高效液相色谱法_

N-羟基琥珀酰亚胺-荧光素-O-乙酸酯柱前衍生HPLC分离荧光检测肽及其水解产物

http://www.100md.com 《广东药学院学报》 2002年第4期

高效液相色谱法,,高效液相色谱法；N-羟基琥珀酰亚胺-荧光素-O-乙酸酯；荧光衍生试剂；肽,1实验部分,2结果与

     摘要探索了N-羟基琥珀酰亚胺-荧光素-O-乙酸酯(SIFA)与氧化型谷胱甘肽(GSSG)、三甘肽(Gly-Gly-Gly)、二甘肽(Gly-Gly)和组成它们的氨基酸-谷氨酸(Glu)、胱氨酸(Cys2)、甘氨酸(Gly)反应的衍生条件以及其衍生物的色谱分离条件。于含pH8.5的0.2 mmol/L H3BO3-Na2B4O7缓冲溶液中衍生25 min，衍生反应液直接进样，采用C18柱，以含42%甲醇的pH5.0 10 mmol/L H3cit-Na2HPO4缓冲溶液为流动相，在λex/λem＝492/513 nm处荧光检测，分离测定了六种肽及其组成氨基酸的衍生物，检出限为0.64～12 fmol。该方法具有流动相体系简单、灵敏度更高等优点。

    关键词高效液相色谱法；N-羟基琥珀酰亚胺-荧光素-O-乙酸酯；荧光衍生试剂；肽

    N-hydroxysuccinimidyl fluorescein-O-acetate as a fluorescent

    derivatizing reagent for oligopeptides and their hydrolysates

    Abstract Some oligopeptides and their hydrolysates such as GSSG, Gly, Gly-Gly, Gly-Gly-Gly, Glu and (Cys)2 were separated and determined by HPLC-fluorescent using SIFA as precolumn derivatizing reagent. The fluorescent properties of SIFA and its derivatives were examined, and the effects of the pH value and CH3OH concentration were studies. In the CH3OH-H2O(7:3,V/V)solution containing pH8.5 H3BO3-Na2B4O7 buffer, SIFA was derivatized at 30 ℃ for 25 min. The resulting fluorescent derivatives were separated on a C18 column using φ(CH3OH)=42% solution containing 10 mmol/L H3cit-Na2HPO4 (pH5.0) as the mobile phase and detected spectrofluorimetrically at 513 nm with excitation at 492 nm. The detection limits was 0.64～12 fmol(S/N=3). ......

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