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 precolumn 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 μg/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.