Chromatography High-resolution Tandem Mass Spectrometry Research Articles

Comprehensive lipidomic profiling by plasma separation cards.

Large-scale lipidomic analyses have been limited by the cost and accessibility of traditional venipuncture sampling. Microsampling techniques offer a less-invasive and more accessible alternative. From a single drop of blood, plasma separation cards (PSC) deliver two volumetric dried plasma samples which are studied here for profiling endogenous blood lipids. Six lots of EDTA-treated human whole blood were used to compare PSC, dried blood spot analyses (DBS), and classic wet plasma extractions. Six replicate extractions were performed for each lot. Nontargeted lipidomics was performed by liquid chromatography-high resolution tandem mass spectrometry. Lipids were annotated by accurate mass/retention time matching and MS/MS spectral library matching using peak intensities for quantitation. Four hundred ninety-eight compounds covering 24 lipid subclasses were annotated. Inter-lot repeatability was evaluated by the percent relative standard deviation (%RSD) for each lot, giving median %RSD values across the lots at 14.6% for PSC, 9.3% for DBS, and 8.6% for wet plasma. Strong correlations of lipid peak intensities between wet plasma and PSCs were observed, but less for DBS. Lipid recovery and stability were comparable between the PSC and DBS samples, with roughly 60% of annotated lipids stable at room temperature after 28 days. Overall, PSCs provide a better alternative for quantitative blood lipidomic analyses compared to dried blood spots. However, problems with lipid stability for samples handled and shipped at room temperature are currently unavoidable outside of a clinical setting. Data transferability and comparability to standard plasma is lipid and lipid class dependent.

Associations between Maternal Polychlorinated Dibenzo-p-dioxin and Dibenzofuran Serum Concentrations and Pulse Pressure in Early Pregnancy: A Cross-Sectional Study.

Pulse pressure (PP) is the difference between systolic blood pressure (SBP) and diastolic blood pressure (DBP), and an independent predictor of cardiovascular risk. Previous research suggests, with different conclusions, that exposure to polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) could affect blood pressure (BP). We conducted a cross-sectional study to determine the association of dioxin exposure with PP in early pregnancy. A total of 305 pregnant women in early pregnancy in Yingcheng, China, recruited from May 2018 to February 2021, were included in this study. We measured 17 congeners of PCDD/Fs in maternal serum via high-resolution gas chromatography tandem high-resolution mass spectrometry. A generalized linear regression model was used to analyze the influencing factors of dioxin exposure and their relationships with PP. The levels of total PCDD/Fs (∑PCDD/Fs) ranged from 163.52 pg/g lipid to 1,513,949.52 pg/g lipid, with a mean of 10,474.22 pg/g lipid. The mean toxicity equivalent (TEQ) of total PCDD/Fs (∑TEQ-PCDD/Fs) was 42.03 pg/g lipid. The ratio of tetrachlorinated to octa-chlorinated congeners in maternal serum was enriched with an increasing number of chlorines. Pregnant women with college and above education had higher concentrations of ∑PCDD/Fs than those with education levels of junior high school and below (β = 0.34, 95% CI: 0.01, 0.67). The adjusted model for ∑TEQ-PCDD/Fs was significantly and negatively associated with PP (β = -1.79, 95% CI: -2.91, -0.68). High levels of dioxins were found in this area, and exposure to dioxins may affect the PP of women in early pregnancy, with health risks.

Non-targeted analysis of co-formulants in antifungal pesticide formulations by gas chromatography-tandem high resolution mass spectrometry

In the present study, six commercial pesticide formulations with antifungal activity were characterized. Thus, two complementary injection methods based on gas chromatography were employed: direct injection (DI) and headspace (HS), both coupled to high resolution mass spectrometry (GC-Q-Orbitrap-MS). The combination of both injection modes allowed the tentatively identification of potential co-formulants. Available analytical standards were acquired for their confirmation, and 21 compounds were successfully confirmed. Finally, the concentration of these co-formulants was calculated, finding the highest value in one of the pesticide formulation, at 218.22 g L−1 for cyclohexanone.Results clearly show that this methodology is suitable for the reliable identification of co-formulants in pesticide formulations, offering high sensitivity, and highlighting that five co-formulants were detected by both injection techniques (DI and HS). Moreover, one of the main advantages of the proposed methods was the great capacity for the elucidation of compounds with similar molecular formula, bearing in mind that up to 8 co-formulants with the same molecular formula C10H14, were well differentiated by retention times between 8.46 (1-methyl-3-propylbenzene) and 10.98 min (1,2,3,4- tetramethylbenzene) in one of the pesticide formulation.Toxicity to human health and the environment has been evidenced for the co-formulants detected, finding compounds with relatively high toxicity, as naphthalene and cyclohexanone.

