High-resolution Accurate Mass Spectrometry Research Articles

Identification of chemical constituents and pharmacokinetic characteristics of Xiaoyan Tuire Granule in rats

Xiaoyan Tuire Granule is a type of Chinese patent medicine that has been proven effective in treating respiratory tract infections. However, while it has been successfully introduced into clinical use, more knowledge is still needed regarding its chemical components and pharmacokinetics. This study investigated the chemical profile in the medicine and rat plasma by ultra high-performance liquid chromatography coupled with Q Exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry (UHPLC-Orbitrap-MS/MS). Subsequently, it developed a validated ultra high-performance liquid chromatography coupled with quadrupole mass spectrometry (UHPLC-MS/MS) method for determining five components in rat plasma after oral administration of Xiaoyan Tuire Granule. As a result, a total of 106 constituents were inferred, including 9 terpenoids, 29 flavonoids, 33 organic acids, 12 phenylpropanoids and 23 other compounds. After administration, 86 compounds were inferred in rat plasma, including 73 prototypes and 13 metabolites. The metabolic pathways were primarily hydrogenation, glucuronic acid conjugation, sulfate conjugation, hydrolysis and methylation. The established method determined the contents of esculetin, esculin, isovitexin, caffeic acid and p-coumaric acid had a good separation, and all the legal verification met the requirements. The pharmacokinetic results indicate that the absorption rate of the five compounds in vivo was rapid, with a Tmax of less than 0.25 h, and the elimination rate was also fast, with a half-time (T1/2) ranging from 1.22 h to 2.19 h. It is worth noting that esculin and esculetin have similar half-time in vivo due to their structural similarities. Among these five compounds, the AUC0-∞ and MRT0-∞ of p-coumaric acid and esculetin were relatively higher, indicating higher exposure and longer residence time of both compounds in vivo. In conclusion, this paper researched the chemical constituents and pharmacokinetics of Xiaoyan Tuire Granule, which provided the reference for further study.

Determination of Anthropogenic Pollutants in Adipose Tissue of the Caspian Seal Pusa caspica Gmelin, 1788 by High-Resolution Accurate Mass Spectrometry.

Tissue contamination with persistent organic pollutants (POPs) in organisms proved possible to comprehensively characterize in a single test by combining gas chromatography and high-resolution accurate mass spectrometry. Adipose tissue samples were collected from two Caspian seals (Pusa caspica Gmelin, 1788) found dead on the Caspian Sea shore in 2020. Organochlorine pesticides, primarily DDT and HCH, and polychlorinated biphenyls (PCBs) were major pollutants found in the Caspian seals. The distribution of metabolites indicated the absence of recent pesticide use. The PCB content was relatively high, but still at the lower limit of the range of values determined previously, as was also the case with pesticides. Chlordanes, polychlorinated naphthalenes, and polybrominated diphenyl ethers were detected in minor quantities and were therefore not considered to be major pollutants of the Caspian seal. The pollutant levels were below a threshold at which a distinct effect on seal health can be expected. High-resolution accurate mass (HRAM) spectrometry was found to provide a convenient tool for both targeted and nontargeted analyses of a wide range of organic pollutants in a single experiment.

Improved multi-food allergen analysis of processed foods using HRAM-LC-MS/MS with an ELISA-validated extraction solution and MS sample prep kit.

Food allergens in processed foods are affected by heating, processing, and the food matrix. To conduct highly reliable tests, extracting allergens into test solutions is necessary for appropriate detection. In addition to the commonly used enzyme-linked immunosorbent assay (ELISA), liquid chromatography-mass spectrometry (LC-MS), which has the advantage of simultaneously detecting multiple allergens in foods, is being increasingly used. When managing food allergens at food manufacturing sites, obtaining the same measured values is desirable, regardless of the analytical method used. Therefore, in this study, we focused on the importance of pretreatment steps for LC-MS when examining food allergens in processed foods, which can be difficult to analyze. The ELISA method uses food extracts optimized for analyzing allergens in processed foods. We developed a high-resolution accurate mass spectrometry (HRAM)-LC-MS/MS method using the same food extract used in the ELISA method and an MS sample preparation kit. Multiple food allergen analysis was performed using 1, 5, 10, and 20ppm of allergen-incurred processed foods. Overall, a strong correlation was observed between the measured values of HRAM-LC-MS/MS and ELISA, demonstrating the applicability of multi-allergen analysis using LC-MS.

