I. TOXIC0L.-TOXIN REVIEWS, 17(1), 85-98 (1998)

ABSTRACTS OF ORAL AND POSTER PRESENTATIONS

AUTOMATED PREPARATION AND ANALYSIS OF MORPHINE IN BIOLOGICAL E U I D S

A l a n Namera*, Miluo Yashiki*, Kanako Okada*, Yasumasa Iwasakr*, Tohru Kojima* and HaJirne Kawakami**, *Department of Legal Medicine, Hiroshima University School of Medicine, 1-2-3, Kasumi, Minamr ku, Hiroshima 734, Japan and **Yokogawa Analqtical Systems Inc , 1-15-5, Naka-cho, Musashini 180, Japan

A method for the automatic analqsis of morptune i n biological fluids u a s debeloped using a PrepStation (Hewlett Pachard, USA) in combination with a gas chromatographimass spectrometer Human interaction is only required to place samples on the auto-sampler tray Recoveq of morphine was more than 90% The calibration curve, using the internal standard method, demonstrated good lineanty throughout the concentration range from 0 01 to 1 0 [ L gjml

for unne and from 0 05 to 1 0 u g/ml for serum The proposed method was applied to some clinical cases

MICROANALYSIS OF C A N N A B I S COMPONENTS A N D THEIR METABOLITES BY ELISA. K. Watanabe”, Y. Tateoka”, T. Matsunaga”, I. Yamamoto‘), Y. Shoyama” & H. Yoshimura’), ’ ) Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa 920- 11, Japan, *) Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Kyushu Uiversity, Fukuoka 812, Japan & 3, Department of Food and Nutrition, Nakamura Gakuen University, Fukuoka 814-01, Japan

The monoclonal antibody (MAb-4A4) against A9-tetrahydrocannabinolic acid (A9-THCA) was produced in mice immunized with A9-THCA-BSA conjugate. Competitive ELISA was established using MAb-4A4, A9-THCA-@alanineBSA conjugate, peroxidase- or alkaline phosphatase-conjugated anti-mouse IgG. The antibody cross-reacted with THC, cannabidiol, cannabinol and 15 kinds of THC metabolites, but not with other biological

85

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86 ABSTRACTS

compounds such as cholesterol, testosterone and anandamide. Thus, the ELISA was not specific for A9-THCA but detected various cannabinoids indicating the advantage of the antibody for screening of cannabinoid related compounds. This system is applicable to detection of cannabinoids and their metabolites in biological samples.

A NEW SCREENING METHOD FOR AMPHETAMINE AND METHAM-

PHETAMINE USING DANSYL CHLORIDE DERIVATIZATION. H. Y A M A D A I . H. IWASAKI ' , K. OGURI' & S. WADA*. I Faculty of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 8 12-81, Japan; Kyushu District Narcotic Control Office, Fukuoka 8 12, Japan.

A new screening method for amphetamines was developed. It consists of derivati- zation with dansyl chloride, extraction of the derivative using a Sep-Pak cartridge, a reversed phase column, and visualization of the fluorescence of the cartridge. Of fifty five compounds examined, amphetamine, methamphetamine and the methylene- dioxy derivatives exhibited strong fluorescence, while related compounds, such as N- ethylamphetamine and fenetylline, were negative or weak positive. Amino acids were negative. In consistent with this, drug-free control urine exhibited no fluores- cence in cartridge. The disadvantage of the present method is that i t is a multi- step procedure and needs 20 - 30 min for screening. However. since the method has a different specificity from the widely used immunochemical method, i t is suggested as a specific screen for amphetamines.

SIMULTANEOUS DETERMINATION OF METHAMPHETAMINE AND p-HYDROXY DESMETHYL BENZPHETAMINE BY HPLC USING POST- COLUMN SIMON'S REACTION Laboratory, Ishikawa Pref Police H Q . 2-1-1 Hirosaka, Kanazawa 920 Japan

