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Article(id=1239250703202447595, tenantId=1146029695717560320, journalId=1205117023404326918, issueId=1239250701583446236, articleNumber=null, orderNo=null, doi=10.16155/j.0254-1793.2024.04.10, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1695744000000, receivedDateStr=2023-09-27, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1773389991807, onlineDateStr=2026-03-13, pubDate=1714406400000, pubDateStr=2024-04-30, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773389991807, onlineIssueDateStr=2026-03-13, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773389991807, creator=13701087609, updateTime=1773389991807, 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=628, endPage=636, ext={EN=ArticleExt(id=1239250703382802673, articleId=1239250703202447595, tenantId=1146029695717560320, journalId=1205117023404326918, language=EN, title=Differential components screening of Bofonis Corium and Bufonis Venenum based on UPLC-Q-Exactive Orbitrap MS couple with chemometrics and its application research*, columnId=1206272757852074373, journalTitle=Chinese Journal of Pharmaceutical Analysis, columnName=Safety Monitoring, runingTitle=null, highlight=null, articleAbstract=
Objective:

To investigate and use the different constituents in Bofonis Corium distinguishing from Bufonis Venenum based on ultra-performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry (UPLC-Q-Exactive Orbitrap MS) coupled with chemometrics.

Methods:

UPLC analysis was performed on a Waters Atlantis T3 (150 mm×2.1 mm, 3 μm) column with gradient elution of acetonitrile and 0.1% formic acid at a flow rate of 0.3 mL·min-1. The column temperature was set at 30 ℃. UPLC-Q-Exactive Orbitrap MS was chosen for data collection in both positive and negative ion modes. The potential differential ions were obtained by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). The Xcalibur 3.0 data processing system was utilized to extract the secondary fragmentation details of the differential ions. Subsequently, the specificity of differential ions was confirmed through the multiple reaction monitoring (MRM) mode of AB SCIEX 6500+ triple quadrupole mass spectrometry. Identification of adulterated Bofonis Corium in Bufonis Venenum was performed by applying the ion pairs with good specificity.

Results:

After the research and verification, a total of five distinction pairs (m/z: 377.5→243.2, 172.1；330.4→170.9, 127.0；313.4→201.2, 171.0；452.4→280.2, 298.2 and 614.7→332.4, 281.3) were discovered, and a method for the examination of Bofonis Corium in Bufonis Venenum was established and methodologically investigated by applying the information of the different ions. The constructed method had reasonable specificity and durability. Each ion pair showed an excellent linear relationship within a specific range, with correlation coefficients above 0.996 7. The RSDs of the precision test were in the range of 1.3%-4.1%. The RSDs of the repeatability test were in the range of 1.3%-3.2%. Five batches of market samples of Bufonis Venenum were determined according to the above process, and two batches of samples showed the presence of Bofonis Corium components but did not exceed the proposed 5% adulteration limit.

Conclusion:

In this study, a rapid method for detecting discrepancy information based on UPLC-Q-Exactive Orbitrap MS combined with chemometrics is presented, and the discrepant information is applied to establish an identification method for adulterated Bofonis Corium in Bufonis Venenum.

, correspAuthors=Yong-qiang LIN, 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=Juan CHEN, Yang JIAO, Bing WANG, Lin WANG, Fei XUE, Ying-ying XIE, Qing-kang YAN, Xiang LI, Yong-qiang LIN), CN=ArticleExt(id=1239250705463177499, articleId=1239250703202447595, tenantId=1146029695717560320, journalId=1205117023404326918, language=CN, title=基于UPLC-Q-Exactive Orbitrap MS结合化学计量学筛选蟾皮区别于蟾酥的差异成分及应用研究*, columnId=1206272758036623764, journalTitle=药物分析杂志, columnName=安全监测, runingTitle=null, highlight=null, articleAbstract=
目的：

应用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱(UPLC-Q-Exactive Orbitrap MS)结合化学计量学对蟾皮区别于蟾酥的差异成分进行研究和应用。

方法：

采用Waters Atlantis T3(150 mm×2.1 mm，3 μm)色谱柱，以乙腈-0.1%甲酸溶液为流动相，进行梯度洗脱，流速0.3 mL·min-1，柱温30 ℃，选用UPLC-Q-Exactive Orbitrap MS分别在正、负离子模式下采集数据，通过主成分分析(PCA)和正交偏最小二乘-判别分析(OPLS-DA)得到候选差异离子，通过Xcalibur 3.0数据处理系统提取差异离子的二级碎片信息，再使用AB SCIEX 6500＋三重四极杆质谱的多反应监测模式(MRM)对候选差异离子进行专属性验证，应用专属性良好的离子对建立蟾酥中掺伪蟾皮的鉴别方法，并进行方法学考察。

结果：

经查找验证，共找到5个可用于识别蟾皮的差异离子对(m/z：377.5→243.2，172.1；330.4→170.9，127.0；313.4→201.2，171.0；452.4→280.2，298.2；614.7→332.4，281.3)，应用差异离子信息建立了蟾酥中蟾皮的检查方法并进行了方法学考察。考察结果表明，所建方法具有较好的专属性、耐用性，各离子对在一定范围内均呈现良好的线性关系，相关系数均大于0.996 7，精密度试验RSD为1.3%~4.1%，重复性试验RSD为1.3%~3.2%，依据上述方法对5批蟾酥市场样品进行测定，结果有2批样品出现蟾皮成分但未超出拟定的5%掺伪上限。

结论：

本研究报道了一种将UPLC-Q-Exactive Orbitrap MS和化学计量学相结合的快速寻找差异信息的方法，并应用差异信息建立了蟾酥中掺伪蟾皮的检查方法。

