Simultaneous Measurement of Tabun, Sarin, Soman, Cyclosarin, VR, VX, and VM Adducts to Tyrosine in Blood Products by Isotope Dilution UHPLC-MS/MS

Abstract

This work describes a new specific, sensitive, and rapid stable isotope dilution method for the simultaneous detection of the organophosphorus nerve agents (OPNAs) tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), VR, VX, and VM adducts to tyrosine (Tyr). Serum, plasma, and lysed whole blood samples (50 µL) were prepared by protein precipitation followed by digestion with Pronase. Specific Tyr adducts were isolated from the digest by a single solid phase extraction (SPE) step, and the analytes were separated by reversed-phase ultra high performance liquid chromatography (UHPLC) gradient elution in less than 2 min. Detection was performed on a triple quadrupole tandem mass spectrometer using time-triggered selected reaction monitoring (SRM) in positive electrospray ionization (ESI) mode. The calibration range was characterized from 0.100–50.0 ng/mL for GB– and VR– Tyr and 0.250–50.0 ng/mL for GA–, GD–, GF–, and VX/VM–Tyr (R2 ≥ 0.995). Inter- and intra-assay precision had coefficients of variation of ≤17 and ≤10%, respectively, and the measured concentration accuracies of spiked samples were within 15% of the targeted value for multiple spiking levels. The limit of detection was calculated to be 0.097, 0.027, 0.018, 0.074, 0.023, and 0.083 ng/mL for GA–, GB–, GD–, GF–, VR–, and VX/VM–Tyr, respectively. A convenience set of 96 serum samples with no known nerve agent exposure was screened and revealed no baseline values or potential interferences. This method provides a simple and highly specific diagnostic tool that may extend the time postevent that a confirmation of nerve agent exposure can be made with confidence.

Mass Spectral Detection of Diethoxyphospho-Tyrosine Adducts on Proteins from HEK293 Cells Using Monoclonal Antibody depY for Enrichment

“…31 Proteins that have no active site serine are more easily modified by nerve agents and organophosphorus pesticides on tyrosine and lysine. 18,21,32−34 HEK293 cells have no endogenous acetylcholinesterase, butyrylcholinesterase, or carboxylesterase, consistent with the absence of serine adducts in our samples.…”

Bridging the gap between sample collection and laboratory analysis: using dried blood spots to identify human exposure to chemical agents

“…Previously, we developed an analytical panel of biomarkers to identify human exposures to organophosphorus nerve agents using plasma or serum 16–18 . This broad sweeping approach accommodated identification of low dose exposures, but required the appropriate handling and storage of whole blood, plasma, or serum to obtain a viable sample for analysis.…”

Exploring adduct formation between human serum albumin and eleven organophosphate ester flame retardants and plasticizers using MALDI-TOF/TOF and LC-Q/TOF

“…However, there is still a debate on the toxic effects and current thought suggests that OP toxicity cannot be attributed entirely to inhibition of AChE (Casida and Quistad 2004; EPA 2015). Recently, investigators have begun to investigate covalent OP adduction of non-AChE protein targets as a possible causative step in other toxic responses (Thompson et al 2010; Schopfer et al 2010; Marsillach et al 2013; Crow et al 2014). Serum albumin was found to form a covalent bond with OPs (Casida and Quistad 2004; Peeples et al 2005; Schopfer et al 2010; Crow et al 2014) and these covalent adducts with blood proteins might provide a new biomarker of OP exposure (Li et al 2007; Williams et al 2007; John et al 2008 and 2010; Crow et al 2014).…”

“…Human serum albumin (HSA) adducts can be used as a biomarker for OP insecticides and chemical warfare agent exposure (Li et al 2007; Schopfer et al 2010; John et al 2010; Crow et al 2014). However, to our knowledge, there is no report in the literature on phosphorylation of HSA by OPE FRs and OPE plasticizers.…”