Mayer Michael, Muscate-Magnussen Angelika, Vogel Horst, Ehrat Markus, Bruin Gerard J M
Institute of Physical Chemistry, Swiss Federal Institute of Technology, Lausanne, Switzerland.
Electrophoresis. 2002 May;23(9):1255-62. doi: 10.1002/1522-2683(200205)23:9<1255::AID-ELPS1255>3.0.CO;2-G.
Highly efficient capillary electrochromatographic separations of cardiac glycosides and other steroids are presented. Employing butyl-derivatized silica particles as stationary phase resulted in a nearly three times faster electroosmotic flow (EOF) compared to capillary electrochromatography (CEC) with octadecyl silica particles. On-column focusing with a preconcentration factor of 180 was performed and separation efficiencies of up to 240,000 plates per meter were obtained. Using label-free standard UV absorbance, detection limits of 10-80 nM were reached for all steroids tested. For screening of cardiac glycosides, e.g., digoxin and digitoxin in mixtures of steroids, CEC was combined with immunoaffinity extraction using immobilized polyclonal anti-digoxigenin antibodies and F(ab) fragments. Simply adding small amounts of antibody carrying particles to the samples and comparing chromatograms before and after antibody addition allowed screening for high affinity antigens in mixtures with moderate numbers of compounds. Under conditions of competing antigens, affinity fingerprints of immobilized anti-digoxigenin and anti-digitoxin antibodies were obtained, reflecting the cross-reactivity of eleven steroids. The method provides high selectivity due to the combination of bioaffinity interaction with highly efficient CEC separation and UV detection at several wavelengths in parallel. This selectivity was exploited for the detection of four cardiac glycosides in submicromolar concentrations in an untreated urine sample.
本文介绍了强心苷和其他甾体类化合物的高效毛细管电色谱分离方法。与使用十八烷基硅胶颗粒的毛细管电色谱（CEC）相比，采用丁基衍生化硅胶颗粒作为固定相，电渗流（EOF）速度快了近三倍。进行了柱上聚焦，预富集因子为180，分离效率高达每米240,000理论塔板数。使用无标记标准紫外吸收法，所有测试甾体类化合物的检测限达到10 - 80 nM。为了筛选甾体混合物中的强心苷，例如地高辛和洋地黄毒苷，CEC与使用固定化多克隆抗地高辛配基抗体和F(ab)片段的免疫亲和萃取相结合。只需向样品中加入少量携带抗体的颗粒，并比较添加抗体前后的色谱图，即可在含有中等数量化合物的混合物中筛选高亲和力抗原。在竞争抗原的条件下，获得了固定化抗地高辛配基和抗洋地黄毒苷抗体的亲和指纹图谱，反映了11种甾体类化合物的交叉反应性。该方法由于生物亲和相互作用与高效CEC分离以及在多个波长下并行紫外检测相结合，具有高选择性。利用这种选择性在未经处理的尿液样本中检测到了亚微摩尔浓度的四种强心苷。
