Chapter-5 Aniline/Genotoxic Impurity Determination in ...shodhganga.inflibnet.ac.in/bitstream/10603/8513/12/12_chapter 5.pdf · Aniline/Genotoxic Impurity Determination in Mesalamine

177

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 Moreover, recently the US-FDA has also published draft guidance for ‘the Genotoxic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.

Aniline/Genotoxic Impurity Determination in Mesalamine Delayed Release Tablets by UPLC

178

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.

Chapter-5

179

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


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].

Chapter-5

181

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


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]

Chapter-5

183

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


184

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.

Chapter-5

185

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


186

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

Chapter-5

187

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


188

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.

Chapter-5

189

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


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

Chapter-5

191

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


192

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]

Chapter-5

193

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]


194

[Figure 5.6 Overlaid specimen chromatograms of standard, sample and spiked

drug product preparation]

[Figure 5.7 Standard chromatogram with its purity plot]

Chapter-5

195

[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)]


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.

Chapter-5

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


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

Chapter-5

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.)


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]

Chapter-5

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:


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

5.10 REFERENCES

[1] Harshal KT and Mukesh CP, “Development and validation of a stability-indicating RP-

UPLC method for determination of rosuvastatin and related substances in pharmaceutical

dosage form” Scientia Pharmaceutica, 2012; 80: 393-406, doi:10.3797/scipharm.1201-09.

[2] Rakshit KT, Mukesh CP, Sushant BJ, “A rapid, stability indicating RP-UPLC method for

simultaneous determination of ambroxol hydrochloride, cetirizine hydrochloride and

antimicrobial preservatives in liquid pharmaceutical formulation” Scientia Pharmaceutica,

2011; 79: 525-543, doi:10.3797/scipharm.1103-19.

[3] Rakshit KT and Mukesh CP, “Development of a stability indicating RP-UPLC method for

rapid determination of metaxalone and its degradation products in solid oral dosage form”

Scientia Pharmaceutica, 2012; 80: 353-366, doi:10.3797/scipharm.1112-08.

[4] Genotoxic and Carcinogenic impurities in drug Substances and Products: Recommended

approaches, FDA Centre for Drug Evaluation and Research, Guidance for Industries

(Draft), 03 December 2008.

[5] Ernst MB, Bernd AH, “Genotoxic activities of aniline and its metabolites and their

relationship to the carcinogenicity of aniline in the spleen of rats” Critical Reviews in

Toxicology, 2005; 35(10): 783-835, doi:10.1080/10408440500442384.

[6] Ken-ichi M, Yukari T, Keiji W and Takehiko N, “Potent genotoxicity of

aminophenylnorharman, formed from non-mutagenic norharman and aniline, in the liver

of gpt delta transgenic mouse” Carcinogenesis, 2003; 24(12): 1985-1993, doi:10.10

93/carcin/bgg170.

[7] Toshihiko K, Yukari T, Naoaki U, Tomohiro K, Hideyuki S, Takashi S and Keiji W,

“Carcinogenicity of aminophenylnorharman, a possible novel endogenous mutagen,

formed from norharman and aniline, in F344 rats” Carcinogenesis, 2004; 25(10): 1967-

1972, doi:10.1093/carcin/bgh189.

[8] Meiping Z, Xinxiang Z, Yuanzong Li, Wenbao Chang, “A new spectrophotometry

method for the determination of aniline in environmental water samples” Analytical

Latters, 2000; 33(14): 3067-3075, doi:10.1080/00032710008543242.

[9] Rahim SA, Ismail ND and Bashir WA, “Spectrophotometric determination of aniline in

aqueous solution by azo-dye formation with diazotized p-nitroaniline” Microchimica

Acta, 1986; 90(5-6): 417-423, doi:10.1007/BF01199282.


204

[10] Zhao X, Suo Y, “Analysis of primary aromatic amines using precolumn derivatization by

HPLC fluorescence detection and online MS identification” J Separation Sci, 2008; 31(4):

646-658, doi:10.1002/jssc.200700400.

[11] Xiao-Lan C, Zhong-Bao L, Ya-Xian Z and Jin-Gou X, “Sensitive fluorimetric method for

the determination of aniline by using tetra-substituted amino aluminium phthalocyanine”

Analytical Chimica Acta, 2004; 505(2): 283-287, doi:10.1016/j.aca.2003.10.067.

