ANALYTICAL METHOD VALIDATION PROTOCOL FOR ASSAY OF PSEUDOEPHEDRINE HYDROCHLORIDE IN CETIRIZINE HYDROCHLORIDE 5 mg AND PSEUDOEPHEDRINE HYDROCHLORIDE 120 mg EXTENDED RELEASE TABLETS BY HPLC Validation P.
ANALYTICAL METHOD VALIDATION PROTOCOL FOR ASSAY OF PSEUDOEPHEDRINE HYDROCHLORIDE IN CETIRIZINE HYDROCHLORIDE mg AND PSEUDOEPHEDRINE HYDROCHLORIDE 120 mg EXTENDED RELEASE TABLETS BY HPLC Validation Protocol No.:FP/001 INDEX Section No./ Table No I II III IV/Table V Table Table Table Table Table Table Table Table Table 10 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16 Table 17 Table 18 Table 19 Table 20 Table 21 Table 22 Table 23 Table 24 VI VII Contents Protocol Approval Page Introduction Scope Objective Acceptance Criteria Experimental Plan & Data Evaluation Sample Sequence for Selectivity Study Preparation of Solutions for Forced Degradation Studies (API) Preparation of Solutions for Forced Degradation Studies (Excipient blend) Preparation of Solutions for Forced Degradation Studies (Cetirizine Hydrochloride mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets) Sample Sequence for Forced Degradation Studies Dilutions for Linearity Sequence for Linearity Sequence for System Precision Sequence for Method Precision Sequence for Intermediate Precision Dilutions for Accuracy Sequence for Accuracy Sample Sequence for Filter Validation Sample Sequence for change in Column Lot Sample Sequence for change in Flow Rate (1.3 mL/minute) Sample Sequence for change in Flow Rate (1.7 mL/minute) Sample Sequence for change in Wavelength (213 nm) Sample Sequence for change in Wavelength (217 nm) Sample Sequence for change in Buffer pH (3.8) Sample Sequence for change in Buffer pH (4.2) Sample Sequence for change in Temperature (45°C) Sample Sequence for change in Temperature (35°C) Sequence for Solution Stability Validation Report Annexure – : Analytical Method I INTRODUCTION: Pseudoephedrine Hydrochloride is a fine, white to off-white crystals or powder Pseudoephedrine Hydrochloride is freely soluble in Water, Methanol and in Hydrochloric acid Pseudoephedrine Hydrochloride is an orally active sympathomimetic amine and exerts a decongestant action on the nasal mucosa Pseudoephedrine Hydrochloride is recognized as an effective agent for the relief of nasal congestion due to allergic rhinitis Cetirizine Hydrochloride mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets are White to off white round, coated, biconvex, bilayer tablets debossed with “106” on one side and “EM” on other side Structure: PSEUDOEPHEDRINE HYDROCHLORIDE Chemical name: [S-(R*,R*)]-a-[1-(methyl amino) ethyl]-benzene Methanol Hydrochloride Molecular formula : C10H15NO·HCl Molecular weight : 201.7 The limits for assay of Pseudoephedrine Hydrochloride in Cetirizine Hydrochloride mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets is not less than 90.0% and not more than 110.0% of the labeled amount II SCOPE: A reverse phase HPLC method for the assay of Pseudoephedrine Hydrochloride in Cetirizine Hydrochloride mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets is considered for validation This protocol is intended for the validation of HPLC method for the Assay of Pseudoephedrine Hydrochloride in Cetirizine Hydrochloride mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets III OBJECTIVE: To validate the HPLC method for the Assay of Pseudoephedrine Hydrochloride in Cetirizine Hydrochloride mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets (Method No.:CPH/ASY-02/0) For Analytical method (wherever indicated) refer to Annexure Instruments to be used: A Waters Alliance HPLC system equipped with quaternary gradient pump, autosampler and a UV / Photo Diode Array detector Column to be used: C18, 150 mm x 4.6 mm, μm (Zorbax SB or Waters symmetry or equivalent) Working Standard to be used: Pseudoephedrine Hydrochloride USP : WS/PEH-USP/03 with Assay: 99.86% (on as is basis) To be used before: 13/05/2008 Impurities to be used for validation: Name Ephedrine Hydrochloride Norpseudoephedrine Hydrochloride Batch No Expiry Date Potency (in % on as is basis) 99.40 99.73 Norephedrine Hydrochloride 99.80 99.85 (By HPLC) Pseudoephedrine Hydrochloride API to be used for validation: Pseudoephedrine Hydrochloride USP B.No.: Mfg Date: Retest Date: Sample to be used for validation: Cetirizine Hydrochloride mg and Pseudoephedrine Hydrochloride 120 mg Extended Release tablets : B No.: Mfg Date: Apr-2007 Excipient Blend sample to be used for validation will be prepared in house as per the manufacturing formula except the active pharmaceutical ingredient Parameters to be validated are as follows: Specificity 1.1 Selectivity 1.2 Forced Degradation Linearity and Range Precision 3.1 System Precision 3.2 Method Precision 3.3 Intermediate Precision Accuracy (% Recovery) Filter Validation Robustness Stability of Analytical Solution IV ACCEPTANCE CRITERIA: Table Validation Parameter Specificity - Specification Range of Study Acceptance Criteria By injecting diluent blank