Article(id=1239250706096517457, tenantId=1146029695717560320, journalId=1205117023404326918, issueId=1239250701583446236, articleNumber=null, orderNo=null, doi=10.16155/j.0254-1793.2024.04.05, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=null, receivedDateStr=null, revisedDate=1709913600000, revisedDateStr=2024-03-09, acceptedDate=null, acceptedDateStr=null, onlineDate=1773389992498, onlineDateStr=2026-03-13, pubDate=1714406400000, pubDateStr=2024-04-30, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773389992498, onlineIssueDateStr=2026-03-13, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773389992498, creator=13701087609, updateTime=1773389992498, updator=13701087609, issue=Issue{id=1239250701583446236, tenantId=1146029695717560320, journalId=1205117023404326918, year='2024', volume='44', issue='4', pageStart='553', pageEnd='736', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1773389991422, creator=13701087609, updateTime=1773390513217, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1239252890213209050, tenantId=1146029695717560320, journalId=1205117023404326918, issueId=1239250701583446236, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1239252890213209051, tenantId=1146029695717560320, journalId=1205117023404326918, issueId=1239250701583446236, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=585, endPage=593, ext={EN=ArticleExt(id=1239250706348175715, articleId=1239250706096517457, tenantId=1146029695717560320, journalId=1205117023404326918, language=EN, title=Eatablishment of HPLC fingerprint of lichens of Thamnolia subuliformis (Ehrh.) W. Culb. and its UHPLC-Q Exactive Focus MS/MS analysis*, columnId=1206272756333736276, journalTitle=Chinese Journal of Pharmaceutical Analysis, columnName=Ingredient Analysis, runingTitle=null, highlight=null, articleAbstract=
Objective:

To establish a quality assessment method for lichens of Thamnolia subuliformis (Ehrh.) W. Culb. based on HPLC fingerprint, and qualitative analysis of the chemical constituents by ultra-high performance liquid chromatography coupled with quadrupole/electrostatic field orbital trap high resolution mass spectrometry (UHPLC-Q Exactive Focus MS/MS).

Methods:

Agilent TC-C18(250 mm×4.6 mm, 5 μm) chromatographic column was used, the mobile phase was methol -0.1% phosphoric acid with gradient elution at the flow rate of 1.0 mL·min-1, the column temperature was 30 ℃, the detection wavelength was 254 nm, and the injection volume was 10 μL. HPLC fingerprints of lichens of Thamnolia subuliformis was established. The software of similarity calculation for traditional Chinese medicines fingerprints (version 2012) was used to establish the fingerprinting of lichens of Thamnolia subuliformis. The chemical constituents were analyzed by UHPLC-Q Exactive Focus MS/MS, The chromatographic separation was performed on a Waters Acquity UPLC BEH C18 (50 mm×2.1 mm, 1.7 μm) column with acetonitrile(A) -0.1% formic acid aqueous solution(B) as mobile phase for gradient elution, the flow rate was 0.3 mL·min-1 and the column temperature was 30 ℃.Mass spectrometry was performed using an electrospray ionization and data was collected in negative ion modes in the range of m/z 80-1 200.

Results:

Eight common peaks were identified from fingerprints of 10 batches of samples. The RSD values of relative retention time of 8 common peaks of chromotograms of samples were all below 0.5% and the similarities were above 0.9. 4 of the identified peaks were further confirmed by UHPLC-Q Exactive Focus MS/MS as squamatic acid, baeomycesic acid, thamnoliadepsides B, barbatinic acid.

Conclusion:

The established method of fingerprint is stable, reliable, and specific, which can be used for quality evaluation of lichens of Thamnolia subuliformis.

, correspAuthors=Wei-feng WANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, 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=Sha-sha LI, Ya-juan LI, Xiao-ting WANG, A-juan MAO, Fan LI, Fang LI, Hong ZHANG, Wei-feng WANG), CN=ArticleExt(id=1239250707929428465, articleId=1239250706096517457, tenantId=1146029695717560320, journalId=1205117023404326918, language=CN, title=太白茶HPLC指纹图谱的建立及UHPLC-Q Exactive Focus MS/MS联用分析*, columnId=1206272756476342615, journalTitle=药物分析杂志, columnName=成分分析, runingTitle=null, highlight=null, articleAbstract=
目的:

建立太白茶药材高效液相色谱(HPLC)指纹图谱分析方法,并运用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱(UHPLC-Q Exactive Focus MS/MS)法对太白茶中的特征峰进行定性分析。

方法:

