CHAPTER 2 A New stability Indicating RP-HPLC method for related substances in Zolmitriptan

CHAPTER 2 A New stability Indicating RP-HPLC method for related substances in Zolmitriptan Chapter-2 Page 68

2.1. INTRODUCTION This chapter deals the method development and validation of new stability indicating RP-HPLC method for related substances in Zolmitriptan. Active ingredients therapeutic activity, review of literature, materials and methods, development trials, validation results and summary and conclusion were covered. Zolmitriptan (S)-4-({3-[2-(dimethylamino)ethyl]-1H-indol-5-yl}methyl)-1,3- oxazolidin-2-one is a selective serotonin receptor agonist of the 1B and 1D sub types drug, used in the acute treatment of migraine attacks with or without aura and cluster headaches. Analytical HPLC method has been developed for Zolmitriptan and impurities. Many HPLC, LC-MS and Chiral methods are observed in publications during method development, some of the methods are mentioned by LC-MS for human plasma and metabolites, A new HPLC method is developed for Zolmitriptan (1-14) and impurities. Figure-2.1 Structure of Zolmitriptan. Chemical name (S)-4-({3-[2-(dimethylamino)ethyl]-1H-indol-5- yl}methyl)-1,3oxazolidin-2-one CAS Registry Number 139264-17-8 Molecular formula C16H21N3O2 Molecular weight 287.357 Therapeutic category Antimigraine Chapter-2 Page 69

Table- 2.1Zolmitriptan impurities details S. No Impurity structure Chemical name Molecular weight Impurity-1 (S)-4-({3-[2- (dimethylamino)ethyl]-1h- indol-5-yl}methyl)- oxazolidin-2-one N-Oxide 303.36 Impurity-2 (S)-4-({3-[2- (dimethylamino)ethyl]-1h- indol-5-yl}methyl)- oxazolidin-2-one 273.33 2.2. REVIEW OF LITERATURE A gradient, reversed-phase liquid chromatographic (RP-LC) assay method was developed for the quantitative determination of Zolmitriptan, used to treat severe migraine headaches. The developed method is also applicable for the related substances determination in bulk drugs. The Chromatographic separation was achieved on a waters Xterra RP18, 250mm 4.6 mm, I.D, 5.0µm column. The gradient LC method employs solutions A and B as mobile phase. The solution a contains a mixture of phosphate buffer (ph 9.85) methanol acetonitrile (702010, v/v/v) and solution B contains a mixture of phosphate buffer( ph 9.85) acetonitrile (3070). The flow rate was 1.0 ml/min and the detection wavelength was 225 nm. In the developed HPLC method, the resolution between Zolmitriptan and its potential impurities, namely impurity- 1, impurity-2 and impurity-3 was found to be greater than 3. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in alkaline medium and oxidative stress conditions. Degradation product formed Chapter-2 Page 70

during base hydrolysis was found to be impurity-3. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.5%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness. The Chromatographic column used was a waters Xterra RP18 250mm 4.6mm column with 5µm particles. The gradient LC method employs solutions A and B as mobile phase. The solution A contains a mixture of 10mM ammonium di hydrogen ortho phosphate, ph adjusted to 9.85 using ammonia solution (buffer) methanol acetonitrile (70 20 10, v/v/v) and solution B contains a mixture of buffer acetonitrile (3070, v/v). The flow rate of the mobile phase was 1.0 ml/min. The HPLC gradient program was set as time/% solution B 0/0, 10/0, 30/55, 35/55 and 36/0 with a post run time of 10 min. The column temperature was maintained at 30 C and the detection was monitored at a wavelength of 225 nm. The injection volume was 10µL. A mixture of water acetonitrile (11) was used as a diluent. A new, accurate and reliable chiral HPLC method was developed for the determination of Zolmitriptan, (4S)-4-[[3-[2-(dimethylamino)ethyl]-1H-indol-5-yl] methyl]-2-oxazolidinone an antimigraine agent and its potential impurities namely (4R)-4-[[3-[2-(dimethylamino)ethyl]- 1H-indol-5-yl] methyl]-2-oxazolidinone [(R)- enantiomer] and (4S)-4-(4-aminobenzyl)-2-oxazolidinone (impurity-1) in pharmaceutical formulations and in bulk drugs. HPLC separation was carried out by normal phase Chromatography with a mobile phase composed of hexane isopropanol methanol diethylamine in the ratio (75 10 15 0.1, v/v/v/v) pumped at a flow rate of 1.0 ml/min on a Chiralpak AD-H column. Zolmitriptan and its potential impurities were baseline resolved in the optimized method. The presence of diethylamine in the mobile phase has played a key role in achieving Chromatographic resolution between the enantiomers and also in enhancing Chromatographic efficiency. The developed method was also found to be selective under exposed conditions UV light and 60 C. The developed method was completely validated and proved to be robust. The values of the limit of detection (LOD) and limit of quantification (LOQ) of (R)-enantiomer and impurity-1 were 100, 250 Chapter-2 Page 71

