Fluorometric method with 1, 2-naphthoquinone-4-sulfonic acid for the determination of cyclohexylamine, a hydrolysation product of cyclamate, was investigated. Optimal conditions for the determination was as follows : A sample solution of cyclohexylamine and 1, 2-naphthoquinone-4-sulfonic acid was heated at 50°for 10 min in the presence of borate buffer (pH 10.3). The reaction solution was shaken with dichloromethane, and the organic layer was dried over anhyd. sodium sulfate and then treated with sodium borohydride. Ethanolic sulfuric acid solution was added to the reduced solution. The developed fluorescence was determined at 397 nm, exciting at 345 nm. This fluorometric method was applied to the determination of cyclohexylamine in the hydrolytic solution of cyclamate by the reported procedure of Johnson, et al., and a satisfactory result was obtained. In this case, coexistence of food additive did not interfere with the determination. Coloured substance, 4-cyclohexylamino-1, 2-naphthoquinone (I) was obtained from the reaction solution of cyclohexylamine and 1, 2-naphthoquinone-4-sulfonic acid. Intense violet fluorescence was observed in the dichloromethane solution of I by reduction with sodium borohydride and then addition of ethanolic sulfuric acid solution.

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Fluorometric determination of cyclohexylamine with 1, 2-naphthoquinone-4-sulfonic acid was applied to the determination of cyclamate in various foods. Cyclamate was hydrolyzed to cyclohexylamine as follows : To 1 ml of a sample solution containing 50-500μg of cyclamate, 0.5 ml of 0.5N sulfuric acid solution and 3 ml of dimethyl sulfoxide were added, heated for 2 hr in a boiling water bath, cooled, and diluted to 25 ml with water. Cyclohexylamine in the solution was estimated by the procedure described in the preceding paper. In the recovery test of cyclamate in various foods, the recovery was satisfactory. Determination of cyclohexylamine in human urine by the fluorometric method with 1, 2-naphthoquinone-4-sulfonic acid was also investigated. Cyclohexylamine was extracted with chloroform from urine basified with 2N sodium hydroxide. The reaction solution of cyclohexylamine and 1, 2-naphthoquinone-4-sulfonic acid was shaken with dichloromethane. The concentrated dichloromethane layer was spotted on a silicagel plate and developed with a mixture of ether : chloroform (1 : 2). Extract of the orange-yellow spot with borate buffer (pH 10.3) was treated with sodium borohydride, and acidified with 0.2 N sulfuric acid solution. The developed fluorescence was estimated. The recovery ratio was 94.3%.

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Lead nitrate (80mg/kg Pb), lead stearate (80mg/kg Pb), and tetraethyl lead (0.3mg/kg Pb) were given to 3 separate groups of rats by a single oral administration. Difference in the distribution and excretion of lead in rats among these 3 kinds of lead compounds was examined. Atomic absorption spectrophotometry was used for the determination of lead in biological materials. After administration of lead nitrate, 92% of lead administered was excreted in the feces within 3 days. The highest distribution of lead was found in the liver (0.169±0.030%), followed by the kidney (0.153±0.018%), leg bone (0.078±0.015%), blood (0.052±0.015%), and rib (0.023±0.006%) at 7 hr after the administration. Lead distributed in the liver, kidney, and blood decreased rapidly, while that in the leg bone and rib increased up to 2 days after the administration, and then decreased slowly. After administration of lead stearate, distribution and excretion of lead were similar to those of lead nitrate. In the case of tetraethyl lead, 62% of lead was excreted in the feces during 8 days. The highest lead distribution was found in the liver throughout the experiment (27.1±3.5% after 7 hr and 12.5±3.4% after 32 days). Lead distributed in the blood, kidney, brain, leg bone, and rib increased up to 3 or 4 days after the administration. On the 32 nd day, lead in the blood and rib was almost 0%, but lead was found in the kidney (1.29±0.39%), brain (0.75±0.36%), and leg bone (3.13±1.35%).

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The study was carried out to obtain the fundamental data about biological action of selenium in order to establish the standard of water quality for supply water. Following the previous examination on the body turnover, time-excretion, and distribution of ⁷⁵Se-labeled sodium selenate after a single oral administration to rats, acute and subacute toxicity of sodium selenate were examined in Wistar rats. Acute oral LD₅₀ value of sodium selenate for female rats was 31.5 mg/kg body weight, and its 95% confidence limit was 30.08-37.46 mg/kg body weight. In acute toxicity tests of sodium selenate, these rats showed decreased spontaneous movement, bristle and violent respiration, and had diarrhea, and then died of respiratory embarrassment with coordinate convulsion. The subacute toxicity of sodium selenate was examined by continuous oral administration of 1 and 5 mg/kg/day for 30 days in male and female rats. Subacute symptoms included a decrease in growth rate, inhibition of food intake, and fecal excretion. The outstanding pathological lesions were enlargement of spleen and kidney, edema of pancrease, degeneration of liver cells, bleeding foci in the liver, and a subacute yellow liver atrophy, and these appeared more evidently in female rats than in males.

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Spectrophotometric determination of ultramicro amount of vanadium based on its catalytic effect in the oxidation of gallic acid has an advantage in rapidness and sensitivity. However, the catalytic method is seriously interfered by the coexistence of trace amounts of copper and iron ions in the sample, and the applicability of the method has been restricted. Therefore, addition of masking agents to improve the method was investigated, and addition of 1 ml of 0.1 M cyclohexanediaminetetraacetic acid (CDTA) solution to the sample almost eliminated the interference by copper and iron ions. In the proposed method, ppb levels of vanadium can be determined in the presence of diverse ions such as copperiron, lead, zinc, nickel, cadmium, chromium, sulfate, chloride, nitrate, etc., at their ppm concentrations. Application of the proposed method was tried for the analysis of vanadium in the water samples from river, swamp, underground water, and hot spring, and the concentrations of vanadium were found in the range of 0.1 to 10 ppb, and the recovery of vanadium was within 90 to 120%.

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In order to know the background concentration of mercury in biological samples, a quantitative method was established by using the technique of mercury-copper amalgam distillation for thermal neutron activation analysis. One of the advantages for this technique is the use of "copper broom" which was made by bundling 50 pieces of copper wire (0.2×50 mm). The other is that mercury was distilled and recovered by heating the Hg-Cu broom in a Pyrex glass tube (5 mm in φ, 7 mm ex φ, 30 cml, one of the ends being closed). It was possible to recover quantitatively 40 mg of carrier mercury in 10 ml of a sample solution with 3-4 bundles of Cu broom. It was found that this method was available for a sample of low mercury concentration. As a result of application to a human venous blood, the value of 23.2 ppb of mercury was obtained by using 0.5 ml of the blood.

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