CHAPTER-7 Development and Validation of a RP-HPLC method for related compund-c in Ziprasidone hydrochloride monohydrate

CHAPTER-7 Development and Validation of a RP-HPLC method for related compund-c in Ziprasidone hydrochloride monohydrate Chapter-7 Page 212

7.1. INTRODUCTION This chapter describes about the Development and validation of a RP-HPLC method for related compound-c in Ziprasidone hydrochloride monohydrate, survey of literature, materials and methods, results and discussion, summary and conclusion were covered. Ziprasidone hydrochloride monohydrate 5-[2-[4-(1,2- benzisothiazol-3-yl)-1-piperazinyl]ethyl]-6-chloro-1,3-dihydro-2h-indol-2-one is the treatment of schizophrenia, and acute mania and mixed states associated with bipolar disorder. Its intramuscular injection form is approved for acute agitation in schizophrenic patients for whom treatment with just Ziprasidone hydrochloride monohydrate is appropriate. Analytical HPLC method has been developed for Ziprasidone hydrochloride monohydrate and impurities. Many HPLC, LC-MS and UPLC methods are observed in publications during method development. An HPLC method is developed for Ziprasidone hydrochloride monohydrate (1 1-11) and related compound-c. Figure- 7.1: Structure of Ziprasidone hydrochloride monohydrate. Chemical name : 5-[2-[4-(1,2-benzisothiazol-3-yl)-1- piperazinyl]ethyl]-6-chloro-1,3-dihydro-2h-indol-2- one CAS Registry Number : 146939-27-7 Chapter-7 Page 213

Molecular formula : C21H21 CLN4OS Molecular weight : 412.936 Therapeutic category : Schizophrenia and acute mania Table- 7.1: Ziprasidone hydrochloride monohydrate impurities details: S. No Impurity structure Chemical name Molecular weight Related compound-c 1 Methyl (-)-(R)-(o- chlorophenyl)- 6,7-dihyrothieno [3,2-c] pyridine-5 (4H)- acetate, hydrogen sulfate 871.85 C42H40Cl2N8O5S2 Related compound-d 2 (3- (benzo[d]isothiazol- 3-yl)-5-(2-(4- (benzo[d] isothiazol-3- yl)piperazil-1-yl) ethyl)-6- chloroindolin-2-one) 545.11 C28H24 ClN5OS2 Chapter-7 Page 214

ZPH Acetone 5-(2-(4-3. O HN N N (benzo[d]isothiazol- 3-yl)piperazin-1- yl)ethyl)-6-chloro-3-453 C24H25ClN4OS Cl N S (prop-1-en-2- yl)indolin-2-one 7.2. REVIEW OF LITERATURE A simple and reliable head space gas chromatographic method has been developed for the determination of residual methyl chloride, ethyl chloride and isopropyl chloride in Ziprasidone hydrochloride monohydrate. The proposed method is based on flame ionization detection technique with DB-624 as stationary phase. Linearity of detector response was established up to 13.5μg/g and the detection limit was 0.8μg/g for methyl chloride, ethyl chloride and 0.9μg/g for isopropyl chloride respectively. No interference of organic solvents used in the synthesis was observed. Performance of the method was assessed by evaluating the recovery, repeatability, reproducibility, linearity and limits of detection and quantification. The proposed method has a potential for application to drug substances which may contains traces of alkyl chloride. Results prove that the validated method was suitable for determining the residual methyl chloride, ethyl chloride and isopropyl chloride in Ziprasidone hydrochloride monohydrate drug substance. To widen the scope of this method, interference of 17 commonly employed solvents in the synthesis has been studied for any possible interference with methyl chloride, ethyl chloride and isopropyl chloride. The potentiality of method has been studied for various drug substances containing possible alkyl chlorides residue present in their drug matrix. Chapter-7 Page 215

