Fatality involving ocfentanil documented by identification of metabolites

The use of new psychoactive substances (NPS) has rapidly increased over the last decade. In the last 4 years, producers increasingly appear to be targeting non‐controlled synthetic opioids, involving fentanyl derivatives such as ocfentanil (OcF). Identification of metabolites is of major importance in the context of NPS use, as it could improve the detection window in biological matrices in clinical and forensic intoxication cases. Hence, this work aims to report a fatality involving OcF documented by the identification of metabolites. A 30‐year‐old woman was found dead at home: an unidentified powder was found near her body and some injection sites were found at the autopsy. Toxicological analyses allowed to determine the presence of OcF in the powder, blood (3.7/3.9 μg/L, peripheral/cardiac) and in other post‐mortem samples. The most relevant potential CYP‐ and UGT‐dependent metabolites of OcF were investigated in vitro using human liver microsome incubation and liquid chromatography coupled with high resolution mass spectrometry, and subsequently confirmed in post‐mortem samples. Four OcF metabolites were produced in vitro (a mono‐hydroxylated OcF, O‐desmethylOcF, a hydroxylated desmethylOcF and a glucuronidated form of the O‐desmethylOcF), and all except the glucuronide were observed in blood and bile post‐mortem samples. Considering the relative intensity of the chromatographic peak areas, O‐desmethylOcF can be suggested to be an abundant metabolite of OcF. Nevertheless, the relevance of O‐desmethylOcF as being a complementary analytical target of OcF for OcF use detection needs further in vivo confirmation, especially through analysis of urines from users.     

Diphenidine,a new psychoactive substance: metabolic fate elucidated with rat urine and human liver preparations and detectability in urine using GC‐MS,LC‐MSn,and LC‐HR‐MSn

Diphenidine is a new psychoactive substance (NPS) sold as a ‘legal high’ since 2013. Case reports from Sweden and Japan demonstrate its current use and the necessity of applying analytical procedures in clinical and forensic toxicology. Therefore, the phase I and II metabolites of diphenidine should be identified and based on these results, the detectability using standard urine screening approaches (SUSAs) be elucidated. Urine samples were collected after administration of diphenidine to rats and analyzed using different sample workup procedures with gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐(high resolution)‐mass spectrometry (LC‐(HR)‐MS). With the same approaches incubates of diphenidine with pooled human liver microsomes (pHLM) and cytosol (pHLC) were analyzed. According to the identified metabolites, the following biotransformation steps were proposed in rats: mono‐ and bis‐hydroxylation at different positions, partly followed by dehydrogenation, N,N‐bis‐dealkylation, and combinations of them followed by glucuronidation and/or methylation of one of the bis‐hydroxy‐aryl groups. Mono‐ and bis‐hydroxylation followed by dehydrogenation could also be detected in pHLM or pHLC. Cytochrome‐P450 (CYP) isozymes CYP1A2, CYP2B6, CYP2C9, and CYP3A4 were all capable of forming the three initial metabolites, namely hydroxy‐aryl, hydroxy‐piperidine, and bis‐hydroxy‐piperidine. In incubations with CYP2D6 hydroxy‐aryl and hydroxy‐piperidine metabolites were detected. After application of a common users’ dose, diphenidine metabolites could be detected in rat urine by the authors’ GC‐MS as well as LC‐MSⁿ SUSA. Copyright © 2016 John Wiley & Sons, Ltd.     

In vitro and in vivo human metabolism of the synthetic cannabinoid AB‐CHMINACA

N‐[(1S)‐1‐(aminocarbonyl)‐2‐methylpropyl]‐1‐(cyclohexylmethyl)‐1H‐indazole‐3‐carboxamide (AB‐CHMINACA) is a recently introduced synthetic cannabinoid. At present, no information is available about in vitro or in vivo human metabolism of AB‐CHMINACA. Therefore, biomonitoring studies to screen AB‐CHMINACA consumption lack any information about the potential biomarkers (e.g. metabolites) to target. To bridge this gap, we investigated the in vitro metabolism of AB‐CHMINACA using human liver microsomes (HLMs). Formation of AB‐CHMINACA metabolites was monitored using liquid chromatography coupled to time‐of‐flight mass spectrometry. Twenty‐six metabolites of AB‐CHMINACA were detected including seven mono‐hydroxylated and six di‐hydroxylated metabolites and a metabolite resulting from N‐dealkylation of AB‐CHMINACA, all produced by cytochrome P450 (CYP) enzymes. Two carboxylated metabolites, likely produced by amidase enzymes, and five glucuronidated metabolites were also formed. Five mono‐hydroxylated and one carboxylated metabolite were likely the major metabolites detected. The involvement of individual CYPs in the formation of AB‐CHMINACA metabolites was tested using a panel of seven human recombinant CYPs (rCYPs). All the hydroxylated AB‐CHMINACA metabolites produced by HLMs were also produced by the rCYPs tested, among which rCYP3A4 was the most active enzyme. Most of the in vitro metabolites of AB‐CHMINACA were also present in urine obtained from an AB‐CHMINACA user, therefore showing the reliability of the results obtained using the in vitro metabolism experiments conducted to predict AB‐CHMINACA in vivo metabolism. The AB‐CHMINACA metabolites to target in biomonitoring studies using urine samples are now reliably identified and can be used for routine analysis. Copyright © 2015 John Wiley & Sons, Ltd.     

