Complete composition analysis of polysaccharides based on HPAEC-PAD coupled with quantitative analysis of multi-components by single marker.

INTRODUCTION: Monosaccharide compositions analysis (MCA) is indispensable for structural characterisations and structure-activity relationships of plant polysaccharides. OBJECTIVES: To develop a concise and direct MCA method, we established a quantitative analysis of the multi-monosaccharaides by single marker (QAMS) by high-performance anion-exchange chromatography with pulsed-amperometric detection (HPAEC-PAD) method. METHODOLOGY: A stable and reproducible HPAEC-PAD method for simultaneous determination of aldoses, ketoses and uronic acids (i.e., l-arabinose, d-xylose, d-ribose, l-rhamnose, d-fucose, d-mannose, d-glucose, d-galactose, d-fructose, d-glucuronic acid and d-galacturonic acid) was established by systematic optimisation of stationary phases, column temperatures and elution programmes. On this basis, the QAMS method was proposed through comprehensive investigations of relative correction factor (RCF) variations under different influencing factors, for example, sample concentrations, flow rates, and column temperatures. RESULTS: Using rhamnose as an internal reference standard, the contents of the other monosaccharide components in polysaccharides from Panax quinquefolium L. and Achyranthes bidentata Bl. samples were simultaneously determined by QAMS, and there was no significant difference between the results from the QAMS and external standard method (t test, P > 0.520). In addition, a MCA fingerprinting of 30 batches of P. quinquefolium polysaccharide was established by HPAEC-PAD, and six common peaks were assigned and determined. CONCLUSIONS: The established HPAEC-PAD-QAMS method was successfully applied to the MCA of polysaccharides from P. quinquefolium and A. bidentata after optimisation of hydrolysis conditions. HPAEC-PAD-QAMS was proposed and established for MCA of plant polysaccharides for the first time.

Experimental design approach for the quantitative analysis of multicomponents by single marker and HPLC fingerprinting of Thunbergia laurifolia aqueous extract.

INTRODUCTION: Thunbergia laurifolia is used in traditional Thai medicine to reduce fever and treat mouth ulcers. However, the quantitative analysis of chemical markers has not yet been officially defined. OBJECTIVE: The objective of this study is to develop a high-performance liquid chromatography (HPLC) method using a design of experiment (DoE) for the quantitative analysis of multicomponents by single marker (QAMS) and fingerprinting of the T. laurifolia aqueous extract. MATERIALS AND METHODS: Critical variables were screened using a two-level fractional factorial design, followed by the optimization of the selected variables using a central composite design. The validated method was applied for quality assessment based on QAMS and fingerprinting of the extract. RESULTS: Optimum conditions of DoE for the analysis of caffeic acid, vicenin-2, and rosmarinic acid were determined. The relative correction factors for caffeic acid and vicenin-2 were calculated using rosmarinic acid as an internal reference standard, and their contents in 30 samples were determined. The differences between the external standard method (ESM) and QAMS were compared. No significant difference was observed in the quantitative determination, proving the consistency QAMS and ESM. HPLC fingerprints of T. laurifolia were established with 8 of 12 characteristic peaks that were structurally characterized using HPLC-diode array detection-electrospray ionization/tandem mass spectrometry. The similarity of the fingerprints in all samples was ≥0.74, and the pattern recognition of the characteristic peaks was satisfied. CONCLUSION: The proposed method efficiently detected multiple components of the T. laurifolia extract. Thus, the method is beneficial in providing references for enhancing the quality control of other herbal medicines.

[Quality control method for phenolic acid components in abandoned stems and leaves of Artemisia selengensis based on UPLC fingerprint combined with quantitative analysis of multi-components by single marker (QAMS)].

