Integration of offline two-dimensional chromatography and mass defect filtering-based precursor ion list data acquisition for targeted characterization of diterpenoid alkaloids in the lateral roots of Aconitum carmichaelii.

The hyphenated technique of offline two-dimensional (2D) chromatography with high resolution mass spectrometry (MS) was an efficient tool for separation and characterization of components in complex systems such as herbal medicines, especially those co-eluting components or isomers. In this study, we constructed the ultra-performance convergence chromatography (UPC2) × reversed phase (RP) chromatographic separation system and developed a mass defect filtering (MDF)-based precursor ion list (PIL) acquisition method to improve the selectivity and sensitivity of this technique, and the systematic characterization of diterpenoid alkaloids in the lateral roots of Aconitum carmichaelii (namely "Fuzi" in Chinese) was used as an example. The constructed offline 2D separation system showed a good orthogonality of 0.77. Besides, the in-house databases for known and predicted C19- and C20-diterpenoid alkaloids were established by molecular design in Compound Discoverer software for MS data matching and filtering, and two MDF windows were further constructed to screen out more potential diterpenoid alkaloids with novel structures and to obtain the PIL (mass range: even values between 298 and 1020 Da, parent mass width: ±100 mDa) for data acquisition by calculating the m/z values of potential ions using mass range and corresponding mass defect in the MDF windows. In addition, an integrative structure interpretation strategy was developed by integrating elemental composition analysis, ring double bond analysis, neutral loss filtering, diagnostic ion filtering and database matching, etc. As a result, a total of 659 components in the lateral roots of A. carmichaelii were exposed and characterized, including 526 potential new compounds. This strategy showed significant advantages in improving the coverage and selectivity of screening, and could also be applied in systematic characterization of components in other herbal medicines.

An integrated strategy for comprehensive characterization of metabolites and metabolic profiles of bufadienolides from Venenum Bufonis in rats.

Comprehensive characterization of metabolites and metabolic profiles in plasma has considerable significance in determining the efficacy and safety of traditional Chinese medicine (TCM) in vivo. However, this process is usually hindered by the insufficient characteristic fragments of metabolites, ubiquitous matrix interference, and complicated screening and identification procedures for metabolites. In this study, an effective strategy was established to systematically characterize the metabolites, deduce the metabolic pathways, and describe the metabolic profiles of bufadienolides isolated from Venenum Bufonis in vivo. The strategy was divided into five steps. First, the blank and test plasma samples were injected into an ultra-high performance liquid chromatography/linear trap quadrupole-orbitrap-mass spectrometry (MS) system in the full scan mode continuously five times to screen for valid matrix compounds and metabolites. Second, an extension-mass defect filter model was established to obtain the targeted precursor ions of the list of bufadienolide metabolites, which reduced approximately 39% of the interfering ions. Third, an acquisition model was developed and used to trigger more tandem MS (MS/MS) fragments of precursor ions based on the targeted ion list. The acquisition mode enhanced the acquisition capability by approximately four times than that of the regular data-dependent acquisition mode. Fourth, the acquired data were imported into Compound Discoverer software for identification of metabolites with metabolic network prediction. The main in vivo metabolic pathways of bufadienolides were elucidated. A total of 147 metabolites were characterized, and the main biotransformation reactions of bufadienolides were hydroxylation, dihydroxylation, and isomerization. Finally, the main prototype bufadienolides in plasma at different time points were determined using LC-MS/MS, and the metabolic profiles were clearly identified. This strategy could be widely used to elucidate the metabolic profiles of TCM preparations or Chinese patent medicines in vivo and provide critical data for rational drug use.

An enhanced strategy integrating offline two-dimensional separation with data independent acquisition mode and deconvolution: Characterization of metabolites of Uncaria rhynchophylla in rat plasma as a case.

