1.0 OBJECTIVE
To lay down the procedure for the Validation of Analytical Test Procedure for Related substances by HPLC based on ICH, USP and In-house Guidelines.
2.0 SCOPE
This SOP is applicable for the Validation of Analytical Test Procedure for Related substances by HPLC in company Name
3.0 BACKGROUND
NIL
4.0 RESPONSIBILITY
4.1 Scientist - Analytical Research to perform the Validation of Test Procedure.
4.2 Group Leader- Analytical Research to check the Validation of Test procedure.
4.3 Head - Analytical Research to review the data and to ensure the overall compliance
4.0 PROCEDURE
5.1 Specificity
5.1.1 During the specificity, following experimental details shall be followed.
5.1.2 To evaluate the specificity of the Test Procedure, demonstrate the non interference of impurities (known), degradant and excipients from each others and from the principal peak. Also inject blank/diluent as per test method and check non-interference of diluent with impurities/degradants and principal peak.
5.1.3 Conduct the following forced degradation studies to obtain degraded samples with 2 to 30% degradation.
5.1.4 Subject the Tablet/Capsule/ oral suspension powder to forced degradation by means of Heat and water stress.
5.1.5 Subject the Tablet/Capsule/ oral suspension powder to forced degradation by means of Acid and Base stress followed by neutralization.
5.1.6 The Tablet / Capsule / oral suspension powder to oxidative conditions such as reflux under oxygen or heating the aqueous solution containing hydrogen peroxide up to 10%.(Note: Do not heat Peroxide Solution over 40°C)
5.1.7 The temperature and strength of the stress solutions needed to be decided by trials to get the sample with required degradation.
5.1.8 Expose the Tablet /Capsule/Oral suspension powder to Visible radiation up to minimum 1.2 million lux hrs and ultra violet radiation up to minimum 200 watts/m2 in photo stability chamber.
5.1.9 Tablet /Capsule/Oral suspension powder to be exposed to a relative humidity of 90% at room temperature by exposing the samples to saturated potassium nitrate solution. Note: Saturated solution of Potassium nitrate will give 90% RH at room temperature.
5.1.10 Simultaneously subject the drug substance and placebo of the respective formulations to all the above stress conditions.
5.1.11 For multi drug product, placebo for the respective formulation containing one drug substances each shall be subjected to forced degradation.
5.1.12 Prepare test solutions using unstressed sample, placebo and the stressed samples as per the test procedure, if necessary by spiking the known impurities and inject into the HPLC system with photo diode array detector (PDA Detector).Record the chromatograms & UV spectra during whole run and measure the responses of all the peaks.
5.1.13 Determine the effective separations of analyte peak from excipients, known impurities and degradation products.
5.1.14 Peak Purity Evaluations: Demonstrate the absence of interference of impurities / degradation products on analyte peak by evaluating peak purity testing using HPLC software.e.g. For Evaluation by HP Chemstation software, “ The purity factor within the threshold limit” and is not less than 990.0For Evaluation by Waters Millennium software, “Purity angle shall be less than the purity threshold”.(Purity flag should be NO)
5.2 Placebo interference:
5.2.1 Evaluate the non interference or extent of interference of placebo in estimation of % of impurities. Prepare a placebo solution in triplicate by following the procedure for test solution using equivalent weight of placebo.
5.2.2 Inject the placebo solutions into the HPLC system following the Test procedures, record the chromatogram and measure the responses of peaks due to placebo, if any.
5.2.3 Estimate the % of peaks other than those due to mobile phase or diluent as % of Test concentrations.
5.2.4 Acceptance Criteria:
No interference from placebo.
5.3 System suitability:
5.3.1 System precision:
The system precision as measured by replicate injections of standard solution indicates the performance of HPLC system under the chromatographic condition
and the day tested. As a part of method validation minimum of 10 injections shall be made, and %RSD of the peak area should be not more than half of the value
of acceptance criteria under system suitability of test method.
5.3.2 Other system suitability parameters: System suitability parameters like Resolution, Theoretical plate counts or tailing Factors shall be evaluated as per the Test procedure. The method is considered to be suitable if all the mentioned parameters are well within the limit as per the Test procedure.
Note: If system suitability parameters mentioned in the Test procedure are observed to be in adequate or difficult to meet the requirements, New system suitability criteria shall be evaluated with proper justification, and the same shall be followed during the validation activity.
5.3.3 Acceptance Criteria:
System suitability parameters shall be achieved.
