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Article(id=1209788337334915693, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1209788321396552074, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2021-1097, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1627228800000, receivedDateStr=2021-07-26, revisedDate=1633968000000, revisedDateStr=2021-10-12, acceptedDate=null, acceptedDateStr=null, onlineDate=1766365616400, onlineDateStr=2025-12-22, pubDate=1644595200000, pubDateStr=2022-02-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1766365616400, onlineIssueDateStr=2025-12-22, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1766365616400, creator=13701087609, updateTime=1766365616400, updator=13701087609, issue=Issue{id=1209788321396552074, tenantId=1146029695717560320, journalId=1189982191388893191, year='2022', volume='57', issue='2', pageStart='251', pageEnd='546', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1766365612600, creator=13701087609, updateTime=1766370652531, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1209809460445451136, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1209788321396552074, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1209809460445451137, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1209788321396552074, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=467, endPage=473, ext={EN=ArticleExt(id=1209788338073113247, articleId=1209788337334915693, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Determination of six effective ingredients in Imperatae Rhizoma concentrated granules by ultra-performance liquid chromatography using two internal references, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

To simultaneously determine the contents of p-coumaric acid, chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, caffeic acid and ferulic acid in Imperatae Rhizoma concentrated granules, an ultra-high performance liquid chromatography (UPLC) with two internal references method (TIRM) was established and validated. Chromatographic separation was achieved on a ZORBAX RRHD Eclipse Plus C18 column (2.1 mm×100 mm, 1.8 μm) using 1.7 mmol·L-1 oxalic acid in water and methanol as mobile phase. The flow rate was 0.4 mL·min-1 and the column temperature was set as 35℃. The relative correction factors (RCFs) of caffeic acid and ferulic acid using p-coumaric acid as internal reference were calculated and the RCFs of 4-caffeoylquinic acid and 5-caffeoylquinic acid were calculated using chlorogenic acid as the internal reference. The TIRM was fully validated for linearity, accuracy, repeatability, stability and recovery so that it could be compared with the external standard method (ESM). The RCFs of 5-caffeoylquinic acid, 4-caffeoylquinic acid, caffeic acid, and ferulic acid were 1.069, 1.022, 1.368, and 1.493, respectively. The TIRM and ESM were used to determine the contents of six ingredients in Imperatae Rhizoma concentrated granules from different manufacturers and the variation between results was within acceptable limits. In conclusion, the newly established TIRM allowed simultaneous determination of six ingredients (p-coumaric acid, chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, caffeic acid, ferulic acid) in Imperatae Rhizoma concentrated granules, providing support for the quality control of this traditional Chinese medicine.

, correspAuthors=Li YANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2022 Acta Pharmaceutica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Yu WANG, Zheng-cai JU, Ting ZHANG, Ya-fu XUE, Li YANG, Zheng-tao WANG), CN=ArticleExt(id=1209788339792777956, articleId=1209788337334915693, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=超高效液相色谱双内参法同时测定白茅根配方颗粒中六种有效成分, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

建立超高效液相色谱方法并采用基于一测多评的双内参法同时测定白茅根配方颗粒中有效成分对香豆酸、绿原酸、5-咖啡酰奎宁酸、4-咖啡酰奎宁酸、咖啡酸、阿魏酸的含量。采用ZORBAX RRHD Eclipse Plus C18（2.1 mm×100 mm，1.8 μm）色谱柱，流动相为1.7 mmol·L-1草酸水-甲醇溶液梯度洗脱，流速0.4 mL·min-1，柱温35℃，进样量1 μL。分别以对香豆酸为内参物，计算咖啡酸和阿魏酸的相对校正因子；以绿原酸为内参物，计算4-咖啡酰奎宁酸和5-咖啡酰奎宁酸的相对校正因子，考察其稳定性并利用相对保留时间法确定各化合物的色谱峰出峰时间。结果显示，5-咖啡酰奎宁酸、4-咖啡酰奎宁酸、咖啡酸和阿魏酸的相对校正因子分别为1.069、1.022、1.368和1.493，在不同条件下稳定性良好。使用外标法和双内参法对18批不同厂家的白茅根配方颗粒进行含量测定，两种方法所得的结果相对误差小于5.0%，表明建立的双内参法同时测定白茅根配方颗粒中6种有效成分可行，为白茅根配方颗粒质量标准建立提供依据。

