Name: hplc coupled with chemical fingerprinting for multi-pattern recognition for identifying the authenticity of clematidis armandii caulis
Uploaded: 2022-11-11T14:40:00
Description: Watch this Scientific Journal Video about HPLC Coupled with Chemical Fingerprinting for Multi-Pattern Recognition for Identifying the Authenticity of Clematidis Armandii Caulis at JoVE.com

This work was supported by the Project of Sichuan Traditional Chinese Medicine Administration (no. 2020JC0088, no. 2021MS203).

1. Methods for chemical fingerprint detection
<ol>
	<li>Chromatographic conditions
	<ol>
		<li>Prepare the acetonitrile (A)/water (B) mobile phase. Set a gradient program as follows in the HPLC software: 0-20 min, 3%A-10%A; 20-25 min, 10%A-13%A; 25-65 min, 13%A-25%A; 65-75 min, 25%A-40%A; 75-76 min, 40%A-3%A; 76-85 min, 3%A-3%A.</li>
		<li>Maintain the flow rate of the mobile phase at 1.0 mL/min.</li>
		<li>Conduct the chromatographic separation on a C18 column (250 mm x 4.6 mm, 5 &#181;m) maintained a...

<ol>
	<li>Chen, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chinese Medicine Specimen Picture Book. , 116 (1935).</li><li>Xiong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lignans+from+the+stems+of+Clematis+armandii+(&amp;#34;Chuan-Mu-Tong&amp;#34;)+and+their+anti-neuroinflammatory+activities.">Lignans from the stems of Clematis armandii (&amp;#34;Chuan-Mu-Tong&amp;#34;) and their anti-neuroinflammatory activities.</a> Journal of Ethnopharmacology. 153 (3), 737-743 (2014).</li><li>Pan, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+lignan+from+the+Chinese+medicinal+plant+Clematis+armandii,+inhibits+A431+cell+growth+via+blocking+p70S6/S6+kinase+pathway.">a lignan from the Chinese medicinal plant Clematis armandii, inhibits A431 cell growth via blocking p70S6/S6 kinase pathway.</a> Integrative Cancer Therapies. 16 (3), 351-359 (2017).</li><li>Chinese Pharmacopoeia Commission. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pharmacopoeia of The People's Republic of China: (Edition 2020). , 38 (2020).</li><li>Wang, D. G., Guo, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Textual+research+on+the+materia+medica+and+authenticity+of+Clematidis+armandii.">Textual research on the materia medica and authenticity of Clematidis armandii.</a> Lishizhen Medicine and Materia Medicine Research. 18 (11), 2696 (2007).</li><li>Fang, Q. M., Zhang, M., Zhong, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+natural+resources+and+exploitation+of+Caulis+Clematidis+Armandii+in+Sichuan.">The natural resources and exploitation of Caulis Clematidis Armandii in Sichuan.</a> West China Journal of Pharmaceutical Sciences. 20 (5), 404-406 (2005).</li><li>Bai, M. N., Dai, Y. X., Wang, Y., Ren, Y. Y., Tan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+on+the+identi&#64257;cation+of+Caulis+Clematidis+Armandii.">Study on the identi&#64257;cation of Caulis Clematidis Armandii.</a> Research and Practice on Chinese Medicines. 31 (3), 13-16 (2017).</li><li>Zhou, P. J., Li, X. F., Fu, D. H., Pu, X. Y., Zhu, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacognosy+identification+of+Ethnomedicine+Clematis+ranunculoides+Franchet.">Pharmacognosy identification of Ethnomedicine Clematis ranunculoides Franchet.</a> Journal of Guangzhou University of Traditional Chinese Medicine. 34 (3), 432-436 (2017).</li><li>Guo, J. L., Tang, Y., Pei, J., Wang, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+on+random+amplified+polymorphic+DNA+and+sequence+characterized+amplified+regions+of+Caulis+Clematidis+Armandii.">Study on random amplified polymorphic DNA and sequence characterized amplified regions of Caulis Clematidis Armandii.</a> Journal of Chengdu Medical College. 6 (4), 283-286 (2011).</li><li>Liu, M. Z., Li, M. N., Yao, H., Liu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+identification+of+Clematidis+Armandii+Caulis+and+its+adulterants+and+closely+related+species+by+ITS2+sequence.">Molecular identification of Clematidis Armandii Caulis and its adulterants and closely related species by ITS2 sequence.</a> Global Traditional Chinese Medicine. 4 (6), 446-450 (2011).</li><li>Liu, J. J., Chen, X., Wei, Z. Q., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+constituents+and+identification+of+the+caules+of+Clematis+armandii+Franch.and+Clematis+montana+Buch.-Ham.">Chemical constituents and identification of the caules of Clematis armandii Franch.and Clematis montana Buch.-Ham.</a> Natural Product Research and Development. 22 (6), 998-1000 (2010).</li><li>Yang, T. R., Xu, Z., Shi, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+four+compounds+from+Clematis+Montana.">Studies on four compounds from Clematis Montana.</a> Journal of Liaoning University of Traditional Chinese Medicine. 10 (4), 142-143 (2008).