Chapter-4 EXPERIMENTAL WORK BY RP-HPLC

Chapter-4 EXPERIMENTAL WORK BY RP-HPLC

4.0 EXPERIMENTAL WORK BY RP-HPLC 4.1. DEVELOPMENT AND VALIDATION OF AN RP-HPLC METHOD FOR THE DETERMINATION OF NIFLUMIC ACID 4.1.1. MATERIALS AND METHODS OF NIFLUMIC ACID Instrumentation The author had attempted to develop a liquid chromatographic method for quantitative estimation of Niflumic acid. Quantitative HPLC was performed on a binary gradient HPLC with Shimadzu LC-10AT and LC-10AT VP series HPLC pumps, with a 20μl sample injection loop (manual),scl-10a VP system controller,spd-10a VP UV-Visible absorbance detector and ELSD(Evaporation Light scattering Detector) were used. The output signal was monitored and integrated using Shimadzu CLASS-VP version 6.12 SP1 software. Phenomenex RP-C18 (250 x 4.6 mm, packed with Luna 5 micron) column was used for the separation. A 20 μl Hamilton injection syringe was employed for sample injection. The mobile phase was degassed by using a Spectra lab DGA 20A3 ultrasonic bath sonicator. A Shimadzu SM 114 electronic balance was used for weighing the materials. Class A Borosil glassware (Borosil, India) was employed for volumetric and general purpose in the entire study. CHEMICALS AND REAGENTS: A pure sample of Niflumic acid Active Pharmaceutical Ingredient (API) was procured from Glenmark Pharmaceutical Ltd., Mumbai, India. Niflumic acid (API) was provided by Glenmark Pharmaceutical Ltd., Mumbai, India.Methanol, acetonitrile & water of HPLC grade were purchased from Rankem (Ranbaxy Fine Chemicals Ltd.), Delhi. Commercial formulation of Niflumic acid [NIFLURIL, Glenmark Pharmaceutical Ltd. and NIFTON of Dr.Reddy s Lab] were procured from the local market of Visakhapatnam. Mobile phase preparation The mobile phase was prepared by taking a mixture containing methanol and water in a ratio 75:25 in to a 500 ml volumetric flask. The same solution was also used as the diluents for preparing further dilutions. 80

Niflumic acid stock and working standard solutions Accurately weighed quantity of 10 mg Niflumic acid was taken in a 10 ml volumetric flask containing 2 ml of the mobile phase. The solution was made up to 10 ml with the mobile phase to give 1 mg/ml followed by sonication for twenty minutes.from the above solution 5 ml was taken and made up to the volume to 50 ml with the mobile phase to give 100 µg/ml of Niflumic acid which is used as stock solution. 4.1.2. OPTIMIZATION OF THE CHROMATOGARPHIC CONDITIONS AND METHOD DEVELOPMENT For developing the method, a systematic study for optimisation of chromatographic condition was taken up. This was done by varying one parameter at a time and keeping all other conditions constant. The following studies were conducted for this purpose. A phenomenex non-polar C 18 (250 mm x 4.6 mm; 5µ) column was chosen as the stationary phase for this study. The mobile phase and the flow rate For ideal separation of drug in isocratic mode different commonly used solvents (water, methanol, acetonitrile etc.) with or without buffers were tried on a phenomenex RP C-18 (250 X 4.6; 5µ) column with varying flow rates (0.8-1.2 ml/min) and wavelength of detections. Linearity and construction of calibration curve The external standard method was applied to estimate the drug in bulk and formulation. A 0.45 µ membrane filter was used for filtration before use. As per the results obtained for the optimization the flow rate was adjusted. During the entire study the column temperature was maintained at 25±1 C. By taking six replicate measurements at different concentration points linearity of the peak area response was estimated. Suitable dilutions of standard niflumic acid drug solution were taken in different 10 ml volumetric flasks with volume make up to the mark with mobile phase to give working dilutions of niflumic acid. Twenty micro litres of each concentration was injected six times in to the HPLC column. Monitoring of the drug in the eluents was detected at its wavelength of maximum and the respective chromatograms were observed. The peak areas of each concentration chromatogram were noted and the graph of concentration over the peak areas was plotted. By applying least squares method the regression of the method was calculated. 81

