Multiresidue analysis of pesticides in vegetables and fruits using two-layered column with graphitized carbon and water absorbent polymer.

A high-throughput multiresidue analysis of pesticides in non-fatty vegetables and fruits was developed. The method consisted of a single extraction and a single clean-up procedure. Food samples were extracted with ethyl acetate and the mixture of extract and food dregs were poured directly into the clean-up column. The clean-up column consisted of two layers of water-absorbent polymer (upper) and graphitized carbon (lower), which were packed in a reservoir (75 ml ) of a cartridge column. The polymer removed water in the extract while the carbon performed clean-up. In a recovery test, 110 pesticides were spiked and average recoveries were more than 95% from spinach and orange. Most pesticides were recovered in the range 70-115% with RSD usually < 10% for five experiments. The residue analyses were performed by the extraction of 12 pesticides from 13 samples. The two methods resulted in similar residue levels except chlorothalonil in celery, for which the result was lower with the proposed method. The results confirmed that the proposed method could be applied to monitoring of pesticide residue in foods.

GC/MS analysis of polybrominated diphenyl ethers in fish collected from the Inland Sea of Seto, Japan.

Development of an analytical method for polybrominated diphenyl ethers (PBDEs) in fish and their concentration in Japanese marine fish were investigated. Fish homogenate was extracted with diethyl ether/hexane (1 + 3). The extract was cleaned up by automated gel permeation chromatography (GPC) and then by mini-column chromatography, which consisted of three layers of silica gel and sulfuric acid-impregnated silica gel. The PBDE fraction was concentrated and injected into a GC/MS with negative chemical ionization (NCl). Recoveries of the 15 individual PBDEs (BDE-15, 28, 37, 47, 66, 71, 75, 77, 85, 99, 100, 119, 153, 154, and 209) each at a fortification level of 4 ng/g lipid were in the range of 88-128% and the relative standard deviations (RSD) were 0.43-7.6% (n = 4). Seven species of marine fish (conger eel, flounder, gray mullet, horse mackerel, red sea bream, sea bass, and yellowtail) were collected from the Inland Sea of Seto, and were analyzed with the developed method. Seven PBDEs (BDE-28, 47, 66, 99, 100, 153, and 154) were detected in all the samples. The most abundant PBDE congener was BDE-47 found in all the samples. Relatively high levels of PBDEs were found in the gray mullets and yellowtails.

[Drying ability of anhydrous sodium sulfate on wet organic solvents after liquid-liquid partition].

Water concentration in organic solvents after liquid-liquid partition was determined by the Karl Fischer titration method. n-Hexane and petroleum ether showed quite low levels of water, such as 0.1 mg/mL. The water concentration in wet ethyl acetate was about 20-30 mg/mL and that in diethyl ether was about 8-10 mg/mL. Anhydrous sodium sulfate absorbed about 20-25% of the water after vigorous mixing with wet ethyl acetate or diethyl ether. Wet acetonitrile extract from wet food, which contained about 60 mg/mL water after salting out with sodium chloride, was not dried at all with anhyfrous sodium sulfate treatment. Spiking n-hexane into wet ethyl acetate or wet diethyl ether was effective to exclude water. Spiking toluene into salted acetonitrile drove out water and dissolved sodium chloride. It can be concluded that the drying ability of anhydrous sodium sulfate towards wet organic solvents is poor, but it is effective in removing suspended water in solvents.

Determination of triazine herbicides in foods with liquid chromatography mass spectrometry.

Eight residual triazine herbicides and three metribuzin metabolites in foods were determined by liquid chromatography mass spectrometry (LC-MS) with an atmospheric pressure chemical ionization (APCI) interface, under both positive and negative ion modes. Herbicides were extracted with acetonitrile, and no cleanup procedure was adopted in this method. Four foods were spiked with eight herbicides and three metabolites at 0.05 ppm. The average recoveries of these herbicides usually ranged from 82 to 99% and the relative standard deviations were usually around 10%. These results suggest that LC-MS with APCI can be used to determine residues of triazine herbicides in foods.

Multiresidue analysis of pesticides in vegetables and fruits using a high capacity absorbent polymer for water.

