Simplified derivatization for determining sphingolipid fatty acyl composition by gas chromatography-mass spectrometry

Abstract

A simple procedure for simultaneously derivatizing non-hydroxy and hydroxy fatty acids prior to GC analysis [I. Ciucanu and F. Kerek, J. Chromatogr., 284 (1984) 179] has been evaluated for its usefulness in determining sphingolipid acyl composition. The method uses methyl iodide in polar aprotic solvents to generate methyl esters of carboxyl groups and methyl ethers of hydroxyl groups. Methylation efficiency is examined as a function of hydroxyl group presence and location in free fatty acids as well as a function of 2-hydroxy fatty acid chain length. Conditions are also reported for efficient saponification and derivatization of sphingolipid fatty acyl chains as is illustrated using bovine brain galactosylceramide.

Fig. 1

Capillary GC profile of derivatized FAMEs from bovine brain GalCer. A FFAP-fused silica capillary column (Quadrex) was used as described in the Methods section.

Fig. 2

Typical fragmentation patterns of non-hydroxy and 2-methoxy fatty acid methyl esters obtained by mass spectral analysis. (A) Tetracosanoic (24:0) methyl ester (mol. wt. 382); (B) 2-methoxy-tetracosanoic methyl ester (mol. wt. 412).

Fig. 3

Fatty acyl composition of bovine brain GalCer. Solid bars represent FAMEs derived from Avanti’s bovine brain GalCer. Cross-hatched bars represent FAMEs derived from Sigma’s Type I and Type II bovine brain GalCer (see Methods). Data are % (w/w). A FFAP–fused-silica capillary column was used as described in the Methods section.