CHAPTER-II - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/71060/10/10_chapter 2.pdf · Chapter-II-HPLC Determination of Solifenacin Succinate impurities by Stability Indicating

CHAPTER-II

Development and Validation of a Specific Stability Indicating

High Performance Liquid Chromatographic Methods for

Related Compounds and Assay of Solifenacin Succinate

Chapter-II-HPLC Determination of Solifenacin Succinate impurities by Stability Indicating HPLC Method

Development and Validation of a Specific Stability Indicating High Performance Liquid

Chromatographic Methods for Related Compounds and Assay of Solifenacin Succinate

2.0 Introduction

Solifenacin succinate (SFS) is a muscarinic receptor antagonist [1], which play an important

role in several major cholinergically mediated functions, including contractions of urinary

bladder smooth muscle and stimulation of salivary secretion, antagonist belongs to

anticholinergics which are used for the treatment of overactive bladder [2-4] and has higher

selectivity for the urinary bladder over salivary glands [5,6]. It acts as a selective antagonist to

the M (3) receptor and works by relaxing the bladder muscles to prevent urgent, frequent, or

uncontrolled urination. Chemically, solifenacin is (1S)-(3R)-1-azabicyclo [2.2.2] oct-3-yl 3,4-

dihydro-1-phenyl-2(1H)-iso-quinolinecarboxylate (1:1) [7] having an empirical formula of

C23H26N2O2.C4H6O4 and molecular weight is 480.55 grams/mole. It is a white to pale-

yellowish-white crystal or crystalline powder. It is freely soluble in water, glacial acetic acid,

dimethyl sulfoxide and methanol. The chemical structure of solifenacin succinate is

represented in Fig.2.1.

Fig.2.1: Chemical structure of solifenacin succinate

It is available in the market under the brand name of VESIcare in the form of 5 mg and 10 mg

tablets manufactured by Astellas Pharma Technologies, Inc. Norman, Oklahoma and

marketed and distributed by Astellas Pharma US, Inc. Deerfield, Illinois. Each tablet contains

lactose monohydrate, corn starch, hypromellose, magnesium stearate, talc, polyethylene

glycol and titanium dioxide with yellow ferric oxide (5 mg) or red ferric oxide (10 mg) as

inert ingredients. It is principally (98%) bound to α1 acid glycoprotein’s of human plasma and

is highly distributed to non-CNS tissues, having a mean steady-state volume of distribution of

600 L. The chemical synthetic route of solifenacin succinate is shown in Fig.2.2.

53


Fig.2.2: Brief synthetic scheme of solifenacin succinate

The literature survey of solifenacin succinate suggested that various analytical methods were

reported for the determination of pure drug substance present in different pharmaceutical

formulation and in various biological fluids. N. Hiren mistri et al [8] developed a LC-ESI-

MS/MS method for the simultaneous quantification of uroselective α1-blocker, alfuzosin and

an antimuscarinic agent, solifenacin in human plasma. A LC-MS method [9] was developed

by Jan Macek to quantitate solifenacin in human plasma. T. Yanagihara [10] et al developed

semi-micro HPLC method for the determination of solifenacin succinate and its major

metabolite in rat plasma. Spectrophotometric methods [11,12] were reported for the

estimation of solifenacin succinate in tablet and dosage forms. R. Seetharaman, KS. Lakshmi,

developed and validated first order derivative spectrophoto metric method for estimation of

solifenacin succinate in pharmaceutical formulation [13]. A validated normal phase HPTLC

method was developed by wankhede [14] for simultaneous analysis of alfuzosin and

solifenacin in tablets. D. Desai et al, developed and validated a stability-indicating HPTLC

method of solifenacin succinate [15]. A normal phase-liquid chromatographic method [16]

was developed to separate and quantify the solifenacin and its three stereoisomers. A stability

indicating UPLC [17] method was developed for the determination of related substances in

solifenacin succinate. Nilesh desai et al [18] developed a stability indicating HPLC method

for determination of solifenacin in bulk formulations. D. Desai, G. Patel, N. Shukla, S. Rajput,

developed a stability-indicating HPLC method for isolation and identification of major base

degradation product [19]. Another two HPLC methods were also reported for the

determination of solifenacin succinate in bulk and pharmaceutical formulations [20,21].

54


The main objective of the present investigation was to develop [22] a stability

indicating reverse phase, gradient liquid chromatographic method for the determination of

solifenacin succinate and its impurities in API and pharmaceutical formulations. The

characterization and determination of three impurities of solifenacin succinate by the

developed method, and assay of API sample, study of forced degradation under stress

condition, to resolve all known impurities that were generated during the forced degradation

studies and perform analytical method validation for the proposed method as per ICH

guideline is carried in this study [23-26].

2.1 Experimental

2.1.1 Instrumentation details

UV-Visible spectrophotometer:

Perkin Elmer UV-Visible spectrophotometer (Model: Lambda 35) was used for the UV

absorption in the range of 200-400 nm.

FT-IR spectrophotometer:

Perkin Elmer FT-IR spectrophotometer (Model: Spectrum GX) controlled with spectrum one

software was used for characterization of different functional groups in the SFS and its related

substances.

NMR spectrometer:

The 1H and 13C experiments were performed on a Bruker Advance DPX-300MHz NMR

spectrometer [Bruker AG, Faellanden, Switzerland] using deuterated Dimethyl sulfoxide

(DMSO-d6) as solvent and tetramethylsilane (TMS) as internal standard.

Mass spectrometer:

Alliance 2695 HPLC and Micromass ZQ-2000 MS (Waters Assoc., Milford, MA, USA),

controlled with Mass Lynx version 4.0 software was used for the identification of oxidative

stress impurities.

LC-MS equipment:

Alliance 2695 model HPLC and Micromass ZQ-2000 MS (Waters Assoc., Milford, MA,

U.S.A.), controlled with Mass Lynx (version 4.0) software, used for impurity identification.

Mass spectrometer was provided with electrospray ionization source in positive ion mode.

The capillary sprayer voltage was 3.5 kV and the sample cone voltage was 25 V. The source

temperature was 120° and the desolvation temperature was 350°C. The desolvation and cone

55


gas flow-rates were set to 100 and 650 L/hr, respectively. The acquisition mass range is m/z

0-1000 at 0.5 s per scan with a 0.1 s inter scan delay.

HPLC system-I:

A Waters alliance HPLC system equipped with 2695 separation module with quaternary

gradient pumps with inbuilt auto injector, 270852 thermostatic compartments and connected

with 2996 photodiode array detector was used for peak purity. This was controlled with

empower chromatography manager software.

HPLC system-II: Shimadzu make 2010 series HPLC system equipped with quaternary

gradient pump, auto sampler, column oven and dual wavelength UV-visible detector

controlled with LC solutions software.

2.1.2 Materials and reagents

Samples of SFS API reference standard as well as impurities were characterized in in-house

research and development laboratory. Solifenacin succinate, reference standard (SFSWS/12,

99.90% potency), test samples, impurity-A (purity-92.14%), impurity-B (purity-99.93%),

impurity-C (purity-91.51%) were obtained from Hetero Drugs Limited and solifenacin

succinate innovator tablets 10 mg vesicare (EI000081, Potency-99.8%) obtained from

Astellas Pharma US. HPLC grade acetonitrile (Merck, India), other analytical grade

chemicals and reagents such as potassium dihydrogen orthophosphate, ammonium formate,

orthophosphoric acid and formic acid were purchased from Qualizen Fine chemicals, India.

High pure water was prepared from Milli ‘Q’ system.

2.2 Characterization of SFS Impurities by Spectral Analysis

2.2.1 Characterization of solifenacin succinate impurity-A

Ultra-Violet absorption spectrum:

About 2.0 mg of impurity-A was accurately weighed and transferred into 100 ml volumetric

flask containing 50 ml of methanol. Sonicated for five minutes to dissolve the sample, then

the solution was diluted to the mark with methanol and mixed well with using cyclomixer to

get the uniform solution (20 µg/ml). The ultra-violet absorption spectrum of impurity-A in

methanol was scanned from 200 to 400 nm. The absorbances of compound at different

wavelength maxima along with interpretation were listed in Table.2.1. The UV-absorption

spectra of the impurity-A obtained is represented in Fig.2.3.

56


Fig.2.3: UV-Absorption spectrum of solifenacin succinate impurity-A

Fourier-transform infrared spectrum:

Weighed accurately about 200 mg of KBr (potassium bromide), which is previously dried at

150°C and cooled, into a mortar and grinded to a fine powder. Added about 2.0 mg of

impurity-A, then mixed perfectly and grind to a uniform powder. Taken a small quantity of

the powder and prepared it as thin semi-transparent disk. FT-IR spectrum (Fig.2.4) of the disk

from 3800 cm-1 to 650 cm-1 was recorded by taking air as reference. The major infrared

frequencies and the respective assignments were listed in Table.2.1.

Fig.2.4: FT-IR spectrum of solifenacin succinate impurity-A

57


Carbon (13C) & proton (1H) magnetic resonance spectrum:

The proton magnetic resonance spectrum and carbon-13 nuclear magnetic resonance spectrum

of impurity-A in CDCl3 at 27°C was obtained on a 300 MHz Bruker Advance NMR

spectrometer. Structural assignments were represented as below and corresponding data listed

in Table.2.1. The structural characterization data of impurity-A was given in Table.2.1 and

the corresponding NMR 1H and 13C spectrums were given in Fig.2.5 & 2.6 respectively.

