Orbitrap Tandem Mass Spectrometry Research Articles

Mailuo Shutong pills inhibit neuroinflammation by regulating glucose metabolism disorders to protect mice from cerebral ischemia-reperfusion injury

Ethnopharmacological relevanceMailuo Shutong Pill (MLST), a traditional Chinese medicine (TCM), has been widely used for clearing heat and detoxifying, eliminating stasis and dredging meridians, dispelling dampness and diminishing swelling. Earlier study found that MLST could improve cerebral ischemic-reperfusion injury, however, the potential mechanism has not been well evaluated. Aim of studyIn this study, a well established and widely used mice model of middle cerebral artery occlusion/reperfusion (MCAO/R) was preformed to evaluate the protective function of MLST on cerebral ischemic-reperfusion injury and further discuss the potential pharmacological mechanisms. Materials and methodsChemical profiling of MLST was analyzed based on Ultra-high-performance liquid chromatography electrospray ionization orbitrap tandem mass spectrometry. ICR mice were challenged by MCAO/R surgery. The protective effect of MLST on MCAO/R injury was evaluated by neurological deficit score, cerebral infarct rate, brain water content, H&E and nissl staining. The blood-brain barrier (BBB) integrity was detected by Evans blue staining. The potential pharmacological mechanism of MLST in treating MCAO/R injury was further elucidated by the methods of proteomics, central carbon targeted metabolomics, as well as Western blot. Immunohistochemistry was used to detect the microglia infiltration, enzyme linked immunosorbent assay (ELISA) kit was explored to evaluate the content of IL-1β, TNF-α and IL-6 in brain tissue, and Western blot was used to detect proteins expression in brain tissue. ResultsA total of 76 chemical compounds have been determined in MLST. MLST effectively protected mice from MCAO/R injury, which was confirmed by lower neurological deficit score, cerebral infarct rate, brain water content and nissl body loss, and improved brain pathology. Meanwhile, MLST upregulated the expression of ZO-1, Occludin and Claudin 5 by downregulating the ratio of TIMP1/MMP9 to suppress the entrance of Evans blue to brain tissue, indicating that MLST maintained the integrity of BBB. Further studies indicated that MLST inhibited the inflammatory level of brain tissue by inhibiting microglia infiltration and downregulating NLRP3 inflammasome signaling pathway. The results of proteomics, Western blot, and central carbon targeted metabolomics confirmed that MLST regulated Glycolysis/Gluconogenesis, Pyruvate metabolism and TCA cycle in brain tissue of mice with MCAO/R. ConclusionMLST inhibits neuroinflammation by regulating glucose metabolism disorders to interfere with immune metabolism reprogramming and inhibit the NLRP3 inflammasome signaling pathway, and finally improve cerebral ischemia-reperfusion injury. This study confirms that MLST is a potential drug for treating Cerebral ischemic stroke.

Enhancing anthocyanin extraction efficiency in vegetables and fruits: a high-speed shear homogenization technology.

To extract anthocyanins with high efficiency, a hypothesis for high-speed shear homogenization extraction (HSHE) method was established through a combination of solvent and ultrasonic-assisted extractions. The efficacy of this hypothesis was demonstrated by performing qualitative and quantitative analyses of 16 anthocyanins extracted from five northern vegetables, and five berry fruits using ultra-high-performance Q-Exactive Orbitrap tandem mass spectrometry. Single-factor experiments were conducted by varying ethanol concentration, temperature, pH and extraction cycles to determine the optimal conditions for this method. Optimal extraction conditions (ethanol 70-80%, 40-50 °C, pH 3-4, performed twice) were determined using an HSHE (5 min, 10 000 rpm, 25 °C) assisted shaker (60 min) and ultrasonication (40 kHz, 160 W cm-2, 30 min, 25 °C) procedure. Compared to the traditional non-HSHE method, the total anthocyanin content obtained through HSHE extraction showed a significant increase, ranging from 1.0 to 3.9 times higher, with purple cabbage exhibiting the most pronounced enhancement in content. More types of anthocyanins were detected in blueberry (9), black bean (7) and raspberry (5), of which malvidin was the major anthocyanin (0.426 g kg-1) in blueberry, having an amount five times than previously obtained. The established HSHE method has been proven to be a superior technique for anthocyanin extraction, with higher extraction efficiency and concentrations. This technique also provides a new avenue for extracting bioactive compounds from diverse food sources, with potential applications in improving the functional properties of food products. © 2024 Society of Chemical Industry.

Metabolic and elemental profiling as potential discriminating features among the black mahlab seeds (Monechma ciliatum) grown in three different regions.