, correspAuthors=林永强, authorNote=null, correspAuthorsNote=
** Tel：13864067104；E-mail：
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Tel：18369277698；E-mail：

, authorsList=陈娟, 焦阳, 汪冰, 王琳, 薛菲, 解盈盈, 闫庆康, 李向, 林永强)}, authors=[Author(id=1239250705958105414, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, orderNo=0, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=chenj7698@163.com, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1239250706050380109, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, authorId=1239250705958105414, language=EN, stringName=Juan CHEN, firstName=Juan, middleName=null, lastName=CHEN, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=1, 2, address=1.Shandong University of Traditional Chinese Medicine, Jinan 250355, China
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Acta Acad Med CPAF，2011，20(12)：1009, articleTitle=Survey on the study of extraction and separation of the effective components from the skin of Bufo bufo gargarizans Cantor, refAbstract=null), Reference(id=1239250720898216897, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, doi=null, pmid=null, pmcid=null, year=2007, volume=42, issue=8, pageStart=858, pageEnd=null, url=null, language=null, rfNumber=[19], rfOrder=36, authorNames=代丽萍, 高慧敏, 王智民, journalName=药学学报, refType=null, unstructuredReference=代丽萍，高慧敏，王智民，等. 蟾皮化学成分的分离与结构鉴定[J]. 药学学报，2007，42(8)：858, articleTitle=蟾皮化学成分的分离与结构鉴定, refAbstract=null), Reference(id=1239250721036628932, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, doi=null, pmid=null, pmcid=null, year=2007, volume=42, issue=8, pageStart=858, pageEnd=null, url=null, language=null, rfNumber=[19], rfOrder=37, authorNames=DAI LP, GAO HM, WANG ZM, journalName=Acta Pharm Sin, refType=null, unstructuredReference=DAI LP，GAO HM，WANG ZM，et al. Isolation and structure identification of chemical constituents from the skin of Bufo bufo gargarizans[J]. 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remark=3.Shandong KangYuanTang Chinese Herbal Medicine Co., Ltd., Jining 272000, China), AuthorCompanyExt(id=1239250705878413632, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, companyId=1239250705861636413, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3.山东康源堂中药饮片股份有限公司，济宁 272000)])], figs=[ArticleFig(id=1239250711800771231, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, language=EN, label=Fig.1, caption=PCA plot of Bufonis Venenum and Bofonis Corium, figureFileSmall=eWsIkrCzN8S50Eq4SMrZHg==, figureFileBig=2FMqJCDB1yG//SmL7ttVbg==, tableContent=null), ArticleFig(id=1239250711905628836, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, language=CN, label=图1, caption=蟾酥和蟾皮的PCA图, figureFileSmall=eWsIkrCzN8S50Eq4SMrZHg==, figureFileBig=2FMqJCDB1yG//SmL7ttVbg==, tableContent=null), ArticleFig(id=1239250712136315577, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, language=EN, label=Fig.2, caption=OPLS-DA and S-plot plot of Bufonis Venenum and Bofonis Corium (red circles represent differential ions), figureFileSmall=VSZoQQlWbbYz9ttfDIr8dQ==, figureFileBig=zaWZXT0HwaaSTjsR4vd5fg==, tableContent=null), ArticleFig(id=1239250712249561790, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, language=CN, label=图2, caption=蟾酥和蟾皮的OPLS-DA和S-plot分析结果图(红色圆形代表差异离子)

A.正离子模式下OPLS-DA图(OPLS-DA chart in positive ion mode)　B.负离子模式下OPLS-DA图(OPLS-DA chart in negative ion mode)　C.正离子模式下S-plot图(S-plot chart in positive ion mode)　D.负离子模式下S-plot图(S-plot chart in negative ion mode)

, figureFileSmall=VSZoQQlWbbYz9ttfDIr8dQ==, figureFileBig=zaWZXT0HwaaSTjsR4vd5fg==, tableContent=null), ArticleFig(id=1239250712371196615, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, language=EN, label=Fig.3, caption=Specificity results of differential ion, figureFileSmall=5wIUrxLfgzLT5MkOoYMMFw==, figureFileBig=MxcSKPTpONFGvIrT4ECNWg==, tableContent=null), ArticleFig(id=1239250712501220049, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, language=CN, label=图3, caption=差异离子专属性结果, figureFileSmall=5wIUrxLfgzLT5MkOoYMMFw==, figureFileBig=MxcSKPTpONFGvIrT4ECNWg==, tableContent=null), ArticleFig(id=1239250714015363797, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, language=EN, label=Tab.1, caption=

Sample information of Bufonis Venenum and Bofonis Corium

, figureFileSmall=null, figureFileBig=null, tableContent=
样品
(sample)		样品编号
(sample No.)		样品来源
(source)
蟾酥(Bufonis Venenum)CS-SD-1~3山东(Shandong)
CS-SC-1~3四川(Sichuan)
CS-JS-1~3江苏(Jiangsu)
CS-DB-1~3东北(Dongbei)
CS-HN-1~2河南(Henan)
CS-YCSC-1~5安徽亳州药材市场(Anhui Bozhou Medicinal Herb Market)
蟾酥对照药材(Bufonis Venenum control)CS-DZYC中国食品药品检定研究院(National Institutes for Food and Drug Control)
蟾皮(Bofonis Corium)CP-SD-1~3山东(Shandong)
CP-SC-1~3四川(Sichuan)
CP-JS-1~3江苏(Jiangsu)
CP-DB-1~3东北(Dongbei)
CP-ZJ-1~3浙江(Zhejiang)
CP-HN-1~3河南(Henan)
CP-SN-1~3陕西(Shaanxi)
CP-SX-1~2山西(Shanxi)
干蟾皮对照药材(Bofonis Corium control)CP-DZYC中国食品药品检定研究院(National Institutes for Food and Drug Control)
), ArticleFig(id=1239250714103444185, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, language=CN, label=表1, caption=

