A compound-specific method for measuring the stable carbon isotopic composition of amino acids: elimination of the effect of isotopic fractionation during derivatization

Abstract Compound-specific isotope analysis (CSIA) of carbon and nitrogen within amino acids by gas chromatography/isotope ratio mass spectrometry (GC/IRMS) has been employed as a powerful tool for estimating the trophic tendency and resource utilization of organisms in food webs, as well as for illustrating the metabolic fate of amino acids and associated energy flux in diverse levels from within a single organism to entire ecosystems. However, the polar functional groups (i.e., carboxyl, amino, and hydroxyl groups) of amino acids should be neutralized by derivatization (i.e., esterification followed by acylation) before injection into a GC/IRMS instrument, which has caused several serious issues. For instance, the toxicity of acylation reagents (e.g., pivaloyl chloride) and the fluorinated derivatives (e.g., derived from trifluoroacetic anhydride) have complicated the use of isotope ratio analysis of amino acids in our studies. Moreover, large isotopic fractionation in 13 C/ 12 C associated with the acylation has considerably reduced the accuracy of the analysis of carbon isotope ratios. In the present study, we therefore developed a new derivatization method to minimize these issues, which can reduce the isotopic fractionation in 13 C/ 12 C by following two sequential reactions for the acylation: the first is acylation, which introduces isotopic fractionation, and the second is replacement of the pivaloyl group with that of pivalic anhydride. Further, the contribution of the 13 C/ 12 C ratios of derivative groups (i.e., isopropyl and pivaloyl groups) can be completely removed by a simultaneous mass balance calculation. Also, the new method is relatively safe, without the use of toxic reagents and the production of fluorinated derivatives. Thus, this derivatization method can be useful for CSIA of amino acids in diverse studies, which promises to provide unprecedented accuracy in understanding the complex physiological, ecological, and geochemical dynamics across aquatic and terrestrial ecosystems.