Preparation of sphingolipid fatty acid methyl esters for determination by gas-liquid chromatography

Sphingolipid fatty acids are first converted to a mixture of free acids and their n-butyl esters by heating the specimen at 85 degree C in aqueous butanolic hydrogen chloride; the butyl esters are then saponified with methanolic potassium hydroxide. After acidification and extraction into hexane, the fatty acids are extracted into a very small volume of aqueous trimethyl(m-trifluorotolyl)ammonium hydroxide (TMTFTH), injection of an aliquot of the TMTFTH extract into the gas chromatograph yields the fatty acid methyl esters by pyrolytic methylation of the quaternary ammonium salts of the fatty acids. The preparation of a specimen ready for the gas--liquid chromatographic (GLC) analysis with quantitative recovery of the sphingolipid fatty acids can be accomplished in less than 2 h. By comparison, none of a number of well-accepted techniques for the release of sphingomyelin fatty acids by hydrolysis or methanolysis released the fatty acids quantitatively in less than 3 h, and all required additional manipulations before GLC analysis.