蟾酥及蟾皮样品信息

, figureFileSmall=null, figureFileBig=null, tableContent=
样品
(sample)		样品编号
(sample No.)		样品来源
(source)
蟾酥(Bufonis Venenum)CS-SD-1~3山东(Shandong)
CS-SC-1~3四川(Sichuan)
CS-JS-1~3江苏(Jiangsu)
CS-DB-1~3东北(Dongbei)
CS-HN-1~2河南(Henan)
CS-YCSC-1~5安徽亳州药材市场(Anhui Bozhou Medicinal Herb Market)
蟾酥对照药材(Bufonis Venenum control)CS-DZYC中国食品药品检定研究院(National Institutes for Food and Drug Control)
蟾皮(Bofonis Corium)CP-SD-1~3山东(Shandong)
CP-SC-1~3四川(Sichuan)
CP-JS-1~3江苏(Jiangsu)
CP-DB-1~3东北(Dongbei)
CP-ZJ-1~3浙江(Zhejiang)
CP-HN-1~3河南(Henan)
CP-SN-1~3陕西(Shaanxi)
CP-SX-1~2山西(Shanxi)
干蟾皮对照药材(Bofonis Corium control)CP-DZYC中国食品药品检定研究院(National Institutes for Food and Drug Control)
), ArticleFig(id=1239250714191524573, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, language=EN, label=Tab.2, caption=

The special property verification results of candidate differential ions

, figureFileSmall=null, figureFileBig=null, tableContent=
监测模式
(monitoring mode)		编号
(No.)		m/ztR/min蟾皮
(Bofonis Corium)		蟾酥
(Bufonis Venenum)
正离子模式(postive ion mode)1102.04282.919 6+-+
2124.0492.389 98+-+
3137.0562.589 66+--
4152.0682.389 98++-
5275.1542.422 93++-
6303.25478.272 1++-
7317.2047.758 42+-+
8317.2046.167 53+-+
9331.2211.567 4+-+
10332.22411.224 8++-
11333.22611.539 5++-
12345.23715.726 5++-
13377.47217.988 1+-
14385.26456.198 4++-
15386.26756.163 9++-
16438.32974.353 7+-+
17443.27356.454 9+-+
18444.27756.454 9+-+
19445.27956.454 9+-+
20445.28944.623 3+-+
21446.29344.507 4+-+
22452.35678.033 2+-
23454.32671.612 0++-
24464.34776.886 9++-
25496.37572.077 8+--
26497.37972.077 8+--
27506.34570.846 6++-
负离子模式(negative ion mode)1135.022.563 38++-
2150.032.379 78+--
3195.7962.086 97++-
4197.7932.086 97+--
5199.792.086 97+--
6273.1013.020 83++-
7311.20165.036 8++-
8313.41663.645 1+-
9315.1468.560 66++-
10315.1467.095 43++-
11317.1970.130 1+-+
12319.20576.118 9++-
13319.20578.212 5++-
14329.1611.569 2+-+
15330.36411.545 0+-+
16330.21344.465 2+-
17421.10718.006 8++-
18438.26873.439 1++-
19452.24771.589 5+-+
20453.2571.589 5+-+
21464.28275.981 8++-
22464.28383.972 8++-
23489.21444.431 3+-+
24490.21844.423 7+-+
25524.29974.763 5+-+
26540.29372.003 0+-+
27541.29672.003 1+-+
28568.32281.526 6++-
29597.26678.872 3+--
30614.70372.081 9+-
), ArticleFig(id=1239250714308965092, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, language=CN, label=表2, caption=

候选差异离子专属性验证结果

, figureFileSmall=null, figureFileBig=null, tableContent=
监测模式
(monitoring mode)		编号
(No.)		m/ztR/min蟾皮
(Bofonis Corium)		蟾酥
(Bufonis Venenum)
正离子模式(postive ion mode)1102.04282.919 6+-+
2124.0492.389 98+-+
3137.0562.589 66+--
4152.0682.389 98++-
5275.1542.422 93++-
6303.25478.272 1++-
7317.2047.758 42+-+
8317.2046.167 53+-+
9331.2211.567 4+-+
10332.22411.224 8++-
11333.22611.539 5++-
12345.23715.726 5++-
13377.47217.988 1+-
14385.26456.198 4++-
15386.26756.163 9++-
16438.32974.353 7+-+
17443.27356.454 9+-+
18444.27756.454 9+-+
19445.27956.454 9+-+
20445.28944.623 3+-+
21446.29344.507 4+-+
22452.35678.033 2+-
23454.32671.612 0++-
24464.34776.886 9++-
25496.37572.077 8+--
26497.37972.077 8+--
27506.34570.846 6++-
负离子模式(negative ion mode)1135.022.563 38++-
2150.032.379 78+--
3195.7962.086 97++-
4197.7932.086 97+--
5199.792.086 97+--
6273.1013.020 83++-
7311.20165.036 8++-
8313.41663.645 1+-
9315.1468.560 66++-
10315.1467.095 43++-
11317.1970.130 1+-+
12319.20576.118 9++-
13319.20578.212 5++-
14329.1611.569 2+-+
15330.36411.545 0+-+
16330.21344.465 2+-
17421.10718.006 8++-
18438.26873.439 1++-
19452.24771.589 5+-+
20453.2571.589 5+-+
21464.28275.981 8++-
22464.28383.972 8++-
23489.21444.431 3+-+
24490.21844.423 7+-+
25524.29974.763 5+-+
26540.29372.003 0+-+
27541.29672.003 1+-+
28568.32281.526 6++-
29597.26678.872 3+--
30614.70372.081 9+-
), ArticleFig(id=1239250714405434090, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, language=EN, label=Tab.3, caption=