S Chinaha & N Takayama, Forensic Science

A high-performance liquid chromatographic method for the sitnultaneous determination of methamphetamine (MA) and p-hydrox) desrnethyl benzphetamine (OHnorBZP) using post-column Simon's reaction (Simon's-HPLC) has been developed MA is used by most stimulant addicts Benzphetamine (BZP), from which OHnorBZP or MA etc are biotransformed, is also being irnponed or used as street drug The detection limit of MA was Ing This data indicated that

the Simon's-HPLC has about 100 times sensitivity in comparison with the thin-

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ABSTRACTS 87

l q e r chromatography using Simon’s reaction (Simon’s-TLC) The detection liniit of OHnorBZP ivas ca.20ng. The calibration curve of MA \$as linear at the range 0 1 - 1 000pgiiiil. and the intermediate precisions of within-run and between-run assals Mere I 1% and 2 12%. respectively. MA i n 13 stimulant addicts’ urine sainplcc was determined by the Simon’s-HPLC and gas chromatography with flame ionization dctection. and these quantiiative values Mere good correlative M A and OIHiiorBZP i n the MA user’s urine spiked OHnorBZP could be analyzed simultaneously M ithout interference peaks. Our method was useful as a substitute of the Slmoil’s- ILC.

SENSITIVE DETERMINATION OF THINNER COMPONENTS IN HUMAN BODY

FLUIDS BY CAPILLARY GAS CHROMATOGRAPHY WITH LOW OVEN

TEMPERATURE. X.-P. Lee”, T. Kumazawa”, K. Satoa), K. Watanabeb’, H. Senob’ and 0.

Suzuki”. ”Department of Legal Medicinc, Showa University School of Medicine, 1-58

Hatanodai, Shinagawa-ku, Tokyo 142, Japan ; b’Department of Legal Medicine, Hamamatsu

University School of Mcdicine, 3600 Handa-cho, Hamamatsu 431 -31, Japan.

A sensitive method is presented for determination of thinner components in human body

fluids by capillary gas chromatography (GC) with low oven temperature for trapping headspace

vapor insidc the capillary column. After heating a whole blood or urinc sample containing

ethyl acetatc, bcnzene, ri-butanol, toluenc, rr-butyl acetate, n-isoamyl acetate and ethyl benzene

as internal standard in a 7.5 ml-vial at YO “C for 30 min, 5 ml of headspace vapor was drawn

into a glass syringe. All vapor was introduced through an injection port in the splitless mode

into a D E h 2 4 middle-bore capillary column with the oven temperature at 5 ” C for trapping

all volatilc compounds, and the oven temperature was programmed up t o 110 “C for detection

of the compounds by GC with a flame ionization detector. The prcsent conditions gave sharp

peaks and good separation of each compound with low background noises. Recoveries of thc

6 compounds were 4.7&75.7 o/o and 3.SCA1.7 % for whole blood and urine samples,

respcctively. The CV values of all compounds in whole blood and urine samples were

3.5-9.5 %. The calibration curves showed linearity in the range 0.78400 ngi0.5 ml wholc

blood or urine; the detection limits were 0.5-5 ngj0.5 ml. The data on actual analysis of

toluene and n-butyl acetate in postmortem blood were also reported.

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88 ABSTRACTS

SENSITIVE DETERMINATION OF CHLOROFORM AND METHYLENE CHLORIDE IN WHOLE BLOOD BY CAPILLARY GAS CHROMATOGRAPHY WITH CRYOGENIC OVEN TEMPERATURE Kanako Watanabe, Akira Ishii, Hiroshi Sen0 & Osamu Suzuki, Department of Legal Medicine, Hamamatsu University School of Medicine, 3600 Handa- cho, Hamamatsu 43 1-3 1, Japan & Hideki Hattori, Department of Legal Medicine, k c h i Medical University, Nagakute-cho, Aichi, 480- 1 1, Japan

A new and sensitive method of gas chromatography (GC), for measurements of chloroform or methylene chloride in whole blood with use of cryogenic oven temperature, is presented. After heating a blood sample containing chloroform and methylene chloride (internal standard, vice versa) in a 7.0-ml vial at 55°C for 20 min, 5 ml of the headspace vapor was drawn into a glass

syringe. All vapor was introduced into an Rtx-Volatiles middle-bore capillary column in the splittless mode at -30°C of oven temperature for trapping

entire amounts of compounds to be analyzed, and the oven temperature was programmed up to 280% for detection of the compounds and for cleaning of

the column. The present conditions gave sharp peaks for hoth chloroform and methylene chloride, and very low background noises for whole blood samples. As much as 1 1 . 5 and 20 0 YO of chloroform and methylene chloride, respectively, which had been added to whole blood in a vial, could be introduced into the GC column. The calibration curves showed linearity in the range of 0.05-5.0 mg/0.5 ml whole blood. The detection limit was about 2

ng0 .5 ml. The coefficients of intra-day and inter-day variations of area ratios of chloroform / methylene chloride, which had been spiked to human blood, were not greater than 1.74 and 8 85 %, respectively. The data on chloroform or methylene chloride in rat blood after inhalation of each compound were also presented

