derivatizing reagent—Dissolve 3 g of vanillin in a mixture
of 95 mL of methanol and 2 mL of sulfuric acid. [Caution—
To avoid splattering, add the sulfuric acid carefully and slowly
with a pipet; do not pour. Allow the mixture of methanol and
sulfuric acid to cool before adding vanillin.]
Standard preparation—Dissolve an accurately weighed
quantity of USP Monensin Sodium RS quantitatively in
methanol to obtain a solution containing the equivalent of
1000 ug of monensin per mL. Dilute an accurately meas-
ured volume of this stock solution quantitatively with Diluent
to obtain a solution containing 20.0 ug of monensin per
mL.
Assay preparation—Transfer about 500 mg of Monensin,
accurately weighed, to a 250-mL flask, add 200.0 mL of Dil-
uent, and shake by mechanical means for 1 hour. Allow the
solids to settle, and dilute an accurately measured volume of
the supernatant quantitatively with Diluent to obtain a solu-
tion containing about 20 jug of monensin per mL.
Resolution solution—Prepare a solution in Neutralized
methanol containing about 1 mg of USP Monensin Sodium
RS and 3 mg of USP Narasin RS per mL. Transfer 2 mL of
this solution to a 200-mL volumetric flask, dilute with Dilu-
ent to volume, and mix.
Chromatographic system (seeSiromatogaeny (621))—The
liquid chromatograph is equipped with a 4.6-mm x 25-cm
column that contains packing L1 and the outlet of which is
attached to a tee, the opposing arm of which is attached to
a tube from which is pumped the Derivatizing reagent, and
the outlet of which is connected to a 2-mL postcolumn re-
action coil maintained at 98°. The outlet of the reaction coil
is connected to a detector set at 520 nm. The Mobile phase
and the Derivatizing reagent flow at the rate of about 0.7 mL
per minute. Chromatograph the Resolution solution, and re-
”
<= cord the peak responses as directed under Procedure: the
ie relative retention times are about 0.9 for monensin B, 1.0
S tion obtained as directed in the Assay exhibits a major peak
— for monensin A, 1.3 for narasin A, and 1.5 for narasin |, the
Dd resolution, R, between the monensin B peak and the
2} monensin A peak is not less than 1.25, and between the
c
S monensin A peak and the narasin A peak is not less than
= 3.5. Chromatograph the Standardpeegnnoy and record
[om the peak responses as directed under Procedure: the tailing
al factor is not more than 1.4, and the relative standard devia-
= tion for replicate injections is not more than 2.0%. [NOTE—
After use,flush the system with methanol.]
Procedure—[NOTE—Use peak areas where peak responses
are indicated.] Separately inject equal volumes (about
200 uL) of the Standard Bee and the Assay prepara-
tion into the chromatograph, record the chromatograms,
and measure the responses for the major peaks, including a
peak for monensin C/D, if present, at a retention time of
about 1.1 relative to that of the main monensin A peak in
the chromatogram obtained from the Assay preparation. Cal-
culate the quantity, in ug, of monensin A in each mg of the
Monensin taken by the formula:
(CFD / 100,000W)(ru / rs)
in which C is the concentration, in ug per mL, of monensin
activity in the Standard preparation, based on the quantity
of USP Monensin Sodium RS taken, its desioviated paren’
in ug per mg, and the extent of dilution, Fis the designated
percentage of monensin A in USP Monensin Sodium RS,D is
the dilution factor used in preparing the Assay preparation,
Wis the quantity, in g, of Monensin taken to prepare the
Assay preparation, and ry and rs are the monensin A peak
responses obtained from the Assay preparation and the Stan-
dard preparation, respectively. Calculate the quantity, in ug,
of monensin B in each mg of the Monensin taken by the
same formula, except that ry is the monensin B peak re-
sponse obtained from the Assay preparation and rs is the
USP 41
monensin A peak response obtained from the Standard prep-
aration. Calculate the quantity, in ug, of monensin C/D in
each mg of the Monensin taken by the same formula, ex-
cept that ry is the monensin C/D peak response obtained
from the Assay preparation. Calculate the potency, in ug of
monensin, in each mg of the Monensin taken by the
ormula:
A+ 0.28B+1.5C/D
in which A is the quantity, in 4g, of monensin A in each mg
of the Monensin taken, as calculated above, and B is the
quantity, in ig, of monensin B in each mg of the Monensin
taken, and C/D is the quantity, in ug, of monensin C/D in
each mg of Monensin taken, as calculated above.
Monensin Granulated
» Monensin Granulated contains Monensin
mixed with suitable diluents, carriers, and inac-
tive ingredients prepared in a granulated form
that is free-flowing and free from aggregates. It
may contain added Monensin Sodium. It con-
tains not less than 140 mg of monensin per g.
Packaging and storage—Preserve in well-closed contain-
ers. Avoid moisture and excessive heat.
Labeling—Label it to indicate that it is for veterinary use
only. Label it also to state that it is for manufacturing, pro-
cessing, or repackaging.
USP Reference standards (11)—
USP Monensin Sodium RS
USP Narasin RS
Identification—The chromatogram of the Assay prepara-
for monensin A and a minor peak for monensin B, the re-
tention times of which correspond to those exhibited in the
chromatogram of the Standard preparation, obtained as di-
rected in the Assay.
Loss on drying (731)—Dry it in vacuum at 60° for 2 hours:
it loses not more than 10% of its weight.
Content of monensin A and B activity—Using the re-
sults of the calculations in the Assay, calculate the percent-
age of monensin A activity in the Monensin Granulated
under test by the formula:
100A
/P
in which A is the potency, in ig per mg, of monensin A in
the Monensin Granulated under test, as determined in the
Assay, and P is the potency, in ug of monensin, in each mg
of the Monensin Granulated under test, as determined in
the Assay: not less than 90% is found. Calculate the per-
centage of monensin A actly plus monensin B activity in
the Monensin Granulated under test by the formula:
100(A
+B)/P
in whichBis the potency, in ug per mg, of monensin B in
the Monensin Granulated under test, as determined in the
Assay, and the other terms are as defined above: not less
than 95% is found.
Assay—
Mobile phase, Neutralized methanol, Diluent, Derivatizing
reagent, Standard preparation, Resolution solution, and Chro-
matographic system—Proceed as directed in the Assay under
Monensin.
Assay preparation—Transfer about 5 g of Monensin Gran-
ulated, accurately weighed, to a 250-mL flask, add 200.0 mL
USP 41
of Diluent, and shake by mechanical means for 1 hour. Allow
the solids to settle, and dilute an accurately measured vol-
ume of the supernatant quantitatively with Diluent to obtain
a solution containing about 20 ug of monensin per mL.
Procedure—Proceed as directed for Procedure in the Assay
under Monensin. Calculate the quantity, in mg, of monensin
A in each g of the Monensin Granulated taken by the
formula:
(CFD / 100,000W)(ru / rs)
in which Cis the concentration, in ug per mL, of monensin
activity in the Standard preparation, based on the quantity
of USP Monensin Sodium RS taken, its org acedpene
in ug per mg, and the extent of dilution, F is the designated
percentage of monensin A in USP Monensin Sodium RS,Dis
the dilution factor used in preparing the Assay preparation,
Wis the quantity, in g, of Monensin Granulated taken to
prepare the Assay preparation, and ry and rs are the monen-
sin A peak responses obtained from the Assay preparation
and the Standard preparation, respectively. Calculate the
quantity, in mg, of monensin B in each g of the Monensin
Granulated taken by the same formula, except that ry is the
monensin B peak response obtained from the Assay prepara-
tion and rs is the monensin A peak response obtained from
the Standard preparation. Calculate the quantity, in mg, of
monensin C/D in each g of the Monensin Granulated taken
by the same formula, except that ry is the monensin C/D
eak response obtained from the Assay preparation. Calcu-
late the potency, in ug of monensin, in each mg of the
Monensin Granulated taken by the formula:
A+ 0.28B+1.5C/D
in whichA is the quantity, in mg, of monensin A in each g
of the Monensin Granulated taken, as calculated above, and
B is the quantity, in mg, of monensinB in each g of the
Monensin Granulated taken, and C/D is the quantity, in mg,
of monensin C/D in each g of Monensin Granulated taken,
as calculated above.
Monensin Type A Medicated Article
ty this monograph not to change until December 1,
2017,
(Prior to December 1, 2017, the current practice of labeling
the article of commerce with the name Monensin Premix
may be continued. Use of the name Monensin Type A
Medicated Article will be permitted as of June 1, 2017;
however, the use of this name will not be mandatory until
December 1, 2017.) " time of 1.1 relative to that of the main monensin A
DEFINITION
Monensin lyps A Medicated Article contains Monensin
Granulated mixed with suitable diluents and inactive in-
gredients. It contains the equivalent of NLT 85.0% and
NMT 115.0% of the labeled amount of monensin.
IDENTIFICATION
e A. The retention times of the major peak for monensin A
and minor peak for monensin B of the Sample solution
correspond to those of the Standard solution, as obtained
in the Assay.
ASSAY
© PROCEDURE
Mobile phase: Methanol, glacial acetic acid, and water
(940:1:60)
Neutralized methanol: Add 1 g of sodium bicarbonate
to 4L of methanol, mix, and filter.
Diluent: Methanol and water (9:1)
Derivatizing pagent Dissolve 3 g of vanillin in a mix-
ture of 95 mL of methanol and 2 mL of sulfuric acid.
Official Monographs / Monensin 2793
[CAuTION—To avoid splattering, add the sulfuric acid
carefully and slowly with a pipet; do not pour. Allow
the mixture of methanol and sulfuric acid to cool
before adding vanillin.]
System suitability solution: 1 mg/mL of USP Monensin
Sodium RS and 3 mg/mL of USP Narasin RS in Neutral-
ized methanol. Dilute 2 mL of this solution with Diluent
to 200 mL.
Standard stock solution: 1000 g/mL of monensin
from USP Monensin Sodium RS in methanol
Standard solution: 20 1g/mL of monensin from Stan-
dard stock solution in Diluent
Sample stock solution: Dilute 5 g of Monensin Type A
Medicated Article in 200.0 mL of Diluent, and shake by
mechanical means for 1 h. Allow the solids to settle.
Sample solution: Nominally 20 g/mL of monensin,
from the clear supernatant of the Sample stock solution,
in Diluent
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 520 nm
Column: 4.6-mm x 25-cm; packing L1. The column
outlet is attached toa tee, the opposing arm is at-
tached to a tube from which is pumped the Derivati-
zing reagent, and the outlet is connected to a 2-mL
postcolumn reaction coil maintained at 98°. The outlet
of the reaction coil is connected to the Detector.
Flow rate: 0.7 mL/min for Mobile phase and Derivati-
zing reagent
Injection volume: 200 pL
System suitability
Samples: System suitability solution and Standard
solution
[Note—The relative retention times for monensin B,
monensin A, narasin A, and narasin | are about 0.9,
1.0, 1.3, and 1.5, respectively.]
Suitability requirements fox
Resolution: NLT 1.25 between the monensin B and al
the monensin A peaks; NLT 3.5 between the monen-
v
sin A and the narasin A peaks, System suitability c<
solution 2)
]
Tailing factor: NMT 1.4, Standard solution )
Relative standard deviation: NMT 2.0%, Standard eo)=
solution 2
[Note—After use, flush the system with methanol.] so)
Analysis Sy
“
Samples: Standard solution and Sample solution
[Note—Use peak areas where peak responses are
indicated.]
Measure the responses for the major peaks, including
a peak for monensin C/D, if present, at a retention
peak in the chromatogram from the Sample solution.
Calculate the quantity, in mg, of monensin A, monen-
sin B, and monensin C/D in each g of Monensin Type
A Medicated Article taken:
Result = (ru/rs) x (Cs x F x D)/(100,000 x W)
tu = peak response of monensin A, monensin B, or
monensin C/D from the Sample solution
rs = peak response of monensin A from the
Standard solution
Cs = concentration of monensin activity in the
Standard solution, based on the quantity of
USP Monensin Sodium RS taken, its
designated potency (\ug/mg) and extent of
dilution (tug/mL)
= designated percentage of monensin A in USP
Monensin Sodium RS
D = dilution factor used in preparing the Sample
solution
 2794 Monensin / Official Monographs USP 41

Ww = quantity of Monensin Type A Medicated Loss on drying (731)—Dry it in vacuum at 60° for 3 hours:
Article taken to prepare the Sample solution it loses not more than 4% of its weight.
(g) Content of monensin A and Bactivity—Using the re-
Calculatethe potency, in mg, of monensin in each g of sults of the calculations in the Assay, calculate the percent-
Monensin Type A Medicated Article taken: age of monensin A activity in the Monensin Sodium under
test by the formula:
Result = (A x Fa) + (Bx Fa) + (C/D X Fen)
100A/P
A = quantity of monensin A in each g of Monensin
Type A Medicated Article taken, as calculated in which A is ie pati. in mgper 9. of monensin A in
previously (mg) the Monensin Sodium under test, as determined in the As-
Fa = biopotency conversion factor for monensin A, say, and Pis the potency, in mg of monensin, in each g of
1.00 the Monensin Sodium under test, as determined in the As-
B = quantity of monensin B in each g of Monensin Say: not less than 90% is found. Calculate the percentage of
Type A Medicated Article taken, as calculated monensin A activity plus monensin B activity in the Monen-
previously (mg) sin Sodium under test by the formula:
Fg = biopotency conversion factor for monensin B,
0.28 100(A
+B)/P
C/D = quantity of monensin C/D in eachg of
Monensin Type A Medicated Article taken, as in which B is the potency, in mg per g, of monensin B in
calculated previously (mg) the Monensin Sodium under test, asSs etined in the As-
Fo = biopotency conversion factor for monensin say, and the other terms are as defined above: not less than
C/D, 1.50 95% is found.
Acceptance criteria: 85.0%-115.0% Assay—
SPECIFIC TESTS Mobile phase, Neutralized methanol, Diluent, Derivatizing
e Loss ON DRYING (731) reagent, Standard preparation, Resolution solution, and Chro-
Analysis: Dry under vacuum at 60° for 2 h. matographic system—Proceed as directed in the Assay under
Acceptance criteria: NMT 10% Monensin.
Assay preparation—Transfer about 100 mg of Monensin
ADDITIONAL REQUIREMENTS Sodium, accurately weighed, to a 100-mL volumetric flask,
e PACKAGING AND STORAGE: Preserve in well-closed contain- dissolve in and dilute with methanol to volume. If necessary,
ers. Avoid moisture and excessive heat. to achieve complete dissolution, sonicate for about 1 min-
© LABELING: Label it to indicate that it is for veterinary use ute, and mix. Dilute an accurately measured volume of this
only. The label bears the statement “Do not feed solution quantitatively with Diluent to obtain a solution con-
undiluted”. taining about 20 ug of monensin per mL.
e USP REFERENCE STANDARDS (11)
Procedure—Proceed as directed for Procedure in the Assay
USP Monensin Sodium RS under Monensin. Calculate the quantity, in mg, of monensin
=
”
USP Narasin RS Ain each g of the Monensin Sodium taken by the formula:
rs
i]
—
D (CFD / 100,000W)(ru / rs)
°
€ in which C is the concentration, in ug per mL, of monensin
Sj Monensin Sodium activity in the Standard preparation, based on the quantity
= of USP Monensin Sodium RS taken, its gesgnated potency,
a C36HeiNaOi (monensin A sodium) 692.85 in 4g per mg, and the extent of dilution; Fis the designated
a) C3sHsgNaO11 (monensin B sodium) 678.83 percentage of monensin A in USP Monensin Sodium RS;D is
a C37HesNaOii (monensin C sodium) 706.88 the dilution factor used in preparing the Assay preparation;
Monensin, sodium salt. Wis the quantity, in g, of Monensin Sodium taken to pre-
Stereoisomer of 2-[2-ethyloctahydro-3’-methyl-5’- pare the Assay preparation; and ry and rs are the monensin A
[tetrahydro-6-hydroxy-6-(hydroxymethyl)-3,5-dimethyl- peak responses obtained from the Assay preparation and the
2H-pyran-2-yl][2,2’-bifuran-5-yl]]-9-hydroxy-B-methoxy-o, Standard preparation, respectively. Calculate the quantity, in
y,2,8-tetramethyl-1,6-dioxaspiro[4.5]decan-7—butanoic mo, of monensin B in each g of the Monensin Sodium
acid sodiumsalt [22373-78-0]. taken by the same formula, except that ry is the monensin B
peak response obtained from the Assay preparation, and rs is
» Monensin Sodium has a potency of not less the monensin A peak response obtained from the Standard
than 800 wg per mg. preparation. Calculate the quantity, in mg, of monensin C/D
in each g of the Monensin Sodium taken by the same
Packaging and storage—Preserve in well-closed contain- formula, except that ry is the monensin C/D peak response
ers. Avoid moisture and excessive heat. obtained from the Assay preparation. Calculate the potency,
Labeling—Label it to indicate that it is for veterinary use in mg of monensin, in each g of the Monensin Sodium
only. Label it also to state that it is for manufacturing, pro- taken by the formula:
cessing, or repackaging.
USP Reference standards (11)— A+0.288
+ 1.5C/D
USP Monensin Sodium RS
USP Narasin RS in which A is the quantity, in mg, of monensin A in each
Identification—The chromatogram of the Assay prepara- of the Monensin Sodium taken, as calculated above; B is the
tion obtained as directed in the Assay exhibits a major peak quantity, in mg, of monensin B in each g of the Monensin
for monensin A and a minor peak for monensin B, the re- Sodium taken; and C/D is the quantity, in mg, of monensin
tention times of which correspond to those exhibited in the a in each g of Monensin Sodium taken, as calculated
chromatogram of the Standard preparation, as obtained in above.
the Assay.
USP 41 Official Monographs / Montelukast 2795

Monobenzone Monobenzone Cream

i N\ - » Monobenzone Cream contains not less than

94.0 percent and not more than 106.0 percent of
the labeled amount of monobenzone (Ci3H1202).
Ci3H1202 200.23 Packaging and storage—Preserve in tight containers, and
Phenol, 4-(phenylmethoxy)-. avoid exposure to temperatures higher than 30°.
p-(Benzyloxy)phenol [103-16-2].
USP Reference standards (11)—
» Monobenzone, dried at 105° for 3 hours, con- USP Monobenzone RS
tains not less than 98.0 percent and not more Identification—Transfer a quantity of Cream, equivalent to
than 102.0 percent of C:3Hi202. about 500 mg of monobenzone, to a centrifuge bottle, add
100 mL of water, and shake until the cream is completely
Packaging and storage—Preserve in tight, light-resistant dispersed. Centrifuge the suspension, decant the superna-
containers, and avoid exposure to temperatures above 30°. tant, wash the residue with water, again centrifuge, and de-
cant the water. Transfer the residue to a separator with the
USP Reference standards (11)—
aid of water, and adjust the volume to about 100 mL. Ex-
USP Monobenzone RS tract with four 25-mL portions of chloroform, filtering the
identification— extracts through a pledget of cotton into a 150-mL flask.
A: Infrared Absorption (197K). Evaporate the chloroform in a current of warm air, and add
B: Ultraviolet Absorption (197U)— 5 mL of pyridine and 3 mL of acetic anhydride to the ay
Solution: 101g per mL. residue. Connect the flask to a reflux condenser, reflux for
10 minutes, cool, and proceed as directed in Identification
Medium: — methanol. test C under Monobenzone, beginning with “Add 100 mL of
Absorptivities at 292 nm, calculated on the dried basis, do water.” It meets the requirements of /dentification test C
not differ by more than 3.0%. under Monobenzone.
C: Transfer about 500 mg of Monobenzone, previously Assay—
dried, to a 150-mL flask fitted with a reflux condenser, em-
ploying a suitable glass joint. Add 5 mL of pyridine and Standardpleparation—erepate as directed in the Assay
3 mL of acetic anhydride, reflux for 10 minutes, and cool. under Monobenzone.
Add 100 mL of water and 6 mL of acetone to the flask, and Assay preparation—Transfer an accurately weighed por-
insert a stopper. Cool the contents of the flask in a refrigera- tion of Cream, equivalent to about 200 mg of
tor for 1 hour, collect the precipitate in a sintered-glass cru- monobenzone, to a suitable container, add 100 mL of meth-
cible, and wash the precipitate with water until no odor of anol, and shake for about 30 minutes. Transfer the mixture
pyridine remains. Dry the precipitate for 16 hours in a vac- to a 200-mL volumetric flask. Rinse the container with two
25-mL portions of methanol, and add the rinsings to the c
uum desiccator over phosphorus pentoxide. The a)
monobenzone acetate so obtained melts between 110° and 200-mL volumetric flask. Dilute with methanol to volume, a)
113° when determined as directed for Class | (see Melting mix, and filter, discarding the first 20 mL of the filtrate. Pi-
=
Range or Temperature {741)). pet 4 mL of this solution into a 100-mL volumetric flask, i}
Melting range, Class | (741): between 117° and 120°. dilute with methanol to volume, and mix. =)
re)
Loss on drying (731)—Dry it at 105° for 3 hours: it loses Procedure—Proceed as directed in the Assay under re}=
not more than 1.0% of its weight. Monobenzone, but use the Assay preparation under Ey
Residue on ignition (281): not more than 0.5%.
Monobenzone Cream. Calculate the quantity, in mg, of no}
monobenzone (C;3H;202) in the portion of Cream taken by a
Assay— the formula: ww

Standard preparation—Dissolve an ae Katey weighed

quantity of USP Monobenzone RS in methanol, and dilute 5000C(Au/ As)
quantitatively, and stepwise if necessary, with methanol to
obtain a solution having a known concentration of about in which C is the concentration, in mg per mL, of USP
40 ug per mL. Monobenzone RS in the Standard preparation and Ay and As
Assay preparation—Transfer about 100 mg of are the absorbances obtained from the Assay preparation
Monobenzone, accurately weighed, to a 100-mL volumetric and the Standard preparation, respectively.
flask, dissolve in and dilute with methanol to volume, and
mix. Pipet 4 mL of this solution into a 100-mL volumetric
flask, dilute with methanol to volume, and mix.
Procedure—With a suitable spectrophotometer, using Montelukast Sodium
methanol as a blank, concomitantly determine the ab-
sorbances of the Standard preparation and the Assay prepa-
S 9
ration at the wavelength of maximum absorbance at about
292 nm. Calculate the quantity, in mg, of Ci3Hi2O2 in the ae as
Lh
SOON ON
portion of Monobenzone taken by the formula: LZ 4
2500C(Ay / As)
in which Cis the concentration, in mg per mL, of USP
Monobenzone RS in the Standard preparation; and Ay and As
are the absorbances obtained from the Assay preparation C3sH3sCINNaO3S 608.17
and the Standard preparation, respectively. Cyclopropaneacetic acid, 1-[[[1-[3-[2-(7-chloro-2-qui-
nolinyl)etheny!]pheny!]-3-[2-(1-hydroxy-
Fe ee propyiithio}metnyls sodium salt,
 2796 Montelukast / Official Monographs USP 41

Sodium 1-{[[(R)-m-[(£)-2-(7-chloro-2-quinolyl)viny!]-c-[o- Cs = concentration of the Standard solution

qa ae roxy-1-methylethyl)phenethyl]benzyl]thio]- (mg/mL)
methyl]cyclopropaneacetate [151767-02-1]. Cu = concentration of the Sample solution (mg/mL)
CasH3¢CINO3S 586.18 Ma = eee weight of montelukast sodium,

Montelukast [138906-92-8); M2 = molecular weight of montelukast

DEFINITION dicyclohexylamine, 767.50
Montelukast Sodium contains NLT 98.0% and NMT 102.0% Acceptance criteria: 98.0%-102.0% on the anhydrous
of C3sH3sCINNaOsS, calculated on the anhydrous and sol- and solvent-free basis
vent-free basis.
IMPURITIES
IDENTIFICATION
e A. INFRARED ABSORPTION (197) Delete the following:
[Nott—Methods described under Infrared Absorption
(197K), (197M), or (197A) may be used.]
°e HEAVY METALS
Diluent: Acetone and water (4:1)
Change to read: Sample solution: Dissolve 0.50 g of Montelukast So-
dium in 20 mL of Diluent.
e B. IDENTIFICATION TESTS—GENERAL, Sodium (191) Reference solution: Dilute 0.5 mL of the Standard Lead
Sample: 100mg Solution, Pee as directed under Heavy Metals
Analysis: Ignite the Sample in a crucible until an almost (231), with Diluent to 20 mL.
white residue is obtained. Take up the residue in 2 mL Blank solution: 20 mL of the Diluent
of water, and filter. Analysis: To each solution, add 2 mL of pH 3.5 Acetate
Acceptance criteria: The filtrate meets the require- Buffer, prepared as directed under Heavy Metals (231).
ments of ®test Ae (cn 1-May-2018) Mix, and add to 1.2 mL of thioacetamide-glycerin base
e C. Meets the requirements of the test for Enantiomeric TS. Mix immediately, and allow to stand for 2 min. Pass
Purity. the solutions through a membrane filter of 0.45-1um
pore size, Compare the spots on the filters obtained
ASSAY rom the different solutions: the brownish-black color of
[Note—Avoid exposure of the samples to light. Use low- the spot resulting from the Sample solution is not more
actinic glassware.] intense than that of the spot resulting from the Refer-
© PROCEDURE ence solution. The test is invalid if the Reference solution
Solution A: Add 1.5 mL of trifluoroacetic acid to 1 L of does not show a brownish-black color compared to the
water. Blank solution.
Solution B: Add 1.5 mL of trifluoroacetic acid to 1 L of Acceptance criteria: NMT 10 ppme ‘otrciai 1-jan-2012)
acetonitrile. © ORGANIC IMPURITIES
Mobile phase: See Table 1. Return to original condi- [Note—Avoid exposure of the samples to light. Use low-
=
”
tions and re-equilibrate the column. actinic glassware.]
is Solution A, Solution B, Mobile phase, Diluent, and
S
= Table 1 ee system: Proceed as directed in the
Dp
°
= Solution A Solution B Impurity solution: 1mg/mL of USP Montelukast for
3 Peak Identification RS in Diluent
= System suitability solution: Transfer 1 mL of the /mpu-
rity solution to a colorless glass vial, and expose to am-
ra
coy 16.0 bient light for approximately 20 min to generate the
cis-isomer of montelukast.
Diluent: Methanol and water (9:1) Sample solution: 1 mg/mL of Montelukast Sodium in
Standard solution: 0.13 mg/mL of USP Montelukast Diluent
Dicyclohexylamine RS in Diluent Sensitivity solution: 0.5 ug/mL of Montelukast Sodium
Sample solution: 0.1 mg/mL of Montelukast Sodium in in Diluent from the Sample solution
Diluent System suitability
Chromatographic system Samples: System suitability solution and Sensitivity
(See Chromatography (621), System Suitability.) solution
Mode: LC Suitability requirements
Detector: UV 238 nm Resolution: NLT 2.5 between the cis-isomer and
Column: 4.6-mm x 5-cm; 1.8-"um packing L11 montelukast; NLT 1.5 between montelukast and the
Column temperature: 30° methylketone impurity, System suitability solution
Flow rate: 1.2 mL/min Signal-to-noise ratio: NLT 10, Sensitivity solution
Injection size: 10 pL Analysis
System suitability Sample: Sample solution
Sample: Standard solution Calculate the percentage of each impurity in the por-
Suitability requirements tion of Montelukast Sodium taken:
Relative standard deviation: NMT 0.73%
Analysis Result = (ru/rz) x 100
Samples: Standard solution and Sample solution
Calculate the percentage of montelukast sodium tu peak response of each impurity from the
(C3sH3sCINNaQs5) in the portion of Montelukast So- Sample solution
dium taken: r sum of all the peak responses from the Sample
solution
Result = (ru/rs) x (Cs/Cu) x (Ma/Mr2) x 100 Acceptance criteria: See Table 2.
Reporting level for impurities: 0.05%
tu = peak area from the Sample solution
rs = peak area from the Standard solution
USP 41 Official Monographs / Montelukast 2797

Table 2 Analysis
Relative Acceptance
Sample: Sample solution
Retention Criteria,
Calculate the percentage of S-enantiomer in the por-
NMT
tion of Montelukast Sodium taken:
4 Result = (ru/rz) x 100

ichael Add ¢ and 9: tu = peak response of the S-enantiomer from the

jon’ 0 Sample solution
nr = sum of the peak responses of the S-
of
enantiomer and montelukast from the
Sample solution
indi — 0.1 Acceptance criteria: NMT 0.2% of the S-enantiomer
Total impu = 0.
* These two impurities are not resolved by the method and need to be SPECIFIC TESTS
integrated together to determine conformance. e WATER DETERMINATION, Method Ia (921): NMT 4.0%
@(1-{[f1 fe Ce)247 Ghilorciaainiolin-2 yethensN phenyl:3: (241 -hydroxy-1-
methylethyl)pheny!]propyl]sulfiny|]methyl]cyclopropyllacetic acid: ADDITIONAL REQUIREMENTS
» [1-[[[(1R)-1-[3-[(Z)-2-(7-Chloroquinolin-2-yl)ethenyl] phenyl]-3-[2-(1-hy- e PACKAGING AND STORAGE: Preserve in tight containers,
droxy-1-methylethyl)phenyl]propy!]sulfanyl|methyl|cyclopropyljacetic acid. protected from light. Store at room temperature.
© 1-[([(1 R)-1-[3-[(1 R)-1-[[[1 -(Carbox:mmetoyicyciopropyimetny/Bultanyi]: 2 e USP REFERENCE STANDARDS (11)
(-<hloroquinain-2-ybethyi}phenyif3.2-( ey -methylethyl)phenyl] USP Montelukast Sodium RS
propyl]sulfany!]methyl]cyclopropyljacetic acid.
4 1-[{[[(1 R)-1-[3-[(1 $)-1-[[[1 -(Carboxymethyl)cyclopropyl]methyl]sulfanyl]-2-
USP Montelukast Dicyclohexylamine RS
(7-chloros uisoite 2 yhathyijphenyn -3-[2-(1“hyarony-1-methylthy)phenyl C3sH3eCINO3S - Cy2H23N =767.50
propyl]sulfanyl]methyl]cyclopropyljacetic acid. USP Montelukast Racemate RS
© [1 -[[[(1 R)-3-(2-Acetylpheny!)-1 -[3-[(E)-2-(7-chloroquinolin-2- USP Montelukast for Peak Identification RS
yl)ethenyl]phenyl]propylsulfany!]methyl]cyclopropyljacetic acid. (montelukast containing sulfoxide impurity, michael
f[1-[[[CR)-1-[3-[(E)-2-(7-Chloroquinolin-2-yl)ethenyl] phenyl]-3-[2-(1- adducts 1 and 2, methylketone impurity, and methyl-
methylethenyl)phenyl]propyl]sulfanyl]methyl]cyclopropylJacetic acid. styrene impurity)
e@ ENANTIOMERIC PURITY
[Note—Avoid exposure of the samples to light. Use low-
actinic glassware.]
Solution A: 2.3 g/L of ammonium acetate in water. Ad-
just with glacial acetic acid to a pH of 5.7. Montelukast Sodium Oral Granules
Solution B: Methanol and acetonitrile (60:40)
Mobile phase: See Table 3. DEFINITION
Montelukast Sodium Oral Granules contain Montelukast So-
Table 3
dium equivalent to NLT 90.0% and NMT 108.0% of the c
labeled amount of montelukast (C3sH36CINO3S). wn
Solution A Solution B [Note—Avoid exposure of samples containing montelukast uv
to light.] =
0 0 i}
IDENTIFICATION =]
e A. ULTRAVIOLET ABSORPTION (197U) }
5 40
Diluent: Methanol and water (3:1) =}
Standard solution: 3.3 ug/mL of USP Montelukast Di- ES)
Diluent: Acetonitrile and water (1:1)
cyclohexylamine RS in Diluent
se}
System suitability solution: 0.1 mg/mL of USP =>
Montelukast Racemate RS in Diluent Sample stock solution: Nominally 0.02 mg/mL of "

Sample solution: 1 mg/mL of Montelukast Sodium in montelukast prepared as follows. Transfer the contents
Diluent of one packet to a suitable volumetric flask, add 66% of
Sensitivity solution: 1 g/mL of Montelukast Sodium in the flask volume of Diluent, shake well, and sonicate for
Diluent from the Sample solution 15 min with occasional shaking. Cool to room tempera-
Chromatographic system ture, dilute with Diluent to volume, and mix well.
(See Chromatography (621), System Suitability.) Sample solution: Nominally 2 g/mL of montelukast in
Mode: LC Diluent from the Sample stock solution. Pass a portion of
Detector: UV 280 nm the resulting solution through a suitable filter of 0.45-
Column: 4.0-mm x 15-cm; 5-m packing L41 um pore size or centrifuge to obtain a clear solution.
Column temperature: 30° Wavelength range: 210-400 nm
Flow rate: 0.9 mL/min Acceptance criteria: The Sample solution exhibits max-
Injection size: 10 uL ima only at the same wavelengths as the Standard
System suitability solution.
Samples: System suitability solution and Sensitivity e B. The retention time of the major peak of the Sample
solution solution corresponds to that of the Standard solution, as
[Note—The relative retention times are 1.0 for obtained in the Assay.
montelukast, which is the R-enantiomer, and 0.7 for ASSAY
the S-enantiomer.] e PROCEDURE
Suitability requirements Diluent: Methanol and water (3:1)
Resolution: NLT 2.9 between the S-enantiomer and Solution A: 0.2% (v/v) trifluoroacetic acid in water
montelukast, System suitability solution Solution B: Methanol and acetonitrile (3:2)
Signal-to-noise ratio: NLT 10 for the montelukast Mobile phase: See Table 1.
peak, Sensitivity solution
 2798 Montelukast / Official Monographs
Table 1
Time Solution A Solution B
min %)
2 Test 1
5 55
12
75
25 52
30 2 acetonitrile
Standard solution: 0.33 mg/mL of USP Montelukast
Dicyclohexylamine RS in Diluent
System suitability solution: Transfer 10 mL of the Stan-
dard solution to a clear 10-mL volumetric flask, add 4 wL
of hydrogen peroxide, and mix well. Expose the flask
for at least 4 h to ambient light or 10 min to a 4 kIx
cool white light. [NoTs—Montelukast is partially con-
verted to the cis-isomer under these conditions.]
Sensitivity solution: 0.33 t4g/mL of USP Montelukast
Dicyclohexylamine RS in Diluent from the Standard
solution
Sample solution: Norainalit 0.24 mg/mL of
montelukast prepared as follows. Transfer the equivalent
of 60 mg of montelukast from the contents of the pack-
ets (NLT 15) to a 500-mL volumetric flask, and aad
250 mL of Diluent. Shake well and sonicate for 30 min,
with occasional shaking. Pass a portion of the resulting
solution through a suitable filter of 0.45-11m pore size or
centrifuge to obtain a clear solution.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 255 nm
Columns
Py Guard: 3.0-mm x 4-mm; packing L11
Analytical: 4.6-mm x 10-cm; 3-4um packing L11
nad
fo Column temperature: 50°
s
sS
Flow rate: 1.5 mL/min
Dd
) Injection volume: 20 uL
re Run time: 2 times the retention time of montelukast
S System suitability
= Samples: Standard solution, System suitability solution,
5 and Sensitivity solution
”" [NoTe—The relative retention times for the cis-isomer
=) and montelukast are about 0.92 and 1.0, respectively.]
Suitability requirements
Resolution: NLT 1.5 between the cis-isomer and
montelukast, System suitability solution
Relative standard deviation: NMT 2.0% for five in-
jections, Standard solution
Signal-to-noise ratio: NLT 10, Sensitivity solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
montelukast (C3sH36CINO3S) in the portion of Oral
Granules taken:
Result = (ru/rs) x (Cs/Cu) x (Mr/Mj2) x 100
tu = peak response from the Sample solution
rs = peak response from the Standard solution
Cs = concentration of USP Montelukast
Dicyclohexylamine RS in the Standard
solution (mg/mL)
Cu = nominal concentration of montelukast in the
Sample solution (mg/mL)
Mz = molecular weight of montelukast, 586.18
Mz = molecular weight of montelukast
dicyclohexylamine, 767.50
USP 41
Acceptance criteria: 90.0%-108.0%
PERFORMANCE TESTS
e DISSOLUTION (711)
Medium: 0.5% (w/v) sodium dodecyl sulfate in water;
900 mL. Do not deaerate.
Apparatus 1: 100 mesh; 50 rpm
Time: 15 min
Solution A: 0.2% (v/v) trifluoroacetic acid in water
Solution B: 0.2% (v/v) trifluoroacetic acid in
Mobile phase: Solution A and Solution B (1:1)
Standard stock solution: 0.33 mg/mL of USP
Montelukast Dicyclohexylamine RS in methanol (equiv-
alent to 0.25 mg/mL of montelukast)
Standard solution: (1/900) mg/mL of montelukast in
Medium from the Standard stock solution, whereLis
the label claim in mg/packet of montelukast
ane solution: Place the entire contents of one
packet in the basket. At the appropriate time point,
pass a portion of the solution under test through a
suitable filter to obtain a clear solution. Discard the
first 10 mL of the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 389 nm
Column: 3,0-mm x 10-cm; 5-um packing L11
Column temperature: 50°
Flow rate: 0.9 mL/min
Injection volume: 25 uL
Run time: 1.5 times the retention time of
montelukast
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 1.5
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
montelukast (C3sH36CINO3S) dissolved:
Result = (ru/rs) x Cs x Vx (1/L) x 100
tu = peak response of montelukast from the Sample
solution
Is = peak response of montelukast from the
Standard solution
Cs = concentration of montelukast in the Standard
solution (mg/mL)
Vv = volume of Medium, 900 mL
L = label claim (mg/packet)
Tolerances: NLT 85% (Q) of the labeled amount of
montelukast (C3sH36CINO3S) is dissolved.
Test 2: If the product complies with this test, the label-
ing indicates that it meets USP Dissolution Test 2.
Medium: 0.5% (w/v) sodium dodecyl sulfate in water;
900 mL
Apparatus 1: 100 mesh; 50 rpm
Time: 15 min
Solution A: 0.07 g/L of monobasic sodium phosphate
Solution B: Acetonitrile
Mobile phase: Solution A and Solution B (45:55). Add
1.33 mL/L of triethylamine and adjust with phosphoric
acid to a pH of 6.7.
Standard stock solution: 0.1 mg/mL of montelukast
from montelukast sodium hydrate prepared as follows.
Transfer a suitable amount of montelukast sodium hy-
drate to an appropriate volumetric flask. Dissolve in
4% of the flask volume of methanol and dilute with
Medium to volume. Determine the water content of
montelukast sodium hydrate at the time of use.
USP 41
Standard solution: 0.004 mg/mL of montelukast in
Medium from the Standard stock solution
somnl solution: Place the entire contents of one
packet in the basket. At the appropriate time point,
centrifuge a portion of the solution under test.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 225 nm
Column: 4.6-mm x 5-cm; 1.8-14m packing L1
Column temperature: 35°
Flow rate: 1 mL/min
Injection volume: 100 pL
Run time: 1.5 times the retention time of
montelukast
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
montelukast (CisHseCINOSS) dissolved:
Result = (ru/rs) x Cs x Vx (1/L) x 100
ry = peak response from the Sample solution
Is = peak response from the Standard solution
Cs = concentration of montelukast in the Standard
solution (mg/mL)
Vv volume of Medium, 900 mL
L = label claim (mg/packet)
Tolerances: NLT 80% (Q) of the labeled amount of
montelukast (C3sH36CINO3S) is dissolved.
Test 3: If the product complies with this test, the label-
ing indicates that it meets USP Dissolution Test 3.
Medium: 0.5% (w/v) sodium dodecyl sulfate in water;
900 mL
Apparatus 2: 50 rpm
Time: 10 min
Solution A: 2.72 g/L of monobasic potassium phos-
phate in water
Mobile phase: Acetonitrile and Solution A (70:30)
Diluent: Acetonitrile and water (50:50)
System suitability solution: Expose a portion of Stan-
dard solution in a clear glass vial to direct room light
for about 30 min.
Standard stock solution: 0.524 mg/mL of USP
Montelukast Dicyclohexylamine RS in Diluent (equiva-
lent to 0.4 mg/mL of montelukast)
Standard solution: 0.0065 mg/mL of USP Montelukast
Dicyclohexylamine RS in Medium from the Standard
stock solution (equivalent to 0.005 mg/mL of
montelukast)
rake solution: Transfer the entire contents of one
packet to the dissolution vessel. At the specified time
point, withdraw 10 mL of sample from the dissolution
vessel. Pass a portion of the solution under test
throughasuitable filter. Discard the first 5 mL of the
filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 281 nm
Column: 4.6-mm x 3-cm; 3-4um packing L1
Column temperature: 40°
Flow rate: 0.8 mL/min
Injection volume: 25 uL
Run time: About 1.5 times the retention time of
montelukast
System suitability
Samples: System suitability solution and Standard
solution
[Note—The relative retention times for Z-isomer and
montelukast are 0.8 and 1.0, respectively.]
Official Monographs / Montelukast 2799
Suitability requirements
Resolution: NLT 2.0 between the Z-isomer and
montelukast, System suitability solution
Tailing factor: NMT 2.0 for montelukast, System
suitability solution
Relative standard deviation: NMT 2.0%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
montelukast (C3sH3sCINO3S) dissolved:
Result = (ru/rs) x Cs x Vx (1/L) x (Ma/M2) x 100
ry = peak response from the Sample solution
rs = peak response from the Standard solution
Cs = concentration of USP Montelukast
Dicyclohexylamine RS in the Standard
solution (mg/mL)
Vv = volume of Medium, 900 mL
E = label claim (mg/packet)
Ma = molecular weight of montelukast, 586.18
Mz = molecular weight of montelukast
dicyclohexylamine, 767.50
Tolerances: NLT 80% (Q) of the labeled amount of
montelukast (C3sH3sCINO3S) is dissolved.
© UNIFORMITY OF DOSAGE UNITS (905)
Procedure for content uniformity
Solution A, Solution B,Rronilephuise and System
suitability: Proceed as directed in Dissolution Test 1.
Standard solution: 26.4 11g/mL of USP Montelukast
Dicyclohexylamine RS in methanol
Sample solution: Nominally 0.02 mg/mL of
montelukast prepared as follows. Transfer the contents
of one packet to a suitable volumetric flask, add 66%
of the flask volume of methanol, shake well, and soni-
cate for 15 min with occasional shaking. Cool to room
temperature, dilute with methanol to volume, and mix c
well. Pass a portion of the resulting solution through a a)
suitable filter of 0.45-uum pore size or centrifuge to ob- uv
tain a clear solution. =
Chromatographic system: Proceed as directed in Dis- i}
solution Test 1, except use an Injection volume of 5 iL. re}
}
Analysis ro}=
Samples: Standard solution and Sample solution 2
Calculate the ene of the labeled amount of ne}
montelukast (C3sH3s6CINO3S) in the packet taken: Ee
a)
Result = (ru/rs) x (Cs/Cu) x (Ma/M2) x 100
tu = peak response from the Sample solution
rs = peak response from the Standard solution
Gs = concentration of USP Montelukast
Dicyclohexylamine RS in the Standard
solution (mg/mL)
Cu = nominal concentration of montelukast in the
Sample solution (mg/mL)
Mn; = molecular weight of montelukast, 586.18
Mz = molecular weight of montelukast
dicyclohexylamine, 767.50
Acceptance criteria: Meet the requirements
IMPURITIES
¢ ORGANIC IMPURITIES
Diluent, Solution A, Solution B, Mobile phase, Stan-
dard solution, System suitability solution, Sensitivity
solution, Sample solution, Chromatographic system,
and System suitability: Proceed as directed in the
Assay.
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of any individual degradation
product in the portion of Oral Granules taken:
Result = (ru/rs) x (Cs/Cu) x (Mn/Mr2z) x (1/F) x 100
 2800 Montelukast / Official Monographs USP 41

tu = peak response of any individual degradation

product from the Sample solution
rs = peak response of montelukast from the Montelukast Sodium Tablets
Standard solution
Gs = concentration of USP Montelukast DEFINITION
Dicyclohexylamine RS in the Standard Montelukast Sodium Tablets contain Montelukast Sodium
solution (mg/mL) equivalent to NLT 92.5% and NMT 107.5% of the labeled
Cu = nominal concentration of montelukast in the amount of montelukast (C3sH36CINO3S).
Sample solution (mg/mL) [Note—Avoid exposure of samples containing montelukast
My — = molecular weight of montelukast, 586.18 to light.]
M2 = molecular weight of montelukast IDENTIFICATION
dicyclohexylamine, 767.50 e A. ULTRAVIOLET ABSORPTION (197U)
F = relative response factor (see Table 2) Diluent: Methanol and water (3:1)
Acceptance criteria: See Table 2. Disregard any peak Standard solution: 0.026 mg/mL of USP Montelukast
with an area less than that of the Sensitivity solution. Dicyclohexylamine RS in Diluent
Sample solution: Nominally 0.02 mg/mL of
Table 2 montelukast prepared as follows. Transfer one Tablet
Relative Relative Acceptance equivalent to 10 mg of montelukast to a suitable volu-
Retention Response Criteria, metric flask, add 25% of the flask volume of water, and
Name Time Factor NMT (%)
let stand for 5-10 min until the Tablet has disinte-
grated. Add 60% of the flask volume of methanol,
Sulfoxide shake well, and sonicate for 70 min with occasional
impuritys® 0.45 1.0 0.8 shaking. Cool to room temperature, dilute with metha-
Montelukast nol to volume, and mix well. Centrifuge a portion of
ketone impurity: 0.71 Te 0.2 the resulting solution to obtain a clear solution.
cis-Isomer4 0.92 1.0 02 Wavelength range: 210-400 nm
Montelukast 1.0 = = Acceptance criteria: The Sample solution exhibits max-
Methylketone fiz. ibis ima only at the same wavelengths as the Standard
impuritye* 1.04 solution.
Michael ny ws} e B. The retention time of the major peak of the Sample
adduct 1ge 1.16 solution corresponds to that of the Standard solution, as
obtained in the Assay.
Michael _ eS
adduct 2he 1.18 ASSAY
Methylstyrene i _ © PROCEDURE
impurity: 1:53 Diluent: Methanol and water (3:1)
Any other Solution A: 0.2% (v/v) Trifluoroacetic acid in water
te
a)
individual Solution B: Methanol and acetonitrile (3:2)
os degradation = Mobile phase: See Table 7.
ne
io] product 1.0 02!
Dp Total impurities = = 1.0
i} Table 1
c These two impurities are not resolved by the method and need to be Solution A Solution B
5 integrated together to determine conformance.
%o’
= 6(1-[[[1 -[3-[(6-2-(7-Chloroquinolin-2-yl)ethen phensl 3 [aac bydroxy:t=
methylethyl)phenyl]propyl]sulfinyl] methyl]cyclopropyllacetic acid.
a
” ©(E)-1-{3-[2-(7-Chloroquinolin-2-yl)vinyl]pheny}}-3-[2-(2-hydroxypropan-2- 55
= yl)phenyl]propan-1-one.
3 [1-[[[C1R)-1 13 1.2-(7-Chloroguinalin 2 yethen \]phenyl]-3-[2-(1-hy-
droxy-1-methylethyl)pheny!]propyl]sulfanyl|methyl|cyclopropylacetic acid.
eThis is a process impurity and ts included in the table for identification
only. This impurity 1s controlled in the drug substance. It is not to be 52
reported for the drug product and should not be included in the total
impurities. 3 48 52
£1 -[[[(1 R)-3-(2-Acetylphenyl)-1 -[3-[(-2-(7-chloroquinolin-2-_
yl)etheny!]phenyl]propyl]sulfanyl]methyl]cyclopropyljacetic acid. Standard solution: 0.52 mg/mL of USP Montelukast
9(1-{[(R)-1-(3-[(R)-1-{[1-(Carboxymethyl)cyclopropyl]methylthio}-2-(7- Dicyclohexylamine RS in Diluent
chloroquinolin-2-yl)ethyl]phenyl)-3-[2-(2-hydroxypropan-2-yl)phenyl] System suitability solution: Transfer 10 mL of the Stan-
propylthio]methyl}cyclopropyljacetic acid.
»(1-{((R)-1-G-[(S)-1-{[1 -(Carboxymethyl)cyclopropylmethylthio}-2-(7- dard solution to a clear 10-mL volumetric flask, add 4 WL
chloros uinolin 2:yDetny phen }-3-[2-(2-hydroxypropan-2-yl)phenyl] of hydrogen peroxide, and mix well. Expose the flask
propylthio]methyl}cyclopropyljacetic acid. for at least 4 _h to ambient light or 10 min to a 4 kIx
'T1-[[[C 2)-1-[3-[(6)-2-(7-Chloroquinolin-2-yl)ethenyl]pheny!]-3-[2-(1- cool white light. [Note—Montelukast is partially con-
methylethenyl)phenyl]propy!|sulfanyl]methyl]cyclopropyljacetic acid. verted to the cis-isomer under these conditions.]
Sensitivity solution: 0.52 g/mL of USP Montelukast
ADDITIONAL REQUIREMENTS Dicyclohexylamine RS in Diluent from the Standard
e PACKAGING AND STORAGE: Preserve in tight containers, solution
protected from light. Store at controlled room Sample solution: Nominally 0.4 mg/mL of montelukast
temperature. prepared as follows. Transfer a number of Tablets equiv-
e LABELING: When more than one Dissolution test is given,
alent to 100 mg of montelukast to a suitable volumetric
the labeling states the test used only if Test 7 is not used. flask, add 70% of the flask volume of Diluent, and soni-
e USP REFERENCE STANDARDS (11) cate for 30 min. Shake for 30 min on a platform shaker.
USP Montelukast Dicyclohexylamine RS Dilute with Diluent to volume and stir for 30 min. Pass
C3sH3eCINO3S + CizH23N 767.50 a portion through a suitable filter of 0.45-um pore size,
discarding the first mL of filtrate. Use the filtrate.
USP 41 Official Monographs / Montelukast 2801

Chromatographic system System suitability

(See Chromatography (621), System Suitability.) Sample: Standard solution
Mode: LC Suitability requirements
Detector: UV 255 nm Tailing factor: NMT 1.5
Columns Relative standard deviation: NMT 2%
Guard: 3.0-mm x 4-mm; packing L11 Analysis
Analytical: 4.6-mm x 10-cm; 3-{um packing L11 Samples: Standard solution and Sample solution
Column temperature: 50° Calculate the percentage of the labeled amount of
Flow rate: 1.5 mL/min montelukast (CisHsgCINO:S) dissolved:
Injection volume: 15 Ll
Run time: 2 times the retention time of montelukast Result = (ru/rs) x Cs x Vx (1/L) x 100
System suitability
Samples: Standard solution, System suitability solution, ty = peak response from the Sample solution
and Sensitivity solution Is peak response from the Standard solution
[NotE—The relative retention times for the cis-isomer Cs concentration of montelukast in the Standard
and montelukast are about 0.92 and 1.0, respectively.] solution (mg/mL)
Suitability requirements Vv volume of Medium, 900 mL

1
Resolution: NLT 1.5 between the cis-isomer and L = label claim (mg/Tablet)
montelukast, System suitability solution Tolerances: NLT 80% (Q) of the labeled amount of
Relative standard deviation: NMT 2% for five injec- montelukast (C3sH3sCINO3S) is dissolved.
tions, Standard solution Test 2: If the product complies with this test, the label-
Signal-to-noise ratio: NLT 10, Sensitivity solution ing indicates that it meets USP Dissolution Test 2.
Analysis Medium: 0.5% (w/v) Sodium dodecyl sulfate in water;
Samples: Standard solution and Sample solution 900 mL
Calculate the percentage of the labeled amount of Apparatus 2: 50 rpm
montelulest (CasHsgCINOSS) in the portion of Tablets Time: 45 min
taken: Solution A: 0.07 g/L of monobasic sodium phosphate
Solution B: Acetonitrile
Result = (ru/rs) x (Cs/Cu) x (Mn/Mi2) x 100 Mobile phase: Solution A and Solution B (45:55). Add
1.33 mL/L of triethylamine and adjust with phosphoric
tu = peak response from the Sample solution acid to a pH of 6.7.
rs = peak response from the Standard solution Standard stock solution: 0.1 mg/mL of montelukast
Cs = concentration of USP Montelukast from montelukast sodium hydrate prepared as follows.
Dicyclohexylamine RS in the Standard Transfer a suitable amount of montelukast sodium hy-
solution (mg/mL) drate to a suitable volumetric flask. Dissolve in 5% of
Cu = nominal concentration of montelukast in the the flask volume of methanol and dilute with Medium
Sample solution (mg/mL) to volume. Determine the water content of
Ma = molecular weight of montelukast, 586.18 montelukast sodium hydrate at the time of use. (ss
M2 = molecular weight of montelukast Standard solution: 0.01 mg/mL of montelukast in Me- a)
dicyclohexylamine, 767.50 dium from the Standard stock solution i}
Acceptance criteria: 92.5%-107.5% Sample solution: Centrifuge a portion of the solution c<
nder test. i)
PERFORMANCE TESTS Chromatographic system |
e DISSOLUTION (711) )
Test 1
(See Chromatography (621), System Suitability.) ro}=
Mode: LC 2
Medium: 0.5% (w/v) Sodium dodecy] sulfate in water; Detector: UV 225 nm so]
900 mL. Do not deaerate. Column: 4.6-mm x 5-cm; 1.8-m packing L1 =P
Apparatus 2: 50 rpm Column temperature: 35°
Aa

Time: 20 min Flow rate: 1 mL/min

Solution A: 0.2% (v/v) Trifluoroacetic acid in water
Solution B: 0.2% (v/v) Trifluoroacetic acid in
acetonitrile
Mobile phase: Solution A and Solution B (1:1)
Standard stock solution: 0.35 mg/mL of USP
Montelukast Dicyclohexylamine RS in methanol (equiv-
alent to 0.27 mg/mL of montelukast)
Standard solution: (1/900) mg/mL of montelukast in
Medium from the Standard stock solution, where L is
the label claim in mg/Tablet of montelukast
Sample solution: Pass a portion of the solution under
test through a suitable filter or centrifuge to obtain a
clear solution.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 389 nm
Column: 3.0-mm x 10-cm; 5-um packing L11
Column temperature: 50°
Flow rate: 0.9 mL/min
Injection volume: 20 uL
Run time: 1.5 times the retention time of
montelukast
Injection volume: 100 pL
Run time: 1.5 times the retention time of
montelukast
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
montelukast (C3sH36CINO3S) dissolved:
Result = (ru/rs) x Cs x Vx (1/L) x 100
tu = peak response from the Sample solution
rs = peak response from the Standard solution
Cs = concentration of montelukast in the Standard
solution (mg/mL)
Vv = volume of Medium, 900 mL
L = label claim (mg/Tablet)
Tolerances: NLT 80% (Q) of the labeled amount of
montelukast (C3sH36CINO3S) is dissolved.
Test 3: If the product complies with this test, the label-
ing indicates that it meets USP Dissolution Test 3.
Megtune 0.5% (w/v) Sodium dodecyl sulfate in water;
0 mL
 2802 Montelukast / Official Monographs USP 41

Apparatus 2: 50 rpm Cs = concentration of USP Montelukast

Time: 30 min Dicyclohexylamine RS in the Standard
Solution A: Acetonitrile and water (80:20) solution (mg/mL)
Solution B: 3% Trifluoroacetic acid in Solution A pre- Cu = nominal concentration of montelukast in the
pared as follows. Transfer 3 mL of trifluoroacetic acid Sample solution (mg/mL)
to a 100-mL volumetric flask and dilute with Solution A Mj = molecular weight of montelukast, 586.18
to volume. M2 = molecular weight of montelukast
Mobile phase: Acetonitrile, water, and Solution B dicyclohexylamine, 767.50
(75: 25: 0.05). Acceptance criteria: Meet the requirements
Standard solution: (1/900) mg/mL of montelukast in
Medium from montelukast sodium hydrate, where L is IMPURITIES
the label claim in mg/Tablet of montelukast. Deter- e@ ORGANIC IMPURITIES
mine the water content of montelukast sodium hy- Diluent, Solution A, Solution B, Mobile phase, Stan-
drate at the time of use. dard solution, System suitability solution, Sensitivity
Sample solution: Pass a portion of the solution under solution, Sample solution, Chromatographic system,
test througha suitable filter to obtain a clear solution. and System suitability: Proceed as directed in the
Chromatographic system Assay.
(See Chromatography (621), System Suitability.) Analysis
Mode: LC Samples: Standard solution and Sample solution
Detector: UV 346 nm Calculate the percentage of ay individual degradation
Column: 4.6-mm x 10-cm; 3-um packing L1 product in the portion of Tablets taken:
Flow rate: 1.5 mL/min
Injection volume: 25 wL Result = (ru/ts) < (Cs/Cu) x (Mr/Mrz) x (1/F) x 100
Run time: NLT 1.5 times the retention time of
montelukast ru = peak response of any individual degradation
System suitability product from the Sample solution
Sample: Standard solution rs = peak response of montelukast from the
Suitability requirements Standard solution
Relative standard deviation: NMT 2.0% Cs = concentration of USP Montelukast
Analysis Dicyclohexylamine RS in the Standard
Samples: Standard solution and Sample solution solution (mg/mL)
Calculate the percentage of the labeled amount of Cu = nominal concentration of montelukast in the
montelukast (C3sH36CINO3S) dissolved: Sample solution (mg/mL)
Mn = molecular weight of montelukast, 586.18
Result = (ru/rs) x Cs x Vx (1/1) x 100 M2 = molecular weight of montelukast
dicyclohexylamine, 767.50
ty = peak response from the Sample solution F = relative response factor (see Table 2)
rs = peak response from the Standard solution Acceptance criteria: See Table 2. Disregard any peak
ms
”w
with an area less than that of the Sensitivity solution.
ro Gs = concentration of montelukast in the Standard
solution (mg/mL)
i
a V volume of Medium, 900 mL Table 2
° L label claim (mg/Tablet)
S Tolerances: NLT 80% (Q) of the labeled amount of Relative Relative Acceptance
G montelukast (C3sH36CINOsS) is dissolved. Retention Response Criteria,
= © UNIFORMITY OF DosAGE UNITS (905) Name Time Factor NMT (%)
a
va)
Procedure for content uniformity Sulfoxide impurity2® 0.45 1.0 2.0
Solution A, Solution B, Mobile phase, and System Montelukast ketone
=)
suitability: Proceed as directed in Dissolution Test 1. impurity 0.71 Te 0.2
Diluent: Methanol and water (3:1) cis-lsomeré 0.92 1.0 0.2
Standard solution: 0.052 mg/mL of USP Montelukast Montelukast 1.0 — _
Dicyclohexylamine RS in Diluent Methylketone _ sss)
Sample solution: Nominally 0.04 mg/mL of impuritye! 1.04
montelukast prepared as follows. Transfer one Tablet
equivalent to 10 mg of montelukast to a suitable volu- Michael adduct _19¢ 1.16 = os
metric flask, add 25% of the flask volume of water, Michael adduct _2h« 1.18 = =
and let stand for 5-10 min until the Tablet has disinte- These two impurities are not resolved by the method and need to be
grated. Add 60% of the flask volume of methanol, integrated together to determine conformance.
shake well, and sonicate for 70 min with occasional el AIL TS Icey 2-(7. Chisroquinolin-2 yDethenyllphen 1]-3-[2-(1-hydroxy-1-
methylethyl)phenyl]propyl]sulfiny!]methyl]cyclopropyllacetic acid.
shaking. Cool to room temperature, dilute with meth-
¢(E)-1 -{3-[2-(7-Chloroquinolin-2-yl)vinyl]phenyl}-3-[2-(2-hydroxypropan-2-
anol to volume, and mix well. Pass a portion of the yl)phenyl]propan-1-one.
resulting solution through a suitable filter or centrifuge 9[7-[[[C R)-1 He ee noreuinoliy 2 seinen |]jpheny!]-3-[2-(1-hy-
to obtain a clear solution. droxy-1-methylethyl)phenyl]propyl]sulfany/ Hmethyllcyelopropyljacetic acid.
Chromatographic system: Proceed as directed in Dis- This 1s a process impurity and is included in the table for identification
solution Test 1, except use an Injection volume of 10 wL. only. This impurity is controlled in the drug substance It is not to be
Analysis reported for the drug product and should not be included in the total
impurities.
Samples: Standard solution and Sample solution
*[1-[[[(1 R)-3-(2-Acetylpheny!)-1 -[3-[(E)-2-(7-chloroquinolin-2-
Calculate the percentage of the labeled amount of yl)etheny!]phenyl]propy|]sulfanyl]methyl]cyclopropyljacetic acid.
montelukast (C3sH3sCINO3S) in the Tablet taken: 9 1-[[((1 R)-1-[3-[(1 R)-1-[[[1-(Carboxymeth: Deyclopropylimethyl sulfanyll 2
(7-chloroquinelinse etyipheny| -3-[2-(1-hydroxy-1-methylethyl)phenyl]
Result = (ru/rs) x (Cs/Cu) x (Mr1/Mrz) x 100 propyl|sultanyl]methyl]cyclopropyljacetic acid.
1 -[[[Q R)-1-[3-[1 $)-1-[[[1-(Carbox: metnyDeyelopropyllmethy isulfariyil-2.
ty = peak response from the Sample solution (diorequinciny 2 pecaylpreny -3-[2-01 yStONys -methylethyl) phenyl]
Is = peak response from the Standard solution propyl]sulfanyl]methyl]cyclopropyljacetic acid.
'(1-[[[C R)-1-[3-[(-2-(7-Chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(1-
methylethenyl)phenyl]propy!]sulfanyl]methyl]cyclopropyllacetic acid.
USP 41 Official Monographs / Montelukast 2803

Table 2 (Continued) Wavelength range: 210-400 nm

Relative Relative Acceptance
Acceptance criteria: The Sample solution exhibits max-
Retention Response Criteria,
ima only at the same wavelengths as the Standard
Name Time Factor NMT (%)
solution.
e B. The retention time of the major peak of the Sample
Methylstyrene solution corresponds to that of the Standard solution, as
impurity: 1.55 — Bs obtained in the Assay.
Any other individual
degradation — ASSAY
roduct 1.0 0.2 © PROCEDURE
Total impurities = = 3.0 Diluent: Methanol and water (3:1)
These two impurities are not resolved by the method and need to be Solution A: 0.2% (v/v) Trifluoroacetic acid in water
integrated together to determine conformance. Solution B: Methanol and acetonitrile (3:2)
>[1-[[[1-[3-[(6-2-(7-Chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(1-hydroxy-1- Mobile phase: See Table 1.
methylethyl)pheny!]propyl]sulfinyl] methy!]cyclopropyljacetic acid.
¢(6)-1-{3-[2-(7-Chloroquinolin-2-yl)vinyl]pheny|}-3-[2-(2-hydroxypropan-2-
y!)phenyl]propan-1-one. Table 1
411-{[[(1 R)-1 -[3-[(Z)-2-(7-Chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(1-hy- Solution A Solution B
droxy-1-methylethyl)phenyl]propyl]sulfany[]methyl]cyclopropyljacetic acid. So %e'
€This is a process impurity and is included in the table for identification
only. This impurity is controlled in the drug substance. It is not to be 2
reported for the drug product and should not be included in the total 55
impurities.
*[1-[[[(1 R)-3-(2-Acetylphenyl)-1 131-2 (Zciloronuinolity2:
yl)ethenyl]phenyl]propyl]sulfanyl]methy!]cyclopropyljacetic acid. 75
91-{[[(1 R)-1-[3-[(1 8)-1-[[[1 -(Carboxymethyl)cyclopropyl]methyl]sulfanyl]-2- Zi
Z-eNerosinain-2 yebylphenyt3 (24) usr -methylethyl)pheny!]
propyl]sulfanyl]methy!]cyclopropyljacetic acid. 48 2
®1-[[[(1 R)-1-[3-[(1 5)-1-[[[1-(Carbox: methyevcoprepylimeliyllsultany!| 2 52
-eNeroauinoin-Z ethypnenyi 3 124) jydioxy: -methylethyl)phenyl)
propyl]sulfanyl]methyl]cyclopropyllacetic acid. Standard solution: 0.33 mg/mL of USP Montelukast
‘[1-[[[C R)-1 -[3-[()-2-(7-Chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(1- Dicyclohexylamine RS in Diluent
methylethenyl)pheny!)propyl]sulfanyl]methyl]cyclopropyljacetic acid. System suitability solution: Transfer 10 mL of the Stan-
dard solution to a clear 10-mL volumetric flask, add 4 wL
ADDITIONAL REQUIREMENTS of hydrogen peroxide, and mix well. Expose the flask
© PACKAGING AND STORAGE: Preserve in tight containers, for at least 4 h to ambient light or 10 min to a 4 kIx
protected from light. Store at controlled room cool white light. [Note—Montelukast is partially con-
temperature.
verted to the cis-isomer under these conditions.]
e LABELING When more than one Dissolution test is given, Sensitivity solution: 0.33 g/mL of USP Montelukast
the labeling states the test used only if Test 7 is not used.
e USP REFERENCE STANDARDS (11) Dicyclohexylamine RS in Diluent from the Standard (eae
solution 4)
USP Montelukast Dicyclohexylamine RS z
C3sH36CINO3S - CizH23N =767.50
Sample solution (for 4-mg Chewable Tablets): Nomi-
nally 0.24 mg/mL of montelukast prepared as follows. =
Transfer 12 Chewable Tablets to a suitable volumetric i)
flask, add 75% of the flask volume of Diluent, and shake =)
vigorously for 60 min. Dilute with Diluent to volume. ro}
Pass a portion of the resulting solution through a suita-
to}=
Montelukast Sodium Chewable Tablets ble filter of 0.45-1um pore size, discarding the first mL of i
3
DEFINITION
filtrate. Use the filtrate. oy
Sample solution (for 5-mg Chewable Tablets): Nomi- “

Montelukast Sodium Chewable Tablets contain Montelukast
Sodium equivalent to NLT 92.5% and NMT 107.5% of
the labeled amount of montelukast (C3sH36CINO3S).
[Nott—Avoid exposure of samples containing montelukast
to light.]
IDENTIFICATION
e A, ULTRAVIOLET ABSORPTION (197U)
Diluent: Methanol and water (3:1)
Standard solution (for 4-mg Chewable Tablets):
0.026 mg/mL of USP Montelukast Dicyclohexylamine RS
in Diluent
Standard solution (for 5-mg Chewable Tablets):
0.033 mg/mL of USP Montelukast Dicyclohexylamine RS
in Diluent
Sample solution: Nominally (1/200) mg/mL of
montelukast, where L is the label claim of montelukast
in mg/Chewable Tablet prepared as follows. Transfer 1
Chewable Tablet to a suitable volumetric flask, add 25%
of the flask volume of water, and let stand for 5-10 min
until the Chewable Tablet has disintegrated. Add 55%
of the flask volume of methanol, shake well, and soni-
cate for 70 min with occasional shaking. Cool to room
temperature, dilute with methanol to volume, and mix
well. Centrifuge a portion of the resulting solution to
obtain a clear solution.
nally 0.25 mg/mL of montelukast prepared as follows.
Transfer 10 Chewable Tablets to a suitable volumetric
flask, add 75% of the flask volume of Diluent, and shake
vigorously for 60 min. Dilute with Diluent to volume.
Pass a portion of the resulting solution through a suita-
ble filter of 0.45-um pore size, discarding the first mL of
filtrate. Use the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 255 nm
Columns
Guard: 3.0-mm x 4-mm; packing L11
Analytical: 4.6-mm x 10-cm; 3-m packing L11
Column temperature: 50°
Flow rate: 1.5 mL/min
Injection volume: 20 uL
Run time: 2 times the retention time of montelukast
System suitability
Samples: Standard solution, System suitability solution,
and Sensitivity solution
[Note—The relative retention times for the cis-isomer
and montelukast are about 0.92 and 1.0, respectively.]
Suitability requirements
Resolution: NLT 1.5 between the cis-isomer and
montelukast, System suitability solution
 2804 Montelukast / Official Monographs USP 41