The importance to clarify the drug metabolites is beyond doubt in view of their potential efficacy and safety. However, due to the complex matrix interference, relatively low content and the co-eluting effect, it is of a great challenge to comprehensively and systematically characterize the metabolites in vivo, especially for the traditional Chinese medicines (TCMs) due to the numerous types of components. In the present study, a comprehensive off-line two-dimensional separation system combining with data independent acquisition (DIA) mode and multi-dimensional data deconvolution method was established for chromatographic separation, data acquisition and data procession of indole alkaloids in rat plasma after intragastrically administrated with the extract of Uncaria rhynchophylla at the dose of 1 g/kg. The orthogonality of the off-line 2D separation system consisting of HILIC for first-dimensional separation and the PRLC for second-dimensional separation was valuated with the "asterisk" equations, and the results showed that off-line 2D separation system had passable orthogonality (A0 = 53.3%). Furthermore, the DIA mode was applied to capture MS/MS spectra in view of its advantage in acquiring MS data, and an effective multi-dimensional deconvolution method integrating the calculation of chemical formula, the extraction of diagnostic ion, the filter of ring double bond (RDB) and the judgement of neutral loss was established to parse the spectra for the complicated DIA data for comprehensive analysis of metabolites in rat plasma. Ultimately, a total of 127 indole alkaloids were tentatively characterized, and the main metabolic pathways were inferred as demethylation, dehydrogenation, hydroxylation and deglycosylation. The off-line two-dimensional separation system was applied for the comprehensive characterization of metabolites in vivo for the first time. This study suggested a new approach to enable the enrichment, separation and analysis of the low content components in vivo.

Chemical profiling of Huashi Baidu prescription, an effective anti-COVID-19 TCM formula, by UPLC-Q-TOF/MS.

Huashi Baidu prescription (HSBDF), recommended in the Guideline for the Diagnosis and Treatment of Novel Coronavirus (2019-nCoV) Pneumonia (On Trials, the Seventh Edition), was clinically used to treat severe corona virus disease 2019 (COVID-19) with cough, blood-stained sputum, inhibited defecation, red tongue etc. symptoms. This study was aimed to elucidate and profile the knowledge on its chemical constituents and the potential anti-inflammatory effect in vitro. In the study, the chemical constituents in extract of HSBDF were characterized by UPLC-Q-TOF/MS in both negative and positive modes, and the pro-inflammatory cytokines were measured by enzyme-linked immunosorbent assays (ELISA) to determine the effects of HSBDF in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The results showed that a total of 217 chemical constituents were tentativedly characterized in HSBDF. Moreover, HSBDF could alleviate the expression levels of IL-6 and TNF-α in the cell models, indicating that the antiviral effects of HSBDF might be associated with regulation of the inflammatory cytokines production in RAW264.7 cells. We hope that the results could be served as the basic data for further study of HSBDF on anti-COVID-19 effect.

A strategy for practical authentication of medicinal plants in traditional Chinese medicine prescription, paeony root in ShaoYao-GanCao decoction as a case study.

Authentication of Chinese medicine materials in prescriptions is extremely difficult due to the complicated chemical matrix. A strategy integrating in-depth profiling, chemical marker selection, and selected detection was established and exemplarily applied to authenticate paeony root in ShaoYao-GanCao decoction. First, an ultra-performance liquid chromatography/linear trap quadrupole-Orbitrap method was developed to probe the chemical compositions of the decoction. Second, 20 batches of decoctions prepared from white paeony root and red paeony root were compared by a metabolomics method, and multistep chemometrics analysis distinguished the chemical markers. Third, an ultra-performance liquid chromatography/QDa-selected ion monitoring method was developed to authenticate the paeony root in decoctions. As a result, 161 compounds were characterized, including 84 triterpene saponins, 42 flavonoids, and 10 monoterpenes. Four chemical markers and paeoniflorin were successfully screened out as chemical markers for white paeony root. The selected ion monitoring method easily differentiated authentic decoction (prepared from white paeony root) from fraud decoction (prepared from red paeony root) by monitoring the above five chemical markers. In conclusion, the strategy was proved effective in authentication of paeony root in ShaoYao-GanCao decoction, and it can also be applied to authenticate other Chinese medicine materials in prescriptions, which will greatly avail the quality enhancement of prescriptions.

