ee VALIDATING CHROMATOGRAPHIC METHODS A Practical Guide DAVID M. BLIESNER \WILEY- Ce icc A JOHN WILEY & SONS, INC., PUBLICATION188 APPENDIX! Determine which system suitability parameters are important to the overall function of the method. © Establish limits for critical parameters. © For column variability ensure all columns used in validation are com- mercially available. © Ensure that three columns from at least two different lots of packing material are obtained and used. © Ensure that a brand new column and an old column (> 500 injections) are obtained and used. © Ensure the retention times are similar on each of the three columns, ®._ System Suitability Determination Description of System Suitability Testing ‘System suitability is the evaluation of the components of an analytical system to show that the performance of a system meets the standards required by a method. A system suitability evaluation usually contains its own set of parameters; for chromatographic assays, these may include tailing factors, resolution, and precision of standard peak areas, and comparison to a confir- mation standard, capacity factors, retention times, theoretical plates, and cal- ibration curve linearity. Where applicable, system suitability parameters are calculated, recorded, and trended throughout the course of the validation. Final values are then determined from this history. Experimental Determination of System Suitability for Assay and Related A and B Where applicable, calculate system suitability (capacity factor, tailing factor, resolution, theoretical plates, and reproducibility) for each chro- matographic run during methods validation. © Identify upper and lower limits for each parameter by analyzing values obtained throughout the validation, © Establish minimum criteria from this range Acceptance Criteria: The following minimum criteria are suggested: K'= 2.0, where k’ is capacity factor i © PS 2, where Tis tailing factor © R> 1.5, where R is resolutionTEMPLATE FOR AN EXAMPLE METHODS VALIDATION PROTOCOL 189 © N-& 1000 plates, where N is theoretical plates per column | % RSD = 2.0%, where % RSD is the percent relative standard deviation 10. Forced Degradation Description of Forced Degradation Forced degradation studies are undertaken to degrade the sample (e.g., drug product or API) deliberately. These studies are used to evaluate an analytical method’s ability to measure an active ingredient and its degradation products without interference, Samples or drug product (spiked placebos) and drug substance are exposed to heat, light, acid, base, and oxidizing agent to produce 10%-30% degradation of the active. The degraded samples are then analyzed using the method to determine if there are interferences with the active or related compound peaks. Experimental Execution of Forced Degradation for Assay and Related A and B ‘The following degradation conditions are recommended as a starting point: Degradation Reaction ‘Typical Conditions Acid hydrolysis, 1 Sample in aqueous acid or acidified solvent at ~0.5 N up to 24 hours (or) 2. Heat/reflux or UV radiation in ~0.5 N HC up to 24 hours. Base hydrolysis, 1. Sample in aqueous base or basic solvent at ~0.5 N up to 24 hours (or) 2. Heav/reflux or UV radiation in ~0.5 N NaOH up to 24 hours. Oxidation 1. Treat with ~3% H;02 up to 24 hours (or) 2. UV irradiation in ~3% HO, up to 24 hours. ht decomposition (photolysis) Expose to high-intensity UV light in suitable increments, up to 24 hours, ‘Thermal decomposition (pyrolysis) Expose to ~100° C heat in suitable increments, up to 24 hours © Obtain or prepare solutions of ~0.5 N HCL, ~0.5 N NaOH, ~3% H;O2, and purified water. © Prepare blank samples for each condition, including light and heat.