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Chapter-5
Aniline/Genotoxic Impurity Determination in Mesalamine
Delayed Release Tablets by UPLC
Overview
The present chapter deals with the low level (high sensitive) determination of Aniline/Genotoxic
impurity in Mesalamine delayed release tablets using the developed and validated, stability
indicating, RP-UPLC method.
5.1 LITERATURE REVIEW
Higher sample throughput with more information per sample may decrease the time to market, an
important driving force in today’s pharmaceutical industry [1-3]. UPLC is a new category of
separation technique based upon well-established principles of liquid chromatography, which
utilizes sub-2 µm particles for stationary phase. These particles operate at elevated mobile phase
linear velocities to affect dramatic increase in resolution, sensitivity and speed of analysis. Owing
to its speed and sensitivity, this technique is gaining considerable attention in recent years for
pharmaceuticals and biomedical analysis. In the present work, this technology has been applied to
the method development and validation study of aniline determination in mesalamine delayed-
release dosage form.
Aniline is the critical genotoxic impurity to be considered for mesalamine drug substance and its
drug product. Moreover, recently the US-FDA has also published draft guidance for ‘the Genotoxic
and Carcinogenic impurities in drug substances and drug products’ [4]. This emphasises the
importance of controlling aniline impurity in drug substances and in drug product as well.
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A genotoxic activity of aniline and its metabolites and their relationship to the carcinogenicity of
aniline in the spleen of rats is reported [5] by Ernst and Bernd. Potent genotoxicity of
aminophenylnorharman, formed from aniline in the liver of gpt delta transgenic mouse is reported
[6] by Ken-ichi Masumura et al. Carcinogenicity of aminophenylnorharman, formed from aniline in
F344 rats had been reported [7] by Toshihiko Kawamori et al. A few analytical methods has also
been reported for quantification of aniline and these are: by spectrometric [8], by azo-dye formation
[9], by fluorescence [10, 11], by alternating-current oscillopolarographic titration [12], by NIR
spectroscopy [13], by GC [14], by HPLC [15, 16] and by LCMS [17, 18].
Aniline determination in mesalamine drug substance by GC is official in US Pharmacopeia [19]
and by HPLC is official in British Pharmacopeia [20].
5.2 THE SCOPE AND OBJECTIVES OF PRESENT STUDY
The comprehensive literature survey revealed that the HPLC methods used for determination of
quality of mesalamine drug substance have low sensitivity, high LOQ and long run time. Moreover,
these reported methods lack the suitable procedure for quantification and estimation of aniline.
According to our literature survey, no UPLC method has yet been reported for the determination
and quantification of aniline from mesalamine delayed-release tables. Thus, it is thought to
undertake the development and validation of reversed phase UPLC method with lower LOQ for the
quantification of aniline in delayed release mesalamine formulation.
The objectives of the present work are as follow:
Development of rapid, stability indicating RP-UPLC method for determination of Aniline in
mesalamine delayed release dosage form.
Forced degradation study.
Lower value of LOQ (limit of quantification) for Aniline determination.
Perform analytical method validation for the proposed method as per ICH guideline.
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5.3 DETERMINATION OF GENOTOXIC IMPURITIES
Ensuring the safety of pharmaceutical products is a primary responsibility of the chemists,
engineers and formulators involved in their manufacture - regardless of whether the products are
intended for commercial purposes or for clinical investigation. When focusing on safety, particular
attention is paid to the quality and purity of the raw materials used in the formulation, especially of
the active pharmaceutical ingredient(s) (API(s)). A drug substance will typically contain a range of
low-level impurity compounds, for example arising as residues of starting materials, reagents,
intermediates, or as side-products generated by the synthetic processes or degradation reactions;
these need to be understood and controlled within tight limits. The drug substance itself is unlikely
to be entirely safe, but a certain level of risk to the patient can be tolerated when weighed against
the anticipated health benefits. This balance between risk and benefit has to be finely judged by
pharmaceutical manufacturers and regulatory authorities on a case-by-case basis. Impurities,
however, are expected to bring no benefits – only risk. Manufacturers must therefore eliminate
them (or at least mitigate the risk associated with them) to the greatest extent possible.
The International Conference on Harmonization (ICH) began to publish definitive guidelines on
impurities in drug substances and drug products in the late 1990s [21, 22]. These guidelines have
been adopted by the regulatory bodies of, among others, the USA, the European Union, and Japan,
and have enjoyed wide support within the pharmaceutical industry over the intervening decade.
Under this guidance, the normal qualification threshold for impurities is 0.15% [1500 parts per
million (ppm)] or 1 mg/day, whichever is lower, for drug substances whose intake is up to 2 g/day.
