Chapter

Applications of High Performance Liquid Chromatography in the Analysis of Herbal Products

Authors:
  • Liverpool John Moores University & Palacký University
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Abstract

High performance liquid chromatography (HPLC), also known as high pressure liquid chromatography, is one of the most popular, modern, powerful, and versatile chromatographic separation techniques that have been routinely used to separate the components in a mixture (e.g., an herbal extract or product), to identify each component (or at least as many components as possible), to quantify separated components, and to obtain the chemical profile or fingerprint of a crude mixture. This chapter focuses on the applications of HPLC techniques in the analysis of various herbal products, and it will present several specific examples of protocols of such analyses. A brief overview of available HPLC techniques and methods will also be presented.

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... Chemometrics is not a single tool, but a wide variety of methods including basic statistics, signal processing, factorial design, calibration, curve fitting, factor analysis, detection, pattern recognition, and neural network (Kumar et al., 2014;Sarker and Nahar, 2015). The term chemometrics was first introduced by the Swedish scientist Svante Wold in 1971, and was simply shown as a collection of applications of mathematical and statistical techniques to retrieve more information from the chromatographic data. ...
... The term chemometrics was first introduced by the Swedish scientist Svante Wold in 1971, and was simply shown as a collection of applications of mathematical and statistical techniques to retrieve more information from the chromatographic data. This term has been officially defined as the science of relating measurements made on a chemical system or process to the state of the system via application of mathematical or statistical methods (Sharaf et al., 1986;Massart et al., 1988;Kumar et al., 2014;Sarker and Nahar, 2015). ...
... Chemometrics can be classified into two main categories: pattern recognition methods (unsupervised and supervised) when a qualitative evaluation is considered, and multivariate calibration for quantitative purposes (Brenton, 2003). Design of experiment, data pre-processing, classification, and calibration are the main practical steps involved in any chemometrics analysis (Sarker and Nahar, 2015). Experimental design primarily screens factors that are important for the success of a process. ...
... High-performance liquid chromatography or high-pressure liquid chromatography (HPLC), is one of the most popular, modern, powerful and versatile chromatographic separation techniques that have been routinely used to separate, identify and quantify components from complex mixtures, e.g. an herbal extracts or products, and to obtain chemical profiles or fingerprints of crude mixtures 15,16 . In a standard HPLC operation, a compact column is usually 2.0-4.6 mm in diameter, and 20-250 mm in length, packed with a stationary phase, e.g. ...
... MS or nuclear magnetic resonance (NMR) spectrometry, has now become quite common, and this hyphenation offers enhanced capability of separating and solving structural problems of complex natural products. 15 15, 16 The quality of MS response invariably F I G U R E 1 Cannabinoids in different matrices analysed by HPLC and UPLC (or UHPLC) depends on a number of factors, e.g. nature of the compounds to be analysed, the solvent and buffer used as the mobile phase, the flow rate, and, of course, the type of interface used, and thus, often creates difficulties in relation to reproducibility of information. ...
... [23][24][25][26][27][28][29][30][31][32][33]35,36 While the UV or UV-PDA-based detection is quite common, ESI-MS/MS detection has been increasingly becoming the method of choice for cannabinoids analysis from C. sativa plant crude extracts by HPLC or UPLC. In an HPLC-UV based method, a 50 mm long monolithic column of C 18 packing with an internal diameter of 4.6 mm, and particle size of 5 μm has recently been used with a linear ACN-water gradient (flow rate: 2 mL/min) to determine THC (12) (at 210 nm) in the plant extract obtained (yield: >26%) by the supercritical extraction method at different pressures (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33), temperature (40-80 C) and ethanol (EtOH) as a co-solvent (0-5%) 23 . ...
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... Chemometrics can be classified into two main categories: pattern recognition methods (unsupervised and supervised) when a qualitative evaluation is considered, and multivariate calibration for quantitative purposes (Brenton, 2003). Design of experiment, data pre-processing, classification, and calibration are the main practical steps involved in any chemometrics analysis (Sarker and Nahar, 2015). Experimental design primarily screens factors that are important for the success of a process. ...
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... HPLC has been used over the years as a separation technique to separate constituents of herbal extracts, for identification and quantification of compounds and establishing the fingerprint profiles of mixtures and crude extracts. Various detection tools such as UV, DAD, MS, FLD are coupled to the HPLC in performing its function efficiently (Sarker and Nahar, 2015). ...
