Analysis of Lipid Classes by HPLC with the Evaporative Light Scattering Detector

Abstract

The evaporative light scattering detector enables the detection and quantitation of all relatively non-volatile lipids. The mixtures of polar and non-polar lipids were separated in one run, in 20 to 25 minutes on Silica Si-100 columns, using consecutive gradients of pentane to diethylether, to chloroform, to methanol containing a large concentration of ammonia.

The flexibility of the method is illustrated by the change in elution patterns following the treatment of the packing material by ammonia. For example, the elution order of phosphatidyl inositol and phosphatidyl choline is reversed and the separation of the former compound from phosphatidyl serine, which is generally difficult, is now accomplished readily.

The weak dependence of the detector sensitivity on the nature of the analytes permits an easy quantitation, as illustrated by the results of the analyses of lipid classes in blood serum, amniotic fluid, beef brain and other natural samples.

The method is particularly useful for the analysis of lecithin and sphingomyelin in the amniotic fluid. The ratio of the concentration of these two compounds is an indicator of lung maturity and could permit an early diagnosis of the respiratory stress syndrome of neonates.