[12] Jun PX, Qing XZ, Xiao KT, Hua HB and Xian FS, “Determination of aniline in

environmental water samples by alternating-current oscillopolarographic titration”

Chinese Chemical Letters, 2007; 18(6): 730-733, doi:10.1016/j.cclet.2007.04.030.

[13] John M, Andrew M, Michael LH and David L, “Determination of aniline in hydrofluoric

acid solutions by NRI spectrometry with a fiber-optic interface” Applied Spectroscopy,

1998; 52(6): 908-911.

[14] Cheing-Tong Y and Jen-Fon J, “Determination of aniline in water by microwave-assisted

headspace solid-phase microextraction and gas chromatography” J Chromatographia,

2004; 59(7-8): 517-520, doi:10.1365/s10337-004-0203-2.

[15] Liu XB, Lin HB, Sun ZQ, Zhang HB, Zhao B, “Study on the oxidation of solutions of

aniline and phenol on Ti/SnO2-Sb2O5 electrode by UV-Vis spectroscopy and HPLC”

Spectroscopy and Spectral Analysis, 2006; 26(6): 1141-1144, pmid:16961252.

[16] Sarafraz YA, Es’haghi Z, “Comparison of hollow fiber and single-drop liquid-phase

microextraction techniques for HPLC determination of aniline derivatives in water” J

Chromatographia, 2006; 63(11-12): 563-569, doi:10.1365/s10337-006-0801-2.

[17] Reig N, Calaf RE, Messegure A, Morato A, Escabros J, Gelpi E, Abian J, “LC-MS ion

maps for the characterization of aniline derivatives of fatty acids and triglycerides in

laboratory-denatured rapeseed oil” Journal of Mass Spectrometry, 2007; 42(4): 527-541,

doi:10.1002/jms.1185.

[18] Mortensen SK, Trier XT, Foverskov A, Petersen JH, “Specific determination of 20

primary aromatic amines in aqueous food simulants by liquid chromatography-

electrospray ionization-tandem mass spectrometry” Journal of Chromatography A, 2005;

1091(1-2): 40-50, pmid:16395791.

[19] Mesalamine, “United States Pharmacopeia” Pharmacopeial Forum, Volume No. 31(2),

Page 424.

[20] Mesalazine, “British Pharmacopeia” BP-2010, Page 2894.

Chapter-5

205

[21] ICH, “Impurities in New Drug Substances” Q3A(R2), International Conference on

Harmonisation, 2006.

[22] ICH, “Impurities in New Drug Products” Q3B(R2), International Conference on

Harmonisation, 2006.

[23] A recent FDA guideline (July 2009) on impurities in generic drugs would effectively

reduce the qualification threshold to 1000 ppm or 1 mg/day for new impurities which were

not present in the reference listed drugs.

[24] European medicines evaluation agency, committee for medicinal products for human use

(CHMP): “Guideline on the limits of genotoxic impurities” CPMP/SWP/5199/02,

London, 28 June 2006.

[25] FDA center for drug evaluation and research, “Genotoxic and carcinogenic impurities in

drug substances and products” Recommended approaches, Guidance for industry (Draft);

03 December 2008.

[26] Delaney EJ, “An impact analysis of the application of the threshold of toxicological

concern concept to pharmaceuticals” Regul Toxicol Pharmacol, 2007; 49(2): 107-124,

doi:10.1016/j.yrtph.2007.06.008.

[27] HumfreyCD, “Recent developments in the risk assessment of potentially genotoxic

impurities in pharmaceutical drug substances” Toxicol Sci, 2007; 100(1): 24-28,

pmid:17656486.

[28] Snodin DJ, “EU guideline on genotoxic impurities needs updating” RAJ Pharma, 2008;

Part I September (593-598) and Part II October (663-670).

[29] DerekIR, “Control of genotoxic impurities in active pharmaceutical ingredients: A review

and perspective” Organi Pro Resea and Develop, 2010; 14(4): 946-959,

doi:10.1021/op900341a.

[30] ICH Guideline: “A standard battery for genotoxicity testing of pharmaceuticals S2B”

International Conference on Harmonisation, 1997.

[31] Int J Pharm Med., 2004; 18 (4). Special edition.

