Solid‐phase extraction–liquid chromatography–tandem mass spectrometry method for the qualitative analysis of 61 synthetic cannabinoid metabolites in urine

This article comprises the development and validation of a protocol for the qualitative analysis of 61 phase I synthetic cannabinoid metabolites in urine originating from 29 synthetic cannabinoids, combining solid‐phase extraction (SPE) utilizing a reversed phase silica‐based sorbent (phenyl) with liquid chromatography–tandem mass spectrometry (LC?MS/MS). Validation was performed according to the guidelines of the German Society of Toxicological and Forensic Chemistry. Sufficient chromatographic separation was achieved within a total runtime of 12.3 minutes. Validation included specificity and selectivity, limit of detection (LOD), recovery and matrix effects, as well as auto‐sampler stability of processed urine samples. LOD ranged between 0.025 ng/mL and 0.5 ng/mL in urine. Recovery ranged between 43% and 97%, with only two analytes exhibiting recoveries below 50%. However, for those two analytes, the LODs were 0.05 ng/mL in urine. In addition, matrix effects between 81% and 185% were determined, whereby matrix effects over 125% were observed for 10 non‐first‐generation synthetic cannabinoid metabolites. The developed method enables the rapid and sensitive detection of synthetic cannabinoid metabolites in urine, complementing the spectrum of existing analytical tools in forensic case work. Finally, application to 61 urine samples from both routine and autopsy case work yielded one urine sample that tested positive for ADB‐PINACA N‐pentanoic acid.     

Performance characteristics of an ELISA screening assay for urinary synthetic cannabinoids

Synthetic cannabinoids are marketed as legal alternatives to cannabis, as routine urine cannabinoid immunoassays do not detect synthetic cannabinoids. Laboratories are challenged to identify these new designer drugs that are widely available and represent a major public health and safety problem. Immunoassay testing offers rapid separation of presumptive positive and negative specimens, prior to more costly and time‐consuming chromatographic confirmation. The Neogen SPICE ELISA kit targets JWH‐018 N‐pentanoic acid as a marker for urinary synthetic cannabinoids. Assay performance was evaluated by analyzing 2469 authentic urine samples with the Neogen immunoassay and liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Two immunoassay cut‐off concentrations, 5 and 10 µg/L, classified samples as presumptive positive or negative, followed by qualitative LC‐MS/MS confirmation for 29 synthetic cannabinoids markers with limits of detection of 0.5–10 µg/L to determine the assay's sensitivity, specificity and efficacy. Challenges at ±25% of each cut‐off also were investigated to determine performance around the cut‐off and intra‐ and inter‐plate imprecision. The immunoassay was linear from 1 to 250 µg/L (r² = 0.992) with intra‐ and inter‐plate imprecision of ≤5.3% and <9%, respectively. Sensitivity, specificity, and efficiency results with the 5 µg/L cut‐off were 79.9%, 99.7%, and 97.4% and with the 10 µg/L cut‐off 69.3%, 99.8%, and 96.3%, respectively. Cross‐reactivity was shown for 18 of 73 synthetic cannabinoids markers evaluated. Good sensitivity, specificity, and efficiency, lack of sample preparation requirements, and rapid semi‐automation documented that the Neogen SPICE ELISA kit is a viable method for screening synthetic cannabinoids in urine targeting JWH‐018 N‐pentanoic acid. Copyright © 2014 John Wiley & Sons, Ltd.     

Screening,quantification, and confirmation of synthetic cannabinoid metabolites in urine by UHPLC–QTOF–MS

