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

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Abstract

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.

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Data Availability

In addition, raw NMR data and R-scripts for processing the data are available at the Texas Data Repository at: https://doi.org/10.18738/T8/HLYCSP.

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Funding

This research was supported by a Department of Energy Basic Energy Sciences research grant DE-SC0022524.

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Authors and Affiliations

  1. Department of Molecular Biosciences, The University of Texas at Austin, 100 East 24th St., Austin, TX, 78712, USA

    David W. Hoffman

  2. Institute for Geophysics, The University of Texas at Austin, J. J. Pickle Research Campus, 10601 Exploration Way, Austin, TX, 78758, USA

    Cornelia Rasmussen

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D.H. and C.R. performed experiments, developed protocols, acquired and analyzed NMR spectra, and contributed to manuscript preparation. All authors reviewed the manuscript.

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Correspondence to David W. Hoffman.

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Hoffman, D.W., Rasmussen, C. Position-specific carbon stable isotope analysis of glyphosate: isotope fingerprinting of molecules within a mixture. Anal Bioanal Chem (2024). https://doi.org/10.1007/s00216-024-05326-5

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