Mieko Kataoka

Effect of cation-exchange pretreatment of aqueous soil extracts on the gas chromatographic-mass spectrometric determination of nerve agent hydrolysis products after tert.-butyldimethylsilylation

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.

Stability of blood carbon monoxide and hemoglobins during heating.

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.

Efficiency of pretreatment of aqueous samples using a macroporous strong anion-exchange resin on the determination of nerve gas hydrolysis products by gas chromatography-mass spectrometry after tert.-butyldimethylsilylation.

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.

Discriminative determination of alkyl methylphosphonates and methylphosphonate in blood plasma and urine by gas chromatography-mass spectrometry after tert.-butyldimethylsilylation.

A method for determining two nerve gas hydrolysis products, alkyl (ethyl, isopropyl and pinacolyl) methylphosphonates (RMPAs) and methylphosphonate (MPA), separately, in human plasma and urine samples was developed, using two different deproteinization procedures. In the first method, the plasma sample was deproteinized by adding a fourfold volume of acetonitrile, followed by passing the supernatant through a Bond Elut strong anion-exchange (SAX) cartridge [fluoride (F(-)) form]. After washing the cartridge with water and methanol, the RMPAs were eluted with a 3% (v/v) solution of methanolic ammonia, and analyzed by gas chromatography-mass spectrometry (GC-MS) after tert.-butyldimethylsilyl (TBDMS) derivatization. The detection yields of TBDMS derivatives of RMPAs were in the range of 69 to 99%, in contrast to the poor yields obtained when only acetonitrile deproteinization pretreatment was used (yield: 13-26%). The yield of the TBDMS derivative of MPA was very low (8%), however. In a the second method, a plasma sample was deproteinized by adding a half volume of 10% (w/v) trichloroacetic acid (TCA), and the resulting supernatant was extracted with diethyl ether to remove TCA, the aqueous fraction was then passed through a Bond Elut SAX cartridge. After washing the cartridge with 0.5% (v/v) methanolic ammonia, MPA was eluted with 3% (v/v) methanolic ammonia. The detection yield of the TBDMS derivative of MPA was nearly quantitative. A pretreatment method using SAX solid-phase extraction was also developed for the cleanup of a urine sample, in which the sample was directly applied to a Bond Elut SAX cartridge, followed by elution of the RMPAs and MPA with 3% (v/v) methanolic ammonia, which were then derivatized and analyzed by GC-MS. The detection yields of TBDMS derivatives of RMPAs and MPA were in the range of 61 to 97%.

Stability and detectability of lachrymators and their degradation products in evidence samples

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.

Blood Cholinesterase Activity Levels of Victims Intoxicated with Sarin in Matsumoto and Tokyo Subway System

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<0.05) lowered in 7 fatalities and 16 nonfatal casualties in Matsumoto and 6 fatalities in Tokyo. The extent of the lowered activity levels was more remarkable in RBC AChE than in plasma BuChE. There were one fatal and 9 nonfatal casualties which RBC AChE activities were significantly lowered even though the counterpart plasma BuChE activities were not lowered.