- Email: [email protected]
Comparison of rapid methods (HPLC, ELISA and Minicolumn) to detect aflatoxin M1 in the milk
PosterSession3G. Methods Dapsone, which was not a carcinogen, showed negative results in the above mentioned tests. LPO and micro viscosity were of no change in comparing with the control. From the consistency of the resulte derived from such tests and their known carcinogenicity, relationship between the carcinogenic activty and particular alteration s in certain cellular biohemical and biophysical parameters might be suspected.
197
IP3G188I SIMULTANEOUS DETERMINATION OF OXAZEPAM ENANTIOMERS AND ITS ANTAGONIST, FLUMAZENIL, IN SERUM BY CHIRALHPLC FOR TOXICOKINETIC STUDY
G. Pautet! , C. Pham-Huy e", N. Chikhi-Chorfi l , H. Galons", J.-M . Wamet" , J.R. Claude 1 . JLab. Toxicologie (EA 207); 2 Lab.
Chimie Organique. FacultePharmacie, Universite Paris5, France Oxazepam (OZP), a tranquillizer, has been used as racemic mix-
1
P3G 1861 DETECTION OF HYDROXYL AND 1 HYDROXYETHYL
ture to treat anxiety and other mental diseases . It is also an active
FREERADICALSUSINGSPINTRAPS FOLLOWED BY GAS CHROMATOGRAPHIC-MASS SPECTROMETRIC DETECTION. APPLICATION TO STUDIES ON ETHANOLato-TRANSFORMATION IN LIVERMICROSOMES OR HIGHLY PURIFIED NUCLEI
metabolite of a number of benzodiazepines. In humans . S(+)-OZP is pharmacologically the active form and the inversion phenomena of enantiomers is also observed. Therefore, it is suitable to analyze the respective OZP enantiomer levels in biological fluids rather than the total OZP cencentrations for therapeutic monitoring and for overdosage treatment by its antagonist, flumazenil. The simultaneous determination of OZP enantiomers and its antagonist, flumazenil, in rabbit serum by enantioselective HPLC using a new Cyclobond-I-2000-SP column is described. After a1kaline extraction with clonazepam as internal standard, OZP enantiomers and flumazenil are separated on the previous column with a new polar organic mobile phase (acetonitrile-absolute ethanol-glacial acetic acid-triethylamine) and detected at 230 nm. Peak resolutions are 2.7 for OZP enantiomers. Their selectivity factor ex is 1.18. The sensitivity is about 10 nglmI for OZP enantiomers and 5 nglml for flumazenil. The coefficients of variation are about 3.9%. The area ratio of two enantiomers is about 1.00 ± 0.05. The development of this new chiral HPLC method is used to determine the toxicokinetic profile of the two OZP enantiomers in the rabbits under the treatment of flumazenil. It is also suitable for pharmacokinetic and toxicological assays in therapeutic monitoring and in overdosage treatment in humans . It can be applied to study the inversion mechanism of the two OZP enantiomers in organism and also the metabolism of a number of benzodiazepines because oxazepam is their active metabolite.
G.D. Castro *, A.M.A. Delgado de Layfio, J.A. Castro . Centro de
Investigaciones Toxicol6gicas (CEITOX) -CITEFAICONICET, Zufriategui 4380, (1603) Villa Martelli, Pcia. de BuenosAires, Argentina Detection of hydroxyl radicals is frequently made by spin trapping procedures followed by ESR detection of the adduct radical formed. ESR equipment however is very expens ive and not frequent in many countries . In this work we describe a procedure where the .OH radical is detected by spin trapping with DMPO followed of derivatization of the adduct formed by silylation and precise and specific identification by gas chromatography/mass spectrometry. The procedure was initially tested against a model system where the .OH was generated by a Fenton reaction (iron plus hydrogen peroxide) either in the presence or the absence of ethanol. In the later case an 1-hydroxyethyl radical was also formed and detected. The usefulness and limitations of this detection system were also tested in a NADPH dependent liver microsomal ethanol metabolizing system (MEOS) and in a liver nuclear ethanol metabolizing system (NEMS ). (Recently reported by our laboratory. G.D. Castro, et al. Experimental Biology Meeting, San Francisco, April, 1998). In both cases, the procedure evidenced to be able to detect the OH-m.fPO adduct formed . The spin trap DMPO was not equally good to detect I-hydroxyethyl radicals in biological systems. However, its detection was possible by GCIMS replacing DMPO by other spin trap, PBN. This spin trap GClMS procedure suffers of equivalent risks of artefacts formation than those previously reported by others for ESR. The method does not require deuterated alcohol for precise identification. Supported by the Consejo Nacional de Investigaciones Cientificas y Tecnicas, (CONICET), Argentina.
