Quantification of 6-Mercaptopurine and Its Metabolites in Patients with Acute Lympoblastic Leukemia Using Dried Blood Spots and UPLC-MS/MS

 

Quantification of 6-Mercaptopurine and Its
Metabolites in Patients with Acute Lympoblastic
Leukemia Using Dried Blood Spots and
UPLC-MS/MS

Supandi Supandi, Supandi

This research aimed to quantitatively bioanalyze 6-mercaptopurine (6-MP),
6-methylmercaptopurine (6-MMP), and 6-thioguanosine-50

-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 (2.1 × 100 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/8 × 108

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/8 × 108

erythrocytes,
which includes concentrations below the hepatotoxic range. The method employed here can thus be
effectively utilized to support therapeutic drug monitoring.

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