7. Stability indicating analytical method development and validation of Ramipril and Amlodipine in capsule dosage form by HPLC.

Transcription

1 7. Stability indicating analytical method development and validation of and in capsule dosage form by HPLC. 7.1 INSTRUMENTS AND MATERIALS USED INSTRUMENTS 1. Shimadzu LC-2010 CHT with liquid chromatograph 2. Millipore (Q-GARD Milli-Q) 3. Remi Laboratory Centrifuge 4. Life Care Equipment Pvt. Ltd. (Sonicator) 5. Chromatographic software: LC Solution 6. Photo stability chamber: - SVI equipments, Germany. 7. Hot air oven: - Labline, India. 8. Analytical Balance: - electronic analytical balance (Shimadzu) 9. ph Meter: - Labindia, India Working standard Table 7.1 Working standard Sr. No. Material Name Potency / Purity 1. working standard 99.9% 2. working standard 99.4% 3. Placebo for and capsules N/A Page 118

2 7.1.3 Chemicals used Table 7.2 Chemicals used Chemicals Grade Manufacturer Acetonitrile HPLC Merck, Rankem Methanol HPLC Merck, Rankem Triethylamine HPLC Rankem NaOH GR Merck Conc. HCl GR Merck Milli-Q Water Milli- Pore In House MARKET FORMULATION USED Table 7.3:- List of Company s formulations used Brand Naprix A Company Name Libbs Pharmaceuticals, Label Claim mg mg 7.2 DEVELOPMENT OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY FOR THE ESTIMATION OF RAMIPRIL AND AMLODIPINE IN CAPSULE DOSAGE FORMS Observation and determination for physical and chemical properties of and. Page 119

3 7.2.1 PHYSICAL PROPERTIES Solubility - Solubility was determined by taking 100 mg of and in 50 ml volumetric flask, adding required quantity of solvent (as per pharmacopoeial procedure) at room temperature and shaken for few minutes. Table 7.4:- Solubility Study of and Solubility Solvent Water Sparingly soluble Slightly soluble Methanol Freely Soluble Freely Soluble Acetonitrile Very Slightly soluble Very Slightly soluble CHEMICAL IDENTIFICATION: - It is the test to identify the material done by various method e.g. Infrared spectroscopy (IR). Identification solely by a single chromatography retention time, for e.g., is not regarded as being specific. However the use of the two chromatographic procedures, where the separation is based on different principles or a combination of tests into a single procedure, such as HPLC- UV (ultraviolet) diode array, HPLC-MS (mass spectroscopy), or GC (Gas Chromatography) and MS generally accepted. If the new drug substance is a salt, identification testing should be specific for the individual ions. An identification test that is specific for the salt itself should suffice. The identification and specification testing or performance of and capsule is done by HPLC- UV (ultraviolet) HIGH PERFORMANCE LIQUID CHROMATOGRAPHY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY- WORK Page 120

4 Table 7.5: - Observation and remarks of method development for and Sr. No. Trails Taken Observation Remarks 1 Acetonitrile:water (60:40 v/v) Flow rate 1.0 ml/min Column:- Inertsil C18 The elution of the main Peak was late. Not Satisfactory 2 Acetonitrile:water (70:30 v/v) Flow rate 1.0 ml/min Column:- Inertsil C18 The elution of the main Peak was late. Not Satisfactory Buffer Acetonitrile 3 (70:30%v/v) Flow rate 1.0 ml/min Buffer: - Sodium dihydrogen phosphate Buffer ph 3.0 Peak was found to be very early and splitting. Not Satisfactory Column:-Inertsil C18 Buffer Acetonitrile (70:30%v/v) Peak shape was found 4 Flow rate 1.0 ml/min Buffer: - Potasium dihydrogen to be good but, impurities merge with Not Satisfactory phosphate Buffer ph 3.0 the main peak Column:- Inertsil C18 Buffer Acetonitrile (70:30%v/v) Peak shape was found 5 Flow rate 1.0 ml/min Buffer: - Potasium dihydrogen to be good but, impurities merge with Not Satisfactory phosphate Buffer ph 3.0 the main peak Column:- Inertsil C8 Buffer Acetonitrile Peak was found to be 6 (70:30%v/v) merged and not better Not Satisfactory Flow rate 1.0 ml/min resolution Page 121

