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A new class of synthetic cannabinoids termed OXIZIDs has recently emerged on the recreational drug market. In order to continue the detection of new drugs in biological specimens, the identification of metabolites is essential. The aim of this study was to elucidate the metabolites of BZO-4en-POXIZID produced in human liver microsomes (HLMs) and human hepatocyte incubations and to compare the results with closely related analogs using the same experimental setup. Each drug was incubated for 1 h in HLM and BZO-4en-POXIZID was also incubated in human hepatocytes for up to 3 h. Subsequently, the incubates were analyzed by liquid chromatography–high-resolution mass spectrometry. BZO-4en-POXIZID metabolites were obtained in the incubation with HLMs and human hepatocytes, via the metabolic pathways of dihydrodiol formation, hydroxylation, reduction of the alkene bond and glucuronidation. The major metabolic pathway was found to be dihydrodiol formation at the pentenyl tail moiety. BZO-POXIZID, 5 F-BZO-POXIZID, BZO-HEXOXIZID and BZO-CHMOXIZID underwent similar metabolism to those reported in the literature, via the metabolic pathways of N-dealkylation, hydroxylation, ketone formation and oxidative defluorination (to alcohol or carboxylic acid). The results suggest that OXIZIDs are mainly metabolized at the N-alkyl moiety and the major metabolic pathways are hydroxylation when the N-alkyl moiety is a simple hydrocarbon, whereas functional-group-specific pathways (dihydrodiol formation and oxidative defluorination) are preferred when the moiety contains specific functional groups (alkene or fluoro), as has been observed for other synthetic cannabinoids. The major metabolites generated via these major metabolic pathways should serve as useful analytical targets for urine analysis. Furthermore, the higher abundance of glucuronidated metabolite suggests that enzymatic hydrolysis of glucuronides may be necessary for urine analysis to increase phase I metabolite concentration and improve detection.

Fiber examination is frequently performed in forensics, and gel permeation chromatography (GPC) is one candidate method for discriminating polyester fibers. Here, the effects of machine washing on weight-average molecular weight (

Automotive glasses are important forensic evidence often recovered from crime scenes. Black ceramic prints on automotive glasses contains various elements in high concentrations. A portable X-ray fluorescence spectrometer (pXRF) allows an instant and nondestructive analysis of various elements. In this study, the Bruker Tracer 5 g was used as the pXRF equipment. The NIST SRM612 glass standard was used to determine the limit of detection (LODs) of a pXRF and its optimal conditions. The acceleration voltages of 15, 30, and 50 kV were appropriate for measuring Si K

The adsorption and photocatalytic degradation of Ethyl methylphosphonate (EMPA) on powdery TiO2 film has experimentally investigated using attenuated total reflection-infrared Fourier transform spectroscopy (ATR-FTIR) in ambient condition. Characteristic IR frequency as P-O-C vibration mode as EtO was observed by EMPA adsorbed at the surface of TiO2. By TiO2 photocatalysis, the adsorbed EMPA was decomposed to methyl phosphonic acid and phosphoric acid. The increment of IR intensity of which is assigned to Ti–O-P-O-Ti of EMPA was accompanied with increasing the IR peak intensity assigned to MPA. About that, we suggest that the appearance of the Ti–O-P-O-Ti of EMPA by the TiO2 photocatalysis is regarded as acceleration of the hydrolysis of EMPA by the surface OH groups of TiO2. The plausible adsorption structure and the photocatalytic reaction mechanism of EMPA at the surface of TiO2 photocatalyst were elucidated.

High-efficiency decomposition technology for volatile chemical warfare agent sarin was developed using a dielectric-barrier discharge (DBD). The power consumptions of the developed device for which the gap lengths of the plasma-generation sites were 1.0–2.0 mm, were found to be 22–27 W, respectively. The DBD driven sarin-decomposition amount per unit time and the decomposition efficiency in the optimized condition were obtained to be 2.05 × 10−3 mg/s and 1.38 × 10−4 mg/W, respectively, measured by ion mobility spectrometer. Sarin-decomposition products were investigated using a gas chromatography–mass spectrometry with tert-butyldimethylsilylation derivatization, indicating that isopropyl methylphosphoric acid (IMPA), methylphosphoric acid (MPA), and phosphoric acid (PA) were generated during sarin decomposition by DBD. The decomposition efficiency at 37 W decreased by 41% molar concentration compared with that at 15 W, but the production levels of IMPA, MPA and PA were changed from 10.0%, 41.5% and 3.9% to 7.2%, 21.3% and 6.4%, respectively.

