Characterization and determination of the major constituents in Belamcandae Rhizoma by HPLC-DAD-ESI-MS(n)

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-MSⁿ) 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-MSⁿ 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.     

Chromatographic fingerprint analysis of Cephalotaxus sinensis from various sources by high-performance liquid chromatography-diodearray detection-electrospray ionization-tandem mass spectrometry

Selective and efficient analytical methods are required not only for quality assurance but also for authentication of Chinese herbal medicine. A simple, rapid and valid fingerprint method has been first carried out for the quality control of Cephalotaxus sinensis by using high-performance liquid chromatography (HPLC) coupled with photodiode array detection (DAD) and electrospray ionisation-tandem mass spectrometry (ESI-MS). The characteristic analytical fingerprints of this plant extract showed 18 common peaks, and out of these, 10 compounds involving 2 new compounds were identified by comparing the retention time, UV and ESI-MS/MS spectrum of each standard with those of each peak separated by on-line HPLC-DAD-MS/MS. Moreover, the effects of collecting locations, harvesting time, storage time, drying methods, and medicinal portions on herbal chromatographic fingerprints were examined by similarity analysis and principal component analysis (PCA) along with markers. Using the reference fingerprint along with markers, the best harvesting time, cultivation location and medicinal part were determined. The results obtained suggest that the chromatographic fingerprint combining similarity evaluation and PCA along with markers or pharmacologically active constituents can efficiently identify raw herb of Cephalotaxus sinensis from different sources, which provide helpful clues to the study of plant's secondary metabolites and benefit quality control.     

Qualitative and quantitative characterization of chemical constituents in Xin-Ke-Shu preparations by liquid chromatography coupled with a LTQ Orbitrap mass spectrometer

Xin-Ke-Shu (XKS), a traditional Chinese medicine (TCM) preparation containing five herbal medicines, has been commonly used for the treatment of coronary heart disease in China. However, the chemical constituents in XKS have not been clarified yet. In order to quickly define the chemical profiles and control the quality of XKS preparations, liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap (LC-LTQ-Orbitrap) mass spectrometry was applied for simultaneous identification and quantification of multi-constituent. A total of 51 compounds, including phenolic acids, isoflavone-C-glycosides, isoflavone-O-glycosides, flavonoids, and triterpenoid saponins, were identified or tentatively deduced on the base of their retention behaviors, MS and MS(n) data, or by comparing with reference substances and literatures. In addition, an optimized LC-ESI-MS method was established for quantitative determination of 15 marker compounds in XKS preparations from 7 independent pharmaceutical companies. The validation of the method, including spike recoveries, linearity, sensitivity (LOD and LOQ), precision, and repeatability, was carried out and demonstrated to be satisfied the requirements of quantitative analysis. This is the first report on the comprehensive determination of chemical constituents in XKS preparations by LC-LTQ-Orbitrap mass spectrometry. The results suggested that the established methods would be a powerful and reliable analytical tool for the characterization of multi-constituent in complex chemical system and quality control of TCM preparations.     

Chemical profiling of Radix Paeoniae evaluated by ultra-performance liquid chromatography/photo-diode-array/quadrupole time-of-flight mass spectrometry

In this study, an ultra-performance liquid chromatography/photo-diode-array/quadrupole time-of-flight mass spectrometry (UPLC-PDA-QTOFMS) based chemical profiling method was established for rapid global quality evaluation of Radix Paeoniae. By virtue of the high resolution, high speed of UPLC and the accurate mass measurement of TOFMS, a total of 40 components including 29 monoterpene glycosides, 8 galloyl glucoses and 3 phenolic compounds were simultaneously separated within 12 min, and identified through the matching of empirical molecular formulae with those of published components in the in-house library, and were further elucidated by adjusted lower energy collision-induced dissociation (CID) mass spectra. Among forty components, five monoterpene glycoside sulfonates were identified as novel components. The established method was successfully applied to rapidly and globally compare the quality of Radix Paeoniae Alba and Radix Paeoniae Rubra, two post-harvesting handled products of Radix Paeoniae. Together with paeoniflorin sulfonate, five newly assigned monoterpene glycoside sulfonates were characteristic markers to detect non-official sulfur dioxide gas fumigated Radix Paeoniae Alba samples. It could be concluded that UPLC-PDA-QTOFMS based chemical profiling is a powerful approach for the global quality evaluation of Radix Paeoniae as well as other herbal medicines.     

