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 pretests for urine proved to be unsuitable for the detection of 5F-MDMB-PICA consumption.

Indole‐ and indazole‐based synthetic cannabinoid receptor agonists (SCRAs), featuring valine or tert‐leucine substituents, are commonly abused new psychoactive substances (NPS). A major metabolic pathway for these SCRAs is hydrolysis of the terminal amide or methylester functionalities. Although these hydrolysis products were already detected as main ingredients in some “legal highs,” these metabolites are often poorly characterized. Here, we report a systematic investigation of the activity of 7 common hydrolysis metabolites of 15 SCRAs featuring scaffolds based on L‐valine or L‐tert‐leucine in direct comparison to their parent compounds. An activity‐based cannabinoid receptor 1 (CB1) bio‐assay was used for activity profiling of SCRAs and their metabolites in a stable HEK293T cell system. The recruitment of β‐arrestin2 to the activated CB1 (each fused to one part of a split Nanoluciferase) was provoked by adding the (putative) SCRAs. Luminescence of the functionally complemented luciferase was monitored by a 96‐well plate‐reader. The major hydrolysis metabolites of 5F‐AB‐PINACA, ADB‐CHMICA, ADB‐CHMINACA, ADB‐FUBICA, and their methyl‐ and ethylester derivatives showed no detectable CB1 activation at concentrations up to 1 μM. On the other hand, metabolites of 5F‐ADB‐PINACA, AB‐CHMINACA, and ADB‐FUBINACA did retain activity, although significantly reduced as compared to the parent compounds (EC50 values >100 nM). Activity‐based characterization of SCRAs and their metabolites at CB1 may not only allow a better insight into the complex interplay between SCRAs and their metabolites in intoxications, but may also allow application of the concept of “activity equivalents” present in biological fluids or, alternatively, in confiscated materials.

Synthetic cannabinoids (SCs) remain one of the largest groups of new psychoactive substances (NPS) on the European drug market. Although the number of new derivatives occurring on the market has dropped in the last two years, newly emerging NPS still represent a challenge for laboratories performing forensic drug analysis in biological matrices. The newly emerged SC 4F‐MDMB‐BINACA has been reported by several law enforcement agencies in Europe and the USA since November 2018. This work aimed at revealing urinary markers to prove uptake of 4F‐MDMB‐BINACA and differentiate from the use of structurally similar SCs. Phase‐I metabolites detected in human urine specimens were confirmed by phase‐I metabolites generated in vitro using a pooled human liver microsomes (pHLM) assay. Seized materials and test‐purchased “legal high” products were analyzed by gas chromatography–mass spectrometry (GC–MS) and liquid chromatography−quadrupole‐time‐of‐flight−mass spectrometry (LC−qToF−MS). Human urine specimens and pHLM assay extracts were measured with liquid chromatography−electrospray ionization−tandem mass spectrometry (LC−ESI−MS/MS) and confirmed by LC−qToF−MS. In January 2019, the Institute of Legal Medicine in Erlangen (Germany) identified 4F‐MDMB‐BINACA in three herbal blends. During the same time period, the described SC was identified in a research chemical purchased online. Investigation of phase‐I metabolism led to the metabolites M10 (ester hydrolysis) and M11 (ester hydrolysis and dehydrogenation) as reliable urinary markers. Widespread distribution on the German drug market was proven by analysis of urine samples from abstinence control programs and by frequent detection of 4F‐MDMB‐BINACA in “herbal blends” and “‘research chemicals” purchased via the Internet.