Verification of exposure to sarin nerve agent through the chemical analysis of red blood cell samples

Organophosphorus nerve agents (OPNAs) are an essential class of internationally banned chemical warfare agents that broadly target a family of enzymes known as serine hydrolases. In the body, OPNAs bind to butyrylcholinesterase (BChE), acetylcholinesterase (AChE), albumin and other proteins to form nerve agent adducts. These adducts can develop reliable, long-term protein biomarkers for human exposure to nerve agents. Red blood cells (RBC) are known targets of OPNAs toxicity effects. Since erythrocytes have a relatively long half-life, they may serve as a valuable tool for OPNA exposure verification. In the present report, a new, simple, and less expensive approach for detection of nerve agent exposure using alkaline hydrolysis of RBC membrane proteins, is evaluated. In this method, the hemoglobin-free ghost of human erythrocyte is prepared from 6 mL of sarin (GB) spiked RBC sample, and then treated with sodium hydroxide solution. In the presence of sodium hydroxide and elevated temperature, isopropyl methylphosphonic acid (IMPA) is released from RBC proteinsarin adducts. Two dimensional Solid-phase extraction (SPE) is employed for clean-up. The monitoring of IMPA using LC-MS/MS demonstrates that GB is added to RBC proteins. In the quantitative analysis of GB adducts, the calibration curve was found to be linear over a range of 10-100 mu g L-1, and the limit of detection was 1 mu g L-1. Our results indicate that membrane proteins of human erythrocyte could be exploited as a surrogate biomarker of nerve agent exposure.