Global detection and analysis of volatile components from sun-dried and sulfur-fumigated herbal medicine by comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry

The sulfur-fumigation process can induce changes in the contents of volatile compounds and the chemical transformation of herbal medicines. Although literature has reported many methods for analyzing volatile target compounds from herbal medicine, all of them are largely limited to target compounds and sun-dried samples. This study provides a comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC×GC-TOF/MS) method based on a chemical profiling approach to identify non-target and target volatile compounds from sun-dried and sulfur-fumigated herbal medicine. Using Chrysanthemum morifolium as a model herbal medicine, the combined power of this approach is illustrated by the identification of 209 and 111 volatile compounds with match quality >80% from sun-dried and sulfur-fumigated Chrysanthemum morifolium, respectively. The study has also shown that sulfur-fumigated samples showed a significant loss of the main active compounds and a more destructive fingerprint profile compared to the sun-dried ones. 50 volatile compounds were lost in the sulfur-fumigated Chrysanthemum morifolium sample. The approach and methodology reported in this paper would be useful for identifying complicated target and non-target components from various complex mixtures such as herbal medicine and its preparations, biological and environmental samples. Furthermore, it can be applied for the intrinsic quality control of herbal medicine and its preparations.     

Evaluation of different internal-diameter column combinations in comprehensive two-dimensional gas chromatography in flavour and fragrance analysis

A series of OV1-OV1701 column sets, in which the two dimensions differ in id and/or film thickness, were adopted to separate components of the volatile and semivolatile fraction of samples of plant origin. In particular two applications are presented: a target analysis approach to determine volatile suspected allergens in a medium-complexity fragrance and in sandalwood essential oil, and a fingerprint analysis approach to compare herbal extracts. Some basic chromatographic parameters of these combinations, i. e. net separation measure (S(GC x GC)), degree of orthogonality and a suitable number of modulations per peak, were also estimated by analysing two test mixtures: FAME (C4:0-C24:0) and suspected volatile allergens. Experimental results show that 0.25 mm homologous id column combination, despite their lower separation power, have good capacity to separate and resolve medium-to-complex fractions from samples of plant origin, demonstrating: (i) good system orthogonality, maximized through proper exploitation of stationary-phase selectivity and (ii) reduced 2-D column overloading effects due to the increased 2-D mass loadability, thus facilitating the analysis of mixtures whose components differ significantly in relative abundance.     

Comparison of micellar electrokinetic capillary chromatography and high performance liquid chromatography on fingerprint of Cnidium monnieri

In our studies, micellar electrokinetic capillary chromatography (MEKC) was employed in fingerprint analysis of Cnidium monnieri for the first time. Average chromatography of 10 batches Cnidium monnieri from Jiangsu province, China, which have long been considered as the original and genuine herbal medicine, was first established as the characteristic fingerprint. Within 25 min the major effective components were separated by 18 mM borate, 12 mM phosphate and 50 mM SDS (pH 9.2) containing 20% methanol. The relative standard deviations of migration times and peak areas were less than 5%. As a new approach of fingerprint, MKCE was compared to the conventional approach-HPLC in our experiments. The fingerprint developed by HPLC comprised 8 peaks that were collected within 40 min. Relative standard deviation (RSD) values of retention times of corresponding peaks in HPLC analysis were very small (maximum 3% and average 0.9%). In conclusion, each two methods had its advantages and disadvantages. Furthermore, besides HPLC, MEKC as a feasible method, could be used in the development of fingerprint of Cnidium monnieri.     

