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

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• 作者：Yasuto Tsuruta ; Kiyoshi Maruyama ; Hirofumi Inoue ; Keiko Kosha ; Yuuko Date ; Nobuyuki Okamura ; Seiji Eto ; Eijiro Kojima
• 关键词：Carnosine ; Urine ; Fluorescence detection ; 4-(5 ; 6-Dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride ; HPLC
• 刊名：Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences
• 出版年：2010
• 出版时间：1 February 2010
• 年：2010
• 卷：878
• 期：3-4
• 页码：327-332
• 全文大小：553 K

文摘

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).