Phase I metabolism of the recently emerged synthetic cannabinoid CUMYL‐PEGACLONE and detection in human urine samples

Indole‐, indazole‐, or azaindole‐based synthetic cannabinoids (SCs), bearing a cumyl substituent are a widespread, recreationally used subgroup of new psychoactive substances (NPS). The latest cumyl‐derivative, CUMYL‐PEGACLONE, emerged in December 2016 on the German drug market. The substance features a novel γ‐carboline core structure, which is most likely synthesized to bypass generic legislative approaches to control SCs by prohibiting distinct core structures. Using liquid chromatography–tandem mass spectrometry and liquid chromatography–high resolution mass spectrometry techniques, the main in vivo phase I metabolites of this new substance were detected. A pooled human liver microsome assay was applied to generate in vitro reference spectra of CUMYL‐PEGACLONE phase I metabolites. Additionally, 30 urine samples were investigated leading to 22 in vivo metabolites. A metabolite mono‐hydroxylated at the γ‐carbolinone core system and a metabolite with an additional carbonyl group at the pentyl side chain were evaluated as highly specific and sensitive markers to proof CUMYL‐PEGACLONE uptake. Moreover, 3 immunochemical assays commonly used for SC screening in urine were tested for their capability of detecting the new drug but failed due to insufficient cross‐reactivity.     

Metabolic characterization of AH‐7921, a synthetic opioid designer drug: in vitro metabolic stability assessment and metabolite identification,evaluation of in silico prediction,and in vivo confirmation

AH‐7921 (3,4‐dichloro‐N‐[(1‐dimethylamino)cyclohexylmethyl]benzamide) is a new synthetic opioid and has led to multiple non‐fatal and fatal intoxications. To comprehensively study AH‐7921 metabolism, we assessed human liver microsome (HLM) metabolic stability, determined AH‐7921's metabolic profile after human hepatocytes incubation, confirmed our findings in a urine case specimen, and compared results to in silico predictions. For metabolic stability, 1 µmol/L AH‐7921 was incubated with HLM for up to 1 h; for metabolite profiling, 10 µmol/L was incubated with pooled human hepatocytes for up to 3 h. Hepatocyte samples were analyzed by liquid chromatography quadrupole/time‐of‐flight high‐resolution mass spectrometry (MS). High‐resolution full scan MS and information‐dependent acquisition MS/MS data were analyzed with MetabolitePilot? (SCIEX) using multiple data processing algorithms. The presence of AH‐7921 and metabolites was confirmed in the urine case specimen. In silico prediction of metabolite structures was performed with MetaSite? (Molecular Discovery). AH‐7921 in vitro half‐life was 13.5 ± 0.4 min. We identified 12 AH‐7921 metabolites after hepatocyte incubation, predominantly generated by demethylation, less dominantly by hydroxylation, and combinations of different biotransformations. Eleven of 12 metabolites identified in hepatocytes were found in the urine case specimen. One metabolite, proposed to be di‐demethylated, N‐hydroxylated and glucuronidated, eluted after AH‐7921 and was the most abundant metabolite in non‐hydrolyzed urine. MetaSite? correctly predicted the two most abundant metabolites and the majority of observed biotransformations. The two most dominant metabolites after hepatocyte incubation (also identified in the urine case specimen) were desmethyl and di‐desmethyl AH‐7921. Together with the glucuronidated metabolites, these are likely suitable analytical targets for documenting AH‐7921 intake. Copyright © 2015 John Wiley & Sons, Ltd.     