Linear relationships of ion pair

, figureFileSmall=null, figureFileBig=null, tableContent=
离子对
(ion pair)m/z		回归方程
(regression equation)		r
377.5→243.2Y=209 900X+2 2220.998 8
330.4→170.9Y=24 828X+1 5800.997 8
313.4→201.2Y=153 524X+5 5550.998 2
452.4→280.2Y=1 420 504X+22 8540.998 2
614.7→332.4Y=7 991X+9760.996 7
), ArticleFig(id=1239250714518680304, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, language=CN, label=表3, caption=

各离子对线性关系

, figureFileSmall=null, figureFileBig=null, tableContent=
离子对
(ion pair)m/z		回归方程
(regression equation)		r
377.5→243.2Y=209 900X+2 2220.998 8
330.4→170.9Y=24 828X+1 5800.997 8
313.4→201.2Y=153 524X+5 5550.998 2
452.4→280.2Y=1 420 504X+22 8540.998 2
614.7→332.4Y=7 991X+9760.996 7
), ArticleFig(id=1239250714636120827, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, language=EN, label=Tab.4, caption=

Results of precision and repeatability test

, figureFileSmall=null, figureFileBig=null, tableContent=
离子对
(ion pair)m/z		RSD/%
精密度(precision)重复性(repeatability)
377.5→243.22.31.3
330.4→170.92.92.9
313.4→201.23.02.3
452.4→280.21.33.0
614.7→332.44.13.2
), ArticleFig(id=1239250714720006912, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, language=CN, label=表4, caption=

精密度和重复性试验结果

, figureFileSmall=null, figureFileBig=null, tableContent=
离子对
(ion pair)m/z		RSD/%
精密度(precision)重复性(repeatability)
377.5→243.22.31.3
330.4→170.92.92.9
313.4→201.23.02.3
452.4→280.21.33.0
614.7→332.44.13.2
), ArticleFig(id=1239250714833253123, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, language=EN, label=Tab.5, caption=

Results of matrix effect examination

, figureFileSmall=null, figureFileBig=null, tableContent=
离子对
(ion pair)
m/z		5%浓度下峰面积
(peak area at 5% concentration)		10%浓度下峰面积
(peak area at 10% concentration)		20%浓度下峰面积
(peak area at 20% concentration)
基质样品
(matrix sample)		基质空白样品
(matrix blank sample)		基质样品
(matrix sample)		基质空白样品
(matrix blank sample)		基质样品
(matrix sample)		基质空白样品
(matrix blank sample)
377.5→243.212 7148 53024 09417 06043 12234 120
330.4→170.93 0681 7254 0973 4516 5206 902
313.4→201.213 9249 17920 41518 35835 12036 716
452.4→280.289 44972 517170 946145 035292 050290 070
614.7→332.41 4019041 8651 8082 5423 616
), ArticleFig(id=1239250714938110730, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, language=CN, label=表5, caption=

基质效应考察结果

, figureFileSmall=null, figureFileBig=null, tableContent=
离子对
(ion pair)
m/z		5%浓度下峰面积
(peak area at 5% concentration)		10%浓度下峰面积
(peak area at 10% concentration)		20%浓度下峰面积
(peak area at 20% concentration)
基质样品
(matrix sample)		基质空白样品
(matrix blank sample)		基质样品
(matrix sample)		基质空白样品
(matrix blank sample)		基质样品
(matrix sample)		基质空白样品
(matrix blank sample)
377.5→243.212 7148 53024 09417 06043 12234 120
330.4→170.93 0681 7254 0973 4516 5206 902
313.4→201.213 9249 17920 41518 35835 12036 716
452.4→280.289 44972 517170 946145 035292 050290 070
614.7→332.41 4019041 8651 8082 5423 616
), ArticleFig(id=1239250715030385423, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, language=EN, label=Tab.6, caption=

Sample determination results

, figureFileSmall=null, figureFileBig=null, tableContent=
样品编号
(sample No.)		峰面积(peak area)
m/z 377.5→243.2m/z 330.4→170.9m/z 313.4→201.2m/z 452.4→280.2m/z 614.7→332.4
CS-YCSC-17 5712 60512 41337 490323
CS-YCSC-211 8132 1596 54727 105906
CS-YCSC-3/////
CS-YCSC-4/////
CS-YCSC-5/////
参比溶液(reference solution)12 7143 06813 92489 4491 401
), ArticleFig(id=1239250715097494290, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239250703202447595, language=CN, label=表6, caption=

样品测定结果

, figureFileSmall=null, figureFileBig=null, tableContent=
样品编号
(sample No.)		峰面积(peak area)
m/z 377.5→243.2m/z 330.4→170.9m/z 313.4→201.2m/z 452.4→280.2m/z 614.7→332.4
CS-YCSC-17 5712 60512 41337 490323
CS-YCSC-211 8132 1596 54727 105906
CS-YCSC-3/////
CS-YCSC-4/////
CS-YCSC-5/////
参比溶液(reference solution)12 7143 06813 92489 4491 401
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基于UPLC-Q-Exactive Orbitrap MS结合化学计量学筛选蟾皮区别于蟾酥的差异成分及应用研究*
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陈娟 1, 2 , 焦阳 2 , 汪冰 2 , 王琳 1, 2 , 薛菲 2 , 解盈盈 2 , 闫庆康 3 , 李向 3 , 林永强 1, 2, **
药物分析杂志 | 安全监测 2024,44(4): 628-636
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药物分析杂志 | 安全监测 2024, 44(4): 628-636
基于UPLC-Q-Exactive Orbitrap MS结合化学计量学筛选蟾皮区别于蟾酥的差异成分及应用研究*
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陈娟1, 2 , 焦阳2, 汪冰2, 王琳1, 2, 薛菲2, 解盈盈2, 闫庆康3, 李向3, 林永强1, 2, **
作者信息
  • 1.山东中医药大学，济南 250355
  • 2.山东省食品药品检验研究院国家药监局胶类产品质量评价重点实验室山东省中药标准创新与质量评价工程实验室 中药配方颗粒共性技术山东省工程研究中心，济南 250101
  • 3.山东康源堂中药饮片股份有限公司，济宁 272000