4.1.3. VALIDATION OF THE PROPOSED METHOD The developed method was validated as per ICH guidelines. Here are the following parameters to be considered for validation. Specificity The chromatograms obtained from the blank solution were compared with those obtained from the drug with the most commonly used additives for accessing specificity of the drug. To prepare the blank solution the additives other than the drug were mixed in the mobile phase. The ratio between drug and additives were maintained in the same manner as that in the commercial formulations available of the drug. The present specificity study used some commonly used additives such as starch, lactose, ethyl cellulose, micro crystalline cellulose, magnesium stearate, hydroxyl propyl methyl cellulose and colloidal silicon dioxide. Before injecting in to HPLC column the mixtures should be filtrated through 0.45µ membrane filter. Precision Precision was determined as both repeatability and intermediate precision, in accordance with ICH guidelines. Repeatability of sample injection was determined as intra day variation and intermediate variation was determined by measurement of inter day variation. For these determinations, three concentrations of the solutions of Niflumic acid API were used (20, 300 and 500 μg/ml). Accuracy Accuracy was best determined by the standard addition method. Previously analyzed samples of Niflumic acid API were added with standard drug solutions and are analyzed by the proposed method. Recovery (%), RSD (%) and bias (%) were calculated for each concentration. Accuracy is reported as percentage bias, which is calculated from the expression %Bias = (measured value - true value) x 100 True value Robustness The concept of robustness of an analytical procedure has been defined by the ICH as a measure of its capacity to remain unaffected by small but deliberate variations in method 82

parameters. The robustness of a method is the ability to remain unaffected by small changes in parameters such as ph of the mobile phase, temperature, % organic solvent strength and buffer concentration etc. To determine the robustness of the method experimental conditions are purposely altered and chromatographic characters are evaluated. Influence of small changes in chromatographic conditions such as change in flow rate (± 0.1ml/min), temperature (±20C), wavelength of detection (±2nm) and water content in mobile phase (±2%) were studied to determine the robustness of the method. Stability of the analytical solution A study to establish bench top stability of the drug solution was conducted. A freshly prepared working standard solution (100 µg/ml of the drug) was analysed immediately and at different time intervals. The tailing factor, theoretical plates and difference in percent assay at different time intervals were calculated Limit of detection (LOD) The limit of detection (LOD) of an analytical method may be defined as the concentration, which gives rise to an instrument signal that is significantly different from the blank. For spectroscopic techniques or other methods that rely upon a calibration curve for quantitative measurements, the IUPAC approach employs the standard deviation of the intercept (Sa), which may be related to LOD and the slope of the calibration curve, b, by LOD = 3 Sa / b Limit of quantitation (LOQ) The LOQ is the concentration that can be quantitated reliably with a specified level of accuracy and precision. The LOQ represent the concentration of analyte that would yield a signal-to-noise ratio of 10. LOQ = 10 Sa / b Where, Sa is the standard deviation of the peak area ratio of analyte to IS (5 injections) of the drugs and b is slope of the corresponding calibration curve. System precision and System suitability System precision and system suitability studies were carried out by injecting six replicates of the working standard solution. The % RSD for the peak areas obtained was calculated. 83

4.1.4. ESTIMATION OF THE DRUG FROM DOSAGE FORMS Satisfactory results obtained with the method development for the assay of niflumic acid have forced the author to attempt its applicability for the determination of the drug in its formulations. The gel was procured from the local market contained niflumic acid and some exipients. The gel equivalent to 3.0 mg of niflumic acid was weighed and transferred to a 100 ml volumetric flask, 60 ml of the mobile phase was added into it and warmed to just melt, sonicated for 20 minutes, cooled and made the volume up to the mark with mobile phase. The resultant solution was filtered with Whatmann filter paper (No.1), and the filtrate was collected for use in the study discarding the first portions of the filtrate. The above solution (20 L) was then injected six times into the column. The mean peak area of the drug was calculated and the drug content in the formulation was calculated by the regression equation of the method. 4.2. DEVELOPMENT AND VALIDATION OF AN RP-HPLC METHOD FOR THE DETERMINATION OF MESALAMINE 4.2.1. MATERIALS AND METHODS Instrumentation The author had attempted to develop a liquid chromatographic method for quantitative estimation of Mesalamine on an isocratic Analytical Technologies HPLC instrument equiped with a phenomenx RP-C18 (250x4.6mm ;5 µ) column, having a 20 μl injection loop and a UV detector set at 230 nm governed by A-4000 software. The UV spectrum of Mesalamine was taken using an SL-159 UV-Visible spectrophotometer. Degassing of the mobile phase was done by using a analytical ultrasonic bath sonicator. A Shimadzu SM 114 electronic balance was used for weighing the materials. Class A Borosil glassware (Borosil, India) was employed for volumetric and general purpose in the entire study. CHEMICALS AND REAGENTS: A pure sample of Mesalamine was obtained from Mankind Pharmaceutical Ltd., Kolkata, India. Methanol & water of HPLC grade were procured from Rankem (Ranbaxy Fine Chemicals Ltd.), Delhi. 84

