高效液相色谱法测定人血中脂溶性维生素的含量

Simultaneous Determination of Fat-Soluble Vitamins A, D3, 25-OH-D3, E and β-Carotene in Human Serum by HPLC

目的建立同时测定人血中5种脂溶性维生素的高效液相色谱法。方法取血清0．5mL,甲醇蛋白沉淀后用正己烷、乙醚分2次提取,提取液氮气吹干,用甲醇-氯仿(3:1)液定容进样。色谱柱:Hypersil ODS(4．0 mm×250 mm);流动相:0-4．0min 甲醇-水(90:10),4．5-9 min甲醇-异丙醇(95:5),10-20 min甲醇-异丙醇(60:40);检测波长:维生素A 325 nm,25-羟基维生素 D3和维生素D3 265 nm,维生素E 290 nm,β-胡萝卜素450 nm;流速:1．0 mL·min-1;柱温:30℃。结果维生素A和β-胡萝卜素在0．1-1．2 mg·L-1,25-羟基维生素D3在10．0-120．0 μg·L-1,维生素D3在20-240μg·L-1,维生素E在2．0-24．0 mg·L-1内线性良好;提取回收率均大于69％,方法回收率均大于92％,日内、日间RSD均小于6％;以本法测定了50例体检儿童血清中5 种脂溶性维生素的浓度。结论本方法快速、灵敏,操作简便易行。

OBJECTIVE To establish a HPLC method for the simultaneous determination of fat-soluble vitamins A, D3, 25-OH-D3, E and ,8-carotene in human serum. METHODS Following liquid-phase extraction with n-hexane and ether from 0.5 mL human serum samples after being deproteinizated with methanol, the extractions were evaporated to dry under a flow of nitrogen and the residues were dissolved in chlomform-methanol （ 1：3） for HPLC assay. These five vitamins were successfully separated on the Hypersil ODS column （4 mm×250 mm） at a flow-rote of 1.0 mL·min^-1, with the mobile phases of methanol-water （90： 10） at 0-4 min, methanol-isopropanol （95：5） at 4.5-9 min and methanol-isopropanol （60：40） at 10-20 rain. The column temperature was set at 30℃. The wavelengths for detection were 325 nm for vitamin A, 265 nm for vitamin D and 25-OH-D3, 290 nm for vitamin E and 450 nm for p-Carotene. RESULTS The method was linear over the range of 0.1-1.2 mg·L-1 for vitamin A and ,β-carotene,20.0-240.0μg·L^-1 for vitamin D3, 10.0-120μg·L^-1 for vitamin 25-OH-D3. and 2.0-24.0 mg·L^-1 for vitamin E. The extraction recoveries were greater than 69% and the method recoveries were greater than 92 % for thesefive vitamins. The RSDs of intra-day and inter-day were less than 6% . Fifty samples of children had been determined with this method. CONCLUSION A simple, rapid and sensitive method is developed for determining simultaneously the concentration of vitamin A, D3, 25-OH-D3, E and ,β-carotene in human serum.