Simultaneous quantification of soman and VX adducts to butyrylcholinesterase, their aged methylphosphonic acid adduct and butyrylcholinesterase in plasma using an off-column procainamide-gel separation method combined with UHPLC–MS/MS

This work describes a novel and sensitive non-isotope dilution method for simultaneous quantification of organophosphorus nerve agents (OPNAs) soman (GD) and VX adducts to butyrylcholinesterase (BChE), their aged methylphosphonic acid (MeP) adduct and unadducted BChE in plasma exposed to OPNA. OPNA-BChE adducts were isolated with an off-column procainamide-gel separation (PGS) from plasma, and then digested with pepsin into specific adducted FGES*AGAAS nonapeptide (NP) biomarkers. The resulting NPs were detected by UHPLC-MS/MS MRM. The off-column PGS method can capture over 90% of BChE, MeP-BChE, VX-BChE and GD-BChE from their respective plasma materials. One newly designed and easily synthesized phosphorylated BChE nonapeptide with one Gly-to-Ala mutation was successfully reported to serve as internal standard instead of traditional isotopically labeled BChE nonapeptide. The linear range of calibration curves were from 1.00-200ngmL-1 for VX-NP, 2.00-200ngmL-1 for GD-NP and MeP-NP (R2≥0.995), and 3.00-200ngmL-1 for BChE NP (R2≥0.990). The inter-day precision had relative standard deviation (%RSD) of