Ultra-high-performance Liquid Chromatography-high-resolution Mass Spectrometry Research Articles

Biotransformation of white and black mustard grains through germination and enzymatic hydrolysis revealed important metabolites for antioxidant properties and cytotoxic activity against Caco-2 cells

Germination and enzymatic hydrolysis are biological processes with well-recognized positive effects on phenolic composition and antioxidant potential. This study aimed to apply those processes to white (Sinapsis alba) and black (Brassica nigra) mustard grains and to analyze the influences on the total phenolic content (TPC); phenolic and peptide profile determined by ultra-performance liquid chromatography–high resolution mass spectrometry (UPLC-HRMS); antioxidant potential (DPPH, ABTS, and FRAP assays); and cytotoxicity against Caco-2, a human colorectal adenocarcinoma cell line. Enzyme combinations for hydrolysis were different for each mustard grain, but for both species, enzymatic hydrolysis and germination showed a positive effect on antioxidant properties. From UPLC-HRMS analysis and molecular network studies, 14 peptides and 17 phenolic compounds were identified as metabolites released from mustard after processes application, which were strongly correlated with increased antioxidant activity. In addition, enzymatic hydrolysis applied in germinated mustard grains for both mustards increased the cytotoxic activity against Caco-2 human colorectal adenocarcinoma cell line.

Margaritaria nobilis L.f. (Phyllanthaceae) Ethanolic Extract: Low Acute Oral Toxicity and Antinociceptive Activity.

Margaritaria nobilis L.f. (Phyllanthaceae), a native Brazilian tree occurring mainly in the Amazon, is used in folk medicine for the treatment of abscesses (bark) and cancer-like symptoms (leaves). The present study evaluates the safety of its acute oral administration and its effects on nociception and plasma leakage. The chemical constitution of the leaf's ethanolic extract is determined by ultra-performance liquid chromatography-high-resolution mass spectrometry (LC-MS. Its acute oral toxicity is evaluated in female rats at a dose of 2000 mg/kg, evaluating the occurrence of deaths and Hippocratic, behavioral, hematological, biochemical, and histopathological changes, as well as food and water consumption and weight gain. Antinociceptive activity is evaluated in male mice with acetic-acid-induced peritonitis (APT) and formalin (FT) tests. An open field (OF) test is performed to verify possible interferences in the animals' consciousness or locomotion. LC-MS analysis shows the presence of 44 compounds classified as phenolic acid derivatives, flavonoids and O-glycosylated derivatives, and hydrolyzable tannins. No deaths or significant behavioral, histological, or biochemical changes are observed in the toxicity assessment. In nociception tests, M. nobilis extract significantly reduces abdominal contortions in APT, demonstrating selectivity for inflammatory components (FT second phase), not interfering in neuropathic components (FT first phase) or consciousness and locomotion levels in OF. Additionally, M. nobilis extract inhibits plasma acetic-acid-induced leakage. These data demonstrate the low toxicity of M. nobilis ethanolic extract, as well as its effectiveness in modulating inflammatory nociception and plasma leakage, possibly related to the flavonoids and tannins present in its composition.

A comparison of feature extraction capabilities of advanced UHPLC-HRMS data analysis tools in plant metabolomics.

Data analysis of ultrahigh performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) is an essential and time-consuming step in plant metabolomics and feature extraction is the fundamental step for current tools. Various methods lead to different feature extraction results in practical applications, which may puzzle users for selecting adequate data analysis tools to deal with collected data. In this work, we provide a comprehensive method evaluation for some advanced UHPLC-HRMS data analysis tools in plant metabolomics, including MS-DIAL, XCMS, MZmine, AntDAS, Progenesis QI, and Compound Discoverer. Both mixtures of standards and various complex plant matrices were specifically designed for evaluating the performances of the involved method in analyzing both targeted and untargeted metabolomics. Results indicated that AntDAS provide the most acceptable feature extraction, compound identification, and quantification results in targeted compound analysis. Concerning the complex plant dataset, both MS-DIAL and AntDAS can provide more reliable results than the others. The method comparison is maybe useful for the selection of suitable data analysis tools for users.

