Identification of non‐reported bupropion metabolites in human plasma

Bupropion and its three active metabolites exhibit clinical efficacy in the treatment of major depression, seasonal depression and smoking cessation. The pharmacokinetics of bupropion in humans is highly variable. It is not known if there are any non‐reported metabolites formed in humans in addition to the three known active metabolites. This paper reports newly identified and non‐reported metabolites of bupropion in human plasma samples. Human subjects were dosed with a single oral dose of 75 mg of an immediate release bupropion HCl tablet. Plasma samples were collected and analysed by LC–MS/MS at 0, 6 and 24 h. Two non‐reported metabolites (M1 and M3) were identified with mass‐to‐charge (m/z) ratios of 276 (M1, hydration of bupropion) and 258 (M3, hydroxylation of threo/erythrohydrobupropion) from human plasma in addition to the known hydroxybupropion, threo/erythrohydrobupropion and the glucuronidation products of the major metabolites (M2 and M4–M7). These new metabolites may provide new insight and broaden the understanding of bupropion's variability in clinical pharmacokinetics. © 2016 The Authors Biopharmaceutics & Drug Disposition Published by John Wiley & Sons Ltd.     

Tentative identification of the phase I and II metabolites of two synthetic cathinones,MDPHP and α‐PBP,in human urine

Cathinone derivatives are one of the more prominent groups of new psychoactive substances in terms of the number of forensic case reports and the variety of chemical structures available. These substances often sold as “bath salts” are classified as psychostimulants. Using liquid chromatography‐high resolution mass spectrometry, the metabolites of two pyrrolidine cathinone derivatives, α‐PBP and the less common MDPHP, were tentatively identified in urine samples collected from patients admitted to hospital following drug intoxications. The major metabolic pathways for α‐PBP and MDPHP were similar to those of their more common analogs (α‐PVP and MDPV). Metabolites arising from hydroxylation, reduction of the carbonyl group to an alcohol, oxidation to form a lactam and subsequent ring‐opening, and a combination of these processes were identified. In addition, biotransformations of the benzodioxole moiety in MDPHP included demethylenation with subsequent methylation and carboxylation of the butyl group. The majority of the hydroxylated metabolites of α‐PBP and MDPHP were found to be glucuronidated. Both α‐PBP and MDPHP undergo extensive metabolism and the chromatographic peak areas of the metabolites were found to be comparable to or exceeded those of the parent substances. Metabolites resulting from demethylenation and subsequent methylation (MDPHP), reduction of carbonyl group (α‐PBP), and oxidation to form a lactam combined with ring‐opening (α‐PBP and MDPHP) were found to be the most useful target analytes for the confirmation of ingestion.     

Synthesis and identification of deschloroketamine metabolites in rats' urine and a quantification method for deschloroketamine and metabolites in rats' serum and brain tissue using liquid chromatography tandem mass spectrometry

Deschloroketamine (2‐(methylamino)‐2‐phenyl‐cyclohexanone) is a ketamine analog belonging to a group of dissociative anesthetics, which have been distributed within the illicit market since 2015. However, it was also being sold as ‘ketamine' misleading people to believe that they were getting genuine ketamine. Dissociative anesthetics have also come to the attention of the psychiatric field due to their potential properties in the treatment of depression. At present, there is a dearth of information on deschloroketamine related to its metabolism, biodistribution, and its mechanism of action. We have therefore carried out a metabolomics study for deschloroketamine via non‐targeted screening of urine samples employing liquid chromatography combined with high‐resolution mass spectrometry. We developed and validated a multiple reaction monitoring method using a triple quadrupole instrument to track metabolites of deschloroketamine. Furthermore, significant metabolites of deschloroketamine, (trans‐dihydrodeschloroketamine, cis‐ and trans‐dihydronordeschloroketamine, and nordeschloroketamine), were synthesized in‐house. The prepared standards were utilized in the developed multiple reaction monitoring method. The quantification method for serum samples provided intra‐day accuracy ranging from 86% to 112% with precision of 3% on average. The concentrations of cis/trans‐dihydronordeschloroketamines and trans‐dihydrodeschloroketamine were lower than 10 ng/mL, nordeschloroketamine and deschloroketamine ranged from 0.5 to 860 ng/mL in real samples. The quantification method for brain tissue provided intra‐day accuracy ranging from 80% to 125% with precision of 7% on average. The concentrations of cis/trans‐dihydronordeschloroketamines and trans‐dihydrodeschloroketamine ranged from 0.5 to 70 ng/g, nordeschloroketamine and deschloroketamine varied from 0.5 to 4700 ng/g in real samples.     

