1.0 OBJECTIVE
To lay down the procedure for the Validation of Analytical Test method for Assay by HPLC based on ICH, USP.
2.0 SCOPE
This SOP is Applicable for validation of Analytical test method for Assay of Tablets/ Capsules/Oral suspension by HPLC in Company Name
3.0 BACKGROUND
Not Applicable
4.0 RESPONSIBILITY
4.1 Analytical research personnel whoever is operating the Equipment.
4.2 Group Leader Analytical research to ensure proper and safe operation of the Equipment.
4.3 Head - Analytical research or his designee to ensure overall compliance.
5.0 PROCEDURE
5.1 System suitability:
5.1.1 System precision:
The precision as measured by replicate injections of homogeneous standard solution indicates the performance of the HPLC instrument under the chromatographic conditions and the day tested. As a part of method validation, a minimum of 10 injections of the standard preparation is recommended.
5.1.1.1 Acceptance criteria:
The % RSD for peak areas of ten replicate injections of standard should be not more than half the value given for system suitability.
5.1.2 Other system suitability parameters:
Prepare resolution solution preparation / any other system suitability preparations as per the test method and evaluate system suitability parameters as required by
the test method.
5.1.2.1 Acceptance criteria:
The system suitability parameters should be within the specified limits in the test method.
Note : If system suitability parameters mentioned in the Test procedure are observed to be inadequate or difficult to meet the requirements, New system
suitability criteria shall be evaluated with proper reason, and the same shall be followed during the Validation activity.
5.2 Linearity of detector response:
5.2.1 Demonstrate the Linearity in the range of 25% to 150% of the target concentration of assay.
5.2.2 Prepare a series of standard solutions (not less than six is recommended) in the above concentration range and inject in duplicate into the HPLC system. For standards of multiple dosages conduct linearity from 25% for lower strength to 150% of higher strength. Plot average peak area versus the actual concentration, in µg/ mL or mg/mL.
5.2.3 Calculate the correlation coefficient (r).
Note : Linearity range shall be altered based on the requirement.
5.2.4 Acceptance criteria:
The correlation coefficient (r) should be not less than 0.999.
Note: In case of discrepancy investigate and explain the reason. Repeat if necessary and report the range of linearity.
5.3 Method Precision:
5.3.1 Prepare six assay preparations as per the test method and inject into the HPLC system. Calculate the % Assay and %RSD.
5.3.2Acceptance criteria:
The method is considered “PRECISE”, if assay results are between 97.0% to 103.0% of labeled amount of drug substances, the relative standard deviation of six assay results is not more than 2.0%.
Note: 1.If the precision fails perform Validation using the product blend prepared by mixing the active ingredient with excipients as per the manufacturing formula.
2. Data generated in this section may also be used to demonstrate system to system variability, Analyst-to-Analyst variability and Column-to-Column variability.
5.3.3 If the method is used for over a range of dosage strength, and if the test preparation is same for all the dosage strength then conduct method precision for highest and lowest dosage strength.
5.3.4 If the test preparation is different from strength to strength then conduct method Precision for all dosage strength.
5.3.5 If the method is used for range of dosage types, (e.g. Tablets/Capsules/reconstituted oral suspension), then conduct method precision for each dosage type.
5.4 Recovery / Accuracy:
5.4.1 Prepare homogeneous tablet / capsule /reconstituted oral suspension having 25%, 50%, 75%, 100% and 150% of the labeled amount of drug substance.
5.4.2 Prepare blend by keeping average weight constant, i.e. if the quantity of active ingredient is reduced, alter the weight of placebo or vice versa to keep the average weight constant. (or) keep the placebo constant (or) weigh active ingredient and placebo individually for each test instead of using blend.
5.4.3 Prepare test solutions in triplicate at each spike level and assay as per the test method. Calculate the % recovery, mg recovered.
Note: Spiked levels of drug substances shall be altered based on the requirement.
5.4.4 Acceptance criteria:
The method is considered “ACCURATE” if the average recovery is between 97.0% and 103.0%.
Note: In case of discrepancy investigate the reason, repeat the experiment if Necessary and explain the reason for discrepancy.