This study established an ultra-performance liquid chromatography(UPLC) fingerprint of abandoned stems and leaves of Artemisia selengensis and quantitative analysis of multi-components by single marker(QAMS) for five phenolic acid components. Waters Acquity UPLC BEH C_(18) chromatography column(2.1 mm×100 mm, 1.7 µm) was used. The gradient elution was carried out with the mobile phase composed of 0.1% phosphoric acid water and acetonitrile at a flow rate of 0.3 mL·min~(-1) and a column temperature at 30 ℃. The detection wavelength was 330 nm, and the injection volume was 2 µL. Similarity evaluation and cluster analysis were conducted on the fingerprint data, and 15 common components in 13 batches of abandoned stems and leaves of A. selengensis were identified. The relative correction factors of ferulic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C were calculated using chlorogenic acid as the internal reference. The QAMS for determining five components in the abandoned stems and leaves of A. selengensis was established. At the same time, the content of these five components was determined using the external standard method(ESM), and the results showed that there were no significant differences in their content determined by the QAMS and the ESM. The results indicated that the content of phenolic acid components in the abandoned stems and leaves of A. selengensis from different varieties and different origins had obvious differences. In addition, the content of phenolic acid components in the abandoned stems and leaves of lignified A. selengensis was significantly higher than that of non-lignified A. selengensis. In summary, QAMS established in this study can be quickly, accurately, and economically used to determine the content of five phenolic acid components in abandoned stems and leaves of A. selengensis, laying a foundation for the resource development and utilization of abandoned stems and leaves of A. selengensis.

Quality evaluation of Hawk tea from different months and regions based on quantitative analysis of multiple components with a single marker (QAMS) combined with HPLC fingerprint.

BACKGROUND: Hawk tea, a medicinal and edible plant, has been consumed for thousands of years in Southwest China. To date, no unified food safety standard for Hawk tea has been established, and systematic research on the quality of Hawk tea is lacking. OBJECTIVE: The aim of this study was to develop a comprehensive evaluation method for the quality of Hawk tea based on inclusions content, high-performance liquid chromatography (HPLC) fingerprinting combined with the quantitative analysis of multiple components with a single marker (QAMS) method. METHODS: The contents of total flavonoids, total phenols, total polysaccharides, and total protein were determined using the colorimetric method. An effective comprehensive evaluation method was established to classify the 16 batches of samples based on HPLC fingerprint analysis combined with similarity analysis (SA), hierarchical cluster analysis (HCA), principal component analysis (PCA), partial least-squares discrimination analysis (PLS-DA), and the QAMS method. RESULTS: Flavonoids were the main chemical components of Hawk tea. The accuracy of the QAMS method was verified by comparing the calculated results with those of the external standard method (ESM). No significant differences were found between the two methods. Additionally, the fingerprint of Hawk tea was also established. CONCLUSION: The method established in this study can be used for the comprehensive quality evaluation of Hawk tea and can also provide a reference for the quality evaluation of other herbal medicines.

Determination of Phytochemical Contents in Extracts from Different Growth Stages of Oroxylum indicum Fruits Using HPLC-DAD and QAMS Methods.

Flavones are major compounds found in several parts of Oroxylum indicum (O. indicum). The quantification of multiple components by one marker (QAMS) method and the high-performance liquid chromatography (HPLC) method were developed for the quantitative analysis of extracts from the young fruits, green mature fruits, dry pod coats and seeds of O. indicum. Oroxin A, oroxin B and chrysin-7-O-glucuronide were identified in the O. indicum extracts. Oroxylin A and 5-hydroxymethylfurfural were isolated and structurally identified from the pod coat and young fruit extracts, respectively. From the HPLC analysis of the seven major flavones in the extracts, baicalin was the major compound in all extracts investigated (0.4-11% w/w of the extract). All flavone contents were low in the young fruit extract (<1% w/w of the extract). The green mature fruit and dry pod coat extracts showed similar constituent compositions. They contained small amounts of baicalin and oroxylin A, which were found only in these two extracts. Oroxylin A could be used as a marker to indicate the maturity of O. indicum fruits, while 5-hydroxymethylfurfural could be used as a marker for the young fruits. Baicalin was found to be a suitable single marker to calculate the contents of all flavones in the O. indicum extracts.

[Quality evaluation of Compound Cheqian Tablets based on UPLC-Q-TOF-MS/MS, network pharmacology and "double external standards" QAMS].