Fig.2.5: 1H NMR spectrum of solifenacin succinate impurity-A

Fig.2.6: 13C NMR spectrum of solifenacin succinate impurity-A

58


Mass spectrum:

The sample solution of concentration 200 µg/ml in methanol was injected into the mass

spectrometer and mass spectrum pattern of impurity-A recorded was given in Fig.2.7 and its

mass fragmentation pattern was given in Fig.2.8. The probable structure of the solifenacin

succinate impurity-A was presented in Fig.2.9.

Fig.2.7: Mass spectrum of solifenacin succinate impurity-A

Fig.2.8: Mass fragmentation pattern of solifenacin succinate impurity-A

59


Fig.2.9: Probable structure of the solifenacin succinate impurity-A

2.2.2 Characterization of solifenacin succinate impurity-B

Ultra-violet absorption spectrum:

A standard solution of concentration 20 µg/ml was prepared by accurately weighed quantity

about 2.0 mg of impurity-B into a 100 ml volumetric flask contained 50 ml of methanol.

Sonicated for five minutes to dissolve sample, then the solution was diluted to the mark with

methanol and mixed well with using cyclomixer to get the uniform solution. The ultra-violet

absorption spectrum of impurity-B in methanol was scanned from 200 to 400 nm. The uv-

absorbances of compound at different wavelength maxima along with interpretation were

listed in Table.2.2. The uv-absorption spectrum of impurity-B was shown in Fig.2.10.

Fig.2.10: UV-absorption spectrum of solifenacin succinate impurity-B

60



About 200 mg of (dried at 150°C and cooled) KBr was weighed accurately, transferred into a

mortar and grinded to a fine powder. Then about 2.0 mg of impurity-B was added, then mixed

perfectly and grind to a uniform powder. A small quantity of the powder was taken and

prepared as thin semi-transparent disk. FT-IR spectrum (Fig.2.11) of the disk from 3800 cm-1

to 650 cm-1 was recorded by taking air as reference. The major infrared frequencies and the

respective assignments were listed in Table.2.2.

Fig.2.11: FT-IR spectrum of solifenacin succinate impurity-B


The proton magnetic resonance spectrum and carbon-13 nuclear magnetic resonance

spectrum of impurity-B in CDCl3 at 27°C is obtained on a 300 MHz Bruker Advance NMR

spectrometer. Structural assignments are represented as below and data listed in Table.2.2.

The NMR 1H and 13C spectrums are given in Fig.2.12 & 2.13 respectively.

61


Fig.2.12: 1H NMR spectrum of solifenacin succinate impurity-B

Fig.2.13: 13C NMR spectrum of solifenacin succinate impurity-B

Mass spectrum:

Accurately weighed about 2.0 mg of impurity-B into a 20 ml volumetric flask, contained 10

ml methanol, sonicated to dissolve the sample and diluted to the volume with diluent and

mixed well. The sample solution injected into the mass spectrometer, and mass spectrum

pattern of the impurity-B was given in Fig.2.14. The obtained mass fragmentation pattern was

given in Fig.2.15. The most probable structure of the impurity-B was presented in Fig.2.16.

62


Fig.2.14: Mass spectrum of solifenacin succinate impurity-B

Fig.2.15: Mass fragmentation pattern of solifenacin succinate impurity-B

Fig.2.16: Probable structure of the solifenacin succinate impurity-B

63


2.2.3 Characterization of solifenacin succinate impurity-C

Ultra-violet absorption spectrum:

About 20 µg/ml solution of impurity-C was prepared in methanol. The ultra-violet spectrum

of impurity-C in methanol was scanned from 200 to 400 nm. The uv-absorbances of

compound at different wavelength maxima along with interpretation were listed in Table.2.3.

The uv-absorption spectrum of the impurity-B was shown in Fig.2.17.

Fig.2.17: UV-absorption spectrum of solifenacin succinate impurity-C


About 2.0 mg of impurity-C was added to 200 mg of grinded to a fine powder of KBr, then

mixed perfectly and grind to a uniform powder. A small quantity of powder sample was taken

and prepared a thin semi-transparent disk, recorded the FT-IR spectrum (Fig.2.18) of the disk

from 3800 cm-1 to 650 cm-1, taking air as reference. The major infrared frequencies and the

respective assignments were listed in Table.2.3.


1H and 1 3C NMR spectra (Fig.2.19 & 2.20) of impurity-C in CDCl3 at 27°C was obtained on

a 300 MHz Bruker Advance NMR spectrometer. Structural assignments were represented in

Table.2.3.

64


Fig.2.18: FT-IR spectrum of solifenacin succinate impurity-C

Fig.2.19: 1H NMR spectrum of solifenacin succinate impurity-C

65


Fig.2.20: 13C NMR spectrum of solifenacin succinate impurity-C

Mass spectrum:

Weighed accurately about 2.0 mg of impurity-C into 20 ml volumetric flask contained 10 ml

methanol. Sonicated to dissolve the sample and diluted to the volume with diluent and mixed

well. This sample solution is injected into the mass spectrometer and the mass fragmentation

pattern was recorded. The mass spectrum and mass fragmentation pattern of the impurity-C

were given in Fig.2.21 & 2.22 respectively. The proposed structure of the impurity-C was

presented in Fig.2.23.

Fig.2.21: Mass spectrum of solifenacin succinate impurity-C

66


Fig.2.22: Mass fragmentation pattern of solifenacin succinate impurity-C

Fig.2.23: Probable structure of the solifenacin succinate impurity-C

2.3 Analytical RP-HPLC Method Development and Optimization

2.3.1 Analytical RP-HPLC method development

Selection of wavelength of maximum absorbance:

The UV-absorption spectra (Fig.2.24) of solifenacin and its impurities were recorded in HPLC

system equipped with photo diode array detector. The absorption maxima of solifenacin and

its impurities were observed at 220 nm. Hence the same wavelength was selected for the

quantification of related impurities in solifenacin succinate.

Selection of stationary phase:

The main objective of this chromatographic method was the separation of critical closely

eluting impurity pairs, i.e. solifenacin and impurity-C, between impurity-A and impurity-B. In

this study, the determination of solifenacin and its impurities was made by employing

different columns to achieve the best separation and resolution. The above said impurities

were eluted very closely to each other by using different stationary phases like CN, C18, C8

and phenyl.

67


Fig.2.24: UV-absorption PDA spectra of SFS and its impurities

Selection of mobile phase:

To meet this, different mobile phases containing buffers like phosphate, sulfate and acetate

with different pH (2.5-5) and using organic modifiers like acetonitrile and methanol in the

mobile phase. The pH of the buffer has played a significant role in achieving the separation

between impurity-A and impurity-B, solifenacin and impurity-C. At higher pH of the buffer in

the mobile phase, the peak shape of solifenacin was very broad, so lower pH was selected for

separation between solifenacin and its three impurities. Here we selected a phosphate buffer

and adjusted pH of the solution to 3.5 with orthophosphoric acid.

Preparation of buffer (Mobile phase-A):

Accurately weighed and transferred 1.36 g (0.01M) of potassium dihydrogen orthophosphate

into two litre beaker contained 1000 ml of water, sonicated for ten minutes and adjusted pH of

this solution to 3.5±0.05 with orthophosphoric acid. The resulting solution is filtered and

degassed through 0.45 µ membrane filter paper.

Preparation of Mobile phase-B:

Prepared a mixture of acetonitrile and water in the ratio of 90:10 (v/v). The resulting solution

is filtered and degassed through 0.45 µ membrane filter paper.

68


Preparation Diluent:

Prepared a mixture of mobile phase-A and acetonitrile in the ratio of 90:10 (v/v).

Mode of elution:

In order to get the clear separation between all the known, unknown impurities as well as

degradants, the gradient method was recommended rather than isocratic method. Since there

was no clear separation between these impurities in isocratic method, the gradient method was

used to assay and process related impurities in solifenacin succinate in the present study.

2.3.2 Optimization of the proposed method

Several trials were made by using different mobile phase ratios, gradient programmes by

varying buffer pH between 2 and 8 with C8, C18, phenyl and cyano stationary phases. Based

on experimental trials it was understood that pH and stationary phases were playing the

critical role in the separation between impurity-C and solifenacin, because of impurity-C

having similar chemical structure (only one oxygen atom) when compared to solifenacin. The

method development experiments were summarized in the Table.2.4. Initially method

development was started with solifenacin standard prepared in acetonitrile as diluent. But, it

was observed that succinic acid peak shape was not good and solifenacin peak was also broad.

Then all the impurities and solifenacin is dissolved in mobile phase-A, but solution was not

clear. Finally the solution was cleared by adding few drops of acetonitrile. In order to dissolve

all impurities and sample, mobile phase-A and acetonitrile was taken as diluent in the ratio of

90:10 (v/v) respectively. Few of HPLC trails with different pH and mobile phase

combinations were given in Fig.2.25 to 2.30 respectively.

Finally the separation of impurities from solifenacin was achieved with waters

symmetry shield RP-18, 150 mm x 4.6 mm, 5 µm column, by using variable mixtures of

mobile phase-A and mobile phase-B as mobile phase in gradient mode. The flow rate of the

mobile phase was 1.0 ml/minute. The column temperature kept at 35°C. In the optimized

conditions solifenacin succinate, impurity-A, impurity-B and impurity-C were well separated

with a resolution of greater than 3.0. For the determination of assay of solifenacin, the above

HPLC gradient programme of related compounds method was optimized with shorter run

time. In the optimized assay method, the resolution between solifenacin and impurity-C was

greater than 2.5 and the peak shape was also symmetrical. The retention of solifenacin in the

optimized assay gradient method was at about 5.5 minutes.

