青蛙七中异黄酮类成分质量控制研究

Quality control study of isoflavones in Iris tectorum Maxim.

目的:依据对青蛙七主要成分的表征结果,建立青蛙七薄层色谱(TLC)定性鉴别方法及一测多评(QAMS)的含量测定方法。方法:首先,采用超高效液相色谱-四极杆-飞行时间质谱联用(UPLC-Q TOF MS/MS)技术对青蛙七的主要成分进行表征,使用ACQUITY UPLC^(Ⓡ)BEH C_(18)(100 mm×2.1 mm,1.7μm)色谱柱,以0.1%甲酸水溶液(A)-乙腈溶液(B)为流动相进行梯度洗脱,正离子模式下采集信息;其次,通过TLC建立定性鉴别方法;最后,建立QAMS方法,采用Agilent 5 TC-C_(18)(250 mm×4.6 mm,5μm)色谱柱,流动相为0.05%磷酸水(A)-乙腈(B),梯度洗脱,以鸢尾黄素为内参物,建立其与鸢尾苷及野鸢尾黄素的相对校正因子,并通过校正因子计算3个异黄酮类成分的含量。同时与外标法(ESM)进行比较,以验证QAMS法的准确性和可行性。结果:UPLC-Q TOF MS共表征出青蛙七中5个含量较高的异黄酮类成分(鸢尾苷、鸢尾甲苷B、鸢尾黄素、野鸢尾黄素、鸢尾黄酮A)。建立了鸢尾苷、鸢尾甲苷B、鸢尾黄素、野鸢尾黄素4个成分的TLC鉴别法。在一定的浓度范围内,鸢尾苷、鸢尾黄素、野鸢尾黄素3个成分线性关系良好(r﹥0.999),平均加样回收率为97.7%~100.8%,RSD为1.1%~2.9%,鸢尾苷、野鸢尾黄素的相对校正因子分别为1.1145、0.8270,在不同试验条件下重现性良好(RSD<5%);采用QAMS法测定的10个不同产地青蛙七中3个成分含量与ESM的实测值之间无显著性差异。结论:以表征的青蛙七药材的成分为基础,选择含量较高且稳定的成分建立的TLC和QAMS方法简便、稳定,可用于青蛙七药材定性定量分析及质量评价。

Objective:To establish methods of thin-layer chromatography(TLC)qualitative identification and quantitative analysis of multi-components by single-marker(QAMS)according to the characterization results of the main components of Iris tectorum Maxim..Methods:Firstly,the main components of Iris tectorum were characterized by ultra performance liquid chromatography tandem time-of-flight mass spectrometry(UPLC-Q TOF MS/MS)technology,using an ACQUITY UPLC^(Ⓡ)BEH C_(18) column(100 mm×2.1 mm,1.7μm)with gradient elution using 0.1%formic acid-water(A)-acetonitrile(B)as mobile phase,and the data were collected in positive ion mode.In addition,the qualitative identification method was established by the TLC.Finally,quantitative analysis was performed on an Agilent 5 TC-C18 column(250 mm×4.6 mm,5μm).The mobile phase was 0.05%phosphoric acid water(A)-acetonitrile(B)with gradient elution.The QAMS method was established by using tectorigenin as an internal standard to establish its relative correction factor with tectoridin and irigenin,and the contents of three isoflavones were calculated by the correction factor.At the same time,the accuracy and feasibility of the QAMS method was verified by the external standard method(ESM).Results:Five isoflavones were characterized(tectoridin,iristectorin B,tectorigenin,irigenin and iristectorigenin A)by UPLC-Q TOF MS/MS.A TLC identification method for four components,tectoridin,iristectorin B,tectorigenin and irigenin was established.In this study,the linearity of the three components of tectoridin,tectorigenin and irigenin were good in a certain concentration range(r>0.999),with the average recoveries of 97.7%-100.8%and the RSDs of 1.1%-2.9%,and the relative correction factors of tectoridin and irigenin were 1.1145 and 0.8270,respectively.And the repeatability of the correction factors was good under different experimental conditions(RSD<5%).There was no significant difference between the contents of the three analytes in ten different batches of Iris tecto⁃rum determined by QAMS and ESM.The contents of tectorigenin,tectoridin and irigenin in 10 batches of Iris tec⁃torum from different habitats determined by QAMS were not significantly different from those determined by ESM.Conclusion:Based on the characterized components of Iris tectorum medicinal materials,the TLC and QAMS methods with high-content and stable ingredients are established.The methods are simple and stable,which can be used for qualitative and quantitative analysis and quality evaluation of Iris tectorum.