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1 Introduction on Development of analytical methods for the determination of related components in pharmaceutical compounds using chromatography technique: Identification and quantification of impurities is a crucial task in pharmaceutical process development for quality and safety. Related components are the impurities in pharmaceuticals which are unwanted chemicals that remain with the active pharmaceutical ingredients (APIs), or develop during sta ility testing, or develop during formulation or upon aging of oth API and formulated APIs to medicines. !he presence of these unwanted chemicals even in small amounts may influence the efficacy and safety of the pharmaceutical products. "arious analytical

methodologies were employed for the determination of related components in pharmaceuticals. !here is a great need for development of new analytical methods for quality evaluation of new emerging drugs. An impurity as defined Harmonisation of y the I#$ (The International Conference on Requirements for Registration of

Technical

Pharmaceuticals for Human Use) guidelines is %Any component of the medicinal product which is not the chemical entity defined as the active su stance or an e&cipient in the product'. Analytical methods for impurities estimation should e sta ility indicating to monitor the sta ility of pharmaceutical dosage forms during the investigational phase of drug development, and once the drug is marketed, the ongoing sta ility studies must e conducted( performed. !he purpose of sta ility testing is to

2 provide evidence on how the quality of a drug su stance or drug product varies with time under the influence of a variety of environmental factors such as temperature, humidity and light, ena les to esta lish a retest period(shelf lives for a drug su stance and a recommended storage condition. )ethods can e developed which measure the amount of drug

remaining, the amount of drug lost (or the appearance of degradation products), or oth. !he development of these methods for pharmaceuticals can e approached from several avenues. Related components, related

su stances, and related impurities terms are synonyms for the term impurities* the use of a ove terms at different phrases means one and the same (i.e. impurities). 1.2 Sources of impurities in pharmaceutical substances: !he origin of impurities in drugs is from various sources and phases of the synthetic process and preparation of pharmaceutical dosage forms. )a+ority of the impurities are characteristics of the synthetic route of the manufacturing process. !here are several possi ilities of synthesi,ing a drug* it is possi le that the same product of different sources may give rise to different impurities. According to the international conference on harmoni,ation (I#$) of technical requirements for registration of

pharmaceuticals for human use, impurities are classified as organic impurities, inorganic impurities and residual solvents. -rganic impurities may arise from starting materials, y products, synthetic intermediates

and degradation products. Inorganic impurities may derived from the

3 manufacturing process and are normally known and identified as reagents, ligands, inorganic salts, heavy metals, catalysts, filter aids and charcoal etc. Residual solvents are the impurities introduced with solvents ./0 12. -f the a ove three types, the num er of possi le inorganic impurities and residual solvents is limited. !hese are easily identified and their physiological effects and to&icity are well known. 3or this reason the limits set y the pharmacopoeias and the I#$ guidelines can guarantee

that the harmful effects of these impurities do not contri ute to the to&icity or the side effects of the drug su stances. !he situation is different with the organic impurities. 4rugs prepared y multi0step

synthesis results in various impurities, their num er and the variety of their structures are almost unlimited and highly dependent on the route and reaction conditions of the synthesis and several other factors such as the purity of the starting material, method of isolation, purification, conditions of storage etc. In addition, to&icity is unknown or not easily predicta le. 3or this reason the I#$ guideline set threshold limit a ove which the identification of the impurity is o ligatory. 1.2.1 Sources of Organic Impurities: -rganic impurities may arise during the manufacturing process and(or storage of the drug su stance. !hese impurities are derived from drug su stance synthetic processes and degradation reactions in drug su stances and drug products. !he process (synthetic process) related impurities can e derived from starting materials, intermediates, reagents,

4 ligands, and catalysts used in the chemical synthesis, as well as products from the side0reactions of the chemical synthesis y0 .52.

4egradation products are derived from the chemical degradation of drug su stances and drug products under storage or stress conditions. !hey may e identified or unidentified, volatile or non0volatile, and include the

following .12. 1.2.1.1 Impurities Originating from Drug Substance Synthetic

Processes: )ost of the drug su stances (low molecular weight) are chemically synthesi,ed. #hemical entities, other than the drug su stance, that are involved or produced in the synthetic process can e carried over to the

final drug su stance as trace level impurities. !hese chemical entities include raw materials, intermediates, solvents, chemical reagents,

catalysts, chemical

y0products, impurities present in the starting materials, and entities formed from those starting material impurities

(particularly those involved in the last steps of the synthesis). !hese impurities are usually referred to as process impurities .52. !he goal of process impurity identification is to determine the structures and origins of these impurities. !his knowledge is critical for improving the synthetic chemical process, in order to eliminate or minimi,e process impurities .62. 1.2.1.2 Starting aterials and Intermediates: locks

7tarting materials and intermediates are the chemical uilding

used to construct the final form of a drug su stance. 8nreacted starting

5 materials and intermediates, particularly those involved in the last steps of the synthesis, can potentially survive the synthetic and purification process and appear in the final product as impurities .9,/:2. 3or e&ample, in the synthesis of tipranavir drug su stance, the %aniline' is the intermediate in the last step of the synthesis. !he similarity etween the

structures of the %aniline' and the final product, it is difficult to totally eliminate it in the su sequent purification step. #onsequently, it appears in the drug su stance at around :./; .//2. 1.2.1.! Impurities in the Starting aterials:

Impurities present in the staring materials could follow the same reaction pathways as the starting material itself, and the reaction products could carry over to the final product as process impurities. <nowledge of the impurities in starting materials helps to identify related impurities in the final product, and to understand the formation mechanisms of these related process impurities .5, /:, //2. -ne such e&ample is the presence of a =0trifluoromethyl positional isomer in >0 trifluoromethyl0alpha0ethyl en,hydrol (flumecinol), due to the presence of =0trifluoromethyl en,ene impurity in the starting material, >0

trifluoromethyl en,ene. A second e&ample involves a ?0methyl analogue present as a trace impurity in tolperisone, due to the presence of ?0 methylpropiophenone in the starting material, =0methylpropiophenone .?2.

6 1.2.1." #eagents$ %igands and &atalysts: !hese chemicals are less commonly found in APIs* however, in some cases they may pose a pro lem as impurities .?, 12. #hemical reagents, ligands, and catalysts used in the synthesis of a drug su stance can e

carried over to the final products as trace level impurities. 3or e&ample, car onic acid chloromethyl tetrahydro0pyran0=0yl ester ( && '(P), which is used as an alkylating agent in the synthesis of a @ lactam drug su stance, was o served in the final product as an impurity. )any chemical reactions are promoted y metal ased catalysts. 3or instance, a Aiegler0Batta catalyst contains titanium, Cru Ds catalyst contains

ruthenium, and AdamDs catalyst contains platinum. In some cases, reagents or catalysts may react with intermediates or final products to form y0products. Pyridine, a catalyst used in the course of synthesis of

ma,ipredone, reacts with an intermediate to form a pyridinium impurity .52. 1.2.1.) *y+Products of the Synthesis: All chemical reactions are not /::; selective* the side0reactions are common during the synthesis of drug su stances. Ey0products from the side reactions are among the most common process impurities in drugs .12. Ey0products can as incomplete e formed through a variety of side reactions, such overreaction, isomerisation, dimerisation,

reaction,

rearrangement, or unwanted reactions

etween starting materials or

intermediates with chemical reagents or catalysts .52.