The Compound Cheqian Tablets are derived from Cheqian Power in Comprehensive Recording of Divine Assistance, and they are made by modern technology with the combination of Plantago asiatica and Coptis chinensis. To investigate the material basis of Compound Cheqian Tablets in the treatment of diabetic nephropathy, in this study, the chemical components of Compound Cheqian Tablets were characterized and analyzed by UPLC-Q-TOF-MS/MS, and a total of 48 chemical components were identified. The identified chemical compounds were analyzed by network pharmacology. By validating with previous literature, six bioactive compounds including acteoside, isoacteoside, coptisine, magnoflorine, palmatine, and berberine were confirmed as the index components for qua-lity evaluation. Furthermore, the content of the six components in the Compound Cheqian Tablets was determined by the "double external standards" quantitative analysis of multi-components by single marker(QAMS), and the relative correction factor of isoacteoside was calculated as 1.118 by using acteoside as the control; the relative correction factors of magnoflorine, palmatine, and berberine were calculated as 0.729, 1.065, and 1.126, respectively, by using coptisine as the control, indicating that the established method had excellent stability under different conditions. The results obtained by the "double external standards" QAMS approximated those obtained by the external standard method. This study qualitatively characterized the chemical components in the Compound Cheqian Tablets by applying UPLC-Q-TOF-MS/MS and screened the pharmacodynamic substance basis for the treatment of diabetic nephropathy via network pharmacology, and primary pharmacodynamic substance groups were quantitatively analyzed by the "double external stan-dards" QAMS method, which provided a scientific basis for clarifying the pharmacodynamic substance basis and quality control of Compound Cheqian Tablets.

Fingerprint combining with quantitative analysis of multi-components by single marker for quality control of Chenxiang Huaqi tablets.

BACKGROUND: Chenxiang Huaqi tablets (CXHQTs) are a traditional Chinese medicine (TCM) commonly used to treat stomach-related diseases. Currently, the ministerial standards do not provide detailed guidance and regulations on the content determination of CXHQTs, and the reported studies only use individual active components as indicators for determining effective ingredients. OBJECTIVE: The present study aimed to propose a methodology for quality control of CXHQTs based on high-performance liquid chromatography (HPLC) fingerprinting combined with the quantitative analysis of multi-components by single marker (QAMS) method. METHODS: HPLC method was used to determine seven active ingredients and performed fingerprint analysis of CXHQTs. To further process chemometric assessment, technical analysis-model including similarity analysis (SA), hierarchical clustering analysis (HCA), principal components analysis (PCA) and orthogonal partial least squares discrimination analysis (OPLS-DA) was set up to differentiate and classify the 20 batches of samples. RESULTS: After comparing the results of QAMS method with the external standard method (ESM), we found there is no significant difference. Besides, the fingerprint of CXHQT was also established. CONCLUSION: HPLC fingerprint combined with the QAMS could be an efficient and selective analysis technique to achieve a qualitative and quantitative evaluation of executing quality processes.

Chemical comparison and discrimination of two plant sources of Angelicae dahuricae Radix, Angelica dahurica and Angelica dahurica var. formosana, by HPLC-Q/TOF-MS and quantitative analysis of multiple components by a single marker.