    • Tel：18369277698；E-mail：

通讯作者:

** Tel：13864067104；E-mail：
Differential components screening of Bofonis Corium and Bufonis Venenum based on UPLC-Q-Exactive Orbitrap MS couple with chemometrics and its application research*
Juan CHEN1, 2 , Yang JIAO2, Bing WANG2, Lin WANG1, 2, Fei XUE2, Ying-ying XIE2, Qing-kang YAN3, Xiang LI3, Yong-qiang LIN1, 2, **
Affiliations
  • 1.Shandong University of Traditional Chinese Medicine, Jinan 250355, China
  • 2.Shandong Institute for Food and Drug Control, NMPA Key Laboratory for Quality Evaluation of Gelatin Products, Shandong Engineering Laboratory for Standard Innovation and Quality Evaluation of TCM, Shangdong Engineering Research Center for Generic Technologies of Traditional Chinese Medicine Formula Granules, Jinan 250101, China
  • 3.Shandong KangYuanTang Chinese Herbal Medicine Co., Ltd., Jining 272000, China
出版时间: 2024-04-30 doi: 10.16155/j.0254-1793.2024.04.10
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摘要
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目的：

应用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱(UPLC-Q-Exactive Orbitrap MS)结合化学计量学对蟾皮区别于蟾酥的差异成分进行研究和应用。

方法：

采用Waters Atlantis T3(150 mm×2.1 mm，3 μm)色谱柱，以乙腈-0.1%甲酸溶液为流动相，进行梯度洗脱，流速0.3 mL·min-1，柱温30 ℃，选用UPLC-Q-Exactive Orbitrap MS分别在正、负离子模式下采集数据，通过主成分分析(PCA)和正交偏最小二乘-判别分析(OPLS-DA)得到候选差异离子，通过Xcalibur 3.0数据处理系统提取差异离子的二级碎片信息，再使用AB SCIEX 6500＋三重四极杆质谱的多反应监测模式(MRM)对候选差异离子进行专属性验证，应用专属性良好的离子对建立蟾酥中掺伪蟾皮的鉴别方法，并进行方法学考察。

结果：

经查找验证，共找到5个可用于识别蟾皮的差异离子对(m/z：377.5→243.2，172.1；330.4→170.9，127.0；313.4→201.2，171.0；452.4→280.2，298.2；614.7→332.4，281.3)，应用差异离子信息建立了蟾酥中蟾皮的检查方法并进行了方法学考察。考察结果表明，所建方法具有较好的专属性、耐用性，各离子对在一定范围内均呈现良好的线性关系，相关系数均大于0.996 7，精密度试验RSD为1.3%~4.1%，重复性试验RSD为1.3%~3.2%，依据上述方法对5批蟾酥市场样品进行测定，结果有2批样品出现蟾皮成分但未超出拟定的5%掺伪上限。

结论：

本研究报道了一种将UPLC-Q-Exactive Orbitrap MS和化学计量学相结合的快速寻找差异信息的方法，并应用差异信息建立了蟾酥中掺伪蟾皮的检查方法。

蟾皮  /  蟾酥  /  化学成分  /  差异成分  /  高分辨质谱  /  化学计量学  /  主成分分析  /  正交偏最小二乘-判别分析
Objective:

To investigate and use the different constituents in Bofonis Corium distinguishing from Bufonis Venenum based on ultra-performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry (UPLC-Q-Exactive Orbitrap MS) coupled with chemometrics.

Methods:

UPLC analysis was performed on a Waters Atlantis T3 (150 mm×2.1 mm, 3 μm) column with gradient elution of acetonitrile and 0.1% formic acid at a flow rate of 0.3 mL·min-1. The column temperature was set at 30 ℃. UPLC-Q-Exactive Orbitrap MS was chosen for data collection in both positive and negative ion modes. The potential differential ions were obtained by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). The Xcalibur 3.0 data processing system was utilized to extract the secondary fragmentation details of the differential ions. Subsequently, the specificity of differential ions was confirmed through the multiple reaction monitoring (MRM) mode of AB SCIEX 6500+ triple quadrupole mass spectrometry. Identification of adulterated Bofonis Corium in Bufonis Venenum was performed by applying the ion pairs with good specificity.

Results:

After the research and verification, a total of five distinction pairs (m/z: 377.5→243.2, 172.1；330.4→170.9, 127.0；313.4→201.2, 171.0；452.4→280.2, 298.2 and 614.7→332.4, 281.3) were discovered, and a method for the examination of Bofonis Corium in Bufonis Venenum was established and methodologically investigated by applying the information of the different ions. The constructed method had reasonable specificity and durability. Each ion pair showed an excellent linear relationship within a specific range, with correlation coefficients above 0.996 7. The RSDs of the precision test were in the range of 1.3%-4.1%. The RSDs of the repeatability test were in the range of 1.3%-3.2%. Five batches of market samples of Bufonis Venenum were determined according to the above process, and two batches of samples showed the presence of Bofonis Corium components but did not exceed the proposed 5% adulteration limit.

