CHAPTER 2 SIMULTANEOUS DETRMINATION OF ANASTROZOLE AND TEMOZOLOMIDE IN TEMOZOLOMIDE CAPSULES 20 MG AND ANASTROZOLE TABLETS 1 MG ANALYTICAL METHOD VALIDATION REPORT FOR ASSAY 43
2.1 Introduction Analytical method of assay for Temozolomide capsules 20 mg and Anastrozole tablets 1 mg is developed on reverse phase Fast LC. The assay method for Anastrozole tablets 1 mg and Temozolomide capsules 20 mg has been subjected for validation. All the parameter has been covered during the validation, which are illustrated in the table no.01.the analytical method validation has been performed as per ICH guideline Q2 (R1). This analytical method validation report is prepared and proved capability of analytical method for its specific nature, accuracy, linearity, preciseness and robust nature. 2.2 Label Claim Anastrozole Tablet 1 mg Each Film coated Tablet consisting of Anastrozole.1 mg Temozolomide capsules 20 mg Each Capsule contains consisting of Temozolomide.20 mg 2.3 References Validation references Analytical Method Validation Program as per ICH guideline Q2 (R1) 44
2.4 Analytical method validation program as per ICH guideline Method validation activities has been conducted as per following validation program, Table No. 01: Validation program as per ICH guideline Parameter Assay Specificity + Accuracy + Detection Limit ( LOD ) + Quantitation Limit (LOQ) + Linearity + Range + Precision Injection repeatability + Analysis repeatability + Intermediate precision + Solution Stability Stability of analytical solutions Robustness Influence of variations of test parameters + + - Signifies that these characteristic are not evaluated. + Signifies that these characteristic are evaluated. 45
2.5 Details of the API, standard, sample and placebo used in validation activity Table No. 02 : Details of the API, standard, sample and placebo Sr. No. Name B.No./Lot No. % Purity as is basis Manufactured date 1 Anastrozole API FX8098 99.7% 2 Temozolomide API IW880085 99.8% 3 Anastrozole working standard WS/C/A87/01 99.95 % (on as is basis) NA 4 Temozolomide working standard TS/C/T23/O1 100.00 % (on as is basis) NA 5 6 Anastrozole Tablet 1 mg Temozolomide Capsule 20 mg TANA 81009 NA NA CTZA91003 NA NA 7 2-Azahyooxanthine Iimpurity standard 474.01.09.03 99.3% NA 8 AIC HCL ImpurityStandard TS-22/T01 98.5% NA 9 TMZ II Impurity Standard TS-22/T03 99.2% NA 10 Anastrozole Impurity A Impurity standard TS-21/A5 99.9% NA 11 Anastrozole Impurity B Impurity standard 0098.056-01 98.9% NA 12 Anastrozole Broomo Compound Impurity standard TS/C/A62/01 99.0% NA 46
2.6 Details of chemicals and reagents used for validation activity Table No. 03: List of chemicals and reagents used Sr.No. Name of Chemicals / Reagents Batch / Lot No. Grade Manufactu rer 1 Acetonitrile SF0SF60511 HPLC grade Merck 2 Ammonium Acetate ME18DO580153 A R grade Merck 3 Triethylamine SC0S590651 Synthesis Merck 4 Purified water - HPLC grade Millipore 5 Orthophosphoric acid 0174 7006-5 A R grade Qualigens 6 Methanol 1036/1 7003-6 HPLC grade Qualigens 2.7 Details of instrument and equipment used for validation activity Table No. 04: List of Instruments and equipments used Sr.No. Name of Instrument / Equipment Make Software 1 Ultimate 3000 Dionex Chromeleone 2 Waters Waters Empower 3 Weighing Balance Mettler Toledo XS205 5 ph meter Metrohm 780 ph Meter 7 Water Purifier Millipore Gradient A10 8 Vacuum pump ACMEVAC 15P166 9 Sonicator PCi 10.5L 250/SPL 2.8 Detail of LC columns used for validation activity : 1 Column : Inertsil C8 Dimentions Serial No. : 25 mm x 5-cm, 4.6-µm : ILU9976500341 and ILU9976500768 47
2.9 Method validation parameters for assay by LC method : SYSTEM SETUP A. Chromatographic Conditions Column OvenTemperature: 25 C Mobile Phase A Ammonium acetate buffer (0.1%) Mobile Phase B Acetonitrile Flow Rate: 1.0 ml/min Injection Volume: 10 µl Detection: UV at 215 nm Gradient Program: Time (min.) %A %B 0 95 05 7.0 95 05 12.0 93 07 17.0 50 50 40.0 30 70 45.0 95 05 55.0 95 05 2.9.1 SPECIFICITY AND SYSTEM SUITABILITY Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expeced to present. For this study first upon a 10 μl of blank solution (Acetonitrile: water 50:50) and placebo was injected and ran with the gradient programme. After this 10 μl of Resolution solution was injected followed by Individual Impurity standard solutions and injected standard solutions in 6 replicate and the % relative standard deviation (% R.S.D) of the response peak areas was calculated. Specificity study solution are prepared as follows, Blank solution: Acetonitrile: water 50:50. 48
