Ingvild Comfort Hvinden, Bent Tore Røen and Helene Thorsen Rønning
a Forsvarets Forskningsinstitutt (FFI), Instituttveien 20, 2007 Kjeller, Norway
Email: Ingvild-comfort.hvinden@ffi.no
The taboo of chemical weapons has existed for decades and was significantly strengthened by the Chemical Weapons Convention (1997). Unfortunately, the willingness to use chemical weapons is on the rise. Riot control agents and chloropicrin have been reported in the Ukrainian war [1, 2]. Novichoks, organophosphorus nerve agents, were used in the politically motivated assassination attempts of Sergei Skripal (2018) and Alexei Navalny (2020). Confident identification of highly toxic compounds therefore remains critical, both for medical, protective, and attributional needs.
Biological matrices, e.g., blood and urine, contain biomarkers necessary for diagnosis of chemical warfare agent exposure. The biomarkers are well suited for liquid chromatography-mass spectrometry (LC-MS) methods [3-5], but the challenge lies in low concentrations (< 250 nM) and complex matrices. Additionally, identification is considered confident only after detection with two or more complementary methods. The aim of this study was therefore to implement and further develop multiple LC-MS methods for identification of Novichok biomarkers in blood and urine.
Blood serum and urine samples were spiked with low levels (< 250 nM) of Novichok agents A-230 and A-232. Sample preparation was focussed on clean-up and preconcentration of specific protein biomarkers, intact agents, and acid degradation products. Blood serum was straightforward to prepare, but the combination of urine and phosphonic acids was challenging and pre-concentration of samples prior to analysis proved difficult. All biomarker types were found in their respective matrices. Reversed phase (C18) was suitable for all compounds, and hydrophilic interaction chromatography (amide) worked well for the phosphonic acids. However, sensitivity remained an obstacle for confident identification across the different methods, and highly targeted MS/MS leaves little room for discovering unknowns.
In conclusion, several LC-MS/MS methods were implemented and further developed, but sensitivity and analytical flexibility are areas of improvement. Capillary- or nano-LC may improve sensitivity and enable more extensive use of high-resolution LC-MS/MS, which would also facilitate detection of unknown compounds. Derivatisation and/or gas chromatography-MS/MS methods may offer additional paths towards sensitive and confident identification of biomarkers of Novichoks and related compounds.
References
1. OPCW. OPCW issues report on third Technical Assistance Visit to Ukraine following an incident of alleged use of toxic chemicals as a weapon. 2025; Available from: https://www.opcw.org/media-centre/news/2025/06/opcw-issues-report-third-technical-assistance-visit-ukraine-following.
2. Russia further intensifies its use of chemical weapons in Ukraine. 2025 30.10.2025]; Available from: https://english.defensie.nl/latest/news/2025/07/04/russia-further-intensifies-its-use-of-chemical-weapons-in-ukraine.
3. Noort, D., et al., Verification of Exposure to Novichok Nerve Agents Utilizing a Semitargeted Human Butyrylcholinesterase Nonapeptide Assay. Chem. Res. Toxicol, 2021. 34(8): p. 1926–1932.
4. Røen, B.T., S.R. Sellevåg, and E. Lundanes, Quantification of Nerve Agent Biomarkers in Human Serum and Urine. Analytical Chemistry, 2014. 86(23): p. 11833–11840.
5. de Bruin-Hoegée, M., et al., On-site detection and laboratory verification of the presence of nerve agent biomarkers using dried blood spots. Forensic Chemistry, 2023. 35: p. 100526.