Purpose: JWH-424, (8-bromo-1-naphthyl)(1-pentyl-1H-indol-3-yl)methanone, is a synthetic cannabinoid, which is a brominated analogue of JWH-018, one of the best-known synthetic cannabinoids. Despite the structural similarity to JWH-018, little is known about JWH-424 including its metabolism. The aim of the study was to compare human liver microsomes (HLM) and the fungus Cunninghamella elegans as the metabolism catalysts for JWH-424 to better understand the characteristic actions of the fungus in the synthetic cannabinoid metabolism. Methods: JWH-424 was incubated with HLM for 1 h and Cunninghamella elegans for up to 72 h. The HLM incubation mixtures were diluted with methanol and fungal incubation mixtures were extracted with dichloromethane and reconstituted in methanol before analyses by liquid chromatography–high-resolution mass spectrometry (LC-HRMS). Results: HLM incubation resulted in production of ten metabolites through dihydrodiol formation, hydroxylation, and/or ipso substitution of the bromine with a hydroxy group. Fungal incubation led to production of 23 metabolites through carboxylation, dihydrodiol formation, hydroxylation, ketone formation, glucosidation and/or sulfation. Conclusions: Generally, HLM models give good predictions of human metabolites and structural analogues are metabolised in a similar fashion. However, major hydroxy metabolites produced by HLM were those hydroxylated at naphthalene instead of pentyl moiety, the major site of hydroxylation for JWH-018. Fungal metabolites, on the other hand, had undergone hydroxylation mainly at pentyl moiety. The metabolic disagreement suggests the necessity to verify the human metabolites in authentic urine samples, while H9 and H10 (hydroxynaphthalene), H8 (ipso substitution), F22 (hydroxypentyl), and F17 (dihydroxypentyl) are recommended for monitoring of JWH-424 in urinalysis.

Drift tube ion mobility spectrometry with a novel atmospheric electron emission (AEE) source was developed for determination of gaseous and blister chemical warfare agents (CWAs) in negative mode. The AEE source was fabricated from an aluminum substrate electrode covered with 1 μm silver nanoparticle-dispersed silicone resin and a thin gold layer. This structure enabled stable tunneling electron emission upon the application of more than 11 V potential under atmospheric pressure. The reactant ion peak (RIP) was observed for the reduced mobility constant ( K

An inter-method variation experiment to evaluate visible spectrophotometric methods for determining carboxyhemoglobin (CO-Hb) in blood was performed in 17 different forensic science laboratories. Three reference samples were prepared and the actual percentages of CO-Hb (%CO-Hb) in reference samples were determined with gas chromatography. The %CO-Hb values in reference samples were measured via four procedures: (1) the “Standard method of chemical analysis in poisoning 2017” (edited by the Pharmaceutical Society of Japan), (2) the isosbestic point method (developed by the Department of Forensic Medicine, Kagawa University), (3) the alkaline condition method, and (4) ISO 27368 with a slight modification. Procedures (1) and (2) gave results approximately the same as the actual values in low-%CO-Hb samples, but both methods underestimated %CO-Hb levels in blood samples with high %CO-Hb. Using a formula reported by Katsumata et al., procedure (3) overestimated %CO-Hb levels in blood samples with low %CO-Hb, while using the modified formula, gave underestimates in all samples. Procedure (4) gave %CO-Hb values relatively close to the actual values. Thus, an accuracy evaluation test was performed for this procedure and the values obtained were close to the actual CO-Hb content levels. Procedure (4) was considered sufficiently accurate and thus recommendable for use in forensic science laboratories.

A sensitive method for determination of fluoridated phosphonates produced by fluoride-mediated regeneration of nerve agent adduct in human serum was developed using gas chromatography-mass spectrometry (GC MS) with large-volume injection. The GC injection was administered using stomach-type spiral injector (LVI, AiSTI SCIENCE) enabling introduction of only target compounds from 50 μL ethyl acetate extract after purging the solvent. For GC MS analysis of sarin (GB), 670 times higher sensitivity, based on limit of detection (LOD, S/N = 3, on extracted ion chromatogram (EIC) at m/z 99), was achieved using this injection (50 μL) compared to that achieved using 1 μL split injection (ratio 20:1). Ethyl (EtGB), isopropyl (GB), n-propyl (nPrGB), isobutyl (iBuGB), pinacolyl (GD), cyclohexyl (GF) methylphosphonofluoridates, and O-ethyl N, N-dimethylphosphoramidofluoridate (GAF) were detected with low LOD (15–75 pg/mL) and sharp peak shapes (high practical plate number (defined as 5.54 x (tR/Wh)2, where tR is the retention time and Wh is the bandwidth at half-height): 1100000–2400000) in GC MS using a polar separation column, electron ionization, and quadruple mass analyzer. During the analysis of fluoridated phosphonate-spiked ethyl acetate extract of solid phase extraction (SPE, Bond Elut NEXUS) from fluoride-mediated regeneration of blank human plasma, LOD (on EIC at m/z 99 except for GAF (m/z 126)) were 25–140 pg/mL with sharp peak shapes. The reaction recoveries in fluoride-mediated regeneration of plasma, which was inhibited by GB, GD, GA, GF, VX, and Russian VX (10 ng/mL), were 49–114% except for GD (10%). The concentration levels of 0.3–1 ng/mL of nerve agents in plasma could be determined.

