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Artemisinin is an important frontline antimalarial. Fast, accurate detection of artemisinin in human serum is of importance in monitoring its clinical pharmaceutical effect. In this work, a strategy using microsensor array coupled with electrochemiluminescence (ECL) imaging technique was developed for detection of artemisinin. The microsensor array was constructed by integrating a patterned indium tin oxide glass plate with two perforated hydrophobic paper covers. By introducing the reactant of p-aminophenylboronic acid, luminol and artemisinin into the microsensor array, artemisinin would oxidize p-aminophenylboronic acid into p-aminophenol, a product which can efficiently inhibit the ECL of luminol. ECL signals decrease linearly with the increase of artemisinin. Based on the decreased ECL signal, artemisinin can be accurately detected. A good linearity (r = 0.994) was observed for artemisinin detection. The detection sensitivity is 0.48 μM for artemisinin. The detection selectivity and stability were also investigated. Results show that the present method shows a good selectivity and stability towards artemisinin detection. To evaluate the applicability of the present strategy for detecting artemisinin in real samples, the artemisinin content in human serum and Artemisia annua samples were analyzed. Results demonstrated that the present strategy shows excellent selectivity with high sensitivity towards artemisinin detection in real samples., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cordyceps sinensis has gained increasing attention due to its nutritional and medicinal properties. Herein, we employed label-free quantitative mass spectrometry to explore the proteome differences between naturally- and artificially-cultivated C. sinensis. A total of 22,829 peptides with confidence ≥95%, corresponding to 2541 protein groups were identified from the caterpillar bodies/stromata of 12 naturally- and artificially-cultivated samples of C. sinensis. Among them, 165 proteins showed significant differences between the samples of natural and artificial cultivation. These proteins were mainly involved in energy production/conversion, amino acid transport/metabolism, and transcription regulation. The proteomic results were confirmed by the identification of 4 significantly changed metabolites, thus, lysine, threonine, serine, and arginine via untargeted metabolomics. The change tendencies of these metabolites were partly in accordance with changes in abundance of the proteins, which was upstream of their synthetic pathways. In addition, the nutritional value in terms of the levels of nucleosides, nucleotides, and adenosine between the artificially- and naturally-cultivated samples was virtually same. These proteomic data will be useful for understanding the medicinal value of C. sinensis and serve as reference for its artificial cultivation., Significance: C. sinensis is a precious and valued medicinal product, the current basic proteome dataset would provide useful information to understand its development/infection processes as well as help to artificially cultivate it. This work would also provide basic proteome profile for further study of C. sinensis., (Copyright © 2018. Published by Elsevier B.V.)

Altered mitochondrial oxidation increases vulnerability to cardiac ischemia/reperfusion (I/R) injury in metabolic disorders. However, the metabolic signaling responsible for the dysfunction remains partly unknown. We sought to test whether or not hypoxic succinate accumulation could inhibit pyruvate dehydrogenase (PDH) activity and subsequently aggravate I/R injury. Results showed that saturated fatty acid palmitate stimulation increased fatty acid oxidation and induced hypoxia in cardiomyocytes, leading to succinate accumulation. Intracellular succinate induced hypoxia inducible factor-1α (HIF-1α) expression and impaired PDH activity via upregulation of pyruvate dehydrogenase kinase 4 (PDK4) expression. Luciferase reporter assay showed that succinate increased PDK4 expression through gene promoter induction in a HIF-1α-dependent manner. Palmitate also induced the release of succinate into extracellular space. By activating GRP91, extracellular succinate induced the translocation of PKCδ to mitochondria and further exacerbated PDH impairment. These results demonstrated that succinate impaired PDH activity via GPR91-dependent and independent pathways. Ginsenoside Rb1 (a major compound isolated from ginseng) and trimetazidine (fatty acid β-oxidation inhibitor) prevented hypoxic succinate accumulation in cardiomyocytes and improved PDH activity by blocking succinate-associated HIF-1α activation and GPR91 signaling. Through improving PDH activity, Rb1 and trimetazidine prevented cardiac acidification, ameliorated mitochondrial dysfunction and thereby reduced apoptosis during hypoxia/reoxygenation insult. In isolated working rat hearts perfused with palmitate and in high-fat diet-fed mice, early intervention of Rb1 and trimetazidine reduced succinate production and resultantly increased heart resistance to ischemia/reperfusion injury. Taken together, our findings demonstrated that early intervention by targeting inhibition of succinate accumulation-induced PDH impairment is an effective strategy to alleviate I/R injury., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This paper describes a simple and miniaturized microdroplet chip (μchip) that is constructed with a silica nanochannel (SNC)-assisted electrode array and a hydrophobic paper cover (SNC&P-μchip). Vertically aligned SNCs with uniform pore size of 2-3 nm in diameter and negatively charged surface can significantly accelerate the mass transport of the positively charged tris(2,2'-bipyridyl) ruthenium (II), resulting in a remarkably enhanced electrochemiluminescence (ECL) signal. The SNC-assisted electrode array was coupled to a low cost paper cover to achieve simultaneous detection of six samples in 1 min. The feasibility and universality of the SNC&P-μchip