雷公藤中雷公藤甲素、雷公藤吉碱和次碱的高效液相色谱-电喷雾串联质谱分析方法

建立了一种高效液相色谱-电喷雾串联质谱法 (HPLC-ESI-MS/MS) 测定雷公藤提取物中雷公藤甲素 (triptolide)、雷公藤吉碱 (wilforgine) 和雷公藤次碱 (wilforine) 3种活性物质的分析方法，比较了雷公藤不同植株部位和不同组织培养产物中3种活性物质的含量差异。分别采用回流和超声两种方法提取。回流提取中，样品经V (甲醇) : V (乙腈) = 1:1溶液回流，采用Supelclean LC-Si固相萃取小柱净化；超声提取中，样品经乙醇超声，用OASIS HLB固相萃取柱净化。采用C₁₈色谱柱分离，乙腈和水作为流动相进行梯度洗脱，采用HPLC-ESI-MS/MS测定。结果显示:在雷公藤植株中，雷公藤甲素含量最高的是不定根，发状根、根皮中含量次之，茎、叶中含量很少；雷公藤次碱含量最高的是发状根，其次是根皮，叶中未检测出；发状根和根皮中雷公藤吉碱的含量相当。在0.01~2 mg/kg添加水平下，3种活性物质的回收率在81%~109%之间，相对标准偏差 (RSD) 为0.4%~1.8% (n = 5)。检出限在0.08~0.12 μg/mL之间。该方法可以准确、快速地对雷公藤提取物及其产品进行质量监控。

Determination of triptolide, wilforgine and wilforine in Tripterygium wilfordii by high performance liquid chromatography-electrospray ionization tandem mass spectrometry

A rapid analytical method for the simultaneous determination of triptolide, wilforgine and wilforine was developed using high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The contents of those three active substances in different tissues and plant tissue culture of Tripterygium wilfordii were compared. The ultrasonic extraction and reflux extraction technologies were used for extraction of those three active substances in T. wilfordii. In the reflux extraction process, the analytes were extracted by V (methanol) : V (acetonitrile) = 1:1 solution, and cleaned up by Supelclean LC-Si solid phase extraction cartridges. During the ultrasonic extraction, the analytes were extracted by ethanol, purified by OASIS HLB SPE column, and separated on a reversed phase C₁₈ column with gradient elution with acetonitrile and water as the mobile phase. The extract was then determined with HPLC-ESI-MS/MS. The results showed that the highest contents of triptolide is detected in adventitious roots, followed by hairy root and root bark, while very low content was detected in stems and leaves. The highest contents of wilforgine content is observed in hairy root, followed by the root bark, and it was not detected in leaves. Wilforine contents determined in hairy root and root bark are similar. When the addition level ranged from 0.01 mg/kg to 2 mg/kg, the recoveries of three active substances of T. wilfordii was 81%-109%, and with RSD ranged from 0.4% to 1.8% (n = 5). The detection limits of the three active substances ranged from 0.08 μg/mL to 0.12 μg/mL. Results indicated that this method is highly efficient, with good sensitivity and accuracy, which can quickly monitor the quality of T. wilfordii extract and its products.