The inhibitory effect of 225 frequently-used traditional Chinese medicines for CYP3A4 metabolic enzyme by isoform-specific probe.

Traditional Chinese medicines (TCMs), have been widely used for the prevention, treatment, and cure of various diseases for thousands of years in China and Asian countries. It is usually applied either alone or in combination with synthetic drugs or other herbs to be more effective. However, the evaluation of TCMs against the main phase I metabolic enzyme CYP3A4 in vitro was limited. In the present study, a high throughput method based on an isoform-specific probe was applied to evaluate the inhibitory effect of 225 frequently-used TCMs on CYP3A4 activity. The results showed that 25 TCM herbs possessed inhibition effect with residual activity below 50%, and four TCMs (Curcumae Rhizoma, Piperis Longi Fructus, Dalbergiae Odoriferae Lignum, Arisaematis Rhizoma Preparatum) had fairly strong inhibition effect with residual activity below 20%. In an attempt to validate the results obtained from isoform-specific probe, the Curcumae Rhizoma with lowest residual activity was further tested to screen main bioactive constituents which possessed significant inhibitive effect. The crude extract of Curcumae Rhizoma was fractionated to investigate the inhibition effect of each fraction, the results showed that fractions 9-13 exhibited obvious inhibitory effect, and the main constituent (curdione) was identified with standard reference. The molecular docking results verified that the inhibiting effect of curdione could be explained that curdione was interacted with 7 amino acid residues to generate the hydrophobic interaction, and also interacted with imidazole to form hydrogen bond. It is anticipated that the results could be used as reference data to avoid drug-drug interaction and guide the clinical application of TCM or prescriptions.

An enhanced strategy integrating offline superimposed two-dimensional separation with mass defect filter and diagnostic ion filter: Comprehensive characterization of steroid alkaloids in Fritillariae Pallidiflorae Bulbus as a case study.

The inherent complexity of traditional Chinese medicines necessitates the application of multi-dimensional information to accomplish comprehensive profiling and confirmative identification of their chemical components. In this study, we display an enhanced strategy by integrating offline superimposed two-dimensional separation (S-2D-LC) with mass defect filter and diagnostic ion filter to comprehensively characterize the alkaloid composition of Fritillariae Pallidiflorae Bulbus (FPB). The superimposed HILIC × RP and UPCC × RP offline two-dimensional liquid chromatography system was constructed with superior orthogonality (R2=0.004 and R2=0.001) for chromatographic separation. In total, 70 fractions were collected after the first-dimensional chromatographic separation (HILIC and UPCC) and then analyzed by the second-dimensional reversed phase (RP) liquid chromatography coupled with Q-TOF/MS/MS in FAST DDA acquisition mode. A four-step interpretation strategy combining mass defect filter with diagnostic ion filter was developed to rapidly characterize alkaloids in Fritillaria species. Ultimately, a sum of 529 Fritillaria alkaloids were characterized from two botanical origins of FPB. The integrated strategy is practical to efficiently expose and comprehensively characterize more trace and isomeric components in complex herbal medicines.

Exploration of tissue distribution of ginsenoside Rg1 by LC-MS/MS and nanospray desorption electrospray ionization mass spectrometry.