Black mahlab (Monechma ciliatum) seed is a rich source of metabolites and minerals and is usually believed to have a similar composition between different areas of cultivation. Until now, no studies have assessed changes in black mahlab seeds (BMSs) to determine those constituents that help to discriminate them according to geographical origin. The present study attempted to compare the metabolomics and elemental profiles of BMSs of different geographical origins and identified the potential markers using ultrahigh-performance liquid chromatography quadrupole Orbitrap tandem mass spectrometry (UHPLC-Q-Orbitrap-MS2 ), and inductively coupled plasma mass spectrometry (ICP-MS) techniques and established the chemometric model to identify the potential markers and discriminate them according to cultivation sites. In this work, data from metabolites analysis by UHPLC-Q-Orbitrap-MS2 and multi-elemental data obtained from ICP-MS were combined with chemometrics for tracing the geographical origin of BMSs. Principal component analysis (PCA) was used to evaluate the overall grouping of samples. In contrast, partial least squares-discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were employed for authentication. PLS-DA and OPLS-DA models were fully validated (R2 Y and Q2 values > 0.5). Variable importance of various projections was applied to obtain valuable data about differential elements (seven markers were identified) and metabolites (23 markers were identified) with high discrimination potential. The outcomes presented in this study serve as an appropriate framework for developing novel discrimination approaches in food origin screening.

Metabolite characterisation of the peptide–drug conjugate LN005 in liver S9s by UHPLC-Orbitrap-HRMS

LN005 is a peptide–drug conjugate (PDC) targeting glucose-regulated protein 78 (GRP78) to treat several types of cancer, such as breast, colon, and prostate cancer. As a new drug modality, understanding its metabolism and elimination pathways will help us to have a whole picture of it. Currently, there are no metabolic studies on LN005; therefore, this study aimed to investigate the metabolism of LN005, clarify its metabolic profile in the liver S9s of different species, and identify the major metabolic pathways and differences between species. The incubation samples were measured by ultra-high performance liquid chromatography combined with orbitrap tandem mass spectrometry (UHPLC-Orbitrap-HRMS). The results showed that LN005 was metabolised by liver S9s, and four metabolites were identified. The main metabolic pathway of LN005 in liver S9s was oxidative deamination to ketone or hydrolysis. Similar metabolic profiles were observed in mouse, rat, dog, monkey, and human liver S9s, indicating no differences between these four animal species and humans. This study provides information for the structural modification and optimisation of LN005 and affords a reference for subsequent animal experiments and human metabolism of other PDCs.

Exploration of material basis: Chemical composition profile, metabolic profile and pharmacokinetic characteristics in Xingbei Zhike granule

Headings ethnopharmacological relevanceXingbei Zhike granule (XBZK), a widely prescribed Chinese patent medicine, is known for its efficacy in clearing lung qi, relieving cough and reducing phlegm, as well as fever, dry and bitter taste, and irritability. Despite its clinical popularity, comprehensive investigations into its chemical composition, in vivo metabolism, and pharmacokinetic characteristics are limited. Aim of the studyThis study investigates the chemical composition, in vivo metabolism, and in vivo dynamics of XBZK to clarify its material basis and pharmacokinetic characteristics. Materials and methodsUltra-high performance liquid chromatography with Orbitrap tandem mass spectrometry (UPLC-Orbitrap-MS) was used to determine the chemical composition and in vivo metabolic profile of XBZK. Additionally, UPLC with triple quadrupole mass spectrometry (UPLC-TQ-MS/MS) was performed to quantify its main components and evaluate its in vivo dynamics in rat plasma. ResultsIn total, 57 components were identified in XBZK. Furthermore, 40 prototype components and 31 metabolites were detected in various biological matrices of rats, including plasma, tissues, bile, feces, and urine. After administration, the area under the curve (AUC) for ephedrine (Eph), pseudoephedrine (Peph), neotuberostemonine (Neo), amygdalin (Amy), and enoxolone (Eno) exhibited a strong linear relationship with the administered dose (r > 0.9) in all rats. And gender-related differences in the absorption of peiminine (Pmn), peimisine (Pms), and chrysin-7-O-glucuronide (Cog) were notable among rats, with male rats showing a dose-dependent pattern of absorption, while female rats exhibited minimal absorption. ConclusionsXBZK contains 57 components, primarily composed of flavonoids, alkaloids, and coumarins. The eight main components were rapidly absorbed and eliminated, with some, such as Eph, Peph, Neo, Amy and Eno, following a linear pharmacokinetic pattern. Furthermore, Pmn, Pms and Cog were well absorbed in male rats, showing a dose-dependent behavior.

