Two fluorescence labeling reagents, 3-and 4-(2-phthalimidyl)benzoyl azide, were designed for primary and secondary alcohols for high performance liquid chromatography.These reagents, prepared from orthophthalaldehyde and aminobenzoic acid in several steps, react with alcohols at 125°C for 30 min to produce the fluorescent derivatives, which can be separated on a reversed-phase column ERC-ODS-1161 with acetonitrile/ water (70 : 30) or acetonitrile/ ethanol (70 : 30) as eluent. The extent of conversion of cholesterol into fluorescent derivative was 100%. The detection limit for the alcohols labeled with 3-(2-phthalimidyl)benzoyl azide was 0.1-0.4 pmol per injection (10 µl). 3-(2-Phthalimidyl)-benzoyl azide was applied for the determination of cholesterol in human serum by high performance liquid chromatography with fluorescence detection. The values of cholesterol in normal human sera were 4.8 -9.4 µmol/ ml.
KeywordsHigh performance liquid chromatography, fluorescence detection, fluorescence labeling reagent, alcohol, cholesterol Acyl azides are well known to react with hydroxyl compounds by heating to form the corresponding carbamic acid esters.' Reagents such as 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxo-quinoxaline-2-carbonyl azide2 and 7-methoxycoumarin-3-or 4-carbonyl azide3 were used as labeling reagents for hydroxyl compounds in high performance liquid chromatography (HPLC). Previously, considerable attention was given to phenylphthalimidines having highly fluorescent properties.4 Some fluorescence labeling reagents having the phenylphthalimidine moiety as a fluorophore were developed for amino compounds5,6 and for thiol compounds.'In the present research, we designed 3-and 4-(2-phthalimidyl)benzoyl azide (abbreviated, m-and p-Phibyl-N3, respectively) as fluorescence labeling reagents for alcohols. In order to investigate their reactivities and sensitivities, we used octanol, nonanol, decanol, l-menthol and cholesterol as model compounds and we applied mPhibyl-N3 for the determination of cholesterol in a small amount of human serum by HPLC with fluorescence detection.
Experimental Chemicals and solvents
All chemicalswere of analytical-reagent grade, unless stated otherwise. Deionized-distilled water was used.Organic solvents were purified by distillation prior to use.Synthesis of m-Phibyl-N3 m-Aminobenzoic acid (0.68 g) in diethyl ether (50 ml) and o-phthalaldehyde (0.67 g) in diethyl ether (100 ml) were mixed and stirred at room temperature for 2 days. 3-(2-Phthalimidyl)benzoic acid separated as a white precipitate and was filtered off. After a solution of 3-(2-phthalimidyl)benzoic acid (0.5 g) in dimethylformamide (15 ml) and diphenylphosphoryl azide (0.56 g) in dimethylformamide (4 ml) was mixed in the presence of triethylamine (0.2 g) at 0 ° C for 2 h, 5% sodium bicarbonate aqueous solution (60 ml) was added. The resulting mixture was shaken with diethyl ether (100 ml), and the aqueous layer was discarded. When the organic layer was washed with cold water (100 ml, twice), some precipitates floating around...