Hybrid Quadrupole-orbitrap High-resolution Mass Spectrometry Research Articles

Exploring the gut microbiota mediated biotransformation of Senecio scandens Buch.-Ham.: Insights from metabolite spectrum with UHPLC-Q-Orbitrap HRMS and bioinformatics analysis of gut microbiota metabolites

Senecio scandens Buch.-Ham., a traditional Chinese medicine commonly used clinically, exhibits various pharmacological properties, including anti-inflammatory, anti-tumor, antiviral, and antibacterial activities. However, its water extracts' chemical components and metabolites are inadequately understood, limiting further research. In this study, the chemical components and metabolism processes of Senecio scandens, both in vivo (plasma, feces, urine, and bile) and in vitro (gut microbiota and liver microsomes), were characterized based on ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap high-resolution mass spectrometry. Additionally, metabolites detectable in fecal samples and intestinal microbiota incubated but absent in liver microsomes were identified as characteristic metabolites of intestinal microbiota. The targets of the characteristic metabolites of intestinal microbiota were collected, followed by exploration of potential pathways through KEGG enrichment analysis. As a result, a total of 133 chemical components were preliminarily identified, including 35 organic acids, 21 alkaloids, 19 flavonoids and their glycosides, 17 phenylpropanoids, 10 jacaranda ketones, and 31 other compounds. Notably, 12 of these were potentially novel compounds. In addition, 39 prototype components in rats and 109 metabolites were identified and characterized, including 102 in vivo and 52 metabolites in vitro (51 in rat gut microbiota and 24 in rat liver microsomes). The main metabolic pathways include oxidation, reduction, hydrolysis, methylation, glucuronidation, sulfonation, and acetylation reactions. Furthermore, KEGG enrichment analysis revealed that the characteristic metabolites of intestinal microbiota may be related to the ErbB, FoxO, mTOR, and MAPK signaling pathways, exhibiting anti-inflammatory and anti-tumor effects. In summary, the chemical components and metabolites of Senecio scandens were comprehensively identified using a rapid and accurate method, providing a scientific basis for the in-depth study of the material basis and its clinical application of Senecio scandens.

Quality assessment and Q-markers discovery in Citri Sarcodactylis Fructus by integrating serum pharmacochemistry and network pharmacology.

Citri Sarcodactylis Fructus (CSF), a common fruit and traditional Chinese medicine (TCM), has been hindered in its further development and research owing to the lack of comprehensive and specific quality evaluation standards. This study aimed to establish clear TCM quality standards related to the therapeutic mechanisms of CSF and to provide a basis for subsequent research and development. Ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap high-resolution mass spectrometry (UPLC-Q-orbitrap HRMS) technology was used to comprehensively identify CSF components and explore their absorbance levels in rat serum. Network pharmacology research methods were employed to investigate the potential mechanisms of action of the identified components in the treatment of major clinical diseases. Subsequently, a combination of HPLC chromatographic fingerprinting for qualitative analysis and multi-index content determination was used to evaluate the detectability of the identified quality markers (Q-markers). Twenty-six prototype components were tentatively characterized in rat serum. Network pharmacology analysis showed six effective components, namely 7-hydroxycoumarin, isoscopoletin, diosmin, hesperidin, 5,7-dimethoxycoumarin, and bergapten, which played important roles in the treatment of chronic gastritis, functional dyspepsia, peptic ulcer, and depression and were preliminarily identified as Q-markers. The results of content determination in 15 batches of CSF indicated significant differences in the content of medicinal materials from different origins. However, compared with the preliminarily determined Q-markers, all six components could be measured and were determined as Q-markers of CSF. The chemical Q-markers obtained in this study could be used for effective quality control of CSF.

Biotransformation of 5-methoxy-N-isopropyl-N-methyltryptamine by zebrafish and human liver microsome with high-resolution mass spectrometry

