4.1 MATERIALS 4.2 EQUIPMENTS

Transcription

1 4.1 MATERIALS 1. Aceclofenac Moraceae Pharma Ltd., Uttarakhand, India 2. Diclofenac Arti Drugs Ltd., Mumbai, India 3. Paracetamol Moraceae Pharma Ltd., Uttarakhand, India 4. Para-aminophenol Hema Pharmaceuticals Ltd., Gujarat, India 5. Telmisartan Systopic Laboratories Ltd., Delhi, India 6. Hydrochlorothiazide Systopic Laboratories Ltd., Delhi, India 7. DSA Supra Chemicals Ltd., Mumbai, India 8. Marketed tablets (Aceclo Plus, Tablets) Aristo Pharmaceuticals Ltd., Mumbai, India (ACF 100 mg + PCM 500 mg FDC tablets) 9. Marketed tablets (Telma H, Tablets) Glenmark Pharmaceuticals Ltd., Mumbai, India (TEL 40 mg + HCTZ 12.5 mg FDC tablets) 10. Methanol HPLC grade Qualigens Fine Chemicals, Mumbai, India 11. Acetonitrile HPLC grade Qualigens Fine Chemicals, Mumbai, India 12. HPLC grade water Thomas Baker Pvt. Ltd., Mumbai, India 13. Potassium dihydrogen phosphate E. Merck Ltd., Mumbai, India 14. Methanol LC-MS grade Fluka Analytial, Sigma- Aldrich, St. Louis, USA 15. Acetonitrile LC-MS grade Fluka Analytial, Sigma- Aldrich, St. Louis, USA 16. Ammonium acetate LC-MS grade Fluka Analytial, Sigma- Aldrich, St. Louis, USA 17. Formic acid LC-MS grade Fluka Analytial, Sigma- Aldrich, St. Louis, USA 18. Sodium Hydroxide S.D. Fine Ltd., Mumbai, India 19. Hydrochloric Acid S.D. Fine Ltd., Mumbai, India 20. Hydrogen Peroxide S.D. Fine Ltd., Mumbai, India 21. Nylon membrane and syringe filter Millipore Corporation, Bedford, MA, USA 4.2 EQUIPMENTS 1. UV-Vis Spectrophotometer Shimadzu 1601 UV Visible Spectrophotometer, Tokyo, Japan 2. FTIR Shimadzu FTIR Spectrophotometer, Tokyo, Japan 3. DSC system Pyris 6 DSC, Perkin Elmer, MA, USA 4. HPLC system LC: LC-10 AT VP Liquid Chromatograph 56

2 Detector: SPD 10 AT VP Shimadzu UV-Visible Pump: Quaternary solvent manager System Controller: Shimazu SCL-10 A VP Software: Class VP 5. UPLC-QTOF-MS system UPLC: Waters Acquity UPLC TM system (Serial No. F09 UPB 920M; Model Code. UPB; Waters Corporation, MA, USA) Detector: Q-TOF tunable MS detector (Synapt; Waters, Serial No. JAA 272; Micromass Limited, Manchester, UK) Column: Waters Acquity UPLC TM BEH C 18 ( mm, 1.7 µm) Pump: Binary solvent manager Software: MassLynx V 4.1 software 6. ph meter Mettler Toledo MP220, Tokyo, Japan 7. Electronic balance Mettler Toledo MP220, Tokyo, Japan 8. Water bath Metrex Scientific Instruments, Pvt. Ltd., New Delhi, India 9. Millipore filtration unit Millipore Corporation, Bedford, MA, USA 10. Vacuum filtration unit Effem Scientific Instruments, Pvt. Ltd., New Delhi, India 11. Sonicator PCI Analytics, Mumbai, India 12. Stability Chamber Nirmal International, New Delhi, India 13. UV Chamber Camag UV Chamber, Muttenz, Switzerland 4.3 PHYSICOCHEMICAL CHARACTERIZATION AND IDENTIFICATION OF DRUGS Identification UV Spectrophotometry: The 10 mg of each drug individually was prepared by dissolving in sufficient methanol to produce 10 ml. One ml of this solution was diluted to 10 ml with methanol. The solution was scanned in the wavelength range of nm. IR Spectrophotometry: IR spectrums of drugs were taken by potassium bromide pellet technique using FTIR. Approximately about 0.5 g of KBr was transferred in to mortar and grind to a fine powder. Exactly 5 mg of test sample was added and mixed perfectly and grinded to a 57

