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電子レンジ加熱・COセンサー法として,150ml容器に照射した粉砕香辛料3gを入れ,密栓し電子レンジにて1分間加熱後,COセンサーにてヘッドスペース中のCO濃度を測定する香辛料中CO濃度の改良測定法を考案した.本法の定量下限は試料3gを用いた場合,0.1ppmであった.照射直後の2種香辛料について本法をこれまで報告された方法と比較したところ,その線量依存性は化学発光法(CL法),GC法及びESR法のものと相関性が高く,本電子レンジ加熱・COセンサー法は,照射試料中のCO検出法として使用できるものと思われた.しかし,照射試料を3ヵ月間,25℃,暗所で放置した場合,黒胡椒(粒・粉末),赤とうがらし共に,揮散によるCO濃度減少により照射の判別は不可能であった.

The analytical detection methods for the irradiation treatment of food are investigated recently as international projects as well as projects carried out in our country to resolve the problems on the international trade and labelling in the market. Several promising methods such as ESR, thermoluminescence, chemical luminescence, impedance, viscosity, gas-measurement, lipid-volatile hydrocarbon, lipid-volatile cyclobutanone, germination, microbiology and so on were proposed and reviewed on the international research program organized by FAO/IAEA.

The germination of wheat provides a reliable test for the identification of wheat that has been subjected to irradiation. Germination success of non-irradiated wheat is not affected by storage time and temperature. Root and shoot lengths are more sensitive to γ-irradiation than the germination percentages. The critical dose that inhibits root elongation varies from 0.15 to 0.5 kGy for imported cultivars and from 0.3 to 1.0 kGy for domestic cultivars. Germination percentage is reduced above 5.0 kGy. The difference in radiosensitivity between imported and domestic cultivars may be the result of differences in moisture content. In addition, storage periods of up to 12 months have little effect on irradiation-induced reduction of root length. Fumigation with methyl bromide inhibits root growth, but this inhibition can be distinguished from γ-irradiation effects by the germination pattern. Most imported wheat samples that were irradiated above 0.5 kGy exhibit root growths of less than 20 mm after 4 days of culturing under the specified conditions. The same behavior was noted in domestic wheat samples that were irradiated at or above 1.0 kGy. This test can thus discriminate between irradiated and non-irradiated wheat even 12 months after γ-irradiation.

Active oxygen species derived from the interaction of potassium bromate (KBrO3), a rat renal carcinogen, with cells from rat kidney and other organs were examined by electron spin resonance spectrometry using the spin trapping agents 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 2,2,6,6-tetramethylpiperidine (TEMP). DMPO-OH, an indicator of hydroxyl radical production, was generated from KBrO3 by kidney cells or homogenate, but not by liver preparations and to only a limited extent by heart and brain homogenates, suggesting relative kidney specificity. To assess what chemical components are responsible for production of DMPO-OH, several physiologically related materials were examined. Glucose, saccharose, albumin and methyl linolate were found not to be involved in the KBrO3 reaction, but reduced glutathione and also ferric ions participated to produce DMPO-OH. In addition, DMPO-OH production derived from the reaction of KBrO3 with kidney homogenate was not affected by superoxide dismutase, catalase or hydroxyl radical scavengers such as DMSO or ethanol, but was effectively inhibited by singlet oxygen scavengers such as histidine and NaN3, implying singlet oxygen production. To assess this possibility, TEMP was used as a trapping agent, and TEMPO, derived from singlet oxygen, was found to be produced by the reaction of KBrO3 with homogenates of kidney, but not of liver. Furthermore, singlet oxygen production was confirmed by studies of chemiluminescence using 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2a]pyrazine-3-one. As a control, DMPO-OH was also demonstrated to be produced by a known singlet oxygen source, toluidine blue plus light. The results thus indicate that singlet oxygen is a very probably candidate for the active oxygen species generated in the specific interaction of KBrO3 with rat kidney cells in vitro. This raises the question of its concern with renal carcinogenicity in vivo.