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Bozhougyiqi-Tang (BZYQT) is of traditional herbal medicine used for enhancement of digestive capacity. An accurate and reliable simultaneous determination using a HPLC-DAD and ESI-MS was developed and validated for the qualitative and quantitative analysis of 9 major compounds, ferulic acid (1), naringin (2), hesperidin (3), decursinol (4), glycyrrhizin (5), saikosaponin A (6), 6-gingerol (7), ginsenoside Rg3 (8), decursin (9), in traditional herbal medicine 'Bozhougyiqi-Tang.' The chromatographic separation of 9 compounds was performed on a SHISEIDO C18 column (250 mm × 4.6 mm i.d., S-5 μm) using gradient elution with 0.1% trifluoroacetic acid and acetonitrile at a flow rate of 1.0 ml/min. The 9 compounds were identified based on peak retention time and UV spectrum and MS data of these compounds. This developed method showed good linearity (R(2) > 0.999). The LOD and LOQ of the major compounds were less than 0.09 and 0.28 μg/ml, respectively. The intra - day and inter - day RSD values were within 2.06% and 1.64%, respectively. The mean recoveries were from 92.10% to 108.56% with less than 1.88%. The results indicated that established method had good precision and accuracy. The new method was successfully applied to the simultaneous analysis of 9 compounds in Bozhougyiqi-Tangs samples.
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Curculiginis Rhizoma (Curculigo orchioides GAERTN.) is a well-known Chinese herbal medicine, as well as an important Rasayana drug in India. Current criteria of quality control on this herb are to quantitatively analyze single compound curculigoside, which fail to comprehensively evaluate quality of this herb. In this paper, a simple and reliable HPLC coupled with diode array detector (DAD) method was developed to evaluate the quality of Curculiginis Rhizoma through establishing chromatographic fingerprint and simultaneously quantitating four phenolic compounds, orcinol glucoside, orcinol, 2,6-dimethoxybenzoic acid and curculigoside. The fingerprint displayed eleven common peaks, and the similarity index of different samples was in a range of 0.890-0.977. Validation of the method was acceptable, with 96.03-102.82% accuracy in recovery test and inter and intra-day precisions were less than 2%. This developed method by having a combination of chromatographic fingerprint and quantitation analysis could be applied to the quality control of Curculiginis Rhizoma.
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The SuoQuan formulae containing Fructus Alpiniae Oxyphyllae has been used to combat the urinary incontinence symptoms including frequency, urgency and nocturia for hundreds of years in China. However, the chemical information was not well characterized. The quality control marker constituent only focused on one single compound in the current Chinese Pharmacopeia. Hence it is prudent to identify and quantify the main constituents in this herbal product. This study aimed to analyze the main constituents using ultra-fast performance liquid chromatography coupled to tandem mass spectrometry (UFLC-MS/MS). Fourteen phytochemicals originated from five chemical classes constituents were identified by comparing the molecular mass, fragmentation pattern and retention time with those of the reference standards. A newly developed UFLC-MS/MS was validated demonstrating that the new assay was valid, reproducible and reliable. This method was successfully applied to simultaneously quantify the fourteen phytochemicals. Notably, the content of these constituents showed significant differences in three pharmaceutical preparations. The major constituent originated from each of chemical class was isolinderalactone, norisoboldine, nootkatone, yakuchinone A and apigenin-4',7-dimethylther, respectively. The variation among these compounds was more than 1000 times. Furthermore, the significant content variation between the two different Suoquan pills was also observed. The proposed method is sensitive and reliable; hence it can be used to analyze a variety of SuoQuan formulae products produced by different pharmaceutical manufacturers.
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A simple, reliable and accurate fingerprinting method has been developed for quality control of YiGongNingXue oral liquid. The separation was done by reverse phase chromatography using Zorbax eclipse XDB C18 column (250mm×4.6mm i.d. with 5.0μm particle size) and detection at 246nm. Methanol (5-95 % in 70 min)-buffer solution (water-phosphoric acid, pH 3.0, 95-0 % in 70 min) as mobile phase for linear gradient elution. The flow rate was 1.0 mL·min-1 and the column temperature was at 25°C. The similarity of 20 batches of YGNX oral liquid was more than 90 %. Also 15 common peaks of chromatogram have been detected, ten of them were identified by comparing fingerprint chromatogram with reference substances. The HPLC fingerprint can be used to control the quality of YiGongNingXue oral liquid.
