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Abstract: Radix Polygoni Multiflori, Rhizoma et Radix Polygoni Cuspidati, and Radix et Rhizoma Rhei are the most frequently used traditional Chinese medicines in the family Polygonaceae. The three herbal medicines (HMs) contain similar types of compounds. In Chinese Pharmacopoeia, five high-performance liquid chromatography (HPLC) coupled with ultraviolet (UV) detection methods have been employed for their quality control. The aim of this study was to develop a simple and conventional strategy, segmental monitoring based on variable wavelength detection (VWD), for simultaneous quantification of phenolic acids, flavonoids, stilbenes and anthraquinones in the three chemically analogous HMs. Compared with the commonly used HPLC-diode array detection (DAD), the proposed method afforded desirable performance on linearity, precision and sensitivity. Additionally, a quadrupole time-of-flight mass spectrometry (QTOF-MS) was applied to the structural confirmation of analytes from complex matrices. Based on the chemical profiles and contents of the analyses, 27 samples from three HMs were well classified using the principal component analysis. The results of this study demonstrated the potential and applicability of segmental monitoring strategy based on VWD for comprehensive quality control of HMs. [Copyright &y& Elsevier]

Abstract: In this work, the metabolite profiles of Danggui Buxue Tang (DBT) in rat bile and plasma were qualitatively described, and the possible metabolic pathways of DBT were subsequently proposed. Emphasis was put on correlative analysis of metabolite profiling in different biological fluids. After oral administration of DBT, bile and plasma samples were collected and pretreated by solid phase extraction. Rapid resolution liquid chromatography coupled to time-of-flight mass spectrometry (RRLC–TOFMS) was used for characterization of DBT-related compounds (parent compounds and metabolites) in biological matrices. A total of 142 metabolites were detected and tentatively identified from the drug-containing bile and plasma samples. Metabolite profiling shows that rat bile contained relatively more glutathione-derived conjugates, more saponins compounds and more diverse forms of metabolites than urine. The metabolite profile in plasma revealed that glucuronide conjugates of isoflavonoids, dimmers, acetylcysteine conjugates and parent form of phthalides, as well as saponin aglycones were the major circulating forms of DBT. Collectively, the metabolite profile analysis of DBT in different biological matrices provided a comprehensive understanding of the in vivo metabolic fates of constituents in DBT. [Copyright &y& Elsevier]

Abstract: A dynamic microdialysis sampling method with liquid chromatography–diode array detection and time-of-flight mass spectrometry (LC–DAD–TOF/MS) analysis was developed to investigate rat microsomal metabolisms of calycosin and formononetin, and their drug–drug interactions. Two hydroxylated metabolites from calycosin, and three hydroxylated or 4′-O-demethylated derivatives from formononetin were detected and identified after co-incubation with microsomes. Calibration curves offered linear ranges of two orders of magnitude with r 2 >0.999 for calycosin, formononetin and daidzein. The quantitative LC method provides a range of 0.028–0.034μg/mL for limits of detection, overall precision less than 5% and accuracy less than 3% by RSD. Besides, calycosin and formononetin were found to produce the depressive effect on the CYP450 enzyme reaction, and inhibit phase I enzyme reaction of each other when they are concurrent. Dynamic microdialysis sampling with LC–DAD–TOF/MS analysis developed in this work is a powerful tool for in vitro metabolism studies of drugs and metabolic interactions. [Copyright &y& Elsevier]

Abstract: A quantitative method by ultra-fast HPLC coupled with time-of-flight mass spectrometry (TOF/MS) has been developed for analysis of 15 major constituents in Danggui Buxue Tang (DBT) preparations. Its performance was compared with a conventional HPLC method with diode array detection/evaporative light scattering detection (DAD/ESLD). Accurate mass measurements within 3ppm error were obtained for all the compounds. The analytical time by an ultra-fast system is 4 times faster than conventional HPLC, the limits of detection by TOF/MS are low to 0.004–0.08ng compared with 1–200ng for HPLC–DAD–ELSD, and acceptable linearity of response was demonstrated over two orders of magnitude (r 2 >0.99) for all analytes. Intra-day reproducibility was below 3% and inter-day values were below 5% R.S.D. Robustness and applicability of the method was validated for the analysis of market DBT real samples, proposing a rapid, sensitive and validated method for routine analysis and quality control of DBT. [Copyright &y& Elsevier]

