Development of a clinical assay to measure chlorinated tyrosine in hair and tissue samples using a mouse chlorine inhalation exposure model.

Chlorine is a toxic industrial chemical with a history of use as a chemical weapon. Chlorine is also produced, stored, and transported in bulk making it a high-priority pulmonary threat in the USA. Due to the high reactivity of chlorine, few biomarkers exist to identify exposure in clinical and environmental samples. Our laboratory evaluates acute chlorine exposure in clinical samples by measuring 3-chlorotyrosine (Cl-Tyr) and 3,5-dichlorotyrosine (Cl2-Tyr) using liquid chromatography tandem mass spectrometry (LC-MS/MS). Individuals can have elevated biomarker levels due to their environment and chronic health conditions, but levels are significantly lower in individuals exposed to chlorine. Historically these biomarkers have been evaluated in serum, plasma, blood, and bronchoalveolar lavage (BAL) fluid. We report the expansion into hair and lung tissue samples using our newly developed tissue homogenization protocol which fits seamlessly with our current chlorinated tyrosine quantitative assay. Furthermore, we have updated the chlorinated tyrosine assay to improve throughput and ruggedness and reduce sample volume requirements. The improved assay was used to measure chlorinated tyrosine levels in 198 mice exposed to either chlorine gas or air. From this animal study, we compared Cl-Tyr and Cl2-Tyr levels among three matrices (i.e., lung, hair, and blood) and found that hair had the most abundant chlorine exposure biomarkers. Furthermore, we captured the first timeline of each analyte in the lung, hair, and blood samples. In mice exposed to chlorine gas, both Cl-Tyr and Cl2-Tyr were present in blood and lung samples up to 24 h and up to 30 days in hair samples.

Use of Hupresin To Capture Red Blood Cell Acetylcholinesterase for Detection of Soman Exposure.

Toxicity from acute exposure to nerve agents and organophosphorus toxicants is due to irreversible inhibition of acetylcholinesterase (AChE) in the nervous system. AChE in red blood cells is a surrogate for AChE in the nervous system. Previously we developed an immunopurification method to enrich red blood cell AChE (RBC AChE) as a biomarker of exposure. The goal of the present work was to provide an alternative RBC AChE enrichment strategy, by binding RBC AChE to Hupresin affinity gel. AChE was solubilized from frozen RBC by addition of 1% Triton X-100. Insoluble debris was removed by centrifugation. The red, but not viscous, RBC AChE solution was loaded on a Hupresin affinity column. Hemoglobin and other proteins were washed off with 3 M NaCl, while retaining AChE bound to Hupresin. Denatured AChE was eluted with 1% trifluoroacetic acid. The same protocol was used for 20 mL of RBC AChE inhibited with a soman model compound. The acid denatured protein was digested with pepsin and analyzed by liquid chromatography tandem mass spectrometry on a 6600 Triple-TOF mass spectrometer. A targeted method identified the aged soman adduct on serine 203 in peptide FGESAGAAS. It was concluded that Hupresin can be used to enrich soman-inhibited AChE solubilized from 8 mL of frozen human erythrocytes, yielding a quantity sufficient for detecting soman exposure.

Mass Spectral Detection of Diethoxyphospho-Tyrosine Adducts on Proteins from HEK293 Cells Using Monoclonal Antibody depY for Enrichment.

Chronic illness from exposure to organophosphorus toxicants is hypothesized to involve modification of unknown proteins. Tyrosine in proteins that have no active site serine readily reacts with organophosphorus toxicants. We developed a monoclonal antibody, depY, that specifically recognizes diethoxyphospho-tyrosine in proteins and peptides, independent of the surrounding amino acid sequence. Our goal in the current study was to identify diethoxyphosphorylated proteins in human HEK293 cell lysate treated with chlorpyrifos oxon. Cell lysates treated with chlorpyrifos oxon were recognized by depY antibody in ELISA and capillary electrophoresis based Western blot. Tryptic peptides were analyzed by liquid chromatography tandem mass spectrometry. Liquid chromatography tandem mass spectrometry identified 116 diethoxyphospho-tyrosine peptides from 73 proteins in immunopurified samples, but found only 15 diethoxyphospho-tyrosine peptides from 12 proteins when the same sample was not immunopurified on depY. The most abundant proteins in the cell lysate, histone H4, heat shock 70 kDa protein 1A/1B, heat shock protein HSP 90 ß, and α-enolase, were represented by several diethoxyphospho-tyrosine peptides. It was concluded that use of immobilized depY improved the number of diethoxyphospho-tyrosine peptides identified in a complex mixture. The mass spectrometry results confirmed the specificity of depY for diethoxyphospho-tyrosine peptides independent of the context of the modified tyrosine, which means depY could be used to analyze modified proteins in any species. Use of the depY antibody could lead to an understanding of chronic illness from organophosphorus pesticide exposure.

