J. Sep. Sci.

2015, 00, 1–16 1

Ambavaram Vijaya Bhaskar Review

Reddy1
Jafariah Jaafar1
Khalid Umar2
Zaiton Abdul Majid1
Azmi Bin Aris2
Juhaizah Talib2
Gajulapalle Madhavi3
1 Department of Chemistry,
Faculty of Science, Universiti
Teknologi Malaysia, Johor,
Malaysia
2 Department of Environmental
Engineering, Faculty of Civil
Engineering, Universiti
Teknologi Malaysia, Johor,
Malaysia
3 Department of Chemistry, Sri
Venkateswara University,
Tirupati, India
Received October 24, 2014
Revised December 12, 2014
Accepted December 16, 2014
Identification, control strategies, and
analytical approaches for the determination
of potential genotoxic impurities in
pharmaceuticals: A comprehensive review
Potential genotoxic impurities in pharmaceuticals at trace levels are of increasing concern to
both pharmaceutical industries and regulatory agencies due to their possibility for human
carcinogenesis. Molecular functional groups that render starting materials and synthetic
intermediates as reactive building blocks for small molecules may also be responsible for
their genotoxicity. Determination of these genotoxic impurities at trace levels requires highly
sensitive and selective analytical methodologies, which poses tremendous challenges on an-
alytical communities in pharmaceutical research and development. Experimental guidance
for the analytical determination of some important classes of genotoxic impurities is still
unavailable in the literature. Therefore, the present review explores the structural alerts of
commonly encountered potential genotoxic impurities, draft guidance of various regulatory
authorities in order to control the level of impurities in drug substances and to assess their
toxicity. This review also describes the analytical considerations for the determination of
potential genotoxic impurities at trace levels and finally few case studies are also discussed
for the determination of some important classes of potential genotoxic impurities. It is
the authors’ intention to provide a complete strategy that helps analytical scientists for the
analysis of such potential genotoxic impurities in pharmaceuticals.

Keywords: Alkyl halides / Control strategies / Regulatory guidance / Toxicology /

Trace analysis
DOI 10.1002/jssc.201401143

1 Introduction

During the manufacturing of active pharmaceutical ingredi-

Correspondence: Dr. Jafariah Jaafar, Department of Chemistry,
Faculty of Science, Universiti Teknologi Malaysia, Johor Bahru,
Johor 81310, Malaysia
E-mail: jafariah@kimia.fs.utm.my
Abbreviations: ALARP, as low as reasonably practicable;
APIs, active pharmaceutical ingredients; APP, (2S)-2-amino-
3-(4-aminophenyl)propan-1-ol; CCP, 1-(3-chloropropyl)-4-
(3-chlorophenyl) piperazine HCl; CDP, 2-chloromethyl-3,
4-dimethoxy pyridine hydrochloride; DEREK, deductive
estimation of risk from existing knowledge; EMA, Eu-
ropean Medicines Agency; FID, flame ionization detector;
HS-GC, headspace gas chromatography; ICH, Interna-
tional Conference on Harmonization; MAP, (4-methyl-
acetophenone)-p-sulfonamide phenylhydrazine hydrochlo-
ride; MCASE, multicomputer-automated structure evalua-
tion; MNA, (S)-methyl-4-nitrophenylalanate hydrochloride;
NPA, (S)-4-nitrophenylalanine hydrate; NPP, (S)-2-amino-3-
(4-nitrophenyl)propanol; PCV, N-phenoxycarbonyl-L-valine;
PhRMA, pharmaceutical research and manufacturers of
America; PGI, potential genotoxic impurity; QL, quanti-
tation limit; R&D, research and development; SHH, (4-
sulfamoylphenyl)hydrazine hydrochloride; TTC, threshold
of toxicological concern; USFDA, U.S. Food and Drug
Administration
ents (APIs) some starting materials, intermediates, reagents,
and reaction byproducts inevitably end up in the final prod-
ucts as impurities [1]. The safety of a drug product is de-
pendent not only on the toxicological properties of the active
drug substance itself, but on the impurities it contains. Or-
ganic impurities that have the potential to induce genetic
mutations, chromosomal breaks, and/or chromosomal rear-
rangements are considered as potential genotoxic impurities
(PGIs), which may cause cancer in humans [2, 3]. Therefore,
there is an ever-increasing interest in controlling and mon-
itoring of PGIs in APIs [4–6]. The process chemists always
try to explore the possible opportunities to avoid the use and
generation of these genotoxic substances in the manufactur-
ing process. However, the complete elimination/prevention
of such impurities is always not achievable [7], and if not ad-
dressed correctly these PGIs could lead to clinical holds or
delays the approval from regulatory agencies. This poses an
imperative challenge on analytical scientists to develop ap-
propriate analytical methodologies to accurately measure as
well as to control the levels of PGIs in pharmaceuticals. In
addition to the process impurities, certain drugs may also
generate PGIs via degradation during the synthetic process,


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2 A. V. B. Reddy et al.
Table 1. PhRMA, USFDA, and EMA recommended acceptable
qualification thresholds for genotoxic impurities in phar-
maceuticals in clinical studies
Duration of exposure Limit (␮g/day)
PhRMA USFDA EMA
Single dose 120 120 120
<14 days 120 60 120
14 days to 1 month 120 60 60
1–3 months 40 20 20
3–6 months 20 10 10
6–12 months 10 5 5
>12 months 1.5 1.5 1.5
storage, formulation of dosage forms, and aging [8, 9]. For
instance, penicillins and cephalosporins are classic examples
for the formation of degradation products [10]. Another exam-
ple is the formation of oxidative degradation products such
as hydroperoxides/epoxides and hydrolytic products such as
anilines are of potential genotoxicity concern. In addition,
the components in excipients may react with APIs or its
counterions and forms a new class of impurities, which
have the genotoxic concern (e.g., halogenated furanones) [11],
this burdens the drug development process with additional
roadblocks.
Regulatory guidance acknowledge that the identified
PGIs are unusually toxic and require lower reporting, iden-
tification, and qualification limits than outlined in the Inter-
national Conference on Harmonization (ICH) guidelines for
typical impurities in drug substances or drug products [12,13].
A guidance on the genotoxic impurities from the European
Medicines Agency (EMA) was issued in 2006 followed by
three rounds of questions and answers, providing a clarifi-
cation on the aspects associated with this guidance [14, 15].
The U.S. Food and Drug Administration (USFDA) draft guid-
ance was subsequently issued in 2008, providing the direction
greatly consistent with the concepts as outlined in the EMA
guidance. Therefore the EMA and USFDA have strengthened
their guidelines to control the limit of genotoxic impurities in
new commercial drugs. Both the agencies have set a thresh-
old of toxicological concern (TTC) of 1.5 ␮g/person/day for
each impurity, which can be considered as an acceptable qual-
ification threshold for supporting a marketing authorization
by EMA and USFDA [16, 17]. As such, pharmaceutical re-
search and development (R&D) has devoted great efforts to
the development of manufacturing processes that can effec-
tively control the PGIs below TTC levels. During the clinical
development stages, a staged TTC applies where greater daily
intake can be allowed for shorter dosing durations as pre-
sented in Table 1 [18, 19]. The methods capable for such a
low level of detection are uncommon in the context of tra-
ditional pharmaceutical analysis, where the typical level of
interest is above 0.05% (equivalent to 500 ppm), which poses
significant challenge on analytical method development
for controlling these impurities. Therefore the usage and

