HPTLC analysis of Withaferine A from an herbal extract and polyherbal formulations

Withania somnifera has been used in Ayurvedic medicine for treatment of depression and inflammation, and as an aphrodisiac. It contains many phytochemicals such as Withaferine A, withanine, anahygrine, tropine, and withanolides. Of these, withaferine A is considered to be the most active compound. Withaferine A was estimated in herbal extract and polyherbal formulations by high performance thin layer chromatography (HPTLC). As there is no official HPTLC protocol for quantitation of the above phytochemicals, an attempt was made to quantify withaferine A in herbal extract and polyherbal formulations produced from Withania somnifera. Precoated silica gel G (aluminium backed) plates were used as stationary phase and toluene:ethyl acetate: formic acid (50 : 15 : 5) was used as mobile phase. Detection and quantification were performed by densitometry at λ 213 nm. The linear range was 1 μg to 3 μg. This HPTLC method was found to be reproducible, accurate, and precise.     

Comparing chemical fingerprints of herbal medicines using modified window target-testing factor analysis

A chromatographic fingerprint of a herbal medicine is essentially its chromatographic spectrum: a characteristic representation of its chemical components, some of which are pharmacologically active. Since a wide variety of factors, such as the geographical location, the harvest season, and the part used can influence the chemical constituents (and therefore the pharmacological activity) of any particular herbal medicine and its products, these fingerprints provide a way to compare and contrast the compositions of different variants of the same herbal medicine. In particular, it is possible to ascertain whether particular components present in one herbal fingerprint are also present in another fingerprint. In this work we use a novel method—modified window target-testing factor analysis (MWTTFA), based on the use of target factor analysis (TFA), fixed-size moving window evolving factor analysis (FSMWEFA) and a Gaussian shape correction to the chromatographic profiles—to achieve this end. To demostrate the strategy, the fingerprints of samples from garlics produced in different geographical locations were compared, as well as the fingerprints of samples taken from above-ground and below-ground parts of Houttuynia cordata Thunb. The results from these comparisons clearly show that four chemical components present in Hunan common edible garlic are absent in Xingping base garlic, while seven components are present in Xingping base garlic but absent in Hunan common edible garlic. Also, eleven components are present in the sample from the above-ground part of Houttuynia cordata Thunb but not in the sample from the below-ground part, while seven components are present in the sample from the below-ground part of Houttuynia cordata Thunb that are not present in the sample from the above-ground part. These interesting conclusions should be very useful for future pharmacological and clinical research into these herbal medicines, and the novel MWTTFA technique can also be used for quality control purposes.     

Chromatographic separation techniques and data handling methods for herbal fingerprints: a review

As herbal medicines have an important position in health care systems worldwide, their current assessment and quality control are a major bottleneck. Over the past decade, major steps were taken not only to improve the quality of the herbal products but also to develop analytical methods ensuring their quality. Nowadays, chromatographic fingerprinting is the generally accepted technique for the assessment and quality control of herbal products. This paper briefly considers the evolution of the regulations and guidelines on the quality control of herbal medicines, and reviews the established analytical techniques for herbal fingerprinting with an emphasis on the most recent developments, such as miniaturized techniques, new stationary phases, analysis at high temperatures and multi-dimensional chromatography. Accessory to the new analytical techniques, the chemometric data handling techniques applied are discussed. Chemometrics provide scientists with useful tools in understanding the huge amounts of data generated by the analytical advances and prove to be valuable for quality control, classification and modelling of, and discrimination between herbal fingerprints.     