A novel method for efficient screening and annotation of important pathway-associated metabolites based on the modified metabolome and probe molecules

植物次生代谢物在抵御生物/非生物胁迫、生物间互作以及信息传递等方面发挥重要作用,次生代谢途径解析对植物分子育种、天然产物合成等方面具有重要意义。液相色谱-高分辨串联质谱(LC-HRMS/MS)为次生代谢物鉴定及途径表征提供了技术手段。非靶向LC-HRMS/MS方法可获得丰富的质谱信号,包括一级质谱和二级质谱(MS, MS/MS),但受质谱数据库规模以及次生代谢物复杂性的制约,次生代谢物注释十分困难。该研究以玉米叶片中苯丙烷途径代谢物为例,发展用于非靶向代谢组数据中重要途径代谢物的高效筛选和注释新方法。首先,利用公共代谢途径数据库及文献获取参与苯丙烷代谢途径的61种修饰反应类型,进而从非靶向实验数据中筛选出修饰代谢组。其次,获取开源串联质谱数据中的苯丙烷类化合物作为探针分子,构建探针分子质谱数据库。将探针分子与修饰代谢组共建分子网络,锁定目标途径代谢物并注释结构。该方法在正、负离子模式下分别筛选出玉米叶片中392个和417个苯丙烷途径候选代谢物,去冗余后共注释出129个代谢物,涉及苯丙烷代谢的主要分支途径,如黄酮途径的8个类黄酮、19个氧苷类黄酮和32个碳苷类黄酮,31个羟基肉桂酸途径代谢物以及22个木脂素途径代谢物;其中26个在PubChem和SciFinder数据库中未见收录。该研究利用探针分子结合修饰组可快速锁定途径代谢物,且有助于快速、准确的网络传播注释,可显著提高目标途径代谢物筛选与注释效率,为植物次生代谢途径的深入解析提供分析手段。

β'-Phenylfentanyl Metabolism in Primary Human Hepatocyte Incubations: Identification of Potential Biomarkers of Exposure in Clinical and Forensic Toxicology.

From 2014 onwards, illicit fentanyl and analogues have caused numerous intoxications and fatalities worldwide, impacting the demographics of opioid-related overdoses. The identification of cases involving fentanyl analogues is crucial in clinical and forensic settings to treat patients, elucidate intoxications, address drug use disorders and tackle drug trends. However, in analytical toxicology, the concentration of fentanyl analogues in biological matrices is low, making their detection challenging. Therefore, the identification of specific metabolite biomarkers is often required to document consumption. β'-Phenylfentanyl (N-phenyl-N-[1-(2-phenylethyl)-4-piperidinyl]-benzenepropanamide) is a fentanyl analogue that was first detected in Sweden in 2017 and has recently reemerged onto the American illicit drug market. There is little data available on β'-phenylfentanyl effects and toxicokinetics and its metabolism is yet to be studied. We aimed to investigate β'-phenylfentanyl human metabolism to identify potential biomarkers of use. To assist in β'-phenylfentanyl metabolite identification, a list of putative reactions was generated using in silico predictions with GLORYx freeware. β'-phenylfentanyl was incubated with cryopreserved 10-donor-pooled human hepatocytes, analyses were performed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS-MS) and data were processed using a partially automated targeted/untargeted approach with Compound Discoverer. We identified 26 metabolites produced by N-dealkylation, oxidation, hydroxylation, O-glucuronidation, O-methylation and combinations thereof. We suggest β'-phenylnorfentanyl (N-phenyl-N-4-piperidinyl-benzenepropanamide) and further metabolites 1-oxo-N-phenyl-N-4-piperidinyl-benzenepropanamide and 1-hydroxy-N-phenyl-N-4-piperidinyl-benzenepropanamide as major biomarkers of β'-phenylfentanyl use. In silico predictions were mostly wrong, and β'-phenylfentanyl metabolic fate substantially differed from that of a closely related analogue incubated in the same conditions, highlighting the value of the experimental assessment of new psychoactive substance human metabolism. In vivo data are necessary to confirm the present results. However, the present results may be necessary to help analytical toxicologists identify β'-phenylfentanyl-positive cases to provide authentic samples.