solution, excipient blend solution, all known impurities, test solution and test solution spiked with impurities The forced degradation studies should be performed using 10N HCl, 10N NaOH and 50% H2O2 Also effect of temperature, humidity and photolysis should be checked The studies shall be performed on API, The Pseudoephedrine Hydrochloride peak and all known impurities should be well resolved from any other peak and from each other The diluent blank, excipient blend and known impurities should not show any peak at the retention time of the Pseudoephedrine Hydrochloride The Pseudoephedrine Hydrochloride peak should be well resolved from any other peak The intent is to create approximately 10% – 30% degradation in at least one condition The peak purities will be demonstrated by a Photo Diode Array (PDA) detector Selectivity Specificity – Forced degradation study excipient blend and tablets separately Linearity and Range 50% to 150% System precision Method precision Intermediate precision Accuracy (%Recovery) Filter validation Robustness – change in column lot Robustness – change in flow rate (± 0.2 mL/ minute) Robustness – change in column oven Temperature (± 5°C) Robustness – change in pH of buffer (± 0.2 unit) Stability of analytical Correlation coefficient should be greater than or equal to 0.999 % RSD of peak areas of ten replicate injections of system suitability solution should not be more than 2.0% and system suitability criteria should pass as per analytical method -% RSD of the results of six test solutions should not be more than 2.0% -% RSD of the results of twelve test solutions (six of Method Precision and six of Intermediate Precision) should not be more than 2.0% 50%, 100% & 150% of Mean recovery at each concentration level should assay concentration be between 98.0% and 102.0% Unfiltered and filtered test There should not be any significant difference solution between filtered and unfiltered test solution 0.45 µm Nylon Filter Paper C18, 150 mm Same make, different serial System suitability criteria should pass as per x 4.6 mm, no analytical method and the % RSD between results µm obtained with changed condition and average result of method precision, should not be more than 2.0% 1.5 mL/ 1.3 mL/minute and System suitability criteria should pass as per minute 1.7 mL/minute analytical method and the % RSD between results obtained with changed condition and average result of method precision, should not be more than 2.0% 40°C 35°C and 45°C 4.0 3.8 and 4.2 System suitability criteria should pass as per analytical method and the % RSD between results obtained with changed condition and average result of method precision, should not be more than 2.0% System suitability criteria should pass as per analytical method and the % RSD between results obtained with changed condition and average result of method precision, should not be more than 2.0% Evaluate the stability upto The analyte is considered stable if there is no days for standard and test significant change in % assay solution solution V EXPERIMENTAL PLAN AND DATA EVALUATION: The analytical method validation will be executed as per the following plan: The experiments may be done as sequential or simultaneous operations Sample sequence of each experiment may be run together or independently with necessary alteration of sample sequence The system suitability parameters should be monitored throughout the validation study Any deviation from the plan shall be evaluated and approved by protocol approving authority Specificity – Selectivity and Forced Degradation: Selectivity: Prepare the diluent blank solution, system suitability solution, test solution as per the analytical method Preparation of excipient blend solution: Weigh and transfer a quantity of excipient blend of about 187.2 mg to a 100 mL of volumetric flask add 70 mL diluent and sonicate for 15 minutes Dilute upto mark with diluent Centrifuge this solution at 8000 rpm for 10 minutes Decant the supernatant solution into another test tube and transfer 10 mL of supernatant solution into another 50 mL volumetric flask and make up the volume with diluent Filter the solution through 0.45 µm nylon membrane filter Preparation of known impurity sample: Preparation of Ephedrine Hydrochloride Impurity: Weigh and transfer accurately about 3.0 mg of Ephedrine Hydrochloride to a 100 mL of volumetric flask add 70 mL diluent and sonicate for 15 minutes Dilute upto mark with diluent and mix Further dilute mL of this solution to 50 mL with diluent and mix Preparation of Nor-ephedrine Hydrochloride Impurity: Weigh and transfer accurately about 3.0 mg of Nor ephedrine Hydrochloride to a 100 mL of volumetric flask add 70 mL diluent and sonicate for 15 minutes Dilute upto mark with diluent and mix Further dilute mL of this solution to 50 mL with diluent and mix Preparation of Nor-Pseudoephedrine Hydrochloride Impurity: Weigh and transfer accurately about 3.0 mg of Nor-Pseudoephedrine Hydrochloride to a 100 mL of volumetric