Shen H, Witowski S R, Boyd B W, Kennedy R T
Department of Chemistry, University of Florida, Gainesville 32611-7200, USA.
Anal Chem. 1999 Mar 1;71(5):987-94. doi: 10.1021/ac9809837.
The separation and detection of biuret complexes of neuropeptides by capillary liquid chromatography with electrochemical detection was explored. Capillaries of 25-micron inner diameter packed with base-resistant, polymer-based reversed-phase particles were used for separation, and C-fiber electrodes were used for detection. Detection at the C-fiber electrode was found to have some differences in relative sensitivity for peptides compared to glassy carbon electrodes used previously. On-column preconcentration of preformed complexes allowed up to 1-microL samples to be injected with minimal band broadening resulting in a 100-fold improvement in concentration detection limit with no effect on mass detection limit. Concentration detection limits ranged from 5 to 59 pM, depending upon the peptide, corresponding to 5-59 amol injected. The low concentration detection limit was possible because of minimal baseline disturbances, minimal formation of unwanted products, and high efficiency of complex formation associated with biuret derivatization. The method was applied to determination of vasopressin and bradykinin in dialysates collected with 5-min sampling frequency from the rat supraoptic nucleus.
研究了采用毛细管液相色谱-电化学检测法分离和检测神经肽的缩二脲络合物。使用内径为25微米、填充有耐碱聚合物基反相颗粒的毛细管进行分离，并使用碳纤维电极进行检测。结果发现，与先前使用的玻碳电极相比，在碳纤维电极上进行检测时，肽的相对灵敏度存在一些差异。对预形成的络合物进行柱上预富集，可注入高达1微升的样品，且峰展宽最小，从而使浓度检测限提高了100倍，而对质量检测限没有影响。浓度检测限在5至59皮摩尔之间，具体取决于肽，相当于注入5 - 59阿托摩尔。能够实现低浓度检测限是因为基线干扰最小、 unwanted产物形成最少以及与缩二脲衍生化相关的络合物形成效率高。该方法应用于以5分钟采样频率从大鼠视上核收集的透析液中血管加压素和缓激肽的测定。
