Quantification of Tenofovir in Human Plasma by Solid-Phase Extraction and High-Performance Liquid Chromatography Coupled With UV Detection

Abstract

Tenofovir is used in the treatment of HIV and hepatitis virus infections. The aim of this work was to develop and validate an high-performance liquid chromatography coupled with ultraviolet detection assay that can be implemented in most laboratories for the purposes of therapeutic drug monitoring and pharmacokinetic studies. Two hundred microliters of plasma sample was used for the assay. Sample processing was carried out with solid-phase extraction. Tegafur was used as the internal standard. The chromatographic separation was achieved on a C18 reverse-phase analytic column with a mobile phase consisting of sodium phosphate buffer (pH: 6.12; 20.0 mM)-acetonitrile-tetrabutylammonium hydroxide solution (pH: 13.64; 1.14 mM) (90.0:10.0:0.3, v/v/v). The detection was at 262 nm, and the column oven was set at 35°C. The linear range of the calibration curve was 20-2000 ng/mL (r > 0.999, n = 6). The absolute extraction recoveries were 97.4% ± 2.5% and 81.6% ± 0.8% for tenofovir and tegafur, respectively. The relative standard deviations were 2.3%-3.3% for the intraday and 2.8%-5.3% for the interday analyses. The accuracy was within 100% ± 7%. The successful application of this method in a pharmacokinetic study in Chinese HIV-infected patients confirmed its robustness and reliability. A validated and reproducible method has been established to quantify the concentration of tenofovir in human plasma by high-performance liquid chromatography coupled with ultraviolet detection.