Quadrupole Orbitrap High-resolution Mass Research Articles

Skin mucus metabolomics provides insights into the interplay between diet and wound in gilthead seabream (Sparus aurata)

The molecular processes underlying skin wound healing in several fish species have been elucidated in the last years, however, metabolomic insights are scarce. Here we report the skin mucus metabolome of wounded and non-wounded gilthead seabream (Sparus aurata) fed with silk fibroin microparticles, a functional additive considered to accelerate the wound healing process. The three experimental diets (commercial diet enriched with 0 mg (control), 50 mg or 100 mg of silk fibroin microparticles Kg−1) were administered for 30 days and thereafter, a skin wound was inflicted. Skin mucus was collected on day 30 of feeding and 7 days post-wounding and subjected to metabolomic analysis by Ultra Performance Liquid Chromatography coupled with a high-resolution quadrupole-orbitrap mass spectrometry. The most enriched metabolite class was amino acids and derivatives, followed by nucleotides, nucleosides and analogues and carbohydrates and their derivatives. Metabolomic profiles revealed that the diet had a more profound effect than wounding in skin mucus. Metabolic pathway analysis of significantly affected metabolites revealed perturbations in the aminoacyl t-RNA biosynthesis in the skin. In particular, skin wound resulted in a decreased methionine level in mucus. Further, silk fibroin supplementation increased methionine level in skin mucus, which correlated with several wound morphometric parameters that characterized the epithelial healing capacity in seabream. The results provided new insight into the physiological consequences of skin wounds and how these processes could be influenced by dietary manipulation.

Identification of the active compounds and their mechanisms of medicinal and edible Heigen based on UHPLC-Q-Exactive Orbitrap MS and network pharmacology

Heigen is the root and rhizomes of Duhaldea Nervosa Anderberg., a traditional medicine food homology herb used to treat rheumatoid arthritis in China,but the potential active components and the underlying mechanism have not been clarified. In this study, the chemical compounds and the components absorbed into the blood from Heigen were assessed using ultra-high performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry. 72 chemical constituents were identified in Heigen, 13 blood prototypical constituents, and 9 metabolites were found in serum samples after intragastric administration of Heigen extracts to rats. Then, using network pharmacology and molecule docking, 10 compounds were assumed to be potentially key active compounds against RA, which may be via the multi-component, multi-target and multi-pathway interaction mechanism of Heigen in the treatment of RA, and also provides a pharmacological basis for the treatment of RA.

Degradation of Aflatoxin B1 in Peanut Oil by Ultraviolet-LED Cold-Light Irradiation and Structure Elucidation of the Degradation Products.

This study aimed to determine the efficiency of ultraviolet (UV)-LED cold light treatment on the degradation of aflatoxin (AF)B1 in peanut oils. The peanut oil samples obtained from different places in China and abroad were determined for AFB1 degradation efficiency of the UV-LED cold-light irradiation method. The degradation products were analyzed by ultra-high performance liquid chromatography coupled to quadrupole orbitrap high-resolution mass spectrometry (UPLC-Q-Exactive MS). The results indicated that the AFB1 content in all peanut oil samples decreased rapidly after 5 min of irradiation. Four main photodegradation products (C18H16O7, C17H14O7, C17H14O7, and C17H14O8) were identified using the established LC-MS method. Their chemical structures were postulated based on the LC-MS data. Also, the degradation pathways were proposed based on the data obtained. Oxidation and reduction reactions were mainly responsible for AFB1-decomposition. The reactions occurred at the furan and lactone rings. These findings demonstrated that UV-LED cold-light irradiation was an effective method for treating AFB1- contaminated peanut oil.

Simultaneous determination of cordycepin and its metabolite 3'-deoxyinosine in rat whole blood by ultra-high-performance liquid chromatography coupled with Q Exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometry and its application to accurate pharmacokinetic studies.

