Hundreds of new psychoactive substances (NPS) covering most drugs-of-abuse classes have been introduced to the recreational drug market in recent years. One class of NPS drugs that has become more common recently is \"designer\" benzodiazepines. Due to a close structural resemblance with prescription benzodiazepines, some of these substances may elicit a positive response (i.e. cross react) in immunoassay screening. Consequently, it is increasingly important to include NPS benzodiazepines during method confirmation to ensure accurate identification of closely-related compounds as well as detection of the benzodiazepines themselves. Here, we present our efforts to develop a screening and confirmation method for detection of 28 NPS benzodiazepines in urine using reversed-phase liquid chromatographic separation in combination with high-resolution mass spectrometry (LC-HRMS). MS was performed in positive electrospray mode on a Thermo Fischer Scientific Q Exactive Orbitrap instrument using either full scan (for screening) or parallel reaction monitoring (for confirmation). We found the lower quantification limit of the method to range from 5 to 50 ng/mL. Analytical precision and accuracy were ≤15% for both screening and confirmation for all except one analyte. The method was used to analyze patient urine samples from routine drug testing and samples from acute intoxication cases presenting in emergency wards. Altogether, 16 of the 28 benzodiazepines (i.e., clobazam, clonazolam, deschloroetizolam, diclazepam, estazolam, etizolam, flubromazepam, flubromazolam, flunitrazolam, 3-hydroxyflubromazepam, 3-hydroxyphenazepam, ketazolam, meclonazepam, metizolam, nifoxipam, and pyrazolam) were detected in the urine samples. The results from patient sample analysis indicate a high prevalence of NPS benzodiazepine use, emphasizing the importance of including novel drugs of abuse in drug testing menus.

The non-psychoactive cannabinoids cannabidiol (CBD) and cannabidiolic acid (CBDA) are available on the market in different forms, mostly for their anti-inflammatory and potential analgesic properties. These substances are prohibited during equine competitions. CBD and CBDA are naturally present in hemp straw, commonly used as a bedding substitute for wheat straw. Unfortunately, horses can eat it, which therefore could lead to a possible risk of positive findings for CBD/CBDA in biological samples after doping control tests. The goals of this study were, first, to provide recommendations on the use of hemp straw before competition and, second, to assess if discrimination between hemp bedding exposure and CBD oil administration is possible. Several CBD equine in vivo studies have been conducted, including one on hemp straw used as bedding and one after administration of CBD oil by topical and sublingual routes. In hemp straw, CBDA was detected in higher quantities than CBD, and other cannabinoids have been observed. After hemp straw exposure, CBDA was also detected in higher quantities than CBD in all urine samples. It appeared that hemp straw should not be used as bedding for equine competition except if a delay of at least 48 h is respected. Regarding the CBD oil product analysis, CBD was the main compound detected. After administration, 7-hydroxy CBD was identified in the urine. In conclusion, based on these data, we highlighted that it could be possible to discriminate the exposure of a horse to hemp straw from an administration of a CBD oil product thanks to the main presence of CBDA.

BACKGROUND: The high virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for coronavirus disease 2019 (COVID-19), has triggered global health and economic concerns. The absence of specific antiviral treatments and the side effects of repurposed drugs present persistent challenges. This study explored a promising antiviral herbal extract against SARS-CoV-2 from selected Thai medicinal plants based on in vitro efficacy and evaluated its antiviral lead compounds by molecular docking.
METHODS: Twenty-two different ethanolic-aqueous crude extracts (CEs) were rapidly screened for their potential activity against porcine epidemic diarrhea virus (PEDV) as a surrogate using a plaque reduction assay. Extracts achieving ≥ 70% anti-PEDV efficacy proceeded to the anti-SARS-CoV-2 activity test using a 50% tissue culture infectious dose method in Vero E6 cells. Molnupiravir and extract-free media served as positive and negative controls, respectively. Potent CEs underwent water/ethyl acetate fractionation to enhance antiviral efficacy, and the fractions were tested for anti-SARS-CoV-2 performance. The fraction with the highest antiviral potency was identified using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Molecular docking analyses of these compounds against the main protease (Mpro) of SARS-CoV-2 (6LU7) were performed to identify antiviral lead molecules. The top three hits were further evaluated for their conformational stability in the docked complex using molecular dynamics (MD) simulation.
RESULTS: The water fraction of mulberry (Morus alba Linn.) leaf CE (WF-MLCE) exhibited the most potent anti-SARS-CoV-2 efficacy with low cytotoxicity profile (CC50 of ~ 0.7 mg/mL), achieving 99.92% in pre-entry mode and 99.88% in postinfection treatment mode at 0.25 mg/mL. Flavonoids and conjugates were the predominant compounds identified in WF-MLCE. Molecular docking scores of several flavonoids against SARS-CoV-2 Mpro demonstrated their superior antiviral potency compared to molnupiravir. Remarkably, myricetin-3-O-β-D-galactopyranoside, maragrol B, and quercetin 3-O-robinobioside exhibited binding energies of ~  - 9 kcal/mol. The stability of each ligand-protein complex of these compounds with the Mpro system showed stability during MD simulation. These three molecules were pronounced as antiviral leads of WF-MLCE. Given the low cytotoxicity and high antiviral potency of WF-MLCE, it holds promise as a candidate for future therapeutic development for COVID-19 treatment, especially considering its economic and pharmacological advantages.