Conclusion:

In this study, a rapid method for detecting discrepancy information based on UPLC-Q-Exactive Orbitrap MS combined with chemometrics is presented, and the discrepant information is applied to establish an identification method for adulterated Bofonis Corium in Bufonis Venenum.

Bofonis Corium  /  Bufonis Venenum  /  chemical constituents  /  differential component  /  high resolution mass spectrometry  /  chemometric  /  principal component analysis  /  orthogonal partial least squares discriminant analysis
陈娟, 焦阳, 汪冰, 王琳, 薛菲, 解盈盈, 闫庆康, 李向, 林永强. 基于UPLC-Q-Exactive Orbitrap MS结合化学计量学筛选蟾皮区别于蟾酥的差异成分及应用研究*. 药物分析杂志, 2024 , 44 (4) : 628 -636 . DOI: 10.16155/j.0254-1793.2024.04.10
Juan CHEN, Yang JIAO, Bing WANG, Lin WANG, Fei XUE, Ying-ying XIE, Qing-kang YAN, Xiang LI, Yong-qiang LIN. Differential components screening of Bofonis Corium and Bufonis Venenum based on UPLC-Q-Exactive Orbitrap MS couple with chemometrics and its application research*[J]. Chinese Journal of Pharmaceutical Analysis, 2024 , 44 (4) : 628 -636 . DOI: 10.16155/j.0254-1793.2024.04.10
正文
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蟾皮和蟾酥均是来源于蟾蜍[中华大蟾蜍(Bufo bufo gargarizans Cantor)或黑眶蟾蜍(Bufo melanostictus Schneider)]的常用中药材[1-2]。蟾皮为其干燥全皮[3]，具有清解热毒、利水消肿、活血通络的功效，临床上被广泛用于治疗恶性肿瘤、毒疮、肠头挺出等疾病[4]。蟾酥是其耳后腺及皮肤腺干燥分泌物，具有解毒、止痛、开窍醒神等功效，主要用于治疗痈疽疔疮、咽喉肿痛、中暑神昏、痧胀腹痛吐泻[5-7]。蟾酥药材一直是稀缺高价的贵细药材，每千克售价可高达60 000元，市售商品药材不断出现掺杂淀粉、泥沙、蛋白、蟾皮的现象[8-9]。而蟾皮每千克市售价约300元，仅为蟾酥的1/200，2种药材差价较大。
蟾皮和蟾酥虽来源于同一动物，但不同的药用部位决定了物质基础的必然差异。历史记载中蟾皮、蟾酥的功能主治不尽相同，并在不同的应用中发挥各自的药用功效，二者掺用、混用必然影响药材发挥治疗作用[10-11]。在蟾酥中掺入蟾皮后，由于二者具有相同的物种来源，在所含物质成分上存在较大的交叉，因此鉴别难度大，特别是当掺杂的蟾酥加工炮制进入制剂后，更加难以通过常规手段进行杂质的辨别。因此寻找到蟾皮区别于蟾酥的差异成分对建立蟾皮专属有效的识别方法非常关键，可为蟾蜍药材的质量评价、掺伪打假及开发利用提供技术支持。《中华人民共和国药典》中收载的蟾酥包括中华大蟾蜍和黑眶蟾蜍2个物种来源，通过前期养殖厂调研发现，黑眶蟾蜍体积较小且产酥量低，因此目前蟾蜍养殖以中华大蟾蜍为主，是主要的市售物种。本文以中华大蟾蜍为研究对象，所建立的方法适用于中华大蟾蜍来源的蟾酥与蟾皮的识别分析。
本研究通过UPLC-Q-Exactive Orbitrap MS法并结合主成分分析(PCA)、正交偏最小二乘-判别分析(OPLS-DA)等化学计量学方法，寻找蟾皮区别于蟾酥的差异离子，进一步用三重四极杆质谱的多反应监测模式(MRM)对差异离子对进行专属性验证，应用专属性良好的离子对建立了蟾酥中掺伪蟾皮的鉴别方法，并进行了方法学考察。
1　仪器、试剂、软件与样品信息
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1.1　仪器
Thermo ScientificTM UltiMateTM 3000超高效液相色谱系统-Q-Exactive质谱联用仪(Thermo Scientific公司)；Triple Quad 6500+高效液相色谱-三重四极杆质谱联用仪(AB SCIEX公司)；XSE205DU十万分之一电子天平(Mettler Toledo公司)；电热恒温水浴锅(上海森信实验仪器有限公司)。
1.2　试剂
甲醇(色谱纯，批号230412)，天津市康科德科技有限公司产品；甲酸(色谱纯，批号E2128234)，上海阿拉丁生化科技股份有限公司产品；乙腈(色谱纯，批号222721)，赛默飞世尔科技(中国)有限公司产品；蒸馏水，广州屈臣氏食品饮料有限公司产品。
1.3　软件
Xcalibur 3.0数据处理系统；SIMCA 14.1 (32-bit)软件；SIEVE 2.2软件(Thermo Scientific公司)；Compound Discoverer 3.1软件(Thermo Scientific公司)。
1.4　样品信息
