Stable Nitrogen Isotope Analysis of Amino Acid Enantiomers by Gas Chromatography/Combustion/Isotope Ratio Mass Spectrometry

Abstract

The analysis of the stable nitrogen isotope compositions of individual amino acid stereoisomers through the use of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is presented. Nitrogen isotopic compositions of single amino acids or of their enantiomers is possible without the labor-intensive and time-consuming preparative-scale chromatographic procedures required for conventional stable isotope analysis. Following hydrolysis and derivatization, single-component isotope analysis is accomplished on nanomole quantities of each of the stereoisomers of an amino acid, utilizing the effluent stream of gas chromatographic separation. Nitrogen isotope fractionation is minimal during acylation of the amino acid, with no additional nitrogen being added stoichiometrically to the derivative. Thus, the isotopic composition of the nitrogen in the derivative is that of the original compound. Replicate stable nitrogen isotope analyses of 11 amino acids, and their trifluoroacetyl (TFA)/isopropyl (IP) ester derivatives, determined by both conventional isotope ratio mass spectrometry (IRMS) and GC/C/IRMS, indicate that the GC procedure is highly reproducible (standard deviations typically 0.3-0.4‰) and that isotopic differences between the amino acid and its TFA/IP derivative are, in general, less than 0.5‰.

Isotope‐ratio detection for gas chromatography

“…Nitrogen isotopic analysis is also based on combustion of analytes, followed obligately by reduction of NO X to N 2 [61][62][63]. Conveniently, the conversion can be accomplished using the same combination of combustion/ reduction reactors that are used for carbon (above) so that both types of analyses can be performed using the same interface.…”

“…An additional problem for N-isotopic analyses is that CO 2 entering the IRMS produces significant ion-currents at m/z 28 and 29 due to CO + fragments. CO 2 must therefore be removed from the carrier gas stream, generally by cryogenic trapping [26,61,63].…”

Sources, Fate and Transformation of Organic Matter in Wetlands and Estuaries

“…Gas chromatography and mass spectrometry (GC-MS) determination on the silylated phenolic compound was proved to be selective and sensitive for detection of gallic acid (Tor et al, 1996). In addition, GC-MS analysis of derivatized amino acid has been proven feasible in biological samples (Macko et al, 1997;Kaspar et al, 2008). However, while amino acid analyses in DOM samples have been commonly performed (e.g.…”

“…This amino acid derivatization method was originally used for stable nitrogen isotope analysis of amino acid by Macko et al, (1997) and then applied for amino acid analysis in DOM samples (Yamashita and Tanoue 2003;Jaffé et al, 2012). Two-hundred microliter aliquots of a 0.05 M solution of each amino acid (50 µmol) were placed in a 3 ml screw-cap vial and evaporated to dryness at 40 ⁰C under a stream of N 2 .…”

“…In the present study, I compared the concentration, fluorescence intensity, and fluorescence yield efficiency of phenolic acid vs. amino acid in order to gain a better idea of the strengths of these constituents in the protein-like fluorophores. We developed an analytical method using GC-MS based on tannin hydrolysis (Tor et al, 1996), protein hydrolysis (Keil and Kirchman, 1991;Yamashita and Tanoue, 2003;Jaffé et al, 2012) followed by different derivatization methods for gallic acid analysis (Tor et al,1996;An et al,2013) and tyrosine analysis (Macko et al,1997). This is a preliminary study on the chemical characterization of OM fluorophores, but through which we want to raise attention on the need for a critical evaluation of the traditionally defined EEM-PARAFAC components.…”