Impurities which exceed this threshold must have their toxicity specifically investigated. Below the
qualification threshold, no investigation is required, although impurities at levels above 1000 ppm
(or 1 mg/day) are expected, at the least, to be identified [23]. While these thresholds are considered
adequate for the general run of process-related impurities, the guideline also recognises that
impurities which are “unusually toxic” are of increased concern and deserving of a qualification
threshold significantly lower than the default values. Subsequent guidance from the European
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180
Medicines Agency (EMEA) [24] and the U.S. Food and Drug Administration (USFDA) [25]
confirmed that the ICH thresholds may not be acceptable for genotoxic or carcinogenic impurities.
This, however, has proved to be more controversial within the industry - initially because of the
lack of clear regulatory guidance, and subsequently because of the increased stringency imposed
when more detailed guidance finally did emerge. Several commentators [26-28] have critically
reviewed the history of the evolving guidance on genotoxic impurities. Genotoxic compounds are
those which cause damage to DNA, for example by alkylation or intercalation, which could lead to
mutation of the genetic code. The terms “genotoxic” and “mutagenic” are usually employed
synonymously by chemists, although there is a subtle distinction [29]. The property, however
named, can be easily demonstrated by subjecting the chemical to standard in Vitro tests [30], the
best known being the Ames mutagenicity test. Whether a given genotoxic compound is also
carcinogenic (the real worry from the safety viewpoint) is more difficult to determine; it relies on
longer-term in vivo studies using animal models, and there is always a question of the extent to
which the results of such studies can be extrapolated to humans. The conservative approach is to
assess known genotoxic compounds as potential carcinogens unless there is experimental evidence
to the contrary.
5.4 THE REGULATORY VIEWPOINT
In December 2002 the Safety Working Party (SWP) of the European Committee for Proprietary
Medicinal Products (CPMP) published a “Position paper on the limits of genotoxic impurities”,
signaling an intention to fill the gap in ICH guidelines. Genotoxic impurities are topic selected for a
joint meeting of the Drug Information Association (DIA) and the EMEA in October 2003, where
scientific and regulatory updates are presented and discussed in the light of case studies. The
outcome of these discussions, along with other industry and regulatory comment, has been
subsequently published in a special edition of the International Journal of Pharmaceutical Medicine
[31]. The EMEA has published a guideline on the subject in June 2006 that finally came into effect
in January 2007 [24].
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The EMEA guideline recommends that any potentially genotoxic impurities (PGIs) in the drug
substance should be identified, either from existing genotoxicity data or through the presence of
“structural alerts”. PGIs should then be dichotomized into those for which there is “sufficient
(experimental) evidence for a threshold-related mechanism” and those “without sufficient
(experimental) evidence for a threshold-related mechanism.” The former category would include
inter alia compounds that induce aneuploidy by interfering with the mitotic spindle, compounds
that interfere with the activity of topoisomerase, and/or compounds that inhibit DNA synthesis. The
limits for impurities with clear evidence for a threshold mechanism can be addressed using methods
similar to those recommended by ICH for setting limits on Class 2 solvents [32]. This approach
calculates a “permitted daily exposure,” which is derived using the “no observed effect level” or,
alternatively, the “lowest observed effect level” from the most relevant animal study and
incorporating a variety of uncertainty factors. Where there is no, or insufficient, evidence that the
genotoxic impurity acts via a threshold-related mechanism, establishing a safe limit is much more
problematic. The EMEA approach has been to adopt a concept originally applied by the USFDA to
contaminants leaching from food packaging materials and known as the “threshold of regulatory
concern” [33]. This principle states that regulators ought not to be concerned with extremely low
levels of contamination where the risk of harm is negligible. Subsequent research has aimed to
quantify such a regulatory threshold, which is re-designated as the “threshold of toxicological
concern” (TTC) [34].
On the basis of an analysis of the carcinogenic potency in rodents of over 700 carcinogens, [35, 36]
it is estimated that exposures less than 0.15 μg/day of these substances are unlikely to increase a
lifetime cancer risk by more than 1 in one million; it is therefore reasonable to regard 0.15 μg/day
as a “virtually safe dose” for all but the most potent carcinogens. The actual limit recommended by
the EMEA is 1.5 μg/day, representing a 1 in 105 excess lifetime cancer risk, the extra latitude being
justified by the presumed benefit to the patient of taking the medicine. It is recognized that certain
classes of compounds, specifically aflatoxin-like, N-nitroso, and azoxy compounds, will require
even tighter control. This type of compound is unlikely to feature in a typical drug substance
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182
synthesis. However, the general TTC limit, being several orders of magnitude lower than the
normal qualification threshold suggested by ICH guidelines, still presents manufacturers with a host
of technical and analytical challenges. The EMEA guideline summarises their recommendations in
the form of a decision tree [Figure 5.1]. Their preferred option is, if possible, to eliminate the
opportunities for the genotoxic impurity(ies) to appear at all. The second preference is for
manufacturers to reduce the level(s) to as low as is reasonably practicable (ALARP principle).
