基于全二维液相色谱的牛黄上清丸指纹图谱研究
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摘要
目的研究牛黄上清丸全二维液相色谱指纹图谱的评价方法,并建立三维指纹图谱,为牛黄上清丸的质量控制提供新方法。方法第一维采用Acquity UPLC HSS CYANO(100 mm×2.1 mm,1.8μm)色谱柱,柱温40℃,流动相为0.1%甲酸水溶液-甲醇,体积流量0.1 mL/min;第二维采用Kinetex Phenyl-Hexyl(50 mm×3 mm,2.6μm)色谱柱,流动相为0.1%甲酸水溶液-乙腈,体积流量1.5 mL/min,柱温40℃,检测波长254 nm,样品采集速率为12.5 Hz;连接第一维与第二维色谱柱的定量环体积为100μL。建立牛黄上清丸的全二维液相色谱指纹图谱,采用欧氏距离、夹角余弦和相关系数法,分别以各共有峰峰体积的中位数、平均数的计算结果为标准对照指纹图谱,对21批市售牛黄上清丸样品进行相似度评价。结果建立了21批市售牛黄上清丸的三维指纹图谱,标定了共有峰18个,并指认了其中的5个共有峰:栀子苷(1号峰)、胡薄荷酮(8号峰)、黄芩苷(9号峰)、欧前胡素(15号峰)、汉黄芩素(16号峰)。结论首次建立了基于全二维液相色谱的牛黄上清丸的指纹图谱及其相似度的评价方法,说明该法可用于中药色谱指纹图谱的建立,该方法也是对传统液相色谱指纹图谱的补充和提高。
Objective To develop a new three-dimensional fingerprinting method and its assessing methods based on comprehensive two-dimensional liquid chromatography using Niuhuang Shangqing Pills(NSP) as an example. The developed method could offer new method for the quality control of NSP. Methods In first dimension, the separation was achieved with an Acquity UPLC HSS CYANO column(100 mm × 2.1 mm, 1.8 μm), methanol-0.1% formic acid in water were used as mobile phases, flow rates were 0.1 m L/min. In second dimension, the separation was achieved with a Kinetex Phenyl-Hexyl column(50 mm × 3 mm, 2.6 μm), acetonitrile-0.1% formic acid in water were used as mobile phases, flow rates were 1.5 mL/min, detection wavelength was set at 254 nm, and acquiring frequency was at 12.5 Hz. Column temperature for each dimension was 40 ℃ and volume of loop linking the two dimensions was 100 μL. Three similarity-calculating methods, Euclidean Distance, Cosine, and Correlation Coefficient, were employed to assess the similarities among the 21 samples on the market using medians with arithmetic means of peak volumes of the common peaks as control fingerprints. Results The three-dimensional fingerprints of 21 batches of NSP samples on the market were developed; Eighteen common peaks were assigned and five of them were identified, which were geniposide(1), pulegone(8), baicalin(9), imperatorin(15), and wogonin(16). Conclusion A three-dimensional fingerprinting method and its assessing methods based on comprehensive two-dimensional liquid chromatography using NSP as an example were successfully developed for the first time, suggesting that it is a feasible method for developing fingerprints for Chinese materia medica. This work improves and supplements the traditional liquid-chromatography fingerprints.
Objective To develop a new three-dimensional fingerprinting method and its assessing methods based on comprehensive two-dimensional liquid chromatography using Niuhuang Shangqing Pills(NSP) as an example. The developed method could offer new method for the quality control of NSP. Methods In first dimension, the separation was achieved with an Acquity UPLC HSS CYANO column(100 mm × 2.1 mm, 1.8 μm), methanol-0.1% formic acid in water were used as mobile phases, flow rates were 0.1 m L/min. In second dimension, the separation was achieved with a Kinetex Phenyl-Hexyl column(50 mm × 3 mm, 2.6 μm), acetonitrile-0.1% formic acid in water were used as mobile phases, flow rates were 1.5 mL/min, detection wavelength was set at 254 nm, and acquiring frequency was at 12.5 Hz. Column temperature for each dimension was 40 ℃ and volume of loop linking the two dimensions was 100 μL. Three similarity-calculating methods, Euclidean Distance, Cosine, and Correlation Coefficient, were employed to assess the similarities among the 21 samples on the market using medians with arithmetic means of peak volumes of the common peaks as control fingerprints. Results The three-dimensional fingerprints of 21 batches of NSP samples on the market were developed; Eighteen common peaks were assigned and five of them were identified, which were geniposide(1), pulegone(8), baicalin(9), imperatorin(15), and wogonin(16). Conclusion A three-dimensional fingerprinting method and its assessing methods based on comprehensive two-dimensional liquid chromatography using NSP as an example were successfully developed for the first time, suggesting that it is a feasible method for developing fingerprints for Chinese materia medica. This work improves and supplements the traditional liquid-chromatography fingerprints.
