Chromatography High-resolution Tandem Mass Spectrometry Research Articles

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.

A comprehensive method to elucidate pyoverdines produced by fluorescent Pseudomonas spp. by UHPLC-HR-MS/MS

Microbial secondary metabolites represent a rich source for drug discovery, plant protective agents, and biotechnologically relevant compounds. Among them are siderophores, iron-chelating molecules, that show a great influence on bacterial community assembly and the potential to control pathogen invasions. One of such a siderophore is pyoverdine that is produced by fluorescent Pseudomonas members and consists of different peptide chains specific to each bacterial species. The identification and structural elucidation of such suites of siderophores remain widely underexplored as general high-throughput analytical protocols are missing. Therefore, a dedicated method was established allowing a rapid localization and structural elucidation of pyoverdines. Liquid bacterial culture samples were purified by an easy small-scale solid-phase extraction (SPE). Ultra-high-performance liquid chromatography high-resolution tandem mass spectrometry (UHPLC-HR-MS/MS) separated highly polar pyoverdines and their derivatives. All ion fragmentation (AIF) generated mass spectra containing the characteristic fragments of the biological precursor of pyoverdine, ferribactin. This led to the revelation of the mass of secreted pyoverdines. Targeted MS/MS experiments at multiple collision energies accomplished the full structure elucidation of the pyoverdine peptide chain. A mass calculator and a fragmentation predictor facilitated greatly the interpretation of MS/MS spectra by providing accurate masses for a straightforward comparison of measured and theoretical values. The method was successfully validated using four well-known pyoverdines with various peptide chains. Finally, the applicability was proven by the analysis of 13 unknown pyoverdines secreted by sampled bacterial cultures. Among these, 4 novel pyoverdine peptide chains were discovered and are herein reported for the first time.Graphical abstract

In silico deconjugation of glucuronide conjugates enhances tandem mass spectra library annotation of human samples

Mass spectral library annotation of liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) data is a reliable approach for fast identification of organic contaminants and toxicants in complex environmental and biological matrices. While determining the exposure of humans or mammals, it is indispensable to include phase I and phase II metabolites (conjugates) along with the parent compounds, but often, tandem mass spectra for these are unavailable. In this study, we present and evaluate a strategy for annotating glucuronide conjugates in LC-HRMS/MS scans by applying a neutral loss search for detection, then truncating the spectra which we refer to as in silico deconjugation, and finally searching these against mass spectral libraries of the aglycones. The workflow was tested on a dataset of in vitro–generated glucuronides of reference standard mixtures and a dataset of 51 authentic urine samples collected from patients with known medication status, acquired on different instrumentations. A total number of 75 different glucuronidated molecular structures were identified by in silico deconjugation and spectral library annotation. We also identified specific molecular structures (sulfonamides, ether bonds, di-glucuronides), which resulted in slightly different fragmentation patterns between the glucuronide and the unconjugated compound. This led to a decreased spectral matching score and in some cases to a false-negative identification. Still, by applying this method, we revealed a reliable annotation of most common glucuronides, leading to a new strategy reducing the need for deconjugation steps or for recording many reference glucuronide spectra for screening approaches.Graphical

Chemical composition analysis, antioxidant activity, and target cell‐based screening of the potential active components in jujube and its fermented product

In this study, the chemical constituents of jujube and jujube fermentation broth were compared by chromatography tandem high-resolution mass spectrometry for the first time. A total of 68 compounds were detected, and six of them were quantified. The results showed that rutin was hydrolyzed and converted into quercetin during the fermentation process. Further, the antioxidant experiments in vitro were carried out, and the IC50 value of the fermentation broth was lower than that of the jujube extract. Twenty-three potential bioactive components from jujube samples were identified by a target cell-based screening method, and triterpenic acid compounds exhibited high cell binding property. These results will help to understand the different biological activities of jujube and its fermented products and will benefit the discovery of new functional components.