To explore the metabolites of 5-Methoxy-N-isopropyl-N-methyltryptamine (5-MeO-MiPT) and unveil its toxicological effects, we examined its metabolic profiles using zebrafish and human liver microsome models. Employing ultra-high-performance liquid chromatography Q Exactive hybrid quadrupole-Orbitrap high-resolution mass spectrometry (UPLC-QE-HRMS), we analyzed samples from intoxicated zebrafish and human liver microsomes. In the zebrafish model, we identified a total of six metabolites. Primary phase I metabolic pathways involved N-Demethylation and Indole-hydroxylation reactions, while phase II metabolism included Glucoside conjugation directly, Glucoside conjugation after Indole-hydroxylation, and Sulfonation following Indole-hydroxylation. In the human liver microsome model, nine metabolites were generated. Major phase I metabolic pathways encompassed N-Demethylation, 5-O-Demethylation, and N-Depropylation, N-Oxidation, Indole-hydroxylation, N-Demethylation combined with Indole-hydroxylation, and 5-O‐Methylation-carboxylation. Phase II metabolism involved Glucoside conjugation after Indole-hydroxylation, as well as Glucoside conjugation after 5-O-Demethylation. Proposed phase I metabolites, such as 5-MeO-MiPT-N-Demethylation (5-MeO-NiPT) and 5-MeO-MiPT-Indole-hydroxylation, alongside the phase II metabolite OH&Glucoside conjugation-5-MeO-MiPT, were identified as effective markers for screening 5-MeO-MiPT intake. This study systematically delineates the phase I and II metabolites of 5-MeO-MiPT, confirming their pathways through in vivo and in vitro extrapolation. Additionally, inclusion of the parent drug itself and OH&Glucoside conjugation-5-MeO-MiPT could serve as valuable confirmation tools.

Bio-based material-edible rosemary induced biodegradation of aflatoxin B1 via altering endogenous protective enzymes signatures in animal-derived foods

Aflatoxin B1 (AFB1) is the most hazardous mycotoxin, posing risks to public health. Utilization of bio-based materials to biodegrade AFB1 is a green strategy to overcome this issue. The investigation aimed to screen for endogenous protective enzymes in bio-based material-edible rosemary based on ultra-high performance liquid chromatography coupled to hybrid quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS)-proteomics and ascertain their impacts on the biodegradation and biotransformation of AFB1, and the trade-offs of multilevel metabolism of the animal-derived foods through untargeted metabolomics. The proteomics results verified that bio-based material-edible rosemary (0.20%, w/w) significantly up-regulated glutathione S-transferase and stimulated the down-regulation of cytochrome P450 1A2 levels via activating AhR nuclear translocation in rosemary-pickled AFB1-contaminated goat meat. Metabolomics results demonstrated that edible rosemary substantially increased histidine and glutathione implicated in the antioxidant status of goat meat. More importantly, edible rosemary with high endogenous protective enzyme content could efficiently biodegrade AFB1 in goat meat. We first unveiled that rosemary could not only efficiently biodegrade AFB1 up to 90.20% (20.00–1.96 μg kg−1) but also elevate the bio-ingestion quality of goat meat. These findings suggest that the bio-based material-rosemary is an efficient and environmentally friendly approach for biodegrading AFB1 and elevating the bio-ingestion composition of goat meat.

Fructus Zanthoxyli extract improves glycolipid metabolism disorder of type 2 diabetes mellitus via activation of AMPK/PI3K/Akt pathway: Network pharmacology and experimental validation

ObjectiveThis study investigated the potential mechanisms behind the beneficial effects of Fructus Zanthoxyli (FZ) against type 2 diabetes mellitus (T2DM) based on network pharmacology and experimental validation. MethodsUltra-high-performance liquid chromatography coupled with hybrid quadrupole-orbitrap high-resolution mass spectrometry, and gas chromatography-mass spectrometry were used to identify the constituents of FZ. Next, the differentially expressed genes linked to the treatment of diabetes with FZ were screened using online databases (including Gene Expression Omnibus database and Swiss Target Prediction online database), and the overlapping genes and their enrichment were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG). Finally, the pathway was verified by in vitro experiments, and cell staining with oil red and Nile red showed that the extract of FZ had a therapeutic effect on T2DM. ResultsA total of 43 components were identified from FZ, and 39 differentially expressed overlapping genes were screened as the possible targets of FZ in T2DM. The dug component-target network indicated that PPARA, PPARG, PIK3R3, JAK2 and GPR88 might be the core genes targeted by FZ in the treatment of T2DM. Interestingly, the enrichment analysis of KEGG showed that effects of FZ against T2DM were closely correlated with the adenosine monophosphate-activated protein kinase (AMPK) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) signaling pathways. In vitro experiments further confirmed that FZ significantly inhibited palmitic acid-induced lipid formation in HepG2 cells. Moreover, FZ treatment was able to promote the AMPK and PI3K/Akt expressions in HepG2 cells. ConclusionNetwork pharmacology combined with experimental validation revealed that FZ extract can improve the glycolipid metabolism disorder of T2DM via activation of the AMPK/PI3K/Akt pathway.