Preparation of Placebo solution for Anastrozole tablets 1 mg: A Placebo about 0.90 gm was weighedand transferred in to 100 ml volumetric flask equivalent excluding 10 mg of Anastrozole, to this flask added 60 ml of equal volumes of Acetonitrile and water respectively and the mixture was subjected to vigorous shaking for 10 min and diluted up to volume with same solvent. Further diluted 10 ml of these solutions to 100 ml with Acetonitrile: water 50:50.. The solution was filtered through 0.45µ nylon filter before analysis. 10 µl of this solutions were injected in to the HPLC system Preparation of Placebo solution for Temozolomide capsules 20 mg: A Placebo about 0.260 gm was weighedand transferred in to 100 ml volumetric flask equivalent excluding 10 mg of Temozolomide, to this flask added 60 ml of equal volumes of Acetonitrile and water respectively and the mixture was subjected to vigorous shaking for 10 min and diluted up to volume with same solvent. Further diluted 10 ml of these solutions to 100 ml with Acetonitrile: water 50:50. The solution was filtered through 0.45µ nylon filter before analysis. 10 µl of these solutions were injected in to the HPLC system. Preparation of Standard solution Standard stock solution was prepared by dissolving accurately 50 mg Anastrozole and 50 mg Temozolomide in 100 ml of equal volumes of Acetonitrile and water. 2 ml of standard stock solution was diluted to 100 ml with Acetonitrile: water 50:50 to obtain each of 10 µg/ml of Anastrozole and Temozolomide. The solution was filtered through 0.45µ nylon filter before analysis. A series of solutions containing Anastrozole and Temozolomide were prepared with mobile phase A and 10 µl of these solutions were injected in to the HPLC system. Preparation of Resolution solution Transferred 2.0 mg of each of AIC HCL (5amino imidazole-4- carboxamidehydrochloride) impurity, 2-Azahypoxanthine impurity and TMZ II impurity and Anastrozole Bromo compound, Anastrozole impurity A and Anastrozole impurity B dissolved in 200 ml of equal volumes of Acetonitrile: water. Further dilute 1 ml of this solution and 2 ml of standard stock solution into 100 ml of Acetonitrile: water (50:50) 49
Preparation of Impurity solutions for Identification. AIC HCL (5amino imidazole-4- carboxamidehydrochloride) impurity solution for Identification Transferred 2.0 mg of AIC HCL (5amino imidazole-4- carboxamidehydrochloride) impurity dissolved in 200 ml of equal volumes of Acetonitrile: water. Further dilute 1 ml of this solution 100 mlvolumetric flask and diluted to volume with Acetonitrile: water (50:50) 2-Azahypoxanthine impurity solution for Identification Transferred 2.0 mg of 2-Azahypoxanthine impurity dissolved in 200 ml of equal volumes of Acetonitrile: water. Further dilute 1 ml of this solution 100 mlvolumetric flask and diluted to volume with Acetonitrile: water (50:50). TMZ II impurity solution for Identification Transferred 2.0 mg of TMZ II impurity dissolved in 200 ml of equal volumes of Acetonitrile: water. Further dilute 1 ml of this solution 100 mlvolumetric flask and diluted to volume with Acetonitrile: water (50:50). Anastrozole Bromo compound impurity solution for Identification Transferred 2.0 mg of Anastrozole Bromo compound impurity dissolved in 200 ml of equal volumes of Acetonitrile: water. Further dilute 1 ml of this solution 100 ml volumetric flask and diluted to volume with Acetonitrile: water (50:50) Anastrozole Impurity A solution for Identification Transferred 2.0 mg of Anastrozole impurity A dissolved in 200 ml of equal volumes of Acetonitrile: water. Further dilute 1 ml of this solution 100 mlvolumetric flask and diluted to volume with Acetonitrile: water (50:50). Anastrozole Impurity B solution for Identification Transferred 2.0 mg of Anastrozole impurity B dissolved in 200 ml of equal volumes of Acetonitrile: water. Further dilute 1 ml of this solution 100 mlvolumetric flask and diluted to volume with Acetonitrile: water (50:50) 50
