B-282 Validation of a Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Quantitation of Voriconazole, Posaconazole, Itraconazole, and Hydroxyitraconazole in Human Plasma

Abstract Background Azole-based antifungals are the first-line therapy for many common mycoses and are increasingly used prophylactically to protect immunocompromised patients. Due to variability in their metabolism and bioavailability, therapeutic drug monitoring is crucial to avoid toxicity while ensuring maximum efficacy. Methods Sample preparation involved protein precipitation with a methanol and internal standard mixture, followed by centrifugation. Isotope-labeled analogues for each analyte served as internal standards. Chromatographic separation was performed using an ACQUITY UPLC BEH C18 Column (30 mm x 2.1 mm, 1.7 µm) with a BEH C18 VanGuard Pre-column (2.1 mm x 5 mm, 1.7 µm) on an ACQUITY UPLC I-Class System. Gradient elution was achieved with Mobile phase A and B consisting of 2 mM ammonium acetate in 0.1% formic acid in H2O and methanol, respectively, at a flow rate of 0.8 mL/min. Azoles were detected using a triple-stage quadrupole mass spectrometer in selected reaction monitoring mode with positive heated electrospray ionization, within a runtime of 3.00 minutes. Results Linearity and calibration ranges for all azoles were verified from 0.05 to 10 µg/mL, with a limit of quantitation at 0.05 µg/mL. Within-run and between-run precisions ranged from 0.6% to 6.7% for all azoles. Accuracy was assessed by comparing results with a reference lab, showing high correlation with an average slope of 1.06, intercept of 0.06, and R value of 0.99. Comparison between two instrument systems also demonstrated similar results, with an R value of 0.99. Dilution was verified by reduced volume injection at a 1:2 dilution, extending reportable ranges up to 20 µg/mL. Carryover studies, conducted by interspersing high concentration and negative samples, passed the EP Evaluator protocol. Matrix-induced ion suppression/enhancement studies revealed no interference from the EDTA plasma matrix, except for hydroxyitraconazole. Consequently, hydroxyitraconazole results are not reported when itraconazole is undetectable, as hydroxyitraconazole was enhanced by over 30% of the spiked concentration due to the sample matrix. Whole blood stability was assessed at 8 hours at room temperature before centrifugation, with results acceptable based on a 20% total allowable error. Conclusion A simple yet reliable liquid chromatography-tandem mass spectrometry method was validated that enables quantification of azoles in EDTA plasma across therapeutic ranges. These new assays have significantly improved result turnaround time and have been successfully implemented in our therapeutic drug monitoring service, making them suitable for routine monitoring of antifungal therapy, especially in severely ill patients.