was evaluated by detecting a series of alkaloidal drugs both in buffers and in human serum. The performance of the SNC&P-μchip was fully validated with respect to linearity (0.9999 > R > 0.9939), sensitivity (limits of detection from 1.799 nM to 11.43 nM), and accuracy (recovery rate between 94.38% and 109.12%). The facile and economic SNC&P-μchip shows promising potential for rapid drug detection in complex biofluids., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A simple, efficient and versatile biosensing platform capable of the multiplexed detection for glucose, lactate and choline was developed by the integration of bipolar electrochemistry and electrochemiluminescence (ECL) imaging. The sensing bipolar electrodes (BPEs) were simply modified via a one-step method adaptable to different enzymes. The biorecognition event happening between the substrate and the corresponding enzyme could be directly reported by the ECL emitted on the same pole from luminol and in situ generated H 2 O 2 . Under optimized conditions, the BPEs array was successfully applied for the determination of glucose, lactate and choline in the ranges of 0.01-1mM, 0.01-1mM and 0.02-5mM, with the LOD of 7.57μM, 8.25μM and 43.19μM, respectively. Owing to the improved stability of in situ generated H 2 O 2 , a whole series of analytes testing could be completed in the same BPE biochip. Subsequently, an array chip consisting of nine BPEs enabled the concomitant detection of glucose, lactate and choline, demonstrating the capability for multifunctional detection of biomolecules. This versatile analytical system could be easily extended to sensitive screening in a miniaturized device and point of care testing., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Baicalin and scutellarin, two flavonoid glucuronic acids isolated from Scutellaria baicalensis, exhibit beneficial effects on glucose homeostasis. Baicalin and scutellarin are similar in structure except scutellarin has an additional hydroxyl at composition C-4'. In this work, we observed that baicalin and scutellarin promoted glucose disposal in mice and in adipocytes. Baicalin selectively increased phosphorylation of AMP-activated kinase (AMPK), while scutellarin selectively enhanced Akt phosphorylation. Both of them increased AS160 phosphorylation and glucose uptake in basal condition. AMPK inhibitor or knockdown of AMPK by siRNA blocked baicalin-induced AS160 phosphorylation and glucose uptake, but showed no effects on scutellarin. In contrast, Akt inhibitor and knockdown of Akt with siRNA decreased scutellarin-stimulated glucose uptake but had no effects on baicalin. The molecular dynamic simulations analysis showed that the binding energy of baicalin to AMPK (-34.30kcal/mol) was more favorable than scutellarin (-21.27kcal/mol), while the binding energy of scutellarin (-29.81kcal/mol) to Akt was much more favorable than baicalin (4.04kcal/mol). Interestingly, a combined treatment with baicalin and scutellarin acted synergistically to enhance glucose uptake in adipocytes (combination index: 0.94-0.046). In conclusion, baicalin and scutellarin, though structurally similar, promoted glucose disposal in adipocytes by differential regulation on AMPK and Akt activity. Our data provide insight that multicomponent herbal medicines may act synergistically on multiple targets., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This work aimed to establish a systematic strategy to enrich and separate quercetin-3-O-β-d-glucuronide (Q3GA) from lotus leaves with macroporous resin and semi-preparative HPLC. Six resins were tested, and LX-5 was chosen as the appropriate resin for Q3GA based on the adsorption and desorption performances. After one-step enrichment, the content of Q3GA increased from 2.15% in crude extract to 52.25% in 30% ethanol fraction with yield of 11.97%. The Q3GA was then isolated from the 30% ethanol fraction by semi-preparative HPLC, and the purity of Q3GA was above 98.00% with yield of 19.76%. These results suggested that the aforementioned strategy was a useful and economic method to enrich and separate Q3GA from lotus leaves. Additionally, the anti-inflammatory effect of Q3GA was evaluated in lipopolysaccharide-treated RAW264.7 macrophages, and the result demonstrated that Q3GA could significantly inhibit LPS-induced NO release in vitro in a dose-dependent manner compared with control group., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Screening and deciphering active natural products of herbal medicines are of great importance for modern drug discovery. In this study, a novel strategy was proposed to rapidly filter ineffective compounds and target the most potential leads. The aim is to answer the key question of what components are responsible for the holistic bioactivity of an herbal product. To support the strategy, the pharmacophore-guided knockout/knockin chromatography was established for the first time. The greatest advantage of this method is that any interesting components could be automatically fished or knocked out. The method validation shows that the herbal extract was accurately reconstructed according to the experimental design. By combining with bioactivity assays, we demonstrated that "functional compound combination (FCC)", which is the core and indispensable effective part, could be discovered from an herbal medicine and suitable as marker compounds for quality control. The applicable objects of the strategy include single herbs, herbal formulas and commercially herbal preparations. As an illustrative case study, the strategy was successfully applied to simultaneously determine active leads and the FCC in Dan-Qi formula which shows excellent free radical scavenging activity. The potential mechanisms of compounds in Dan-Qi formula reacting with three different free radicals were systematically reported for the first time. This strategy was expected to unveil the mystery of herbal medicines and inspire a natural product-based drug discovery., (Copyright © 2016 Elsevier B.V. All rights reserved.)