7 1.2.1., Products of over+reaction: In many cases the least or previous steps of the syntheses are not selective enough and the reagents attack the intermediate not only at the desired site. 3or e.g. in the synthesis of nanodralone decanoate, the last step of the synthesis is the decanoylation of the /5 F-$ group. In the course of overreaction the reagents also attacts the =ene0 > o&o group leading to an enol ester0 type impurity (>, /5G0 dihydro&yestra0>, H0 diene disdecanoate) .?, 62. 1.2.1.- Products of side reactions: 7ome of the frequently occurring side reactions (which are unavoida le in drug synthesis) are well0 known to the synthetic chemist* other which lead to trace level impurities have to e detected and elucidated during

impurity profiling. !he formation of diketopipera,ine derivative is a typical side reaction in peptide synthesis .?2. 1.2.1.. Impurities Originating from Degradation of the Drug

Substance: Impurities can also e formed y degradation of the end product

during manufacturing of ulk drugs. 4egradation products resulting from storage or formulation to different dosage forms or aging are common impurities in the medicines .12. !he definition of degradation product in the I#$ guideline is a molecule resulting from a chemical change in the su stance rought a out y overtime and(or action of e.g. Iight, y reaction with e&cipient and(or the

temperature, p$ or water or

8 intermediate container closure system .?, /?2. 3or e&ample in the case of aspartame, in the presence of moisture, hydrolysis occurs to form the degradation products i.e. I0 aspartyl0 I0 Phenyalanine and >0 en,yl010 car o&ymethyl ?, H0diketopiera,ine. !hird degradation product is also known, G0I0 aspartyl0I0phenylalanine methyl ester. Aspartame

degradation also occurs during prolong heat treatment .?2. 1.2.2 /nantiomeric Impurities: !he ma+ority of therapeutic chiral drugs used as pure enantiomers are natural products. !he high level of enantio selectivity of their iosynthesis e&cludes the possi ility of the presence of enantiomeric impurities .12. In the case of synthetic chiral drugs, the racemates which are usually marketed, if the pure enantiomer is administered, the antipode is considered to e an impurity. !he reason for its presence can e either the incomplete enantio selectivity of the syntheses or incomplete resolution of the enantiomers of the racemate .>, />2. Although the I#$ guideline e&cludes enantiomeric impurities, pharmacopoeias consider them as ordinary impurities .?2. A single enantiomeric form of chiral drug is now considered as an improved chemical entity that may offer a etter pharmacological profile

and an increased therapeutic inde& with a more favoura le adverse reaction profile. $owever, the pharmacokinetic profile of levoflo&acin (70 Isomeric form) and oflo&acin (R0 isomeric form) are compara le,

suggesting the lack of advantages of single isomer in this regard .?, 12. !he

9 prominent single isomer drugs, which are eing marketed, include

levoflo&acin (70oflo&acin), leval uterol (R0al uterol), esomepra,ole (70 omepra,ole). !ypical e&amples of drugs containing enantiomeric impuritiesJ a) 4e&chlorophenarmine maleate (R enantiomer impurity allowed B)! :.H;) ) !imolol maleate (R enantiomer impurity allowed B)! /;) c) #lopidogrel sulphate (R enantiomer impurity allowed B)! /;) In general, an individual API may contain all of the a ove0mentioned types of organic impurities at levels varying from negligi le to significant level .12. 1.! #equirement for control of impurities: Impurities often posses unwanted pharmacological or to&icological effects y which any enefits from their administration may e outweighed .?2. Impurities will have different disastrous efficacy, different

ioavaila ility, adverse effects and to&ic effects. In case of chiral impurities one isomer may produce the desired therapeutic activities, while the other may e inactive or in worst cases, produce unwanted effects, for e&ample

consider the tragic case of the racemic drug of n0phthalyl0glumatic acid imide that was marketed in the /91:Ds as the sedative !halidomide. Its thereapeutic activity resided e&clusively in the R0(K)0enantiomer. It was discovered only after several hundred irths of malformed infants that the 70(K)0enantiomer was teratogenic. It is not only that one enantiomer reacts

10 and the other does not ut also in some instances different enantiomers

can have different effects as shown in !a le /./. 'able: 1.1 /0amples of pharmaceutical products$ and its effect of chirality
&ompound !halidomide R0isomer 70isomer Ear iturates R0isomer R,70isomer -pirates 7,R0isomer 7,R0isomer Ia etalol R,R0isomer 40isomer Pencillamine I0isomer !o&ic Eeta0 locker Anti0arthritic Bon0addictive cough mi&ture Alpha0 locker #onvulsant Barcotics 7leep inducing, anti0nausea 4epressant Isomer 70isomer /ffect !eratogenic

1.!.1 Pharmacopoeial Status: !he quality of a chemical active su stance with respect to organic impurities is controlled y a set of tests within a pharmacopoeial

monograph. Individual monographs are periodically updated to keep pace with scientific progress and regulatory developments. 3ollowing the revised I#$ L>A (R?) impurity testing guideline ma+or pharmacopoeias will continue pu lishing new or revised relevant monographs and general chapters. Active su stances found to contain an organic impurity not detected y the relevant pharmacopoeial tests prescri ed elow are not of

11 pharmacopoeial quality, unless the amount and the nature of this impurity are compati le with C)P ./=2. !wo general chapters (M=11N O M/:61N) of the 87 Pharmacopoeia (87P) deal with organic impurity testing. #oncepts and definitions are clearly descri ed although different terminology from that of I#$ is used. 8ntil now, one of three types of tests in orderedJ /. A chromatographic purity test coupled with a non0 specific assay ?. A chromatographic purity0 indicating method that also serves as an assay >. A specific limit test for known impurities, a procedure that requires reference standards for these impurities ./H2. In the future, new and revised 87P individual monographs will include tests that actually control specified and unspecified organic impurities. Phere different routes of synthesis yield different impurity profile, different analytical procedures will e proposed. All specified impurities ulk pharmaceutical chemicals is

will e separately limited, with a further limit of :./:; for any unspecified (unknown) impurity. !otal impurities a ove the disregard limit should e

less than /.:;. 87P also proposes that a suita le test for detecting impurities that may have should een introduced from e&traneous sources

e employed in addition to tests provided in a specific monograph

./>, /=, /H2.

12 !he Quropean #ommission decided that the principles and terminology of the revised I#$ L>A should e implemented in the Quropean oth new and

Pharmacopoeia (QP) monographs of the active su stances*

already pu lished ./12. A new general chapter concerning the control of impurities in pharmaceutical su stances was introduced in the fifth edition of the QP, while a revision of the monograph entitled 7u stances for Pharmaceutical 8se has also een done. According to the policy of QP

control of the relevant organic impurities in synthetic drug su stances is often accomplished y the test of related su stances. #urrently, it is a

limit test (comparison of the peak areas), ut will progressively e changed to utili,e a quantitative acceptance criterion .>, /52. 7ome individual monographs already satisfy this demand. )ore tests are ordered, if the general test does not control a given impurity or there are other special reasons ./12. Potential impurities with a defined structure that are known to e detected y the tests in a monograph, are not known to ut

e present in medicinal su stances a ove the

identification threshold, are referred to as detecta le impurities. !hey are limited y a general acceptance criterion ./52. QP individual monographs

pu lished in the new format include a separate section in which all impurities (specified and detected) are listed. 8nidentified specified impurities are not listed in this section, ut their specific acceptance

criteria along with appropriate analytical characteristics (e.g., retention time) are reported in the te&t, wherever it is applica le ./>2.