Field-induced wooden-tip electrospray ionization mass spectrometry for high-throughput analysis of herbal medicines

This study demonstrates the first application of field-induced wooden-tip electrospray ionization (ESI) mass spectrometry (MS) for high-throughput analysis of herbal medicines. By application of an opposite and sample-contactless high voltage on the MS inlet rather than wooden tips, a high-throughput analysis device is easily set up, and a relatively fast analysis speed of 6 s per sample was successfully achieved. In addition, fast polarity switching between positive and negative ion detection mode is readily accomplished, which provides more complete chemical information for quality assessment and control of herbal medicines. By using the proposed method, various active ingredients present in different herbal medicines were rapidly detected, and the obtained mass spectra were served as the samples' fingerprints for tracing the origins, establishing the authenticity, and assessing the quality consistency and stability of herbal medicines. Our experimental results demonstrated that field-induced wooden-tip ESI-MS is a desirable method for high-throughput analysis of herbal medicines, with promising prospects for rapidly differentiating the origin, determining the authenticity, and assessing the overall quality of pharmaceuticals.     

Établissement du profil chromatographique liquide non aqueux des métabolites phytochimiques apolaires des phytomédicaments

Chromatographic profiling of plant metabolites is therefore a good tool for quality control of such herbal medicinal products. Our objective was to propose a protocol for sample preparation and liquid chromatographic profiling of non-polar metabolites for quality assessment of African herbal medicinal products. The methodology is based on the chemometric assessment of liquid chromatographic profiles of non-polar metabolites issued from several batches of leaves of Combretum micranthum and Mitracarpus scaber. Metabolic profiling is carried out by non-aqueous liquid chromatography on porous carbon graphite, coupled with mass spectrometry, after extraction with dichloromethane and removal of chlorophyll. Our method using liquid chromatography, coupled to mass spectrometry can detect non-polar metabolites already identified in the two herbal drugs. Chemometric data analysis of chromatographic profiles using the PLS-discriminant analysis with or without orthogonal signal correction, allowed a distinction between the two herbal drugs.     

Direct analysis of herbal powders by pipette-tip electrospray ionization mass spectrometry

Conventional electrospray ionization mass spectrometry (ESI-MS) is widely used for analysis of solution samples. The development of solid-substrate ESI-MS allows direct ionization analysis of bulky solid samples. In this study, we developed pipette-tip ESI-MS, a technique that combines pipette tips with syringe and syringe pump, for direct analysis of herbal powders, another common form of samples. We demonstrated that various herbal powder samples, including herbal medicines and food samples, could be readily online extracted and analyzed using this technique. Various powder samples, such as Rhizoma coptidis, lotus plumule, great burdock achene, black pepper, Panax ginseng, roasted coffee beans, Fructus Schisandrae Chinensis and Fructus Schisandrae Sphenantherae, were analyzed using pipette-tip ESI-MS and quality mass spectra with stable and durable signals could be obtained. Both positive and negative ion modes were attempted and various compounds including amino acids, oligosaccharides, glycosides, alkaloids, organic acids, ginosensides, flavonoids and lignans could be detected. Principal component analysis (PCA) based on the acquired mass spectra allowed rapid differentiation of closely related herbal species.     

Similarity analyses of chromatographic herbal fingerprints: A review

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.     

Slurry sampling electrothermal atomic absorption spectrometric determination of copper in herbal medicine samples with a molybdenum tube atomizer

An ultrasonic slurry sampling electrothermal atomic absorption spectrometric method with a molybdenum tube atomizer has been developed for the determination of copper in herbal medicine samples. Glycerol solution (10%) was used as the slurry medium. The optimum pyrolysis temperature was 760 °C. The detection limit was 72 fg (3×S/N ratio). Matrix element interference was investigated and it was found that glycerol as a chemical modifier eliminated the interference. The amounts of copper in herbal medicines determined by the method proposed are in good accordance with those measured in dissolved acid-digested samples. The method enables rapid calibration, and simple and rapid analysis of copper in herbal medicine samples at low cost.     