, correspAuthors=杨莉, authorNote=null, correspAuthorsNote=
*杨莉, Tel: 86-21-51322506, Fax: 86-21-51322519, E-mail:
, copyrightStatement=版权所有©《药学学报》编辑部2022, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=ZEKEZA6yFlnobEhBupnT0g==, magXml=GaAFzkyv+DhzG6AAy+GjSg==, pdfUrl=null, pdf=8EnZfObPES5z0awZNkQUYA==, pdfFileSize=721099, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=20//fmk24lLwv2k2ZC4Rmw==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=mvjS9525WoDgq3/aPySSHQ==, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=王煜, 鞠政财, 张婷, 薛亚甫, 杨莉, 王峥涛)}, authors=[Author(id=1209809051085574686, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1209788337334915693, orderNo=0, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=null, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1209809051244958255, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1209788337334915693, authorId=1209809051085574686, language=EN, stringName=Yu WANG, firstName=Yu, middleName=null, lastName=WANG, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=1, 3, address=1. The MOE Key Laboratory for Standardization of Chinese Medicines and SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
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2. Shanghai R&D Centre for Standardization of Chinese Medicines, Shanghai 201203, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1209809054348743437, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1209788337334915693, authorId=1209809054084502252, language=CN, stringName=杨莉, firstName=莉, middleName=null, lastName=杨, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=1, 2, *, address=1.上海中医药大学中药研究所, 中药标准化教育部重点实验室, 国家中医药管理局中药新资源与质量评价重点实验室, 上海 201203
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Acta Pharm Sin (药学学报), 2019, 54: 1661-1666., articleTitle=Simultaneous quantitative analyses of eight components in Artemisia capillaris Thunb standard decoction based on a quantitative method of multi-components with a single-marker, refAbstract=null), Reference(id=1209809063332942263, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1209788337334915693, doi=null, pmid=null, pmcid=null, year=2021, volume=56, issue=null, pageStart=557, pageEnd=564, url=http://www.yxxb.com.cn:8081/aps/CN/abstract/abstract18098.shtml, language=null, rfNumber=[22], rfOrder=21, authorNames=Yao J, Sun XG, Dong R, journalName=Acta Pharm Sin (药学学报), refType=null, unstructuredReference= Yao J , Sun XG , Dong R et al . Simultaneous quantitative analyses of six components in Astragalus membranaceus based on HPLC-CAD and quantitative analysis of multi-components with a single-marker[J]. 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Acta Pharm Sin (药学学报), 2012, 47: 930-933., articleTitle=Simultaneous quantitative analysis of four lignanoids in Schisandra chinensis by quantitative analysis of multicomponents by single marker, refAbstract=null), Reference(id=1209809063563629012, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1209788337334915693, doi=null, pmid=null, pmcid=null, year=2015, volume=50, issue=null, pageStart=480, pageEnd=485, url=http://www.yxxb.com.cn:8081/aps/CN/abstract/abstract15300.shtml, language=null, rfNumber=[24], rfOrder=23, authorNames=Wang JJ, Zhang L, Guo Q, journalName=Acta Pharm Sin (药学学报), refType=null, unstructuredReference= Wang JJ , Zhang L , Guo Q et al . Quantitative analysis of seven phenolic acids in eight Yinqiao Jiedu serial preparations by quantitative analysis of multi-components with single-marker[J]. 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AnalyteCalibration curverLinear range/μg·mL-1LOD/ng·mL-1LOQ/ng·mL-1
p-Coumaric acidy = 13.37x + 5.5470.999 90.048-651448
5-Caffeoylquinic acidy = 4.875x - 0.7650.999 50.104-11032104
Chlorogenic acidy = 5.222x - 0.6430.999 50.070-1052170
Caffeic acidy = 9.690x + 1.3140.999 80.027-56827
4-Caffeoylquinic acidy = 5.039x + 1.1880.999 80.046-951446
Ferulic acidy = 9.032x + 0.5600.999 80.117-6058117
), ArticleFig(id=1209809058169753663, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1209788337334915693, language=CN, label=Table 1, caption=

The calibration curve, linear range, regression, LOD, LOQ of 6 compounds

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AnalyteCalibration curverLinear range/μg·mL-1LOD/ng·mL-1LOQ/ng·mL-1
p-Coumaric acidy = 13.37x + 5.5470.999 90.048-651448
5-Caffeoylquinic acidy = 4.875x - 0.7650.999 50.104-11032104
Chlorogenic acidy = 5.222x - 0.6430.999 50.070-1052170
Caffeic acidy = 9.690x + 1.3140.999 80.027-56827
4-Caffeoylquinic acidy = 5.039x + 1.1880.999 80.046-951446
Ferulic acidy = 9.032x + 0.5600.999 80.117-6058117
), ArticleFig(id=1209809058299777100, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1209788337334915693, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
InstrumentColumnRelative retention time
S/1S/2S/3S/4S/6
Agilent 1290Eclipse Plus C180.3970.6760.7360.7601.306
Endeavorsil C180.4020.6820.7330.7871.290
Waters HSS C180.3620.6670.7340.7601.272
ShimadzuEclipse Plus C180.4030.6840.7330.7611.292
LC-40D XSEndeavorsil C180.3990.6710.7160.7901.239
Waters HSS C180.3730.6730.7260.7501.293
Mean0.3890.6760.7300.7681.282
RSD%4.4680.9671.0282.1361.849
), ArticleFig(id=1209809058417217626, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1209788337334915693, language=CN, label=Table 2, caption=

Relative retention time of the 6 components. S: p-Coumaric acid; 1: 5-Caffeoylquinic acid; 2: Chlorogenic acid; 3: Caffeic acid; 4: 4-Caffeoylquinic acid; 6: Ferulic acid