[32] ICH Guideline: “Impurities - guideline for residual solvents” Q3C(R4), International

Conference on Harmonisation, 2009.

[33] Rulis AM, “De minimis and the threshold of regulation” In food protection technology;

Felix, C. W., Ed.; Lewis Publishers: Chelsea, Michigan, USA, 1986; pp 29-37.


206

[34] Munro IC, Renwick AG, Danielewska-Nikiei B, “The threshold of toxicological concern

(TTC) in risk assessment” Toxicol Lett, 2008; 180(2): 151-156, doi:10.1016/j.toxlet.20

08.05.006.

[35] Cheeseman MA, Machuga EJ, Bailey AB, “A tiered approach to threshold of regulation”

Food Chem Toxicol, 1999; 37(4): 387-412, doi:10.1016/S0278-6915(99)00024-1.

[36] Kroes R, Renwick AG, Cheeseman M, Kleiner J, Mangelsdorf I, Piersma A et al.

“Structure-based thresholds of toxicological concern (TTC): Guidance for application to

substances at low levels in the diet” Food Chem Toxicol, 2004; 42(1): 65-83. doi:10.10

16/j.fct.2003.08.006.

[37] Questions and answers on the CHMP guideline on the limits of genotoxic impurities,

Revision-1; EMEA/CHMP/SWP/43 1994/2007, CHMP safety working party: London, 26

June 2008.

[38] Muller L, Mauthe RJ, Riley CM, Andino MM, De Antonis D, Beels C et al., “A rationale

for determining, testing, and controlling specific impurities in pharmaceuticals that possess

potential for genotoxicity” Regul Toxicol Pharmacol, 2006; 44(3): 198-211, doi:10.10

16/j.yrtph.2005.12.001.

[39] Syntagon Newsletter, “A pragmatic approach to controlling potentially genotoxic

impurities for phase-1” June 2009; Issue 2, www.syntagon.com/PGI.aspx.

[40] Kirkland D, Snodin D, “Setting limits for genotoxic impurities in drug substances:

Threshold-based and pragmatic approaches” Int J Pharm Med, 2004; 18(4): 197-207.

[41] Gisbert JP, Gomollon F, Mate J, Pajares JM, “ Role of 5-Aminosalicylic acid (5-ASA) in

treatment of inflammatory bowel diseas, A systematic review” Dig Dis Sci, 2002; 47: 471-

488, doi:10.1023/A:1017987229718.

[42] Klotz U, “The role of aminosalicylates at the beginning of the new millennium in the

treatment of chronic inflammatory bowel disease” Eur J Clin Pharmacol, 2000; 56: 353-

362, doi: 10.1007/s002280000163.

[43] De Vos M, “Clinical Pharmacokinetics of Slow Release Mesalazine” Clin

pharmacokinetics, 2000; 39: 85-97, doi:10.2165/00003088-200039020-00001.

[44] Pappercorn MA, Goldman P, “Distribution studies of salicylazosulfapyridine and its

metabolites” Gastroenterology, 1973; 64(2): 240-245, pmid:4405598.

[45] Haagen NO, Bondesen S, “Kinetics of 5-aminosalicylic acid after jejunal installation in

man” Br J Clin Pharmacol, 1983; 16(6): 738-740, pmid:6661362.

http://www.syntagon.com/PGI.aspx

Chapter-5

207

[46] Schröder H, Compbell DES, “Absorption, metabolism and excretion of

salicylazosulfapyridine in man” Clin Pharmacol Ther, 1983; 13(4): 539-51,

pmid:4402886.

[47] Goodman and Gilman’s, ‘The Pharmacological Basis of Therapeutics’ 8th

ed., Pergamon,

1990, p-650.

[48] JF Reynolds (Ed.) Martindale, “The Extra Pharmacopoeia” Royal Pharmaceutical

Society, London, 1996, p-1227.

[49] ICH, “Validation of Analytical Procedure, Text and Methodology” Q2(R1), International

Conference on Harmonization, IFPMA, Geneva, 2005.

Documents

Chapter-5 Aniline/Genotoxic Impurity Determination in ...shodhganga.inflibnet.ac.in/bitstream/10603/8513/12/12_chapter 5.pdf · Aniline/Genotoxic Impurity Determination in Mesalamine