Analysis of the bioactive constituents of ChanSu in rat plasma by high performance liquid chromatography with mass spectrometric detection

A plasma pharmacochemistry analysis of the bioactive constituents in rat plasma after oral administration of ChanSu was performed. High performance liquid chromatography coupled with the electrospray ionization mass spectrometry techniques of HPLC/ESI-IT-MS/MS and RRLC/ESI-Q-TOF-MS were used for the chemical profiling of samples of dosed plasma, control plasma and ChanSu extract. Comparative analysis of the resulting chemical profiles revealed 20 prototype compounds and 4 metabolites derived from ChanSu as potential bioactive components. By MS/MS analysis and accurate molecular weight assessments, the majority of these bioactive components (19 prototype compounds and 2 metabolites) were structurally identified. Moreover, seven were confirmed by comparing their retention behaviors using MS and MS/MS analysis. This study proposes a series of potential bioactive components of ChanSu, which we hope will lead to new drug discovery based on ChanSu and other traditional Chinese medicines.     

Structure elucidation of in vivo and in vitro metabolites of mangiferin

The in vivo and in vitro metabolism of mangiferin was systematically investigated. Urine, plasma, feces, contents of intestinal tract and various organs were collected after oral administration of mangiferin to healthy rats at a dose of 200mg/kg body weight. For comparison, mangiferin was also incubated in vitro with intestinal flora of rats. With the aid of a specific and sensitive liquid chromatography coupled with electrospray ionization tandem hybrid ion trap mass spectrometry (LC-ESI-IT-MS(n)), a total of thirty-three metabolites of mangiferin were detected and their structures were tentatively elucidated on the basis of the characteristics of their precursor ions, product ions and chromatographic retention times. The biotransformation pathways of mangiferin involved deglycosylation, dehydroxylation, methylation, glycosylation, glucuronidation and sulfation.     

Development of the chromatographic fingerprint of herbal preparations Shuang-Huang-Lian oral liquid

A simple, reliable and reproducible method, based on high performance liquid chromatography (HPLC), for developing chromatographic fingerprint of complex herbal medicine Shuang-Huang-Lian (SHL) oral liquid was described. Ten batches of SHL obtained from different pharmaceutical factories were used to establish the fingerprint. In addition, the contents of baicalin and chlorogenic acid, which are two marker constituents in the preparations, were also determined. Chromatographic fingerprint, together with the contents of the markers were applied for quality control of SHL. SHL comprises three kinds of medicinal herbs: Fols Lonicerae, Radix Scutellariae and Fructus Forsythiae. According to State Food and Drug Administration (SFDA) requirement, the chromatographic fingerprints of Fols Lonicerae, Radix Scutellariae and Fructus Forsythiae, the raw materials of SHL preparations, were also established. The data of fingerprints of SHL and its raw herbs established by HPLC were all processed with two kinds of mathematic methods including correlation coefficient and cosine value of vectorial angle to validate their similarities. In conclusion, fingerprints of Radix Scutellariae, Fols Lonicerae and Fructus Forsythiae are suitable for identification, differentiation of their geographic origins and quality control. The similarity of 10 batches of SHL oral liquid was more than 0.988, which showed the preparations from different pharmaceutical factories were consistent.     

Fingerprint analysis of the fruits of Cnidium monnieri extract by high-performance liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry

A method incorporating high-performance liquid chromatography (HPLC) with electrospray ionization (ESI) and tandem mass spectrometry (MS), with parallel analysis by HPLC with UV detection using a diode-array detector (DAD) was developed for the qualitative characterization of coumarin and chromone constituents in the fruits of Cnidium monnieri. The chromatographic separations were performed on a Diamonsil C18 column (4.6 mm x 200 mm, 5 microm) with water with 50 mM ammonium acetate and 2% acetic acid (A) and acetonitrile (B) as the mobile phase. According to the characteristic UV spectra, the information of molecular weight and structure provided by ESI-MS/MS, 13 coumarin and 7 chromone components were detected and identified. This method is rapid and reliable for identification of the constituents in the complex herbal system, and the fragmentation patterns proposed could be extended to the similar compounds.     