The recent biological invasion of box tree moth Cydalima perspectalis on Buxus trees has a major impact on European boxwood stands through severe defoliation. This can hinder further regrowth and threaten survival of populations. In a mesocosm approach and controlled larval density over a 2-month period, responses of B. sempervirens essential and specialized metabolites were characterized using metabolomics, combining 1H-NMR and LC-MS/MS approaches. This is the first metabolome depiction of major Buxus responses to boxwood moth invasion. Under severe predation, remaining green leaves accumulate free amino acids (with the noticeable exception of proline). The leaf trans-4-hydroxystachydrine and stachydrine reached 10-13% and 2-3% (DW), while root content was lower but also modulated by predation level. Larval predation promoted triterpenoid and (steroidal) alkaloid synthesis and diversification, while flavonoids did not seem to have a relevant role in Buxus resistance. Our results reveal the concomitant responses of central and specialized metabolism, in relation to severity of predation. They also confirm the potential of metabolic profiling using 1H-NMR and LC-MS to detect re-orchestration of metabolism of native boxwood after severe herbivorous predation by the invasive box-tree moth, and thus their relevance for plant-insect relationships and ecometabolomics.

In the current investigation, total phenolics and flavonoids of the methanolic extract obtained from the trunk bark of Acacia cyanophylla Lindl. were quantified by LC-HRMS technique. DPPH and ABTS reagents were employed to assay the antioxidant potential. The anti-tyrosinase and anti-α-amylase potentials were also assayed. The findings revealed that thirteen polyphenolic compounds were detected in the methanolic extract with trans-taxifolin (23.2 g/kg), as the major constituent. A. cyanophylla extract displayed a higher activity with DPPH test (IC50=10.14±1.00 μg/mL) than with ABTS (IC50=15.27±2.09 μg/mL). The same extract also exhibited interesting α-amylase inhibitory action (IC50 value of 4.00±0.17 μg/mL). Moreover, methanolic trunk bark extract exerted strong anti-tyrosinase capacity with an IC50 of 5.12±0.41 μg/mL in comparison to kojic acid (IC50=10.22±0.85 μg/mL) used as positive control. The antioxidant, anti-tyrosinase and anti-α-amylase potentials of the methanolic extract of A. cyanophylla trunk bark were reinforced by in silico molecular docking analyses, which confirmed the results of the in vitro tests.

Broccoli (Brassica oleracea L. var. italica Plenck) is a widely consumed vegetable, very popular due to its various nutritional and bioactive components. Since studies on the lipid components of broccoli have been limited so far, the aim of the present work was the study of free fatty acids (FFAs) present in different broccoli parts, aerial and underground. The direct determination of twenty-four FFAs in broccoli tissues (roots, leaves, and florets) was carried out, using a liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method in a 10 min single run. Linolenic acid was found to be the most abundant FFA in all different broccoli parts in quantities ranging from 0.76 to 1.46 mg/g, followed by palmitic acid (0.17-0.22 mg/g) and linoleic acid (0.06-0.08 mg/g). To extend our knowledge on broccoli\'s bioactive components, for the first time, the existence of bioactive oxidized fatty acids, namely hydroxy and oxo fatty acids, was explored in broccoli tissues adopting an HRMS-based lipidomics approach. 16- and 2-hydroxypalmitic acids were detected in all parts of broccoli studied, while ricinoleic acid was detected for the first time as a component of broccoli.

