Mieko Kanamori-Kataoka

DEVELOPMENT OF AN ON-SITE DETECTION METHOD FOR CHEMICAL AND BIOLOGICAL WARFARE AGENTS

We evaluated commercially available, portable, on-site equipment for chemical warfare agent detection (a gas detection tube, ion mobility spectrometer, surface acoustic wavelength detector, flame photometric detector, photoionization detector, Fourier-transformed infrared spectrometer and a gas chromatograph-mass spectrometer) using authentic, vaporized chemical-warfare agents from the standpoint of their qualitative detection characteristics, detection limits, response times, frequency of false alarms and residubility on the devices. False alarms and the strong adsorption of agents by the devices are typical drawbacks of such equipment. As a screening method for biological warfare agents, on-site methods using flow cytometry, bioluminescence assay, and lateral flow immunoassay were developed.

Sensitive Monitoring of Volatile Chemical Warfare Agents in Air by Atmospheric Pressure Chemical Ionization Mass Spectrometry with Counter-Flow Introduction

A new method for sensitively and selectively detecting chemical warfare agents (CWAs) in air was developed using counter-flow introduction atmospheric pressure chemical ionization mass spectrometry (MS). Four volatile and highly toxic CWAs were examined, including the nerve gases sarin and tabun, and the blister agents mustard gas (HD) and Lewisite 1 (L1). Soft ionization was performed using corona discharge to form reactant ions, and the ions were sent in the direction opposite to the airflow by an electric field to eliminate the interfering neutral molecules such as ozone and nitrogen oxide. This resulted in efficient ionization of the target CWAs, especially in the negative ionization mode. Quadrupole MS (QMS) and ion trap tandem MS (ITMS) instruments were developed and investigated, which were movable on the building floor. For sarin, tabun, and HD, the protonated molecular ions and their fragment ions were observed in the positive ion mode. For L1, the chloride adduct ions of L1 hydrolysis products were observed in negative ion mode. The limit of detection (LOD) values in real-time or for a 1 s measurement monitoring the characteristic ions were between 1 and 8 μg/m(3) in QMS instrument. Collision-induced fragmentation patterns for the CWAs were observed in an ITMS instrument, and optimized combinations of the parent and daughter ion pairs were selected to achieve real-time detection with LOD values of around 1 μg/m(3). This is a first demonstration of sensitive and specific real-time detection of both positively and negatively ionizable CWAs by MS instruments used for field monitoring.

Determination of ricin by nano liquid chromatography/mass spectrometry after extraction using lactose‐immobilized monolithic silica spin column

Ricin is a glycosylated proteinous toxin that is registered as toxic substance by Chemical Weapons convention. Current detection methods can result in false negatives and/or positives, and their criteria are not based on the identification of the protein amino acid sequences. In this study, lactose-immobilized monolithic silica extraction followed by tryptic digestion and liquid chromatography/mass spectrometry (LC/MS) was developed as a method for rapid and accurate determination of ricin. Lactose, which was immobilized on monolithic silica, was used as a capture ligand for ricin extraction from the sample solution, and the silica was supported in a disk-packed spin column. Recovery of ricin was more than 40%. After extraction, the extract was digested with trypsin and analyzed by LC/MS. The accurate masses of molecular ions and MS/MS spectra of the separated peptide peaks were measured by Fourier transform-MS and linear iontrap-MS, respectively. Six peptides, which were derived from the ricin A-(m/z 537.8, 448.8 and 586.8) and B-chains (m/z 701.3, 647.8 and 616.8), were chosen as marker peptides for the identification of ricin. Among these marker peptides, two peptides were ricin-specific. This method was applied to the determination of ricin from crude samples. The monolithic silica extraction removed most contaminant peaks from the total ion chromatogram of the sample, and the six marker peptides were clearly detected by LC/MS. It takes about 5 h for detection and identification of more than 8 ng/ml of ricin through the whole handling, and this procedure will be able to deal with the terrorism using chemical weapon.