Relative standard deviation: NMT 2% for five injec- L = label claim (mg/Chewable Tablet)
tions, Standard solution Tolerances: NLT 80% (Q) of the labeled amount of
Signal-to-noise ratio: NLT 10, Sensitivity solution montelukast (C3sH3sCINO3S) is dissolved.
Analysis Test 2: If the product complies with this test, the label-
Samples: Standard solution and Sample solution ing indicates that it meets USP Dissolution Test 2.
Calculate the percentage of the labeled amount of Medium: 0.5% (w/v) Sodium dodecyl sulfate in water;
montelukast (C3sH3sCINO3S) in the portion of Chew- 900 mL
able Tablets taken: Apparatus 2: 50 rpm
Time: 45 min
Result = (ru/rs) x (Cs/Cu) x (Mr/My2) x 100 Solution A: 0.07 g/L of monobasic sodium phosphate
Solution B: Acetonitrile
ru = peak response from the Sample solution Mobile phase: Solution A and Solution B (45:55). Add
Is = peak response from the Standard solution 1.33 mL/L of triethylamine and adjust with phosphoric
Cs = concentration of USP Montelukast acid to a pH of 6.7.
Dicyclohexylamine RS in the Standard Standard stock solution: 0.1 mg/mL of montelukast
solution (mg/mL) from montelukast sodium hydrate prepared as follows.
Cy = nominal concentration of montelukast in the Transfer a suitable amount of montelukast sodium hy-
Sample solution (mg/mL) drate to an appropriate volumetric flask. Dissolve in
M1 = molecular weight of montelukast, 586.18 4% of the flask volume of methanol and dilute with
Mrz = molecular weight of montelukast Medium to volume. Determine the water content of
dicyclohexylamine, 767.50 montelukast sodium hydrate at the time of use.
Acceptance criteria: 92.5%-107.5% Standard solution: 0.005 mg/mL of montelukast in
Medium from the Standard stock solution
PERFORMANCE TESTS
e DISSOLUTION (711) Sample solution: Centrifuge a portion of the solution
under test.
Test 1 Chromatographic system
Medium: 0.5% (w/v) Sodium dodecyl sulfate in water; (See Chromatography (621), System Suitability.)
900 mL. Do not deaerate. Mode: LC
Apparatus 2: 50 rpm Detector: UV 225 nm
Time: 20 min Column: 4.6-mm x 5-cm; 1.8-1um packing L1
Solution A: 0.2% (v/v) Trifluoroacetic acid in water Column temperature: 35°
Solution B: 0.2% (v/v) Trifluoroacetic acid in Flow rate: 1 mL/min
acetonitrile Injection volume: 100 uL
Mobile phase: Solution A and Solution B (1:1) Run time: 1.5 times the retention time of
Standard stock solution (for 4-mg Chewable Tab- montelukast
lets): 0.30 mg/mL of USP Montelukast Dicyclohexy- System suitability
lamine RS in methanol (equivalent to 0.23 mg/mL of Sample: Standard solution
montelukast) Suitability requirements
ie
a)
Standard stock solution (for 5-mg Chewable Tab-
rm lets): 0.35 mg/mL of USP Montelukast Dicyclohexy-
Relative standard deviation: NMT 2.0%
J Analysis
— lamine RS in methanol (equivalent to 0.27 mg/mL of
ia) Samples: Standard solution and Sample solution
iS) montelukast) Calculate the percentage of the labeled amount of
iS Standard solution: (1/900) mg/mL of montelukast in
Sj Medium from the Standard stock solution, where L is
montelukast (C3sH36CINO3S) dissolved:
= the label claim in mg/Chewable Tablet of montelukast Result = (ru/rs) x Cs x Vx (1/L) x 100
[3 Sample solution: Pass a portion of the solution under
A) test throughasuitable filter or centrifuge to obtain a tu = peak response from the Sample solution
=) clear solution. Is = peak response from the Standard solution
Chromatographic system Cs = concentration of montelukast in the Standard
(See Chromatography (621), System Suitability.) solution (mg/mL)
Mode: LC Vv = volume of Medium, 900 mL
Detector: UV 389 nm L = label claim (mg/Chewable Tablet)
Column: 3.0-mm x 10-cm; 5-um packing L11 Tolerances: NLT 70% (Q) of the labeled amount of
Column temperature: 50° montelukast (C3sH36CINO3S) is dissolved.
Flow rate: 0.9 mL/min e UNIFORMITY OF DosAGE UNITS (905)
Injection volume: 50 uL Procedure for content uniformity
Run time: 1.5 times the retention time of Solution A, Solution B, MobsPasar: and System
montelukast suitability: Proceed as directed in Dissolution Test 1.
System suitability Diluent: Methanol and water (3:1)
Sample: Standard solution Standard solution (for 4-mg Chewable Tablets):
Suitability requirements 0.026 mg/mL of USP Montelukast Dicyclohexylamine
Tailing factor: NMT 1.5 RS in Diluent
Relative standard deviation: NMT 2% Standard solution (for 5-mg Chewable Tablets):
Analysis 0.033 mg/mL of USP Montelukast Dicyclohexylamine
Samples: Standard solution and Sample solution RS in Diluent
Calculate the percentage of the labeled amount of Sample solution: Nominally (L/200) mg/mL of
montelukast (C3sH36CINO3S) dissolved: montelukast, where Lis the label claim of montelukast
in mg/Chewable Tablet prepared as follows. Transfer 1
Result = (ru/rs) x Cs x Vx (1/L) x 100 Chewable Tablet to a suitable volumetric flask, add
25% of the flask volume of water, and let stand for
ru = peak response from the Sample solution 5-10 min until the Chewable Tablet has disintegrated.
rs = peak response from the Standard solution Add 55% of the flask volume of methanol, shake well,
Cs = concentration of montelukast in the Standard and sonicate for 70 min with occasional shaking. Cool
solution (mg/mL)
to room temperature, dilute with methanol to volume,
Vv = volume of Medium, 900 mL and mix well. Pass a portion of the resulting solution
USP 41 Official Monographs / Morantel 2805

throughasuitable filter or centrifuge to obtain a clear Table 2

solution. Relative Relative Acceptance
Chromatographic system: Proceed as directed in Dis- Retention Response Criteria,
solution Test 1, except use an Injection volume of 10 wL. Name Time Factor NMIT (%)
Analysis
Samples: Standard solution and Sample solution Sulfoxide
impurity2> 0.45 1.0 1.5
Calculate the percentage of the labeled amount of
moreelibae (C3sH36CINO3S) in the Chewable Tablet Montelukast
taken: ketone impurity* 0.71 17 0.2
cis-Isomer# 0.92 1.0 0.2
Result = (ru/s) x (Cs/Cu) X (Mr/M,2) x 100 Montelukast 1.0 = =
Methylketone ah ay
tu = peak response from the Sample solution impurityet 1.04
rs = peak response from the Standard solution
Cs = concentration of USP Montelukast Michael a _
Dicyclohexylamine RS in the Standard adduct 19 1.16
solution (mg/mL) Michael _ id
Cy = nominal concentration of montelukast in the adduct 2he 1.18
Sample solution (mg/mL) Methylstyrene _ _
M, — = molecular weight of montelukast, 586.18 impurity'e 1:55)
Mz = molecular weight of montelukast Any other
dicyclohexylamine, 767.50 individual _
Acceptance criteria: Meet the requirements degradation
|product 1.0 0.2
IMPURITIES
Total impurities — = 2.0
e ORGANIC IMPURITIES
Diluent, Solution A, Solution B, Mobile phase, Stan- These two impurities are not resolved by the method and need to be
integrated together to determine conformance.
dard solution, System suitability solution, Sensitivity [1 -[([1 $B-1(6-2-7-Chloroquinolin-2-y)ethenyl}phenyl}-3.[2-(1 -hydroxy-1-
solution, Sample solution, Chromatographic system, methylethyl)pheny!]propyl]sulfinyl]methyl]cyclopropyljacetic acid.
and System suitability: Proceed as directed in the ¢(£)-1-{3-[2-(7-Chloroquinolin-2-yl)vinyl]phenyl}-3-[2-(2-hydroxypropan-2-
Assay. yl)phenyl]propan-1-one.
Analysis 8[1-[[[(1 R)-1-[3-[(Z)-2-(7-Chloroquinolin-2-yl)ethenyl]pheny!]-3-[2-(I -hy-
Samples: Standard solution and Sample solution droxy-1-methylethyl)pheny!]propyl]sulfany! methyl}cyclopropyllacetic acid.
Calculate the percentage of any individual degradation This is a process impurity and is included in the table for identification
product in the portion of Chewable Tablets taken: only. This impurity is controlled in the drug substance. It is not to be
reported for the drug product and should not be included in the total
impurities.
Result = (ru/rs) x (Cs/Cu) x (Mri/M,2) x (1/F) x 100 ‘1 -[[[(1 R)-3-(2-Acetylphenyl)-1 -[3-[(E)-2-(7-chloroquinolin-2-
yl)ethenyl]pheny!]propyl]sulfanyl]methyl]cyclopropyljacetic acid.
ty = peak response of any individual degradation Cc
91-[[[(1 R)-1-[3-[(1 R)-1-[I[1-(Carboxymethyl)cyclopropyl]methyl]sulfanyl]-2- nn
product from the Sample solution G-eNeroguincin-2yhetylpheryl 3240 hydroxy] Ymethylethy)phenyl] a]
rs = peak response of montelukast from the propyl]sulfanyl]methyl]cyclopropyljacetic acid.
Standard solution 41-[[[(1R)-1-[3-[(1 5)-1-[[[1-(Carboxymethyl)cyclopropyl]methyl|sulfanyl]-2- =
ate Limp yethy erent eZ “ycrony-t-methylethy phenyl co}
Cs = concentration of USP Montelukast propyl|sulfanyl]methyl]cyclopropyljacetic acid. |
Dicyclohexylamine RS in the Standard *[1-[[[C R)-1-[3-[(-2-(7-Chloroquinolin-2-yl)ethenyl] phenyl]-3-[2-(1- °
solution (mg/mL) so}
methyletheny!)phenyl]propyl]sulfany!]methyl]cyclopropyljacetic acid. mm
Cy = nominal concentration of montelukast in the i}
Sample solution (mg/mL) ADDITIONAL REQUIREMENTS mo]
=a
Mn = molecular weight of montelukast, 586.18 © PACKAGING AND STORAGE: Preserve in tight containers, a
Mz = molecular weight of montelukast protected from light. Store at controlled room
dicyclohexylamine, 767.50 temperature.
F = relative response factor (see Table 2) © LABELING When more than one Dissolution test is given,
Acceptance criteria: See Table 2. Disregard any peak the labeling states the test used only if Test 7 is not used.
with an area less than that of the Sensitivity solution. © USP REFERENCE STANDARDS (11)
USP Montelukast Dicyclohexylamine RS
C3sH3eCINO3S - CizH23N =767.50

Morantel Tartrate

Ci2HigN2S - CyHeOs 370.42

Pyrimidine, 1,4,5,6-tetrahydro-1-methyl-2-[2-(3-methyl-
2-thienyl)ethenyl]-, (E)-,[R-(R*,R*)]-2,3-
dihydroxybutanedioate (1:1).
(£)-1,4,5,6-Tetrahydro-1-methy|-2-[2-(3-methyl-
2-thienyl)vinyl]pyrimidine tartrate (1:1) | [26155-31-7].
Morantel [20574-50-9].
 2806 Morantel / Official Monographs
» Morantel Tartrate contains not less than
96.4 percent and not more than 101.5 percent of
Ci2HieN2S + C4HoOc, calculated on the dried
basis.
Packaging and storage—Preserve in well-closed, light-re-
sistant containers. Store at 25°, excursions permitted be-
tween 15° and 30°.
Labeling—Label it to indicate it is for veterinary use only.
USP Reference standards (11)—
USP Morantel Tartrate RS
Clarity and color of solution—Dissolve and dilute 0.25 g
to 25.0 mL in carbon dioxide-free water. The solution is
clear and yellow to greenish yellow in color.
Identification—
A: Infrared Absorption (197K).
wet It meets the requirements of the test for Tartrate
C: The retention time of the morantel peak in the chro-
matogram of the Test solution corresponds to that in the
chromatogram of Standard solution 1, as obtained in the
test for Related compounds.
Melting temperature (741): 167° to 172°.
pH (791): between 2.8 and 3.9.
Solution—Dissolve and dilute 0.25 g to 25.0 mL in carbon
dioxide-free water.
Loss on drying (731)—Dry it at 100° to 105° to constant
weight: it loses not more than 1.5% of its weight.
Residue on ignition (281): not more than 0.1%.
Delete the following:
Es
“
a
Ss
°Heavy metals, Method I/ (231)—not more than 20 ppm.
© (Official 1-jan-2018)
Related compounds—[NoTE—Conduct this test without
— exposure to daylight, and with the minimum necessary ex-
D Co2H25N304S +» HCI
° posure to artificial light.]
is Mobile phase—Mix 3.5 mL of triethylamine and 850 mL
Sj of water. Adjust with phosphoric acid to a pH of 2.5. Add
= 50 mL of tetrahydrofuran and 100 mL of methanol, and
a mix.
”
=) Tartrate solution—Prepare a solution containing about
0.15 mg of tartaric acid per mL in Mobile phase.
Standard solution 1—Dissolve an accurately weighed
quantity of USP Morantel Tartrate RS in Mobile phase to ob-
tain a solution having a known concentration of about
5.0 ug per mL.
Standard solution 2—Dilute 2.0 mL of Standard solution 1
to 100.0 mL with Mobile phase.
System suitability solution—Expose 10 mL of Standard so-
lution 1 to daylight for 15 minutes before injection.
Test solution—Dissolve an accurately weighed quantity of
Morantel Tartrate in Mobile phase to obtain a solution hav-
ing a concentration of about 0.5 mg per mL.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 226-nm detector
and a 4.6-mm x 25-cm column that contains 5-um packing
L1. The flow rate is about 0.75 mL per minute. Chromato-
graph the Tartrate solution, Standard solution 1, and the Sys-
tem suitability solution, and record the peak areas as directed
for Procedure: using the System suitability solution, the resolu-
tion, R, between morantel and its preceding peak ((Z)-iso-
mer) is not less than 2. The relative retention times are
about 0.8, 1.0, and 1.2 for the morante! (Z)-isomer,
morantel, and the morantel 4-methyl isomer (1-methyl-2-
[(E)-2-(4-methylthiophen-2-yl)ethenyl]-1,4,5,6 tet-
rahydropyrimidine), respectively.
USP 41
Procedure—Separately inject equal volumes (about 20 wL)
of the Tartrate solution, Standard solution 1, Standard solution
2, and the Test solution into the chromatograph, record the
chromatograms, and measure the areas for the major peaks.
Disregarding the tartrate peak and any peak in the chromat-
ogram of the Test solution less than the area of the principal
peak in the chromatogram of Standard solution 2, calculate
the area percentage of each impurity, relative to morantel,
in the portion of Morantel Tartrate taken by the formula:
100(Cs
/ Cu)(n/ 15)
in which Cs and Cy are the concentrations of morantel tar-
trate, in mg per mL, of Standard solution 71 and the Test
solution, respectively; and r, and rs are the peak areas of
each individual impurity and morantel obtained from the
Test solution and Standard solution 1, respectively: not more
than 3% of the morantel 4-methyl isomer is found; not
more than 0.5% of any other individual impurity is found;
and not more than 1% of total other individual impurities is
found.
Assay—
Dissolve 0.280 g in 40 mL of anhydrous acetic acid. Ti-
trate with 0.1 N perchloric acid VS, determining the
endpoint potentiometrically (see Titrimetry (541)). One mL
of 0.1 N perchloric acid is equivalent to 37.04 mg of
CraHisN2S + CaeOc.
Moricizine Hydrochloride
" 6 ‘ |
SZ
i o; 7 He
463.98
Carbamic acid, [10-[3-(4-morpholinyl)-|-oxopropyl]-10H-phe-
nothiazin-2yl]-, ethyl ester, hydrochloride;
Ethyl 10-(3-morpholinopropionyl)phenothiazine-2-carba-
mate, hydrochloride [29560-58-5].
DEFINITION
Moricizine Hydrochloride contains NLT 98.0% and NMT
102.0% of moricizine hydrochloride (C22H2sN3O4S - HCl),
calculated on the anhydrous and alcohol-free basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
e C. IDENTIFICATION TESTS—GENERAL, Chloride (191): Meets
the requirements
ASSAY
e PRoceDuRE: Use low-actinic glassware. Protect the solu-
tions containing moricizine hydrochloride from light.
Mobile phase: A mixture of acetonitrile, triethylamine,
glacial acetic acid, and water (420:1:20:580) containing
5 mM sodium 1-octane sulfonate
Diluent: Acetonitrile and 0.02 N hydrochloric acid
(42:58)
Internal standard solution: 5 mg/mL of butamben in
Diluent
Standard solution: 1 mg/mL of USP Moricizine Hydro-
chloride RS in Diluent prepared as follows. Transfer a
suitable amount of USP Moricizine Hydrochloride RS to
a suitable volumetric flask, add Internal standard solution
to fill about 20% of the total volume, and dilute with
Diluent to volume.
USP 41
Sample solution: 1 mg/mL of Moricizine Hydrochloride
prepared as follows. To a suitable amount of Moricizine
Hydrochloride in a 50-mL volumetric flask add Internal
standard solution to fill about 20% of the total volume,
and dilute with Diluent to volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; packing L7
Column temperature: 35°
Flow rate: 2.5 mL/min
Injection volume: 10 pL
System suitability
Sample: Standard solution
[NotE—The relative retention times for moricizine, bu-
tamben, the reverse Mannich product, and the amide
hydrolysis product are about 0.6, 1.0, 1.7, and 2.0,
respectively.]
Suitability requirements
Resolution: NLT 2 between moricizine and butamben
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of moricizine hydrochloride
(C22H2sN304S - HCl) in the portion of Moricizine Hydro-
chloride taken:
Result = (Ru/Rs) x (Cs/Cu) x 100
Ru = ratio of the moricizine peak area response to
the butamben peak area response from the
Sample solution
Rs = ratio of the moricizine peak area response to
the butamben peak area response from the
Standard solution
Gs = concentration of USP Moricizine Hydrochloride
RS in the Standard solution (mg/mL)
Cu = concentration of Moricizine Hydrochloride in
the Sample solution (mg/mL)
Acceptance criteria: 98.0%-102.0% on the anhydrous
and alcohol-free basis
OTHER COMPONENTS
© CONTENT OF CHLORIDE
Sample solution: Transfer 400 mg of Moricizine Hydro-
chloride to a conical flask and add 75 mL of methanol.
Swirl to dissolve. Add 5 mL of glacial acetic acid and
3 drops of eosin Y TS.
Analysis: Titrate the Sample solution with 0.1 N silver
nitrate VS to a pink endpoint. Each mL of 0.1 N silver
nitrate is equivalent to 3.546 mg of chloride.
Acceptance criteria: 7.49%-7.80% on the anhydrous
and alcohol-free basis
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.1%
Delete the following:
°e HEAVY METALS, Method I! (231): NMT 10 U9/Qe cotiaa! 1-
Jan-2018)
© ORGANIC IMPURITIES
Mobile phase: A mixture of acetonitrile, triethylamine,
and water (420:1:580) containing 5 mM sodium 1-oc-
ts sulfonate. Adjust with glacial acetic acid to a pH of
Diluent: Acetonitrile and 0.02 N hydrochloric acid
(42:58)
Official Monographs / Moricizine 2807
Internal standard solution: 0.1 mg/mL of butamben in
Diluent
Standard stock solution: 0.10 mg/mL of USP
Moricizine Hydrochloride RS in Diluent
Standard solution: 2.0 g/mL of USP Moricizine Hydro-
chloride RS prepared as follows. Transfer a suitable vol-
ume of Standard stock solution to a suitable volumetric
flask, add Internal standard solution to fill 5% of the
total volume, and dilute with Diluent to volume.
[Note—Protect the solution from light.]
Sample solution: 1 mg/mL of Moricizine Hydrochloride
in eat prepared as follows. Transfer a suitable
amount of Moricizine Hydrochloride to a suitable volu-
metric flask, add Internal standard solution to fill 5% of
the total volume, and dilute with Diluent to volume.
[Note—Protect the solution from light.]
Chromatographic system: Proceed as directed in the
Assay except for the following:
Run time: Five times the elution time of moricizine
Injection volume: 20 uL
System suitability
Sample: Standard solution
[Note—The relative retention times for moricizine and
butamben are about 0.6 and 1.0, respectively.]
Suitability requirements
Resolution: NLT 2 between moricizine and butamben
Relative standard deviation: NMT 5%
Analysis
Samples: Standard solution and Sample solution
Measure the responses for all the peaks except the sol-
vent peak.
Calculate the percentage of each impurity in the por-
tion of Moricizine Hydrochloride taken:
Result = (Ru/Rs) x (Cs/Cu) x 100
Ru = ratio of the peak area response of each
impurity to the butamben peak area (=
response from the Sample solution 4)
Rs = ratio of the peu area response of moricizine ao]
to the peak area response of butamben from =
the Standard solution i}
Cs = concentration of USP Moricizine Hydrochloride =)
re}
RS in the Standard solution (mg/mL) as
Cu = concentration of the Sample solution (mg/mL) »
Acceptance criteria: Disregard any impurity of less i}
than 0.1%. =a
Any impurity eluting before moricizine: NMT 0.25%
a
Any impurity: eluting after moricizine: NMT 0.20%
Total of all impurities: NMT 1.5%
e LIMIT OF ALCOHOL
Standard solution: 0.1184 mg/mL of dehydrated alco-
hol in water prepared as follows. Transfer 6.0 mL of de-
hydrated alcohol to a 100-mL volumetric flask and di-
lute with water to volume. Dilute 5.0 mL of this
solution in a 100-mL volumetric flask with water to vol-
ume. Further dilute 5.0 mL of this solution in a third
100-mL volumetric flask with water to volume.
Sample solution: Transfer 1 g of Moricizine Hydrochlo-
ride to a 50-mL glass-stoppered centrifuge tube, add
19.0 mL of water, and sonicate to dissolve. Transfer
1.0 mL of 3 N ammonium hydroxide to the tube, insert
the stopper, and shake the tube by mechanical means
for 30 min. Centrifuge, draw off a portion of the clear
supernatant, and pass throughasuitable filter of 0.5-
uum or finer pore size.
 2808 Moricizine / Official Monographs USP 41

Chromatographic system flask, dilute with 0.1 N hydrochloric acid to volume, and
(See Chromatography (621), System Suitability.) mix.
Mode: GC Standard solution: 8 ug per mL.
Detector: Flame ionization
Column: 4-mm x 1.8-m glass; supporting $2 Medium: 0.1 N hydrochloric acid.
Temperatures B: Shake a Tablet with 10 mL of methanol until it disinte-
Column: 150° grates, and filter: the filtrate responds to Identification test C
Injector port: 170° under Moricizine Hydrochloride.
Detector block: 170° Dissolution (711)—
Carrier gas: Helium Medium: 0.1 N hydrochloric acid; 900 mL.
Flow rate: 50 mL/min Apparatus 2: 50 rpm.
Injection volume: 5 uL
System suitability Time: 30 minutes.
Sample: Standard solution Procedure—Determine the amount of moricizine hydro-
Suitability requirements chloride (C22H2sN304S- HCI) dissolved from UV absorbance
Relative standard deviation: NMT 3% at about 267 nm of filtered portions of the solution under
Analysis test, suitably diluted with Dissolution Medium, in comparison
Samples: Standard solution and Sample solution with a Standard solution having a known concentration of
Calculate the percentage of alcohol (C,HsOH) in the USP Moricizine Hydrochloride RS in the same medium.
portion of Moricizine Hydrochloride taken: Tolerances—Not less than 75% (Q) of the labeled amount
of moricizine hydrochloride (C22H2sN304S - HCl) is dissolved
Result = (ru/rs) x (Cs/Cu) x 100 in 30 minutes.
tu = peak response of alcohol from the Sample Uniformity of dosage units (905): meet the require-
solution ments.
rs = peak response of alcohol from the Standard Limit of degradation products—
solution Mobile phase—Dissolve 1.08 g of sodium 1-octanesulfon-
Gs = concentration of alcohol in the Standard ate in 580 mL of water, add 420 mL of acetonitrile, 20 mL
solution (mg/mL) of glacial acetic acid, and 1 mL of triethylamine. Adjust with
Cy = concentration of Moricizine Hydrochloride in 5 N sodium hydroxide to an apparent pH of 4.5. Mix, and
the Sample solution (mg/mL) filter through a filter having a porosity of 0.5 um or finer.
Acceptance criteria: NMT 0.25% Make adjustments if necessary (see System Suitability under
Chromatography (621)).
SPECIFIC TESTS Diluent—Prepare a mixture of 0.02 N hydrochloric acid
e Loss ON DRYING (731) and acetonitrile (58:42).
Analysis: Dry at 105° for 4 h.
Acceptance criteria: NMT 1.0% Internal standard solution—Prepare a solution of butam-
e WATER DETERMINATION, Method | (921): NMT 1.0% ben in Diluent containing about 0.2 mg per mL.
te
”
e CLARITY OF SOLUTION Standard solution—Prepare a solution of USP Moricizine
ot Sample solution: Dissolve 1 g in 30 mL of methanol, Hydrochloride RS in Diluent containing 0.10 mg per mL.
i}
— sonicating for 5 min if necessary. Transfer 5.0 mL of this solution to a 50-mL volumetric flask,
io) add 20.0 mL of Internal standard solution, dilute with Diluent
° Acceptance criteria: The solution is not less clear than
= an equal volume of methanol contained ina similar ves- to volume, and mix. [NoTE—Protect this solution from light.]
S sel and examined similarly. Test solution—tTransfer 10 Tablets to a 1000-mL flask, add
3 500.0 mL of Diluent, sonicate until the Tablets are disinte-
re ADDITIONAL REQUIREMENTS grated, and then shake by mechanical means for 30 min-
a) ¢ PACKAGING AND STORAGE: Preserve in tight containers. utes. Filter this solution, discarding the first 10 mL of the
=] ¢ USP REFERENCE STANDARDS (11) filtrate. Transfer 25.0 mL of the filtrate to a 50-mL volumet-
USP Moricizine Hydrochloride RS ric flask, add 20.0 mL of Internal standard solution, dilute
with Diluent to volume, and mix. [NoTE—Protect this solu-
tion from light.]
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 254-nm detector
Moricizine Hydrochloride Tablets and a 4.6-mm x 25-cm column that contains packing L7
and is maintained at a constant temperature of about 35°.
» Moricizine Hydrochloride Tablets contain not The flow rate is about 2.5 mL per minute. Chromatograph
the Standard solution, and record the peak responses as di-
less than 90.0 percent and not more than rected under Procedure: the relative retention times are
110.0 percent of the labeled amount of about 0.6 for moricizine and 1.0 for butamben, the resolu-
moricizine hydrochloride (C22H2sN304S « HCl). tion, R, between the moricizine peak and the butamben
eak is not less than 2, and the relative standard deviation
Packaging and storage—Preserve in tight containers. ‘or replicate injections is not more than 5%.
USP Reference standards (11)— Procedure—Separately inject equal volumes (about 20 uL)
USP Moricizine Hydrochloride RS of the Standard solution and the Test solution into the chro-
Identification— matograph, record the chromatograms for a period of time
A: Ultraviolet Absorption (197U)— that is five times the elution time of moricizine, and meas-
ure the responses for the peaks, except for any that elute
Test solution—Transfer a portion of finely ground Tablets, before moricizine. Calculate the percentage of each impurity
equivalent to about 50 mg of moricizine hydrochloride, to a peak that elutes after butamben in the portion of Moricizine
250-mL volumetric flask, add about 100 mL of 0.1 N hydro- Hydrochloride taken by the formula:
chloric acid, shake by mechanical means for 15 minutes, di-
lute with 0.1 N hydrochloric acid to volume, and mix. Filter 1000(C
/ L)(R;/ Rs)
a portion of this solution, discarding the first 10 mL of the
filtrate. Transfer 10 mL of the filtrate to a 250-mL volumetric in which C is the concentration, in mg per mL, of USP
Moricizine Hydrochloride RS in the Standard solution, L is the
USP 41
labeled amount, in mg, of moricizine hydrochloride in each
Tablet, R; is the ratio of the peak areas of an individual im-
purity peak to the butamben peak obtained from the Test
solution, and Rs is the ratio of the peak areas of the
moricizine peak to the butamben peak obtained from the
Standard solution. The first impurity eluting after the butam-
ben peak is not more than 0.50%, and the second impurity
eluting after butamben is not more than 0.25%.
Assay—
Mobile phase, Diluent, Internal standard solution, Standard
preparation, and Chromatographic system—Proceed as di-
rected in the Assay under Moricizine Hydrochloride.
Assay preparation—Transfer an accurately counted num-
ber of Tablets, equivalent to about 4000 mg of moricizine
hydrochloride, to a 2000-mL flask, add 1000.0 mL of Dilu-
ent, and sonicate until the Tablets have disintegrated. Shake
by mechanical means for 30 minutes. Filter a portion of this
solution, discarding the first 10 mL of the filtrate. Cover the
filter funnel with a watch glass to minimize evaporation of
the solvent. Transfer 25.0 mL of the filtrate and 20.0 mL of
Internal standard solution to a 100-mL volumetric flask, di-
lute with Diluent to volume, and mix. [NoTE—Protect this
solution from light.]
Procedure—Proceed as directed for Procedure in the Assay
under Moricizine hyeraeniae Calculate the quantity, in
mo of moricizine hydrochloride (C22H2sN30.S- HCI) in each
Tablet by the formula:
4000(C
/ N)(Ru
/ Rs)
in whichCis the concentration, in mg per mL, of USP
Moricizine Hydrochloride RS in the Standard preparation, N
is the number of Tablets taken, and Ru and Rs are the ratios
of the peak area responses of the moricizine peak to the
butamben peak obtained from the Assay preparation and
the Standard preparation, respectively.
Morphine Sulfate
2N—CH.
i
( nh
\ © H,S0, © 5H0
(Ci7HigNO3)2 > H2SO4-5H20 758.83
Morphinan-3,6-diol, 7,8-didehydro-4,5-epoxy-17-methyl,
(5a,6c)-, sulfate (2:1) (salt), pentahydrate.
7,8-Didehydro-4,5a-epoxy-17-methylmorphinan-3,6a-diol
sulfate (2:1) (salt) pentahydrate ~[6211-15-0].
Anhydrous 668.77 [64-31-3].
» Morphine Sulfate contains not less than
98.0 percent and not more than 102.0 percent of
(Ci7zHigNO3)2- H2SOx4, calculated on the anhy-
drous basis.
Packaging and storage—Preserve in tight, light-resistant
containers. Store up to 40° as permitted by the manufac-
turer.
USP Reference standards (11)—
USP Morphine Sulfate RS
identification—
A: Infrared Absorption (197K): dried at 145° for 1 hour.
B: To 1 mg in a porcelain crucible or small dish add
0.5 mL of sulfuric acid containing, in each mL, 1 drop of
formaldehyde TS: an intense purple color is produced at
once, and quickly changes to deep blue-violet (distinction
from codeine, which gives at once an intense violet-blue color,
Official Monographs / Morphine 2809
and from hydromorphone, which gives at first a yellow to
brown color, changing to pink and then to purplish red).
C: To a solution of 5 mg in 5 mL of sulfuric acid in a test
tube add 1 drop of ferric chloride TS, mix, and heat in
boiling water for 2 minutes: a blue color is produced, and
when 1 drop of nitric acid is added, it changes to dark red-
brown (codeine and ethylmorphine give the same color reac-
tions, but hydromorphone and papaverine do not produce this
color change).
D: A solution (1 in 50) responds to the tests for Sulfate
(191).
Specific rotation (781S): between -107° and —-109.5°.
Test solution: the equivalent of 20 mg per mL, in water.
Acidity—Dissolve 500 mg in 15 mL of water, add 1 drop of
methyl red TS, and titrate with 0.020 N sodium hydroxide:
not more than 0.50 mL is required to produce a yellow
color.
Water Determination, Method | (921): between 10.4%
and 13.4% is found.
Residue on ignition (281): not more than 0.1%, from
500 mg.
Chloride—To 10 mL of a solution (1 in 100) add 1 mL of
2N nitric acid and 1 mL of silver nitrate TS: no precipitate
or turbidity is produced immediately.
Ammonium salts—Heat 200 mg with 5 mL of 1 N sodium
hydroxide on a steam bath for 1 minute: no odor of ammo-
nia is perceptible.
Limit of foreign alkaloids—Dissolve 1.00 g in 10 mL of
1 N sodium hydroxide in a separator, and shake the solution
with three successive portions of 15, 10, and 10 mL of chlo-
roform, passing the chloroform solutions through a small
filter previously moistened with chloroform. Shake the com-
bined chloroform solutions with 5 mL of water, separate the
chloroform layer, and carefully evaporate on a steam bath
to dryness. To the residue add 10.0 mL of 0.020 N sulfuric
acid, and heat gently until dissolved. Cool, add 2 drops of c
“
methyl red TS, and titrate the excess acid with 0.020 N U
sodium hydroxide: not less than 7.5 mL is required (1.5%).
Es
Assay— i}
Mobile phase—Dissolve 0.73 g of sodium 1-heptanesul- =|
}
fonate in 720 mL of water, add 280 mL of methanol and a=
10 mL of glacial acetic acid, mix, filter, and degas. Make
iy
adjustments if necessary (see System Suitability under Chro- ae
matography (621)). 33
Standard preparation—Dissolve an accurately weighed
quantiyy of USP Morphine Sulfate RS in Mobile phase, and
ilute quantitatively, and stepwise if necessary, with Mobile
phase to obtain a solution having a known concentration of
about 0.24 mg per mL. Prepare a fresh solution daily.
System suitability preparation—Dissolve suitable quantities
of USP Morphine Sulfate RS and phenol in Mobile phase to
obtain a solution containing about 0.24 and 0.15 mg per
mL, respectively.
Assay preparation—Transfer about 24 mg of Morphine
Sulfate, accurately weighed, to a 100-mL volumetric flask,
dissolve in and dilute with Mobile phase to volume, and mix.
Chromatographic system (see Chromatography (621))—The
liquid chromatograph is equipped with a 284-nm detector
and a 3.9-mm x 30-cm column that contains packing L1.
The flow rate is about 1.5 mL per minute. Chromatograph
the Standard preparation and the System suitability prepara-
tion, and record the peak responses as directed for Proce-
dure: the relative retention times are about 0.7 for phenol
and 1.0 for morphine sulfate; the resolution, R, between
phenol and morphine sulfate is not less than 2.0; the tailing
factor for the morphine sulfate peak is not more than 2.0;
and the relative standard deviation for replicate injections of
the Standard preparation is not more than 2.0%.
Procedure—Separately inject equal volumes (about 25 j1L)
of the Standard preparation and the Assay preparation into
the chromatograph, record the chromatograms, and meas-
 2810 Morphine / Official Monographs
ure the responses for the major peaks. Calculate the quan-
tity, in m9, of (Ci7Hi9NO3)2- H2SO4 in the portion of Mor-
phine Sulfate taken by the formula:
100C(ru/ rs)
in which C is the concentration, in mg per mL, of anhy-
drous morphine sulfate in the Standard preparation, as deter-
mined from the concentration of USP Morphine Sulfate RS
corrected for moisture content by a titrimetric water deter-
mination; and ry and rs are the peak responses obtained
from the Assay preparation and the Standard preparation, re-
spectively.
Morphine Sulfate Extended-Release
Capsules
DEFINITION
Morphine Sulfate Extended-Release Capsules contain NLT
90.0% and NMT 110.0% of the labeled amount of mor-
phine sulfate pentahydrate [(Ci7HigNO3)2 + H2SO4 + 5H20].
IDENTIFICATION
eA.
Standard solution and Sample solution: Prepare as di-
rected in the Assay.
Analysis: Inject 10 uL each of the Standard solution and
the Sample solution using the Chromatographic system
except for the Injection volume in the Assay.
Acceptance criteria: The UV absorption spectrum of
the morphine peak of the Sample solution and of the
Standard solution exhibits maxima and minima at the
same wavelengths, as obtained in the Assay.
e B. The retention time of the major peak of the Sample
"
solution corresponds to that of the Standard solution, as
<=
a obtained in the Assay.
s
-
D ASSAY
° © PROCEDURE
< Diluent: Water. Adjust with phosphoric acid to a pH of
5 3.6.
= Buffer solution: 13.8 mg/mL of monobasic sodium
a phosphate
”“
=) Solution A: Acetonitrile, triethylamine, Buffer solution,
and water (25: 0.5: 100: 874.5). Adjust with phosphoric
acid to a pH of 3.6.
Solution B: Acetonitrile
Mobile phase: See Table 1.
Table 1
Solution A Solution B
1 Dosage Forms, Method B Procedure, observing the fol-
1
System suitability solution: 400 ug/mL of USP Mor-
phine Sulfate RS and 10 g/mL each of USP Morphine
Related Compound A RS and USP Morphine Related
CompoundB RS (pseudomorphine) in Diluent
Standard solution: 1.0 mg/mL of USP Morphine Sul-
fate RS in Diluent
Sample stock solution: Transfer a weighed portion of
the contents from NLT 20 Capsules, nominally equiva-
lent to 250 mg of morphine sulfate pentahydrate, to a
100-mL volumetric flask. Add 5 mL of methanol and
mix well for NLT 30 min with gentle swirling about
USP 41
every 5 min. Add Diluent up to half of the flask volume
and sonicate for NLT 5 min to dissolve. Dilute with Dilu-
ent to volume.
Sample solution: peat 1.0 mg/mL of morphine
sulfate pentahydrate from the Sample stock solution in
Diluent. Pass through a suitable filter and use the clear
filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 245 nm. For Identification A, use a diode
array detector in the range of 200-400 nm.
Columns
Guard: Packing L1
Analytical: 3.9-mm x 30-cm; 10-um packing L1
Flow rate: 2 mL/min
Injection volume: 40 uL
System suitability
Samples: System suitability solution and Standard
solution
Suitability requirements
Resolution: NLT 2.0 between the morphine related
compound A and morphine sulfate peaks, System suit-
ability solution
Relative standard deviation: NMT 2.0%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of mor-
phine sulfate pentahydrate [(Ci7Hi3NO3)2 - H2SO4
-
5H20] in the portion of Capsules taken:
Result = (ru/rs) x (Cs/Cu) x (Mu/M,2) x 100
tu = peak response from the Sample solution
Is = peak response from the Standard solution
G = concentration of USP Morphine Sulfate RS in
the Standard solution (mg/mL), calculated on
the anhydrous basis
Cu = nominal concentration of morphine sulfate
pentahydrate in the Sample solution (mg/mL)
Mn, — = molecular weight of morphine sulfate
pentahydrate, 758.83
Mz = molecular weight of anhydrous morphine
sulfate, 668.77
Acceptance criteria: 90.0%-110.0%
PERFORMANCE TESTS
e DISSOLUTION (711)
Test 1
pH 7.5 phosphate buffer: 6.8 mg/mL of monobasic
potassium phosphate and 1.6 mg/mL of sodium hy-
droxide. Adjust with phosphoric acid or 2 N sodium
hydroxide to a pH of 7.5.
Medium: Prepare as directed in Dissolution (711), Pro-
cedure, Apparatus 1 and Apparatus 2, Delayed-Release
lowing exceptions. Perform Acid Stage testing, using
500 mL of 0.1 N hydrochloric acid for 1 h; and per-
form Buffer Stage testing, using 500 mL of pH 7.5 phos-
phate buffer for NLT 8 h.
Apparatus 1: 100 rpm
Times: 1, 4, 6, and9h
Mobile phase: Methanol, glacial acetic acid, and
water (280:10:720), containing 0.73 g of sodium
1-heptanesulfonate for each 1.01 L of the solvent
mixture
System suitability solution: 0.1 mg/mL each of phe-
nol and USP Morphine Sulfate RS in Mobile phase
Standard solution: USP Morphine Sulfate RS in pH
7.5 phosphate buffer to obtain a solution with a known
concentation corresponding to that of the Sample
solution
 3 2 P0 e0n i c /i Olflfii nM
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i lU ln i in t s / m g . [ N o t re e l aT rthie e vt ee nt ti imfoeon2 sr - p h e n y -
l a c e t aa nmp die nd iec iG al lr a ie nb o0 u. at8n d 1 . 0 ,
I D E N T I F I C A T I O N r e s p e ]c t i v e l y .
e A ,T H I N - CL HA RY O E RM A T O G R A P H Y S u i t a br ie l qi ut i y r e m e n t s
S o l u At :iA oc ne t 0o. n1M ec , i t ra ic ci ad ,n0 d. 1Ms o - R e s o l u Nt Li2 o T. nb0 :e t w2 e- e p n h e n y laa nc de
d i cu i mt r( a2t :e 1 : 1 ) p e n i c Gi ,lS l yi sn stu ei tm a sb io ll iu tt yi o n
S t a n sdo al ru t d Pi ro en p:a a s or le u ct oi n o nt a ti hnei n g C o l euf m f inc i Ne nL 1cTy0 :t0 h0e o r ep lt ai tc ea sl ,
A ) e q u i vo af1l 2e , n Pt0 e 0n 0i c iG lU lni in t fs r/ oUm mS L P S t a n sdo al ru td i o n
< =
o h P e n i c iG lP loi tn a sR sSi i n S uo ml u At .i o n T a i lf ia nc gt oNr M :2 .T0S ,t a n s do al r u td i o n
S s S a m spo ll e u t Ni o nm :i n1 a2 l, Pl0ey0n i0 c iG lU ln ii nt s / R e l a st t i va en dd eav ri da tNi M o2n T.: 0S %t ,a n d a r
J
D p m fL r Po emn i c iG lP lo i tn a s f osIrin uj m e ci tnSioolnu t i o n s o l u t i o n
i} A A n a l y s i s
i J C h r o m a t soy gs rt ae pm h i c
iS S a m p lS e t sa n:s do al ru atd in S odna m spo ll eu 1t, i o n
( S e e e Y y ( 6e 2 1T )h ,i n - CL ha ry o e rm a t o - S a m spo ll eu 2t ,o i orSn a m sp o ll eu 3t i o n
= r a p h y . P e r ft oh Are sm so an1y 0c o n t a wi hn eei trirssre e p r e -
a A a c o r b 0 e. n2 t5l sa-yome fcrm h r o m a ts oi lgi r c aa p h si ec n at seb d e ii nna gs i n g l e c -o dn ot sa ein indf e n, r
e c -
a l
= g e ml i x t u r e e s s a or n y1 ,0c o n t a wi hn eetrrhslea b se tl a t eh se
A p p l i c v ao tl iu o 2m n0
ue L: q u a n ot fpi etn yi c G i il nla ig ni v ve on l oufcmo en s t i -
D e v e ls oo pl ivs ney g ns tt Te oml :u de in oe x, aa n ed , t u ts eo dl u tUi sotenh.ien d i v rieds uutaloldt se t e r m i
g l a cai ca el at ci ic( d9 0 : 2 5 : 4 ) t h Ue n i f oo rfDm oi t s Uyan gi aet n s t dh ae v e ro aft gh ee
S p rr ae ya g 1 :eS nt ta T r cS h r e s ua lstt hs Ae s sv aa y l u e .
S p rr ae ya g2 :e lno td T i nS d ei l u1 ti en 1d 0w i tw ha t e r C a l c ut lh pae e t er c eo nftth ale a g be enl ue dm obfP ee r n i -
A n a l y s i s c i l l Gi Un n ii tn ts h ce o n t ao iri nnt eh pre o r to ifco on n -
S a m p lS e t s a n : sdo al ru a td in oSdna m spo ll eu t i o n s t i t su ot le ud tt ai ko en n :
P l atc hepe l ai tnaes u i t ca bh lr eo m a t co hg ar ma bp eh ri .c
D e v et lh co e hp r o m ai ntt h o Dege rv ae lms oo pl iv ne ng t R e s u= (lr tu / xr (s C) s /xCP yx )1 0 0
s y s ut n et tmi hl se o l vf er nohtn atms o vt e h rde e - f o u r t h s
o ft h le e n og ft h p el a tRe e. m toh vpe lea f t e r to hm e t u =p e r a ek s p fo rn o S
s m a
e m spo ll eu 1t oi ro n
c h a m mb ae tr r hk,se o l vf e r on na
t t n,a dl lt ooa wi r - d r y . S a m sp o ll eu 2 t i o n
S p rt ah pye l a wt ie tS hp rr ae ya g1 e f no tl l bo yw S ep dr a y r s =p e a r ek s p fo rn t os hme Se t a n sdo al ru td i o n
r e a g2 .eP ne nt i c iG la lpi pn ea a sa rw sh is tp eoo tna G s = c o n c e n ot fr U aSPtPe in oi cn iG lP loi tn a sR sS i u
p u r b p la ec k g r o u n d . i n t h Se t a n s do al r u ( td imo gn / m L )
A c c e pc t r ia t ne T rc ih
eaR e:;v a lo ufte h pee n i c G i l l i n C u = n o m ic no an l c e n ot fS r aa tm sip oollneu 1 t oi ro n
s p o ft h Se a m spo ll e u ct io or nr e ts ot p ho aon fttd hs e S a m spo ll eu 2 t (i Poenn i cGiUl nl ii nt s / m L )
S t a ns do al ur tdi o n . P = p o t eo n fp ec nyi c G i il nlUi S nP Pe n i c iG l l i n
P o t a sR sS( i P eu nmi cGiUl nl ii nt s / m g )
A S S A Y C a l c ut lh pae ot t e e innPc e yn ,i c iG lU ln i in t so / ft mh ge ,
e P R O C E D U R E P e n i c iG lP loi tn a s f osIrin uj m e ct ta ik oe nn :
S o l u At :i0 o. n0 M 1m o n o b p o a ts ai spc sh io us mp h a t e
M o b pi hl ae sM ee :t h aa nnS do o ll u A t (i 4o 0n : 6 0 ) R e s = u (lr tu / xr (s C) s /xCP u )
S y s stu ei tm a bs io ll iu tt y0i.o1mn g : /e a m ocLfhU S P
P e n i c iG lP loi tn a sR sSai nu2dm- p h e n y li a n c e t a tm ui d =ep e a r ek s p fo rn o Ss m a
e m spo ll eu 3t i o n
w a t e r I s =p e a r ek s p fo rn t os hmeSe t a ns do al r u td i o n
G s = c o n c e n ot fr U aSPtP e in oi cn iG lP loi tn a sR sS i u
i nt h Se t a n sdo al r u ( td imo gn / m L )
U S4 P1 O f f i M
c ioa n
l o g/ Pr ean p
i chi3sl2l 0i n1

C y = c o n c e n ot fPr ean ti ic i
G
o lPn lo i tn a s
f os ri u m S t a n sdo al ru td Pi ro en p:a a s or le u ct oi n o nt a ti hnei n g
I n j e ci nt si onn sa o pl u 3t ( i om n g / m L ) e q u i vo af1l 2e , n Pt
0 e 0n 0i c iG lU lni in t fs r/ oUm mS L P
P = p o t eo n fp ec nyi c G i il nlUi SnP Pe n i c G i l l i n P e n i c iG lP loi tn a sR sS i inS uo ml u A t i o n
P o t a sR sS( iP eu nmi cGi l l ie n e S a m spo ll e u t Si ho ana :kp eo r to iif to,cn o n t a i n i n g
A c c e pc tr ia t ne c r9 ie0a .
: 0 % -o f1 t h2 le0a b. e0l % e d n o m i n1 a0 l0 l,Pye0 n 0i 0 c iG Ul ln ii ntw si ,t8 h
m oLfS o l u -
n u m obfP ee nr i c iG Ul nl i ntW s h. eP er n ei c iG Pl lo it na s - t i oAn
s i fu omIr n j e cc to ino tnsa o i nd csii ut rmi taht aeasp o - C h r o m a t soy gs rt ae pm h i c
t e no cfNy L 1T3 3
a 5
n N
d M1 T
5 P9 e 5n i c i
GUl ln ii nt s / ( S se ep y m a yv( o 6 2g 1T r)h ,ai np- CL ha ry o e rm a t o -
m g . r a p h y .
H s e e t0 e.c2o5 lka- yomefcrm h r o m a ts o i lgi r
c aa p h i
P E R F O TR EMS AT N S C E g e ml i x t u r e
' U N I F OO RFDM OI S T UAY NGI(ET9 S0 5 M) e: et th rs ee q u i r e - A p p l i v c a o tl iu 2 om n0
pe L:
m e n P t e
s r. ft oh Are sm soa n1y 0 c o n t a wi hn ei trirs se D e v e ls oo pl ivs ney g ns tt Te om l:u de in oe x, aa n ed ,
r e p r e as seb ne tii enadgs i n g l c e -o dn ot saa ein indf e, r g l a cai ca el at ci ic( d9 0 : 2 5 : 4 )
n e c e s os na1 r0 cyo,n t a wi hn e et rrhslea b se tl a t eh se S p rr ae ya g1 :e Snt ta T r cS h
q u a n ot fpi etn yi c iGil nla ig ni v ve on l ou fcm o en s t i t u t eS dp rr ae ya g2 :e lno td T i nS d ei l u1 ti en 1d 0w i tw ha t e r
s o l u tUi sotenh.ien d i v ri edsuutaloldt se t e r t m
h Ue in ni e- A n a l y s i s
f o r mo ifDt oy s Ua ngi aet n st dh ae v e ro a ft gh reee s ual st s S a m p lS e t sa n: sdo al ru atd in S odna m sp o ll eu t i o n
t h Ae s sv aa ly u e . P l atc hepel ai tnaes u i t ca bh lr eo m a t co hg ar ma bp eh ri .c
D e v et lh co e hp r o m ai ntt h o Dege rv a
e lms oo pl iv ne gn t
S P E C TI EF S I TC S s y s ut n et tmi h
l seo l vf er nohtn atms o vt e h rd e e - f o u r t h
' I N J E C AT I NIODMN P S L AD NR TP UERGDo p( u 1 )S
c,pte s c i f i c o ft h le e n og ft h pel a tR e e. m toh vpe lea tf e r t o hm e
T e s tCs o, m p l eat nce d ln a erosifstsoy l u t iA ottn hs te: i om fe c h a m mb ae tr r hk,se o l vf e r on na
t tn,a dl lt ooa wi r - d r y .
u s ie t ,m e et th rse e q u i r e m e n t s . S p rt ah ypel a wt ie tS hp rr ae ya g1 ef no tl l bo yw S ep d
r a y
e B A C T EE RN ID AO LTT OE X(S I 8T5N )ISt:c o n t a NiM n0 s.T0 1 r e a g2 .eP ne nt i c iG a l lp i pn ea a sa rw sh is tp eoo tna
U S EPn d o tU on x i i t n s
P e/ n1i 0c 0 iG U
l ln ii nt s . p u r b p la ec k g r o u n d .
' S T E R IT LE IS(TT7Y S1 )_ I :t m e et th rse e q u i rwe hm e e nn t s A c c e pc t r ia t ne T rc ih
eRa er:v a lo ufte h p ee n i c G i l l i n
t e s at sed di r e icnTt ee sfd to Srt e r io l fti ht Pye r o dt uoB ce t s p oo ft h Se a m spo ll eu ct io or nr e ts ot p ho a
on fttd hs e
E x a m iM n ee m
d ,
bF r
i l at rna e
t i o n .
S t a n s do al ur td i o n .
e C R Y S T A (L 6L 9I 5NM) Ie: TetYth rse e q u i r e m e n t s
e P ( H7 9 1 ) A S S A Y
S a m sp o ll e u tA isoo nl :u ct oi o n nt a 6i 0m n ig n/g m L . ' P R O C E D U R E
W h ep ra ec k f ao dgr ie sdp e nu s tie hnsego ,l u ct oi no sn t i - S t a n sdo al ru td Pi ro en p:a asd ri er e icnlt oe dd o mA es t- r i c
t u ta esdd i r e icn tt he ld ea b e l i n g . s a y A n t (i4b2 i5 So)tt,ai ncPdsr ae rp d a r ua st iiUno Sgn ,P
A c c e pc t r ia t ne 5rc i.ea0 :- W7 .h 5e i .tirs lea b ea ls e d P e n i c iG lP loi tn a sR Ss .i u m
c o n t a si on d i ciii gu t rmta th epe, iHs b e t w6 e . a0e nn d S a m spo ll e u t Ci oo nn s: tP ie tn iu ct iGelP lo i tn a s f os ri u m
5 O r aSl o l u at sdi io rn e icn tt he ld ea b e ul si niBgnu gf Bf .e 1r
e L o sO sN D R Y (I 7N 3G1 ) ( s eA en t i b i o t i cA ss s(Ma8 i1y c M) r,e odabiinSaado ll u - j e s
S a m p 1l 0em0: og fP e n i c iG lP loi tn a s f osIrin uj me c t i o n t i o nS so ,l u t iD oi nl sau )st.ue i t aa lb il qet uooo bt t aa i n a )
A n a l y Ds ritsyh:Se a m ipnalc ea p i l l a r yb -o st tt ol pe p se or le ud ct oi on nt a ni on mi in n2 g a0 lP0le 0ny i c iG Ul ln ii nt s /~ ~
u n dve ar c aut6u0fm o 3r h . m L . E
A c c e pc t r ia t ne rcNieaM1 : T. 5 % A n a l y s i s iS}
e P A R T I C M UA LT A I TNTI EEN RJ E C(T7I 8O 8NM)S e : et th rsee - S a m p lS e t s a n :sdo al ru a
td in oSdna m spo ll eu t i o n ]
q u i r ef mo se r mn at ls l -i vn jo el cut mi eo n s }
P i p2e mt oLft h Se a m spo ll e u it ni toe ona oc fht w o r e}
e O T HR EE R Q U I R E I t Mm Ee NetTthSrse:e q u i ri neL ma e b en lt -s g l a s s - s t1 o 2p 5p ce-ormne iL fdcl,aa slPk sr .o ca e i -d E=y
sd e
i n( g7 )L ,a b ea lnLs da b e fl o iIr n jg e c Pt ra ob dl ue c t s . r e c it nel do d o mAestsr ai yc A n( t4 i2 bP 5 ir
) oo, tc ie dc aus }
r e ,
u s i on ngo eft hf el a ts kops e r ft oh Bre lm a n k ¥
A D D I TR IE OQ NUAILR E M E N T S D e t e r m i n a t i o n .
' P A C K A A GNSIDTN OGR AP G r eE s:ae sdr e v es c r i niP ba ec dk - C a l c ut lh pae e t er c eo nftth a le ag be e nl u e dm obfP ee r n i -
a g i an ngS dt o rR ae gq eu i r( e6 m5 e I9 n)n j,t e scP tai co kn a g i n gc ,i l l Gi Un n ii tn ts h pe o r to ifP oe nn i c iG lP loi tn a s s i u m
P a c k fa ogcr io nn sg t i t u t i o n . f o Or r aS lo l u tt ai ko en n :

C h a tnorge ea d : R e s =u (l Bt) -x F [x 1 /x ( V
Dx
1 0)0 ]

U SR PE F E RS ET NA CNE (D 1A 1R) D S B = v o l ouf0m. e0N1s o d ti h ui m

o s u l f a t e
c o n si u
n t mh Be
e l daDn ek t e r m (i m
n L
a t
) i o n
@ ( C 1N- M a y - 2 0 1 8 )
U SP Pe n i c iG lP lo i tn a sR sS i u m = v o l ouf0m. e0 N1s o d ti h ui m o s u l f a t e
c o n si u n t mh Ie
en da c t i av n
a Ttdiit or na t i o n
( m L )
F = e q u i v fa al ceatnsoccrayl c ui lnta htcee hd a p t e r
P e n i c G
i lU lni i
n to f/0 m
. 0LN1s o d i u m
t h i o s uc lof n
a ts ebuytmh e
Se td a n d a r d
P e n i cG
i Pl o
l it na f
s o
sOrir ua
S m
l
o l u t i o n s o l u t i o n )
D = n o m ic no an lc e n ot fpr ean ti ic G io ilnnlt ihn e
D E F I N I T I O N S a m spo ll eu (t Pieon ni cGiUl nl ii nt s / m L )
P e n i c iG lP lo i tn a s
f osOrir uaS mlo l u i ts aido rnmy i x to uf r e V = v o l oufSm ae m spo ll e u ut is ofe nodtr h e
P e n i c i
GPl lo i tn a sa sn o
id u
nomerm o sr u ei t ba ub fl fe e r s , I n a c t i av naTtdi it or na(t mi Lo n)
c o l od ri sl ,u ef nl ta sv o,a rnsp d,r e s e r vI tac toinv te N
as iL
. nT s A c c e pc tr ia t ne c
r9 ie
0a .
: 0 % - 1 3 0 . 0 %
9 0 .a 0n % Nd M1 T 3 0 o.ft0h l%e a b e nl u e dm obfP ee rn i -
c i l l Gi Un n i wt sh ce onn s t ai std ui tr e idcntt he ldea b e l i nPg .E R F O TR EMS AT N
S C E
e U N I F OO RFDM oI sT Ua
Y NGI(ET9 S0 5 )
I D E N T I F I C A T I O N F o sro l ip das c k i ans gi en dg l ce -o un nt ia ti Mn ee er ts s
:
e A .T H I N - CL HA RY O E RM A T O G R A P H Y t h re e q u i r e m e n t s
S o l u At :iA oc ne t 0o. n1Mec i, t ra ic ci ad ,n0 d. 1Ms o - e D E L I V E V RO ALB(UL6 ME9 8EM) e: et th rse e q u i r e m e n t s
d i cu i mt r( a2t :e 1 : 1 )
 3 2 0
P e2n i c /i Olflf ii ncM ioa n
l o g r a p h s U S4 P1

S P E C TI EF S
I T
C S ! = v o l ouf0m. e0N1s o d tih ui m o s u l f a t e
' P H
( 7 9 1 ) c o n si u nI mn ae c dt i av n
aT tdi it or nao tfti ho e
n
S a m sp o ll e
u t Ci oo nn s: ta isdt iu rt eeicn tt he ld
ea b e l i n g . S a m spo ll eu (t im oL n)
A c c e pc t r iat ne 5rc i.ea5 :- 7 . 5 EP = f a c at soc ra l c ui ln la ot de od m Ae st rs iac y
e @ W A TD EE T R E R M (I 9 N2 A1M T )e,I tO
| :hN oNd M1 T . 0 % A n t i b (
i o4 t2 i5
C ca
) sl
, c u l a t i o n s
C u = n o m ic no an l c e n ot fpr ean ti ic iG
o ilnnlt ihn e
A D D I TR IE OQNUAIL R E M E N T S S a m spo ll eu (t Pi eonni cGiUl nl i nt s / m L )
e P A C K A
A GNSIDTN OGR AP G
r eE s:ientr iv gech ot n t a i n e r s A
. c c e pc t
r ia t ne c
r9 ie
0a .
: 0 % - 1 2 0 . 0 %
e U SR P
E F E RS ET NA CNE (
D 1A 1R) D S
U SP Pe n i c iG lP lo i tn a sR sS i u m P E R F O TR EMS AT N
S C E
e D I S S O (L 7U 1T 1I )O N
M e d i pu H 6m. p
:0 h o s p
b uh fa(f tse erR ee a g eI nn t
d is c, a
t o r a
s , nS do l u t i o Sn os l u Bt ui9f
o 0nfms0
e )L
r;
A p p a 2r :a7 t5ru ps m
P e n i cG
i Pl o
l it na s
T a
s bi lu emt s T i m 6e 0:m i n
S t a n sdo al ru t d 4i o
0Pn0e :n i c iG lU lni in t fs r/ oUm mS
L P
D E F I N I T I O N P e n i c iG lP lo i tn a sR sSi niMu em d i u m
P e n i c G
i lP lo i tn a s
T as bi luc em
o tnstNa Li9T
n 0 .a 0n % N
d M T S a m spo ll e u t Ui soanef :i l t pe ro er dto iftohnse o l u t i o
1 2 0 o.ft0h l%e a b e nl u e dm obfP ee nri c iG Ul ln ii nt s . u n dt ee srt .
S o l u At :iAo1 n- i n -s 1 o l0 u0ot0fip oo nl y o x y e t h
I D E N T I F I C A T I O N ( 2 3 l a) u re yt lhi en wr a t e r
e A .T H I N - CL HA RY E O RM A T O G R A P H Y S o l u Bt: iD oi ns s 2o l0 g ov feh y d r o x hy yl da rm oicnh e
D i l u eA nc te :t 0o. n1M ec ,i t ra ic ci ad ,n0 d. 1Ms o d i u m r i di ne5 m oL fS o l u At ,ai nod na d wda tt eomr a k e
c i t r(a2t :e 1 : 1 ) 1 0 m0 L0 .
S t a n sdo al ru t d 1i 2 o n, :P0 e 0n 0 i c iG lU lni in t fs r/ om mL B u f f 2 e r6m: g / o fm s oL d hi yu dm r oa xn3id. d1me g / m
U SP Pe n i c iG lP loi tn a sR sSi i n D ui ml u e n t o fs o d ai cue mti nawt ae t e r
S a m spo ll e u t Ni oo m n :i n1 a2 l, Pl5ey0n i0 c iG Ul ln ii nt s / F e r nr i ct rs ao tl eu t Si u o ns :p2 e3g n3o dff e r rn ii ct r a t e
G r f r To am b li enD ti sl u Pe an tst .sh r oa su ug i ht a b l e i n a b o6u 0tm0 oL fw a t e a rd2,d. m 8 oL fs u l f au cr ii dc ,
ilter. s t iu rn t ti lhf ee r rn ii ct ri sad ti es s o al vd1 edm d ,oL fp o l y -
C h r o m a ts oy gs rt ae pm h i c o x y e t (h 2y 3 ll a)eu nre ye
t lh de ir l, w
u ti et w ha tt eo r
( S eC eh r o m a (t 6o 2g1T r)h a,i p n -hCLyha ry o e rm a t o - 1 0 m0 L0a, nm di x .
g r a p h y . ) i A p p a r Aa ut tu o s ma: na at li(yc F zi eg1ru)cr oe n s i os ft i n
A d s o r b 0 e. n2 t5 l :a- yomefcrm h r o m a ts o c aa p h 1i)acl i q us ia dm p (l 2ea) rp ,r o p o r p
i lgi r t iu om(n3pis), un igt -
g e ml i x t u r e a b ls ep e c t r o p eh q o ut o i wmp ieptmtehaed t
r s cf hl oe wd
A p p l i v c ao tl iu o 2m n0
pe L: c e la l sna dn a l cy as pi as b ait4 l i8 tn0ym( ,4 a) m e ao n f s
D e v e ls oo pl ivs n ey n
gs tt Te om l:u de in oe x, aa n ed , r e c o rs dp ie nc gt r o p rh eo at doaimnnedgta/ s rcoiorcm -
g l a caica el at ci ic( d9 0 : 2 5 : 4 ) p u tf eo drr a t r ea t r iae n vcadal l c u l aa n t(id5oa) nm ,a n i -
S p rr ae ya g1 :e Snt ta T r cS h f o lc do n s i os ftt hi cen o g m p oi n l leu ns tit nrtsa ht ee d
a =
5 S p rr ae ya g2 :e lno td T i nS d ei l u1 ti ne1d 0w i tw ha t e r f i g u r e .
i } A n a l y s i s
S a m p lS e t sa n :s do al r ua td in Sodna m sp o ll eu t i o n m L / m i n u t e
D d T h ne u m br ee pr rse s e n t
2) A p pt lh S ye a m spo ll eu a t into dnh Se t a n sdo al ru ttd oi o n 1 ) 0 . 4 2
mS t h p el a tp el ,ai cnaes u i t ca bh lr e o m a t co hg ar ma -
t h re e a g ae sfn otl sl o w s :
p h i c
5 b e ra , n dd e v et lh coe hp r o m a ut soitgn hrgDeae vm e, l - ( 1 H) y d r o x y l a m i n (
A i r _ _ _ 0 . 6 0
e 2 ) _ _ 0 . 3 2
P= o p is no gl vs ey ns ttu en mt ti, h l se o l vf er nohtn at s h y d r o c sh oll ou rt i od ne ;
a m o vt e h rde e - of ft o uh lre et nh ogs fet h p el a tR ee .- ( 2 A) c e tb a u ft fe e r ; e o 0 . 3 2