Cordycepin from Cordyceps possesses excellent pharmacological properties, including anti-inflammation and anti-tumor effects, therefore representing a potential alternative medicine. However, doubts about the pharmacokinetic results of cordycepin had been raised in the previous study due to its rapid deamination. The organic solvent methanol was immediately added to terminate the degradation of cordycepin in anticoagulated blood samples and enable the accurate evaluation of pharmacokinetics in vivo. A sensitive and selective ultra-high-performance liquid chromatography coupled with Q Exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometry method was developed and validated to simultaneously determine cordycepin and its deamination metabolite 3'-deoxyinosine using 2-chloroadenosine as an internal standard in rat whole blood. The calibration curves of cordycepin and 3'-deoxyinosine showed excellent linearity within the concentration range of 1.05-10 000.00 ng/ml with acceptable accuracy, precision, selectivity, recovery, matrix effect, and stability. This method was successfully applied to the pharmacokinetic study of cordycepin and its metabolite in rat blood. The effect of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride on the pharmacokinetics of cordycepin was investigated. In summary, the reliable pharmacokinetic parameters of cordycepin and its deamination metabolite 3'-deoxyinosine in rat blood were successfully elucidated. Erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride considerably prolonged the half-life of cordycepin in vivo.

Investigation of the protection from liver injury and pharmacokinetics of baijiu with fermented ginseng in rat and zebrafish models

Ginsenosides in Shen Quan baijiu (SQJ) with fermented ginseng were identified by ultrahigh-performance liquid chromatography coupled to quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap MS). Their pharmacokinetic behavior in rats and effects on zebrafish liver were investigated, and their anti-alcoholic and liver-protecting ingredients in SQJ were further explored. Twenty rats were randomly divided into five groups and administered with different samples. Serum samples were collected at different time points within 48 h, and the ethanol content in the rat serum was detected by gas chromatography (GC). The ginsenosides of SQJ and commercial ginseng baijiu were identified by UHPLC-Q-Exactive Orbitrap MS to explore their differential components. An alcoholic liver disease (ALD) zebrafish model was established for the detection of malondialdehyde (MDA) content and glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) activities. For the rat model, ethanol was not detected in the serum of the SQJ group at the 3rd h. In addition, the half-life of alcohol metabolism and the time to reach the maximum ethanol concentration in serum were both short. For the zebrafish model, the active ingredients in SQJ significantly enhanced the GSH-PX and SOD activities and reduced the MDA production in the liver. MS results revealed that the most abundant component of SQJ was ginsenoside Rg5/Rk1. Compared with those in commercial ginseng baijiu, the types of ginsenoside compound and the content of rare ginsenosides in SQJ increased significantly, especially ginsenoside Rg5/Rk1. This work was the first to use two biological models, rat and zebrafish. The findings serve as a basis for elucidating the protective mechanism of fermented ginseng components against ALD.

Rapid separation and characterization of the in vitro metabolites of moscatilin by ultra-high performance liquid chromatography coupled to hybrid quadrupole orbitrap tandem mass spectrometry.

Moscatilin, a bioactive ingredient isolated from Dendrobium moscatum, has been demonstrated to have excellent anti-cancer activity. The goals of the present study were to investigate the metabolic profiles of moscatilin and to identify and characterize its metabolites. In vitro studies were performed by incubating moscatilin (10 μM) with rat, dog, monkey, and human liver microsomes (0.5 mg protein/ml) to generate the metabolites. An analytical method of liquid chromatography combined with hybrid quadrupole orbitrap high-resolution mass spectrometry in full mass/data-dependent tandem mass spectrometry scan was utilized to separate and identify the metabolites in accordance with their accurate masses, formulas, and tandem mass spectrometry fragment ions determination. A total of six phase I metabolites were detected and structurally characterized. The phase I metabolic pathways of moscatilin were hydroxylation, demethylation, and dehydrogenation. In glutathione-supplemented liver microsomes, nine glutathione conjugates were detected and identified. Our results demonstrated that moscatilin was susceptible to bioactivation with the result of ortho quinone and quinone-methide intermediates. The present study provided an overview of the in vitro metabolic profiles of moscatilin, which will aid in the understanding of the efficacy and safety of this active compound.

Effect of Germination on the Avenanthramide Content of Oats and Their in Vitro Antisensitivity Activities.