Paraoxonase activity against nerve gases measured by capillary electrophoresis and characterization of human serum paraoxonase (PON1) polymorphism in the coding region (Q192R).

An analytical method for determining paraoxonase activity against sarin, soman and VX was established. We used capillary electrophoresis to measure directly the hydrolysis products: alkyl methylphosphonates. After enzymatic reaction of human serum paraoxonase (PON1) with nerve gas, substrate was removed with dichloromethane, and alkyl methylphoshphonates were quantified by capillary electrophoresis of reversed osmotic flow using cationic detergent and sorbic acid. This method was applied to the characterization of human serum PON1 polymorphism for nerve gas hydrolytic activity in the coding region (Q192R). PON1-192 and PON1-55 genotypes were determined by their gel electrophoretic fragmentation pattern with restriction enzymes after polymerase chain reaction (PCR) of blood leukocyte genomic DNA. Frequencies of genotypes among 63 members of our institutes with PON1-192 and PON1-55 were 9.5% ((192)QQ), 30.1% ((192)QR) and 44.4% ((192)RR), and 82.5% ((55)LL), 17.5% ((55)LM) and 0% ((55)MM), respectively. (192)Q and (192)R enzymes were purified from the respective genotype human plasma, using blue agarose affinity chromatography and diethyl amino ethane (DEAE) anion exchange chromatography. V(max) and K(m) were measured using Lineweaver-Burk plots for hydrolytic activities against sarin, soman and VX at pH 7.4 and 25 degrees C. For sarin and soman, the V(max) for (192)Q PON1 were 3.5- and 1.5-fold higher than those for (192)R PON1; and k(cat)/K(m) for (192)Q PON1 were 1.3- and 2.8-fold higher than those for (192)R PON1. For VX, there was little difference in V(max) and k(cat)/K(m) between (192)Q and (192)R PON1, and VX hydrolyzing activity was significantly lower than those for sarin and soman. PON1 hydrolyzed sarin and soman more effectively than paraoxon.

Ion mobility spectrometric analysis of vaporous chemical warfare agents by the instrument with corona discharge ionization ammonia dopant ambient temperature operation.

The ion mobility behavior of nineteen chemical warfare agents (7 nerve gases, 5 blister agents, 2 lachrymators, 2 blood agents, 3 choking agents) and related compounds including simulants (8 agents) and organic solvents (39) was comparably investigated by the ion mobility spectrometry instrument utilizing weak electric field linear drift tube with corona discharge ionization, ammonia doping, purified inner air drift flow circulation operated at ambient temperature and pressure. Three alkyl methylphosphonofluoridates, tabun, and four organophosphorus simulants gave the intense characteristic positive monomer-derived ion peaks and small dimer-derived ion peaks, and the later ion peaks were increased with the vapor concentrations. VX, RVX and tabun gave both characteristic positive monomer-derived ions and degradation product ions. Nitrogen mustards gave the intense characteristic positive ion peaks, and in addition distinctive negative ion peak appeared from HN3. Mustard gas, lewisite 1, o-chlorobenzylidenemalononitrile and 2-mercaptoethanol gave the characteristic negative ion peaks. Methylphosphonyl difluoride, 2-chloroacetophenone and 1,4-thioxane gave the characteristic ion peaks both in the positive and negative ion mode. 2-Chloroethylethylsulfide and allylisothiocyanate gave weak ion peaks. The marker ion peaks derived from two blood agents and three choking agents were very close to the reactant ion peak in negative ion mode and the respective reduced ion mobility was fluctuated. The reduced ion mobility of the CWA monomer-derived peaks were positively correlated with molecular masses among structurally similar agents such as G-type nerve gases and organophosphorus simulants; V-type nerve gases and nitrogen mustards. The slope values of the calibration plots of the peak heights of the characteristic marker ions versus the vapor concentrations are related to the detection sensitivity, and within chemical warfare agents examined the slope values for sarin, soman, tabun and nitrogen mustards were higher. Some CWA simulants and organic solvents gave the ion peaks eluting at the similar positions of the CWAs, resulting in false positive alarms.