In the work reported here, a screening procedure was developed for the detection and identification of RMPAs (nerve agent degradation products) after pentafluorobenzylation using liquid chromatography-tandem mass spectrometry (LC-MS/MS). With this method, all RMPAs, including highly hydrophilic types such as methylphosphonic acid (MPA) and ethyl methylphosphonic acid (EMPA), were sufficiently retained in commonly used reversed-phase columns (retention times: 15.7 and 11.0 min.), and the presence of RMPAs was determined more efficiently than with the conventional direct LC-MS/MS method. The detection limits of RMPAs using this approach (33 ng) were mostly superior to those observed with direct LC-MS/MS (74 ng) and gas chromatography-mass spectrometry (GC-MS) after pentafluorobenzylation (1.1 μg). The applicability of newly developed method toward real samples was evaluated via recovery tests involving urine/serum and wipe tests on various surfaces.

The Raman spectra of twenty-two chemical warfare agents (CWAs) were measured: eleven nerve agents and their precursor, five blister agents, three lachrymators, one choking agent, and one vomit agent, in liquid or solid state in colorless transparent vials were analyzed using a portable Raman spectrometer, Xantus-2 from Rigaku Corporation, equipped with selectable excitation lasers (785 nm and 1064 nm). With 785 nm excitation, characteristic Raman spectra composed of many sharp peaks were observed for twenty CWAs, but nitrogen mustard 3 (HN3) and adamsite (DM) did not show particular peaks owing to broad and intense mountain-like baselines. With 1064 nm excitation, Raman spectra similar to those with 785 nm excitation were observed for the twenty CWAs, where the wavenumbers of the peak tops and comparative heights were similar to those with 785 nm excitation. Characteristic Raman spectra with several sharp peaks could be even obtained for HN3 and DM with 1064 nm excitation. The resolutions of the peaks in the spectral region below 1000 cm−1 were higher with 785 nm excitation than those with 1064 nm excitation. In contrast, those above 1000 cm−1 were almost compatible with both excitations. The heights of the peaks in the spectral region lower than 1000 cm−1 were significantly higher with 785 nm excitation than those with 1064 nm excitation, but those higher than 1000 cm−1 were almost compatible with both excitations. The CWAs could be discriminated based on the Raman spectra showing respective unique fingerprint patterns, even among six alkyl methylphosphonofluoridate congeners. Structural assignment to Raman bands observed in the spectra was also proposed. The influence of mixing with gasoline to match the quality of library search was examined for seven representative CWAs. With 785 nm excitation, the hit quality index (HQI) of sarin was higher than 50% when the concentration (V/V) was higher than 25%. Meanwhile, with 1064 nm excitation, HQI of sarin was higher than 50% even when the concentration was as low as 15%. With 785 nm excitation, the HQI of L1 was higher than 50% when the concentration was higher than 80%. However, with 1064 nm excitation, the HQI of L1 was higher than 50% when the concentration was 20%. Measurements with 1064 nm excitation seemed superior in identifying CWAs in a gasoline mixture using the library search. The Raman spectra with 785 nm and 1064 nm excitation were compared in the measurement in the amber glass containers.

Currently, atmospheric plasmas are applied to the desorption and ionization of surface adhesive materials for rapid and highly sensitive analysis. One widely used chemical ionization method applies protons generated from atmospheric water molecules to a desorbed sample; however, a detailed study of the ionization mechanism has not been addressed thus far. Therefore, this paper proposes an ionization method that adds a small, optimized amount of hydrogen to the helium plasma for generating protons in the sample ionization source. Using this technique, mass spectrometry was performed on the sample surface deposits of 2-isopropylpyridine and 4-isopropylaniline. The hydrogen flow rate was optimized to the addition ratio of 2%, and the lowest limit of detection achieved is 0.1 and 42 pmol for 2-isopropylpyridine and 4-isopropylaniline, respectively. By optimizing the hydrogen addition flow rate, the optimum number of protons for sample ionization was attained, and it became possible to realize control of proton content in the sample, irrespective of humidity and surrounding environmental factors during mass spectrometry.

The surface of a commercially available silicon nitride (SiN) substrate was modified with gold (Au) nanoparticle and then with either lactosyl derivative or monoclonal antibody. Each of the derived SiN chips carrying Au-lactose or Au-antibody was evaluated in the reflectometric interference spectroscopic (RIfS) analyses of Ricinus communis lectin (RCA 120 ) and toxin (RCA 60 ). The RIfS analysis using the Au-lactose/SiN chip enabled us to detect the plant lectin at concentrations less than 5–10 ng/mL as well as the toxin (ricin) sealed carefully in a test sample at concentration about 1 μg/mL. The RIfS analysis using the Au-antibody/SiN chip was useful for the facile and direct screening of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain in the presence of possible interfering materials. The overall detection was completed within 2.5 h-cultivation time without any purification process for the pathogenic bacterium.

