Diethyl 4-nitrophenyl Phosphate Research Articles

A MOF-doped molecularly imprinted polymer/MOF hybrid gel incorporating with pH-buffering sodium acrylate for practical detoxification of organophosphorus nerve agents

The fatal toxicity of organophosphorus nerve agents (ONAs) and the realistic condition of chemical attack occurring place trigger the urgent need to develop catalyst materials with degrading ability under the natural environment without bulk water and bases. Here, we strategically design a molecularly imprinted polymer (MIP)/MOF hybrid catalyst material by integrating enzyme-mimic MIP with Zr-MOF UiO-66-NH2 which demonstrate promising catalytic performance for degradation of ONAs. Furthermore, sodium acrylate functions as a hydrophilic and alkaline functional unit together with pyridine-amidoxime based functional monomers (PAAO) to copolymerize into molecularly imprinted polymeric network. The basic sodium carboxylate groups were installed near the active sites to provide appropriate alkaline microenvironment, non-bulk water and acid buffer capacity for the catalysis in pure water. The resultant MIP/MOF composite catalyst demonstrate outstanding catalytic activities in both liquid and solid phase hydrolysis of ONAs. Specifically, the half-life of liquid phase hydrolysis catalyzed by MIP/UiO-66-NH2-0.5 achieves 9.4 min for diethyl-4-nitrophenyl phosphate (DENP) and 10 min for dimethyl 4-nitrophenyl phosphate (DMNP), and solid-state hydrolysis present rapid degradation efficiency with a half-life of 28.6 min. In addition, composite catalyst MIP-Ag/UiO-66-NH2-0.5 that retains substrate selectivity of MIP, enables the simultaneous degradation of two types of organophosphorus substrates (phosphate and thiophosphate) by MIP and MOF in a synergistic way. Summarily, this work displays promising potential of MIP/MOF hybrid enzyme-like catalyst as effective and practical material for the practical protection and detoxification of organophosphorus nerve agents.

An efficient bioinspired functional micellar nanoreactor for dephosphorylation reactions

An efficient bioinspired catalytic system for dephosphorylation reactions should combine imidazole reaction sites with a hydrophobic region that favors intimate interaction with the substrate. In this work, we show that the catalytic activity of long-chain alkylimidazoles such as 1-tetradecylimidazole (ImR) that are poorly soluble in aqueous medium is greatly improved by the use of mixed micelles of ImR with cetyltrimethylammonium bromide (CTAB). The size, zeta potential, degree of protonation of ImR and the catalytic activity in dephosphorylation of diethyl 2,4-dinitrophenyl phosphate (DEDNPP) were found to be functions of the CTAB/ImR ratio and the pH of the medium, with optimal catalytic activity at pH 6 and at low CTAB/ImR ratios (3/1 and 2/1). For 3/1 CTAB/ImR, the ImR-catalyzed dephosphorylation of DEDNPP was 9x106-fold faster than the spontaneous hydrolysis of DEDNPP and ca. 102-fold faster than the reaction catalyzed by free imidazole. The same 3/1 CTAB/ImR mixed micelles were also shown to accelerate the dephosphorylation of the pesticide Paraoxon (diethyl 4-nitrophenylphosphate). The relative structural simplicity of the components of these mixed micellar aggregates, combined with the ability to promote significant accelerations of the rates of dephosphorylation reactions, point to mixed micellar aggregates of this type as prototypes for the design of efficient nanoreactors for performing alkylimidazole-catalyzed dephosphorylation reactions.

Determination of Picomolar Concentrations of Paraoxon in Human Urine by Fluorescence-Based Enzymatic Assay.