Specimen chromatograms for specificity are as follows : Figure No. 4 : Blank solution chromatogram in specificity Figure No.5 : Standard solution chromatogram in specificity Figure No. 6 : Resolution solution chromatogram in specificity 51
Figure No. 7 : Placebo Temozolomide chromatogram in specificity Figure No. 8: Placebo Anastrozole chromatogram in specificity. Figure No. 9 : Temozolomide chromatogram for Identification in specificity 52
Figure No. 10 : Anastrozole chromatogram for Identification in specificity Figure No. 11 : 2-Azahypoxanthine impurity chromatogram for Identification in specificity Figure No. 12 : TMZ II Impurity chromatogram for Identification in specificity 53
Figure No. 13 : AIC HCL Impurity chromatogram for Identification in specificity Figure No. 14 : Anastrozole Impurity A chromatogram for Identification in specificity Figure No. 15 : Anastrozole Impurity B chromatogram for Identification in specificity 54
Figure No. 16 : Anastrozole Bromo Compound chromatogram for Identification in specificity 55
Table No. 05 : System suitability of specificity study Sr.No System suitability parameter Results Acceptance criteria 1 2 3 4 5 6 7 8 9 10 Resolution between2- Azahypoxanthine and AIC Impurity Resolution between Temozolomide and TMZ-II impurity Resolution between ANZ Impurity A and Anastrozole Resolution between ANZ impurity B and ANZ Bromo Impurity RSD Anastrozole peak from five replicate Injections of standard. Asymmetry for Anastrozole Theoretical Plates for Anastrozole RSD Temozolomide peak from five replicate Injections of standard. Asymmetry for Temozolomide. Theoretical Plates for Temozolomide 2.00 NLT 2.0. 4.46 NLT 2.0. 7.04 NLT 2.0. 2.24 NLT 2.0. 0.2 NMT 2.0%. 1.9 NLT 2.0. 172812 NLT 3000 0.1 NMT 2.0%. 1.6 NLT 2.0. 7239 NLT 3000 Conclusion : Assay method for Temozolomide capsules 20 mg and Anastrozole tablets 1 mg is said to specific as per assessment of the specificity study. 56
2.9.2 ACCURACY Accuracy was carried out at 50.0%, 100.0% and 150.0% of the target concentration of Anastrozole (10.0 µg per ml) and Temozolomide (10.0 µg per ml).solutions were prepared in triplicate at each level. Accuracy study solution are prepared as follows, For blank solution, standard solution preparation, refer the solution preparation under specificity parameter. For system suitability table refer specificity parameter. Preparation of accuracy solution as per follows : Preparation of solutions for Anastrozole tablets 1 mg: A Placebo about 0.90 gm was weighed and transferred in to 100 ml volumetric flask equivalent excluding 10 mg of Anastrozole, then weighed and added specified quantity of Anastrozole API (quantity as per Table given below) to the same volumetric flask.to this flask added 60 ml of equal volumes of Acetonitrile and water respectively and the mixture was subjected to vigorous shaking for 10 min and diluted up to volume with same solvent. Further diluted 10 ml of these solutions to 100 ml with Acetonitrile: water 50:50.. The solution was filtered through 0.45µ nylon filter before analysis. 10 µl of this solutions were injected in to the HPLC system Preparation of solutions for Temozolomide capsules 20 mg: A Placebo about 0.270 gm was weighedand transferred in to 100 ml volumetric flask equivalent excluding 10 mg of Temozolomide, then weighed and added specified quantity of Anastrozole API (quantity as per Table given below) to the same volumetric flask. to this flask added 60 ml of equal volumes of Acetonitrile and water respectively and the mixture was subjected to vigorous shaking for 10 min and diluted up to volume with same solvent. Further diluted 10 ml of these solutions to 100 ml with Acetonitrile: water 50:50. The solution was filtered through 0.45µ nylon filter before analysis. 10 µl of these solutions were injected in to the HPLC system 57
Table No. 06 : Preparation of accuracy levels as per following table: For Anastrozole First dilution Second Dilution Level No. % Level Prep No. Wt.of Placebo (mg) Wt.of API (mg) Diluted to (ml) Pipetted (ml) Diluted to (ml) Level 1 Level 3 Level 5 50 100 150 1 0.90 25.0 100 2 100 2 0.90 25.0 100 2 100 3 0.90 25.0 100 2 100 1 0.90 50.0 100 2 100 2 0.90 50.0 100 2 100 3 0.90 50.0 100 2 100 1 0.90 75.0 100 2 100 2 0.90 75.0 100 2 100 3 0.90 75.0 100 2 100 For Temozolomide First dilution Second Dilution Level No. % Level Prep No. Wt.of Placebo (mg) Wt.of API (mg) Diluted to (ml) Pipetted (ml) Diluted to (ml) Level 1 Level 3 Level 5 50 100 150 1 0.490 25.0 100 2 100 2 0.490 25.0 100 2 100 3 0.490 25.0 100 2 100 1 0.490 50.0 100 2 100 2 0.490 50.0 100 2 100 3 0.490 50.0 100 2 100 1 0.490 75.0 100 2 100 2 0.490 75.0 100 2 100 3 0.490 75.0 100 2 100 58