Synthetic cannabinoids are one of the most significant groups within the category new psychoactive substances (NPS) and in recent years new compounds have continuously been introduced to the market of recreational drugs. A sensitive and quantitative screening method in urine with metabolites of frequently seized compounds in Norway (AB‐FUBINACA, AB‐PINACA, AB‐CHMINACA, AM‐2201, AKB48, 5F‐AKB48, BB‐22, JWH‐018, JWH‐073, JWH‐081, JWH‐122, JWH‐203, JWH‐250, PB‐22, 5F‐PB‐22, RCS‐4, THJ‐2201, and UR‐144) using ultra‐high pressure liquid chromatography–quadrupole time of flight–mass spectrometry (UHPLC–QTOF–MS) has been developed. The samples were treated with ß‐glucuronidase prior to extraction and solid‐phase extraction was used. Liquid handling was automated using a robot. Chromatographic separation was achieved using a C18‐column and a gradient of water and acetonitrile, both with 0.1% formic acid. Each sample was initially screened for identification and quantification followed by a second injection for confirmation. The concentrations by which the compounds could be confirmed varied between 0.1 and 12 ng/mL. Overall the validation showed that the method fulfilled the set criteria and requirements for matrix effect, extraction recovery, linearity, precision, accuracy, specificity, and stability. One thousand urine samples from subjects in drug withdrawal programs were analyzed using the presented method. The metabolite AB‐FUBINACA M3, hydroxylated metabolite of 5F‐AKB48, hydroxylated metabolite of AKB48, AKB48 N‐pentanoic acid, 5F‐PB‐22 3‐carboxyindole, BB‐22 3‐carboxyindole, JWH‐018 N‐(5‐hydroxypentyl), JWH‐018 N‐pentanoic acid, and JWH‐073 N‐butanoic acid were quantified and confirmed in 2.3% of the samples. The method was proven to be sensitive, selective and robust for routine use for the investigated metabolites.     

Analysis of 30 synthetic cannabinoids in oral fluid using liquid chromatography‐electrospray ionization tandem mass spectrometry

In recent years, the analysis of synthetic cannabinoids in human specimens has gained enormous importance in the broad field of drug testing. Nevertheless, the considerable structural diversity among synthetic cannabinoids already identified in ‘herbal mixtures’ hampers the development of comprehensive analytical methods. As the identification of the main metabolites of newly appearing substances is very laborious and time‐consuming, the detection of the parent compounds in blood samples is the current approach of choice for drug abstinence testing. Whenever blood sampling is not possible however, the need for alternative matrices arises. In this article, we present a fully validated liquid chromatography‐electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) method for the analysis of 30 synthetic cannabinoids in oral fluid samples collected with the Dräger DCD 5000 collection device. The method proved to be suitable for the quantification of 28 substances. The limits of detection were in the range from 0.015 to 0.9 ng/ml, while the lower limits of quantification ranged from 0.15 to 3.0 ng/ml. The method was successfully applied to 264 authentic samples during routine analysis. A total of 31 samples (12%) was tested positive for at least one of the following synthetic cannabinoids: AM‐694, AM‐2201, JWH‐018, JWH‐019, JWH‐081, JWH‐122, JWH‐203, JWH‐210, JWH‐250, JWH‐307, MAM‐2201, and RCS‐4. Given that stabilization of the collection pads after sampling is warranted, the collection device provides satisfactory sensitivity. Hence, whenever blood sampling is not possible, the Dräger DCD 5000 collection device offers a good tool for the analysis of synthetic cannabinoids in oral fluid in the broad field of drug testing. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of a selection of synthetic cannabinoids and metabolites in urine by UHPSFC‐MS/MS and by UHPLC‐MS/MS

