Comparison of High-Performance Liquid Chromatographic and Microbiological Methods for Determination of Voriconazole Levels in Plasma

Abstract

A new selective high-performance liquid chromatography (HPLC) method with UV detection for the determination of the investigational triazole voriconazole in human plasma by using acetonitrile precipitation followed by reverse-phase HPLC on a C 18 column was compared with a simple agar well diffusion bioassay method with Candida kefyr ATCC 46764 as the assay organism. Pooled plasma was used to prepare standard and control samples for both methods. The results of analyses with spiked serum samples (run as unknowns) were concordant by the bioassay and HPLC methods, with expected values being obtained. HPLC demonstrated an improved precision (3.47 versus 12.12%) and accuracy (0.81 versus 1.28%) compared to those of the bioassay method. The range of linearity obtained by both methods (from 0.2 to 10 g/ml for HPLC and from 0.25 to 20 g/ml for the bioassay) includes the range of concentrations of voriconazole (from 1.2 to 4.7 g/ml) which are considered clinically relevant. Although either methodology could be used for the monitoring of patient therapy, the smaller variability observed with HPLC compared to that observed with the bioassay favors the use of HPLC for pharmacokinetic studies.

Plasma Voriconazole Estimation by HPLC

“…Quantification of azoles by various analytical techniques such as high performance liquid chromatography (HPLC) [5,9,14], liquid chromatography-tandem mass spectrometry methods (LC-MS) [15] and microbiological methods [16] have been reported. HPLC methods are specific and sensitive for plasma voriconazole concentrations however, special columns and sample preparation with liquid phase extraction and/or tedious precipitation steps are involved [5,17].…”

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

“…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].…”

Therapeutic Drug Monitoring of Voriconazole: Comparison of Bioassay with High-Performance Liquid Chromatography

“…In this study, a specific clinical isolate of C. kefyr (voriconazole-hyper susceptible strain, MIC ≤ 0.015 µg/mL) was used as the test organism. Previous experiments involving bioanalytical methods have used some types of reference strains provided by American type culture collection (24,34) or applied a mutant of C. albicans, constructed by targeted deletions of genes in their studies (36). According to the present study, use of in-house clinical isolates of C. kefyr susceptible to voriconazole, which provided well-defined and symmetric zones of growth inhibition, is suitable as the test organism.…”