Table No. 07 : % Recovery at difference levels for Anastrozole Level No. % Level wrt. working conc. Conentration added in µg per ml Conentration recovered in µg per ml % Recovery Unrounded % Assay Rounded 5.1 5.18 101.55 101.6 1 50 5.1 5.19 101.74 101.7 5.1 5.19 101.73 101.7 10.2 10.24 100.35 100.3 2 100 10.2 10.28 100.75 100.7 10.2 10.24 100.40 100.4 15.3 15.03 98.25 98.3 3 150 15.3 15.11 98.73 98.7 15.3 15.03 98.25 98.3 Mean % Recovery 100.2 Minimum % Recovery 98.3 Maximum % Recovery 101.7 STDev 1.4 RSD 1.4 59
Table No. 08 : % Recovery at difference levels for Temozolomide Level No. % Level wrt. working conc. Conentration added in µg per ml Conentration recovered in µg per ml % Recovery Unrounded % Assay Rounded 4.95 4.95 99.91 99.9 1 50 4.95 4.94 99.77 99.8 4.95 4.96 100.27 100.3 9.9 9.90 99.99 100.0 2 100 9.9 9.90 99.99 100.0 9.9 9.85 99.49 99.5 14.85 14.79 99.63 99.6 3 150 14.85 14.79 99.59 99.6 14.85 14.79 99.63 99.6 Mean % Recovery 99.8 Minimum % Recovery 99.6 Maximum % Recovery 100.3 STDev 0.251 RSD 0.3 60
Specimen chromatograms of accuracy study are given below, Figure No. 17: Anastrozole chromatogram in accuracy at 50% level Figure No. 18 : Anastrozole chromatogram in accuracy at 100% level Figure No. 19 : Anastrozole chromatogram in accuracy at 150% level 61
Figure No. 20 : Temozolomide chromatogram in accuracy at 50% level. Figure No. 21 : Temozolomide chromatogram in accuracy at 100% level Figure No. 22 : Temozolomide chromatogram in accuracy at 150% level 62
Table No. 09 : Mean results of accuracy study for Anastrozole Sr. No. Parameter Results Acceptance criteria 1 Individual % recovery. % Recovery obtained between 98.3 to 100.2 Between 97.0% to 103.0%. 2 Mean % recovery of all recovery levels. 3 RSD of recovery of all five recovery levels. 100.2 Between 98.0% to 102.0%. 1.4 Not more than 2.0%. Table No. 10 : Mean results of accuracy study for Temozolomide Sr. No. Parameter Results Acceptance criteria 1 Individual % recovery. % Recovery obtained between 99.6 to 100.3 Between 97.0% to 103.0%. 2 Mean % recovery of all recovery levels. 3 RSD of recovery of all five recovery levels. 99.8 Between 98.0% to 102.0%. 0.3 Not more than 2.0%. Conclusion: Assay method for Temozolomide capsules 20 mg and Anastrozole tablets 1 mg is found accurate as recovery results and individual recoveries are well within the acceptance criteria. 63
2.9.3 LOD AND LOQ SENSITIVITY The Quantitation limit of an individual analytical procedure is the lowest amount of analyte or known impurity in a sample which can be quantitatively determined with suitable precision and accuracy. The detection limit of an individual analytical procedure is the lowest amount of analyte or known impurities in a sample which can be detect but not quantified. Determination of LOD and LOQ solution : For LOD and LOQ concentration determination, solutions containing Anastrozole and Temozolomide of different concentration injected and detected signal to noise ratio instrumentally. Table No. 11: The Concentrations of LOD and LOQ level experiment. Concentration For LOD Concentration For LOQ Anastrozole 0.092 µg/ ml 0.075 µg/ ml Temozolomide 0.17 µg/ ml 0.16 µg/ ml Table No. 12: Signal to Noise Ratio for Anastrozole Table No. 13: Signal to Noise Ratio for Temozolomide 64
2.9.4 LINEARITY AND RANGE The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. Linearity was carried out with minimum 8 concentration levels in duplicate within a range of LOQ to 150% of the target concentration of analyte by using test sample. Linearity and range of assay method from LOQ to 150.0% of working concentration of anastrozole (Coonc.10.00 µg per ml) and Temozolomide (Coonc.10.00 µg per ml). Evaluation Table No. 14 : Linearity study data for Anastrozole No of Linearity levels % Level wrt. Working conc. Concentraton in ppm Area of Anastrozole 1 0.16 % 0.016 24152 2 10 % 1.011 100016 3 20 % 2.022 200224 4 50 % 5.055 501898 5 75 % 7.583 752937 6 100 % 10.110 1005739 7 125 % 12.638 1253672 8 150 % 15.165 1506657 Table No. 15: Linearity study data for Temozolomide No of Linearity % Level w.r.t. Working conc. Concentration in ppm Area of Temozolomide 1 0.17 % 0.017 3273 2 10 % 1.051 198599 3 20 % 2.102 392978 4 50 % 5.255 990627 5 75 % 7.883 1488360 6 100 % 10.510 1982557 7 125 % 13.138 2480228 8 150 % 15.765 2975332 65