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Objective: To study the anxiolytic activity of Jessica- a polyherbal formulation, in rats. Methods and Material: Swiss albino rats of either sex weighing between 180g-210g were used. The standard anxiolytic, diazepam (1mg and 2mg/kg) standard polyherbal drug, Streswin (100 and 200mg/kg) and the test drug, Jessica (100mg and 200mg/kg) were suspended in 2% gum acacia and administered orally. In acute study the vehicle and test drug were given 40 min prior to experiment, while in chronic study they were given daily once for 10 days. The anxiolytic effect of Jessica (100 and 200mg/kg) was evaluated by using Despair swim test (DST), Elevated Plus maze (EPM), Actophotometer and Rota rod apparatus the animals were explored on the above models on 1st, 5th, 10th day of the treatment plan. Results: It was observed that Jessica (100 and 200mg/kg) has increased the immobility time in DST, increased the total no of entries and time spent in open arm in EPM, increased the percentage reduction in locomotion in Actophotometer and decreased the time spent on revolving rod in Rota rod on 5 th day and 10 th day. All the results were significant when compared to control group and the effect was similar to that of standard drug diazepam (1 and 2mg/kg) and standard polyherbal formulation Streswin (100 and 200mg/Kg). Conclusion: Jessica has exhibited anxiolytic activity comparable to diazepam and streswin. So, it can be used as a novel therapeutic strategy for anxiety.
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A simple and precise high performance liquid chromatography (HPLC) method was developed and validated for simultaneous trace analysis of the content of pharmacologically important active curcuminoids: curcumin (C), demethoxycurcumin (DMC) and bisdemethoxy curcumin (BDMC) in a novel standardized herbal preparation derived from the hexane soluble extract of Curcuma longa, tentatively assigned the generic nomenclature "herbal medicament" (HM). HPLC separation of active constituents was achieved on a Chromolith (100 x 4.6 mm, 2 μm, Merck) column using water-acetonitrile-acetic acid (60:40:1, v/v/v) as mobile phase, with UV detection at 425 nm. The method was validated for linearity, limit of detection (LOD), limit of quantification (LOQ), robustness, recovery, precision and accuracy. An empirical equation enabling calculation of the percentage content of DMC and BDMC from a calibration curve of C, without relying on reference standards of DMC and BDMC, is proposed on the basis of available evidence, suggesting that pharmacologically important curcuminoids may be accurately reported as proportions of a single constituent assayed quantitatively. Hence, quantitative estimation of trace constituents on the basis of empirical evidence for proportionate composition is suggested as a means of quantifying the key constituents in herbal preparation intended for pharmacological intervention in patients requiring treatment with HM.
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Chemical fingerprint and metabolic fingerprint of Danhong injection and its raw materials (Radix Salviae miltiorrhizae and Flos carthami) were analyzed by HPLC-UV and HPLC-MS techniques. HPLC profiles, established as their characteristic fingerprints, were employed to assess the consistency and difference between Danhong injection and its raw materials. Ten batches of Danhong injections were analyzed under the same HPLC conditions. The results showed a good similarity. To determine the major constituents of Danhong injection for quality control, 19 major chromatographic peaks were characterized. Through comparison of the HPLC profiles of Danhong injection with its raw materials, it was found that they are greatly different, which indicated the changes of major constituents in the course of preparation procedure. In addition, to explore the in vivo metabolism of the major active constituents, the rat plasma was analyzed by HPLC techniques after intravenous administration of Danhong injection at different time intervals. The results indicated that danshensu, protocatechuic aldehyde, hydroxysafflor yellow A, rosmarinic acid and salvianolic acid B might be important for biological activity of Danhong injection. It could be concluded that chemical fingerprint combined with metabolic fingerprint is a useful means of controlling the quality and clarifying the possible mechanism of Danhong injection.
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Essentials in Modern HPLC Separations discusses the role of separation in high performance liquid chromatography (HPLC). This up-to-date reference systematically covers new developments in types and characteristics of stationary phases, mobile phases, and other factors of this technique that influence separation of compounds being analyzed. The volume also considers the selection process for stationary and mobile phases in relation to the molecules being separated and examined, as well as their matrices. The book includes a section on the contemporary applications of HPLC, particularly the analysis of pharmaceutical and biological samples, food and beverages, environmental samples, and more. Discusses key parameters in HPLC separation Describes interrelation between various HPLC features (solvent pressure, separation, detection) Includes a large number of references.
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Plant medicines are gaining more and more attention all over the world. Many modern analytical approaches have been introduced to evaluate the quality of medicinal plants, and significant amount of measurement data has been produced. Therefore, the application of chemometrics in the field of medicinal plants is spontaneous and necessary. Comprehensive methods and hyphenated techniques associated with chemometrics used for extracting useful information and supplying various methods of data processing are now more and more widely used in medicinal plants. This review focuses on the applications of chemometrics in quality evaluation of medicinal plants in the authenticity, efficacy and safety evaluation.