ANALYSIS FOR PESTICIDE COMPONENTS BY PULSE HEATlNG - GAS CHROMATOGRAPHY MASS SPECTROMET'RY. T. Takayasu, T. Ohshima, T. Kondo, M. Ohtsuji & Y. Sato, Department of Legal Medicine, Kanazawa University Faculty of Medicine, School of Medicine. 13-1 Takara-machi, Kanazawa 920-8640. Japan

Analysis for pesticide components, xylene, o-dichlorbenzene (DCB), cresol and DDVP (an organophosphate) has been attempted with 3 /*I of biological specimens by pulse

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ABSTRACTS 89

heating (Py 1- gas chromatography - mass spectrometry (GC-MS) without pretreatment. A model JHP-3 of Curie-point pyrolyzer and JMS-DX-303 system for mass spectrometer with a DB- 17 widebore column were used for analysis. Electron impact and positive ion detection modes were utilized for MS. Three it1 of specimen were analyzed by GC-MS within 15 niin (Ref.: J Clin Forensic Med. 2, 65, 1995). Xylene, DCB, cresol and DDVP were separately detected by Py-GC-MS. The regression lines for these 4 components in the blood were shown to be linear between peak height and their concentrations, 0.1 to 20 pglml (for xylene, DCB, cresol): 0.2 to 20 pglml (for DDVP). Recoveries of DDVP, cresol. DCB. xylene at the concentration of I.Opg/ml by Py-GC-MS were 91.4-101% in the blood and 96.2-103% in the urine. Lower detection limits for xylene, DCB, cresol and DDVP were 0.15,0.15,0.15 and 0.45 ng/ injection (3 pl), respectively. By Py-GC-MS, xylene and DDVP could be successfully detected in the urine in an emergency medical care case. These results indicate that the simple and rapid Py-GC-MS provides a useful method for analyzing small molecule pesticide components in forensic toxicology and emergency medical practice.

DETECTION OF SARIN HYDROLYSIS PRODUCTS FROM BRAIN TISSUES IN ACUTE SARIN POISONING CASES. T. Takaton, M Nakajima, H. Niyma, and H. Iwase, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan

Y. Matsuda, M Nagao, Graduate School of Medicine,

We detected the sann hydrolysis products from brain tissues stored in the formalin fixative for 2 years in acute sann poisoning victims in the Tokyo subway. Sann-bound acetylcholinesterase (AChE) was solubilized from formalin-fixed cerebellum, punfied with immunoaffinity chromatography, and digested with tqpsin. Then, sarin hydrolysis products bound to AChE were released by alkaline phosphatase digestion. and released sarin hydrolysis products were subjected to trimethylsilyl derivatizanon (TMS) and detected by gas chromatography-mass spectrometry. The peaks of rnk 275 and m t 740, which are specific to TMS-methylphosphonic acid, were observed within the range of retenhon time as that of the authentic methylphosphonic acid. We could not detect isopropylmethylphosphonic acid from the formalin-fixed cerebellum of these sarin victims, suggesting that the isopropoxy group of isopropylmethylphosphonic acid would be chemically hydrolysed during storage. These procedures u ill be useful for the forensic diagnosis of protein-bound toxic agents.

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90 ABSTRACTS

HIGHLY SENSITIVE QUANTlTATION O F METHAMPHETAMINE

NEW EUROPIUM CHELATE LABEL. Hiroko Kimura, Masahiro Mukaida* Jingli Yuan**, Guilan Wang** Kazuko Matsumoto**and Akua Mori***, Department of Forensic Medicine, Juntendo University School of Medicine, Hongo, Tokyo 113. * Department of Forensic Medicine, National Defense Medical College, **Department of Chemistry, Waseda University, ***Department of Legal Medicine, Teikyo University School of Medicine.

A simple and hghly sensitive time-resolved fluoroimmunoassay is described for the quantitation of methamphetamine (MA). Newly synthesized europium complex (BHHCT-ELI) was used as labeling material and MA was quantitated by one step and two step assay variations of competitive immunoassay. The minimum detection limits of MA were 1 ng/ml (25 pglassay) and 1 pg/ml (25 fg/assay), respectively. These are ten to one thousand times superior than any other immunoassay method and have enough detectability to measure a small quantity of MA in a segment of hair. We assayed 34 urine samples and there was a good correlation between this method and previously established gas chromatography (r=0.954). Intra-assay CV is about 3-6% at eight different concentrations (n=4). In our assay, the use of new fluorescent labeling makes highly sensitive and selective detection possible without interfering specificity of the anti-MA.

