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Pharmaceutical Analysis—Drug Purity Determination

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 Pharmaceutical Analysis—Drug Purity Determination
Dilip Kumar Singh, Sheena Sharma, Aarzoo Thakur, Sanjay Kumar, and Saranjit Singh, National Institute of Pharmaceutical
Education and Research (NIPER), S.A.S. Nagar, Punjab, India
© 2018 Elsevier Inc. All rights reserved.

Introduction 1
Is “Determination of Purity” Akin to “Searching for Impurities”? 1
Superimposition of the Terms “Purity” and “Impurities”: The Official Perspective 1
Understanding the Terms “Absolute”, “Highest”, “Optimal”, “Relative”, and “Technique-Based” Purity 3
“Purity” Versus “Potency” 3
“Purity” in Relation to the Terms “Content” and “Assay” 4
What Happens to “Purity” When Pharmaceuticals Undergo Aging on Storage Through Their Shelf-Life? 5
Purity as a Determinant of Drug Substance and Product Quality, Efficacy and Safety 5
Purity Evaluations During Different Stages of Drug Discovery, Development and Commercialization 5
Techniques Used in Purity Determination 7
Determination of “Absolute” and “Relative” Purity 8
Determination of Absolute/Inherent Purity 8
Determination of Relative Purity 9
Case Studies Highlighting Absolute and Relative Purity Determination 10
Examples of Absolute Purity Determination 10
Summary 11
Further Reading 11

Introduction

To initiate discussion on the topic, it is very important to highlight what exactly is the meaning of the term “purity.” In context of
pharmaceuticals, the simplest meaning of “purity” is “the absence of impurities in a drug substance or a drug product”. The
definitions of “purity” given in various texts are outlined in Table 1.
The context of this text is to rightly place the meaning of “purity” with respect to quality assessment of pharmaceuticals, and
discuss its determination, including techniques and calculations involved.

Is “Determination of Purity” Akin to “Searching for Impurities”?

By definition of “purity” as given above, being “pure” means absence of impurities, contaminants and extraneous matter of any
type. Therefore, determination of purity in true sense is not the same as searching and accounting for minutia. Yet, it depends upon a
particular situation, whether concern is only in the determination of absolute purity of the material or also simultaneously on the
impurities/contaminants/extraneous matter present. For example, pharmacopoeial authorities usually are responsible for the
preparation of primary drug reference standards. At the time of sourcing of raw material, these agencies seek detailed analysis of
the material’s purity along with impurities, residual solvents, contaminants, etc. While quantitative information on the impurities/
contaminants in the established standard remains in the files of standard setting agency, the purity value is passed over to the
customers. This example is similar to the case of gold, where a karatmeter measurement is shown to the buyer as evidence of purity.
The same machine also gives composition of the alloy, when the metal is of lesser purity. This important information is normally
not shared with the customers, and is retained by the goldsmith for its record. Therefore, it suggests that the determination of purity
can be segregated from searching for minutia. However, they may also be dealt together, for which another example is the
establishment of mass balance (see later discussion).

Superimposition of the Terms “Purity” and “Impurities”: The Official Perspective

With respect to the above-stated issue, confusion exists in pharmacopoeias and even regulatory guidelines with respect to merging
of the meaning of terms “purity” and “impurities.” Rather situations exist, where “purity” has been sought to be established by the
way of testing for impurities. An example here is of Japanese Pharmacopoeia (JP), where limiting of impurities/contaminants is
done by multiple tests under the nomenclature “Purity tests.” The compendium lists 44 tests (Table 2) to define “purity” of
pharmacopoeial articles. The same pharmacopoeia also mentions the heading “Purity” in individual monographs, under which
relevant tests for impurities/contaminants are listed. Incidentally, International Conference on Harmonisation (ICH) quality

Encyclopedia of Analytical Science, 3rd Edition https://doi.org/10.1016/B978-0-12-409547-2.14551-2 1

2 Pharmaceutical Analysis—Drug Purity Determination

Table 1 Definitions of “purity”

Source Definition

Wikipedia Absence of impurity or contaminants in a substance

(https://en.wikipedia.org/wiki/Purity)
Web Dictionary
(https://www.merriam-webster.com/dictionary/purity)
Good Practices in the Manufacture and Quality Control of Drugs (Annex 2, 22nd
Report, 1969)
(http://www.who.int/medicines/services/expertcommittees/pharmprep/
20111208_QASterminologyDB.pdf)
Drug and Therapeutics Committee Training Course-Participant’s Guide (http://
www.who.int/medicines/technical_briefing/tbs/Participant-s-Guide-All-
Sessions.pdf)
Quality Assurance for Pharmaceuticals
(MDS-3: Managing Access to Medicines and Health Technologies, Chapter 19)
(http://apps.who.int/medicinedocs/documents/s19596en/s19596en.pdf )
Japanese Pharmacopoeia,17th Edition (General Notices)
The International Pharmacopoeia, 7th Edition, 2017: Note for Guidance on
Organic Impurities in Active Pharmaceutical Ingredients and Finished
Pharmaceutical Products (http://apps.who.int/phint/pdf/b/10.3.4.Note-for-
guidance-on-organic-impurities-in-active-ph_.pdf)
Food and Drug Administration, HHS (21 CFR 600.3: Definitions) (https://ntp.
niehs.nih.gov/iccvam/suppdocs/feddocs/fda/fda_21_600_3.pdf)
The quality or state of being pure
The degree to which other chemical or biological entities are present in any
substance
Drug substance: The medicine contains no contaminants.
Drug product: The medicine is not contaminated with potentially harmful
substances.
In addition to the API, most pharmaceuticals are made with ingredients added
for bulk, consistency, or color that should not contain potentially harmful
contaminants or microorganisms. The product should not have significant
quantities of other products from cross-contamination
The test to detect impurities/contaminants in drugs, and it, as well as other
requirements in each monograph, specifies the purity of the drug usually by
limiting the kind/nature and quantity of the impurities/contaminants. The
impurities/contaminants subject to the purity test are those supposed to
generate/contaminate during the manufacturing process or storage, including
hazardous agents such as heavy metals, arsenic, etc. If any foreign
substances are used or supposed to be added, it is necessary to perform tests
to detect or limit the presence of such substances
Purity is a critical attribute of active pharmaceutical ingredients (APIs) and
finished pharmaceutical products (FPPs), which potentially affects their safety
and efficacy. Therefore, API and FPP monographs in The International
Pharmacopoeia (Ph. Int.) shall contain specifications for purity which include
requirements for the control of impurities, wherever possible. To control
relevant organic impurities, individual monographs will contain a stability-
indicating test entitled “Related substances.” This test may be supplemented
by a specific test where a given impurity is not adequately controlled by the
related substances test or where there are particular reasons (for example,
safety reasons a genotoxic/mutagenic or an enantiomeric impurity) requiring
specific control
Purity means relative freedom from extraneous matter in the finished product,
whether or not harmful to the recipient or deleterious to the product. Purity
includes but is not limited to relative freedom from residual moisture or other
volatile substances and pyrogenic substances