5.4 Limit of detection and quantitation:
5.4.1 Establish the Limit of detection and Quantitation for all known impurities and identified Degradants found to be present in normal samples or accelerated stability samples Limit of detection and quantitation will be established based on Signal to noise ratio (or) by standard deviation of the response and the slope.
Note: If drug product contains known impurity then Limit of detection and Limit of Quantitation shall be established on Placebo by considering the equivalent blend weight and Theoretical average weight/ fill weight for calculating the % impurity at LOQ and LOD level.
5.4.2 Limit of detection and Limit of Quantitation can be established for known impurities either by injecting individual solutions or by injecting an impurity blend solutions
5.4.3 Derive the approximate concentration, which will give signal to noise ratio 3 based on impurities standard solution (on blend/placebo). Prepare impurities /
degradants in that concentration range and inject into the HPLC system following conditions described in test procedure.
5.4.4 Determine the Limit of detection of an impurity / degradant by identifying the Concentration which gives signal to noise ratio 3.
5.4.5 Determine the Limit of Quantitation of an impurity / degradant by identifying the Concentration which gives signal to noise ratio 10.
5.4.6 Limit of detection and quantitation (Based on the Residual standard deviation of the response and the slope):
5.4.7 Inject solutions (NLT 6) and roughly find the detection limit and Quantitation limit based on the formula given below.
3.3 σ 10 σ
Detection Limit =-------- Quantitation Limit =--------
S S
σ – Residual standard deviation of the response (Standard error)
S – Slope of calibration curve
5.4.8 Perform Precision at LOQ by preparing 6 test solution having impurities/ degradant at the level of about the limit of quantitation Inject into the HPLC
System. Calculate the relative standard deviation for % impurity, which should be not more than 15.0%.
5.4.9 Perform accuracy at LOQ level on three samples by spiking all known Impurities at LOQ Concentration. Calculate % recovery.Acceptance criteria: NLT 70.0% & NMT 130.0% of the added amount
5.4.10 The attempts made for evaluations of Limit of detection and Limit of Quantitation shall be documented and presentable.
5.5 Linearity of Detector response:
5.5.1 Demonstrate the linearity of detector response for all known available impurities and Degradants. Linearity can be established for impurities /Degradants either by injecting Individual solutions or by injecting impurity blend solutions.
5.5.2 In case the impurity peak is eluting very closely to an analyte peak, (RRT about 0.9 to 1.1) conduct the study in presence of analyte.
5.5.3 To demonstrate the linearity of detector response for impurities / degradants, prepare not less than six solutions with concentrations ranging from LOQ level to 150% of the target concentration or at level of impurity / degradant in the drug product as per the specifications.
5.5.4 Plot average peak area versus the actual concentration in mg/mL.
5.5.5 Acceptance Criteria: The response is considered to be linear if correlation coefficient (r) derived from the least squares fit the data is not less than 0.98.
5.5.6 In case of any discrepancy, investigate the reason for the discrepancy and shall be justified. Repeat if necessary and establish the range within which the response is linear.
5.6 Method Precision:
5.6.1 Prepare sample using tablet / capsule/oral suspension powder as per the Test method, prepare stock solution having all known impurities and degradant.
5.6.2 Prepare not less than six test solutions as per the test procedure but spiking with known impurity stock solution having impurities at about target concentration or at specified level (e.g. at allowed % levels in drug product) on drug product concentration.
5.6.3 Inject the solutions into the HPLC system, record the chromatogram and measure the peak response
5.6.4 Acceptance Criteria: The method is considered “precise” if the RSD of %individual impurities / degradant is not more than 10.0%.
NOTE: 1) Data generated in this section may be used to represent one part in System to system variability, Analyst to Analyst variability, Column to Column variability and Laboratory to Laboratory variability.
2) If impurities are not sufficient, change the test dilution without affecting the final concentration.
5.7 Recovery / Accuracy:
5.7.1 Prepare homogeneous tablet / capsule blend or constitute the oral suspension as per the test procedure. Prepare stock solution having all known impurities and degradants.
5.7.2 Prepare sample solutions in triplicate by spiking test preparation from LOQ to 150% of target concentration or at specified level of each impurity / degradant.
5.7.3 Prepare the standard solutions as per the test method and inject into the HPLC system.
5.7.4 Analyze sample solutions as per the test procedure and calculate the % recovery and average recovery of each impurity at LOQ to 150% spiked levels in comparison with unspiked samples.
