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Food poisoning is frequently caused by the accidental ingestion of toxic plants with strong morphological similarities to edible plants. The plant, false helleborine (Veratrum album), is commonly involved in such incidents. Cases of poisoning by toxic plants require rapid and accurate identification for appropriate medical treatment or forensic investigation in the event of death. However, morphological examinations require experience in systematic botany, and chemical analysis of natural compounds can be challenging. This study aimed to develop a sensitive, rapid, and specific method for identifying V. album and V. stamineum. We developed and optimized a loop-mediated isothermal amplification (LAMP) detection method. Total genomic DNA was extracted from approximately 10 mg (dry weight) of leaf from 26 specimens comprising the target plants, allied plants, and other causative plants of poisoning. These samples were analyzed the LAMP assay, and mixed samples, cooked samples, and simulated gastric contents were also tested. The limit of detection of our LAMP assay was 0.08 ng of target DNA, and the assay was specific to V. album and V. stamineum. The LAMP assay could be performed in approximately 30 min. V. album was also successfully identified in mixed samples, cooked samples, and simulated gastric contents. In addition, the LAMP assay signal could be effectively assessed with the naked eye. The present LAMP assay is a highly sensitive, rapid, and specific method for identifying V. album and V. stamineum.

Food poisoning is frequently caused by the accidental ingestion of toxic plants that possess strong morphological similarities to edible plants. False helleborine (Veratrum album) is one of the most common plants involved in such accidents. In cases of poisoning by toxic plants, rapid and accurate identification, usually based on the morphological or chemical analysis of plant parts, is required for appropriate medical treatment or forensic investigation. However, morphological examinations require experience in systematic botany because the samples are fragmentary, and chemical analysis of natural compounds can be difficult. In this study, we developed a TaqMan real-time PCR method using trnH-psbA and trnL-trnF that could be carried out in 30-60min. The lower detection limit was less than 10pg of DNA and the primer sets were specific to V. album and Veratrum stamineum. Mixed samples, cooked samples, and simulated gastric contents were successfully identified, and a multiplex assay of two regions was also possible. These results indicate that the TaqMan real-time PCR analysis is a very effective method to detect small samples of V. album and V. stamineum accurately and rapidly in poisoning cases.

Species identification from extracted DNA is sometimes needed for botanical samples. DNA quantification is required for an accurate and effective examination. If a quantitative assay provides unreliable estimates, a higher quantity of DNA than the estimated amount may be used in additional analyses to avoid failure to analyze samples from which extracting DNA is difficult. Compared with conventional methods, real-time quantitative PCR (qPCR) requires a low amount of DNA and enables quantification of dilute DNA solutions accurately. The aim of this study was to develop a qPCR assay for quantification of chloroplast DNA from taxonomically diverse plant species. An absolute quantification method was developed using primers targeting the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene using SYBR Green I-based qPCR. The calibration curve was generated using the PCR amplicon as the template. DNA extracts from representatives of 13 plant families common in Japan. This demonstrates that qPCR analysis is an effective method for quantification of DNA from plant samples. The results of qPCR assist in the decision-making will determine the success or failure of DNA analysis, indicating the possibility of optimization of the procedure for downstream reactions.

A semi-automated bacterial spore detection system (BSDS) was developed to detect biological threat agents (e.g., Bacillus anthracis) on-site. The system comprised an aerosol sampler, micro-fluidic chip-A (for spore germination and cell lysis), micro-fluidic chip-B (for extraction and detection of genomic DNA) and an analyzer. An aerosol with bacterial spores was first collected in the collection chamber of chip-A with a velocity of 300 l/min, and the chip-A was taken off from the aerosol sampler and loaded into the analyzer. Reagents packaged in the chip-A were sequentially applied into the chamber. The genomic DNA extract from spore lyzate was manually transferred from chip-A to chip-B and loaded into the analyzer. Genomic DNA in chip-B was first trapped on a glass bead column, washed with various reagents, and eluted to the detection chamber by sequential auto-dispensing. Isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) with fluorescent measurement was adopted to amplify and detect target DNA. Bacillus subtilis was the stimulant of biological warfare agent in this experiment. Pretreatment conditions were optimized by examining bacterial target DNA recovery in the respective steps (aerosol collection, spore germination, cell lysis, and DNA extraction), by an off-chip experiment using a real-time polymerase chain reaction quantification method. Without the germination step, B. subtilis spores did not demonstrate amplification of target DNA. The detection of 104 spores was achieved within 2 h throughout the micro-fluidic process.