Degradation of Isotianil in Water and Soil: Kinetics, Degradation Pathways, Mechanisms, and Ecotoxicity Assessments.

Pesticides are transported and transformed in soil and can enter surface water through various pathways. They undergo hydrolysis, oxidation, and photoconversion in surface water. Isotianil is a new fungicide that effectively controls rice blast. However, there are limited reports on its degradation. Herein, the hydrolysis and photolysis of isotianil in water and its degradation in soil samples from five provinces of China were investigated. The degradation products of isotianil were identified using ultrahigh-performance liquid chromatography-Q exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry, and four compounds were discovered for the first time. The degradation pathways of isotianil were inferred, and the reaction active site and degradation mechanism of isotianil were clarified based on density functional theory calculations. The ecotoxicity of the degradation product M118 (aminobenzonitrile) was found to be moderate toward Daphnia magna, which was predicted and confirmed by Ecological Structure Activity Relationships and the experiment, respectively. The results of this study will contribute to a better understanding of the fate of isotianil in the environment.

Effect of biological sex and short-term high-fat diet on cellular proliferation, ribosomal biogenesis, and targeted protein abundance in murine articular cartilage

ObjectiveTo identify factors contributing to sex-differences in OA risk by evaluating the short-term effect of high-fat (HF) diet on sex-specific changes in cartilage cell proliferation, ribosomal biogenesis, and targeted extra-cellular and cellular protein abundance. Materials and methodsKnee cartilage was harvested to the subchondral bone from 20-week-old female and male C57BL/6J mice fed a low-fat or HF diet for 4 weeks and labeled with deuterium oxide for 1, 3, 5, 7, 15, or 21 days. Deuterium enrichment was quantified in isolated DNA and RNA to measure cell proliferation and ribosomal biogenesis, respectively. Protein concentration was measured using targeted high resolution accurate mass spectrometry. ResultsHF diet increased the maximal deuterium incorporation into DNA from approximately 40 to 50%, albeit at a slower rate. These findings, which were magnified in female versus male mice, indicate a greater number of proliferating cells with longer half-lives under HF diet conditions. HF diet caused distinct sex-dependent effects on deuterium incorporation into RNA, increasing the fraction of ribosomes undergoing biogenesis in male mice and doubling the rate of ribosome biogenesis in female mice. HF diet altered cartilage protein abundance similarly in both sexes, except for matrilin-3, which was more abundant in HF versus LF conditions in female mice only. Overall, HF diet treatment had a stronger effect than sex on cartilage protein abundance, with most changes involving extracellular matrix and matrix-associated proteins. ConclusionsShort-term HF diet broadly altered cartilage matrix protein abundance, while sex-dependent effects primarily involved differences in cell proliferation and ribosomal biogenesis.

Profiling of bioactive compounds identified in functional gummies by metabolomics analysis using High-Resolution Accurate Mass-Spectrometry

The consumers demand for healthier and fortified products received much attention these days. The main aim of this study was to prepare functional gummies that contain beneficial bioactive compounds such as flavonoids, terpenoids, polyphenols, and phenolics. The gummies were prepared using varying concentrations of jamun pulp and pear pomace powder, which are both known for their health benefits. Optimization of the prepared gummies was done on the basis of their antioxidant profile. The bioactive compounds found in the gummies were characterized by High-Resolution Accurate Mass-Spectrometry. These bioactive compounds were divided into different categories, such as phenolic acids, flavonoids, as well as terpenoids. Analytical characterization was employed to identify both known and unknown bioactive compounds and metabolites in the optimized product. The results showed that the optimized gummies (FG3) exhibited desirable sensory, textural, and functional properties. FG3 contains a substantial amount of dietary fibre and bioactive compounds, including flavonoids, terpenoids, phenolics, triterpenes, which exhibits health benefits such as anti-cancerous, anti-inflammatory etc., Overall, the developed gummies provide a promising solution to address health-related issues.

Viral Infection Leads to a Unique Suite of Allelopathic Chemical Signals in Three Diatom Host-Virus Pairs.

