HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ASSAY FOR 3'-AMINO-3'-DEOXYTHYMIDINE IN PLASMA, WITH 9-FLUORENYL METHYL CHLOROFORMATE AS THE DERIVATIZATION AGENT

We have developed a sensitive high-performance liquid chromatographic assay for the determination of the zidovudine metabolite 3'-amino-3'-d eoxythimidine (AMT) using fluorescence detection and sensitivity in th e picomolar range. Plasma was diluted with 0.05 M sodium phosphate buf fer pH 7.2 and subsequently prepared for analysis using solid-phase ex traction. AMT was derivatized with 9-fluorenyl methylchloroformate and chromatographed using a reversed-phase system. The mobile phase consi sted of acetonitrile-0.01 M potassium phosphate buffer (pH 7) (32:68, v/v). The fluorescence of the column effluent was monitored at 262 nm (excitation) and 306 nm (emission). Good resolution of AMT from endoge nous plasma components was obtained. Within- and between-day variabili ty was less than 10%. The limit of quantitation was 0.9 mu g/l. The as say was successfully applied to the determination of AMT in human plas ma and in plasma of mice treated with zidovudine.