Ultra Performance Liquid Chromatography (UPLC) was employed to develop a rapid and robust method for the analysis of Ziprasidone hydrochloride monohydrate, both as a drug substance and in the final dosage forms. The application of this method in stability analyses was verified. Tests were carried out according to ICH/FDA guidelines, European Pharmacopeia, and United States Pharmacopeia rules, which take into account factors such as specificity, linearity, accuracy, and precision. Separation was performed on an acquity UPLC BEH phenyl 1.7μm column with a simple mobile phase, consisting of acetonitrile and water adjusted to ph 2.0 with ortho-phosphoric acid. Using this mobile phase and gradient elution, the separation was completed with in 5 min. This method is very sensitive, and allows performing simultaneous identification, assay, and determination of impurities and related substances in one injection. A reverse phase HPLC method is described for the determination of Ziprasidone hydrochloride monohydrate in bulk and pharmaceutical dosage forms. Chromatography was carried out on an ODS C18 column using a mixture of methanol and phosphate buffer (55:45 v/v) as the mobile phase at a flow rate of 1mL/min. Detection was carried out at 314nm. The retention time of the drug was 4.522 min. The method produced linear responses in the concentration range of 0.5-30 μg /ml of Ziprasidone hydrochloride monohydrate. The method was found to be applicable for determination of the drug in capsules. Ziprasidone is known as a novel "a typical" or "second-generation" antipsychotic drug. A sensitive and reproducible method was developed and validated for determination of Ziprasidone hydrochloride monohydrate and its major impurities, which are significantly different in polarity. The separation is performed on a Waters spherisorb octadecylsilyl column (5.0 μm, 250 x 4.6 mm I.D.) using a gradient with mobile phase A [buffer:acetonitrile] [80:20, v/v] and mobile phase B [buffer:acetonitrile] [10:90, v/v] at a working temperature of 25 C. The buffer was 0.05 M KH2PO4 solution with an addition of 10 ml triethylamine /Lsolution, adjusted to ph 2.5 with ortho phosphoric acid. The flow rate was 1.5 ml/min, and the elute was monitored at 250 nm using a diode array detector. Chapter-7 Page 216

Optimization of the experimental conditions was performed using partial least squares regression, for which four factors were selected for optimization: buffer concentration, buffer ph, triethylamine concentration, and temperature. The proposed validated method is convenient and reliable for the assay and purity control in both raw materials and dosage forms. 7.3. OBJECTIVE The main objective of this research work is to develop method for separation of related compound-c and Ziprasidone Acetone impurity in Ziprasidone hydrochloride monohydrate. After review of so many literature is reveals that the reported methods are not reported for the separation of these two impurities in any where. 7.4. MATERIALS AND METHODS 7.4.1. Reagents & Chemicals. a. Water : Merck b. Acetonitrile HPLC grade : Merck c. Mono basic potassium phosphate : Merck d. Potassium hydroxide : Merck e. Methanol : Merck f. Hydrochloric acid : Merck 7.4.2. Drug substances: Ziprasidone hydrochloride monohydrate and related compound-c and related compound-d samples were received from M/S Aurobindo Laboratories, Hyderabad(A.P),India. Chapter-7 Page 217

7.4.3. Instrument details: The High performance Liquid Chromatography using Waters HPLC instrument having quaternary pumps including auto injector. This HPLC connected with PDA detector, make Waters. All the components are controlled with Empower2 software. 7.4.4. Method development: Development trials were performed with all neutral buffer salts and different make HPLC columns but finally the chromatographic conditions were optimized with the potassium phosphate salt, acetonitrile and methanol with simple gradient program. 7.4.4.1. Wave length Selection: The UV spectrums were generated for Ziprasidone hydrochloride monohydrate and related compound-c using with photo diode array detector (PDA). Ziprasidone hydrochloride monohydrate and its impurities were found to have varying absorption maxima over a range of wave length. But it was found that at about 229nm, Ziprasidone hydrochloride monohydrate and its impurities were found to have optimum UV absorption. Therefore, 229nm was selected for the study and quantification of Ziprasidone hydrochloride monohydrate and it s related impurities. Figure- 7.2: UV Spectra of (a) Ziprasidone hydrochloride monohydrate, (b) related compound-c (c) related compound-d. Chapter-7 Page 218

7.4.4.2. Selection of mobile phase and stationary phase: Ziprasidone hydrochloride monohydrate and related compound-c were found that different functional groups, shows different affinities with mobile phases and stationary phase. A different column with different selectivity provides good separation for method development. Two parameters were chosen to get required resolutions and separations and symmetrical peaks for Ziprasidone hydrochloride monohydrate and impurities. i.e., Selection of the mobile phase and column. 7.4.4.3. Selection of Mobile phase: Ziprasidone hydrochloride monohydrate acetone, related compound-c were co-eluted using with different mobile phases. Ziprasidone hydrochloride monohydrate and the impurities of Ziprasidone hydrochloride monohydrate were having wide range of polarities and the separation of these impurities mainly depends on the column stationary phase. An gradient method was mobile phase of buffer is 0.02M ammonium dihydrogen phosphate in water ph adjusted to 9.85 and acetonitrile and methanol was suitable for the separation of Ziprasidone hydrochloride monohydrate and its related substances. Mobile phase was degassed and filtered through 0.22 µm millipore filter paper. 7.4.4.4. Selection of stationary phase: Separation was achieved with Zorbax RX C8 150 x 4.6 mm I.D.,5.0µm column. Different stationary phases were studies for the separation of Ziprasidone hydrochloride monohydrate such as C18 and C8 using the mobile phase specified. The experimentation was started using Waters Symmetry C18, 250 x 4.6mm,I.D., 5.0µm, column. Trail-1: The complete experiment details are as follows. Column : Waters symmetry C18, 250 x 4.6mm I.D.,5.0µm Mobile Phase-A : 0.25% phosphoric acid mixed 2.50 g of ortho phosphoric acid in 1000 ml of water. Mixed well, filtered and degassed Chapter-7 Page 219