69


Fig.2.25: HPLC chromatogram of SFS spiked with its three impurities



70


Fig.2.28: HPLC development trials of SFS spiked with its three impurities



71


2.3.3 Optimized chromatographic separation

The chromatographic separation was achieved by injecting 10 µl in gradient mode using

symmetry shield RP-18, 150 x 4.6 mm, 5 µm, at 35°C and the components were detected at

220 nm with a flow rate of 1.0 ml/min for 40 minutes. Gradient programme for related

compounds was time/% mobile phase-B: 0.01/20, 20/40, 30/40 and 32/20 with a post run time

of 8 minutes, whereas for assay the gradient programme was maintained as time/% mobile

phase-B: 0.01/30,7/60 and 10/30 with a post run time of 5 minutes. The typical retention

times for solifenacin, impurity-A, impurity-B and impurity-C were about 16.5, 4.5, 6.2 and

18.5 minutes respectively, and the developed method was found to be specific for solifenacin

and its three impurities.

2.3.4 Identification of degradants by LC-MS

After completion of RP-HPLC method development for related compounds, then performed

preliminary degradation studies for identification and evaluation of degradation pathways and

major degradation impurities. The major degradation was observed in oxidative stress

condition. The major degradant formed in oxidative stress condition was identified by LC-

MS.

Selection of LC-MS conditions & identification of oxidative impurity by LC-MS:

Based on the HPLC conditions, ammonium formate was selected for buffer instead of

potassium dihydrogen orthophosphate, which was due to nonvolatile nature of phosphate

buffer. The chromatographic separation was achieved on waters symmetry shield RP-18

150 mm x 4.6 mm, 5 µm column by performing gradient programme, employs variable

solutions of mobile phase-A and mobile phase-B used as mobile phase. The mobile phase-A

contained 0.01 M ammonium formate in water, adjusted to pH: 3.5±0.05 with formic acid

solution and mobile phase-B contained mixture of acetonitrile and water in the ratio of 90:10

(v/v). All chromatographic conditions were same as used in LC-MS except flow rate i.e 0.9

ml/minute. The injection volume was 20 µl. A mixture of mobile phase-A and acetonitrile in

the ratio of 90:10 (v/v) used as diluent.

For the identification of impurity-A, impurity-B, SFS sample was analyzed and the ESI

mass spectrum of impurities eluted at RRT 0.28 and 0.37 (Fig.2.31) in positive ion mode

showed a molecular ion peaks at m/z 208, 210 [(mH)+], indicating the molecular weights of

these compounds were 207 and 209 respectively. From this data we concluded that these two

impurities were intermediates, which are used in manufacturing process of solifenacin and

72


these two confirmed with photo diode array detector by comparing the spectra of known

standards. ESI mass spectrum of major impurity formed during the oxidative stress condition

at RRT 1.10 in positive ion mode showed a molecular ion peak at m/z 379 [(mH)+] indicating

the molecular weight of the compound as 378. The mass spectrum of this impurity was shown

in Fig.2.32 This molecular ion mass were 16 mass units higher than that of solifenacin

indicating that the probability to the formation of N-oxide. The same impurity was also

formed in base degradation, but in smaller quantities.

Fig.2.31: LC-MS chromatogram of solifenacin succinate and its impurities

Fig.2.32: Mass spectrum of impurity-C at RRT~1.1

73


2.4 Specificity and Forced Degradation

2.4.1 Specificity

Specificity is the ability to assess unequivocally the analyte in the presence of components

which may be expected to be present. Typically these might include impurities, degradant,

matrix, etc. Lack of specificity of an individual analytical procedure may be compensated by

other supporting analytical procedures. Specificity can be determined for instance by spiking

pure substances with excipients and/or impurities and /or degradation products and to

compare the test results with those of pure substances.

Forced degradation studies were performed to provide an indication of the stability

indicating property and specificity of the proposed RP-HPLC method for solifenacin, in the

presence of its impurities. The HPLC chromatograms of blank, solifenacin and spiked sample

chromatograms were shown in Fig.2.33 to 2.35. No interferences were observed due to blank

at the retention time of impurity-A, impurity-B, impurity-C and solifenacin succinate. The

elution order of impurity-A, impurity-B, impurity-C obtained from individual solution and

test & impurity blend solution were matched. The typical retention time of solifenacin is

about 16.5 minutes. The retention times of succinic acid, impurity-A, impurity-B and

impurity-C are at about 1.8, 4.5, 6.1 & 18.4 minutes respectively.

Fig.2.33: Typical HPLC chromatogram of blank solution

74


Fig.2.34: Typical HPLC chromatogram of solifenacin succinate

Fig.2.35: HPLC chromatogram of SFS spiked with 0.15% of impurity-A,B&C

2.4.2 Forced degradation

The drug was allowed to degrade under some various environmental and chemical conditions

for the study of extent of degradation and stability of the drug under the same conditions. The

experimental details for the forced degradation under the investigation were presented below.

Ambient condition (25±2°C):

Transferred about 2.0 g SFS sample in to petridish and spread uniformly and the petridish was

kept in laboratory conditions (at 25±2°C) for 96 hours and analyzed for, FT-IR and related

compounds and assay. It was observed that the sample was stable under ambient condition.

Thermal degradation:

About 2.0 g SFS sample was transferred into petridish, spread uniformly and it was kept in

oven at 105°C for 10 days, after that removed the sample from oven and cooled to room

temperature and then analyzed. At the end of the stipulated time period, removed the sample

75


from oven and analyzed for description, FT-IR and related compounds and assay. It was

found that the sample was stable under thermal condition.

Exposure to humidity (90% RH):

About 2.0 g SFS sample was taken into petridish and spread uniformly. Then the petridish

was kept in desiccator containing saturated ammonium chloride solution (to obtain 90% RH)

for 10 days. At the end of the stipulated time period, removed the sample from desiccator and

analyzed for description, FT-IR and related compounds, assay. Sample was found to be stable

under humidity conditions.

Exposure to photo light (Photolysis):

Transferred about 2.0 g SFS sample into petridish and spread uniformly. Then the petridish

was kept in photo light chamber and exposed it to light for three times cycle to 1.2 million

LUX hours and 200 Watt-Hours/Sq.mts. At the end of the stipulated time period, removed the

sample from photolytic chamber and analyzed for description, FT-IR and related compounds

and assay, and found that the sample was stable under photolytic condition.

Hydrolysis (Water degradation):

Weighed and transferred accurately 50 mg of SFS sample into a 100 ml volumetric flask,

added 10 ml of water and heated the solution at 80°C for 4 hrs. At the end of the exposure

time period, cooled the solution and made up to the volume with diluent and mixed well and

analyzed for description, FT-IR, related compounds and assay. It was observed that sample

was stable under photolytic condition.

Acid hydrolysis (Acid degradation):

About 50 mg of SFS sample was weighed and transferred accurately into a 100 ml volumetric

flask, added 10 ml of 0.1N HCl and heated the solution at 80°C for 4 hrs. At the end of the

exposure, cooled the solution and made up to the volume with diluent and mixed well. Taken

this solution and analyzed for description, FT-IR, related compounds and assay. The sample

was stable under acid hydrolysis.

Base hydrolyses (Base dégradation) :

Weighed and transferred accurately 50 mg of SFS sample into a 100 ml volumetric flask,

added 10 ml of 0.1N NaOH and heated the solution at 80°C for 4 hours. At the end of the

exposure, cooled the solution and made up to the volume with diluent and mixed well. Taken

this solution and analyzed for description, FT-IR, related compounds and assay. It was found

76


that the SFS was stable under base hydrolysis.

Oxidation: Weighed and transferred accurately 50 mg of SFS sample into a 100 ml

volumetric flask, added 10 ml of 10% H2O2 and heated the solution at 80°C for 2 hours. At

the end of the exposure, cooled the solution and made up to the volume with diluent and

mixed well. This solution was analyzed for description, FT-IR, related compounds and assay.

It was observed that SFS degraded under oxidative conditions. Typical HPLC degradation

chromatograms were shown in Fig.2.36 to 2.41 respectively. The summary of forced

degradation studies were given in the Table.2.5.

During the forced degradation studies, it was observed that, SFS was not degraded

significantly under the ambient (25+2°C), thermal (105°C for 10 days), photolytic (1.2 million

LUX Hrs & 200Watt-hrs/Sq.mts), humidity, acid (0.1N HCl-4hrs at 80°C) and base (0.1N

NaOH-4hrs at 80°C) hydrolysis. Significant degradation was observed in oxidative stress

condition.

Fig.2.36: Typical HPLC chromatogram of acid (0.1N HCl) stressed solution

Fig.2.37: Typical HPLC chromatogram of base (0.1N NaOH) stressed solution

77


Fig.2.38: Typical HPLC chromatogram of oxidative (10%H2O2) solution

Fig.2.39: Typical HPLC chromatogram of water hydrolysis solution

Fig.2.40: Typical HPLC chromatogram of thermal stressed solution

78


Fig.2.41: Typical HPLC chromatogram of photo light stressed solution

The peak purity test was carried out for SFS sample for each stress condition by using with

PDA detector. In each stressed condition the single point threshold of SFS was less than that

of peak purity index value. Assay studies were carried out for stress samples against qualified

reference standard and the mass balance (%assay + %impurities + %degradation products)

was calculated. Assay was also calculated for bulk sample by spiking all three impurities

(impurity-A, B & C) at the specification level (i.e. 0.15 % of impurity-A, B and C with

respect to analyte concentration, which was 0.5 mg/ml). The mass balance data of forced

degradation samples were shown in Table.2.6.

2.5 Analytical Method Validation

2.5.1 Preparation of solutions

Preparation of working standard stock solution (200 µg/ml):

Accurately weighed and transferred 500 mg of SFS reference standard into a 100 ml

volumetric flask containing 50 ml diluent, sonicated for ten minutes and diluted up to the

mark with diluent and mixed homogeneously by kept on cyclo mixer for five minutes.