HPLC-DAD-MS(n) analysis and HPLC quantitation of chemical constituents in Xian-ling-gu-bao capsules

In this study, a systematic method was established for the global quality control of Xian-ling-gu-bao capsules (XLGB), a popular six-herb Traditional Chinese Patent Medicine (TCPM) for the treatment of osteoporosis. Both qualitative and quantitative analyses were conducted. In qualitative analysis part, a fast and sensitive method based on high-performance liquid chromatography coupled with diode-array detection and electrospray ionization tandem mass spectrometry (HPLC-DAD-MS(n)) was established for rapid separation and sensitive identification. Samples were separated on a Waters Symmetry C(18) column (250mm×4.6mm, 5μm) by gradient elution using acetonitrile (A) and water-formic acid (B; 0.03%, v/v) as mobile phase at a flow rate of 1.0ml/min. Based on the mass spectra, UV spectra and retention time, 47 compounds were identified or tentatively characterized, including 27 flavonols (all from Epimedii Herba, the major component herb), 4 coumarins, 3 flavonones, 1 chalcone, 3 isoflavones, 1 coumestrol, 3 triterpenoid saponins, 1 iridoid, 3 steroidal saponins, and 1 phenolic acid. Among them, 18 compounds were confirmed by comparing with reference standards. In quantitative analysis part, 10 major compounds in 18 batches of XLGB were simultaneously determined by HPLC/UV detected at 270nm. The method was validated with respect to intra- and inter-day precision, repeatability and stability, with RSD less than 1.0%, 1.5%, 2.9% and 1.8%, respectively. All the 10 analytes showed good linearity in wide linear ranges (r(2)=0.9999), and their average recoveries varied between 97.8% and 104.9%.     

Qualitative and quantitative analysis of steroidal saponins in crude extracts from Paris polyphylla var. yunnanensis and P. polyphylla var. chinensis by high performance liquid chromatography coupled with mass spectrometry

High performance liquid chromatography coupled with electrospray ionization multi-stage tandem mass spectrometry (HPLC–ESI-MSⁿ) and triple quadrupole mass spectrometric detection (HPLC–ESI-MS/MS), respectively, had been employed for the simultaneous identification and quantification of steroidal saponins in the rhizomes of Parispolyphylla var. yunnanensis and P. polyphylla var. chinensis, which are the qualified plants of “Chonglou” in Chinese. The HPLC experiments were performed by means of a reversed-phase C-18 column and a binary mobile phase system consisting of 0.1% aqueous formic acid and acetonitrile under gradient elution conditions. The characteristic fragmentation patterns of diosgenin- and pennogenin-type steroidal saponins were investigated using ESI-MSⁿ in negative ion mode. The MSⁿ data of the [M−H]⁻ ions provided structural information on the sugar sequence of the oligosaccharide chains and the aglycones of steroidal saponins. As a result, ten and seven saponins were determined in P. polyphylla var. yunnanensis and P. polyphylla var. chinensis, respectively, including four unknown compounds. One unknown compound was tentatively identified as diosgenin-3-O-α-l-rhamnopyranosyl(1 → 4) [α-l-rhamnopyranosyl(1 → 2)]-β-d-glucopyranoside and the aglycones of the other three new compounds were reported from Chonglou for the first time. The developed HPLC–ESI-MS/MS method was validated and found to be satisfactorily linear, selective and robust. The limits of detection (LODs) and quantitation (LOQs) ranged, respectively, from 0.5 to 10 ng/mL and 2 to 34 ng/mL depending on six various compounds. The intra- and inter-day precisions of the method were evaluated and were less than 5.0%. Recoveries ranged from 92% to 104% for all compounds. The established quality evaluation method was successfully used for simultaneous quantification of six predominant steroidal saponins in the rhizomes of these two Paris species.     

Isolated perfused lung extraction and HPLC-ESI-MS(n) analysis for predicting bioactive components of Saposhnikoviae Radix

A novel strategy for predicting bioactive components in traditional Chinese material herb was proposed, using isolated perfused rat lung (IPL) extraction and high performance liquid chromatography\tandem mass spectrometry (HPLC-MS(n)) analysis. The hypothesis is that when the IPL is perfused with the extract of Saposhnikoviae Radix (ESR), the potential bioactive components in the ESR should selectively combine with the receptor or channel of lung, by changing the pH of perfused liquid, the combining components would be eluated and then detected by HPLC-ESI-MS(n). Five compounds were detected in the desorption eluate of IPL; among these compounds, two potential bioactive compounds, prim-O-glucosylcimifugin (2) and 4'-O-β-D-glucosyl-5-O-methylvisamminol (4) were identified by comparing with the chromatography of the standard sample, and three other compounds, i.e. cimifugin (1), 5-O-methylvisamminol (3) and sec-O-glucosylhamaudol (5) were determined by analysis of the structure clearage characterization of mass spectrometry. The application of IPL extraction coupled with HPLC-ESI-MS(n) for predicting potential bioactive components of TCMs is rapid, convenient, operational, economic and reliable.     

