Toshiaki Shinohara

Determination of blood cyanide by headspace gas chromatography with nitrogen-phosphorus detection and using a megabore capillary column

Abstract A method for the determination of cyanide in blood using head-space capillary gas chromatography (HS-GC) with nitrogen-phosphorus detection (NPD) was developed. The separation efficiency and sensitivity for hydrogen cyanide (HCN) were improved by using a GS-Q megabore capillary column and by injection of 500 μl of HS gas with a splitting ratio of 5. No interference or column deterioration was observed. Optimum HS conditions were found to be incubation of 50°C for 30 min in the presence of 10% phosphoric acid as an acidifying reagent. The distribution coefficient ( k ) was 75.7 and 158.9 for HCN acetonitrile (CH 3 CN; candidate for internal standard), respectively. HCN showed a different vaporization behaviour to CH 3 CN with respect to temperature and matrix effect. The salting-out effect was relatively small for HCN and CH 3 CN. An increase in the blood content in the liquid phase resulted in a significant increase in k for HCN. The calibration graph was linear over a wide concentration range of cyanide, and the limit of determination was 1 ng ml −1 (R.S.D. 10%). Because a considerable amount of CH 3 CN was detected in control blood, the direct calibration method could be used instead of the internal standard method using CH 3 CN. The detectable amount of cyanide was significantly decreased during the preincubation of cyanide and blood at 2°C. Thiocyanate in blood showed a positive interference. Contents determined by this HS-GC method agreed closely with those obtained by the microdiffusion-spectrophotometric method for blood samples from fire victims.

Structure-activity relationship of reversible cholinesterase inhibitors including paraquat

The inhibitory effect of paraquat on cholinesterase activity was investigated in comparison with four paraquat derivatives, six monoquaternary ammoniums and six anticholinergic drugs. Inhibitor concentrations to cause 50% inhibition (I50) and Hill coefficients for three enzymes, human erythrocyte acetylcholinesterase (AChE), Electrophorus electricus AChE and human plasma butyrylcholinesterase (BuChE) were measured. The results obtained were as follows. The I50 for erythrocyte AChE was similar to the I50 for eel AChE. Secondary to edrophonium, diethylparaquat, paraquat, morfamquat and monoquat showed lower I50 for AChE, and possessed higher inhibition selectivity (IS), expressed as the ratio of I50 for BuChE to I50 for erythrocyte AChE. However, diquat showed higher I50 for AChE and lower IS, similar to the other monoquaternary ammoniums. A negative correlation was observed between log [I50 for erythrocyte AChE] and log [IS], among paraquat and its derivatives, monoquaternary ammoniums and anticholinergic drugs, respectively. With respect to Hill coefficients, these inhibitors could be classified into four groups, [1] competitive inhibitors: diquat, edrophonium, choline, tetramethylammonium and trimethylphenylammonium, [2] inhibitors showing negative cooperativity: paraquat, diethylparaquat, morfamquat, d- tubocurarine, atropine, gallamine and nicotine, [3] moderate type inhibitors: monoquat, hexamethonium and decamethonium. [4] the other type inhibitors showing positive cooperativity for erythrocyte AChE: tetraethylammonium and ethyltrimethylammonium.

Size fractionation of oligosaccharides by liquid chromatography on a cation-exchange column

Oligosaccharides, labelled with 2-aminopyridine at their reducing ends, were satisfactorily fractionated according to the sugar sizes on Shodex RSpak DC-613, a cation-exchange resin column (Na+ form), with water-acetonitrile as the eluent in the presence of sodium acetate or triethylammonium acetate buffer. For the fractionation of sugar samples, dextran hydrolyzates, chitin oligomers and oligosaccharide moieties of ovomucoid were used. The oligosaccharides were strongly adsorbed to the resin column with solvents containing less water and at lower temperature, and were eluted in order of increasing molecular size above the critical concentration of acetonitrile. Baseline separation of a dextran hydrolyzate up to oligomers having 20 glucose units was observed by gradient elution. The separation efficiency and elution pattern were investigated by changing the buffer concentration, mobile phase pH and temperature.

SOME IMMUNOCHEMICAL PROPERTIES OF Lea -AND Leb-ACTIVE SUBSTANCES IN HUMAN URINE

Lea‐ and Leb‐active substances present in normal human urine were isolated by sequential membrane filtration, column chromatography with DEAE‐Sephadex (A‐25), and gel filtration of Bio‐Gel (P‐6). Lea‐active substance with the highest activity was obtained in the P‐1 fraction (0.8 mg/1) from urine of a non‐secretor type by the final gel filtration. Leb‐active substance with the highest activity was also obtained in the P‐1 fraction (0.2 mg/1) from urine of a secretor type. The P‐1 of the non‐secretor inhibited Lea‐anti‐Lea haemagglutination minimally at 4 μg/ml, and the P‐1 of the secretor inhibited Leb‐anti‐Leb haemagglutination at 1 μg/ml. Both P‐1 fractions possessing Lea or Leb activity were characteristic in having a much higher fucose content than other isolated fractions, and seemed to be complex carbohydrates with molecular weights of the order of 4–5 × 103.

Chromogen Formation and Epimerization of N-Acetylhexosamines on Paper impregnated with Potassium Tetraborate

Kuhn and Krüger1 have shown that under mild alkaline conditions, N-acetylglucosamine is partly converted into three chromogens which react directly with an acid p-dimethylaminobenzaldehyde reagent to produce violet colours. Roseman and Comb2, and Kuhn and Brossmer3, have reported that under alkaline conditions, N-acetylglucosamine and N-acetylmannosamine are converted into each other by epimerization. Fujii and Kushida4 have recently reported that N-acetylgalactosamine is epimerized to N-acetyltalosamine under mild alkaline conditions. In a previous paper5 from this laboratory, it was demonstrated that N-acetylhexosamines can be specifically detected on paper chromatograms by the use of the potassium tetraborate and p-dimethylaminobenzaldehyde spray reagents. This communication describes chromogen formation and epimerization of the N-acetylhexosamines on paper impregnated with potassium tetraborate.