High-performance Liquid Chromatography Fingerprint Method Research Articles

Quality evaluation of WuweiZhenju tablets based on HPLC fingerprint, content determination and chemical pattern recognition analysis

AbstractObjectiveTo establish a method for the high-performance liquid chromatography (HPLC) fingerprint and content determination of Wuwei Zhenju Tablets, and combine with chemical pattern recognition analysis to provide basis for its quality evalution.MethodsBased on HPLC, taking the chromatographic peak of puerarin as the reference peak, the fingerprints of 10 batches of Wuwei Zhenju Tablets were established and the similarity evaluation was carried out to determine the common peaks. SPSS 26.0 and SIMCA 14.1 software were used to evaluate the overall quality of Zhenju Tablets by cluster analysis and principal component analysis. The contents of chlorogenic acid, puerarin, daidzin, isochlorogenic acid B, isochlorogenic acid A and aurantio-obtusin in the samples were determined by the same method.ResultsTwenty-six common peaks were identified in the fingerprints of 10 batches of Wuwei Zhenju Tablets, and the similarity evaluation was greater than 0.985. Ten chromatographic peaks were identified as neochlorogenic acid, chlorogenic acid, caffeic acid, puerarin, daidzin, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, aurantio-obtusin and betaine. Combined with cluster analysis and principal component analysis, 10 batches of samples can be clustered into two categories, which can distinguish different Zhenju tablets. The model established in pattern recognition research can accurately identify Wuwei Zhenju tablets, and the prediction results are ideal. The six index components had a good linear relationship in their respective concentration ranges. The contents of chlorogenic acid, puerarin, daidzin, isochlorogenic acid B, isochlorogenic acid A and aurantio-obtusin in 10 batches of Wuwei Zhenju Tablets were 2.902–5.819, 3.693–49.680, 1.449–10.654, 2.768–7.724, 2.941–6.343 and 0.071–0.089 mg g−1, respectively.ConclusionThe HPLC fingerprint and multi-component content determination method of Wuwei Zhenju Tablets were established for the first time. The method is simple, efficient and accurate, and can effectively provide experimental basis for the quality control and evaluation of the preparation.

Quality Evaluation of Market Acacia catechu by Fingerprint-Chemical Pattern Recognition

Acacia catechu (L.f.) Willd, a leguminous plant, is included in the 2020 edition of the Chinese Pharmacopoeia and is mainly used to treat eczema, mouth ulcers, diarrhea, bruising, and traumatic hemorrhage. However, there are imported and domestic Acacia catechu samples available in China, and their quality and price are very different, which seriously affects the safety and stability of their clinical application. Importantly, there is no simple and effective method for identifying or classifying grades of Acacia catechu. In this study, 47 batches of commercial Acacia catechu were used for identifying or classifying grades of Acacia catechu using high performance liquid chromatography (HPLC) combined with chemometric analysis. Firstly, gradient elution was adopted with 0.05% phosphoric acid water (A)-methanol (B) as the mobile phase to establish chromatographic conditions. The HPLC chromatograms of 47 batches of Acacia catechu samples were analyzed by the “Similarity Evaluation System for Chromatographic Fingerprint of TCM” software (version 2012A). The common peaks of Acacia catechu were identified to evaluate the similarity. Based on the determination results of fingerprint chromatographic peak area, the quality of the collected Acacia catechu was evaluated by chemometric methods such as CA, PCA, and OPLS-DA. The results showed that the collected Acacia catechu samples were significantly divided into three categories. The first-class samples were all imported Acacia catechu except S9 sample, which was domestic Acacia catechu; the second-class samples were partly domestic Acacia catechu and partly imported Acacia catechu; and the third-class samples were all domestic Acacia catechu. Moreover, OPLS-DA of 47 batches of samples showed that the contents of catechin and the total contents of catechin and epicatechin could be used as key indicators for assessing the quality of Acacia catechu. The developed HPLC fingerprint and quantitative analysis method of multi-indicator components can be used for classification and quality evaluation of market Acacia catechu, which has a significant reference value for developing Acacia catechu grade quality standards.

