4-(5,6-Dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl Chloride as a Fluorescent Labeling Reagent for Determination of Amino Acids in High-Performance Liquid Chromatography and Its Application for Determination of Urinary Free Hydroxyproline

✍ Scribed by Yasuto Tsuruta; Hirofumi Inoue

• Publisher: Elsevier Science
• Year: 1998
• Tongue: English
• Weight: 106 KB
• Volume: 265
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1998.2899

✦ Synopsis

A highly sensitive fluorescent labeling reagent, 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride (DMS-Cl), for determination of amino acids by HPLC has been developed. DMS-Cl reacted with amino acids in the basic medium to produce the corresponding fluorescent sulfonamides (excitation and emission wavelength: 318 and 406 nm in aqueous acetonitrile, respectively). When the reactivity of DMS-Cl was investigated by means of reversedphase HPLC using hydroxyproline (Hyp) and alanine (Ala) as model compounds, the reaction of Hyp was completed within 5 min at 25°C and that of Ala within 15 min at 70°C. The efficiency of conversion of Hyp into the fluorescent derivative was about 100%. The detection limits (signal-to-noise ratio ‫؍‬ 3) of almost all amino acids were less than 5 fmol/injection. When the concentration of urinary free Hyp was measured with HPLC using DMS-Cl, the concentration (mean ؎ SD, n ‫؍‬ 10) was 3.8 ؎ 2.76 M (2.7 ؎ 1.71 nmol/mg creatinine).