Analytical method validation

AMV Parameter Accuracy, Precision, Specificity, Limit of detection, Robustness etc.

1. ROSHAN H. KHETADE Quality Assurance Department

2. C ontent Introduction Strategy for the validation of method. Verification of standard method. Validation of non-routine method. Quality control plan. Implementation of routine analysis Transferring validated routine method Revalidation. Parameters for method validation.

3. Introduction Method validation is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. From method validation can be judge the quality, reliability and consistency of analytical result. It is an integral part of any good analytical practice.

4. Analytical method need to be validated or revalidation Before their introduction into routine use. Whenever the conditions change for which the method has been validated (e.g. instrument with different characteristic or sample with different matrix.) Whenever the method changed and the change is outside the original scope of the method.

5. The USP has published specific guidelines for method validation for compound evaluation. USP define eight step for validation Accuracy Precision Specificity Limit of detection Limit of quantitation Linearity and range Ruggedness Robustness

6. Strategy for the validation of method The validity of a specific method should be demonstrated in laboratory experiment using samples or standards that are similar to unknown samples analyzed routinely. The preparation and execution should follow a validation protocol, preferably in a step-by-step instruction format. Steps for complete method validation are as follows:

7. Develop a validation protocol ,an operating procedure , or a validation master plan for the validation. For a specific validation project , define owners and responsibilities. Develop a validation project plan. Define the application ,purpose and scope of the method. Define the performance parameter and acceptance criteria. Define validation experiments. Verify relevant performance characteristics of equipment.

8. Quality material, e.g. standards and reagents for purity, accurate amount, and sufficient stability . Perform pre-validation experiment. Adjust method parameter / Acceptance criteria if necessary. Perform full internal(and external)validation experiment. Develop SOPs for executing the method in the routine. Define criteria for validation. Define type and frequency of system suitability tests and /analytical quality control checks for the routine. Document validation experiments and result in the validation report.

9. Validation Report Objective and scope of the method. Summary of methodology. Type of compounds and matrix. All chemical reagents, reference standards,QC sample with purity, grade, their source, and detailed instruction on their preparation. Procedure for quality check of standards and chemicals used. Safety precautions. A plan and procedure for method implementation from the method development lab to routine analysis. Method parameters.

10. Critical parameter taken from robustness testing. Listing of equipment and its function and performance requirements. e.g.: cell dimensions, column temperature. Detailed conditions on how the experiments were conducted, including sample preparation. Statistical procedures and representative calculations. Procedures for Qc routine analysis. E.g. System suitability. Representative plots , e.g. chromatograms, spectra , and calibration curve. Method acceptance limit performance data.

11. The expected uncertainty of measurement results. Criteria for revalidation. The person who developed and validated the method. References Summary and conclusion. Approval with name, tittle, date, and signature of those responsible for the review and approval of the analytical test procedure.

12. Verification of standard method There are two important requirement : The standard’s method validation data are adequate and sufficient to meet the laboratory’s method requirements. The laboratory must be able to match the performance data as described in the standard.

13. Evaluation and validation of standard method: Define scope of user method Define validation parameter and limits Standard method fits scope, parameter and limit Perform part or full validation Define and perform system suitability testing N o yes

14. Step for validating complete analytical procedure S ampling Sample preparation A nalysis Calibration Data evaluation Reporting

15. Quality control plan F or any method that will be used for routine analysis, a QC plan should be developed. The plan should ensure that the method, together with the equipment, delivers consistently accurate result. Plan may include recommendation for the following: Selection, handling and testing of QC standards. Type and frequency of equipment checks and calibrations Type and frequency of system suitability testing Type of frequency of QC sample Acceptance criteria for equipment checks, system suitability test and QC sample analysis.

16. Transferring validated routine method Validated routine method are transferred between laboratories at the same or different sites when contract laboratories offer services for routine analysis in different area or when product are manufactured in different area. When validated routine methods are transferred between laboratories and sites. Their validated state should be maintained to ensure the same reliable result in the receiving laboratory.

17. The transfer should be controlled by a procedure. The recommended step are Designate a project owner Develop a transfer plan Define transfer tests and acceptance criteria Describe rationale for tests Train receiving lab operators in transferring lab on equipment, method , critical parameters, and troubleshooting Repeat two critical method validation test in routine lab Analyze at lest three sample in transferring and receiving lab Document transfer result

18. Revalidation Revalidation is necessary whenever a method changed and the new parameter lies outside the operating range. For example the operating range of the column temperature has been specified to be between 30°C and 40°C, the method should be revalidated if, for whatever reason, the new operating parameter is 41°C.

19. Define and document change Change fits the method’s scope, parameter and limit Perform part or full revalidation Define and perform system suitability testing yes No Flow diagram for revalidation

20. Method validation

21. PRECISION The precision of on analytical procedure express the closeness of agreement (degree of scatter) between a sampling of the same homogenous sample under the prescribed condition. Precision may be considered at three level : repeatability, intermediate precision, reproducibility. Precision should be investigated using homogeneous authentic sample. The precision of on analytical procedure is usually expressed as the various standard deviation of coefficient of variation of a serious of measurement.