INTRODUCTION: Angelica dahurica(BZ) and Angelica dahurica var. formosana(HBZ) are two plant sources of Angelicae dahuricae Radix. Although BZ and HBZ are commonly used herbal medicines with great medicinal and dietary values, study on their phytochemicals and bioactive compositions is limited. OBJECTIVE: To compare the chemical compositions of BZ and HBZ and find the chemical makers for discrimination and quality evaluation of the two botanical origins of Angelicae dahuricae Radix. METHODOLOGY: A high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry method was established for chemical profiling of BZ and HBZ. Then, a quantitative analysis of multiple components by a single marker method was developed for simultaneous determination of nine bioactive coumarins (xanthotoxol, oxypeucedanin hydrate, byakangelicin, xanthotoxin, bergapten, oxypeucedanin, phellopterin, imperatorin and isoimperatorin). Moreover, chemometrics were performed to compare and discriminate BZ and HBZ samples. RESULTS: A total of 30 coumarins compounds were identified, and the chemical compositions in BZ and HBZ were quite similar. The quantitative analysis showed that there were significant differences in the contents of bioactive coumarins, and the chemometric analysis indicated five coumarins (xanthotoxol, xanthotoxin, bergapten, phellopterin and isoimperatorin) were responsible for the significant differences between BZ and HBZ, which could be used as chemical markers to distinguish the two original plant sources of Angelicae dahuricae Radix. CONCLUSION: The present work provided useful information for understanding the chemical differences between BZ and HBZ and also provided feasible methods for quality evaluation and discrimination of herbal medicines originating from multiple botanical sources.

Quantification of Chemical Groups and Quantitative HPLC Fingerprint of Poria cocos (Schw.) Wolf.

(1)Objective: In this study, a quantitative analysis of chemical groups (the triterpenoids, water-soluble polysaccharides, and acidic polysaccharides) and quantitative high liquid performance chromatography (HPLC) fingerprint of Poria cocos (Schw.) Wolf (PC) for quality control was developed. (2) Methodology: First, three main chemical groups, including triterpenoids, water-soluble polysaccharides, and acidic polysaccharides, in 16 batches of PC were evaluated by ultraviolet spectrophotometry. Afterward, the quantitative fingerprint of PC was established, and the alcohol extract of PC was further evaluated. The method involves establishing 16 batches of PC fingerprints by HPLC, evaluating the similarity of different batches of PC, and identifying eight bioactive components, including poricoic acid B (PAB), dehydrotumulosic acid (DTA), poricoic acid A (PAA), polyporenic acid C (PAC), 3-epidehydrotumulosic acid (EA), dehydropachymic acid (DPA), dehydrotrametenolic acid (DTA-1), and dehydroeburicoic acid (DEA), in PC by comparison with the reference substance. Combined with the quantitative analysis of multi-components by a single marker (QAMS), six bioactive ingredients, including PAB, DTA, PAC, EA, DPA, and DEA, in PC from different places were established. In addition, the multivariate statistical analyses, such as principal component analysis and heatmap hierarchical clustering analysis are more intuitive, and the visual analysis strategy was used to evaluate the content of bioactive components in 16 batches of PC. Finally, the analysis strategy of three main chemical groups in PC was combined with the quantitative fingerprint strategy, which reduced the error caused by the single method. (3) Results: The establishment of a method for the quantification of chemical groups and quantitative HPLC fingerprint of PC was achieved as demonstrated through the quantification of six triterpenes in PC by a single marker. (4) Conclusions: Through qualitative and quantitative chemical characterization, a multi-directional, simple and efficient routine evaluation method of PC quality was established. The results reveal that this strategy can provide an analytical method for the quality evaluation of PC and other Chinese medicinal materials.

[Chemical pattern recognition of Atractylodes chinensis from different producing areas and establishment of quantitative analysis of multi-components by single marker (QAMS) method for four components].

This study established the fingerprint and combined it with chemical pattern recognition to evaluate the quality of Atractylodes chinensis samples from different producing areas and then employed the quantitative analysis of multi-components by single marker(QAMS) method to verify the feasibility and applicability of the established method in the quality evaluation of A. chinensis. The fingerprints of A. chinensis samples were constructed via high performance liquid chromatography(HPLC) to evaluate the inter-batch consistency. With the quality control component atractylodin as the internal reference, the relative correction factors(RCFs) were established for atractylenolide Ⅰ, atractylenolide Ⅲ, and ß-eudesmol and the content of the four components was calculated. The external standard method was used to verify the accuracy of QAMS method. The quality of A. chinensis was further evaluated by similarity analysis, clustering analysis, and principal component analysis. The fingerprints of 13 batches of samples were calibrated with 21 common peaks, and 4 common peaks were identified with the similarities all above 0.9. The RCFs established with atractylodin as the internal reference represented good reproducibility under different experimental conditions. Specifically, the RCFs of atractylenolide Ⅰ, atractylenolide Ⅲ, and ß-eudesmol in A. chinensis were 2.091, 4.253, and 6.010, respectively. QAMS and ESM showed no significant difference in the results, indicating that the QAMS method established in this study was stable and reliable. Thus, HPLC fingerprint combined with QAMS can be used for the quality evaluation of A. chinensis, providing a basis for comprehensive and rapid quality evaluation of A. chinensis.

