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Developing analytical methods for the chemical components of natural medicines remains a challenge due to its diversity and complexity. Miao-Fu-Zhi-Tong (MFZT) granules, an ethnic Yi herbal prescription, comprises 10 herbs and has been clinically applied for gouty arthritis (GA) therapy. Herein, a series of chemical profiling strategies including in-house library matching, molecular networking and MS/MS fragmentation behavior validation based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) were developed for qualitative analysis of MFZT granules. A total of 207 compounds were identified or characterized in which several rare guanidines were discovered and profiled into alkyl substituted or cyclic subtypes. Moreover, network pharmacology analysis indicated that MFZT's anti-gout mechanism was mostly associated with the nuclear factor kappa-B (NF- κ B) signaling, nucleotide oligomerization domain (NOD)-like signaling and rheumatoid arthritis pathways, along with the synergistic effect of 84 potential active compounds. In addition, a quantitative analytical method was developed to simultaneously determine the 29 potential effective components. Among them, berberine, pellodendrine, 3-feruloylquinic acid, neoastilbin, isoacteoside and chlorogenic acid derivatives at higher concentrations were considered as the chemical markers for quality control. These findings provide a holistic chemical basis for MFZT granules and will support the development of effective analytical methods for the herbal formulas of natural medicines. [ABSTRACT FROM AUTHOR]

This study presents a comprehensive investigation into the potential anticancer properties and chemical constituents of an unexplored plant (Grewia velutina) extracts. Three distinct extracts of hexane (GVH), chloroform (GVC) and methanol(GVM) of the plant were used to check their anticancer activity against four prominent cancer cell lines (HepG2, DU145, Hela, A549), and the results were compared with the standard chemotherapeutic agent doxorubicin. Anticancer assays revealed that the GVC extract exhibited highest activity than others, with IC50 values of 61.06 µg/mL, 47.87 µg/mL, 88.76 µg/mL, and 87.99 µg/mL against HepG2, DU145, Hela, and A549 cell lines, respectively and reflected a comparable activity to doxorubicin, highlighting its potential as an effective anticancer agent. With the help of chemical profiling data, molecular docking studies were performed and this study suggested that ionol, present in GVC extract is the most active compound against all the cancerous cells. [ABSTRACT FROM AUTHOR]

In the present study, the chemical profiling of methanol (MSC) and ethyl acetate (EASC) extracts of Sargassum cristaefolium was investigated using UPLC-ESI-Q-TOF-MSE analysis and the results showed the presence of 146 compounds belongs to various class of bioactive compounds. As well, the LC-QqQ-MS analysis was used to quantify the compounds present in the extracts of S. cristaefolium. The in vitro cytotoxic activity showed that the MSC and EASC have excellent growth inhibitory activity against prostate cancer and induce apoptosis in DU145 prostate cancer cells by generating oxidative stress and arresting the cell cycle progression via upregulating Bax, p53, and caspase 3. The in vivo analysis evidenced that the MSC and EASC inhibit tumor growth by upregulating the p53 and caspase 3 in BALB/c mice. Together, the compounds present in the solvent extracts of S. cristaefolium could be a potential source for the treatment of prostate cancer. [Display omitted] • UPLC-Q-TOF-MSE analysis used for the identification of anticancer compounds. • p53-mediated signalling pathway is prominent in skin melanoma during apoptosis was confirmed with Gene Expression Omnibus database (GEO). • EASC and MSC generates oxidative stress in prostate cancer cells. • Induce apoptosis by inhibiting the p53-mediated signalling pathway. • EASC and MSC inhibits the tumor growth in BALB/c mice by upregulating the p53 and caspase 3 expression. [ABSTRACT FROM AUTHOR]