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J. Sep. Sci. 2015, 00, 1–16
generation of potential genotoxic chemicals in any new manu-
facturing processes should be minimized whenever possible,
but the complete removal of PGIs from the drug substances
or drug products is often impractical. This necessitates that
the PGIs should be tested in the final products to ensure
the product quality and patient safety. Moreover, from the
process understanding point of view, the analytical methods
that are capable of measuring the PGIs at trace levels are
needed to monitor the fate of PGIs during chemical synthe-
sis. This in turn allows for the establishment of effective PGI
control strategies. In response to the USFDA recent quality-
by-design initiative, the pharmaceutical industry has been
struggling to gain process understanding and to implement
the process controls. This requires analytical chemists to de-
liver sensitive and robust analytical methods to support pro-
cess development, thereby an effective control strategy can
be drafted and implemented to ensure the API quality with
respect to PGIs concentration [20]. Furthermore, in cases
where multiple structurally similar PGIs are identified, they
are assumed to have same mode of action and similar mech-
anisms for toxicity, and might exert effects in an additive
manner. Therefore EMA recommended that the appropriate
individual limit should be applied to the sum of structurally
similar PGIs. Hence the TTC becomes the total exposure to
all of the related compounds and the sum of the genotoxic
impurities should not exceed 1.5 ␮g/day [21]. These consid-
erations pose tremendous a challenge on analytical method
sensitivity, and lower the analytical testing limit by several
folds beyond an already very low limit, which brings addi-
tional challenges to the analytical scientists in pharmaceutical
R&D.
Ideally, conventional analytical instrumentation in the
pharmaceutical analysis such as HPLC with UV detection
(for typical nonvolatile analytes) or GC with flame ioniza-
tion detection (FID; for volatile small molecules) should be
employed as the choice of techniques for PGIs analysis, but
these are often inadequate for accurate determination of ana-
lytes at low ppm levels depending on the properties of an-
alytes and sample matrices. In the past few years, much
progress has been attained in the development of highly
sensitive analytical methods for the analysis of various PGIs
in pharmaceuticals [22]. Although conventional HPLC–UV
methods are sometimes viable options [23], recently hyphen-
ated MS has gained popularity in this field [24–28] due to
its superior sensitivity and specificity. Because of the ad-
vantage of mass-selective detection, MS methods are gen-
erally less prone to interference compared to the nonspe-
cific detectors such as UV detector. Therefore, the efforts
required for method development regarding the PGIs anal-
ysis was greatly reduced. With regard to the ionization tech-
niques in LC–MS, API, which includes ESI and APCI are the
most popular tools because of their simplicity and reliabil-
ity. Consequently, in the past few years analytical scientists
in the pharmaceutical industry have strived to develop an-
alytical strategies to meet the regulatory guidance [29]. As
a result, various types of sample introduction methodolo-
gies, different chromatographic separation tools, and various
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J. Sep. Sci. 2015, 00, 1–16
kinds of detectors have been explored and demonstrated as
useful approaches [30–35]. An increasing number of publica-
tions with regard to individual genotoxic impurity or a spe-
cific class of impurities has appeared in the recent literature
[36–38].
However, practical guidance for the analytical determi-
nation of some paramount classes of PGIs is still lacking.
Therefore, this review is focused to explore recent advances
in the analysis of some important classes of PGIs that are
commonly encountered during the process development. It
also encompasses the trends in recent literature, together
with several practical approaches developed in the authors’
laboratory in recent years. As such, this article is organized
into the following main sections: (i) genotoxic impurities
identification, assessment, and control strategies, (ii) struc-
tural alerts of commonly encountered PGIs and various reg-
ulatory guidance issued to control their limit in final APIs,
(iii) toxicology assessment and analytical strategies includ-
ing the selection of analytical technique for the analysis of
PGIs, and (iv) case studies for the determination of some im-
portant classes of PGIs including hydrazines, alkyl halides,
phenols, and benzene derivatives. It is the authors’ inten-
tion to provide an industrial perspective with respect to the
identification, assessment, and control strategies for the PGIs
that guides the analytical communities in the pharmaceutical
industry.
2 Genotoxic impurities assessment
and control
Usually genotoxic impurities can be identified by any of the
following methods. (i) Already known genotoxins, (ii) pos-
sessing similar functional groups with known genotoxins,
(iii) testing positive by genotoxicity assays, (iv) marked as a po-
tential genotoxin by one of many computer-based structure–
activity software programs.
The main focus of the risk assessment is to monitor the
impurities that arise particularly in the penultimate and final
synthetic stages. If the genotoxic impurity is formed during
the API synthesis, elimination may be achieved by changing
the synthetic route and reagents used. However, this may not
be feasible as the API synthesis is quite complicated and lim-
ited based on the chemistry and available reagents making
these approaches impractical. Another means of genotoxic
impurity control in this situation is the implementation of
additional purification steps to the synthetic route to get rid of
the impurities. However, this is not effective if the genotoxic
impurity forms after the API synthesis (e.g., API degradation
or reactions with excipients/containers). Hence the impu-
rity levels are still needed to be measured and monitored in
pharmaceuticals [39].
The genotoxic impurity testing point can vary and needs
to be determined with scientific basis and supporting data
[40]. The testing strategy for PGIs analysis is shown in Fig. 1.
For instance, an impurity that has formed during the API