The application of digital image recognition to the analysis of two-dimensional fingerprints

High-performance liquid chromatography (HPLC) fingerprint has been commonly used in the quality control and assessment of herbal medicines, and two-dimensional (2D) fingerprint obtained by means of HPLC-diode array detector (HPLC/DAD) can provide higher reliability. In this paper, an approach to the analysis of the 2D HPLC/DAD fingerprints, which was based on digital image recognition techniques, was developed for the first time. First, wavelet transform was employed to eliminate noise signal in the 2D fingerprint, and then the 2D fingerprint was converted into grayscale image. Second, several features of the image were calculated, and hierarchical clustering. This approach was applied to the qualitative analysis of the different samples of coptis chinensis, and the clustering result of samples was all highly consistent with the real situation. Based on the densities in grayscale image, three components in standard samples were quantitative analyzed, and the obtained correlation coefficients between concentration and grayscale density were more than 0.999. Our study indicated that the analysis of the 2D HPLC/DAD fingerprint was successful based on the idea and techniques of digital image recognition techniques, and this proposed approach provided a new pathway for the analysis of two-dimensional spectrums.     

Quality assessment of Fructus xanthii based on fingerprinting using high-performance liquid chromatography

Because almost every traditional Chinese medicine (TCM) is a multicomponent system, QC of TCMs always involves various difficulties. As a current popular quality assessment approach, focusing on qualitative and quantitative analysis of certain compounds contained in herbal medicine has been widely used for the sake of expediency rather than being a practical and realistic way. However, this method does not take the existence of other constituents into account. Comparatively, the chromatographic fingerprint of the components is a more suitable approach to holistically assess the quality of herbal drugs. Fructus xanthii is a well-known herbal drug listed in all editions of the Chinese Pharmacopoeia. However, there is no quality evaluation method given in its monograph, even for the above-mentioned expediency. This paper reports an HPLC fingerprinting method for quality evaluation of F. xanthii. The HPLC profiles of 27 batches of commercial samples were further analyzed using chemometric methods, including similarity evaluation and principal component analysis. As a result, the established HPLC fingerprint contained 23 characteristic peaks; therein, 13 peaks were unambiguously assigned by comparing their retention times and UV spectra with those of reference compounds, and five peaks were tentatively identified on the basis of their MS/MS fragmentation patterns and UV spectra. Moreover, it could be clearly observed that caffeoylquinic acid and its analogs predominate in F. xanthii. Except for three samples identified as outliers, 24 other commercial samples displayed similar HPLC profiles, indicating that the quality of the herbs from different markets is stable and consistent.     

Application of headspace solid-phase microextraction (HS-SPME) and comprehensive two-dimensional gas chromatography (GC x GC) for the chemical profiling of volatile oils in complex herbal mixtures

The coupling of headspace solid-phase microextraction (HS-SPME) with comprehensive two-dimensional gas chromatography (GC x GC) was shown to be a powerful technique for the rapid sampling and analysis of volatile oils in complex herbal materials. When compared to one-dimensional (1-D) GC, the improved analytical capabilities of GC x GC in terms of increased detection sensitivity and separation power were demonstrated by using HS-SPME/GC x GC for the chemical profiling (fingerprinting) of essential/volatile oils contained in herbal materials of increasing analytical complexity. More than 20 marker compounds belonging to Panax quinquefolius (American ginseng) can be observed within the 2-D contour plots of ginseng itself, a mixture of ginseng and another important herb (P. quinquefolius/Radix angelicae sinensis), as well as a mixture of ginseng and three other herbs (P. quinquefolius /R. angelicae sinensis/R. astragali/R. rehmanniae preparata). Such analytical capabilities should be important towards the authentication and quality control of herbal products, which are receiving increasing attention as alternative medicines worldwide. In particular, the presence of Panax in the herb formulation could be readily identified through its specific peak pattern in the 2-D GC x GC plot.     

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.     

Quantitative analysis combined with chromatographic fingerprint and antioxidant activities for the comprehensive evaluation of Compound Danshen Tablets

The composition of traditional Chinese medicine is extremely complex, so it is difficult to ensure quality consistency. We took Compound Danshen Tablets as the object of the study, by using high‐performance liquid chromatography to establish multiwavelength fusion fingerprints. Characteristic fingerprints of 30 batches of samples were generated at four wavelengths and evaluated by systematic quantified fingerprint method. An on‐line antioxidant determination method was used for the determination of the antioxidant components in Compound Danshen Tablets. The fingerprint analysis of the marker compounds can reflect the content of the marker compounds, which were determined by using the external standard method. This study elucidated that multiwavelength fusion fingerprint profiles and multiple markers compound analysis in conjunction with the assay of antioxidant activity offered a reliable and efficient approach to quantitatively evaluate the quality consistency of the traditional Chinese medicine and herbal preparations.     