flask add 70 mL diluent and sonicate for 15 minutes Dilute upto mark with diluent and mix Further dilute mL of this solution to 50 mL with diluent and mix Preparation of Test solution spiked with known Impurities: Weigh powder equivalent to 60.0 mg of Pseudoephedrine Hydrochloride and transfer to 100 mL of volumetric flask, add 70 mL of diluent to flask Sonicate for 15 minutes Make up to volume with diluent and mix Centrifuge this solution at 8000 rpm for 10 minutes Decant the supernatant solution into another test tube and transfer 10 mL of supernatant solution and mL each of impurity stock solution into another 50 mL volumetric flask and make up the volume with diluent Filter the solution through 0.45 µm nylon membrane filter Perform the analysis on HPLC equipped with Photo Diode Array detector The sequence of injections for selectivity is given in table Table 2: Sample Sequence for Selectivity Study Sr No Sample Name No of Injections 10 Diluent blank System suitability solution Diluent blank Excipient blend solution Ephedrine Hydrochloride Impurity Nor ephedrine Hydrochloride Impurity Nor-Pseudoephedrine Hydrochloride Impurity Test solution Test solution spiked with Known impurities Bracketing standard 1 1 1 Data evaluation: Record each chromatogram All the injections will be processed at the wavelength provided in the method and selectivity will be demonstrated with regards to non-interference from diluent blank, excipient blend solution and known impurities with Pseudoephedrine Hydrochloride peak 1.1.2 Forced Degradation – Experiment: Degrading agents will be added separately to Pseudoephedrine Hydrochloride API, Excipient blend and Cetirizine Hydrochloride mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets Degrading agents will be 10N HCl (Acid degradation), 10N NaOH (Base degradation), 50% Hydrogen peroxide (Oxidative degradation), Heat (60°C) Humidity (75% R.H.) and Photolysis Forced degradation samples will be prepared as follows as given in table 3, & Sr No Table 3: Preparation of Solutions for Forced Degradation Studies (API) Degrading Condition Sample Preparations Agents Untreated -Weigh accurately about 60 mg of API and transfer into 100 mL volumetric sample flask, add 70 mL of diluent, sonicate to dissolve and dilute to volume with diluent and mix Dilute 10 mL of this solution to 50 mL with diluent and mix Acid 10N HCl Weigh accurately about 60 mg of API and transfer into 100 mL volumetric degradation flask, add 70 mL diluent, sonicate to dissolve Add 10 mL of 10N HCl (aqueous) to the flask Keep the flask to room temperature for 24 Hrs Neutralize this solution with 10N NaOH (aqueous) and dilute to volume with diluent and mix Dilute 10 mL of this solution to 50 mL with diluent and mix A blank solution should be prepared in similar way Base 10N NaOH Weigh accurately about 60 mg of API and transfer into 100 mL volumetric degradation flask, add 70 mL diluent, sonicate to dissolve Add 10 mL of 10N NaOH (aqueous) to the flask Keep the flask to room temperature for 24 Hrs Neutralize this solution with 10N HCl (aqueous) and dilute to volume with diluent and mix Dilute 10 mL of this solution to 50 mL with diluent and mix A blank solution should be prepared in similar way Peroxide 50% H2O2 Weigh accurately about 60 mg of API and transfer into 100 mL volumetric degradation flask, add 70 mL, diluent sonicate to dissolve Add 10 mL of 50% H2O2 Sr No Condition Degrading Agents Heat degradation (Solid state) 60°C Heat degradation (solution state) Humidity degradation 60°C Photolytic degradation 75% R.H 1.2 million lux hours Sample Preparations (aqueous) to the flask Heat the flask at 80°C for Hours Cool the flask to room temperature and dilute to volume with diluent and mix Dilute 10 mL of this solution to 50 mL with diluent and mix A blank solution should be prepared in similar way Weigh about 1.0 g of API and transfer in a Petri dish Keep it at 60°C in hot air oven Withdraw after 24 hours Weigh accurately about 60 mg of treated API and transfer into 100 mL volumetric flask, Add 70 mL diluent, sonicate to dissolve Dilute to volume with diluent and mix Dilute 10 mL of this solution to 50 mL with diluent and mix Weigh accurately about 60 mg of API and transfer into 100 mL volumetric flask, add 70 mL of diluent, sonicate to dissolve Keep the flask at 60°C for hours Cool the flask to room temperature and dilute to volume with diluent and mix Dilute 10 mL of this solution to 50 mL with diluent and mix Weigh about 1.0 g of API and transfer in a Petri dish Keep it at 75% R.H Withdraw the sample after 24 hours Weigh accurately about 60 mg of the treated API and transfer into 100 mL volumetric flask, add 70 mL diluent sonicate to dissolve and dilute to volume with diluent and mix Dilute 10 mL of this solution to 50 mL with diluent and mix Weigh about 1.0 g of API and transfer in a Petri dish Keep it in photolytic chamber for a sufficient period to give total exposure of about 1.2 million lux hours After the desired exposure