A rapid UPLC-MS/MS assay for the simultaneous measurement of fluconazole, voriconazole, posaconazole, itraconazole, and hydroxyitraconazole concentrations in serum.

BACKGROUND: Triazole antifungals are essential to the treatment and prophylaxis of fungal infections. Significant pharmacokinetic variability combined with a clinical need for faster turnaround times has increased demand for in-house therapeutic drug monitoring of these drugs, which is best performed using mass spectrometry-based platforms. However, technical and logistical obstacles to implementing these platforms in hospital laboratories have limited their widespread utilization. Here, we present the development and validation of a fast and simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to measure fluconazole, voriconazole, posaconazole, itraconazole, and hydroxyitraconazole in human serum suitable for incorporation into a hospital clinical laboratory.

METHODS: Serum samples (20 µL) were prepared using protein precipitation in the presence of deuterated internal standards. Chromatographic separation was accomplished using reversed phase UPLC and analysis was performed using positive-mode electrospray ionization and collision-induced dissociation MS.

RESULTS: Total analytical run time was 3 min. All analytes demonstrated linearity (r2>0.998) from 0.1 to 10 µg/mL (1-100 µg/mL for fluconazole), acceptable accuracy and precision (%DEV<15% and %CV<15% at all levels tested), suitable stability under relevant storage conditions, and correlated well with reference laboratory results.

CONCLUSIONS: A simple and rapid UPLC-MS/MS method for monitoring multiple triazole antifungals was developed with a focus on the needs of hospital laboratories. The assay is suitable for clinical utilization and management of patients on these medications.