Characterization of Low-Level D-Amino Acid Degradation Impurities Using Liquid Chromatography-High Resolution Tandem Mass Spectrometry

Characterization of Low-Level D-Amino Acid Degradation Impurities Using Liquid Chromatography-High Resolution Tandem Mass Spectrometry

Non-Targeted Analysis of Co-Formulants in Antifungal Pesticide Formulations by Gas Chromatography-Tandem High Resolution Mass Spectrometry

Non-Targeted Analysis of Co-Formulants in Antifungal Pesticide Formulations by Gas Chromatography-Tandem High Resolution Mass Spectrometry

Optimization of the Everolimus Intermediate Preparation Process: Impurity Identification Followed by One-Pot Transformation to the Desired Product

An investigation of the everolimus intermediate PG-EVR preparation process resulted in the identification of three impurities PG-D,E,F constantly formed as byproducts in large amounts. Their structures were reliably established based on two-dimensional (2D) NMR and liquid chromatography–high-resolution tandem mass spectrometry (LC–HRMS/MS) data as dialkylated rapamycin derivatives. Finding that one of these impurities PG-E is hydrolyzable to the target everolimus intermediate resulted in optimized technology based on changing the selectivity of byproduct formation to PG-E as the major one followed by its one-pot quantitative transformation to PG-EVR. The yield of the everolimus intermediate increased by 10% under mild conditions, while lower amounts of reagents and solvents were required. The active pharmaceutical ingredient (API) that meets the requirements of the European Pharmacopoeia was prepared from thus obtained intermediate with no need for high-performance liquid chromatography (HPLC) purification, indicating a significant increase in the efficiency of large-scale production using new technology.

Vertical Inheritance Facilitates Interspecies Diversification in Biosynthetic Gene Clusters and Specialized Metabolites.

ABSTRACTWhile specialized metabolites are thought to mediate ecological interactions, the evolutionary processes driving chemical diversification, particularly among closely related lineages, remain poorly understood. Here, we examine the evolutionary dynamics governing the distribution of natural product biosynthetic gene clusters (BGCs) among 118 strains representing all nine currently named species of the marine actinobacterial genus Salinispora. While much attention has been given to the role of horizontal gene transfer (HGT) in structuring BGC distributions, we find that vertical descent facilitates interspecies BGC diversification over evolutionary timescales. Moreover, we identified a distinct phylogenetic signal among Salinispora species at both the BGC and metabolite level, indicating that specialized metabolism represents a conserved phylogenetic trait. Using a combination of genomic analyses and liquid chromatography–high-resolution tandem mass spectrometry (LC-MS/MS) targeting nine experimentally characterized BGCs and their small molecule products, we identified gene gain/loss events, constrained interspecies recombination, and other evolutionary processes associated with vertical inheritance as major contributors to BGC diversification. These evolutionary dynamics had direct consequences for the compounds produced, as exemplified by species-level differences in salinosporamide production. Together, our results support the concept that specialized metabolites, and their cognate BGCs, can represent phylogenetically conserved functional traits with chemical diversification proceeding in species-specific patterns over evolutionary time frames.

Metabolism of third generation synthetic cannabinoids using zebrafish larvae.