USING TIME-RESOLVED IMMUNOFLUOROMETFUC ASSAY WITH A

QUATITATIVE ANALYSIS OF 4-CHLOROPHENYLPJPERIDINE

ANALOGS DERIVED FROM HALOPERIDOL IN BIOLOGICAL

SAMPLES K Igarashi, T Matsumoto, T Yokoi, F Kasuya and M Fukui.

Faculty of Pharmaceutical Sciences. Kobegakuin University, Nishi-ku, Kobe

651-21, JAPAN

We have developed the determination of 4-chloro-phenylpipendine analogs

which are structurally similar to the dopaminergic pro-neurotoxin, I methyl-4-

phenyl-I,2,3$-tetrahydropyridine (MPTP) using HPLC and GC-MS system

Four metabolites derived from haloperidol in biological samples, 4-(4-

chloropheny1)piperidinol (CPHP), 4-(4-~hlorophenyl)pyridine (CPP), 1 -

methyl-4-(4-chlorophenyl)pipendinol (MCPHP) and 1 -methyl-4-(4-

chloropheny1)pyridinium species (MCPP+), were extracted by using

chloroform under alkaline condition and Sep-pak CN cartridge CPHP only

was analyzed as N-isopropylformate derivative CPHP, CPP and MCPHP in

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ABSTRACTS 91

samples were determined with GC-MS-SIU system MCPP' mas determined

using HPLCifluorescence detection with O D S column These methods icere

applied in the biological samples from rats follovving 10 administration of

halopendol

UNUSUAL POISONING CASE BY LAXATIVE -TOXICOLOGICAL ANALYSIS OF BISACODYL AND ITS METABOLITE- Keiko Kudo. Chiaki Miyazaki. Ryo Kadoya. Tohru Irnamura. Narurni

Jitsufuchi and Noriaki Ikcda. Faculty of Medicine. K y u s h u University, Japan

A 11-year-old boy was hospitalized because of scvere diarrhea with the volume of more than 5 I . Although he could survive by the addition of intravenous fluid with proper care ot electrolyte balance, his diarrhea continued. Based on thc careful observation of thc patient and his family in hospital. surreptitious administration of poison by his mother was suspected. and toxicological analysis was carried out on his stool and white powder given by his mother. Bisacodyl was found in the powder. and the metabolite of bisacodyl was found in the stool. In order to quantitate bisacodyl metabolite in his urine, stool and serum, we devised a sensitive and reliable method. The sample was incubated with p -glucuronidase at 37"Cfor 2 hours, bisacodyl metabolite was effectively extracted with f -butyl methyl ether. and then derivatized by methylation and subjected to gas chromatography/mass spectrometry (GCI'MS). Bisphenol A was used as an internal standard. The calibration curve was linear in the concentration range from 1 nglO.2 ml (or g) to 1000 ngiO.2 ml (or g), and the lower limit of detection was 1 ng/0.2 ml (or 9) . The concentrations of bisacodyl metabolite in the urine. stool and serum of the patient were determined.

IMMUNOHISTOCHEMICAL INVESTIGATION OF PULMON.4RY SURF4CTANT IN FATALITIES RELATED TO POISONING B-L Zhu. L Quan. S Oritani, K Ishida.

M Q Fujita. M Ogawa* and H Maeda, Department of Legal Medicine and *Hospital

Department o f Pharmacy. Osaka Citv University Medical School. Asahi-machi 1-4-54. Abeno, 545 Osaka, Japan

In order to verify forensic pathological significance o f pulmonary surfactant in interpretation of respiratory failure o r distress for the fatal mechanisms in poisoning. an immunohistochemical investigation was performed Surfactant apoprotein 4 h a s most

intensely observed in poisoning with pancuronium bromide (muscle relaxant) and i n

fatalities from inhalation o f petroleum (butane) gas In carbon monoxide poisoning, the increase o f surfactant was more evident in cases of inhalation o f automobile exhaust fumes in the cabin with relativelv lower carboxyhemoglobin levels Fatal poisoning with

