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.
- Dried blood spot
- Ultra performance liquid chromatography tandem mass spectrometry