Table 2 List of purity tests mentioned in 17th edition, Japanese Pharmacopoeia (Unnecessary test items are recommended to be omitted depending on the
nature of drug)

S. No. Purity tests S. No. Purity tests S. No. Purity tests S. No. Purity tests

1 Color 12 Carbonate 23 Chromium 34 Arsenic

2 Odor 13 Bromide 24 Bismuth 35 Free phosphoric acid
3 Clarity and/or color of solution 14 Iodide 25 Tin 36 Foreign matters
4 Acidity or alkalinity 15 Soluble halide 26 Aluminum 37 Related substances
5 Acidity 16 Thiocyanate 27 Zinc 38 Isomer
6 Alkalinity 17 Selenium 28 Cadmium 39 Optical isomer
7 Chloride 18 Cationic salts 29 Mercury 40 Multimers
8 Sulfate 19 Ammonium 30 Copper 41 Residual solvent
9 Sulfite 20 Heavy metals 31 Lead 42 Other impurities
10 Nitrate 21 Iron 32 Silver 43 Residue on evaporation
11 Nitrite 22 Manganese 33 Alkaline earth metals 44 Readily carbonizable substances

guidance viz., ICH Q2(R1) also defines the term “Purity tests,” which reads as: “to ensure that all the analytical procedures
performed allow an accurate statement of the content of impurities of an analyte, i.e., related substances test, heavy metals, residual
solvents content, etc.”
 Pharmaceutical Analysis—Drug Purity Determination 3

An interesting development has happened in recent editions of United States Pharmacopeia (USP). In earlier editions, the
monographs had “Assay” and a separate test under the name “Chromatographic purity,” which was meant to monitor impurities in
drug substances or drug products. Recently, the name of this test has been changed to “IMPURITIES,” which reflects the exact
realization that test for “Purity” is not the one that targets impurities. In most other compendia, the same test is rightly named as test
for “Related substances.” Even there exist instances where additional tests are found for specific and named impurities, separate to
tests for “IMPURITIES” or “Related substances,” wherever the latter tests are not sufficient to control them. Typical example is the
monograph on perindopril erbumine in European Pharmacopoeia (Ph. Eur.), where a Test for Impurity A and Stereochemical purity
are given separate to test for “Related substances,” which is meant to monitor seven impurities.
According to ICH guideline Q3A, the quantitative assessment of impurities in a pharmaceutical has multiple and separate roles
to play like: (i) fixing of their specifications, (ii) evaluation of whether they are within limits of specifications, (iii) identification of
impurities needing characterization or qualification, and (iv) setting up of mass balance. In no way, these processes can be said to be
measurement of “purity” of a substance of interest, if we go by the definition discussed in the previous section. Therefore, in our
opinion, the term “Purity test(s)” needs to be replaced suitably, like done by United States Pharmacopeia Convention (USPC), to
make the definition more exact, reflecting the true focus on impurities/contaminants.

Understanding the Terms “Absolute”, “Highest”, “Optimal”, “Relative”, and “Technique-Based” Purity

“Absolute” and “highest” terms have been used as synonyms, and reflect equal states of purity. These are normally employed to
describe purity of the reference standard material that: (i) is comprehensively characterized; (ii) carries an absolute value; (iii) is
used without further testing (provided stored under conditions consistent with the supplier’s recommendations), and (iv) is widely
enforced by International compendial agencies for checking the purity of chemically similar test materials. General Chapter 5.12 of
European Pharmacopoeia (Ph. Eur.) section 2: Terminology is more explicit in defining the primary reference standard: “A standard
designated or widely acknowledged as having the highest metrological qualities and whose property value is accepted without
reference to other standards of the same property or quantity, within a specified content.” As per World Health Organization
(WHO), a purity of 99.5% or higher is desirable, which is calculated on the basis of the material in its anhydrous form or free from
volatile substances. As a general requirement, reference standards need to possess high degree of purity even when they are to be
used for identification, assay and calibration purposes.
The term “optimal” purity is usually applicable to drug materials synthesized through drug discovery and different stages of
development. These are synthesized to a purity level in accordance with the intended requirements. Even at the stage of marketing of
commercial scale batches, it is usually not practical for industry to achieve “absolute” purity for bulk drug substances, and hence
quality or purity achieved at the best can be said to be “optimal”. Such materials are usually meant to meet minimum quality
standards prescribed in compendia or in-house purity specifications.
The “relative” purity is established in relation to a primary or secondary reference standard or an in-house developed working
standard. Typical techniques where relativity against standards is established for the test samples are HPLC, UPLC, etc. that are
currently used extensively for the purpose.
When no reference standard is used in HPLC or UPLC analysis and normalized area values are used to assign the purity, it is
better to employ the term “technique-based” purity. Such purity may be usually higher than the actual purity value, as the result
does not account for the minutia. The content of the latter are otherwise during the establishment of the reference standard.

“Purity” Versus “Potency”

A further question that arises is how “purity” is related to “potency”. The latter is defined as definite biological activity shown by a
definite quantity of a drug standard or even the drug substance. By simple logic, if the drug standard has 100% purity, and it shows
certain potency, the lowering of purity by 2% shall mean lowering of potency by the same percentage, provided the dose response
curve is linear.
The definitions of “potency,” as they appear in the various regulatory and non-regulatory texts, are listed in Table 3. According to
WHO, when taken as a determinant or aspect of medicine quality, “potency” is defined as the correct amount of active ingredient
present, usually between 95% and 110% of the labeled amount. Of course, this definition pertains to actives in drug products,
where such broad percentages are the norm. Hence “potency” here is correlated to “assay” and cannot be truly related to “purity.” It
has also been reported in another sense that potency of a hygroscopic compound may decrease over the period due to moisture
gain, but the “purity” determined by a technique like HPLC may tend to remain the same, as it targets the UV-absorbing analyte and
not the moisture. So it means “purity” determination is strongly governed by the technique and the method employed.
The term “potency” is also routinely defined with respect to analytical quality. In a way, it reflects “absolute purity”. It is usually
calculated by mass balance approach, wherein sum of the % content of impurities, loss on drying (LOD) (water þ volatiles), residue
on ignition (ROI), etc. is subtracted from 100%. This is correct in principle, as the drug remaining after subtraction, is mainly
responsible for eventual biological activity. In the same context, the term “potency” is also used to describe absolute purity for
primary/secondary/working reference standards that are used for assay or other quantitative purposes. Even, alternate terms are used
to describe the potency of the standards, for example, USP uses the nomenclature as “Assigned Value”, “Property Value” or
4 Pharmaceutical Analysis—Drug Purity Determination

Table 3 Definitions of “potency”

