Chemical composition and antioxidant activity of the Amazonian fruit Ambelania acida Aubl.

Aqueous and hydroalcoholic extracts from the pulp of Ambelania acida Aubl. (Apocynaceae) fruits were subjected to analysis through UHPLC-HRMS and antioxidant potential using the TPC, DPPH, ABTS, FRAP, and ORAC assays. A putative identification of the compounds carried out by comparison of the fragmentation spectra revealed the predominance of the monoterpene indole alkaloids tabersonine, pseudocopsinine, ajmalicine, and strictosidine. Additionally, gallic acid, caffeic acid, citric acid, 3-O-p-coumaroylquinic acid, chlorogenic acid, catechin, ellagic acid, eschweilenol C (ellagic acid deoxyhexoside), and sucrose were identified. In face of the phenolic compounds observed, hydroalcoholic extract showed a higher antioxidant activity compared to the aqueous extract, observed at TPC (108.85 mg GAE/100g), FRAP (0.73 µmol Fe2SO4/g), DPPH (1221.76 µmol TE/g), ABTS (3460.00 µmol TE/g), and ORAC assays (120.47 µmol TE/g). These findings underscore the abundant presence of bioactive compounds, including phenolics and alkaloids, in an edible Amazonian fruit.

Identification and Characterization of Rotigotine Degradation Products by HPLC Coupled DAD and CAD Detectors and HRMS Through Q-Orbitrap and Electrospray Ionization.

Rotigotine (RTG) is a dopamine agonist used in the treatment of Parkinson's disease. As it is susceptible to oxidation, stability studies must be carefully designed for the identification and characterization of all possible degradation products. Here, RTG degradation was evaluated according to the International Conference on Harmonization guidelines under various stress conditions, including acidic and basic hydrolysis, oxidative, metallic, photolytic, and thermal conditions. Additionally, more severe stress conditions were applied to induce RTG degradation. Significant degradation was only observed under oxidative and photolytic conditions. The samples were analyzed by high performance liquid chromatography coupled to photodiode array detectors, charged aerosol, and high-resolution mass spectrometry. Chromatographic analyses revealed the presence of eight substances related to RTG, four of which were already described and were qualified impurities (impurities B, C, K and E) and four new degradation products (DP-1 - DP-4), whose structures were characterized by high-resolution mass spectrometry through Q-Orbitrap and electrospray ionization. In the stress testing of the active pharmaceutical ingredient in solid form, significant RTG degradation was observed in the presence of the oxidative matrix. The results corroborate the literature that confirm the high susceptibility of RTG to oxidation and the importance of using different detectors to detect degradation products in forced degradation studies.

Chemical Profile of Ocotea delicata (Lauraceae) Using Ultra High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry-Global Natural Products Social Molecular Networking Workflow.

Ocotea, the largest genus in the Lauraceae family, encompasses numerous species of scientific interest. However, most Ocotea species have only been described morphologically. This study used an untargeted metabolomics workflow with UHPLC-HRMS and GNPS-FBMN to provide the first chemical evaluation of the polar specialized metabolites of O. delicata leaves. Leaves from three O. delicata specimens were extracted using ultrasound-assisted extraction with 70% ethanol. Among the examined samples, 44 metabolites, including alkaloids and flavonoids, were identified. In contrast to other Ocotea species, O. delicata has a wider diversity of kaempferol derivatives than quercetin. The biomass of the specimens showed a significant correlation with the chemical profile. The similarity among specimens was mostly determined by the concentrations of quinic acid, kaempferol glycosides, and boldine. The evaluated specimens exhibited chemical features similar to those of species classified as New World Ocotea, with the coexistence of aporphine and benzylisoquinoline alkaloids.

Systematic characterization of Lysergic Acid Diethylamide metabolites in Caenorhabditis elegans by ultra-high performance liquid chromatography coupled with high-resolution tandem mass spectrometry.

