A Validated Multi-Analyte Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Therapeutic Drug Monitoring and Targeted Toxicological Assessment of 54 Drugs and Metabolites in Human Serum.

Therapeutic drug monitoring and targeted toxicological assessment require quantitative methods capable of covering chemically diverse drugs over clinically relevant concentration ranges. We developed and validated a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of 54 analytes in human serum, including antiepileptic drugs, antidepressants, antipsychotics, benzodiazepines, antifungal agents, theophylline, and selected clinically relevant metabolites. Serum samples were prepared by protein precipitation with perchloric acid. Chromatographic separation was achieved on an ACQUITY UPLC BEH C18 column using water/methanol mobile phases containing 2 mM ammonium acetate. Detection was performed in polarity-switching selected reaction monitoring mode using two transitions per analyte and 28 isotope-labeled or structurally related internal standards. The total run time was 8.5 min. Analyte-specific calibration models and validated concentration ranges covered 0.1-22,500 ng/mL, depending on the analyte. The method demonstrated acceptable selectivity, calibration performance, accuracy, precision, matrix effects, and stability under routine handling and storage conditions for analytes with available data. Within-run and between-run precision did not exceed 14.3% and 13.9%, respectively. Matrix effects ranged from -8.35% to +10.25%. Application to 120 remnant patient serum samples supported the feasibility of multiplex measurement of drugs from several therapeutic classes in a single analytical run. The method is suitable for routine serum-based therapeutic drug monitoring and for targeted toxicological interpretation of a predefined analyte panel.