2. means to “TO CHECK OR PROVE THE VALIDITY OF” According to FDA – “ The goal of validation is to establish a documented evidence which provides a high degree of assuarance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes.”

3. Definition:  By ISO : Confirmation by examination and provision of evidences that the particular requirements for a specified intended use are fulfilled.  Another definition as given in the Handbook for the Quality Assurance of Metrological Measurements : Method validation consists of documenting the quality of an analytical procedure, by establishing adequate requirements for performance criteria, such as accuracy, precision, detection limit, etc. and by measuring the values of these criteria

4. A. The ISO/IEC Guide 25 includes a chapter on the validation of methods with a list of nine validation parameters. B. The International Conference on Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceuticals for Human Use has given a text on the validation of analytical procedures, including definitions for eight validation characteristics. C. The U.S. Environmental Protection Agency (U.S. EPA) prepared a guidance for methods development and validation under the Resource Conservation and Recovery Act (RCRA). D. The American Association of Official Analytical Chemists (AOAC) E. The U.S Environmental Protection Agency (EPA) F. The U.S. Food and Drug Administration (U.S. FDA)

5. 1.Before their introduction into routine use that is a new method of analysis is going to be used in a laboratory or in field analysis. 2.Whenever the conditions change for which the method has been validated (e.g., instrument with different characteristics) 3.Whenever the method is changed, and the change is outside the original scope of the method. 4.When quality control indicates an established method is changing with time. 5.In order to demonstrate the equivalence between two methods (e.g., a new method and a standard)

6. 1. Specificity 2. Precision 3. Accuracy 4. Linearity 5. Range 6. Detection limit 7. Quantitation limit 8. Robustness

7. The terms selectivity and specificity are often used interchangeably. It is the ability to measure unequivocally the desired analyte in the presence of interference, such as synthetic precursors, excipients, enantiomers, and known (or likely) degradation products that may be expected to be present in the sample matrix.

8. Precision is the degree of agreement among individual results It is expressed as the relative standard deviation (RSD). The measured standard deviation can be subdivided into three categories: repeatability, intermediate precision, and reproducibility.

9. 1. Repeatability : Repeatability is obtained when the analysis is carried out in one laboratory by one operator using one piece of equipment over a relatively short time span. At least 5 or 6 determinations of,three different matrices at,two or three different concentrations should be done and the relative standard deviation calculated. 2. Intermediate precision : Intermediate precision is long-term variability of the measurement process and is determined by comparing the results of a method run within-laboratory variations : usually on different days, by different analysts and with different equipment. 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.

10. 3. Reproducibility : Reproducibility as defined by ICH represents the precision obtained between laboratories. The objective 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 and by using operational and environmental conditions that may differ from but are still within the specified parameters of the method (inter laboratory tests).

11. True value

12. Definition : Accuracy is the degree of agreement of test results generated by the analytical method with the true value, or the closeness of the results obtained by the procedure to the true value. Accuracy should be established across the specified range of the analytical procedure.

13. True value

14. Accurate and precise

15. Definition : The linearity of an analytical method is its ability to elicit test results (response) that are directly, or by means of well-defined mathematical transformation, are proportional to the concentration of analytes in samples within a given range.

16. A series of samples should be prepared in which the analyte concentrations span 80–120% of the expected concentration range. A minimum of five concentrations should be used. The response should be—directly or by means of a well-defined mathematical calculation should be proportional to the concentrations of the analytes. If there is a linear relationship, test results should be evaluated by appropriate statistical methods.

17. CALCULATION OF LINEARITY (graphically) Graphical presentations of linearity plot of a caffeine sample using HPLC. Response versus log amount Response versus amount

18. Range is an expression of the lowest and highest levels of analyte that have been demonstrated to be determinable for the product. The specified range is normally derived from linearity studies. The range is normally expressed in the same units as the test results (e.g., percentage, ppm) obtained by the analytical method.

