Characterisation and identification of chemical constituents in aqueous extract of Fomes officinalis Ames based on ultrahigh-performance liquid chromatography tandem quadrupole-Orbitrap high-resolution mass spectrometry.

INTRODUCTION: Fungal species are an attractive resource for physiologically functional food and drug precursor. Fomes officinalis Ames, a medicinal fungus, is traditionally used as a folk medicine in traditional Chinese medicine prescription for the therapy of cough and asthma. The water-soluble substances in Chinese herbal medicines are likely to play an important physiological function. However, information on probing and identifying chemical components of the aqueous extract of Fomes officinalis Ames (AFO) remains unknown. OBJECTIVE: This study was conducted to screen and characterise the chemical components of AFO. MATERIAL AND METHODS: An effective and sensitive ultrahigh-performance liquid chromatography tandem quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-HRMS) method with the Full MS/PIL/dd-MS2 acquisition approach was applied for the profiling of chemical components in AFO. An HSS T3 column was used for component separation, and a strategy of simultaneous targeted and untargeted multicomponent characterisation was implemented. Multiple identification approaches were used, including accurate molecular mass and elemental composition matching, literature and database searching, and fragmentation rules elucidation. RESULTS: A total of 115 components, including 20 amino acids and derivatives, six nucleobases, nine nucleosides, 75 dipeptides, two tripeptides, and three other components, were tentatively identified. Among them, the targeted exploring method screened six nucleobases and nine nucleosides including modified nucleosides. To our best knowledge, this is the first time a report has been done on the presence of the 115 compounds in AFO. CONCLUSION: Profiling and characterisation compounds of AFO enriched its material basis, which would lay the foundation for improving potential medicinal and nutritional values and effecting comprehensive quality control of Fomes officinalis Ames.

Rapid identification of chemical components in Xuelian granule by UHPLC-Q-orbitrap-HRMS based on enzyme activity in vitro.

BACKGROUND: Xuelian granule (XL), a traditional Chinese medicine (TCM) formula, has been used for the treatment of diabetic nephropathy for a long time as a hospital preparation. Because the active ingredients in the XL that can help to treat diabetic nephropathy are still unclear, which limits the interpretation for its pharmacological mechanism, further development and subsequent study on the material basis of its efficacy. METHODS: In this study, a screening method based on inhibition activity against aldose reductase (AR) was employed for activity-directed chemical analysis of XL using ultra-high performance liquid chromatography combined with quadrupole-orbitrap high resolution mass spectrometry (UHPLC-Q-orbitrap-HRMS) technique. RESULTS: A total of 178 compounds, including 46 terpenes, 47 organic acids, 25 flavonoids, 29 phenylethanoid glycosides, and 31 other types, were tentatively identified from XL which might responsible for its AR inhibition activity. CONCLUSION: This is the first study for a systematic, rapid, and accurate qualitative analysis of XL. This research provides a scientific and experimental basis for further researches on pharmacodynamics material basis and quality control of XL.

Comprehensive Identification of Chemical Fingerprint and Screening of Potential Quality Markers of Aloe vera (L.) Burm. f. from Different Geographical Origins via Ultra-High-Performance Liquid Chromatography Hyphenated with Quadrupole-Orbitrap-High-Resolution Mass Spectrometry Combined with Chemometrics.

An integrated strategy was developed for the systematic chemical fingerprint and chemometrics analysis for the quality assessment of Aloe vera (L.) Burm. f. The ultra-performance liquid chromatography fingerprint was established, and all common peaks were tentatively identified by using ultra-high-performance liquid chromatography hyphenated with quadrupole-orbitrap-high-resolution mass spectrometry. Afterwards, the datasets of common peaks were subjected to hierarchical cluster analysis, principal component analysis and partial least squares discriminant analysis to holistically compare the differences. The results revealed that the samples were predicted to fall into four clusters, which were related to four different geographical locations. Using the proposed strategy, aloesin, aloin A, aloin B, aloeresin D and 7-O-methylaloeresin A were rapidly determined to be the potential characteristic quality markers. Finally, five screened compounds in 20 batches of samples were simultaneously quantified, and their total contents were ranked as follows: Sichuan province > Hainan province > Guangdong province > Guangxi province, which suggests that geographical origins may be an important factor affecting the quality of A. vera (L.) Burm. f. This new strategy can not only be used to explore possibly the latent active substance candidates for pharmacodynamic studies, but it is also an efficient analytical strategy for other complex traditional Chinese medicine systems.

