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

Abstract

El método de extracción en fase sólida (SPE) utilizado por 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] para el fraccionamiento de fosfolípidos hepáticos (PL) en fosfatidilcolina (PC), fosfatidiletanolamina (PE), fosfatidilserina (PS) y fosfatidilinositol (PI) se modificó para mejorar su eficacia de separación. Una mezcla de estándares de PL, que contenía PC, PE, PS y PI, se separó en clases individuales utilizando minicolumnas de aminopropilo, siguiendo el método de Banni et al. (2001). Las fracciones eluidas obtenidas se analizaron posteriormente mediante cromatografía en capa fina (TLC) y se identificaron las clases de PL. La TLC reveló la co-elución de PC y PE, la de PE con PS y la no elución de PI. El método SPE se modificó posteriormente para obtener un fraccionamiento correcto de los PL en PC, PE, PS y PI. Las principales modificaciones consistieron en el aumento de los volúmenes de disolvente para la elución de PC, PE y PS, y una mezcla de disolventes diferente para permitir la elución de PI. La eficacia del método SPE modificado se comprobó mediante TLC, utilizando tanto patrones como muestras de músculo, mostrando una elución correcta de PC, PE, PS y PI.

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.