Phase I metabolism of the carbazole‐derived synthetic cannabinoids EG‐018, EG‐2201, and MDMB‐CHMCZCA and detection in human urine samples

Synthetic cannabinoids (SCs) are a structurally diverse class of new psychoactive substances. Most SCs used for recreational purposes are based on indole or indazole core structures. EG‐018 (naphthalen‐1‐yl(9‐pentyl‐9H‐carbazol‐3‐yl)methanone), EG‐2201 ((9‐(5‐fluoropentyl)‐9H‐carbazol‐3‐yl)(naphthalen‐1‐yl)methanone), and MDMB‐CHMCZCA (methyl 2‐(9‐(cyclohexylmethyl)‐9H‐carbazole‐3‐carboxamido)‐3,3‐dimethylbutanoate) are 3 representatives of a structural subclass of SCs, characterized by a carbazole core system. In vitro and in vivo phase I metabolism studies were conducted to identify the most suitable metabolites for the detection of these substances in urine screening. Detection and characterization of metabolites were performed by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) and liquid chromatography–electrospray ionization–quadrupole time‐of‐flight–mass spectrometry (LC–ESI–QToF–MS). Eleven in vivo metabolites were detected in urine samples positive for metabolites of EG‐018 (n = 8). A hydroxypentyl metabolite, most probably the 4‐hydroxypentyl isomer, and an N‐dealkylated metabolite mono‐hydroxylated at the carbazole core system were most abundant. In vitro studies of EG‐018 and EG‐2201 indicated that oxidative defluorination of the 5‐fluoropentyl side chain of EG‐2201 as well as dealkylation led to common metabolites with EG‐018. This has to be taken into account for interpretation of analytical findings. A differentiation between EG‐018 and EG‐2201 (n = 1) uptake is possible by the detection of compound‐specific in vivo phase I metabolites evaluated in this study. Out of 30 metabolites detected in urine samples of MDMB‐CHMCZCA users (n = 20), a metabolite mono‐hydroxylated at the cyclohexyl methyl tail is considered the most suitable compound‐specific consumption marker while a biotransformation product of mono‐hydroxylation in combination with hydrolysis of the terminal methyl ester function provides best sensitivity due to its high abundance.     

In vitro metabolism of the lignan (−)‐grandisin,an anticancer drug candidate,by human liver microsomes

(?)‐grandisin is a tetrahydrofuran lignan that displays important biological properties, such as trypanocidal, anti‐inflammatory, cytotoxic, and antitumor activities, suggesting its utility as a potential drug candidate. One important step in drug development is metabolic characterization and metabolite identification. To perform a biotransformation study of (?)‐grandisin and to determine its kinetic properties in humans, a high performance liquid chromatography (HPLC) method was developed and validated. After HPLC method validation, the kinetic properties of (?)‐grandisin were determined. (?)‐grandisin metabolism obeyed Michaelis‐Menten kinetics. The maximal reaction rate (V_max) was 3.96 ± 0.18 µmol/mg protein/h, and the Michaelis‐Menten constant (K_m) was 8.23 ± 0.99 μM. In addition, the structures of the metabolites derived from (?)‐grandisin were characterized via gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐mass spectrometry (LC‐MS) analysis. Four metabolites, 4‐O‐demethylgrandisin, 3‐O‐demethylgrandisin, 4,4′‐di‐O‐demethylgrandisin, and a metabolite that may correspond to either 3,4‐di‐O‐demethylgrandisin or 3,5‐di‐O‐demethylgrandisin, were detected. CYP2C9 isoform was the main responsible for the formation of the metabolites. These metabolites have not been previously described, demonstrating the necessity of assessing (?)‐grandisin metabolism using human‐derived materials. Copyright © 2015 John Wiley & Sons, Ltd.     

Studies on the metabolism of the fentanyl‐derived designer drug butyrfentanyl in human in vitro liver preparations and authentic human samples using liquid chromatography‐high resolution mass spectrometry (LC‐HRMS)