Ecophysiological stress and the grazing of diatoms are known to elicit the production of chemical defense compounds called oxylipins, which are toxic to a wide range of marine organisms. Here we show that (1) the viral infection and lysis of diatoms resulted in oxylipin production; (2) the suite of compounds produced depended on the diatom host and the infecting virus; and (3) the virus-mediated oxylipidome was distinct, in both magnitude and diversity, from oxylipins produced due to stress associated with the growth phase. We used high-resolution accurate-mass mass spectrometry to observe changes in the dissolved lipidome of diatom cells infected with viruses over 3 to 4 days, compared to diatom cells in exponential, stationary, and decline phases of growth. Three host virus pairs were used as model systems: Chaetoceros tenuissimus infected with CtenDNAV; C. tenuissimus infected with CtenRNAV; and Chaetoceros socialis infected with CsfrRNAV. Several of the compounds that were significantly overproduced during viral infection are known to decrease the reproductive success of copepods and interfere with microzooplankton grazing. Specifically, oxylipins associated with allelopathy towards zooplankton from the 6-, 9-, 11-, and 15-lipogenase (LOX) pathways were significantly more abundant during viral lysis. 9-hydroperoxy hexadecatetraenoic acid was identified as the strongest biomarker for the infection of Chaetoceros diatoms. C. tenuissimus produced longer, more oxidized oxylipins when lysed by CtenRNAV compared to CtenDNAV. However, CtenDNAV caused a more statistically significant response in the lipidome, producing more oxylipins from known diatom LOX pathways than CtenRNAV. A smaller set of compounds was significantly more abundant in stationary and declining C. tenuissimus and C. socialis controls. Two allelopathic oxylipins in the 15-LOX pathway and essential fatty acids, arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) were more abundant in the stationary phase than during the lysis of C. socialis. The host-virus pair comparisons underscore the species-level differences in oxylipin production and the value of screening more host-virus systems. We propose that the viral infection of diatoms elicits chemical defense via oxylipins which deters grazing with downstream trophic and biogeochemical effects.

Mass Spectrometry-Based Method to Measure Aflatoxin B1 DNA Adducts in Formalin-Fixed Paraffin-Embedded Tissues.

Aflatoxin B1 (AFB1) is a potent human liver carcinogen produced by certain molds, particularly Aspergillus flavus and Aspergillus parasiticus, which contaminate peanuts, corn, rice, cottonseed, and ground and tree nuts, principally in warm and humid climates. AFB1 undergoes bioactivation in the liver to produce AFB1-exo-8,9-epoxide, which forms the covalently bound cationic AFB1-N7-guanine (AFB1-N7-Gua) DNA adduct. This adduct is unstable and undergoes base-catalyzed opening of the guanine imidazolium ring to form two ring-opened diastereomeric 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy-aflatoxin B1 (AFB1-FapyGua) adducts. The AFB1 formamidopyrimidine (Fapy) adducts induce G → T transversion mutations and are likely responsible for the carcinogenic effects of AFB1. Quantitative liquid chromatography-mass spectrometry (LC-MS) methods have shown that AFB1-N7-Gua is eliminated in rodent and human urine, whereas ring-opened AFB1-FapyGua adducts persist in rodent liver. However, fresh frozen biopsy tissues are seldom available for biomonitoring AFB1 DNA adducts in humans, impeding research advances in this potent liver carcinogen. In contrast, formalin-fixed paraffin-embedded (FFPE) specimens used for histopathological analysis are often accessible for molecular studies. However, ensuring nucleic acid quality presents a challenge due to incomplete reversal of formalin-mediated DNA cross-links, which can preclude accurate quantitative measurements of DNA adducts. In this study, employing ion trap or high-resolution accurate Orbitrap mass spectrometry, we demonstrate that ring-opened AFB1-FapyGua adducts formed in AFB1-exposed newborn mice are stable to the formalin fixation and DNA de-cross-linking retrieval processes. The AFB1-FapyGua adducts can be detected at levels comparable to those in a match of fresh frozen liver. Orbitrap MS2 measurements can detect AFB1-FapyGua at a quantification limit of 4.0 adducts per 108 bases when only 0.8 μg of DNA is assayed on the column. Thus, our breakthrough DNA retrieval technology can be adapted to screen for AFB1 DNA adducts in FFPE human liver specimens from cohorts at risk of this potent liver carcinogen.

