VALIDATION OF 6-MERCAPTOPURINE AND 6-THIOGUANINE IN DRIED BLOOD SPOT BY ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY
Objective: This study aimed to obtain an optimal and validated method of analyzing 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) simultaneously
in dried blood spot samples using ultra high-performance liquid chromatography-tandem mass spectrometry.
Method: Separation was performed with a 1.7-Î¼m amide column, which had a mobile phase with a flow rate of 0.2 mL/min and comprised 0.2%
formic acid in water, 0.1% formic acid in acetonitrile, and methanol with a gradient elution. Detection was performed using Waters Xevo TQD.
Result: This method was linear with a range of 25â€“1000 ng/mL for 6-MP and 6-TG, with consecutive r values of â‰¥0.996 and â‰¥0.995, respectively. The
intra- and inter-day % difference value and coefficient of variation for the accuracy and precision were not more than 15% and 20%, respectively, at
a concentration lower limit of quantitation.
Conclusion: This method fulfilled the requirements of the European Medicines Agency guideline for validation.
2. National Cancer Institute. What You Need To Know About Leukemia.
U.S: Department of Health and Human Services; 2015.
3. Rini TA, Aisyi M, Sari Y, Edi ST. Characteristics of acute lymphoblastics
leukemia in children at dharmais cancer hospital 2000 â€“ 2008. Ind J
4. Katzung BG. Basic and Clinical Pharmacology. 10th ed. USA: McGraw-
5. Polifka J, Friedman J. Teratogen update: Azathioprine and 6-mercaptopurine.
6. Su Y, Hon YY, Chu Y, Van de Poll ME, Relling MV. Assay of
6-mercaptopurine and its metabolites in patient plasma by highperformance
liquid chromatography with diode-array detection.
J Chromatogr B Biomed Sci Appl 1999;732:459-68.
7. Al-Ghobashy MA, Hassan SA, Abdelaziz DH, Elhosseiny NM, Sabry NA,
Attia AS, et al. Development and validation of LC-MS/MS assay for
the simultaneous determination of methotrexate, 6-mercaptopurine and
its active metabolite 6-thioguanine in plasma of children with acute
lymphoblastic leukemia: Correlation with genetic polymorphism.
J Chromatogr B Analyt Technol Biomed Life Sci 2016;1038:88-94.
8. European Medicines Agency. Guideline on Bioanalytical Method
Validation. Available from: http://www.ema.europa.eu/docs/en_GB/
[Last accessed on 2015 Oct 12].
9. Wagner M, Tonoli D, Varesio E, Hopfgartner G. The use of mass
spectrometry to analyze dried blood spots. Mass Spectrom Rev
10. Zochowska D, Zegarska J, Hryniewiecka E, Samborowska E,
Jazwiec R, Tszyrsznic W, et al. Determination of concentrations of
azathioprine metabolites 6-thioguanine and 6-methylmercaptopurine
in whole blood with the use of liquid chromatography combined with
mass spectrometry. Transplant Proc 2016;48:1836-9.
11. Patel D. Matrix effect in a view of LC-MS/MS: An overview. Int J
Pharm Bio Sci 2011;2:559-64.
12. Potawale RS, Gilhotra RM, Gabhe SY. Liquid chromatography tandemmass
spectrometry method development and validation for simultaneous
analysis of paracetamol, guaifenesin, phenylephrine hydrochloride,
chlorpheniramine maleate, and ambroxol hydrochloride in bulk and in
tablet dosage form. Asian J Pharm Clin Res 2018;11:375-82.
13. Srikanth I, Prameela RA. Development and validation of liquid
chromatography coupled with tandem mass spectrometry method for
estimation of lenvatinib in human plasma. Asian J Pharm Clin Res
14. Alvi SN, Hammami MM. Measurement of cortisol in human plsma
and urine by ultra performance liquid chromatography-tandem mass
spectrometry. Asian J Pharm Clin Res 2018;11:199-203.