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.     

Study of the in vitro and in vivo metabolism of the tryptamine 5‐MeO‐MiPT using human liver microsomes and real case samples

The synthetic tryptamine 5‐methoxy‐N‐methyl‐N‐isopropyltryptamine (5‐MeO‐MiPT) has recently been abused as a hallucinogenic drug in Germany and Switzerland. This study presents a case of 5‐MeO‐MiPT intoxication and the structural elucidation of metabolites in pooled human liver microsomes (pHLM), blood, and urine. Microsomal incubation experiments were performed using pHLM to detect and identify in vitro metabolites. In August 2016, the police encountered a naked man, agitated and with aggressive behavior on the street. Blood and urine samples were taken at the hospital and his premises were searched. The obtained blood and urine samples were analyzed for in vivo metabolites of 5‐MeO‐MiPT using liquid chromatography–high resolution tandem mass spectrometry (LC–HRMS/MS). The confiscated pills and powder samples were qualitatively analyzed using Fourier transform infrared (FTIR), gas chromatography–mass spectrometry (GC–MS), LC‐HRMS/MS, and nuclear magnetic resonance (NMR). 5‐MeO‐MiPT was identified in 2 of the seized powder samples. General unknown screening detected cocaine, cocaethylene, methylphenidate, ritalinic acid, and 5‐MeO‐MiPT in urine. Seven different in vitro phase I metabolites of 5‐MeO‐MiPT were identified. In the forensic case samples, 4 phase I metabolites could be identified in blood and 7 in urine. The 5 most abundant metabolites were formed by demethylation and hydroxylation of the parent compound. 5‐MeO‐MiPT concentrations in the blood and urine sample were found to be 160 ng/mL and 3380 ng/mL, respectively. Based on the results of this study we recommend metabolites 5‐methoxy‐N‐isopropyltryptamine (5‐MeO‐NiPT), 5‐hydroxy‐N‐methyl‐N‐isopropyltryptamine (5‐OH‐MiPT), 5‐methoxy‐N‐methyl‐N‐isopropyltryptamine‐N‐oxide (5‐MeO‐MiPT‐N‐oxide), and hydroxy‐5‐methoxy‐N‐methyl‐N‐isopropyltryptamine (OH‐5‐MeO‐MiPT) as biomarkers for the development of new methods for the detection of 5‐MeO‐MiPT consumption, as they have been present in both blood and urine samples.     

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.     

Elucidation of the metabolites of the novel psychoactive substance 4‐methyl‐N‐ethyl‐cathinone (4‐MEC) in human urine and pooled liver microsomes by GC‐MS and LC‐HR‐MS/MS techniques and of its detectability by GC‐MS or LC‐MSn standard screening approaches

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‐MSⁿ standard urine screening approaches at least after overdose. Copyright © 2014 John Wiley & Sons, Ltd.     

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

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

The in vivo and in vitro phase I metabolism of FYL‐67, a novel oxazolidinone antibacterial drug,studied by LC‐MS/MS