ng/ml and 30, 1000 ng/ml, respectively, for 10 _l injection volume. The validated method yielded good results regarding selectivity, linearity, precision, accuracy and ruggedness. Zolmitriptan sample solution and mobile phase are found to be stable for at least 24 h. The proposed method was found to be suitable and accurate for the quantitative determination of Zolmitriptan and its impurities namely (R)- enantiomer and impurity-1 in bulk drugs and commercial formulations. The Chromatographic conditions were optimized using a amylose based chiral stationary phase Chiralpak AD-H (250mm 4.6mm,I.D., 5.0µm, Daicel make) which was safeguarded with a 1 cm long guard column. The mobile phase was hexane isopropanol methanol diethylamine (7510150.1, v/v/v/v). The flow rate was set at 1.0 ml/min. The column was maintained at 25 C and the detection was carried out at a wavelength of 225 nm. The injection volume was 10µL. Cellulose based chiral stationary phases Chiralcel OD-H and Chiralcel OJ-H (Daicel make) were also employed during method development. All calculations concerning the quantitative analysis were performed with external standardization by measurement of peak areas. A simple, rapid and sensitive High-Performance Lliquid Chromatographic (HPLC) method has been developed to quantify Zolmitriptan in plasma using an isocratic system with fluorescence detection. The method included a single-step liquid liquid extraction with methyl tertiary butyl ether. HPLC separation was carried out by reversed phase Chromatography with a mobile phase composed of 0.05% (v/v) triethylamine in water(adjusting to ph 2.75 with 85% phosphoric acid) and acetonitrile (928, v/v), pumped at flow rate of 1.5 ml/min. Fluorescence detection was performed at 225 nm (excitation) and 360 nm (emission). The calibration curve for Zolmitriptan was linear from 0.2 to 40 ng/ml. The validation method yielded good results regarding linearity, precision, accuracy, specificity and recoveries. The values of the limit of detection (LOD) and limit of quantification, respectively. The method was sensitive, simple and repeatable enough to be used in pharmacokinetic studies. Analyses were performed on an inertsil ODS-3 reverse column (5.0µm column, 4.6mm 200mm, I.D.) purchased from GL Sciences Inc. Chapter-2 Page 72

(Japan). The mobile phase was composed of 0.05% (v/v) triethylamine in water (adjusting to ph 2.75 with 85% phosphoric acid) and acetonitrile (928, v/v). The flow rate was set at 1.5 ml/min, and the total run time was 7 min. The column was maintained at 40 C. Fluorescence detection was (LOQ) were 20 and 40 pg/ml. (Picograms per milliliter) performed at an excitation wavelength of 225 nm and an emission wavelength of 360 nm. 2.3. OBJECTIVE To best of our knowledge, all HPLC reported methods are chiral and reverse phase methods for the determination of Zolmitriptan and its impurities, for impurity-1 and impurity -2, no single method reported for its determination in Zolmitriptan. The present research work objective is to develop and single method for new stability indicating RP-HPLC method for both impurities determination in Zolmitriptan. 2.4. MATERIALS AND METHODS 2.4.1. Reagents & Chemicals a. Water HPLC grade b. Acetonitrile HPLC grade Merck c. Methanol Merck d. Dipotassium hydrogen orthophosphate Merck 2.4.2. Drug Substances Zolmitriptan, impurity-1 and impurity-2 are gift samples received from M/S Aurobindo Pharma Ltd, Hyderabad(A.P), India. 2.4.3. 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 instrument. All the components are controlled with Empower2 software. Chapter-2 Page 73

2.4.4. 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 dipotassium hydrogen phoshate salt, acetonitrile and methanol with simple gradient program. 2.4.4.1. Wave length Selection The UV spectrums were generated for Zolmitriptan, impurity-1and impurity- 2 using with Photo diode array detector (PDA). Zolmitriptan and its impurities were found to have varying absorption maxima over a range of wavelength. But it was found that at about 225 nm, Zolmitriptan and its impurities were found to have optimum UV absorption. Therefore, 225nm was selected for the study and quantification of Zolmitriptan and it s related impurities. Figure-2.2 UV Spectrum of Zolmitriptan. Figure-2.3 UV Spectrum of impurity-1. Figure-2.4 UV Spectrum of impurity-2. Chapter-2 Page 74

