基于HPLC-Q-TOF-MS/MS定性分析当归中多种香豆素类成分
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摘要
目的:建立高效、稳定的HPLC-Q-TOF-MS/MS方法,系统、全面地定性分析当归中多种香豆素成分,为当归质控标准的完善、香豆素的开发及临床应用提供依据。方法:通过调整液相色谱柱种类、温度、流动相、流速、样品浓度等条件,建立可高效分离当归提取液中香豆素成分的HPLC-Q-TOF-MS/MS,并结合对照品、精确相对分子质量、极性、裂解规律、参考文献等鉴定当归中多种香豆素类成分。结果:该研究建立了一种高效、稳定的香豆素分离方法,并鉴定出14种香豆素成分,发现其存在大量的同分异构体,其中珊瑚菜素、王草酚等香豆素成分较少作为当归成分被报道;同时通过分析香豆素类成分的主要碎片离子,发现香豆素类成分在质谱中主要通过母核侧链的甲氧基键或苯甲醚键的断裂以及侧链上其他基团的丢失而裂解,总结出香豆素成分的一般裂解规律。结论:当归中含有大量的香豆素类成分,将其深入定性、定量分析可进一步完善当归的质量标准,并为香豆素的开发及当归的临床应用提供参考。
Objective: To systematically and comprehensively analyze coumarin components in Angelicae Sihensis Radix by an efficient and stable HPLC-Q-TOF-MS/MS method,in order to offer the theoretical basis to develop coumarin,establish the quality control standard and apply in clinic. Method: The separation effect of coumarin components was extracted by adjusting the column, temperature, mobile phase, flow rate, sample concentration and other conditions,and various coumarin components in Angelicae Sihensis Radix were identified by corresponding standards,precise molecular mass,polarity,pyrolysis pattern. Result: In this study,a highefficiency and stable coumarin separation method was established that can be used to separate complex components,and 14 coumarin components were identified in this study,including phellopterin and osthenol that were rarely reported as effective components in Angelicae Sihensis Radix. Major fragment ions of coumarin components were analyzed. The cleavage in methoxy bond or anisole bond on the parent nucleus was the primary pattern for coumarin components,which was summarized for detecting unknowing coumarins. Conclusion: Abundant coumarins were contained in Angelicae Sihensis Radix. Further qualitative and quantitative analysis of coumarins are conducive to improving the quality standards of Angelicae Sihensis Radix, and providing reference for the development of coumarins and clinical application of Angelicae Sinensis Radix.
Objective: To systematically and comprehensively analyze coumarin components in Angelicae Sihensis Radix by an efficient and stable HPLC-Q-TOF-MS/MS method,in order to offer the theoretical basis to develop coumarin,establish the quality control standard and apply in clinic. Method: The separation effect of coumarin components was extracted by adjusting the column, temperature, mobile phase, flow rate, sample concentration and other conditions,and various coumarin components in Angelicae Sihensis Radix were identified by corresponding standards,precise molecular mass,polarity,pyrolysis pattern. Result: In this study,a highefficiency and stable coumarin separation method was established that can be used to separate complex components,and 14 coumarin components were identified in this study,including phellopterin and osthenol that were rarely reported as effective components in Angelicae Sihensis Radix. Major fragment ions of coumarin components were analyzed. The cleavage in methoxy bond or anisole bond on the parent nucleus was the primary pattern for coumarin components,which was summarized for detecting unknowing coumarins. Conclusion: Abundant coumarins were contained in Angelicae Sihensis Radix. Further qualitative and quantitative analysis of coumarins are conducive to improving the quality standards of Angelicae Sihensis Radix, and providing reference for the development of coumarins and clinical application of Angelicae Sinensis Radix.
引文
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[20] ZHAO A H,ZHANG Y B,YANG X W. Simultaneous determination and pharmacokinetics of sixteen Angelicae dahurica coumarins in vivo by LC-ESI-MS/MS following oral delivery in rats[J]. Phytomedicine,2016,23(10):1029-1036.
[21] SONG G S,JIN M M,DU Y F,et al. UPLC-QTOF-MS/MS based screening and identification of the metabolites in rat bile after oral administration of imperatorin[J]. J Chromatogr B,2016,1022(11):21-29.
[22] BAI Y,LI D H,ZHOU T T,et al. Coumarins from the roots of Angelica dahurica with antioxidant and antiproliferative activities[J]. J Funct Food,2016,20(7):453-462.