Transferred 4.0 ml of this solution into 100 ml volumetric flask and diluted to the volume

with diluent, mixed homogeneously by using on cyclo mixer for five minutes.

Preparation of working standard reference solution (20 µg/ml):

Accurately weighed and transferred 2.0 mg of SFS reference standard into a 100 ml

volumetric flask containing 50 ml diluent, sonicated for ten minutes and diluted up to the

mark with diluent and mixed homogeneously by using cyclo mixer for five minutes.

79


Preparation of impurity stock solution (30 µg/ml): Accurately weighed and transferred

each 3.0 mg of impurity-A, impurity-B and impurity-C into a 100 ml volumetric flask

containing 50 ml diluent. Sonicated for five minutes and diluted to the mark with diluent and

mixed homogeneously by using cyclo mixer for five minutes.

Preparation of standard solution (0.75 µg/ml of impurities & 0.5 µg/ml of standard):

Transferred 2.5 ml of impurity stock solution (30 µg/ml) and 0.25 ml of working standard

solution (200 µg/ml) into 100 ml volumetric flask and then diluted to the volume with diluent

and mixed homogeneously by using cyclo mixer for five minutes.

Preparation of system suitability solution (500 µg/ml of test & 0.75 µg/ml of impurities):

Accurately weighed and transferred 50 mg of test sample into a 100 ml volumetric flask

containing 50 ml diluent, sonicated for 10 minutes and added 2.5 ml of impurity stock

solution and then diluted to the mark with diluent and mixed homogeneously by using cyclo

mixer for five minutes.

Preparation of test solution (500 µg/ml): Accurately weighed and transferred 50 mg of the

test sample into a 100 ml volumetric flask containing 50 ml of diluent, sonicated for 10

minutes and diluted to the mark with diluent and mixed homogeneously by using on cyclo


Preparation of assay standard solution (100 µg/ml): Accurately weighed and transferred 10

mg of SFS reference standard into a 100 ml volumetric flask containing 50 ml diluent,

sonicated for 10 minutes and diluted to the mark with diluent and mixed homogeneously.

Preparation of assay test solution (100 µg/ml): Accurately weighed and transferred 10 mg

of SFS test sample into a 100 ml volumetric flask containing 50 ml diluent, sonicated for 10

minutes and diluted to the volume with diluent and mixed homogeneously by kept on cyclo


Preparation of SFS tablet solution (100 µg/ml): Thirty tablets were crushed to fine powder

by mortar and pestle. Sample powder equivalent to about 50 mg of SFS was weighed and

transferred to 100 ml volumetric flask, 70 ml of diluent was added and sonicated for 30

minute with intermittent swirling, diluted to volume with diluent (0.5 mg/ml of solifenacin

succinate) and filtered with 0.45 μm nylon membrane filter. Evaluated the resolution between

SFS and impurity-C (N-Oxide). The system suitability parameters like, resolution between

closely eluting impurities, theoretical plates and tailing factor of SFS was shown in Table.2.7.

Typical spiked chromatogram of SFS with its impurities was shown in Fig.2.42.

80


Fig.2.42: Typical HPLC chromatogram of system suitability solution

2.5.2 Detection limit (DL) & Quantitation limit (QL)

The DL and QL for solifenacin, impurity-A, impurity-B and impurity-C were estimated at a

signal-to-noise ratio of 3:1 and 10:1 respectively, by injecting a series of diluted solutions

with known concentrations [19]. The typical DL and QL chromatograms were displayed in

Fig.2.43 & 2.44 respectively. Precision study was also carried at the QL level by injecting six

replicates of solifenacin spiked with impurity-A, impurity-B and impurity-C blend and

calculated % RSD for the peak areas.

Fig.2.43: Typical HPLC chromatogram of DL solution

81


Fig.2.44: Typical HPLC chromatogram of QL solution

Based on the S/N ratio obtained from DL solution prepared the QL solution and calculated

the S/N ratio. The detection and quantitation limits values based on signal to noise ratio

values of solifenacin, impurity-A, B and C was shown in Table.2.8. The % RSD calculated

for area of each impurity and solifenacin. The precision data of these three impurities and

solifenacin at quantitation level was shown in Table.2.9.

2.5.3 Linearity

Preparation of linearity stock solutions:

Linearity of solifenacin, impurity-A, impurity-B and impurity-C solutions were prepared by

taking different aliquots ranging from 0.25, 0.40, 0.45, 0.50, 0.60, 0.75 ml of impurity stock

and reference solutions into separate 20 ml volumetric and made up to the volume with

diluent and mixed homogeneously by kept on cyclo mixer for five minutes The above

mentioned at seven concentrations levels from QL to 150% of the specification level (i.e 0.15

%) were injected into the chromatographic column and the area of each peak was calculated,

and a calibration curve was drawn by plotting area impurities against the concentration

expressed in percentage. From the calibration curve correlation coefficient, slope, y-intercept

and residual sum was calculated and shown in Table.2.10 to 2.17, which confirmed good

linearity between peak areas and concentration. Typical different concentrations of HPLC

linearity chromatograms of three impurities at different concentrations were displayed in

Fig.2.45 to 2.50 respectively. The linearity curves (Fig.2.51 to 2.54) and residual graph for

impurities A, B, C and solifenacin were shown in Fig.2.55 to 2.58 respectively.

82


Fig.2.45: HPLC chromatogram of SFS and its impurities at QL level

Fig.2.46: HPLC chromatogram of solifenacin and its impurities at 50% level

Fig.2.47: HPLC chromatogram of solifenacin and its impurities at 80% Level

83





84


Fig.2.51: Calibration curve of solifenacin succinate impurity-A

Fig.2.52: Calibration curve of solifenacin succinate impurity-B

Fig.2.53: Calibration curve of solifenacin succinate impurity-C

1927 15383

24391

28070 31552 37319

47048

0

10000

20000

30000

40000

50000

0.000 0.050 0.100 0.150 0.200 0.250

Peak

Are

a

Concentration(%)

Linerity graph of solifenacin succinate impurity-A Average area Vs Concentration

y=210020x-325 R2=0.9997

3342 25411

41404 46816 52071

63213 79118

0

20000

40000

60000

80000

100000

0.000 0.050 0.100 0.150 0.200 0.250

Peak

Are

a

Concentration(%)

Linerity graph of solifenacin succinate impurity-B Average area Vs Concentration

3708

14269

23662 27278 30361 35010

45181 y =201872x- 461 R² = 0.998

0

10000

20000

30000

40000

50000

0.000 0.050 0.100 0.150 0.200 0.250

Peak

Are

a

Concentration(%)

Linerity graph of solifenacin succinate impurity-C Average area Vs Concentration

y=353463x-711 R² = 0.9998

85


Fig.2.54: Calibration curve of solifenacin succinate

Fig.2.55: Residual plot of solifenacin succinate impurity-A

Fig.2.56: Residual plot of solifenacin succinate impurity-B

3848 9882

16397

18302 20684

24087 29890

y = 201556x+20 R² = 0.9987

0 5000

10000 15000 20000 25000 30000 35000

0.000 0.050 0.100 0.150 0.200

Peak

Are

a

Concentration(%)

Linerity graph of solifenacin succinate impurity-C Average area Vs Concentration

152

-44 -486

42 374

-160

118

-3500 -2500 -1500 -500 500

1500 2500 3500

0 1 2 3 4 5 6 7 8 Res

idua

ls

Order of residuals

Residual plot of solifenacin succinate impurity-A

518

-388 -301 -191 -237

301 300

-5207

-3207

-1207

793

2793

4793

0 1 2 3 4 5 6 7 8 Res

idua

ls

Order of residuals

Residual plot of solifenacin succinateimpurity-B

86


Fig.2.57: Residual plot of solifenacin succinate impurity-C

Fig.2.58: Residual plot of solifenacin succinate

2.5.4 Range

The range of an analytical procedure normally was derived from the linearity studies, interval

between the upper and lower concentration for which the analytical procedure has

demonstrated a suitable level of precision, accuracy, and linearity. The lower and upper range

of impurity-A, impurity-B and impurity-C were shown in Table.2.18 to 2.19 respectively. For

an impurity, the range should be determined from the reporting level to 120% of the

specification.

132

-410 -102

486 541

-866

221

-3000

-2000

-1000

0

1000

2000

3000

0 1 2 3 4 5 6 7 8

Res

idua

ls

Order of residuals

Residual plot solifenacin succinate impurity-C

-203 -216

253 142 508

-120 -363

-2000

-1000

0

1000

2000

0 1 2 3 4 5 6 7 8 Res

idua

ls

Order of residuals

Residual plot for Solifenacin succinate

87


2.5.5 Accuracy

Accuracy of the impurities was carried out in triplicate at 0.075 % (0.375 µg/ml), 0.15% (0.75

µg/ml) and 0.225% (1.125 µg/ml) (50, 100 and 150%) levels of the SFS concentration (0.5

mg/ml or 500 µg/ml). The percentage of mean recoveries in three replicates of all the

impurities at 50, 100, 150% and QL levels were found to be in the range of 97.61-105.31%.

The mean recovery results were shown in Table.2.20. The accuracy chromatograms were

displayed in Fig.2.59 to 2.62.

Fig.2.59: HPLC accuracy chromatogram of SFS test spiked with its three impurities at QL concentration Level

Fig.2.60: HPLC chromatogram of SFS test spiked with its three impurities at 50%

88




2.5.6 Precision & Intermediate precision

The system precisions of the impurities were checked by injecting six replicate runs of SFS

and its impurities at concentration of 0.1% and 0.15% respectively. The % RSD was

calculated for area of each impurity. The precision of the method was checked by injecting six

individual preparations of solifenacin test sample and calculated known and unknown

impurities content from six sample preparations. The intermediate precision of the method

was also evaluated by using different day, different lots of column and a different instrument

in the same laboratory. The % RSD for area of each impurity from system precision, method

precision and intermediate precision was calculated and results were summarized in

Table.2.21 to 2.23.