Chemical profiling of composite prescription caraway and quantification of three pairs isomeric components in caraway administered rat plasma by tandem mass spectrometry.

Caraway, a well-known traditional Uyghur medicine, has been used to treat vitiligo for centuries. Its biological effects on melanin synthesis of caraway have been investigated. However, beyond psoralen and isopsoralen alone, no further chemical component of caraway has been revealed. In this study, ultra-high performance liquid chromatography coupled with hybrid quadrupole orbitrap mass spectrometry was employed to comprehensively characterize the chemical components present in caraway. Based on accurate mass measurements, key fragmental ions and comparison with reference standards, 75 chemical components were identified in caraway. Moreover, a tandem mass spectrometry method was developed and validated for quantitative analysis of three pairs isomeric components, namely psoralen/isopsoralen, bavachin/isobavachalcone and bavachromene/isobavachromene in rat plasma. Psoralen, isopsoralen, bavachin, and isobavachalcone showed linearity with concentration ranging of 1.0-500.0 ng/ml. The linear ranges for bavachromene and isobavachromene were 0.2-500.0 ng/ml. The accuracies were in ranges of 85%-115% with coefficient of variation errors of less than 15%. Furthermore, the method was applied to quantify the three pairs isomeric components in rats after oral administration of caraway.

Comprehensive chemical profile and quantitative analysis of the Shabyar tablet, a traditional ethnic medicine prescription, by ultra-high-performance liquid chromatography with quadrupole-orbitrap high-resolution mass spectrometry.

The Shabyar tablet is commonly used as a traditional ethnic medicine prescription for the treatment of night blindness, poor vision, and headaches. However, the chemical components of the Shabyar tablet have not been holistically explored, which seriously hinders the discovery of the activity. This study qualitatively and quantitatively investigated the overall chemical profile of the Shabyar tablet using ultra-high-performance liquid chromatography hyphenated with quadrupole-orbitrap high-resolution mass spectrometry. Altogether, 170 chemical components, including 59 flavonoids, 78 organic acids, 12 anthranones, three anthraquinones, one naphthalene, and 17 other compounds were tentatively identified and attributed, with 40 among these being unambiguously characterized in comparison with their corresponding authentic standards. To further determine the major representative constituents of the Shabyar tablet, a quantitative method was used for the simultaneous analysis of 33 characteristic components in Shabyar samples. The results were validated in terms of linearity, precision, repeatability, stability, and recovery. This newly developed approach could be successfully employed for evaluating the holistic quality of crude extracts and Chinese medicines in the Shabyar compound tablet and provide a solid chemical foundation for additional investigations on in vivo pharmacodynamics and therapeutic mechanisms to identify the potential effective components of traditional medicines.

Phenolic Compounds and Bioactivities from Pomegranate (Punica granatum L.) Peels.

Pomegranate peels, which are normally processed as the main byproduct of pomegranate juice production, are worthy of being researched and utilized for the aim of economic and environmental benefits. In a phytochemical investigation of the peels of Punica granatum L., 10 phenolic compounds containing a common hexahydroxy diphenol moiety were isolated. Three of them were identified for the first time and named as pomegranatins A-C, and from the other seven known ones, two of them were obtained from pomegranate peels for the first time. Their structures were determined via extensive spectroscopic analysis. Besides, for the sake of preliminarily comprehending their biological activities, in vitro antimicrobial, antioxidant, as well as antitumor assays were detected. In the DPPH antioxidant assay, six compounds presented significant free radical scavenging ability. Two compounds exhibited moderate antimicrobial activities against Candida albicans; one compound could inhibit the proliferation of both C. albicans and Escherichia coli within limits. Four compounds possessed weak antitumor activity toward the Hela cell line without taking into account the bioavailability of ellagitannins. Overall, these results provided further information on the structural diversity of bioactive compounds present in pomegranate peels, as well as on their biological activities.

Simultaneous Quantification of Four Compounds in Rat Plasma by HPLC-MS/MS and Its Application to Pharmacokinetic Study after Oral Administration of Pomegranate Flowers.