Synthetic cannabinoids are the second largest group of new psychoactive substances reported by the United Nations Office on Drugs and Crime in the last decade and case reports bring attention to its high potency effects and its severe toxicity, including fatalities. Moreover, synthetic cannabinoids are usually entirely metabolized and metabolic pathways for many new generation synthetic cannabinoids are still unknown. In this study, the metabolism of five third generation synthetic cannabinoids was evaluated using zebrafish (Danio rerio) larvae as 24-h in vivo model studied within 5 days after fertilization. The studied synthetic cannabinoids were MMB-CHMICA, ADB-CHMICA, ADB-CHMINACA, MDMB-CHMCZCA, and NNL-3, and the respective metabolites were identified by liquid chromatography-high resolution tandem mass spectrometry. Eleven, six, fourteen, eleven, and four metabolites were identified for MMB-CHMICA, ADB-CHMICA, ADB-CHMINACA, MDMB-CHMCZCA, and NNL-3, respectively, and metabolic pathways have been proposed. The use of zebrafish larvae, with a high degree of physiological and genetic homology to humans, is an emerging tool very useful for the identification of metabolic pathways of psychoactive substances. Results obtained in this study compared well with metabolites obtained previously for the same target molecules or structural analogous after in vitro incubation with human or rat hepatocytes. Thus, potential biomarkers for the evaluated compounds are the O-demethylated metabolite for MMB-CHMICA; the oxidative deamination to hydroxyl metabolite for ADB-CHMICA; hydroxyl metabolites at cyclohexylmethyl, tert-butyl, and indazole moieties for ADB-CHMINACA; hydroxyl metabolites at carbazole core, tert-butyl, or cyclohexylmethyl tail moieties for MDMB-CHMCZCA; and amide hydrolyzed, defluorinated, and dihydroxilated metabolite for NNL-3.

Serum Untargeted UHPLC-HRMS-Based Lipidomics to Discover the Potential Biomarker of Colorectal Advanced Adenoma.

BackgroundAs a key precancerous lesion, colorectal advanced adenoma (CAA) is closely related to the occurrence and development of colorectal cancer (CRC). Effective identification of CAA-related biomarkers can prevent CRC morbidity and mortality. Lipids, as an important endogenous substance, have been proved to be involved in the occurrence and development of CRC. Lipidomics is an advanced technique that studies lipid metabolism and biomarkers of diseases. However, there are no lipidomics studies based on large serum samples to explore diagnostic biomarkers for CAA.MethodsAn integrated serum lipid profile from 50 normal (NR) and 46 CAA subjects was performed using ultra-high performance liquid chromatography tandem high-resolution mass spectrometry (UHPLC-HRMS). Lipidomic data were acquired for negative and positive ionization modes, respectively. Differential lipids were selected by univariate and multivariate statistics analyses. A receiver operator characteristic curve (ROC) analysis was conducted to evaluate the diagnostic performance of differential lipids.ResultsA total of 53 differential lipids were obtained by combining univariate and multivariate statistical analyses (P < 0.05 and VIP > 1). In addition, 12 differential lipids showed good diagnostic performance (AUC > 0.90) for the discrimination of NR and CAA by receiver operating characteristic curve (ROC) analysis. Of them, the performance of PC 44:5 and PC 35:6e presented the outstanding performance (AUC = 1.00, (95% CI, 1.00–1.00)). Moreover, triglyceride (TAG) had the highest proportion (37.74%) as the major dysregulated lipids in the CAA.ConclusionThis is the first study that profiled serum lipidomics and explored lipid biomarkers with good diagnostic ability of CAA to contribute to the early prevention of CRC. Twelve differential lipids that effectively discriminate between NR and CAA serve as the potential diagnostic markers of CAA. An obvious perturbation of TAG metabolism could be involved in the CAA formation.

LC-HRMS/MS study of the prodrug ciclesonide and its active metabolite desisobutyryl-ciclesonide in plasma after an inhalative administration to horses for doping control purposes.

Ciclesonide (CIC) is the first inhaled highly potent corticosteroid that does not cause any cortisol suppression. It has been developed for the treatment of asthma in human and more recently in equine. CIC is the active compound of Aservo® EquiHaler® (Boehringer Ingelheim Vetmedica GmbH), the pre-filled inhaler generating a medicated mist based on Soft Mist™ technology. This prodrug is rapidly converted to desisobutyryl-ciclesonide (des-CIC), the main pharmacologically active compound. Due to its anti-inflammatory properties, CIC is prohibited for use in horse competitions. To set up an appropriate control, the determination of detection times and screening limits are required. Therefore, a highly sensitive analytical method based on supported liquid extraction (SLE) combined with liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) was developed to detect CIC and its active metabolite des-CIC in plasma. The lower limit of detection of CIC and des-CIC was approximately 1 pg/ml in plasma. After a pilot study conducted on a single horse at the recommended dose (eight actuations twice daily corresponding to 5.5mg/day for the first 5days, followed by 12 actuations once daily corresponding to 4.1mg/day in the last 5days), the same protocol was applied in the main study using six horses. In all horses, CIC and des-CIC levels were less than 5 and 10pg/ml, respectively, at 36 h after the end of the administration. The outcome of this risk assessment study should be useful to draw any recommendations for horse competitions.