, figureFileSmall=null, figureFileBig=null, tableContent=
InstrumentColumnRelative retention time
S/1S/2S/3S/4S/6
Agilent 1290Eclipse Plus C180.3970.6760.7360.7601.306
Endeavorsil C180.4020.6820.7330.7871.290
Waters HSS C180.3620.6670.7340.7601.272
ShimadzuEclipse Plus C180.4030.6840.7330.7611.292
LC-40D XSEndeavorsil C180.3990.6710.7160.7901.239
Waters HSS C180.3730.6730.7260.7501.293
Mean0.3890.6760.7300.7681.282
RSD%4.4680.9671.0282.1361.849
), ArticleFig(id=1209809058597572713, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1209788337334915693, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Sample No.Chlorogenic acid (S1)
ESM		5-Caffeoylquinic acid4-Caffeoylquinic acidp-Coumaric acid (S2)
ESM		Caffeic acidFerulic acid
ESMTIRMESMTIRMESMTIRMESMTIRM
S10.4740.5370.5330.4500.4550.9420.2030.2070.1700.174
S20.4660.5320.5270.4550.460.9270.2040.2080.1670.172
S30.4590.5210.5170.4410.4460.9130.1990.2030.1650.170
S40.4750.5420.5380.4510.4570.9450.2070.2110.1710.176
S50.4700.5350.5310.4390.4450.9370.2010.2050.1740.178
S60.4640.5270.5220.4290.4350.9170.1980.2020.1760.18
S70.4610.4930.4880.4150.420.8950.1960.2000.1750.179
S80.4540.5230.5180.4480.4530.9090.2000.2040.1750.180
S90.4610.5240.5200.4540.460.9150.2020.2060.1780.182
S100.4380.5010.4960.4360.4420.8710.1920.1960.1690.173
S110.5960.4110.4060.3870.3930.5620.1500.1550.1220.125
S120.5030.4360.4310.3760.3830.5020.1170.1210.0840.087
S130.3920.3430.3380.2990.3050.4780.1230.1260.0840.087
S140.2490.2930.2880.2320.2390.1860.0630.0660.0370.038
S150.4020.3740.3690.3230.3290.4930.1180.1230.0660.068
S160.3160.2840.280.2580.2650.2450.1080.1120.0540.056
S170.3690.3370.3320.3060.3120.3850.1030.1070.0560.058
S180.3360.3040.2980.2740.2820.4040.1120.1150.0530.055
RE%01.1371.67002.6522.816
), ArticleFig(id=1209809058731790453, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1209788337334915693, language=CN, label=Table 3, caption=

The contents of 6 components in Imperatae Rhizoma concentrated granules by two methods/mg·g-1. ESM: External standard method; TIRM: Two internal references method

, figureFileSmall=null, figureFileBig=null, tableContent=
Sample No.Chlorogenic acid (S1)
ESM		5-Caffeoylquinic acid4-Caffeoylquinic acidp-Coumaric acid (S2)
ESM		Caffeic acidFerulic acid
ESMTIRMESMTIRMESMTIRMESMTIRM
S10.4740.5370.5330.4500.4550.9420.2030.2070.1700.174
S20.4660.5320.5270.4550.460.9270.2040.2080.1670.172
S30.4590.5210.5170.4410.4460.9130.1990.2030.1650.170
S40.4750.5420.5380.4510.4570.9450.2070.2110.1710.176
S50.4700.5350.5310.4390.4450.9370.2010.2050.1740.178
S60.4640.5270.5220.4290.4350.9170.1980.2020.1760.18
S70.4610.4930.4880.4150.420.8950.1960.2000.1750.179
S80.4540.5230.5180.4480.4530.9090.2000.2040.1750.180
S90.4610.5240.5200.4540.460.9150.2020.2060.1780.182
S100.4380.5010.4960.4360.4420.8710.1920.1960.1690.173
S110.5960.4110.4060.3870.3930.5620.1500.1550.1220.125
S120.5030.4360.4310.3760.3830.5020.1170.1210.0840.087
S130.3920.3430.3380.2990.3050.4780.1230.1260.0840.087
S140.2490.2930.2880.2320.2390.1860.0630.0660.0370.038
S150.4020.3740.3690.3230.3290.4930.1180.1230.0660.068
S160.3160.2840.280.2580.2650.2450.1080.1120.0540.056
S170.3690.3370.3320.3060.3120.3850.1030.1070.0560.058
S180.3360.3040.2980.2740.2820.4040.1120.1150.0530.055
RE%01.1371.67002.6522.816
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超高效液相色谱双内参法同时测定白茅根配方颗粒中六种有效成分
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王煜 1, 3 , 鞠政财 1 , 张婷 3 , 薛亚甫 1 , 杨莉 1, 2, * , 王峥涛 1, 2
药学学报 | 研究论文 2022,57(2): 467-473
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药学学报 | 研究论文 2022, 57(2): 467-473
超高效液相色谱双内参法同时测定白茅根配方颗粒中六种有效成分
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王煜1, 3, 鞠政财1, 张婷3, 薛亚甫1, 杨莉1, 2, * , 王峥涛1, 2
作者信息
  • 1.上海中医药大学中药研究所, 中药标准化教育部重点实验室, 国家中医药管理局中药新资源与质量评价重点实验室, 上海 201203
  • 2.上海中药标准化研究中心, 上海 201203
  • 3.上海中医药大学交叉科学研究院, 穆拉德中药现代化研究中心, 上海 201203

通讯作者:

*杨莉, Tel: 86-21-51322506, Fax: 86-21-51322519, E-mail:
Determination of six effective ingredients in Imperatae Rhizoma concentrated granules by ultra-performance liquid chromatography using two internal references
Yu WANG1, 3, Zheng-cai JU1, Ting ZHANG3, Ya-fu XUE1, Li YANG1, 2, * , Zheng-tao WANG1, 2
Affiliations
  • 1. The MOE Key Laboratory for Standardization of Chinese Medicines and SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
  • 2. Shanghai R&D Centre for Standardization of Chinese Medicines, Shanghai 201203, China
  • 3. Murad Research Center for Modernized Chinese Medicine, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
出版时间: 2022-02-12 doi: 10.16438/j.0513-4870.2021-1097
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摘要
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建立超高效液相色谱方法并采用基于一测多评的双内参法同时测定白茅根配方颗粒中有效成分对香豆酸、绿原酸、5-咖啡酰奎宁酸、4-咖啡酰奎宁酸、咖啡酸、阿魏酸的含量。采用ZORBAX RRHD Eclipse Plus C18（2.1 mm×100 mm，1.8 μm）色谱柱，流动相为1.7 mmol·L-1草酸水-甲醇溶液梯度洗脱，流速0.4 mL·min-1，柱温35℃，进样量1 μL。分别以对香豆酸为内参物，计算咖啡酸和阿魏酸的相对校正因子；以绿原酸为内参物，计算4-咖啡酰奎宁酸和5-咖啡酰奎宁酸的相对校正因子，考察其稳定性并利用相对保留时间法确定各化合物的色谱峰出峰时间。结果显示，5-咖啡酰奎宁酸、4-咖啡酰奎宁酸、咖啡酸和阿魏酸的相对校正因子分别为1.069、1.022、1.368和1.493，在不同条件下稳定性良好。使用外标法和双内参法对18批不同厂家的白茅根配方颗粒进行含量测定，两种方法所得的结果相对误差小于5.0%，表明建立的双内参法同时测定白茅根配方颗粒中6种有效成分可行，为白茅根配方颗粒质量标准建立提供依据。

白茅根配方颗粒  /  对香豆酸  /  绿原酸  /  一测多评  /  超高效液相色谱

To simultaneously determine the contents of p-coumaric acid, chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, caffeic acid and ferulic acid in Imperatae Rhizoma concentrated granules, an ultra-high performance liquid chromatography (UPLC) with two internal references method (TIRM) was established and validated. Chromatographic separation was achieved on a ZORBAX RRHD Eclipse Plus C18 column (2.1 mm×100 mm, 1.8 μm) using 1.7 mmol·L-1 oxalic acid in water and methanol as mobile phase. The flow rate was 0.4 mL·min-1 and the column temperature was set as 35℃. The relative correction factors (RCFs) of caffeic acid and ferulic acid using p-coumaric acid as internal reference were calculated and the RCFs of 4-caffeoylquinic acid and 5-caffeoylquinic acid were calculated using chlorogenic acid as the internal reference. The TIRM was fully validated for linearity, accuracy, repeatability, stability and recovery so that it could be compared with the external standard method (ESM). The RCFs of 5-caffeoylquinic acid, 4-caffeoylquinic acid, caffeic acid, and ferulic acid were 1.069, 1.022, 1.368, and 1.493, respectively. The TIRM and ESM were used to determine the contents of six ingredients in Imperatae Rhizoma concentrated granules from different manufacturers and the variation between results was within acceptable limits. In conclusion, the newly established TIRM allowed simultaneous determination of six ingredients (p-coumaric acid, chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, caffeic acid, ferulic acid) in Imperatae Rhizoma concentrated granules, providing support for the quality control of this traditional Chinese medicine.