5 Buffer: - Potasium dihydrogen phosphate Buffer ph 3.0 Column:- Hypersil BDS C8 Buffer Acetonitrile 7 (70:30%v/v) Flow rate 1.0 ml/min Buffer: - Potasium dihydrogen phosphate Buffer ph 2.5 Peak was found to be very late due to lower flow rate Not Satisfactory Column:- Hypersil BDS C8 Buffer Acetonitrile (70:30%v/v) Peak was found to be 8 Flow rate 1.2 ml/min Buffer: - Potasium dihydrogen earlier than previous but Resolution was Not Satisfactory phosphate Buffer ph 2.5 not better Column:- Hypersil BDS C8 Buffer Acetonitrile 9 (70:30%v/v) Flow rate 1.2 ml/min Buffer: - Potassium dihydrogen phosphate, 1ml triethylamine/ltr, ph 2.5 Resolution was better but peak shape of was not good Not Satisfactory Column:- Hypersil BDS C8 Buffer Acetonitrile (70:30%v/v) 10 Flow rate 1.2 ml/min Buffer: - Potassium dihydrogen phosphate, 5ml Better Resolution with good peak shape Satisfactory triethylamine/ltr, ph 2.5 Column:- Hypersil BDS C8 Page 122

6 Table 7.6:- Optimized Condition ware found to be as follows:- Column: Hypersil, BDS, C8, 150 mm x 4.6mm, 5µ. Flow rate: Detection: 1.2 ml/minute At 210 nm Column Temperature: 55 C Injection Volume: 10 µl Runtime: 25 minutes mau / PDA Multi / minutes Figure 7.1:-Chromatogram indicating untreated sample preparation of and Preparation of buffer:- Buffer: Dissolve 3.42 g of potassium dihydrogen phosphate in 1000ml of water add 5 ml Triethylamine and adjust with 10% Ortho-phosphoric acid to a ph of 2.5, and mix. Mobile phase: Use Buffer and Acetonitrile in the ratio of 70:30, sonicate and filter through 0.45µ filter Diluent: Water: Methanol in the ratio of 50:50 Page 123

7 7.2.4 QUANTITATIVE DETERMINATION OF RAMIPRIL AND AMLODIPINE CAPSULES Procedure Sample preparation: Remove as completely as possible and weigh the content not less than 20 capsules and mix. Transfer and weigh accurately about granules equivalent to 12.5mg of and 25mg of into 500ml volumetric flask. Add about 30ml of diluent and shake mechanically to disperse the granules till granules dissolve completely and then add about 300ml of diluent and sonicate for 45 minutes with intermittent shaking. Allow to attain room temperature, make up the volume with diluent and centrifuge the solution at 3000rpm for 15minutes. Filter the solution through 0.45µ membrane filter (Nylon). Standard preparation: Stock solution of (equivalent to 0.25mg/ml) and (equivalent to 0.50mg/ml) were prepared in diluent. Each of 5 ml, standard stock solution of and, were transferred using A-grade bulb pipettes into 50ml volumetric flask and made up to the volume with diluent to yield final concentration 25µg/ml and 50µg/ml for and, respectively. Separately inject equal volume of the standard preparation and assay preparation in to the chromatograph, record the chromatogram, and measure the responses for the major peak due to and. Calculate the Assay in the formulation by taking following formula: %Assay (): Mean Sample Area of /Mean Standard Area of *Standard weight of /100*5/50*500/sample weight*avg. weight / Lable Claim of *Potency of Standard of %Assay (): Mean Sample Area of /Mean Standard Area of *Standard wt of /100*5/50*500/Sample weight*408.92/567.1*avg. weight/ Label Claim of *Potency of Standard of Page 124

