Cullum M E, Zile M H
Anal Biochem. 1986 Feb 15;153(1):23-32. doi: 10.1016/0003-2697(86)90055-2.
A single method is described for quantitation of 14 retinoids found in biological material. The method consists of reversed-phase HPLC, internal standardization, and carrier extraction procedures with three synthetic retinoids. Primary standardization of HPLC uv detector is achieved using tritiated all-trans-retinoic acid, all-trans-retinol, all-trans-retinyl palmitate, and all-trans-retinyl acetate. Extraction methods are standardized by correlating the uv absorbance of retinoids at 340 nm with radioactivity of tritiated retinoids of known specific activity. Quantitation of 10 pg of tritiated or 5 ng of nonradioactive retinoid per 0.1 g sample in a polarity range from 4-oxo-retinoic acid to retinyl stearate can be achieved in a single, 50-min chromatographic run. A single HPLC pump, a C18 reversed-phase analytical column, a multistep three-solvent gradient, and inexpensive solvents based on methanol, water, and chloroform comprise this cost-effective chromatographic system. Our primary standardization method allows investigators employing different procedures to compare results between laboratories by standardizing the HPLC uv detector with commercially available tritiated retinoids. With this method we were able to quantitate nanomolar amounts of endogenous retinoic acids and retinyl esters, that "HPLC uv only" conditions usually would not detect in the circulation and liver of rats under physiological conditions.
本文描述了一种用于定量生物材料中14种类视黄醇的方法。该方法包括反相高效液相色谱法、内标法以及使用三种合成类视黄醇的载体萃取程序。高效液相色谱紫外检测器的初步标准化是通过使用氚标记的全反式视黄酸、全反式视黄醇、全反式视黄醇棕榈酸酯和全反式视黄醇乙酸酯来实现的。萃取方法通过将类视黄醇在340 nm处的紫外吸光度与已知比活度的氚标记类视黄醇的放射性相关联来进行标准化。在单次50分钟的色谱运行中，每0.1 g样品中可实现对10 pg氚标记或5 ng非放射性类视黄醇的定量，其极性范围从4-氧代视黄酸到视黄醇硬脂酸酯。该经济高效的色谱系统由一个高效液相色谱泵、一根C18反相分析柱、一个多步三溶剂梯度以及基于甲醇、水和氯仿的廉价溶剂组成。我们的初步标准化方法允许采用不同程序的研究人员通过使用市售的氚标记类视黄醇对高效液相色谱紫外检测器进行标准化，从而在不同实验室之间比较结果。通过这种方法，我们能够定量纳摩尔量的内源性视黄酸和视黄醇酯，而在生理条件下，“仅高效液相色谱紫外检测”条件通常无法在大鼠的循环系统和肝脏中检测到这些物质。
