基于UPLC特征图谱和QAMS对怀牛膝药材质量控制的应用研究

Study on quality control of Achyranthes bidentata Bl.based on UPLC and QAMS

目的:建立中药材怀牛膝成分的超高效液相(UPLC)特征图谱和一测多评法(QAMS)的实验方法,对其质量控制与评价进行研究。方法:以怀牛膝药材为研究对象,用β-蜕皮甾酮作为内参物,采用Waters BEH C_(18)(1.7μm,2.1 mm×100 mm)色谱柱,在柱温30°C,流速0.2 mL/min,检测波长250 nm条件下,以乙腈(A)-0.1%甲酸水(B)混合溶液进行梯度洗脱,建立5-羟甲基糠醛、杯苋甾酮与β-蜕皮甾酮的RCF,并计算含量;同时以ESM法测定10批怀牛膝药材中5-羟甲基糠醛、杯苋甾酮、β-蜕皮甾酮3种成分的含量,比较QAMS法计算值与ESM法实测值的差异。结果:完成了怀牛膝多指标UPLC特征图谱的建立,并标定了12个共有峰,指认了其中的5-羟甲基糠醛、杯苋甾酮和β-蜕皮甾酮3个特征峰,10批样品特征图谱与对照特征图谱的相似度>0.920;建立的QAMS法计算的含量值与EMS法实测值之间差异无统计学意义,RCF的重现性良好。结论:本实验研究建立的怀牛膝药材UPLC-QAMS法对怀牛膝药材中的5-羟甲基糠醛、杯苋甾酮、β-蜕皮甾酮3种成分可进行有效的定性与定量测定,该方法科学有效、简便省时、结果可靠。

Objective:To establish the characteristic atlas for Achyranthes bidentata Bl.based on ultra-performance liquid chromatography(UPLC)fingerprint and quantitative analysis of multi-components by single marker(QAMS)technique,and evaluate the value of this technique in quality control of Achyranthes bidentata Bl.extraction.Methods:Achyranthes bidentata Bl.was used as the research material,and β-ecdysterone as internal reference.Waters BEH C_(18)(1.7μm,2.1 mm×100 mm)column,by which temperature was set at 30°C,with flow rate being at 0.2 ml·min^(-1) and detection wavelength being at 250 nm,was used to elute Achyranthes bidentata Bl using acetonitrile(A)-0.1%formic acid water(B)mixture solution in gradient fashion,and relative correction factor(RCF)for 5-hydroxymethylfurfural,calicosterone andβ-ecdysterone was established.The contents of 5-hydroxymethylfurfural,calicosterone andβ-ecdysterone in 10 batches of Achyranthes bidentata Bl.were determined by ESM method,and the differences between the calculated values by QAMs and those measured by ESM were compared.Results:The characteristic atlas of Achyranthes bidentata Bl.was established based on multi index UPLC,with 12 common peaks demarcated.Three characteristic peaks of 5-hydroxymethylfurfural,calicosterone andβ-ecdysterone were identified from the common peaks.The similarity between the characteristic atlas of 10 batches of samples and that of control was more than 0.920.There was no significant difference between the content value calculated by QAMS and the measured value by EMS method.Better reproducibility of RCF was observed.Conclusion:The UPLC-QAMS method can be used for the qualitative and quantitative determination of 5-hydroxymethylfurfural,calicosterone andβ-ecdysterone in Achyranthes bidentata Bl.,and this technique is scientific,effective,simple,time-saving and reliable.