Factors affecting liquid chromatography tandem-mass spectrometry analysis of 6-mercaptopurine and 6-thioguanine in dried blood spots

Objective: This study aimed to determine the effects of the method of internal standard addition, spotting volume, paper type, and sample storage temperature on 6-mercaptopurine, and 6-thioguanine on liquid chromatography tandem-mass spectrometry (LC-MS/MS) bioanalysis methods using dried blood spot (DBS). Methods: Blood samples were spotted on CAMAG DBS paper and a Perkin Elmer 226 sample collection device (paper) and extracted into methanol containing 5-fluorouracil as an internal standard. The separation was performed on a water acquity ultra high-performance LC BEH Amide 1.7 µm (2.1 mm×100 mm) column with a mobile phase of 0.2% formic acid in water - 0.1% formic acid in acetonitrile methanol with gradient elution at a flow rate of 0.2 mL/min. Results: The step at which the internal standard was added (blood, spot on DBS card, or extraction solution) affected the chromatogram. Differences in paper types and blood volumes significantly affected (p<0.05) the percent coefficient of variation, whereas differences in blood hematocrit significantly affected the peak area ratio. Conclusion: The method of internal standard addition affected the chromatograms in this study. The best chromatogram was observed when the Internal Standard was added to the extracting solution. The card type also affected the analysis, so it is recommended to use the same card during sample analysis.