Metabolism of the tryptamine‐derived new psychoactive substances 5‐MeO‐2‐Me‐DALT, 5‐MeO‐2‐Me‐ALCHT,and 5‐MeO‐2‐Me‐DIPT and their detectability in urine studied by GC–MS,LC–MSn,and LC‐HR‐MS/MS

Many N,N‐dialkylated tryptamines show psychoactive properties and were encountered as new psychoactive substances. The aims of the presented work were to study the phase I and II metabolism and the detectability in standard urine screening approaches (SUSA) of 5‐methoxy‐2‐methyl‐N,N‐diallyltryptamine (5‐MeO‐2‐Me‐DALT), 5‐methoxy‐2‐methyl‐N‐allyl‐N‐cyclohexyltryptamine (5‐MeO‐2‐Me‐ALCHT), and 5‐methoxy‐2‐methyl‐N,N‐diisopropyltryptamine (5‐MeO‐2‐Me‐DIPT) using gas chromatography–mass spectrometry (GC–MS), liquid chromatography coupled with multistage accurate mass spectrometry (LC–MSⁿ), and liquid chromatography‐high‐resolution tandem mass spectrometry (LC‐HR‐MS/MS). For metabolism studies, urine was collected over a 24 h period after administration of the compounds to male Wistar rats at 20 mg/kg body weight (BW). Phase I and II metabolites were identified after urine precipitation with acetonitrile by LC‐HR‐MS/MS. 5‐MeO‐2‐Me‐DALT (24 phase I and 12 phase II metabolites), 5‐MeO‐2‐Me‐ALCHT (24 phase I and 14 phase II metabolites), and 5‐MeO‐2‐Me‐DIPT (20 phase I and 11 phase II metabolites) were mainly metabolized by O‐demethylation, hydroxylation, N‐dealkylation, and combinations of them as well as by glucuronidation and sulfation of phase I metabolites. Incubations with mixtures of pooled human liver microsomes and cytosols (pHLM and pHLC) confirmed that the main metabolic reactions in humans and rats might be identical. Furthermore, initial CYP activity screenings revealed that CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were involved in hydroxylation, CYP2C19 and CYP2D6 in O‐demethylation, and CYP2C19, CYP2D6, and CYP3A4 in N‐dealkylation. For SUSAs, GC–MS, LC‐MSⁿ, and LC‐HR‐MS/MS were applied to rat urine samples after 1 or 0.1 mg/kg BW doses, respectively. In contrast to the GC–MS SUSA, both LC–MS SUSAs were able to detect an intake of 5‐MeO‐2‐Me‐ALCHT and 5‐MeO‐2‐Me‐DIPT via their metabolites following 1 mg/kg BW administrations and 5‐MeO‐2‐Me‐DALT following 0.1 mg/kg BW dosage. Copyright © 2017 John Wiley & Sons, Ltd.     

Interactions of phenethylamine‐derived psychoactive substances of the 2C‐series with human monoamine oxidases

Psychoactive substances of the 2C‐series (2Cs) are phenethylamine‐derived designer drugs that can induce psychostimulant and hallucinogenic effects. Chemically, the classic 2Cs contain two methoxy groups in positions 2 and 5 of the phenyl ring, whereas substances of the so‐called FLY series contain rigidified methoxy groups integrated in a 2,3,6,7‐tetrahydrobenzo[1,2‐b:4,5‐b’]difuran core. One of the pharmacological features that has not been investigated in detail is the inhibition of monoamine oxidase (MAO). Inhibition of this enzyme can cause elevated monoamine levels that have been associated with adverse events such as agitation, nausea, vomiting, tachycardia, hypertension, or seizures. The aim of this study was to extend the knowledge surrounding the potential of MAO inhibition for 17 test drugs, which consisted of 12 2Cs (2C‐B, 2C‐D, 2C‐E, 2C‐H, 2C‐I, 2C‐N, 2C‐P, 2C‐T‐2, 2C‐T‐7, 2C‐T‐21, bk‐2C‐B, and bk‐2C‐I) and five FLY analogs (2C‐B‐FLY, 2C‐E‐FLY, 2C‐EF‐FLY, 2C‐I‐FLY, and 2C‐T‐7‐FLY). The extent of MAO inhibition was assessed using an established in vitro procedure based on heterologously expressed enzymes and analysis by hydrophilic interaction liquid chromatography–high resolution tandem mass spectrometry. Thirteen test drugs showed inhibition potential for MAO‐A and 11 showed inhibition of MAO‐B. In cases where MAO‐A IC₅₀ values were determined, values ranged from 10 to 125 μM (7 drugs) and from 1.7 to 180 μM for MAO‐B (9 drugs). In the absence of detailed clinical information on most test drugs, it is concluded that a pharmacological contribution of MAO inhibition cannot be excluded and that further studies are warranted.     

