We examined two unknown red dyes (designated as red dyes “A” and “B”) from a dried strawberry package with a label that indicated the presence of food red No. 40 (R40). Red dye “A” was identified as trisodium 3-hydroxy-4-[(2'-methoxy-5'-methyl-4'-sulfonatophenyl)azo]-2,7-naphthalenedisulfonate (CSA-R) by HPLC, UV-VIS spectra and MS spectra. This compound is one of the four reported subsidiary colors of R40. Detailed analyses of red dye “B” by MS and NMR demonstrated that its structure was disodium 3-hydroxy-4-[(2'-methoxy-5'-methyl-4'-sulfonatophenyl)azo]-2-naphthalenesulfonate. Red dye “B” is a structural isomer of R40, that has not been reported previously. Our results suggest that the two minor red dyes were subsidiary colors contained in R40, which had been added to the dried strawberries.
Although the difference in allergenicity between landlocked and anadromous salmon is little understood, only anadromous salmon are recommended to be labeled in the current allergen labeling system. This study was designed to examine the allergenic potency of landlocked species (yamame) and anadromous species (sakuramasu) of masu salmon Oncorhynchus masou masou, with special reference to parvalbumin, a known major fish allergen. Analysis of the heated extracts by SDS-PAGE suggested that yamame contains parvalbumin in the muscle at considerably higher levels, as compared with sakuramasu. In accordance with this, the parvalbumin content in the muscle of yamame (1.8–7.8 mg/g), determined by visible-light ELISA, was significantly higher than that of sakuramasu (0.28–0.52 mg/g). Furthermore, fluorescence ELISA experiments showed that the heated extract from yamame reacts with serum from fish-allergic patients more strongly than that from sakuramasu. Three parvalbumin isoforms (PA-I, -II and -III) were individually purified from yamame and sakuramasu by gel filtration and reverse-phase HPLC. Based on the retention times in reverse-phase HPLC and the molecular weights estimated by MALDI/TOF-MS, PA-I, -II and -III from yamame were judged to be identical with PA-I, -II and -III from sakuramasu, respectively. Taken together, our data indicate that landlocked masu salmon (yamame) is more allergenic than anadromous salmon (sakuramasu).
The components of commercial sodium stearoyl lactylate (SSL), purchased in Japan, were determined and identified using thin layer chromatography (TLC) and liquid chromatography with mass spectroscopy (LC-MS). Stearoyl lactate (SL) and stearoyl-2-lactylate (SLL) were purified using TLC and silica gel chromatography to obtain standards. The results show that SSL consisted of lactic acid (8.4%), stearic acid (15%), SL (57%), and SLL (13%). The total amounts of free lactic acid, lactic acid derived from SL and lactic acid derived from SLL were determined using LC-MS. The mean value was approximately equal to that determined using the JECFA method. This is the first study to determine and identify the components of SSL purchased in Japan, using TLC and LC-MS.
Plasticizers in 101 samples of polyvinyl chloride (PVC) toys on the Japanese market were surveyed. No phthalates were detected in designated toys, though bis(2-ethylhexyl)phthalate, diisononyl phthalate, diisobutyl phthalate, dibutyl phthalate, diisodecyl phthalate and benzyl butyl phthalate were detected in more than half of other toys. 2,2,4-Tributyl-1,3-pentanediol diisobutylate, o-acetyl tributyl citrate, adipates and diacetyl lauroyl glycerol, which are alternative plasticizers to phthalates, were detected. The results of structural analysis confirmed the presence of di(2-ethylhexyl)terephthalate, tributyl citrate, diisononyl 1,2-cyclohexanedicarboxylate and neopentyl glycol esters; these have not previonsly been reported in Japan. There appears to be a shift in plasticizers used for designated toys from phthalates to new plasticizers, and the number of different plasticizers is increasing.
A test method of methanol content in detergent using a headspace-GC method was established. A 1 g aliquot of test sample was mixed with 0.4 mg of 2-propanol and made up to 20 mL with water. Then, 5 mL of test solution was placed in a headspace vial. The vial was sealed and incubated for 30 min at 60°C, then the headspace gas was analyzed by GC-FID. The recovery from spiked 1 mg/g of methanol was 95.6–100.6%. The determination limit was 0.1 mg/g. Using this method, the methanol content in 14 kinds of detergents was quantified. Methanol was detected from two detergents at the levels of 0.13 and 0.27 mg/g.
Tetrodotoxin (TTX) was administered to artificially hybridized specimens of the pufferfish Takifugu rubripes and Takifugu porphyreus to investigate toxin accumulation in hybrids and TTX transfer/accumulation profiles in the pufferfish body. In test fish administered TTX-containing feed homogenate at a dose of ∼400 MU/fish by oral gavage using a syringe (OGA group), the toxin content (MU/g tissue) of the digestive tract rapidly decreased and that of the liver increased from 1 to 24 h after administration. From 24 to 120 h, the toxin content of the liver decreased gradually, and the toxin appeared in the skin. On the other hand, intramuscularly administered TTX (400 MU/fish) was rapidly transferred to the liver and skin via the blood, and only a little toxin remained in the muscle even at 1 h (IMA group). The total amount of toxin remaining in the whole body (% of administered toxin) was 31–45% in the OGA group, and 42–74% in the IMA group; the scores in the OGA group were generally lower than those in the IMA group. In both OGA and IMA groups, the greatest amount of toxin accumulated in the liver (23–52%) after 8 h, followed by the skin (11–21%) after 72 h. The TTX administration experiment, especially using the oral gavage administration method, revealed that skins and livers of ‘torama’ pufferfish hybrid are endowed with TTX-accumulating ability, but the muscles are not, and that TTX taken up from toxic feed to the pufferfish body is transferred first to the liver and then to the skin via the blood.