Exposure to chemical warfare agents (CWAs) can be verified by detecting their degradation products in biological samples. However, the half-lives of these CWA degradation products in the body are short. CWAs are known to combine with biological macromolecules and the covalently bound compounds are called adducts. The residence time of adducts in the human body is longer than that of the corresponding CWA degradation products. Therefore, development of analytical methods to detect these adducts is a more realistic way of verifying CWA exposure. In this mini review, we describe the present state of research on analytical methods for identifying CWA adducts, and mainly introduce our research on nerve gas adducts using a direct method. Novel analytical methods for verifying low level exposure to nerve gases use liquid chromatography-mass spectrometry (LC-MS). Butyrylcholinesterase (BuChE) has been purified by affinity chromatography and sodium dodecylsulfate poly-acrylamide gel electrophoresis. The purified enzyme is inhibited by nerve gases (sarin, VX and soman) and digested by chymotrypsin, and then the peptides are analyzed by LC-MS and LC tandem MS. The peptide fragment that is combined with nerve gas is detected by LC-MS. Furthermore, the chemical structure of the adduct peptide characterized by MS provides structural information about the analyte. The detection limit of BuChE or BuChE adduct is estimated at 4 ng/injection (53 fmol/injection). If at least 1% of BuChE activity is inhibited by nerve gas, the adduct can be detected at a level of 1% BuChE inhibition in the victim’s serum.

Introduction : In cases of unknown poisoning, forensic toxicologists are required to elucidate the cause of death, and one of the most noticeable poisons is cyanide. We report on the artifactual production of cyanide from coffee and tea drinks in the presence of nitrite, which is frequently adulterated to lead to the fault result in drug testing. Methods : Cyanide was measured by head-space gas chromatography using porous polymer capillary GS-Q column and a nitrogen-phosphorus detector. Samples were mixed with sodium nitrite in a glass vial under acidic conditions of 10% phosphoric acid or 0.6 M sodium phosphate buffer (pH 5.0). After incubation for 30 min at 50 °C, the resulting head-space was injected into the gas chromatograph. Hydrogen cyanide was eluted at 2.2 min, and the peak area was converted for measuring cyanide concentration in the liquid reaction mixture. From the 50 °C incubation mixture of commercial coffee drink (or tea drink) and nitrite (50 mM), several g/mL and sub-g/mL of cyanide was produced in the presence of 10% phosphoric acid and pH 5.0 buffer, respectively. Cyanide was also produced from the reaction mixture of nitrite and polyphenol compounds such as chlorogenic acid and catechins, which are included in coffee and tea drinks, but not from a monophenol mixture. The amount of cyanide produced was dependent on substrate concentrations of nitrite and polyphenol, pH, temperature and incubation time. Conclusion : Forensic toxicologists should pay attention to this type of artifactual production of cyanide.

The Tokyo subway sarin gas attack in 1995 and the US postal anthrax letter attack in 2001 presented a threat of chemical and biological terrorism all over the world. To realize safe and secure society, the authority should establish a more strengthened crisis management system at national level for civil defense. Detection and identification of the causative agents is very important for countering chemical and biological terrorism from the standpoint of damage minimization. Various kinds of measuring technologies have been used for the on-site detection and laboratory identification of chemical and biological warfare agents (CBWAs). This review highlights the physicochemical and toxic properties of CBWAs, detection and identification requirements for crisis management, and the present status of detection technologies. Efficiency of mass spectrometry is compared with that of other analytical technologies from the standpoint of countering chemical and biological terrorism.

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).

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.

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.