American ginseng is a commonly consumed herbal medicine in the United States and other countries. Ginseng saponins are considered to be its active constituents. We have previously demonstrated in an in vitro experiment that human enteric microbiota metabolize ginseng parent compounds into their metabolites. In this study, we analyzed American ginseng saponins and their metabolites in human plasma, urine and feces samples by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). Six healthy male volunteers ingested 1 g of American ginseng twice a day for 7 days. On day 7, biological samples were obtained and pretreated with solid phase extraction. The ginseng constituents and their metabolites were characterized, including 5 ginseng metabolites in plasma, 10 in urine, and 26 in feces. For the plasma, urine and feces samples, the levels of ginsenoside Rb1 (a major parent compound) were 8.6, 56.8 and 57.7 ng/mL, respectively, and the levels of compound K (a major metabolite) were 58.4 ng/mL, 109.8 ng/mL and 10.06 μg/mL, respectively. It suggested that compound K had a remarkably high level in all three samples. Moreover, in human feces, ginsenoside Rk1 and Rg5, Rk3 and Rh4, Rg6 and F4 were detected as the products of dehydration. Further studies are needed to evaluate the pharmacological activities of the identified ginseng metabolites., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Hydrolysis plays an important role in the metabolic transformations of lactones. Analysis of lactones and their hydrolyzed metabolites in biological samples is challenging, because unexpected hydrolysis or reversed dehydration may occur depending on the test environments. In this work, a supercritical fluid chromatography hybrid triple-quadrupole mass spectrometry (SFC-QQQ MS) method has been proposed for simultaneous analysis of ginkgolides and hydrolyzed metabolites. The SFC utilizes carbon dioxide as the mobile phase, avoiding hydrolysis of ginkgolides that always happens during reversed phase liquid chromatographic detection. Compared with normal phase liquid chromatography, the SFC provides good resolutions especially for ginkgolide with similar structures. This SFC-QQQ MS method was validated in linearity, precision, accuracy, and stability for ginkgolides and metabolites. Then this method was successfully applied to pharmacokinetic study of 3 ginkgolides and their 6 hydrolyzed metabolites after intravenous administration of total ginkgolide extract. Ginkgolides and metabolites showed different clear rates and excluded in 2-4h. The developed SFC-QQQ MS method allows accurate determination of ginkgolides and metabolites with high resolutions, and can be extended to analysis of other water-unstable compounds., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Quality standardization of herbal medicines (HMs) is an important task with great challenges. Selection of abundant compounds as markers is currently a major approach for the quality control of HMs; however, such marker compounds are irrelevant to the bioactivities in many cases. Taking Lycoridis Radiatae Bulbus (LRB) as an example, we proposed a universal strategy to identify the effective combinatorial markers (ECMs) that are representative of the bioactivities of HMs, and took them as chemical markers for quality standardization. Fingerprinting and quantification were employed to find out the common components in various batches of medicines. The contribution of each common compound to the overall bioactivity was determined through fingerprint-bioactivity modeling, which based on the absolute quantification of each compound and the acetylcholinesterase (AChE) inhibitory activity of LRB. Two most effective compounds, ungerimine and galanthamine, were therefore proposed as ECMs. Interestingly, these two compounds could synergistically inhibit AChE. This approach demonstrated its strong advantage of the bioactivity relevant quality assessment when compared with conventional methods. And the success of applying this ECMs-based method to the quality assessment of unknown LRB samples proved that our approach was reliable and reproducible. In conclusion, this approach is not only useful for the bioactivity relevant quality control of HMs but also helpful for the discovery of ECMs as new drug candidates., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Generation of a high-purity fraction library for efficiently screening active compounds from natural products is challenging because of their chemical diversity and complex matrices. In this work, a strategy combining high-resolution peak fractionation (HRPF) with a cell-based assay was proposed for target screening of bioactive constituents from natural products. In this approach, peak fractionation was conducted under chromatographic conditions optimized for high-resolution separation of the natural product extract. The HRPF approach was automatically performed according to the predefinition of certain peaks based on their retention times from a reference chromatographic profile. The corresponding HRPF database was collected with a parallel mass spectrometer to ensure purity and characterize the structures of compounds in the various fractions. Using this approach, a set of 75 peak fractions on the microgram scale was generated from 4mg of the extract of Salvia miltiorrhiza. After screening by an ARE-luciferase reporter gene assay, 20 diterpene quinones were selected and identified, and 16 of these compounds were reported to possess novel Nrf2 activation activity. Compared with conventional fixed-time interval fractionation, the HRPF approach could significantly improve the efficiency of bioactive compound discovery and facilitate the uncovering of minor active components., (Copyright © 2013 Elsevier B.V. All rights reserved.)

