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Wastewater (WW)-based epidemiology (WBE) is a powerful tool for screening and surveillance of drugs (of abuse) or new psychoactive substances (NPSs) in larger population. Since the drug market changes frequently, it is crucial for WBE to define screening and surveillance biomarkers considering drug metabolism and (microbial) stability. The aims of the presented work were first to identify metabolites, potentially serving as a WBE biomarker of five deschloroketamine derivatives (DCKDs) in rat feces samples after oral administration in addition to already known urinary metabolites, and second to elucidate the microbial biotransformation and WW stability of five DCKDs and their metabolites detected in urine and feces. Microbial biotransformation and stability of DCKD and their metabolites in WW were assessed by incubating parent compounds at 0.1 mg/L or rat urine or rat feces samples in freshly collected, untreated, influent WW over a period of 24 h. All samples were analyzed using liquid chromatography-high-resolution tandem mass spectrometry. All parent compounds, seven Phase I, and one Phase II metabolite were detected in rat feces samples. After WW incubations, all tested DCKD and their metabolites were still detectable at least in trace amounts, but particularly, peak areas of the Phase II N- and O-glucuronides showed a markable decrease. This is in line with previous findings where Phase II conjugates were identified to be unstable in WW and thus not recommended as a WW biomarker. Hence, incubations demonstrated that the five DCKD and most of their metabolites were sufficiently stable in WW influent and can thus be used as analytical targets in the context of WBE., (© 2024 The Author(s). Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Sewage-based epidemiology using influent wastewater is used to estimate the consumption trends of (illicit) drugs over a short or long period of time in a subpopulation. The current study aimed to develop two separate methods for the quantitative analysis of selected drugs of abuse (DOA) and cognitive enhancers in influent wastewater using reversed-phase (RP) or hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS). The performance of RP and HILIC column was evaluated. A simple solid phase extraction was used for sample preparation. Short runtimes of 10 and 15 min on the RP and the HILIC column, respectively, allowed sufficient throughput. A six-point calibration was used for quantification with calibration ranges between 10 and 100 ng/L for all analytes except for benzoylecgonine (BZE, 30-300 ng/L). Method validation was performed according to ICH guideline M10. Analytes such as amphetamine (AMPH), BZE, cocaethylene (CE), cocaine (COC), ethyl sulfate, 4-hydroxy-3-methoxymethamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine, methylphenidate (MPH), and ritalinic acid (RA) were included in method development and validation. Two different column types were necessary for sufficient chromatographic resolution. The analytical setup allowed detection of all other analytes at concentration levels between 1 ng/L for methylphenidate to 10 ng/L for amphetamine. A method for the detection and quantification of DOA, cognitive enhancers, and their biomarkers in wastewater was successfully developed and validated. Moreover, six proof-of-concept samples were analyzed in which AMPH, BZE, COC, MDMA, MPH, and RA were identified and further quantified., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

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., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

The synthetic cannabinoid receptor agonists (SCRAs) (quinolin-8-yl 4-methyl-3-(morpholine-4-sulfonyl)benzoate [QMMSB]) and (quinolin-8-yl 4-methyl-3-((propan-2-yl)sulfamoyl)benzoate [QMiPSB], also known as SGT-46) are based on the structure of quinolin-8-yl 4-methyl-3-(piperidine-1-sulfonyl)benzoate (QMPSB) that has been identified on seized plant material in 2011. In clinical toxicology, knowledge of the metabolic fate is important for their identification in biosamples. Therefore, the aim of this study was the identification of in vitro Phase I and II metabolites of QMMSB and QMiPSB in pooled human liver S9 fraction (pHLS9) incubations for use as screening targets. In addition, the involvement of human monooxygenases and human carboxylesterases (hCES) was examined. Analyses were performed by liquid chromatography coupled with high-resolution tandem mass spectrometry. Ester hydrolysis was found to be an important step in the Phase I metabolism of both SCRAs, with the carboxylic acid product being found only in negative ionization mode. Monohydroxy and N-dealkyl metabolites of the ester hydrolysis products were detected as well as glucuronides. CYP2C8, CYP2C9, CYP3A4, and CYP3A5 were involved in hydroxylation. Whereas enzymatic ester hydrolysis of QMiPSB was mainly catalyzed by hCES1 isoforms, nonenzymatic ester hydrolysis was also observed. The results suggest that ester hydrolysis products of QMMSB and QMiPSB and their glucuronides are suitable targets for toxicological screenings. The additional use of the negative ionization mode is recommended to increase detectability of analytes. Different cytochrome P450 (CYP) isozymes were involved in the metabolism; thus, the probability of drug-drug interactions due to CYP inhibition can be assessed as low., (© 2022 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