1
P3G 187 1 COMPARISON OF RAPID METHODS (HPLC, ELISA AND MINICOLUMN) TO DETECT AFLATOXIN M1 IN THE MILK
M.A. Abdel- Wahhab *, S.A. Nada 1 . Mycotoxins CentralLab.and J Pharmacology Dept. NationalResearch Centre, Dokki, Cairo, Egypt
High Pressure Liquid Chromatography (HPLC) , Chemoselective Immobilization and detection of AFM1 (CSID-M1), and Enzyme linked Immunosorbent (ELISA) were compared for detection of AFM1 in the three kinds of milk (whole milk, low fat milk, and skim milk). Milk samples were spiked with AFMI at five concentrations (2.0, 1.0,0.5,0.25, and 0.125 ppb) . Among the three methods , CSID-M1 and HPLC methods were accurate and detected AFM 1 at concentration as low as 0.125 ppb in milk samples . ELISA detected AFM1 at concentration of > 0.25 ppb. This study demonstrates that the CSID-M1 minicolurnn method is rapid and accurate, coast-effective, and user friendly. This method is particularly useful for the field screening of milk and predict exposure to AFB 1.
IP3G189I
A SIMPLEHIGH-PERFORMANCE UQUID CHROMATOGRAPHIC METHOD FORTHE DETERMINATION OF URINARY N-ACETYL-8-(N-METHYLCARBAMOYL)-CYSTEINE (AMCC), THE MERCAPTURIC ACIDOF N,N-DIMETHYLFORMAMIDE (DMF)
L. Maestri *, S. Ghittori, M.lmbriani, L. Rolandil , JResearch
Centre for Physiopathology and Occupational Safety, "Salvatore Maugeri" Foundation, IRCCS, Pavia; "S. Matteo" Hospital, Pavia, Italy N,N-Dimethylformamide (DMF), a widely used solvent, is rapidly metabolized in vivo, mainly to the hydroxylated compound N-(hydroxymethyl)-N-methylformamide (HMMF) . The biological monitoring of occupational exposure to DMF is currently performed by the gas-chromatographic determination of urinary N-methylformamide (MF), another metabolite of DMF. An alternative metabolic pathway has been suggested, which involves the conjugation of MF with glutathione and the excretion of N-acetyl-S-(N-methylcarbamoyl)-cysteine (AMCC), the mercapturic acid of DMF. We have developed a high-performance liquid chromatographic (HPLC) method for the determination of urinary AMCC. After acidification, urine samples were purified by solid phase extraction (SPE) cartridges and directly injected onto a Waters 600E HPLC system equipped with a Bio-Rad Aminex Ion Exclusion HPX-87H column (maintained at 25°C) and a UV detector (wavelength = 196 om) . The isocratic run was performed with a 2.4 roM sulphuric acid solution delivered at a flow-rate of 0.6 mllmin. Under these conditions, the peak of AMCC was free from interfering compounds and eluted at 20.2 min. The limit of detection of AMCC
197
IP3G188I SIMULTANEOUS DETERMINATION OF OXAZEPAM ENANTIOMERS AND ITS ANTAGONIST, FLUMAZENIL, IN SERUM BY CHIRALHPLC FOR TOXICOKINETIC STUDY
G. Pautet! , C. Pham-Huy e", N. Chikhi-Chorfi l , H. Galons", J.-M . Wamet" , J.R. Claude 1 . JLab. Toxicologie (EA 207); 2 Lab.
Chimie Organique. FacultePharmacie, Universite Paris5, France Oxazepam (OZP), a tranquillizer, has been used as racemic mix-
1
P3G 1861 DETECTION OF HYDROXYL AND 1 HYDROXYETHYL
ture to treat anxiety and other mental diseases . It is also an active
FREERADICALSUSINGSPINTRAPS FOLLOWED BY GAS CHROMATOGRAPHIC-MASS SPECTROMETRIC DETECTION. APPLICATION TO STUDIES ON ETHANOLato-TRANSFORMATION IN LIVERMICROSOMES OR HIGHLY PURIFIED NUCLEI
metabolite of a number of benzodiazepines. In humans . S(+)-OZP is pharmacologically the active form and the inversion phenomena of enantiomers is also observed. Therefore, it is suitable to analyze the respective OZP enantiomer levels in biological fluids rather than the total OZP cencentrations for therapeutic monitoring and for overdosage treatment by its antagonist, flumazenil. The simultaneous determination of OZP enantiomers and its antagonist, flumazenil, in rabbit serum by enantioselective HPLC using a new Cyclobond-I-2000-SP column is described. After a1kaline extraction with clonazepam as internal standard, OZP enantiomers and flumazenil are separated on the previous column with a new polar organic mobile phase (acetonitrile-absolute ethanol-glacial acetic acid-triethylamine) and detected at 230 nm. Peak resolutions are 2.7 for OZP enantiomers. Their selectivity factor ex is 1.18. The sensitivity is about 10 nglmI for OZP enantiomers and 5 nglml for flumazenil. The coefficients of variation are about 3.9%. The area ratio of two enantiomers is about 1.00 ± 0.05. The development of this new chiral HPLC method is used to determine the toxicokinetic profile of the two OZP enantiomers in the rabbits under the treatment of flumazenil. It is also suitable for pharmacokinetic and toxicological assays in therapeutic monitoring and in overdosage treatment in humans . It can be applied to study the inversion mechanism of the two OZP enantiomers in organism and also the metabolism of a number of benzodiazepines because oxazepam is their active metabolite.