3 uniform mixture. A small quantity of that powder was taken and compressed in to a thin semitransparent pellet by applying about 8 tons pressure under vacuum. Differential scanning colorimetry (DSC): DSC was done using Perkin Elmer DSC system. The nitrogen flow rate was maintained at 20mL/min and the heating rate was 10 C/min. DSC thermogram of drug samples were obtained in the temperature range of 50 to 300 C. Mass spectrometry: The Mass spectrometry was done by using Waters Synapt Quadrupole time-of-flight mass spectrometer. The 100 ng/ml each of drug was prepared in methanol: water (50:50, v/v) individually. The solution was filtered through 0.20 µm nylon syringe filter and injected in to the Q-TOF-MS system for analysis. The identification of the compounds was done by selected Q-TOF-MS conditions. The optimum values for compound-dependent parameters like trap collision energy and transfer collision energy were set to obtain the fragmentation information. The accurate mass and composition of the precursor ions and fragment ions were calculated using the MassLynx V 4.1 software incorporated in the instrument. 4.4 ANALYTICAL METHOD DEVELOPMENT AND VALIDATION Development of UPLC-QTOF-MS method for simultaneous estimation of aceclofenac and paracetamol and their degradation products UPLC-QTOF-MS technique was preferred over the HPLC-UV technique because, 1. It provides the required sensitivity, speed of analysis and resolution. 2. UPLC column contains smaller particles (1.7 µm) and operated under high pressure (up to psi). 3. UPLC-QTOF-MS allows the fragementation of the compounds with higher accuracy and precision, which is ultimately helpful in structure elucidation and identification of drugs and their degradation products. 58

4 On the basis of information provided by literature, that there was no method available for simultaneous determination of aceclofenac and paracetamol without demonstrating its separation from their major degradation products, diclofenac and para-aminophenol in pharmaceutical formulations. Hence an analytical method was developed for simultaneous determination of aceclofenac and paracetamol along with their known degradation products. Q-TOF-MS conditions Mass spectrometry was performed on a Waters Synapt Q-TOF Premier (Micromass MS Technologies, Manchester, UK) mass spectrometer. The capillary voltage, sampling cone voltage, extraction cone voltage, source temperature, desolvation temperature, cone gas flow, desolvation gas flow, trap gas flow, and source gas flow were set to 3.0 kv, 40 V, 4 V, 80ºC, 350ºC, 50 L/h, 800 L/h, 1.50 ml/min, and 0.50 ml/min, respectively for all the compounds. The Q-TOF Premier TM was operated in positive ionization mode with resolution over 8500 mass with 1.0 min scan time. The accurate mass and composition of the precursor ions and fragment ions were calculated using the MassLynx V 4.1 software incorporated in the instrument. Argon was employed as the collision gas at a pressure of mbar. Quantitation was performed using MS/MS transitions, m/z for aceclofenac, for diclofenac, for paracetamol, for paraaminophenol with a scan time of 1.0 min and 0.02 seconds inter-scans per transition. The optimum values for compound-dependent parameters like trap collision energy and transfer collision energy were set to 12 and 6 V, respectively for aceclofenac, 16 and 6 V for diclofenac, 18 and 6 V for paracetamol, and 6 and 4 V for para-aminophenol to obtain the fragmentation information. UPLC conditions UPLC was performed with a Waters Acquity UPLC TM system (Serial No. F09 UPB 920M; Model Code. UPB; Waters Corporation, MA, USA) equipped with a binary solvent manager, an auto-sampler, column manager and a tunable MS detector (Synapt; Waters, Serial No. JAA 272; Micromass Limited, Manchester, UK). Chromatographic separation was performed on a Waters Acquity UPLC TM BEH C 18 ( mm, 1.7 µm) column. Different mobile phase 59

