A high-throughput LC-MS/MS assay for therapeutic drug monitoring of combined immunosuppressive and antimicrobial therapy.

UNLABELLED: This study established a rapid and reliable LC-MS/MS method for the simultaneous determination of six therapeutically relevant drugs: meropenem, vancomycin, voriconazole, linezolid, tacrolimus, and cyclosporine A. A key methodological advance is the first systematic characterization of the specific SRM transition of commercially available 13C₂,d₄-cyclosporine A, and its first application as an internal standard for this multiplex assay. METHODS: The mass spectrometry parameters were optimized using a univariate approach to define the qualitative and quantitative ion transitions. Blood samples were extracted with methanol and quantified via the internal standard method, with calibration curves established for concurrent monitoring of the six target drugs. The developed method underwent comprehensive validation for selectivity, matrix effects, LLOQ, carryover, accuracy, precision, stability, dilution integrity, and calibration curve linearity. Its preliminary clinical applicability was assessed via pilot comparison with routine clinical reference methods: chemiluminescence immunoassay for immunosuppressants and conventional LC-MS/MS for antimicrobials. RESULTS: The analytical method demonstrated excellent selectivity, with no detectable interfering peaks at the expected retention times for all analytes and internal standards. Matrix effects and carryover were minimal. In the accuracy test, both intra- and inter-batch accuracies ranged from 87.04% to 113.04%. The coefficients of variation (CV) for precision were within ±12.41%. Stability was satisfactory. The recoveries for all drugs were between 88.30% and 106.43%. Good linearity was demonstrated across the concentration ranges, with correlation coefficients (r) > 0.99. The method comparison agreement ranged from 86.47% to 111.53%. CONCLUSION: The specific SRM transition of 13C₂,d₄-cyclosporine A was characterized for the first time, and a robust LC-MS/MS method was established for the simultaneous quantification of the six target drugs in human whole blood.