Estimated daily intake of plasticizers in 1-week duplicate diet samples following regulation of DEHP-containing PVC gloves in Japan.

Duplicate hospital diet samples obtained over 1 week in 2001 were analysed to estimate the daily intake of plasticizers and the results were compared with those obtained in 1999. The plasticizers quantified in this study were: dibutyl phthalate, butylbenzyl phthalate (BBP), di(2-ethylhexyl) phthalate (DEHP), diisononyl phthalate (DINP), di(2-ethylhexyl) adipate (DEHA), diisononyl adipate (DINA) and O-acetyl tributyl citrate (ATBC). Dipropyl, dipentyl, dihexyl and dicyclohexyl phthalate were also analysed but not detected. The analytical procedure for this follow-up study was essentially the same as in the previous one. Detection limits were 0.1-15.6 ng g(-1) for each plasticizer. One-week duplicate diet samples provided by three hospitals in three remote prefectures of Japan were analysed as individual meals. DEHP was detected at 6-675 ng g(-1) in 62 of 63 meals, significantly lower levels compared with those detected in 1999. Levels of DEHA and DINP also decreased. The mean intake of plasticizers estimated from all samples was 160 microg DEHP day(-1), 12.5 microg DEHA day(-1), 4.7 microg DINP day(-1) and 3.4 microg BBP day(-1). Levels of DINA were relatively high in meals from one hospital: in those meals, the average daily intake was 1338 microg day(-1). Those of ATBC were also higher in meals from another hospital: the average daily intake was 1228 microg day(-1). The sources of DINA and ATBC can be cling-film or sausage packaging.

[Modification of HPLC conditions for the determinations of raw materials, intermediates and subsidiary colors in 5 kinds of food azo colors].

Modification of HPLC conditions for the determinations of raw materials, intermediates and subsidiary colors (organic impurities) in 5 kinds of food azo colors were studied in order to analyze them simply and rapidly. The organic impurities were determined by HPLC using L-column ODS and a gradient system (0.02 mol/L ammonium acetate solution for 10 min, and a gradient with a mixture of acetonitrile and water (7:3)). The organic impurities in 163 samples of azo colors subjected to inspection in fiscal year 1999 were determined under the proposed HPLC conditions. It was found that the contents of organic impurities in 163 samples were less than the limit of Japan's Specifications and Standards for Food Additives, 7th Edition.

[Contents of eleven phthalates and di(2-ethylhexyl) adipate in retail packed lunches after prohibition of DEHP-containing PVC gloves for cooking purposes].

Ten samples of retail packed lunches purchased from convenience stores were determined for 11 phthalates and di(2-ethylhexyl) adipate (DEHA) in August 2000, 2 months after the prohibition of DEHP-containing PVC gloves in Japan. Each homogenized sample was extracted with acetonitrile, partitioned with n-hexane, and cleaned up using Florisil and PSA columns. Phthalates in the extract were determined by GC/MS (SIM). The limits of detection were 14.9 ng/g for di(2-ethylhexyl) phthalate (DEHP) and 18.6 ng/g for dibutyl phthalate (DBP). Levels of phthalates in packed lunch samples were 45 to 517 ng DEHP/g (198 ng/g, average), ND to 90 ng DEHA/g, and ND to 10.0 ng BBP/g. Diisononyl phthalate (DINP) was detected in one sample at 76 ng/g. Average DEHP level in ten samples was 4% of that in 1999. The contents of other phthalates were also reduced. DBP was not detected in any sample. Recovery of deuterated isomers added as surrogates was 27.9% for DNP-d4, and 40.6 to 101.5% for the other phthalates.

[Determination of bisphenol A in foods using GC/MS].