Integrating UPLC-HR-MS/MS, Network Pharmacology, and Experimental Validation to Uncover the Mechanisms of Jin'gan Capsules against Breast Cancer.

In the theory of traditional Chinese medicine (TCM), “liver-qi” stagnation and heat-induced toxicity represent the main etiologies of breast cancer. Recently, several TCMs with heat-clearing and detoxification efficacy have shown inhibitory effects on breast cancer. Jin’gan capsules (JGCs), initially approved to treat colds in China, are a heat-clearing and detoxification TCM formula. However, the anticancer activity of JGCs against breast cancer and its underlying mechanisms remain unclear. First, we assessed the antiproliferative activity of JGCs in breast cancer cell lines and evaluated their effects on cell apoptosis and the cell cycle by flow cytometry. Furthermore, we identified the potential bioactive components of JGCs and their corresponding target genes and constructed a bioactive compound–target interaction network by ultra-performance liquid chromatography–high-resolution tandem mass spectrometry (UPLC-HR-MS/MS) and network pharmacology analysis. Finally, the underlying mechanism was investigated through gene function enrichment analysis and experimental validation. We found that JGCs significantly inhibited breast cancer cell growth with IC50 values of 0.56 ± 0.03, 0.16 ± 0.03, and 0.94 ± 0.09 mg/mL for MDA-MB-231, MDA-MB-468, and MCF-7, respectively. In addition, JGC treatment dramatically induced apoptosis and S phase cell cycle arrest in breast cancer cells. Western blot analysis confirmed that JGCs could regulate the protein levels of apoptosis- and cell cycle-related genes. Utilizing UPLC-HR-MS/MS analysis and network pharmacology, we identified 7 potential bioactive ingredients in JGCs and 116 antibreast cancer targets. Functional enrichment analysis indicated that the antitumor effects of JGCs were strongly associated with apoptosis and the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. Western blot analysis validated that JGC treatment markedly decreased the expression levels of p-JAK2, p-STAT3, and STAT3. Our findings suggest that JGCs suppress breast cancer cell proliferation and induce cell cycle arrest and apoptosis partly by inhibiting the JAK2/STAT3 signaling pathway, highlighting JGCs as a potential therapeutic candidate against breast cancer.

UHPLC-HRMS-based serum lipisdomics reveals novel biomarkers to assist in the discrimination between colorectal adenoma and cancer.

The development of a colorectal adenoma (CA) into carcinoma (CRC) is a long and stealthy process. There remains a lack of reliable biomarkers to distinguish CA from CRC. To effectively explore underlying molecular mechanisms and identify novel lipid biomarkers promising for early diagnosis of CRC, an ultrahigh-performance liquid chromatography tandem high-resolution mass spectrometry (UHPLC-HRMS) method was employed to comprehensively measure lipid species in human serum samples of patients with CA and CRC. Results showed significant differences in serum lipid profiles between CA and CRC groups, and 85 differential lipid species (P < 0.05 and fold change > 1.50 or < 0.67) were discovered. These significantly altered lipid species were mainly involved in fatty acid (FA), phosphatidylcholine (PC), and triacylglycerol (TAG) metabolism with the constituent ratio > 63.50%. After performance evaluation by the receiver operating characteristic (ROC) curve analysis, seven lipid species were ultimately proposed as potential biomarkers with the area under the curve (AUC) > 0.800. Of particular value, a lipid panel containing docosanamide, SM d36:0, PC 36:1e, and triheptanoin was selected as a composite candidate biomarker with excellent performance (AUC = 0.971), and the highest selected frequency to distinguish patients with CA from patients with CRC based on the support vector machine (SVM) classification model. To our knowledge, this study was the first to undertake a lipidomics profile using serum intended to identify screening lipid biomarkers to discriminate between CA and CRC. The lipid panel could potentially serve as a composite biomarker aiding the early diagnosis of CRC. Metabolic dysregulation of FAs, PCs, and TAGs seems likely involved in malignant transformation of CA, which hopefully will provide new clues to understand its underlying mechanism.