采用Agilent TC-C18(250 mm×4.6 mm,5 μm)色谱柱,以甲醇(A)-0.1%甲酸水溶液(B)为流动相,梯度洗脱,流速1.0 mL·min-1,柱温30 ℃,检测波长254 nm,进样量10 μL。采用“中药色谱指纹图谱相似度评价系统2012年版”,建立指纹图谱并进行相似度评价。化学成分分析选用UHPLC-Q Exactive Focus MS/MS技术,采用Waters Acquity UPLC BEH C18(50 mm×2.1 mm,1.7 μm)色谱柱,以乙腈(A)-0.1%甲酸水(B)为流动相,梯度洗脱,流速0.3 mL·min-1,柱温30 ℃;质谱条件为电喷雾离子源(ESI),负离子模式扫描,扫描范围m/z 80~1 200。

结果:

建立的太白茶特征指纹图谱中,标定共有峰8个,10批太白茶药材相似度均≥0.9,各批次之间共有峰的相对保留时间RSD均<0.5%。对8个共有峰中的4个峰进行了结构指认,依次为鳞片衣酸、羊角衣酸、thamnoliadepsides B、坝巴酸。

结论:

所建立的HPLC指纹图谱方法稳定可靠,专属性好,结合UHPLC-Q-Exactive Focus-MS/MS的定性分析,可用于太白茶药材的整体质量综合评价。

, correspAuthors=王卫锋, authorNote=null, correspAuthorsNote=
** Tel:13572021669;E-mail:
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Tel:15829736183;E-mail:

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figureFileSmall=1HT3TJY8o2AJA07q0hRz4w==, figureFileBig=8DHLQaFYqZ63Ip89G8Lykg==, tableContent=null), ArticleFig(id=1239250715282043678, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250706096517457, language=CN, label=图2, caption=总离子流图, figureFileSmall=1HT3TJY8o2AJA07q0hRz4w==, figureFileBig=8DHLQaFYqZ63Ip89G8Lykg==, tableContent=null), ArticleFig(id=1239250715386901284, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250706096517457, language=EN, label=Fig.3, caption=MS/MS spectra of common peak 4(A),peak 6(B),peak 7(C),and peak 8(D), figureFileSmall=01oCK39SbabgRpTNtHSQMA==, figureFileBig=Cml3YL8KNBFbXrx3kNoSwQ==, tableContent=null), ArticleFig(id=1239250715483370279, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250706096517457, language=CN, label=图3, caption=共有峰峰4(A)、峰6(B)、峰7(C)及峰8(D)的二级质谱图, figureFileSmall=01oCK39SbabgRpTNtHSQMA==, figureFileBig=Cml3YL8KNBFbXrx3kNoSwQ==, tableContent=null), 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Relative peak areas of common peaks in the fingerprint of 10 batches of lichens of Thamnolia subuliformis

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峰号
(peak No.)
相对峰面积(relative peak area)
S1S2S3S4S5S6S7S8S9S10
10.045 90.017 40.016 80.017 40.025 90.016 80.017 80.017 90.018 10.020 4
20.151 90.007 80.005 00.006 30.006 40.036 70.015 00.037 40.050 30.056 2
30.125 00.061 70.026 10.033 30.021 60.085 50.093 20.054 60.059 70.085 4
41.000 01.000 01.000 01.000 01.000 01.000 01.000 01.000 01.000 01.000 0
50.040 60.017 70.020 10.021 40.016 40.030 20.027 20.037 20.029 20.038 5
60.518 10.794 20.641 60.810 60.623 00.738 00.474 00.739 40.732 00.799 9
70.039 90.013 10.002 60.002 80.004 70.007 90.014 80.001 30.011 70.017 6
80.023 00.012 80.010 00.019 70.011 60.015 30.013 40.016 40.014 20.021 7
), ArticleFig(id=1239250716133487431, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250706096517457, language=CN, label=表1, caption=

10批太白茶指纹图谱共有峰的相对峰面积

, figureFileSmall=null, figureFileBig=null, tableContent=
峰号
(peak No.)
相对峰面积(relative peak area)
S1S2S3S4S5S6S7S8S9S10
10.045 90.017 40.016 80.017 40.025 90.016 80.017 80.017 90.018 10.020 4
20.151 90.007 80.005 00.006 30.006 40.036 70.015 00.037 40.050 30.056 2
30.125 00.061 70.026 10.033 30.021 60.085 50.093 20.054 60.059 70.085 4
41.000 01.000 01.000 01.000 01.000 01.000 01.000 01.000 01.000 01.000 0
50.040 60.017 70.020 10.021 40.016 40.030 20.027 20.037 20.029 20.038 5
60.518 10.794 20.641 60.810 60.623 00.738 00.474 00.739 40.732 00.799 9
70.039 90.013 10.002 60.002 80.004 70.007 90.014 80.001 30.011 70.017 6
80.023 00.012 80.010 00.019 70.011 60.015 30.013 40.016 40.014 20.021 7
), ArticleFig(id=1239250716242539338, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250706096517457, language=EN, label=Tab.2, caption=

Relative retention time of common peaks in the fingerprint of 10 batches of lichens of Thamnolia subuliformis

