Rapid Simultaneous Determination of 11 Synthetic Cannabinoids in Urine by Liquid Chromatography-Triple Quadrupole Mass Spectrometry

Synthetic cannabinoids are a series of synthetic substances that mimic the effects of natural cannabinoids and produce a much stronger toxicity than natural cannabinoids, which have become the most abused family of new psychoactive substances. A solid-phase extractive-liquid chromatography-triple quadrupole/linear ion trap mass spectrometry method was developed to determine 11 synthetic cannabinoids in rat urine. The factors affecting recovery were optimized, and Oasis HLB was selected to extract synthetic cannabinoids simultaneously. The results showed that the linear correlation coefficients of the synthetic cannabinoids ranged from 0.993 to 0.999, and the limit of quantitation ranged from 0.01 to 0.1 ng/mL, and the spiked recoveries ranged from 69.90% to 118.39%. This method has the advantages of good purification ability, simple operation, and good reproducibility, and can be used for the high sensitivity analysis of various synthetic cannabinoids in urine.

Phase I-metabolism studies of the synthetic cannabinoids PX-1 and PX-2 using three different in vitro models

Purpose Synthetic cannabinoids (SCs), highly metabolized substances, are rarely found unmodified in urine samples. Urine screening relies on SC metabolite detection, requiring metabolism knowledge. Metabolism data can be acquired via in vitro assays, e.g., human hepatocytes, pooled human liver microsomes (pHLM), cytochrome P450 isoforms and a fungal model; or in vivo by screening, e.g., authentic human samples or rat urine. This work describes the comprehensive study of PX-1 and PX-2 in vitro metabolism using three in vitro models. 5F-APP-PICA (PX-1) and 5F-APP-PINACA (PX-2) were studied as they share structural similarity with AM-2201, THJ-2201 and 5F-AB-PINACA, the metabolism of which was described in the literature. Methods For SC incubation, pHLM, cytochrome P450 isoenzymes and the fungal model Cunninghamella elegans LENDNER (C. elegans) were used. PX-1 and PX-2 in vitro metabolites were revealed comprehensively by liquid chromatography–high-resolution mass spectrometry measurements. Results In total, 30 metabolites for PX 1 and 15 for PX-2 were detected. The main metabolites for PX-1 and PX-2 were the amide hydrolyzed metabolites, along with an indole monohydroxylated (for PX-1) and a defluorinated pentyl-monohydroxylated metabolite (for PX-2). Conclusions CYP isoforms along with fungal incubation results were in good agreement to those obtained with pHLM incubation. CYP2E1 was responsible for many of the metabolic pathways; particularly for PX-1. This study shows that all three in vitro assays are suitable for predicting metabolic pathways of synthetic cannabinoids. To establish completeness of the PX-1 and PX-2 metabolic pathways, it is not only recommended but also necessary to use different assays.

Towards the development of a chemical lure to improve the management of invading rat populations

<p>Introduced species, such as Rattus norvegicus and Rattus rattus,have contributed to the extinction of many native animals and plants in New Zealand(NZ). Current strategies exist to monitor, manage and eradicate pest species. However, these haven’t always been completely successful and tools to detect small or invading densities remain to be developed. One possible new method to address this problem is the application of chemical attractants (lures). Recently, a major urinary protein (MUP) has been shown in male miceto act as a sexual attractant. MUPs modulate the release of volatile attractants and have potential to act as attractants themselves. Our aim was to determine if a similar MUP(s) and associated volatiles are present in the urine of rats, with the prospect of creating a chemical lure to use in rat detection and eradication. Using Gas Chromatography/Mass Spectrometry, potential volatiles in rat urine have been identified. Analysis of rat urine by gel electrophoresis has shown MUPs present in both sexes. A 22.4 kDa MUP in Rattus norvegicushas been synthesised and expressed in E.coliusing recombinant DNA technology. Preliminary steps have been made towards the production of a MUP based on ship rat DNA sequence. Future behavioral trials are needed to investigate whether the synthesised protein, in the presence or absence of the urinary-derived volatiles, is a sexual attractant.</p>

Towards the development of a chemical lure to improve the management of invading rat populations

<p>Introduced species, such as Rattus norvegicus and Rattus rattus,have contributed to the extinction of many native animals and plants in New Zealand(NZ). Current strategies exist to monitor, manage and eradicate pest species. However, these haven’t always been completely successful and tools to detect small or invading densities remain to be developed. One possible new method to address this problem is the application of chemical attractants (lures). Recently, a major urinary protein (MUP) has been shown in male miceto act as a sexual attractant. MUPs modulate the release of volatile attractants and have potential to act as attractants themselves. Our aim was to determine if a similar MUP(s) and associated volatiles are present in the urine of rats, with the prospect of creating a chemical lure to use in rat detection and eradication. Using Gas Chromatography/Mass Spectrometry, potential volatiles in rat urine have been identified. Analysis of rat urine by gel electrophoresis has shown MUPs present in both sexes. A 22.4 kDa MUP in Rattus norvegicushas been synthesised and expressed in E.coliusing recombinant DNA technology. Preliminary steps have been made towards the production of a MUP based on ship rat DNA sequence. Future behavioral trials are needed to investigate whether the synthesised protein, in the presence or absence of the urinary-derived volatiles, is a sexual attractant.</p>

2C-B-Fly-NBOMe Metabolites in Rat Urine, Human Liver Microsomes and C. elegans: Confirmation with Synthesized Analytical Standards