Psychedelic compounds have gained renewed interest for their potential therapeutic applications, but their metabolism and effects on complex biological systems remain poorly understood. Here, we present a systematic characterization of Lysergic Acid Diethylamide (LSD) metabolites in the model organism Caenorhabditis elegans using state-of-the-art analytical techniques. By employing ultra-high performance liquid chromatography coupled with high-resolution tandem mass spectrometry, we putatively identified a range of LSD metabolites, shedding light on their metabolic pathways and offering insights into their pharmacokinetics. Our study demonstrates the suitability of Caenorhabditis elegans as a valuable model system for investigating the metabolism of psychedelic compounds and provides a foundation for further research on the therapeutic potential of LSD.

Investigations into Annona fruit consumption as a potential source of dietary higenamine intake in the context of sports drug testing.

Higenamine is prohibited in sports as a ß2 -agonist by the World Anti-Doping Agency. As a key component of a great variety of plants, including the Annonaceae family, one aim of this research project was to evaluate whether the ingestion of Annona fruit could lead to higenamine adverse analytical findings. Single-dose administration studies including three Annona species (i.e., Annona muricata, Annona cherimola, and Annona squamosa) were conducted, leading to higenamine findings below the established minimum reporting level (MRL) of 10 ng/mL in urine. In consideration of cmax values (7.8 ng/mL) observed for higenamine up to 24 h, a multidose administration study was also conducted, indicating cumulative effects, which can increase the risk of exceeding the applicable MRL doping after Annona fruit ingestion. In this study, however, the MRL was not exceeded at any time point. Further, the major urinary excretion of higenamine in its sulfo-conjugated form was corroborated, its stability in urine was assessed, and in the absence of reference material, higenamine sulfo-conjugates were synthesized and comprehensively characterized, suggesting the predominant presence of higenamine 7-sulfate. In addition, the option to include complementary biomarkers of diet-related higenamine intake into routine doping controls was investigated. A characteristic urinary pattern attributed to isococlaurine, reticuline, and a yet not fully characterized bismethylated higenamine glucuronide was observed after Annona ingestion but not after supplement use, providing a promising dataset of urinary biomarkers, which supports the discrimination between different sources of urinary higenamine detected in sports drug testing programs.

Coordination chemistry applied to anti-doping analysis: Cobalt quantification as its diethyldithiocarbamate complex by liquid chromatography coupled to high resolution tandem mass spectrometry.

Cobalt was included on the World Anti-Doping Agency Prohibited List in 2015 due to its effect on stimulus of erythropoiesis via stabilization of hypoxia-inducible factor. Although it has proven benefits for performance enhancement, the unavailability of inductively coupled plasma-mass spectrometry on routine of the accredited laboratories is a factor that reduces its applicability in anti-doping analysis. Therefore, an analytical method for quantification of urinary cobalt as its diethyldithiocarbamate complex by liquid chromatography coupled with high-resolution tandem mass spectrometry was developed and validated. Palladium was proposed as internal standard and rhodium as a complexation control. A microwave-assisted acid digestion of the urine samples was essential, not only to eliminate the matrix effect but mainly to avoid the non-specific bond of cobalt to endogenous molecules. A linear method was obtained over the studied range from a negative urine control to a spiked concentration of 25 ng/mL, with an estimated limit of quantification of 2.5 ng/mL, and an adequate combined standard uncertainty of 11.4%. Considering that all reagents are commercially available, the proposed strategy is feasible to be included on routine sample preparation. Monitoring urinary cobalt concentrations globally opens the perspective to support the anti-doping system to define a suitable threshold value and to understand its potential misuse by athletes seeking for performance improvement.

Antioxidant Capacity, Antitumor Activity and Metabolomic Profile of a Beetroot Peel Flour.