The metabolic fate of two new psychoactive substances − 2‐aminoindane and N‐methyl‐2‐aminoindane − studied in vitro and in vivo to support drug testing

The aim of this study was to characterize the in vitro and in vivo metabolism of 2‐aminoindane (2,3‐dihydro‐1H‐inden‐2‐amine, 2‐AI), and N‐methyl‐2‐aminoindane (N‐methyl‐2,3‐dihydro‐1H‐inden‐2‐amine, NM‐2‐AI) after incubations using pooled human liver microsomes (pHLMs), pooled human liver S9 fraction (pS9), and rat urine after oral administration. After analysis using liquid chromatography coupled to high‐resolution mass spectrometry, pHLM incubations revealed that 2‐AI was left unmetabolized, while NM‐2‐AI formed a hydroxylamine and diastereomers of a metabolite formed after hydroxylation in beta position. Incubations using pS9 led to the formation of an acetyl conjugation in the case of 2‐AI and merely a hydroxylamine for NM‐2‐AI. Investigations on rat urine showed that 2‐AI was hydroxylated also forming diasteromers as described for NM‐2‐AI or acetylated similar to incubations using pS9. All hydroxylated metabolites of NM‐2‐AI except the hydroxylamine were found in rat urine as additional sulfates. Assuming similar patterns in humans, urine screening procedures might be focused on the parent compounds but should also include their metabolites. An activity screening using human recombinant N‐acetyl transferase (NAT) isoforms 1 and 2 revealed that 2‐AI was acetylated exclusively by NAT2, which is polymorphically expressed.     

Metabolic fate of the new synthetic cannabinoid 7’N‐5F‐ADB in rat,human, and pooled human S9 studied by means of hyphenated high‐resolution mass spectrometry

New psychoactive substances (NPS) are an important issue in clinical/forensic toxicology. 7’N‐5F‐ADB, a synthetic cannabinoid derived from 5F‐ADB, appeared recently on the market. Up to now, no data about its mass spectral fragmentation pattern, metabolism, and thus suitable targets for toxicological urine screenings have been available. Therefore, the aim of this study was to elucidate the metabolic fate of 7’N‐5F‐ADB in rat, human, and pooled human S9 (pS9). The main human urinary excretion products, which can be used as targets for toxicological screening procedures, were identified by Orbitrap (OT)‐based liquid chromatography–high resolution‐tandem mass spectrometry (LC–HRMS/MS). In addition, possible differentiation of 7’N‐5F‐ADB and 5F‐ADB via LC–HRMS/MS was studied. Using the in vivo and in vitro models for metabolism studies, 36 metabolites were tentatively identified. 7’N‐5F‐ABD was extensively metabolized in rat and human with minor species differences observed. The unchanged parent compound could be found in human urine but metabolites were far more abundant. The most abundant ones were the hydrolyzed ester (M5), the hydrolyzed ester in combination with hydroxylation of the tertiary butyl part (M11), and the hydrolyzed ester in addition to glucuronidation (M30). Besides the parent compound, these metabolites should be used as targets for urine‐based toxicological screening procedures. Two urine‐paired human plasma samples contained mainly the parent compound (c = 205 μg/L, 157 μg/L) and, at a higher abundance, the compound after ester hydrolysis (M5). In pS9 incubations, the parent compound, M5, and M30 were detectable among others. Furthermore, a differentiation of both compounds was possible due to different retention times and fragmentation patterns.     