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The efficacy and quality of commercially manufactured "Maha Varthikava watee" preparations may differ from its authentic preparation prepared as per original recipe. Therefore, a study was carried out to investigate whether the physico-chemical properties, HPLC and UV chromatographic and spectrophotometric measurements can be used for standardization of the above drug. Purified herbals used in the original recipe of "Maha Varthikava watee", were finely powdered and mixed. The mixture was ground together with three juices extracted from leaves of three different species and bee's honey to make pills. Five commercial samples purchased from the market were compared with the authentically prepared samples. Physical properties, chemical properties and chromatographic and spectrophotometric measurements were considered as tools for standardization. For the HPLC and UV spectrophotometry, ethanol extraction was used. Data were analyzed at 0.05 significant levels. No significant differences were observed in any commercial samples with regard to fiber content, acid insoluble ash, dichloromethene extract, ethyl acetate extract and methanol extract while the weight of pills was significantly different. The pH values and loss on drying were significantly different only in two commercial samples. Specific gravity and ash values were found to be different only in one sample. Hexane extract value was significantly different in one sample. In HPLC chromatograms, one main peak was more prominent in three commercial samples. In UV spectrophotometric measurement, two commercial samples had a λ max of 287-290 nm while other three samples had a λ max of 340-345 nm. Hence, these measurements can be used for the standardization of "Maha Varthikava watee".
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There are twenty-one compounds of steroidal sapogenins in Chinese Dioscorea L., the type of 3β-hydroxy sapogenin exists in most species, only a few species contain the type of 3α-hydroxy sapogenin. Fifty-nine compounds of steroidal saponin are separated from thirteen species of Chinese Dioscorea, and which can be divided into four types. Seventeen species belonging to Sect. Stenophora of Dioscorea contain steroidal sapogenin (mostly diosgenin). Based on consulting the literatures, constituents of steroidal sapogenins and steroidal saponins in different species of Dioscorea and chemical structures of these compounds are summarized and reviewed.
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John Dolan answers questions about two LC practices: column regeneration after a reversed-phase gradient has been run, and which degassing technique is best.
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The batch-to-batch quality consistency needs to be controlled to ensure the efficacy and safety of botanical drug products. Chromatographic fingerprinting is a powerful tool for characterizing complex chemical systems such as botanical drugs. This paper presented the combined use of a proposed weighting algorithm and multivariate statistical analysis on fingerprint data to achieve quality consistency evaluation of botanical drug products. A set of fingerprint data following multivariate normal distribution was simulated based on the real fingerprint data of a botanical drug product, Danshen injection. The weight of each peak was set based on its batch-to-batch peak variation. A principal component analysis (PCA) model was established, and the Hotelling T2 and DModX statistics were applied to evaluate quality consistency. The effect of weighting on the monitoring capability of the two statistics was evaluated through monitoring simulated peak area variations. The results showed that both the two statistics were more sensitive to the characteristic peaks with smaller batch-to-batch peak variations. Hence the peaks with larger variability will be permitted to have a wider tolerance range of variation, while a more narrow range of variation was seen for the peaks with smaller variability among batches. Peak weighting combined with multivariate statistical analysis has overcome some drawbacks of the fingerprint similarity analysis method, and is worthy of application and recommendation for the quality consistency evaluation of botanical drug products.
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Contudol capsule, a combination of four herbal extracts namely Boswellia serrata, Curcuma longa, Moringa oleifera and Zingiber officinale was chosen for the study. Two lots of Contudol capsule were analyzed qualitatively and quantitatively by applying various classical and instrumental methods. Along with general test parameters of capsules like average weight, filled average weight, uniformity of weight, disintegration time, blends of both the capsule lots were tested for loss on drying, total ash content, acid insoluble ash, extractive values, pH by appropriate standardized classical and instrumental methods. Active components like Boswellic acid content was found to be around 129.15 and 131.33 mg per capsule by titration method in respective lots, whereas, spectrophotometric analysis for Curcumin and titrimetric analysis of Tannin content showed 20.806 mg, 20.005 mg and 2.063 mg, 1.883 mg per capsule respectively in corresponding lots. Presence of gingerol was well illustrated by modern sophisticated HPTLC technique, where, in Diethyl ether: Hexane (7:3) system, after spraying with VSR, resembling blue colored spots were observed at Rf about 0.20, 0.40 and 0.70 in reference standard, extract powder and blend of the capsule. Heavy metals like Arsenic, Mercury, Lead and Cadmium were found to be at the ppm level within the limits set by AYUSH, by Inductively Coupled Plasma spectrometry. Microbiological testing was carried out as per the USP/ BP guidelines and findings were complying with the pharmacopoeial specified values. In this way, by considering these entire test parameters one can very well establish and maintain the quality of polyherbal formulations.