Some commercially available portable on-site detection equipment (detection paper, gas detection tube, ion mobility spectrometer, surface acoustic wavelength detector, flame photometric detector, gas chromatograph–mass spectrometer) were evaluated using authentic vaporized chemical-warfare agents, from the standpoint of their qualitative detection characteristics, detection limits, response time, 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.

The CheckLite™ 250 Plus portable bioluminescence assay system for measuring bacterial adenosine 5'-triphosphate (ATP) was investigated for its performance with respect to the field detection of bacteria in bioterrorism incidents. Vegetative bacteria, Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), Staphylococcus aureus, and Yersinia pseudotuberculosis gave considerably high luminescence in a dose-dependent manner with responses of 104-105 relative light units (RLU) per 107 cells, whereas spore forms of B. subtilis showed considerably low luminescence with 214 RLU per 109 spores. Typical white powder materials such as wheat flour, sugar, and bovine serum albumin at concentrations of 0.1 and 0.5% (w/v) gave only negligible luminescence (lower than 400 RLU), and did not change the luminescence of E. coli cells significantly (42-102% of the control). The luminescence of B. subtilis spores increased considerably to over 104 RLU per 107 spores by pretreatment consisting of 37°C incubation for 30 min in nutrient broth medium containing 4 mM L-alanine. The increased luminescence by this pretreatment was not changed considerably (42-145% of the control) in the presence of the above tested white powder materials (0.1 and 0.5%).

A gas chromatographic-mass spectrometric method for the determination of S-methyl-L-cysteine sulfoxide (1), S-propyl-L-cysteine sulfoxide (2), and S-propenyl-L-cysteine sulfoxide (3), specific marker compounds in the genus Allium, is described. The target amino acids were converted to the tert-butyldimethylsilyl derivatives. The products were silylated on the amino and carboxyl groups and on an additional oxygen atom and were separated on a nonpolar capillary column. That incorporation of three tert-butyldimethylsilyl groups had occurred was verified by mass spectrometry, which gave an m/z 302 fragment as base peak (amino acid side chain eliminated ion) and m/z 436 (1), 464 (2), or 462 (3) as major peaks (tert-butyl function eliminated ion), by electron impact ionization. The detection limits for 1 and 2 under selected ion monitoring at m/z 436 (1) and m/z 464 (2), respectively, were determined to be 0.3 and 1.8 ng per injection. To clean up the analytes from the solvent extract of onion, as a representative food material, onion, the sample solution was subjected to combined solid phase extraction. The eluate from a Sep-Pak C(18) cartridge was applied to a Bond Elut SCX cartridge (H(+) form), followed by washing with 0.1 M hydrochloric acid and elution with 0.5 M ammonia. From a simulated matrix solution containing 5% sucrose, 1 and 2 were extracted quantitatively, and the detection yield was approximately 75%. The contents of 1, 2, and 3 in commercial onion were estimated to be 0.3, 3.1, and 3.0 mg, respectively, per gram of fresh weight.

A highly sensitive and specific real-time field-deployable detection technology, based on counterflow air introduction atmospheric pressure chemical ionization, has been developed for a wide range of chemical warfare agents (CWAs) comprising gaseous (two blood agents, three choking agents), volatile (six nerve gases and one precursor agent, five blister agents), and nonvolatile (three lachrymators, three vomiting agents) agents in air. The approach can afford effective chemical ionization, in both positive and negative ion modes, for ion trap multiple-stage mass spectrometry (MS(n)). The volatile and nonvolatile CWAs tested provided characteristic ions, which were fragmented into MS(3) product ions in positive and negative ion modes. Portions of the fragment ions were assigned by laboratory hybrid mass spectrometry (MS) composed of linear ion trap and high-resolution mass spectrometers. Gaseous agents were detected by MS or MS(2) in negative ion mode. The limits of detection for a 1 s measurement were typically at or below the microgram per cubic meter level except for chloropicrin (submilligram per cubic meter). Matrix effects by gasoline vapor resulted in minimal false-positive signals for all the CWAs and some signal suppression in the case of mustard gas. The moisture level did influence the measurement of the CWAs.

