Simultaneous determination of free and N-acetylated polyamines in urine by semimicro high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labeling reagent

• Published in: Analytical biochemistry Vol. 339; no. 2; pp. 191 - 197
• Main Authors: Inoue, Hirofumi, Fukunaga, Keiko, Munemura, Sayaka, Tsuruta, Yasuto
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.04.2005
• Subjects: 4-(5,6-Dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride; Acetylation; Adult; Aged; Chromatography, High Pressure Liquid - methods; Female; Fluorescent Dyes - analysis; Fluorometric detection; Humans; Male; Microchemistry - methods; Middle Aged; N-Acetylated polyamine; Neoplasms - urine; Phthalimides; Polyamine; Polyamines - urine; Reference Values; Reproducibility of Results; Semimicro HPLC; Sensitivity and Specificity; Urine; Urine; 4-(5,6-Dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride; Polyamine; Semimicro HPLC; Fluorometric detection; N-Acetylated polyamine
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2005.01.008

We have developed a simple and highly sensitive semimicro high-performance liquid chromatographic method for the simultaneous determination of free and N-acetylated polyamines in urine. Polyamines and N-acetylated polyamines were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride to produce fluorescent sulfonamides. The labeling reaction was carried out at 50 °C for 15 min at pH 9. The fluorescent derivatives were separated on a reversed-phase column with a gradient elution using water–acetonitrile–methanol at 50 °C and detected by fluorescence measurement at 318 nm (excitation) and 406 nm (emission). The detection limits (signal-to-noise ratio = 3) of the polyamines and N-acetylated polyamines were 0.7–4.5 fmol/injection. The within-day and day-to-day relative standard deviations were 3.2–7.9 and 3.0–7.7%, respectively. Significant differences were found in the urinary excretion of polyamines between cancer patients and normal subjects.