隔山消的指纹图谱建立、化学模式识别分析及多指标含量测定
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篇名: 隔山消的指纹图谱建立、化学模式识别分析及多指标含量测定
TITLE: Establishment of fingerprint ,analysis of chemical pattern recognition and multi-index content determination of Cynanchum auriculatum
摘要: 目的 建立隔山消药材的指纹图谱并进行化学模式识别分析,同时测定其中4种成分的含量。方法采用高效液相色谱(HPLC)法。色谱柱为ACEUExcelC18,流动相为乙腈-0.1%磷酸溶液(梯度洗脱),流速为1.2mL/min,检测波长为210nm,柱温为40℃,进样量为10μL。以青阳参苷元为参照,采用《中药色谱指纹图谱相似度评价系统(2012版)》绘制16批隔山消药材的HPLC指纹图谱并进行相似度评价,确定共有峰。采用SPSS26.0软件和SIMCA14.0软件进行聚类分析、主成分分析和正交偏最小二乘-判别分析,以变量重要性投影(VIP)值大于1为标准筛选影响隔山消药材质量的差异性成分;采用相同的HPLC法测定丁香酸、去酰基萝藦苷元、白首乌二苯酮和青阳参苷元的含量。结果16批隔山消药材中共有29个共有峰,相似度为0.723~0.998;共指认了4个共有峰,分别为丁香酸(7号峰)、去酰基萝藦苷元(9号峰)、白首乌二苯酮(13号峰)、青阳参苷元(15号峰)。聚类分析结果显示,16批隔山消药材可聚为3类,其中S1为一类,S3为一类,S2、S4~S16为一类。主成分分析结果显示,5个主成分的累计方差贡献率为88.706%,分类结果与聚类分析结果一致。正交偏最小二乘-判别分析结果显示,VIP值大于1的共有峰由大到小依次为20号峰、10号峰、25号峰、12号峰、15号峰(青阳参苷元)、21号峰、14号峰、16号峰、26号峰、22号峰、17号峰。丁香酸、去酰基萝藦苷元、白首乌二苯酮和青阳参苷元检测质量浓度的线性范围分别为0.7153~45.7780、2.3794~152.2810、0.6420~41.0850、14.5416~930.6620µg/mL(R2均大于0.999);定量限分别为0.3577、0.4759、0.6420、2.4236μg/mL,检测限分别为0.1460、0.1641、0.2488、0.8333μg/mL;精密度、重复性、稳定性(24h)试验的RSD均小于3%;平均加样回收率分别为99.11%(RSD=2.00%,n=9)、98.54%(RSD=2.21%,n=9)、96.33%(RSD=2.54%,n=9)、95.96%(RSD=2.93%,n=9);含量分别为17.12~147.80、95.23~583.10(S8低于定量限)、16.91~210.88、211.68~3587.15(S1低于定量限)μg/g。结论所建HPLC指纹图谱和含量测定方法稳定、可靠、准确度高且重复性好,结合化学模式识别分析可用于隔山消药材的质量控制。
ABSTRACT: OBJECTIVE To establis h the fingerprint of Cynanchum auriculatum,to conduct its chemical pattern recognition analysis,and to determine the contents of four components at the same time. METHODS High performance liquid chromatography (HPLC)method was adopted. The determination was performed on ACE UExcel C 18 column with mobile phase consisted of acetonitrile-0.1% phosphoric acid solution (gradient elution )at the flow rate of 1.2 mL/min. The determination wavelength was set at 210 nm,and the column temperature was 40 ℃. The sample size wa s 10 μL. Taking qingyang shengenin as the reference ,HPLC fingerprints of 16 batches of C. auriculatum medicinal materials were drawn and similarity was evaluated by using the Similarity Evaluation of Chromatographic Fingerprints of Traditional Chinese Medicine (2012 edition), and the common peaks were determined. SPSS 26.0 software andSIMCA 14.0 software were used for cluster analysis ,principal component analysis and orthogonal partial least squares- discriminant analysis. The differential components affecting the quality of C. auriculatum were screened by taking the value of variable importance in projection (VIP)greater than 1 as the standard ;same HPLC method was used to determine the contents of syringic acid ,acyl asclepiadelenin ,baishouwubenzophenone and qingyang shengenin. RESULTS There were 29 common peaks in 16 batches of C. auriculatum ,with a similarity of 0.723-0.998. Four common peaks were identified ,namely syringic acid (peak 7),acyl asclepiadoidin (peak 9),baishouwubenzophenone(peak 13)and qingyang shengenin(peak 15). The results of cluster analysis showed that 16 batches of C. auriculatum could be clustered into three categories ,among which S 1 were grouped into one category,S3 were grouped into one c ategory,S2,and S 4-S16 were grouped into one category. The results of principal component analysis showed that the cumulative variance contribution rate of the five principal components was 88.706%,and the classification results were consistent with the results of cluster analysis. The results of orthogonal partial least squares-discriminant analysis showed that the common peaks (from large to small )with VIP value greater than 1 were peak 20,peak 10,peak 25,peak 12, peak 15(qingyangshengenin),peak 21,peak 14,peak 16,peak 26,peak 22 and peak 17. The linear ranges of syringic acid,acyl asclepterin,baishouwubenzophenone and qingyangshengenin were 0.715 3-45.778 0,2.379 4-152.281 0,0.642 0- 41.085 0,14.541 6- 930.662 0 µg/mL respectively (all R 2>0.999). The quantitative limits were 0.357 7,0.475 9,0.642 0 and 2.423 6 μg/mL;the detective limits were 0.146 0,0.164 1,0.248 8 and 0.833 3 μg/mL,respectively. RSDs of precision ,repeatability and stability (24 h)tests were less than 3%;the average recoveries were 99.11%(RSD=2.00%,n=9),98.54%(RSD=2.21%,n=9), 96.33%(RSD=2.54%,n=9)and 95.96%(RSD=2.93%,n=9);the contents were 17.12-147.80,95.23-583.10(S8 below the quantitative limit ),16.91-210.88 and 211.68-3 587.15(S1 below the quantitative limit )μg/g,respectively. CONCLUSIONS Established HPLC fingerprint and the method of content determination are stable ,reliable,accurate and reproducible. Combined with analysis of chemical pattern recognition ,it can be used for the quality control of C. auriculatum .
期刊: 2022年第33卷第06期
作者: 孙佳,勾健,秦兰,刘亭,潘洁,韦宇,刘春花
AUTHORS: SUN Jia,GOU Jian,QIN Lan,LIU Ting,PAN Jie,WEI Yu,LIU Chunhua
关键字: 隔山消;高效液相色谱法;指纹图谱;化学模式识别分析;含量测定
KEYWORDS: Cynanchum auriculatum ;high performance liquid chromatography ;fingerprint;analysis of chemical pattern
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