Thiabendazole (TBZ) is a benzimidazole antibacterial agent used as an anthelminthic for livestock. 5-Hydroxythiabendazole (5-OH TBZ) is a metabolite formed by hydroxylation of TBZ in an animal's body. The recovery of 5-OH TBZ from animals by extraction in conventional TBZ analysis, which employs a strongly alkaline (pH 11.0) buffer, is as low as 50%. In order to establish a method for simultaneous analysis of TBZ and 5-OH TBZ with high recovery, we examined the optimum pH of a buffer for extraction, a suitable solvent for extraction, and a clean-up column. As a result, it was found that the optimum pH of the buffer was 9.0 and that suitable solvents were ethyl acetate (AcOEt) for pig muscle and liver, and MeCN for pig fat and cow's milk. Among the three types of solid phase extraction (SPE) clean-up columns investigated, the highest recovery for both chemicals was achieved by using an alumina N (ALN) column.

Studies to develop a GC/MS (SIM) method for the determination of 34 pesticides including carbamates and organonitrogen pesticides in foods with solid-phase extraction (SPE) were conducted.Three kinds of SPE columns, a porous diatomaceous earth cartridge column, a diatomite column with a mini C18 cartridge column and a Florisil cartridge column were used successively to clean-up the extract from foods as an alternative to a separatory funnel for liquid-liquid partition. The handling was simpler than the conventional method and gave high recoveries of the pesticides.In GC/MS (SIM), 2 to 4 different fragment ions were selected for each pesticide so that the GC/MS (SIM) identification could be done more accurately for multi-residue analysis of carbamate and organonitrogen pesticides. This simpler method was applicable for 29 pesticides out of 34 with almost the same recoveries and detection limits as the conventional method.

GC/MS with selected ion monitoring (GC/MS (SIM)) has been developed as a screening method for the determination of 14 pesticides residues including cyhexatin and 2, 4, 5-T in nuts. The pesticides were extracted with acetone from nuts, and then partitioned between n-hexane and acetonitrile. Clean-up was conducted by solid-phase extraction (SPE) with a Bond Elut Florisil column. Most of the pesticides were eluted in a mixture of n-hexane and acetone (17:3) from the column, and the eluate was examined by GC/MS (SIM). Cyhexatin and 2, 4, 5-T were analyzed after derivatization and SPE clean-up. Recoveries of 14 pesticides from 6 kinds of nuts were mostly over 80%. This method is effective for multi-residue analysis of pesticides in nuts.

Residual levels of permethrin in foods and tableware after fumigation (permethrin: 1g) in a room were investigated. The residual levels of permethrin were 19.0μg/g in rice, 1.5-19.0μg/g in crackers, bread and tomato and 0.2-48μg/cm2 in tableware. In the case of packed bread and wrapped lettuce, permethrin was not detected. When the foods and tableware were kept for 10 days at room temperature after fumigation, the levels of permethrin were 52.4-88.4% of the initial levels. Almost all residual permethrin in the rice and tableware was removed by washing with water or by washing with detergent using a sponge.

The fate of cyanogenic compounds in butter beans and baby lima beans (Phaseolus lunatus L.) during the manufacturing process of bean paste on a laboratory scale was investigated. Linamarin, the main cyanogenic glycoside in the beans, was analyzed by FID-GC after trimethylsilylation and free cyanide was analyzed by ECD-GC using the head-space method.The changes of linamarin and free cyanide contents showed similar decreasing tendencies in both beans though there was a six-fold difference of cyanogen content between butter beans and baby lima beans. Linamarin and free cyanide decreased greatly during the soaking and refining processes, but they decreased little during the boiling process. The percentage of residual cyanogen in the final bean paste was 0.3% in both kinds of beans. Linamarin was the main cyanogenic component in the original beans and at each step.