Decoction regulating phytochemicals’ micromorphology changes and anti-inflammation activity enhancements originated from herb medicine supermolecules

BackgroundMahuang Fuzi decoction (MGF) is composed of three herb medicines that has been clinically used to treat inflammatory diseases for a long history. At present, more and more active phytochemicals’ aggregations have been found during the thermodynamic process of herb medicine decoction, and revealing the clinical efficacy of herb medicine through supramolecular strategies is the focus of current research. However, it is not clear whether decoction induced supermolecules’ morphological changes to modify activity.MethodsDynamic light scattering (DLS) and field emission scanning electron microscopy (FESEM) were used to analyze the micromorphology of MGF, MGF SA (MGF supermolecules), and MIX (physical mixture of MGF single decoction). The interaction and thermodynamic parameters of single herbs in a decoction were investigated by Isothermal titration calorimetry (ITC). The phytochemicals were systematically analyzed by ultra high performance liquid chromatography-Q Exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry (UHPLC-Q-Orbitrap HRMS). Under the safe dose on RAW264.7 cells, NO, IL-6 and TNF-α were determined by Enzyme-Linked ImmunoSorbent Assay (ELISA) method. NF-κB p65 translocation from the cytoplasm into the nucleus was examined using the immunofluorescence assay and the western blot, respectively. Furthermore, Metabolomics was used to discover potential biomarkers and the associated metabolic pathways of MGF SA treatment.ResultsThere were nanoscale aggregations in MGF, and the micromorphology of the extracted MGF SA consisted of uniform particles; while the MIX micromorphology had no uniformity. ITC showed that the interaction MH-GC and FZ-GC were a spontaneous exothermic reaction, indicating that their phytochemicals had the property of self-assembly. Though the micromorphology between MGF, MGF SA, and MIX was obviously different, UHPLC-Q-Orbitrap HRMS results displayed that the main phytochemicals of MGF and MIX had nearly the same components. Interestingly, MGF and MGF SA could significantly inhibit the production of NO, and had better inhibition effect on the expression of nuclear protein NF-κB p65 than MIX, among which MGF SA had the best effect. Further investigation indicated that the perturbance of metabolic profiling in RAW264.7 inflammatory cells was obviously reversed by MGF SA.ConclusionsThe decoction enriched the key active phytochemicals and regulated the formation of homogeneous nanoparticles in MGF SA. The supermolecules in MGF SA significantly enhanced its anti-inflammatory activity, primarily affecting the NF-κB signaling pathway and the biosynthesis and metabolism of arginine in RAW264.7 inflammatory cells. Current study displayed that co-decocting herbal medicine were beneficial to the treatment of diseases than the mixture of the single herbs’ extraction.Graphical

Lipid composition, caloric content, and novel oxidation products from microbial communities within seasonal pack ice cores

Sea ice is a critical feature of polar environments with importance for coastal ecosystems. Along the West Antarctic Peninsula (WAP), climate change is causing decreases in seasonal sea ice duration and extent. Organic carbon within sea ice is an important aspect of southern ocean carbon cycling but less studied than water column or sedimentary carbon reservoirs. Though conditions within sea ice can be extreme, phytoplankton are able to flourish and serve as a key food source for higher trophic levels, especially in the spring and fall. A portion of this sea ice phytoplankton biomass is composed of lipids, which are both calorically dense and useful as tracers of biological processes within the sea ice. To better understand both the trophic value and the diversity of lipids present within sea ice we employed high-resolution accurate-mass mass spectrometry to analyze ice core samples from six sites collected along the peninsula in November 2018. Using untargeted methods we annotated 1,173 intact lipid species across 14 classes of intact polar lipids, triacylglycerols, and pigments. We compared lipids’ physical distribution within sea ice cores and found they are highly geographically and physically heterogenous within sites. Ratios between intact polar lipid classes show little signs of phytoplankton nutrient stress; a finding consistent with internally nutrient replete sea ice brines. We found key differences in the composition between pack ice versus fast ice and observed chlorophyll a to be inconsistently correlated with triacylglycerol content. We determined the mean caloric content of lipids within pack ice (4.45 ± 3.47 kJ m−2) and found that caloric densities from the lipids alone were in the range of total water column energy content. Lastly, we show evidence of fatty acid hydroxy fatty acid triacylglycerols within the ice matrix and discuss possible biochemical sources of these novel biomarkers in an ocean system. These results shed light on the chemical diversity of a dynamic and ecosystem relevant carbon pool.

