Journal of Analytical Toxicology, 2016;40:419–426 doi: 10.1093/jat/bkw049 Advance Access Publication Date: 23 June 2016 Article
Article
Lewisite Metabolites in Urine by Solid Phase Extraction—Dual Column Reversed-Phase Liquid Chromatography—Isotope Dilution Tandem Mass Spectrometry Jason D. Palcic1,*, Stephen F. Donovan2, Janet S. Jones1, E. Lindsay Flagg1, Redentor A. Salonga1, Walter E. Mock1, and Victor S. Asirvatham1 1
Florida Department of Health, Bureau of Public Health Laboratories, 1217 N. Pearl St, Jacksonville, FL 32202, USA, and 2NMS Labs, 3701 Welsh Rd, Willow Grove, PA 19090, USA *Author to whom correspondence should be addressed. Email: jason.palcic@flhealth.gov
Abstract Lewisite (2-chlorovinyldichloroarsine) is a chemical warfare agent developed during World War I. A quantitative method using solid phase extraction (SPE) followed by dual column liquid chromatography (LC)—isotope dilution tandem mass spectrometry (MS-MS) was developed for the determination of (2-chlorovinyl)arsonic acid (CVAOA), a metabolite of Lewisite, in human urine. The sample was treated with hydrogen peroxide to oxidize any (2-chlorovinyl)arsonous acid (CVAA) that remained in the trivalent arsenic oxidation state. There was 1.19% (arsenic purity) of bis-(2-chlorovinyl)arsinic acid (BCVAOA), a minor Lewisite metabolite, in the stock CVAA material. The high-throughput method qualitatively assessed BCVAOA simultaneously utilizing normalphase silica SPE followed by reversed-phase C18 LC for an orthogonal separation. The chromatographic method results in a 5.8-min cycle time with adequate retention (k′ = 2.4) of CVAOA. The mass spectrometer was operated in positive electrospray ionization mode with quantitative m/z 186.9→61.0 and confirmation 186.9→91.0 mass transitions. This selective method demonstrated linearity, accuracy and reproducibility for the clinically relevant calibration range (25–3,200 µg/L as CVAA). The method detection limit was 3.3 µg/L as CVAA from a 10 µL injection. This LC–MS-MS emergency response method has a throughput of >240 samples (2.5 extracted 96-well plates) per day.
Introduction The Organisation for the Prohibition of Chemical Weapons (OPCW), Chemical Weapons Convention (CWC) listed Lewisite (L) as a Schedule I chemical warfare agent (1). Lewisite was mass produced by several countries during World War I (2) and the destruction of stockpiles has been overseen by the OPCW. However, a few countries have not ratified the CWC and their stockpiles remain unaccounted for. Lewisite is a highly toxic vesicant (3, 4) that can be relatively easily synthesized by the catalytic reaction of arsenic trichloride and acetylene (5, 6). The relative ease of
synthesis and availability of stockpiled agent makes Lewisite a chemical threat that could be used by terrorist organizations to endanger public health. In the early 1990s, Lewisite ordnance rounds were unearthed in the Spring Valley neighborhood in Washington, DC, former site of American University Experimental Station (7, 8). The extent to which people were exposed to chemical warfare agent(s) or their breakdown products was not fully known. Although during soil excavations, some individuals reported burning eyes and respiratory irritation, which would be consistent with Lewisite exposure from the
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420 chemical munitions found. Clinical testing was limited to inorganic arsenic and total arsenic in urine and hair, and tested 40 individuals with arsenic levels within the normal reference range. Koryagina et al. (9) demonstrated the time-dependent urinary excretion profile of rats subcutaneously injected with Lewisite, with and without antidotal therapy. Lewisite urinary biomarkers persist in the body for a few days after an exposure; however, the highest concentrations are detected within the first 24 hours. Lewisite metabolite detection is complex, because the catalytic reaction forms multiple products; (2-chlorovinyl)dichloroarsine (L1, Cl2AsC2H2Cl), bis(2-chlorovinyl)chloroarsine (L2, ClAs(C2H2Cl)2), tris(2-chlorovinyl)arsine (L3, As(C2H2Cl)3), byproducts and isomers. Weapons-grade Lewisite munitions contain, on average, 64.0% (wt%) L1, 28.5% L2 and 98% pure) were purchased from Thermo Fisher Scientific (Fair Lawn, NJ). The US Army Medical Research Institute of Chemical Defense (Aberdeen Proving Ground, MD) provided semi-purified hydrolyzed Lewisite to the Centers for Disease Control and Prevention (CDC) National Center for Environmental Health (NCEH). An aqueous stock CVAA standard was provided to the Florida Department of Health, Bureau of Public Health Laboratories, a member of the Laboratory Response Network—Chemical (LRN-C). The concentration was determined by diluting (1:200) the stock material and measuring total arsenic against a National Institute of Standards and Technology traceable standard purchased from High Purity Standards (Charleston, SC). An additional 1:20 dilution was applied with ICP–MS diluent (2% nitric acid; 10 µg/ L iridium, m/z 193 as an internal standard) to be within the 5–4,000 µg/L calibration range. Total arsenic was measured by ICP–MS at m/z 91 using a PerkinElmer (Shelton, CT) DRCII with 1.2 mL/min oxygen (research grade >99.999% Airgas, Radnor, PA) in the dynamic reaction cell (RPa = 0, RPq = 0.75). A 100 µL aliquot of the 1:200 diluted stock standard was further diluted with 300 µL of mobile phase (11.5 mM tetrabutylammonium hydroxide, 5 mM succinic acid, 2% isopropyl alcohol and pH 5.5) and 100 µL of water and analyzed by an Agilent Technologies (Santa Clara, CA) 1200 series LC and 8800 ICP–MS-MS using conditions described (25) with and without the addition of hydrogen peroxide. A 4.6 × 150-mm, 5-µm RP-amide column from SigmaAldrich (Milwaukee, WI) at 1.0 mL/min was used for better resolution of L1 metabolites, and a 32-min run-time was used to elute the L2 metabolite(s). The unoxidized sample was used to determine the extent of CVAA oxidation to CVAOA. The stock standard was determined to be predominately CVAA, gem-CVAOA was not detected. The oxidized sample was used to determine the arsenic purity: 91.38% CVAA (+CVAOA), 2.97% gem-CVAA and 1.19% BCVAA (+BCVAOA), with the remainder as inorganic arsenic, to correct for the stock standard concentration. The concentration was also corrected for the molecular/elemental arsenic ratio (i.e., CVAA, 170.4/74.9). The calculated concentrations were 2.132, 0.0693 and 0.0350 mg/mL for CVAA, gem-CVAA and BCVAA, respectively. Analytical standards for CVAA (25, 50, 100, 200, 400, 800, 1,600 and 3,200 µg/L) and quality controls (QC) for CVAA (75, 300 and 1,200 µg/L) were prepared in base urine. After volumetric preparation of matrix-matched calibrators and QC, they were subsequently stored at ≤–70°C in cryogenic vials until use. The base urine is defined as urine from laboratory volunteers collected with informed consent. The collection protocol was described in detail elsewhere (25) and approved by the Florida Department of
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Lewisite Metabolites in Urine by Solid Phase Extraction Health Institutional Review Board (IRB). Isotopically labeled CVAA-13C2D2 was synthesized by Los Alamos National Laboratory (Los Alamos, NM). The concentration was determined at 1.023 mg/ mL of CVAA-13C2D2 (99.17% pure, as arsenic) by LC–ICP–MS-MS, analogous to CVAA as described above. The gem-CVAA-13C2D2 was not detected, and only trace (
