title: 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.
abstract: 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.
authors.affiliation: Division of Clinical Pharmacology, Department of Medicine, BH18, Lab 218-226, Centre Hospitalier Universitaire Vaudois and University of Lausanne, CH-1011 Lausanne, Switzerland. LaurentArthur.Decosterd@chuv.ch
authors.forename: Laurent Arthur
authors.initials: LA
authors.lastname: Decosterd
authors.forename: Bertrand
authors.initials: B
authors.lastname: Rochat
authors.forename: Benoît
authors.initials: B
authors.lastname: Pesse
authors.forename: Thomas
authors.initials: T
authors.lastname: Mercier
authors.forename: Frédéric
authors.initials: F
authors.lastname: Tissot
authors.forename: Nicolas
authors.initials: N
authors.lastname: Widmer
authors.forename: Jacques
authors.initials: J
authors.lastname: Bille
authors.forename: Thierry
authors.initials: T
authors.lastname: Calandra
authors.forename: Boris
authors.initials: B
authors.lastname: Zanolari
authors.forename: Oscar
authors.initials: O
authors.lastname: Marchetti
mesh_terms: D000077612:Anidulafungin; D000935:Antifungal Agents / Q000097:blood*; D001774:Blood Chemical Analysis / Q000379:methods*; D000077336:Caspofungin; D002853:Chromatography, Liquid; D054714:Echinocandins / Q000097:blood; D006801:Humans; D017964:Itraconazole / Q000031:analogs & derivatives / Q000097:blood; D055666:Lipopeptides; D011743:Pyrimidines / Q000097:blood; D053719:Tandem Mass Spectrometry; D014230:Triazoles / Q000097:blood; D065819:Voriconazole
chemical_list: D000935:Antifungal Agents; D054714:Echinocandins; D055666:Lipopeptides; D011743:Pyrimidines; D014230:Triazoles; C084794:hydroxyitraconazole; D017964:Itraconazole; C101425:posaconazole; D000077612:Anidulafungin; D000077336:Caspofungin; D065819:Voriconazole