Commercial formulations of Mesalamine, ASACOL Tablets of Actavis Pharma were obtained from the local market of Visakhapatnam. Mobile phase preparation The mobile phase was prepared by taking a mixture containing methanol and water in a ratio 50:50 in to a 500 ml volumetric flask. The same solution was also used as the diluents for preparing further dilutions. Mesalamine stock and working standard solutions Accurately weighed quantity of 10 mg Mesalamine was taken in a 10 ml volumetric flask containing 5 ml of the mobile phase. The solution was made up to 10 ml with the mobile phase to give 1 mg/ml followed by sonication for twenty minutes. From the above solution 2.5 ml was taken and made up to the volume to 50 ml with the mobile phase to give 50 µg/ml of Mesalamine which is used as stock solution. 4.2.2. OPTIMIZATION OF THE CHROMATOGARPHIC CONDITIONS AND METHOD DEVELOPMENT For developing the method, a systematic study for optimisation of chromatographic condition was taken up. This was done by varying one parameter at a time and keeping all other conditions constant. The following studies were conducted for this purpose. A phenomenex non-polar C 18 (250 mm x 4.6 mm; 5µ) column was chosen as the stationary phase for this study. The mobile phase and the flow rate For ideal separation of drug in isocratic mode different commonly used solvents (water, methanol, acetonitrile etc.) with or without buffers were tried on a phenomenex RP C-18 (250 X 4.6; 5µ) column with varying flow rates (0.8-1.2 ml/min) and wavelength of detections. Linearity and construction of calibration curve Accurately weighed 10 mg of mesalamine working standard was taken into a 10 ml volumetric flask, added with 7 ml of diluent and sonicated to dissolve it completely and made volume up to the mark with the same solvent. Further 0.1 ml of the above stock solution was pipetted into a 10 ml volumetric flask and diluted up to the mark with diluent. Mixed well and filtered through 0.45 mm filter. The calibration curve was plotted with the seven 85

concentrations ranging from 20 to 50 mg/ml working standard solutions. Chromatogram was recorded thrice for each dilution. Calibration solutions were prepared daily and analyzed immediately after preparation. The linearity of an analytical method is its ability to elicit test results that are directly proportional to the concentration of analytes in samples within a given range or proportional by means of well-defined mathematical transformations. Linearity may be demonstrated directly on the test substance (by dilution of a standard stock solution) and/or by using separate weighings of synthetic mixtures of the test product components, using the proposed procedure. 4.2.3. VALIDATION OF THE PROPOSED METHOD The developed method was validated as per ICH guidelines. Here are the following parameters to be considered for validation. Specificity The chromatograms obtained from the blank solution were compared with those obtained from the drug with the most commonly used additives for accessing specificity of the drug. To prepare the blank solution the additives other than the drug were mixed in the mobile phase. The ratio between drug and additives were maintained in the same manner as that in the commercial formulations available of the drug. The present specificity study used some commonly used additives such as starch, lactose, ethyl cellulose, micro crystalline cellulose, magnesium stearate, hydroxyl propyl methyl cellulose and colloidal silicon dioxide. Before injecting in to HPLC column the mixtures should be filtrated through 0.45µ membrane filter. Precision Precision was determined as both repeatability and intermediate precision, in accordance with ICH guidelines. Repeatability of sample injection was determined as intra day variation and intermediate variation was determined by measurement of inter day variation. For these determinations, three concentrations of the solutions of mesalamine API were used. Accuracy Accuracy was best determined by the standard addition method. Previously analyzed samples of mesalamine API were added with standard drug solutions at three different levels 86

and are analyzed by the proposed method. Recovery (%) and RSD (%) were calculated for each concentration. Robustness The concept of robustness of an analytical procedure has been defined by the ICH as a measure of its capacity to remain unaffected by small but deliberate variations in method parameters. The robustness of a method is the ability to remain unaffected by small changes in parameters such as ph of the mobile phase, temperature, % organic solvent strength and buffer concentration etc. To determine the robustness of the method experimental conditions are purposely altered and chromatographic characters are evaluated. Influence of small changes in chromatographic conditions such as change in flow rate (±0.1 ml/min), temperature (±2 C), wavelength of detection (±2 nm) and water content in mobile phase (±2%) were studied to determine the robustness of the method. Stability of the analytical solution A study to establish bench top stability of the drug solution was conducted. A freshly prepared working standard solution (50 µg/ml of the drug) was analysed immediately and at different time intervals. The tailing factor, theoretical plates and difference in percent assay at different time intervals were calculated. Limit of Detection and Limit of Quantification The limit of detection (LOD) of an analytical method may be defined as the concentration, which gives rise to an instrument signal that is significantly different from the blank. For spectroscopic techniques or other methods that rely upon a calibration curve for quantitative measurements, the IUPAC approach employs the standard deviation of the intercept (Sa), which may be related to LOD and the slope of the calibration curve, b, by LOD = 3 Sa / b The LOQ is the concentration that can be quantitated reliably with a specified level of accuracy and precision. The LOQ represents the concentration of analyte that would yield a signal-to-noise ratio of 10. LOQ = 10 Sa / b Where, Sa is the standard deviation of the peak area ratio of analyte to IS (5 injections) of the drugs and b is slope of the corresponding calibration curve. 87