Quantification of Microcystin-LR in Human Urine by Immunocapture Liquid Chromatography Tandem Mass Spectrometry.

Microcystins are toxins produced by many cyanobacteria species, which are often released into waterways during blue-green algal blooms in freshwater and marine habitats. The consumption of microcystin-contaminated water is a public health concern as these toxins are recognized tumor promoters and are hepatotoxic to humans and animals. A method to confirm human exposures to microcystins is needed; therefore, our laboratory has developed an immunocapture liquid chromatography tandem mass spectrometry (LC-MS/MS) method targeting the conserved adda portion of microcystins for the quantitation of a prevalent and highly toxic congener of microcystin, microcystin-LR (MC-LR). An acute exposure method was initially evaluated for accuracy and precision by analyzing calibrators and quality control (QC) samples ranging from 0.500 to 75.0 ng/mL in urine. All calibrators and QC samples characterized were within 15% of theoretical concentrations. An analysis of acutely exposed mouse urine samples using this method identified MC-LR levels from 10.7 to 33.9 ng/mL. Since human exposures are anticipated to result from low-dose or chronic exposures, a high-sensitivity method was validated with 20 calibration curves and QC samples ranging from 0.0100 to 7.50 ng/mL. Relative standard deviations (RSDs) and inaccuracies of these samples were within 15%, meeting United States Food and Drug Administration (FDA) guidelines for analytical methods, and the limit of detection was 0.00455 ng/mL. In conclusion, we have developed a method which can be used to address public health concerns by precisely and accurately measuring MC-LR in urine samples.

High-Confidence Qualitative Identification of Organophosphorus Nerve Agent Adducts to Human Butyrylcholinesterase.

In this study, a data-dependent, high-resolution tandem mass spectrometry (ddHRMS/MS) method capable of detecting all organophosphorus nerve agent (OPNA) adducts to human butyrylcholinesterase (BChE) was developed. After an exposure event, immunoprecipitation from blood with a BChE-specific antibody and digestion with pepsin produces a nine amino acid peptide containing the OPNA adduct. Signature product ions of this peptic BChE nonapeptide (FGES*AGAAS) offer a route to broadly screen for OPNA exposure. Taking this approach on an HRMS instrument identifies biomarkers, including unknowns, with high mass accuracy. Using a set of pooled human sera exposed to OPNAs as quality control (QC) materials, the developed method successfully identified precursor ions with <1 ppm and tied them to signature product ions with <5 ppm deviation from their chemical formulas. This high mass accuracy data from precursor and product ions, collected over 23 independent immunoprecipitation preparations, established method operating limits. QC data and experiments with 14 synthetic reference peptides indicated that reliable qualitative identification of biomarkers was possible for analytes >15 ng/mL. The developed method was applied to a convenience set of 96 unexposed serum samples and a blinded set of 80 samples treated with OPNAs. OPNA biomarkers were not observed in convenience set samples and no false positive or negative identifications were observed in blinded samples. All biomarkers in the blinded serum set >15 ng/mL were correctly identified. For the first time, this study reports a ddHRMS/MS method capable of complementing existing quantitative methodologies and suitable for identifying exposure to unknown organophosphorus agents.

Immunopurification of Acetylcholinesterase from Red Blood Cells for Detection of Nerve Agent Exposure.

