Department of Pharmaceutical Analysis, College of Pharmacy, Chung-Ang University, Seoul, South Korea.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Dec 15;879(32):3887-95. doi: 10.1016/j.jchromb.2011.10.040. Epub 2011 Nov 4.
A simple, rapid and robust high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) method was established for the species discrimination and quality evaluation of Radix Bupleuri through the simultaneous determination of ten saikosaponins, namely saikosaponin-a, -b(1), -b(2), -b(3), -b(4), -c, -d, -g, -h, and -i. These compounds were chromatographed on an Ascentis(®) Express C18 column with a gradient elution of acetonitrile and water containing 0.1% acetic acid at a flow rate of 1.0 mL/min. Saikosaponins were monitored by ELSD, which was operated at a 50°C drift tube temperature and 3.0 bar nebulizer gas (N(2)) pressure. The developed method was validated with respect to linearity, intra- and inter-day accuracy and precision, limit of quantification (LOQ), recovery, robustness and stability, thereby showing good precision and accuracy, with intra- and inter-assay coefficients of variation less than 15% at all concentrations. Furthermore, a high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) method was developed to certify the existence of ten saikosaponins, as well as to confirm the reliability of ELSD. The extraction conditions of saikosaponins from Radix Bupleuri were also optimized by investigating the effect of extraction methods (sonication, reflux and maceration) and various solvents on the extraction efficiencies for saikosaponins. Sonication with 70% methanol for 40 min was found to be simple and effective for extraction of major saikosaponins. This analytical method was applied to determine saikosaponin profiles in 20 real samples consisting of four Bupleurum species, namely B. falcatum, B. chinense, B. sibiricum and the poisonous B. longiradiatum. It was found that three major saikosaponin-a, -c and -d were the major constituents in B. falcatum, B. chinense, and B. longiradiatum, while one major saikosaponin (saikosaponin-c) was not identified from B. sibiricum. In addition, no saikosaponin-b(3) was detected in B. longiradiatum samples, indicating that the toxic B. longiradiatum may be tentatively distinguished from officially listed Bupleurum species (B. falcatum and B. chinense) based on their saikosaponin profiles. Overall the simultaneous determination of ten saikosaponins in Radix Bupleuri was shown to be a promising tool to adopt for the discrimination and quality control of closely related Bupleurum species.
建立了一种简单、快速、稳健的高效液相色谱-蒸发光散射检测（HPLC-ELSD）方法，用于通过同时测定十种柴胡皂苷，即柴胡皂苷-a、-b(1)、-b(2)、-b(3)、-b(4)、-c、-d、-g、-h 和 -i，对柴胡进行种属鉴别和质量评价。这些化合物在 Ascentis(®)Express C18 柱上进行色谱分离，采用乙腈和含 0.1%乙酸的水作为流动相进行梯度洗脱，流速为 1.0 mL/min。柴胡皂苷通过 ELSD 进行监测，漂移管温度为 50°C，雾化器气体（N(2)）压力为 3.0 巴。该方法在线性、日内和日间精密度和准确度、定量限 (LOQ)、回收率、稳健性和稳定性方面进行了验证，结果表明该方法具有良好的精密度和准确度，所有浓度下的日内和日间变异系数均小于 15%。此外，还建立了高效液相色谱-电喷雾电离质谱（HPLC-ESI-MS）方法，用于证明十种柴胡皂苷的存在，并确认 ELSD 的可靠性。还通过考察不同提取方法（超声、回流和浸渍）和不同溶剂对柴胡皂苷提取效率的影响，优化了从柴胡中提取柴胡皂苷的提取条件。结果发现，70%甲醇超声 40 min 是提取主要柴胡皂苷的简单有效的方法。该分析方法用于测定由四种柴胡属植物（北柴胡、狭叶柴胡、西伯利亚柴胡和有毒的黑柴胡）组成的 20 个真实样本中的柴胡皂苷谱。结果发现，三种主要的柴胡皂苷-a、-c 和 -d 是北柴胡、狭叶柴胡和黑柴胡的主要成分，而西伯利亚柴胡中未鉴定出一种主要的柴胡皂苷（柴胡皂苷-c）。此外，黑柴胡样品中未检测到柴胡皂苷-b(3)，表明有毒的黑柴胡可能可以根据其柴胡皂苷谱与官方列出的柴胡属植物（北柴胡和狭叶柴胡）进行初步区分。总之，同时测定柴胡中的十种柴胡皂苷被证明是一种有前途的工具，可以用于密切相关的柴胡属植物的鉴别和质量控制。