5.5 Linearity of Test Method:
5.5.1 Plot a linearity graph using average “mg” added versus average “mg” found in recovery section and plot a graph for average mg added versus mg found. Evaluate the co-efficient of correlation.
5.5.2 Acceptance criteria:
The coefficient of correlation shall be NLT 0.999. If the correlation coefficient is less than 0.999, report the range at which the method is linear.
5.6 Ruggedness:
5.6.1 System to System variability:
5.6.1.1 Conduct System to System variability on two HPLC systems (of same or different Manufacturer) by the same analyst and using same column. Analyse six assay Preparations using tablets / capsules / reconstituted oral suspension as directed under precision as per the test method.
5.6.1.2 Calculate the assay results as % of labeled amount of drug substance, % RSD of assay results and overall %RSD for both the systems.
5.6.1.3 Acceptance criteria:
The method is considered rugged for system to system variability, if the assay results are between 97.0% and 103.0% of labeled amount of drug substance, over all RSD for assay results is not more than 2.0% for both the systems.
5.6.2 Column to Column variability:
5.6.2.1 Conduct Column to Column variability on two HPLC columns (of same or different
Manufacturers) by the same analyst and using the same HPLC system. Analyse six assay preparations using tablets / capsule / reconstituted oral suspension as directed under precision as per the test method.
5.6.2.2 Calculate the assay results as % of labeled amount of drug substance, % RSD of assay results and overall %RSD for both the columns.
5.6.2.3 Acceptance criteria:
The method is considered rugged for column-to-column variability, if the assay results are between 97.0% and 103.0% of labeled amount of drug substance; over all %RSD for assay results is not more than 2.0% for both the Columns.
5.6.3 Analyst to Analyst variability:
5.6.3.1 Conduct Analyst-to-Analyst variability on two analysts using the same HPLC system and column. Analyse six assay preparations using tablets / capsule / reconstituted oral suspension as directed under precision as per the test method.
5.6.3.2 Calculate the assay results as % of labeled amount of drug substance, % RSD of assay results and overall %RSD for both the Analysts.
5.6.3.3 Acceptance criteria:
1) The method is considered rugged for Analyst-to-Analyst variability, if the assay results are between 97.0% and 103.0% of labeled amount of drug substance; overall %RSD for assay results is not more than 2.0% for both the analysts.
2) Perform F&T test between Analyst to Analyst, either F or T test should pass.Note: It is not considered necessary to study the variations (days, columns,
equipment and analysts) individually. The use of an experimental design (matrix) is encouraged.
5.6.4 Bench top stability of standard and test preparations:
5.6.4.1 Establish the stability of standard and test solutions on bench top for a period of 2 days
5.6.4.2 Prepare standard and test solution in duplicate as per the test method and keep them on bench top. Inject standard and test solutions into the HPLC system following the conditions described in the test method at initial, 1day and 2 days. Calculate % Assay of both standard and test solutions against fresh standard each time.Note: Take potency of standard as initial % Assay of standard.
5.6.4.3 Acceptance criteria:
The solutions are considered “STABLE” if the difference in % assay results from initial to 1 day and 2 days is not more than 2.0.
5.6.4.4 If the solutions are found to be not stable for 1 day, inject and calculate the % assay at shorter time intervals, eg. At 1, 2, 3, hours interval, establish the period of time during which the solutions are stable.
5.6.5 Refrigerator stability of standard and test preparations:
5.6.5.1 Establish the stability of standard and test solutions in refrigerator for a period of about 2 days.
5.6.5.2 Prepare standard and test solutions in duplicate as per the test method and keep them in refrigerator. Inject standard and test solutions into the HPLC system following the conditions described in Test Method at initial, 1 day and 2 days. Note: Allow the samples to come at room temperature before injecting.
5.6.5.3 Calculate % assay of both standard and test solutions against a fresh standard each time.
5.6.5.4 Acceptance criteria:
The solutions are considered “STABLE” if the difference in % assay results from initial to 1 day and 2 days is not more than 2.0.
5.6.5.5 If the solutions are found to be not stable for 1 day, inject and calculate the % assay at shorter time intervals, eg. At 1, 2, 3, hours interval, and establish the period of time during which the solutions are stable.