American ginseng is a widely used natural product. Ginseng products are usually taken orally, and human intestinal microflora may metabolize ginsenosides. Existing publications report the metabolite fates of ginsenosides. However, investigations on the comprehensive metabolic profile of American ginseng extract are absent because of the chemical complexity and limitation of analytical methods. In this work, we studied the biotransformation and metabolic profile of American ginseng extract by human intestinal microflora. Human fecal microflora was prepared from a healthy Chinese man and then anaerobically incubated with American ginseng sample at 37 °C for 24 h. A rapid and simple liquid-liquid extraction method was used for sample pretreatment. A highly sensitive and selective liquid chromatography/quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) method was used to characterize ginsenosides and related metabolites in the reaction samples. The LC-Q-TOF-MS provides superior data quality and advanced analytical capabilities for profiling, identifying, and characterizing complex metabolites in matrix-based biological samples. A total of 25 metabolites were detected, 13 of which were undoubtedly assigned by comparison with reference compounds, and 12 others were tentatively identified. The three most abundant metabolites are 20S-ginsenoside Rg3, ginsenoside F2 and compound K. The main metabolic pathways of ginseng saponins are deglycosylation reactions by intestinal microflora through stepwise cleavage of sugar moieties. Subsequent dehydration reactions also occur. Protopanaxadiol- and oleanane-type triterpenoids are easy to metabolize. The intestinal microbiota may play an important role in mediating the metabolism bioactivity of American ginseng., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Various analytical technologies have been developed for quantitative determination of marker compounds in herbal medicines (HMs). One important issue is matrix effects that must be addressed in method validation for different detections. Unlike biological fluids, blank matrix samples for calibration are usually unavailable for HMs. In this work, practical approaches for minimizing matrix effects in HMs analysis were proposed. The matrix effects in quantitative analysis of five saponins from Panax notoginseng were assessed using high-performance liquid chromatography (HPLC). Matrix components were found to interfere with the ionization of target analytes when mass spectrometry (MS) detection were employed. To compensate the matrix signal suppression/enhancement, two matrix-matched methods, standard addition method with the target-knockout extract and standard superposition method with a HM extract were developed and tested in this work. The results showed that the standard superposition method is simple and practical for overcoming matrix effects for quantitative analysis of HMs. Moreover, the interference components were observed to interfere with light scattering of target analytes when evaporative light scattering detection (ELSD) was utilized for quantitative analysis of HMs but was not indicated when Ultraviolet detection (UV) were employed. Thus, the issue of interference effects should be addressed and minimized for quantitative HPLC-ELSD and HPLC-MS methodologies for quality control of HMs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Plant-based medicines become increasingly popular over the world. Authentication of herbal raw materials is important to ensure their safety and efficacy. Some herbs belonging to closely related species but differing in medicinal properties are difficult to be identified because of similar morphological and microscopic characteristics. Chromatographic fingerprinting is an alternative method to distinguish them. Existing approaches do not allow a comprehensive analysis for herbal authentication. We have now developed a strategy consisting of (1) full metabolic profiling of herbal medicines by rapid resolution liquid chromatography (RRLC) combined with quadrupole time-of-flight mass spectrometry (QTOF MS), (2) global analysis of non-targeted compounds by molecular feature extraction algorithm, (3) multivariate statistical analysis for classification and prediction, and (4) marker compounds characterization. This approach has provided a fast and unbiased comparative multivariate analysis of the metabolite composition of 33-batch samples covering seven Lonicera species. Individual metabolic profiles are performed at the level of molecular fragments without prior structural assignment. In the entire set, the obtained classifier for seven Lonicera species flower buds showed good prediction performance and a total of 82 statistically different components were rapidly obtained by the strategy. The elemental compositions of discriminative metabolites were characterized by the accurate mass measurement of the pseudomolecular ions and their chemical types were assigned by the MS/MS spectra. The high-resolution, comprehensive and unbiased strategy for metabolite data analysis presented here is powerful and opens the new direction of authentication in herbal analysis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Sample pretreatment is a key step in bioanalytical process because of possible interference and matrix effects in mass spectrometry analysis. In this work, a novel strategy towards high speed and sensitivity was developed combining in vivo microdialysis (MD) sampling, turbulent-flow chromatography (TFC), and liquid chromatography-mass spectrometry (LC-MS). The procedures of cleanup, preconcentration, and separation were completed on-line in one step within 10min. During the MD optimization procedure, 1% hydroxypropyl-β-cyclodextrin (HP-β-CD) was used to improve the relative recovery of the analyte. Untreated MD samples were directly injected, and a TFC precolumn was flushed for 1min with aqueous phase of 4mL/min flow rate to desalt and concentrate biosamples. The retained analytes were then back-flushed by a switching valve onto a fast LC column (4.6mm×50mm, 1.8μm) for separation. Another diverter valve was employed to prevent the HP-β-CD that interferes with the ESI process from entering the MS. Puqietinone, a lipophilic alkaloid from Fritillaria puqiensis, was used as a case for validation. Results showed that the limit of quantification for puqietinone was 0.10ng/mL, and good linearity (R(2)=0.9993) was maintained over the range of 1.02-200.02ng/mL. Accuracy and precision were satisfactory within the range of the standard curve. This approach was able to effectively eliminate the influences of matrix effect and carry-over as the injection volume increased up to 20μL. The developed method was successfully applied to pharmacokinetic study of puqietinone after intravenous administration to rat. Results demonstrate the potential of using MD with TFC-LC/MS for in vivo monitoring experiments., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A dynamic microdialysis sampling method with liquid chromatography-quadrupole time-of-flight mass spectrometry (Q-TOF-MS) was developed for rapid and sensitive analysis of the metabolite profile of Panax notoginseng extract (PNE) in rat bile. In vivo studies in male Sprague-Dawley rats were performed with microdialysis probes implanted into the bile duct before bile samples were collected from 0 to 12h. Metabolites of PNE were identified using dynamic adjustment of the fragmentor voltage to produce structure-relevant fragment ions. The mass accuracy of precursor and fragment ions was typically within 5 ppm of the theoretical values. We identified 7 compounds: 4 parent compounds (notoginsenoside R1, ginsenosides Rg1, Rb1, and Rd) and 3 metabolites (ginsenosides Rg2, Rh2, and compound K). Data from this study suggest that this microdialysis technique could be used in notoginseng saponin metabolic animal studies., (Published by Elsevier B.V.)

As one of the most commonly used medicinal plants, ginseng has been an attractive model herb for study. A wide range of analytical methods has been used to characterize its constituents. However, less effort has been devoted to the rare ginseng saponins, especially their isomers and sugar linkages. In this study, we used segmental monitoring and diagnostic ion filtering to characterize ginseng saponins by rapid liquid chromatography with time-of-flight mass spectrometry (LC-TOF-MS). By using selected diagnostic ions, specific groups of ginseng saponins were readily extracted from the complicated matrix. 20(R) and 20(S) stereo-saponins were differentiated using the peak abundance ratio of [M-H(2)O+H](+) to [M-2H(2)O+H](+). The fragmentation behavior of ginsenosides was first reported in negative ion mode by MS/MS with high-energy collision-induced dissociation, producing rules to determine sugar numbers, positions and linkages. Using the rules, we identified and compared the nontarget ginseng saponin profiling of raw and steamed American ginseng roots and berries. We characterized 70 saponins in the samples. Our strategy can be extended to screen and characterize other rare ginseng saponins and their metabolites., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Spatholobus suberectus is a widely used herb in traditional medicine for the treatment of blood stasis syndrome and related diseases. In this work, a potential ultrasonic/microwave assisted extraction (UMAE) method was developed for efficient sample pretreatment, and a diagnostic ion filtering strategy with liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) was established for rapid characterization of flavonoids in S. suberectus. The factors of UMAE influencing the extraction yield of flavonoids of S. suberectus were evaluated. The optimal conditions were determined as: microwave power of 300 W, extraction time of 450 s, 70% methanol as extraction solvent, solvent to solid ratio of 20 mL/g, ultrasound power of 50 W, extraction temperature of 80 °C, and one extraction cycle. Compared with commonly used extraction methods, UMAE showed higher efficiency and shorter extraction time for sample preparation. Subsequently, the major diagnostic ions and fragmentation pathways of flavonoids in Q-TOF-MS were summarized with available reference compounds. Using a new diagnostic ion filtering strategy, a rapid screening and identification of thirty-eight compounds was achieved in real S. suberectus samples. The results of this study clearly demonstrate the potential of UMAE for efficient extraction and LC-Q-TOF-MS for rapid and sensitive structural elucidation of flavonoids in S. suberectus, and open perspectives for similar studies on other medicinal herbs., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Ginseng occupies a prominent position in the list of best-selling natural products in the world. Compared to the long history of use and widespread research on Asian ginseng, the study of American ginseng is relatively limited. In the past decade, some promising advances have been achieved in understanding the chemistry, pharmacology and structure-function relationship of American ginseng. To date, there is no systematic review of American ginseng. In this review, the different structures of the ginsenosides in American ginseng are described, including naturally occurring compounds and those resulting from steaming or biotransformation. Preclinical and clinical studies published in the past decade are also discussed. Highlighted are the chemical and pharmacological