As a countermeasure against terrorism involving highly toxic chemical warfare agents, the rapid identification of the causative toxic substances is extremely important. This symposium review describes analytical methods the author's group has developed for detecting nerve gases after either high level or low level exposure. As a method for assessing human exposure to high levels of nerve gases, a technology that detects nerve gas hydrolysis products, i.e., strong anion exchange extraction-tert-butyldimethylsilyl derivatization-selectable one-dimensional or two-dimensional GC-MS, is explained. As a method for assessing human exposure to low levels of nerve gases, two technologies that detect adducts of nerve gas with blood cholinesterase, i.e., adduct purification-enzymatic digestion-LC/MS and fluoride-mediated regeneration-solid phase extraction-large volume introduction GC-MS, are explained.

Lactosides having either an amino-triethylene glycol or an azido-triethylene glycol were designed and synthesized, and the two derivatives were immobilized onto silicon nitride (SiN) surfaces. When a click reaction was applied for the immobilization of the azido-sugar, a Ricinus communis lectin (RCA120) was detected with a higher response by reflectometric interference spectroscopy (RIfS). When an N-hydroxysuccinimide (NHS) method was applied for the sugar immobilization, the response was less than that of the click one. The response of bovine serum albumin (BSA) as the negative control was negligible, but the lactose-SiN chip prepared by the click method suppressed nonspecific binding more effectively than did the chip from the NHS method. Next, we examined an antibody-immobilized SiN chip prepared by the click reaction. The detection response was, however, lower than that of the lactose-SiN chip, meaning that the sugar-chip by the click reaction was superior to the antibody-chip. Finally, to detect Shiga toxins from Escherichia coli O157:H7, globotrisaccharide (Gb3) with an azido-triethylene glycol was synthesized and immobilized onto the SiN chip by the click reaction. The Gb3-SiN chips enabled us to detect the toxins at concentrations less than 100 ng/mL. RCA120, horse gram, gorse lectins and BSA showed no response to the Gb3-SiN chip, showing a high specificity for the toxin.

Identification and quantification method for chemical warfare agents (CWAs) was developed using a continuous and standalone type hazardous gas detection instrument (MKS AIRGARD® Air Analyzer), Fourier transform infra-red (FT-IR) spectroscopy system which uses a multi pass gas cell, a Stirling-cooled mercury cadmium telluride detector, and advanced data processing including an exponentially weighted moving background and chemical identification algorithm. Although FT-IR spectrum of sarin vapor prepared in ambient air included absorbance bands of carbon dioxide and water remaining due to incomplete background correction, characteristic absorbance bands were clearly observed in the fingerprint region (700–1400 cm−1) and around 2950 cm−1. n-Hexane was used not only as dissolving solvent for volatile CWAs but also as an interfering field matrix compound. The instrument provided a “GB” alarm against n-hexane (1320 mg/m3) vapor containing dilute sarin (more than 0.44 mg/m3). The height of the band peak at 1021 cm−1 increased proportionally with the concentration. Vapors of soman, tabun, cyclohexylsarin, VX and RVX, which were prepared in ambient air, provided correct alarms, and showed FT-IR spectra similar to that of sarin. The vapors of sulfur mustard, nitrogen mustard 3, Lewisite 1, cyanogen chloride and phosgene provided correct alarms with characteristic FT-IR features, which are different from those of nerve agents. The vapors of volatile CWAs except for VX and RVX provided correct alarm even in the presence of high level of n-hexane. The vapors of unregistered CWAs and mixture vapor of nerve agents were also examined.

We determined new potential parameters for a calculation of ion mobility in air using the MOBCAL program. In our model, we regard air as a virtual uni-atomic molecular gas. And the Lennard-Jones parameters for various kinds of atoms in molecular ions were determined with simple and systematic methods. Using these parameters, we have calculated the mobilities of product ions from chemical warfare agents, normal saturated hydrocarbons, and illicit drugs. Most of the calculation results showed good agreement with the experimental values. Therefore this calculation method will be useful for understanding of the ion mobility spectrometry in air.

