Aripiprazole is a quinolinone derivative with the chemical name 7-[4- [4-(2,3-dichlorophenyl)-1-piperazinyl]butoxy]-3,4-dihydro-2(1H)

147 5.0 INTRODUCTION Aripiprazole is a quinolinone derivative with the chemical name 7-[4- [4-(2,3-dichlorophenyl)-1-piperazinyl]butoxy]-3,4-dihydro-2(1H) quinolinone. Aripiprazole is a psychotropic agent belonging to the chemical class of benzisoxazole derivatives and is indicated for the treatment of schizophrenia. Aripiprazole is a selective monoaminergic antagonist with high affinity for the serotonin Type 2 (5HT2), dopamine Type-2(D2),1 and 2 adrenergic and H1 histaminergic receptors. Aripiprazole acts as an antagonist at other receptors, but with lower potency. Antagonism at receptors other than dopamine and 5HT2 with similar receptor affinities may explain some of the other therapeutic and side effects of Aripiprazole. Aripiprazole's antagonism of histamine H1 receptors may explain the somnolence and antagonism of adrenergic A1 receptors may explain the orthostatic hypotension. 5.1 NEED FOR NEW ANALYTICAL METHOD : The different analytical techniques have been reported so far for the determination of this drug in biological samples as well as in pharmaceutical formulations include LC MS/MS method for the determination of Aripiprazole and its metabolites have been previously published 179-185. Also, the determination of aripiprazole in plasma by RP-HPLC 186-190 and determination of aripiprazole in bulk drug and solid dosage forms by RP-HPLC method 191-193 have also been published. In one of the articles, study on a high-performance liquid chromatographic method for the separation of substances related to aripiprazole 194-196 was described where in only two Impurities; Imp-B

148 & Imp-D were disclosed. In the present study, Imp-A, Imp-B, Imp-C, Imp-D and Imp-E were considered for the validation Recently, we have developed a process for synthesis of Aripiprazole in our laboratory. During the development of an analytical procedure, the HPLC method was developed for the determination of In-House synthesized Aripiprazole and the Impurities arising during its manufacturing. In the present study, we described a reverse phase high performance column liquid chromatography method for the separation and quantification of process related Impurities of Aripiprazole. The accuracy, precision, limit of detection (LOD), limit of quantification (LOQ) and robustness of the method was determined in accordance with ICH guidelines and found to be suitable for quality assurance of Aripiprazole. 5.2 EXPERIMENTAL 5.2.1 Chemicals Reference standard of Aripiprazole and five Impurities namely Imp-A, Imp-B, Imp-C, Imp- D and Imp-E were synthesized and characterized in M/s Inogent Laboratories Private Limited, Hyderabad, India. The commercial samples of Aripiprazole being manufactured by M/s Inogent Laboratories Private Limited, Hyderabad, India. All reagents used were of analytical reagent grade unless stated otherwise. Milli Q water, HPLC grade Acetonitrile and HPLC grade Trifluoroacetic acid (TFA) were purchased from Merck (Darmstadt, Germany)

149 5.2.2 Equipment The HPLC system was equipped with quaternary gradient pumps with auto sampler and auto injector (Model: Alliance 2695, Make :Waters, Milford, MA,USA ) connected with photo diode array detector (PDA Model : 2996, Make :Waters, Milford, MA,USA) controlled with Empower software ( Make: Waters, Milford, MA, USA). 5.2.3 Preparation of standard and sample solutions The sample and standartd concentration for assay by HPLC were 100 µg/ml and 50 µg/ml, 75 µg/ml, 100 µg/ml, 125 µg/ml, 150 µg/ml and 200 µg/ml were prepared to coduct the linearity studies. Stock solutions of Aripiprazole (500 µg/ml) and all Impurities (500 µg/ml) were prepared in mobile phase for related substances by HPLC. The spiked solutions were prepared by taking 0.05 µg/ml, 0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml, 1.0 µg/ml, 1.25 µg/ml and 1.5 µg/mlof all Impurities in mobile phase from stock solutions for the evaluation of limit of detection, limit of quantification and linearity in accordance with ICH guidelines. Aripiprazole sample solution from stock is spiked with 0.05 µg/ml, 0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml, 1.0 µg/ml, 1.25 µg/ml and 1.5 µg/ml of all Impurities to establish accuracy, precision and robustness. The quantities of Impurities were calculated from their peak areas.