diversity and potential structural-activity relationship of ginsenosides. The goal is that this article is a useful reference to chemists and biologists researching American ginseng, and will open the door to agents in drug discovery., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (LC/ESI-QTOF-MS/MS) was performed to study the fragmentation behaviors of steroidal alkaloids from Fritillaria species, the antitussive and expectorant herbs widely used in traditional Chinese medicine. We propose, herein, a strategy that combining key diagnostic fragment ions and the relative abundances and amounts of major fragment ions (the ions exceeding 10% in abundance) to distinguish different sub-classes of Fritillaria alkaloids (FAs). It was found that hydrogen rearrangement and induction effects result in ring cleavage of the basic skeletons occurred in the MS/MS process and produced characteristic fragment ions, which are useful for structural elucidation. This method was finally used to investigate the primary steroidal alkaloids in the extracts of eight major Fritillaria species. As a result, 41 steroidal alkaloids (29 cevanine type, 1 jervine type, 6 veratramine type and 5 secosolanidine type alkaloids) were selectively identified in these Fritillaria species. Twenty-six compounds were unambiguously identified by comparing with the reference compounds and 15 compounds were tentatively identified or deduced according to their MS/MS data. Logical fragmentation pathways for different types of FAs have been proposed and are useful for the identification of these types of steroidal alkaloids in natural products especially when there are no reference compounds available., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A method based on the on-line turbulent-flow chromatography and fast high-performance liquid chromatography/mass spectrometry (TFC-LC/MS) was developed for sensitive and high throughput pharmacokinetic study of traditional Chinese medicines (TCMs). In this method, an on-line extraction column (Waters Oasis HLB) and a fast HPLC column with sub-2 microm particle size (Agilent Zorbax StableBond-C(18), 4.6 mm x 50 mm, 1.8 microm) in a column-switching set-up were utilized. HLB is a reversed-phase extraction column with hydrophilic-lipophilic balanced copolymer (2.1 mm x 20 mm, 25 microm particle size), which will exhibit some turbulent-flow properties at a high-flow rate. The method combines the speed and robustness of turbulent-flow extraction and the sensitivity and separation efficiency of fast HPLC-MS to analyze multiple and trace constituents of TCMs in plasma matrix. This method was successfully applied for pharmacokinetic study of verticine, verticinone and isoverticine, the chemical markers of Fritillaria thunbergii, after oral administration of total steroidal alkaloids extract of F. thunbergii to rats. Each plasma sample was analyzed within 7 min. The method demonstrated good linearity (R>0.999) ranged from 0.505 to 96.0 ng/mL with satisfactory accuracy and precision, and the lower limit of quantifications of verticine, verticinone and isoverticine were estimated to be 0.120, 0.595 and 0.505 ng/mL, respectively. These results indicate that the proposed method is fast, sensitive, and feasible for pharmacokinetic study of TCMs., (2009 Elsevier B.V. All rights reserved.)

The fact that the effects of herbal medicines (HMs) are brought about by their chemical constituents has created a critical demand for powerful analytical tools performing the chemical analysis to assure their efficacy, safety and quality. Liquid chromatography coupled to mass spectrometry (LC-MS) is an excellent technique to analyze multi-components in complex herbal matrices. Due to its inherent characteristics of accurate mass measurements and high resolution, time-of-flight (TOF) MS is well-suited to this field, especially for qualitative applications. The purpose of this article is to provide an overview on the potential of TOF, including the hybrid quadrupole- and ion trap-TOF (QTOF and IT-TOF), hyphenated to LC for chemical analysis in HMs or HM-treated biological samples. The peculiarities of LC-(Q/IT)TOF-MS for the analysis of HMs are discussed first, including applied stationary phase, mobile-phase selection, accurate mass measurements, fragmentation and selectivity. The final section is devoted to describing the applicability of LC-(Q/IT)TOF-MS to routine analysis of multi-components, including target and non-target (unknown) compounds, in herbal samples, emphasizing both the advantages and limitations of this approach for qualitative and quantitative purposes. The potential and future trends of fast high-performance liquid chromatography (HPLC) (e.g. rapid resolution LC and ultra-performance LC) coupled to (Q)TOF-MS for chemical analysis of HMs are highlighted.

Harpagoside, a major bioactive iridoid glucoside in genus Scrophularia, has been widely used in clinical practice for the treatment of pain in the joints and lower back for its neuroprotective and anti-inflammation activities. To investigate the pharmacokinetics and hepatobiliary excretion, an in vivo microdialysis method coupled with high performance liquid chromatography was developed to monitor the concentration of harpagoside in blood and bile. The harpagoside bile-to-blood distribution ratio (AUC(bile)/AUC(blood)) up to 986.28+/-78.46 significantly decreased to 6.41+/-0.56 or 221.20+/-18.92 after co-administration of cyclosporin A or verapamil. The results indicated that harpagoside went through concentrative elimination from the bile which was probably regulated by P-glucoprotein, providing possible clinical trials of co-administration of transporter inhibitors to decrease drug efflux, thus to enhance the curative effects.

