MSACL 2018 US Abstract

Topic: Troubleshooting

Low Extraction Rate of 6-Methylmercaptopurine in RBC

Soo Young Moon (Presenter)
Seoul National University Hospital

Authorship: Soo Young Moon(1, 2), Ji-hyun Lim(2), Eun-hee Kim(2), Kyoung Hoon Lee(3), Sang Hoon Song(1, 2), Junghan Song(3)
(1)Department of Laboratory Medicine, Seoul National University Hospital (2)Institute of Biomedical Research, Seoul National University College of Medicine, Seoul (3)Department of Laboratory Medicine, Seoul National University Bundang Hospital, Seongnam, Korea

Short Abstract

For measuring thiopurine nucleotides, acid hydrolysis step is required. Recovery rate of 6-methylmercaptopurine was 1~3% according to comparison of peak chromatogram before and after extraction. By using isotope-substituted internal standard, final concentration was not overly affected.

Long Abstract


6-thioguanine (6TG) and 6-methyl mercaptopurine (6MMP) nucleotides, which are intracellular metabolite of the drugs azathioprine and mercaptopurine, should be hydrolyzed for measurement. Heating in strong acid is required for complete hydrolysis, but conformational change of analytes might be accompanied, which may lead to inappropriate extraction and varying matrix effect. In this study, extraction efficiency and matrix effect of 6TG and 6MMP was evaluated.


Adding perchloric acid, RBC was hemolyzed and protein was precipitated, then supernatant was heated at 100°C for 1 hour (1). Chromatic separation was performed using Agilent 1200 series adopting Eclipse plus C18 column (3.5 µm, 4.6×100 mm, Agilent Technologies) in 5 minutes of gradient elution of 0.1% formic acid added water and acetonitrile. 6TG and 6MMP was quantified by Triple Quadrupole 6490 in positive ion mode using isotope substituted analytes as internal standard.

Matrix effect was evaluated by post-extraction method (2) including 3 sets of experiments: analytes and IS added to water (set1), to hemolysate after extraction (set2), and to RBC before extraction (set3). Comparing chromatographic peak area and final concentrations between these sets enabled calculation of matrix effect (ratio of set2 to set1), extraction recovery (ratio of set3 to set2), and process efficiency (ratio of set3 to set1).


As a result, the matrix effect of 6TG was around 200% and extraction recovery was about 50% resulting in process efficiency of around 100% when calculated by peak area. As IS was affected to a similar degree, final concentrations were not affected by matrix effect or extraction recovery. The degree of ionization enhancement and low extraction recovery was greater for 6MMP. The matrix effect was more than 200% and extraction recovery around 1%, which produced process efficiency of about 3%. However, like 6TG, the effects were also true for IS and final concentrations were not significantly affected.


In summary, recovery rate of 6MMP was markedly affected by extraction process although final concentration could be corrected by using isotope-substituted. Considerable uncertainty of 6MMP due to variable recovery rate in extraction process remained as a problem.

References & Acknowledgements:

(1) Shipkova M, Armstrong VW, Wieland E, Oellerich M. Differences in nucleotide hydrolysis contribute to the differences between erythrocyte 6-thioguanine nucleotide concentrations determined by two widely used methods. Clin Chem. 2003;49(2):260-8.

(2) Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem. 2003;75(13):3019-30.

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