Development of N-acetyl methyl ester derivatives for the determination of δ13C values of amino acids using gas chromatography-combustion-isotope ratio mass spectrometry

A novel derivatization procedure, N-acetyl methyl (NAG ME) esterification, was developed to improve the accuracy and precision of amino acid delta(13)C value determination using gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS). Standard mixtures of 15 protein amino acids were converted to NACME and N-acetyl-isopropyl (NAIP) esters; the latter established derivative was employed for comparison purposes. Both procedures yielded baseline-resolved peaks for all 15 amino acids when GC columns coated with polar stationary phases were employed. For NACME esters, the methylation conditions governed reaction yields, with highest yields observed when a I h, 70 degrees C methylation procedure (anhydrous MeOH/acetyl chloride, 25:4, v/v) was performed. The mean derivatization yields expressed relative to an underivatized coinjected standard (n-nonadecane) for both NACME and NAIP esters were identical. likewise, the mean kinetic isotope effects (KIEs) were not significantly different (KIE(NACME) = 1.036; KIE(NAIP) = 1.038) and were shown in both cases to be reproducible. The mean reproducibility obtained from 15 replicates (3 x batches of 5) of both derivatives was strong (mean STDV(NACME) = 0.3 parts per thousand and STDV(NAIP) = 0.4 parts per thousand). The isotopic robustness of both derivatization procedures was observed over a concentration range of 52 500 mu g of amino acid. NACME esters displayed low errors (+/- 0.6 parts per thousand for phenylalanine to +/- 1.1 parts per thousand for serine) due to the higher sample-to-derivative carbon ratio of this derivative. Finally, the integrity of the new NACME procedure was confirmed through analysis of diet and bone collagen amino acids of rats reared on C(3) or C(4) diets, which indicated the high degree of both accuracy and precision of the delta(13)C values obtained for individual amino acids.