13 $owever, previous QP monographs were not having a related su stances test in the new e&plicit style are to e read and interpreted according to

the recent amendments. 4uring the coming years, QP individual monographs now pu lished in the old format will e revised to contain

related su stances tests and lists on specified and other detecta le impurities. )onographs containing tests for related su stances !I# will also e revised .>, />, /1, /52. 1.!.2 Pharmacopoeial norms for the enantiomeric impurities: E.P ?::/ has recommended following norms for the enantiomerically pure drug su stance. It descri es the way in which the stereochemistry of a su stance is identified and(or controlled. /0 )any medicinal su stances that contain one or more chiral centers and that are already on the market have een made availa le for ased on

pharmaceutical use as racemic mi&tures with little known a out the iological activities of the separate isomers. !his has een reflected in the monograph in the pharmacopoeia and a test to show that the su stance is a racemic mi&ture has not usually een included unless it was known

that at least one of the separate enantiomers was also availa le commercially. Bevertheless, with increasing concern y regulatory

authorities for su stances to e made availa le as single isomers, tests for enantiomeric composition will ecome more common .92. &hemical definition (monographs other than those of the Quropean pharmacopoeia)

14 /0In the case of su stances containing a single chiral centre, the descriptor R(R7)D0is included at the appropriate position in the chemical definition of the su stances to indicate a racemic mi&ture. ?0 3or su stances containing multiple chiral centres and comprising mi&ture of all possi le stereomers the term Rall0recD has een used, for

e&ample Isoaminile. In those few su stances e&isting as diastereomeric mi&tures, that is where in one or more centers the stereochemistry is e&plicit ut in other centers it is not, each centre is defined either as the

specific (R)0 or (7) F configuration , or as racemic (R7)0, respectively. 'ests: >0 In future, when a monograph descri es an enantiomer, it will include oth a test for specific optical rotation under identification and a test using methods such as chiral chromatography, to control enantiomeric purity. =0 Phen oth the racemic mi&ture and the enantiomer are availa le, the

monograph for the racemic mi&ture will specify a test for angle of rotation together with a cross reference under identification. !he test for angle of rotation will normally specify limits of K:./:S to 0:./:Sin order to limit the presence of optically active impurities and demonstrate equal proportions of the enantiomers. H0 Phen only the racemic mi&ture is availa le, the monograph for the racemic mi&ture will simply specify a test for angle of rotation .9, /62.

15 1.!.! I&( 1uideline: According to I#$ Cuideline, each impurity must respect to e investigated with

oth chemistry and safety aspects. !he former include

identification (structural characteri,ation), reporting and quantitation using suita le analytical procedures, while the latter include a process of acquiring and evaluating data concerning the iological safety of an

impurity (qualification). Individually listed impurities, limited with specific acceptance criteria, are referred to as specified and they can identified or unidentified. 8nspecified impurities are limited y a general acceptance criterion. A decision tree for the identification and qualification along with the corresponding thresholds, which are dependent on the ma&imum permitted daily dose ()44), is given list of organic impurities must synthetic drug su stanceJ 0 Qach specified identified or unidentified impurity 0 Any unspecified impurity 0 !otal impurity 7pecified unidentified impurities are referred to y an appropriate y I#$. 7umming up, the following e either

e presented in the specification of a

qualitative analytical description (e.g. relative retention time) .>, =, /?, />, /92. Eelow are the I#$ topics, codes of quality guidelines.

16

'able: 1.2 %ist of topics$ codes and corresponding quality guidelines developed by I&(
!opics ( #ode L/A(R?) L/E L/# L/4 L/Q L/3 L?(R/) L>A(R?) L>E(R?) L>#(R=) L=E L=E ABBQT / Luality guidelines 7ta ility !esting of Bew 4rug 7u stances and Products 7ta ility !estingJ Photo sta ility !esting of Bew 4rug 7u stances and Products 7ta ility !esting for Bew 4osage 3orms Anne& to the I#$ $armonised !ripartite Cuideline on 7ta ility !esting for Bew 4rugs and Products Eracketing and )atri&ing 4esigns for 7ta ility !esting of 4rug 7u stances and 4rug Products Qvaluation of 7ta ility 4ata 7ta ility 4ata Package for Registration in #limatic Aones III and I" "alidation of Analytical ProceduresJ !e&t and )ethodology Impurities in Bew 4rug 7u stances Impurities in Bew 4rug Products ImpuritiesJ Cuideline for Residual 7olvents Qvaluation and Recommendation of Pharmacopoeial !e&ts for 8se in the I#$ Regions Qvaluation and Recommendation of Pharmacopoeial !e&ts for 8se in the I#$ Regions on Residue on Ignition(7ulphated Ash Ceneral #hapter Qvaluation and Recommendation of Pharmacopoeial !e&ts for 8se in the I#$ Regions on !est for Q&tracta le "olume of Parenteral Preparations Ceneral #hapter Qvaluation and Recommendation of Pharmacopoeial !e&ts for 8se in the I#$ Regions on !est for Particulate #ontaminationJ 7u 0"isi le Particles Ceneral #hapter

L=E ABBQT ?

L=E ABBQT >

L=E ABBQT =A Qvaluation and Recommendation of Pharmacopoeial !e&ts for 8se in the I#$ Regions on )I#R-EI-I-CI#AI QTA)IBA!I-B of Bon07terile ProductsJ )icro ial Qnumerations !ests Ceneral #hapter L=E ABBQT =E Qvaluation and Recommendation of Pharmacopoeial !e&ts for 8se in the I#$ Regions on )icro iological Q&amination of Bon07terile ProductsJ !est for 7pecified )icro0-rganisms Ceneral #hapter Qvaluation and Recommendation of Pharmacopoeial !e&ts for 8se in the L=E ABBQT =# I#$ Regions on )icro iological Q&amination of Bon07terile ProductsJ Acceptance #riteria for Pharmaceutical Preparations and 7u stances for Pharmaceutical 8se Ceneral #hapter L=E ABBQT 1 Qvaluation and Recommendation of Pharmacopoeial !e&ts for 8se in the I#$ Regions on 8niformity of 4osage 8nits Ceneral #hapter Qvaluation and Recommendation of Pharmacopoeial !e&ts for 8se in the

L=E ABBQT 5

17
I#$ Region on 4issolution !est Ceneral #hapter L=E ABBQT 6 Qvaluation and Recommendation of Pharmacopoeial !e&ts for 8se in the I#$ Regions on 7terility !est Ceneral #hapter Qvaluation and Recommendation of Pharmacopoeial !e&ts for 8se in the I#$ Regions on !a let 3ria ility Ceneral #hapter Qvaluation and Recommendation of Pharmacopoeial !e&ts for 8se in the I#$ Regions on Polyacrylamide Cel Qlectrophoresis Ceneral #hapter "iral 7afety Qvaluation of Eiotechnology Products 4erived 3rom #ell Iines of $uman or Animal -rigin Luality of Eiotechnological ProductsJ Analysis of the Q&pression #onstruct in #ells 8sed for Production of R04BA 4erived Protein Products Luality of Eiotechnological ProductsJ 7ta ility !esting of Eiotechnological( Eiological Products 4erivation and #haracterisation of #ell 7u strates 8sed for Production of Eiotechnological(Eiological Products #ompara ility of Eiotechnological(Eiological Products 7u +ect to #hanges in !heir )anufacturing Process 7pecificationsJ !est Procedures and Acceptance #riteria for Bew 4rug 7u stances and Bew 4rug ProductsJ #hemical 7u stances 7pecificationsJ !est Procedures and Acceptance #riteria for Eiotechnological(Eiological Products Cood )anufacturing Practice Cuide for Active Pharmaceutical Ingredients Pharmaceutical 4evelopment Luality Risk )anagement Pharmaceutical Luality system 2uality Implementation 3or4ing 1roup on 2.$ 25 and 216 Luestions O Answers

L=E ABBQT 9

L=E ABBQT /:

LHA(R/) LHE LH# LH4 LHQ L1A L1E L5 L6(R?) L9 L/: L6(L9(L/: LOAs

1." &ontrol of Organic Impurities: Control of the organic impurities in new drug substances is based on the Maximum ail! ose and total dail! inta"e #T I) of the impurities . !a le /.>

provides the I#$ threshold for control of the organic impurities in new drug su stances. 4epending on whether the )a&imum 4aily 4ose is higher or lower than ?g, organic impurities in a new drug su stance at (or greater than) :.:H; or :./; requires identification. #ontrol of organic

18 impurities in new drug products are outlined in !a le /.=. Eased on the )a&imum 4aily 4ose, the identification thresholds for organic impurities in new drug products are divided into = groups to give more consideration to low dose drug products. 3or most new drug products, the )a&imum 4aily 4ose is etween /: mgF? g(day, therefore, any impurities at :.?; or greater would have to e identified .H, 5 and /?2. 'able: 1.! Organic impurity 'hreshold in ne7 drug substances based on I&(2!8 9"$ ) and 15:
)a&imum daily dose
1

Reporting !hreshold :.:H;


2$!

