VALIDATION OF ANALYTICAL METHODS Presented by Dr. A. Suneetha Dept. of Pharm. Analysis Hindu College of Pharmacy
According to FDA,validation is established documented evidence which provides a high degree of assurance that a special process will consistently produce a product meeting its pre-determined specifications & quality attributes.
Purpose of validation Method is suitable for its intended purpose to get consistent, reliable & accurate data. Identification of sources & quantization of potential errors. Satisfy FDA requirements. Establish proof that a method can be used for decision making.
Advantages Increased output. Reduction in rejections & reworking. Cost effective. Avoidances of capital expenditure. Reduced testing in-process & in finished goods. Easier maintenance of equipment.
Methods need to be validated or re-validated as follows: a. Before their introduction into routine use. b. Whenever the conditions change for which the method has been validated. c. Whenever the method is changed & the change is outside the original scope of the method. d. When QC indicates an established method is changing with time. e. In order to demonstrate the equivalence b/w two methods.(eg,a new mtd & a std)
Steps involved in method validation : 1. Develop a validation protocol or operating procedure for the validation. 2. Define the application, purpose & scope of the method. 3. Define the performance parameters & acceptance criteria. 4. Define validation experiments. 5. Verify relevant performance characteristics of equipment.
6. Qualify materials. 7. Perform pre-validation experiments. 8. Adjust method parameters or/& acceptance criteria if necessary. 9. Perform full internal(& external)validation experiments. 10. Develop SOP s for executing the method on the routine.
11. Define criteria for re-validation. 12. Define type & frequency of system suitability tests &/or analytical quality control(aqc)checks for the routine. 13. Document validation experiments & results in the validation report. During method validation,the parameters, acceptance limits & frequency of ongoing system suitability tests or QC checks should be defined.
Types of validation 1. Analytical method validation 2. Equipment validation 3. Process validation 4. Cleaning validation 5. Raw material validation 6. Finished material validation
Analytical method validation Analytical validation ensures that the selective analytical method will give reproducible & reliable results adequate for intended use.
Parameters for method validation The parameters as defined by the ICH & other organizations are as follows; 1. Selectivity & Specificity 2. Precision 3. Accuracy 4. Linearity 5. Range 6. LOD & LOQ 7. Ruggedness & Robustness.
Selectivity & Specificity USP defines selectivity of an analytical method as its ability to measure accurately an analyte in the presence of interference such as synthetic precursors,excipients, enantiomers & known degradation products that may be expected to be present in the sample matrix.
SELECTIVITY 1. If an analytical procedure is able to separate & resolve the various components of a mixture & detect analyte qualitatively. 2. Restricted qualitative detection of the components of a sample. SPECIFICITY 1. If the method determines or measures quantitatively the component of interest in the sample matrix without separation. 2. Restricted quantitative measurement of on or more analyte.
Precision Precision of a method is the extent to which the individual test results of multiple injections of a series of standards agree. The measured standard deviation can be sub divided into; 1. Repeatability 2. Intermediate precision 3. Reproducibility
Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision. Intermediate precision expresses variations within laboratories, such as different days, different analysts, different equipment, and so forth. The objective of intermediate precision validation is to verify that in the same laboratory the method will provide the same results once the development phase is over.
Reproducibility expresses the precision between laboratories (collaborative studies usually applied to standardization of methodology). The objective of reproducibility is to verify that the method will provide the same results in different laboratories. The reproducibility of an analytical method is determined by analyzing aliquots from homogeneous lots in different laboratories with different analysts.
Accuracy The extent to which test results generated by the method & the true value agree. The true value for accuracy assessment can be obtained in several ways:- 1. To compare results of the method with results from an established reference method. 2. By analyzing a sample with known concentrations. 3. Comparing the measured value with the true value as supplied with the material.
Linearity Ability to elicit test results that are directly, or by means of well-defined mathematical transformation,proportional to the concentration of analytes in samples with in a given range. Linearity is determined by a series of 3 to 6 injections of 5 or more standards whose concentrations span 80 to 120 % of the expected concentration range.
Range It is the interval b/w the upper & lower levels that have been demonstrated with precision,accuracy,linearity using the method as written. Expressed in the units %,ppm.
LOD & LOQ LOD 1. Lowest amount of analyte in a sample that can be detected but not necessarily quantities. 2. Estimation by S/N ratio of 3 : 1. LOQ 1. Lowest amount of analyte in a sample that can be quantified with suitable accuracy & precision. 2. Estimation by S/N ratio of 10 : 1.
Robustness ICH defines the robustness of an analytical procedure as a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters. It provides an indication of the procedure s reliability during normal usage. Robustness tests examine the effect that operational parameters have on the analysis results.
For the determination of a method s robustness, a number of method parameters, such as ph, flow rate, column temperature, injection volume, detection wavelength or mobile phase composition, are varied within a realistic range, and the quantitative influence of the variables is determined. If the influence of the parameter is within a previously specified tolerance, the parameter is said to be within the method s robustness range. It is recommended to consider the evaluation of a method s robustness during the development phase, and any results that are critical for the method should be documented
Ruggedness Ruggedness is defined by the USP as the degree of reproducibility of results obtained under a variety of conditions, such as different laboratories, analysts, instruments, environmental conditions, operators and materials.
Equipment validation It includes; 1. Design qualification(dq) 2. Installation qualification(iq) 3. Operational qualification(oq) 4. Performance qualification(pq)
Design qualification Defined as Providing documented evidence that the design of the facility & equipments meet the requirements of the user specification & GMP.
Installation qualification Establishing confidence that process equipment & ancillary systems are capable of consistently operating within established limits & tolerances.
Operational qualification Defined as Providing documented verification that the systems & sub-systems perform as intended throughout all anticipated operating ranges. This step proceeds after the IQ has been performed. Define the specification & acceptance criteria.
Performance qualification Process PQ : Establishing confidence that the process is effective & reproducible. Product PQ : Establishing confidence through appropriate testing that the finished product produced by a specific process meets all release requirements for functionality & safety.
Process validation It is the process to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Types of validation: Four types; 1. Prospective validation 2. Retrospective validation 3. Concurrent validation 4. Revalidation.
Prospective validation : Employed when historical data is not available or is not sufficient & in process & finished product testing are not adequate to ensure reproducibility or high degree of compliance to product likely attributes. Such validation is conducted prior to release of either new product or product made under revised / new manufacturing process where revision may effect the product characters.
Retrospective validation : Provides trend of comparative result. This is acceptable provided specific test results generated by reliable analytical method on no. of samples are available to allow statistical analysis. Concurrent validation : Based on the information generated during implementation of a system. It verifies the quality characteristics of a particular batch & provide assurance that the same quality would be attained again when subsequent batches are manufactured & analyzed under similar conditions.
Revalidation Analytical methods require revalidation if: 1. The method parameters have to be changed to maintain the original performance and the change is outside the tolerance allowed by USP 2. New compounds are analyzed that are not within the method s original scope. 3. The sample matrix changes.
Any such modification should be documented following a change control procedure and the method should be revalidated. As part of the procedure the reason for the change should be defined and the change should be authorized for implementation and documented. Performance tests should be justified and documented, and the change formally released.
Reference Pharmaceutical Process Validation Robert A. Nash Alfred H. Wachter. www.chem-agilent.com. www.autostream.com.
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