5F‐MDMB‐PICA metabolite identification and cannabinoid receptor activity

According to the European Monitoring Center for Drugs and Drug Addiction (EMCDDA), there were 179 different synthetic cannabinoids reported as of 2017. In the USA, 5F‐MDMB‐PINACA, or 5F‐ADB, accounted for 28% of cannabinoid seizures 2016–2018. The synthetic cannabinoid, 5F‐MDMB‐PICA, is structurally similar to 5F‐MDMB‐PINACA with an indole group replacing the indazole. Limited data exist from in vivo or in vitro metabolic studies of these synthetic cannabinoids, so potential metabolites to identify use may be missed. The goals of this study were to (a) investigate 5F‐MDMB‐PICA and 5F‐MDMB‐PINACA in vitro metabolism utilizing human hepatocytes; (b) to verify in vitro metabolites by analyzing authentic case specimens; and (c) to identify the potency and efficacy of 5F‐MDMB‐PICA and 5F‐MDMB‐PINACA by examining activity at the CB₁ receptor. Biotransformations found in this study included phase I transformations and phase II transformations. A total of 22 5F‐MDMB‐PICA metabolites (A1 to A22) were identified. From hepatocyte incubations and urine samples, 21 metabolites (B1 to B21) were identified with 3 compounds unique to urine specimens for 5F‐MDMB‐PINACA. Phase II glucuronides were identified in 5F‐MDMB‐PICA (n = 3) and 5F‐MDMB‐PINACA (n = 5). For both compounds, ester hydrolysis and ester hydrolysis in combination with oxidative defluorination were the most prevalent metabolites produced in vitro. Additionally, the conversion of ester hydrolysis with oxidative defluorination to pentanoic acid for the first time was identified for 5F‐MDMB‐PICA. Therefore, these metabolites would be potentially good biomarkers for screening urine of suspected intoxication of 5F‐MDMB‐PICA or 5F‐MDMB‐PINACA. Both 5F‐MDMB‐PICA and 5F‐MDMB‐PINACA were acting as full agonists at the CB₁ receptor with higher efficacy and similar potency as JWH‐018.     

Automated optimization of XCMS parameters for improved peak picking of liquid chromatography–mass spectrometry data using the coefficient of variation and parameter sweeping for untargeted metabolomics

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.     

Analysis of AM‐2201 and metabolites in a drugs and driving case

This case report describes the analysis of AM‐2201 in plant material and its metabolites in human urine obtained from an operator of a motor vehicle in the United States. The samples were taken from the driver because of his illegal driving activities and, his subsequent erratic behaviour. The AM‐2201 was extracted from a sample of plant material by sonicating it in methanol, after which an aliquot was taken and diluted with aqueous phosphate buffer (pH 6) and extracted by solid‐phase extraction using a C8/aminopropyl SPE cartridge. The cartridges were washed, dried, and eluted with ethyl acetate‐ methanol solvent system. The urine sample was hydrolyzed with β‐glucuronidase at pH 6.8 before being diluted with aqueous phosphate buffer (pH 6) and extracted with the same type of SPE cartridge. After evaporation of the eluates, the samples were dissolved in mobile phase for analysis by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Analysis of the plant material determined the concentration to be 0.05% (AM2201) by mass of dry material. The concentration of AM‐2201 (AM2201‐N‐(4‐hydroxypentyl ) metabolite in the urine was found to be 3.1 ng/ml. The urine also contained 109 ng/ml of delta‐9‐tetrahydrocannabinol carboxylic acid but no other drugs including JWH‐018 metabolites. Copyright © 2013 John Wiley & Sons, Ltd.     

