Determination of lipids in animal tissues by high-performance thin-layer chromatography with densitometry

A high-performance thin-layer chromatographic method with densitometry has been developed, optimized, and used to quantify changes in renal and hepatic lipids after chemical insult. The lipids were extracted from tissues with 2:1 (v/v) chloroform-methanol, Petroleum ether (b.p. 40-60 degreesC)-diethyl ether-acetic acid, 80 + 20 + 3 (v/v), was used as mobile phase to separate neutral lipids (cholesterol, triacylglycerol, and cholesterol ester); oleic acid methyl ester was used as internal standard. Phospholipids (sphingomyelin, L-alpha -phosphatidylcholine, L-a-phosphatidylserine, L-alpha -phosphatidylinositol, cardiolipin, and L-a-phosphatidylethanolamine) were separated with methanol-chloroform-n-propanol-methyl acetate-43 mm KCI, 10 + 25 + 25 + 25 + 9 (v/v), as mobile phase and with galactosecerebrosides as internal standards. After chromatography bands were visualized by charring with manganese chloride-sulfuric acid at 110 degreesC. Quantification was performed by scanning densitometry and integration. Application of the optimized method was demonstrated by quantification of renal and hepatic lipids from rats treated with a nephrotoxin, The detection limit was 20 ng lipid and the sample throughput was 20-24 samples per plate.