The active, poisonous constituents in Taxus baccata, the yew plants, are taxine alkaloids whose main action is suggested to be a block of calcium and sodium channels. The main alkaloids are taxine B (30%) and taxine A (1.3%). Symptoms can include bradycardia, bradypnea, diastolic, and cardiac standstill. The current investigation reports the analytical toxicology of human blood and urine to confirm a suspected ingestion of yew needles. This includes the qualitative detection of several yew ingredients, including the main alkaloids, the validated quantification of 3,5-dimethoxyphenol, and the discussion of suitable analytical targets. After analyzing human specimens and yew needle extracts using the developed procedures, the five alkaloids 1-deotaxine B, taxicatin, taxine A, taxine B, and taxine I could be detected and tentatively identified. Finally, taxine A and B can be recommended as analytical targets besides 3,5-dimethoxyphenol., (© 2022 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Studies on the tissue distribution of the new synthetic opioid U-47700 and its main metabolite N-desmethyl-U-47700 revealed about sixfold higher metabolite concentrations in pig brain as compared with the parent compound. To better assess the toxic potential of this drug, the aim of this study was to assess the in vitro μ-opioid receptor (MOR) activation potential of the main metabolites of U-47700, N-desmethyl-U-47700, and N,N-bisdesmethyl-U-47700, using a live cell-based reporter assay based on NanoLuc Binary Technology®. Cells stably expressing human MOR and β-arrestin 2 (βarr2), each fused via a flexible linker to two complementary inactive subunits of the nanoluciferase, were seeded on poly-d-lysine-coated 96-well plates and treated with N-desmethyl-U-47700, N,N-bisdesmethyl-U-47700, U-47700, or hydromorphone as reference standard. MOR activation results in functional complementation of the nanoluciferase, which can be assessed via luminescence monitoring. The potency of the metabolites is lower than that of U-47700 (EC 50 of 186 nM for U-47700, 3770 nM for N-desmethyl-U-47700, and >5 μM for N,N-bisdesmethyl-U-47700). The maximal efficacy (E max ) observed (relative to hydromorphone, set arbitrarily at 100%) decreased from 183% to 127% and 39.2% for U-47700, N-desmethyl-U-47700, and N,N-bisdesmethyl-U-47700, respectively. Thus, the loss of one or two methyl groups reduced the MOR activation potential, which was more pronounced if both methyl groups were removed. It is thus anticipated that the impact on MOR exerted by the higher metabolite concentration in brain has only little-if any relevance for the strong toxic effects of U-47700., (© 2021 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Colchicum autumnale, which can be mistaken for Allium ursinum, contains the alkaloid colchicine potentially leading to life-threatening up to fatal intoxications. We report two cases of acute intoxications with unexplained circumstances. Using the authors' systematic screening approaches, colchicine could be detected in blood plasma and urine samples using liquid chromatography coupled to linear ion trap mass spectrometry (LC-ITMS n ) and high-resolution tandem mass spectrometry (LC-HRMS/MS). Metabolites of colchicine could be identified in urine for confirmation of screening results. Gas chromatography-mass spectrometry (GC-MS) analysis was also conducted, but colchicine could not be detected. Furthermore, colchicine concentration was estimated via LC-HRMS/MS in plasma samples. Results of the systematic screening indicated the ingestion of colchicine from both subjects. In both cases, the parent compound was detected in blood plasma and urine using the LC-HRMS/MS and LC-ITMS n system. An O-demethylation metabolite was identified in urine samples of both subjects using LC-HRMS/MS; the N-deacetylation product was also found in urine samples of both cases via LC-HRMS/MS and LC-ITMS n . The use of LC-ITMS n resulted only in the detection of the O-demethylation product in case 2. Plasma concentrations were estimated at 2.5 ng/ml and 4.7 ng/ml for cases 1 and 2, respectively. We demonstrated the detection of this highly toxic alkaloid in blood plasma and urine using a time-saving and reliable clinical systematic screening. Furthermore, we identified metabolites of colchicine being rarely discussed in literature, which can be used as additional screening targets., (© 2021 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Recently, we presented a strategy for analysis of eight biomarkers in human urine to verify toxic mushroom or Ricinus communis ingestions. However, screening for the full panel is not always necessary. Thus, we aimed to develop a strategy to reduce analysis time and by focusing on two sets of analytes. One set (A) for biomarkers of late-onset