title: Direct quantitation of methyl phosphonate adducts to human serum butyrylcholinesterase by immunomagnetic-UHPLC-MS/MS.
abstract: Hydrolysis of G- and V-series organophosphorus nerve agents (OPNAs) containing a phosphorus-methyl bond yields a methylphosphonic acid (MeP) product when adducted to human butyrylcholinesterase (BChE). The MeP adduct is considered a sign of "aging" and results in loss of the o-alkyl identifier specific to each nerve agent. After aging has occurred, common therapeutics such as oximes cannot reactivate the cholinesterase enzyme and relieve cholinergic inhibition. Until now, a direct, quantitative method for determination of the MeP adduct to BChE was unavailable. Aged adducts in serum samples were processed by immunomagnetic separation of BChE by antibody conjugated bead, isotope-dilution, pepsin digestion, followed by UHPLC separation and detection by conventional electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Ions were detected in selected reaction monitoring (SRM) mode, and transition m/z 874.3 → 778.3 was used for quantitation. The analytical response ratio was linearly proportional to the serum concentration of MeP-adducted peptide (MeP-P) over the nominal concentration range of 2.0-250 ng/mL, with a coefficient of determination of R(2) ≥ 0.997. Intrarun accuracy, expressed as %Relative Error (%RE), was ≤13.5%, 16.3%, and 3.20% at 2.0, 16, and 250 ng/mL, respectively; the corresponding precision expressed as %RSD was ≤11.9%, 6.15%, and 3.39%. Interday %RSD was ≤7.13%, 5.69%, and 1.91%. Recovery of MeP-P from serum was ≥68% across the validated concentration range, and contributions from matrix effects were minimal. The method provides a direct, quantitative measurement of MeP-P found in clinical samples suspected of nerve agent exposure and subjected to such post-sampling stresses as elevated temperature and extended shipping.
authors.affiliation: Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention , Atlanta, Georgia 30341, United States.
authors.forename: Melissa D
authors.initials: MD
authors.lastname: Carter
authors.forename: Brian S
authors.initials: BS
authors.lastname: Crow
authors.forename: Brooke G
authors.initials: BG
authors.lastname: Pantazides
authors.forename: Caroline M
authors.initials: CM
authors.lastname: Watson
authors.forename: Jerry D
authors.initials: JD
authors.lastname: Thomas
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Blake
authors.forename: Rudolph C
authors.initials: RC
authors.lastname: Johnson
mesh_terms: D002091:Butyrylcholinesterase / Q000378:metabolism*; D002619:Chemical Warfare Agents / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D063065:Organophosphonates / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D044967:Serum / Q000737:chemistry / Q000201:enzymology; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002619:Chemical Warfare Agents; D063065:Organophosphonates; D009943:Organophosphorus Compounds; D010446:Peptide Fragments; C032627:methylphosphonic acid; D002091:Butyrylcholinesterase