Identification of isomers and subsidiary colors in commercial Fast Green FCF (FD&C Green No. 3, Food Green No. 3) by liquid chromatography-mass spectrometry and comparison between amounts of the subsidiary colors by high-performance liquid chromatography and thin-layer chromatography-spectrophotometry.

The existence of five of the six expected isomers in commercial Fast Green FCF (G3: Food Green No. 3, FD&C Green No. 3, CAS No. 2353-45-9, C.I. No. 42053), the main product of which is m,m-G3 and the sub-products of which are presumed to be m,p-G3, o,m-G3, p,p-G3, o,p-G3 and o,o-G3, was confirmed using LC/MS, and the levels of the isomers, m,m-G3, m,p-G3, p,p-G3, o,m-G3 and o,p-G3, were determined by analytical HPLC. The existence of seven subsidiary colors that were decomposed from G3 was also confirmed using LC/MS. The levels of the subsidiary colors in ethanol extracts from TLC were determined by HPLC and spectrophotometry, and these results were compared. It was clear that the values determined by TLC-spectrophotometry were higher than those by HPLC. It was recommended that the levels of subsidiary colors in G3 should be determined by HPLC.

Determination and confirmation of five phenolic antioxidants in foods by LC/MS and GC/MS.

Identification and determination of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), nordihydroguaiaretic acid (NDGA), propyl gallate (PG) and tert-butylhydroquinone (TBHQ) by means of LC/MS and GC/MS were examined. These five phenolic antioxidants were detected as their pseudo-molecular ions [M-H]- by LC/MS using a Shim-pack FC-ODS column with drying gas. Moreover, BHA, BHT and TBHQ were detected based on their mass fragment ions by GC/MS. Decomposition of TBHQ, NDGA and PG during analysis could be prevented by the addition of L-ascorbic acid (AsA) to the extraction solvent. All five antioxidants were extracted from nikuman, olive oils, peanut butter, pasta sauce and chewing gum with a mixture of acetonitrile-2-propanol-ethanol (2:1:1) containing 0.1% AsA (AsA mixture), which had been cooled in a freezer and filtered. One part filtrate and 5 parts water were mixed and placed on a Mega-Bond Elut C18 cartridge, except in the case of chewing gum. Lipids in foods were removed on a C18 cartridge by washing with 5 mL of 5% acetic acid, and antioxidants were eluted with 5 mL of AsA mixture. The antioxidants spiked into nikuman, olive oil, peanut butter, pasta sauce and chewing gum were successfully identified and their concentrations determined by LC/MS, and GC/MS with good recoveries.

[Identification and determination of the isomer and subsidiary color in food blue no. 2 (indigo carmine) by LC/MS and HPLC].

CFR and JECFA specify that the total color in FD&C Blue No. 2 (B2; Indigo Carmine, Indigotine, Food Blue No. 2) is not less than 85%, its isomer (B2iso) in B2 is not more than 18%, and its subsidiary color (B2sub) in B2 is not more than 2% (CFR) or 1% (JECFA). Japan's Specifications and Standards for Food Additives, 7th Edition, specifies that the total color in B2 is not less than 85.0% and other color materials in B2 are not detected by paper chromatography. LC/MS and HPLC were employed to identify and determine the main component (B2m) of B2, B2iso, and B2sub. The pseudo molecular ions (B2m and B2iso: [M-2Na+H]-, m/z=421; B2sub: [M-Na]-, m/z=341) of each color material were obtained and identified by LC/MS based on their absorptions and mass spectra. The contents of B2iso and B2sub in B2 samples (certified samples from fiscal year 1998 to fiscal year 2002) were determined by HPLC using calibration curves for the standards of B2m and B2iso. The contents of B2iso in most samples were less than 10%, and the contents of B2sub in all samples were not more than 1%. All of them were within the regulatory limits set by the CFR and JECFA.

[Estimation of daily intake of phenols in hospital meal samples].