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Embelia ribes burm f., also known as Vidanga, is one of the oldest herbs in Indian traditional medicine. It is used mainly as an anthelmintic, carminative and stimulants. A selective, precise and accurate High Performance Liquid Chromatography (HPLC) method has been developed for the quantification of Embelin in Vidanga churna formulations. The chromatographic separation was performed on Chromatopak Peerless basic C 18 column (250 mm L x 4.6 mm ID Column; packing size-5µ) with a mobile phase Methanol: Phosphate Buffer pH 3.0 (adjusted with 5% v/v acetic acid) in 90:10 proportion at flow rate was 1.4 ml/min. Densitometric analysis was carried out in the absorbance mode at 291 nm. Developed HPLC method showed good regression (r 2 = 0.9988 ± 0.0012) and the recovery of Embelin was in the range of 99.6 – 102.2%. The limit of detection and limit of quantitation were found to be 1.94 µg/ml and 5.891µg/ml respectively. The method was validated for precision, recovery, limit of detection and limit of quantitation as per ICH guidelines. The proposed HPLC method was found to be simple, precised and accurate and can be used for the quantitative and chemical fingerprint analysis of Vidanga.
Article
The qualitative and quantitative analyses by HPLC and LC-DAD-MS/MS were conducted for the quality consistency evaluation of Compound Kushen Injection (CKI). For fingerprint analysis, 8 peaks were selected as the characteristic peaks in the HPLC chromatograms of 27 different batches of CKI samples and their similarities were more than 0.90, except the four samples. For the typical HPLC profile, peak identification was carried out by HPLC and LC-MS/MS analyses and as a result, 21 chromatographic peaks were identified or characterized based on the comparison of retention time and UV spectra with authentic compounds as well as by summarized MS fragmentation rules. Six of these identified compounds, including oxymatrine, oxysophocarpine, N-methylcytisine, matrine, sophocarpine, and trifolirhizin, were simultaneously determined and the results showed that the total amounts of six marker compounds were variable, to some extent, which was deduced to be related to the quality of the crude materials and the manufacturing process employed. Supplemental materials are available for this article. Go to the publisher's online edition of the Journal of Liquid Chromatography & Related Technologies to view the supplemental file.
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Sample preparation is a crucial bottleneck in the whole analytical process. Solid-phase sorbents (SPSs) have aroused increasing interest in research on sample preparation, as they have key roles in obtaining high clean-up and enrichment efficiency in the analysis of trace targets present in complex matrices. The objective of this review is to provide a broad overview of the recent advances and applications of SPSs in sample preparation prior to chromatographic analysis, during the period 2008–13. We include SPSs, such as molecularly-imprinted polymers, carbon nanomaterials, metallic nanoparticles and metal organic frameworks, focusing on solid-phase extraction, solid-phase microextraction, matrix solid-phase dispersion and stir-bar sorptive extraction of typical pollutants in environmental, biological, food and pharmaceutical samples. We propose remaining challenges and future perspectives to improve development of new SPSs and to apply them further.
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This study was designed for simultaneous determination of 13 chemical marker compounds, namely, protocatechuic acid, chlorogenic acid, caffeic acid, liquiritin, hesperidin, apigetrin, rosmarinic acid, oxypeucedanin hydrate, byakangelicin, apigenin, glycyrrhizin, nobiletin, and 6-gingerol in Gwakhyangjeonggi-san (GJS: Huoxiang-zhengqi-san in Chinese). A quantitative analytical method was developed based on HPLC-PDA with validation in terms of precision, accuracy, and repeatability, and successfully employed for quality evaluation of GJS samples with the help of chemometric techniques such as principal component analysis (PCA) and hierarchical clustering analysis (HCA). The correlation coefficient for the linear regression was > 0.9994. The intra-day and inter-day precision was < 3.0% of the relative standard deviation (RSD) value, and the recovery was in the range 92.5-107.0%, with RSD values < 4.0%, and the repeatability was < 3.0% of RSD. Variations in the quantity were observed in GJS products from different origins, which were classified by PCA and HCA. The quantitative and chemometric analyses indicate the necessity for consistency in GJS production for the purpose of quality control.