A single extraction and a single clean-up procedure was developed for multi-residue analysis of pesticides in non-fatty vegetables and fruits. The method involves the use of a high capacity absorbent polymer for water as a drying agent in extraction from wet food samples and of a graphitized carbon column for clean-up. A homogeneously chopped food sample (20 g) and polymer (3 g) were mixed to absorb water from the sample and then 10 min later the mixture was vigorously extracted with ethyl acetate (100 ml). The extract (50 ml), separated by filtration, was loaded on a graphitized carbon column without concentration. Additional ethyl acetate (50 ml) was also eluted and both eluates were concentrated to 5 ml for analysis. The procedure for sample preparation was completed within 2 h. In a recovery test, 107 pesticides were spiked and average recoveries were more than 80% from asparagus, orange, potato and strawberry. Most pesticides were recovered in the range 70-120% with usually less than a 10% RSD for six experiments. The results indicated that a single extraction with ethyl acetate in the presence of polymer can be applied to the monitoring of pesticide residues in foods.

Determination of N-methylcarbamate pesticides in foods using an accelerated solvent extraction with a mini-column cleanup.

Residual N-methylcarbamate pesticides in food were determined by accelerated solvent extraction (ASE) and HPLC with post-column fluorescence. Pesticides were extracted with acetonitrile at 100 degrees C under 2000 psi pressure in less than 20 min. Extracts were cleaned-up with a carboxylic acid mini-column eluted with 10% or 30% acetone in hexane. Eight foods were spiked with 17 pesticides at 0.2 ppm. The average recoveries of these pesticides were 70-100% and the relative standard deviations were < 10%. These results suggested that ASE can be used to extract residues of N-methylcarbamate pesticides in foods.

Determination of organophosphorus pesticides in foods using an accelerated solvent extraction system.

Residual organophosphorus pesticides in foods were determined by accelerated solvent extraction (ASE), gel permeation chromatography and GC-FPD. Pesticides were extracted at 100 degrees C under 1500 psi pressure in less than 20 min. Wet samples were extracted after mixing with Extrelut drying agent. Four foods were spiked with 19 pesticides at 0.1 ppm or less. The average recoveries of these pesticides were 80-90% and the precision was < 10%. Both methamidophos and acephate gave 37-50% recoveries and 6-40% precision. These results suggested that ASE can be used to extract residues of organophosphorus pesticides in foods.

Aspergillus ochraceus Production of 5,6-Dihydropenicillic Acid in Culture and Foods.

The production of 5,6-dihydropenicillic acid (DHPA) and penicillic acid (PA) by Aspergillus ochraceus was studied. DHPA has been revealed to be genotoxic in the Drosophila DNA-repair test in our previous report. The two compounds were produced by various type strains of A. ochraceus and related strains of Aspergillus in nutrient cultures. When A. ochraceus cells were inoculated into nutrient medium, PA was rapidly produced followed by a steady increase of DHPA and a decrease in the PA level. On the contrary, A. ochraceus produced almost exclusively PA in potato dextrose agar medium, which is low in nutrients. A. ochraceus cells produced DHP A when inoculated into grain, especially in rice flour at 20 to 24°C, and the DHPA level increased as the incubation was prolonged. These results suggested that PA was converted to DHPA under highly nutritive conditions. In vitro mutagenicity tests, the Ames test and the umu test, showed that DHPA was not mutagenic.

Mutagenicity of 5,6-Dihydropenicillic Acid (DHPA) in Drosophila melanogaster and Bacterial Conversion of Penicillic Acid to DHPA.

We studied the mutagenicity of 5,6-dihydropenicillic acid (DHPA) by means of the Drosophila wing-spot test. DHPA (10 mg/g of medium) significantly increased the number of small single and twin spots. Since twin spots were exclusively caused by mitotic crossing-over, the results demonstrated that DHPA can induce chromosome recombination in Drosophila somatic cells. Penicillic acid (PA) was converted to DHPA by an incubation with Agrobacterium radiobactor . The conversion occurred with other spoilage bacteria, such as Pseudomonas aeruginosa and P. cepacia . PA was completely converted to DHPA within a 24-h incubation with P. aeruginosa or P. cepacia in medium containing yeast extract. The results suggested that mutagenic DHPA is produced by environmental bacteria when foods are contaminated by fungi which produce PA.

