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 β-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 chloroform-methanol (1:3) for HPLC assay. These five vitamins were successfully separated on the Hypersil ODS column (4 mm×250 mm) at a flow-rate 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 min. 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 β-Carotene.RESULTS The method was linear over the range of 0.1～1.2 mg·L^-1 for vitamin A and β-carotene, 20.0～240.0 μg·L^-1 for vitamin DsJO.O-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 these five 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.