In our previous study, FYL‐67, a novel linezolid analogue with the morpholinyl ring replaced by a 4‐(pyridin‐2‐yl)‐1H‐pyrazol‐1‐yl group, was demonstrated to own an excellent activity against Gram‐positive organisms,such as methicillin‐resistant Staphylococcus aureus (MRSA). However, metabolic biotransformation was not investigated. This study was performed to identify the phase I metabolites of FYL‐67 using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). The chemical structures were confirmed by comparison with corresponding chemical standards obtained internal. Primary elucidation of the metabolic pathway of FYL‐67 in vitro was performed using liver preparations (microsomes and hepatocytes) from rats and humans, and SD (Sprague Dawley, rat, rattus norvegicus) rats were used for the study of in vivo approach. To the end, two metabolites (M₁ and M₂) were detected after in vitro as well as in vivo experiments. Based on LC‐MS/MS analyses, the metabolites were demonstrated to be 5‐(aminomethyl)‐3‐(3‐fluoro‐4‐(4‐(pyridin‐2‐yl)‐1H‐pyrazol‐1‐yl)phenyl)oxazolidin‐2‐one (M₁) and 3‐(3‐fluoro‐4‐(4‐(pyridin‐2‐yl)‐1H‐pyrazol‐1‐yl)phenyl)‐5‐(hydroxymethyl)oxazolidin‐2‐one (M₂). Amide hydrolysis at acetyl group of FYL‐67 leading to the formation of M₁ was observed and suggested to play a major role in both in vivo and in vitro phase I metabolism of FYL‐67. M₁ was demonstrated to undergo a further oxidation to form M₂. In addition, the results indicated no species difference existing between rats and humans. The outcomes of our research can be utilized for the development and validation of the analytical method for the quantification of FYL‐67 as well as its metabolites in biological samples. Furthermore, it is helpful to conduct studies of pharmacodynamics and toxicodynamics. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

LC‐MS/MS detection of unaltered glucuronoconjugated metabolites of metandienone

The aim of this study was to evaluate the direct detection of glucuronoconjugated metabolites of metandienone (MTD) and their detection times. Metabolites resistant to enzymatic hydrolysis were also evaluated. Based on the common mass spectrometric behaviour of steroid glucuronides, three liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) strategies were applied for the detection of unpredicted and predicted metabolites: precursor ion scan (PI), neutral loss scan (NL), and theoretical selected reaction monitoring (SRM) methods. Samples from four excretion studies of MTD were analyzed for both the detection of metabolites and the establishment of their detection times. Using PI and NL methods, seven metabolites were observed in post‐administration samples. SRM methods allowed for the detection of 13 glucuronide metabolites. The detection times, measured by analysis with an SRM method, were between 1 and 22 days. The metabolite detected for the longest time was 18‐nor‐17β‐hydroxymethyl‐17α‐methyl‐5β‐androsta‐1,4,13‐triene‐3‐one‐17‐glucuronide. One metabolite was resistant to hydrolysis with β ‐glucuronidase; however it was only detected in urine up to four days after administration. The three glucuronide metabolites with the highest retrospectivity were identified by chemical synthesis or mass spectrometric data, and although they were previously reported, this is the first time that analytical data of the intact phase II metabolites are presented for some of them. The LC‐MS/MS strategies applied have demonstrated to be useful for detecting glucuronoconjugated metabolites of MTD, including glucuronides resistant to enzymatic hydrolysis which cannot be detected by conventional approaches. Copyright © 2016 John Wiley & Sons, Ltd.     

Detection of the recently emerged synthetic cannabinoid 5F–MDMB‐PICA in ‘legal high’ products and human urine samples

Indole or indazole‐based synthetic cannabinoids (SCs) bearing substituents derived from valine or tert‐leucine are frequently abused new psychoactive substances (NPS). The emergence of 5F–MDMB‐PICA (methyl N‐{[1‐(5‐fluoropentyl)‐1H–indol‐3‐yl]carbonyl}‐3‐methylvalinate) on the German drug market is a further example of a substance synthesized in the context of scientific research being misused by clandestine laboratories by adding it to ‘legal high’ products. In this work, we present the detection of 5F–MDMB‐PICA in several legal high products by gas chromatography–mass spectrometry (GC–MS) analysis. To detect characteristic metabolites suitable for a proof of 5F–MDMB‐PICA consumption by urine analysis, pooled human liver microsome (pHLM) assays were performed and evaluated using liquid chromatography–tandem mass spectrometry (LC–MS/MS) and liquid chromatography quadrupole time‐of‐flight mass spectrometry (LC‐QToF‐MS) techniques to generate reference spectra of the in vitro phase I metabolites. The in vivo phase I metabolism was investigated by the analysis of more than 20 authentic human urine specimens and compared to the data received from the pHLM assay. Biotransformation of the 5‐fluoropentyl side chain and hydrolysis of the terminal methyl ester bond are main phase I biotransformation steps. Two of the identified main metabolites formed by methyl ester hydrolysis or mono‐hydroxylation at the indole ring system were evaluated as suitable urinary biomarkers and discussed regarding the interpretation of analytical findings. Exemplary analysis of one urine sample for 5F–MDMB‐PICA phase II metabolites showed that two of the main phase I metabolites are subject to extensive glucuronidation prior to renal excretion. Therefore, conjugate cleavage is reasonable for enhancing sensitivity. Commercially available immunochemical pre‐tests for urine proved to be unsuitable for the detection of 5F–MDMB‐PICA consumption. Copyright © 2017 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.     