Studies on Novel Diagnostic and Predictive Biomarkers of Intrahepatic Cholestasis of Pregnancy Through Metabolomics and Proteomics.

BackgroundIntrahepatic cholestasis of pregnancy (ICP) usually occurs in the third trimester and is associated with increased risks in fetal complications. Currently, the exact mechanism of this disease is unknown. The purpose of this study was to develop potential biomarkers for the diagnosis and prediction of ICP.MethodsWe enrolled 40 pregnant women diagnosed with ICP and 40 healthy pregnant controls. The number of placental samples and serum samples between the two groups was 10 and 40 respectively. Ultra-performance liquid chromatography tandem high-resolution mass spectrometry was used to analyze placental metabolomics. Then, we verified the differentially expressed proteins and metabolites, both placental and blood serum, in the first, second, and third trimesters.ResultsMetabolomic analysis of placental tissue revealed that fatty acid metabolism and primary bile acid biosynthesis were enriched. In the integrated proteomic and metabolomic analysis of placental tissue, peroxisomal acyl-CoA oxidase 1 (ACOX1), L-palmitoylcarnitine, and glycocholic acid were found to be three potential biomarkers. In a follow–up analysis, expression levels of both placental and serum ACOX1, L-palmitoylcarnitine, and glycocholic acid in both placenta and serum were found to be significantly higher in third-trimester ICP patients; the areas under the ROC curves were 0.823, 0.896, and 0.985, respectively. Expression levels of serum ACOX1, L-palmitoylcarnitine, and glycocholic acid were also significantly higher in first- and second-trimester ICP patients; the areas under the ROC curves were 0.726, 0.657, and 0.686 in the first trimester and 0.718, 0.727, and 0.670 in the second trimester, respectively. Together, levels of the three aforementioned biomarkers increased the value for diagnosing and predicting ICP (AUC: 0.993 for the third, 0.891 for the second, and 0.932 for the first trimesters).ConclusionsL-palmitoylcarnitine, ACOX1, and glycocholic acid levels taken together may serve as a new biomarker set for the diagnosis and prediction of ICP.

Chromatographic-mass spectrometric analysis of the urinary metabolite profile of chlorphenesin observed after dermal application of chlorphenesin-containing sunscreen.

Chlorphenesin is an approved biocide frequently used in cosmetics, and its carbamate ester is an approved skeletal muscle relaxant in certain countries for the treatment of discomfort related to skeletal muscle trauma and inflammation. A major urinary metabolite is 4-chlorophenoxy acetic acid (4-CPA), also known as para-chlorophenoxyacetate, which is also employed as a target analyte in sports drug testing to detect the use of the prohibited nootropic stimulant meclofenoxate. To distinguish between 4-CPA resulting from chlorphenesin, chlorphenesin carbamate, and meclofenoxate, urinary metabolite profiles of chlorphenesin after legitimate use were investigated. Human administration studies with commercially available sunscreen containing 0.25% by weight of chlorphenesin were conducted. Six study participants dermally applied 8g of sunscreen and collected urine samples before and up to 7days after application. Another set of six study participants applied 8g of sunscreen on three consecutive days, and urine samples were also taken for up to 5days after the last dosing. Urine specimens were analyzed using liquid chromatography-high resolution (tandem) mass spectrometry, and urinary metabolites were identified in accordance with literature data by accurate mass analysis of respective precursor and characteristic product ions. In accordance with literature data, chlorphenesin yielded the characteristic urinary metabolites, chlorphenesin glucuronide, chlorphenesin sulfate, and 3-(4-chlorophenoxy)-2-hydroxypropanoic acid (4-CPP), as well as the common metabolite 4-CPA. 4-CPA and 4-CPP were observed at similar abundances, with urinary concentrations of 4-CPA reaching up to ~1500 and 2300 ng/mL after single and multiple sunscreen applications, respectively. 4-CPA is a common metabolite of meclofenoxate, chlorphenesin, and chlorphenesin carbamate. Monitoring the diagnostic urinary metabolites of chlorphenesin provides conclusive supporting evidence of whether chlorphenesin or the prohibited nootropic meclofenoxate was administered.

