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The chemical components of Huanglian Decoction were identified by ultra-performance liquid chromatography-quadrupole-time-of-flight-tandem mass spectrometry(UPLC-Q-TOF-MS/MS) technology. The gradient elution was conducted in Agilent ZORBAX Extend-C_(18) column(2.1 mm×100 mm, 1.8 μm) with the mobile phase of 0.1% formic acid aqueous solution(A)-acetonitrile(B) at a flow rate of 0.3 mL·min~(-1) and the column temperature of 35 ℃. The MS adopted the positive and negative ion mode of electrospray ionization(ESI), and the MS data were collected under the scanning range of m/z 100-1 500. Through high-resolution MS data analysis, combined with literature comparison and confirmation of reference substances, this paper identified 134 chemical components in Huanglian Decoction, including 12 alkaloids, 23 flavonoids, 22 terpenes and saponins, 12 phenols, 7 coumarins, 12 amino acids, 23 organic acids, and 23 other compounds, and the medicinal sources of the compounds were ascribed. Based on the previous studies, 7 components were selected as the index components. Combined with the network pharmacology research and analysis me-thods, the protein and protein interaction(PPI) network information of the intersection targets was obtained through the STRING 11.0 database, and 20 core targets of efficacy were screened out. In this study, UPLC-Q-TOF-MS/MS technology was successfully used to comprehensively analyze and identify the chemical components of Huanglian Decoction, and the core targets of its efficacy were discussed in combination with network pharmacology, which laid the foundation for clarifying the material basis and quality control of Huanglian Decoction.
Subject(s)
Tandem Mass Spectrometry , Network Pharmacology , Chromatography, High Pressure Liquid , Drugs, Chinese Herbal/chemistry , TechnologyABSTRACT
This study aimed to establish a method for positioning six chromatographic peaks occurred in HPLC profile of Gastrodiae Rhizoma. The "liner calibration with two reference substances" (LCTRS) method was used to calculate the retention time so as to assist in positioning of chromatographic peaks in terms of the prediction accuracy of retention time and the coincidence rate of chromatographic column. A total of 24 C18 chromatographic columns from different brands and types available were used to determine the retention times of six components in Gastrodiae Rhizoma, then the average retention time of each component was obtained as standard retention time (SRT). Parishin E (peak 3) and Parishin A (peak 6) were simultaneously taken as reference substance to forecast the retention time of the other four components by using the LCTRS method. Four different C18 columns were employed to verify the method. Meanwhile, for the purpose of comparison, the relative retention time (RRT) method was applied to forecast the retention time, by using Parishin E as the single reference substance. The comparison between LCTRS and RRT methods indicated that the former was more accurate in predicting the retention time and more applicable in utilization of chromatographic columns. This study demonstrated that the LCTRS method shows the superior performance in positioning of chromatographic peak, and therefore has a good prospect of application.
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OBJECTIVE：To establish the m ethod for simultaneous determination of 8 kinds of ginsenosides in Panax quinquefolium broken pieces. METHODS ：HPLC-DAD method was used to determine the contents of ginsenoside Rg 1，Re，Rb1， Rc，Ro，Rb2，Rb3，Rd in P. quinquefolium broken pieces. The determination was performed on Agilent 5 TC-C18 column with mobile phase consisted of acetonitrile- 0.2％ phosphoric acid water solution （gradient elution ）at the flow rate of 1.0 mL/min. The column temperature was set at 30 ℃. The detection wavelength was set at 203 nm，and sample size was 10 μL. Ginsenoside Re and ginsenoside Rb 2 were used as control ，liner calibration with two-reference substances correction was used to predict the retention time of other 6 components，and was compared with the relative retention time method. Using ginsenoside Re as control ， above components were quantified by the relative correction factor method ，and the results were compared with the external standard method. RESULTS ：The contents of ginsenoside Rg 1，Re，Rb1，Rc，Ro，Rb2，Rb3，Rd were 10.59-12.78，2.160-2.768， 27.492-38.880，3.154-4.018，3.368-4.080，0.343-0.755，0.961-1.415，5.857-6.923 mg/g. The accuracy of two-reference substances linear correction method to predict the retention time of components was higher ，and the absolute deviation of the predicted retention time was lower than that of the relative retention time method. There was no significant difference between the relative correction factor method and the external standard method ，and relative error was ＜3％ . CONCLUSIONS ：Established two-reference substances for determination of multiple components can be used for qualitative and quantitative analysis of 8 kinds of ginsensides in P. quinquefolium broken pieces simultaneously and accurately.