Profiling of synthesis‐related impurities of the synthetic cannabinoid Cumyl‐5F‐PINACA in seized samples of e‐liquids via multivariate analysis of UHPLC−MSn data

Vaping of synthetic cannabinoids via e‐cigarettes is growing in popularity. In the present study, we tentatively identified 12 by‐products found in a pure sample of the synthetic cannabinoid Cumyl‐5F‐PINACA (1‐(5‐fluoropentyl)‐N‐(2‐phenylpropan‐2‐yl)‐1H‐indazole‐3‐carboxamide), a prevalent new psychoactive substance (NPS) in e‐liquids, via high‐resolution mass spectrometry fragmentation experiments (HRMS/MS). Furthermore, we developed a procedure to reproducibly extract this synthetic cannabinoid and related by‐products from an e‐liquid matrix via chloroform and water. The extracts were submitted to flash chromatography (F‐LC) to isolate the by‐products from the main component. The chromatographic impurity signature was subsequently assessed by ultra‐high‐performance liquid chromatography coupled to mass spectrometry (UHPLC–MS) and evaluated by automated integration. The complete sample preparation sequence (F‐LC + UHPLC–MS) was validated by comparing the semi‐quantitative signal integrals of the chromatographic impurity signatures of five self‐made e‐liquids with varying concentrations of Cumyl‐5F‐PINACA [0.1, 0.2, 0.5, 0.7 and 1.0% (w/w)], giving an average relative standard deviation of 6.2% for triplicate measurements of preparations of the same concentration and 10.5% between the measurements of the five preparations with different concentrations. Lastly, the chromatographic signatures of 14 e‐liquid samples containing Cumyl‐5F‐PINACA from police seizures and Internet test purchases were evaluated via hierarchical cluster analysis for potential links. For the e‐liquid samples originating from test purchases, it was found that the date of purchase, the identity of the online shop, and the brand name are the critical factors for clustering of samples.     

Unambiguous identification and characterization of a long‐term human metabolite of dehydrochloromethyltestosterone

In doping control analysis, the characterization of urinary steroid metabolites is of high interest for a targeted and long‐term detection of prohibited anabolic androgenic steroids (AAS). In this work, the structure of a long‐term metabolite of dehydrochloromethyltestosterone (DHCMT) was elucidated. Altogether, 8 possible metabolites with a 17α‐methyl‐17β‐hydroxymethyl – structures were synthesized and compared to a major DHCMT long‐term metabolite detected in reference urine excretion samples. The confirmed structure of the metabolite was 4α‐chloro‐18‐nor‐17β‐hydroxymethyl‐17α‐methyl‐5α‐androst‐13‐en‐3α‐ol.     

Chemical profiling of the synthetic cannabinoid MDMB‐CHMICA: Identification,assessment, and stability study of synthesis‐related impurities in seized and synthesized samples

In this work, the most discriminating synthesis‐related impurities found in samples from seizures and controlled synthesis of the synthetic cannabinoid MDMB‐CHMICA (methyl (S)‐2‐(1‐(cyclohexylmethyl)‐1H‐indole‐3‐carboxamido)‐3,3‐dimethylbutanoate) were characterized. Based on 61 available powder samples of MDMB‐CHMICA, 15 key‐impurities were assessed, isolated in larger quantities via flash chromatography and structurally elucidated and characterized via high resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Apart from verifying the relation of the impurities to the major component, the interpretation of their chemical structures with distinct structural elements provided first insights into the manufacturing process and the precursor compounds used. Following liquid chromatography mass spectrometry analysis of the 15 key‐impurities, the 61 seized samples of MDMB‐CHMICA were evaluated and classified via multivariate data analysis based on the corresponding relative peak areas. In a second part of this work, stability tests and multiple controlled syntheses of MDMB‐CHMICA were carried out to better understand variations in impurity signatures and to assess the significance of variations in the impurity patterns of seized samples. The last coupling step of the amino acid with 1‐(cyclohexylmethyl)‐1H‐indole‐3‐carboxylic acid was performed using the coupling agents oxalyl chloride, thionyl chloride, and HATU. Furthermore, the impact of reaction time and temperature on the impurity profile were investigated. Overall, eight new impurities were found in the controlled syntheses and two degradation products of MDMB‐CHIMCA were found in the course of the stability tests. Replicates of a synthesis conducted on the same day showed similar impurity signatures; on different days they showed discriminable signatures. The use of different coupling reagents or conditions gave clearly distinguishable impurity signatures.     