Method Development and Validation for Simultaneous Detection of Corticosteroids, Small Peptides, SARMs and Quaternary Ammonium Drugs in Camel Urine for Doping Control Applications

Background: Detection and identification of a wide range of drugs, including small peptides, corticosteroids, selective androgen receptor modulators (SARMs), and quaternary ammonium drugs (QADs), are imperative across several domains, particularly in anti-doping analysis, given the potential misuse of these substances in animal sports, there is an urgent demand for an allencompassing screening method to effectively identify these compounds. Objective: To develop a robust and sensitive high-resolution method for simultaneous screening of small peptides, corticosteroids, SARMs, and QADs using liquid chromatography accurate mass spectrometry in post-race urine samples. This method integrates a streamlined single-stage extraction approach, significantly enhancing efficiency and adaptability for screening drugs across various classes. Methods: The method development and validation involved a comprehensive solid-phase extraction protocol, which included a sequential elution for corticosteroids, small peptides, SARMs, and QADs. Chromatographic separation was achieved using a reverse-phase C18 column, and the analysis was performed using liquid chromatography - high-resolution accurate mass spectrometry. Results: The developed method was validated using a selection of drugs representing each class. The method exhibited robustness and sensitivity, enabling the simultaneous screening of the specified drug classes. The flexibility inherent in the proposed extraction and analysis method allows for seamless integration of new drug candidates eliminating the need for method redevelopment. Conclusion: A versatile and effective screening method was successfully developed and validated for the simultaneous detection of small peptides, corticosteroids, SARMs, and QADs. The method's ability for retrospective analysis of emerging drugs using full scan HRMS enhances its utility. This method holds great promise across various fields where accurate and comprehensive drug screening is imperative.

Anti-filarial efficacy of Centratherum anthelminticum: unravelling the underlying mechanisms through biochemical, HRAMS proteomics and MD simulation approaches.

Traditionally, Centratherum anthelminticum (CA) has been reported to be a potent anti-filarial, however no reports are available detailing its mechanism of action against filarial parasites. In this study, we have evaluated the anti-filarial activity of CA against lymphatic filarial parasites Setaria cervi using ex vivo biochemical, proteomics and in silico approaches. The motility and viability of the parasites decreased significantly after treatment with CA concentrations of ≥125 μg mL-1. An increase in lipid peroxidation (51.92%), protein carbonylation (48.99%), NADPH oxidase (88.88%) activity and decrease in the glutathione (GSH) (-39.23%), glutathione reductase (GR) (-60.17%), and glutathione S-transferase (GST) (-50.48%) activity was also observed after CA treatment. The proteomics analysis was performed by two-dimensional gel electrophoresis and high-resolution accurate mass spectrometry (HRAMS). In total, 185 proteins were differentially expressed (DEPs) following CA treatment. The major DEPs were mostly involved in tRNA processing, biosynthetic processes, metabolic activities, protein transport, the tricarboxylic acid cycle, protein translation, and stress response. The UPLC-ESI-MS/MS analysis of CA extract revealed the presence of 40 bioactive compounds. Further the docking analysis showed 10 CA bioactive compounds to have high binding affinity towards antioxidant proteins of filarial parasites. Additionally, MD simulation studies showed stable interactions (RMSF ≤ 10 Å) of 3-O-methylquercitin, quinic acid, gentisic acid, and vanillin with filarial antioxidant enzymes/proteins. To our knowledge, this is the first report detailing the molecular mechanism of anti-filarial activity of CA, which can be further evaluated for the development of new anti-filarial formulations.

Evaluation of the Mechanisms Involved in the Development of Bladder Toxicity following Exposure to Occupational Bladder Cancer Causative Chemicals Using DNA Adductome Analysis.

Occupational exposure to aromatic amines (AAs) is an important risk factor for urinary bladder cancer. This study aimed to evaluate the toxicity of AAs and analyze the carcinogenic mechanisms in rat bladder by comprehensive analysis of DNA adducts (DNA adductome). DNA was extracted from the bladder epithelia of rats treated with AAs, including acetoacet-o-toluidine (AAOT) and o-toluidine (OTD), and adductome analysis was performed. Principal component analysis-discriminant analysis revealed that OTD and AAOT observed in urinary bladder hyperplasia could be clearly separated from the controls and other AAs. After confirming the intensity of each adduct, four adducts were screened as having characteristics of the OTD/AAOT treatment. Comparing with the in-house DNA adduct database, three of four candidates were identified as oxidative DNA adducts, including 8-OH-dG, based on mass fragmentation together with high-resolution accurate mass (HRAM) spectrometry data. Therefore, findings suggested that oxidative stress may be involved in the toxicity of rat bladder epithelium exposed to AAs. Consequently, the administration of apocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, in six-week-old rats fed with 0.6% OTD in their diet resulted in simple hyperplastic lesions in the bladder that were suppressed by apocynin. The labeling indices of Ki67, γ-H2AX, and 8-OHdG were significantly decreased in an apocynin concentration-dependent manner. These findings indicate that oxidative stress may have contributed to the development of urinary cancer induced by OTD.