syndromes, such as phalloides syndrome or the syndrome after castor bean intake. Another set (B) for biomarkers of early-onset syndromes, such as pantherine-muscaria syndrome and muscarine syndrome. Both analyses should be based on hydrophilic-interaction liquid chromatography coupled with high-resolution mass spectrometry (MS)/MS (HILIC-HRMS/MS). For A, urine samples were prepared by liquid-liquid extraction using dichloromethane and subsequent solid-phase extraction of the aqueous supernatant. For B urine was precipitated using acetonitrile. Method A was validated for ricinine and α- and β-amanitin and method B for muscarine, muscimol, and ibotenic acid according to the specifications for qualitative analytical methods. In addition, robustness of recovery and normalized matrix factors to matrix variability measured by urinary creatinine was tested. Moreover, applicability was tested using 10 urine samples from patients after suspected mushroom intoxication. The analytes α- and β-amanitin, muscarine, muscimol, and ibotenic acid could be successfully identified. Finally, psilocin-O-glucuronide could be identified in two samples and unambiguously distinguished from bufotenine-O-glucuronide via their MS 2 patterns. In summary, the current workflow offers several advantages towards the previous method, particularly being more labor-, time-, and cost-efficient, more robust, and more sensitive., (© 2021 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Numerous case reports of intoxications with nutmeg seeds (Myristica fragrans, Houtt.) can be found in literature often following their abuse, as psychotropic effects were described after ingestions of large doses. The successful detection of the main ingredients of the nutmeg seeds essential oil elemicin, myristicin, and safrole, as well as their metabolites in human urine by gas chromatography coupled to mass spectrometry (GC-MS) was already described. The aim of this study was to investigate the detectability of the main ingredients of nutmeg seeds and their metabolites in human blood and urine samples using liquid chromatography coupled to linear ion trap mass spectrometry (LC-LIT-MS n ) and liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS/MS) after nutmeg seed abuse. Sample material of three individuals was retrospectively investigated after a systematic screening approach indicated an intoxication with nutmeg seeds as a likely cause of symptoms. Metabolic patterns in plasma and urine using GC-MS were comparable with those described in earlier publications. Investigations using hyphenated liquid chromatography techniques lead to the detection of myristicin and safrole, as well as further metabolites not described using GC-MS and revealed sulfation as an additional Phase II metabolic pathway. These results might help to detect or confirm future intoxications with nutmeg seeds by using LC-MS techniques., (© 2021 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Due to the dynamic market involving synthetic cannabinoids (SCs), the determination of analytical targets is challenging in clinical and forensic toxicology. SCs usually undergo extensive metabolism, and therefore their main metabolites must be identified for the detection in biological matrices, particularly in urine. Controlled human studies are usually not possible for ethical reasons; thus, alternative models must be used. The aim of this work was to predict the in vitro and in vivo metabolic patterns of 7-azaindole-derived SCs using 1-(5-fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-pyrollo[2,3-b]pyridin-3-carboxamide (cumyl-5F-P7AICA) as an example. Different in vitro (pooled human liver S9 fraction, pooled human liver microsomes, and pig liver microsomes) and in vivo (rat and pig) systems were compared. Monooxygenase isoenzymes responsible for the most abundant phase I steps, namely oxidative defluorination (OF) followed by carboxylation, monohydroxylation, and ketone formation, were identified. In both in vivo models, OF/carboxylation and N-dealkylation/monohydroxylation/sulfation could be detected. Regarding pHS9 and pig urine, monohydroxylation/sulfation or glucuronidation was also abundant. Furthermore, the parent compound could still be detected in all models. Initial monooxygenase activity screening revealed the involvement of CYP2C19, CYP3A4, and CYP3A5. Therefore, in addition to the parent compound, the OF/carboxylated and monohydroxylated (and sulfated or glucuronidated) metabolites can be recommended as urinary targets. In comparison to literature, the pig model predicts best the human metabolic pattern of cumyl-5F-P7AICA. Furthermore, the pig model should be suitable to mirror the time-dependent excretion pattern of parent compounds and metabolites., (© 2020 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