Nerve agents and organophosphorus pesticides make a covalent bond with the active site serine of acetylcholinesterase (AChE), resulting in inhibition of AChE activity and toxic symptoms. AChE in red blood cells (RBCs) serves as a surrogate for AChE in the nervous system. Mass spectrometry analysis of adducts on RBC AChE could provide evidence of exposure. Our goal was to develop a method of immunopurifying human RBC AChE in quantities adequate for detecting exposure by mass spectrometry. For this purpose, we immobilized 3 commercially available anti-human acetylcholinesterase monoclonal antibodies (AE-1, AE-2, and HR2) plus 3 new monoclonal antibodies. The monoclonal antibodies were characterized for binding affinity, epitope mapping by pairing analysis, and nucleotide and amino acid sequences. AChE was solubilized from frozen RBCs with 1% (v/v) Triton X-100. A 16 mL sample containing 5.8 µg of RBC AChE was treated with a quantity of soman model compound that inhibited 50% of the AChE activity. Native and soman-inhibited RBC AChE samples were immunopurified on antibody-Sepharose beads. The immunopurified RBC AChE was digested with pepsin and analyzed by liquid chromatography tandem mass spectrometry on a 6600 Triple-TOF mass spectrometer. The aged soman-modified PheGlyGluSerAlaGlyAlaAlaSer (FGESAGAAS) peptide was detected using a targeted analysis method. It was concluded that all 6 monoclonal antibodies could be used to immunopurify RBC AChE and that exposure to nerve agents could be detected as adducts on the active site serine of RBC AChE.

Monoclonal Antibody That Recognizes Diethoxyphosphotyrosine-Modified Proteins and Peptides Independent of Surrounding Amino Acids.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are irreversibly inhibited by organophosphorus pesticides through formation of a covalent bond with the active site serine. Proteins that have no active site serine, for example albumin, are covalently modified on tyrosine and lysine. Chronic illness from pesticide exposure is not explained by inhibition of AChE and BChE. Our goal was to produce a monoclonal antibody that recognizes proteins diethoxyphosphorylated on tyrosine. Diethoxyphosphate-tyrosine adducts for 13 peptides were synthesized. The diethoxyphosphorylated (OP) peptides cross-linked to four different carrier proteins were used to immunize, boost, and screen mice. Monoclonal antibodies were produced with hybridoma technology. Monoclonal antibody depY was purified and characterized by ELISA, western blotting, Biacore, and Octet technology to determine binding affinity and binding specificity. DepY recognized diethoxyphosphotyrosine independent of the amino acid sequence around the modified tyrosine and independent of the identity of the carrier protein or peptide. It had an IC50 of 3 × 10-9 M in a competition assay with OP tubulin. Kd values measured by Biacore and OctetRED96 were 10-8 M for OP-peptides and 1 × 10-12 M for OP-proteins. The limit of detection measured on western blots hybridized with 0.14 µg/mL of depY was 0.025 µg of human albumin conjugated to YGGFL-OP. DepY was specific for diethoxyphosphotyrosine (chlorpyrifos oxon adduct) as it failed to recognize diethoxyphospholysine, phosphoserine, phosphotyrosine, phosphothreonine, dimethoxyphosphotyrosine (dichlorvos adduct), dimethoxyphosphoserine, monomethoxyphosphotyrosine (aged dichlorvos adduct), and cresylphosphoserine. In conclusion, a monoclonal antibody that specifically recognizes diethoxyphosphotyrosine adducts has been developed. The depY monoclonal antibody could be useful for identifying new biomarkers of OP exposure.

Development and validation of a high-throughput online solid-phase extraction-liquid chromatography-tandem mass spectrometry method for the detection of gonyautoxins 1&4 and gonyautoxins 2&3 in human urine.

Paralytic shellfish toxins (PSTs), including gonyautoxins and saxitoxins, are produced by multiple species of microalgae and dinoflagellates, and are bioaccumulated by shellfish and other animals. Human exposure to PSTs typically occurs through ingestion of recreationally harvested contaminated shellfish and results in nonspecific symptomology. Confirmation of exposure to PSTs has often relied on the measurement of saxitoxin, the most toxic congener; however, gonyautoxins (GTXs), the sulfated carbamate derivatives of saxitoxin, may be present in shellfish at higher concentrations. To improve identification of PST exposures, our group has developed an online solid phase extraction hydrophilic interaction liquid chromatography method to identify GTX1-4 in human urine with tandem mass spectrometry. The reportable range varied for each analyte, with all falling within 0.899 and 250 ng/mL in urine with precision <15% and >85% accuracy as determined for all quality control samples. This new online method quantitates GTX1-4 following exposures to PSTs, supporting the work of public health authorities.