Cyanide and thiocyanate levels were determined by head space gas chromatography and the spectrophotometric Konig method respectively, for blood and salivary samples collected from 40 healthy adult volunteers. Blood cyanide (mean±standard deviation : 0.27±0.07μм), plasma thiocyanate (111.2±92.1μм), salivary cyanide (0.66±0.52μм) and salivary thiocyanate (1655±841μм) levels were significantly higher in the group of 20 tobacco smokers than in the group of 20 nonsmokers (0.17±0.04μм, 33.5±25.4μм, 0.38±0.26μм, 542±406μм). Statistical correlations were observed between the blood cyanide and the salivary thiocyanate levels (γ=0.64), between the blood cyanide and the plasma thiocyanate levels (γ=0.54), and between the plasma thiocyanate and the salivary thiocyanate levels (γ=0.46). It is concluded that not only plasma and salivary thiocyanate levels but also blood cyanide level can be suitable indices for distinguishing smokers from nonsmokers.

The efficiency of pretreatment of aqueous soil extracts using a cation-exchange resin has been investigated by gas chromatographic-mass spectrometric (GC-MS) determination of nerve agent hydrolysis products after tert.-butyldimethylsilyl (TBDMS) derivatization. An aqueous solution containing methylphosphonic acid (MPA) and its monoalkyl esters, ethyl methylphosphonic acid, isopropyl methylphosphonic acid and pinacolyl methylphosphonic acid, was dried, and these phosphonic acids were derivatized with N-methyl-N-(tert.-butyldimethylsilyl)trifluoro-acetamide and analyzed by GC-MS. The yields of TBDMS derivatives were significantly decreased by the addition of calcium and magnesium ions to an aqueous solution (approximately 0.5 mM) before derivatization. The extent of lowered yields was related to the hydrophilicity of phosphonic acids. MPA and its monoalkyl esters were spiked into soil samples (sand, alluvial soil and volcanic ash soil), extracted with distilled water, dried, silylated and applied to GC-MS. The yields of TBDMS derivatives of monoalkyl esters from soil samples were low (3-42%) and MPA derivative was scarcely detected (yield: < 0.7%). By desalting the aqueous soil extract by passage through a strong cation-exchange resin, the yields of TBDMS derivatives of monoalkyl esters were significantly improved (12-69%) and MPA derivative was detected (yield: 2-36%). The extent of improved yields was related to the concentrations of divalent metal cations in aqueous soil extracts. In combination with desalting by the cation-exchange resin, GC-MS after TBDMS derivatization enables detection of nerve agent hydrolysis products in soils at sub-ppm (0.2 microgram/g) concentrations.

To verify the exposure to nerve gas, a method for detecting human butyrylcholinesterase (BuChE)-nerve gas adduct was developed using LC-electrospray mass spectrometry (ESI-MS). Purified human serum BuChE was incubated with sarin, soman or VX, and the adduct was purified by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and digested in gel by treatment with chymotrypsin. The resulting peptide mixture was subjected to LC-ESI-MS. From the chymotryptic digest of untreated human BuChE, one peak corresponding to the peptide fragment containing the active center serine residue was detected on the extracted ion chromatogram at m/z 948.5, and the sequence was ascertained to be "GESAGAASVSL" by MS/MS analysis. From the chymotryptic digest of the human BuChE-sarin adduct, a singly charged peptide peak was detected on the extracted ion chromatogram at m/z 1,069.5, and the sequence was ascertained to be "GEXAGAASVSL" by MS/MS analysis (X denotes isopropylmethylphosphonylated serine). The difference in molecular weight (120.0 Da) between the active center peptide fragments corresponding to the untreated BuChE and BuChE-sarin adduct was assumed to be derived from the addition of an isopropyl methylphosphonyl moiety to the serine residue. The formation of human BuChE adducts with soman, VX and an aged soman adduct was confirmed by detecting the respective active center peptide fragments using LC-ESI-MS. To apply the established method to an actual biological sample, human serum was incubated with VX, and the adduct was purified by procainamide affinity chromatography followed by SDS-PAGE. After chymotryptic in gel digestion, the ethylphosphonylated active center peptide fragment could be detected, and the structure of the residue was ascertained by LC-ESI-MS analysis.

