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Dual element (15N/14N, 13C/12C) isotope analysis of glyphosate and AMPA by derivatization-gas chromatography isotope ratio mass spectrometry (GC/IRMS) combined with LC/IRMS

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Abstract

To assess sources and degradation of the herbicide glyphosate [N-(phosphonomethyl) glycine] and its metabolite AMPA (aminomethylphosphonic acid), concentration measurements are often inconclusive and even 13C/12C analysis alone may give limited information. To advance isotope ratio analysis of an additional element, we present compound-specific 15N/14N analysis of glyphosate and AMPA by a two step derivatization in combination with gas chromatography/isotope ratio mass spectrometry (GC/IRMS). The N-H group was derivatized with isopropyl chloroformate (iso-PCF), and remaining acidic groups were subsequently methylated with trimethylsilyldiazomethane (TMSD). Iso-PCF treatment at pH <10 gave too low 15N/14N ratios indicating an incomplete derivatization; in contrast, too high 15N/14N ratios at pH >10 indicated decomposition of the derivative. At pH 10, and with an excess of iso-PCF by 10–24, greatest yields and accurate 15N/14N ratios were obtained (deviation from elemental analyzer-IRMS: −0.2 ± 0.9 % for glyphosate; −0.4 ± 0.7 % for AMPA). Limits for accurate δ15N analysis of glyphosate and AMPA were 150 and 250 ng injected, respectively. A combination of δ15N and δ13C analysis by liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) (1) enabled an improved distinction of commercial glyphosate products and (2) showed that glyphosate isotope values during degradation by MnO2 clearly fell outside the commercial product range. This highlights the potential of combined carbon and nitrogen isotopes analysis to trace sources and degradation of glyphosate.

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Acknowledgments

This work was supported by the German Academic Exchange Service (DAAD) by a fellowship scholarship to E.M. and by the German National Science Foundation (DFG) withhin the priority program SPP 1315. The authors thank Martina Höche, Ramona Brejcha, and Harald Lowag for assistance with LC/IRMS and support in characterization of in-house laboratory standards.

Note Added in Proof

After submission of the manuscript, the following additional optimization of our method was accomplished for samples containing a high background (10 gl−1) of glutamate, corresponding to an excess of 2 · 104 of this foreign nitrogen-containing compound. (i) To adjust the pH to 10, 300 μl instead of 50 μl borate buffer were added in order to provide sufficient buffer capacity to counter the effect of glutamate. (ii) To provide also a sufficient excess of derivatization agent, 400 μl of 1.0 M isopropyl chloroformate in hexane were added. (iii) As vortex time, 6 min instead of 2 min, was chosen to allow additional time for glyphosate molecules to partition into the aqueous phase (see ESM Fig. S4). This extra time was found to be necessary to compensate for the slower reaction of glyphosate due to competition by glutamate for partitioning and reaction with the derivatization agent. (iv) In the subsequent second derivatization step, 300 μl instead of 50 μl of 2.0 M trimethyl silyl diazomethane (absolute 600 μmol) were added to provide again a sufficient excess of derivatization agent. (v) To enable, finally, separation of glyphosate- and AMPA-derivatives from the interfering glutamate-derivative, a Rtx-5 amine column (30 m; 0.32 μm inner diameter; 1 μm film thickness, supplied by Restek GmbH, Bad Homburg, Germany), was used with the following temperature program: the initial temperature of 80 °C was held for 1 min, then increased to 150 °C at 10 °C min−1, held again for 1 min, and then ramped to 230 °C at 3 °C min−1. The final temperature was held for 2 min (see GC chromatogram in ESM Fig. S5).

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Authors and Affiliations

  1. Helmholtz Zentrum München, Institute of Groundwater Ecology, Ingolstädter Landstrasse 1, 85764, Neuherberg, Germany

    Emmanuel O. Mogusu & Martin Elsner

  2. Instrumental Analytical Chemistry, University of Duisburg-Essen, Universitätstraße 5, 45141, Essen, Germany

    J. Benjamin Wolbert, Dorothea M. Kujawinski & Maik A. Jochmann

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  1. Emmanuel O. Mogusu
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  2. J. Benjamin Wolbert
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  3. Dorothea M. Kujawinski
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  4. Maik A. Jochmann
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  5. Martin Elsner
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Correspondence to Martin Elsner.

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Mogusu, E.O., Wolbert, J.B., Kujawinski, D.M. et al. Dual element (15N/14N, 13C/12C) isotope analysis of glyphosate and AMPA by derivatization-gas chromatography isotope ratio mass spectrometry (GC/IRMS) combined with LC/IRMS. Anal Bioanal Chem 407, 5249–5260 (2015). https://doi.org/10.1007/s00216-015-8721-3

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