150 5.2.4 Chromatographic conditions The mobile phase was prepared by mixing 0.2% TFA in water and Acetonitrile (55:45; v/v), filtered through 0.45 µm PTFE filter and degassed by using ultra-sonicator for 15 min prior to use. The system was equilibrated for 30 min and analysis was carried out under isocratic conditions using a flow rate of 1.0 ml min -1 at 40 o C temperature. Chromatograms were recorded at 217 nm. Fig: 5.1. Structure of Aripiprazole O N H O N N Cl Cl Chemical Name : 7-[4-[4-(2,3-dichlorophenyl)piperazinyl] butoxy]- 3,4-dihydro- 2(1H)- quinolinone Molecular weight(amu) Molecular formula CAS Registry Number Therapeutic Catogory : : : : 448.39 C23H27Cl2N3O2 [129722-12-9] Antipsychotic Table 5.1 Impurities of Aripiprazole Impurity Chemical structure & Name Origin of Impurity

151 Imp-A Imp-B Unreacted1,4 Dibromobutane, raw material for the preparation of SM-2 leads to formation of this Impurity under dry conditions, possible bi product. Also, the KSM for Aripiprazole in different synthetic route. Starting material-1 (SM-1)for the synthesis of Aripiprazole. N NH Cl Cl 1-(2,3-dichlorophenyl)piperazine Imp-C O N H O O H N O Process related Impurity, carried from SM-2. Imp-D 7,7'-(butane-1,4-diylbis(oxy)) bis(3,4-dihydroquinolin-2(1h)-one) O N H O Br Starting material-2 (SM-2) for the synthesis of Aripiprazole. Imp-E 7-(4-bromobutoxy)-3,4 dihydroquinolin-2(1h)-one O N H O N N + Br- Cl Cl 8-(2,3-dichlorophenyl)-8-Aza-5- Azoniaspiro(4.5) Decane Bromide 7-(4-iodobutoxy)-3,4- dihydroquinolin-2(1h)-one I Process related Impurity, carried out from SM-2. 5.3 VALIDATION OF ANALYTICAL METHOD

152 5.3.2 Specificity The specificity of the HPLC method for Aripiprazole was carried out in the presence of its Impurities namely Imp-A, Imp-B, Imp-C, Imp-D and Imp-E. Stability indicating property and specificity of the proposed method was conduced with the aid of forced degradation studies for Aripiprazole bulk drug Intentional degradation was attempted to stress condition of UV light (254 nm), heat (105 C), acid (1.0 N HCl, 60 C ), base (0.5N NaOH ) and oxidation (3.0% H2O2 60 C ) to evaluate the ability of the proposed LC method to separate Aripiprazole from its degradation products. The study period was 24 Hrs for heat and light studies, for the acid and base it was 48 Hrs, for oxidation it was 2 Hrs. Peak purity test was carried out for the Aripiprazole peak by using Diade Array Detector (DAD or PDA) in stress samples. Assay studies were carried out for stress samples against qualified Aripiprazole reference standard. Assay was also calculated for Aripiprazole sample by spiking all five Impurities at the specification level (i.e. 0.15 %). 5.3.3 Precision The precision for assay method was evaluated for Aripiprazole test sample against a qualified primary reference standard. Six independent assays were conducted and calculated the % RSD for replicate assay determinations. Six individual preparations of Aripiprazole (0.5 mg/ml) spiked with 0.15% of Imp-A, Imp-B, Imp-C, Imp-D and Imp-E were