We propose detecting a fragment ion (Ph2As(+)) using counter-flow introduction atmospheric pressure chemical ionization ion trap mass spectrometry for sensitive air monitoring of chemical warfare vomiting agents diphenylchloroarsine (DA) and diphenylcyanoarsine (DC). The liquid sample containing of DA, DC, and bis(diphenylarsine)oxide (BDPAO) was heated in a dry air line, and the generated vapor was mixed into the humidified air flowing through the sampling line of a mass spectrometer. Humidity effect on the air monitoring was investigated by varying the humidity of the analyzed air sample. Evidence of the in-line conversion of DA and DC to diphenylarsine hydroxide (DPAH) and then BDPAO was obtained by comparing the chronograms of various ions from the beginning of heating. Multiple-stage mass spectrometry revealed that the protonated molecule (MH(+)) of DA, DC, DPAH, and BDPAO could produce Ph2As(+) through their in-source fragmentation. Among the signals of the ions that were investigated, the Ph2As(+) signal was the most intense and increased to reach a plateau with the increased air humidity, whereas the MH(+) signal of DA decreased. It was suggested that DA and DC were converted in-line into BDPAO, which was a major source of Ph2As(+). Graphical Abstract ᅟ.

The threat of biological and chemical terror acts remains a growing worldwide concern. There is therefore a need to develop appropriate technology for the detection of chemical and biological warfare agents, with early identification intended for use by first responders. Here, we disclose the developed autonomous air sampling and detection system for evaluation of the presence of chemical and biological warfare agents that can be harmful to the population. The current device utilizes the designed mist generator-assisted air collection system (338 l min−1) and biosensing chip technologies, such as electrochemical measurement, Au nanoparticle-based localized surface plasmon resonance, and rapid microfluidic chip PCR for detection of minute concentrations lower than the mean lethal dose (LD50) of nerve gases (sarin and VX), toxic proteins (BTX/A/Hc and ricin), and pathogens (anthrax simulant). An operation time of only 5–15 min is needed for the collection and detection; sample preparation is already integrated into the system without the need for direct human intervention. In addition to the system’s sensitivity and ease of use, its portability makes it highly beneficial for first responders, which could aid in immediate risk assessment and mitigation of on-site events. A portable device that can detect dangerous chemical and biological warfare agents at less than the mean lethal dose has been developed. Chemical and biological terrorism represents an increasing threat worldwide. Although various sensors have been developed for detecting toxins and pathogenic agents, there remains a need for a portable device that first responders can use to perform initial risk assessments. Masato Saito of Osaka University in Japan and co-workers have achieved this by combining three detection strategies in one device. The device samples air from the environment and then uses electrochemical measurements to detect nerve gases, gold nanoparticles to detect toxic proteins, and a PCR chip to detect pathogens. Molecules of interest can be detected in 5 to 15 minutes, and the device can be transported in a 30-centimeter cubic case.

A field-portable gas chromatograph-mass spectrometer (Hapsite ER system) was evaluated for the detection of chemical warfare agents (CWAs) in the vapor phase. The system consisted of Tri-Bed concentrator gas sampler (trapping time: 3s(-1)min), a nonpolar low thermal-mass capillary gas chromatography column capable of raising temperatures up to 200°C, a hydrophobic membrane-interfaced electron ionization quadrupole mass spectrometer evacuated by a non-evaporative getter pump for data acquisition, and a personal computer for data analysis. Sample vapors containing as little as 22μg sarin (GB), 100μg soman (GD), 210μg tabun (GA), 55μg cyclohexylsarin (GF), 4.8μg sulfur mustard, 390μg nitrogen mustard 1, 140μg of nitrogen mustard 2, 130μg nitrogen mustard 3, 120μg of 2-chloroacetophenone and 990μg of chloropicrin per cubic meter could be confirmed after Tri-Bed micro-concentration (for 1min) and automated AMDIS search within 12min. Using manual deconvolution by background subtraction of neighboring regions on the extracted ion chromatograms, the above-mentioned CWAs could be confirmed at lower concentration levels. The memory effects were also examined and we found that blister agents showed significantly more carry-over than nerve agents. Gasoline vapor was found to interfere with the detection of GB and GD, raising the concentration limits for confirmation in the presence of gasoline by both AMDIS search and manual deconvolution; however, GA and GF were not subject to interference by gasoline. Lewisite 1, and o-chlorobenzylidene malononitrile could also be confirmed by gas chromatography, but it was hard to quantify them. Vapors of phosgene, chlorine, and cyanogen chloride could be confirmed by direct mass spectrometric detection at concentration levels higher than 2, 140, and 10mg/m(3) respectively, by bypassing the micro-concentration trap and gas chromatographic separation.

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.

The plasmid encoding His-tagged organophosphorus hydrolase (OPH) cloned from Sphingobium fuliginis was modified to be transferred back to this bacterium. The replication function of S. amiense plasmid was inserted at downstream of OPH gene, and S. fuliginis was transformed with this plasmid. The transformant produced larger amount of active OPH with His-tag than E. coli.