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.)

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

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-MS n , 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., (© 2019 The Authors Drug Testing and Analysis Published by John Wiley & Sons Ltd.)

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., (© 2018 John Wiley & Sons, Ltd.)

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., (© 2018 John Wiley & Sons, Ltd.)

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 IC 50 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., (© 2018 John Wiley & Sons, Ltd.)

Multiple new psychoactive substances (NPS) are released into the recreational drug market each year. One NPS drug class that has become more common in recent years is that of the benzodiazepines (designer benzodiazepines, DBZ). Several metabolism studies have been performed to improve their bioanalytical detection via the best target. These studies have shown the presence of parent glucuronides and, as polymorphisms have been noted for the catalyzing enzymes (UDP-glucuronyltransferases) responsible for glucuronide conjugation reactions, it is important to keep this in mind when interpreting DBZ cases in clinical and/or forensic toxicology. Therefore, the aim of this study was to determine the UDP-glucuronyltransferases (UGTs) responsible for parent compound conjugation of nine DBZ to facilitate interpretation of related cases. Clonazolam, deschloroetizolam, etizolam, flubromazolam, flunitrazolam, metizolam, nifoxipam, nitrazolam, and pyrazolam were incubated with pooled human liver microsomes (pHLM) or 13 different human UGTs. The samples were analyzed using liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). Glucuronide conjugates of flunitrazolam and nifoxipam were only detected in pHLM, suggesting that these reactions are performed by dimer complexes of several UGTs or complexes between UGTs and other metabolizing enzymes contained in pHLM. Nitrazolam or pyrazolam glucuronides were not detected. Glucuronidation of clonazolam, deschloroetizolam, etizolam, flubromazolam, and metizolam was catalyzed exclusively by UGT1A4. The conjugation of the majority of the DBZ was performed by the UGT isoform 1A4 for which polymorphisms have been described. This underlines the importance of taking glucuronidation polymorphism into consideration when interpreting intoxication cases., (© 2018 John Wiley & Sons, Ltd.)

Fast and comprehensive qualitative and quantitative methods preferably by gas chromatography-mass spectrometry (GC-MS) and/or liquid chromatography-mass spectrometry (LC-MS) are needed to support the (differential) diagnosis of acute poisonings in emergency toxicology. One option is a commercially available qualitative screening solution based on LC-MS n (Bruker Daltonik Toxtyper™, TT). Identified and toxicologically relevant compounds should be quantified to assess severity of poisonings. The aim of the present study was to test the TT system for quantification simultaneous with the screening process in blood plasma exemplified for 22 relevant drugs and two active metabolites. A standard liquid-liquid extraction was used for sample work-up followed by 1:5 dilution of the final extracts. They were analyzed using the TT system consisting of a Bruker amaZon speed ion trap and a Thermo Fisher Dionex Ultimate 3000 LC system. Plasma levels were assessed using full-scan data and an electronically stored five-point calibration. The calibration model was linear for the studied ranges and could be used for at least two months. The method was validated according to international guidelines. The acceptance criteria recommended for emergency toxicology for accuracy and precision were fulfilled for all tested compounds, but bromazepam, lorazepam, oxycodone, and prothipendyl could reliably be determined only above the therapeutic range. In conclusion, the presented procedure allowed the combination of a comprehensive LC-MS n screening with fast automated assessment of plasma levels for emergency toxicology of tested compounds., (© 2018 John Wiley & Sons, Ltd.)

Over the past ~8 years, hundreds of unregulated new psychoactive substances (NPS) of various chemical categories have been introduced as recreational drugs through mainly open online trade. This study was performed to further investigate the human metabolic pattern of the NPS, or designer benzodiazepines flubromazolam and pyrazolam, and to propose analytical targets for urine drug testing of these substances. The urine samples originated from patient samples confirmed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) analysis to contain flubromazolam or pyrazolam. The LC-HRMS/MS system consisted of a YMC-UltraHT Hydrosphere C18 column (YMC, Dinslaken, Germany) coupled to a Thermo Scientific (Waltham, MA, USA) Q Exactive Orbitrap MS operating in positive electrospray mode. The samples were analyzed both with and without enzymatic hydrolysis using β-glucuronidase. Besides the parent compounds, the main urinary excretion products were parent glucuronides, mono-hydroxy metabolites, and mono-hydroxy glucuronides. In samples prepared without hydrolysis, the most common flubromazolam metabolites were 1 of the mono-hydroxy glucuronides and 1 of the parent glucuronides. For pyrazolam, a parent glucuronide was the most common metabolite. These 3 metabolites were detected in all samples and were considered the primary targets for urine drug testing and confirmation of intake. After enzymatic hydrolysis of the urine samples, a 2-19-fold increase in the concentration of flubromazolam was found, highlighting the value of hydrolysis for this analyte. With hydrolysis, the flubromazolam hydroxy metabolites should be used as target metabolites., (Copyright © 2017 John Wiley & Sons, Ltd.)