Identification of essential components of Houttuynia cordata by gas chromatography/mass spectrometry and the integrated chemometric approach

Starting with Biller-Biemann's work [J.E. Biller, K. Biemann, Anal. Lett. 7 (1974) 515], various kinds of approaches have been proposed to extract GC/MS data to obtain pure components responses. In this paper, an integrated chemometric approach is proposed, which combine four sequential steps, data pretreatment, component perception, resolution and component identification, and then the proposed approach is manipulated to analyze the essential oils of a herbal medicine named Houttuynia cordata (HC). On the basis of the selective information obtained from both chromatograms and mass spectra, the proposed integrated chemometric approach can resolve the two-way GC/MS responses matrix into pure chromatograms and mass spectra without any model assumption on the peak shape. The resolution results obtained from HC samples demonstrate the performance of the proposed approach and indicate that it may be a promising one for analyzing complex chromatograms.     

Comprehensive chemical profiling of Guizhi Fuling capsule by the combined use of gas chromatography-mass spectrometry with a deconvolution software and rapid-resolution liquid chromatography quadrupole time-of-flight tandem mass spectrometry

Herbal formulations are complex natural mixtures. Researchers usually tend to focus more on analysis of nonvolatile components but pay less attention to volatile compounds. In this study, an analytical strategy combining two approaches was established for comprehensive analysis of herbal formulations. Guizhi Fuling capsule (GFC), a drug approved by the FDA to enter phase II clinical trial for treatment of primary dysmenorrhea, was taken as a case for analysis. Gas chromatography–mass spectrometry (GC‐MS) with automated mass spectral deconvolution and identification system (AMDIS) led to rapid identification of 48 volatile components including four acetophenones, three fatty acid esters, 13 phenylpropanoids and 19 sesquiterpenes. Most of them were found from Guizhi. The volatile oils of Guizhi have been proved to exhibit many pharmacological activities. This is helpful in understanding the pharmacological mechanism of GFC. Furthermore, AMDIS turned out to be efficient and reliable for analysis of complex herbal formulations. Rapid‐resolution liquid chromatography (RRLC) coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (ESI‐Q‐TOF MS/MS) allowed the identification of 70 nonvolatile components including six acetophenones, 12 galloyl glucoses, 31 monoterpene glycosides, three phenols and 12 triterpene acids. Fragmentation behaviors of assigned components, especially triterpene acids, which are hard to identify by low‐resolution MS, were first investigated by TOF MS/MS. Characteristic ions and typical loss of assigned triterpene acids were summarized. Combinatorial use of GC‐MS‐AMDIS and RRLC‐ESI‐Q‐TOF MS/MS could be of great help in global qualitative analysis of GFC, as well as other herbal products. Copyright © 2012 John Wiley & Sons, Ltd.     

气相色谱-质谱指纹图谱在甄别真假卷烟上的应用

运用固相微萃取／气相色谱-质谱法建立卷烟挥发性和半挥发性物质的指纹图谱。通过指纹图谱相似度评价软件,对12批正品卷烟和1个假冒品卷烟的指纹图谱进行了相似度计算,结果表明：12批样品间相似度很好,而假冒品卷烟与正品卷烟的相似度很低,该方法为探讨卷烟质量稳定和甄别真假卷烟提供了一种简便易行的方法模式。     

A novel and rapid HPGPC-based strategy for quality control of saccharide-dominant herbal materials: Dendrobium officinale,a case study

