Development and validation of a multi-matrix LC-MS/MS method for quantification of 20 drugs in human postmortem and porcine surrogate matrices.

Alternative matrices are increasingly used in postmortem toxicology when conventional specimens such as femoral blood are unavailable. However, quantitative multi-matrix methods covering a wide range of tissues remain limited. This study describes the development and fit-for-purpose validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantification of 20 forensically relevant drugs in human and porcine postmortem matrices. Analytes and internal standards were extracted by methanol-based protein precipitation, followed by reversed-phase chromatography gradient separation and quantitation using a linear ion trap consisting of a quadrupole mass filter. A solvent calibration curve was used to analyze all matrices in a single analytical run. Validation was performed according to a modified fit-for-purpose approach focusing on accuracy, precision, selectivity, carry-over, matrix effect, dilution feasibility, and stability. Acceptance criteria were fulfilled for 89.3% of analyte-matrix combinations under standard criteria and 96.7% after predefined fit-for-purpose adjustments. Porcine matrices showed analytical performance comparable to human tissues, supporting their use as surrogate matrices for method development and validation. The developed LC-MS/MS method enables quantification of drugs across a broad range of alternative postmortem matrices using a single extraction and calibration approach. The method is intended for fast, reliable, and resource-efficient multi-matrix studies rather than absolute quantification in individual matrices and provides a practical tool for the investigation of postmortem redistribution processes.