Note: Spiked levels of Impurities shall be altered based on the requirement.
5.7.5 Acceptance Criteria:
The method is considered “ACCURATE”, if the recovery of each individual impurity is between 85.0% and 115.0%.
5.7.6 If in case of discrepancy, investigate the reason, repeat the experiment, if necessary and explain the reason for discrepancy
5.8 Linearity of test method:
Note: Data generated under Recovery/accuracy shall be used to conduct linearity of test method.
5.8.1 Draw the graph between Ave. µg/mL added versus Ave. µg/mL found for each impurity and calculate the correlation coefficient.
5.8.2 Acceptance criteria:
Correlation coefficient (r) for each known impurity shall be NLT 0.98
5.9 Ruggedness:
5.9.1 System to System variability:
5.9.1.1 Conduct System to System variability study on two HPLC systems (of the same or different manufacturer) by the same analyst and using the same column.
5.9.1.2 Prepare homogeneous tablet / capsule blend or constitute the oral suspension as per the test procedure. Prepare stock solution having all known impurities and degradant.
5.9.1.3 Prepare not less than six test solutions as per the test procedure but by spiking with impurity stock solution(s) to get solution having Impurities at about Target concentration or at the specified levels (i.e. at allowed % levels in drug product) on drug product concentration.
5.9.1.4 Inject the solutions into the HPLC system as per the test procedure, record the
Chromatograms and measure the peak responses.
5.9.1.5 Acceptance Criteria:
The test procedure is considered rugged for system to system variability, if the RSD of peak areas of individual known impurity (spiked) is NMT 10.0% for both the systems
5.9.2 Column to Column variability:
5.9.2.1 Conduct Column to Column variability study on two columns (of the same or different manufacturer) by the same analyst and using the same HPLC system.
5.9.2.2 Proceed as directed in point no 4.9.1.2 to 4.9.1.4.
5.9.2.3 Acceptance Criteria: The test procedure is considered rugged for Column to Column variability, if the RSD of % individual impurity (spiked) is NMT 10.0% for both the Columns.
5.9.3 Analyst to Analyst variability:
5.9.3.1Conduct Analyst to Analyst variability study by two analysts at different days using same HPLC systems and the same column.
5.9.3.2 Proceed as directed in point No. 5.9.1.2 to 5.9.1.4.
5.9.3.3 Acceptance Criteria: The test procedure is considered rugged for Analyst to Analyst variability, if the RSD of % individual impurity (spiked) is NMT 10.0% for both the analysts. 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.
5.9.4 Bench top Stability of standard and test solutions:
5.9.4.1 Establish the Bench top stability of test solutions and standard solution(s), if any, which will be used in estimation of % impurities, over a period of 2-days.
5.9.4.2 Prepare standard solution, test solution and test solution spiked with impurities as per the test procedures and keep them on Bench top for a period of 2 days
5.9.4.3 Inject the solutions into HPLC system at initial, 1day and 2days ”, following the conditions described in the test procedure. Calculate the % of known individual impurities and % of total impurities at initial ,1day and 2days as per the test procedure against the freshly prepared standard solution wherever applicable.
5.9.4.4 Acceptance Criteria:
I) The spiked sample and unspiked sample is considered to be “STABLE” The difference in % of known, highest unknown and total impurities shall be:
Not more than ± 0.05, if the % of impurities are less than or equal to 0.3%;
Not more than ± 0.1, if the % of impurities is > 0.3% and ≤ 0.8%;
Not more than ± 0.2, if the % of impurities is > 0.8% and ≤ 1.5%;
Not more than ± 0.4, if the % of impurities is > 1.5% and ≤ 3.0%;
Not more than ± 0.6, if the % of impurities is > 3.0%.
when compared with that of initial.
II)The standard preparation is considered to be stable if, the % assay difference from initial is not more than 3.0. If the solutions are not found to be stable for 1day, conduct the study at shorter time intervals e.g. at 1, 2, 3 hours interval and establish the period of time during which the solutions will be stable.
5.9.5 Refrigerator Stability of standard and test solutions:
5.9.5.1 Establish the Refrigerator stability of test solutions and standard solution(s), if any, which will be used in estimation of % impurities, over a period of 2-days.
5.9.5.2 Prepare standard solution, test solution and test solution as per the test procedures by spiking with the impurity solution, and keep them in refrigerator for a period of 2 days.