Imperatae Rhizoma concentrated granules  /  p-coumaric acid  /  chlorogenic acid  /  quantitative determination analysis multi-component by a single-marker  /  UPLC
王煜, 鞠政财, 张婷, 薛亚甫, 杨莉, 王峥涛. 超高效液相色谱双内参法同时测定白茅根配方颗粒中六种有效成分. 药学学报, 2022 , 57 (2) : 467 -473 . DOI: 10.16438/j.0513-4870.2021-1097
Yu WANG, Zheng-cai JU, Ting ZHANG, Ya-fu XUE, Li YANG, Zheng-tao WANG. Determination of six effective ingredients in Imperatae Rhizoma concentrated granules by ultra-performance liquid chromatography using two internal references[J]. Acta Pharmaceutica Sinica, 2022 , 57 (2) : 467 -473 . DOI: 10.16438/j.0513-4870.2021-1097
正文
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中药配方颗粒是由单味中药饮片经提取浓缩制成的具有统一规格、统一剂量、统一质量标准的新型配方用药[1]。随着中药现代化的快速发展以及国家标准的出台, 配方颗粒产业的规模逐步扩大[2]。规模的扩大会积极带动各单味药配方颗粒国家标准的推进更新, 对于系统性地实现中药配方颗粒安全性、有效性的整体质量控制具有深刻的历史意义[3]。
白茅根是禾本科植物白茅Imperata cylindrica Beauv. var. major (Nees) C. E. Hubb.的干燥根茎, 具有凉血止血、清热利尿的功效[4]。白茅根始载于《神农本草经》, 称之为“茅根”, 列为中品, 其化学成分主要为糖类、苯丙素类、有机酸类、黄酮类、色原酮类、甾醇类、三萜类以及内酯类, 其中苯丙素类为其特征性成分[5-12]。在现代药理研究中发现, 白茅根具有止血、利尿、抑菌、降血脂、降血糖、抗炎、抗肿瘤、抗氧化等药理作用, 与传统功效相符[10]。
目前, 2020版《中国药典》白茅根项下缺乏含量测定相关标准[4], 现行质量控制方法难以全面反映白茅根的质量差异。对已有文献进行检索发现白茅根控制指标成分多选用绿原酸, 采用HPLC或UPLC建立绿原酸单一或多指标的定量检测方法[13, 14], 然而实验结果提示, 单一指标无法全面评价中药配方颗粒质量。白茅根配方颗粒中存在大量苯丙酸类成分, 有着数量多、含量高的特点, 是其发挥抑菌、抗炎生物活性的主要物质基础[15], 故考虑引用一测多评法对白茅根配方颗粒中该类有效成分进行同步测定。一测多评法(quantitative determination analysis multi-component by a single-marker, QAMS) 是通过测定一个成分实现多个成分同步定量的质量评价模式[16]。考虑到白茅根配方颗粒中对香豆酸与咖啡酸、阿魏酸结构接近, 绿原酸与5-咖啡酰奎宁酸、4-咖啡酰奎宁酸结构接近, “一测”无法准确评估各成分, 为保证一测多评法的准确性, 采用两个内参物的策略建立多组分测定的分析方法(双内参法), 可对临床用药的精确性和安全性提供指导[17]。
本研究首次对白茅根配方颗粒的提取工艺、色谱条件、以及相对校正因子(relative correction factor, RCF) 的稳定性等多方面进行考察, 建立了以对香豆酸、绿原酸为内参物, 同时测定白茅根配方颗粒中6种有效成分(咖啡酸、对香豆酸、4-咖啡酰奎宁酸、5-咖啡酰奎宁酸、绿原酸、阿魏酸) 含量的方法。该方法经过方法学考察后应用于测定18批不同厂家白茅根配方颗粒中6种有效成分的含量。本研究为白茅根配方颗粒质量标准的制订提供可靠依据, 并为白茅根的品质评价、生产栽培以及资源开发等提供相关支撑。
材料与方法
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仪器与试剂  安捷伦公司Agilent 1290超高效液相色谱仪(四元泵1290 Flexible Pump, 进样器1290 Vialsampler, 柱温箱1290 MCT, 检测器1290 DAD FS); 岛津公司LC-40D XS (二元泵LC-40D XS Pump, 进样器SIL-40C XS, 柱温箱CTO-40S, 检测器SPD-40); BSA124S-CW型电子分析天平、BSA323S型电子分析天平(北京赛多利斯科学仪器有限公司, 中国); SCQ-5201数控超声波清洗器(上海声彦超声波仪器有限公司, 中国); 色谱柱ZORBAX RRHD Eclipse Plus C18 (2.1 mm × 100 mm, 1.8 µm) (安捷伦科技有限公司, 美国), Endeavorsil C18 (2.1 mm × 100 mm, 1.8 µm) (北京迪科马科技有限公司, 中国), ACQUITY UPLC HSS C18 (2.1 mm × 100 mm, 1.8 µm) (沃特世科技有限公司, 美国)。分析级甲醇(国药集团化学试剂有限公司, 中国); 色谱级甲醇(Fisher Scientific, 加拿大); 乙二酸(批号: BMZ235, 上海毕得医药科技股份有限公司, 中国); 超纯水由MILLI-Q超纯水制备仪制备(Millipore, 美国)。对香豆酸对照品(批号: 202105110008) 购自国药集团化学试剂有限公司, 纯度≥ 98.0%; 4-咖啡酰奎宁酸(批号: 3208) 购自上海诗丹德标准技术服务有限公司, 纯度≥ 98.0%; 5-咖啡酰奎宁酸(批号: 20-2009)、绿原酸(批号: 25-1004)、阿魏酸(批号: 08-2001)、咖啡酸(批号: 30-2001) 均购自上海中医药大学标准化中心, 纯度≥ 98.0%。
