黄金蝉若虫体蛋白水解液的荧光标记及电喷雾质谱鉴定

Application of 2-[2-(7H-Dibenzo[a,g]-carbazol-7-yl)-ethoxyl]ethyl Chloroformate as A Pre-column Derivatization Reagent for Determination of Amino Acids from Hydrolyzed Cicada by High Performance Liquid Chromatography with Fluorescence Detection and Mass Sp

采用2-[2-(7H-二苯并[a,g]咔唑-乙氧基)-乙基]氯甲酸酯(DBCEC-Cl)作为柱前衍生试剂,于室温下,在硼酸钠缓冲液(pH9.0)中,金蝉若虫体蛋白水解液中氨基酸的完全衍生化在5min即可完成。在HypersilBDSC18色谱柱(200mm×4.6mm,5μm)上,采用梯度洗脱实现了各衍生物的完全基线分离。最佳荧光检测为λex/λem=300/395nm。在线串联的ESI/MS正离子质谱数据显示:所有衍生物均给出m/z338.5,294.5的特征碎片离子峰。荧光条件下的线性回归系数大于0.9992;检出限为2.6～24fmol。本方法灵敏、可靠、重现性好。对实际黄金蝉若虫体蛋白水解液中氨基酸的测定,结果满意。

By using 2-[2-（7H-dibenzo[a,g]carbazole-ethoxy）-ethyl chloroformate （DBCEC-Cl） as a sensitive pre-column derivatization reagent,a simple method for evaluating the chemical compositions of protein amino acids from the hydrolyzed cicada sample has been developed.Studies on derivatization conditions indicated that the labeling reaction proceeded rapidly and smoothly in the presence of a base catalyst （pH 9.0） in acetonitrile to give the corresponding sensitively fluorescent derivatives with an excitation maximum at λex 300 nm and an emission maximum at λem 395 nm.The separation of amino acid derivatives was carried out on a reversed phase Hypersil BDS-C18（200 mm×4.6 mm×5 μm） column in combination of a gradient elution.The complete baseline resolution for 20 amino acid derivatives was achieved within 65 min.Identification of amino acid derivatives was carried out by the online post-column ion trap/mass spectrometry with electrospray ion source （ESI source） in positive ion mode.Excellent linear response was observed,with coefficients of 0.9992.Fluorescent detection limits calculated from 25 pmol injection,at a signal-to-noise ratio of 3,were 2.6-24 fmol.The established method for the determination of amino acids from cicada sample was satisfactory.