5F‐ADB (methyl 2‐{[1‐(5‐fluoropentyl)‐1H‐indazole‐3‐carbonyl] amino}‐3,3‐dimethylbutanoate) is a frequently abused new synthetic cannabinoid that has been sold since at least the end of 2014 on the drug market and has been classified as an illicit drug in most European countries, as well as Turkey, Japan, and the United States. In this study, the in vitro metabolism of 5F‐ADB was investigated by using pooled human liver microsomes (HLMs) assay and liquid chromatography‐high‐resolution mass spectrometry (LC–HRMS). 5F‐ADB (5 μmol/L) was incubated with HLMs for up to 3 hours, and the metabolites were identified using LC–HRMS and software‐assisted data mining. The in vivo metabolism was investigated by the analysis of 30 authentic urine samples and was compared to the data received from the in vitro metabolism study. Less than 3.3% of the 5F‐ADB parent compound remained after 1 hour of incubation, and no parent drug was detected after 3 hours. We identified 20 metabolites formed via ester hydrolysis, N‐dealkylation, oxidative defluorination, hydroxylation, dehydrogenation, further oxidation to N‐pentanoic acid and glucuronidation or a combination of these reactions in vitro. In 12 urine samples (n = 30), 5F‐ADB was detected as the parent drug. Three of the identified main metabolites 5F‐ADB carboxylic acid (M20), monohydroxypentyl‐5F‐ADB (M17), and carboxypentyl ADB carboxylic acid (M8) were suggested as suitable urinary markers. The screening of 8235 authentic urine samples for identified 5F‐ADB metabolites in vitro resulted in 3135 cases of confirmed 5F‐ADB consumption (38%).

CUMYL-4CN-BINACA(1-(4-cyanobutyl)-N-(2-phenylpropan-2-yl)-1H-indazole-3-carboxamide) is a recently introduced indazole-3-carboxamide-type synthetic cannabinoid (SC) that was detected in herbal incense seized by of the Council of Forensic Medicine, Istanbul Narcotics Department, in May 2016 in Turkey. Recently introduced SCs are not detected in routine toxicological analysis; therefore, analytical methods to measure these compounds are in demand. The present study aims to identify urinary marker metabolites of CUMYL-4CN-BINACA by investigating its metabolism in human liver microsomes and to confirm the results in authentic urine samples (n = 80). In this study, 5 μM CUMYL-4CN-BINACA was incubated with human liver microsomes (HLMs) for up to 3 hours, and metabolites were identified using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Less than 21% of the CUMYL-4CN-BINACA parent compound remained after 3 hours of incubation. We identified 18 metabolites that were formed via monohydroxylation, dealkylation, oxidative decyanation to aldehyde, alcohol, and carboxylic acid formation, glucuronidation or reaction combinations. CUMYL-4CN-BINACA N-butanoic acid (M16) was found to be major metabolite in HLMs. In urine samples CUMYL-4CN-BINACA was not detected; CUMYL-4CN-BINACA N-butanoic acid (M16) was major metabolite after β-glucuronidase hydrolysis. Based on these findings, we recommend using M16 (CUMYL-4CN-BINACA N-butanoic acid), M8 and M11 (hydroxylcumyl CUMYL-4CN-BINACA) as urinary marker metabolites to confirm CUMYL-4CN-BINACA intake.

In May 2016, a new type of synthetic indazole-3-carboxamide cannabinoid (CUMYL-4CN-BINACA) was detected in seized plant material submitted to the Istanbul Council of Forensic Medicine by the National Police Office. The major ingredient in this material was purified using preparative liquid chromatography, and its structure was identified using liquid chromatography-high-resolution mass spectrometry (LC-HR/MS), gas chromatography-electron ionization/mass spectrometry (GC-EI/MS), nuclear magnetic resonance (NMR) spectroscopy and Fourier transform-infrared spectroscopy (FT-IR). Using HR-MS, the molecular formula of the compound was determined to be C22H24N4O (MW = 360.1950). The 1H and 13C-NMR and FT-IR spectrometric data revealed that the structure of compound was 1-(4-cyanobutyl)-N-(2-phenylpropan-2-yl)-1H-indazole-3-carboxamide (CUMYL-4CN-BINACA). After identification, it was quickly added to our generic drug list, and an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method was developed to determine its presence in blood samples. This study reports on the identification of CUMYL-4CN-BINACA in plant material using LC-HR/MS, GC-EI/MS, NMR and FT-IR as well as a validated method for quantification of CUMYL-4CN-BINACA in post-mortem blood samples by UPLC-MS-MS analysis. The quantification method has been validated in terms of linearity (0.1-50 ng/mL), selectivity, intra- and inter-assay accuracy and precision (CV < 15%), recovery (94-99%), limit of detection (0.07 ng/mL) and limit of quantification (0.1 ng/mL). Matrix effects, stability and process efficiency were also assessed. The method has been applied to 2,350 post-mortem blood samples from the autopsy cases in the Morgue Department of the Council of Forensic Medicine (Istanbul, Turkey) between 1 July 2016 and 31 December 2016.

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MDMB-4en-PINACA (methyl (S)-3,3-dimethyl-2-(1-(pent-4-en-1-yl)-1H-indazole-3-carboxamido)butanoate) is a recently emerged synthetic cannabinoid in Turkey. MDMB-4en-PINACA was detected in herbal material investigated by the Council of Forensic Medicine, Istanbul Narcotics Department in Turkey in April 2019. MDMB-4en-PINACA was added to the drug abuse list and quickly reported in biological samples after its first detection. In this study, the in vitro metabolism of MDMB-4en-PINACA was investigated by using a pooled human liver microsomes (HLMs) assay and liquid chromatography–high-resolution mass spectrometry (LC–HRMS). MDMB-4en-PINACA (5 μmol/L) was incubated with HLMs for up to 1 h, and the metabolites were identified using LC–HRMS and software-assisted data mining. The in vivo metabolism was investigated by the analysis of 22 authentic urine samples and compared to the data received from the in vitro metabolism study. Less than 7.5% of the MDMB-4en-PINACA parent compound remained after the 1 h incubation. We identified 14 metabolites, which were formed via double bond oxidation, ester hydrolysis, N-dealkylation, hydroxylation, dehydrogenation and further oxidation to N-pentanoic acid or a combination of these reactions in vitro. In 10 urine samples (total n = 22), MDMB-4en-PINACA was detected as the parent drug. Three of the identified main metabolites, double bond oxidation in combination with ester hydrolysis and hydroxylation metabolite (M3), MDMB-4en-PINACA butanoic acid (M14) and monohydroxypentyl-MDMB-4en-PINACA (M12), were suggested as suitable urinary markers. In vitro screening of 2,150 authentic urine samples for these identified MDMB-4en-PINACA metabolites resulted in 56 cases of confirmed MDMB-4en-PINACA consumption (2.6%).