A method for determining thiodiglycol (TDG), a mustard gas hydrolysis product in water, serum and urine samples using gas chromatography–mass spectrometry (GC–MS) after tert-butyldimethylsilylation (TBDMS) is described. Quantitation of TDG was performed by measuring the respective peak area on the extracted ion chromatogram of m/z 293, using an internal standard, the TDG homologue, thiodipropanol, peak area of which was measured as m/z 321. The presence of salts in the sample solution not only suppressed the loss of TDG by vaporization during the evaporation of water, but also facilitated the rate of production of di-silylated derivative, bis(tert-butyldimethylsilyoxylethyl)sulfide (TDG-(TBDMS)2). Under the pretreatment conditions used, in which 0.5 ml of water sample supplemented with 100 μM potassium chloride was evaporated to dryness under reduced pressure, followed by reaction with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide at 60 °C for 1 h, TDG-(TBDMS)2 was reproducibly detected with about a 55% recovery and a limit of detection (LOD, scan mode, S/N = 3) of 5.4 ng/ml. TDG was also determined by GC–MS from a 0.5 ml serum sample (after perchloric acid deproteinization) and from a 0.1 ml urine sample, after TBDMS derivatization. The LOD was determined to be 7.0 and 110 ng/ml for serum and urine, respectively.

In order to confirm that diisopropylfluorophosphate (DFP) phosphorylates the active site serine residue in alpha-chymotrypsin, a peptide containing the phosphorylated active site was analyzed by liquid chromatography (LC)-electrospray mass spectrometry (ESI-MS). After reduction with dithiothreitol and subsequent alkylation with acrylamide, alpha-chymotrypsin was digested by treatment with trypsin. Tryptic digest was subjected to LC-ESI-MS. Nearly all the peptide fragments were identified by comparison with fragments predicted from as tryptic digest of alpha-chymotrypsin. From the tryptic digest of native alpha-chymotrypsin, a doubly protonated peptide peak which corresponded to the peptide fragment containing the active site serine residue was detected on a selected ion chromatogram at m/z 1265.0, and the sequence was determined to be "DAMICAGASGVSSCMGDSGGPLVCK". From the tryptic digest of DFP-inhibited alpha-chymotrypsin, the doubly protonated peptide peak was detected on a selected ion chromatogram at m/z 1347.0. The difference in mass number (82 in a doubly charged ion) of active site peptide fragments between the native and DFP inhibited alpha-chymotrypsins was assumed to be the result of phosphorylation of the serine residue with a diisopropylphosphoryl moiety. A total of +164 Da mass shifts of y-series fragment ions from the y(8) to y(21) positions in the active site peptide of the DFP inhibited alpha-chymotrypsin was observed, in comparison with the native alpha-chymotrypsin. Thus, the phosphorylation site in alpha-chymotrypsin could be unequivocally identified to be at the serine residue which is located at position 47, from the N-terminus of the alpha-chymotrypsin C-chain.

The effects of heating on hemoglobin (Hb) and carbon monoxide (CO) levels in human blood were investigated by in vitro experiments. Head-space gas chromatography (HS-GC) using a molecular sieve 5A stationary phase and thermal conductivity detection was adopted for the measurement of CO gas, and spectrophotometric methods were used for the measurement of various Hb forms, protein and heme contents. Deteriorated absorbance spectra were observed for heat-treated blood samples, and double wavelength spectrophotometry was proven to give wrong percent saturation of carboxyhemoglobin content (% CO-Hb). The blood sample taken from one fatal fire casualty gave significantly higher % CO-Hb measured spectrophotometrically, compared to that by HS-GC. Control blood or purified Hb solution, which was saturated with CO in designated extent, was heated in a sealed vial. Under the incubation below 54 degrees C, all Hb forms were stable, except for oxyhemoglobin (Hb-O(2)), which was partially oxidized to met-hemoglobin (Met-Hb). In contrast, under the incubation at 65 degrees C, Met-Hb was denatured completely to be insoluble, and Hb-O(2) was partially denatured via Met-Hb formation. CO-Hb was resistant against heating. The difference of heat susceptibility and precipitability among Hb forms resulted in artificial increase of % CO-Hb. During heating, spontaneous CO was produced from blood.