γ-Irradiation at a practical dose level of allspice and cinnamon generates a principal signal (signal I, g-value: 2.0048-2.0050) and a minor signal (signal II) at 30G lower field from signal I in the electron spin resonance spectrum. Signal I, which was not increased in red pepper by photo-exposure, was increased in allspice and cinnamon by γ-irradiation, heating and even photo-exposure. Signal II was generated only by γ-irradiation, was little influenced by humidity and was stable for a long time. The ESR method with signal II was applicable to detection of allspice and cinnamon irradiated at 5kGy or more for up to 6 months after irradiation, as well as allspice irradiated at 10kGy or more and cinnamon at 5kGy or more for up to a year. However, signal intensities of signal II differed to some extent between allspice and cinnamon, and even between varieties of cinnamon.Signals I and II were both enhanced after extraction with methanol. Since the rate of increase in signal I was obviously distinct from that of signal II, the radicals corresponding to these signals were presumed to be located at different positions of the matrix of the spice. The methanolic extracts did not yield a major component common to the spices giving signal II.

A method for the direct determination of linamarin, a cyanogenic glycoside in beans, bean paste products and cassava flour was improved. A sample was extracted with a mixture of acetonitrile and water, cleaned up by using a solid-phase extraction cartridge, and trimethylsilylated, then linamarin was quantitated by FID-GC and confirmed by GC/MS. The detection limit of linamarin was 1μg/g in beans, 0.25μg/g in dry bean paste and 0.1μg/g in raw paste. The contents of linamarin in beans, bean paste products and cassava flour were surveyed. The contents of linamarin determined by the proposed method were in good accordance with those found by the headspace GC method.

A simple and rapid determination method of methyl isothiocyanate (MITC) in wine was developed. MITC in wine was extracted with ethyl acetate and the extract was subjected to NPD-GC and GC/MS. The determination limits were 0.02ppm with NPD-GC, 0.22ppm with FPD-GC (S-mode) and 0.05ppm with GC/MS. Recoveries from wine spiked with 0.044 and 0.088ppm of MITC were 89.4% and 83.5%, respectively, from white wine and 81.6% and 75.1%, respectively, from red wine.MITC in wine imported from Italy was detected in the range of 0.087 to 0.349ppm in three brands out of 30 examined.

The addition of borates to foods is not permitted in Japan. However, agar may contain boron derived from boric acid. In order to study whether or not the bioavailability of agar-bound boron is different from that of free boric acid, rats that had been subjected to a 16hr fasting period were given a 5% (w/v) suspension of agar, a 2% carboxymethylcellulose solution containing boric acid, or a boric acid solution, by oral intubation. For 24hr after the treatment, only water was given to the animals ad libitum, then the rats were killed. The amount of boron excreted in the urine within 24hr was significantly higher in the three treated groups than in the control group that received only distilled water. However, no significant differences were observed among the three boron-treated groups. The difference in the urinary boron content between the boron-treated groups and the control group was equivalent to approximately two-thirds of the administered dose. No significant differences in the boron contents of the feces and the cecum contents were detected among the four groups. Consequently, there appeared to be no difference in gastrointestinal absorption and urinary excretion of boron between boron in agar and free boric acid.

A simultaneous analytical method for residual synthetic antibacterials in fish and meat has been developed. Synthetic antibacterials were extracted with acetonitrile, followed by partition with hexane. Extracts were analyzed by the screening method using HPLC with stepwise gradient elution. Detected synthetic antibacterials were quantitatively analyzed by HPLC under optimum conditions, and UV spectra were taken for further qualitative analysis.The lower limit of determination was 10-50ppb for each synthetic antibacterial. The results of a collaborative study with 10 laboratories indicated that the proposed method gives accurate and reproducible data.

The electron spin resonance (ESR) spectrometry spectra of white (WP), black (BP) and red (Capsicum annuum L. var. frutescerns L., RP) peppers each had a principal signal with a g-value of 2.0043, and the intensities of the principal signals were increased not only by γ-irradiation but also by heating. Irradiated RP also showed a minor signal -30G from the principal one, and the intensity of the minor signal increased linearly with increasing dose from 10 to 50kGy. Since the minor signal was observed in RP irradiated at 10kGy and stored for one year, but did not appear either after heating or after exposure to this signal is unique to γ-irradiated RP and should therefore be useful for the identification of γ-irradiated spices of Capsicum genus, such as paprika and chili pepper. The computer simulation of the ESR spectra suggested that the minor signal should be assigned to methyl radical and the principal signal mainly to a combination of phenoxyl and peroxyl radicals. Such minor signals were found in γ-irradiated allspice and cinnamon among 10 kinds of other spices.