Qing-Fei-Pai-Du decoction (QFPDD) is a Chinese medicine compound formula recommended for combating corona virus disease 2019 (COVID-19) by National Health Commission of the People's Republic of China. The latest clinical study showed that early treatment with QFPDD was associated with favorable outcomes for patient recovery, viral shedding, hospital stay, and course of the disease. However, the effective constituents of QFPDD remain unclear. In this study, an UHPLC-Q-Orbitrap HRMS based method was developed to identify the chemical constituents in QFPDD and the absorbed prototypes as well as the metabolites in mice serum and tissues following oral administration of QFPDD. A total of 405 chemicals, including 40 kinds of alkaloids, 162 kinds of flavonoids, 44 kinds of organic acids, 71 kinds of triterpene saponins and 88 kinds of other compounds in the water extract of QFPDD were tentatively identified via comparison with the retention times and MS/MS spectra of the standards or refereed by literature. With the help of the standards and in vitro metabolites, 195 chemical components (including 104 prototypes and 91 metabolites) were identified in mice serum after oral administration of QFPDD. In addition, 165, 177, 112, 120, 44, 53 constituents were identified in the lung, liver, heart, kidney, brain, and spleen of QFPDD-treated mice, respectively. These findings provided key information and guidance for further investigation on the pharmacologically active substances and clinical applications of QFPDD. [ABSTRACT FROM AUTHOR]

Honey is a natural food often used as an auxiliary material in Traditional Chinese Medicine (TCM). Cortex Mori (CM) is a commonly used Chinese medicinal material that requires honey preparation before use. Our research discovered that various factors including honey temperature, color intensity, moisture content, viscosity, TPC, 5-HMF content, and FRAP all increased over time during the honey refining process. To systematically study the excipients that play a unique role in TCM processing, this study focuses on honey as the representative and CM as the model drug. The overall components of honey-processed CM, stir-fried CM, and raw CM were determined using the fingerprint method, resulting in 36 chromatographic peaks with significant differences. The principal component analysis (PCA) results showed significant differences among honey-fried, stir-fried, and raw CM. Orthogonal partial least squares-discriminant analysis (OPLS-DA) and artificial neural network (ANN) were used to screen the important differential components. Network pharmacology and molecular docking technology were used to correlate the efficacy of the marker components. Oxyresveratrol, kuwanon G, and morusin were identified as potential markers. This study proved that the addition of honey as an excipient can affect the chemical composition of the drug itself, thereby impacting its efficacy. [ABSTRACT FROM AUTHOR]

Astragali Radix (AR) is one of the most popular herbal medicines in traditional Chinese medicine (TCM). Wild AR is believed to be of high quality, and substitution with cultivated AR is frequently encountered in the market. In the present study, two types of ARs (wild and cultivated) from Astragalus membranaceus (Fisch.) Bge. and A. membranaceus var. mongholicus (Bge.) Hsiao, growing in different regions of China, were analyzed by NMR profiling coupled with multivariate analysis. Results showed that both could be differentiated successfully and cultivation patterns or growing years might have greater impact on the metabolite compositions than the variety; the metabolites responsible for the separation were identified. In addition, three extraction methods were compared and the method (M1) was used for further analysis. In M1, the extraction solvent composed of water, methanol, and chloroform in the ratio of 1 : 1 : 2 was used to obtain the aqueous methanol (upper layer) and chloroform (lower layer) fractions, respectively, showing the best separation. The differential metabolites among different methods were also revealed. Moreover, the sucrose/glucose ratio could be used as a simple index to differentiate wild and cultivated AR. Meanwhile, the changes of correlation pattern among the differential metabolites of the two varieties were found. The work demonstrated that NMR-based non-targeted profiling approach, combined with multivariate statistical analysis, can be used as a powerful tool for differentiating AR of different cultivation types or growing years. [ABSTRACT FROM AUTHOR]

Cimicifugae Rhizoma (Sheng ma) is a Ranunculaceae herb belonging to a composite family and well known in China. has been widely used in traditional Chinese medicine. The Pharmacopoeia of the People׳s Republic of China contains three varieties ( Cimicifuga dahurica (Turcz.), Cimicifuga foetida L. and Cimicifuga heracleifolia Kom.) which have been used clinically as “Sheng-ma”. However, the chemical constituents of three components of “Sheng-ma” have never been documented. In this study, a rapid method for the analysis of the main components of “Sheng-ma” was developed using ultra-high performance liquid chromatography with quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS). The present study reveals the major common and distinct chemical constituents of C. dahurica , C. foetida and C. heracleifolia and also reports principal component and statistical analyses of these results. The components were identified by comparing the retention time, accurate mass, mass spectrometric fragmentation characteristic ions and matching empirical molecular formula with that of the published compounds. A total of 32 common components and 8 markers for different “Sheng-ma” components were identified. These findings provide an important basis for the further study and clinical utilities of the three “Sheng-ma” varieties. [ABSTRACT FROM AUTHOR]

