Multiplex Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Quantification in Human Plasma of Fluconazole,Itraconazole, Hydroxyitraconazole,Posaconazole, Voriconazole,Voriconazole-N-Oxide,Anidulafungin, and Caspofungin

Therapeutic drug monitoring (TDM) may contribute to optimizing the efficacy and safety of antifungal therapy because of the large variability in drug pharmacokinetics. Rapid, sensitive, and selective laboratory methods are needed for efficient TDM. Quantification of several antifungals in a single analytical run may best fulfill these requirements. We therefore developed a multiplex ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method requiring 100 μl of plasma for simultaneous quantification within 7 min of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, caspofungin, and anidulafungin. Protein precipitation with acetonitrile was used in a single extraction procedure for eight analytes. After reverse-phase chromatographic separation, antifungals were quantified by electrospray ionization-triple-quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. Deuterated isotopic compounds of azole antifungals were used as internal standards. The method was validated based on FDA recommendations, including assessment of extraction yields, matrix effect variability (<9.2%), and analytical recovery (80.1 to 107%). The method is sensitive (lower limits of azole quantification, 0.01 to 0.1 μg/ml; those of echinocandin quantification, 0.06 to 0.1 μg/ml), accurate (intra- and interassay biases of −9.9 to +5% and −4.0 to +8.8%, respectively), and precise (intra- and interassay coefficients of variation of 1.2 to 11.1% and 1.2 to 8.9%, respectively) over clinical concentration ranges (upper limits of quantification, 5 to 50 μg/ml). Thus, we developed a simple, rapid, and robust multiplex UPLC-MS/MS assay for simultaneous quantification of plasma concentrations of six antifungals and two metabolites. This offers, by optimized and cost-effective lab resource utilization, an efficient tool for daily routine TDM aimed at maximizing the real-time efficacy and safety of different recommended single-drug antifungal regimens and combination salvage therapies, as well as a tool for clinical research.Differences in oral drug bioavailability, altered volumes of distribution, drug-drug interactions, impaired hepatic and/or renal drug clearance, and the genetic background of hepatic metabolism contribute to the large intra- and interindividual variability of pharmacokinetics of antifungal agents in patients with life-threatening fungal infections. A given drug dosing regimen can yield very different plasma concentrations: subtherapeutic drug exposure may result in a lack of response to therapy and emergence of fungal resistance, while overexposure may increase the risk of toxicity (21, 48, 57, 59). There is therefore increasing clinical evidence of the usefulness of therapeutic drug monitoring (TDM), which allows real-time adjustment of antifungal dosing aimed at optimizing the individual drug''s pharmacokinetic/pharmacodynamic profile. This may be especially helpful for severely ill patients with multiple organ dysfunctions and comedications, when drug absorption is uncertain, in infections not responding to therapy, or when severe toxicity is suspected (1, 11, 24, 35, 46, 50, 55). Tentative recommendations for timing of measurement and therapeutic concentration intervals in TDM for azole antifungals have been proposed (1). For echinocandins, experimental in vitro and in vivo studies have determined the pharmacokinetic/pharmacodynamic parameters associated with therapeutic success. Clinical studies are ongoing for the assessment of the relationships between exposure to echinocandins and efficacy or safety. Moreover, combinations of these compounds with azoles or polyenes are used for salvage therapy of refractory infections and are being investigated for first-line therapy (13, 18, 31).Analytical methods using liquid chromatography coupled with triple-quadrupole mass spectrometry (LC-MS/MS) have been reported for the quantification of single antifungals in biological fluids, including fluconazole (FLC) (14, 34, 49), itraconazole (ITZ) (39, 62) and its active metabolite (hydroxyitraconazole [OH-ITZ]) (4, 6, 27, 53), voriconazole (VRC) (2, 15, 26, 54, 63), posaconazole (PSC) (10, 40a, 44, 52), caspofungin (CASPO) (7, 16, 40), and anidulafungin (ANI) (13). LC-MS/MS assays were recently reported for the simultaneous quantification of posaconazole, voriconazole, isavuconazole, caspofungin, anidulafungin, and micafungin in different peripheral blood compartments (19) and of itraconazole, voriconazole, and posaconazole in blood (3).Ultra-performance liquid chromatography coupled with tandem mass spectrometry (ULPC-MS/MS) allows the selective and sensitive quantification of structurally unrelated drugs in a single analytical run, resulting in substantial reductions in analytical time, turnaround time, and costs. An analytical method using a simple extraction procedure followed by simultaneous quantification of multiple antifungal agents would be most efficient for rapidly providing TDM results, including those for patients receiving combination antifungal therapy (13, 18, 31), and for maximizing lab resource utilization.We aimed at developing and validating a simple, sensitive, and robust multiplex UPLC-MS/MS method for the simultaneous analysis in human plasma of six recommended and frequently used antifungal drugs and two metabolites: fluconazole, itraconazole and its active metabolite (hydroxyitraconazole), posaconazole, voriconazole, voriconazole-N-oxide (VRC-NO; the principal metabolite of voriconazole, produced by cytochrome P450 2C19 [CYP2C19] [23, 41]), caspofungin, and anidulafungin.