A liquid chromatography tandem mass spectrometric method on in vitro nerve agents poisoning characterization and reactivator efficacy evaluation by determination of specific peptide adducts in acetylcholinesterase

• A proteolysis-based LC–MS/MS method for quantitatively differentiation of modified and unmodified undecapeptides of nerve agents exposed AChE was developed.
• This method can be applied in characterization of nerve agent poisoning and evaluation on the efficacy of reactivator in vitro.
• Less than 1% inhibition rate of AChE can be precisely determined by this method.

The terroristic availability of highly toxic nerve agents (NAs) highlights the necessity for a deep understanding of their toxicities and effective medical treatments. A liquid chromatography tandem mass spectrometry (LC–MS/MS) method for a characterization of the NAs poisoning and an evaluation on the efficacy of reactivators in in vitro was developed for the first time. After exposure to sarin or VX and pepsin digestion, the specific peptides of acetylcholinesterase (AChE) in a purified status, i.e. undecapeptide “GESAGAASVGM” in free, unaged, or aged status was identified and quantified. A key termination procedure is focused to make the reaction system “frozen” and precisely “capture” the poisoning, aging and spontaneous reactivation status of AChE, and the abundance of such specific peptides can thus be simultaneously measured. In our established method, as low as 0.72% and 0.84% inhibition level of AChE induced by 0.5 nM sarin and VX can be detected from the measurement of peptide adducts, which benefits a confirmation of NAs exposure, especially at extremely low levels. Comparing with conventional colorimetric Ellman assays, our method provides not only enzyme activity and inhibition rate, but also the precise poisoning status of NAs exposed AChE. Based on the full information provided by this method, the efficacy of reactivators, such as HI-6, obidoxime and pralidoxime, in the typical treatment of NAs poisoned AChE in in vitro was further evaluated. Our results showed that this method is a promising tool for the characterization of NAs poisoning and the evaluation of reactivator efficacy.