[Quality evaluation of Psoraleae Fructus based on QAMS method and TLC identification].

The present study established a determination method of Psoraleae Fructus by quantitative analysis of multi-components by the single marker(QAMS) and further improved the thin-layer chromatography(TLC) method. The QAMS method was established by UPLC with psoralen as the internal marker, and the content of psoralenoside, isopsoralenoside, psoralen, and isopsoralen was simultaneously determined. As revealed by the comparison with results of the external standard method, the QAMS method was accurate and feasible. According to the current quality standards of Psoraleae Fructus, the TLC method was further optimized and improved, and bakuchiol was added for identification based on the original TLC method with psoralen and isopsoralen as indicators. This study provides a reference for improving the quality control method of Psoraleae Fructus.

[Determination of content of nine components in Xinnaojian preparations from different manufacturers by QAMS].

This study established the ultra-performance liquid chromatography(UPLC) fingerprint of Xinnaojian preparations. With epicatechin gallate as the internal reference substance, a quantitative analysis of multi-components by single marker(QAMS) method for determining the content of nine components(gallic acid, epigallocatechin, catechin, caffeine, epicatechin, epigallocatechin gallate, gallocatechin gallate, epicatechin gallate, and catechin gallate) in Xinnaojian preparations was established. The content determined by the external standard method(ESM) and QAMS method was compared to evaluate the feasibility and accuracy of QAMS method. The results showed that the standard curves of nine components had good linear relationship within the test concentration ranges. The average recoveries were 87.57%-107.4%, and the RSD was 1.5%-2.9%. Except epigallocatechin, the other components showed good repeatability under different experimental conditions. Epigallocatechin could meet the requirements in the same instrument and at the same wavelength. The results generally showed no significant difference between QAMS and ESM. The content of 9 components varied between the samples from different manufacturers, while it showed no significant difference between the samples from the same manufacturer. In summary, the UPLC fingerprint combined with QAMS method is feasible and accurate for determining the content of the nine components, which can be used for rapid quality evaluation of Xinnaojian preparations.

[Quality evaluation of Ginseng Radix et Rhizoma based on UPLC characteristic chromatogram and quantitative analysis of multi-components by single marker (QAMS)].

Based on UPLC characteristic chromatogram and quantitative analysis of multi-components by single marker(QAMS), the content of seven types of ginsenosides in Ginseng Radix et Rhizoma was simultaneously determined, and the quality of Ginseng Radix et Rhizoma was evaluated by the principal component analysis(PCA). The chromatographic separation was performed on the Acquity UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 µm) with the mobile phase of acetonitrile-water for gradient elution at the flow rate of 0.3 mL·min~(-1), the column temperature of 30 ℃, the detection wavelength of 203 nm, and the injection volume of 2 µL. The UPLC chromatogram was established with 19 batches of Ginseng Radix et Rhizoma samples from three producing areas by Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine(version 2012). Thirteen characteristic peaks were determined and seven components were identified. SPSS 26.0 was used to conduct PCA on the characteristic peak areas. With the peak of ginsenoside Rb_1 as reference peak S, ginsenoside Rb_1 showed good durability of relative correction factor as compared with other ginsenosides. The QAMS method for the determination of seven ginsenosides in Ginseng Radix et Rhizoma was established. There was no significant difference in results between the QAMS method and the external standard method. As revealed by the results of PCA and the determination of the total content of seven ginsenosides, the four batches of Ginseng Radix et Rhizoma numbered S19, S18, S1, and S2 were of superior quality. The characteristic chromatogram and QAMS method for the determination of seven ginsenosides in this study were convenient and accurate, which greatly shortened the analysis time and improved the analysis efficiency. The findings of this study are expected to provide a basis for the overall quality evaluation of Ginseng Radix et Rhizoma.

