N-羟基琥珀酰亚胺-3-吲哚乙酸酯柱前衍生高效液相色谱分离及荧光测定肽及其水解物

本文首次报道了一种新的羧酸活性酯试剂,N-羟基琥珀酰亚胺-3-吲哚乙酸酯作为柱前荧光衍生试剂,流动相为10mmol/L柠檬酸-Na₂HPO₄－pH 3.6的甲醇/水(20/80,V/V)溶液,C₁₈柱,于λ_cx/λ_cm＝278nm/355nm处进行荧光检测,高效液相色谱分离测定了谷胱甘肽、二甘肽、谷氨酸、胱氨酸、甘氨酸.当S/N＝3时,检出限为0.2至2.5Pmol.

Analysis of Peptides and Amino Acids by Pre-column Derivatization High Performance Liquid Chromatography with Fluorescence Detection

Anew activated ester fluorescence derivatization reagent, N-hydroxysucci-nimidyl-3-indolylacetate(SIIA) is used for analysis of small peptides and their hydrolysates by HPLCand fluorescence detection. In a one-step procedure, SIIAreacts with peptides and amino acids to form stable derivatives. Studies on derivatization conditions demonstrate ex-cellent derivative properties of the reagent. Maximal yields are observed with a molar reagent excess of approximately twenty five or greater and heated at 60℃ for 10 min in pH 8.0 buffer. SIIAderivatives of glutathionide, diglycine, glutamic acid, cystine and glycine are separated on C₁₈, reverse-column with the mobile phase of CH₃OH/H₂O (20/80, V/V)containing 10mmol/LpH 3.6 citric acid-Na₂HPO₄ buffer. They are detected at λ_cx/λ_cm＝278 nm/355 nm with the detection limits ranged from 0. 2 pmol to 2. 5 pmol.