Evaluation of the Orbitrap Ascend Tribrid Mass Spectrometer for Shotgun Proteomics.

Mass spectrometry (MS)-based proteomics is a powerful technology to globally profile protein abundances, activities, interactions, and modifications. The extreme complexity of proteomics samples, which often contain hundreds of thousands of analytes, necessitates continuous development of MS techniques and instrumentation to improve speed, sensitivity, precision, and accuracy, among other analytical characteristics. Here, we systematically evaluated the Orbitrap Ascend Tribrid mass spectrometer in the context of shotgun proteomics, and we compared its performance to that of the previous generation of Tribrid instruments─the Orbitrap Eclipse. The updated architecture of the Orbitrap Ascend includes a second ion-routing multipole (IRM) in front of the redesigned C-trap/Orbitrap and a new ion funnel that allows gentler ion introduction, among other changes. These modifications in Ascend hardware configuration enabled an increase in parallelizable ion injection time during higher-energy collisional dissociation (HCD) Orbitrap tandem MS (FTMS2) analysis of ∼5 ms. This enhancement was particularly valuable in the analyses of limited sample amounts, where improvements in sensitivity resulted in up to 140% increase in the number of identified tryptic peptides. Further, analysis of phosphorylated peptides enriched from the K562 human cell line yielded up to ∼50% increase in the number of unique phosphopeptides and localized phosphosites. Strikingly, we also observed a ∼2-fold boost in the number of detected N-glycopeptides, likely owing to the improvements in ion transmission and sensitivity. In addition, we performed the multiplexed quantitative proteomics analyses of TMT11-plex labeled HEK293T tryptic peptides and observed 9-14% increase in the number of quantified peptides. In conclusion, the Orbitrap Ascend consistently outperformed its predecessor the Orbitrap Eclipse in various bottom-up proteomic analyses, and we anticipate that it will generate reproducible and in-depth datasets for numerous proteomic applications.

Systematic Characterization of the Chemical Components of Canna edulis Ker-Gawl Based on UHPLC Q-Exactive Orbitrap MS Technology

Canna edulis Ker-Gawl is a versatile crop that integrates food, energy, and feed in China. The rhizome is a traditional Chinese medicine with a long history. However, there are few studies on chemical components. Crude plant extracts are a complex mixture of several biologically active secondary metabolites. Therefore, rapid and accurate identification and quantification are critical in phytochemical analysis. An efficient approach based on ultrahigh-performance liquid chromatography coupled with Q-Exactive Orbitrap tandem mass spectrometry (UHPLC Q-Exactive Orbitrap MS) was used to analyze the chemical components systematically. We used retention time, accurate molecular weight, parent peaks, fragment peaks, fragmentation characteristics, comparative analysis with the literature, reference standards, and standard databases, including ChemSpider, mzVault, MassBank, and mzCloud. A total of 54 chemical constituents were identified from the methanol extract of the rhizome, including 36 organic acids (with six phenolic acids), three phenols, one sugar (and its derivatives), one coumarin, one triterpenoid, ten N-containing organic compounds (including seven amino acids), and two other compounds. Moreover, three compounds were quantitatively analyzed and a methodological study of the three components was carried out. The established method provided satisfactory precision and accuracy, acceptable recovery, good linearity, and a reasonable detection limit. The UHPLC Q-Exactive Orbitrap MS method was used for the first time to accurately, quickly, and systematically characterize the compounds of the rhizome, revealing the pharmacodynamic substances and providing a theoretical basis for its quality control, in-depth research, and development.

Screening quality markers (Q-markers) of Xiaoer Chaige Tuire Oral Liquid by in vitro sequential metabolism and in vivo biopharmaceutical analysis

