Development of N-Acetyl Methyl Ester Derivatives for the Determination of δ13C Values of Amino Acids Using Gas Chromatography-Combustion- Isotope Ratio Mass Spectrometry

• Published in: Analytical chemistry (Washington) Vol. 79; no. 23; pp. 9082 - 9090
• Main Authors: Corr, Lorna T, Berstan, Robert, Evershed, Richard P
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.12.2007
• Subjects: Analytical chemistry; Chemistry; Chromatographic methods and physical methods associated with chromatography; Exact sciences and technology; Gas chromatographic methods; Spectrometric and optical methods; Phenylalanine; Serine; Combustion; Isotopic composition; Derivatization; Replica method; Esterification; Protein; Gas chromatography; Ester; Accuracy; Isotope effect; Reproducibility; Aminoacid; Chlorides; Robustness; Methylation; Mass spectrometry; Comparative study
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac071223b

A novel derivatization procedure, N-acetyl methyl (NACME) esterification, was developed to improve the accuracy and precision of amino acid δ13C 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 1 h, 70 °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 (KIENACME = 1.036; KIENAIP = 1.038) and were shown in both cases to be reproducible. The mean reproducibility obtained from 15 replicates (3 × batches of 5) of both derivatives was strong (mean STDVNACME = 0.3‰ and STDVNAIP = 0.4‰). The isotopic robustness of both derivatization procedures was observed over a concentration range of 52 500 μg of amino acid. NACME esters displayed low errors (±0.6‰ for phenylalanine to ±1.1‰ 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 C3 or C4 diets, which indicated the high degree of both accuracy and precision of the δ13C values obtained for individual amino acids.