Direct detection using the Drosophila DNA-repair test and isolation of a DNA-damaging mycotoxin, 5,6-dihydropenicillic acid, in fungal culture.

The Drosophila DNA-repair test was used in an attempt to detect fungal production of DNA-damaging mycotoxins without an extraction process. 29 species of fungi, 13 Aspergillus, 12 Penicillium and four Fusarium were inoculated directly to a Drosophila medium, and the larvae were then bred in the mouldy medium. Production of DNA-damaging mycotoxin was detected directly by counting the decrease in the survival of DNA-repair-deficient flies. With the direct detection method, Aspergillus ochraceus, A. parasiticus and A. versicolor produced DNA-damaging mycotoxins. The same results were obtained with the mouldy medium extract using the standard DNA-repair test. The direct detection method was convenient for surveying the fungal production of DNA-damaging mycotoxins. The extracts of A. parasiticus and A. versicolor contained aflatoxin B1 and sterigmatocystin, respectively. The DNA-damaging compound in the extract of A. ochraceus was isolated and purified to clear, colourless 'needles'. With nuclear magnetic resonance-mass spectroscopy spectra, the compound was confirmed to be 5,6-dihydropenicillic acid, the DNA-damaging potency of which has not been previously reported.

The effects of "yusho" type PCB on triglyceride lipase and fatty acid composition.

Yusho type polychlorinated biphenyl (Y-PCB) was prepared in order to study lipid disorder of the PCB poisoning "yusho." Y-PCB caused hypertriglyceridemia and hypercholesterolemia in the rat. Triglyceride lipase (TGL) activities in liver and postheparin serum were suppressed by Y-PCB administration, whereas it was not suppressed in fat. In in vitro experiments, Y-PCB did not suppress TGL in either liver or fat. Although Y-PCB increased fatty acids in the liver 2.5-fold, the relative proportion of fatty acids composition was not changed so remarkably as that in absolute concentrations. These findings suggested that liver is the target of Y-PCB -mediated alteration of lipid metabolism.

Suppressive effects of coffee on the SOS responses induced by UV and chemical mutagens.

SOS-inducing activity of UV or chemical mutagens (AF-2, 4NQO and MNNG) was strongly suppressed by instant coffee in Salmonella typhimurium TA1535/pSK1002. As decaffeinated instant coffee showed a similarly strong suppressive effect, it would seem that caffeine, a known inhibitor of SOS responses, is not responsible for the effect observed. The suppression was also shown by freshly brewed coffee extracts. However, the suppression was absent in green coffee-bean extracts. These results suggest that coffee contains some substance(s) which, apart from caffeine, suppresses SOS-inducing activity of UV or chemical mutagens and that the suppressive substance(s) are produced by roasting coffee beans.

Biological effect of PCBs, PCQs and PCDFs present in the oil causing yusho and yu-cheng.

Male Sprague-Dawley rats were daily given orally for 22 days a regimen consisting of polychlorinated biphenyls (PCBs), 1 mg/day; polychlorinated quaterphenyls (PCQs), 1 mg/day; polychlorinated dibenzofurans (PCDFs), 10 micrograms/day; or a mixture of PCBs, PCQs and PCDFs (Mix-1, 1 mg + 1 mg + 10 micrograms/day). Female Cynomolgus monkeys were daily administered PCBs (5 mg), PCQs (5 mg) or a mixture (Mix-2) containing 5 mg PCBs + 20 micrograms PCDFs for 20 weeks. The PCBs, and PCDFs had the components of PCBs, PCQs and PCDFs similar to those contained in Japanese yusho oils, respectively. The PCB-treated rats and monkeys showed hepatic hypertrophy, immunosuppression and increased drug-metabolizing enzyme activities in hepatic microsomes, but were devoid of the dermal symptoms characteristic of yusho. PCQs caused an increase in drug-metabolizing enzyme activities in hepatic microsomes and immunosuppression in monkeys, but these effects were much smaller than those found with PCBs treatment. On the other hand, treatment with PCDF or Mix-1 or Mix-2 caused hypertrophy of the liver, immunosuppression, increase in drug-metabolizing enzyme activities of hepatic microsome to much greater extent than observed with PCBs, being more than 100 times as effective as PCBs. In addition PCDFs and the mixtures containing PCDFs caused weight loss and thymic atrophy. PCDFs and Mix-2-treated monkeys showed the dermal symptoms that are characteristic of yusho patients but were not observed in monkeys treated with PCBs and PCQs alone. These results suggest that PCDFs are the primary causative agent of yusho.

