Yuuko Date

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

Determination of free hydroxyproline and proline in human serum by high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)phenylsulfonyl chloride as a pre-column fluorescent labelling reagent

A fluorescent labelling reagent, 4-(5,6-dimethoxy-2-phthalimidinyl)phenylsulfonyl chloride, was designed for the determination of amines by precolumn HPLC and was applied to the simultaneous determination of hydroxyproline and proline in serum. The reagent reacted with hydroxyproline and proline at 30 degrees C for 10 min to produce the fluorescent derivatives, which were separated on a reversed-phase column by gradient elution with phosphate buffer (1 mmol l-1, pH 7) and acetonitrile and detected by fluorescence measurement at 315 nm (excitation) and 385 nm (emission). The detection limits (signal-to-noise ratio = 3) for both hydroxyproline and proline were 10 fmol per injection. The within-day (n = 10) and day-to-day (n = 5) relative standard deviations using human sera were less than 2.16% and 2.75%, respectively, for hydroxyproline and less than 2.30% and 3.25%, respectively, for proline. The concentrations of free hydroxyproline and proline in normal human sera (n = 13) were 5.6-18.0 and 137.6-252.6 mumol l-1, respectively. The proposed method was also applied to the determination of hydroxyproline and proline in sera from patients with chronic renal failure. The mean concentrations of hydroxyproline and proline in chronic renal failure were about 2.6 and 1.6 times higher, respectively, than those in normal human sera.

Sensitive determination of carnosine in urine by high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labeling reagent

A simple and highly sensitive high-performance liquid chromatography procedure was developed for the determination of carnosine in urine. Carnosine was derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70 degrees C for 15 min in borate buffer (20 mmol l(-1), pH 9.0) to produce fluorescent sulfonamides. After hydrolysis of the reaction mixture with formic acid at 100 degrees C for 15 min, the fluorescent derivative of carnosine was separated on a reversed-phase column with a linear gradient elution using solvents of (A) acetate buffer (0.1 mmol l(-1), pH 7.0) and (B) acetonitrile at a flow-rate of 1.0 ml/min and was detected at excitation and emission wavelengths of 318 and 400 nm, respectively. The detection limit of carnosine was 4 fmol at a signal-to-noise ratio of 3. The within-day and day-to-day relative standard deviations were 2.7-4.6% and 0.4-5.2%, respectively. The concentration of carnosine in normal human urine was found to be 4.6-125 nmol (mg creatinine)(-1) (mean+/-SD: 21.6+/-26.6 nmol (mg creatinine)(-1), n=20).

Determination of malondialdehyde by high-performance liquid chromatography using 4-(2-phthalimidyl)benzohydrazide as a pre-column fluorescent labelling reagent

A high-performance liquid chromatographic method was developed for the determination of malondialdehyde after conversion into 1-[4-(2-phthalimidyl)-benzoyl]pyrazole with the fluorescent labelling reagent 4-(2-phthalimidyl)benzohydrazide. The labelling reaction was carried out at 50 °C for 60 min in the presence of phosphoric acid. The extent of conversion of malondialdehyde into the fluorescent derivative was approximately 100%. The fluorescent derivative was separated on an ODP-50 column with elution using 40% aqueous acetonitrile and detected by fluorescence at 320 nm (excitation) and 385 nm (emission). The detection limit (signal-to-noise ratio = 3) was 8 fmol per injection (20 µl). The relative standard deviations for within-day and day-to-day precision were 3.18 and 3.53%(0.5 µmol l–1, n= 8), respectively. The method was applied to the determination of malondialdehyde generated from isolated rat hepatocytes stimulated with tert-butyl hydroperoxide.

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.

Determination of paroxetine in serum treated with simple pretreatment by pre-column high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labeling reagent

The therapeutic drug monitoring of paroxetine could be used to optimize the pharmacological treatment of depressed patients. A simple and sensitive high-performance liquid chromatography procedure was developed for the determination of paroxetine in serum. After simple pretreatment of serum (50 μL) with acetonitrile and o-phthalaldehyde, paroxetine was derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70°C for 20 min in borate buffer (0.1 mol/L, pH 8.0) to produce a fluorescent product. The derivative was separated on a reversed-phase column at 40°C for stepwise elution with (A) acetic acid (10 mmol/L) and (B) acetonitrile. The flow rate was 1.0 mL/min. The fluorescence intensity was monitored at excitation and emission wavelengths of 320 and 400 nm, respectively. The within-day and day-to-day relative standard deviations were 3.0-3.4 and 2.7-8.3%, respectively. The detection limit of paroxetine was 8.3 fmol at a signal-to-noise ratio of 3. As the proposed method that only requires a small quantity of serum (50 μL) is simple, sensitive and reproducible, it would be useful for clinical and biochemical research as well as drug monitoring.