System precision and System suitability System precision and system suitability studies were carried out by injecting six replicates of the working standard solution. The % RSD for the peak areas obtained was calculated. 4.2.4. ESTIMATION OF THE DRUG FROM DOSAGE FORMS Satisfactory results obtained with the method development for the assay of mesalamine have prompted the author to attempt its applicability for the estimation of the drug in its formulations. Preparation of sample solution and recovery study From Tablet dosage forms Twenty mesalamine tablets were weighed and the average weight was calculated. Accurately sample equivalent to 10 mg of mesalamine was weighed and transferred into a 100 ml volumetric flask. Fifty milliliters of diluent was added and sonicated to dissolve it completely and made volume up to the mark with the same diluent. Mixed well and filtered through 0.45 mm filter. Further 0.1 ml was pipette out of the above stock solution into a 10 ml volumetric flask and diluted up to the mark with diluent. Mixed well and filtered through 0.45 mm filter. An aliquot of this solution was injected into HPLC system. Peak area of mesalamine was measured for the determination. 88

4.3. DEVELOPMENT AND VALIDATION OF AN RP-HPLC METHOD FOR THE DETERMINATON OF MELOXICAM 4.3.1. MATERIALS AND METHODS Instrumentation The author had attempted to develop a liquid chromatographic method for quantitative estimation of Meloxicam. Quantitative determination was performed on chromatographic system comprised of Analytical technologies ltd UV 2230 UV vis detector. Data integration was carried out using A-4000 version software. Samples were injected into Develosil ODS HG-5 RP C18 (5 μm, 15 cm 4.6 mm i.d) column. An Analytical technologies ltd.sonicator was used for enhancing the dissolution of the compounds. A Wenster digital ph meter was used for ph adjustment.class A Borosil glassware (Borosil, India) was employed for volumetric and general purpose in the entire study. CHEMICALS AND REAGENTS: Pure Meloxicam (MEL) used as working standards, was purchased from Yarrow chem. Products, Mumbai, India. Tablets containing 15 mg and 7.5 mg of meloxicam (MUVERA15 and MOVAC) were obtained from Apollo Pharmaceuticals Pvt. Ltd, Visakhapatnam, India and used within their shelf life period. Acetonitrile and water (HPLC-grade) were purchased from Merck, India. All other chemicals and reagents employed were of analytical grade, and purchased from Merck, India.dia. Anhydrous sodium dihydrogen phosphate and Orthophosphoric acid was purchased from Sd fine-chem ltd; Mumbai Preparation of phosphate buffer (ph 3.4) 1.452gms of anhydrous di-potassium hydrogen phosphate, 7.601gms of anhydrous sodium dihydrogen phosphate and 4.8gms of sodium chloride was accurately weighed and were dissolved in small amount of distilled water and sonicated for 15 min and volume made to 1000ml with distilled water and ph was adjusted to 3.4 with ortho phosphoric acid and the resulting solution was filtered using 0.45μ filter paper. Preparation of the mobile phase A mixture consisting of HPLC grade acetonitrile and phosphate buffer (ph 3.4) in a ratio 60: 40 was prepared in a 500 ml flask and used as the mobile phase and the diluent as well. 89

Preparation of stock and working standard solutions of Meloxicam The stock solution was prepared by transferring 100 mg of Meloxicam into 100 ml volumetric flask. Then it was added with small amount of diluent [acetonitrile: water (50:50)], and the mixture was sonicated to dissolve and made up to volume with mobile phase. From this stock solution different concentrations were prepared to give final concentrations of 20 120 μg/ml for standard calibration curve. 4.3.2. OPTIMIZATION OF THE CHROMATOGARPHIC CONDITIONS AND METHOD DEVELOPMENT For developing the method, a systematic study for optimisation of chromatographic condition was taken up. This was done by varying one parameter at a time and keeping all other conditions constant. The following studies were conducted for this purpose. A develosil non-polar C 18 (250 mm x 4.6 mm; 5µ) column was chosen as the stationary phase for this study. The mobile phase and the flow rate In order to effect ideal separation of the drug under isocratic conditions, mixtures of commonly used solvents like water, methanol and acetonitrile with or without different buffers in different combinations were tested as mobile phases on a develosil RP-C18 (250x4.6mm; 5 µ) column stationary phase with varying flow rate ( 0.8-1.2 ml / min) and wavelength of detections. Linearity and Construction of calibration curve The quantitative determination of the drug was accomplished by the external standard method. The mobile phase was filtered through a 0.45 µ membrane filter before use. The flow rate of the mobile phase was adjusted to the result of optimized method. The column was equilibrated with the mobile phase for at least 30 min prior to the injection of the drug solution. The column temperature was maintained at 25±1 0 C throughout the study. Linearity of the peak area response was determined by taking 6 replicate measurements at different concentration points. Working dilutions of Meloxicam in different range were prepared by taking suitable dilutions of the standard solutions in different 10 ml volumetric flasks and diluted up to the mark with the mobile phase. Twenty µl of the dilutions were injected six 90