Application of TTC concepts is - according to this decision tree - only third favourite [17].
[Figure 5.1 EMEA decision tree for assessment of acceptability of genotoxic impurities]
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One issue which the EMEA guideline has originally failed to address is appropriate limits for
investigational drugs. At early stages of development the process understanding which is required
to control trace-level impurities would likely be limited. On the other hand, clinical subjects are
typically exposed to investigational drugs only for limited periods - certainly not the “lifetime”
which is assumed in the TTC derivation. The industry group Pharmaceutical Research and
Manufacturers of America (PhRMA) have therefore proposed a “staged TTC” approach for the
intake of genotoxic impurities over various periods of exposure during clinical trials [37].
Extrapolating from the established acceptable intake over a lifetime, and resetting the acceptable
risk to 1 in 106 (since clinical trial subjects may be volunteers who derive no specific benefit from
the drug), they have proposed limits of up to 120 μg/day for exposures lasting up to one month,
decreasing to 10 μg/day for > 6-12 month exposures. (In all cases a maximum limit of 0.5% would
be observed on quality grounds.) This staged approach is subsequently endorsed by the EMEA in a
2008 “Question and Answer” document clarifying their original guidance [38], albeit with reduced
limits for each duration of exposure. Similar limits are proposed by the USFDA in their draft
guideline published in December 2008 [25]. A summary of the current recommendations from both
authorities, as well as PhRMA, is provided in Table 5.1.
Table 5.1 Proposed allowable daily intake (μg) for genotoxic impurities of unknown
carcinogenic potential during clinical development
Single
dose
NMT 14
days
14 days
to
1 month
1-3
months
3-6
months
6-12
months
>12 months
PhRMA
recommendation 120 120 120 40 20 10 1.5
EMEA limits 120 60 60 20 10 5 1.5
Draft USFDA limits 120 120 60 20 10 5 1.5
An example of this “staged TTC” approach has been presented by Syntagon in a recent web
newsletter [39]. They evaluated the synthetic route to an investigational API [Figure 5.2] intended
for a phase I study with a duration of 20 days and a daily dose of approximately 100 mg. They have
identified two reagents, benzene and thiourea, as known genotoxins, and the iodo- intermediate 3 as
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potentially genotoxic. For the initial clinical study they set specifications of NMT 0.06% w/w
(equivalent to 60 μg/day) for all three.
[Figure 5.2 Four last chemical steps in the manufacture of an API [39]]
It could be argued that the available compound-specific data for benzene and thiourea should have
been consulted in preference to applying a staged TTC. In the case of benzene, this would have
justified a higher limit of 800 μg/day (0.80% w/w) for the short study, but they defaulted to the
lower level as it is easily controlled for using standard analytical techniques (HPLC-UV and GC-
FID). It is possible to argue that the “staged” levels can be equally valid for approved drugs which
are intended to be administered only for short periods [40]. This suggestion, however, has been
specifically dismissed by the USFDA on the grounds that a drug may be used multiple times by the
same individual, or may be used outside of its approved indication. However, applicants may
provide the agency with a detailed rationale to support higher limits on a case-by-case basis - for
example if the patient is likely to be exposed to higher levels of the potential genotoxic impurity
(PGI) from other sources or if the drug is intended to treat a life-threatening condition. The
EMEA’s 2008 Question and Answer document made a number of other welcome clarifications.
The guideline need not be applied retrospectively to existing authorised products so long as
the manufacturing procedure remains essentially unchanged, unless newly acquired
knowledge flags a potential problem.
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It is not necessary to apply ALARP principles to genotoxic impurities which are controlled
below the TTC, unless they have structures of very high concern. (This, as pointed out by
Snodin et al. [28], actually contradicts the original guideline.)
Absence of a “structural alert” based on a well performed assessment allows the impurity
concerned to be classified as nongenotoxic, without the requirement for specific testing.
A negative Ames test (properly conducted) would be sufficient to overrule any structural
alerts and allow an impurity to be classified as nongenotoxic.
