目的 采用超高效液相色譜串聯(lián)四極桿靜電場軌道阱質譜法（UPLC-Q-Exactive Orbitrap-MS/MS）對五神湯中的化學成分進行鑒定，進而對五神湯中京尼平苷酸、秦皮甲素、咖啡酸、木犀草苷、β-蛻皮甾酮和毛蕊花糖苷 6種成分進行含量測定。方法 UPLC-Q-Exactive Orbitrap-MS/MS 法鑒定五神湯中的化學成分，采用 ACQUIFY UPLCTMBEH C₁₈ 色譜柱（100 mm×2.1 mm，1.7 μm）進行分離，以0.1%甲酸-乙腈為流動相進行梯度洗脫，檢測波長為 254 nm，柱溫30 ℃；電噴霧離子源（ESI）全掃模式；根據一級質譜中的分子離子，推測化合物的相對分子質量和元素組成；再利用二級質譜信息，與對照品及文獻報道中化合物信息庫進行比對，推測五神湯的體外化學成分。HPLC 法檢測五神湯中京尼平苷酸、秦皮甲素、咖啡酸、木犀草苷、β-蛻皮甾酮和毛蕊花糖苷，Aglient Eclipse XDB-C₁₈色譜柱（150 mm×4.6 mm，5 mm），流動相為0.1%磷酸水（A）-乙腈（B），梯度洗脫，檢測波長254 nm，體積流量1.0 mL·min^-1，柱溫30 ℃，進樣量10 μL。結果 共檢測到五神湯中的71種化學成分，主要包括黃酮類、酚酸類、三萜皂苷類以及香豆素類；6種成分在濃度范圍內線性關系良好，平均加樣回收率為89.6%～93.0%。京尼平苷酸、秦皮甲素、咖啡酸、木犀草苷、β-蛻皮甾酮和毛蕊花糖苷在10批樣品中的平均質量濃度依次為 2.92、2.48、1.72、1.38、2.08、1.77 mg·g^-1。結論 建立的 UPLC-Q-Exactive Orbitrap-MS/MS、HPLC法可用于五神湯化學成分的鑒定和含量測定。

Objective To establish a method for the identification of chemical constituents in Wushen Decoction (WSD) by UPLC-QExactive orbitrap-MS/MS, then, establish a method for the determination of six components of WSD: geniposidic acid, esculin, caffeic acid, luteoloside, β -ecdysterone and verbascoside by HPLC.Methods Identification of chemical components in WSD by UPLC-Q-Empirical Orbitrap MS/MS method, the separation was performed on an ACQUIFY UPLCTMBEH C₁₈ column (100 mm×2.1 mm, 1.7 μm), the detection wavelength was 254 nm and the column temperature was 30 ℃ . Mass spectrum conditions: electrospray ion source (ESI) full sweep mode. The mobile phase was eluted by gradient elution with 0.1% formic acid water and acetonitrile at 254 nm. Infer the relative molecular weight and elemental composition of the compound based on the molecular ions in the primary mass spectrometry. By utilizing the secondary mass spectrometry information and comparing it with the reference material and the compound information library in literature, the in vitro chemical composition of WSD can be inferred. HPLC method was used to detect geniposidic acid, aesculin, caffeic acid, luteoloside, β -ecdysterone and verbascoside, Aglient Eclipse XDB-C₁₈ column (150 mm×4.6 mm, 5 mm), mobile phase consisting of 0.1% phosphoric acid water (A) - acetonitrile (B), gradient elution, detection wavelength of 254 nm, volume flow rate of 1.0 mL·min^-1, column temperature of 30 ℃, injection volume of 10 μL.Results A total of 71 chemical components were detected in WSD, including flavonoids, phenolic acids, triterpenoid saponins and coumarins, according to primary, secondary and control samples by mass spectrometry. The linear relationships of the six components were good in the concentration range, and the average recoveries were 89.6%—93.0%. The average contents of geniposidic acid, aesculin, caffeic acid, luteoloside, β-ecdysterone and verbascoside in ten batches of samples were 2.92, 2.48, 1.72, 1.38, 2.08, and 1.77 mg·g^-1, respectively.Conclusion The established UPLC-Q-Exactive Orbitrap-MS/MS and HPLC method are stable, reliable and can be used for the identification and content determination of the chemical constituents of WSD.

R286.02

遼寧省自然科學基金面上項目（2021-MS-361）