Characteristic chromatogram and index components content of substance benchmark of Xuanfu Daizhe Decoction / 中国中药杂志

The methods for determining the characteristic chromatogram and index components content of Xuanfu Daizhe Decoction were established to provide a scientific basis for the quality evaluation of substance benchmarks and preparations. Eighteen batches of Xuanfu Daizhe Decoction were prepared with the decoction pieces of different batches and of the same batch were prepared respectively, and the HPLC characteristic chromatograms of these samples were established. The similarities of the chromatographic fingerprints were analyzed. With liquiritin, glycyrrhizic acid, 6-gingerol, ginsenoside Rg_1, and ginsenoside Re as index components, the high performance liquid chromatography was established for content determination with no more than 70%-130% of the mass average as the limit. The results showed that there were 19 characteristic peaks corresponding to the characteristic chromatograms of 18 batches of Xuanfu Daizhe Decoction, including 8 peaks representing liquiritin, 1,5-O-dicaffeoylqunic acid, ginsenoside Rg_1, ginsenoside Re, 1-O-acetyl britannilactone, ginsenoside Rb_1, glycyrrhizic acid, and 6-gingerol, and the fingerprint similarity was greater than 0.97. The contents of liquiritin, glycyrrhizic acid, 6-gingerol, and ginsenosides Rg_1 + Re in the prepared Xuanfu Daizhe Decoction samples were 0.53%-0.86%, 0.61%-1.2%, 0.023%-0.068%, and 0.33%-0.66%, respectively. Except for several batches, most batches of Xuanfu Daizhe Decoction showed stable contents of index components, with no discrete values. The characteristic chromatograms and index components content characterized the information of Inulae Flos, Ginseng Radix et Rhizoma, Glycyrrhizae Radix et Rhizoma, and Zingiberis Rhizoma Recens in Xuanfu Daizhe Decoction. This study provides a scientific basis for the further research on the key chemical properties of substance benchmark and preparations of Xuanfu Daizhe Decoction.

UHPLC-Q-Exactive Orbitrap MS/MS-based rapid identification of chemical components in substance benchmark of Kaixin San / 中国中药杂志

Ultra-performance liquid chromatography-quadrupole-electrostatic field Orbitrap mass spectrometry(UHPLC-Q-Exactive Orbitrap MS/MS) was used for rapid identification of the chemical components in Kaixin San substance benchmark. The gradient elution was performed through a Waters ACQUITY~(TM) BEH C_(18) column(2.1 mm×150 mm, 1.7 μm) with water-acetonitrile as mobile phase, a column temperature of 30 ℃, a flow rate of 0.3 mL·min~(-1), and a sample size of 1 μL. The scanning was performed in the negative ion mode. The complex component groups in Kaixin San substance benchmark were quickly and accurately identified and clearly assigned based on the comparison of the retention time and MS data with those of the reference substance as well as the relative molecular weight of the same or similar components in the mass spectrum database and literature. A total of 77 compounds were identified, including 26 saponins, 13 triterpenoid acids, 20 oligosaccharide esters, 5 xanthones, and 13 other compounds. The qualitative method established in this study can systematically, accurately, and quickly identify the chemical components in Kaixin San substance benchmark, which can provide a basis for the further analysis of its active components in vivo and the establishment of its quality control system.

UPLC fingerprint and determination of five components of substance benchmark of classical prescription Shentong Zhuyu Decoction / 中国中药杂志

Fingerprints of 18 batches of substance benchmark of Shentong Zhuyu Decoction(SZD) were established by UPLC under the following conditions: Waters Sun Fire C_(18) column(3.0 mm×150 mm, 3.5 μm), column temperature of 35 ℃, gradient elution with mobile phase of acetonitrile(A)-0.1% phosphoric acid aqueous solution(B) at the flow rate of 0.4 mL·min~(-1), and detection by wavelength switching. A total of 16 common peaks were identified. The similarities among the fingerprints were calculated by Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine(2012 Edition) and the result showed they were in the range of 0.911-0.988. Based on the 16 common peaks, cluster analysis(CA), principal component analysis(PCA), and partial least square discriminant analysis(PLS-DA) all categorized the 18 batches of samples into two groups(S1, S2, S5-S8, S14, and S17 in one group, and S1, S2, S5-S8, S14, and S17 in another), and 11 most influential components were screened. Five known components with great difference among samples(hydroxysafflor yellow A, ferulic acid, benzoic acid, ecdysone, and ammonium glycyrrhizinate) were determined. The combination of multi-component content determination and fingerprints can reflect the overall cha-racteristics of the primary standards of SZD, which is simple, feasible, reproducible, and stable. This study can serve as a reference for the quality control of the primary standards of SZD.

