人红细胞中巯嘌呤甲基转移酶活性的HPLC测定法

High-performance liquid chromatographic assay of thiopurine methylthansferase activity in human red blood cells

目的建立人红细胞中巯嘌呤甲基转移酶(thiopurinemethyltransferase,TPMT)活性的HPLC测定法,为初步考察汉族健康人及自身免疫病患者TPMT活性分布状况及TPMT遗传多态性对硫唑嘌呤药动学影响提供方法学基础。方法以S腺苷L甲硫氨酸(SadenosyleLmethionine,SAM)作为甲基供体,6巯基嘌呤(6mercaptopurine,6MP)作为酶反应底物,TPMT催化6MP生成6甲基巯基嘌呤(6methylmercaptopurine,6MeMP),采用二氯甲烷异丙醇(80∶20)萃取6MeMP,常温下氮气吹干,120μL流动相复溶,10～50μL进样分析。色谱条件ShimpackCLCODS(6mm×150mm,5μm)柱;流动相甲醇水三乙胺(24∶756∶04),磷酸调至pH7,流速为10mL·min-1;紫外检测,λ=290nm。结果该方法在50～2000ng·mL-1内线性良好,相关系数r=09996,斜率变异系数RSD=473%(n=7);高、中、低浓度质控样本的批内、批间RSD在111%～429%之间,回收率在949%～1032%之间(n=7)。结论该方法灵敏、准确、重现性好,满足临床研究需要。

OBJECTIVE To develop a HPLC assay for the determination of thiopurine methyltransferase (TPMT) activity in red blood cells. METHODS Based on the TPMT-catalysed effection,6-MP was conversed to 6-methylmercaptopurine(6-MeMP) with S-adenosyle-L-methionine (SAM) as methyl donor. The 6-MeMP was extracted with dichloromethane-2-propanol (80/20). 6-MeMP was quantitated by the reversed-phase high performance liquid chromatography (HPLC) using CLC-ODS column (6 mm×150 mm,5 μm). The mobile phase consisted of methanol-water-triethylamine (24∶75.6∶0.4) and adjusted to pH 7 with orthophosphoric acid. The flow rate was at 1.0 mL·min^-1 and the detection wavelength was at 290 nm.RESULTS The linear concentration range was 50～2 000 ng·mL^-1 red blood cells lysate, with good corelation of coefficient 0.999 6.The coefficient of variation of slope was 4.73% (n=7). The RSDs of check samples were below 4.29% for inter-and intra-run validation. The recoveries were between 94.9% and 103.2% for low, middle and high concentration check samples, respectively (n=7).CONCLUSION The method was sensitive,accurate,precise and specific.It was suitable for the determination the TPMT activity in red blood cells and clinical study.