Source Definition

Wikipedia (https://en.wikipedia.org/wiki/Potency_(pharmacology))
Web dictionary (https://www.merriam-webster.com/dictionary/potency)
Drug and Therapeutics Committee Training Course-Participant’s Guide (http://
www.who.int/medicines/technical_briefing/tbs/Participant-s-Guide-All-
Sessions.pdf)
Japanese Pharmacopoeia, 17th Edition (General Notices)
Food and Drug Administration, HHS (21 CFR 600.3: Definitions) (https://ntp.
niehs.nih.gov/iccvam/suppdocs/feddocs/fda/fda_21_600_3.pdf)
ICH Q6B (https://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/
Guidelines/Quality/Q6B/Step4/Q6B_Guideline.pdf)
Glossary of Terms Used in Medicinal Chemistry (IUPAC recommendations 1998)
(https://doi.org/10.1351/pac199870051129)
Stability Versus Potency Testing: The Madness is in the Method (https://pdfs.
semanticscholar.org/f5ec/ba586c26e185c53d73c094432763401226f5.pdf)
It is a measure of drug activity expressed in terms of the amount required to
produce an effect of given intensity
The ability or capacity to achieve or bring about a particular result
The correct amount of active ingredient is present, usually between 95% and
110% of the labeled amount
The unit used for expressing the potency of the JP Drugs is recognized as the
quantity of drug. Usually it is expressed by a definite quantity of a definite
standard substance which shows a definite biological activity, and differs
according to each drug. The units are determined, in principle, by comparison
with each reference standard by means of biological methods
The specific ability or capacity of the product, as indicated by appropriate
laboratory tests or by adequately controlled clinical data obtained through the
administration of the product in the manner intended, to effect a given result
The measure of the biological activity using a suitably quantitative biological
assay (also called potency assay or bioassay), based on the attribute of the
product which is linked to the relevant biological properties
Potency is the dose of drug required to produce a specific effect of given
intensity as compared to a standard reference
Potency is defined as the concentration of the drug in a compounded
preparation. Potency tests are known as quantitative tests and are designed
to determine how much of the drug is in the sample

“Calculation Value”, Ph. Eur. employs the expression “Assigned Content”, whereas British Pharmacopoeia (BP) uses the term
“Declared Content”. The compendia recommend that when no value is highlighted on the label, the potency/purity of the reference
standard shall be assumed as 100%.
The term “potency” is even employed in defining assay or purity of antibiotics, hormones, vaccines and other biologicals, which
is determined usually through bioassays. In such case, potency is normally expressed through International units (IU) to harmonize
with globally employed terminology. However, SI units or no units can be found, on case by case basis, depending on available
biological, medical and physicochemical information.

“Purity” in Relation to the Terms “Content” and “Assay”

It may be of relevance to cite here an interesting example of the concept of “purity” and how it differentiates from the term
“content”. If a manufacturer uses a drug substance of 99.9% purity to prepare tablets containing labeled amount of 20, 40, and
60 mg, the assay for drug substance in the tablets of three strengths will only yield “content” (20, 40, and 60 mg), whereas the initial
drug purity would remain the same as 99.9% in all the three formulations. By this example, “purity” means a “true value” of the
drug added, whereas “assay” only determines the content of the drug in tablets of different strengths.
So a new question arises—what then is a real utility of “assay”? Whether it represents “purity” in some situations, and “content”
in others? Let us first take the case of drug substance. An assay done on drug substance against a primary reference standard will tend
to yield the “content”, but the same is more exactly a “relative purity”. Another issue then arises—what about “absolute or inherent
purity” of a drug substance? Can we determine the “absolute purity”, as is done in the case of gold using a karatmeter? Yes, there are
certain techniques and methods, as discussed later, which can be employed for the purpose. Such “assay” methods target the
“purity” of the drug substance, which is theoretically anticipated to be 100%. Due to this reason most compendial monographs on
drug substances provide for assay limits of 98%–102%, considering an analytical error of 2% RSD.
So what about “purity” determination of drug products? Can we only determine drug content in drug products or some (assay!)
methods can also yield “purity”, in difference to the example of tablets of three strengths discussed above? In preparing the drug
product, we take certain amount of drug substance(s) of known purity, in accordance with regulatory or in-house specifications.
This active is deliberately mixed with excipients, along with mechanical transformation like tabletting, coating, etc. to create a
delivery system with desired functionality. To quantitate the drug strength in formulation so created, it has to be quantitavely
extracted for the analysis. Accordingly, most assay methods involve some sort of extraction procedure. There is also associated issue
of content uniformity in drug products, which may vary with each unit of the dosage form. Accordingly, the approach of
pharmacopoeias is to define limits of drug that must be present in a formulation to ensure efficacy of the dosage form. The limits
are set based on therapeutic window of the drug, and usually range from 90% to 110%; 90% to 105%, or 95% to 105%. These
percentages denote “percent assay” of the drug relative to the labeled amount, and not really the “purity.” Considering these
contentions, none of the compendia practically correlates “assay” in terms of “purity” in case of the drug products.
 Pharmaceutical Analysis—Drug Purity Determination 5

We cannot still say that “purity” doesn’t apply to drug products. The latter also have to be free from impurities, degradation
products, contaminants and extraneous matter or have to be controlled to the specified limits. The impurities and degradation
products are controlled in compendial monographs, even in the case of drug products, through tests for related substances, while
the contaminants and extraneous matter are anticipated to be controlled through stringent good manufacturing practices (GMP).
Here, it may be of interest to add that USP has recently changed the stance to control separately synthetic impurities in drug
substance and degradation products in drug product monographs, unlike Ph. Eur. or BP that do not make such segregation and
provide a common list of impurities at the end of the monograph.
Usually, “purity” of drug substances is achieved through repeated crystallization or other efficient purification methods; while
the “purity” in drug products is built-in by taking highest quality (“pure”) drug substances, excipients and all other components.
This substantiates that “purity” is essentially an absolute virtue of raw materials, and not the products formulated from their use.

What Happens to “Purity” When Pharmaceuticals Undergo Aging on Storage Through Their Shelf-Life?

The change on storage of both drug substances and drug products is called “degradation” or “decomposition.” The same may be
physical and/or chemical. The physical change, unless it is due to internal and external mechanical stress (like tablet chipping,
breakage, swelling, etc.), can also result from chemical interactions among formulation components (color change, precipitation,
spotting/mottling of tablets, etc.). Physical change usually reduces the customer appeal, and if it is due to chemical interactions, it
may even be a safety concern. In difference to physical change, chemical change influences both efficacy and safety, and may result
in loss of “purity” and lowering of drug “content,” with simultaneous rise in the degradation products. Usually, in the case of drug
substances, degradation leads to loss of “purity,” while the drug degradation in products can lower drug “content” or “% assay”
value. Yet we can broadly claim that purity of the drug product has been influenced, as there is simultaneous rise in degradation
impurities, a corollary to discussion in the above section.

