SIMULTANEOUS ANALYTICAL METHOD DEVELOPMENT OF 6-MERCAPTOPURINE AND 6-METHYLMERCAPTOPURINE IN DRIED BLOOD SPOT USING ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY

Abstract

Objective: 6-mercaptopurine (6-MP) is a chemotherapeutic agent in the antimetabolite class. It has to go through the metabolic pathway to form
6-methyl MP (6-MMP). This study aimed to obtain an optimum and validated method for the analysis of 6-MP and 6-MMP in dried blood spot (DBS)
samples simultaneously and to evaluate the potential for future drug concentration monitoring in DBS samples.
Methods: The quality control and calibration curves were made by spotting 40 μL blood on DBS paper and dried for 3 hrs. DBS papers were cut with a
diameter of 8 mm and extracted with acetonitrile-methanol (1:3) containing internal standard 5-fluorouracil (5-FU). Separation was performed with
waters acquity ultra performance liquid chromatography BEH C18 column of 1.7 μm (2.1×100 mm) with a mobile phase consisting of 0.1% formic
acid in water 0.1% formic acid in acetonitrile with gradient elution and a flow rate of 0.2 mL/minute. Mass detection was performed using Waters
Xevo TQD with positive electrospray ionization (ESI) for 6-MP and 6-MMP and negative ESI for 5-FU in the multiple reaction monitoring mode.
Results: The detection rates of 6-MP, 6-MMP, and 5-FU were 153.09>119.09, 167.17>126.03, and 129.09>42.05, respectively. This method was linear
with the range at 26-1000 ng/mL for 6-MP and 13-500 ng/mL for 6-MMP with consecutive r≥0.998 and ≥0.999, respectively. The % relative error
value and % relative standard deviation for accuracy and precision of intraday and interday were not more than 15% and not more than 20% at the
lower limit of quantification concentration, respectively.
Conclusions: This method fulfilled the requirements of selectivity, linearity, carry over, and matrix effects referring to the European Medicines Agency
guidelines.