• Flavonoids and polyphenols are the main components type of Loropetalum chinensis (R.Br) oliv leaves (Bhjm). • Bhjm and ellagic acid (EA) exerted a therapeutic effect against 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced gastrointestinal mucosa damage (GMD) in terms of inflammation and mucosal repair. • Molecular mechanism was investigated by multiomics approaches. • Bhjm and EA may exert therapeutic effects by regulating key gene klf4, hist1h2ba , and differential lipid metabolites, including phosphatidylcholine (PC) and triglyceride (TG). Loropetalum chinensis (R.Br) Oliv (Bhjm), a Chinese folk herbal medicine, was traditionally used in the treatment of wound bleeding and skin ulcers. A new drug named JIMUSAN granules used for gastrosia was developed by our group, and clinical trials have been approved. However, as the principal herb, the material basis and underlying mechanisms of Bhjm in attenuating gastrointestinal mucosa damage (GMD) remain to be systemically illuminated. An integrated strategy was used to explore the therapeutic effects and mechanisms of Bhjm and ellagic acid (EA) on GMD zebrafish, using network pharmacology, transcriptomics, lipidomics, and real-time quantitative PCR (RT-qPCR) verification. First, network pharmacological analysis was used to infer the major effective constituents and targets of Bhjm. Ultra high performance liquid chromatography-linear ion trap/orbitrap high resolution mass spectrometry (UHPLC-LTQ-Orbitrap HRMS) and ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) were employed to identify the chemical constituents and quantify the different types of constituents. Second, zebrafish model of GMD was established by using 2,4,6-trinitrobenzenesulfonic acid (TNBS) to evaluate the efficacy of Bhjm and EA. The potential mechanism was examined by integrated transcriptomics and lipidomics analysis. Finally, validation tests were implemented using RT-qPCR. In this study, targets indentified by network pharmacology were related to inflammation and mucosal damage. Ten representative components that interacted with these targets were simultaneously determined by UHPLC-MS/MS. Sixty four compounds were identified or tentatively characterized, most of which were flavonoids and polyphenols. Bhjm and EA alleviated mucosal damage and reduced inflammation in a TNBS-induced zebrafish GMD model, indicating that EA was the main active compounds. Eight common differentially expressed genes were downregulated by Bhjm and EA, as determined by transcriptomics analysis. Lipidomics analysis confirmed 12 differential lipids, including phosphatidylcholine (PC) and triglyceride (TG). Further network enrichment analysis demonstrated that differential lipid metabolism was regulated by klf4 and hist1h2ba , and were validated by RT-qPCR. In our study, the chemical profile of Bhjm was clarified. Moreover, the GMD repair effect and the mechanism of Bhjm and EA was comprehensively analyzed for the first time, involving inflammation and lipid metabolism. Collectively, these findings will be significantly helpful for deeply exploring the clinical application value of Bhjm. [Display omitted] [ABSTRACT FROM AUTHOR]

Fusarium graminearum, a pathogen of wheat and maize, not only reduces grain yield and degrades quality but also produces mycotoxins in the infected grain. Focus has been on mycotoxins because of the human and animal health hazards associated with them. In addition to work done on mycotoxins, chemical profiling of F. graminearum to identify other compounds produced by this fungus remains critical. With chemical profiling of F. graminearum the entire chemistry of this fungus can be understood. The focus of this work was to identify chlorinated compounds produced by F. graminearum. Various chlorinated compounds were detected and their role in F. graminearum is yet to be understood. [ABSTRACT FROM AUTHOR]

The present study was designed to develop and validate a sensitive and reliable ultra high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) method to separate and identify the chemical constituents of Qixue Shuangbu Tincture (QXSBT), a classic traditional Chinese medicine (TCM) prescription. Under the optimized UPLC and QTOF/MS conditions, 56 components in QXSBT, including chalcones, triterpenoids, protopanaxatriol, flavones and flavanones were identified and tentatively characterized within a running time of 42 min. The components were identified by comparing the retention times, accurate mass, and mass spectrometric fragmentation characteristic ions, and matching empirical molecular formula with that of the published compounds. In conclusion, the established UPLC-QTOF/MS method was reliable for a rapid identification of complicated components in the TCM prescriptions. [ABSTRACT FROM AUTHOR]

