GC/EI-MS Analysis of Amino Acids Derivatized with Pentafluorobenzyl Bromide

The determination of amino acids by gas chromatography-mass spectrometry (GC-MS) using a one-step derivatization with pentafluorobenzyl bromide (PFB-Br) in an aqueous acetone phase is described. Although GC-MS analysis of amino acids by pentafluorobenzyl (PFB) derivatization has been reported previously, its application to non-proteinogenic free amino acids, which play important biological roles in various metabolic pathways, has not been reported. In this study, 6 non-proteinogenic amino acids (sarcosine, 2-aminobutyric acid, 3-aminoisobutyric acid, /3-alanine, 2-aminoadipic acid, and cystine) as well as 19 proteinogenic amino acids, with the exception of arginine, were PFB derivatized and analyzed by GC-MS using both electron ionization (EI) and positive-ion chemical ionization (PICI) modes. Two derivative peaks were observed for each amino acid with primary amino groups, which were identified as di-and/or tri-substituted derivatives (i.e., NH(PFB)-CHR-COO(PFB) or N(PFB)(2)- CHR-COO(PFB)) based on their EI-and PICI-mass spectra. The ratio of the tri-substituted to the sum of the di-and tri-substituted derivative peaks was highest in glycine and /3-alanine and lowest in branched amino acids, suggesting a dependence on steric hindrance around amino groups. Based on the identification of PFB derivative peaks, their retention indices on (5 %-phenyl)-methylpolysiloxane liquid phase capillary column, target and qualifier ions for selected ion monitoring (SIM) analysis on GC-EI-MS were determined to compile a quantification database.