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92 ABSTRACTS

sedative-hypnotics showed no remarkable increase of surfactant The above-described observations suggest the pulmonary surfactant (apoprotein A) may be a possible indicator of respiratory distress (asphyxiation) of peripheral origins (agony) and the agonal periods

THE POSSIBILITY OF ABSORPTION OF A SIGNIFICANT AMOUNT OF

INTUBATION-RELATED LIDOCAINE FROM THE TRACHEA DURING

PROLONGED CARDIOPULMONARY RESUSCITATION F Moriya & Y

Hashimoto, Department of Legal Medicine, Kochi Medical School, Nankoku, Kochi

783, Japan

The purpose of this study was to determine whether intubation-related lidocaine is

absorbed from the trachea during the artificial circulation of cardiopulmonary

resuscitation. The tissue distribution of lidocaine was investigated in four individuals

whose heart beat was never restored during cardiopulmonary resuscitation In Cases 1

and 2 where heart massage had been continued for 5 min, 0.102-1 02 n idL and 0 163-

0 195 m a of lidocaine were detected in the blood left in the heart and inferior vena

cava, respectively, a large proportion of the lidocaine detected in these blood samples

may have diffused from the trachea after cessation of cardiopulmonary resuscitation

since no lidocaine was detected in the brain, liver and femoral muscle In C,ase 3 where

cardiopulmonary resuscitation had been performed for 60 min, tracheal lidocaine was

thought to have been absorbed during cardiopulmonary resuscitation because 0 167-

0 340 mg/L or mgikg lidocaine was detected in the cerebrospinal fluid, liver, hdney and

femoral muscle Case 4 in which cardiopulmonary resuscitation had been continued for

60 min did not show an increase in the lidocaine concentration in the brain, liver and

femoral muscle, although 0 135-1.41 mg/L or mgikg lidocaine was detected in the

inferior vena cava blood and lungs, a possible reason for lack of absorption of lidocaine

from the trachea may have been that effective blood circulation was not obtained during

cardiopulmonary resuscitation because of bleeding and pulmonary collapse. The

pattern of tissue distribution of lidocaine gives useful information on the state of

decedents during cardiopulmonary resuscitation

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A E S T R A C T S

Analysis of Illicit Drugs by Ion Mobility Spectrometry

93

Ahihiro Mihi. Michiahi Tastuno Munehiro Katagi, May umi Nishiha\w and I Iitoshi

Tsuchihashi

Forensic Science Laboratorq Osaha Prefectural Police Headquarters

3-18, I-Chome, Hornmachi, Chuo-\\ord. Osaha 541. Japan

Tracec of illegal drugc \%ere detectable bc IMS from a residue on a pipe ujed tor

smoking drugs as \\ell as a suspect's clothing The detection limits of

methamphetamine(MA) cocaine and heroin were 0 3 0 5 and 3 0 ng, respecti\el\ In

analSsis of biological samples, the use of an appropriate internal standard, such as tri-ii-

hexklamine. was \er) effectike in iinproi ing reliabilib and repea tab ih Ho\re\er to

date this inethod in IMS had newr been utilized Using this technique umple and

rapid screenings were developed for MA and amphetamine-bpe drugs in both urine and

hair The cutoffle\el u a c set at 0 5

&mL M A could be discriminated froin its analogs including ephedrine and MDMA

In terms of IMS detection of MA in hair digestion of hair cpecimens prior t o

measurements was verq etfscti\e in unbinding M A f'rom the hair inatrix and efticientlb

vaporizing the drug for it? eftectix detection MA incorporated in the hair Ma5 seini-

quantitativel! detected b! IMS s i t h a detection limit o f 0 5 ng mg using 2-mg hair

Although IMS does not hold for identification there are certain advantages in using thic

technique in t\hich no immunoasa) cross-reactlrig \\ith other amphetamine-t\pe

drugs is obserced

MA in urine \bas semi-quantitativeli detectable

SIMULTANEOUS DETERMINATION OF TRIAZOLOBENZODIAZEPINE DRlJGS BY CAPILLARY COLUMN GAS CHROMATOGRAPHY WITH ELECTRON CAPTURE DETECTION. Masaru Terada', Yuki lijima', Yoshiyuki Sakano', Tatsuo Shinozukab. Junichi Yanagidah, Einosuke Tanaka' and Shogo Misawa', 'Department of Legal Medicine, Osaka University Medical School bDepartment of Legal Medicine, School of Medicine, Keio University 'Department of Legal Medicine, Institute of C o m m u n i t y Medicine, University of Tsukuba