Ginsenoside Rg1 (Rg1) was one of the dominent active components in several Panax medicinal species as Panax notoginseng and Panaxginseng with diversified bioactivities. However, the study on tissue distribution of Rg1 remained limited and needed to be further explored for elucidation of its spatial distribution. In the present study, a LC-MS/MS combined with nanospray desorption electrospray ionization (DESI) mass spectrometry method was developed for exploration of tissue distribution of Rg1 at different time points after intravenous administration to rats. Furthermore, a MS inlet-heat method was developed to improve the imaging efficacy of Rg1 in brain tissue. The results obtained from LC-MS/MS analysis indicated that kidney possessed the highest tissue concentration, followed by liver, lung, spleen, heart and brain. Meanwhile, the elimination of Rg1 was swift within 1 h. For the spatial distribution of Rg1 by DESI-MS, Rg1 mainly accumulated in the pelvis section of kidney. Meanwhile, the imaging result of brain implied that Rg1 might be distributed in the pons and medulla oblongata region of brain at 15 min after intravenous administration. It is anticipated that the data on tissue distribution of Rg1 could provide references for further probing its efficacy and drug development.

A systematic strategy integrating solid-phase extraction, full scan range splitting, mass defect filter and precursor ion list for comprehensive metabolite profiling of Danqi Tongmai tablet in rats.

In vivo metabolite profiling of herbal medicines remains a challenge due to the complex chemical composition and drastic interference from biological matrix. In this study, a systematic strategy was established for comprehensive metabolite profiling of Danqi Tongmai (DQTM) tablet, a combination of salvianolic acids and notoginsenosides, in rats after oral administration. This strategy was composed of six steps. Firstly, the rat plasma and tissue samples were collected at multiple time points to increase the representativeness of samples. Secondly, different sample preparation methods were systematically investigated including protein precipitation, liquid-liquid extraction and solid-phase extraction to obtain superior extraction efficiency for both salvianolic acids and notoginsenosides. Thirdly, the MS acquisition method was optimized by splitting the full scan range into two separate segments to improve the detection capability for minor components. Fourthly, an extended polygonal mass defect filter (EP-MDF) model was constructed to filter potential metabolites of salvianolic acids and notoginsenosides, and remove large amounts of interference ions. Fifthly, ion intensity-based time point-staggered precursor ion list (IITPS-PIL) was generated to trigger more targeted MS/MS acquisition for potential metabolites at the highest concentration. Finally, the absorbed prototypes and metabolites were comprehensively characterized by reference standards and MS/MS fragmentation. The proposed strategy significantly improved the detection ability for trace prototypes and metabolites in vivo. A total of 370 components, including 94 prototypes (38 confirmed with reference standards) and 276 metabolites, were tentatively characterized in rat plasma and tissue samples after oral administration of DQTM. Collectively, this paper provided an applicable reference for comprehensive metabolite profiling of herbal medicines in complex biological samples.

Systematic comparison of metabolic differences of Uncaria rhynchophylla in rat, mouse, dog, pig, monkey and human liver microsomes.

Metabolites have a close relationship with the efficacy and safety of herbal medicines. However, ubiquitous matrix interferences, complex co-elution, and minor or trace amounts in plasma restrict the comprehensive identification of metabolites. In this study, an efficient strategy comprising a mass defect filter and time-staggered targeted ion lists was established to characterize the metabolites of alkaloids of Uncaria rhynchophylla (UR) for the systematic comparison of metabolic differences in rat, mouse, dog, pig, monkey and human liver microsomes. The mass defect filter model effectively decreased interfering ions by 63-68%, and time-staggered precursor ion lists significantly increased the number of triggered MS/MS fragmentation by 65-120% in liver microsomes of six species. Ultimately, a total of 165 metabolites in the liver microsomes of six species were tentatively characterized, and the main metabolic pathways were demethylation, isomerization, hydrolysis, oxygenation and dehydrogenation. The results showed that the mouse liver microsomes exhibited metabolic behavior most similar to human metabolism of UR alkaloids. We hope that these results provide basic data for further investigation of UR metabolism in different species, and that the strategy can provide a reference for metabolite characterization of herbal medicines in complex biological matrix. Graphical abstract.