Mobile phase-b Sample Preparation : Flow rate Oven temperature Injection volume Elution : Mixed acetonitrile and water in the ratio of 95 and 5 and sonicate to degassed 0.5mg/mL : 0.9 ml/ min : 30 o C : 20µL : Gradient Gradient program Time in min Mobilephase- A Mobilephase- (%) B (%) 0 85 15 20 70 30 25 65 35 30 60 40 45 60 40 46 85 15 50 85 15 55 85 15 Figure-7.3: Blend Chromatogram by using waters Symmetry C18, 250 x 4.6mm I.D., 5.0µm column and trail-1 method conditions. Chapter-7 Page 220

Observation: Related compound-c is not stable in trail-1method conditions, hence waters symmetry C18, 250 x 4.6mm,I.D., 5.0µm, column and trail-1 method conditions not suitable for the separation of related compound-c. Trail-2: The complete experiment details are as follows. Column Buffer : : Zorbax RX C8 150 x 4.6 mm I.D., 5.0µm column Dissolved 6.8 g/l of monobasic potassium phosphate in water and adjust with 5N potassium hydroxide to a ph of 6.0 Mobile Phase : Acetonitrile: methanol : buffer ( 11:1:8) Wavelength : 229 nm Flow rate Diluent Elution : : : 1.0 ml/min Methanol, water and hydrochloric acid (20:5:0.01) Isocratic Sample preparation Injection volume Run time : : : 0.5mg/mL 20µL 60min Figure-7.4: Blend Chromatogram by using Zorbax RX C8 150 X 4.6 mmi.d., 5.0µm column and trail-2 method conditions. Chapter-7 Page 221

Observation: Related compound-c and ZPH Acetone are coeluting. Hence, the isocratic elution and trail-2 method conditions is not suitable for the separation of related compound-c. Trail-3: The complete experiment details are as follows. Column Buffer(Mobile phase-a) : Zorbax RX C8 150 x 4.6 mm I.D., 5.0μm column : Dissolve 6.8 g/l of monobasic potassium phosphate in water and adjust with 5N potassium hydroxide to a ph of 6.0 Mobile phase-b : Buffer and acetonitrile in the ratio of 75:25 v/v Sample preparation : 5 mg in 25 ml of diluent Wavelength : 229 nm Flow rate : 1.0 ml/ min Oven temperature : 40 o C Diluent Elution Injection volume Runtime : 100% acetonotrile : Gradient : 20μL : 60 min Gradient program Mobile phase- Mobile phase B Time in min A(%) (%) 0 65 35 5 50 50 10 50 50 23 20 80 35 20 80 45 50 50 60 65 35 Chapter-7 Page 222

Figure-7.5: Blend Chromatogram by using Zorbax RX C8 150 X 4.6 mm I.D., 5.0µm column and trail-3 method conditions. Conclusion: Method needs to modify for getting to reduce blank peaks and baseline noise. Based on the above study on stationary phase, it was concluded related compound-c in Ziprasidone hydrochloride monohydrate were well separated from each other in Zorbax RX C8 150 X 4.6 mm I.D., 5.0µm column. 7.4.5. Optimized method: Based on the above study, the below mentioned HPLC parameters was chosen for the separation and quantification of related compound-c and Ziprasidone hydrochloride monohydrate. Column : Zorbax RX C8 150 x 4.6 mm I.D., 5.0μm column Buffer preparation Mobile phase-a Mobile phase-b Sample preparation Auto sampler Wavelength : ddissolved 6.8 g/l of mono basic potassium phosphate in adjust with 5N of potassium hydroxide to a ph 6.0 : A degassed mixture of buffer: methanol: acetonitrile in the ratio of 40:15:45 (v/v/v) : Acetonitrile (100%) : 45 mg in 100 ml of diluent 5 o C : 229 nm Chapter-7 Page 223