5.6.6 Bench top stability of mobile phase:
5.6.6.1 Establish the bench top stability of mobile phase for a period of about 2 days.
5.6.6.2 Prepare the mobile phase as per the test method and keep it on Bench top in well closed condition. Evaluate system suitability parameters and perform Assay on two samples by following the conditions described in the test method at initial, 1 day and 2 days.
5.6.6.3 Prepare system suitability solutions and test solutions in duplicate as per the test method. Prepare test solution each time from the same sample.
5.6.6.4 Acceptance criteria:
The mobile phase is considered to be “STABLE” if all the system suitability values are within the acceptable criteria set forth in the test method and % assay results should meet the specification. The difference in average % Assay results of the same sample from initial is not more than 2.0.
5.6.7 Refrigerator stability of system suitability solution(s) other than standard Preparation:
5.6.7.1 Establish the stability of system suitability solution, if any, other than standard preparation in refrigerator for a period of about 1day to 4 weeks or more.
5.6.7.2 Prepare the system suitability solution(s), other than the standard preparation as per the test method, and keep in refrigerator. Inject system suitability solution into the HPLC system following the conditions described in test method at initial, after 1, 2 days or more.
Note: Allow the system suitability solution to come at room temperature before injecting.
5.6.7.3 Acceptance criteria:
The solutions are considered “STABLE”, if the system suitability criteria passes.
5.7 Robustness:
5.7.1 Filter Validation:
5.7.1.1 To demonstrate that the filtration does not affect the analysis results, validate different filters at least two types of filters before use.
5.7.1.2 Prepare standard and test solutions in triplicate as per the test method. Centrifuge a portion of test solution and filter portion of test solution through individual filters. Inject unfiltered standard solution, centrifuged test solution and filtered test solution
Into the HPLC system under the test conditions. Calculate the % assay for Centrifuged and Filtered test solutions using unfiltered standard.
5.7.1.3 Acceptance criteria:
The filter is considered acceptable, if difference in % assay results between centrifuged and filtered test solutions is not more than 2.0.
5.7.2 Effect of Variation in mobile phase composition:
5.7.2.1 Prepare two mobile phases having 90% to 110% of the method organic phase composition. Prepare system suitability solution (e.g. Resolution solution, Standard
preparation, etc) and test preparation in duplicate as per the test method and inject into the HPLC system using both mobile phases. Evaluate the system suitability parameters as required by the test method and % Assay of two test samples for both the mobile phases.
Note: If the mobile phase contains more than two organic phases, conduct variability individually and verify one at a time.
5.7.2.2 Acceptance criteria:
The method is considered robust for variation organic phase,
I) if all the system suitability parameters shall meet the acceptance criteria set forth
in the test method.
II) The difference in % Assay with respect to 100% variation should be NMT 2.0
5.7.2.3 If any of the acceptance criteria fails, narrow the organic phase composition range and establish the allowable range of variation of organic phase composition.
5.7.3 Effect of variation of pH in mobile phase:
5.7.3.1 Prepare two mobile phases having the buffer pH with +/- 0.2 of the method pH.
Prepare system suitability solution (e.g. Resolution solution, Standard preparation, etc) and test preparation in duplicate as per the test method and inject into the HPLC system using both mobile phases. Evaluate the system suitability
parameters as required by the test method and % Assay of test samples for boththe mobile phases.
5.7.3.2 Acceptance criteria:
The method is considered robust for variation of pH,
I) if all the system suitability parameters meet the acceptance criteria set forth in the test method.
II) The difference in % Assay with respect to 100% variation should be NMT 2.0
5.7.3.3 If any of the acceptance criteria fails, narrow the pH range and establish the allowable range of variation of pH in mobile phase.
5.7.4 Effect of variation in flow rate:
5.7.4.1 Prepare system suitability solutions (e.g. Resolution solution, standard preparation, etc), and test preparation in duplicate as per the test method and inject into the HPLC system with +/-0.2 ml of method flow. Evaluate the system suitability parameters as required by the test method and % Assay for both the flow rates.
5.7.4.2 Acceptance criteria:
The method is considered robust for variation of flow rate
I) if all the system suitability parameters meet the acceptance criteria set forth in the test method.