22. REPEATABILITY Repeatability expressed the precision under interval of time repeatability is also term termed intra-assay precision. I NTERMEDIATE PRECISION Intermediate precision express within laboratories variation different days, different analysts, different equipment etc. REPRODUCABILITY Reproducibility express the precision between laboratories collaborative studies usually applied to standardization of methodology.

23. ACCURACY The agreement between the test result obtained by the proposed method and the true value. It expresses the correctness of the method. It is expressed as percentage by the assay of known amount of substance. Accuracy also evaluated by recovery studies in which amount of drug is added to previously analyzed pharmaceutical preparation of the drug and tested for the recovery of the added drug.

24. Should be established across specified range of analytical procedure. Should be assessed using a minimum of 3 concentration levels. Each in triplicate (total of 9 determination) Should be reported as Percent recovery of known amount or The difference between the mean assay result and the accepted value The absolute error is a measure of the accuracy of the measurement, it is then calculated as Absolute error = mean error (true value – measured value )/true value x 100

25. LIMIT OF DETECTION It can be defined as the lowest amount of the analyte in a sample that can be detected but not necessarily quantified under the standard experiment condition. How is it determined ? Based on visual inspection Detection limit is determined by the analysis of sample with known concentration of analyte and by establishing the minimum level at which the analyte can be reliably detected. Based on standard deviation of the black Measurement of the magnitude of analytical background response is performed by analyzing an appropriate number of black sample and calculating the standard deviation of these response

26. Based on the slop of the calibration curve A specific calibration curve is study using sample containing and analyte in the range of limit of detection the residual standard deviation of a regression line or the standard deviation of y-intercept of regression lines may be used as the standard deviation.

27. LIMIT OF QUANTITATION The quantitation limit of an indivial analytical procedure is the lowest amount of analyze in a sample which can be quantitatively determine with suitable precision and accuracy. The quantitative limit is parameter of quantities assay for low week of compound in sample matrices and is used particularly for the determination of impurities and /or degradation product. It express as the concentration of analyte (e.g. part per million) in the sample

28. SPECIFICITY Is the ability to measure the analyte in the present of other component which may be expected to be present typically this might include impurities, degradants, matrix etc. Lack of specificity of an individual procedure may be compensated by other supporting analytical procedure. An investigation of specificity should be conducted the validation of identification tests, the determination of impury and assay.

29. LINEARITY It indicates the ability to produce the result that are directly proportional to the concentration of analyte in sample within a given range (Y =mx+c). The following parameter should be determine Correlation coefficient y-intercept (c) Slop of the regression line (m)

30. RANGE The range of analytical procedure is the interval between the upper and lower level of analyte (including these level) that have been demonstrated with a suitable level of precision, accuracy and linearity. The range is normally expressed in the same units as the test results(E.g . percentage, parts per million) obtained by the analytical method.

31. The following minimum specified range should be consisted: For the assay of a drug substance or a finished (drug) product: normally from 80-120 % of the test concentration. For content uniformity, covering a minimum of 70-130 % of the test concentration ,unless a wider more appropriated range, based on the nature of the dosage form (e.g. metered dose inhalers),is justified. For dissolution testing +/- % over the specified range.

32. ROBUSTNESS The robustness of analytical method is measure of its capacity to remain unaffected by small but deliberated variation in method parameter and provide an indicated of it reliably during normal usage. Robustness examines the effect of variation in operational parameter on the analysis result for the determination of a method robustness, parameter like variation in detector wavelength are varied within a realistic range and the quantitative influence of the variable id determine.

33. If the influence of the parameter is within a previously specified tolerance , the parameter is said to be within the method robustness range. Acceptance criteria % RSD should not be more than 2%.

34. RUGGEDNESS It is a measure of method reproducibility under variable conditions within specified test parameter of test method. The following are the typical method parameter need to tested during method validation. Analyst to analyst variability. Column to column variability. System to system variability. Different days different laboratory. Stability of solution of mobile phase.(at least for 48 hours)

35. STABILITY Stability testing is an integral part of many analytical procedure. t he tests are based on the concept that the equipment, electronics, analytical operation and sample to be analyzed constituent an integral system that can be evaluated as such. Determination: Reputability, Tailing factor (T), Capacity factor (k’),Resolution(R), and Theoretical plates(N).

36. ICH/ USP VALIDATION REQUIRMENT AND PARAMETER USP Specificity Linearity and range Accuracy Precision Limit of detection Limit of quantitation Ruggedness robustness ICH Specificity Linearity Range Accuracy Precision Repeatability Intermediate precision Reproducibility Limit of detection Limit of quantitation

37. Reference Pharmaceutical process validation An international third edition revised and expanded volume 129 edited by Robert A. Nash ,Alfred H . Watcher page no. 542-559.