Two different analytical techniques, ultra‐high performance supercritical fluid chromatography‐tandem mass spectrometry (UHPSFC‐MS/MS) and reversed phase ultra‐high performance liquid chromatography‐tandem mass spectrometry (UHPLC‐MS/MS), were used for the determination of two synthetic cannabinoids and eleven metabolites in urine; AM‐2201 N‐4‐OH‐pentyl, AM‐2233, JWH‐018 N‐5‐OH‐pentyl, JWH‐018 N‐pentanoic acid, JWH‐073 N‐4‐OH‐butyl, JWH‐073 N‐butanoic acid, JWH‐122 N‐5‐OH‐pentyl, MAM‐2201, MAM‐2201 N‐4‐OH‐pentyl, RCS‐4 N‐5‐OH‐pentyl, UR‐144 degradant N‐pentanoic acid, UR‐144 N‐4‐OH‐pentyl, and UR‐144 N‐pentanoic acid. Sample preparation included a liquid‐liquid extraction after deconjugation with ß‐glucuronidase. The UHPSFC‐MS/MS method used an Acquity UPC^{2 TM} BEH column with a mobile phase consisting of CO₂ and 0.3% ammonia in methanol, while the UHPLC‐MS/MS method used an Acquity UPLC® BEH C₁₈ column with a mobile phase consisting of 5 mM ammonium formate (pH 10.2) and methanol. MS/MS detection was performed with positive electrospray ionization and two multiple reaction monitoring transitions. Deuterated internal standards were used for six of the compounds. Limits of quantification (LOQs) were between 0.04 and 0.4 µg/L. Between‐day relative standard deviations at concentrations ≥ LOQ were ≤20%, with biases within ±19%. Recoveries ranged from 40 to 90%. Corrected matrix effects were within 100 ± 10%, except for MAM‐2201 with UHPSFC‐MS/MS, and for UR‐144 N‐pentanoic acid and MAM‐2201 N‐4‐OH‐pentyl with UHPLC‐MS/MS. Elution order obtained by UHPSFC‐MS/MS was almost opposite to that obtained by UHPLC‐MS/MS, making this instrument setup an interesting combination for screening and confirmation analyses in forensic cases. The UHPLC‐MS/MS method has, since August 2014, been successfully used for confirmation of synthetic cannabinoids in urine samples revealing a positive immunoassay screening result. Copyright © 2015 John Wiley & Sons, Ltd.     

Doping control analysis of four JWH‐250 metabolites in equine urine by liquid chromatography–tandem mass spectrometry

JWH‐250 is a synthetic cannabinoid. Its use is prohibited in equine sport according to the Association of Racing Commissioners International (ARCI) and the Fédération Équestre Internationale (FEI). A doping control method to confirm the presence of four JWH‐250 metabolites (JWH‐250 4‐OH‐pentyl, JWH‐250 5‐OH‐pentyl, JWH‐250 5‐OH‐indole, and JWH‐250 N‐pentanoic acid) in equine urine was developed and validated. Urine samples were treated with acetonitrile and evaporated to concentrate the analytes prior to the analysis by liquid chromatography–tandem mass spectrometry (LC–MS/MS). The chromatographic separation was carried out using a Phenomenex Lux^® 3 μm AMP column (150 x 3.0 mm). A triple quadrupole mass spectrometer was used for detection of the analytes in positive mode electrospray ionization using multiple reaction monitoring (MRM). The limits of detection, quantification, and confirmation for these metabolites were 25, 50, and 50 pg/mL, respectively. The linear dynamic range of quantification was 50–10000 pg/mL. Enzymatic hydrolysis indicated that JWH‐250 4‐OH‐pentyl, JWH‐250 5‐OH‐pentyl, and JWH‐250 5‐OH indole are highly conjugated whereas JWH‐250 N‐pentanoic acid is not conjugated. Relative retention time and product ion intensity ratios were employed as the criteria to confirm the presence of these metabolites in equine urine. The method was successfully applied to post‐race urine samples collected from horses suspected of being exposed to JWH‐250. All four JWH‐250 metabolites were confirmed in these samples, demonstrating the method applicability for equine doping control analysis.     

Metabolic patterns of JWH‐210, RCS‐4, and THC in pig urine elucidated using LC‐HR‐MS/MS: Do they reflect patterns in humans?

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 Δ⁹‐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.     

