正相高效液相色谱检测人血清中生育酚异构体

目的:建立人血清中生育酚异构体检测方法。方法:取血清400μl,乙醇去蛋白后,正己烷-乙酸乙酯萃取,正己烷定容,进样50μl,以正己烷-叔丁基甲醚(93:7)为流动相,流速为1.5ml/min,在Inertsilsilica100-A,3μm,4.6mm×250mm硅胶柱中分离,激发波长295nm,发射波长330nm下检测定量。结果:四种生育酚异构体最低检测限分别为1.0、1.0、0.5、0.5ng/ml,在5ng/ml￣5μg/ml范围内线性良好,相关系数大于0.999,变异系数<5%,回收率在95%￣105%之间。结论:该方法分离度佳,定量准确,能满足人体血清生育酚检测需要,适于科研和临床应用。

ANALYSIS OF HUMAN SERUM TOCOPHEROL ISOMERS BY NORMAL-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC PROCEDURE

Objective: A high-performance liquid chromatographic (HPLC) procedure with a fluorescence detector was developed to rapidly separate α,β,γ,δ- tocopherol isomers in human serum Methods: The HPLC system consisted of Inertsil silica column (100-A, 3μm,4.6mm×250mm) and 7% (v/v) methyl-tert- butyl ether in n-hexane as mobile phase . Prior to HPLC, the serum sample wa deproteined by ethanol (BHT 0.0625%) and the tocopherol isomers were efficiently extracted in thei original isomeric conformations using n-hexane-ethyl acetate (5:1) in the presence of 2,6-bi-buty p-methylphenol (BHT). Result: The quantification limits, defined as the lowest quantitatively measurable concentration of the different compounds (ng/ml) are calculated according to the experiment:α-tocophero 1.0,β-tocopherol 1.0,γ-tocopherol 0.5,δ-tocopherol 0.5. The recovery rates are between 95%~105% Correlation coefficients are over 0.999 when the concentration is between 5 ng/ml~5 μg/ml. Conclusion This technique is suitable for assay of tocopherol isomers in human serum at all ages.