实验材料  此次供研究使用的18批白茅根配方颗粒样品分别购自上海万仕诚药业有限公司、上海雷允上药业有限公司、江阴天江药业有限公司、四川新绿色药业科技发展有限公司、北京康仁堂药业有限公司。
UPLC色谱条件   Agilent 1290超高效色谱仪; ZORBAX RRHD Eclipse Plus C18 (2.1 mm × 100 mm, 1.8 µm) 色谱柱; 流动相为甲醇(A)-1.7 mmol·L-1草酸水溶液(B), 梯度洗脱0~1.5 min, 5% A; 1.5~6 min, 5%~9% A; 6~9 min, 9%~17% A; 9~17 min, 17% A; 17~20 min, 17%~35% A; 检测波长为308 nm; 体积流量0.4 mL·min-1; 柱温35 ℃; 进样量1 μL。
混合对照品溶液制备  精密称取各对照品适量, 用50%甲醇制成每1 mL中含0.3 mg的对香豆酸、0.1 mg的4-咖啡酰奎宁酸、0.2 mg的5-咖啡酰奎宁酸、0.1 mg的绿原酸、0.1 mg的阿魏酸和0.1 mg的咖啡酸的混合溶液, 作为白茅根配方颗粒对照品溶液。
供试品溶液制备  取白茅根配方颗粒约1.0 g, 精密称定, 置具塞锥形瓶中, 精密加入50%甲醇40 mL, 密塞, 称定重量, 超声处理(功率300 W, 频率40 kHz) 15 min, 取出, 放冷, 再称定重量, 用50%甲醇补足减失的重量, 摇匀, 静置, 取上清液过0.22 μm微孔滤膜, 即得。
方法学考察
专属性  取空白溶液、混合对照品溶液、白茅根配方颗粒供试品溶液分别进样测定, 比较色谱图。
线性范围、定量限和检测限  分别精密称取对香豆酸、4-咖啡酰奎宁酸、5-咖啡酰奎宁酸、绿原酸、阿魏酸、咖啡酸对照品各3.00、1.00、2.20、1.40、0.60、0.56 mg, 加50%甲醇制成质量浓度分别为0.30、0.10、0.22、0.14、0.060和0.056 mg·mL-1的混合溶液, 对半稀释得到7个不同浓度对照品溶液。浓度由低到高依次进样测定。分别以对照品溶液的浓度为横坐标(X), 峰面积为纵坐标(Y), 绘制标准曲线, 并得到回归方程、线性范围、相关系数。将混合对照品溶液逐级稀释, 以信噪比为10∶1确定定量限(LOQ), 以信噪比为3∶1确定检测限(LOD)。
精密度精密  吸取同一供试品溶液, 于一天内连续进样6次, 记录色谱图测定峰面积, 计算各待测物含量的RSD值。
重复性  取同一批白茅根配方颗粒按“供试品溶液制备”项下方法平行制备6份供试品进样测定, 计算各待测物含量的RSD值。
稳定性  取同一供试品溶液, 在室温下, 于0、2、4、6、8、12、24和48 h进样, 记录峰面积并计算各化合物含量。
加样回收率  取白茅根配方颗粒样品约0.5 g, 精密称定, 分别按照各成分已知含量的50%、100%和150%加入各对照品适量。按“供试品溶液制备”项下方法制备供试品溶液, 每个浓度水平平行3份, 并按“UPLC色谱条件”项下进样测定。
双内参法
RCF的计算  将“线性范围、定量限和检测限”项下7个不同浓度系列混合对照品溶液进样并记录峰面积, 以公式fsi = fs/fi = As·Cs/Ai·Ci (fsi是待测化合物i的相对校正因子, As是内参化合物峰面积, Cs是内参化合物浓度; Ai是待测化合物i的峰面积, Ci是待测化合物i的浓度) 计算6个化合物的RCF, 按照双内参法, 分别以绿原酸为内参化合物S1, 计算不同浓度点下5-咖啡酰奎宁酸、4-咖啡酰奎宁酸的RCF的平均值; 以对香豆酸为内参物S2, 计算不同浓度点下咖啡酸、阿魏酸的RCF的平均值, 比较RSD值。
RCF稳定性考察  按照“混合对照品溶液”项下制备对照品溶液, 分别考察在不同厂家超高效液相色谱仪(Agilent 1290, 岛津LC-40D XS) 上使用三根不同厂家的C18色谱柱(ZORBAX RRHD Eclipse Plus C18, Endeavorsil C18, ACQUITY UPLC HSS C18) 对RCF稳定性的影响, 记录峰面积, 按照“RCF的计算”计算各化合物RCF的平均值并比较RSD值; 考察温度对RCF稳定性的影响, 采用eclipse plus C18色谱柱进行色谱分离, 分别记录在30、35和40 ℃下各化合物的峰面积, 计算其RCF的平均值和RSD值; 考察流速对RCF稳定性的影响, 分别记录在0.38、0.4和0.42 mL·min-1不同流速条件下各成分的峰面积, 计算6个化合物的RCF的平均值及RSD值; 考察流动相草酸浓度对RCF稳定性的影响, 记录以不同浓度1.5、1.7和2 mmol·L-1草酸水为流动相时各化合物的峰面积变化, 计算RCF的平均值和RSD值。