引文
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[20]张艳海,金燕,王峥涛.在线二维多中心切割液相色谱法测定三七、人参及其相关产品中8种人参皂苷[J].中草药,2017,48(5):894-901.
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[22]周文杰.RP-HPLC法测定牛黄上清丸中盐酸小檗碱的含量[J].中国药师,2009,12(2):264-265.
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[25]吴芳,乔蓉霞,杨瑞瑞,等.牛黄上清丸(片、胶囊)挥发油特征图谱研究[J].中成药,2012,34(7):1388-1390.
[26]Murphy R E,Schure M R,Foley J P.Effect of sampling rate on resolution in comprehensive two-dimensional liquid chromatography[J].Anal Chem,1998,70(8):1585-1594.
[27]Thekkudan D F,Rutan S C,Carr P W.A study of the precision and accuracy of peak quantification in comprehensive two-dimensional liquid chromatography in time[J].J Chromatogr A,2010,1217(26):4313-4327.
[28]Dugo P,Cacciola F,Kumm T,et al.Comprehensive multidimensional liquid chromatography:Theory and applications[J].J Chromatogr A,2008,1184(1/2):353-368.
[29]Bedani F,Schoenmakers P J,Janssen H G.Theories to support method development in comprehensive two-dimensional liquid chromatography-a review[J].JSep Sci,2012,35(14):1697-1711.
[30]Wang D D,Liang J,Yang W Z,et al.HPLC/qTOF-MS-oriented characteristic components data set and chemometric analysis for the holistic quality control of complex TCM preparations:Niuhuang Shangqing pill as an example[J].J Pharm Biomed Anal,2014,89:130-141.
[31]Qiao X,Song W,Ji S,et al.Separation and detection of minor constituents in herbal medicines using a combination of heart-cutting and comprehensive twodimensional liquid chromatography[J].J Chromatogr A,2014,1362:157-167.
[2]张娟娟,孙巍,张磊,等.复方丹参滴丸及原药材指纹图谱研究进展[J].药物评价研究,2017,40(6):859-865.
[3]刘昌孝.基于中药质量标志物的中药质量追溯系统建设[J].中草药,2017,48(18):3669-3676.
[4]佘一鸣,胡永慧,韩立云,等.中药质量控制的研究进展[J].中草药,2017,48(12):2557-2563.
[5]Liu C X,Cheng Y Y,Guo D A,et al.A new concept on quality marker for quality assessment and process control of Chinese medicines[J].Chin Herb Med,2017,9(1):3-13.
[6]Stoll D R,Li X,Wang X,et al.Fast,comprehensive two-dimensional liquid chromatography[J].JChromatogr A,2007,1168(1/2):3-43.
[7]Dugo P,Herrero M,Kumm T,et al.Comprehensive normal-phase×reversed-phase liquid chromatography coupled to photodiode array and mass spectrometry detection for the analysis of free carotenoids and carotenoid esters from mandarin[J].J Chromatogr A,2008,1189(1/2):196-206.
[8]Erni F,Frei R W.Two-dimensional column liquid chromatographic technique for resolution of complex mixture[J].J Chromatogr A,1978,149:561-569.
[9]Wang Y,Kong L,Lei X Y,et al.Comprehensive twodimensional high-performance liquid chromatography system with immobilized liposome chromatography column and reversed-phase column for separation of complex traditional Chinese medicine Longdan Xiegan Decoction[J].J Chromatogr A,2009,1216(11):2185-2191.
[10]Wang C,Wang S W,Fan G,et al.Screening of antinociceptive components in Corydalis yanhusuo W.T.Wang by comprehensive two-dimensional liquid chromatography/tandem mass spectrometry[J].Anal Bioanal Chem,2010,396(5):1731-1740.