Pomegranate flowers (PFs) were reported to possess various biological activities such as antidiabetic, anti-inflammatory and hepatoprotective activities, and using to treat diabetes. Although chemical constituents and pharmacological activities of PFs have been studied, unfortunately, there was no report on the pharmacokinetic profile of PFs in vivo. In this study, a selective high-performance liquid chromatography triple quadrupole tandem mass spectrometry (HPLC-QQQ-MS/MS) method was developed and validated for simultaneous quantification of four compounds (corilagin, ellagic acid, gallic acid and brevifolincarboxylic acid) in rat plasma after oral administration of PFs. The good linearity concentration ranges for the four analytes were from 2.5 to 3000 ng/mL with coefficient value R2 > 0.99 in calibration curves. The intra- and inter-day accuracy of the four analytes was in the range of 85.33-102.50%, with relative standard deviation (RSD) of <14.81%. The stability results showed that accuracy of the four analytes was in the range of 81.88-104.74%, with RSD of <14.86%. The validation method was successfully applied to pharmacokinetic profiles of the four analytes in rats after oral administration of PFs extract. This pharmacokinetic study can provide better understanding to clarify in vivo mechanisms of PFs and may facilitate its further development as therapeutic agent.

Spectrum-effect relationship between UPLC fingerprints and antidiabetic and antioxidant activities of Rosa rugosa.

In this study, the antidiabetic and antioxidant properties of the chemical constituents of Rosa rugosa Thunb. (R. rugosa) was evaluated through analysis of spectrum-effect relationship. The ultra-performance liquid chromatography (UPLC) fingerprints of 21 batches of R. rugosa were evaluated by similarity analysis (SA) and hierarchical clustering analysis (HCA). The 28 common components were identified by ultra-high-performance liquid chromatography coupled to quadrupole-orbitrap high resolution mass spectrometry (UHPLC-Q-orbitrap-HRMS/MS). Meanwhile, the antidiabetic activities and antioxidant activities of 21 batches of R. rugosa were estimated in vitro. Besides, four chemometrics named principal component analysis (PCA), grey correlation analysis (GRA), partial least squares regression (PLSR) and the bivariate correlations analysis (BCA) were applied to construct spectrum-effect relationship between the UPLC fingerprints and biological activities of R. rugosa. The spectrum-effect relationship study revealed that di-O-galloyl-HHDP-glucoside, galloyl-HHDP-glucoside and avicularin were more relevant to antidiabetic activity. Di-O-galloyl-HHDP-glucoside, galloyl-HHDP-glucoside and ellagic acid were the main antioxidant components of R. rugosa. The current bioassay and spectrum-effect relationships are proper for associating sample quality with the active ingredient, and our finding would provide foundation and further understanding of the quality evaluation and quality control of R. rugosa.

Chemical Components and Hepatoprotective Mechanism of Xwak Granule in Mice Treated with Acute Alcohol.

OBJECTIVE: To evaluate the hepatoprotective mechanism of Xwak granule (Xwak) in treatment of mice with alcoholic liver injury via activating ERK/NF-κB and Nrf/HO-1 signaling pathways. METHODS: The chemical composition of Xwak was tested by liquid chromatography coupled with mass spectrometry (LC-MS). Herein, 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay and 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical tests were performed in vitro. The hepatoprotective effect of Xwak was assessed at different concentrations (1.5, 3, and 6 g/kg) in a mouse model of alcoholic liver injury. RESULTS: Totally, 48 compounds, including 16 flavonoids, 8 tannins, 9 chlorogenic acids, and 15 other compounds, were identified from Xwak. Xwak showed to have a satisfactory antioxidant activity in vitro. In a group of Xwak-treated mice, the serum levels of alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) were decreased compared with a group of the mouse model of alcoholic liver injury. In addition, the levels of antioxidant enzymes, such as glutathione peroxidase (GSH-PX), total superoxide dismutase (T-SOD), and catalase (CAT), were noticeably increased and the levels of malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), transforming growth factor-ß (TGF-ß), and interleukin-6 (IL-6) were markedly reduced in the liver of mice. The state of oxidative stress in the mouse model of alcoholic liver injury was improved after treatment with Xwak. The improvement of inflammation-mediated disruption may conducive to the Xwak activity in the control of liver injury. The signals of p-ERK1/2, p-NF-κB, COX-2, iNOS, CYP2E1, Nrf, and HO-1 were significantly induced in the liver of mice after treatment with Xwak. CONCLUSIONS: The abovementioned findings indicated that the hepatoprotective mechanism of Xwak could be achieved by activating ERK/NF-κB and Nrf/HO-1 signaling pathways to alleviate oxidative stress and inflammatory.

