Quality control method for commercially available wild Jujube leaf tea based on HPLC characteristic fingerprint analysis of flavonoid compounds

Flavonoids are the main active components of natural medicinal plants with many physiological functions. In this study, an HPLC fingerprinting method based on the distribution and relative amount of 11 bioactive flavonoids was established for the quality evaluation of commercially available wild Jujube leaf tea (JLT) from China. Separation of the crude flavonoid extract was achieved on a column filled with C₁₈ material with a high carbon content. The flavonoids in wild JLT were identified based on UV spectroscopy and accurate mass measurements by TOF‐MS. Twenty‐one batches of practical samples collected from different habitats were analyzed by using the developed HPLC method to construct the HPLC characteristic fingerprint of wild JLT. Then, combined with clustering and similarity analyses, the HPLC characteristic fingerprint was used for the authentication and quality evaluation of commercial wild JLT. Results indicated that the proposed HPLC characteristic fingerprint reflected the inherent characteristics of wild JLT collected from different regions. Authenticity identification and quality control of commercially available wild Jujube tea were achieved based on the HPLC characteristic fingerprint analysis. This new approach to bioactive component profiling provided a promising reference method for the quality evaluation of commercial wild flower and plant tea.     

丹皮药材的高效液相色谱-质谱/质谱研究

采用高效液相色谱．质谱／质谱建立了丹皮药材的指纹图谱方法。丹皮药材中的主要成分得到较好分离,指纹峰重现性好,通过多维联用技术获得各指纹峰的保留时间、分子量及结构信息,推测出15个指纹峰的可能组成。结果表明：所建立的丹皮指纹图谱信息量丰富,对丹皮药材的化学表征及质量评价有重要的参考价值。     

HPLC Fingerprint with Multi-components Analysis for Quality Consistency Evaluation of Traditional Chinese Medicine Si-Mo-Tang Oral Liquid Preparation

Si-Mo-Tang(SMT) oral liquid preparation, a traditional Chinese medicine, was prepared from four crude herbal drugs, Fructus Aurantii Submaturus, Radix Aucklandiae, Semen Arecae and Radix Linderae Aggregatae. A combinative method using HPLC fingerprint and quantitative analysis was developed and validated for quality consistency evaluation of SMT. Individual HPLC chromatograms were evaluated against the mean chromatogram generated via a similarity evaluation computer program. Data from chromatographic fingerprints were also processed with principal component analysis(PCA) and hierarchical cluster analysis(HCA). Additionally, six components (naringin, isonaringin, hesperidin, neohesperidin, norisoboldine and potassium sorbate) in SMT were simultaneously determined to interpret the quality consistency. For fingerprint analysis, 20 peaks were selected as the characteristic peaks to evaluate the similarities of 26 SMT collected from different manufacturers. Among the 20 characteristic peaks, 10 peaks were assigned to be naringin, hesperidin, neohesperidin, isonaringin, neoeriocitrin, tangeretin, nobiletin, norisoboldine, 5-(ethoxymethyl)furan-2-carbaldehyde and potassium sorbate, respectively. The results of similarity analysis, PCA and HCA, indicate that the samples from different manufacturers were consistent with each other in composition. The results from the quantitative data show that the contents of six compounds were significantly different in SMT oral liquid preparations from different manufacturers. The combinative method of chromatographic fingerprint with quantitative analysis developed here offered an efficient way for the quality consistency evaluation of the traditional Chinese medicine SMT.     

Application of LC/MS and ICP/MS for establishing the fingerprint spectrum of the traditional Chinese medicinal preparation Gan-Lu-Yin