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Liquid Chromatography 3
synthesis may only require testing in the intermediate (and
not the final product) that immediately follows the introduc-
tion of the impurity. The synthetic process may significantly
purge the impurity during various reaction steps such as pro-
cessing and purification. Further testing is required in later
intermediates, only if the amount of impurity in the first
intermediate tested exceeds a predetermined level. This pre-
determined level can be found by spiking studies that relate
impurity survival throughout the synthesis. Obviously, the
testing point and impurity level specifications will vary on
a case-by-case basis. The additional work involved in con-
trolling the genotoxic impurities level is the development
and validation of analytical methods, which can significantly
impact the drug development timeline. Because of this, a
strong risk assessment approach is needed to be applied for
the developmental process that balances patient safety with
the control of PGIs. Otherwise, it needs more time and re-
sources and there is a chance to delay the drug to reach the
market.
It is relatively easy to perform a risk assessment on the
synthetic route and identification of all theoretical PGIs that
could be presented in the final API. Such approaches produce
a significant number of theoretical PGIs and additional chem-
ical reasoning should be used to evaluate their significance,
particularly if they arise at an early stage in a long synthetic
route [41]. This kind of exercise can be applied based on the
known reactivity of a particular PGI in conjunction with the
known downstream chemistry. Some industry experts have
raised their concerns toward risk assessment of the synthetic
processes, namely, Snodin et al. argued that highly reactive
reagents (possessing a structural alert for genotoxicity) that
are utilized in the early stages of complex synthetic pathways
are unlikely to be carried over to the final API [42]. Similarly,
Pierson et al. reasoned that most PGIs raised from reagents
or synthetic intermediates are likely to be eliminated or sig-
nificantly reduced by the downstream chemistries and pro-
cesses [41]. In the recent EMA questions and answer update,
the EMA does describe different scenarios based on where
the impurity is introduced into the synthetic route. If the
impurity is introduced before the final stage of the synthe-
sis then it can be omitted from the final API specification.
In contrast, if the impurity is introduced into the final stage
of the synthesis then it should be included in the final API
specification.
3 Structural alerts of commonly
encountered PGIs
Knowledge about the chemical functional groups that cause
DNA mutations have been used to develop computer-based
programs such as deductive estimation of risk from exist-
ing knowledge (DEREK), multicomputer-automated struc-
ture evaluation (MCASE), and TOPKAT for the prediction
of potential genotoxicity [43–45]. A positive in silico result
from a chemical structure infers potential genotoxicity. This
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4 A. V. B. Reddy et al.
potential alert is usually investigated by a bacterial reverse
mutation test such as the “Ames test” by which DNA re-
active genotoxins can be identified [46]. Conversely, a clear
negative result in an appropriate genotoxicity test usually in-
dicates the absence of genotoxicity [47]. For the purpose of
deciding whether a given impurity possesses a genotoxicity
risk as well as to control the PGIs below TTC level, Mueller
et al. [48] has classified the impurities into groups based on
their respective genotoxicity potentials: Class 1 impurities are
known to be both genotoxic and carcinogenic; Class 2 impu-
rities are genotoxic but with unknown carcinogenic potential;
Class 3 impurities are structural alerts with unknown geno-
toxic potential (namely PGIs) and for which the structures
are unrelated to the API structure; Class 4 impurities are
structural alerts but share the alerting structure with the API;
Class 5 impurities are not structural alerts, they are controlled
as ordinary impurities covered by ICH Q3 guidelines [49, 50]
(Fig. 2).

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J. Sep. Sci. 2015, 00, 1–16
Figure 1. Decision tree for PGIs testing strategy
and assessment.
Some of the commonly encountered potentially geno-
toxic structural moieties are illustrated in Fig. 3 [51]. One
important group among them is alkylating agents, such
as alkyl halides [52], alkyl sulfonates [53], and other re-
lated structures. These molecules are generally used as
reagents or might be generated during the chemical synthe-
sis. These result in cytotoxicity with the effect of inhibition
of the cell growth, initiation of programmed cell death, or
apoptosis, examples include: ␤-propiolactone, dimethyl sul-
fate, diepoxybutane, anticancer drugs. In addition, aromatic
amines and nitro compounds do not directly form cova-
lent bonds with DNA bases, but these molecules undergo
metabolic activation to arylnitrenium ions, which will then
react with DNA bases and become genotoxic, the example
includes: 2-naphthylamine [54, 55]. Acrylates are Michael
acceptors that are susceptible to nucleophilic additions,
although recent data demonstrated that ethyl acrylate is
nongenotoxic in humans [56]. Epoxides and aziridines can
Figure 2. Genotoxic impurities control and clas-
sification strategy.
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J. Sep. Sci. 2015, 00, 1–16 Liquid Chromatography 5

Figure 3. Structural alerts of com-

monly encountered PGIs.

alkylate DNA via ring-opening reactions [57, 58]. Some hy- Table 2. Drug substance impurity thresholds
droperoxides can result in oxidative damage to DNA, and
their degradation products may react with DNA. Hydrazines Maximum Reporting Identification Qualification
are also known genotoxic impurities and potential human daily dosea) thresholdb),c) thresholdc) thresholdc)
carcinogens [59]. Figure 3 is by no means an exhaustive list
ࣘ2 g/day 0.05% 0.10% or 1.0 0.15% or 1.0
of PGIs, but is presented to show the representative structural
mg/day mg/day
features of some crucial PGIs, those frequently required for
(whichever (whichever
the analysis during the chemical synthesis of drugs. is lower) is lower)
>2 g/day 0.03% 0.05% 0.05%

4 Regulatory aspects a) The amount of drug substance administered per day.

There are several regulatory guidelines and position papers
focused on controlling the amount of PGIs in pharmaceuti-
cals using specified limits. Different organizations from in-
dustry and regulatory authorities have developed guidelines
specifically addressing genotoxic impurities. Pharmaceutical
regulatory agencies such as USFDA, ICH, and EMA have
raised concerns on the presence of PGIs in APIs and issued
the guidelines to control their limits in APIs. To address
their concerns, R&D scientists have to identify the PGIs in
advance during the process development, develop the proper
analytical methods, and also need to demonstrate the control
strategies of synthetic processes leading to PGIs. The accep-
tance criterion for impurities in the drug substances should
be set lower than the qualified level to protect the patients
(Table 2).
4.1 ICH guidelines
According to ICH guidance, the impurities in drug sub-
stances and drug products are potentially toxic and give no
b) Higher reporting threshold should be scientifically justified.
c) Lower threshold can be appropriate if the impurities are un-
usually toxic.
benefits to the patients. For instance, ICH Q3A15 regulates
the impurities in new drug substances with thresholds for
reporting, identification, and qualification of impurities [60].
ICH Q3B16 and ICH Q3C17 are equivalent guidelines for
impurities in new drugs, which control residual solvents. De-
pending on the potential risk to human health, residual sol-
vents are categorized into three classes [61]. Class I solvents
should be avoided, class II solvents have permitted daily ex-
posure limits, and class III solvents have no health-based
exposure limits if the daily exposure is ࣘ50 mg/day. ICH
Q3D is currently under development and will include ele-
ments and limits for heavy metal impurities [62]. Currently
released ICH guidelines for impurity limits are not suitable
for most genotoxic impurities. The main issue related to geno-
toxic impurities is that the synthesis of drug substances often
requires the use of reactive materials that have the capacity
to interact with human DNA and cause mutations, as well as
cancer even at extremely lowest levels. Therefore, genotoxic