An analytical method using GC/MS was developed for bisphenol A (BPA) in foods and BPA was determined in canned foods and fresh foods such as vegetables, fruit and meat. BPA was extracted with acetone from the samples and the extract was concentrated at under 40 degrees C in vacuo to afford an aqueous solution, which was washed with hexane after alkalization and extracted with 50% diethyl ether-hexane after acidification. Extracts were cleaned up on a PSA and/or a C18 cartridge column, and BPA was derivatized with heptafluorobutyric anhydride and determined by GC/MS (SIM). This method was applicable to the detection and determination of BPA residues in food samples at the level of 1 ng/g. Among canned foods, BPA was found in 6 corned beef, 1 chicken, 9 sweet corn and 3 bean samples at the levels of 17-602 ng/g, 212 ng/g, 2.3-75 ng/g and 3.5-26 ng/g, respectively. BPA was also detected in 1 retort soup and 1 retort pack product at the levels of 11 ng/g and 86 ng/g, respectively. As for dairy products, BPA was not detected in butter and milk. Among fresh foods, BPA was detected in 2 fish and 3 liver samples at the levels of trace (tr)-6.2 ng/g and tr-2.2 ng/g, respectively. In vegetables, fruits and chocolates, a trace level of BPA was detected in only 1 chocolate. Traces of BPA were also detected in 3 samples of 6 boxed lunches.

Determination of clethodim and its oxidation metabolites in crops by liquid chromatography with confirmation by LC/MS.

A method was developed for determination of the herbicide clethodim (C0) and its oxidation metabolites clethodim sulfoxide (C1) and clethodim sulfone (C2) in agricultural products. Upon extraction, both C0 and C1 were oxidized to C2 by m-chloroperoxybenzoic acid, and C2 was determined by liquid chromatography (LC). The C2 peak was confirmed by liquid chromatography/mass spectrometry (LC/MS) with electrospray ionization (ESI). Recoveries of C0 from radish, tomato, onion, sweet potato, kidney bean, carrot, cabbage, and lettuce ranged from 91 to 118% following fortification at 0.05-1.0 ppm. The detection limit of C2 in crops was 0.01 ppm (S/N > 3). The fortified samples of onion, sweet potato, kidney bean, and carrot were confirmed by LC/MS (ESI), and the peak of C2 was detected.

Di(2-ethylhexyl) phthalate contamination of retail packed lunches caused by PVC gloves used in the preparation of foods.

Plasticizer contamination of foods sold in retail packed lunches and set lunches in restaurants was determined by GC/MS. The phthalate esters were as follows: diethyl, dipropyl, dibutyl, dipentyl, dihexyl, butylbenzyl, dicyclohexyl, di(2-ethylhexyl), dioctyl, diisooctyl (mixture of isomers) and diisononyl (mixture). Di(2-ethylhexyl) adipate was also determined. Sixteen packed lunches and ten set lunches were analysed, and in all samples the concentration of di(2-ethylhexyl) phthalate (DEHP) was the highest, at 0.80-11.8 mg/kg in packed lunches and 0.012-0.30 mg/kg in set lunches. The DEHP content of five packed lunches exceeded 1.85 mg, which is the EU tolerable daily intake (TDI) for a person of 50 kg body weight. Foodstuffs that were components of the packed lunches were taken from the factory at each step of preparation and phthalates were determined. For example, chicken contained 0.08 mg/kg DEHP when uncooked, 13.1 mg/kg after frying and 16.9 mg/kg after packing. Disposable PVC gloves used in the preparation of foods were apparently the source of high DEHP concentrations. The gloves used during cooking or packaging were sprayed with 68% (w/w) ethanol to sterilize them. PVC gloves from the factory contained 22 or 41% by weight of DEHP. To confirm the link with the contamination problem, samples of boiled rice, croquette and boiled dry radish were handled in the laboratory with PVC gloves containing 30% (w/w) DEHP. DEHP migration levels of 0.05 mg/kg in rice or 0.33 mg/kg in croquette, and 11.1 mg/kg in radish were found. The alcohol sprayed onto the gloves increased the migration of DEHP to 2.03 mg/kg in rice, 2.45 mg, kg in croquette, and 18.4 mg/kg in radish.

Simultaneous determination of residues of emamectin and its metabolites, and milbemectin, ivermectin, and abamectin in crops by liquid chromatography with fluorescence detection.