A rapid screening method was developed for the determination of the toxic volatile anions, cyanide and azide, in beverages. This method consisted of a microdiffusion extraction combined with spectrophotometry using the Konig cyanide reaction and ferric azide complex formation in conjugation with cerium azide oxido-reduction. The time required to achieve full recovery in the extraction of hydrogen cyanide and hydrazoic acid from samples was considerably shortened by increasing the diffusion temperature from 25 degrees C to 40 degrees C. The time required to achieve saturated color development in the Konig cyanide reaction was also shortened by increasing incubation temperature to 40 degrees C. The interference in both azide color reactions was examined for volatile compounds. Cyanide interfered only in the case of ferric azide complex formation. Sulfide, sulfate, nitrite, and acetic acid interfered in both the color reactions. The established method gave a detection limit of 6 microM for cyanide and 0.5mM for azide, and it required only 1 h to determine both anions. Cyanide and azide disappeared by evaporation from beverages during 25 degrees C storage under open conditions in a pH-dependent manner as a function of their respective pKa values of 9.2 and 4.6.

Detection and identification of alkyl methylphosphonate (RMPA) and methylphosphonate (MPA) are performed to verify the existence of nerve gases by gas chromatography-mass spectrometry (GC-MS) after tert-butyldimethylsilylation (TBDMS). However, it is sometimes difficult to detect RMPA and MPA in soils. This study examines the relationship between the pedological characteristics and the aqueous extraction recoveries and TBDMS derivatization yields of ethyl-, isopropyl- and pinacolyl methylphosphonate and MPA for 21 soil samples. The aqueous extraction recoveries were measured directly by capillary electrophoresis. Andosols showed low extraction recoveries, while Regosols and Fluvisols showed high recoveries. RMPA were extracted with higher recoveries than MPA from all soils. MPA could not be extracted from Andosols. Within the pedological characteristics, phosphate absorption coefficients showed a strong negative correlation with the extraction recoveries of all phosphonates. The levels of RMPA and MPA in aqueous soil extracts were also determined for eight soils by GC-MS after TBDMS. Compared to the aqueous extraction recoveries, the yields of TBDMS derivatives were low. Strong anion exchange led to a significant improvement in derivatization yields. The efficiencies of TBDMS derivatization were inversely correlated with the levels of alkaline earth metals extractable from soils when the three soils that possessed high total carbon were excluded.

A forensic investigation was carried out on one poisoning case, where cyanide was first detected in an evidence sample of a canned coffee drink. A more complete study revealed that it had been adulterated with isobutyl nitrite (IBN) and not cyanide. We examined the detectivity of IBN and related compounds by headspace gas chromatography and capillary electrophoresis. IBN decomposes to isobutyl alcohol (iBuOH) and nitrite in aqueous solution, and under higher temperature and more acidic conditions, the rate of this reaction becomes more rapid. IBN was also produced by the esterification of iBuOH with nitrite below pH 5. Cyanide was produced in a coffee solution by the addition of nitrite below pH 6. An IBN-spiked canned coffee drink solution was stored at 4 degrees C and periodically analyzed for IBN, iBuOH, nitrite, nitrate, and cyanide. Since the IBN level decreased rapidly, iBuOH was produced in an almost 90% molar yield. Nitrite production reached a maximum of 40% molar recovery on the first day and then gradually disappeared. The nitrate level reached a plateau of approximately 60% molar recovery. Cyanide was also detected, and its level at the 14th day was approximately 0.26% molar recovery. These findings suggest that, in a coffee drink solution, IBN undergoes hydrolysis to produce iBuOH and nitric acid, which is oxidized to nitrate and also produces cyanide through the nonspecific oxidation of organic compounds under acidic conditions.