A strategy integrating parent ions list-modified mass defect filtering-diagnostic product ions for rapid screening and systematic characterization of flavonoids in Scutellaria barbata using hybrid quadrupole-orbitrap high-resolution mass spectrometry

In this study, full scan (FS)-parent ions list (PIL)-higher energy collision induced dissociation (HCD)-MS/MS (FS-PIL-HCD-MS/MS) was used to acquire the chemical profile of flavonoids in Scutellaria barbata. Mass defect filtering (MDF) induced subtype classification and diagnostic product ions (DPIs) dominated structural confirmation were integrated into an effective strategy for the systematic screening and identification of the flavonoids. An in-house flavonoid MS database based on molecular design was established to construct a modified triangle MDF algorithm for progressive screening and subtype classification. The obtained results demonstrated that the modified MDF was capable of simplifying the workload in formula editing and subsequent screening process, and distinguishing different subtypes. The fragmentation behaviors of eleven reference standards were evaluated to obtain the MS2 fragmentation pathway and DPIs which can provide a criterion to eliminate false-positive results and judge the target flavonoids with the exact number and position of substituents for the first time. Structure confirmation was characterized by comparing with the reference substance, searching the database, and analyzing DPIs. To distinguish some isomers, ClogP (the calculated lipophilicity parameter) was adopted. As a result, 127 target flavonoids, including 30 flavone/flavonol aglycones, 10 flavanone/flavanonol aglycones, 49 flavone/flavonol monoglycosides, 16 flavanone/flavanonol monoglycosides, 21 flavone/flavonol diglycosides and 1 flavanone/flavanonol diglycoside, were ultimately identified or tentatively characterized based on the MS fragmentation pathway and DPIs analysis. This study provides a novel MDF method with improved subtype classification and develops a novel strategy for the progressive screening, subtype classification and systematic characterization of complex components in herbal medicines.

Chemical constituents, anti-hyperuricemic and anti-gouty arthritis activities of extract of Herpetospermum caudigerum

In this paper, we studied the anti-hyperuricemia and anti-gouty arthritis effects of the ethyl acetate extract of Herpetospermum caudigerum (EH), and applied ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap HRMS) technology to analyze and identify the chemical components of EH. EH can dramatically lower serum uric acid (UA) levels in the hyperuricemia mice model induced by potassium oxazine (PO). In addition, in the gouty arthritis rats model established by sodium urate, the ankle swelling degree, serum uric acid (UA), IL-1β, and TNF-α related inflammatory cytokines all decreased by treating with EH. Consequently, EH appears to have anti-hyperuricemia and anti-gouty arthritis activities, making it a viable option for anti-gout drugs without hepatic and renal side effects, and its effective substances of anti-gout can be further illustrated by UPLC-Q-Orbitrap HRMS analysis of 48 compounds.

Identification of the Metabolites of Scutebarbatine A in Rat Plasma, Bile, Urine, and feces by Using Ultra-high-performance Liquid Chromatography Coupled with Q Exactive Hybrid Quadrupole-orbitrap High-resolution Mass Spectrometry.

Scutebarbatine A is a new neo-Clerodane Diterpenoid Alkaloids from Scutellaria barbata, which has many pharmacological effects, such as anti-tumor, antibacterial, and anti-inflammatory. However, there are no studies on the metabolism of Scutebarbatine A. The objective of this study is to explore the metabolism of Scutebarbatine A in the body, the bile, plasma, urine, and feces samples of rats after administration were investigated. The biological samples were investigated using ultra-high-performance liquid chromatography coupled with Q Exactive hybrid quadrupole-orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap-HRMS). A total of 20 metabolites were identified: 16 phase I metabolites and 4 phase II metabolites. The main metabolic pathways were hydrolysis, oxidation, hydrogenation, dehydration, and combination with sulfate. This study further elucidates the metabolism of Scutebarbatine A in rats and provides a reference for the study of its pharmacodynamic material basis and pharmacological mechanism.