Organophosphate (OP) pesticides are widely used in the agricultural field and in the prevention of pest infestation in private and public areas of cities. Despite their unquestionable utility, several of these compounds demonstrate toxic effects to the environment and human health. In particular, the occurrence of some organophosphate pesticides is correlated to the incidence of nervous system disorders, especially in children. The detection of pesticide residues in the human body represents an important task to preserve human health. In our work we propose the use of esterase-based biosensors as a viable alternative to the expensive and time-consuming systems currently used for their detection in human fluids. Using the esterase-2 activity, coupled with a fluorescence inhibition assay, we are able to detect very low concentration levels of diethyl (4-nitrophenyl) phosphate (paraoxon) in the range of the femtomole (fmol). Method robustness tests indicate the stability of esterase-2 in a diluted solution of 4% human urine, and we are able to accurately determine concentration levels of paraoxon in the range from 0.1 to 2 picomoles (pmol). The system sensitivity for OP detection is calculated at 524 ± 14.15 fmol of paraoxon recognized at 10% of inhibition, with an estimated limit of quantification of 262 ± 8.12 pmol mL−1. These values are comparable with the most recent analysis methods based on mass spectrometry carried out on human samples for pesticide detection. This research represents a starting point to develop cheap and fast testing methods for a rapid screening of toxic substances in human samples.

Rapid hydrolysis of nerve agent simulants by molecularly imprinted porous crosslinked polymer incorporating mononuclear zinc(II)-picolinamine-amidoxime module

Effective catalyst material capable of rapid organophosphate (OP) detoxification is highly imperative for practical application. Herein, taking advantage of molecular imprinting technique with solvothermal method, molecular imprinted porous crosslinked polymers (MIPCPs) incorporating mononuclear zinc(II)-picolinamine-amidoxime (Zn-PAAO) functional module is developed as organophosphorus hydrolase (OPH) mimic to realize rapid OP hydrolysis in minute level. The synergy of monopyridyl with amidoxime of Zn-PAAO generates a reactive Lewis acidic zinc(II) ion and a strong free oxime nucleophile at the active center of MIPCPs. The as-fabricated MIPCP-Zn-PAAO displays extremely high catalytic ability for both phosphotriester and phosphodiester hydrolysis under mild condition (pH = 9, 30 °C). Advantageously, the observed pseudo first rate constant (kobsMIPCP) of MIPCP-Zn-PAAO-80% for diethyl 4-nitrophenyl phosphate (DENP, a nerve agent stimulant) hydrolysis is 2 × 104-fold higher than that of self-hydrolysis. The maximum velocity (Vmax) of MIPCP-Zn-PAAO-80% for DENP hydrolysis is even 6.9 times higher than that of the reported free organophosphorus hydrolase (OPH), representing the highest level among current polymer enzyme mimics. Our work provides a new promising strategy for effective OP catalytic degradation.

Reclaimable La: ZnO/PAN nanofiber catalyst for photodegradation of methyl paraoxon and its toxicological evaluation utilizing early life stages of zebra fish (Danio rerio)

In the present work, La: ZnO/PAN and ZnO/PAN nanofibers were designed and evaluated as a catalyst for the photodegradation of diethyl (4-nitrophenyl) phosphate (MP). The solid catalyst was characterized by various analytical techniques. Hexagonal and wurtzite crystal structure of the fabricated rod-shaped La-ZnO nanoparticles were substantiated from the XRD pattern. The catalytic activity of ZnO nanorods enhanced in the presence of La due to their surface plasmon resonance (SPR) effect. Real-time experiment carried out in the wastewater sample spiked with MP showed 100 %degradation efficiency at 150 min as quantified by HPLC. The product of degradation (4-nitrophenol) was directly converted to tertiary compound (p-amino phenol) eschewing the intermediate as attested by GC–MS analysis. Acute toxicity of MP was evaluated utilizing early stages of zebra fish and the degraded product was found to be less toxic for the aquatic living system.

Theoretical investigation of the neutral hydrolysis of diethyl 4-nitrophenyl phosphate (paraoxon) in aqueous solution.

In this work the neutral or spontaneous hydrolysis of paraoxon, one of the most popular organophosphate pesticides, in aqueous solution was investigated at the DFT and MP2 levels of theory, using a combination of local solvation of the phosphoryl group with explicit water molecules, and treating the long range solvent effects using continuum solvation model. In contrast to the alkaline hydrolysis, the neutral hydrolysis takes place in two steps, through an AN + DN mechanism, with formation of a pentacoordinate phosphorane intermediate. The reaction has activation free energies of 31.8 and 1.9kcal mol-1 for the first and second steps, respectively, and has an overall reaction free energy of -9.3kcal mol-1, computed at the MP2/6-311++G(2d,2p)//B3LYP/6-31+G(d) level of theory. The reaction proceeds through a sequence of proton transfer processes from the attacking water molecule and ends with the protonation of the nitrophenolate leaving group. Explicit description of the local solvating water molecules is essential to describe the proton transfer processes along the reaction coordinate and to stabilize the pentacoordinate intermediate formed. The neutral hydrolysis is very slow and has an overall rate constant of 3.05 × 10-11s-1, computed at the MP2/6-311++G(2d,2p)//B3LYP/6-31+G(d) level of theory. This result, in conjunction with the sensitivity of the rate constant to the experimental conditions, indicates that the hydrolysis of paraoxon in aqueous solution can be even slower than predicted experimentally.