Abstract: A novel on-line technique for stacking and sweeping of long sample plugs with simultaneous determination of charged analytes in the plant (protocatechuic aldehyde, rosmarinic acid, danshensu, salvianolic acid B, and protocatechuic acid) by the nonionic microemulsion electrokinetic chromatography (MEEKC) is presented. The preconcentration efficiency provided about 9–28-fold for stacking and 7–14-fold for sweeping in the enhancements of LOD. The effects of oil phase, Brij-35 and buffer concentrations on stacking and sweeping efficiency were examined in order to optimize the two methods. In nonionic MEEKC, the effect of the type of oil and buffer contents on preconcentration mechanism is often sophisticated. This study had demonstrated that the oil type and buffer content in nonionic microemulsion indeed markedly altered the affinity of microemulsion with analytes. Finally, in comparison to the stacking method, the most apparent disadvantages of the sweeping method were the relatively high limits of detection and poor peak shapes. [Copyright &y& Elsevier]

Abstract: A herb-combined prescription, mainly derived from roots of Salvia miltiorrhiza and Panax notoginseng, has been widely used for improving coronary or cerebral circulation in China as well as in Western countries. Multiple commercially available preparations, known as Fufang Danshen preparations (FDPs), produced by various manufacturers with the raw materials from different sources, pose a serious challenge to the quality control of this herb medicine. Previous pharmacological studies identify three types of bioactive components correlated with the clinical effect of those herb preparations. Those mainly include four phenolic acids, four saponins and four diterpenoid quinones. In this report, by using high performance liquid chromatography (HPLC) coupled with diode array and evaporative light scattering detectors (DAD–ELSD), we developed an improved quality control method for those herb medicines. A simultaneous separation and quantification of the 12 components was performed on a C18 column, in which the mobile phase consisted of (A) 0.1% aqueous formic acid and (B) acetonitrile using a gradient elution. The optimum detection wavelength was set at 281nm, the drift tube temperature of ELSD was set at 113°C, the nitrogen flow rate at 3.1L/min, and the gain=4. All calibration curves showed good linear regression (r 2 >0.9927) within test ranges. The method developed showed good precision and accuracy with overall intra- and inter-day variations of 0.64–4.79% and 0.69–4.96%, respectively, and the overall recoveries of 93.50–107.69% for the 12 compounds analyzed. This method was successfully applied to quantify the twelve components in ten commercial samples from three formulas by seven independent manufacturers. This readily available, low-cost and reliable HPLC–DAD–ELSD method improved the quality control of traditional Chinese medicinal preparations consisting of complex compounds with different structures such as FDPs. [Copyright &y& Elsevier]

Abstract: Spinosin, a major bioactive herbal ingredient isolated from Semen Ziziphi Spinosae, plays an important role in sedation and hypnosis. However, the pharmacokinetic behavior of spinosin in special sites has not been reported. Microdialysis (MD) technique, as a continuous, realtime monitoring sampling technique, is very suitable for the evaluation of the disposition of diverse drugs. To obtain more useful information on spinosin, an in vivo microdialysis sampling technique with High Performance Liquid Chromatography–mass spectrograph (HPLC–MS) method was developed to investigate the pharmacokinetics of spinosin and its interaction with cyclosporin A (CsA) in the brain, blood and bile of rats. The method was validated in terms of selectivity, linearity and sensitivity, and showed advantages in monitoring the pharmacokinetic behavior of drugs. The results revealed that CsA has obvious effects on the pharmacokinetic process of spinosin. When co-administered, the area under the curve (AUC) of spinosin in blood, bile and brain increased from 205.70 to 673.51mgmin/L, 7.77×104 to 1.25×105 mgmin/L, and 2.09 to 5.58mgmin/L, respectively. The t 1/2 values of spinosin in blood, bile and brain also changed from 48.07 to 95.04min, from 97.20 to 152.21 and from 42.18 to 73.83min, respectively. These results demonstrated that the CsA decreased the efflux of spinosin through the inhibition of P-glycoprotein (P-gp) efflux transporter and it might be used as a group of P-gp substrate. Other transporters or pathways may also be involved in the metabolism of spinosin. [Copyright &y& Elsevier]