89


2.5.7 Robustness

The chromatograms for the deliberate change in chromatographic conditions in the study of

robustness such as flow rate from 1.0 ml/min to 0.8 ml/min (Fig.2.63) and 1.2 ml/min

(Fig.2.64), column temperature from 35°C to 33°C (Fig. 2.65) and 37°C (Fig.2.66), pH of

the buffer from 3.5 to 3.3 (Fig.2.67) and 3.7 (Fig.2.68) and organic phase composition in the

mobile phase-B composition from 100% to 95% (Fig.2.69) and 105% (Fig.2.70) were

recorded. The method was demonstrated to be robust over an acceptable working range of its

operational parameters as shown in Table.2.24 to 2.32.

Fig.2.63: HPLC robustness chromatogram of SFS spiked with its three impurities at 100% concentration level with flow rate: 0.8 ml.min-1

Fig.2.64: HPLC robustness chromatogram of SFS spiked with its three impurities at 100% concentration level with flow rate: 1.2ml.min-1

90


Fig.2.65: HPLC robustness chromatogram of SFS spiked with its three impurities at

100% concentration level with column temperature: 33°C


100% concentration level with column temperature: 37°C

Fig.2.67: HPLC robustness chromatogram of SFS spiked with its three impurities at 100% concentration level with mobile phase pH: 3.3±0.05

91



100% concentration level with mobile phase pH: 3.7±0.05

Fig.2.69: HPLC robustness chromatogram of SFS spiked with its three impurities at 100% concentration with mobile phase-B in the ratio of 85:10, v/v

Fig.2.70: HPLC robustness chromatogram of SFS spiked with its three impurities at 100% concentration with mobile phase-B in the ratio of 95:10, v/v

92


2.5.8 Solution stability and mobile phase stability

The solution stability of SFS and its three impurities was carried out by leaving spiked sample

solutions in tightly capped volumetric flasks at room temperature for 48 hrs. Content of each

impurity was estimated for every 12 hrs interval up to 48 hrs. The mobile phase stability was

also carried by analyzing freshly prepared sample solutions in stored mobile phase at bench

top for six days and observed the results with precision study. No significant changes were

observed in the content of impurities of SFS i.e. impurity-A, impurity-B and impurity-C

during the solution stability and mobile phase stability experiments. The mobile phase and

solution stability results were presented in Table.2.33 & 2.34 respectively.

2.5.9 Relative response factor (RRF)

The relative response factor for each impurity was established by injecting known

concentrations i.e 0.75 µg/ml (0.15%) of impurity-A, impurity-B and impurity-C and

solifenacin. The RRF for the above said impurities were calculated by comparing area of

solifenacin. The RRF values for all the impurities found to be between 1.08 and 1.74. The

chromatographic data, including retention times, relative retention times and relative response

factors for the three impurities were tabulated in Table.2.35.

2.6 Solifenacin succinate batch analysis

Using the above validated method, few commercial solifenacin succinate API batch samples

and vesicare tablets were analyzed for related compounds and assay. In this gradient

programmes blank and placebo interferences were not observed at retention time of

solifenacin succinate in tablet analysis. The obtained results of API and tablet data were

furnished in Table.2.36 & 2.37 respectively. The typical HPLC chromatograms of solifenacin

succinate API batches were shown in Fig.2.71 to 2.73. The typical HPLC chromatogram of

vesicare tablet and assay chromatogram was shown in Fig.2.74 & 2.75 respectively.

93


Fig.2.71: Typical HPLC chromatogram of SFS B.No-446

Fig.2.72: Typical HPLC chromatogram of SFS B.No-SFS0030509

Fig.2.73: Typical HPLC chromatogram of SFS B.No-SFS004 (crude)

94


Fig.2.74: Typical HPLC chromatogram of solifenacin succinate tablet (VESIcare)

Fig.2.75: Typical HPLC assay chromatogram of solifenacin succinate (API)

2.7 Results and Discussions

The presence of impurities in bulk drug can have a significant impact on the quality and

safety of the drug. Therefore, it is necessary to study the impurity profile of the API to be

used in the manufacturing of a drug product. During the analysis of laboratory batches of

solifenacin succinate, three impurities were detected. These three impurities are identified

and characterized with UV, FT-IR, NMR, LC-MS and Mass spectrometry. The structures of

these impurities were identified, names of these impurities designated as impurities-A, B and

C with chemical names given as 1-Phenyl-3,4-dihydroisoquinoline, (1S)-1-Phenyl-1,2,3,4-

tetrahydro isoquinoline and (1S)-3,4-Dihydro-1-phenyl-2-(1H)-isoquinoline carboxylic acid

(3R)-1-aza bicyclo [2.2.2]oct-3-yl ester N-oxide respectively. Simple and precise analytical

RP-HPLC gradient method was developed for the determination of related impurities and to

find out assay of SFS API samples. In the developed HPLC methods no blank interferences

95


were observed due to diluents or solvent or reagents.The chromatographic separation was

achieved by injecting 10 µl in gradient mode using symmetry shield RP-18, 150 x 4.6 mm, 5

µm column, at oven temperature of 35°C and the components were monitored at 220 nm with

a flow rate of 1.0 ml/min for 40 minutes. The gradient programme for related compounds was

Time/%Mobile phase-B: 0.01/20, 20/40, 30/40 and 32/20 with a post run time of 8 minutes.

Whereas for assay the gradient programme was optimized with shorter run time as

Time/%Mobile phase-B: 0.01/30,7/60 and 10/30 with a post run time of 5 minutes. The

typical retention times for solifenacin, impurity-A, impurity-B and impurity-C were about

16.5, 4.5, 6.2 and 18.5 minutes respectively and the developed method was found to be

specific for solifenacin and its three impurities. RT, RRT and RRF for the impurities A, B and

C were found to be 4.5, 0.27 and 0.26; 6.2, 0.38 and 0.39; 18.5, 1.12 and 1.14 respectively.

LC-MS method also described for the identification of oxidative stress products. ESI mass

spectrum of major impurity formed during the oxidative stress condition at RRT 1.1 in

positive ion mode showed a molecular ion peak at m/z 379 [(mH)+] indicating the molecular

weight of the compound as 378. This molecular ion mass was 16 mass units higher than that

of solifenacin and this indicates that the probability to the formation of N-oxide. The same

impurity was also formed in base degradation, but in smaller quantities.

The specificity and forced degradation of SFS was determined in presence of

its impurities, by the developed RP-HPLC method and no interferences were observed from

blank at the retention times of impurity-A, impurity-B, impurity-C and solifenacin succinate.

The elution order of impurity-A, impurity-B, impurity-C obtained from individual solutions

and impurity blend solutions are in same elution order with same retention times. During the

forced degradation studies, it was observed that, solifenacin sample was not degraded

significantly under the ambient (25+2°C), thermal (105°C for 10 days), photolytic (1.2

million LUX Hrs & 200 Watt-hrs/Sq.mts, humidity), acid (0.1N HCl, 4hrs at 80°C) and base

(0.1N, NaOH, 4hrs at 80°C) hydrolysis. Significant degradation was observed in oxidative

stress condition. The peak purity test was carried out for solifenacin in each stress condition

by using PDA detector. In each stressed condition the single point threshold of SFS was less

than that of peak purity index value. The mass balance data of forced degradation samples of

solifenacin was found to between 100.1 to 101.7%.

Assay studies were carried out for stress samples against qualified reference

standard and the mass balance (%assay+%impurities+%degradation products) was calculated.

Assay was also calculated for bulk sample by spiking all these three impurities at the

96


specification level (i.e. 0.15% of impurity-A, B and C with respect to analyte concentration,

which is 0.5 mg/ml). The detection limit (DL), quantitation limit (QL) and precision study at

QL (%RSD, 0.90-4.53) level was also carried for solifenacin spiked with impurity-A,

impurity-B and impurity-C and calculated the % RSD for the peak areas. The detection limits

of impurity-A, B & C are 0.003, 0.002 and 0.007% respectively. The quantitative limits of

these three impurities are 0.01, 0.01 and 0.02% respectively. The developed method showed

good precision (less than 5.0%) and accuracy (97.9 to 104.1%) at QL level. A calibration

curve was drawn by plotting peak area of impurities against the concentration expressed in

percentage (QL, 50, 80, 90, 100, 120 and 150% levels), from the calibration curve correlation

coefficient, slope, y-intercept and residual sum was calculated. The coefficient of correlation

was found to be 0.9993-0.9999. The percentage mean recoveries of three replicates of all the

impurities at 50, 100, 150% and QL levels were found to be in the range of 98.36-107.24. The

intermediate precision of the method was also evaluated by using different day, different lots

of column and a different instrument in the same laboratory. Calculated the % RSD of area of

each impurity for system precision, method precision and intermediate precision was found to

be within the limits. The method was demonstrated to be robust over an acceptable working

range of its operational parameters. Solution stability and mobile phase stability studies also

carried out and these solutions are stable up to 48hrs. Different samples of solifenacin

succinate API batches, crude samples tablets were analyzed by using the proposed method,

among these, all three impurities were detected in few batches, impurity-C only was detected

in VESIcare and few API batches. The chromatographic assay method was used for few API

and tablet dosage forms.