Metabolic study of cafestol using in silico approach, zebrafish water tank experiments and liquid chromatography high-resolution mass spectrometry analyses

Coffee is one of the most consumed beverages worldwide. Cafestol is an endogenous coffee diterpene present in raw coffee beans and also found in hot beverages, with several biological activities. However, there is still little information on this molecule after ingestion of coffee infusion.Zebrafish (Danio rerio) is a promising in vivo model for metabolic studies due to the annotation of mammalian orthologs to encode enzymes related to drug metabolism. Experiments using Zebrafish Water Tank (ZWT) model produce more significant number of metabolites for molecular investigation in a cleaner matrix than other classical models, such as purified hepatocytes.This work aimed to investigate the biotransformation of cafestol by the ZWT model using ultra-performance liquid chromatography coupled to hybrid quadrupole-orbitrap high-resolution mass spectrometry equipped with electrospray ionization (UPLC-HRMS) supported by in silico approach using SMARTCyp, Way2Drug and XenoSite Softwares.Twenty-five metabolites of cafestol were proposed by in silico analysis, in which 5 phase I metabolites were confirmed in the ZWT by UPLC and MS/HRMS investigation: 6-hydroxy-cafestol, 6,12-dihydroxy-cafestol, 2-oxo-cafestol, 6-oxo-cafestol and one isomer whose position in the carboxyl group was not determined. These metabolites were observed during 9 h of the experiment, whose contents were associated with the behavioral responses of the fish.

Protein Biomarkers of Bovine Defective Meats at a Glance: Gel-Free Hybrid Quadrupole-Orbitrap Analysis for Rapid Screening.

An understanding of biological mechanisms that could be involved in the stress response of animal cattle prior to slaughter is critical to create effective strategies aiming at the production of high-quality meat. The sarcoplasmic proteome of directly extracted samples from normal and high ultimate pH (pHu) meat groups was studied through a straightforward gel-free strategy supported by liquid chromatography hybrid quadrupole-Orbitrap high-resolution mass spectrometry (LC-HRMS) analysis. A stepped proteomic pipeline combining rapid biomarker hunting supported by qualitative protein Mascot scores followed by targeted label-free peptide quantification revealed 26 descriptors that characterized meat groups assayed. The functional study of the proposed biomarkers suggested their relevant role in metabolic, chaperone/stress-related, muscle contractility/fiber organization, and transport activities. The efficiency, flexibility, rapidity, and easiness of the methodology proposed can positively contribute to the creation of innovative proteomic alternatives addressing meat quality assessment.

Analysis of the characteristic compounds of Citri Sarcodactylis Fructus from different geographical origins.

Citri Sarcodactylis Fructus (CSF) is widely used as a food ingredient and a traditional Chinese medicine. In China, CSF is cultivated in many places, including Sichuan, Guangdong, Zhejiang, and Fujian provinces. The types and chemical contents of CSF from different origins may vary greatly due to the difference in climate and environmental conditions. Therefore, comparing the chemical composition of CSF from various places is vital. To rapidly select potential characteristic compounds for differentiating CSF from different origins. Thirty-one batches of CSF samples from different regions were analysed using ultra-performance liquid chromatography with hybrid quadrupole-orbitrap high-resolution mass spectrometry. Thereafter, chemometric methods, including principal component analysis (PCA) and orthogonal partial least squares discrimination analysis (OPLS-DA), were employed to find differential metabolites among the CSF samples from various origins. PCA revealed 77.9% of the total variance and divided all CSF samples into three categories corresponding to their origins. OPLS-DA displayed better discrimination of CSF from different sources, with R2 X, R2 Y, and Q2 of 0.801, 0.985, and 0.849, respectively. Finally, 203 differential metabolites were obtained from CSF from different origins using the variable importance in projection of the OPLS-DA model, 30 of which were identified, and five coumarin compounds were selected as marker compounds discriminating CSF from different origins. This work provides a practical strategy for classifying CSF from different origins and offers a research foundation for the quality control of CSF.

An Entire Process Optimization Strategy for Comprehensive In Vivo Metabolite Profiling of Prucalopride in Rats Based on Ultra-Performance Liquid Chromatography With Q-Exactive Hybrid Quadrupole-Orbitrap High-Resolution Mass Spectrometry.