[Display omitted] • Identification of bioactives in n butanol extract of the Justicia vahlii was performed using UPLC-QTOF-MS and GC–MS analysis. • The acute oral toxicity test in mice and MTT assay confirmed the non-toxic nature of the extract. • The extract showed antioxidant based anti-inflammatory potential in dose dependent manner. • The extract showed significant in vitro enzyme inhibition potential. Justicia vahlii Roth. (acanthaceae) is an important medicinal food plant used in pain relief and topical inflammation. The present study aimed to evaluate phytochemical composition, toxicity, anti-inflammatory, antioxidant and enzyme inhibition potential of n- butanol extract of J. vahlii (BEJv). The extract prepared through maceration was found rich in total phenolic contents (TPC) 196.08 ± 6.01 mg of Gallic acid equivalent (mg GAE/g DE) and total flavonoid contents (TFC) 59.08 ± 1.32 mg of Rutin equivalent (mg RE/g DE). The UPLC-Q-TOF-MS analysis of BEJv showed tentative identification of 87 compounds and 19 compounds were detected in GC–MS analysis. The HPLC-PDA quantification showed the presence of 14 polyphenols amongst which kaempferol (3.45 ± 0.21 µg/ mL DE) and ferulic acid (2.31 ± 1.30 µg/ mL DE) were found in highest quantity. The acute oral toxicity study revealed the safety and biocompatibility of the extract up to 3000 mg/kg in mice. There was no effect of BEJv on human normal liver cells (HL 7702) and very low cytotoxic effect on liver cancer cells (HepG2) and breast cancer cells (MCF-7). In anti-inflammatory evaluation, the BEJv treated groups showed significant inhibition (p < 0.001) of late phase carrageenan induced paw edema at 400 mg/kg and increased the levels of oxidative stress markers; catalase, superoxide dismutase (SOD) and glutathione (GSH) while decreased the inflammatory markers; interleukin-1beta (IL-β) and tumor necrosis factor alpha (TNF-α) in paw tissue of mice. BEJv displayed highest results in Ferric reducing antioxidant power (FRAP) assay 97. 21 ± 2.34 mg TE (trolox equivalent)/g DE, and highest activity 3.32 ± 0.31 mmol ACAE (acarbose equivalent)/g D.E against α-glucosidase. Docking study showed good docking score by the tested compounds against the various clinically significant enzymes. Conclusively the current study unveiled J. vahlii as novel non-toxic source with good antioxidant-mediated anti-inflammatory potential which strongly back the traditional use of the species in pain and inflammation. [ABSTRACT FROM AUTHOR]

This paper reviews a body of work published by the authors over a number of years aimed at providing a complementary “signature” to conventional chemical profiling of methylamphetamine. The work, beginning in 2009, was undertaken because of the changing nature of clandestine methylamphetamine manufacture which has seen a dramatic rise in production and a major shift to a higher quality product. As manufacturing methods changed and methylamphetamine purity increased some conventional drug profiling techniques became less useful as an intelligence tool. For instance, the value of the organic impurity profile, a mainstay of conventional profiling, decreases as there are fewer impurities present in seized samples. However, whilst many seizures may contain less conventional chemical intelligence another property of the methylamphetamine molecule is always available, i.e. the light element stable isotope ratios. The stable isotope ratios of carbon, hydrogen and nitrogen are an integral part of the methylamphetamine molecule itself. This review describes previous work by the authors and by researchers in other national laboratories to investigate and implement the use of stable isotope ratios as an everyday methylamphetamine profiling tool. It also looks at how powerful this technique can be when used to compare samples from different seizures. [ABSTRACT FROM AUTHOR]

Over the past six years the Brazilian Federal Police has undertaken major efforts in order to implement and to develop its own drug chemical profiling program. This paper aims to provide a broad perspective regarding the managerial strategies and some examples of subsequent technical issues involved in the implementation of such a project. Close collaboration with local drug enforcement and investigation teams, establishment of proper worldwide partnerships with well recognized institutions in the field of drug analysis and the attainment of suitable funding and human resources are shown to be key success factors. Some preliminary results concerning the chemical profile of cocaine seizures in Brazil during this process are presented. [ABSTRACT FROM AUTHOR]