II) The difference in % Assay with respect to 100% variation should be NMT 2.0
5.7.4.3 If any of the acceptance criteria fails, narrow the flow rate and establish the allowable range of variation in column oven temperature.
5.7.5 Effect of variation in column temperature:
5.7.5.1 Prepare system suitability solutions (e.g. Resolution solution, standard preparation ,etc), and test preparation in duplicate as per the test method and inject into the HPLC system with +/- 5°C of method column temperature. Evaluate the system
suitability parameters as required by the test method and % Assay for both the column temperatures.
Note : If the column oven specification limit does not allow to conduct the above variability, then conduct possible variation within the column oven specification limit.Consider the ambient temperature as 25°C.
5.7.5.2 Acceptance criteria:
The method is considered robust for variation in column temperature
I) if all the system suitability parameters meet the acceptance criteria set forth inthe test method.
II) The difference in % Assay with respect to 100% variation should be NMT 2.0
5.7.5.3 If any of the acceptance criteria fails, narrow the temperature range and establish the allowable range of variation in column oven temperature.
5.8 Specificity:
5.8.1 Interference from Degradants:
5.8.1.1 A test method should be “Specific” for the analyte. Therefore, demonstrate the Non – interference of impurities (Known), degradant and excipients on analyte.
5.8.1.2 Conduct the following degradation studies to obtain the degraded samples with 2% to 30% degradation wherever possible.
5.8.1.3 Subject the Tablet powder / Content of Capsule / reconstituted oral suspension to forced degradation by means of Heat, Humidity, and water stress.
Note: saturated solution of Potassium nitrate will give 90% RH at room temperature.
5.8.1.4 Subject the Tablet powder / Content of Capsule / reconstituted oral suspension to forced degradation by means of Acid and Base stress followed by neutralization if necessary.
5.8.1.5 Subject the Tablet powder / Content of Capsule / reconstituted oral suspension to forced degradation by means of peroxide test, treating with aqueous solution containing Hydrogen Peroxide up to 10%.(Note: Do not heat Peroxide Solution over 40°C)
5.8.1.6 Expose the Tablet powder / Content of Capsules / reconstituted oral suspension to intense ultraviolet radiation (both at longer and shorter wavelength), up to minimum 200 watts/m2 and visible light radiation up to minimum 1.2 million lux hrs of exposure.
5.8.1.7 The temperature and the strength of the stress solution needs to be decided by experimenting to get the sample of required degradation.
5.8.1.8 Simultaneously subject the placebo (excipients mixture as per the manufacturing formula) to Heat, Humidity, Ultraviolet radiation, Water, Acid, Base and Peroxide stress conditions.
5.8.1.9 For multi drug product, placebo formulation containing one drug substance each shall be subjected to forced degradation.
5.8.1.10 Prepare test solutions using unstressed sample and placebo and the stressed samples and placebo as per the test method, if necessary by spiking with known
impurities, and inject into the HPLC system with Photo diode array detector(PDA Detector). Record the chromatograms and UV spectra during the whole run
and measure the response of all the peaks.
5.8.1.11 Demonstrate the effective separation of the analyte peak and the internal standard if any, from the known impurities, Degradants, and the peak due to components of placebo mixture.
5.8.1.12 Also demonstrate the absence of interference of impurities / degradant on analyte peak(s) and on internal standard peak if any, by evaluating peak purity using HPLC software.e.g. For Evaluation by Chemstatation software, “ The purity factor shall be within the threshold limit”, and is not less than 990.0 For Waters Empower software, “ Purity angle shall be not more than the purity threshold” i.e. Purity flag should be “No”.
Note: All requirements of the software are to be met while evaluating peak purity.
5.8.2 Placebo Interference:
5.8.2.1 Perform assay in triplicate on weight of placebo, equivalent to the amount present in portion of test preparation as per the test method.
5.8.2.2 Acceptance criteria:
No interference from placebo.
5.9 NOTE:
I) This SOP is applicable for Blend Assay, Content uniformity and Uniformity of dosage units.
II) Point No.: 4.8.1 is not applicable for Blend Assay, Content uniformity and Uniformity of dosage units.
6.0 REFERENCES
NIL
7.0 VERSION HISTORY
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