Fundamental properties of a touchable high‐power pulsed microplasma jet and its application as a desorption/ionization source for ambient mass spectrometry

Plasma-based ambient desorption/ionization mass spectrometry (ADI-MS) has attracted considerable attention in many fields because of its capacity for direct sample analyses. In this study, a high-power pulsed microplasma jet (HPPMJ) was developed and investigated as a new plasma desorption/ionization source. In an HPPMJ, a microhollow cathode discharge is generated in a small hole (500 µm in diameter) using a pulsed high-power supply. This system can realize a maximum power density of 5 × 10(8) W/cm(3). The measured electron number density, excitation temperature and afterglow gas temperature of the HPPMJ were 3.7 × 10(15) cm(-3), 7000 K at maximum and less than 60 °C, respectively, which demonstrate that the HPPMJ is a high-energy, high-density plasma source that is comparable with an argon inductively coupled plasma while maintaining a low gas temperature. The HPPMJ causes no observable damage to the target because of its low gas temperature and electrode configuration; thus, we can apply it directly to human skin. To demonstrate the analytical capacity of ADI-MS using an HPPMJ, the plasma was applied to direct solid sample analysis of the active ingredients in pharmaceutical tablets. Caffeine, acetaminophen, ethenzamide, isopropylantipyrine and ibuprofen were successfully detected. Application to living tissue was also demonstrated, and isopropylantipyrine on a finger was successfully analyzed without damaging the skin. The limits of detection (LODs) for caffeine, isopropylantipyrine and ethenzamide were calculated, and LODs at the picogram level were achieved. These results indicate the applicability of the HPPMJ for high-sensitivity analysis of materials on a heat-sensitive surface.

Development of a Gas-Cylinder-Free Plasma Desorption/Ionization System for On-Site Detection of Chemical Warfare Agents

A gas-cylinder-free plasma desorption/ionization system was developed to realize a mobile on-site analytical device for detection of chemical warfare agents (CWAs). In this system, the plasma source was directly connected to the inlet of a mass spectrometer. The plasma can be generated with ambient air, which is drawn into the discharge region by negative pressure in the mass spectrometer. High-power density pulsed plasma of 100 kW could be generated by using a microhollow cathode and a laboratory-built high-intensity pulsed power supply (pulse width: 10-20 μs; repetition frequency: 50 Hz). CWAs were desorbed and protonated in the enclosed space adjacent to the plasma source. Protonated sample molecules were introduced to the mass spectrometer by airflow through the discharge region. To evaluate the analytical performance of this device, helium and air plasma were directly irradiated to CWAs in the gas-cylinder-free plasma desorption/ionization system and the protonated molecules were analyzed by using an ion-trap mass spectrometer. A blister agent (nitrogen mustard 3) and nerve gases [cyclohexylsarin (GF), tabun (GA), and O-ethyl S-2-N,N-diisopropylaminoethyl methylphosphonothiolate (VX)] in solution in n-hexane were applied to the Teflon rod and used as test samples, after solvent evaporation. As a result, protonated molecules of CWAs were successfully observed as the characteristic ion peaks at m/z 204, 181, 163, and 268, respectively. In air plasma, the limits of detection were estimated to be 22, 20, 4.8, and 1.0 pmol, respectively, which were lower than those obtained with helium plasma. To achieve quantitative analysis, calibration curves were made by using CWA stimulant dipinacolyl methylphosphonate as an internal standard; straight correlation lines (R(2) = 0.9998) of the peak intensity ratios (target per internal standard) were obtained. Remarkably, GA and GF gave protonated dimer ions, and the ratios of the protonated dimer ions to the protonated monomers increased with the amount of GA and GF applied.