Abstract: Belamcandae Rhizoma, derived from the rhizome of Belamcanda chinensis (L.) DC., has been used as traditional Chinese medicine for the treatment of coughing and pharyngitis. However, there have been few studies dealing with the systematic analysis of the bioactive constituents in Belamcandae Rhizoma. In this work, high performance liquid chromatography–diode array detection–electrospray ionization multiple-stage mass spectrometry (HPLC-DAD-ESI-MS n ) combined with liquid chromatography–time of flight-mass spectrometry (HPLC–TOF/MS) was established for profiling and characterization of multi-constituent in Belamcandae Rhizoma. The ESI-MS n fragmentation behaviors of the authentic references were proposed for aiding the structural identification of components in the extract. Thirty-five flavonoids, including 30 isoflavones and five xanthones, were identified or tentatively identified by comparing their retention times, UV and MS spectra with those of authentic compounds or literature data. Twelve of the identified compounds (neomangiferin, mangiferin, tectoridin, iristectorin B, iristectorin A, iridin, tectorigenin, iristectorigenin A, irigenin, irisflorentin, irilone and dichtomitin) were determined by HPLC–DAD using a C18 column. The results indicated that the developed analysis method could be employed as a rapid, effective technique for structural characterization of chemical constituents in herbal medicine. This work is expected to provide comprehensive information for the quality evaluation of Belamcandae Rhizoma, which would be a valuable reference for the further study and development of this herb and related medicinal products. [Copyright &y& Elsevier]

Jitong Ning Tablet (JTNT), a traditional Chinese herbal formula, consists of Eucommia ulmodies oliv , Angelicae pubescentis radix , Aconiti radix cocta , Corydalis yanhusuo w.t. wang , Glycyrrhizae radix et rhizoma , Paeoniae radix rubra and Radix puerariae . It has been demonstrated to show protective effects on ankylosing spondylitis and anti-inflammatory effects. The chemical compositions of JTNT, playing a key role in quality control, remain unknown. In this study, an ultra-performance liquid chromatography combined with quadrupole time of flight mass spectrometry (UPLC-Q–TOF–MS) method in both positive and negative ion mode was established to investigate the chemical constituents of JTNT formula. In total, 162 compounds including flavonoids, triterpenoids, coumarins, alkaloids, phenylpropionic acids, lignans, terpenoids, and organic acids were detected, 152 of which were unambiguously or tentatively identified by comparing their retention times and accurate mass measurement with reference compounds and data in literatures. Our results would benefit quality control and chemical basis for JTNT. [ABSTRACT FROM AUTHOR]

The root and rhizome of Panax notoginseng (PNG) are used as folk medicine. Recent studies have reported PNG to possess immunomodulatory, cardioprotective, hepatoprotective, anti-diabetic and anticancer activities among a host of other pharmacological effects. The main active constituents responsible for these pharmacological effects are saponins. It has also been proven that the chemical constituents of steamed PNG differs from the raw form. Traditional methods of separating individual components in crude extracts are usually tedious, almost irreproducible and time-consuming. In this study, an automated multi-step preparative separation system, known as Sepbox afforded a quick, reproducible and fast separation of saponins from PNG. With Sepbox, a total of 11 saponins of high purity were obtained in a short period of time. The separated compounds were identified as notoginsenosides R1, T5, ginsenosides Rb1, Rg1, Rg2, Rh1, Rh4, Rd, 20 (S) −Rg3 and a mixture of ginsenosides Rk1 and Rg5. [ABSTRACT FROM AUTHOR]

Most Aconitum species, also known as aconite, are extremely poisonous, so it must be identified carefully. Differentiation of Aconitum species is challenging because of their similar appearance and chemical components. In this study, a universal strategy to discover chemical markers was developed for effective authentication of three commonly used aconite roots. The major procedures include: (1) chemical profiling and structural assignment of herbs by liquid chromatography with mass spectrometry (LC-MS), (2) quantification of major components by LC-MS, (3) probabilistic neural network (PNN) model to calculate contributions of components toward species classification, (4) discovery of minimized number of chemical markers for quality control. The MS fragmentation pathways of diester-, monoester-, and alkyloyamine-diterpenoid alkaloids were compared. Using these rules, 42 aconite alkaloids were identified in aconite roots. Subsequently, 11 characteristic compounds were quantified. A component–species modeling by PNN was then established combining the 11 analytes and 26-batch samples from three aconite species. The contribution of each analyte to species classification was calculated. Selection of fuziline, benzoylhypaconine, and talatizamine, or a combination of more compounds based on a contribution order, can be used for successful categorization of the three aconite species. Collectively, the proposed strategy is beneficial to selection of rational chemical markers for the species classification and quality control of herbal medicines. [ABSTRACT FROM AUTHOR]

