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 δ 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 α ‐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 δ ¹³ C value determinations, ranging from ±0.6‰ for phenylalanine, leucine and isoleucine to ±1.1‰ for serine and glycine. Copyright © 2007 John Wiley & Sons, Ltd.

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