, figureFileSmall=null, figureFileBig=null, tableContent=
峰号
(peak No.)
相对保留时间(relative retention time)
S1S2S3S4S5S6S7S8S9S10RSD/%
10.211 40.211 30.211 30.211 20.211 60.211 70.211 20.211 50.211 60.211 30.17
20.350 70.350 70.350 60.350 40.351 10.351 20.350 70.350 90.351 00.350 70.11
30.699 80.699 90.699 30.698 70.700 20.700 50.700 40.701 10.701 30.700 90.074
41.000 01.000 01.000 01.000 01.000 01.000 01.000 01.000 01.000 01.000 00.00
51.131 31.131 31.131 01.131 71.131 31.130 81.129 91.130 41.130 51.130 60.028
61.258 81.258 91.258 81.259 91.258 91.258 21.257 21.257 01.256 61.257 30.056
71.404 71.404 71.404 71.404 61.104 41.403 41.401 31.401 41.401 41.401 70.11
81.581 61.581 01.581 51.583 61.580 81.579 91.577 51.577 01.577 21.577 30.12
), ArticleFig(id=1239250716334814031, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250706096517457, language=CN, label=表2, caption=

10批太白茶指纹图谱共有峰的相对保留时间

, figureFileSmall=null, figureFileBig=null, tableContent=
峰号
(peak No.)
相对保留时间(relative retention time)
S1S2S3S4S5S6S7S8S9S10RSD/%
10.211 40.211 30.211 30.211 20.211 60.211 70.211 20.211 50.211 60.211 30.17
20.350 70.350 70.350 60.350 40.351 10.351 20.350 70.350 90.351 00.350 70.11
30.699 80.699 90.699 30.698 70.700 20.700 50.700 40.701 10.701 30.700 90.074
41.000 01.000 01.000 01.000 01.000 01.000 01.000 01.000 01.000 01.000 00.00
51.131 31.131 31.131 01.131 71.131 31.130 81.129 91.130 41.130 51.130 60.028
61.258 81.258 91.258 81.259 91.258 91.258 21.257 21.257 01.256 61.257 30.056
71.404 71.404 71.404 71.404 61.104 41.403 41.401 31.401 41.401 41.401 70.11
81.581 61.581 01.581 51.583 61.580 81.579 91.577 51.577 01.577 21.577 30.12
), ArticleFig(id=1239250716460643154, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250706096517457, language=EN, label=Tab.3, caption=

Similarity evaluation results of 10 batches of lichens of Thamnolia subuliformis

, figureFileSmall=null, figureFileBig=null, tableContent=
编号(peak No.)S1S2S3S4S5S6S7S8S9S10R
S11.0000.9710.9850.9700.9890.9810.9910.9790.9810.9760.987
S20.9711.0000.9940.9990.9920.9990.9730.9990.9990.9990.997
S30.9850.9941.0000.9941.0000.9970.9900.9970.9980.9930.999
S40.9700.9990.9941.0000.9920.9980.9700.9990.9980.9990.996
S50.9890.9921.0000.9921.0000.9950.9900.9960.9970.9920.998
S60.9810.9990.9970.9980.9951.0000.9811.0001.0000.9990.999
S70.9910.9730.9900.9700.9900.9811.0000.9800.9810.9730.987
S80.9790.9990.9970.9990.9961.0000.9801.0001.0000.9990.999
S90.9810.9990.9980.9980.9971.0000.9811.0001.0000.9990.999
S100.9760.9990.9930.9990.9920.9990.9730.9990.9991.0000.997
R0.9870.9970.9990.9960.9980.9990.9870.9990.9990.9971.000
), ArticleFig(id=1239250716557112150, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250706096517457, language=CN, label=表3, caption=

10批太白茶相似度评价结果

, figureFileSmall=null, figureFileBig=null, tableContent=
编号(peak No.)S1S2S3S4S5S6S7S8S9S10R
S11.0000.9710.9850.9700.9890.9810.9910.9790.9810.9760.987
S20.9711.0000.9940.9990.9920.9990.9730.9990.9990.9990.997
S30.9850.9941.0000.9941.0000.9970.9900.9970.9980.9930.999
S40.9700.9990.9941.0000.9920.9980.9700.9990.9980.9990.996
S50.9890.9921.0000.9921.0000.9950.9900.9960.9970.9920.998
S60.9810.9990.9970.9980.9951.0000.9811.0001.0000.9990.999
S70.9910.9730.9900.9700.9900.9811.0000.9800.9810.9730.987
S80.9790.9990.9970.9990.9961.0000.9801.0001.0000.9990.999
S90.9810.9990.9980.9980.9971.0000.9811.0001.0000.9990.999
S100.9760.9990.9930.9990.9920.9990.9730.9990.9991.0000.997
R0.9870.9970.9990.9960.9980.9990.9870.9990.9990.9971.000
), ArticleFig(id=1239250716666164055, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250706096517457, language=EN, label=Tab.4, caption=

