Noriko Tsunoda

Determination of Aconitum alkaloids in blood and urine samples. I. High-performance liquid chromatographic separation, solid-phase extraction and mass spectrometric confirmation

Determination of four toxic Aconitum alkaloids, aconitine, mesaconitine, hypaconitine and jesaconitine, in blood and urine samples has been established using high-performance liquid chromatography (HPLC) combined with ultraviolet absorbance detection, solid-phase extraction and mass spectrometry (MS). These alkaloids were hydrolyzed rapidly in alkaline solution (half lives (t1/2)<one day), were stable in solutions of acetonitrile, tetrahydrofuran and diluted hydrochloric acid (t1/2>five months) and were unstable in solutions of methanol and ethanol (t1/2<one month). These alkaloids were separated on an octadecylsilica column with isocratic elution using a solvent mixture of tetrahydrofuran and 0.2% trifluoroacetic acid (14:86, v/v), which was found to be the optimal solvent of the elution systems examined. Calibration curves with UV detection were linear on injection of amounts ranging from 2.5 to 500 ng, and the limit of detection was 1 ng (S/N=3). These four alkaloids in aqueous solution were recovered almost totally by solid-phase extraction using the styrene polymer resin, Sep-Pak Plus PS-1, and were eluted using a mixture of acetonitrile and hydrochloric acid. These Aconitum alkaloids were confirmed by HPLC coupled with fast atom bombardment MS, giving their protonated molecular ions as base peaks. These alkaloids were detected by HPLC with UV detection from blood samples spiked with more than 50 ng ml(-1) of alkaloids, but were not detectable from urine samples spiked with 5 microg ml(-1) of alkaloids because of severe sample interference.

Simultaneous determination of the herbicides glyphosate, glufosinate and bialaphos and their metabolites by capillary gas chromatography-ion-trap mass spectrometry

Abstract A sensitive gas chromatographic (GC)—ion-trap mass spectrometric (IT-MS) method has been developed to determine simultaneously the herbicides glyphosate, glufosinate and bialaphos and their major metabolises. A single-step derivatization is achieved at 80°C for 30 min with the reagent N-methyl-N-( tert. -butyldimethylsilyl)trifluoroacetamide in dimethylformamide, which introduces the tert. -butyldimethylsilyl group at active hydrogens and gives only a single peak for each compound. The derivatives of three herbicides, their metabolises and nineteen amino acids were simultaneously chromatographed and well separated in a single run on a DB-1 fused-silica capillary column. Each tert. -butyldimethylsilylated derivative produces an easily interpretable mass spectrum dominated by unique M - 15, M - 57, M - 85 and M - 159 fragment ions. The limits of detection were estimated to be 10–20 ng for glyphosate and glufosinate and their metabolises, and 500 ng for bialaphos, by GC-IT-MS. On the other hand, using GC analysis with flame ionization detection, glyphosate and glufosinate and their metabolises were detectable at levels of approximately 100 ng, but bialaphos could not be detected at a level of 5000 ng.

Determination of blood cyanide by headspace gas chromatography with nitrogen-phosphorus detection and using a megabore capillary column

Abstract A method for the determination of cyanide in blood using head-space capillary gas chromatography (HS-GC) with nitrogen-phosphorus detection (NPD) was developed. The separation efficiency and sensitivity for hydrogen cyanide (HCN) were improved by using a GS-Q megabore capillary column and by injection of 500 μl of HS gas with a splitting ratio of 5. No interference or column deterioration was observed. Optimum HS conditions were found to be incubation of 50°C for 30 min in the presence of 10% phosphoric acid as an acidifying reagent. The distribution coefficient ( k ) was 75.7 and 158.9 for HCN acetonitrile (CH 3 CN; candidate for internal standard), respectively. HCN showed a different vaporization behaviour to CH 3 CN with respect to temperature and matrix effect. The salting-out effect was relatively small for HCN and CH 3 CN. An increase in the blood content in the liquid phase resulted in a significant increase in k for HCN. The calibration graph was linear over a wide concentration range of cyanide, and the limit of determination was 1 ng ml −1 (R.S.D. 10%). Because a considerable amount of CH 3 CN was detected in control blood, the direct calibration method could be used instead of the internal standard method using CH 3 CN. The detectable amount of cyanide was significantly decreased during the preincubation of cyanide and blood at 2°C. Thiocyanate in blood showed a positive interference. Contents determined by this HS-GC method agreed closely with those obtained by the microdiffusion-spectrophotometric method for blood samples from fire victims.

Determination of Aconitum alkaloids in blood and urine samples. II. Capillary liquid chromatographic-frit fast atom bombardment mass spectrometric analysis.

Determination of fourteen alkaloids, toxic Aconitum alkaloids, aconitine, mesaconitine, jesaconitine, hypaconitine and deoxyaconitine, and their hydrolysis products, benzoylaconines and aconines, have been established using capillary liquid chromatography (LC) fast atom bombardment mass spectrometry (FAB-MS) with a frit interface. Protonated molecular ions were observed as base peaks in the FAB-MS for these fourteen alkaloids. All the alkaloids were simultaneously quantified with linear gradient LC elution by solvent mixture of acetonitrile and 0.3% trifluoroacetic acid using selected ion monitoring of the protonated molecular ions. The calibration curves of these alkaloids were linear in injection amounts ranging from 5 to 500 pg, and their detection limits were 1 pg per injection (S/N=3). Solid-phase extraction using Sep-Pak Plus PS-1 was also investigated to clean-up and concentrate alkaloids in blood and urine samples, and showed satisfactory recoveries. This capillary LC-frit-FAB-MS method enables determination of low levels of Aconitum alkaloids in blood and urine samples, coupled with solid-phase extraction.

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.

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.