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

Abstract:

:The current article describes a new assay to measure thiopurine methyltransferase (TPMT) activity from red blood cells. This method is based on the measurement of the reaction product 6-methylmercaptopurine (6-MMP) by high-performance 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/100 microL packed red blood cells, respectively. 6-MMP was identified and measured by a Zorbax CN column installed in an HPLC system. The chromatograms were resolved using a mobile phase consisting of 40 mmol/L sodium phosphate buffer (pH 3) and methanol in a gradient from 1% to 20% of methanol. Under these conditions 6-MMP is well resolved from substrates (6-mercaptopurine 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 radiochemical method, a linear correlation was obtained between both procedures ( y = 0.99x + 0.33; x-axis, radiochemical assay; y-axis, HPLC-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 simpler than the previously described ones.

journal_name

Ther Drug Monit

authors

Menor C,Fueyo JA,Escribano O,Cara C,Fernández-Moreno MD,Román ID,Guijarro LG

doi

10.1097/00007691-200110000-00007

subject

Has Abstract

pub_date

2001-10-01 00:00:00

pages

536-41

issue

5

eissn

0163-4356

issn

1536-3694

journal_volume

23

pub_type

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