Comprehensive characterisation of phenolics from Nitraria sibirica leaf extracts by UHPLC-quadrupole-orbitrap- MS and evaluation of their anti-hypertensive activity.

ETHNOPHARMACOLOGICAL RELEVANCE: For more than ten scores years, the leaves and fruits of Nitraria sibirica have been used as a natural remedy for indigestion, irregular manes, and hypertension in the Middle East and Central Asia, especially, are recommended for hypertension treatment in the northwest region, China. AIM OF THE STUDY: we aimed to support the traditional usage of N. sibirica leaves as pharmaceuticals or dietary supplements in treatment of hypertension by investigating their chemical constituents and anti-hypertensive activity. METHODS: We identified the chemical composition of N. sibirica leaves ethanolic purified extract (NSL-EPE) using UHPLC-quadrupole-orbitrap-MS, and quantified the main chemical constituents by an analytical method established and validated. We also evaluated anti-hypertensive activity of NSL-EPE using spontaneously hypertensive rats (SHR): blood pressure was measured weekly by non-invasive blood pressure (NIBP) measurements; hemodynamic parameters, biochemical and clinical chemistry variables in plasma, serum and kidney tissue were measured after 10 weeks of treatment with NSL-EPE as well. RESULTS: UHPLC-quadrupole-orbitrap-MS analysis identified 52 compounds, of which 40 compounds were reported for the first time in N. sibirica. 11 phenolic compounds further quantitatively analyzed, among which the most abundant compound was found to be clovin (8.8%). Systolic blood pressure decreased progressively from the second treatment week compared to that in non-treated SHRs. The plasma endothelin, aldosterone, angiotensin II levels were significantly increased, while the level of NOX was significantly decreased; glutathione to oxidized glutathione ratio, superoxide dismutase and total catalase levels in the kidney tissue were markedly accelerated, while malondialdehyde level was significantly reduced in NSL-EPE treated SHRs. Moreover, the serum cholesterol, triglyceride, blood uria nitrogen and creatinine were attenuated in NSL-EPE treated SHRs (P < 0.05), but in sharp contrast to those values in the water-treated SHRs. CONCLUSION: This study screened out leading compounds from N. sibirica and offered a new understanding of the antihypertensive properties of N. sibirica leaves, by which inhibit oxidative stress-induced endothelial dysfunction and improve lipid profiles.

Simultaneous determination of loading capacity and selectivity in preparative off-line two-dimensional separation: An application for purification of corilagin from Pomegranate flower extracts.

This work describes the development of capacity orthogonal chromatography (COC), a new technique for simultaneously determining the loading capacity and orthogonality during the construction of two-dimensional (2D) separations. Three steps were required for the construction of a COC based on the correlation between the selectivity factor (α) and both orthogonality and loading capacity. (1) α values of the impurities-target compound were used to normalize the retention of the impurities around the target compound. (2) α values were input into four quadrants of a coordinate system to identify correlations between orthogonality and loading capacity. α values of the impurities must be greater in the first dimension than the second dimension, with iterated analyses performed until an αmax is obtained for the two purification methods. (3) Touch-peak separation using the first-dimensional αmax was performed and the target compound was collected. The co-eluted impurities are further separated in the second dimension. To test the efficiency of this technique, a COC using two methods on a standard C18 column was developed to purify corilagin from pomegranate flower extract. Despite its low abundance, 288 mg of corilagin was obtained by COC and further purified by LH-20 gel chromatography to obtain the compound with an 80.0% recovery and 98.4% purity. Compared to COC, the purity of corilagin independently obtained using the same purification methods and identical loading capacity was poor (60.1% and 61.6%). These results indicate that COC is a useful tool for extending loading capacity in the development of preparative 2D separations.

Identification and quantification of Meiguihua oral solution using liquid chromatography combined with hybrid quadrupole-orbitrap and triple quadrupole mass spectrometers.