In this study, a method, based on an ultraperformance liquid chromatography coupled with high-field quadrupole orbitrap high-resolution mass spectrometry (UHPLC-QE-HF-HRMS) platform, was established for the trace determination of three major avenanthramides (AVNs). The MS conditions for determining the AVNs were optimized, and the cracking methods of avenanthramides were analyzed. The linear range of the results and the correlation coefficient were 1–2000 μg/L and >0.996, respectively. Further, the established method was employed for the determination of the AVN contents of oats at different germination times, and the results indicated that the AVN contents of Zaohua and Bayou oats increased 19.26 and 6.09 times, respectively, after germination. The total AVN content of both oat varieties reached a maximum on the fifth day of germination (153.51 ± 4.08 and 126.30 ± 3.33 μg/g for the Zaohua and Bayou oats, respectively). Furthermore, this study investigated the antiallergic and antioxidant activities of the germinated oats via hyaluronidase inhibition and 2,2-diphenyl-1-picrylhydrazyl (DPPH)-scavenging assays. The antiallergic and DPPH-scavenging abilities of the ungerminated forms of both oat varieties were weaker. However, on the fifth day of germination, the inhibition rate of anthranilamide hyaluronidase reached 72.7% and 67.3% for the Zaohua and Bayou oat varieties, respectively. The antiallergic abilities of the oats increased significantly on the fifth day of germination in terms of their antiallergic capacities and DPPH clearance (82.67% and 77.64% for the Zaohua and Bayou oats, respectively), and the two indicators exhibited similar trends. These findings demonstrated that AVNs exhibit good antisensitivity and antioxidation properties, and the antisensitivity effect correlated positively with the AVN content.

An Improved Isotope Labelling Method for Quantifying Deamidated Cobratide Using High-Resolution Quadrupole-Orbitrap Mass Spectrometry.

Protein deamidation can severely alter the physicochemical characteristics and biological functions of protein therapeutics. Cobratide is a non-addictive analgesic with wide clinical acceptance. However, the Asn residue at position 48 from the N-terminus of the cobratide amino acid sequence (N48) tends to degrade during purification, storage, and transport. This characteristic could severely affect the drug safety and clinical efficacy of cobratide. Traditional methods for quantitating deamidation reported in previous research are characterised by low efficiency and accuracy; the quality control of cobratide via this method is limited. Herein, we developed an improved 18O-labelling method based on the detection of a unique peptide (i.e., the protein fragment of cobratide containing the N48 deamidation hotspot after enzymolysis) using an Orbitrap high-resolution mass spectrometer to quantify deamidated cobratide. The limits of detection and quantification of this method reached 0.02 and 0.025 μM, respectively, and inter- and intra-day precision values of the method were <3%. The accuracy of the 18O-labelling strategy was validated by using samples containing synthesised peptides with a known ratio of deamidation impurities and also by comparing the final total deamidation results with our previously developed capillary electrophoresis method. The recoveries for deamidation (Asp), deamidation isomerisation (iso-Asp), and total deamidation were 101.52 ± 1.17, 102.42 ± 1.82, and 103.55 ± 1.07, respectively. The robustness of the method was confirmed by verifying the chromatographic parameters. Our results demonstrate the applicability of the 18O-labelling strategy for detecting protein deamidation and lay a robust foundation for protein therapeutics studies and drug quality consistency evaluations.

Renshen Baidu Powder Attenuated Intestinal Inflammation and Apoptosis in Ulcerative Colitis Rats through the Inhibition of PI3K/AKT/NF-κB Signaling Pathway.