8 7.2.5 METHOD VALIDATION Determination of and as API and in Capsule dosage form based on the separation using Isocratic-reversed phase HPLC with UV detection. Validation of proposed developed method is done and parameters of validation are as follows:- Specificity Linearity and range. Accuracy (Recovery) Precision Method Precision (Repeatability) Intermediate precision (Ruggedness) Solution stability Filter study Robustness System suitability SPECIFICITY: a) Check for interference from blank, placebo and impurities. Placebo Preparation for and Capsules ( mg):- Weigh and transfer accurately about mg placebo in 500 ml volumetric flask. Add about 30ml of diluent and shake mechanically. Then add about 300ml of diluent and sonicate for 45 minutes with intermittent shaking. Allow to attain room temperature, make up the volume with diluent and centrifuge the solution at 3000rpm for 15minutes. Filter the solution through 0.45µ membrane filter (Nylon). NOTE: Maintain the temperature of sonicator below 15 C. Sample Preparation for and Capsules ( mg):- Remove as completely as possible and weigh the content not less than 20 capsules and mix. Transfer and weigh accurately about granules equivalent to 12.5mg of and 25mg of into 500ml volumetric flask. Add about 30ml of diluent and shake mechanically to disperse the granules till granules dissolve completely and then add about 300ml of diluent and sonicate for 45 minutes with intermittent shaking. Allow to attain room temperature, make up the volume with diluent and centrifuge the solution at 3000rpm for 15minutes. Filter the solution through 0.45µ membrane filter (Nylon). NOTE: Maintain the temperature of sonicator below 15 C Page 125

9 Acceptance Criteria: 1) There should not be any interference from blank, placebo peaks with main peak. 2) The peak purity index for the main peak in standard preparation, sample preparation and sample spiked with impurities should be equal to or more than ) Absolute difference between assay of spiked sample and sample as such should not be more than 2.0%. b) Check for the interference from degradation product by stress study: Procedure: Subject API of and, Formulation and its placebo to acid, base, oxidation and thermal degradation. For each degradation, prepare a blank accordingly. The stress conditions shall be adjusted such that minimum 10% to maximum 30% degradation is achieved in at least two conditions. Stress conditions shall be as follows: 1. Acid degradation : Sample shall be refluxed for 12 hours in 5N HCl 2. Base degradation : Sample shall be refluxed for 12 hours in 5N NaOH 3. Oxidative degradation: Sample shall be refluxed for 12 hours in 30% H 2 O 2 4. Thermal degradation : Sample shall be heated at 100 C for 72 hours. Inject blank for each stress condition with their respective stressed placebo preparation, stressed API preparation and stressed sample preparation. Check for the separation of degraded impurities from msain peak. Check peak purity for the main peak in all the degraded sample preparation. Acceptance Criteria: 1. Degradation impurities in all degraded sample preparation should be separated from the main peak. 2. Peak purity for the main peak in all degraded sample preparation should be equal to or more than Page 126

10 LINEARITY AND RANGE: A) Linearity: The linearity of an analytical method is its ability to elicit test results that are directly, or by well defined mathematical transformation, proportional to the concentration of analyte in sample within a given working range. B) Range: The range of an analytical method is the interval between the upper and lower levels of analyte (including these levels) that have been demonstrated to be determined with precision, accuracy and linearity using the method as written. The range is normally expressed in the same units as test results (e.g. percent, part per million) obtained by the analytical method. Preparation of linearity solution: Stock solution: Weigh and transfer accurately about 25 mg of working standard and 70 mg of Besilate (equivalent to 50 mg of ) into 100 ml volumetric flask,. Dissolve and dilute with diluent and mix. Further dilute this stock solution as describe below. Table 7.7:- Dilutions for linearity Linearity Level Stock solution (ml) Diluted to (ml) Conc. (ppm) Concentration with respect to test concentration (%) Page 127