In this study, a beetroot peel flour was made, and its in vitro antioxidant activity was determined in aqueous (BPFw) and ethanolic (BPFe) extracts. The influence of BPFw on breast cancer cell viability was also determined. A targeted betalain profile was obtained using high-resolution Q-Extractive Plus Orbitrap mass spectrometry (Obrtitrap-HRMS) alongside untargeted chemical profiling of BPFw using Ultra-High-Performance Liquid Chromatography with High-Resolution Mass Spectrometry (UHPLC-HRMS). BPFw and BPFe presented satisfactory antioxidant activities, with emphasis on the total phenolic compounds and ORAC results for BPFw (301.64 ± 0.20 mg GAE/100 g and 3032.78 ± 55.00 µmol T/100 g, respectively). The MCF-7 and MDA-MB-231 breast cancer cells presented reductions in viability when treated with BPFw, showing dose-dependent behavior, with MDA-MB-231 also showing time-dependent behavior. The chemical profiling of BPFw led to the identification of 9 betalains and 59 other compounds distributed amongst 28 chemical classes, with flavonoids and their derivates and coumarins being the most abundant. Three forms of betalain generated via thermal degradation were identified. However, regardless of thermal processing, the BPF still presented satisfactory antioxidant and anticancer activities, possibly due to synergism with other identified molecules with reported anticancer activities via different metabolic pathways.

Chemical variability of Copaifera langsdorffii Desf. from environmentally contrasting populations.

Copaifera langsdorffii Desf. is recognised as one of most famous medicinal and economic species of Copaiba, occurring in several distinct biomes. An untargeted metabolomic approach was used to evaluate the chemical variability of C. langsdorffii from contrasting climates biomes (Atlantic Rainflorest and the semiarid Cerrado). Metabolomic analysis enabled the identification of 11 compounds, including glycosylated flavonoids and galloylquinic acid derivatives. Multivariate analysis highlighted that Cerrado population had a significantly higher concentrations of galloylquinic acid derivatives in comparison to the rainforest biome. Meanwhile, Atlantic Rainforest populations presented higher content of flavonols. Semiarid biome, reduced the concentration of flavonoids, mainly concerning quercetin and kaempferol derivatives, however, in this biome flavonoids were more diverse. Both chemical classes presented relevance to be used as geographical origin chemical markers by qualitative and quantitative features.

Assessing limits of detection in qualitative methods: A simple implementation of logistic regression in a web-based R Shiny application environment and its potential in toxicology and doping control.

The estimation of limits of detection (LOD) for solely qualitative methods in analytical chemistry may prove challenging because all the approaches with which chemists are familiar require some type of numeric data input. The best model to describe the binary response in these methods (detected/not detected) is a logistic model; however, these models are not easily handled by most of the laboratories and generally demand expensive statistical software packages. In this work, the advantages of applying this approach are discussed and its implementation using commercial spreadsheet software is demonstrated. A free online application based on the R environment using shinyapps was developed and its application was validated and discussed with a dataset of 57 different target compounds analyzed in urine according to the requirements of the World Anti-Doping Agency (WADA). This tool allows free, extremely quick, and easy determinations of LOD in qualitative analyses as well as the determination of the probabilities of detection in any given concentration.

Minor red blood cell antigen phenotyping of athletes sampled in international competitions.

Blood transfusion is performed by cheating athletes to rapidly increase oxygen delivery to exercise muscles and enhance their performance. This method is banned by the World Anti-doping Agency (WADA). Heterologous or allogenic blood transfusion happens when blood from a different person is transfused. The method used to detect this type of doping is based on flow cytometry, by identifying variations in blood group minor antigens present on the red blood cells' surface. Transfusion practices have regained interest since the introduction of human recombinant erythropoietin detection method. It has been reported that the number of occurrences of two athletes sharing an identical phenotype in the same sport was five times higher than the theoretical populational probability. The present work describes the prevalence of 10 erythrocytes surface antigens in a population of 261 athletes from all five continents. The matching phenotype per sport is also described.

A simple quinoline salt derivative is active in vitro against Plasmodiumfalciparum asexual blood stages and inhibits the development of cerebral malaria in murine model.