Flow rate : 1.0 ml/min Oven temperature Injection volume Run time : 35 o C : 20μL : 65min Diluent : Methanol: water : hydrochloric acid (80:20:0.04) Elution : Gradient Gradient programme : Mobile phase-a Mobile phase-b Time in min (%) (%) 0 100 0 20 100 0 35 60 40 50 60 40 55 100 0 65 100 0 a) Reference stock solution: Weighed about each 18 mg of Ziprasidone hydrochloride monohydrate working standard and related compound-d into 100 ml volumetric flask, dissolved and diluted to volume with diluent and mixed well (0.18mg/mL). b) Reference Solution (0.2%): Diluted 0.5 ml of reference stock solution to 100 ml with diluent (0.0009 mg/ml). c) Preparation of Sample solution: Accurately weighed 45 mg of sample into 100 ml volumetric flask, dissolved and diluted to volume with diluent. Procedure: Injected all above solutions once and reference solution six times and calculated the system suitability parameters i.e. the theoretical plates, Tailing factor, %RSD for reference solution. System suitability criteria: % RSD for six replicate injections of reference solution should be not more than 10.0. Chapter-7 Page 224

The tailing factor for Ziprasidone hydrochloride monohydrate peak reference solution should be not more than 1.5. The number of theoretical plates for Ziprasidone hydrochloride monohydrate peak reference solution should be not less than 3000. Table -7.2: Specification: S. No Name of the impurity Specification 01 Related compound-c Not more than 0.20% Calculation: calculate the impurity using below formula: Related compound- C area in test solution 1 Related compound C = --------------------------------------------------------- X ------ X 0.2 Avg. area of Ziprasidone peak in reference solution RRF Total impurities: % known impurities + % other unknown impurities calculated by area normalization. RRF for related compound-c: 0.42 RRT for related compound-c: 2.70 Figure- 7.6: A typical HPLC Chromatogram of diluent. Chapter-7 Page 225

Figure- 7.7: A typical HPLC Chromatogram of reference solution (0.2%). Figure- 7.8: A typical HPLC Chromatogram of Ziprasidone hydrochloride monohydrate as such sample. Figure- 7.9: A typical HPLC Chromatogram of Ziprasidone hydrochloride monohydrate and all impurities blend solution. Chapter-7 Page 226

7.5. RESULTS AND DISCUSSION 7.5.1. Method validation: Analytical method validation was performed as per ICH and USFDA guidelines with specificity, precision, accuracy, linearity, limit of detection, limit of quantification, ruggedness and robustness. 7.5.1.1. Related substances by HPLC: 7.5.1.2. System suitability: a) Reference stock solution: Weighed about each 18 mg of Ziprasidone hydrochloride monohydrate working standard and related compound-d into 100 ml volumetric flask, dissolved and diluted to volume with diluent and mixed well (0.18mg/mL). b) Reference solution (0.2%): Diluted 0.5mL of reference stock solution to 100 ml with diluent (0.0009 mg/ml). c) Preparation of sample solution: Transferred 45mg of sample into 100mL volumetric flask, dissolved and diluted to volume with diluent. Injected all above solutions once and calculated the system suitability parameters i.e. the resolution between adjacent peaks, Tailing factor and tangent for Ziprasidone hydrochloride monohydrate. Conclusion: Under optimized Chromatographic conditions, related compound-d and Ziprasidone hydrochloride monohydrate, were separated well, retention times being about 20.08 and 5.70 min, respectively. The system suitability results are given in table-7.3. Table- 7.3: System suitability results: S. No Name Retention Relative retention % RSD Theoretical Tailing time(min) time(min) plates(n) factor( T) 01 Ziprasidone reference solution 5.70 1.00 0.5 6050 1.10 02 Related compound- D 20.08 3.52 2.4 ------ ----- Chapter-7 Page 227