It is acceptable to assume that an identified PGI is in fact genotoxic without specifically
testing it.
Unidentified impurities which occur below the ICH identification threshold (0.10% or 1
mg/day intake) need not be considered further.
Identified impurities, even those below the identification threshold, should always be
screened for structural alerts.
When more than one genotoxic impurity is present, the TTC value of 1.5 μg/day can be
applied to each, provided the impurities are not structurally related. Where structurally
related PGIs are present, the 1.5 μg/day limit should be applied to the whole group.
5.5 MESALAMINE
Mesalamine (5-aminosalicylic acid, 5-ASA), a therapeutically active moiety of Sulfasalazine [41-
43] is routinely employed in the treatment of inflammatory bowel disease like ulcerative colitis and
Crohn’s disease. Orally administrated mesalamine is rapidly and almost completely absorbed from
the small intestine [44-46]. Mesalamine formulations which are able to deliver the intact drug to the
lower intestine are nowadays successfully used [47, 48]. The purity evaluation of mesalamine in
drug product by determination of related substances is a critical step in examination of the safety
and quality of the drug product. Aniline is the critical genotoxic impurity to be considered for
mesalamine drug substance and its drug product. Chemical structure and UV spectra of mesalamine
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and aniline are shown in [Figure 5.3]. Molecular formula of Mesalamine is C7H7NO3 with
molecular weight 153.135 g/mole. Its melting point is 283°C and reported predicted pKa is 15.48.
Aniline; Phenylamine; Aminobenzene; Benzenamine
Mesalamine; Mesalazine; 5-Aminosalysilic acid; 5-ASA
[Figure 5.3 Aniline and mesalamine chemical structure with its UV spectra]
Absorption
20 to 30% absorbed following oral administration. 10 to 35% absorbed from the colon (rectal
suppository) - extent of absorption is determined by the length of time the drug is retained in the
colon.
5.6 EXPERIMENTAL
5.6.1 Materials and reagents
Mesalamine delayed-release tablets, placebo and related aniline impurity standard (purity 98.7 %)
are provided by Dr. Reddy’s Laboratory Ltd., Hyderabad. Acetonitrile of HPLC grade is obtained
from J.T.Baker (NJ, USA), HPLC grade 1-octane sulphonic acid sodium salt is obtained from
RANKEM (RFCL ltd., Delhi, India), di-potassium hydrogen orthophosphate purified, GR grade
orthophosphoric acid, GR grade sodium hydroxide pallets and GR grade hydrochloric acid are
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obtained from Merck (Mumbai, India). 0.2 µm nylon 66 membrane filter and 0.2 µm nylon syringe
filter is manufactured by Pall life science ltd. (India). 0.2 µm PVDF syringe filter is manufactured
by Millipore (India). Ultra pure water generated by a Milli-Q system (Millipore, Milford, MA,
USA).
5.6.2 Equipments
Acquity UPLCTM
system (Waters, Milford, USA), consists of a binary solvent manager, sample
manager and PDA (photo diode array) detector. System control, data collection and data processing
are accomplished using Waters EmpowerTM
-2 chromatography data software. Cintex digital water
bath is used for specificity study. Photo stability study is carried out in a photo-stability chamber
(SUNTEST XLS+, ATLAS, Germany).
5.6.3 Preparation of mobile phase and its gradient program
Buffer preparation
1.74 g of di-potassium hydrogen orthophosphate (K2HPO4) and 2.2 g of 1-octane sulphonic acid
sodium salt is dissolved in one liter of milli-Q water. The pH of this solution is adjusted to 6.0 with
orthophosphoric acid and then filtered through 0.2 µm pall nylon membrane filter. The buffer
preparation is found stable with respect to pH and visual clarity up to 48h.
Mobile phase: Mixture of buffer (pH 6.0) and acetonitrile at a ratio of 900:100 (v/v) respectively.
It is filtered through 0.2 µ nylon filter and degassed before use.
5.6.4 Diluent preparation
1N hydrochloric acid and 1N sodium hydroxide solution in the ratio of 50:50 (v/v) is mixed and
used as a diluent.
5.6.5 Impurity standard solution preparation (approx 0.156 µg/mL solution)
Impurity standard solution is prepared by dissolving standard substances in methanol to obtained
solution containing 7.5 µg/mL of aniline in methanol. Five millilitres of 7.5 µg/mL of aniline
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solution is transferred to 250 mL volumetric flask, diluted to volume with the diluent and mixed
well.
