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The 2,4-xylenol method was modified and a gas-liquid chromatographic (GLC) method was developed for nitrate and nitrite determinations in several foods. Either a flame ionization (FID) or an electron capture detector (ECD) can be used. Proteins and fats are removed from warm alkaline water with zinc sulfate and filtered. The filtrate is evaporated to dryness, redissolved in water, and reacted with 2,4-xylenol in the presence of sulfuric acid to form 6-nitro-2,4-xylenol. Interfering chlorides are precipitated with silver sulfate and the nitrosylenol is extracted with hexane, concentrated, and injected. Nitrite in the filtrate is distilled at pH 5, collected in alkaline solution, and dried. The residue is oxidized to nitrate with permanganate in the presence of sulfuric acid, and then chromatographed in the same manner as nitrate. Recoveries from several foods were 83--100% for nitrate and 80--100% for nitrite. The limit of sensitivity was 0.1 ppm for both residues.

A fluorescent substance was found in imported fruit candy. In the elution test of the fluorescent substance, it reacted in the same manner as fluorescent dyes. Further pursuit revealed that the fluorescence was derived from the grape flavor preparation added during manufacture and the fluorescence spectra of the candy and the grape flavor were quite coincident with that of methyl anthranilate. Methyl anthranilate contents in the candy and grape flavor were determined gas chromatographically to be 40.0 and 33,000 ppm, respectively, and it was calculated that the addition level of grape flavor to the candy was about 0.12%.

Attempts were made to develop a sensitive and reproducible method to determine nitrite in cod roe. Two diazotation-coupling reaction methods were considered; (a) the method defined by the Ministry of Health and Welfare of Japan (Method 1) and (b) the reference method of ISO (Method 2). Since the nitrite content in cod roe was much less than in meat products, Method 2 was modified to make it suitable for microanalysis at 1 ppm level as NO 2 . Modifications included reducing volumes of color-development solutions and making changes in the color development process, thus making the color intensity four times as great as before. Carrying out corrections with both reagent and water blanks made the effect of the blank on measured values negligible. Recoveries of nitrite at 20- and 2-ppm levels were 94.7 and 88.1%, respectively, reproducibility being ± 7 .9%, as the coefficient of variation. The obtained values by the modified method were, on the average, higher than those of the original method by 37.1%. Nitrite contents obtained by Method 1 were lower than those by the original Method 2. These low values might be attributed to loss of nitrite during extraction from the sample without pH adjustment, since the measured value showed a remarkable increase by addition of alkaline solution before extraction. Nitrite contents in imported cod roe were within the range 0.16-1.03 ppm expressed as NO 2 .

The toxicity of xanthene dyes were studied by various interaction between the dyes and the components in vital body. (1) An increase in the amount of Rose Bengale adsorbed on the gill of fish was followed by the increase in red corpuscle number, and it was assumed that the death of fish in xanthene dye solution was due to anoxemia. (2) Binding capacity of xanthene dyes with bovine serum albumin decreased in the following; Rose Bengale, Phloxine, Erythrosine, Eosine and Uranine. This order was quite coincident with the toxicity compared by TLm values. (3) From the results of rec-assay test by use of Bacillus subtilis, it was confirmed that Phloxine and Rose Bengale had DNA-damaging capacity related to the mutagenecity.

An isotachophoretic method was developed for the determination of EDTA in foods imported by Japan. Skimmed samples of dressings, mayonnaise, or margarine were chromatographed on an anion exchange column, and interfering organic acids were eluted with water and 0.01N HCl. EDTA was eluted with 0.2N HCl and reacted with ferric chloride to form a stable EDTA--Fe complex. Electrophoresis was carried out with dilute HCl containing 0.05% Triton and beta-alanine (pH 3.5) as the leading electrolyte and 0.01M caproic acid as terminating electrolyte. Since uncoupled EDTA showed more than one zone, it was reacted with ferric chloride to form EDTA-Fe complex which showed only a single zone on an isotachopherogram. More than 90% of EDTA spiked at 100 or 1000 ppm level as disodium salt was recovered from the above mentioned three types of food. Detection limit was 10 ppm as disodium EDTA.

Diacetyl tartaric acid ester of diglyceride was directly extracted from dairy or nondairy coffee cream powder under acidic conditions with ethyl acetate; then the extract was saponified with methanolic potash. After acidification with HCl, free fatty acid was removed with ether and the reaction mixture was absorbed on an anion exchange column. Tartaric acid was eluted with 2N HCl-acetone (1+1). An aliquot of the trimethylsilyl (TMS) derivative of the eluate was injected into a gas chromatograph with flame ionization detection, and a 1.5% SE-30 column. Recoveries of diacetyl tartaric acid ester of diglyceride at 50, 200, and 2000 ppm were 85.6-99.5%.