色谱峰定位  将“混合对照品溶液”项下溶液在两台色谱仪Agilent 1290、岛津LC-40D XS以不同厂家C18色谱柱ZORBAX RRHD Eclipse Plus C18、Endeavorsil C18、ACQUITY UPLC HSS C18进样测定。采用两点校正法, 以Agilent 1290上使用eclipse plus C18色谱柱的对香豆酸和绿原酸的保留时间为标准建立校正方程, 计算6种苯丙酸类化合物的理论时间, 与检测时间相比较, 并以相对误差(relative error, RE) 为评价指标; 采用保留时间差法以对香豆酸为内参物S, 计算其余5种化合物的保留时间差以及RSD值; 采用相对保留时间法以对香豆酸为内参物S, 计算其余5种化合物的相对保留时间以及RSD值。
样品含量测定  将收集得到的18批白茅根配方颗粒样品按“供试品溶液制备”项下方法处理得供试品溶液, 并按“UPLC色谱条件”项下进样检测, 采用双内参法分别以绿原酸为内参计算5-咖啡酰奎宁酸、4-咖啡酰奎宁酸的含量; 以对香豆酸为内参计算咖啡酸、阿魏酸的含量, 与外标法测得的含量结果进行比较, 并以RE进行评价。
结果
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1 方法学考察
1.1 专属性
通过对比空白溶液、混合对照品溶液和供试品溶液色谱图, 考察方法专属性, 6个苯丙酸类化合物与相邻各色谱峰分离度均大于1.5, 色谱图如图 1所示。结果表明, 该方法专属性良好。
1.2 线性范围、定量限和检测限
6个成分的线性回归方程、相关系数、检测限和定量限结果见表 1, 各成分在线性范围内均显示出良好的线性关系。
1.3 精密度
测得6种化合物对香豆酸、5-咖啡酰奎宁酸、绿原酸、咖啡酸、4-咖啡酰奎宁酸、阿魏酸的含量(mg·g-1) RSD分别为1.092%、0.274%、1.033%、0.636%、0.185%和0.377%, 均小于2%, 表明精密度良好。
1.4 重复性
测得样品中对香豆酸、5-咖啡酰奎宁酸、绿原酸、咖啡酸、4-咖啡酰奎宁酸和阿魏酸的含量(mg·g-1) RSD分别为1.526%、0.768%、1.583%、1.919%、0.541%和0.591%, 均小于2%, 表明方法重复性良好。
1.5 稳定性
结果显示对香豆酸、5-咖啡酰奎宁酸、绿原酸、咖啡酸、4-咖啡酰奎宁酸和阿魏酸6种有效成分的含量(mg·g-1) RSD分别为1.487%、0.945%、1.613%、1.449%、0.181%和1.516%, 表明供试品溶液在48 h内稳定性良好。
1.6 加样回收率
6种成分对香豆酸、5-咖啡酰奎宁酸、绿原酸、咖啡酸、4-咖啡酰奎宁酸和阿魏酸的平均回收率分别为98.37%、97.69%、93.98%、106.53%、99.47%和103.7%, RSD均小于2%, 表明方法准确度较高。
2 RCF的计算
按照双内参法, 以绿原酸为内参化合物S1, 计算5-咖啡酰奎宁酸和4-咖啡酰奎宁酸的RCF为1.069和1.022; 以对香豆酸为内参物S2, 计算咖啡酸和阿魏酸的RCF分别为1.368和1.493, RSD均小于2%。
2.1 RCF的稳定性
本次研究先后考察了不同高效液相色谱仪、色谱柱、柱温、流速、草酸浓度对6种有效成分的影响, 在确保分离度大于1.5的情况下, 对该6种化合物的RCF进行了稳定性实验。
2.1.1 不同超高效液相色谱仪及色谱柱的影响
不同超高效液相色谱仪及色谱柱条件下, 计算5-咖啡酰奎宁酸和4-咖啡酰奎宁酸的RCF平均值为1.067和1.026, 咖啡酸和阿魏酸的RCF平均值分别为1.438和1.462, RSD值均小于5%, 表明不同的超高效液相色谱仪和色谱柱对RCF没有显著影响, 方法稳定性好。
2.1.2 柱温的影响
不同柱温条件下, 5-咖啡酰奎宁酸和4-咖啡酰奎宁酸的RCF平均值为1.055和1.028, 咖啡酸和阿魏酸的RCF平均值分别为1.393和1.531, RSD均小于1%, 表明各化合物RCF在30~40 ℃的稳定性良好。
2.1.3 流速的影响
不同流速条件下, 5-咖啡酰奎宁酸和4-咖啡酰奎宁酸的RCF平均值为1.054和1.028, 咖啡酸和阿魏酸的RCF平均值分别为1.392和1.524, 结果RSD值均小于1%, 表明各化合物RCF在0.38~0.42 mL·min-1流速范围内稳定性良好。
2.1.4 草酸浓度的影响
在不同草酸浓度条件下, 计算5-咖啡酰奎宁酸、4-咖啡酰奎宁酸的RCF平均值为1.055和1.008, 咖啡酸、阿魏酸的RCF平均值分别为1.424和1.527, RSD值均小于1%, 表明各成分RCF在流动相为不同草酸浓度(1.5~2.0 mmol·L-1) 时稳定性良好。
2.2 待测组分色谱峰定位
为了确定白茅根配方颗粒中6种有效成分的出峰时间, 分别采用两点校正法、保留时间差法和相对保留时间法确定各待测组分出峰时间[18]。结果显示, 以两点校正法进行色谱峰定位时, RE部分值大于5%。进一步以对香豆酸为内参物S, 分别采用保留时间差法和相对保留时间法定位各色谱峰。不同仪器不同色谱柱对保留时间差法影响较大, RSD大于10%, 很难准确进行定位; 以相对保留时间法进行计算时, RSD小于5%, 差异较小, 较两点校正法更优, 表明使用相对保留时间法对白茅根配方颗粒中6种有效成分进行峰定位具有可行性。最终确定以对香豆酸为内参S, 计算各化合物相对保留时间平均值, 各化合物详细结果见表 2。
3 外标法和双内参法含量测定比较