A high-throughput UHPLC-MS/MS method for the quantification of five aged butyrylcholinesterase biomarkers from human exposure to organophosphorus nerve agents.

Organophosphorus nerve agents (OPNAs) are toxic compounds that are classified as prohibited Schedule 1 chemical weapons. In the body, OPNAs bind to butyrylcholinesterase (BChE) to form nerve agent adducts (OPNA-BChE). OPNA-BChE adducts can provide a reliable, long-term protein biomarker for assessing human exposure. A major challenge facing OPNA-BChE detection is hydrolysis (aging), which can continue to occur after a clinical specimen has been collected. During aging, the o-alkyl phosphoester bond hydrolyzes, and the specific identity of the nerve agent is lost. To better identify OPNA exposure events, a high-throughput method for the detection of five aged OPNA-BChE adducts was developed. This is the first diagnostic panel to allow for the simultaneous quantification of any Chemical Weapons Convention Schedule 1 OPNA by measuring the aged adducts methyl phosphonate, ethyl phosphonate, propyl phosphonate, ethyl phosphoryl, phosphoryl and unadducted BChE. The calibration range for all analytes is 2.00-250. ng/mL, which is consistent with similar methodologies used to detect unaged OPNA-BChE adducts. Each analytical run is 3 min, making the time to first unknown results, including calibration curve and quality controls, less than 1 h. Analysis of commercially purchased individual serum samples demonstrated no potential interferences with detection of aged OPNA-BChE adducts, and quantitative measurements of endogenous levels of BChE were similar to those previously reported in other OPNA-BChE adduct assays.

Supplemental Learning in the Laboratory: An Innovative Approach for Evaluating Knowledge and Method Transfer.

The Multi-Rule Quality Control System (MRQCS) is a tool currently employed by the Centers for Disease Control and Prevention (CDC) to evaluate and compare laboratory performance. We have applied the MRQCS to a comparison of instructor and computer-led pre-laboratory lectures for a supplemental learning experiment. Students in general chemistry and analytical chemistry from both two- and four-year institutions performed two laboratory experiments as part of their normal laboratory curriculum. The first laboratory experiment was a foundational learning experiment in which all the students were introduced to Beer-Lambert's Law and spectrophotometric light absorbance measurements. The foundational learning experiment was instructor-led only, and participant performance was evaluated against a mean characterized value. The second laboratory experiment was a supplemental learning experiment in which students were asked to build upon the methodology they learned in the foundational learning experiment and apply it to a different analyte. The instruction type was varied randomly into two delivery modes, participants receiving either instructor-led or computer-led pre-laboratory instruction. The MRQCS was applied and determined that no statistical difference was found to exist in the QC (quality control) passing rates between the participants in the instructor-led instruction and the participants in the computer-led instruction. These findings demonstrate the successful application of the MRQCS to evaluate knowledge and technology transfer.

Naturally Occurring Genetic Variants of Human Acetylcholinesterase and Butyrylcholinesterase and Their Potential Impact on the Risk of Toxicity from Cholinesterase Inhibitors.

Acetylcholinesterase (AChE) is the physiologically important target for organophosphorus toxicants (OP) including nerve agents and pesticides. Butyrylcholinesterase (BChE) in blood serves as a bioscavenger that protects AChE in nerve synapses from inhibition by OP. Mass spectrometry methods can detect exposure to OP by measuring adducts on the active site serine of plasma BChE. Genetic variants of human AChE and BChE do exist, but loss of function mutations have been identified only in the BCHE gene. The most common AChE variant, His353Asn (H322N), also known as the Yt blood group antigen, has normal AChE activity. The most common BChE variant, Ala567Thr (A539T) or the K-variant in honor of Werner Kalow, has 33% reduced plasma BChE activity. The genetic variant most frequently associated with prolonged response to muscle relaxants, Asp98Gly (D70G) or atypical BChE, has reduced activity and reduced enzyme concentration. Early studies in young, healthy males, performed at a time when it was legal to test nerve agents in humans, showed that individuals responded differently to the same low dose of sarin with toxic symptoms ranging in severity from minimal to moderate. Additionally, animal studies indicated that BChE protects from toxicants that have a higher reactivity with AChE than with BChE (e.g., nerve agents) but not from toxicants that have a higher reactivity with BChE than with AChE (e.g., OP pesticides). As a corollary, we hypothesize that individuals with genetic variants of BChE may be at increased risk of toxicity from nerve agents but not from OP pesticides.