A comparison of the combined solvent-extraction gas liquid chromatography (GLC) method with the AOAC method was carried out by use of two lots of processed cheese manufactured from Samsoe and Cheddar cheeses, containing 14.5 and 72.3 ppm of sorbic acid, respectively. On the whole, higher values were obtained by AOAC method, the ratio to theoretical values ranging from 98.6 to 135.9%. Such results indicate that substances other than sorbic acid might have reacted with 2-thiobarbituric acid to develop color. Contrary to this, results by the combined solvent-extraction GLC method were within the range of 97.5 to 106.2% of the theoretical values. The difference of the determined values by the two methods was significant at the 5% level for the lot containing 72.5 ppm of sorbic acid. The advantages of the combined method of solvent-extraction and GLC over the AOAC method include rapidity, simplicity and accuracy.

Ortho-, poly (pyro- and higher)- and meta-phosphates can be separated from each other with two dimensional thin layer chromatography, the detection level being 1 μg as P 2 O 5 . For determination of ortho- and poly (including polymeta-)-phosphates, an ion-exchange column chromatography was used. A single concentration elution with 0.2 M potassium chloride was effective for separating of orthophosphates from polyphosphates, and the residual polyphosphates could be recovered with 6 N hydrochloric acid. This system was used for a survey of polyphosphates in 61 samples of soft drinks sold in Osaka. Results showed that the total phosphate contents within the range of 0 - 0.420 g of P 2 O 5 /kg. Only three samples contained polyphosphates. The highest being 0.261 g of polyphosphate/kg in a soft drink containing 20% mango juice.

Tertiary-butylhydroquinone (TBHQ) is extracted from foods with ethyl acetate, the solvent is evaporated, the residue is dissolved in n-hexane-ethyl acetate (99 + 1), and the TBHQ is selectively transferred into an aqueous layer (2% sodium chloride) as a separation from food interferences. Finally, TBHQ is reextracted with n-hexane-ethyl acetate (1 + 1), concentrated, and determined by flame ionization gas-liquid chrmoatography. Dried fish, frozen shrimp, vegetable oils, and edible fats were analyzed by the method. More than 85% was recovered when the samples were fortified with 100 ppm TBHQ. Detection limit was 1.0 ppm TBHQ.

Reason of acute toxicity to fish caused by coal-tar dyes and their industrial effluents were studied from the three points of view. 1) Partition coefficient (n-octanol/water) was determined on several coal-tar dyes, and it was found that the larger the ratio was, the stronger was toxicity to fish. 2) As a result of determination about existent form of xanthene dyes under photo-irradiation, it was clarified that dyes in solution were in the form of excited triplet state and halogen in dye molecule was liberated in free form. 3) When coal-tar dyes were treated with sodium hypochlorite, harmful chloroform was detected from dye solution. Chloroform and its homologous compounds showed the low TLm value and had the serious effect on fish.

Borate was directly chelate-extracted from foods with 5% 2-ethyl-1,3-hexanediol (EHD) in n-hexane-n-butyl acetate mixture (8 + 2), from which borate was selectively transferred into 1% NaOH, since EHD-chelated boron did not react with curcumin to develop color. Finally, an aliquot of the alkaline solution was acidified with HCl and reacted with curcumin in a rotary evaporator. Color development was increased by heating for 8 min at 80 degrees C under reduced pressure of 16 mm Hg. Frozen shrimp and prawns (peeled and with shells) and salted jelly fish were analyzed by the proposed method. Results were compared with the contemporary official method of Japan based on curcumin reaction on an incinerated sample. Over 90% of the boric acid was recovered by the proposed method when samples were fortified with 20 ppm boric acid. Recoveries were superior to those of the official method especially for shrimp and prawns with shells and salted jelly fish. Detection limit of boric acid is 1 ppm. Moreover, the method requires only about 1 hr for analysis of one sample, making it suitable for routine analysis.

A simple and rapid fluorometric method for determining benzo (a) pyrene in foods was developed. Benzo (a) pyrene is extracted from foods with n-hexane:ether mixture (4:1), purified through a column of activated alumina and determined fluorometrically. An excitation wavelength of 295 nm and emission wavelength of 403 nm were used for calculating concentrations of benzo (a) pyrene. The peak height at 403 nm and baseline between 392 and 418 nm were employed to derive a standard curve for quantitating benzo (a) pyrene. A calibration curve for between 0.04 - 4 ng/ml of benzo (a) pyrene was used. Recoveries of benzo (a) pyrene from 14 kinds of food spiked at levels of 20 and 2ppb were within the range of 79.5 - 93.8% and 50.0 - 80.6%, respectively. The entire procedure takes only one hour with the detection limit being 0.1 ppb. Benzo (a) pyrene detected was reconfirmed by thin-layer chromatography.