Metabolic profiles of Sri Lankan cassava mosaic virus-infected and healthy cassava (Manihot esculenta Crantz) cultivars with tolerance and susceptibility phenotypes

BackgroundCassava mosaic disease (CMD) of cassava (Manihot esculenta Crantz) has expanded across many continents. Sri Lankan cassava mosaic virus (SLCMV; family Geminiviridae), which is the predominant cause of CMD in Thailand, has caused agricultural and economic damage in many Southeast Asia countries such as Vietnam, Loas, and Cambodia. The recent SLCMV epidemic in Thailand was commonly found in cassava plantations. Current understanding of plant–virus interactions for SLCMV and cassava is limited. Accordingly, this study explored the metabolic profiles of SLCMV-infected and healthy groups of tolerant (TME3 and KU50) and susceptible (R11) cultivars of cassava. Findings from the study may help to improve cassava breeding, particularly when combined with future transcriptomic and proteomic research.ResultsSLCMV-infected and healthy leaves were subjected to metabolite extraction followed by ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS/MS). The resulting data were analyzed using Compound Discoverer software, the mzCloud, mzVault, and ChemSpider databases, and published literature. Of the 85 differential compounds (SLCMV-infected vs healthy groups), 54 were differential compounds in all three cultivars. These compounds were analyzed using principal component analysis (PCA), hierarchical clustering dendrogram analysis, heatmap analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation. Chlorogenic acid, DL-carnitine, neochlorogenic acid, (E)-aconitic acid, and ascorbyl glucoside were differentially expressed only in TME3 and KU50, with chlorogenic acid, (E)-aconitic acid, and neochlorogenic acid being downregulated in both SLCMV-infected TME3 and KU50, DL-carnitine being upregulated in both SLCMV-infected TME3 and KU50, and ascorbyl glucoside being downregulated in SLCMV-infected TME3 but upregulated in SLCMV-infected KU50. Furthermore, 7-hydroxycoumarine was differentially expressed only in TME3 and R11, while quercitrin, guanine, N-acetylornithine, uridine, vorinostat, sucrose, and lotaustralin were differentially expressed only in KU50 and R11.ConclusionsMetabolic profiling of three cassava landrace cultivars (TME3, KU50, and R11) was performed after SLCMV infection and the profiles were compared with those of healthy samples. Certain differential compounds (SLCMV-infected vs healthy groups) in different cultivars of cassava may be involved in plant–virus interactions and could underlie the tolerance and susceptible responses in this important crop.

Effects of Different Storage Conditions on Lipid Stability in Mice Tissue Homogenates.

Lipids are biomolecules involved in numerous (patho-)physiological processes and their elucidation in tissue samples is of particular interest. However, tissue analysis goes hand in hand with many challenges and the influence of pre-analytical factors can intensively change lipid concentrations ex vivo, compromising the results of the whole research project. Here, we study the influence of pre-analytical factors on lipid profiles during the processing of homogenized tissues. Homogenates from four different mice tissues (liver, kidney, heart, spleen) were stored at room temperature as well as in ice water for up to 120 min and analyzed via ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). Lipid class ratios were calculated since their suitability as indicators for sample stability has been previously illustrated. Only approx. 40% of lipid class ratios were unchanged after 35 min, which was further reduced to 25% after 120 min during storage at room temperature. In contrast, lipids in tissue homogenates were generally stable when samples were kept in ice water, as more than 90% of investigated lipid class ratios remained unchanged after 35 min. Ultimately, swift processing of tissue homogenates under cooled conditions represents a viable option for lipid analysis and pre-analytical factors require more attention to achieve reliable results.

Therapeutic Mechanism of Baicalin in Experimental Colitis Analyzed Using Network Pharmacology and Metabolomics.