A high sensitive method for simultaneous determination of triazolobenzodiazepine drugs(estazo1am. alprazolam, etizolam, triazolam)) was developed using non-polar fused

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94 ABSTRACTS

silica capillary column( IS m x 0.32 mm i.d.. f i l m thickness 0.25 pm. DB-I gas chromatography with electron capture detection (GC-ECD). The etizolam and triamlani were not separated by our gas chromatographic condition because of a similar chemical structure and molecular weight of these drugs. The identification of the etizolani and

triazolam mixture was very difficult using capillary GC-mass spectroniet?(MS) because of the same retention times and similar mass spectra. However. the both drugs are identified by thin layer chromatography-GC-MS. high performance liquid chromatography(HPLC)-photodiode array detection, HPLC-MS and negative chemical ionization-GC tandem MS, respectively. Using non-polar fused silica capillary column GC-ECD, the sensitivity of triazolobenzodiazepinedrugs showed a higher sensitivity and

detectedat least in a. 8-1 I and 160-220 fold lower concentration than GC-NPD and GC- FID, respectively. The average recoveries of triazolobenzodiazepine drugs from serum containing 20 ng and 50 ng were 92.94% and 93.96%. respectively. Satisfactory recoveries of triazolobenzodiazepine drugs in serum indicated this method applicable for

the determination of these compounds in human biological materials. The detection limits of estazolam, alprazolam, etizolam and triazolam were a. 9- 12 pg, This method could be used to determineconcentrationas low as u. 6-9 nginll of these drugs in serum.

HEAD-SPACE GAS CHROMATOGRAPHY IN TOXICOLOGICAL ANALYSIS -RELATIONSHIP AMONG PHYSICAL PROPERTIES, ANALYTICAL CONDITIONS AND BLOOD LEVELS OF TOXIC SUBSTANCES-. Y . Scto, h4. Kataoka & N. Tsunoda, National Rcscarch Institute ol Police Scienccs, 6 , Sanban-cho, Chiloda-ku, Tok!o 102. Japail

In head-space g:~? cliroiiiatograplii~ (HS-GC) anal) lor \ olatilc substance\ in blood, the detection limit I S mainly dctcnnincd by both GC scnsiti\ i ty and

thc partition coeflicicnt ( k , dcfincd as thc ratlo of anal),tc concenti-ation i n the liquid phase to that in the gascous phase) The detcction Iiiiiits wci-c measurcd

for typical t m i c substanccs (h!drogcn c!,anidc (HCN), chloroform (CL) , carbon inono\ide (CO), cthunol (EtOl-1) undci- thc l.ollo\\,ing HS-GC conditions (HS tcnipcraturc, k. GC col tiiiiii. CC detector): 50°C. 76. GS-Q. NPD; W ' C , 8.2, DB-WAX, FID; room tcmp.. 0.021, Molccular .;ic\e 5A, TCD; 37°C. 1756, DB-WAX, FID. Thc dctcction l i i i i i t s (npi i i i l ) nci-e e\pcriiiicnrally obtained to bc I (HCN), 20 (CL), 200 (CO) and I600

(EtOH), rcspcctiicly, and tar Iowcr than the blood IcicIs (iiigiii i l) of 3 (HCN, Icthal). 30 (CL, Icthal). 100 (CO. leth:il) and 500 (EtOH. impaircd

driving), rcspecti\cly. Thcrcl'orc. i t is imporcant to design ,uiJylical

conditions, so as LO pct sulficicnt detccrton semiti\ it! cttnsidcnng their knit

blood levels

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ABSTRACTS 95

RAPID QUANTITATIVE ANALYSIS OFOLFANDRIN IN HUMAN BLOOD BY HIGH PEWORMA NCE LIQUID CHROMATOGRAPHY

Ahra Namera, Miho Yashihi, Kanako Okada, Yasumasa Iuasahi and Tohru KOJimd

Department of Legal Medicine, Hiroshima Unicersity School of Medicine, 1-2-3, Kasumi, Minami-ku, Hiroshima 734, Japan

A simple dnd rapid methcxi lor quantildtion of oleandnn in human blood w a s developed using Ewtrelut column extraction in combination with high performance liquid chromatography Oleandnn W A S eluted uith cthyl dcetate from Extrelut column and a n a l y ~ e d hy HPLC Separation of oleandrin from endogeneous impunties has generally satisfactory with the use of a rebersed-phase column The oleandnn showed excellent linearity in the range of 0 05 - 10 f i gig and the detection limit ma5 0 02 LL gig lor human blood The recobery o f oleandnn, which had been spiked Kith human blood, was more than 90%