Authentication of the Bilberry Extracts by an HPLC Fingerprint Method Combining Reference Standard Extracts.

A simple and fast high-performance liquid chromatography (HPLC) fingerprint method combining reference standard extract for the identification of bilberry extract was developed and validated. Six batches of bilberry extract collected from different manufactures were used to establish the HPLC fingerprint. Other berry extracts—such as blueberry extracts, mulberry extracts, cranberry extracts, and black rice extracts—were also analyzed for their HPLC chromatograms. The fingerprints of five batches of bilberry extract showed high similarities, while one batch was distinguished from others. Additionally, the content of anthocyanin Cyanidin-3-O-glucoside (Cy-3-glc) in each berry extract was analyzed and compared. The results indicate that this HPLC fingerprint method, combining reference standard extracts, could be used for the authentication and quality control of bilberry extracts.

Qiang-Gan formula extract improves non-alcoholic steatohepatitis via regulating bile acid metabolism and gut microbiota in mice

Ethnopharmacological relevanceQiang-Gan formula is a traditional Chinese medicine formula, which has been widely used in treating liver diseases in China. Aim of the studyTo investigate the effect of Qiang-Gan formula extract (QGE) on non-alcoholic steatohepatitis (NASH) and its underlying possible mechanisms. Materials and methodsThe high-performance liquid chromatography finger-print method was used for the quality control of chemical components in QGE. Methionine- and choline-deficient diet-induced NASH mice were administrated with QGE via gavage for four weeks. Phenotypic parameters including liver histological change as well as serum levels of alanine transaminase (ALT), aspartate transaminase (AST) were detected. Bile acid profile in the serum, liver and fecal samples was analyzed by gas chromatography-mass spectrometer technique, and fecal microbiota was detected by 16S rDNA sequencing. Expression of liver G protein-coupled bile acid receptor 1 (TGR5), farnesiod X receptor (FXR), tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) as well as molecules in nuclear factor kappa B (NF-κB) pathway was assayed by immunohistochemistry staining, RT-qPCR, or Western blot, respectively. ResultsQGE alleviated liver inflammation, reduced serum ALT and AST levels and liver TNF-α and IL-1β expression in NASH mice. It also decreased liver and serum BA concentration and increased fecal lithocholicacid (LCA) production in this animal model. QGE altered the structure of gut microbiota, predominantly increased LCA-producing bacteria Bacteroides and Clostridium in NASH mice. In addition, the expression of liver TGR5 but not FXR was increased, and the molecules in NF-κB pathway were decreased in QGE-treated NASH mice. ConclusionsQGE was effective in preventing NASH, possibly by regulation of gut microbiota-mediated LCA production, promotion of TGR5 expression and suppression of the NF-κB activation.

Quality evaluation of bee pollens by chromatographic fingerprint and simultaneous determination of its major bioactive components

Bee pollens constitute a large number of flavonoids and thus possess great medicinal value. However different varieties of bee pollen flavonoids vary with different species and their content also differ greatly in different region. Herein, the aim of present research is to establish a method based on high performance liquid chromatography (HPLC) for quantitative analysis of flavonoids compounds and chemical fingerprint analysis of bee pollen. Five batches of rape bee pollen collected from different region of China and particularly six bee pollen species obtained in Anhui were used to establish the fingerprint. The feasibility and advantages of the used HPLC fingerprint were verified for its similarity evaluation by systematically comparing chromatograms with professional analytical software. The similarities of liquid chromatography fingerprints for five batches of rape bee pollen were more than 0.994 while six batches of different species of bee pollen were lower than 0.810. In quantitative analysis, the six compounds showed good regression (R ≥ 0.9964) within the test ranges, and all the values for the RSD were lower than 2%. The developed HPLC fingerprint method was found simple, reliable, and it was validated for the quality control and identification of bee pollen. Additionally, simultaneous quantification of six flavonoids ingredients in the bee pollen samples was conducted to reveal the variation in their content. The results indicated that the HPLC fingerprint, as a characteristic distinguishing method combining similarity evaluation and quantification analysis, can be successfully used to assess the quality and also to identify the authenticity of bee pollen.