[11]Liu Z,Zhu D,Qi Y,et al.Elucidation of steroid glycosides in Anemarrhena asphodeloides extract by means of comprehensive two-dimensional reversedphase/polyamine chromatography with mass spectrometric detection[J].J Sep Sci,2012,35(17):2210-2218.
[12]Wang S,Wang C,Zhao X,et al.Comprehensive twodimensional high performance liquid chromatography system with immobilized liposome chromatography column and monolithic column for separation of the traditional Chinese medicine Schisandra chinensis[J].Analytica Chimica Acta,2012,713:121-129.
[13]Li D,Schmitz O J.Use of shift gradient in the second dimension to improve the separation space in comprehensive two-dimensional liquid chromatography[J].Anal Bioanal Chem,2013,405(20):6511-6517.
[14]Li D,Schmitz O J.Comprehensive two-dimensional liquid chromatography tandem diode array detector(DAD)and accurate mass QTOF-MS for the analysis of flavonoids and iridoid glycosides in Hedyotis diffusa[J].Anal Bioanal Chem,2014,407(1):231-240.
[15]Qu L,Xiao Y,Jia Z,et al.Comprehensive twodimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry for chemical constituents analysis of tripterygium glycosides tablets[J].J Chromatogr A,2015,1400:65-73.
[16]Wang S,Wang Q,Qiao X,et al.Separation and characterization of triterpenoid saponins in Gleditsia sinensis by comprehensive two-dimensional liquid chromatography coupled with mass spectrometry[J].Planta Med,2016,82(18):1558-1567.
[17]Cao J L,Wang S S,Hu H,et al.Online comprehensive two-dimensional hydrophilic interaction chromatography×reversed-phase liquid chromatography coupled with hybrid linear ion trap orbitrap mass spectrometry for the analysis of phenolic acids in Salvia miltiorrhiza[J].JChromatogr A,2017,1536:216-227.
[18]Zhou W J,Guo Z M,Yu L,et al.On-line comprehensive two-dimensional liquid chromatography tandem mass spectrometry for the analysis of Curcuma kwangsiensis[J].Talanta,2018,186:73-79.
[19]朱靖博,籍立新,萧伟,等.反相二维色谱制备甘草中黄酮类化合物[J].中草药,2018,49(9):2033-2040.
[20]张艳海,金燕,王峥涛.在线二维多中心切割液相色谱法测定三七、人参及其相关产品中8种人参皂苷[J].中草药,2017,48(5):894-901.
[21]中国药典[S].一部.2015.
[22]周文杰.RP-HPLC法测定牛黄上清丸中盐酸小檗碱的含量[J].中国药师,2009,12(2):264-265.
[23]范红.牛黄上清丸中多指标成分含量测定方法研究[J].中国医药导刊,2011,13(6):1074-1075.
[24]杨小军,丁永辉.RP-HPLC法测定牛黄上清丸中栀子苷、芍药苷、黄芩苷、小檗碱的含量[J].西部中医药,2016,29(2):27-33.
[25]吴芳,乔蓉霞,杨瑞瑞,等.牛黄上清丸(片、胶囊)挥发油特征图谱研究[J].中成药,2012,34(7):1388-1390.
[26]Murphy R E,Schure M R,Foley J P.Effect of sampling rate on resolution in comprehensive two-dimensional liquid chromatography[J].Anal Chem,1998,70(8):1585-1594.
[27]Thekkudan D F,Rutan S C,Carr P W.A study of the precision and accuracy of peak quantification in comprehensive two-dimensional liquid chromatography in time[J].J Chromatogr A,2010,1217(26):4313-4327.
[28]Dugo P,Cacciola F,Kumm T,et al.Comprehensive multidimensional liquid chromatography:Theory and applications[J].J Chromatogr A,2008,1184(1/2):353-368.
[29]Bedani F,Schoenmakers P J,Janssen H G.Theories to support method development in comprehensive two-dimensional liquid chromatography-a review[J].JSep Sci,2012,35(14):1697-1711.
[30]Wang D D,Liang J,Yang W Z,et al.HPLC/qTOF-MS-oriented characteristic components data set and chemometric analysis for the holistic quality control of complex TCM preparations:Niuhuang Shangqing pill as an example[J].J Pharm Biomed Anal,2014,89:130-141.
[31]Qiao X,Song W,Ji S,et al.Separation and detection of minor constituents in herbal medicines using a combination of heart-cutting and comprehensive twodimensional liquid chromatography[J].J Chromatogr A,2014,1362:157-167.
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