Chloroquine (CQ) was the most effective and widely used drug for the prophylaxis and treatment of severe and non-severe malaria. Although its prophylactic use has led to resistance to P. falciparum in all endemic countries, CQ still remains the drug of choice for the treatment of vivax malaria. Otherwise, the speed in which parasite resistance to available antimalarials rises and spreads in endemic regions points to the urgent need for the development of new antimalarials. Quinoline derivatives have been used as a tool in the search for new drugs and were investigated in the present study in an attempt to produce a HIT compound to avoid the cerebral malarial (CM). Seven compounds were synthesized, including three quinoline derivate salts. The cytotoxicity and antiplasmodial activity were assayed in vitro, highlighting compound 3 as a HIT, which also showed interaction with ferriprotoporphyrin IX similarly to CQ. Physicochemical and pharmacokinetic properties of absorption were found to be favorable when analyzed in silico. The in vivo assays, using the experimental cerebral malaria (ECM) model, showed important values of parasite growth inhibition on the 7th day-post infection (Q15 15 mg/kg: 76.9%, Q30 30 mg/kg: 90,1% and Q50 50 mg/kg: 92,9%). Compound 3 also showed significant protection against the development of CM, besides hepatic and renal parameters better than CQ. In conclusion, this quinoline derivative demonstrated promising activity for the treatment of malaria and was able to avoid the development of severe malaria in mice.

Chemophenetic study of Ocotea canaliculata (Lauraceae) by UHPLC-HRMS and GNPS.

The metabolic fingerprint of a non-volatile fraction of Ocotea canaliculata (Rich.) Mez (Lauraceae) leaves was determined by UHPLC-HRMS analysis. Twenty-four compounds were suggestively identified by GNPS-FBMN. The results revealed a large production of flavonoids, mainly flavones and flavanones, a chemical class poorly described in the Ocotea genus. Within the identified compounds, four are being described for the first time in this genus. The major metabolite detected was astilbin, with a concentration corresponding to 23.2 ± 1.58% of the extracts. The expressive content of astilbin also highlights it as a chemical marker for the species. As a species that is classified as a complex, qualitative and semi-quantitative features obtained through the O. canaliculata flavonoid fingerprint can be further used for a more precise circumscription and species-specific characterization.

Metabolic study of cafestol using in silico approach, zebrafish water tank experiments and liquid chromatography high-resolution mass spectrometry analyses.

Coffee is one of the most consumed beverages worldwide. Cafestol is an endogenous coffee diterpene present in raw coffee beans and also found in hot beverages, with several biological activities. However, there is still little information on this molecule after ingestion of coffee infusion. Zebrafish (Danio rerio) is a promising in vivo model for metabolic studies due to the annotation of mammalian orthologs to encode enzymes related to drug metabolism. Experiments using Zebrafish Water Tank (ZWT) model produce more significant number of metabolites for molecular investigation in a cleaner matrix than other classical models, such as purified hepatocytes. This work aimed to investigate the biotransformation of cafestol by the ZWT model using ultra-performance liquid chromatography coupled to hybrid quadrupole-orbitrap high-resolution mass spectrometry equipped with electrospray ionization (UPLC-HRMS) supported by in silico approach using SMARTCyp, Way2Drug and XenoSite Softwares. Twenty-five metabolites of cafestol were proposed by in silico analysis, in which 5 phase I metabolites were confirmed in the ZWT by UPLC and MS/HRMS investigation: 6-hydroxy-cafestol, 6,12-dihydroxy-cafestol, 2-oxo-cafestol, 6-oxo-cafestol and one isomer whose position in the carboxyl group was not determined. These metabolites were observed during 9 h of the experiment, whose contents were associated with the behavioral responses of the fish.

Variable Data Independent Acquisition and Data Mining Exploring Feature-Based Molecular Networking Analysis for Untargeted Screening of Synthetic Cannabinoids in Oral Fluid.