采用双内参法分别以绿原酸为内参计算5-咖啡酰奎宁酸、4-咖啡酰奎宁酸的含量; 以对香豆酸为内参计算咖啡酸、阿魏酸的含量, 与外标法测得的含量结果进行比较, 详细结果见表 3。结果显示, 18批样品均能检测出6种有效成分, 两种方法测得结果RE小于5%, 表明双内参法可行, 结果准确度较高。
讨论
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本次实验对配方颗粒的苯丙酸类成分提取工艺进行了系统的优化, 以峰面积与称样量的比值为评价依据。考察了不同提取溶剂, 结果显示随着甲醇含量升高, 极性较大的化合物受到的影响大, 色谱峰形变差, 在考虑最大化提取并保证峰形的情况下选择50%甲醇为提取溶剂。比较超声提取和回流提取, 结果显示二者并无明显差异, 择优超声为提取方法。为了使配方颗粒能够被完全提取, 考察不同料液比, 以完全提取为目的选择了1∶40为最终料液比。考察不同提取时间, 结果表明不同提取时间无明显差异, 最终选择15 min为提取时间。前期研究过程中发现, 白茅根配方颗粒中含有大量的糖类成分, 由于大极性基团易与水分子结合, 白茅根配方颗粒极易吸潮结块, 因此在储存管理上, 应当严格控制其湿度以及使用密封器减少与空气中的水接触, 避免吸潮对质量的影响[19]。
对6个苯丙酸类成分进行了光谱分析, 发现该类化合物均在306~330 nm有最大吸收, 考虑到峰形、分离度以及峰面积, 且含量最高的内参化合物对香豆酸在308 nm波长下峰面积最大。经检验, 308 nm条件下各化合物均能达到基线分离, 灵敏度良好, 故选择了308 nm为检测波长。
实验中发现咖啡酸与4-咖啡酰奎宁酸出峰时间较为接近, 存在一定程度的分离难度。流动相对分离度影响较大, 因此尝试添加不同缓冲盐进行优化, 结果显示草酸水分离效果更佳且峰形较好, 故选择草酸水溶液为流动相。
参考QAMS法指南, 应选择配方颗粒中相对易得、含量较高、药理作用相对明确的成分作为内参物[20]。根据实验经验以及文献检索, 待测成分的结构越接近, 计算结果更为准确, 更具有参考意义[21-24]。前期预试发现, 该6种有效成分的结构特点仅用一个内参物计算得出的部分化合物RCF远大于或远小于1, 为保证结果的准确性, 本次研究采用两个内参物的策略, 对白茅根配方颗粒中6个有效成分进行了分类, 分别以更易获得、含量较高且具有一定活性的绿原酸、对香豆酸为内参进行RCF的计算, 结果发现, RCF在此条件下均接近1, RSD小于5%。为验证RCF的稳定性, 考察了不同厂家仪器、色谱柱、柱温、流速和草酸浓度, 结果显示RSD均小于5%, 表明以该策略建立双内参法可行。
同时用外标法和双内参法对18批来自5个不同厂家的白茅根配方颗粒进行含量测定, 结果显示, 不同厂家生产的配方颗粒中6种有效成分的含量存在差异; 同一厂家不同批次之间的含量差异较小, 其中绿原酸、5-咖啡酰奎宁酸和4-咖啡酰奎宁酸含量接近。本研究方法可用于测定白茅根药材中6种有效成分的含量, 制定相应的含量限度, 为白茅根药材及其饮片制剂等的质量控制和质量标准提升提供依据。
本次研究首次引用双内参法, 在能够减少检测成本、节约时间、简化流程, 避免单一指标的局限性的基础上, 实现同步对白茅根配方颗粒中6种有效成分的含量测定, 以期为白茅根配方颗粒乃至白茅根药材饮片提供质量参考依据。
作者贡献: 王煜和杨莉设计实验; 王煜、鞠政财、薛亚甫进行实验数据采集; 杨莉进行药材收集; 王煜、张婷、王峥涛和杨莉撰写和修改论文。
利益冲突: 无利益冲突。
基金
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  • 上海市科技成果转化与产业化项目(17DZ1920100)
  • 国家自然科学基金资助项目(81530096)
文献
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2022年第57卷第2期
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文章信息
doi: 10.16438/j.0513-4870.2021-1097
  • 接收时间：2021-07-26
  • 首发时间：2025-12-22
  • 出版时间：2022-02-12
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出版历史
  • 收稿日期：2021-07-26
  • 修回日期：2021-10-12
基金
上海市科技成果转化与产业化项目(17DZ1920100)
国家自然科学基金资助项目(81530096)
作者信息
    1.上海中医药大学中药研究所, 中药标准化教育部重点实验室, 国家中医药管理局中药新资源与质量评价重点实验室, 上海 201203
    2.上海中药标准化研究中心, 上海 201203
    3.上海中医药大学交叉科学研究院, 穆拉德中药现代化研究中心, 上海 201203

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*杨莉, Tel: 86-21-51322506, Fax: 86-21-51322519, E-mail:
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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