BACKGROUND: Fluconazole, itraconazole, posaconazole and voriconazole are four triazole antifungal drugs administered for the prevention and in the treatment of invasive fungal infections. Therapeutic drug monitoring of these antifungals in human plasma is advised. METHODS: After protein precipitation by adding methanol/ZnSO(4) containing ketoconazole as internal standard (IS) the analytes were isolated from matrix and enriched by solid-phase extraction (SPE) and were separated on an Allure PFP HPLC column, maintained at 60°C. The retention times were 1.12, 1.09, 1.09, 1.15 and 1.94 min for fluconazole, voriconazole, posaconazole, itraconazole and ketoconazole, respectively. Cycle time was 3 min, injection to injection. The analytes were monitored by means of a tandem mass spectrometer. Selected reaction monitoring (SRM) in positive ion mode was used for quantitation. RESULTS: The presented method has limits of detection of 2-10 ng/mL, a lower limit of quantitation of 14 ng/mL and linearities ≥0.999 for the different analytes. The intra- and interday correlation coefficients of variation were <8.1 and <9.8%, respectively. The accuracy ranged from 85 to 106%. CONCLUSIONS: A rapid, sensitive, and robust HPLC-MS/MS method for quantifying fluconazole, itraconazole, posaconazole and voriconazole levels in human plasma was validated and successfully applied to samples from antifungal treated patients.
BACKGROUND:Fluconazole, itraconazole, posaconazole and voriconazole are four triazole antifungal drugs administered for the prevention and in the treatment of invasive fungal infections. Therapeutic drug monitoring of these antifungals in human plasma is advised. METHODS: After protein precipitation by adding methanol/ZnSO(4) containing ketoconazole as internal standard (IS) the analytes were isolated from matrix and enriched by solid-phase extraction (SPE) and were separated on an Allure PFP HPLC column, maintained at 60°C. The retention times were 1.12, 1.09, 1.09, 1.15 and 1.94 min for fluconazole, voriconazole, posaconazole, itraconazole and ketoconazole, respectively. Cycle time was 3 min, injection to injection. The analytes were monitored by means of a tandem mass spectrometer. Selected reaction monitoring (SRM) in positive ion mode was used for quantitation. RESULTS: The presented method has limits of detection of 2-10 ng/mL, a lower limit of quantitation of 14 ng/mL and linearities ≥0.999 for the different analytes. The intra- and interday correlation coefficients of variation were <8.1 and <9.8%, respectively. The accuracy ranged from 85 to 106%. CONCLUSIONS: A rapid, sensitive, and robust HPLC-MS/MS method for quantifying fluconazole, itraconazole, posaconazole and voriconazole levels in human plasma was validated and successfully applied to samples from antifungal treated patients.