Detection of acyl-coenzyme A thioester intermediates of fatty acid beta-oxidation as the N-acylglycines by negative-ion chemical ionization gas chromatography-mass spectrometry.

An analytical method for the separation and quantitation of acyl-CoA thioesters by gas chromatography-mass spectrometry is described. The method utilizes glycine aminolysis of the acyl-CoA thiolesters, esterification with pentafluorobenzyl bromide followed by gas chromatographic separation, and detection by negative chemical ionization mass spectrometry of the N-acylpentafluorobenzyl glycinates. The glycine aminolysis provides over 100-fold discrimination against oxygen esters and obviates the difficulty of removing trace contaminants of free fatty acids. The limit of detection of the described methodology for palmitoyl-CoA has been found to be 300 fmol, which improves at shorter chain lengths. Baseline separation was obtained for a standard mixture of seven acyl-CoAs (60 pmol injected) containing butyryl-CoA, hexanoyl-CoA, octanoyl-CoA, decanoyl-CoA, lauroyl-CoA, myristoyl-CoA, and palmitoyl-CoA. The above procedure is also applicable to the alpha-beta unsaturated and 3-hydroxyacyl-CoA derivatives, making it possible to quantify all of the intermediates in fatty acid oxidation, except the 3-ketoacyl-CoAs, in a single procedure.