withdraw the sample Weigh accurately about 60 mg of treated API and transfer into 100 mL volumetric flask, add 70 mL of diluent sonicate to dissolve Dilute to volume with diluent and mix Dilute 10 mL of this solution to 50 mL with diluent and mix Table 4: Preparation of Solutions for Forced Degradation Studies (Excipient Blend) Sr No Condition Untreated Excipient blend Degrading Agents Acid degradation 10N HCl Base degradation 10N NaOH Peroxide degradation 50% H2O2 Sample Preparations Weigh accurately about 187.2 mg of excipient blend and transfer into 100 mL volumetric flask, Add 70 mL of diluent sonicate for 15 minutes and make up to volume with diluent and mix Centrifuge this solution at 8000 rpm for 10 minutes Decant the supernatant solution into another test tube and transfer 10 mL of supernatant solution into another 50 mL volumetric flask and make up the volume with diluent Filter the solution through 0.45 µm nylon membrane filter Weigh accurately about 187.2 mg of excipient blend and transfer into 100 mL volumetric flask, Add 70 mL diluent sonicate for 15 minutes Add 10 mL of 10N HCl (aqueous) to the flask Keep the flask to room temperature for 24 Hrs Neutralize this solution with 10N NaOH (aqueous) and make up to volume with diluent and mix Centrifuge this solution at 8000 rpm for 10 minutes Decant the supernatant solution into another test tube and transfer 10 mL of supernatant solution into another 50 mL volumetric flask and make up the volume with diluent Filter the solution through 0.45 µm nylon membrane filter Weigh accurately about 187.2 mg of excipient blend and transfer into 100 mL volumetric flask, Add 70 mL diluent sonicate for 15 minutes Add 10 mL of 10N NaOH (aqueous) to the flask Keep the flask to room temperature for 24 Hrs Neutralize this solution with 10N HCl (aqueous) and make up to volume with diluent and mix Centrifuge this solution at 8000 rpm for 10 minutes Decant the supernatant solution into another test tube and transfer 10 mL of supernatant solution into another 50 mL volumetric flask and make up the volume with diluent Filter the solution through 0.45 µm nylon membrane filter Weigh accurately about 187.2 mg of excipient blend and transfer into 100 mL volumetric flask, Add 70 mL diluent sonicate for 15 minutes Add 10 mL of 50% H2O2 (aqueous) to the flask Heat the solution at 80°C for hours Cool Heat degradation (Solid state) 60°C Heat degradation 60°C the flask to room temperature and dilute to volume with diluent and mix Centrifuge this solution at 8000 rpm for 10 minutes Decant the supernatant solution into another test tube and transfer 10 mL of supernatant solution into another 50 mL volumetric flask and make up the volume with diluent Filter the solution through 0.45 µm nylon membrane filter Keep about 2.0 g of excipient blend and transfer in a Petri dish Keep it at 60°C in hot air oven Withdraw after 24 hours Weigh accurately about 187.2 mg of the treated sample and transfer into 100 mL volumetric flask, add 70 mL of diluent, sonicate 15 minutes and make up to volume with diluent and mix Centrifuge this solution at 8000 rpm for 10 minutes Decant the supernatant solution into another test tube and transfer 10 mL of supernatant solution into another 50 mL volumetric flask and make up the volume with diluent Filter the solution through 0.45 µm nylon membrane filter Weigh about 187.2 mg of excipient blend and transfer into 100 mL volumetric flask, add 70 mL of diluent and sonicate for 15 minutes Keep the Weigh and crush 20 tablets to a fine powder Use this sample powder for the preparation of Forced Degradation study Table 5: Preparation of Solutions for Forced Degradation Studies (Cetirizine Hydrochloride mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets) Sr No Condition Untreated Sample Degrading Agents Acid degradation 10N HCl Base degradation 10N NaOH Peroxide degradation 50% H2O2 Heat degradation (solid state) 60°C Heat degradation (solution 60°C Sample Preparations Weigh tablet powder equivalent to 60.0 mg of Pseudoephedrine Hydrochloride and transfer to 100 mL of volumetric flask, add 70 mL of diluent to flask and sonicate for 15 minutes Make up to volume with diluent and mix Centrifuge this solution at 8000 rpm for 10 minutes Decant the supernatant solution into another test tube and transfer 10 mL of supernatant solution into another 50 mL volumetric flask and make up the volume with diluent Filter the solution through 0.45 µm nylon membrane filter Weigh tablet powder equivalent to 60.0 mg of Pseudoephedrine Hydrochloride and transfer to 100 mL of volumetric flask Add 70ml of diluent, sonicate for 15 minutes add 10 mL of 10N HCl (aqueous) to the flask Keep the flask to room temperature.for 24 Hrs Neutralize this solution with 10N NaOH (aqueous) and make up to volume with diluent and mix Centrifuge this solution at 8000 rpm for 10 minutes Decant the supernatant solution into another test tube and transfer 10 mL of supernatant solution into another 50 mL volumetric flask