</li><li>Yan, L. H., Xu, L. Z., Zhou, Z. M., Yang, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+constituents+from+stems+of+Clematis+armandii(I).">Chemical constituents from stems of Clematis armandii(I).</a> Chinese Traditional and Herbal Drugs. 38 (3), 340-342 (2007).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+uses,+phytochemistry,+pharmacology,+and+toxicology+of+Akebiae+Caulis+and+its+synonyms:+A+review.">Traditional uses, phytochemistry, pharmacology, and toxicology of Akebiae Caulis and its synonyms: A review.</a> Journal of Ethnopharmacology. 277, 114245 (2021).</li><li>Ye, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diuretic+effect+and+material+basis+of+Clematidis+Armandii+Caulis+in+rats.">Diuretic effect and material basis of Clematidis Armandii Caulis in rats.</a> China Journal of Chinese Materia Medica. 44 (9), 1889-1894 (2019).</li><li>Kuang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consistency+study+of+Caulis+Clematidis+Armandii+and+its+traditionally+used+products+and+counterfeit+species+based+on+efficacy+and+pharmacognosy.">Consistency study of Caulis Clematidis Armandii and its traditionally used products and counterfeit species based on efficacy and pharmacognosy.</a> Pharmacology and Clinics of Chinese Materia Medica. 36 (3), 115-121 (2020).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+assessment+of+Asarum+heterotropoides+var.+mandshuricum+based+on+HPLC+multi-component+determination+and+multiple+pattern+recognition+method+of+fingerprint.">Quality assessment of Asarum heterotropoides var. mandshuricum based on HPLC multi-component determination and multiple pattern recognition method of fingerprint.</a> Chinese Traditional and Herbal Drugs. 53 (1), 238-243 (2022).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+multi-components+of+Paeoniae+Alba+Radix+based+on+fingerprints+and+chemical+pattern+recognition.">Determination of multi-components of Paeoniae Alba Radix based on fingerprints and chemical pattern recognition.</a> Chinese Traditional and Herbal Drugs. 53 (1), 231-237 (2022).</li><li>Li, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+investigation+for+raw+and+processed+products+of+Euodiae+Fructus+based+on+high-performance+liquid+chromatography+fingerprints+and+chemical+pattern+recognition.">Comparative investigation for raw and processed products of Euodiae Fructus based on high-performance liquid chromatography fingerprints and chemical pattern recognition.</a> Chemistry &amp; Biodiversity. 18 (8), 2100281 (2021).</li><li>Dong, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+on+suitable+distribution+areas+of+Kawa+Kimichi+produced+in+Sichuan+province-based+on+remote+sensing+and+GIS-A+case+study+of+Clematis+Armandii.">Study on suitable distribution areas of Kawa Kimichi produced in Sichuan province-based on remote sensing and GIS-A case study of Clematis Armandii.</a> Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology. 17 (11), 2398-2404 (2015).</li><li>Tang, S. W., Pei, J., Li, F. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+histological+identification+of+Chuanmutong.">Morphological and histological identification of Chuanmutong.</a> West China Journal of Pharmaceutical Sciences. 15 (1), 19-21 (2000).</li><li>Li, B., Chen, X., Fang, Q. M., Zhang, B. J., Wei, Z. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+Research+of+Chuanmutong.">Quality Research of Chuanmutong.</a> Journal of Sichuan of Traditional Chinese Medicine. 27 (6), 58-60 (2009).</li><li>Wu, W. J., Wan, M. M., Cao, Y. H., Tan, R., Song, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+on+the+Quality+Standard+of+Chuanmutong.">Research on the Quality Standard of Chuanmutong.</a> Chinese Archives of Traditional Chinese Medicine. 33 (2), 313-315 (2015).</li><li>Wei, Z. Q., Chen, X., Li, B., Dong, X. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+stigmasterol+in+Clematis+armandii+Franch+and+Clematis+montana+Buch-Ham.+by+HPLC.">Determination of stigmasterol in Clematis armandii Franch and Clematis montana Buch-Ham. by HPLC.</a> Lishizhen Medicine and Materia Medica Research. 20 (3), 574-575 (2009).</li><li>Qing, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+oleanolic+acid+in+Caulis+clematidis+armandii+by+HPLC.">Determination of oleanolic acid in Caulis clematidis armandii by HPLC.</a> West China Journal of Pharmaceutical Sciences. 21 (3), 273-274 (2006).</li><li>Yang, S. D., Wang, R., Fu, D. Y., Guo, J. J., Tan, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+on+oleanolic+acid+in+caulis+clematidis+armandii+by+microwave+assisted+extraction-HPLC/MS.">Determination on oleanolic acid in caulis clematidis armandii by microwave assisted extraction-HPLC/MS.</a> Anhui Academy of Agricultural Sciences. 38 (13), 6929-6931 (2010).</li></ol>

