China Military Institute of Chinese Materia Medica, 302 Military Hospital of China, Beijing, P. R. China.
J Sep Sci. 2010 Jun;33(10):1518-27. doi: 10.1002/jssc.200900751.
A fast ultra-performance liquid chromatography-evaporative light scattering detection (UPLC-ELSD) method was established for simultaneous quantification of seven components in natural Calculus bovis (C. bovis) and its substitutes or spurious breeds. On a Waters Acquity UPLC BEH C(18) column, seven analytes were efficiently separated using 0.2% aqueous formic acid-acetonitrile as the mobile phase in a gradient program. The evaporator tube temperature of ELSD was set at 100 degrees C with the nebulizing gas flow-rate of 1.9 L/min. The results showed that this established UPLC-ELSD method was validated to be sensitive, precise and accurate with the LODs of seven analytes at 2-11 ng, and the overall intra-day and inter-day variations less than 3.0%. The recovery of the method was in the range of 97.8-101.6%, with RSD less than 3.0%. Further results of PCA on the contents of seven investigated analytes suggested that compounds of cholic acid, deoxycholic acid and chenodeoxycholic acid or cholesterol should be added as chemical markers to UPLC analysis of C. bovis samples for quality control and to discriminate natural C. bovis sample and its substitutes or some spurious breeds, then normalize the use of natural C. bovis and ensure its clinical efficacy.
建立了一种快速超高效液相色谱-蒸发光散射检测（UPLC-ELSD）方法，用于同时定量测定天然牛黄及其替代品或假冒品种中的七种成分。在 Waters Acquity UPLC BEH C(18)柱上，采用 0.2%甲酸-乙腈水溶液作为流动相，在梯度程序下可有效分离七种分析物。ELSD 的蒸发器管温度设置为 100°C，雾化气体流速为 1.9 L/min。结果表明，该建立的 UPLC-ELSD 方法具有较高的灵敏度、精密度和准确度，七种分析物的 LOQs 为 2-11ng，日内和日间总变异小于 3.0%。该方法的回收率在 97.8-101.6%范围内，RSD 小于 3.0%。进一步对七种被调查分析物含量的 PCA 结果表明，应在 UPLC 分析中添加胆酸、脱氧胆酸和鹅去氧胆酸或胆固醇等化合物作为化学标志物，以控制天然牛黄样品的质量，并区分天然牛黄样品及其替代品或一些假冒品种，然后规范天然牛黄的使用，确保其临床疗效。