[23] SHI X W,WANG S,QIAN Q,et al. Herb-herb pharmacokinetic interaction between Glehniae Radix and Ophiopogonis Radix in rats using superimposed multiple product ion(SMPI)LC-HR-MS/MS[J]. Rsc Adv,2017,7(47):29732-29741.
[24] ZHAO A H,ZHANG Y B,YANG X W. Simultaneous determination and pharmacokinetics of sixteen Angelicae dahurica coumarins in vivo by LC-ESI-MS/MS following oral delivery in rats[J]. Phytomedicine,2016,23(10):1029-1036.
[25] YANG A H,CHEN J X,MA Y T,et al. Studies on the metabolites difference of psoralen/isopsoralen in human and six mammalian liver microsomes in vitro by UHPLCMS/MS[J]. J Pharm Biomed Anal,2017,141(8):200-209.
[26] YANG Y F,ZHANG L,ZHANG Y B,et al. Simultaneous assessment of absorption characteristics of coumarins from Angelicae Pubescentis Radix:in vitro transport across Caco-2 cell and in vivo pharmacokinetics in rats after oral administration[J]. J Chromatogr B,2017,1060(5):308-315.
[27]徐厚谦,颜春鲁,张永花,等.当归补血汤通过PI3K/Akt通路对AngⅡ诱导肥大心肌细胞的保护作用[J].中国实验方剂学杂志,2018,24(2):135-139.
[28]杨艺萱,张兆芳,刘卉,等.当归红芪超滤物对辐射后心肌成纤维细胞的增殖和胶原分泌的影响[J].中国实验方剂学杂志,2018,24(12):136-142.
[2]王雁梅,任京力,朱吾元,等.不同炮制方式对当归中有效成分含量的影响[J].中国实验方剂学杂志,2014,20(7):42-45.
[3]黄明军,孙耀志,高松,等. LC-MS分析当归六黄汤中主要成分[J].中国实验方剂学杂志,2016,22(9):63-67.
[4] Kim A N,Cho J L,Son N R,et al. Development of analytical method for determination of baicalin,eleutheroside E,and ligustilide by LC-MS/MS in raw materials of health functional foods[J]. J Food Saf,2017,32(3):193-198.
[5] HU P Y,LIU D,QIN Z,et al. Elucidation of transport mechanism of paeoniflorin and the influence of ligustilide,senkyunolide I and senkyunolide A on paeoniflorin transport through Mdck-Mdr1 cells as bloodbrain barrier in vitro model[J]. Molecules,2016,21(3):1137-1149.
[6] MA W,WANG W H,PENG Y,et al. Ultra-high performance liquid chromatography with tandem mass spectrometry method for the simultaneous quantitation of five phthalides in rat plasma:application to a comparative pharmacokinetic study of Huo Luo Xiao Ling Dan and herb-pair extract[J]. J Sep Sci,2016,39(11):2057-2067.
[7] LI W X,TANG Y P,QIAN Y F,et al. Comparative analysis of main aromatic acids and phthalides in Angelicae Sinensis Radix,Chuanxiong Rhizoma,and FoShou-San by a validated UHPLC-TQF-MS/MS[J]. J Pharm Biomed Anal,2014,99(11):45-50.
[8] Bartnik M,Slawinska-Brych A,Zurek A,et al.8-methoxypsoralen reduces Akt phosphorylation,induces intrinsic and extrinsic apoptotic pathways, and suppresses cell growth of SK-N-AS neuroblastoma and SW620 metastatic colon cancer cells[J]. J Ethnopharmacol,2017,207:19-29.
[9] Franklin C,Cesko E,Hillen U,et al. Modulation and apoptosis of neutrophil granulocytes by extracorporeal photopheresis in the treatment of chronic graft-versushost disease[J]. PLo S One,2017,10(8):1124-1136.
[10] OUYANG J Y,JIANG H L,et al. Isoimperatorin ameliorates osteoarthritis by downregulating the mammalian target of rapamycin C1 signaling pathway[J]. Mol Med Rep,2017,16(6):9636-9644.
[11] Wijerathne C U B,Seo C S,Song J W,et al.Isoimperatorin attenuates airway inflammation and mucus hypersecretion in an ovalbumin-induced murine model of asthma[J]. Int Immunopharmacol,2017,49(6):67-76.
