Labelled microbial culture as a calibration medium for (13) C-isotope measurement of derivatized compounds: application to tert-butyldimethylsilyl amino acids.

Abstract

Compound-specific stable carbon isotope analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is widely used in studies of environmental or biological functioning. In the case of derivatized molecules, a calibration might be required due to added non-analyte carbon and in some cases non-stoichiometric recovery by the mass spectrometer. Two biological materials of known isotopic composition were produced by microbial cell cultures on either (13) C-labelled glucose or non-labelled glucose as sole source of carbon. Subsequent hydrolyzed amino acids were derivatized as tert-butyldimethylsilyl (tBDMSi) derivatives and analyzed by GC/C/IRMS. The (13) C-enrichment measurements were used as a direct calibration to calculate the original (13) C/(12) C ratios of individual amino acids. We tested this calibration on both known and unknown samples. For the main proteinogenic amino acids we could determine the number of non-analyte added carbon atoms and assess the non-stoichiometrical recovery of tBDMSi carbon atoms, due to their incomplete oxidation in the combustion step of GC/C/IRMS. The calibration enabled the determination of the natural abundances (δ(13) C values) of amino acids with an average accuracy of ±1.1 ‰. We illustrate the application of the calibration to determine the (13) C/(12) C ratios of amino acids, and the associated uncertainty, in biological and plant materials. The analysis of a labelled microbial cell culture offers a straightforward, rapid and reliable estimate of non-analyte carbon contribution to stable isotope composition. We recommend this method as a calibration or a control in artificial or natural (13) C-tracing experiments. Copyright © 2016 John Wiley & Sons, Ltd.