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

Abstract

A novel derivatization procedure, N-acetyl methyl (NACME) esterification, was developed to improve the accuracy and precision of amino acid delta13C 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 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 per thousand and STDV(NAIP) = 0.4 per thousand). The isotopic robustness of both derivatization procedures was observed over a concentration range of 52,500 microg of amino acid. NACME esters displayed low errors (+/-0.6 per thousand for phenylalanine to +/-1.1 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 C3 or C4 diets, which indicated the high degree of both accuracy and precision of the delta13C values obtained for individual amino acids.

Tracing Carbon Sources through Aquatic and Terrestrial Food Webs Using Amino Acid Stable Isotope Fingerprinting

“…The samples were stored at −18°C. To volatize the AAs, we followed the derivatization procedure by Corr et al [22] methylating the dried samples with acidified methanol and subsequently acetylating them with a mixture of acetic anhydride, triethylamine and acetone (NACME: N -acetyl methyl ester derivatives). As a precautionary measure to reduce oxidation of amino acids during derivatization, we flushed and sealed reaction vials with N 2 gas prior to the methylation and acetylation reactions.…”

Assessing the potential of amino acid <sup>13</sup>C patterns as a carbon source tracer in marine sediments: effects of algal growth conditions and sedimentary diagenesis

“…For the remaining amino acids, we found no consistent isotope effects of purification indicating that δ 13 C AA values of purified and nonpurified samples are comparable except for Asx. The derivatization procedure, which serves to convert the nonvolatile amino acids to volatile derivatives, was modified from Corr et al (2007) as described by Larsen et al (2013). Briefly, the dried samples were methylated with acidified methanol and subsequently acetylated with a mixture of acetic anhydride, triethylamine, and acetone, forming N-acetyl methyl ester derivatives.…”

“…As a precautionary measure to reduce the oxidation of amino acids, we flushed and sealed reaction vials with N 2 gas prior to methylation and acetylation. Another modification from Corr et al (2007) was that ice baths in that protocol were substituted here with solid aluminum blocks at room temperature. We used known δ 13 C values of pure amino acids prepared and analyzed under the same conditions as the samples to calculate correction factors specific to each amino acid to account for carbon addition and fractionation during derivatization.…”

Comparison of liquid chromatography–isotope ratio mass spectrometry (LC/IRMS) and gas chromatography–combustion–isotope ratio mass spectrometry (GC/C/IRMS) for the determination of collagen amino acid δ13C values for palaeodietary and palaeoecological reconstruction

“…[30][31][32] Amongst them, we believe that the N-acetyl methyl ester (NACME) derivative, which adds the theoretically lowest number of carbon atoms per AA (one carbon atom to the C-termini and hydroxyl groups, and two carbon atoms to the N-termini or the hydroxyl moiety), is the optimal choice in terms of reducing analytical error. [33,34] For many years, N-trifluoroacetyl isopropyl (TFA/IP) esters have been widely used for the GC/C/IRMS analysis of AAs, primarily because of their stability, facile production, and favourable chromatography. [30] As a halogen-bearing derivative, however, TFA/IP has been reported to be detrimental to GC/C/IRMS performance, [26,30,35] but it is still widely employed as a common derivative for the stable isotope analysis of AAs by GC/C/IRMS.…”