Article
Sample preparation is still considered as the bottleneck of the whole analytical procedure, and efforts has been conducted towards the automation, improvement of sensitivity and accuracy, and low comsuption of organic solvents. Development of online sample preparation techniques (SP) coupled with liquid chromatography (LC) is a promising way to achieve these goals, which has attracted great attention. This article reviews the recent advances on the online SP-LC techniques. Various online SP techniques have been described and summarized, including solid-phase-based extraction, liquid-phase-based extraction assisted with membrane, microwave assisted extraction, ultrasonic assisted extraction, accelerated solvent extraction and supercritical fluids extraction. Specially, the coupling approaches of online SP-LC systems and the corresponding interfaces have been discussed and reviewed in detail, such as online injector, autosampler combined with transport unit, desorption chamber and column switching. Typical applications of the online SP-LC techniques have been summarized. Then the problems and expected trends in this field are attempted to be discussed and proposed in order to encourage the further development of online SP-LC techniques.
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In this study, a fingerprint of steroid saponins, the major bioactive constituents in the crude extracts from Dioscorea zingiberensis C. H. Wright (DZW), has been established for the first time by combined use of the following two methods: high-performance liquid chromatography coupled with evaporative light scattering detector (HPLC-ELSD) and the simultaneous characterization of the steroid saponins by high-performance liquid chromatography coupled with electrospray ionization-mass spectrometry and quadrupole tandem time-of-fight mass analyzers detection (HPLC-ESI-Q/TOF). All HPLC analyses were carried out on a Welchrom C18 column (250mm×4.6mm I.D., 5μm) with a mobile phase composed of water and acetonitrile under gradient elution. There were 68 common characteristic peaks in the fingerprints, in which 12 of them were confirmed by comparing their mass spectra and retention times with those of the reference compounds. In order to identify other unknown peaks, their fragmentation behaviors characteristic of the major groups of steroid saponins from DZW with six types of aglycone skeletons were discussed in detail, and possible MS/MS fragmentation pathways were proposed for aiding the structural identification of these components. According to the summarized fragmentation patterns, these peaks were tentatively assigned by matching their empirical molecular formula with those of the published compounds, or by elucidating their quasi-molecular ions and fragment ions referring to available literature information when the reference standards were unavailable. As a result, 22 new steroid saponins were found in DZW for the first time. In addition, the quantitative analysis of the nine (except for the reference compounds A, B, and C) known peaks was accomplished at the same time which indicated that there was a great variability in the amount of these active compounds in different batches in the crude extracts. This approach could demonstrate that the fingerprint could be considered to be a suitable tool to comprehensively improve the quality control of DZW. The identification and structural elucidation of the peaks in the fingerprint may provide important experimental data for further pharmacological and clinical researches.
Article
Rheumatoid arthritis is historically linked with disability and no cure for the disease has been found. This has encouraged a renaissance of traditional medicine for the management of this condition in Mauritius. There are a number of traditional herbal medicines for the treatment of rheumatoid arthritis. This study aimed to determine the antioxidant and anti-inflammatory activities, as well as the phytocomposition, of the locally available polyherbal formulations used in the management of rheumatoid arthritis; particularly as no previous studies have reported on their biological activities. Flavonoids and proanthocyanidins were the predominant phenolic compounds identified in the four selected herbal formulations (A, B, C and D) and two elixirs (Harpago and Hemidesmus). The Trolox equivalent antioxidant capacity and hypochlorous acid assays showed scavenging potentials of 0.20 to 0.49 mmol trolox equivalent/g and IC50 values ranging from 0.03 to 0.37 mg/mL, respectively. Similarly, from the herbal formulations, formulation C had the highest reducing power (0.55 ± 0.02 mmol Fe(II)/g), while formulation B was a potent iron (II) ion chelator. The herbal formulations also showed anti-inflammatory activity in a dose-dependent manner, with formulation B exhibiting a calculated IC50 value of 0.30 ± 0.01 mg/mL. Hemidesmus was a more potent inhibitor of xanthine oxidase than Harpago, with an IC50 value of 0.30 ± 0.04 μL/mL. Thus, the xanthine oxidase inhibitory and antioxidant effects of the herbal formulations may partially explain their general anti-inflammatory properties, corresponding to their use in the treatment of rheumatoid arthritis in Mauritian folk medicine.
Article
Objective To establish a validated high performance liquid chromatography (HPLC) for the fingerprint analysis of Codonopsis Radix and for the determination of lobetyolin. Methods HPLC coupled with diode array detection method was employed to establish the fingerprint profile and quantitative determination of lobetyolin in Codonopsis Radix. Principal component analysis method was employed to analyze the 52 Codonopsis Radix samples. Results The reference chromatogram was generated with 25 common peaks showing good separation from adjacent peaks. Conclusion Statistical analysis of the obtained data demonstrates that the developed HPLC fingerprint combined with chemometric is a reliable method for the similar evaluation and quality assessment of Codonopsis Radix, and other traditional Chinese herbs.
