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  • 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

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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 Simons reaction (Simons-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 Simons-HPLC and gas chromatography with flame ionization dctection. and these quantiiative values Mere good correlative M A and OIHiiorBZP i n the MA users urine spiked OHnorBZP could be analyzed simultaneously M ithout interference peaks. Our method was useful as a substitute of the Slmoils- 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 ; bDepartment 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 55C 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 -30C 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 I...