2.4.4.2. Selection of mobile phase and stationary phase Zolmitriptan, impurity-1 and impurity-2 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 Zolmitriptan and impurities. i.e., selection of the mobile phase and column. 2.4.4.3. Selection of Mobile phase Impurity-1 and impurity-2 impurities were coeluted using with different mobile phases. Zolmitriptan is triptan derivative and the impurities of Zolmitriptan were having wide range of polarities and the separation of these impurities mainly depends on the column stationary phase. A gradient method was mobile phase of buffer is 0.02M Ammonium dihydrogen phosphate in water ph adjusted to 9.85 and acetonitrile and methanol were suitable for the separation of Zolmitriptan and its related substances. Mobile phase was degassed and filtered through 0.22 µm millipore filter paper. 2.4.4.4. Selection of stationary phase Separation was achieved with Waters Xterra RP18 250 x 4.6 mm I.D., 5.0µm column. Different stationary phases were studies for the separation of Zolmitriptan such as C8, Phenyl, and Cyano using the mobile phase specified. The experimentation was started using Xterra RP18 250 X 4.6 mm I.D., and 5.0µm column. Trail-1 The complete experiment details are as follows. Column Buffer Mobile phase-a Xterra RP18, 250 X 4.6 mm I.D., 5.0µm column Di potassium hydrogen ortho phosphate, ph adjusted to 9.85 using ammonia solution Prepare a filtered and degassed mixture of buffer Chapter-2 Page 75

Mobile phase-b Prepare a filtered and degassed mixture of buffer acetonitrile in the ratio of 30 70 Wavelength 225 nm Flow rate 1.2 ml/ min Oven temperature 35 C Diluent Mixture of water and acetonitrile in the ratio of 1 1 Elution Injection volume Gradient programme Gradient 10µL Mobile Mobile Time in min Phase A (%) phase B (%) 0 70 30 10 70 30 30 0 100 40 0 100 42 70 30 50 70 30 Figure-2.5Typical HPLC Chromatogram of Zolmitriptan using Xterra RP18, 250 X 4.6 mm, I.D., 5.0 µm column and trail-1method conditions. Chapter-2 Page 76

Observation Separation observed between impurity-1 & impurity-2. But Peak shapes are not good. After Zolmitriptan main peak base line is drifting upper side. Conclusion Method needs to modify for better peak shape and base line. Trail-2 The complete experiment details are as follows. Column Buffer Mobile phase-a Mobile phase-b Xterra RP18, 250 X 4.6 mm, I.D., 5.0µm column 10 mm dipotassium hydrogen ortho phosphate, ph adjusted to 9.85 using ammonia solution Prepare a filtered and degassed mixture of buffer Acetonitrile Wavelength 225 nm Flow rate 1.2 ml/ min Oven temperature 35 C Diluent Mixture of water and acetonitrile in the ratio of 1 1 Elution Injection volume Gradient 10µL Gradient programme Time in min Mobile Mobile Phase A (%) Phase B (%) 0 60 40 10 60 40 30 0 100 40 0 100 42 60 40 50 60 40 Chapter-2 Page 77

Figure-2.6 Typical HPLC Chromatogram of Zolmitriptan using Xterra RP18, 250 X 4.6 mm, I.D., 5.0µm column and trail-2 method conditions. Observation Low Separation observed between impurity-1& impurity-2. But Peak shapes are not good for all peaks. After Zolmitriptan main peak base line is drifting upper side. All peaks are eluting before 7 min. Conclusion Method needs to modify for getting to reduce baseline noise. Trail-3 The complete experiment details are as follows. Column Buffer Mobile phase-a Mobile phase-b Xterra RP18, 250 X 4.6 mm, I.D., 5.0µm column 10 mm ammonium di hydrogen ortho phosphate, ph adjusted to 9.85 using ammonia solution Prepare a filtered and degassed mixture of buffer and methanol in the ratio of 9010 Mixture of acetonitrile buffer methanol in the ratio of 70246 Wavelength 225 nm Flow rate 1.2 ml/ min Oven temperature 40 C Diluent Mixture of water and acetonitrile in the ratio of 1 1 Chapter-2 Page 78

Elution Injection volume Gradient programme Gradient 10µL Time in min Mobile Mobile phase A (%) phase B (%) 0 90 10 10 75 25 30 10 90 40 10 90 42 90 10 55 90 10 Figure-2.7 Typical HPLC Chromatogram of Zolmitriptan using Xterra RP18, 250 X 4.6 mm, I.D., 5.0µm column and trail-3 method conditions. Observation Separation observed between Impurity-1& Impurity-2. Sharp peak shapes are observed. After Zolmitriptan main peak base line noise observed. Conclusion Method needs to modify for getting to reduce baseline noise. Chapter-2 Page 79

Trail-4 The complete experiment details are as follows. Column Buffer Mobile phase-a Mobile phase-b Xterra RP18, 250 X 4.6 mm, I.D., 5.0µm column 10 mm ammonium di hydrogen ortho phosphate, ph adjusted to 9.85 using ammonia solution Prepare a filtered and degassed mixture of buffer and methanol in the ratio of 9010 Mixture of acetonitrile water in the ratio of 7030. Wavelength 225 nm Flow rate 1.2 ml/ min Oven temperature 40 C Diluent Elution Injection volume Gradient programme Mixture of mobile phase-a mobile phase-b in the of ratio (11) Gradient 10µL Time in min Mobile Mobile phase A (%) phase B (%) 0 90 10 10 90 10 20 35 90 30 35 90 33 90 10 36 90 10 Chapter-2 Page 80