2.8 Conclusion

A simple gradient HPLC method was developed for quantification of SFS and related

compounds and assay is precise, accurate, rapid and specific. The developed method was

stability indicating and can be conveniently used by quality control department to determine

the related substance of regular SFS samples and stability samples.

97


Tables:

Table.2.1: UV, FT-IR & NMR structural data of solifenacin succinate impurity-A

UV-absorption data

Wavelength Absorbance Band

204.10nm 0.3485 E1 band of aromatic ring

247.36nm 0.1031 B band of aromatic ring

FT-IR Assignment data

Frequency(cm-1) Assignment

3060 & 3026 =C-H stretching of aromatic ring

2939 & 2839 C-H-stretching of CH2

1607&1566 C=N Stretching

1481 & 1445 C=C stretching in aromatic ring

1317 & 1306 C-H bendings in CH2

1229 C-N stretching

1158,1053 & 1020 In plane bending vibrations of C-H in aromatic ring

761, 744 & 699 Out of plane bending vibrations of C-H in aromatic ring 13C & 1H Assignment data

Proton No. Multiplicity Chemical shifts

(ppm) Carbon No Chemical shift

H-2´,6´(2H) m 7.58-7.61 C-1 166.65

H-3’,4’,5’8(4H) m 7.40-7.42 C-4a, 8a 138.26-138.47

H-5,6,7(3H) m 7.24-7.26 C-1’ 130.16

H-3(2H) m 3.81-3.86 C-4´ 128.78

H-4(2H) t 2.76-2.81 C-2´,6´,6 128.23-128.29

--- --- --- C-8 127.62

--- --- --- C-5 127.35

--- --- --- C-7 126.90

--- --- --- C-3 126.06

--- --- --- C-4 47.12

--- --- --- 25.79

98


Table.2.2: UV, FT-IR & NMR structural data of solifenacin succinate impurity-B

UV Assignment data


206.34nm 1.6782 E2 band of aromatic ring

261.50nm 0.0897 β band of aromatic ring


Wave Number(cm-1) Assignment

3428 & 3326 NH stretching

3063 & 3043 =C-H stretching of aromatic ring

2966 & 2923 C-H-stretching of CH2

1492 & 1452 C=C stretching in aromatic ring

1366 C-H bendings in CH2

1292 C-N stretching

1125 & 945 In plane bending vibrations of C-H in aromatic ring

749, 737 & 706 Out of plane bending vibrations of C-H in aromatic

ring 13C & 1H Assignment data

Proton No. Multiplicity Chemical shifts

(ppm) Carbon No Chemical shift

H-2´,3´,4´,5´,6´ (5H) m 7.25-7.36 C-8a 144.81

H-6,8(2H) m 7.15-7.16 C-1´ 138.21

H-7(1H) m 7.02-7.07 C-4a 135.35

H-5(1H) d 6.746.77 C-3´5´,5 128.88-128.93

H-1(1H) s 5.11 C-2´,6´ 128.30

H-3e(1H) m 3.24-3.32 C-8 128.00

H-3a,4e(2H) m 3.02-3.15 C-4´ 127.26

H-4a(1H) m 2.80-2.89 C-7 126.14

C-6 125.53

C-1 62.02

C-3 42.20

C-4 29.71

99


Table.2.3: UV, FT-IR & NMR structural data of solifenacin succinate impurity-C

UV Assignment data


212.02nm 2.2016 K band of aromatic ring


Wave Number(cm-1) Assignment

3060 & 3025 C-H stretching in aromatic ring

2949 C-H stretching in CH2

1684 C=O stretching

1427 C=C stretching in aromatic ring

1348 & 1319 CH2 bending

1228 & 1202 C-N stretching

1121 & 1095 C-O stretching

1031,998 7 933 In plane bending vibrations of C-H in aromatic ring

887, 750 & 697 Out of plane bending vibrations of C-H in aromatic ring 13C & 1H Assignment Data

Proton No. Multiplicity Chemical

shifts(ppm)

Carbon No Chemical shift

H-10,11,12,13,14 (5H) m 7.18-7.20 C-15 153.79 H-5,8(2H) m 6.65 C-8a 141.86 H-6,7(2H) m 6.14-6.38 C-4a,9 134.23-134.55 H-1(1H) s 5.11 C11,13 128.46 H-2’e,3’(2H) m 3.83-3.91 C-10,14 127.94 H-3,2’a, 8’,6’e(6H) m 3.37-3.46 C-5,8 127.18 H-4,6’a(3H) m 2.76-2.97 C-6,7 126.84 H-4’,5’e(2H) m 2.15-2.24 C-12 125.92 H-5’a,7’(3H) m 1.94-2.02 C-2’ 70.08 C-3’ 68.86 C-6’ 61.75 C-8’ 60.84 C-1 57.39-58.23 C-3 45.69-46.67 C-4’ 38.15-38.73 C-4 24.17 C-5’ 22.46 C-7’ 22.38

100


Table.2.4: Summary of HPLC method development trials of solifenacin succinate

S.No. Mobile phase/Stationary phase Resolution/Critical area

1

Shimpack XRODS-II column and the mobile

phase-A contained 10 mM KH2PO4, pH of the

buffer was adjusted to 7.0 with TEA.

Acetonitrile was used as mobile phase B,

employing a binary-gradient program.

There was no separation between

impurity-A&B and solifenacin and

impurity-C (Fig.2.25).

2

The mobile phase contained buffer 10 mM

KH2PO4 with 0.1% v/v Triethylamine

(pH.3.0) and acetonitrile (60:40, v/v). Analysis

performed on a hypersil C8 (250 mm×4.6 mm

i.d., 5μm)


impurity-A & B (Fig.2.26).

3

The mobile phase contained 10 mM

ammonium formate, adjusted pH to 3.0 with

formic acid-acetonitrile-methanol in the ratio

of 52.5:37.5:10, (v/v/v).


impurity-A & B and negative peak

observed at impurity-A (Fig.2.27)

4

Mobile phase-A:0.01M KH2PO4, adjusted pH-

3.5 with H3PO4, mobile phase-B: acetonitrile

and water in the ratio of 90:10, (v/v), Gradient

Programme:time:%B:0.01/10, 15/60,

25/80,30/40, 40/40

Solifenacin succinate and N-Oxide

(impurity-C) eluted closely.

Corresponding chromatogram is

shown in Fig.2.28.

5 Mobile Phase-A:0.01M KH2PO4, adjusted

pH-3.5 with H3PO4, Mobile Phase-B: ACN

and Water in the ratio of 90:10, (v/v) Gradient

Programme :time: %B: 0.01/10, 10/50, 25/60,

40/80, 45/40, 50/40

Solifenacin succinate and N-Oxide

(impurity-C) eluted closely.

Corresponding chromatogram is

shown in Fig.2.29.

6 Mobile phase-A&B: Same as above and

slightly gradient program changed. Gradient

programme: time/%B: 0.01/20, 20/55, 35/80,

45/20, 50/20,


solifenacin and impurity-C. The

corresponding chromatogram was

shown in Fig.2.30.

101


Table.2.5: Forced degradation data (related compounds) of solifenacin succinate

Name of the sample

Stressed conditions and

period of exposure

% of imp-A

% of imp-B

% of imp-C

% of MSUI

% of TI

Mother sample (As such) ---- ND ND ND 0.04 0.04

Ambient sample Week days ND ND ND 0.04 0.04

Thermal sample Week days ND ND ND 0.04 0.04

Humidity sample

Week days ND ND ND 0.04 0.04

Photo light 1.2 million LUX Hrs

& 200Watt-hrs/Sq.mts

ND ND ND 0.04 0.04

Water hydrolysis

Heated at 80°C in H2O for 4hrs ND ND ND 0.04 0.04

Acid hydrolysis Heated at 80°C in 2M HCl for 4hrs ND 0.05 ND 0.05 0.10

Base hydrolysis Heated at 80°C in 2M NaOH for 4 hrs. ND 0.02 0.39 0.15 0.79

Oxidation Heated at 80°C in 10% H2O2 for 2hrs. 0.06 ND 8.15 0.42 10.07

Note: ND-Not Detected

102


Table.2.6: Mass balance (HPLC, Assay) data of solifenacin succinate

Type of sample

Stressed conditions and period of

exposure

Assay % w/w

%Total

impurities

Mass balance

Observations

Mother sample (as such) - 100.0 0.12 100.1 -

Ambient 25°C+ 2°C, 10 days 100.2 0.11 100.3 No significant Degradation

Thermal Heated at 105°C for 10 days 100.0 0.12 100.1 No significant

Degradation

Humidity 90%RH, 10 days 100.0 0.12 100.1 No significant Degradation

Photo light (Photolysis)

1.2million LUX Hrs &

200Watt.Hrs/Sq.mts 100.3 0.11 100.4 No significant

Degradation

Water hydrolysis

Heated at 80°C in H2O for 4 hrs 100.8 0.06 100.9 No significant

Degradation

Acid hydrolysis Heated at 80°C in 0.1N HCl for 4 hrs 100.7 0.10 100.8 No significant

Degradation

Base hydrolysis Heated at 80°C in 0.1N NaOH for 4 hrs

100.9 0.79 101.7 Unknown impurities

are observed.

Oxidation Heated at 80°C in 10% H2O2 for 2 hrs 90.7 10.07 100.8

Impurity-A, C and Unknown impurities are

observed.