Identification and quantification of 32 bioactive compounds in Lonicera species by high performance liquid chromatography coupled with time-of-flight mass spectrometry

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 0.006–0.355 μg/ml, respectively. All calibration curves showed good linear regression (r² ≥ 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.     

Rapid simultaneous quantification of five active constituents in rat plasma by high-performance liquid chromatography/tandem mass spectrometry after oral administration of Da-Cheng-Qi decoction

A rapid, sensitive and specific liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI/MS/MS) method has been developed and validated for simultaneous determination of five active constituents (including magnolol, honokiol, rhein, emodin and aloe-emodin) from Da-Cheng-Qi decoction (DCQD) in rat plasma. After the addition of gliquidone as the internal standard (IS), plasma samples were prepared by one-step protein precipitation using methanol and separated by HPLC on a short reversed phase C(18) column packed with smaller particles (100 mm x 3.0 mm, 3.5 microm) using a mobile phase of methanol-0.1% formic acid aqueous solution (70:30, v/v). Analytes were determined in a triple-quadrupole mass spectrometer in the selected reaction-monitoring (SRM) mode using electrospray source with negative mode. The method was proved to be rapid, sensitive, specific, accurate and reproducible and has been successfully applied to the determination of the five compounds in rat plasma after oral administration of low dose DCQD for pharmacokinetic study.     

Rapid separation and characterization of active flavonolignans of Silybum marianum by ultra-performance liquid chromatography coupled with electrospray tandem mass spectrometry

Ultra-performance liquid chromatography (UPLC) interfaced with the electrospray ionization (ESI) tandem mass spectrometer (MSⁿ) was developed for the simultaneous determination of silychristins A (1) and B (2), silydianin (3), silybins A (4) and B (5), and isosilybins A (6) and B (7), major bioactive flavonolignans in silymarin, a herbal remedy derived from the milk thistle Silybum marianum. In this study, the seven major active flavonolignans including the diastereomers 1/2, 4/5, and 6/7 were completely separated using UPLC with an ACQUITY UPLC C₁₈ column and a MeOH/water/formic acid mobile phase system. The collision-induced dissociation (CID) MSⁿ spectra of these flavonolignans were studied systematically using ESI-MS. The results with the present methodology show that UPLC–MSⁿ can be useful for general screening of active natural products from plant extracts and for the specific quality control of silymarin.     

Characterization of flavonoids in the extract of Sophora flavescens Ait. by high-performance liquid chromatography coupled with diode-array detector and electrospray ionization mass spectrometry

A method coupling high-performance liquid chromatography (HPLC) with diode-array detector (DAD) and electrospray ionization mass spectrometry (ESI) was established for the separation and characterization of flavonoids in Sophora flavescens Ait. Based on the chromatographic separation of most flavonoids present in S. flavescens Ait., a total of 24 flavonoids were identified. Fourteen compounds were unambiguously identified comparing experimental data for retention time (t(R)), UV and MS spectra with those of the authentic compounds: 3',7-dihydroxy-4'-methoxy-isoflavone (13), trifolirhizin (14), kurarinol (18), formononetin (19), 7,4'-dihydroxy-5-methoxy-8-(gamma,gamma-dimethylallyl)-flavanone (22), maackiain (21), isoxanthohumol (23), kuraridine (26), kuraridinol (27), sophoraflavanone G (30), xanthohumol (31), isokurarinone (33), kurarinone (35) and kushenol D (38), and additional 10 compounds were tentatively identified as kushenol O (10), trifolirhizin-6'-malonate (15), sophoraisoflavanone A (20), norkurarinol/kosamol Q (24), kushenol I/N (25), kushenol C (28), 2'-methoxykurarinone (29), kosamol R (32), kushecarpin A (34) and kushenol A (37) by comparing experimental data for UV and MS spectra with those of literature. Furthermore, fragmentation pathways in positive ions mode of 24 flavonoid compounds of types of flavanone, flavanonol, flavonol, chalcone, isoflavone, isoflavanone and ptercocarpane were summarized. Some common features, such as CH(3)., H(2)O, CO, CO(2), C(3)O(2) and C(2)H(2)O losses, together with Retro-Diels-Alder fragmentations were observed in the prenylated flavonoids in S. flavescens Ait. The loss of the lanandulyl chain was their characteristic fragmentation, which might help deducing the structure of unknown flavonoid compounds. The present study provided an approach to rapidly characterize bioactive constituents in S. flavescens Ait.     

