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 chromatographytandem mass spectrometry (UPLC-MS/MS) method requiring 100 l 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 g/ml; those of echinocandin quantification, 0.06 to 0.1 g/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 g/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 comedi...

Therapeutic drug monitoring of voriconazole: validation of a novel ARK™ immunoassay and comparison with ultra-high performance liquid chromatography

“…These techniques are accurate, precise and allow the simultaneous analysis of multiple antifungal drugs. However, these methods also include some disadvantages, such as the limited availability of chromatographic instruments in the core clinical laboratory, the need for skilled laboratory technicians, the use of (sometimes) time consuming sample preparation steps and the need [20][21][22][23][24][25][26][27][28][29] LOQ, limit of quantification; TAT, turn-around-time ( = plasma pretreatment+analysis time). Within-run (n = 21), between-run imprecision and accuracy (n = 40, 25 days) were evaluated using quality control material and a plasma pool and were all found acceptable ( < 7% at all tested concentrations, Table 2).…”

“…In Table 1, an overview is given of the analytical methods that are suitable for measuring VRC in plasma, including bioassays [8][9][10][11], HPLC [12][13][14][15][16][17][18][19] and LC-MSMS [20][21][22][23][24][25][26][27][28][29] methods. Currently, chromatographic methods are predominantly used, as can also be derived from proficiency testing results [30].…”

A New HPLC Validated Method for Therapeutic Monitoring Of Triazoles in Human Plasma: First Results in Leukaemic Patients

“…They also used linezolid as IS, an antibiotic which may be co-administered in clinical practice, while the use of flavone, as we set in our methodology, makes our method more applicable to the analysis of immunocompromized patients. More recently LC-MS/MS methods have been developed to the purpose of antifungal TDM [25][26][27][28][29][30][31][32], but MS facilities are not always available in standard hospital laboratories. Our developed method, based on HPLC-UV system, reveals a good performance to this aim.…”

“…Several reported the use of high performance liquid chromatography coupled with ultraviolet determination (HPLC-UV) [4,[13][14][15][16][17][18][19][20][21][22][23][24]. More recently, liquid chromatographic methods based on mass spectrometry (LC-MS/MS) detection have been developed to this purpose [25][26][27][28][29][30][31][32], although MS facilities are not always available in standard hospital laboratories, because they are expensive compared with a simple HPLC system. reached from patients and approval for the research was obtained from local ethics committee.…”

An on-line solid phase extraction procedure for the routine quantification of caspofungin by liquid chromatography-tandem mass spectrometry

“…This compound may be suited, but was obtained from the manufacturer of caspofungin and is not commercially available; thus the method cannot easily be reproduced. Decosterd et al (9) rely on stable isotope labeled voriconazole for internal standardization. This is questionable as well, since molecular weight and molecular structure of this compound is completely different from that of caspofungin.…”