Quantitative analysis of plasma neutral glycosphingolipids by high performance liquid chromatography of their perbenzoyl derivatives.

A quantitative high performance liquid chromatography method for the analysis of neutral glycosylceramides as their perbenzoyl derivatives has been devised. Samples containing more than 2.5 nmol each of mono-, di-, tri-, and tetraglycosylceramide are benzoylated with 10% benzoyl chloride in pyridine at 37degrees C for 16 hr. The products are separated from excess reagents by solvent distribution and injected onto a pellicllar silica gel (Zipax) column (2.1 mm X 50 cm). The derivatives are eluted with a 10 min linear gradient of 2-17% ethyl acetate in hexane at 2 ml/min and absorbance at 280 nm is recorded. The detector response was proportional to the weight of sample used (2-30 nmol) and the lower limit of detection was about 70 pmol. The procedure has been applied to the quantitative analysis of erythrocyte and plasma glycolipids. As little as 0.5 ml of plasma can be used for analysis. The relative standard deviation of repetitive analyses ranged between 2.0% for glucosylceramide to 5.4% for galactosyllactosylceramide.