目的 建立杜仲Eucommia ulmoides不同部位的高效薄层色谱（high performance thin layer chromatography，HPTLC）、高效薄层质谱鉴别方法（TLC/MS）和高效液相色谱（HPLC）指纹图谱，比较杜仲皮、叶、雄花、种子的差异，寻找差异性成分，并互相验证，用于杜仲的质量控制。方法 通过优化供试品制备、展开剂和显色剂等条件，建立杜仲皮的HPTLC，并将该方法应用于杜仲皮、叶、雄花、种子的区分，寻找差异性斑点；利用QTOF/MS对4个部位的HPTLC斑点进行鉴定，明确差异性成分；采用HPLC建立杜仲皮、叶、雄花、种子的指纹图谱，采集多批次数据，利用偏最小二乘判别分析（partial least squares discriminant analysis，PLS-DA）筛选对4个部位区分贡献较大的差异性成分，与薄层色谱和薄层质谱鉴定结果互相验证。结果 自建HPTLC鉴别方法优于各国药典，可清晰地实现4个部位的区分；利用QTOF/MS对主要斑点进行分析，共鉴定27个成分，其中10个成分通过对照品确认；基于HPLC的PLS-DA多元统计分析筛选出14个差异性成分（VIP>1）。松脂醇二葡萄糖苷在HPTLC、TLC/MS和HPLC检测技术下均鉴定为杜仲皮的特异性成分；ulmoidoside A、ulmoidoside B、ulmoidoside C、ulmoidoside D是种子中稳定的差异性标志物；对于叶和花的鉴别，3种鉴别方式所得到的差异性标志物差别较大，叶中共找到7个标志物，花中共找到9个标志物。结论 建立的高效薄层色谱、薄层质谱和HPLC指纹图谱方法均可实现杜仲皮、叶、雄花、种子的区分，联用技术寻找到的差异性成分为杜仲及其中成药质量控制指标的选择奠定基础。

Objective To establish the high performance thin layer chromatography (HPTLC), thin layer chromatography-mass spectrometry (TLC/MS) and high performance liquid chromatography (HPLC) fingerprint for the identification of bark, leaf, flower and seed of Duzhong (Eucommia ulmoides Oliv.) and differential components were filtrated and verified for the quality control of E. ulmoides. Methods HPTLC method was established by optimizing the sample preparation, mobile phase and derivatization reagent and applied to identify the bark, leaf, male flower, seed of E. ulmoides based on the specific spots and/or peaks. HPTLC spots in four parts were then identified through quadrupole time of flight mass spectrometry (QTOF/MS) to find the differential components. HPLC was used to establish the fingerprints of bark, leaf, flower and seed of E. ulmoides. Partial least squares discriminant analysis (PLS-DA) was used to screen the chemical markers that contributed greatly to the differentiation of the 4 parts based on multiple batches of data and the results were verified with TLC and TLC/MS. Results HPTLC identification method is the best compared to the method reported in pharmacopoeia from other countries, and can clearly distinguish the four parts. Additionally, the main spots were analyzed by QTOF/MS, and a total of 27 components were identified, 10 of which were confirmed by reference substances. Fourteen differential components (VIP > 1) were screened out by PLS-DA based on HPLC. According to the results obtained by HPTLC, TLC/MS and HPLC, pinoresinol diglucoside were selected as the characteristic component of bark, ulmoidoside A, ulmoidoside B, ulmoidoside C, ulmoidoside D as the robust markers in seed. The differential markers obtained by these three methods varied widely, with seven markers in leaf, nine markers in flower in total. Conclusion HPTLC, TLC/MS, HPLC methods established could be used to effectively distinguish bark, leaf, flower and seed of E. ulmoides, and the screened differential components lay a solid foundation for the selection of quality control indexes of E. ulmoides and its related Chinese patent medicines.

R286.2

国家自然科学基金青年项目（82003940）；岐黄工程首席科学家项目（2020）