Identification !hreshold2$! :./ or /.:mg(day intake (whichever is lower) :.:H;

Lualification !hreshold2$! :./H; or /.:mg(day (whichever is lower) :.:H;

U?g(day N ?g(day

:.:>;

;oteJ
?0

/0

!he amount of drug su stance administered per day

$igher reporting thresholds should e scientifically +ustified Iower thresholds can e appropriate if the impurity is unusually to&ic

>0

'able: 1." Organic impurity 'hreshold in ne7 drug products based on I&( 2!* 9"$ 12 and 15:
Reporting !hresholds )a&imum 4aily 4ose / U/ g N/g

!hreshold?,> :./; :.:H;

Identification !hresholds

19
)a&imum 4aily 4ose/ M / mg /mg F /:mg N/: mg 0 ? g N?g !hreshold?,> /.:; or H Vg !4I, whichever is lower :.H; or ?: Vg !4I, whichever is lower :.?; or ? mg !4I, whichever is lower :./:;

Lualification !hresholds )a&imum 4aily 4ose / M /: mg /: mg 0 /:: mg N/:: mg 0 ? g N?g

!hreshold?,> /.:; or H: Vg !4I, whichever is lower :.H; or ?:: Vg !4I, whichever is lower :.?; or > mg !4I, whichever is lower :./H;

;ote: !4I F total daily intake


/0

!he amount of drug su stance administered per day !hresholds for degradation products are e&pressed either as a percentage

?0

of the drug su stance or as total daily intake (!4I) of the degradation product. Iower thresholds can e appropriate if the degradation product is unusually to&ic.
>0

$igher thresholds should e scientifically +ustified.

1.) Stability testing of ;e7 Drug substances and Drug Products: Analytical methods for impurities estimation should e sta ility

indicating to monitor the sta ility of pharmaceutical dosage forms during the investigational phase of drug development, and once the drug is marketed, for the ongoing sta ility studies which must e conducted (

performed. !he purpose of sta ility testing is to provide evidence on how the quality of a drug su stance or drug product varies with time under the influence of a variety of environmental factors such as temperature,

20 humidity and light, ena les to esta lish a retest period(shelf lives for a drug su stance and a recommended storage condition. )ethods can e

developed which measure the amount of drug remaining, the amount of drug lost (or the appearance of degradation products), or oth. !he e&pert Porking Croup of the International Conference on Harmonisation of the Technical Requirements for Registration of Pharmaceuticals for Human Use developed a guideline on sta ility testing for registration application within the Quropean 8nion, Wapan and the 8nited 7tates. !he goal of the I#$ sta ility guideline was to e&emplify the core sta ility data package required for new drug su stances and products in the Quropean 8nion, Wapan and the 8nited 7tates such that the data generated in any of the regions is mutually accepta le in the other two. !he guideline applies to the information required for the registration applications of new molecular entities and drug products, ut not to a reviated or a ridged

applications, clinical trail applications, and so on. !he test conditions were selected ased on the climatic conditions in three areas so that test

data provides evidence on the variation in quality with time under the influence of a variety of representative environmental factors. !hese data in turn allow recommended storage conditions and shelf lives to esta lished. 1.).1 Drug substance: !he primary sta ility studies for the drug su stance show that it will remain within specification during the retest period. Iong0term (/?0 e

21 month) and accelerated (10month) testing are performed on at least three atches. Eatches can e manufactured at a minimum of pilot scale, ut

should use the same synthetic route and a method of manufacture that simulates the final process to e used at manufacturing scale. In addition, supporting sta ility on la oratory0scale quality of the quality of F (a) )aterial used in preclinical and clinical studies ( ) )aterial to e made at a manufacturing scale. !he first three atches made post approval should also e placed on long0 term sta ility using the product registration protocol. !esting should cover physical, chemical, and micro iological properties suscepti le to change during storage and likely to affect product quality, safety, and(or efficacy. "alidated sta ility0indicating methods should replicates to limits should e used. !he num er of atches may e su mitted. !he

atches placed on sta ility should

e representative of the

e run depends on the results of validation studies, and e derived from material used in preclinical and clinical oth individual and total upper limits for impurities

studies, including

and degradation products. !he length of the studies and the storage conditions should cover storage, shipment, and su sequent use, although use of the same conditions as for the drug product will facilitate comparative review and assessment. -ther conditions should e included

as scientifically +ustified. !emperature0sensitive drugs should e stored at the la eled long0term storage temperature, and accelerated testing should

22 e conducted at /HS# a ove the designated long0term storage temperature with appropriate relative humidity conditions. At the time of regulatory su mission, a minimum of /? months at ?HS# X ?S#(1:; R$ X H (long term) and 1 months at =:S# X ?S# and (5H; R$ X H; (accelerated) is required. If significant changes are noted at the elevated temperature, additional testing at an intermediate condition, such as >:S# X ?S#(1H; R$ X H; should e conducted. !he registration

application should include a minimum of 1 months of data from a /?0 month study at the intermediate condition. 7ignificant change at =:S# and 5H; R$ is defined as failure to meet specification. Iong0term0testing should e continued to cover all retest periods. Bormally, testing under

long0term conditions is performed every > months for the first year, every 1 months for the second year, and then annually. #ontainers employed in the long0term sta ility study should e the same or simulate actual

packaging used for storage and distri ution. As the application is pending review, accumulated sta ility data should intermediate temperature data may e su mitted. Accelerated or

e used to support shipping

conditions and evaluate the effect of short0term e&cursions outside the la el storage conditions. Eecause long0term sta ility is used to esta lish appropriate retest periods, it should e noted that the degree of inter atch varia ility affects the confidence that a future atch will remain within specifications for the entire retest period. As a rule, determination of the time at which the 9H;

23 one0sided confidence limit for the mean degradation curve intersects the accepta le lower specification limit is accepta le, com ining data into one overall estimate to account for varia ility. Eefore com ining the data, apply appropriate statistical tests (e.g., p test) to e sure it is allowa le. If inappropriate to com ine data, the retest period may depend on the minimum time a atch is actually measured to remain in specification.

!he nature of the degradation relationship determines the need for transformation of the data for linear regression analysis. !his relationship can generally e fitted to a linear, quadratic, or cu ic function on an

arithmetic or logarithmic scale. 7tatistical methods can e used to test the goodness of fit of the data on all atches and com ined atches, where appropriate, to the assumed

degradation curve. If the data show little degradation or varia ility, a retest period can e +ustified without statistical analysis and a limited

e&trapolation of real0time data may e undertaken when supported y the accelerated data. Any e&trapolation must e +ustified, ecause it assumes eyond the degradation

that the same mechanism of degradation will continue o served data* this evaluation should include assay,

products, and any other appropriate attri utes. !he storage temperature range should e ased on the sta ility data

and used in accordance with the national or regional requirements. 7pecific la eling requirements should e stated, particularly for drugs that

24 cannot free,e* terms such as am ient and room temperature should avoided. 1.).2 Drug Product: !he sta ility program for the drug product should e ased on e

knowledge of the drug su stance and e&perience from e&perimental and clinical formulations. 8nless specifically noted in this section, the requirements for drug su stances also apply to drug products.