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6 A. V. B. Reddy et al.
impurities should be avoided, and if this is not possible they
must be reduced below a defined threshold.
4.2 USFDA draft guidance
In December 2008, the USFDA published a draft guidance
entitled “Genotoxic and Carcinogenic Impurities in Drug
Substances and Products-Recommended Approaches” [63].
Recently, it has been replaced by the ICH M7 guideline
on “Assessment and Control of DNA Reactive Impurities
(PGIs) in Pharmaceuticals to Limit Potential Carcinogenic
Risk.” The USFDA guidelines have provided the specific rec-
ommendations regarding the safety qualification of impu-
rities with known/suspected genotoxicity. These guidelines
describe a variety of ways to characterize and reduce the po-
tential cancer risk associated with the patient exposure to
genotoxic and carcinogenic impurities. The recommended
approaches include (i) prevention of genotoxic and carcino-
genic impurities formation, (ii) reduction of genotoxic and
carcinogenic impurities level (maximum daily allowable tar-
get of 1.5 ␮g/day), (iii) additional characterization of geno-
toxic and carcinogenic risk, and (iv) considerations for flex-
ibility in approaches to better support appropriate impurity
specifications.
4.3 EMA guidelines
The EMA is one of the pioneering regulatory bodies to im-
pose detailed guidelines to handle genotoxic impurities. In
June 2006, the EMA’s CHMP published the final guideline
on the limits of genotoxic impurities. The document applies
to the genotoxic impurities in new drug substances. It also ap-
plies to new applications for existing active substances, where
assessment of the route of synthesis, process control, and im-
purity profile does not provide reasonable assurance. EMA
guidelines on the limit of PGIs are classified into two cate-
gories. (i) PGIs with sufficient (experimental) evidence for a
threshold-related mechanism, these are to be regulated using
methods outlined in ICH Q3C (R4) for class 2 solvents [64]
and (ii) PGIs without sufficient (experimental) evidence for
a threshold-related mechanism, these are to be controlled in
accordance with “as low as reasonably practicable” (ALARP)
principle. Although the second approach is acceptable in
most instances, sufficient mechanistic data that allows the
assessment of threshold mechanism is still lacking. Hence,
the EMA guidelines proposed the use of “TTC” concept,
which refers a threshold exposure level to compounds that
do not pose a significant risk of carcinogenicity or other toxic
effects.
A TTC value of 1.5 ␮g/day intake of PGIs is considered to
be associated with an acceptable risk [65]. The concentration
limit in ppm of a PGI permitted in drug substance is the
ratio of TTC in microgram per day and daily dose in gram
per day. The TTC approach benefits the consumers, indus-
try, and regulators by avoiding unnecessary toxicity testing

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J. Sep. Sci. 2015, 00, 1–16
and safety evaluations. This guideline has summarized its
recommendations in the form of a decision tree in which
the preferred option is to eliminate PGIs, second preference
is to apply ALARP principle, and the final alternative is the
TTC approach. EMA also released “Question and Answer”
document with clarifications for the questions raised in its
original guidance [66].
4.4 PhRMA approach
The Pharmaceutical Research and Manufacturing Associa-
tion (PhRMA) formed a task force in 2004 to discuss about
testing, classification, qualification, and toxicological risk as-
sessment of PGIs in pharmaceuticals [48]. It stipulates that,
some structurally alerting functional groups are known to
be involved in reactions with DNA. These DNA-affecting
functional groups were categorized into three groups viz.,
Group 1: aromatic groups, for example, N-hydroxyaryls, N-
acylated aminoaryls, aza-aryl N-oxides, aminoaryls and alky-
lated aminoaryls, purines or pyrimidines, intercalators, etc.,
Group 2: alkyl and aryl groups, for example, aldehydes, N-
methylols, N-nitrosamines, nitro compounds, carbamates,
epoxides, aziridines, propiolactones, propiosultones, beta
haloethyl, hydrazines, and azo compounds, Group 3: het-
eroaromatic groups includes Michael-reactive acceptors, alkyl
esters of phosphonates or sulfonates, haloalkenes, primary
halides (alkyl and aryl-CH2 ). PhRMA categorized the impu-
rities into five classes. Class 1: impurities known to be both
genotoxic (mutagenic) and carcinogenic. These impurities
represent the most serious risk and the default preference
is to eliminate them by modifying the process. If this is not
possible, the TTC concept must be employed as a last option.
Class 2: impurities known to be genotoxic (mutagenic) but
with unknown carcinogenic potential. These impurities are to
be controlled using TTC principles. Class 3: impurities con-
taining alerting structures, unrelated to the structure of the
API, and of unknown genotoxic (mutagenic) potential. This
group includes impurities with functional moieties that can
be linked to genotoxicity based on the structure. Class 4: im-
purities containing alerting structures related to the API. This
group includes impurities that contain an alerting functional
moiety that is shared with the parent structure. Class 5: im-
purities with no alerting structures shall be treated as normal
impurities and controlled according to the ICH guidelines.
If class 3 or 4 compounds are genotoxic or not tested, they
are moved into class 2, and if they are nongenotoxic, they are
considered as class 5.
4.5 European pharmacopoeia guidelines
European pharmacopoeia required a practical approach to
elaborate or revise monographs on PGIs control. It says
that, the products that received the marketing authorization
after the issuance of the EMA guidelines [67] have to be
evaluated for the presence of PGIs and this should be the
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J. Sep. Sci. 2015, 00, 1–16
basis for a new monograph [68]. The ICH S9 guideline on
nonclinical evaluation for anticancer pharmaceuticals [65]
also states that, “for genotoxic impurities, several approaches
have been used to set limits based on the increase in life-
time risk of cancer.” Such limits are not appropriate for
pharmaceuticals intended to treat patients with advanced
cancer and justification should be considered to set higher
limits.
5 Toxicology assessments
Toxicology assessment should be done by pharmaceutical
scientists and toxicologists to identify PGIs and their entry
into the synthetic process [69–71]. In addition, the synthetic
route must be reviewed by a team of process chemists, an-
alytical chemists, and toxicologists to identify likely reaction
byproducts and their potential for carry through to the APIs.
Utilizing the experts opinion, PGI alert structures can be
identified among the compounds for which no data is avail-
able using either of the commercial software applications
including MDL-QSAR [72], MC4PC [73], and DEREK for
Windows [74]. Articles containing several structural alerts
were published [75–79]. However, due to the uncertain rel-
evance of the structural alerts, regulatory action cannot be
solely based on the presence of a particular functional group.
The accuracy of the predicted genotoxicity should be evaluated
case by case based on the available literature and genotoxicity
test results.
If PGIs are introduced as starting materials and/or
reagents or synthesized in the form of an intermediate or reac-
tion byproduct, different approaches must be considered for
the identification of structural alerts. Compounds that yield
negative results in the alert assessment are placed in Class
5 and no additional action beyond normal impurity monitor-
ing is needed. For any positive result (Classes 3 and 4), the
samples must be submitted for in vitro mutagenicity testing,
usually with the Ames or mini-Ames test [80]. If the muta-
genicity test is negative, this overrides the alert assessment
and the compound is placed in Class 5. Compounds giving a
positive mutagenicity results are placed in Class 2 [81]. The
need for toxicology assessment depends on the PGIs entry (as
starting materials or as intermediates or as reagents) in the
process and opportunities for their removal. For byproducts
that are known to be genotoxic, a toxicology limit is obtained
regarding acceptable levels in the API. For byproducts that
have alerting structures, isolation or preparation of material
may be required for mini-Ames testing. In general, theoret-
ical byproducts that are not anticipated to be formed are not
assessed. This is consistent with EMA guidance recommend-
ing that the assessment of genotoxicity could be limited to
those impurities that might be reasonably expected on the
basis of the chemical reactions and conditions involved [82].
Scientific judgment is required to balance the potential for
impurity formation and carry through with consideration of
safety risk that would be caused by the presence of the impu-
rity in the API.