11 Procedure: Determine linearity at five levels over the range of 15 ppm (60% of test concentration) to 35 ppm (140% of test concentration). Inject the solutions in duplicate and plot a graph of mean area vs. concentration (%). Calculate correlation co-efficient (r), y-intercept, slope of regression line and residual sum of squares. Acceptance criteria: The correlation coefficient value should not be less than over the working range ACCURACY STUDY (RECOVERY): The accuracy of an analytical method is the closeness of test results obtained by that method to the true value. The true value is that result which would be observed in the absence of error. Accuracy may often be expressed as percent recovery by assay of known, added amounts of analyte. Accuracy is a measure of the exactness of the analytical method that is true for all practical purpose. Perform accuracy at 3 levels in triplicate, viz. 60%, 100% and 140% of the test concentration for 1mg and 4mg tablets. Procedure: Accuracy for and capsules: Stock solution: Weigh and transfer accurately about 125mg of working standard and 350 mg of Besilate working standard (equivalent to 250 mg of ) into a 500ml volumetric flask, dissolve and dilute with diluent and mix. Level-1 (60%): Weigh and transfer accurately about mg placebo in three different 500 ml volumetric flask and add to each flask 3.0 ml of stock solution and mix well. Add 30ml of diluent and sonicate for 45 minutes with intermittent shaking, then Cool to room temperature, make up the volume with diluent and mix. Filter the solution through 0.45µ Nylon filter. Level-2 (100%): Weigh and transfer accurately about mg placebo in three different 500 ml volumetric flask and add to each flask 5.0 ml of stock solution and mix well. Add 30ml of diluent and sonicate for 45 minutes with intermittent shaking, then Cool to Page 128

12 room temperature, make up the volume with diluent and mix. Filter the solution through 0.45µ Nylon filter. Level-3 (140%): Weigh and transfer accurately about mg placebo in three different 500 ml volumetric flask and add to each flask 7.0 ml of stock solution and mix well. Add 30ml of diluent and sonicate for 45 minutes with intermittent shaking, then Cool to room temperature, make up the volume with diluent and mix. Filter the solution through 0.45µ Nylon filter PRECISION: The precision of an analytical method is the closeness of agreement (degree of scatter) between series of measurements obtained from multiple sampling of the same homogeneous sample under prescribed condition. a) Method precision (Repeatability): Repeatability expresses the precision under the same operating conditions over a short interval of time. Procedure: Perform assay of six sample preparations under same conditions for and capsules as per the method under same conditions. Determine the assay. Calculate individual assay value, mean assay value, 95% confidence interval and % RSD Acceptance Criteria: The RSD of assay of six sample preparations should not be more than 2.0%. b) Intermediate Precision (Ruggedness): Intermediate precision expresses within-laboratory precision on a different day, by a different analyst, different HPLC instrument and different column lot using same lot of sample as specified under precision. Page 129

13 Procedure: Repeat the procedure followed for method precision on a different day, by a different analyst, using different column lot, using a different HPLC system. Also calculate individual assay value, mean assay value, 95% confidence interval and % RSD. Compare mean assay value with the average assay value obtained in method precision study. Record the difference of the average assays obtained. Acceptance Criteria: 1) The RSD of assay of six sample preparations should not be more than 2.0%. 2) The difference in the mean assay value obtained in intermediate precision study and method precision study should not be more than 2.0 % SOLUTION STABILITY: Prepare standard and sample as per method and determine the % assay (Initial). Store the standard and sample solution up to 48 hours at Room temperature and determine the %assay in stored standard and sample preparation after 12 hours and 24 hours storage against freshly prepared standard. The % assay of standard and sample preparation calculated after 24 hours and 48 hours storage will be compared with the initial value. Note: If required, performed solution stability at 2-8 C temperature based on result obtained from solution stability at 25 C. Acceptance criteria: The difference in the initial value of % assay and the values obtained at 12 hours and 24 hours of % assay should not be more than 2.0% ROBUSTNESS: Procedure: Change following parameters, one by one and observe their effect on system suitability test and assay. Page 130