Analysis on UPLC Fingerprint and Determination of Multi-index Components in Pipa Qingfeiyin / 中国实验方剂学杂志

Objective:To establish the ultraperformance liquid chromatography （UPLC） fingerprint of Pipa Qingfeiyin substance benchmark， and to establish a quantitative analysis method for simultaneous determination of the contents of five index components， so as to provide reference for the quality control and evaluation of this famous classical formula. Method:ACQUITY UPLC^® CSH^TM C₁₈ column （2.1 mm×100 mm， 1.7 μm） was used with mobile phase of acetonitrile （A）-0.1% formic acid aqueous solution （B） for gradient elution （0-7 min， 5%-7%A； 7-11 min， 7%-8%A； 11-22 min， 8%-14%A； 22-30 min， 14%-15%A； 30-35 min， 15%-25%A； 35-42 min， 25%-40%A； 42-45 min， 40%-50%A； 45-50 min， 50%-60%A）， the flow rate was 0.35 mL·min^-1， the column temperature was 25 ℃， the detection wavelengths were 278 nm and 248 nm. UPLC fingerprints of 15 batches of Pipa Qingfeiyin substance benchmark were established， and the "Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine" software （2012 edition） was used for similarity analysis， and the common peaks were assigned. Cluster analysis （CA）， principal component analysis （PCA） and orthogonal partial least squares discriminant analysis （OPLS-DA） were used to evaluate the fingerprint data. UPLC fingerprint method was used to simultaneously determine the contents of five components in the substance benchmark. Result:The method validation of fingerprint and determination method was good， the similarities between 15 batches of Pipa Qingfeiyin substance benchmark and their control fingerprint were ≥0.997， 23 common peaks were identified and 11 chromatographic peaks were identified. CA， PCA and OPLS-DA divided 15 batches of the substance benchmark into two groups. The linear relationship of phellodendrine hydrochloride， chlorogenic acid， berberine hydrochloride， palmatine hydrochloride and ammonium glycyrrhizinate was good in a certain range of concentration （<italic>R</italic>²>0.999）， their average recovery was 96.47%-101.16%， and the contents of these five components in the substance benchmark were 0.87-2.00， 1.53-5.95， 18.45-33.97， 3.87-6.29， 1.02-4.12 mg·g^-1， respectively. Conclusion:The established UPLC fingerprint and multi-index component content determination methods have strong specificity， good resolution and high sensitivity， it can be characterized except for the Ginseng Radix et Rhizoma flavor， which can provide reference for the quality control and evaluation of Pipa Qingfeiyin compound preparation.

Establishment of UPLC-PDA Fingerprint of Baoyuantang Substance Benchmark and Identification of Its Chemical Constituents / 中国实验方剂学杂志

Objective:To establish the fingerprint of Baoyuantang substance benchmark， and to analyze and identify the common peaks. Method:A total of 15 batches of Baoyuantang substance benchmark were prepared， ultra performance liquid chromatography-diode array detector method （UPLC-PDA） was used to establish the fingerprint of the substance benchmark， and the methodology was developed. The chromatographic conditions were as follows：ACQUITY UPLC BEH Shield C₁₈ column （2.1 mm×100 mm， 1.7 μm）， mobile phase of 0.05% formic acid solution （A） and 0.05% formic acid acetonitrile solution （ B） for gradient elution （0-0.5 min， 5%-19%B； 0.5-6 min， 19%B； 6-10 min， 19%-27%B； 10-20 min， 27%-45%B； 20-20.1 min， 45%-95%B； 20.1-23 min， 95%B）， the flow rate of 0.4 mL·min^-1， the column temperature of 30 ℃， the detection wavelength at 203 nm and 260 nm， and the injection volume of 2 μL. Similarity evaluation system of traditional Chinese medicine fingerprint （2012 edition） was used to establish the fingerprint and generate the control fingerprint. The chemical constituents of Baoyuantang substance benchmark were identified by comparison of standard substances and UPLC-electrospray ionization tandem mass spectrometry （UPLC-ESI-MS/MS） with full information tandem mass spectrometry （MS^E） and scanning range of <italic>m</italic>/<italic>z</italic> 50-1 200. Result:The similarities of 15 batches of Baoyuantang substance benchmark were above 0.90 by comparing with the control fingerprint. There were 37 common peaks， 22 of which were identified through UPLC-ESI-MS/MS， including liquiritin， violanthin， ginsenoside Rg₁， ginsenoside Rb₁， ginsenoside Re and so on. These components were all from Astragali Radix， Ginseng Radix et Rhizoma， Zingiberis Rhizoma Recens and Glycyrrhizae Radix et Rhizoma. Conclusion:This method is accurate， stable and reliable， which will basically reflect the overall chemical composition characteristics of Baoyuantang， and it provides experimental basis for development of the granules of this famous classical formulas.