=) m o tv he pel aft re t o hmce h a m mb ae trr hk ,se o l v e n t ( 3 3) . N3 s u l f au cr ii dc ; a

f r o na t n,a dl lt ooa wi r - S d rp yrt . ah pyel a wt ie tS hp r a y ( 4 F) e r nr i ct rs ao tl eu t i o n . @ ) _ 0 . 3 2
r e a g1 ef no tl l bo yw S e p d rr ae ya g2 .eP ne tn i c iG l l i n
( 4 ) 0 . 6 0
a p p ea a sa r
w sh is tp eoo tn a p u r b p l a ec k g r o u n d . S p e c t r o p h o g t o m _e _ t e0 r. O 6 0
A c c e pc t r ia t ne Trc ih
eRa e;:v a lo ufte h pe e n i c iGl l i n 4 8 n0 m
s p f o tr to hm Se a m sp o ll eu ct io or nr e ts ot p ho afn tr
d s
o m
t h Se t a n s do al ur td i o n .
A S S A Y
¢ P R O C E D U R E S a m p l e r
S t a n sdo al ru d
t Pi ro en p:a asd ri er e icn lt oe d o mA es t- r i c (1) 0 . 4 2 R a t 4e 0:
s a y A n t (i 4b2 i5So)tt,ai ncPdsr ae rp da rua st iiUno S
gn P
, A i r 0 . 6 0 p ehro u r
A b s o r b a n c e@ ) 0 . 3 2
P e n i c G
i lP loi tn a sR Ss .i u m m o n i t o r
S a m spo ll e u t Ni oo m
n :i n2 a0 lP0 l
e 0ny i c iG Ul ln ii nt s /
2 0 . 3 2
m L p, r e p aasfro el dl Po lw a
s N.c L
e5TT a b li ena th si g h - a
s p eg el adbs ls e nj adcreorn t aa im nei an sg vu or le ud m e (2) 0 . 3 2
o fB u f Bf .e 1ra, n bdl ef no 4
d
r + m 1i n
D i
. l au st ue i t a b l e
a l i qw ui ot B tuh f Bf .e 1r . (4) 0 . 6 0
A n a l y Ps ri o s :ca esd ei dr e icn lt oe d o m Ae st s
r ia cy A n - B l a n k2
t i b i o( t 4i 2c s5
P r) o, c e ud su irg nel g
,a s s - s t1 o 2p 5p e- rme Ld , 4 8 n0 m
c o n if cl aa sl k s .
C a l c ut lh p ae te er c eo nftth a le ag be enl ue dm obfP ee r
n i - S p e c t r o p h o t o m e t e r
c i l l Gi Un n ii tnts h pe o r to ifT oa nb lt ea tk se n : F i g 1u r e
R e s =
u (l Bt / ) x ( F / x2() 1 / xC 1
u )0 0 A n a l y Ws ii t s :hh se a m lpi lnpee u m pw ai tnt e ghr ,e
o t hl ei nr p
e su m p t hie rni ergs p e rc et ai gv ea e nnttdsh ,e
B = v o l ouf0m. e0N1s o d ti h ui m
o s u l f a t e s p e c t r o ps he atot4t 8o n0 m mes ,tt ae nr d ta h r se
dy is z- e
c o n si unB ml eaDndek t e r m (i m
n a
L t) i o n t eu mn t ai sl t e aa b d ys o rb ba as n e hlc aiebs
n ee ee sn t a b
l i s hTe rd a. n ps of re tr oi fto hn Se
s t a n sdo al ru a td in odn
U S4 P1 O f f i cM ioa n
l o g/ Pr ean p
i chi3sl 2l 0
i n3

t h Se a m sp o ll eu tt ois oa n m pc lu p e asr n,pdl ai cn te h e S p rr ae ya g1 :e Snt ta T r cS h

s a m p Sl tea t rr h
.tse a m p al n e crd o, n dd u e tc et r m i n a t Si po r nr a
se ya g2 :e lno td T i nSd ei l u1 ti en 1d 0 w i t w h a t e r
o ft h Se t a n s do al ru atd intodhn Se a m spo ll ue tt iy op ni ,- S p rr ae ya g3 :e 5n 0m t g /o fm p -L d i m e t h y l a m i n
c a l al tt
y h rea to ef4 0 p e h r,u s ia nr ga t oi foa b o2 u : 1t a l d e i hn my ed te h a n o l
f o sr a m ap n l wd
e a ts ihm e . A n a l y s i s
C a l c ut lh pae e t er c eo nftth ale ag be e al m e do oufP ne t n i - S a m p lS e t sa n :sdo al ru 1td, iS ot na n s do al r u 2td,ia onn d
c i l lGi Un n idtiss s o l v e d : S a m spo ll eu t i o n
P r o ca esd ei dr e icntt he ce d h a p Dt ee rv .et lh co e hpr o -
R e s u= (l At y /xAC ssx) V (x 1 / xL 1) 0 0 m a t ou gn trti a h
l semo l vh ea nms to vt e h rd e e - of fo u r t h s
t h le e n og ft h pel a tRe e. m toh p veleaft re t o hm e
= a b s o ro bft ah n Se c
a em spo ll eu t i o n c h a m mb ae tr r hk
,se o l vf e r on nat t n,a dl lt ooa wi r - d r y .
A s = a b s o ro bftah n Se c
t ea n sdo al ru td i o n E x a mt i h penl ea ut ne ds eh or ra tn -ldo n g - w a v e l e n g
G s c o n c e n ot fr U aSPtPe in oi cn iG lP lo i tn a sR sS i u m U lVi g hn to, t ti hnpego s i to ift ohnsesp o tS sp .rt ah ye
i nt h Se t a n sdo al ru (tdPi eon ni cGiU lnl ii tn s / p l a wt ie tS hp rr ae ya g7 ef no tl l bo y wS ep drr ae ya g 2 .e n t
m L ) P e n i c iG al lp i pn ea asa r
w sh is tp eo o tna p u i av ce k -
Vv = v o l oufMm ee d i9 u0 m m0,L g r o uS np drt.ah y leo c a ot ft i oh sne p ov ti ss u a wl ii tz eh d
L = l a b ce ll a( iP me n i cGiU lnl ii tn s / T a b l e t ) U lV i g wh it tS hp rr ae ya g3 .eP nr to c aa pi pn eea a sa r s
T o l e r a Nn Lc 7T
e 0
s( :%
Q o) ft h le a b eal m
e do ouf n t b r i yg eh tl sl po owt .
P e n i c iG Ul ln iiinstd si s s o l v e d . A c c e pc t r ia t ne T rc iheaR e:;v a lo ufte h pee n i c G i l l i n
e U N I F OO RFDM OI S T UYA NGI(ET9 S0 5 M) e
: te ht e s p of tr t o hmSe a m spo ll eu ct o i or nr e ts otp ho afn trd o s m
r e q u i r e m e n t s S t a n s do al r u 1td. iT ohRn erv a lo ufte h pe r o c sa pi on te
f r to hmSe a m sp o ll eu ct io or nr e ts ot p ho afn trd Sos tm a n -
A D D I TR IE OQ NUAILR E M E N T S d a sr od l u 2t .i o n
e P A C K A A GNSIDTN OGR AP G r eE s:ientr iv gechot n t a i n e r s .
' U SR PE F E RS ET NA CNE (D 1A 1R) D S A S S A Y
U SP P
e n i c iG lP lo i tn a sR sS i u m ¢ P R O C E D U R E
S t a n sdo al ru t d Pi ro en p:a asd ri er e icn lt oe d o mA es t- r i c
s a y A n t (i4b2 i5So)tt,ai ncPdsr ae rp da rua st iiUno S gn ,P
P e n i c Gi lP loi tn a sR Ss .i u m
S a m spo ll e u t Pi ro en p:a asd ri er e icn lt oe d o mA es t- r i c
P e n i cG i Pl r l i o nc a i n e s a y A n t (i 4b2iA 5 o)st,sPi arc yes p a r ea xt ci doe inps,ts o l v e
1 0m 0
g o fP e n i c G
i lP lr ion c ian2i .n m
0e oL fm e t h a n o l ,
C H , a nd di l wu it tBeuh f Bf .e (1r s eA en t i b i o t i cA s - M i c r o

u eo e
H O . oO C c

p
s a y( s
8 1t) oo) b t aa si on l u ct oi n o nt a 2i 0
n Pi0 en0n gi c i l -
0 l i nG U n i t s / m L .
a
i a s A n a l y Ps ii ps2e:mt oLft h Se a m spo ll e u it ni toe ona oc fh
t wg ol a s s - s t1 o 2p 5p ce-ormne iLfd cl,aa slUk ss .oe no ef
t h et sope e r ft oh Bre lm aD ne kt e r m iP nra ot cai e .C o
so en d
5 8 8 . 7 2 d i r e icnlt oe d o mAestsrai yc A n( t4 i2 b5 i) o, t i c S ss )
a )
C i s H i s- NC 2iO 30 H4 82- SH
0 N2 20 0 2
P r o c e d u r e .
C r 6 H i - sC Ni 23 0H 42 S0 N 2 0 2 5 7 0 . 7 1 C a l c ut lh pae o
t t
e e innPce yn ,i c iG lU ln i in t so f/t mh ge , E
4 - T h i a - 1 - a z a b i c y c l o [a 3c .i 32d ,. , 30- ]d hi e- p tP ae nn ie c-iG2lP -lr iconac r
ta abi konexeny :l i c 5
m e t h y l - 7 - o x o - 6 - [ (2 p5 h - (e 2n 0y ,l 5a 0c, e, t6 yB l) )- a
, m i n o - ] , F
c o m p w oi u t
2 -hn (dd i e t h y l4 a- mai mn io n) e o tb he ynl z o a t Re e s = u ( l tB) -x F x 1 / ( x VD x )1 0 0 }
( 1 : m1 o) n o h y d r a t e ;
r= o: }
( 2 5 , 5 R , 6 R ) - 3 , 3 - D i m e t h y l - 7 - o xBo - 6= -v (o 2l-oupf0mh. ee0Nn1sy ol da ti c heui tmo as mu li fd ao t) e-b i e}
4 - t h i a - 1 - a z a b i c y c l o [ a3c.ic2do . 0m ]-h e p t a n ec - o2 n - c siaun tr mhb Bee
o lxdayDnlekit ce r m (i mn L a t) i oa s n
p o uw ni t d
2 h- ( d i e t h y lp a- mai mn io n ) eo(tb1he: y1nl)z oI a t e= v o l ouf0m. e0N1s o d ti h ui mo s u l f a t e
m o n o h [y 6d 1 r3 a0 t - 6e 4 - 9 ] . c o n si unt mh /e en da c t i av n a Ttdiit or na t i o n
A n h y d [ 5r 4o- u3 s5 - 3 ] . ( m L )
F = e q u i v fa al ceatnsoccrayl c ui ln Pa rt oe cd e i nd u r e
D E F I N I T I O N t h ce h a p( Pt ee n ri cGiUl nl ii nt osf0/. m0N L1
P e n i c iG lP lr ion c ha aiasnp eo t eo n fN cLy 9T0P 0 e n i c G i l l i n s o d ti h ui mo s uc lof n a ts ebuytmh ee d
U n i t as n/ N dm gM1 T 0 P5 e 0n i c iG lU lni in t s / m g . S t a n sdo al r u td i o n )
D = n o m ic no an l c e n ot fPr ean ti ic G io ilnnlt ihn e
I D E N T I F I C A T I O N S a m spo ll eu (t Pieon ni cGiUl nl ii nt s / m L )
e A .T H I N - CL HA R Y O E RM A T O G R A P H Y Vv = v o l ouftm h eSe a m spo ll e u ut is ofe nodtr h e
S o l u At :iA oc ne t 0o. n1M ec ,i t ra ic ci ad ,n0 d. 1Ms o - I n a c t i av na Ttdi it or na( t mi Lo n)
d i cu i mt r( a2t:e 1 : 1 ) A c c e pc tr ia t ne c 9
r ie 0a 0: - P 1e n0 i 5c iG 0 lU lni in t s / m g
S t a n sd o al ru 1dt: iP or ne pa s a or le u ct oi n o nt a ti hn ei n g
e q u i vo af1l 2 e n, Pt0e 0n i0 c Gi lU ln iin t sf /r mo ULmS, P S P E C TI EF S I T C S
P e n i c iG lP loi tn a sR sSi i n S uo ml u At .i o n
S t a n sdo al ru 2dt: i5 om n g / o fm U S LP Pr o c Ha yi dn r e o -
c h l o Rr S i ndS eo l u A t i o n C h a tn orge ea d :
S a m spo ll e u t Ni oo n m :i n1 a2 l, Pl0ey0n i0 c iG Ul ln ii nt s /
m fL r Po emn i c G i lP lr ion c ianSi on leu At i o n ' C O N TO E FP EN NTI C G I AL LNPIDRN O C A I N E
C h r o m a t soy gs rt ae pm h i c S o l u At :iP oh no s pa hc oid rdi il c u1 ti en 1d 0w i tw ha t e r
( S eC eh r o m a (t 6o 2g1 T r)h a,i pn -hCLyha ry oe r m a t o - M o b pi hl ae sD ei : s s 1o l 4g ov fem o n o b p o a ts ai sc s i u m
g r a p h y . ) h o s p ah na6 dt. ge 5 o ft e t r a b u t hy yl da rmo mx o- n i
M o dT eL :
C i d e4, 0i n%
w a t ie nr7 ,0 m0 oL fw a t Ae dr j
. wu is 1tt h
N
A d s o r b 0 e. n2 t5 l :a-yome fcrm h r o m a ts oi lgi r c aa p hpio ct a sh sy id ur mot oxa ip dHo ef7 . 0a, n ddi l uwt ie t h
g e ml i x t u r e w a tt eo1r 0 m0 L0M. i 5x 0 m0 oL ft h is so l u t2i 5o mn0 ,L
A p p l i c v ao tl iu o 2mn0ue L: o fa c e t o n ia tnr2di 5lme0 o,Lfw a t Ae d r j. wu is 1tt h
N
D e v e ls oo pl ivs ney g ns tt Te om l:u de in oe x, aa n ed , p o t a sh sy id ur mo r xS io ld ueAt ti oao p
n H o f7 . +5
g l a caica el at ic ci( d9 0 : 2 5 : 4 ) 0 . 0 a5 n, p da st sh r oa su ug iht fai bl tl eer .
 3 2 0
P e4n i c /i Olflfii nM
c ioa n
l o g r a p h s U S4 P1

S t a n sdo al r
u t
d0i .om
8n g
: /
o fm
U S
LP P
e n i c i
GPl lo i-n f o M
r e m bF r i lat rni aseut is oe npd e, r ft oh pre rm o c e a sd u
t a s sR iSau nm0d. 5m 4g /o fm
U SLP Pr o c Ha yi dn re o - d i r e icn tt he cd
e h a pw ti ett rh f eo l l o e w ix nc ge p t i o n
c h l o Rr Si ndM eo b pi h l a e s e U sF el uA it odw h ih ca bh s e e a nd ds e u fdf is ct ie er ni tl e
S y s stu ei m t a bs io ll iu tt y2i .om 4n g: / o fm
U SLP Pe n i c i l - p e n i c i tl oli in na ac st tei hvp eae tn ei c Gi ,lal nis ndw i tr hl e
i n V P o t a sR sSi in M uo mb pi hl ae s M eit.xh ree s u l t a n t v e s us ne tlsi ol l u i ts ci o o nm p bl e ef t fo ierl et e r i n g .
s o l u wt ii t
oS nht a n s do al r u (td1i :o 3n ) . ' C R Y S T A (L 6L 9I5N M) Ie: TetYth rse e q u i r e m e n t s
S a m spo ll e u t Ti ro an n:7s fm0 ge or fP e n i c iG lP lr io n- ' P H ( 7 9 1 )
c a it noae5 0 -v m o lL u mf el at sA rk i.d 3cd0m oLfM o b i l e S a m spo ll eu t Ai so an t: u rs ao tl eu ctd oi on nt a ai bn oi u n g
p h a s oe n, i tc oda it se s oal nvd edi ,l uw ti etM ho b pi h l ae s e 3 0 m0 g /o fPm e L n i c iG lP lr ion c ianwi a n t e e r
t ov o l u m e . A c c e pc t r ia t ne 5rc i.ea0 :- 7 . 5
C h r o m a t soy gs rt ae pm h i c ' W A TD E E TR E R M (I 9 N2 A1M T )e,ItO I :hN 2o . d 8 % - 4 . 2
( S eC eh r o m a t ( 6o 2g1Sr)y a,s pStuhei ytm a b i l i t y . )
M o d Le C: A D D I TR IE OQ NU AI LR E M E N T S
D e t e c Ut o V
2 r3 :n5 m ' P A C K A A GNSIDTN OGR AW Gh Eei :tirs ie n t e fno dur e sie
dn
C o l u 3m .n 9: x-3 m0 m- 1 c m0 ;-p 4a m c kL i
1 n g p r e p ai r n ji en cgdt o a bs lfaeog r epm rs e, s ae sdr iv re e icn t e
F l ro awt 1e :m L / m i n P a c k aa gnSidtn og rR ae gq eu i r( e6 m5 Ie 9 n)n j,te scP tai co kn -
I n j e cv toi lo un 1m 0 ue L: a g i n ga , f o cr o n s t i t u t i o n .
S y s stu ei m t a b i l i t y e L A B E L WI h N Gei t:irs ie n t e fno dur e si edn p r e p ai nr - i n g
S a m p lS e t s a n
: sdo al r u a
td in Sodny s stu ei m t a b i l i t y j e c t da bo ls efa ogr etm h s le
,a b se tl a t eh isatits s t e roi rl e
s o l u t i o n m u bs es
t u b j et c of tu er d tp hr eor c e ds us ritinhngpegr e p -
[ N o t re e l aT rt he i ve
t ee nt ti imfoeonpsr r o c ap ie nn ei ,- a r a to ifio nn j e c dt o a bs lfaeogr em s .
c i l lGi ,na np de n i c V
i al rl ae
i nb o0 u
. 4t
1 ,. 0a, n1 d. 5 ,
r e s p e c t i v e l y . ] C h a tnorge ea d :
S u i t a br iel qi ut yi r e m e n t s
R e s o l u Nt Li2 o T. nb0 :e t wp ee n ei n c iG la lnip d
ne n i c i ¢l U
- SR P E F E RS ET NA CNE ( D 1A 1R) D S
l i Vn ,S y s stu ei mt a sb io ll iu tt yi o n
t e ( C I1 N- M a y - 2 0 1 8 )
R e l a st t i va en dd eav ri da tNi M o 3nT.: f0 o %
pr e n i c i l - U SP P e n i c iG lP loi tn a sR sS i u m
l i nG p o t a s Ss ti au nmsd,o al r u td i o n U SP P e n i cV i Pl lo itn a sR sS i u m
A n a l y s i s U SP Pr o c Ha yi dn re o c Rh Sl o r i d e
S a m p lS et sa : n s do al ru atd in S
odna m sp o ll eu t i o n
C a l c ut lh pae e
t er c eP ntpt ae g n eG (eC i e H i s N 2 0 , S )
i nt h pe o r to ifP oe nn i c iGlP lr ion c ta ai kne en :
R e s u= (l r tu / xr (s C) s /xCG us )
P e n i cG
i Pl r
l io nc Ia n
i n
t er a m a m
t u =p e r
a e
k s p oofpne sn e
i c G
i f
l lri ton hmSe a m p l e
s o l u t i o n I n f u s i o n
P a

p e ar ek s p oofpne sn ei c G i fl lri t
on hm e
"

v A
co S t a n sdo al ru td i o n » P e n i c iG lP lr ion c Ia i n nt er a mI a n fm u m
i ss a
i or
[ o s
3 C s = c o n c e n ot frU aSPtP i lP loi tn a sR sS i u ams u s p eo nfPse in i
e in oi cn G o cn iG lP lr ion c i anais nu ei t a b
dD i nt h Se t a n sdo al ru (td imo gn / m L ) y e q r oi i avl be hl ieIct lm e a.c yo n toa nio enrm o r
fo} G u = c o n c e n ot fPr ean ti ic iG
o lPnlr ion c ianti hn ee b u f f de ir s ,p e rp sr ae ns te sr ,vaa ntt d ihv ie cs k, e
& S a m spo ll eu t i(o mn g c/e 1rm-a 4Lp r)- 2e0 1 7 )
G s = c o n to e
fp en nt i c G
i il nlUi S
nP Pe n i c iG l l i n a g e nI ttc so .n t na oil tne ssts h a
9 n
0 p
. e0 r ca enn dt
= P o t a sR sS( i%u )m n o mt o tr he a1 n 1 5p .e 0r co eft nh ltea b e l e d
a C a l c ut lh pae e n N 2 Oa2 m
t er c eo nfpt raogc e(a Ci in 3e H 2i 0 ) o oufp n e nti c Gi .
l l i n
rm) t h pe o r to ifP oe nn i c G
i lP lr ion c ta ai kne en :
| P a c k a a gnsidtn ogr a g e i nwP erl el s- c de l
ir osvsp eeo ds
R e s u= (l r tu / xr (s C) s /xC( uM )r i /x M1 ,02 0) b l se y r i n g e s .
L a b e l i int tg oi n Ld ai tcb haeiattiltes f o vr e t e r ui sn ea r y
r u =p e a r ek s p oofpn rs oe c fa ri t on hemSe a m p l e o n l y .
s o l u t i o n
r s =p e r a ek s p oo fpn rs oe c fa ri t o mSe t a n d a rUd SR Pe f e r
n he s e t na c n ed
( a 1 r 1 d ) s
s o l u t i o n U SP P e n i c iG lP lo i tn a sR sS i u m
C s = c o n c e n ot fr U aS PtPri oo cn Ha yi dn re o c h l o Ur SiP d Pe ne i c iG lP lr ion c Ra Si n e
R Si nt h Se t a n sdo al ru ( td imo gn / m L ) I d e n t i f i c a at pio or nto ifi tTo,en r qau ni svtfaoel r e n t
G u = c o n c eon Pf te nr i ac iG
t lPilr ioon n
c i an ti hn ee a b o1 u0t0 ,P e0 n0i 0c iG Ul nl i nt soa ,t e st tu b ae d , 2d 5m L
S a m spo ll eu t i(o mn g / r imr- sLp r)- 2e0 1 7 ) o fm e t h aa nnsodhl a, A k el .lt oos we p a ra antudes t,e h e
M n = m o l e cw ue li oag frph rto c a2 i3n 6e ., 3 2 m e t h laa nyao estrl ht ee s to l u tP iro en pa. aS rt ea n sdo al ru d -
M z = m o l e cw ue li oagfrph rt o c ha yi dn re o c h l o rt ii doo enfU, SP P e n i c iG lP lr ion c Ra Sii nnm ee t h ca onn ot la i n
2 7 2 . 7 8 a b o4 u. m t
5 p g e mr L A. p ps le y p a r 1a 0u t eL o lfey a sc oh l u -
A c c e pc t r ia t ne Sc r ieeTae:a b1 l. e t i ot noa t h i n - cl ha yr eo rm a t pol g a (t res aeC peh hr io cm a t
r a p{ h6 y2 c 1 )o )a wt ie t ad 0
h . 2 5l a-y ome frcm h r o m a t o
T a b1 l e
g r a ps ih l iig cceaml i x t Au lr l et .oh swe p ot todsr ya , n d
d e v et lh coe h p r o m ai nt a so ogl rvs a ey nmstct oe nms i os ft i
P e n i c iG l l i n 5 1 . 0 % - 5 9 . 6 a % m i x to u fb ru et a in so ol p, ra ol pc yo ahl c o le ,t ao n wed a, t e r
P r o c a i n e 3 7 . 5 % - 4 3 . 0 ( % 4 : 4 :u 2 n t: ti2 hl)seo l vf er nohtnatms o va eb d ot uh r t e e -
f o u ro tfthhsle e n og ft h p el a tRe e. m toh pvelea ft re o m
e B A C T EE RN ID AO LTT OE X(S I 8T 5N )WS: h et r h leea b se tl a t e s t h de e v e l co hp ai mn mbg a e tr h,kse o l vf er on nt a t n,adl l o w
t h a P et n i c iG lP lr i on ci as s it n e reoi rlt eh iat tm u b s est u b - t h se o l vt eoen vt a p o Er xa pt to e h.speel attoie o d vi an p e o r
j e c t oef du r tp hr eo rc e ds us ri tin hngpegr e p a or fa i nt -i oi na c l o sc eh da m f obar eb ro1 u5m ti n u at n e lsdo ,c t a th ee
J e c t da bo ls efa ogr eimt cs o, n t a NiM n0 s.T0U1 S EPn d o - s p o t sh R:erv a l u a encsdo l oo frt sh te wp or i n cs ip po atl s
t o x Ui nn i t P/e 1n i0 c0 iG Ul nl i nt s . o b t a fi rn toe hm dtee ss to l u ct io or nr et sotp hooonsbde-
' S T E R IT LE IS(TT7Y S1 )W : h et r h leea b se tl a t eh saP et n i c i l tl ai ni f n er dto hmSe t a n sdo al ru d t i o n .
G P r o ci as s it en reii t lme e, et th rse e q u i r I ef tm het ene st ts .
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W a tD e e rt e r m iM ne at| th ( i9o 2od1n)n ,:o mt o tr he a n u c tt ob eE x a m ie nx ec d te o,up stFel u Ti hd i o g l yMc eo - l l a t e

1 . 4 2% 0m , oL fa m i x to uft ro el ua ennmdee t h (a7 n: 3o )l d i u a n m Sd o y b e a nD -i C g eM
a sseted icino un m t a ai nn i n g
b e iunsgie nd p l ao cfme e t h i an tnhtoeilt r av te is os ne l . a m o oufs nt etr ip le en i c i sl lu if nf ai tsc eoi en na tc t ti hv ea t e
A s s a y a Psd ri or ec ucet n eedAd
den rt i b i o t i c A s - M pie cn ri oc G
ib ilinlea ailcn vh e s s e l .
s a y( 8s 1 e) ,x p e lt lh cie n o gn t oef1 n sty sr io nfIg ne t r a m aPm (H-7 9 1 )b :e t w5 e . a0e nn7 d. 5 .
m a Ir nyf u is ni tao ohn i g h -g sl pa be s lse ed nj ad cre or n t a i nP ie nn gi cGia np dr o c ca oi nn te(e wnh t i eti ssrp er e p a r e d
4 9 9 m. oL 0fB u f Bf .e a1r n 1 d. m0 oL fp o l y s 8o 0r a,b na d t e f r po emn i c iG lp lr ion c aa n ii sn
dl ea b ef lo ver ed t e r ui sn ea r y
b l e n fdo i3r tn og 5 m i n u At le lst o .os wt af no a dr b o u t o n l y ) a Dp i o rl tuo it
fi toe
,en q u i vt aoal be o n3tu0t0 , 0 0 0
1 0m i n u at n ed sdi ,l au tna ec c u r ma et ea ls yvu or leoudf m e P e n i c iG Ul ln i ntw si ,tw ha tt eoo rb t aa vi o n l ouf1m 0me L ,
t h ae q u ep oh uaq ssu e a n t i ta a nts dit ve eplwwyii t Bs uhef Bf .e r c e n t r ia fnurdgee m, ao nvd dei s ct ah rse du p e r nR ae ts au ns t- .
1 t oo b t aa Ti ensD ti l u ht ia ov nai cn og n c e n a t rs as tu imo ep n de t
n hdse e d i i mn 1e 0m n toLfw a t ce er n, t r ia fnu r deg -e ,
t ob ee q ut aotl h me e d di oa lsneev oe flt h Se t a n d a r d . m o a v ned di s ct ah rsedu p e r nDa rtt yah s ne t
e .d ii mna e n t
v a c du e us m i ccc oa nt to ars ii ln igicen afl go 1r 8h o ua rta s
t e m p e n r o aet xu cr ee e2 5d i T .nhdger imeadt e m r ie ae lt s
t h re e q u i ro eft mhteeensf tto Psre n i c iG la l nip ndr o c a i n e
c o n t ue n tdP se e nri c iG Pl lr io nc a[ i Nn Oe .T e aRp eo sr et i r ov ne
P e n i cG i Pl rl io nc Ian ij ne ec t a b l eo ft h der imeadt e fr oitrahtlee sf to Cr r y s t a l l i n i t y . ]
S u s p e n s i o n O t hr ee rq u i r e m me eetnth t rse es q uIi tru enm d/e nen-rt s
j e c t ai onI n dsm p l aD nr tPu erg d o d ( u1 c) .t s
» P e n i c iG lP lr ion c Ia nij ne ec St ua bs lp eei sna s i Ao s
n s a y
s t e r si u
l es p eo nfP se in io cnGi l,Pl ri on c oar i, n e S t a n pd ra er pd a r a t Ui SoP P
en n i Uc s
G
i lPi loni tgn a s s i u m
w h el ra b e ef lo ver ed t e r ui snoean rl s t e r i l e Rm Se p,t rAr sei spca aasd yri er Ae nfc( ott4Sri2etb5dai) no.pdtraie rcp sda ru an tdlieoo rdno -
o yfy
p e n i c iG p l lr io nc ai niW n ae t, f e
o Irrn j e cat ni d o n A s sp ar ye p a 1r (a wt ih ioetinsrr ee p r e as seb en i tni egnad
c o n t oa n i onerm
s o sr u ei t ba ub fl fed ei rs sp e r s sa inn tg sl ,ec -o dno ts ae i n e ra l )lo ft Wh wie it th hd dr raaww a b l e
o rs u s p ea ng de inan t nsga d
,s u i t ap br le se e r v ac -o n t oeftn htIesn j e c St ua sb lp ee nu s ia nos gnu i, t ha by lp eo -
t i v I et .m ac yo n tp ar io nc ha yi dn re o c ihnal o rd ie dr e nm ei ec a d l nsedy r i a n gndedi, l q u tu ea n t i tw ai tt i v e l y
c o n c e n nt oret a xt ci eo e2n .dp0ie nr gc ae nn td , B u f Bf .e t1r oo b t aa si on l u ct oi on nt a ai bn oi2 un0gtP0 e0n i -
m ac yo n toa nio enrm o sr u ei t sa tba lb ei lI it z e rcosift l l Gi Un n i pt esmr L P.i p2e mt oL ft h is so l u it ni teo ona c h
. wg ol a s s - s t1 o 2p 5p ce-ormne iLfd cl,aa sl k s .
c o n t na oil tne ssts h a9 n0 p . e0 r ca ennndto mt o r e A s s pa rye p a r2 a( twi ho tne hrleea b se tl a t eh qse u a n t i t y
t h a1 n1 5p .e 0r co eft nh ltea b eal me do ouf n t o fp e n i c Gi pl lr ion c ianai gn iev v e n o l oufImn ej e c St ua sb -l e
p e n i c Gi ,lt lhilena b eal me do bu e nintnolgte s s p e n s i o n a) na cD ciu lr m ua t et ee
a ls y vu or leoudfImn e j e c t -
t h a3 n0 0 ,P e0 n 0i 0 c iG lU ln ii p
nt esm
r oL rp e r a b lS eu s p eq nu sa in o t in tw ai tBtiuhvf eBf l.et7yr oo b t aa i n
c o n t a i n e r . s o l u ct oi on nt a ai bn oi2 u n0g tP0e 0n i c iG lU ln ii p nt esmr L .
P i p2e .tm 0 oLft h is so l u it ni teo ona oc fh t wg ol a s s - s t oi p= -
P a c k a a gnsidtn ogr a g e i nsP irn eg sl e m duv loets ie p- e r e1 d2 , 5 c-o mn iLf cl aa sl k s .
o r-r a )
p l e - cd oo nstea ip nr ee rf se o,rfTa b y | lpoyerT y I pl l gel a is ns , P r o c e d u ra esd i Pr re fcootcPrere doe cd eu dn udlr eoe dr o -u v
ar e f r i g e r a t o r . m e t Ar si sc a y A n( t4 i2 bC5 ia) ol. tc iut clh qsaeut ae n ti ni t y , =
L a b e l i inti sgi n W t ehfne o dvrree e td e r ui snoean rl ty h, e P e n i c iG Ul ln ii nit nst ,h ce o n t a oi rin n et rh p,e o r to ifI on n- f=o ])
l a b se osl t a t e s . j e c t Sa u b ls ep et na sk ieb onyt n
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i n w h iL ics th h le a b eq lu eadn itnPi etn yi c iG Ul ln ii nit nst ,h e a
u s RPe f e r s et n a c n ed( a1 r 1 d ) s c o n t a oi rin net rh v,e o l oufImn ej e c St ua bs lp eet na sk ie on n ;a
@ ( C IN- M a y - 2 0 1 8 )
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U SP Pe n i c iG lP loi tn a sR sS i u m A s sp ar ye p a 1r , ao tro ifA osn sp ar ye p a 2r ,ao tnti hobena s i s
U S P Pr o c Ha yi dn re o c Rh Sl o r i d e o ft h le a b eq lu ea dn itn ti htceyo n t a oi rinn t e rh p,e o r to if o n
o eS u s p et na sk ire eon sn , p e c ta invt deh ele yx ,t oe fn t
I d e n t i f ir ce as tp tiooo tn hnd/es dI e nt t i ft ie csuat nt id oen r i l u t i o n .
P e n i c iG lP lr io nc a i n e .
C r y s t a l(l 6i 9n(5iw)thyi eti srp er e p fa rr poe emdn i c G i l l i n
p r o c a an dii snl ea b ef lo ver ed t e r ui snoean rl y )m :e et th se
r e q u i r te hmdeer ni rteesds, ip dru ee p aasdr ie rde icntt he d e
t e sf to Pre n c iGl al inpndr o c ca oi nn tebe en i tu sns ge d .
B a c t eE rn ida ol tT oe x( s i 8t 5n )c s o In t na oi mtn os r e
P e n i cG i Pl r l io nc fa oi Irn je e c t a b l e
t h a 0 .n 0U1 S E Pn d o tU o n ix
p tei 1rn 0P 0e n i c iG Ul ln ii nt s . S u s p e n s i o n
S t e r iT le is(t t7 y s1 )m e Iettth rse e q u i rwe hm e e nn t s
t e s at sed di r e fc otM r e ed m bF r i lat rn ua etn i doT nee sfr ot Sr t e - » P e n i c G i lP lr ion c fao iIr n je e c St ua bs lp ee n s i o
r i l io t fty h Pe r o dt uob ceEt x a m ie nx ec dte o,up sta ep o r t i o ins a s t e r mi i l ex to Puf er ne i c G i Pl rl oi cn aa n i nd e
o fs p e c fi rm e
oe a
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e i
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i r
vt ao3l 0 0 t, 0 0 0o
e n no erm o sr u ei t ba ub fl fede i
r s ,p e ros rsa un st -s ,
P e n i c G i Ul nl i nti sn ,s to efta hdme i n iv m o l u su mp me e
c i f i pe de n da i g en ngat nsp d,r e s e r vI tac to inv te as i. n s
i n t h Te a b2 l, Me i n iQ u m au nm ttobi et U ys fe o dEr a cM he -
d i u am n,t dou s Fel u Ai td ow h ih ca bhs e e a n d ds e n o
u fdf i c i e n t l t
e st s h a
9 n0 p . e 0 r ca ennndto mt o tr he a n
ei hv pea etn ei c iGlal intn dos w i r l 1 1 5p .e 0r co e
s t e rpi el ne i c i tl oli in na ac st t ft nh lte a b eal m e do oufp ne nti c i l l i n
t h vee s us ne tlsi ol l u i ts ci oo nm p l b eef t fo irele t e I rf ti nh ge. G ,t h le a b eal me do bu e nintnolgte sts h a n
I n j e c St ua s b lp ee cnosn it o laenic ni tsuh siFenl ,uDi.Idf i t c o n - 3 0 0 ,P e0 n0i 0 c iG lU ln ii p
nt escr o n t ao irp ne er r
t a i cn as r b o x y m e t sh oy dl ica uedsmldu, lf uf li scot ise ereni tl e m oL fc o n s t i S t u u
s t
p ee dn s i o n .
c a r b o x y m e tt hoFyl lu Aci oderFll lu u Di tld oda is se s to hl ve e
c a r b o x y m e t sh oy d l bc
i eeu flmfoli rul etl eoI rf isit nd
e go. e
n os t P a c k a a gnsidtno gr a g e i nsP ir n eg sl e o -rr
m duv olest i
e -
d i s s co ol m v ep l ep tr eo lcaye sd,ei dr e fc otDrei dr Ie nc ot c u l a p- l e - cd oo nst ea ip nr ee rf seo,rfTa y b | lpoyerT y I pl l gel a s s .
t i oo nft h Ce u l tMu er ed ui nu dTm ee sfr otSrt e r io lfti ht Pye r o d -
 3 2 P0 e6n i c /i Ol flfii ncM ioa n
l o g r a p h s U S4 P1

C h a tnorge ea d : t h a1 n2 0p .e 0r co eft nh lte a b eal me o

d uo fn t s
P e n i c iG U
l ln ii nat nso dfd i h y d r o s t r e p
U SR Pe f e r
s e
t n
a c
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d
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) s ( C a t H a i N 7 O i 2 ) .
t e
( C 4N1 - M a y - 2 0 1 8 )
U SP Pe n i c iG lP loi tn a sR sS i u m P a c k a a gnsidtno gr a g e i nwP erl el s - cedlr io svs pe od s,
U S P Pr o c Ha yi dn re o c Rh Sl o r i d e b l se y r i n g e s .
I d e n t i f i rc ea s t pi tootn hd/eIs d e\ nt t i ft ie csuat n t id o en rL a b e l i int tg oi n Ld ai tcb haeiattiltes i n t e fno vdr ee t de r i
P e n i c iG lP lr ion c a i n e . n a r u ys oen l y .
p (H 7 9 1 )b :e t w5 e . a0e nn7 d. 5w, h ce o nn s t ai std iu -t e dU SR Pe f e r s e t n a c n e d
( a 1 r 1 d ) s
r e c it net dh lea b e l i n g . U S D Pi h y d r o s tS ru le fpRatSt oe m y c i n
W a tD e e t r e r m iM ne at| th( i 9o 2od1n)b ,:e t w2 e. e8n % U SP P e n i c G i lP loi tn a sR sS i u m
a n 4d . 2 % . U SP P e n i c iG lP lr ion c Ra Si n e
O t hr ee rq u i r e mme eetnth t rse es q uIi trf eo Brm aec nt te I s- d e n t i f i c a t i o n
r i ael n d o t ao nxS d it en r siul n i tdPy ee nri c iG lP lr ion c Ian ij ne ce t - A :I t r e s p tootn hd/es d e n t i ft ie csuat nt idPoeennri c iG l l i n
a b lS eu s p e \nt sm ie oean tl, ss t oh re e q u i ru enm dIe e nnjrte cs - P r o c Ia in nt er a mI na f mu m s ia o rn .y
t i o an snI dm p l aD nr tPu erg d o d (u 1ca ) t nsUdn i f oorf m i t y B :P l aa cp eo r to ifi to,en q u i vt aoal be n o1tu 0tm0 og f
D o s Ua ngi(et9s 0 5 ) . d i h y d r o s t irnaes pe tp oa mr ay a t d
c 2oid r
0n
m,,oLfc h l o r o -
A s s a y f o r a mn 2d 0m oL fw a t ea rns,dh abk yme e c h a mn ei a c an ls
S t a n pd ra er pd a r a t Ui SoP Pen n i Uc s G
i lPi loni tgn a s s if uo 1 mr 5m i n u At le lst o .os we p a ra a ntd d ei ,s ct ah rle do w e r
R S p, r e pa asd ri er e fc otSretd a n pdr ae rp da ru an tdli eoo drn o - c h l o rl oa f y eR
o rer. mp te haeetx t r a wc it ati 2 ho n0 -p m o rL -
m e t Ar si s c a y A n( t4 i2 b5 i) o. t i c s t i oo nfc h l o r o d if so cr amtr,hdceih nl go rl oa yf eU or s
r.tm eh e
A s sp ar ye p a 1r (a w t ih ioetinsrr ee p r e as seb ne tii enadg a q u el ao yuaesstr ht ee ss to l u tP iro en pa. aS rt ea n sdo al ru -d
s i n g l e c -o dn ot sa ei n e r ) P e nCi oc iG n lPslrti oi t neet i oo nfU SD Pi h y d r o s tS r
n ctfauo ir u le fpRatSit noew ma ytcceoirnn -
i m e c Sa ub slp eea nsds iir oe incn tt he ld ea b e lWi in gt .h d rt aa w i n6 i.nm 5g pg e mr L A. p ps le y p a r 3a 0 u
t eL o lfe ya sc oh l u -
a l l o ft h we i t h d rc ao wn ta eb u nsl tieasns,gu i t ha by lp eo d e tr i-ot noa t h i n - cl ha yr eo rm a tp ol ga(trseae p e h i c
m i nce e a d l nsedy r i a n gndedi, l qu tu ea n t i tw ai ttBiuhvf ef le yrC h r o m a t ( 6o 2g c 1r)oa)a pwt hie y atd 0 h . 2 5 l a-yome frm
B . t1 oo b t aa si on l u ct oi on nt a ai bn oi2un0t g P0e 0n i c iG l l i cn h r o m a ts o i lgig r ceamal ip xh tiAu clr l et .
oh swep ot tods r y ,
U n i pt esmr L P.i p2emt oL ft h is so l u it ni teo oa n c o fht w o a n dde v et lh coe h p r o m ai nta so ogl rvs a ey nmstct oen ms i s t
g l a s s - s t1 o 2p 5p ce-ormne iLfd cl,aa slk s . i n og fa m i x to ufn r- e p r ao lp cy ol wh ao tl p,e yr r, i da i nn d e ,
g l a caica el a t ic ci( d1 5 : 1 2u :n t1ti0h l :se2o ) l vf er nohtn at s
A s s pa rye p a r2 a( twi ho tne hrlee a b se tl a t eh qse u a n t i tmy o va eb d ot uh t r e e - of ft o uh lre et nh ogs ft h p el a tR ee .-
o fp e n i c iG lp lr ion c i anaigniev ve on l oufcmo en s t i t u t me o d tv he pe l a ft re to hmde e v e l co hp ai mn mbg ae trr hk ,s eo l -
s u s p e n s i o nP )e n iC c oiGnlP ls r it on c
ifato iIur nt jeee c t a bv leefn rt o na tn,adl lt oh s weo l vt eoen vt a p o Sr pa rtt aeh y.e
S u s p ea nsdsi ir oe incn tt he ld ea b e lD ii nl ga u .tna ec c u r a t pe ll aywt ie t a rh e a gp er ne tp ba y dr ie sd s o 2lgvo ifnn ig n h y d
m e a s vu or leoudftm he ceo n s t i nt ju et cestdua s b lp ee n s ii no1 n0 m0 oL fa l c oah noadld d 2 i 0 nm goL fg l a cai ca el t i c
a l
q u a n t i tw ai tt B iuhvf eBf l.e ty1r oo b t aa si on l u ct oi on nt a i na ic in hdg e, t a h t pel aatt1 e 1 f0 o 1r 0 m i n u at n e esd x, a m i n
f s a b o2u0tP0 e 0n i c iG Ul ln ii pnt esmr L P.i p 2e mt oL ft h i s t h ce h r o m a tt o h Regrv ra alamu ns ec d:o lo oftr h per i n c i p
% s o u l iu ne tne oa oc fht wg ol a s s - s t1 o 2p 5p ce-ormne iLdc,a ls p o tb t a fi rn t oe hmdt ee ss to l u ct o i or nr et sotp hoons de
i } jasks.
io.) o b t a fi rn t oe hmdSe t a n sdo al ru d t i o n .
} P r o c e d u ra esd i Pr r e fcootc Prere doe cd eu dn udlreoe r d o - W a tD e e r t e r m iM ne at| th( i 9o 2od1n) n ,:o mt o tr he a n
i<j m e t Ar si sc a y A n( t4 i2 bC 5 i)a o.l tc iut clh qsaeut ae n ti ni t y , 1 . 4 2% 0m , oL fa m i x to uft ro el ua ennmdee t h (a7 n: 3o )l
iS P e n i c iG Ul ln ii nit nst ,h ceo n t a oi r inn t
e r h p,e o r to ifco on n - b e i un sgie n d t ht ei t r av te is oisnnep ll ao cfme e t h a n o l
2 s t i t iu nt je ed c st ua s b lp ee tn askibeoy tn nh fe o r m u l a :
A s sf ao pyre n i c Gi l lP irn ao sdc ier e e fcdotpre nd i c i l l
r o
a ) ( L 2/D ) ( -A| )\ ( B G u n dA en r t i b i o t i cA ss s (Ma 8iy 1cse)rx, op be ilt alh lcie n o gn -
= t e notf1 s s y r io nfIg ne t r a mI a n fm u mis na i taorohnyi g h -
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U n i pt esmr L o ,fA s spa ry e o 1n oiroifAns nsp ar ye p a r a ta ic oc nu r ma et ea ls yvu or leoudftm h eae q u ep oh uaqssu ea n t i
2 o nt h bea soifts h le a b eq lu ea dn itnti htceyo n t ao iri nn e rt i v ea lnysdt e p ww ii t Bs uhef Bf .e t1r oo b t aa Ti e nsD ti l u t i o
t h pe o r to ifcoonn s t i nt ju et cestdua s b lp eet na sk ire eon -n, h a v ai cn og n c e n ot fpr ean ti ic G io lan lsi sn ut omb eee dq u a l
s p e c t ia vnetdlh yee,x t o ef dn it l u t i o n . t ot h me e d di oa lsneev oe flt h Se t a n d a r d .
A s sf ao dyr i h y d r o s t r e pa tsd oi rm eyfc c ot ri
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v o l oufpme en i c i sl ul fi fn iatcsoie en na tc t ti hvpeae tn ei c i l l
» P e n i c iG lP lr ion c aa n i Dnd ie h y d r o s t r e G cpo tn o t a tm hiyen creDeidi n ln tu. ht ise so l u qt ui ao nn t i tw ai tti hv
S u l fIa nt et r a mI a n fm u m i ss aai sorunys p eo nf s Bi uof Bn f .e t3r oo b t aa Ti ensD ti l u ht ia ov nai cn og n c e n ot fr a
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P e n i c iG lP lr ion c aa n
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n cgey l i n d e r s .
c o n ts au i tn ga eb ll leai nntgdh i c k aegne inI ntt gs .
c o n t na oil tne ssts h a
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t tjo eem cyt c
Sauibs n pl et
ena skbie yotn nh fe o r m u l a :
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» P e n i c iG lP lr ion c aa n i Dnd ie h y d r o s t r ienwp htiLi oc m h h le a bi eqnlu ea dn ti niP te yn i, c iGUl nl i nit ns ,
ts y c
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s i o nfP e n i c iG lP lr ion c ianaisno el u o tf i D oi nh y c-o n c e n ti nrP ae nt ii coiGnUl ,ln ii pnt esmr L o ,ft h Ae s s a y
d r o s t r e Spu tl ofi m an Wty a
ec ti
f eonIrrn j e c t i o pnr ,e p a r oa nt ih boean s,oi fts h le a b eq lu eadn itn ti htpeyo r -
a n cdo n t oa n ionerms o sr u ei t ba ub fl fepe r es ,- tt ii ooannfI,nntdjh oee ct Sthtuaeebsrrlpa emertasenasdskeaiefonnittndnhh eeee xrd te oei fdnn i.t l u -
s e r v a ta i nvd dei ss, p e or rss u i ns gp ea ng de in nt s gA .s sf ao dyr i h y d r o s t r e pa tsd oi rm eyfc oct rie dn P
I t m ac yo n tP ar io nc Ha yi dn re o c ihnal co or ni -dt e h teu r b i d ai sm sef ato d yrr i ch y d r o s tu rne dAp ent r-o m y c i n
c e n t r na oteti xo cn e e2 . dp0ie nr gc a e nni ttdm
, a yt i b i o t i c sA s Ms i(ac8yr1 su)o s,b iiananagl c c u r ma et ea ls yu r e d
c o n toa nio enrm o sr u ei t sa tba lb ei lI it cz eo rns -. v o l ou fImn ej e c St ua bs lp eed ni sl iuq otu ena dn t i tw ai tti hv e l y
t a i nn osl te sts h a9 n0 p. e0 r ca ennndto mt o r e w aatttee oy ri e alT de sD ti l u ht ia ov nai cn og n c e n at sr- a t i o n
ob ee q ut aotl h me e d di oa lsneev oe flt h Se t a n -
t h a1 n1 5p .e 0r co eft nh lte a b eal me o d uo fn t s a r d .
P e n i c iG lU ln ii nat nso dfd i h y d r o s t r e p t o m y c i n
( C a r H a i N 7 O 1 2 ) .
P a c k a a gnsidtn ogr a g e i nsP irn eg sl e o - rr
m duv oles
t ie -
p l e - dt oi sgceh o,t n t a i n e r s .
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P e n i cG
i Pl rl oi n
c a i n e ,
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(¢C I1N - M a y - 2 0 1 8 ) a m e t hI an js eo c n
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U SP Pe n i c iG lP loi tn a sR sS i u m p e n so ifP o e nn i c iG lP lr ion c aa n i nd e
I d e n t i f ir ce as tp tiooo Indnde sn It it ft ie cs A at a
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Si toeen r Di il eh y d r o -
u n dP e e nri c G
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C h l o r p h e M a n li er aaa tn
m Deide,n xe a m e Stu hs ap s e no sn i eoM na. l e i Sa m i
i naW ta etf eo Irrn j e c I tt ci o nn .t oa n i oners 4 )
B a c t eE rn ida ol tT oe xs ( i 8
t 5 n )sc o In t na oi mt no s r e m o sr u ei t ba ub fl fepe r es ,s e r vaa ntd idi vs ep se ,ru v s -
t h a 0 .n 0U1 S E Pn d o tU o n ix
p te i 1rn 0P 0e n i c iG Ul ln ii nt s . =
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t e s at sed di r e fc otM re ed m bF r i lat rn ua etn i doT ne
e sfr to Sr t e - c a i Hn ye d r o c ih nal co o r ni c d e en nt o
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ax t- i o= n
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o fs p e c fi rm e oe a
mn c o h n t ae iq n u ei r vt ao3l 0 e n0 t, 0 0 0 m o sr u ei t sa tba lb ei lI it cz o e rn st.na oil tne ssts h a n= 2
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d i a u n mt dou s Fel u Aitd ow h ih ca b hs e e a nd ds e u fdf i c i etn h t le a b eal me o d uo fnP etn si c iG lU ln ii nat nso df w
s t e rpi el ne i c i tl oli in na ac st t ei hv pea etn ei c iGlal intn dos w i r l d i h y d r o s t( rC e 2 p1 Ht 4o ima Nyn7ncdOoil1tne2s )s ,
t h vee s us ne tlsi ol l u i ts ci oo nm p l b eef tfo irele t e I rf ti nh ge. t h a 9 n 0 p . e 0 r ca ennndto mt o tr he a1 n1 0p .e 0 r -
I n j e c St ua s b lp eecno sn it loaenic ni tsuh siFenl ,u D i td ow h i c hc e o n ft h le a b eal me o d uo fnc t h l s o r p h e n i r a
h a bs e e a n d ds e u fdf i pc ei ne in ct i tl oli in na ac st t ei hvpea et ne -
i c i l lGian nt dos w i tr hl vee s us ne tlsi ol l u i ts ci o o nm p l e tme a l e ( C a it se H - iC g4C HI 4a N O2no 4
fd d
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n go .n t ca ai rn bs o x y m e t sh oy dl ic ue ml ,l u l o s e a m e t h ( a C s
2 2
o Hn 2 e9 F O s ) .
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f i l t e I rf iit nd go. e
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r e c ft oeDrdi r Ie nc to c u lo aft th ieC ou nl t Mu ree d ui nu dTmee sr t L a b e l i int tg oi n Ld ai tcb haeiattiltes i n t e fno vdree tde r i -
f o Srt e r io lfti th Pye r o dt uob ceEt x a m ie nx ec dte o,up stFel u i d n a r u y
s oen l y .
T h i o g l yMc eo ld lciao u tn em
t a ai na n im nogoufs n t etr i l e
p e n i c i sl ul fi fn iatcsoie en na tc t ti hvpeae tn ei c G i il nle ian c h
v e s s e l . C h a tnorge ea d :
P (H 7 9 1 )b :e t w5 e . a0e nn8 d. 0 .
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A s sf aopyre n i c G i l l i n U SC Ph l o r p h e M na il reR aS tm e i n e
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m e t Ar s
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c a y A n( t4 i2 b5 i) o. t i c s @ ( C 1N- M a y - 2 0 1 8 )
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o fI n j e c St ua bs lp eeq nu sa in o t in tw ai tt Biuhvf eBf l.e t7yr oo b - U SP Pe n i c iG lP lr ion c Ra Si n e
t a ia ns o l u ct oi on nt a ai bn oi2un0t g P0e 0n i c iG lU ln ii p nt es r I d e n t i f i c a t i o n
m L P.i p2emt oL ft h is so l u it ni teo ona oc fht wg ol a s s - s t o Ap :T- r a n sw fie tr h,aei od fw a t ae pr o, r to ifto hnI en j e c t -
p e r e1 d2 , 5 c-o mn L if cl aa sl k s . a b lS eu s p e fn rs ei sm
oh nil ,yxa enfddr ef er ao i mbr u b b l e s ,
P r o c e d u ra esd i Pr r e fcootc
Prere doe cd eu dn udlreoe r d o - e q u i vt aoal be o n4t
u0t0 , P e0 n 0i c0 G
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m e t Ar si sc a y A n( t4 i2 b5e i)x o,ctienitpchtsBe l aDnekt e r m si e- p a r aa t d 5od r
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Prere doe cd eu dn ud/r eoe dr o -
l o wc eh rl o rl oa fy to e hrr rm oa ub go4hu g ot fa n h y sd or -o um se t Ar si sc a y A n( t4 i2 b5e i)x o,c tienitpchtB se l aD ne kt e r m i
d i su u ml fsa ut p e p oo rna tp el de do gfg el t a ws os oc lo ,l l e c t n- a t ti ooa nd0 d. 1m oLf1 . N 2 h y d r o ca hc liiodmr mi ec d i -
i n tg hf ei l t ir naa t 1e 0 0 v-o ml Lu mf el at sRrk e i. pc te haeetx - a t e bl ey f to hr 1ee 0 .m 0oL f0 . 0N1i o d Vi Sn C.e a l c ut lh ae t e
t r a c wt i ott nhw 2o 5 -p m o rL t oi fco hn l
s o r o c fo om rbm i, n qiu na gn ti niP te yn i, c iG Ul ln ii nit nst ,h pe o r to ifI on nj e c t a b l
t hf ei l t ri antt eh s1e 0 0 v-o ml Lu mf el at sDrkii. lcwu ti etc hh l o -S u s p etna sk b ie o
ytn nh fe o r m u l a :
r o f to ov
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m di, x[ .N O T E t Rh a
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p h af so /red e n t i ft ie csB a.t t]P ir oen pa Sa tr ae n ds ao rl du t i o n ( L 2/ D ) (-| A) ( B
o fU SP Pe n i c iG lP lr ion c Ra Sii nnc eh l o r coofn ot ra mi n i n g
a b o4 u. m t5 pg e mr L A. p ps le y p a r 1a 0 w
t eL o lfey a sc oh l u - i nw h iL ics th h le a b eq lu ea dn ti niP te yn i, c iG Ul ln ii nit ns ,
t i ot noa t h i n - cl h a yr eo r m a t pol g a (t res aeC peh hr io cm a t toh gve -o l oufImn ej e c St ua s b lp eet na sk iea onn n ,Ddi s t h e
r a p( h6 y2 c 1 )o )a wt ie t ad 0h . 2 5l a-y ome frcm h r o m a t oc -o n c e n ti nrP ae nt ii coiGnUl ,ln ii pnt esmr L o,ft h Ae s s a y
g r a ps ih l ii g c
ce lam i x t Au lr l et .
oh swe p ot todsr ya , n d p r e p a r oa nt ih boean s,oi fts h le a b eq lu ea dn itnti ht peyo r -
d e v et lh coe h p r o m ai nta so ogl rvs a eynmstct oe nms i os ft i tni goo nfI n j e c St ua bs lp ee tnask i ae onntndh ee x t oe fdn it l u -
a m i x to ufb ru et a in so ol p, ra ol pc yo al h c o le ,t ao n wed a, t e rt i o an ,n tdh oe t ht eer ra mr ase sd e f it nh e rd e i n .
( 4 : 4 :u 2n t: ti2 h l)seo l vf er nohtn atms o va eb d ot uh r t e e - A s sf ao dyr i h y d r o s t r e pa tsd oi rm eyfc octri e dn
f o u ro tfthhsle e n og ft h pel a tRe e . m to h v pelea ft re o m t h te u r b i d ai sm sef ato d yrr i h c y d r o s tu rn edApent r-o m y
t h de e v e l co hp ai mn mbg ae trr hk ,se o l vf er on na t t n,a dl l o w t i b i o t i c sA s Ms i(ac8yr1suo) s,b iiana nal g c c u r ma e t ea ls yu r
t h se o l vt eoenvta p o Er xa pt to e h.speel at toie o d vi a n e p o r sv o l ou f mn e e S u a s p e fn rs eis m oh nil ,yxa enfddr e e
i na c l o s c eh da m f obar eb r o1 u5mti n u at n e lsdo ,c t a th ee f r ao i mbr u b b dli el us tq,eu da n t i t w a ti it wv hae ltt yeoy ri e al d
s p o t sh R:e-v a l u a en csdo l oo frt sh te wp or i n cs ip po atl s T e sD ti l u ht ia ov nai cn og n c e n o tf d r i a ht i y od nr o s t r e
o b t a fi rn toe hm dtee ss to l u ct o i or nr et sotp hooonsbde- a s s ut m ob eee dq ut aotl h me e d di oa lsneev oe flt h Se t a n -
t a i f n er dt
o hmSe t a n sdo al ru d t i o n . d a r d .
B :D i l t u th aee q u ep oh uar sse et a fi r n e/o ddme n t i f i c aAt s i osfn ao cy rh l o r p h e m na il re aam ti en e
t e sA tw i tw ha tt eoo rb t aa tie nss to l u ct oi on nt a ai bn oi u n gt I n t e sr t n aa ln sd oa lr ud t i o an s oP lru eot p fi ao r n e
5 m og fd i h y d r o s tp re m er p L Pt.ro em payaScrtiean n d a rbd r o m p h e mna il rei a naw mta i ethneaerv ai cn og n c e n t r a
s o l u ot fiU oSnD Pi h y d r o s tS ru le fpRatSit noew ma yt ce ir n o fa b o7 um t p g e mr L .
c o n t a 6i. nm 5i p g
n egmr L A. p ps l e py a r a3t 0ue lL o yfe a c h
s o l u tt oia to hn i n - cl h a yr eo r m a tp ol ga(trseaeCp ehhrioc m a - S t a n p d ra erpda r a t i ao nan c cD ui rswase toeillgvy he e d
t o g r (a6p2 hc1 y) o )a wt ie t ad 0
h . 2 5 l a- yomefcrm h r o m a t q uo a- n ot fU i tS CyPh l o r p h e M n a i l r eR aSi tmn weia ntteeo r
g r a ps ih l iig cceaml i x t Au lr let o .h swe p ot tods r ya, nd de - o b t aa si tn os co kl u ht a i ov ani kn n g oc w o nn c e n ot fr a t i
v e lt oh pce h r o m ai nta so ogl rvs a ey nmstct oe nms i os fat i n ag b o6 um t p g e mr LT .r a n 5s .f m 0e orLft h is so l u tt oai o n
m i x to ufn r- ep r ao lp cyo lwh ao tl pe , yr r, i dai nng del ,a cai ca el - 5 0 -c m e nL t r ti uf buAegd .e5 d. m 0 oLfI n t e sr tn aa ln s do al ru -d
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a b ot uh t r e e - of ft o uh lre et nh ogs ft h pel a tRe e. m to h ve e a p H o fa b o1 u0 A t
. d2 d5 .m 0oL fh e x a pn le ast c,hece a p
p l a ft re t o hmde e v e l co hp ai mn mbg ae trr hk ,se o l vf er on nt t ,o nt h teu b se h, af ko ae r b o2 u m i t n u at n e csde ,n t r iU fs ueg e
a na dl lt oh wse o l vt eoenvta p o Sr pa rtt aeh py.e l awt ie t a h t h ue p ph ee rx al n a yeaestr h Se t a n pdr ae rp da r a t i o n
S a l
f e r e a gp er ne tp ba y dr ie sds o 2l gvo fi nn ig n h yi nd1 r0im0n L A s sp ar ye p a r a t i ao nan c cT ur ramanets a eflsevyuro r
l -e d
r m o fa l c oah noadld d 2 i 0nm g La f y a c ia a cl e at ci ichd e, a t th e u mo e fI n j e c St ua s b lp ee fn rs ei sm oh nil ,yxa enfddr ef er o m
i ]
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i n w h it ch the e ra mr ase sd e f it nh e rd e i n . t ob ee q ut aotl h me e d di oa lsneevo ef lt h Se t a n dA a dr dd .
t oe a tc eh sd ti l u ot fit oh Sne t a n adq au ra dn ot fU i tS NyPe o -
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i ss aai sorunys p eo nfP se in io cn i
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1 1 0p .e 0r co eft nh lte a b eal me do oufhny td r o -
c o r t ia sc oe nt( eaC t2 e3 H 3 2 0 6 ) . P a c k a a gnsidtno gr a g e i n dP ir sep sosesyr ar bvi le
n eg e
t h a t r we e l l - c ol no ts ae id n e r s .
P a c k a a gnsidtno gr a g e i nwP erl el s- ce lor onvst eea di L n -a b e l i int tg oi n Ld ai tcb haeiattiltes f o vr e t e r ui sn ea r y
e r s . o n l y .
L a b e l i int tg oi n Ld a i tcbhaeiattil
tes i n t e fno vdr ee tde r i -U SR Pe f e r s e
t n a cn e d
( a 1 r1 d) s
n a ru ys oen l y . U S NPo v o b RiS o c i n
U SR Pe f e r s e t n a cn ed
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U S HPy d r o c oA rc te itR saSot ne e W a tD e e t r e r m iM ne at| th ( i
9o 2od1n)n ,:o mt o tr he a n
U S NP e o m S yu c l fiRa S
nt e 1 . 0 2% 0m , oL fa m i x to uft ro el ua ennmdee t h (a7 n: o 3 )l
U SP Pe n i c iG lP lo i tn a sR sS i u m b e iun sgie nd p l ao cfme e t h i antnh toeilt r av te is os ne l .
U S PPo l y mB Sy uxl ifRna St e
A s sf ao pyre n i c Gi l lP irn ao sdc ier e e fcdotpre nd i c i l l
W a tD e e rt e r m iM ne at| th ( i9o 2od1nn) ,:o mt o tr he a n G u n dA en r t i b i o t i c A s s (Ma 8iy 1cse)rx
, ocbteioupa stl e
1 . 02% 0m , oL fa m i x to uft ro el ua ennmdee t h (a7 n: o 3 )l S t a p h y la ou cr oe A cuTcs NC
u osC1. 2 6a 9 st 2h tee so tr g a n i
b e iunsgie nd p l ao cfme e t h i antnh toeilt r av te is os ne l . P r e pt ah r ie en o c bu ygl r u om wt ih on e rg g a ant3i 2st mo
A s sf ao pyre n i c Gi l lP irn ao sdc ier e e fcdotpre nd i c i l l 3i 5nf o 2r 4h o uor nM s e d 1 it u ow m
h i h ca sb h e e a nd da e d
G u n dA en r t i b i o t i cA s s (Ma 8iy 1csu)rs, oi abn i nag ac lc u - s o l u ot fin oo nv o b si oo dc ici uon mn ,t a ti hneeiq nu gi vo af l e
U S4 P1 O f f i cM ioa n
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2 . m5 og fn o v o b pi eom rc tLih nah ta bs e fe inl t t e rhe rd o u g h M o d Le C:

a m e m bf irl a the a
n
r veai 0n g. 2 p - o r mo ssi ot yh ,t a th e D e t e c Ut o V
2 r2 :n0 m
m e d cio un tmta hieenq su i vo af 1l 0 eu ngo tfn o v o bp i eo c r i n C o l u 4m .n 6: x-1 m0 m - 5 c m- ;p4 am c kL i 1 n g
m L U. s ae ni n o c cu ol mu pm o os fi a tb io5oumntoL fs t o c ! F l ro awt 1e :m L / m i n
s u s p ei nn es ai c1o h0 n m0 oL fM e d 1 i. E ux m p te hlce o n t e n t I s n j e cv toi lo un 1m 0 pe L:
o fa s y r io nfIg ne t r a mI a n fm u mis nai taorohnyi g h - s p e e S d y s stu ei m t a b i l i t y
g l a bs ls e nj ad cre or i a l1 n. m i0 noLgfp o l y s o8 r0ab na dt e S a m p lS e y s st :u ei mt a sb io ll iu tat yin S
o dnt a n d a r d
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t os t af no d 1r 0m i n u at n ed sdi ,l ua tna ec c u r amt ee la ys u r e d[ N o t re e l aT rt hie ve t ee nt ti imfoeon2sr - p h e n y -
v o l ouftm heae q u ep oh uaq ssu ea n t i ta na dts it vee pl wy i s e l a c e t aa nmp die nd iec G i al rl aie nb o0 u.at8n d 1 . 0 ,
w i tB uh f Bf .e t1r oo b t aa Ti ensD ti l u ht ia ov nai cn og n c e n t r a r- e s p e c t i v e l y . ]
t i oo nfp e n i c G i la lsi s n ut omb eee dq ut aotl h me e d di oa sn e S u i t a br ie l qi ut yi r e m e n t s
l e v oe flt h Se t a n d a r d . R e s o l u Nt Li2 o T. nb0 :e t w2 e- e p nh e n y laa nc de t a m
A s sf ao ny r o v o b i o c aisdni r P e fcrotnroeocdveo eb di o c i pne n i c Gi ,lS l yi sn stu ei tm a bs io ll iu tty i o n
u n dA en r t i b i o t i cA ss s (Ma 8iy 1cse)rx, op be ilt alh lcie n o gn - C o l euf m f inc i Ne nL 1 cTy0 :t0 h0e o r ep lt ai tc ea sl ,
t e no tfass y r io nfIg ne t r a mI a n fm umis nai taorh onyi g h - S t a n s do al r u td i o n
s p eb el de nj ad creorn t a 1i .nm 0 i oL
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o ft h ae q u ep oh uaass de s du f f i pc ei ne in ct i tl oli in an ca ts ie - A n a l y s i s
v a t eh p ee n i c G i tl hl ie nr ae indn di, l u qt uea n t i taa n t id v e l y S a m p lS e t s a :ns do al r uatd in oSdna m spo ll eu t i o n
s t e p ww ii t Bs uhef Bf .e t6
r oo b t aa Ti ens Dt i l u th i ao nv i n C a l c ut lh pae ot te e on fpc ey n i c G i s
l lo idn ii nuP m
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m e d di oa lsneev oe flt h Se t a n d[ aN rod T. E t h SiT ste so tr e t a k e n :
D i l u at ti3 o7fn o 3r 0 m i n ua tn e adls lt oocw o ob le f uo sr -e
i ni gt t foi l t
l h ce y l i no d n te hrpesl a t e s . ] R e s u = (l r tu / xr (s C) s /xCP u )

r u = p e ar ek s p oofpne sn ei c G i fl lr i o
tn hm Se a m p l e
s o l u t i o n
r s =p e a r ek s p oofpne sn ei c G i lf lri ton hm e
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i nt h Se t a n sdo al r u (td imo gn / m L )
a O N a * C u = c o n c e n ot fPr ean ti ic iG o lSnl oi nd iintu h me
3 S a m spo ll e u (t imo gn / m L )
S9 N H y
wk P = p o t eo fnp ec nyi c G i il nlUi S nP Pe n i c iG l l i n
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H H
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() )7] a9- m1, i) n o ] - ,
m o n o ss aol dt ;i u m S a m spo ll e u t 6i 0 omn g: / o fPm e L
n i c iG lS l oi nd iin u m= )
M o n o s( o2 d5 ,i 5uR m, 6 R ) - 3 , 3 - d i m e t h y l w- a7 t- eo rx o - 6 - ( 2 - p h e n y -
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l a c e t a m i d o ) - 4 - t h i a - 1 - a z a b i c y Ac cl co [e 3pc.tr2ia.t n
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a t [e 6 9 - 5 7 - 8 ] . J
e L o sO sN D R Y (I 7N 3G1 ) 2 ]
S a m p 1l 0em0: og fP e n i c G i lS l oi nd i u m a
D E F I N I T I O N A n a l y Ds rity s h:Se a m ipnalc ea p i l l a r yb -o st tt ol7 pe) p e r
P e n i c Gi lS l oi d
n hi auaspm o t eo n fN cLy 1T5 a 0 n 0N d M T u n da ev ra c auta up r m e s sN u M r5 eT
m omfm e r c u r y
1 7 P5 e 0n i c iG lU ln i in t s / m g . a t6 0 f o 3rh .
I D E N T I F I C A T I O N A c c e pc t r iat ne rcNieaM1 : T . 5 %
e S T E R IT LE IS(T T7Y S1 )W : h et r h leea b se tl a t eh sa
P et n i c i l l i n
e A .I N F R AA BR SE O D R( P1 T9 I7 OK N) G S o di i ss u
t e mr ii tlme e, et th rse e q u i rwe hm e nn t s
' B ,I D E N T I FT I EC SA T T IS O N(G1 E9 1N S )oE ,dR iAMu Lem e: t s t e s at sed di r e icnTt ee sfdto Srt e r io l fti ht Pye r o dt u oB ce t
t h re e q u i r e m e n t s E x a m iM nee md b ,F r
i lat rn a et i o n .
A S S A Y ¢ B A C T EE RN ID AO LTT OE X(S I 8T5N )WS: h et r h leea b se tl a t e s
¢ P R O C E D U R E t h aP et n i c G
i lS l oi nd i i ss u
t emr oi r
ilt em u b s e t
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S o l u At :i0 o. n0 M 1m o n o b p o a ts ai sp c sh io us mp h a t te of u r tp hr eorc e ds us ri tin hngpegr e p a or fai nt ji eo cn t a b l e
M o b pi hl ae sM ee :t h aa nnS do ll u At (i 4o 0n : 6 0 ) d o s fa og r eimt cs o, n t a NiM n
0 s.T0U1 S E Pn d o t o x i n
S y s stu ei tm a bs io ll iu tt y0i. o1mn g : /e a mc oLfhU S P U n i t sP e/ n1i 0c 0iG Ul nl i nt s .
P e n i c iG lP loi tn a sR sSai nu2dm- p h e n y li a n c e t Aa m D iD d Ie TR IEOQNUAIL R E M E N T S
w a t e r ¢ P A C K A A GNSIDTN OGR AP G r eE s:ientr iv gech ot n t a i n e r s .
S t a n sdo al ru t d0i .o1mn g: /o fm U S LP Pe n i c G i lP lo i- n
t a s sR iSi unwm a t e S rh .aa ksne e e td odei ds s oT lh vi es. e L A B E L WI h
N Gei t
: irs ie n t e fno dur esiednp r e p ai nr -i n g
s o l u ct oi no nt aa b P 0e n i c iG lU ln iin t s / m L . j e c t da bo ls efa og retm hsle,a b se tl a t eh siatits s t e roi rl e
i no1su 6t
m u b s et
s u b j et cof tu er dtp hreor c e ds us ri tin hngpegr e p -
S a m spo ll e u t 0i. o1mn g: / o fPm e L
n i c G i lS l oi nd iin u m a r a to ifionnj e c dt o a bs lfaeogr em s .
w a t e r
C h r o m a t soygs rt ae pm h i c
( S e
C h
e r o m a t(o 6g r2 aS1py)hs ,
yStu ei m
t a b i l i t y . ) C h a tnorge ea d :

U SR PE F E RS ET NA C NE D
( 1A 1R) D S
@ { C 1N- M a y - 2 0 1 8 )
U SP Pe n i c G
i lP loi tn a sR sS i u m
U SP Pe n i c G
i lS l oi nd Ri Su m
 3 2 1
P e2n i c /i Olflfii nM
c ioa n
l o g r a p h s U S4 P1

