Determination of the phospholipid composition of bovine muscle by high performance liquid chromatography with emphasis on the choline and ethanolamine plasmalogens

Abstract

Bovine muscle phospholipids contain large proportions of plasmalogens. A method for the separation of the plasmalogens from the corresponding diacyl compounds by high performance liquid chromatography (HPLC) has not been available although HPLC has many other advantages for lipid separations. The separation of phospholipid classes by HPLC was modified in order to separate hydrolysis products of plasmalogens from other phospholipids. The choline and ethanolamine glycerophospholipids were collected from the first HPLC separation, hydrolyzed at room temperature with 50 m m HCl in chloroform-methanol-water (50:47·5:2·5, by vol), and rechromatographed. In bovine semimembranosus muscle, the content of each phospholipid determined by phosphorus assay was 38·2, 21·4, 10·6, 17·9, 4·3, 2·4, and 5·2% for acid-stable choline glycerophospholipids, choline plasmalogens, acid-stable ethanolamine glycerophospholipids, ethanolamine plasmalogens, inositol glycerophospholipids, serine glycerophospholipids, and sphingomyelin, respectively. This HPLC method efficiently separated the phospholipid species, especially the lyso compounds corresponding to plasmalogens. The gradient of the solvent system composed of hexane-2-propanol (3:2 by vol), A, and hexane-2-propanol (3:2 by vol) with 5·5% water, B, was adjusted. For the gradient elution for the phospholipid class separation, the initial solvent ratio was 50% B, and B was increased to 100% at 10·5 min and decreased to 50% at 22 min, whereas, for resolution of lyso compounds from diacyl-glycero-3-phosphocholine and diacyl-glycero-3-phosphoethanolamine, the initial solvent ratio was 50% B, then 100% B at 17 min, and 50% B at 38 min. With HPLC separation of phospholipid classes and mild acid hydrolysis of choline and ethanolamine glycerophospholipids prior to rechromatography, it is possible to recover quantitatively all of the phospholipid components for any desired assay or determination of composition.