Linearity graph of concentration in µg per ml against area response, plotted on basis of above information is as follows, Figure No. 23 : Anastrozole Linearity plot of concentration in µg per ml against area responce in assay Figure No. 24 : Temozolomide Linearity plot of concentration in µg per ml against area responce in assay Table No. 16: Results of Linearity study S.R.NO. Anastrozole Temozolomide 1 The linear regression equation 997514x + 7103.2 1,984,462.43x - 1,121.41 2 correlation coefficient 0.999 0.999 Conclusion : Assay method for Temozolomide capsules 20 mg and Anastrozole tablets 1 mg is found linear within the specified range as LOQ to 150% of working level of Anastrozole and Temozolomide 66
2.9.5PRECISION : The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Injection repeatability Analysis repeatability Intermediate precision Injection repeatability Injection repeatability is established at the start of each experiment. For blank solution, standard solution preparation, refer the solution preparation under specificity parameter. For system suitability table refer specificity parameter. Analysis repeatability Analysis repeatability for assay carried out with six preparations of assay samples. Precision sample solution : Six sample prepared as per follows, Preparation of sample solution for Anastrozole tablets 1 mg: A sample about 0.90 gm was weighedand transferred in to 100 ml volumetric flask (equivalent to 10 mg of Anastrozole), to this flask added 60 ml of equal volumes of Acetonitrile and water respectively and the mixture was subjected to vigorous shaking for 10 min and diluted up to volume with same solvent. Further diluted 10 ml of these solutions to 100 ml with Acetonitrile: water 50:50.. The solution was filtered through 0.45µ nylon filter before analysis. 10 µl of this solutions were injected in to the HPLC system. Preparation of Sample solution for Temozolomide capsules 20 mg: A sample about 0.270 gm was weighedand transferred in to 100 ml volumetric flask (equivalent 10 mg of Temozolomide), to this flask added 60 ml of equal volumes of Acetonitrile and water respectively and the mixture was subjected to vigorous shaking for 10 min and diluted up to volume with same solvent. Further diluted 10 ml of these solutions to 100 ml with Acetonitrile: water 50:50. The solution was filtered through 0.45µ nylon filter before analysis. 10 µl of these solutions were injected in to the HPLC system. 67
Evaluation Table No. 17: Results of precision study for Anastrozole Lable Claim of Anastrozole % Assay of Anastrozole Sample 1 1.00 99.74 Sample 2 0.99 99.33 Sample 3 0.99 99.49 Sample 4 1.00 100.09 Sample 5 1.01 100.84 Sample 6 1.00 100.39 Mean 99.98 S.D 0.57 %RSD 0.57 Table No. 18 : Results of precision study for Temozolomide Lable Claim of Temozolomide % Assay of Temozolomide Sample 1 19.09 95.44 Sample 2 19.01 95.05 Sample 3 19.04 95.20 Sample 4 19.16 95.78 Sample 5 19.30 96.49 Sample 6 19.21 96.07 Mean 95.67 S.D 0.55 %RSD 0.57 68
Specimen chromatograms are given below, Figure No. 25 : Anastrozole sample chromatogram in Precision study. Figure No. 26 : Temozolomide sample chromatogram in Precision study. Conclusion: Assay method for Temozolomide capsules 20 mg and Anastrozole tablets 1 mg is found precise during analysis repeatability as RSD is well within acceptance criteria. 69