A Strategy for Quality Control of Vespa magnifica (Smith) Venom Based on HPLC Fingerprint Analysis and Multi-Component Separation Combined with Quantitative Analysis

As a folk medicine of the Jingpo minority in Yunnan province, the venom of Vespa magnifica has been commonly used for the treatment of rheumatoid arthritis. Quality standardization of the wasp venom is a necessary step for its pharmaceutical research and development. To control the quality of the wasp venom, a method based on high-performance liquid chromatography (HPLC) was developed for chemical fingerprint analysis. In the chromatographic fingerprinting, chemometrics procedures, including similarity analysis (SA), hierarchical clustering analysis (HCA), and principal component analysis (PCA), were applied to classify 134 batches (S1–S134) of wasp venom from different origins. The HPLC fingerprint method displayed good precision (Relative standard deviation, RSD < 0.27%), stability (in 16 h, RSD < 0.34%), and repeatability (RSD < 1.00%). Simultaneously, four compounds (VMS1, VMS2, VMS3, and VMS4) in the wasp venom were purified and identified. VMS1 was 5-hydroxytryptamine, and the other compounds were three peptides that were sequenced as follows: Gly–Arg–Pro–Hyp–Gly–Phe–Ser–Pro–Phe–Arg–Ile–Asp–NH2 (VMS2), Ile–Asn–Leu–Lys–Ala–Ile–Ala–Ala–Leu–Ala–Lys–Lys–Leu–Leu–NH2 (VMS3), and Phe–Leu–Pro–Ile–Ile–Gly–Lys–Leu–Leu–Ser–Gly–Leu–Leu–NH2 (VMS4). The quantifications for these components were 110.2 mg/g, 26.9 mg/g, 216.3 mg/g, and 58.0 mg/g, respectively. The results of this work indicated that the combination of the chemical fingerprint and quantitative analysis offers a reasonable way to evaluate the quality of wasp venom.

Fingerprinting and multi-indicator quantitative analysis of Mongolian drug Digeda-4 decoction

To establish the high performance liquid chromatography (HPLC) fingerprint for Digeda-4 decoction (DGD-4D), determine the contents of aesculetin, geniposide, picroside Ⅰ, picroside Ⅱ and ellagicacid in DGD-4D, and provide the scientific foundation for quality control of DGD-4D. The analysis was performed on Diamonsil(2) C₁₈ (4.6 mm×250 mm,5 μm) column, with methanol-0.1% phosphoric acid aqueous solution as mobile phase for gradient elution. The flow rate was 1.0 mL·min⁻¹; injection size was 10 μL; temperature was maintained at 30 °C, and the detection wavelength was set at 254 nm. The common mode of DGD-4D HPLC fingerprint was established, and the hidden information was analyzed by Chemometrics. Chromatographic peaks for DGD-4D were identified by HPLC and quantitative analysis was conducted for characteristic peaks. There were 17 common peaks in the fingerprints and the similarity of the fingerprints was over 0.9 in all 15 batches. The samples were broadly divided into four kinds by principal component analysis and clustering analysis. Four marker compounds were verified by partial least squares discriminant analysis, and No. 9, 12 and 14 peaks were identified as geniposide, picroside Ⅱ, and picroside Ⅰ respectively. The average recoveries were in the range of 95.91%-97.31%. The HPLC fingerprint method for content determination is reliable, accurate, rapid, simple, and reproducible, and can be used as one of the effective methods to control the quality of DGD-4D.