5.9.5.3 Inject the solutions into HPLC system at initial, 1day and 2days ”, following the conditions described in the test procedure. Calculate the % of known individual impurities and % of total impurities at initial, 1day and 2days as per the test procedure against the freshly prepared standard solution wherever applicable
5.9.5.4 Acceptance Criteria:
I) The spiked sample and unspiked sample is considered to be “STABLE” The difference in % of known, highest unknown and total impurities shall be:
Not more than ± 0.05, if the % of impurities are less than or equal to 0.3%;
Not more than ± 0.1, if the % of impurities is >0.3% and ≤0.8%;
Not more than ± 0.2, if the % of impurities is > 0.8% and ≤ 1.5%;
Not more than ± 0.4, if the % of impurities is > 1.5% and ≤ 3.0%;
Not more than ± 0.6, if the % of impurities is > 3.0%.
when compared with that of initial.
II) The standard preparation is considered to be stable if, the % assay difference from initial is not more than 3.0. If the solutions are not found to be stable for 1day, conduct the study at shorter time intervals e.g. at 1, 2, 3 hours interval and establish the period of time during which the solutions will be stable.
5.9.6 Bench top stability of mobile phase:
5.9.6.1 Establish the bench top stability of mobile phase, for a period of 2 days.
5.9.6.2 Prepare mobile phase as per the test procedure and keep it on bench top for period of 2 days in well-closed condition.
5.9.6.3 Prepare Blank, system suitability solution (e.g. Resolution solution, standard solution etc.) and Test solution by spiking with impurity solution. Inject into the HPLC system by following the conditions described in test procedure at initial, 1 day and 2 day.
5.9.6.4 Acceptance Criteria:
1.All the system suitability should be within the limits specified in the test procedure.
2.RRT’s of known impurities shall be comparable with the initial values
5.9.7 Refrigerator stability of system suitability solution other than standard Preparation:
5.9.7.1 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.
5.9.7.2 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.
5.9.7.3 Acceptance Criteria:
All the system suitability values shall be within the limits specified in the test procedure.
5.10 Robustness:
5.10.1 Effect of variation in mobile phase composition:
5.10.1.1 Prepare two mobile phases having 90% and 110% method organic phase concentrations. Prepare system suitability solutions (e.g. Resolution solution,
standard preparation) as per the test procedure and inject into the HPLC system using both the mobile phases. Evaluate the system suitability values as required
by the test procedure for both mobile phases.
5.10.1.2 Prepare a test solution spiked with known impurity blend of target concentration or at specified level. Inject into the HPLC system in both the mobile phases
5.10.1.3 Acceptance Criteria:
The method is considered robust for the variation of organic phase composition, if all the system suitability values meets the acceptance criteria set forth in the test procedure, if all known impurities are separated from analyte, from each other and from the excipients peak, Relative Retention times are comparable and the difference in % of known impurities shall be:
Not more than ± 0.05, if the % of impurities are less than or equal to 0.3%;
Not more than ± 0.1, if the % of impurities is >0.3% and ≤0.8%;
Not more than ± 0.2, if the % of impurities is > 0.8% and ≤ 1.5%;
Not more than ± 0.4, if the % of impurities is > 1.5% and ≤ 3.0%;
Not more than ± 0.6, if the % of impurities is > 3.0%.
when compared with those obtained with mobile phase having 100% organic phase composition.
5.10.1.4 If any of the criteria is not met, narrow the organic phase composition range and establish the allowance range of variation for organic phase composition.
Note: If method contains more than one organic phase then conduct variability individually and verify one at a time.
5.10.2 Effect of variation of pH of mobile phase:
5.10.2.1 Prepare two mobile phases having buffer with ±0.2 of the method pH. Prepare system suitability (e.g. Resolution solution, standard preparation, etc) as per the test procedure and inject into the HPLC system using both mobile phases. Evaluate the system suitability values as required by the test procedures for both the mobile phases.
5.10.2.2 Prepare a test solution spiked with known impurity blend solution at Target concentration or at specified level. Inject into the HPLC system in both the mobilephases.
5.10.2.3 Acceptance Criteria:
The method is considered robust for the variation of pH, if all the system suitability values meets the acceptance criteria’s set forth in the test procedure, if all known impurities are separated from analyte, from each other and from the excipients peaks, Relative Retention times are comparable and the difference in % of known
impurities shall be:
Not more than ± 0.05, if the % of impurities are less than or equal to 0.3%;
Not more than ± 0.1, if the % of impurities is >0.3% and ≤0.8%;
Not more than ± 0.2, if the % of impurities is > 0.8% and ≤ 1.5%;
Not more than ± 0.4, if the % of impurities is > 1.5% and ≤ 3.0%;
Not more than ± 0.6, if the % of impurities is > 3.0%.
when compared with those obtained with mobile phase having method pH.
