Evidence of nerve agent VX exposure in rat plasma by detection of albumin-adducts in vitro and in vivo

VX is a highly toxic organophosphorus nerve agent that reacts with a variety of endogenous proteins such as serum albumin under formation of adducts that can be targeted by analytical methods for biomedical verification of exposure. Albumin is phosphonylated by the ethyl methylphosphonic acid moiety (EMP) of VX at various tyrosine residues. Additionally, the released leaving group of VX, 2-(diisopropylamino)ethanethiol (DPAET), may react with cysteine residues in diverse proteins. We developed and validated a microbore liquid chromatography-electrospray ionization high-resolution tandem mass spectrometry (mu LC-ESI MS/HR MS) method enabling simultaneous detection of three albumin-derived biomarkers for the analysis of rat plasma. After pronase-catalyzed cleavage of rat plasma proteins single phosphonylated tyrosine residues (Tyr-EMP), the Cys(34)(-DPAET)Pro dipeptide as well as the rat-specific LeuProCys(448)(-DPAET) tripeptide were obtained. The time-dependent adduct formation in rat plasma was investigated in vitro and biomarker formation during proteolysis was optimized. Biomarkers were shown to be stable for a minimum of four freeze-and-thaw cycles and for at least 24 h in the autosampler at 15 degrees C thus making the adducts highly suited for bioanalysis. Cys(34)(-DPAET)Pro was superior compared to the other serum biomarkers considering the limit of identification and stability in plasma at 37 degrees C. For the first time, Cys(34)(-DPAET)Pro was detected in in vivo specimens showing a time-dependent concentration increase after subcutaneous exposure of rats underlining the benefit of the dipeptide disulfide biomarker for sensitive analysis.

Automated summary

A microbore liquid chromatography-electrospray ionization high-resolution tandem mass spectrometry method was developed to detect three albumin-derived biomarkers for the analysis of VX nerve agent exposure. The stability and identification limit of the biomarkers were evaluated, and a dipeptide disulfide biomarker (Cys(34)(-DPAET)Pro) showed the highest performance. In vivo experiments confirmed the time-dependent concentration increase of Cys(34)(-DPAET)Pro after subcutaneous exposure in rats.