Synthesis, Biological Evaluation, and Docking Studies of Novel Bisquaternary Aldoxime Reactivators on Acetylcholinesterase and Butyrylcholinesterase Inhibited by Paraoxon.

Nerve agents and oxon forms of organophosphorus pesticides act as strong irreversible inhibitors of two cholinesterases in the human body: acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8), and are therefore highly toxic compounds. For the recovery of inhibited AChE, antidotes from the group of pyridinium or bispyridinium aldoxime reactivators (pralidoxime, obidoxime, HI-6) are used in combination with anticholinergics and anticonvulsives. Therapeutic efficacy of reactivators (called “oximes”) depends on their chemical structure and also the type of organophosphorus inhibitor. Three novel oximes (K131, K142, K153) with an oxime group in position four of the pyridinium ring were designed and then tested for their potency to reactivate human (Homo sapiens sapiens) AChE (HssACHE) and BChE (HssBChE) inhibited by the pesticide paraoxon (diethyl 4-nitrophenyl phosphate). According to the obtained results, none of the prepared oximes were able to satisfactorily reactivate paraoxon-inhibited cholinesterases. On the contrary, extraordinary activity of obidoxime in the case of paraoxon-inhibited HssAChE reactivation was confirmed. Additional docking studies pointed to possible explanations for these results.

Enzymeless PEDOT-based electrochemical sensor for the detection of nitrophenols and organophosphates

PEDOT:PSS modified electrodes were prepared via one-step electropolymerization and employed as electrochemical sensor for the detection of nitrophenols and organophosphorus compounds. The electrocatalytic behavior of PEDOT towards the redox reactions of the nitrophenol isomers were studied by electrochemical and spectroscopy techniques and a detection mechanism is proposed. The detection of the isomers was possible by the analysis of the oxidation peaks of the intermediates generated by the direct reduction of the nitro groups and the analytical performance was evaluated in terms of the individual analysis of each isomer and in a multi-component solution. It was found that the simultaneous detection of the isomers is feasible, although the quantification is only possible of 3- and 4-NPh in the multi-component sample. In addition, hazardous organophosphate pollutants such as diethyl 2,4-dinitrophenyl phosphate (DEDNPP) and diethyl 4-nitrophenyl phosphate (Paraoxon) were also detected by the study of the electrochemical reduction of the nitro groups presented and as observed by the nitrophenol molecules and some analytical parameters were analyzed. The results presented herein suggest the construction of a rapid and straightforward electrochemical sensor for these important pollutants, in a micromolar detection.

Reactivity of inorganic α-nucleophiles in acyl group transfer processes in water and surfactant micelles: I. Systems based on organic complexes of tribromide anion

Systems based on organic complexes of tribromide anion generate upon dissolution in water nucleophile–oxidant couple HOBr/BrO– and accelerate hydrolysis of ethyl 4-nitrophenyl ethylphosphonate, diethyl 4-nitrophenyl phosphate, and 4-nitrophenyl p-toluenesulfonate by a factor of 15–90 in the presence of cationic surfactant micelles. As in water, hypobromite ion in surfactant micelles acts as α-nucleophile, and the magnitude of the α-effect almost does not change in going from water to micelles. Micellar effects of surfactants are determined by the nucleophilicity of hypobromite ion in surfactant micelles and by solubilization of the substrate and BrO–, which largely depend on the counterion concentration in the micelle surface layer. The main factor responsible for the observed acceleration is increased reactant concentration in the micellar pseudophase.

Novel approaches to mitigating parathion toxicity: targeting cytochrome P450-mediated metabolism with menadione.