Air monitoring for illegal drugs including new psychoactive substances: A review of trends,techniques and thermal degradation products

The detection of illicit psychotropic substances in both indoor and outdoor air is a challenging analytical discipline, and the data from such investigation may provide intelligence in a variety of fields. Applications of drug monitoring in air include providing data on national and international drug consumption trends, as monitored by organisations such as the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) and the United Nations Office on Drugs and Crime (UNODC). Air monitoring enables mapping of illicit drug manufacturing, dealing or consumption in cities and the identification of emergent compounds including the recent proliferation of new psychoactive substances (NPS). The rapid spread of NPS has changed the global drug market with greater diversity and dynamic spread of such compounds over several nations. This review provides an up to date analysis of key thematic areas within this analytical discipline. The process of how illicit psychotropic substances spread from emission sources to the atmosphere is considered alongside the sampling and analytical procedures involved. Applications of the technique applied globally are reviewed with studies ranging from the analysis of individual dwellings through to major international air-monitoring campaigns providing evidence on global drug trends. Finally, we consider thermal breakdown products of illicit psychotropic substances including NPS that are released upon heating, combustion or vaping and related potential for exposure to these compounds in the air.     

Liquid chromatography–tandem mass spectrometry screening method using information‐dependent acquisition of enhanced product ion mass spectra for synthetic cannabinoids including metabolites in urine

The total number of synthetic cannabinoids (SCs) – a group of new psychoactive substances (NPS) – is increasing every year. The rapidly changing market demands the latest analytical methods to detect the consumption of SCs in clinical or forensic toxicology. In addition, SC metabolites must also be included in a screening procedure, if detection in urine is asked for. For that purpose, an easy and fast qualitative liquid chromatography—tandem mass spectrometry (LC?MS/MS) urine screening method for the detection of 75 SCs and their metabolites was developed and validated in terms of matrix effects, recovery, and limits of identification for a selection of analytes. SC metabolites were generated using in vitro human liver microsome assays, identified by liquid chromatography?high resolution tandem mass spectrometry (LC?HRMS/MS) and finally included to the MS/MS spectra in‐house library. Sample preparation was performed using a cheap‐and‐easy salting‐out liquid–liquid extraction (SALLE) after enzymatic hydrolysis. Method validation showed good selectivity, limits of identification down to 0.05 ng/mL, recoveries above 80%, and matrix effects within ±25% for the selected analytes. Applicability of the method was demonstrated by detection of SC metabolites in authentic urine samples.     

Toxicokinetics and analytical toxicology of the abused opioid U‐48800 — in vitro metabolism,metabolic stability,isozyme mapping,and plasma protein binding

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 (t_1/2), and the intrinsic (CL_int) and hepatic clearance (CL_h). 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 t_1/2 and CL_int 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.     

Characterization and in vitro phase I microsomal metabolism of designer benzodiazepines: An update comprising flunitrazolam,norflurazepam, and 4'‐chlorodiazepam (Ro5–4864)

The number of newly appearing benzodiazepine derivatives on the new psychoactive substances (NPS) drug market has increased over the last couple of years totaling 23 ‘designer benzodiazepines’ monitored at the end of 2017 by the European Monitoring Centre for Drugs and Drug Addiction. In the present study, three benzodiazepines [flunitrazolam, norflurazepam, and 4′‐chlorodiazepam (Ro5–4864)] offered as ‘research chemicals' on the Internet were characterized and their main in vitro phase I metabolites tentatively identified after incubation with pooled human liver microsomes. For all compounds, the structural formula declared by the vendor was confirmed by gas chromatography?mass spectrometry (GC–MS), liquid chromatography?tandem mass spectrometry (LC MS/MS), liquid chromatography?quadrupole time of flight?mass spectrometry (LC?QTOF?MS) analysis and nuclear magnetic resonance (NMR) spectroscopy. The metabolic steps of flunitrazolam were monohydroxylation, dihydroxylation, and reduction of the nitro function. The detected in vitro phase I metabolites of norflurazepam were hydroxynorflurazepam and dihydroxynorflurazepam. 4’‐Chlorodiazepam biotransformation consisted of N‐dealkylation and hydroxylation. It has to be noted that 4′‐chlorodiazepam and its metabolites show almost identical LC–MS/MS fragmentation patterns to diclazepam and its metabolites (delorazepam, lormetazepam, and lorazepam), making a sufficient chromatographic separation inevitable. Sale of norflurazepam, the metabolite of the prescribed benzodiazepines flurazepam and fludiazepam, presents the risk of incorrect interpretation of analytical findings.     