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Liquid Chromatography 7
6 Considerations for analytical testing
6.1 Analytical strategies
Analysis of PGIs in pharmaceuticals is the challenging task
because they must be controlled at levels significantly lower
than 0.01–0.03%. Ideally, the analytical procedure should al-
low the detection limits in the range of 1.0–5.0 ppm (0.0001–
0.0005% w/w). The analysis at such lower levels require not
only sensitive analytical instruments, but also place higher
demands on selectivity because more number of other or-
ganic impurities may be present at lower concentrations, for
example, from excipients. The relatively large amount of API
may also interfere with low-level impurities. Furthermore,
it is very difficult to analyze all PGIs with a single ana-
lytical procedure because they contain different functional
groups and come from different sources. In addition, due
to their highly reactive nature most of the PGIs can eas-
ily react during extraction, sample preparation, or analysis
and may cause inaccurate results. Also, some of the geno-
toxic impurities are low-molecular-weight compounds that
are quite volatile and therefore they might disappear during
the sample preparation step. Depending on the nature and
quantity of the genotoxic impurity being investigated, care-
ful investigation must be needed for the selection of appro-
priate analytical techniques (or a combination of analytical
techniques).
Numerous articles have appeared in the scientific litera-
ture mainly for the analysis and testing of PGIs in APIs. But,
the general API determination methods are not suitable for
the determination of PGIs due to their lower quantitation lim-
its (QLs). In the analytical point of view, the actual QL should
be much more sensitive than the PGIs concentration limit. In
addition to this, certain drug substances may generate PGIs
via degradation or storage and are to be separated from pro-
cess impurities to achieve specificity in the analysis [83–86].
Besides the sensitivity and specificity, the other challenges
in PGIs determination includes (i) diverse structural types of
PGIs that require the application of various analytical tech-
niques, (ii) PGIs without structural features are not favorable
to common analytical detectors, (iii) chemically reactive or
unstable PGIs lead to low recovery and poor sensitivity, and
require special handling techniques, (iv) interference of sam-
ple matrix resulted by enhanced test concentration of API to
achieve lower detection limits [87]. Sample solubility is also
an important issue, because high concentrations of samples
are often required to enable the analysis of low-level geno-
toxic impurities in the sample. Achieving high solubilities
can often be a significant challenge, and there is also a risk
of the sample precipitating out of the solution especially if
the sample solution needs to be cooled to slow the degra-
dation. In addition to the solubility requirements, param-
eters such as detection limit, quantification limit, linearity
and range, accuracy, and solution stability are to be estab-
lished as per ICH Q2(R1) validation guidelines [87]. How-
ever, the extent of validation depends on the purpose of the
study.
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8 A. V. B. Reddy et al.
6.2 Selection of analytical techniques
The selection of an analytical technique for the determination
of PGIs can be divided into two groups based on their volatil-
ity. HPLC with UV detection should be considered as first
choice for the analysis of nonvolatile PGIs due its simplic-
ity and availability [88]. However, HPLC–UV may not offer
sufficient sensitivity for certain PGIs in trace level analysis.
If PGIs offer insufficient UV response, UHPLC or ultrafast
LC can be used because they have enhanced UV-detector
sensitivity. UHPLC methods are more sensitive than HPLC
because of the holistic design with advanced technology in the
pump, detector, and autosampler, which has evolved from the
HPLC [89,90]. As a brief description, UHPLC is just a special
variant of HPLC, but two properties makes UHPLC more
practical and in use worldwide, that is, having high-quality
small porous packing material and the capability of very high
pressure. The speed, resolution, and sensitivity of the detec-
tor cannot be compromised as compared to the traditional
HPLC. Now it is possible to take the full advantage of chro-
matographic principles to run separations using speed, supe-
rior resolution, and sensitivity afforded by smaller particles.
When PGIs lack chromophores, evaporative light-scattering
detection is the alternative choice. But, this detector is limited
in sensitivity and dynamic range. Refractive index detector
and fluorescence detector are the other alternative detectors
used in HPLC. However, lower QL establishment is always a
challenging task in the PGIs analysis.
As the trend toward smaller and smaller analyte con-
centrations progresses, conventional analytical methods are
becoming less effective to address the PGIs at trace levels.
Hence, attention in the last few years has shifted to the appli-
cation of hyphenated techniques as preferred choice for the
identification and characterization of PGIs at trace level. Hy-
phenation of GC and HPLC/UHPLC with MS significantly
improves the method sensitivity and makes the methods
more rapid [91, 92]. Broadly, the available hyphenated instru-
ments are GC, LC, or CE on the front end and MS, NMR, or IR
on the detection side, for example, GC–MS, LC–MS, CE–MS,
LC–NMR, LC–NMR/MS, CE–NMR, LC–IR, etc. In particular,
LC–MS instruments are commercialized in multiple variants.
These hyphenated instruments are slowly penetrating into
the analytical laboratories worldwide for the characterization
of new emerging impurities [93–95].
MS detectors are highly selective and sensitive, which are
capable of minimizing the issues caused by the interferences
in the sample matrices. However, hyphenated instruments
are expensive, differ from vendor to vendor, and therefore
they need optimization of instrumental parameters while
transferring the methods between the development stages
to receiving laboratories [96]. In combination with FID, GC is
the standard choice for the analysis of small molecule PGIs.
Headspace gas chromatography (HS-GC) methods allow the
determination and identification of volatile low-molecular-
weight organic compounds, volatile degradation products,
and other volatile compounds. HS-GC–FID can be used to im-
prove sensitivity of the analytes and minimize interferences