[Note: Working concentration of aniline is 0.0005 % with respect to Mesalamine]
5.6.6 Sample solution preparation (30,000 µg/mL solution)
Twenty tablets are crushed to fine powder. An accurately weighed portion of the powder equivalent
to 750 mg of mesalamine is taken in 25 mL volumetric flask. About 12.5 mL of 1N hydrochloric
acid is added to this flask and sonicated in an ultrasonic bath for 20min. This solution is then
diluted up to the mark with 1N sodium hydroxide solution and mixed well. It is then filtered
through 0.2 µ PVDF syringe filter and the filtrate is collected after discarding first few millilitres.
5.6.7 Placebo solution preparation
Twenty placebo tablets are crushed to fine powder. An accurately weighed portion of the powder
equivalent to 750 mg of mesalamine is taken in 25 mL volumetric flask. About 12.5 mL of 1N
hydrochloric acid is added to this flask and sonicated in an ultrasonic bath for 20min. This solution
is then diluted up to the mark with 1N sodium hydroxide solution and mixed well. It is then filtered
through 0.2 µ PVDF syringe filter and the filtrate is collected after discarding first few millilitres.
5.6.8 Chromatographic conditions
The chromatographic condition is optimized using a column Reprosil Gold 100, C18-XBD 50 mm
x 2.0 mm, 1.8 µm. The separation of aniline from mesalamine is achieved by Isocratic elution. The
injection volume is 7 µl and flow rate is 0.5 mL/min. The detection is carried out at 200 nm and
column oven temperature is set at 50°C. The stress degraded samples and the solution stability
samples are analyzed using a PDA detector covering the range of 195-400 nm.
5.6.9 Method Validation
The RP-UPLC method described herein has been validated for Assay determination of Aniline.
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5.6.9.1 System suitability
System suitability parameters are measured so as to verify the system performance. Standard
preparation is consider for the system suitability. All important characteristics including capacity
factor, peak tailing and theoretical plate number are measured. The similarity factor for the peak of
aniline in the duplicate standard preparation is measured. Related standard deviation for the peak
areas of aniline for five replicate (standard solution) injections is also measured. All these system
suitability parameters covered the system, method and column performance.
5.6.9.2 Specificity
Forced degradation studies are performed to demonstrate selectivity and stability indicating
capability of the proposed method. The powdered sample of tablets is exposed to acidic [1N HCl (5
mL), 60°C, 6h], alkaline [1N NaOH (5 mL), 60°C, 6h], strong oxidizing [6% w/v H2O2 (5 mL),
60°C, 6h], hydrolysis [water (5 mL), 60°C, 12h] and photolytic [1.2 million Lux hours] degradation
conditions. Also, placebo of tablets is exposed to above all stress conditions to identify source of
degradation peaks. The entire exposed samples are analyzed by the proposed method with photo
diode array detector.
5.6.9.3 Precision
Precision is investigated using sample preparation procedure for six real samples (with spiked
impurity in standard concentration level) of tablets and analyzing by proposed method.
Intermediate precision study is performed with different column, different instrument, and different
day by another analyst. Precision is also performed at LOQ and 100% of standard concentration
level. The mean of percentage impurity (n=6) and the relative standard deviation is calculated for
aniline impurity.
5.6.9.4 Accuracy
To confirm the accuracy of the proposed method, recovery experiments are carried out by standard
addition technique. Six different levels (LOQ, 50%, 70%, 100%, 130% and 150%) of impurity
Aniline/Genotoxic Impurity Determination in Mesalamine Delayed Release Tablets by UPLC
190
standard are added to pre-analyzed tablet samples in triplicate. The percentage recoveries of aniline
at each level (LOQ to 150% of standard concentration) and each replicate are determined. The
mean of percentage recoveries (n=18) and the relative standard deviation is calculated.
5.6.9.5 Limit of detection (LOD) and limit of quantification (LOQ)
The LOD and LOQ of aniline impurity are determined by using signal to noise ratio method as
defined in International Conference on Harmonization (ICH) guideline [46]. Increasingly dilute
solution of aniline impurity is injected into the chromatograph and signal to noise (S/N) ratio is
calculated at each concentration.
5.6.9.6 Linearity
Linearity is demonstrated from LOQ to 150% of standard concentration using minimum seven
calibration levels (LOQ, 25%, 50%, 75%, 100%, 125% and 150%) for aniline impurity standard.
The method of linear regression is used for data evaluation. Peak area of standard compound is
plotted against respective concentrations. Linearity is described by regression equation, correlation
coefficient and Y-intercept bias.
5.6.9.7 Robustness
The robustness as a measure of method capacity to remain unaffected by small, but deliberate
changes in chromatographic conditions is studied by testing influence of small changes in flow rate
(± 0.05 mL/min), column oven temperature (45°C to 55°C) and pH of buffer (pH 5.9 to pH 6.1).