7.5.1.3. Specificity: a) Thermal degradation: Accurately weighed 1 gm of Ziprasidone hydrochloride monohydrate sample is taken and kept under thermal condition i.e., at 105 C for 7days and sample collected after 48hours and sample analysed related compound -C by HPLC and checked for % degradation and determine the peak purity of main peak. Observation: Ziprasidone hydrochloride monohydrate sample is stable under thermal condition. b) Photo degradation: Weighed 1 gm of sample is taken and kept in UV chamber i.e., at 254 nm for 48 hours and sample collected after 48 hours and sample analyzed. Observation: A Ziprasidone hydrochloride monohydrate sample is stable under photo condition. c) Acid hydrolysis: Sample was dissolved in 0.1N HCl at room temperature and collected the sample after 30minutes. The 30 minutes sample was analyzed for related compound -C by HPLC and checked for % degradation and determine the peak purity of main peak. Observation: Ziprasidone hydrochloride monohydrate sample is stable under acid hydrolysis. d) Base hydrolysis: Sample was dissolved in 0.1N NaOH at room temperature and collected the sample after 1hr. The 1hr sample was analyzed for related compound -C by HPLC and checked for % degradation and determine the peak purity of main peak. As 1 hour sample was found at RRT 5.2 impurity only observed as 0.06% and all other individual unspecified impurities eluting after Ziprasidone hydrochloride monohydrate are detected less than disregard limit. Hence, sample was dissolved in 0.1N NaOH and reflux at 70 o C. Collected the sample after 12 hours and analyzed for related compound-c by HPLC. Chapter-7 Page 228

Calculated the % degradation of related compound-c, unspecified impurity and determine the peak purity of Ziprasidone hydrochloride monohydrate peak. Observation: Ziprasidone hydrochloride monohydrate was degraded under base hydrolysis. e) Oxidation degradation: Sample was dissolved in 3% H2O2 in dark at room temperature up to 24 hours and samples were analyzed for related compound-c by HPLC and checked for % degradation and determine the peak purity of main peak. Note: Initial sample was dissolved in formic acid and found Ziprasidone hydrochloride monohydrate main peak is completely degraded. Hence, experiments were conducted without co-solvent for Water, base, acid and oxidation. Observation: Ziprasidone hydrochloride monohydrate was degraded to under peroxide solution. f) Water hydrolysis: Sample was dissolved in water and refluxed at 70 C temperature for 12 hours. 12 th hour, sample was analyzed for related compound-c by HPLC and checked for % degradation and determine the peak purity of main peak. Observation: Ziprasidone hydrochloride monohydrate was not degraded to under water hydrolysis. Conclusion: Related compound-c found to be not degraded under the all stress study conditions. Un specified impurity eluting after Ziprasidone hydrochloride monohydrate peak: Found to be degraded unspecified impurities eluting after Ziprasidone hydrochloride monohydrate peak in base degradation. The un specified impurity was found to be degraded drastically from not detected to 1.02% at about RRT 6.07 minutes and 0.90% at about RRT 5.00 minutes under stressed condition of base hydrolysis (0.1N NaOH) at 70 o C temperature. Chapter-7 Page 229

Ziprasidone hydrochloride monohydrate: However, Ziprasidone hydrochloride monohydrate peak found drastically degraded under stressed condition of 0.1N HCl and 0.1N NaOH and not degraded under the stress condition of UV, Visible, heat, water hydrolysis and oxidation. The degradation results are given in below table - 7.4. Table- 7.4: Ziprasidone hydrochloride monohydrate degradation data : Stressed condition Time (hrs) % Purity Thermal degradation 7 x 24hrs 99.87 Photo degradation 7 x 24hrs 99.85 Acid hydrolysis 0.5hrs 97.75 Base hydrolysis 12hrs 53.13 Oxidation degradation 24hrs 90.0 Water hydrolysis 12 hrs 99.90 Figure- 7.10: A typical HPLC Chromatogram of thermal degradation sample. Chapter-7 Page 230

Figure- 7.11: A typical HPLC Chromatogram of photo degradation sample. Figure- 7.12: A typical HPLC Chromatogram of acid degradation sample. Figure-7.13: A typical HPLC Chromatogram of base degradation sample. Chapter-7 Page 231

Figure- 7.14: A typical HPLC Chromatogram of oxidation degradation sample. Figure- 7.15: A typical HPLC Chromatogram of water hydrolysis degradation sample. 7.5.1.4. Limit of Detection and Limit of Quantification: a) LOQ solution-1 preparation (0.05%): Transferred 5.0µL of related compound- C stock solutions into 10mL volumetric flask, dissolved and diluted to volume with diluent. b) LOQ solution-2 preparation: Transferred 5.0µL of related compound-c stock solutions into 10mL volumetric flask, dissolved and diluted to volume with diluent. Chapter-7 Page 232