Detection of proteinous toxins using the Bio-Threat Alert system, part 3: effects of heat pretreatment and interfering substances

We previously reported that the Guardian Bio-Threat Alert (BTA) system could detect (detection limit: about 0.1 μg/ml) staphylococcal enterotoxin B (SEB), botulinum toxins (BTX) A and B, and ricin, with no interference by white-powdered materials or colored matrices. In this study, the capability of the BTA system was further assessed. With 10 min of preheating at 60°C, all toxins could be detected, but with preheating at 80°C, BTX A and B and ricin became undetectable. About 20% SEB could be detected after heating at 80°C, but this detection ability was completely removed after heating at 100°C. The effects of chemicals usually used for decontamination, such as sodium hypochlorite, hydrogen peroxide, formaldehyde, and sodium nitrite, on the detectability of SEB, BTX A, or ricin in the BTA system were also tested. The concentrations giving 50% line intensity for SEB, BTX A, and ricin were 3.1, 11, and 15 μM for sodium hypochlorite and 88, 210, and 60 mM for formaldehyde, respectively. The addition of hydrogen peroxide or sodium nitrite did not decrease the detectability even when used at high concentrations.

Decontamination of nerve agents by immobilized organophosphorus hydrolase

Organophosphorus hydrolase (OPH; EC 3.1.8.1) is known to be capable of hydrolyzing a variety of organophosphorus compounds, such as sarin and paraoxon. We have developed a nerve agent decontamination method using OPH. The gene that encodes OPH was cloned from the bacterial strain Sphingobium fuliginis ATCC 27551, and several OPH gene fusion plasmids were constructed. Escherichia coli was utilized as the expression host for the resulting plasmids. The activities of the recombinant OPH enzymes expressed (KGU0060, KGU0092, and KGU0094) were determined by measuring paraoxon hydrolysis activities. The recombinant OPH enzymes that lacked the signal peptide regions (KGU0092 and KGU0094) were remarkably activated by zinc ion; while the OPH enzyme that contained the signal peptide region (KGU0060) was activated by both zinc and cobalt ions, although the specific activity of this enzyme was much lower than that of KGU0092 or KGU0094. The pH profile demonstrated that the OPH enzymes effectively hydrolyzed the substrate under alkaline conditions. We applied the recombinant OPH to the degradation of nerve agents; it hydrolyzed sarin, tabun, cyclohexyl sarin, and soman with high activities, but not O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX). The enzyme was immobilized to a nickel-chelating sepharose resin. Similarly, all nerve agents could be hydrolyzed by the immobilized enzyme except VX. Our results suggest the possibility for developing a powerful and ecological nerve agent detoxication system using OPH.

Detection of proteinous toxins using the Bio-Threat Alert system, part 4. Differences in detectability according to manufactural lots and according to toxin subtypes

In a series of experiments, we have tested the usefulness and limitations of the Guardian Bio-Threat Alert (BTA) system for detection of staphylococcal enterotoxin B (SEB), botulinum toxins (BTXs) A and B, and ricin. In this report, the BTA system has been further evaluated for toxin subtypes and the detection ability of manufactural lots of the BTA strips. The SEB strips failed to detect staphylococcal enterotoxin A, C, and D; the BTX strips generally failed to detect BTXs C, D, E, and F, but one lot showed positive results for BTXs C and D with very low sample values. Differences were observed in sample values at 1 μg/ml for all main toxins according to the different manufactural strip lots: 3.9-fold difference for SEB, 6.3-fold difference for BTX A, 10.9-fold difference for BTX B, and 6.4-fold difference for ricin. The ricin strips showed high cross reactivity toward RCA120. The BioWarfare Agent Detection Devices system showed much lower sensitivity than the BTA system for BTX and ricin (detection limit: about 10 μg/ml).