Compounds from the N-benzylphenethylamine (NBPEA) class of novel psychoactive substances are being increasingly utilized in neurobiological and clinical research, as diagnostic tools, or for recreational purposes. To understand the pharmacology, safety, or potential toxicity of these substances, elucidating their metabolic fate is therefore of the utmost interest. Several studies on NBPEA metabolism have emerged, but scarce information about substances with a tetrahydrobenzodifuran (“Fly”) moiety is available. Here, we investigated the metabolism of 2-(8-bromo-2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b’]difuran-4-yl)-N-(2-methoxybenzyl)ethan-1-amine (2C-B-Fly-NBOMe) in three different systems: isolated human liver microsomes, Cunninghamella elegans mycelium, and in rats in vivo. Phase I and II metabolites of 2C-B-Fly-NBOMe were first detected in an untargeted screening and identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Several hypothesized metabolites were then synthesized as reference standards; knowledge of their fragmentation patterns was utilized for confirmation or tentative identification of isomers. Altogether, thirty-five phase I and nine phase II 2C-B-Fly-NBOMe metabolites were detected. Major detected metabolic pathways were mono- and poly-hydroxylation, O-demethylation, oxidative debromination, and to a lesser extent also N-demethoxybenzylation, followed by glucuronidation and/or N-acetylation. Differences were observed for the three used media. The highest number of metabolites and at highest concentration were found in human liver microsomes. In vivo metabolites detected from rat urine included two poly-hydroxylated metabolites found only in this media. Mycelium matrix contained several dehydrogenated, N-oxygenated, and dibrominated metabolites.

Investigation of Bemethyl Biotransformation Pathways by Combination of LC–MS/HRMS and In Silico Methods

Bemethyl is an actoprotector, an antihypoxant, and a moderate psychostimulant. Even though the therapeutic effectiveness of bemethyl is well documented, there is a gap in knowledge regarding its metabolic products and their quantitative and qualitative characteristics. Since 2018, bemethyl is included to the Monitoring Program of the World Anti-Doping Agency, which highlights the challenge of identifying its urinary metabolites. The objective of the study was to investigate the biotransformation pathways of bemethyl using a combination of liquid chromatography-high-resolution mass spectrometry and in silico studies. Metabolites were analyzed in a 24 h rat urine collected after oral administration of bemethyl at a single dose of 330 mg/kg. The urine samples were prepared for analysis by a procedure developed in the present work and analyzed by high performance liquid chromatography–tandem mass spectrometry. For the first time, nine metabolites of bemethyl with six molecular formulas were identified in rat urine. The most abundant metabolite was a benzimidazole–acetylcysteine conjugate; this biotransformation pathway is associated with the detoxification of xenobiotics. The BioTransformer and GLORY computational tools were used to predict bemethyl metabolites in silico. The molecular docking of bemethyl and its derivatives to the binding site of glutathione S-transferase has revealed the mechanism of bemethyl conjugation with glutathione. The findings will help to understand the pharmacokinetics and pharmacodynamics of actoprotectors and to improve antihypoxant and adaptogenic therapy.

Altered metabolic pathways elucidated via untargeted in vivo toxicometabolomics in rat urine and plasma samples collected after controlled application of a human equivalent amphetamine dose

Amphetamine is widely consumed as drug of abuse due to its stimulating and cognitive enhancing effects. Since amphetamine has been on the market for quite a long time and it is one of the most commonly used stimulants worldwide, to date there is still limited information on its effects on the metabolome. In recent years, untargeted toxicometabolomics have been increasingly used to study toxicity-related pathways of such drugs of abuse to find and identify important endogenous and exogenous biomarkers. In this study, the acute effects of amphetamine intake on plasma and urinary metabolome in rats were investigated. For this purpose, samples of male Wistar rats after a single dose of amphetamine (5 mg/kg) were compared to a control group using an untargeted metabolomics approach. Analysis was performed using normal and reversed phase liquid chromatography coupled to high-resolution mass spectrometry using positive and negative ionization mode. Statistical evaluation was performed using Welch’s two-sample t test, hierarchical clustering, as well as principal component analysis. The results of this study demonstrate a downregulation of amino acids in plasma samples after amphetamine exposure. Furthermore, four new potential biomarkers N-acetylamphetamine, N-acetyl-4-hydroxyamphetamine, N-acetyl-4-hydroxyamphetamine glucuronide, and amphetamine succinate were identified in urine. The present study complements previous data and shows that several studies are necessary to elucidate altered metabolic pathways associated with acute amphetamine exposure.

Comprehensive and Rapid Identification of Astilbin Metabolites in Rats Based on Multiple Metabolite Templates Combined with UHPLC-Q-ExactiveMass Spectrometry

Background : Astilbin, a dihydroflavonoid compound widely found in plants, exhibits a variety of pharmacological activities and biological effects. However, little is known about the metabolism of this active compound in vivo, which is very helpful for elucidating the pharmacodynamic material basis and application of astilbin. Objective: To establish a rapid profiling and identification method for metabolites in rat urine, faeces and plasma using a UHPLC-Q-Exactive mass spectrometer in negative ion mode. Methods: In this study, a simple and rapid systematic strategy and 7 metabolite templates, which were established based on previous reports, were utilized to screen and identify astilbin metabolites. Results: As a result, a total of 72 metabolites were detected and characterized, among which 33 metabolites were found in rat urine, while 28 and 38 metabolites were characterized from rat plasma and faeces, respectively. These metabolites were presumed to be generated through ring cleavage, sulfation, dehydrogenation, methylation, hydroxylation, glucuronidation, dehydroxylation and their composite reactions. Conclusion: This study illustrated the capacity of the sensitive UHPLC-Q-Exactive mass spectrometer analytical system combined with the data-mining methods to rapidly elucidate the unknown metabolism. Moreover, the comprehensive metabolism study of astilbin provided an overall metabolic profile, which will be of great help in predicting the in vivo pharmacokinetic profiles and understanding the action mechanism of this active ingredient.