Multiplexed analysis of amino acids in mice brain microdialysis samples using isobaric labeling and liquid chromatography-high resolution tandem mass spectrometry

We developed a new multiplexed reversed phase liquid chromatography-high resolution tandem mass spectrometric (LC-MS/MS) method. The method is based on isobaric labeling with a tandem mass tag (TMT10-plex) and stable isotope-labeled internal standards, and was used to analyze amino acids in mouse brain microdialysis samples. The TMT10-plex labeling of amino acids allowed analysis of ten samples in one LC-MS/MS run, significantly increasing the sample throughput. The method provides good chromatographic performance (peak half-width between 0.04–0.12 min), allowing separation of all TMT-labeled amino acids with acceptable resolution and high sensitivity (limits of detection typically around 10 nM). The use of stable isotope-labeled internal standards, together with TMT10-plex labeling, ensured good repeatability (relative standard deviation ≤ 12.1 %) and linearity (correlation coefficient > 0.994), indicating good quantitative performance of the multiplexed method. The method was applied to study the effect of d-amphetamine microdialysis perfusion on amino acid concentrations in the mouse brain. All amino acids were reliably detected and quantified, indicating that the method is sensitive enough to detect low concentrations of amino acids in brain microdialysis samples.

Combining OSMAC Approach and Untargeted Metabolomics for the Identification of New Glycolipids with Potent Antiviral Activity Produced by a Marine Rhodococcus.

Natural products of microbial origin have inspired most of the commercial pharmaceuticals, especially those from Actinobacteria. However, the redundancy of molecules in the discovery process represents a serious issue. The untargeted approach, One Strain Many Compounds (OSMAC), is one of the most promising strategies to induce the expression of silent genes, especially when combined with genome mining and advanced metabolomics analysis. In this work, the whole genome of the marine isolate Rhodococcus sp. I2R was sequenced and analyzed by antiSMASH for the identification of biosynthetic gene clusters. The strain was cultivated in 22 different growth media and the generated extracts were subjected to metabolomic analysis and functional screening. Notably, only a single growth condition induced the production of unique compounds, which were partially purified and structurally characterized by liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). This strategy led to identifying a bioactive fraction containing >30 new glycolipids holding unusual functional groups. The active fraction showed a potent antiviral effect against enveloped viruses, such as herpes simplex virus and human coronaviruses, and high antiproliferative activity in PC3 prostate cancer cell line. The identified compounds belong to the biosurfactants class, amphiphilic molecules, which play a crucial role in the biotech and biomedical industry.

Targeted and Untargeted Mass Spectrometry Reveals the Impact of High-Fat Diet on Peripheral Amino Acid Regulation in a Mouse Model of Alzheimer's Disease.