Multi-Attribute Method (MAM) Analytical Workflow for Biotherapeutic Protein Characterization from Process Development to QC.

The multi-attribute method (MAM) has emerged significantly in recent years to support biotherapeutic protein characterization from process development to the QC environment. MAM is a liquid chromatography mass spectrometry (LC-MS) based peptide mapping approach, which combines the benefits from liquid chromatography coupled to high resolution accurate mass mass spectrometry (LC-HRAM MS), enabling direct assessment of protein sequence and product quality attributes with site specificity. These product quality attributes may impact efficacy, safety, stability, and process robustness. MAM is intended to replace conventional analytical approaches as it offers a more streamlined strategy for parallel monitoring of multiple attributes in a single analysis with high sensitivity and confidence, and ultimately supports more robust Quality by Design (QbD) approaches and faster decision cycles for biotherapeutic development. MAM consists of three main stages. The first stage is sample digestion, which typically entails proteolytic digestion of the protein. The second stage is reversed-phase chromatographic separation of the generated peptides and detection by HRAM MS in two phases. During MAM Phase I (discovery phase), data-dependent acquisition (DDA) MS/MS is performed to enable confident identification of peaks and development of a peptide workbook. During MAM Phase II (monitoring phase), full MS acquisition is only carried out for the monitoring of predefined product quality attributes (PQAs). The third stage is data processing, which entails analysis and reporting for each of the two phases including evaluation of sequence coverage, assessment of PQAs and peptide workbook creation during phase I, and targeted monitoring of predefined product attributes and new peak detection (NPD) during phase II. The latter is a comparative analysis that uses a base peak alignment algorithm to determine any non-monitored differences between the LC-MS chromatograms of a test sample and a reference standard. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: In-solution sample digestion Alternate Protocol: Automated sample digestion Basic Protocol 2: Reversed-phase chromatographic separation and detection by HRAM-MS (RPLC-HRAM MS) Basic Protocol 3: Data processing and reporting.

Automated silylation of flavonoids using 3D printed microfluidics prior to chromatographic analysis: system development.

Flavonoids are a class of secondary plant metabolites with low molecular weights. Most flavonoids are highly polar and unsuitable for gas chromatographic analyses. Derivatization is commonly used to make them amenable to gas chromatography by altering their physicochemical properties. Although highly effective, derivatization techniques introduce extra preparation steps and often use hazardous chemicals. The aim of this study was to automate derivatization (specifically, silylation) by developing 3D printed microfluidic devices in which derivatization of flavonoids can occur. A microfluidic device was designed and 3D printed using clear polypropylene. Quercetin and other flavonoids (TED 13 and ZTF 1016) isolated from plant extracts were silylated with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) at room temperature both in batch and in continuous flow. All the samples were analyzed using Fourier transform infrared (FTIR) spectroscopy, gas chromatography combined with mass spectrometry (GC-MS), and high-resolution accurate mass spectrometry (HR-MS). Interestingly, the HR-MS results showed that the flow method was about 25 times more efficient than the batch method for quercetin samples. The TED 13 flavonoid was completely derivatized in the flow method compared to the batch method where the reaction was incomplete. Similar results were observed for ZTF 1016, where the flow method resulted in a four times derivatized compound, while the compound was only derivatized once in batch. In conclusion, 3D printed microfluidic devices have been developed and used to demonstrate a semi-automated, inexpensive, and more efficient natural product derivatization method based on continuous flow chemistry as an alternative to the traditional batch method.

Comprehensive metabolite profiling of trimetazidine in camels using high-resolution accurate mass spectrometry: Implications for doping control.