14 Change flow rate by 10%. (i.e ml/minute and 1.32 ml/minute) Change the minor components in the mobile phase by 2% absolute or 30% relatively whichever is lower. Change the column temperature by +5 C. (i.e. 50 C and 60 C) Change in mobile phase ph by 0.2unit (i.e. ph 2.3 and ph 2.7) Change in column lot. Acceptance criteria: 1. System suitability criteria should meet the requirement as per method. 2. Difference in assay of normal condition and varied condition should not be more than 2% SYSTEM SUITABILITY: Perform the system suitability test as per method before performing any parameter METHOD VALIDATION - RESULTS Specificity a) Check for interference from blank, placebo and known Impurity Procedure: A blank, standard solution, placebo preparation, test solution were prepared and injected. The assay of unspiked sample, spiked sample and mean assay of spiked sample was calculated and recorded in Table-7.7. The peak purity of main peak in test solution was determined and recorded in Table-7.7. Table 7.8: - Peak purity data of degradation Sample Peak purity % Assay %Difference preparation Amlodi Amlodi Amlodi pine pine pine Placebo Unspiked Sample Spiked Sample Page 131

15 0.3 Detector A (210nm) + Capsules 0001 Retention Time Minutes Figure 7.2: - Chromatogram of Blank-Diluent uv PDA Figure 7.3: - Chromatogram of Placebo used for tablet formulation Detector A(210nm) + Capsules Rep5 Name Retention Time Minutes Figure 7.4: - Chromatogram of Sample as Such Page 132

16 mau 100 PDA Multi / / Figure 7.5: - Chromatogram of Sample treated with 0.1N HCl reflux for 30 mins at 100 C. minutes mau / PDA Multi / Figure 7.6: - Chromatogram of Sample treated with 0.1N NaOH for 5 mins at room temperature minutes mau 100 PDA Multi / / Figure 7.7: - Chromatogram of Sample treated with 5% H 2 O 2 at 100 C for 15minutes minutes Page 133

17 mau / PDA Multi / Figure 7.8: - Chromatogram of Sample treated with 100 C for 72 hours (Thermal Degradation) minutes mau / PDA Multi / Figure 7.9: - Chromatogram of Sample placed in photo light chamber for 1.2 million lux hours minutes Figure: 7.10 Typical HPLC chromatogram of Acid hydrolysis degraded sample preparation of Formulation (1), API (2) and API (3) Page 134

18 Figure: 7.11 Typical HPLC chromatogram of Alkali hydrolysis - degraded sample preparation of Formulation (1), API (2) and API (3) Figure: 7.12 Typical HPLC chromatogram of Oxidative hydrolysis - degraded sample preparation of Formulation (1), API (2) and API (3) Figure: 7.13 Typical HPLC chromatogram of Thermal - degraded sample preparation of Formulation (1), API (2) and API (3) Page 135

19 Acceptance Criteria: 1) There should not be any interference from blank, placebo peaks and impurity peaks with main peak (Active). 2) All impurity peaks should be well separated from each other and the main peak. 3) Peak purity for the main peak in reference solution, sample solution should be more than ) The difference of assay between unspiked and spiked sample should not more than 2.0% Conclusion There is no interference of blank, placebo with the main peak. All impurity peaks are well separate from each other and main peak. The peak purity results obtained are well within the acceptance criteria. Hence method is specific. a) Check for interference from forced degradation study: Table 7.9:- Degradation condition for API and API and Formulation Sample Name API, API and Formulation Acid Degradation (0.1N HCl at 100 C for 30 minutes) API, API and Formulation Base Degradation (0.1 N NaOH at room temperature for 5 minutes) API, API and Formulation Oxidative degradation (5% H 2 O 2 at 100 C for 15minutes) API, API and Formulation Thermal Degradation (100 C for 72 hours) API, API and Formulation Photo Degradation (1.2 million lux hours) Peak purity index Page 136