Daily intakes of 12 phenols which are possible endocrine disruptors were estimated in hospital meals from 2000 to 2001. 4-Nonylphenol (4-NP (mix)) and bisphenol A (BPA) were detected at levels of 5.0 to 19.4 ng/g and 0.2 to 1.1 ng/g, respectively. 4-tert-Butylphenol, 4-n-pentylphenol, 4 -tert-pentylphenol, 4-n-hexylphenol, 4-n-heptylphenol and 4-tert-octylphenol were detected at levels of 0.1 to 2.4 microg/g. The daily intakes of 4-NP (mix) and BPA were 5.8 microg/day and 0.42 microg/day, respectively. The daily intakes of other phenols were less than 1 microg/day.

[Studies on the contents of water-soluble chlorides and water-soluble sulfates in food color aluminum lakes].

Japan's Specifications and Standards for Food Additives, 7th Edition (JSFA-VII) does not set limits for total contents of water-soluble chlorides and water-soluble sulfates (water-soluble inorganic salts) in food color aluminum lakes (FC-Als). However, the regulatory limit is 2% in JECFA and CFR. We used column switching suppressor-type ion chromatography (CSS-IC) for determination of anions. The total contents of water-soluble inorganic salts in FC-Als (112 qualified samples) were determined using the modified CSS-IC from fiscal year 1998 to fiscal year 2003. Total contents of water-soluble inorganic salts in four samples exceeded 2%. From an international point of view, it is desirable that the total content of water-soluble inorganic salts in FC-Al should again be regulated in Japan.

[Estimated production by the official inspection of tar colors (including aluminum lakes) in fiscal year 2003].

There were 197 official inspections of tar colors and their lakes in fiscal year 2003, the two of their samples were rejected, and the other were qualified. Total production amount of tar colors that passed inspection in Japan in fiscal year 2003 reached 147.9 tons. Tar color production amounts were described by month and by manufacturer. The food tar color produced in the largest amount was Food Yellow No. 4, accounting for 41.7% during this period.

Metabolism of grape seed polyphenol in the rat.

The metabolism of grape seed polyphenol (GSP) has been investigated in rats by high-performance liquid chromatography analysis of the serum and urinary concentrations of the GSP metabolites (+)-catechin (CT), (-)-epicatechin (EC), 3'-O-methyl-(+)-catechin, and 3'-O-methyl-(-)-epicatechin. The serum concentration of these four metabolites reached a maximum 3 h after the oral administration of GSP. The urinary excretion of these GSP metabolites accounted for 0.254% (w/w) of the administered dose of GSP (1.0 g/kg), and the majority of these metabolites were excreted within 25 h of oral administration. The serum concentration and urinary excretion of these metabolites were also compared after the oral administration of different GSP monomers (gallic acid, CT, and EC), normal GSP, and the high molecular weight components of GSP (GSPH). No metabolites were detected in the serum of rats given GSPH. The urinary percentage excretion of the GSP metabolites derived from the respective monomers (CT or EC) did not vary with the administration of different substances (CT or EC, GSP, or GSPH). Taken together, these results suggest that only the monomers of GSP are absorbed and metabolized.

[An experimental proficiency test for ability to screen 104 residual pesticides in agricultural products].

An experimental proficiency test program for ability to screen 104 residual pesticides in agricultural products has been conducted. Eight Japanese laboratories joined the program. Items tested in the present study were limit of detection, internal proficiency test (self spike) and external proficiency test (blind spike). All 104 pesticides were well detected and recovered from agricultural foods in the internal proficiency test. However, the results of the external proficiency test did not completely agree with those of the internal proficiency tests. After 5 rounds of the blind spike test, the ratio of the number of correctly detected pesticides to that of actually contained ones (49 total) ranged from 65% to 100% among laboratories. The numbers of mistakenly detected pesticides by a laboratory were 0 to 15. Thus, there was a great difference among the laboratories in the ability to screen multiresidual pesticides.

