Quinolin-8-yl 4-methyl-3-(piperidine-1-sulfonyl)benzoate (QMPSB) and quinolin-8-yl 4-methyl-3-(piperidine-1-carbonyl)benzoate (QMPCB, SGT-11) are synthetic cannabinoid receptor agonists (SCRAs). Knowing their metabolic fate is crucial for the identification of toxicological screening targets and to predict possible drug interactions. The presented study aimed to identify the in vitro phase I/II metabolites of QMPSB and QMPCB and to study the contribution of different monooxygenases and human carboxylesterases by using pooled human liver S9 fraction (pHLS9), recombinant human monooxygenases, three recombinant human carboxylesterases, and pooled human liver microsomes. Analyses were carried out by liquid chromatography high-resolution tandem mass spectrometry. QMPSB and QMPCB showed ester hydrolysis, and hydroxy and carboxylic acid products were detected in both cases. Mono/dihydroxy metabolites were formed, as were corresponding glucuronides and sulfates. Most of the metabolites could be detected in positive ionization mode with the exception of some QMPSB metabolites, which could only be found in negative mode. Monooxygenase activity screening revealed that CYP2B6/CYP2C8/CYP2C9/CYP2C19/CYP3A4/CYP3A5 were involved in hydroxylations. Esterase screening showed the involvement of all investigated isoforms. Additionally, extensive non-enzymatic ester hydrolysis was observed. Considering the results of the in vitro experiments, inclusion of the ester hydrolysis products and their glucuronides and monohydroxy metabolites into toxicological screening procedures is recommended.

The two fentanyl homologs cyclopropanoyl-1-benzyl-4´-fluoro-4-anilinopiperidine (4F-Cy-BAP) and furanoyl-1-benzyl-4-anilinopiperidine (Fu-BAP) have recently been seized as new psychoactive substances (NPS) on the drugs of abuse market. As their toxicokinetic and toxicodynamic characteristics are completely unknown, this study focused on elucidating their in vitro metabolic stability in pooled human liver S9 fraction (pHLS9), their qualitative in vitro (pHLS9), and in vivo (zebrafish larvae) metabolism, and their in vitro isozyme mapping using recombinant expressed isoenzymes. Their maximum-tolerated concentration (MTC) in zebrafish larvae was studied from 0.01 to 100 µM. Their µ-opioid receptor (MOR) activity was analyzed in engineered human embryonic kidney (HEK) 293 T cells. In total, seven phase I and one phase II metabolites of 4F-Cy-BAP and 15 phase I and four phase II metabolites of Fu-BAP were tentatively identified by means of liquid chromatography high-resolution tandem mass spectrometry, with the majority detected in zebrafish larvae. N-Dealkylation, N-deacylation, hydroxylation, and N-oxidation were the most abundant metabolic reactions and the corresponding metabolites are expected to be promising analytical targets for toxicological analysis. Isozyme mapping revealed the main involvement of CYP3A4 in the phase I metabolism of 4F-Cy-BAP and in terms of Fu-BAP additionally CYP2D6. Therefore, drug-drug interactions by CYP3A4 inhibition may cause elevated drug levels and unwanted adverse effects. MTC experiments revealed malformations and changes in the behavior of larvae after exposure to 100 µM Fu-BAP. Both substances were only able to produce a weak activation of MOR and although toxic effects based on MOR activation seem unlikely, activity at other receptors cannot be excluded. Keywords In vitro and in vivo metabolism • Metabolic stability • LC-HRMS/MS • Zebrafish larvae • In vitro µ-opioid receptor activity Electronic supplementary material The online version of this article (

Flubromazolam is widely known as highly potent designer benzodiazepine (DBZD). Recently, the two flubromazolam-derived new psychoactive substances (NPS) clobromazolam and bromazolam appeared on the drugs of abuse market. Since no information concerning their toxicokinetics in humans is available, the aims of the current study were to elucidate their metabolic profile and to identify the isozymes involved in their phase I and II metabolism. In vitro incubations with pooled human liver S9 fraction were performed and analyzed by liquid chromatography coupled to orbitrap-based high-resolution tandem mass spectrometry (LC–HRMS-MS). Biosamples after ingestion of bromazolam allowed the identification of metabolites in human plasma and urine, as well as determination of bromazolam plasma concentrations by LC–HRMS-MS using the standard addition method. In total, eight clobromazolam metabolites were identified in vitro, as well as eight bromazolam metabolites in vitro and in vivo. Predominant metabolic steps were hydroxylation, glucuronidation, and combinations thereof. Alpha-hydroxy bromazolam glucuronide and bromazolam N-glucuronide are recommended as screening targets in urine. Bromazolam and its alpha-hydroxy metabolite are recommended, if conjugate cleavage is part of the sample preparation procedure. The bromazolam plasma concentrations were determined to be 6 and 29 µg/L, respectively. Several cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) isozymes were shown to catalyze their metabolic transformations. CYP3A4 was involved in the formation of all phase I metabolites of both NPS, while UGT1A4 and UGT2B10 catalyzed their N-glucuronidation. Several UGT isoforms catalyzed the glucuronidation of the hydroxy metabolites. In conclusion, the determined bromazolam plasma concentrations in the low µg/L range underlined the need for sensitive analytical methods and the importance of suitable urine screening procedures including DBZD metabolites as targets. Such an analytical strategy should be also applicable for clobromazolam.