Accelerated and long0term data should e provided on three atches of the same formulation and dosage form in the containers and closure proposed for marketing. !his revision of the I#$ guideline specifies only solid oral dosage forms, and it states that two of the three atches placed on

sta ility should e at least pilot scale, ut that a third may e smallerYfor e&ample, ?H,:::0H:,::: ta lets or capsules. As with drug su stance, at least /? months of long0term sta ility data should e su mitted at the atches of of drug

time of regulatory filing. Phen possi le, manufacture sta ility the finished product using identifia ly different atches

su stance. 4ata on la oratory0scale accepta le as primary sta ility data,

atches of drug product is not ut may e su mitted as supportive

information, as may data on associated formulations or packaging. If required, preservative efficacy testing and assays on stored samples should e performed to determine content and efficacy of antimicro ial etween release and shelf0life specifications for e supported y preservative efficacy

preservatives. 4ifferences

antimicro ial preservatives should

25 testing. Iimits for tests such as dissolution and particle si,e require reference to results of ioavaila ility and clinical atches. 7torage at high relative humidity is important for solid oral dosage forms, ut is not necessary for products such as solutions, suspension,

and so on, stored in containers designed to provide a permanent water arrier. Iow relative humidity (/:0?:;) is appropriate for products of high water content stored in semi permea le containers. !esting of unprotected drug product can e a useful part of stress testing and package

evaluation, as can studies in related packaging materials. If a product needs to e reconstituted or diluted, sta ility in the final form should also e addressed. 1., Stress testing route to the development of stability+indicating analytical methods <SI8 s=: 7tudies were under taken to elucidate the intrinsic sta ility of the drug su stance. 7uch testing is part of the development strategy and is normally carried out under more severe conditions than those used for accelerated testing. A more detailed description of stress testing is provided near the eginning of the I#$ sta ility guideline, under the

%4rug su stance' headingJ %7tress testing of the drug su stance can help identify the likely degradation products, which in turn can help esta lish the degradation pathways and the intrinsic sta ility of the molecules and validate the sta ility indicating power of the analytical procedures used'.

26 !he nature of the stress testing will depend on the individual drug su stance and drug product involved. 7tress testing is likely to e carried out on single atch of drug

su stance. It should include the effect of temperatures (in /:Z # increments (e.g., H:Z#, 1:Z# etc.) a ove that for accelerated testing), humidity (e.g., 5H; R$ or greater) where appropriate, o&idation, and photolysis on the drug su stance. !he testing should also evaluate the suscepti ility of the drug su stance to hydrolysis across wide range of p$ values when in solution or suspension. Photo sta ility testing should e

an integral part of stress testing. !he standard conditions for photo sta ility testing are descri ed in I#$ L/E. Q&amining the degradation products under stress conditions is useful in esta lishing the degradation pathways, developing and validating suita le analytical procedures. $owever, it may not e necessary to een

e&amine specifically for certain degradation products if it has

demonstrated that they are not formed under accelerated or long term storage conditions. Results from these studies will form an integral part of the information provided y the regulatory authorities. !he description of stress testing

was slightly modified in the revised sta ility guideline from the original description in I#$ L/A. !he original L/A description contains this additional paragraphJ

27 7tress testing is conducted to provide data on forced decomposition products and decomposition mechanisms for the drug su stance. !he severe conditions that may e encountered during distri ution can e

covered y stress testing of definitive atches of drug su stance. !he I#$ definition of stress testing for drug product shown elowJ 7tudies undertaken to asses the effect of severe conditions on the drug product. 7uch studies include photo sta ility testing (I#$ L/E) and specific testing on certain products (e.g., metered dose inhalers, creams, emulsions, refrigerated aqueous liquid products). 3rom the I#$ definition, it is now clear that there is now a (regulatory) differentiation etween %accelerated testing' and %stress testing'. 7tress

testing is distinguished y oth severity of the conditions and the focus or intent of the results. 7tress testing, which is also often referred as %forced degradation,' is an investigation of the %intrinsic sta ility' characteristics of the molecule, providing the foundation for developing and validating analytical methods and for developing sta le formulations. 7tress testing studies are intended to disco$er sta ility issues, and are therefore predicti$e in nature. 7tress testing studies are not part of the %validated' formal sta ility program. Rather, pharmaceutical stress testing is a research investigation requiring a scientific e&pertise and +udgment. It is interesting to consider some of the conditions that have historically een employed to consider in the stress testing of

pharmaceuticals, documented

oth in the %Analytical Profiles of 4rug

28 7u stances' .?:2 and can y 7ingh and Eakshi .?/2. Acidic stress conditions

e found to vary from :./ B $#l at =:Z# for / week (with %negligi le

degradation') .??2, to :./ B $#l at 1HZ# for ?/ days (5/.1; degradation) .?/2, to :./ B $#l at /:HZ # for ? months (with %considera le degradation'), to = B $#l under reflu&ing conditions for ? days (11; degradation) .?>2, to 1.H B $#l at /:6Z # for ?= hr (H:; degradation), to concentrated $#l at room temperature (H1.H; degradation) .?=2. 7imilar elevated temperatures, times, and ase strength have een employed for

asic stress conditions. 3or e&ample, conditions can e found to vary from :./ B Ba-$ at =:Z# for / week (with negligi le degradation) .??2, to :./ B Ba-$ at 1HZ# for ?/ days (16; degradation) .?/2, to :./ B Ba-$ under reflu&ing conditions for ? days (16; degradation) .?>2, to / B Ba-$ under oiling conditions for > days (5.?; degradation) .?H2, to H B Ba-$ under reflu&ing conditions for = hr (/::; degradation) .?12. In terms of o&idative degradation studies, hydrogen pero&ide has een employed at strengths

from :.>; to >:; .?52. 7tudies were often conducted at elevated temperatures, e.g., >5Z# for 1 hr .>; hydrogen pero&ide, 1:; degradation .?62, H:Z# for 5? hr (>; hydrogen pero&ide, 1.1; degradation), and even reflu&ing conditions for >: min (>; hydrogen pero&ide, e&tensive degradation) .?12 or 1 hr (/:; hydrogen pero&ide, no significant degradation) .?92. As these e&amples illustrate that historically there has een

tremendous variation in the conditions employed in acid( ase and

29 o&idative stress testing studies. !here has also een tremendous variation in defining the appropriate %endpoint' of the stress testing studies, i.e., length of time (and temperature) or amount of degradation that is sufficient to end the stress e&posure. Perhaps the most dramatic varia ility in stress testing conditions is o served in the photo0 stressing of drugs .>:2, where the lamps and e&posures range from short wavelength $g arc lamps (?H= nm, 8"# range), fluorescent light, artificial light, halogen lamps to &enon lamps. !he varia ility of e&posure to type of light during pharmaceutical photo0sta ility studies has also documented >=2. 3rom the information provided a ove, it is apparent that stress0testing conditions have varied greatly from compound to compound and from investigator to investigator. Q&tremely harsh conditions have een een

y surveys of practices in the pharmaceutical industry .>/0

commonly used in the past to ensure degradation, even if the conditions far e&ceeded plausi le e&posures. )ore recently, several articles relevant to stress testing have appeared in the pharmaceutical literature. A paper y 7ingh and Eakshi .?/2 in

?::: provides the most thorough collection of references to various degradation studies of drug products, documenting the diversity of conditions and approaches to stress testing. !his paper attempts to

provide a classification system (Q&tremely la ile, "ery la ile, Ia ile, 7ta le) ased on a defined systematic approach. It is not clear from the