Novel psychoactive substances (NPS) are constantly emerging in the drug market, and synthetic cannabinoids (SCs) are included in this NPS family. Forensic laboratories often struggle with these continually emerging SCs, forcing them to develop an untargeted workflow to incorporate these psychoactive drugs in their procedures. Usually, forensic laboratories select analytical methods based on targeted mass spectrometry (MS) technologies for strictly tracking already known NPS. The appropriate way to tackle unknown substances is to develop pipelines for untargeted analysis that include LC-HRMS analytical methods and data analysis. Once established, this strategy would allow drug testing laboratories to be always one step ahead of the new trends concerning the "designer drugs" market. To address this challenge an untargeted workflow based on mass spectrometry data acquisition and data analysis was developed to detect SCs in oral fluid (OF) samples at a low concentration range. The samples were extracted by mixed-mode solid-phase extraction and analyzed by Liquid Chromatography - High-Resolution Mass Spectrometry (LC-HRMS). Tandem mass spectra (MS2) were recorded performing a variable isolation width across a mass range of all theoretical precursor ions (vDIA) after the chromatographic separation. After raw data processing with the MSDial software, the deconvoluted features were sent to GNPS for Feature-Based Molecular Networking (FBMN) construction for nontargeted data mining. The FBMN analysis created a unique integrated network for most of the SCs assessed in the OF at a low level (20 ng/mL). These results demonstrate the potential of an untargeted approach to detect different derivatives of SCs at trace levels for forensic applications.

Is zebrafish (Danio rerio) water tank model applicable for the assessment of glucocorticoids metabolism? The budesonide assessment.

Knowledge of the metabolic profile is essential for doping control analysis in sport since most drugs are excreted after an elaborate biotransformation process. Currently, Zebrafish Water Tank (ZWT) model has been applied to investigate the metabolism of different doping agents. Nevertheless, the class of glucocorticoids has not been subjected to this model for metabolism studies. In the present work, budesonide (BUD) was applied as a pilot to investigate the metabolic pathways of glucocorticoids in the ZWT model. The BUD biotransformation in ZWT model was compared to the described metabolism in humans. Samples from ZWT experiments were collected after BUD administration and analyzed by Liquid Chromatography coupled to High Resolution Mass Spectrometry (LC-HRMS). Following the identification and characterization of all significant metabolites described for humans, it was observed that the ZWT was able to produce in a relevant amount the main target for doping control purposes: the 6ß-hydroxy BUD. In addition, prior knowledge about the lack of butyrylcholinesterase activity in the zebrafish organism was considered for the evaluation for the formation of the 16α-hydroxy prednisolone, the most intense BUD metabolite in human urine. Biotransformation of BUD by ZWT focused on metabolites with the acetal fraction preserved, including the intermediate metabolite for the 16α-hydroxy prednisolone pathway. However,analternative metabolic pathway for the complete biotransformation of the 16α-hydroxy prednisolone intermediate was not observed, leading to the absence of the major human metabolite in the ZWT model. The findings reported in this study elucidate for the first time the application and limitations of the ZWT model to evaluate the metabolism of other glucocorticoids.

Synthesis and biological activity of novel 4-aminoquinoline/1,2,3-triazole hybrids against Leishmania amazonensis.

