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.
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 reversed-phase HPLC using hydroxyproline (Hyp) and alanine (Ala) as model compounds, the reaction of Hyp was completed within 5 min at 25 degrees C and that of Ala within 15 min at 70 degrees 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 microM (2.7 +/- 1.71 nmol/mg creatinine).
Tsuruta
Y
Y
Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Hiroshima, Japan. tsuruta@fupharm.fukuyama-u.ac.jp
Inoue
H
H
D000596:Amino Acids / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D006909:Hydroxyproline / Q000652:urine*; D010797:Phthalimides / Q000737:chemistry*; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
C117624:4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride; D000596:Amino Acids; D005456:Fluorescent Dyes; D010797:Phthalimides; D006909:Hydroxyproline