The sample collection for research is the first key step to constructing multi-pattern recognition in identifying the authenticity of Chinese medicinal materials. Through market research, we found that Sichuan Ya&#39;an, Liangshan, and Leshan are the main production areas of wild resources of Chuanmutong. The related varieties of the same genus also have the same geographical distribution6,20; CC, DC, DE, XS, and SMT are often misused as Chuangmutong<sup class="x...

Chuanmutong, dry Caulis of Clematis armandii Franch. or Clematis montana Buch.-Ham., is a traditional Chinese medicine commonly used in clinics1,2,3. It is used for treating urinary problems, edema, sores on the tongue and mouth, decreased milk secretion, joint stiffness, and muscle pain caused by damp heat4. Chuanmutong has always been obtained from wild varieties, mainly distributed in southwest China, especially in Sichuan, where the best quality can be found5,6. It is difficult to distinguish between authentic varieties and their closely related adulterants due to their similar characteristics7,8,9,10. The quality standard of Chuanmutong in the 2020 edition of Chinese Pharmacopoeia only stipulates the properties, microscopic identification, and thin-layer identification without content determination, which cannot effectively identify adulterants, and hence has potential risks. Moreover, there are few reports comparing and identifying Chuanmutong and related plants. Consequently, a quality control method to ensure the authenticity of Chuanmutong is worthy of further study.
The chemical constituents of Chuanmutong are mainly composed of oleanane-type pentacyclic triterpenoids and their glycosides, flavonoids, and organic acids11,12,13,14. Among them, oleanolic acid, &#946;-sitosterol, stigmasterol, and ergosterol have diuretic effects of different intensities, which may be potential pharmacodynamic substances for promoting diuresis and relieving stranguria15,16. Chemical fingerprints are obtained by separating and detecting many chemical components contained in samples by high-performance liquid chromatography (HPLC), gas chromatography (GC), etc. Adopting appropriate statistical analysis methods to analyze the characteristics of Chuanmutong can determine the overall quality control and scientific identification of traditional Chinese medicine17,18,19.
In this study, 10 batches of Chuanmutong authentic varieties and five batches of adulterants were collected. Their quality was compared and analyzed by the HPLC fingerprint method combined with multi-pattern recognition, including cluster analysis (CA), principal component analysis (PCA), orthogonal partial least-squares discrimination analysis (OPLS-CA), and content determination of the pharmacodynamic component. This protocol establishes a method for identifying authentic varieties with high specificity, a new strategy for the scientific identification of authentic varieties and adulterants of Chinese medicinal materials.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid</td>
			<td>Zhiyuan Chemical Reagent Co., Ltd., Tianjin, China</td>
			<td>2017381038</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich&#160; Trading Co., Ltd., Shanghai, China</td>
			<td>WXBD5243V</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Sitosterol</td>
			<td>Meisai Biological Technology Co., Ltd., Chongqing, China</td>
			<td>20210201</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;C18 column</td>
			<td>Yuexu Material Technology Co., Ltd., Shanghai, China</td>
			<td>Welch Ultimate LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Chuanmutong</td>
			<td>Guoqiang Chinese Herbal Pieces Co., Ltd., Sichuan, China&#160;</td>
			<td>19020103</td>
			<td>CMT-1</td>
		</tr>
		<tr>
			<td>Chuanmutong</td>
			<td>Hongya Wawushan Pharmaceutical Co., Ltd., Sichuan, China&#160;</td>
			<td>200701</td>
			<td>CMT-2</td>
		</tr>
		<tr>
			<td>Chuanmutong</td>
			<td>Hongpu Pharmaceutical Co., Ltd., Sichuan, China&#160;</td>
			<td>200701</td>
			<td>CMT-3</td>
		</tr>
		<tr>
			<td>Chuanmutong</td>
			<td>Hongpu Pharmaceutical Co., Ltd., Sichuan, China&#160;</td>
			<td>200901</td>
			<td>CMT-4</td>
		</tr>
		<tr>
			<td>Chuanmutong</td>
			<td>Xinrentai Pharmaceutical Co., Ltd., Sichuan, China&#160;</td>
			<td>210701</td>