Identification and analytical characterization of four synthetic cannabinoids ADB‐BICA,NNL‐1, NNL‐2, and PPA(N)‐2201

Since the first appearance as psychotropic drugs in illegal markets in 2008, the spread of synthetic cannabinoids is becoming a serious problem in many countries. This paper reports on the analytical properties and structure elucidation of four cannabimimetic derivatives in seized material: 1‐benzyl‐N ‐(1‐carbamoyl‐2,2‐dimethylpropan‐1‐yl)‐1H ‐indole‐3‐carboxamide (ADB‐BICA, 1 ), N ‐(1‐carbamoylpropan‐1‐yl)‐1‐(5‐fluoropentyl)‐1H ‐pyrrolo[2,3‐b]pyridine‐3‐carboxamide (NNL‐1, 2 ), (4‐benzylpiperazin‐1‐yl)(1‐(5‐fluoropentyl)‐1H ‐indol‐3‐yl)methanone (NNL‐2, 3 ), and N ‐(1‐carbamoyl‐2‐phenylethyl)‐1‐(5‐fluoropentyl)‐1H ‐indazole‐3‐carboxamide (PPA(N)‐2201, 4) . The identifications were based on liquid chromatography‐quadrupole‐time‐of‐flight‐mass spectrometry (LC‐QTOF‐MS), gas chromatography‐mass spectrometry (GC‐MS), Fourier transform infrared spectroscopy (FT‐IR), and nuclear magnetic resonance (NMR) spectroscopy. No chemical or pharmacological data about compounds 1–3 have appeared until now, making this the first report on these compounds. The GC‐MS data of 4 has been reported, but this study added the LC‐MS, FT‐IR, and NMR data for additional characterization. Copyright © 2016 John Wiley & Sons, Ltd.     

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

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

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.     

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

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

Targeted and non‐targeted drug screening in whole blood by UHPLC‐TOF‐MS with data‐independent acquisition

High‐resolution mass spectrometry (HRMS) is widely used for the drug screening of biological samples in clinical and forensic laboratories. With the continuous addition of new psychoactive substances (NPS), keeping such methods updated is challenging. HRMS allows for combined targeted and non‐targeted screening. First, peaks are identified by software algorithms, and identifications are based on reference standard data. Attempts are made to identify the remaining unknown peaks with in silico and literature data. However, several thousand peaks remain where most are unidentifiable or uninteresting in drug screening. The aims of the study were to apply a combined targeted and non‐targeted screening approach to authentic driving‐under‐the‐influence‐of‐drugs (DUID) samples (n = 44) and further validate the approach using whole‐blood samples spiked with 11 low‐dose synthetic benzodiazepine analogues (SBAs). Analytical data were acquired using ultra‐high‐performance liquid chromatography coupled with a time‐of‐flight mass spectrometer (UHPLC‐TOF‐MS) with data‐independent acquisition (DIA). We present a combined targeted and non‐targeted screening, where peak deconvolution and filtering reduced the number of peaks to inspect by three orders of magnitude, down to four peaks per DUID sample. The screening allowed for tentative identification of metabolites and drugs not included in the initial screening; 3 drugs and 14 metabolites were tentatively identified in the authentic DUID samples. Running targeted‐screening true‐positive identifications through the filters retained 73% of identifications. In the non‐targeted screening, nine of the spiked SBAs were identified in the concentration range of 0.005–0.1 mg/kg, of which three were tentatively identified at concentrations below those reported in the literature. Copyright © 2016 John Wiley & Sons, Ltd.     

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

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

Synthesis of deuterium‐labelled paclitaxel and its hydroxyl metabolite

This paper describes the synthesis of deuterium‐labelled paclitaxel and its hydroxyl metabolite. Paclitaxel labelled with ²H was obtained in four steps using the commercially available [²H₅]benzoic chloride as the stable labelled reagent with a 40% overall yield. The hydroxyl metabolite labelled with ²H was prepared starting from deuterium‐labelled paclitaxel in six steps with a 42% overall yield based on unrecovered starting material. Copyright © 2011 John Wiley & Sons, Ltd.     