Dereplication of bioactive constituents of the genus hypericum using LC-(+,−)-ESI-MS and LC-PDA techniques: Hypericum triquterifolium as a case study

Utilizing liquid chromatography–electro spray ionization-mass spectrometry (LC–(+,−)-ESI-MS) and liquid chromatography-photodiode array detection (LC-PDA) techniques, a dereplication strategy for the analysis of the secondary metabolites constituents of the genus Hypericum has been developed. From the crude methanolic extract of the aerial parts of H. triquetrifolium (leaves, stems, and flowers) and on the basis of their UV-profiles, chromatographic retention times and (+,−)-ESI-MS (TIC and SIM) mass spectral data, seven known (1–7) compounds were dereplicated fairly rapidly. The compounds were classified into three structural classes: phloroglucinols: hyperfirin and adhyperfirin; naphthodianthrones: hypericin, pseudo-hypericin, proto-hypericin, and protopseudo-hypericin; and the flavonoid rutin.     

Development of an in-line HPLC fingerprint ion-trap mass spectrometric method for identification and quality control of Radix Scrophulariae

Chromatographic fingerprinting has been widely accepted as a crucial method for qualitative and quantitative analyses of bioactives within traditional Chinese medicine. A fingerprint provides detailed information, specific for any given herb, thus facilitating the quality control measures of a given traditional Chinese medicine. In this article, quality assessment of Radix Scrophulariae was achieved by using high performance liquid chromatography combining diode-array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI/MS). Eight batches of sample obtained from different origins in China were used to establish the fingerprint and quantitative analyses. By comparing the retention times, UV and MS spectral data with reference standards, four characteristic peaks in the chromatograms were confirmed as corresponding to acetoside, angoroside C, cinnamic acid, and harpagoside. In addition, other two characteristic peaks were tentatively identified, following the literature interpretation of HPLC-ESI-MS and LC-MS/MS (affording structural information) to be sibirioside A and scrophuloside B₄, respectively. The results indicated that the newly developed HPLC-DAD-MS fingerprint method would be suitable for quality control of Radix Scrophulariae.     

Characterization of phenolic compounds in the Chinese herbal drug Artemisia annua by liquid chromatography coupled to electrospray ionization mass spectrometry

A simple and rapid method has been established for the screening of the main phenolic compounds in Artemisia annua by LC-DAD-ESI-MS(n). A total of 40 phenolic compounds were identified or tentatively characterized in the methanol extract of A. annua, including 8 C-glycosyl flavonoids, 5 O-glycosyl flavonoids, 3 flavonoid aglycones, 21 quinic acid derivatives, 2 benzoicacid glucosides and 1 coumarin. The C-glycosyl flavonoids were reported from A. annua for the first time and they were found to be a new type of main constituents, and might be responsible for its antioxidant and antiviral activity. Quinic acid derivatives were also found to be the major constituents of A. annua.     

Qualitative and quantitative analysis of Fructus Corni using ultrasound assisted microwave extraction and high performance liquid chromatography coupled with diode array UV detection and time-of-flight mass spectrometry

The first successful combination of ultrasound assisted microwave extraction (UAME) with liquid chromatography analysis is described for the quality evaluation of Fructus Corni, a commonly used traditional Chinese medicine (TCM). Due to their multifarious biological activities, seven representative bioactive constituents (two phenolics and five iridoids) were chosen as targets for the quality assessment. The chromatographic separation was performed on a C18 Aq column with gradient elution using methanol and aqueous solution containing 0.2% acetic acid. The quantitative method developed was validated and successfully applied to determine the seven markers in 12 batches of Fructus Corni extract from various habitats. Significant variations were demonstrated in the contents of seven compounds. Further 13 components were tentatively identified by online TOF mass analysis.