Increasing numbers of new psychoactive substances (NPS) among them fentanyl derivatives has been reported by the European monitoring centre for drugs and drug addiction (EMCDDA). Butyrfentanyl is a new fentanyl derivative whose potency ratio was found to be seven compared to morphine and 0.13 compared to fentanyl. Several case reports on butyrfentanyl intoxications have been described. Little is known about its pharmacokinetic properties including its metabolism. However, knowledge of metabolism is essential for analytical detection in clinical and forensic toxicology. Therefore, in vitro and in vivo phase I and phase II metabolites of butyrfentanyl were elucidated combining liquid chromatography with a qTOF high resolution mass spectrometer. Human liver microsomes and recombinant cytochrome P450 enzymes (CYP) were used for in vitro assays. Authentic blood and urine samples from a fatal intoxication case were available for in vivo comparison. Butyrfentanyl was shown to undergo extensive metabolism. Six pathways could be postulated with hydroxylation and N ‐dealkylation being the major ones in vitro . In vivo , hydroxylation of the butanamide side chain followed by subsequent oxidation to the carboxylic acid represented the major metabolic step in the authentic case. Initial screening experiments with the most relevant CYPs indicated that mainly CYP2D6 and 3A4 were involved in the primary metabolic steps. Altered CYP2D6 and CYP3A4 status might cause a different metabolite pattern, making the inclusion of metabolites of different pathways recommendable when applying targeted screening procedures in clinical and forensic toxicology. Copyright © 2016 John Wiley & Sons, Ltd.     

The detection of the urinary metabolites of 1‐[(5‐fluoropentyl)‐1H‐indol‐3‐yl]‐(2‐iodophenyl)methanone (AM‐694), a high affinity cannabimimetic,by gas chromatography – mass spectrometry

AM‐694 (1‐[(5‐fluoropentyl)‐1H‐indol‐3‐yl]‐(2‐iodophenyl)methanone), a synthetic indole‐based cannabimimetic, was first reported to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) via the Early Warning System (EWS) by Irish authorities in 2010. Using gas chromatography–mass spectrometry (GC‐MS), we have identified six AM‐694 metabolites in post‐ingestion samples. The metabolites were tentatively identified as products of (1) hydrolytic defluorination, (2) carboxylation, (3) monohydroxylation of N‐alkyl chain, and (4) hydrolytic defluorination combined with monohydroxylation of N‐alkyl chain. The parent compound was not detected. The excretion of major metabolites was observed up to 117 h following administration. One metabolite (a product of hydrolytic defluorination) was also identified in urine samples from two individuals admitted to hospital suffering from suspected drug overdoses. Copyright © 2012 John Wiley & Sons, Ltd.     

In vitro metabolism of the novel synthetic opioid agonist cyclopropylfentanyl and subsequent confirmation in authentic human samples using liquid chromatography‐high resolution mass spectrometry

Novel synthetic opioids (NSOs) are a class of novel psychoactive substances (NPS) that are growing in popularity and presenting a significant public health risk. Included in this class are derivatives of the highly potent analgesic, fentanyl. Cyclopropylfentanyl (CycP‐F) was first reported to the EU Early Warning System in August 2017, and was subsequently linked to more than 100 deaths in the US alone. Limited pharmacological, pharmacokinetic or toxicological data is available for many emerging NSOs; however we can expect novel fentanyl analogues to present limited detection windows, short onset, narrow therapeutic indices and the potential for very high potency. Knowledge of the metabolism of these drugs is essential for the identification of analytical targets for their detection. Therefore in vitro metabolites of CycP‐F were produced using human liver microsomal incubations. Metabolites formed were elucidated using liquid chromatography‐high resolution accurate mass analysis (LC‐HRAM). Identified metabolites were added to our accurate mass screening database for NPS which was utilised for subsequent screening analysis. CycP‐F and metabolites were identified in two human blood case samples. Eleven metabolites were identified in vitro, with the major metabolites produced via N‐dealkylation, monohydroxylation and N‐oxidation. Analysis of the positive case samples identified four in vivo metabolites, all of which were observed in vitro. The major metabolite identified in vitro and in vivo was the N‐dealkylated nor‐metabolite; two further mono‐hydroxylated and one dihydroxylated metabolite were detected in vivo.     

In vitro characterization of potential CYP‐ and UGT‐derived metabolites of the psychoactive drug 25B‐NBOMe using LC‐high resolution MS