			<td>CMT-5</td>
		</tr>
		<tr>
			<td>Chuanmutong</td>
			<td>Haobo Pharmaceutical Co., Ltd., Sichuan, China&#160;</td>
			<td>210401</td>
			<td>CMT-6</td>
		</tr>
		<tr>
			<td>Chuanmutong</td>
			<td>Xinrentai Pharmaceutical Co., Ltd., Sichuan, China&#160;</td>
			<td>200901</td>
			<td>CMT-7</td>
		</tr>
		<tr>
			<td>Chuanmutong</td>
			<td>Wusheng Pharmaceutical Group Co., Ltd., Sichuan, China&#160;</td>
			<td>201201</td>
			<td>CMT-8</td>
		</tr>
		<tr>
			<td>Chuanmutong</td>
			<td>Limin Chinese Herbal Pieces Co., Ltd., Sichuan, China&#160;</td>
			<td>201001</td>
			<td>CMT-9</td>
		</tr>
		<tr>
			<td>Chuanmutong</td>
			<td>Yuhetang Pharmaceutical Co., Ltd., Sichuan, China</td>
			<td>210501</td>
			<td>CMT-10</td>
		</tr>
		<tr>
			<td>Clematis argentilucida (Levl. et Vant.) W. T. Wang</td>
			<td>Madzi Bridge, Sanlang Township, Tianquan County, Sichuan, China&#160;</td>
			<td>-</td>
			<td>CC</td>
		</tr>
		<tr>
			<td>Clematis apiifolia var. obtusidentata Rehd. et Wils.</td>
			<td>Heilin Village, Qiliping Township, Hongya County, Sichuan, China&#160;</td>
			<td>-</td>
			<td>DC</td>
		</tr>
		<tr>
			<td>Clematis peterae Hand.-Mazz.</td>
			<td>Huangmu Village, Huangmu Township, Hanyuan County, Sichuan, China&#160;</td>
			<td>-</td>
			<td>DE</td>
		</tr>
		<tr>
			<td>Clematis gouriana Roxb. Var. finetii Rehd. et Wils</td>
			<td>Mixedang Mountain, Huangwan Township, Emei County, Sichuan, China&#160;</td>
			<td>-</td>
			<td>XS</td>
		</tr>
		<tr>
			<td>Clematis finetiana Levl. et Vaniot.</td>
			<td>Wannian Village, Huangwan Township, Emei County, Sichuan, China&#160;</td>
			<td>-</td>
			<td>SMT</td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>Haozhuang Hengping Scientific Instrument Co., Ltd., Shanghai,&#160; China&#160;</td>
			<td>FA1204</td>
			<td></td>
		</tr>
		<tr>
			<td>Ergosterol</td>
			<td>Meisai Biological Technology Co., Ltd, Chongqing, China</td>
			<td>20210201</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Kelon Chemical Co., Ltd., Chengdu, China</td>
			<td>2021112602</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl acetate</td>
			<td>Zhiyuan Chemical Reagent Co., Ltd., Tianjin, China</td>
			<td>2017042043</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Kelon Chemical Co., Ltd., Chengdu, China</td>
			<td>2016062901</td>
			<td></td>
		</tr>
		<tr>
			<td>High performance liquid chromatography</td>
			<td>Agilent, USA.</td>
			<td>1260</td>
			<td></td>
		</tr>
		<tr>
			<td>IBM SPSS Statistics version 26.0</td>
			<td>International Business Machines Corporation, USA</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich&#160; Trading Co., Ltd., Shanghai, China</td>
			<td>WXBD6409V</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Kelon Chemical Co., Ltd., Chengdu, China</td>
			<td>202010302</td>
			<td></td>
		</tr>
		<tr>
			<td>n-butyl alcohol</td>
			<td>Zhiyuan Chemical Reagent Co., Ltd., Tianjin, China</td>
			<td>2020071047</td>
			<td></td>
		</tr>
		<tr>
			<td>Petroleum ether</td>
			<td>Zhiyuan Chemical Reagent Co., Ltd., Tianjin, China</td>
			<td>2020090125</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoric acid</td>
			<td>Comeo Chemical Reagent Co., Ltd., Tianjin, China</td>
			<td>20200110</td>
			<td></td>
		</tr>
		<tr>
			<td>SESCF-TCM version 2012</td>
			<td>National Pharmacopoeia Commission, China</td>
			<td>-</td>
			<td>http://114.247.108.158:8888/login</td>
		</tr>
		<tr>
			<td>Stigmasterol</td>
			<td>Meisai Biological Technology Co., Ltd., Chongqing, China</td>
			<td>20210201</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloromethane</td>
			<td>Sinopharm Group Chemical Reagent Co., Ltd., Shanghai, China</td>
			<td>20200214</td>
			<td></td>
		</tr>
		<tr>
			<td>Umetrics SIMCA version 14.1.0.2047</td>
			<td>Umetrics, Sweden</td>
			<td>-</td>
			<td>https://www.sartorius.com/en/products/process-analytical-technology/data-analytics-software/mvda-software/simca/simca-free-trial-download</td>
		</tr>
		<tr>
			<td>Ultrapure water machine</td>
			<td>Youpu Ultrapure Technology Co., Ltd., Sichuan, China</td>
			<td>UPH-II-10T</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>Kunshan Hechuang Ultrasound Instrument Co., Ltd., Jiangsu, China</td>
			<td>KH3200E</td>
			<td></td>
		</tr>
	</tbody>
</table>