Prucalopride was widely used for chronic constipation, which is difficult to be adequately relieved by laxatives in adult patients in clinic. Due to the difficulty in metabolite identification, metabolic process of prucalopride had not been investigated in vivo. In this study, an efficient strategy was proposed for comprehensive metabolite profiling of prucalopride after oral administration in rat plasma, urine, and feces samples. This strategy was composed of five steps. First, the samples at multiple time points after oral administration were collected to increase the representativeness of the samples. Second, different sample preparation methods were investigated to obtain superior extraction efficiency. Third, the raw data of test sample and blank sample were acquired using ultra-performance liquid chromatography with Q-Exactive hybrid quadrupole–orbitrap high-resolution accurate mass spectrometry under the positive and negative full-scan/dd MS2 mode. Fourth, combined mass defect filter with background subtraction model in soft of compound discovery, all peaks were constructed to filter potential metabolites after retention time alignment and ion filtration, which could remove large amounts of interference ions. Besides, it can predict potential biotransformation, promoting to understand how to metabolize the drug. This provides multiple possibilities and prevents us conjecturing the potential metabolites blindly. Finally, the verification procedure was implemented through exporting the structure and MS2 spectrum to the analytical tool of Mass Frontier. The proposed strategy significantly improved the targeted detection and identification for metabolites in vivo. A total of 47 metabolites were tentatively characterized in the plasma, urine, and feces samples after oral administration of prucalopride. This study could provide a valuable reference for systematic metabolite profile of drug in vivo.

Establishment of a Target, Suspect, and Functional Group-Dependent Screening Strategy for Organophosphate Esters (OPEs): "Into the Unknown" of OPEs in the Sediment of Taihu Lake, China.

Current environmental monitoring studies are generally confined to several target organophosphate esters (OPEs), and there is a lack of strategies for comprehensively screening all potential OPEs in environmental samples. Here, an effective and accurate strategy was developed for the target, suspect, and functional group-dependent screening of OPEs by the use of ultrahigh-performance liquid chromatography-Q Exactive hybrid quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS), and this strategy was applied for the analysis of n = 74 sediment samples (including 23 surface sediment samples and 51 sediment core samples) collected from Taihu Lake (eastern China) in 2019. In these analyzed samples, we successfully identified n = 35 OPEs, and 23 of them were reported in this region for the first time. In addition, this strategy also presented other interesting findings, i.e., (1) OPE concentrations decreased with increasing distance from the coast of the lake; (2) the newly identified 3-hydroxyphenyl diphenyl phosphate (meta-OH-TPHP) was not statistically significantly correlated with triphenyl phosphate (TPHP; r = 0.02494, p = 0.9101) but with resorcinol bis(diphenyl phosphate) (RDP) (r = 0.9271, p < 0.0001) and three other OPEs; and (3) the summed concentrations of aryl OPEs (∑arylOPEs) in sediment core samples exhibited significantly increasing trends as the depth decreased. Collectively, this study provided an effective strategy that was successfully applied for comprehensive screening of OPEs in the sediments of Taihu Lake, and this strategy could have promising potential to be extended to other environmental matrices or samples.

Validated comprehensive metabolomics and lipidomics analysis of colon tissue and cell lines

Current untargeted approaches for metabolic fingerprinting of colon tissue and cell lines lack validation of reproducibility and/or focus on a selection of metabolites as opposed to the entire metabolome. Yet, both are critical to ensure reliable results and pursue a fully holistic analysis. Therefore, we have optimized and validated a platform for analyzing the polar metabolome and lipidome of colon-derived cell and tissue samples based on a consecutive extraction of polar and apolar components. Peak areas of selected targeted analytes and the number of untargeted components were assessed. Analysis was performed using ultra-high performance liquid-chromatography (UHPLC) coupled to hybrid quadrupole-Orbitrap high-resolution mass spectrometry (HRMS). This resulted in an optimized extraction protocol using 50% methanol/ultrapure water to obtain the polar fraction followed by a dichloromethane-based lipid extraction. Using this comprehensive approach, we have detected more than 15,000 components with CV < 30% in internal quality control (IQC) samples and were able to discriminate the non-transformed (NT) and transformed (T) state in human colon tissue and cell lines based on validated OPLS-DA models (R2Y > 0.719 and Q2 > 0.674). To conclude, our validated polar metabolomics and lipidomics fingerprinting approach could be of great value to reveal gastrointestinal disease-associated biomarkers and mechanisms.

