Article pubs.acs.org/ac
High-Throughput Immunomagnetic Scavenging Technique for Quantitative Analysis of Live VX Nerve Agent in Water, Hamburger, and Soil Matrixes Jennifer S. Knaack,†,¶ Yingtao Zhou,† Carter W. Abney,‡ Samantha M. Prezioso,§ Matthew Magnuson,∥ Ronald Evans,⊥ Edward M. Jakubowski,⊥ Katelyn Hardy,†,◆ and Rudolph C. Johnson*,† †
National Center for Environmental Health, Division of Laboratory Sciences, Emergency Response and Air Toxicants Branch, Centers for Disease Control and Prevention, 4770 Buford Highway, MS F44, Chamblee, Georgia 30341, United States ‡ Oak Ridge Institute for Science and Education Fellow, Centers for Disease Control and Prevention, 4770 Buford Highway, MS F44, Chamblee, Georgia 30341, United States § IHRC, Incorporated, Centers for Disease Control and Prevention, 2 Ravinia Drive, Suite 1260, Atlanta, Georgia, United States ∥ Environmental Protection Agency, 26 West Martin Luther King Drive, Mailstop NG-16, Cincinnati, Ohio 45268, United States ⊥ U.S. Army Edgewood Chemical Biological Center, E3150 RDCB-DRT-A, 5183 Blackhawk Road, Aberdeen Proving Ground, Maryland 21010-5424, United States ABSTRACT: We have developed a novel immunomagnetic scavenging technique for extracting cholinesterase inhibitors from aqueous matrixes using biological targeting and antibody-based extraction. The technique was characterized using the organophosphorus nerve agent VX. The limit of detection for VX in high-performance liquid chromatography (HPLC)-grade water, defined as the lowest calibrator concentration, was 25 pg/mL in a small, 500 μL sample. The method was characterized over the course of 22 sample sets containing calibrators, blanks, and quality control samples. Method precision, expressed as the mean relative standard deviation, was less than 9.2% for all calibrators. Quality control sample accuracy was 102% and 100% of the mean for VX spiked into HPLC-grade water at concentrations of 2.0 and 0.25 ng/mL, respectively. This method successfully was applied to aqueous extracts from soil, hamburger, and finished tap water spiked with VX. Recovery was 65%, 81%, and 100% from these matrixes, respectively. Biologically based extractions of organophosphorus compounds represent a new technique for sample extraction that provides an increase in extraction specificity and sensitivity.
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AChE protection against OP adduction by acting as a bioscavenger for OPs.7 OP adducts to BuChE can be used as a biomarker of exposure.8,9 Many quantitative methods have been developed for assessing OPNA levels in environmental and food samples. These methods can be categorized as either nonspecific methods, such as the enzyme-linked immunosorbent assay that does not distinguish between OP compounds, or specific methods, such as gas chromatography coupled to mass spectrometry (GC/MS) that provides compound identification. A variety of nonspecific biosensors utilizing AChE activity to generate chemical and electrical signals have been developed for the analysis of OP pesticides10−12 and nerve agents13−15 in environmental and food matrixes. Limits of detection as low as 0.5, 4.6, 20, and 20 μg/mL have been reported for sarin, soman, VX, and Russian VX.16 Lower limits of detection, between 2517 and 50 ng/mL,18 have been reported for dimethyl methylphosphonate, an OPNA mimic.
rganophosphorus nerve agents (OPNAs) represent a large class of chemical warfare agents with anticholinesterase activities. All agents are highly toxic synthetic alkylphosphonic esters with VX being the most toxic having an estimated human LD50 value of 10 mg on the skin of a 70 kg person.1 Unlike G-series agents such as sarin and tabun, Vseries agents like VX are persistent in the environment and less volatile.2,3 For these reasons, VX poses a significant environmental health threat and serves as an area denial chemical weapon. Subchronic exposure to levels of only 2.25 μg VX/kg/ day, corresponding to 5% of the acute LD50 dose, has been shown to induce behavioral effects in rats.4 So it is imperative that detection methods for VX and other OPNAs have limits of detection in the nanogram per milliliter range for environmental restoration following an OPNA release and to ensure sufficient decontamination to risk-based goals. OPNA poisoning symptoms include miosis, twitching, seizures, and increased secretions, and severe exposures can result in death.5,6 These symptoms derive from inhibition of acetylcholinesterase (AChE) via covalent adduct formation at the enzyme active site which allows constant activation of cholinergic receptors. Butyrylcholinesterase (BuChE), a naturally occurring protein found in plasma, is believed to provide © 2012 American Chemical Society
Received: August 31, 2012 Accepted: October 22, 2012 Published: November 5, 2012 10052
dx.doi.org/10.1021/ac3025224 | Anal. Chem. 2012, 84, 10052−10057
Analytical Chemistry
Article