Purity as a Determinant of Drug Substance and Product Quality, Efficacy and Safety

A patient of a particular disease, present anywhere on the planet, deserves to receive same high quality drug product for an early
recovery. Hence the International concept of “one drug, one quality” deserves to be implemented globally. The “quality” of the drug
product is directly linked to the purity of the drug substance, and quality of excipients and the packaging material. It also depends
heavily upon compliance to conditions of storage during distribution and use. ICH Q6A defines the “quality” as the suitability of
either a drug substance or drug product for its intended use. This term includes three major attributes, viz., identity, strength, and
purity. The “purity” in true sense provides assurance of the quality of the drug substance and drug product, both at release and
during shelf life, as discussed in the previous section.
The linkage of “purity” to “efficacy” is direct. The use of low purity drug substance raw material; its intentional or even
unintentional use in lesser quantity during the product manufacture, and/or its decay during transport, distribution and storage,
may result in lowering of assay or potency of the active drug in the pharmaceutical product. For example, stereoisomeric SS form of
ethambutol is used in formulations to obtain the desired “efficacy.” As the RS form is inactive, therefore, it is controlled as an
impurity in the SS form of active drug. Against these, the stereochemical RR form of drug is toxic and hence is a safety concern.
Therefore, it must be absent in the drug substance or even the drug product. Similarly, degradation products generated from the drug
in the drug product, or interaction products that can form due to interaction of drug with excipients and packaging material, may
also prove to be of toxic nature, leaving influence on safety of the product.
The challenge to quality, efficacy and safety, owing to sacrificed purity of the drug used in dosage forms, is viewed seriously by
International regulatory. The agencies have forced the manufacturers in multiple instances to recall the products from the market
due to purity issues. An example is recall of Pfizer’s nelfinavir mesylate formulation owing to conversion of mesylate to ethyl
methane sulfonate, a genotoxic impurity, which forms on interaction with residual ethanol, used routinely for cleaning manufactur-
ing surfaces. Lately, the United States Food and Drug Administration (USFDA) has alerted health care professionals and patients of
voluntary recall of plenty of drug products containing valsartan, used to treat high blood pressure and heart failure. This recall was
forced due to detection of N-nitrosodimethylamine (NDMA), an impurity that is classified as a probable human carcinogen (a
substance that could cause cancer). There had been similar concern earlier with respect to low purity of heparin, which resulted in
multiple deaths in the United States. There are many other recent examples, as listed in Table 4.

Purity Evaluations During Different Stages of Drug Discovery, Development and Commercialization

As discussed earlier, purity assessment of drug candidates is done repeatedly, albeit to different specifications, during the journey of
discovery, development and marketing. There is no compound synthesized in target discovery (identification and validation) stage
of early drug discovery, and hence no purity determination comes into the picture. In the synthesis and screening stage, where focus
is on identification of actives and confirmation of hits, emphasis is on high throughput screening (HTS) based assays and, therefore,
high compound purity is not the focus. The target purity generally lies between 85% and 95%, based on internal policy of a
6 Pharmaceutical Analysis—Drug Purity Determination

Table 4 Additional examples of recalls of drug products by USFDA

Recall initiation
Product Volume Declared reason Class Recalling firm date

Allergy Relief Diphenhydramine HCl 369,012 cartons This product is being recalled due to low III P & L Development, 17 April 2017
25 mg, Antihistamine, Dye-Free, out of specification assay results at the LLC
24 Softgels 9 month time point
NitroGlycerin (1 mg/5 mL) 1 mg in 5% 320 syringes Subpotent drug: Found to be below the I Advanced Pharma 9 June 2017
Dextrose Inj, USP QS 5 mL (200 mg per specification for labeled assay Inc.
mL), 5 mL Sterile single dose syringe,
packaged in 8  5 (Forty) syringes per
box
Decitabine for Injection, 50 mg per vial 3222 vials Failed impurities/degradation III InvaGen 5 March 2018
specifications: Failure to water content Pharmaceuticals,
and impurity Inc.
Triamcinolone Acetonide Lotion, USP 4128 bottles Failed impurities/degradation III Akorn, Inc. 9 March 2018
0.1%, 60 mL bottle specifications: High out of specification
results for an impurity.
Chlorhexidine Gluconate Oral Rinse, 56,1800 unit Subpotent drug: Product crystallization III Akorn, Inc. 29 March
0.12%, 15 mL unit dose cups packaged dose cups with accompanying low out of 2018
in 100-count (10  10) cups per case specification results for chlorhexidine
pack assay
OraVerse (Phentolamine Mesylate) 8509 boxes Failed impurities/degradation: This recall III Septodont Inc. 13 April 2018
Injection, 0.4 mg/1.7 mL, packaged in a has been initiated due to an out-of-
box of 10 cartridges of 0.4 mg/1.7 mL specification (OOS) result that was
each obtained for related substance
(Phentolamide), a known degradation
product impurity at the 15 month
stability test point
Fluocinolone acetonide topical solution, 27,803 bottles Failed impurities and degradation II Teva 5 July 2018
USP, 0.01%, 60 mL bottle specifications and subpotent drug: Out- Pharmaceuticals
of-specification (OOS) test results for USA
below assay and above specification for
degradants
Valsartan, USP, 40 mg Tablets, 30-count – cGMP Deviations: Carcinogen impurity II Prinston 17 July 2018
bottle detected in API used to manufacture Pharmaceutical
drug product Inc.
Valsartan and hydrochlorothiazide (HCTZ) 56,603 bottles cGMP Deviations: Carcinogen impurity II Teva 17 July 2018
tablets, USP 80 mg/12.5 mg tablets, detected in API used to manufacture Pharmaceuticals
90 count-count bottle drug product United States
Diltiazem HCl Extended-Release Capsules, 11,799 bottles Failed impurities/degradation III Mylan 10 September
USP 120 mg, 100-count bottle specifications: Out of specification test Pharmaceuticals 2018
results obtained during routine stability Inc., United States
testing for related compound
Montelukast Sodium Tablets, 10 mg, 98,016 bottles Discoloration: A complaint was received II Hetero Labs Limited, 26 September
30-count bottles from a pharmacist for the presence of India 2018
blue specks on tablets
Pantoprazole Sodium Delayed-Release 158,466 bottles Discoloration: Presence of dark II Jubilant Generics 9 October
Tablets, USP, 40 mg, 90-count bottle discoloration or brown spots on the Limited, India 2018
edges of the tablets

