一种简易检测人红细胞中6-硫鸟嘌呤浓度的HPLC法

A Simple and Rapid HPLC Method for Determination of 6 - Thioguanine Concentration in Human Leukemic Cells

目的:建立一种简易的反相高效液相色谱法检测人红细胞中6-硫鸟嘌呤的浓度。方法:以焦性没食子酸为内标,用70％高氯酸沉淀红细胞裂解液中的蛋白质,上清液在100℃水浴45min后,用HPLC分析。色谱柱为Hypersil BDS C_(18),流动相为水-乙腈-三乙胺(磷酸调至pH3.2)(99:0.8:0.2),流速为1.0mL·min^(-1),紫外检测波长为340nm,进样量为20μL。结果:6-硫鸟嘌呤最低检测限为0.02 mg·L^(-1)(S/N=3),线性范围在0.025～1mg·L^(-1),标准曲线回归方程Y=0.1085X+0.004 11(r=0.9999),平均回收率为96.0％～105.5％。结论:本测定方法简便快速,可满足6-硫鸟嘌呤和6-骁基嘌呤药代动力学研究和临床监测的要求。

Objective: A simple and rapid HPLC method was developed for the determination of 6 - thioguanine (6 -TG) in human leukemic celis. Method: Protein in-erythrocyte sample was precipitated by perchloric acid. The 6 -TG nucleotides were hydrolyzed to their own bases by heating the sample for 45 min at 100×. The analy-sis involved a C18column as stationary phase and water - acetonitrile - triethylamine (99: 0. 8: 0. 2) , adjusted to pH 3. 2 with phosphoric acid, as mobile phase. The flow rate was 1. 0 mL ·min-1, UV detection wavelength was at 340nm, and injection volume was 20 μL. Pyrogallic acid was used as the internal standard. Results: The lim-it of detection was 0. 02 mg · L-1. The calibration curve was linear between 0. 025 - 1 mg · L -1 with a correlation coefficient of 0. 999 9. The recoveries of 6 - TG samples were 96. 0% - 105. 5% . Conclusion: The method is e-conomic, simple and rapid. It can be applied to pharmacokinetic study of 6 - TG.