Identification results of commom peaks

, figureFileSmall=null, figureFileBig=null, tableContent=
峰号
(peak No.)
tR/min分子式
(formula)
测定值
(measured)
m/z
理论值
(actual)
m/z
碎片离子
(fragment ion)
m/z
误差
(error)/10-6
名称
(compound)
453.551C19H18O9389.088 6389.087 8389.087 0、345.098 6、327.088 6、225.040 9、181.051 0、163.040 4、137.061 12.06鳞片衣酸(squamatic acid)
667.411C19H18O8373.093 8373.092 9373.093 2、181.051 1、163.040 5、137.061 2、119.050 52.41羊角衣酸(baeomycesic acid)
775.222C18H18O7345.098 9345.098 0345.098 9、181.051 2、163.040 6、137.061 2、119.050 72.61thamnoliadepsides B
884.698C19H20O7359.114 7359.113 6359.114 9、181.051 2、163.040 5、137.061 2、119.050 63.07坝巴酸(barbatinic acid)
), ArticleFig(id=1239250716750050140, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250706096517457, language=CN, label=表4, caption=

共有峰成分鉴定结果

, figureFileSmall=null, figureFileBig=null, tableContent=
峰号
(peak No.)
tR/min分子式
(formula)
测定值
(measured)
m/z
理论值
(actual)
m/z
碎片离子
(fragment ion)
m/z
误差
(error)/10-6
名称
(compound)
453.551C19H18O9389.088 6389.087 8389.087 0、345.098 6、327.088 6、225.040 9、181.051 0、163.040 4、137.061 12.06鳞片衣酸(squamatic acid)
667.411C19H18O8373.093 8373.092 9373.093 2、181.051 1、163.040 5、137.061 2、119.050 52.41羊角衣酸(baeomycesic acid)
775.222C18H18O7345.098 9345.098 0345.098 9、181.051 2、163.040 6、137.061 2、119.050 72.61thamnoliadepsides B
884.698C19H20O7359.114 7359.113 6359.114 9、181.051 2、163.040 5、137.061 2、119.050 63.07坝巴酸(barbatinic acid)
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太白茶HPLC指纹图谱的建立及UHPLC-Q Exactive Focus MS/MS联用分析*
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李莎莎 1 , 李雅娟 2 , 王晓婷 1 , 毛阿娟 1 , 李凡 1 , 李芳 1 , 张红 1 , 王卫锋 1, **
药物分析杂志 | 成分分析 2024,44(4): 585-593
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药物分析杂志 | 成分分析 2024, 44(4): 585-593
太白茶HPLC指纹图谱的建立及UHPLC-Q Exactive Focus MS/MS联用分析*
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李莎莎1 , 李雅娟2, 王晓婷1, 毛阿娟1, 李凡1, 李芳1, 张红1, 王卫锋1, **
作者信息
  • 1.陕西省中医药研究院,西安 710003
  • 2.陕西中医药大学,咸阳 712046
  • Tel:15829736183;E-mail:

通讯作者:

** Tel:13572021669;E-mail:
Eatablishment of HPLC fingerprint of lichens of Thamnolia subuliformis (Ehrh.) W. Culb. and its UHPLC-Q Exactive Focus MS/MS analysis*
Sha-sha LI1 , Ya-juan LI2, Xiao-ting WANG1, A-juan MAO1, Fan LI1, Fang LI1, Hong ZHANG1, Wei-feng WANG1, **
Affiliations
  • 1.Shaanxi Provincial Academy of Traditional Chinese Medicine, Xi’an 710003, China
  • 2.Shaanxi University of Chinese Medicine, Xianyang 712046, China
出版时间: 2024-04-30 doi: 10.16155/j.0254-1793.2024.04.05
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目的:

建立太白茶药材高效液相色谱(HPLC)指纹图谱分析方法,并运用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱(UHPLC-Q Exactive Focus MS/MS)法对太白茶中的特征峰进行定性分析。

方法:

采用Agilent TC-C18(250 mm×4.6 mm,5 μm)色谱柱,以甲醇(A)-0.1%甲酸水溶液(B)为流动相,梯度洗脱,流速1.0 mL·min-1,柱温30 ℃,检测波长254 nm,进样量10 μL。采用“中药色谱指纹图谱相似度评价系统2012年版”,建立指纹图谱并进行相似度评价。化学成分分析选用UHPLC-Q Exactive Focus MS/MS技术,采用Waters Acquity UPLC BEH C18(50 mm×2.1 mm,1.7 μm)色谱柱,以乙腈(A)-0.1%甲酸水(B)为流动相,梯度洗脱,流速0.3 mL·min-1,柱温30 ℃;质谱条件为电喷雾离子源(ESI),负离子模式扫描,扫描范围m/z 80~1 200。