Causal agents of yusho.

Through the analysis of polychlorinated biphenyls (PCBs), polychlorinated quaterphenyls (PCQs), and polychlorinated dibenzofurans (PCDFs) retained in the blood of Japanese and Taiwanese patients who had been poisoned by ingesting contaminated cooking oils, in the blood of workers occupationally exposed to PCBs, and in the blood of unexposed individuals, it was strongly suggested that PCDFs might be the responsible compounds for the development of yusho disease. To confirm this, animal experiments were done. The dermal signs resembling those of yusho were observed among the cynomolgus monkeys that received a PCDF mixture consisting of PCDF components similar to those in the yusho oils. In addition, significant thymic atrophy and decreased body weight gain were noted in the rats fed PCDFs. However, the administration of PCBs or PCQs alone failed to cause such signs in the monkeys or rats. As a result, it became clear that PCDFs were the main causative agents in the pathogenesis of yusho disease.

Causal agents of yusho.

Through the analysis of polychlorinated biphenyls (PCBs), polychlorinated quaterphenyls ( PCQs ), and polychlorinated dibenzofurans ( PCDFs ) retained in the blood of Japanese and Taiwanese patients who had been poisoned by ingesting contaminated cooking oils, in the blood of workers occupationally exposed to PCBs, and in the blood of unexposed individuals, it was strongly suggested that PCDFs might be the responsible compounds for the development of yusho disease. To confirm this, animal experiments were done. The dermal signs resembling those of yusho were observed among the cynomolgus monkeys that received a PCDF mixture consisting of PCDF components similar to those in the yusho oils. In addition, significant thymic atrophy and decreased body weight gain were noted in the rats fed PCDFs . However, the administration of PCBs of PCQs alone failed to cause such signs in the monkeys or rats. As a result, it became clear that PCDFs were the main causative agents in the pathogenesis of yusho disease.

Effect of polychlorinated biphenyls and polychlorinated quaterphenyls in Cynomolgus monkey (Macaca fascicularis).

Female Cynomolgus monkeys (Macaca fascicularis) with P-KC-400, Y-PCB, PY-PCB or polychlorinated quaterphenyls (PCQ) received a daily dose of 5 mg for 20 weeks, and some monkeys received a daily dose of 10 mg of Y-PCB or 0.5 mg of PCQ. The chemical compositions of the polychlorobiphenyls (PCB) used for the oral administration were as follows: P-KC-400, PCB from which polychlorodibenzofurans (PCDF) have been removed from Kanecklor 400, largely contains tri- and tetrachlorobiphenyls and no PCDF. Whereas, Y-PCB and PY-PCB, PCB with constituents similar to PCB ingested by yusho patients, largely contain penta- and hexachlorobiphenyls, in addition, PCDF of 400 ppm was present only in Y-PCB, but not in PY-PCB. There were immunosuppression, enlargement and histopathological changes of the liver (such as interstitial inflammation, and proliferation of epithelial cells of biliary duct, etc.) in the groups fed P-KC-400 and PY-PCB (free of PCDF). In the group fed Y-PCB (with PCDF), there were more apparent decreases in body weight, immunosuppression, fatty liver and histopathological changes than in the groups P-KC-400 and PY-PCB. In addition, there were hair loss, acneform eruptions, edema of the eyelid, congestion and abscess of the Meibomian gland, and cornifications of the skin, characteristic dermatological findings of yusho disease.