Objective Renshen Baidu Powder (RBP) is a famous classic compound of traditional Chinese medicine (TCM) and is commonly used for treating ulcerative colitis (UC). However, the pharmacological mechanism of RBP in treating UC remains unclear. This study investigates the possible mechanism of RBP for UC treatment by network pharmacological analysis and rat validation. Methods First, the main chemical constituents of RBP were identified using ultrahigh-performance liquid chromatography quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-HRMS). Then, we obtained targets of identified compounds from the SwissTargetPrediction database and targets associated with UC from GeneCards database. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the metabolism-related signaling pathways affected by RBP. Hematoxylin-eosin (HE) staining was used to observe the pathological change of colon in UC rats after treating RBP, and terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP Nick end labeling (TUNEL) staining was used to detect apoptosis after RBP treatment. The enzyme-linked immunosorbent assay (ELISA) was employed to evaluate cytokine levels of TNF-α, IL-1β, and IL-6. The protein expressions of Bax, Bcl-2, PI3K, AKT, and NF-κB in colonic tissue were detected using immunohistochemistry (IHC). Real-time quantitative polymerase chain reaction (RT-QPCR) was employed to evaluate mRNA expression of PI3K, AKT, and NF-κB. Results We found a total of 24 main compounds and 329 potential targets related to UC. According to KEGG results, 3 main pathways were identified as responsible for UC, including PI3K-AKT, HIF-1, and VEGF signaling pathway. Animal experiments showed that RBP treatment significantly attenuated colon damage in rats with UC. Mechanistically, RBP could inhibit PI3K/AKT/NF-κB pathway; decrease cell apoptosis; and downregulate the expression of TNF-α, IL-1β, and IL-6. Conclusions This study demonstrated that RBP may exert anti-inflammatory and antiapoptotic therapeutic benefits in UC by regulating the PI3K/AKT/NF-κB signaling pathways, providing a scientific basis for understanding the mechanism of RBP against UC.

Rapid analysis of serum components and metabolites of Sanzi San by high performance liguid chromatography-quadrupole/ electrostatic field orbitrap high resolution mass spectrometry

蒙药三子散由诃子、川楝子、栀子3味药材等比例组成,其临床用药主要采用口服给药方式,药物在体内的吸收、分布、代谢、排泄过程与药物发挥药理作用和疗效的产生密切相关,因此考察灌胃给药后的入血成分有助于阐明三子散的药效物质基础。研究采用血清药物化学研究思路,将Wistar大鼠分成空白组和给药组,给药组给予三子散水提物,腹主动脉取血,离心制备血清样品,采用高效液相色谱-四极杆/静电场轨道阱高分辨质谱(HPLC-Q/Orbitrap HRMS),在SHIMADZU GIST C18色谱柱(150 mm×4.6 mm, 5 μm)上进行色谱分离;以甲醇和0.1%(v/v)甲酸水溶液为流动相进行梯度洗脱,柱温30 ℃,流速0.5 mL/min,进样量10 μL,采用加热电喷雾电离(HESI)源,正、负离子同时扫描。通过比对三子散含药血清和空白血清的图谱差异,查阅数据库、各类成分体内代谢途径、三子散成分的相关文献,采用Xcalibur 3.0软件进行峰提取、峰匹配等质谱数据处理,结合Compound Discover 3.0软件对化合物代谢途径的预测分析和裂解规律的推断,解析三子散水提液经大鼠灌胃后血清中的原型成分和代谢产物。结果表明,在给药大鼠血清样品中鉴定出55种入血成分,其中41种原型成分,14种代谢产物。入血的原型成分主要为鞣质类、环烯醚萜类和小分子酚酸类。该研究较为全面地阐释了三子散水提液在大鼠血中的移行成分,有助于揭示三子散的药效物质基础,为该药的临床应用提供参考。

Transcriptome and metabolite profiling analyses reveal the molecular mechanism underlying the characteristic accumulation of anthocyanins and flavonols in Fritillaria unibracteata and F. delavayi.

As an important medicinal herb with high profit, Sichuan Fritillaria is on the verge of extinction because of unreasonable exploitation, but the information on flavonoids which possess diverse medicinal activities was almost unknown. Here, two representative Sichuan Fritillaria species (F. unibracteata and F. delavayi) were studied with UHPLC-Q-Orbitrap HRMS (ultra-high performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry). Anthocyanins mainly accumulate in flowers especially in F. unibracteata (8.22 mg/g; mainly consisting of cyanidin 3-O-rutinoside), with trace amounts in leaves and stems, but to the lowest levels in bulbs. Flavonols accumulates considerably in all tissues of the two Fritillaria species except bulbs, to the highest level in the flower of F. delavayi (28.94 mg/g; mainly consisting of quercetin glycosides). Further transcriptomic and RT-qPCR (real-time quantitative Polymerase Chain Reaction) analysis revealed the anthocyanin-specific biosynthetic unigenes displayed the highest expression levels in the flower of F. unibracteata while most of flavonol-specific biosynthetic unigenes showed the highest transcript abundance in the flower of F. delavayi, but to the lowest expression levels in the bulbs of the two species, accounting for the tissue- and species-specific accumulation of anthocyanins and flavonols. Moreover, the functions of FcMYB90, FcTT8 and FcMYB4 was carefully studied in tobacco. These results prove that several MYB and bHLH transcription factors coordinately controlled the direction of the phenylpropanoid metabolic flux which finally resulted in the characteristic accumulation pattern of anthocyanins and flavonols in Sichuan Fritillaria. This work provides a good foundation and novel insights for the further industrial development, resource utilization and breeding of Sichuan Fritillaria.