company. This level of purity is considered appropriate for in-house registration of the synthesized compounds. Usually, purity at
this stage is determined using a generic analytical method, mainly HPLC/UPLC with UV detection. Owing to large number of
synthesized compounds, the new focus is “time is money,” therefore, open access fast purity assessment techniques are making
inroads in process chemistry laboratories, where chemists themselves carry out analysis on reaction mixtures with minimum
training. The assessment of enantiomeric purity of the chiral compounds is also very useful at this stage.
The HTS leads to short listing of candidates that can be taken forward for preclinical studies and exploration of drugability. This
is a part of the process for identification and selection of the optimized chemical lead. At the stage of identification, purity target is
improved to 95%–98%, with no single impurity at a level of >1%. Owing to smaller number of compounds, the validated generic
methods among those pre-developed for polar, mid polar or non-polar compounds (also called global methods) are employed for
 Pharmaceutical Analysis—Drug Purity Determination 7

the purity assessment. To ensure data consistency, same global methods are employed across all different sites. All those candidate
molecules that show low purity are subjected to re-purification; otherwise the compound is dropped to save time. The available
analytical data are collated for chemical candidate registration purpose.
Once lead candidates are recognized, further optimization of selected lead synthesis is done in accordance with in-house
specifications of purity and quality, which are fixed based on regulatory insisted reporting and identification thresholds of
impurity(ies). The target purity at this stage is further improved to fall between 98.0% and 99.0%, with no single impurity at a
level of >0.5%.
As discussed above, till this time of discovery stage, generic chromatographic methods yielding % purity are relied upon for
candidate optimization. The purity assessment is mainly done without the use of any primary/secondary/working reference
standards. However, for sending the “key compound” for in vitro and in vivo toxicity studies, it is a trend in industry these days
to estimate “absolute” purity through applicable techniques (differential scanning calorimetry (DSC), quantitative nuclear magnetic
resonance (qNMR), etc.) as well as the “mass balance” approach. Further, as the discovery process moves into early development,
and the compounds that show favorable efficacy along with optimum toxicity have been identified, the latter are synthesized in
scaled-up quantities based on the pharmacological action and dose assignment and/or amounts required for toxicity formulation
development, stability evaluation and acute toxicity studies. Another objective is to evaluate feasibility of GMP manufacture. At this
stage, a primary purity determination method is developed using quality by design (QbD) approach and is duly validated as per
regulatory guidelines. Purity monitoring is done on pre to pre-final (N-2), pre-final (N-1) and final step API, to keep a tab on
carryover or generation of side products during late stages of synthesis, which can influence the quality of GMP material. The purity
requirement remains the same at >98% with individual impurity limit of <0.5%. Stress stability studies are also made part of this
early development stage, which helps in the development of stability-indicating methods that are used for the purity evaluation of
accelerated and long-term stability testing samples. Simultaneous to separation and identification of minutiae, their thorough
structural characterization is done using isolation and spectral analysis, or through use of sophisticated hyphenated techniques.
Once the structure is available, the prediction of their physicochemical properties and potential of toxicity, including genotoxicity,
becomes possible, for which help is taken of “in silico” software like DEREK, TOPKAT, ADMET Predictor™, etc.
In the next stages of development, the control of impurities and degradation products becomes more stringent and gets aligned
to ICH Q3 series of guidances and even ICH M7(R1) for setting of release and shelf-life specifications. Although, these guidelines are
applicable only to registration batches of drug substances and drug products, but prior adoption increases the self-assurance of
stakeholders during dossier submission to the regulatory agencies, because of strict scrutiny these days for the assurance of quality,
efficacy and safety. The products, post approval, are marketed with these initially set specifications, which are improved based on
experience gained over the period. In case of generic drugs and products, the purity assessment is usually guided by comparison with
innovator and/or compendial standards, whenever available.

Techniques Used in Purity Determination

In practical sense, any quantitative analytical technique and tool can be employed for determination of “purity.” The selection of
technique depends upon the purpose and stage of drug discovery and development. Usually, in initial high throughput discovery
environment, the future fate of molecules is unknown, so primary emphasis is on obtaining “technique-based” purity values using
HTS techniques. At this stage, the quantity of analyte is mostly small; the intention is not to establish absolute or the relative purity,
and even no efforts are made to prepare their reference standards. So purity determination is mostly done through area normal-
ization values given by HPLC or any other chromatographic technique. However, current emphasis is on the use of dual- or multi-
detection systems, viz., LC-UV-ELSD (Evaporative Light Scattering Detector), LC-UV-CAD (Charged Aerosol Detector),
LC-UV-CLND (Chemiluminescent Nitrogen Detector), LC-UV-MS, LC-UV-ELSD-MS, etc. Owing to better speed of analysis, the
trend is to employ UPLC against HPLC along with the mentioned detector combinations. Overall, UPLC-UV-MS is the most
popular technique, which is currently in widespread use for the purpose of high-throughput purity assessment in discovery stage.
Along with the above, one of the major interest of pharmaceutical companies within discovery stage is to estimate chiral purity of
the synthesized key compounds. Again, being an early stage, emphasis usually is given to technique-based chiral purity. Historically,
this has been done using chiral HPLC stationary phases with UV detection. Alternatively, capillary electrophoresis (CE) has also
been used as a substitute to HPLC, due its advantage to separate chiral compounds through employment of micellar electrokinetic
chromatography (MEKC) and capillary electrochromatography (CEC) modes. Over the years, modern chiroptical techniques like
optical rotatory dispersion (ORD) and circular dichroism (CD) have been inducted for the purpose, as they are more accurate and
can be easily coupled with HPLC, CE, etc. In more recent times, reliance is being made on supercritical fluid chromatography (SFC),
which comes with an advantage of better peak resolution (at desired impurity levels) due to increased diffusivity of SFC eluents.
In the next, early-development stage, an in-house “standard” is developed for various compounds selected to be taken forward.
The purpose of such standard is manifold, viz., confirmation of identity, determination of purity, assay, etc. The standard, which is
obtained through multiple purification of the candidate material, is subjected to absolute purity determination by “mass balance”
approach, using analytical techniques like RP-HPLC, ion chromatography, gas chromatography (GC), thermogravimetric analysis
(TGA), Karl Fischer titration, inductive coupled plasma-mass spectrometry (ICP-MS), etc. The absolute purity value is even verified
by independent techniques like DSC and NMR.
8 Pharmaceutical Analysis—Drug Purity Determination

At this stage, defining chiral purity is a regulatory requirement, therefore, other than the above discussed chiral purity techniques,
DSC and NMR are included for the purpose.
The availability of the standard allows “relative” purity monitoring and quantitative calculation at all later stages, including
scale-up of the candidate compound. This is done by simply using HPLC equipped with a suitable detector. Of course, the LC
method ought to be developed employing the QbD approach and shall be duly validated.
The same techniques are even used for purity determination during development of generic drugs. At this late stage, the primary/
secondary reference standards for “relative” purity determination of compendial drugs are usually available from pharmacopoeial
agencies, so in-house development of primary standards is avoided, except the working standards.

Determination of “Absolute” and “Relative” Purity

“Absolute” purity is usually determined in situations where inherent purity of the drug substances is of interest. Examples include
assigning of purity to reference substances, calculation of dosages used for toxicological testing, etc. In contrast, “relative” purity
concept applies to all methods that are based on comparison with reference substances of known purity. The determination of these
two kinds of purity is discussed in more details below.

