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  • Title: 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.
    Author: Inoue H, Iguchi H, Kono A, Tsuruta Y.
    Journal: J Chromatogr B Biomed Sci Appl; 1999 Mar 19; 724(2):221-30. PubMed ID: 10219662.
    Abstract:
    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.
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