Quantification of 6-mercaptopurine and its metabolites in patients with acute lympoblastic leukemia using dried blood spots and UPLC-MS/MS

This research aimed to quantitatively bioanalyze 6-mercaptopurine (6-MP), 6-methylmercaptopurine (6-MMP), and 6-thioguanosine-5'-monophosphate (6-TGMP) in dried blood spots (DBS) prepared from a small volume of acute lymphoblastic leukemia (ALL) patients. Analytes on the DBS card were extracted using 90% methanol with 5-fluorouracil (5-FU) as an internal standard. Analytical separation was performed on a Waters Acquity® UPLC BEH AMIDA column of 1.7 µm (2.1x100 mm) with a mobile phase mixture of 0.2% formic acid in water and 0.1% formic acid in acetonitrile-methanol, with gradient elution and a flow rate of 0.2 mL/min. Mass detection of 6-MP, 6-MMP, 6-TGMP, and 5-FU showed m/z values of 153.09 > 119.09, 167.17 > 126.03, 380.16 > 168.00, and 129.09 > 42.05, respectively. This DBS method had a run time of 5 min and yielded a linear calibration curve over a range of 25.5-1020 ng/mL for 6-MP, 6-MMP, and 6-TGMP. Analyte analysis in 22 of 24 ALL patients showed that the measured value of 6-TGMP as an active metabolite was in the range of 29-429 pmol/8x10⁸ erythrocytes. Five of 22 patients had concentrations in a therapeutic range, which indicates that the treatment is effective, while 17 of 24 patients had concentrations below the therapeutic range, which indicates that a treatment dose adjustment is needed. The measured value of 6-MMP, an inactive metabolite, was in the range of 28-499 pmol/8x10⁸ erythrocytes, which includes concentrations below the hepatotoxic range. The method employed here can thus be effectively utilized to support therapeutic drug monitoring.