Simultaneous determination of voriconazole, posaconazole, itraconazole and hydroxy-itraconazole in human plasma using LCMS/MS

Introduction: Invasive fungal infections are an increasing cause of mortality and morbidity in high risk patient populations such as those on immunosuppressive therapy. Triazole antifungals are recommended for the prevention and treatment of such infections. The aim of this study was to develop and validate a simple, sensitive and robust LCMS/MS method for the simultaneous analysis in human plasma of three frequently used antifungal drugs: voriconazole, posaconazole, and itraconazole. Methods: Precipitation reagent, containing deuterated internal standards, is added to 50 mu L of plasma. The vials are vortexed before centrifugation. The organic supernatant is transferred to a polypropylene vial and 1 mu L is injected into the Waters Acquity (R) Ultra Performance Liquid Chromatography system coupled with a Waters Acquity (R) TQ Detector system. Chromatographic separation is achieved on a BEH C-18 column using gradient elution with mobile phases consisting of 2 mM ammonium acetate with 0.1% formic acid in water and methanol. Run time is < 5 min between injections. Results: The evaluation of the LCMS/MS triazole method showed good precision (intra-assay CVs < 6.7%, inter-assay CVs < 8.3%). The lower limit of quantitation for all antifungal triazoles tested was 0.10 mg/L. Passing Bablok comparisons of voriconazole (n = 50) and posaconazole (n = 50) showed good correlation with the current HPLC method (Voriconazole LCMS = 0.94(HPLC) + 0.03, r(2) = 0.99; Posaconazole LCMS = 1.18(HPLC) -0.04, r(2) = 0.95). Passing Bablok comparisons of itraconazole and hydroxy-itraconazole (n = 18) showed good agreement with an external referral laboratory's antifungal LCMS/MS method (Itraconazole LCMS = 1.00(referral lab) + 0.01, r(2) = 0.99; Hydroxy-Itraconazole LCMS = 1.05(referral lab) + 0.04, r(2) = 0.99). External quality assurance samples for posaconazole and voriconazole (n = 12, UK NEQAS Antifungal Pilot Panel) were assayed 'blind' and results were in good agreement with consensus mean values (both r(2) = 0.99). Conclusion: The rapid pre-analytical sample preparation procedure, short chromatographic time, limit of quantitation and linear range make this LCMS/MS method suitable for determination of plasma voriconazole, posaconazole, itraconazole and hydroxy-itraconazole levels in a high throughput laboratory.