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Factorial design-assisted reversed phase-high performance liquid chromatography method for simultaneous determination of fluconazole, itraconazole and terbinafine.

ABSTRACT: A 2³ full factorial design model was used for the development of a new high performance liquid chromatography method with UV detection to estimate three antifungal drugs simultaneously. Fluconazole (FLU), itraconazole (ITR) and terbinafine (TRH) are co-administered for severe fungal infections. They have been determined using MOS-1 Hypersil C₁₈ column and an isocratic eluent; methanol 95% and phosphate buffer 5% with 0.001% triethylamine. The pH was adjusted to 7, and the flow rate was 0.7 ml min^-1. The three drugs were separated within less than 7 min at 210 nm. The developed method gave a linear response over 5-80 µg ml^-1, 5-50 µg ml^-1 and 1-50 µg ml^-1 for FLU, ITR and TRH, respectively. It showed detection limits of 0.88, 0.29 and 0.20 µg ml^-1 and quantification limits of 2.66, 0.88 and 0.60 µg ml^-1 for the three drugs, respectively. The design of the experiment facilitated the optimization of different variables affecting the separation of the three drugs. The sensitivity of the designed method permitted the simultaneous estimation of ITR and TRH in spiked human plasma successfully.

SUBMITTER: Roshdy A

PROVIDER: S-EPMC8103232 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 ?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.

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Factorial design-assisted reversed phase-high performance liquid chromatography method for simultaneous determination of fluconazole, itraconazole and terbinafine.

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