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The synthetic benzimidazole opioid etazene (which has a 70-times higher analgesic activity than morphine), a recreational drug, has gained popularity as a novel psychoactive substance (NPS) on the illegal/darknet market; however, no experimental information is available at the molecular level on the binding mechanism and putative binding site of etazene and its metabolites at the µ-opioid receptor (MOR). In the present study, we investigated the metabolism of etazene in human liver microsomes using ultra-high-performance liquid chromatography–mass spectrometry (UHPLC–MS). We also explored the possibilities of MOR activation by etazene and its metabolites by studying their binding mechanisms and interaction profiles at an active-state MOR model via molecular docking, binding free energy calculations, and all-atom molecular dynamics (MD) simulations. The putative metabolites of etazene were also predicted using the ADMET Predictor 10.1. The molecular docking studies and free energy calculations showed that etazene and its metabolites (M1, M2, and M5–M7) exhibited strong predicted binding affinity at MOR and showed overlapped binding orientation with MOR-bound agonist BU72, which was co-crystallized in the MOR X-ray crystal structure (PDB ID: 5C1M). MD also confirmed the stability of the MOR–etazene and MOR–M6 complexes. These results suggest that etazene and its metabolites may act as strong MOR agonists, highlighting the necessity of experimental validation. The insights from this study, such as key interactions between etazene and its metabolites and the MOR, will allow authorities to predict potential analogs and clarify the target–protein interactions associated with this illicit substance, granting advanced or rapid reactions to confiscating or banning potential emerging drugs.

Aspalathus linearis (Burm.f.) R. Dahlgren, commonly known as rooibos tea, was consumed traditionally by the indigenous South African inhabitants as an herbal remedy. Beside antioxidant properties, it displays antiallergic, antispasmodic, and hypoglycemic activities. An ultra-high-performance liquid chromatography method coupled with photodiode array and mass spectrometry detectors were developed for the determination of 14 phenolic constituents from leaves and stems of A. linearis. The efficient separation was performed within 30 min at a temperature of 30 °C by using C-18 column as the stationary phase and water/acetonitrile with 0.05% formic acid as the mobile phase. Method validation for linearity, repeatability, limits of detection, and limits of quantification was achieved. The limits of detection from 0.2–1 μg/mL were reported for the standard compounds. Their total content varied substantially (1.50–9.85 mg/100 mg sample) in 21 dietary supplements. The presence of regioisomers and diastereomers which co-elute on a variety of stationary phases make separation for quantification purposes challenging. This method was found to be efficient in providing low retention times and excellent resolution for this type of phytochemicals. The established method is suitable for chemical fingerprint analysis of A. linearis and cost-effective for quality control of rooibos tea products.

Morphine and codeine are the two principal opiates found in the opium poppy (Papaver somniferum L.) and are therapeutically used for pain management. Poppy seeds with low opiates are primarily used for culinary purposes due to their nutritional and sensory attributes. Intentional adulteration of poppy seeds is common, often combined with immature, less expensive, exhausted, or substituted with morphologically similar seeds, viz., amaranth, quinoa, and sesame. For a safer food supply chain, preventive measures must be implemented to mitigate contamination or adulteration. Moreover, the simultaneous analysis of P. somniferum and its adulterants is largely unknown. Pre- and post-processing further complicate the alkaloid content and may pose a significant health hazard. To address these issues, two independent methods were investigated with eight botanically verified and fifteen commercial samples. Microscopical features were established for the authenticity of raw poppy seeds. Morphine, codeine, and thebaine quantities ranged from 0.8–223, 0.2–386, and 0.1–176 mg/kg, respectively, using LC-QToF. In most cases, conventional opiates have a higher content than papaverine and noscapine. The analytical methodology provided a chemical profile of 47 compounds that can be effectively applied to distinguish poppy seeds from their adulterants and may serve as an effective tool to combat ongoing adulteration.

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