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With a rising demand of cocaine over the last years, it is likely that unregulated new psychoactive substances with similar effects such as indatraline ((1R,3S)-3-(3,4-dichlorophenyl)-N-methyl-2,3-dihydro-1H-inden-1-amine) and troparil (Methyl (1R,2S,3S,5S)-8-methyl-3-phenyl-8-azabicyclo[3.2.1]octane-2-carboxylate) become popular as well. Both substances share a similar pharmacological profile as cocaine, while their potency is higher, and their duration of action is longer. This study investigated their metabolic fate in rat urine and incubations using pooled human liver S9 fraction (pHLS9). Indatraline formed two phase I and four phase II metabolites, with aromatic hydroxylation and glucuronidation being the main metabolic steps. All metabolites were detected in rat urine, while the parent compound was not detectable. Although low in abundance, indatraline metabolites were well identifiable due to their specific isotopic patterns caused by chlorine. Troparil formed four phase I and three phase II metabolites, with demethylation being the main metabolic step. Hydroxylation of the tropane ring, the phenyl ring, and combinations of these steps, as well as glucuronidation, were found. Phase I metabolites were detectable in rat urine and pHLS9, while phase II metabolites were only detectable in rat urine. [ABSTRACT FROM AUTHOR]

New synthetic opioids are an increasing challenge for clinical and forensic toxicologists that developed over the recent years. Desmethylmoramide (DMM), a structural analogue of methadone, is one of the most recent appearances on the drug market. This study investigated its metabolic fate in rat and pooled human liver S9 fraction (pHLS9) to allow the identification of suitable urinary screening targets beyond the parent compound. The analysis of rat urine after the administration of DMM revealed five metabolites, which were the result of pyrrolidine ring or morpholine ring hydroxylation and combinations of them. Additionally, an N′,N‐bisdesalkyl metabolite was formed. Incubations of DMM using pHLS9 revealed a pyrrolidine hydroxy metabolite, as well as an N‐oxide. No Phase II metabolites were detected in either rat urine or incubations using pHLS9. The metabolism of DMM did in part comply with that of its archetype dextromoramide (DXM). Although morpholine ring hydroxylation and N‐oxidation were described for DXM and detected for DMM, phenyl ring hydroxylation was not found for DMM but described for DXM. An analysis of 24 h pooled rat urine samples after DMM administration identified the hydroxy and dihydroxy metabolite as the most abundant excretion products, and they may, thus, serve as screening targets, as the parent compound was barely detectable. [ABSTRACT FROM AUTHOR]

Tryptamines represent a group of hallucinogenic new psychoactive substances with increasing prevalence. Unfortunately, only limited data concerning their toxicology and bioanalysis are available as tryptamines are not included in routine screening procedures in many laboratories. In order to expand the current knowledge, we report a non-fatal clinical toxicology case involving the synthetic tryptamine 4-HO-MET (4-hydroxy- N -methyl- N -ethyl-tryptamine, 3-{2-[ethyl(methyl)amino]ethyl}-1H-indol-4-ol, metocin or methylcybin). As only blood of the intoxicated patient was available, our systematic blood plasma screening approaches based on gas chromatography–mass spectrometry (GC–MS) and liquid chromatography (LC) coupled to low-resolution linear ion trap mass spectrometry (ITMSn) or high-resolution tandem mass spectrometry (HRMS-MS) were conducted. The ingestion of the synthetic tryptamine 4-HO-MET could be revealed by blood plasma analysis using both LC-based systematic screening approaches. However, 4-HO-MET was not detected by GC–MS. Furthermore, the detection of metabolites, which may be used to confirm an intake of the parent compound 4-HO-MET, was only successful using LC–HRMS-MS most probably due to its increased sensitivity compared to LC–ITMSn. A total of four metabolites were detected in blood, including N -demethyl-, oxo- and hydroxy-4-HO-MET, as well as the N -oxide. Finally, LC–HRMS-MS analysis revealed a plasma concentration of 193 ng/mL for 4-HO-MET using the standard addition method. The presented data may help clinical and forensic toxicologists with the interpretation of future cases involving synthetic tryptamines, especially if only blood samples are available. [ABSTRACT FROM AUTHOR]

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 phase 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 the ingestion of bromazolam allowed the identification of metabolites in human plasma and urine as well as the 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 uridine 5'-diphospho-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 micrograms per liter 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. [ABSTRACT FROM AUTHOR]

Prerequisites for the reliable identification of substances in terms of forensic and clinical toxicology or doping control include knowledge about their metabolism and their excretion patterns in urine. N -Ethyl- N -propyltryptamine (N -ethyl- N -[2-(1H-indol-3-yl)ethyl]propan-1-amine, EPT) is an N,N- dialkylated tryptamine derivative, sold as new psychoactive substance, and supposed to act as a partial agonist at the 5-HT 2A receptor. The aims of the presented study were to elucidate in vitro metabolites of EPT after incubations with pooled human liver S9 fraction (pS9) and in vivo metabolites excreted into rat urine. Finally, suitable analytical target compounds should be identified. Analysis of pS9 incubations using liquid chromatography–high-resolution tandem mass spectrometry revealed EPT metabolites formed after N -dealkylation as well as alkyl and aryl hydroxylation and formation of a hydroxy sulfate. Investigations using rat urine after oral dosing showed that the metabolic pathways of EPT shifted from in vitro hydroxylation of the alkyl amine group to an increased in vivo hydroxylation of the indole ring with several N -dealkyl metabolites. A glucuronic acid conjugate after hydroxylation of the indole ring was additionally found in vivo. The parent compound could not be detected in the rat urine samples. Therefore, analytical methods using mass spectrometry should include hydroxy-EPT and two hydroxy-EPT glucuronide isomers for reliable identification. [ABSTRACT FROM AUTHOR]