c) LOD solution-1 preparation: Transferred 3.3mL of above LOQ solution-2 stock solutions into 10mL volumetric flask, dissolved and diluted to volume with diluent. Injected all above solutions and calculated the Limit of detection and Limit of quantification for each impurity. Conclusion: The LOD for related compound-c was found 0.013% respectively. The LOQ for related compound-c was found to be 0.05 % respectively. The results are summarized in the table - 7.5. Table- 7.5: Limit of detection and Limit of Quantification data: Concentration Related compound-c (%) LOD 0.013 LOQ 0.05 7.5.1.5. Precision and accuracy at Limit of Quantification level: a) Related compound-c stock Solution preparation: Transferred 45mg of related compound-c into 100mL volumetric flask, containing 20mL of diluent dissolved and diluted to volume with diluent. Prepared six times the solution as mentioned above and inject all the above solutions each preparation once, calculated the % RSD for six preparations for impurity. Accuracy: b) Sample + related compound-c stock solution preparation: Accurately weighed 45mg of sample into 100mL volumetric flask, dissolved in 50mL of diluent and added 50µL of related compound-c dissolved and diluted to volume with diluent. c) Sample solution preparation: Weighed 45mg of sample into 100mL volumetric flask, dissolved and diluted to volume with diluent. Chapter-7 Page 233

Prepared three times the solution as mentioned above and inject each preparation once and calculated the % recovery for related compound-c at Limit of Quantification level. Conclusion: The repeatability and recovery at the LOQ concentrations for related compound-c were 3.7% and 105.7% respectively. The results are summarized in the table- 7.6. Table- 7.6: Precision and accuracy at Limit of Quantification level data: S. No Impurity % RSD (n=6) % Recovery (n=3) 1 Related compound-c 3.7 105.7 7.5.1.6. Linearity: a) Linearity solution-1 (0.049%): Transferred 5µL related compound-c stock solution into 10mL volumetric flask, containing 5mL of diluent dissolved and diluted to volume with diluent. b) Linearity solution-2 (0.099%): Transferred 10µL of related compound-c stock solution into 10mL volumetric flask, containing 5mL of diluent dissolved and diluted to volume with diluent. c) Linearity solution-3(0.148%): Transferred 15µL of related compound-c stock solution in to 10mL volumetric flask, containing 5mL of diluent dissolved and diluted to volume with diluent. d) Linearity solution-4 (0.198%): Transferred 20µL of related compound-c stock solution into 10mL volumetric flask, containing 5mL of diluent dissolved and diluted to volume with diluent. e) Linearity solution-5 (0.247%): Transferred 24.7µL of related compound-c stock solution into 10mL volumetric flask, containing 5mL of diluent dissolved and diluted to volume with diluent. f) Linearity solution-6(0.297%): Accurately transferred 29.7µL of related compound-c stock solution into a 10mL volumetric flask, containing 5mL of diluent dissolved and diluted to volume with diluent. Injected all above Chapter-7 Page 234

solutions each preparation once and calculated the Linearity parameters i.e. correlation coefficient, slope and intercept for impurity. Conclusion: Linearity established for related compound-c at 0.049%, 0.099%, 0.148%, 0.198%, 0.247% and 0.297%. The correlation coefficient (r) are more than 0.99. The above result reveal that method is linear, results are summarized in purity wise and presented in table-7.7. Table- 7.7: Ziprasidone related compound-c linearity data: S. No Level (%) Concentration (%) Area of related compound-c 1 LOQ 0.049 5236 2 50 0.099 10845 3 75 0.148 15787 4 100 0.198 21248 5 125 0.247 25794 6 150 0.297 31249 Correlation coefficient(r) 0.9997 Slope 104026.7 Y-Intercept 363.2 % 100 Y-Intercept 1.70 Chapter-7 Page 235

Figure- 7.15: Ziprasidone related compound-c linearity graph. 7.5.1.7. Accuracy: a) Accuracy solution-1 preparation (LOQ): Accurately weighed 45mg of sample into 100 ml volumetric flask, dissolved in 30mL of diluent and added 50µL impurity stock solution, dissolved and diluted to volume with diluent. Three solutions prepared as mentioned above. b) Accuracy solution-2 preparation- (50%): Weighed 45 mg of sample into 100 ml volumetric flask, dissolved in 30 ml of diluent and added 100µL of impurity stock solution, dissolved and diluted to volume with diluent. Three solutions prepared as mentioned above. c) Accuracy solution-3 preparation (100%): Transferred 45 mg of sample into 10 ml volumetric flask, dissolved in 5mL of diluent and added 200µL of impurity stock solution, dissolved and diluted to volume with diluent. Three solutions prepared as mentioned above. d) Accuracy solution-4 preparation (150%): Accurately weighed 45mg of sample into 10 ml volumetric flask, dissolved in 5mL of diluent and added 300µL of impurity stock solution, dissolved and diluted to volume with diluent. Three solutions prepared as mentioned above. Chapter-7 Page 236