times each concentration in to the column. The drug in the eluents was monitored at its wavelength of maximum and the corresponding chromatograms were obtained. From the chromatograms the mean peak areas were noted and a plot of concentrations over the peak areas was constructed. The regression of the plot was computed by least squares method. 4.3.3. VALIDATION OF THE PROPOSED METHOD The developed method was validated as per ICH guidelines. Here are the following parameters to be considered for validation. Specificity The chromatograms obtained from the blank solution were compared with those obtained from the drug with the most commonly used additives for accessing specificity of the drug. To prepare the blank solution the additives other than the drug were mixed in the mobile phase. The ratio between drug and additives were maintained in the same manner as that in the commercial formulations available of the drug. The present specificity study used some commonly used additives such as starch, lactose, ethyl cellulose, micro crystalline cellulose, magnesium stearate, hydroxyl propyl methyl cellulose and colloidal silicon dioxide. Before injecting in to HPLC column the mixtures should be filtrated through 0.45µ membrane filter. Precision Precision was determined as both repeatability and intermediate precision, in accordance with ICH guidelines. Repeatability of sample injection was determined as intraday variation and intermediate variation. For these determinations, single concentration (20 μg/ml) at different time intervals and different days, of the solution of Meloxicam API was used. Accuracy Accuracy was best determined by the standard addition method. Previously analyzed samples of Meloxicam API were added with standard drug solutions and are analyzed by the proposed method. Recovery (%), RSD (%) and bias (%) were calculated for each concentration. Accuracy is reported as percentage bias. 91

Robustness The concept of robustness of an analytical procedure has been defined by the ICH as a measure of its capacity to remain unaffected by small but deliberate variations in method parameters. To determine the robustness of the method experimental conditions are purposely altered and chromatographic characters are evaluated. Influence of small changes in chromatographic conditions such as change in flow rate (± 0.1 ml/min), wavelength of detection (±2 nm) and acetonitrile content in mobile phase (±2%) were studied to determine the robustness of the method. Stability of the analytical solution A study to establish bench top stability of the drug solution was conducted. A freshly prepared working standard solution (100 µg/ml of the drug) was analysed immediately and at different time intervals. The tailing factor, theoretical plates and difference in percent assay at different time intervals were calculated. Limit of Detection and Limit of Quantification The limit of detection (LOD) of an analytical method may be defined as the concentration, which gives rise to an instrument signal that is significantly different from the blank. For spectroscopic techniques or other methods that rely upon a calibration curve for quantitative measurements, the IUPAC approach employs the standard deviation of the intercept (Sa), which may be related to LOD and the slope of the calibration curve, b, by LOD = 3 Sa / b The LOQ is the concentration that can be quantitated reliably with a specified level of accuracy and precision. The LOQ represents the concentration of analyte that would yield a signal-to-noise ratio of 10. LOQ = 10 Sa / b Where, Sa is the standard deviation of the peak area ratio of analyte to IS (5 injections) of the drugs and b is slope of the corresponding calibration curve. System precision and System suitability System precision and system suitability studies were carried out by injecting six replicates of the working standard solution. The % RSD for the peak areas obtained was calculated. 92

4.3.4. ESTIMATION OF THE DRUG FROM DOSAGE FORMS Satisfactory results obtained with the method development for the assay of meloxicam have prompted the author to attempt its applicability for the estimation of the drug in its formulations. Preparation of sample solution and recovery study From Tablet dosage forms Twenty tablets were accurately weighed and crushed to a fine powder in a mortar in each of the marketed formulation separately. An amount of the powder equivalent to 100 mg was transferred into a 100 ml volumetric flask and 10 ml of diluent was added to it followed by 10 ml of 0.1 N NaOH. The mixture was sonicated to dissolve the exipients and then made up to volume with mobile phase. Following 15 min of mechanical shaking, it was kept in an ultrasonic bath for 15 mins, and the solution was filtered through a 0.45 μm filter paper. Suitable aliquots (1 ml each) of the filtered solution were transferred to 50 ml volumetric flasks and made up to volume with mobile phase to yield six concentrations of Meloxicam (20 μg/ml). A 20 μl volume of the sample solution was injected into the chromatographic system, six times, under optimized chromatographic conditions. The peak areas were measured at 268 nm and concentrations in the samples were determined by interpolation from standard calibration curve of each drug previously obtained. 93