Accidental or intentional exposures to parathion, an organophosphorus (OP) pesticide, can cause severe poisoning in humans. Parathion toxicity is dependent on its metabolism by the cytochrome P450 (CYP) system to paraoxon (diethyl 4-nitrophenyl phosphate), a highly poisonous nerve agent and potent inhibitor of acetylcholinesterase. We have been investigating inhibitors of CYP-mediated bioactivation of OPs as a method of preventing or reversing progressive parathion toxicity. It is well recognized that NADPH-cytochrome P450 reductase, an enzyme required for the transfer of electrons to CYPs, mediates chemical redox cycling. In this process, the enzyme diverts electrons from CYPs to support chemical redox cycling, which results in inhibition of CYP-mediated biotransformation. Using menadione as the redox-cycling chemical, we discovered that this enzymatic reaction blocks metabolic activation of parathion in rat and human liver microsomes and in recombinant CYPs important to parathion metabolism, including CYP1A2, CYP2B6, and CYP3A4. Administration of menadione to rats reduces metabolism of parathion, as well as parathion-induced inhibition of brain cholinesterase activity. This resulted in inhibition of parathion neurotoxicity. Menadione has relatively low toxicity and is approved by the Food and Drug Administration for other indications. Its ability to block parathion metabolism makes it an attractive therapeutic candidate to mitigate parathion-induced neurotoxicity.

New copper(II) complexes including pyridine-2,5-dicarboxylic acid: synthesis, spectroscopic, thermal properties, crystal structure and how these complexes interact with purified PON 1 enzyme

We report the synthesis of two square-pyramidal copper(II) complexes, [Cu(2,5-pydc)(2-aepy)(H2O)]·H2O, 1, and [Cu(2,5-pydc)(2-ampy)(H2O)]·H2O, 2 (2-aepy = 2-(aminoethyl)pyridine, 2-ampy = 2-(aminomethyl)pyridine, 2,5-pydc = pyridine-2,5-dicarboxylic acid or isocinchomeronic acid). The synthesized complexes have been characterized by X-ray diffraction, FT-IR, elemental, and thermal analysis techniques. The crystal structure of 1 was established by X-ray analysis. Powder X-ray diffraction analysis showed that the complexes are pure. The inhibition of human serum paraoxonase 1 (PON 1, EC 3.1.8.1) enzyme with these complexes were investigated. We used diethyl 4-nitrophenyl phosphate as a substrate to measure the paraoxonase activity of PON 1 enzyme spectrophotometrically. Complexes 1 and 2 decreased the in vitro PON 1 activity with different inhibition mechanisms. Complexes 1 and 2 inhibited paraoxonase activity of this enzyme as competitively and noncompetitively, respectively.

Identification of products in the reaction of 2-[(hydroxyimino)methyl]-1,3-dimethylimidazolium iodide with diethyl 4-nitrophenyl phosphate in alkaline medium

Products of the reaction of 2-[(hydroxyimino)methyl]-1,3-dimethylimidazolium iodide with diethyl 4-nitrophenyl phosphate in alkaline medium have been identified by electronic spectroscopy, one-(1H, 31P, 13C) and two-dimensional (1H–1H COSY, 1H–31P HMBC) NMR techniques, and NMR titration. 2-[(Hydroxyimino)-methyl]-1,3-dimethylimidazolium iodide has been found to act as nucleophile which is likely to be converted into 2-cyano-1,3-dimethylimidazolium. The other products are 4-nitrophenol and diethyl hydrogen phosphate. Other reaction paths, such as nucleophilic catalysis of the hydrolysis of diethyl 4-nitrophenyl phosphate by 2-[(hydroxyimino)methyl]-1,3-dimethylimidazolium iodide and formation of a stable phosphorylated product, have been ruled out.

Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication

Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation.