Enhanced stability of blood matrices using a dried sample spot assay to measure human butyrylcholinesterase activity and nerve agent adducts.

Dried matrix spots are safer to handle and easier to store than wet blood products, but factors such as intraspot variability and unknown sample volumes have limited their appeal as a sampling format for quantitative analyses. In this work, we introduce a dried spot activity assay for quantifying butyrylcholinesterase (BChE) specific activity which is BChE activity normalized to the total protein content in a sample spot. The method was demonstrated with blood, serum, and plasma spotted on specimen collection devices (cards) which were extracted to measure total protein and BChE activity using a modified Ellman assay. Activity recovered from dried spots was ∼80% of the initial spotted activity for blood and >90% for plasma and serum. Measuring total protein in the sample and calculating specific activity substantially improved quantification and reduced intraspot variability. Analyte stability of nerve agent adducts was also evaluated, and the results obtained via BChE-specific activity measurements were confirmed by quantification of BChE adducts using a previously established LC-MS/MS method. The spotted samples were up to 10 times more resistant to degradation compared to unspotted control samples when measuring BChE inhibition by the nerve agents sarin and VX. Using this method, both BChE activity and adducts can be accurately measured from a dried sample spot. This use of a dried sample spot with normalization to total protein is robust, demonstrates decreased intraspot variability without the need to control for initial sample volume, and enhances analyte stability.

Quantification of metabolites for assessing human exposure to soapberry toxins hypoglycin A and methylenecyclopropylglycine.

Ingestion of soapberry fruit toxins hypoglycin A and methylenecyclopropylglycine has been linked to public health challenges worldwide. In 1976, over 100 years after Jamaican vomiting sickness (JVS) was first reported, the cause of JVS was linked to the ingestion of the toxin hypoglycin A produced by ackee fruit. A structural analogue of hypoglycin A, methylenecyclopropylglycine (MCPG), was implicated as the cause of an acute encephalitis syndrome (AES). Much of the evidence linking hypoglycin A and MCPG to these diseases has been largely circumstantial due to the lack of an analytical method for specific metabolites. This study presents an analytical approach to identify and quantify specific urine metabolites for exposure to hypoglycin A and MCPG. The metabolites are excreted in urine as glycine adducts methylenecyclopropylacetyl-glycine (MCPA-Gly) and methylenecyclopropylformyl-glycine (MCPF-Gly). These metabolites were processed by isotope dilution, separated by reverse-phase liquid chromatography, and monitored by electrospray ionization tandem mass spectrometry. The analytical response ratio was linearly proportional to the concentration of MCPF-Gly and MCPA-Gly in urine from 0.10 to 20 µg/mL with a correlation coefficient of r > 0.99. The assay demonstrated accuracy ≥80% and precision ≤20% RSD across the calibration range. This method has been applied to assess exposure to hypoglycin A and MCPG as part of a larger public health initiative and was used to provide the first reported identification of MCPF-Gly and MCPA-Gly in human urine.

Simplified Method for Quantifying Sulfur Mustard Adducts to Blood Proteins by Ultrahigh Pressure Liquid Chromatography−Isotope Dilution Tandem Mass Spectrometry.

