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-CI), for determination of amino acids by HPLC has been developed. DMS-C1 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-CI 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 DIMS-CI, the concentration (mean +/- SD, n = 10) was 3.8 +/- 2.76 mu M (2.7 +/- 1.71 nmol/mg creatinine). (C) 1998 Academic Press.