Optimisation of derivatisation procedures for the determination of δ¹³C values of amino acids by gas chromatography/combustion/isotope ratio mass spectrometry

Abstract

Compound-specific stable carbon isotope analysis of amino acids by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is a highly selective and sensitive method for probing the biosynthetic/diagenetic pathways, pool size and turnover rates of proteins, previously intractable to bulk isotope analyses. However, amino acids are polyfunctional, non-volatile compounds which require derivatisation prior to GC analysis. While a wide range of derivatives exist for the GC analysis of amino acids only a handful have been utilised for their GC/C/IRMS analysis. Significantly, none of those derivatives currently employed appear completely satisfactory and a thorough assessment of their relative utility is lacking. Seven derivatives (three previously reported and four novel) for obtaining delta(13)C values of amino acids via GC/C/IRMS analysis were compared. More specifically, standard mixtures of 15 protein amino acids were converted into N-acetylmethyl (NACME) esters, N-acetyl n-propyl (NANP) esters, N-acetyl i-propyl (NAIP) esters, N-trifluoroacetyl-i-propyl (TFA-IP) esters, N-pivaloyl methyl (NPME) esters, N-pivaloyl n-propyl (NPNP) esters and N-pivaloyl i-propyl (NPIP) esters. Each derivative was assessed with respect to its applicability to carbon isotope determinations of all the common alpha-amino acids, reaction yield, chromatographic resolution, stability, analyte-to-derivative carbon ratio, kinetic isotope effects and errors associated with their carbon isotope determinations. The NACME derivative was concluded to be the preferred derivative mainly due to the highest analyte-to-derivative carbon ratio being achieved, resulting in the lowest analytical errors for amino acid delta(13)C value determinations, ranging from +/-0.6 per thousand for phenylalanine, leucine and isoleucine to +/-1.1 per thousand for serine and glycine.

Current challenges in compound-specific stable isotope analysis of environmental organic contaminants

“…Acetylation is suitable for derivatization of hydroxyl and amino groups, and is frequently used for steroids [118], carbohydrates [119], and amino acids [120]. Reactions are usually performed with excess acid (e.g., acetic acid) or an anhydride mixed with pyridine [121] or n-methylimidazole [119] as promoters.…”

Contraction intensity and feeding affect collagen and myofibrillar protein synthesis rates differently in human skeletal muscle

“…The salt-precipitated total collagen protein pool and the washed myofibrillar pellet were then hydrolyzed in 6 M HCl (110°C for 18 h). The constituent amino acids in the hydrolyzates were purified by cation exchange resin (Dowex AG-50W; Bio-Rad), dried down in a SpeedVac as described above, and finally derivatized as their N-acetyl n-propyl esters using our own optimized protocol for previously applied procedures (22). Leucine and phenylalanine concentrations.…”

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.…”