Determination of Absolute/Inherent Purity

According to International Union of Pure and Applied Chemistry (IUPAC), the determination of absolute/inherent purity is
possible only by analytical methods that yield results dependent upon the evaluation of concentration (amount) using some
fundamental physical constant(s), and/or universal quantity(ies) associated with the material under test. For example, a unique
chemical reaction associated with a functional group (e.g., titration); solubility (phase solubility analysis) and melting point are
among a few important intrinsic properties of the drug substances, which have been used routinely to determine “absolute” purity.
More modern tools used for the purpose include DSC and qNMR. Additionally, good reliance is made on “mass balance” approach.
Titrations (acid-base, oxidative, precipitation, etc.) are classical methods, which are conventionally used in majority of phar-
macopoeias for the “assay” of drug substance. These yield absolute purity only when the drug substance is in highly pure form
(100%), and is almost free from all type of related impurities and contaminants, in particular, containing the same functional
group. Therefore, they are normally used for establishing purity of the candidate reference material, when it is under the certification
process. The advantages of the technique include saving of time and labour, and high precision.
Solubility as a critical intensive property of any drug substance gives only crude assessment of the drug substance purity. The
constancy of solubility is only indicative that the material is pure. Instead, phase-solubility analysis has been used historically in the
application of precise solubility measurements, and by that virtue it had been exploited for absolute purity determination for drug
substances. This analysis applies to all species of compounds that are crystalline solids and the ones that form stable solutions, and
is not readily applicable to compounds that form solid solutions with impurities. Purity determination by this technique provides a
totally independent assessment and does not require a fully characterized reference standard or identity of impurities. This
technique in real sense exploits the differences in solubility of the targeted substance and its impurities, and mostly solubility is
not known, especially in the case of the latter. Even there are several other associated limitations of this technique like: (a) liquid
samples are poorly suited to the method; (b) acids, bases, and their salts that may be involved in either general acid-base
equilibrium or irreversible solvolysis reactions with a solvent may not be analyzed in that solvent, and (c) solids that exist in
different polymorphic form and/or mixtures of enantiomers are not suitable to the method, etc. Owing to these issues, this
technique is better used now-a-days for purification purposes than for “absolute” purity determination.
Melting point determination and DSC techniques depend upon thermodynamic behaviour of the drug substance. A sharp
melting point of a substance is indicative of good purity, but does not give an “absolute” quantitative assessment. Hence DSC is
more popular, as it allows more precise quantitative value. In case of DSC, the purity is determined indirectly using modified Van’t
Hoff equation, which relates amount of impurities to melting point depression:
RTo X 1
Ts ¼ To  
DHf F
where Ts ¼ observed sample temperature; To ¼ fusion temperature of main components; R ¼ gas constant; X ¼ molar fraction of
impurities; Hf ¼ fusion heat of main components, and F ¼ fusion fraction of temperature Ts. In this equation, Ts is measured as the
F function. If Ts is plotted against 1/F, X is obtained from the slope.
The limitation of thermal techniques is that the sum of impurities must be <2% (or drug substance purity should be >98%),
and impurities must have a eutectic behaviour (i.e., solubles in the liquid phase, and insolubles in the solid phase).
In recent times, qNMR has emerged as a popular and more acceptable tool for the determination of absolute purity. It is
considered to be a better alternative to all other classical analytical methods discussed above. The fundamental concept of qNMR is
that the signal intensity in the NMR spectrum for a specific proton in a drug substance is directly proportional to the number of
nuclei responsible for that particular resonance. The only requirement is availability of a well-resolved assignable and integrable
signal in the spectrum. Any commercially available pure compound can be used as an internal calibrant, provided it is soluble in the
 Pharmaceutical Analysis—Drug Purity Determination 9

same solvent and has at least one NMR resonance separate from those of the analyte being quantified. These days, qNMR has
advanced to a great extent by introduction of the internal calibrant pulse through software only, i.e., electronic referencing technique
(The Electronic REference To access In vivo Concentrations (ERTIC) method, Amplitude-corrected Referencing Through Signal
Injection (ARTSI) method, etc.). The equation for calculation of purity by qNMR method is given as:
nIC  Intt  MWt  mIC
Purity ð%Þ ¼  PIC
nt  IntIC  MWIC  ms
where MWt ¼ molecular weight of the target analyte; MWIC ¼ molecular weight of the internal calibrant; mS ¼ weight of the
sample; mIC ¼ weight of the internal calibrant; Intt ¼ area (integral) of the target analyte resonance signal being used for purity
estimation; IntIC ¼ Area (integral) of the internal calibrant resonance being used for purity estimation; nt ¼ number of protons of
the target analyte that give rise to Intt; nIC ¼ number of protons that give rise to IntIC, and PIC ¼ purity of the internal calibrant, as
percent value.
The mass balance approach is further added in the testing protocol to add to the confidence in the determined absolute purity
values. It is rather considered to be a “must-do” experimentation along with the orthogonal technique(s) employed. It requires
precise accounting for all types of impurities, residual solvents, water, and other minutiae, and their sum is subtracted from 100. The
following is the governing relationship:

Purity ð%Þ ¼ 100%  ½% impurities þ % water þ % residual solvents þ % other

where % impurities is the total amount of related substances, chiral impurities, degradation products, etc., typically determined by
chromatography and % other is the total amount of inorganic components like elemental impurities determined by modern
instrumental techniques, viz., ICP-MS or inductively coupled plasma-optical emission spectrometry (ICP-OES). Additionally, %
other consists of the amount of salt/counter ions that are measured by ion chromatography. In absence of these techniques, the
content of inorganic impurities can be determined by residue on ignition/sulphated ash tests. The amount of each component
subtracted in the above equation is expressed on a weight percent basis.
The impurity/degradation-product standards can be used to establish a mass balance more accurately, as their availability helps
in the quantitative determination through corrected response factors. If the determination of content of impurities has been based
on chromatographic area percent values, the following equation should be used to more accurately reflect purity, provided that the
level of related impurities is significant (>1%):

100%  % impurities 100%  ½% water þ % residual solvents þ % others

Purity ð%Þ ¼ 100%  
100 100

The purity determined through the above given equations is also called an “experimental purity,” because it is calculated against
experimental data.
The purity assessed on theoretical basis is called “theoretical purity”. The theoretical purity is the calculated amount of the base
form of active in drug substance that may be available in commerce as hydrate, solvate or the salt form.
MWBase form
Theoretical Purity ð%Þ ¼ 100% 
MWDrug substance

where, MWBase form is the molecular weight of the active in base form, MWDrug substance is the molecular weight of hydrate, solvate or
the salt form of the active.