A liquid chromatographic (LC) method was developed for the determination of emamectin and its metabolites (8,9-Z-isomer, N-demethylated, N-formylated, and N-methylformylated emamectin) in various crops. The analytes were extracted with acetone, cleaned up on cartridge columns (C18 and NH2), derivatized with trifluoroacetic anhydride and 1-methylimidazole, and determined by LC with fluorescence detection. Because radish inhibited the formation of the fluorescent derivatives, an additional Bond Elut PRS cartridge was used in the cleanup of Japanese radish samples. During sample preparation, N-formylated emamectin partially degraded to emamectin B1b and emamectin B1a, and the 8,9-Z-isomer partially degraded to N-demethylated emamectin. Therefore, emamectin and its metabolites were determined as total emamectin, i.e., their sum was estimated as emamectin benzoate. Their recoveries from most crops were approximately 80-110% with the developed method. The detection limits for the analytes in vegetables were 0.1-0.3 parts per trillion (ppt). The results for these compounds were confirmed by LC/mass spectrometry (LC/MS; electrospray ionization mode). Because the fluorescent derivative of emamectin was undetectable by LC/MS, the results for the analyte were confirmed by using a sample solution without derivatization. Limits of detection by LC/MS were similar to the fluorescence detection limits, 0.1-0.3 ppt in vegetables. In addition to the emamectins, milbemectin, ivermectin, and abamectin were also determined by the developed method.

Eleven phthalate esters and di(2-ethylhexyl) adipate in one-week duplicate diet samples obtained from hospitals and their estimated daily intake.

Plasticizers in duplicate diet samples obtained over 1 week were analysed in order to estimate daily intake. The phthalate esters were as follows: diethyl, dipropyl, dibutyl, dipentyl, dihexyl, butylbenzyl, dicyclohexyl, di(2-ethylhexyl), dioctyl, diisooctyl (mixture of isomers) and diisononyl (mixture). Di(2-ethylhexyl) adipate was also determined. Homogenized samples of composite meals were extracted with acetonitrile, lipids were removed by extraction into n-hexane and the acetonitrile layer was cleaned using Florisil and Bondesil PSA dual layer column. Phthalates were determined by GC/MS (SIM). Phthalate recovery from the fortified food mixture by this method was 62.5-140.8%. Quality assurance as assessed by three laboratories indicated coefficient of variance in the levels of detected phthalates in same lot samples as below 10%. Detection limits were 0.1-23 ng/g for each phthalate. One-week duplicate diet samples provided by three hospitals in three remote prefectures of Japan were analysed as individual meals. In all 63 samples, DEHP was present at the highest level among all phthalates in the range 10-4400 ng/g. The intake of plasticizers estimated from all samples was 519 microg DEHP/day, 86 microg DEHA/day, 65 microg DINP/day, and 4.7 microg BBP/day. Calculated DEHP in 2-day samples out of 21 days exceeded EU TDI for a person of 50 kg body weight (1850 microg per day). Disposable PVC gloves used during the preparation of meals were suspected as the source of the high DEHP content. One-day intake of the other phthalates and DEHA was below 7% of TDI in all cases. High concentrations of DEHP (5990 ng/g) was found in baby food used in quality assurance work. The source of contamination was the PVC-tube used during production and was effectively reduced by replacing the tube by one made of stainless steel.

Structural determination of the subsidiary colors in food blue No. 1 (brilliant blue FCF) aluminum lake detected by paper chromatography.

One of eight Food Blue No. 1 aluminum lakes (B-1Als) used in the official inspection of coal-tar colors in fiscal year 1999 had a violet sub-spot during paper chromatography and was rejected. To clarify the orgin of the sub-spot, the violet subsidiary color (Sub-V) was isolated from the sample. On the basis of NMR and MS analyses and ion chromatography, the structure of the subsidiary color was elucidated to be 2-[[4-[N-ethyl-N-(3- sulfophenylmethyl)amino]phenyl][4-hydroxyphenyl]methylio]benzenesulfonic acid. The relative content of Sub-V to that of m,m-B-1 in the rejected sample was determined to be 39.5% by HPLC. The relative contents in other submitted samples of B-1Al were in the range of 1.1-3.6%.

[Preparation of HPLC test solutions for organic impurities in aluminum lakes of food red no. 40 (allura red AC) and food yellow no. 5 (sunset yellow FCF)].

The HPLC determination of organic impurities in Food Red No. 40 aluminum lakes (R-40Als) as directed by Japan's Specifications and Standards for Food Additives, 7th Ed. (JSFA-VII), has problems, such as reproducibility and low recovery. ICP analyses suggested that the problem was caused by the aluminum in the test solution. In the improved method for preparation of the test solution, aluminum was precipitated as a hydroxide gel by boiling with 1% aqueous ammonia. After centrifugation, the supernatant was used for the HPLC analysis of the organic impurities in the lakes. Recoveries of organic impurities were more than 85% from R-40Al spiked at the 0.1 and 1.0% levels of R-40. The proposed method was also adapted for Food Yellow No. 5 aluminum lakes.