Whole grapefruit were exposed to 5, 15, 30, 50, and 100 krad doses of gamma-radiation. Seeds were removed and half-embryos, consisting of one cotyledon and embryo axis, dissected from the surrounding tissue. Nonirradiated half-embryos germinated faster than intact or partially dissected (outer seed coat removed) seeds. Shoot elongation was also quicker, occurring within 6 days. Irradiated half-embryos over 15 krad doses showed markedly reduced root growth and shoot elongation was almost totally retarded. Differences between irradiated and nonirradiated half-embryo were not affected when variety, harvest date, and fruit storage conditions were varied. This test could thus discriminate between irradiated and non irradiated grapefruit.

The photosensitizers pheophorbide a and pyropheophorbide a, derived from chlorophyll in salted green vegetables and chlorella, were determined by high performance liquid chromatography under the following operating conditioms: μ-Bondapak C-18 reverse-phase separation with methanol-2% phosphate solution (90: 10v/v) as the mobile phase, and UV detection at 415nm. A flow rate ot 1.0ml/min at room temperature gave a good base line separation of pheophorbide a and pyropheophorbide a (retention times of 12.1 and 14.8min, respectively).The method was applied to the rapid monitoring of poisonous chlorophyll derivatives in vegetables and chlorella. Pheophorbide a was detected in fresh vegetables, but pyropheophorbide a was not. In salted vegetables stored for a long time (more than 3 months), almost all the chlorophyll had been converted to pyropheophorbide a. In commercial chlorella, pheophorbide a was detected to a far greater extent than pyropheophorbide a.

Mise en evidence, par chromatographie liquide haute performance, d'une oxydation du glutathion dans les pamplemousses, consecutive a leur irradiation. Ce groupe de composes ne semble pas pouvoir etre utilise comme indicateur de l'irradiation du fait de son instabilite

Hydride generation-flame atomic-absorption spectrometry (HG-FAAS) was used as a continuous detection system for arsenic in the eluate from high-performance liquid chromatography (HPLC). Four arsenic species (arsenite, arsenate, mono-methylarsonate and dimethylarsinate) were detected separately with the HPLC-HG-FAAS system equipped with an anion-exchange column. When hijiki (Hizikia fusiforme) extract was examined, arsenate was found predominantly and arsenite and dimethylarsinate were also detected. Liver supernatant fraction obtained from mice administered orally with arsenite was also studied with the HPLC-HG-FAAS system equipped with a gel permeation column. In addition to free or low-molecular-weight ligand-bound arsenic, high-molecular-weight protein-bound arsenic fractions were also detected.

Spraying of the fluorescence spot on a thin-layer plate with a non-volatile and viscous organic solvent after development enhanced the fluorescence. The elucidation of the physicochemical factors that affect such an enhancing effect was investigated. The effect was not related to the transmissibility of light through the thin layer but to the properties of the spraying reagent (polarity, viscosity and acidity). The appropriate reagents for 5-dimethylamino-1-naphthalenesulphonamides (DANS-amines) are non-polar, viscous and non-acidic solvents such as a mixture of liquid paraffin and n -hexane, which enhanced the fluorescence internsity 10-fold. With benzo( a )pyrene, the use of a solvent such as this liquid paraffin mixture enhanced the fluorescence intensity 35-fold.

The duration of the half-embryo test used for identification of gamma-irradiated grapefruit was shortened by increasing germination temperature to 35°C. Maximum shooting percentages were reached within 3 days. Gibberellin application reduced the required incubation time to 2 days. Half-embryos extracted from irradiated orange and lemon gave similar results to those of grapefruit. This half-embryo test is proposed as an identification method for irradiated citrus. Assessment can be made after 3 to 4 days when shooting percentage is greater than 50%.