One of the main challenges posed by the emergence of new psychoactive substances is their identification in human biological samples. Trying to detect the parent drug could lead to false‐negative results when the delay between consumption and sampling has been too long. The identification of their metabolites could then improve their detection window in biological matrices. Oxidative metabolism by cytochromes P450 and glucuronidation are two major detoxification pathways in humans. In order to characterize possible CYP‐ and UGT‐dependent metabolites of the 2‐(4‐bromo‐2,5‐dimethoxy‐phenyl)‐N‐[(2‐methoxyphenyl)methyl]ethanamine (25B‐NBOMe), a synthetic psychoactive drug, analyses of human liver microsome (HLM) incubates were performed using an ultra‐high performance liquid chromatography system coupled with a quadrupole‐time of flight mass spectrometry detector (UHPLC‐Q‐TOF/MS). On‐line analyses were performed using a Waters OASIS HLB column (30 x 2.1 mm, 20 µm) for the automatic sample loading and a Waters ACQUITY HSS C18 column (150 x 2 mm, 1.8 µm) for the chromatographic separation. Twenty‐one metabolites, consisting of 12 CYP‐derived and 9 UGT‐derived metabolites, were identified. O‐Desmethyl metabolites were the most abundant compounds after the phase I process, which appears to be in accordance with data from previously published NBOMe‐intoxication case reports. Although other important metabolic transformations, such as sulfation, acetylation, methylation or glutathione conjugation, were not studied and artefactual metabolites might have been produced during the HLM incubation process, the record of all the metabolite MS spectra in our library should enable us to characterize relevant metabolites of 25B‐NBOMe and allow us to detect 25B‐MBOMe users. Copyright © 2015 John Wiley & Sons, Ltd.     

Phase I metabolism of the highly potent synthetic cannabinoid MDMB‐CHMICA and detection in human urine samples

Among the recently emerged synthetic cannabinoids, MDMB‐CHMICA (methyl N ‐{[1‐(cyclohexylmethyl)‐1H ‐indol‐3‐yl]carbonyl}‐3‐methylvalinate) shows an extraordinarily high prevalence in intoxication cases, necessitating analytical methods capable of detecting drug uptake. In this study, the in vivo phase I metabolism of MDMB‐CHMICA was investigated using liquid chromatography‐electrospray ionization‐tandem mass spectrometry (LC‐ESI‐MS/MS) and liquid chromatography‐electrospray ionization‐quadrupole time‐of‐flight‐mass spectrometry (LC‐ESI‐Q ToF‐MS) techniques. The main metabolites are formed by hydrolysis of the methyl ester and oxidation of the cyclohexyl methyl side chain. One monohydroxylated metabolite, the ester hydrolysis product and two further hydroxylated metabolites of the ester hydrolysis product are suggested as suitable targets for a selective and sensitive detection in urine. All detected in vivo metabolites could be verified in vitro using a human liver microsome assay. Two of the postulated main metabolites were successfully included in a comprehensive LC‐ESI‐MS/MS screening method for synthetic cannabinoid metabolites. The screening of 5717 authentic urine samples resulted in 818 cases of confirmed MDMB‐CHMICA consumption (14%). Since the most common route of administration is smoking, smoke condensates were analyzed to identify relevant thermal degradation products. Pyrolytic cleavage of the methyl ester and amide bond led to degradation products which were also formed metabolically. This is particularly important in hair analysis, where detection of metabolites is commonly considered a proof of consumption. In addition, intrinsic activity of MDMB‐CHMICA at the CB₁ receptor was determined applying a cAMP accumulation assay and showed that the compound is a potent full agonist. Based on the collected data, an enhanced interpretation of analytical findings in urine and hair is facilitated. Copyright © 2016 John Wiley & Sons, Ltd.     

Pharmacological and metabolic characterisation of the potent σ1 receptor ligand 1′‐benzyl‐3‐methoxy‐3H‐spiro[[2]benzofuran‐1,4′‐piperidine]

Objectives The pharmacology and metabolism of the potent σ1 receptor ligand 1′‐benzyl‐3‐methoxy‐3H‐spiro[[2]benzofuran‐1,4′‐piperidine] were evaluated. Methods The compound was tested against a wide range of receptors, ion channels and neurotransmitter transporters in radioligand binding assays. Analgesic activity was evaluated using the capsaicin pain model. Metabolism by rat and human liver microsomes was investigated, and the metabolites were identified by a variety of analytical techniques. Key findings 1′‐Benzyl‐3‐methoxy‐3H‐spiro[[2]benzofuran‐1,4′‐piperidine] (compound 1 ) is a potent σ1 receptor ligand (K_i 1.14 nM) with extraordinarily high σ1/σ2 selectivity (>1100). It was selective for the σ1 receptor over more than 60 other receptors, ion channels and neurotransmitter transporters, and did not interact with the human ether‐a‐go‐go‐related gene (hERG) cardiac potassium channel. Compound 1 displayed analgesic activity against neuropathic pain in the capsaicin pain model (53% analgesia at 16 mg/kg), indicating that it is a σ1 receptor antagonist. It was rapidly metabolised by rat liver microsomes. Seven metabolites were unequivocally identified; an N‐debenzylated metabolite and a hydroxylated metabolite were the major products. Pooled human liver microsomes formed the same metabolites. Studies with seven recombinant cytochrome P450 isoenzymes revealed that CYP3A4 produced all the metabolites identified. The isoenzyme CYP2D6 was inhibited by 1 (IC50 88 nM) but did not produce any metabolites. Conclusions 1′‐Benzyl‐3‐methoxy‐3H‐spiro[[2]benzofuran‐1,4′‐piperidine] is a potent and selective σ1 receptor antagonist, which is rapidly metabolised. Metabolically more stable σ1 ligands could be achieved by stabilising the N‐benzyl substructure.     