Determination of Relative Purity

In contrast to absolute purity, “relative purity” means purity that is established against a reference standard of same material.
Generally, it can be determined by two ways. The first is an “interpolation method”, wherein purity of the test analyte is determined
by interpolating its signal/response from a calibration plot, which is established by using a reference standard. In this case, the
relative purity is determined through the following equation:
ST CRS
Purity ð%Þ ¼   correction factor  RSPurity ð%Þ
SRS CT
where, ST ¼ signal of test sample; SRS ¼ signal of reference standard; CRS ¼ concentration of reference standard; CT ¼ concentration
of test sample, and RSPurity ¼ % purity of reference standard. The correction factor in this relationship means molecular weight
correction, if the analyte is in the form of hydrate, solvate, salt, etc. (see Case Study V later in the text).
The second approach is the “comparative method,” in which signal or response of the test analyte is compared to the one
produced by reference standard of similar amount or concentration. The above equation becomes simplified in this case. Overall,
the sample preparation methodology for the test sample and the reference standard has to be the same, so as to avoid ingress of error
due to method difference.
10 Pharmaceutical Analysis—Drug Purity Determination

Case Studies Highlighting Absolute and Relative Purity Determination

Examples of Absolute Purity Determination
Case Study I: The drug substance is a salt but not a hydrate or solvate (Ketamine hydrochloride).
MW of Ketamine hydrochloride ¼ 274.19 g/mol
MW of Ketamine (free base) ¼ 237.73 g/mol
Ketamine hydrochloride sample characterization results:
Related compound A: 0.5% w/w, Water: 0.7% w/w, Residual solvents: 0.8% w/w, Chloride: 12.9% w/w, Residue on ignition (ash):
0.2% w/w

Purity ð%Þ ¼ 100%  ½0:5% þ 0:7% þ 0:8% þ 12:9% þ 0:2% ¼ 84:9%

Case Study II: The drug substance is a salt and hydrate (Lincomycin monohydrochloride monohydrate).
MW of Lincomycin monohydrochloride monohydrate ¼ 461.01 g/mol
MW of Lincomycin (anhydrous free base) ¼ 406.55 g/mol
Lincomycin monohydrochloride monohydrate sample characterization results:
Related compound A: 0.4% w/w, Related compound B: 0.3% w/w, Water: 5.5% w/w, Residual solvents: 0.6% w/w, Chloride: 6.8%
w/w, Residue on ignition (ash): 0.3% w/w

Purity ð%Þ ¼ 100%  ½0:4% þ 0:3% þ 5:5% þ 0:6% þ 6:8% þ 0:3% ¼ 86:1%

Case Study III: Purity calculation for the “Key Compound X” (not a hydrate/solvate/salt) at discovery stage, when reference
standards of impurities are not available.
MW of “Key Compound X”: 385.23 g/mol
“Key Compound X” sample characterization results:
HPLC purity: 99.3%, Total related impurities: 0.7% (using area normalization method), Water: 3.5% w/w, Residual solvents:
2.2% w/w, Residue on ignition (ash): 0.6% w/w

100%  0:7% 100%  ½3:5% þ 2:2% þ 0:6%

Purity ð%Þ ¼ 100%   ¼ 93:04%
100 100

Case Study IV: Absolute purity estimation of artesunate sodium using 1H qNMR.
Targeted analyte ¼ Artesunate sodium
Internal calibrant ¼ Caffeine
Deuterated solvent ¼ Deuterated methanol (CD3OD)
Molecular weight of artesunate sodium (MWt) ¼ 406.40 g/mol
Molecular weight of caffeine (MWIC) ¼ 194.19 g/mol
Weight of the sample (mS) ¼ 4.06 mg
Weight of the internal calibrant (mIC) ¼ 1.94 mg
Area (integral) of the artesunate sodium resonance signal being used for purity estimation (Intt) ¼ 1
Area (integral) of the caffeine resonance being used for purity estimation (IntIC) ¼ 3.00
Number of protons of the target analyte that give rise to Intt (nt) ¼ 0.99
Number of protons of internal calibrant that give rise to IntIC (nIC) ¼ 3
Purity of the internal calibrant (PIC) ¼ 99.90%
1
H NMR spectrum of mixture of artesunate sodium and internal calibrant (caffeine) is shown in Fig. 1.
3  0:99  406:40  1:94
Purity ð%Þ ¼  99:90 ¼ 98:90%
1  3:00  194  4:06

An example of relative purity determination

Case Study V: Purity estimation for amlodipine besilate as “amlodipine” using RP-HPLC method.
Standard solution preparation (0.05% w/v): Weighed 25 mg of the amlodipine besilate reference standard (RS) into 50 mL
volumetric flask, dissolved and the volume was made up with the mobile phase. 2.0 mL of this solution was diluted further to
20 mL in a volumetric flask with the mobile phase.
Test solution preparation (0.05% w/v): Weighed 25 mg of the amlodipine besilate into 50 mL volumetric flask, dissolved and the
volume was made up with the mobile phase. 2.0 mL of this solution was added to a 20 mL volumetric flask and the volume was
made up with the mobile phase.
Average area of amlodipine in test solution (ST) ¼ 1214301
Average area of amlodipine in reference solution (SRS) ¼ 1215345
Actual weight of amlodipine besilate primary reference standard for standard solution preparation (WRS) ¼ 25.23 mg
Actual weight of amlodipine besilate sample for test sample preparation (WT) ¼ 25.13 mg
 Pharmaceutical Analysis—Drug Purity Determination 11

(A) (B) 7 1a
4a 4b
3b
H
7 N
H H
8a 8
3a H H 6
H
4
N
6 8b
3 H 1
O 5 8
H N 2
2 1 O 9 9a 9 5 O
15 10
O H 9b 4 3
14 H 9a
H H-1a signal N
O 11 O
13
12
Na
of caffeine
16 17 C O 3a
O 19
O
H C
18

H-14 signal of
artesunate
sodium

Fig. 1 1H NMR spectra of artesunate sodium (A) and caffeine (B). Methine proton (position H-14) of artesunate sodium at d 5.51 ppm was used as purity
calculation signal. Whereas a singlet arising due to methyl proton of caffeine at d 3.51 ppm corresponding to position H-1a was selected for purity estimation.

% Purity of amlodipine besilate reference standard on “as is” basis (RSPurity) ¼ 99.3%
Molecular weight of amlodipine ¼ 408.9 g/mol
Molecular weight of amlodipine besilate ¼ 567.1 g/mol
ST WRS 2 ml 50 ml 20 ml 408:9
Purity ð%Þ ¼       RSPurity ð%Þ
SRS 50 ml 20 ml WT 2 ml 567:1
1214301 25:23 mg 2 ml 50 ml 20 ml 408:9
Purity ð%Þ ¼       99:3% ¼ 71:82%
1215345 50 ml 20 ml 25:13 mg 2 ml 567:1

Summary

In this chapter, true meaning of the term “purity” is explained and critically discussed how it is different from searching for
impurities. The chapter also provides understanding of differentiation of “purity” from other routinely used terms like potency,
assay, content, bioassay, etc. The roles of “absolute” purity, “relative” purity and “technique-based” purity are discussed, including
their advantages during different stages of drug discovery and development. The techniques employed for determination of purity
are outlined. Selected case studies are also cited, highlighting calculations involved in determination of “absolute” and “relative”
purity in variety of situations.