Baicalin is an important active flavonoid isolated from the roots of Scutellaria baicalensis (S. baicalensis), a well-known traditional Chinese herb used in treating inflammatory bowel disease (IBD). The objectives of this study were to assess the potential benefit of baicalin in experimental colitis, as well as to investigate metabolic biomarkers of experimental colitis in conjunction with network pharmacology. Using a widely utilized network pharmacology technique, baicalin's targets and pathways were predicted. Simultaneously, experimental colitis was induced by intrarectal administration of TNBS. Histopathology examinations were performed to confirm pathological changes. Plasma samples were examined by using an untargeted metabolomics technique based on ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) to screen differential metabolites and associated metabolic pathways. Additionally, network pharmacology and integrated analysis of metabolomics were used to identify the primary targets. Through network pharmacology research, tumor necrosis factor (TNF), interleukin 6 (IL6), serine/threonine-protein kinase (AKT1), and other 7 proteins were found to be the main targets of baicalin against IBD. The untargeted metabolomics results showed that 47 metabolites in glycerophospholipids and sphingolipid metabolism were involved as key pathways in the experimental colitis model group. 19 metabolites, including Sphingomyelin (SM d42:2, SM d42:1, SM d34:1), Lysophosphatidic acids (LPA 18:4), 1-Palmitoylglycerophosphocholine, and 17(18)-EpETE were demonstrated as key metabolites for baicalin to exert effects. Moreover, udp-glucose ceramide glucosyltransferase (UGCG), sphingomyelin synthase 1 (SGMS1), and sphingosine kinase (SPHK1) were predicted as sphingolipids-linked targets of baicalin against experimental colitis by integrative analysis. Based on these results, it implies that sphingolipid metabolism and sphingolipid signaling pathway might be acted as therapeutic mechanism for baicalin against experimental colitis.

Metabolite profiling and bioactivity of Cicerbita alpina (L.) Wallr. (Asteraceae, Cichorieae)

Cicerbita alpina (L.) Wallr. is a perennial herbaceous plant in the tribe Cichorieae (Lactuceae), Asteraceae family, distributed in the mountainous regions in Europe. In this study, we focused on the metabolite profiling and the bioactivity of C. alpina leaves and flowering heads methanol-aqueous extracts. The antioxidant activity of extracts, as well as inhibitory potential towards selected enzymes, involving in several human diseases, including metabolic syndrome (α-glucosidase, α-amylase, and lipase), Alzheimer's disease, (cholinesterases: AChE, BchE), hyperpigmentation (tyrosinase), and cytotoxicity were assessed. The workflow comprised ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS analysis revealed more than 100 secondary metabolites, including acylquinic, acyltartaric acids, flavonoids, bitter sesquiterpene lactones (STLs), such as lactucin, dihydrolactucin, their derivatives, and coumarins. Leaves showed a stronger antioxidant activity compared to flowering heads, as well as lipase (4.75 ± 0.21 mg OE/g), AchE (1.98 ± 0.02 mg GALAE/g), BchE (0.74 ± 0.06 mg GALAE/g), and tyrosinase (49.87 ± 3.19 mg KAE/g) inhibitory potential. Flowering heads showed the highest activity against α-glucosidase (1.05 ± 0.17 mmol ACAE/g) and α-amylase (0.47 ± 0.03). The obtained results highlighted C. alpina as a rich source of acylquinic, acyltartaric acids, flavonoids, and STLs with significant bioactivity, and therefore the taxon could be considered as a potential candidate for the development of health-promoting applications.

A comprehensive study of the effect of drying methods on compounds in Elaeagnus angustifolia L. flower by GC-MS and UHPLC-HRMS based untargeted metabolomics combined with chemometrics

The effect of drying methods on the variations of volatile, semi-volatile, and involatile compounds in Elaeagnus angustifolia L. (E.A.) flower was comprehensively investigated by using gas chromatography-mass spectrometry (GC-MS) and ultrahigh performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) based untargeted metabolomics combined with our recently developed automatic data analysis software AntDAS for the first time. About 21 volatile compounds, 44 semi-volatile compounds, and 17 involatile compounds were identified and showed significant differences among drying methods. Chemometric methods like principal components analysis (PCA), and partial least squares-discriminant analysis (PLS-DA) were used in combination with heatmap analysis to reveal the relationship between compound compositions and drying methods. Finally, it was concluded that room-temperature drying is more conducive to preserving volatile compounds in E.A. flower, and hot air drying is better for preserving semi-volatile and involatile compounds. This work can provide guidelines for industrial processes of E.A. flower.

Temporal metabolic profiling of bone healing in a caprine tibia segmental defect model.