[Clean-up procedure with ion-exchange mini column in the analysis of flusulfamide in agricultural products by HPLC].

A clean-up procedure with an ion-exchange column in the analysis of flusulfamide by HPLC was examined. Pesticide in the sample was extracted with methanol following liquid-liquid partition with ethyl acetate. The ethyl acetate fraction was cleaned up with silica gel column chromatography. The eluate from the silica gel column was further cleaned up with SAX + PSA mini column, then determined by HPLC. Carotenoids and interfering peaks were removed by washing the combined mini columns with 10 mL of 20% acetone-containing n-hexane and 5 mL of acetone, and flusulfamide was eluted with 35 mL of acetone.

[Estimated production by the official inspection of tar colors (including aluminum lakes) in fiscal year 2000].

There were 176 official inspections of tar colors and their lakes in fiscal year 2000, and 175 samples were qualified. The quantity of tar colors that passed inspection in Japan in fiscal year 2000 reached 137.5 tons. Tar color production is estimated by month and by manufacturer. The food tar color produced in the largest quantity was Food Yellow No. 4, accounting for 43.4% during this period.

[Studies on rejected food yellow no. 5 (sunset yellow FCF) aluminum lake].

One out of two sunset yellow FCF aluminum lakes (Y-5Als) did not comply with the specifications in JSFA-VII in the official inspection of tar colors in fiscal year 2000. A sub-spot was detected in the paper chromatography test. This rejected sample was analyzed by HPLC for the subsidiary color, raw materials and intermEdiates in Y-5. The sub-spot was identified as sulfanilic acid azo R salt color, and its content was estimated at 4.5% as the content of Y-5 in Y-5Al being 100.0%.

Liquid chromatographic determination of emamectin, milbemectin, ivermectin and abamectin in crops and confirmation by liquid chromatography-mass spectrometry.

Emamectin, milbemectin, ivermectin and abamectin are similar macrocyclic lactone chemicals used as an acaricides or parasiticides. We developed a simultaneous analytical method for determining the residual amounts of these compounds and emamectin metabolites in crops. A sample extracted with acetone was cleaned up with Bond Elut C18 and NH2. The sample was then fluorescence-derivatized with trifluoroacetic anhydride and 1-methylimidazole in acetonitrile. The analyte was measured by HPLC with fluorescence detection using an octadecylsilyl column with 3 microm particle size and gradient elution. In most crops, their recoveries by the developed method were ca. 80-110%. The detection limits of the analytes in vegetables were 0.1-0.3 ppt. Using the developed method, we surveyed the residues of these compounds in 20 commercial crops in Osaka, Japan. The result of the surveillance was that emamectin benzoate of 0.2-6.7 ppb was detected in nine cases and milbemectin of 16.7-279.3 ppb was detected in four cases. The detected samples were confirmed by LC-electrospray ionization (ESI) MS. The limit of detection by LC-ESI-MS was similar to the fluorescence detection level of 0.1-0.3 ppt in vegetables except for milbemectin.

High-performance liquid chromatographic determination with photodiode array detection of ellagic acid in fresh and processed fruits.

A high-performance liquid chromatographic (HPLC) procedure based on an isocratic elution with photodiode array detection has been developed for a simple and rapid determination of ellagic acid (EA) in fresh and processed fruits. The homogenized sample was refluxed with methanol and then the extract was refined using a solid-phase cartridge before HPLC. We analyzed EA in 40 kinds of fresh fruits and 11 kinds of processed fruits by the developed method. EA was found in several berries, fueijoa, pineapple and pomegranate. This is the first occurrence of the detection of EA in bayberry, fueijoa and pineapple.

Determination of phenolic acids in fruit juices by isocratic column liquid chromatography.

A simple and rapid analytical method of five phenolic acids, gallic, chlorogenic, caffeic, ellagic and ferulic acid, which are naturally occurring bioactives, were determined in fruit juices by isocratic LC using photodiode array UV detection. The sample was pre-treated by solid-phase extraction (a combination of Sep-Pak Plus tC18 and Bond Elut PSA).