2.9.6 INTERMEDIATE PRECISION Intermediate precision is the precision repeated under difference conditions such as by different analyst, on different day, on different instrument and by using different Fast LC column of same brand. Intermediate Precision is performed by following different conditions, 1. Different analyst 2. Different day 3. Different instrument 4. Different LC column of same dimension and make Intermediate precision study solution are prepared as follows, For this study first 10 μl of blank solution (Acetonitrile: water 50:50) and placebo was injected and ran with the gradient programme. After this 10 μl of Resolution solution was injected followed by Individual Impurity standard solutions and injected standard solutions in 6 replicate and the % relative standard deviation (% R.S.D) of the response peak areas was calculated. Preparation of Placebo solution for Anastrozole tablets 1 mg: A Placebo about 0.90 gm was weighed and transferred in to 100 ml volumetric flask equivalent excluding 10 mg of Anastrozole, to this flask added 60 ml of equal volumes of Acetonitrile and water respectively and the mixture was subjected to vigorous shaking for 10 min and diluted up to volume with same solvent. Further diluted 10 ml of these solutions to 100 ml with Acetonitrile: water 50:50. The solution was filtered through 0.45µ nylon filter before analysis. 10 µl of these solutions were injected in to the HPLC system. Preparation of Placebo solution for Temozolomide capsules 20 mg: A Placebo about 0.270 gm was weighed and transferred in to 100 ml volumetric flask equivalent excluding 10 mg of Temozolomide, to this flask added 60 ml of equal volumes of Acetonitrile and water respectively and the mixture was subjected to vigorous shaking for 10 min and diluted up to volume with same solvent. Further diluted 10 ml of these solutions to 100 ml with Acetonitrile: water 50:50. The solution was filtered through 0.45µ nylon filter before analysis. 10 µl of these solutions were injected in to the HPLC system. 70
Preparation of Standard solution Standard stock solution was prepared by dissolving accurately 50 mg Anastrozole and 50 mg Temozolomide in 100 ml of equal volumes of Acetonitrile and water. 2 ml of standard stock solution was diluted to 100 ml with Acetonitrile: water 50:50 to obtain each of 10 µg/ml of Anastrozole and Temozolomide. The solution was filtered through 0.45µ nylon filter before analysis. and 10 µl of these solutions were injected in to the HPLC system. Preparation of Resolution solution Transferred 2.0 mg of each of AIC HCL (5amino imidazole-4- carboxamidehydrochloride) impurity, 2-Azahypoxanthine impurity and TMZ II impurity and Anastrozole Bromo compound, Anastrozole impurity A and Anastrozole impurity B dissolved in 200 ml of equal volumes of Acetonitrile: water. Further dilute 1 ml of this solution and 2 ml of standard stock solution into 100 ml of Acetonitrile: water (50:50) Preparation of sample solution for Anastrozole tablets 1 mg: A sample about 0.350 gm was weighed and transferred in to 100 ml volumetric flask (equivalent to 10 mg of Anastrozole), to this flask added 60 ml of equal volumes of Acetonitrile and water respectively and the mixture was subjected to vigorous shaking for 10 min and diluted up to volume with same solvent. Further diluted 10 ml of these solutions to 100 ml with Acetonitrile: water 50:50.. The solution was filtered through 0.45µ nylon filter before analysis. 10 µl of this solutions were injected in to the HPLC system. Prepared six sample preparations for this study. Preparation of Sample solution for Temozolomide capsules 20 mg: A sample about 0.270 gm was weighed and transferred in to 100 ml volumetric flask (equivalent 10 mg of Temozolomide), to this flask added 60 ml of equal volumes of Acetonitrile and water respectively and the mixture was subjected to vigorous shaking for 10 min and diluted up to volume with same solvent. Further diluted 10 ml of these solutions to 100 ml with Acetonitrile: water 50:50. The solution was filtered through 0.45µ nylon filter before analysis. 10 µl of these solutions were injected in to the HPLC system. Prepared six sample preparations for this study. 71
Evaluation Table No. 19 : Results of Intermediate precision study for Anastrozole Lable Claim of Anastrozole % Assay of Anastrozole Sample 1 1.02 101.68 Sample 2 1.00 100.29 Sample 3 0.99 99.29 Sample 4 1.00 100.50 Sample 5 1.01 100.74 Sample 6 1.01 100.60 Mean 100.52 S.D 0.77 %RSD 0.76 Table No. 20 : Results of Intermediate precision study for Temozolomide Lable Claim of Temozolomide % Assay of Temozolomide Sample 1 19.46 97.30 Sample 2 19.19 95.97 Sample 3 19.00 95.02 Sample 4 19.23 96.17 Sample 5 19.28 96.40 Sample 6 19.25 96.26 Mean 96.19 S.D 0.74 %RSD 0.76 72
