In-depth comparison of the metabolic and pharmacokinetic behaviour of the structurally related synthetic cannabinoids AMB-FUBINACA and AMB-CHMICA in rats.

Synthetic cannabinoids receptor agonists (SCRAs) are often almost completely metabolised, and hence their pharmacokinetics should be carefully evaluated for determining the most adequate biomarker in toxicological analysis. Two structurally related SCRAs, AMB-FUBINACA and AMB-CHMICA, were selected to evaluate their in vivo metabolism and pharmacokinetics using male Sprague-Dawley rats. Brain, liver, kidney, blood (serum) and urine samples were collected at different times to assess the differences in metabolism, metabolic reactions, tissue distribution and excretion. Both compounds experimented O-demethyl reaction, which occurred more rapidly for AMB-FUBINACA. The parent compounds and O-demethyl metabolites were highly bioaccumulated in liver, and were still detected in this tissue 48 h after injection. The different indazole/indole N-functionalisation produced diverse metabolic reactions in this moiety and thus, different urinary metabolites were formed. Out of the two compounds, AMB-FUBINACA seemed to easily cross the blood-brain barrier, presenting higher brain/serum concentrations ratio than AMB-CHMICA.

How to perform spectrum-based LC-HR-MS screening for more than 1,000 NPS with HighResNPS consensus fragment ions.

INTRODUCTION: The ever-changing market of new psychoactive substances (NPS) poses challenges for laboratories worldwide. Analytical toxicologists are constantly working to keep high-resolution mass spectrometry (HR-MS) screening libraries updated for NPS. This study sought to use the online crowd-sourced HighResNPS database for spectrum comparison screening, thereby broadening its utility to all HR-MS instruments. METHOD: HighResNPS allows formation of a set of consensus fragment ions for a NPS and prioritises among multiple entries of collision-induced fragment ions. A subset of 42 NPS samples was analysed in data-independent acquisition (DIA) and data-dependent acquisition (DDA) modes on two different instruments. HighResNPS-computed spectra were generated with either Absolute (all fragment ions set to 100%) or Fractional (50% intensity reduction of former fragment ion) intensity. The acquired NPS data were analysed using the consensus library with computed ion intensities and evaluated with vendor-neutral screening software. RESULTS: Overall, of the 42 samples, 100% were identified, with 88% identified as the top candidate. Three samples had the correct candidate proposed as the second highest ranking NPS. In all three of those samples, the top proposed candidate was a positional isomer or closely related compound. Absolute intensity assignment provided identical scoring between the top two proposed compounds in two samples with DIA. DDA had a slightly higher identification rate in the spectra comparison screening with fractional intensity assignment, but no major differences were observed. CONCLUSION: The fractional intensity assignment was slightly more advantageous than the absolute assignment. It was selective between proposed candidates, showed a high identification rate and had an overall higher fragmentation scoring. The candidates proposed by the HighResNPS library spectra comparison simplify the determination of NPS for researchers and toxicologists. The database provides free monthly updates of consensus spectra, thereby enabling laboratories to stay at the forefront of NPS screening by LC-HR-MS with spectra screening software.

In vitro and in vivo metabolism and detection of 3-HO-PCP, a synthetic phencyclidine, in human samples and pooled human hepatocytes using high resolution mass spectrometry.

The new psychoactive substance (NPS) 3-HO-PCP, a phencyclidine (PCP) analog, was detected in a law enforcement seizure and in forensic samples in Denmark. Compared with PCP, 3-HO-PCP is known to be a more potent dissociative NPS, but no toxicokinetic investigations of 3-HO-PCP are yet available. Therefore, 3-HO-PCP was quantified in in vivo samples, and the following were investigated: plasma protein binding, in vitro and in vivo metabolites, and metabolic targets. All samples were separated by liquid chromatography and analyzed by mass spectrometry. The unbound fraction in plasma was determined as 0.72 ± 0.09. After in vitro incubation with pooled human hepatocytes, four metabolites were identified: a piperidine-hydroxyl-and piperidine ring opened N-dealkyl-COOH metabolite, and O-glucuronidated- and O-sulfate-conjugated metabolites. In vivo, depending on the sample and sample preparation, fewer metabolites were detected, as the O-sulfate-conjugated metabolite was not detected. The N-dealkylated-COOH metabolite was the main metabolite in the deconjugated urine sample. in vivo analytical targets in blood and brain samples were 3-HO-PCP and the O-glucuronidated metabolite, with 3-HO-PCP having the highest relative signal intensity. The drug levels of 3-HO-PCP quantified in blood were 0.013 and 0.095 mg/kg in a living and a deceased subject, respectively. The 3-HO-PCP concentrations in deconjugated urine in a sample from a living subject and in post-mortem brain were 7.8 and 0.16 mg/kg, respectively. The post mortem results showed a 1.5-fold higher concentration of 3-HO-PCP in the brain tissue than in the post mortem blood sample.

