Thiopurine methyl transferase activity: new extraction conditions for high-performance liquid chromatographic assay

A new liquid-liquid extraction is described for thiopurine methyl transferase (TPMT, EC 2.1.1.67) activity determination: the use of a pH 9.5 NH4Cl buffer solution, before adding the solvent mixture, allows more rapid extraction, avoiding a centrifugation step, and reduces the global cost of analysis. After the extraction step, 6-methylmercaptopurine, synthesised during the enzymatic reaction, is determined by a liquid chromatographic assay. Analytical performance of the assay was tested on spiked erythrocyte lysates. The linear concentration range was 5-250 ng ml(-1) (r greater than or equal to 0.997, slope=1.497, intercept = -0.367). The recoveries were 82.8, 89.9 and 82.2% for 75, 125 and 225 ng ml(-1), respectively. The coefficients of variation were less than or equal to 6.1% for within-day assay (n=6) and less than or equal to 9.5% for between-day assay precision (n=6; 14 days). TPMT activity was determined in a French adult Caucasian population (n=70). The results ranged from 7.8 to 27.8 nmol h(-1) ml(-1) packed red blood cells and the frequency distribution histogram is similar to that previously published. (C) 1999 Elsevier Science BN. All rights reserved.