System suitability parameters are measured for above all experiments.
5.6.9.8 Stability of standard and sample preparation
Stability of standard and sample solution is established by storage of sample solution (duplicate
preparation) and standard solution at ambient temperature for 24h. Sample solution stability is
demonstrated by spiking impurity standard in pre-analyzed tablet sample. Standard and sample
solutions are re-analyzed in between duration and after 24h. Percentage difference is calculated
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against fresh injected sample solution and system suitability requirements are measured for the
standard preparation.
5.6.9.10 Filter compatibility
Filter compatibility is performed for nylon 0.2 µ syringe filter (Pall Life sciences) and PVDF 0.2 µ
(Millipore) syringe filter. To confirm the filter compatibility in proposed method, filtration recovery
experiments are carried out by sample filtration technique. The working concentration level
impurity standard is added to pre-analyzed tablet sample in duplicate. Spiked impurity samples are
filter through both syringe filter and percentage difference is calculated against centrifuged sample.
5.7 RESULTS AND DISCUSSION
5.7.1 Method Development and Optimization
The important criteria for development of successful RP-UPLC method for determination of aniline
from the mesalamine drug product are: the method should be able to determine aniline in single run
and it should be accurate, reproducible, robust, stability indicating, free of interference from
blank/placebo/degradation product/other impurities and straightforward enough for routine use in
quality control laboratory testing.
To develop the stability indicating method, first the retention behavior of aniline compound with
change in percentage of organic solvent (acetonitrile) and with change in pH of buffer is studied on
Reprosil Gold 100 C18-XBD column (50 mm x 2.0 mm, 1.8 µ). 1-octane sulphonic acid sodium
salt ion pair reagent is used in buffer preparation to improve the peak shape and avoid the other
substances co-elution at the same retention time in RP chromatography. The buffer pH 6.0 is found
more appropriate for robust peak shape and RT (retention time) performance of interested aniline
substance. The final isocratic run is chosen with regards to the peak resolution and analysis time
(total run time 5min for one injection) as well. The flow rate of 0.5 mL/min is optimized with
regard to the back pressure and analysis time as well. Detection wavelength 200 nm is selected for
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aniline due to higher detector response at this nm. Diluent is optimized based on solubility of
aniline and mesalamine.
5.7.2 Analytical Parameters and Validation
After satisfactory development of method it is subjected to method validation as per ICH guideline
[49]. The method is validated to demonstrate that it is suitable for its intended purpose by
performing system suitability, accuracy, precision, linearity, robustness, ruggedness, solution
stability, LOD, LOQ, filter compatibility and stability indicating capability.
5.7.2.1 System suitability
The percentage RSD of area count of six replicate injections is below 2.0%. The results of system
suitability are presented in Table 5.2. Low values of % RSD of replicate injections indicate that the
system is precise. Results of other system suitability parameters such as capacity factor, tailing
factor, similarity factor (between two standard preparation), and theoretical plates are presented in
Table 5.2. As seen from this data, the acceptable system suitability parameters are: relative standard
deviation of replicate injections is not more than 1.5, similarity factor should be in between 0.95 to
1.05 and theoretical plates are not less than 5000. Overlaid chromatograms of replicate standard
injections are presented in Figure 5.4. Results of system suitability parameters from different
studies are presented in Table 5.2.
[Figure 5.4 Overlaid chromatograms of replicate standard injection]
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Table 5.2 System suitability results
Condition
USP
tailing
factor
USP
theoretical
plates
Capacity
factor
Similarity
factor of
standard
% RSD of
standard
area
Precision 1.06 7643 9.2 1.02 1.5
Intermediate Precision 1.06 7825 9.2 1.03 1.2
At 0.45mL/min flow rate 1.10 7596 10.3 0.96 0.9
At 0.55mL/min flow rate 1.10 8386 8.3 0.97 1.7
At 45°C Column oven temp. 1.13 7411 9.7 1.01 0.8
At 55°C Column oven temp. 1.04 7704 8.8 0.98 0.9
With Buffer pH 5.9 1.03 7740 9.2 1.02 1.2
With Buffer pH 6.1 1.12 7799 9.2 1.01 1.3
USP = United state pharmacopeia
5.7.2.2 Specificity
Typical overlaid chromatograms are presented in Figure 5.5, which show separation of aniline
compound from blank and placebo. Overlaid specimen chromatograms of standard, drug product
(sample) and spiked sample (aniline spiked in drug product) are presented in Figure 5.6. No
interference is observed at the RT of aniline due to blank and placebo (without mesalamine drug).
