Direct Quantitation of Methyl Phosphonate Adducts to Human Serum Butyrylcholinesterase by Immunomagnetic-UHPLC-MS/MS
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- 作者：Melissa D. Carter ; Brian S. Crow ; Brooke G. Pantazides ; Caroline M. Watson ; Jerry D. Thomas ; Thomas A. Blake ; Rudolph C. Johnson
- 刊名：Analytical Chemistry
- 出版年：2013
- 出版时间：November 19, 2013
- 年：2013
- 卷：85
- 期：22
- 页码：11106-11111
- 全文大小：239K
- 年卷期：v.85,no.22(November 19, 2013)
- ISSN：1520-6882
文摘
Hydrolysis of G- and V-series organophosphorus nerve agents (OPNAs) containing a phosphorus鈥搈ethyl bond yields a methylphosphonic acid (MeP) product when adducted to human butyrylcholinesterase (BChE). The MeP adduct is considered a sign of 鈥渁ging鈥?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鈥搕andem mass spectrometry (ESI-MS/MS). Ions were detected in selected reaction monitoring (SRM) mode, and transition m/z 874.3 鈫?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鈥?50 ng/mL, with a coefficient of determination of R<sup>2sup> 鈮?0.997. Intrarun accuracy, expressed as %Relative Error (%RE), was 鈮?3.5%, 16.3%, and 3.20% at 2.0, 16, and 250 ng/mL, respectively; the corresponding precision expressed as %RSD was 鈮?1.9%, 6.15%, and 3.39%. Interday %RSD was 鈮?.13%, 5.69%, and 1.91%. Recovery of MeP-P from serum was 鈮?8% 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.