5.10.2.4 If any of the criteria is not met, narrow the pH range and establish the allowance range of variation of pH.
5.10.3 Effect of variation in Flow rate:
5.10.3.1 Prepare system suitability (e.g. Resolution solution, standard preparation, etc) as per the test procedure and inject into the HPLC system with ± 0.2 mL of the method flow rate. Evaluate the system suitability values as required by the test procedures for both the flow rates.
5.10.3.2 Prepare a test solution spiked with known impurity blend solution at Target concentration or at specified level. Inject into the HPLC system in both the flow rates.
5.10.3.3 Acceptance Criteria: The method is considered robust for the variation in flow rates, if all the system
suitability values meets the acceptance criteria set forth in the test procedure, if all known impurities are separated from analyte, from each other and from the excipient peak, Relative Retention times are comparable and the difference in % of known impurities shall be:
Not more than ± 0.05, if the % of impurities are less than or equal to 0.3%;
Not more than ± 0.1, if the % of impurities is >0.3% and ≤0.8%;
Not more than ± 0.2, if the % of impurities is > 0.8% and ≤ 1.5%;
Not more than ± 0.4, if the % of impurities is > 1.5% and ≤ 3.0%;
Not more than ± 0.6, if the % of impurities is > 3.0%.
when compared with those obtained with method flow rate.
5.10.3.4 If any of the criteria is not met, narrow the flow range and establish the allowance range of variation of flow.
5.10.4 Effect of variation in Column Temperature:
5.10.4.1 Prepare system suitability (e.g. Resolution solution, standard preparation, etc) as per the test procedure and inject into the HPLC system with ± 5°C of the method Column Temperature. Evaluate the system suitability values as required by the test procedures for both the Column Temperatures.
5.10.4.2 Prepare a test solution spiked with known impurity blend solution at target concentration or at specified level. Inject into the HPLC system in both the column
temperatures.
5.10.4.3 Acceptance Criteria:
The method is considered robust for the variation in column temperatures, if all the system suitability values meets the acceptance criteria’s set forth in the test
procedure, if all known impurities are separated from analyte, from each other and from the excipients peak, Relative Retention times are comparable and the difference in % of known impurities shall be:
Not more than ± 0.05, if the % of impurities are less than or equal to 0.3%;
Not more than ± 0.1, if the % of impurities is >0.3% and ≤0.8%;
Not more than ± 0.2, if the % of impurities is > 0.8% and ≤ 1.5%;
Not more than ± 0.4, if the % of impurities is > 1.5% and ≤ 3.0%;
Not more than ± 0.6, if the % of impurities is > 3.0%.
when compared with those obtained with method column temperature. 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.
5.10.5 Filter Validation:
5.10.5.1 If the filters are involved in the test Procedure for filtration of test solutions, To demonstrate that the filtration does not affect the analysis results, at least validate two types of filters during validation.
5.10.5.2 Prepare standard solution if any, as per the test procedure without filtration. Prepare test solutions preferably using aged samples or an accelerated sample or forced degradation samples and if necessary spiking with known impurities.
5.10.5.3 Centrifuge the portion of test solutions and filtered portions of test and standard solution(s) through individual filters. Inject filtered and unfiltered standard solutions and centrifuged & filtered test solutions into HPLC system under the test conditions.
5.10.5.4 Calculate the % of known impurities, % of total impurities for centrifuged and filtered test preparations using unfiltered standard preparation into consideration wherever required.
5.10.5.5 Acceptance Criteria:
The filters are considered to be acceptable if the difference in centrifuged to filtered samples for % of known and % of total impurities shall be:
Not more than ± 0.05, if the % of impurities are less than or equal to 0.3%;
Not more than ± 0.1, if the % of impurities is >0.3% and ≤0.8%;
Not more than ± 0.2, if the % of impurities is > 0.8% and ≤ 1.5%;
Not more than ± 0.4, if the % of impurities is > 1.5% and ≤ 3.0%;
Not more than ± 0.6, if the % of impurities is > 3.0%.
6.0 REFERENCES
ICH guidelines - Validation of Analytical Procedures Text and Methodology Q2(R1)
7.0 VERSION HISTORY
8.0 ANNEXURES
NIL
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