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., (Copyright © 2018 John Wiley & Sons, Ltd.)

Reliable, sensitive, and comprehensive urine screening procedures by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS) with low or high resolution (HR) are of high importance for drug testing, adherence monitoring, or detection of toxic compounds. Besides conventional urine sampling, dried urine spots are of increasing interest. In the present study, the power of LC-HR-MS/MS was investigated for comprehensive drug testing in urine with or without conjugate cleavage or using dried urine spots after on-spot cleavage in comparison to established LC-MS n or GC-MS procedures. Authentic human urine samples (n = 103) were split in 4 parts. One aliquot was prepared by precipitation (UP), one by UP with conjugate cleavage (UglucP), one spot on filter paper cards and prepared by on-spot cleavage followed by liquid extraction (DUSglucE), and one worked-up by acid hydrolysis, liquid-liquid extraction, and acetylation for GC-MS analysis. The 3 series of LC-HR-MS/MS results were compared among themselves, to corresponding published LC-MS n data, and to screening results obtained by conventional GC-MS. The reference libraries used for the 3 techniques contained over 4500 spectra of parent compounds and their metabolites. The number of all detected hits (770 drug intakes) was set to 100%. The LC-HR-MS/MS approach detected 80% of the hits after UP, 89% after UglucP, and 77% after DUSglucE, which meant over one-third more hits in comparison to the corresponding published LC-MS n results with ≤49% detected hits. The GC-MS approach identified 56% of all detected hits. In conclusion, LC-HR-MS/MS provided the best screening results after conjugate cleavage and precipitation., (Copyright © 2017 John Wiley & Sons, Ltd.)

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-MS n ), 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-MS n , 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., (Copyright © 2017 John Wiley & Sons, Ltd.)

Wastewater-based epidemiology (WBE) employs the analysis of wastewater to detect and quantify drug use and discharge within a community. In this work, transformation products (TP) by microbes in the environment were identified after incubations in wastewater and an isolated microbial strain. The microbial strain was isolated from an enrichment culture of wastewater supplemented with 3,4-methylenedioxy-pyrovalerone, and identified by matrix assisted laser desorption - time of flight mass spectrometry as Pseudomonas putida (P. putida). Five pyrrolidinophenone-type psychoactive substances (PPPS) were then incubated in wastewater and in P. putida tryptic soy broth (TSB) growth cultures. TPs were identified using liquid chromatography coupled to mass spectrometry techniques. All TPs observed in P. putida TSB growth cultures were also identified in wastewater incubations. The main TP for all PPPSs in P. putida TSB growth cultures, and two PPPSs incubated in wastewater, were the N-desalkyl-carboxy-TPs. The study showed P. putida TSB growth cultures used for identification of TPs in wastewater, represent parts of the microbial community. With data provided in this type of experiments more information will be available to select targets for monitoring drug use by WBE. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