Single standard to determine multi-components (SSDMC) is a practical pattern for quality evaluation of herbal medicines (HMs). However, it remains challenging because of potential inconsistency of relative response factors (RRF) on different instruments. In this work, the effects of two key roles, i.e., ultraviolet (UV) wavelength and standard solution concentrations, on reproducibility of RRF were investigated. The effect of UV wavelength on reproducibility of RRF was studied by plotting the relationship of the peak area ratios (internal standard vs analyte) to wavelengths. The preferable wavelength should be set at the flat parts of the curve. Optimized 300 nm produced a 0.38% RSD for emodin/emodin-8- O -β- D -glucopyranoside on five instruments, much lower than 2.80% obtained from the maximum wavelength at 290 nm. Next, the effects of standard solution concentrations of emodin on its response factor (RF) were investigated. For one single point method, low concentration less than 49b/k resulted in significant variations in RF. For emodin, when the concentration is higher than 7.00 μg mL −1 , a low standard deviation (SD) value at 0.13 was obtained, while lower than 7.00 μg mL −1 , a high SD at 3.71 was obtained. The developed SSDMC method was then applied to determination of target components in 10 Polygonum cuspidatum samples and showed comparable accuracy to conventional calibration methods with deviation less than 1%. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop a convenient method without pretreatments for nontarget discovery of interested compounds. The segment and exposure strategy, coupled with two mass spectrometer data acquisition methods was firstly proposed for screening the saponins in extract of Panax notoginseng (Sanqi) via high-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (HPLC-QTOF/MS). By gradually removing certain major or moderate interference compounds, the developed segment and exposure strategy could significantly improve the detection efficiency for trace compounds. Moreover, the newly developed five-point screening approach based on a modified mass defect filter strategy and the visual isotopic ion technique was verified to be efficient and reliable in picking out the interested precursor ions. In total, 234 ginsenosides including 67 potential new ones were characterized or tentatively identified from the extract of Sanqi. Particularly, some unusual compounds containing the branched glycosyl group or new substituted acyl groups were firstly reported. The proposed integrated strategy held a strong promise for analyses of the complex mixtures. [ABSTRACT FROM AUTHOR]

Many analytical methods have been developed to characterize ginsenosides in ginseng. Relatively less attention has been paid to the malonyl ginsenosides, amino acids and polysaccharides in various processing ginsengs. In this study, malonyl ginsenosides were characterized by LC–Q-TOF/MS. In positive mode, the most abundant ions at m / z 425.38 were observed corresponding to the protopanoxadiol-type ginsenosides. A rich diagnostic ion at 835.48 was shown representing the malonyl ginsenosides with at least two glucosides. Twelve malonyl ginsenosides were rapidly screened using 835.48–835.49 to restructure ion chromatograms. In negative mode, besides the high deprotonated ion, a neutral loss of 44 Da (CO 2 ) was found. High-energy collision-induced dissociation at 50 V produced the most abundant product ion [M−H-malonyl] − by a neutral loss of 86 Da. Determination of 17 common amino acids was performed on an automatic amino acid analyzer. Arginine, glutamic acid, and aspartic acid were abundant. The contents of amino acids were 9.1% in fresh ginseng and 3.1% in black ginseng. Phenol-sulfuric acid method was applied to analysis of polysaccharides. The contents of polysaccharides were 29.1% in fresh ginseng and 11.1% in black ginseng. The optimal growth age for the accumulation of constituents was supposed to be 5–6 years. In conclusion, the contents of malonyl ginsenosides, amino acids, and polysaccharides, based on decreasing order, ranked as follows: fresh ginseng > frozen ginseng > white ginseng > stoved ginseng > red ginseng > black ginseng. Processing should be paid more attention for the quality control of ginseng products. [ABSTRACT FROM AUTHOR]

