Kazuya Kohashi

Sensitive derivatization reagents for hydroxyl and amino compounds for thin-layer or high-performance liquid chromatography with fluorescence detection

Abstract Three fluorescent derivatization reagents for compounds having hydroxyl and/or amino groups are described. 4-(2-Phthalimidyl)benzoyl chloride, 3-(2-phthalimidyl)benzoyl chloride and 3-(2-phthalimidyl)-4-methoxybenzoyl chloride, prepared from the corresponding phthalimidylbenzoic acid, were stable at room temperature and condensed quantitatively with alcohols, amines and amino acids in the presence of alkali under mild conditions to give strongly fluorescent derivatives. The derivatives were separated by thin-layer and high-performance liquid chromatography.

Phthalimidylbenzenesulphonyl chlorides as fluorescence labelling reagents for amino acids in high-performance liquid chromatography

This paper deals with the preparations of 4-(N-phthalimidyl) benzenesulphonyl chloride and 2-methoxy-5(N-phthalimidyl) benzenesulphonyl chloride as fluorescent derivatization reagents for amines and amino acids and their reactivities towards amino compounds using thin-layer chromatography (TLC) and HPLC

Simultaneous determination of serum and urinary hydroxyproline and proline by liquid chromatography using two fluorescent labeling reagents

A method for the simultaneous determination of free serum and total urinary hydroxyproline (Hyp) and proline (Pro) concentrations by liquid chromatography (LC) using two fluorescent labeling reagents was developed. Serum Hyp and Pro, after treatment with o-phthalaldehyde, were derivatized with 4-(2-phthalimidinyl)phenylsulfonyl chloride (Phisyl-Cl). Imino acids in hydrolysed urine were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)phenylsulfonyl chloride (DPS-Cl) after treatment with o-phthalaldehyde and cleanup on a Bond Elut C 18 column. The two reaction mixtures were combined and mixed with dichloromethane to extract the excess of the reagents, and then the aqueous layer (10 pl) was subjected to LC. The Phisyl and DPS derivatives of imino acids were separated on a reversed-phase column by gradient elution with phosphate buffer (5 mM, pH 2.5) and acetonitrile at 35°C and detected by fluorescence measurement at 300 nm (excitation) and 400 nm (emission). The detection limits (s:n=3) for Hyp and Pro were 30 and 40 fmol/injection, respectively, for Phisyl derivatives and 5 fmol/injection for both DPS derivatives. The within-day and day-to-day relative standard deviations for Hyp and Pro in serum and urine were <2.8%. The recoveries of Hyp and Pro added to serum and urine were 97.6-103.6%.

Simultaneous determination of cholesterol and cholestanol in human serum by high-performance liquid chromatography using 3-(5,6-methylenedioxy-2-phthalimidyl)benzoyl azide as precolumn fluorescent labelling reagent

A fluorescent labelling reagent, 3-(5,6-methylenedioxy-2-phthalimidyl) benzoyl azide, designed for the determination of alcohols by precolumn high-performance liquid chromatography, has been applied to the simultaneous determination of cholesterol and cholestanol in human serum. The reagent reacts with cholesterol and cholestanol at 140 degrees C for 10 min to produce the fluorescent derivatives, which can be separated on a reversed-phase column with acetonitrile-ethanol-water (60:35:7.5, v/v) as eluent. The detection limits for cholesterol and cholestanol were 45 and 50 fmol per injection (20 microliters), respectively. The values of cholesterol and cholestanol in normal human sera were 135-212 mg/dl and 137-928 micrograms/dl, respectively.

Simultaneous determination of imidazoleacetic acid and nτ-and nπ-methylimidazoleacetic acids in human urine by high-performance liquid chromatography with fluorescence detection

A sensitive method for the simultaneous determination of urinary imidazoleacetic acid and N tau- and N pi-methylimidazoleacetic acids which employs high-performance liquid chromatography with fluorescence detection is described. The compounds were converted into the corresponding fluorescent esters by reaction with 4-bromomethyl-7-methoxycoumarin. These derivatives are separated by liquid chromatography on a Radial-Pak silica column. The detection limits for imidazoleacetic acid and N tau-and N pi-methylimidazoleacetic acids in urine are 15, 10 and 20 pmol/ml, respectively. The 24-h urinary excretion of imidazoleacetic acid and N tau-and N N pi-methylimidazoleacetic acids by healthy persons was 5.7-39.9, 4.3-24.6 and 1.5-19.3 nmol/mg of creatinine, respectively.

Kinetics and Mechanism of Zinc Ion-Mediated Degradation of Cephalosporins in Tromethamine Solution

Earlier studies of the hydrolysis and aminolysis of penicillin, in the presence of zinc ion and tromethamine (Tris), revealed a very rapid catalysis mediated by a ternary complex in which the metal ion brought the reactants into close proximity in a suitable configuration for reaction. In the present work similar studies with a group of cephalosporins show not only much slower rates of reaction but a different mechanism in which the zinc ion–tromethamine complex functions as a nucleophile in a bimolecular reaction. Evidence for the differences in mechanism includes not only the different dependence of rate upon tromethamine concentration, but comparable rates of reaction of methyl esters of a penicillin and a cephalosporin and the reaction products observed by high-performance liquid chromatography.