BackgroundXiaoer Chaige Tuire Oral Liquid (XCT) is a preparation composed of 7 traditional Chinese medicines including Bupleuri Radix, Puerariae Lobatae Radix, Scutellariae Radix, Gypsum Fibrosum, Artemisiae Annuae Herba, Paeoniae Radix Alba and Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle in proportion. According to traditional Chinese medicine theory, it has the function of dispelling wind evil and relieving exterior syndrome, clearing summer heat and dampness, and reducing internal heat. So, it is indicated for pediatric upper respiratory tract infection caused by exogenous wind-heat. Modern pharmacological studies have indicated that XCT has a variety of activities such as anti-inflammation and antivirus. PurposeTo screen potential quality markers (Q-markers) of XCT by tracking in vivo bioactive compounds concomitantly using in vitro sequential metabolism and in vivo biopharmaceutical analysis. MethodsIn vitro metabolic models including artificial gastric juice, intestinal juice, intestinal microbiota, Caco-2 cell monolayer and liver S9 were employed to simulate metabolism of main compounds of XCT in the body. High performance liquid chromatography with diode-array detection (HPLC-DAD) was used to quantitatively determine main components of XCT preparation and its sequential metabolism samples. Ultra performance liquid chromatography with QExactive Orbitrap tandem mass spectrometry (UPLC-QExactive-HF-x-Orbitrap-MS) was used to qualitatively determine in vivo components of XCT preparation in rat plasma and metabolites obtained with liver S9 fraction of rats. ResultsTwenty-five compounds were identified from the preparation of XCT. Sequential in vitro metabolism studies indicated that most of these compounds except baicalin and baicalein were stable in artificial gastric juice, albiflorin, glycyrrhizic acid, gallic acid and baicalein were unstable in artificial intestinal juice, daidzin, liquiritin and genistin were hydrolyzed into their aglycones daidzein, liquiritigenin and genistein by intestinal microbiota, and 7 compounds thereout including benzoic acid, puerarin, 3′-methoxypuerarin, paeoniflorin, scopoletin, daidzein and liquiritigenin were shown to be well absorbed with Caco-2 cell monolayer model. These 7 compounds were demonstrated to be metabolized via hydroxylation and glycosylation by liver S9 system. Ten components of XCT preparation including puerarin, baicalin, wogonoside, benzoic acid, daidzein, baicalein, wogonin, oroxylin A, isoscopoletin and isoliquiritigenin were identified from rat plasma by in vivo biopharmaceutical analysis. Most of the compounds screened with both in vitro and in vivo metabolic studies were shown to be active against inflammation and influenza virus. ConclusionsA screening strategy for potential quality markers (Q-markers) of XCT preparation based on tracking in vivo bioactive compounds using the combination of in vitro sequential metabolism and in vivo biopharmaceutical analysis was established. With this strategy, a total of 12 compounds including puerarin, daidzein, benzoic acid, baicalin, baicalein, wogonoside, wogonin, oroxylin A, 3′-methoxypuerarin, paeoniflorin, scopoletin and liquiritigenin were screened to be potential Q-markers of XCT, which provides a material basis for quality control and development of XCT.

Development of Chemical Isotope Labeling Liquid Chromatography Orbitrap Mass Spectrometry for Comprehensive Analysis of Dipeptides.

Dipeptides have recently attracted considerable attention due to their newly found biological functions and potential biomarkers of diseases. Global analysis of dipeptides (400 common dipeptides in total number) in samples of complex matrices would enable functional studies of dipeptides and biomarker discovery. In this work, we report a method for high-coverage detection and accurate relative quantification of dipeptides. This method is based on differential chemical isotope labeling (CIL) of dipeptides with dansylation and liquid chromatography Orbitrap tandem mass spectrometry (LC-Orbitrap-MS). An optimized LC gradient ensured the separation of dansyl-dipeptides, including positional isomers (e.g., leucine- and isoleucine-containing dipeptides). MS/MS collision energy in Orbitrap MS was optimized to provide characteristic fragment ion information to sequence dansyl-dipeptides. Using the optimized conditions, a CIL standard library consisting of retention time, MS, and MS/MS information of a whole set of 400 dansyl-dipeptides was constructed to facilitate rapid dipeptide identification. For qualitative analysis of dipeptides in real samples, IsoMS data processing software's parameters were tuned to improve the coverage of dipeptide annotation. Data-dependent acquisition was also carried out to improve the reliability of dipeptide identification. As examples of applications, we successfully identified a total of 321 dipeptides in rice wines and 105 dipeptides in human serum samples. For quantitative analysis, we demonstrated that the intensity ratios of the peak pairs from 96% of the dansyl-dipeptides detectable in a 1:1 mixture of 12C- and 13C-labeled rice wine samples were within ±20% of an expected value of 1.0. More than 90% of dipeptides were detected with a relative standard deviation of less than 10%, showing good performance of relative quantification.