and make up the volume with diluent Filter the solution through 0.45 µm nylon membrane filter Weigh tablet powder equivalent to 60.0 mg of Pseudoephedrine Hydrochloride and transfer to 100 mL of volumetric flask Add 70ml of diluent, sonicate for 15 minutes add 10 mL of 10N NaOH (aqueous) to the flask Keep the flask to room temperature for 24 Hrs Neutralize this solution with 10N HCl (aqueous) and make up to volume with diluent and mix Centrifuge this solution at 8000 rpm for 10 minutes Decant the supernatant solution into another test tube and transfer 10 mL of supernatant solution into another 50 mL volumetric flask and make up the volume with diluent Filter the solution through 0.45 µm nylon membrane filter Weigh tablet powder equivalent to 60.0 mg of Pseudoephedrine Hydrochloride and transfer to 100 mL of volumetric flask Add 70ml of diluent sonicate for 15 minutes, add 10 mL of 50% H2O2 (aqueous) to the flask Heat the solution at 80°C for hours Cool the flask to room temperature and make up to volume with diluent and mix Centrifuge this solution at 8000 rpm for 10 minutes Decant the supernatant solution into another test tube and transfer 10 mL of supernatant solution into another 50 mL volumetric flask and make up the volume with diluent Filter the solution through 0.45 µm nylon membrane filter Weigh about 2.0 g of tablet powder and transfer in a Petri dish Keep it at 60°C in hot air oven Withdraw after 24 hours Weigh treated tablet powder equivalent to 60.0 mg of Pseudoephedrine Hydrochloride and transfer to 100 mL of volumetric flask Add 70ml of diluent, sonicate for 15 minutes make up to volume with diluent and mix Centrifuge this solution at 8000 rpm for 10 minutes Decant the supernatant solution into another test tube and transfer 10 mL of supernatant solution into another 50 mL volumetric flask and make up the volume with diluent Filter the solution through 0.45 µm nylon membrane filter Weigh tablet powder equivalent to 60.0 mg of Pseudoephedrine Hydrochloride and transfer to 100 mL of volumetric flask, add 70 mL of diluent and sonicate for 15 minutes Keep the flask at 60°C for hours Cool Note : 494.4 mg of tablet blend is equivalent to 120 mg of Pseudoephedrine Hydrochloride Perform the above analysis on HPLC equipped with Photo Diode Array detector The sequence of injections for forced degradation is given in table Table 6: Sample Sequence for Forced Degradation Studies Sample Name Diluent blank System suitability solution Blank – HCl Blank – NaOH Blank – H2O2 Excipient blend – Untreated Excipient blend – Heat (Solid state) Excipient blend Heat (Solution state) Excipient blend – Humidity Excipient blend – Photolytic degradation Excipient blend – HCl Excipient blend – NaOH Bracketing standard – Excipient blend – H2O2 Bracketing standard – No of Injections 1 1 1 1 1 2 Note : Sequence for Forced Degradation of Pseudoephedrine Hydrochloride API and Tablets is carried out as per given sequence replacing Excipient blend with API & Tablets respectively 1.2.1 Data evaluation: Record each chromatogram The peak purity will be determined for the Pseudoephedrine Hydrochloride peak The degradation if any will be compared with untreated sample The peaks due to degradation and of excipient blend should be well separated from the Pseudoephedrine Hydrochloride peak Linearity and Range: 2.1 Experiment: Prepare a series of standard preparations (five preparations) of Pseudoephedrine Hydrochloride, over a range starting from 50% to at least 150% of the specified limits of assay 2.1.1 Preparation of linearity stock solutions: Stock solution preparation of Pseudoephedrine Hydrochloride: Weigh accurately about 60 mg of Pseudoephedrine Hydrochloride working standard to a 100 mL volumetric flask Add 30 mL of diluent & sonicate for 15 minutes to dissolve Dilute to volume with diluent and mix The preparation of linearity solutions is given in table Inject each of the linearity preparation in triplicate and then take average area count for calculations Table 7: Dilutions for Linearity The Linearity level Sample concentration Amount of stock solution preparation of Pseudoephedrine Hydrochloride to be transferred (mL) Volume made up to (mL) with Diluent Concentration of Pseudoephedrine Hydrochloride (ppm) 50 50 50 50 50 60 90 120 150 180 Level – 50% 5.0 mL Level – 75% 7.5 mL Level – 100% 10.0 mL Level – 125% 12.5 mL Level – 150% 15.0 mL sequence of injections for linearity is given in table Table 8: Sequence for Linearity Sample Name Diluent blank System suitability solution Linearity Level Linearity Level Linearity Level Bracketing standard Linearity Level Linearity Level Bracketing standard 2.2 No of Injections 3 3 Data evaluation: Plot a linearity graph of average area at each level against the concentration (%) and determine the correlation coefficient The range of the analytical method in concentration (%) will be reported 3.1 3.1.1 Precision: System Precision: Experiment: Make ten replicate injections of system suitability solution into