We developed a method to analyze the fingerprint spectrum qualitatively and quantitatively for the traditional Chinese herbal medicinal preparation Gan-Lu-Yin with HPLC combined with photodiode array detection, and MS, and to identify the preparation's 14 main components including baicalin, baicalein, oroxylin A-7-O-glucuronide, wogonin-7-O-glucuronide, wogonin, and oroxylin A in Radix Scutellariae; naringin and neohesperidin in Aurantii fructus; liquiritigenin, liquiritin, and glycyrrhizic acid in Radix Glycyrrhizae. In LC/UV assay, a Cosmosil 5C18-MS-II column was used as the stationary phase, and a gradient of potassium dihydrogen phosphate, ACN, and water as the elute solution. The UV detection wavelengths were 250 and 280 nm. In LC/MS assay, a gradient of phosphoric acid, ACN, and water was used as the elute solution, and electrospray positive ion mode ((+)-ESI) as the analytic mode. In order to explore the distribution of trace metal elements effectively in Gan-Lu-Yin, a microwave digestion method was used for sample treatment, and an inductively coupled plasma MS assay was used to analyze fingerprint spectra of the inorganic metals in Gan-Lu-Yin. Combined with fingerprint spectra of organic compounds by LC/UV and LC/MS, it was expected to provide effective quality control in the production of Gan-Lu-Yin.     

Fingerprint Analysis of Euonymus alatus (Thuhb) siebold by LC–DAD and LC–ESI–MS

A liquid chromatography coupled with photodiode array detection and electrospray ionization tandem mass spectrometry method was first developed for a chemical fingerprint analysis of Euonymus alatus (Thuhb) siebold (EAS) and rapid identification of major compounds in the fingerprints. Fingerprint profiles were found to be consistent for the herbs acquired from different locations, but the relative abundance of peaks was varied. Twelve peaks were chosen as the common peaks. Quercetin and rutin were detected by comparing the retention times, MS and UV spectra with the standards. The relative retention time and relative peak area of the 12 peaks in the fingerprint were calculated by setting the quercetin as the reference peak. The experimental data were used for similarity calculation and hierarchical clustering analysis. By comparing the UV and MS spectra data with those of the authentic standards and literature, five main peaks in the fingerprints were identified. Finally, five medicinal portions of the herb (leaf, fruit, stem, pterygium and root) were also analyzed by this method. It was found that there were similar chemical components in different parts of this herb but the contents were very different. The developed fingerprint assay was specific and could be readily utilized for comprehensive evaluation of EAS, as well as to distinguish different medicinal portions.     

Development of the Fingerprints for the Quality Evaluation of Scutellariae Radix by HPLC-DAD and LC-MS-MS

A high-performance liquid chromatography coupled with photodiode array detection and electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS²) method was firstly developed for a chemical fingerprint analysis of Scutellariae Radix and rapid identification of major compounds in the fingerprints. The experimental data for chromatography were used for hierarchical clustering analysis and similarity calculation, and those for UV and MS spectra were applied for identifying characteristic peaks. Twenty samples including S. baicalensis Georgi., S. viscidula Bge. and S. amoena C. H. Wright were classified into three groups. By comparing the UV and MS spectra data with those of the authentic standards and literature, 20 main peaks in the fingerprints were identified for the first time. The developed fingerprint assay was specific and could be readily utilized for comprehensive evaluation of Scutellariae Radix.     

Quantitative fingerprinting based on the limited‐ratio quantified fingerprint method for an overall quality consistency assessment and antioxidant activity determination of Lianqiao Baidu pills using HPLC with a diode array detector combined with chemometric methods

Lianqiao Baidu pills are widely used herbal medicinal preparation that were analyzed to develop a quality consistency technique. The characteristic fingerprints of 28 batches of Lianqiao Baidu pill samples were established at five wavelengths and simultaneously assessed by using a limited‐ratio quantified fingerprint method using 15 marker compounds. The principal component analysis and fingerprinting results were compared, and the qualitative classification of the samples by principal component analysis agreed with their quantitative evaluation by the limited‐ratio quantified fingerprint method. Furthermore, the antioxidant activities of the samples were surveyed and determined using a 2,2‐diphenyl‐1‐picrylhydrazyl radical‐scavenging approach. A relationship between the common peaks in the fingerprints and the antioxidant activities was established using a partial least squares model. The relationship can be used both to determine the antioxidant activities of the Lianqiao Baidu pill preparations in vitro and as a reference for the selection of active constituents for sample quality classification. The classification results for the samples based on principal component analysis agreed with the quantitative evaluation by the limited‐ratio quantified fingerprint method, which demonstrated that the method can be applied to the holistic quality control of traditional Chinese medicine and herbal preparations.     