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

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

Improved oxytocin analysis from human serum and urine by orbitrap ESI‐LC‐HRAM‐MS

Native circulating oxytocin (OT) levels in non‐pregnant/non‐lactating/non‐medicated humans are very low (≤ 8 pg/mL). The lower limit of detection (LLOD) of our previous liquid chromatography mass spectrometry (LC–MS) method (10–25 pg/mL) precluded their quantification in serum and urine. Thus, we sought to improve the LC–MS sensitivity of OT measurements in these matrices by hydrophobic tagging and solid phase extraction (SPE). In the former approach, OT was reduced then alkylated with N‐alkyl acetamide (C12, C14, C16, and C18) tags or derivatized using sulfonyl chloride‐based reagents. In the latter approach, native OT in serum and urine was concentrated by offline SPE using gradient acetonitrile washings after first crashing with acetonitrile. Peak urinary eluate fractions were further concentrated online then analyzed by orbitrap‐based LC–MS with electrospray ionization. All hydrophobic OT derivatives had lower sensitivity than native OT. Washing with a water‐acetonitrile gradient during SPE improved the LLOD of OT in spiked serum to 2.5 pg/mL, while adding a subsequent online‐concentration step improved the LLOD in spiked urine to 1–5 pg/mL and allowed us to detect OT in urine from lactating women. We were unable to improve the sensitivity of OT measurements by hydrophobic tagging or by derivatization using sulfonyl chloride‐based reagents. However, we were successful in improving the sensitivity of native OT measurements in serum and urine 2‐ and 5‐fold, respectively, from our previous orbitrap‐based LC–MS method. Offline SPE was mandatory for both matrices and a subsequent online‐concentration step was required for urine.     

Testing for AB‐PINACA in human hair: Distribution in head hair versus pubic hair

Synthetic cannabinoid receptor agonists were first identified in herbal products in 2008 advertised as a legal replacement for cannabis. These herbal incense are usually called “spice” and among these, one product in particular has gained popularity: AB‐PINACA (N‐[(2S)‐1‐Amino‐3‐methyl‐1‐oxobutan‐2‐yl]‐1‐pentyl‐1H‐indazole‐3‐carboxamide). This drug has been discovered to have a stronger binding to human cannabinoid CB₁ and CB₂ receptors than ?⁹‐THC.While some articles have been published regarding the presence of AB‐PINACA in biological fluids such as blood and urine, none reports the presence of AB‐PINACA in hair. We have developed and validated a method for detection of AB‐PINACA in hair using a liquid chromatography?tandem mass spectrometry system and applied it to head and pubic hair obtained in a case of intoxication. The validation procedure demonstrated a limit of detection and a limit of quantification of 0.5 and 1 pg/mg, respectively and acceptable linearity, repeatability, and reproducibility. AB‐PINACA tested positive in the blood (5.7 ng/mL) and less than 1 ng/mL was found in urine. The analysis of the hair specimens resulted in an unusual distribution of the drug between head and pubic hair. AB‐PINACA was identified at a higher concentration in head hair (195 pg/mg) versus in pubic hair (5 pg/mg). The very low concentration of AB‐PINACA in the urine after consumption, due to rapid metabolism, could explain this infrequent distribution, as pubic hair can be contaminated by urine. In any case, it cannot be excluded that the high concentration in head hair may be due to environmental contamination.     

Identification of the designer steroid Androsta‐3,5‐diene‐7,17‐dione in a dietary supplement

A liquid chromatography‐mass spectrometry (LC–MS) screen for known anabolic‐androgenic steroids in a dietary supplement product marketed for “performance enhancement” detected an unknown compound having steroid‐like spectral characteristics. The compound was isolated using high performance liquid chromatography with ultraviolet detection (HPLC–UV) coupled with an analytical scale fraction collector. After the compound was isolated, it was then characterized using gas chromatography with simultaneous Fourier Transform infrared detection and mass spectrometry (GC–FT–IR–MS), liquid chromatography–high resolution accurate mass–mass spectrometry (LC–HRAM–MS) and nuclear magnetic resonance (NMR). The steroid had an accurate mass of m/z 285.1847 (error?0.57 ppm) for the protonated species [M + H]⁺, corresponding to a molecular formula of C₁₉H₂₄O₂. Based on the GC–FT–IR–MS data, NMR data, and accurate mass, the compound was identified as androsta‐3,5‐diene‐7,17‐dione. Although this is not the first reported identification of this designer steroid in a dietary supplement, the data provided adds information for identification of this compound not previously reported. This compound was subsequently detected in another dietary supplement product, which contained three additional active ingredients.