共收集蟾酥样品20批，其中15批来源明确的样品用于差异离子对查找(14批来自中华大蟾蜍养殖基地，1批为中国食品药品检定研究院对照药材)，另有5批来自药材市场，用于样品测定；收集蟾皮样品24批，23批来自中华大蟾蜍养殖基地，经山东省食品药品检验研究院汪冰主任药师鉴定来源均为中华大蟾蜍，1批为中国食品药品检定研究院对照药材，见表1。
2　方法与结果
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2.1　差异离子的查找
2.1.1　供试品溶液制备
蟾酥、蟾皮粉碎，取蟾酥细粉25 mg、蟾皮细粉1 g，精密称定，分别置具塞回流瓶中，精密加入甲醇20 mL，称量，加热回流1 h，放冷，再称量，用甲醇补足减失的量，摇匀，滤过，取续滤液，即得。
2.1.2　色谱-质谱条件
2.1.2.1　色谱条件
采用Waters atlantis T3(150 mm×2.1 mm，3 μm)色谱柱，以乙腈(A)-0.1%甲酸溶液(B)为流动相，梯度洗脱(0~90 min，3%A→70%A)，进样量5 μL，流速0.3 mL·min-1，柱温为30 ℃。
2.1.2.2　高分辨质谱条件
电喷雾离子源(ESI)，正、负离子模式检测。一级质谱参数设置：分辨率70 000，AGC target 1×105，Maximum IT 100 ms，扫描范围m/z 90~1 350；二级质谱参数设置：分辨率17 500，AGC target 1×105，Maximum IT 50 ms，Loop count 5，归一化碰撞能量(NCE/stepped)30、40、60，Minimum AGC 8×103，毛细管温度350 ℃，鞘气流速55 arb，辅助气体流速15 arb，喷雾电压3.6 kV。
2.1.3　高分辨质谱的数据采集处理及化学计量学分析
采用高分辨质谱对蟾皮和蟾酥甲醇提取物进行数据采集，高分辨质谱采集的数据导入SIEVE 2.2软件进行峰对齐和框架化，将离子精确质荷比、保留时间、峰面积等信息提取出来，并对每个离子对进行编号。
数据导入SIMCA 14.1(32-bit)软件进行PCA和OPLS-DA。将数据柏拉图标准化(Pareto scaling)后，去除离群值，得到PCA图(图1)，图中可以看出正、负离子模式下蟾皮、蟾酥均存在明显的分离趋势，表明蟾酥和蟾皮在成分上存在差异。
为进一步探究蟾皮和蟾酥之间的差异成分，构建了OPLS-DA模型(见图2)。以变量重要性投影值(projection value of variable importance，VIP)>1和可靠性/相关性[p(corr)]>0.8为条件进行筛选，正离子模式下得到27个候选差异离子，负离子模式下得到30个候选差异离子，结果见表2，需进一步验证各个离子的专属性，选择专属性好的离子建立蟾酥中掺伪蟾皮的鉴别方法。
2.2　差异离子的筛选及应用
通过计量学得到的差异离子并不能直接用于待测样品的识别，还需使用高灵敏度的三重四极杆质谱对离子在蟾酥和蟾皮实际样品中的检出情况进行分析验证，选择专属性好，响应高，色谱行为良好的离子对信息建立蟾皮的识别方法，并进行方法学验证。
2.2.1　色谱-质谱条件
2.2.1.1　色谱条件
采用Waters BEH C18 (100 mm×2.1 mm，1.7 μm)色谱柱，以乙腈(A)-10 mmol·L-1乙酸铵溶液(B)为流动相，梯度洗脱(0~21 min，30%A→90%A)，流速0.3 mL·min-1，柱温30 ℃，进样量5 μL。
2.2.1.2　质谱条件
电喷雾离子源(ESI)，正/负离子多反应监测(MRM)，喷雾电压5 500 V/-4 500 V，雾化器温度(TEM)550 ℃，雾化气(GS1)压力345 kPa，辅助气(GS2)压力345 kPa，气帘气压力207 kPa。
2.2.2　专属性验证
通过Xcalibur软件提取候选差异离子的二级碎片信息，按“2.2.1”项下条件进行试验。经验证，正离子模式下的27个候选差异离子，有22个在蟾皮、蟾酥中均可检出，仅响应值差别较大；有3个离子仅在蟾皮中检出，但响应值较低，检测难度大；有2个离子在蟾皮样品中均可检出，在蟾酥样品中均未检出，且响应良好。
负离子模式下的30个候选离子，有23个在蟾皮、蟾酥中均可检出，仅响应值差别较大；有4个离子仅在蟾皮中检出，但响应值较低，检测难度大；有3个离子在蟾皮样品中均可检出，在蟾酥样品中均未检出，且响应良好，结果见表2。
汇总结果，正、负离子模式下共有5个离子对显示出良好的专属性、色谱行为和响应值，适用于检测，这5个离子对m/z分别为377.5→243.2，172.1；330.4→170.9，127.0；313.4→201.2，171.0；452.4→280.2，298.2；614.7→332.4，281.3。专属性结果见图3。
2.2.3　耐用性试验
对上述验证得到的5个离子对继续考察在3种不同品牌色谱柱上的耐用性，包括Agilent HSS C18(100 mm×2.1mm，1.8 μm)色谱柱，Waters BEH C18 (100 mm×2.1 mm，1.7 μm)色谱柱，Thermo BDS C18(100 mm×2.1 mm，2.2 μm)色谱柱。结果采用3根色谱柱均得到了与“2.2.2”项下相同的验证结果，表明耐用性良好。
2.2.4　线性关系考察
取蟾酥、蟾皮的对照药材，按不同比例混合后，按照“2.1.1”项下方法提取，分别制得蟾酥药材中含5%、8%、10%、20%、30%、40%、50%蟾皮药材的系列溶液，以掺入量(X，%)为横坐标，每个母离子下响应较高的子离子峰面积值(Y)为纵坐标，计算线性方程，结果见表3，线性关系良好。
2.2.5　精密度和重复性试验
称取干蟾皮对照药材25 mg，按“2.1.1”项下方法制备精密度考察用供试品溶液，按“2.2.1”项下条件连续进样6次，以每个母离子下响应较高的子离子峰面积计算RSD，测得各离子对峰面积的RSD均<5%，结果见表4，表明进样和仪器精密度良好。
取蟾酥对照药材、蟾皮对照药材，平行制备6份20%掺伪供试溶液，按“2.2.1”项下条件进样测定，以每个母离子下相应较高的子离子峰面积计算RSD均<5%，结果见表4，表明该分析方法重复性较好。