Exploring the effective components and potential mechanisms of Zukamu granules against acute upper respiratory tract infections by UHPLC-Q-Exactive Orbitrap-MS and network pharmacology analysis

Acute upper respiratory tract infections (AURTIs) are common diseases of respiratory system, which are caused by adenoviruses and generate the mix of severe clinical presentation. Zukamu granules (ZKMG), a traditional Chinese medicine (TCM) prescription within Health Commission of Xinjiang Uygur Autonomous Region possesses anti-influenza virus, antibacterial and anti-inflammatory effects that exerts therapeutic effects in treatment of AURTIs. However, the main effective chemical components and their corresponding action mechanisms have not been clarified. Therefore, ultra-performance liquid chromatography coupled with quadrupole Exactive orbitrap tandem mass spectrometry (UHPLC-Q-Exactive Orbitrap MS) and network pharmacology were used to detect and identify potentially effective components in ZKMG as well as uncover their pharmacological mechanisms against AURTIs. As a result, a total of 265 components from all 12 composed herbal medicines were characterized based on self-built database and fragmentation patterns, of which 38 compounds were unambiguously confirmed using reference standards. Then, the compound-target-pathway network was constructed that implied potential therapeutic mechanisms of ZKMG on AURTIs. Compounds noscapine, cryptopine, steviol-19-O-glucoside, N-methylnarcotine, allocryptopine, naringenin, boldine, methyl rosmarinate with related targets EGFR, PTGS2, IL2, MMP9, TNF, AKT1, PIK3CA, F3 were considered as the key components and targets. Besides, the results also indicated that PI3K-Akt, AGE-RAGE, PD-L1, HIF-1 signaling pathways contributed significantly to the therapeutic effects of ZKMG on AURTIs. Overall, ZKMG could have an effect on AURTIs based on multicomponent, multitarget, and multichannel mechanisms of action as well as this method provides guiding significance for the further development of TCM treatment.

Dynamic changes in the metabolite profile and taste characteristics of loose-leaf dark tea during solid-state fermentation by Eurotium cristatum

Loose-leaf dark tea fermented by Eurotium cristatum has increasingly gained favor in the market due to its unique fungal aroma and soft mouthfeel. In this study, the dynamic changes in the metabolite profile and taste characteristics of loose-leaf dark tea were investigated. Ninety-two critical metabolites were identified during fermentation by ultraperformance liquid chromatography coupled with Q-Exactive Orbitrap tandem mass spectrometry (UPLC-Q-Exactive Orbitrap/MS). Among these metabolites, catechin derivatives were converted to numerous other metabolites, such as fuzhuanin A and teadenol A, by the metabolic activity of E. cristatum, which were significantly increased in the later stages of fermentation. Taste evaluation and targeted metabolomics demonstrated that the intensities of astringency, bitterness, umami and sourness of the tea infusions gradually weakened with declines in the contents of ester-catechins and amino acids, while the intensities of color, sweetness and stale taste gradually developed during fermentation with increases in the contents of theabrownins and tea polysaccharides. These results revealed the important contribution of E. cristatum to the formation of the compounds that impart the unique taste of loose-leaf dark tea and provide a chemical basis for quality control during dark tea processing.

An innovative impurity profiling of esmolol hydrochloride injection using UPLC-MS based multiple mass defect filter, chemometrics and in-silico toxicity prediction

Esmolol hydrochloride injection is indicated for the rapid control of ventricular rate in patients with atrial fibrillation or atrial flutter in perioperative, postoperative, or other emergent circumstances where short term control of ventricular rate with a short-acting agent is desirable. The potential toxic impurities in pharmaceuticals at micro levels or trace levels are of increasing concern to both pharmaceutical industries and regulatory agencies, due to their potential risk to human health. The impurity investigation of esmolol remains incomplete. Thereby, efficient impurities monitoring and potential toxicity assessment should be emphasized to assure drug safety. Impurity profiling methods of esmolol were developed using ultraperformance liquid chromatography plus Q-Exactive Orbitrap tandem mass spectrometry (UPLC-QE-MS) based multiple mass defect filter and chemometrics. Impurities were characterized by both UPLC-QE-MS and reference substance comparison. The toxicities of esmolol impurities were predicted by employing quantitative structure–activity relationship. The results showed that a total of 20 impurities were detected and identified using the above integrated strategy, 14 impurities (EP2-3, EP5-6, EP8-9, EP12-13, EP15-20) have firstly found. EP20 was predicted as hepatotoxic and mutagenic using QSAR model, and its hepatotoxicity were verified in vivo. The EP1 contents showed maximum volatility in all batches, and varied by sample. EP1 and its accompanying product of methanol were measured. This UPLC-MS/MS based chemometrics strategy is useful for monitoring the manufacturing process and quality control of esmolol hydrochloride injection.

UHPLC-ESI-QE-Orbitrap-MS based metabolomics reveals the antioxidant mechanism of icaritin on mice with cerebral ischemic reperfusion.

