Service de Biochimie, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, Paris, France.
Service de Biochimie, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, Paris, France.
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Apr 15;1113:91-97. doi: 10.1016/j.jchromb.2019.03.014. Epub 2019 Mar 14.
Thiopurines are drugs widely used for the treatment of autoimmune conditions, inflammatory bowel disease or acute lymphoblastic leukemia. Determination of thiopurine methyltransferase activity (TPMT), a major determinant of thiopurines toxicity, has been suggested before implementing thiopurine treatment. An ultraperformance liquid chromatography (UPLC) method was developed and validated for the quantification of TPMT enzyme activity based on the conversion of 6-mercaptopurine (6-MP) to 6-methylmercaptopurine (6-MMP) using S-adenosyl-L-methionine (SAM) as methyl donor in red blood cell lysates (RBC). This method was improved from a previous laborious high performance liquid chromatography (HPLC) method, using a lower volume of injection and with a shorter runtime. After incubation and protein precipitation 6-MMP was separated on a HSS-T3 (2.1 × 50 mm, 1.8 μm) column and monitored by UV detection (290 nm). A change on the organic solvent used to dissolve 6-MP resulted in a reduction of interference by endogenous or non-enzymatic methylated 6-MMP. A full validation of the 6-MMP assay was performed according to the FDA and EMA guidelines. The method was linear from 0.125 to 2 nmol/mL, with acceptable values of accuracy and precision. The method was applied in 106 patients treated with thiopurines whose TPMT activity was previously quantified by HPLC. Evaluation through Bland-Altman plot showed that TPMT activities were in agreement between both methods.
硫嘌呤类药物被广泛用于治疗自身免疫性疾病、炎症性肠病或急性淋巴细胞白血病。在实施硫嘌呤治疗之前，建议测定硫嘌呤甲基转移酶活性（TPMT），这是硫嘌呤毒性的主要决定因素。本研究建立并验证了一种超高效液相色谱（UPLC）法，用于测定红细胞裂解物（RBC）中 TPMT 酶活性，该方法基于 S-腺苷-L-蛋氨酸（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）。与之前繁琐的高效液相色谱（HPLC）法相比，该方法采用了更小的进样量和更短的运行时间，对方法进行了改进。孵育和蛋白沉淀后，用 HSS-T3（2.1×50mm，1.8μm）柱分离 6-MMP，并通过紫外检测（290nm）进行监测。改变溶解 6-MP 的有机溶剂可减少内源性或非酶促甲基化 6-MMP 的干扰。根据 FDA 和 EMA 指南对 6-MMP 测定法进行了全面验证。该方法在 0.125 至 2 nmol/mL 范围内呈线性，具有可接受的准确度和精密度值。该方法应用于 106 例接受硫嘌呤治疗的患者，这些患者的 TPMT 活性之前已通过 HPLC 进行了定量。通过 Bland-Altman 图评估表明，两种方法的 TPMT 活性具有一致性。