A field-portable gas chromatography-mass spectrometry (GC-MS) system (Hapsite ER) was evaluated for the detection of nonvolatile V-type nerve agents (VX and Russian VX (RVX)) in the vapor phase. The Hapsite ER system consists of a Tri-Bed concentrator gas sampler, a nonpolar low thermal-mass capillary GC column and a hydrophobic membrane-interfaced electron ionization quadrupole mass spectrometer evacuated by a non-evaporative getter pump. The GC-MS system was attached to a VX-G fluoridating conversion tube containing silver nitrate and potassium fluoride. Sample vapors of VX and RVX were converted into O-ethyl methylphosphonofluoridate (EtGB) and O-isobutyl methylphosphonofluoridate (iBuGB), respectively. These fluoridated derivatives were detected within 10 min. No compounds were detected when the VX and RVX samples were analyzed without the conversion tube. A vapor sample of tabun (GA) was analyzed, in which GA and O-ethyl N,N-dimethylphosphoramidofluoridate were detected. The molar recovery percentages of EtGB and iBuGB from VX and RVX vapors varied from 0.3 to 17%, which was attributed to variations in the vaporization efficiency of the glass vapor container. The conversion efficiencies of the VX-G conversion tube for VX and RVX to their phosphonate derivatives were estimated to be 40%. VX and RVX vapors were detected at concentrations as low as 0.3 mg m-3 . Gasoline vapor was found to interfere with the analyses of VX and RVX. In the presence of 160 mg m-3 gasoline, the detection limits of VX and RVX vapor were increased to 20 mg m-3 . Copyright © 2017 John Wiley & Sons, Ltd.

A target analysis method for the sensitive and discriminative determination of the nerve agent hydrolysis products alkyl methylphosphonic acids as their tert-butyldimethylsilyl (TBDMS) derivatives was developed using a combination of selectable one- and two-dimensional (1D/2D) GC–MS, and applied to the analysis of samples with significant interfering matrices. After sample drying, the alkylmethylphosphonic acids and methylphosphonic acid (MPA) were converted to TBDMS derivatives by addition of N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide with heating, and subjected to 1D/2D GC–MS. The apparatus consisted of an initial low thermal mass DB-5 column and a second DB-17 column together with an electron ionization quadrupole mass spectrometer, offering simple and flexible switching between one- and two-dimensional GC–MS analysis in a single GC–MS system. Using 1D/2D GC–MS, analytes that do not co-elute with matrix components can be separated using 1D GC mode alone. Only those parts of the chromatogram that are negatively affected by the co-elution of matrix components need to be transferred and separated with 2D GC. Quantitation can be performed by a combination of both separations and mass spectrometric detection. The TBDMS derivatives of ethyl-, isopropyl-, isobutyl-, pinacolyl-, and cyclohexyl-MPA (cHMPA) and MPA itself were well separated within 3 min and determined in 1D GC–MS mode with detection limits of around 10 ng/ml of reaction mixture (except for the cHMPA derivative, whose mass spectrum contained noisy background peaks). In 2D-GC–MS mode, where each 0.04 min elution window from the 1D GC was subjected to heart-cut (H/C) and transferred to the second column after back-flushing the first column, the peak for the cHMPA TBDMS derivative was isolated and afforded a clean mass spectrum within 6 min. The recoveries of all the derivatives on 2D GC from 1D GC were estimated to be over 66%, and the detection limits were around 10 ng/ml of reaction mixture. In the presence of urine extract, the target compounds were not detected as separated peaks in 1D GC–MS mode (except for isobutyl-MPA), and quantification based on extracted ion monitoring could not be achieved. However, 2D GC–MS of the H/C fractions of the target derivatives gave single peaks with well-defined mass spectra, and the recoveries of the derivatives were over 70% except for cHMPA (31% at 1.25 μg/ml). Phosphonic acids could be detected at less than 60 ng/ml. Sulfuric acid and phosphoric acid also negatively affected the determination of alkyl methylphosphonic acid TBDMS derivatives in 1D GC–MS, and the MPA-TBDMS-derivative peak was completely obscured by the large sulfuric-acid-derivative peak. However, under 1D/2D GC–MS conditions, baseline separation of the MPA derivative and sulfuric acid derivative was achieved, enabling highly sensitive MPA detection at 20 ng/ml.

Anion exchange liquid chromatography (LC) with inductively coupled plasma–mass spectrometry (ICP–MS) was used for simultaneous determination of glyphosate and glufosinate (phosphonic and amino acid group-containing herbicides; PAAHs) and their hydrolysis products, aminomethylphosphonic acid (AMPA) and 3-methylphosphinicoacetic acid (MPPA), in biological samples. The target compounds were separated using an anion exchange resin column and gradient elution with sodium carbonate and sodium hydroxide system. The chromatographic eluates were passed through a membrane suppressor system and monitored by phosphorus-specific detection at m/z 31. PAAHs and their hydrolysis products were baseline separated from each other within 40 min of elution time, and phosphoric acid did not interfere with detection. The detection limits in serum samples were 0.1–0.7 µg/ml; those in urine samples, 0.2–1.6 µg/ml for the four compounds. The spiked recoveries for the four compounds were over 91 % in serum and urine samples. The detection of the compounds was not subject to interference from sample matrix components. The present method would be useful for toxicological analysis of PAAHs and their products in actual forensic practice. To our knowledge, this is the first trial to establish an LC–ICP–MS method for analysis of PAAHs and their products in biological samples .