Bone tissue engineering is an emerging field of regenerative medicine, with a wide array of biomaterial technologies and therapeutics employed. However, it is difficult to objectively compare these various treatments during various stages of tissue response. Metabolomics is rapidly emerging as a powerful analytical tool to establish broad-spectrum metabolic signatures for a target biological system. Developing an effective biomarker panel for bone repair from small molecule data would provide an objective metric to readily assess the efficacy of novel therapeutics in relation to natural healing mechanisms. In this study we utilized a large segmental bone defect in goats to reflect trauma resulting in substantial volumetric bone loss. Characterization of the native repair capacity was then conducted over a period of 12 months through the combination of standard (radiography, computed tomography, histology, biomechanics) data and ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) metabolic profiling. Standard metrics demonstrated that samples formed soft callus structures that later mineralized. Small molecule profiles showed distinct temporal patterns associated with the bone tissue repair process. Specifically, increased lactate and amino acid levels at early time points indicated an environment conducive to osteoblast differentiation and extracellular matrix formation. Citrate and pyruvate abundances increased at later time points indicating increasing mineral content within the defect region. Taurine, shikimate, and pantothenate distribution profiles appeared to represent a shift toward a more homeostatic remodeling environment with the differentiation and activity of osteoclasts offsetting the earlier deposition phases of bone repair. The generation of a comprehensive metabolic reference portfolio offers a potent mechanism for examining novel biomaterials and can serve as guide for the development of new targeted therapeutics to improve the rate, magnitude, and quality of bone regeneration.

PS-C19-3: HYDROCHLOROTHIAZIDE WAS NOT ASSOCIATED WITH PHOTOSENSITIVE OR PHOTOTOXIC REACTIONS AND DNA-DAMAGE IN VIVO AND IN VITRO (THE HCTOX-STUDY)

Objective: Pharmacoepidemiologic studies associated the use of HCT with an increased risk of skin cancer, resulting in a decrease of HCT prescriptions, in turn leading to worsening of blood pressure therapy in a significant proportion of patients. However, whether HCT causes skin cancer remains elusive. Hence, we aimed to examine the photosensitive and phototoxic potential of HCT in vivo in a randomized, placebo-controlled trial. To further enlighten the pathophysiologic mechanisms of carcinogenesis and phototoxicity caused by HCT in vitro, we conducted a series of laboratory experiments. Design and method: A randomized, double-blind, placebo-controlled clinical trial to assess the phototoxic properties of HCT was conducted, assigning 30 healthy normotensive adult volunteers in a 2:1 ratio to either HCT 25 mg daily or placebo once daily for 15 days. The skin photosensitivity by phototesting, office blood pressure, serum 25-hydroxyvitamin D (25(OH)D) status and urinary excretion of thymidine-dimers, i.e. cyclobutan-dimers by ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) following whole-body irradiation were assessed. To further assess the pathophysiologic mechanisms of possibly HCT induced photosensitivity, human keratinocytes (HaCaT) were incubated with HCT and then irradiated with UV-B radiation (311 nm one burst of 100 J/cm2). rt-PCR-testing and western blots were performed to analyze reactive oxygen species, inflammation, and carcinogenesis. Results: All 30 participants were adherent to the protocol, which was confirmed by UHPLC-HRMS analysis of serum and plasma. Skin photosensitivity to exposure of UV-A and UV-B radiation did not change in both groups (HCT vs. placebo: MED-UVB change 0.0 J/cm2 vs. 0.2 J/cm2; p = 0.06). No thymidine-dimers were detectable in either group. Systolic blood pressure decreased in both groups (-5.2 mmHg vs. 5.4 mmHg; p = 0.94), as did diastolic blood pressure (-4.3 mmHg vs. -1.9 mmHg; p = 0.34). Serum 25(OH)D increased in both groups (3.1 ng/ml vs. 1.2 ng/ml; p = 0.52). HCT in combination with a single high-intensity bursts of UV-B radiation was not associated with increased expression of inflammatory proteins, chronic inflammation and reactive oxygen species. Conclusions: HCT was not associated with increased photosensitivity for UV-A or UV-B radiation in healthy volunteers. Moreover, no relevant DNA-damages as measured by UHPLC-HRMS were detectable in either group. The combination of HCT treatment and a single burst of UV-B did not increase inflammatory markers. HCT appeared to be safe in healthy volunteers and was not associated with photosensitivity or DNA-damages in vivo.

