ANALISIS 6-MERKAPTOPURIN DAN 6-TIOGUANIN DALAM DRIED BLOOD SPOT DENGAN KROMATOGRAFI CAIR-TANDEM SPEKTROMETRI MASSA
Keywords:
6-mercaptopurine, 6-thioguanine, analysis, dried blood spotAbstract
6-Mercaptopurine (6-MP) is a chemotherapeutic drug that belongs to a class of purine antagonist antimetabolite used for acute lymphoblastic leukemia. 6-Mercaptopurine has to go through the metabolic pathway to become 6-Thioguanin nucleotide(active metabolites). This study aimed to analyzing 6-Mercaptopurine and 6-Thioguanine (6-TG) simultaneously in Dried Blood Spot (DBS) samples using liquid chromatography-tandem mass spectrometry. The quality control and calibration curve solutions were made by spot 60 µL at DBS CAMAG paper and were dried for 3 hours. DBS papers were cut and extracted with methanol containing internal standard 5-fluorouracil (5-FU). Separation performed with Waters Acquity™ UPLC BEH Amide column 1.7 µm (2.1 x 100 mm) with a mobile phase consists of 0.2 % formic acid in water – 0.1% formic acid in acetonitrile – methanol with gradient elution and flow rate 0.2 mL/minutes. Mass detection was done using Waters Xevo TQD with positive electrospray ionization (ESI) for 6-MP and 6-TG and negative ESI for 5-FU in Multiple Reaction Monitoring mode. Detection of 6-MP, 6-MMP, 5-FU respectively are 153,09 > 119,09; 168,09 > 107,06; 129,15 > 42,05. This method is linear with the range 25-1000 ng /mL for 6-MP and 6-TG with consecutive r value is ≥ 0,996 and ≥ 0,995. Diff value percentage and coefficient of variation (CV) for accuracy and precision of intra-day and inter-day are not more than ±15% and not more than ±20% at a concentration LLOQ. This method fulfill the requirements of validation that refers to the European Medicines Agency guideline.References
Al-Ghobashy, M. A., Hassan, S. A., Abdelaziz, D. H., Elhosseiny, N. M., Sabry, N. A.,
Attia, A. S., & El-Sayed, M. H. (2016). Development and validation of LCMS/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. Journal of Chromatography B:
Analytical Technologies in the Biomedical and Life Sciences, 1038, 88–94.
Déglon, J., Thomas, A., Mangin, P., & Staub, C. (2012). Direct analysis of dried blood
spots coupled with mass spectrometry: Concepts and biomedical applications.
Analytical and Bioanalytical Chemistry, 402(8), 2485–2498.
Evans, C., Arnold, M., Bryan, P., Duggan, J., James, C. a, Li, W., & Zane, P. (2015).
Implementing Dried Blood Spot Sampling for Clinical Pharmacokinetic
Determinations: Considerations from the IQ Consortium Microsampling Working
Group. The AAPS Journal, 17(2), 292–300.
Katzung, B. G., Susan, B., Masters, T., & Anthony, J. (2012). Basic and clinical
Pharmacology (Twelfth Ed). Mc. Graw Hill Medical.
Lockwood, W., 2015,Leukemia: AML, CML, ALL and CLL, (Cml), 1–32.
Rini, T. A., Aisyi, M., Sari, Y., & Edi, S.T. (2009). Karakteristik Leukemia Limfoblastik
Akut pada Anak di Rumah Sakit Kanker Dharmais 2000-2008. Ind J Cancer, 4, 4:
-140.
Spooner, N., Lad, R., & Barfield, M. (2009) Dried blood spots as a sample collection
technique for the determination of pharmacokinetics in clinical studies:
considerations for the validation of a quan- titative bioanalytical method. Analytical
Chemistry., 81(4), 155.
Su, Y., Hon, Y. Y., Chu, Y., Poll, M. E. C., & Relling, M. V. (1999). Assay of 6-
mercaptopurine and its metabolites in patient plasma by high-performance liquid
chromatography with diode-array detection. Jounal of Chromatography B:
Analytical Technologies in the Biomedical and Life Sciences, 732, 459–468.
Zochowska, D., Zegarska, J., Hryniewiecka, E., Samborowska, E., Jazwiec, R.,
Tszyrsznic, W.,&Paczek, L. (2016). Determination of Concentrations of
Azathioprine Metabolites 6-Thioguanine and 6-Methylmercaptopurine in Whole
Blood With the Use of Liquid Chromatography Combined With Mass Spectrometry.
Transplantation Proceedings, 48(5), 1836–1839.