Icaritin (ICT) has been previously demonstrated to display protective effects against cerebral ischemic reperfusion (I/R) by inhibiting oxidative stress, but the mechanism remains unclear. This study aimed to explore the mechanism from the perspective of metabolomics. A mice cerebral artery occlusion/reperfusion (MCAO/R) model was explored to mimic cerebral ischemic reperfusion and protective effect of ICT was assessed by neurologic deficit scoring, infarct volume and brain water content. Ultra-high-performance liquid chromatography electrospray ionization orbitrap tandem mass spectrometry (UHPLC-ESI-QE-Orbitrap-MS) based metabolomic was performed to explore potential biomarkers. Brain tissue metabolic profiles were analyzed and metabolic biomarkers were identified through multivariate data analysis. The protein levels of Nrf2, HO-1 and HQO1 were assayed by western blot. The release of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) were detected using corresponding assay kits. The results showed that after ICT treatment, the neurological deficit, cerebral infarction area, brain edema and the level of MDA in brain tissue of MCAO/R mice were significantly reduced. Meanwhile, ICT enhanced the activity of SOD, CAT and GSH-Px. Western blot results confirmed that ICT up-regulated the protein levels of antioxidant-related protein including Nrf2, HO-1 and NQO1. According to the metabolomic profiling of brain tissues, clear separations were observed among the Sham, Model and ICT groups. A total of 44 biomarkers were identified, and the identified biomarkers were mainly related to linoleic acid metabolism, arachidonic acid metabolism, alanine, aspartate and glutamate metabolism, arginine biosynthesis, arginine and proline metabolism, D-glutamine and D-glutamate metabolism, taurine and hypotaurine metabolism and purine metabolism, respectively. At the same time, the inhibitory effect of ICT on arachidonic acid and linoleic acid in brain tissue, as well as the promoting effect on taurine, GABA, NAAG, may be the key factors for the anti-neurooxidative function of mice after MCAO/R injury. Our results demonstrate that ICT has benefits for MCAO/R injury, which are partially related to the suppression of oxidative stress via stimulating the Nrf2 signaling and regulating the production of arachidonic acid, linoleic acid, taurine, GABA, NAAG in brain tissue.

Identification of volatile and nonvolatile compounds in Citri Reticulatae Pericarpium Viride using GC-MS, UPLC-Q-Exactive Orbitrap-MS, and HPLC-PDA.

Gas chromatograph-mass spectrometer (GC-MS), ultra-high-performance liquid chromatograph-Q-Exactive Orbitrap tandem mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS), and high-performance liquid chromatography-photodiode array detection (HPLC-PDA) were used to qualitatively and quantitatively analyze the chemical component of Citri Reticulatae Pericarpium Viride "Geqingpi" (GQP). First of all, the volatile components of GQP are identified by GC-MS. Totally 56 volatile components were determined, and γ-Terpinene (33.39%) and D-Limonene (22.95%) were the main terpenes. Secondly, UHPLC-Q-Exactive Orbitrap-MS was used for identifying nonvolatile compositions and 42 compositions were identified totally, including 23 flavonoids, nine organic acids, three coumarins, two alkaloids compounds, and five other compounds, among which nine of the determined constituents were detected for the first time in GQP. Thirdly, the content of seven main constituents in GQP was quantitatively analyzed via HPLC-PDA, which were synephrine, hesperidin, limonin, nobiletin, HMF, tangeretin, and 5-HPMF. Further investigation for quantitative analysis of seven bioactive compounds suggested that the concentration of hesperidin in GQP approximately was 16.0% (160.78 ± 0.95 mg·g-1), which was far higher than the standard for identification and quality control of CRPV in Chinese Pharmacopoeia (2020 edition) that "the content of hesperidin shall not be less than 5.0%." The phytochemicals of GQP were elucidated in this study, which might be supporting information for identification between GQP and Citri Reticulatae Pericarpium Viride "Sihuaqingpi" (SHQP) and provided a scientific basis for the further active ingredient for pharmacological research and development prospects of GQP.

Identification and characterization of the metabolites of moscatilin in mouse, rat, dog, monkey and human hepatocytes by LC-Orbitrap-MS/MS combined with diagnostic fragment ions and accurate mass measurements.