[Simultaneous determination of glucosinolates and flavonoids in leaves of Moringa oleifera by quantitative analysis of multi-components by single-marker (QAMS)].

To establish a method for simultaneously determining the content of four glucosinolates and five flavonoids in leaves of Moringa oleifera via quantitative analysis of multi-components by single-marker(QAMS) and verify the feasibility and applicability of the established method. The glucosinolates and flavonoids were analyzed via Waters Acquity UPLC HSS T_3 column(2.1 mm × 100 mm, 1.8 µm). The gradient elution was carried out with the mobile phase composed of 0.1% formic acid-acetonitrile and 0.3% formic acid at the flow rate of 0.4 mL·min~(-1) and the column temperature of 40 ℃. The wavelengths for the detection of glucosinolates and flavonoids were 225 nm and 260 nm, respectively. With 4-O-(α-L-rhamnoyloxy)-benzyl glucosinolate and vecenin-2 as internal reference substances, the relative correction factors of four glucosinolates and five flavonoids were respectively calculated for determining the content of the 9 ingredients in leaves of M. oleifera. To verify the accuracy and feasibility of QAMS, we used internal standard method(ISM) and external standard method(ESM) to determine the content of glucosinolates and flavonoids, respectively. The t-test results showed that there was no significant difference in the content of glucosinolates obtained by ISM and QAMS methods or in the content of flavonoids obtained by ESM and QAMS methods. The content of glucosinolates and flavonoids varied among M. oleifera of different varieties and from different producing areas. The total glucosinolates and total flavonoids had the highest content in the Indian variety while the lowest content in the variety ■Honghe No. 1'. The established QAMS method is rapid, simple and accurate and can be used for simultaneous determination of glucosinolates and flavonoids in the leaves of M. oleifera. This study provides experimental data for the quality control and utilization of M. oleifera leaves.

Extraction and Analysis of Six Effective Components in Glycyrrhiza uralensis Fisch by Deep Eutectic Solvents (DES) Combined with Quantitative Analysis of Multi-Components by Single Marker (QAMS) Method.

Deep eutectic solvents (DESs) are green organic solvents that have broad prospects in the extraction of effective components of traditional Chinese medicine. This work employed the quantitative analysis of multi-components by a single marker (QAMS) method to quantitatively determine the six effective components of glycyrrhizic acid, liquiritin, isoliquiritin apioside, liquiritigenin, isoliquiritin, and glycyrrhetinic acid in Glycyrrhiza uralensis, which was used for comprehensive evaluation of the optimal extraction process by DESs. First, Choline Chloride: Lactic Acid (ChCl-LA, molar ratio 1:1) was selected as the most suitable DES by comparing the extraction yields of different DESs. Second, the extraction protocol was investigated by extraction time, extraction temperature, liquid-to-material ratio, molar ratio, and ultrasonic power. The Box-Behnken design (BBD) combined with response surface methodology (RSM) was used to investigate the optimal DES conditions. The result showed that the best DES system was 1.3-butanediol/choline chloride (ChCl) with the molar ratio of 4:1. The optimal extraction process of licorice was 20 mL/g, the water content was 30%, and the extraction time was 41 min. The comprehensive impact factor (z) was 0.92. At the same time, it was found that the microstructure of the residue extracted by the eutectic solvent was more severely damaged than the residue after the traditional solvent extraction through observation under an electron microscope. The DES has the characteristics of high efficiency and rapidity as an extraction solution.

Carotenoid Contents of Lycium barbarum: A Novel QAMS Analyses, Geographical Origins Discriminant Evaluation, and Storage Stability Assessment.