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J. Sep. Sci. 2015, 00, 1–16
Figure 4. Decision tree for the selection of analytical technique in
PGIs analysis.
from the sample matrix. Headspace injection is desirable be-
cause it minimizes potential contamination of the injector or
column by avoiding the introduction of a large quantity of
API. More recently, GC coupled with MS (GC–MS or GC–
MS/MS) has gained attention for the confirmation and iden-
tification purposes, highlighting the flexibility of this tech-
nology [97–100]. If PGIs are labile, possess no chromophores,
and have reactive functional groups, they can be derivatized to
form detectable species (e.g., hydrazine derivatizes benzalde-
hyde to form 1,2-dibenzylidenehydrazine) [101]. Derivatiza-
tion reagent can be selected based on the functional groups
in the analyte. Derivatization helps in stabilization, incorpo-
ration of a unique structural moiety, enhancing fluorescence,
ionization for mass detection, and volatization for GC. The
proposed decision tree for the selection of analytical tech-
nique is shown in Fig. 4.
MS-based methods generally provide additional robust-
ness and ruggedness compared to the techniques such as UV
alone, due to their high specificity and sensitivity. Highly sen-
sitive Q-TOF mass spectrometers provide higher resolution
and mass accuracy that enables the unambiguous identifica-
tion of unknown trace impurities, making them very useful
for PGIs analysis. Usually MS-based methods are often se-
lected for the impurity profiling of APIs during process de-
velopment, while UV-based methods are generally selected
for the testing of PGIs in QC laboratories at manufacturing
sites.
7 Case studies for the determination of
some crucial PGIs
The following sections provide the analytical strategies for
the analysis of some important classes of PGIs that have
been carried out recently in our research laboratory.
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J. Sep. Sci. 2015, 00, 1–16
7.1 Analysis of hydrazines and related analogues
Hydrazines are commonly used as starting materials in the
synthesis of some pharmaceuticals. Some N-alkyl, N-aryl,
and N-acyl analogues have been subjected to extensive tox-
icological evaluations [88]. Hydrazine adducts with DNA and
the mechanism of adduction could include the formation of
methyldiazonium ions or methyl free radicals. In addition, it
seems that the hydrazines react with endogenous formalde-
hyde to produce formaldehyde hydrazones [91]. In a study,
20 hydrazine derivatives were found to induce a direct DNA
damage in Escherichia coli and 16 of them (80%) were Ames
positive as well [88, 92, 96]. Other methods have reported that
hydrazine, methyl hydrazine, 1,1-dimethylhydrazine, and 1,2-
dimethylhydrazine and other analogues are carcinogenic in
rodents and possibly in human. Besides this, it was also found
that the hydrazine derivatives like hydralazine and its hy-
drochloride salts were tumorigenic in rodents [92, 96].
A study was reported by Hmelnickis et al. [97] for
the separation of six polar impurities including the hy-
drazine and 1,1,1-trimethylhydrazinium bromide impurities
in mildronate API using HILIC method. Another sensi-
tive analytical method was developed by Liu et al. [98] us-
ing HILIC for the direct determination of hydrazine and
Figure 5. Specificity chromatogram of SHH, MAP, and celecoxib. Chromatographic separation was achieved on Symmetry C18 column
(150 mm × 4.6 mm, 3.5 ␮m) kept at 40⬚C, using the mobile phase of 5.0 mM ammonium acetate-acetonitrile in the ratio of 30:70 (v/v), with
a flow rate of 0.7 mL/min, and the injection volume of 10 ␮L [103].

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Liquid Chromatography 9
1,1-dimethylhydrazine in a pharmaceutical intermediate at
LOQ concentration of 200 ppm. In addition, several research
groups used nitrogen-selective detection for the characteriza-
tion of hydrazine analogues [99, 100], but Kirtley et al. [101]
commented on the poor sensitivity of nitrogen phospho-
rous detection and they found inadequate LOD to address
the analytical issues. Static headspace techniques combined
with quadrupole electron ionization (EI) detection with se-
lected ion monitoring (SIM) have been routinely used by
Sun et al. [102]. These authors reported a generic in situ
derivatization HS-GC–MS method for the determination
of hydrazine at low concentrations. Unlike alkyl halides,
hydrazine analogues commonly needed derivatization as one
of the main analytical strategy toward enhancing the sensi-
tivity and addressing volatility.
To overcome the difficulties in the analysis of hydrazine
analogues, a simple and sensitive LC–MS/MS method
was developed for the simultaneous determination of
two hydrazine PGIs namely (4-sulfamoylphenyl)hydrazine
hydrochloride (SHH) and (4-methyl-acetophenone)-
p-sulfonamide phenylhydrazine hydrochloride (MAP) in
celecoxib without any derivatization [103]. Better separation
was achieved with symmetry C18 (150 mm × 4.6 mm,
3.5 ␮m) column in positive ESI using multiple-reaction
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10 A. V. B. Reddy et al. J. Sep. Sci. 2015, 00, 1–16

Figure 6. Specificity chromatogram of ritonavir and its three phenol impurities viz., phenol, 4-nitrophenol, and NPV. Chromatographic
conditions: Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 ␮m) kept at 35⬚C, mobile phase consisted of 5.0 mm ammonium
acetate–0.05% ammonia in methanol 30:70 (v/v), mobile phase flow was 0.2 mL/min, and the injection volume was 8 ␮L [106].