Injected each above preparation once and calculated the % recovery for Ziprasidone related compound-c. Conclusion: The percentage recovery of related compound-c in Ziprasidone hydrochloride monohydrate samples is shown in table - 7.8. Table- 7.8: % Recovery in accuracy : Concentration Related compound-c (%) LOQ 105.7 50% 95.8 100% 95.5 150% 95.7 7.5.1.8. Precision: a) Sample preparation: Weighed 45mg of sample into 100mL volumetric flask,dissolved and diluted to volume with diluent. b) Sample + LOQ Solution spiked preparation: Accurately weighed 45mg of sample into a 100mL volumetric flask, dissolved in 30mL of diluent added 200µL of impurity stock solution dissolved and diluted to volume with diluent. Prepared the solution six times as mentioned above. Injected all above sample preparations and calculated the % RSD for impurity. Conclusion: The precision of the related substance method was checked by injecting six individual preparations of Ziprasidone hydrochloride monohydrate spiked with 0.20% related compound-c. The % R.S.D of the area for of related compound-c was calculated. The results was summarized in the table-7.9, Chapter-7 Page 237

Table- 7.9: Precision data: S. No Preparation Related compound-c area % 1 1 0.1858 2 2 0.1862 3 3 0.1958 4 4 0.1927 5 5 0.1954 6 6 0.1879 Average 3.83099 Standard deviation 0.0909 %RSD 2.37 7.5.1.9. Robustness: Flow variation: a) Sample solution preparation: Weighed 5 mg of sample into 100mL volumetric flask, dissolved and diluted to volume with diluent. b) Sample + 0.20% spiked preparation: Transferred 45 mg of sample into 100mL volumetric flask, dissolved in 5 ml of diluent added 200µL of impurity stock solution dissolved and diluted to volume with diluent. Injected the above sample solution at flow rates 0.8mL/min and at 1.2mL/min and observed the system suitability parameters and impurities relative retention times and compared with 1.0mL/min results. Temperature variation: a) Sample solution preparation: Transferred 45mg of sample into 100mL volumetric flask, dissolved and diluted to volume with diluent. b) Sample + 0.20% spiked preparation: Accurately weighed 45mg of sample into 100mL volumetric flask, dissolved in 30mL of diluent added 200µL of impurity stock solution dissolved and diluted to volume with diluent Injected the above sample solution at temperature 30 C and at 40 C and observed the system suitability parameters and impurities relative retention times and compared with 35 C results. Chapter-7 Page 238

ph variation: a) Sample solution preparation: Weighed 45mg of sample into 100mL volumetric flask, dissolved and diluted to volume with diluent. b) Sample + 0.20% spiked preparation: Accurately weighed 45mg of sample into 100mL volumetric flask, dissolved in 30 ml of diluent added 200µL of impurity stock solution dissolved and diluted to volume with diluent. Injected the above sample solution at ph 5.8 and at 6.2 and observed the system suitability parameters and impurities relative retention times and compared with 6.0 results. Conclusion: The results are summarized in the table-7.10. Table -7.10: Robustness data: Parametr 30 C 40 C 0.8 1.2 ph at ph at As ml/min ml/min 5.8 6.2 such Tailing factor for Ziprasidone 1.14 0.84 1.03 1.02 1.25 1.08 1.10 reference solution % RSD for Ziprasidone 0.3 1.4 1.7 2.2 1.8 2.4 0.5 reference solution % RSD for Ziprasidone related compound-d 1.9 3.3 1.4 1.0 2.6 2.22 2.4 7.5.1.10. Solution stability: Sample solution preparation: Accurately weighed 45mg of sample into a 100mL volumetric flask, dissolved and diluted to volume with diluent. Injected the solution for 0 hrs(initial), 12hrs, 24 hrs and 48 hrs and performed the impurity content. Conclusion: Related compound-c is not increased and other impurities are also not observed during the solution stability and mobile phase stability experiments when performed using the related substance method. The solution stability and mobile Chapter-7 Page 239

