高效液相色谱法检测体外酶促反应体系中底物3-脱氧葡萄糖醛酮  

Determination of 3-deoxyglucosone as a substrate in enzymic reaction in vitro by HPLC

作　　者：唐文敏[1] 江国荣[2] 张露蓉[2] 

机构地区：[1]南京中医药大学,江苏南京210029 [2]南京中医药大学附属苏州中医院,苏州中医药研究所,江苏苏州215003

出　　处：《中国医院药学杂志》2009年第15期1297-1299,共3页Chinese Journal of Hospital Pharmacy

基　　金：江苏省中医药管理局科研课题资助项目(编号:2003-2004);苏州市高层次人才资助项目(编号:2004-33)

摘　　要：目的:建立体外酶促反应体系中底物3-脱氧葡萄糖醛酮(3-deoxyglucosone,3-DG)的高效液相色谱检测方法。方法:应用2,3-二氨基萘柱前衍生,采用C18色谱柱(4.6mm×250mm,5μm)为固定相,50mmol.L-1磷酸盐缓冲液(pH7.4)-甲醇-乙腈(70∶5∶25)为流动相,检测波长为Ex/Em=271/503nm的条件,测定体外酶促反应体系中底物3-DG的含量。结果:3-DG峰呈尖峰,分离较好,理论塔板数为6459;在1～5mg·mL-1范围内线性关系良好(Y=332.40X+34.50,r=0.9992);平均加样回收率为77.17%,RSD为8.98%(n=5)。结论:该方法可初步应用于体外酶促反应体系中底物3-DG的含量测定,并以3-DG的消耗量来反映体系中酶的活性。OBJECTIVE To establish a method for the determination of the 3-deoxyglucosone（3-DG） in the system of 3-deoxyglucosone reductase enzymic reaction in vitro by HPLC. METHODS The determination was carried out on a C18 （4. 6 minx 250 mm, 5 μm） column by pre derivation with Na2 HPO4·12H2 O-Nail2 PO4·2H2O buffer solution（pH 7. 4）-methanol-methanol （70：5：25） as the mobile phase, the flow-rate was 1 mL. rain-1 and detected at the fluorescence excitation/emission wavelengths of 271/503 nm. RESULTS 3-BC- was well resolved（ the number of theoretical plates was 6 459）. The calibration curve was in good linearity over the rang of 1-5 m·g·L^-1 for 3-DG. The equation was Y= 332. 40X＋ 34. 50（r= 0. 999 2）. The average recovery of 3-DG were 77. 17%（RSD= 8. 98%,n = 5）. CONCLUSION The method could be used for the determination of the 3-DG and it can be used to reflex the enzyme activity by detecting the 3-DG as a substrate in the system of 3-DG reductase enzymic reaction in vitro.

关 键 词：高效液相色谱法 酶促反应 3-脱氧葡萄糖醛酮 

分 类 号：R927.2[医药卫生—药学]