Optimization of recombinant β‐glucuronidase hydrolysis and quantification of eight urinary cannabinoids and metabolites by liquid chromatography tandem mass spectrometry

Prolonged urinary cannabinoid excretion in chronic frequent cannabis users confounds identification of recent cannabis intake that may be important in treatment, workplace, clinical, and forensic testing programs. In addition, differentiation of synthetic Δ9‐tetrahydrocannabinol (THC) intake from cannabis plant products might be an important interpretive issue. THC, 11‐hydroxy‐THC (11‐OH‐THC) and 11‐nor‐9‐carboxy‐THC (THCCOOH) urine concentrations were evaluated during previous controlled cannabis administration studies following tandem alkaline/E. coli β‐glucuronidase hydrolysis. We optimized recombinant β‐glucuronidase enzymatic urinary hydrolysis before simultaneous liquid chromatography tandem mass spectrometry (LC–MS/MS) quantification of THC, 11‐OH‐THC, THCCOOH, cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), tetrahydrocannabivarin (THCV) and 11‐nor‐9‐carboxy‐THCV (THCVCOOH) in urine. Enzyme amount, incubation time and temperature, buffer molarity and pH were optimized using pooled urine samples collected during a National Institute on Drug Abuse, Institutional Review Board‐approved clinical study. Optimized cannabinoid hydrolysis with recombinant β‐glucuronidase was achieved with 2000 IU enzyme, 2 M pH 6.8 sodium phosphate buffer, and 0.2 mL urine at 37°C for 16 h. The LC–MS/MS quantification method for hydrolyzed urinary cannabinoids was validated per the Scientific Working Group on Toxicology guidelines. Linear ranges were 1–250 μg/L for THC and CBG, 2–250 μg/L for 11‐OH‐THC, CBD, CBN, THCV and THCVCOOH, and 1–500 μg/L for THCCOOH. Inter‐batch analytical bias was 92.4–112.4%, imprecision 4.4–9.3% CV (n = 25), extraction efficiency 44.3–97.1% and matrix effect ?29.6 to 1.8% (n = 10). The method was utilized to analyze urine specimens collected during our controlled smoked, vaporized, and edible cannabis administration study to improve interpretation of urine cannabinoid test results.     

Phase I metabolism of the highly potent synthetic cannabinoid MDMB‐CHMICA and detection in human urine samples

Among the recently emerged synthetic cannabinoids, MDMB‐CHMICA (methyl N ‐{[1‐(cyclohexylmethyl)‐1H ‐indol‐3‐yl]carbonyl}‐3‐methylvalinate) shows an extraordinarily high prevalence in intoxication cases, necessitating analytical methods capable of detecting drug uptake. In this study, the in vivo phase I metabolism of MDMB‐CHMICA was investigated using liquid chromatography‐electrospray ionization‐tandem mass spectrometry (LC‐ESI‐MS/MS) and liquid chromatography‐electrospray ionization‐quadrupole time‐of‐flight‐mass spectrometry (LC‐ESI‐Q ToF‐MS) techniques. The main metabolites are formed by hydrolysis of the methyl ester and oxidation of the cyclohexyl methyl side chain. One monohydroxylated metabolite, the ester hydrolysis product and two further hydroxylated metabolites of the ester hydrolysis product are suggested as suitable targets for a selective and sensitive detection in urine. All detected in vivo metabolites could be verified in vitro using a human liver microsome assay. Two of the postulated main metabolites were successfully included in a comprehensive LC‐ESI‐MS/MS screening method for synthetic cannabinoid metabolites. The screening of 5717 authentic urine samples resulted in 818 cases of confirmed MDMB‐CHMICA consumption (14%). Since the most common route of administration is smoking, smoke condensates were analyzed to identify relevant thermal degradation products. Pyrolytic cleavage of the methyl ester and amide bond led to degradation products which were also formed metabolically. This is particularly important in hair analysis, where detection of metabolites is commonly considered a proof of consumption. In addition, intrinsic activity of MDMB‐CHMICA at the CB₁ receptor was determined applying a cAMP accumulation assay and showed that the compound is a potent full agonist. Based on the collected data, an enhanced interpretation of analytical findings in urine and hair is facilitated. Copyright © 2016 John Wiley & Sons, Ltd.     