the HPLC using the method of analysis The sequence of injections for system precision is given in table Table 9: Sequence for System Precision Sample Name Diluent blank System suitability solution 3.1.2 No of Injections 10 Data evaluation: Calculate and report standard deviation and % RSD of peak areas of 10 replicate injections and also report theoretical plates and tailing factor for the last injection of system suitability solution 3.2 Method Precision: 3.2.1 Experiment: Prepare six test solutions of Pseudoephedrine Hydrochloride in Cetirizine Hydrochloride mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets and inject into the HPLC as per the analytical method The sequence of injections for method precision is given in table 10 Table 10: Sequence for Method Precision Sample Name Diluent blank System suitability solution Test solution - Test solution - Test solution - Test solution - Test solution - Bracketing standard-1 Test solution - Bracketing standard-2 3.2.2 No of Injections 2 2 2 2 Data evaluation: The % assay of Pseudoephedrine Hydrochloride will be calculated and reported along with the standard deviation and % RSD of the six test solutions 3.3 Intermediate Precision: 3.3.1 Experiment: Prepare six test solutions of Pseudoephedrine Hydrochloride in Cetirizine Hydrochloride mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets of the same lot (as used in 3.2) using a different analyst on a different column and inject into a different HPLC system (other than that used in 3.2) on different day as per the analytical method The sequence of injections for intermediate precision is given in table 11 Table 11: Sequence for Intermediate Precision Sample Name Diluent blank System suitability solution Test solution - Test solution - Test solution - Test solution - Test solution -5 Bracketing standard - Test solution - Bracketing standard - 3.3.2 Data evaluation: No of Injections 2 2 2 2 The % assay of Pseudoephedrine Hydrochloride will be calculated and reported along with the % RSD of the twelve test solutions (six of method precision & six of intermediate precision) Accuracy (% Recovery): 4.1 Experiment: Weigh and transfer accurately, in triplicate at each level, about 187.2 mg of excipient blend and add to it Pseudoephedrine Hydrochloride API at 50%, 100% and 150% of the working concentration Working (assay) concentration of Pseudoephedrine Hydrochloride is 120 ppm Prepare the solutions as per the analytical method The preparation of accuracy solutions is given in table 12 Inject each of the samples in triplicate and then take average area count for calculations Table 12: Dilutions for Accuracy Level First level Second level Third level Sample Name Rec-50% / Rec-50% / Rec-50% / Rec-100% / Rec-100% / Rec-100% / Rec-150% / Rec-150% / Rec-150% / Amount of Pseudoephedrine Hydrochloride API to be weighed (mg) 30 30 30 60 60 60 90 90 90 Make up volume in mL 100 100 100 100 100 100 100 100 100 Further transfer 10 mL of each of the above solution to 50 mL and dilute with diluent and mix The sequence of injections for accuracy is given in table 13 Table 13: Sequence for Accuracy Sample Name Diluent blank System suitability solution Rec-50% / Rec-50% / Rec-50% / Bracketing standard - Rec-100% / Rec-100% / Rec-100% / Bracketing standard - Rec-150% / Rec-150% / Rec-150% / Bracketing standard - No of Injections 3 3 3 3 4.2 Data evaluation: The percent actual concentration and theoretical concentration will be calculated and percent recovery at each level will be calculated and reported along with percent mean recovery at each level Filter Validation: 5.1 Experiment: Weigh accurately tablet powder equivalent to 60 mg of Pseudoephedrine Hydrochloride and transfer to 100 mL volumetric flask Add about 70 mL of diluent and sonicate for 15 minutes Dilute to volume with the diluent and mix Centrifuge a portion of the resulting solution at about 8000 rpm for about 10 minutes Decant the supernatant solution into another test tube and transfer 10 mL of supernatant solution into another 50 mL volumetric flask and make up the volume with diluent Use this solution as unfiltered test solution Dilute 10 mL of remaining portion of the supernatant solution to 50 mL with diluent and filter it through 0.45 µm nylon membrane filter paper Fill vials of this solution Use this as filtered test solution Analyze all filtered and unfiltered solutions in single sequence The sequence of injections for filter validation is given in table 14 Table 14: Sample Sequence for Filter Validation Sample Name Diluent blank System suitability solution Unfiltered test solution Filtered test solution – Filtered test solution – Filtered test solution – Filtered test solution – Filtered test solution – Bracketing standard No of Injections 1 1 1 5.2 Data evaluation: The % assay of Pseudoephedrine Hydrochloride in Cetirizine Hydrochloride mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets with filtered and unfiltered test solution will be calculated and reported