C h r o m a t soy gs rt ae pm h i c
P e n i cG
iSl lo idnfio I
urnm j e c t i o n ( S eC eh r o m a t ( 6o 2g1Sr)y a
,s pStuhei ym
t a b i l i t y . )
M o d Le C:
D e t e c Ut oV
2 r2 :n0 m
D E F I N I T I O N
C o l u 4m .n 6: x-1 m0 m- 5 c m- ;pu am c kL i 1 n g
P e n i c G
i lS l oi nd fio Iur nmj e ci sts it eo rnPi el ne i c G
i S
l lo i- n F l ro awt 1e :m L / m i n
d i ou arms t e r mi il ex to ufP re ne i c iG lS l oi nd ai nuNdmL T I n j e cv toi lo un 1m u 0e L :
4 . a 0 n% N d M5 T . o0 fS % o d Cii u t rmoa f tw e h, i N c hM T S y s stu ei m t a b i l i t y
0 . 1 m5 a%byer e p l ba y c
C ie tdAr ic ciI dt c. o n t N a iL nTs S a m p lS e y s st :u ei m t a sb io ll iu ta t yin So dnt a n d a r d
9 0 .a n0d % N M1 T 2 0 o.ft0h l%e a b eal me do oufp ne t n i - s o l u t i o n
c i l lGi .nI na d d i tw i ho e n
i t ,r
c oe n t S a io nd s Cii u t rmiat t e , [ N o t re e l aT rthie e vt ee nt ti imfoeon2sr - p h e n y -
h a asp o t eo n fN cLy 1T4 a 2 n 0N d M1 T 6 P6 e 7n i c G i l l i n l a c e t aa nmp die nd iec iG al lr a ie nb o0 u. at 8 n1 d. 0 ,
U n i t s / m g . r e s p e c t i v e l y . ]
I D E N T I F I C A T I O N S u i t a br iel qi ut yi r e m e n t s
' A .T H I N - CL HA RY E O RM A T O G R A P H Y R e s o l u Nt Li2 oT. nb0 :e t w2 e- ep n h e n y laa nc de
S o l u At :iA oc ne t 0o. n1M ec , i t ra ic ci ad ,n0 d. 1Ms o - p e n i c Gi ,lSl yi sn stu ei m t a sb io ll iu tt yi o n
d i cu i mt r( a2t:e 1 : 1 ) C o l euf m f inc i Ne nL 1 cTy0 :t0 h0e o r ep lt ai tc ea sl ,
S t a n sdo al ru t d Pi ro en p:a a s or le u ct oi no t n a ti hnei n g S t a n s do al r u td i o n
e q u i vo af1l 2 e n , Pt0e 0n i0 c iG lU lni in t fs r/ o U
m mS
L P T a i lf ia nc gt oNr M :2 .T0S ,t a n sdo al ru td i o n
P e n i c iG lP lo i tn a sR sSi i nS uo ml u At .i o n R e l a st t i va en dd eav ri da tNi M o2n T .: 0S %t ,a n d a r
S a m spo ll e u t Ni oo m n :i n1 a2 l, Pl0ey0n i0 c iG Ul ln ii nt s / s o l u t i o n
m fL r Po emn i c iG lS l oi nd fio Iur nmj e ci tnS ioolnu A t i o n A n a l y s i s
C h r o m a t soy gs rt ae pm h i c S a m p lS e t s a n :s do al ru atd in S odna m spo ll e u 1t, i o n
( S e5 e e e
y( 6 2 e 1T )h ,i n - CL ha ry o e rm a t o - S a m sp o l l eu 2t ,i
o orS n a m sp o ll eu 3t i o n
r a p h y . P e r ft oh Are sm soan1y 0c o n t a wi hn ei trirs sre e p r e -
A d s o r 0 b . e 2 n t 5
l a: -yome fcrm h r o m a ts oi lgi rc aa p h si ec n at se b d e ii nna gs i n g l c e o - d n ot saa ein indf en, r
e c -
g e ml i x t u r e e s s a or n y1 ,0 c o n t a wi hn e et rrhslea b se tl a t eh se
A p p l i c v ao tl iu o 2m n0pe L: q u a n ot fpi etn yi c iGil nla ig ni v ve on l ou fcmo en s t i -
D e v e ls oo pl ivs n ey gns tt Te oml :u de in oe x, aa n e d , t u ts eo dl u tUi sotenh.ien d i v rie ds uutaloldt se t e r m i
g l a caica el at ci ic( d9 0 : 2 5 : 4 ) t h Ue n i f oorfDm o i ts Uyan gi aet n st dh ae v e ro a ft gh ee
S p rr ae ya g1 :e Snt ta r T cS h r e s ua lstt s
h A e s sv aa y
l u e .
S p rr ae ya g 2 :e lno td T i nS d ei l u1 ti ne1d 0w i tw ha t e r C a l c ut lh pae e t er c eo nftth ale ag be e al m e do oufpne tn -
A n a l y s i s i c i l lGi nn t h ce o n t ao iri nnt eh pre o r to ifc oo nn s t i -
S a m p lS e t s a n : sdo al r ua td in oSdna m spo ll eu t i o n t u ts eo dl u tt ai ko en n :
P l atc hepel ai tnaes u i t ca bh lr eo m a t co hg ar ma bp eh ri .c R e s = u (lr tu / xr (s C) s /xC Pu )x 1 0 0
D e v et l h ceo hp r o m au ts i otnghgDre eav me ls oo lp -i n g
v e n s ty s ut n et tmi hl se o l vf er nohtn atms o v te hd r e e - t u =p e a r e k s p fo rn o Ss ma
e m spo ll e u 7t oi ro n
<e f o u ro tfthhsle e n og ft h p el a tRe e. m to h v pe lea t e S a m spo ll eu 2t i o n
a f r to hmce h a m mb ae trr hk ,se o l vf er on na t t n,a dl l o w r s p e r a ek s p fo rn tos hm eSe t a n s do al r u td i o n
L s
_ t oa i r - S d rp yrt . ah pyel a wt ie tS hp rr ae ya g1fe onl t-
D p l o wbeySd p rr ae ya g2 .eP ne nt i c iG la lpi pn ea a sa r s
C s c o n c e n ot fr U aSPtP e in oi cn i G lP lo i tn a sR sS i u
3 i nt h Se t a n s do al r u( td im o gn / m L )
¢ w h is tp eo o tna p u r b p l a ec k g r o u n d . C u = n o m ic no an l c e n ot fS r aa tm sipoollneu 1t oi ro n
G) A c c e pc t r ia t ne T c
r iheRa e;:v a lo ufte h ep e n i c iGl l i n S a m spo ll eu 2 t (i Poe nn i cGiUl nl i nt s / m L )
2 " if pr t o hmSe a m spo ll eu ct o i or nr e ts otp ho afn trd o s m P: = p o t eo fnp ec nyi c G i il nlUi SnP Pe n i c iG l l i n
rs t h Se t a ns do al ur tdi o n . P o t a sR sS( iP eu nmi cGiUl nl ii nt s / m g )
a )
a A S S A Y C a l c ut lh pae o t e t e innPc e yn ,i c iG lU lni in t s i n/t mh ge ,
e P R O C E D U R E p o r to ifP oe nn i c i
G lS l oi n
d fi o I
ur n m j e ct ta ik oe nn :
S o l u At :i0 o. n0 M 1m o n o b p oa ts ai spc sh io us mp h a t e R e s = u (lr tu / xr (s C) s /xCPu )
M o b pi hl ae sM ee :t h aa nnS do o ll u A t (i 4o 0n : 6 0 )
S y s stu ei m t a bs io ll iu tt 0yi. o1mn g : /e m a c oLfhU S P t u =p e r a ek s p fo rn o Ss me
a m spo ll eu 3t i o n
P e n i c iG lP lo i tn a sR sSa inu2dm- p h e n y li a n c e t a lm si d =ep e r a ek s p fo rn tos hm eSe t a n s do al r u td i o n
w a t e r C s = c o n c e n ot fr
U aSPtP
e in oi cn iG lP loi tn a sR sS i u
S t a n sdo al ru td0i. o1mn g : /o fm U S
LP Pe n i c iG Pl lo i-n i nt h Se t a n sdo al ru (td im o gn / m L )
t a s sR iSi unwm a t eS rh .aa ksne e e td odei ds s oT lh vi es. C u = c o n c e n ot fPr ean ti ic iG
o lSnl oi nd fio ur m
s o l u ct oi no t
n aa bi no1su6Pt 0e n i c iG lU ln iin t s / m L . I n j e ci nt Si ao n
m spo ll e
u 3t (
i om n g / m L )
S a m sp oll eu 1 t (i wo hni eti srr ee p r e as seb en itni egnad P
= p o t eo n fp ec nyi c G i il nlUi SnP Pe n i c iG l l i n
s i n g l c e o- nd to as ieCn oe nr s) t:P ie tn iu ct i GelS l oi nd i u m P o t a sR sS( iP eu nmi cGiUl nl ii nt s / m g )
f o Ir n j e ca tsdi io rn e icntt he le da b e lWi in gt .ha ldlo rf a w A c c e pc t r iat ne r9c ie 0a .: 0 % -o f1 t h2le0a b. e0l %e d
t h we i t h d rc ao w n ta ebu nsl tieasnh,gy p o dn ee re md i l e
c a m o oufp ne nt i c Gi .lWl h i neP ern ei c iG lS l oi nd fio ur m
a ns dy r i a n gndedi, l w u ti etw ha tt eoo rb t aa si on l u t i o n I n j e cc to ino tnsa o i nd csii u t rmiat th ea ,asp o t eo n f c y
c o n t a in noi mn ig n1 a6Pl0eln yi c G i lU ln iin t s / m L . N L 1T 4 a 2 n 0N d M1 T 6 P6 e 7n i c iG lU lni in t s / m g .
S a m sp o ll eu 2t (i wo hn te hrlee a b se tl a t eh seg e e t
o fp e n i c G i il nla ig ni v ve on l oufcmo e n s t i st ou lt ue -d P E R F O TR EMS AT N S C E
t i o n C) o: n s tP ie tn iu ct G ielS l oi nd fio Iur nmj e ca ts i o n ' U N I F OO RFDM OI ST UA Y NGI(ET9 S0 5 M) e: et th rse e q u i r e
d i r e icn tt he ldea b e lD ii nl gau .st ue i t aa lb il qeo ufto ht e m e n P t es r. ft oh Are sm so an1y 0c o n t a wi hn ei trirsse
c o n s t si ot lu ut wte iid tow nha tt eoo rb t aa si onl u t i o n r e p r e as seb ne tii ennadgs i n g l c e -o dn ot saa ein indf e, r
c o n t a in n oi mn i g n1 a6Pl0eln yi c iG lU ln i in t s / m L . n e c e s os n a1 r0cyo,n t a wi hn e et rrhslea bse tl a t eh se
S a m sp o ll eu 3t (i wo hni et cro en t sa o i nd csii ut rma t e ) : q u a n ot fpi etn yi c G i il nla ig ni v ve on l ou fcm o en s t i t u
T r a n as b f eo5ru0m t og ft h P ee n i c G i lS l oi nd fio Iurn m- s o l u tU i sotenh .ien d i v rieds uutal oldt se t e r t mh Ue in ni e-
j e c tt ioao 5n 0 0 v-o ml L u mf el at sa rk id
, acdb o4u 0tm0 L f o r mo ifDt oy s Ua n gi aet nstdh ae v e ro fat gh reee s ua lst s
o fw a t e a rn s
,dh at kod ei s s oD li vl ew u. t
i etw ha tt eo r t h Ae s sv aa yl u e .
v o l ua mn m edi, x .
U S4 P1 O f f i cM ioa n
l o g/ Pr ean p
i chi3sl 2l 1
i n3

S P E C TI EF S
I C
T S S a m spo ll e u t 2i .om 5n g : /o fPm e L n i c V i il nlM ion b i l e
' I N J E C AT N I IODMN P
S L AD Nr TP uErGDo p( u1 )c S ,pte sc i f i c p h a s e
T e s tCs o, m p l eat necdln a erosi f
stsoy l u t iA ottn hs t:e i om fe C h r o m a t soy gs rt ae pm h i c
u s e
i t ,m e et th rse e q u i r e m e n t s . ( S eC eh r o m a t (o 6g r2 aS1 py)hs, yStu ei tm a b i l i t y . )
C R Y S T A (L 6L 9I 5N M) Ie: TetYth rse e q u i r e m e n t s M o d Le C:
S T E R IT LE IS(TT7Y S1 )_ I t: m e et th r se e q u i rwe hm e nn t s D e t e c Ut V o2 r5 :n4 m
t e s at sed di r e icnTt ee sfdto Srt e r io lfti th Pye r o dt uoB ce t C o l u 4m - n x:m3 m 0 - pc am c; kL i1 n g
E x a m iM nee md b ,F r i l at rna e t i o n . F l ro awt 1e :m L / m i n
e B A C T EE RN ID AO LTT OE X(S I 8T5N )ISt:c o n t a NiM n0 s.T0 1 I n j e cv toi lo un 1m 0 ue L:
U S EPn d o tU on xi i t n sP e/ n1i 0c 0iG Ul ln ii nt s . S y s stu ei m t a b i l i t y
P (H 7 9 1 ) S a m p lS e y s st :u ei mt a sb io ll iu tat yin S
o dnt a n d a r d
S a m spo ll e u tA i so onl:u ct oi n o nt a 6i 0n m ign / g m L s o l u t i o n
A c c e pc t r ia t ne 5c r .iea0 :- W7 .h 5e i .tirs lea b ea ls e d [ N o t re e l aT rthe i evt ee nt ti imfoeonpsr - h y d r o x -
c o n t a si on d i cnii gu t rmat th ep
e, iHs b e t w6 e . a0e nn d y p e n i cV i,p le ln ii nc Gi l,al ni npde n i c iVla lriaen b o u t
0 . 40 ,. 8a , n 1 d. 0r ,e s p e c t i v e l y .
L o s
O sN
D R Y (I 7N 3G1 ) S u i t a br ie l qi ut i y r e m e n t s
S a m p 1l 0em0: og fP e n i c iG lS l oi nd fio Iur nmj e c t i o n R e s o l u Nt L i3 oT. nb0 :e t wp ee nei n c Gi la lnip dne n i c i l -
A n a l y Ds ritsyh:Se a m ipnal a e e e ep e e blo e t tdl e l i Vn ,S y s stu ei m t a sb io ll iu tt yi o n
u n dve ar c auta up r m e s Ns Mu rT5 em omfm e r ca tu r y C o l euf m f inc i Ne nL 1T8 t0 h0e o r ep lt ai tcSeaysls, -
6 0f o 3
r h . t es mu i t a bs io ll iu tt yi o n
A c c e pc t r ia t ne c
rNie aM1: T
. 5 % R e l a st t i va en dd eav ri da tNi M o 1n T.: f0 o %
pr e n i c i l -
e P A R T I C M UA LT AI TNTI EEN RJ E C (T 7I 8O 8NM)S :e et th rsee - l i nV p o t a s Ss ti au nms d,o al r u td i o n
q u i r ef mo ser mn at ls l -i vn jo el cu t mi eo n s A n a l y s i s
e L A B E (L7 I)L ,Na Gb ea lnLsda b e fl oiIr n gj e c Pt ra ob dl ue c t s : S a m p lS et s a : n s do al ruatd in oSdna m spo ll eu t i o n
M e et th rse e q u i r e m e n t s C a l c ut lh aep to et eo nfp ce yn i c V i pl lo it na si snPie un mi ,-
c i l lVi Un n i t s
i n/t mh pge o, r to ifP oe nn i c V
i tl al ik ne n :
A D D I TR IE OQ NU AI LR E M E N T S
' P A C K A A GNSIDTN OGR AP G r eE s:ae sdr ev e s c ri niP ba ec dk - R e s u= (l r tu / xr (s C) s /xCP u )
a g i an ngS dt o rR ae gq eu i r( e6 m5 Ie 9 n)n j,te scP tai co kn a g i n g ,
P a c k a f ogcrio nn sg t i t u t i o n . r u = s uo mft h pe e ar ek s p o n
fp s
- e s
h y d r o x y p Vean nip d ce in il cl V
iiflnlr it
on hm e
S a m sp o ll eu t i o n
C h a tnorge ea d : f s = s uo mft h pe e r a ek s p oo fnp -s e s
h y d r o x y p Vean nip d ce in il cl V
iiflnlr it
on hm e
u sR P
E F E RS ET NA CNE (
D 1A 1R) D S S t a n sdo al r u td i o n
@ ( C 1N- M a y - 2 0 1 8 ) C s = c o n c e n ot fr U aSPtPe in oi cn V
iPl lo itn a sR sS i u m
U SP P
e n i c iG lP loi tn a sR sS i u m i nt h Se t a n sdo al r
u (
td imo gn / m L )
C u = c o n c e n ot fPr ean ti i cV ioilnnlt ihnSe a m p l e i c
s o l u (t imo gn / m L ) 4 )
P = p o t eo n fp ec nyi c V i il nlU i SnP Pe n i c V i l l i n a ]
P o t a sR sS( i P eu nmi cViU l nl ii nt s / m g ) =
P e n i cV
i l l i n A c c e pc tr ia t ne c1
r ie
5a 2 : 5 P- e 1n i7 c 8V iUl0 ln i in t s / }m g
S s
I M P U R I T I E S }
e L i mOiFPt H E N O X AY CA IC DE T I C
a
=
iS)
M o b ip hl aes Ae c: e t o n gi lt ar ciailcaeel,at ci ic ad ,n d 3
w a t (e3 r5 : 1 : 6 5 ) =
D i l u epn H 6t .:p6 h o s p b uh fa(f tse eRr ee a g eI nn td is c, a - "

t o ra s , nS do l u t i o Sn os l uBt ui fo fn es r )
S t a n sdo al ru d t 0i. o1mn g : / o fmp hL e n o x ay cai cnd e t i c
C i s H i s N 2 0 s S 3 5 0 . 3 9 D i l u e n t
4 - T h i a - 1 - a z a b i c y c l o [a 3c .i 32d ,. , 30- ]d hi e- pSt aa m n sepo-ll2 e u- tc2ia0or .nmb 0 :og x / oy fPm
l eiLn ci c V
i il nlD ii nl u e n t .
m e t h y l - 7 - o x o - 6 - [ ( [p2 h5 e- n( 2o ax ,y 5a0 c, e, t U
6 Byst)leh])is-sao; ml iu otn io
nt o]h n-de a,yp r e p a r e d .
( 2 5 , 5 R , 6 R ) - 3 , 3 - D i m e t h y l - 7 - o xC oh -r6 o- m( a 2 - t spoyhgsert n aoe pxm h y ai cc e t a m i d o ) -
ee ae c i d e ( S eC eh r o m a t ( 6o 2g1Sr)y a,s pStuhei ytm a b i l i t y . )
- 0 8 - 1 ] . M o d Le C:
D e t e c Ut o V2 r5 :n4 m
D E F I N I T I O N C o l u 4m .n 6: x-2 m5 m - c5 m -;p a m c kL i 1 n g
P e n i cV i hl la aispn o t eo n fN cLy 1T5 a 2 n
5N d M1 T 7 P8 e0n i - F l ro awt 1e :m L / m i n
c i l lVi Un n i t s / m g . I n j e cv toi lo un 2m 0 pe L:
S y s stu ei m t a b i l i t y
I D E N T I F I C A T I O N S a m p Sl te a:n sdo al ru td i o n
e A .I N F R AA BR S E OD R( P1 T9 I7 OK N) S u i t a b ri le i qt yu i r e m e n t s
S a m p Dl o e
n o:d tr y . T a i l fia nc gt oNr :M1 .T5
A c c e pc t r ia t ne M
rc ie
ea e:t th rse e q u i r e m e n t s R e l a st t i va en dd eav ri da tNi M o 2n T.: 0 %
A n a l y s i s
A S S A Y S a m p lS et s a : n s do al r u atd in S
odna m sp o ll eu t i o n
¢ P R O C E D U R E C a l c ut lh pae e t er c eo nfpt ha eg ne o x ay cai cndte hte i c
M o b pi hl ae sA e c e: t o n gi lt ar ciailcaeel,at ci ic ad ,n d p o r to ifP oe nn i c V i tl al ik ne n :
w a t( e3 r55 0. :765 5: 0 )
S y s stu ei tm a bs io ll iu tt y2i .om 5n g
: /e a m ocLfhU S P R e s = u (lr tu / xr (s C) s /xC1u 0) 0
P e n i c iG lP lo i tn a sR sSai nuUdmSP Pe n i c V i Pl lo itn a s s i u m
R Si nM o b pi h l ae s e r u = p e aa rk eo fap h e n o x ay caifcdre t ot hmi ec
S t a n sdo al ru t d 2i .om5n g: /o fm U SLP Pe n i cV i Pl lo i- n S a m spo ll eu t i o n
t a s sR iSi un Mm o b pi h l ae s e
 3 2 1
P e4n i c /i Olflfii nM
c ioa n
l o g r a p h s U S4 P1

r s = p e aa rk eo a f p h e n o x ay ca if c der tto ihmce s o l u tt ih or noa su ug iht fai bl lto eef0r . 5 o- rfui mn pe or r e

S t a n sdo al ru td i o n s i z e .
C s = c o n c e n ot fp r h a te i n oo nx ay cai cndte hte i c C h r o m a t soy gs rt ae pm h i c
S t a n s do al r u ( td im o gn / m L ) ( S eC eh r o m a t ( 6o 2g1Sr)y a,s pStuhei ymt a b i l i t y . )
G y = c o n c e n ot fPr ean ti i c Vioilnnlt ihnSe a m p l e M o d Le C:
s o l u ( t im o gn / m L ) D e t e c Ut o V
2 r5 :n4 m
A c c e pc t r ia t ne crNieaM0: T . 5 % C o l u 4m - n x:m3 m0 - pc am c; kL i 1 n g
e L I MOI FpT - H Y D R O X YVP E N I C I L L I N F l ro awt 1e :m L / m i n
M o b pi hl ae s S ey ,s stu ei m t a bs io ll iu ttSyito an n, d a r d I n j e cv toi lo un 1m 0 Le L:
s o l u tSi ao m n s,p o ll eu tC i ho rn ,o m a t soy gs rt ae pm hS iy cs stu ei m t a b i l i t y
a n Sd y s stu ei tm a b iP lri ot cya :esd ei dr e icntt he e d S a m p lS e y s st :u ei tm a bs io ll iu taty in S
odnt a n d a r d
A s s a y . s o l u t i o n
A n a l y s i s [ N o t re e l aT rthie e vt ee nt ti imfoeonpsr - h y d r o x -
S a m p Sl ae m:sp o ll eu t i o n y p e n i cV i,p le ln ii nc Gi ,lal ni npde n i c iVla lriaen b o u t
C a l c ut lh pae e t er c eo nfpt- ah gy ed r o x yVpi net nh ie c i l l0 i. n 40 ,. 8a , n1 d. r0 e
, s p e c t i v e l y ]
p o r to ifP oe nn i c V i tl al ik ne n : S u i t a br ie l qi ut i y r e m e n t s
R e s o l u Nt L i3 oT. nb0 :e t wp ee n ei n c iG la lnip dne n i c i
R e s = u (lr tu / xr 1r )0 0 l i Vn ,S y s stu ei m t a sb io ll iu tt yi o n
C o l euf m f inc i Ne nL 1cTy8 :t0 h0e o r ep tl ia fct oae rls
t u p e a r ek s p oofpn -s hey d r o x yVpf ern oi mc i l l i pn e n i c Vi ,lS lyi sn stu ei m t a sb io ll iu tt yi o n
t h Se a m spo ll eu t i o n R e l a st ti va en dd eav ri da tNi M o1n . T: 0S%t,a n d a r
rr = s uo m ft h pe e r a ek s p oo n fp s- e s s o l u t i o n
h y d r o x y p Vean nip d ce in il cl V
ii lnl i n A n a l y s i s
A c c e pc t r ia t ne rcNieaM5 : T. 0 % S a m p lS e t s a n:s do al r u a
td in oSdna m spo ll eu t i o n
C a l c ut lh pae t e er c eo nftth ale g a be e nl u e dm obfP ee r n i -
S P E C TI EF S I T C S c i l lViUn n ii tnts h pe o r to ifP oe n i c V i fl olOri rn aS lu s -
e C R Y S T A (L 6L 9I 5N M)Ie: TetYth rs ee q u i r e m e n t s p e n st ia o k en n :
e P H ( 7 9 1 )
S a m sp o ll e u t Pi ro en p:a as ru es p e cn osnitoa ni n i n g R e s = u (lr tu / xt (s C) s /xCP ux )1 0 0
3 0m g /
o fPm e L
n i c V
i il nlw ian t e r
A c c e pc t
r ia t ne 2c
r i.ea5 : - 4 . 0 t u = s uo mft h pe e r a ek s p o fnp s- e s
e W A TD E
E T
R E R M (I 9 N2 A1M T ItIO:hN oNd M2 T
)e, . 0 % h y d r o x y p Vean nip d ce in il cl V
iiflnlr it on hm e
S a m spo ll eu t i o n
A D D I TR IE OQ NU AI LR E M E N T S s uo mft h pe e r a ek s p o fnp s- e s
' P A C K A A GNSIDTN OGR AP G r eE s:ientr iv gec hotn t a i n e r s l .s h y d r o x y p Vean nip d ce in il cl V
iiflnlr it on hm e
e L A B E L LI aNbiGtet:loi n d i tc ha iattites t ob eu s ie nd
t h e S t a n s do al ru td i o n
m a n u f oa fnc ot nu pr ae r de rn uto g en r
s
l ya .l G s = c o n c e n ot fr U aSPtPe in oi cn V i Pl lo itn a sR sS i u
e U SR P
E F E RS ET NA CNE D
( 1A 1R) D S i nt h Se t a ns do al r u ( td imo gn / m L )
a e U SP Pe n i c iG lP lo i tn a sR sS i u m C u = n o m ic no an l c e n ot fpr ean ti ic V ioilnnlt ih ne
r a U SP Pe n i cVi Rl lS i n S a m spo ll eu (t Pi eon ni cViU l nl ii nt s / m L )
s
U SP Pe n i c V
iP l lo itn a sR sS i u m P = p o t eo fnp ec nyi c V i il nlU i SnP P e n i cV i l l i n
aD
-) P o t a sR s
S( i
P eu nmi cViU l nl i
i nt s / m g )
= A c c e pc t
r iat ne r9
c ie
0a .
: 0 % - 1 2 0 . 0 %
)
= P E R F O TR EMS AT N S C E
a P e n i cV
i fl olOr
irn a
S lu s p e n s i oe n
U N I F OO RFDM OI S
T UY
A NGI(ET9 S0 5 )
F os ro l ip das c k i ans gi en g
d l ce o- u n nt ia ti Mn ee er t
s :
s
= ) D E F I N I T I O N t h re e q u i r e m e n t s
P e n i cV
i fl ol Ori rn aS lu s p ei sna sd ir mo e ne i c i l le iDn E L I V V
y in x to ufP r E RO ALB(UL6 ME
9 8EM) e: et th rse e q u i r e m e n
Vw i to rhw i t ho on ouertm o sr u ei t ba ub fl fece or ls o, r s ,
f l a v oa rnss d
,u s p e na dg ie nnI tgtc so .n t N
a iL 9 0 . 0 % S P E C TI EF S
nTs I T
C S
a nN d M1 T 2 0 o.ft0h % le a b e nl eu dm obfP ee nri c V
i l l i n P H
( 7 9 1 )
U n i wt sh ce o nn s t ai std ui tr e cd t e d . S a m spo ll e
u t Ci oo nn s: ta isdt iu rt eeicn tt he ld
ea b e l i n
A c c e pc tr ia t ne 2rc i.ea0 :- 4 . 0
I D E N T I F I C A T I O N ' W A TD EE TR E R M (I9 N2 A1M T )e,ItO
| :hN o
N dM1 T . 0 %
e A . T hr ee t e nt ti iomofe tn h pee n i c V i pl le ioanft k h e
S a m spo ll eu ct io or nr e ts otp ho oan fttd hsSe t a n s do al ur -d A D D I TR IE OQ NU AI LR E M E N T S
t i o an s
,o b t a iintn heAeds s a y . ' P A C K A A GNSIDTN OGR AP r G eE s:ientr iv gechot n t a i n e r
e L A B E LI I t mN G ab:yel a b ei ln te ed ro fmt sh we e i og fh t
A S S A Y p e n i cV i cl lo in n t at hi en reied
n ia nd ,d i tt ooi roi n s to ef a d
e¢ P R O C E D U R E U n i to s nt, h bea s ti hs a1 t6 P0 e 0n i c V i Ul nl iiantr see q u i v
M o b pi hl ae sA e c e: t o n gi lt ar ciailcaeel,at ci icad ,n d a l et no1 t m og fp e n i c Vi .l l i n
w a t( e3 r55 0. :765 5: 0 ) e U SR PE F E RS ET NA CNE (D 1A 1R) D S
S y s stu ei m t a bs io ll iu tt y2i .om5n g
: /e a m ocLfh U S P . U SP Pe n i c iG lP loi tn a sR sS i u m
P e n i c iG lP loi tn a sR sSai nuUdmSP Pe n i c V iP l lo itn a s s i u U mSP Pe n i c V i Rl lS i n
R Si n M o b pi hl a e s e U SP Pe n i c V i Pl lo itn a sR sS i u m
S t a n sdo al ru t d 2i .om5n g: /o fm U S LP Pe n i c V i Pl lo i- n
t a s sR iSi un Mm o b pi hl a e s e
S a m spo ll e u t Ci oo nn s: tP ie tn iu ct Viefl ol Ori rn aS lu s -
p e n sa isd oi nr e icn tt he ld ea b e lT ir na gn.ass fu ei rt a b l e
a l i q fu ro et s,mh il yxa enfddr ef er aoi mbr u b b al en sd , P e n i c V i Tl al bi l n e t s
c o n t a ai bn oi4 un0t g0 ,P e0 n 0i c0 Vi Ul nl iitntoass u i t a -
b l ve o l u mf el at sDrkii. lcw u ti etM ho b pi hl a et sov eo l - D E F I N I T I O N
u m e a ,
n md it xoo b t aa si on l u ct oi o n nt a ni on mi in ng a Ple ln iy c V i Tl al ib nlc eo tn stNa Li9T n 0 .a 0n % Nd M1 T 2 0 o.f 0 %
a b o4u 0tP0 e 0n i c V i Ul ln ii nt sP /a msa psL o. r to ift oh n i s t h le a b e nl u e dm obfP ee nri c V i Ul nl ii tn s .
U S4 P1 O f f i M
c ioa n
l o g/ Pr ean p
i chi3sl2l 1
i n5

I D E N T I F I C A T I O N S P E C TI EF S
I T
C S
e A . T hr ee t e nt ti iomofetn h pee n i c V ipl le ioan ftk h e e W A TD E
E TR E R M (I 9 N2 A1M T
)e,I tO
I :hN o
N dM3 T
. 0 %
S a m spo ll eu ct io or nr e ts ot
p ho oan fttd hsSe t a ns do al ur -d
t i o an s,o b t a i in tn heAed
s s a y . A D D I TR IE OQ NU AI LR E M E N T S
e P A C K AA GNSIDTN OGR AP G r eE s:ientr iv gech ot n t a i n e r s .
A S S A Y e L A B E L TI h NT Gea :b l me a t bsyel a b ei ln te ed ro mft sh e
' P R O C E D U R E w e i og fph e tn i c V i cl lo inn t a t hi en reie ndia nd ,d i tt ooi ro n
M o b pi hl ae sA e
c e: t o n gi lt ar ciailcaeel,at ci icad ,n d i n s to efU an di to sn
t, h bea sti hs a1 t6 P0 e 0n i c V
i Ul n
l ii nt s
w a t( e3 r55 0. :7 65 5: 0 ) a r ee q u i vt ao1 l m e nogtfp e n i c Vi .l l i n
S y s stu ei tm a bs io ll iu tt y2i .om 5n g : /e m a ocLfhU S P e U SR P E F E RS ET NA CNE (D 1A 1R) D S
P e n i c iG lP lo i tn a sR sSai nuUdmSP P e n i cV i Pl lo itn a s s i u U mSP Pe n i c iG lP lo i tn a sR sS i u m
R Si n M o b pi hl a e s e U SP P e n i cV i Rl lS i n
S t a n sdo al ru d t 2i .om
5n g: / o fm U S LP P
e n i c V i Pl lo i- n U SP P e n i cV i Pl lo itn a sR sS i u m
t a s sR iSi un Mm o b pi h l a
e s e
S a m spo ll e u t Ni oo n m :i n4 a0 lP0el 0nyi c V i Ul ln iin t s / m L ,
i n M o b pi h l aed si es s of lrvfoeimdn ep l oy w d T ea rb le edt s
( N 2L 0T )S .h af ko are b o5 um t i nP . a sa ps o r to iftohn e
s o l u tt ih or noa s uu gi htf ai lbto lef0r e. 5 -o 1rf 4i mn pe or r e
s i z e . P e n i cV i Bl e l in nz a t h i n e
C h r o m a t soygs rt ae pm h i c
( S eC eh r o m a t ( 6o 2g1Sr)y a,s pStuhei ym
t a b i l i t y . ) tooO
< n
M o d Le C: fe)
i li e sN . o A N
D e t e c Ut o V2 r5 :n4 m 0 . N

C o l u 4m - n x :m3 m0 - pc am c; kL i 1 n g
F l ro awt 1e :m L / m i n
= H H
; S

I n j e cv to i lo un 1m 0 pe L:
S y s stu ei m t a b i l i t y ( C i g H i + e CN i2 sO Hs a9
S o)4 N21 2. 1 2
S a m p lS e y s st :u ei mt a bs io ll iu taty in Sodnt a n d a r d 4 - T h- ia az -a1 b i c y c l o [ 3 . 2 . 0a ]c ih3de,,p3 t- a n e - 2
s o l u t i o n d i m e t h y l - 7 - 0 x o - 6 - [ ([ 22 5- -p (5h2aea,,n ,o x y a
[ N o t re e l aT rthie e vt ee nt ti imfoeonpsr - h y d r o x - 6 8 ) ]c - o, m wp idtN .h
, N - b i s ( p h e n y l m e t h y l
y p e n i cV i,p le ln ii nc Gi ,lal ni pnde n i c iVla lriaen b o u t e t h a n e (d2 i: 1a )m. i n e
0 . 40 ,. 8a, n 1 d. c0 e, p e c t i v e l y s ) ( 2 5 , 5 R , 6 R ) - 3 , 3 - D i m e t h y l - 7 - o x o
S u i t a br ie l qi ut yi r e m e n t s 4 - t h -i aa z- a1 b i c y c l o [ 3 . 2 . 0a ]c hi ed p t a n e - 2
R e s o l u Nt Li3 oT. nb0 :e t wp ee n ei n c iG la lnip dne n i c i l c
- o m p w oi utN h, n Nd - d i b e n z y (l 2e :t 1h) y l e n e d
l i nV ,S y s stu ei m t a sb io ll iu tt yi o n [ 5 9 2 8 - 8 4 - 7 ] .
C o l euf m f inc i Ne nL 1T8 t0 h0e o r ep lt ai tcS eaysls, - T e t r a h y1 d0 r1a 3t .[e 26 13 6 9 0 - 5 7 - 3 ] .
t es mu i t a bs io ll iu tty i o n
R e l a st t i va en dde av ri da tNi M o 1nT pr e n i c i l»-P e n i c V
.: f0 o % i Bl le inn z ahta haspi on t
e eo fnn co yt =
l i nV p o t a s Ss ti au nms d,o al r
u td i o n l e sts h a1 n0 a
6 n
0 ndo mt o tr he a1 n2 P4 e 0n i c i al )l i n u v
A n a l y s i s V U n i pt esmr g .
S a m p lS e t s a n:s do al rua td in oSdna m spo ll eu t i o n
C a l c ut lh pae e t er c eo nftth ale ag be e nl u e dm obfP ee r n i -P a c k a a g nsidtno gr a g e i ntP i rgceh otsn et ar iv nee r s .
c i l lViUn n ii tnts h pe o r to ifT oa nb lt ea tk se n : |
U SR Pe f e r s e t na c n e d
( a 1 r 1 d ) s
U SP Pe n i c V i Pl lo itn a sR sS i u m K o }
R e s = u (lr tu / xr (s C) s /xC Pu )1 x 0 0 =
2
C r y s t a (l 6l 9i5 n)m i:et et y th rse e q u i r e m e n t s m. e ]
t y = s uo m ft h pe e r a ek s p oo n fp s- e s p (H 7 9 1 )b :e t w4 e . a0e nn6 d. 5i ,na s u s p ecno sn it oa ni a n=-
h y d r o x y p Vean nip d ce in il cl V
iifl nlr iton hm e i n ag b o3 u0mt g p e mr L .
S a m spo ll eu t i o n W a tD e e rt e r m iM ne at| th ( i 9o 2od1n)b ,:e t w5 e. e0n %
r s = s uo m ft h pe e r a ek s p o fnp s - e s a n 8d. 0 % .
h y d r o x y p Vean nip d ce in il cl V
iifl nlr iton hm e
S t a ns do al r u td i o n P e n i cV i cl ol n i nt e n t a b T or4 ua0 mtnga s,cf ceu rr a t e l y
G s = c o n c e n ot fr U aSPtP e in oi cn V i Pl lo itn a sR sS i u wm e i g thoae 1d 0, 0 v-o ml Lu mf el at sark d i, mcd e t h tao n o l
i nt h Se t a n s do al r u ( td im o gn / m L ) v o l ua mn m edi, xC .o n c o m di et ta en r tt hmlaeiybn se o r b a n c e
C u = n o m ic no an l c e n ot fpr ean ti ic V ioilnnlt ih ne o ft h is so l u at in oodfan s i m i l ap r rl ye p Sa tr ae ndsd oal ru dt i o n
S a m spo ll eu (t Pi eon ni cViU l nl ii nt s / m L ) e e w i ta hb o3u0m t og f U S PP e n i c V i Pl lo itn a sR sS i u m
P = p o t eo n fp ec nyi c V i il nlU i SnP P e n i cV i l l i n a tt h we a v e lo efmn a g tx hi a bm suo mra btaa bn oc 2 ue7t 6
P o t a sR sS ( iP eu nmi cViU l nl i nt s / m g ) p i D n e t e r t hm pei enrec eo nfp te a n ig ceVi tl la ik bne y
tn h e
A c c e pc tr ia t ne c r9 ie0a .: 0 % - 1 2 0 . 0 % o r m u l a :
P E R F O TR EMS AT N S C E P (/ aas )u
e D I S S O (
L 7
U 1
T 1I )O N
M e d iW ua mt 9
e: r0 m
;0 L i nw h iPics th h pe e r c e cnotnatogefpen
e nt i c V
i il nlt ih n e
A p p a 2r :a5t0ru ps m U SP P
e n i c V
i P
l lo itn a s
R Ss a
,i nua d
mya na dsa r te h ae b s o r p -
T i m 4e 5
:
m i n t i v i ot ifte hs seo l u ot ft i oh sne p e c ai nmtdeh Sne t a n d a r d
S t a n sdo al ru t
d Ui oSPnP
e :n i c V
i Pl lo itn a sR sSi i
n u m s o l u tr ie os np ,e c tb iev te w l6ye2 : e.an 3n 7%d 2 .i s f 5 o% u n d .
M e d i u m A s s a y
S a m spo ll e
u t Si a
o nm :pp elDrie s s o l( u7 t1 iD1 oi) nl. u t e S t a n pd ra er pd a r a t ia o sd ni r e P fcroteSreptdaa rn e d a r d
w i tM he d i
ifn
u em c,e s ts oaa cr oy n, c e n tt hr iaas tt i o pn r e p a r u an tdlieoo r dn o mAestsr ai yc A n( t4 i2 b5 u i)s o,i tn ig c s
s i m it lota hr Se t a n s do al ur td i o n . U SP Pe n i cV i Pl lo itn a sR Ss .i u m
T o l e r a Nn Lc 7T e 5
s( :%
Q o)ft h le a b eal m e do ouf n t A s sp ar ye p a r a t i oa qn us a nDotifPisetsn yio clV i vl el i n
p e n i c V
i (l C
l ii ns H i si s Nd 2i Os 0
s o
s l
S v) e d .
B e n z ai tn1h. iN 0 ns eo d hi yu dm r o t oxo ib dt aae si on l u t i o n
e U N I F OO RFDM OI S
T UY
A NGI(ET9 s0 5 M) e
: te ht e c o n t a 2i 0 n Pi0en0n gi c V
i Ul nl ii pnt esmr L P .i p2e mt oL ft h i s
r e q u i r e m e n t s s o l u it ni tao gon l a s s - s t1 o 2p 5p ce-ormne iLfd cl,aa sa
lk n, d
u s aest h Ae s sp ar ye p a fr oaIrtn ia oc nt i av n
at td
i it ro an t i o n .
 3 2 P1 e6n i c /i Olflfii nM
c ioa n
l o g r a p h s U S4 P1

D i l au t u e ao fPe e n i cV i Bl le inn z aq tu ha in nt ie t a t i Pv een li yc V i Ul nl ii pnt esmr L i ,n t h Ae s sp ar ye p a fr oaIrtn ai co tni -

w i tw ha tt eoo rb t aa si un s p e cnosnit oa n2i 0 n Pi0 en0n gi c i l vl ai nt ia ontn d
i t r aot n it oh nbea soi fts h ve o l ou fOm r eaS lu s -
V U n i pt esmr L P.i p2e mt oLft h is s u s p ei nn sta i og lo an s s - p e n st ia ok ane nntdh ee x t oe fdn itl u t i o n .
s t o p p 1e 2 r e5 cd-o, mn iLf cl aa sa
lk n, uds aest h Ae s sp ar ye p a -
r a t fi oo trn h Be l ad ne kt e r m i n a t i o n .
P r o c e d u ra esd i Pr r e fcootc Prere doe cd eu dn udlr eoe dr o -
m e t Ar si sc a y A n( t4 i2 be 5 i)x o,c tienitpchtIsen a c t i av n a td i o n
t i t r ao tfti ho sne p e c tioom me tinhtae d d i ot f2i .o m 0n oL f P e n i cV i Pl o l it na s s i u m
1 . N0 s o d hi yu dmr o Cx aildc eut.lh pae ot t e e innPc eyn ,i c i l l i n
: a ip e mr gi ,n t h P ee n i c V
o r m u l a :
i Bl le inn z atta hk b ie n
ytn eh e
¢
( A (I )/ B( -2 D ) a O e
i nw h iD ic htsh ce o n c e n ti nr ma tpg ie mor L n o,,ft h Ae s s a y
p r e p a fr oaIrtn ia oc nt i av nat td
i it or naot nit oh nbea s oi fts h e
w e i og fPhe tn i c Vi Bl le inn z attahki aennntedh ee x t oe fn t C i s H i 7 K N 2 0 5 S 3 8 8 . 4
d i l u t i o n . 4 - T h i a - 1 - a z a b i c y c l o [a 3c .i 32d ,. , 30- ]d hi e-
m e t h y l - 7 - o x o - 6 - [ ( mp o h en no op xoy ta ac
s a l t[ , 2 5 - ( 2 0 , 5 0 , 6 8 ) ] - ;
M o n o p o (t 2a5 s, s5 i R ,u 6m R ) - 3 , 3 - d i m e t
y a c e t a m i d o ) - 4 - t h i a - 1 - a z a b i c
P e n i cV
i Bl e S ilu ns ep e n ast [i
l in nz aO tr ha e1 o
3 2 n- 9 8 - 9 ] .
D E F I N I T I O N
»P e n i cV i Bl le inn z aO tr haSilun se p e cnosni- o Pne n i c V
iP l lo itn a sh sa ai
spu om t eo n
fN cLy1T3 a
8 n
0Nd M T
t a i nn osl te sts h a9 n0 p. e0 r ca ennndto mt o r e 1 6 P1 e 0n i c Vi Ul ln iin t s / m g .
t h a1 n2 0p .e 0r co eft nh lte a b e nl u e dm obf e r I D E N T I F I C A T I O N
P e n i cV i Ul ln ii pnt esmr LI t.c o n t oa n
i onerm
s o r 'eA .I N F R AA BR SE O D R(P1 T9 I7 OK N)
s u i t ba ub fl fece or ls o,dris s, p e rf sl aa nv ot a rsns,d , e B . T hr ee t e nt ti iomoften h pee n i c V ip l le ioanftk h Se a m -
p r e s e r v a t i v e s . p l se o l u ct o i or nr e ts ot p ho aon tftd hsSe t a ns do al ur tdi o n
a so b t a iintn heAeds s a y .
P a c k a a gnsidtno gr a g e i ntP irgcehos tn et r a ia v nnee dr se, C
s t oi rnae r e f r i g e r a t o r . D i l u eG nl ty :c ae rn widan t( e2 r5 : 1 4 )
L a b e l mi anybg el a Ib tei lnte ed ro mft sh we e i og fph et n - S o l u At :i1 o0nm6 g / o fms oL d ci au rm b io nnw aa tt ee r
icilV l ic no n t a ti hn ee rdie nia nd ,d i tt ooi roi nn s to efUan di t s , S o l u Bt: i1 o2nm0 g / o fm s oL d siu ul fmi in Dd ie l u e n t ,
= o nt h bea sti hs a1 t6 P0 e 0n i c V i Ul ln iiantr see q u i vt ao l e n t p r e p aasfro el dl Do iw s .s so lo vd esiu ul fmi inDd ie l u e n t
a 1 m og fp e n i c Vi .l l i n u s i an bg o 4u 5 to f% t hf ei n v a lo l au nmh de a At l. lt oo w
i ]
U SR Pe f e r s e t n a c n ed( a 1 r 1 d ) s c o o a
l ,
n d di l uw t
i et
D ih l u
t eotn htf ei n va lo l u m e .
a ) U SP P e n i cV i Pl lo itn a sR sS i u m S o l u Ct :i1 o5nm0 g /o ftm a Lr t aa cr ii cndw a t e r
re} S a m spo ll e u t 0i. og 1 no :fP e n i c V i Pl lo itn a s i ns i u m
rs U n i f oo rfdm oi stuayngi(et 9s 0 5 ) 2 m oLfw a t e r
Co F O SRU S P E PN AS CI KOIANNSG IEN DG L E C -O UN NT IA TIm Ne EeR tS s: A n a l y s i s
= t h re e q u i r e m e n t s . P a 1r :t A d1 dm oL fS o l u At ti oto h n Se a m sp o ll eu t i o
a. D e l i v v e ro al b ( u6l m
9e 8e)m :e et th rse e q u i r e m e n t s a. nh de a t .
= . p (H 7 9 1 )b :e t w6 e. a0e nn7 d. 0 . P a 2r :t T ot h he os to l u ft ir oPo namr1 ta d 0d. 0m 5oLf
S o l u Bt. i o n
A s s a y P a 3r :t C o o t hl me i x tf ur rPo eam r2 it n i c ew da ta enrd
S t a np d aer p d o ea sd ie r e fc otSret da n d a r d a d 2dm oL fS o l u Ct .iA ol nlt oos wt a n d .
p r e p a r u an tdlieoo rdn o mAestsr ai yc A n( t4 i2 b5 u i)s o,i tn ig c sA c c e pc t r ia t ne M c
r iea e:t th rse e q u i rf eo Prm aer nt ts s
U SP Pe n i c V i Pl lo itn a sR Ss .i u m 1 , 2 , a n3 d
A s s pa ry e p a r a t ia onan cs c uD rimal etu eat les vyuo r l -e d P a 1r :t N po r e c i ips if to ar t me e d .
u mo e fOraS l u s p e fn rs ei so mh nil ,yxa enfddr ef er ao i mr P a 2r :t N po r e c i ips f i to ar tme e d .
b u b b ql ue as n,t i tw ai tt1i . hvN0esl oy d hi yu dm r o t oxo ib d- e P a 3r :t A w h ip tr ee c i ips if to ar tme e d .
t a ia ns o l u ct oi n o nt a 2i 0n Pi 0 en0n gi c V i Ul nl ii pnt esmr L .
P i p2e mt oL ft h is so l u it ni tao ogn l a s s - s t1 o 2p 5p e- rme L Ad ,S S A Y
c o n if cl aa sl ak n, u ds aest h Ae s sp ar ye p a fr oaIrtn ia oc tn i v a' -P R O C E D U R E
t i oan nt id t r a Dt ii ol nua. tna ec c u r ma et ea ls yvu or leoudf m e M o b pi hl ae sA e c e: t o n gi lt ar ciailcaeel,at ci icad ,n d
O r aS lu s p eq nu sa in o t in tw ai ttwi hav tet leooyrb t aa siu ns - w a t( e3 r55 0. :765 5: 0 )
p e n sc io on nt a 2i 0 n Pi0en0n gi c Vi Ul n l ii pnt esmr L P.i p e t S y s stu ei m t a bs io ll iu tt 2 yi .o m5n :g /e a m ocLfhU S P
2 m oL ft h is su s p ei nn sta igo o l an s s - s t1 o 2p 5p ce-ormneiLd- , P e n i c iG lP loi tn a sR sSai nuUdmSP P e n i cV i Pl lo itn a s s
c a fl l a sak n, uds aest h Ae s sp ar ye p a fr oatrth iBeol nad ne k- R Si n M o b pi hl a e s e
t e r m i n a t i o n . S t a n sdo al ru t d 2i .o m5n :g / o fm U S LP Pe n i c V i Pl lo i- n
P r o c e d u ra esd i Pr r e fcootc Prere doe cd eu dn ud/r eoe dr o - t a s sR iSi un Mm o b pi h l a e s e
m e t Ar si s c a y A n( t4 i2 b5e i)x o,c tienitpchtIsen a c t i av n a td i o Sn a m spo ll e u t 2i .om 5n g : / o fPm e L n i cV i Pl lo itn a s s i
t i t r at tooi omn ti htae d d i ot f2i .o m 0n oL f1 . N 0s o d i u m i n M o b pi h l ea s e
h y d r o Cx ail dc eut.lh qaeut ae n ti niP te yn i, c V i Ul nl ii itn ns , C h r o m a t soy gs rt ae pm h i c
e a cm hoL ft h Oe r aS lu s p e tnaski beoytn nh fe o r m u l a : ( S Se h e r o m ay (t 6o2 g1S r )y ,a
s Stpu ei m
t a b i l i t y . )
M o d e :
( T 2/ D ) 1 F ) B - D e t e c Ut o V
2 r5 :n4 m
C o l u 3m .n 9: x-3 m0 m- 1 c m 0 ;-p ua m c kL i 1 n g
i n w h iTic ts h h le a b eq lu ea dn ti niP te yn i, c V i Ul nl ii pnt es r F l ro awt 1e :m L / m i n
m L i ,n t h Oe r aS lu s p e na sn i Ddi os tn h,ce o n c e n ti nr a t i o In n, j e cv toi lo un 1m 0pe L:
U S4 P1 O f f i M
c ioa n
l o g/ Pr ean p
i chi3sl 2l 1
i n7

S y s stu ei m t a b i l i t y A n a l y s i s
S a m p lS e y s st :u ei tm a sb io ll iu tatyin Sodnt a n d a r d S a m p Sl ae m:spo ll e u t i o n
s o l u t i o n C a l c ut lh pae et er c eo nfp t- ah gy ed r o x yVpi net nh iec i l l i
[ N o t rt e l aT rthie e vt ee nt ti imfoeonpsr - h y d r o x - p o r to ifP oe nn i c V
i Pl lo itn a st s
a ki eu nm :
y p e n i cV i,p le ln ii nc Gi ,lal ni pnde n i c iVla lriaen b o u t
0 . 40 ,. 8a , n 1 d. 0r ,e s p e c t i v e l y . R e s u= (l r tu / xr 1 r )0 0
S u i t a br ie l qi ut yi r e m e n t s
R e s o l u Nt Li3 o T. nb0 :e t wp ee n ei n c Gi la lnip dne n i c i l t - u = p - h y d r o x yVpp een a ri e
kc si pl fo
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ee
l i Vn , S y s stu ei m t a sb io ll iu tt yi o n S a m spo ll eu t i o n
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s o l u t i o n Vp e a r ek s p of nr sto ehmS se a m spo ll e u t i o n
A n a l y s i s A c c e pc t r ia t ne crNieaM5 : T . 0 %
S a m p lS e t sa n:s do al r ua td in oSdna m spo ll eu t i o n
C a l c ut lh pae t o et eo n fp ec nyi c V i pl lo it na si sn i P ue mn ,- S P E C TI EF S I C T S
icilV l iU n n i t s i n/t mh pge o, r to ifP oe nn i c V i Pl ol it na s -' O P T IR CO AT LA ( T7 I 8 1OS5 NpS e) c,Ri o f it ca t i o n
s i tu am k e n : S a m sp o ll e u t 1i 0om n g: / o fPm e L n i cV i Pl lo itn a s i ns i u m
c a r db io o nx i dwe a- tf er er e
R e s = u (lr ty / xr (s C ) s /xCP u ) A c c e pc t r iat ne + rc i2ea 2:t 0o+ 2 3 5
' C R Y S T A (L 6L 9I 5NM) Ie: TetYth rse e q u i r e m e n t s
r u = s uo m ft h pe - h y d r o x yVpa e nnp di e nc ii cli ll il¢ni Pn H( 7 9 1 )
Vp e r a ek s p of nr st o ehm S se a m spo ll eu t i o n S a m spo ll e u t 3i 0 om n g: /o fPm e Ln i c V i Pl lo itn a s i ns i u m
f s = s uo mft h pe - h y d r o x yVpa ennp dei nc ii cli ll il n i nw a t e r
Vp e r a ek s p of nr st o ehmS se t a n s do al r u td i o n A c c e pc t r ia t ne 4rc i.ea0 :- 7 . 5
G s = c o n c e n ot fr U aSPtPe in oi cn V iPl lo itn a sR sS i ue mL o s O sND R Y (I 7N 3G1 )
i nt h Se t a n s do al r u ( td im o gn / m L ) S a m p 1l 0em0: og fP e n i c V i Pl lo itn a s s i u m
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S a m sp o ll eu ( t imo gn / m L ) u n dve ar c aut6u 0fm o 3rh .
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( P e n i cVi le l i n a e
A c c e pc t r ia t ne rc
1 ie 3a 8: 0 -P e1 n 6i c1 V l lni in t s / Am D
i 0U g D I TR IE OQ NU AI LR E M E N T S
' P A C K A A GNSIDTN OGR AP G r eE s:ientr iv gech ot n t a i n e r s .
I M P U R I T I E S ' L A B E L LI aN biGtet:loi n d i tc ha iattites t ob eu s ie nd t h e
e L I MOIFPT H E N O X AY CA IC DE T I C m a n u f oa fnc ot nu pr ae r de rn uto gen srl ya .l
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w a t( e3 r5 : 1 : 6 5 ) U SP Pe n i c iG lP loi tn a sR sS i u m
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b l be u f f ce or ls o,f rl sa ,v po r se ,s e r vaa ntsd i uv se p s ,e n Bd e i) n g
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l ii w
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S u i t a b ri le i qt u
y i r e m e n t s I D E N T I F I C A T I O N
T a i l fia nc gt o Nr : M1 .T5 e A . T hr ee t e nt ti iomofe tn h p ee n i c V i pl le ioanftk h e
R e l a st t i va en dd eav ri da tNi M o 2nT .
: 0 % S a m sp o ll eu ct io or nr e ts ot p ho oan fttd hsSe t a n s do al ur -d
A n a l y s i s t i o an s,o b t a i in tn heAeds s a y .
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o dna m spo ll e u t i o n
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p o r to ifP oe nn i c V i P
l lo itn a st s a ki eu nm : e P R O C E D U R E
M o b pi hl ae sA e c e: t o n gi lt ar ciailcaeel,at ci icad ,n d
R e s = u (lr tu / xr (s C) s /xC1u 0) 0 w a t( e3 r55 0. :765 5: 0 )
S y s stu ei m t a bs io ll iu tt y2i .om 5n g: /e m a c oLfhU S P
t u = p h e n o x ay caipcdee a rt e ki sc p fo rn tos hm
ee e n i c i Gl Pl io nt a sR sSai nuUdmS PPe n i c iVlPl oi nt a s s i u m
S a m spo ll eu t i o n R Si n M o b pi hl a e s e
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e e S t a n sdo al ru d t 2i .om 5n g : / o fmU S LP Pe n i c V i Pl lo i- n
S t a n sdo al ru td i o n t a s sR iSi un Mm o b pi h l ae s e
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S t a n s do al ru ( td im o gn / m L ) O r aSl o l u at sdi io rn e icntt he le da b e lT ir na gn .ass fu ei tr -
C u = c o n c e n ot fPr ean ti i c Vio P
ln lo itn a s
i nst ih ue m a b la el i qc uo on tt a ni on mi in n4g a0 l0 l,Pye0 n 0i c0 V i l l i n
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e L i mOiFpt - H Y D R O X YVP E N I C I L L I N i n ng o m i n4 a0 lP0le 0ny i c V i Ul ln iin t sP /a m sa psL o. r t i o n
M o b pi hl ae s S ey ,s st u ei m t a bs io ll iu ttSyito an n , d a r d o ft h is so l u tt i h or noa s uu gi htf ai lbto lef0re . 5 o- ru m
s o l u tSi ao m n s,po ll e u tCi ho r n ,o m a ts oy gs r t ae mp ,hf ii ncpe or sr i ez e .
aS y s stu ei tm a b iP lri ot c ya :e
sd ei dr e icn tt he d
e
s a y .
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c ioa n
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C h r o m a t soy gs rt ae pm h i c I D E N T I F I C A T I O N
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t a b i l i t y . ) e A . T hr ee t e nt ti iomofetn h p ee n i c V ipl le ioan ftk h e
M o d Le C: S a m sp o ll eu ct io or nr e ts otp ho aon tftd hsSe t a n s do al ur -d
D e t e cU t o
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:
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1 n g
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I n j e cv toi lo un 1m 0pe L: e P R O C E D U R E
S y s stu ei m t a b i l i t y M o b pi hl ae sA e c e: t o n gi lt ar ciailcaeel,at ci icad ,n d
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s o l u t i o n S y s stu ei m t a bs io ll iu tt 2 yi .om5n g : /e a m ocLfhU S P
[ N o t re e l aT rt hie ve t ee nt ti imfoeonpsr - h y d r o x - P e n i c iG lP lo i tn a sR sSa inuUdmSP Pe n i c V i Pl lo itn a s s i
y p e n i cV i,p le ln ii nc Gi ,lal ni npde n i c iVla lriaen b o u t R Si n M o b pi h l ae s e
0 . 40 ,. 8a, n1 d. r0 e , s p e c t i v e l y ) S t a n sdo al ru td2i .om 5n g: / o fmU S LP Pe n i cV i Pl lo i- n
S u i t a br ie l qi ut i y r e m e n t s t a s sR iSi un Mm o b pi hl a e s e
R e s o l u Nt L i3 oT. nb0 :e t wp ee nei n c Gi la lnip dne n i c i l -S a m spo ll e u t Ti ro an n:as pf oe rr to iff oi nn ep loy w d e r
l i Vn ,S y s stu ei m t a sb io ll iu ttyi o n T a b l(e Nt 2Ls 0T)c ,o n t a inn oi mn i g na ab lo4lu0yt0 , 0 0
C o l euf m f inc i Ne nL 1cTy8 :t0 h0e o r ep lt ai tcSeaysls, - P e n i cV i Ul nl ii tn soa ,s u i t va ob l eu mf el at sDrkii. lc u t e
t es mu i t a bs io ll iu tty i o n w i tM ho b pi hl a et sov eo l um mitexo, o b t aa si onl u t i o
R e l a st t i va en dd eav ri da tNi M o 1n T.: 0S %t ,a n d a r d c o n t a ni on mi in na g ab lo4lu 0 ytP0e 0n i c V iUl ln i in t s / m
s o l u t i o n a n sdh af ko ae r b o5 um t i nP .a sa ps o r to ift oh nis so l u -
A n a l y s i s t i ot nh r oa su ug iht fai bl lto eef0r . 5 o- rfui mn pe or r e
S a m p lS e t s a n : sdo al ru a td in oSdna m spo ll eu t i o n s i z e .
C a l c ut lh p ae te er c eo nftth a le ag be enl u e dm obfP ee r n i - C h r o m a t soy gs rt ae pm h i c
c i l lViUn n ii tn ts h pe o r to ifP oe nn i c V i Pl lo itn a s s i u m ( S eC eh r o m a t ( 6o 2g1Sr)y a,s pStuhei ym t a b i l i t y . )
f o Or r aS lo l u tt ai ko en n : M o d Le C:
D e t e c Ut o V
2 r5 :n4 m
R e s u= (l r tu / xr (s C) s /xCP ux )1 0 0 C o l u 4m - n ~ :
m3 m0 - pc am c; kL i 1 n g
F l ro awt 1e :m L / m i n
t u = s uo m ft h pe e r a ek s p oo n fp s- e s I n j e cv toi lo un 1m 0 Le L:
h y d r o x y p Ve an nip d ce in il cl V
iiflnlr it on hm e S y s stu ei m t a b i l i t y
S a m spo ll eu t i o n S a m p lS e y s st :u ei m t a bs io ll iu ta
t yin oSdnt a n d a r d
r s = s uo m ft h pe e r a ek s p o fnp s - e s s o l u t i o n
h y d r o x y p Vean nip d ce in il cl V
iiflnlr it on hm e [ N o t re e l aT rt hie ve t ee nt ti imfoeonpsr - h y d r o x -
S t a n s do al r u td i o n y p e n i cV i,p le ln ii nc Gi , lal ni pnde n i c iVla lriaen b o u t
C s = c o n c e n ot fr U aSPtP e in oi cn V i Pl lo itn a sR sS i u m 0 . 40 ,. 8a , n 1 d.r0 e, s p e c t i v e l y
i nt h Se t a n sdo al r u ( td imo gn / m L ) S u i t a br ie l qi ut i y r e m e n t s
C u = n o m ic no an l c e n ot fpr ean ti ic V ioilnnlt ih n e R e s o l u Nt L i3 oT. nb0 :e t wp ee nei n c G i la lnip dne n i c i l
S a m spo ll eu (t Pi eonni cViU l nl ii nt s / m L ) l i Vn ,S y s stu ei m t a bs io ll iu tty i o n
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5
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pr e n i c i l
l i nV p o t a s Ss ti au nms d,o al r u td i o n
= P E R F O TR EMS AT N S C E A n a l y s i s
e U N I F OO RFDM oI s T UYa NGI(ET9 S0 5 ) S a m p lS et sa : n s do al r uatd in S
odna m spo ll eu t i o n
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a l e D E L I V V E RO ALB(UL6 M 9 8EM) e: et th rse e q u i r e m e n t s
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m ab yel a b ei lnte ed ro mft sh we e i og fphe tn i c V i l l i n M o b pi hl ae sM ee :t h aa nnB do u fl(f 6 e r5 : 3 5 )
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I C
T S
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A S S A Y ¢ P A C K A A GNSIDTN OGR AP G r eE s:ientr iv gech ot n t a i n e r s ,
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0 . 2m 5oLft h y bm lo u TlSe . e U SR P E F E RS ET NA CNE ( D 1A 1R) D S
A n a l y Tsiit sru:a nt eda e s tr r oe fn a im t r wo ig t 0e .hn1M U SP Pe n t a Im si edt ih niR eoS n a t e
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a n u dst eh i snp l ao cfte h Se t a n sdo al ru std pi eo cn i f i e
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r ie ea e:t th rse e q u i r e m e n t s
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A n a l y wt ai v c ae ll e2 n7 gn8t mh :
A c c e pc t r ia t ne Acr biesa :o r p tdi o nv o
i dtti if efb se yr A S S A Y
m o tr he a3 n. 0c %a ,l c u ol nat th dee rd ib ea ds i s . ' P R O C E D U R E
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A S S A Y d r o c h il no5 r0mi odLfge l a caica el at ci ic d .
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( 9 4 : 3 : 3 )
( 9 4 : 3 : 3 ) V i s u a l i Hz e a ta ihtpoeln a:i tnae no v ae tn 1 0 f5 o 1r 5
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v i eu wn ds eh ro r t - w aU vlVei gl het n. g t h A c c e pc t r ia t ne Trc ih
eta eo: t oa fla n oyr d i in ma pr uy r i
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c iea e:t th rs ee q u i r e m e n t s t i eos b s e dr ov e ne osdet x c 1e .e 0d % .
S P E C TI EF S I C T S S P E C TI EF S I CT S
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s l i gd ha tr k e n i n g A n a l y Ds r ia syta: p r e s ns ouetr xe c e e5 dmi om nf g
e L o sO sND R Y (I 7N 3G1 ) m e r ca tu1 r0 yt0 oc o n s wt ea in gt h t .
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m e r ca tu6 r0 tyoc o n s wt ea in gt h t .
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A D D I TR IE OQ NUAILR E M E N T S
c o n t a i n e r s .
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A GNSIDTN OGR AP r
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c o n t a i n e r s . U S PPe n t a R
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( 1A 1R) D S
U S PPe n t a Rz So c i n e
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l o g/ r
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M o d Le C:
P e n t a az on Acd ci e e a m i n o p Dhe tee n
n t c Ut oV2 r8 :n0 m
C o l u 4m .n 6: x-2 m5 m - 1
c m0 ;-p ua m
c kL i3 n g
T a b l e t s F l ro awt e1 :. m2 L / m i n
I n j e cv toi lo un 2m 0 ue L:
D E F I N I T I O N S y s stu ei m t a b i l i t y
P e n t a a z on Acd c
i n
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S a m p lS et s a :n s do al rua td in oSdna m spo ll eu t i o n
I D E N T I F I C A T I O N C a l c ut lh aep t e e onft h nle a e b e al m
e do ouf n t
e * o oC H R t O M A TI OD G E NR TA I PFTHIE C ISACTT I O N p e n t a (z Coi c9 Hi 2ni 7 eNtnO h)pe o r to ifT oa nb l e t s
2 0 1 t a k e n :
D i l u eC nht l: o r ao nfmdoe rt mh (a1 n: o 1 )l
S t a n sdo al ru Adt :i1 omn g /o fm
U S
LPPe n t a Rz S
o c i n e R e s =
u (lr tu / xr (s C
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i nD i l u e n t
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o h im Sen ae m p l e
p h Re Sin nD i l u e n t s o l u t i o n
S a m spo ll e u t Ti ro an n:as qf ue ar n ot ffi itn yb e la yn i s i e l ds =p e r a ek s p oo fpn es ne t a f z ro c o
t h im en e
T a b l ne to sm ,i ne aq l u il vyt aoal be n o5 tum to g ’ S t a n s do al r u td i o n
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o so u pi h
t ae - n ,
G s = c o n c e n ot fr U aSPtPei no tn a R z S oi nct ih ne e
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s o l ti dos se t t Ul es t.eh se u p e r n a t a n t . C u = n o m ic no a n l c e n ot fp r ae tn it oa inz n t o hc ei n e
C h r o m a t soygs rt ae pm h i c S a m spo ll eu ( t imo gn / m L )
D e v e ls oo pl ivs n ey n
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S p rr ae ya g eD ni st s: 3 o l0 m v0eog f p l a t ic nhi lc o irn i d e [ N o T e M
t hiteni imb mee it zwt ee h ae d nd i ot fti ho e n
1 0 m0 oLfw a ta enrad d 1d 0 m0 o L f p o t a si o s di iu dm e D i l uae nnt d thi en j e co tfti hoSen a m spo ll eu tt oip or ne -
s o l u (t 6ii no1 n0 0 ) . v e sn it g n i fh iy cd ar no otl fa y sc ies t a m ti opn- o p h e n
A n a l y Es vi as p : ot rh saeotl ev iencn ot oscli ,r c u l aa it ri. n g a m i n o p h e n o l . ]
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nn iodsl o,p r o p y l -
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' P E N T A Z O C I N E s e r f v e o hmAe s sf ao Pry e n t a iz mo mc ei dn iewait te hl =
r dt y
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a m i (n9 e6 0 : 4 0 : 2 ) m i n i hmy id zr eo ol fa y sc ies t a m tiopn -o ap mh ie nn o = p h e
D i l u eM net t: h aa nn0do. l0 N3s 5 u l f au cr i( cd 1 : 1 )
S t a n sd taosrcodkl u t 0i .om 5n g: / o fm U S L P
n o al n mdi x . t =}
o
C h r o m a t soy gs rt ae pm h i c
P e n t a R z S oi ncD ii nl ue e n t ( S eC eh r o m a t ( 6o 2g1Sr)y a,s pStuhei ytm a b i l i t y . )m e ]
i )
S t a n sdo al ru t dTi ro an n:1s 0f . em r0oL ft h Se t a n d a r d M o d Le C: =
s t os co kl u tt oia o1 n2 5 s-e mp L a r Aa td 3odr0m. oLf D e t e c Ut o V
2 r5 :n4 m
a )

w a ta enr5dm oLfs o d ci au rm b o s on la ut(t e1i :o 1n 0 ) . C o l u 4m .n 6: x-2 m5 m- 1c 0m -; p j a u m

c kL i3 n g
E x t rw aic t6t h0m oLfc h l o r ao nfpdoa rst m sh ce h l o r o - F l ro awt e1 :. m4 L / m i n
f o lr amy te hr r o f iul gtp ehar p ce or l, l e tc htf ei ln tgir na t e I n j e cv toi lo un 1m 0 ue L:
a 1 0 0 v-o ml Lu mf el at sDrkii. lcw u ti et c h l o rt oo f o r m S y s stu ei m t a b i l i t y
v o l au nmm dei x . S a m p Sl te a: n sdo al r u td i o n
S a m spo ll e u t Ti ro an n:as nfa e mr o nu onm ti ne a q ul il vy a - S u i t a b ri le i qt u y i r e m e n t s
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t a fn ot ur s i en t h Ae s sf ao Ary c e t a m iM ni on pi hm ei nz .e
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' U N I F OO RFDM oI s T UY
a NGI(ET9 S0 5 ) U S AP c e t a m Ri Sn o p h e n
P r o c e f odcruo rn etu en ni t f o r m i t y U S PPe n t a R z So c i n e
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M o b pi hl ae sT ee :t r a h y dp rh oo fs uparhca o inadr,,ni dc
0 . 0M 0 m5 o n o b s a o s d p i hc
u om s p ( 5h 0a :t 1e : 9 5 0 )
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U S AP c e t a m Ri Sinn Do i pl hu ee nn t D E F I N I T I O N
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a m i n o R Spt oha seu ni t va ob l eu mf el at sA rk id
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a n md it xod i s s to hl ave c e e t a m iD n i lo wuptihet eh n .
e * H a Cd H Ra O M A TI OD G E N R TAI PFT H IE C
ISACTT I O
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S a m sp tloseco kl u t Ti ro an n:1 s Tf ae brtl oea t1 0 0 - m L R Si n D i l u e n t
v o l u mf el at sa rk di, 5cd 0m oL fD i l u a e nntsd,o n i c a t e S t a n sdo al ru Bdt: i6 o5m n g /o fm U S LA Ps p iRr Sii n n
f o 3r 0m i n D .
i l w u t i etD hi l ut eovn ot l ua mne m di
, x . D i l u e n t
P a sa ps o r to ift oh n is so l u tt ih or noa u p g a hpf iel r
ter, S a u s opl e u t Si ho n aa :ka eu o ff i n ep l oy w d e r
c o v e tr hifenu gn wn i e tl
a wh a tg cl ahas n s d di s c a r d i nT ga b l ne to sm ,i ne a q u l il vyt aoal be n o2tu5m t og f
t hf ei r fs te mw oL ft hf ei l t r a t e . p e n t a a z on 6cd 5i mn0 oe
g fa s p i w r ii nt1, h0m oL fD i l u -
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s o l u wt i otM nho b pi h l aet so1e 0 m0 a L n p da ts hs is so - s e t t l e .
l u t it ohn r oa um ge hm bf irl a to efn
0
r . e 5 o- rf1i mn e r C h r o m a t soy gs rt ae pm h i c
p o sr iez e . D e v e ls oo pl ivs n ey ngs tt Ee tmh :y l . amc ee tt ha ta en ,o
C h r o m a t soy gs rt ae pm h i c a n fdo r am ci i(cd9 0 : 5 : 5 )
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M o d Le C: 3 0 m0 og fp l a t ci hn il co irn i 1 d0 e m0 oL fw a t e a rn ,d
D e t e c Ut o V
2 r2 : n0 m a d 1d 0 m0 oLfp o t a si s o di siu odmle u (t 6ii no1 n0 0 ) .
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F l ro awt e1 :. m5 L / m i n c i r c u al iar Pt. li natgc hepe l ai tn te h de e v e l co hp ai m n g-
I n j e cv toi lo un 1m 0ye l: b e ra, na df t de er v e l tohpeli antrge e, m i o t , av ne d
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s S a m p Sl ye s:stu ei m t a sb io ll iu tt yi o n o u g ihn w l ya cri mr c u al iar a t, in nedgx a mt i h penl ea t e
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= S u i t a br ie l qi ut i y r e m e n t s t h pe l a wt ie tS hp rr ae ya g e n t .
S R e s o l u Nt L i7 oTb ne: t wp ee e n t n a a z on cd i n e A c c e pc t r ia t ne cT
r iewap :o
r i n cs ip poaftlrs t o hm Se a m -
= a c e t a m i n o p h e n p l se o l u ct io or nr ei snR prv oa nl uds ei zs a e, ,ni dn t e n s i t
[ 3 R e l a st t i va en dd eav ri da tNi M o 2n T.: f0o t %r h e o fc o lw oir t hh pe e n t a sz po ooc ftS i t n a e n s do al r u td i o
a )
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A n a l y s i s r e s p e c t i v e l y .
S a m p lS e t s a n :s do al r u atd in S
odna m sp o ll eu t i o n
C a l c ut lh p ae te er c eo nftth a le ag be e al m e do ouf n t A S S A Y
p e n t a ( z o C ci is nH ae2 n7adNc Oe )t a m i n o p he eP nR O C E D U R E
( C s H is n t N hOpezo )r to ifT oa nb lt ea tk se n : D i l uA e: Mn e t t h aa nnwdoa lt( e1 :r 1 )
D i l uB e: Mn e t t h aa nn6doN hl y d r o ca hc li( od1 r: i 1 )c
R e s u= (lr tu / xr (s C) s /xC1y 0) 0 eC :n W a t m e re ,t h aa nn 6 odNl h,y d r o ca hc li od r
6 : 1 : 1 )
t u =p e ra ek s p oo fpn es ne t a oz ro c i n e C h r o m a t coo gl ru a Um spn
a eh2
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rs = p e a
r ek s p oofpn es ne t a oz ro c i n e f u st eoad b oa u1 t0 0 l-e mn ogmft5h - tm u mb hi an vg -
a c e t a m fi rn t oo hmpSe ht a
e nnsdo al r u td i o n i n ag s t o p actto hcbeko t otftoh m is se c t Pi lo anac. e
C s = c o n c e n ot ftr haaetp ip or no pU rS iPa t e p l e do gfg el atws o s ao ttl h be o t otfto hm5 e - pmo mr -
R e f e rS et na cn e
(d Ua P
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dn t a z
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U S AP c e t a m Ri S in)n t
o hpSeht ea nn d a r d t i to yfs u l f ao cn iic dca t i o n - r ee xs tic oanhb ae na gk ee r ,
s o l u (
t imo gn / m L ) a n wd a ts hh rteie mw ei st w ah t de ir s , c a tr hdweia ntg e r
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n l
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1

a c e t a m i in tn hoSe pa h
m se
po n
ll eu t i o n D i l uB ,ean ntadl lt oosw t af no1 drh . D e c ta hnaet
c i d
( m g / m L ) w a si fh i ti;s c o l oy re el dol ro rw a nr ge ep , te h aistst e p
A c c e pc t
r ia t ne rc
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a
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I M P U R I T I E S d e c a n ut nat ti i h
l wo
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P r o p (u2 c2 t
7 M)s e
: te htre e q u i r e m e n t s s l u orf rt y h we a s rhe es iidnDn i l uA e. Wn a t ts hh ceo l -
u mw n i t2 h5m oLfD i l uA e. n t
A D D I TR IE OQ NU AI LR E M E N T S S t a n sd o al ru Adt :i1o 8u n g /o fmU LSS Pa l i cA ycl iR i cdS
@ P A C K A A GNSIDTN OGR AP G r eE s:ientr ivg ehl ti ,g h t - r e s i s it na0 n. t1N s o d hi yu dm r o x i d e
c o n t a i n e r s .
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S t a n sdo al ru Bdt :i6 o2 nj . 51 g o/ fU m LS PPe n t a z o c i nt ae n Rt e . p te haaetc e at ci icw da s hf i o unm rgo tr iem e s .