Sulfur mustard binds to reactive cysteine residues, forming a stable sulfur-hydroxyethylthioethyl [SHETE]adduct that can be used as a long-term biomarker of sulfur mustard exposure in humans. The digestion of sulfur mustard-exposed blood samples with proteinase K following total protein precipitation with acetone produces the tripeptide biomarker [S-HETE]-Cys-Pro-Phe. The adducted tripeptide is purified by solid phase extraction, separated by ultra high pressure liquid chromatography, and detected by isotope dilution tandem mass spectrometry. This approach was thoroughly validated and characterized in our laboratory. The average interday relative standard deviation was ≤ 9.49%, and the range of accuracy was between 96.1 and 109% over a concentration range of 3.00 to 250. ng/mL with a calculated limit of detection of1.74 ng/mL. A full 96-well plate can be processed and analyzed in 8 h, which is 5 times faster than our previous 96-well plate method and only requires 50 µL of serum, plasma, or whole blood. Extensive ruggedness and stability studies and matrix comparisons were conducted to create a robust, easily transferrable method. As a result, a simple and high-throughput method has been developed and validated for the quantitation of sulfur mustard blood protein adducts in low volume blood specimens which should be readily adaptable for quantifying human exposures to other alkylating agents.

Simultaneous measurement of tabun, sarin, soman, cyclosarin, VR, VX, and VM adducts to tyrosine in blood products by isotope dilution UHPLC-MS/MS.

This work describes a new specific, sensitive, and rapid stable isotope dilution method for the simultaneous detection of the organophosphorus nerve agents (OPNAs) tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), VR, VX, and VM adducts to tyrosine (Tyr). Serum, plasma, and lysed whole blood samples (50 µL) were prepared by protein precipitation followed by digestion with Pronase. Specific Tyr adducts were isolated from the digest by a single solid phase extraction (SPE) step, and the analytes were separated by reversed-phase ultra high performance liquid chromatography (UHPLC) gradient elution in less than 2 min. Detection was performed on a triple quadrupole tandem mass spectrometer using time-triggered selected reaction monitoring (SRM) in positive electrospray ionization (ESI) mode. The calibration range was characterized from 0.100-50.0 ng/mL for GB- and VR-Tyr and 0.250-50.0 ng/mL for GA-, GD-, GF-, and VX/VM-Tyr (R(2) ≥ 0.995). Inter- and intra-assay precision had coefficients of variation of ≤17 and ≤10%, respectively, and the measured concentration accuracies of spiked samples were within 15% of the targeted value for multiple spiking levels. The limit of detection was calculated to be 0.097, 0.027, 0.018, 0.074, 0.023, and 0.083 ng/mL for GA-, GB-, GD-, GF-, VR-, and VX/VM-Tyr, respectively. A convenience set of 96 serum samples with no known nerve agent exposure was screened and revealed no baseline values or potential interferences. This method provides a simple and highly specific diagnostic tool that may extend the time postevent that a confirmation of nerve agent exposure can be made with confidence.

Quantitation of five organophosphorus nerve agent metabolites in serum using hydrophilic interaction liquid chromatography and tandem mass spectrometry.

Although nerve agent use is prohibited, concerns remain for human exposure to nerve agents during decommissioning, research, and warfare. Exposure can be detected through the analysis of hydrolysis products in urine as well as blood. An analytical method to detect exposure to five nerve agents, including VX, VR (Russian VX), GB (sarin), GD (soman), and GF (cyclosarin), through the analysis of the hydrolysis products, which are the primary metabolites, in serum has been developed and characterized. This method uses solid-phase extraction coupled with high-performance liquid chromatography for separation and isotopic dilution tandem mass spectrometry for detection. An uncommon buffer of ammonium fluoride was used to enhance ionization and improve sensitivity when coupled with hydrophilic interaction liquid chromatography resulting in detection limits from 0.3 to 0.5 ng/mL. The assessment of two quality control samples demonstrated high accuracy (101-105%) and high precision (5-8%) for the detection of these five nerve agent hydrolysis products in serum.

An enhanced butyrylcholinesterase method to measure organophosphorus nerve agent exposure in humans.