Capillary electrophoresis fingerprinting coupled with chemometrics to evaluate the quality consistency and predict the antioxidant activity of Sanhuang tablet as part of its quality control

A capillary electrophoresis fingerprint was constructed for Sanhuang tablet, a Chinese traditional patent medicine, that was commonly used in clinical practice, where the isosceles trapezoid method was first applied for the optimization of background electrolyte solution, and the resolution index was performed to assess the experimental conditions; furthermore, a novel linear quantitative fingerprint method was established for accurate qualitative and quantitative discrimination of the test samples from diverse commercial brands. The fingerprint analysis coupled with quantitative determination of two components was employed to elucidate that the quality consistency of the products was relatively good within one manufactory, but poor among different companies for the 30 batches of samples. In addition, the fingerprint–efficacy relationship between chemical components and antioxidant activity in vitro was investigated using partial least squares analysis, and the calibration and prediction of the antioxidant activity of the selected samples via fingerprint data were presented with the desired results. This work illustrates that the proposed fingerprint analysis based on linear quantitative fingerprint method can be applied for the quality evaluation of traditional Chinese medicine and herbal preparations as part of their quality control, and the constructed mathematical model is particularly suitable for depicting the fingerprint–efficacy relationship.     

Simultaneous determination of polyphenols and triterpenes in pomegranate peel based on high‐performance liquid chromatography fingerprint by solvent extraction and ratio blending method in tandem with wavelength switching

Traditionally, pomegranate (Punica granatum L.) has been consumed as fresh fruit or as pomegranate juice. Pomegranate peel, the dried husk of P· granatum, is an important herbal medicine for treating diarrhea, hemostasis and insect‐induced abdominal pain in China. However, the quality control methods for pomegranate peel remain unsatisfactory. In this work, a new HPLC‐based qualitative and quantitative method for quality control of pomegranate peel was developed and validated for the simultaneous determination of polyphenols and triterpenes (including punicalagins A and B, ellagic acid, oleanolic acid and ursolic acid) by solvent extraction and ratio blending method in tandem with wavelength switching. The average recoveries were 98.07–100.61% with relative standard deviation no more than 4.27%. In addition, the fingerprint analysis was conducted to interpret the consistency of the quality test. Thirteen characteristic peaks were selected to evaluate the similarities of 16 batches of pomegranate peel. The similarities of samples were all more than 0.80, indicating that the samples from different areas of China were consistent. The results demonstrated that quantitative analysis and the HPLC fingerprint as a characteristic distinguishing method combining similarity evaluation can be successfully used to assess the quality and to identify the authenticity of pomegranate peel.     

Chemical fingerprint analysis of Phellodendri Amurensis Cortex by ultra performance LC/Q-TOF-MS methods combined with chemometrics

Ultra performance LC with quadrupole TOF MS (UPLC/Q‐TOF‐MS) fingerprinting is first developed for the identification of the major components of Phellodendri Amurensis Cortex (PAC). The PAC samples are separated using a Waters ACQUITY UPLC BEH C18 (2.1×50 mm, 1.7 μm) by linear gradient elution using water (containing 0.2% formic acid) and acetonitrile (containing 0.2% formic acid) as the mobile phase. Ten batches of PAC are selected to construct the UPLC/Q‐TOF‐MS fingerprint. Sixteen common peaks in the fingerprint are obtained, ten of which are tentatively identified, with reference to the literature data, as phellodendrine, magnoflorine, tetrahydropjatrorrhizine, menisperine, tetrahydropalmatine, jatrorrhizine, palmatine, berberine, obacunone, and limonin. Chemometric methods are also employed to evaluate the variation of herbal drugs and other closely related herbs based on the characteristics of peaks in the UPLC/Q‐TOF‐MS profiles. The developed fingerprint assay is a powerful method that may be used to conduct quality control of PAC.     

Combinational numeral fingerprint spectra of Glycyrrhiza and analysis of common peak ratio invariableness in HPLC

To overcome the instability of traditional Chinese medicine (TCM) fingerprint spectra (FPS) and to build up absolute identification standard of TCM, the construction method of combinational numeral fingerprint spectra (CNFPS) was set up. The analysis of invariableness based on CNFPS was carried out. It can be used as absolute quantitative standard to identify the difference of TCM samples. According to this method, the HPLC FPS of components extracted from Glycyrrhiza root samples by ethanol was analyzed. Stable results and accurate quality evaluation were obtained perfectly. The common peak ratio invariableness is an absolute standard in identifying TCM samples, which is better than the similarity method of the FPS.     

