Direct quantitation of methyl phosphonate adducts to human serum butyrylcholinesterase by immunomagnetic-UHPLC-MS/MS

Anal Chem. 2013 Nov 19;85(22):11106-11. doi: 10.1021/ac4029714. Epub 2013 Nov 8.

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Butyrylcholinesterase / metabolism*
  • Chemical Warfare Agents / analysis*
  • Chromatography, High Pressure Liquid / methods*
  • Humans
  • Immunomagnetic Separation / methods*
  • Organophosphonates / metabolism*
  • Organophosphorus Compounds / metabolism*
  • Peptide Fragments / analysis
  • Serum / chemistry
  • Serum / enzymology
  • Spectrometry, Mass, Electrospray Ionization / methods
  • Tandem Mass Spectrometry / methods*

Substances

  • Chemical Warfare Agents
  • Organophosphonates
  • Organophosphorus Compounds
  • Peptide Fragments
  • methylphosphonic acid
  • Butyrylcholinesterase