along with the absolute difference between unfiltered and filtered test solution Robustness: 6.1 Experiment: Prepare two test solution of the same lot (as used in 3.2 and 3.3) of Pseudoephedrine Hydrochloride in Cetirizine Hydrochloride mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets as per analytical method Inject this solution along with the blank and system suitability solution along different chromatographic conditions as shown below: 6.1.1 Change in column lot (same make, different serial no.) 6.1.2 Change in flow rate (± 0.2 mL/minutes) 6.1.3 Change in wavelength (± nm) 6.1.4 Change in column oven temperature (± 5°C) 6.1.5 Change in pH of buffer (± 0.2 units) The sequence of injections for robustness study is given in table 15 to table 23 6.1.1 Change in Column Lot: [Normal Experimental Condition: C18, 150 mm x 4.6 mm, µm (Zorbax SB or Waters symmetry) Sr No.: Table 15: Sample sequence for change in column lot 6.1.2 Sample Name No of Injections Diluent blank System suitability solution Test solution-1 Test solution -2 Bracketing standard This experiment can be performed as a part of intermediate precision study Change in Flow Rate: (Normal Experimental Condition: 1.5 mL/minute) Table 16: Sample sequence for change in flow rate (1.3 mL/minute) Sample Name Diluent blank System suitability solution Test solution -1 Test solution -2 Bracketing standard No of Injections 1 Table 17: Sample sequence for change in flow rate (1.7 mL/minute) Sample Name Diluent blank System suitability solution Test solution -1 Test solution -2 Bracketing standard 6.1.3 No of Injections 1 Change in Wavelength: (Normal Experimental Condition; 215 nm) Table 18: Sample sequence for change in wavelength (213 nm) Sample Name Diluent blank System suitability solution Test solution -1 Test solution -2 Bracketing standard No of Injections 1 Table 19: Sample sequence for change in wavelength (217 nm) Sample Name Diluent blank System suitability solution Test solution -1 Test solution -2 Bracketing standard No of Injections 1 ] 6.1.4 Change in pH of Buffer: (Normal Experimental Condition; pH 4.0) Table 20: Sample sequence for change in pH of buffer (3.8) Sample Name Diluent blank System suitability solution Test solution -1 Test solution -2 Bracketing standard No of Injections 1 Table 21: Sample sequence for change in pH of buffer (4.2) Sample Name Diluent blank System suitability solution Test solution -1 Test solution -2 Bracketing standard 6.1.5 No of Injections 1 Change in Temperature: (Normal Experimental Condition; 40°C) Table 22: Sample sequence for change in column oven temperature (35°C) Sample Name Diluent blank System suitability solution Test solution -1 Test solution -2 Bracketing standard No of Injections 1 Table 23: Sample sequence for change in column oven temperature (45°C) Sample Name Diluent blank System suitability solution Test solution -1 Test solution -2 Bracketing standard 6.2 No of Injections 1 Data evaluation: System suitability is to be reported for each experiment % RSD between mean of method precision results and two results obtained with changed condition is to be reported for each experiment Stability of Analytical Solution: 7.1 Experiment: Prepare the Pseudoephedrine Hydrochloride standard solution, and test solution of Pseudoephedrine Hydrochloride in Cetirizine Hydrochloride mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets on st, 3rd, 5th, 6th and 7th day of experiment Store these test solutions at room temperature for every time interval upto th day Analyze these test solutions on th day with freshly prepared test solution Prepare the Pseudoephedrine Hydrochloride standard solution freshly at the time of analysis and calculate the assay of Pseudoephedrine Hydrochloride in the sample The sequence of injections for solution stability for the initial analysis is given in table 24 Table 24: Sequence for solution stability Sample Name Diluent blank Freshly prepared standard solution 7th day prepared standard 6th day prepared standard 5th day prepared standard 3rd day prepared standard 1st day prepared standard Freshly prepared test solution 7th day prepared test solution 6th day prepared solution 5th day prepared solution 3rd day prepared solution Bracketing standard -1 1st day prepared solution Bracketing standard -2 No of Injections 1 1 1 1 1 2 7.2 Data evaluation: The test solution for Pseudoephedrine Hydrochloride in Cetirizine Hydrochloride mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets is said to be stable up to the time point till which there is no significant change in % assay VI VALIDATION REPORT: Prepare the validation report using the following headers: • Introduction • The instruments and samples used including standards • Validation results and Discussion with following headings: Specificity Selectivity Forced Degradation Linearity and Range Precision System Precision Method Precision Intermediate Precision Accuracy (% Recovery) Filter validation Robustness Stability of Analytical