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

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

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.     

Development and validation of an LC‐MS/MS method after chiral derivatization for the simultaneous stereoselective determination of methylenedioxy‐methamphetamine (MDMA) and its phase I and II metabolites in human blood plasma

3,4‐Methylenedioxymethamphetamine (MDMA, ecstasy) is a racemic drug of abuse and its two enantiomers are known to differ in their dose‐response curves. The S‐enantiomer was shown to be eliminated at a higher rate than the R‐enantiomer. The most likely explanation for this is a stereoselective metabolism also claimed in in vitro studies. Urinary excretion studies showed that the main metabolites in humans are 4‐hydroxy 3‐methoxymethamphetamine (HMMA) 4‐sulfate, HMMA 4‐glucuronide and 3,4‐dihydroxymethamphetamine (DHMA) 3‐sulfate. For stereoselective pharmacokinetic analysis of phase I and phase II metabolites in human blood plasma useful analytical methods are needed. Therefore the aim of the presented study was the development and validation of a stereoselective liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method for the simultaneous quantification of MDMA, 3,4‐methylenedioxyamphetamine, DHMA, DHMA 3‐sulfate, HMMA, HMMA 4‐glucuronide, HMMA 4‐sulfate, and 4‐hydroxy 3‐methoxyamphetamine in blood plasma for evaluation of the stereoselective pharmacokinetics in humans. Blood plasma samples were prepared by simple protein precipitation and afterwards all analytes were derivatized using N‐(2,4‐dinitro‐5‐fluorophenyl) L‐valinamide resulting in the formation of diastereomers which were easily separable on standard reverse phase stationary phases. This simple and fast method was validated according to international guidelines including specificity, recovery, matrix effects, accuracy and precision, stabilities, and limits of quantification. The method proved to be selective, sensitive, accurate and precise for all tested analytes except for DHMA. Copyright © 2014 John Wiley & Sons, Ltd.     

Synthesis,radio‐LC–MS analysis and biodistribution in mice of 99mTc–NIM–BAT

S,S′‐bis‐trityl‐N‐BOC‐1,2‐ethylenedicysteamine (S,S′‐bis‐trityl‐N‐BOC–BAT) was conjugated to 2‐nitroimidazole (NIM) through a propylene spacer in order to provide a precursor for a potential technetium‐99 m labelled hypoxia tracer. For labelling with technetium‐99 m, a two‐step one‐pot procedure was developed consisting of deprotection of the ligand by heating in mild acidic conditions and subsequent exchange labelling in the presence of SnCl₂, tartrate and ^99mTcO. The labelling reaction mixture was analyzed using electrospray radio‐LC–MS and the observed mass spectrum corresponding to the main radiometric peak was in accordance with the predicted structure of oxo–Tc(V)–NIM–BAT. ^99mTc–NIM–BAT was purified using RP–HPLC and its biodistribution was evaluated in normal mice at 10 min and 4 h p.i. ^99mTc–NIM–BAT was cleared from plasma mainly by hepatobiliary excretion. Copyright © 2003 John Wiley & Sons, Ltd.     