19. Definition : Limit of Detection is the smallest quantity of an analyte that can be detected, but not necessarily quantified. Approaches to calculation : 1. visual evaluation 2. signal to noise ratio 3. standard deviation of the response and the slope of the calibration curve

20. 1. visual evaluation - DL is determined by the analysis of a series of samples with known concentrations and establishing the minimum level at which the analyte can be reliably detected. 2. signal to noise ratio - For instrumental procedures that exhibit background noise, it is common to compare measured signals from samples with known low concentrations of analyte with those of the blank samples. The minimum concentration at which the analyte can reliably be detected is established using an acceptable signal - to - noise ratio of 2 : 1 or 3 : 1. 3. standard deviation of the response and the slope of the calibration curve DL=3 σ /S where σ is the standard deviation of the response and S is the slope of the calibration curve Calculation

21. Definition : Quantitation limit is the lowest concentration of an analyte in a sample that may be determined with acceptable accuracy and precision Approaches to calculation : 1. visual evaluation 2. signal to noise ratio 3. standard deviation of the response and the slope of the calibration curve QL=10 σ /S where σ is the standard deviation of the response and S is the slope of the calibration curve

22. Definition : Robustness is the ability of the procedure to provide analytical results of acceptable accuracy and precision under a variety of conditions. e.g., different flow rate, column temperature, injection volume, detection wavelength, or mobile phase composition in case of HPLC Robustness tests examine the effect, operational parameters have on the analysis results. If the influence of the parameter is within a previously specified tolerance the parameter is said to be within the method’s robustness range

23. 1. Develop a validation protocol or operating procedure for the validation. 2. Define the application, purpose, and scope of the method. 3. Define the performance parameters and acceptance criteria. 4. Define validation experiments. 5. Verify relevant performance characteristics of equipment. 6. Qualify materials (e.g., standards and reagents). 7. Perform prevalidation experiments. 8. Adjust method parameters or/and acceptance criteria if necessary.

24. 9. Perform full internal (and external) validation experiments. 10. Develop SOPs for executing the method in the routine. 11. Define criteria for revalidation. 12. Define type and frequency of system suitability tests and/or analytical quality control (AQC) checks for the routine. 13. Document validation experiments and results in the validation report.

25. The scope of the method and its validation parameters and acceptance criteria should be defined early in the process. These can be defined by finding answers to the following questions :- What analytes should be detected? What is the expected concentration range? What are the sample matrices? Are there interfering substances expected and if so, should they be detected and quantified? Are there any specific legislative or regulatory requirements? Should information be qualitative or quantitative?

26. What are the required detection and quantitation limits? What precision and accuracy is expected? How robust should the method be? For example, should the method work at a specific room temperature or should it run independently from room temperatures? Which type of instrument should be used? Will the method be used in one specific laboratory or should it be applicable all laboratories in your organization? What skills should the anticipated users of the method have? Continued..

27. 1. Description of the method. 2. Objective and scope of the method (applicability, type). 3. Summary of methodology, including sampling procedures. 4. Type of compounds and matrix. 5. All chemicals, reagents, mobile phases, reference standards, quality control samples with purity, grade, their source, or detailed instructions on their preparation. 6. Procedures for quality checks of standards and chemicals used Safety precautions.

28. 7. A plan and procedure for method implementation from method development lab to routine. 8.Method parameters. 9.Critical parameters taken from robustness testing. 10.Listing of equipment and its functional and performance requirements (e.g., cell dimensions, baseline noise, column temperature range). For a complex equipment a picture or schematic diagrams may be useful. 11.Detailed conditions on how the experiments were conducted, including sample preparation. The report must be detailed enough to ensure that it can be reproduced by a competent technician with comparable equipment. 12.Statistical procedures and representative calculations. 13.Procedures for quality control in the routine (e.g., system suitability tests) with acceptance criteria. Contents of a validation report continued..1

29. 14. Representative plots (e.g., chromatograms, spectra, and calibration curves). 15.Method-acceptance limit performance data. 16.The expected uncertainty of measurement results. 17.Criteria for revalidation. 18.The person who developed and initially validated the method. 19.Approval with names, titles, date, and signature of those responsible for the review and approval of the analytical test procedure. 20.The expected uncertainty of measurement results. 21.Criteria for revalidation. 22.The person who developed and initially validated the method. 23.References (if any). 24.Approval with names, titles, date, and signature of those responsible for the review and approval of the analytical test procedure. Contents of a validation report continued..2