5 containing acetonitrile-water or methanol-water along with modifiers such as ammonium acetate and or formic acid were tested to obtain the best chromatographic conditions. Optimized chromatographic conditions The mobile phase for UPLC analysis consisted of acetonitrile 2mM ammonium acetate (40:60, v/v) which was filtered through 0.45 µm membrane filter and degassed by sonication. For isocratic elution, the flow rate of the mobile phase was kept at 0.25 ml/min and 10 µl of sample solution was injected in each run. The total chromatographic run time was 2.0 min. The temperature for column and auto-sampler were maintained at 40 and 4ºC, respectively and the pressure of the system was in the range of 2450 to 2500 psi with delta value below 50. 2mM ammonium acetate buffer The 77 mg of ammonium acetate was taken in 500 ml volumetric flask and volume was made up to 500 ml with HPLC water. Mixed well and sonicated for 5 min in sonicator. 0.1% formic acid solution The 0.5 ml of formic acid was taken in 500 ml volumetric flask and volume was made up to 500 ml with HPLC water. Mixed well and sonicated for 5 min in sonicator. Preparation of mobile phase The 200 ml of LC-MS grade acetonitrile was mixed with 300 ml of HPLC water (40:60, v/v) in a 500 ml reagent bottle. The solution was filtered though 0.45 µm nylon membrane filter and sonicated for 5 min. Washing solutions For weak wash and seal wash, 50 ml of LC-MS grade acetonitrile was mixed with 450 ml of HPLC water (10: 90, v/v) in a 500 ml reagent bottle. For strong wash, 450 ml of LC-MS grade acetonitrile was mixed with 50 ml of 2 mm ammonium acetate buffer (90: 10, v/v) in a 500 ml reagent bottle. The solutions were sonicated for 5 min. Preparation of standard solutions 50 mg each of aceclofenac, diclofenac, paracetamol, and para-aminophenol were weighed accurately and transfer to 50 ml volumetric flasks separately. The powders were then 60

6 dissolved with approximately 25 ml of methanol and ultrasonicated for 5 min. The final volume was made up with methanol. The solutions were further diluted with methanol: water (50:50, v/v) to give a series of standard solutions containing required concentrations for each compound. Preparation of sample solutions Twenty tablets were weighed accurately and powdered. Powder equivalent to 50 mg of aceclofenac and 250 mg of paracetamol was taken and transferred to a 50 ml volumetric flask. The powder was dissolved with approximately 25 ml of methanol and ultrasonicated for 5 min. The final volume was made up with methanol. This solution was filtered through 0.45 µm nylon membrane filter to remove all the excipients. The resultant filtrate was further diluted with methanol: water (50:50, v/v) to give a sample solution containing 100 ng/ml of aceclofenac and 500 ng/ml of paracetamol. Validation of the method The developed method was validated according to ICH validation guidelines. The validation parameters addressed were linearity and range, limit of detection and quantitation, precision, accuracy, robustness, and specificity. Linearity and range Different standard concentrations each of aceclofenac and paracetamol in the range of ng/ml, diclofenace ng/ml (specification limit: 0.2 ng/ml for working concentration of 100 ng/ml of aceclofenac), and para-aminophenol ng/ml (specification limit: 2.5 ng/ml for working concentration of 500 ng/ml of paracetamol) were prepared separately in methanol: water (50:50, v/v). The solutions were filtered through 0.20 µm nylon syringe filter and injected in to the UPLC/Q-TOF-MS system for analysis. Average peak area at each concentration level was subjected to linear regression analysis with the least squares method. Linearity was described by slope, intercept and correlation coefficient obtained from regression equations. 61

7 Limits of detection and quantitation Standard stock solutions were diluted appropriately to obtain concentrations for the estimation of the limit of detection (LOD) and the limit of quantitation (LOQ) based on signal to noise (S/N) ratio of 3:1 and 10:1, respectively. Precision The intraday precision was assessed by performing six analysis using tablet sample solution containing 100 ng/ml of aceclofenac and 500 ng/ml of paracetamol, spiked with 0.2 ng/ml of diclofenac and 2.5 ng/ml of para-aminophenol. Similarly interday precision was assessed by performing replicate analysis using same concentration of all the analytes for three consecutive days under the same experimental conditions. The intermediate precision of the method was also evaluated by a different analyst in the same laboratory under the same experimental conditions. The mean of percentage recoveries and the RSD (%) was calculated. Accuracy The accuracy of the method was determined by standard addition technique. Three different levels (50, 100, and 150%) of standards were added to pre-analyzed tablet sample in six replicates and the mixtures were re-analyzed by the proposed method. The percentage recoveries of all the compounds at each level and each replicate were determined. The mean of percentage recoveries and the RSD (%) was calculated. Robustness The robustness of an analytical procedure refers to its ability to remain unaffected by small and deliberate variations in the method parameters and provides an indication of its reliability for routine analysis. The robustness was determined by analyzing the sample solution containing 100 ng/ml of aceclofenac and 500 ng/ml of paracetamol, spiked with 0.2 ng/ml of diclofenac and 2.5 ng/ml of para-aminophenol under a variety of conditions of the method parameters, such as flow rate, mobile phase composition, column temperature and injection volume. The mean of percentage recoveries and the RSD (%) was calculated from six replicates. 62