153 considered for the evaluation of precision of the related substance method and %RSD of peak responses for each Imp-A, Imp-B, Imp-C, Imp-D, and Imp-E were calculated for six replicate injections. The intermediate precision for the proposed LC evaluated was also evaluated by employing different equipment and different analyst in the same laboratory. 5.3.4 Limit of detection (LOD) and Limit of quantification (LOQ) The limit of detection (LOD) and Limit of quantification (LOQ) were determined by measuring the magnitude of analytical background. The LOD and LOQ were determined from slopes of linear regression curves. The LOD and LOQ for Imp-A, Imp-B, Imp-C, Imp-D and Imp-E were determined by injecting a series of dilute solutions with known concentrations. 5.3.5 Linearity Test solutions to evaluate linearity for the assay method were prepared from Aripiprazole stock solutions at five concentration levels from 50 to 150 % of assay analyte concentration i.e, 25, 50, 75, 100, 125, 150 and 200 μg/ml respectively. The peak area versus concentration data was treated by least-squares linear regression analysis. Test solutions to evaluate linearity for the related substance method were prepared by diluting stock solutions to the required concentrations. The solutions were prepared at six concentration

154 levels from LOQ to 200 % (0.5 μg/ml) of the specification level ( 0.01, 0.05, 0.1, 0.15, 0.2, 0.25 and 0.3 %). Both assay and related substances methods were carried out for 3 consecutive days in the same concentration range. The % RSD value for the slope and Y-intercept of the calibration curve was calculated. 5.3.6 Accuracy The accuracy of the assay method was evaluated in triplicate at seven concentration levels, i.e. 25, 50, 75, 100, 125, 150 and 200 µg/ml. The percentage of recoveries was calculated from the slope and Y-intercept of the calibration curve obtained from linearity studies. The accuracy study of Impurities was carried out in triplicate at, 0.1, 0.15, 0.2, 0.25 and 0.3% of the Aripiprazole analyte concentration (500 µg/ml). The percentages of recoveries for Impurities were calculated from the slope and Y-intercept of the calibration curve obtained from linearity studies. 5.3.7 Robustness The robustness of the developed LC method was evaluated by intentinally altered chromatographic conditions and the resolution between Aripiprazole and its Impurities namely Imp-A, Imp-B, Imp-C, Imp-D and Imp-E was recorded.

155 The conditions which were altered deliberately were Flow rate of the mobile phase,temperature of the column oven and % of the Organic strength. For conducting flow rate of the mobile phase was 1.0 ml/min. The effect of flow rate on the resolution was studied at the flow rates of 0.8 and 1.2 ml/min respectively, instead of 1.0 ml/min. The effect of the column temperature on resolution was studied at 35 C and 45 C instead of 40 C. The effect of the percent organic strength on resolution was studied by varying acetonitrile by 5 to +5%, while other mobile phase components were held constant. 5.3.8 Solution stability and mobile phase stability The solution stability of Aripiprazole in the assay method was carried out by leaving both the test solutions of sample and reference standard in tightly capped volumetric flasks at room temperature for 48 Hrs. The same sample solutions were assayed for 6 Hrs interval up to the study period of 48 Hrs. The mobile phase stability was also carried out by assaying the freshly prepared sample solutions against freshly prepared reference standard solution for 6 Hrs interval up to 48 Hrs. Mobile phase prepared was kept constant during the study period. The % RSD for the assay of Aripiprazole was calculated during mobile phase and solution stability experiments. The solution stability of Aripiprazole and its Impurities in the related substance method was carried out by leaving spiked sample solutions in tightly capped volumetric flasks at room temperature for 48 Hrs. Content of Imp-A, Imp-B, Imp-C, Imp - D and Imp-E were

