quadrupole-Orbitrap Mass Spectrometer Research Articles

Alkaloid profiling of the new species Corydalis huangshanensis and other 13 medicinal plants in genus Corydalis.

Corydalis DC., the largest genus of Papaveraceae, comprises numerous species known for their abundant alkaloid content and historical use in clinical medicine. Recently, a new species of genus Corydalis named Corydalis huangshanensis Lu Q. Huang & H. S. Peng was discovered in the Huangshan Mountains of Anhui Province, China. To compare the chemical characteristics of C. huangshanensis and other 13 Corydalis species, aiming to elucidate the potential medicinal value of this new species. The chemical constituents of C. huangshanensis and other 13 medicinal plants of genus Corydalis were analyzed using ultra-high-performance liquid chromatography Q-Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer (Q-Orbitrap) mass technology. The differences in the alkaloids in the 14 species were distinguished by chemometrics. The mass spectrometry fragmentation information and relative content of 72 alkaloids were obtained. Orthogonal partial least squares discriminant analysis (OPLS-DA) and cluster heat mapping analysis showed that these 14 species were divided into two groups. The clustering relationship between C. huangshanensis and C. decumbens (Thunb.) Pers. was similar, exhibiting similar chemical compositions and characteristics. These results indicate the potential pharmacological effects of C. huangshanensis. This study enhances our understanding of the chemical classification of Corydalis and provides a basis for speculations on the medicinal value of C. huangshanensis.

Proprotein Convertase Subtilisin/Kexin Type 9 Induction in COVID-19 Is Poorly Associated with Disease Severity and Cholesterol Levels.

SARS-CoV-2 infection was shown to induce proprotein convertase subtilisin/kexin type 9 (PCSK9) plasma levels in sepsis. Here, we investigate the association between serum PCSK9 levels and disease severity. PCSK9 was measured in serum of 55 controls, 40 patients with moderate and 60 patients with severe COVID-19 disease. Serum PCSK9 was elevated in moderate COVID-19 compared to controls and further increased in severe cases. PCSK9 levels were not associated with C-reactive protein, bacterial superinfections, interventions, or survival in patients with severe COVID-19. PCSK9 regulates circulating cholesterol levels, and 15 cholesteryl ester (CE) species and free cholesterol (FC) were quantified by direct flow injection analysis using a high-resolution hybrid quadrupole-Orbitrap mass spectrometer. Most CE species with shorter fatty acid chains were decreased in severe compared to moderate COVID-19, and none of the CE species were correlated with PCSK9 in patients with severe COVID-19. Levels of all CE species negatively correlated with C-reactive protein in severe COVID-19 patients. Notably, FC was induced in severe compared to moderate COVID-19. The FC/CE ratio correlated positively with inflammatory markers and was associated with non-survival. The current study suggests that the imbalance between CE and FC levels is associated with disease severity and mortality in patients with COVID-19.

Improved Data Acquisition Settings on Q Exactive HF-X and Fusion Lumos Tribrid Orbitrap-Based Mass Spectrometers for Proteomic Analysis of Limited Samples.

Deep proteomic profiling of complex biological and medical samples available at low nanogram and subnanogram levels is still challenging. Thorough optimization of settings, parameters, and conditions in nanoflow liquid chromatography-tandem mass spectrometry (MS)-based proteomic profiling is crucial for generating informative data using amount-limited samples. This study demonstrates that by adjusting selected instrument parameters, e.g., ion injection time, automated gain control, and minimally altering the conditions for resuspending or storing the sample in solvents of different compositions, up to 15-fold more thorough proteomic profiling can be achieved compared to conventionally used settings. More specifically, the analysis of 1 ng of the HeLa protein digest standard by Q Exactive HF-X Hybrid Quadrupole-Orbitrap and Orbitrap Fusion Lumos Tribrid mass spectrometers yielded an increase from 1758 to 5477 (3-fold) and 281 to 4276 (15-fold) peptides, respectively, demonstrating that higher protein identification results can be obtained using the optimized methods. While the instruments applied in this study do not belong to the latest generation of mass spectrometers, they are broadly used worldwide, which makes the guidelines for improving performance desirable to a wide range of proteomics practitioners.

Plasma drug screening using paper spray mass spectrometry with integrated solid phase extraction.