A rapid high-performance liquid chromatography-mass spectrometry (HPLC-MS) method was developed and validated for simultaneous quantification of 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol in rat plasma after oral administration of ginger oleoresin. Plasma samples extracted with a liquid-liquid extraction procedure were separated on an Agilent Zorbax StableBond-C(18) column (4.6 mm x 50 mm, 1.8 microm) and detected by MS with electrospray ionization interface in positive selective ion monitoring (SIM) mode. Calibration curves (1/x(2) weighted) offered satisfactory linearity (r(2)>0.995) in a wide linear range (0.0104-13.0 microg/mL for 6-gingerol, 0.00357-4.46 microg/mL for 8-gingerol, 0.00920-11.5 microg/mL for 10-gingerol and 0.00738-9.22 microg/mL for 6-shogaol). The lower limit of quantification (LLOQ) was in a range of 3.57-10.4 ng/mL. The analytes and internal standard can be baseline separated within 6 min. Inter- and intra-day assay variation was less than 15%. This developed method was successfully applied to pharmacokinetic studies of ginger oleoresin after oral administration to rats. Glucuronide of 6-gingerol was determined after beta-glucuronidase hydrolysis for more information, and the intestinal glucuronidation was further confirmed by comparison of plasma samples of hepatic portal vein and femoral vein.

A high-performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) method has been developed for qualitative and quantitative analysis of isoflavonoids and saponins, as well as for the quality control of Radix Astragali and its preparations. The selectivity, reproducibility and sensitivity are compared with HPLC with diode array detection (DAD) and evaporative light scattering detection (ELSD). Limits of detection and quantification fell in ranges of 9-40 and 23-103 ng/mL for 13 analytes with a injection of 10 microL samples, and all calibration curves showed good linear regression (r(2)>0.9938) within the test range. The assay was successfully utilized to analyze the 13 marker components in 20 samples of Radix Astragali products. The quantitative results demonstrated that samples from different localities and manufacturers showed different quality. Advantages, in comparison with conventional HPLC-DAD-ELSD, are that reliable identification of target compounds could be achieved by accurate mass measurements (<3 ppm) along with characteristic retention time, extracted ions chromatograms using a narrow mass window for quantification ensure that the chromatographic peaks are free from background or co-elutes interference, and the great enhancement in selectivity and sensitivity allows identification and quantification of low levels of constituents in complex Radix Astragali matrixes.

A liquid chromatography-electrospray ionization-mass spectrometry method has been developed and validated for identification and quantification of four major bioactive saponins in rat plasma after oral administration of extraction of saponins from Flos Lonicerae, i.e., macranthoidin B, macranthoidin A, dipsacoside B, and macranthoside B. Plasma samples were extracted with solid-phase extraction, separated on a Shim-pack CLC-ODS column and detected by MS in negative selective ion monitoring mode. Calibration curves offered linear ranges of two orders of magnitude with r(2)>0.999. The method showed the low limit quantification of 7.72, 6.06, 7.16, and 1.43 ng/mL for macranthoidin B, macranthoidin A, dipsacoside B, and macranthoside B, respectively. The inter- and intra-CV precision (R.S.D.) were all within 10% and accuracy (% bias) ranged from -10 to 10%. The overall recovery was more than 70%. This developed method was subsequently successfully applied to pharmacokinetic profiles of the four saponins in rats. After oral administration of extraction of saponins in rats, the concentration-time course was found to be the double peaks of curve.

A novel fast high-performance liquid chromatography (HPLC) method coupled with diode array detection (DAD) and time-of-flight mass spectrometry (TOF/MS) was developed for qualitative and quantitative analysis of Radix Astragali products. The potential of fast HPLC on 1.8-microm particles was compared with the performance of HPLC on conventional 5-microm particles columns. Significant advantages of fast HPLC include high-speed chromatographic separation, four times faster than HPLC with conventional columns, and great enhancement in sensitivity with limits of detection low to 0.001 ng. With dynamic adjustment of fragmentor voltage in TOF/MS, an efficient transmission of the ions was achieved to obtain the best sensitivity and abundant fragmentation. By accurate mass measurements within 5 ppm error for each molecular ion and subsequent fragment ions, a reliable identification and differentiation of six major saponins including two groups of isomers and twelve main isoflavonoids was described here for the first time. For quantitative analysis by fast HPLC-TOF/MS, linearity of response over two orders of magnitude was demonstrated (r(2)>0.99) for all analytes. Intra-day reproducibility was below 3% RSD and inter-day values were below 5% RSD. A good correlation (slope=1.1108, r(2)=0.9853) was observed for accuracy test. It is concluded that the fast and sensitive HPLC-DAD-TOF/MS is powerful in qualitative and quantitative analysis of complex herbal medicines in terms of time savings, sensitivity, selectivity, precision, accuracy as well as increasing sample throughout and lowering solvent consumption.