Application of quadrupole-Orbitrap high-resolution mass spectrometry in rapid screening and identification of synthetic dyes in herbal medicines

In this work, a method combining ultra-high performance liquid chromatography and hybrid quadrupole-Orbitrap high-resolution mass spectrometry (HR-MS) was developed and validated for use in the simultaneous screening, identification, and quantification of 21 synthetic dyes in herbal medicines. To optimize the chromatographic conditions, we used a combined Full mass scan and data-dependent MS/MS (Full MS/dd-MS2) approach in positive and negative ion mode. Under this mode, selected ions with given fragmentation energy were subjected to a dd-MS2 scan following a Full MS scan. The selectivity of this method was effectively improved using 70,000 full width at half maximum mass resolution and narrow mass window (typically 5 ppm), and a single injection was sufficient for simultaneous identification and quantification of 21 synthetic dyes within 10 min. The combined method was fully validated and complies with all criteria for selectivity, sensitivity, calibration curve linearity, accuracy, precision, recovery, matrix effect, and stability. All analytes showed excellent linear relationships as all the coefficients of determination (r2) are greater than 0.9978 over wide ranges of concentrations (e.g. 1.0-500 ng/mL for sunset yellow). The validated method was employed to detect synthetic dyes in herbal medicines and was demonstrated to provide a reliable technical basis for drug regulation and public health protection.

Tissue distribution profiles of multiple major bioactive components in rats after intravenous administration of Xuebijing injection by UHPLC-Q-Orbitrap HRMS.

Xuebijing injection (XBJI) is a traditional Chinese medicine prescription extracted from five Chinese herbs. Hydroxysafflor yellow A, oxypaeoniflorin, ferulic acid and benzoylpaeoniflorin are the main bioactive ingredients of XBJI. This paper presents an application of ultra-high-performance liquid chromatography-Q-exactive hybrid quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) to quantify four compounds of XBJI in rats various tissues for tissue distribution studies. The analytes were separated on a Waters Acquity UHPLC® BEH C18 column with a gradient mobile phase consisting of acetonitrile-water (containing 0.1% formic acid) at a flow rate of 0.2 mL/min. Mass spectrometric detection was performed by parallel reaction monitoring via a heated electrospray ionization source under the negative ionization mode. The method was validated in various tissue samples, and has demonstrated great performance for rapidity, accuracy, high sensitivity and selectivity. It was successfully applied to the tissue distribution studies of XBJI after intravenous administration to rats. It was also the first study to investigate the tissue distribution of XBJI in rats and we found that the concentrations of four compounds were high in kidney, liver, stomach and intestine. The clinical use of XBJI should focus on its pharmacodynamics and safety studies in these tissues.

A validated multi-matrix platform for metabolomic fingerprinting of human urine, feces and plasma using ultra-high performance liquid-chromatography coupled to hybrid orbitrap high-resolution mass spectrometry

In recent years, metabolomics has surfaced as an innovative research strategy in human metabolism, whereby selection of the biological matrix and its inherent metabolome is of crucial importance. However, focusing on a single matrix may imply that relevant molecules of complementary physiological pathways, covered by other matrices, are missed. To address this problem, this study presents a unique multi-matrix platform for polar metabolic fingerprinting of feces, plasma and urine, applying ultra-high performance liquid-chromatography coupled to hybrid quadrupole-Orbitrap high-resolution mass spectrometry, that is able to achieve a significantly higher coverage of the system's metabolome and reveal more significant results and interesting correlations in comparison with single-matrix analyses. All three fingerprinting approaches were proven ‘fit-for-purpose’ through extensive validation in which a number of endogenous metabolites were measured in representative quality control samples. For targeted and untargeted validation of all three matrices, excellent linearity (coefficients of determination R2 ≥ 0.99 or 0.90 respectively), recovery and precision (coefficients of variance ≤ 15% or 30% respectively) were observed. The potential of the platform was demonstrated by subjecting fecal, urine and plasma samples (collected within one day) from ten healthy volunteers to metabolic fingerprinting, yielding respectively 9 672, 9 647, and 6122 components. Orthogonal partial least-squares discriminant analysis provided similar results for feces and plasma to discriminate according to gender (p-value, R2(X), R2(Y) and Q2(Y)), suggesting feces as an excellent alternative biofluid to plasma. Moreover, combining the different matrices improved the model's predictivity, indicating the superiority of multi-matrix platforms for research purposes in biomarker detection or pathway elucidation and in the selection of the most optimal matrix for future clinical purposes.