Quantification and isotope abundance determination of 13C labeled intracellular sugar metabolites with hydrophilic interaction liquid chromatography.

Metabolic flux analysis (MFA) using stable isotope labeled tracers is a powerful tool to estimate fluxes through metabolic pathways. It finds applications in studying metabolic changes in diseases, regulation of cellular energetics, and novel strategies for metabolic engineering. Accurate and precise quantification of the concentration of metabolites and their labeling states is critical for correct MFA results. Utilizing an ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) system, an analytical method for simultaneously quantifying the concentration of sugar metabolites and their mass isotopologue distribution (MID) was developed. The method performs with good linearity and coefficient of determination (R2) > 0.99, while the detection limit ranged from 0.1 to 50 mg L-1. Seven sugar metabolites were detected in a labeled Brevibacterium flavum sample using the method. The detected quantities ranged from 6.15 to 3704.21 mg L-1, and 13C abundance was between 12.77% and 66.67% in the fermentation fluid and 16.28% and 91.93% in the bacterial body. Overall, the method is efficient, accurate, and suitable for analysis of labeled sugar metabolites in 13C MFA studies.

Comparison of Phytochemical Profiles of Wild and Cultivated American Ginseng Using Metabolomics by Ultra-High Performance Liquid Chromatography-High-Resolution Mass Spectrometry

American ginseng (Panax quinquefolius L.) has been recognized as a valuable herb medicine, and ginsenosides are the most important components responsible for the health-beneficial effects. This study investigated the secondary metabolites responsible for the differentiation of wild and cultivated American ginsengs with ultrahigh-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS)-based metabolomic approach. An in-house ginsenoside library was developed to facilitate data processing and metabolite identification. Data visualization methods, such as heatmaps and volcano plots, were utilized to extract discriminated ion features. The results suggested that the ginsenoside profiles of wild and cultivated ginsengs were significantly different. The octillol (OT)-type ginsenosides were present in greater abundance and diversity in wild American ginsengs; however, a wider distribution of the protopanaxadiol (PPD)-and oleanolic acid (OA)-type ginsenosides were found in cultivated American ginseng. Based on the tentative identification and semi-quantification, the amounts of five ginsenosides (i.e., notoginsenoside H, glucoginsenoside Rf, notoginsenoside R1, pseudoginsenoside RT2, and ginsenoside Rc) were 2.3-54.5 fold greater in wild ginseng in comparison to those in their cultivated counterparts, and the content of six ginsenosides (chicusetsusaponin IVa, malonylginsenoside Rd, pseudoginsenoside Rc1, malonylfloralginsenoside Rd6, Ginsenoside Rd, and malonylginsenoside Rb1) was 2.6-14.4 fold greater in cultivated ginseng compared to wild ginseng. The results suggested that the in-house metabolite library can significantly reduce the complexity of the data processing for ginseng samples, and UHPLC-HRMS is effective and robust for identifying characteristic components (marker compounds) for distinguishing wild and cultivated American ginseng.

Global Metabolomics Discovers Two Novel Biomarkers in Pyridoxine-Dependent Epilepsy Caused by ALDH7A1 Deficiency.

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive developmental and epileptic encephalopathy caused by pathogenic variants in the ALDH7A1 gene (PDE-ALDH7A1), which mainly has its onset in neonates and infants. Early diagnosis and treatment are crucial to prevent severe neurological sequelae or death. Sensitive, specific, and stable biomarkers for diagnostic evaluations and follow-up examinations are essential to optimize outcomes. However, most of the known biomarkers for PDE lack these criteria. Additionally, there is little discussion regarding the interdependence of biomarkers in the PDE-ALDH7A1 metabolite profile. Therefore, the aim of this study was to understand the underlying mechanisms in PDE-ALDH7A1 and to discover new biomarkers in the plasma of patients using global metabolomics. Plasma samples from 9 patients with genetically confirmed PDE-ALDH7A1 and 22 carefully selected control individuals were analyzed by ultra high performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). Two novel and reliable pyridoxine-independent diagnostic markers, 6-hydroxy-2-aminocaproic acid (HACA) and an isomer of C9H11NO4, were identified. Furthermore, a possible reaction mechanism is proposed for HACA. This study demonstrates the capability of global metabolomics in disease screening to detect established and novel biomarkers.

