Shoji Takitani

Spectrodensitometric determination of trichothecene mycotoxins with 4-(p-nitrobenzyl)pyridine on silica gel thin-layer chromatograms

A simple method for the detection and spectrodensitometric determination of a number of trichothecene mycotoxins on silica gel layers based on a colour reaction between 4-(p-nitrobenzyl)pyridine and the 12,13-epoxy group in the trichothecene nucleus is described. The detection limits for the twelve trichothecenes examined were 0.025--0.2 micrograms per spot. Further, six of the twelve trichothecenes could be determined spectrodensitometrically in the range from ca. 0.05--0.2 to 10 micrograms per spot with a coefficient of variation of ca. 5%.

High-performance liquid chromatographic method for determining trichothecene mycotoxins by post-column fluorescence derivatization

The method described here is based on a separation of deoxynivalenol, nivalenol and fusarenon-X on a C18 column using aqueous acetonitrile, and successive post-column fluorescence derivatization involving an alkaline decomposition to form formaldehyde and modified Hantzsch reaction with methyl acetoacetate and ammonium acetate (lambda ex = 370 nm and lambda em = 460 nm). By this method, 5-10 ng of the standard trichothecenes could be determined. By employing a clean-up procedure with a florisil column and a Sep-Pak CN cartridge, 61.4-96.9% recoveries were obtained for deoxynivalenol and nivalenol added to corn, wheat and barley at concentration levels of 0.05-1 ppm.

High-performance liquid chromatographic determination of cyanide in human red blood cells by pre-column fluorescence derivatization.

A method for the determination of cyanide in human red cells has been developed. Cyanide was extracted from red cells by adding water and methanol, and then derivatized with 2,3-naphthalene-dialdehyde and taurine to give a fluorescent product, which was determined by reversed-phase high-performance liquid chromatography with fluorescence detection. The recovery of cyanide from red cells was ca. 83%, and the limit of detection was 100 pmol/ml. The mean concentrations of red cell cyanide from ten smokers and from ten non-smokers were 705 and 466 pmol/ml, respectively. The method was also applicable to whole blood.

Fluorodensitometric determination of trichothecene mycotoxins with nicotinamide and 2-acetylpyridine on a silica gel layer

Abstract A fluorodensitometric method for the determination of trichothecene mycotoxins on silica gel thin-layer plates based on a fluorescence reaction of the epoxy group with nicotinamide and 2-acetylpyridine is described. The limits of detection for the five trichothecenes examined were 20–50 ng per spot with visual inspection under UV light (360 nm). Further, these trichothecenes could be determined fluorodensitometrically in the range from ca. 10–25 ng per spot to 1500 ng per spot with a coefficient of variation of about 10%.

Spectrofluorimetric determination of cyanide in blodd and urine with naphthalene-2,3-dialdehyde and taurine

Abstract Cyanide is separated from biological bluids by microdiffusion using 1 M acetate buffer (pH 5.2) as an acidifying agent, followed by the fluorimetric determination of the cyanide with naphthalene-2,3-dialdehyde and taurine. The recovery and limit of detection of cyanide in blood and urine are about 70–80% and 0.03 nmol ml−1, respectively. The proposed method for cyanide was successfully adapted for application to blood and urine from smokers and non-smokers.

Fluorometric determination of cyanide with 2,3-naphthalenedialdehyde and taurine

A method is proposed for the fluorometric determination of cyanide based on a fluorogenic reaction with 2,3-naphthalenedialdehyde and taurine at basic pH. As little as 10 pmole of cyanide in 500 mul of sample can be determined.

Formation of a diazoquinone-type mutagen from acetaminophen treated with nitrite under acidic conditions

Acetaminophen (paracetamol) showed mutagenicity to Salmonella typhimurium TA100 and TA98 either with or without S9 mix when treated with excess nitrite in acidic solution. The mutagen was isolated and identified as 4-acetylamino-6-diazo-2,4-cyclohexadienone. Mutation tests and product analysis by high-performance liquid chromatography, however, suggested that this type of mutagen was hardly formed in the digestive tract of normal subjects.

High-performance liquid chromatography of the anti-tumour agent triethylenethiophosphoramide and its metabolite triethylenephosphoramide with sodium sulphide, taurine and o-phthalaldehyde as pre-column fluorescent derivatization reagents

The method described is based on the reaction of triethylenethiophosphoramide (ThioTEPA) and triethylenephosphoramide (TEPA), through their ethyleneimine groups, with sodium sulphide, taurine and o-phthalaldehyde to give fluorescent products, and separation of the derivatives by reversed-phase high-performance liquid chromatography. The method was successfully applied to the determination of ThioTEPA and TEPA in rabbit plasma samples after clean-up with an Extrelut 3 column. The recoveries of ThioTEPA and TEPA from plasma were 66.1-80.3% and the limits of determination in plasma were ca. 10 and 20 ng/ml, respectively.

Application of an anhydrotrypsin-immobilized precolumn for selectvie separation of peptides having arginine or lysine at theinr C-termini by column-switching high-performance liquid

A column-switching high-performance liquid chromatographic (CS-HPLC) system which consisted of an anhydrotrypsin (AHT)-immobilized diol—silica precolumn and a reversed-phase analytical column was developed for the selective separation of peptides having Arg or Lys at their C-termini. Tuftsin (Thr-Lys-Pro-Arg) could be enriched almost quantitatively on the precolumn when loadd with water as a carrier solvent and the precolum was washed with 10–30 mM acetate buffer (pH 5.0). An investigation of the affinity characteristics of 55 peptides to the AHT precolumn showed that among twelve peptides having Arg or ArgNH2 at their C-termini and more than four amino acid residues, ten were retained almost quantitatively on the precolumn, and eight out of nine peptides having Lys at their C-termini were less retained. The peptide having d-Arg at its C-termini was not retained. However, twelve out of thirty peptides having no Arg or Lys at their C-termini were also retained, but the retention was greatly decreased, in contrast to the Arg peptides, when the precolumn was washed with 20mM calcium chloride solution. The results indicate that the CS-HPLC system equipped with an AHT precolumn offers new selectivity in the HPLC separation of peptides.

Preparation of anhydrotrypsin-immobilized diol silica as a selective adsorbent for high-performance affinity chromatography of peptides containing arginine or lysine at their C-termini

SummaryAnhydrotrypsin (AHT), a catalytically inert derivative of trypsin in which the active site serine residue was converted to dehydroalanine residue by chemical modification, was immobilized onto diol silica through the activation with trifluoroethanesulfonyl chloride, and an AHT-diol-silica column was used for high-performance affinity chromatography separation of peptides containing arginine or lysine at their C-termini from the others. Improved separation in terms of speed was accomplished.