ANALYSIS OF 6-MERCAPTOPURINE AND 6-METHYLMERCAPTOPURINE IN DRIED BLOOD
SPOTS USING LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY AND ITS
APPLICATION IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA PATIENTS
Objective: To analyze and validate 6-mercaptopurine (6-MP) and 6-methylmercaptopurine (6-MMP) in dried blood spots (DBS) using liquid
chromatography-tandem mass spectrometry (LC/MS-MS).
Methods: Bio-sampling dried blood spot with DBS-CAMAG® paper diameter of 8 mm and extracted with acetonitrile-methanol (1:3) containing
internal standard 5-fluorouracil (5-FU). Separation was performed with C18 column Acquity® 1.7 μm (2.1 mm × 100 mm), with a mobile phase mixture
of 0.1% formic acid in water 0.1% formic acid in acetonitrile with gradient elution and flow rate 0.2 ml/min. Mass detection was Waters Xevo TQD
with positive electrospray ionization (ESI) for 6-MP, 6-MMP and negative ESI for 5-FU in multiple reaction monitoring modes. The ions of 6-MP was
detected at m/z 153.09->119.09, 6-MMP at m/z 167.17->126.03, and 5-FU at m/z 129.15->42.05.
Results: This method fulfill the requirements of selectivity, linearity, lower limit of quantification, accuracy, precision, carry-over, matrix effects,
and stability which refers to the european medicines agency (EMEA) guidelines. The linearity of 0.99 was 26-1000 ng/mL for 6-MP and 6-MMP,
respectively. The validated method was applied to two childhood ALL maintenance phase. Retrieved 6-MP levels of 10.37 pmol/8×108 erythrocytes,
respectively. The levels of 6-MMP gained 16.59 pmol/8×108 erythrocytes, respectively.
Conclusion: The developed LC/MS-MS method is valid to analysis 6-MP and 6-MMP in DBS simultaneous in vitro according to EMEA guidelines. The
method was successfully applied to authentic capillary blood samples from two childhood patients with ALL in the maintenance phase.
Keywords: 6-mercaptopurine, 6-methylmercaptopurine, Dried blood spots, Acute lymphoblastic leukemia.