结果:

建立的太白茶特征指纹图谱中,标定共有峰8个,10批太白茶药材相似度均≥0.9,各批次之间共有峰的相对保留时间RSD均<0.5%。对8个共有峰中的4个峰进行了结构指认,依次为鳞片衣酸、羊角衣酸、thamnoliadepsides B、坝巴酸。

结论:

所建立的HPLC指纹图谱方法稳定可靠,专属性好,结合UHPLC-Q-Exactive Focus-MS/MS的定性分析,可用于太白茶药材的整体质量综合评价。

太白茶  /  高效液相色谱  /  指纹图谱  /  质量评价  /  超高效液相色谱-四极杆/静电场轨道阱高分辨质谱  /  定性分析
Objective:

To establish a quality assessment method for lichens of Thamnolia subuliformis (Ehrh.) W. Culb. based on HPLC fingerprint, and qualitative analysis of the chemical constituents by ultra-high performance liquid chromatography coupled with quadrupole/electrostatic field orbital trap high resolution mass spectrometry (UHPLC-Q Exactive Focus MS/MS).

Methods:

Agilent TC-C18(250 mm×4.6 mm, 5 μm) chromatographic column was used, the mobile phase was methol -0.1% phosphoric acid with gradient elution at the flow rate of 1.0 mL·min-1, the column temperature was 30 ℃, the detection wavelength was 254 nm, and the injection volume was 10 μL. HPLC fingerprints of lichens of Thamnolia subuliformis was established. The software of similarity calculation for traditional Chinese medicines fingerprints (version 2012) was used to establish the fingerprinting of lichens of Thamnolia subuliformis. The chemical constituents were analyzed by UHPLC-Q Exactive Focus MS/MS, The chromatographic separation was performed on a Waters Acquity UPLC BEH C18 (50 mm×2.1 mm, 1.7 μm) column with acetonitrile(A) -0.1% formic acid aqueous solution(B) as mobile phase for gradient elution, the flow rate was 0.3 mL·min-1 and the column temperature was 30 ℃.Mass spectrometry was performed using an electrospray ionization and data was collected in negative ion modes in the range of m/z 80-1 200.

Results:

Eight common peaks were identified from fingerprints of 10 batches of samples. The RSD values of relative retention time of 8 common peaks of chromotograms of samples were all below 0.5% and the similarities were above 0.9. 4 of the identified peaks were further confirmed by UHPLC-Q Exactive Focus MS/MS as squamatic acid, baeomycesic acid, thamnoliadepsides B, barbatinic acid.

Conclusion:

The established method of fingerprint is stable, reliable, and specific, which can be used for quality evaluation of lichens of Thamnolia subuliformis.