Evaluation of the in-vitro inhalation uptake of MDMB-4en-PINACA using a smoking simulation chamber

Synthetic cannabinoids are typically sold as herbal smoking mixtures impregnated with psychoactive substances, being the main route of administration through smoking inhalation. In this study, a simulation chamber has been designed for the estimation of the inhalation uptake of synthetic cannabinoids during conventional herb smoking using a dedicated smoking machine. MDMB-4en-PINACA was selected as model compound because of it was one of the most popular synthetic cannabinoids in the late 2020. Silicone tube and Gamble’s solution were employed as simulants of human trachea and interstitial lung fluids, respectively. Simulants were extracted with methanol and the obtained extracts were analysed by liquid chromatography-fluorescence detection with a limit of detection of 0.7 µg L−1. The percentage of lung inhalation uptake was significative, ranged from 24.5 to 34.1 % of the MDMB-4en-PINACA vaporized amount. Moreover, MDMB-4en-PINACA thermal degradation products were identified by liquid chromatography-high resolution tandem mass spectrometry and a major thermolytic pathway was proposed. Thus, the developed smoking simulation chamber is a versatile tool that allows the rapid and simple estimation of the inhalation uptake of synthetic cannabinoids during smoking.

Chiral analysis of glycerol phosphates - can bacteria biosynthesize heterochiral phospholipid membranes?

Phosphatidylglycerol (1,2-diacyl-sn-glycero-3-phospho-glycerol) (PG) is one of the most abundant lipids in bacteria. However, the chirality of the carbon atom on glycerol phosphate is different between the three kingdoms, Archaea, Bacteria, and Eukarya. Archaea membranes consist of phospholipids with glycerol-1-phosphate (G1P) in the S configuration, whereas phospholipids of the other two kingdoms contain glycerol-3-phosphate (G3P) having R stereochemistry. In the present study, GC/MS and LC/MS methods sensitively detected G3P and G1P from four bacterial strains (Bacillus amyloliquefaciens, B. subtilis, Clavibacter michiganensis, and Geobacillus stearothermophilus). Strain selection was carried out based on a GenBank search that revealed bacterial sequences associated with both enzymes involved in glycerol-phosphate synthesis, i.e., glycerol-3-phosphate dehydrogenase and glycerol-1-phosphate dehydrogenase. The detection of G1P and G3P was made by comparing the retention times of synthetic standards with those of analyzed samples. The structures of both glycerol phosphates were confirmed by selected ion monitoring (SIM) at m/z 171.006. The total concentration of G3P and G1P was around 30 µM, with a ratio of G3P to G1P of 4:1. We showed that PG was the most abundant phospholipid in all four bacteria by using the following analytical techniques and chromatographic modes: hydrophilic interaction liquid chromatography (HILIC), reversed-phase high-performance liquid chromatography high-resolution electrospray ionization tandem mass spectrometry (RP-HPLC/HR-ESI tandem MS) in negative and positive ionization modes, and an enzymatic cleavage by phospholipase C. By using chiral chromatography, the presence of both enantiomers in the glycerol backbone of some molecular species of PG was revealed. These results allow us to conclude that the bacteria examined here produce both enantiomer glycerol phosphates.

Elucidation of in Vitro Chlorinated Tyrosine Adducts in Blood Plasma as Selective Biomarkers of Chlorine Exposure.