Quinoline and 1,2,3-triazoles are well-known nitrogen-based heterocycles presenting diverse pharmacological properties, although their antileishmanial activity is still poorly exploited. As an effort to contribute with studies involving these interesting chemical groups, in the present study, a series of compounds derived from 4-aminoquinoline and 1,2,3-triazole were synthetized and biological studies using L. amazonensis species were performed. The results pointed that the derivative 4, a hybrid of 4-aminoquinoline/1,2,3-triazole exhibited the best antileishmanial action, with inhibitory concentration (IC50) values of ~1 µM against intramacrophage amastigotes of L. amazonensis , and being 16-fold more active to parasites than to the host cell. The mechanism of action of derivative 4 suggest a multi-target action on Leishmania parasites, since the treatment of L. amazonensis promastigotes caused mitochondrial membrane depolarization, accumulation of ROS products, plasma membrane permeabilization, increase in neutral lipids, exposure of phosphatidylserine to the cell surface, changes in the cell cycle and DNA fragmentation. The results suggest that the antileishmanial effect of this compound is primarily altering critical biochemical processes for the correct functioning of organelles and macromolecules of parasites, with consequent cell death by processes related to apoptosis-like and necrosis. No up-regulation of reactive oxygen and nitrogen intermediates was promoted by derivative 4 on L. amazonensis -infected macrophages, suggesting a mechanism of action independent from the activation of the host cell. In conclusion, data suggest that derivative 4 presents selective antileishmanial effect, which is associated with multi-target action, and can be considered for future studies for the treatment against disease.

In vitro and in vivo antiplasmodial activity of novel quinoline derivative compounds by molecular hybridization.

Chloroquine (CQ) has been the main treatment for malaria in regions where there are no resistant strains. Molecular hybridization techniques have been used as a tool in the search for new drugs and was implemented in the present study in an attempt to produce compound candidates to treat malarial infections by CQ-resistant strains. Two groups of molecules were produced from the 4-aminoquinoline ring in conjugation to hydrazones (HQ) and imines (IQ). Physicochemical and pharmacokinetic properties were found to be favorable when analyzed in silico and cytotoxicity and antiplasmodial activity were assayed in vitro and in vivo showing low cytotoxicity and selectiveness to the parasites. Candidates IQ5 and IQ6 showed important values of parasite growth inhibition in vivo on the 5th day after infection (IQ5 15 mg/kg = 72.64% and IQ6 15 mg/kg = 71.15% and 25 mg/kg = 93.7%). IQ6 also showed interaction with ferriprotoporphyrin IX similarly to CQ. The process of applying condensation reactions to yield imines is promising and capable of producing molecules with antiplasmodial activity.

Chemosystematic evaluation of Amazonian freshwater sponges.

Freshwater sponges can be considered a promising new source of bioactive compounds for the pharmaceutical industry; however, the research on their chemical composition is still in the incipient stage. We evaluated the most endemic Amazonian freshwater sponge species from the Drulia and Metania genera by untargeted metabolomic approaches, based on UHPCL-HRMS, in order to identify chemical markers and explore the diversity of specialized metabolites. The use of untargeted approaches allowed us to observe subsets of metabolites that enabled the characterization of, not only each genus, but also, of each species. Freshwater sponge species presented themselves as rich sources of fatty acids and sterols, which were putatively identified. These metabolites were suggested as chemical markers for further targeted metabolomic studies.

UHPLC-HRMS/MS on untargeted metabolomics: a case study with Copaifera (Fabaceae).

Untargeted metabolomics is a powerful tool in chemical fingerprinting. It can be applied in phytochemistry to aid species identification, systematic studies and quality control of bioproducts. This approach aims to produce as much chemical information as possible, without focusing on any specific chemical class, thus, requiring extensive chemometric effort. This study aimed to evaluate the feasibly of an untargeted metabolomics method in phytochemistry by a study case of the Copaifera genus (Fabaceae). This genus contains significant medicinal species used worldwidely. Copaifera exploitation issues include a lack of chemical data, ambiguous species identification methods and absence of quality control for its bioproducts. Different organs of five Copaifera species were analysed by UHPLC-HRMS/MS, GNPS platform and chemometric tools. Untargeted metabolomics enabled the identification of 19 chemical markers and 29 metabolites, distinguishing each sample by species, plant organs, and biome type. Chemical markers were classified as flavonoids, terpenoids and condensed tannins. The applied method provided reliable information about species chemodiversity using fast workflow with little sampling size. The untargeted approach by UHPLC-HRMS/MS proved to be a promising tool for species identification, pharmacological prospecting and in the future for the quality control of extracts used in the manufacture of bioproducts.