R Si n D i l uCe n t W a ws ihtw ha tu e n tr5i .l m 0 oLft h we a tw eargs ihva e s
S a m sp tloseco kl u At :iT or na n as pf oe rr to iff oi nn e n e g l i rg ei sb lp w eo nh ssee u nb s t fi ot5ru .tm 0e oL
df
p o w fd re oNrmL 2T0 f r e s ph o l yw dT ea r b len e dto sm, i - S a m spo ll ue tai nocnda, r rti he rd ot uh Ag en ha l fy os ri s
n a l el qy u i vt aoal be n o2tu5mt og f p e n t a za on c di n e p e n t a z o c i n e .
6 5 m0 og fa s p ifr ri noN mL2 T0 f i n ep loy w d T ea r b le e dt s ,S a m sp o ll e u t Ti oao sn u: i t 5 a b0l -fe lm a Las kd 0 d. g4o f
t oa s u i t 2 a b5l 0ef -l amsAL k d. 1 d0 0 m. oL 0 fD i l uA , e n t S t r o bna gs lia ycn, i o n - er ex scai hnn 2 adn 5mg oLeft h e
a n sdh abk yme e c h a mn ei a cf aon 2rls 0m i n C .e n t r i f u gs e o l u ut in odtnee s rS t .h abk yme e c h a mn ei a cf aon 1rls5
a s u i t qa ub a l en ft oi5r tm yi n . m i nA .l lt oos we t t al en u , ds te h cel esa ur p e r n a t a n t .
S a m sp tloseco kl u Bt: iT or na n 2s 5f .em r0oLft h cel e a r I n s t r uc mo en nd ti at li o n s
s u p e r fn r a t
oS am
a nm t spt loseco kl u A t ti oot nh per e - M o d UeV :- V i s
p a rCehdr o m a t coo gl rufamopnlh,libocfy w iev1de 0 - m L A n a l y wt ai v c ae ll e2 n9 gn6tm (h m: a x a ib m s ou rmb -
p o r t oi fDo in ls uA e,c no tl l e tc hteeilnugia nat e 2 5 0 - m L a n cf eo t)r h aen a l oy fas si ps i r4 i0nn8 ; m ( m a x i m u m
v o l u mf el ta csr oki nc t a 1i 0n .m i 0noLgf2 . N 5s o d i u m a b s o r fb oatrnh acenea )l oy fsp ie sn t a z o c i n e
h y d r o Dxiil dwu eti .etw ha tt eov ro l ua mn m edi, x . C e l l1: . c m
S a m sp o ll eu At :iP oi np4e mt oLfS a m sp t loseco kl u t i o n B l a n0 k. :1N s o d hi yu dm r fo oxtrih adene a l oy fas si -s
Bi n t a o1 0 0 v-o ml L u mf el at sa rk in, dcdi l w u ti e0t .h 1N p i r it nh;ce h l o rl oa fy feo rrr t om hmre e a gb el naftno kr
s o d hi yu dm r o t oxv io dl eua mn m edi, x . t h aen a l oy fsp ie sn t a z o c i n e
S a m sp o ll eu Bt :iP oa nst sh r ot uh cg e o h l auf mt Sen ra m - A n a l y s i s
is t os co kl u Bt fii ov5ne - pm oLr t oi fDo in ls uB ,ef no lt - S a m p lS e t s a : n s do al ru Atd,iS ot na n sdo al r u Btd, ia onn d
l o w be y1d 0m oLfw a t Ce or l. lt eh ceetl u ia nta e S a m spo ll e u t i o n
1 0 0 v-o ml L u mf el at sa rk i
n
, dcdi l u w ti et w ha tt eo r F o ars p i tr ri na ,n 1s .f m 0e roL ft h Se a m spo ll eu tt oia o n
v o l u m e . 2 5 -v m o lL u mf el atcsrokinct a 1i. nm 0 i oLnfsg o d i u m
I n s t r uc mo en nd ti at li o n s h y d r os ox li ud(t1eii no1 n0 )a, ns dw i rA ll. lt oos wt a n d
M o d Ue V : f o 1r 0m i n D i. l w u ti et w h a tt eov ro l ua mn e m di, x .
A n a l y wt ai v c ae ll e2 n9 gn6tm (h m: a x a ib m s ou rmb - D e t e r t hm aei bnseo r ob fta hnSec ae mssp o ll eu a t in odn
a n cf eo t)r h aen a l oy fas si ps i r2 i7nn8 ; m( m a x i m u m S t a n s do al r u Atdai go an it nh sBetl af no tkr h aen a l oy fs i s
a b s o r fb oatr nh acenea )l oy fsp ie sn t a z o c i n e a s p i r i n .
C e l l 1: c m C a l c ut lh ae e t e a o ft he le a b e al m e do oufa ns pt i -
B l a n0 k. : 1N s o d hi yu dm r fo oxtrih adene a l oy fas si -s r i (
n C y H dgi Os xs )o l v e d :
p i r iD ni; l uCef no ttr h aen a l oy fsp ie sn t a z o c i n e
A n a l y s i s R e s u= (l At u /xA( sC )s /x LV) (x M n / xM 1y 0z 0)
S a m p l
S e
t s
a n
:s do al r
u Atd,iS ot na n s do al r
u Btd, i o n
S a m spo ll eu At ,ia onnSd a m spo ll e u Bt i o n A u = a b s o rfbratonhmc Se e
a m sp o ll eu t i o n
C a l c ut lh pae e
t er c eo nfa ts ap ig(reCi sn H i sn tO h.pe)o r - A s = a b s o rfb ra S
on t
mcaens do al ur A td i o n
t i oo nfT a b lt ea tk se n : C s = c o n c e n ot fr
U aSStP
a il oi cnA ycl iR
i d
cS
i n
S t a n s do al r
uAtd (i om n g / m L )
R e s =
u (l At u /xA( sC )s /xC( uM) r / xM 1r 02 0) L = l a b eal me do oufa ns pt i(r mi gn / T a b l e t ) c
V v = v o l ou fMm ee d i9 u0 m m0,L
A u = a b s o rfbrao Sn mac mesp o ll eu A t i o n M a =m o l e c w ue li a
og fr
ahs tp i r1 i8n 0, . 1 6 = ]
A s = a b s o rfbrao Sntmcaen s do al r uA td i o n M 2 = m o l e cw ue li aog fr
sha tl i ca yc lii1dc ,3 8 . 1 2 =
C s = c o n c e n ot frU aSStPa il oi cnA y cl iR
i cdSi n F o pr e n t a zt or ca i n 5
s
n fe. e,0r -p om r Lt oi oft nhsSe a m p l} e
S t a n s do al r
uAtd (
i ogn / m L ) s o l u tSi to a u Btd,ai nod nw a tt eos re ra vste h e = |
n ,n s do al r
C u = n o m ic no an lc e n ot far sa ptiiirn S oi n a m p l e r e a gb el naitnn ktt oh rseee p a 1
re}
r a2 t5 es-e mp aL r a t o r s .
s o l uA
t (i ogn / m L ) T oe a c s eh p a ra ad t1do0mr oL fa f i l t se ro el du (t1 i o n 5 S
M n =m o l e c
w ue li o
ag fr
ahs tp i r1 i8n 0, . 1 6 i n 4 0 0o f 0b )r o m o c p ur re ipsndloiell gu lt ae cai ca el t im ce ]
M , 2 _ = m o l e cw ue li oag fr
sha tl i ca yc lii1dc 3
, 8 . 1 2 a c (i 1di n 5 0 a) n 2d0 .m 0oLfc h l o r Io nf sot e rrhmte. a3 a
C a l c ut lh pae et er c eo nfpt eangtea (z C
o c
i is nHi e2n 7 N O )s t o p ap en srdh, ag ke en ft ol1 rym i na ,c c u r ta it me el dy .
t h pe o r to ifT oa nb lt ea tk se n : A l lt oh lwea y te ros se p a ra antddee, t e r t m h ei n e
a b s o r ob ft a hncecl e cas hr l o rl oa fy eo f rrrsmo m
R e s u= (l At u /xA( sC )s /xC1u 0) 0 S t a n sdo al r u Btd ai on tndh Se a m spo ll eu at gi oa nit nh se t
c h l o rl oa fy fo e rrr tom hmre e a gb el an nt k .
A u = a b s o rfb ra o Sn m
ac mespo ll e u Bt i o n C a l c ut lh pae e
t er c eo nftth a le ag be eal me do ouf n t
A s = a b s o rfb ra o Sn t
mc ae n s do al r
u Btd i o n p e n t a (
z o
C c
i is nHde2i 7s s
N oO l) v e d :
C s = c o n c e n ot fr U aS PtPei no tn a z R oSi nc i n e
S t a n sdo al ru Btd (i ou ng / m L ) R e s u= (l At u /xA( sC )s /x LV) x
1 0 0
C u = n o m ic no an lc e n ot fp r e a tn it oa inz n o c i n e
S a m sp o ll eu Bt (i ogn / m L ) A u = a b s o rfb rat onhmc
Se ea m spo ll e u t i o n
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: 0 % -e 1 a co1 fh
t0 h. l ea 0- % A s = a b s o rfbrao Snmtc aen s do al r
u Btd i o n
b e l ae md o uo fn
a st p si(r Ci sn H as nOpd 4e)n t a z o c i n Ces = c o n c e n ot fr
U aS PtPei n
o tn a z R oSi nc i n e
( C i s H 2 7 N O ) S t a n s do al ru Btd (i om ng / m L )
L = l a b e al me do oufpne tn t a ( z mo gc /i Tn ae b l e t )
P E R F O TR EMS AT N
S C E Vv = v o l oufMm ee d i9 u0 m m0,L
¢ D I S S O (
L 7U 1T 1I )O N T o l e r a Nn Lc 8T
e 0s( :%
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Q o) ft hl ea -
A p p a 1r : a8 t0ru ps m e l ea d m o oufa ns pt i(r Ci o n H ag rOde ,i s) s o l v e d .
T i m 3 e 0 :m i n e U N I F OO RFDM oI s T UYa NGI(ET9 S0 5 M) e : te ht e
S t a n sd oal ru Adt :i1o 5ng / om fUL SS P a l i cA y cl iR
i cdS r e q u i r e m e n t s
i n0 . 1N s o d hir uy m c l i a n l e e
S t a n sdo al ru Bdt: i1 o3g n / o mfU LSP Pe n t a R z S o c iI nMe P U R I T I E S
i nd i l gu lt ae cai ca el at ci (ci 1di n 5 0 ) ' N O N A SSPA IL RI IC NY L A T E S
S t r o bna gs lia y cn, i o n - e r ex sci Mhn :i aa x
n
s ug iet a b l e F e r rc ih cl o r i rd ee a- gu eT r noe
a tma :i x to uf8 rme oL f
q uo a fa nyi o n - e r ex scwi h
in t
a1 hn0v go el uo fm e s f e r rc ih cl o sr oi ld ue(t 6ii no1 n0 a) n 4d 2m oL f0 . 0N 5
i l u tg el a c ai ac l e at ci (ci 1di n5 0 )a , n sdh af ko 2e r 0 h y d r o ca hc l i ado ,
dr 6d
i0 gc o fu r e Da i. s s to hlue v re e a
m i n A .l lt oh rwee s ti osn e t t al en ,dd e c ta hnsetu p e r n a - b y s w i r a l inwndgi t ht oh au ei tod fh e a at n, a dd j tu hs e
t
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r e s u ls to il nu gti f in oe nc ,e s ws ia t6r N hy h, y d r o c h l o r Si ca m sp o ll eu At :iCo rn u1 sT h a b il n e1 t0m oL fD i l u e n t

a c it doa p H o f3 . 2P .r e po antr hede ao fy u s e . S o n i fco aar tb eo2 um t i na ,nf idl t e r .
S t a n sd taosrcodkl u t 1i o5 g n0 : / o mfs aLl i ca ycl ii ndc S a m sp o ll eu Bt :iE ov na p o5 rm aoLtfSe a m spo ll eu A t i o n
c h l o r o f o r m t o d r y n oe n sa ss t eb aa mtu hn da e s r t r oe fna im t r o g e
S t a n sdo al ru t d Pi io pn5e:mt oL fS t a n sdt aosrco d kl u - D i s s to hlrev ee s ii nd0 u. em 2 oL fD i l u e n t .
t i oi nn ta o5 0 -v m o lL u mf el ta csr oki nc t a 1i 0nm ioLnf g C h r o m a t soygs rt ae pm h i c
m e t h a2 dn ro olo p,fhsy d r o ca hc l i ado ,
nr 1d
i 0m
c oLfa ( S eC eh r o m a t (o 6g r2 aT1ph)hi , yn - CL ha ry o e rm a t o -
s o l u (t1 ii no1 n0 o) fg l a ca ica el at ci ic nde t h eA rd. d g r a p h y . )
c h l o rt oovf oo lr um a mn e mdi, x . A d s o r b 0 e. n2 t5 l :a- yomefcrm h r o m a ts o i lgi rc aa p
S a m sp o ll eu t i o n g e ml i x t u r e
I n s ae sr mt a pl ll e do gfg el atws o s oa l b ot vh see t e m A p p l i v c ao tl iu o 1m n 0
ueL :S, t a n sdo al ru A td ai on nd
c o n s t ro ifac 2t 0ix-o2 n. 5 c- hc rm o m a tt ou gb r e ,a p h Si ac m spo ll eu At ;i5 wo Ln S, t a ns do al r u Btd ai on Snd a m p l
a n udn i f op ra mwc likyt a mh i x to ufa rbe o1 gu ot f s o l u Bt i o n
c h r o m a ts oi g l ir ceaeapor uh
atsinh0cd. m 5 oLf 5 M D e v e ls oo pl ivs n ey n gs tt 1e -m B:u t wa an to e al rn, ,d
h o s p h a co irDd ii. rc e ac tb loty hv ilesa y ep r a, ac ski m i - g l a cai ca el at ci ci( d7 0 : 2 0 : 1 0 )
a rm i x to ufa rbe o3 u g ot fc h r o m a ts oi g l ir cae po uh si Sc p rr ae ya g eF n o lt i: n - C Pi ho c e a Tn lS
of tlo elu l bo yw e d
e a r at nh 2dm oLfF e r rc ih cl o r i rd ee a- gu Ter no ae ta . s o d hi yu dm r o s ox li ud(t1eii no1 n0 )
q u a n nt oi mt iy ne a q lu i l vyt ao5l 0 em n og tfa s p if nr no m A n a l y Ds e i sv :et l h ce
o hp r o m a i tn toh D ge er va eml so p
f i n ep l oy w d T ea rb leaeddt1ds 0m oL fc h l o r so t fi ro r m ,s o l vs eyn stut n et tmi lh se o l vh ea nms to va eb d ot uh rt e e -
f o 3r m i na ,nt dr a n ts oft eh cre h r o m a t aod g- r a p hf io cu ro tfthhsle e n og ft h pel a tRe e. m toh v pe lea t e
s o r p c t io ol nwu im tnh aei od f5 m oL fc h l o r o f o r mf .r t o hmce h a m mb ae tr r hk
,se o l vf e r on nta t n,d dr y
P a s5 s0m oL fc h l o ri ons fe ov reprmoa rl t ti h o nr so u g h u n da e c ur r ro efwn a t a ri rm S. p rt ah pye l awt ie tS hp r a y
t h ce o l ur m i nnts ,het ei op ft h ce h r o m a t to ugbre a p rh e ia cg e n t .
w i t c h l o r oa fn dodir sm c, t ahreeld u aI ftt e h. ep u r p l e A c c e pc t r ia t ne S c
r iaea m:spo ll eu A ta i on nSd a m spo ll ue -
z o r n ee a ct hh e be so t ot fto hm teu b dei,s ct ahr c eod l - t i oB ne x h is bp iohtt as v ti hn seg a Rm ;vea l u a en asdp -
u m n a , n r de p te hatete sw ti t a sh m a lq lu ea rn otf i t y p r o x i tm ha set ae sm l i yez ae sn sdh a ap ste hse ri er s p e c -
o w d eT ar bel d e t s . t i vS et a n sdo al ru d t i o n s .
E l u tt heae d s o s ra b l iecadycl iii dnc ta o1 0 0 v- oml Lu m e t -
r i fc l a cs ok n t a 2i 0nm ioLnfmg e t h aa nn4do d rl oo pf s A S S A Y
h y d r o ca hc libodypr ai sc sti wn1og 0 -p m o rL t oi fao n s ' P R O C E D U R E
s o l u (t1 ii no1 n0 o) fg l a ca ica el a t ci ic ndw a t e r - s a t u D -i l u eM ne tt: h aw an to eal rn, p ,d h o s pa hc oi rd i c
r a te et dh ea rn,tdh e3 n0m oL fc h l o r o t fh or ro mu ,g h ( 5 0 0 : 5 0 0 : 1 )
t h ce o l ua mndndi, l t u th eee l u w a it e tc hh l o rt oo f o r mS o l u At :iP or ne pa fa ir l et ea rnedd d e g a ms is xe tbd uy r e
v o l u m e . d i s s o 6l v7mi5 g no gfs o d 1i -u omc t a n eas nu dl f o n
I n s t r uc mo en nd ti at li o n s 4 2 m6 og fa n h y dd ir boasus osi d c pi hu om s ip n h a t e
M o d Ue V : 6 2 m5 oL fw a t e a rn m,di x .
A n a l y wt ai v c ae ll e3 n0 gn 6 tm (h m: a x i m u m M o b pi hl ae sAe d:4 d7 m5 oL fm e t h aa nn1do0ml oL f
"
a b s o r b a n c e ) p h o s pa hc oit rdoS io cl u At .i o n
a a S t r ao n n i g o n - e r ex sciThnr:aa nn 3s gf 0g
e oe frs t r o n g
o C e l l 1 : m
i J A n a l y Cs oi n s :c o m di et ta en rtt m hlaeiybn se o r ob fa n c e as n i o n - e r ex scti ohna 2a n5 g0 e b-e ma L kWe ar t .s hhe
aD b o t th h Se a m sp o ll eu a t in Sodnt a n s do al ur td ui so ina n, g r e s wi in tt hw 2o 0 0 p-o mr L t oi fwo an ts d e re ,c a nt t h ie n
¢
) s o l vm ei nxtto uft rh e se a cm oe m p o as sti ht oai ftto hn e w a ta e f tree ar c w ha s Wh a. ws ihtt hw 2o 0 0 p-o mr L t i o n
o fd i l gu lt ae cai ca el at ci (ci 1di n2 0 )d ,e c a nt t hf i
ei rns gt
5 S t a n sdo al r
A c c e pc t
u atdsit oh bne l a n k .
r ia t ne 3 rc i.ea 0:t %h ;ae b s o ro bft ah nSe ca em - w a s ah n, fd i l tw e ir t h aei od fs u c t i o n .
Ps p l se o l u dt io oe n nos et x c te he oadftt h Se t a n s do al ur -d S t a n sd taosrcodkl u t 0i .om 2n g: / o fm U S LNP a l o x o
[ a t i oa n ,n nye c e sa sd aj ru ysbte mi e mn na g tfdo he
r a v i n g R Si nD i l u e n t
w
=) u s e a sd m a ls la emr p l e . S t a n sdo al ru t dTi ro an n:1 s f0 me0rog fU S P Pe n t a z o
R St oa 5 0 -v m o lL u mf el at sDrkii. sc s ion a l vb eo3 u0mt L
A D D I TR IE OQ NUAILR E M E N T S o fD i l u eA ndt5 d .. m0 oLft h Se t a n sdt aosrco d kl u t i o n ,
e P A C K A A GNSIDTN OGR AP G r eE s:ientr ivg ehl ti ,g h t - r e s i s ta annd dti l w u ti etD ih l ut eovn ot l u m e .
c o n t a i n e r s . S a m sp o ll e u t Ti ro an n:as nfa e mr o nu o nm ti ne a q ul il vy a
e U SR PE F E RS ET NA C NE D ( 1A 1R) D S l e nt to1 0 m0 og fp e n t a f z ro c oN imL2 n Te0
T a b lt eoa t s
U SA Ps p iRr Si n 1 0 0 v-o ml L u mf el at sa rk i
n, acd d 5d0 .m 0oLfD i l u e n t .
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U SS P a l i cA y cl iRi cdS F i l ti en rta og l a s s - s ct oonpifpcl eaa r sA
lke d. da db o u t
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3 0
m i n .
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( S eC eh r o m a t ( 6o 2g1Sr)y a,s pStuhei ytm a b i l i t y . )
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d Hx i nd je e cv toi lo un 2m 0ue L:
e q u i vt aoN l eL 9nT t0 .a 0n %N
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a m o u o fnp t
e ns t a ( z o
C ci i9 nH ae2 n7ndNa Ol )o x o n e S a m p Sl te a:n s do al r u td i o n
( C i s H 2 i N O s ) . [ N o t re e l aT rthi e e
vt ee nt ti imfoeonn
sr a l o ax no d
n e
p e n t a az roaec bi o0 n u.e a
t3 n1 d. 0r ,e s p e c t i v e l y
I D E N T I F I C A T I O N S u i t a br iel qi ut yi r e m e n t s
o A . R e s o l u Nt Li6 o Tb ne: t wp ee e n t n a az on cd i n e
D i l u eC nht l: o r ao nfmdoe rt mh (a1 n: o 1 )l n a l o x o n e
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U S LPPe n t a z o c i n Re e l a st t i va en dd eav ri da tNi M o 2n T
.: 0 %
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l o g/ r
P ea np th asz3 o2 c2i 5n e

A n a l y s i s S y s stu ei mt a b i l i t y
S a m p lS e t s a n:sdo al ru a td in S
odna m sp o ll eu t i o n S a m p Sl tea:n s do al r u td i o n
C a l c ut lh pae et er c eo nftth ale ag be e al m e do ouf n t [ N o t re e l aT rt hie ve t ee nt ti imfoeonn sr a l o ax no dn e
p e n t a ( z o C ci ig nH ae2 n7ndNa Ol )o (x Co in se H i2 n i N O , ) p e n t a az roaecbi o0n u.e at 3 n1 d. 0r ,e s p e c t i v e l y . ]
t h pe o r to ifT oa nb lt ea tk se n : S u i t a br iel qi ut yi r e m e n t s
R e s o l u Nt L i6 oTb ne: t wp ee e n t n a az on cd i n e
R e s = u (l r tu / xr (s C) s /xC1u 0) 0 n a l o x o n e
R e l a st ti va en dd eav ri da tNi M o 2n T
.: 0 %
t y =p e r a ek s p oofpn es ne t a oz ron ca il no ex o n e A n a l y s i s
f r t o hm Se a m spo ll eu t i o n S a m p lS e t s a n:sdo al ru a td in oSdna m spo ll eu t i o n
r s =p e a r ek s p oofpn es ne t a oz ron ca il no ex o n e C a l c ut lh p
ae te er c eo nftth a le ag be e al m
e do ouf n t
f r to hmSe t a n sdo al ru td i o n p e n t a ( z o C cy is nH ae2 n7ndNa Ol )o (x Co in se H i2 n i N O x )
G = c o n c e n ot ftr haaetpiporno pU rS iPa t e t h pe o r to ifT oa nb lt ea tk se n :
P e n t a R z S oo crUi SnNPea l o Rx Siontnh ee
S t a n s do al r u (td im o gn / m L ) R e s = u (lr tu / xr (s C) s /xC1u 0) 0
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( C r oN H O2 s; ) f r t o hmSe t a n sdo al r u td i o n
C s = c o n c e n ot ftr ha aet piporno pU rS iPa t e
P E R F O TR EMS AT N S C E P e n t a z R oSo crUi SnNPea l o Rx Sion tn hee
¢ D I S S O ( L 7U 1T 1I )O N S t a n s do al r u (td im o gn / m L )
M e d iW ua m t e
9: r0 m
;0 L G u = n o m ic no an l c e n ot fp r ae tn it oa n
oz ro c i n e
A p p a 2r :a5 t0ru ps m n a l o i xnto hnSe ea m spo ll eu ( t imo gn / m L )
T i m 4e 5:m i n A c c e pc t
r ia t ne rc
M iea te: htre e q u i r e m e n t s
D e t e c Ut V 2o 7r n9: (m c o r r feo car tb esd o ra bt3a 0n 5c e
n m A D D I TR IE OQ NU AI LR E M E N T S
S t a n sdo al ru t d Di io sn s:ao sluvi et a a bml oe oufUn St P ' P A C K A A GNSIDTNOGR AP G r eE s:ientr ivg ehl ti ,g h t - r e s i s t a
P e n t a R z S oi nca im nie n i v mo l u oumf0m. e 1N h y d r o - c o n t a i n e r s .
c h l oa rcii(c da b 2o 5 um tg / md iL l)u , qt ui an ng t i t a t ei vU e SlR yPE F E RS ET NA CNE (D 1A 1R) D S
a n sdt e p ww ii tws h ae t e r . U S NP a l o Rx So n e
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t e s st u, i t da ib ll uyw ti te hMd e d ii f nu em c,e s s a r y .
A n a l y Ds e i st :e r t hmleia nb eal m e do oufpne tn t a z o c i n e
( C i s Hd 2i 7s N
s o
O
i nl)
t vh eSeda m spo ll e
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s o wn i t h Se t a n s do al ur tdi o n .
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i o n fom
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P r o c e f odcruo rn etu en ni t f o r m i t y sta esi to en rsi ol le u ot fiP oe nn t a iz no c i 4
P e n t a Iz no jc e ici n n e
D i l u eM ne tt: h aw an to eal rn,p,dh o s pa hc oi rd i c W a tf e
o Irrn j e c pt ir oe n p,wa ir te hdaei od fL a c At ci ci d .
( 5 0 0 : 5 0 0 : 1 ) I tc o n t N a iL 9nTs5 .a 0n % N
d M1 T 0 5 o.ft0h l%e a b e l e ]d
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4 2 m6 ogfa n h y d ir bo as us o
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c pi hu om s ip n h a t e E y
I D E N T I F I C A T I O N n o ]
6 2 m5 oL fw a t e
a rn m,di x . >
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p h o s pa hc oit rdoS io cl u At .i o n C h a tnorge ea d :
S t r ao n n ig o n - e r ex sciThnr:aa nn 3s gf 0ge oe frs t r o n g
a n i o n - e r ex scti ohan 2
a n5 g0 e b-e ma L kWe ar t.s hhe e A . 4 T rh eet e nt ti iomofe tn h pe e n t a zp oe aco kfit nh e
r e s wi in tt hw 2o 0 0 p-o mr L d re ,c a n t i n gS a m spo ll eu ct io or nr e ts otp ho oan fttd hs eS t a n sd oa lru d-
t oi fwo an ts e
t h we a ta ef tree ar c w ha s Wh a. ws ih tt hw 2o 0 0 - m L t i o an s ,o b t a iintn heAeds sa au ys .e s i
p o r t oi fdo in ls gu lt ae caica el at ci ic( 1di n2 0 )d ,e -
c a n tt ihfnei gr ws ta s ah n, fdi l tw e ir t h aei od f D e l te htfeeo l l o w i n g :
s u c t i o n .
S t a n sd taosrcodkl u t 0i .o m 2n :g / o fm
U S LNP a l o x o n e
R Si nD i l u e n t 4 eB .I D E N T I F I C ANT II TO RN O BGO AER SN(G EO1A SU 8N 1SI )C
S t a n sdo al ru t dTi ro an n:1s f0 m e0 rogfU S P S t a n sdo al r u tdDi io sn s:5
o l0mv og
e fU SP Pe n t a z o c i n
P e n t a R z Sot coa i5 n0 e -v m o lL u mf el at sDrkii. sc s ion l v e R S i n 2 5m oL f0 . 0N1h y d r o ca hc li onda rs ie cp a r a t o r ,
a n udst eh ii snp l ao cfte h Se t a ns do al ur stdpi eo cn i f i e d
a b o3 u0mt oL fD i l u A e ndt5 d
.. m0 oL ft h Se t a n d a r d i nt h ceh a p t e r .
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u tA i vo on :l ou fImn ej e ce tq iuo in vt ao l e n t
s t o p p c ye lr i enAdd d
e 2rd5. .m 0oL fD i l u Se no tn .i c a t e 5 0
m o
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( S eC eh r o m a t ( 6o 2g1Sr)y a,s pStuhei ytm a b i l i t y . )4 eB . T h Ue V s p e c otftr hupe me n t a p z oe oc
a fi
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h ee
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ue L:
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oy fm
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t a b i l i t y
S a m p Sl een ss i: st io vl iu ta
t yin Sodnt a n sdo al ru td i o n
S u i t a br ie l qi ut yi r e m e n t s
¢ P R O C E D U R E R e l a st ti va en dd eav ri da tNi M o5n .
T; 0S %t ,a n d a r
4 S o l uA : t 1i 5omn sMo d bi ou r mia nwt ae t Ae d r j. u s t S o l u t i o n
w i t1 h0N s o d hi yu dm r o t oxa ip doH ef1 0 . 0 . S i g n a l - rta ot -i Nn
o :o
L1iT0 sS, ee n s i s
t io vl iu tt yi o n
S o l u Bt: iM oen t h a n o l A n a l y s i s
M o b pi hl ae sS ee T:e a b1 l. e S a m p lS e t a s n
:sdo al r
uatd in S
odna m spo ll eu t i o n
C a l c ut lh pae e
t e
r c eo nfet aa cig m
hep u irnti htpeyo r -
T a b1 l e t i oo nfI n j e ct ta ik oe nn :
S o l uA
t i o n S o l uB
t i o n
R e s u= (l r tu / xr (s C) s /x G1u 0) 0
M e
t u =p e ar k
e s p oofen as c ie mh p u fr ri ot h ym e
S a m spo ll e
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t im oan i n t )
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n m:2 g
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C h r o m a t soy gs rt ae pm h i c R e l a t i v e A c c e p t a n c
( S eC eh r o m a t ( 6o 2g1Sr)y a,s pStuhei ytm a b i l i t y . ) R e t e n t i o nC r i t e r i a ,
M o d Le C:
D e t e c Ut o V
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; p- au cm kL i 1 n g P e n t a z e c i n e
C o l tu e mm np e r 4a 0t u r e :
F l ro awt e0 :. m5 L / m i n
I n j e cv toi lo un 2m 0
pe L: A nu yn s p e c i f i e d
S y s stu ei tm a b i l i t y
n o d S a m p Sl tea:n s do al r u td i o n 3 5
5 S u i t a b ri le i qt u
y i r e m e n t s ' ( 2 R , 6 R , 1 1 R ) - 1 , 2 1, | -3 d, i4 m, e5 t, h6 y- !H -e 2x ,a 6h -y md er to
J T a i l fia nc gt oNr ;M2 .T 0 b e n z a z o c i n - 8 - o l .
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.: 0 % > ( 2 R , B
6 R S, 1c1Gi a m n se l 1 - di i m t e t ha y l - 1 , 2 , 3
i J A n a l y s i s a h y d r o - 2 , 6 - m e t h a n o b e n z o | d j a z e c i n -
i} S a m p lS e t s a n: sdo al ru atd in Sodna m spo ll eu t i o n
= C a l c ut lh pae et er c eo nftth a le ag be eal me do ouf n t A U S P 4 I
p e n t a ( z Co c i is nHi e2nt 7h pNe oO r) to ifi on nj e c t i o nS P E C TI EF S I CT S
i S
t a k e n : e B A C T EE RN ID AO LTT OE X(S I 8T 5N )SN: M5 .TU8 S EPn d o -
= t o x Ui nn i t osfp/ em ng t a z o c i n e
R e s u= (l r tu /«r (s C) s /xC1y 0) 0 e P H ( 7 9 14).: 0 - 5 . 0
t u =p e r a ek s p oo fpn es ne t a f z o r c t
o i e ae m p l 'e O T HR EE RQ U I R E
hm Sn { t Mm Ee NetTthSrse:e q u i ri neI mn je enc t- s
s o l u t i o n t i o an snI dm p l aD nr tPu erg do d ( u1 c) .t s
r s =p e r a ek s p oofpn es ne t a f z o r cto ihm ne e A D D I TR IE OQ NUAILR E M E N T S
S t a n sdo al ru td i o n
C s = c o n c e n ot fr U aSPtPei no tn a R z S o
i nct ih ne e
S t a n sdo al r u ( td imo gn / m L ) C h a tnorge ea d :
C y = n o m ic no an lc e n ot fp r ae t n it oa inz nto hc ei n e
S a m sp o ll eu ( t ino gn a/u m s eLa r) ' P A C K A A GNSIDTN OGR AP G r eE s:iensr iv ne g l o e r -m du ol s-e
A c c e pc t r ia t ne r9
c ie
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I M P U R I T I E S
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e U SR P
E F E RS ET NA C NE (
D 1A 1R) D S
4 eO R G AI MN PI UCR I T I E S @ ( C 1 N1 - M a y - 2 0 1 8 )
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z S
o c i n e
C h r o m a ts oy gs rt Pa e rmp o:hcaie
sdcei dr e icntt he d
e
A s s a y .
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i nD i l u e n t
S t a n sdo al ru td 0i .o 0 n m:0 g
2 / o fmU SLP Pe n t a z o c i n e
R Si nD i l u e n t
U S4 P1 O f f i M
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t i vs et a n dd ea vri daf to rri eo pnl ii cn aj te eci ts ni oo mt

n so r e
t h a 5 n. 0 % .
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e mal eby 2o
s u0 Lt )
o ft h Se t a n sdo al r uatd in todnh Tee s to l u it ni tto onh ce h r o -
Il 7 H m a t o g ar namdpe ha ,st u h rer ees p o f ontrshemesa jp oe ra k s .
C a l c ut lh paeetr ec e n ot afn gi et r i l o ta rci iandt c eh pteio cr -
HO. S y O H
t i oo nfP e n t Ae ct it c
d
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c e na tn ndo mt o tr he a1 n0 0p .e 5r co ef n t I r o n 1 U. g5so ifs np ge c ip mr eo nca ,e sd ei dr e ic nt h
e de
C y 4 H 2 3 N 3 0 1 0 . t e sf to Ir r ou nn dE ed er At ci icTd .hc eo lo oftr ht ee ss to l u t i o n
i s n o dt e e tp he tar hn ao tft h seo l u ct oi n o nt a ti hnseit an ng -
P a c k a
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ers.
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U SP Pe n t Ae ct iR c
dS 1 . m 5 oLf1 N s o d hi yu dmr o ax nis dw ietr,od l i s s to hl e v e
I d e n t i f Iin cf ar At a bri seo o
dn r,(p 1t 9i 7o Kn ) . s p e c iA mde1dn0m. oL f0 . 1N a m m ot nh ii o uc y maa nn da t e ,
R e s io dn i ug en i (t2 i8 o1 n ) :o mt o tr he a 0 n. 2 % . m i xA . da db o4 u0mt oL fm e t eh tyh l ky el t om ni exa,,n d
a l lt ohw l ea y te ros se p a rT ai t rew a.it t0e h. 0Nf5 e r rai mc -
m o n s iu lu fVmaSts,et i r cr io nn gt i n Au s ot uh tseilt yr .a t i o n
D e l te htfeeo l l o w i n g : p r o c et eh a de sq ,u ep oh uat ssu er fn rs co om l o rt loyees ls l o w ,
a n t dho er g ap nh i ar sce em ac oi ln osr Al s et sh see.n d pi so i n t
H e ma evt yaM le stI,ih( o2 3d 1 )0 : . 0'(0o t 5 i 1c -i%aj ta.n - 2 0 1 a
8 ) p p r o sa tc o th phet ed
i t,r a mt ii oxan,n , adl lt oh w lea y te ro s
L i mo if n ti t r i l o ta r ci i a cd e t i c s e p a rAa dt0ed.. 1 i- nm cL r eo mf0e .n 0Ntf5se r rai cm m o -
n i su uml fV aStm,e i x a i n ag dl l o tw hilena gy t e ros se p a r a t e
C u p ar ci ec ts ao tl eu t i o n2 0 gDo ifSs us o pa lc ve eti an t ea f t ee ar ac dh d i tu in totin hl,oe r g al na yitecur r fn rs co oml o r -
a m i x to uf8r 0em0 oLfw a ta enr1d 0m oLfg l a caica el t i c l e st sop i n Ek a . cm hoL f0 . 0Nf5 e r rai cm m os n u lif au t me
a c i Ad d. j wu is 1tt hN s o d hi yu dm r ot oxa ip dH o ef4 . 2 ,
d i l w u ti etw ha tt eoo rb t a1 i0 n0 m 0oL fs o l u tai nof ind l,t e r . c o n si sue m q ue id vt ao1l 9e . n tm6 6og8fC i 4 H 2 3 N 3 O r 0 .
M o b pi h l ea s e aPm ri ex pto a uf1r r6ee 0 m 0oL fw a t e r ,
4 0m oL fg l a cai ca el at ci ic3d 0 , .m 4oL f0 . 5Md o d e c y l - (ey
4 )
t r i e t h y l p ha om sm poahnnai 2dtu0mem , oL fC u p ar ci ec t a t e = x
s o l u tA i do jn wu. is 1tt Nh s o d hi yu dm r o t oxa ip doH ef4 . 0 , P e n t o b a r b i t a l z
d i l w u ti etw ha tt eoo rb t a 2 i0 nm 0 0 oL fs o l u tf ii lo t ne ,r
t h r oafui gl the ar v ai 0n .g5 -o \rf 1i mnpe or r o sai ntdd ye, -
g a sM. a ak de j u s i tf nm ee cn ets(ss eaS ery ys St u ei mt a b i l i t y ow H N e r a|)
u n dC eh rr o m a t ( 6o 2g1 r) a) .p h y M e N H P =
S t osc tk a n sd oa lrudt i o na bT or5 ua0m t
n sog fn ie trr i l o - a e 2
t r i a ca ec ti aid cc, c u r wa et ie l g tyhoae 1d 0, 0 v-o ml Lu m e t r i c HC: cH, 3 e 5
f l a sd ki ,l uw ti etC uh p ar ci ec ts ao tl ue tt oiv oo n l ua mn medi, x . 7)
S t a n sd oal ru dt i o n1 . Tm 0 r oLaftn hsSeft eosrc tk a n d a C r dHi i g N 2 0 3 2 2 6 . 2 7
s o l u tt oia o2 n5 -v m o lL u mf el at sdrkii,l cuw ti etC h u p ar ci ec - = a H , 3 H , 5 H ) - P y r5 i- me it dh iy nl e- t5 r- i( o1 n- em ,e t
t a ts eo l u tt oiv oo nl ua mn m edi, x T .h is s o l u ct oi no t n a i n s t y l )@ - ), - ;
a b o0 u. t0m 2og fn i t r i l o ta rci ipa de c mer tL i .c ( 4 ) - 5 - E t h y l - 5 - ( 1 - am ce i[t d7h 6y -l 7b 4u -t 4y ]l .) b a r
T e ss to l u t i o na bT or2 u g
a ont fPs ef ne trAe ct iiadcc, c u -
r a t ew ley i g th oae 1d 0, 0 v-o ml Lu mf el at sA rk i.dacdb o u t D E F I N I T I O N
7 0m oL fC u p ar ci ec ts ao lt ue tai nos ndw ,i tr odl i s s oS lo vnei. - P e n t o b ca or nb t iN a
t a iL 9lnTs8 .a 0n % Nd M1 T 0 2 o.f 0 %
c a ti fe n, e c e s ts oda ir sy s,oD li vlew u. ti etC u h p ar ci ec ts ao lt ue - C i H i sc Na 2l c O u3ol,n at th deerd ib ea ds iWs h. et r h mee a -
t i ot nov o l ua mn e mdi, x . t e r ii sall a b ea lsie nd t e sno dl e fe oldvry e t e ru is nePa,er ny -
R e s o l su ot li uo tn i o n1 . Tm 0 roLaftn hsSeft eosrc tk a n d a rt do b a r cb oi nt ta N a
l iL 9nT s7 .a 0n % Nd M1 T 0 2 o.f 0 %
s o l u tt oia o2 n5 -v m o lL u mf el at sdrkii,l cuw ti etT eh s to l u t i o n C i H i s c Na 2
l c
O u
3 o l, na
t th dee rd i b e a d
s i s .
t ov o l ua mn m edi, x . I D E N T I F I C A T I O N
C h r o m a t soy gs r(t ase epCmehh ir co m a t( o6 g2 r1 a) p) h e AyT.I hN eF R AA BR S E O D R( P1 T9 I7 OS N)
l i q uc ihd r o m a i t s eo qg uriawpp ipt ahe2 h d9 0 d- e nt m e c t o r S a m spo ll e u t 7ii on 1n 0 : 0
a na d4 . 6 x-2 m5 m-c co ml tu h mac nto n t 5 a i- npusam c k i n gM e d i C uh ml : o r o f o r m
L 1 t h ah ta bs e h e in g dh el ay c t i ( v c a trl ebo doa ndo if n g e B . T hr ee t e nt ti iomofetn h me a jp oe roa ft k h Se a m p l e
a b o3 u0 t%T)h. f el ro aw it s ea b o1 um tpL e mr i n uE t q ue i.l i - s o l u ct io or nr e ts ot p ho a on fttd hsSe t a ns do al ur tadis o n ,
b r a tt hece o l bu ypm ans s ii n ns ge ,q u ew n a tc m ee re, ,t h a n o o l b
, t a i in tn heAeds s a y .
a n wda tf e
o arr b o1 u5mti n ue ta e
c as
h n,tdh e
M n
o b i l e
p h af s o are b o4 u5 mti n u Ct e h sr .o m a tth o Reeg sro al up thiAo Sn S A Y
s o l u tai no rnde, c to h r pe
de r a ek s p oa snd si er se fc otPrer do - ' P R O C E D U R E
c e d ut rhere:e s o l u R ,tbi eo nt ,wp e ne tnae ct ia nn di t r i l o - M o b pi hl ae s0 e. :
i dc 0M1m o n o b p o a ts ai spc sh i
o u
s m-
t r i a ca ec tiiis dnc ol te st s h 2a .n 0a , n tdh r ee l a rt ie vt ee n t i o n p h aat ne a dc e t o n(i 6t 5r :i A3l d5e j) t.
u hs p
et H
t o3 . 5 .
t i ma er sae b o0 u. ft 6o pr e n t ae ct ia i dcn 1d. f0 o nri t r i l o - S t a n sdo al ru td0i. o1mn g : / o fm U S LP Pe n t o b Ra S r b i t a l
t r i a ca ec ti iCd c.h r o m a tt hoSe g t ar nas dpo alhur td
ai nor nde ,- i n M o b pi h l a
e s e
c o rt dh pe e r a ek s p oa snd si er s e fc otPr er do c e td hur ere le a: -
 3 2 2
P e8 n t o b /aO rf bf ii M
ct io
aa ln
l o g r a p h s U S4 P1

S a m sp tloseco kl u t1 i mo ng : / o fP m eLn t o b ia n r b i t a lr s = p e aa rk e
f oa pr e n t o b fa r bt o ihm te a l
M o b pi hl ea( ss eo n iu nc taditiles s o l v e d ) S t a n sdo al r u td i o n
S a m spo ll e u t Ti ro an n:1s 0f .em r0oL ft h Se a m sp t lo ec k C s = c o n c e n ot fr U aSPtPei no tn o b R a Sri nbt ih te a l
s o l u tt oi
a o1 n0 0 v-o ml L u mf el at sark i
n
, dcdi l w
u ti et h S t a n sdo al r u (td imo gn / m L )
M o b pi hl aet sov eo l u m e . C u = c o n c e n ot fPr ea nt ti oo bnian t r bh Sie at am lp l e
C h r o m a t soy gs rt ae pm h i c s o l u (t imo gn / m L )
( S eC eh r o m a t ( 6o 2g1Sr)ya,s pStuhei ym
t a b i l i t y . ) F =r e l a rt ie vse p foa nc sot efot rh ie m p u (r sie te y
M o d Le C: I m p u Tr ai bt1 l)y e
D e t e c Ut o V
2 r1 :n4 m A c c e pc t
r iat ne S
rc iee/ae:m p u Tr ai bt1 l.y e
C o l u 4m .n 6: x-2 m5 m- 5 c m - ;pu am c kL i 1 n g
F l ro awt 1e :m L / m i n I m p u Tr ai b1t lye
I n j e cs ti zi eo1:n0
w L
S y s stu ei m t a b i l i t y R e l a t iR ve el a t iA vc ec e p t a n
S a m p Sl tea:n sdo al r u td i o n R e t e n Rt e i os n p o n
C rsi et e r i a ,
S u i t a br ie l qi ut i y r e m e n t s N a m e T i m e F a c t o rN M( T % )
C o l euf m f inc i Ne nL 1cT5 y :, t0 h0e 0o r ep tl ia ct ae sl 6 - I m i n o - 5 - e t h y l - 5 -
T a i lf ia nc gt oNr M :1 .T5 ( 1 - m eb t u th yy l! )
R e l a st t i va en dd eav ri da tNi M o 2n T
.: f0o %r b a r b i at cu ir di c 0 . 3 9 1 . 5 0 . 2
p e n t o b a r b i t a l 5 - E t h y l - 5 - ( 1 - e t h y l -
A n a l y s i s p r o pb ya lr )b i t u r i c
S a m p lS et s a :n s do al r
u atd in S
odna m sp o ll eu t i o n a c i d e 0 . 9 3 1 . 0 0 . 1
C a l c ut lh pae et er c eo nfCt 1a ;g He ii snt Nh 2 pe oO rs t i o Pn e n t o b a r b i t a l 1 . 0 =
o fP e n t o b ta ar kb ei nt :a l 5 - E t h y l - 5 - ( 1 , 3 -
R e s u= (l rt u /x (s )C s /xC1 u 0) 0 d i m e t h y l b u t y ! )
b a r b i at cu ir di c A S 0 . 9 0 . 3
t u = p e aa rk ef ar t
o hm Se a m spo ll eu t i o n U n k ni mo p wu n r i t i e s 1 . 0 0 . 1
t s = p e aa rk ef ar t
o hmSe t a n sdo al ru td i o n T o t a l = = 0 . 5
C s = c o n c e n ot frU aSPtPei no tn o b Ra S
ri nbt ih te a l 2 W h et rh meea t e i rs li aa bl ea lsie nd t e sno dl efe oldvry e t e ruis neta,hr ey
S t a n sdo al ru ( td imo gn / m L ) l i moi f5t - e t h y l - 5 - ( b1 a- re bt ihatyculiirspd3irc.o 0p %y ,
l )
C u = c o n c e n ot fPr ea nt ti oo bnian tr hb Sei at m
a lp l e
s o l u ( t im o gn / m L ) S P E C TI EF S I CT S
A c c e pc t r ia t ne r9
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: 0 % -o f1C 0 1 ;2 H. i0 %N 2 eO L3 o sO sND R Y (I 7N3 G1 D) r:ays a m apt1l 0e f5 o 2r h :i t
os n
t h de r ib ea ds ia s n;9d 7 . 0 % -o f1C 0 i ;2 H. i0 %N 2 O 3l o s eN s M1 T
os n . o0 fi %t sw e i g h t .
t h de r ib ea ds iw s h, et rh me
e a t e i rs li aa bl ea lsi en d
- A D D I TR IE OQ NUAILR E M E N T S
t e n sd o el d
fe ol vry e t e r ui sn ea r y ' P A C K A A GNSIDTN OGR AP Gr eE s:ientr iv gechotn t a i n e r
e U SR PE F E RS ET NA CNE (D 1A 1R) D S
I M P U R I T I E S
te U SP Pe n t o b Ra Sr b i t a l
I n o r g I a m pn u
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i) D e l te htfeeo l l o w i n g :
=
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Ps oH E AM VE YT AM LeS t,I ]h( o2 3d 1 N
) :M2 T i c i a 1 -P
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a r
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[ 5 j a n - 2 0 1 8 ) C r i H i y 2N 42 8N .a 2O 53
O r g aI n
m i
p u
c r i t i e s 2 , 4 , 6 ( 1 H , 3 H , 5 H )5 -- Pe yt rh iy m l -i 5d -i (n 1e -t mr
=) e P R O C E D U R E b u t y lm ) o- ,n o ss aol td . i u m
M o b pi hl ae sP er :e pa asd ri er e ic nt h e Ades s a y . S o d 5i -ue mt h y l - S - ( 1 - m e t h[ y5 l7 b- u3 t3 y- l0 )]
S t a n sdo al ru t d 0i .o 0n m0: 1g /o fm U S LP Pe n t o b a r b i t a l
R Si n M o b pi hl a e s e »P e n t o b S a ro bdicitou an lmt na oil tne ssts h a n
S a m spo ll e u t1 i m o ng : / o fPm eLn t o b ian r M bo ib ti al le9 8 p . e0 r ca ennndto mt o tr he a1 n0 2 p. e 0r co efn t
p h a s e C i i H i 7 cN a2l Nc au oO l na
s
t th,dee rd i be a ds i s .
C h r o m a t soy gs rt ae pm h i c W h e t r
h meea t e i rs l
i a
a bl ea lsie nd t e sno dl ee ld y
( S eC eh r o m a t ( 6o 2g1Sr)y a ,s pStuhei ym
t a b i l i t y . )
M o d Le C: f o vr e t e r u is nePa ,e r n y t o b S
a ro bdicitou an lmt a i
D e t e c Ut o V
2 r1 :n4 m n ol te st s h a 9 n 7 p. e0 r ca enn dto mt o tr he a n
C o l u 4m .n 6: x-2 m5 m- 5 c m- ;pu am c kL i 1 n g 1 0 2p .e 0r co efC n 1t H i z Nc 2 a lN cau O ol s n
a t, e d
F l ro awt 1e :m L / m i n t h de r ib ea ds i s .
I n j e cs t i zi eo1:n0
p L
S y s t eE m e P a c k a a gnsidtno gr a g e i ntP i rgcehotsn et ar iv nee r s
S a m p Sl te a: n s do al r u td i o n U SR Pe f e r s e t na c n ed( a1 r 1 d ) s
S u i t a br ie l qi ut i y r e m e n t s U SP Pe n t o b Ra S r b i t a l
C o l euf m f inc i Ne nL1cTy5 :, t0 h0e 0o r ep tl ia ct ae sl
T a i lf ia nc gt oNr M :1 .T5 C o m p l eo tfs eo nl eu st si1 . og0nw i t1 M h0i
m x oL fc a r -
R e l a st t i va en dd eav ri da tNi M o 1nT 5: .f o0 r% b o dn i o x i dwe a- tf are fer te1:emr i n ut th seeo, l u i ts ci lo en a r
e n t o b a r b i t a l a nf dr ef er o u nm d i s ss ool li vd .e d
A n a l y s i s I d e n t i f i c a t i o n
S a m p lS e t s a n: sdo al ru a td in oSdna m spo ll eu t i o n A :U l t r a vA ib os l oe rt(p 1 t i9 o 7n U )
C a l c ut lh p ae te er c eo nfat naiygm e p u irn ti htpeyo r - S o l u t1i 0uo ngp :e mr L .
t i oo nfP e n t o b ta ar kb ei nt :a l
M e d id iu lmua:t m e m o h ny i d ru (o m
1 xi ni2 d0 e0 ) .
R e s u = (l r tu / xr (s C
) s /x C( u1 ) / xF 1) 0 0 B :T hr ee t e nt ti iomofetn h me a jp oe ria n tk h ce h r o m a
g r oa ftm h Ae s sp ar ye p a c r ao tr ir oe nts ot
p ho iannttd hs e
t u = p e aa rk fe oa ar n iym p u fr ri to h ymSe a m p l e
s o l u t i o n
U S4 P1 O f f i M
c ioa n
l o g/ P
r ea npt ho sb a3 r2 b 2i 9t a l

c h r o m ao t ft o h Segt ra an pmdr ae rp da ra asot bi t o na i,in n e d R e l a t i vR ee l a t i v e

t h Aes s a y . C o m p o u Rn ed t e n t iR oe ns p o n Ls ie m i t
C :I g n ia tbe o2u 0tm0g t :h re e s ie df uf ee r w v ei st ch e s N a m e T i m e F a c t o r ( % )
o r a n md e et th rse e q u i ro eft mhteeensftt ossSr o d i u mU n k ni mo p wu n r i t i e s = 1 . 0 0 . 1
1 9 1 ) . T o t a l = 0 . 5
P (H 7 9 1b )e :t w9 e . a8e nn1 d1 .i 0nt, h seo l u pt ir oe np a r *e Wd h te hrme ae t e i rs li aa bl ea lsie nd t e sno dl efe oldvry e t e r uis neta,hrl eyi m i t
i n t ht ee sf to Cr o m p l eo tfs e o ln uet si so n . o f5 - e t h y l - 5 - ( b1 a- re bt ihatyculiirspd3
irc.o p0 y%l )
L o so sn d r y (i 7n 3g 1 i)t a t1D 0rf5 yo 6rh o u irt ls o: s e s
n o mt o tr he a3 n. o5 fi %t sw e i g h t . A s s a y [t Nh vO
e a Tlf E uo Ler o Uso ssnd e r y o i nb gt a ai t n e d
t h se a tm ie am se t h pe r e p a or fat th Tieeos nto l u it nt i oh ne
t e sf to Rr e l ac t oe md p o a u n tn dh A de ss sp ar ye p a ir nat th ie o n
D e l te htfeeo l l o w i n g : A s s a y . ]
M o b pi hl ae s S et ,a n pdr ae rp da r aa nt Cid ho rn ,o m a t o g r a p
H e ma e vt yaM le st, I Ih( o2 3d 10 ) .: 0 c0o t t3i1c % 4i a0 i.
0 - 2 0 1 8 S) y s ~t fe rm o:acsde ees dc ri nit bh Aee sd su an ydP ee rn t o b a r b i
R e l act oe m d p o u n d s t a l .
M o b pi h l e a s e aPsdri er pe ia c ntr heeAdes s a y . A s sp ar ye p a r a t i ao bn o1T u1rtm0a ogn fPs ef ne tr o b a r b i -
S t a n sd oalru dt i o na naD ci cs us rowalet veielgqyhu ea d n - t a lS o d ia uc cm u, r wa et ie g l tyhoae 1d 0, 0 v- o ml Lu m e t r i c
t i to yfU SP Pe n t o b Ra S ri nbM io tbapilhl aeasn ed d, i l u t e f l a sak d , ad b o8 u0mt oL fM o b pi hl ae s a en s,do n i ucn at ti e l
q u a n t i t aa tnsidtv e lpiyw f n,ie sc ee s ws ia tMr hy o ,b ip l h ae s e d i s s o Dl iv le wud ti. etM h o b pi hl a et sov eo l ua mn m edi, x .
t oo b t aa si onl u ht a i ov nai kn n g oc w o nn c e n o tf a r ab to iu oTt nr a n 1s 0f . em r0oL ft h is so l u tt oai o1 n0 0 v-o ml Lu m e t r i c
0 , 0m 0 1pg e mr L . f l a sD ki. l wu ti etM ho b pi hl a et sov eo l ua mn m edi, x .
T e ss to l u t i o na bT or1ua1t nm0s ogf fPe er n t o b Sa or -b i t Pa rl o c e d u r e i n Sj e cp q tuavar ola lt u(e ma
l eb
y 1s
o 0 u Lt )
d i ua mc ,c u r wa et ie g l tyhoae 1d 0, 0 v-o ml L u mf el at srk i, c o ft h Se t a n pdr ae rp da r a an ttdh i Ao
e sn sp ar ye p a ir na tt oi o n
a d adb o8u0m t oL f M o b ip lhe a s a ens,do n i ucn at dti ie ls - t h ce h r o m a tr oe g c tor hracedph hr ,o m a ta on mgd er aa sm -s ,
s o l vD ei dl .wu ti etM ho b pi h l a et sov eo l ua mn m edi, x . u r te h re e s p of on trshemesa jp oe ra Ck as l. c ut lh q ae tu ea n -
C h r o m a tsoy gs r(t a se epCmehh ir co m a t( o6 g2 r1 a) p) th i ytTiy nh , m ego ,fC i i H i zi nNt 2h pN e oar Qto sifPoenn t o b a r b i t a
l i q uc ihd r o m a i t s eoqgu ri a wp i
ppathe2h d1 4 d- e nt m e c t oS ro d ti auk b me y tn h fe o r m u l a :
a na d4 . 6 x-2 m5 m-c co ml tu h ma c nt
o n t 5a i- npu sam c k i n g
L 1T . hf el ro aw it s ea b o1 u. m t0 p L e mr i n uC t he r. o m a t o - ( 2 4 8 . 2 5 / 2 2 /6r s.) 2 7 ) 1 0 0 0 C ( r
g r at ph S he t a n s do al ur tdai no rnde, c to h r pe
de r a ek s p o n s e s
a sd i r e fc otPrer do c e td hucera ep :a fca ic ttk yo i,rs n , ol te s s i nw h i2 c4 h8 a . n 2 2d
5 2 6 a. r2te 7 h me o l e cw ue li ago rh f t s
t h 2a .n 5t ;h ce o l euf mf in ci is ne onl cte ysts h a1 n5 , t0 h0 e0o -p e n t o b s a ro bd iaitnuap dmle n t o b ar re bs ip t e ca C
tl ii,s v e l y ;
r e t ipc la al tte hst e;a i lfi an cgits n o ro mt o tr he 1a .n 5a; n d t h ce o n c e n ti nr ma tp g ie mor L n o,,fU SP Pe n t o b Ra S ri nb i t a l
t h r ee l a st it va e n dd ea vri daf to rri eop nl ii cn aj te eci ts ni o nt s t h Se t a np dr ae rp da raa ntr d i
y ao nnr sd ;a r te h pe e a a rk e a s
m o tr he a1 n5 . 0 % . o b t a fi rn t oe hmdAe s sp ar ye p a ra anttdhi Seo tn a n pd rae rp da -
r a t iro en s, p e c t i v e l y .
P r o c e d u r e i n Sj e cp q ta
uvar ola lt u(e ml a e y
b 1s o 0Lu lt ) =
o ft h Se t a n s do al r u atd inToden s to l u it ni tt o onh ce h r o m a t o - 4 )
m o ]
r a p rh e, c to hr ced h r o m a ta on m gd er a s mt us
h ar e
,r ee a s
o rt h me a jp oe ra Ck a s .l c ut lh pae t e er c eo nfat naiygm ep u - E
n ien U peo r to ifPoenn t o b S a ro bdititau aklb me ytn h e iS
o r m u l a : P e n t o b S
a o
r b
dI el c t i o ni|}
iintuja m
@
( 2 4 8 . 2 5 / 2 2 6 . 2 /7r s) ) ( 1 0 , 0 0» 0
P /e An (t Co /bWS
a) ro( bndirIitn uaj e ci sta si to en rsi ol le u E-=y
lm
i n w h i2 c4 h8 a . n2 2d5 2 6 a. r2te 7h me o l e cw ue li ago rhf t s
t i oo nfP e n t o b S a ro bdiiitnau u i t sa obl l- e a a
as lm }
s v e l yv ;e n Pt e. n t o b a m rabb yies tu ab ls t fi ottru ht ee d a l
p e n t o b s a ro bd iaitnuap dmle n t o b ar re bs ip et ca F tli i,
t h r ee l a r
t ie vse p foa nc sot efot rh ie m p u ar ci ct oy rt d ot ih ne g e q u i v a a lm eoaoutfPne tn t o b S a ro bd iiftouarml ,
t a b bl ee l oC w
i t;s h ce o n c e n ti nr ma tp g ie mor L n o,
,fU S P a d j u so tft mh e pe n
H T.
t hI en j e cc to ino tnta hi en s
P e n t o b R a S
ri nbt ih t
Se at la ns do al ur tdW ii os tn h; we e i gi nh t , e q u i vo afnl oel ten sts h a 9 n 2 .p 0e r cae nn ntdo t
m go ,fP e n t o b S a ro bd iiotun a
t mlh d
,er ib ea ds iu ss, e t od
p r e pt ah Treees ot l u tr ,i iotsnh ;pe e aa rk e f oa ar n iym p u r i m t yo t r he a1 n0 8p .e 0r co eft nh tle a b e al m e do u n t
i nt h Tee s to l u tai nor nd f oa pr e n t o b a r o
si s;t h pe e aa rk e fC tia 4l H i z 7 N 2 N a Q O 3 .
b i
i nt h Se t a ns do al ur ttd ihoienm :p u r mi te ite ehtrse e q u i r e -
m e ng ti svi en tn h tea b bl ee l o w : P a c k a a gnsidtn ogr a g e i nsP ir neg sl e oe r-
rm duv o
lets ie -
p l e - cd oo ns tea ip nr ee rf seo,rfTa y b | lpg yel a sT s h.I en j e c t i o n
m ab yep a c k i an 5g 0e - dc mo nL t a i n e r s .
R e l a t i v Ree l a t i v e
L a b e l i lna g b ie nl td iT cth ahetat eth sIe n j e ci stn i oot tnob e
C o m p o u n R ed t e n t iR oe n s p o n Lsi em i t
u s ief i dt c o n t aap ir ne sc i p i t a t e .
N a m e T i m e F a c t o r ( % )
6 - I m i n o - 5 - e t ah by o
l0 -u.5t3- 9( 1 - 1 . 5 0 . 2
m e t h y l - b u t y l ) b a r b i - C h a tnorge ea d :
t u r ai cc i d
5 - E t h y l - 5 - ( 1 -a ebt oh0 uy. lt9- 3 1 . 0 0.1 U SR Pe f e r
s e
t n
a c
n e
d
( a
1 r
1 d
) s
p r o p y l ) b a r b i t u r i c @ ( C 1 N7 - M a y - 2 0 1 8 )
a c i d * U SP Pe n t o b Ra S r b i t a l
P e n t o b a r b i t a l 1 . 0 = _ I d e n t i f i rc eat te i nt toi in
omofetn hTmeha ejp oe ria n k
5 - E t h y l - 5 - ( 1 ,a3 b- o1 u. t
5 0 . 9 0 . 3 t h ce h r o m ao t ft o
h Aegs rspaar mye p a r c ao tr ir oe nts op o n d s
d i m e t h yb la br -u t y ! ) t h ia ntt h ce h r o m ao ftt o h Segt ra an pmdr ae rp da ra as t i o n ,
b i t uar ci ic d o b t a i in tn heAed
s s a y .
* W h te hrme ae t e i rs li aa bl ea lsie nd t e sno dl efe od
l vry e t e r uis neta,hrl eyi m i t B a c t eE rn ida ol tT oe x( si 8t 5n )sc o In t na oi mtn os r e
o f5 - e t h y l - 5 - ( b1 a- re bt ihatyculiirspd3irc.o 0p %y . l ) t h a0 .n U8 S EPn d o tU o n ixp ti
em r
n og fp e n t o b sa or -b i t a l
d i u m .
 3 2 3
P e0 n t o b /a O rf bf ii cMt ia
oa ln
l o g r a p h s U S4 P1

p (H 7 9 1 )b :e t w9 e . a0e nn1d0 . 5 . A S S A Y
O t hr ee rq u i r e m me eetnth t rse es q uii tru enm d/e e nn-rt es P R O C E D U R E
j e c t a i onIn dsm p l aD nr tPu erg d o d (u1 c) .t s S o l u At :1i o g n/ oLfp e r c h al co ir di c
M o b ip hl aes Me : e t h at n e t o rl a, h y d a cr eo tf o un ri at n
r i,
A s s a y
a nS do l u A t (i2 o2: n. 51 :5 8: 0 )
M o b ip lh ea s e a fP ir le tp aea n rrddeeed g a ps H s3 .e 5d S y s stu ei m t a bs io ll iu tt y0i .o n 0 m:2 g 4 /o fcm a Lf f e i n e
m i x t o f u0 r. e0 M
1m o n o b p o a ts ai spc sh io us mpa hn aadc t- e a n 0d . 0m4 g 8 / o fm U SLP Pe n t o x iR fSiyn M l lo ib ni el e
e t o n i (t 6r 5i l: e3
M 5a) ak. de j u s itf nm ee cn et s(s s ea S ery ys t e m p h a s e
S u i t a bu i nl d iCtehy rr o m a (t 6o 2g1 r) )a .p h y S t a n sdo al ru t d 0i .o 0nm 5:g / o fmU S LP Pe n t o x i f y l
S t a n pd ra erpda r a t i ao nan c cD ui rswasetoe illgvyhe e d R Si n M o b pi hl a e s e
q u a n ot fU i tSPyPe n t o b Ra S ri nbM io tbapilhl ae sa end,di l u t e S a m spo ll e u t 0i .o 0nm :5g /o fPm e Ln t o x ii fn My o l l- i n
q u a n t i ta a tn sidtv ee lpiywf n,ie sc ee s ws ia tMr ho y ,b ip lh ae s e b i lpe h a s e
t oo b t aa si onl u ht ia ov nai kn g n oc w o nn c e n o tf a r ab to iu ot n C h r o m a t soy gs rt ae pm h i c
0 . 1m g p e mr L . ( S eC eh r o m a t ( 6o 2g1Sr)y a,s pStuhei ym t a b i l i t y . )
A s sp ar ye p a rq au at ni to intdsaitl iau vst eue i t v a bo lle u m e M o d Le C:
o fI n j e cw tii tMo hon b pi h l aet soo eb t aa si onl u ht a i ov ani n g D e t e c Ut o V2 r7 :n 3 m
k n oc wo n n c e n ot fa r ab to0 iu . o1m
tn g p e mr L . C o l u 4m .n 6: x-2 m5 m - 5c -m ’;pu am c kL i 1 n g
C h r o m a t soy gs r(t a se epCmehh ir co m a t( o6 g2 r1 a) p) h yTF hl ero awt e0 :. m7 L / m i n
l i q uc ihd r o m a i t s eo qguri a wp ippt ahe2h d1 4 d- e nt m e c t o r I n j e cv toi lo un 1m 0 pe L:
a na d4 . 6 x-2 m5 m-c co ml tu h mac nto n t a 5i - np sa m c k i n gS y s stu ei m t a b i l i t y
L 1T . hf el ro aw it s ea b o1 u. m 0 pL e mr i n uC t he r. o m a t o -S a m p lS e
t y s st :u ei m t a bs io ll iu ta ty in S
odnt a n d a r d
g r at ph S he t a n pdr ae rp da r aa ntrideo cn to, h r pe
d e ra e k- s o l u t i o n
s p o na sd ei sr e fc otPrer do c e td hucera ep :a fca ic ttk Kyo i,rs , S u i t a br iel qi ut yi r e m e n t s
n ol te st s h 2a .n 5t ;h ce o l euf mf n i ci is ne onl cte yst s h a n R e s o l u Nt Li1 o T0 n.b:0e t wc ae fe f na e in pndee n t o x i
1 5 , t0 h0e 0o r ep lt ai tcteahstle;a i lf ian cgits n o ro mt o tr he a n f y l l i
S nye s
, st u ei t
m a b
s io l
l iu tt y i o n
1 . 5a; nt dh ree l a st it va e n dd ea vr idaf ot rri eop nl ii cn aj et ce - R e l a st t i v a en dd eav ri da tNi M o2n T .: 0S %t ,a n d a r
t i oi ns ns o mt o tr he a2 n. 0 % . s o l u t i o n
A n a l y s i s
P r o c e d u r e i n Sj e cp q tuava r ola lt u( e ma
l eb
y 1 s
o u 0 Lt ) S a m p lS e t s a n :s do al r u a td in oSdna m spo ll eu t i o n
o ft h Se t a n pdr ae rp da ra an ttdhi Aeo sn sp ar ye p a ir na tt oi o n C a l c ut lh pae e t er c eo nf pt ea n gt eo x i(f Cy i l ls iHn ie s N
t h ce h r o m a tr o e g c tor hracedph hr ,o m a ta on m gd er aa sm -s , i nt h pe o r to ifP oe nn t o x it fa yk le lni :n e
u r te h re e s p o f ontrshemesa jp oe ra C k s a .l c ut lh paeet re -
c e n to a fC gi e; H i 7 Nt 2 h peN oar Qto3ifIion nj e ct ta i kobenyn R e s = u (lr tu / xr (s C) s /xC1u 0) 0
t h fe o r m u l a :
r u =p e r a ek s p fo rn tos hm eSe a m sp o ll eu t i o n
1 0 0 ( 2 4 8 . 2 5C /u 2) (2 r sr6) u. / 2 7 ) ( C s r/s =p e r a ek s p fo rn t os hmeSe t a n sdo al r u td i o n
C s = c o n c e n ot fr U aSPtPei not no x iR fSiyntl h l ie n e
a l i n w h i2 c4 h8 a . n 2 2d5 2 6 a. r2te 7 h me o l e cw ue li ago rh f t s S t a n sdo al ru ( td imo gn / m L )
i s p e n t o b s a ro bd iaitnuapdmle n t o b ar re bs ip te ca Ctl isi,sv e l y ; C y = c o n c e n ot fPr ea nt tio ox nii fn ty hlSel ai mn ep l e
f o t h ce o n c e n ti nr ma tp g ie mor L n o,,fU SP Pe n t o b Ra S ri nb i t a l s o l u ( t im o gn / m L )
i }
t h Se t a n pdr ae rp da rCa iyts ti hof eni n; ca lo n c e n ti nr ma tg i o nA ,c c e pc t r ia t ne r9 c ie 8a . : 0 % - 1 0 2 . 0 %
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e A .I N F R AA BR S E O
D R( P1 T9 I7 OK N) i n M o b ip lh ae s e
e B . T h r ee t e nt ti iomofe tn h me a jp oe roa ftk h Se a m p l e S a m spo ll e u t 3i o 5gn0 :/ o mfP eLn t o x ii fn My ol bl i nl e
s o l u ct io or nr e ts otp ho aon tftd hsSe t a n s do al ur tadiso n , p h a s e
o b t a iintn heAeds s a y . C h r o m a ts oy gs rt Pa e rmp o:hcaiesdcei dr e icntt he ed
A s se ax yc fe o ptr th feo l l o w i n g :
I n j e cv toi lo un 2m 0 pe L:
R ut ni m e N :L5 T t i mt eh sre e t e nt ti ifmooenr
p e n t o x i f y l l i n e
U S4 P1 O f f i cM ioa n
l o g/ Pr ea n pt h
o xs i 3f 2y 3
l l1 i n e