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OBJECTIVE: To propose a holistic strategy for quality control of Chinese patent medicines, and establish an HPLC analytical method for Tongzhi Surunjiang preparation according to the strategy. METHODS: The strategy contained three steps.The first step was multi-wavelength chromatographic detection.The second step was multivariate analysis for identification and assay. The third step was to establish substitute reference substance method.The preparations were extracted by ultrasound with methanol, chromatography was performed on ODS column with gradient elution with acetonitrile and 0.1%phosphoric acid aqueous solution.The detection wavelengths were set at 270, 350, 410 and 440 nm.The radar chart, HCA heatmap, principal component analysis and cosine similarity were used for data analysis.At last, linear calibration using two reference substances, relative retention time method and PDA spectrum method were used for peak identification, and relative correction factor method was used for quantitative analysis. RESULTS: The multi-components determination method and fingerprint analysis met the method validation requirements. Data analysis showed that there were some differences among the samples of different manufacturers. Strong characteristic peaks for classification were gallic acid, chebulinic acid, chebulea fructus, sennae folium, sennae folium and crocin.CONCLUSION: The method is specific, with low cost, and could be used to accurately control the quality of Tongzhi Surunjiang preparation.
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Objective To determinate the contents of α-pinene, β-pinene, eucalyptol and linalool in Baeckea frutescens by reference substances method and reference extract method respectively; To explore the feasibility of replacing single component reference by control extracts in assay. Methods The GC system consisted of a quartz column DB-5 (60 m×0.25 mm×0.25 μm); The temperature programming rose from 80 ℃ (15 min) to 90 ℃ by 1 ℃/min, lasting 2 min, then 10 ℃/min to 110 ℃, then 25 ℃/min to 240 ℃, lasting 8 min in the end; The temperature of the entrance of capillary vessel column was 250 ℃, and the temperature of the detector was 250 ℃. Results α-pinene, β-pinene, eucalyptol and linalool were in a good linear relationship within each concentration scope (r≥0.999). The average recovery rates were in the range of 96.5%–102.2%. The results of t-test demonstrated that there is no significant difference between the two methods. Conclusion The reference extract method can be used as a quality evaluation pattern for Baeckea frutescens.
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OBJECTIVE: To establish the HPLC fingerprint of Swertia chirayita, and validate the feasibility of the double standard linear correction method using two reference substances with PDA assistance. METHODS: The fingerprint of Swertia chirayita was established by HPLC, and similarity analysis and two-dimensional clustering analysis were used for quality evaluation. Linear calibration using two reference substances with PDA assistance was used for identification of chromatographic peak. RESULTS: Ten chromatographic peaks were selected as common peaks in the fingerprint of 10 batches of Swertia chirayita. Preliminary evaluation of the quality was made by chemometrics evaluation. The precision of predicting retention time by linear calibration using two reference substances was superior to the relative retention time method. Linear calibration using two reference substances with PDA assistance could qualify the chromatographic peaks better. CONCLUSION: Linear calibration using two reference substances is more accurate and suitable for more chromatographic columns than relative retention time method. PDA assistance can enlarge the scope of application of linear calibration using two reference substances.
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Substitute reference substance method is an effective approach for quality control of multiple components in accordance with the characteristics of traditional Chinese medicines. The purpose of the guideline is to guide the establishment of substitute reference substance method, prove the conformance of the method to the requirements for testing, and standardize the study method and its application in national drug standards. The topics of the guideline include the definition and classification of substitute reference substance method, the principles and approaches of quantitative analysis, the identification and confirmation of chromatographic peaks, and technical requirements. When substitute reference substance method is used for fingerprint identification or multiple components assay in traditional Chinese medicines, the analytical method can be validated following the guideline.
