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 hepati c lipids after chemical insult. The lipids were extracted from tissues with 2:1 (v/v) chloroform-methanol, Petroleum ether (b.p. 40-60 degreesC)-dieth yl ether-acetic acid, 80 + 20 + 3 (v/v), was used as mobile phase to separa te neutral lipids (cholesterol, triacylglycerol, and cholesterol ester); ol eic acid methyl ester was used as internal standard. Phospholipids (sphingo myelin, L-alpha -phosphatidylcholine, L-a-phosphatidylserine, L-alpha -phos phatidylinositol, cardiolipin, and L-a-phosphatidylethanolamine) were separ ated with methanol-chloroform-n-propanol-methyl acetate-43 mm KCI, 10 + 25 + 25 + 25 + 9 (v/v), as mobile phase and with galactosecerebrosides as inte rnal standards. After chromatography bands were visualized by charring with manganese chlor ide-sulfuric acid at 110 degreesC. Quantification was performed by scanning densitometry and integration. Application of the optimized method was demo nstrated by quantification of renal and hepatic lipids from rats treated wi th a nephrotoxin, The detection limit was 20 ng lipid and the sample throug hput was 20-24 samples per plate.