Glycosyl-[60]fullerenes were first used as decontaminants against ricin, a lactose recognition proteotoxin in the Ricinus communis family. A fullerene glycoconjugate carrying two lactose units was synthesized by a [3 + 2] cycloaddition reaction between C60 and the azide group in 6-azidohexyl β-lactoside per-O-acetate. A colloidal aqueous solution with brown color was prepared from deprotected bis(lactosyl)-C60 and was found stable for more than 6 months keeping its red color. Upon mixing with an aqueous solution of Ricinus communis agglutinin (RCA120), the colloidal solution soon caused precipitations, while becoming colorless and transparent. In contrast, a solution of concanavalin A (Con A) caused no apparent change, indicating that the precipitation was caused specifically by carbohydrate–protein interactions. This notable phenomenon was quantified by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the results were discussed in terms of detection and decontamination of the deadly biological toxin in the Ricinus communis family.

We investigated the distribution of azide in blood and solid tissues in rats that were orally administered with fatal or toxic doses of sodium azide. We also determined the stability of azide in human blood in vitro. Azide concentrations were measured by gas chromatography–mass spectrometry after pentafluorobenzyl derivatization. The rats died immediately after administration of 100 mg sodium azide per kilogram of body weight. Azide was detected in blood and stomach at concentrations of >100 μg/ml, with lower concentrations in solid tissues, such as the liver, kidney, heart, and lung. The rats survived after administration of 25 mg sodium azide per kilogram of body weight. Azide was detected in blood until 3 h after administration. These results suggest that blood is the most useful specimen for detecting azide poisoning. We conducted in vitro experiments for the stability of azide by spiking it into human blood collected from healthy volunteers. After spiking human blood with 5 μg/ml of sodium azide, the azide concentration decreased in the red blood cell fraction, but not in the plasma fraction at 4 °C. We investigated the disappearance of azide from red blood cells using hemoglobin molecules with different heme oxidation and ligand binding states at 23 °C. The azide concentration was unaffected by carboxyhemoglobin or methemoglobin, but was decreased to 30 % of the original level by oxyhemoglobin. The disappearance of azide in the presence of oxyhemoglobin was suppressed by methemoglobin, but not by cyanmethemoglobin. These results suggest that azide is oxidized by oxyhemoglobin, and is stabilized by methemoglobin via strong ligand binding.

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 × 108 W/cm3. The measured electron number density, excitation temperature and afterglow gas temperature of the HPPMJ were 3.7 × 1015 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. Copyright © 2014 John Wiley & Sons, Ltd.

Dimethyl methylphosphonate (DMMP) removal by a continuous photocatalytic reaction system at different levels of humidity (RH) was investigated for the purpose of allowing rescue operations at disaster sites over an extended period of time when applied to gas masks or other protective gear. With a high flow rate of air (10 L/min), around 85% of the 0.17 μmol/L DMMP was successfully eliminated by this photocatalytic reaction system regardless of relative humidity. On the other hand, the formation of intermediates on the TiO2 surface indicated that photocatalytic activity was dependent on relative humidity, and during a 5-h test period, the ratio of photocatalytic DMMP conversion reached 74.5% at 100% RH. DMMP adsorption onto the TiO2 surface was found to be quite strong; DMMP removal was observed despite low humidity and dark conditions

The hydrolytic activities of organophosphorus hydrolase (OPH) toward nerve agents were investigated by using mutant OPH enzymes constructed by site-directed mutagenesis of the gene. The amino acids at position 136, 254, and 257 are known to influence the enzyme activity. The nucleotide sequencing of the OPH gene cloned in our previous study revealed that the deduced amino acid at position 254 was tyrosine (Tyr) instead of histidine (His) that was reported to reside at this position by other research groups. In the present study, such key amino acids were substituted to construct five mutant enzymes, and their hydrolytic activities were compared with that of the wild type enzyme. The activity assays proved that the substitution of Tyr at 254 to His led to the remarkable enhancement of the activities toward paraoxon and also toward VX, which could be decomposed by the wild type OPH only slightly. This mutant enzyme hydrolyzed most of the nerve agents almost completely, and approximately half of the VX during 20 min when the enzyme was activated with 10 mM CoCl2. The cobalt ion could activate the mutant OPH more efficiently than zinc ion, but the difference of the activation ability was not significant when other mutant OPH enzymes were activated with these divalent metal ions. The present results suggest that a practical decontamination system for nerve agents could be established using the mutant OPH with enhanced activity.