Drug overdoses have risen dramatically in recent years. We developed a simple nontargeted method using a disposable paper spray cartridge with an integrated solid phase extraction column. This method was used to screen for ~160 fentanyl analogs, synthetic cannabinoids, other synthetic drugs, and traditional drugs of abuse in over 300 authentic overdose samples collected at emergency departments in Indianapolis. A solid phase extraction step was implemented on the paper spray cartridge to enable subnanograms per milliliter synthetic drugs screening in plasma. Analysis was performed on a quadrupole orbitrap mass spectrometer using the sequential window acquisition of all theoretical fragment ion spectra approach in which tandem mass spectrometry was performed using 7 m/z isolation windows in the quadrupole. Calibration curves with isotopically labeled internal standards were constructed for 35 of the most frequently encountered synthetic and traditional illicit drugs by US toxicology labs. Additional qualitative-only drugs in a suspect screening list were also included. Limits of detection in plasma for synthetic cannabinoids ranged from 0.1 to 0.5 and 0.1 to 0.3ng/mL for fentanyl and its analogs and between 1 and 5ng/mL for most other drugs. Relative matrix effects were evaluated by determining the variation of the calibration slope in 10 different lots of biofluid and found to be between 3% and 20%. The method was validated on authentic overdose samples collected from two emergency departments in Indianapolis, Indiana, from suspected or known overdoses. Commonly detected synthetic drugs included fentanyl related substances, designer benzodiazepines such as flubromazolam, and the synthetic cannabinoid 5F-PB-22.

Discovery of a series of portimine-A fatty acid esters in mussels

Vulcanodinium rugosum is a benthic dinoflagellate known for producing pinnatoxins, pteriatoxins, portimines and kabirimine. In this study, we aimed to identify unknown analogs of these emerging toxins in mussels collected in the Ingril lagoon, France. First, untargeted data acquisitions were conducted by means of liquid chromatography coupled to hybrid quadrupole-orbitrap mass spectrometry. Data processing involved a molecular networking approach, and a workflow dedicated to the identification of biotransformed metabolites. Additionally, targeted analyses by liquid chromatography coupled to triple quadrupole mass spectrometry were also implemented to further investigate and confirm the identification of new compounds. For the first time, a series of 13-O-acyl esters of portimine-A (n = 13) were identified, with fatty acid chains ranging between C12:0 and C22:6. The profile was dominated by the palmitic acid conjugation. This discovery was supported by fractionation experiments combined with the implementation of a hydrolysis reaction, providing further evidence of the metabolite identities. Furthermore, several analogs were semi-synthesized, definitively confirming the discovery of these metabolization products. A new analog of pinnatoxin, with a molecular formula of C42H65NO9, was also identified across the year 2018, with the highest concentration observed in August (4.5 μg/kg). The MS/MS data collected for this compound exhibited strong structural similarities with PnTX-A and PnTX-G, likely indicating a substituent C2H5O2 in the side chain at C33. The discovery of these new analogs will contribute to deeper knowledge of the chemodiversity of toxins produced by V. rugosum or resulting from shellfish metabolism, thereby improving our ability to characterize the risks associated with these emerging toxins.

Exploring Inflammatory Asthma Phenotypes: Proteomic Signatures in Serum and Induced Sputum.

Asthma drug responses may differ due to inflammatory mechanisms triggered by the immune cells in the pulmonary microenvironment. Thus, asthma phenotyping based on the local inflammatory profile may aid in treatment definition and the identification of new therapeutic targets. Here, we investigated protein profiles of induced sputum and serum from asthma patients classified into eosinophilic, neutrophilic, mixed granulocytic, and paucigranulocytic asthma, according to inflammatory phenotypes. Proteomic analyses were performed using an ultra-performance liquid chromatography (ultra-HPLC) system coupled to the Q Exactive Hybrid Quadrupole Orbitrap Mass Spectrometer. Fifty-two (52) proteins showed significant differences in induced sputum among the groups, while only 12 were altered in patients' sera. Five proteins in the induced sputum were able to discriminate all phenotypic groups, while four proteins in the serum could differentiate all except the neutrophilic from the paucigranulocytic inflammatory pattern. This is the first report on comparative proteomics of inflammatory asthma phenotypes in both sputum and serum samples. We have identified a potential five-biomarker panel that may be able to discriminate all four inflammatory phenotypes in sputum. These findings not only provide insights into potential therapeutic targets but also emphasize the potential for personalized treatment approaches in asthma management.