Figure-2.8 Typical HPLC Chromatogram of Zolmitriptan using Xterra RP18, 250 X 4.6 mm, I.D., 5.0µm column and trail-4 method conditions. Observation Separation observed between impurity-1& impurity-2. But Peak shapes are not good for all peaks. After Zolmitriptan main peak more blank peaks observed. 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 that impurity-1, impurity-2 and Zolmitriptan were well separated from each other in column Waters Xterra RP18, 250 X 4.6 mm I.D., 5.0µm column. 2.4.5. Optimized method Column Xterra RP18, 250 X 4.6 mm, I.D., 5.0µm column Buffer preparation 0.02M dipotassium hydrogen phoshate in water ph adjusted to 9.85 +0.02 with KOH Solution Mobile phase-a Mix buffer and acetonitrile in the ratio of 9010 Mobile phase-b Acetonitrile buffer methanol in the ratio of 70246 Sample preparation 5 mg in 10 ml of diluent Wavelength 225 nm Flow 1.2 ml/min Oven temperature 40 C Injection volume 10 µl Chapter-2 Page 81

Diluent Elution Gradient programme Water and acetonitrile(11) Gradient Mobile phase A Mobile phase B Time in min (%) (%) 0 95 5 10 95 5 12 90 10 30 60 40 40 60 40 47 40 60 55 40 60 57 95 5 65 95 5 Preparation of Sample solution Weighed about 5 mg of sample into 10 ml volumetric flask, dissolved and diluted to volume with diluent. Preparation of standard solution Weighed 5 mg of sample into 10mL volumetric flask, dissolved and diluted to volume with diluent. Preparation of Impurity-1 stock solution Transferred 5 mg of impurity-1 into 10mL volumetric flask, dissolved and diluted to volume with diluent. Preparation of Impurity-2 stock solution Transferred 5 mg of impurity-2 into 10mL volumetric flask, dissolved and diluted to volume with diluent. Preparation of system suitability solution Weighed about 5mg of Zolmitriptan standard into 10 ml volumetric flask and 15µL of impurity-1 stock solution and impurity-2 stock solution dissolved and diluted to volume with diluent. Procedure After equbilrated the column, separately injected 10µL of diluent as a blank, system suitability solution, 10µL of standard solution and test solution in the Liquid Chromatograph. Eliminate peaks due to the blank. System suitability criteria The resolution between impurity-1 and Zolmitriptan from system suitability solution should be not less than 10.0. Chapter-2 Page 82

The tailing factor for Zolmitriptan should be not more than 1.5. The number of theoretical plates for Zolmitriptan should be not less than 50000. Table- 2.2 Specification S. No Name of the impurity Specification 01 Impurity-1 Not more than 0.15% 02 Impurity-2 Not more than 0.15% 03 Any other impurity Not more than 0.10% 04 Total impurities Not more than 0.50% Calculation calculate the impurity using below formula Known impurities % area obtained for known impurity /RRF Total impurities % known impurities + % other unknown impurities calculated by area normalization. RRF for Impurity-1 0.49 RRF for Impurity-2 0.98 Figure- 2.9 A typical HPLC Chromatogram of diluent. Chapter-2 Page 83

Figure-2.10 A typical HPLC Chromatogram of system suitability solution. Figure 2.11 A typical HPLC Chromatogram of Zolmitriptan sample. Conclusion Based on the above study, the below mentioned HPLC parameters were chosen for the separation, quantification of Impurity-1, Impurity- 2, and Zolmitriptan. Chapter-2 Page 84

2.5. RESULTS AND DISCUSSION 2.5.1. Method validation Analytical method validation was performed as per ICH (15-17) and USFDA guidelines with specificity, precision, accuracy, linearity, limit of detection, limit of quantification, ruggedness and robustness. 2.5.1.1. Related substances by HPLC 2.5.1.2. System suitability a) Preparation of impurity-1 stock solution Transferred 5mg of impurity-1 into 10 ml volumetric flask, dissolved and diluted to volume with diluent. b) Preparation of impurity-2 stock solution Transferred 5mg of impurity-2 into 10 ml volumetric flask, dissolved and diluted to volume with diluent. c) Preparation of Sample solution Weighed about 5mg of sample into 10mL volumetric flask, dissolved and diluted to volume with diluent. d) Preparation of sample + all impurities spiked About 5 mg of sample into 10 ml volumetric flask, dissolved in 5 ml of diluent and added 10µL of each impurity stock solution 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 each impurity. Results and discussion Under optimized Chromatographic conditions, impurity-1, impurity-2, and Zolmitriptan, were separated well, retention times being about 17.9, 18.5, and 26.3 min, respectively. The system suitability results are given in table 2.3. Table- 2.3 System suitability results S. No Name Retention Relative Resolution, Theoretical Tailing time retention time (Rs) plates (N) factor (min) (min) (T) 01 Impurity-1 7.49 0.28 --- --------- ------ 02 Impurity-2 18.5 0.70 ---- -------- ------ 03 Zolmitriptan 26.3 1.00 26.85 124034 0.86 Chapter-2 Page 85