8 Specificity The samples were chromatographed to determine the extent to which mobile phase components and excipients could contribute to the interference with the analytes. This was done by comparing the chromatograms of each analyte with chromatograms obtained from blank solution. Specificity of the developed method was also assessed by performing forced degradation studies. The stress conditions employed for the degradation study includes acid hydrolysis (1 N HCl), alkali hydrolysis (1 N NaOH), oxidation (3% H 2 O 2 ) and light carried out as per ICH Q1B (ICH Q1A R2, 2005). For acid, base and oxidation induced degradation, 5 ml solutions each of aceclofenac and paracetamol (each 200 ng/ml), separately were dissolved in 5 ml of methanolic solution of 1N HCl, 1N NaOH, and hydrogen peroxide (H 2 O 2, 3% v/v), respectively in 10 ml volumetric flask. These solutions were kept at room temperature (25 C) for 1 h and analysis was performed by the proposed method. For photochemical degradation, 10 ml solutions each of aceclofenac and paracetamol (each 100 ng/ml), separately were taken in 10 ml volumetric flask and exposed to UV light in UV chamber at 254 nm for 24 h and analysis was performed by the proposed method. Analysis of marketed tablets Twenty tablets were weighed accurately and powdered. Powder equivalent to 50 mg of aceclofenac and 250 mg of paracetamol was taken and transferred to a 50 ml volumetric flask. The powder was dissolved with approximately 25 ml of methanol and ultrasonicated for 5 min. The final volume was made up with methanol. This solution was filtered through 0.45 µm membrane filter to remove all the excipients. The resultant filtrate was further diluted with methanol: water (50:50, v/v) to give a sample solution containing 100 ng/ml of aceclofenac and 500 ng/ml of paracetamol. This solution was finally filtered through 0.20 µm nylon syringe filter and injected in to the UPLC/Q-TOF-MS system for analysis. The amount of aceclofenac and paracetamol in tablets was determined by calibration equations obtained from the respective calibration plot. Stability studies on marketed tablets The ongoing stability studies on marketed tablets were performed as per ICH Q1A (R2) guidelines. Tablets from single batch were stored in stability chambers at 30 ± 2 C/65 ± 5% RH 63

9 (Relative humidity) as long-term condition for 12 months and 40 ± 2 C/75 ± 5% RH as accelerated condition for 6 months with testing frequency of every 3 months. The tablets were evaluated for stability-relevant test parameters such as assay of drugs and presence of their related degradation products. Assay of drugs from tablets during stability study was performed by applying the same procedure as described in the section analysis of marketed tablets. The amount of aceclofenac and paracetamol in tablets during storage was calculated by extrapolating the values of peak area from the respective calibration plots. The amounts of diclofenac and para-aminophenol were calculated with respect to peak area of aceclofenac and paracetamol, respectively. The stability study data were evaluated and analyzed by statistical methodology recommended by ICH guidelines. The ultimate purpose of the stability evaluation was to monitor and confirm the established shelf life of tablet dosage form. Determination of shelf life As mentioned in ICH guidelines, an appropriate approach for shelf life estimation is to analyze a quantitative attribute (e.g., assay, degradation products) and determine the earliest time at which the 95 % confidence limit for the mean intersects the proposed acceptance criterion. For an attribute known to decrease with time (i.e. assay %) the one-sided 95 % lower confidence limit should be compared to the acceptance criterion. Hence the shelf life was determined by calculating the label claim (%) of drugs in the tablets at various time intervals of long term stability study. For the determination of the shelf life of tablet product, the graph was plotted with assay values against time intervals of long term testing for both the drugs. Extrapolation to extend the shelf life beyond the period covered by long term stability data was proposed up to 30 months Development and validation of UPLC-QTOF-MS method for simultaneous estimation of telmisartan and hydrochlorothiazide and their degradation products On the basis of literature, there was no method available for simultaneous determination of telmisartan and hydrochlorothiazide without demonstrating its separation from their degradation products in their combination products. Hence an analytical method was developed for 64