The standard chromatogram with its purity plot is presented in Figure 5.7. In all the degradation
conditions, no interference is observed at the RT of aniline [Figure 5.8]. It’s indicating that method
is specific for the aniline substance in mesalamine drug product. Therefore, the method is specifics
and suitable for routine work.
[Figure 5.5 Overlaid specimen chromatograms of specificity study]
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[Figure 5.6 Overlaid specimen chromatograms of standard, sample and spiked
drug product preparation]
[Figure 5.7 Standard chromatogram with its purity plot]
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[Figure 5.8 Overlaid specimen chromatograms of spiked sample and stress conditions]
5.7.2.3 Precision
The average % impurity (n=6) and % RSD results are shown in Table 5.3. Precision (at LOQ and at
100% level) results are shown in Table 5.3 along with intermediate precision data. Low values of
RSD, indicates that the method is precise. Overlay chromatograms of precision at LOQ and
precision at 100% are presented in Figure 5.9 and 5.10.
[Figure 5.9 Overlaid chromatograms of precision study (at LOQ level)]
Aniline/Genotoxic Impurity Determination in Mesalamine Delayed Release Tablets by UPLC
196
[Figure 5.10 Overlaid chromatograms of precision study (at 100% level)]
Table 5.3 Precision at LOQ, precision at 100% and intermediate precision results@
Impurity Precision at LOQ Precision at 100% Intermediate precision
%impurity# %RSD* %impurity
# %RSD* %impurity
# %RSD*
Aniline 0.00005591 1.98 0.0005253 0.75 0.0005136 0.97
# Average of six determinations; * Determined on six values;
@ Demonstrated by spiking known impurities into sample.
5.7.2.4 Accuracy
The accuracy by recovery is performed on aniline impurity, from LOQ to 150% of standard
concentration. Recovery of aniline is performed in triplicate for each level. The results of recoveries
(LOQ, 50%, 70%, 100%, 130% and 150%) for aniline are shown in Table 5.4. The amount
recovered is within ±10% of amount added which indicates that the method is accurate and also
there is no interference due to excipients present in the tablets. Overlay chromatograms of accuracy
are presented in Figure 5.11.
Table 5.4 Accuracy results for aniline
Aniline Recovery (triplicate determination at each level)
At LOQ At 50% At 70% At 100% At 130% At 150%
Mean % 106.75 102.24 98.20 99.42 97.14 97.04
% R.S.D.* 0.96 1.14 1.57 0.50 0.76 0.11
* Determined on three values.
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197
[Figure 5.11 Overlaid specimen chromatograms of accuracy study]
5.7.2.5 LOD and LOQ
The concentration (in µg/mL) with signal to noise ratio of at least 3 is taken as LOD and
concentration with signal to noise of at least 10 is taken as LOQ, which meets the criteria defined
by ICH guidance [49]. The LOD and LOQ result of aniline substance is presented in Table 5.5.
Table 5.5 Limit of detection and limit of quantification
Aniline Concentration (ng/mL) % Impurity (w.r.t. 5-ASA) Signal to Noise Ratio
LOD 5.5 0.000018 2.9
LOQ 18 0.00006 10.4
5.7.2.6 Linearity
The response is found linear for aniline from LOQ to 150% of standard concentration. This test is
performed on seven different levels of aniline solution, which gave us a good confidence on
analytical method with respect to linear range. For aniline substance the correlation coefficient is
greater than 0.999. Correlation coefficients and linearity equations of impurity and Y- intercept bias
Aniline/Genotoxic Impurity Determination in Mesalamine Delayed Release Tablets by UPLC
198
are presented in Table 5.6. Overlaid linearity chromatograms and linearity curves are presented in
Figure 5.12 and 5.13 respectively.
Table 5.6 Linearity results (determined on LOQ, 25%, 50%, 75%, 100%, 125% and 150%)
Substance Linearity range
(µg/mL)
Correlation
Coefficient (r2)
Linearity (Equation) Y- intercept
bias
Aniline 0.018 to 0.225 0.9998 y =155496(x) - 429.33 -1.865
[Figure 5.12 Overlaid specimen chromatograms of linearity study]
[Figure 5.13 Linearity curve of aniline impurity]
y = 155496x - 429.33
R² = 0.9998
0.00
5000.00
10000.00
15000.00
20000.00
25000.00
30000.00
35000.00
40000.00
0.000 0.050 0.100 0.150 0.200 0.250
Are
a
Con. in ppm
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199
5.7.2.7 Robustness
No significant effect is observed on system suitability parameters such as tailing factor, capacity
factor and theoretical plates of aniline compound, when small but deliberate changes are made to
chromatographic conditions. The results are presented in Table 5.2 along with system suitability
parameters. Thus, the method is found to be robust with respect to variability in variable conditions.