Meiguihua oral solution (MOS), a classical Chinese medicinal formula, was approved by the China Food and Drug Administration for production. However, the quality evaluation of MOS has not been reported. In this present study, qualitative and quantitative analysis of MOS were conducted by ultra high performance liquid chromatography coupled to quadrupole-orbitrap high resolution mass spectrometry (UHPLC-Q-orbitrap-HRMS) and high performance liquid chromatography-tri-quadrupole linear ion trap mass spectrometry (HPLC-tri-Q-LIT-MS). Totally 46 phenolic compounds (21 flavonoids and 25 tannins) were identified in the MOS, among them 14 polyphenols were not reported in raw plant materials of MOS. The simultaneous quantification of ten compounds including gallic acid, quercetin-3-O-sophoroside, ellagic acid, sophoraflavonoloside, hyperoside, isoquercitrin, avicularin, astragalin, quercitrin and juglanin, which were completed in 16 min in the negative electrospray ionization (ESI) mode under multiple reaction monitoring (MRM) method. Linearity was reached fine determination coefficient (r2 > 0.9995). Precisions, repeatability, stability (inter-day and intra-day), and recovery were validated and the relative standard deviations (RSD) were less than 2.9%, 4.7%, 3.6% and 1.79%, respectively. This result proved the high sensitivity and efficiency of the method. The quantitative and qualitative analysis of MOS would provide the substantial basis for further quality control and medicinal values.

A comprehensive study of pomegranate flowers polyphenols and metabolites in rat biological samples by high-performance liquid chromatography quadrupole time-of-flight mass spectrometry.

Pomegranate flowers is an ancient medicine that has commonly been used to treat various diseases such as diabetes. However, no reports are available on the metabolic profile of pomegranate flowers in vivo. In the present study, with the aid of HPLC-Q-TOF-MS2, 67 compounds were identified in pomegranate flowers extract, including 18 ellagitannins, 14 gallic acid and galloyl derivatives, five anthocyanins and 18 flavonoids. Seven compounds were firstly identified. In vivo, 22 absorbed compounds and 35 metabolites were identified in rat biosamples (urine, feces, plasma and tissues) after orally administered with pomegranate flowers extract. This result showed that not all compounds abundant in pomegranate flowers extract could be absorbed well in plasma and tissues. This finding also suggested a potential correlation between study on metabolic profile of these compounds in vivo and study on strategy of screening bioactivity of the isolates with in vitro cell systems evaluation. Notably, mono-glucuronide conjugated metabolite of ellagitannin compound (corilagin) was firstly identified. In addition, this is first report to identify phase II conjugate metabolites of ellagitannins in vivo after oral administration of ellagitannins-rich extracts (or foods). Thus, characterizing its multiple constitution, absorption and metabolic fate of these compounds in vivo is helpful to better analyze the active components in pomegranate flowers.

Quality Evaluation of the Traditional Medicine Majun Mupakhi ELA via Chromatographic Fingerprinting Coupled with UHPLC-DAD-Quadrupole-Orbitrap-MS and the Antioxidant Activity In Vitro.

By merging a high-performance liquid chromatography diode array detector (HPLC-DAD) method with high-performance thin-layer chromatography (HPTLC), an assay was developed for chemical fingerprinting and quantitative analysis of traditional medicine Majun Mupakhi ELA (MME), and constituent compounds were identified using HPLC coupled with UHPLC-DAD-Quadrupole-Orbitrap-MS method. In addition, the antioxidant capacity of MME was assessed based on the ability of components to scavenge radicals using in vitro method. Using a HPLC-DAD method with HPTLC easily validated the chemical fingerprinting results and quantified three characteristic components, namely, gallic acid (1), daidzein (2), and icariin (3), in commercial MMEs. The three compounds presented excellent regression values (R2 = 0.9999) in the ranges of the test and the method recovery was in the range from 100.49% to 100.68%. The fingerprints had 27 common characteristic peaks, of which 13 were verified by rapid UHPLC-DAD-Q-Orbitrap-MS analysis. In vitro antioxidant assays rapidly assessed and contrasted antioxidant activity or the free radical scavenging activity of the main polyphenolic classes in MMEs, and the antioxidant capacity was mostly affected by the presence of gallic acid. Thus, this study establishes a powerful and meaningful approach for MME quality control and for assessing in vitro antioxidant activity.

Qualitative Analysis of Polyphenols in Macroporous Resin Pretreated Pomegranate Husk Extract by HPLC-QTOF-MS.

