Quantification of 3'-amino-3'-deoxythymidine, a toxic catabolite of 3'-azido-3'-deoxythymidine (zidovudine) in human plasma by high-performance liquid chromatography using precolumn derivatization with fluorescamine and fluorescence detection.

A specific and sensitive high-performance liquid chromatographic (HPLC) assay with precolumn derivatization and fluorescence detection was developed for the determination of 3'-amino-3'-deoxythymidine (AMT), a recently characterized toxic catabolite of 3'-azido-3'-deoxythymidine (zidovudine, AZT). After extraction with tert.-butyl methyl ether-1-butanol (6:4, v/v) and back-extraction into basified aqueous phase, the solution was neutralized with phosphate buffer and the compound derivatized with fluorescamine. Fluorescamine-labeled AMT was chromatographed on a reversed-phase C18 column using a mixture of phosphate buffer-methanol-acetonitrile (47:48:5, v/v/v) as mobile phase and fluorescence detection (excitation wavelength 265 nm, emission wavelength 475 nm). The limit of quantification was 3 ng/ml using a 200-microliters plasma sample. The recovery of the extraction procedure averaged at 94.4% in the range of 3-200 ng/ml. The HPLC analysis time required per sample was 16 min. Replicate analyses of quality control samples (5-80 ng/ml) gave satisfactory intra- and inter-assay precision (coefficient of variation varied from 1.9 to 6.7%) and accuracy.