The knowledge of pharmacokinetic (PK) properties of synthetic cannabinoids (SCs) is important for interpretation of analytical results found for example in intoxicated individuals. In the absence of human data from controlled studies, animal models elucidating SC PK have to be established. Pigs providing large biofluid sample volumes were tested for prediction of human PK data. In this context, the metabolic fate of two model SCs, namely 4-ethylnaphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-210) and 2-(4-methoxyphenyl)-1-(1-pentyl-indol-3-yl)methanone (RCS-4), was elucidated in addition to Δ 9 -tetrahydrocannabinol (THC). After intravenous administration of the compounds, hourly collected pig urine was analyzed by liquid chromatography-high resolution mass spectrometry. The following pathways were observed: for JWH-210, hydroxylation at the ethyl side chain or pentyl chain and combinations of them followed by glucuronidation; for RCS-4, hydroxylation at the methoxyphenyl moiety or pentyl chain followed by glucuronidation as well as O-demethylation followed by glucuronidation or sulfation; for THC, THC glucuronidation, 11-hydroxylation, followed by carboxylation and glucuronidation. For both SCs, parent compounds could not be detected in urine in contrast to THC. These results were consistent with those obtained from human hepatocyte and/or human case studies. Urinary markers for the consumption of JWH-210 were the glucuronide of the N-hydroxypentyl metabolite (detectable for 3-4 h) and of RCS-4 the glucuronides of the N-hydroxypentyl, hydroxy-methoxyphenyl (detectable for at least 6 h), and the O-demethyl-hydroxy metabolites (detectable for 4 h). Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Monitoring population drug use through wastewater-based epidemiology (WBE) is a useful method to quantitatively follow trends and estimate total drug consumption in communities. Concentrations of drug biomarkers might be low in wastewater due to dilution; and therefore analysis of pooled urine (PU) is useful to detect consumed drugs and identify targets of illicit drugs use. The aims of the study were (1) to screen PU and urinated soil (US) samples collected at festivals for illicit drug excretion products using hyphenated techniques; (2) to develop and validate a hydrophilic interaction liquid chromatography - mass spectrometry / mass spectrometry (HILIC-MS/MS) method of quantifying urinary targets of identified drugs in wastewater; and (3) to conduct a 24 h stability study, using PU and US to better reflect the chemical environment for targets in wastewater. Cocaine (COC) and ecstasy-like compounds were the most frequently detected illicit drugs; an analytical method was developed to quantify their excretion products. Hydroxymethoxymethamphetamine (HMMA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), HMMA sulfate (HMMA-S), benzoylecgonine (BE), and cocaethylene (CE) had 85-102% of initial concentration after 8 h of incubation, whereas COC and ecgonine methyl ester (EME) had 74 and 67% after 8 h, respectively. HMMA showed a net increase during 24 h of incubation (107% ± 27, n = 8), possibly due to the cleavage of HMMA conjugates, and biotransformation of MDMA. The results suggest HMMA as analytical target for MDMA consumption in WBE, due to its stability in wastewater and its excretion as the main phase I metabolite of MDMA. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Many N,N-dialkylated tryptamines show psychoactive properties in humans and the number of derivatives involved in multidisciplinary areas of research has grown over the last few decades. Whereas some derivatives form the basis of a range of medicinal products, others are predominantly encountered as recreational drugs, and in some cases, the areas of therapeutic and recreational use can overlap. In recent years, 5-methoxy-N,N-diallyltryptamine (5-MeO-DALT) has appeared as a new psychoactive substance (NPS) and 'research chemical' whereas 4-acetoxy-DALT and the ring-unsubstituted DALT have only been detected very recently. Strategies pursued in the authors' laboratories included the preparation and biological evaluation of previously unreported N,N-diallyltryptamines (DALTs). This report describes the analytical characterization of 17 DALTs. Fifteen DALTs were prepared by a microwave-accelerated Speeter and Anthony procedure following established procedures developed previously in the authors' laboratories. In addition to DALT, the substances included in this study were 2-phenyl-, 4-acetoxy-, 4-hydroxy-, 4,5-ethylenedioxy-, 5-methyl-, 5-methoxy-, 5-methoxy-2-methyl-, 5-ethoxy-, 5-fluoro-, 5-fluoro-2-methyl-, 5-chloro-, 5-bromo-, 5,6-methylenedioxy-, 6-fluoro-, 7-methyl, and 7-ethyl-DALT, respectively. The DALTs were characterized by nuclear magnetic resonance spectroscopy (NMR), gas chromatography (GC) quadrupole and ion trap (EI/CI) mass spectrometry (MS), low and high mass accuracy MS/MS, photodiode array detection, and GC solid-state infrared analysis, respectively. A comprehensive collection of spectral data was obtained that are provided to research communities who face the challenge of encountering newly emerging substances where analytical data are not available. These data are also relevant to researchers who might wish to explore the clinical and non-clinical uses of these substances. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

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., (Copyright © 2016 John Wiley & Sons, Ltd.)