G.D. Castro *, A.M.A. Delgado de Layfio, J.A. Castro . Centro de
Investigaciones Toxicol6gicas (CEITOX) -CITEFAICONICET, Zufriategui 4380, (1603) Villa Martelli, Pcia. de BuenosAires, Argentina Detection of hydroxyl radicals is frequently made by spin trapping procedures followed by ESR detection of the adduct radical formed. ESR equipment however is very expens ive and not frequent in many countries . In this work we describe a procedure where the .OH radical is detected by spin trapping with DMPO followed of derivatization of the adduct formed by silylation and precise and specific identification by gas chromatography/mass spectrometry. The procedure was initially tested against a model system where the .OH was generated by a Fenton reaction (iron plus hydrogen peroxide) either in the presence or the absence of ethanol. In the later case an 1-hydroxyethyl radical was also formed and detected. The usefulness and limitations of this detection system were also tested in a NADPH dependent liver microsomal ethanol metabolizing system (MEOS) and in a liver nuclear ethanol metabolizing system (NEMS ). (Recently reported by our laboratory. G.D. Castro, et al. Experimental Biology Meeting, San Francisco, April, 1998). In both cases, the procedure evidenced to be able to detect the OH-m.fPO adduct formed . The spin trap DMPO was not equally good to detect I-hydroxyethyl radicals in biological systems. However, its detection was possible by GCIMS replacing DMPO by other spin trap, PBN. This spin trap GClMS procedure suffers of equivalent risks of artefacts formation than those previously reported by others for ESR. The method does not require deuterated alcohol for precise identification. Supported by the Consejo Nacional de Investigaciones Cientificas y Tecnicas, (CONICET), Argentina.
1
P3G 187 1 COMPARISON OF RAPID METHODS (HPLC, ELISA AND MINICOLUMN) TO DETECT AFLATOXIN M1 IN THE MILK
M.A. Abdel- Wahhab *, S.A. Nada 1 . Mycotoxins CentralLab.and J Pharmacology Dept. NationalResearch Centre, Dokki, Cairo, Egypt
High Pressure Liquid Chromatography (HPLC) , Chemoselective Immobilization and detection of AFM1 (CSID-M1), and Enzyme linked Immunosorbent (ELISA) were compared for detection of AFM1 in the three kinds of milk (whole milk, low fat milk, and skim milk). Milk samples were spiked with AFMI at five concentrations (2.0, 1.0,0.5,0.25, and 0.125 ppb) . Among the three methods , CSID-M1 and HPLC methods were accurate and detected AFM 1 at concentration as low as 0.125 ppb in milk samples . ELISA detected AFM1 at concentration of > 0.25 ppb. This study demonstrates that the CSID-M1 minicolurnn method is rapid and accurate, coast-effective, and user friendly. This method is particularly useful for the field screening of milk and predict exposure to AFB 1.
IP3G189I
A SIMPLEHIGH-PERFORMANCE UQUID CHROMATOGRAPHIC METHOD FORTHE DETERMINATION OF URINARY N-ACETYL-8-(N-METHYLCARBAMOYL)-CYSTEINE (AMCC), THE MERCAPTURIC ACIDOF N,N-DIMETHYLFORMAMIDE (DMF)
L. Maestri *, S. Ghittori, M.lmbriani, L. Rolandil , JResearch
Centre for Physiopathology and Occupational Safety, "Salvatore Maugeri" Foundation, IRCCS, Pavia; "S. Matteo" Hospital, Pavia, Italy N,N-Dimethylformamide (DMF), a widely used solvent, is rapidly metabolized in vivo, mainly to the hydroxylated compound N-(hydroxymethyl)-N-methylformamide (HMMF) . The biological monitoring of occupational exposure to DMF is currently performed by the gas-chromatographic determination of urinary N-methylformamide (MF), another metabolite of DMF. An alternative metabolic pathway has been suggested, which involves the conjugation of MF with glutathione and the excretion of N-acetyl-S-(N-methylcarbamoyl)-cysteine (AMCC), the mercapturic acid of DMF. We have developed a high-performance liquid chromatographic (HPLC) method for the determination of urinary AMCC. After acidification, urine samples were purified by solid phase extraction (SPE) cartridges and directly injected onto a Waters 600E HPLC system equipped with a Bio-Rad Aminex Ion Exclusion HPX-87H column (maintained at 25°C) and a UV detector (wavelength = 196 om) . The isocratic run was performed with a 2.4 roM sulphuric acid solution delivered at a flow-rate of 0.6 mllmin. Under these conditions, the peak of AMCC was free from interfering compounds and eluted at 20.2 min. The limit of detection of AMCC