Profiling of pyrrolizidine alkaloids using a retronecine-based untargeted metabolomics approach coupled to the quantitation of the retronecine-core in medicinal plants using UHPLC-QTOF

Pyrrolizidine alkaloids (PA) are secondary metabolites of high toxicological relevance. Several PA quantitative methodologies were developed based on a limited number of certified standards, including time consuming solid phase extraction (SPE) purification steps. Herein, we shed light on the variability of PA in herbal extracts and propose a quantification methodology based on ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) for the evaluation of the total PA content as retronecine-equivalents (RE) directly from crude matrices. Particularly in the focus of the investigation were Alkanna spp. (Boraginaceae), which possess a wide range of pharmaceutical properties. In addition, a comparative PA screening of crude and SPE enriched extracts was performed and PA-containing plants from Fabaceae and Compositae families were included to demonstrate universal applicability. In total, 105 PA were identified using HRMSe experiments, specific MS/MS fragmentation PA patterns, a customized in-house library and literature data. Among them, 18 glycosidic PA derivatives were reported for the first time in literature. Using a hierarchical clustering approach, PA distribution in herbal extracts was shown to be family-dependent and significantly different among species. This was further supported by the results of the total PA concentrations, obtained using a retronecine/heliotridine/internal standard-based targeted UHPLC-HRMS quantification method, which varied from 8.64 ± 0.08–3096.28 ± 273.72 μg RE/g extract dry weight in shoots extracts of Alkanna spp. and leaves extracts of Crotalaria retusa L. respectively. Worth mentioning is that the procedure allowed to quantify PA in Alkanna spp. If the procedure based on 35 specific PA recommended by European regulations had been used, results would have been equal to zero for the four species since none were observed in Alkanna spp. Finally, by combining the RE results with the corresponding dereplication results, a customized correction factor for each extract (ranging from 2.12 to 2.48) was assessed leading to a more accurate estimate of the PA content regardless of the molecular weight of each PA. The present methodology will facilitate PA quantification directly from crude extracts and avoid the underestimation the real PA content due to limited availabilty of authentic reference compounds in botanical extracts used in phytomedicines or food supplements/cosmetics.

Identification of etazene (etodesnitazene) metabolites in human urine by LC-HRMS.

Etazene (or etodesnitazene) is a novel and highly active synthetic opioid belonging to the rapidly evolving and emerging group of "nitazenes." Etazene metabolites were identified through analysis of a human urine sample. The sample was obtained from a 25-year-old man who attempted suicide by taking a new psychoactive substances (NPS) cocktail purchased online and was analyzed by ultrahigh performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). Etazene metabolites were predicted with BioTransformer 3.0, and the exact masses were added to the inclusion list. Eight possible metabolites were identified in the urine sample. N- and O-deethylation were identified as the predominant metabolism routes, resulting in M1 (O-deethylated etazene; most abundant metabolite based on the peak area), M2 (N-deethylated etazene), and M3 (N,O-dideethylated etazene) metabolites. Less abundant hydroxylated products of these deethylated metabolites and etazene were also found. Additionally, in the analysis without β-glucuronidase treatment, M1- and M3-glucuronide phase II metabolites were found. As N- and O-deethylated products seem to be the predominant urinary metabolites, the detection of these metabolites in urine can be useful to demonstrate etazene exposure.

Metabolomic Profiles in Starved Light Breed Horses during the Refeeding Process.

Simple SummaryUnwanted horses and other equids continue to be a global issue throughout which the prevalence of malnourished equids persists as a welfare concern. Nutritional studies evaluating the refeeding or rehabilitation process are limited, and little data exists to explain how metabolic function changes during refeeding. Therefore, the objective of this study was to assess changes in metabolite profile (metabolome) of emaciated horses throughout rehabilitation via refeeding. Horses were fed specific diets over the rehabilitation period, and blood samples were collected and analyzed throughout. Comparison of equine within a malnourished state and equine during the refeeding process showed decreases in potentially toxic chemical compounds related to liver, kidney, and muscle function as well as shifts related to changing energy demands. This study concludes that the refeeding and rehabilitation process results in an overall change in the equine metabolome.The large population of emaciated horses continues to be an issue troubling the equine industry. However, little is known regarding the collection of equine metabolites (metabolome) during a malnourished state and the changes that occur throughout nutritional rehabilitation. In this study, ten emaciated horses underwent a refeeding process, during which blood samples were collected for a blood chemistry panel and metabolomics analysis via ultrahigh performance liquid chromatography–high resolution mass spectrometry (UHPLC-HRMS). Significant differences among blood chemistry analytes and metabolite abundance during the critical care period (CCP; Days 1–10 of rehabilitation) and the recovery period (RP; the remainder of the rehabilitation process) were observed. Potentially toxic compounds, analytes related to liver, kidney, and muscle function, as well as energy-related metabolites were altered during the refeeding process. The combination of blood chemistry and metabolomics analyses on starved equine during rehabilitation provide vital biological insight and evidence that the refeeding process has a significant impact on the equine metabolome.