Quality control of medicinal herbs Fructus gardeniae, Common Andrographis Herb and their preparations for their active constituents by high-performance liquid chromatography-photodiode array detection-electrospray mass spectrometry

Dehydroandrographolide, andrographolide and geniposide are the main active constituents of many herbal medicines, e.g., Fructus gardeniae, Common Andrographis Herb. They are used as the markers to control the quality of such herbal medicines and their herbal preparations. In this paper, a simple and sensitive high-performance liquid chromatographic (HPLC) method coupled with photodiode array detection (DAD) and electrospray mass spectrometry (ESI/MS) were developed to determine the three compounds simultaneously in extracts of medicinal herbs and herbal preparations produced by different companies. The extracts were separated on a C₁₈ reversed phase HPLC column, with a gradient solvent system, the time for the separation of the three target analytes was 10 min. The abundance ions were recorded using selected ion monitoring (SIM) mode with m/z 297.3, 297.3 and 411.1 for dehydroandrographolide, andrographolide and geniposide, respectively. The limit of detection for dehydroandrographolide, andrographolide and geniposide were 20, 30 and 150 ng mL⁻¹, respectively. The proposed method was successfully applied to the determination of the contents of the compounds in related to medicinal herbs and preparations.     

Determination of aristolochic acids in medicinal plant and herbal product by liquid chromatography-electrospray-ion trap mass spectrometry

This paper describes a liquid chromatography-electrospray-ion trap mass spectrometry (LC-ES-ITMS) method for the determination of aristolochic acid I and II (AA-I and AA-II) in medicinal plants and Chinese herbal remedies. A reversed phase C₁₈ column with gradient elution was utilized. The effects of mobile phase additives, acetic acid and ammonium acetate, on LC separation and ES ionization were investigated. For both AA-I and AA-II, the [M+NH₄]⁺ ion was found to be the precursor ion for target MS/MS analysis. The MS/MS product ion, [M+H−44]⁺, was used for the quantitative measurement of AA-I and AA-II. The linearity was good from 0.03 to 5 μg ml⁻¹ and good correlation (r²=0.999) over the range examined was determined for both AA. The detection limit based on a signal-to-noise ratio of three was 0.012 and 0.015 μg ml⁻¹ for AA-I and AA-II, respectively. Various Chinese herbal remedies obtained from renal failure patients and medicinal plants were examined by this newly developed method.     

Solid‐phase microextraction based method for determination of essential oils components in herbal beverages

A method employing the direct immersion solid‐phase microextraction followed by GC‐MS analysis is presented for the determination of essential oils components in herbal tea infusions, i.e. their direct content in the liquid phase. The extraction performances were compared using five different microextraction fibres. Significant parameters affecting sorption process such as sample amount, sorption and desorption time and temperature, stirring speed, pH adjustment and effect of ionic strength were optimised and discussed. By optimising the key parameters, a detection limits (LOD = S/N × 3) for ten target marker compounds were obtained in the range from 5.3 to 48.2 ng/mL with recoveries ranged between 93.03 and 100.50%. Intra‐day and inter‐day repeatability at three concentration levels were found to be 1.1–15.3 and 7.2–15.5% RSD, respectively. Finally, the optimised procedure enabling a rapid and simple analysis of essential oils was applied for the direct determination of these compounds in ten herbal tea infusions.     

Evaluation of capillary electrophoresis for the analysis of rooibos and honeybush tea phenolics

Rooibos and honeybush are popular herbal teas produced from the shrubs of Aspalathus linearis and Cyclopia spp., respectively, which are indigenous to South Africa. Both herbal teas are rich in polyphenols and their consumption is associated with several health benefits, partly ascribed to their phenolic constituents. Quantification of phenolics in extracts and teas for quality control and research purposes is generally performed using HPLC, although dedicated and often species‐specific methods are required. CE offers an attractive alternative to HPLC for the analysis of phenolics, with potential benefits in terms of efficiency, speed and operating costs. In this contribution, we report quantitative CZE methods for the analysis of the principal honeybush and rooibos phenolics. Optimal separation for honeybush and rooibos phenolics was achieved in 21 and 32 min, respectively, with good linearity and repeatability. Quantitative data for extracts of “unfermented” and “fermented” rooibos and two honeybush species were statistically comparable with those obtained by HPLC for the majority of compounds. The developed methods demonstrated their utility for the comparison of phenolic contents between different species and as a function of manufacturing processes, thus offering cost effective, although less sensitive and robust, alternatives to HPLC analysis.     