Given the standard substances of zeaxanthin and its homologues obtained from Lycium barbarum L. (LB) are extremely scarce and unstable, a novel quantitative analysis of carotenoids by single marker method, named QAMS, was established. Four carotenoids including lutein, zeaxanthin, ß-carotene, and zeaxanthin dipalmitate were determined simultaneously by employing trans-ß-apo-8'-carotenal, a carotenoid component which did not exist in LB, as standard reference. Meanwhile, ß-carotene, another carotenoid constituent which existed in LB, was determined as contrast. The QAMS methods were fully verified and exhibited low standard method difference with the external standard method (ESM), evidenced by the contents of four carotenoids in 34 batches of LB samples determined using ESM and QAMS methods, respectively. HCA, PCA, and OPLS-DA analysis disclosed that LB samples could be clearly differentiated into two groups: one contained LB samples collected from Ningxia and Gansu; the other was from Qinghai, which was directly related to the different geographical location. Once exposed under high humidity (RH 75 ± 5%) at a high temperature (45 ± 5 °C) as compared with ambient temperature (25 ± 5 °C), from day 0 to day 28, zeaxanthin dipalmitate content was significantly decreased, and ultimately, all the decrease rates reached about 80%, regardless of the storage condition. Our results provide a good basis for improving the quality control of LB.

Exploitation of HPLC Analytical Method for Simultaneous Determination of Six Principal Unsaturated Fatty Acids in Oviductus Ranae Based on Quantitative Analysis of Multi-Components by Single-Marker (QAMS).

As one of the featured products in northeast China, Oviductus Ranae has been widely used as a nutritious food, which contains a variety of bioactive unsaturated fatty acids (UFAs). It is necessary to establish a scientific and reliable determination method of UFA contents in Oviductus Ranae. In this work, six principal UFAs in Oviductus Ranae, namely eicosapentaenoic acid (EPA), linolenic acid (ALA), docosahexaenoic acid (DHA), arachidonic acid (ARA), linoleic acid (LA) and oleic acid (OA), were identified using UPLC-MS/MS. The UFAs identified in Oviductus Ranae were further separated based on the optimized RP-HPLC conditions. Quantitative analysis of multi-components by single-marker (QAMS) method was implemented in content determination of EPA, ALA, DHA, ARA and OA, where LA was used as the internal standard. The experiments based on Taguchi design verified the robustness of the QAMS method on different HPLC instruments and chromatographic columns. The QAMS and external standard method (ESM) were used to calculate the UFA content of 15 batches of Oviductus Ranae samples from different regions. The relative error (r < 0.73%) and cosine coefficient showed that the two methods obtained similar contents, and the method validations met the requirements. The results showed that QAMS can comprehensively and effectively control the quality of UFAs in Oviductus Ranae which provides new ideas and solutions for studying the active components in Oviductus Ranae.

[Qualitative and quantitative analysis on triterpenoids in Ligustri Lucidi Fructus].

Triterpenoids are one of the most active constituents in Ligustri Lucidi Fructus, but only oleanolic acid has been mostly studied. In recent years, a growing number of studies have shown that other triterpenes from Ligustri Lucidi Fructus also have various biological activities, so it is necessary to build up a detailed profile of the triterpenoids in Ligustri Lucidi Fructus. The chromatographic separation was performed on a C_(18) column(4.6 mm×250 mm, 5 µm) with mobile phase of methanol-acetonitrile-0.2% formic acid for gradient elution. The detection wavelength was set at 210 nm, with a flow rate of 0.5 mL·min~(-1), and the column temperature of 25 ℃. The HPLC fingerprint of triterpenoids in Ligustri Lucidi Fructus was built by testing 21 batches of samples from different sources. The structures of the total 15 common chromatographic peaks were elucidated with UHPLC-ESI-Orbitrap-MS/MS technique and six of them were identified as tormentic acid, pomolic acid, maslinic acid, botulin, oleanolic acid and ursolic acid by comparison to the reference substances. Under the same chromatographic condition, four main triterpenes(podocarboxylic acid, hawthorn acid, oleanolic acid and ursolic acid) were quantified and the results of system adaptability and methodology investigation all met the requirements of content determination. Meanwhile, with oleanolic acid(A) as the internal reference substance, quantitative analysis of multi-components by single marker(QAMS) method was used to analyze the above four components. The relative correction factor of oleanolic acid(B), hawthorn acid(C) and ursolic acid(D) to oleanolic acid was f_(B/A)=1.12, f_(C/A)=1.02 and f_(D/A)=0.88, respectively, and the relative retention values of these three to oleanolic acid was RRV_(B/A)=0.46, RRV_(C/A)=0.70 and RRV_(D/A)=1.03, respectively. The contents determined by two methods were similar. In conclusion, the method built in this paper is proved to be simple, reliable and specific for the simultaneous qualitative and quantitative analysis of the triterpenoids in Ligustri Lucidi Fructus, which can lay foundation for further assays of the triterpenoids in Ligustri Lucidi Fructus and the relative products.