Phase I metabolism of the carbazole‐derived synthetic cannabinoids EG‐018, EG‐2201, and MDMB‐CHMCZCA and detection in human urine samples

Synthetic cannabinoids (SCs) are a structurally diverse class of new psychoactive substances. Most SCs used for recreational purposes are based on indole or indazole core structures. EG‐018 (naphthalen‐1‐yl(9‐pentyl‐9H‐carbazol‐3‐yl)methanone), EG‐2201 ((9‐(5‐fluoropentyl)‐9H‐carbazol‐3‐yl)(naphthalen‐1‐yl)methanone), and MDMB‐CHMCZCA (methyl 2‐(9‐(cyclohexylmethyl)‐9H‐carbazole‐3‐carboxamido)‐3,3‐dimethylbutanoate) are 3 representatives of a structural subclass of SCs, characterized by a carbazole core system. In vitro and in vivo phase I metabolism studies were conducted to identify the most suitable metabolites for the detection of these substances in urine screening. Detection and characterization of metabolites were performed by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) and liquid chromatography–electrospray ionization–quadrupole time‐of‐flight–mass spectrometry (LC–ESI–QToF–MS). Eleven in vivo metabolites were detected in urine samples positive for metabolites of EG‐018 (n = 8). A hydroxypentyl metabolite, most probably the 4‐hydroxypentyl isomer, and an N‐dealkylated metabolite mono‐hydroxylated at the carbazole core system were most abundant. In vitro studies of EG‐018 and EG‐2201 indicated that oxidative defluorination of the 5‐fluoropentyl side chain of EG‐2201 as well as dealkylation led to common metabolites with EG‐018. This has to be taken into account for interpretation of analytical findings. A differentiation between EG‐018 and EG‐2201 (n = 1) uptake is possible by the detection of compound‐specific in vivo phase I metabolites evaluated in this study. Out of 30 metabolites detected in urine samples of MDMB‐CHMCZCA users (n = 20), a metabolite mono‐hydroxylated at the cyclohexyl methyl tail is considered the most suitable compound‐specific consumption marker while a biotransformation product of mono‐hydroxylation in combination with hydrolysis of the terminal methyl ester function provides best sensitivity due to its high abundance.     

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.     

Frequency of new psychoactive substances in hair and urine samples of individuals subject to drug testing in driving license regranting—A toxicological perspective

In recent years, numerous new psychoactive substances (NPS) have emerged on the illicit drug market. The assumed non-detectability of these drugs is often a key motivation for individuals subject to drug testing, such as those in driving license regranting programs. In these programs, NPS are not routinely tested for, and thus, subjects who have to prove abstinence from common drugs of abuse might switch to NPS to avoid positive drug tests. The aim of the study was to determine the frequency of these substances in the hair and urine samples of individuals undergoing drug testing in driving license regranting. A total of 1037 samples (577 hair and 460 urine samples) collected from 949 subjects between February 2017 and December 2018 were retrospectively analyzed for designer drugs and synthetic cannabinoids by liquid chromatography–quadrupole-time-of-flight–mass spectrometry (LC-QTOF-MS). For a more sensitive analysis of synthetic cannabinoids and their metabolites, additional testing was performed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Overall, 42 hair and two urine samples, which were obtained from 40 subjects, tested positive for NPS resulting in a frequency of 4.2%. While synthetic cannabinoids were detected in all cases, designer drugs were only found in three of these cases. With regard to the 577 hair samples analyzed, 7.3% screened positive, whereas only 0.4% of the 460 tested urine samples contained NPS. The results of this study indicate that synthetic cannabinoid use seems to be popular among this population, and therefore, testing for synthetic cannabinoids should be requested more often preferably using hair analysis.     

The detection of the urinary metabolites of 3-[(adamantan-1-yl)carbonyl]-1-pentylindole (AB-001), a novel cannabimimetic, by gas chromatography-mass spectrometry

3‐[(Adamantan‐1‐yl)carbonyl]‐1‐pentylindole (AB‐001), a synthetic cannabimimetic, was identified in head shop products in Ireland in 2010. German authorities also reported it to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) via the Early Warning System (EWS) in 2011. As indole‐derived cannabimimetics, such as JWH‐018, JWH‐073, and JWH‐250, undergo extensive metabolism, it was expected that AB‐001 would behave similarly. To include it in our toxicological screening protocols, we have identified its urinary metabolites in humans following oral administration. The major metabolites were found to be adamantane mono‐hydroxylated and adamantane mono‐hydroxylated/N‐dealkylated products. No parent compound was found in urine, and metabolites were detectable for up to 160 h following administration. Copyright © 2011 John Wiley & Sons, Ltd.     