Thamnolia subuliformis (Ehrh.) W. Culb.  /  HPLC  /  fingerprinting  /  quality evaluation  /  UHPLC-Q Exactive Focus MS/MS  /  qualitative analysis
李莎莎, 李雅娟, 王晓婷, 毛阿娟, 李凡, 李芳, 张红, 王卫锋. 太白茶HPLC指纹图谱的建立及UHPLC-Q Exactive Focus MS/MS联用分析*. 药物分析杂志, 2024 , 44 (4) : 585 -593 . DOI: 10.16155/j.0254-1793.2024.04.05
Sha-sha LI, Ya-juan LI, Xiao-ting WANG, A-juan MAO, Fan LI, Fang LI, Hong ZHANG, Wei-feng WANG. Eatablishment of HPLC fingerprint of lichens of Thamnolia subuliformis (Ehrh.) W. Culb. and its UHPLC-Q Exactive Focus MS/MS analysis*[J]. Chinese Journal of Pharmaceutical Analysis, 2024 , 44 (4) : 585 -593 . DOI: 10.16155/j.0254-1793.2024.04.05
太白茶,别名雪茶、地茶,为雪地茶Thamnolia subuliformis (Ehrh.) W. Culb.或地茶Thamnolia vermicularis (Sw.) Ach. ex Schaer.的地衣体,主要生长在高寒地带,如陕西太白山、云南、西藏等地,具有清热解毒、安神养心、清心明目的功效[1-3]。在《全国中草药汇编》[4]及《中药大辞典》[5]中其名为雪茶,民间常作汤剂或泡茶饮,主要用于治疗中暑、心烦口渴、肺热咳嗽、咽喉肿痛、虚劳骨蒸、癫痫狂躁、失眠、目涩等症。2015年版《陕西省药材标准》[6]中收录的太白茶为雪地茶Thamnolia subuliformis (Ehrh.) W. Culb.的地衣体。现代研究表明,太白茶是一类独特的由共生菌和共生藻组成的复合生物有机共生体,其代谢产物主要分为初生代谢物和次生代谢物。初生代谢物主要包括多糖、氨基酸等物质,次生代谢物主要为地衣酸类,具有抗菌、调节免疫、抗衰老、抗癌等药理活性[7-9],并被用于辅助治疗阿尔兹海默症、高血压、肥胖和神经衰弱等症[10-12]。但目前国内外关于太白茶的研究报道较少,质量控制方面的研究更是稀缺,因此,本实验拟通过建立太白茶的HPLC指纹图谱,并采用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱(UHPLC-Q Exactive Focus MS/MS)技术对其共有峰成分进行定性分析,以期为太白茶的质量综合评价提供可行方法,并为该药材后续药效物质基础研究提供依据。
UltiMate 3000型超高效液相色谱仪和Q Exactive Focus型质谱仪(Thermo Fisher Scientific公司);Peak View数据处理软件(Thermo Fisher Scientific公司);BT25S型十万分之一电子分析天平和BS210S型万分之一电子分析天平(北京赛多利斯科学仪器有限公司);DFT-200A粉碎机(温岭市林大机械有限公司);KQ-5200E台式超声波清洗器(昆山市超声仪器有限公司)。
对照品:苔色酸(批号CHB210121)、松萝酸(批号CHB201214),纯度≥98%,购自成都克洛玛生物科技有限公司;黑茶渍素(批号OK201912B),纯度≥98%,购自北京沃凯生物科技有限公司。甲醇、乙腈、甲酸为色谱级;水为屈臣氏蒸馏水;其他试剂均为分析纯。
10批太白茶样品,其中云南省2批(香格里拉编号S3;丽江编号S4),四川阿坝1批(编号S5),陕西省7批(周至县编号S2;眉县编号S10;太白县5批分别编号S1、S6、S7、S8、S9),经陕西省中医药研究院杨智锋主任药师鉴定为管枝衣科植物雪地茶Thamnolia subuliformis(Ehrh.) W. Culb.的地衣体。
精密称取苔色酸、松萝酸、黑茶渍素的对照品适量,分别置10 mL量瓶中,以三氯甲烷约1 mL溶解,加甲醇定容至刻度,分别配制成质量浓度为0.2、0.5、0.2 mg·mL-1的对照品储备液,备用。
取太白茶样品(粉碎,过60目筛)0.5 g,精密称定,置100 mL具塞三角瓶中,加入甲醇25 mL,称量,超声(功率400 W,频率40 kHz)提取30 min,放置至室温,再称量,用甲醇补足减失的量,0.22 μm微孔滤膜滤过,即得。
采用Agilent TC-C18(250 mm×4.6 mm,5 μm)色谱柱,以甲醇(A)-0.1%甲酸水溶液(B)为流动相,梯度洗脱(0~10 min,30%A→45%A;10~80 min,45%A→80%A;80~90 min,80%A→95%A),流速1.0 mL·min-1,柱温30 ℃,检测波长254 nm,进样量10 μL。
取“2.1.2”项下供试品溶液(样品编号S6)适量,按照“2.2.1”项下色谱条件连续进样测定6次,以4号色谱峰的保留时间和峰面积为参照,计算出各共有峰的相对保留时间的RSD≤0.13%(n=6)、相对峰面积的RSD≤1.8%(n=6),表明仪器的精密度良好。