Several DNA extraction methods have been officially introduced to detect genetically modified soybeans, but the choice of DNA extraction kits depend on the nature of the samples, such as grains or processed foods. To overcome this disadvantage, we examined whether the GM quicker kit is available for both grains and processed foods. We compared GM quicker with four approved DNA extraction kits in respect of DNA purity, copy numbers of lectin gene, and working time. We found that the DNA quality of GM quicker was superior to that of the other kits for grains, and the procedure was faster. However, in the case of processed foods, GM quicker was not superior to the other kits. We therefore investigated an unapproved GM quicker 3 kit, which is available for DNA extraction from processed foods, such as tofu and boiled soybeans. The GM quicker 3 kit provided good DNA quality from both grains and processed foods, so we made a minor modification of the GM quicker-based protocol that was suitable for processed foods, using GM quicker and its reagents. The modified method enhanced the performance of GM quicker with processed foods. We believe that GM quicker with the modified protocol is an excellent tool to obtain high-quality DNA from grains and processed foods for detection of genetically modified soybeans.
An analytical method of ethephon in feeds by GC-FPD was developed. Ethephon was extracted with ethyl acetate–hydrochloric acid (100 : 1) from feed samples. The extract was treated with added trimethylsilyldiazomethane in acetone–acetic acid (99 : 1) and this methylation procedure was repeated three times. Methylated ethephon was cleaned up on a graphitized carbon mini-column and a silicagel mini-column, and determined by GC-FPD. A method performance study in 7 laboratories was conducted with three kinds of samples spiked with ethephon at 10 mg/kg, 1 mg/kg and 0.5 mg/kg. The recovery of ethephon ranged from 81.8% to 90.8% (the reproducibility standard deviation (RSDr) were within 11%) and HorRat values were 0.58 to 0.94. The limit of detection (S/N≧3) and the limit of quantitation (S/N≧10) of ethephon in samples except hay were 0.02 mg/kg and 0.05 mg/kg, respectively. In the case of hay, the corresponding values were 0.2 mg/kg and 0.5 mg/kg, respectively.
An improved analysis method for 2-mercaptoimidazoline in rubber products containing chlorine was developed. 2-Mercaptoimidazoline (20 μg/mL) is detected by means of TLC with two developing solvents in the official method. But, this method is not quantitative. Instead, we employed HPLC using water–methanol (9 : 1) as the mobile phase. This procedure decreased interfering peaks, and the quantitation limit was 2 μg/mL of standard solution. 2-Mercaptoimidazoline was confirmed by GC-MS (5 μg/mL) and LC/MS (1 μg/mL) in the scan mode. For preparation of test solution, a soaking extraction method, in which 20 mL of methanol was added to the sample and allowed to stand overnight at about 40°C, was used. This gave similar values to the Soxhlet extraction method (official method) and was more convenient. The results indicate that our procedure is suitable for analysis of 2-mercaptoimidazoline. When 2-mercaptoimidazoline is detected, it is confirmed by either GC/MS or LC/MS.
The aluminium (Al) content of 105 samples, including bakery products made with baking powder, agricultural products and seafoods treated with alum, was investigated. The amounts of Al detected were as follows (limit of quantification: 0.01 mg/g): 0.01–0.37 mg/g in 26 of 57 bakery products, 0.22–0.57 mg/g in 3 of 6 powder mixes, 0.01–0.05 mg/g in all three agricultural products examined, 0.03–0.90 mg/g in 4 of 6 seafood samples, 0.01–0.03 mg/g in 3 of 11 samples of instant noodles, 0.04–0.14 mg/g in 3 of 4 samples of vermicelli, 0.01 mg/g in 1 of 16 soybean products, but none in soybeans. Amounts equivalent to the PTWI of a 16 kg infant were detected in two samples of bakery products, two samples of powder mixes and one sample of salted jellyfish, if each sample was taken once a week. These results suggest that certain foods, depending on the product and the intake, might exceed the PTWI of children, especially infants.
Supercritical fluid extraction (SFE) was applied to extraction of pesticides from vegetables and fruits. Residues were extracted from homogenized samples mixed with water-absorbent polymer with supercritical carbon dioxide in a stainless steel tube, followed by elution with acetone. Co-extractives were removed by means of mini-column clean-up. Measurement was performed by GC-MS/MS. Calibration was achieved by preparing matrix-matched calibration standards to counteract matrix effects. With the Japanese method validation guideline as a reference, the method was assessed in 5 agricultural products spiked with 334 pesticides at 0.01 and 0.1 μg/g. Compounds at each level were extracted from 2 samples on 5 separate days. The trueness of the method for 189 pesticides in all samples was 70–120%, and the repeatability and within-run reproducibility were also consistent with the guideline. The trueness of the method for the other 71 pesticides was in the range of 50–70%, though the repeatability and within-run reproducibility were satisfactory. This method is available as a multiresidue analysis method for vegetables and fruits.