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-acyl-pentafluorobenzyl 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 α-β 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. © 1992.