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).
Tsuruta
Yasuto
Y
Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima 729-0292, Japan. tsuruta@fupharm.fukuyama-u.ac.jp
Maruyama
Kiyoshi
K
Inoue
Hirofumi
H
Kosha
Keiko
K
Date
Yuuko
Y
Okamura
Nobuyuki
N
Eto
Seiji
S
Kojima
Eijiro
E
D000328:Adult; D002138:Calibration; D002336:Carnosine / Q000737:chemistry / Q000652:urine*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D007202:Indicators and Reagents; D057230:Limit of Detection; D008297:Male; D010797:Phthalimides / Q000737:chemistry*; D012015:Reference Standards; D013696:Temperature; D013997:Time Factors; D055815:Young Adult
C117624:4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D010797:Phthalimides; D002336:Carnosine