The astounding exhaustiveness and speed of the Astral mass analyzer for highly complex samples is a quantum leap in the functional analysis of microbiomes

BackgroundBy analyzing the proteins which are the workhorses of biological systems, metaproteomics allows us to list the taxa present in any microbiota, monitor their relative biomass, and characterize the functioning of complex biological systems.ResultsHere, we present a new strategy for rapidly determining the microbial community structure of a given sample and designing a customized protein sequence database to optimally exploit extensive tandem mass spectrometry data. This approach leverages the capabilities of the first generation of Quadrupole Orbitrap mass spectrometer incorporating an asymmetric track lossless (Astral) analyzer, offering rapid MS/MS scan speed and sensitivity. We took advantage of data-dependent acquisition and data-independent acquisition strategies using a peptide extract from a human fecal sample spiked with precise amounts of peptides from two reference bacteria.ConclusionsOur approach, which combines both acquisition methods, proves to be time-efficient while processing extensive generic databases and massive datasets, achieving a coverage of more than 122,000 unique peptides and 38,000 protein groups within a 30-min DIA run. This marks a significant departure from current state-of-the-art metaproteomics methodologies, resulting in broader coverage of the metabolic pathways governing the biological system. In combination, our strategy and the Astral mass analyzer represent a quantum leap in the functional analysis of microbiomes.DtQfodvXmxPWcwvjCbxEj1Video

Ultra-fast label-free quantification and comprehensive proteome coverage with narrow-window data-independent acquisition.

Mass spectrometry (MS)-based proteomics aims to characterize comprehensive proteomes in a fast and reproducible manner. Here we present the narrow-window data-independent acquisition (nDIA) strategy consisting of high-resolution MS1 scans with parallel tandem MS (MS/MS) scans of ~200 Hz using 2-Th isolation windows, dissolving the differences between data-dependent and -independent methods. This is achieved by pairing a quadrupole Orbitrap mass spectrometer with the asymmetric track lossless (Astral) analyzer which provides >200-Hz MS/MS scanning speed, high resolving power and sensitivity, and low-ppm mass accuracy. The nDIA strategy enables profiling of >100 full yeast proteomes per day, or 48 human proteomes per day at the depth of ~10,000 human protein groups in half-an-hour or ~7,000 proteins in 5 min, representing 3× higher coverage compared with current state-of-the-art MS. Multi-shot acquisition of offline fractionated samples provides comprehensive coverage of human proteomes in ~3 h. High quantitative precision and accuracy are demonstrated in a three-species proteome mixture, quantifying 14,000+ protein groups in a single half-an-hour run.

Leaching of biochar at different stages of anaerobic digestion: Leaching behaviors, leachate chemodiversity and effects

Biochar addition is recognized as a promising way to increase the potential of anaerobic digestion. However, few studies have investigated the interaction between biochar leaching in complex, practical anaerobic digestion systems and the negative effects that biochar leachate may have on the anaerobic digestion process. The traditional measurement methods are relatively simple, but may underestimate the complexity of dissolved organic matter in biochar leachate. In this study, the leaching behaviors of biochar and the influence of biochar leachate on anaerobic digestion were studied at the molecular level by applying ultra-performance liquid chromatography online coupled with a hybrid quadrupole orbitrap mass spectrometer (UPLC Orbitrap MS/MS) test analysis and various molecular information-based analytical methods such as van Krevelen diagrams and by conducting a series of anaerobic digestion experiments. Dissolved organic matter leaching was higher in buffered saline solution and methanol than in ultrapure water and the dissolved organic matter mainly consisted of lipids and proteins. In the lactic acid solution, a large amount of dissolved organic matter was leached from the biochar, dominated by low molecular weight dissolved organic matter such as CHO and CHON, and the lower the pH, the more was leached, suggesting that lactic acid may have an inhibitory effect on anaerobic digestion. The maximum methane production amount and microbial abundance especially key methanogenic archaea such as Methanosaeta in the sample with biochar leachate addition were lower than those for the other samples. This meant that the dissolved organic matter in the biochar leachate might inhibit the growth of microorganisms and adversely affect the acetic acid methanogenesis process. The results from this study showed that a greater number of substances with more complex structures were leached out than had been detected in previous studies. When applying biochar to anaerobic digestion in the future, it is recommended that biochar is leached first to remove toxic and detrimental substances to prevent possible inhibitory effects.