were prepared in 2 mL deep 96-well plates and 250 μL shallow 96-well plates (VWR, Radnor, PA). These plates were sealed with either Eppendorf adhesive foils or Easy Pierce 20 μm healsealing foils (Fisher Scientific, Fair Lawn, NJ). Sample mixing in 96-well format was carried out on an Eppendorf MixMate plate mixer (VWR, Radnor, PA). IMSc extraction was performed on a KingFisher automated magnetic bead processor with deepwell head attachment (95041-912, VWR, Radnor, PA) fitted with a deepwell comb (83007-594, VWR, Radnor, PA). Extracted samples were filtered on a 10 kDa multiscreen Ultracel-10 filter plate (Millipore MAUF01010 purchased from Fisher Scientific, Fair Lawn, NJ) fitted with a 96-well Advion collection plate (Ithaca, NY). Plate centrifugation was conducted on an Eppendorf 5810R with a microplate bucket rotor (Fisher Scientific, Fair Lawn, NJ). Samples were incubated at 37 °C in a Precision (Winchester, VA) water bath. Hamburger meat (80% lean/20% fat) was purchased locally from a Food Lion grocery store (Edgewood, MD), and Sassafras sandy loam soil was furnished by the ECBC Environmental Toxicology Branch (Aberdeen Proving Ground, MD). Roughly extracted hamburger and soil extracts were directly injected in an HPLC from GC vials with inserts (Agilent Technologies, Santa Clara, CA). Finished tap water was obtained from laboratory sinks at the CDC (Chamblee, GA). VX calibration curves and quality control (QC) materials were prepared by diluting 10 ppm VX into Tedia HPLC-grade water to create 100 and 1 ng/mL spiking solutions. These solutions were then spiked into HPLC-grade water to create calibrators at 0.025, 0.090, 0.310, 1.13, and 4.00 ng/mL. QC materials were also made in HPLC-grade water at 2.0 ng/mL (high-QC) and 0.25 ng/mL (low-QC). Unspiked HPLC-grade water served as a negative control. Extractions were conducted using 96-well deep V-bottom KingFisher microplates (11388− 566 VWR, Radnor, PA) containing 500 μL aliquots of each standard, QC, and negative control. Isotopically labeled internal standard corresponding to the VX-adducted peptide was added to each calibrator, QC, blank, and sample following filtration and prior to chromatography at a working concentration of 550 ng/mL in 0.6% formic acid. Magnetic beads were prepared using a previously described method for clinical samples8 and scaled up for larger bead volumes. Briefly, 2 mL of protein G-conjugated magnetic beads was transferred to a 15 mL Falcon tube (Fisher Scientific, Fair Lawn, NJ). A DynaMag-15 magnet (Invitrogen, Carlsbad, CA) was used to separate magnetic beads from the storage solution, and the storage solution was removed. Beads were washed three times in 4 mL of 1× PBS and incubated in 400 μL of a 1 mg/mL solution of monoclonal BuChE antibody in PBST overnight on a Dynal sample mixer (Invitrogen, Carlsbad, CA). The antibody solution was then removed and the beads were washed in two 4 mL aliquots of triethanolamine buffer. Antibody was cross-linked to the beads by incubating the beads in 4 mL of DMP in triethanolamine buffer (5.4 g/L) for 30 min at room temperature with rotation on the Dynal sample mixer. The DMP solution was removed, and beads were incubated in 4 mL of TBS for 15 min with rotation at room temperature followed by three 2 mL PBST washes. Beads were then conjugated to BuChE proteins by incubating them for 2 h with rotation in 10 mL of pooled human serum that had been previously characterized as free from exposure to VX. Finally, beads were washed three times in 2 mL of PBST and stored in PBST at 4 °C until use.
Methods that provide compound identification generally rely on GC/MS and provide lower limits of detection than nonspecific biosensors. Because OPNAs can hydrolyze upon contact with water, measurements of degradation products can be made as indicators for OPNAs. Limits of detection for degradation products of VX and Russian VX have been reported as low as 10 ng/mL in water.19 Analysis of OPNAs has also been made by measuring acid hydrolysis products with limits of detection between 4.3 and 11 ng/mL.20 Here we describe a novel, sensitive technique termed “immunomagnetic scavenging” (IMSc) coupled high-performance liquid chromatography with tandem mass spectrometry (HPLC/MS/MS) for extracting OP compounds from aqueous matrixes. In this method, BuChE-conjugated magnetic beads scavenge OP compounds by forming stable adducts that are extracted by immunomagnetic separation and enzymatically digested into peptides that were analyzed by HPLC/MS/MS for BuChE−OP adducts. The method was characterized for sensitivity and precision using VX as a model nerve agent that does not readily hydrolyze in water.3 IMSc/HPLC/MS/MS provides compound identification, allows for compound quantitation, and produces a lower detection limit for VX than other analytical methods.
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MATERIALS AND METHODS The 10 ppm VX (O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate, CAS 50782-69-9) in isopropyl alcohol was provided to the Centers for Disease Control and Prevention (CDC) by Lawrence Livermore National Laboratory (Livermore, CA) through interagency agreement number DW89-92261601 with the Environmental Protection Agency. Peptides corresponding to the BuChE active site with the sequence FGESAGAAS and the corresponding stable isotope-labeled peptide (13C3D315N) were synthesized at Los Alamos National Laboratory (Los Alamos, NM). Stable isotope-labeled (VX−BuChE−13C4D615N) and unlabeled (VX−BuChE) VX-adducted peptides were also synthesized at Los Alamos National Laboratory. VX and stable isotopically labeled VX (2H5−VX) used in direct analysis of hamburger and soil extracts were provided by the U.S. Army Edgewood Chemical Biological Center (ECBC) (Aberdeen Proving Ground, MD). Monoclonal antibodies against human BuChE from clone 3E8 (HAH0020102, Thermo Fisher Affinity BioReagents, Rockford, IL) were purchased from Fisher Scientific (Fair Lawn, NJ). The following materials were purchased from Sigma-Aldrich (St. Louis, MO): triethanolamine buffer solution containing 0.2 M triethanolamine with magnesium ions, EDTA, and
