Automated reversed-phase chromatographic analysis of etoposide and teniposide in plasma by using on-line surfactant-mediated sample clean-up and column-switching.
An automated high-performance liquid chromatographic method for the plasma assay of two neutral drugs, etoposide and teniposide, involving direct plasma injection is presented. The problematic nature of protein precipitation has been circumvented by adding the anionic surfactant sodium dodecyl sulphate to the plasma at a final concentration of 38 mM. Plasma samples are loaded on to a clean-up column with an aqueous mobile phase with which the analyte(s) is (are) retained, whereas the solubilized plasma proteins are flushed to waste. Next, the retained compounds are eluted from the clean-up column on to the analytical column by using the chromatographic mobile phase with a higher elution capacity. The column-switching technique is used to achieve an automated assay. At least 10 ml of plasma, representing 100 repeated injections of 100 microliters or five repeated injections of 2 ml, can pass through the clean-up column without increasing the back-pressure. The recovery increased considerably from 10-30% to 90-95% on adding surfactant to the plasma samples prior to the analysis. The relative standard deviation of the proposed clean-up procedure is 3.5% (n = 6) for both drugs measured at the 2 micrograms/ml level without using an internal standard. The limit of determination with 100-microliters injections is 0.10-0.15 microgram/ml for ultraviolet detection and is seven times lower with electrochemical detection. Teniposide was determined in patients' plasma and the results agreed well with those obtained by the conventional procedure involving manual liquid-liquid extraction prior to chromatographic analysis.