2.2.6　检测限
以响应值最低的离子对m/z 614.7→281.3作为检测离子进行观察，含0.1%蟾皮的蟾酥溶液，上述离子的信噪比约为3∶1，故检测限为0.1%。
2.2.7　基质效应考察
取蟾酥、蟾皮对照药材，按不同比例混合后，按照“2.1.1”项下方法提取，分别制得蟾酥药材中含5%、10%、20%蟾皮药材的系列溶液，再以等量溶剂代替蟾酥基质，制备相应的系列溶液。比较在不同浓度下，蟾酥基质对各目标离子响应值的影响，结果见表5。
2.2.8　判定限度
2020年版《中华人民共和国药典》四部“0212药材和饮片检定通则”中，药材饮片的药屑及杂质通常规定不得过3%的限度[12]；已批准的掺伪补充检验方法如“阿胶中猪皮源成分检查项补充检验方法”“鹿角胶中驴皮源成分补充检验方法”等[13-14]，均规定掺伪上限为5%。本文考虑到取酥时难免会带入少量的蟾蜍皮肤组织，所以参考补充检验方法的限度原则，拟定蟾皮掺入上限为5%。
由于存在较明显的基质效应，实验中以中国食品药品检定研究院发行的蟾皮对照药材为对照物质，以蟾酥对照药材为基质，制备掺入5%蟾皮的蟾酥溶液作为参比溶液，照本方法的离子对进行检测，测得样品的各离子对峰面积均大于参比溶液的峰面积时视为检出，以避免误判。
2.2.9　样品测定结果
取蟾酥对照药材细粉23.75 mg，蟾皮对照药材细粉1.25 mg，置同一具塞回流瓶中，精密加入甲醇20 mL，称量，加热回流1 h，放冷，再称量，用甲醇补足减失的量，摇匀，滤过，取续滤液，作为参比溶液。
取市场收集的5批蟾酥样品，按“2.1.1”项方法制备供试品溶液，按“2.2.1”项下条件进行测定，结果有2批样品出现蟾皮成分离子信号，但峰面积小于参比溶液的峰面积，其余3批样品未检测出蟾皮成分(表6)。
3　结论
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蟾皮中化学成分复杂，文献报道已知化合物100多个，包含蟾蜍二烯羟酸内酯类、吲哚生物碱类、蟾蜍环酰胺和小分子环肽类、甾体及其他类化合物[15-19]，且有很多同分异构体。本研究综合Compound Discoverer软件、在线数据库以及相关参考文献等多种方式对目标差异离子对的成分进行结构推测，但均未获得可信度较高的结果。一方面原因可能是因为现有的质谱数据库对蟾蜍来源的化合物结构收录数量有限；另一方面，这些差异化合物很有可能是未被报道的新化合物。可见蟾皮中依然有一部分物质未被揭晓和应用，说明蟾皮的化学物质基础具有潜在的研究价值，后续可以通过收集大量蟾皮样品，有针对性地进行分离纯化及结构鉴定。
本文采用四极杆-静电场轨道阱高分辨质谱无差别采集待测样品的成分信息，再借助化学计量学分析手段对成分信息进行分类和过滤，筛选得到的差异离子并不能直接用于待测样品的识别，还需经过高灵敏度的三重四极杆质谱对离子对在多批次实际样品中的检出情况进行方法学验证，经验证的差异离子方可用于蟾皮的识别。因此，数据采集、分析和验证是发掘蟾皮特异成分过程中必不可少的3个环节。只有对这3个环节全面把控，才能够保证获取的差异离子信息的准确性和可靠性，从而提高数据的应用价值和意义，为蟾皮的筛查识别提供可靠的决策支持。本文报道的差异离子对，其色谱行为、响应值、专属性、线性、精密度、重复性及耐用性均良好，适用于常规检测，为蟾酥以及含有蟾酥的中成药制剂的掺伪研究提供了指标成分选择，为蟾酥药材的质量控制提供了数据基础。
基金
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  • *山东省重点研发计划（重大科技创新工程）项目(2022CXGC010511)
  • 山东省重点研发计划（重大科技创新工程）项目(2021CXGC010511)
  • 泉城产业领军人才支持计划创新团队项目(MRJT2105)
  • “新高校20”项目科研带头人工作室(202228096)
  • 国家重点研发计划-中医药现代化专项，National Key R&D Program of China(2023YFC3504102)
  • 国家药品监管科学体系建设重点项目(RS2024Z006)
文献
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doi: 10.16155/j.0254-1793.2024.04.10
  • 接收时间：2023-09-27
  • 首发时间：2026-03-13
  • 出版时间：2024-04-30
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  • 收稿日期：2023-09-27
基金
*山东省重点研发计划（重大科技创新工程）项目(2022CXGC010511)
山东省重点研发计划（重大科技创新工程）项目(2021CXGC010511)
泉城产业领军人才支持计划创新团队项目(MRJT2105)
“新高校20”项目科研带头人工作室(202228096)
国家重点研发计划-中医药现代化专项，National Key R&D Program of China(2023YFC3504102)
国家药品监管科学体系建设重点项目(RS2024Z006)
作者信息
    1.山东中医药大学，济南 250355
    2.山东省食品药品检验研究院国家药监局胶类产品质量评价重点实验室山东省中药标准创新与质量评价工程实验室 中药配方颗粒共性技术山东省工程研究中心，济南 250101
    3.山东康源堂中药饮片股份有限公司，济宁 272000

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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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