[Research and verification of quality evaluation method of Epimedii Folium based on quantitative analysis of multi-components by single marker].

The quality control of Epimedii Folium, composed of diverse constituents, is single at present. In view of this, an eva-luation method of 13 chemical constituents based on quantitative analysis of multi-components by single marker(QAMS) was established to further explore the composition differences of raw products and alcohol extracts in different batches and the influence of alcohol extraction on the composition, so as to provide a reference for improving the quality evaluation and control of Epimedii Folium. The fingerprints of different batches of Epimedii Folium were constructed by ultra-high performance liquid chromatography(UPLC) to evaluate the inter-batch consistency. The changes of the flavonoids in Epimedii Folium during alcohol extraction were analyzed based on determined levels and heat map, and the reasons for the changes were preliminarily discussed. With icariin, the quality control component recorded in the Chinese Pharmacopoeia, as the internal reference, the stability of the relative correction factors of chemical components under different conditions was investigated to obtain the relative correction factors. Then the determination results of QAMS and the external standard method were compared to verify the accuracy of QAMS. The results revealed that all batches of Epimedii Folium met the requirements specified in the Chinese Pharmacopoeia, and the fingerprints of Epimedii Folium from the same place of origin exhibited a high similarity. Raw products and alcohol extracts of Epimedii Folium could be clearly distinguished by prenylated flavonoids, which are potential biomarkers for quality control. Additionally, the glycoside hydrolysis in the alcohol extraction was preliminarily explored. The QAMS method has good accuracy, durability, and repeatability in determining 13 chemical components in Epimedii Folium under different experimental conditions. No significant difference in the results obtained by the two methods was observed. This study can provide a reference for comprehensive, rapid and reasonable quality evaluation of Epimedii Folium.

[Content determination of five flavonoids in Tibetan medicine Rhododendron anthopogonoides by quantitative analysis of multi-components by single marker (QAMS)].

To establish a quantitative analysis of multi-components by single marker(QAMS) method for five flavonoids in Rhododendron anthopogonoides and verify its feasibility and applicability in the medicinal materials of R. anthopogonoides. With hyperoside as the internal reference, relative correction factors(RCF) of rutin, quercetin, quercitrin and kaempferol were established by high-performance liquid chromatography(HPLC) analysis. RCFs were used to calculate the content of each component, system durability and relative retention time. Simultaneously, QAMS and external standard method(ESM) were used to determine the content of five flavonoids in 12 batches of R. anthopogonoides from different origins. The results were statistically analyzed to verify the accuracy and feasibility. The fingerprints and cluster analysis data of R. anthopogonoides analyzed and discussed differences among the batches. According to the results, the RCFs of rutin, quercetin, quercetin and kaempferol in R. anthopogonoides were 1.242 6, 0.990 5, 0.535 0, and 0.781 3, respectively. The RCFs represented a good reproducibility under different experimental conditions. Besides, there was no significant difference between QAMS and ESM. Besides, the fingerprint and cluster analysis data showed the consistency between the classification and with the origin distribution of the herbs. In conclusion, the QAMS method shows a good stability and accuracy in the quality control of R. anthopogonoides.