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> or =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 < or =6.1% for within-day assay (n=6) and < or =9.5% for between-day assay precision (n=6; 14 days). TPMT activity was determined in a French adult Caucasian population (7 =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.
Ganiere-Monteil
C
C
Laboratoire de Pharmacologie Clinique, Institut de Biologie, Centre Hospitalier Universitaire, Nantes, France.
Pineau
A
A
Kergueris
M F
MF
Azoulay
C
C
Bourin
M
M
D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D008780:Methyltransferases / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
D008780:Methyltransferases; C022745:thiopurine methyltransferase