Synaptic proteins in pre‐symptomatic Alzheimer’s disease: biomarkers for early detection of Cognitive Decline

AbstractBackgroundSynaptic proteins in the cerebrospinal fluid (CSF) may reveal changes in the pre‐symptomatic stages of Alzheimer’s disease (AD), thus may be candidate biomarkers for early detection of the disease.MethodThe PREVENT‐AD cohort includes symptom‐free (upon enrolment) elderly participants who are at risk of developing AD from their family history. We used enzyme‐linked immunosorbent assay kits to assess CSF samples from 129 such participants for the “classical” AD biomarkers total tau, phosphorylated (181) tau and Aβ42. We also used neuroimaging data (MRI, PET) and neuropsychological assessments (MMSE, RBANS) as potential indicators disease progression. We then used in the CSF samples to measure the soluble synaptic biomarkers ADAM 22 (post‐synaptic), ADAM23 (pre‐synaptic). Immunoprecipitated SYT1 (pre‐synaptic) from CSF were analyyzed using high‐resolution selected ion monitoring analyses on a quadrupole–orbitrap mass spectrometer Q Exactive. Statistical analysis of the association of these markers with evidence of disease in analyses were done included sex and APOE e4 status as covariates.ResultAmong participants who remained cognitively unimpaired, we observed significant correlations between baseline CSF ADAM 22 levels and t‐tau (R2 = 0.22, p < 0.0001), p‐tau (R2 = 0.22, p < 0.0001), and Aβ42 (R2 = 0.06, p = 0.01488). We also found similarly suggestive correlations also between CSF ADAM 23, CSF SYT1 and the same disease markers. Covariate analyses suggested little or no variation in the associations between these synaptic proteins with t‐tau and p‐tau by sex, APOE e4 status, negative PET amyloid positivity (standardized uptake value ratio ≤ 1.37) and negative CSF total tau positivity (≤ 335pg/ul). PET amyloid positivity was significantly associated with ADAM22 and p‐tau interactions whereas SYT1 was significantly associated with t‐tau and p‐tau in CSF tau‐positive participants. In linear regression analyses, baseline CSF ADAM22 levels correlated with the language cognitive performance trajectory slopes estimated over the course of 5 to 8 years on the RBANS (R2 = 0.04, p = 0.03912). We found no significant interaction between other baseline CSF synaptic protein levels and other subscales of the RBANS.ConclusionCSF synaptic protein levels show promising correlation with landmark AD proteins and emerging cognitive deficits.

Quantitative serum proteome analysis using tandem mass tags in dogs with epilepsy

This study included four groups of dogs (group A: healthy controls, group B: idiopathic epilepsy receiving antiepileptic medication (AEM), group C: idiopathic epilepsy without AEM, group D: structural epilepsy). Comparative quantitative proteomic analysis of serum samples among the groups was the main target of the study. Samples were analyzed by a quantitative Tandem-Mass-Tags approach on the Q-Exactive-Plus Hybrid Quadrupole-Orbitrap mass-spectrometer. Identification and relative quantification were performed in Proteome Discoverer. Data were analyzed using R. Gene ontology terms were analyzed based on Canis lupus familiaris database. Data are available via ProteomeXchange with identifier PXD041129. Eighty-one proteins with different relative adundance were identified in the four groups and 25 were master proteins (p < 0.05). Clusterin (CLU), and apolipoprotein A1 (APOA1) had higher abundance in the three groups of dogs (groups B, C, D) compared to controls. Amine oxidase (AOC3) was higher in abundance in group B compared to groups C and D, and lower in group A. Adiponectin (ADIPOQ) had higher abundance in groups C compared to group A. ADIPOQ and fibronectin (FN1) had higher abundance in group B compared to group C and D. Peroxidase activity assay was used to quantify HP abundance change, validating and correlating with proteomic analysis (r = 0.8796). SignificanceThe proteomic analysis of serum samples from epileptic dogs indicated potential markers of epilepsy (CLU), proteins that may contribute to nerve tissue regeneration (APOA1), and contributing factors to epileptogenesis (AOC3). AEM could alter extracellular matrix proteins (FN1). Illness (epilepsy) severity could influence ADIPOQ abundance.