30 article on what asis (scientific or otherwise) the classification system was devised* however, the paper does define %endpoints' to stressing (al eit, fairly harsh endpoints), allowing for the conclusion that a particular compound may e regarded as %sta le' under a certain set of conditions. In /99? (and again in /99=), Eoccardi provided some needed guidance on o&idative stress testing y asserting that most pharmaceutical

o&idative degradation was the result of auto&idation and that hydrogen pero&ide was not a very good reagent to mimic auto&idation processes .>H,>12. Eoccardi was the first to descri e the use of radical initiators such as a,o isiso utyronitrile (AIEB) for o&idative pharmaceutical stress testing, and he provided a simple procedure with mild conditions, which he termed as %!he AIEB !est.' In /991, Eaertschi .>52 presented and discussed an approach to stress testing that had defined limits of harshness and e&posure time. In /996, Peiser .>62, while discussing the role of stress testing in analytical method development, suggested a set of conditions for performing stress testing that was argua ly milder than many of the historical studies cited a ove. In ?::/, Alsante et al. .>92 provided a guide to stress testing studies that suggested defined limits to the stress conditions of / B $#l and / B Ba-$ for a ma&imum of / week at room temperature. In ?::?, the views of the Pharmaceutical Research and )anufacturerDs Association (PhR)A) were summari,ed in an article on forced degradation studies pu lished in Pharmaceutical !echnology .=:2. !he PhR)A article did not discuss specifics of conditions of stress,

31 ut rather focused more on what kinds of stress testing should e

performed for drug su stances and products and on the regulatory requirements. Recent pu lications on the topic of stress testing(forced degradation studies reveal that there is still a tremendous varia ility in the conditions employed. A few e&amples will e discussed here, although this discussion is not intended to e an e&haustive review of the literature. A degradation study of haloperidol utili,ed / ) $#l and / ) Ba-$ (reflu&ed for H hr), and >:; hydrogen pero&ide (5:Z# for H hr) for the most stressful conditions of the study .=/2. !hese conditions appear to have een chosen to ena le production of known degradation products (si& degradation products shown) to facilitate $PI# method validation efforts. A degradation study of i uprofen produced /> degradation products, several of which had never een detected efore .=?2. In this study,

o&idative studies were carried out utili,ing potassium permanganate (:.:H )) at room temperature up to /1 hr in :.H ) Ba-$* up to >>; hydrogen pero&ide at room temperature for ?? hr* and potassium dichromate (:./ B) at room temperature up to /= days in :.H ) $#l. 7olid0state studies utili,ed H:Z# up to 6 months and /::Z# up to /1 hr to detect volatile degradation products. An B)R study of the aqueous degradation of isophosphoramide mustard was conducted in uffered aqueous solutions

in the p$ range of /0/> .=>2. !he degradation of sumatriptan in :./ B $#l, :./ B Ba-$, and in >; hydrogen pero&ide was studied using I#()7 and

32 I#()7()7 .==2. !he solutions were heated at 9:Z# for >: min to 9 hr. Photosta ility was assessed y e&posure to 8" irradiation at ?H= min for

?= hr (no indication of irradiation intensity). A study of the ma+or o&idative degradation products of 7#$ H1H9? was conducted y e&posure of the

drug su stance in the solid state to /H:Z# for /? days with identification of the ma+or products using I#0)7 and I#0B)R .=H2. 7ingh et al. descri e stress degradation studies of ornida,ole .=12 and pra,osin, tera,osin, and do&a,osin .=52 under conditions designed to e in %alignment' with the

I#$ 7ta ility guideline (L/AR). In the case of ornida,ole, significant degradation was seen under acidic conditions of :./ ) $#l to H ) $#l at 6:Z# for /?05? hr, although no degradation products were detected (presuma ly 7tudies under ecause of degradation to non0chromophoric products). asic conditions of :./ ) Ba-$ at oth 6:Z# and =:Z#

revealed complete degradation at time ,ero. )ilder studies were then conducted at p$ 6 and =:Z#. -&idative studies involved >; and >:; hydrogen pero&ide at room temperature for ?= and =6 hr, with losses of 6; and H>; of the parent, respectively. Photo0degradation studies utili,ed -ption ? of the I#$ photosta ility guideline with e&posures up to >: days at 5::: lu& (over H million lu&0hr e&posure). 7imilar conditions were employed for pra,osin, tera,osin, and do&a,osin. In these recent e&amples of stress testing studies, it is apparent that there is still a great diversity of conditions employed to induce degradation, although the diversity is argua ly less than was o served prior to pu lication of the I#$ guidance.

33 !his continued diversity of approach could e interpreted in a couple of

ways. -ne interpretation is that stress0testing studies are inherently a research undertaking, and, therefore, fle&i ility and scientific +udgment are required, leading to diverse conditions and approaches. Another interpretation is that there is (appropriately or inappropriately) very little guidance (either regulatory or in the scientific literature) on the specification of the conditions or appropriate endpoints of pharmaceutical stress testing. 1.- 'echniques employed in literature reports for the development of SI8 s: If one critically evaluates the literature reports, titrimetric, spectrophotometric and chromatographic techniques have een commonly employed in the analysis of sta ility samples. 1.-.1 'itrimetric and spectrophotometric In these methods, usually the o +ective is the analysis of drug alone in the matri& of impurities, degradation products, impurities, etc., and also other drugs in case of the com ination products. !heir advantage is low cost and simplicity, though some times they are not sensitive. 4ue to limitation of specificity there are hardly any reports these days on the use for the assay of sta ility samples. $owever, a few reports involving derivative spectroscopy have een pu lished lately. 1.-.2 &hromatographic Eecause of very nature of requirement of separation of multiple components during analysis, chromatographic methods have taken

34 precedence over conventional methods of analysis. -ther than separation of multiple components, the advantage of chromatographic methods is that these possess greater accuracy and sensitivity for even small quantities of degradation products performed. "arious chromatographic methods that have een used are thin0layer chromatography (!I#), high0

performance thin0layer chromatography ($P!I#), gas chromatography (C#), $PI# ($igh Performance Iiquid #hromatography) and newer technique like capillary electrophoresis (#Q). In comparison, $PI# has een very widely employed. It has gained

popularity in sta ility studies due to its high0resolution capacity, sensitivity and specificity. Bon0volatile, thermally unsta le or polar(ionic compounds can also e analy,ed y this technique. !herefore, most of the 7IA)s have een esta lished using $PI#. 1.-.! Steps involved during the development of stability+indicating analytical methods <SI8 s=: A 7IA) is a quantitative analytical procedure used to detect a decrease in the amount of the active pharmaceutical ingredient (API) present due to degradation. According to 34A guidelines, a 7IA) is defined as a

$alidated anal!tical procedure that accuratel! and precisel! measures acti$e ingredients #drug substance or drug product) free from potential interferences li"e degradation products% process impurities% excipients% or other potential impurities , and the 34A recommends that all assay procedures for sta ility studies e sta ility0indicating .=62. 4uring

35 sta ility studies, liquid chromatography (I#) is used routinely to separate and quantitate the analytes of interest. necessary for implementing a 7IA)J !here are three components sample generation, method

development, and method validation. Step 1: 1eneration of the Sample: 7tressing the API in oth solutions and in solid0state form generate the sample that contains the products most likely to form under most realistic storage conditions, which is in turn used to develop the 7IA). In simplest terms, the goal of the 7IA) is to o tain aseline resolution of all the

resulting products (the API and all the degradation products) with no coelutions. 7amples should e stored in appropriate vessels that allow sampling at timed intervals and that protect and preserve the integrity of the sample. !hermo stated and humidity0controlled ovens should also Cenerally, the goal of these studies is to degrade the API more than this and relevant compounds can e employed.

y H0?: ;. Any

e destroyed, or irrelevant

degradation products produced (for e&ample, degradation products of the degradation products). Any less, and important products might e missed. Q&perience and data

o tained from studies performed previously on related compounds also should e used when developing new protocols.

