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Untargeted and targeted analysis of sarin poisoning biomarkers in rat urine by liquid chromatography and tandem mass spectrometry

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Abstract

Chemical warfare agents continue to pose a real threat to humanity, despite their prohibition under the Chemical Weapons Convention. Sarin is one of the most toxic and lethal representatives of nerve agents. The methodology for the targeted analysis of known sarin metabolites has reached great heights, but little attention has been paid to the untargeted analysis of biological samples of victims exposed to this deadly poisonous substance. At present, the development of computational and statistical methods of analysis offers great opportunities for finding new metabolites or understanding the mechanisms of action or effect of toxic substances on the organism. This study presents the targeted LC-MS/MS determination of methylphosphonic acid and isopropyl methylphosphonic acid in the urine of rats exposed to a non-lethal dose of sarin, as well as the untarget urine analysis by LC-HRMS. Targeted analysis of polar acidic sarin metabolites was performed on a mixed-mode reversed-phase anion-exchange column, and untargeted analysis on a conventional reversed-phase C18 column. Isopropyl methylphosphonic acid was detected and quantified within 5 days after subcutaneous injection of sarin at a dose of 1/4 LD50. A combination of generalized additive mixed models and dose-response analysis with database searches using accurate mass of precursor ions and corresponding MS/MS spectra enabled us to propose new six potential biomarkers of biological response to exposure. The results confirm the well-known fact that sarin poisoning has a significant impact on the victims’ metabolome, with inhibition of acetylcholinesterase being just the first step and trigger of the complex toxicodynamic response.

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Availability of data and material

Additional supporting research data for this article, in full-scan mode, provided as mzXML format files in MassIVE Repository, may be accessed at MassIVE MSV000087738, https://doi.org/10.25345/C54V71

Code availability

All scripts codes are available at GitHub [42, 43].

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Acknowledgements

We are very grateful to Goncharov N.V. from the Research Institute of Hygiene, Occupational Pathology and Human Ecology Federal State Unitary Enterprise, Federal Medical Biological Agency, for his valuable insights on writing this article. The authors thank George Varziev (CEO, InterAnalyt—General Distributor of Shimadzu—Russia) for providing the Shimadzu LCMS-IT-TOF.

Source of biological material

Rat urine were provided by the Research Institute of Hygiene, Occupational Pathology, and Human Ecology Federal State Unitary Enterprise (Leningrad region, Russia).

Funding

This work was supported by the Russian Science Foundation (Grant No. 19-13-00057) for Lomonosov Moscow State University.

Author information

Author notes

  1. M. F. Vokuev, Т. М. Baygildiev and I. V. Plyushchenko contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, Lomonosov Moscow State University, 119991, Moscow, Russia

    M. F. Vokuev, Т. М. Baygildiev, I. V. Plyushchenko, Y. A. Ikhalaynen, R. L. Ogorodnikov, I. K. Solontsov, А. V. Braun, I. V. Rуbalchenko & I. A. Rodin

  2. Laboratory for the Chemical and Analytical Control of the Military Research Centre, 105005, Moscow, Russia

    А. V. Braun & I. V. Rуbalchenko

  3. Research Institute of Hygiene, Occupational Pathology and Human Ecology Federal State Unitary Enterprise, Federal Medical Biological Agency of Russia, Kuz’molovsky g/p, 188663, Leningrad Region, Russia

    E. I. Savelieva

  4. Department of Epidemiology and Evidence Based Medicine, I.M. Sechenov First Moscow State Medical University, Moscow, Russia

    I. A. Rodin

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  1. M. F. Vokuev
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  2. Т. М. Baygildiev
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  3. I. V. Plyushchenko
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  9. I. V. Rуbalchenko
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Contributions

Writing—original draft preparation, review, and editing: Т.М. Baygildiev, M.F. Vokuev; I.V. Plyushchenko. Targeted analysis: M.F. Vokuev, R.L. Ogorodnikov, I.K. Solontsov. Metabolomic profiling: Т.М. Baygildiev, M.F. Vokuev, I.V. Plyushchenko, Y.A. Ikhalaynen. Сonducting a toxicological experiment: E.I. Savelieva; Supervision: А.V. Braun, I.V. Rуbalchenko. Funding acquisition and encouraging to the research: I. A. Rodin. All authors have approved the final version of the manuscript.

Corresponding author

Correspondence to M. F. Vokuev.

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Ethics approval

All experiments with animals were conducted in accordance with Russian National Standards which is based on The European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (ETS 123).

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The authors declare no competing interests.

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Supplementary information

Table S1 The results of the evaluation of signal correction methods by three criteria. Figure S1. Weighted (w = 1/c2) calibration curve for the IMPA determination in rat urine. Figure S2. Weighted (w = 1/c2) calibration curve for the MPA determination in rat urine. Figure S3. The chromatogram of the mixture of MPA and IMPA with a concentration of 1 mg L−1. Figure S4. RLA plots for datasets after QC-XGB in positive and negative polarities. Figure S5.Dose-response curves for selected metabolites for raw data.

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Vokuev, M.F., Baygildiev, Т.М., Plyushchenko, I.V. et al. Untargeted and targeted analysis of sarin poisoning biomarkers in rat urine by liquid chromatography and tandem mass spectrometry. Anal Bioanal Chem 413, 6973–6985 (2021). https://doi.org/10.1007/s00216-021-03655-3

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