Simultaneous Analysis of 6-Mercaptopurine, 6-Methylmercaptopurine, and 6-Thioguanosine-5’-monophosphate in Dried Blood Spot Using Ultra Performance Liquid Chromatography Tandem Mass Spectrometry

https://doi.org/10.22146/ijc.31116

Supandi Supandi(1*), Yahdiana Harahap(2), Harmita Harmita(3), Rizka Andalusia(4)

(1) Department of Pharmacy, Faculty of Pharmacy and Science UHAMKA, Jl. Delima II Klender Jakarta 13460, Indonesia
(2) Faculty of Pharmacy, Universitas Indonesia, Jl. Campus UI Depok 16424, Indonesia
(3) Faculty of Pharmacy, Universitas Indonesia, Jl. Campus UI Depok 16424, Indonesia
(4) Department of Research and Development of Dharmais Cancer Hospital, Jl. S. Parman Kav. 84-86 Slipi Jakarta 11420, Indonesia
(*) Corresponding Author

Abstract


6-Mercaptopurine is a chemotherapeutic agent of the antimetabolite class. This study aims to analyze simultaneous validation of 6-mercaptopurine (6-MP), 6-methylmercaptopurine (6-MMP), and 6-thioguanosine-5’-monophosphate (6-TGMP) in dried blood spot (DBS) using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). An accurate volume of 60 μL blood was spotted onto DBS-CAMAG paper and then extracted using methanol 90% (v/v) containing an internal standard of 5-fluorouracil (5-FU). Separation was performed using a Waters Acquity UPLC BEH AMIDA column 1.7 μm (2.1 x 100 mm) with a mobile phase mixture of 0.2% (v/v) formic acid in water−0.1% (v/v) formic acid in acetonitrile-methanol with gradient elution and flow rate of 0.2 mL/min. Mass detection was done using Waters Xevo TQD with positive electrospray ionization (ESI) for 6-MP, 6-MMP, 6-TGMP and negative ESI for 5-FU, in multiple reaction monitoring mode. Detection rates of 6-MP, 6-MMP, 6-TGMP and 5-FU were m/z 153.09 > 119.09; 167.17 > 126.03; 380.16 > 168.00); 129.09 > 42.05, respectively. This method is linear across the range 25.5–1020 ng/mL for 6-MP, 6-MMP and 6-TGMP. This method is valid for the in vitro simultaneous analysis of 6-MP, 6-MMP and 6-TGMP in DBS, based on European Medicine Agency guidelines.

Keywords


6-mercaptopurine; dried blood spot; simultaneous; method validation

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References

[1] Hunger, S.P., and Mullighan, C.G., 2015, Acute lymphoblastic leukaemia in children, N. Engl. J. Med., 375 (16), 1541–1552.

[2] Stork, L.C., Matloub, Y., Broxson, E., La, M., Yanofsky, R., Sather, H., Hutchinson, R., Heerema, N.A., Sorrell, A.D., Masterson, M., Bleyer, A., and Gaynon, P.S., 2010, Oral 6-mercaptopurine versus oral 6-thioguanine and veno-occlusive disease in children with standard-risk acute lymphoblastic leukaemia: Report of the Children’s Oncology Group CCG-1952 clinical trial, Blood, 115 (14), 2740–2748.

[3] Beaumais, T.A., Fakhoury, M., Medard, Y., Azougagh, S., Zhang, D., Yakouben, K., and Jacqz-Aigrain, E., 2011, Determinants of mercaptopurine toxicity in paediatric acute lymphoblastic leukaemia maintenance therapy, Br. J. Clin. Pharmacol., 71 (4), 575–584.

[4] Dervieux, T., Meyer, G., Barham, R., Matsutani, M., Barry, M., Boulieu, R., Neri, B., and Seidmans, E., 2005, Liquid chromatography-tandem mass spectrometry analysis of erythrocyte thiopurine nucleotides and effect of thiopurine methyltransferase gene variants on these metabolites in patients receiving azathioprine/6-mercaptopurine therapy, Clin. Chem., 51 (11), 2074–2084.