Trimetazidine and its metabolites are prohibited substances in sports. With a growing number of adverse findings in human athletes, it is crucial to develop doping control strategies that include screening for trimetazidine in animal sports. This study aims to detect and characterize trimetazidine and its metabolites for doping control in camel racing. Camel urine and plasma samples were collected from four healthy animals following a single oral dose of trimetazidine. In vitro investigations were conducted using camel liver samples. Liquid-liquid extraction and solid-phase extraction techniques were employed for the extraction of trimetazidine metabolites from plasma and urine matrices. The metabolites were analyzed using a Thermo Orbitrap Exploris LC-MS system with optimized settings to achieve maximum sensitivity and accurate mass measurements. Comprehensive metabolite profiling of trimetazidine in camels revealed the identification of seven phase I and five phase II metabolites. Phase I metabolites were primarily formed through dealkylation, while phase II metabolites were dominated by glucuronide conjugation of demethylated trimetazidine. The findings provided insights into the distinct metabolic pathways and biotransformation patterns of trimetazidine in camels under the experimental conditions. The developed method enables detection and characterization of trimetazidine and its metabolites in camels. The identified metabolites have the potential to serve as marker metabolites for trimetazidine abuse in camel racing. This study provides valuable insights into the metabolism of trimetazidine in camels.

Association of maternal metals exposure, metabolites and birth outcomes in newborns: A prospective cohort study

BackgroundMaternal exposure to metals may pose a risk to the health of newborns, however, the underlying mechanisms remain ambiguous. Herein, we aimed to investigate the influence of metals exposure on birth outcomes and reveal the importance of metabolites in the exposure-outcomes association by using metabolomics methods. MethodsIn our study, 292 mother-pairs were included who were recruited from the affiliated hospitals of Nanjing Medical University between 2006 and 2011. We measured fifteen metals (mercury, lead, vanadium, arsenic, zinc, cadmium, rubidium, copper, cobalt, iron, molybdenum, strontium, thallium, magnesium and calcium) and metabolites in maternal second trimester serums by using inductively coupled plasma mass spectrometry and ultra-high performance liquid chromatography high resolution accurate mass spectrometry, respectively. A multi-step statistical analysis strategy including exposome-wide association study (ExWAS) model, variable selection models and multiple-exposure models were performed to systematically appraise the associations of individual and mixed metals exposure with birth outcomes. Furthermore, differential metabolites that associated with metals exposure and birth outcomes were identified using linear regression models. ResultsMetal’s levels in maternal serums ranged from 0.05 μg/L to 1864.76 μg/L. In the ExWAS model, maternal exposure to arsenic was negatively associated with birth weight (β = 188.83; 95% CI: −368.27, −9.39), while maternal mercury exposure showed a positive association (β = 533.65; 95%CI: 179.40, 887.90) with birth weight. Moreover, each unit increase in mercury (1 ng/mL-log transformed) was associated with a 1.82 week-increase (95%CI: 0.85, 2.79) in gestational age. These findings were subsequently validated by variable selection models and multiple exposure models. Metabolomic analysis further revealed the significant role of 3-methyladenine in the relationship between arsenic exposure and birth weight. ConclusionThis study provides new epidemiological evidence indicating the associations of metals exposure and neonatal birth outcomes, and emphasizes the potential role of metabolite biomarkers and their importance in monitoring adverse birth outcomes.

A highly sensitive method for determination of tacrolimus in peripheral blood mononuclear cells by nano liquid chromatography–high resolution accurate mass spectrometry

The determination of intracellular tacrolimus concentration in peripheral blood mononuclear cells (PBMCs) is crucial for assessing the effect-site concentration of tacrolimus. Analytical methods previously reported required a minimum of 3 mL of whole blood sample for measuring the tacrolimus concentration. In this study, we developed a highly sensitive method using EASY nLC 1200 combined with Q Exactive orbitrap mass spectrometer for detecting tacrolimus in PBMCs, requiring only 0.5–2 mL of sample. Furthermore, we compared two primary normalization methods for PBMCs tacrolimus concentration using Passing–Bablok regression, Bland–Altman analysis, Spearman's rank correlation, and Mountain plot. The newly established method was employed to compare tacrolimus concentrations in whole blood and PBMCs among 194 lung transplant recipients. The developed method exhibited high sensitivity with a lower limit of quantitation at 5 pg/mL, and excellent intra- and inter-days accuracy and precision. The comparison between different normalization methods for PBMCs tacrolimus concentration revealed a strong correlation between PBMCs count and intracellular protein amount within these cells. This finding suggests that both PBMCs count and intracellular protein amount can be used for normalizing intracellular tacrolimus levels and can be mutually converted. However, a weaker correlation was observed between PBMCs and whole-blood tacrolimus concentrations in lung transplant recipients, warranting further investigation. The method reported herein enables the quantification of PBMCs tacrolimus concentration using smaller volumes of whole blood samples, which has significant implications for both patients and laboratory personnel.