36 'able: 1.) lists some common conditions used in conducting forced degradation studies for drug su stances .=92.
Sample condition 7olid ( 1: 0 5:Z# 7olid ( 1: 0 5:Z# ( 5H; R$ 7olid ( simulated sunlight 'ime > /0posure 5 F /: days /: days ? F > weeks & I#$ confirmatory e&posure :./ to ? B $#l solutions either at R! or at 1: 0 5:Z# :./ to / B Ba-$ solutions either at R! or at 1: 0 5:Z# 4ilute hydrogen pero&ide (:./ to 1;) at R! or at 1: 0 5:Z# 7olution in Pater or at 1: 0 5:Z# / F > days / F > days / F > days / F > days

Step 2: Developing the %&

ethod:

After the sample is generated through the use of a properly designed and e&ecuted forced degradation, it can e used to develop the I# method. !hese days, I# method development often is performed on gradient systems capa le of automated column and solvent switching, and temperature control. 7ystems and software that automate the process,

some with decision making uilt0in, also have een reported .H:2. 7couting e&periments often are run, and then conditions are chosen for further optimi,ation. Resolving power, specificity, and speed are key

chromatographic method attri utes to

e kept in mind during method

development. $owever, e&cellent resources are availa le to anyone not already schooled in the art .H/2.

37 Selectivity during Method Development: 7electivity can e manipulated y any one or a com ination of different factors that include solvent composition, type of stationary phase in column, and mo ile phase uffers and p$. #hromatographers for the

most part are comforta le changing solvents and column stationary phases to generate a separation. $owever, advances in I# column

technology recently have made possi le the use of p$ as a true selectivity tool for the separation of ionisa le compounds .H?, H>2. chemistry columns take advantage of the est of !hese hy rid

oth the silica and

polymeric column worlds. !hey are manufactured using a classical sol0gel synthesis that incorporates car on in the form of methyl groups, resulting in columns that are mechanically strong, with high efficiency, and operate over an e&tended p$ range. !he graphics in 3igure/./ illustrate why p$ can e such a useful tool. Acidic compounds are more retained at low p$* while asic

compounds are more retained at higher p$ (neutral compounds are of course unaffected). At p$ values used traditionally (p$ =06)* a slight

change in p$ would result in a dramatic shift in retention (up0slope or down0slope of curve). $owever, y operating at p$ e&tremes, not only is e e&ploited in method

there a /:0>:0fold difference in retention that can development, the method can

e made more ro ust as well, a desira le Indeed, the selectivity differences

outcome with validation in mind.

38 afforded y a change in p$ are the equivalent to a ?:; change in the

organic solvent composition, and often are underutili,ed. ?ig1.1: #everse @ phase retention behavior as p( is varied

/valuating Specificity during

ethod Development:

Another key parameter to evaluate during method development is specificity. The United &tates Pharmacopoeia #U&P) and International

#onference on $armonisation (I#$) guidelines define specificity as the a ility of a method to assess unequivocally the analyte of interest in the presence of potential interferences .H=, HH2. In the past, it has een

accepta le to evaluate resolution, peak shape, and tailing factors to measure and document specificity. $owever, starting with U&P '(% and as

39 a direct result of the I#$ process, it was recommended that a peak purity test ased upon photodiode0array (P4A) detection or mass spectrometry

()7) e used to demonstrate that a given peak was pure. )odern P4A technology is a powerful tool for evaluating specificity. P4A detectors can collect spectra across a range of wavelengths* at each data point collected across a peak, and through software manipulations involving multidimensional vector alge ra, they compare each of the spectra to determine peak purity. In this manner, P4A detectors today can distinguish minute spectral and chromatographic differences not readily o served y simple overlay comparisons .H10H62.!o e successful,

three components are requiredJ A 8" chromophore, or some a sor ance in the wavelength range selected 7ome degree of chromatographic resolution. 7ome degree of spectral difference.

3igure /.? shows an e&ample of a partial reversed0phase I# separation, where, y the appearance, the peaks certainly are well0resolved, sharp,

and symmetrical. An e&amination of peak ? indicated the peak was pure. $owever, a close e&amination of the spectral information related to peak one reveals a different situation. In 3igure /.>, the calculated peak purity (in green) is plotted against the noise threshold (in lue), oth superimposed on the

red chromatographic trace. !he purity plot clearly indicates a co0elution

40 in the front of the peak as the purity plot e&ceeds the threshold, requiring more method development. P4A detectors can evaluating peak purity, governed e limited on occasion in

y the three required components

mentioned previously, as well as the noise of the system and disparate levels of a sor ance responses. ?ig 1.2: &hromatogram sho7ing the separation of t7o components seemingly pure

!hat is, the more similar the spectra, and the lower the relative a sor ances, the more difficult it can e to distinguish co0eluted

compounds. )ass spectrometric detection overcomes many of these limitations and in most la oratories* it has ecome the detection method

of choice for even routine method development. )7 has come a long way from the days in which many companies had a dedicated central )7 la and staff. )odern mass spectrometers are

41 smaller, simpler, and operate from the same software used to operate the chromatographic system or other detectors commonly utili,ed, decreasing the learning curve. )7 can provide unequivocal peak purity information, e&act mass, and structural and quantitative information depending upon the type of instrument used. ?ig: 1.! 'he calculated pea4 purity <in green= is plotted against the noise threshold <in blue=$ both super imposed on the red chromatographic trace.

)7 is also a very useful tool to track peaks as they move around in response to selectivity manipulations in method development. $owever, only the com ination of oth P4A and )7 on a single instrument and

software platform provides the type of valua le orthogonal information required when evaluating specificity and developing 7IA)s.

42 Step !: Aalidation of SI8 s 4ifferent methods have different requirements when it comes to validation. !he U&P recogni,es four method categories and defines the analytical performance characteristics that must each method type .H92J #ategory / J Analytical methods for the quantification of ma+or components of ulk drug su stances or active ingredients. #ategory ? J Analytical methods for the determination of impurities in ulk drug su stances or degradation compounds. #ategory > J Analytical methods for the determination of e measured to validate

performance characteristics. #ategory = J Identification tests

!a le /.1 summari,es each category and the analytical performance characteristics that must e investigated. 7IA) falls into the quantitative division of #ategory ?, and as such, all analytical performance parameters must e determined, e&cept for the ecause

limit of detection, limit of quantification would apply instead,

7IA)s need to e quantitative. According to I#$ guideline on validation of analytical methods .1:2, the ob)ecti$e of an anal!tical procedure is to demonstrate that it is suitable for its intended purpose* -ne should keep in mind that stress testing methods are screening methods used to help in understanding the degradation chemistry of the drug and therefore, do

43 not need to (nor, in general, can they) control methods. 'able 1.,: Data elements required for assay validation <as per USP=
Analytical performance characteristics Accuracy Precision 7pecificity 4etection limit Luantitation limit Iinearity Range Assay #ategory I Luantitative [es [es [es Bo Bo [es [es [es [es [es Bo [es [es [es Iimit tests \ Bo [es [es Bo Bo \ \ [es \ \ \ \ \ Bo Bo [es Bo Bo Bo Bo Assay #ategory II Assay #ategory III Assay #ategory I"

e validated to the e&tent of final

B)ay e required, depending on the nature of the specific test !he concepts in the I#$ guideline on validation of analytical methods are a good starting point for validation of stress testing methods, however, the overall validation should e significantly a reviated when

compared to the validation of final control methods, as stress testing methods are investigational methods. Accuracy normally should not e a

pro lem with stress testing methods as long as the response of the detector is linear and samples are completely dissolved prior to analysis. !he specificity of the methods can not e fully validated ecause one does not know all of the possi le degradation products during initial stress testing. 7pecificity can e addressed y using any known impurities and

degradation products produced in the method development samples.