取“2.1.2”项下供试品溶液(样品编号S6)适量,分别于室温放置0、2、4、8、10、12 h,按照“2.2.1”项下色谱条件进样测定,以4号色谱峰的保留时间和色谱峰面积为参照,计算出8个共有峰的相对保留时间的RSD≤0.81%(n=6),相对峰面积的RSD≤3.0%(n=6),表明供试品溶液在室温放置12 h内稳定性良好。
取同一批次(样品编号S6)药材粉末6份,每份0.5 g。按照“2.1.2”项下方法制备供试品溶液,再按照“2.2.1”项下色谱条件进样测定,以4号色谱峰的保留时间和色谱峰面积为参照,计算出8个共有峰的相对保留时间的RSD≤0.11%(n=6) ,相对峰面积的RSD≤3.1%(n=6),表明本方法重复性良好。
取10批次太白茶样品,按照“2.1.2”项下方法制备供试品溶液,再按照“2.2.1”项下色谱条件进样测定,记录色谱图。采用《中药色谱指纹图谱相似度评价系统(2012年版)》,以编号S6的样品色谱图为参照图谱,时间窗宽度为0.1,以中位数法生成对照指纹图谱(R),得到的10批太白茶药材HPLC叠加图谱均有8个共有峰,见图1。以4号色谱峰为参照峰,计算10批太白茶样品共有峰的相对峰面积及相对保留时间,结果见表12
表1可知,10批太白茶色谱峰中有8个共有峰。以最大峰面积的4号峰为对照峰,峰面积设为1,每个太白茶图谱中的其他共有峰峰面积与之峰面积相除得到相对峰面积。同理,表2则提供了8个共有峰的保留时间,由表中数据可知,每个太白茶色谱图中共有峰保留时间的标准差(SD)均<1,说明这些峰的选取符合共有峰的特点。
采用《中药色谱指纹图谱相似度评价系统(2012年版)》对10批样品进行相似度评价,结果见表3。软件相似度计算是采用向量夹角余弦法,相似度结果在0.9~1.0,符合要求。由表中数据可知,各批次样品S1~S10的指纹图谱与对照指纹图谱的相似度均≥0.970,符合指纹图谱的技术要求,表明10批药材之间具有较好的一致性,本方法可用于综合评价太白茶药材的整体质量。
采用Waters Acquity UPLC BEH C18 (50 mm×2.1 mm,1.7 μm)色谱柱,以乙腈(A)-0.1%甲酸水(B)为流动相,梯度洗脱(0~8 min,10%A→45%A;8~15 min,45%A→60%A;15~20 min,60%A→95%A;20~22 min,95%A),流速0.3 mL·min-1,柱温30 ℃,进样量1 μL。采用加热电喷雾离子源(ESI),负离子监测模式,喷雾电压为3.0 kV,毛细管温度350 ℃,雾化温度320 ℃。扫描方式为一级全扫描/数据依赖型二级质谱扫描(Full MS/dd-MS2),Full MS分辨率70 000,dd-MS2分辨率17 500,扫描范围m/z 100~1 500,碰撞能量(CE)设定15 eV。数据分析采用Xcalibur 4.0软件。
取“2.1.1”与“2.1.2”项下对照品储备液和供试品溶液,按“2.3.1”项下色谱-质谱条件进行测定,得到总离子流图,见图2,结合对照品以及查阅文献相关报道[13-17],对指纹图谱中的一些共有峰的结构归属进行判断,详细信息见表4。太白茶中的主要成分为地衣酸类成分,主要包括二苯并呋喃类、缩酚酸及其衍生物和单本环衍生物,采用已有对照品比对,鉴定出太白茶药材中的单苯环取代物苔色酸、缩酚酸类黑茶渍素和二苯并呋喃类松萝酸,然而在质谱中三者的响应值较低,且在HPLC中色谱峰也很小,几乎检测不到,故未加入指纹图谱中的共有特征峰。而图1的共有峰峰4、峰6、峰7及峰8的响应值相对较高,分离度较好,采用制备液相色谱仪按照“2.2.1”项下色谱条件对共有峰峰4、峰6、峰7及峰8进行分离纯化,采用UHPLC-Q Exactive Focus MS/MS进行定性分析,指认其均属于缩酚酸类化合物,依次为鳞片衣酸、羊角衣酸、thamnoliadepsides B、坝巴酸,其二级质谱图见图3
单本环取代物在负离子模式下主要以[M-H]-的准分子离子峰形式存在,发生裂解时,母核易出现连续丢失CO2、H2O等小分子。保留时间为4.19 min时,根据结构和对照品二级碎片,离子鉴定化合物为苔色酸,一级质谱出现准分子离子峰为m/z 167.035 0[M-H]-,根据数据库匹配出分子式为C8H8O4,二级质谱中失去一分子H2O,产生m/z 149.024 6 [M-H-H2O]-,或直接失去1个CO2,获得m/z 123.045 4[M-H-CO2]-,再失去一分子H2O,得到m/z 105.034 8[M-H-CO2-H2O]-,其裂解途径见图4
缩酚酸类化合物发生裂解时,一方面母核易出现CO、CO2、CH3等中性分子的连续丢失或组合丢失,另一方面通过打破八元环过渡态氢重排的酯键与水杨酸部分A和苯酚部分B进行竞争,断裂为A和B 2个部分,再由A或B部分连续丢失CH3、CO、CO2或组合丢失[13]。以对照品黑茶渍素为例,其保留时间为16.59 min时,一级质谱出现准分子离子峰m/z 373.094 3[M-H]-,经计算分子式为C19H18O8m/z 195.067 3[M-H-C9H7O4]-为相应酯键断裂的B部分,m/z 181.051 0[M-H-C9H7O4-CH3]-m/z 163.040 8[M-H-C9H7O4-CH3-H2O]-m/z 119.050 5[M-H-C9H7O4-CH3-H2O-CO2]-则分别是由B部分失去CH3、H2O和CO2所形成的;而m/z 177.019 8[M-H-C10H11O4-H2O]-m/z 133.029 9[M-H-C10H11O4-H2O-CO2]-m/z 105.035 0[M-H-C10H11O4-H2O-CHO]-则分别是由相应酯键断裂的A部分连续失去H2O、CO2和CHO所形成的[14-15],其裂解途径见图5
