title: 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.
abstract: 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-1for VX-NP, 2.00-200ngmL-1for GD-NP and MeP-NP (R2≥0.995), and 3.00-200ngmL-1for BChE NP (R2≥0.990). The inter-day precision had relative standard deviation (%RSD) of <8.89%, and the accuracy ranged between 88.9-120%. The limit of detection was calculated to be 0.411, 0.750, 0.800 and 1.43ngmL-1for VX-NP, GD-NP, MeP-NP and BChE NP, respectively. OPNA-exposed quality control plasma samples were characterized as part of method validation. Investigation of plasma samples unexposed to OPNA revealed no baseline values or interferences. Using the off-column PGS method combined with UHPLC-MS/MS, VX-NP and GD-NP adducts can be unambiguously detected with high confidence in 0.10ngmL-1and 0.50ngmL-1of exposed human plasma respectively, only requiring 0.1mL of plasma sample and taking about four hours without special sample preparation equipment. These improvements make it a simple, sensitive and robust PGS-UHPLC-MS/MS method, and this method will become an attractive alternative to immunomagnetic separation (IMS) method and a useful diagnostic tool for retrospective detection of OPNA exposure with high confidence. Furthermore, using the developed method, the adducted BChE levels from VX and GD-exposed (0.10-100ngmL-1) plasma samples were completely characterized, and the fact that VX being more active and specific to BChE than GD was re-confirmed.
authors.affiliation: State Key Laboratory of NBC Protection for Civilian, Beijing 102205, China; Laboratory of Analytical Chemistry, Research Institute of Chemical Defence, Beijing 102205, China.
authors.forename: Chang-Cai
authors.initials: CC
authors.lastname: Liu
authors.affiliation: State Key Laboratory of NBC Protection for Civilian, Beijing 102205, China; Laboratory of Analytical Chemistry, Research Institute of Chemical Defence, Beijing 102205, China.
authors.forename: Gui-Lan
authors.initials: GL
authors.lastname: Huang
authors.affiliation: State Key Laboratory of NBC Protection for Civilian, Beijing 102205, China. Electronic address: fhxihl@163.com.
authors.forename: Hai-Ling
authors.initials: HL
authors.lastname: Xi
authors.affiliation: State Key Laboratory of NBC Protection for Civilian, Beijing 102205, China; Laboratory of Analytical Chemistry, Research Institute of Chemical Defence, Beijing 102205, China. Electronic address: liu_shilei@lacricd.com.
authors.forename: Shi-Lei
authors.initials: SL
authors.lastname: Liu
authors.affiliation: Laboratory of Analytical Chemistry, Research Institute of Chemical Defence, Beijing 102205, China.
authors.forename: Jing-Quan
authors.initials: JQ
authors.lastname: Liu
authors.affiliation: Laboratory of Analytical Chemistry, Research Institute of Chemical Defence, Beijing 102205, China.
authors.forename: Hui-Lan
authors.initials: HL
authors.lastname: Yu
authors.affiliation: State Key Laboratory of NBC Protection for Civilian, Beijing 102205, China; Laboratory of Analytical Chemistry, Research Institute of Chemical Defence, Beijing 102205, China.
authors.forename: Shi-Kun
authors.initials: SK
authors.lastname: Zhou
authors.affiliation: Laboratory of Analytical Chemistry, Research Institute of Chemical Defence, Beijing 102205, China.
authors.forename: Long-Hui
authors.initials: LH
authors.lastname: Liang
authors.affiliation: Laboratory of Analytical Chemistry, Research Institute of Chemical Defence, Beijing 102205, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Yuan
mesh_terms: D002091:Butyrylcholinesterase / Q000097:blood* / Q000302:isolation & purification; D002619:Chemical Warfare Agents / Q000493:pharmacokinetics*; D002800:Cholinesterase Inhibitors / Q000097:blood* / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D004867:Equipment Design; D005782:Gels / Q000737:chemistry; D006801:Humans; D057230:Limit of Detection; D009943:Organophosphorus Compounds / Q000097:blood* / Q000302:isolation & purification; D009946:Organothiophosphorus Compounds / Q000097:blood* / Q000302:isolation & purification; D011342:Procainamide / Q000737:chemistry; D012999:Soman / Q000097:blood* / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
chemical_list: D002091:Butyrylcholinesterase; D002619:Chemical Warfare Agents; D002800:Cholinesterase Inhibitors; D005782:Gels; D009943:Organophosphorus Compounds; D009946:Organothiophosphorus Compounds; D011342:Procainamide; D012999:Soman; C032627:methylphosphonic acid; C009680:VX-agent
keywords: BChE adduct; Chemical warfare agent; MS; OPNA exposure; Organophosphorus nerve agents; Retrospective detection; UHPLC-ESI-MS