Qualitative and quantitative characterization of natural saccharides, especially polysaccharides, in herb materials remains a challenge due to their complicated structures and high macromolecular masses. Currently available methods involve time-consuming and complicated operations, and present poor specificity. Here, a novel and rapid high-performance gel permeation chromatography (HPGPC)-based approach is described for quality assessment of saccharide-dominant herbal materials by simultaneous qualitative and quantitative analysis of saccharide components. Dendrobium officinale, one of the rarest tonic herbs worldwide, was employed as the model herb in this study. First, a HPGPC fingerprint based on the molecular weight distribution of its carbohydrate components was established for qualitative identification of D. officinale. Then, HPGPC-guided dominant holistic polysaccharide marker was separated using ultra-filtration followed by HPGPC determination for quantitative evaluation of D. officinale. The experimental results suggest that this method is more efficient, stable, and convenient compared with the currently available methods for authentication and quality evaluation of D. officinale, and we expect the method will have similar advantages when used for quality control of other saccharide-dominant herbal materials and products. Graphical Abstract

The characteristic HPGPC fingerprint of Dendrobium officinale compared with other confused Dendrobium species     

Internal standard mass spectrum fingerprint: A novel strategy for rapid assessing the quality of Shuang-Huang-Lian oral liquid using wooden-tip electrospray ionization mass spectrometry

In this study, an internal standard (IS) wooden-tip electrospray ionization mass spectrometry (ESI-MS) fingerprint method was developed for rapid quality assessment and control of Shuang-Huang-Lian (SHL) oral liquid, a famous herbal preparation registered by Chinese Pharmacopoeia. Sharp wooden tips with tip-end o.d. of 150–200 μm, which are similar to the diameter of commercially available ESI emitter, were used as solid substrates to extract samples and induce electrospray for mass spectrometric analysis. Various active ingredients present in SHL oral liquid, including organic acids, flavonoids, and phenylethanoid glycosides, etc., were simultaneously detected without any sample pretreatment and chromatographic separation. Perfluorooctanesulfonic acid was used as an IS compound to calculate the content fluctuations of active ingredients, and principal component analysis was applied to the obtained fingerprints to achieve systematically and comprehensively quality assessment of investigated samples. The quality stability and consistency were successfully assessed, the sources of different manufacturers were traced, and the qualified, short expired and long expired products were also distinguished unambiguously. Our experimental data demonstrated that IS ambient mass spectrum fingerprint is a simple and efficient approach for rapid quality assessment and control of herbal medicines.     

Modified secured principal component regression for detection of unexpected chromatographic features in herbal fingerprints

Secured principal component regression is modified for the qualitative analysis of chromatographic fingerprint data sets of herbal samples with residual concentrations. After chromatographic shift-correction and autoscaling are performed on the data, this modified secured principal component regression (msPCR) can detect unexpected chromatographic features in various herbal fingerprints. The successful application of msPCR to two real herbal medicines of Erigeron breviscapus from different geographical origins and Ginkgo biloba from various sources or vendors demonstrates that the proposed method can detect reasonably unexpected features differing from the regulars or not being modeled. From a chemical point of view, the causes have also been explained to corroborate the results. Moreover, it presents a viable approach for the qualitative evaluation of diverse herbal objects with a regular class of chromatographic fingerprints.     

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.     

Quality assessment of Cinnamomi Ramulus by the simultaneous analysis of multiple active components using high‐performance thin‐layer chromatography and high‐performance liquid chromatography

A novel and improved method for the quality assessment of Cinnamomi Ramulus was developed and completely validated. The method was established using fingerprint technology and simultaneous quantitative determination of six main marker compounds including coumarin, cinnamic alcohol, cinnamic acid, 2‐methoxy cinnamic acid, cinnamaldehyde, and 2‐methoxy cinnamaldehyde in the herbal medicine for the first time. A newly developed high‐performance thin‐layer chromatography method, which achieved simultaneous definition of five marker components by comparing the colors and retardation factor values of the bands in high‐performance thin‐layer chromatography, was first used for the authentication of Cinnamomi Ramulus. The fingerprints of 26 batches of herbal samples from different regions of China showed very similar chromatographic patterns that were evaluated by similarity analysis and hierarchical clustering analysis. In addition, six marker compounds were simultaneously determined using single standard to determine multiple components by the relative response factors. Compared with the external standard method, the new quantitative method was validated to determine multiple compounds in 26 batches of Cinnamomi Ramulus samples. All results demonstrated that the simple and rapid method could be effectively utilized for the quality control of Cinnamomi Ramulus.     