Chlorine is a widely available industrial chemical and involved in a substantial number of cases of poisoning. It has also been used as a chemical warfare agent in military conflicts. To enable forensic verification, the persistent biomarkers 3-chlorotyrosine and 3,5-dichlorotyrosine in biomedical samples could be detected. An important shortfall of these biomarkers, however, is the relatively high incidence of elevated levels of chlorinated tyrosine residues in individuals with inflammatory diseases who have not been exposed to chlorine. Therefore, more reliable biomarkers are necessary to distinguish between endogenous formation and exogeneous exposure. The present study aims to develop a novel diagnostic tool for identifying site-specific chlorinated peptides as a more unambiguous indicator of exogeneous chlorine exposure. Human blood plasma was exposed in vitro to various chlorine concentrations, and the plasma proteins were subsequently digested by pronase, trypsin, or pepsin. After sample preparation, the digests were analyzed by liquid chromatography tandem mass spectrometry (LC–MS/MS) and liquid chromatography high-resolution tandem mass spectrometry (LC–HRMS/MS). In line with other studies, low levels of 3-chlorotyrosine and 3,5-dichlorotyrosine were found in blank plasma samples in this study. Therefore, 50 site-specific biomarkers were identified, which could be used as more unambiguous biomarkers for chlorine exposure. Chlorination of the peptides TY*ETTLEK, Y*KPGQTVK, Y*QQKPGQAPR, HY*EGSTVPEK, and Y*LY*EIAR could already be detected at moderate in vitro chlorine exposure levels. In addition, the latter two peptides were found to have dichlorinated fragments. Especially, Y*LY*EIAR, with a distinct chlorination pattern in the MS spectra, could potentially be used to differentiate exogeneous exposure from endogenous causes as other studies reported that this part of human serum albumin is nitrated rather than chlorinated under physiological conditions. In conclusion, trypsin digestion combined with high-resolution MS analysis of chlorinated peptides could constitute a valuable technique for the forensic verification of exposure to chlorine.

Unveiling the Phytochemical Profile and Biological Potential of Five Artemisia Species.

The Artemisia L. genus comprises over 500 species with important medicinal and economic attributes. Our study aimed at providing a comprehensive metabolite profiling and bioactivity assessment of five Artemisia species collected from northeastern Romania (A. absinthium L., A. annua L., A. austriaca Jacq., A. pontica L. and A. vulgaris L.). Liquid chromatography–tandem high-resolution mass spectrometry (LC-HRMS/MS) analysis of methanol and chloroform extracts obtained from the roots and aerial parts of the plants led to the identification of 15 phenolic acids (mostly hydroxycinnamic acid derivatives), 26 flavonoids (poly-hydroxylated/poly-methoxylated flavone derivatives, present only in the aerial parts), 14 sesquiterpene lactones, 3 coumarins, 1 lignan and 7 fatty acids. Clustered image map (CIM) analysis of the phytochemical profiles revealed that A. annua was similar to A. absinthium and that A. pontica was similar to A. austriaca, whereas A. vulgaris represented a cluster of its own. Correlated with their total phenolic contents, the methanol extracts from both parts of the plants showed the highest antioxidant effects, as assessed by the DPPH and ABTS radical scavenging, CUPRAC, FRAP and total antioxidant capacity methods. Artemisia extracts proved to be promising sources of enzyme inhibitory agents, with the methanol aerial part extracts being the most active samples against acetylcholinesterase and glucosidase. All Artemisia samples displayed good antibacterial effects against Mycobacterium tuberculosis H37Ra, with MIC values of 64–256 mg/L. In conclusion, the investigated Artemisia species proved to be rich sources of bioactives endowed with antioxidant, enzyme inhibitory and anti-mycobacterial properties.

Quantitative Determination of Whey Protein to Casein Ratio in Infant Formula Milk Powder.

This study was aimed to establish a method for quantitatively determining the ratio of whey protein in the total protein of infant formula by respectively selecting two characteristic peptides from whey protein and casein and calculating the ratio between the characteristic peptides. A nanoliter high-performance liquid chromatography tandem high-resolution mass spectrometry (Q Exactive) was used to simultaneously detect the characteristic peptides of two main whey proteins and two main caseins. The characteristic peptides were calculated, predicted, and screened using the ExPASy website, and peptide information was confirmed by database retrieval after the analysis by using a high-resolution mass spectrometer. The matrix effect was compensated by comparing the characteristic peptides in whey protein with those in casein protein, in which isotope internal standards were not required. The influence of the changes of the protein content in whey protein and casein on the detection method was eliminated by the calculation formula designed by ourselves. In this detection method, the sample was stable in the total protein concentration range of between 0.1 and 0.4 mg/ml. In the simulated industrial processing environment, with desalted whey powder, the recovery rate was 98.63–113.33% under different spiked levels with good reproducibility (RSD<8%). The RSDs of intraday and interday precisions were 2.03–9.35% and 0.61–11.02%, respectively. The different processing procedures of samples had no significant impact on the detection of whey protein (RSD% for milk samples treated by different processing techniques was 2.97%). The quantitation method of whey protein was applied to evaluate the whey protein content in different brands of commercially available milk powder. In summary, the proposed method was applicable for quantitative analysis of whey proteins in the infant formula.