Method for analysis of tannic acid and its metabolites in biological samples: application to tannic acid metabolism in the rat.

A new analytical method for measuring tannic acid (TA) using tannase was developed and applied to the investigation of TA metabolism in the rat following oral administration at a dose of 1.0 g/kg. The proposed method for TA determination was based on the enzymatic hydrolysis of TA to gallic acid (GA) and subsequent determination by HPLC. TA metabolites were determined by HPLC. 4-O-Methylgallic acid (4-OMGA), pyrogallol (PY), and resorcinol (RE) were detected in serum. TA was excreted into urine as GA (0.01%), 4-OMGA (0.10%), PY (0.24%), and RE (2.06%) and into feces as TA (62.74%), GA (0.19%), PY (0.02%), and RE (0.76%) within 54 h after oral administration. It was suggested that >60% of TA remained unchanged but that some was hydrolyzed to GA by tannase in the intestine and further metabolized to 4-OMGA, PY, and RE.

[Determination of low-level pesticide residues in agricultural products by ion-trap GC/MS/MS].

The objective of this study was to elucidate the utility of ion-trap GC/MS/MS for the analysis of pesticides in extracted matrices from various agricultural products. Identification and quantitative analysis of pesticides in matrices were performed by quadrupole GC/MS and ion-trap GC/MS/MS. Chlorpyrifos was added to the matrix of spinach, soybean in the pod or corn, and aldrin, dieldrin, endrin, alpha-BHC, beta-BHC, gamma-BHC, delta-BHC, p,p'-DDD, p,p'-DDE, o,p'-DDT and p,p'-DDT were added to each matrix of green tea, black tea or oolong tea. Although most of the pesticides in the matrix could not be determined by quadrupole GC/MS-Scan analysis at 0.1 microgram/mL, every pesticide was identified from the mass spectrum using ion-trap GC/MS/MS at the same concentration. The quantitation limit of every pesticide in each matrix by ion-trap GC/MS/MS analysis was higher than that by GC/MS-SIM analysis. The calibration curves obtained by GC/MS/MS were linear in the range of 0.01-0.25 microgram/mL of each pesticide. The recoveries of each pesticide from four kinds of samples spiked at the levels of 0.01 ppm to 0.02 ppm in extracts were 61.2-138.3% with SD values in the range from 1.2 to 15.4%. This study revealed that ion-trap GC/MS/MS was useful for the identification and quantitative analysis of low-level pesticides residues in matrices of agricultural products.

[Estimated production by the official inspection of tar colors (including aluminum lakes) in fiscal year 2002].

There were 157 official inspections of tar colors and their lakes in fiscal year 2002, and all of their samples were qualified. Total production amount of tar colors that passed inspection in Japan in fiscal year 2002 reached 113.6 tons. Tar color production amounts were described by month and by manufacturer. The food tar color produced in the largest amount was Food Yellow No. 4, accounting for 42.5% during this period.

[Studies on flavianic acids as color reference standard for thin-layer chromatography].

Various flavianic acids were prepared and examined for the preparation for color reference standard for thin-layer chromatography (TLC). Their analytical data were: IR spetra, their specific absorptions were same; no impurities were detected; specific odor-free. It was clear that the prepared flavianic acids were useable as color reference standards for TLC.

[Studies on eight color reference standards for thin-layer chromatography].

Eight color NIHS reference standards were studied on IR spectra and thin-layer chromatography (TLC). Their IR spectra and TLCs were determined. They were also authorized as reference standards for TLC.

[Migration of di(2-ethylhexyl) phthalate from polyvinyl chloride tubes used in preparation of foods].

