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.

[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.

[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.

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.

[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.

[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.

Rapid Determination of Mono-, Di- and Tri-isopropyl Citrate in Foods by Gas Chromatography.

A simple and rapid method for determining mono-, di- and tri-isopropyl citrates in foods was developed. Isopropyl citrates in butter and milk powder were extracted with ethyl acetate under acid condition and in edible oil with hexane. The ethyl acetate containing isopropyl citrates was evaporated and the residue was taken up in hexane. The isopropyl citrates in hexane were extracted into acetonitrile. After evaporation of the solvent, isopropyl citrates were methylated with diazomethane, and they were determined by gas chromatography. Recoveries of mono-, di- and tri-isopropyl citrates from edible oil, butter and milk powder by this method were more than 92.9%, 95.7% and 94.8%, respectively. The detection limits of isopropyl citrates were 1 µg/g of sample.