Effects of mono and dinuclear copper(II) complexes derived from non-steroidal anti-inflammatory drug naproxen on human serum paraoxanase1 (PON1) activity

The inhibition of human paraoxonase 1 (PON1, EC 3.1.8.1) enzyme, with two Cu(II) complexes derived from naproxen was investigated by using the paraoxonase activity method with diethyl 4-nitrophenyl phosphate as substrate. The complexes [Cu 2 (µ-nap) 4 (3-pic) 2 ] (1) and [Cu(nap) 2 (H 2 O)(4-pic) 2 ] (2) decreased the in vitro PON1 activity with different inhibition mechanisms. The inhibition mechanism of complex 1 was uncompetitive whereas complex 2 was noncompetitive inhibitors. In this study, complexes mentioned above showed effective inhibitory activity on PON1. IC 50 values for [Cu 2 (µ-nap) 4 (3-pic) 2 ] (1) and [Cu(nap) 2 (H 2 O)(4-pic) 2 ] (2) were of 0.109 mM and 0.103 mM for PON1, respectively. K i values for complex 1 and complex 2 were of 0.116 mM and 0.121 mM for PON1, respectively.

Amplification of single molecule translocation signal using β-strand peptide functionalized nanopores.

Changes in ionic current flowing through nanopores due to binding or translocation of single biopolymer molecules enable their detection and characterization. It is, however, much more challenging to detect small molecules due to their rapid and small signal signature. Here we demonstrate the use of de novo designed peptides for functionalization of nanopores that enable the detection of a small analytes at the single molecule level. The detection relies on cooperative peptide conformational change that is induced by the binding of the small molecule to a receptor domain on the peptide. This change results in alteration of the nanopore effective diameter and hence induces current perturbation signal. On the basis of this approach, we demonstrate here the detection of diethyl 4-nitrophenyl phosphate (paraoxon), a poisonous organophosphate molecule. Paraoxon binding is induced by the incorporation of the catalytic triad of acetylcholine esterase in the hydrophilic domain of a short amphiphilic peptide and promotes β-sheet assembly of the peptide both in solution and for peptide molecules immobilized on solid surfaces. Nanopores coated with this peptide allowed the detection of paraoxon at the single molecule level revealing two binding arrangements. This unique approach, hence, provides the ability to study interactions of small molecules with the corresponding engineered receptors at the single molecule level. Furthermore, the suggested versatile platform may be used for the development of highly sensitive small analytes sensors.

ZnII/pyridyloxime complexes as potential reactivators of OP-inhibited acetylcholinesterase: In vitro and docking simulation studies

In order to investigate the ability of metal complexes to act as reactivators of organophosphorus compounds (OP)-inhibited acetylcholinesterase (AChE), we have synthesized and crystallographically characterized three novel mononuclear ZnII complexes formulated as [ZnCl2{(4-py)CHNOH}2] (1), [ZnBr2{(4-py)CHNOH}2] (2) and [Zn(O2CMe)2{(4-py)CHNOH}2]∙2MeCN (3∙2MeCN), where (4-py)CHNOH is 4-pyridinealdoxime. Their reactivation potency was tested in vitro with a slight modification of the Ellman's method using Electric eel acetylcholinesterase and the insecticide paraoxon (diethyl 4-nitrophenyl phosphate) as inhibitor. The activity of the already reported complex [Zn2(O2CPh)2{(4-py)CHNOH}2]·2MeCN (4·2MeCN) and of the clinically used drug obidoxime 1,1′-[oxybis(methylene)]bis{4-[(E)- (hydroxyimino)methyl]pyridinium} was also examined. The results of the in vitro experiments demonstrate moderate reactivation of the metal complexes compared to the drug obidoxime. On the other hand, it is clearly shown that the metal complex is the responsible molecular entity for the observed activity, as the reactivation efficacy of the organic ligand (4-pyridinealdoxime) is found to be inconsequential. Docking simulation studies were performed in the light of predicted complex-enzyme interactions using the paraoxon-inhibited enzyme along with the four ZnII complexes and obidoxime as a reference reactivator. The results showed that the three mononuclear metal complexes possess the required characteristics to be accommodated into the active site of AChE, while the entrance of the dinuclear ZnII compound is unsuccessful. An interesting outcome of docking simulations is the fact that the mononuclear compounds accommodate into the active site of AChE in a similar mode as obidoxime.