156 estimated for every 6 Hrs interval up to 48 Hrs. The mobile phase stability was also carried by analyzing the freshly prepared sample solutions for every 6 Hrs interval up to the study period of 48 Hrs.. Content of Imp-A, Imp-B, Imp-C, Imp - D and Imp-E were evaluated. 5.4 RESULTS AND DISCUSSION 5.4.1 Optimization of chromatographic conditions Based on the spectra of all known Impurities, the wavelength for monitoring the Impurities were chosen as 217 nm. The Ultra Violet (UV) absorption spectra of Aripiprazole and its Impurities recorded in HPLC system using photo diode array detection. The UV absorption maxima of Aripiprazole and Impurities are about 217 nm UV absorptions. Therefore, wavelength 217 nm is selected for the separation and quantification of related Impurities in Aripiprazole. The purpose of this study is to separate Aripiprazole from Imp-A, Imp-B, Imp-C, Imp-D and Imp-E and the degradation products formed by stress degradation studies. The Impurities were found merging with Aripiprazole and also co-eluted each other by employing various stationary phases such as C18, C8 and cyano as well as different mobile phases.

157 Fig: 5.2 UV absorption spectra of Aripiprazole and Impurities 5.4.2 Selection of stationary phase and mobile phase: Experiment - 1 Column : Inertsil C18 250 X 4.6, 5 microns ( GL Sciences)

158 Mobile Phase Flow rate Wavelength : 0.2 % TFA in water - acetonitrile 55:45 (v/v). : 1.0 ml/min : 217 nm Diluent : Mobile phase Elution : Isocratic Column Temp. : 40 o C Injection volume : 20 μl Inertsil C 18 Column Fig: 5.3 Typical chromatogram of Aripiprazole and its Impurities with Inertsil C18 column Conclusion: The resolution between Impurity B and Aripiprazole is very poor and hence the column is not suitable for separation.

159 Experiment - 2 Column Mobile Phase Flow rate Wavelength : Inertsil C8 250 X 4.6, 5 microns ( GL Sciences) : 0.2 % TFA in water - acetonitrile 55:45 (v/v). : 1.0 ml/min : 217 nm Diluent : Mobile phase Elution : Isocratic Column Temp. : 40 o C Injection volume : 20 μl Inertsil C 8 Column Fig: 5.4 Typical chromatogram of Aripiprazole and its impurities with Inertsil C8 column Conclusion: Impurity B is merged with Aripiprazole and hence selectivity of the column to be verified. Experiment - 3

160 Column : Inertsil Phenyl, 250 mmx4.6 mm, 5 microns Mobile Phase Flow rate Wavelength : 0.2 % TFA in water - acetonitrile 55:45 (v/v). : 1.0 ml/min : 217 nm Diluent : Mobile phase Elution : Isocratic Column Temp. : 40 o C Injection volume : 20 μl Fig: 5.5 Typical chromatogram of Aripiprazole and its Impurities with Inertsil Phenyl column Conclusion : The resolution between all Impurities from Aripiprazole is greater than 2.0 and peak shapes are found to be symmetrical. Experiment - 4 Column : Inertsil Cyano, 250X4.6 mm,5 microns

161 Mobile Phase Flow rate Wavelength : 0.2 % TFA in water - acetonitrile 55:45 (v/v). : 1.0 ml/min : 217 nm Diluent : Mobile phase Elution : Isocratic Column temp. : 40 o C Run time : 25 min Injection volume : 20 μl Inertsil CN Column Fig: 5.6 Typical chromatogram of Aripiprazole and its Impurities with Inertsil CN column Conclusion: The resolution between Impurity B & Impurity C was less than 1.5 and the order of elution was altered. Hence this method is not suitable