A pretreatment procedure, using a macroporous strong anion-exchange resin (MSA) has been established for the determination of nerve gas hydrolysis products by gas chromatography-mass spectrometry (GC-MS) after tert.-butyldimethylsilyl (TBDMS) derivatization. Aqueous solutions of methylphosphonic acid (MPA) and three alkyl methylphosphonic acids (AMPAs) (ethyl, isopropyl and pinacolyl methylphosphonic acid), were retained on the MSA column, and then quantitatively eluted with 0.1 M hydrochloric acid. The neutralized column eluate was dried, and MPA and AMPAs were derivatized with N-methyl-N-(tert.-butyldimethylsilyl)-trifluoroacetamide and analyzed by GC-MS. The column eluate was also analyzed in order to determine the exact hydrolysis product levels by capillary electrophoresis using borate and benzoate buffer (pH 6). The MSA pretreatment was examined for the clean-up of aqueous extracts of three types of soils and an aqueous solution containing 10% sucrose, which is regarded as model for a typical soft drink, after spiking with MPA and AMPAs. MPA and AMPAs were quantitatively recovered in the MSA eluate fraction from those samples, except for MPA from volcanic acid and alluvial soils. The yields of TBDMS derivatives were remarkably improved, compared with for which no pretreatment was used and also for those in which a strong cation-exchange resin was used. The achieved detection limits of MPA and AMPAs ranged from 0.12 to 0.18 microg/g of soil (S/N=3). The established MSA method was applied to the pretreatment of spiked sea water, two types of beverages, Pepsi Cola and canned coffee. Although the yields of TBDMS derivatives of MPA and AMPAs in sea water (in a range between 44 and 96%) and AMPAs in Pepsi Cola (in a range between 58 and 92%) were rather high, those for MPA in the Pepsi Cola (27%) and those for MPA and AMPAs in the canned coffee (in a range between 5 and 17%) were low.

Sarin gas attack caused by AUM SHINRIKYO, Japanese cult, gave us great shock toward illegal usage of chemical warfare agent. Eighteen people died and more than fifty hundreds had received medical treatment, in Matsumoto Incident (June 27, 1994) and in Tokyo Subway Incident (March 20, 1995). We have performed toxicological tests for the blood samples drawn from 7 (Matsumoto) and 11 (Tokyo) dead and 38 (Matsumoto) injured people. Both red blood cell (RBC) acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BuChE) activities of victim’s blood samples have been measured by modified Ellman’s method using specific substrates acetylthiocholine and butyrylthiocholine, respectively. Compared to the control values of healthy donors, both RBC AChE and plasma BuChE activities were significantly (p

The detectability and stability of lachrymators [2-chloroacetophenone (CN), o-chlorobenzylidene malononitrile (CS) and synthetic capsaicin (nonivamide)] were investigated using dichloromethane extraction followed by gas chromatography-mass spectrometry. Ultrasonication at 40°C slightly improved the extraction yields of nonivamide to almost quantitative yield, compared to low yields (30 to 40%) of CN and CS. In terms of the stability of low spiked concentrations (6 to 7 _g), from the surface of glass and stainless steel (60 to 100 cm square), CN rapidly disappeared, CS disappeared gradually, and nonivamide was almost quantitatively recovered. The three lachrymators in absorbent cotton (0.3 g) gradually disappeared. From water (20 mL), nonivamide gradually disappeared and was undetectable after eleven days. CN decreased gradually, and instead, acetophenone appeared. CS disappeared rapidly, and o-chlorobenzaldehyde, o-chlorobenzyl alcohol, and ochlorobenzoic acid were produced. The stability of high spiked concentrations of CN and CS in water (0.5 to 0.6 mg per 20 mL) was also investigated using liquid chromatography. CN was quantitatively recovered. The concentrations of CS decreased to about an8 % recovery level within 2 h. Concomitantly, o-chlorobenzaldehyde appeared at a concentration of about 80% recovery.