Molecular networking and collision cross section prediction for structural isomer and unknown compound identification in plant metabolomics: a case study applied to Zhanthoxylum heitzii extracts

Mass spectrometry-based plant metabolomics allow large-scale analysis of a wide range of compounds and the discovery of potential new active metabolites with minimal sample preparation. Despite recent tools for molecular networking, many metabolites remain unknown. Our objective is to show the complementarity of collision cross section (CCS) measurements and calculations for metabolite annotation in a real case study. Thus, a systematic and high-throughput investigation of root, bark, branch, and leaf of the Gabonese plant Zhanthoxylum heitzii was performed through ultra-high performance liquid chromatography high-resolution tandem mass spectrometry (UHPLC-QTOF/MS). A feature-based molecular network (FBMN) was employed to study the distribution of metabolites in the organs of the plants and discover potential new components. In total, 143 metabolites belonging to the family of alkaloids, lignans, polyphenols, fatty acids, and amino acids were detected and a semi-quantitative analysis in the different organs was performed. A large proportion of medical plant phytochemicals is often characterized by isomerism and, in the absence of reference compounds, an additional dimension of gas phase separation can result in improvements to both quantitation and compound annotation. The inclusion of ion mobility in the ultra-high performance liquid chromatography mass spectrometry workflow (UHPLC-IMS-MS) has been used to collect experimental CCS values in nitrogen and helium (CCSN2 and CCSHe) of Zhanthoxylum heitzii features. Due to a lack of reference data, the investigation of predicted collision cross section has enabled comparison with the experimental values, helping in dereplication and isomer identification. Moreover, in combination with mass spectra interpretation, the comparison of experimental and theoretical CCS values allowed annotation of unknown features. The study represents a practical example of the potential of modern mass spectrometry strategies in the identification of medicinal plant phytochemical components.

Soil-derived Streptomyces sp. GMR22 producing antibiofilm activity against Candida albicans: bioassay, untargeted LC-HRMS, and gene cluster analysis

Biofilm-forming fungi, Candida albicans, are currently a serious problem in infectious disease cases. Soil bacteria Streptomyces sp. GMR22 have a large genome size and antifungal metabolites against C. albicans, but its potential antibiofilm activity is not clearly defined. The aims of this study were to determine the antibiofilm activity of GMR22 against C. albicans, identify the main constituents of active extracts, and investigate the biosynthesis gene clusters encoding the enzymes related to metabolism pathways. Antifungal and antibiofilm measurements were performed using in vitro assays on C. albicans ATCC 10231. Main constituents of active extracts were analyzed using untargeted Liquid Chromatography tandem High-Resolution Mass Spectrometry (LC-HRMS). RAST software was applied to investigate the gene clusters of the biosynthesis pathways based on whole genome sequences. Chloroform extract of GMR22 has antifungal and antibiofilm properties at 13–420 μg/mL with palmitic acid (C16H32O2, 273.27028 Da), a saturated fatty acid as a major constituent (42.74). Streptomyces sp. GMR22 has 53 subsystems related to fatty acids biosynthesis (Fab) FAS II. The Kyoto Encyclopedia of Gene and Genome map of Fab revealed 10 of 21 (47.6%) gene clusters encode enzymes related to Fab. There were six gene clusters encoding the enzymes related to the hexadecenoic acid (palmitic acid) biosynthesis pathways: 6.4.12; FabD, FabH, FabF, FabG, FabI and 1.14.192. Each enzyme was encoded by 3–14 genes. These results confirmed that soil Streptomyces sp. GMR22 bacterium has remarkable biotechnological potential by producing fatty acids which are mostly palmitic acid as an active antibiofilm agent against C. albicans.

UHPLC-ESI-QqTOF Analysis and In Vitro Rumen Fermentation for Exploiting Fagus sylvatica Leaf in Ruminant Diet.