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

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

Investigating the ability of the microbial model Cunninghamella elegans for the metabolism of synthetic tryptamines

Tryptamines can occur naturally in plants, mushrooms, microbes, and amphibians. Synthetic tryptamines are sold as new psychoactive substances (NPS) because of their hallucinogenic effects. When it comes to NPS, metabolism studies are of crucial importance, due to the lack of pharmacological and toxicological data. Different approaches can be taken to study in vitro and in vivo metabolism of xenobiotica. The zygomycete fungus Cunninghamella elegans (C. elegans) can be used as a microbial model for the study of drug metabolism. The current study investigated the biotransformation of four naturally occurring and synthetic tryptamines [N,N‐Dimethyltryptamine (DMT), 4‐hydroxy‐N‐methyl‐N‐ethyltryptamine (4‐HO‐MET), N,N‐di allyl‐5‐methoxy tryptamine (5‐MeO‐DALT) and 5‐methoxy‐N‐methyl‐N‐isoporpoyltryptamine (5‐MeO‐MiPT)] in C. elegans after incubation for 72 hours. Metabolites were identified using liquid chromatography–high resolution–tandem mass spectrometry (LC–HR–MS/MS) with a quadrupole time‐of‐flight (QqTOF) instrument. Results were compared to already published data on these substances. C. elegans was capable of producing all major biotransformation steps: hydroxylation, N‐oxide formation, carboxylation, deamination, and demethylation. On average 63% of phase I metabolites found in the literature could also be detected in C. elegans. Additionally, metabolites specific for C. elegans were identified. Therefore, C. elegans is a suitable complementary model to other in vitro or in vivo methods to study the metabolism of naturally occurring or synthetic tryptamines.     

Ultra‐fast LC–MS/MS in therapeutic drug monitoring: Quantification of clozapine and norclozapine in human plasma

A novel approach to high‐throughput, targeted liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis has been developed. A single chromatographic system can be used for the analysis of a range of 20 drugs and metabolites with a total analysis time of 36 s (one 96‐well plate of prepared samples per hour). To demonstrate the applicability of this approach to quantitative analysis, a method has been validated for the therapeutic drug monitoring of clozapine and norclozapine following automated extraction from human plasma. Chromatographic retention times were 11.4 and 12.4 s for norclozapine and clozapine, respectively (for both analytes the chromatographic peak width was less than 1 s). Comparison with a conventional LC–MS/MS method (5 min analysis time) showed excellent agreement. This new approach offers analysis times more akin to flow‐injection analysis, but is likely to be more widely applicable because of chromatographic resolution from residual matrix components and isobaric interferences.     

Detection of stanozolol O‐ and N‐sulfate metabolites and their evaluation as additional markers in doping control

Stanozolol (STAN) is one of the most frequently detected anabolic androgenic steroids in sports drug testing. STAN misuse is commonly detected by monitoring metabolites excreted conjugated with glucuronic acid after enzymatic hydrolysis or using direct detection by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). It is well known that some of the previously described metabolites are the result of the formation of sulfate conjugates in C17, which are converted to their 17‐epimers in urine. Therefore, sulfation is an important phase II metabolic pathway of STAN that has not been comprehensively studied. The aim of this work was to evaluate the sulfate fraction of STAN metabolism by LC‐MS/MS to establish potential long‐term metabolites valuable for doping control purposes. STAN was administered to six healthy male volunteers involving oral or intramuscular administration and urine samples were collected up to 31 days after administration. Sulfation of the phase I metabolites commercially available as standards was performed in order to obtain MS data useful to develop analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) to detect potential sulfate metabolites. Eleven sulfate metabolites (M‐I to M‐XI) were detected and characterized by LC‐MS/MS. This paper provides valuable data on the ionization and fragmentation of O‐ sulfates and N‐ sulfates. For STAN, results showed that sulfates do not improve the retrospectivity of the detection compared to the previously described long‐term metabolite (epistanozolol‐N ‐glucuronide). However, sulfate metabolites could be additional markers for the detection of STAN misuse. Copyright © 2016 John Wiley & Sons, Ltd.     