Ketamine analogues: Comparative toxicokinetic in vitro-in vivo extrapolation and quantification of 2-fluorodeschloroketamine in forensic blood and hair samples.

Recently, the new psychoactive substance (NPS) ketamine analogue 2-fluoro-deschloroketamine (2FDCK) was observed in driving-under-the-influence-of-drugs whole blood samples and in a forensic hair investigation case in Denmark. The molecular structure variations among the NPS subgroups may alter the metabolic fate and drug potency, thereby posing a threat for drug users. This study reports quantification of 2FDCK in whole blood samples and forensic hair and compares the following toxicokinetic parameters: unbound fraction (fu) and in vitro-in vivo-extrapolation (IVIVE) of hepatic clearance for ketamine, norketamine, 2FDCK, methoxetamine and deschloroketamine. The fu was investigated with ultrafiltration, while clearance studies were conducted at 1 µM with pooled human liver microsomes. Samples were analysed by liquid chromatography and mass spectrometry. For the first time, 2FDCK was determined in a concentration range between 0.005 and 0.48 mg/kg in blood samples. Segmental hair analysis demonstrated 2FDCK at concentrations from 0.007 to 0.034 ng/mg throughout the three investigated segments. Toxicokinetic comparison of the five compounds gave a fu between 0.54 and 0.84, with ketamine being the most bound and deschloroketamine being the least bound, in accordance with the logP of the compounds. Conversely, a negative correlation was observed between the molecular weight of the halogen in the ortho-position and IVIVE hepatic clearance. The IVIVE of hepatic clearance, CLparallel-tube, gave values from 18.1 to 5.44 mL/min/kg for ketamine and methoxetamine, respectively. The deschloroketamine IVIVE was disregarded due to low drug elimination under the experimental conditions used. This study provides a basis for toxicokinetic understanding of ketamine analogues.

In Vitro Metabolism and Hepatic Intrinsic Clearance of the Synthetic Cannabinoid Receptor Agonist JWH-122 and Its Four ω-Halogenated Analogues.

The number of new psychoactive substances (NPS) emerging on the illicit drug market has increased over the last decade. Halogenation of existing illicit drugs is a particular trend, with the purpose of both circumventing the law and altering the toxicodynamic and toxicokinetic profiles of the compounds. This study investigates the in vitro impact of JWH-122 ω-halogenation (fluoro, chloro, bromo and iodo) on the metabolism, apparent intrinsic hepatic clearance and analytical targets for detecting drug consumption. Metabolite profiling was conducted with pooled human liver microsomes, suspended rat hepatocytes and pooled human hepatocytes. The in vitro half-life was also determined in pooled human hepatocytes. All samples were analysed by liquid chromatography/high-resolution mass spectrometry. All compounds, except for JWH-122, showed high formation rates of phase I metabolites, predominantly ω-COOH and methylnaphthyl hydroxylation metabolites. Phase II metabolites were ω-O-glucuronides, methylnaphthyl O-glucuronides and ω-glutathione conjugates. The relative ion intensity of the glutathione conjugates increased with the ω-halogen size, with I-JWH-122 having the highest intensity. Stability studies gave a low half-life and a high intrinsic hepatic clearance for JWH-122 (1305 mL/min/kg) and MAM-2201 (1408 mL/min/kg). Cl-, Br- and I-JWH-122 showed increasing half-life with increasing ω-halogen size, with intrinsic clearance values of 235-502 mL/min/kg. The recommended analytical targets for consumption of JWH-122 or ω-halogenated JWH-122 analogues are the ω-COOH metabolites for unspecific profiling and the methylnaphthyl hydroxylated metabolites to distinguish the compounds. Furthermore, ω-halogenation with larger halogens appears to increase the intrinsic hepatic stability, thereby prolonging exposure and possibly the duration of action.