A multiparametric liquid chromatography-tandem mass spectrometry method has been developed for the simultaneous quantification of 11 antifungal drugs and their metabolites in human plasma. This method addresses the critical need for therapeutic drug monitoring in the treatment of invasive fungal infections, which are increasingly prevalent among immunocompromised patients and those in intensive care units. The method quantifies flucytosin, fluconazole, itraconazole, hydroxy-itraconazole, posaconazole, isavuconazole, voriconazole, voriconazole-N-oxide, anidulafungin, caspofungin, and micafungin. Key challenges in method development included optimising mass spectrometer settings, chromatographic conditions, and sample preparation techniques to ensure accurate, sensitive, and specific detection. Validation of this method was conducted in accordance with the guidelines set by the USA Food and drug administration and the European Medicines Agency covering linearity, precision, accuracy, selectivity, matrix effect, and stability. The method exhibited robust performance with intra- and inter-assay precision under 10 % and average accuracy for intra- and inter-assay comparison of −2.35 % and 0.80 %, respectively. Limits of detection (0.002 to 0.110 mg/L) and a quantification range between 0.005 and 200 mg/L make this method suitable for clinical TDM applications. The ability to simultaneously analyse eleven antifungals and their metabolites within a single 5-minute run enhances its utility in clinical settings, particularly for critically ill patients who may experience significant pharmacokinetic variations. The method requires only 100 µL of plasma, demonstrating good analytical performances rendering it a valuable tool for optimising antifungal therapy and improving patient outcomes in ICU management.
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