Article
Wu-tou decoction (WTD), a traditional Chinese medicine (TCM) formula, is composed of Aconiti Radix Cocta, Ephedrae Herba, Paeoniae Radix Alba, Astragali Radix and Glycyrrhiza Radix Preparata, and it has been used for more than a thousand years to treat rheumatic arthritis, rheumatoid arthritis and pain of joints, while the active constitutions of WTD are unclear. In this research, an ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) method in both positive and negative ion mode was established to investigate the major constitutions in WTD. A Waters ACQUITY UPLC BEH C18 column was used to separate the aqueous extract of WTD. Acetonitrile and 0.1% aqueous formic acid (v/v) were used as the mobile phase. 74 components including alkaloids, monoterpene glycosides, triterpene saponins, flavones and flavone glycosides were identified or tentatively characterized in WTD based on the accurate mass within 15ppm error and tandem MS behavior. All the constitutions were also detected in the corresponding individual herbs. These results will provide a basis for further study in vivo of WTD and the information of potential new drug structure for treating rheumatic arthritis and rheumatoid arthritis.
Article
Herbal medicines are becoming again more popular in the developed countries because being "natural" and people thus often assume that they are inherently safe. Herbs have also been used worldwide for many centuries in the traditional medicines. The concern of their safety and efficacy has grown since increasing western interest. Herbal materials and their extracts are very complex, often including hundreds of compounds. A thorough understanding of their chemical composition is essential for conducting a safety risk assessment. However, herbal material can show considerable variability. The chemical constituents and their amounts in a herb can be different, due to growing conditions, such as climate and soil, the drying process, the harvest season, etc. Among the analytical methods, chromatographic fingerprinting has been recommended as a potential and reliable methodology for the identification and quality control of herbal medicines. Identification is needed to avoid fraud and adulteration. Currently, analyzing chromatographic herbal fingerprint data sets has become one of the most applied tools in quality assessment of herbal materials. Mostly, the entire chromatographic profiles are used to identify or to evaluate the quality of the herbs investigated. Occasionally only a limited number of compounds are considered. One approach to the safety risk assessment is to determine whether the herbal material is substantially equivalent to that which is either readily consumed in the diet, has a history of application or has earlier been commercialized i.e. to what is considered as reference material. In order to help determining substantial equivalence using fingerprint approaches, a quantitative measurement of similarity is required. In this paper, different (dis)similarity approaches, such as (dis)similarity metrics or exploratory analysis approaches applied on herbal medicinal fingerprints, are discussed and illustrated with several case studies.
Article
Context: Ginger, the fresh rhizome of Zingiber officinale Rosc. (Zingiberaceae), has been used worldwide; however, for a long time, there has been no standard approbated internationally for its quality control. Objective: To establish an efficacious and combinational method and pattern recognition technique for quality control of ginger. Methods: A simple, accurate and reliable method based on high-performance liquid chromatography with photodiode array (HPLC-PDA) detection was developed for establishing the chemical fingerprints of 10 batches of ginger from different markets in China. The method was validated in terms of precision, reproducibility and stability; and the relative standard deviations were all less than 1.57%. On the basis of this method, the fingerprints of 10 batches of ginger samples were obtained, which showed 16 common peaks. Coupled with similarity evaluation software, the similarities between each fingerprint of the sample and the simulative mean chromatogram were in the range of 0.998-1.000. Then, the chemometric techniques, including similarity analysis, hierarchical clustering analysis and principal component analysis were applied to classify the ginger samples. Results and conclusion: Consistent results were obtained to show that ginger samples could be successfully classified into two groups. This study revealed that HPLC-PDA method was simple, sensitive and reliable for fingerprint analysis, and moreover, for pattern recognition and quality control of ginger.
Article
This study aims to identify and discriminate between two commonly confused traditional Chinese medicines, Epimedium wushanense and Epimedium koreanum, using pattern recognition aided fingerprint analysis of their secondary metabolites. Samples of the two species were collected during different stages of their growth period. The HPLC generated chromatographic data were analyzed using principal components analysis (PCA) and hierarchical cluster analysis (HCA). Two major clusters were formed, each consisting of a single species. The entire dataset was then divided into two: a training set and a test set. Supervised pattern recognition techniques, soft independent modeling by class analogy (SIMCA) and back propagation artificial neural network (BP-ANN), were performed. SIMCA failed to predict one sample, whereas BP-ANN precisely predicted the whole test set. In conclusion, fingerprint analysis assisted by pattern recognition techniques is a potential strategy for the authentication and differentiation of species used in herbal medicines. (C) 2011 Elsevier Ltd. All rights reserved.