Comparative result of precision and intermediate precision results Table No. 21 : Comparison between % Assay in precision study and intermediate precision study for anastrozole Sr.No. % Assay values in Precision for anastrozole (Analyst I) % Assay values in Intermediate Precision for anastrozole (Analyst II) 1 99.74 101.68 2 99.33 100.29 3 99.49 99.29 4 100.09 100.50 5 100.84 100.74 6 100.39 100.60 Average 99.98 100.52 SD 0.57 0.77 RSD 0.57 0.76 % Absolute difference 0.5 For Temozolomide Sr.No. % Assay values in Precision for Temozolomide (Day I) % Assay values in Intermediate Precision for Temozolomide (Day II) 1 95.44 97.30 2 95.05 95.97 3 95.20 95.02 4 95.78 96.17 5 96.49 96.40 6 96.07 96.26 Average 95.67 96.19 SD 0.55 0.74 RSD 0.57 0.76 % Absolute difference 0.5 73
Specimen chromatograms are given below, Figure No. 27 : Anastrozole sample chromatogram in Intermediate Precision study. Figure No. 28 : Temozolomide sample chromatogram in Intermediate Precision study. Conclusion: Assay method for Temozolomide capsules 20 mg and Anastrozole tablets 1 mg is found rugged during analysis of precision and intermediate precision as % absolute difference is well within acceptance criteria. 74
2.9.7 SOLUTION STABILITY Solution stability study for standard solution has been conducted for 72 hours by keeping the solution on bench top at normal illuminated laboratory condition. 1. Sample solution stability at bench top condition (At normal illuminated laboratory condition) This study has done for 72 hours for standard and sample solutions. Solution stability study solutions are prepared as follows : For standard solution preparation, refer the solution preparation under specificity parameter For system suitability table refer specificity parameter. Standard solution at initial time point : Calibration standard solution from specificity is used for solution stability. Test solution at initial time point : Test solution from specificity is used for solution stability. Standard and test solution stability summary : Solution stability study for standard and test solution has been established by calculating % relative difference of the standard area and test area at each time interval with respect to initial solutions area. 75
Evaluation Table No. 22 : Solution stability study of standard solution at bench top condition for Anastrozole (i.e. at normal illuminated laboratory condition) as given below Solution stability for Standard solution Status for standard solution Area response for Anastrozole peak % Recovery Relative % Difference Initial 1002568 100.0 6 hours 1003613 100.1 0.21 24 hours 1002037 99.9 0.05 72 hours 1003776 100.1 0.24 Table No. 23 : Solution stability study of Sample solution at bench top condition for Anastrozole (i.e. at normal illuminated laboratory condition) as given below Solution stability for Standard solution Status for Sample solution Area response for Anastrozole peak % Recovery Relative % Difference Initial 1011034 100.0 6 hours 1009787 99.9 0.12 24 hours 998827 98.8 1.21 72 hours 1006823 99.6 0.42 76
Table No. 24: Solution stability study of Sample solution at bench top condition for Temozolomide (i.e. at normal illuminated laboratory condition) as given below Solution stability for Standard solution Status for Sample solution Area response for Temozolomide peak % Recovery Relative % Difference Initial 1981865 100.0-6 hours 1976779 99.7 0.26 24 hours 1977816 99.8 0.20 72 hours 1977832 99.8 0.20 Table No. 25: Solution stability study of Sample solution at bench top condition for Temozolomide (i.e. at normal illuminated laboratory condition) as given below Solution stability for Standard solution Status for Sample solution Area response for Temozolomide peak % Recovery Relative % Difference Initial 1956086 100.0-6 hours 1963322 100.4 0.74 24 hours 1956890 100.0 0.08 72 hours 1968727 100.6 1.29 Results: % Relative difference between area of standard solution upto 72 hours on bench top under normal illuminated laboratory condition with respect to area of initial area is within limit i.e. less than 2.0%. % Relative difference between areas of test solution up-to 72 hours on bench top under normal illuminated laboratory condition with respect to area of initial test solution area is within limit i.e. less than 2.0%. Conclusion : Standard solution is stable up-to 72 hours at normal illuminated laboratory condition on bench top. Test solution is stable up-to 72 hours at normal illuminated laboratory condition on bench top. 77