Ultrasonic assisted activation of persulfate for the treatment of spent porous biochar: Degradation of adsorbed PFOA and adsorbent regeneration

In this work, the regeneration of spent biochar adsorbent and the degradation of adsorbed PFOA by the system of ultrasonic assisted activation of persulfate were explored. The effects of ultrasound, persulfate and biochar on PFOA removal were investigated through a series of regeneration and degradation experiments. Meanwhile, the degradation mechanism and reaction pathways of PFOA were investigated with electron paramagnetic resonance (EPR) and high-resolution quadrupole orbitrap mass spectrometer. The results showed that PFOA adsorbed by biochar could be degraded efficiently with the synergistic effect of ultrasound and persulfate. The degradation of PFOA in biochar could be finished in 4 h, while the defluorination took longer, with a defluorination efficiency of 50.6% after 4 h and 99.6% after 10 h. In this process, PFOA was degraded and defluorinated gradually by thermal decomposition caused by cavitation bubbles and degradation caused by the attack of SO4•ˉ and •OH. Two different free radicals controlled different degradation pathways, with SO4•ˉ playing a dominant role. In this system, the regeneration of the waste biochar adsorbent was achieved by eliminating PFOA, which could be recycled more than three times. The application potential of the system was evaluated using real water sample, and it was found that the removal of PFOA in real water sample was satisfactory even with the influence of inorganic ions and organic matter, indicating that it has promising application prospects. This work provided new insight into the degradation of adsorbed PFOA and adsorbent regeneration.

Non-targeted analysis and risk assessment of intentionally and non-intentionally added substances migrating from the emerging biodegradable food contact material poly(butylene adipate-co-terephthalate)/modified starch blend film

Emerging biodegradable food contact materials (FCMs) are omnipresent in food handling and processing. However, similar to conventional FCMs, small molecules can migrate into the food matrix during contact. Extensive evaluation of the migration of chemicals, added intentionally and non-intentionally, from FCMs into food is a crucial step in the safety assessment of such materials. In this study, non-targeted screening was first performed to analyze migrants from poly(butylene adipate-co-terephthalate)/modified starch (PBAT/MFS) FCM using gas chromatography–mass spectrometry and ultra-performance liquid chromatography-Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer. Sixteen volatile migrants were identified, and 134 non-volatile compounds were screened for dissolution precipitates and food simulants using a self-built and commercially-available databases. The risk ranking for migrants revealed that 13 small molecules had high scores and were assigned as Risk I substances of great concern. Among them, pesticide residues and mycotoxins non-intentionally introduced from MFS blending are of great concern.

Screening of volatile organic compounds (VOCs) from liquid fungal cultures using ambient mass spectrometry

The potential of fungi for use as biotechnological factories in the production of a range of valuable metabolites, such as enzymes, terpenes, and volatile aroma compounds, is high. Unlike other microorganisms, fungi mostly secrete secondary metabolites into the culture medium, allowing for easy extraction and analysis. To date, the most commonly used technique in the analysis of volatile organic compounds (VOCs) is gas chromatography, which is time and labour consuming. We propose an alternative ambient screening method that provides rapid chemical information for characterising the VOCs of filamentous fungi in liquid culture using a commercially available ambient dielectric barrier discharge ionisation (DBDI) source connected to a quadrupole-Orbitrap mass spectrometer. The effects of method parameters on measured peak intensities of a series of 8 selected aroma standards were optimised with the best conditions being selected for sample analysis. The developed method was then deployed to the screening of VOCs from samples of 13 fungal strains in three different types of complex growth media showing clear differences in VOC profiles across the different media, enabling determination of best culturing conditions for each compound-strain combination. Our findings underline the applicability of ambient DBDI for the direct detection and comparison of aroma compounds produced by filamentous fungi in liquid culture.Graphical abstract

The Functional Characterization of DzCYP72A12-4 Related to Diosgenin Biosynthesis and Drought Adaptability in Dioscorea zingiberensis.