黄芪药材的指纹图谱研究方法的建立

用反相高效液相色谱－紫外－质谱联用技术对黄芪药材进行指纹图谱研究，为阐明不同产地药材的异同性，建立黄芪质量的国际统一标准奠定基础，黄芪的总提取物各类成分得到很好分离，紫外和质谱两种检测器可对不同特性化合物的检测进行互补，获得相对充分的指纹图谱信息。     

Fingerprint Analysis of Flos Carthami by Pressurized CEC and LC

A fingerprint analysis of Flos Carthami was performed using a standardized capillary electrochromatography (CEC) procedure. This procedure was first used to establish the electrochromatographic profile of genuine Flos Carthami from Tacheng, Xinjiang, China. This Flos Carthami fingerprint was then used to identify and assess the consistency of raw herbs from different sources in China. The study of a limited number of samples from ten different sources demonstrated a reasonable consistency among their CEC fingerprints relative to that of the genuine sample. Using this technique, we can readily distinguish the fingerprint of Flos Carthami from that of Stigma Croci, a possible substitute in traditional Chinese medicine, and Flos Hemerocallis, a commercial adulterant. A method based on high performance liquid chromatography (HPLC) is described to establish fingerprints of Flos Carthami simultaneously. The fingerprints obtained with HPLC consist of 21 common peaks within 65 min while 43 common peaks obtained with CEC. CEC showed better performance on fingerprinting of hydroxysaffloryellow A and its neighboring peaks, which contained more chemical information than that of HPLC. It was proven that CEC could be a feasible and effective method for development of fingerprint of TCM based on the comparison with HPLC.     

Development of high-performance liquid chromatographic fingerprint for the quality control of Rheum tanguticum Maxim. ex Balf

A novel, accurate and valid fingerprint method was developed by using high-performance liquid chromatography-photodiode array detection (HPLC-DAD) to control the quality of Rheum tanguticum Maxim. ex Balf. By comprehensively comparing chromatograms of all samples, our result revealed that the chromatographic fingerprint we developed combining similarity evaluation could efficiently identify and distinguish raw materials of R. tanguticum from different sources. Besides, 21 characteristic peaks in chromatogram of R. tanguticum were unambiguously confirmed by comparison of reference compounds based on their retention time (t(R)) and UV spectra.     

Fingerprint and multicomponent quantitative analysis for the quality evaluation of Sibiraea angustata leaves by HPLC-DAD and HPLC-ESI-MS/MS combined with chemometrics

Sibiraea angustata leaves, known as a traditional Tibetan medicine, have been specially used in the treatment of indigestion and obesity. In the study, a simple and sensitive high-performance liquid chromatography (HPLC) method with a diode array detector (DAD) was established to solve the problem of lacking quality standard of S. angustata leaves, including the fingerprint analysis and quantification of six characteristic components. The analytical method was validated for linearity, repeatability, stability, recovery, and specificity. Seventeen raw samples and 1 processed sample of S. angustata leaves were collected from different locations of China to establish the fingerprint. The chemometric methods, including similarity analysis (SA), principal component analysis (PCA), and hierarchical clustering analysis (HCA), were applied to distinguish the 18 batches of S. angustata samples. The results successfully sorted these samples into five clusters and kept in line with each other. According to the result of the fingerprint analysis, 21 peaks were extracted to be the common peaks and most of them were identified by mass spectrometry (MS) with electron-spray ionization (ESI) in the negative mode. Meanwhile, the loading plot of PCA further indicated that the peaks of neochlorogenic acid, chlorogenic acid, ferulic acid, rutin, hyperin, and isoquercitrin played a greater role in the discrimination among the 21 peaks. So the six components mentioned above were investigated as index constituents to evaluate the quality of S. angustata leaves from different locations. The study demonstrated that the developed new method was a beneficial approach for authentication and quality evaluation of S. angustata leaves.     

