Comparison of 6‐mercaptopurine with 6‐thioguanine for the analysis of thiopurine S‐methyltransferase activity in human erythrocyte by LC–MS/MS

Abstract

Thiopurines (TPDs) are first-line drugs in treating neuromyelitis optica spectrum disorders (NMOSD). Evaluation of thiopurine S-methyltransferase activity (TPMT), a major determinant of TPD toxicity, before TPD treatment using 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) as substrate was suggested. However, the equivalent of the two substrates in TPMT activity evaluation was unknown, and an alternative substrate was required in TPMT activity evaluation in patients who were already taking 6-MP or 6-TG. Before evaluating the agreement of 6-MP and 6-TG in TPMT activity measurement in patients with NMOSD, the affinity of the two substrates for the active center of TPMT should be established. A computer-based simulation indicated that 6-MP and 6-TG had similar affinities for the two active sites of TPMT. According to the guidelines, an LC-MS/MS method was developed and validated to evaluate the TPMT activity in human erythrocyte hemolysate using 6-MP or 6-TG as substrates via 1 h incubation at 37°C. The method was applied in 81 patients with NMOSD. Evaluated by Bland-Altman plot, 6-methylmercaptopurine and 6-methylthioguanine represented TPMT activities were in agreement with each other. Further studies are warranted to confirm the results.

Development and validation of a UPLC-UV method for the quantification of thiopurine methyltransferase enzyme activity in human erythrocytes

“…The retention time of 6-MMP was 1.89 min (Figure 1), and the total run time was 9 min. It represents a significant decrease from the run time of the previous HPLC method (40 min), and it is in line with the available LC-MS/MS methods (23)(24)(25)(26), with run times around 7 min.…”

“…In a second time, several HPLC methods with UV and fluorescence detection have been published (15)(16)(17)(18)(19)(20)(21)(22). More recently, the use of LC-MS/MS methods to measure TPMT activity has emerged (23)(24)(25)(26). Most methods are based on the enzymatic methylation of 6-MP by TPMT in red blood cell lysates with Sadenosyl-L-methionine (SAM) as methyl donor.…”