反相高效液相色谱法同时测定血清中维生素A,D_3,E

Simultaneous determination of retinol,cholecalciferol and α-tocopherol concentration in human serum by RP-HPLC

目的建立RP-HPLC法测定血清中脂溶性维生素A,D3,E的含量.方法血清经甲醇沉淀蛋白后,以正己烷提取,经氮气吹干,甲醇定容后进样分析.色谱柱:Hypersil ODS 5μm,250×4.0mm;流动相:甲醇-异丙醇(95:5,v/v);流速:1mL/min;检测波长:维生素A 325nm,维生素D3265nm,维生素E 292nm;柱温:25℃.结果三种维生素的提取回收率均大于85%,方法回收率均大于90%;日内、日间相对标准偏差均小于7%.线性范围:维生素A 0.2～2.0μg/mL(r=0.9987),维生素D340～400ng/mL(r=0.9994),维生素E 4.0～30.0μg/mL(r=0.9988).结论本法操作快速、灵敏、简便易行.

OBJECTIVE To establish an RP-HPLC assay for the determination of retinol, cholecalciferol and a-tocopherol in human serum. METHODS The serum sample was extracted with n-hexane after being deproteinizated with methanol. The n-hexane solution was evaporated to dryness under a flow of nitrogen at room temperature. The residue was redissolved in methanol, and the solution was determined by RP-HPLC. Retinol,cholecalciferol and α-tocopherol was separated on Hypersil ODS 5μm,250 ×4.0mm column and detected at 325nm,265nm and 292nm by using methanol：isopropanol （95：5 ,v/v） as mobile phase when the column temperature was 25℃. RESULTS The ranges of calibration curves of retinol, cholecalciferol and α-tocopherol were 0.2～ 2.0μg/mL（ r = 0. 9987） ,40 ～ 400ng/mL （ r = 0. 9994） and 4.0 ～30.0μg/mL（ r = 0.9988 ） respectively. The extracted recovery of retinol, cholecalciferol and α- tocopherol were more than 85% ,and the method recovery were more than 90%. RSD of intra-day and inter-day were less than 7%. CONCLUSION The method is simple, rapid and sensitive.