Ma Jing, Sies Christiaan W, Pike Linda S
Specialist Chemistry, Canterbury Health Laboratories, Christchurch, New Zealand.
Clin Biochem. 2018 Apr;54:100-105. doi: 10.1016/j.clinbiochem.2018.02.002. Epub 2018 Feb 6.
Identification of patients with thiopurine S-methyltransferase (TPMT) deficiency prior to thiopurine drug therapy has become routine clinical practice worldwide. To measure TPMT activity, traditional radiochemical assays have been replaced by chromatographic methods.
Inspired by the increasing number of isotope labelled sources that may be of benefit for the TPMT assay, a new LC-MS/MS method for TPMT activity was developed and validated. Isotope labelled d3-S-adenosyl-l-methionine (d3-SAM) was selected for the enzymatic methylation of mercaptopurine during sample incubation; d3-6-methylmercaptopurine (d3-6-MMP) with d2-2, 8-hypoxanthine as the internal standard was quantified to ascertain individual TPMT activity.
The validation of the analytical part of this method showed good linearity (coefficient of determination 0.9999 in the range of 1-500 ng/mL) with the intra-and inter-day impression CV% between 7.6% and 9.1% and 3.7% and 9.2%, respectively. Recovery ranged from 94.9% to 112.3%. The specificity of the enzymatic reaction was validated by using 108 clinical check samples. After compared with traditional radiochemical assay and genotype results, all homozygous and heterozygous deficiency clinical checks fitted into the nominal groups, inter-batch and intra-batch impression CV% were between 2.3% and 9.7%.
With the inclusion of isotope labelled substrate, interfering non-enzymatic methylation no longer results in potential false assignment of abnormal patients. Furthermore, the method can be applied to patients who have already been prescribed thiopurine drugs. This new LC-MS/MS is therefore a favourable clinical routine application to test TPMT activity, as it shows excellent performance in identifying patients with TPMT deficiency.
在硫嘌呤类药物治疗前识别硫嘌呤甲基转移酶（TPMT）缺乏的患者已成为全球常规临床实践。为了测量TPMT活性，传统的放射化学分析法已被色谱法所取代。
受可能对TPMT检测有益的同位素标记源数量增加的启发，开发并验证了一种新的用于TPMT活性检测的液相色谱-串联质谱（LC-MS/MS）方法。在样品孵育期间，选择同位素标记的d3-S-腺苷-L-甲硫氨酸（d3-SAM）用于巯嘌呤的酶促甲基化；以d2-2,8-次黄嘌呤为内标对d3-6-甲基巯嘌呤（d3-6-MMP）进行定量，以确定个体TPMT活性。
该方法分析部分的验证显示出良好的线性（在1-500 ng/mL范围内的决定系数为0.9999），日内和日间精密度CV%分别在7.6%和9.1%以及3.7%和9.2%之间。回收率在94.9%至112.3%之间。通过使用108份临床对照样品验证了酶促反应的特异性。与传统放射化学分析法和基因型结果比较后，所有纯合子和杂合子缺乏临床对照均符合标称组，批间和批内精密度CV%在2.3%和9.7%之间。
由于加入了同位素标记底物，干扰性的非酶促甲基化不再导致对异常患者的潜在错误分类。此外，该方法可应用于已经开具硫嘌呤类药物的患者。因此，这种新的LC-MS/MS是一种用于检测TPMT活性的良好临床常规应用方法，因为它在识别TPMT缺乏患者方面表现出优异的性能。