The potential of direct analysis in real time as a forensic tool for carfentanil profiling

Carfentanil, a strong anesthetic, has been misused both as a recreational drug and as an incapacitating weapon due to its high potency. Therefore, chemical profiling of carfentanil aimed at gathering forensic intelligence is important. Previously, we showed that it is possible to create a highly significant synthesis route classification model based on chemical profiling of carfentanil samples by gas chromatography-mass spectrometry (GC–MS) and ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). In this study, we go further and investigate whether Direct Analysis in Real Time (DART) coupled to HRMS can be used for rapid detection of carfentanil, and whether the data from this technique provide enough information to identify the synthesis route used to produce a carfentanil sample. From the DART-HRMS data, we found key components previously identified as synthesis route specific, as well as the active substance. We also created a synthesis-route classification model, indicating that DART-HRMS data provides information that is critical for forensic intelligence gathering.

Determination of free amino acids in Eriocheir sinensis by ultra-high performance liquid chromatography-high resolution mass spectrometry

游离氨基酸不仅是一类重要的营养物质,更与中华绒螯蟹独特的滋味和香味密切相关。对游离氨基酸含量进行测定,能为中华绒螯蟹产品的品质评价和风味研究提供有价值的信息。该工作通过优选提取溶剂、优化仪器测定参数,最终使用5%(v/v)高氯酸水溶液提取目标物,采用XDB-C18色谱柱(100 mm×4.6 mm, 1.7 μm),以0.1%(v/v)甲酸水溶液-乙腈为流动相进行梯度洗脱,在电喷雾正离子电离、选择离子扫描模式下进行检测,外标法定量,建立了超高效液相色谱-高分辨质谱(UHPLC-HRMS)测定中华绒螯蟹中17种游离氨基酸含量的方法。结果表明,17种氨基酸在10.0~200.0 mg/L范围内线性关系良好,相关系数(R2)≥0.9990,仪器检出限为0.3 mg/L,仪器定量限为1.0 mg/L。在中华绒螯蟹可食部分中按三水平进行加标回收试验,各目标物的回收率在78.4%~105.3%之间。通过对仪器和方法精密度进行评估,发现17种氨基酸测定仪器重复性的RSD≤4.2%,方法重复性试验的RSD≤5.2%,再现性试验的RSD≤11.4%,精密度数据均在合理范围内。利用该研究建立的检测方法对20个中华绒螯蟹样本进行了实际样品检测,总结了17种游离氨基酸在蟹肉、蟹膏、蟹黄中的分布特点,验证了该方法是一种前处理简便、选择性好、准确度高的检测技术。

Differential Radiomodulating Action of Olea europaea L. cv. Caiazzana Leaf Extract on Human Normal and Cancer Cells: A Joint Chemical and Radiobiological Approach.

The identification of a natural compound with selectively differential radiomodulating activity would arguably represent a valuable asset in the striving quest for widening the therapeutic window in cancer radiotherapy (RT). To this end, we fully characterized the chemical profile of olive tree leaf polyphenols from the Caiazzana cultivar (OLC), autochthonous to the Campania region (Italy), by ultra-high-performance liquid chromatography–high-resolution mass spectrometry (UHPLC-HR-MS). Oleacein was the most abundant molecule in the OLC. Two normal and two cancer cells lines were X-ray-irradiated following 24-h treatment with the same concentration of the obtained crude extract and were assessed for their radioresponse in terms of micronucleus (MN) induction and, for one of the normal cell lines, of premature senescence (PS). Irradiation of pre-treated normal cells in the presence of the OLC reduced the frequency of radiation-induced MN and the onset of PS. Conversely, the genotoxic action of ionising radiation was exacerbated in cancer cells under the same experimental conditions. To our knowledge, this is the first report on the dual action of a polyphenol-rich olive leaf extract on radiation-induced damage. If further confirmed, these findings may be pre-clinically relevant and point to a substance that may potentially counteract cancer radioresistance while reducing RT-associated normal tissue toxicity.