As first-line antimalarials used in the artemisinin combination therapy, artemisinin drugs exert their action inside red blood cells. However, the blood pharmacokinetic characteristics of artemisinin drugs have not been fully revealed owing to their built-in chemical instability initiated by Fe2+ released from hemoglobin, with limited information on their metabolites. In this study, liquid chromatography tandem high-resolution mass spectrometric (LC-HRMS) methods were developed for the quantification of two representative artemisinin drugs (artemisinin, ART; dihydroartemisinin, DHA) and their respective metabolite (deoxyartemisinin, D-ART; dihydroartemisinin glucuronide, DHA-Glu) in rat blood/plasma. The blood samples were pretreated with the stabilizer (0.4 m potassium dichromate and 3% EDTA-2Na). The methods displayed excellent specificity, linearity, accuracy and precision for ART (17.7-709.2 nm) and its metabolite D-ART (18.8-751.9 nm), and the linear range was 40.0-4,000.0 nm for both DHA and DHA-Glu. The methods were successfully applied to the pharmacokinetic studies of ART and DHA in rats. The blood-to-plasma ratio was 0.8-1.5 for ART, 1.0-1.5 for D-ART, 1.2-2.2 for DHA and 0.9-1.3 for DHA-Glu, which was time dependent. The results indicated that artemisinin drugs and their metabolites showed a high but different blood-to-plasma ratio, which should be considered when optimizating their dosing regimens or evaluating their clinical outcomes.

Tubulin-associated unit (tau) has an important role in the pathogenesis and the diagnosis of Alzheimer\'s disease (AD) and other tauopathies. In view of the diversity of tau proteoforms, antibody-free methods represent a good approach for unbiased quantification. We adapted and evaluated the single-pot, solid-phase-enhanced sample-preparation (SP3) protocol for antibody-free extraction of the tau protein in cerebro-spinal fluid (CSF) mimic and in human brain. A total of 13 non-modified peptides were quantified by high-resolution mass spectrometry (HRMS) after digestion of tau by trypsin. We significantly improved the basic SP3 protocol by carefully optimizing the organic solvents and incubation time for tau binding, as well as the digestion step for the release directly from the SP3 beads of the 13 tau peptides. These optimizations proved to be primarily beneficial for the most hydrophilic tau peptides, increasing the sequence coverage of recombinant tau. Mean recovery in CSF mimic of the 13 non-modified peptides was of 53%, with LODs ranging from 0.75 to 10 ng/mL. Next, we tested the optimized SP3 protocol on pathological tau extracted from the soluble fraction from an AD brain sample (middle frontal gyrus). We could successfully identify and quantify biologically relevant tau peptides including representative peptides of two isoforms and two phospho-peptides (pTau217 and pTau181).

Due to the different price and high quality, halal meat such as beef can be adulterated with non-halal meat with low price to get an economical price. The objective of this research was to develop an analytical method for halal authentication testing of beef meatballs (BM) from dog meat (DM) using a non-targeted metabolomics approach employing liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and chemometrics. The differentiation of authentic BM from that adulterated with DM was successfully performed using partial least square-discriminant analysis (PLS-DA) with high accuracy (R2X = 0.980, and R2Y = 0.980) and good predictivity (Q2 = 0.517). In addition, partial least square (PLS) and orthogonal PLS (OPLS) were successfully used to predict the DM added (% w/w) in BM with high accuracy (R2 > 0.990). A number of metabolites, potential for biomarker candidates, were identified to differentiate BM and that adulterated with DM. It showed that the combination of a non-targeted LC-HRMS Orbitrap metabolomics and chemometrics could detect up to 0.1% w/w of DM adulteration. The developed method was successfully applied for analysis of commercial meatball samples (n = 28). Moreover, pathway analysis revealed that beta-alanine, histidine, and ether lipid metabolism were significantly affected by dog meat adulteration. In summary, this developed method has great potential to be developed and used as an alternative method for analysis of non-halal meats in halal meat products.

Dolichyl phosphates (DolP) are ubiquitous lipids that are present in almost all eukaryotic membranes. They play a key role in several protein glycosylation pathways and the formation of glycosylphosphatidylinositol anchors. These lipids constitute only ~0.1% of total phospholipids, and their analysis by reverse phase (RP) liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is challenging due to their high lipophilicity (log P > 20), poor ionization efficiency, and relatively low abundance. To overcome these challenges, we have introduced a new approach for DolP analysis by combining trimethylsilyldiazomethane (TMSD)-based phosphate methylation and HRMS analysis. The analytical method was validated for its reproducibility, sensitivity, and accuracy. The established workflow was successfully applied for the simultaneous characterization and quantification of DolP species with different isoprene units in lipid extracts of HeLa and Saccharomyces cerevisiae cells.