Development and Validation of a High-Performance Liquid Chromatography Assay for Voriconazole

Abstract

An analytical method for the determination of voriconazole (UK-109,496; Pfizer) in plasma was developed and validated. The method utilizes solid-phase extraction technology and high-performance liquid chromatography. The lower limit of quantitation is 0.2 g/ml, and the range of linearity tested was 0.2 to 10 g/ml.Voriconazole (VRC; 496 [C 16 H 14 N 5 OF 3 ]; Pfizer Pharmaceuticals) is a novel broad-spectrum triazole antifungal that is used in the treatment of a wide range of opportunistic fungal infections, including aspergillosis (4). VRC is marketed in formulations for administration both orally (tablet) and intravenously. Previously described assays for VRC include a bioassay procedure and two different high-performance liquid chromatography (HPLC) methods. The former lacks the required sensitivity, and the latter HPLC methods either lacked the necessary sensitivity (4) or were lengthy and technically difficult (6). This assay includes the use of an internal standard (UK-115,794) and sample preparation by solid-phase extraction (SPE). Validation guidelines published by Shah et al. (5) were used to determine the method's accuracy, precision, reproducibility, and specificity.Pfizer Research and Development, Sandwich, United Kingdom, provided VRC and internal-standard powders. The HPLC system (Beckman Coulter, Fullerton, Calif.) consisted of a 168 diode array detector, a 126 solvent pump, a 508 autosampler, an IBM NT-based computer work station, and 32 Karat software.Stock and working VRC standards (1,000, 100, and 10 g/ ml, respectively) and stock and working internal standards (1,000 and 100 g/ml, respectively) were all made in methanol (MeOH) and stored at Ϫ20°C. Calibration standards (0.2, 0.5, 1, 2, 4, 6, 8, and 10 g/ml) were prepared in pooled plasma from the VRC working standards on the day of the analysis.Plasma controls (0.2, 0.5, 4, and 8 g/ml) were made in batches and frozen for analyses over a period of time from an independent weighing of VRC powder. Five hundred microliters of each blank, standard, or control was pipetted into an appropriately labeled tube. Ten microliters of a 100-g/ml internal standard was added to each tube except the "blankblank" tube. All samples were buffered with 700 l of 0.2 M borate buffer (pH 9.0). Samples were extracted by SPE with C 18 , 100 mg, 1-ml Bond Elut columns (Varian, Inc., Harbor City, Calif.). The columns were conditioned with separate washings in the following order: 1 ml of MeOH, 1 ml of H 2 O, and 1 ml of 0.2 M borate buffer (pH 9.0). The buffered plasma samples were added to each respective column. After the columns completely drained, they were washed with separate and independent washings of the following reagents: 1 ml of 0.2 M borate buffer, followed by 1 ml of MeOH-H 2 O (50:50, vol/vol). Inside the vacuum manifold glass chamber, microcentrifuge tubes were positioned for collection of each eluted sample from its respective SPE column. One milliliter of the eluent, MeOH-glacial acetic acid (99:1, vol/vol), was added to each column. The collected elua...

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