Development and validation of a reliable method for thiopurine methyltransferase (TPMT) enzyme activity in human whole blood by LC–MS/MS: An application for phenotypic and genotypic correlations
© 2017 Elsevier B.V. A liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed for the determination of thiopurine methyltransferase (TPMT) activity in human whole blood lysate, based on conversion of 6-mercaptopurine (6-MP) by TPMT to 6-methylmercaptopurine (6-MMP) using S-ad...
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th-mahidol.417512019-03-14T15:02:44Z Development and validation of a reliable method for thiopurine methyltransferase (TPMT) enzyme activity in human whole blood by LC–MS/MS: An application for phenotypic and genotypic correlations Supaporn Wiwattanakul Santirhat Prommas Nuttawut Jenjirattithigarn Siwalee Santon Apichaya Puangpetch Samart Pakakasama Usanarat Anurathapan Chonlaphat Sukasem Mahidol University Faculty of Medicine, Ramathibodi Hospital, Mahidol University Biochemistry, Genetics and Molecular Biology Chemistry © 2017 Elsevier B.V. A liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed for the determination of thiopurine methyltransferase (TPMT) activity in human whole blood lysate, based on conversion of 6-mercaptopurine (6-MP) by TPMT to 6-methylmercaptopurine (6-MMP) using S-adenosyl-L-methionine (SAM) as the methyl donor. This method was improved from the previous laborious method for washing of red cell lysate preparation to develop whole blood EDTA lysate. In addition, the TPMT incubation was optimized and the chromatography was performed in a short runtime of 7 min on a C18-column by detection via triple quadrupole mass spectrometry. The MS/MS was optimally tuned to monitor mass to charge a ratio (m/z) for 6-MMP 167.2 → 151.9 and the isotope 6-MMP-d3 with m/z of 170.5 → 152.2 were applied as an internal standard. The calibration curve covered the range of 2.5–360 ng/ml and the correlation coefficient was greater than 0.999. The accuracy of this method was determined in four concentrations of control of quality that ranged between 99.33 and 106.33%. The intra-assay coefficient of variation (CV) was less than 4.41% and the inter-assay was less than 5.43%. This method developed for measuring TPMT by LC–MS/MS is a reliable, safe, and simple with a small volume requirement (100 μl of whole blood EDTA). The assay was used to study TPMT activity in 132 Thai children with a range from 29.0 to 89.1 nmol 6-MMP/g Hb/h with means and median values of TPMT activity 55.9 ± 12.47 nmol 6-MMP/g Hb/h and 54.2 nmol 6-MMP/g Hb/h. The genotype-phenotype association of TPMT was evaluated for common ethnic Thai single nucleotide polymorphisms (SNP) in 30 samples and demonstrated good concordance. 2018-12-21T06:40:32Z 2019-03-14T08:02:44Z 2018-12-21T06:40:32Z 2019-03-14T08:02:44Z 2017-10-25 Article Journal of Pharmaceutical and Biomedical Analysis. Vol.145, (2017), 758-764 10.1016/j.jpba.2017.07.039 1873264X 07317085 2-s2.0-85029308665 https://repository.li.mahidol.ac.th/handle/123456789/41751 Mahidol University SCOPUS https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85029308665&origin=inward |
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Biochemistry, Genetics and Molecular Biology Chemistry Supaporn Wiwattanakul Santirhat Prommas Nuttawut Jenjirattithigarn Siwalee Santon Apichaya Puangpetch Samart Pakakasama Usanarat Anurathapan Chonlaphat Sukasem Development and validation of a reliable method for thiopurine methyltransferase (TPMT) enzyme activity in human whole blood by LC–MS/MS: An application for phenotypic and genotypic correlations |
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© 2017 Elsevier B.V. A liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed for the determination of thiopurine methyltransferase (TPMT) activity in human whole blood lysate, based on conversion of 6-mercaptopurine (6-MP) by TPMT to 6-methylmercaptopurine (6-MMP) using S-adenosyl-L-methionine (SAM) as the methyl donor. This method was improved from the previous laborious method for washing of red cell lysate preparation to develop whole blood EDTA lysate. In addition, the TPMT incubation was optimized and the chromatography was performed in a short runtime of 7 min on a C18-column by detection via triple quadrupole mass spectrometry. The MS/MS was optimally tuned to monitor mass to charge a ratio (m/z) for 6-MMP 167.2 → 151.9 and the isotope 6-MMP-d3 with m/z of 170.5 → 152.2 were applied as an internal standard. The calibration curve covered the range of 2.5–360 ng/ml and the correlation coefficient was greater than 0.999. The accuracy of this method was determined in four concentrations of control of quality that ranged between 99.33 and 106.33%. The intra-assay coefficient of variation (CV) was less than 4.41% and the inter-assay was less than 5.43%. This method developed for measuring TPMT by LC–MS/MS is a reliable, safe, and simple with a small volume requirement (100 μl of whole blood EDTA). The assay was used to study TPMT activity in 132 Thai children with a range from 29.0 to 89.1 nmol 6-MMP/g Hb/h with means and median values of TPMT activity 55.9 ± 12.47 nmol 6-MMP/g Hb/h and 54.2 nmol 6-MMP/g Hb/h. The genotype-phenotype association of TPMT was evaluated for common ethnic Thai single nucleotide polymorphisms (SNP) in 30 samples and demonstrated good concordance. |
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Mahidol University |
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Mahidol University Supaporn Wiwattanakul Santirhat Prommas Nuttawut Jenjirattithigarn Siwalee Santon Apichaya Puangpetch Samart Pakakasama Usanarat Anurathapan Chonlaphat Sukasem |
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Supaporn Wiwattanakul Santirhat Prommas Nuttawut Jenjirattithigarn Siwalee Santon Apichaya Puangpetch Samart Pakakasama Usanarat Anurathapan Chonlaphat Sukasem |
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Supaporn Wiwattanakul |
title |
Development and validation of a reliable method for thiopurine methyltransferase (TPMT) enzyme activity in human whole blood by LC–MS/MS: An application for phenotypic and genotypic correlations |
title_short |
Development and validation of a reliable method for thiopurine methyltransferase (TPMT) enzyme activity in human whole blood by LC–MS/MS: An application for phenotypic and genotypic correlations |
title_full |
Development and validation of a reliable method for thiopurine methyltransferase (TPMT) enzyme activity in human whole blood by LC–MS/MS: An application for phenotypic and genotypic correlations |
title_fullStr |
Development and validation of a reliable method for thiopurine methyltransferase (TPMT) enzyme activity in human whole blood by LC–MS/MS: An application for phenotypic and genotypic correlations |
title_full_unstemmed |
Development and validation of a reliable method for thiopurine methyltransferase (TPMT) enzyme activity in human whole blood by LC–MS/MS: An application for phenotypic and genotypic correlations |
title_sort |
development and validation of a reliable method for thiopurine methyltransferase (tpmt) enzyme activity in human whole blood by lc–ms/ms: an application for phenotypic and genotypic correlations |
publishDate |
2018 |
url |
https://repository.li.mahidol.ac.th/handle/123456789/41751 |
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1763489843986300928 |