The still increasing number of drugs of abuse, particularly the so-called new psychoactive substances (NPS), poses an analytical challenge for clinical and forensic toxicologists but also for doping control. NPS usually belong to various classes such as synthetic cannabinoids, phenethylamines, opioids, or benzodiazepines. Like other xenobiotics, NPS undergo absorption, distribution, metabolism, and excretion processes after consumption, but only very limited data concerning their toxicokinetics and safety properties is available once they appear on the market. The inclusion of metabolites in mass spectral libraries is often crucial for the detection of NPS especially in urine screening approaches. Authentic human samples may represent the gold standard for identification of metabolites but are often not available and clinical studies cannot be performed due to ethical concerns. However, numerous alternative in vitro and in vivo models are available. This trends article will give an overview on selected models, discuss current studies, and highlight recent developments.

The growing number of new synthetic opioids (NSO) on the new psychoactive substances (NPS) market bears new challenges in toxicology. As their toxicodynamics and particularly their toxicokinetics are usually unknown, impact on human health is not yet fully understood. Detection of the 2 NSO cyclopentanoyl-fentanyl (CP-F) and tetrahydrofuranoyl-fentanyl (THF-F) was first reported in 2016. Both were involved in several fatal intoxication cases, but no detailed information about their toxicological characteristics is available so far. The main purpose of this study was therefore to investigate the in vitro toxicokinetics and in vivo analytical toxicology of CP-F and THF-F by means of liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). These studies included metabolic stability, phase I and II metabolism, isozyme mapping, plasma protein binding and detectability in LC-HRMS/MS standard urine screening approaches (SUSA) using rat urine samples. In total, 12 phase I metabolites of CP-F and 13 of THF-F were identified, among them 9 metabolites described for the first time. Overall, N-dealkylations, hydroxylations and dihydroxylations were the main metabolic reactions. The cytochrome P450 (CYP) isozymes mainly involved were CYP2D6 and CYP3A4, leading to elevated drug levels and intoxications in CYP2D6 poor metabolizers. CP-F showed a high plasma protein binding of 99%, which may increase the risk of toxicity by simultaneous intake of other highly bound drugs. Detectability studies showed that neither the parent compounds nor their metabolites were detectable in rat urine using LC-HRMS/MS SUSA. However, a more sophisticated analytical strategy was successfully applied and should be used for analytical confirmation of an intake of CP-F and/or THF-F.

Due to the risk of new synthetic opioids (NSOs) for human health, the knowledge of their toxicokinetic characteristics is important for clinical and forensic toxicology. U‐48800 is an NSO structurally non‐related to classical opioids such as morphine or fentanyl and offered for abuse. As toxicokinetic data of U‐48800 is not currently available, the aims of this study were to identify the in vitro metabolites of U‐48800 in pooled human liver S9 fraction (pS9), to map the isozymes involved in the initial metabolic steps, and to determine further toxicokinetic data such as metabolic stability, including the in vitro half‐life (t1/2), and the intrinsic (CLint) and hepatic clearance (CLh). Furthermore, drug detectability studies in rat urine should be done using hyphenated mass spectrometry. In total, 13 phase I metabolites and one phase II metabolite were identified. N‐Dealkylation, hydroxylation, and their combinations were the predominant metabolic reactions. The isozymes CYP2C19 and CYP3A4 were mainly involved in these initial steps. CYP2C19 poor metabolizers may suffer from an increased U‐48800 toxicity. The in vitro t1/2 and CLint could be rated as moderate, compared to structural related compounds. After administration of an assumed consumer dose to rats, the unchanged parent compound was found only in very low abundance but three metabolites were detected additionally. Due to species differences, metabolites found in rats might be different from those in humans. However, phase I metabolites found in rat urine, the parent compound, and additionally the N‐demethyl metabolite should be used as main targets in toxicological urine screening approaches.

Intake of synthetic cannabinoids (SC), one of the largest classes of new psychoactive substances, was reported to be associated with acute liver damage but information about their hepatotoxic potential is limited. The current study aimed to analyze the hepatotoxicity including the metabolism-related impact of JWH-200, A-796260, and 5F-EMB-PINACA in HepG2 cells allowing a tentative assessment of different SC subclasses. A formerly adopted high-content screening assay (HCSA) was optimized using a fully automated epifluorescence microscope. Metabolism-mediated effects in the HCSA were additionally investigated using the broad CYP inhibitor 1-aminobenzotriazole. Furthermore, phase I metabolites and isozymes involved were identified by in vitro assays and liquid chromatography–high-resolution tandem mass spectrometry. A strong cytotoxic potential was observed for the naphthoylindole SC JWH-200 and the tetramethylcyclopropanoylindole compound A-796260, whereas the indazole carboxamide SC 5F-EMB-PINACA showed moderate effects. Numerous metabolites, which can serve as analytical targets in urine screening procedures, were identified in pooled human liver microsomes. Most abundant metabolites of JWH-200 were formed by N-dealkylation, oxidative morpholine cleavage, and oxidative morpholine opening. In case of A-796260, most abundant metabolites included an oxidative morpholine cleavage, oxidative morpholine opening, hydroxylation, and dihydroxylation followed by dehydrogenation. Most abundant 5F-EMB-PINACA metabolites were generated by ester hydrolysis plus additional steps such as oxidative defluorination and hydroxylation. To conclude, the data showed that a hepatotoxicity of the investigated SC cannot be excluded, that metabolism seems to play a minor role in the observed effects, and that the extensive phase I metabolism is mediated by several isozymes making interaction unlikely.

No abstract