Wiwattanakul Supaporn, Prommas Santirhat, Jenjirattithigarn Nuttawut, Santon Siwalee, Puangpetch Apichaya, Pakakasama Samart, Anurathapan Usanarat, Sukasem Chonlaphat
Division of Pharmacogenomics and Personalized Medicine, Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand; Laboratory for Pharmacogenomics, Somdech Phra Debaratana Medical Center (SDMC), Ramathibodi Hospital, Bangkok, 10400, Thailand.
Division of Pediatric Hematology-Oncology, Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand.
J Pharm Biomed Anal. 2017 Oct 25;145:758-764. doi: 10.1016/j.jpba.2017.07.039. Epub 2017 Aug 4.
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 7min 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 2.5-360ng/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 (100μl of whole blood EDTA). The assay was used to study TPMT activity in 132 Thai children with a range from 29.0 to 89.1nmol 6-MMP/g Hb/h with means and median values of TPMT activity 55.9±12.47nmol 6-MMP/g Hb/h and 54.2nmol 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.
建立了一种液相色谱-串联质谱（LC-MS/MS）法，用于测定人全血裂解物中的硫嘌呤甲基转移酶（TPMT）活性，该方法基于TPMT以S-腺苷-L-甲硫氨酸（SAM）作为甲基供体将6-巯基嘌呤（6-MP）转化为6-甲基巯基嘌呤（6-MMP）。该方法是在之前繁琐的红细胞裂解物制备洗涤方法基础上改进而来，用于制备全血EDTA裂解物。此外，对TPMT孵育条件进行了优化，并通过三重四极杆质谱检测，在C18柱上以7分钟的短运行时间进行色谱分析。MS/MS经过优化调整，以监测6-MMP的质荷比（m/z）为167.2→151.9，并将m/z为170.5→152.2的同位素6-MMP-d3用作内标。校准曲线覆盖2.5 - 360ng/ml范围，相关系数大于0.999。该方法在四种质量控制浓度下的准确度在99.33%至106.33%之间。批内变异系数（CV）小于4.41%，批间变异系数小于5.43%。这种通过LC-MS/MS测定TPMT的方法可靠、安全且简单，所需样品量少（100μl全血EDTA）。该检测方法用于研究132名泰国儿童的TPMT活性，范围为29.0至89.1nmol 6-MMP/g Hb/h，TPMT活性的均值和中位数分别为55.9±12.47nmol 6-MMP/g Hb/h和54.2nmol 6-MMP/g Hb/h。在30个样本中评估了TPMT常见泰国民族单核苷酸多态性（SNP）的基因型-表型关联，结果显示具有良好的一致性。