10 simultaneous determination of telmisartan and hydrochlorothiazide along with their degradation products. Again the ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry was chosen to provide for required fast, high resolution separations having the necessary sensitivity and associated advantages over the other analytical techniques. Q-TOF-MS conditions Mass spectrometry was performed on a Waters Synapt Q-TOF Premier (Micromass MS Technologies, Manchester, UK) mass spectrometer. The capillary voltage, sampling cone voltage, extraction cone voltage, source temperature, desolvation temperature, cone gas flow, desolvation gas flow, trap gas flow, and source gas flow were set to 3.0 kv, 40 V, 4 V, 80ºC, 350ºC, 50 L/h, 800 L/h, 1.50 ml/min, and 0.50 ml/min, respectively for all the compounds. The Q-TOF Premier TM was operated in positive ionization mode with resolution over 8500 mass with 1.0 min scan time. The accurate mass and composition of the precursor ions and fragment ions were calculated using the MassLynx V 4.1 software incorporated in the instrument. Argon was employed as the collision gas at a pressure of mbar. Quantitation was performed using MS/MS transitions, m/z for telmisartan, for hydrochlorothiazide, and for DSA with a scan time of 1.0 min and 0.02 seconds inter-scans per transition. The optimum values for compound-dependent parameters like trap collision energy and transfer collision energy were set to 12 and 6 V, respectively for telmisartan, 16 and 6 V for hydrochlorothiazide and DSA to obtain the fragmentation information. UPLC conditions UPLC was performed with a Waters Acquity UPLC TM system (Serial No. F09 UPB 920M; Model Code. UPB; Waters Corporation, MA, USA) equipped with a binary solvent manager, an auto-sampler, column manager and a tunable MS detector (Synapt; Waters, Serial No. JAA 272; Micromass Limited, Manchester, UK). Chromatographic separation was performed on a Waters Acquity UPLC TM BEH C 18 ( mm, 1.7 µm) column. Different mobile phase containing acetonitrile-water or methanol-water along with modifiers such as ammonium acetate and or formic acid were tested to obtain the best chromatographic conditions. 65

11 Optimized chromatographic conditions The mobile phase for UPLC analysis consisted of acetonitrile 2mM ammonium acetate (50:50, v/v) which was filtered through 0.45 µm membrane filter and degassed by sonication. For isocratic elution, the flow rate of the mobile phase was kept at 0.2 ml/min and 10 µl of sample solution was injected in each run. The total chromatographic run time was 3.0 min. The temperature for column and auto-sampler were maintained at 40 and 4ºC, respectively and the pressure of the system was in the range of 2450 to 2500 psi with delta value below 50. Preparation of mobile phase 250 ml of LC-MS grade acetonitrile was mixed with 2500 ml of 2 mm ammonium acetate buffer (50:50, v/v) in a reagent bottle. The solution was filtered though 0.45 µm nylon membrane filter and sonicated for 5 min. Preparation of standard solutions 50 mg each of telmisartan, hydrochlorothiazide, and DSA were weighed accurately and transfer to 50 ml volumetric flasks separately. The powders were then dissolved with approximately 25 ml of methanol and ultrasonicated for 5 min. The final volume was made up with methanol. The solutions were further diluted with methanol: water (50:50, v/v) to give a series of standard solutions containing required concentrations for each compound. Preparation of sample solutions Twenty tablets were weighed accurately and powdered. Powder equivalent to 25 mg of telmisartan and 5 mg of hydrochlorothiazide was taken and transferred to a 50 ml volumetric flask. The powder was dissolved with approximately 25 ml of methanol and ultrasonicated for 5 min. The final volume was made up with methanol. This solution was filtered through 0.45 µm nylon membrane filter to remove all the excipients. The resultant filtrate was further diluted with methanol: water (50:50, v/v) to give a sample solution containing 500 ng/ml of telmisartan and 100 ng/ml of hydrochlorothiazide. Validation of the method The developed method was validated according to ICH validation guidelines. The validation parameters addressed were linearity and range, limit of detection and quantitation, precision, accuracy, robustness, and specificity. 66