Book
During the past three decades, chemometrics has emerged as an important sub-discipline of analytical chemistry. This book provides a practical application-oriented introduction to the univariate and multivariate methods that are widely used in chemometrics. Multivariate methods and procedures are introduced without a heavy emphasis on their theoretical background. Rather, the aim of the book is to illustrate these methods through practical examples in a style that makes the material accessible to a broad audience of non experts.
Article
Huang-Lian-Jie-Du-Tang (HLJDT), comprising Coptidis Rhizoma, Scutellariae Radix, Phellodendri Cortex and Gardeniae Fructus, is one of the commonly used Chinese medicine formulas for clearing heat and detoxifying. Quality control of the herbal complex like Chinese medicine formulas still remains a challenge. The successful approval of botanical drug Veregen by FDA indicated the importance of quantitative analysis in quality control of herbal medicines. In this study, an effective quantitative method based on conventional HPLC-DAD was developed for simultaneous determination of fourteen major ingredients (seven alkaloids, four flavonoids, three terpenes) in HLJDT. The established method was well validated in terms of linearity, sensitivity, precision, accuracy and stability and then successfully applied to quality evaluation of commercial HLJDT samples. The developed method can quantitatively determine up to 70% of the chemicals of commercial HLJDT sample and effectively revealed the significant variation in the quality of the commercial HLJDT samples collected from different locations.
Article
Context: Coptidis Rhizoma-Euodiae Fructus couple (CEC) is a classic traditional Chinese medicine preparation consisting of Coptidis Rhizoma and Euodiae Fructus at the ratio of 6:1, and used to treat gastro-intestinal disorders. Alkaloids are the main bioactive component. This research provides comprehensive analysis information for the quality control of CEC. Objective: To develop a high-performance liquid chromatography-diode array detection fingerprint for chemical composition characteristics of CEC and its products. Materials and methods: The samples were separated with a Gemini C18 column by using gradient elution with water-formic acid (100:0.03) and acetonitrile as mobile phase. Flow rate was 1.0 mL/min and detection wavelength was 250 nm. Similarity analysis and principal component analysis (PCA) were employed to evaluate quality consistencies of analytes. Mean chromatograms and correlation coefficients of analytes were calculated by the software "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine". Results: Fingerprint chromatogram comparison determined 20 representative general fingerprint peaks, and the fingerprint chromatogram resemblances are all better than 0.988. Consistent results were obtained to show that CEC and its related samples could be successfully divided into three groups. Contribution plots generated by PCA were performed to interpret differences among the sample groups while peaks which significantly contributed to classification were identified. Seven bioactive constituents in the samples were verified by quantitative analysis. Discussion and conclusion: The chromatographic fingerprint with similarity evaluation and PCA assay combined with quantification of seven compounds could be utilized as a quality control method for the herbal couple.
Article
For standardization of natural product drugs, single chemical entities, “marker compounds,” may be used as potency standards in high performance liquid chromatography (HPLC) analysis. Using well-characterized marker compounds, conventional pharmaceutical manufacturing criteria for assay and content uniformity may be applied. These marker compounds may be used to help identify herbal materials, set specifications for raw materials, standardize botanical preparations during all aspects of manufacturing processes, and obtain stability profiles. HPLC analysis for marker compounds may provide additional information in the form of “chromatographic fingerprints.” Examples discussed include valerian (Valeriana officinalis) and feverfew (Tanacetum parthenium). The benefits and limitations of using markers are discussed. An intent of this paper is to illustrate how, in certain instances, chromatographic fingerprinting and markers can by used to make consistent herbal drug products.
Article
This chapter provides the novice and the experienced analyst with an overview of sample preparation techniques focusing on solid dosage forms. It describes the “best practices” in the “dilute and shoot” approach, and the “tricks of the trade” in grinding, mixing, sonication, dilution and filtration of drug products. Selected case studies of sample preparations for assays and impurity testing are used to illustrate the strategies, trade-offs and potential pitfalls encountered during method development. Future trends in sample preparation are also discussed.
Article
Immunol, a polyherbal formulation was evaluated for its safety and effect on the immune system of rats. The rats were administered a single oral dose of 20 mL kg-1 b.wt. while others were given repeated doses of 1, 5 and 10 mL kg-1 b.wt., orally for 28 days. Immunol treatment did not elicit any abnormalities on the body weight gain, food consumption, neurological assessment, hematology and clinical chemistry parameters, organ weights, gross organ pathology and histopathology and humoral immunity, but it enhanced the cell mediated immunity. It was concluded that Immunol was safe at single oral dose of 20 mL kg-1 b.wt. and repeated dose of 10 mL kg-1 b.wt. in rats for 28 days.