Fatal intoxication by 5F–ADB and diphenidine: Detection,quantification, and investigation of their main metabolic pathways in humans by LC/MS/MS and LC/Q‐TOFMS

Despite the implementation of a new blanket scheduling system in 2013, new psychoactive substance (NPS) abuse remains a serious social concern in Japan. We present a fatal intoxication case involving 5F–ADB (methyl 2‐[1‐(5‐fluoropentyl)‐1H–indazole‐3‐carboxamido]‐3,3‐dimethylbutanoate) and diphenidine. Postmortem blood screening by liquid chromatography/quadrupole time‐of‐flight mass spectrometry (LC/Q‐TOFMS) in the information‐dependent acquisition mode only detected diphenidine. Further urinary screening using an in‐house database containing NPS and metabolites detected not only diphenidine but also possible 5F–ADB metabolites; subsequent targeted screening by LC/tandem mass spectrometry (LC/MS/MS) allowed for the detection of a very low level of unchanged 5F–ADB in postmortem heart blood. Quantification by standard addition resulted in the postmortem blood concentrations being 0.19 ± 0.04 ng/mL for 5F–ADB and 12 ± 2.6 ng/mL for diphenidine. Investigation of the urinary metabolites revealed pathways involving ester hydrolysis (M1) and oxidative defluorination (M2), and further oxidation to the carboxylic acid (M3) for 5F–ADB. Mono‐ and di‐hydroxylated diphenidine metabolites were also found. The present case demonstrates the importance of urinary metabolite screening for drugs with low blood concentration. Synthetic cannabinoids (SCs) fluorinated at the terminal N‐alkyl position are known to show higher cannabinoid receptor affinity relative to their non‐fluorinated analogues; 5F–ADB is no exception with high CB₁ receptor activity and much greater potency than Δ⁹‐THC and other earlier SCs, thus we suspect its acute toxicity to be high compared to other structurally related SC analogues. The low blood concentration of 5F–ADB may be attributed to enzymatic and/or non‐enzymatic degradation, and further investigation into these possibilities is underway.     

Identification of AB‐FUBINACA metabolites in authentic urine samples suitable as urinary markers of drug intake using liquid chromatography quadrupole tandem time of flight mass spectrometry

Synthetic cannabinoids are a group of psychoactive drugs presently widespread among drug users in Europe. Analytical methods to measure these compounds in urine are in demand as urine is a preferred matrix for drug testing. For most synthetic cannabinoids, the parent compounds are rarely detected in urine. Therefore urinary metabolites are needed as markers of drug intake. AB‐FUBINACA was one of the top three synthetic cannabinoids most frequently found in seizures and toxicological drug screening in Sweden (2013–2014). Drug abuse is also reported from several other countries such as the USA and Japan. In this study, 28 authentic case samples were used to identify urinary markers of AB‐FUBINACA intake using liquid chromatography quadrupole tandem time of flight mass spectrometry and human liver microsomes. Three metabolites suitable as markers of drug intake were identified and at least two of them were detected in all but one case. In total, 15 urinary metabolites of AB‐FUBINACA were reported, including hydrolxylations on the indazole ring and the amino‐oxobutane moiety, dealkylations and hydrolysis of the primary amide. No modifications on the fluorobenzyl side‐chain were observed. The parent compound was detected in 54% of the case samples. Also, after three hours of incubation with human liver microsomes, 77% of the signal from the parent compound remained. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Cyp3a11‐mediated testosterone‐6β‐hydroxylation decreased,while UGT1a9‐mediated propofol O‐glucuronidation increased,in mice with diabetes mellitus

The db/db mouse is one of the most popular animal models for type 2 diabetes mellitus, but changes in the activities of important P450s and UGTs are still not completely clear. This study was designed to investigate the alterations of major hepatic cytochrome P450s and UDP‐glucuronyltransferase enzymes in db/db mice. Mouse liver microsomes (MLMs) were obtained from male db/db mice and their wild type littermates. After incubation of the substrates separately with MLMs, the samples were pooled and analysed by high‐throughput liquid chromatography–tandem mass spectrometry system for the simultaneous study of nine phase I metabolic reactions and three glucuronidation conjugation reactions to determine the activity of the metabolic enzymes. Compared with normal controls, the Cl_int estimate for testosterone‐6β‐hydroxylation was lower (46%) (p < 0.05), while the V_max and Cl_int estimates for propofol O‐glucuronidation were 5‐fold higher (p < 0.01) in the liver microsomes from db/db mice. There was no significant difference in phase I metabolic reactions of phenacetin‐O‐deethylation, coumarin‐7‐hydroxylation, bupropion‐hydroxylation, omeprazole‐5‐hydroxylation, dextromethorphan‐O‐demethylation, tolbutamide‐4‐hydroxylation, chlorzoxazone‐6‐hydroxylation and midazolam‐1‐hydroxylation and in glucuronidation reactions of estradiol 3‐O‐glucuronidation, and 3‐azido‐3‐deoxythymidine glucuronidation. The data suggest that, in db/db mice, the activity of Cyp3a11, catalysing testosterone‐6β‐hydroxylation, decreased, while the activity of UGT1a9, catalysing propofol O‐glucuronidation, increased. Copyright © 2016 John Wiley & Sons, Ltd.     