Abstract: Bupleuri Radix has been widely used in traditional Chinese medicine. In the current herbal market, the species Bupleurum marginatum Wall. ex DC. is the main source of Bupleuri Radix. Although Bupleuri Radix from the roots of B. marginatum grown wild in the North West of Hubei province has higher quality compared with those from other regions according to the previous investigations, the exhaustive exploitation driven by increasing demand has drastically reduced the wild resource. As a result, germplasm evaluation and quality resource exploration are important for the sustainable utilization and cultivation of B. marginatum. A preliminary study indicated differences in the tissue structure of B. marginatum grown in different areas of North Western Hubei province. In the current study, various tissues of the roots of B. marginatum grown in different areas of North Western Hubei were subjected to laser microdissection and analyzed by microscopy and ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC–Q-TOF-MS). The results show that wild plants from Maqiao Town, Baokang County contain the most saikosaponins distributed mainly in cork, cortex and phloem. This study provides key chemical information for evaluating the quality of B. marginatum roots. [Copyright &y& Elsevier]

Background: Traditional Chinese medicine (TCM) is regarded as a large database containing hundreds to thousands of chemical constituents that can be further developed as clinical drugs, such as artemisinin in Artemisia annua. However, effectively exploring novel candidates is still a challenge faced by researchers.Purpose: In this work, an integrated strategy combining chemical profiling, molecular networking, chemical isolation, and activity evaluation (CMCA strategy) was proposed and applied to systematically characterize and screen novel candidates, and Forsythiae fructus (FF) was used as an example.Study Design: It contained four parts. First, the chemical compounds in FF were detected by ultra-high-performance liquid chromatography-mass spectrometry (UPLC/Q-TOF MS) with data-dependent acquisition, and further, the targeted compounds were screened out based on an in-house database. In the meantime, the representative MS/MS fragmentation behaviors of different chemical structure types were summarized. Second, homologous constituents were grouped and organized based on feature-guided molecular networking, and the nontargeted components with homologous mass fragmentation behaviors were characterized. Third, the novel compounds were isolated and unambiguously identified by nuclear magnetic resonance (NMR). Finally, the anti-angiotensin-converting enzyme 2 (ACE2) activities of isolated chemical constituents were further evaluated by in vitro experiments.Results: A total of 278 compounds were profiled in FF, including 151 targeted compounds and 127 nontargeted compounds. Among them, 16 were unambitiously identified by comparison with reference standards. Moreover, 25 were classified into potential novel compounds. Two novel compounds were unambiguously identified by using conventional chromatographic methods, and they were named phillyrigeninside D (peak 254) and forsythenside O (peak 155). Furthermore, the ACE2 activity of the compounds in FF was evaluated by modern pharmacological methods, and among them, suspensaside A was confirmed to present obvious anti-ACE2 activity.Conclusion: Our work provides meaningful information for revealing potential FF candidates for the treatment of COVID-19, along with new insight for exploring novel candidates from complex systems. [ABSTRACT FROM AUTHOR]

Woodfordia fruticosa (L.) Kurz is a widely used plant in traditional medicine systems. The tribal communities of Amarkantak, Madhya Pradesh (India) are using this plant for the treatment of general weakness, blood related complications like blood deficiency, blood purification and for the treatment of symptoms related to sickle cell disease (SCD). SCD is a genetic disease with life threatening complications. In the absence of any drugs without any side effects, the alternative plant based therapies that may either reduce/ reverse the sickling of the red blood cells can be safe and effective therapeutic agents. We evaluated W. fruticosa extracts for phytoconstituents, anti-oxidant and anti-inflammatory properties. Anti-sickling properties of the extracts were evaluated by estimation of reverse sickling, polymerization inhibition and osmotic fragility assays. Chemical profiling of the methanol extract was done using LC-MS analysis. Phytochemicals such as alkaloids, steroids, tannin, and saponins were present in all the extracts. Methanol extract displayed maximum reversal (66 ± 1%) of sickled Red blood Cells (RBC) and significantly inhibited Hb polymerization. The hexane and methanol extracts led to minimum hemolysis of sickled RBC in the osmotic fragility assays. Total tannin (365 ± 2.4 TAE) content was highest in acetone extract, while the total flavonoid and phenolic content (156.9 ± 2.0 QE) and (113.7 ± 0.7 GAE) were highest in methanol extract. The methanol extract displayed minimum IC 50 (8.1 ± 1.5) in 2, 2-diphenylpicrylhydrazyl (DPPH) while the acetone extract had minimum IC 50 (215.8 ± 5.7) in 2,2′-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay. The hexane extract displayed maximum Ferric reducing anti-oxidant power (FRAP) value (1.5 ± 0.5 mM Fe(II)/mg dry weight) that were higher than methanol and aqueous extracts (1.45 ± 0.1, 1.45 ± 0.05 mM Fe(II)/mg dry weight). The methanol extract provided maximum RBC protection from hemolysis (73.8 ± 0.8%). Maximum Lipoxygenase (LOX) inhibition was observed by the acetone and methanol extracts at 400 μg/mL while the hexane extract displayed maximum Xanthine oxidase (XO) inhibition (57.0 ± 0.5%). LC-MS profiling of the methanol extract identified several secondary metabolites that might be responsible for the observed activities. The results validate the traditional use of W. fruticosa and present us with potential compounds for further development of novel anti sickling agents. [ABSTRACT FROM AUTHOR]