Organophophorous Ester Degradation by Chromium(III) Terephthalate Metal–Organic Framework (MIL-101) Chelated to N,N-Dimethylaminopyridine and Related Aminopyridines

Porous materials based on chromium(III) terephthalate metal organic frameworks (MOF) MIL-101(Cr) and their complexes with dialkylaminopyridines (DAAP) were synthesized via a DAAP-MOF complexation, and tested for hydrolytic degradation of organophosphorous esters such as diethyl 4-nitrophenyl phosphate (paraoxon). Elemental analysis, TGA, XRD, FT-IR, TEM, SEM, and nitrogen adsorption measurements indicated that the DAAP units were incorporated into MIL-101 pores by complexation, keeping the parent framework intact. The DAAP-MOF enabled facile paraoxon hydrolysis in water/acetonitrile mixtures under ambient conditions (100% conversion after 24 h at pH 10). The MOF-DAAP complexes showed synergistic effects, being 7-fold and 47-fold more active than the parent MIL-101 or DAAP materials, respectively. The high hydrolysis reaction turnover was realized by simultaneous action of the Lewis acid Cr(III) center of the MOF as well as the electron-rich nucleophile, DAAP. This study demonstrates a simple and efficient method of generating catalytically active MOF materials for environmental detoxification as well as defensive applications.

Reactivity of the H2O2/B(OH)3/HO- system in the decomposition of 4-nitrophenyl esters of diethylphosphonic and diethylphosphoric acids

The kinetics of the decomposition of 4-nitrophenyl diethylphosphonate and 4-nitrophenyl diethyl phosphate by the H2O2/B(OH)3/HO– system with wide variation of the pH and the initial concentrations of H2O2 and B(OH)3 (water, 25°C) was studied. Additions of boric acid either weakly accelerate the reaction or retard it, depending on the ratio of the initial concentrations of hydrogen peroxide and boric acid and on the nature of the substrate. The peroxoborate anions (HO)3BOOH–, (HO)2B(OOH) 2 – , and (HO)3BOOB(OH) 3 2– anions generated by this system do not affect the decomposition rate of the substrates. In both substrates linear relationships were found between the observed rate constants k and the concentrations of peroxide anion.

Peroxyhydrolysis of 4-nitrophenyl diethyl phosphate in micellar systems based on imidazolium gemini surfactants

The nucleophilic reactivity of HO– and HOO– ions in the presence of imidazole gemini surfactants toward 4-nitrophenyl diethyl phosphate (NPDEP) was studied. It was shown that in the micelles of these detergents, as in water, an α effect is realized for the hydroperoxide ion. Its magnitude, characterized by the ratio of the second-order rate constants k 2(HOO-) m /k 2(HO-) m for the reaction with NPDEP in the micellar pseudophase, reaches ~100. Comparable micellar effects are observed at concentrations of the gemini surfactants not less than an order of magnitude lower than in the case of their monomeric analogs.

Identification of phosphorylated butyrylcholinesterase in human plasma using immunoaffinity purification and mass spectrometry

Paraoxon (diethyl 4-nitrophenyl phosphate) is an active metabolite of the common insecticide parathion and is acutely toxic due to the inhibition of cholinesterase (ChE) activity in the nervous systems. The inhibition of butyrylcholinesterase (BChE) activity by paraoxon is due to the formation of phosphorylated BChE adduct, and the detection of the phosphorylated BChE adduct in human plasma can serve as an exposure biomarker of organophosphate pesticides and nerve agents. In this study, we developed an immunoaffinity purification and liquid chromatography–mass spectrometry (LC–MS) strategy for identifying phosphorylated BChE in human plasma treated by paraoxon. BChE was captured by biotinylated anti-BChE polyclonal antibodies conjugated to streptavidin magnetic beads. Western blot analysis showed that the antibody was effective to recognize both native and modified BChE with high specificity. Using a purified BChE protein, we initially identified the exact phosphorylation site on the serine residue (S198) with a 108Da modification by both MS/MS and accurately measured parent ion masses and quantified the extent of phosphorylation on S198 following paraoxon treatment to be >99.9%. Then, the phosphorylated BChE peptide in paraoxon-treated human plasma following immunoaffinity purification was successfully identified based on the accurate measured mass and retention time information initially obtained from the purified BChE protein. Thus, immunoaffinity purification combined with LC–MS represents a viable approach for the detection and quantification of phosphorylated BChE as an exposure biomarker of organophosphates and nerve agents.