A strategy for screening and identifying mycotoxins in herbal medicine using ultra‐performance liquid chromatography with tandem quadrupole time‐of‐flight mass spectrometry

The objective of this study was to develop an effective strategy for screening and identifying mycotoxins in herbal medicine (HM). Here, Imperatae Rhizoma, a commonly used Chinese herb, was selected as a model HM. A crude drug contaminated with fungi was analyzed by comparing with uncontaminated ones. Ultra‐performance LC coupled to tandem quadrupole TOF‐MS (UPLC–Q‐TOF‐MS) with collision energy function was applied to analyze different samples from Imperatae Rhizoma. Then, MarkerLynx^TM software was employed to screen the excess components in analytes, compared with control samples, and those selected markers were likely to be the metabolites of fungi. Furthermore, each of the accurate masses of the markers obtained from MarkerLynx^TM was then searched in a mycotoxins/fungal metabolites database established in advance. The molecular formulas with relative mass error between the measured and theoretical mass within 5 ppm were chosen and then applied to MassFragment^TM analysis for further confirmation of their structures. With the use of this approach, five mycotoxins that have never been reported in HM were identified in contaminated Imperatae Rhizoma. The results demonstrate the potential of UPLC–Q‐TOF‐MS coupled with the MarkerLynx^TM software and MassFragment^TM tool as an efficient and convenient method to screen and identify mycotoxins in herbal materials and aid in the quality control of HM.     

Simple quality assessment approach for herbal extracts using high performance liquid chromatography-UV based metabolomics platform

A lack of adequate or accepted research methodology has been a major obstacle to study herbal medicines. In this study, instead of the prevalent hyphenated chromatographies, common high performance liquid chromatography equipped with ultraviolet detector (HPLC-UV) and multivariate statistical analysis were utilized to assess the qualities of total flavones of sea buckthorn (TFS), an 85% ethanol extract of the sea buckthorn berries. Two complementary HPLC-UV methods were developed, validated and combined to comprehensively determine the ingredients in TFS. Principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) of the combined analytical data showed that the six batches of TFS could be well differentiated. Hierarchical cluster analysis (HCA) using Ward's minimum variance method of the PLS-DA loading matrix demonstrated the known ingredients (quercetin, kaempferol, isorhamnetin, oleanolic acid and ursolic acid) and three unknown ingredients in TFS significantly contributed to the quality differences. A PLS regression model indicated that the results of the present method correlated well with the content of total flavones, which is now the quality control approach of TFS. Results from this study indicated that the proposed method is reliable for the quality reassessment of some widely used herbal extracts.     

Rapid determination of aristolochic acids I and II in herbal products and biological samples by ultra-high-pressure liquid chromatography-tandem mass spectrometry

Aristolochic acids (AAs) are a mixture of structural-related compounds, in which aristolochic acid I (AA I) and aristolochic acid II (AA II) are reported to be correlated with Aristolochic acid nephropathy (AAN). In this work, a rapid and sensitive ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to determine AA I and AA II in herbal products and biological fluids. By using gradient elution with a mobile phase composed of a mixture of 10 mM ammonium formate buffer (pH 3.0) and acetonitrile, AAs could be determined within 10 min. Under optimum UHPLC-MS/MS conditions, the limit of detections was 0.14 and 0.26 ng mL⁻¹ for AA I and AA II, respectively. Run-to-run repeatability and intermediate precision of peak area for AA I and AA II were less than 5.74% relative standard deviation (RSD). Accuracy was tested by spiking 10, 100 and 1000 ng mL⁻¹ in rat serum and the recoveries were within 76.5-92.9%. Matrix effects were within 78.8-127.7%. The developed method was successfully applied to determine AA I and AA II in several herbal products and to investigate their pharmacokinetic behavior in female Wister rats. The result shows that the developed UHPLC-MS/MS method is efficient, sensitive, and accurate for the determination of AA I and AA II in herbal products and biological samples.     