phase stability experiment data confirms that the sample solutions and mobile phases used during the related substance determination were stable for at least 48 hours. The results are summarized in the table-7.11. Table- 7.11: Solution stability data: Duration Related compound-c (%) Sample solution initial 0.1858 After 12 hrs 0.1784 After 24 hrs 0.1811 After 48 hrs 0.1802 Table -7.12: Mobile phase stability data: Duration Related compound-c (%) Sample solution initial 0.1858 After 12 hrs 0.1794 After 24 hrs 0.1848 After 48 hrs 0.1891 7.5.1.11. Batch analysis: Using the above validated method, Ziprasidone hydrochloride monohydrate sample was analyzed and the data is furnished in table- 7.13. Table- 7.13: Batch analysis data Lot Number Related compound-c 001 0.1858 7.6. SUMMARY AND CONCLUSION The simple isocratic HPLC method for quantification of related compound-c in Ziprasidone hydrochloride monohydrate. Related compound-c is precise, accurate, rapid and specific. The method was fully validated showing satisfactory data for all the method validation parameters tested. The developed method can be used for regular samples and stability samples analysis also. Chapter-7 Page 240

7.7. REFERENCES 1. Daniel oakowiecki, and Krzysztof cal.development of rapid and robust stability-indicating method for analysis of Ziprasidone hydrochloride and freebase as drug substance and in medicines by UPLC. Pelpioska 19, 83-200 starogard gdaoski, poland. Hallera107, 80-416 Gdaosk, Poland. 2. T.Kaleemullah1, Mansur Ahmed, Hemant Kumar sharma, KVSN Raju, M.Narendra Kumar. Development and validation of gas chromatography method for low level detection of residual methyl chloride, ethyl chloride and isopropyl chloride in Ziprasidone hydrochloride scholars Research Library Der Pharma Chemica, 2011, 3 (6):390-399 ISSN 0975-413XCODEN(USA): PCHHAX. 3. B. Sudha Rani and P. Venkata Reddy. Volume 3 (2006), Issue 3, Pages 169-72,DOI:10.1155/2006/252570. Volume 3 (2006), Issue 3, Pages 169-172, doi:10.1155/2006/252570. 4. Medikondu Kishorea, Y.Hanumantharao. A validated spectrophotometric methods for the determination of Ziprasidone as hydrochloride in bulk and pharmaceutical formulations. International Journal of Chemical and analytical Science 2010, 1(7),151-153, ISSN: 0976-1209. 5. A. Singh, B. M. Rao, G. R. Deshpande, S. Sangaraju, M.K. Srinivasu, M. Lalitha Devi, P. V. V. Satyanarayana and K.B.Chandrasekhar. A rapid stability- Indicating LC method for Ziprasidone hydrochloride. Volume 65,numbers 3-4 (2007),191-196,DOI:10.1365/s10337-006-0125-2. 6. S.Uma Devi, E. Pushpa Latha, C.V.Nagendra Kumar Guptha, P. Ramalingam, Development and validation of HPTLC method for estimation of Ziprasidone hydrochloride in bulk and pharmaceutical dosage forms. ISSN 2229 6867. 7. Ganji ramanaiah, Dr.D.Ramachandran,G.srinivas, Jayapal gowardhane, Purnachanda rao. Development and validation of a rapid uv-spectroscopic method for the estimation of Ziprasidone hydrochloride monohydrate in drug substance and its dosage forms. Issn- 0975-1491 vol 4, issue 2, 2012. 8. M.Gnana ruba priya, M. Karikalan, S. Asadulla, S. Rajesh, J. Siva rama krishna and S. Vijay sridhar. Development and method validation using HPLC for assay Chapter-7 Page 241

of Ziprasidone capsule,ijpsr (2011), vol. 2, issue 9(research), ISSN: 0975-8232. 9. K. Srinivasa rao, K.Srinivas, S. Satyanarayana raju, new RP-HPLC method For the estimation of Ziprasidone hydrochloride In pharmaceutical dosage forms. Vol. 7, Issue 8, 2008. 10. J.D.Chudasama, K.P.Channabasavaraj, J. S. Modiya, T. T. Mani.Development and validation of three new assay methods for Ziprasidone hydrochloride. monohydrate using UV-Spectrophotometry, IJPRD, 2011; Vol 3(3): 8; May 2011 (57-63) International Standard Serial Number 0974 9446. 11. Shilpi agarwal1,vinod K. Gupta2, Barkha Singhal3, potentiometric assay of antipsychotic drug (Ziprasidone hydrochloride) in pharmaceuticals, serum and urine..1, J. Electrochem. Sci., 6 (2011) 3036 3056. Chapter-7 Page 242