162 Based on the data, the proper chromatographic separation of Aripiprazole from the Impurities was achieved on an Inertsil Phenyl 250 mm 4.6 mm, 5 μm column. The mobile phase was prepared by mixing 0.2 % TFA in water - acetonitrile 55:45 (v/v). The successful separation was achieved by employing the flow rate of 1.0 ml/ min for the mobile phase, the column was maintained at 40 C temperature and the resulted peak shape of the Aripiprazole was found to be symmetrical. In optimized chromatographic conditions Aripiprazole, Imp-A, Imp-B, Imp-C, Imp-D and Imp E were separated with resolution greater than 2.0, typical retention time were about 8.1, 6.0, 7.1, 8.8, 9.8, and 11.5 min, respectively. The system suitability results are given in Tables 5.3 and 5.4 for assay and related substances methods respectively. The developed LC method was found to be specific for Aripiprazole and its five Impurities namely Imp-A, Imp-B, Imp-C, Imp-D, and Imp-E. 5.4.3 Results of forced degradation studies During forced degradation studies, it was observed that the degradation was not observed in Aripiprazole sample when subjected to stress conditions like photolytic and aqueous hydrolysis. Aripiprazole was degraded to Imp-B under base hydrolysis and was degraded to Imp-B and Imp-C under oxidation. Peak purity test results obtained by using PDA detector confirmed that the Aripiprazole peak is homogenous and pure in all the analyzed stress samples. The assay of Aripiprazole is unaffected in the presence of Imp-A, Imp-B, Imp-C, Imp-D, and Imp-E and its degradation products

163 confirms the stability indicating power of the method. The summary of forced degradation studies is given in Table-5.2. Table 5.2 Forced Degradation Data of Aripiprazole Stress condition Content, in % Imp-A Imp-B Imp-C Imp-D Imp-E Unknown RRT 0.88 0.74 1.09 1.2 1.41 -- % Degradation % Mass balance (% Assay + % Impurities) Normal ND 0.05 ND ND ND 0.01 -- 99.90 Thermal degradation ND 0.04 ND ND ND 0.08 0.14 99.76 Photo degradation ND 0.04 ND ND ND 0.06 0.10 99.83 Oxidation 1.24 0.15 ND ND ND 10.53 13.81 99.63 Acid hydrolysis Base hydrolysis 0.14 2.53 ND 0.24 ND 2.95 9.16 99.2 0.47 0.09 ND ND ND 0.66 1.78 99.69

164 Fig: 5.7 Typical chromatograms of Aripiprazole (a) sample (b) Acid degradation and (c) Peroxide degradation

165 5.5 Validation of the Analytical Method 5.5.1 System Suitability The system suitability test solution was injected into HPLC for Assay by HPLC and evaluated the Resolution and Relative standard deviation for replicate injections. The system suitability test solution was injected into HPLC for related substance and chromatographic parameters like resolution; number of theoretical plates and tailing factor were evaluated. Table 5.3 System Suitability Results of Assay by HPLC S.No Resolution between Aripiprazole & Imp-C Area of Aripiprazole Peak 1 2.20 7182557 2 2.22 7198863 3 2.19 7196583 4 2.24 7198465 5 2.21 7205497 6 2.22 7201469 Avgerage 2.21 7197239 Std.dev. 0.018 7825.85 % RSD 0.79 0.11

166 Table 5.4 System Suitability for Related Substances by HPLC Parameter Imp-A Imp-B Aripiprazole Imp-C Imp-D Imp-E Rt 6.075 7.212 8.184 8.904 9.862 11.521 RRT 0.88 0.74 1 1.09 1.2 1.41 Rs ---- 4.3 3.2 2.2 2.8 4.6 N 11093 10014 11255 11580 13644 15028 Asymmetry factor 1.3 1.2 1.2 1.2 1.2 1.2 Selectivity -- 1.18 1.13 1.09 1.11 1.17 K 302.7 359.6 408.2 444.2 492.1 575.0 5.5.2 Precision The % RSDof assay of Aripiprazole during the assay method precision study was within 0.08% and the % R.S.D for the area Imp- A, Imp-B, Imp-C, Imp-D, and Imp-E in related substance method precision study was within 1.35%. The %RSD of the assay results obtained in the intermediate precision study was within 1.2% and the % RSD for the area of Imp-A, Imp-B, Imp-C, Imp-D and Imp-E were well within 2.6%, conforming good precision of the method Table 5.5 Precision Data of Aripiprazole and Impurities Compound Rt RRT % RSD (n=6) Impurity-A 6.075 0.88 1.35 Impurity-B 7.212 0.74 0.69 Aripiprazole (Assay) 8.184 1.00 0.08 Impurity-C 8.904 1.09 1.07 Impurity-D 9.862 1.20 0.22 Impurity-E 11.521 1.41 0.61

