Highly sensitive determination of N-terminal prolyl dipeptides, proline and hydroxyproline in urine by high-performance liquid chromatography using a new fluorescent labelling reagent, 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride.
A highly sensitive pre-column HPLC method for simultaneous determination of prolyl dipeptides, Pro and Hyp in urine was developed. The analytes were labelled with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70 degrees C for 20 min. The derivatives separated on tandem reversed-phase columns by a gradient elution and were monitored with fluorescence detection at 318 nm (excitation) and 392 nm (emission). The detection limits for prolyl dipeptides, Pro and Hyp were 1-5 fmol/injection (S/N = 3). Urine samples were treated with o-phthalaldehyde, followed by purification on a Bond Elut C18 column before conducting the labelling reaction. Pro-Hyp, Pro-Gly and Pro-Pro were identified as prolyl dipeptides in urine. The within-day and between-day relative standard deviations were 1.5-4.8 and 1.7-5.8%, respectively. The concentrations of Pro-Hyp, Pro-Gly, Pro-Pro, Pro and Hyp in normal human urine were 97.6 +/- 28.2, 2.74 +/- 1.48, 2.08 +/- 1.13, 6.71 +/- 3.34 and 2.30 +/- 1.59 nmol/mg creatinine, respectively.
Inoue
H
H
Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Hiroshima, Japan.
Iguchi
H
H
Kono
A
A
Tsuruta
Y
Y
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004151:Dipeptides / Q000652:urine*; D005456:Fluorescent Dyes; D006801:Humans; D006909:Hydroxyproline / Q000652:urine*; D010797:Phthalimides; D011392:Proline / Q000652:urine*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
C117624:4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride; D004151:Dipeptides; D005456:Fluorescent Dyes; D010797:Phthalimides; D011392:Proline; D006909:Hydroxyproline