S y s stu ei tm a b i l i t y t a r a, dP du r i W f ia e tid nes rm a plolr t ia o nnt dsr i , t ut ro a t e

S a m p lS ey s s: tu ei mt a bs io ll iu ta odnt a n d a r d m a ak sem o poa ts h
ty in S tA e d.i dn c r e avs o i nlg uo f mP ue rsi f i e d
s o l u t i o n W a tt eomr a akp ee n t o x il ifq yu iltd lh iias nt p eo u r a b l e .
S u i t a br ie l qi ut yi r e m e n t s T r a n ts hfcee or n t oe ftn htmeso r ts at re ,p aw n i qsd ue a n -
R e s o l u Nt Li1 o T0 n.b:0e t wc ae fe f na e in pnde n t o x i t-i t a t it voae cl ay ,l i b br oat tt A el ded e .d n o Puu g r ihf i e d
f y l l iS nye s, stu ei m t a sb io ll iu tt yi o n W a tt eob rr it nof gi n va lo l ua mn e md,iw xe l l .
R e l a st t i va en dd eav ri da tNi M o5n T.: 0S %t ,a n d a r d
s o l u t i o n A S S A Y
A n a l y s i s ' P R O C E D U R E
S a m p lS et s a n :s do al ru atd in Sodna m spo ll eu t i o n S o l u At :i5 o0n m mM o n o b p o a ts ai spc sh io us mp h a t e
M e a st u h aerr eeoafa sl lt h pe e ai kn t sh Se a m spo ll ue - b u f fae dr j, u ws it tpe hh d o s pa hc oit rdoa i p cH o f3 . 2
t i o en ,x c fe optrth oa ftp e n t o x i f y l l i n e . M o b ip hl aes Ae c: e t o n ai n tSrdo il lu eA t ( i 2o 0n : P8 0
a )s .s
C a l c ut lh pae e t er c eo nfet aa i cg mhep u irnti htpeyo r - t h r oa u fgi hl to ef0r . 4 5p -o u sr imez ae ,n dde g a s .
t i oo nfP e n t o x it fa yk le ln i:n e I n t e sr t n aa ln sdo al ru d t 1i o 0jn01: g o/ fcm aL f f ien i n e
M o b pi hl a e s e
R e s = u (lr tu / xr (s C) s /xC1u 0) 0 S t a n sd taosrcodkl u t 2i 0 om n g: / o fmU S LP Pe n t o x i f y l -
l i nR e Si n M o b pi hl a e s e
t y =p e r a ek s p oofen as c ie m
h p u fr ri o t hym e S t a n sdo al ru t d Pi io pn1e:.tm 0 oL fS t a n sdt aosrco d kl u -
S a m spo ll eu t i o n t i oi nn ta o1 5 -c m o nL ic ce an lt r ti uf buaegn,ead d 9dm L
r s =p e a r e k s p oofpn esn et o x if fry t ol hm
l ei n e o fd e i o nw i a t z eM rdit.xh se a m fpo l3r es0 i na v o r t e x
S t a n sdo al ru td i o n m i x ea rn c,de n t rf io f3r u0mg iea nt1 2 x 5 g 0.P i p e t
G s = c o n c e n ot fr U aSPtPei not no x iR fS iyntl lh ie n e 5 0 w lo ft h se u p e r in na tatso ae np ta br oar to es i l i c a t e
S t a ns do al r u (td igo / n m L ) c u l tt uu rbdeei ,l uw ti et5 h7w5oLf M o b pi hl ae sa en ,d
C u = c o n c e n ot fPr ea nt tio ox nii fn ty hlSel ai mn ep l e a d 6d 2u5l o fI n t e sr t n aa ln sdo al ru td t oio ob nt aa si on-
s o l u (t \i 4 o ng / m L ) l u t ih oa nv ai nn og m ic no an l c e n ot fr 8 a0gt / i o mfn L
A c c e pc tr ia tne c r ie a p e n t o x iaf ny5d l 0lg i / n oemfc aLf f e i n e .
I n d i v ii md pu ua rli N t i Me0 sT .: 2 % S a m spo ll e u t Si h o nat:kh eo r o bu yhg ah e nl ay
d bc oh t -
T o tiamlp u r i N t iMe0 sT .: 5 % t l oe fO r aS lu s p e Pn is pi1eo.tnm 0 .oLfO r aS lu s p e n s i o n
i n ta o1 5 -c m o nL ic ce an lt r ti uf buaegn,ead d 9 dm oLf
S P E C TI EF S I C T S d e i o nw ia t z eM rdit.xh se a m fpo l3r es0 i na v o r t e x
e C O M P L EO TFSEO NL EU S(T S 6I 4O1N) m i x e a rnc,de n t rf io f3r u0mg iea nt1 2 x 5g 0.P i p e t
S a m sp o ll e u t 1 i oign 5: 0m oL fc a r db io o nx i d e - f r e5 e0 u Lo ft h se u p e r in na tatso ae np ta br oar to es i l i c a t e
w a t e r c u l tt uu rbdeei ,l w u ti et5 h7u5oLf M o b pi hl ae sa en ,d
A c c e pc t r ia t ne rMc iea e:t th rse e q u i r e m e n t s a d 6d 2u5l o fI n t e sr t n aa ln sdo al ru td t oio ob nt aa si on-
A C I D I T Y l u t ih oa nv ai nn og m ic no an lc e n ot fr 8 a0 u t gi /o fnm L
S a m spo ll e u t 1 i oign 5: 0m oL fc a r db io onx i d e - f r ep ee n t o x iaf ny5d l 0lg i / n oe mfc aLf f e i n e .
w a t e r C h r o m a t soy gs rt ae pm h i c
A n a l y Ts o it sh S:e a m spo ll eu a t id1o dd
n r oo fp ( S eC eh r o m a t ( 6o 2g1Sr)y a,s pStuhei ytm a b i l i t y . ) &
b r o m o bt lh u T ySe m. o l M o d Le C: a l
A c c e pc tr ia t ne c rNieaM0: .Tm 2 oLf0 . 0N1s o d i u m D e t e c Ut V o2 r8 :n0 m a ]
h y d r ios rx ei qd ueti op r er do da cu oc lc eo hr a n g e . C o l u 4m .n 6: x-2 m5 m- 5c -m 1;pu am c kL 1i n g Ss
e L O SO SND R Y (I 7N 3G1 ) F l ro awt e1 :. m0 L / m i n
A n a l y Ds r i usy n: dve ar c aut6u0 fm o 3r h . I n j e cv toi lo un 1m 0 pe L: |
A c c e pc t r ia t ne rcNieaM0 : T . 5 % 2
S y s stu ei m t a b i l i t y K e ]
S a m p Sl te a: n s do al r u td i o n m i
A D D I TR IE OQ NUAILR E M E N T S i )
[ N o t re e l aT rt hie ve t ee nt ti imfoeoncsr a f f a e in nde m o l
¢ P A C K A A GNSIDTN OGR AP G r eE s:ienwr ev le l - c l o s e d p e n t o x ia fr yae lb lo0 i un. te4a 2n1 d. 0r ,e s p e c t i v e7zl) sy . ]
c o n t a i n e r s .
e U SR PE F E RS ET NA CNE (D 1A 1R) D S S u i t a b
r ie l qi ut i
y r e m e n t s
U SP Pe n t o x iR fSy l l i n e R e s o l u Nt Li1 o T0 n.b:0e t wp ee ne t no x iaf ny d l l i n e
c a f f e i n e
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R e l a st t i va en dd eav ri da tNi M o 2n T
.: f0o r%r e p l i c a t e
P e n t o x iCf oy lm lpi onO eu r an ld e dA ni an lj ey cs ti iso n s
S u s p e n s i o n S a m p lS e t sa n :sdo al ru a td in oSdna m spo ll eu t i o n
C a l c ut lh pae e t er c eo nftth a le ag be eal me do ouf n t
D E F I N I T I O N p e n t o x i( fC yil sl Hi ini e gnt Nh p 4e oO r3 t)o ifO or naS lu s -
P e n t o x iCf o y lml p i noOe u r aSn luds ep d e cno sn it oNa niL nTs p e n st ia ok n e n :
9 0 .a 0n % Nd M1 T 1 0 o.ft0h l %e a b eal me do ouf n t
p e n t o x i( fC y)l 3l Hi in se N « O s ) . R e s =u (l Rt u /xR( sC )s /xC1u 0) 0
P r e pP ae rn te o x iCf o y lml p i noOe u r aSn lud s ep d e 2n 0m
s ig o/n
m aL sf o l l( os weP seh a r m a cC eo u mt pi oc u a ln d i n g R Nu o = n ps etbaeakri - r as t oi fop e n t o x it foty hl el i n e
i l eP r e p a r( a7 t9 i5 o) n) s. i n t e rs nta al n fd ra t or hdm Se a m spo ll eu t i o n
R s = p e ra ek s p r oa n t oisfpo ee n t o x it foty hl el i n e
i n t e rs nta al n fd ra tor hmdSe t a n sdo al ru td i o n
P e n t o x ie fxy tl el ni dn e d - r e l e a s e C s = c o n c e n ot frU aSPtPei not no x iR fSiyn tl lh ie n e
t a b le eq tu si ?vt ao l e n t 2 go p f
e n t o x i f y l l i n e S t a n sdo al ru (td \i 1o ng / m L )
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t o taa lm o tuobn ept r e p aP rl eatd c he.re e q u n i ru emd b e' rP (H 7 9 15 ).: 9 - 7 . 7
o fP e n t o x ie fxy tl el ni dn e dt a- br lei enlat e
ss ua is te ma bo lr e -
 3 2 3
P e2 n t o x i/ Of fy fl ilcMiiona en
l o g r a p h s U S4 P1

A D D I TR IE OQ NU AI LR E M E N T S T o l e r a np ce er sc e oT n fh
t he
a lega eb seal me do ouf n t
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c -m
c o n t a iS nt eoi rrnaser. e f r i goe rar tc a to on rt r ro lo lo e md c e p t Ta an b2cl.ee
t e m p e r a t u r e .
e B E Y O ND DA -T UENs: M E9 T0
d a ay fs t te hrde a t o enw h i c h T i (m heo u r s ) A m o d ui n s st o l v e d
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e L A B E L LI aN biGtet: loi n d i tc ha iatt ites t ob e w e ls lh a k e n 4 b e t w3e 0ea % nn 5d 5 %
b e f uo sr ea
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e U SR PE F E RS ET NA C NE D
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T E S2 T tI hf pe r o dc uo cm tp wl ii ttehhsit es s tt ,h lea b e l i n
i n d i ct ah tiatetm se eUt SsD Pi s s o lT eu s2t .ti o n
M e d iw u a tm e9: r0m;0L .
A p p a r 2 :a7 t5ru ps m .
P e n t o x iE fx ytl el ni dn ee T
d a- bR le el te sa 6s,:1e0
T i m 1e ,s a
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e eo
n ol te sts h a 5 p. e0 r ca ennndto mt o tr he a n C iT 3o Hl ied sri Nas 4snp oOace lt3etrvshceteedioTnmfshtephaeselegca eibcsfeoail neme dftdo oootufrhnm
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1 0 5p .e 0r co eft nh lte a b eal m
e do oufp ne tn t o xf io l- l ot aw bi ln eg.
f y l l( iCn ye 3 H i g N 4 O 3 ) .

P a c k a
a gnsidtno gr a g e i nwP erl el s- ce lor onvst eea di n - T i (m he o u r s ) A m o d ui n s st o l v e d
e r sP. r o tf erc loti mg hat n,s dt o br e
e t w1 e
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L a b e l il an bge il niTdn hig ce t ah Dteie ss s o lT eu stw tii ot nh 6 b e t w3e 5ea % nn 6d 0 %
w h it ch phe r o dc uo c m p
t l i e s . 1 0 b e t w5e 3ea % nn 7d 8 %
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( a 1 r 1 d ) s 2 0 n ol te sts h a8 n 0 %
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I d e n t i f i c a t i o n T E S3 T tI hf pe r o dc uo cm tp wl ii ttehhsit es s tt ,h lea b e l i
A :I n f r Aa br se do r (p 1 t i9 o7n K ) i n d i ct ah tiat etm se eUt SsD Pi s s o lT eu s3t .ti o n
T e ss tp e c i m e pn o w F nid one
fteer lwtyeh ra 5 Tn a b l e t s . M e d iw a u tm e 9: r0m ; 0L .
( Ac o a sr cs re em eanb yeu s te od s e p a tr hapet oe w fd re orm A p p a r 1 :a1 t0 r u0p s m .
t h tea b fl ie tl m - ci fon ae tc ie ns gTs r a ra yn a.s n)fa ec rc u r a t e l Tyi m 2e ,s8 ,:1 2 a , n 2d h 0 o u r s .
w e i gp h o re td
o ifto hnpe o w de eq r u i, vt aoal be o n tu t P r o c e d u ra esd i Pr e r fcootTcreeeds7 e.t d
a l 2 0 m0 og fp e n t o x i tf oya l1 l5i - nc em
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m he o u r s ) A m o dui n
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5 r e s ii dn au be o1 u5m t oLfm e t h y c hl leo nrt eir da en t,s ofa e r 2 b e t w1e 5ea % nn 3d 5 %
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qa A l lt oh lwea y te ros se p a rt ar ta en ts, hf mee er t h y c hl le onr ei d e
1 2 b e t w7e 5ea % nn 9d 5 %
a ) l a y ea r n,p da st sh r oa fu ug nh np eal cf i a l l e wd i ta hn h y -
=) d r osu os d siu ul fmca ot el ,l e tc htf ei ln tgir naa tse m a l l 2 0 n ol te sts h a8 n 5 %
b e a kEevr a. p ot rh saeotl eu wt ii to nh aei od fa c u r ro ef n t
a i tr od r y na etas bs o3 u5 tD .i s s to hlrev ee s is doou be t a i n eT dE S4 T tI hfpe r o dc uo cm tp wl ii ttehhsit se s tt ,h lea b e l i
i n8 t o 1 0 m oL fe t h ea rn,tdh ce h ni il nla ni c be a ti fh , i n d i ct ah tiatetm se eUt SsD Pi s s o lT eu s4t .ti o n
n e c e s ts oi a rn yd ,
cu r cy es t a l lC io zla lt eih coertn y. s o
t anl s M e d iw u a tm e9: r0m;0L .
f i l tp ear p e
w ra ,ws ih ta hb o2 um toL fc o l e td h ea rn,adl l o w
t oa i r - dP rr ye. pa am ri ex to ufa rbe o1u .t ( 5 w% /o wft )h e A p p a 2
r :a5 t0rupsm .
c r y s it nap los t a sb sr io um mi d e . T i m 1e ,s
8 ,:a n 2d 4 h o u r s .
B :T h r ee t e nt ti iomofetn h me a jp oeria ntk h ce h r o m a t Po r- o c e d u ra esd i Pr r e fcootc
Treeeds1 e.t d
g r oa ftm h A es s pa ry :e p a c r ao tr ir oe nts otp ho iannttd hs e T o l e r a np ce er sc e oT n fh
t h e
a lega eb seal me do ouf n t
c h r o m a o f tt o
h Segt ra anpmdr ae rp ad ra asot bi t o na i,in n e dC i 3 H id gi Ns 4s o Oa lt3t vh t
ee di ms ep se c ic f oi ne dft oot rh m
e
t h Aes s a y . f o l l ot aw bi ln eg.
D i s s o ( l u7 t1 i1 o) n
T E S1 T t I hf pe r o dc uo cm tp wl ii ttehhsit es s tt ,h le a b e l i n g T i (m he o u r s ) A m o dui n s st o l v e d
i n d i ct ah tiatetms e eUt Ss D Pi s s o lT eu st1 .ti o n i L b e t w0e a % e nn2d 0 %
M e d i
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9: r0 ;
m0 oL r1 0 m
0 L0 . 8 b e t w3e 5ea % nn 6d 0 %
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2 :a1 t0 r
u0s
p m . 2 4 n ol te sts h a8n 0 %
T i m 1e ,s
4 ,:8 , a n 1d 2
h o u r s .
P r o c e d u r et h aD e me to oeufCrnimt3i Hn ie d si sN -4 O 3 T E S5 T l tI hf pe r o dc uo cm tp wl ii ttehhsit se s tt ,h le a b e l i
s o l b v eye dm p l oU ya Vib ns go ra pttt hiweoa nv e lo ef n g ti nh d i ct ah tiatetm se eUt SsD Pi s s o lT eu s5t .ti o n
m a x i a bm suo mra btaa bn oc 2ue7tn4 o m n f i l t eproerdt i o n s M e d iw u a tm e9: r0m;0L .
o ft h seo l u u t in odtnee s r
st u, i t da ib ll uyw tie tMd he d i ifu m ,
n e c e s i sn ca or ym ,p aw ri iat sSh o t na n sd oal ru d ht ai ov ani n g A p p a r
2 :a
7 t
5 ru ps m .
k n oc wo nn c e n ot fr U aSPtPei not no x iR fSiyntl lh s ie na e m e T i m 1e ,s
2 ,:4 ,6 , a n 2d 0h o u r s .
M e d i u m . P r o c e d u ra esd i Pr r e fc
ootc
Tr eee sd
1 e,ted x c te oup st e
t h we a v e lo efmn a g tx hi
a bm suo mra btaa bn o2 c ue
6tn4 m
i n s to ef2a 7d n4 m .
U S4 P1 O f f i cM ioa nl o g/ Pr ea n pt ho xs i 3f 2y l3 l3i n e

T o l e r a np ce er sc e oT
n fh
t h
e
a lega eb se al m
e do ouf n t M e d iw u
a tm e
9: r0m
;0L .
C i 3 H id gi Ns 4
s o
O
a l
t3
t vh e
tedi ms ep se c ic f oi n
e dft o
ot r
h e
m A p p a 2r :a5 t0rupsm .
f o l l ot aw bi ln eg. T i m 1e ,s3 ,:6 ,1 2 a, n 1d 8
h o u r s .
P r o c e d u ra esd i Pr r e fcootc
Treee ds1 e,ted x c te oup st e
T i (m he o u r s ) A m o dui n s st o l v e d t h we a v e lo efmn a g tx hia bm suo mra btaa bn oc 2ue3tn0 m
1 b e t w5e a
%e nn2
d 5 % i n s to ef2a 7
d n4 m .
2 b e t w1e 0ea %
nn 3d 5 % T o l e r a np ce er sc e oT
n fh
t h
a
e le
ga eb se al m
e do ouf n t
4 b e t w2e 0ea n
%
n5d 0 % C i 3 H id gi Ns 4
s o
O
a l
t3t vh e
te di ms ep se c ic f oi ne d
ft ot r
h m
e
6 b e t w3e 0ea %nn 6d 0 % f o l l ot aw bi ln eg.
2 0 n ol te sts h a8 n 0 %
T i (m he o u r s ) A m o d ui n s st o l v e d
T E S6 T t I hf pe r o dc uo cm tp wl ii ttehhsit es s tt ,h le a b e l i n g 1 b e t w0e a %e nn2d 0 %
i n d i ct ah tiatetm se eUt SsD Pi s s o lT eu s6t .ti o n 3 b e t w2e 0ea % nn 4d 0 %
M e d is u i m u: lg aa st tfe rldiu (ci w
d i t eh no zu yt m e s ) ; 6 b e t w3e 0ea % nn 6d 0 %
9 0m0L . 1 2 b e t w5e 0ea % nn 8d 0 %
A p p a 2r :a5 t0r ups m . 1 8 n ol te st s h a8 n 0 %
T i m 2e ,8s ,:1 2 a
, n 2d 4
h o u r s .
P r o c e d u ra esd i Pr r e fcootc
Treee ds1 e.t d T E S1 T0 t Ih p fe r o dc uo cm tp wl ii ttehhsit es s tt ,h l ea b e l -
T o l e r a np ce er sc e oT n fh
t h e
a lega eb se al me do ouf n t i n ig n d i ct ah tiatetm se eUt SsD Pi s s o lT eu st1 ti0 .o n
C i 3 H id gi Ns 4 s o O
a l
t3
t vh e
tedi ms ep se c ic f oi ne d ft ot r
h m
e M e d iw a u tm e9: r0m;0L .
f o l l ot aw bi lne g. A p p a r 2 :a7 t5ru ps m .
T i m 1e ,s
6 ,:1 2 a
, n 2d 0
h o u r s .
T i (m heo u r s ) A m o d ui n
s st o l v e d P r o c e d u ra esd i Pr re fcootcTreee ds1e.t d
2 B e t w e1 e0na %n d ’ 3 0 % T o l e r a np ce er sc e oTn fht hae lega eb seal me do ouf n t
8 b e t w4e 0ea %
nn 6d 0 % C i 3 H id si Ns 4s O
oa lt
3t vh e
tedi ms ep se c icf oi n
e dft o
ot r
h e
m
1 2 b e t w5e 5ea %
nn 7d 5 % f o l l ot aw bi ln eg.
2 4 n ol te st s h a8 n 5 %
T i (m he o u r s ) A m o dui n s st o l v e d
T E S7 T t I hf pe r o dc uo cm tp wl ii ttehhsit es s tt ,h lea b e l i n g i n o mt o tr he a2n 0 %
i n d i ct ah tiatem
t se eUt SsD Pi s s o lT ue ts7 .it o n 6 b e t w3e 5ea % nn 6d 5 %
M e d i
w a
u tm e
9: r0m;0L . 1 2 b e t w6e 0ea % nn 9d 0 %
A p p a 2r :a5 t0ru ps m . 2 0 n ol te sts h a8 n 0 %
T i m 1e ,s3 ,:8 , a n 1d 8
h o u r s . je
w n
P r o c e d u ra esd i Pr r e fcootc
Tr eee d
s7 e.t d U n i f oo rfdm oi stuayn gi(et9 s0 5m )e :te htre e q u i r e -x e ]
T o l e r a np ce er sc e oT n fh
t h a lega eb seal me do ouf n t m e n t s .
e
C h r o m a t pougrri at py h i c =
C i 3 H id gi Ns 4s O oa l
t3t vh t
ee d
i ms ep se c ic f oi ne d ft ot r
h e
m iS
f o l l ot aw bi ln eg. s xe t, r a sco tl iu nt gi o =n ],
P e r c h al co sir odi lc u tMi oo bn p,i hl ae E
a n Sdy s stu ei m t a bs iol li u t yt i o an sd P i r eeicpntta herAe s -r 5
de e}
T i (m heo u r s ) A m o dui n s st o l v e d s a y . rie y
1 n o mt o tr he a2n 5 % S t a n sd oa lru dt i o na naD ci cs us rowaletve ielgqyhu e n i- }
a d
t i to yfU SP Pe n t o x iR fSiynEl xl ti rn aescotliu nct go
i on nt a i n >i n g
2: b e t w2e 5ea % nn 4d 5 % v v

8 b e t w5e 5ea % nn 7d 5 % a na m o oufmn e tt h ea qnutoaol0l . o8 ft% h t eo t va lo l u m e

t ob eu s e ad n,d di l qu ut ae n t i t a a tn sidt v ee lpiyw f n,iescee s -
1 8 n ol te st s h a8n 0 % s a r wy i, t
E xh t r a scotliu ntt goio ob nt aa si on l u ht ia ov nai n g
k n oc w o nn c e n ot fa r ab to0iu.ot9 1n 6gp e mr L .
T E S8 T tI hfpe r o dc uo cm tp wl ii ttehhsit se s tt h , lea b e l i n Tg e ss to l u t i o n1 0T .mr 0a oL fnt shffei er dsrit l u fti il ot nr a t e
i n d i ct ah tiatetm se eUt SsD Pi s s o lT eu s8t .ti o n f r to hmAe s sp ar ye p a tr oaa t2 i5 o -vn m o lL u mf el at sdrki i,- c
M e d iw u a tm e9: r0m;0L . l u tw ei tE xh t r a sc otliunttgoiv oon l ua mn e m di, xT .hf e i n a l
A p p a r 2 :a7 t5ru os m . c o n c e n ot fpr ea nt ti oox nii nft yh lis l so il nu iets ai bo no u t
0 . 3m 2 p g e mr L .
T i m 1e ,s2 ,:4 ,1 0 a, n 1d 6 h o u r s .
C h r o m a t soy gs r(t a se epCmehh ir co m a t( o6 g2 r1 a) p) h y
P r o c e d u ra esd i Pr r e fcootcTreee ds7 e.t d P r o ca e sd ei dr e icn tt he Ades s aC yh. r o m a tt ho Se tg arn a- p h
T o l e r a np ce er sc e oTn fht e ha lega eb seal me do ouf n t d a sr odl u tai no rnde, c to h r pe
de a r ek s p o f onprse en st o x i f y l -
C i 3 H id gi Ns .s o Oa lt3t vh etedi ms ep se c ic f oi ne dft ot rh m e l i na esd i r e fc otPrer do c e td hur ere el a; st it va e n dd ea vri da t i o n
f o l l ot aw bi ln eg. f o rr e p l ii cn aj te eci ts ni o mnt so tr he a5 n. 0 % .
P r o c e d u r e i n Sj e cq p tuavar ola lt u( e mal eby 1so 0 u
p Lt )
T i (m he o u r s ) A m o d ui n s st o l v e d o ft h Se t a n s do al r u atd in todh
n Tee s to l u it ni tt o onh ce h r o -
1 b e t w1e 0ea % nn 2d 0 % m a t o g ar naadp l lht o,h wce h r o m at t or ouf ngi vr t ei
a mm e s
2 b e t w1e 5ea % nn 3d 5 % l o n tg he a tr n
h re e t e nt ti iomofetn h pee n t o x ip fe ya lkl .i n e
4 b e t w2e 5ea % nn 4d 5 % R e c to hrcedh r o m a ta on m gd er a s am l u
slt r
,h pe e ra e k-
s p o nf sr et o shmTee s to l u tei xo c n t,e hp fatotpr e n t o x i f y l l i n
1 0 b e t w5e 5ea % nn 7d 5 %
a l c u tl ha e tp ee r c e on fet aa gc i emh p u irn ti htpeyo r to if o n
1 6 n ol te sts h a8 n 0 % T a b lt ea tks be ytn h fe o r m u l a :

T E S9 T t I hf pe r o dc uo cm tp wl ii ttehh sit es s tt ,h le a b e l i n g 3 1 2 Cr (s )r , /
i n d i ct ah tiatetm se eUt SsD Pi s s o lT eu s9t .ti o n
i n w h i Cics th h ce o n c e n ti nr ma tp g ie mor L n o,fU S P
P e n t o x iRf Siyntl lh Sie t
n ea n s do al ur trd; ii s ot nh;pe e ra e k-
 3 2 3
P e4 n t o x i/ Of fy fl ilcMiiona en
l o g r a p h s U S4 P1

s p o fn o ser ea c i m h p u or bi tt ay fi rn t oe hmdTee ss to l u t i o n ; P e p p eO ri lm i n t 1 0 m0 L
a nr sdi s t h pe e a r e k s p foo n pr es ne t o x io fby tl al fi rnn eoe m d P e p p e ri nmc io na trp ,so ew d e r 1 0 g
t h Se t a n s do al ur td ni oomtn o : tr he a0 n. o3 fa% ni yn d i v i d uA la cl o ha so ul f, f i qc ui ea nn tt oim ta yk e 1 0 0 m 0L
i m p ui rs fi ot uy a n dn n;do mt o tr he a1 n. o0 ft%o t ia ml p u r i -
t i ei ssf o u n d . M a c e tr hape te ep p el re am vi f erns et
,a esmd u ac spho s s i b l
A s s a y f r os tm eam ns c do a r p s eo lw yd f e or1 r e
h idn 5
, 0 m0 oL f
P e r c h al co isr doi cl u t i o n1 . D g0oi fps es ro cl hvaleco ir di c p u r i wf ai t e de
a rn t,dh e s tn r o en xg pl ryt eh sesAm d.t dh e
i n1 0 0 m 0oL fw a t e a rn m,di x . m o i sm ta ,c e r l ea attv eoe9ds 0 m0 oL fa l c o a h on aldl, l o w
M o b pi h l ea s e aPf ir let pa e ranedrddee g a ms is xe toduf r e t h me i x tt uos rt eaf no 6dr h w i tf hr e q aug ei nt taF ti li -o n .
P e r c h al co sir odi lc u ta ic oe nt ,o n t i te rt irl ae h, y da rn o df u r at ne r,a, nt dot hf ei l t ra a dttedho ei l a, n ad da dl c ot ho o l
m e t h (a8 n0 o: 1l 5 :M2 a . 5ak :de2j )u ,s i tf nm ee cn et s(ss ea er y m a tk hepe r o dm ue ca ts1 u0 rm0 eL0 .
S y s St u ei m t a bu i nl diCteh y rr o m a (t 6o 2g1 r) a ) .p h y A S S A Y
E x t r a sc ot li nu gt i o an m iP xr teo ufpw raa ertaeenr a dl c o - e C O N TO E FP NE T P P EO RI ML I N T
h o (l 7 : 3 ) . S a m p 5l. em 0 :oLfS p i r i t
S y s stu ei m t a bs io lli ut t y i o na bT or2ua0m t
n sog fUe Sr P A n a l y Ts ri as n:ts hfSee ar m tp oal Be a b cb oo tct kgl re a, d -
P e n t o x iRf Say ln aldib no1 e u0m t og fc a f f eei an c ae ,c
h c u - u a tt eo8d % A. d1 d. m 0 oL fk e r o sa ennmdei ,xA .da d
r a t e lwy e i g thoae 2d 5, -v m o lL u mf el at sA rk id
. 0cd. m 2 oL f s a t u rc aa tl eccdih ul m o sr oi ldueta ic oi nd ,iw fii t he y
hd -
m e t h aa nnsodw li t,r hlf el a ts od e h a n o l . d r o c h a lc oi a
k i s t r ti hbmeu et t rd i
l
, cm to o
fsi lt
l h be u o
l fb
t h beo t t l e .
D i l wu ti etE xh t r a scotliu ntt goiv oon l ua mn medi, xP .i p e tR o t ta htbeeo t vt il ge o r ot uoes nl ys mu ir xe ian ngtd,h e n
3 .m
0 oLft h re e s u ls to il nu git ni tao o5 o lL u m e t r i c a da
n 0 -v m sdu f f iq cu ia enontf ti htcey a l cc ih ul m o sr oi ldu e-
f l a sd ki ,l u
w ti et
E xh t r a sc ot liunttgoiv oon l ua mn e
m di, x . t i ot nob r itn hgse e p a or iali tn ett doh ne e oc fk
t h beo t -
S t a n pd ra er pd a r a t i ao nan c cD ui rswaseto i ellgvyhe e d t l eC. e n t ra itaf bu o g1eu5t 0r 0pf m o 5r m i na ,n rde a d
q u a n ot fU
i tSPyPe n t o x iR fSiynEl xl ti rn aescotliu nct go i on n- t h ve o l oufo mi ilen t h se t eSmu.b t f ri avd cei tv i sf io or n s
t a i nai na
n gm o oufmne tt h ea qnutoaol 0l . o8 ft%h t eo t a l t h ke e r o as de dn a ee nd m,du l t ti hpreley m a in nui mn -g
v o l tuobm e ue s e ad n,d di l qu ut ae n t i t aa tn sidtv ee lpiywf ,i s e b e o rfd i v i sb i y4o .nt2s oo b t ta hi ven o l ui nmm eL o,,f
n e c e s ws ia t
Er xh
y t, r ascotliu ntt goio ob nt aa si onl u ht ai v
o -n p e p p eo iril mn 1i 0nm0toL ft hS ep i r i t .
i n ag k n oc wo nn c e n ot fa r ab to0iu.ot0nm4 p g
8 e mr L . A c c e pc t r ia t ne 9cr i.ea0: - m
1 1L . 0
A s sp ar ye p a r a tainfo dinn epW l oye windgoefht e
r w e r S P E C TI EF S I CT S
t h a2 nT0 a b l Te rt as n. as n
fa ec rc u r wa et ei lgpyh
o re td o if o n e A L C OD H
E TO EL R M IM NeA tTI IhI
(o6O 1d
N 1,7) 9
: . 0 % - 8
t h pe o w de eq r u i, vt aoal be n o4tu0m
t og fp e n t o x i tf oy l l i on fCe ,2 H s O H
a 5 0 -v m o lL u mf el at sPrkii. pc
0e.tm4 oLfm e t h ian ntt ooh l e
f l a sak n,s dw i fr olar tl e a1 s mt i n uAt dea d
.b o3u0m
t oL f A D D I TR IE OQ NUAILR E M E N T S
E x t r asco tl iu ntagi no sndo, n i fc oa6r t0 me i n uw ti eto s c c a - ' P A C K A
h A GNSIDTN OGR AP G r eE s:ientr iv gechot n t a i n e r
s i o sn wa li r ol fit nhfgel a sAk d . a dna d d i t 1i 5omn oL a fEl x - p r o t ef cr tloei mgd h t .
t r a c st oi ln ug ta i lo lnt o
,ocw o ot lor o to em m p e rd ai t l uu tr ee ,
S a l w i tE xh t r a scotliu ntt goiv oon l ua mn m edi, xC .e n t r oirf u g e
i d p a st sh r o a su ug iht f ai bl tlReere. s ea rp vo er to ift oh nfi is r s t
a d i l u ft oiprorn e p a or fat th Tieeos nto l u it n it oh C ne h r o m a t o -
fe]
g r a pp hu ri ticets yPt .i p 3e .tm 0 oL ft h cel e sa or l u it ni tao on
io) 5 0 -v m o lL u mf el at sdrkii,l cuw ti et E xh t r a scotliu ntt goiv o ln - P e r f l u b r o n
re}
& u m e
a ,
n mdi x .
5 C h r o m a t soy gs r(t a se epCmehh ir co m a t( o6 g2 r1 a) p) h yT h e
= l i q uc ihd r o m a i t s eo qguri a wp i
ppathe2 h d7 3 d- e nt m e c t o r
oe a na d4 . 6 x-2 m5 m-c co ml tu h mac nto n t pa ai cn ksL i1 .n g
a l
= T hf el ro aw it s ea b o0 u. m t7 pL e mr i n uCt he r. o m a t o Cg e rB ar 4 Fp i9h 78 . 9 6
t h Se y s stu ei m t a sb oi l iu tayi n o rnde, c to hr ped e ra ek s p o n Os ce ts a1 n- eb , r o m o - 1 , 1 , 2 , 2 , 3 , 3 , 4 , 4 , 5
a sd i r e fc otPrer do c e td hurere es : o l u R ,tbi eo t n ,wc ae fe f ne i n 8e - h e p t a d e c a f l u o r o - .
a np de n t o x ii sfn yoll tel stis nh ea1 0n . C0 h . r o m a tt hoe g 1r -aB pr ho m o h e p t a d e c a f l u o r o o c t a
S t a n pd ra e rp da r aa tn ridoe nc ,to hr ped e a r ek s p of onr s e s P e r f l u obr ro oo cm t[ i y4dl2e 3 - 5 5 - 2 ] .
p e n t o x ia fsdy il rl eifcnotePr er do c e td hurere el a: st it va en d a r d
d e v i af t o rri eop nl ii cn aj te eci ts ni o mnt so tr he a2 n. 0 % . » P e r f l cu ob nr tonanoil tne sst s h a 9 n 8 .a 0n ndo t
P r o c e d u r e i n Sj e cp q tu
ava r ola lt u(e ma
l eby 1s
o 0 u
w Lt ) m o t r he a1 n0 0p .e 0r co efC ng tB r F 1 7 .
o ft h Se t a n pdrae rp da ra an ttdhi Aeo sn sp ar ye p a ir na tt oi o n
t h ce h r o m a tr oe g c tor hracedph hr ,o m a ta on mgd er aa sm -sP ,a c k a a gnsidtno gr a g e i ntPi rg he l tis,g eh r t -vr ee s i
u r te h re e s p o f ontrshemesa jp oe ra C k s a .l c ut lh qae t u ea n - c o n t a i n e r s .
t i t iy n, m go ,fp e n t o x i( fCy il 3l Hi i ni es
n t Nh «
pe o
O rs t)o if o n U SR Pe f e r s et na cn e d( a1 r1 d) s
T a b lt eat ksbe ytn h fe o r m u l a : U SP Pe r f l Ru Sb r o n
8 3 3 C 1( 5r u) / I d e n t i f i c a t i o n
A :R e c to hrI edRa b s o r ps tpi eo cn tu sri unagmg ,ac se l l .
i n w h i Cics t h h ce o n c e n ti nr ma tg p ie mor Ln o,
,fU S P T h sep e c st oor b ut ma ie nx ehdi m b iat xs oi n mal a
tyt h se a m e
P e n t o x iR fSiyntl h l Sie t
n ea n pdr ae rp da raa ntr d iy ao nnr s;
d w a v e l ae stn hg oatftahs si m ip lra er p a or faU tSi P Peo rn -
a r te h pe e r a ek s p oo nb st ea s fi rn ote hmdAe s sp ar ye p a r a tf il ou nb R rS . o n
a n tdh Se t a np dr ae rp d a rr ae ts ip oe nc ,t i v e l y . B :T hr ee t e nt ti iomofe tn h me a jp oe ria n tk h ce h r o m a
g r oa ftm h tee ss tp e c io mb et na a,isdn ie rde icntt heAeds -
s a yc, o r r e ts ot
p ho a
on f
td
U sS P Pe r f l RuS bs, r
i mo in l a r l y
c h r o m a t o g r a p h e d .
S p e c gi rf ai vc( i 8 4t 1y b
) :e t w1 e. e 9an2 n21 d. 9 2 5 .
P e p p eS r p im rii n t t C h r o m a tpougr ri at py h i c
D E F I N I T I O N C h r o m a tsoy gs r(t a se epCmehh ir co m a t( o6 g2 r1 a) p) h
P e p p eS r
p im rciio tnn t
t ai nien as c
1
, h0m0L N, L9 T. m
0 a
L n d g a cs h r o m a i t
s eoq gu ri a wp ippt
ahe
shpdl ii nt j e cp to iro tn
N M1 1T .m 0oLfp e p p eo irl m . i n t w i at shp l ri at t ir o a, no gf1 e: 4 t o51 : 1 a0 f0 l, a m e - i o n i
U S4 P1 O f f i M
c ioa n
l o g/ Pr ea r pf hl 3
us t2 r 3e 5
n

d e t e ca t noa dr0 ,. 2 5x 6 - 0 m c -
mo m l cu om anwt ie t ad h B a c t eE rn ida ol tT oe x( si 8t 5n )s c o In t na oi mtn os r e
1 - fu i mlo mfp h aG s2 eH. y d ri sou gs eae n sdt h cea r rg ia esr . t h a 0 .n U5 S EPn d o tU o n i x
p ti
em r
n oL fP e r f l Pu rt or te en i n -
T h ce h r o m a i t s po rg or g a r pt ha
om ma i m nettdhaceion l u mT ny p A Me i c r o sI pn h j ee crSteua ssb lp ee n s i o n .
t e m p e ar t3 a 5tf uo 7r
r mei n u tt he t e
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p r o d Su tc ae ns d
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or no stc eo innc e n t r a t i o n s
o f0 . 40 ,. 81 ,. 01 ,. 2a, n 1 d. m
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A s sp ar ye p a r a t i o n e a Ec qo h un it la
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je earctt e- C h r o m a tpougr ri at py h i c
a b lS eu s p et nors o i too e
nm m p e raa ntmdui e rxe a f ,c ohar t
l e a 5s mt i n ut t oe en ss au hr o e m o g se uns ep e on Vu sesin ot n . S o l u At i oPn r ae f pi la trea e rneddde g a ms is xe tod uf r e
t h ceo n t a ai ntedrra ,n 5s f0 e0 ra- l ui Lq iu no tst oe s p a r a t e5 . m 0 oL fm o r p hw oi lt9 ih9nm5eoL fw a t ea rna,dd j u s t
t u b D e is l. t
u th cee o n t oefen at c t
s h u wb ie t w ha tt eo2 rm L , w i tp hh o s pa hc oit rdoa ip cH o f7 . 0 .
a n ad d 1d 0u l o fD i l ua tn etd i rf eoa ag me n t . S o l u B t i oPn r a e
f ip la traeenrdd e ed g a ms is xe tod uf r e
P r o c e de ua roc efh t h tTe uo bc eo sn t a ti hnBeil na g n k m e t h a cn eot lo , n ia tnrt die lte r, a h y (d1 r: o1 f :M 1ua)r .ka en
p r e p a r Sa tt ai n o pndr,ae rp d
a r aa tn A i dos nspsar,ye p a r a t i ao d n ,j u s i tf nm ee cn et s(s s eaS ery ys St u ei mt a bu inl d iC tehy r
r o -
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S m, 6 2a 1p ) h) y.
3 0m i n u at ce cs u, rta it mefelodym r, a x i c omlduo ermv e l o p -M o b p i lh ea s ve a r Ui mas ibex l et ou fSr oe l su A
t ai onSndo -
m e nTt h.B el ap nr ke p a r Sa tt ai n o pndr,ae rp d a r aa tnAi dso -n s l, u t iB ao snd i r e fc otCrehd r o m a ts oy gs r(t a se epSm eh
y i s tc e m
s ap yr e p a ar ratetr ie oaintd ee nd t i Uc sa litlnhy g B.e l ap nrke p aS -u i t a bu inl diCtehyrr o m a (t 6o 2g1 r) a ) .p h y
r a t is oentt,h ae b s o re bq autnaozcleer Do e. t e r t h m ei n e S t a n sdo al ru t1 d i oDn i s asnao clc vu er wa et ei lg yh e d
a b s o ro bfeaanoc cfh t eh Se t a n pdr ae rp da raa nttdih oe n s q u a n ot fU i tSPyPe r g oM le is dy eRl Sia n t m e t h aa nndod i l- ,
A s sp ar ye p a ir na1 t-iccoe mnl wl i s t a sh u i t sa p b le ec t r o pl hu o tq eu- a n t i t aa tn sidtv e lpiyw f n,ie sc ee s ws ia tm r hye ,t h a n o l
t o m ea tta w e ar v e lo ef5n 4gn0tm h U.s il ni g n er ae rg r e s s ti ooo nb ,t aa si on l u ht a i ov ani kn n g oc w o nn c e n ot fa r ab to iu ot n
a n a lt yh dze ae to ab t a fio ner ea doc fh t h Se t a n pdrae rp da r 3a 0 -u gp e mr L .
t i o nC sa. l c ut lh caeot re r e lc oa et fi fo insc li oe pan etny,, d- i n t e r -S t a n sdo al ru 2td i oDn i 1l 0u.mt0oL e fS t a n s do al r u td i o n
c e vp at l ute hsce:o r r e l c oa et fi fo iins cn ioletenstts h a 0 .n 9 9 57 .t o5 0m wL i tm he t h a n o l .
C a l c ut lh qaeut ae n ti ni mt gyo ,,fp r o ti enei an cm hoL ft h e T e ss to l u t i o na bT or6ua0m tn s
ogffP ee rr g oM le is dy el -
I n j e c St ua s b lp ee bn yts hifeoo nr m u l a : a t ea ,c c u r wa et ie g l tyhoae 1d 0, -v mo l L u mf el at sdrki i,s c-
1 0 [ ( yA- ui n t e r c e p t ) / s l o p es] o li vn ae nd di l uw ti etm he t h taovn oo ll ua mn m edi, x .
C h r o m a t soy gs rt( ae s pemS heu ir c a n (y 6 ,2 1 ) )
i n w h i1 ci0sht h dei l u ft ai co tnao n r A;diys t h ae b s o r b aTn hc l eie q uc ihd r o m a i ts eo qg uria w piptpahhe2 d8 0 d- e n- m
o ft h Ae s sp ar y e p a rt ahtceia ol nc :u lq ua at neotdfip tr yo t e i tn e c a t onard4 . 6 x-2 m5 m-c co ml tu h mac nto n t ba ai sn es -
i n t h Ien j e c St ua s b lp eei snbs ei to wn8e a e n 1nd 2m p ge r d e a c t ip va ac tkL ei1 dT .n hgf el ro aw it s ea b o1 um tpL e r
m L . m i n uT thece.o l tu em mn p e irs ma at iu nr tea at4 i0 nTe.hd e
c h r o m ai t s po rg or g a rpa h asf om lml e o wd s .

T i m e S o l uA t i oS no l u Bt i o n
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t h a o ftp e r g o fl ri t d
o ehmT,ee ss to l u i ts ni oo m nt o tr he a n
» P e r g oM le is dy ecl oa nt tena oil tne sst s h a n t h pe e r g op le ir adeke s p oo bn ts aefi rn oeS m dt a n s do al r u -d
9 7 . 5 r ca ennndto mt o tr he a1 n0 2 p. e 0r co efn tt ie
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td i o n
C i g H - 2 C6 HN 42 OcS 3a lS c, u ol n
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t a sn ta y
c o n t a i n e r s . D i l u e n t 5 mD g io sfms eo tl hv iei on 5n 0im0n oLef0 . 0 1
U SR Pe f e r s e t n a cn ed( a1 r 1 d) s N h y d r o ca hc liAdod.rd 5i 0 c m0 oLfm e t h aa nn modil x, .
U SP Pe r g oM le is dy eRl Sa t e M o b pi l
h ea s e a Ps ro le upotafi0 ro. en0 M 0s9 o d i u m
U SP Pe r g oS lu il df eoR xS i d e 1 - o c t a n ecsounltfao1in.nm a
0itoLnefgg l a cai ca el at ci ic d
( 8 8 ) - 8 - [ ( M e t h y l s u l f i n y l ) m e tp heLyr. P! r ] -e 6pa -fa ipr lretoeaprnyedd ld e- gD a- ms
e irs xge t
ood luft ihr n
ise seo l. u -
I d e n t i f /in cf ar Ata bri seo odn r,(p 1t 9i 7o Kn ) . t i o mn e
, t h aa nnaodcle ,t o n( i2 t: r1 i:Ml1 a e) a.k de j u s itf m e n t s
o i
( s eS ey s St u ei mt a bu inl diCtehyrr o m a t o g r a p h
S p e c ri of t i c a ( t7 i
8 1o 5bnSe) t: w- e1 e7a nn d 2 a3 t2 0 .
T e s to l u t1i 0om n p g: e mr Li ,nd i m e t h y l f o r m a Rm eis do el s. u ot li uo tn i o n a bD oi4 usm s togofUl v S PePe r g o l i d e
L o s o snd r y (i 7n 3g 1 i)t i n vD ar cyaut1u 0 mf5 o r S u l f oR xSai nd8d em og fU SP Pe r g oM le is dy eRl Sia n t e
1 h o ui trl :o s ne os mt o tr he a0 n. o5 fi %t sw e i g h t . 5 0m oL fD i l u e n t .
R e s io dn i ug en i (t2 i8 o1n n)o m:t o tr he a0 n. 1 % . S t a n pd ra er pda r a t i ao nan c cD ui rswasetoe illgvyhe e d
q u a n ot fUi tS PyPe r g oM le is dy eRl Sia n Dt ie l u a e nndt d,i l u t e
q u a n t i ta a tn sidt v ee lpiywf n,ie sc ee s ws ia t Dr ihy l, ut eoon bt -
t a ia ns o l u ht a
i ov nai kn n
g oc wo nn c e n ot far ab to iu ot n
0 . 1m 3 p
g e mr L .
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c ioa nl o g r a p h s U S4 P1

A s sp ar ye p a r a t i ao bn o6 u T. r
mt
5 aognfPse fr eg ro l i d et i v e tl oya ,c a l i b bro at tt A el deds. d u f f i Vc ei he ifn cot lOr er a l
M e s y la ac tc eu ,r wa et ie g l tyh oae 5d 0, -v m o lL u mf el at srk i, c S o l u tt oib or nitnhgpe r e p a tr oa ft i in o va lno l ouf m e
d i s s io nla vnde di l wu itt ehD i l ut eovn ot l ua mn m edi, x . 2 0 m L a, n md iw xe l l .
C h r o m a tsoy gs rt( ae spCem heh ie ca m any a ( r6 e 2 r 1 ) a )
p
T hl ei q uc i hd r o m a i t s eoqgu ri wapi ptpahhe2 d8 0 d-e n- m I D E N T I F I C A T I O N
t e c a t onard4 . 6 x-2 m5 m-c co ml tu h mac nto n t ba ai sn es - e A . T hr ee t e nt ti iomofe tn h pe e r g op le ioa dft keh Se a m -
d e a c t ip va ac tkL ei7 dT .n h
gf el ro aw it s ea b o1 um tpL e r p l seo l u ct io or nr e ts ot p ho a on fttd hsSe t a ns do al ur tdi o n
m i n uT thece.o l tu em mn p e irs ma at iu nr tea at4 i0 n e. d a so b t a iintn heAeds s a y .
C h r o m a tt ho Reeg sro alsp uo th l iu otani no rnde, c to hr pe
de a kA S S A Y
r e s p oa snd si er s e fc otrPe rd o c e td hurere es :o l uR ,tb ieo -n , e P R O C E D U R E
t w e pe e nr g os lu ild fe oa xnipdde er g ios ln io ldte est s h a n S o l u At :i0 o. n5M s o d oi cu tma n e s su oll fu oa t nin aodtn e
1 2 . 0C .h r o m a tt hoSegt r a na dpp arhre dp a r aa nt rideo cn o, r d e (r2 : 9a8 d) j, u ws it gte lhda cai ca el at c i c
it d
oa p H
t h pe e r a ek s p oa snd si er s e fc otPr er do c e td hut era ie l:f iancg - of 2 .
t oi rs n o mt o tr he a 1 .n 5a; n t dh r ee l a s t it va e n dd ea vr ida t i oMn o b pi hl aesA e c e: t o n ai ntSrdoil lu eAt (i 5o 0n : 5 0 )
f o rr e p l ii cn aj te eci ts ni o mnt so tr he a2 n. 0 % . D i l u eM net t: h aa nn0 do . 0lN1h y d r o ca hc li od r i c
P r o c e d u r e i n Sj e cq p tuava r ola lt u(e mal e
by 1s
o 0L
u Lt ) ( 5 0 : 5 0 )
o ft h Se t a n pdr ae rp da ra anttdhi Aeo sn sp ar ye p a ir na tt oi o n S t a n sd taosrcodkl u t 1i .om 0n g : /o fm U S LP Pe r g o l i
t h ce h r o m a tr o e cg tor h racedph hr ,o m a ta on mgd er aa sm -s , M e s y Rl Sia n t m ee t h pa rn eo pli an lr oew d- a c t i n i c
u r te h re e s p o f on trshepese r g o p le ia kd C sae. l c ut lh ae t e g l a s s w a r e
q u a n ti nimt gyo ,,fC i s H - a Cs HN 4 2 iSOnt3h p Se o r to ifP oe nr - S t a n sdo al ru t d iP or ne spf :ia vsr eoe l u to ifko n n so w n
g o l M i ed es y tl aa ktbeeytn h fe o r m u l a : c o n c e n to rfa abt o2i u0o 1t ,n0 s5, ,2 , a n1 dg / o mf L
p e r g om le is dy e bl yqa ut ae n t i td ai tl iu tvt e h
i Snle tgy a n -
5 0 C / (r sr) u d a sr td os co kl u wt i otM nho b pi hl ae s U es l.eo w - a c t i n
g l a s s w a r e .
i n w h i Cics t h h ce o n c e n ti nr ma tp g ie mor Ln o,
,fU SP Pe r - S a m sp o ll e u t Ti ro an n:5 s f0we0Lor fO r aS lu s p e n s i o
g o l M i de es y Rl Sia n t he Se t a n pdr ae rp da r aa ntr d iyao nnr d
s; V e t e rtioan 5a - r yvm oLl u mf el at sa rk i
n,dcdi l w u ti et h
a r te h pe e r a ek s p oo nb st ea s fi rn t oe hmdAe s sp ar ye p a r a t i D o in l ut eovn ot l uF mu er t .d hi el ua r tna el i qo uftohtseo l u -
a n t dh Se t a n pdr ae rp da rr ae ts ip oe nc ,t i v e l y . t i ow ni tM ho b pi hl a et soo eb t aa si on l u wt ii t ao nn
h o m -
i n ac l o n c e n ot f1 r a0gt / i o mfnp eL r g o Ul sile d oe w. -
a c t ig nli ac s s w a r e .
C h r o m a t soygs rt ae pm h i c
( S eC e h r o m a t (o 6g r2 aS1 py)hs, yStu ei tm a b i l i t y . )
P e r g oC loi md p e oO u r an ld e d M o d Le C:
S u s p e V n e s t i e o r n i , n a r y D e t e c Ut o V
2 r2 :n 3 m
C o l u 4m .n 6: x-1 m5 m- 5c -m 1;pu am c kL i 1 n g
D E F I N I T I O N C o l tu emm np e r 4a 0t u r e :
a P e r g oCl oi dm ep oO u r aSn luds ep ed Vn es ti eo rnci,on na tr ay i n sF l ro awt 1e :m L / m i n
@ N L 9T 0 .a 0n % Nd M1 T 1 0 o.ft0h l%e a b e al m e do ouf n t I n j e cv toi lo un 2m 5we L:
% p e r g o( lC ij do e H 2 s N 2 S ) . S y s stu ei m t a b i l i t y
c S
D P r e pP ae rr eg oCl oi d m ep oO u r aSn luds ep ed Vn es ti eo rni ,n a r Sy ,a m p lS e t s a n :s do al ru tda i noSd n as m sp o ll eu t i o n
i} 1 m g / amsfLo ,l l( os weP seh a r m a cC e ou mt ip co a ul n d iS n u igt a b ri le i qt yu i r e m e n t s
= N o n s t Pe rr ei pl ae r{ a7 t9 i5 o) n) s . T a i l fia nc gt oNr :M2 .T0S ,t a n s do al ru (td 2 i uo0 gn /
S m L )
= P e r g o( al Psi ed re g oM le is dy el a t e ,
R e l a st t i va en dde av ri da tNi M o 2n T
.: f0o % rr e p l i c a
[omy i n j e c tS ito an ns s,do al ru (td 2 i to0un g / m L )
a l U S P ) 2 0m (g 2 6 .m 1 q1 ) C o r r e cl oa et fif oi ncN iLe0 Tn. t9 :l9 i5 n,er ae rg r e s s i
= V e h if co Olr re aS lu s p e nNs F i o n , 1 0m L o ft h Se t a n sdo al ru td i o n s
V e h if co Olr re a
S ol l u t Ni oFn , 1 0m L R e s o l u Nt Li2 oT . 0nS,:a m spo ll eu t i o n
A n a l y s i s
C o n na e ne cm tp ct ayl ,i b r3 a 5t - eLdmu, leL or ci kn j e cs ty i- o n S a m p lS e t s
a n :s do al r u tda i noSdn as m spo ll e u t i o n
r i ntgote h pe o o r fta f l u i d - d ic so pn en ne sRcie tn omg ro. v e G e n e arr ae tg r e e cs us rio vofpene h a ek i vg eh rtps eu rs -
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S o l u Bt: iA o c en t o n i t r i l e
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C i g H 3- C
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2 Or s
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7 a B ] c] -o, m pw oi u
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( 2 5 , 3 a S , 7 a 5 ) - 1 - [ ( S ) - N - [ ( S ) - 1 - C a r b o x y b u t y l j a l a n y l 2] 0h e x a h y
2 - i n d o l i n ae cci a 1d -r, eb toeh x
sy ty!ec
l rio
, c m pw oi u
t hn d
t e r t - b u (t1 y: l
[1 a1) m0 i7 n1 e3 3 - 3 6 - 8 ] . 3 2
D E F I N I T I O N
P e r i n dE orp or ui cl
mo in nt e
N
a iL 9nT s8 .a 0n %
N
d M T 8 0
1 0 2 o.fp0e %r i n de or p br ui(m
l Cii ns eH+ 3C 24 NH 2i Oi sN ) , 0
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n h y d
b ars oi su .s
I D E N T I F I C A T I O N 0
e A .I N F R AA BR SE O
D R(P1 T9 I7 OK N)
e B . T hr ee t e nt ti iomofetn h me a jp oeroa ftk h Se a m p l e D i l u eS no tl u: Bt ai onSndo l u A t (i 2o 0n : 8 0 )
s o l u ct io or nr e ts ot p ho oan ftS
d y
s s stu ei mt a sb io ll iu ttyi o n S y s stu ei m
t a bs it losi c o
t kyl u At :i0 o. n0m 3 g /o fm L
1 , a so b t a i in tn het ed
e sf to Lr i moifPt e r i n dR oe pl ra it le d
U SI Pm i d aR zSi onD li el u e n t
C o m pI . o u n d
S y s stu ei mt a bs it losi cto kyl u Bt: i0 o. n0m 3g /e m a cL h
o fU SP Pe r i n dE orp or uiRlmS i U
, nSPePe r i n dR oe pl ra it le
A S S A Y C o m pBo R SuU, nSPd Pe r i n dR oe pl raC it lo
e dm p Co u
' P R O C E D U R E R S U
, SP Pe r i n dR oe pl raC
it lo
e dm pDoR Sua
, nn Ud
dS P
v oe2 fs o d 1i -u hme p t a n ie n s u l fP oe nr ai n
B u f f De ir s: s 0o .l g
9 t dR
e oe pl raC it lo
e dm pF R o iuS nDni dl u e n t
1 oL fw a ta enrad d1 dm oL ft r i e t h yA ld ajmwuiisn tte h. S y s stu ei m t a bs io ll iu tt Tyi ro an n:5s mf ee Lra oc fhS y s -
a s o l u ot fip oe nr c h al co iar din wcd a t( e1 :rt1 oa) p oH f t es mu i t a bs it losicot kly u A
t ai on Sndy s stu ei m t a bs it lo ict k
s o l u Bt ti oao n5 0 -v m o lL u mf el t a asr knid d
ci l wu ti et h
D i l ut eovn ot l uP ma set s.h r oasu ug i ht fai bl lto eef0r . 4 5 -
U S4 P1 O f f i cM ioa n
l o g/ r
P ea rpi hn s
d3 o2p 4r 1i l

u p
m o sr iez de ,i s ct ahfreidr 3s tm oL f i l t r aa tn eud,s e T a b2 l
( C
e o n t i n u e d )
t h cel ef ai rl t r a t e . R e l a t iR ve el a t iA vc ec e p t a n c e
S t a n sd taosrcodkl u t 0i .o 0nm 3 :g / o fmU SLP Pe r - R e t e n R
t e
i s
o n
p o n
C rsi e
t e r i a ,
i n d o Ep rr iol u Rm Siin Dn i el up ern et p aasfro el dl o w s . N a m e T i m e F a c t o r
N M( T % )
D i s s aoslu vi et qa ub al en ot fU
i tSPyPe r i n dE orp or ui lm i n e
R Si n 8 0o f% t ht eo t va lo l oufDmi e l u se no tn ,i fc oar t e P e r i n dr oe pl ra it el d
a b o5 um ti na ,nd di l u w ti etD i h l ut eovn ot l u m e . c o m p C os u n d 0 . 7 4 0 . 9 6 0 . 1
S t a n sdo al ru t d 0i .o 0
n m:0 g3 / o fm
U S LP Pe r i n d o p r Pi el r i n rd eo lp ar ti el d
E r b u Rm Siin Dn i el uf ern t ot hmSe t a n s dt aosrco kdl u t i o n c. o m p D o¢ u n d 0 . 8 5 0 . 9 8 0 . 1
P a st sh r oa su ug i ht fai bl lto eef0r . 4 5 p- o1 sr4 iemzae n d P e r i n de orp br ui lm i n1 e. 0 = =
d i s ct ahfr eidr 3s tm oL f i l t r a t e . l s o p r o p y l 0 . 4 0
S a m sp o ll eu t 3i mo ng : /o fm P eLr i n dE orp br uii l mn i n ep e r i n d o p r i l e1 . 2 2 1 . 0 0 _ ( pir fe s e n t )
D i l up ern et p aasfro el dl Do iw s .s ao sluvi et qa ub al en t i tP e r i n dr oe pl ra it el d
o fP e r i n dE orp br uii lmn 8i n 0o ef%
t ht eo t va lo l ou m e c o m pF fo u n d 1 . 3 8 0 . 8 5 0 . 2
D i l u se n
o tn ,i fc oa5r tm ei na ,nd di l u
w ti et
D i
h l ut eo n t P e r i n d o p r i l
v o l uP ma set s.h r a
os uu gi htf a
i lbto elf0
r e . 4 5p -o u
r e
m i m i d a z o l i d i n o n e 0 . 1 5
s i zae nd di s ct ahfrei dr 3s tm oL f i l t r a t e . a n a _
l o
_ g s 1 . 6 5 1 . 0 0 ( i fp r e s e n t )
C h r o m a t soygs rt ae pm h i c
( S eC eh r o m a t ( 6o 2g1Sr)y a,s pStuhei ym
t a b i l i t y . ) A ni yn d i v i d u a l
u n s p e c i f i e d = =
M o d Le C:
D e t e c Ut oV
2 r1 :n0 m i m p u r i t y 0 . 1 0
T o t ia ml p u r i t i e s d s a e s 1
C o l u 4m .n 0: x-2 m5 m - 4c -m 1;p1 am c kL i7 n g
C o l tu emm np e r 6a 0t u r e : a l m i di saq zu oa ln et i
u st iatnthge
t eed sf to Lr i moi fPt e r i n R
d oe pl ra it le d
F l ro awt 1e :m L / m i n C o m pA oa u nI n
dm d
i d aa zniosdil n
e c l iun td heteda bf lo eir d e n t i f i c a t i o n
p u r p oon sl ey s .
I n j e cv toi lo un 2m 0 ue L: » ( 2 5 , 3 a S , 7 a 5 ) - 1 - { ( S ) - 2 - [ ( S ) - 1 - HC -a r b o x y b u
S y s stu ei m t a b i l i t y i n d o l e - 2 -a cc ai dr .b o x y l i c
S a m p Sl ye s:stu ei m t a bs io ll iu tty i o n ‹ ( S ) - 52 -, {5 (a3 5 , 9 a S , 1 0 a S ) - 3 - M e t h y l - 1 , 4 - d i o x o
S u i t a b ri le i qt u
y i r e m e n t s a j i n d o l - 2 ( 1 Ha )
c i- dy .l } p e n t a n o i c
T a i l fia nc gt oNr :M1 .Tf5 o tr h pe e r i n dp oe pa rki l 4 ( S ) - 2 - { ( 3 5 , 5 a S , 9 a 5 , 1 0 a R ) - 3 - M e t h y l - 1 , 4 -
R e l a st t i va en dd eav ri da tNi M o 5n T
.: f0o %
tr h pee r - a J i n d o l - 2 ( 1 Ha )
c i- dy .l } p e n t a n o i c
i n d o p re ial k ' ( 2 5 S , 3 a S , 7 a S ) - 1 - { ( 5 ) - 2 - [ ( 5 ) - 1 - i s o p r o p o x
n o y l } o c t a h y d r o - 1 aH c-i id n. d o l e - 2 - c a r b o x y l i c
A n a l y s i s
f ( S ) - 2E -t {h y( l3 5 , 5 a S , 9 a S , 1 0 a S ) - 3 - m e t h y l - 1 , 4 -
S a m p lS e t s a n:s do al r
u atd in Sodna m sp o ll eu t i o n a j i n d Ho ) l - 2y (l 1} p e n t a n o a t e .
C a l c ut lh pae et er c eo nfet aa c ig m
hep u irn ti htpeyo r - 8 ( 2 S , 3 a S , 7 a S ) - 1 - ( ( 2 5 ) - 2 - ( ( 5 R S ) - 3 - c y c l o h e
t i oo nfP e r i n dE or p br uitlma ik n e ne : p r o p y l i m i d a z o l i d i n - 1 - y l ) p r o p aa cn iod y. l ) o c t a h
* T o t ia ml p u r ii nt ci lae lussl dp ee c iaf niu d
en ds p e i c mi pf ui re aid n
t iidme is d -
R e s =
u (lr tu / xr (s C) s /xC( u1 )/ xF 1) 0 0 a z ofl re to hmt ee sf to Lr i moi fPt e r i n R d oe pl raCit loe dm pA oa u nI n
dm di d a z -
o l eP. e r i n dr oe lp ar cti eo
l dm pAo i s nu onitnd c l u d e d . ( 3
f u =p e r a ek s p oofen as c ie m
h p u fr ri to y hm e a )
S a m spo ll eu t i o n ' L I MOIFPT E R I N R D OE PL RACIT OLE MD P AOA UNINDMDI D A Za ~O] L E
r s =p e r a ek s p oo fpn es rei n de or p br ui flm ri on me S o l u At :iP orno ca e sd ei d
r e icn Ot r
e dg aI n
m pi ucr i t i e E
s .
t h Se t a n s do al r
u td i o n S o l u Bt: iA o
c en t o n i t r i l e ey
C s = c o n c e n ot fr U aSPtPei ro inn dE orp or ui Rlm Si n e M o b pi hl ae sS ee T:e a b3 l. e 3
i}
C u
i nt h Se t a n sdo al r
u (
td imo gn / m L )
= c o n c e n ot fPr ea rt ii nodEnorp br uii l mntih ne e T a b3 l e
=a
i y
n a Ro E
]
S a m spo ll eu ( t imo gn / m L )
F =r e l a r
t ie vse p foa nc s(t eso erT ea b2 l) e S o l u A
t i o n S o l u Bt i o
a )
A c c e pc t
r ia t ne Src ieeTae:a b2 l. De i s r ep ge aalrke dss s
t h a0 n. 0 5 % . 8
8
T a b2 l e
R e l a t iR ve el a t iA vc ec e p t a n c e
8 3
| R e t e n t
R i
e o
s n
p o n
C s
r ie t e r i a ,
N a m e T i m e F a c t o r N M( T% ) 3
I m i d a z o l e ? 0 . 0 8 = a s
D i l u eS no tl u: Bt ai onSndo l u At (i 1o 7
n : 8 3 )
P e r i n dr oe pl ra it el d S t a n sd taosrcodkl u t 0i .o 2
n
m5:g /
o fm
U S
LP Pe r -
c o m pB ofi u n d 0 . 4 2 1 . 2 0 0 . 3 i n d o Rp er il la Ct o
e dm p AoR S
ua n0 dd
. 1m g /
o fm L
@ I m i d ias zq ou l
a en t iu ts aittnhegt eed sf to Lr i moi fPt e r i n R
d oe pl ra it le d U SI Pm i d aR zSi onD li el u Se no tn .ii c
f nae tc ee s s a r y .
C o m pA oa u nI n
dm d
i d aa zniosdil ne c l iuntd heteda bf lo eir d e n t i f i c a t i o nS t a n sdo al r u td 1i 0
om n g: /o fm U S LP Pe r i n d o p r i l
p u r p oon sl ey .s
E r b u R mS i
0, .n 0em 2a 5i on fU t .SP Pe r i n dR oe pl ra it le d
» ( 2 5 , 3 a S , 7 a 5 ) - 1 - { ( S ) - 2 - [ ( 5 ) - 1 - HC -a r b o x y b u t y l a m i n o ] p r o p a n o y l } o c t a h y d r o - 1
i n d o l e - 2 -a cc ai rd .b o x y l i c C o m p Ao
R Sua
, nn 0d
d, 0 1m g /o fm
U S
LI Pm i d aR zS o l e
¢ ( S ) - 2 - { ( 31 50 ,a 55 aS 5) ,- 93 a- SM ,e t h y l - 1 , 4, -2 d- i o x o d e c ia nhD yi
gd er on
p ptry e r apzaa isfnro
o e[l 1dl To rw as n. as wf e ri g h e d
a j i n d oH l ) - y2 l( }1 p7 ea cn it da .n o i c a m oo fu Un St P Pe r i n dE orp or ui Rlm Sai nna de s u i t a b l e
4 ( 5 ) - 2 - { ( 3 5 , 5 a S , 9 a S , 1 0 a R ) - 3 - M e t h y l - 1 ,a 4 m - doio u o fS
x not dtae nc sadth ayosrdcordklo up tyt oria aso zuniin to va[ bo1 ,ll2eu- m e t -
a j i n d o l - 2 ( 1 Ha ) c i- dy .l } p e n t a n o i c r i fc l a sak n, d di s s wo il t vs eh
o n i ci an Dt ii lo uen eq nu ti v a -
1 o x o p e n t a n -l2 e- n
' ( 2 5 , 3 a S , 7 a S ) - 1i -s {o( pS r) o- 2p -o [x (yS -) 1- - ty tol6a m0oi fn%t oh]f epi nr vao lpo al- uD mi le wu. ti etD ih l ut eo n t
n o y l } o c t a h y d r o - 1 aH c-i id n. d o l e - 2 - c a r b o x y l i v c o l up ma e st s,h r oa su ug iht fai bl tlo eef0 r . 4 5p -o ur m e
1 ( S ) - 2E -t (
h y( l3 5 ,105aaS5),-9 3a -5 m, e t h y l - 1 , 4 - d i o x o d es ci az ha e y,ndd drio sp c yt rahafrez
idri 3sn tm
o [oL 1 f, i2l -t r a t e .
a j i n d H
o )
l - 2
y (l 1} p e n t a n o a t e .
s a m ps lo el u t 1i 0
om n g: /o fm
P eLr i n dE or po rb iu
9 ( 2 5 , 3 a S , 7 a S ) - 1 - ( ( 2 S ) - 2 - ( ( S R S ) - 3 - c y c l o h e x y l - 2 - ( c y c l o h e x y l i m i n o ) - 4 - o x o - 5 -
i lnm i n e
p r o p y l i m i dy ap zro ol pi ad i n no -yH1li sn od co t l a e - i il cu e n t
h 2ya -ec cir dao.r-b1o x y l
» T o t ia ml p u r ii nt ci lae luslsdp ee c iaf niu d
en ds p e i c mi pf ui re aid n
t iidme is d -
a z o fl re o
t hmt ee sf to Lr i moi fPt e r i n R d oe pl raCit loe dm p Aoa unI n dm di d a z -
o l eP , e r i n dr oe lp ar cti o
el dm pA i
o nu
sonitnd c l u d e d .
 3 2 4
P e2 r i n d/ Oo fpf ri iM
c lioa n
l o g r a p h s U S4 P1