Establishment and validation of analytical methods for 15 hazardous drugs by UPLC-Q/Orbitrap-HRMS

BackgroundCytotoxic drug residues in pharmacy intravenous admixture services (PIVAS) have always been a major problem for pharmaceutical workers and the PIVAS environment,which is not only pollutes the PIVAS environment, but also causes serious harm to the life and health of the staff. This study aimed to establish an ultra-high performance liquid chromatography quadrupole orbitrap high resolution mass spectrometry (UPLC-Q/Orbitrap-HRMS) method for the rapid detection and monitor of 15 cytotoxic drugs.MethodsUPLC-Q/Orbitrap-HRMS method was used to establish a rapid detection method for 15 cytotoxic drugs such as cytarabine, gemcitabine and so on. The daily precision and accuracy of this method were verified by injecting four concentrations of standard solution on the same day, and the same four concentrations of standard solution were injected within three days respectively to verify the daily precision of this method. The signal-to-noise ratio (SNR) of 10:1 was calculated as the limit of quantity. The mixed standard solution of 15 cytotoxic drugs with concentrations of 0.5, 1, 3, 10, 30, 100, 300, and 1,000 ng/mL was configured and detected by this method for linearity and range.The stability of this method was investigated using a mixture of 15 drugs (15MIX) standard solutions at high concentration (300 ng/mL) and low concentration (10 ng/mL) at room temperature for 12 and 24 hours, respectively. A standard solution of each drug, 15MIX and blank solution were taken to verify the exclusivity of the method.ResultsThe results showed that the method had good specificity, and the intraday precision of all drugs was less than 10% and the intraday precision was less than 15%. At the same time, the standard curve had good linearity, R2 was greater than 0.99, and the limit of quantification of most drugs was about 1 ng/mL.ConclusionsIn this study, an UPLC-Q/Orbitrap-HRMS method was established for the rapid detection of 15 cytotoxic drugs, providing technical support for the monitoring of cytotoxic drug residues in PIVAS, which is of great significance for environmental contamination mornitoring as well as occupational exposure alert.

Multi-residue analysis of 206 pesticides in grass forage by the one-step quick, easy, cheap, effective, rugged, and safe method combined with ultrahigh-performance liquid chromatography quadrupole orbitrap mass spectrometry.

A novel method for detecting pesticide multi-residue in grass forage (alfalfa and oat) was established based on the one-step automatic extractionand purification technology of quick, easy, cheap, effective, rugged, and safe combined with ultrahigh-performance liquid chromatography quadrupole Orbitrap high-resolution mass spectrometry. The crushed sample was extracted with acetonitrile with 1% acetate, followed by a cleanup step with a primary-secondary amine, octadecylsilane, and graphitized carbon black. The extraction and purification were carried out using the one-step automatic pretreatment equipment. The target pesticides were acquired in positive ion electrospray ionization mode and full scan/data dependent secondary scan mode. The calibration curve shows good linearity over the corresponding concentration range, with the coefficient of determination greater than 0.99. The screening detection limits were 0.5-50μg/kg, and the limit of quantification for the 206 pesticides was set at 1-50μg/kg. At the spiking levels of one, two, and 10 times of limit of quantification, more than 95% of pesticides had recovery between 70-120%, with a relative standard deviation ≤20%. The method was proved to be simple, rapid, high-sensitivity, and could be routinely used for the high throughput screening and quantitative analysis of pesticide residues in alfalfa and oat.

