Archives of Toxicology

, Volume 78, Issue 9, pp 508–524 | Cite as

Retrospective detection of exposure to nerve agents: analysis of phosphofluoridates originating from fluoride-induced reactivation of phosphylated BuChE

  • Marcel J. van der SchansEmail author
  • Martine Polhuijs
  • Corry van Dijk
  • Carla E. A. M. Degenhardt
  • Kees Pleijsier
  • Jan P. Langenberg
  • Hendrik P. Benschop
Organ Toxicity and Mechanisms


The utility was explored of a new approach to detect retrospectively exposure to nerve agents by means of conversion of the inhibitor moiety bound to the active site of the enzyme BuChE in plasma with fluoride ions into a phosphofluoridate which is subsequently analyzed by means of gas chromatography (GC). This quantifies ≥0.01% inhibition of BuChE and identifies the structure of the inhibitor except for the original leaving group. A three-tiered approach was followed involving the five classical nerve agents GA, GB, GF, GD, and VX, as well as the active metabolite of parathion, i.e., paraoxon: in vivo experiments in rhesus monkeys after iv administration of a sign-free dose of agent and concomitant in vitro experiments in plasma of rhesus monkeys and humans should allow an assessment of in vivo retrospectivity in humans. A systematic investigation was performed in order to find a single set of reaction conditions which yields a maximum amount of phosphofluoridate for all nerve agents. Fluoride-induced reactivation at 25°C at a final concentration of 250 mM KF during 15 min in a pH-range between 4 and 6 appears to be effective. The in vitro decrease with time in reactivatibility of inhibited BuChE in plasma from humans and rhesus monkeys was largely due to aging of the phosphyl moiety, except for VX where spontaneous reactivation was a major cause. The decrease followed first-order except for a biphasic course in the case of GF in human and rhesus monkey plasma as well as of GD in rhesus plasma. In vitro half-lifes in human plasma ranged between ca. 14 h for GB and ca. 63 h for GA. A comparison of the in vivo data from rhesus monkeys and the in vitro data is complicated by the observation that the in vivo decrease with time of fluoride-reactivated phosphofluoridate is biphasic for all nerve agents. The terminal in vivo phase pertains to a small fraction of the amount of initially regenerated phosphofluoridate but is responsible for a considerable degree of retrospectivity, ranging between 14 and 56 days for GF and GB, respectively. The new procedure can be used in a variety of practical applications, e.g., (i) biomonitoring in health surveillance at exposure levels that are several orders of magnitude lower than presently possible; (ii) diagnosis in case of alleged exposure to nerve agents in time of war or after terrorist attacks; (iii) in forensic cases against suspected terrorists that have handled organophosphate anticholinesterases; and (iv) in research applications such as investigations on lowest observable effect levels of exposure to nerve agents.


Biomarker Biomonitoring Butyryl cholinesterase Fluoride ions Low-level exposure Nerve agents Organophosphates Reactivation Retrospective detection 







Butyrylthiocholine iodide


O,O-diethyl phosphorofluoridate


O,O-diisopropyl phosphorofluoridate




Chemical ionisation


Cooled injection system


Chemical warfare


O-ethyl methylphosphonofluoridate, ethylsarin


O-ethyl dimethylphosphoramidofluoridate, fluorotabun


O-ethyl dimethylphosphoramidocyanidate, tabun


O-isopropyl methylphosphonofluoridate, sarin


Gas chromatography


O-pinacolyl methylphosphonofluoridate, soman


O-cyclohexyl methylphosphonofluoridate, cyclohexyl sarin


Liquid chromatography


Mass spectrometry


Mass selective detection


Multiple switchable intelligent controller


Detection nitrogen phosphorus selective detection


Pulsed flame photometric detection


Thermodesorption cold trap


Thermodesorption autosampler


O-ethyl S-2-diisopropylaminoethyl methylphosphonothioate



The Ministerium der Verteidigung of Germany is acknowledged for funding this project.


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Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Marcel J. van der Schans
    • 1
    Email author
  • Martine Polhuijs
    • 1
  • Corry van Dijk
    • 1
  • Carla E. A. M. Degenhardt
    • 1
  • Kees Pleijsier
    • 1
  • Jan P. Langenberg
    • 1
  • Hendrik P. Benschop
    • 1
  1. 1.Department of Medical CountermeasuresTNO Prins Maurits LaboratoryRijswijkThe Netherlands

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