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This study was aimed to establish a separation method for neochlorogenic acid reference substances from Lonicera japonica. Refined neochlorogenic acid inL. japonica water extract was separated and concentrated by HPD200A macroporous resin, which was isolated and purified by medium-low-pressure preparative chromatography and determined by HPLC. The structure was identified by various spectroscopic data including ESI-MS,1H-NMR and13C-NMR. The results showed that the optimal purification technology conditions were as follows: washed with 5BV of water, collected elution, concentration, drying; neochlorogenic acid crude products were eluted with acetonitrile-0.5% formic acid solution (10:90) with the flow rate of 20 mL·min-1; and the detection wavelength was 326 nm. The contents of the prepared neochlorogenic acid reached to 98.86% and the yield was 89.1%. It was concluded that the method was effective for the preparation of neochlorogenic acid with high purity. It can be used to prepare the reference substances for quantitative analysis and content determination of Chinese materia medica.
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OBJECTIVE: To establish the HPLC fingerprint of Semen Strychni by chemometrics and the method of linear calibration using two reference substances (LCTRS). METHODS: The fingerprint of Semen Strychni was established by HPLC, using similarity analysis, 2-dimensional clustering analysis and principal component analysis for quality evaluation. LCTRS was used for identification of chromatographic peak. RESULTS: Fourteen chromatographic peaks were selected as common peaks in the fingerprints of 10 batches of Semen Strychni, and the chromatographic peaks of loganic acid, chlorogenic acid, strychnine and brucine were selected as characteristic peaks. By means of chemometrics evaluation, 10 batches of Semen Strychni were classified by their origins. The precision of predicting retention time by LCTRS was superior to the relative retention time method. CONCLUSION: LCTRS is more accurate for predicting retention time and suitable for more kinds of chromatographic columns than relative retention time method. In addition, the established fingerprint is specific, and can be used for the quality evaluation of Semen Strychni.
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This review concerns the definitions and appropriate analytical characterisations of herbal reference standards within the framework of regulatory requirements. It describes currently applicable rules and regulations, as well as future issues relating to the European Pharmacopoeia and United States Pharmacopoeia. It provides an update on the use and availability of pharmacopoeial (EP and USP) herbal reference standards since our last review was published in 2009. The continuing challenges facing manufacturers, suppliers and analysts are discussed on the basis of exemplary reference substances for herbal products in medicinal and food products. The article also reviews the special aspects of Brazilian stipulations (Brazilian Pharmacopoeia, Anvisa) by comparison with European regulations. The term herbal products as used throughout this article refers to herbal drugs, herbal preparations and finished herbal medicinal products unless a different meaning is obvious from the context. More specific terms are used where necessary.
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Objective To establish a stable and reliable HPLC method and fingerprint reference substance for the measurement of the fingerprint, the practical quality control, and assay of the water-soluble components of Salvia miltiorrhiza. Methods The HPLC was run on C 18 columns with methanol-1.0 % glacial acetic acid solution as mobile phase in gradient elution mode at a flow rate of 1.0 mL/min. The chromatographic system suitability, the gradient elution mode, mobile phase acidity, and the effect of column type on fingerprint repeatability were tested. Results The HPLC fingerprints of the water-soluble extract of reference S. miltiorrhiza were obtained with very good resolution under the established chromatographic system. Fifteen peaks in the chromatograms were selected for the fingerprint identification and quality control of S. miltiorrhiza. The quality of ten batches of S. miltiorrhiza samples from different hibitats were assessed by comparing their chromatographic fingerprints with the reference fingerprints obtained at the same time, and the similarity showed no difference, eventhough the column filler was changed. Conclusion Because the inherent complexity of medicinal material components has been reflected by chromatographic fingerprints, there are many factors affecting the fingerprint repeatability for the changes of column type. The results of the quality assessment of S. miltiorrhiza, using fingerprints from different columns, are not all coincidence. In order to obtain the comparable and repeatable results in different laboratories, it is much practical with both a defined chromatographic system suitability and a fingerprint reference substance.