4.4. DEVELOPMENT AND VALIDATION OF AN RP-HPLC METHOD FOR THE DETERMINATION OF CLOPIDOGREL 4.4.1. MATERIALS AND METHODS Instrumentation The author had attempted to develop a liquid chromatographic method for the quantitative estimation of clopidogrel. The chromatographic system used was an Analyti-cal Technologies Ltd UV 2230 UV-Vis detector. Datawere integrated with A-4000 version software. Sam-ples were injected into a Develosil ODS HG-5 RPC18 (15 cm 4.6 mm, i.d. 5 µm) column. An Analytical Technologies Ltd. sonicator was used to enhance dissolution of the compounds. A Wenster digital ph meter was used to adjust the ph. A 20 μl Hamilton injection syringe was employed for sample injection. Class A Borosil glassware (Borosil, India) was employed for volumetric and general purpose in the entire study. CHEMICALS AND REAGENTS: Pure clopidogrel, used as a working standard, wasobtained from Aurobindo Pharma, Hyderabad, India.Tablets containing clopidogrel (Clopilet and Plavix )were obtained from Apollo Pharmaceuticals Pvt. Ltd.,Visakhapatnam, India, and used within their shelf life.acetonitrile and water (HPLC grade) were purchased from Merck, India. All other chemicals and reagentsemployed were of analytical grade and purchased from Desai Chemicals, Visakhapatnam, India. Preparation of phosphate buffer (ph 2.85) Accurately weighed di-potassium hydrogen ortho-phosphate and potassium dihydrogen ortho-phosphate were dissolved in small amount of distilled water and sonicated for 15 min and volume made to final required with distilled water and ph was adjusted to 2.85 with glacial acetic acid and the resulting solution was filtered using 0.45μ filter paper. Preparation of Mobile Phase The mobile phase was prepared by mixing acetonitrile and phosphate buffer with ph 2.85 in the ratio of 65:35 % (v/v) and degassed by ultra-bath sonicator for 30 min and passed through 0.45 μ filter paper. 94

Preparation of Stock Solution The stock solution was prepared by transferring100 mg of clopidogrel into a 100-mL volumetric flask, to which a small amount of diluent was added; the mixture was sonicated to dissolution and made up to volume with the mobile phase. Final concentrations of 10 60 µg/ml were prepared from the stock solution for calibration of the standard curve. 4.4.2. OPTIMIZATION OF THE CHROMATOGARPHIC CONDITIONS AND METHOD DEVELOPMENT For developing the method, a systematic study for optimisation of the chromatographic condition was taken up. This was done by varying one parameter at a time and keeping all other conditions constant. The following studies were conducted for this purpose. A develosil non-polar C 18 (250 mm x 4.6 mm;5µ) column was chosen as the stationary phase for this study. The mobile phase and the flow rate For ideal separation of drug in isocratic mode different commonly used solvents (water, methanol, acetonitrile etc.) with or without buffers were tried on a phenomenex RP C-18 (250 X 4.6; 5µ) column with varying flow rates (0.8-1.2 ml/min) and wavelength of detections. Linearity and construction of calibration curve The external standard method was applied to estimate the drug in bulk and formulation. A 0.45 µ membrane filter was used for filtration before use. As per the results obtained for the optimization the flow rate was adjusted. During the entire study the column temperature was maintained at 25±1 C. By taking six replicate measurements at different concentration points linearity of the peak area response was estimated. Suitable dilutions of standard Clopidogrel drug solution were taken in different 10 ml volumetric flasks with volume make up to the mark with mobile phase to give working dilutions of clopidogrel. Twenty micro litres of each concentration was injected six times in to the HPLC column. Monitoring of the drug in the eluents was detected at its wavelength of maximum and the respective chromatograms were observed. The peak areas of each concentration chromatogram were noted and the graph of concentration over the peak areas was plotted. By applying least squares method the regression of the method was calculated. 95

4.4.3. VALIDATION OF THE PROPOSED METHOD The developed method was validated as per ICH guidelines. Here are the following parameters to be considered for validation. Specificity The chromatograms obtained from the blank solution were compared with those obtained from the drug with the most commonly used additives for accessing specificity of the drug. To prepare the blank solution the additives other than the drug were mixed in the mobile phase. The ratio between drug and additives were maintained in the same manner as that in the commercial formulations available of the drug. The present specificity study used some commonly used additives such as starch, lactose, ethyl cellulose, micro crystalline cellulose, magnesium stearate, hydroxyl propyl methyl cellulose and colloidal silicon dioxide. Before injecting in to HPLC column the mixtures should be filtrated through 0.45µ membrane filter. Precision Precision was determined as both repeatabilityand intermediate precision, in accordance with ICH guidelines. The repeatability of sample injection was determined as intra-day and intermediate variation. For these determinations, a single concentration (40 µg /ml) of clopidogrel API was tested at different intervals and on different days. Accuracy Accuracy was best determined by the standard addition method. Samples of clopidogrel previously analyzed for active pharmaceutical ingredient (API) were added with standard drug solutions and analyzed by the proposed method. Recovery (%), relative standard deviation (RSD) (%) and bias (%) were calculated for each concentration. Robustness The concept of the robustness of an analytical procedure has been defined by the ICH as a measure of its capacity to remain unaffected by small but deliberate variations in method parameters. To determine the robustness of the method, experimental conditions are purposely altered, and chromatographic character-istics are evaluated. The influence of small changes in chromatographic conditions, such as in flow rate (±0.1 ml /min), the wavelength 96