C h r o m a t soy gs rt ae pm h i c S y s stu ei m t a bs io ll iu t2ty: i3 ogn / o mfU LSP Pe r -

( S eC eh r o m a t ( 6o 2g1Sr)y a,s pStuhei ym
t a b i l i t y . ) i n d o Ep rr ibl u Rm Siin Dn iel uf ern o St y m s stu ei m
t a b i l i
M o d Le C: s o l u 1t i o n
D e t e c t o r s S a m spo ll e u t 3i mo ng : / o fPm e Lr i n dE orp br uii lmn i n
P e r i n dr e
o lp arcti o
el dm pA o : Uu V 2n 1dn0 m D i l up ern et p aasfro el dl Do iw s .s ao sluvi et sa bp lae n t y
I m i d a zU oV2l 2e n
5: m o fP e r i n dE orp or uii lmn 8i n 0o ef%
t ht eo t va lo l o u j m e
C o l u 4m .n 6: x-2 m5 m - 5c -m u;pu am c kL i7 n g D i l u se no tn ,i fc oa5r tm ei na ,nd di l w u ti etD h
i l ut eo n t
T e m p e r a t u r e s v o l uP ma se t s.
h r oa s uu gi htf a i lbto elf0r e. 4 5p -o 1 r e
m
C o l u 6m0 n : s i zae n d di s ct ahfrei dr 3s tm oL f i l t r a t e .
S a m cpo lo el 5e r : C h r o m a t soy gs rt ae pm h i c
F l ro awt 1e :m L / m i n ( S eC eh r o m a t ( 6o 2g1Sr)y a,s pStuhei ytm a b i l i t y . )
I n j e cv toi lo un 5m 0 ue L: M o d Le C:
S y s stu ei m t a b i l i t y D e t e c Ut o V
2 r1 :n0 m
S a m p Sl tea:n sdo al r u td i o n C o l u 4m .n 6: x-2 m5 m - 5 c m- ;pu am c kL i 1 n g
[ N o t re e l aT rt hie ve t ee nt ti imfoeonisr m i d aa zn odl e C o l tu emm np e r 6a 0t u r e :
p e r i n dr eo lp arcti o el dm p Aoa ru1en. a0d n1 d. 4 , F l ro awt 1e :m L / m i n
r e s p e c t i v e l y . ] I n j e cv toi lo un 2m 0 Le L:
S u i t a b i rl ie t qy u i r e m e n t s S y s stu ei m t a b i l i t y
T a i l fia nc gt o Nr : M1 .Tf8 o pr e r i n dr eo lp arcti o el dm - S a m p Sl ye s:stu ei m t a sb io ll iu t2
t yi o n
p o uAn d S u i t a b ri le i qt yu i r e m e n t s
R e l a st t i va en dd eav ri da tNi M o 5n T.: f0 o %
pr e r - T a i l fia nc gt oNr :M2 .T0
i n d o rp er li alct o e dm p Ao u n d R e l a st t i va en dd eav ri da tNi M o 5n T .: 0 %
A n a l y s i s A n a l y s i s
S a m p lS e t a s n :s do al r u atd in S
odna m spo ll eu t i o n S a m p Sl ae m:sp o ll eu t i o n
C a l c ut lh pae e t er c eo nfpte ar gi en dr eo lp arcti oel dm - C a l c ut lh pae e t er c eo nfp te ar gi e n dr eo lp arcti oel dm -
p o uAn o rd i m i d ai nzt oh ple oe r to ifPoe nr i n d o p r i l p o u | inn tdh pe o r to ifPoe nr i n dE orp br uitl ma ik ne ne :
E r b u tma ik ne ne :
R e s u
= (l r tu / xr 1
7 )0 0
R e s u
=(lr tu / xr (s C) s /xC1u 0) 0
t u =p e r a ek s p oo fpn es rei n rd eo lp ar ti el d
t u =p e r a ek s p oo fpn es rei n dr eo lp ar ti el d c o m p| f o ru tonhmdSe a m spo ll eu t i o n
c o m p Aoo ru i mn idd af zr ot o lhmeSe a m p l e t r = t o t oa fall lp e ar ek s p of nr st o ehm S
se a m p l e
s o l u t i o n s o l u t i o n
a

p e ar ek s p oo fpn es re i n dr eo lp ar ti el d A c c e pc t r ia t ne crNieaM0: T. 1 %
I

c o m pA o o u irmn idd af zr oto lhmS ee t a n d a r d

s o l u t i o n S P E C TI EF SI T C S
G s = c o n c e n ot fr U aSPtPei roi nn dR oe pl ra it le d e @ W A TD E TR E R M IM NeA tTIhI a( oO9 d
N
2 1, N
) :M1 T . 0 % ;
C o m p AoR Su o rn
U S dI Pm i d aR zSi not lh ee 3 . 0 0 % f-o a4r m. o5 n0o%h y d r a t e
F = f S t a n s do al r
u (
td im o gn / m L ) ' O P T IR CO AT LA T
S pI eOc N
Ri o
f, it ca (t 7
i o
8 n1 S )
Q a S a m sp o ll e u t 1i 0om n g: / o fPm e Lr i n dE orp or uii lmn i n
3 C u = c o n c e n ot fPr ea rt ii nodEnorp br uii l mntih ne e
=
S a m spo ll e u(t im o gn / m L ) e t h a n o l
D a A c c e pc t r ia t ne c
r i6eat6: o 6 9a t2, 0
A c c e pc tr ia tne cr ie a
r= P e r i n d r o
e l
p arctio e
l dm pA o: N u n M0dT . 2 5 %
{o} A D D I TR IE OQ NU AI LR E M E N T S
I m i d a zNo M l0eT .: 1 %
= ' L I MOIFPT E R I N R D OE PL RACIT OLE MD P| O U N D ' P A C K A A GNSIDTN OGR AP G r eE s:ientr iv gech ot n t aait n e r
a [ N o t e P er re il n
a ctd eoo dm
p rp| iiosltuh en
e pd i om fe r c o n t r ro l o lto eemdm p e r a t u r e .
a p e r i n d(o2p5r ,i 3l a: 5 S , 7 a S ) - 1 - { ( S ) - 2e -U[ S (RRPE) F- E1 R
-S eETtNAhCNoExD
( y1A -1R1) D- oS x -
= )
o p e n t a n - 2 - y l a m i n o ] p r o p a n o yU lSI}Pmo ic dt aRa zSh oy ld er o - 1 H - i n d o l e -
2 - c a r ba oc xi dy .l ]i c U SP Pe r i n dE orp br ui Rlm Si n e
S o l u At :iD oi ns s 5o glo vfpe o t a sp sh io us mpm ho ant oe - U SP Pe r i n dR oe pl ra C it lo
e dm p AoR S u n d
b a si in 1c 9 0m 0oLfw a t Ae d r j. wu is tt rh i e t h ty ol a m i n2 e a
3 e a s O r nH - e i n d| o l e - 2a c- ic da .r b
C o H i s 1N6 O9 2. 2
a p Ho f6 . 5a 0n ad d 1d 0 m0 oLfa c e t o n i t r i l e .
S o l u Bt: iA oc en t o n i t r i l e U SP Pe r i n dR oe pl raC it lo
e dm p B o R Su n d
( 2 5 , 3 a S , 7 a S ) - 1 - { ( S ) - 2 - [ ( S
M o b pi hl ae sS ee T:e a b4 l. e
n o y l } o c t a h y d r o - 1 aH c-i id n. d o l e -
C i z H 2 s N3 24 00 s. 54 1
T a b4l e U SP Pe r i n dR oe pl raC
it lo
e dm p CoR S
u n d
S o l u A
t i o n S o l u Bt i o n ( S ) - 2 - { ( 3 5 , 5 a 5 , 9 a 5 S , 1 0 a S ) -
% o d e c a h y d r o p y r a zH i) n- oy [l 1} ,p 2e -n at ]
a
1 7 a c i d .
C i z H 2 6 3N 22 20 .4 4 0
1 7
U SP Pe r i n dR oe pl raC
it lo
e dm p DoR u
S n d
( 5 ) -
10 a2
k )-
- 3 {
- M e(t h3y l 5
- 1,
, 4
2 0 o u e c a l y l r, o2 p- y a jr ia nHzd)io-ilye
-l2l}( p1 e n t
8 3 a c i d .
3 1 7 C i z H 2 63N 22 20 .4 4 0
U SP Pe r i n dR oe pl ra C
it lo
e dm pF o R Su n d
D i l u eS no tl u: Bt ai n o Sndo l u A t (i 1o 7
n : 8 3 ) ( S ) - E2 t-h {y (l 3 5 , 5 a 5 , 9 a S , 1 0 a 5 ) - 3
S y s stu ei m t a bs io ll iut1t:y i3 om n g / o fm
U S
LP Pe r - o d e c a h y d r o p y r a z iH n) -o y[ l1} ,p 2e n- ta a]
i n d o E p rr iol u Rm Siin Dni el u e[ nNt O. T Es o T l uhi tsii so n C i s H 3 03N5200.44 5
u s ie n /d d e n t i ft ie csB a.t t] i o n
U S4 P1 O f f i M
c ioa n
l o g/ Pr ea r pi hn ds3o 2p 4
r i3 l

A n a l y s i s
S a m p lS e t s a n:sdo al ru a
td in oSdna m spo ll eu t i o n
P e r i n d
E or pbr uiT mla ib n
l e
e t s C a l c ut lh pae e
t er c eo nftth ale ag be eal me do oufp ne t
r -
i n d o e
p rr ibl u (m Cii ns eH - 3C 2a NH 2iiOnits N
h p)
eo r t i o n
D E F I N I T I O N o fT a b lt ea tk se n :
P e r i n dE orp or uiTlma ib n
lc eeo tnstNa Li9T
n 0 .a 0n % N
d M T
1 1 0 o.ft0h l%e a b eal me do oufp ne t r i n de or p br ui lm i n e R e s =
u (lr tu / xr (s C) s /xC1u 0) 0
( C i s H+ 3C 2a NH 2i Oi sN ) .
t u =p e r a ek s p fo rn t os hm
eSe a m spo ll eu t i o n
I D E N T I F I C A T I O N r s p e ar ek s p fo rn t os hm
eSe t a n s do al ru td i o n
e A . T h Ue a V b s o r sp pte icootftnrhame a jp oe roa ft k h e G s c o n c e n ot fr U aSPtPei ro inn dE or pb ru iuRlmS i n e
S a m spo ll eu et xi ho inmb ai tx ai nmmdai n aittm h ase a m e i nt h Se t a n sdo al ru ( td imo gn / m L )
w a v e l ae st n hg oto shftesh ce o r r e s pp e o oan ftk
d hi en g C u = n o m ic no an lc e n ot fpr ea rt ii no dno p r i l
S t a n ds ao lr ud ta isoo b
n ,t a i intn heAed
s s a y . e r b ui mnt ih n Se a
e m spo ll eu ( t im o gn / m L )
e B . T hr ee t e nt ti iomofe tn h me a jp oe roa ft
k h Se a m p l e A c c e pc t r ia t ne rc
9 ie
0a :. 0 % - 1 1 0 . 0 %
s o l u ct i
o or nr e ts ot
p ho a
on t
ftd hsSe t a n s do al ur ta
d iso n ,
o b t a i intn heAeds s a y . P E R F O TR EMS AT N S C E
e D I S S O (
L 7U 1T 1I )O N
A S S A Y M e d i0 u
. 1N
m h :y d r o ca hc l
i 9do ;0
r mi0 c
L
' P R O C E D U R E A p p a 2r :a5 t0ru ps m
S o l u At :iD oi ns s o l v8 oe fs o d 1i -u hm e p t a n e s uT li fm o3en0: m- i n
a t ien1 L o fw a ta enar d d d1 m oLft r i e t h yA lda -m i n eS .o l u At :iP or no ca e sd ei dr e icn tt he Ad
es a y .
j u sw ti t
a sh o l u ot fpi e
o rn c h al co iar din wcd a t( e1 :rt1 o) M o b pi hl ae sA ec e : t o n ai nt Srdoi ll ueAt (i 3
o n5 0 : 6 5 0 )
a p H o f2 . 0 . S t a n sd taosrcodkl u t 0i .o 5nm 5:g / o fmU SLP Pe r -
M o b pi hl ae sA e c e: t o n ai ntSrdoil lu eAt (i 3o 8n : 6 2 ) i n d o Ep r io lb uR mSi ina nc ee t o n i t r i l e
D i l u eA nc t e t: o n ai ntSrdoi ll ueAt (i 4o 0n : 6 0 ) S t a n sdo al ru td Pi ro en p:s ao r l ue to ifUo SnP sPe r i n d o p r i l
S t a n sdo al ru td0i .o 0n m8:g / o fmU S LP Pe r i n d o p r i l E r b u Rm Siin Mn e e d fi rut o mhmSe t a n sdt aosrco d kl u -
E r b u Rm Siin Dn i el up e rn et p aasfro el dl Do iw s .s ao l v e t i o wn ,i ft ihn ca o
l n c e n tf rr aT otamib2ol. ne s
s u i t qa ub al en ot fU
i tSPyPe r i n dE or po rb iuRlmSi in n e
8 0o f%
t ht eo t va lo l oufDmi e
l u se o
n tn ,i fc oa5r tm ei n ,
a nd di l w u ti etD h
i l ut eovn ot l uP ma set s.h r o a s uu gi ht a - T a b2 l e
b lf ei l to ef0r . 4 5p -o su r iemza e nd di s ct ahfreidr 3s tm L T a b Sl t
e r
t e n g t h C o n c e n t r a t i o n
o fi l t r a t e .
S a m spo ll e u t Ni oo mn :i n e qa ul il vyt ao0l .e 0nm8 tg / m L
o fp e r i n de orp br uii l mn D ii nl uep e rn et p aasfro el dl o w s .
W e ia ngd th r a n ts hf ne ru m obfT ea r b li en tta so u i t a -
b l ve o l u mf el at sarksii, nc d i ci naT ta eb1dl. e
S a m spo ll e
u t Pi ao san ps: o r to ift ohnseo l u u
t in od ne r c
T a b1 l e t e st th r oa su ug iht fai bl tlaeenr d di s ct ahfreidr 1s tm L a )
a )
N u m obf e r V o l u m e t r i co f i l t r a t e .
T a b Sl t
e r
t e n g t Th a b l e t s F l a s k C h r o m a t soy gs rt ae pm h i c =
( m q ) ( N L T ) _ _ ( m L ) ( S eC eh r o m a t t a b i l i t y . )r o }
( 6o 2g1Sr)ya,s pStuhei ym
M o d Le C: p o
2 2 0 5 0 0
D e t e c Ut o
V2 r1 :n
5 m i}
4 1 0 5 0 0 a
=
C o l u 4m .n 6: x-1 m5 m - 5c -m 1;pu am c kL i7 n g i )
8 1 0 1 0 0 0 C o l tu emm np e r 5a 0t u r e : 3
A dD di l ut eoan bt o 7u 0 to f%t hf el a vs o k l us mh ea ,k e
F l ro awt e1 :. m2 L / m i n o
I n j e cv toi lo un 1m 0ep 0:L
m e c h a nf io ac r ba ol6 l
u0myt ia nt1 8 r0p ma ,ns do n i fc oar t e R ut ni m eN :L1 T. t6 i mt eh sre e t e nt ti iomofen
2 0m i nD . i l wu ti etD h
i l ut eovn ot l uP ma set s.h r oasu ugi ht a - p e r i n d o p r i l
b lf ei l to ef0r . 4 5 p- o1 sr1 iemzae nd di s ct ahfreidr 3s tm oL f S y s stu ei m t a b i l i t y
f i l t r a t e . S a m p Sl tea:n s do al r u td i o n
C h r o m a t soy gs rt ae pm h i c S u i t a b ri le i qt u
y i r e m e n t s
( S Ce he r a m e y t y ( e6 g2 1S
n )y
a ,sp St)u ei tm a b i l i t y . ) T a i l fia nc gt oNr :M1 .T5
M o d Le : R e l a st t i va en dd eav ri da tNi M o2nT.: 0 %
D e t e c Ut V o2 r1 :
n0 m A n a l y s i s
C o l u 4m - n x:m2 m 5 - 4 c m- ;pu am c kL i7 n g S a m p lS et s a :n sdo al r
ua td in oSdna m spo ll eu t i o n
T e m p e r a t u r e s C a l c ut lh pae et er c eo nftth a le ag be eal me do oufp ne t
r -
C o l u 6m0 n : i n d o ep rr ibl u (m Cii n9 eH- 3C 24 NH 2idO1 i ssN s) o l v e d .
S a m cpo lo e
l 5e r :
F l ro awt 1e :m L / m i n = (l r tu / xr (s )G /xL V) x
R e s u 1 0 0
I n j e cv toi lo un 2m 0 ue L:
R ut ni m e N :L2 T. t5 i mt eh sre e t e nt ti iomofen t u =p e a
r e
k s p fo rn t
os hm
eSe a m sp o ll eu t i o n
p e r i n d o p r i l r s =p e a
r e
k s p fo rn t
os hm
eSe t a n s do al ru td i o n
S y s stu ei m t a b i l i t y G s = c o n c e n ot ft
r ha Set ti ao nnsdo al ru t
d i o n
S a m p Sl te a:n sdo al ru td i o n ( m g / m L )
S u i t a br ie l qi ut iy r e m e n t s L = l a b ce ll a(i mm g / T a b l e t )
T a i lf ia nc gt oNr M:2 .T0 Vv = v o l oufMm ee d i9 u0 m m0,L
R e l a st t i va en dd eav ri da tNi M o 2n T
.: 0 % T o l e r a Nn Lc 8T e 5
s( :%
Q o) ft h le a b eal me do oufp ne t
r -
i n d o e
p rr ibl u (m Cii n9 eH- 3C 24 NH 21
iOs1
d siN s) s o l v e d .
¢ U N I F OO RFDM OI S
T UA
Y NGI(ET9 S0 5 M) e: te ht e
r e q u i r e m e n t s
 3 2 4
P e4 r i n d/ Oo fpf ri icM iloa n
l o g r a p h s U S4 P1

I M P U R I T I E S r s =p e r a ek s p oo fpn es rei n de or pbr uifm l ri on me

' O R G AI N
M PI UCR I T I E S t h Se t a n s do al r u td i o n
S o l u At :iP or no ca e sd ei dr e icn tt he Ad
es s a y . C s = c o n c e n ot fr U aSPtPei ro inn dE orp br ui Rl m Si n e
S o l u Bt: iA o
c en t o n i t r i l e i nt h Se t a ns do al r u (td imo gn / m L )
M o b pi hl ae sS ee T:e a b3 l. e G y = n o m ic no an lc e n ot fpr ea rt ii no dno p r i l
e r b u i mnt ih n Se a
e m spo ll e u (t imo gn / m L )
T a b3 l e F =r e l a rt ie vse p foa nc sft oeoerr a ic nh d i v i d u a l
i m p u (r sieTteay b4 l) e
S o l uA
t i o n S o l u Bt i o n A c c e pc tr ia t ne S
c
r ieeTae:a b4 l. De i s r ep ge aalrke dss s
t h a0 n. 1 % .
2 0
T a b4l e
3
R e l a t i Rv ee l a t iA vc ec e p t a
5 R e t e n tR ie os np o n
C rs i et e r i a
N a m e T i m e F a c t o r N M( T
% )
i m i d a z o l e s 0 . 0 8 =
P e r i n dr oe p
l r
a t
i el d
c o m pB oe u n d 0 . 4 2 1 . 2 1 2 . 0
P e r i n dr oe pl ra it el d
D i l u eS no tl :u Bt ai n o Sndo l u A t (i 2o 0n : 8 0 ) c o m p C o< u n d 0 . 7 4 0 . 9 7 0 . 5
S y s stu ei m t a bs it losi co t kyl u At :i0 o. n0m 3g / o fm L P e r i n dr oe pl ra it el d
U S I Pm i d aR zSi o n D li el u e n t
c o m p Do¢ u n d 0 . 8 5 0 . 9 8 0 . 5
S y s stu ei m t a bs it losico t kyl u Bt: i0 o. n1m 2g /e m a cL h
o fU SP Pe r i n dR oe pl ra C it lo
e dm p CoR u Sa n UddS P P e r i n d o p r i l cath
P e r i n R d eo lp arCti oel dm pDoR u Si nDn i dl u Ie nni tt .i a l l ye r b u m i n e 1 . 0
a d 8d 0o f% t ht eo t va lo l oufDmi el u se no tn ,i fc oa5r t e P e r i n dr oe lp ar ti el d
m i na ,n d di l wu ti etD h i l ut eovn ot l u m e . c o m pF o e u n d 1 . 3 8 0 . 8 6 3 . 0
S y s stu ei m t a bs io ll iu tt yAi co cn u: r wa et iea lgbyho u t A nu yn s p e c i f i e de y ties
1 . m
5 e g a oc fhU SP Pe r i n dR oe pl raC it lo
e dm p B o u n idm p u r i t y 0 . 2
R Sa n Ud SP Pe r i n dR oe pl ra C it lo
e dm pF o R Siu n nta od T o t ia ml p u r i t i e s 1 . 5
5 0 -v m o lL u mf el at sA rk id
. 3cd0m oL fD i l uae nns d to n i - 2 I m i d ias zg oi lvf eo inr d e n t i fo inc laa ytni isdno no qtu a n t ui st iat nth gei sd
c a ft oe 5r m i nT r. a n 5s .f m 0e re
L a oc fh S y s stu ei tm a b i l pi rt oy c e d u r e .
s t os co kl u At ,iS oy ns stu ei mt a bs it losicot kyl u Bt, ia onn d » ( 2 5 S , 3 a S , 7 a S ) - 1 - { ( 5 ) - 2 - [ ( 5 ) - 1 - H -C a r b
S t a n sdt aosrco d kl u tD iio ln w
u. ti et
D ih l ut eovn ot l u m e . i n d o l e - 2 -a cc a i dr .b o x y l i c
S t a n sd taosrcodkl u t 0i .o 0nm 5:g / o fm U S LP Pe r - ¢ ( 5 ) - 2 - { ( 3 5 , 5 a 5 , 9 a S , 1 0 a S ) - 3 - M e t h y l
i n d o E p rr iol u Rm SiinDn iel up ern et p aasfr o el dl o w s . a j i n d )o l- -y 2l(}1 p ea cn it da. n o i c
D i s s ao sluvi et qa ub a l en ot fUi tSPyPe r i n dE orp or ui lm i9a (n 5e ) - 2 - { ( 130 5a ,R 5) a- 53 ,-,9M4ae -
5t ,
dh iy ol x- o1 d e c a h y d r o p y
Pe ] i n d o l - 2 ( 1 Ha ) c i- dy .l } p e n t a n o i c
i s R S
i n 8 0o f %t ht eo t va lo l oufDmi e l u se n
o tn ,i fc oa5r t e e{ 5 e t 2y -|{ ( 3 5 , 5 a 5 , 9 a 5 S , 1 0 a 5 ) - 3 - m e t h y l
i ] m i na ,nd di l w
u ti et
D ih l ut eovn ot l u m e . a J i n d Ho )
l - 2y (l 1} p e n t a n o a t e .
D S t a n sdo al ru td 0i .o 0 n m:0 g5 /o fm U S LP Pe r i n d o p r f iT ol ti am lp u r ei xt ci le psu edr ei sn dr eo lp arcti eol dm pF oa u
np d
ne rd i n d o p r
E r b u Rm Siin Dni el uf e rn tot hmSe t a n s dt aosrco kdl u t i o rn e. l act oe dm pB o. u n d
i J P a st sh r oa su ug iht fai bl tlo eef0 r . 4 5p -o u sr ie mzae n d
9 d i s ct ahfreidr 3s tm oLf i l t r a t e . A D D I TR IE OQNUAILR E M E N T S
= S a m spo ll e u t Ni oo nm :i ne qa ul il vyt ao2l ein tr o f o' P A C K AA GNSIDTN OGR AP Gr eE s:ienar iv re- tc io gnh tt a i n e
[ o m
a )
p e r i n de or p br uii m ln D ii nl uep ern et p aasfro el dl o w s . P r o tf erc h ot em aa tn m do i s t u r e .
FS T r a n as qf ue ar n et qi ut iy vt aoal be n o2tu0mt og f B G e U SR P E F E RS ET NA C NE ( D 1A 1R) D S
i n d o e p rr ibl u fm ri opn moe w d T ea r b le(edNt2 0Ls) Ti n t o U SI Pm i d aR zS o l e
a t e st tu b Pe i. p 1e 0t .m 0oL fD i l ui en ntt toh t ee st tu b e , U SP Pe r i n dE orp br ui Rlm Si n e
s o n i fc oaar tb e o1u0mt i na ,nv do r ft oear xb o1 um t i n . U SP Pe r i n dR oe pl raC it lo
e dm p B o R Su n d
P a st sh r oa su ug iht fai bl tlo eef0r . 4 5 p- o1 sr4 iemzae n d ( 2 S , 3 a S , 7 a S ) - 1 - { ( 5 ) - 2 - [ ( S
d i s ct ahfreidr 3s tm oLf i l t r a t e . n o y l } o c t a h y d r o - 1 aH c-i id n. d o l e -
C h r o m a t soy gs rt ae pm h i c C i 7 H 2 g3 N4 20 0. s4 1
( S eC eh r o m a t ( 6o 2g1Sr)ya,s pStuhei ymt a b i l i t y . ) U SP Pe r i n dR oe pl ra C it lo
e dm p CoR S u n d
P r o ca e sd ei dr e icn tt he Aeds s ae yx ,c fe o ptr th Re u n ( 5 ) - 2 - { ( 3 5 , 5 a S , 9 a S , 1 0 a S ) - 3
t i m [e .N o T re u tnTi im hs dee e t e r bm y ti hngere ad d i - o d e c a h y s i ,r 2o -p dy jri anHzd)io- nly e-l 2l} (!p1 e n t
e n ft r o
T am b3 l. ]e a c i d .
S y s stu ei m t a b i l i t y C i z H 2 6 3N 22 20 .4 4 0
S a m p Sl ye s:stu ei m t a sb io ll iu ttyi o n U SP Pe r i n dR oe pl raC
it lo
e dm p DoR u
S n d
S u i t a b ri le i qt u
y i r e m e n t s ( S ) - 2 - { ( 3 5 , 5 a 5 , 9 a 5 , 1 0 a R ) - 3
T a i l fia nc gt o Nr : M1 .Tf5 o tr h pe e r i n dp oe par ki l o ds a n y c o p, y
2 -a a cj i
i nHn d)ao-t
ly -tl 2}( p1 e n t
R e l a st t i va en dd eav ri da tNi M o 5n T
.: f0o %
tr h pe e r - a c i d .
i n d o p re ia l k C i 7 H 2 6 3N 22 20 .4 4 0
A n a l y s i s U SP Pe r i n dR oe pl raC
it lo
e dm pF o
R Su n d
S a m p lS e t s a n :sdo al r
ua td in oSdna m spo ll eu t i o n ( S ) - E2 t-h {y (l 3 5 , 5 a 5 , 9 a S , 1 0 a 5 ) - 3
C a l c ut lh p ae te er c eo nfet aa c ig m
hep u irnti htpeyo r - o d e c a h y d r o p y r a zH i) n- oy [l 1} ,p 2e -n at
t i oo nfT a b lt ea tk se n : C i s H 3 03N5200.44 5
R e s =
u (lr tu / xr (s C) s /xC( u1 )/ xF 1) 0 0
t u =p e a
r e
k s p oofen as cie m
h p u fr ri t
o hy
me
S a m spo ll eu t i o n
U S4 P1 O f f i M
c ioa n
l o g/ r
P e
a rp ph hs e 3n 2a 4z 5i n e

R Sa, n 0d . 0 m0 g 2 /o fmU S LPPe r p h eR ne al za i t en de

C o m pB o R Siu nSno dl u At i o n
P e r p h e n a z i n e S t a n sdo al ru td 0i .o 0 n m:0 g
2 /o fmU S
LP Pe r p h e n a z i n
R Si n S o l u A
t i o n
S a m spo ll e u t 2i mo ng : /o fm
P eL r p h ei nnS ao z
l ui tn ieo n
A
D i l us tae mdsp o ll eu t 0i .o 0 n m:0 g
2 /
o fm
P eL r p h e n a -
z i ni neS o l u At fi ro nto hmSe a m spo ll eu t i o n
C h r o m a t soy gs rt ae pm h i c
( S eC eh r o m a t ( 6o 2g 1Sr)ya,s pStuhei ytm a b i l i t y . )
M o d Le C:
C a H 2 6 C I N 3 O S 4 0 3 . 9 7 D e t e c Ut o V
2 r4 :n5 m
P i p e r a z i 4
n -
e e
[ t
3 h- a( n2 o- lc ,h l o r o - 1 0 H - p h Ce on ol tu h4mi.na 6: z x-
i2 nm5-m - c4 m -;p a m c kL i7 n g
1 0 - y l ) p r o p y l - ] ; C o l tu e mm np e r 3a 0t u r e :
4 - [ 3 - ( 2 - C h l o r o p h e n o t h i a z i n - 1 0 F
- yl lro )awtp e1r :.o m3p Ly l/ ]m- i1 n- p i p e r a z i n e e t h a -
n o [l 5 8 - 3 9 - 9 ] . I n j e cv toi lo un 1m 0ue L:
S y s stu ei m t a b i l i t y
D E F I N I T I O N S a m p lS ey s s: tu ei tm a sb io ll iu ta
tyin Sodnt a n d a r d
P e r p h ec no an zt N
ia n
iL 9n
eT s
8 .a 0n %
N
d M1 T
0 2 o.fp0e %
r - s o l u t i o n
p h e n a
( z
C 2
i :
n He a s cC aI lNc 3u O
ol Sn
a
t )th d,eerd ib ea ds i s . [ N o t e T a bS 2lf eeo re
r e l a rt ie vt ee nt ti im oe sn . ]
I D E N T I F I C A T I O N S u i t a br ie l qi ut i y r e m e n t s
e A .I N F R AA BR SE O D R (P 1T 9I 7O MN ) R e s o l u Nt Li3 o T. nb0 :e t wp ee er n p h ea nna p dez ri -n e
e B . T hr ee t e nt ti iomofe tn h me a jp oe roa ft k h De i l u t e d p h e n a r ez li a n ct eeo dm pB o , S uy ns stdu ei tm a b i l i t y
s a m sp o ll eu ct oi or nr e ts ot p ho oan fttd hsSe t a n s do al ur -d s o l u t i o n
t i o an s,o b t a i in tn het ed
e sf to Or r g aI mn pi ucr i t i e s . T a i lf ia nc gt oNr M :2 .T5S ,t a n s do al r u td i o n
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t er c eo nfitm ap gu e ri in tt hipe os r to if o n
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P ep t u si nhW ae tfse o Irrn j e c pt ir oe np, a r ei ndw h i Cci s th h ce o n c e n ti nrj au pgt eimro Lno ,,fU SP Pe r -
w i tth h aei od fC i t Ar ic ciI tdc.o n t na oil tne ss s p h e n aR zSi nit nh See t a n pdr ae rp d a r aa n t Aidya
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U S PPe r p h eR nS a z i n e P a c k a a gnsidtno gr a g e i nwP erl el s - cel lirogvsh et -d r,
[ N o t e T th hrfeo o lu lgophwr oio nuc gt e pd ru or te es c, ts i s tc aon nt t a i n e r s .
t e so tra s ssa py e c i tmheUe nSs R Pe , f e rS et na cn eda an r dd ,
s o l u tc io on nts a ti hn eibmnyc, g o n d utch t pe ir n o cg e d u rUe SsR Pe f e r s e t n a c n ed
( a 1 r1 d ) s
w i t hd o e lu aut yn ,ds eur b dl iug eho tdru, s ilno gw - a c t i n Ui S c P Pe r p h eR nS a z i n e
g l a s] s w a r e . [ N o t E T th hrfe o lu lgophwr oio nuc gt e d p ur or te es c, t
t e so tra s ssa py e c i tmheUenS s R Pe, f e rS et na cn eda anr dd ,
I d e n t i f i c 1a m t iwL oi n tm he Dt ih tlao5 unmtoLeA l. p p l sy o l u tc io on nt sa ti hn eib mnyc, g o n d ut ch t pe i r o n cg e d u r
5 u le a oc fth h is so l u a t inaodsn o l u ot fiU oSnP Pe r p h e n a -
v w
z i nR eSi n m e t h ca onn ot la1 i mn g p
i e nm rg tL oa s u i t a b l we i t hd o e lu aut yn ,ds eu rb d l iug e ho td
ru, s ilno gw - a c t i n
h . 2 5 - mg ml a s] s w a r e .
a
a t h i n - cl ha yr eo rm a t p ol g a tcr eoa, ap wth ieiatdc0
i ] l a yoefcr h r o m a ts o i lgig r
ce alaD.pe hv iet clh co e hp r o m a tI od -e n t i f im ce aettthirseoe nq u Ii trf eo trm he/ edn etn st i f i
D g r ianams o l vs eyn stct oe nms i osfat mi in xg to ufa rc ee t o cn ae t ti eo su n t n dP ee rr p h eI n ja ezc it in oe n .
2) a n ad m m o h ny i d ru o(mx 2 0 i 0du :ne t1ti)hl se o l vf er no tn t U n i f oo rfdm oi stuayn gi(et 9s 0 5 )
¢
5 h am s o va eb d o1 u5ct mA .i r -t dh rpely a ta e n,sdp rl ai yg h t l y F O OR R A S LO L U PT AI CO KNI ANSG IEN DG L EC -O UN NT IA TI N E R S
w i at sh o l u ot fi o nd o p la ac tipidrn ei cp ba ydr ie sds o l v i mn eg et th rse e q u i r e m e n t s .
2 1 0 m0 og fc h l o r o pa lc ai tnd1 i mn oiLfc1 N h y d r o c h l oD re il c i v e
[ a v ro alb(ul6me9e8 )
a c i ad d, d i 2n g5m oL fp o t a si s o di siu odmle u (t 4i no1 n0 0 ) ,
w n F O OR R A
S LO L U PT AI CO KNIANMG UEL DT I P CL OE N- TU NA I TN E R
=) d i l u wti it nhw ga tt eo1r 0m 0L a, n ad d d 0i .n 5m g 0oL ff o r -
m ia cc i td h:R erv a lo ufte h per i n cs ip p oa t bl t a fi rn oe m d m e et th rse e q u i r e m e n t s .
t h Ie n j e cc toi ro rn e ts ot p ho aon tbd ts a fi rn t oe hmdSe t a n - L i mo ifpt e r p h es nu al zf io nx ei d e
d a sr od l u t i o n . M o b pi hl ae Rs ee s, o lsuo tl iu otSni to an ,np dr ae rp d a r a t i
B a c t eE rn ida ol tT oe xs ( i 8
t 5 n )c
so I n t na oi mtn os r e a n Cdh r o m a t so yg sr ta ep mh i a scP
d ir ro ecicnett e heAed
ds -
t h a3 n5 .U 7S EPn d o tU o n ixptiesnm r og fp e r p h e n a z si any e. .
p (H 7 9 1 )b :e t w4 e . a2e nn5 d. 6 . T e sp tr e p a r a t i ao nan c cT ur ramanetseaflsep yuro rr et d i o
O t hr ee rq u i r e m me eetnth t r
se es q uii tru enm dIe e nn-rt os fO r aS lo l u te iq ou ni, vt aoal be n o1tu6m t og fp e r p h e n
j e c t a i onIn dsm p l aD nr tPu erg d o d (u1 c) t. s z i n te oa
, 2 0 0 v-o ml L u mf el at sdrkii, scs ion l a vnde di l u t e
w i tm he t h taovn oo ll um mi e xa ,nf idl t e r .
A s s a y
P r o c e d ua rv eo ln(ujamebec1o t0 u Lto) ft h Tee s t
A c i d - as lo clo uh to il o n1 0m T roLafh ny sd fr eo rca hc li od rp ir ce p a ir na ttt ohi ce o hn r o m a tr o e c g tor hracedph hr ,o m a t
t oa 1 0 0 f0 l-a cm s okLn t a 5i 0n m0 i oL
n fag l c oah no dl g r aam n,md e a st u h per ee r
a ek s p o Cn as le cs ut. lh paeet re -
3 0 m0 oL fw a t De ir l. w u ti etw h a tt eov ro l u m e . c e n to a fp ge er p h es nu a l fz oii nxtnihedpeeo r to ifOor na l
P a l l ac hd li ousrmoi dl eu t i o n 1 0D m0 i osg fsp oa ll vl ea d Si ou lm u tt ai koben ytn h fe o r m u l a :
c h l o irnai md ie x to uf1 rme oLfh y d r o ca hc liaodnr di c
5 0m oL fw a ti e na r1 0 0 v-o ml Lu mf el at shrkei, ac toi n an g 1 0 0/ 5( )r
s t eb aa mt ohe f f se oc lt u tC io oodn li. ,l uw ti etw ha tt eo r
v o l ua mn e m di
, xS . t oi rn ae na m bb e o tr ta ln u eds we i t h i ni n w h i1 ; ics th h pe e a r e k s p oofpn es re p h es nu a l fz oi xni ed
3 0d a y Os .n t h de ao yfu s et ,r a n 5s f0me trLoa 5 0 0 - m L( r e l ar te itv ee nt ti iomofean b o0 u. 7t 2a )nr;,di s t h se uo m f
v o l u mf el at sa rk i
d
, 4cdm oL fh y d r o ca hc liaodnr4 di . g1c o f t h re e s p o fn a l slt eh pse e a kn so m :t o tr he a5 n. o0 fp%e r -
a n h y ds ro od aui csue mt da it le w u, ti et
w ha tt eov ro l ua mn ed,p h e n a s uz li fnio sexf io du en d .
m i x . A s s a y
S t a n pd ra erpda r a t i ao nan c cD ui rswaseto i ellgvyhe e d M o b pi h l e a s e aPf ir let pea ranedrdd ee g a ms is xe toduf r
q u a n ot fU i tS PyPe r p h eR nSi anA zc ii nd e- as lo cl ou th t oio ol n 0 . 0 M 1 a m m oa n c eit au a ctmee t, o n ia tnrmdi el t e ,h a n o l
o b t aa si on l u ht a i ov nai kn ng oc w o nn c e n ot fr a ab to iu ot n( 4 8 : 3 9 A : d 1j w3u i)s gt
. lh a cai ca el at ci cit doa p H o f4 . 5 .
1 5u0gp e mr L . M a ak de j u s i tf nm ee cn ets(ss eaS ery ys St u ei mt a bu inl di te y r
C h r o m a t ( 6o 2g1 r) a ) .p h y
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l o g/ r
P e
a rp ph hs e 3n 2a 4z 7
i n e

R e s o l su ot li uo t n i o ns u Di it saq s bu alo nelt voi fet i e s U n i f oo rfdm oi stuayngi(et 9s 0 5 )

b r o m p h e mna il reaaanmtUdi eS PnPee r p h eR nSi an z i n e F O SRY R PU AP C K I ANSG IEN DG L CE -O UN NT IA TIm Ne EetR th S s:
e
m e t h taoon bot laa si on l u ht a i ov nik nn goc wo n nc e n t r a r t e iq o u ni s r e m e n t s .
o fa b o4u0 ut gp e m r L a n 8dy gp e mr L r ,e s p e c t i v e l D y .e l i v v e ro al b(ul6me9e8 )
S t a n pd ra er pda r a t i ao nan c cD ui rswasetoeillgvyhe e d F O SR Y R PU AP C K I AN MG UEL DT I P CL OE N- TU NA Im TNe EetR th S s:e
q u a n otfU i tS PyPe r p h eR nSi an mz ei tn he ad in lo uqltu,ea n -r e q u i r e m e n t s .
t i t a t iav n e sldty ,e pi wf nie sc ee s ws ia tm r hye ,t h taoon bot la i n
a s o l u ht a i ov nai kn ng oc w o nn c e n ot fa r ab to8iu.out0ng A s s a y
p e mr L a, nf id l t e r . A c i d - as lo cl ou a ht oin Poldna l l ac hd li ousrmoi dl eu t i o n P r e
A s sp ar ye p a r a t i ao nan c cT ur ramanetseaflsep yuro rr e- dp a ar sed i r e icntt he Ae d s su any dP ee rr p h eI n n ja ezc it in oe n .
t i oo nfO r aS l o l u te iq ouni, vt aoal be n o1tu6m t ogfp e r - S t a n pd ra er pd a r a t i ao nan c cD ui rswasetoe illgvyhe e d
h e n a zt ioa n2 e 0, 0 v-o ml L u mf el at sdrkii, scs ion l a vnde d i - q u a n ot fU i tS PyPe r p h eR nSi anA zc ii nd e - as lo cl ou tht ooi l
o n
j u twe i tm he t h taovn oo ll ua mn e mdi, xD .i l u t e o b t aa si on l u ht a i ov nai kn n g oc wo nn c e n ot fa r ab to iu ot n
q u a n t i t aa tn sidtv ee lpiyw f n,ie sc ee s ws ia tm r hye ,t h tao n o 1 l 6u0gp e mr L .
o b t aa si on l u ht a i ov nai cn og n c e n ot fa r a b to8iu.1ot01ng A s sp ar ye p a r a t i ao nan c cT ur ramanetse aflsevyuro rl -e d
p e mr L a, nf idl t e r . u mo e fS y r e u qp u, i vt aoal be o n6 tum tog fp e r p h e tn oa z i n e
C h r o m a t soy gs r(t ase epCmehh ir co m a t( o6 g2 r1 a) p) h a 2 yT 5h -ve m o lL u mf el at sdrkii,l cuw ti etw ha tt eov ro l ua mn ed ,
l i q uc ihd r o m a i t s eo qguri awp p ipathe2h d5 4 d- e nt me c t om ri xT . r a n 1s f0me tL r oa 1 2 5 s-e mp aL r aa d t 2od 5rm, oL f
a na d3 . 9 x- 3m0m-c co ml tu h ma c nto n t pa ai cn ksL i1 n1 g . w a t ae dr j, wu i s ta h m m o h ny i d ru o tmoxa i p dH o ef1 0t o
T hf el ro aw it s ea b o1 u. m t
5 p L e mr i n uCt he r. o m a t o 1g 1 ra, anepdx ht rw ai cf tt oh u2 r0 -p m o rL t oi fco h n ls o r foi lf - o r m ,
t h Re e s o ls uo tl iu otani no rnde, c to hr ped e r a ek s p oa sn s e st e r t i nh egex t r ta ch tr soa un gh hy d s ro od suiusul fm aE tv ea. p -
d i r e fc otPrer do c e td hur ere el a: rt e i vt ee nt ti imaoerns ae b o u ot r att hece o m b e ix nt re oadcna ts st eb aa mt w hi tt h aei d
0 . f6o br r o m p h e an ni1d.rf0ao prm ei rn peh e an n a tzdhie n oe fa; s t r oe fna im t r to oa g eb no5 u m t L C. o m p tlh ee v t ae p o -
r e s o l uR ,tbi eo nt ,wb er eo nm p h e an ni d pre ar mp h i en ne ar za it niw eoi nt ha op u p lt i co fah te ia ato ,n ddi s st ah rve ee s i d u e
i s n ol te st s h a 3 .n 0C. h r o m a tt hoSe r g or taeup ro bhrpda r ei n 1 5 .m 0oL fA c i d - as lo cl uo th f iio ollnt ,ei frn ien cg e s s a r y .
t i oa n ,n r de c to hr ped e a r ek s p oa snd si er s e fc otPrer do c e - P r o c e d 1u 0r .me0e L aM oci fh tx h Ae s sp ar ye p a r a t i o n
d u r te h:t ea i lfi an cgits n o ro mt o tr he 3 a .n 0a; n tdh r ee l a t i a v ent dh Se t a n pdr ae rp da w r a i t1 ih5 o.mn0oLfP a l l a d i u m
s t a n dd ea vri daf to rri eop nl ii cn aj te eci ts ni oo mt n so tr he a n c h l o sr oi lduetf i lo itnf en,r e c e s as n a crdoy n, c o m di ett ae nr t- l y
2 . 0 % . m i tn heae b s o r ob fta hn ecs se o els u t ai go an isa n ,r se ta g e n t
P r o c e d u r e i n Sj e cp q tuavar ola lt u( e ml a eyb 1so 0 pu Lt ) b l a in nk1 , - cce ml al tst h we a v e lo ef mn a g tx hi a bm suo m r b -
o ft h Se t a n pdr ae rp da ra anttdhi Aeo sn sp ar ye p a ir na tt oi o an n ac te a b o4u 8tn0 m w ,i t a sh u i t sa p b le ec t r o p h o t o m e
t h ce h r o m a tr o e cg tor hrace
dph hr ,o m a ta on mgd er aa sm -s C a, l c ut lh qaeut ae n ti nimt gyo , ,fp e r p h e n a z i n e
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t h le a b eal me do oufpne tr p h e n a z i n e e S o te h fe o l l op wr io nc ge p d ru or tes esac,mt -
( C H a i2 6 C I N 3 O S ) . p l e ts h, Re e f e rS et na c n eda anr s dod l, u tc io on nts a i n i n g
t h eb y m c, o n d ut ch t pe i
r n o c g ewd iu tr hde o eslu aut yn, d e r
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r e s i sc to an ntt a i n e r s .
I D E N T I F I C A T I O N
U SR Pe f e r s e t n a cn e d
( a 1 r 1 d ) s e A .
U S P Pe r p h eR nS a z i n e S t a n sdo al ru d t1 i mo ng : / o fm U S LPPe r p h eR nS i an z i n e
[ N o t t T th hrf eoo l ul og wphir o
noguc t e dp u
r o r te es c, t m e t h a n o l
t e so tra s ssa py e c i tm heUe nSs R P,e f e rS et na cn eda anr dd , S a m spo ll e u t Si ho n aa :k
p oe r to iff oi nn ep loy w d e r e d
s o l u tc io onn ts a ti hn eib mnyc, g o n d utch t pe i
r no c g e d u r eTsa b l ne ot ms i, ne a q l u il vyt ao5l m e nogtfp e r p h e n a z i n
w i t hd o e lu aut yn ,ds eur b dl iug eho tdru, s il no gw - a c t i n i cw i t1 h0m oL fc h l o r Fo i fl o t eerrvm, a. p ot r hf a
ei t
l ter a t e
g l a s s w a r e . ] o na s t eb aa mt n eh a tr old yr y n a e sndsdi, s s to hl ve e
I d e n t i f i 1c 0a m toL ifw o a ntt eoa rA
v d o d l ou fSm y er u p , r e s ii nd5 ume oL fm e t h a n o l .
e q u i vt aoal be o n4tum t ogfp e r p h e rn ea nz adilenk rea , l i n e A d s o r b 0 e. n2 t5 l :a- yome fcrm h r o m a ts o i lgi rc aa p h i c
b yd r o p aw did si oet fs i oo nd hi yu dm r o t oxa ip dHo ef1 1t o el
1 2 a, ne dx t rw ai ct ft o
h u5 r- pm oLr t oi fco h n ls o r oc fo o mr -m ,A g o l i cv ao tlt uo5mnp eL :
b i n ti hneegx t r ta ch tr soa u b geohdfa n h y ds ro od suiusu l -m D e v e ls oo pl ivs ney g ns tt Ae cm e: ta on n ad em m o n i u m
f a ti nea f u n in ne tal ob e a kE evr a. p ot rh ea ex tt er o a cna t s h y d r o( x 2 0i 0d :e 1 )
s t eb aa mt n eh a tr old yr y n aen sd ds i, s s to hl rev ee s ii dn u e l o d o p la ac ti i dD :ni is sc 1o l0 m v0 eog fc h l o r o p l a t i n i c
4 m oLfm e t h at n h so
eo ll :u st oio ob nt a ri en se pdtootn hd es a c i nd1 m oL f1 N h y d r o ca hc li A do .rd2id5cm oLf
I d e n t i ft ie csuat nt idPoeen rr p h eI n ja ezc it n i oe n . p o t a si s o di siu odmle u (t 4i no1 n0 0d )i ,l w u ti etw ha t e r
t o1 0 m0L a, n ad d 0d. 5m 0oL ff o r am ci i cd .
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oa en
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S p rr ae ya g eJ n o dt o: p la actii dn i c A n a l y s i s
A n a l y s i s S a m p lS et s a :n sdo al rua td in S
odna m spo ll e u t i o n
S a m p lS e t sa n:sdo al ru a td in oSdna m sp o ll eu t i o n D e t e rt hm pei enrec eo nftth a le ag be eal me do ouf n t
D e v eu ls o itnphgDe e v e ls oo pl ivsneygn stut n et tmi h
l e p e r p h e( nC a2 z; iH n2 e6d iC sI sN o3l Ov Se )d .
s o l vf er nohtn atms o v1 e5c dm A.i r -t dhrpely a ta en, d T o l e r a Nn Lc 7eT5 s( :%
Q o) ft h le a b e al m e do oufp ne rt -
s p rl ai yg hwt il t
yS hp rr ae ya g e n t . p h e n a( zC i2 i n He 2 6 i sCd iI sN s3o Ol Sv )e d .
A c c e pc t r ia t ne T
c eRa e-:v a lo ufte h per i n cs ipp oa tl e U N I F OO RFDM OI ST UA
r ih Y NGI(ET9 S0 5 M) e : te ht e
f r to hm Se a m sp o ll eu ct io or nr e ts ot p ho afn tr
d to
s hm e r e q u i r e m e n t s
S t a n s do al ur td i o n .
A D D I TR IE OQ NU AI LR E M E N T S
A S S A Y e P A C K AA GNSIDTN OGR AP G r eE s:ientr ivg ehl ti ,g h t - r e s i
e P R O C E D U R E c o n t a i n e r s .
S o l u At :iT or na n 1s f0me oLr fh y d r o ca hc lit odoar i c e U SR PE F E RS ET NA CNE (D 1A 1R) D S
1 0 0 0 f l-a cm
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g l c oah no dl U S PPe r p h eR nS a z i n e
3 0 m0 oLfw a t De ir l. w u ti etw ha tt eov ro l u m e .
S o l u Bt: iD oi ns s 1 o l0mv0
g eo fp a l l ac dh l i ouirmnai d e
m i x to uf1 rme oL fh y d r o ca hc liaodnr5di0mc oL f
w a ti nea r1 0 0 v-o ml Lu mf el at shrkei, ac toi n
ansg t e a m
b a t ohe f f se oc lt u tC io oodnli.,l wu ti etw ha tt eov ro l -
u m ea ,n m di xS .t oi rn ae na m bb oe trta lneu ds we i t h i nP e r p h ea nnAadmz ii tn rei p t y l
3 0d a yOs .tn h de ao yfu s et ,r a n 5s f0me trLoa 5 0 0 - mH L y d r o c Th al bol re it ds e
v o l u mf el at sa
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p ty yd lr io nc eh l
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i n S o l u At i o n
S a m spo ll e u t Ni oo n m :i n1 a6jl01l gyo/ fpm e L r p h e n aa - m o uo fnp t
e r s p h e( nC a2 z1 Hi 2n zea6 nC Id N 3
z i np er e p aasfro el dl To rw as n. as pf oe rr to ifpo on w d e ra ,m i t r i h p ty yd lr io nce(h Cl zo or- HHi 2
Cd l3e )N .
e q u i vt ao4l me nogftp e r p h ef nr aoNzmLi2 T n0f ei n e l y
p o w dT ea br lete do ta sg ,l a s s - s ct oon pifpcl eaa sr Plkie-. d P a c k a
a gnsidtno gr a g e i nwP erl el s- ce lor onvst eea di
p e itn tt ohf el a 2s k5m oL fS o l u At ,s
i oh nabk yme e - ers.
c h a n mi ce aafl on 3rs0m i na ,nc de n t r aip fo ur gtoeif o n U SR Pe f e r s e t na c n e d
( a 1 r 1 d ) s
t h me i x t Uu s
r teeh.cel e sa ur p e r fn l au t
i da . n t U SA Pm i t r i Hp t y yd lr io nc eRh Sl o r i d e
I n s t r uc mo en nd ti at li o n s U S PPe r p h eR nS a z i n e
M o d V ei s: [ N o t e T th hrf eoo l ul og wphir o
noguc t
e d
p r
u o
r te es c, t
A n a l y wt ai vc ae ll eMn ag x t hia :bm suo mra bt a n c te e so tra s ssa py e c i tmheUenSR s Pe
, f e rS et na cn eda an rdd ,
a b o4 u8tn0 m s o l u tc io on nts a ti hn eibmnyc,
g o n d utch tpe i r n
o g
c e d u r
ra) C e l l :1 m w i t hd o
e lu au
t yn ,ds eu rb d
l iug eho td
ru, s ilno gw - a c t i n
c o e A n a l y s i s g l a s s w a r e . ]
f o S a m p lS e
t s
a n
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o Sndo, l u - I d e n t i f i c a at pio or nto ifpTo o
r
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t i oB n e q u i vt aoal be o
n4tu0mt ogfp e r p h e tn oaa 1z i
0 n0 e- , m L
3 M i 1x0 .m 0 eL a oc fht h Se a m spo ll eu at into dnh Se t a n - v o l u mf el ta csr oki nc t a ai bn oi5un0mgt oL fa l c o Ah go il t. a t
5 d a sr od l u wt i ot1 nh5 .m 0oL fS o l u Bt.Fi iol nti ef nr ,e c - f o 2r 0m i n u at e dasdl, c ot hovo ol l um mi e xa ,,nf d
i l to err
= e s s a ar ny d,de t e rt h m aei bn se o r ob fta hn ecs soe els u - c e n t r iS fe up ga er p.a rt ee plt aywrS oet a n sd oal ru dtc io onn -s
t i o an gs a ia nr se ta gb el na nt k . t a i n0 i. nm4g pge m r oLfU S P Pe r p h eR nSaa nzUdiSn Pe
2 . C a l c ut lh pae et er c eo nftthale g a be eal m e do oufp ne t r - A m i t r i Hp y t yd lr io nc eRh Slr, o e sr pi edc eit nia vl ec lo ySh ,e
o lp .-
a y p h e n a ( zC i2 1n He 2 6i nCt Ih pNe o3 rO toSifT)oa nb l e t s a r a ta epl py5 lw yLo ft ht ee ss to l u at in 5odun lo fe a Sc th a n -
t a k e n : d a sr od l u tt oai to hn i n - cl ha yr eo rm a tp ol ga(r ts eae p e h i
C h r o m a ( t 6o 2g c1r)o a)a pwt hie y
atd 0
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ceamal ip xh tiDu cer ev .et lh co e hp r o m a t
g r ua sm ia ns go l vs eyn stct oe nms i os fat mi in xg to ufc ry e-
A u = a b s o ro bft ah n Se ca em spo ll eu t i o n C l o h e e xt ah any ce
l e,t a tn dedi , e t h y( l8 a5 m: i2u n5 te: i5l )
A s = a b s o ro bft ah nSe c t ae n sdo al ru td i o n t h se o l vf er nohtn atms o va eb d o1 u5ct m R. e m to h v pe lea t e
C s = c o n c e n ot fU r aS PtPei ro p n h eR nSi an tzhie n e f r t o hmde e v e l co hp ai mnabgi er -rf d, o r2r y0
m i n u a t e n sd ,
S t a n s do al r u (td igo /n m L ) e x a mt i h pe
nl ea ut ne ds eh ro r t - w aU vlVei gl htethn:R egr t h
C y = n o m cio nn ac el n ot fp
r ae tr ip oh nei nnt ah ze i nv e
a l ou fet sh per i n cs ip po oat lbs t a fi rn toe hmdt ee ss to l u t i
S a m spo ll e u( t iu ogn / m l ) c o r r et sotp hooo
ns b
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rmi0Lc .
e D I S S O (
L 7U 1T 1I )O N A p p a r 2 :a t5 u0r sp m .
M e d i0 u . 1Nm h :y d r o ca hc li 9do ;0 r mi0 cL
A p p a 2r :a5 t0ru ps m T i m e 6: 0 m i n u t e s .
T i m 4 e 5
:m i n P r o c e d u r et op o[ tNe ond T te iEca rli e
Dn tauhsreeee -
S t a n sdo al ru td A i ko nn :oc w o nn c e n ot fr U aSPtPeir o- n c o v oe pfr ey r p n ewn ha mezu n il nt iienp jle ec at ri m eo na s d e
p h e n aR z Si niM nee d i u m f r aov m
i a n l , om o tr he a t nww oi t h d s r ha o w b u
a lem d sa d e
S a m spo ll e u t Pi ao san ps: o r to ift ohnseo l u u t in od ne rf r a o mns y i n vg ila elD. e
] t e r t hm aei mn oe uo fnp t e sr p h e n
t e st th r oa su ug iht f ai bl tlDe eri .l wu ti etM he d tioa u m z i an ned a m i t r i hp ty yd lr io nceihnsl oo lru iit ndfi ieo lnt e r e d
c o n c e n st irm aittlotia hro a on ftt h Se t a ns do al ur tdi o n p. o r t oi fto hn se so l u u t in odtnee s rit n,c o m p aw ri it a sh o n
I n s t r uc mo en nd ti at li o n s S t a n sd oal ru dht a i ov nk
i nn goc w o nn c e n to rfUa StP Pi e ro -n
M o d Ue V : p h e n aR zSa in nUdeSA Pm i t r i Hp t y yd lr io nc eRh Sil n o t hr ei d
A n a l y wt ai v c ae ll eMn ag x t hia :bm suo mra bt a n c es a m e d ia sud im r, e fc otPrer do c ie ndt uh Arese s a y .
a b o2 u5tn7 m T o l e r a n l ecstes hs a7 nN5(o % Qto) ft h le a b e l e d
a m o uo fn p te sr p h e(n Ca zz ii Hn 2e sa CnaIdmN i3 t Or Si )p t y
h y d r o c (h Cl 2o or- HH i 2C
d i3I
es N
d) i s s oi nl6v 0 me id n u t e s .
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l o g/ r
P ea tp rh os3l 2
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U n i f o r
o m
fd iot sy ua n gi(et9 s m :e te htree q u i r e -i
0 5 ) m m u n w ii t pz eher d
t uv sa sci cs iu nt c ehh ae ta c h
m e nf to rs
C o n tu enn it f owr im tri het sy pt eopc et r p h e n a 1 z i. n2me5cL o n t na oil tne sst s h at h
n ae m o ouf n t
a nt doa m i t r i hp yt y d lr io nc eh l o r i d e .
i m m gul no betu olb iee nq u i vt ao2l 5 em noLtf
A s s a y
M o b pi h l ea s e a fP ir let pae anrdrde eed g a ms is xe tod uf r e
h u mh ay np e r si em r mIut um m n.acey o n t0 a. i3 n
w a t ae cr e, t o n im ter ti hl eaa,nn m
ode l t, h a n e asc ui ld f o nM iac y ae sa sst a b i la ig zei n an tn
gi ,tdc o n t aa i n s
( 4 9 0 : 3 1 0M :a2 ak0 de s
0 j: u2 s)i tf.nm ee cn et s(ss eaS ery ys t e m u i t p
a r
b el se e r v a t i v e .
S u i t a bu inl diCtehyrr o m a t ( 6o 2g1 r) a ) .p h y
P a c k a a gnsidtno gr a g e a taP tre em sp ee rbr e va -et u r e
S t a n pd ra er pda r a t i ao nan c cD ui rswaseto e illgvyhe e td w e2 eann8 d .
q u a n ot fUi tS PyPe r p h eR nSi an mz ei tn he aa nndodill ,u t e
q u a n t i tw ai ttmi hev et lh ytaoon bot laa si on l u ht a i ov ani n g E x p i r da at t i oeen x pT i hr dea at itis n
eo o
n l ta t te hr a
3 n
k n oc wo n n c e n ot fa r a b to0 iu. om
t
8n pge m r (l S o l uP )t .i o ny e aa rf st de ar t
o fei s s fu r
e o
m am n u f a cc to uls rdt eo rr ’a sg e
T r a n 4s Jfme ogr fU SA Pm i t r i Hp y a r i d( e5 3 ,y e a r s ) .
t yd lr io nc eRh Stl oo
5 0 -v m o l ,e cit nh rgea t oi fot h le a b e l e d L a b e l i int tg os t La tt
L u mf el at /srkb i ahebiatie
ts nl o it n t e fno idr ne tdr a -
a m o ui nnm tgo,,fa m i t r i hp ty yd lr io nceth otl holera ib de el v e de n ion uj es c t i o n .
a m o ui nnm tgo,,fp e r p h ep neTarazb il Ane d te5.d. m0 oL f
S o l u Pt ai n
o 2nd 0m oL f0 . N
2 a c e at ci icsd h, a k
a ens,do n i -
c a t eod i s s to hl Ue
v S
e R Pe f e rS et na c
n e
d Dairl dwu st
i .et h
m e t h taovn oo ll ua mne
m di
, xP .i p 2e 5tm oLft h is so l u t i o n
i n t
a o1 0 0 v-o ml Lu mf el at sdrkii,l cw
u ti et
a mh i x to u
f r e P e t r o l a t u m
m e t h aa nn0do. l0N a4 c e at ic ci( d3 : t2 ov ) o l ua mn e md ,i x
t oo b t aa Si tn a n pdr ae rp da h r aa tv ii k onnngoc w o nn c e n t rD aE - F I N I T I O N
t i oonfas b o2 u0 ut g
o fU S PPe r p h eR nSpaezmri L an ne d P e t r oi sla ap ut ru immf ii ex dto ufs re em i sh oy ldi rd o c a r b o n
a b o2 u0 ut/ go fU SA Pm i t r i Hp y t yd lr io nc eRh Sl p e o mr Li .d e o b t a fi rn o pe emd t r oI tl me a uc myo .n ta as ui in t a b l e
A s s ap yr e p a r a t i o1 n0 T a tb Tltreoaat2ns s5 f0ev-ro ml uL - s t a b i l i z e r .
m e t r f lia scak ,d 1d 0 m0 oL f0 . N 2 a c e at ci icad ,n sdh at kh ee
m i x tu u n trtiehl Te a b lhe at dvs ie s i n t eAg drmadet te dh .a n Io M l P U R I T I E S
t ov o l um mi exa ,nf id l t De ri . l a u tna ec c u r ma et ea ls yu r eI dn o r g I am n p u i r c i t i e s
v o l (uV m; e Lo )ft h cel ef ai rl t qr au ta e n t i tw ai tatimhvie xl -y' R E S IOD N IU GE N I (T 2I 8O 1N )
t u ro efm e t h aa nn0do. l0N a4 c e at ci ci( d3 : t2 oo) b t aa i n S a m p 2 l eg :
A n a l y Hs ei t sa :
htSe a m ipn al no e p p e on r c eo lrp al ia tn -
s o l u (t Vim 4o Lnc )o n t a ai bn oi2un0 pt
gg o fp e r p h e n a z i n e
p e mr L a, nf d i l tt e hr r oa um ge hm bf irl tae n r .e i n du imso hv ae Br u n fs le a nm e .
A c c e pc t r ia t ne Irctvieoa l: a t iw lii tz ehe som u
i t at ni n g
C h r o m a tsoy gs r(t a se epCmehh ir co m a t ( o6 g2 r1 a) p) h yTa hc re oi d o a r ny di e lNd sM0 T . o1 fr%e s i d u e .
l i q uc ihd r o m a i t s eoqgu ri a wp ipptahe2h d5 4 d- e nt me c t oO rr g aI n m ip u c r i t i e s
a na d3 . 9 x-3m0 m-c co ml tu h mac nto n t pa ai cn ksL i1 .n g e P R O C E OD RU GR AA E NC
: I C D S
T hf el ro aw it s ea b o1 um tp L e mr i n ua tniesda , d j u s t e d S a m spo ll e u t 2i 0o g.n o0 : fP e t r oi ln 1a 0tm0u oL m fa
u n t ti h l r ee l a rt ie vt ee nt ti imfoeonpsr e r p h ea nna dz m i -n e 1 i n2 m i x to ufn re eu t r a l ic zoaeh ndow dla t Ae gr i. t a(wotns e
t r i p t ay rl aei b n eo1 ua tn1 d. 5r ,e s p e c tC ihv re lo ym. a t o g r t ha op rho ua gn hhdel tay ot ,
b o i l i n g . iS]
t h Se t a n pdr ae rp d a r aa ntrideo cn to, h r pe
de r a ek s p oa sn s e s A n a l y As id1 sdm : oLfp h e n o l p ThS ta, hntad i lt er ai tne
d i r e fc otPrer do c e td hurere el a: st it vae n dd ea vri dai stn io ot n r a p iw dil t 0y.h 1N s o d hi yu dm r o V Sx w,i idtveh
i g o r o) =u s
m o tr he a2 n. f0o r%r e p l ii cn aj te ec ta i notd nhsre,e s o l u t i o n a, g i t at toti hopenr o d uo cfats ih oapnri pne kn d p o i n= t ,
R , b e t wp ee er n p h ea nna dmz ii tnr ei i ps nt o yl lte istns he a n n o t ti hncego l co hr a inn tg h e ae l c o h o lla -y w e ra .t reo } r
4 . A c c e pc t r ia t ne c rNieaM4: T 0t 0L o f0 . 1N s o d hi yu- m a @
P r o c e d u r e i n Sj e cp q tauvar ola lt u(e ml
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o ft h Se t a n pdr ae rp da ra an ttdhi Ae o sn sp ar ye p a ir na tt oi o n 3
a
t h ce h r o m a tr oe g c tor hracedph hr ,o m a ta on mgd er aa sm -sS ,P E C TI EF S I T C S a )