The distribution of so-called new psychoactive substances (NPS) as substitute for common drug of abuse was steadily increasing in the last years, but knowledge about their toxicodynamic and toxicokinetic properties is lacking. However, a comprehensive knowledge of their toxicokinetics, particularly their metabolism, is crucial for developing reliable screening procedures and to verify their intake, e.g., in case of intoxications. The aim of this study was therefore to tentatively identify the metabolites of the methylphenidate-derived NPS isopropylphenidate (isopropyl 2-phenyl-2-(2-piperidyl) acetate, IPH), 4-fluoromethylphenidate (methyl 2-(4-fluorophenyl)-2-(piperidin-2-yl) acetate, 4-FMPH) and 3,4-dichloromethylphenidate (methyl 2-(3,4-dichlorophenyl)-2-(piperidin-2-yl) acetate, 3,4-CTMP) using different in vivo and in vitro techniques and ultra-high performance liquid chromatography–high-resolution mass spectrometry (UHPLC-HRMS/MS). Urine samples of male rats were analyzed, and the transfer to human metabolism was done by using pooled human S9 fraction (pS9), which contains the microsomal fraction of liver homogenisate as well as its cytosol. UHPLC-HRMS/MS analysis of rat urine revealed 17 metabolites for IPH (14 phase I and 3 phase II metabolites), 13 metabolites were found for 4-FMPH (12 phase I metabolites and 1 phase II metabolite) and 7 phase I metabolites and no phase II metabolites were found for 3,4-CTMP. pS9 incubations additionally indicated that all investigated substances were primarily hydrolyzed, resulting in the corresponding carboxy metabolites. Finally, these carboxy metabolites should be used as additional analytical targets besides the parent compounds for comprehensive mass spectrometry–based screening procedures. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Accurate peak picking and further processing is a current challenge in the analysis of untargeted metabolomics using liquid chromatography–mass spectrometry (LC–MS) data. The optimization of these processes is crucial to obtain proper results. This study investigated and optimized the detection of peaks by XCMS, a widely used R package for peak picking and processing of high‐resolution LC–MS metabolomics data by their coefficient of variation using neat standard solutions of drug like compounds. The obtained results were additionally verified by using fortified pooled plasma samples. Settings of the mass spectrometer were optimized by recommendations in literature to enable a reliable detection of the investigated analytes. XCMS parameters were evaluated using a comprehensive parameter sweeping approach. The optimization steps were statistically evaluated and further visualized after principal component analysis (PCA). Concerning the lower concentrated solution in methanol samples, the optimization of both mass spectrometer and XCMS parameters improved the median coefficient of variation from 24% to 7%, retention time fluctuation from 9.3 seconds to 0.54 seconds, and fluctuation of the mass to charge ratio (m/z) from m/z 0.00095 to m/z 0.00028. The number of parent compounds and their related species annotated by CAMERA increased from 88 to 113 while the total amount of features decreased from 3282 to 428. Optimized MS settings such as increased resolution led to a higher specificity of peak picking. PCA supported these findings by showing the best clustering of samples after optimization of both mass spectrometer and XCMS parameters. The results implied that peak picking needs to be individually adapted for the experimental set up. Reducing unwanted variation in the data set was most successful after combining high resolving power with strict peak picking settings. [ABSTRACT FROM AUTHOR]

Synthetic cathinones belong to the most often seized new psychoactive substances on an international level. This study investigated the toxicometabolomics, particularly the in vitro metabolism of 2-(methylamino)-1-(4-methylphenyl)-1-pentanone (4-MPD) and 2-(ethylamino)-1-(4-methylphenyl)-1-pentanone (4-MEAP) in pooled human liver microsomes (pHLM) using untargeted metabolomics techniques. Incubations were performed with the substrates in concentrations ranging from 0, 12.5, and 25 µM. Analysis was done by means of high-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS/MS) in full scan only and the obtained data was evaluated using XCMS Online and MetaboAnalyst. Significant features were putatively identified using a separate parallel reaction monitoring method. Statistical analysis was performed using Kruskal-Wallis test for prefiltering significant features and subsequent hierarchical clustering, as well as principal component analysis (PCA). Hierarchical clustering or PCA showed a distinct clustering of all concentrations with most of the features z-scores rising with the concentration of the investigated substances. Identification of significant features left many of them unidentified but revealed metabolites of both 4-MPD and 4-MEAP. Both substances formed carboxylic acids, were hydroxylated at the alkyl chain, and formed metabolites after combined hydroxylation and reduction of the cathinone oxo group. 4-MPD additionally formed a dihydroxy metabolite and a hydroxylamine. 4-MEAP was additionally found reduced at the cathinone oxo group, N-dealkylated, and formed an oxo metabolite. These findings are the first to describe the metabolic pathways of 4-MPD and to extend our knowledge about the metabolism of 4-MEAP. Findings, particularly the MS data of the metabolites, are essential for setting up metabolite-based toxicological (urine) screening procedures. [ABSTRACT FROM AUTHOR]

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., (© 2019 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)