hplc coupled with chemical fingerprinting for multi-pattern recognition for identifying the authenticity of clematidis armandii caulis

A method for identifying Chinese medicinal materials and their related adulterants was constructed by taking Clematidis Armandii Caulis (Chuanmutong, a universally used traditional Chinese medicine) as an example. Ten batches of genuine Chuanmutong varieties and five batches of related adulterants were analyzed and compared based on the high-performance liquid chromatography (HPLC) fingerprints combined with chemometrics, including cluster analysis (CA), principal component analysis (PCA), and orthogonal partial least-squares discrimination analysis (OPLS-DA). In addition, the content of &#946;-sitosterol was determined. The control chemical fingerprint of Chuanmutong was established, and 12 common peaks were identified. The similarity between the fingerprint of 10 batches of genuine Chuanmutong varieties and the control fingerprint was 0.910-0.989, while the similarity of five batches of adulterants was only 0.133-0.720. Based on the common peaks in the chromatogram, 15 batches of samples were classified into three content levels by PCA, and were aggregated into four categories by CA, achieving a clear distinction between authentic Chuanmutong and adulterants of Chuanmutong. Further, seven differential components that can effectively identify authentic Chuanmutong and adulterants of Chuanmutong were found through OPLS-DA. The &#946;-sitosterol content of 10 batches of genuine Chuanmutong varieties was 97.53-161.56 &#181;g/g, while the &#946;-sitosterol content of the five batches of adulterants varied greatly, among which the &#946;-sitosterol content of Clematis peterae Hand.-Mazz. and Clematis gouriana Roxb. Var. finetii Rehd. et Wils. was significantly lower than that of authentic varieties of Chuanmutong. The HPLC index component content and chemical fingerprint multi-pattern recognition method established in this study provide a new strategy for effectively identifying authentic Chinese medicinal materials and related adulterants.