for detection (±2 nm)and acetonitrile content in the mobile phase (±2%), were studied to determine the robustness of the method. Stability of the analytical solution A study to establish bench top stability of the drug solution was conducted. A freshly prepared working standard solution (50 µg/ml of the drug) was analysed immediately and at different time intervals. The tailing factor, theoretical plates and difference in percent assay at different time intervals were calculated. Limit of Detection and Limit of Quantification The limit of detection (LOD) of an analytical method may be defined as the concentration, which gives rise to an instrument signal that is significantly different from the blank. For spectroscopic techniques or other methods that rely upon a calibration curve for quantitative measurements, the IUPAC approach employs the standard deviation of the intercept (Sa), which may be related to LOD and the slope of the calibration curve, b, by LOD = 3 Sa / b The LOQ is the concentration that can be quantitated reliably with a specified level of accuracy and precision. The LOQ represents the concentration of analyte that would yield a signal-to-noise ratio of 10. LOQ = 10 Sa / b Where, Sa is the standard deviation of the peak area ratio of analyte to IS (5 injections) of the drugs and b is slope of the corresponding calibration curve. System precision and System suitability System precision and system suitability studies were carried out by injecting six replicates of the working standard solution. The % RSD for the peak areas obtained was calculated. 4.4.4. ESTIMATION OF THE DRUG FROM DOSAGE FORMS Satisfactory results obtained with the method development for the assay of clopidogrel have prompted the author to attempt its applicability for the estimation of the drug in its formulations. 97

Preparation of sample solution and recovery study 1. From Tablet dosage forms Twenty tablets of each marketed formulation were accurately weighed and crushed to a fine powder in a mortar. Then, 100 mg of the powder were transferred into a 100-mL volumetric flask, to which 25 ml of diluent were added, followed by 10 ml of o-phosphoric acid. The mixture was sonicated to dissolve the excipients and then made up to volume with mobile phase. After 15 min of mechanical shaking, the solution was maintained in an ultrasonic bath for 15 min and then filtered through0.45- µm filter paper. Suitable aliquots of the filtered solution were transferred to a volumetric flask and made up to volume with mobile phase (40µg/mL) to yield six concentrations of clopidogrel. A 20- µl volume of the sample solution was injected into the chromatographic system six times under optimized chromatographic conditions. The peak areas were measured at 225 nm and the concentrations in the samples were determined by interpolation from previously obtained calibration plots for each drug. 98

4.5. DEVELOPMENT AND VALIDATION OF AN RP-HPLC METHOD FOR THE DETERMINATON OF SIMVASTATIN 4.5.1. MATERIALS AND METHODS Instrumentation The author had attempted to develop a liquid chromatographic method for quantitative estimation of Simvastatin. The chromatographic system (Model No.1575) used comprised of Hitachi L2130 with UV-Vis detector. Data integration was carried out using A- 4000 version software. Samples were injected into Develosil ODS HG-5 RP C18 (5µm, 15cmx4.6mm i.d) column. An Analytical technologies ltd.sonicator was used for enhancing the dissolution of the compounds. A Wenster digital ph meter was used for ph adjustment. A Shimadzu SM 114 electronic balance was used for weighing the materials. Class A Borosil glassware (Borosil, India) was employed for volumetric and general purpose in the entire study. Chemicals and reagents: Pure Simvastatin used as working standards, was obtained from Aurobindo pharma, Hyderabad, India. Tablet containing Simvastatin (Biosim 20) was obtained from Apollo Pharmaceuticals Pvt. Ltd, Visakhapatnam, India and used within its shelf life period. Acetonitrile and water (HPLC-grade) were purchased from Loba chem.., Mumbai, India. All other chemicals and reagents employed were of analytical grade, and purchased from Desai chemicals, Visakhapatnam India. Preparation of phosphate buffer (ph 3.0) Accurately weighed di-potassium hydrogen ortho-phosphate and potassium dihydrogen ortho-phosphate were dissolved in small amount of distilled water and sonicated for 15 min and volume made to final required with distilled water and ph was adjusted to 3.0 with O- Phosphoric acid and the resulting solution was filtered using 0.45μ filter paper. Mobile phase preparation The mobile phase was prepared by taking a mixture containing Acetonitrile and phosphate buffer (PH 3.0) in a ratio 85:15 in to a 500 ml volumetric flask. The same solution was also used as the diluents for preparing further dilutions. 99