2.5.1.3. Specificity a) Acid hydrolysis Dissolved 50mg of sample in 50mL of 5N HCl solution and kept for 24 hours at 60 C with continuous stirring and analysed after 24 hours. Observation Zolmitriptan sample is stable under acid hydrolysis. b) Base hydrolysis Dissolved 50mg of sample in 50mL of 0.1N NaOH solution and kept for 8 hours at 60 C with continuous stirring and analysed after 8 hours. Observation Zolmitriptan was degraded under base hydrolysis. c) Oxidation degradation Dissolved 50mg of sample in 50mL of 0.01% Peroxide solution and kept for 3 hours at 60 C with continuous stirring and analysed after 1 hour. Observation Zolmitriptan was degraded to under peroxide solution. d) Thermal degradation About 1gm of Zolmitriptan sample is taken and kept under thermal condition i.e., at 105 C for 7days and sample collected after 48 hours and sample analyzed. Observation Zolmitriptan sample is stable under thermal condition. e) Photo degradation About 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 Zolmitriptan sample is stable under photo condition. f) Water hydrolysis Dissolved 50mg of sample in 100mL of water and kept for 24 hours at 70 C with continuous stirring and injected after 24 hours. Observation Zolmitriptan was not degraded to under water hydrolysis. Conclusion Zolmitriptan samples are stable in thermal, photo degradation, acid hydrolysis and water hydrolysis. Zolmitriptan was degraded in oxidation solution and base hydrolysis. All samples are analysed and found that degradation peaks are separated from known impurities and Zolmitriptan. Peak purity were established with PDA detector and proved that Zolmitriptan peak is pure in all above conditions. The studies are summarized in table 2.4. Chapter-2 Page 86

Table- 2.4 Zolmitriptan degradation data Stressed condition Time (hrs) % Purity Acid hydrolysis 24 97.75 Base hydrolysis 8 53.13 Oxidation degradation 3 90.00 Thermal degradation 7 x 24 99.87 Photo degradation 2 x 24 99.85 Water hydrolysis 24 99.90 Figure- 2.12 A typical HPLC Chromatogram of acid degradation sample. Figure- 2.13 A typical HPLC Chromatogram of base degradation sample. Chapter-2 Page 87

Figure- 2.14 A typical HPLC Chromatogram of oxidation degradation sample. Figure- 2.15 A typical HPLC Chromatogram of thermal degradation sample. Figure- 2.16 A typical HPLC Chromatogram of photo degradation sample. Chapter-2 Page 88

Figure- 2.17 A typical HPLC Chromatogram of water hydrolysis degradation sample. 2.5.1.4. Limit of Detection and Limit of Quantification a) LOD/LOQ solution-1 preparation (0.05%) Transferred about 5µL of each impurity stock solutions into 10mL volumetric flask, dissolved and diluted to volume with diluent. b) LOQ solution-2 preparation Transferred 4.0µL of impurity-1, and 2.0µL of impurity-2, stock solutions into 10 ml volumetric flask, dissolved and diluted to volume with diluent. c) LOD solution-1 preparation Transferred 3.3 ml of above LOQ solution-2 stock solutions into 10 ml 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 impurity -1, and impurity-2 were found to be 0.005 % and 0.007 % respectively. The LOQ for impurity -1, and impurity-2 were found to be 0.015 % and 0.02 % respectively. The results are summarized in the below table-2.5. Chapter-2 Page 89

Table- 2.5 Limit of detection and Limit of Quantification data Conc Impurity -1 Impurity -2 LOD 0.005 0.007 LOQ 0.015 0.02 2.5.1.5. Precision and accuracy at Limit of Quantification level a) Solution preparation 40µL of impurity-1 and 20µL of impurity-2 transferred into 100 ml volumetric flask, containing 50 ml of diluent dissolved and diluted to volume with diluent. Prepared six times the solution as mentioned above and injected all the above solutions each preparation once, calculated the % RSD for six preparations for each impurity. Accuracy b) Sample + All impurities Solution preparation 50mg of sample, transferred into 100mL volumetric flask, dissolved in 50mL of diluent and added 40µL of impurity-1, and 20µL of impurity-2 dissolved and diluted to volume with diluent. c) Sample solution preparation About 50mg of sample transferred into 100mL volumetric flask, dissolved and diluted to volume with diluent. Prepared three times the solution as mentioned above and injected each preparation once and calculated the % recovery for each impurity at Limit of Quantification level. Conclusion The repeatability and recovery at the LOQ concentrations for impurity-1, and impurity-2 were 2.37%, 2.8 % and 94.72%, 95.87% respectively. The results are summarized in the table 2.6. Chapter-2 Page 90