The γ-irradiation and heating of Kamaboko, a staple fish meat cake in Japan, resulted in the formation of free radicals (g = 2.0030 ∼ 2.0049) measurable at a room temperature by an electron spin resonance spectrometer. These free radicals were retained in protein mainly and increased with rise of dose of γ-ray or temperature. The radical productivity of amino acid by heating was high in L-lysine, L-tryptophan and L-phenylalanine. Among them L-lysine displayed the greatest amount of radical formation.

The ESR intensities (g=2.0042 ∼ 2.0053) of free radicals in L-lysine and casein pyrolysates increased with increase of the heating time. In L-lysine pyrolysate (200°C, 20 min), the fraction insoluble in organic solvents and water accounted for 48.1% of the ESR intensity of the whole pyrolysate; the component responsible appeared to be a product of condensation polymerization, but the elemental analysis data indicated a smaller value for nitrogen than that expected for polylysine. The pyrolysis of casein brought about denatured products insoluble in water. The free radicals formed were stable for a week in distilled water and were unaffected by digestion with pepsin or pancreatin (37°C for 3 hr).

The proposed simultaneous determination of thiols and disulfides requires 4- (aminosulfonyl)- 7-fluor-2,1,3,-benzoxadiazole (ABD-F) as the pre-column derivatization reagent for thiols and ammonium 7-fluoro-2,1,3,-benzoxadiazole-4-sulfonate (SBD-F) for disulfides followed by chromatography. The thiols and disulfides in solution are treated with ABD-F at 60°C for 5 min in pH 9.3 borate buffer containing 1 nM disodium-EDTA. After removal of excess of ABD-F with ethyl acetate, the remaining disulfides in the aqueous phase are treated with SBD-F at 60°C for 20 min in the presence of tri-n-butylphosphine, a reducing agent. The ABD-thiols and SBD-thiols thus produced are separated by reversed-phase chromatography and detected by fluorimetry (380- nm excitation, 510 - nm emission). SBD-cystein, SBD-homocystein, ABD-homocysteine, ABD-cysteine, SBD-glu- tathione, ABD-homocystein, SBD-N-acetylcystein, ABD-glutatione and ABD-N-acetylcysteine are well separated by linear gradient elution from 0.15 M H3PO4/CH3CN (96:4) to 0.15 M H3PO4/CH3CN (85:15) over 30 min followed by isocratic elution with 0.15 M H3PO4/CH3CN (85:15) fro 10 min. The detection limits for the derivatives are in the range 0.09–0.9 pmol. When the method was applied to the determination of thiols and disulfides in rat tissues, cystein (0.75 μmol g-1) and cystine (0.62 μmol g-1) were obtained in kidney and reduced glutathione (1.4–3.4 μmol g-1) was observed in liver, spleen, heart and testicle.

The reaction mechanism of the fluorometric determination of dulcin with sodium nitrite was studied and estimated as follows : Dulcin produces 4-ethoxybenzenediazonium (4EBD) ion via. p-phenetidine by the action of nitrous acid. The ion itself decomposes in part to the substances possessing the active methylene neighbouring to a carbony group. Finally, 4EBD ion reacts with the active methylene in the presence of an alkaline solution containing nitrous oxide to form the essential fluorescent compound, 1, 3-bis (4-ethoxyphenyl)-5-tetrazolone.

The fluorometric method for determination of formaldehyde by means of Hantzsh reaction reported by Sidney Belman was improved and a new determination method of formaldehyde in food was established.To 5ml of formaldehyde solution in a test tube is added 1ml of acetylacetone reagent and the test tube is heated in a boiling water bath for 10 minutes. After cooling to room temperature, 4ml of ethyl-alcohol is added. The solution gives a fluorescence spectrum with an excitation maximum at 416mμ and an emission maximum 500mμ.A linear line relationship between formaldehyde concentration and fluorescence intensity was obtained within the range of 0.05-0.5μg/ml.Influence of other substances on the reaction was studied in detail. This method was successfully applied to the determination and the identification of formaldehyde in several foods.