Further Reading
Grady, L. T.; Hays, S. E.; King, R. H.; Klein, H. R.; Mader, W. J.; Wyatt, D. K.; Zimmerer, R. O., Jr. Drug Purity Profiles. J. Pharm. Sci. 1973, 62, 456–464.
Rompay, J. V. Purity Determination and Evaluation of New Drug Substances. J. Pharm. Biomed. Anal. 1986, 4, 125–132.
Johnson, C. A. Purity Requirements From a Pharmacopoeial Point of View. J. Pharm. Biomed. Anal. 1986, 4, 565–571.
Repta, A. J.; Bansal, P. Phase Solubility Analysis Employing Solubility Product Relationships: Purity Determination of Monobasic Amines and Their Salts. J. Pharm. Sci. 1972, 61,
1069–1075.
 12 Pharmaceutical Analysis—Drug Purity Determination

Görög, S. The Sacred Cow: The Questionable Role of Assay Methods in Characterizing the Quality of Bulk Pharmaceuticals. J. Pharm. Biomed. Anal. 2005, 36, 931–937.
Hofer, J. D.; Olsen, B. A.; Rickard, E. C. Is HPLC Assay for Drug Substance a Useful Quality Control Attribute?J. Pharm. Biomed. Anal. 2007, 44, 906–913.
Singh, D. K.; Sahu, A.; Kumar, S.; Singh, S. Critical Review on Establishment and Availability of Impurity and Degradation Product Reference Standards, Challenges Faced by the Users,
Recent Developments, and Trends. TrAC Trends Anal. Chem. 2018, 101, 85–107.
Pauli, G. F.; Chen, S. N.; Simmler, C.; Lankin, D. C.; Gödecke, T.; Jaki, B. U.; Friesen, J. B.; McAlpine, J. B.; Napolitano, J. G. Importance of Purity Evaluation and the Potential of
Quantitative 1H NMR as a Purity Assay: Miniperspective. J. Med. Chem. 2014, 57, 9220–9231.
Mahajan, S.; Singh, I. P. Determining and Reporting Purity of Organic Molecules: Why qNMR. Magn. Reson. Chem. 2013, 51, 76–81.
W
Weber, M.; Hellriegel, C.; Rück, A.; Sauermoser, R.; Wüthrich, J. Using High-Performance Quantitative NMR (HP-qNMR ) for Certifying Traceable and Highly Accurate Purity Values of
Organic Reference Materials With Uncertainties <0.1%. Accred. Qual. Assur. 2013, 18, 91–98.
Westwood, S.; Choteau, T.; Daireaux, A.; Josephs, R. D.; Wielgosz, R. I. Mass Balance Method for the SI Value Assignment of the Purity of Organic Compounds. Anal. Chem. 2013, 85,
3118–3126.
Giron, D.; Goldbronn, C. Place of DSC Purity Analysis in Pharmaceutical Development. J. Therm. Anal. 1995, 44, 217–251.
Mathkar, S.; Kumar, S.; Bystol, A.; Olawoore, K.; Min, D.; Markovich, R.; Rustum, A. The Use of Differential Scanning Calorimetry for the Purity Verification of Pharmaceutical Reference
Standards. J. Pharm. Biomed. Anal. 2009, 49, 627–631.
Bruening, C. F.; Kline, O. L. Rapid Determination of the Relative Purity of Vitamin B12 (Cyanocobalamin) in Pharmaceutical Products. J. Pharm. Sci. 1961, 50, 537–538.
Lemoff, A.; Yan, B. Dual Detection Approach to a More Accurate Measure of Relative Purity in High-Throughput Characterization of Compound Collections. J. Comb. Chem. 2008, 10,
746–751.
Letot, E.; Koch, G.; Falchetto, R.; Bovermann, G.; Oberer, L.; Roth, H. J. Quality Control in Combinatorial Chemistry: Determinations of Amounts and Comparison of the “Purity” of
LC-MS-Purified Samples by NMR, LC-UV and CLND. J. Comb. Chem. 2005, 7, 364–371.
Marín, A.; Sharman, G.; Burgess, M.; Reutter, C.; Espada, A. Standardization of Analytical Methodology and Procedures for Purity Assessment of Small Molecules in Drug Discovery. In
Instrumental Methods for the Analysis and Identification of Bioactive Molecules; Jayprakasha, G. K., Patil, B. S., Pellati, F., Eds.; ACS Symposium Series American Chemical Society:
Washington, DC, 2014; vol. 1145, pp 125–149.
WHO, Expert Committee on Specifications for Pharmaceutical Preparations, WHO Technical Report Series, No. 943, Forty-First Report, Annex 3- General Guidelines for the
Establishment, Maintenance and Distribution of Chemical Reference Substances, Geneva, Switzerland (2006).
ICH, Q2(R1) Validation of Analytical Procedures: Text and Methodology, Geneva, Switzerland (2005).
ICH, Q3A(R2) Impurities in New Drug Substances, Geneva, Switzerland (2006).
ICH, Q3B(R2) Impurities in New Drug Products, Geneva, Switzerland (2006).
ICH, Q3C(R6) Impurities: Guideline for Residual Solvents, Geneva, Switzerland (2016).
ICH, Q3D Guideline for Elemental Impurities, Geneva, Switzerland (2014).
ICH, Q6A Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances, Geneva, Switzerland (1999).
ICH, M7(R1) Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk, Geneva, Switzerland (2017).
Ahuja, S. (1998) Impurity Evaluation of Pharmaceuticals. New York: Dekker.
Görög, S. (ed.) (2000) Identification and Determination of Impurities in Drugs. Amsterdam: Elsevier.
United States Pharmacopeia (USP 41-NF 36), The United States Pharmacopeial Convention, Rockville, MD, USA (2018).
European Pharmacopoeia 9.0, European Directorate for the Quality of Medicines (EDQM), Strasbourg, France (2018).
British Pharmacopoeia, Medicines and Healthcare products Regulatory Agency (MHRA), London, UK (2018).
Japanese Pharmacopoeia, 17th Edition, Ministry of Health, Labour and Welfare (MHLW), Japan (2016).
The International Pharmacopoeia, 7th Edition, World Health Organization (see at http://apps.who.int/phint/2017/index.html#p/home).
Ahuja, S., Alsante, K. M. (2004) Handbook of Isolation and Characterization of Impurities in Pharmaceuticals. Elsevier (see at https://www.sciencedirect.com/bookseries/separation-
science-and-technology/vol/5/suppl/C).

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