Lunte S M, Mohabbat T, Wong O S, Kuwana T
Center for Bioanalytical Research, University of Kansas, Lawrence 66046.
Anal Biochem. 1989 Apr;178(1):202-7. doi: 10.1016/0003-2697(89)90380-1.
Naphthalenedialdehyde (NDA) in the presence of cyanide (CN) reacts with primary amines to produce fluorescent cyano[f]benzoisoindole (CBI) derivatives. These derivatives have been shown to be substantially more stable than the corresponding o-phthalaldehyde derivatives. However, one drawback of this method is that compounds derivatized at more than one site exhibit quenching, precluding the use of fluorescence detection. The CBI derivatives have been found to be electroactive and are oxidized at a modest oxidation potential (+750 mV). Electrochemical detection is especially useful for the analysis of compounds containing more than one primary amine site because the response is not attenuated as it is in fluorescence detection. Desmosine and isodesmosine were of particular interest because of their importance in elastic fiber and the lack of highly sensitive HPLC methods for the determination of these compounds. Both of these compounds react with NDA/CN to produce electrochemically active derivatives. The combination of derivatization with NDA/CN and electrochemical detection was found to be linear over three orders of magnitude. Detection limits for CBI-lysine and CBI-desmosine were 100 fmol at a S/N of 2. Amino acids in elastin were quantitated using this method. The results correlate well with what has been reported previously in the literature. A significant advantage of the use of liquid chromatography with electrochemical detection with precolumn derivatization with NDA/CN for the analysis of desmosine and isodesmosine is that they can be separated and quantitated individually using this method. In addition, the unique voltammetry of multiderivatized CBI-amino acids can be used to verify peak purity.
萘二醛（NDA）在氰化物（CN）存在下与伯胺反应生成荧光氰基[f]苯并异吲哚（CBI）衍生物。这些衍生物已被证明比相应的邻苯二甲醛衍生物稳定得多。然而，该方法的一个缺点是在多个位点衍生化的化合物会出现淬灭现象，这使得荧光检测无法使用。已发现CBI衍生物具有电活性，且在适度的氧化电位（+750 mV）下被氧化。电化学检测对于分析含有多个伯胺位点的化合物特别有用，因为其响应不像荧光检测那样会衰减。由于去甲缬氨酸和异去甲缬氨酸在弹性纤维中的重要性以及缺乏用于测定这些化合物的高灵敏度HPLC方法，它们受到了特别关注。这两种化合物都与NDA/CN反应生成具有电化学活性的衍生物。发现NDA/CN衍生化与电化学检测的组合在三个数量级范围内呈线性。CBI - 赖氨酸和CBI - 去甲缬氨酸的检测限在信噪比为2时为100 fmol。使用该方法对弹性蛋白中的氨基酸进行了定量。结果与文献中先前报道的结果相关性良好。使用NDA/CN进行柱前衍生化并结合电化学检测的液相色谱法分析去甲缬氨酸和异去甲缬氨酸的一个显著优点是，使用该方法可以分别对它们进行分离和定量。此外，多衍生化的CBI - 氨基酸独特的伏安法可用于验证峰纯度。
