During method development the method parameters are finalized and tested to ensure accuracy and ruggedness. GLP method development follows the method feasibility study and requires an additional three to four days.
Below is a brief summary of the related method development activities:
• Suitable internal standards will either be provided by Sponsor (i.e., labeled internal) or acquired by Enthalpy Analytical, Inc.
• LC/MS/MS parameters are optimized.
• Full sets of calibration standards are prepared in matrix (i.e., rat, dog, monkey plasma, etc.) with an appropriate anticoagulant (to be specified by Sponsor). Calibration standards must have a back-calculated concentration within +/- 20% at the lower limit of quantitation and +/- 15% for all other concentrations.
• Quality control samples are prepared in matrix. Quality control samples must be within +/- 20% of their nominal concentration at the lower limit of quantitation and +/- 15% for all other concentrations.
• Selectivity is verified by extracting blank matrix and demonstrating that there are no chromatographic peaks greater than 20% of the LLOQ at or around the expected retention time of the target analytes or internal standard.
• Calibration standards and quality control samples are extracted and analyzed to ensure the method is ready for validation over the required analytical range.
• An informal development report is issued detailing the results from development activities. This report is issued as soon as possible after completion of development activities. The contents of this report are summarized in the validation report.
