Determination of the enantiomeric purity of synthetic peptides by gas chromatography-mass spectrometry

Abstract

Hydrolysis using 2H-labelled HCl and H2O, derivatization of free amino acids as N.O.S-trifluoroacetyl isobutyl esters, separation by gas chromatography on a chiral stationary phase and detection by mass spectrometry in selected-ion monitoring mode have been used in order to determine the enantiomeric purity of several synthetic peptides. Chromatographic separation has been optimized for proline, whose two enantiomers are difficult to resolve under standard conditions. Electron impact and methane chemical ionization mass spectra and chromatographic resolution of unnatural amino acids, such as 3-(1-naphthyl)-alanine and p-chlorophenylalanine, are reported. For both natural and unnatural amino acids selected-ion monitoring of the different fragmentation peaks has been carried out. The results are interpreted from the point of view of whether or not the fragments contain a hydrogen atom on the alpha-carbon, and a comparison between electron impact and methane chemical ionization has been carried out. The main advantage of the latter method is that a quasimolecular ion can be observed for all the amino acids studied.