20 Linearity and Range Procedure System suitability was performed as per method. Linearity was determined at five levels over the range of 60% to 140% of test concentration. A standard Linearity solution was prepared to attain concentration of 60%, 80%, 100%, 120%, and 140% of the test concentration. Each linearity solution was injected in triplicate. The mean area at each level is calculated and a graph of mean area versus concentration is plotted. The correlation co-efficient (r), Y-intercept, slope of regression line, residual sum of squares are calculated and recorded. Table 7.10:- Linearity data of Level % Concentrat ion w.r.t.test Concentrat ion Concentr ation (µg/ml) Actual % Concentration w.r.t.test Concentration Area Mean Area Correlation coefficient (r) Slope of regression line Y-intercept Residual sum of squares Page 137

21 Linearity of Mean Area y = x R 2 = (%) Concentration w.r.t. Test Concentration Figure 7.14:- Standard linearity curve of by developed method Table 7.11:- Linearity data of Level % Conc. W.r.t. Test Conc. Concentrat ion (µg/ml) Actual %Co nc.w.r.t.test Conc. Area Mean Area Correlation coefficient (r) Slope of regression line Y-intercept Residual sum of squares Page 138

22 Linearity of Mean Area y = x R 2 = (%) Concentration w.r.t. Test Concentration Figure 7.15:- Standard linearity curve of by developed method Acceptance criteria: The correlation coefficient value should not be less than Conclusion The areas obtained are directly proportional to the concentration of analyte in the sample. The method is linear in the specified range Precision A) Repeatability Procedure: Six replicate injections of reference solution were injected. SD, %RSD was calculated. Page 139

23 Table 7.12:- System precision data - Analyst-1 Injection No. Area Mean % RSD Acceptance criteria: % RSD of six replicate injections should not more than 2.0% Conclusion: The results obtained lies well within the acceptance criteria. B) Intermediate Precision (Ruggedness) Procedure: The procedure followed for method precision was repeated on a different day; by different analyst, using a different HPLC system and different column lot number. Individual assay value, mean assay value, SD, %RSD and 95% confidence interval of result was calculated and recorded. Also the difference in the mean assay value of method precision and intermediate precision was found and recorded. Page 140

24 Table 7.13:- Intermediate precision data Analyst- 2 Injection No. Area Mean % RSD Acceptance Criteria: i) RSD of intermediate precision should not more than 2.0% ii) The difference between assay of method precision and intermediate precision should not be more than 2.0% Conclusion The results obtained are well within the acceptance criteria. Therefore method is precise. Comparison:- Table 7.14:- Precision data for and Replicate Repeatability Intermediate Precision Repeatability Intermediate Precision Page 141

25 Statistics-Set level Mean % RSD Statistics-Overall Mean % RSD % Difference of two Means Accuracy Procedure: System suitability was performed as per method. The accuracy of the method was established at three levels in the range of % of the working concentration of sample. Calculated amount of working standard was added in placebo to attain 60%, 100% and 140% of the working concentration. Triplicate preparation was prepared for each level and each preparation was injected in duplicate. % Recovery, mean recovery and %RSD was calculated at each level and recorded. Table 7.15:- Accuracy data for Amount Recovery Level Replicate Added (µg/ml) Found (µg/ml) % Recovery Mean % RSD Level-1 (60%) Level-2 (100%) Level-3 (140%) Page 142

26 Table 7.16:- Accuracy data for Amount Recovery Level Replicate Added (µg/ml) Found (µg/ml) % Recovery Mean % RSD Level-1 (60%) Level-2 (100%) Level-3 (140%) Acceptance Criteria: Recovery at each level and % mean recovery should be between with % RSD should not be more than 2.0% Conclusion: The %recovery at each level, % mean recovery and % RSD at each level meets the established acceptance criteria. Hence the method is accurate in the specified range Robustness Procedure: The robustness of the method was established by making deliberate minor variations in the following method parameters. Page 143