INTRODUCTION: Pomegranate (Punica granatum L.) husk is a traditional herbal medicine abundant in phenolic compounds and plays some roles in the treatment of oxidative stress, bacterial and viral infection, diabetes mellitus, and acute and chronic inflammation. OBJECTIVE: Identification and determination of polyphenols in macroporous resin pretreated pomegranate husk extract by high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF-MS). METHODS: The total polyphenols of pomegranate husk were prepared by ethanol extraction followed by pretreatment with HPD-300 macroporous resin. The polyphenolic compounds were qualitatively analysed by HPLC-QTOF-MS in negative electrospray ionisation (ESI) mode at different collision energy (CE) values. RESULTS: A total of 50 polyphenols were detected in the extract of pomegranate husk, including 35 hydrolysable tannins and 15 flavonoids with distinct retention time, fragmentation behaviours and characteristics, and the accurate mass-to-charge ratios at low, moderate and high CE values. Of these, we identified nine compounds for the first time in the pomegranate husk, including hexahydroxydiphenoyl-valoneoyl-glucoside (HHDP-valoneyl-glucoside), galloyl-O-punicalin, rutin, hyperoside, quercimeritrin, kaempferol-7-O-rhahmano-glucoside, luteolin-3'-O-arabinoside, luteolin-3'-O-glucoside, and luteolin-4'-O-glucoside. To validate the specificity and accuracy of mass spectrometry in the detection of polyphenols, as compared to the fragmentation pathways of granatin B in detail, including the HHDP-valoneyl- glucoside was first identified from pomegranate husk. CONCLUSION: The study provides evidence for the quality control and development of novel drugs based on polyphenols from the pomegranate husk. Copyright © 2017 John Wiley & Sons, Ltd.

Chemical Fingerprint Analysis and Quantitative Analysis of Rosa rugosa by UPLC-DAD.

A method based on ultra performance liquid chromatography with a diode array detector (UPLC-DAD) was developed for quantitative analysis of five active compounds and chemical fingerprint analysis of Rosa rugosa. Ten batches of R. rugosa collected from different plantations in the Xinjiang region of China were used to establish the fingerprint. The feasibility and advantages of the used UPLC fingerprint were verified for its similarity evaluation by systematically comparing chromatograms with professional analytical software recommended by State Food and Drug Administration (SFDA) of China. In quantitative analysis, the five compounds showed good regression (R² = 0.9995) within the test ranges, and the recovery of the method was in the range of 94.2%-103.8%. The similarities of liquid chromatography fingerprints of 10 batches of R. rugosa were more than 0.981. The developed UPLC fingerprint method is simple, reliable, and validated for the quality control and identification of R. rugosa. Additionally, simultaneous quantification of five major bioactive ingredients in the R. rugosa samples was conducted to interpret the consistency of the quality test. The results indicated that the UPLC fingerprint, as a characteristic distinguishing method combining similarity evaluation and quantification analysis, can be successfully used to assess the quality and to identify the authenticity of R. rugosa.

Characterization and identification of chemical compositions in the extract of Artemisia rupestris L. by liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry.

Liquid chromatography coupled to negative electrospray ionization (ESI) tandem mass spectrometry (MS/MS) employing a time-of-flight tandem mass spectrometer was used in the structural determination of phenolic compounds and sesquiterpenoids occurring in the extract from Artemisia rupestris L. A total of 91 compounds including chlorogenic acid derivatives, flavonoids (aglycone, O-glycosyl, C-glycosyl and C,O-glycosyl), 2-phenoxychromones and guaiane sesquiterpenoids were identified by comparing the retention time and fragmentation behavior with reference standards or according to accurate mass measurement and the characteristic fragmentation at low and high collision energy. Most of these compounds were reported in Artemisia rupestris L. for the first time. Meanwhile, the proposed pathway and the major diagnostic fragmentation of 2-phenoxychromone and rupestonic acid were investigated to trace 2-phenoxychromone and rupestonic acid derivatives in crude plant extracts. According to these rules, we have successfully characterized five potential novel compounds including three 2-phenoxychromones (6-demethoxy-4'-O-methylcapillarisin-O-hexosylglucuronide, 6-demethoxy-4'-O-methylcapillarisin-O-pentosylhexoside and 6-demethoxy-4'-O-methylcapillarisin-O-deoxyhexosylhexoside) and two sesquiterpenoids (hexosyl-glycurinide-rupestonic acid and hexoside-rupestonic acid).