167 5.5.3 Limit of detection and limit of quantification The limit of detection of all the Impurities namely Imp-A, Imp-B, Imp-C, Imp-D and Imp-E was achieved at 20-50 ng/ml for 20 μl injection volume. The limit of quantification for all five Impurities namely Imp-A, Imp-B, Imp-C, Imp-D and Imp-E was achieved at 50-140 ng/ ml for a 20 μl injection volume. The precision at the LOQ concentrations for Imp-A, Imp-B, Imp-C, Imp-D and Imp-E were below 2.9%. Table 5.6 LOD and LOQ Data for Impurities Compound Rt RRT LOD ( %) LOQ(%) Impurity-A 6.075 0.88 0.009 0.028 Impurity-B 7.212 0.74 0.007 0.022 Aripiprazole 8.184 1.00 -------- ----- Impurity-C 8.904 1.09 0.008 0.024 Impurity-D 9.862 1.20 0.005 0.015 purity-e 11.521 1.41 0.003 0.010 5.5.4 Linearity The calibration plot for linearity of the assay method was obtained over the calibration ranges tested i.e. 25 200 μg/ml and correlation coefficient obtained was greater than 0.9999. Linearity was checked for the assay method over the same concentration range for 3 consecutive days. The % RSD values of the slope and Y-intercept of the calibration curves were 0.03 and 1.83, respectively. The result

168 shows that an excellent correlation existed between the peak area and concentration of the analyte. Table 5.7 Linearity data for Assay of Aripiprazole Conc. in APZ peak area mg/ml 0.0254 1803121.0 0.0512 3653048.0 0.0756 5380380.0 0.1010 7193797.0 0.1256 8924271.0 0.1503 10675515.7 0.1993 14142826.3 Correl. Coefficient 0.999998 Slope 70919530.2 Y-Intercept 16453.3 The calibration plot for Linearity study of the related substance method was obtained over the calibration ranges tested, i.e., 0.01 % to 0.3% for Impurity Imp-A, Imp-B, Imp-C, Imp-D and Imp-E. The correlation coefficient obtained was greater than 0.998. Linearity was checked for the related substance method over the same concentration range for 3 consecutive days. The % RSD values of the slope and Y-intercept of the calibration curves were 0.15 and 2.64, respectively. The above result show that an excellent correlation existed between the peak area and the concentration of Imp-A, Imp-B, Imp-C, Imp-D and Imp-E. Table 5.8 Linearity data for impurities of Aripiprazole

169 S.No Conc. (%) Impurity -A Impurity -B Impurity -C Impurity -D Impurity -E 1 0.01 5927.0 6561 13585 18802 5643 2 0.05 22073.0 22989 55529 80916 25440 3 0.10 42345.0 44638 106936 152997 50287 4 0.15 65227.0 66681 158751 227923 73520 5 0.20 85210.0 88761 212932 308115 97990 6 0.25 108554.0 112816 265380 383979 123235 7 0.30 126627.0 132618 325016 455092 147216 Correlation co-efficient 0.99971 0.99990 0.99978 0.99991237 0.99996621 Y-Intercept 1248.09 1313.03 1175.73 3733.8 928.6 Slope 421911.7 439502.6 1065942 1511025.9 487576.5

170 Fig: 5.8 Linearity Plots for Impurities of Aripiprazole: Impurity-A, Impurity-B, Impurity-C, Impurity-D and Impurity-E

171 5.5.5 Accuracy The recovery for assay of Aripiprazole in bulk drug samples was ranged from 99.8 to 100.0 %. The percentage recovery of all five Impurities in Aripiprazole samples varied from 97.2 to 104.6 %. The details of the study was listed in the table 5.9. Table 5.9 Accuracy data of Aripiprazole and impurities Compound Rt RRT % Recovery (n=3) Impurity-A 6.075 0.88 97.2-99.8% Impurity-B 7.212 0.74 99.4-104.6% Aripiprazole 8.184 1.00 98.0-100.0% Impurity-C 8.904 1.09 99.8-100.7% Impurity-D 9.862 1.20 99.6-104.2% Impurity-E 11.521 1.41 99.5-100.8% 5.5.6 Robustness The robustness of the methods was successfully verified and in all the deliberately varied chromatographic conditions such as flow rate, column temperature and composition of organic solvent, the resolution between critical pair, i.e. Aripiprazole and Imp-B was greater than 2.2, illustrating the robustness of the method. The results obtained from all the deliberatrly varied condition were summarized in the table 5.10.

