Reversed-phase high-performance liquid chromatographic approach to determine total lymphocyte concentrations of 6-thioguanine, methylmercaptopurine and methylthioguanine in humans.

A reversed-phase high-performance liquid chromatographic (HPLC) procedure was developed to quantify intracellular lymphocyte 6-thioguanine, methylmercaptopurine and methylthioguanine. The free base of each metabolite was obtained by acid hydrolysis, which allowed for a total determination of thiopurine metabolites. 6-Thioguanine was analyzed on an octadecylsilane column using acetonitrile-10 mM sodium phosphate (11:89), pH 7, containing 0.06% tetrabutylammonium chloride. 6-Thioguanine was oxidized with potassium permanganate, and fluorescence was measured at 330 nm excitation and 410 nm emission. Methylmercaptopurine and methylthioguanine were separated on a cyanopropylsilane column using methanol-40 mM sodium phosphate (22:78), pH 2.7, and detected by ultraviolet absorbance at 314 and 290 nm, respectively.