Kwong E, McErlane K M
J Chromatogr. 1986 Apr 25;377:233-42. doi: 10.1016/s0378-4347(00)80778-1.
An efficient high-performance liquid chromatographic (HPLC) separation for digoxin and its metabolites has been developed. Quantitation of digoxin at plasma levels was possible after the column effluent was passed through a fluorogenic post-column reactor. A study of the optimum post-column conditions was undertaken using a combination of ascorbic acid, hydrogen peroxide and hydrochloric acid, which was known to induce fluorescence in the digoxin molecule. Digoxin and its metabolites were separated on a 15 cm X 4.6 mm I.D., 3-microns reversed-phase (C18) HPLC column using methanol--ethanol--isopropanol--water (52:3:1:45) as the mobile phase at a flow-rate of 0.3 ml/min. A solution of 1.1 X 10(-3) M hydrogen peroxide in a 0.1% ascorbic acid solution and concentrated hydrochloric acid were added into the post-column reactor through a peristaltic pump at a combined flow with a flow-rate of 0.23 ml/min. The mixture was passed into the 20-m reaction coil maintained at 79 +/- 1 degrees C. The resulting digoxin fluorophore was monitored with a fluorescence detector. Detector responses were linear from 1.5 to 10 ng injected on-column. The overall performance demonstrated that this system has the sensitivity, linearity and stability desired in a digoxin plasma level determination. The total chromatographic time including the postcolumn derivatization step was about 40 min.
已开发出一种高效的用于地高辛及其代谢物的高效液相色谱（HPLC）分离方法。柱流出物通过荧光柱后反应器后，就可以对血浆中的地高辛进行定量分析。使用抗坏血酸、过氧化氢和盐酸的组合对柱后最佳条件进行了研究，已知该组合可诱导地高辛分子产生荧光。地高辛及其代谢物在一根15 cm×4.6 mm内径、3微米的反相（C18）HPLC柱上分离，流动相为甲醇 - 乙醇 - 异丙醇 - 水（52:3:1:45），流速为0.3 ml/min。将1.1×10（-3）M过氧化氢在0.1%抗坏血酸溶液中的溶液和浓盐酸通过蠕动泵以0.23 ml/min的组合流速加入柱后反应器。混合物进入保持在79±1℃的20米反应盘管。用荧光检测器监测生成的地高辛荧光团。检测器响应在柱上进样1.5至10 ng范围内呈线性。整体性能表明该系统在地高辛血浆水平测定中具有所需的灵敏度、线性和稳定性。包括柱后衍生步骤在内的总色谱时间约为40分钟。