44 Precision (repeata ility) of the assay of the main component can evaluated e

y preparing a limited num er of assay samples and using

simple statistics to estimate the standard deviation. Qstimation of intermediate precision and reproduci ility should normally not e

necessary for stress testing methods. 4etection and quantitation limits for degradation products can e determined y using parent compound and e similar.

assuming that the response of all degradation products will

Although there is no requirement to reach any specific detection limit, a reasona le is :./; since the aim of stress testing is to detect the ma+or degradation products in samples which is appro&imately /: F ?:; degraded. !he linearity of the method should e validated over ranges for e

oth assay and impurity determination. A typical assay range might

from H:; to /H:; of nominal sample concentration, while a typical range for impurity determination might cover a range from the quantitation limit to a few percent. If one wishes to quantitate impurities vs. the parent peak, then linearity (range) should e demonstrated from the quantitation limit to at least /?:; of nominal sample concentration. -ne of the most important aspects of stress testing is the analysis of samples using a suita le analytical method, which, in many cases, is reverse0phase $PI#. !his necessitates the development of an $PI# method capa le of measuring oth the loss of parent compound as well

the levels of degradation products or impurities formed in stress conditions.

45 1.-." #ole of )ass ass *alance during SI8 development:

alance correlates the measured loss of a parent drug to the

measured increase in the amount of degradation products. It is a good quality control check on analytical methods to show that all degradation products are adequately detected and do not interfere with quantitation of the parent drug (i*e*% sta ility0indicating methods). Regulatory agencies use mass alance to assess the appropriateness of the analytical method

as a sta ility0indicating method and determine whether all degradents have een accounted for .1/2. In mass alance calculations, the loss of parent drug or the amount of drug remaining is determined from a sample assay, and the measured increase in degradation products is determined y a related su stances alance is to

method. !he fundamental approach for determining mass

quantitate the decomposition peaks using degradation methods and then reconcile the measured loss in the parent drug with the amount of degradation products. If the loss in potency can for e reasona ly accounted alance is

y the amount of degradants measured, then mass

achieved. !he assessment of degradation in pharmaceutical products involves two aspects of analytical measurement. 3irstly, a specific or selective analytical method must e availa le for accurate assay of parent drug e

compound, in order to measure any loss. 7econd, methodology should

in place for quantification of the degradation products formed. Ideally,

46 when degradation occurs, the measured amount of parent drug lost should correlate well with the measured increase in degradation products. !his correlation is referred to as %mass alance' .1?2. )ore recently, the

International #onference on $armoni,ation (I#$) has provided definition of %mass alance* material alance' as followsJ The process of adding together the assa! $alue and le$els of degradation products to see how closel! these add up to +,,- of initial $alue% with due consideration of the margin of anal!tical precision* !he concept is useful scientific guide for evaluating data, ut it is not

achieva le in all circumstances. !he focus may instead e on assuring the specificity of the assay, the completeness of the investigation of route of degradation, and the use, if necessary, of identified degradents as indicators of the e&tent of degradation via particular mechanism .1>2. !he analyst must alance time and resource demands to provide the

information necessary to understand degradation without going to e&treme measures of quantify components of little interest. )ass alance in pharmaceutical analysis is very important for several reasons. Ey demonstrating the degardative losses of parent drug correlate well with the measured increase in degradation products unaccounted for. #onversely, if one o serves, for e&ample, a ?:; loss of parent drug ut

only measures a H; increase in degradation products, it is likely that additional degradation products formed are not accurately determined y

the given method(s). Eecause unknown degradation products could

47 potentially e to&ic or otherwise compromise the safety of drug, it is

important to have methods that detect all ma+or degradation products. !hus, safety is the ma+or reason for the study of mass alance. )ass alance is also useful in method validation .1=2. In order to

demonstrate that analytical methods are sta ility0indicating, unstressed and stressed materials are often compared. Any increase in degradation product that correlates well with loss of parent drug, aids in

demonstrating that the methods can accurately assess degradation. )ass alance is also important in understanding alternative degradation pathways .1H2. 3or e&ample, consider a situation where oth acid0

cataly,ed and o&idative degradation produces a su stantial loss of parent compound in stress0testing studies. If good mass the acid0cataly,ed degradation, further work to alance is achieved for

ut not for the o&idative degradation,

etter understand the o&idative degradation pathway(s) is

warranted. It may e that the poor mass alance in the latter case results from important o&idative products that are unaccounted for or from structures, which need to e more fully elucidated to understand response factor differences. )ass alance is an important consideration in

assessing degradation pathways of pharmaceutical products. -ften, response factor differences etween degradation products and the parent alance pro lems. RR3s should,

compound are responsi le for mass therefore,

e incorporated, when possi le, in the quantification of

degraded samples.

48 1.-.) 8pplication of SI8 s: 7ta ility studies are used to esta lish the re0test period for the active ingredient F that is the length of time it can analy,ing immediately e stored and used without

efore use F and the shelf life of the finished

product. !he release and shelf life specifications for the product may differ to accommodate degradation of active ingredient or other accepta le changes, which may occur on storage. !he International #onference on $armoni,ation (I#$) drug sta ility test guideline L/A (R?) requires that analysis of sta ility samples should e done through the use of validated

sta ility0indicating analytical methods (7IA)s). Additional guidance is given only for photo sta ility testing. It also recommends carrying out the stress testing on drug su stance to esta lish its inherent sta ility characteristics and to support the suita ility of proposed analytical procedure. !he validated 7IA)s will e used e&tensively for testing the

sta ility samples of oth drug su stance as well as drug product. 1.. Scope and ObCectives of research 7or4: !he present research work focuses on the development of novel sta ility0indicating analytical methods for some active pharmaceutical ingredients and few of their dosage forms. !he work also includes the validation of the developed methods as per I#$ requirements and demonstrates the suita ility of developed methods to asses the sta ility of 'able 1.- 8PIs$ its chemical name$ structure and therapeutic activity
S.;o API and its chemical Structure 'herapeutic activity

49

name ;ateglinide B0(trans0=0 isopropylcyclohe&yl0 car onyl)040 phenylalanine

HN OH O O

Anti0dia etic drug

#anolaDine (R7)0B0(?,10 dimethylphenyl)0?0.=0.?0 hydro&y0>0(?0 metho&ypheno&y)propyl2 pipera,in0/0yl2 acetamide

H
N N O N OH O

Anti Fanginal and anti0 ischemic agent

COOH

>

*rimonidine tartarate H0 romo010(?0 imida,olidinylideneamin o) quino&aline tartarate 'olterodine tartarate (R)0B, B0di isopropyl0>0 (?0hydro&y0H0 methylphenyl)0>0 phenyl)0>0Phenyl propanamine I0hydrogen tartarate Eafirlu4ast

NH HN

Br N

O H

.
HO H COOH

!reatment of open0angle glaucoma or ocular hypertension

OH H H3 C H N . HO

COOH OH H COOH

8rinary urge incontinence

CH3 N

]>0.?0)etho&y0=0 (toluene0?0 sulfonylaminocar onyl)0 en,yl20/0methyl0/$0 indol0H0yl^0car amic acid cyclo pentyl ester 'adalafil (1R, /?aR)0 ?,>,1,5,/?,/?a0 he&ahydro0?0methyl010 (>,=0methylene dio&yphenyl) pyra,ino(/_, ?_J/,1) pyrido0(>,=0 )indole0/,=0dione

N H

OCH3 H3C O H N S O O

!reatment of asthma

N N

N H

Qnhances erectile function

O O

active pharmaceutical ingredient (API). !he list of active pharmaceutical compounds taken for research study was listed in !a le /.5.

50 Bovel sta ility indicating I# methods were developed to determine the related components in different classes of pharmaceutical compounds which includes Bateglinide (anti0dia etic), Ranola,ine (Anti anginal), Erimonidine !artrate (used in treatment of open0angle glaucoma), !olterodine tartrate (used in treatment of urinary urge incontinence and other symptoms related to unsta le ladder) , Aafirlukast (used in

treatment of asthma) and !adalafil (used in treatment of erectile dysfunction). !he developed methods were validated according to

regulatory norms. 7tress testing was conducted according to I#$. )ass alance and sta ility studies were also conducted. !he developed methods can e successfully implemented during the quality monitoring and also

well employed for the assessment of quality during its storage and sta ility.

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