[12]仝家羽,赵嵘,代云桃,等.当归标准汤剂质量评价体系的建立[J].中国实验方剂学杂志,2017,23(7):18-23.
[13] TIAN W Q,CAI J,XU Y Y,et al. Determination of xanthotoxin using a liquid chromatography-mass spectrometry and its application to pharmacokinetics and tissue distribution model in rat[J]. Int J Clin Exp Med,2015,8(9):15164-15172.
[14] Ngo L,Tran P,Ham S H,et al. Simultaneous determination of imperatorin and its metabolite xanthotoxol in rat plasma and urine by LC-MS/MS and its application to pharmacokinetic studies[J]. J Chromatogr B,2017,1044/1045:30-38.
[15] WANG Y,YANG H J,MA Y Y. Simultaneous determination of three furanocoumarins by UPLC/MS/MS:application to pharmacokinetic study of Angelica dahurica Radix after oral administration to normal and experimental colitis-induced rats[J]. Molecules,2017,22(3):109-123.
[16] HUO H L,JIA P P,ZHANG X X,et al. Tentative identification of new metabolites of cnidilin by liquid chromatography-mass spectrometry[J]. J Chromatogr B,2015,995(5):85-92.
[17] HUO H L,YU S H,X Z LIU,et al. Simultaneous and sensitive determination of eight coumarins in rat bile and urine after oral administration of Radix Angelicae Dahuricae extract by liquid chromatography-electrospray ionization-mass spectrometry[J]. Acta Chromatogr,2013,25(2):201-219.
[18] YUAN L,ZHANG Y Q,LIAO M,et al. UHPLC-Q-TOFMS/MS-based screening and characterization of metabolites of cnidilin in human liver microsomes[J].Biomed Chromatogr,2017,31(11):3992.
[19] Lien N,Tran P,Ham S H,et al. Simultaneous determination of imperatorin and its metabolite xanthotoxol in rat plasma and urine by LC-MS/MS and its application to pharmacokinetic studies[J]. J Chromatogr B,2017,1044(9):30-38.
[20] ZHAO A H,ZHANG Y B,YANG X W. Simultaneous determination and pharmacokinetics of sixteen Angelicae dahurica coumarins in vivo by LC-ESI-MS/MS following oral delivery in rats[J]. Phytomedicine,2016,23(10):1029-1036.
[21] SONG G S,JIN M M,DU Y F,et al. UPLC-QTOF-MS/MS based screening and identification of the metabolites in rat bile after oral administration of imperatorin[J]. J Chromatogr B,2016,1022(11):21-29.
[22] BAI Y,LI D H,ZHOU T T,et al. Coumarins from the roots of Angelica dahurica with antioxidant and antiproliferative activities[J]. J Funct Food,2016,20(7):453-462.
[23] SHI X W,WANG S,QIAN Q,et al. Herb-herb pharmacokinetic interaction between Glehniae Radix and Ophiopogonis Radix in rats using superimposed multiple product ion(SMPI)LC-HR-MS/MS[J]. Rsc Adv,2017,7(47):29732-29741.
[24] ZHAO A H,ZHANG Y B,YANG X W. Simultaneous determination and pharmacokinetics of sixteen Angelicae dahurica coumarins in vivo by LC-ESI-MS/MS following oral delivery in rats[J]. Phytomedicine,2016,23(10):1029-1036.
[25] YANG A H,CHEN J X,MA Y T,et al. Studies on the metabolites difference of psoralen/isopsoralen in human and six mammalian liver microsomes in vitro by UHPLCMS/MS[J]. J Pharm Biomed Anal,2017,141(8):200-209.
[26] YANG Y F,ZHANG L,ZHANG Y B,et al. Simultaneous assessment of absorption characteristics of coumarins from Angelicae Pubescentis Radix:in vitro transport across Caco-2 cell and in vivo pharmacokinetics in rats after oral administration[J]. J Chromatogr B,2017,1060(5):308-315.
[27]徐厚谦,颜春鲁,张永花,等.当归补血汤通过PI3K/Akt通路对AngⅡ诱导肥大心肌细胞的保护作用[J].中国实验方剂学杂志,2018,24(2):135-139.
[28]杨艺萱,张兆芳,刘卉,等.当归红芪超滤物对辐射后心肌成纤维细胞的增殖和胶原分泌的影响[J].中国实验方剂学杂志,2018,24(12):136-142.