Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Research Center of TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; University of Chinese Academy of Sciences, Beijing 100049, China.
Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Research Center of TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; Shenzhen Baoan Authentic TCM Therapy Hospital, Shenzhen 518101, China.
J Chromatogr A. 2022 Nov 22;1684:463554. doi: 10.1016/j.chroma.2022.463554. Epub 2022 Oct 6.
The hyphenated technique of offline two-dimensional (2D) chromatography with high resolution mass spectrometry (MS) was an efficient tool for separation and characterization of components in complex systems such as herbal medicines, especially those co-eluting components or isomers. In this study, we constructed the ultra-performance convergence chromatography (UPC) × reversed phase (RP) chromatographic separation system and developed a mass defect filtering (MDF)-based precursor ion list (PIL) acquisition method to improve the selectivity and sensitivity of this technique, and the systematic characterization of diterpenoid alkaloids in the lateral roots of Aconitum carmichaelii (namely "Fuzi" in Chinese) was used as an example. The constructed offline 2D separation system showed a good orthogonality of 0.77. Besides, the in-house databases for known and predicted C- and C-diterpenoid alkaloids were established by molecular design in Compound Discoverer software for MS data matching and filtering, and two MDF windows were further constructed to screen out more potential diterpenoid alkaloids with novel structures and to obtain the PIL (mass range: even values between 298 and 1020 Da, parent mass width: ±100 mDa) for data acquisition by calculating the m/z values of potential ions using mass range and corresponding mass defect in the MDF windows. In addition, an integrative structure interpretation strategy was developed by integrating elemental composition analysis, ring double bond analysis, neutral loss filtering, diagnostic ion filtering and database matching, etc. As a result, a total of 659 components in the lateral roots of A. carmichaelii were exposed and characterized, including 526 potential new compounds. This strategy showed significant advantages in improving the coverage and selectivity of screening, and could also be applied in systematic characterization of components in other herbal medicines.
离线二维（2D）色谱与高分辨率质谱（MS）的组合技术是一种有效的工具，可用于分离和鉴定复杂体系（如草药）中的成分，特别是那些共洗脱成分或异构体。在本研究中，我们构建了超高效汇聚色谱（UPC）×反相（RP）色谱分离系统，并开发了基于质量亏损过滤（MDF）的前体离子列表（PIL）采集方法，以提高该技术的选择性和灵敏度，并以乌头属侧根中的二萜生物碱的系统表征为例。构建的离线 2D 分离系统表现出良好的正交性（0.77）。此外，通过分子设计在 Compound Discoverer 软件中为已知和预测的 C-和 C-二萜生物碱建立了内部数据库，用于 MS 数据匹配和过滤，并进一步构建了两个 MDF 窗口，以筛选出具有新型结构的更多潜在二萜生物碱，并获得 PIL（质量范围：偶数值在 298 和 1020 Da 之间，母质量宽度：±100 mDa），用于通过计算潜在离子的 m/z 值，在 MDF 窗口中使用质量范围和相应的质量亏损来进行数据采集。此外，通过整合元素组成分析、环双键分析、中性丢失过滤、诊断离子过滤和数据库匹配等方法，开发了一种综合结构解析策略。结果，共鉴定出乌头属侧根中的 659 个成分，包括 526 个潜在的新化合物。该策略在提高筛选的覆盖度和选择性方面具有显著优势，也可应用于其他草药成分的系统表征。
