Improvement of a solid phase extraction method for separation of animal muscle phospholipid classes

Abstract

The solid phase extraction (SPE) method used by Banni et al. (2001) [Banni, S., Carta, G., Angioni, E., Muru, E., Scanu, P., Melis, M. P., et al. (2001). Distribution of conjugated linoleic acid and metabolites in different lipid fraction in the rat liver. Journal of Lipid Research, 42, 1056–1061] for fractionation of liver phospholipids (PLs) into phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) was modified to improve its separation efficiency. A mixture of PL standards, containing PC, PE, PS and PI, was separated into individual classes by using aminopropyl minicolumns, following the method of Banni et al. (2001). The obtained eluted fractions were further analysed by thin layer chromatography (TLC) and PL classes were identified. TLC revealed the co-elution of PC and PE, that of PE with PS and no elution of PI. The SPE method was subsequently modified in order to obtain a correct PL fractionation into PC, PE, PS and PI. The principal modifications consisted of increased solvent volumes for PC, PE and PS elution, and a different solvent mixture to allow PI elution. The effectiveness of the modified SPE method was checked by TLC, using both standards and muscle samples, showing a correct elution of PC, PE, PS and PI.