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 storagetemperature on 6-mercaptopurine, and 6-thioguanine on liquid chromatography tandem-mass spectrometry (LC-MS/MS) bioanalysis methods usingdried blood spot (DBS).Methods: Blood samples were spotted on CAMAG DBS paper and a Perkin Elmer 226 sample collection device (paper) and extracted into methanolcontaining 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 aflow 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. Differencesin paper types and blood volumes significantly affected (p