色谱 ›› 2021, Vol. 39 ›› Issue (5): 478-487.DOI: 10.3724/SP.J.1123.2020.10011

• 研究论文 • 上一篇    下一篇

利用在线加压溶剂提取-超高效液相色谱-离子阱-飞行时间-质谱法定性分析片仔癀化学成分组

李玮1, 蒋珍珍2, 李菡1, 屠鹏飞1, 宋青青1,*(), 于娟2,*(), 宋月林1   

  1. 1.北京中医药大学中药学院, 中药现代研究中心, 北京 100029
    2.漳州片仔癀药业股份有限公司, 福建 漳州 363000
  • 收稿日期:2020-10-20 出版日期:2021-05-08 发布日期:2021-03-31
  • 通讯作者: 宋青青,于娟
  • 作者简介:Tel:(0596)2301615,E-mail:13906945397@163.com(于娟).
    Tel:(010) 64286100,E-mail:song_qingq@163.com(宋青青);
  • 基金资助:
    国家自然科学基金项目(82003911);国家自然科学基金项目(81773875);国家自然科学基金项目(81973444);漳州片仔癀药业股份有限公司.

Chemome profiling of Pien-Tze-Huang by online pressurized liquid extraction-ultra-high performance liquid chromatography-ion trap-time-of-flight mass spectrometry

LI Wei1, JIANG Zhenzhen2, LI Han1, TU Pengfei1, SONG Qingqing1,*(), YU Juan2,*(), SONG Yuelin1   

  1. 1. Modern Research Center for Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China
    2. Zhangzhou Pien-Tze-Huang Pharmaceutical Co., Ltd., Zhangzhou 363000, China
  • Received:2020-10-20 Online:2021-05-08 Published:2021-03-31
  • Contact: SONG Qingqing,YU Juan
  • Supported by:
    National Natural Science Foundation of China(82003911);National Natural Science Foundation of China(81773875);National Natural Science Foundation of China(81973444);Zhangzhou Pien-Tze-Huang Pharmaceutical Co., Ltd.

摘要:

片仔癀(Pien-Tze-Huang)是由三七、牛黄、蛇胆、麝香等名贵中药经加工精制而成的中药制剂。片仔癀中的三七皂苷、胆汁酸以及麝香酮等主要化学成分已被深入研究,然而其全方化学成分组成尚未被整体阐明。该文建立了在线加压溶剂提取-超高效液相色谱-离子阱-飞行时间质谱(online PLE-UHPLC-IT-TOF-MS)法,快速、直接分析片仔癀化学成分组。将少量片仔癀粉末(0.3 mg)均匀地平铺于预柱芯尾端,之后用正相硅胶填充预柱芯,滤膜密封后构成提取池。提取池装入预柱套后置于70 ℃的柱温箱内,连接于UHPLC-IT-TOF-MS分析系统。通过引入一个二位六通电子阀,将整个分析过程自动在提取相和洗脱相间切换。提取相用时3 min,以0.1%(v/v)甲酸水为提取溶剂,流速为0.2 mL/min;洗脱相以0.1%(v/v)甲酸水和乙腈为流动相进行梯度洗脱,IT-TOF-MS检测。通过与对照品、相关文献和自建中药数据库对照,并总结相关质谱裂解规律,从片仔癀中共检测到73个化学成分,初步鉴定并归属了其中71个,36个来源于三七,15个来源于蛇胆,9个来源于牛黄,11个可能来源于牛黄与蛇胆,另有2个结构无法确定。该研究深入解析了片仔癀的化学成分组,为其质量分析提供了丰富的信息。同时,该文构建的online PLE-UHPLC-IT-TOF-MS分析系统为中药复杂体系快速、直接分析提供了可靠的工具。

关键词: 在线加压溶剂提取, 片仔癀, 化学成分组, 质谱裂解途径, 来源归属

Abstract:

Pien-Tze-Huang is one of the most famous traditional Chinese medicine prescriptions and consists of several precious medicinal materials, such as Notoginseng Radix et Rhizoma, Bovis Calculus, Snake Gall, and Moschus. However, its formula has not been completely revealed. It is mainly applied for the treatment of acute and chronic viral hepatitis, carbuncle, and boils caused by blood stasis, unknown swelling, bruises, and various inflammation disorders. The chemical composition of Pien-Tze-Huang is extremely complicated. Thus far, extensive attention has been paid to the principal chemical families in Pien-Tze-Huang, such as ginsenosides, bile acids, and muscone derivatives. Comprehensive chemical profiling, although of immense importance for systematic quality control, has not been achieved. Therefore, we configured a platform, namely online pressurized liquid extraction-ultra-high-performance liquid chromatography-ion trap-time-of-flight mass spectrometry (online PLE-UHPLC-IT-TOF-MS), to characterize the chemical profile of Pien-Tze-Huang in detail as well as to conduct source attribution, aiming to clarify the chemome of Pien-Tze-Huang and to provide a reliable method for quality assessment. A sub-microgram amount of Pien-Tze-Huang powder (0.3 mg) was placed in a hollow guard column, which was subsequently filled with clear silica gel. Filter membranes were used to seal the extraction vessel. The vessel was then placed in an adapted guard column holder and maintained in a thermal column oven (70 ℃). Metal tubing was used to connect the outlet of the guard column holder to the mass spectrometer. The extraction phase was maintained for 3 min by employing 0.1%(v/v) formic acid aqueous solution as the extraction solvent with a flow rate of 0.2 mL/min. Moreover, a six-port two-position electronic valve was introduced to automatically switch the system from extraction to elution phases. Within the elution phase, 0.1%(v/v) formic acid aqueous solution and acetonitrile composed the mobile phase, and the extracts were eluted with a gradient program. Because of the elevated temperature and pressure, the physical and chemical properties of water, especially polarity and solubility, were modified. Therefore, warm water could be an eligible green solvent to achieve wide polarity-spanned extraction. In addition, IT-TOF-MS was employed to acquire tandem mass spectrometry information. The mass fragmentation pathways of saponins and bile acids were carefully studied. Finally, according to authentic compounds, mass fragmentation pathways, reference information in the literature, and accessible databanks, a total of 73 signals were observed from Pien-Tze-Huang, of which 71 components were tentatively identified and assigned. Among them, 36 were from Notoginseng Radix et Rhizoma, 15 from Snake Gall, and 9 from Bovis Calculus, while the occurrences of the other 11 components were synergistically contributed by both Bovis Calculus and Snake Gall, through retrieving the in-house chemical database that was built by considering all accessible chemical information from Notoginseng Radix et Rhizoma, Bovis Calculus, Snake Gall, and Moschus. The other two compounds were assigned as unknown compounds. However, none of the components were assigned to Moschus because they mainly contained hydrophobic compounds, such as cycloketones, cholesterol, and sterols, among others, and it was difficult to detect them with the current measurement program. The extraction efficiency of online PLE was assessed by comparing it with the efficiency obtained from ultrasonication at the same time. According to base peak ion current chromatograms (BPCs) and mass spectrometry information, the efficiency of online PLE was greater than that of ultrasonic extraction, even through direct analysis. Online PLE-UHPLC-IT-TOF-MS is not only a tool fit for the concept of green analytical chemistry, but also a reliable analytical pipeline for the direct characterisation of other complicated matrixes. Above all, this study clarified the chemome of Pien-Tze-Huang and provided meaningful information for the quality control of this famous TCM prescription.

Key words: online pressurized liquid extraction, Pien-Tze-Huang, chemome profiling, mass fragmentation pathways, source attribution

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