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

• Published in: Journal of chromatography. B, Biomedical sciences and applications Biomedical sciences and applications, 1999-04, Vol.727 (1), p.235-239
• Main Authors: Ganiere-Monteil, C, Pineau, A, Kergueris, M.F, Azoulay, C, Bourin, M
• Format: Article
• Language: English
• Subjects: 6-Methylmercaptopurine; Adult; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, High Pressure Liquid - methods; Enzymes; Enzymes and enzyme inhibitors; Erythrocytes - enzymology; Fundamental and applied biological sciences. Psychology; Humans; Methyltransferases - blood; Reproducibility of Results; Sensitivity and Specificity; Spectrophotometry, Ultraviolet; Thiopurine methyl transferase; Transferases
• ISSN: 0378-4347; 1387-2273
• DOI: 10.1016/S0378-4347(99)00083-3

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 NH 4Cl 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≥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 ≤6.1% for within-day assay ( n=6) and ≤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.