五神汤化学成分鉴定及京尼平苷酸、秦皮甲素、咖啡酸、木犀草苷、β-蜕皮甾酮和毛蕊花糖苷定量检测

Identification of chemical constituents and content determination of geniposidic acid,aesculin,caffeic acid,luteoloside,β-ecdysterone,and verbascoside in different batches of Wushen Decoction

目的采用超高效液相色谱串联四极杆静电场轨道阱质谱法(UPLC-Q-Exactive Orbitrap-MS/MS)对五神汤中的化学成分进行鉴定,进而对五神汤中京尼平苷酸、秦皮甲素、咖啡酸、木犀草苷、β-蜕皮甾酮和毛蕊花糖苷6种成分进行含量测定。方法UPLC-Q-Exactive Orbitrap-MS/MS法鉴定五神汤中的化学成分,采用ACQUIFY UPLCTMBEH C18色谱柱(100 mm×2.1 mm,1.7μm)进行分离,以0.1%甲酸-乙腈为流动相进行梯度洗脱,检测波长为254 nm,柱温30℃;电喷雾离子源(ESI)全扫模式;根据一级质谱中的分子离子,推测化合物的相对分子质量和元素组成;再利用二级质谱信息,与对照品及文献报道中化合物信息库进行比对,推测五神汤的体外化学成分。HPLC法检测五神汤中京尼平苷酸、秦皮甲素、咖啡酸、木犀草苷、β-蜕皮甾酮和毛蕊花糖苷,Aglient Eclipse XDB-C18色谱柱(150 mm×4.6 mm,5 mm),流动相为0.1%磷酸水(A)-乙腈(B),梯度洗脱,检测波长254 nm,体积流量1.0 mL·min^(−1),柱温30℃,进样量10μL。结果共检测到五神汤中的71种化学成分,主要包括黄酮类、酚酸类、三萜皂苷类以及香豆素类;6种成分在浓度范围内线性关系良好,平均加样回收率为89.6%~93.0%。京尼平苷酸、秦皮甲素、咖啡酸、木犀草苷、β-蜕皮甾酮和毛蕊花糖苷在10批样品中的平均质量浓度依次为2.92、2.48、1.72、1.38、2.08、1.77 mg·g^(−1)。结论建立的UPLC-Q-Exactive Orbitrap-MS/MS、HPLC法可用于五神汤化学成分的鉴定和含量测定。

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,1.7μm),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 10μL.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.