Table- 2.6 Precision and accuracy at Limit of Quantification level data S. No Impurity % RSD (n=6) % Recovery (n=3) 1 Impurity-1 2.37 94.72 2 Impurity-2 2.8 95.87 2.5.1.6. Linearity a) Linearity solution-1 (0.015%, 0.02%) Transferred 1.5µL impurity-1 and 2 µl impurity-2 into 10mL volumetric flask, containing 5mL of diluent dissolved and diluted to volume with diluent. b) Linearity solution-2(0.0375%) 3.75µL of each impurity transferred into 10mL volumetric flask, containing 5 ml of diluent dissolved and diluted to volume with diluent. c) Linearity solution-3(0.075%) 7.5µL of each impurity transferred into 10mL volumetric flask, containing 5mL of diluent dissolved and diluted to volume with diluent. d) Linearity solution-4(0.1125%) 11.25µL of each impurity transferred into 10mL volumetric flask, containing 5mL of diluent dissolved and diluted to volume with diluent. e) Linearity solution-5 (0.15%) 15µL of each impurity transferred into 10mL volumetric flask, containing 5mL of diluent dissolved and diluted to volume with diluent. f) Linearity solution-6 (0.1875%) 18.75µL of each impurity transferred into 10mL volumetric flask, containing 5mL of diluent dissolved and diluted to volume with diluent. g) Linearity solution-6 (0.225%) 22.5µL of each impurity transferred into 10mL volumetric flask, containing 5mL of diluent dissolved and diluted to volume with diluent. Injected all above solutions each preparation once and calculated the Linearity parameters i.e. correlation coefficient, slope and intercept for each impurity. Chapter-2 Page 91

Conclusion Linearity established for impurity-1 and impurity-2 at 0.015%, 0.0375%, 0.075%, 0.1125%, 0.15%, 0.1875% and 0.225% the correlation coefficient (r) are more than 0.99. The above result reveal that method is linear results are summarized in purity wise. Table- 2.7 Zolmitriptan impurity-1 linearity data S. No Level (%) Concentration (%) Area of impurity-1 1 LOQ 0.015 3.84676 2 25 0.0375 9.50341 3 50 0.075 18.52881 4 75 0.1125 27.49278 5 100 0.15 36.69261 6 125 0.1875 46.31791 7 150 0.225 55.23372 Correlation coefficient(r) 1.0OO Slope 244.73 Y-Intercept 0.174 % Y-Intercept 0.47 Chapter-2 Page 92

Figure- 2.18 Zolmitriptan impurity-1 linearity graph. Table- 2.8 Zolmitriptan impurity-2 linearity data S. No Level (%) Concentration (%) Area of impurity-2 1 LOQ 0.02 2.81356 2 25 0.0375 5.56316 3 50 0.075 11.09347 4 75 0.1125 16.50863 5 100 0.15 22.1304 6 125 0.1875 27.92508 7 150 0.225 34.13726 Correlation coefficient(r) 0.9998 Slope 151.28 Y-Intercept -0.284 % Y-Intercept -1.28 Chapter-2 Page 93

Figure- 2.19 Zolmitriptan impurity-2 graph. 2.5.1.7. Accuracy a) Accuracy solution-1 preparation (LOQ) Weighed about 5mg of sample into 10mL volumetric flask, dissolved in 5 ml of diluent and added 1.5µL and 2 µl of each impurity stock solution, dissolved and diluted to volume with diluent. Three solutions prepared as mentioned above. b) Accuracy solution-2 preparation- (50%) 5 mg of sample, transferred into 10mL volumetric flask, dissolved in 5mL of diluent and added 7.5µL of each impurity stock solution, dissolved and diluted to volume with diluent. Three solutions prepared as mentioned above. c) Accuracy solution-3 preparation (100%) Weighed accurately 5 mg of sample into 10 ml volumetric flask, dissolved in 5 ml of diluent and added 15µL of each impurity stock solution, dissolved and diluted to volume with diluent. Three solutions prepared as mentioned above. d) Accuracy solution-4 preparation (150%) Transferred 5mg of sample into 10mL volumetric flask, dissolved in 5 ml of diluent and added 22.5µL of each impurity stock solution, dissolved and diluted to volume with diluent. Three solutions prepared as mentioned above. Chapter-2 Page 94