Recent research regarding amino acid metabolism has shown that there may be a link between obesity and Alzheimer's disease (AD). This work reports a metabolomics study using targeted and untargeted mass spectrometry-based metabolomic strategies to investigate this link. Targeted hydrophilic interaction liquid chromatography-triple quadrupole mass spectrometry and untargeted reversed-phase liquid chromatography-high resolution tandem mass spectrometry assays were developed to analyze the metabolic changes that occur in AD and obesity. APPSwe/PS1ΔE9 (APP/PSEN1) transgenic mice (to represent familial or early-onset AD) and wild-type littermate controls were fed either a high-fat diet (HFD, 60% kcal from lard) or a low-fat diet (LFD, 10% kcal from lard) from 2 months of age or a reversal diet (HFD, followed by LFD from 9.5 months). For targeted analyses, we applied the guidelines outlined in the Clinical and Laboratory Standards Institute (CLSI) LC-MS C62-A document and the U.S. Food and Drug Administration (FDA) bioanalytical method validation guidance for industry to evaluate the figures of merit of the assays. Our targeted and untargeted metabolomics results suggest that numerous peripheral pathways, specifically amino acid metabolism and fatty acid metabolism, were significantly affected by AD and diet. Multiple amino acids (including alanine, glutamic acid, leucine, isoleucine, and phenylalanine), carnitines, and members of the fatty acid oxidation pathway were significantly increased in APP/PSEN1 mice on HFD compared to those on LFD. More substantial effects and changes were observed in the APP/PSEN1 mice than in the WT mice, suggesting that they were more sensitive to an HFD. These dysregulated peripheral pathways include numerous amino acid pathways and fatty acid beta oxidation and suggest that obesity combined with AD further enhances cognitive impairment, possibly through aggravated mitochondrial dysfunction. Furthermore, partial reversibility of many altered pathways was observed, which highlights that diet change can mitigate the metabolic effects of AD. The same trends in individual amino acids were observed in both strategies, highlighting the biological validity of the results.

TOCSY, hydrogen decoupling and computational calculations to an unequivocal structural elucidation of a new sesquiterpene derivative and identification of other constituents from Praxelis sanctopaulensis.

Praxelis genus comprises 24 species, however, only two species of this genus have been chemically investigated. Here we investigated Praxelis sanctopaulensis, a native plant from Brazil, that occurs mainly in Cerrado regions. The goal was to identify the specialised metabolites from P. sanctopaulensis, and compare with those described from Praxelis and Chromolaena species. The phytochemical study of P. sanctopaulensis was performed through different chromatography techniques, including high-performance liquid chromatography (HPLC), gas chromatography flame ionisation detector (GC-FID), and ultra-high-performance liquid chromatography high-resolution tandem mass spectrometry (UHPLC-HRMS/MS). The structures of the compounds were established based on spectroscopic analysis, total correlated spectroscopy (TOCSY), hydrogen decoupling and computational calculations was used to an unequivocal structural elucidation of a new sesquiterpene. The antioxidant activity was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP), and antimicrobial assay was performed by the microdilution method. Comparison of the flavonoids described P. sanctopaulensis was carried out using principal component analysis. The phytochemical investigation of P. sanctopaulensis led to the isolation of a pair of diastereomers, praxilone A and praxilone B. Seven known compounds were isolated from this species, another 14 fatty acids were detected in hexane fraction, and 26 compounds were identified from ethyl acetate fraction. All these compounds are being described for the first time in this species, with the exception of viridifloric acid. The ethyl acetate fraction showed potent antioxidant activity. Forty-seven compounds are described from P. sanctopaulensis. The combination of different techniques of nuclear magnetic resonance (NMR) spectroscopy and computational calculations allowed the unequivocal structure elucidation of a new cadinene. The clustering analysis showed similarities between the flavonoids identified in P. sanctopaulensis and in Chromolaena species.

In Vitro Metabolic Fate of the Synthetic Cannabinoid Receptor Agonists QMPSB and QMPCB (SGT-11) Including Isozyme Mapping and Esterase Activity.