Lefetamine‐derived designer drugs N‐ethyl‐1,2‐diphenylethylamine (NEDPA) and N‐iso‐propyl‐1,2‐diphenylethylamine (NPDPA): Metabolism and detectability in rat urine using GC‐MS,LC‐MSn and LC‐HR‐MS/MS

N‐Ethyl‐1,2‐diphenylethylamine (NEDPA) and N‐iso‐propyl‐1,2‐diphenylethylamine (NPDPA) are two designer drugs, which were confiscated in Germany in 2008. Lefetamine (N,N‐dimethyl‐1,2‐diphenylethylamine, also named L‐SPA), the pharmaceutical lead of these designer drugs, is a controlled substance in many countries. The aim of the present work was to study the phase I and phase II metabolism of these drugs in rats and to check for their detectability in urine using the authors’ standard urine screening approaches (SUSA). For the elucidation of the metabolism, rat urine samples were worked up with and without enzymatic cleavage, separated and analyzed by gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐high resolution‐tandem mass spectrometry (LC‐HR‐MS/MS). According to the identified metabolites, the following metabolic pathways for NEDPA and NPDPA could be proposed: N‐dealkylation, mono‐ and bis‐hydroxylation of the benzyl ring followed by methylation of one of the two hydroxy groups, combinations of these steps, hydroxylation of the phenyl ring after N‐dealkylation, glucuronidation and sulfation of all hydroxylated metabolites. Application of a 0.3 mg/kg BW dose of NEDPA or NPDPA, corresponding to a common lefetamine single dose, could be monitored in rat urine using the authors’ GC‐MS and LC‐MSⁿ SUSA. However, only the metabolites could be detected, namely N‐deethyl‐NEDPA, N‐deethyl‐hydroxy‐NEDPA, hydroxy‐NEDPA, and hydroxy‐methoxy‐NEDPA or N‐de‐iso‐propyl‐NPDPA, N‐de‐iso‐propyl‐hydroxy‐NPDPA, and hydroxy‐NPDPA. Assuming similar kinetics, an intake of these drugs should also be detectable in human urine. Copyright © 2014 John Wiley & Sons, Ltd.     

Toxicokinetics of new psychoactive substances: plasma protein binding,metabolic stability,and human phase I metabolism of the synthetic cannabinoid WIN 55,212‐2 studied using in vitro tools and LC‐HR‐MS/MS

The new psychoactive substance WIN 55,212‐2 ((R)‐(+)‐[2,3‐dihydro‐5‐methyl‐3‐(4‐morpholinylmethyl)pyrrolo‐[1,2,3‐de]‐1,4‐benzoxazin‐6‐yl]‐1‐napthalenylmethanone) is a potent synthetic cannabinoid receptor agonist. The metabolism of WIN 55,212‐2 in man has never been reported. Therefore, the aim of this study was to identify the human in vitro metabolites of WIN 55,212‐2 using pooled human liver microsomes and liquid chromatography‐high resolution‐tandem mass spectrometry (LC‐HR‐MS/MS) to provide targets for toxicological, doping, and environmental screening procedures. Moreover, a metabolic stability study in pooled human liver microsomes (pHLM) was carried out. In total, 19 metabolites were identified and the following partly overlapping metabolic steps were deduced: degradation of the morpholine ring via hydroxylation, N‐ and O‐dealkylation, and oxidative deamination, hydroxylations on either the naphthalene or morpholine ring or the alkyl spacer with subsequent oxidation, epoxide formation with subsequent hydrolysis, or combinations. In conclusion, WIN 55,212‐2 was extensively metabolized in human liver microsomes incubations and the calculated hepatic clearance was comparably high, indicating a fast and nearly complete metabolism in vivo. This is in line with previous findings on other synthetic cannabinoids. Copyright © 2016 John Wiley & Sons, Ltd.