HPLC Fingerprint Analysis and Multi-component Determination of Substance Benchmark of Yiweitang / 中国实验方剂学杂志

Objective:To establish high performance liquid chromatography （HPLC） fingerprint and multi-component determination for the substance benchmark of Yiweitang， and to evaluate its quality in combination with chemical pattern recognition method. Method:Fifteen batches of substance benchmark of Yiweitang were prepared， the "Chinese medicine chromatographic fingerprint similarity evaluation system" （2012 edition） was used to calculate similarity. Cluster analysis， principal component analysis and orthogonal partial least squares-discriminant analysis were employed to handle the common peaks for evaluating the quality difference among 15 batches of the substance benchmark. The contents of catalpol， verbascoside and methylophiopogonanone A were determined with mobile phase system of acetonitrile-phosphoric acid solution at detection wavelengths of 210 nm and 334 nm. Result:There were 22 common peaks in HPLC fingerprint of the substance benchmark， among them， peaks 1， 9， 12， 14-17， 19 and 20 belonged to Rehmanniae Radix， peaks 3， 4， 6， 7 and 21 belonged to Glehniae Radix， peaks 5 and 22 belonged to Ophiopogonis Radix， peaks 2 and 18 belonged to Polygonati Odorati Rhiaoma， peak 8 was the common peak of Ophiopogonis Radix and Rehmanniae Radix， peak 10 was shared by Ophiopogonis Radix， Polygonati Odorati Rhiaoma<italic> </italic>and<italic> </italic>Rehmanniae Radix， peak 11 was the common peak of these four herbs， and peak 13 was shared by Polygonati Odorati Rhiaoma and Rehmanniae Radix. The similarities between HPLC fingerprints of 15 batches of the substance benchmark and the control fingerprint were all >0.90， the samples could be divided into four categories by three chemical pattern recognition methods. Quantitative analysis showed that the contents of catalpol， verbascoside and methylophiopogonanone A among 15 batches of samples ranged from 0.37% to 1.14%， 0.002% to 0.054% and 0.016% to 0.079%， respectively. Conclusion:The established fingerprint and determination for the substance benchmark of Yiweitang have good separation and high accuracy， which reflect the overall chemical composition characteristics of Yiweitang， and can provide experimental basis for the further development and quality control of the compound preparations of this famous classical formula.

Quality Value Transmitting of Substance Benchmark of Xiebaisan / 中国实验方剂学杂志

Objective:Based on fingerprint， index component content and dry extract yield， a quality evaluation method for substance benchmark of Xiebaisan was established to study the key quality attributes， to explore the quantitative transfer relationship between decoction pieces and substance benchmark， and to preliminarily formulate the quality standard of substance benchmark of Xiebaisan. Method:The substance benchmark of Xiebaisan was prepared according to the records of ancient formulas， fingerprints of 15 batches of decoction pieces and substance benchmarks were collected by high performance liquid chromatography （HPLC） and the index components were determined with the mobile phase of acetonitrile-0.05% phosphoric acid solution for gradient elution. The dry extract yield， fingerprint similarity and transfer rate of index components were combined to study the quantity value transmitting. Result:Ten characteristic peaks were identified in fingerprint of the substance benchmark and two characteristic peaks from stir-fried Mori Cortex， four characteristic peaks from baked Lycii Cortex， four characteristic peaks from Glycyrrhizae Radix et Rhizoma Praeparata cum Melle. Mulberroside A， liquiritin and glycyrrhizic acid were used as index components for the determination， the contents of mulberroside A， liquiritin and glycyrrhizic acid in substance benchmark of Xiebaisan were 2.69%-4.26%， 0.09%-0.17% and 0.09%-0.16%， and their transfer rates were （31.37±4.14）%， （36.12±4.03）% and （12.25±0.88）%， respectively. The similarity of fingerprint of substance benchmarks was good， the fingerprint similarities of 14 batches of substance benchmarks and control fingerprint were >0.9. The dry extract yield of substance benchmark of Xiebaisan ranged from 8.09% to 11.29%. Conclusion:The established quality evaluation method of substance benchmark of Xiebaisan is scientific and reasonable， and the transfer process of decoction pieces to substance benchmarks is stable and controllable. The preliminary quality standard of the substance benchmark can provide basis and reference for the development of modern preparations of Xiebaisan in the future.

