Fluorescent assay of total serum cholesterol, with use of gas-liquid chromatography to study saponification efficiency

We describe a fluorescent determination of total cholesterol in serum for which the accuracy and precision are comparable to that for the method of Abell-Kendall, a method of generally accepted accuracy. By the use of quality reagents and the rigorous exclusion of water, the strong fluorophor that develops on reacting concentrated sulfuric acid with cholesterol can be used to quantitatively determine the total cholesterol in serum. We used gas-liquid chromatography to monitor the extent of saponification of the cholesterol esters, because we found them to have fluorescent efficiencies that differed from that of free cholesterol. Sodium methoxide in methanol/methylene chloride (1/3 by vol) was shown by gas-liquid chromatography to very effectively saponify the cholesterol esters in serum.