Highly efficient and simultaneous production of thirteen taxanes from Taxus × media and mining of their new bioactivity

Taxus serves as a primary natural source of paclitaxel, a valuable anticancer drug. Developing an efficient and synchronous separation and purification process for the abundant taxanes in Taxus, as well as mining their potential bioactive, can not only improve the production efficiency of paclitaxel but also further reduce the production cost. In this study, the abundant taxanes in the leaves and branches of Taxus × media, as well as the intermediates obtained during the purification process, were analyzed by high-performance liquid chromatography (HPLC) in combination with high-resolution accurate-mass spectrometry (LC-HR-MS/MS). Subsequently, thirteen taxanes were successfully separated and purified to a purity of over 98% with a recovery rate of 61% in a pilot-scale production. Notably, the purity and recovery of paclitaxel were increased to over 99.2% and 78.2%, respectively. Interestingly, taxuspine M was identified for the first time to exhibit superior binding properties with poly ADP-ribose polymerase 1 (PARP1) and NRH:quinone oxidoreductase 2 (NQO2) proteins, which were highly associated with breast cancer, through virtual screening across the human disease therapeutic protein in the therapeutic target database (TTD). Moreover, taxuspine M demonstrated significant anti-proliferative ability against the Michigan Cancer Foundation-7 (MCF-7) cell line with an IC50 value of 195.5 nM, indicating its potential as an anticancer agent. This study provides an improved scalable process for the highly efficient and comprehensive utilization of plant-source pharmaceutical resources.

Characterization of Extensive 2-Fluorodeschloroketamine (2F-DCK) Metabolism in Pooled Human Liver Microsomes (pHLM), Urine, and Hair from an Addicted Patient Using High-Resolution Accurate Mass (HRAM) Spectrometry.

2-Fluoro-deschloroketamine (2F-DCK) is a ketamine derivative involved in acute intoxications and deaths. The aim of this study is to investigate its metabolism using pHLM, and to apply it to authentic samples (urine, hair, seized materials) from a drug user. 2F-DCK (100 µM) incubates with pHLM were analyzed by LC-HRAM (Q-Exactive, Thermo Fisher Scientific®) according to a previously published protocol. Spectra annotation was performed using Compound Discoverer® software and the metabolic scheme was drawn using Chemdraw software. Urine (200 µL) and hair (previously decontaminated using dichloromethane and segmented into 3 segments: A 0-3 cm, B 3-6cm, C 6-9cm) were extracted with a mixture of hexane/ethyl acetate (1:1) and chloroform/isopropanol (4:1). 10 µL of both reconstituted residues were analyzed by LC-HRAM. Hair was also analyzed by LC-MS-MS (TSQ Vantage, Thermo Fisher Scientific®) for 2F-DCK and DCK quantification. The two presumed 2F-DCK crystals consumed by the patient were dissolved in methanol (1 mg/mL) and 10 µL were analyzed by LC-MS-MS (Quantum Access Max, Thermo Fisher Scientific®). Twenty-six putative 2F-DCK metabolites were identified, 15 being reported for the first time. Thirteen metabolites were detected in pHLM, 10 confirmed in both the patient's urine and hair, and all found in at least one of the 2 samples. 23 metabolites were detected in urine, and 20 in hair. Our research confirms the reliability of nor-2F-DCK as a target analyte and suggest OH- dihydro-nor-2F-DCK and dehydro-nor-2F-DCK as new target analytes in urine and hair, respectively. This is the first study to report deschloroketamine as a 2F-DCK metabolite using pHLM and to determine its concentrations in hair (A/B/C, 885/1500/1850 pg/mg) following chronic use. Finally, the two seized crystals contained 2F-DCK at 67% and 96% with traces of deschloroketamine (0.4% and 0.6%) related to cross-contamination by container exchange.