Effects of airborne fluoride on soil and vegetation

The study was initiated by the sudden uncontrolled release of airborne fluorides in 2005 into the environment from aluminium smelter factory that caused damage of vegetation. Samples of corn leaves and corn male flower heads with visible symptoms of fluoride intoxication had been collected in autumn 2005. Increased contents of total fluoride, which exceeded the maximum allowable content of fluorine in feeding stuffs, including meadow grass, were detected. During continuation of the study some commercially available herbal teas and plants used for preparing herbal teas infusions, collected in the field in 2010, were investigated to investigate possible uptake of fluoride from the soil. Nettle (Urtica dioica) has been found to be a promising passive bioindicator for monitoring phytotoxic effects of fluoride in the soil on the vegetation. Good correlation between labile free fluoride in the soil and total fluoride in the nettle has been found, while total fluoride in the soil, soil pH and the dominant wind direction were also proven as important factors influencing the uptake of fluoride by the nettle.     

Inhibitory effect of eleven herbal extracts on advanced glycation end-products formation and aldose reductase activity

The formation of advanced glycation end-products(AGEs) and aldose reductase(AR) activity have been implicated in the development of diabetic complications. Our study sought to characterize the capacities of eleven herbal extracts against the formation of AGEs and the AR activity. An ultrahigh performance liquid chromatography and tandem mass spectrometry(UPLC–MS/MS) method was used for the detection of AR activity and the screening of AR inhibitors in this research. The amount of sorbitol from each analyte was directly detected using the multiple reaction monitoring mode and the sorbitol level could be reduced via the addition of an inhibitor. Moreover, the BSA/glucose(fructose) system was applied to investigate their inhibitory activities of AGEs formation in glycation model reactions.Compared with other screened herbs used in our study, Flos Sophorae Immaturus and Radix Scutellariae seemed to be more effective on inhibiting the formation of AGEs and AR activity. The inhibiting capacities of herbal extracts against AR activity and AGEs formation may be correlated with the bioactive components of the herbal extracts. The differences were correlated with the amount of polyphenol and flavonoid components. In the study, we have investigated the potential anti-hyperglycemic bioactivity of eleven herbal extracts in vitro, which could provide a reference for further in vivo research in the prevention and treatment of diabetic complications.     

Determination of Estragole in Fennel Herbal Teas by HS-SPME and GC–MS

Estragole, a volatile phenylpropanoid contained in a variety of edible herbs, has been demonstrated to be genotoxic and carcinogenic, and its addition as a flavoring substance to foodstuffs has been banned by the regulatory bodies of the European Union. Fast and accurate analytical methods for its determination in herbs are thus necessary to assess the dietary exposure of this substance in humans and, in particular, to sensitive groups. In the present study, headspace solid-phase microextraction (HS-SPME) combined with gas chromatography–mass spectrometry (GC–MS) was applied for determination of estragole in infusions from different widely used commercial herbal teas based on Foeniculum vulgare (fennel) seeds. The optimized HS-SPME extraction conditions involved the use of a polydimethylsiloxane fiber exposed to the herbal infusion for 20 min at 50°C followed by GC–MS analysis. The method was fully validated for linearity, sensitivity, accuracy, and precision and applied to real samples; the level of estragole in infusions of commercial fennel seed teas was found to be within 50–250 µ`^?1.     

气相色谱/质谱-化学计量学法分析测定药对桃仁-红花挥发油

药对是中药配伍中最基本、最常用的形式,具有中药配伍的基本特点。药对化学是复方化学的核心内容。联用色谱和化学计量学方法是分析中药复方复杂体系的有效工具。采用GC/MS法分离测定了药对桃仁-红花、单味药桃仁和红花的挥发油成分,并对其重叠色谱峰采用化学计量学解析法进行了分辨,得到药对和各单味药的纯色谱曲线和质谱。药对桃仁-红花、单味药桃仁和红花分辨出的色谱峰,通过质谱库对其进行定性,分别得到84、27和52个定性结果,占总含量的92.06%、89.43%和94%。实验结果表明:桃仁-红花挥发油成分与单味药桃仁和红花的存在较大差别,也不是两者挥发油成分之加和。