Identification and quantification of predominant metabolites of synthetic cannabinoid MAB‐CHMINACA in an authentic human urine specimen

An autopsy case in which the cause of death was judged as drug poisoning by two synthetic cannabinoids, including MAB‐CHMINACA, was investigated. Although unchanged MAB‐CHMINACA could be detected from solid tissues, blood and stomach contents in the case, the compound could not be detected from a urine specimen. We obtained six kinds of reference standards of MAB‐CHMINACA metabolites from a commercial source. The MAB‐CHMINACA metabolites from the urine specimen of the abuser were extracted using a QuEChERS method including dispersive solid‐phase extraction, and analyzed by liquid chromatography–tandem mass spectrometry with or without hydrolysis with β‐glucuronidase. Among the six MAB‐CHMINACA metabolites tested, two predominant metabolites could be identified and quantified in the urine specimen of the deceased. After hydrolysis with β‐glucuronidase, an increase of the two metabolites was not observed. The metabolites detected were a 4‐monohydroxycyclohexylmethyl metabolite M1 (N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐((4‐hydroxycyclohexyl)methyl)‐1H–indazole‐3‐carboxamide) and a dihydroxyl (4‐hydroxycyclohexylmethyl and tert‐butylhydroxyl) metabolite M11 (N‐(1‐amino‐4‐hydroxy‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐((4‐hydroxycyclohexyl)methyl)‐1H–indazole‐3‐carboxamide). Their concentrations were 2.17 ± 0.15 and 10.2 ± 0.3 ng/mL (n = 3, each) for M1 and M11, respectively. Although there is one previous in vitro study showing the estimation of metabolism of MAB‐CHMINACA using human hepatocytes, this is the first report dealing with in vivo identification and quantification of MAB‐CHMINACA metabolites in an authentic human urine specimen.     

Tentative identification of the metabolites of (1‐(cyclohexylmethyl)‐1H‐indol‐3‐yl)‐(2,2,3,3‐tetramethylcyclopropyl)methanone,and the product of its thermal degradation,by in vitro and in vivo methods

Synthetic cannabinoids (SCs), mimicking the psychoactive effects of cannabis, consist of a vast array of structurally diverse compounds. A novel compound belonging to the SC family, (1‐(cyclohexylmethyl)‐1H‐indol‐3‐yl)‐(2,2,3,3‐tetramethylcyclopropyl)methanone (named TMCP‐CHM in this article) contains a cyclopropane ring that isomerizes during the smoking process, resulting in a ring‐opened thermal degradant with a terminal double bond in its structure. Metabolites of TMCP‐CHM were tentatively identified in vitro (after incubation of the parent substance with S9 pooled human liver fraction) and in vivo (rat experimental model) studies by accurate‐mass liquid chromatography–tandem mass spectrometry (LC–MS/MS). For the identification of the degradant metabolites, and to study biotransformation of parent substance in the human, urine and hair samples from patients, who had ingested the compound and were subsequently admitted to hospital with drug intoxications, were analyzed. Products of mono‐, di‐, trihydroxylation, carboxylation, and carboxylation combined with hydroxylation of TMCP‐CHM and its degradant were detected in human urine. Metabolism of the degradant included addition of water to the terminal double bond followed by dehydration and formation of a cyclic metabolite. Degradant metabolites prevailed in comparison with metabolites of the parent substance in each metabolite group examined, except carboxylation. N‐Dealkylated metabolites found in human urine originated only from the degradant. Most of the hydroxy metabolites were detected in human urine in both the free form and as glucuronides. The detection of monohydroxylated (M1.1‐M1.3, M/A1.10) and carboxylated/hydroxylated (M4.2, M/A4.3) metabolites of TMCP‐CHM and the hydrated form of the monohydroxylated metabolite of the degradant was found to be convenient for routine analysis.     