二苯并呋喃类发生裂解时,一方面母核连续脱去CH3、CO等中性小分子,另一方面该类化合物在高温高热环境中易发生逆Diels-Alder(RDA)重排,多形成m/z 84(C4H4O2)和m/z 112(C5H4O3)特征片段[16]。保留时间为15.98 min时,根据结构和对照品二级碎片离子鉴定其为松萝酸[16-17],一级质谱出现准分子离子峰m/z 343.083 3[M-H]-,计算出分子式为C18H16O7,在二级质谱中失去1个CH3得到m/z 328.060 0[M-H-CH3]-、再连续失去2个CH3、CH2得到m/z 313.036 8[M-H-2CH3]-m/z 299.093 6[M-H-2CH3-CH2]-;或直接发生RDA重排得到m/z 259.061 9[M-H-C4H4O2]-m/z 231.067 0[M-H-C5H4O3]-,其裂解途径见图6
本实验对不同提取溶剂(甲醇、乙醇、三氯甲烷)进行考察时,发现甲醇作为提取溶剂时,得到的峰最多;继续考察不同比例(50%、60%、70%、80%、100%)的甲醇,结果以100%甲醇为提取溶剂时,仪器响应较高,图谱基线较为平稳,得到峰较多,故选择100%甲醇作为提取溶剂。分别考察超声法和回流法2种提取方法,发现大部分成分的提取效率相当,仅个别成分如4号峰加热回流法相比超声提取响应值稍大,综合考虑超声提取更加简便,因此选择了超声法。
建立指纹图谱时先采用全波长(190~400 nm)对供试品溶液进行扫描,发现在254 nm下各色谱峰均得到清晰的表达,因此选择254 nm作为太白茶的检测波长。前期也比较了甲醇-水、甲醇-0.1%磷酸水溶液、甲醇-0.1%甲酸水溶液、乙腈-水、乙腈-0.1%磷酸水溶液和乙腈-0.1%甲酸水溶液等不同流动相系统。结果发现,以甲醇-0.1%磷酸水溶液为流动相进行梯度洗脱时,各成分的色谱峰分离效果较好,而在UHPLC-Q Exactive Focus MS/MS分析时则以乙腈-0.1%甲酸水溶液为流动相进行梯度洗脱时,各成分的色谱峰形较好。因此在指纹图谱建立及UHPLC-Q Exactive Focus MS/MS分析时分别选择这2种流动相进行洗脱。
本实验建立太白茶指纹图谱,确定了8个共有峰;对10批样品指纹图谱的相似度进行评价,结果表明与对照指纹图谱有良好的相似性。根据色谱峰的分离情况,4号色谱峰分离度较好,峰面积相对较大,且位置居中,故选择该峰为参照峰。采用UHPLC-Q Exactive Focus MS/MS分析鉴别共有峰时,分别以正负离子模式对各成分进行检测,发现各成分均在负离子模式下响应较高,这可能与太白茶中的主要成分为地衣酸类有关,故最终采用负离子模式进行样品测定。
对太白茶中特征性成分的离子碎片信息进行采集分析,结合文献及数据分析,指认了其指纹图谱中4个成分。由于太白茶中主要成分的对照品比较难获得,通过制备液相色谱及质谱技术所得化合物的碎片离子峰对4个共有峰进行了推测,尚有4个共有峰未鉴定,后续研究可进一步通过制备色谱分离纯化后对其结构进行确定。
综上所述,本研究建立的太白茶药材HPLC指纹图谱,同时采用UHPLC-Q Exactive Focus MS/MS技术,对指纹图谱中的4个共有特征峰进行了鉴定分析,为太白茶药材的质量评价提供了依据,也为后续太白茶药效物质基础研究提供了参考。
  • *陕西省中医药管理局中医药全省性专款专项项目(2021-QYZL-01)
  • 陕西省中医药研究院“苗圃培育计划”项目(2021-16)
  • 陕西省中医药研究院“苗圃培育计划”项目(2021-21)
  • 陕西省中医药管理局传承创新暨“秦药”开发重点科学研究项目(2021-02-GJ-021)
  • 陕西省重点研发计划一般项目(2022SF-301)
  • 陕西省重点研发计划一般项目(2024SF-YBXM-481)
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2024年第44卷第4期
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doi: 10.16155/j.0254-1793.2024.04.05
  • 首发时间:2026-03-13
  • 出版时间:2024-04-30
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基金
*陕西省中医药管理局中医药全省性专款专项项目(2021-QYZL-01)
陕西省中医药研究院“苗圃培育计划”项目(2021-16)
陕西省中医药研究院“苗圃培育计划”项目(2021-21)
陕西省中医药管理局传承创新暨“秦药”开发重点科学研究项目(2021-02-GJ-021)
陕西省重点研发计划一般项目(2022SF-301)
陕西省重点研发计划一般项目(2024SF-YBXM-481)
作者信息
    1.陕西省中医药研究院,西安 710003
    2.陕西中医药大学,咸阳 712046

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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

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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