The structure of the fluorescent compound (A) obtained from the reaction of dulcin and sodium nitrite was previously presumed to be 1, 3-bis (4-ethoxyphenyl)-5-tetrazolone. In this paper, (2-bromoethoxy) phenyl derivative (B) which has the same structural skeleton of compound A was synthesized and analized by the single crystal X-ray diffraction method to determine the structure more conclusively. The structure of compound B was discussed on the basis of the result of the two-dimensional analysis. The structure of the fluorescent compound A was consequently concluded as 1, 3-bis (4-ethoxyphenyl)-5-tetrazolone.

The simplified method for determination of ammonia and volatile amines, especially methylamines formed from trimethylamine oxide (TMAO) by γ irradiation, was established.The method involved the extraction procedure by means of Conway's micro diffusion apparatus and the determination by TLC-fluorodensitometry of DNS-derivatives as well as by gas-liquid chromatography.The satisfactory quantitative results were obtained using ammonia, monomethylamine and dimethylamine in the concentration of 1×10-7-3×10-6mol/ml and trimethylamine in the concentration of 8×10-8-1×10-5mol/ml.

The binding potential of ochratoxin A (Oct. A), a carcinogenic mycotoxin, to protein was investigated by using bovine serum albumin (BSA) and proteinous food. An interaction between Oct. A and BSA was suggested in the aqueous system, where fluorescence with high intensity was observed. This was also confirmed by thin-layer chromatography on a microporous cellulose acetate membrane. Measurements of the extractability of Oct. A with chloroform showed that the binding ability was high in the range of pH 4.0 to 6.0. The major binding type was presumed to be ionic, based on a comparison with the extractability of ochratoxin A methyl ester. The binding parameters determined by an equilibrium dialysis method were n=2.38±0.17 for the number of binding sites and K=7.67±0.39 (×105/M) for the affinity constant in phosphate buffer at pH 7.4. The binding was weakened with increasing ionic strength of the solution and with increasing content of methanol. On the basic of these results, the first extraction procedure for Oct. A by the A. O. A. C. method, which gave a low extractability of 29% for Katsuobushi, dried bonito meat rich in protein, was modified to obtain a high recovery. The addition of 2M sodium chloride and further acidification by addition of 4 volumes of 0.5M phosphoric acid resulted in an increase of the extractability to 85%.

The determination of diethylene glycol (DEG), added illegally to some imported wines, was investigated. The usage of the Extrelut-20 column, filled up with diatomaceous earth, was useful in eliminating interfering substances and water for analyses of many samples without column damage in gas-liquid chromatography (GLC) and for GC/MS measurement with a capillary column. The detection limit of DEG was 5ppm in wine by this method, involving clean-up and GLC with a 20% PEG 20M column or a wide-bore capillary column coated with 50% phenyl methyl silicon. The method was also useful for analysis of DEG in other beverages such as grape juice.

In order to obtain the chemical indexes for degree of decomposition in frozen shrimps, an organoleptic test and the measurement of pH, volatile base nitrogen (VBN) and trimethyl amine nitrogen (TMA-N) were performed on raw shrimps. At the stage of primary decomposition, the amounts of VBN and TMA-N increased to 30mg% and 3.9mg% respectively, but the exact index for pH was not obtained.From the above result and the experiment for imported frozen shrimps, the chemical index at the stage of primary decomposition was decided to be 30mg% in VBN. However, an organoleptic test should be also carried out with this index to confirm the degree of decomposition.It was assumed that the decomposition of imported shrimps occurred when they were exposured in the air before freezing, or thawed on account of some troubles of a refrigerator. Because any remarkable changes of pH, VBN and TMA-N were not observed in frozen shrimps stored at -23°C for 2.5 years, or in raw shrimps kept in ice over 10 days before freezing.As for the change of ATP and the related compounds in frozen shrimps kept at -23°C for 2.5 years, the amount of hypoxanthine and inosine increased and that of ATP decreased considerably. In addition, the improved method in thin-layer chromatography of ATP and the related compounds was established.