Chemical Constituent Analysis of Ranunculus sceleratus L. Using Ultra-High-Performance Liquid Chromatography Coupled with Quadrupole-Orbitrap High-Resolution Mass Spectrometry.

Ranunculus sceleratus L.(RS) has shown various pharmacological effects in traditional Chinese medicine. In our previous study, the positive therapeutic effect on α-naphthylisothiocyanate induced intrahepatic cholestasis in rats was obtained using TianJiu treatment with fresh RS. However, the chemical profile of RS has not been clearly clarified, which impedes the research progress on the therapeutic effect of RS. Herein, an ultra-high performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) method was developed to rapidly separate and identify multiple constituents in the 80% methanol extract of RS. A total of sixty-nine compounds (19 flavonoids, 22 organic acids, 6 coumarins, 4 lignans, 14 nitrogenous compounds, and 4 anthraquinones) were successfully characterized. A total of 12 of these compounds were unambiguously identified by standard samples. Their mass spectrometric fragmentation pathways were investigated. It is worth noting that flavonoids and lignans were identified for the first time in RS. In this study, we successfully provide the first comprehensive report on identifying major chemical constituents in RS by UHPLC-Q-Orbitrap HRMS. The obtained results enrich the RS chemical profile, paving the way for further phytochemical study, quality control, and pharmacological investigation of RS.

Integrated Transcriptomic and Metabolomic Analyses Reveal the Mechanisms Underlying Anthocyanin Coloration and Aroma Formation in Purple Fennel.

The color and aroma are the significant traits of vegetables and fruits, but the metabolic and molecular mechanisms underlying anthocyanin accumulation and aroma formation remain almost unknown in fennel (Anethum foeniculum L.), which is a crucial vegetable crop and grown widely for aromatic leaves and bulbs. Here, ten major anthocyanins identified and quantified by ultra-high performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) were mainly responsible for the coloration of purple fennel leaf. With the application of GC-MS, it was found that the reduced volatile phenylpropanoids including isoeugenol, trans-isoeugenol, and apiol chiefly account for the characteristic aroma changes of the purple fennel. Moreover, the characteristic anthocyanin coloration and aroma formation in purple fennel were systematically studied with the integrated transcriptomics and metabolomics. The critical genes associated with the biosynthesis and regulation of anthocyanins and volatile phenylpropanoids were isolated and studied carefully in transiently transfected tobacco cells and transgenic tomato plants. Together with the results of UHPLC-Q-Orbitrap HRMS, RT-qPCR, and yeast two hybrid (Y2H), it is proved that the metabolic flux redirection of phenylpropanoid pathway primarily regulated by a functional MYB-bHLH-WD40 complex consisting of AfTT8, AfMYB7, and AfTTG1 accounts for the characteristic anthocyanin coloration and aroma formation in purple fennel leaf. The systematic understanding of the anthocyanin accumulation and aroma formation will assist in the improvement of fennel resource utilization and breeding.

Evaluation of potential thiamazole exposure of owners of orally treated hyperthyroid cats.

The objective of this study was to evaluate the presence of traces of thiamazole in the urine of owners of hyperthyroid cats treated with antithyroid drugs. Urine was collected from 24 owners of hyperthyroid cats, five human patients treated with thiamazole and five healthy humans without any contact with antithyroid drugs. All owners of hyperthyroid cats were asked to fill out a questionnaire. Urine of hyperthyroid cats was collected by spontaneous micturition. All urine samples were stored at -20°C until analysis by ultra-high-performance liquid chromatography coupled to high-resolution quadrupole Orbitrap mass spectrometry. These owners were assessed to have a lot of contact with their cat. Adherence to antithyroid medication handling guidelines was rather poor. High concentrations of thiamazole were detected in all feline samples (median concentration 2818 ng/ml; range 104-15,127) and in the urine of all human patients treated with thiamazole (median concentration 4153 ng/ml; range 1826-5009). No thiamazole was detected in the urine of owners of hyperthyroid cats (limit of detection 3.88 ng/ml; limit of quantification 11.75 ng/ml). The results regarding the potential exposure of owners of hyperthyroid cats to antithyroid drugs are reassuring. Nevertheless, prudence is still warranted when administering antithyroid drugs. Whether these results can be extrapolated to the use of transdermal application requires further investigation.