Injected each above preparation once and calculated the recovery for each impurity at each level. Conclusion The percentage % recovery of all impurities in Zolmitriptan samples is shown in below table - 2.9. Table- 2.9 % Recovery accuracy data Concentration Impurity-1(%) Impurity-2(%) LOQ 94.72 95.87 50% 101.65 101.34 100% 99.47 101.15 150% 96.27 102.95 2.5.1.8. Precision a) Sample preparation Weighed 5mg of sample, transferred into 10mL volumetric flask, dissolved and diluted to volume with diluent. b) Sample + LOQ Solution spiked preparation Weighed accurately 5 mg of sample into 10 ml volumetric flask, dissolved in 5 ml of diluent added 10µL of each 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 each impurity. Conclusion The precision of the related substance method was checked by injecting six individual Preparations of Zolmitriptan spiked with 0.015% and 0.02% of impurity- 1and impurity-2. The % RSD of the area for each of impurity-1 and impurity-2 were calculated. The results were summarized in table 2.10. Chapter-2 Page 95

Table- 2.10 Precision data S. No Preparation Impurity-1 area Impurity-2 area 1 1 3.87906 2.82410 2 2 3.86330 2.86965 3 3 3.94440 3.00782 4 4 3.74790 2.94437 5 5 3.69799 2.81110 6 6 3.85330 2.97090 Average 3.83099 2.90466 Standard deviation 0.0909 0.08 % RSD 2.37 2.80 2.5.1.9. Robustness Flow variation a) Sample solution preparation Weighed about 5mg of sample into 10mL volumetric flask, dissolved and diluted to volume with diluent. b) Sample + 0.15% spiked preparation Weighed accurately 5mg of sample into 10mL volumetric flask, dissolved in 5 ml of diluent added 10µL of each impurity stock solution dissolved and diluted to volume with diluent. Injected the above sample solution at flow rates 1.0mL/min and at 1.4mL/min and observed the system suitability parameters and impurities relative retention times and compared with 1.2mL/min results. Temperature variation a) Sample solution preparation Weighed accurately 5mg of sample into 10mL volumetric flask, dissolved and diluted to volume with diluent. b) Sample + 0.15% spiked preparation Weighed accurately 5mg of sample into 10mL volumetric flask, dissolved in 5mL of diluent added 10µL of each impurity stock solution dissolved and diluted to volume with diluent Chapter-2 Page 96

Injected the above sample solution at temperatures 35 C and at 45 C and observed the system suitability parameters and impurities relative retention times and compared with 40 C results. ph variation a) Sample solution preparation 5mg of sample, transferred into 10mL volumetric flask, dissolved and diluted to volume with diluent. b) Sample + 0.15% spiked preparation 5mg of sample, transferred into 10mL volumetric flask, dissolved in 5mL of diluent added 10µL of each impurity stock solution dissolved and diluted to volume with diluent. Injected the above sample solution at ph 9.65 and at 10.05 and observed the system suitability parameters and impurities relative retention times and compared with 9.85 results. Conclusion The results are summarized in the table -2.11. Table- 2.11 Robustness data Parameter 35 C 45 C 1.2 ml/min Theoretical plates for Zolmitriptan Tailing factor for Zolmitriptan Resolution between impurity-2 and Zolmitriptan 1.4 ml/min ph at 9.65 ph at 10.05 140218 132720 141200 124853 127114 147114 0.925 0.946 0.944 0.966 0.857 0.993 24.32 24.76 23.45 23.73 25.79 22.34 2.5.1.10. Solution stability Sample solution preparation Weighed about 5mg of sample into 10mL volumetric flask, dissolved and diluted to volume with diluent. Chapter-2 Page 97

Injected the solution for 0hrs(Initial sample data), 12hrs, 24 hrs and 48 hrs and performed the impurity content. Conclusion Impurity-1, and impurity-2 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 phase stability experimental 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 given below. Table- 2.12 Solution stability data Duration Impurity-1 (%) Impurity-2 (%) Any other impurity (%) Total impurities (%) SS Initial 0.16 0.13 0.05 0.34 After 12 hrs 0.16 0.13 0.05 0.34 After 24 hrs 0.15 0.14 0.05 0.34 After 48 hrs 0.16 0.13 0.05 0.34 Note SS Initial- Sample solution initial. Table- 2.13 Mobile phase stability data Duration Impurity-1 (%) Impurity-2 (%) Any other impurity (%) Total impurities (%) SS Initial 0.16 0.13 0.05 0.34 After 12 hrs 0.16 0.13 0.05 0.34 After 24 hrs 0.15 0.14 0.05 0.34 After 48 hrs 0.16 0.13 0.05 0.34 2.5.1.11. Batch analysis Using the above validated method, Zolmitriptan sample was analyzed and the data is furnished in table 2.14. Chapter-2 Page 98

