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目的建立一种测定1-氯-2,4-二硝基苯(CDNB)高效液相色谱分析方法,并以1-氯-2,4-二硝基苯为探针测定大鼠肝微粒体中谷胱甘肽硫转移酶(GST)活性并进行体外动力学分析。方法色谱条件:Welch Materials UltimateTMXB C18反相柱(4.6mm×250 mm,5μm),流动相:乙腈-水(7∶3),流速0.8 mL.min-1,柱温30℃,检测波长238 nm。实验方法:1-氯-2,4-二硝基苯与大鼠肝微粒体在37℃温孵7 min后,冰乙腈终止反应。反应液离心取上清液过滤后进行HPLC分析,通过Sigma Plot软件作图求算Vmax、Km及代谢清除率(CLint)值。结果 1-氯-2,4-二硝基苯的Rt=6.0 min,峰形良好,且无内源性干扰。最低检测限为1.0μmol.L-1,线性范围:2.5～100.0μmol.L-1。日内、日间精密度均小于10%。5 d内于室温及-20℃下较稳定。方法回收率为99.38%～108%。动力学分析表明,不同浓度1-氯-2,4-二硝基苯在0.02 mg.mL-1蛋白浓度下孵育7 min,测得动力学参数:Vmax为85.45 nmol.min-1.(mg protein)-1;Km为15.09μmol.L-1;CLlint为5.66 mL.min-1.(mg protein)-1。结论该方法稳定可靠,灵敏度高,能准确快速测定谷胱甘肽硫转移酶活性,可用于其体外动力学研究。
OBJECTIVE To establish a high performance liquid chromatographic method for the determination of 1-chloro-2,4-dinitrobenzene (CDNB) and to determine the rat liver microsomes with 1-chloro-2,4-dinitrobenzene Glutathione S-transferase (GST) activity and in vitro kinetic analysis. Methods The chromatographic conditions were as follows: Welch Materials UltimateTM XB C18 reversed-phase column (4.6 mm × 250 mm, 5 μm) with a mobile phase of acetonitrile-water (7: 3) at a flow rate of 0.8 mL · min- . Experimental Methods: After 1-chloro-2,4-dinitrobenzene was incubated with rat liver microsomes at 37 ℃ for 7 min, the reaction was stopped by ice-acetonitrile. The reaction mixture was centrifuged and the supernatant was filtered for HPLC analysis. Vmax, Km and CLint values were calculated by Sigma Plot software. Results The Rt of 1-chloro-2,4-dinitrobenzene was 6.0 min with a good peak shape and no endogenous interference. The minimum detection limit was 1.0μmol.L-1, linear range: 2.5 ~ 100.0μmol.L-1. Days, day precision are less than 10%. 5 d at room temperature and -20 ℃ more stable. The recovery rate was 99.38% ~ 108%. Kinetic analysis showed that the kinetic parameters were as follows: Vmax was 85.45 nmol.min-1 (mg / mL) when different concentrations of 1-chloro-2,4-dinitrobenzene were incubated for 7 min at a concentration of 0.02 mg.mL- protein) -1; Km was 15.09μmol.L-1; CLlint was 5.66mL.min-1. (mg protein) -1. Conclusion The method is stable, reliable and sensitive. It can accurately and rapidly determine the activity of glutathione S-transferase and can be used for in vitro kinetics study.