Dioscorea zingiberensis is a perennial herb famous for the production of diosgenin, which is a valuable initial material for the industrial synthesis of steroid drugs. Sterol C26-hydroxylases, such as TfCYP72A616 and PpCYP72A613, play an important role in the diosgenin biosynthesis pathway. In the present study, a novel gene, DzCYP72A12-4, was identified as C26-hydroxylase and was found to be involved in diosgenin biosynthesis, for the first time in D. zingiberensis, using comprehensive methods. Then, the diosgenin heterogenous biosynthesis pathway starting from cholesterol was created in stable transgenic tobacco (Nicotiana tabacum L.) harboring DzCYP90B71(QPZ88854), DzCYP90G6(QPZ88855) and DzCYP72A12-4. Meanwhile, diosgenin was detected in the transgenic tobacco using an ultra-performance liquid chromatography system (Vanquish UPLC 689, Thermo Fisher Scientific, Bremen, Germany) tandem MS (Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer, Thermo Fisher Scientific, Bremen, Germany). Further RT-qPCR analysis showed that DzCYP72A12-4 was highly expressed in both rhizomes and leaves and was upregulated under 15% polyethylene glycol (PEG) treatment, indicating that DzCYP72A12-4 may be related to drought resistance. In addition, the germination rate of the diosgenin-producing tobacco seeds was higher than that of the negative controls under 15% PEG pressure. In addition, the concentration of malonaldehyde (MDA) was lower in the diosgenin-producing tobacco seedlings than those of the control, indicating higher drought adaptability. The results of this study provide valuable information for further research on diosgenin biosynthesis in D. zingiberensis and its functions related to drought adaptability.

In Vitro Antimicrobial and Antibiofilm Properties and Bioaccessibility after Oral Digestion of Chemically Characterized Extracts Obtained from Cistus × incanus L., Scutellaria lateriflora L., and Their Combination.

Periodontal diseases are oral inflammatory diseases ranging from gingivitis to chronic periodontitis. Porphyromonas gingivalis is one of the major pathogens responsible for severe and chronic periodontitis. Plant extracts with antimicrobial activity could be considered possible alternatives to chlorhexidine, an antiseptic substance used in oral hygiene thatcan cause bacteria resistance. Here, two commercial extracts obtained from Cistus × incanus L. and Scutellaria lateriflora L. were chemically characterized usingUltra-High-Performance Liquid Chromatography (UHPLC) coupled with a Q-Exactive Hybrid Quadrupole Orbitrap Mass Spectrometer. The extracts were studied for their bioaccessibility after simulated in vitro oral digestion, their antimicrobial activity against P. gingivalis, their protective effects against cellular invasion by P. gingivalis, and their antibiofilm activity. The extracts were found to contain very complex mixtures of polyphenols, which were quite stable after in vitro simulated oral digestion and demonstrated mild, dose-dependent inhibitory activity against P. gingivalis growth. This activity increased with the combination of the two extracts. Moreover, the combination of the extracts induced a reduction in P. gingivalis HaCaT invasiveness, and the reduction in biofilm came to around 80%. In conclusion, a combination of C. incanus and S. lateriflora showed promising effects useful in the treatment of gingivitis.

The roles of serine hydrolases and serum albumin in alisol B 23-acetate hydrolysis in humans.

Introduction: Alisol B 23-acetate (AB23A), a major bioactive constituent in the Chinese herb Zexie (Rhizoma Alismatis), has been found with multiple pharmacological activities. AB23A can be readily hydrolyzed to alisol B in mammals, but the hydrolytic pathways of AB23A in humans and the key enzymes responsible for AB23A hydrolysis are still unrevealed. This study aims to reveal the metabolic organs and the crucial enzymes responsible for AB23A hydrolysis in human biological systems, as well as to decipher the impact of AB23A hydrolysis on its biological effects. Methods: The hydrolytic pathways of AB23A in human plasma and tissue preparations were carefully investigated by using Q-Exactive quadrupole-Orbitrap mass spectrometer and LC-UV, while the key enzymes responsible for AB23A hydrolysis were studied via performing a set of assays including reaction phenotyping assays, chemical inhibition assays, and enzyme kinetics analyses. Finally, the agonist effects of both AB23A and its hydrolytic metabolite(s) on FXR were tested at the cellular level. Results: AB23A could be readily hydrolyzed to form alisol B in human plasma, intestinal and hepatic preparations, while human butyrylcholinesterase (hBchE) and human carboxylesterases played key roles in AB23A hydrolysis in human plasma and tissue preparations, respectively. It was also found that human serum albumin (hSA) could catalyze AB23A hydrolysis, while multiple lysine residues of hSA were covalently modified by AB23A, suggesting that hSA catalyzed AB23A hydrolysis via its pseudo-esterase activity. Biological tests revealed that both AB23A and alisol B exhibited similar FXR agonist effects, indicating AB23A hydrolysis did not affect its FXR agonist effect. Discussion: This study deciphers the hydrolytic pathways of AB23A in human biological systems, which is very helpful for deep understanding of the metabolic rates of AB23A in humans, and useful for developing novel prodrugs of alisol B with desirable pharmacokinetic behaviors.