Table.2.7: System suitability data of solifenacin succinate

System suitability parameters for related compounds Acceptance criteria Resolution criteria USP resolution

Not Less than 2.0 Between impurity-A & impurity-B 8.53

Between solifenacin & impurity-C 3.62

System suitability parameters for assay compounds

Retention time of solifenacin succinate 6.0minutes Asymmetry 1.32 Not more than 2.0

Theoretical plates 18664 Not less than 5000

103


Table.2.8: DL & QL S/N ratio data of solifenacin succinate

Component Concentration of DL solution (%)

S/N Ratio Concentration of QL solution (%) S/N Ratio

Impurity-A 0.0034 4.11 0.01 12.33 Impurity-B 0.0025 4.10 0.01 17.31 Impurity-C 0.0068 4.24 0.02 15.36

Solifenacin succinate 0.0064 3.72 0.02 11.10

Table.2.9: QL precision data of solifenacin succinate

Replicates Area of solifenacin Area of impurity-A

Area of impurity-B

Area of impurity-C

1 3943 1934 3329 3614 2 3837 1954 3334 3771 3 3765 1893 3362 3740 4 3928 1896 3338 3426 5 4035 1902 3271 3373 6 3982 1901 3326 3634

Average 3915 1913 3327 3593 % RSD 2.51 1.29 0.90 4.53

Table.2.10: Linearity data of solifenacin succinate impurity-A

Level Concentration in (%)

Area of replicate-1

Area of replicate-2

Area of replicate-3 Average %RSD

Level-1 0.010 1934 1954 1893 1927 1.61 Level-2 0.075 15475 15385 15290 15383 0.60 Level-3 0.120 24381 24421 24372 24391 0.11 Level-4 0.135 28010 28039 28161 28070 0.29 Level-5 0.150 31554 31550 31551 31552 0.01 Level-6 0.180 37173 37345 37440 37319 0.36 Level-7 0.225 46947 47146 47051 47048 0.21 Correlation coefficient 0.9998 Y-intercept (c) -325 %Y-intercept -1.03 Slope(m) 210020

104


Table.2.11: Residual summary data of solifenacin succinate impurity-A

Table.2.12: Linearity data of solifenacin succinate impurity-B

Level Conc.

(%)

Area of

replicate-1

Area of

replicate-2

Area of

replicate-3 Average %RSD

Level-1 0.010 3329 3334 3362 3342 0.53 Level-2 0.075 25547 25453 25232 25411 0.64 Level-3 0.120 41415 41298 41500 41404 0.24 Level-4 0.135 46891 46856 46702 46816 0.21 Level-5 0.150 52119 51858 52235 52071 0.37 Level-6 0.180 63415 62961 63264 63213 0.37 Level-7 0.225 79159 79341 78854 79118 0.31 Correlation coefficient 0.9999 Y Intercept (c) -711 % of Y Intercept -1.36 Slope(m) 353463

Residual summary Conc. (%)

(with respect to test conc)

Mean response achieved

Response calculated thru trend line

equation

Residual(Response practical-response theoretical)

Residual square

0.010 1927 1775 152 23104

0.075 15383 15427 -44 1936

0.120 24391 24877 -486 236196

0.135 28070 28028 42 1764

0.150 31552 31178 374 139876

0.180 37319 37479 -160 25600

0.225 47048 46930 118 13924

Residual sum of squares 442400

Trend line equation y=210020x-325

Scale of graph -3500 to + 3500

10% of 100% concentration response 3155

105


Table.2.13: Residual summary data of solifenacin succinate impurity-B

Residual summary Conc. (%) (with respect to test conc)


Response calculated thru

trend line equation

Residual (Response

practical-response theoretical)

Residual square

0.010 3342 2824 518 268324

0.075 25411 25799 -388 150544

0.120 41404 41705 -301 90601

0.135 46816 47007 -191 36481

0.150 52071 52308 -237 56169

0.180 63213 62912 301 90601

0.225 79118 78818 300 90000



Scale of graph -5207 to+5207


Table.2.14: Linearity data of solifenacin succinate impurity-C

Level Conc. (%) Area of replicate-1

Area of replicate-2


Level-1 0.020 3614 3771 3740 3708 2.24 Level-2 0.075 14182 14303 14322 14269 0.53 Level-3 0.120 23674 23872 23440 23662 0.91 Level-4 0.135 27363 27205 27267 27278 0.29 Level-5 0.150 30294 30600 30190 30361 0.70 Level-6 0.180 35071 35093 34865 35010 0.36 Level-7 0.225 45186 45422 44935 45181 0.54

Correlation coefficient 0.99931 Y Intercept (c) -461 % of Y Intercept -1.52 Slope(m) 201872

106


Table.2.15: Residual summary data of solifenacin succinate impurity-C

Residual summary Conc. (%)

(with respect to test conc)



trend line equation

Residual (Response practical-response

theoretical) Residual square

0.020 3708 3576 132 17424

0.075 14269 14679 -410 168100

0.120 23662 23764 -102 10404

0.135 27278 26792 486 236196

0.150 30361 29820 541 292681

0.180 35010 35876 -866 749956

0.225 45181 44960 221 48841



Scale of graph -3000 to +3000


Table.2.16: Linearity data of solifenacin succinate

Level Concentration in (%)

Area of replicate-1

Area of replicate-2


Level-1 0.020 3943 3837 3765 3848 2.33 Level-2 0.050 9954 9845 9847 9882 0.63 Level-3 0.080 16463 16450 16279 16397 0.63 Level-4 0.090 18121 18502 18282 18302 1.05 Level-5 0.100 20749 20632 20671 20684 0.29 Level-6 0.120 24154 24059 24049 24087 0.24 Level-7 0.150 29980 29989 29701 29890 0.55 Correlation coefficient 0.99936 Y Intercept (c) 20 % of Y Intercept 0.10 Slope(m) 201556

107


Table.2.17: Residual summary data of solifenacin succinate

Residual summary

Conc. (%) (with respect to test conc)



trend line equation

Residual (Response practical-response

theoretical)

Residual square

0.020 3848 4051 -203 41209

0.050 9882 10098 -216 46656

0.080 16397 16144 253 64009

0.090 18302 18160 142 20164

0.100 20684 20176 508 258064

0.120 24087 24207 -120 14400

0.150 29890 30253 -363 131769


Trend line equation y=201556x+20

Scale of graph -2068 to +2068


Table.2.18: Range (lower level) summary data of solifenacin succinate

Replicates Area of solifenacin

Area of impurity-A

Area of impurity-B

Area of impurity-C

1 3943 1934 3329 3614

2 3837 1954 3334 3771

3 3765 1893 3362 3740

4 3928 1896 3338 3426

5 4035 1902 3271 3373

6 3982 1901 3326 3634

Average 3915 1913 3327 3593

STDEV 2.51 24.80 30.12 162.3

% RSD 2.51 1.29 0.90 4.53

108


Table.2.19: Range (upper level) summary data of solifenacin succinate


Area of impurity-A

Area of impurity-B

Area of impurity-C

1 29980 46947 79159 45186

2 29989 47146 79341 45422

3 29701 47051 78854 44935

4 29711 46884 78946 45277

5 29711 47063 78899 45176

6 29817 47145 78684 45026

Average 29818 47039 78981 45170

% RSD 0.46 0.22 0.30 0.39

Table.2.20: Accuracy of solifenacin succinate impurity-A, impurity-B and impurity-C

% of Recovery of impurity-A

Level Preparation-01 Preparation-02 Preparation-03 Mean 95% confidence interval of mean

Level-1 98.94 100.00 97.87 98.94 97.73-100.14

Level-2 104.13 102.56 103.70 103.46 102.55-104.38

Level-3 101.42 101.28 99.50 100.73 99.52-101.94

Level-4 102.84 103.41 101.90 102.72 101.85-103.58

% of Recovery of impurity-B

Level-1 100.00 100.00 101.00 100.33 99.68-100.99

Level-2 98.67 97.61 98.80 98.36 97.62-99.10

Level-3 104.92 102.66 100.80 102.79 100.46-105.13

Level-4 100.13 100.67 99.91 100.24 99.79-100.68

% of Recovery of impurity-C

Level-1 104.08 103.06 102.55 103.23 102.35-104.11

Level-2 104.63 104.22 103.81 104.22 103.76-104.68

Level-3 102.59 105.31 103.54 107.24 102.25-105.38

Level-4 103.94 102.80 101.06 102.60 100.96-104.24

%Recovery range of solifenacin in Assay Method

50% level 100% level 150% level

99.6-100.4 100.0-100.1 100.0-100.2

109


Table.2.21: System precision data of solifenacin, imp-A, imp-B and imp-C


Area of impurity-A

Area of impurity-B

Area of impurity-C

1 19920 30158 52782 30147

2 19968 30129 52725 30074

3 20082 30067 52745 29909

4 19952 30151 52793 30418

5 19812 30116 52604 29745

6 19902 30053 52778 30069

Average 19939 30112 52738 30060

STDEV 88.68 43.47 70.33 227.17

% RSD 0.44 0.14 0.13 0.76

Table.2.22: Method precision data of solifenacin succinate

Preparations % of impurity-A

% of impurity-B

% of impurity-C

% of MSUI

% of TI

Precision prep’n-01 0.011 0.010 0.019 0.047 0.101

Precision prep’n-02 0.010 0.010 0.020 0.048 0.102

Precision prep’n-03 0.010 0.010 0.021 0.048 0.103

Precision prep’n-04 0.010 0.010 0.024 0.048 0.107

Precision prep’n-05 0.011 0.010 0.020 0.048 0.104

Precision prep’n-06 0.010 0.010 0.021 0.048 0.103

Average 0.010 0.010 0.021 0.048 0.103

STDEV 0.0005 0.0000 0.0017 0.0004 0.0021

% RSD 5.00 0.00 8.10 0.83 2.04

Confidence interval

of mean from 0.010 0.010 0.020 0.048 0.103

Confidence interval

of mean to 0.011 0.010 0.022 0.048 0.105

110


Table.2.23: Intermediate precision data of solifenacin succinate


% of impurity-B

% of impurity-C

% of MSUI

% of TI

Inter precision prep’n-01 0.012 0.010 0.021 0.047 0.104 Inter precision prep’n-02 0.011 0.011 0.023 0.050 0.110 Inter precision prep’n-03 0.011 0.010 0.022 0.046 0.102 Inter precision prep’n-04 0.011 0.010 0.023 0.046 0.104 Inter precision prep’n-05 0.011 0.011 0.022 0.049 0.108 Inter precision prep’n-06 0.011 0.011 0.018 0.046 0.100 Average 0.011 0.011 0.022 0.047 0.105 STDEV 0.0004 0.0005 0.0019 0.0018 0.0037 % RSD 3.64 4.55 8.64 3.83 3.52 Confidence interval of