Chromatographic fingerprint of Chuanmutong and similarity analysis (SA) 
The RSD values of the relative retention time of precision, repeatability, and stability were below 0.46%, 1.65%, and 0.53%, respectively; the RSD values of the relative peak area were below 4.23%, 3.56%, and 3.96%, respectively. As shown in Figures 1A,B, there were 12 distinct common peaks (from peak 1 to peak 12) in the HPLC fingerprints in the 10 authentic Chuanmutong samples. S...

Watch this Scientific Journal Video about HPLC Coupled with Chemical Fingerprinting for Multi-Pattern Recognition for Identifying the Authenticity of Clematidis Armandii Caulis at JoVE.com

HPLC Coupled with Chemical Fingerprinting for Multi-Pattern Recognition for Identifying the Authenticity of Clematidis Armandii Caulis

Here, we present a protocol to establish high-performance liquid chromatography (HPLC), coupled with chemical fingerprint multi-pattern recognition, which provides a new strategy for effectively identifying the genuine varieties of Clematidis Armandii Caulis and its adulterants.

HPLC Coupled with Chemical Fingerprinting for Multi-Pattern Recognition for Identifying the Authenticity of Clematidis Armandii Caulis

A method for identifying Chinese medicinal materials and their related adulterants was constructed by taking Clematidis Armandii Caulis (Chuanmutong, a ...

This protocol can provide a new strategy for effectively identifying the genuine materials of Clematidis armandii caulis and its adulterants. This method can be used in medicinal material quality research and it provides a reference for studying the quality standards of other Chinese medicinal materials. To begin sample preparation grind the raw material to uniform particle size by passing them through a nylon mesh with an appropriate inner diameter.Transfer accurately weighed two grams of the ground raw material into a stoppered 50 milliliter conical flask before adding 50 milliliters of methanol into the flask. Put the stopper on the flask in ultrasonic heated at 600 watts and 40 kilohertz for 30 minutes. At the end of the incubation, strain the medicinal extract solution through the filter paper.After straining, transfer four milliliters of the methanol solution containing the medicinal extracts into a 10 milliliter volumetric flask. Then add six milliliters of water to it and mix the solution. Allow the solution to settle for 10 minutes.For HPLC analysis of the samples. Start by preparing the mobile phases and set the HPLC gradient program as described in the manuscript. Filter the sample through a 0.45 micron micro porous filter membrane and subject it to HPLC analysis.For each sample, perform analysis six times a day. To evaluate the stability of the sample solution, analyze the same sample solution stored at room temperature for 0, 2, 4, 6, 8, 12, and 24 hours after preparation. Take six replicates of the same sample and detect its fingerprint in HPLC.Import the relevant data into the software named Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine or SESCF TCM version 2012. Import the retention time and peak area for all 10 batches of authentic Chuanmutong samples as well as five batches of adulterants into SESCF TCM for analysis. In the main menu, click on the set reference spectrum and go to the parameter settings window to set the reference chromatographic fingerprint of the authentic species of Chuanmutong as the reference.Next, set control spectrum generation method as the median method with the time window width of 0.5. Then, click multi-point calibration in the main menu, select the peaks and then select the peak matching option as marked peaks. Finally, click Calculate Similarity to perform a similarity analysis of the samples based on the reference chromatogram fingerprints of Chuanmutong.To perform systemic cluster analysis open relevant statistical analysis software, and click on file to import the data of 10 batches of authentic samples and five batches of adulterants into the software. In the menu, Under the Classification tab go to click Analysis and System Clustering. Select the common peak areas of 10 batches of authentic samples and five batches of adulterants as variables.Set the number of clusters to four. Next, click on Method to select the Clustering Method as intergroup connection. Choose the measurement interval as Pearson correlation and use the Okay tab to draw the cluster analysis map.For principle component analysis or PCA, open the data analysis software. Click on file on the menu, and create a new regular project. Import the peak area of 12 common peaks in an Excel or similar spreadsheet from the HPLC system.Hit the Finish button to complete the data import. To set the model type with PCA, Click on New to create a new model and then fit the data using autofit and add tabs. Go to scores to get the PCA score map.To use the Orthogonal Partial Least Squares Discrimination Analysis or OPLSDA. Generate the PCA score map as demonstrated earlier. Set the model type with the OPLSDA by clicking New and New As model one Before going to scale in selecting par for all, Then hit on AutoFit in scores to generate the OPLSDA score map.To estimate the variable importance and projection go to the data analysis software menu and go to Analyze followed by setting the number to 200 under per mutations. Obtain the R square and Q square values from the OPLSDA score and click on VIP and VIP predictive to get the VIP map. HPLC fingerprints of the 10 authentic Chuanmutong samples showed 12 distinct common peaks of which the relatively large and well resolved.Peak number 10 was used as the reference peak to estimate the relative retention times of the rest peaks. The authentic samples demonstrated similarity degrees very close to one implying high similarity in good consistency. However, the similarity degrees between the five batches of adulterants and the control Chuanmutong were quite low.The HPLC fingerprints of the five adulterants were observed to differ from the authentic samples, mainly in the region having retention times ranging from 28 to 55 minutes. PCA showed an obvious similarity between the authentic varieties and their difference from the adulterants, except for the adulterant CC.Similar results were also obtained from cluster analysis when the classification distance was 20. The score matrix generated from OPLSDA indicated no intersection between the sample points of authentic samples and the adulterants.Again, adulterant CC resembled the authentic varieties more than the other adulterants. According to the VIP map the peaks having values greater than one were identified as the main marker components causing the difference between authentic samples and the adulterants. The content of beta-sitosterol, an active component found in Chuanmutong was detected in all five batches of adulterants, but the content varied greatly.This protocol establishes various analysis methods to reflect all the information about medicinal materials from multiple angles and in all around the way to estimate the authenticity of Chuanmutong medicinal materials.