Table- 2.14 Batch analysis data Lot Number Impurity-1 Impurity-2 Any other impurity Total impurities 001 0.16 0.13 0.05 0.34 2.6. SUMMARY AND CONCLUSION 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 Zolmitriptan 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 Zolmitriptan was observed under basic 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 products formed under stress conditions was achieved on a Xterra RP18, 250 X4.6 mm, I.D., 5.0µm column, using buffer 0.02M dipotassium hydrogen phoshate in water ph adjusted to 9.85 with KOH Solution. Using mixed mobile phase-a(buffer acetonitrile in the ratio of 9010) and mobilephase - B(acetonitrile buffer methanol in the ratio of 70246). The developed HPLC method was validated with respect to linearity, accuracy, precision, specificity and robustness. The developed HPLC method to determine the related substances can be used to evaluate the quality of regular production samples and stability samples of Zolmitriptan. This study provides the simple and comprehensive Liquid Chromatography method to evaluate Zolmitriptan, process related impurities and impurities originated from forced degradation studies to the best of our knowledge, the validated stability indicating Liquid Chromatography method which separates all the impurities disclosed in this investigation was not studied elsewhere. Chapter-2 Page 99

2.7. REFERENCES 1. A stability indicating LC method for Zolmitriptan,J Pharm Biomed Anal.2005; 39503 9Mallikarjuna Rao B, Srinivasu MK, Sridhar G, Rajender Kumar P, Chandra Sekhar KB, Aminul I. 2. A validated chiral LC method for the determination of Zolmitriptan and its potential Impurities J Pharm Biomed Anal 37 (2005) 453 460, M.K. Srinivasu, B. Mallikarjuna Rao, G. Sridhar, P. Rajender Kumar, K.B. Chandrasekhar, Aminul Islam. 3. High-performance Liquid Chromatographic analysis of Zolmitriptan in human plasma using fluorescence detection, J Pharm Biomed Anal 35 (2004) 639 645,Jun Chen, Xin- Guo. Jiang, Wen-Ming Jiang, Ni Mei, Xiao-Ling Gao, Qi- Zhi Zhang. 4. Oldman AD, Smith LA, McQuay HJ, Moore RA. A systematic review of treatments for acute migraine. Pain. 2002;97247 57. [PubMed] 5. Yates R, Nairn K, Dixon R, Seaber E. Preliminary studies of the pharmacokinetics and tolerability of Zolmitriptan nasal spray in healthy volunteers. J Clin Pharmacol. 2002;421237 43. [PubMed] 6. Yates R, Nairn K, Dixon R, Kemp JV, Dane AL. Pharmacokinetics, dose proportionality and tolerability of single and repeat doses of a nasal spray formulation of zolmitriptan in healthy volunteers. J Clin Pharmacol. 2002;421244 50. [PubMed] 7. Chen X, Liu D, Luan Y, Jin F, Zhong D. Determination of Zolmitriptan in human plasma by liquid chromatography-tandem mass spectrometry method Application to a pharmacokinetic study. J Chromatogr B. 2006;83230 5. 8. Yu L, Wen Y, Song Z, Mu D, Su L, Yang Y. Determination of Zolmitriptan and its pharmacokinetics in human plasma after intranasal administration using LC-MS. Fenxi Ceshi Xuebao. 2006;2567 70. 9. He H, Meng H, Zhou Y, Li B, Li X. Determination of Zolmitriptan in human plasma by RP-HPLC with liquid-liquid extraction. Yaowu Fenxi Zazhi. 2005;25323 5. Chapter-2 Page 100

10. Yao J, Qu Y, Zhao X, Hu L, Zhu R, Li H, et al. Determination of Zolmitriptan in human plasma by high performance liquid chromatography-electrospray mass spectrometry and study on its pharmacokinetics. J Chinese Pharm Sci. 2005;1425 8. 11. Zang Z, Xu F, Tian Y, Li W, Mao G. Quantification of Zolmitriptan in plasma by high-performance liquid chromatography-electrospray ionization mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2004;813227 33. 12. Clement EM, Franklin M. Simultaneous measurement of Zolmitriptan and its major metabolites N-desmethylzolmitriptan and Zolmitriptan N-oxide in human plasma by high-performance liquid chromatography with coulometric detection. J Chromatogr B Analyt Technol Biomed Life Sci. 2002;766339 43. 13. Kilic B, Özden T, Toptan S, Özilhan S. Simultaneous LC-MS-MS determination of Zolmitriptan and its active metabolite N-desmethylzolmitriptan in human plasma. Chromatographia. 2007;66129 33. 14. Hu Y, Yao T, Wang X. HPLC determination of Zolmitriptan and its related substances. Zhejiang Daxue Xuebao, Yixueban. 2004;3337 40. 15. Q1A (R2) ICH Harmonized Tripartite Guideline. Geneva, Switzerland 2003, Feb. 16. Q2A. ICH Harmonized Tripartite Guideline. Geneva, Switzerland 1994, Oct. 17. Q2B. ICH Harmonized Tripartite Guideline. Geneva, Switzerland 1996, Nov. Chapter-2 Page 101