Moscatilin, a bibenzyl derivative from the stem of Dendrobium loddigesii, has been shown to have anticancer activity. The aim of this study was to identify and characterize the possible in vitro metabolites of moscatilin generated from hepatocytes. The metabolites generated in the hepatocytes of mouse, rat, dog, monkey and human were identified and characterized employing ultra-high-performance liquid chromatography coupled with quadrupole Orbitrap tandem mass spectrometry (LC-Orbitrap-MS/MS) based on diagnostic fragment ions and accurate mass measurements. A total of 18 metabolites were identified, among which seven were phase I and 11 were phase II metabolites. The plausible structures of the metabolites and the probable biotransformation pathways were proposed based on the diagnostic fragment ions, chemical formula and mass fragmentation pattern, as well as the accurate masses. The majority of phase I metabolites were generated by demethylation and hydroxylation, while phase II metabolites were mainly generated by glucuronidation, glutathione conjugation and sulfation. Our study first expounded the metabolites of moscatilin in mouse, rat, dog, monkey and human hepatocytes and provided a foundation for a further pharmacokinetic and toxicity study. More importantly, LC-Orbitrap-MS/MS combined with diagnostic fragment ions and accurate mass measurements has been proved to be an effective method for the rapid identification of bibenzyl derivatives and their metabolites.

Rapid separation and characterization of the in vitro metabolites of moscatilin by ultra-high performance liquid chromatography coupled to hybrid quadrupole orbitrap tandem mass spectrometry.

Moscatilin, a bioactive ingredient isolated from Dendrobium moscatum, has been demonstrated to have excellent anti-cancer activity. The goals of the present study were to investigate the metabolic profiles of moscatilin and to identify and characterize its metabolites. In vitro studies were performed by incubating moscatilin (10 μM) with rat, dog, monkey, and human liver microsomes (0.5 mg protein/ml) to generate the metabolites. An analytical method of liquid chromatography combined with hybrid quadrupole orbitrap high-resolution mass spectrometry in full mass/data-dependent tandem mass spectrometry scan was utilized to separate and identify the metabolites in accordance with their accurate masses, formulas, and tandem mass spectrometry fragment ions determination. A total of six phase I metabolites were detected and structurally characterized. The phase I metabolic pathways of moscatilin were hydroxylation, demethylation, and dehydrogenation. In glutathione-supplemented liver microsomes, nine glutathione conjugates were detected and identified. Our results demonstrated that moscatilin was susceptible to bioactivation with the result of ortho quinone and quinone-methide intermediates. The present study provided an overview of the in vitro metabolic profiles of moscatilin, which will aid in the understanding of the efficacy and safety of this active compound.

Identification, in silico screening, and molecular docking of novel ACE inhibitory peptides isolated from the edible symbiot Boletus griseus-Hypomyces chrysospermus

The aim of this study is to find angiotensin-Ⅰ-converting enzyme inhibitory (ACEI) peptides from the edible symbiot Boletus griseus-Hypomyces chrysospermus (BGHC). The peptides from the methanol extract of BGHC were identified through ultraperformance liquid chromatography quadrupole Orbitrap tandem mass spectrometry analysis coupled with de novo sequencing by using Peak Studio software. Subsequently, two ACEI peptides, being KYPHVF and LFRPE, were screened on the basis of PeptideRanker and the AHTpin online server. The in silico absorption, distribution, metabolism, excretion and toxicities (ADMET) of the two peptides were predicted with AdmetSAR software, and the two peptides showed good absorption and nontoxicity. Meanwhile, the ACEI mechanism of KYPHVF and LFRPE was analyzed through molecular docking with SYBYL-X 2.0 software. The molecular docking study revealed that the total scores of the interactions of KYPHVF and LFRPE with ACE were 12.53 and 11.34, respectively. KYPHVF and LFRPE could establish hydrogen bonds and hydrophobic interactions with the active site of ACE. Furthermore, the ACEI activities of KYPHVF and LFRPE were determined in vitro with the IC50 values of 5.99 and 58.15 μmol/L, respectively.

Multi-DNA Adduct and Abasic Site Quantitation In Vivo by Nano-Liquid Chromatography/High-Resolution Orbitrap Tandem Mass Spectrometry: Methodology for Biomonitoring Colorectal DNA Damage.