Chemical fingerprint analysis for quality assessment and control of Bansha herbal tea using paper spray mass spectrometry

Chemical fingerprinting methodology is an approach for quality assessment and control of herbal medicines and related products based on the holistic chemical profile obtained by various analytical techniques. This study demonstrates the first application of paper spray mass spectrometry (PS-MS) as a chemical fingerprinting methodology for tracing the origins, establishing the authenticity, and assessing the overall quality of a famous herbal product, Bansha herbal tea (BHT). A negative ion PS-MS spectrum yielded the best chemical profiling information and was most appropriate for fingerprint analysis of BHT. In addition to the identification of active ingredients, various compounds present in BHT were simultaneously detected without any sample pretreatment and chromatographic separation, providing valuable information for the quality assessment and control of this herbal product. According to the principal component analysis of the PS-MS fingerprints, two sources of commercially available BHT products made by different manufacturers were easily differentiated. Qualified and expired products from the two manufacturers were also successfully distinguished, and the consistency of the quality between the manufacturers was assessed. Our experimental data demonstrated that the PS-MS chemical fingerprinting is a simple, rapid, and robust methodology for pharmaceutical analysis, with promising prospects for quality assessment and control of herbal medicines and related products with high-throughput.     

High-performance thin layer chromatography for quality control of multicomponent herbal drugs: example of cangzhu xianglian san

Due to their complexity, multicomponent herbal drugs pose enormous analytical challenges for quality control (QC). Although they may have traditionally been used for hundreds of years, the information about their chemical composition is often still limited. Selecting suitable markers to monitor the identity and potency of the mixture is, therefore, difficult. There is also the possibility of natural variability for each plant. This paper illustrates a pragmatic and practical approach to QC of a multicomponent herbal drug by HPTLC. Cangzhu xianglian xan (CXS), composed of the herbal drugs Coptis rhizome, Aucklandia root, and Atractylodes rhizome (30 + 20 + 60, w/w/w), is used as an example. A characteristic fingerprint can be generated for CXS with toluene-ethyl acetate-methanol-isopropanol-water (60 + 30 + 20 + 15 + 3, v/v/v/v/v) mobile phase on HPTLC silica gel 60 conditioned with ammonia. While the corresponding monograph of the Chinese Veterinary Pharmacopoeia focuses only on the detection of berberine, one of the principal components of Coptis rhizome, the proposed method of identification determines the presence of all three components in the drug after derivatization with anisaldehyde reagent. The same method can also be used to quantitatively determine the content of berberine by scanning densitometry. This paper provides details about the validation of the qualitative and quantitative determinations.     

Quality fluctuation detection of an herbal injection based on biological fingerprint combined with chemical fingerprint

Herbal injection is one of the most important preparations of traditional Chinese medicine. More than 130 types of herbal injections are used clinically for 400 million patients annually with total sales of over four billion US dollars per year. However, the current quality control (QC) methods relying mainly on chemical fingerprints (CF) can hardly ensure quality and safety of the herbal injections with complex chemical composition and have resulted in an increase in serious adverse drug reactions. In this study, a comprehensive approach for the QC of a controversial herbal injection Shuang-Huang-Lian lyophilized powder (SHL) was established based on the quality fluctuation detection by a combination of CF and biological fingerprint (BF). High-performance liquid chromatography and the impedance-based xCELLigence system were applied to establish the CF and BF, respectively. In addition, multivariate analysis was performed to evaluate the discriminant ability of the two methods. The results showed that being subjected to environmental influence like oxygen/air, high temperature, and extreme illumination could lead to quality fluctuation of SHL. The combination of chemical and biological fingerprint method is a more powerful tool for the QC of SHL because it can clearly discriminate different groups of abnormal samples. This method can be used for the detection of quality fluctuation of SHL and can provide reference for the quality control of other herbal injections.