Fig.2.1: Chemical structure of solifenacin succinate

Transcription

1 CHAPTER-II Development and Validation of a Specific Stability Indicating High Performance Liquid Chromatographic Methods for Related Compounds and Assay of Solifenacin Succinate

2 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 C 23 H 26 N 2 O 2.C 4 H 6 O 4 and molecular weight is grams/mole. It is a white to paleyellowish-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

3 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

4 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 Instrumentation details UV-Visible spectrophotometer: Perkin Elmer UV-Visible spectrophotometer (Model: Lambda 35) was used for the UV absorption in the range of 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 1 H and 13 C 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

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

6 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

7 Carbon ( 13 C) & proton ( 1 H) magnetic resonance spectrum: The proton magnetic resonance spectrum and carbon-13 nuclear magnetic resonance spectrum of impurity-a in CDCl 3 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 1 H and 13 C spectrums were given in Fig.2.5 & 2.6 respectively. Fig.2.5: 1 H NMR spectrum of solifenacin succinate impurity-a Fig.2.6: 13 C NMR spectrum of solifenacin succinate impurity-a 58

8 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

9 Fig.2.9: Probable structure of the solifenacin succinate impurity-a 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 uvabsorbances 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 Fig.2.10: UV-absorption spectrum of solifenacin succinate impurity-b 60

10 Fourier-transform infrared spectrum: 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 Carbon ( 13 C) & proton ( 1 H) magnetic resonance spectrum: The proton magnetic resonance spectrum and carbon-13 nuclear magnetic resonance spectrum of impurity-b in CDCl 3 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 1 H and 13 C spectrums are given in Fig.2.12 & 2.13 respectively. 61

11 Fig.2.12: 1 H NMR spectrum of solifenacin succinate impurity-b Fig.2.13: 13 C 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 The obtained mass fragmentation pattern was given in Fig The most probable structure of the impurity-b was presented in Fig

12 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

13 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 Fig.2.17: UV-absorption spectrum of solifenacin succinate impurity-c Fourier-transform infrared spectrum: 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. Carbon ( 13 C) & proton ( 1 H) magnetic resonance spectrum: 1 H and 1 3 C NMR spectra (Fig.2.19 & 2.20) of impurity-c in CDCl 3 at 27 C was obtained on a 300 MHz Bruker Advance NMR spectrometer. Structural assignments were represented in Table

14 Fig.2.18: FT-IR spectrum of solifenacin succinate impurity-c Fig.2.19: 1 H NMR spectrum of solifenacin succinate impurity-c 65

15 Fig.2.20: 13 C 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 Fig.2.21: Mass spectrum of solifenacin succinate impurity-c 66

16 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 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

17 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

18 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 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

19 Fig.2.25: HPLC chromatogram of SFS spiked with its three impurities Fig.2.26: HPLC chromatogram of SFS spiked with its three impurities Fig.2.27: HPLC chromatogram of SFS spiked with its three impurities 70

20 Fig.2.28: HPLC development trials of SFS spiked with its three impurities Fig.2.29: HPLC development trials of SFS spiked with its three impurities Fig.2.30: HPLC development trials of SFS spiked with its three impurities 71

21 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 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 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

22 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

23 2.4 Specificity and Forced Degradation 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 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

24 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 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

25 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

26 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% H 2 O 2 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

27 Fig.2.38: Typical HPLC chromatogram of oxidative (10%H 2 O 2 ) solution Fig.2.39: Typical HPLC chromatogram of water hydrolysis solution Fig.2.40: Typical HPLC chromatogram of thermal stressed solution 78

28 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 % 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 Analytical Method Validation 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

29 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 mixer for five minutes. 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 mixer for five minutes. 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

30 Fig.2.42: Typical HPLC chromatogram of system suitability solution 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

31 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 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

32 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

33 Fig.2.48: HPLC chromatogram of solifenacin and its impurities at 100% Level Fig.2.49: HPLC chromatogram of solifenacin and its impurities at 120% Level Fig.2.50: HPLC chromatogram of solifenacin and its impurities at 150% Level 84

34 Peak Area Linerity graph of solifenacin succinate impurity-a Average area Vs Concentration y=210020x-325 R 2 = Concentration(%) Fig.2.51: Calibration curve of solifenacin succinate impurity-a Peak Area Linerity graph of solifenacin succinate impurity-b Average area Vs Concentration y=353463x R² = Concentration(%) Fig.2.52: Calibration curve of solifenacin succinate impurity-b Peak Area Linerity graph of solifenacin succinate impurity-c Average area Vs Concentration y =201872x R² = Concentration(%) Fig.2.53: Calibration curve of solifenacin succinate impurity-c 85

35 Peak Area Linerity graph of solifenacin succinate impurity-c Average area Vs Concentration y = x+20 R² = Concentration(%) Fig.2.54: Calibration curve of solifenacin succinate Residuals Residual plot of solifenacin succinate impurity-a Order of residuals Fig.2.55: Residual plot of solifenacin succinate impurity-a Residuals Residual plot of solifenacin succinateimpurity-b Order of residuals Fig.2.56: Residual plot of solifenacin succinate impurity-b 86

36 3000 Residual plot solifenacin succinate impurity-c 2000 Residuals Order of residuals Fig.2.57: Residual plot of solifenacin succinate impurity-c 2000 Residual plot for Solifenacin succinate Residuals Order of residuals Fig.2.58: Residual plot of solifenacin succinate 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. 87

37 2.5.5 Accuracy Accuracy of the impurities was carried out in triplicate at % (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 %. The mean recovery results were shown in Table The accuracy chromatograms were displayed in Fig.2.59 to 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

38 Fig.2.61: HPLC chromatogram of SFS test spiked with its three impurities at 100% Fig.2.62: HPLC chromatogram of SFS test spiked with its three impurities at 150% 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

39 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 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

40 Fig.2.65: HPLC robustness chromatogram of SFS spiked with its three impurities at 100% concentration level with column temperature: 33 C Fig.2.66: HPLC robustness chromatogram of SFS spiked with its three impurities at 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±

41 Fig.2.68: HPLC robustness chromatogram of SFS spiked with its three impurities at 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

42 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 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 The chromatographic data, including retention times, relative retention times and relative response factors for the three impurities were tabulated in Table 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 The typical HPLC chromatogram of vesicare tablet and assay chromatogram was shown in Fig.2.74 & 2.75 respectively. 93

43 Fig.2.71: Typical HPLC chromatogram of SFS B.No-446 Fig.2.72: Typical HPLC chromatogram of SFS B.No-SFS Fig.2.73: Typical HPLC chromatogram of SFS B.No-SFS004 (crude) 94

44 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

45 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 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

46 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, ) 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, 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 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 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