目的 建立不同产地八角莲中6种有效成分（鬼臼毒素、4’-去甲基鬼臼毒素、去氧鬼臼毒素、去氢鬼臼毒素、鬼臼毒酮、山荷叶素）的定性与化学计量学方法确定的3种差异成分（鬼臼毒素、山荷叶素、去氢鬼臼毒素）和鬼臼毒酮的定量分析方法。方法 以0.1%甲酸水溶液-乙腈为流动相，梯度洗脱，Waters BEH C₁₈柱进行色谱分离，使用Q Exactive OrbitrapMS和电喷雾离子源（ESI）进行正、负离子模式下的质谱扫描分析，根据标准品质谱数据，对不同产地八角莲化学成分进行鉴定；结合主成分分析（PCA）和正交偏最小二乘法-判别分析（OPLS-DA）确定差异性特征成分；使用Waters BEH C18色谱柱，以0.1%甲酸水-乙腈为流动相进行梯度洗脱，应用QqQ MS，在正离子模式下，以MRM配对离子方式，同时对不同产地八角莲中有显著性差异的3个化学成分以及鬼臼毒酮进行定量测定。结果 结合标准品质谱信息，确定收集自四川产地的八角莲样品不含鬼臼毒酮，其余6个产地的八角莲样品中均鉴定到6种有效成分（鬼臼毒素、4’-去甲基鬼臼毒素、去氧鬼臼毒素、去氢鬼臼毒素、鬼臼毒酮、山荷叶素）。PCA表明，不同产地的八角莲样品间存在差异。通过OPLS-DA项下的变量重要性投影（VIP）分析筛选出鬼臼毒素、山荷叶素、去氢鬼臼毒素可能是影响不同产地八角莲质量差异的标志物。定量结果表明，4种化学成分均在4 min内出峰且分离良好，鬼臼毒素、山荷叶素、去氢鬼臼毒素、鬼臼毒酮分别在1～200、10～1 000、1～1 000、1～1 000 ng·mL^-1线性关系良好（r＞0.996），平均加样回收率为97.3%～102.8%，RSD均小于8.3%。7个产地的4种差异成分中，河南产地的鬼臼毒素含量明显高于其他产地（P＜0.01） ，湖南产地的山荷叶素含量明显高于其他产地（P＜0.01） ，4’-去甲基鬼臼毒素与去氧鬼臼毒素在各产地中的含量差别较小。结论 结合化学计量学建立的快速、准确、灵敏的多指标成分定量方法可有效区分不同产地的八角莲，为八角莲药材的质量评价与控制提供参考。

Objective To establish a qualitative and quantitative analysis method for six active components (podophyllotoxin 4'- demethylpodophyllotoxin, deoxypodophyllotoxin, dehydropodophyllotoxin, podophyllotoxin, and diphyllin) in Dysosma versipellis from different habitats, and to determine three differential components (podophyllotoxin, hyperin and dehydropodophyllotoxin) and podophyllotoxin by chemometrics. Methods The qualitative analysis of the chemical components of D. versipellis from different habitats was performed using Q Exactive Orbitrap MS and electrospray ionization (ESI) in positive and negative ion modes, and components were identified based on the standard mass spectrometry data. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were combined to determine the differential characteristic components. Quantitative analysis of three chemical components with significant differences and podophyllotoxin in D. versipellis from different habitats was performed using QqQ MS in the positive ion mode with MRM paired ion mode. Results Combined with the standard mass spectrometry, it was determined that the samples from Sichuan did not contain podophyllotoxin, and six kinds of pharmacodynamic components were identified in the samples of other six habitats. PCA showed that there were differences among the samples of D. versipellis from different habitats. Through the VIP analysis the OPLS-DA, it was screened out that podophyllotoxin, diphyllin, and dehydropophyllotoxin might be the markers affecting the quality difference of D. versipellis from different habitats. The quantitative results showed that the four components eluted within 4 min and were well separated. Podophyllotoxin, diphyllin, dehydropodophyllotoxin, and podophyllxin had a good linear relationship in the range of 1－200, 10－1 000, 1－1 000, and 1－1 000 ng·mL^-1, respectively (r > 0.996), and the average spiking recovery rate was 97.3%－102.8%, and RSD was less than 8.3%. Among the four differential components in the seven habitats, the content of podophyllotoxin in the samples from Henan was significantly higher than that in other habitats (P < 0.01), and the content of diphyllin in the samples from Hunan was significantly higher than that in other habitats (P < 0.01). The content differences of 4'-demethylpodophotoxin and deoxypodophyllotoxin in each producing area were small. Conclusion The rapid, accurate, and sensitive multi-index component quantification method established by combining stoichiometry can effectively distinguish the D. versipellis from different habitats and provides a reference for the quality evaluation and control of the D. versipellis medicinal materials.

R284.1

国家自然科学基金项目青年科学基金项目（82204938）;河南省医学科技攻关计划联合共建项目（LHGJ20240427）