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目的:建立一种快速、灵敏、高效的方法,检测儿童急性淋巴细胞白血病红细胞中硫鸟嘌呤核苷酸浓度。方法:应用反相高效液相色谱(RT-HPLC)技术,测定急性淋巴细胞白血病患儿红细胞内6-巯嘌呤(6-MP)及其代谢产物[硫鸟嘌呤核苷酸、6-硫代黄嘌呤(6-TX)、6-甲基巯基嘌呤(6-MMP)]浓度。色谱柱为Resolve C18,柱温40℃。流动相A为0.2%的乙酸溶液,流动相B为甲醇。梯度洗脱程序为在16min内流动相B的浓度从0上升到80%。流速1.0mL/min。检测波长为0～12.5min用290nm检测6-硫鸟嘌呤(6-TG)、6-MP和6-TX,12.5min时切换为340nm检测6-MMP。结果:进样体积为10μL时,6-TG和6-TX的线性范围为0～20nmol/mL(r=0.9993,r=0.9980)。硫鸟嘌呤核苷酸浓度与测定后第9天的白细胞计数呈负相关(P<0.01)。结论:RT-HPLC法简便、准确检测6-MP代谢物浓度,有助于个体化的治疗。
Objective: To establish a rapid, sensitive and efficient method for the determination of thioguanine nucleotide in erythrocytes of children with acute lymphoblastic leukemia. Methods: The contents of 6-mercaptopurine (6-MP) and its metabolite [thioguanine nucleotide, 6-thio-substituted nucleoside] in erythrocytes of children with acute lymphoblastic leukemia were determined by reverse-phase high performance liquid chromatography Xanthine (6-TX), 6-methylmercaptopurine (6-MMP)] concentrations. The column was Resolve C18 and the column temperature was 40 ℃. Mobile phase A was a 0.2% acetic acid solution and mobile phase B was methanol. The gradient elution program was such that the mobile phase B concentration rose from 0 to 80% within 16 min. Flow rate 1.0 mL / min. Detection of 6-thioguanine (6-TG), 6-MP and 6-TX at 290 nm with a detection wavelength of 0-12.5 min and detection of 6-MMP by switching to 340 nm at 12.5 min. Results: The linear range of 6-TG and 6-TX was 0 ~ 20nmol / mL (r = 0.9993, r = 0.9980) when the injection volume was 10μL. The thioguanine nucleotide concentration was negatively correlated with the white blood cell count at day 9 after the assay (P <0.01). Conclusion: RT-HPLC method is simple and accurate to detect 6-MP metabolite concentration, which is helpful for individualized treatment.