Highlights: [•] A MSPD method for extraction and clean-up of ginkgo components. [•] UHPLC–QQQ-MS for simultaneous analysis of 18 ginkgo components. [•] Direct quantification of 10 flavonol glycosides without hydrolysis. [•] Analysis of the 12 Ginkgo biloba tablets available in the global market. [Copyright &y& Elsevier]

Highlights: [•] Three chemical constituents from Salvia miltiorrhiza were extracted by new designed TSURE. [•] Optimized TSURE offered good extraction efficiency that was comparable or even better than conventional methods. [•] TSURE method was extended to the quality control of Salvia miltiorrhiza. [•] Application of TSURE to other herbal medicines was also explored and similar results were discovered. [•] Mechanisms of TSURE were discussed in detail. [ABSTRACT FROM AUTHOR]

Highlights: [•] HPLC–QTOF MS method was developed for comprehensive analysis of Shenfu injection. [•] A total of 44 compounds were characterized by HPLC–QTOF MS. [•] Twenty-four major alkaloids and ginsenosides were quantified by HPLC–MS. [•] Similarity analyses for Shenfu injection demonstrated high quality consistency. [ABSTRACT FROM AUTHOR]

Abstract: In this study, an efficient strategy based on bioassay-guided fractionation, high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC-ESI-Q/TOF-MS) and high-speed counter-current chromatography (HSCCC) was established to screen and purify bioactive compounds from Chinese herbal medicines (CHMs). This screening system was efficient and successfully applied to reveal anti-prostate cancer candidates from Puerariae thomsonii Flos. As a result, an active fraction with strong in vitro anti-prostate cancer activity was obtained, and the main compounds in the fraction were purified by HSCCC, giving 82mg of tectoridin, 36mg of tectorigenin-7-O-[β-d-xylopyranosyl-(1→6)-β-d-glucopyranoside and 64mg of tectorigenin. Among them, tectorigenin, possessing the highest anti-prostate cancer activity with IC50 value of 0.08μM, has priority to be lead compound. The results of this work demonstrated that the developed method was efficient and could be employed for the rapid screening, identification and purification of active components from CHMs. [Copyright &y& Elsevier]

Abstract: Trace constituents are widely present in complex mixtures, and trace analysis is challenging because of the unpredictable matrix. In this work, a high-component filtering strategy was developed for improved analysis of trace constituents in complex sample by liquid chromatography–mass spectrometry (LC–MS). Using a specifically designed chromatographic apparatus, the high-abundant fractions were filtered prior to LC–MS analysis. The samples complexity was reduced and the sample-loading amount for the rest low-level fractions can be considerably increased. The application of this approach was illustrated with an analytically challenging sample, a traditional Chinese herbal medicine named Compound Danshen Sample. We observed that the loss rate for 12 analytes during the filtering procedure ranged from 6.54 to 26.11%, but showed a stable repeatability with RSD<3.79%. The proposed filtering method with quadrupole time-of-flight mass spectrometritry (Q-TOF/MS) enhanced the detection capacity, offering a comprehensive characterization of 133 compounds in Compound Danshen Samples. The quantification sensitivity was also improved in trace analysis, allowing six low compounds that cannot be quantified by the traditional methods to be tested by the filtering method. It can be predicted that the qualitative and quantitative trace analysis will be greatly improved when the loading samples is increased resulting from the filtration of high-level targets. The proposed strategy is promising to monitor trace constituents in diverse complex mixtures in the analytical field of pharmaceutics, metabonomics and environments. [Copyright &y& Elsevier]