monitoring mode. To achieve the proper separation of SHH
and MAP from celecoxib, different mobile phase solvents at
various compositions were evaluated, 5.0 mM ammonium
acetate-acetonitrile (30:70, v/v) was found to be the most
suitable composition at a flow rate of 0.7 mL/min. The
method showed good linearity over the concentration range
of 0.1–2.5 ppm for SHH and 0.06–2.5 ppm for MAP PGIs.
The LOD and LOQ of SHH are 0.03 and 0.1 ppm, for MAP
they are 0.02 and 0.06 ppm, respectively. The method has
good recovery in the range of 95.0–104.0% for both the
analytes. The developed method was also applied for the
determination of SHH and MAP PGIs in three formulation
batches of celecoxib and the specificity chromatogram is
shown in Fig. 5.
7.2 Analysis of phenol impurities in ritonavir
The available data regarding the genotoxicity and car-
cinogenicity of phenols and its analogue impurities are
inconclusive. But the reported data (RIVM 2001) on animal
testing suggested that the phenol can act as a tumor pro-
moter. ATSDR (2008) noted that “under certain conditions,
especially at high doses” phenols have the potential to exhibit
genotoxicity [104, 105].
In the present study, we have developed an UHPLC–
MS/MS method for the simultaneous determination of phe-
nol, 4-nitrophenol, and N-phenoxycarbonyl-L-valine (PCV) in
ritonavir. Initially the method development was started with
LC–MS/MS for the determination of three closely related
phenol impurities namely phenol, 4-nitrophenol, and PCV.
But the separation and peak shapes are not satisfactory with
LC–MS/MS method. Therefore as the next option, UHPLC–
MS/MS was chosen for the simultaneous determination of
three phenol impurities in ritonavir. Good separation was
achieved on Acquity UPLC BEH C18 column (2.1 mm ×
100 mm, 1.7 ␮m) using gradient elution of 0.05% ammo-
nia in methanol (30:70, v/v) and 5.0 mM ammonium acetate
buffer at a flow rate of 0.2 mL/min. Several attempts were
made with different C18 UHPLC columns (Inertsil ODS-3,
50 mm × 2.1 mm, 2.0 ␮m and Zorbax XDB C-18, 50 mm × 4.6
mm, 1.8 ␮m) using the gradient elution, but phenol and PCV
were merged together, 4-nitrophenol was coeluted with riton-
avir using the above columns excluding the UPLC BEH C18
column. The calibration curves showed good linearity over


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J. Sep. Sci. 2015, 00, 1–16 Liquid Chromatography 11

Figure 7. Specificity chromatogram and mass spectra of 2-chloromethyl-3,4-dimethoxy pyridine hydrochloride and pantoprazole. Chro-
matographic conditions: Hypersil BDS C18 column (50 mm × 4.6 mm, 3.0 ␮m) kept at 40⬚C, mobile phase consisted of 10 mm ammonium
acetate/acetonitrile 79:21 v/v at a flow rate of 1.0 mL/min, and the injection volume of 20 ␮L [117].

the concentration range of 0.3–1.5 ppm for phenol and 0.1–
1.5 ppm for both 4-nitrophenol and PCV. The method has
very low LOD and LOQ and the accuracy lies between 97.8 and
103.2% for all the three impurities. The developed method
was successfully applied for the formulation batches of riton-
avir to determine its phenol impurities [106]. The specificity
chromatogram is shown in Fig. 6.
7.3 Analysis of alkyl halides
Owing to the electrophilic nature of alkylating agents, they
can introduce lesions at nucleophilic centers of DNA. Strong
acid–base interactions during the process of drug salt forma-
tion produce alkylating agents such as alkyl halides and alkyl
esters of alkyl sulfonic acids. As the salt formation is a com-
mon method in drug formulation processes, alkyl halides
exist as impurities in several drug substances and leads to
mutagenicity [107,108]. The nucleophilic attack mechanisms
of alkylating compounds determine their reactivity against
DNA [109].
Historically, analysts have relied on the volatility of alkyl
halides and developed the GC methodologies with FID for
their separation and quantification. GC still remained as the
preferred technique in many cases, but is now more routinely
used in combination with more sensitive and selective detec-
tion techniques, such as GC–MS or GC–ECD. HPLC also
remained as a key separation technique for those analytes
that are insufficiently volatile and/or too thermally labile for
reliable GC analysis. While many historical references em-
ployed HPLC with single-wavelength UV detection, the more
recent literature employs a variety of MS techniques [110].
Somewhat surprisingly, there are few reports in the literature
using SFC for the analysis of alkylating agents. Similarly, few
methods of CZE have been reported for the analysis of alkyl
halides.
Ellison et al. [111] reported a HS-GC–ECD method for
the determination of 23 genotoxic alkyl/aryl halides as po-
tential impurities in API. The method was generically vali-
dated with two different APIs, thereafter it was validated for
every additional API using spiked batches. The detection lim-
its for the 23 analytes ranged from 2.5 to 290 ng/mL, and
the results were greatly correlated with the results of Skett
et al. [112]. Another HS-GC method for the determination
of volatile alkylating impurities in the antiemetic, azasetron
hydrochloride was reported by Wang [113]. Further, Frost
et al. [114] reported a GC–FID method for the determination
of trace levels of pharmaceutical process impurities of toxi-
cological concern, including volatile alkylating agents, benzyl
chloride and chloroethyl methyl ether. The authors have uti-
lized a powerful combination of automated headspace and
solid-phase microextraction to concentrate the analytes. Lee
et al. [115] demonstrated that the HPLC–ESI/MS gave bet-
ter responses than APCI and was used to measure N,N-
dimethylaminoethyl chloride in diltiazem hydrochloride us-
ing SIM mode. Clarke [116] used HPLC–MS to determine


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12 A. V. B. Reddy et al.
three residual alkyl bromides (A, B, and C) in a secondary
amine API. The method gave acceptable LODs for all the
three impurities with LODs of 10 ng/g and the method was
validated as well as used to analyze and release API for clinical
use.
In our study, a sensitive and selective LC–MS/MS
method was developed for the trace level determination of
2-chloromethyl-3,4-dimethoxy pyridine hydrochloride (CDP),
a PGI in pantoprazole sodium drug substance [117]. The
separation was achieved on Hypersil BDS C18 (50 mm ×
4.6 mm, 3.0 ␮m) column using 10 mM ammonium acetate in
1000 mL of water as buffer. The mobile phase was optimized
in the ratio of buffer/acetonitrile (79:21, v/v) to achieve bet-
ter separation and high sensitivity. Trace level of ammonium
acetate is added to the mobile phase to enhance the ioniza-
tion and detection. The method was validated according to
ICH guidelines and was able to quantify CDP impurity at
0.3 ppm. The specificity chromatogram of CDP impurity and
pantoprazole is shown in Fig. 7.
Another simple and sensitive LC–MS/MS method was
developed for the determination of 1-(3-chloropropyl)-4-(3-
chlorophenyl) piperazine HCl (CCP), a process-related impu-
rity in trazodone [118]. The separation was achieved on C18
Symmetry (100 mm × 4.6 mm, 3.5 ␮m) column interfaced
with a triple quadruple tandem mass spectrometer operated
in the multiple-reaction monitoring mode. Positive ESI was
employed as the ionization source and the mobile phase used
was 5.0 mM ammonium acetate/acetonitrile (30:70, v/v). The
LOD and LOQ were found to be 0.01 and 0.03 ppm, re-
spectively, with linearity ranging from 0.03 to 2.5 ppm. The
method was fully validated by following ICH guidelines to
assess LOD, LOQ, linearity, precision, accuracy, specificity,