5.7.2.8 Stability of standard and sample solution
Sample solution does not show any appreciable change in impurity value when stored at ambient
temperature up to 24h. Percentage impurity result of 12h and 24h is compared with freshly prepared
sample solution. Percentage difference in impurity is calculated with respect to freshly injected
sample solution. Results are presented in Table 5.7. Standard solution does not show any unknown
peak during this 24h study and also fulfill the requirements of system suitability. Standard solution
stability results are presented in Table 5.8.
Table 5.7 Sample solution stability results
Impurity
Initial Sample Sample After 12 hours Sample After 24 hours
Imp. in % Imp. in % Difference in % Imp. in % Difference in %
Spl-1 Spl-2 Spl-1 Spl-2 Spl-1 Spl-2 Spl-1 Spl-2 Spl-1 Spl-2
Aniline 0.000505 0.000502 0.000506 0.000500 0.000001 0.000002 0.000496 0.000498 0.000009 0.000004
Table 5.8 Standard solution stability results
I.D. Initial After 12 hrs. After 24 hrs.
Area %RSD# Area %RSD
$ Area %RSD
@
Aniline 22449 0.78 22246 0.79 22356 0.73
# - Determined on five replicate injections.
$ - Determined on five initial and 12h standard injections. (% RSD of six Inj.)
@ - Determined on five initial, 12h and 24h standard injections. (% RSD of seven Inj.)
Aniline/Genotoxic Impurity Determination in Mesalamine Delayed Release Tablets by UPLC
200
5.7.2.9 Filter compatibility
Filter compatibility of PVDF and Nylon syringe filter (0.2 µ) is performed on duplicate sample
preparation. Filtered sample solution does not show significant changes in aniline percentage with
respect to centrifuged samples. Difference in impurity percentage result is presented in Table 5.9.
In displayed result difference in % of impurity is not observed more than 0.000007%, which
indicates that both syringe filter having a good compatibility with sample solution. Overlay
chromatograms are presented in Figure 5.14.
Table 5.9 Filter compatibility results
Impurity
Centrifuged PVDF syringe filter 0.2µ
(Millipore)
Nylon syringe filter 0.2µ
(Pall Life Sciences)
Imp. in % Imp. in % Difference in % Imp. in % Difference in %
Spl-1 Spl-2 Spl-1 Slp-2 Spl-1 Spl-2 Spl-1 Spl-2 Spl-1 Spl-2
Aniline 0.000503 0.000493 0.000498 0.000487 0.000005 0.000006 0.000496 0.000487 0.000007 0.000006
[Figure 5.14 Overlaid specimen chromatograms of filter compatibility study]
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201
5.8 CALCULATION FORMULA
5.8.1 Impurity (% w/w)
Calculated the quantity, in mg, of Aniline in mesalamine dosage form, using the following formula:
Where,
Cstd = Concentration of standard solution in mg/mL
Cs = Concentration of sample solution in mg/mL
Rs = Compound peak response obtained from the sample preparation
Rstd = Compound peak response (mean peak area) obtained from the standard preparation
5.8.2 Relative standard deviation (% RSD)
It is expressed by the following formula and calculated using Microsoft excel program in a
computer.
Where,
SD= Standard deviation of measurements
= Mean value of measurements
5.8.3 Accuracy (% Recovery)
It is calculated using the following equation:
Aniline/Genotoxic Impurity Determination in Mesalamine Delayed Release Tablets by UPLC
202
5.9 CONCLUSION
A novel RP-UPLC method is successfully developed and validated for determination of aniline
impurity from mesalamine drug product. The total run time is 5min, within which drug and their
degradation products are separated from aniline. Method validation results have proved that the
method is selective, precise, accurate, linear, rugged, robust and stability indicating with low LOD
and LOQ. Sample solution stability and filter compatibility is also established. This method can
successfully be applied for routine analysis and stability study of mesalamine delayed release drug
product. Thus, the method provides high throughput solution for determination of aniline in the
mesalamine delayed-release formulation with excellent selectivity, precision and accuracy. This
method can also be applied for quantifying the trace levels of aniline from drug substances, drug
products and different type of samples.
Note: Intellectual Property Management (IPM) clearance number for the present research
work is: PUB-00041-10.
Chapter-5
203
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