Quantification of zidovudine and its monophosphate in cell extracts by on-line solid-phase extraction coupled to liquid chromatography

A simple and rapid analytical method for the simultaneous quantification of zidovudine (AZT) and its monophosphate (AZTMP) in cell extracts has been developed using high-performance liquid chromatography (HPLC) with on-line solid-phase extraction and 2-aminoethyl-3 '-azido-2 ',3 '-dideoxythymidin-5 '-yl phosphodiester sodium salt as internal standard (IS). The cell extract samples were directly injected on a short reversed-phase precolumn using an aqueous buffer containing an ion-pairing reagent as a mobile phase. Under these conditions, the analytes were retained on the C precolunm whereas the proteins were discarded. The analytes were then transferred onto the analytical column by increasing the strength of the eluent. The calibration curve was linear over a concentration range of 0.5-100 mu g/ml. Inter- and intra-day accuracy and precision results satisfied the accepted criteria for bioanalytical validation. This method was used to study the decomposition pathway of a model pronucleotide in an in vitro approach. (C) 2007 Elsevier B.V. All rights reserved.