Metabolites of 5F‐AKB‐48, a synthetic cannabinoid receptor agonist,identified in human urine and liver microsomal preparations using liquid chromatography high‐resolution mass spectrometry

New types of synthetic cannabinoid designer drugs are constantly introduced to the illicit drug market to circumvent legislation. Recently, N‐?(1‐Adamant?yl)‐?1‐?(5‐?fluoropentyl)‐?1H‐?indazole‐?3‐?carboxamide (5F‐AKB‐48), also known as 5F‐APINACA, was identified as an adulterant in herbal products. This compound deviates from earlier JHW‐type synthetic cannabinoids by having an indazole ring connected to an adamantyl group via a carboxamide linkage. Synthetic cannabinoids are completely metabolized, and identification of the metabolites is thus crucial when using urine as the sample matrix. Using an authentic urine sample and high‐resolution accurate‐mass Fourier transform Orbitrap mass spectrometry, we identified 16 phase‐I metabolites of 5F‐AKB‐48. The modifications included mono‐, di‐, and trihydroxylation on the adamantyl ring alone or in combination with hydroxylation on the N‐fluoropentylindazole moiety, dealkylation of the N‐fluoropentyl side chain, and oxidative loss of fluorine as well as combinations thereof. The results were compared to human liver microsomal (HLM) incubations, which predominantly showed time‐dependent formation of mono‐, di‐, and trihydroxylated metabolites having the hydroxyl groups on the adamantyl ring. The results presented here may be used to select metabolites specific of 5F‐AKB‐48 for use in clinical and forensic screening. Copyright © 2014 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.     

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

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

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.     

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

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

Toxicokinetics 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.     

Quantification of six cannabinoids and metabolites in oral fluid by liquid chromatography‐tandem mass spectrometry

Δ⁹‐Tetrahydrocannabinol (THC) is the most commonly analyzed cannabinoid in oral fluid (OF); however, its metabolite 11‐nor‐9‐carboxy‐THC (THCCOOH) offers the advantage of documenting active consumption, as it is not detected in cannabis smoke. Analytical challenges such as low (ng/L) THCCOOH OF concentrations hampered routine OF THCCOOH monitoring. Presence of minor cannabinoids like cannabidiol and cannabinol offer the advantage of identifying recent cannabis intake. Published OF cannabinoids methods have limitations, including few analytes and lengthy derivatization. We developed and validated a sensitive and specific liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method for THC, its metabolites, 11‐hydroxy‐THC and THCCOOH quantification, and other natural cannabinoids including tetrahydrocannabivarin (THCV), cannabidiol (CBD), and cannabigerol (CBG) in 1 mL OF collected with the Quantisal device. After solid‐phase extraction, chromatography was performed on a Selectra PFPP column with a 0.15% formic acid in water and acetonitrile gradient with a 0.5 mL/min flow rate. All analytes were monitored in positive mode atmospheric pressure chemical ionization (APCI) with multiple reaction monitoring. Limits of quantification were 15 ng/L THCCOOH and 0.2 µg/L for all other analytes. Linear ranges extended to 3750 ng/L THCCOOH, 100 µg/L THC, and 50 µg/L for all other analytes. Inter‐day analytical recoveries (bias) and imprecision at low, mid, and high quality control (QC) concentrations were 88.7‐107.3% and 2.3‐6.7%, respectively (n = 20). Mean extraction efficiencies and matrix effects evaluated at low and high QC were 75.9–86.1% and 8.4–99.4%, respectively. This method will be highly useful for workplace, criminal justice, drug treatment and driving under the influence of cannabis OF testing. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.