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

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 878; no. 3; pp. 327 - 332
• Main Authors: Tsuruta, Yasuto, Maruyama, Kiyoshi, Inoue, Hirofumi, Kosha, Keiko, Date, Yuuko, Okamura, Nobuyuki, Eto, Seiji, Kojima, Eijiro
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.02.2010; Elsevier
• Subjects: 4-(5,6-Dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride; Adult; Analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Calibration; Carnosine; Carnosine - chemistry; Carnosine - urine; Chromatography, High Pressure Liquid - methods; Female; Fluorescence detection; Fluorescent Dyes - chemistry; Fundamental and applied biological sciences. Psychology; General pharmacology; HPLC; Humans; Hydrolysis; Indicators and Reagents; Limit of Detection; Male; Medical sciences; Pharmacology. Drug treatments; Phthalimides - chemistry; Reference Standards; Temperature; Time Factors; Urine; Young Adult; Urine; 4-(5,6-Dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride; Carnosine; HPLC; Fluorescence detection; Biological fluid; Fluorescence detector; Peptides; HPLC chromatography; Determination; Fluorescent labelling; Chlorides; Dipeptides; Quantitative analysis
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2009.11.038

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 °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 °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).