Sadao Uchiyama

Simultaneous determination of thiols and disulfides by high-performance liquid chromatography with fluorescence detection

Abstract The proposed simultaneous determination of thiols and disulfides requires 4- (aminosulfonyl)- 7-fluor-2,1,3,-benzoxadiazole (ABD-F) as the pre-column derivatization reagent for thiols and ammonium 7-fluoro-2,1,3,-benzoxadiazole-4-sulfonate (SBD-F) for disulfides followed by chromatography. The thiols and disulfides in solution are treated with ABD-F at 60°C for 5 min in pH 9.3 borate buffer containing 1 nM disodium-EDTA. After removal of excess of ABD-F with ethyl acetate, the remaining disulfides in the aqueous phase are treated with SBD-F at 60°C for 20 min in the presence of tri-n-butylphosphine, a reducing agent. The ABD-thiols and SBD-thiols thus produced are separated by reversed-phase chromatography and detected by fluorimetry (380- nm excitation, 510 - nm emission). SBD-cystein, SBD-homocystein, ABD-homocysteine, ABD-cysteine, SBD-glu- tathione, ABD-homocystein, SBD-N-acetylcystein, ABD-glutatione and ABD-N-acetylcysteine are well separated by linear gradient elution from 0.15 M H3PO4/CH3CN (96:4) to 0.15 M H3PO4/CH3CN (85:15) over 30 min followed by isocratic elution with 0.15 M H3PO4/CH3CN (85:15) fro 10 min. The detection limits for the derivatives are in the range 0.09–0.9 pmol. When the method was applied to the determination of thiols and disulfides in rat tissues, cystein (0.75 μmol g-1) and cystine (0.62 μmol g-1) were obtained in kidney and reduced glutathione (1.4–3.4 μmol g-1) was observed in liver, spleen, heart and testicle.

Fluorescence enhancement in thin-layer chromatography by spraying viscous organic solvents

Abstract Spraying of the fluorescence spot on a thin-layer plate with a non-volatile and viscous organic solvent after development enhanced the fluorescence. The elucidation of the physicochemical factors that affect such an enhancing effect was investigated. The effect was not related to the transmissibility of light through the thin layer but to the properties of the spraying reagent (polarity, viscosity and acidity). The appropriate reagents for 5-dimethylamino-1-naphthalenesulphonamides (DANS-amines) are non-polar, viscous and non-acidic solvents such as a mixture of liquid paraffin and n -hexane, which enhanced the fluorescence internsity 10-fold. With benzo( a )pyrene, the use of a solvent such as this liquid paraffin mixture enhanced the fluorescence intensity 35-fold.

Hydride generation-flame atomic-absorption spectrometry as an arsenic detector for high-performance liquid chromatography.

Hydride generation-flame atomic-absorption spectrometry (HG-FAAS) was used as a continuous detection system for arsenic in the eluate from high-performance liquid chromatography (HPLC). Four arsenic species (arsenite, arsenate, monomethylarsonate and dimethylarsinate) were detected separately with the HPLC-HG-FAAS system equipped with an anion-exchange column. When hijiki (Hizikia fusiforme) extract was examined, arsenate was found predominantly and arsenite and dimethylarsinate were also detected. Liver supernatant fraction obtained from mice administered orally with arsenite was also studied with the HPLC-HG-FAAS system equipped with a gel permeation column. In addition to free or low-molecular-weight ligand-bound arsenic, high-molecular-weight protein-bound arsenic fractions were also detected.

Germination test for identification of gamma-irradiated wheat

Abstract The germination of wheat provides a reliable test for the identification of wheat that has been subjected to irradiation. Germination success of non-irradiated wheat is not affected by storage time and temperature. Root and shoot lengths are more sensitive to γ-irradiation than the germination percentages. The critical dose that inhibits root elongation varies from 0.15 to 0.5 kGy for imported cultivars and from 0.3 to 1.0 kGy for domestic cultivars. Germination percentage is reduced above 5.0 kGy. The difference in radiosensitivity between imported and domestic cultivars may be the result of differences in moisture content. In addition, storage periods of up to 12 months have little effect on irradiation-induced reduction of root length. Fumigation with methyl bromide inhibits root growth, but this inhibition can be distinguished from γ-irradiation effects by the germination pattern. Most imported wheat samples that were irradiated above 0.5 kGy exhibit root growths of less than 20 mm after 4 days of culturing under the specified conditions. The same behavior was noted in domestic wheat samples that were irradiated at or above 1.0 kGy. This test can thus discriminate between irradiated and non-irradiated wheat even 12 months after γ-irradiation.

Determination of Diethylene Glycol in Wine by Use of an Extrelut Column

The determination of diethylene glycol (DEG), added illegally to some imported wines, was investigated. The usage of the Extrelut-20 column, filled up with diatomaceous earth, was useful in eliminating interfering substances and water for analyses of many samples without column damage in gas-liquid chromatography (GLC) and for GC/MS measurement with a capillary column. The detection limit of DEG was 5ppm in wine by this method, involving clean-up and GLC with a 20% PEG 20M column or a wide-bore capillary column coated with 50% phenyl methyl silicon. The method was also useful for analysis of DEG in other beverages such as grape juice.