Determination of thiopurine methyltransferase activity in human erythrocytes by high-performance liquid chromatography: Comparison with the radiochemical method

The current article describes a new assay to measure thiopurine methyltrans ferase (TPMT) activity from red blood cells. This method is based on the me asurement of the reaction product 6-methylmercaptopurine (6-MMP) by highper formance liquid chromatography (HPLC). 6-MMP is extracted by ethyl acetate With recoveries of 85%, 80%, 80%, and 92% for 50, 250, 500, and 1,000 ng/10 0 muL packed red blood cells, respectively. 6-MMP was identified and measur ed by a Zorbax CN column installed in an HPLC system. The chromatograms wer e resolved using a mobile phase consisting of 40 mmol/L sodium phosphate bu ffer (pH 3) and methanol in a gradient from 1% to 20% of methanol. Under th ese conditions 6-MMP is Well resolved from substrates (6-mereaptopurine and S-adenosyl-L-methionine) and endogenous peaks. When the TPMT activity from 20 patients was measured by the HPLC-linked assay and the classic radioche mical method, a linear correlation was obtained between both procedures (y = 0.99x + 0.33; x-axis, radiochemical assay; y-axis, HPI-C-linked assay; r = 0.98). In conclusion, the current report describes a new, reliable, safe, and nonradioactive method to measure TPMT activity that is shorter and sim pler than the previously described ones.