u r te h re e s p o f ontrshemesa jp oe ra Ck as l . c ut lh qae u t ea n - C O L O R
t i t iy n, m go ,fp e r p h e( nC a2 z; iH n2 esi nCe Ia NT c 3ah Ob Sl e) t S t a n sdo al ru tdFie or nrc :ih cl o C r iSa d necdo b a l t o u s
t a k be ytn h fe o r m u l a : c h l o C r iS( d3 e. 8 : 1 . 2 )
S a m p1 l e 0 :g
0 . 2 51 (0 C ) / (V aV )a (r/sr )u / A n a l y Ms ei t ls h:t Se a m op nal se t eb aa mt ah n, pd o u r
5 m oL ft h lei q ui in dta co l e a r -1g 5
l a-xs 1
m
s 5
m 0 - m m
i n w h i Cics th h ce o n c e n ti nrua gpt eimro Lno ,,fU SP Pe r - t e st tu b ke e, e pt ihpne eg x e lm e a lt tue md .
p h e n aR z Si nit nh See t a np dr ae rp d a rVaai ts ti hoven o, l u m e ,A c c e pc t r ia t ne T
rc ih
eawe:a rm me ,l lt ieq d ius in do t
i n m L o ,ft h Ae s sp ar y e p a rV a;i ts ti hoven o, l ui nmm eL o,,f d a r tk h e r a
5 mn oL ft h Se t a n sdo al r u itdnai so in m i l a r
t hf ei l t tr aa t kef eo tnr h Ae s sp ar ye p a r aa ntr idyao nnr ,d
sa r e t u b te h;ce o m p ao rft ih s te ow bnoe i mn a g idnr ee -
t h re e s p oo fnH sePee e s er eg e p et aokbst a fi rn toe hm de f l e clt ae a nd gt a ia nw sh tibtaec k g r a o n tudhnpe de t, r o -
A s sp ar ye p a ar n ad tt hi Seo tn a np dr ae rp d a rr ae ts ip oe nc ,t i v e ll ya .ttuumb e ihne gl d id r e ac gt la yit nh sbe ta c k g r o u n d
C a l c ut lh qaeut ae n ti ni mt gyo ,,fa m i t r i hp t y yd lr io nc eh l o ra i ts du eca hna n gt lh eat th ei srne of l u o r e s c e n c e .
( C a o- HH 2C 3I t N)a kbe y tn h se a fm oe r m ru el aa d,a im ni g- e S P E CG I FR IA CV( I 8 4T 1Y0) .: 8 1 5 a- t6 0 0 . 8 8 0
t r i p t hy yl id nre o c ih nl so torefipa ded er p h e n a z i n e . e M E L TR IA NNOGGR T EE M P E RC Al a T/ /sU/(sR7 E4 1,3) 8: - 6 0
¢ C O N S I S T E N C Y
A p p a r a A pt eu ns e: t rf io tmtw eeidtt a eph r
o l i cs oh n e de -
s h a mp ee tdpa ll u nw ge ei rg 1h 5ig 0 ,nh ga v ai dn eg-
t a c h sat be tleileop ft h fe o l l od wi im ne gn t s hit eoi onp fs :
P e r t uIs m
s im s
Gul n
o b
e u l i n t h ce o h
n aeas na n go lf3e0 t ,h pe o io nftt h t ei ips
t r u n ct aoa td ei da m oef0t .e 3r+801 . 0 m2 m 5t ,h be a s e
o ft ht ei i ps 8 . 3+ 08 . 0m5 i m
n d i a m ea tne
t dh
r e
,
» P e r t uIs m
s imsGul no be cu ol in nf tootr hme s l e n og ft h t ei ips 1 4 .+90 4. 0m 5 m .
r e g u l oa ftt ih oFe nDc s Ao n c eb ri no il on( gsg ie ce s T hr ee m a i pn oi nr gto ift ohnce o hn aeas na n go lf9 e0 ,
B i o l o( g1 i0c 4sI 1ti )s a)s .t e r in l oe ,n p y rs o -g e n i is 2c 8 i m nh emi gah nthd,a as m a x i d im aum amet t e r
l u t io of gn l o b ud l ei rn sif vre t od hmbe l o o d t h be a os fe6 5m mT .hc eo n t af io ntr ehtree ssa trf el a t -
b o t mt eo tm c ay ll i nt dh ea rtr s1e 0+06 m im nd i a m e -
p l a o sf am da u hl tu md a o nn wo rhhs oa vb ee e n
 3 2 5
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t e ar n Nd L6 T5m im n h e i gTh ht ae . ryce o n s t or fu c tW eh diP te et r o l a t u m 8 6g 0

a tl e a 1s t. 6 (- 1 m6 m - g ma eu tg aae l) n a,dr pe r o v i d e Td om a k e 1 0 g0 0
w i wt eh l l - f wi ta tt ien rg c-, ot vi eg rh st .
S a m p Pl eet :r o l a t u m M e tl htSet e aA rl ycl oa hnoWdlh i Wt aet xo g e ot nah se tr e a m
A n a l y Ps li astc:here e q u n i ru e mdobfc eo r n t ai in an ne r s b a t th h, ea nd tdh Ceh o l e s at n es rd
t oiu lrn ,t ci lo m p l e t e
o v e bnr,i tn h g a e nma dq u a n otfti ht Sey a m tp oal e d i s s oA l vdtedhd We
. h iP te et r o la antm dui mxR,. e m o v e
t e m p e or fa 8 t+2 u2 r. a5
e n p,d o tu hr Se a m ip n ltoeon e f r to hmbe a t ah n,s d t iu rn t ti hl me i x tc uo rn eg e a l s .
o rm o or fte h ce o n t a fi inl el tri onws g,i t 6 h imn omft h e
r i mC .o ot ol2 5+ 2 . 5o v ae pr e r oi foN dL 1 T6 h ,p r o -
t e c f
t er ddo rma f Tt sw.
hob e f to hrt eee s pt ,l atc he e
c o n t ai inanwe ar tsb ea rt a th2 5+ 0 . 5I f t. h re o o m
t e m p e irs ba et l u2 or3 ew.o 5ra b o2 v6 .e a5 d , j tu s h te W h iP te e t r o l a t u m
t e m p e or ft a ht ceu or tneoe 2 5+ 0 .b 5yp l a ci tii nn g
t h we a tb ea rt h . D E F I N I T I O N
W i t hd o i su tt u tr hbseiu nr gfo aftchese u b s tu an nd ce er W h iP te et r oi sla ap t u ru immf ii ex dto ufs re em i sh oy ld ird o -
t e s pt ,l atchece o n t ao in t nh epere n e t rt ao bm l ee t, e rc a r bo ob nt sa fi rn o pe emd t r o al new d uh mo ,ol rnl ey a r l y
a n ldo wt eh r ce o un n et ti h l t ei jp u st to u ct hh tee os p d e c o l oI tr mi a zceyod n. ta a s ui in t sa tba lb ei l i z e r .
s u r fo aftc hetee ss tu b s t a taa sn pc o2e t5 - m3 f 8mr o m
t h ee d og fte h ceo n t a Ai dn je tur hs.zete rs eo t tai nn d g I M P U R I T I E S
q u i cr ke ll yet ahspeel u n tg he e r
h ,o
n ilt fd r ef eo 5rs . I n o r g I am n p i u r c i t i e s
S e c tu hrpeel u n a g en rrde, at dht eo t pa el n e t r a t ie oR nE S IO DN IU GEN I (T 2I 8O 1N )
f r o t hmsec a lMe a. tk heroerme o trr iea el sa, c s oh S a m p 2 l eg :
s p a tc he a tdth ei srne oo v e r l oa fpt p h ae
i rneogaf s A n a l y Hs ei t a
s h :t Se a m ipnal no e p pe on r c eo lrp al ia nt -
p e n e t r Wa h t ietor hnpee.e n e t r e ax tc ie2oe0n md m s , i n du imso hv ae fr l a m e .
u s aes e p a cr oa nt te ao fit nhteeerss tu b s t f oaern ac ce h A c c e pc t r ia t ne Irct vieoa l: a t iw lii tz ehe som ui t at ni n g
t r i aRl .e ta hdpe e n e t tr oat th nie eo an r0 e. 1sm t mC . a l - a c r oi d
d a o n ry di e lNd sM0 T . 0o f5r e% s i d u e .
c u l ta ht ae v e ro aft gh tee h roerme o rr eea d ia nng ds ,O r g aI n m p i uc r i t i e s
c o n d f uu rc tttrh ieatrloas t o t oa fl1 i0f t h ien d i v i d u¢ aP lR O C E OD RU GR AA E NC: I D C S
r e s ud li tf sff err to hmae v e rb aymg oe tr he a+ n3 % . S a m spo ll e u t 2i 0o g. n o:0fW h iP te et r oi ln a t u m
A c c e pc t r ia t ne Tc
r ih efa e
i: n aa lv e ro a ft ghteer i ia sl s 1 0 m0 oLfa 1 i n 2 m i x to ufn re eu t r a l ic zoaeh ndo dl
N L1 T0 .m0 am n N d M3 0T .m 0mi , n d i c aac to in ns gi s - w a t Ae gr i. t ap t e o a n he de ta otb o i l i n g .
t e nv ca ylo uf1e 0 0 - 3 0 0 . A n a l y As id1 sdm: oL fp h e n o l p ThS ta, hntadilt er ai tne
¢ A L K A L I N I T Y r a p iw dil 0ty.h 1N s o d hi yu dm r o V Sx w,i idt veh i g o r o
S a m p 3 l e5 :g a g i t at toti hopenr o d uo cfa ts ih oapnri pne kn d p o i n t
A n a l y Is ni ts r: ot dh Sue a c em ip n lta eso u i t ba eb al k e e r , n o t ti hncego l co hr a inntg h e ae l c o h o lla -y w e ra .t e
a d d 1 0 m0 oLfb o i lw ia nt gc e or v, ea rn ,p dl a oc n ae A c c e pc t r ia t ne rcNieaM4 : T 0u 0Lo f0 . 1N s o d hi yu- m
s t i r hr io nt g- pm la ait ne ta attih n be oe id lp ion igo nf t d r o xi s ir de qe u i r e d .
a ) w a t Ae fr t.5emr i na ,l lt oh wpe h a ts ose es p a rDa rt ea .w
ites a co a s s e wr oa ltsehh,pee t - S P E C TI EF S I C T S
a o f tf h se e p a rw aa ttieen dtr
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= ) w a t e a rn a,d dt dh we a s h tiotn hg ceas s s e Tr o ot lh ee . S t a n sdo al ru tdFie or nrc :ih cl o C r iSa dnewda t e r
p o o wl ae sd h ia nd1gdds r , oo fp h e n o l p ThS t, h a l e (i1 n. 6 : 3 . 4 )
< a nb do i l . S a m p1 l e 0 :9
Sj A c c e pc t r ia t ne Trc ih
esa e:o l u dt i o oen no
satc q ua i r e A n a l y Ms ei t l
s h:t Se a m op n al se t eb aa mt ah n, pd o u r
= p i cn ok l o r . 5 m oL ft h l ei q ui in dta co l e a r -1g 6 l a-xs sm1 5 m 0 - m
Pe e A c i p Iif t yh a:e d d i ot fp i oh ne n o l p Th Sit nth hat ele e s ti n b a c t e r i to elstotug bikece,a el pt ih pne eg t r o l a t u m
= ) f o Arl k a l pi r n io tdynu opc ie cnsok l oa rd,0 d. 1m oLf m e l t e d .
m e t o h ry la Tn Sg. e A c c e pc t r iat ne T rc iheawe:a rm me ,l lt ieq d ius in do t
A c c e pc t r ia t ne crN ier
oa e: od rp i c n ok li sopr r o d u c e d d. a r tk he a 5
r mn oL ft h Se t a ns do al r u itdnai so in m i l a r
e F I XO EI DL FS A, T AS N
, R DO S I N t u bte h;ce o m p ao rft ih s
te owbnoe i mn a
g idnr ee -
S a m p 1 l e 0 :g f l e cl ti eg adh gt a ia nw sh tib taec k g r a o n tudhnpe d
e t, r o
A n a l y Ds ii gs te: hs Set a m wp il t5e h0m oLf5 N s o d i u m l a t iu b m b ee ih ne gld id r e ac gt a l yit nh sbeta c k g r o
h y d r o a tx1 i0 df0 oe3 r 0 m i Sn e. p a trh a we at tel e
a yr e r , a ts u ca hna n gt lh ea t th ei srne of l u o r e s c e n c e .
a na dc i di tiw fiy t5 Nh s u l f au cr ii dc . e S P E CG I FR IA CV( I 8 4T 1Y 0) .: 8 1 5 a- t6 0 0. 8 8 0
A c c e pc t r ia t ne c d t st ee pra r a t 'e sM .E L TR IA NNOGGR
rN ieooa i: lo yrs o l mi a T EE M P E RC Al T a/ /sU](sR7 E4 ,1 3 )8 - 6 0
' C O N S I S T E N C Y
A D D I TR IE OQ NU AI LR E M E N T S A p p a r a A pt eu ns e: t rf io tmtweei t dat eph ro l i cs oh ne e d -
¢ P A C K A A GNSIDTN OGR AP G r eE s:ienwr ev le l - c l o s e d s h a mp ee tdpa ll u nw ge ei rg 1h 5ig 0,nh ga v ai dn eg-
c o n t a i n e r s . t a c h sat be tle ileop ft h fe o l l od wi im ne gn t s hit e
oi onp fs :
e L A B E L LI aNbiGtet:loi n d i tc ha ne t ea am nepdr o p o r t i o tn h ce o h n aeas na n go lf3 e0 t ,h pe o io nftt ht ei i ps
o fa n ay d ds te adb i l i z e r . t r u n ct aoa td ei da m oef0t .e 3r+801 . 0 m2 m 5t , h be a s e
o ft ht ei i ps 8 . 3+ 08 . 0m5 i m n d i a m ea tne t dr
h ,e
l e n og ft h t ei ips 1 4 .+90 4. 0m 5 m .
T hr ee m a i pn oi r n gto iftohnce o hn aeas na n go lf9 e0 ,
is 2 8im
pb e
m l ag nhhd,
a as m a x i
d im aum amet t e r
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t lr io cl a t u m t h be a os fe6 5m mT .hc eo n t af io n tr eh tree ssa tr f el a t -
b o t t mo emtceaydll i nt dh ea rrt se1+60m i0 mnd i -
D E F I N I T I O N a m e at n e Nd
r L6 T5m im n h e i gTh ht ae . ryce o n -
P r e pH ay rd er o p
P he it lr ioaclsfao tl ul m
o w s . s t r u oc fat tel ed a 1s t. 6 (- 1m6 m- g maeut g aa enla)dr e
p r o v wi id wteehdl l - f w i ta tt ien rg c-, ot vi eg rh st .
S a m p Wl he i: P te et r o l a t u m
C h o l e s t e r o l 3 0 9 A n a l y Ps li astc:here e q u n i ru emdobfc eo r n t ai in an ne r
S t e aAr ly cl o h o l 3 0 g o v e an n,bdr i tn h g a e nma dq u a n ot fPi e t t y r ot loaa t u
W h iWt ae x 8 0 g t e m p e or f8 a t2+ 2u . r5 Pe o . t
u hrPe e t r oi ln atotonu em
o rm o or fte h ce o n t a fi inl el tr
i onw
s gi
, t 6
h imn omft h e
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l o g/ r
P h
a ep nh as z o 3p 2y 5r 1i d i n

r i mC .o to ol2 5+2 . 5 o v ae pr e r oi foN dL 1 T6h ,p r o - D E F I N I T I O N

t e c f t er do rma f Tt sw.h o b e f to hrt eee s pt ,l atche e P h e n a z o Hp yy dr ri od cichnoleno trN aiiLd9nTes8 .a 0n %d
c o n t ai inanwe ar tsb ea rat th2 5 + 0 . I 5f t h.re o o m N M1 T 0 2 o.fp0 h %e n a z o hp yydrr io dc ih nl eo r i d e
t e m p e irs ba et l u2 ro3 ew.o 5ra b o2 v6 e . a5 d ,j tu hs t e ( C i H +HiC ic HN a, ls c u ol n a
t th deerd ib ea ds i s .
t e m p e or fta htceu ortneoe 2 5+ 0 .b 5yp l a ci tii nn g
t h we a tb ea rt h . I D E N T I F I C A T I O N
W i t hd o i su tt u tr hbseiu nr gfo aftc hese u b s tu an nd ce er' A .I N F R AA BR SE O D R(P1 T9 I7 OK N)
t e s pt ,l atc hece o n t ao intnhepere n e t rt ao bm lee t, ee r B .U L T R A A V BI O S L O ER(TP1T9 I7 OU N)
a n ldo wt eh r ce o un n et ti hlt ei jp u st to u ct hh tee os p M e d iS uu lm f : au cr i ndca l c o( 1hi on 3l 6 0 )
s u r fo aftc het ee ss tu b s t a ta sn pc o2 e t5 - m3 f8mr o m S a m spo ll e u t 5i g o n/: o mfP L h e n a z o Hp yy dr ri od -i n e
t h ee d og fte h ceo n t a Ai dn je tur hs.zete sr eo t tai nn gd c h l o irn iM dee d i u m
q u i cr ke ll yet ahspeel u n tg he e rh ,n
o ilt fd r ef eo 5rs . A c c e pc tr ia t ne c M
r iea e:t th rse e q u i r e m e n t s
S e c tu hrpeel u n ag e n rrde, a t dht eo t pa el n e t r a t i oC n. T hr ee t e nt ti iomofe tn h pe h e n a z o pp ey oarftki hd ei n e
f r t o hms ec a lMe a . tk hero erme o trr iea el sa, c s oh S a m spo ll eu ct io or nr e ts ot p ho oanf ttd hsSe t a ns do al ur -d
s p a tc he atdth ei sr ne oo v e r l oa fp t p h ai
erneogaf s t i o an s,o b t a i in tn heAed s s a y .
p e n e t r Wa h t ietor hnpee.e n e t re ax tc ie2oe0nmd m s , e D .I D E N T I FT IEC SA TT I S O NGC E h N e mEI iRd ceA naLtl,i f i c a t i o
u s a es e p a cr oa nt te ao fit nhteeerss tu b s ft oaernac ce h T e s tC sh , l o (r 1i 9d 1eM) e: et th rs ee q u i r e m e n t s
t r i aRl .e ta hdpe e n e t tr oat th nie eo an r0 e. 1sm t mC . a l -
c u l ta th aee v e ro aft gh teeh roerm e o rr eea d ia n ng ds , A S S A Y
c o n df uu rc tttrh ieatrloas t o t oa fl1 i0f t h ien d i v i d ue aPl R O C E D U R E
r e s ud li tf sff err to hmae v e rb aymg oe tr he a+ n3 % . S o l u At :i2 o0nm a M m m oa nc ei tiua nwm tae t e r
A c c e pc t r ia t ne Trc ih
efa ei: n aa lv e ro a ft ghteer i ia sl s S o l u Bt : iA oc n e t o n i t r i l e
N L1 T0 .m0 am n N d M3 0T .m 0 mi , n d i c aac to in ns gi s - M o b pi hl ae sS ee T:e a b7 l. e
t e nv ca ylo uf1e 0 0 - 3 0 0 .
' A L K A L I N I T Y T a b1 l e
S a m p3
l e
5 :g S o l uA
t i o n S o l u Bt i o n
A n a l y Is ni ts r
: ot dh Sue a
c e
m ip n lta e
so u i t ba eb a
l e
k e r ,
a d d 1 0 m0 oL fb o i lw ia ntgce or v, ea rn p ,dl a oc nae
0 5
s t i r hr io nt g- pm la ait ne ta attih n be oe id lp ion iog nf t
w a t Ae fr t.5emr i n a ,l lt oh wpe h a ts ose es p a rDa rt ea .w 9 5
o f tf h se e p a rw aa ttieendr ta co a s s e wr oa ltsehh,pee t - 1 5
r o l af tu ur tmw hi ettr hw 5o 0 -p m o rL t oi fbo on is l i n g
w a t ea rn a ,d d tdh we a s h tiotn hgceas s s e Tr ot lh ee. 2 8 3 0
p o o wl ae sd h ia nd1gdds r, oo fp h e n o l p ThS t, h a l e i n 3 3 0
a nb do i l .
5 5.
A c c e pc t r ia t ne T
rc ih
eas e:o l u dt io oe n ns
o at c q ua i r e
p i cn ok l o r . 4 0 9.
A C I D I T Y
D i l u eA nc et t: o n ai ntwrdai tl( ee1 r0 : 9 0 ) ic
S a m p F li e n sa:ol l u ot fit oh tnee sf to Arl k a l ii fnt iht ey , a )
a d d i ot fi
p oh ne n o l p Th Sp t hr ao ld e nu io
pc nie n
cdko l o r S t a n sdo al ru t d 0i .o 0nm :3g /o fm U S LP Ph e n a z o p yI r i -
d i nH ey d r o c Rh Sil no D ir li ud een t
A n a l y Ts ot
i sh S:e a m pa l de0 d., 1m oL fm e t oh r y a l n g e
S a m spo ll e u t 0i .o 0nm 3:g / o fm P hL e n a z o Hp yy -r i d i n
c
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A c c e pc t r ia t ne rc
N ieo
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d . r o c h il n o
D i
r l
i u
d ee n t = ]
C h r o m a t soy gs rt ae pm h i c i}
¢ F I XO EI DL FS A, T AS N ,R DO S I N
( S e
C eh r o m a t
( 6o 2g 1
S r)y a
, s p
St u
h ei ym
t a b i l i t y . ) }
=
S a m p 1l 0e : i
M o d Le C:
A n a l y Ds ii gs te: hs Se t a m wp il t5eh0m oLf5 N s o d i u m
D e t e c Ut o V
2 r8 :n0 m bo}
h y d r o a tx1 i0 df0 oe 3r 0m i nS .e p a tr haweta et e r a
l a y ea r n,a dc i di tiw f iy t5 Nh s u l f au cr ii dc . C o l u 4m .n 6: x-2 m5 m - 5 c m- ;pu am c kL i 1 n g vw

A c c e pc t r ia t ne c d t st ee pra r a t e s F. l ro awt 1e :m L / m i n
rN ieooai:lo yrs o l mi a
I n j e cv toi lo un 2m 0Le L:
A D D I TR IE OQ NU AILR E M E N T S S y s stu ei tm a b i l i t y
e P A C K A A GNSIDTN OGR AP G r eE s:ienwr ev le l - c l o s e d S a m p Sl te a:n sdo al ru td i o n
c o n t a i n e r s . S u i t a b ri le i qt u y i r e m e n t s
e L A B E L LI aN biGtet:loi n d i tc ha n m nepdr o p o r t i o nT a i l fia nc gt oNr :M2 .T0
et ea a
o fa n ay d ds te adb i l i z e r . R e l a st t i va en dd eav ri da tNi M o0n T .: 7 3 %
A n a l y s i s
S a m p lS et sa : n sdo al r ua td in oSdna m spo ll eu t i o n
C a l c ut lh pae e t er c eo nfpt aa og n e pa n has yr dy r o c h l o -
r i d( eC y ; - H H1 Ci iINn)ts h pe o r to f i Po hn e n a z o p y r i d
P h e n a z o Hp yydrr io dc ih nl eo r i dH yed r o c th al koe rn i: d e
R e s u= (l r tu / xr (s C) s /xC1u 0) 0

o r p r o &l l y
ou

as I kl e s i p e a
r e
k s p fo rn tos hm
eSe a m sp o ll eu t i o n
l s p e r
a ek s p fo rn t
os hmeSe t a n sdo al ru td i o n
H N NO S N H G s = c o n c e n ot fr
U aS P
tPh
i e
o n a z o p y r i d i n e
H y d r o c Rh Sil not r h Se
i td ae n sdo al ru td i o n
C y H + i HiC NI s 2 4 9 . 7 0 ( m g / m L )
2 , 6 - P y r i d 3i -n (e pd hi ea nmmyi o ne zo, oh )y- d, r o c h l C
ln a o ur i =dc eo ;n c e n ot fP r a
h te in oa nz o p y r i d i n e
2 , 6 - D i a m i n o - 3 - (m po hn eon hy yl da rz oo c ) ph yl ro ir di idn H
ee y d r o c ihn tl hoSerai mdspeo ll eu t i o n
[ 1 3 6 - 4 0 - 3 ] . ( m g / m L )
A c c e pc t r ia t ne c
r9 ie
8a .
: 0 % -o 1nt 0 h de2r .i be0a ds% i s
 3 2 5
P h
2 e n a z o /pO fy fri icM idoain
l noe g r a p h s U S4 P1

I M P U R I T I E S T a b2 l e
e R E S IOD N
IU GE N I (T 2I 8O 1NN
) :M0 T
. 2 % R e l a t i v eA c c e p t a n
e W A T E R - I SN USBOSLT UA BN LC EE S
R e t e n t i oC nr i t e r i a ,
S a m p 2 l eog f:P h e n a z o Hp yy dr ri od ci hn le o r i d e N a m e T i m e N M( T % )
A n a l y Ds ii ss s:to hl Sev ae m ipn 2l 0e m0 oLfw a t e h re ,a t
t ob o i l ia nngtd,h eh n e ia nta c o v ec ro en dt ao ina n e r 2 , 6 - D i a m i n o p y r i0 d. i3 n7 e 0 . 2
s t eb aa mft oh1 r h .F i l tt e hr r oa tu ag r hfe id n, e - p o r o Ps hi et ny a, z o p y r i d i n 1e . 0 0 =
s i n t e r ec dr -u gc liwba als ets
s ,hh o r ow ui gw t hah lt ye r , I n d i v ui nd su pa el c i f i e d
a n ddr a yt1 0 t5 oc o n s wt ea in gt h t . i m p u r i t y 0 . 1 0
A c c e pc t r ia t ne T c
r iheawe:e i og fth htre e s id dou ee s T o t ia ml p u r i t i e s = 2 . 0
n o et x c 0 e e. d o1 ft% h we e i og fPh ht e n a z o Hp yy -r i d i n e
d r o c h tl ao kr ei nd .e
S P E C TI EF S
I C
T S
e L o sO sN
D R Y (I 7N 3G1 )
D e l te htfeeo l l o w i n g : A n a l y Ds r
ia syt1: 0 f5 o 4rh .
A c c e pc t r ia t ne rcNieaM1
: T . 0 %
oH E AM VE YT AM LeS t,I Ih( o2 3d 1 N
) :M2 T
0p o c mo tei c i a .
J a n - 2 0 1 8 ) A D D I TR IE OQ NU AI LR E M E N T S
' O R G A I NM PI UC R I T I E S ' P A C K A
A GNSIDTN OGR AP G
r eE s:ientr iv gech ot n t a i n e r
S o l u At ,Si oo ln u Bt, M
i o
o nb pi hl ae a
s e
n D, e U SR PE F E RS ET NA C NE (
di l u e n t : D 1A 1R) D S
P r o ca e sd ei dr e icn tt he Ad es s a y . U S PPh e n a z o Hp yy dr ri od ciRhnSle o r i d e
S e n s i s t i
o vl iu tt 0yi .o 2ng5: / ema L oc fhU S PPh e n a z o -
p y r i Hd yi d n er o c Rh Sla on 2rd,i 6d -e d i a m ii nn o p y r i d i n e
D i l u e n t
S t a n sdo al ru t d 0i . o n0 :m 0 0g 5/o fm U S LP Ph e n a z o p y r i -
d i nH ey d r o c Rh Sla on 0rd.i 0dm0e 1g / o fm2 ,L6 - d i a mPi h - e n a z o Hp yyd rr io dc i Th anl beol re
n o p y ri in Ddi il nu ee n t
S a m sp o ll e u t 0i .o m 5n :g / o fmP hL e n a z o Hp yy -r i D d iE nF eI N I T I O N
d r o c h il noD r i li ud ee n t P h e n a z o Hp yy dr ri od ciThnale bolcreoitndsteNa Li9T n0 . 0 %
C h r o m a ts oy gs rt Pa e rmp o:hcaie sdcei dr e icn tt he e
d a n N d M1 T 1 0 o.ft0h l%e a b e al m e do oufpnh te n a z o p
A s se ax yc fe o ptr th Dee t e c t o r . d i nh ey d r o c (h Cl io sr« HiH1dC iel N) s.
D e t e c Ut o V2 r4 :n0 m
S y s st u ei m t a b i l i t y I D E N T I F I C A T I O N
S a m p l S ee n ss :i st io vl iu ta o dnt a n sdo al ru td i o n e A . T h Ue V
t yin S s p e c otftr hupe mh e n a z o pp ey oarft ki hd ei n
S u i t a br ie l qi ut i y r e m e n t s S a m spo ll eu ct o i or nr e ts ot
p ho oanf ttd hsSe t a ns do al ur -d
T a i lf ia nc gt oNr M :1 .Tf5 o tr h pe h e n a z o p y r i d t iio n
an s
,eo b t a i in tn heAed s s a y .
p e a Sk t, a n sdo al r u td i o n e B . T h r ee t e nt ti iomofe tn h me a jp oe roa ftk h Se a m p l e
R e l a st t i va en dd eav ri da tNi M o 3n T .: f0 o %
tr h e s o l u ct io or nr e ts otp ho aon tftd hsSe t a n s do al ur tadis o n ,
i s p h e n a z o ppeya S rk ti, a
d n i sdno ael ru td i o n o b t a i in tn heAeds s a y .
i s S i g n a l - r ta ot i-Non:Lo3 Ti0 f so tre h pe h e n a z o p y r i -
i ]
i n pe e aS ke ,n s i st io vl iu tt yi o n A S S A Y
= ) e P R O C E D U R E
3 A n a l y s i s
S o l u At :i2 o 0nm a M m m oa nc ei tiunawt mae t e r
iS S a m p lS e t sa n :sdo al ru a td in oSdna m spo ll eu t i o n
S C a l c ut lh p ae te er c eo nf2t,a6 g- ed i a m ii nnt oh pe y r iS do il nu e Bt : iA oc en t o n i t r i l e
= p o r to ifPo hn e n a z o Hp yy dr ri od cithnalekoe rn i: d e M o b pi hl ae sS ee T:ea b1 l. e
a .
2 ) R e s u= (lr tu / xr (s C) s /xC1u 0) 0 T a b1 l e
> }
r y = p e ar ek s p oo f2n ,s 6e - d i a m if nr oop my r i d i n e S o l uA
t i o n S o l u Bt i o n
t h Se a m spo ll eu t i o n % o
l s =p e a r ek s p oo f2n ,s 6e - d i a m if nr oopmy r i d i n e 9 5 , 5
t h Se t a n sdo al ru td i o n 9 5
C s = c o n c e n ot f2r ,a 6 t -i d
o in a m ii nnt oh pe y r i d i n e 5 0 5 0
S t a n sdo al ru (td imo gn / m L ) 2 0 5 0
C u = c o n c e n ot fr P ah te in oa nz o p y r i d i n e 0
H y d r o c ihn tl hoSerai mdspeo ll eu t i o n
3 3 3 0
( m g / m L )
C a l c ut lh pae e
t er c eo nfat nai yg
n de i v ui nd su pa el c i f i e d 3 5 .
i m p u irnti htpeyo r to ifPo hn e n a z o p y r i d i n e 9 5
H y d r o c th al koe rn i: d e
D i l u eA n
c e
t t: o n ai n
t wrd ai t
l( ee1 r0 : 9 0 )
R e s =u (lr tu / xt (s C) s /xC1u 0) 0 S t a n sdo al ru t d 0i .o 0nm :3g /o fmU S LP Ph e n a z o p y
d i nH ey d r o c Rh Sil noD ir li ud ee n t
r u =p e ra ek s p oo fen as ice nh d i v ui nd su pa el c i f i e Sd a m sp tloseco kl u t Ni oo m n :i n0 .a m 3l gl y/o fmp hL e n -
i m p u fr ri t
o hy
m Se a m sp o ll eu t i o n a z o p y h r iy d ir no ec fh r l oN rmL 2
iTfd0i en e lpy o w d e
r s =p e ar ek s p oofpn hs ee n a z o fp ry t or hmi ed i n e T a b li en D ti sl u p
e nr te, p aasfro el dl To rw as n. ass fu ei rt a -
S t a n sdo al ru td i o n b lae m o ouft nh p te o w tdoa es ur i t va ob l eu mf el at srk i.
C s = c o n c e n ot frU aS PtPhi eo n a z o p y r i d i n eA dD di l ue eq nu ti vt ao7 l e5on %
ft h f el a vs ko l au nm de
H y d r o c Rh Sil not r h Si
e td ae n sdo al ru td i o n s o n i fc oa1rt5me i n A .l lt oh sweo l u tt oic o not lor o o m
( m g / m L ) t e m p e rd ai t
l u
wu tir et
De i
h, l ut eovn ot l ua mn ed ,
C u = c o n c e n ot fP r ah te in oa nz o p y r i d i n e c e n t r i f u g e .
H y d r o c ihn tl hoSerai mdspeo ll eu t i o n S a m spo ll e u t Ni oo m
n :i n
e qa ul il vyt ao0l .e 0n
m 3tg / m
( m g / m L ) o fp h e n a z o hp yy drrio dc ihnnDl e io lr ufie r
dn e
t
ot hm e
A c c e pc t r ia t ne S
rc ieeaTe:a b2 l. De i s r ea gn i
ay rm dp u - S a m sp t loseco kl u t i o n
r i tpy e al ke ssts h a0 n. 0 5 % .
U S4 P1 O f f i cM ioa n
l o g/ r
P h
a ep nh ds i m 3e 2
t 5
r a
3 z i

C h r o m a t soy gs rt ae pm h i c C h r o m a t s oy gs rt Pa e rmp o:hcaie sdcei dr e icn tt he d

e
( S eC eh r o m a t
( 6o 2g1Sr)y a,s pStuhei ym
t a b i l i t y . ) A s se ax yc fe optr th De e t e c t o r .
M o d Le C: D e t e c Ut o V
2 r4 :n0 m
D e t e c Ut V o
2 r8 n :0 m F .o I rd e n t i fA i,uc s o no d e S y s stu ei m
a atedi i t a b i l i t y
a r r da ey t e i cn tt horera no gf2e 0 0 -n 6m 0. 0 S a m p l S ee n ss :
i st io vl iu ta
t yin S
o dnt a n s do al r u td i o n
C o l u 4m .n 6: x-2 m5 m- 5 c m- ;pu am c kL i1 n g S u i t a b ri le i qt u
y i r e m e n t s
F l ro awt 1e :m L / m i n T a i l fia nc gt oNr :M1 .Tf5 o tr h pe h e n a z o p y r i d i
I n j e cv toi lo un 2m 0 pe L: p e a Sk t, a n sdo al r u td i o n
S y s stu ei tm a b i l i t y R e l a st t i va en dd eav ri da tNi M o 3n T.: f0 o %
tr h e
S a m p Sl te a: ns do al r u td i o n p h e n a z o ppe ya S rk ti, ad nisdno ael ru td i o n
S u i t a b i rl ie t qy u i r e m e n t s S i g n a l - rta ot -i Non :oL3iT0 fs oetr h pe h e n a z o p y r i
T a i l fia nc gt oNr :M2 .T 0 d i np e aS ke ,n s i st io vl iu tt yi o n
R e l a st t i va en dd eav ri da tNi M o 1n T
.: 0 % A n a l y s i s
A n a l y s i s S a m p lS et s a :n s do al rua td in S
odna m spo ll eu t i o n
S a m p lS e t s a n :sdo al ru atd in S
odna m sp o ll eu t i o n C a l c ut lhFae e t ec e onfaa nigyn ed i v ui nd su pa e l c i f i e d
C a l c ut lh pae t e er c eo nftth ale g a be eal me do ouf n t i m p u i rn ti htpeyo r to ifto hnTea b lt ea tk se n :
C e e h y d r o c (h Cl io :r+ HiHidCieiINn) ts h e
p o r to f iT oa nb lt ea tk se n : R e s = u (lr tu / xr (s C) s /xC1u 0) 0

R e s =
u (lr tu / xr (s C) s /xC1u 0) 0 t u = p e a
r ek s p oofen as c ie nh d i v ui nd su pa el c i f i e d
i m p u fr ri to hymSe a m spo ll eu t i o n
t u =p e a r ek s p fo rn t os hm
e Se a m spo ll eu t i o n r s =p e ar ek s p oo fpn hs ee n a z o fp ry tor hmi ed i n e
t s =p e r a ek s p fo rn t os hm
eSe t a n sdo al r u td i o n S t a n s do al r
u td i o n
G = c o n c e n ot fr U aS PtPhi eo n a z o p y r i d i nC es = c o n c e n ot fr U aS tpPi e o nn e s ea ee
n
H y d r o c Rh Sil no t rh Sie td ae n sdo al ru td i o n H y d r o c Rh Sil no t r
h Se i td ae n sdo al r
u td i o n
( m g / m L ) ( m g / m L )
C u = n o m ic no an l c e n ot fp r ha te in oa nz o p y r i d Ci un e= n o m ic no an l c e n ot fp r ha te in oa nz o p y r i d i n
h y d r o c ihntl ho Ser ai mdspeo ll eu t i o n h y d r o c ihn tl hoSer ai mdspeo ll eu t i o n
( m g / m L )
A c c e pc tr ia t ne c
r9 ie0a .
: 0 % - 1 1 0 . 0 % A c c e pc t
r ia t ne Sc
(mg/mL)
r ieeTae:a b2 l. De i s r ea gn i ayrm dp u -
r i tpy e al ke ssts h a0 n. 0 5 % .
P E R F O TR EMS AT N
S C E
' D I S S O (
L 7U 1T 1I )O N
M e d iW ua m t e9
: r0 m
;0 L T a b2l e
A p p a 2r : a5 t0ru ps m R e l a t i v eA c c e p t a n c e
T i m 4 e 5:m i n R e t e n t i oC nr i t e r i a ,
S t a n sdo al ru t d Ui oS PnPh: e n a z o Hp yy dr ri od ci hn le o r i dN ea m e T i m e N M( %T )_
R Si n M e d i u m 2 , 6 - D i a m i n o p y r i 0 d.i 3n 7 e ? = _ (os
S a m sp o ll e u t F ii ol tnp e:o r r t oi fto h n sseo l u u
t in od ne r P h e n a z o p y r i d i n 1e . 0 0 = w a )
t e sa t ns du i t da ib ll wuy ti etM he d tioa uc m o n c e n t r a t i o n
I n d i v ui nd su pa el c i f i e d s e
a)
t h ias st i m it lota hr a
o ftt h Se t a ns do al ur td i o n . c=
i m p u r i t y 0 . 2
I n s t r uc mo en nd ti at li o n s i}
M o d Ue V : T o t ia ml p u r i t i e s = 2 . 0 3
A n a l y wt ai v c ae ll e4 n2 gn2 tmh : 2 F o ird e n t i fo in cl T
ay th. ieao snr pee r o ci em sp su r mi ot in ei sit no
t r
h e d i}
A n a l y s i s d r us ug b s ta anandrcneeo it n c l iuntd het edo t ia ml p u r i t i e s . t= o| )
»
S a m p lS e t sa n :s do al r ua td in S
odna m sp o ll eu t i o n A D D I TR IE OQ NU AILR E M E N T S a ]
C a l c ut lh qae ut ae n ot fpi h t y e n a z o hp yy drrio dc ih nl' eoP -A C K A A GNSIDTN OGR AP G r eE s:ientr iv gec ho tn t a i n e ra"sr .
r i d( eC i i - H Hi Ci I dN i)ss s ob ly uv se idUn aV
g b s o r p t i So tn oa rtce o n t r ro lo ltoeemdm p e r a t u r e .
f r t o hm Se a m spo ll eu it n ic oo n m p aw ri it sh Soe tn a n -e U SR PE F E RS ET NA C NE ( D 1A 1R D S
d a sr odl u t i o n . U S P Ph e n a z o Hp yy dr ri od ciRhnSleo r i d e
T o l e r a Nn Lc 7e T5 s( :% Q o) ft h le a b eal me do ouf n t
P e e nN aa elheay yd r o c (h Cl io ;r- HiHydCieilN
s )s
d i s s o l v e d .
e U N I F OO RFDM oI s
T UY
a NGI(ET9 S0 5 M) e
: te ht e
r e q u i r e m e n t s P h e n d i mT ea tr rt a
r a
z ti en e
I M P U R I T I E S
' O R G AI N
M PI UCR I T I E S c H S s 2 on
S o l u At ,S
i o l
n u Bt, M
i o
o nb pi hl ae a
s e
n D,
di l u e n t : : Ho
Ny a 8
P r o ca e sd ei dr e icn tt he Ad
es s a y .
S e n s i s t io vl iu tt 0yi .o 2u
n 5:g /o fmU LSP Ph e n a z o p y r i - o H OO
d i nH ey d r o c Rh Sil no D ir li ud een t
S t a n sdo al ru td 0i .o 0n m0: 1g /o fm U SLPPh e n a z o p Cy yr 2i -H- i C 4z H
N e O O e 3 4 1 . 3 6
d i nH ey d r o c Rh Sil no D ir li ud een t M o r p h 3o ,l 4i -n de i, m e t h (y 2l 5- -2t -r [paRhn-se()nR -y* ,,l R- *, ) ] -
S a m sp o ll e u t Ni oo m n :i n 0 .a m5l gl y/ o fm
p hL e n a z o p2 y, r3 -- d i h y d r o (x1 y: b1 )u ;t a n e d i o a t e
i d i hn ye d r o c fh rl oN r mL2i Td0
f ie n ep loy w d Tea rb e - d( 2 5 , 3 5 ) - 3 , 4 - D i m e t L h- y( +l )- -2t(-a1pr: th1re)a nt ye l
l e ti sn D i l u p e nr te, p aasfro el dl To rw as n. assfu ei rt a b l e [ 5 0 - 5 8 - 8 ] .
a m o ouft nh p
te o w tdoaes ur i t va ob l eu mf el at srk i. c
A dD di l ue eq nu ti vt ao6 l e0on f% t h f el a vs ko l au nm de D E F I N I T I O N
s o n i fc oa1rt5me i nA .l lt oh swe o l u tt oic oo not l
or o o mP h e n d i mT ea tr r t cra oaz n
tietnN
a eiL 9nTs8 .a 0n % N
d M T
t e m p e a r and tdi ul w
r
u tie etD ih l ut eovn ot l uC me ne t. r i - 1 0 2 o.fp0 h %e n d i mt ea tr tr ( ra aCztiie 2n- HeC i4 7H Ne O
O o ) ,
f u tg heseo l u a t indodin l t u th see u p e r wn ia t Dt ihal nu t- c a l c u ol n
at th dee rd ib ea ds i s .
e n ttoo b t 0a .i m5n g / o fmp hL e n a z o p y r i d i n e
h y d r o c h l o r i d e .
 3 2 5P 4
h e n d i m / eO ft fri acM izoa in
l no eg r a p h s U S4 P1

I D E N T I F I C A T I O N C h r o m a t soy gs rt ae pm h i c
e A .I N F R AA B
R S
E D
O R( P1 T9 I7 OK N) M o d e :
e B ,U L T R A AV B I OS LO ER(TP1T9I7 OU N) D e t e c Ft lo ari m:o e n i z a t i o n
S a m spo ll e u t1 i mo ng : /i nm m eL t h a n o l C o l u 2m 5n x-:0m . 2 5 c a-p mi lm c loa lr yut mh inen ,
-
A c c e pc t r ia t ne cM
r iea e:t th rse e q u i r e m e n t s s i dw ea lo lfw h ii s cc ho a wt ie t ad 0h . 4 -f "i ulo mmf
e C ,I D E N T I FT IEC SA T T IS O NGT aE rNt E (r 1R
a t9A e1L ), l i q up ihd aGs 1e
C a r rg ia es Hr: e l i u m
A S S A Y T e m p e r a t u r e s
' P R O C E D U R E I n j e cp to ir ot2 n:5 0
S a m spo ll e u t Ti ro an n:as nfa ec rc u r wa et ei lg yh e d C o l u 1m 4n0 :
a m o ouf5n 0tm0 og fP h e n d i mT ea tr r t tra oaz tie n e D e t e c 2t 8o 0r :
o e b e a kae nrd d,i s s io nl5 v0me oLfg l a cai ca el t i c I n j e cv toi lo un 1m. e u0 :L
a c i d . I n j e ct ty ip oSe n p:l ria tt i1 o 0, 0 : 1
A n a l y As id1 sdd: r oo fcp r y sv ti aolT l eSt tot h Se a m p l e A n a l y s i s
s o l u tai n ot ndi ,t rwa it t
0
e .h 1N p e r c h al co i rV idS
tc oa S a m p Sl ae m:spo ll eu t i o n
g r ee en nd p oP ie nr tfa.bo lra mnd ke t e r m ia nna dt i o n , [ N o t rE e t Te h nt te
i imfoeontsr h De- t hi rseooam n e r
d
m a ak nen ye c e sc os ra rr ey cE ta icm o hnoL.f0 . 1N p e r - t h Le- e r yi tsh oramo reae rb o8 u. at 5 n 9dm i n ,
c h l oa rc iiics d
e q u i vt ao3l 4e .n m1 t og
4 fp h e n d i m e t r ra e- s p e c t i v e l y . ]
z i nt ae r t (r aC tie 2 - HC i4 zH Ne O c ) . P r e f eu rs a i nbag lney l e c t ir notneig crd aet to er r , m i n
A c c e pc t r ia t ne cr9 ie
8a .: 0 % -o 1nt 0 h de2r . ib e0a ds% i s t h ae r eo aaf sl pl e ai knt sh ce h r o m a t o g r a m
C a l c ut lh pae e t er c eo nftth a Le-g ee r yi tsh orimon te hr e
I M P U R I T I E S S a m sp o ll eu tt ai ko en n :
e R E S IO DN
IU GEN I (T 2I 8O 1NN) :M0 T . 1 %
' C H L O AR NIS D
DU EL F CA hT lE o,(r 2i 2
d e
1 ) R e s u
= (l r tu / xr 1
r )0 0
S a m p 1l .e 0: g
A c c e pc t r ia t ne 0
c
r i.ea 0: 3t 5 h Se% a; m sp h l oen wo s t u = p e aa rk eo a
ft h Le- e r yi tsh or pmo e a
r k
m o cr hel o tr hi a dc eno r r e ts op 0 o . n5m d
0oLsf0 . 0 N2 0 r r = s uo mft h aer eo afts h Le- e r yi tsh or pmo e a
r k
h y d r o ca hc li do .r i c a n tdh De - t hi rse oo pm ee ar k
e C H L O AR NIS DU EL F SA uT lEf(,a 2t 2e 1 ) A c c e pc t r ia t ne c
rNieaM0
: T. 1 %
S a m p 1l .e 0: g
A c c e pc t r ia t ne 0
cr i.ea 0: t1 h%Se ;a m sp hl oen woms o r eS P E C TI EF S I T C S
s u l fta ht a
ce no r r e ts op 0 o. n1m d0oLsf0 . 0 N2s 0 u l f u r ei cM E L TR IA NNOG GRT EE M P E (R 7A4 T11U) 8R : 2E -w1i 8t 8h
a c i d . d e c o m p ob su tithtreia onbng ,
ee t wb ee e g i
n na nn id n g
e no dfm e l td io nengos et x c 3e e. d
e O P T IR CO AT LA T
S pI eOc N
Ri o
f, it ca (t 7i 8
o n1 S )
D e l te htfeeo l l o w i n g : S a m spo ll eu t 1i o0 mn0 :g / i nwm aLt e r
A c c e pc t r ia t ne +
c
r i3
eat2: o+ 3 6
eH E AM VE YT (A 2 L3 S1 N
) ;M1 T
0
p p m
( o rMt ie
-c Ji aal r - 2 0 1 8 ) e P HG o t 3) .: 0 - i4na. s0 o, l u (t1 ii no4 n0 )
= ' O R G AI N M PI UCR I T I E S e L o s
O sN
D R Y (I 7N 3G1 )
a S t a n sdo al ru td Ai ona nq: u es oo lu usct oi n o nt a i n i n gA n a l y Ds r
itsyoc: o n s wt ea in agtt1h 0t 5 .
i v
1 0 m0 g /o fm U S LPPh e n d i mT ea tr rt Rra S az ti en e A c c e pc tr ia t ne rcNieaM0: T . 5 %
a S a m spo ll e u t 1i o0 m
n0 :g /o fm P hL e n d i mT ea tr -r a z i n e
2 t r ai tn we a t e r A D D I TR IE OQ NUAILR E M E N T S
5 C h r o m a t soy gs rt ae pm h i c ¢ P A C K AA GNSIDTN OGR AP G r eE s:ientr iv gech ot n t a i n e r
3 ( S eC eh r o m a t ( 6o 2g1Tr)h a,i pn h
- CLyha ry o
e rm a t o - e U SR PE F E RS ET NA C NE D( 1A 1R) D S
ne g r a p h y . ) U S PPh e n d i mT ea tr r t Rra S
az tie n e
2 ) M o d Te L: C
= A d s o r b 0 e. n2 t5l :a- yomefcrm h r o m a ts oi lgi rc aa p h i c
g e ml i x t u r e
A p p l i v c ao tl iu 1
om n0pe L:
D e v e ls oo pl ivs ney g ns tt Ae cm e: t mo en te h, aa nn od l ,P h e n d i mT ea tr r t Car aa z pt i sen uel e
a m m o h ny i
d ru o
(mx
5 0
i :d 5e 0 : 1 )
A n a l y s i s D E F I N I T I O N
D e v et lh co e hp r o m ai nt a so u gi r t caa bhml ae m w ib te hr P h e n d i mT ea tr r t Cra az tpiesncueol ne tsNa Li9Tn 5 .a 0n %d
t h De e v e ls oo pl ivsneyg n stut n et tmi h
l seo l vf er no tn t N M1 T 0 5 o.ft0h l%e a b e al m e do oufpnh te n d i m e t
h a ms o va eb d ot uh tr e e - of ft o uh lre et nh ogs ft h e t a r t (r aC t) e2 H+ Ci z
4 7H Ne OO c ) .
p l a tR e e. m toh pvelea ft re t o hmce h a m a bi e r -r d ,r y ,
v i e u wn ds eh ro r t - w aU vlVei gl haet n, og dbt sh e r v eI D E N T I F I C A T I O N
t h le o c a ot fti oh snep o tE sx .p to hspeel att oie o d i n e o A
v a p io nar cs l o s c eh da m b e r . A n a l y Ss hi s aa :k
q u
e a n ot fCi a t y p sc uo l
n te en not ms i, -
A c c e pc t r ia t ne Yc
r ieeal :sl po owat ps p ae tta hrse a m e n a l el qy u i vt ao3l e 0mn0 tog fp h e n d i mt ea t r tr raa zt
l o c a ta ist oh nsesp o ot bs s e ur nv deUedlVri g hat n,t dh e w i t5 h0m oL fw a t Fe irl .t a e rn,t dr a n ts hff e
ei rl t tr aoat e
R rv a lo ufte h se p o ftt h Se a m sp o ll eu ct io or nr e s p o n 2 d s
0 0 s-e mp aL r Aa td3odm r .oL f1 2 .N5s o d hi yu dm r o x
t ot h a
o t
ft h Se t a ns do a
l ur td
ai n
o nnd ,o
o t hs ep rios t i d ea, ne dx t rw ai cttt hw 5o 0 -p m
o rL t oi fco hn ls o r o -
o b t a i n e d . f o rE mx .t rt ah cceto m b cih nl eo dre ox ft or iarnacm t s
e L - E r yI tS hO rM oE R 2 5 0 s-e mp L a rw ai ttt ohwr1o 5 -p m o rL t oi f0o .n N
5s
S a m sp o ll e u t Di io sn s:3o .lg0vo efP h e n d i m e t r a zh i
y dn re o ca hc li ado ,nreidvc a p ot r h cea ot m e b aiq nu ee d-
T a r t irn a2 t5me oL fs o d hi yu dm r o s ox li ud(t1ei no n o ue sx t r o a cna ts st eb aa mt ohd r y n De is ss s.to hl ev e
2 0 i )na s u i t sa eb pl ae r Aa td 2od5 rm. oLfs o d hi yu- m r e s ii nd5 ume oLfa c e t ao n aed d , 5d 0m oLfa n h y -
d r o xs io d l ue (t1 ii no2 )n s, w i ral n, adl ltohwpe h e n d i - d r oe ut sht eotr h seo l u tOi osn nt .a n dp ih neg n, d i m e
m e t r ab za its ones ee p a rD ai ts ect.ah rle d o w ae lr k, a l i n e z i nh ey d r o c chr ly os tr aiol d ul te
Fi i.z lettsehrper e c i p i t
l a y ea r n,c do l lt e hcuet p pl e a yrec re ,n t r i i ff nu eg ci -
n g , w a ws ih ta hn h y d e tr hoeaurns
,ddr ayt1 0 5 .
e s s atrooy b
, t aa ci lne lai rq u i d . A c c e pc t r ia t ne Tc
r ih
eape:h e n d i m he yt drr ao zc ih nl
t i dc er y s st aool bs t a mi en alett1d 8 9 - b1u 9
tt 3
h e ,
U S4 P1 O f f i M
c ioa n
l o g/ r
P h
a ep nh ds i m 3e 2
t r
5 a5 z i

r a nbg ee t wt eh be en g i na nn e
id no gdfm e l td io neg s S t a n sdo al ru d
tA i ko nn :oc wo nn c e n ot fr U aS tPi o n
n o et x c 2e e. d P h e n d i mT ea tr rt Rra Sazs,tii emn ielp arr el p
y aastr he ed
e B .I D E N T I FT IEC SA T
T S
I O NG
T a
E rNt E
(r 1
R
a t
9
A e1L ), S a m spo ll e
u t i o n
S a m spo ll eu t Fii ol tan ep: ro r to iftohnse o l u u t in od ne r
A S S A Y t e s t .
' P R O C E D U R E C h r o m a t soy gs rt ae pm h i c
M o b pi hl ae sD ei : s s 1o .lg 1 vo efs o d 1i -u hme p t a n e s (u Sl e-C eh r o m a t ( 6o 2g1Sr)y a,s pStuhei ym t a b i l i t y . )
f o n ia n t5 e7 m5 oL fw a t e a rd 4 ,d 0 m0 oL fm e t h a n o l M o d Le C:
a n 2d 5m oLfd i l au tc e at ci ci( d1 i 4n1 0 0 a) n,mdi x . D e t e c Ut o V
2 r1 :n0 m
A d j wu is t gt lh a caica el at ci ict doa p H o f3 . +0 0 . i1 f , C o l u 4m - n x:m1 m 5 - pc am c; kL i1 n5 g
n e c e s Ps aa str shy .r oa um ge hm bf ir l a to efn
0r .
e 4 5 - 1 m F l ro awt 1e :m L / m i n
p o sr iez a e ,n d de g a s . I n j e cv toi lo un 5m 0 ue L:
D i l u eD ni tl : au ct ee at ci ci( d1i 4n 1 0 0 m) e, t h aa nn od l , S y s stu ei m t a b i l i t y
w a t( e2 .r45 0:5: 7 . 5 ) S a m p Sl te a:n sdo al ru td i o n
I n t e sr t n aa ln sdo al ru d t 0i. o1mn g : / o fsm aLl i c y l a m iS du ei t a br iel qi ut yi r e m e n t s
i nD i l u e n t R e l a st ti va en dd eav ri da tNi M o 3n T.: f0 r% to hm r e e
S t a n sdo al ru t d 0i .o m7n :g / o fmU S LPPh e n d i m e t r a rz eip nl eii cn aj te ec t i o n s
T a r t Rr S ai ntI en t e sr t n aa ln sdo al ru d t i o n A n a l y s i s
S a m sp o ll e u t Ri e o nm :oa svc eo , m p la esp to es ls iy b l e , S a m p lS et s a : n s do al r u atd in S
odna m sp o ll eu t i o n
t h ce o n t oe fN n tL2sT0C a p s ua lnewdse ,ia gc ch u r a t e l Cy a. l c ut lh pae e t er c eo nftthale g a be eal me do ouf n t
M it xh ce o m b cio nn e t eda nnt drs a, n as n fa ec rc u r a t e l yR u e n a i mt ae rgt a( r aeC tt;e i2 - e
HC i4 7H Ned OOi sc s) o l v e d
w e i gq h u ae ndot fti ht pey o w de eq r u i, vt ao3l 5emn g t c o m p ao rft ih r se eo sn p o fnt sh mee a s jp oe ra k s
o fp h e n d i mt ea t r tr rtaaoazt 5
ei ,0
n e-v m o lL u m e t r i c o b t a fi rn t oe hmdSe a m sp o ll eu a t into dnh Se t a n d a r d
f l a sAk d. 2 d5m oL fI n t e sr t n aa lns do al ur tdai nosndo, n i - s o l u t i o n .
c a ft oe 1r 5m i nC .o o t hlse o l u tt oir o on t o em m p e r a - T o l e r a Nn Lc 7e T0 s( :% Q o) ft h le a b e al m e do ouf n t
t u r de i, l w u ti etI nh t e sr tn a a ln sdo al ru td
t oiv oon l u m e , p h e n d i mt ea tr tr ( ra aCztiie 2n- HeC ia zH Nei sOO c )
m i xa ,nf id l tte hr r oa um ge hm bf irl a to efn
0r .e 4 5 - u m d i s s o l v e d .
p o sr iez e . e U N I F OO RFDM O I S T UYA NGI(ET9 S0 5 M) e : te hte
C h r o m a t soygs rt ae pm h i c r e q u i r e m e n t s
( S eC eh r o m a t ( 6o 2g1Sr)y a,s pStuhei ytm a b i l i t y . )
M o d Le C: A D D I TR IE OQ NU AI LR E M E N T S
D e t e c Ut oV 2 r5 :n6 m ' P A C K AA GN SIDTN OGR AP G r eE s:ientr iv gec hotn t a i n e r s .
C o l u 3m .n 9:x-3 m0 m- pc am c; kL i 1 n g e U SR P E F E RS ET NA CNE (
D 1A 1R) D S
F l ro awt 1e :m L / m i n U S PPh e n d i mT ea tr rt Rra S
az ti en e
I n j e cv toi lo un 2m 0 pe L:
S y s stu ei tm a b i l i t y
S a m p Sl te a: ns do al r u td i o n
[ N o t rt e l T a rtheie vt ee nt ti imfoeonssr a l i c yal na d m i d e
e
p h e n d i mt ea tr tr arara z 0e
t .ie a5n ne1 d. 0r ,e s p e c t i v P e lhy .e ] n d ei mT ea tr r t Tar aa zbtilene et s 4
a m g

S u i t a br iel qi ut yi r e m e n t s
R e s o l u Nt L i3 oT. nb0 :e t wt e h aeen na lay nti dne t e r - D E F I N I T I O N =
n a sl t a n dp ea ark ds P h e n d i mT ea tr rt Tra aazbtilecneoetnstNa Li9T
n 0 .a 0n %d s
R e l a st ti va en dd eav ri da tNi M o 1n T.: 0 % N M1 T
1 0 o.ft0h l%e a b eal me do oufpnh te n d i m e t
f o )r a z
A n a l y s i s t a r t (
r aC t ye 2 + HC i4 7H Ne O
O c ) . '
S a m p lS e t s a n:s do al r
u atd in S
odna m sp o ll eu t i o n
C a l c ut lh pae e
t er c eo nftth ale g a be eal me do ouf n t I D E N T I F I C A T I O N e S
p h e n d i mt ea tr tr ( ra aCztjie 2n- HeC 14 7H Nei OnOt ¢h )
e A . +i
p o r to ifCo an p s tu alk ee sn : A n a l y Ss hi s aa :kq ue a n ot ffi itn yep l oy w d T ea r b lee dt s ,
n o m i ne a q ul il vyt ao3l e 0m n0 togfp h e n d i mt ea rt -r a z i
R e s = u (l Rt u /xR( sC )s /xC1u 0) 0 t r a tw ei, t5 h0m oLfw a t Fe i rl . t ae rn,t dr a n ts hff ei lr-
t r a tt oae 2 0 0 s-e mp aL r Aa td3odm r .oLf1 2 .N5s o -
R y =p e a r ek s p roa nt os i fope h e n d i m e t r a z i nd ei hu ym d r oa xn die dx et ,rw ai cttt hw 5o 0 -p m o rL t i o n s
t a r t tr oat theien t e sr nt aa ln fd ra t o
r hm
d e o fc h l o r o E xf to rrt amh cc.
e to m b cih nl eo dre ox f- o r m
S a m spo ll eu t i o n t r a ic nta s2 5 0 s-e mp L a rw ai ttt ohwr1o 5 -p m o rL t i o n s
R s =p e r a ek s p roa nt os i fope h e n d i m e t r a z i n o fe0 . N5 h y d r o ca hc li ado ,nr ei dvc a p ot rh ca e o t em -
t a r t tr oat th ieen t e sr nt aa ln fd ra to r hdm e b i nae qd u ee xo tu rso a cn a ts st e baa m tth od r y n e s s .
S t a n s do al r u td i o n D i s s to hlrev ee s ii d n5ume oL fa c e t ao n aed d,5d 0m L
C s = c o n c e n ot fr U aS PtPhi e o n d i mT ea tr rt ra az ti eno efa n h y d e tr hot eoutr sh seo l u tOi osn nt .a n dp ihn eg n, -
R Si nt h Se t a n s do al r u (
td im o gn / m L ) d i m e t r h ya dz r i on c e ch r ly os tr aioldul etFi i.z le ttsehr e
pre-
C u = n o m ic no an l c e n ot p fr ai t icimoae nt r a z i n e c i p i twa a t ews,ih ta hn h y d e tr hoeaurns ,d dr a yt1 0 5 .
t a r t ir nat th eSe a m spo ll e u (t imo gn / m L ) A c c e pc t r ia t ne Tc
r iheape:h e n d i m he ytdrr ao zc ih nl eo -
A c c e pc t r ia t ne 9c
r ie5a .: 0 % - 1 0 5 . 0 % r i dc er y s st aoo l bs t a mi en alett 1d 8 9 - b1u 9 tt 3h e ,
P E R F O TR EMS AT N S C E r a nbg ee t wt e h be en g i na nn e id n og dfm e l td io neg s
@ D I S S O (
L 7U 1T 1I )O N n o et x c 2e e. d
M e d iW ua m t e9: r0 m ;0 L e B .I D E N T I FT IEC SA TT I S O NGT aE rNt E (r 1Ra t9A e1L ),
A p p a 1r : a1 t0 ur0sp m A S S A Y
T i m 6
e 0
:m i n e¢ P R O C E D U R E
S o l u At :i0 o. n0 M 2 m5 o n o b p oa ts ai spc sh io us mp h a tM eo b pi hl ae sD ei : s s 1o .l1gvo efs o d 1i -u h me p t a n e s u l
A d j wu is 1tt N
h p o t a sh sy id ur mo t oxa ip dH e f o n ia n t
5 e7 m5 oLfw a t e a rd , 4d 0 m0 oLfm e t h a n o l
of 7 . 5 : a n 2d 5m oL fd i l au ct e at ci ic( d1 i 4n 1 0 0 a) n,mdi x .
M o b pi hl ae sA e c e: t o n ai nt Srdoi ll ueAt (i 6o 5n : F3i 5l -) . A d j wu is gtt lh a cai ca el at ci ict doa p H
o f3 . +00 . 1i f ,
t e r a, n d de g a s . n e c e s Ps aa str shy .r oa um ge hm bf irl a to efn
0r .
e 4 5 - 4 m
p o sr iez ae ,n dde g a s .
 3 2 5P 6
h e n d i m /eO ft fri aM
c izoain
l no eg r a p h s U S4 P1

D i l u eD ni tl :
au ct ee at c
i i
c( d1 i 4n 1 0 0 m) e, t h aa nn od l , A n a l y s i s
w a t( e2 .r45 0:5: 7 . 5 ) S a m p lS e t s
a n:sdo al ru atd in oSdna m spo ll eu t i o n
I n t e sr tn aa ln sdo al ru d t 0i. o1mn g : / o fsm aLl i c y l a m i Cd ae l c ut lh pae e
t er c eo nftthale g a be eal me do ouf n t
i nD i l u e n t p h e n d i mt ea tr tr (ra C a ztj ie2 nz- eH
C ia zH Ned OiO so- )
S t a n sdo al ru d t 0i .om 7n g : /o fm U S LPPh e n d i m e t r a sz oi lniv e n ec do m p aw ri it sh Soe tn a n s do al ur td i o n .
T a r t Rr aSi tnI en t e sr tn aa ln sdo al ru td i o n T o l e r a Nn Lc 7Te 0s(:%Q o)ft h le a b eal me do ouf n t
S a m sp o ll eu t Ti ro an n:as pf oe rr to iff oi nn ep l oy w d e r e pd h e n d i mt ea tr tr ( ra aCztiie zn- HeC i4 7H Nei sOO o )
T a b lfe rt oN s mL2 T0 T a b l n e to sm ,i ne a q ul il vyt ao l e n t d i s s o l v e d .
3 5m og fp h e n d i mt ea t r tr rtaaoaz
t5 ei ,0
n e
-v mo lL u - o U N I F OO RFDM OI ST UA Y NGI(ET9 S0 5 M) e : te ht e
m e t frl ia csAk .d2 d5m oLfI n t e sr t n aa lns do al ur td i o n , r e q u i r e m e n t s
a ns do n i fc oa1r t5me i nC .o o t hlseo l u tt oir o on o m
t e m p e rd ai t Ie nh, t e sr tn aa ln sdo al ru ttd oi o n A D D I TR IE OQ NU AI LR E M E N T S
l uwu tir et
v o l um mi exa ,n pda st sh r oa um ge hm bf irl a to efnr e ' P A C K A A GNSIDTN OGR AP G r eE s:ienwr ev le l - c l o s e d
0 . 4 5 p- o1 sr1 ie mz e . c o n t a i n e r s .
C h r o m a t soy gs rt ae pm h i c e U SR PE F E RS ET NA CNE (D 1A 1R) D S
( S eC eh r o m a t ( 6o 2g1Sr)y a,s pStuhei ytm a b i l i t y . ) U S PPh e n d i mT ea tr r t Rra S az tie n e
M o d Le C:
D e t e c Ut o V
2 r5 :n 6 m
C o l u 3m .n 9: x-3 m0 m- pc am c; kL i 1 n g
F l ro awt 1e :m L / m i n
I n j e cv toi lo un 2m 0 ue L: P h e n eS lu zl if n a e t e
S y s st u ei m
t a b i l i t y

2 x
S a m p Sl tea:n sdo al r u td i o n
[ N o t re e l aT rthie e vt ee nt ti imfoeonssr a l i c yal na d m i d e a ej N H' ; H , S O ,
p h e n d i mt ea tr tr arara z 0et .ie a5n ne1 d. 0r ,e s p e c t i v e l y . ] S
S u i t a br iel qi ut yi r e m e n t s
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