25C‐NBOMe and 25I‐NBOMe metabolite studies in human hepatocytes,in vivo mouse and human urine with high‐resolution mass spectrometry

25C‐NBOMe and 25I‐NBOMe are potent hallucinogenic drugs that recently emerged as new psychoactive substances. To date, a few metabolism studies were conducted for 25I‐NBOMe, whereas 25C‐NBOMe metabolism data are scarce. Therefore, we investigated the metabolic profile of these compounds in human hepatocytes, an in vivo mouse model and authentic human urine samples from forensic cases. Cryopreserved human hepatocytes were incubated for 3 h with 10 μM 25C‐NBOMe and 25I‐NBOMe; samples were analyzed by liquid chromatography high‐resolution mass spectrometry (LC‐HRMS) on an Accucore C18 column with a Thermo QExactive; data analysis was performed with Compound Discoverer software (Thermo Scientific). Mice were administered 1.0 mg drug/kg body weight intraperitoneally, urine was collected for 24 h and analyzed (with or without hydrolysis) by LC‐HRMS on an Acquity HSS T3 column with an Agilent 6550 QTOF; data were analyzed manually and with WebMetabase software (Molecular Discovery). Human urine samples were analyzed similarly. In vitro and in vivo results matched well. 25C‐NBOMe and 25I‐NBOMe were predominantly metabolized by O‐demethylation, followed by O‐di‐demethylation and hydroxylation. All methoxy groups could be demethylated; hydroxylation preferably occurred at the NBOMe ring. Phase I metabolites were extensively conjugated in human urine with glucuronic acid and sulfate. Based on these data and a comparison with synthesized reference standards for potential metabolites, specific and abundant 25C‐NBOMe urine targets are 5’‐desmethyl 25C‐NBOMe, 25C‐NBOMe and 5‐hydroxy 25C‐NBOMe, and for 25I‐NBOMe 2’ and 5’‐desmethyl 25I‐NBOMe and hydroxy 25I‐NBOMe. These data will help clinical and forensic laboratories to develop analytical methods and to interpret results. Copyright © 2016 John Wiley & Sons, Ltd.     

Different in vitro and in vivo tools for elucidating the human metabolism of alpha‐cathinone‐derived drugs of abuse

In vitro and in vivo experiments are widely used for studying the metabolism of new psychoactive substances (NPS). The availability of such data is required for toxicological risk assessments and development of urine screening approaches. This study investigated the in vitro metabolism of the 5 pyrrolidinophenone‐derived NPS alpha‐pyrrolidinobutyrophenone (alpha‐PBP), alpha‐pyrrolidinopentiothiophenone (alpha‐PVT), alpha‐pyrrolidinohexanophenone (alpha‐PHP), alpha‐pyrrolidinoenanthophenone (alpha‐PEP, PV8), and alpha‐pyrrolidinooctanophenone (alpha‐POP, PV9). First, they were incubated with pooled human liver microsomes (pHLM) or pooled human liver S9 fraction (pS9) for identification of the main phase I and II metabolites. All substances formed hydroxy metabolites and lactams. Longer alkyl chains resulted in keto group and carboxylic acid formation. Comparing these results with published data obtained using pHLM, primary human hepatocytes (PHH), and authentic human urine samples, PHH provided the most extensive metabolism. Second, enzyme kinetic studies showed that the initial metabolic steps were formed by cytochrome P450 isoforms (CYP) CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 resulting in pyrrolidine, thiophene or alkyl hydroxy metabolites depending on the length of the alkyl chain. The kinetic parameters indicated an increasing affinity of the CYP enzymes with increase of the length of the alkyl chain. These parameters were then used to calculate the contribution of a single CYP enzyme to the in vivo hepatic clearance. CYP2C19 and CYP2D6 were mainly involved in the case of alpha‐PBP and CYP1A2, CYP2C9 and CYP2C19 in the case of alpha‐PVT, alpha‐PHP, alpha‐PEP, and alpha‐POP.     