Background: Traditional Chinese herbal patches (TCHPs) characterized by good patient adherence and few side effects have been widely used in the clinic. However, their bioactive transdermal components, which are key to the quality and efficacy, have rarely been investigated.Purpose: Establishing an approach to probe the bioactive transdermal components of TCHPs, using Gutong patch (GTP) as a case study.Study Design and Methods: GTP constituents were identified using ultrahigh-pressure liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS). The identified GTP constituents were transferred to an ultrahigh-pressure liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS) method in a dynamic multiple reaction monitoring (dMRM) mode for sensitively targeted analysis of transdermal components present at low concentrations. The ex vivo transdermal components were rapidly probed using a percutaneous permeation model and the release kinetics of marker components was assessed to evaluate the release behavior of GTP. The in vivo transdermal components were then studied in a rat model of arthritis and the changes in concentration of 15 representative bioactive transdermal components with time were well revealed.Results: A total of 120 phytochemical constituents have been identified in GTP extracts and 61 ex vivo transdermal components were targetedly detected. The release kinetics of GTP were evaluated by eight marker components and fitted to a zero-order kinetic model. In vivo, 62 and 40 transdermal components were observed in the skin and subcutaneous tissues of arthritic rats, respectively. The concentration-time changes of 15 representative transdermal components with documented bioactivities have been successfully exhibited after GTP administration.Conclusion: We established a feasible approach to probe the bioactive transdermal components of TCHPs efficiently. The integration of mass spectrometry profiling and targeted detection in dMRM mode enabled a comprehensive investigation of phytochemical constituents and their transdermal delivery, thus addressing the challenge of direct probing of diverse transdermal components present at low concentrations. This approach could be used to rapidly probe the bioactive components and understand the mechanism of TCHPs. [ABSTRACT FROM AUTHOR]

The co-elution, pervasive isomers, and frequently occurring in-source fragmentation definitely hinder the comprehensive characterization of the multiple components from traditional Chinese medicine (TCM) by liquid chromatography/mass spectrometry. Herein, a dimension-enhanced strategy, by utilizing an ion mobility quadrupole time-of-flight mass spectrometer coupled to ultra-high performance liquid chromatography (UHPLC/IM-QTOF-MS) with the ability of offering four-dimensional information (retention time, Rt; drift time or collision cross section, CCS; accurate mass of precursors; and accurate MS2 product ions), is presented. Shuanghe Decoction (SHD) is a classic tonifying formula composed of nine herbs, however, little is known to its chemical complexity, hitherto. Good chromatographic separation was achieved on an HSS T3 column, and by the Vion™ IMS-QTOF high-resolution mass spectrometer, data-independent MSE and High-Definition MSE (HDMSE) in both the negative and positive electrospray ionization modes were employed for acquiring the collision-induced dissociation-MS2 data. A self-built database, including 642 known compounds, was established, which assisted to perform the automated peak annotation by UNIFI™. By reference to 55 compounds, totally 236 components were identified or tentatively characterized from SHD with 4D data recorded. The combination of negative and positive modes could yield more information complementary to structural elucidation. Compared with the conventional MSE, HDMSE exhibit of merits in the provision of CCS, better separation and detection those easily co-eluting components, and reduction of the false positives in the identification results. Conclusively, the combination of HDMSE acquisition, in-house library, and UNIFI-facilitated automated peak annotation, renders a potent approach dedicated to deconvoluting the chemical composition of complex systems like TCM formulae. [ABSTRACT FROM AUTHOR]