Three samples of polyvinyl chloride products for food use were examined for di(2-ethylhexyl) phthalate (DEHP) migration levels under the conditions described in the Japanese Food Sanitation Law. These samples were two kinds of tubes and one stretched film, containing 12 to 41% DEHP by weight. DEHP migration levels from them were very low, all below 0.1 ppm, into 3 kinds of food simulant; water, 4% acetic acid and 20% ethanol. However, high levels of DEHP migrated into n-heptane, 12 ppm from the stretched film sample and more than 800 ppm from the tube samples. Migration from the 2 tubes was higher than 150 ppm, the limit of residues after evaporation in containers and wraps. Though the limit of residues after evaporation is not set for equipment used in the preparation of foods, the tested tubes were considered to be unsuitable for uses in which they come into direct contact with oils, fats or oily foods.

[Simultaneous determination of N-methylcarbamates and their metabolites in citrus fruits by HPLC avoiding use of halogenic solvents].

Twenty-one N-methylcarbamates (NMCs) and 12 of their metabolites or isomers in citrus fruits were simultaneously determined avoiding use of dichloromethane. NMCs in lemon, orange, and grapefruit were extracted with acetone, then the acetone was evaporated off and sodium chloride was added before extraction with ethyl acetate. The extract was evaporated and the residue was cleaned up on a combined mini-column set of Supelclean ENVI-Carb and Mega Bond Elut SAX cartridges. NMCs were determined by HPLC with post-column reaction and fluorescence detection. All of the NMCs in the orange sample were determined without interfering peaks. However 8 NMCs in lemon extract and 10 NMCs in grapefruit extract were not detected because interfering peaks appeared at similar retention times to those of the NMCs. These NMCs were determined using LC/MS (SIM) and were well recovered. Eighty-three data sets obtained by HPLC and LC/MS showed good similarity, with r2 = 0.9178. Recoveries were 60.1 to 97.8% for major NMCs at a fortification level of 0.1 ppm. The limit of detection by HPLC was 0.005 ppm NMCs in samples and a similar level applied to LC/MS.

[Contents of plasticizers in cap-sealing for bottled food and their migration into food].

Plasticizers in cap-sealing for bottled foods were analyzed. Twenty-three domestic samples and 80 imported samples, a total of 103 samples, were tested. Among them, 93 contained chloride, and 62 contained di(2-ethylhexyl) phthalate (DEHP), diisodecyl phthalate, O-acetyl tributyl citrate, diacetyllauroyl glycerol, di(2-ethylhexyl) adipate, diisononyl phthalate or dicyclohexyl phthalate. Twelve samples with DEHP-containing caps were further tested for plasticizer levels in the foods. Higher DEHP was detected in oily and fluid food specimens. However, the intake of plasticizers calculated based on usual food consumption did not exceed the tolerable daily intake level in any of the cases. A food sample containing the highest level of DEHP was stored under various conditions, and DEHP that migrated from the cap-sealing into the food was determined. Shaking the bottles increased migration of DEHP into foods.

[Estimated production by the official inspection of tar colors (including aluminum lakes) in fiscal year 2001].

There were 162 official inspections of tar colors and their lakes in fiscal year 2001, and 162 samples were qualified. Total production amount of tar colors that passed inspection in Japan in fiscal year 2001 reached 134.8 tons. Tar color production amounts were described by month and by manufacturer. The food tar color produced in the largest amount was Food Yellow No. 4, accounting for 43.9% during this period. Their yearly total productions were decreased from 238.7 to 134.8 tons during 12 years.

[Determination and survey of hexachlorobenzene in food red nos. 104 (phloxine) and 105 (rose bengale) by GC/MS].

The contents of hexachlorobenzene (HCB) in Food Red Nos. 104 and 105 samples certified in fiscal years 1998-2001 were determined by GC/MS. All 32 samples contained HCB ranging from 0.2 to 32.9 mg/kg. The daily intakes of HCB based on the production of food colors was estimated to be from 0.018 to 1.456 ng, and their average (0.441 ng) corresponded to 0.0052% of the ratio for the Tolerable Daily Intake (8.5 micrograms for 50 kg body weight) of HCB developed by the IPCS.