Evaluation of Chinese medicinal herbs fingerprinting by HPLC-DAD for the detection of toxic aristolochic acids

Aristolochic acids are known to contribute to various renal disorders; therefore, expanding the availability of analytical methodology to detect these compounds is important in order to assess the quality of Chinese herbal medicines in which they can be found. Twelve medicinal herbal samples were procured from various sources and extracted in duplicate prior to a "fingerprint" analysis using conventional HPLC-DAD. Multivariate analysis was performed on the entire chromatographed fingerprints. The resulting output was a partial least-square discriminant analysis model, which was able to evaluate the potential presence of aristolochic acids I and II as well as providing an individual herbal "fingerprint". The results of this study provide evidence that the presence of aristolochic acids contained within certain herbal extractions could be detected using a simple method, although some limitations apply to this method for quality control, since newly detected samples for aristolochic acid (positives) will need further confirmation with purity checks or MS hyphenation.     

Improved quality assessment of proprietary Chinese medicines based on multi‐chemical class fingerprinting

Our present study constitutes the first successful attempt to employ eight distinctive chemical groups of compounds for the quality evaluation of a complex traditional Chinese herbal medicine (TCHM) material: BaZhen YiMu (BZYM). Due to the complexity of its matrix, which is composed of nine different herbal ingredients, five representative chemical groups encompassing representative bioactive markers were initially chosen as targets for the quality assessment of this preparation. Furthermore, with the aid of LC‐ESI‐MS, three additional chemical groups were characterized. In summary, a total of nineteen markers belonging to eight different chemical groups were selectively displayed in the chromatographic fingerprint of BZYM preparation. With this fingerprint, the overall quality of any BZYM preparation can be comprehensively authenticated. The chromatographic separation was performed on an HP C₁₈ AQ column with a gradient elution of ACN and aqueous solution containing 0.1% phosphoric acid at the optimal detection wavelength of 230 nm. The established method was rigorously validated with respect to the ICH guidelines and represents the most extensive and facile HPLC quality control technique for this formulation. Compared with the conventional method of using a single or only a few markers of the same chemical group, this technique provides a new dimension for TCHM quality control.     

Fingerprint analysis of Ginkgo biloba leaves and related health foods by high-performance liquid chromatography/electrospray ionization-mass spectrometry

A fingerprint analysis method was developed for Ginkgo biloba leaves and was successfully used for quality evaluation of related health foods by HPLC with electrospray ionization MS. Fifteen samples of G. biloba leaves, which were collected from 15 different locations in China, were analyzed and identified in this study. By both peak analysis and similarity analysis of the fingerprint chromatograms, variation of constituents was easily observed in the leaves from different sources. By comparison with batches of authentic leaves, the authenticity, and quality consistency of related health foods in different matrixes were effectively estimated. It is important to mention that studying a wide range of authentic leaves from various habitats made the quality evaluation of commercial products more convincing and reasonable. The fingerprint-based strategy of the developed method should provide improved QC of G. biloba leaves and products.     

Quality assessment of radix salviae miltiorrhizae

This paper describes an improved quality assessment method for Radix Salviae Miltiorrhizae (Root of Salvia miltiorrhiza BGE.) which was established using chromatographic fingerprinting and quantification of multiple marker compounds in the crude drug. High-performance thin-layer chromatography (HPTLC) fingerprinting of water-soluble phenolics and nonpolar tanshinones was performed separately and the authentication of Radix Salviae Miltiorrhizae was achieved by comparing the fingerprints of the samples with those of the reference crude drug and by comparing the Rf values of the bands in TLC fingerprints with those of reference compounds. HPLC fingerprints were obtained by simultaneous separation of phenolics and diterpenoids in Radix Salviae Miltiorrhizae. The HPLC fingerprints of seven batches of samples from different regions of China showed similar chromatographic patterns, and seven peaks were selected as characteristic peaks. The relative retention time of these characteristic peaks in the HPLC fingerprints was established as an important parameter for the identification of this herbal medicine. The pharmacologically active marker compounds salvianolic acid B, rosmarinic acid, and tanshinone IIA in herbal medicine were quantitatively determined using reverse-phase HPLC techniques. The HPLC quantitation methods of the three marker compounds were validated and the measurement uncertainty, which is important for setting the proposed content limit of the marker compounds in herbal medicine, were further evaluated.