A sensitive and reliable high-performance liquid chromatography-mass spectrometry (HPLC-MS) was developed and validated for simultaneous quantification of five main bioactive components, i.e., calycosin-7-O-beta-D-glucoside, ononin, astragaloside IV, astragaloside I and ferulic acid in rat plasma after oral administration of Danggui Buxue Tang (DBT) extract. Plasma samples were extracted with solid-phase extraction (SPE) separated on an Inertsil ZORBAX C(18) column and detected by MS with electrospray ionization (ESI) interface in negative selective ion monitoring (SIM) mode. Calibration curves offered linear ranges of two orders of magnitude with r(2)>0.99. The method had the lower limit quantification of 0.55, 0.46, 1.07, 1.12 and 4.6 ng/mL for calycosin-7-O-beta-D-glucoside, ononin, astragaloside IV, astragaloside I and ferulic acid, respectively, with precision less than 10%. The RSD of intra- and inter-day variations ranged from 2.10% to 6.19% and 2.37% to 6.72%. This developed method was subsequently applied to pharmacokinetic studies of the five compounds in rats successfully.

A liquid chromatographic method was developed for simultaneous determination of two main types of bioactive compounds: four phytoecdysones and eight triterpenoids in radix achyranthis bidentatae (RAB), i.e., polypodine B (1), ecdysterone (2), 25-R inokosterone (3), 25-S inokosterone (4), ginsenoside Ro (5), chikusetsusaponin IVa (6), zingibroside R1 (7), chikusetsusaponin IVa ethyl ester (8), 28-deglucosyl-chikusetsusaponin IVa (9), PJS-1 (10), 28-deglucosyl-chikusetsusaponin IVa butyl ester (11), and oleanolic acid (12). Optimum separations were obtained with a Zorbax C18 column, using a gradient elution with 0.08% aqueous formic acid (containing 5% isopropyl alcohol) and acetonitrile as mobile phase. Phytoecdysones were detected by diode array detector (DAD) at 242 nm, whereas triterpenoids were monitored by evaporative light scattering detector (ELSD) connected in series with DAD, temperature for the drift tube was 110 degrees C and the nitrogen flow rate was 3.2 L min(-1). The identity of the analytes was confirmed using retention times, ultraviolet absorbance and mass spectral data in comparison with reference compounds. The method was validated for acceptable precision (intra- and inter-day variation < or = 4.87%), accuracy (recovery > or = 88.9%) and sensitivity (LOD < or = 0.43 microg mL(-1) (DAD) and 26.0 microg mL(-1) (ELSD), LOQ < or = 0.97 microg mL(-1) (DAD) and 46.5 microg mL(-1) (ELSD), respectively). This rapid and reliable method was applied for the analysis of four cultivated and ten commercial samples. The results demonstrated that the method is suitable for routine analysis and quality control of RAB.

Interaction of a commonly used combined prescription of Danggui Buxue Decoction (CPDBD) with protein was studied by microdialysis coupled with HPLC-DAD-MS. Nine compounds in CPDBD were unequivocally identified by comparing with their t(R), MS data and UV spectra with those of reference compounds, and simultaneously quantified. Microdialysis recoveries and binding degrees of 20 compounds in CPDBD with bovine serum albumin (BSA) were determined. Recoveries of microdialysis sampling ranged from 66.9 to 91.5% with RSD below 3.0%, and the binding degrees of those to BSA ranged from 6.3 to 59.8% (0.3mM BSA) and from 6.9 to 86.6% (0.6mM BSA). The results were determined at pH 7.4 and the influence of different pH value was investigated. Furthermore, the binding degrees of eight reference compounds were determined separately under the same conditions, indicating a significant effect of the interaction of compounds with each other on their binding degrees to BSA. By comparing their binding degrees with BSA with those of proven active compounds in CPDBD, i.e. chlorogenic acid (3), ferulic acid (6), ononin (12) and calycosin (16), other five compounds were found to possess potential activities, which were tentatively identified as calycosin-7-O-beta-D-glucoside-6-O-malonate (9), senkyunolide I or H (10), formononetin-7-O-beta-D-glucoside-6-O-malonate (17), and two unknown compounds.

A method, high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors (HPLC-DAD-ELSD), was developed to evaluate the quality of Radix Astragali through a simultaneous determination of six major active isoflavonoids and four main saponins. The wavelength at 280 nm was chosen to determine six isoflavonoids: calycosin-7-O-beta-D-glucoside (1), ononin (2), (6alphaR, 11alphaR)-9,10-dimethoxypterocarpan-3-O-beta-D-glucoside (3), (3R)-2'-hydroxy-3',4'-dimethoxyisoflavan-7-O-beta-D-glucoside (4), calycosin (5), and formononetin (6); and ELSD connected after DAD was employed to determine four saponins: astragaloside IV (7), astragaloside II (8), astragaloside I (9), and acetylastragaloside I (10). This assay was fully validated with respect to precision, repeatability and accuracy. The proposed method was successfully applied to quantify the ten components in eleven samples from different localities in China; significant variations were demonstrated in the content of these compounds in the samples from different areas. This simple, rapid, low-cost and reliable HPLC-DAD-ELSD method is suitable for routine quantitative analysis and quality control of traditional Chinese medicines (TCMs) consisting of bioactive multi-components with different structures such as Radix Astragali.