A sensitive high-performance liquid chromatographic method for the determination of 6-mercaptopurine in plasma using precolumn derivatization and fluorescence detection.

A sensitive high-performance liquid chromatographic (HPLC) method for measuring plasma concentrations of 6-mercaptopurine (6-MP) is described. After protein precipitation with 5-sulfosalicylic acid, samples are subjected to precolumn derivatization using the thiol-reactive fluorophore monobromobimane (mBrB). The drug-mBrB adduct is then resolved by isocratic elution from a C18 reversed-phase support and quantified by fluorescence detection. Recovery of 6-MP after protein precipitation was consistently > 85% and the drug-mBrB adduct was found to be stable for at least 2 weeks at room temperature. With plasma samples containing 30 nM 6-MP, the assay displayed within-run (n = 6) and between-day (n = 6) coefficients of variation of 2.2 and 10.6%, respectively. The limit of detection for 6-MP in plasma was 3 nM (500 pg/ml) and the standard curve was linear up to 3 microM. Using this method, we have observed that 6-MP is stable in heparinized whole blood for at least 24 h provided samples are maintained on ice. Since this method requires few manipulations during sample preparation and is readily adaptable to automated techniques, it may prove useful in the routine clinical laboratory setting.