An optimization of SPME/semi-micro LC system for emergency toxicology screening in human fluids

Mahko Hayashida. Kyoko Uekusa. Kciko Hirakawa, Makoto Nihira. Youkichi Ohno and Kiyokatu Jinno* Department of Legal Medicine, Nippon i l ed ica l School Faculty of Matenals Science. Toyohahi University of Technology*

The solid-phase microcutmuon (SPME) is a rapid and simple sample preparation technique. An interface for SPME / semi-rmcro LC ha5 been developed and i t s performance has been evalualed by analyvng organophosphorus pesticides in human fluids of the pauents admitted to Cnucal Care Medical Center, Nippon Medical School. To apply SPME i semi-micro LC system tor emergency toxicology screening. an optimii.atlon ol benwdiaztpines, which have the same basic structure. should be performed as the first step. In thc second stcp, an opumimuon of a letv drugs, M hich have different structures. should be performed. In this study an opnmmtion of henzodiaxpines for SPME I semi-micro LC system was performed. I80min. e\trdcQon temperature : 6 O r , concentration of salt : saturated, flush \olume . 6 rmcro liter.

desorption m e : 30min. Except the amount of acetonitnle llushed into the desorption device, the parameters for henLodiwepines were as same aq those for organophosphorus pesucidcs in the prebi- ous stud). Although the extraction and the desorption umc is relauvcly long in the e\perimental conditions. the charactensucs of SPMUsemi-micro LC system are considered to bc useful lor emer- gencj toxicology screcning.

Parameters were as follou s , cxtraction ume

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96 ABSTRACTS

HEADSPACE SOLID-PHASE MICROEXTRACTION (SPME) AND GC-

ECD DETERMINATION OF DINITROANILINE HERBICIDES IN

SURFACE WATER AND BIOLOGICAL FLUIDS Fuyu Guan, Kanako

Watanabe, Akira Ishii, Hiroshi Seno & Osamu Suzuki, Department of Legal

Medicine, Hamamatsu University School of Medicine, 3600 Handa-cho,

Hamarnatsu 43 1-3 I , Japan

A simple, fast and sensitive method has been established for headspace

SPME and GC-ECD determination of six dinitroaniline herbicides in surface

water, human blood and urine Urine or surface water (1 0 mL) spiked with

herbicides, in the presence of 0.28 g of Na2S04, was pre-heated at 70°C for

10 min, then subject to headspace SPME at 70°C for another 30 min, while

spiked blood (0 5 mL) dIluted with water (0 5 mL) was treated at 90°C in the

same way Headspace SPME yielded clean extracts from urine or blood,

which could be directly analyzed by GC-ECD. The recoveries of the

herbicides from water or urine ranged from 35 to 58%, as measured at the

concentrations of 3.50 and ! C ng/mL, while those from blood va:ied fro=

3.2 to 7 2%, at the concentrations of 6.0 and 20 ng/O 5 mL blood The peak

areas of the extracted herbicides were proportional to their concentrations in

the range from 0 1 to 10 ng/mL urine or water, or from 1 to 60 ng/0 5 mL

blood The intra- and inter-day coefficients of variation were within 10% for

most of the analytes With the above method, a dosed herbicide was

successh~lly separated and determined from rats’ blood The developed

method is more sensitive to the herbicides and simpler than the previously

reported ones

EVALUATION OF BLOOD CYANIDE CONCENTRATION IN AUTOPSY MATERIALS APRELIMINARY STUDY S Oritani, L Quan, M Q Fujita, K Isluda, B-L Zhu, and H Maeda, Department of Legal Medicine, Osaka City Umversity Medical School, A s h - m a c h 1-4-54, Abeno, 545 Osaka, Japan We routinely investigated cyamde concentrations in forensic autopsy cases on an automated head-space gas chromatographyhass spectrometry system The blood cyamde levels were detemuned at 0-3 5 ,u g/ml In fire victims, lugher blood cyamde

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ABSTRACTS 97

levels were often associated with higher carboxyhemoglobin The difference in cyanide levels between the left and right heart blood showed a tendency to correlate with the total hemoglobin contents In a preliminary experimental study, blood cyanide level was increased by hemoconcentration, and the increase of carboxyhemoglobin level seemed to reduce the recovery of added cyanide Thus it is suggested that hrther investigation is required for forensic toxicological evaluation of blood cyanide levels, especially with regard to the influence of total hemoglobin and carboxyhemoglobin