A comprehensive and high-throughput screening method for multiple prohibited substances by UPLC-QE Plus-HRMS and HPLC-QQQ-MS in human urine for doping control.

Since the World Anti-Doping Agency's (WADA) Prohibited List is updated on an annual basis, screening methods must be continually adapted to align with these changes. In accordance with Technical Document-MRPL 2022, a newly combined, comprehensive, rapid and high-throughput doping control screening method has been developed for the analysis of 350 substances with different polarities in human urine using ultra-high performance liquid chromatography coupled with Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer (UPLC-QE Plus-HRMS) and ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometer (UPLC-QQQ-MS). The limits of detection were in the range of 0.12-50 ng mL-1 for beta-2 agonists, hormone and metabolic modulators, narcotics, cannabinoids and glucocorticoids, 0.1-14 ng mL-1 for the manipulation of blood and blood components, beta blockers, anabolic agents and hypoxia-inducible factor (HIF) activating agents, and 2.5-100 000 ng mL-1 for substances of Appendix A, diuretics & masking agents and stimulants. The sample preparation consisted of two parts: one is the dilute & shoot part analyzed in UPLC-QQQ-MS, another is a mixture of the dilute & shoot part and a liquid-liquid extraction part of hydrolyzed human urine analyzed in UPLC-QE Plus-HRMS in full scan mode with polarity switching and parallel reaction monitoring (PRM) mode. The method has been fully validated for doping control purposes. All the substances were compliant with WADA's required 1/2 minimum requirement performance level (MRPL) or minimum reporting level (MRL), and this method was successfully used in the 2022 Beijing Winter Olympic Games and Winter Paralympic Games for anti-doping purpose.

A high-resolution mass spectrometry-based methodology for characterization and identification of methylophiopogonanone B metabolites from cryopreserved hepatocytes and liver microsomes.

Methylophiopogonanone B (MOB), one of the homoisoflavonoids isolated from Ophiopogon japonicus, has been demonstrated to possess antioxidative and antitumor activities. The aim of this work was to investigate the metabolism of MOB using liver microsomes and hepatocytes. MOB was individually incubated with rat, monkey, and human hepatocytes to generate the metabolites. To investigate the bioactivation pathways, MOB was incubated with liver microsomes in the presence of glutathione (GSH). All the metabolites were detected and identified using LC with a quadrupole Orbitrap mass spectrometer. Under the current conditions, nine metabolites were identified in hepatocyte incubations. Of these metabolites, M7 derived from hydroxylation was identified as the most abundant metabolite in hepatocyte incubation. MOB was metabolized via demethylation, hydroxylation, and glucuronidation. In liver microsomes, five GSH conjugates were detected and identified. MOB was subjected to bioactivation through demethylation yielding M9, which further formed quinone-methide and ortho-quinone intermediates, followed by GSH conjugation. This work is the first to study the metabolism of MOB, which will help us understand its disposition and efficacy.

Kinetics, Thermodynamics and Mechanism of Enzymatic Degradation of Zearalenone in Degummed Corn Oil.

The kinetics and thermodynamics of the enzymatic degradation of zearalenone (ZEN) in degummed corn oil were investigated by analyzing the impacts of temperature, pH, ZEN hydrolase dosage and ZEN concentration on the initial reaction rate. The kinetic study found that the maximum reaction rate was 0.97 μmol × kg−1 min−1, the Michaelis constant (Km) was 11,476 μmol × kg−1 and the Michaelis equation was V = 0.97[S]/(11,476 + [S]). The thermodynamic study showed that the activation energy (Ea) was 70.37 kJ·mol−1, the activation enthalpy change of the reaction (ΔH) > 0, the free energy of activation (ΔG) > 0 and the activation entropy change (ΔS) < 0, indicating the reaction could not be spontaneous. The reaction mechanism of ZEN was studied by a hybrid quadrupole orbitrap mass spectrometer. It was found that ZEN first generated the intermediate G/L/D/W-ZEN+H2O, followed by generating the intermediate W-ZEN-H2O under the action of a degrading enzyme. Then, the lactone bond was opened to produce C18H24O6, and finally the decarboxylation product C17H24O4 formed automatically.