Quantification in the subnanomolar range of phospholipids and neutral lipids by monodimensional thin-layer chromatography and image analysis

Abstract

A sensitive, rapid, and simple method is presented for the simultaneous quantitation of the major phospholipids and neutral lipids using an image analyzer after separation by one-dimensional thin-layer chromatography. A clear-cut separation of the lipids was achieved on one EDTA-impregnated chromatoplate with five step-wise developments. An image was acquired, and the integrated optical densities of the individual spots were quantitated by a camera-equipped image analyzer against an internal standard of cholesteryl formate. Calibration curves for each lipid followed linear or hyperbolic functions permitting quantification of as little as 0.015 nmol of lysophosphatidylcholine and as much as 7.50 nmol of triglyceride from a single application. Quantitation of the major lipids in human plasma by TLC and image analysis and standardized enzymatic methods were well correlated. The method is suitable for routine analysis of biological samples having lipid profiles as different as rat hepatocyte subcellular fractions, and very low density lipoproteins secreted by rat hepatocyte suspensions.