mean from 0.011 0.010 0.021 0.047 0.103

Confidence interval of

mean to 0.011 0.011 0.023 0.049 0.108

Table.2.24: Robustness data of solifenacin succinate at flow rate 0.8 ml. min-1


% of impurity-B

% of impurity-C

% of MSUI % of TI

Robustness prep’n-01 0.011 0.010 0.023 0.045 0.103 Robustness prep’n-02 0.012 0.010 0.021 0.045 0.101 Robustness prep’n-03 0.010 0.010 0.019 0.045 0.099 Average 0.011 0.010 0.021 0.045 0.101 STDEV 0.0006 0.0000 0.0020 0.0000 0.0020 % RSD 5.45 0.00 9.52 0.00 1.98

111


Table.2.25: Robustness data of solifenacin succinate at flow rate 1.2 ml. min-1


% of impurity-B

% of impurity-C

% of MSUI % of TI

Robustness prep’n-01 0.010 0.010 0.024 0.043 0.100



Average 0.010 0.010 0.022 0.043 0.098

STDEV 0.0006 0.0000 0.0021 0.0006 0.0021

% RSD 6.00 0.00 9.55 1.40 2.14

Table.2.26: Robustness data of solifenacin succinate at column temperature at 33°C


% of impurity-B

% of impurity-C

% of MSUI

% of TI




Average 0.010 0.010 0.021 0.045 0.101

STDEV 0.0006 0.0000 0.0012 0.0006 0.0015

% RSD 6.00 0.00 5.71 1.33 1.49

Table.2.27: Robustness data of solifenacin succinate at column temperature at 37°C


% of impurity-B

% of impurity-C

% of MSUI

% of TI




Average 0.010 0.010 0.021 0.048 0.104

STDEV 0.0000 0.0000 0.0006 0.0006 0.0006

% RSD 0.00 0.00 2.86 1.25 0.58

112


Table.2.28: Robustness data of solifenacin succinate at buffer pH 3.3±0.05


% of impurity-B

% of impurity-C

% of MSUI

% of TI




Average 0.010 0.010 0.022 0.041 0.096

STDEV 0.0000 0.0000 0.0006 0.0006 0.0006

%RSD 0.00 0.00 2.73 1.46 0.63

Table.2.29: Robustness data of solifenacin succinate at buffer pH 3.7±0.05


% of impurity-B

% of impurity-C

% of MSUI

% of TI


Table.2.30: Robustness data of SFS at mobile phase-B in the ratio of 95:10, v/v


% of impurity-B

% of impurity-C

% of MSUI

% of TI


113


Table.2.31: Robustness data of SFS at mobile phase-B in the ratio of 85:10, v/v


% of impurity-B

% of impurity-C

% of MSUI

% of TI


Table.2.32: Robustness data of solifenacin succinate with column lot variation


% of impurity-B

% of impurity-C

% of MSUI

% of TI


Table.2.33: Mobile phase stability data of solifenacin succinate


% of impurity-B

% of impurity-C

% of MSUI

% of TI


114


Table.2.34: Test solution stability data of solifenacin succinate

Name of the

impurity

Initial average

content %

After 12 hrs average

content %

After 24 hrs average content %

After 36 hrs average content %

After 48 hrs average

content % Impurity-A 0.010 0.010 0.011 0.011 0.011 Impurity-B 0.010 0.010 0.010 0.010 0.010 Impurity-C 0.020 0.020 0.021 0.021 0.021 MSUI 0.048 0.048 0.049 0.049 0.050 TI 0.103 0.103 0.106 0.105 0.106

Table.2.35: RT, RRT and relative response factor of solifenacin succinate impurities

Component Retention time (RT)

Relative retention time (RRT)

Relative response factor (RRF)

Impurity-A 4.5 0.27 0.26 Impurity-B 6.2 0.38 0.39 Impurity-C 18.5 1.12 1.14

Table.2.36: Solifenacin succinate API sample analysis data (values in %)

S.No. Batch No./Lot Impurity-A Impurity-B Impurity-C

1 01 Not Detected Not Detected 0.03

2 02 Not Detected Not Detected Not Detected

3 Crude 0.32 0.48 0.30

4 Tablet-VESIcare Not Detected Not Detected 0.21

Table.2.37: Solifenacin succinate assay results of brand and generic drugs data

S.No. Name of the sample Label claimed % Assay 1 Vesicare 5 mg 4.94 mg 2 Vesicare 10 mg 10.3 mg 3 Solifenacin succinate(Generic) 5 mg 5.11 mg

4 Solifenacin succinate(Generic) 5 mg 5.06 mg



115


References

[1] A. Ohtake, C. Saitoh, H. Yuyama, M. Ukai, H. Okutsu, Y. Noguchi, T. Hatanaka, M.

Suzuki, S. Sato, M. Sasamata, K. Miyata.; Biological and pharmaceutical bulletin,

2007, 30(1), 54-58.

[2] A.J. Wein.; Expert opinion on investigational drugs, 2001, 10(1), 65-83.

[3] G. Haeusler, H. Leitich, M. Van Trotsenburg, A. Kaider, CB. Tempfer.; Obstetrics and

gynecology, 2002, 100(5 Pt 1), 1003-16.

[4] R. Basra, C. Kelleher.; Journal of therapeutics and clinical risk management, 2008,

4(1), 117-128.

[5] Ken Ikeda, Seiji Kobayashi, Mami Suzuki, Keiji Miyata, Makoto Takeuchi,

Toshimitsu Yamada, Kazuo Honda.; Archives of pharmacology, 2002, 366(2), 97-103.

[6] M.C. Michel, M. Oelke, N. Zinner.; Drug discovery today, therapeutic strategies,

2005, 2(1), 1-6.

[7] S. Budavari.; The Merck Index, 14th ed., Merck & Co. Inc., White house station, NJ.

2006, 1484.

[8] N. Hiren Mistri, G. Arvind G. Jangid, Ashutosh Pudage, M. Dhiraj Rathod, S. Pranav

Shrivastav.; Journal of chromatography B, 876, 236-244.

[9] Jan Macek, Pavel Ptacek, Josef Klima.; Journal of chromatography B, 2010, 878(31),

3327-3330.

[10] T. Yanagihara, T. Aoki, Y. Soeishi, T. Iwatsubo, H. Kamimura.; Journal of

chromatography B, 2007, 859(2), 241-245.

[11] G. Divya Teja, Ch. Deva Dasu, P.Srinivasa Babu, P. Ravisankar.; Journal of chemical

and pharmaceutical sciences, 2013, 6(3), 195-198.

[12] Lokesh Singh, Sanju Nanda.; Pharmaceutical methods, 2011, 2(1), 21-24.

[13] R. Seetharaman, KS. Lakshmi.; International journal of research in pharmaceutical and

biomedical sciences, 2011, 2(3), 1052-1057.

[14] S.B. Wankhede, Kratika Somani, S.S. Chitlange.; International journal of chem tech

research, 2011, 3(4), 2003-2010.

[15] D. Desai, G. Mehta, D. Ruikar, R. Jain, S. Rajput.; Asian journal of pharmaceutical

and biological research, 2011, 1(3), 310-316.

116


[16] B.S. Landge, A.S. Jadhav, B.S. Dahale, C.N. Niphade, Ch. Lakshmi Narayana, B.V.

Gaikwad, T.V. Mathad.; Chromatography research international, 2011, 1-7.

[17] S. Radha Krishna, B.M. Rao, N. Someswara Rao.; Journal of chromatographic

science, 2010, 48, 807-810.

[18] Nilesh Desai, Syed Sajjad Hussen, S.G. Vasanthraju, A. Karthik, N. Udupa.;

International journal of pharmacy and pharmaceutical sciences, 2011, 3(1), 70-74.

[19] D. Desai, G. Patel, N. Shukla, S. Rajput.; Acta chromatographica, 2012, 24(3), 399-

418.

[20] S.K. Raul1, B.V.V. Ravi Kumar, A.K. Patnaik.; International journal of bioassays,

2012, 01(12), 210-213.

[21] K. Nageswara Rao, S. Ganapaty, A. Lakshmana Rao.; Pharmanest, 2012, 3(5), 366-

374.

[22] ICH Guideline Q7A.; good manufacturing practice guide for active pharmaceutical

ingredients, 2005.

[23] ICH Q2 (R1) validation of analytical procedures:, text and methodology, 2005.

[24] ICH draft guidelines on validation of analytical procedures: definitions and

terminology, federal register, IFPMA, Switzerland, 1995, 11260.

[25] ICH, stability testing of new drugs substances and products (Q1AR), International

conference on harmonization, IFPMA, Geneva, 2000.

[26] ICH Q1A (R2), Stability testing of new drug substances and products, International

conference on harmonization, 2003.

117

Documents

CHAPTER-II - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/71060/10/10_chapter 2.pdf · Chapter-II-HPLC Determination of Solifenacin Succinate impurities by Stability Indicating