超高效液相色谱-四极杆飞行时间质谱法测定银黄颗粒中化合成分和主要成分

Qualitative and guantitative analysis of yinhuang granules using ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry

建立了超高效液相色谱-四极杆飞行时间质谱（UHPLC-QTOF-MS）测定银黄颗粒中活性成分的定性和定量分析方法。银黄颗粒经甲醇萃取后,采用RRHD SB-C18超高效液相色谱柱进行分离,以0.5%甲酸水溶液-乙腈为流动相进行梯度洗脱,21种主要化学成分在25 min内得到良好的分离。化合物的定性分析通过QTOF-MS的精确分子量测定和碰撞诱导解离（CID）实验进行,其中14个化合物得到了推导鉴定,12个化合物通过与对照品比较保留时间、精确分子量和多级质谱信息得到了结构确证。定量分析通过提取目标化合物的准分子离子峰色谱图（精确至±0.001Da）进行,12种目标化合物的线性相关系数r≥0.9961;检出限和定量限分别在0.01~0.07μg/m L和0.04~0.24μg/m L范围。方法用于测定20批银黄颗粒,银黄颗粒中12种活性成分的质量分数在0.66~62.1 mg/g范围。采用主成分分析（PCA）对这20批次银黄颗粒样品进行来源追踪,并对其质量的稳定性和一致性进行了评价。PCA得分图可将4个厂家的银黄颗粒明显区分开来,所建立的分析方法可有效用于银黄颗粒的质量评价与控制。

An ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry（ UHPLC/ QTOF-MS） method was established for qualitative and quantitative analysis of the active ingredients presenting in Yinhuang granules,which was a famous herbal preparation registered by Chinese Pharmacopoeia. Yinhuang Granules samples were extracted with methanol,and the extracts were separated by an RRHD SB-C18 column with 0. 5% formic acid aqueous solution and acetonitrile as mobile phase for gradient elution. 21 major components in Yinhuang granules were separated within 25 minutes,and 14 of the 21 constituents were identified via accurate mass measurements by QTOF-MS and collision induced dissociation（ CID） experiments of QTOF-MS /MS. Among them,12 constituents were identified by the comparison of their retention time and MS data with those of reference substances. Quantitative analysis of the 12 active ingredients was performed based on the extracted ion chromatograms of the exact pseudo-molecular ions（ with a 0. 01 Da window）. The method showed good linearity,with correlation coefficients（ r） no less than 0. 9961 for the 12 investigated analytes. Good accuracy and desirable stability were also obtained. The limits of detection and quantification were in the ranges of 0. 01 ~ 0. 07 μg/ m L and 0. 04- 0. 24 μg/ m L,respectively. The developed method was then used for determination of the contents of 12 active components present in 20 batches of Yinhuang granules samples. The results showed that the concentrations of the 12 investigated active components ranged from 0. 66 to 62. 1 mg/ g. Principal component analysis（ PCA） was performed to evaluate the quality stability and consistency and trace the origins of the 20 batches of investigated Yinhuang granules samples,and the results showed that four brands of Yinhuang granules produced by different manufactures are easy to differentiate. All the experimental results demonstrated that our developed UHPLC-QTOF-MS method was a desirable approach for quality assessment and control of Yinhuang granules.