Research

JoVE Journal

Chemistry

Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides

We describe a protocol for the preparation of hybrid materials based on highly porous coordination polymer coatings on the internal surface of macroporous ...

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins ...

Sulfate Separation by Selective Crystallization with a Bis-iminoguanidinium Ligand

A simple and effective method for selective sulfate separation from aqueous solutions by crystallization with a bis-guanidinium ligand, ...

Determining the Chemical Composition of Corrosion Inhibitor/Metal Interfaces with XPS: Minimizing Post Immersion Oxidation

An approach for acquiring more reliable X-ray photoelectron spectroscopy data from corrosion inhibitor/metal interfaces is described. More specifically, ...

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography (HPLC)

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography HPLC

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil ...

Chemical Precipitation Method for the Synthesis of Nb2O5 Modified Bulk Nickel Catalysts with High Specific Surface Area

Chemical Precipitation Method for the Synthesis of Nb2O5 Modified Bulk Nickel Catalysts with High Specific Surface Area

We demonstrate a method for the synthesis of NixNb1-xO catalysts with sponge-like and fold-like nanostructures. By varying the Nb:Ni ratio, a series of ...

Methionine Functionalized Biocompatible Block Copolymers for Targeted Plasmid DNA Delivery

Reversible addition-fragmentation chain transfer (RAFT) polymerization integrates the advantages of radical polymerization and living polymerization. This ...

Extraction of Lignin with High &#946;-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the ...

A Two-Step Pyrolysis-Gas Chromatography Method with Mass Spectrometric Detection for Identification of Tattoo Ink Ingredients and Counterfeit Products

Tattoo inks are complex mixtures of ingredients. Each of them possesses different chemical properties which have to be addressed upon chemical analysis. ...

Tuning the Acidity of Pt/ CNTs Catalysts for Hydrodeoxygenation of Diphenyl Ether

We herein present a method for the synthesis of HNbWO6, HNbMoO6, HTaWO6 solid acid nanosheet modified Pt/CNTs. By varying the weight of various solid acid ...