ANALYTICAL METHOD VALIDATION

1. ANALYTICAL METHOD VALIDATION

2. Method Validation • Validation of analytical procedures is the process of determining the suitability of a given methodology for providing useful analytical data. • Validation is the formal and systematic proof that a method compiles with the requirements for testing a product when observing a defined procedures.

3. Method validation is the process of demonstrating that analytical procedures are suitable for their intended use and that they support the identity, strength, quality, purity and potency of the drug substances and drug products • Method validation is primarily concerned with: • identification of the sources of potential errors • quantification of the potential errors in the method • A method validation describes in mathematical and quantifiable terms the performance characteristics of an assay

4. Examples of Methods That Require Validation Documentation • Chromatographic Methods - HPLC, GC, TLC, GC/MS, etc. • Pharmaceutical Analysis - In support of CMC. • Bioanalytical Analysis - In support of PK/PD/Clinical Studies. • Spectrophotometric Methods – UV/VIS, IR, NIR, AA, NMR, XRD,MS • Capillary Electrophoresis Methods - Zone, Isoelectric Focusing • Particle Size Analysis Methods - Laser, Microscopic, Sieving, SEC, etc.

5. Purpose of Method Validation • Identification of Sources and Quantitation of Potential errors • Determination if Method is Acceptable for Intended Use • Establish Proof that a Method Can be Used for Decision Making • Satisfy FDA Requirements

6. What is not Analytical Method Validation? • Calibration The Process of Performing Tests on Individual System Components to Ensure Proper function For example) HPLC Detector calibration • Wavelength Accuracy/ Linear Range/ Noise Level

7. System Suitability Test to verify the proper functioning of the operating system, i.e., the electronics, the equipment, the specimens and the analytical operations. • Minimum Resolution of 3.0 between the analyte peak and internal standard peaks • Relative Standard Deviation of replicate standard injections of not more than 2.0%

8. System Suitability Validation Calibration Pump Injector Detector Data System Analyst Method Sample

9. Validation Development Optimization Method Life Cycle

10. The objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose ICH Guideline for Industry Q2A, Text on Validation of Analytical Procedures March 1995

11. Specificity/Selectivity • Ability of an analytical method to measure the analyte free from interference due to other components. • Selectivity describes the ability of an analytical method to differentiate various substances in a sample • Original term used in USP • Also Preferred by IUPAC • Also used to characterize chromatographic columns • Degree of Bias (Used in USP) • The difference in assay results between the two groups • the sample containing added impurities, degradation products, related chemical compounds, placebo ingredients • the sample without added substances

12. Specificity: Impurities Assay • Chromatographic Methods • Demonstrate Resolution • Impurities/Degradants Available • Spike with impurities/degradants • Show resolution and a lack of interference • Impurities/Degradants Not Available • Stress Samples • For assay, Stressed and Unstressed Samples should be compared. • For impurity test, impurity profiles should be compared.

13. Forced Degradation Studies • Temperature (50-60℃) • Humidity (70-80%) • Acid Hydrolysis (0.1 N HCl) • Base Hydrolysis (0.1 N NaOH) • Oxidation (3-30%) • Light (UV/Vis/Fl) Intent is to create 10 to 30 % Degradation

14. Linearity • Ability of an assay to elicit a direct and proportional response to changes in analyte concentration.

15. Linearity Should be Evaluated • By Visual Inspection of plot of signals vs. analyte concentration • By Appropriate statistical methods • Linear Regression (y = mx + b) • Correlation Coefficient, y-intercept (b), slope (m) • Acceptance criteria: Linear regression r2 > 0.95 Requires a minimum of 5 concentration levels

16. Range • Acceptable range having linearity, accuracy, precision. • For Drug Substance & Drug product Assay • 80 to 120% of test Concentration • For Content Uniformity Assay • 70 to 130% of test Concentration • For Dissolution Test Method • +/- 20% over entire Specification Range • For Impurity Assays • From Reporting Level to 120% of Impurity Specification for Impurity Assays • From Reporting Level to 120% of Assay Specification for Impurity/Assay Methods

17. Accuracy • Closeness of the test results obtained by the method to the true value.

18. Accuracy • 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 determinations) • Should be reported as: • Percent recovery of known amount added or • The difference between the mean assay result and the accepted value

19. Accuracy Data Set (1 of 3)

20. Precision • The closeness of agreement (degree of scatter) between a series of measurements obtained from multiple samplings of the same homogeneous sample. • Should be investigated using homogeneous, authentic samples.

21. Precision… Considered at 3 Levels • Repeatability • Intermediate Precision • Reproducibility

22. Repeatability • Express the precision under the same operating conditions over a short interval of time. • Also referred to as Intra-assay precision • Should be assessed using minimum of 9 determinations • (3 concentrations/ 3 replicates) or

23. Intermediate Precision • Express within-laboratory variations. • Expressed in terms of standard deviation, relative standard deviation (coefficient of variation) and confidence interval. • Depends on the circumstances under which the procedure is intended to be used. • Studies should include varying days, analysts, equipment, etc.

24. Repeatability & Intermediate Precision Mean = 100.5 RSD = 0.24% Mean = 99.5 RSD = 0.36%

25. Reproducibility • Definition: Ability to reproduce data within the predefined precision • Determination: SD, RSD • Repeatability test at two different labs.

26. Detection Limit (LOD)/ Quantitation Limit (LOQ) • LOD • Lowest amount of analyte in a sample that can be detected but not necessarily quantitated. • Estimated by Signal to Noise Ratio of 3:1. • LOQ • Lowest amount of analyte in a sample that can be quantified with suitable accuracy and precision. • Estimated by Signal to Noise Ratio of 10:1.

27. LOD and LOQ Estimated by • Based in Visual Evaluations - Used for non-instrumental methods • Based on Signal-to Noise-Ratio - 3:1 for Detection Limit - 10:1 for Quantitation Limit • Based on Standard Deviation of the Response and the Slope

28. LOD and LOQ Estimated by • S = slope of calibration curve • s = standard deviation of blank readings or standard deviation of regression line Validated by assaying samples at DL or QL

29. Robustness • Definition: Capacity to remain unaffected by small but deliberate variations in method parameters • Determination: Comparison results under differing conditions with precision under normal conditions • Examples of typical variations in LC • Influence of variations of pH in a mobile phase • Influence of variations in mobile phase composition • Different columns (different lots and/or suppliers) • Temperature • Flow rate

30. Degree of reproducibility of test results under a variety of conditions • Different Laboratories • Different Analysts • Different Instruments • Different Reagents • Different Days • Etc. • Expressed as %RSD

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