Adaptation of solid phase extraction to an automated column switching method for online sample cleanup as the basis of a facile and sensitive high-performance liquid chromatographic assay for paclitaxel in human plasma

An improved method for assaying paclitaxel in human plasma by high-performa nce liquid chromatography (HPLC) with UV detection at 227 nm has been devel oped by adapting previously reported sample preparation methods and chromat ographic conditions to facilitate semi-automated sample cleanup using a col umn switching technique. Manual sample manipulations were limited to isolat ing the drug and internal standard from plasma (1.0 mi) by liquid-liquid ex traction using tert-butyl methyl ether. The sample extract was initially lo aded onto a short cartridge column containing a cyanopropyl stationary phas e. During the predetermined time interval that the drug and internal standa rd eluted from the cartridge, 1.50-2.20 min, a fully automated 6-position s witching valve was used to direct the effluent onto an octylsilica analytic al column. The same mobile phase, composed of acetonitrile-methanol- ammoni um acetate buffer (pH 5.0;20 mM) (76:19:105, v/v/v) and delivered at flow r ate of 1.0 ml/min, was used for both separations. The overall retention tim es of paclitaxel and the internal standard were 10.9 and 18.1 min, respecti vely. The analytical method was thoroughly validated for quantitating pacli taxel in plasma at concentrations ranging from 6 to 586 nM (5-500 ng/ml). T he lowest concentration of paclitaxel measured with acceptable day-to-day a ccuracy (100.2%) and precision (RSD 11.7%, n = 21, 5 months) was 6 nM (5 ng /ml). The sensitivity and selectivity of the assay proved to be more than a dequate for monitoring steady-state plasma concentrations of the drug when administered to cancer patients as a 96 h continuous intravenous infusion i n combination with other anticancer agents, such as doxorubicin and topotec an. Moreover, the heart-cutting procedure prevented the problematic introdu ction of interfering nonpolar plasma components onto the analytical column, thereby enhancing sample throughput while decreasing the technical demands of the assay. The method was found to be extremely reproducible and robust during extended use for the routine analysis of plasma specimens acquired from several clinical trials. (C) 1999 Elsevier Science B.V. All rights res erved.