172 Table 5.10 Robustness data Parameter Imp-A Imp-B APZ Imp-C Imp-D Imp-E Rt 6.075 7.212 8.184 8.904 9.862 11.521 RRT 0.88 0.74 1 1.09 1.20 1.41 Different Flow, ml/min 0.8 0.87 0.73 1 1.10 1.21 1.41 1.2 0.86 0.73 1 1.09 1.20 1.42 Different Organic Ratio, 90% &110 % of Acetonitrile 40 0.86 0.75 1 1.11 1.19 1.38 50 0.86 0.74 1 1.08 1.21 1.41 Different Column Temperature, o C 38 0.87 0.73 1 1.07 1.20 1.40 42 0.87 0.74 1 1.08 1.22 1.41 5.5.7 Solution stability and mobile phase stability The % RSD (n=6) of the assay of Aripiprazole during solution stability experiments were within 0.8 %. No significant changes were observed in the content of Impurities namely Imp-A, Imp-B, Imp-C, Imp-D and Imp-E during solution stability and mobile phase stability experiments when performed using the related substance method. The solution stability and mobile phase stability experiment data confirms that the sample solutions and mobile phases used during assay and the related substance determination were stable for at least 48 Hrs.

173 Conclusion The RPLC method developed for quantitative and related substance determination of Aripiprazole is precise, accurate, rapid and specific. The method was fully validated showing satisfactory data for all the method validation parameters tested. The developed method is a stability indicating and can be conveniently used by quality control department to determine the related substance and assay in regular Aripiprazole production samples and also stability samples.

174 Summary Drug analysis play very Important role in creating the basis for high efficient drug theraphy by giving analytical support to synthetic, biotechnological, pharmacological, pharmaceutical technological, clinical research to find the most efficacious drug material and its optimal dosage form. Impurity profiling can be defined as a group of analytical activities aiming at identification/structural elucidation and quantitative determination of organic and inorganic Impurities as well as residual solvents in bulk drugs and pharmaceutical formulations. The DMF (Drug Master File) holder or the ANDA (Abbrevated New Drug Application) applicant should summarise those actual and potential Impurities most likely to arise during synthesis, purification and storage of the drug substance. This summary should be based on sound scientific appraisal of chemical reactions involved in the synthesis and Impurities associated with raw materials that could contribute to the Impurity profile of the drug substance and also about possible degradation products. The studies (e.g. NMR,IR and MS) conducted to charecterise the structure of actual Impurity or degradation product present in the drug substance at the appearent level of 0.1% or above(calculated using the response factor of drug substance) should be described. Hence, the bulk drug manufacturer should include documented evidence for degradation products and Impurities with the validated

175 analytical method for quantification, along with structural elucidation reports before getting the registration or marketing approval. This subject or topic for the research acitivity is selected based on the increasing needs of the phamaceutical industry in developing suitable analytical methods. Among the various other available techniques, the scope of work was focused on the modern chromatographic techniques such as HPLC which are accurate, precise and sophisticated techniques and are having wide spectrum of application in pharmaceutical industry. Literatures were reviewed and it was felt that there is a need to develop new, simple, specific, reliable analytical methods for Rabeprazole Sodium, Rabeprazole Sulfide, Aripiprazole and isolation and charecterisation of synthetic route indicative Impurity in Lansoprazole drug substance which are covered in Chapter 2 to Chapter 5. Chapter 2: A Validated, Stability-Indicating, LC Method for Rabeprazole Sodium The present study describes the development of a simple, economic and time efficient stability indicating reversed phase high performance liquid chromatographic (RP-HPLC) method for Rabeprazole in the presence of its Impurities and degradation products generated from forced decomposition studies. The drug substance was subjected to stress conditions of acid hydrolysis, oxidation, photolysis and thermal degradation. The degradation of Rabeprazole was observed under acid hydrolysis, peroxide and water