The circulating plasma metabolome of Neoparamoeba perurans-infected Atlantic salmon (Salmo salar)

Metabolomics can provide insights into the dynamic small-molecule fluctuations occurring in response to infection and has become a valuable tool in studying the pathophysiology of diseases in recent years. However, its application in fish disease research is limited. Here, we report the circulating plasma metabolome of Atlantic salmon (Salmo salar) experimentally infected with Neoparamoeba perurans—the causative agent of amoebic gill disease (AGD). Plasma samples were collected from fish with varying degrees of infection inferred from an external gross morphological score of gill pathology (i.e., gill score [GS] 1 -- GS3), where a higher GS indicates advanced infection stage. Uninfected fish (GS0) served as the control. Typical pathologies associated with AGD infection, such as hyperplastic lesions and lamellar fusion, were evident in infected gill samples. Plasma metabolites were identified by ultra-performance liquid chromatography coupled with a high-resolution quadrupole-orbitrap mass spectrometer. Identification of compounds were performed at four levels of certainty, where level 1 provided the most accurate compound identity. A total of 900 compounds were detected in the samples of which 143 were annotated at level 3, 68 on level 2b, 74 on level 2a, and 66 on level 1. Versus GS0, GS1 showed the highest number of significantly affected metabolites (104), which decreased with a higher GS. Adrenaline and adenosine were the two Level 1 compounds significantly affected by AGD regardless of GS, with the former increasing and the latter decreasing in infected fish. Hippuric acid significantly increased in GS1 and GS2, while the tryptophan metabolite indole-3-lactic acid decreased in response to the initial stage of infection but returned to basal levels at a higher GS. There were ten significantly affected metabolic pathways: Eight of which were significantly downregulated while two were downregulated in GS1 relative to GS0. The super-pathway of purine nucleotide salvage was enriched both within the upregulated metabolites in GS1vsGS0 and the down-regulated metabolites in GS3vsGS1. This is the first report on the circulating plasma metabolome of AGD infected salmon, and the results show that low infection levels resulted in a more dramatic metabolomic dysregulation than advanced infection stages. The metabolites identified are potential biological markers for the systemic physiological impact of AGD.

Rapid determination of 93 banned industrial dyes in beverage, fish, cookie using solid-supported liquid–liquid extraction and ultrahigh-performance liquid chromatography quadrupole orbitrap high-resolution mass spectrometry

Banned industrial dyes are composed of a large number of chemicals with diverse physical and chemical properties, making their simultaneous determination a challenging task. A one-step extraction and purification of 93 banned industrial dyes from beverage, fish and cookie sample methods was proposed by using solid supported liquid–liquid extraction (SLE). The extract was analyzed by ultrahigh-performance liquid chromatography quadrupole orbitrap high–resolution mass spectrometry (UPLC-Q-Orbitrap-HRMS). The quantitative and qualitative mode adopts Q-Orbitrap-HRMS full scan MS (full scan MS1) and data-dependent MS/MS (dd-MS2) acquisition mode. The mass resolution was screened under 70,000 FWHM for full-scan MS1 and 35,000 FWHM for dd-MS2. Linearity was observed in the range of 0.01 ∼ 0.5 μg/mL and the limits of quantification were 0.04 ∼ 0.2 mg/kg for 93 dyes. The average recoveries were 70.5–105.8%, with RSD ≤ 10%. The proposed method has the ability to simultaneously screen many banned dyes in foods with high throughput, sensitivity and reliability.

