目的 建立不同產(chǎn)地八角蓮中6種有效成分（鬼臼毒素、4’-去甲基鬼臼毒素、去氧鬼臼毒素、去氫鬼臼毒素、鬼臼毒酮、山荷葉素）的定性與化學(xué)計量學(xué)方法確定的3種差異成分（鬼臼毒素、山荷葉素、去氫鬼臼毒素）和鬼臼毒酮的定量分析方法。方法 以0.1%甲酸水溶液-乙腈為流動相，梯度洗脫，Waters BEH C₁₈柱進(jìn)行色譜分離，使用Q Exactive OrbitrapMS和電噴霧離子源（ESI）進(jìn)行正、負(fù)離子模式下的質(zhì)譜掃描分析，根據(jù)標(biāo)準(zhǔn)品質(zhì)譜數(shù)據(jù)，對不同產(chǎn)地八角蓮化學(xué)成分進(jìn)行鑒定；結(jié)合主成分分析（PCA）和正交偏最小二乘法-判別分析（OPLS-DA）確定差異性特征成分；使用Waters BEH C18色譜柱，以0.1%甲酸水-乙腈為流動相進(jìn)行梯度洗脫，應(yīng)用QqQ MS，在正離子模式下，以MRM配對離子方式，同時對不同產(chǎn)地八角蓮中有顯著性差異的3個化學(xué)成分以及鬼臼毒酮進(jìn)行定量測定。結(jié)果 結(jié)合標(biāo)準(zhǔn)品質(zhì)譜信息，確定收集自四川產(chǎn)地的八角蓮樣品不含鬼臼毒酮，其余6個產(chǎn)地的八角蓮樣品中均鑒定到6種有效成分（鬼臼毒素、4’-去甲基鬼臼毒素、去氧鬼臼毒素、去氫鬼臼毒素、鬼臼毒酮、山荷葉素）。PCA表明，不同產(chǎn)地的八角蓮樣品間存在差異。通過OPLS-DA項下的變量重要性投影（VIP）分析篩選出鬼臼毒素、山荷葉素、去氫鬼臼毒素可能是影響不同產(chǎn)地八角蓮質(zhì)量差異的標(biāo)志物。定量結(jié)果表明，4種化學(xué)成分均在4 min內(nèi)出峰且分離良好，鬼臼毒素、山荷葉素、去氫鬼臼毒素、鬼臼毒酮分別在1～200、10～1 000、1～1 000、1～1 000 ng·mL^-1線性關(guān)系良好（r＞0.996），平均加樣回收率為97.3%～102.8%，RSD均小于8.3%。7個產(chǎn)地的4種差異成分中，河南產(chǎn)地的鬼臼毒素含量明顯高于其他產(chǎn)地（P＜0.01） ，湖南產(chǎn)地的山荷葉素含量明顯高于其他產(chǎn)地（P＜0.01） ，4’-去甲基鬼臼毒素與去氧鬼臼毒素在各產(chǎn)地中的含量差別較小。結(jié)論 結(jié)合化學(xué)計量學(xué)建立的快速、準(zhǔn)確、靈敏的多指標(biāo)成分定量方法可有效區(qū)分不同產(chǎn)地的八角蓮，為八角蓮藥材的質(zhì)量評價與控制提供參考。

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

國家自然科學(xué)基金項目青年科學(xué)基金項目（82204938）;河南省醫(yī)學(xué)科技攻關(guān)計劃聯(lián)合共建項目（LHGJ20240427）