12 Linearity and range Different standard concentrations each of telmisartan and hydrochlorothiazide in the range of ng/ml, and DSA ng/ml (specification limit: 0.1 ng/ml for working concentration of 100 ng/ml of hydrochlorothiazide) were prepared separately in methanol: water (50:50, v/v). The solutions were filtered through 0.20 µm nylon syringe filter and injected in to the UPLC/Q- TOF-MS system for analysis. Average peak area at each concentration level was subjected to linear regression analysis with the least squares method. Linearity was described by slope, intercept and correlation coefficient obtained from regression equations. Limits of detection and quantitation Standard stock solutions were diluted appropriately to obtain concentrations for the estimation of the limit of detection (LOD) and the limit of quantitation (LOQ) based on signal to noise (S/N) ratio of 3:1 and 10:1, respectively. Precision The intraday precision was assessed by performing six analyses using tablet sample solution containing 500 ng/ml of telmisartan and 100 ng/ml of hydrochlorothiazide, spiked with 0.1 ng/ml of DSA. Similarly interday precision was assessed by performing replicate analysis using same concentration of all the analytes for three consecutive days under the same experimental conditions. The intermediate precision of the method was also evaluated by a different analyst in the same laboratory under the same experimental conditions. The mean of percentage recoveries and the RSD (%) was calculated. Accuracy The accuracy of the method was determined by standard addition technique. Three different levels (50, 100, and 150%) of standards were added to pre-analyzed tablet sample in six replicates and the mixtures were re-analyzed by the proposed method. The percentage recoveries of all the compounds at each level and each replicate were determined. The mean of percentage recoveries and the RSD (%) was calculated. 67

13 Robustness The robustness was determined by analyzing the sample solution containing 500 ng/ml of telmisartan and 100 ng/ml of hydrochlorothiazide, spiked with 0.1 ng/ml of DSA under a variety of conditions of the method parameters, such as flow rate, mobile phase composition, column temperature and injection volume. The mean of percentage recoveries and the RSD (%) was calculated from six replicates. Specificity The samples were chromatographed to determine the extent to which mobile phase components and excipients could contribute to the interference with the analytes. Specificity of the developed method was also assessed by performing forced degradation studies. For acid, alkali and oxidation induced degradation, 5 ml solutions each of telmisartan and hydrochlorothiazide (each 200 ng/ml), separately were dissolved in 5 ml of methanolic solution of 1N HCl, 1N NaOH, and hydrogen peroxide (H 2 O 2, 3% v/v), respectively in 10 ml volumetric flask. These solutions were kept at room temperature (25 C) for 1 h and analysis was performed by the proposed method. For photochemical degradation, 10 ml solutions each of telmisartan and hydrochlorothiazide (each 100 ng/ml), were taken separately in 10 ml volumetric flask and exposed to UV light in UV chamber at 254 nm for 24 h and analysis was performed by the proposed method. Analysis of marketed tablets Twenty tablets were weighed accurately and powdered. Powder equivalent to 25 mg of telmisartan and 5 mg of hydrochlorothiazide was taken and transferred to a 50 ml volumetric flask. The powder was dissolved with approximately 25 ml of methanol and ultrasonicated for 5 min. The final volume was made up with methanol. This solution was filtered through 0.45 µm nylon membrane filter to remove all the excipients. The resultant filtrate was further diluted with methanol: water (50:50, v/v) to give a sample solution containing 500 ng/ml of telmisartan and 100 ng/ml of hydrochlorothiazide. The amount of telmisartan and hydrochlorothiazide in tablets was determined by calibration equations obtained from the respective calibration plot. 68

14 Stability studies on marketed tablets The stability studies on marketed tablets were performed as per ICH Q1A (R2) guidelines. Tablets from single batch were stored in stability chambers at 30 ± 2 C/65 ± 5% RH (Relative humidity) as long-term condition for 12 months and 40 ± 2 C/75 ± 5% RH as accelerated condition for 6 months with testing frequency of every 3 months. The tablets were evaluated for stability-relevant test parameters such as assay of drugs and related impurity. Assay of drugs from tablets during stability study was performed by applying the same procedure as described in the section analysis of marketed tablets. The amount of drugs and its related impurity in tablets during storage was calculated by extrapolating the values of peak area from the respective calibration plots. The stability study data were evaluated and analyzed by statistical methodology recommended by ICH guidelines. The ultimate purpose of the stability evaluation was to monitor and confirm the established shelf life of tablet dosage form. Determination of shelf life As mentioned in ICH guidelines, an appropriate approach for shelf life estimation is to analyze a quantitative attribute (e.g., assay, degradation products) and determine the earliest time at which the 95% confidence limit for the mean intersects the proposed acceptance criterion. For an attribute known to decrease with time (i.e., assay %) the one-sided 95% lower confidence limit should be compared to the acceptance criterion. Hence the shelf life was determined by calculating the label claim (%) of drugs in the tablets at various time intervals of long term stability study. For the determination of the shelf life of tablet product, the graph was plotted with assay values against time intervals of long term testing for both the drugs. Extrapolation to extend the shelf life beyond the period covered by long term stability data was proposed up to 30 months. 69