2.9.8 ROBUSTNESS 9.8.1 Influence of variation of test parameters Table No. 26 : List of parameters studied with deliberated changes Sr. No. Parameters Deliberate changes 1 Flow rate of mobile phase. 0.9 And 1.1 (i.e. ± 0.1 ml per minute). 2 Change in column temperature. 23 C and 27 C (i.e. ± 2.0 C). For preparation of blank, standard and placebo solutions refer the solution preparation under specificity parameter and for test solution refer precision parameter. Robustness 1 Table No. 27: Results of Robustness at the flow rate of 0.9 ml /mim for sample solution. Sr. No. % Assay of Anastrozole % Assay of Temozolomide 1 99.86 98.75 2 100.46 99.06 3 100.47 97.63 Mean % Assay value for Robustness Study Mean % Assay value for Precision Study 100.26 98.48 99.98 95.67 % Absolute difference 0.54 1.33 78
Robustness 2 Table No. 28 : Results of Robustness at the flow rate of 1.1 ml /mim for sample solution. Sr. No. % Assay of Anastrozole % Assay of Temozolomide 1 99.45 99.88 2 100.90 100.03 3 100.17 100.05 Mean % Assay value for Robustness Study Mean % Assay value for Precision Study 100.17 98.38 99.98 95.67 % Absolute difference 0.44 1.12 Robustness 3 Table No. 29 : Results of Robustness at the column oven temperature 23 C sample solution. Sr.No. % Assay of Anastrozole % Assay of Temozolomide 1 98.02 98.63 2 97.72 98.66 3 98.53 97.78 Mean % Assay value for Robustness Study Mean % Assay value for Precision Study 98.09 98.35 99.98 95.67 % Absolute difference 1.87 1.08 79
Robustness 4 Table No. 30 : Results of Robustness at the column oven temperature 23 C sample solution. Sr.No. % Assay of Anastrozole % Assay of Temozolomide 1 99.94 98.90 2 100.11 98.41 3 100.87 97.93 Mean % Assay value for Robustness Study Mean % Assay value for Precision Study 100.30 98.41 99.98 95.67 % Absolute difference 0.34 1.20 Conclusion: Robustness studies signified that the results of the method remained unaffected by small, deliberate changes in the flow rate and column temperature. Specimen chromatograms are given below, Robustness I :- High flow rate Figure No. 29 : Resolution chromatogram in Robustness I study. 80
Figure No. 30: Mix Standard chromatogram in Robustness I study. Figure No. 31: Anastrozole sample chromatogram in Robustness I study. Figure No. 32 : Temozolomide sample chromatogram in Robustness I study. 81
Robustness II: - Low flow rate Figure No. 33 : Resolution chromatogram in Robustness II study. Figure No. 34 : Mix Standard hromatogram in Robustness II study. Figure No. 35 : Anastrozole Sample chromatogram in Robustness II study. 82
Figure No. 36 : Temozolomide Sample chromatogram in Robustness II study. Robustness III :- High column Temperature. Figure No. 37 : Resolution chromatogram in Robustness III study. Figure No. 38 : Mix Standard chromatogram in Robustness III study. 83
Figure No. 39 : Temozolomide Sample chromatogram in Robustness III study. Figure No. 40 : Anastrozole Sample chromatogram in Robustness III study. Robustness IV :- Low column Temperature. Figure No. 41 : Resolution chromatogram in Robustness IV study. 84
Figure No. 42: Mix Standard chromatogram in Robustness IV study. Figure No. 43: Anastrozole Sample chromatogram in Robustness IV study. Figure No. 44: Temozolomide Sample chromatogram in Robustness IV study. 85
2.9.9. CONCLUSION The advantages of the proposed method involve a simple procedure for sample preparation and relatively short time of analysis. Apart from this, it can be used for assays of Anastrozole and Temozolomide in biological fluids or in pharmacokinetic investigations. The proposed method was validated by testing its linearity, accuracy, precision, limits of detection, and limit of quantitation. Robustness and stability of solutions. The results of the analysis of pharmaceutical dosage forms by the proposed methods are highly reproducible, reliable, and are in good agreement with the label claims of the drug. The additives usually present in the pharmaceutical formulations of the assayed samples did not interfere with Anastrozole and Temozolomide. It may be said that the proposed methods are precise, sensitive, and accurate, so that these can be used as standard Pharmacopeial methods for the simultaneous determination of Anastrozole and Temozolomide using the HPLC systems with PDA detector. 86