4-methyl-N-ethcathinone (4-MEC), the N-ethyl homologue of mephedrone, is a novel psychoactive substance of the beta-keto amphetamine (cathinone) group. The aim of the present work was to study the phase I and phase II metabolism of 4-MEC in human urine as well as in pooled human liver microsome (pHLM) incubations. The urine samples were worked up with and without enzymatic cleavage, the pHLM incubations by simple deproteinization. The metabolites were separated and identified by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-high resolution-tandem mass spectrometry (LC-HR-MS/MS). Based on the metabolites identified in urine and/or pHLM, the following metabolic pathways could be proposed: reduction of the keto group, N-deethylation, hydroxylation of the 4-methyl group followed by further oxidation to the corresponding 4-carboxy metabolite, and combinations of these steps. Glucuronidation could only be observed for the hydroxy metabolite. These pathways were similar to those described for the N-methyl homologue mephedrone and other related drugs. In pHLM, all phase I metabolites with the exception of the N-deethyl-dihydro isomers and the 4-carboxy-dihydro metabolite could be confirmed. Glucuronides could not be formed under the applied conditions. Although the taken dose was not clear, an intake of 4-MEC should be detectable in urine by the GC-MS and LC-MS(n) standard urine screening approaches at least after overdose., (Copyright © 2014 John Wiley & Sons, Ltd.)

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-MS(n) 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.)

3',4'-Methylenedioxy-alpha-pyrrolidinobutyrophenone (MDPBP), a designer drug of the pyrrolidinophenone-type, was first seized in Germany in 2009. It was also identified in 'legal high' samples investigated in the UK. Therefore, the aim of the presented work was to identify its in vivo and in vitro phase I and II metabolites using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-ion trap mass spectrometry (LC-MS(n) ). Furthermore, detectability of MDPBP in rat and human urine using standard urine screening approaches (SUSA) by GC-MS and LC-MS(n) was studied. The metabolites were isolated either directly or after enzymatic cleavage of conjugates by solid-phase extraction (C18, HCX). The metabolites were then analyzed and structures proposed after GC-MS (phase I) and LC-MS(n) (phase II). Based on these identified metabolites, the following main metabolic steps could be proposed: demethylenation followed by methylation of one hydroxy group, aromatic and side chain hydroxylation, oxidation of the pyrrolidine ring to the corresponding lactam as well as ring opening to the corresponding carboxylic acid. Furthermore, in rat urine after a typical user's dose as well as in human urine, mainly the metabolites could be detected using the authors' SUSA by GC-MS and LC-MS(n) . Thus, it should be possible to monitor an application of MDPBP assuming similar toxicokinetics in humans. Finally, CYP2C19 and CYP2D6 could be identified as the isoenzymes mainly responsible for demethylenation., (Copyright © 2013 John Wiley & Sons, Ltd.)

Naphyrone (1-naphthalen-2-yl-2-pyrrolidin-1-yl-pentan-1-one; naphthylpyrovalerone, β-naphyrone) is a cathinone designer drug and was marketed as replacement for the synthetic cathinone derivative mephedrone. Meanwhile, naphyrone is also classified as a controlled drug in several countries. Therefore, the aim of this study was to identify the metabolites of naphyrone in rat urine using gas chromatography-mass spectrometry techniques and to show its detectability in urine samples. The following metabolic steps could be detected in rat urine: oxidation of the pyrrolidine ring to the corresponding lactam, hydroxylation of the propyl side chain and the naphthyl ring, degradation to the primary amines after opening of the pyrrolidine ring, and combinations of these steps. Assuming similar kinetics, an intake of naphyrone should be detectable in human urine mainly via its metabolites., (Copyright © 2013 John Wiley & Sons, Ltd.)

In the last two decades, a large number of new drugs from several drug classes have appeared on the illicit drug market. While some of these compounds have meanwhile been scheduled as controlled substances, the majority of them are (still) sold as so-called 'legal highs', mostly via the Internet. At the time they appear on the market the metabolism of these drugs is generally unknown. Therefore, it must be studied in order to obtain data necessary for analytical method development as well as toxicological risk assessment. In vitro metabolism studies of new designer drugs can be performed for identification and structure elucidation of new designer drug metabolites or to assess the qualitative and quantitative involvement of certain enzymes in the metabolism of a particular drug. In this review, the value of the following enzyme preparations for in vitro metabolism studies of new designer drugs will be discussed: liver microsomes, recombinant cDNA-expressed enzymes, liver cytosol, S9 mix, and hepatocytes. This will cover the major metabolic enzymes: cytochrome P450 monooxygenases, flavin-monooxygenases, monoamine oxidases, UDP-glucuronyltransferases, sulfotransferases, and catechol-O-methyltransferases. Important analytical aspects such as the value of mass spectrometric techniques will also be covered., (Copyright © 2011 John Wiley & Sons, Ltd.)