Abstract
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<0.05) the percent coefficient of variation, whereas differences in blood hematocritsignificantly affected the peak area ratio.Conclusion: The method of internal standard addition affected the chromatograms in this study. The best chromatogram was observed whenthe Internal Standard was added to the extracting solution. The card type also affected the analysis, so it is recommended to use the same card duringsample analysis.
Highlights
The dried blood spot (DBS) method has been used as a sampling technique in therapeutic drug monitoring of some drugs in clinical practice
Several factors should be considered when a DBS method is developed and validated. These include the small blood volume, varying hematocrit (HT) values in patients undergoing chemotherapy, the presence of metabolites resulting from metabolism, and the different paper types to be used in the analysis
The results suggested that some of the IS were permanently absorbed into the cards, which decreased the amount of IS that was extracted, injected into the ultra-high-performance LC (UPLC), and detected by MS/MS
Summary
The dried blood spot (DBS) method has been used as a sampling technique in therapeutic drug monitoring of some drugs in clinical practice. This sampling method offers several advantages over venipuncture, such as more convenient for the patient because it is less invasive than taking blood using a sterile lancet needle on the fingers, low volume, easy storage, storage stability, and reduced risk of infection [1,2]. Several factors should be considered when a DBS method is developed and validated These include the small blood volume, varying hematocrit (HT) values in patients undergoing chemotherapy, the presence of metabolites resulting from metabolism, and the different paper types to be used in the analysis. The spread of the blood drop on the DBS card is influenced by the HT of the blood; high HT forms smaller spots and vice versa
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