Epidemiological and mechanistic studies suggest that processed and red meat consumption and tobacco smoking are associated with colorectal cancer (CRC) risk. Several classes of carcinogens, including N-nitroso compounds (NOCs) in processed meats and heterocyclic aromatic amines (HAAs) and polycyclic aromatic hydrocarbons (PAHs) in grilled meats and tobacco smoke, undergo metabolism to reactive intermediates that may form mutation-inducing DNA adducts in the colorectum. Heme iron in red meat may contribute to oxidative DNA damage and endogenous NOC formation. However, the chemicals involved in colorectal DNA damage and the paradigms of CRC etiology remain unproven. There is a critical need to establish physicochemical methods for identifying and quantitating DNA damage induced by genotoxicants in the human colorectum. We established robust nano-liquid chromatography/high-resolution accurate mass Orbitrap tandem mass spectrometry (LC/HRAMS2) methods to measure DNA adducts of nine meat and tobacco-associated carcinogens and lipid peroxidation products in the liver, colon, and rectum of carcinogen-treated rats employing fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues. Some NOCs form O6-carboxymethyl-2'-deoxyguanosine, O6-methyl-2'-deoxyguanosine, and unstable quaternary N-linked purine/pyrimidine adducts, which generate apurinic/apyrimidinic (AP) sites. AP sites were quantitated following derivatization with O-(pyridin-3-yl-methyl)hydroxylamine. DNA adduct quantitation was conducted with stable isotope-labeled internal standards, and method performance was validated for accuracy and reproducibility. Limits of quantitation ranged from 0.1 to 1.1 adducts per 108 bases using 3 μg of DNA. Adduct formation in animals ranged from ∼1 in 108 to ∼1 in 105 bases, occurring at comparable levels in fresh-frozen and FFPE specimens for most adducts. AP sites increased by 25- to 75-fold in the colorectum and liver, respectively. Endogenous lipid peroxide-derived 3-(2-deoxy-β-d-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG) and 6-oxo-M1dG adduct levels were not increased by carcinogen dosing but increased in FFPE tissues. Human biomonitoring studies can implement LC/HRAMS2 assays for DNA adducts and AP sites outlined in this work to advance our understanding of CRC etiology.

Cell metabolomics study of ginkgo flavone aglycone combined with doxorubicin against liver cancer in synergy

Ultra-high-performance liquid chromatography-Q exactive orbitrap tandem mass spectrometry(UHPLC-QEOrbitrap-MS/MS) was used to explore the inhibitory effect and mechanism of ginkgo flavone aglycone(GA) combined with doxorubicin(DOX) on H22 cells. The effects of different concentrations of GA and DOX on the viability of H22 cells were investigated, and combination index(CI) was used to evaluate the effects. In the experiments, control(CON) group, DOX group, GA group, and combined GA and DOX(GDOX) group were constructed. Then the metabolomics strategy was employed to explore the metabolic markers that were significantly changed after combination therapy on the basis of single medication treatment, and by analyzing their biological significance, the effect and mechanism of the anti-tumor effect of GA combined with DOX were explained. The results revealed that when 30 μg·mL~(-1) GA and 0.5 μmol·L~(-1) DOX was determined as the co-administration concentration, the CI value was 0.808, indicating that the combination of GA and DOX had a synergistic anti-tumor effect. Metabolomics analysis identified 23 metabolic markers, including L-arginine, L-tyrosine and L-valine, mostly amino acids. Compared with the CON group, 22 and 17 metabolic markers were significantly down-regulated after DOX treatment and GA treatment, respectively. Compared with the DOX and GA groups, the treatment of GA combined with DOX further down-regulated the levels of these metabolic markers in liver cancer, which might contribute to the synergistic effect of the two. Five key metabolic pathways were found in pathway enrichment analysis, including glutathione metabolism, phenylalanine metabolism, arginine and proline metabolism, β-alanine metabolism, and valine, leucine and isoleucine degradation. These findings demonstrated that the combination of GA and DOX remarkably inhibited the viability of H22 cells and exerted a synergistic anti-tumor effect. The mechanism might be related to the influence of the energy supply of tumor cells by interfering with the metabolism of various amino acids.

Validated Screening Method for 81 Multiclass Veterinary Drug Residues in Food via Online-Coupling High-Throughput Planar Solid-Phase Extraction to High-Performance Liquid Chromatography-Orbitrap Tandem Mass Spectrometry.

Current screening capabilities for veterinary drugs (VDs) in foods are limited, requiring time-consuming and expensive trace-level analyses. For the first time, a high-throughput planar solid-phase extraction (HTpSPE) cleanup, controlled by UV/vis/FLD imaging, was developed for screening 81 VDs from 6 different groups (glucocorticoids, anthelmintics, antiparasitics, coccidiostats, nonsteroidal anti-inflammatory drugs, and antibiotics) in 4 different matrices (honey, pig muscle, cow milk, and chicken eggs). It consumed 13 times less solvent and was more eco-friendly and 5 times faster than routine methods. The VDs were automatically eluted using the autoTLC-LC-MS interface, separated online on a high-performance liquid chromatography column via a 10-min gradient, and detected by Orbitrap high-resolution tandem mass spectrometry. The screening method was validated according to the latest European Commission Implementing Regulation 2021/808. Most VDs except penicillins and cephalosporins were detected at the 5-μg/kg level in pig muscle, cow milk, and chicken eggs and 25-μg/kg level in honey.