27 a) Flow rate was changed by 10%. [Used flow rate 1.32 ml/min and 1.08 ml/min] b) The organic phase ratio of the mobile phase was changed. Change Mobile phase composition by ± 2% absolute Normal Condition:-Buffer: Acetonitrile (70:30) Change Condition: - Buffer: Acetonitrile (72:28) Buffer: Acetonitrile (68:32) c) Column oven temperature was changed by 5 C. [Used column oven temperature 50 C and 60 C] d) PH change by 0.2 unit absolute [ PH 2.3 and 2.7 ] The effect of changes was observed on system suitability values and recorded. Table 7.17:- Results of Robustness parameter Condition % RSD %Recovery %Difference in Assay LIMIT NMT to 102% NMT 2% 1) Change in Flow rate Normal Condition (1.2 ml per minute) Change in flow rate by ml per minute (1.08 ml per minute) Change in flow rate by ml per minute (1.32 ml per minute) Page 144

28 2) Change in Mobile Phase composition Normal Condition Buffer: Acetonitrile(70:30) Change in organic phase Ratio by -2.0 Buffer: Acetonitrile(68:32) Change in organic phase Ratio by +2.0 Buffer: Acetonitrile (72:28) ) Change in column oven temperature Normal Condition (55 C) Change in oven temperature by - 5 C (50 C) Change in oven temperature by +5 C (60 C) ) Change in Ph Normal Condition PH Change in -0.2 unit (2.3) Change in +0.2 unit (2.7) Page 145

29 Acceptance criteria: - System suitability should be passes. Conclusion: - System suitability passes. The data indicate that there is no significant difference between the results obtained under normal conditions and varied method parameters. Therefore method is robust Solution stability: Procedure The sample solution was prepared at test concentration and initial assay was determined. Solution was stored up to 24 hours at room temperature and assay was determined at 12 hours and 24 hours against freshly prepared standard. The assay obtained at different time intervals was compared with the initial assay value and recorded. The results are recorded. Table 7.18:- Solution stability data Time Assay (%) % Difference Initial After 12 Hours After 24 Hours Acceptance criteria: The difference in Assay values should not be more than 2.0% Conclusion: The results obtained at 24 hours at room temperature are well within the acceptance criteria. Therefore, the sample is stable in solution form up to 24 hours at room temperature. Therefore, the sample is stable in solution form up to 24 hours at about 25 C. Page 146

30 System suitability Procedure System suitability parameters were calculated at the start of study of each validation parameter. The values of system suitability results obtained during the entire study are recorded. Table 7.19:- System suitability test results for for all validation parameters Parameter % RSD Tailing Factor Theoretical plates LIMIT NMT 2.0 NMT 2.0 NLT ) Specificity )Linearity )Method Precision )Intermediate Precision )Accuracy )Solution Stability Initial After 12 Hours After 24 Hours Page 147

31 Table 7.20:- System suitability test results for for all validation parameters Parameter % RSD Tailing Factor Theoretical plates LIMIT NMT 2.0 NMT 2.0 NLT ) Specificity )Linearity )Method Precision )Intermediate Precision )Accuracy )Solution Stability Initial After 12 Hours After 24 Hours Conclusion The system suitability parameters are well within acceptance criteria. Therefore the system and chromatographic conditions were suitable during each validation parameter. Page 148

32 RESULTS AND DISCUSSSION Validation and dissolution profile of analytical method for determination of Assay of and capsules was performed for the parameters including - Specificity, Linearity and Range, Precision System precision, Method Precision, Intermediate precision (Ruggedness), Accuracy, Robustness, Solution stability. The summary of results obtained is appended below. Table 7.21:- Summary of validation parameters Parameters Acceptance criteria Results Specificity 1) There should not be any interference from blank, placebo peaks and impurity peaks with main peak (Active). 2) All impurity peaks should be well separated from each other and the main peak. 3) Peak purity for the main peak in reference solution, sample solution and sample spiked with impurity solution should be more than ) The difference of assay between unspiked and spiked sample should not more than 2.0% There is no interference from blank, placebo and impurities. Specificity Sample peak purity =0.999 Spiked sample = = Assay difference = 0.2 % = 0.2 % a) Acid degradation Peak purity=1.000 (For both) b) Base degradation Peak purity= 0.999(For both) c) H 2 O 2 degradation Peak purity=1.000 (For both) d) Thermal degradation Peak purity= (For Page 149

