Publication: 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
10
Issued Date
2017-10-25
Resource Type
ISSN
1873264X
07317085
07317085
Other identifier(s)
2-s2.0-85029308665
Rights
Mahidol University
Rights Holder(s)
SCOPUS
Bibliographic Citation
Journal of Pharmaceutical and Biomedical Analysis. Vol.145, (2017), 758-764
Suggested Citation
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. Journal of Pharmaceutical and Biomedical Analysis. Vol.145, (2017), 758-764. doi:10.1016/j.jpba.2017.07.039 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/41751
Research Projects
Organizational Units
Authors
Journal Issue
Thesis
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
Other Contributor(s)
Abstract
© 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.