[5] Dipiro, J.T., Talbert, R.L., Yee, G.C., Matzke, G.R., Wells, B.G., and Posey, L.M., 2008, Pharmacotherapy A Pathophysiologic Approach, 7th Ed., McGraw-Hill, New York, 733–735.

[6] Al-Ghobashy, M.A., Hassan, S.A., Abdelaziz, D.H., Elhosseiny, N.M,, Sabry, N.A., Attia, A.S., and El–Sayed, M.H., 2016, 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 leukaemia: Correlation with genetic polymorphism, J. Chromatogr. B, 1038, 88–94.

[7] Schmiegelow, K., and Bretton-Meyer, U., 2001, 6-Mercaptopurine dosage and pharmacokinetics influence the degree of bone marrow toxicity following high-dose methotrexate in children with acute lymphoblastic leukaemia, Leukemia, 15 (1), 74–79.

[8] Kirchherr, H., Shipkova, M., and von Ahsen, N., 2013, Improved method for therapeutic drug monitoring of 6-thioguanine nucleotides and 6-methylmercaptopurine in whole-blood by LC/MSMS using isotope-labeled internal standards, Ther. Drug Monit., 35 (3), 313–321.

[9] Hawwa, A.F., Millership, J.S., Collier, P.S., and McElnay, J.C., 2009, Development and validation of an HPLC method for the rapid and simultaneous determination of 6-mercaptopurine and four of its metabolites in plasma and red blood cells, J. Pharm. Biomed. Anal., 49 (2), 401–409.

[10] Adaway, J.E., and Keevil, B.G., 2012, Therapeutic drug monitoring and LC-MS/MS, J. Chromatogr. B., 883-884, 33–49.

[11] Déglon, J., Thomas, A., Mangin, P., and Staub, C., 2012, Direct analysis of dried blood spots coupled with mass spectrometry: Concepts and biomedical applications, Anal. Bioanal. Chem., 402 (8), 2485–2498.

[12] De Kesel, P.M., Sadones, N., Capiau, S., Lambert, W.E., and Stove, C.P., 2013, Hemato-critical issues in quantitative analysis of dried blood spots: Challenges and solutions, Bioanalysis, 5 (16), 2023–2041.

[13] Sharma, A., Jaiswal, S., Shukla, M., and Lal, J., 2014, Dried blood spots: Concepts, present status, and future perspectives in bioanalysis, Drug Test. Anal., 6 (5), 399–414.

[14] Wilhelm, A.J., den Burger, J.C., and Swart, E.L., 2014, Therapeutic drug monitoring by dried blood spot: Progress to date and future directions, Clin. Pharmacokinet., 53 (11), 961–973.

[15] European Medicines Agency (EMEA), 2011, Committee for Medicinal Products for Human Use (CHMP): Guideline on Bioanalytical Method Validation, European Medicines Agency, London.

[16] Jager, N.G., Rosing, H., Schellens, J.H., and Beijnen, J.H., 2014, Procedures and practices for the validation of bioanalytical methods using dried blood spots: A review, Bioanalysis, 6 (18), 2481–2514.

[17] Kang, J.S., 2012, “Principles and Applications of LC-MS/MS for the Quantitative Bioanalysis of Analytes in Various Biological Samples” in Tandem Mass Spectrometry – Applications and Principles, Prasain, J., (Ed.), InTech, 441–492.

[18] Leung, K.S., and Fong, B.M., 2014, LC-MS/MS in the routine clinical laboratory: Has its time come, Anal. Bioanal. Chem., 406 (9-10), 2289–2301.

[19] Chambers, A.G., Percy, A.J., Yang, J., Camenzind, A.G., and Borchers, C.H., 2013, Multiplexed quantitation of endogenous proteins in dried blood spots by multiple reaction monitoring-mass spectrometry, Mol. Cell. Proteomics, 12 (3), 781–791.

[20] Matuszewski, B.K., Constanzer, M.L., and Chavez-Eng, C.M., 2003, Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS, Anal. Chem., 75 (13), 3019–3030.

[21] Li, W., and Tse, F.L., 2010, Dried blood spot sampling in combination with LC-MS/MS for quantitative analysis of small molecules, Biomed. Chromatogr., 24 (1), 49–65.



DOI: https://doi.org/10.22146/ijc.31116

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