A gas-liquid chromatographic (GLC) method was developed for the detection and determination of thiourea in citrus peels. After the peel is extracted with ethyl ether, the ether extract is adsorbed on sodium sulfate together with water. Thiourea is recoverd from both the sodium sulfate and the peel residue with ethyl acetate-acetone(2+1). The extracted mixture is cleaned on an alumina column, the eluate is concentrated under vacuum, and thiourea is extracted from the concentrate with sodium carbonate solution. GLC was carried out on the prepared benzoyl derivative of thiourea. The average recoveries of thiourea from lemon peel were 85.3, 93.1, and 97.6% at the fortification levels of 1, 10, and 100 ppm, respectively. The detection limit was low as 0.08 ppm.

Improved 4-aminoantipyrine (4-AA) colorimetry was developed for the detection of minute quantities of residual hydrogen peroxide in several kinds of food. Hydrogen peroxide in the sample was stabilized with potassium bromate and extracted with cold methanol. The methanol extract was diluted with phosphate buffer, protein was eliminated with zinc sulfate, and the extract was reacted with phenol, 4-AA, and peroxidase to the stable quinoneimine dye. The weak color solution was purified with Florisil column chromatography, concentrated, and determined by colorimetry. Recoveries of hydrogen peroxide from samples fortified at 0.5, 2, and 10 ppm ranged from 70.7 +/- 8.9 to 98. 5 +/- 1.2%. Color development was linear with amount of H2O2 from 0.5 to 20 micrograms, corresponding to 0.05-2 ppm in samples.

Forty-four kinds of organic nitrogen compounds as well as 37 kinds of dyes which contained nitrogen were subjected to TLm test by use of Himedaka (Oryzias latipes) and the results were compared with their partition coefficients between n-octanol and water. Concerning organic nitrogen compounds, such good correlation was observed among them that the larger the partition coefficient was, the smaller was the TLm value. Their acute toxicity to fish was supposed to be revealed after their passage through cell membrane of fish to be accumulated in the body. As far as coal-tar dyes and dyestuffs were concerned, there was a correlation between partition coefficient and TLm value. Methylene blue and rose bengale showed strong acute toxicity to fish and it was assumed that high affinity of these dyes to gill was responsible to depressing the function of gill, to make fish suffer from anoxemia and die.

A simple and systematic procedure for determination of 1,2-dibromo-3-chloropropane (DBCP), 1,3-dichloropropene (DCP), chlorobutol (TCB) and seven related compounds in citrus fruits was established. Since these compounds have extremely high volatility, the distillation apparatus for volatile oil determination was used. These compounds were trapped into cyclohexane (more than 94.2-98.4% at spiking levels of 0.5-50 ppm) by this distillation. The distillate was purified by Florisil column chromatography. All compounds except TCB were eluted with n-hexane:ether(93:7) and TCB was eluted with ether. Each eluate was injected into an ECD-gas chromatograph directly for determination. Their recoveries from lemon, orange and grapefruit were within 91.5-98.6% at the spiking levels of 0.01-1 ppm.

The acute toxicity of photodecomposed products of Erythrosine, Eosine, Phloxine and Rose Bengale were studied, since it was found that toxicity of these dyes to fish increased after the dyes had been photoirradiated. Photodecomposed products of the dyes were isolated and identified with UV, IR, NMR spectra and the acute toxicity of those compounds were determined by TLm test. As results of these studies, it became clear that the toxicity of photodecomposed organic products (dehalogenated compounds of dyes) were lower than the mother compounds. The increases in toxicity of the xanthene dyes by photo-irradiation were attributed to the liberated halogens by irradiation.

In Japan artificial colors and pigments can be used for food packages if they are processed so that the artificial color or pigment will not be eluted to contaminate food. Ten samples of cardboard and five samples of wrapping paper were subjected to the elution test. One hundred cm 2 of cardboard or paper was immersed in water at 40 C and held for 30 min. Artificial color was eluted from all samples of whole cardboard and, when it was divided into three parts (surface, inner part and reverse side) to do the elution test separately, artificial color was eluted from all parts except with one sample when color was eluted only from inner part. By comparing R f values on a paper chromatogram as well as its maximal absorbance with color standards, the eluted color was identified to be croceine orange. Congo red and croceine orange were eluted each from one sample of wrapping paper.