Solution System suitability data – that obtained during different days of analytical method validation • (including robustness) Summary • Conclusion Figures should include chromatograms of diluent blank, standard preparation, excipient blend, test solution, chromatograms of selectivity, forced degradation study and linearity graph of Pseudoephedrine Hydrochloride ANNEXURE – VII ANALYTICAL METHOD: (Method No.: CPH/ASY-02/0) Chromatographic conditions: Column : C18, 150 mm X 4.6 mm, µm (Zorbax SB, Waters symmetry or equivalent) Buffer : Weigh accurately and transfer 11.5 g of Ammonium dihydrogen orthophosphate to a 1000 ml volumetric flask Add about 980 ml of water, dissolve and dilute to volume with water Add 1.0 ml of Triethylamine to the 1000 ml of buffer solution prepared Adjust the pH to 4.0 ± 0.05 with Mobile Phase Wavelength Flow Rate Column temperature Injection volume Run time Blank solution Diluent Orthophosphoric acid Filter and degas the Buffer : For isocratic system, prepare a mixture of Methanol and buffer in the proportion 10: 90 respectively Mix well Filter through 0.45 µ Nylon membrane filter paper and degas prior to use : 215 nm : 1.5 ml / minute : 40°C ± 2°C : 10 μl : 10 minutes : Use 0.1N Hydrochloric acid as blank : Use 0.1N Hydrochloric acid as diluent Preparation of Pseudoephedrine HCl Standard Stock Solution: Weigh accurately about 30 mg of Pseudoephedrine HCl working standard and transfer to a 50 ml volumetric flask Add 30 ml of diluent and sonicate to dissolve Dilute to volume with diluent and mix (Dilution scheme: 30 mg → 50 ml) Preparation of Pseudoephedrine HCl Standard working Solution: Transfer 10.0 ml of Pseudoephedrine HCl standard stock solution into a 50 ml of volumetric flask and dilute to volume with the diluent Mix well (Dilution scheme: 10.0 ml → 50 ml) Preparation of Test Solution: Weigh and crush 20 tablets to a very fine powder Transfer an accurately weighed 240 mg of compressed tablet powder and 247.2 mg of coated tablet powder i.e equivalent to 60 mg of Pseudoephedrine into a 100 ml volumetric flask Add about 70 ml of diluent and sonicate for 15 minutes Dilute up to mark with diluent Centrifuge this solution at 8000 rpm for 10 minutes Decant the supernatant solution into another test tube and transfer 10.0 ml of supernatant solution into another 50 ml volumetric flask and make up the volume with diluent Filter the solution through 0.45 µm nylon membrane filter (Compressed tablets: Dilution scheme: 240 → 100 ml/ 10.0 ml → 50 ml) (Coated tablets: Dilution scheme: 247.2 → 100 ml/ 10.0 ml → 50 ml) System Suitability Solution: Use Pseudoephedrine HCl standard working solution as system suitability solution Procedure: Separately inject equal volumes of blank, five replicate injections of system suitability solution (Pseudoephedrine HCl standard working solution) Then inject two injections of test solution and record the chromatograms Disregard any peak due to blank in the test solution Calculate % RSD of five replicate injections of system suitability solution (Pseudoephedrine HCl standard working solution) Check tailing factor and theoretical plates of the peak in the chromatogram obtained with 5th injection of system suitability solution (Pseudoephedrine HCl standard working solution) The limits are as below, 1) Theoretical plates should be not less than 3000 2) Tailing factor should be less than 2.0 3) % RSD should be not more than 2.0% Note: Inject system suitability solution (Bracketing) in two replicates after completion of sequence and after injecting 10 injections of Test Solution, whichever is earlier Check the RSD of bracketing standards with earlier injected standards Injection scheme: Sr No Solutions to be injected No of injections Diluent Blank solution System suitability solution (Pseudoephedrine HCl standard working solution) Test Solution Bracketing standard (system suitability solution) after completion of sequence and / or after injecting 10 injections of test solution whichever is earlier Calculations: % Assay = Where, AT AS WS WT AW P AT AS X WS 50 X 10 50 X 100 X WT 50 X 10 P 120 X AW Average peak area of Pseudoephedrine in test solution Mean peak area of Pseudoephedrine in system suitability solution Weight of Pseudoephedrine HCl working standard in mg Weight of sample taken Average weight of Tablets Assay of Pseudoephedrine HCl working standard in % on as is basis Express the results up to two decimals ... method for the assay of Pseudoephedrine Hydrochloride in Cetirizine Hydrochloride mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets is considered for validation This protocol is... intended for the validation of HPLC method for the Assay of Pseudoephedrine Hydrochloride in Cetirizine Hydrochloride mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets III OBJECTIVE:... validate the HPLC method for the Assay of Pseudoephedrine Hydrochloride in Cetirizine Hydrochloride mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets (Method No.:CPH/ASY-02/0)