Determination of Thiopurine S‑methyltransferase Activity using Reverse‑Phase High‑Performance Liquid Chromatography Assay with Ultraviolet Detection: Reference Values for the Indian Population

Abstract

Objective: To determine thiopurine S‑methyltransferase (TPMT) activity and the range of TPMT activity values for the Indian population.Methods: An isocratic reversed‑phase high‑performance liquid chromatography method with ultraviolet detection for the determination of TPMT activity in red blood cells was developed and validated. TPMT activity (nmol of 6‑methylmercaptopurine [6‑MMP]/h/ml erythrocytes) was measured based on the conversion of 6‑mercaptopurine to 6‑MMP with S‑adenosyl‑l‑methionine. The stability of erythrocyte lysate at −20°C and the whole‑blood specimens for TPMT activity were determined at 4°C. TPMT enzyme activity was measured in 150 patients who were not on azathioprine (AZA), and population difference in TPMT enzyme activity was evaluated.Results: The assay was linear from 1.0 to 60.14 nmol 6‑MMP concentrations. The chromatogram peaks obtained were baseline separated for 6‑MMP and the internal standard. The bias and precision of low‑, medium‑, and high‑quality controls (QC) were within acceptable limits. Stock and lysate specimens were found to be stable in −20°C. The Indian populations have a considerable number of patients with low‑ and intermediate‑TPMT activity. The wide range of TPMT activity in the study population (3.85–35.68 nmol/h/ml erythrocytes) is suggestive of high inter‑individual variability in the formation of 6‑thioguanine nucleotide formation, and therefore clinical efficacy and safety of AZA.Conclusion: A simple isocratic method was developed and validated for measuring TPMT activity in erythrocytes. Measuring TPMT activity before initiating AZA may help to decide the initial dose of AZA in patient management.

Keywords: 6‑mercaptopurine; 6‑methylmercaptopurine; azathioprine; Thiopurine S‑methyltransferase