Chromatographic analysis of endogenous retinoids in tissues and serum
Chromatographic analysis of endogenous retinoids in tissues and serum
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DOI：
10.1016/s0003-2697(02)00662-0
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发表时间：
2003-04-01
影响因子：
2.9
通讯作者：
Nau, H
中科院分区：
文献类型：
--
作者：
Schmidt, CK;Brouwer, A;Nau, H
我们提出了一种可靠、高灵敏度和通用的方法，用于同时测定各种生物基质中内源性极性（酸性）和极性（视黄醇、视网膜和视黄醇酯）类维生素a。用异丙醇从组织或血浆中单次液体提取类维生素a后，通过使用氨基丙基相的自动固相萃取将极性类维生素a与极性类维生素a和中性脂分离。在真空浓缩至干燥并在适当的溶剂中重组后，得到的馏分被注射到两种不同的高效液相色谱（HPLC）系统中。极性类维生素a在RP18色谱柱（2.1 min ID）上进行分析，使用由甲醇和水组成的缓冲梯度和柱上聚焦的大体积进样。用由乙腈、氯仿和甲醇组成的非水梯度在正孔RP18柱上分离极性类维生素a。两种高效液相色谱系统都与紫外检测相结合，类维生素a根据适当的内部标准进行定量。对该方法的回收率、精密度、鲁棒性、选择性和分析物稳定性进行了验证。用400多份血清和200 mg组织，全反式维甲酸的检出限分别为0.15 ng/ml和0.3 ng/g。视黄醇对应值分别为1.2 ng/ml和2.4 ng/g。该方法成功地应用于小鼠、大鼠和人体组织和血清样品。(C) 2003 Elsevier Science（美国）版权所有。
We present a reliable, highly sensitive, and versatile method for the simultaneous determination of endogenous polar (acidic) and apolar (retinol, retinal, and retinyl esters) retinoids in various biological matrices. Following a single liquid extraction of retinoids from tissues or plasma with isopropanol, polar retinoids are separated from apolar retinoids and neutral lipids via automated solid-phase extraction using an aminopropyl phase. After vacuum concentration to dryness and reconstitution of the residue in appropriate solvents, the obtained fractions are injected onto two different high-performance liquid chromatography (HPLC)-systems. Polar retinoids are analyzed on a RP18 column (2.1 min ID) using a buffered gradient composed of methanol and water and on-column-focusing large-volume injection. Apolar retinoids are separated on a normal-bore RP18 column using a nonaqueous gradient composed of acetonitrile, chloroform, and methanol. Both HPLC systems are coupled with UV detection, and retinoids are quantitated against appropriate internal standards. The method was validated with regard to recovery, precision, robustness, selectivity, and analyte stability. Using 400 mul serum or 200 mg tissue, the limits of detection for all-trans-retinoic acid were 0.15 ng/ml or 0.3 ng/g, respectively. The corresponding values for retinol were 1.2 ng/ml or 2.4 ng/g, respectively. This method was successfully applied to mouse, rat, and human tissue and serum samples. (C) 2003 Elsevier Science (USA). All rights reserved.