Simvastatin stock and working standard solutions Accurately weighed quantity of 100 mg Simvastatin was taken in a 100 ml volumetric flask containing 10 ml of the mobile phase. The solution was made up to 100 ml with the mobile phase to give 1 mg/ml followed by sonication for twenty minutes. From the above solution various volumes were taken to prepare working standard solutions in the range of 10-100 µg/ ml for Simvastatin drug sample. 4.5.2. OPTIMIZATION OF THE CHROMATOGARPHIC CONDITIONS AND METHOD DEVELOPMENT For developing the method, a systematic study for optimisation of chromatographic condition was taken up. This was done by varying one parameter at a time and keeping all other conditions constant. The following studies were conducted for this purpose. A phenomenex non-polar C 18 (250 mm x 4.6 mm; 5µ) column was chosen as the stationary phase for this study. The mobile phase and the flow rate For ideal separation of drug in isocratic mode different commonly used solvents (water, methanol, acetonitrile etc.) with or without buffers were tried on a Develosil ODS HG-5 RP C-18 (250 X 4.6; 5µ) column with varying flow rates (0.8-1.2 ml/min) and wavelength of detections. Linearity and construction of calibration curve The external standard method was applied to estimate the drug in bulk and formulation. A 0.45 µ membrane filter was used for filtration before use. As per the results obtained for the optimization the flow rate was adjusted. During the entire study the column temperature was maintained at 25±1 C. By taking six replicate measurements at different concentration points linearity of the peak area response was estimated. Suitable dilutions of standard Simvastatin drug solution were taken in different 10 ml volumetric flasks with volume make up to the mark with mobile phase to give working dilutions of Simvastatin. Twenty micro litres of each concentration was injected six times in to the HPLC column. Monitoring of the drug in the eluents was detected at its wavelength of maximum and the respective chromatograms were observed. The peak areas of each concentration chromatogram were 100

noted and the graph of concentration over the peak areas was plotted. By applying least squares method the regression of the method was calculated. 4.5.3. VALIDATION OF THE PROPOSED METHOD The developed method was validated as per ICH guidelines. Here are the following parameters to be considered for validation. Specificity The chromatograms obtained from the blank solution were compared with those obtained from the drug with the most commonly used additives for accessing specificity of the drug. To prepare the blank solution the additives other than the drug were mixed in the mobile phase. The ratio between drug and additives were maintained in the same manner as that in the commercial formulations available of the drug. The present specificity study used some commonly used additives such as starch, lactose, ethyl cellulose, micro crystalline cellulose, magnesium stearate, hydroxyl propyl methyl cellulose and colloidal silicon dioxide. Before injecting in to HPLC column the mixtures should be filtrated through 0.45µ membrane filter. Precision Precision was determined as both repeatability and intermediate precision, in accordance with ICH guidelines. Repeatability of sample injection was determined as intraday variation and interday variation. For these determinations, single concentration (30 μg/ml) at different time intervals and different days, of the solution of Simvastatin API was used. Accuracy Accuracy was best determined by the standard addition method. Previously analyzed samples of Simvastatin API were added with standard drug solutions at three different concentration levels and are analyzed by the proposed method. Recovery (%), RSD (%) was calculated for each concentration. Robustness The concept of robustness of an analytical procedure has been defined by the ICH as a measure of its capacity to remain unaffected by small but deliberate variations in method 101

parameters. To determine the robustness of the method experimental conditions are purposely altered and chromatographic characters are evaluated. Influence of small changes in chromatographic conditions such as change in flow rate (± 0.1ml/min), wavelength of detection (±2nm) and acetonitrile content in mobile phase (±2%) were studied to determine the robustness of the method. Stability of the analytical solution A study to establish bench top stability of the drug solution was conducted. A freshly prepared working standard solution (50 µg/ml of the drug) was analysed immediately and at different time intervals. The tailing factor, theoretical plates and difference in percent assay at different time intervals were calculated. Limit of Detection and Limit of Quantification The limit of detection (LOD) of an analytical method may be defined as the concentration, which gives rise to an instrument signal that is significantly different from the blank. For spectroscopic techniques or other methods that rely upon a calibration curve for quantitative measurements, the IUPAC approach employs the standard deviation of the intercept (Sa), which may be related to LOD and the slope of the calibration curve, b, by LOD = 3 Sa / b The LOQ is the concentration that can be quantitated reliably with a specified level of accuracy and precision. The LOQ represents the concentration of analyte that would yield a signal-to-noise ratio of 10. LOQ = 10 Sa / b Where, Sa is the standard deviation of the peak area ratio of analyte to IS (5 injections) of the drugs and b is slope of the corresponding calibration curve. System precision and System suitability System precision and system suitability studies were carried out by injecting six replicates of the working standard solution. The % RSD for the peak areas obtained was calculated. 102

Estimation of the drug from dosage forms Satisfactory results obtained with the method development for the assay of simvastatin have prompted the author to attempt its applicability for the estimation of the drug in its formulations. Preparation of sample solution and recovery study From Tablet dosage forms Twenty tablets were accurately weighed and crushed to a fine powder in a mortar for the marketed formulation. An amount of the powder equivalent to 100 mg was transferred into a 100 ml volumetric flask and 15 ml of diluent was added to it followed by 10 ml of methanol. The mixture was sonicated to dissolve the exipients and then made up to volume with mobile phase. Following 15 min of mechanical shaking, it was kept in an ultrasonic bath for 15 mins, and the solution was filtered through a 0.45 μm filter paper. Suitable aliquots of the filtered solution were transferred to a volumetric flask and made up to volume with mobile phase to yield six concentrations of Simvastatin (30 μg/ml). A 20 μl volume of the sample solution was injected into the chromatographic system, six times, under optimized chromatographic conditions. The peak areas were measured at 236 nm and concentrations in the samples were determined by interpolation from calibration plots of each drug previously obtained. 103