Verification of nerve agent exposure in human serum

Nerve agents are the most toxic chemical warfare agents known. They have been used at several occasions despite that the Chemical Weapon Convention (CWC) prohibits the production, storage, and use of these chemicals. Nerve agents affect the nervous system after they are absorbed through inhalation or skin exposure. On suspicion of nerve agent exposure, it is vital to get this verified and get the right treatment fast for the exposed persons. The study that is presented in this thesis, deals with the development of a method to determine nerve agent exposure by the analysis of human blood serum. The method is based on three parts; isolation of Butyrylcholinesterase (BuChE), enzymatic digestion of the protein, and analysis of the target peptide by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Magnetic beads coated with antibodies, specific for BuChE, were used for the isolation of BuChE in serum. Enzymatic digestion was evaluated and optimized by examining the denaturation conditions, enzyme, and digestion solution. Analyses of the target peptide were performed on LC-MS with switch technique and trap-column with backflush. Cross-linking of the antibodies showed a significant increase in the yield of BuChE from human serum. The optimized enzymatic digestion conditions were 0.2 mg/mL pepsin in 2 % formic acid. Pepsination resulted in a nonapeptide FGAS198AASAG with m/z 796.3477, which is suitable for LC-MS analysis. S198 is the binding site for the nerve agents, which makes it the target peptide. The LC conditions were optimized focusing on the timing in the switching technique, column dimensions and the mobile phase composition. The reduction of the inner diameter of the separation column, from 2 mm to 1 mm, resulted in a 10-fold increase in the peak intensity of the BuChE target peptide. The mobile phase delivered by the loading pump was optimized to 2.5 % formic acid and 2.0 % acetonitrile (ACN). In the mobile phase delivered by the analytical pump, 2.5 % formic acid and a gradient from 5-90 % ACN ...