Quinolin-8-yl 4-methyl-3-(piperidine-1-sulfonyl)benzoate (QMPSB) and quinolin-8-yl 4-methyl-3-(piperidine-1-carbonyl)benzoate (QMPCB, SGT-11) are synthetic cannabinoid receptor agonists (SCRAs). Knowing their metabolic fate is crucial for the identification of toxicological screening targets and to predict possible drug interactions. The presented study aimed to identify the in vitro phase I/II metabolites of QMPSB and QMPCB and to study the contribution of different monooxygenases and human carboxylesterases by using pooled human liver S9 fraction (pHLS9), recombinant human monooxygenases, three recombinant human carboxylesterases, and pooled human liver microsomes. Analyses were carried out by liquid chromatography high-resolution tandem mass spectrometry. QMPSB and QMPCB showed ester hydrolysis, and hydroxy and carboxylic acid products were detected in both cases. Mono/dihydroxy metabolites were formed, as were corresponding glucuronides and sulfates. Most of the metabolites could be detected in positive ionization mode with the exception of some QMPSB metabolites, which could only be found in negative mode. Monooxygenase activity screening revealed that CYP2B6/CYP2C8/CYP2C9/CYP2C19/CYP3A4/CYP3A5 were involved in hydroxylations. Esterase screening showed the involvement of all investigated isoforms. Additionally, extensive non-enzymatic ester hydrolysis was observed. Considering the results of the in vitro experiments, inclusion of the ester hydrolysis products and their glucuronides and monohydroxy metabolites into toxicological screening procedures is recommended.

Identification of 24-O-β-d-Glycosides and 7-Deoxy-Analogues of Okadaic Acid and Dinophysistoxin-1 and -2 in Extracts from Dinophysis Blooms, Dinophysis and Prorocentrum Cultures, and Shellfish in Europe, North America and Australasia.

Two high-mass polar compounds were observed in aqueous side-fractions from the purification of okadaic acid (1) and dinophysistoxin-2 (2) from Dinophysis blooms in Spain and Norway. These were isolated and shown to be 24-O-β-d-glucosides of 1 and 2 (4 and 5, respectively) by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and enzymatic hydrolysis. These, together with standards of 1, 2, dinophysistoxin-1 (3), and a synthetic specimen of 7-deoxy-1 (7), combined with an understanding of their mass spectrometric fragmentation patterns, were then used to identify 1–5, the 24-O-β-d-glucoside of dinophysistoxin-1 (6), 7, 7-deoxy-2 (8), and 7-deoxy-3 (9) in a range of extracts from Dinophysis blooms, Dinophysis cultures, and contaminated shellfish from Spain, Norway, Ireland, Canada, and New Zealand. A range of Prorocentrum lima cultures was also examined by liquid chromatography–high resolution tandem mass spectrometry (LC–HRMS/MS) and was found to contain 1, 3, 7, and 9. However, although 4–6 were not detected in these cultures, low levels of putative glycosides with the same exact masses as 4 and 6 were present. The potential implications of these findings for the toxicology, metabolism, and biosynthesis of the okadaic acid group of marine biotoxins are briefly discussed.

Anabolic treatments in bovines: quantification of plasma protein markers of dexamethasone administration.

The aim of this study was to profile plasma proteome responses in bulls experimentally treated with dexamethasone at anabolic dosage. Illicit use of active substances in animal husbandry remains a matter of concern in Europe. Corticosteroids are probably one of the most widespread growth promoter family illegally used in beef cattle and veal calves. Testing for corticosteroids relies on detection of drug residues or their metabolites in biological fluids or tissues. Their indirect detection by mapping altered physiological parameters may overcome limits linked to route of administration, dosage, biotransformation and elimination kinetics that can lower residual drug concentration, hampering official controls. A set of 11 proteins proposed in literature as potential markers of anabolic treatments with dexamethasone, was quantified in bovine plasma by targeted proteomics based on liquid chromatography-high resolution tandem mass spectrometry. Among investigated proteins, sex hormone-binding globulin (SHBG), histidine-rich glycoprotein (HRG) and paraoxonase-1 (PON1) were found to be biomarkers of treatment. To investigate further such biomarkers, an additional group of veal calves was experimentally treated with dexamethasone at anabolic. These animals also demonstrated a significant alteration in SHBG, HRG and PON1 concentration, suggesting that quantification of plasma markers have the potential to detect animals illegally exposed to dexamethasone.