Organophosphorus nerve agent (OPNA) adducts to butyrylcholinesterase (BChE) can be used to confirm exposure in humans. A highly accurate method to detect G- and V-series OPNA adducts to BChE in 75 µL of filtered blood, serum, or plasma has been developed using immunomagnetic separation (IMS) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS). The reported IMS method captures > 88 % of the BChE in a specimen and corrects for matrix effects on peptide calibrators. The optimized method has been used to quantify baseline BChE levels (unadducted and OPNA-adducted) in a matched-set of serum, plasma, and whole blood (later processed in-house for plasma content) from 192 unexposed individuals to determine the interchangeability of the tested matrices. The results of these measurements demonstrate the ability to accurately measure BChE regardless of the format of the blood specimen received. Criteria for accepting or denying specimens were established through a series of sample stability and processing experiments. The results of these efforts are an optimized and rugged method that is transferrable to other laboratories and an increased understanding of the BChE biomarker in matrix.

Performance of a novel high throughput method for the determination of VX in drinking water samples.

VX (O-ethyl-S-(2-diisopropylaminoethyl) methylphosphonothioate) is a highly toxic organophosphorus nerve agent, and even low levels of contamination in water can be harmful. Measurement of low concentrations of VX in aqueous matrixes is possible using an immunomagnetic scavenging technique and detection using liquid chromatography/tandem-mass spectrometry. Performance of the method was characterized in high-performance liquid chromatography (HPLC)-grade water preserved with sodium omadine, an antimicrobial agent, and sodium thiosulfate, a dechlorinating agent, over eight analytical batches with quality control samples analyzed over 10 days. The minimum reportable level was 25 ng/L with a linear dynamic range up to 4.0 µg/L. The mean accuracies for two quality control samples containing VX at concentrations of 0.250 and 2.00 µg/L were 102 ± 3% and 103 ± 6%, respectively. The stability of VX was determined in five tap water samples representing a range of water quality parameters and disinfection practices over a 91 day period. In preserved tap water samples, VX recovery was between 81 and 92% of the fortified amount, 2.0 µg/L, when analyzed immediately after preparation. Recovery of VX decreased to between 31 and 45% of the fortified amount after 91 days, indicating hydrolysis of VX. However, the preservatives minimized the hydrolysis rate to close to the theoretical limit. The ability to detect low concentrations of VX in preserved tap water 91 days after spiking suggests applicability of this method for determining water contamination with VX and utility during environmental remediation.

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

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.

Novel dual-mode immunomagnetic method for studying reactivation of nerve agent-inhibited butyrylcholinesterase.

A novel immunomagnetic method has been developed for the simultaneous measurement of organophosphorus nerve agent (OPNA) adducts to butyrylcholinesterase (BuChE) and free OPNAs in serum. This new approach, deemed dual-mode immunomagnetic analysis (Dual-Mode IMA), combines immunomagnetic separation (IMS) and immunomagnetic scavenging (IMSc) and has been used to measure the effectiveness of cholinesterase reactivators on OPNA-inhibited BuChE in serum. BuChE inhibited by the nerve agent VX, uninhibited BuChE, and unbound VX were measured up to 1 h after the addition of oxime reactivators pralidoxime (2-PAM) and obidoxime. IMS experiments consisted of extracting BuChE and VX-BuChE serum adducts using antibutyrylcholinesterase monoclonal antibodies conjugated to protein-G ferromagnetic particles. In a parallel set of experiments using IMSc, BuChE-coated magnetic beads were used to extract free VX from protein-depleted serum. Adducts from both IMS and IMSc were analyzed using a published IMS liquid chromatography tandem mass spectrometry (IMS-LC-MS/MS) protocol, which has also been demonstrated with other OPNAs. By applying this Dual-Mode IMA approach, 2-PAM was observed to be more potent than obidoxime in reactivating VX-adducted BuChE. VX-BuChE peptide concentrations initially measured at 19.7 ± 0.7 ng/mL decreased over 1 h to 10.6 ± 0.6 ng/mL when reactivated with 2-PAM and 14.4 ± 1.2 ng/mL when reactivated with obidoxime. These experiments also show that previously published IMS-LC-MS/MS analyses are compatible with serum treated with oximes. Dual-Mode IMA is the first immunoaffinity method developed for the simultaneous measurement of OPNA adducted BuChE, unadducted BuChE, and free nerve agent in serum and is a promising new tool for studying reactivator effectiveness on cholinesterases inhibited by nerve agents.