STABLE CARBON ISOTOPE ANALYSIS OF AMINO-ACID ENANTIOMERS BY CONVENTIONAL ISOTOPE RATIO MASS-SPECTROMETRY AND COMBINED GAS-CHROMATOGRAPHY ISOTOPE RATIO MASS-SPECTROMETRY

The application of a combined gas chromatography/isotope ratio mass spectrometry (GC/IRMS) method for stable carbon isotope analysis of amino acid enantiomers is presented. This method eliminates the numerous preparative steps integral to the isolation of amino acids and amino acid enantiomers from protein hydrolyzates that precede delta-C-13 analysis by conventional isotope ratio mass spectrometry. Unlike hydrocarbons, amino acids require derivatization prior to GC/IRMS analysis. Replicate delta-C-13 analyses of trifluoroacetyl (TFA) isopropyl ester derivatives of 22 amino acids by IRMS revealed that the derivatization process is reproducible, with an average error (1 standard deviation) of 0.10% +/- 0.09%. The average analytical error for analysis of amino acid derivatives by GC/IRMS was 0.26% +/- 0.09%. In general, absolute differences between IRMS and GC/IRMS analyses were less than 0.5%. The derivatization process introduces a distinct, reproducible isotopic fractionation that is constant for each amino acid type. The observed fractionations preclude direct calculation of underivatized amino acid delta-C-13 values from their respective TFA isopropyl ester delta-C-13 compositions through mass balance relationships. Derivatization of amino acid standards of known stable carbon isotope compositions in conjunction with natural samples, however, permits computation of the original, underivatized amino acid delta-C-13 values through use of an empirical correction for the carbon introduced during the derivatization process.