高效液相色谱法同时测定微量全血中维生素A和E  

SIMULTANEOUS DETERMINATION OF VITAMIN A AND E IN MICRO AMOUNT OF HUMAN WHOLE BLOOD BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

作　　者：胡燕[1] 王希希[1] 孙成均[1] 

机构地区：[1]四川大学华西公共卫生学院,成都610041

出　　处：《现代预防医学》2009年第1期120-121,124,共3页Modern Preventive Medicine

摘　　要：[目的]建立反相高效液相色谱法(RP-HPLC)同时测定微量全血样品中维生素A和E的方法。[方法]取全血50μl,用无水乙醇沉淀蛋白,再加入正己烷漩涡提取维生素A和E,取一定量正己烷层经氮气流挥干,残渣加甲醇溶解后用高效液相色谱法分析,标准曲线法定量。对血样的提取条件和色谱柱选择、流动相比例、流速、柱温等实验条件进行了优化,确定了最佳分析条件。本法色谱分离条件为:分离柱为ZorbaxC8色谱柱(4.6mm×15cm,5μm);流动相为甲醇-水(98+2);流速为0.70ml/min;柱温为50℃;紫外检测波长:维生素A为325nm,维生素E为292nm。[结果]维生素A和E标准曲线的相关系数均大于0.999;相对标准偏差(RSD)﹤5%。对于50μl全血,本法的检出限维生素A为0.02μg/ml;维生素E为0.08μg/ml。维生素A和E的加标回收率分别为93.2%～104%和80.4%～84.9%。[结论]所建立的方法快速、简便、灵敏、准确,已成功地测定了微量全血中维生素A和E。[Objective] To establish a method to determine vitamin A and vitamin E in the microamount of human blood by high performance liquid chromatography simultaneously. [ Methods ] Fifty microliter of human blood was added to a 1.5 ml Eppendorf centrifuge tube and then 200 μl of anhydrous alcohol was added to precipitate protein, the mixture was vortex shaken and then vitamin A and E were extracted with 1 000μl of hexane. 800μl of the hexane was taken into another Eppendorf centrifuge tube and blown to dryness with gentle N2 gas. The residue was redissolved with 200 μl of methanol and subjected to injection into HPLC for analysis. The optimized chromatographic conditions were as follows： analytical column： Cs （Zorbax, 4.6 mm×15cm, 5μm）; mobile phase： methanol-water （98±2）; flow rate： 0.70ml/min; column temperature： 50℃; detection wavelength： 325 nm for vitamin A and 292nm for vitamin E. [ Results ] The range of correlation coefficients for the standard curves was bigger than 0：999 ; the detection limit of the method was 0.02μg/ml of vitamin A and 0.08μg/ml of vitamin E .The recoveries of vitamins A and E in human blood ranged from 93.2% to 104% and 80.4% to 84.9% respectively. [Conclusion] The method is sensitive, accurate and has been applied to the determination of vitamin A and E in the micro amount of human whole blood.

关 键 词：维生素A 维生素E 高效液相色谱法 全血 

分 类 号：R446.11[医药卫生—诊断学]