Venenum bufonis: An overview of its traditional use, natural product chemistry, pharmacology, pharmacokinetics and toxicology.

ETHNOPHARMACOLOGICAL RELEVANCE: The animal medicine of Venenum Bufonis (VB), a product of the secretions of Bufo gargarizans Cantor or B. melanostictus Schneider, has long been used as a traditional Chinese medicine (TCM) for the treatment of sunstroke and faint, acute filthy disease - abdominal pain or vomiting and diarrhea, etc. AIM OF THE REVIEW: This review is aimed at providing the comprehensive and up-to-date information of VB as regards its ethnopharmacological uses, constituents and their metabolism, pharmacokinetics, pharmacology and toxicology, all of which could be used as fundamental data for future research as well as development of new drugs. MATERIALS AND METHODS: The information and data about the studies of VB were collected from scientific journals, material medica, historical documents, library, and electronic databases (PubMed, Google Scholar, Science Direct, Researchgate, Web of Science and CNKI). RESULTS: To date, about 142 bufadienolides and 16 indole alkaloids have been isolated from VB in total. The extract and isolated compounds showed a wide range of in vitro and in vivo pharmacologic effects, such as cardiotonic, anti-tumor, antinociceptive, anti-inflammatory, anesthetic and antimicrobial activities. Especially, bufadienolides have been extensively studied due to its powerful anti-tumor activities against various cancer cells. Furthermore, their metabolites and metabolic pathways were concluded in detail, and the main metabolic pathways of bufadienolides were hydroxylation, 3-isomerization, 3-keto, 16-hydrolyzation, 3-O-sulfate and 3-O-glucuronide. CONCLUSIONS: Although VB possesses significant anti-tumor effect against various cancer cell lines, the development of new drugs still remains to be a challenge due to its pharmacodynamic effects in vivo, druggability and toxicology. The main problem lies in its side effects in vivo, poor bioavailability, fast metabolism, cardiotoxicity and neurovirulence. Besides, studies on its metabolism and toxicology in vitro and in vivo, as well as clinical trials should be further conducted for the new drug development and the establishment of optimal dosage of consumption of its administration.

Simultaneous Quantitation of a Novel α1/ß1-Blocker TJ0711 and Its Two Metabolites in Dog Plasma Using LC-MS/MS and Its Application to a Pharmacokinetic Study after Intravenous Infusion.

TJ0711∙HCl, which is a novel α1/ß1 adrenoceptor blocking agent with a ratio of 1:1 for α1/ß1, is designed to treat and prevent perioperative hypertension. M1 and M3 were identified as important metabolites in vitro for either antihypertension activity or the major metabolite production. In order to obtain a pharmacokinetic profile of both TJ0711 and its metabolites, a rapid, selective, and reliable LC-MS/MS method was developed and validated for simultaneous determination of TJ0711 and two metabolites in beagle dog plasma via efficiently separating two interferential metabolites M16 and M4 from M1 and M3, respectively. Chromatographic separation was achieved on a Waters CORTECS C18⁺ column (2.1 × 100 mm, 2.7 µm). The mass spectrometric detection was carried out in positive ion MRM mode with ESI⁺ source. Protein precipitation was used in sample preparation and provided good recovery without a matrix effect. Good linearity was observed at the ranges of 0.5⁻100 ng/mL for TJ0711 and M3, 0.1⁻20 ng/mL for M1. Additional validation results were within the acceptance limits followed U.S. FDA guidelines for bioanalytical method validation. This method was successfully applied to an intravenous infusion pharmacokinetic study of TJ0711 at dosing rates of 3, 6, and 12 µg/kg/min in anesthetized beagle dogs for the first time. TJ0711 and its two metabolites exhibited effective proportionality in the dosage of 3 to 12 µg/kg/min. Neither TJ0711 nor its metabolites showed significant differences in pharmacokinetic parameters such as t1/2, CL, and Vss among three dose groups.