Determination of (13)C natural abundance of amino acid enantiomers in soil: methodological considerations and first results.

Abstract

The application of a combined gas chromatography-combustion/isotope ratio mass spectrometry (GC-C/IRMS) method for stable carbon isotope analysis of amino acid enantiomers in soil samples is presented. Triplicate delta(13)C analyses of pentafluoropropionyl (PFP) isopropyl ester derivatives of 27 amino acid enantiomers revealed that discrimination of (13)C during derivatization is different for different amino acid enantiomers and different amounts. Injection of increasing amounts of amino acid derivatives showed that the isotopic signal varied up to 10 per thousand for D-aspartic acid. Correction for the delta(13)C signal of underivatized amino acid enantiomers is possible for all investigated amino acid enantiomers using logarithmic functions. Operating the GC-C/IRMS system in the split-mode (split ratio 1:12) is possible but resulted in a higher isotopic discrimination. The detection limit approached 3 ng for some amino acid enantiomers in the splitless mode, while the lower limit of routine determination exceeded 10 ng injection amount. The upper limit at which accurate stable isotope values were obtained was 200 ng injection amount. Compound-specific delta(13)C analysis of alanine, valine, aspartic and glutamic acid showed that the D-forms were enriched in (13)C relative to the L-forms, suggesting that microbes significantly contributed to the formation of the D-enantiomers in soil.