Analysis on quality value transmitting of substance benchmark of Houpo Wenzhong Decoction / 中国中药杂志

By preparing 15 batches of lyophilized powder samples of substance benchmark in Houpo Wenzhong Decoction,the fingerprint,index component content and extract rate were determined,and the characteristic peaks,the range of similarity with the reference map,the content range and transfer rate range of magnolol,hesperidin,glycyrrhizic acid and pinocembrin,the extract rate range and the change range were clarified. The results showed that the similarity between the fingerprint of substance benchmark and the reference map R generated from the 15 batches of substance benchmark samples was higher than 0. 90. The assignment of the characteristic peaks in the full prescription's fingerprint of the herbs except Poria cocos was clarified. Nineteen characteristic peaks were assigned,and 12 characteristic peaks were assigned by the reference substance,of which 4 were from Magnolia ocinalis Cortex,5 from Exocarpium Citri Rubrum,2 from Radix aucklandiae,3 from Glycyrrhiza Radix et Rhizoma,4 from Semen Alpiniae Katsumadai,and one from Rhizoma Zingiberis and Zingiber officinale Roscoe. The index component content range and transfer rate range were 0. 80%-1. 14% and 20. 25%-39. 61% for hesperidin,0. 49%-0. 79% and 23. 09%-33. 87%for glycyrrhizic acid,0. 03%-0. 07% and 3. 55%-10. 09% for pinocembrin,0. 15%-0. 38% and 8. 08%-24. 35% for magnolol. The extract rate range and the change range were22. 60%-25. 57% and 12. 67%-23. 68% respectively. In this study,we introduced the concepts of index component content,fingerprint,extract rate,explored the transfer relation of quality value transmitting of substance benchmark in Houpo Wenzhong Decoction,and initially established the quality standard of Houpo Wenzhong Decoction,all of which would provide ideas for the development and research of similar prescriptions.

Research Strategies and Key Problems Analysis over Substance Benchmark of Famous Classical Formulas / 中国实验方剂学杂志

With continuous introduction of relevant national policies on famous classical formulas, the research of famous classical formulas is popular all over the country. Different from other new drugs, in the research and development process of famous classical formulas, substance benchmark is earlier than the product, suggesting that the research and development of substance benchmark is of great significance. Based on previous work of the authors, content of substance benchmark of famous classical formulas was analyzed, which was included in the document <italic>The Management Regulation of Simplified Registration and Approval over Chinese Herbal Medicine Compound Preparations of Ancient Famous Classical Formulas</italic> released by the National Medical Products Administration in May 2018. In this paper, the significance of substance benchmark development was described, a five-stage of research strategy was proposed, covering the prescription textual research and historical evolution, the collection and quality evaluation of medicinal materials, the processing method and quality evaluation of decoction pieces, the preparation and quality research of substance benchmark, the drafting and formulating of quality standard over substance benchmark. At the same time, some suggestions were put forward to the feasibility of compound preparations development over famous classical formulas, the implementation difficulty of resource evaluation over Chinese medicinal materials, and the irrationality on the quality correlation of Chinese medicinal materials. All of these are expected to provide reference and enlightenment for the development and policy officially landed over ancient famous classical formulas.

Application of DESI-MSI in Quality Control of Banxia Xiexintang / 中国实验方剂学杂志

Objective::To prepare 15 batches of Banxia Xiexintang substance benchmark and lyophilized powder from different places, and the lyophilized powder was analyzed by ultra-high performance liquid chromatography with diode array detection (UHPLC-DAD) and desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) in order to investigate the advantages of DESI-MSI in quality control of famous classical formulas. Method::Taking Banxia Xiexintang as the research model, fingerprints of the substance benchmark and lyophilized powder were established by UHPLC-DAD, and the content of index components and the yield of dry extract were also investigated. Meanwhile, as the research carrier, the lyophilized powder corresponding to Banxia Xiexintang was dissolved in methanol and dotted on qualitative filter paper with 5 μL quantitative capillary, and fixed it on the slide to make samples. The samples were analyzed on a DESI-MSI system in positive and negative ion mode with methanol-formic acid (1 000∶1, flow rate of 3 μL·min-1) as spray solvent, N2 as spray gas (pressure of 0.5 MPa). The scanning range was 100-1 200 Da, the spatial resolution was 300 μm, the spray voltage was 3 kV, the sampling cone voltage was ±40 V, incidence angle of sprayer was 60 degree, its collection angle was 10 degree, the ion source temperature was 120 ℃. Result::DESI-MSI could not only detect the index components of liquiritin, baicalin and wogonoside, as well as the common peaks of liquiritin apioside, berberine and glycyrrhizic acid, but also analyzed them semi-quantitatively, the analysis results were basically consistent with UHPLC-DAD. At the same time, DESI-MSI could detect 16 other components from Glycyrrhizae Radix et Rhizoma, Coptidis Rhizoma, Scutellariae Radix, Jujubae Fructus and Ginseng Radix et Rhizoma, such as licoricesaponin G2, palmatine, coptisine, rutin and ginsenoside Rg1, and present their relative content visually. The qualitative analysis ability of DESI-MSI was much better than UHPLC-DAD. Conclusion::DESI-MSI can be used as the quality control method for substance benchmark and lyophilized powder and dispensing granules of famous classical formulas with advantages of high sensitivity, strong analytical ability, no complex sample processing, qualitative and relative content analysis of complex samples without reference substance.