In recent years, animal husbandry has aimed at improving the conditions of livestock animals useful for humans to solve environmental and health problems. The formulation of animal feeds or supplements based on antioxidant plant compounds is considered a valuable approach and an alternative for livestock productivity. Forest biomass materials are an underestimated source of polyphenolic compounds whose sustainable recovery could provide direct benefits to animals and, indirectly, human nutrition. In this context, an alcohol extract from leaves of Fagus sylvatica L. was first investigated through an untargeted ultra-high-performance liquid chromatography–high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) approach. Then, it was fractionated into a fatty acid-rich and a polyphenolic fraction, as evidenced by total lipid, phenol, and flavonoid content assays, with antiradical and reducing activity positively correlated to the latter. When tested in vitro with rumen liquor to evaluate changes in the fermentative parameters, a significant detrimental effect was exerted by the lipid-rich fraction, whereas the flavonoid-rich one positively modulated the production of volatile fatty acids (i.e., acetate, butyrate, propionate, etc.).

Study on the Absorption and Conduction Properties of Vanisulfane in Tobacco

The purposes of this study were to explore the systemic properties of vanisulfane in tobacco plant and to provide a reference for the rational use of vanisulfane in the field. After the tobacco plants were treated by hydroponics and foliar spraying, the contents of vanisulfane in root and stem leaf were detected by high-performance liquid chromatography tandem high-resolution mass spectrometry (UPLC-HRMS), and the position of vanisulfane in root and stem leaf was real-time observed through fluorescence two-photon confocal microscope. UPLC-HRMS results showed that the contents of vanisulfane in root and stem leaf gradually increased with the extension of processing time, and after 12 h treatment, the contents of vanisulfane in root and stem leaf reached the maximum levels of 31.95 and 0.215 mg/kg, respectively. In addition, fluorescence two-photon confocal microscope results showed that vanisulfane could observe in the root and stem leaf. These results showed that vanisulfane had excellent upward and downward of systemic in tobacco plants, which is helpful to guide a reference for the rational use of vanisulfane in the field.

Profiling Urinary Sulfate Metabolites With Mass Spectrometry.

The study of urinary phase II sulfate metabolites is central to understanding the role and fate of endogenous and exogenous compounds in biological systems. This study describes a new workflow for the untargeted metabolic profiling of sulfated metabolites in a urine matrix. Analysis was performed using ultra-high-performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) with data dependent acquisition (DDA) coupled to an automated script-based data processing pipeline and differential metabolite level analysis. Sulfates were identified through k-means clustering analysis of sulfate ester derived MS/MS fragmentation intensities. The utility of the method was highlighted in two applications. Firstly, the urinary metabolome of a thoroughbred horse was examined before and after administration of the anabolic androgenic steroid (AAS) testosterone propionate. The analysis detected elevated levels of ten sulfated steroid metabolites, three of which were identified and confirmed by comparison with synthesised reference materials. This included 5α-androstane-3β,17α-diol 3-sulfate, a previously unreported equine metabolite of testosterone propionate. Secondly, the hydrolytic activity of four sulfatase enzymes on pooled human urine was examined. This revealed that Pseudomonas aeruginosa arylsulfatases (PaS) enzymes possessed higher selectivity for the hydrolysis of sulfated metabolites than the commercially available Helix pomatia arylsulfatase (HpS). This novel method provides a rapid tool for the systematic, untargeted metabolic profiling of sulfated metabolites in a urinary matrix.

Identification and characterization of Fe3O4/peroxodisulfate advanced oxidation products from sulfameter

Sulfonamides (SAs) are one of the most widely used antibiotics and their residuals in the environment could cause some negative environmental issues. Advanced oxidation such as Fenton-like reaction has been widely applied in the treatment of SAs polluted water. Degradation rates of 95%-99.7% were achieved in this work for the tested 8 SAs, including sulfisomidine, sulfameter (SME), phthalylsulfathiazole, sulfamethoxypyridazine, sulfamonomethoxine, sulfisoxazole, sulfachloropyridazine, and sulfadimethoxine, in the Fe3O4/peroxodisulfate (PDS) oxidation system after the optimization of PDS concentration and pH. Meanwhile, it was found that a lot of unknown oxidation products were formed, which brought up the uncertainty of health risks to the environment, and the identification of these unknown products was critical. Therefore, SME was selected as the model compound, from which the oxidation products were never elucidated, to identify these intermediates/products. With liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS), 10 new products were identified, in which 2-amino-5-methoxypyrimidine (AMP) was confirmed by its standard. The investigation of the oxidation process of SME indicated that most of the products were not stable and the degradation pathways were very complicated as multiple reactions, such as oxidation of the amino group, SO2 extrusion, and potential cross-reaction occurred simultaneously. Though most of the products were not verified due to the lack of standards, our results could be helpful in the evaluation of the treatment performance of SAs containing wastewater.