Molecular, Chemical and Microscopic Analysis of Medicinal Plant (&amp;lt;i&amp;gt;Cymbopogon distans&amp;lt;/i&amp;gt;) from Its Adulterants

Morphological resemblance among Cymbopogon distans species and their adulterants which are procured from different markets in the form of dried or fresh plant tissues represents a serious problem for quality and safety of medicinal plants, as it supports frauds for substitution. In order to assure the quality control of C. distans species, DNA barcode, microscopic identification and High Performance liquid chromatography (HPLC) fingerprint were synergistically used to discriminate C. distans from its adulterants. In this work, the internal transcribed spacer 2 (ITS2) was chosen for distinguishing C. distans from their usual adulterants from 5 provinces of China. Sequences were obtained after removal of the 5.8S and 28S sections. A multiple sequence alignment was finalized. Results exhibited that ITS2 performed well, with 100% of genera being accurately distinguished. Additionally, finding indicates that the upper epidermis in leaf of C. distans was composed of one layer of wide elongated cells called Bulliform cells whereas in C. distans the upper epidermis consist of one layer cell, thus these feactures are very important for the anatomy identification. The HPLC fingerprint method was also developed, the similarities of 6 batches of C. distans samples were all more than 0.93, indicating that the samples from different geographical origins shared similar HPLC fingerprints. And the similarities between C. distans, C. citratus, C. flexuosus and Imperata cylindrica were all less than 0.93, suggesting that there was significance difference between C. distans and its adulterants. Finally, it was concluded that the DNA barcode, HPLC fingerprint and microscopic methods could effectively authenticate the quality of C. distans from their adulterants and can provide accurate and reliable information to tackle the complex quality issue of C. distans in markets. This is the first report of detailed analysis of the C. distans for effective quality and safety.

Fingerprint profiles of flavonoid compounds from different Psidium guajava leaves and their antioxidant activities.

Flavonoids are the main active components in Psidium guajava leaves and have many multi-physiological functions. In this study, the flavonoid compositions were identified in the Psidium guajava leaves samples using a high-performance liquid chromatography with time-of-flight electrospray ionization mass spectrometry method. A high-performance liquid chromatography fingerprint method, combined with chemometrics, was used to perform a quality assessment of the Psidium guajava leaves samples. The eight identified flavonoid compounds including rutin, isoquercitrin, quercetin-3-O-β-d-xylopyranoside, quercetin-3-O-α-l-arabinopyranoside, avicularin, quercitrin, quercetin, and kaempferol were used as the chemical markers. The antioxidant activity of 15 batches of samples was examined using three different methods, and the results revealed the Psidium guajava leaves samples that had higher contents of the flavonoid compounds, glycoside and aglycone, possessed the highest antioxidant capacities. Consequently, a combination of chromatographic fingerprints and chemometric analyses was used for a quality assessment of Psidium guajava leaf tea and its derived products, which can lay the foundation for the development of plant tea resources or other herbs.

Correlation analysis between quality characteristics and commercial specifications of Panacis Quinquefolii Radix

This study was aimed to analyze the correlation between commercial specifications of Panacis Quinquefolii Radix and quantitative indexes of sevent kinds of ginsenosides (ginsenosides Rg¹, Re, Rb¹, Rc, Rb₂, Rb₃, Rd) contained in Panacis Quinquefolii Radix by using high performance liquid chromatography (HPLC), explore the correlation between the characteristics of the traditional Panacis Quinquefolii Radix specifications and modern chemical quantitative indicators, and provide a theoretical basis for the quality grade evaluation of Panacis Quinquefolii Radix. The HPLC fingerprint method was used to analyze 40 batches of Panacis Quinquefolii Radix. A total of 19 peaks were marked, and the similarity was above 0.900 for all samples. On this basis, processing methods, product specifications, contents of 7 components, and the total contents of ginsenoside Rg¹, Re and Rb¹ were used as the original variables for cluster analysis and principal component analysis. The results showed great correlation between the quality of Panacis Quinquefolii Radix and the information on their origins, but the difference was less with the characteristics of traditional commercial specifications, indicating some limitations in the division of commercial specifications of Panacis Quinquefolii Radix. The results revealed the intrinsic relationship between the product specifications, traditional qualitative indexes, and quantitative indexes of chemical components of Panacis Quinquefolii Radix, providing a new idea for the objective comprehensive evaluation of the quality of Panacis Quinquefolii Radix.

