ANALYSIS OF IN-VIVO LEVELS OF ACYL-THIOESTERS WITH GAS-CHROMATOGRAPHYMASS-SPECTROMETRY OF THE BUTYLAMIDE DERIVATIVE

The analysis of the in vivo level and composition of acyl-thioester po ols, such as acyl-ACP, has been accomplished by selective formation of acyl-butylamides and subsequent analysis by gas chromatography/mass s pectrometry. The acyl-butylamide derivative was synthesized by direct aminolysis with n-butylamine. The reaction was specific for thioester- linked acyl groups and 90% conversion was achieved with acyl-ACP and a cyl-CoA in aqueous solution. Electron ionization mass spectra exhibite d two intense diagnostic ions mit 115 and 128 common to butylamides of saturated and monounsaturated fatty acids. Mixtures of butylamides wi th fatty acid moieties ranging between C-4 and C-20 were analyzed by s elective ion monitoring gas chromatography/mass spectrometry set to 11 5 and 128 amu. The limit for the quantitative analysis of the long-cha in 18:0- and 18:1-butylamides was 1.5 pmol and the detection limit was less than 0.5 pmol. The utility of this method was demonstrated by an alysis of two model systems: standard acyl-CoA mixtures and in vivo le vels of spinach leaf acyl-ACP. A purification protocol based on DE 52 anion exchange chromatography was necessary in order to separate spina ch acyl-ACP and acyl-CoA from tissue extracts. The acyl composition ob tained from total spinach leaf acyl-ACP by selective ion monitoring ga s chromatography/mass spectrometry of the butylamide derivatives match ed with the direct analysis of the same sample by urea-polyacrylamide gel electrophoresis and subsequent scanning densitometry of the anti-s pinach ACP immunoblot. (C) 1995 Academic Press, Inc.