Chemical profiling and quantification of XueBiJing injection, a systematic quality control strategy using UHPLC-Q Exactive hybrid quadrupole-orbitrap high-resolution mass spectrometry

To clarify and quantify the chemical profiling of XueBiJing injection (XBJ) rapidly, a feasible and accurate strategy was developed by applying ultra high performance liquid chromatography-Q Exactive hybrid quadrupole-orbitrap high resolution accurate mass spectrometry (UHPLC-Q-Orbitrap HRMS). A total of 162 components were characterized, including 19 phenanthrenequinones, 33 lactones, 28 flavonoids and 12 phenolic acids and 51 other compounds. Among them, 38 major compounds were unambiguously quantified by comparing with reference standards. Meanwhile, 38 representative compounds were simultaneously detected in XBJ samples by Q-Orbitrap HRMS. Satisfactory linearity and correlation coefficient were achieved with wide linear range. The precisions, repeatability, stability and recovery were meeting requirements. The validated method was successfully applied for simultaneous determination of 38 bioactive compounds in 10 batches XBJ samples. In addition, the similarity evaluation of fingerprintings was applied to assess the quality of XBJ. And the results were evaluated by multiple statistical strategies and five compounds might be the most important chemical markers for chemical quality control of XBJ. Finally, a rapid and simple UPLC-MS/MS method was developed for determination of five markers in XBJ sample. This research established a high sensitive and efficient strategy for integrating quality control, including identification and quantification of XBJ.

Broad screening of illicit ingredients in cosmetics using ultra-high-performance liquid chromatography-hybrid quadrupole-Orbitrap mass spectrometry with customized accurate-mass database and mass spectral library

Comprehensive identification and quantitation of 100 multi-class regulated ingredients in cosmetics was achieved using ultra-high-performance liquid chromatography (UHPLC) coupled with hybrid quadrupole-Orbitrap high-resolution mass spectrometry (Q-Orbitrap HRMS). A simple, efficient, and inexpensive sample pretreatment protocol was developed using ultrasound-assisted extraction (UAE), followed by dispersive solid-phase extraction (dSPE). The cosmetic samples were analyzed by UHPLC-Q-Orbitrap HRMS under synchronous full-scan MS and data-dependent MS/MS (full-scan MS1/dd-MS2) acquisition mode. The mass resolution was set to 70,000 FWHM (full width at half maximum) for full-scan MS1 and 17,500 FWHM for dd-MS2 stage with the experimentally measured mass deviations of less than 2ppm (parts per million) for quasi-molecular ions and 5ppm for characteristic fragment ions for each individual analyte. An accurate-mass database and a mass spectral library were built in house for searching the 100 target compounds. Broad screening was conducted by comparing the experimentally measured exact mass of precursor and fragment ions, retention time, isotopic pattern, and ionic ratio with the accurate-mass database and by matching the acquired MS/MS spectra against the mass spectral library. The developed methodology was evaluated and validated in terms of limits of detection (LODs), limits of quantitation (LOQs), linearity, stability, accuracy, and matrix effect. The UHPLC-Q-Orbitrap HRMS approach was applied for the analysis of 100 target illicit ingredients in 123 genuine cosmetic samples, and exhibited great potential for high-throughput, sensitive, and reliable screening of multi-class illicit compounds in cosmetics.

Analytical strategy for determination of known and unknown destruxins using hybrid quadrupole-Orbitrap high-resolution mass spectrometry.

An analytical strategy based on a hybrid quadrupole-Orbitrap mass spectrometry was proposed for the simultaneous screening of known destruxins and characterization of potential members of this class of secondary metabolites, in order to evaluate the metabolite production of entomopathogenic fungi used as biocontrol agents. Initially, the fragmentation pathway of the known and commercially available destruxin A was established combining high resolution mass spectrometry (HRMS) and multiple stage MS data in order to obtain the strategy for the characterization of other destruxins for which reference standards were not available. Nineteen known destruxins including A, B, C, D, Ed, F, A1, B1, Ed1, A2, B2, D2, A3, DesmA, DesmB, DesmC, DesmB2, and two chloro-derivatives (Cl and E2 chlorohydrin) were unequivocally identified in Metarhizium brunneum using the proposed strategy. In addition, four unknown destruxins, namely C1, Ed2, G, and G1, were structurally elucidated and characterized forthe first time in this fungal strain.