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 |
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Authors: | Inoue Hirofumi Fukunaga Keiko Munemura Sayaka Tsuruta Yasuto |
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Affiliation: | Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima 729-0292, Japan. |
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Abstract: | 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 degrees 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 degrees 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. |
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Keywords: | Semimicro HPLC Fluorometric detection 4-(5,6-Dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride Polyamine N-Acetylated polyamine Urine |
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