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J. Sep. Sci. 2015, 00, 1–16
Figure 8. Specificity chromatogram
and mass spectra of 1-(3-choropropyl)-
4-(3-chlorophenyl)piperazine and tra-
zodone. Chromatographic conditions:
C18 symmetry column (100 mm ×
4.6 mm, 3.5 ␮m) kept at 40⬚C, mobile
phase consisted of 5.0 mM ammonium
acetate-acetonitrile in the ratio of 30:70
(v/v at pH 5.0 ± 0.05) at a flow rate of
0.8 mL/min (split down to 0.2 mL/min
into the MS source), and the injection
volume of 10 ␮L [118].
and robustness. The specificity chromatogram of the CCP
impurity and trazodone is shown in Fig. 8.
7.4 Analysis of benzene derivatives in zolmitriptan
Benzene and its derivatives have the probability to increase
the likelihood of DNA damage [119]. Several compounds of
this group are capable of reacting with DNA either directly
or after metabolic transformation and become potent mu-
tagens and carcinogens [120, 121]. Therefore, in the present
study we have developed a selective and sensitive UHPLC–
MS/MS method for the simultaneous determination of
four PGIs, namely, (2S)-2-amino-3-(4-aminophenyl)propan-
1-ol, (S)-4-nitrophenylalanine hydrate, (S)-2-amino-3-(4-
nitrophenyl)propanol, and (S)-methyl-4-nitrophenylalanate
hydrochloride (APP, NPA, NPP, and MNA respectively). The
method development was started with LC–MS/MS, but no
proper separation was achieved using LC–MS/MS as well as
the peaks were merged with each other due to their structural
similarity. Therefore, further method development was car-
ried out using UHPLC–MS/MS, in which all the four PGIs
viz., APP, NPA, NPP, and MNA were separated with better
resolution. The method utilized Hypersil BDS C8 column
(50 mm × 4.6 mm, 3.0 ␮m) with ESI in selected ion record-
ing mode for the quantitation of four PGIs. The method
was able to quantitate APP at 0.1 ppm and NPA, NPP, and
MNA at 0.15 ppm with respect to 5.0 mg/mL of zolmitrip-
tan. The proposed method was validated for its specificity,
linearity, accuracy, and precision by following the ICH guide-
lines. The method was linear in the range of 0.1–2.0 ppm
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J. Sep. Sci. 2015, 00, 1–16
Figure 9. Specificity chromatogram of zolmitriptan and its four PGIs, that is, APP, NPA, NPP, and MNA. Chromatographic conditions:
Hypersil BDS C8 column (50 mm × 4.6 mm, 3.0 ␮m) kept at 35⬚C, gradient elution using 5.0 mM ammonium acetate as solution A and
acetonitrile/methanol (90:10, v/v) as solution B at time/%solution A; 0/95, 6/95, 9.0/80, 10/80, 13/70, 14/70, 20/95 using the flow rate of
0.5 mL/min, and the injection volume of 8 ␮L [122].
for APP and 0.15–2.0 ppm for NPA, NPP, and MNA and the
LOD, LOQ values were found very low with >0.999 correla-
tion coefficient [122]. The accuracy of the method was ranged
between 98.1 and 102.8% for four PGIs. The impurities were
not found in the three studied pure and formulation batches
of zolmitriptan. The specificity chromatogram of impurity
and pantoprazole is shown in Fig. 9.
8 Conclusions
The identification and control of PGIs in a synthetic pro-
cess is always challenging, owing to their evolving nature
and variable points of entry. Therefore, synthetic routes must
be screened for the identification of structural alerts, which
causes genotoxicity. If PGIs are found, alternative synthetic
routes should be adopted to control them in the final APIs. If
this is not technically feasible, the safety limits must be fixed
based on the TTC concept. These limits generally come into
lower levels and need analytical determinations with adequate
selectivity and sensitivity. Rapid developments of extremely
sensitive and selective analytical methodologies are crucial
to monitor PGIs at very low levels. Synthetic and analytical
chemistry is required to balance the risk and cost during
the development of drug substances. In recent years, MS
detection coupled with GC or HPLC played a critical role

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Liquid Chromatography 13
in the trace analysis of PGIs in various stages of preclini-
cal and clinical drug development. It is not surprising that
the vast majority of the methods end up using MS detec-
tion because of the unparallel specificity and sensitivity of
this technique. Because of their highly reactive nature, the
direct analysis of most PGIs is difficult and results in poor
recovery and sensitivity. Therefore, chemical derivatization
and coordination ion spray MS have been found as alterna-
tive analytical strategies for stabilizing and enhancing ana-
lyte detectability. Therefore, the trend clearly shows a def-
inite shift of the PGIs analysis from conventional way of
structure elucidation to the use of modern hyphenated tech-
niques. The strategies including regulatory guidance, flow
charts, decision trees, and toxicology assessment approaches
are developed for the better understanding and to explain
the risks and liabilities associated with PGIs throughout
the development process. By coupling the above practical
strategies with hyphenated MS instrumentation, PGIs anal-
ysis will experience a major leap forward over the next few
years.
This review has presented the recent analytical meth-
ods carried out in our laboratory for the quantitative analysis
of selected PGIs in pharmaceuticals. The case studies dis-
cussed in this review for the determination of some impor-
tant classes of PGIs including hydrazines and its analogues in
celecoxib, phenol impurities in ritonavir, benzene derivatives
in zolmitriptan, and finally alkyl halides in pantoprazole and
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14 A. V. B. Reddy et al.
trazodone provided a better understanding of PGIs-related
risk and assessment, such an attention is ultimately focused
on analytical control of those specific PGIs that pose a the-
oretical risk of presence in the final drug substance. All the
developed methods were fully validated for their linearity,
specificity, accuracy, precision, and robustness. The LOD and
LOQ values of all the developed methods were achieved be-
low the TTC threshold concentration. Some of the developed
methods were successfully applied for the determination of
corresponding PGIs in formulation batches of APIs. The de-
veloped methods were proved to be capable for monitoring
of the mentioned PGIs in corresponding drug substances
during their synthesis/manufacturing.
To offer a product to society that is devoid of PGIs
or acceptable to regulatory agencies, avoiding the usage of
raw materials, reagents, solvents having potential genotoxic
alerts is the most idealistic approach. But, in real life sce-
nario, it would be a difficult and complex activity to any
chemist. Therefore, it is worth to conclude the review with
the USFDA statement “Although marketed medicinal prod-
ucts are required to be safe, safety does not mean zero
risk. A safe product is one that has reasonable risks, given
the magnitude of the benefits expected and the alternatives
available.”
The authors gratefully acknowledged the postdoctoral fi-
nancial support by the Research University Grant, Universiti
Teknologi Malaysia, and the Ministry of Education Malaysia
for providing LRGS Grant on Water Security entitled “Pro-
tection of Drinking Water: Source Abstraction and Treatment
(203/PKT/6720006).”
The authors have declared no conflicts of interest.
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