Assay of fluconazole by high-performance liquid chromatography with a mixed-phase column

Abstract

A mixed-phase liquid chromatographic column was used to assay fluconazole in plasma, serum, and cerebrospinal fluid. The assay was linear from 0.2 to 20 tLg/ml, with an average coefficient of variation of less than 5%. The partitioning of the drug between serum and cerebrospinal fluid was determined for 34 patients.The method was demonstrated to be suitable for both pharmacokinetic studies and monitoring of patients receiving treatment with this antifungal agent.Fluconazole is part of a growing family of triazole antifungal drugs which exhibit a broad spectrum of activity. It has desirable pharmacologic properties, including a relatively long half-life, the ability to be administered either orally or parenterally, and good penetration into cerebrospinal fluid (CSF) (1,3,5). With respect to side effects, it is superior to imidazole antifungal drugs introduced earlier (8). It has already been approved by the U.S. Food and Drug Administration for the treatment of certain systemic fungal infections and is currently in clinical use in the United States and Europe as well as in clinical trials for other applications. A continuing increase in systemic fungal infections concomitant with growing immunosuppressed population established the relevance of fluconazole in the drug treatment arena. ' Several assays are currently in use for determining fluconazole concentrations in biologic specimens. Both gas-liquid chromatographic (2, 4) and high-performance liquid chromatographic (HPLC) (7) assays have been reported. Bioassay techniques are also available (6).The assay described in this report is an HPLC method that is done with a m-ixed-phase column and UV spectrophotometric detection. It has been developed for determining drug concentrations in serum, plasma, and cerebrospinal fluid (CSF).MATERIALS AND METHODS Instrumentation. A model 5020 (Varian Instruments, Walnut Creek, Calif.) liquid chromatograph with a variablewavelength UV detector (Varian UV-50 or equivalent) was used. A Varian mixed-phase PTHAA-5 liquid chromatographic column (15 cm long; 4-mm inner diameter) was used.Reagents. Fluconazole and internal standard UK-54,373 [2 -(2,4 -difluorophenyl) -1,3 -bis(lH -1,2,4 -triazol -1 -yl) -2-propanol; Fig. 1 Chromatographic conditions. The mobile phase was prepared by mixing acetonitrile with filtered 0.051 M monobasic phosphate buffer adjusted to pH of 3.0 at 15:85 (vol/vol). The flow rate was 0.9 ml/min. The UV detector was set at a wavelength of 210 nm. The HPLC apparatus was operated at room temperature (25 to 28°C).Standards and controls. Stock standards at concentrations of 1 and 100 ,ug/ml in methanol were prepared by placing appropriate amounts of fluconazole in polypropylene tubes with screw-cap tops and then adding 0.10 ml of methanol. Working serum standards with fluconazole concentrations of 0.0, 0.2, 0.5, 1.0, 2.0, 2.5, 5.0, 10.0, 15.0, 20.0, 40.0, 50.0, and 100 ,ug/ml were prepared by placing various amounts of the two stock standard solutions in 15-ml glass tubes, taking the methanol to near dryness, an...

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Interactions between Triazoles and Amphotericin B against Cryptococcus neoformans

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