Measurement of δ¹³C values of soil amino acids by GC–C–IRMS using trimethylsilylation: a critical assessment

Abstract

In this study, we evaluated trimethylsilyl (TMS) derivatives as derivatization reagents for the compound-specific stable carbon isotope analysis of soil amino acids by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). We used non-proteinogenic amino acids to show that the extraction-derivatization-analysis procedure provides a reliable method to measure δ(13)C values of amino acids extracted from soil. However, we found a number of drawbacks that significantly increase the final total uncertainty. These include the following: production of multiple peaks for each amino acid, identified as di-, tri- and tetra-TMS derivatives; a number of TMS-carbon (TMS-C) atoms added lower than the stoichiometric one, possibly due to incomplete combustion; different TMS-C δ(13)C for di-, tri- and tetra-TMS derivatives. For soil samples, only four amino acids (leucine, valine, threonine and serine) provide reliable δ(13)C values with a total average uncertainty of 1.3 ‰. We conclude that trimethylsilyl derivatives are only suitable for determining the (13)C incorporation in amino acids within experiments using (13)C-labelled tracers but cannot be applied for amino acids with natural carbon isotope abundance until the drawbacks described here are overcome and the measured total uncertainty significantly decreased.

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

“…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. METHODS: 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.…”

“…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.…”

“…RESULTS: 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.…”

“…Silylation is a commonly used method for polar molecule analysis by GC, especially in the measurement of amino acids by GC/C/IRMS . One argument for the use of silylation in 13 C analysis is that this derivatization reaction is unlikely to involve any significant isotope fractionation effect that would affect the δ 13 C value of the final derivatives, because the chemical reaction does not involve carbon‐containing bonds in the analyte, but only Si–O or Si–N bonds .…”

“…Therefore, determination of the δ 13 C values of individual AAs could shed light on their role in both the carbon and the nitrogen cycles. The GC/C/IRMS 13 C analysis of amino acids has been used both in artificially labelling experiments to study 13 C metabolic flux and in natural abundance …”