Position-specific carbon stable isotope analysis of glyphosate: isotope fingerprinting of molecules within a mixture

Glyphosate [N-(phosphonomethyl) glycine] is a widely used herbicide and a molecule of interest in the environmental sciences, due to its global use in agriculture and its potential impact on ecosystems. This study presents the first position-specific carbon isotope (¹³C/¹²C) analyses of glyphosates from multiple sources. In contrast to traditional isotope ratio mass spectrometry (IRMS), position-specific analysis provides ¹³C/¹²C ratios at individual carbon atom positions within a molecule, rather than an average carbon isotope ratio across a mixture or a specific compound. In this work, glyphosate in commercial herbicides was analyzed with only minimal purification, using a nuclear magnetic resonance (NMR) spectroscopy method that detects ¹H nuclei with bonds to either ¹³C or ¹²C, and isolates the signals of interest from other signals in the mixture. Results demonstrate that glyphosate from different sources can have significantly different intramolecular ¹³C/¹²C distributions, which were found to be spread over a wide range, with δ¹³C Vienna Peedee Belemnite (VPDB) values of -28.7 to -57.9‰. In each glyphosate, the carbon with a bond to the phosphorus atom was found to be depleted in ¹³C compared to the carbon at the C2 position, by 4 to 10‰. Aminomethylphosphonic acid (AMPA) was analyzed for method validation; AMPA contains only a single carbon position, so the ¹³C/¹²C results provided by the NMR method could be directly compared with traditional isotope ratio mass spectrometry. The glyphosate mixtures were also analyzed by IRMS to obtain their average ¹³C/¹²C ratios, for comparison with our position-specific results. This comparison revealed that the IRMS results significantly disguise the intramolecular isotope distribution. Finally, we introduce a ³¹P NMR method that can provide a position-specific ¹³C/¹²C ratio for carbon positions with a C-P chemical bond, and the results obtained by ¹H and ³¹P for C3 carbon agree with one another within their analytical uncertainty. These analytical tools for position-specific carbon isotope analysis permit the isotopic fingerprinting of target molecules within a mixture, with potential applications in a range of fields, including the environmental sciences and chemical forensics.