The specific differences on TiO 2 photocatalytic decomposition rates of vaporized isopropyl methylphosphonofluoridate, sarin (GB), r GB as a real Chemical Warfare Agent (CWA) and vaporized dimethyl methylphosphonate (DMMP), r DMMP as CWA simulant in gas phase have been investigated by Gas chromatography/mass spectrometry (GC/MS) analysis in ambient. The concentration of GB molecules adsorbed at the surface of TiO 2 in the dark, C GB , was 28 times larger than C DMMP in the dark. The r GB was then 3.3 times faster than the r DMMP . The results were explained by taking into account faster hydrolysis of GB molecules at the surface of TiO 2 and the larger diffusion constant of GB in the gas phase than DMMP molecules. The effect of F − adsorption at the surface of TiO 2 as fluoridated surface on the TiO 2 photocatalysis was also studied.

We have detected biological toxins using localized surface plasmon resonance (LSPR) and synthetic glycosyl ceramides (β-lactoside, globosyl trisaccharide (Gb3), or GM1 pentasaccharide) attached to gold (Au) nanoparticles. The particle diameters ranged from 5-100 nm. The detection sensitivity for three toxins (ricin, Shiga toxin, and cholera toxin) was found to depend not only on the attached glycoside but also on the diameter of the Au nanoparticles. For the detection of ricin, the 20-nm β-lactoside-coated Au nanoparticle exhibited the highest LSPR response, whereas 40-nm Gb3- and GM1-coated Au nanoparticles gave the best results for Shiga toxin and cholera toxin, respectively. In addition, a blocking process on the nanoparticle surface greatly improved the detection sensitivity for cholera toxin. The LSPR system enabled us to detect ricin at 30 ng/mL, Shiga toxin at 10 ng/mL, and the cholera toxin at 20 ng/mL.

The TiO2 photocatalytic decomposition of ethyl-S-dimethylaminoethyl methylphosphonothiolate (VX) as a real chemical warfare agent (CWA) in liquid phase has been investigated by gas chromatography/mass spectrometry (GC/MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. The TiO2 photocatalytic decomposition rate, rVX, was accelerated with increase in alkalinity. The several kinds of by-products such as diisopropylamine, ethyl methylphosphonic acid, oxygenated VX and partially oxidized VX were observed. On the basis of the by-products, the TiO2 photocatalysis were proposed as the P–S, C–N cleavage and oxygenation of VX molecule carried out mainly. The difference between rVX and vaporized dimethyl methylphosphonate (DMMP), rDMMP as CWA simulant was also investigated and the rVX was 30 times faster than the rDMMP. On the basis of the decomposition mechanism of VX and DMMP molecules, the results of the acceleration of rVX in alkaline condition and of the comparison of rVX and rDMMP were also discussed.

Japan has experienced numerous incidents of chemical disasters and terrorist attacks. Here we review the history of changes in countermeasures against such incidents. Since 2004, the Civil Protection Law, more formally known as the "Law Concerning the Measures for Protection of the People in Armed Attack Situations etc" was enacted to fully prepare the nation for chemical terrorism. According to this law, the Japanese government must carry out Civil Protection Exercises on an annual basis to gauge response. Problem areas that remain are chosen and addressed one by one until they are judged to be resolved in subsequent exercises.

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.

Two types of biotin-tagged glycopolymers carrying lactose or glucose in clusters along the polyacrylamide backbone were prepared and subjected to decontamination analyses with the plant toxin ricin. A buffer solution containing the toxin was treated with one glycopolymer followed by streptavidin-magnetic particles. Supernatant solutions were analyzed with surface plasmon resonance and capillary electrophoresis, and revealed that the lactose glycopolymer “captured” this toxin more effectively than the glucose polymer. Free toxin was not detectable in the supernatant after treatment with the glycopolymer and magnetic particles; >99% decontamination was achieved for this potentially fatal biological toxin.

A series of sugar-modified porous silica monoliths with different sugar ligands (β-lactoside, β-N-acetyllactosaminide, β-d-galactoside, β-d-N-acetylgalactosaminide and β-d-glucoside) and linkers were prepared and evaluated using plant toxins and lectins including ricin and a Ricinus communis agglutinin (RCA(120)). Among these sugar monoliths, a lactose monolith carrying a triethylene glycol spacer adsorbed ricin and RCA(120) with the highest efficiency. The monolith showed no binding with albumin, globulin, and lectins from Jack beans, Osage orange, Amur maackia and wheat germ. All these data support the utility of the lactose-modified monolith as a tool for adsorption and decontamination of plant toxins.