Phenethylamine‐derived new psychoactive substances 2C‐E‐FLY, 2C‐EF‐FLY,and 2C‐T‐7‐FLY: Investigations on their metabolic fate including isoenzyme activities and their toxicological detectability in urine screenings

Psychoactive substances of the 2C‐series are phenethylamine‐based designer drugs that can induce psychostimulant and hallucinogenic effects. The so‐called 2C‐FLY series contains rigidified methoxy groups integrated in a 2,3,6,7‐tetrahydrobenzo[1,2‐b:4,5‐b']difuran core. The aim of the presented work was to investigate the in vivo and in vitro metabolic fate including isoenzyme activities and toxicological detectability of the three new psychoactive substances (NPS) 2C‐E‐FLY, 2C‐EF‐FLY, and 2C‐T‐7‐FLY to allow clinical and forensic toxicologists the identification of these novel compounds. Rat urine, after oral administration, and pooled human liver S9 fraction (pS9) incubations were analyzed by liquid chromatography?high‐resolution tandem mass spectrometry (LC?HRMS/MS). By performing activity screenings, the human isoenzymes involved were identified and toxicological detectability in rat urine investigated using standard urine screening approaches (SUSAs) based on gas chromatography (GC)?MS, LC?MSⁿ, and LC?HRMS/MS. In total, 32 metabolites were tentatively identified. Main metabolic steps consisted of hydroxylation and N‐acetylation. Phase I metabolic reactions were catalyzed by CYP2D6, 3A4, and FMO3 and N‐acetylation by NAT1 and NAT2. Methoxyamine was used as a trapping agent for detection of the deaminated metabolite formed by MAO‐A and B. Interindividual differences in the metabolism of the 2C‐FLY drugs could be caused by polymorphisms of enzymes involved or drug–drug interactions. All three SUSAs were shown to be suitable to detect an intake of these NPS but common metabolites of 2C‐E‐FLY and 2C‐EF‐FLY have to be considered during interpretation of analytical findings.     

Identification of etazene (etodesnitazene) metabolites in human urine by LC-HRMS

Etazene (or etodesnitazene) is a novel and highly active synthetic opioid belonging to the rapidly evolving and emerging group of “nitazenes.” Etazene metabolites were identified through analysis of a human urine sample. The sample was obtained from a 25-year-old man who attempted suicide by taking a new psychoactive substances (NPS) cocktail purchased online and was analyzed by ultrahigh performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). Etazene metabolites were predicted with BioTransformer 3.0, and the exact masses were added to the inclusion list. Eight possible metabolites were identified in the urine sample. N- and O-deethylation were identified as the predominant metabolism routes, resulting in M1 (O-deethylated etazene; most abundant metabolite based on the peak area), M2 (N-deethylated etazene), and M3 (N,O-dideethylated etazene) metabolites. Less abundant hydroxylated products of these deethylated metabolites and etazene were also found. Additionally, in the analysis without β-glucuronidase treatment, M1- and M3-glucuronide phase II metabolites were found. As N- and O-deethylated products seem to be the predominant urinary metabolites, the detection of these metabolites in urine can be useful to demonstrate etazene exposure.     

Metabolic profiling of synthetic cannabinoid 5F‐ADB by human liver microsome incubations and urine samples using high‐resolution mass spectrometry

5F‐ADB (methyl 2‐{[1‐(5‐fluoropentyl)‐1H‐indazole‐3‐carbonyl] amino}‐3,3‐dimethylbutanoate) is a frequently abused new synthetic cannabinoid that has been sold since at least the end of 2014 on the drug market and has been classified as an illicit drug in most European countries, as well as Turkey, Japan, and the United States. In this study, the in vitro metabolism of 5F‐ADB was investigated by using pooled human liver microsomes (HLMs) assay and liquid chromatography‐high‐resolution mass spectrometry (LC–HRMS). 5F‐ADB (5 μmol/L) was incubated with HLMs for up to 3 hours, and the metabolites were identified using LC–HRMS and software‐assisted data mining. The in vivo metabolism was investigated by the analysis of 30 authentic urine samples and was compared to the data received from the in vitro metabolism study. Less than 3.3% of the 5F‐ADB parent compound remained after 1 hour of incubation, and no parent drug was detected after 3 hours. We identified 20 metabolites formed via ester hydrolysis, N‐dealkylation, oxidative defluorination, hydroxylation, dehydrogenation, further oxidation to N‐pentanoic acid and glucuronidation or a combination of these reactions in vitro. In 12 urine samples (n = 30), 5F‐ADB was detected as the parent drug. Three of the identified main metabolites 5F‐ADB carboxylic acid (M20), monohydroxypentyl‐5F‐ADB (M17), and carboxypentyl ADB carboxylic acid (M8) were suggested as suitable urinary markers. The screening of 8235 authentic urine samples for identified 5F‐ADB metabolites in vitro resulted in 3135 cases of confirmed 5F‐ADB consumption (38%).