Abstract: In the present study, rapid resolution liquid chromatography was coupled with quadrupole time-of-flight tandem mass spectrometry (RRLC–Q-TOF–MS) to identify the absorbed components and metabolites in rat urine after oral administration of Buyang Huanwu decoction (BYHWD). After oral administration of BYHWD, urine samples were collected and pretreated by solid phase extraction. The mass measurements were accurate within 5ppm of error for all the protonated molecules, and subsequent fragment ions offered higher quality structural information for interpretation of the fragmentation pathways of various compounds. A total of 50 compounds were detected in rat urine samples within 20min, including 12 parent compounds and 38 metabolites. Except for three prototype components (Hydroxysafflor yellow A, Paeoniflorin, and Amygdalin), the metabolites identified mainly came from Radix Astragali, Radix Angelicae Sinensis, and Rhizoma Chuanxiong. The results indicated that glucuronidation and sulfation were the major metabolic pathways of isoflavonoids, while glutathione conjugation, glucuronidation and sulfation were the main metabolic pathways of phthalides. No saponin-related metabolites were detected. The present study provided important structural information on the metabolism of BYHWD. Furthermore, the results of this work have demonstrated the feasibility of the RRLC/ESI-Q-TOF–MS approach for rapid and reliable characterization of metabolites from herbal medicines. [Copyright &y& Elsevier]

Abstract: A rapid resolution liquid chromatography coupled with electrospray ionization (ESI) time-of-flight mass spectrometry method was developed and validated for quantitative analysis of 6-gingerol in plasma and various tissues. Liquid–liquid extraction was employed as sample preparation technique. Biological samples were separated on an Agilent Zorbax StableBond-C18 column (4.6mm×50mm, 1.8μm) and detected by TOF/MS with electrospray ionization (ESI) interface in positive ion mode. Calibration curves (1/x 2 weighted) offered satisfactory linearity (r 2 >0.995) within the test range. The lower limit of quantification in different matrices was in a range of 10–100ng/mL. Inter- and intra-day precision were in the range of 0.91–11.90% and 0.75–10.23%, respectively. Recoveries in plasma, urine and tissues ranged from 72.5% to 90.4%. Glucuronide of 6-gingerol, the major metabolite of 6-gingerol, was further determined after β-glucuronidase hydrolyzation. This developed method was successfully applied to pharmacokinetics, tissue distribution and excretion studies of 6-gingerol after oral or intraperitoneal administration in rats. [Copyright &y& Elsevier]

Abstract: Flos Lonicerae, referred to the flower buds of several medicinal Lonicera species, is a commonly used traditional Chinese herbal medicine. A multi-component-assay quality control method, using high performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry (HPLC-ESI/TOF MS), has been developed for the simultaneous identification and quantification of 32 bioactive compounds in Flos Lonicerae. The limits of detection (LOD) and quantification (LOQ) were in the range of 0.002–0.089 and 0.006–0.355μg/ml, respectively. All calibration curves showed good linear regression (r 2 ≥0.99) within the test ranges. The overall intra- and inter-day precisions of analytes were less than 3.47% for peak area and 0.38% for retention time. The recoveries were from 85.4% to 101.6%. The validated method was applied to assay of 32 compounds in 8 medicinal Lonicera species. Furthermore, six unknown chromatographic peaks were tentatively characterized. It was demonstrated that the HPLC-ESI/TOF MS method was suitable for quality control of Lonicera species, owing to the advantages of accurate mass analysis, resolving power, enhanced selectivity and high sensitivity. [Copyright &y& Elsevier]

Abstract: A novel strategy for predicting bioactive components in traditional Chinese medicines (TCM) using live cell extraction and high performance liquid chromatography–diode array detection–mass spectrometry (HPLC–DAD–MS) analysis was proposed. The hypothesis is that when cells are incubated together with the extract of TCM, the potential bioactive components in the TCM should selectively combine with the cells, and the relative concentrations of the cell-combining components in the suspension medium should decrease, while the cell-combining components would be detectable in the extract of denatured cells. The identities of the cell-combining components could be determined by HPLC–DAD–MS analysis. Using the proposed approach, the potential bioactive components of Danggui Buxue decoction, a commonly used TCM for anaemia, and its compositions, Radix Angelica Sinensis and Radix Astragli for endothelial cells, were investigated. Six compounds in the extract of Danggui Buxue decoction were detected as the components selectively combined with endothelial cells, among them two were contributed by Radix Angelica Sinensis, and four by Radix Astragli. The identities of four of the six potential bioactive compounds were elucidated as ononoside, calycosin, 3-butylphthalide and ligustilide by HPLC–DAD–MS analysis. The results indicate that the proposed approach may be applied to predict the bioactive candidates in TCM. [Copyright &y& Elsevier]