Determination of glyphosate exposure in the Iberian hare: A potential focal species associated to agrosystems

Glyphosate is the most used herbicide worldwide. It is a small and highly polar pesticide whose physicochemical properties makes its analytical determination difficult. Here, a procedure based on liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) was developed for glyphosate determination in samples of gastric content from wildlife. Iberian hare (Lepus granatensis), a herbivorous mammal species, strongly associated to agrosystems was selected as model species. The procedure involves direct analysis of sample without derivatization or instead of neither further cleaning steps. The procedure was validated by inter-day accuracy and precision studies with gastric content of hare spiked with glyphosate at ecologically relevant concentrations for the species (0.1–6 μg/g), and with 1 μg/g of isotopically labelled internal standard (glyphosate-2-13C,15N). Finally, glyphosate residues in hunted animals from pesticide-treated and pesticide-free areas (n = 75 and 28, respectively), as well as from hares found dead in the field (n = 11) were analysed. The linearity of both standards in extraction solutions and procedural calibration curves with spiked samples was similar, both with determination coefficients (r2) higher than 0.99. Satisfactory recoveries in spiked samples were achieved within the range of 95% to 118% (CV ≤ 20%). The limit of detection of glyphosate in hare gastric content was 0.03 μg/g. Prevalence of glyphosate in hunted animals from pesticide-treated areas ranged between 9 and 22%, increasing to 45% in animals found dead. The glyphosate concentrations detected in the gastric content of hares ranged from 0.11 to 16 μg/g. No residues were detected in animals from pesticide-free areas. In practice, the developed methodology may be particularly useful in the context of research and other work on the exposure in wildlife of one of the most used pesticides nowadays.

Phytochemical and multi-biological characterization of two Cynara scolymus L. varieties: A glance into their potential large scale cultivation and valorization as bio-functional ingredients

Artichoke leaf (Cynarae folium) extracts are used as traditional herbal medicinal products to treat a wide range of human ailments, being widely commercialized as nutraceutical or pharmaceutical preparations. In the current study, the hydromethanolic dried leaf extracts of Cynara scolymus L. var. major Brotero and C. scolymus L. var. redonensis N.H.F. Desp. were phytochemically and biologically investigated. The liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) metabolite profiling showed a complex composition, with phenolic acids (mostly mono- and di-caffeoylquinic acids), flavonoids and sesquiterpene lactones as the most representative classes. The strong antioxidant activity of the two C. scolymus varieties was evidenced in DPPH [64.84–65.21 mg trolox equivalents (TE)/g] and ABTS (86.39–95.55 mg TE/g) radical scavenging, cupric (160.49–171.07 mg TE/g) and ferric (71.47–78.95 mg TE/g) reducing capacity, metal chelating and phosphomolybdenum assays. In addition, the two extracts also displayed anti-enzymatic effects, as assessed in cholinesterase, tyrosinase, glucosidase and amylase tests. Lastly, the artichoke samples (at the concentration of 20 μg/mL) proved a very potent inhibition of the production of several pro-inflammatory cytokines, namely interleukin (IL)-1β [7.55–15.75% of lipopolysaccharide (LPS) + cells], IL-8 (11.72–13.46% of LPS + cells) and tumor necrosis factor (TNF)-α (4.07–10.35% LPS + cells), in LPS-stimulated human neutrophils. Overall, the results of our study indicate that the two C. scolymus varieties could be regarded as a rich source of biologically active compounds, opening thus the perspectives for their future large scale cultivation and valorization as bio-functional ingredients with putative antioxidant, anti-enzymatic and anti-inflammatory effects.