Characteristic fragmentations of nitroaromatic compounds (NACs) in Orbitrap HCD and integrated strategy for recognition of NACs in environmental samples

Nitroaromatic compounds (NACs) are high of concern due to their mutagenicity, and carcinogenicity to organisms. Here, we attempted to establish a novel searching-validation-evaluation workflow that is tailored to recognize unknown NACs in environmental samples using liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry (LC-Orbitrap-HRMS). We studied the fragmentation process of NAC standards in Orbitrap higher-energy collision dissociation (HCD) cells and observed that the mass loss of NO was the most prevalent among all NAC standards at both low and medium levels of collision energy. Thus, neutral loss of NO was considered as a diagnostic fragment of nitro groups and was used to screen out NACs in environmental samples. This technique is mass-loss-dependent, which enhances the recognition efficiency of NACs. Candidates exported from the PubChem compound database were further evaluated to obtain possible structures. This strategy was applied for the analysis of 24 surface soil, and we tentatively discovered two novel NACs in the analyzed samples. The semi-quantification results demonstrated that the concentrations of novel NACs were comparable to those of the ten targeted NACs in soil samples. This study provides an integrated strategy for the recognition of known and unknown NACs, which could be extended to other environmental matrices.

Pharmacokinetics and anti-liver fibrosis characteristics of amygdalin: Key role of the deglycosylated metabolite prunasin

BackgroundAmygdalin (Amy) is a cyanoside and is one of the chief active ingredients in Persicae Semen, Armeniacae Semen Amarum, and Pruni Semen. Amy has extensive and remarkable pharmacological activities, including against anti-hepatic fibrosis. However, the pharmacokinetic and anti-liver fibrosis effects of Amy and its enzyme metabolite prunasin (Pru) in vivo have not been studied and compared, and studies on Pru are limited. PurposeTo investigate the pharmacokinetic characteristics and anti-liver fibrosis effect of Amy and its metabolite Pru in vivo and in vitro, and elucidate whether the metabolism of Amy in vivo for Pru is activated. MethodsPru was prepared from Amy via the enzymatic hydrolysis of β-glucosidase, and isolated by silica gel column chromatography. An efficient and sensitive ultrahigh-performance liquid chromatography-Q exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometry was developed and validated to determine simultaneously Amy and Pru in rat plasma after dosing intravenously and orally for pharmacokinetic studies. The affinities of Amy and Pru for β-glucosidase were compared by enzyme kinetic experiments to explain the possible reasons for the differences in pharmacokinetic behavior. In vitro, the inhibitory effects of Amy and Pru on hepatic stellate cell activation and macrophage inflammation on JS1 and RAW 264.7 cells were determined. In vivo, the ameliorative effects of Amy and Pru on liver fibrosis effects were comprehensively evaluated by CCl4-induced liver fibrosis model in mice. ResultsThe standard curves of Amy and Pru in rat plasma showed good linearity within the concentration range of 1.31–5000.00 ng/ml, with acceptable selectivity, carry-over, detection limit and quantification limits, intra- and inter-day precision, accuracy, matrix effect, and stability. The Cmax and AUC(0−∞) of Pru (Cmax = 1835.12 ± 268.09 ng/ml, AUC(0−∞) = 103,913.17 ± 14,202.48 ng•min/ml) were nearly 79.51- and 66.22-fold higher than those of Amy (Cmax = 23.08 ± 5.08 ng/ml, AUC(0−∞) = 1569.22 ± 650.62 ng•min/ml) after the oral administration of Amy. The oral bioavailability of Pru (64.91%) was higher than that of Amy (0.19%). The results of enzyme hydrolysis kinetics assay showed that the Vmax and Km of Pru were lower than those of Amy in commercial β-glucosidase and intestinal bacteria. In vitro cellular assays showed that Amy and Pru were comparable in inhibiting the NO production in the RAW264.7 cell supernatant and the mRNA expression of α-SMA and Col1A1 in JS1 cells. Amy and Pru were also showed comparable activity in ameliorating CCl4-induced liver fibrosis in mice. ConclusionThe pharmacokinetic characteristics of Amy and Pru in rat plasma were significantly different. After the separate gavage of Amy and Pru, Amy was absorbed predominantly as it's metabolite Pru, whereas Pru was absorbed predominantly as a prototype. The anti-liver fibrosis effects of Amy and its deglycosylated metabolite Pru were comparable in vivo and in vitro. The deglycosylated activated metabolite Pru of Amy plays an important role in anti-liver fibrosis. These findings will facilitate the further exploitation of Amy and Pru.