The role of analytical pharmacotoxicology in adressing the main functions of the National Early Warning System on NPS

In compliance with the European Council Decision 2005/387/JHA, the Italian AntiDrug Policies Department designated the National Center for Addiction and Doping of the Istituto Superiore di Sanità (ISS) to organize and manage the National Early Warning System (NEWS) on New Psychoactive Substances (NPS) to promote a rapid exchange of information on NPS within Italy and between Italy and the EU. The analytical pharmacotoxicology Unit of the Center supports NEWS with screening and confirmation of NPS presence in conventional and non-conventional biological matrices from acute and fatal intoxication cases. Furthermore, yearly, pure standards of new NPS are tested and provided to Laboratories from collaborative centres and proficiency testing on NPS in oral fluid and hair are organized. Different analytical technologies, including high-sensitivity gas chromatography-mass spectrometry (GC-MS) and tandem mass spectrometry (GC-MS-MS), liquid chromatography-tandem mass spctrometry (LC-MS-MS) and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) are used for a screening and confirmation analysis of classic drugs, prescription medicines and new psychoactive substances in and their metabolites in blood, plasma, urine, hair and oral fluid from intoxication cases and clinical trials. Sample preparation involves both liquid-liquid or solid phase extraction. Dilute and shoot methodology is also applied. During 2021 and first months of 2022, around 80 different psychoactive substances, including new synthetic opioids and/or metabolites were identified in urine from 296 spanish patients with a history of opioid use disorder; 12 classic drugs and NPS were detected in hair from 300 mexican pregnant women; cocaine, THC, heroin, methadone, ketamine and mephedrone and metabolites were determined in urine from 76 italian prisoners; JWH-122 and JWH-210, UR-144 were quantified in oral fluid from consumers and finally some cases of intoxications with fentanyl, norfentanyl and analogs were also disclosed. Furthermore, two exercises of Proficiency testing for NPS in oral fluid and hair were organized. Samples from both matrices were prepared in cooperation with Comedical (Trento, Italy). For the 2021 round classic drugs together with 5-fluoro CUMYL-PeGACLONE, 5-fluoro MDMB-PICA, AB-FUBINACA,5F-ADB, Ethylone, Euthylone, Furanylfentanyl, Ocfentanyl, Isobutyryl fentanyl, Fentanyl and Isotonitazene were included. For the 2022 round, classic drugs plus 5-MMPA, Methoxpropamine, Brorphine, Butonitazene, Etodesnitazene, Flunitazene, 4-fluoro MDMB-BUTICA, 5-fluoro CUMYL—PICA, 5-fluoro EDMB-PICA, 3-Methylmethcathinone, ADB-4en-PINACA, MDMB-4en-PICA were comprises. About 20 different pharmacotoxicological national laboratories participated in the exercised with very variable quali-quantitative results. Although the NPS phenomenon is not uniform, it is well known as a global problem, concerning all the region of the world. Due to the mutating nature of the phenomenon, we believe that investments in toxicological and forensic analytical data sources are strongly needed. Differently from the past when psychotropic drugs occupied the fingers of two hands, in the 21th century, more than 1000 NPS have been identified. Not all of them found the same diffusion in the illicit market, but many of them are temporary adulterant of substitute of controlled compounds or finally fake drugs. The values of accurate and immediately available toxicological and forensic data are crucial to the implementation of national and international early warning system on NPS. Indeed, the drug related severe intoxications and fatalities are seldom associated with the consumption of one substance alone. Drug consumption patterns are often characterized by polyconsumption of both classical drugs and NPS and laboratories supporting emergency rooms, forensic toxicologies and police forces must be enabled to identify the substance/s causing the acute or chronic intoxication or a fatality.