176 hydrolysis. The drug was found to be stable to other stress conditions attempted. Successful separation of the drug from the synthetic Impurities and degradation products formed under stress conditions was achieved on a C8 column using mixture of water, acetonitrile and triethylamine (55: 45: 0.25,v/v/v) and the ph was adjusted to 6.9 with trifluoroacetic acid. The developed HPLC method was validated with respect to linearity, accuracy, precision, specificity and robustness. The developed HPLC method to determine the related substances and assay can be used to evaluate the quality of regular production samples and stability samples of Rabeprazole sodium. This study provides the simple and comprehensive LC method to evaluate Rabeprazole Sodium, process related Impurities and Impur.ities originated from forced degradation studies To the best of our knowledge, the validated stability indicating LC method which separates all the Impurities disclosed in this investigation was not studied else where. This work was published in Chromatographia;, 68,2008,275-280 Chapter 3 : A Validated RPLC Method for Advanced Intermediate of Rabeprazole Sodium The present study describes the development of reversed phase liquid chromatographic (RPLC) method for advanced intermediate of Rabeprazole Sodium (RBS-1) in the presence of its Impurities. Successful separation of RBS-1 from the synthetic Impurities achieved on a Inertsil ODS3V C18, 150 mm x 4.6 mm column. The

177 developed HPLC method was validated with respect to linearity, accuracy, precision, specificity and ruggedness. To ensure the quality of Rabeprazole Sodium, it is required to qualify the advanced intermediate of Rabeprazole Sodium and this study provides the comprehensive testing procedure to qualify the advanced intermediate of Rabeprazole sodium.to the best of our knowledge, validated stability indicating LC method which separates all the impurities disclosed in this investigation was not studied else where. This work was published in Chromatographia; 69, 2009,381-384 Chapter 4 : Detection, Isolation and Characterization of principal synthetic route indicative Impurity in Lansoprazole The present work provides the detailed information of the potential Impurity which is likely to be formed during the synthesis of Lansoprazole, a substituted benzimidazole, 2-[[[3-methyl-4-(2,2,2- trifluoroethoxy)-2-pyridyl] methyl] sulfinyl] benzimidazole, a compound that inhibits gastric acid secretion. The present work will be helpful for those who manufactures Lansoprazole in commercial scale in the regulatory environment for identification and quantification of the potential Impurity namely Des- (trifluoroethoxy)lansoprazole in the commercial samples of Lansoprazole. This work was Accepted for publication and will be published soon in E-Journal of Chemistry. Chapter 5:

178 Stress Degradation Studies on Aripiprazole and Development of a Validated Stability Indicating LC Method The present sudy describes the development of a stability indicating reversed phase high performance liquid chromatographic (RP - HPLC) method for Aripiprazole in the presence of its Impurities and degradation products generated from forced decomposition studies. The drug substance was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. The degradation of Aripiprazole was observed under acid hydrolysis and peroxide. The drug was found to be stable to other stress conditions attempted. Successful separation of the drug from the synthetic Impurities and degradation product formed under stress conditions was achieved on a Inertsil phenyl column using mixture of 0.2 % Trifluoroacetic acid buffer and acetonitrile (55: 45,v/v ). The developed HPLC method was validated with respect to linearity, accuracy, precision, specificity and robustness. The developed HPLC method to determine the related substances and assay determination of Aripiprazole is simple,comprehensive and can be used to evaluate the quality of regular production samples. It can be also used to test the stability samples of Aripiprazole. To the best of our knowledge,the validated stability indicating LC method which separates all the Impurities disclosed in this investigation was not reported else where. This work was published in Chromatographia; 68, 2008,635-640