Analysis of ethyl glucuronide in hair samples by liquid chromatography‐electrospray ionization–tandem mass spectrometry (LC‐ESI‐MS/MS)

Many different biomarkers can be used to evaluate ethanol intake. Ethyl glucuronide (EtG) is a direct phase II and minor metabolite of ethanol formed through the UDP‐glucuronosyl transferase‐catalyzed conjugation of ethanol with glucuronic acid. Its investigation is of interest in both clinical and forensic contexts because of the wide window of detection. A sensitive LC‐MS/MS procedure has been developed and fully validated according to the guidelines of forensic toxicology for the analysis of EtG in hair. Sample preparation and chromatographic separation were thoroughly optimized. The analysis was performed in the multiple reaction monitoring mode using the transitions m/z 221 → 203 (for the quantification) and 221 → 85 or 75 (for the qualification) for EtG, and m/z 226 → 208 (for quantification) and 226 → 75 or 85 (for qualification) for EtG‐D5, used as the internal standard. Analyses were carried out using an Inertsil ODS‐3 column (100 × 3 mm i.d., 3 µm particle size) and a mobile phase composed of formic acid and acetonitrile. Various SPE cartridges and solvents were tested in order to obtain the highest recoveries and cleanest extracts. The assay linearity of EtG was confirmed over the range from 20 to 2500 pg mg^?1, with a coefficient of determination (R²) above 0.99. The lower limit of quantitation (LLOQ) was 20 pg mg^?1 and the limit of detection was 10 pg mg^?1. Intra‐ and inter‐day assays were less than 15% except at the LLOQ (20%). The analytical method was applied to 72 post‐mortem hair samples. EtG concentration in the hair ranged from 0 to 653 pg mg^?1 hair. Copyright © 2012 John Wiley & Sons, Ltd.     

Syntheses,analytical and pharmacological characterizations of the ‘legal high’ 4‐[1‐(3‐methoxyphenyl)cyclohexyl]morpholine (3‐MeO‐PCMo) and analogues

New psychoactive substances (NPS) are commonly referred to as ‘research chemicals’, ‘designer drugs’ or ‘legal highs’. One NPS class is represented by dissociative anesthetics, which include analogues of the arylcyclohexylamine phencyclidine (PCP), ketamine and diphenidine. A recent addition to the NPS market was 4‐[1‐(3‐methoxyphenyl)cyclohexyl]morpholine (3‐MeO‐PCMo), a morpholine analogue of 3‐MeO‐PCP. Although suspected to have dissociative effects in users, information about its pharmacological profile is not available. From clinical and forensic perspectives, detailed analytical data are needed for identification, especially when facing the presence of positional isomers, as these are frequently unavailable commercially. This study presents the analytical and pharmacological characterization of 3‐MeO‐PCMo along with five additional analogues, namely the 2‐ and 4‐MeO‐PCMo isomers, 3,4‐methylenedioxy‐PCMo (3,4‐MD‐PCMo), 3‐Me‐PCMo and PCMo. All six arylcyclohexylmorpholines were synthesized and characterized using chromatographic, mass spectrometric and spectroscopic techniques. The three positional isomers could be differentiated and the identity of 3‐MeO‐PCMo obtained from an internet vendor was verified. All six compounds were also evaluated for affinity at 46 central nervous system receptors including the N‐methyl‐d ‐aspartate receptor (NMDAR), an important target for dissociative anesthetics such as PCP and ketamine. In vitro binding studies using (+)‐[3‐³H]‐MK‐801 in rat forebrain preparations revealed moderate affinity for NMDAR in the rank order of 3‐Me >3‐MeO > PCMo >3,4‐MD > 2‐MeO > 4‐MeO‐PCMo. 3‐MeO‐PCMo was found to have moderate affinity for NMDAR comparable to that of ketamine, and had an approximate 12‐fold lower affinity than PCP. These results support the anecdotal reports of dissociative effects from 3‐MeO‐PCMo in humans.