Quality evaluation of the leaves of Magnolia officinalis var. biloba using high‐performance liquid chromatography fingerprint analysis of phenolic compounds

The high-performance liquid chromatography fingerprint method is a simple and reliable technique to evaluate the quality of leaves of Magnolia officinalis Rehd.et Wils. var. biloba Rehd.et Wils. We used the following bioactive phenolic constituents as reference compounds: rutin, afzelin, hyperoside, isoquercitrin, quercetin-3-O-α-l-rhamnoside, honokiol and magnolol. The conditions of an Agilent 1200 HPLC were: YMC-Pack-ODS-AQ column (250 × 4.6 mm id S-5 μm, 12 nm), mobile phase acetonitrile and 0.2% phosphoric acid in a gradient elute mode, flow rate 1.0 mL/min, detection wavelength 280 nm and column temperature 30°C. The analytical method was validated in terms of linearity, stability, repeatability, precision and recovery tests. While performing fingerprint analysis, we identified 11 peaks as characteristic peaks and assessed the similarities of 17 samples collected from different geological regions of China. The peak areas were used to evaluate the variation in the chemical composition of the tested samples. For this purpose, we performed hierarchical cluster analysis of the peak areas. Our results indicate that simultaneous determination of multiple ingredients could be done through chromatographic fingerprint analysis. Therefore, this high-performance liquid chromatography fingerprint method was readily utilized to evaluate the quality of leaves of M. officinalis var.biloba, which are used in several traditional herbal preparations.

Discrimination of Semen cassiae from two related species based on the multivariate analysis of high-performance liquid chromatography fingerprints.

A simple and efficient high-performance liquid chromatography fingerprint method was developed to discriminate Semen cassiae from two related species: Cassia obtusifolia L. (CO) and Cassia tora L. (CT), the seeds of which are abbreviated as COS and CTS, respectively. 22 major bioactive ingredients in 42 samples (20 COS and 22 CTS) collected from different provinces of China were identified. The statistical methods included similarity analysis and partial least-squares discriminant analysis. The pattern analysis method was specific and could be readily used for the comprehensive evaluation of Semen cassiae samples. Therefore, high-performance liquid chromatography fingerprint in combination with pattern analysis provided a simple and reliable method for discriminating between COS and CTS.

Quality control of tuirejieduling granules using high-performance liquid chromatography fingerprint method and simultaneous determination of four main active ingredients

Background:The Tuirejieduling granule is a compound preparation made from four kinds of Chinese medicines. It is effective for anti-inflammation, antivirus, defervescence and anti-bacterium; however, its quality control standards have remained unknown.Objective:To establish a simple and accurate fingerprint method for quality control of the Tuirejieduling granule.Materials and Methods:The methanol extract of the Tuirejieduling granule was used for the fingerprint analysis and the four selected active ingredients (epigoitrin, phillyrin, saikosaponin A and glycyrrhetinic acid) in the extract were determined. The fingerprint method was performed on an Amethyst C18-P chromatography column by gradient elution with acetonitrile and aqueous phase (containing 0.5% H3PO4 (v/v), pH 3.0).Results:Under the optimal chromatographic condition, twenty peaks were chosen as fingerprint peaks of the Tuirejieduling granules extractions. The similarities of 10 batches of Tuirejieduling granule was more than 0.99. This indicates that the different batches of Tuirejieduling granules were under the consistent quality control. Good linear behaviors over the investigated concentration ranges were obtained with the values of R2 higher than 0.99 for four studied active ingredients. The recoveries for spiked samples were in the range of 96.2–105.5%. The developed method was successfully applied to determine the contents of active constituents in different batches of Tuirejieduling granule.Conclusion:The HPLC fingerprint was proved to be a reliable method for the quality control of Tuirejieduling granule.