Degradation of malathion and phenthoate by glutathione reductase in wheat germ.

Residual malathion in wheat was estimated at a lower value when analysis was performed by extraction with acetone after addition of water to swell the wheat, according to the Japanese Bulletin Method. The supernatant of the wheat homogenate showed degradation not only of malathion but also of phenthoate. Malathion and phenthoate were not degraded by the boiled supernatant of the wheat homogenate. It was presumed for this reason that glutathione reductase (GR; EC 1.6. 4.2) in the wheat degraded malathion. The following results were obtained: (1) GR originating in wheat could degrade malathion and phenthoate. (2) The degradation of malathion by the GR was inhibited by excessive GSSG. (3) There was a high correlation between GR activity and malathion degradation activity of the supernatant of wheat homogenates. It is likely that GR acted on the specific structure of malathion and phenthoate, the S=P-S bond, and the blanch structure bonding with the sulfur atom. Following the above, extraction with acetone after addition of water (the Japanese Bulletin Method) should be replaced by extraction with pure organic solvent and without addition of water for swelling.

Determination of the levels of isoflavonoids in soybeans and soy-derived foods and estimation of isoflavonoids in the Japanese daily intake.

The levels of 6 kinds of isoflavonoids found in 11 domestic and imported soybeans, and 12 kinds of soybean-based processed foods in Japan were systematically analyzed, and the Japanese daily intake of isoflavonoids from those foods was estimated. The total isoflavonoids (daidzein, glycitein, and genistein) were analyzed with acid hydrolysis and the intact isoflavonoids (daidzein, glycitein, genistein, daidzin, glycitin, and genistin) were analyzed without hydrolysis. This was followed by cleanup with an ODS cartridge column and determined by liquid chromatography with a diode array detector. The highest content of isoflavonoids was found in kinako (a roasted soybean powder) and the lowest was found in soy sauce. The contents and composition of the isoflavonoids in the 11 soybeans varied by species and country of origin. The level of isoflavonoids found in the processed foods varied by manufacturing method or ingredients. The percentage of aglycone tended to be higher in miso (fermented soybean paste) and soy sauce, which are heated and fermented during the manufacturing process. Japanese daily intake of isoflavonoids from soybeans and soybean-based processed foods was estimated as 27.80 mg per day (daidzein 12.02 mg, glycitein 2.30 mg, and genistein 13.48 mg).

[Studies on procedure for sample preparation of ellagic acid in several kinds of foodstuffs].

A sample preparation for ellagic acid in several kinds of foodstuffs, which is included in the List of Existing Food Additives as natural antioxidants in Japan, functioning as an antioxidant, was studied. The solid samples were refluxed with methanol, and then the extract was refined using a solid-phase cartridge. The liquid samples were directly pretreated by solid-phase extraction. On the other hand, test solutions for cooking oils and fats were inapplicable to solid-phase extraction in this work, because the recovery tests from samples spiked with ellagic acid gave low recoveries. Consequently, they were prepared by modified frozen method using methanol or acetonitrile-2-propanol-ethanol (2:1:1). The recoveries from tested foodstuffs spiked with ellagic acid (50 micrograms/g) were 75.2-96.9%. The limits of quantification for ellagic acid were 0.05 microgram/g by the proposed method.

[Studies on rejected food blue no. 1 (Brilliant Blue FCF) aluminum lake].

One of the brilliant blue FCF aluminum lakes (B-1Als) in the official inspection of coal-tar dyes in fiscal year 1999 was rejected. The results of tests of the rejected sample were submitted to JSFA-VII except for a violet sub-spot by paper chromatography. Its visual spectrum had same lambda max 630 nm as B-1 standard and a very small shoulder at 580 nm in comparing to B-1 standard. HPLCs detected at 625 nm of subsidiary dyes and 580 nm were performed on the rejected sample of B-1Al. A sub-V in HPLCs of th sample which eluted at 37.6 min had its relative peak area of 7.9% at 580 nm and 0.24% at 625 nm, and its peak ratio (at 580 nm/at 625 nm) was extremely large. Consequently, it was presumed that the sub-V in those HPLCs corresponded to the violet sub-spot in paper chromatography.