柱前衍生 HPLC-UV 法测定柴胡中柴胡皂苷含量 秦雪梅，李媛媛，魏学红，郭小青，张丽增 （山西大学化学化工学院，山西太原 ） 摘要： 目的：建立一种柱前酸化反应的 HPLC 方法测定柴胡中柴胡皂苷的含量，并验证其酸化 反应机理。方法： HPLC-UV 法测定 SSa 、 SSd 酸化衍生物的含量，检测波长分别为 240 、 250nm ，流动相：乙腈 - 水 (40:60, v:v) ，流速 : 1.0 mL/min ，色谱柱为 Hypersil ODS C 18 ；再通过 LC-TOF/MS 技术来验证其酸化反应的机理。结果：温度和酸度对转化产物种类的影响较大， 验证了 SSa 、 SSd 酸化反应的产物分别为 SSb 1 、 SSb 2 。结论：所建立的柱前酸化衍生 HPLC-UV 法可用于柴胡中 SSa 、 SSd 的含量测定；柱前酸化反应破坏 SSa 、 SSd 中的氧桥键并形成共轭双 键，在适宜温度下，使其定向转化为 SSb 1 、 SSb 2 ，从而使检测波长红移至 240 、 250nm 。 柴胡皂苷 SSa 、 SSd 的 HPLC 图谱 酸化反应后柴胡皂苷 TOF/MS 图谱 Xuemei Qin, Yuanyuan Li,et al.(2007) “Validation of a New Optimized Method for the Determination of Saikosaponin in Bupleurum Chinense DC. by Liquid Chromatography Time-of-Flight Mass Spectrometry (LC- TOF/MS)” （ In Press ） 仪器： Agilent G1969A LC/MSD-TOF-MS ，正电子 喷雾离子化器。 Alltima ODS-C 18 柱 (4.6×250 mm) 。 色谱流动相为乙腈 -0.1% 乙酸水溶液。 (1) (2) Waters 高效液相色谱仪 酸化反应机理： 酸化反应： (3)

Validation of a New Optimized Method for the Determination of Saikosaponin in Bupleurum Chinense DC. by Liquid Chromatography Time-of-Flight Mass Spectrometry (LC-TOF/MS) Xuemei Qin, Yuanyuan Li, Xuehong Wei, Xiaoqing Guo, Lizeng Zhang School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan , China Abstract: Objective: To establish a new optimized method for the determination of Saikosaponin in Bupleurum chinense DC. and validate the mechanism of the acidification reaction. Method: An acidification reaction mechanism was investigated and validated by LC-TOF/MS technique. Some possible effects were discussed in detail, such as temperature, reaction time, acidity and quantity of base. Saikosaponin a(SSa) and Saikosaponin d(SSd) were determined by HPLC coupled with UV detector at 240 and 250nm with a mobile phase of acetonitrile - water (40:60, v:v) at a flow rate of 1.0 mL·min-1on a Hypersil ODS C18 column. Results:. The extraction was treated by 4%HCl-CH3OH. Temperature and acidity had some influence on the product of acidification reaction. The reaction was stopped immediately when 2% KOH-CH 3 OH was added. Two major derivatives SSa and SSd were quantified. As shown in Eq. (1) and (2), SSa turns to different products in different temperature. When the reaction temperature was down to 40 ℃, SSa turned to SSb1 only; when it was up to 40 ℃ and down to 80 ℃, SSa would turn to SSb 1 and SSg. Eq. (3) shows that SSd turns to SSb2 only. ►The HPLC Picture of Bupleurum Chinense DC. The reaction condition was at room temperature (25 ℃ ), 4% HCl- methanol, 4 hours, and acid : alkali (1:1.5). The UV-HPLC system consisted of a Hypersil ODS-C18 column (200 mm×4.6 mm), mobile phases were CH3CN and H2O (40: 60), the effluent was delivered at 1.0 mL/min, and the UV absorbance was monitored at 240 nm and 250 nm. ►Acidification Reaction Equation: ►The TOF/MS Picture of Bupleurum Chinense DC. TOF-MS data were recorded with an Agilent G1969A LC/MSD- TOF-MS equipped with Spray electro-spray ionization source. A generic positive electro-spray ionization method was used. Capillary: 4000v, sample cone: 100v, extraction cone voltages: 250V, desolvation: 11 L/min, nebulization flows: 55 psig, gas temperatures: 340 ℃. An Alltima ODS-C18 column (4.6×250 mm) was used. The LC mobile phases were CH3CN for solvent A and CH3COOH/H2O (1:1000, v/v) for solvent B. Conclusion: A rapid, accurate and sensitive HPLC method was set up for determining the content of saikosaponins. Validation result was consist with the present report. SSa and SSd could be regarded as the main components that determine the quality of the phytomedicine.