33 Linearity and Range Precision Repeatability i.e.method precision 1) Correlation coefficient should be NLT over working range (60%-140%) 1) % RSD should not be more than 2.0%. both) e) Photo degradation Peak purity= (For both) Correlation coefficient- = = % Assay = 103.3%, RSD = 1.0% % Assay = 102.2%, RSD = 0.8% Intermediate Precision Accuracy 1) RSD should not be more than 2.0% 2) The difference between assay of method precision and intermediate precision should not be more than 2.0% 1) Recovery at each level and % mean recovery should be between % with % RSD should not be more than 2.0% % Assay = 104.3%, RSD = 0.6% % Assay = 103.1%, RSD = 1.9% Recovery at each level = 99.5% % RSD = 0.1 % Recovery at each level = 98.6% % RSD = 0.15 % Page 150

34 Robustness System suitability should complies Meets the requirement 1) By change in flow rate Normal condition % RSD - NMT 2.0 % Recovery - Between 98 and 102 % % RSD = 0.1 % Recovery =104.3% % RSD = 0.1 % Recovery =101.5% a) 1.08ml/min % RSD - NMT 2.0 % Recovery - Between 98 and 102 % b) 1.32ml/min % RSD - NMT 2.0 % Recovery - Between 98 and 102 % % RSD = 0.1 % Recovery =104.3% % RSD = 0.1 % Recovery =101.5% % RSD % Recovery % % RSD % Recovery % 2) By change in mobile phase composition Buffer: ACN(68:32) % RSD - NMT 2.0 % Recovery - Between 98 and 102 % % RSD % Recovery % % RSD % Recovery % Page 151

35 Buffer: ACN(72:28) % RSD - NMT 2.0 % Recovery - Between 98 and 102 % % RSD % Recovery % % RSD % Recovery % 3) By change in column oven temperature Change in oven temperature by - 5 C (50 C) % RSD - NMT 2.0 % Recovery - Between 98 and 102 % % RSD % Recovery % % RSD - 0.2% Recovery % Change in oven temperature by + 5 C (60 C) 4) Change in PH Change in -0.2 unit (2.3) % RSD - NMT 2.0 % Recovery - Between 98 and 102 % % RSD - NMT 2.0 % Recovery - Between 98 and 102 % % RSD % Recovery % % RSD % Recovery % % RSD % Recovery % % RSD - 0.3% Recovery % Page 152

36 Change in +0.2 unit (2.7) % RSD - NMT 2.0 % Recovery - Between 98 and 102 % % RSD % Recovery % % RSD % Recovery % Solution stability Initial (Room Temperature) Difference in assay value from initial value should not be more than 2.0% % Assay = 105.2%, % Assay = 104.5%, 12 Hrs. (Room Temperature) Difference in assay value from initial value should not be more than 2.0% % Assay = % % difference = 0.2 % Assay = 103.3%, % difference = 1.2 % Page 153

37 24 Hrs. (Room Temperature) System Suitability Difference in assay value from initial value should not be more than 2.0% Should meet the system suitability parameters at the start of study of each validation parameter % Assay = 104.3% % difference = 0.9 % Assay = %, % difference = 1.1 % Complies Discussion: The observations and result obtained for each validation parameter including Specificity, Linearity and Range, Precision Method Precision (Repeatability), Intermediate precision (Ruggedness), Accuracy, Robustness, Solution stability and System suitability lie well within the acceptance criteria. Page 154