THE DISTRIBUTION KIKETICS OF MT, PGP AKD GST I N RAT KIDNEY F O L L O & I X REPEATED r\DMIKISTRATIOK OF CADMIUM. A. Itoh. M. Hayashi, M. Hoshida and Y.Q. Feng. Department of Legal Medicine. Toho L'niversity. School of Medicine. Tokyo, Japan. 113

In order to investigate the defcnse mechanisms of the body against Cd accumulation, the Cd concentration i n various organs and the distribulion of WT. PGP and GST in the kidneys of r a t s receiving repeated o r s i n g l e administration of Cd were analyzed. In the single group, the concentration of Cd was highest i n the liver. followed by, in descending order, the kidney. Iung,heart and blood. In the repeat group, the concentration of Cd was high- est in the kidney, followed by, in descending order, the liver. heart. lung and blood. The results of immunohistological analyses showed that MT was dctec- ted in a wide area of the kidney ranging from the proximal t o distal tuhu- les in the single group. In contrast, in the repetitive group. MT was more strongly expressed in the distal tuhules of the kidnek than i n the proximal tubules. and the level of MT was highest two weeks af[er administration. Both PGP and GST(Ya,Yp) were strongly expressed i n [hc proximal t u b u l e s n f the kidney in the single group. and were expressed more strongly in i h i , single group than in the repetitivc group. The above findings suggest [ha[ the body possesses a strong defcnse mecahnism against Cd accumulation.

SENSITIVE ANALYSIS OF THIOSLJLFATE AND FQLYSIILFIDES I N BODY FLUIDS BY GC/FPD. Y.Asai". K.Clbukata". M.Sakata", T.Tsukuda" and K.1 kemoto". "Fac. of Pharm. Sci., Health Sci. Univ. of HOKKAIDO. "SAPPORO CITY General Hospital.

The sensitive analytical methods of thiosulfate and polysulfide in urine and plasma using GCiFPD are presented. Thiosulfate was alkylated with pentafluorobenzylbromide in ascorbic acid and sodium chloride solution. The alkylated thiosulfate was oxidized with

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98 ABSTRACTS

hydrogen peroxide, followed by concentration under nitrogen gas. Polysulfide were alkylated with pentafluorobenzylbromide under tetradecyldirnethylbenzylarnrnonium as a counter-ion. The alkylated thiosulfate and polysulfide were analyzed quantitatively by GCIFPD. The lower limit of thiosulphate and polysulfide were 0.00Surnol/ml and 0. Ing/ml respectively. The proposed method was sensitive enough to detect thiosulfate and polysulfide in plasma and urine of hydrogen sulfide and/or polysulfide poisoning.

A N AUTOPSY CASE OF HYPNOTIC' DRUG POISONING ACCOMPANIED WITH

HYPERTHERMIA - Motohiko Y.AMAZ.AKI' ". Masaru TERADA". Ynshiaki OGURA". Yoichi

MITSUKUNI" and Masao YOSHIMURA" . "Department of Legal Medicine. Osaka University

Medical School. "Osaka M ed I ca I Ex an1 i ncr'\ Office. Japan

A W-year-old male. I n thc hepiiiiiiiig 0 1 one Febi-liar). he was found dead on his bed at home by

the farnily. I n inquest by tlic policL>. ic'ctiil lemperature showed hyperthermic condition ie. 38.4 "c. Autopsy was performed on thc \ m e day to find out the cause of death. Autopsy findings were as

follows: he wa\ 170 cm 1311 ;tnd wighctl X I .O kg. Findings were consistent with acute death,

and recogniLed white-colored grain i n the gastric contents. Phenobarbital. chlorpromazine and

pi-omethazine were detected 111 concentration of 47. 1.7 and 4.4 ;L d m l in the heart blood

qxcimcn respectivelq. The GNI\C 1,1 death was determined as hypnotic di-ug poisoning by the

\ynergistic effect of these 3 hind\ 0 1 drugs on the hasi.; of autopsy findings. histological findings

m d drug analysis finding\. Caw report\ of hypnotic drug poisoning accompanied with

hypcnhermia are coinpararivcl! ~ r c . and the above ca5e seemed to be an interesting one.

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