Synthetic cannabinoid receptor agonists (SCRAs) are one of the largest groups of new psychoactive substances (NPS). Yet, another novel analog started spreading on the NPS market around 2021. Soon after, the substance could be analytically characterized in herbal material as ADB‐HEXINACA, an SCRA containing a hexyl‐substituted tail on the indazole core. Here, we present suitable urinary markers to prove the consumption of this analog, a case report of acute polydrug intoxication and data on its prevalence in Germany. Anticipated phase I metabolites were detected in 12 authentic urine samples that were collected for abstinence control and analyzed by ultra‐performance liquid chromatography coupled to a time‐of‐flight mass spectrometer (UPLC‐qToF‐MS). The results of in vivo samples were confirmed by analysis of in vitro incubates with pooled human liver microsomes (pHLMs). Forensic samples were used to assess the prevalence of ADB‐HEXINACA. Thirty‐two phase I metabolites were detected in the authentic urine samples. The main metabolites resulted from amide hydrolysis in combination with either monohydroxylation or ketone formation at the hexyl moiety (M15 and M26), the monitoring of which is recommended as a proof of consumption. ADB‐HEXINACA was detected in 3.5% of SCRA positive urine samples collected for abstinence control in Freiburg up to December 2022 and in 5.5% of the SCRA positive blood/serum samples. The hexyl substituent of ADB‐HEXINACA allows for the detection of specific urinary biomarkers suggested as analytical targets to confirm its prior intake. ADB‐HEXINACA had a rather low prevalence in Germany, alternating months of higher prevalence with periods of total absence.

In order to detect the abuse of substances in sports, the knowledge of their metabolism is of undisputable importance. As in vivo administration of compounds faces ethical problems and might even not be applicable for non-approved compounds, cell-based models might be a versatile tool for biotransformation studies. We co-incubated HepG2 cells with metandienone and D₃-epitestosterone for 14 days. Phase I and II metabolites were analyzed by HPLC-tandem mass spectrometry, and confirmed by GC-MS. The metandienone metabolites formed by HepG2 cells were comparable to those renally excreted by humans. HepG2 cells also generated the two long-term metabolites 17β-hydroxymethyl-17α-methyl-18-nor-androst-1,4,13-trien-3-one and 17α-hydroxymethyl-17β-methyl-18-nor-androst-1,4,13-trien-3-one used in doping analyses, though in an inverse ratio compared to that observed in human urine. In conclusion, we showed that HepG2 cells are suitable as model for the investigation of biotransformation of androgens, especially for the anabolic androgenic steroid metandienone. They further proved to cover phase I and II metabolic pathways, which combined with a prolonged incubation time with metandienone resulted in the generation of its respective long-term metabolites known from in vivo metabolism. Moreover, we showed the usability of D₃-epitestosterone as internal standard for the incubation. The method used herein appears to be suitable and advantageous compared to other models for the investigation of doping-relevant compounds, probably enabling the discovery of candidate metabolites for doping analyses.

The recent change from the popular carboxamide to an acetamide (ATA) linker scaffold in synthetic cannabinoid receptor agonists (SCRAs) can be interpreted as an attempt to circumvent legal regulations, setting new analytical challenges. Metabolites of N-cyclohexyl-2-(1-pentyl-1 H-indol-3-yl)acetamide: CH-PIATA, the second ATA type SCRA detected in the EU, were investigated in urine and serum samples by LC–HRMS-MS and LC–MS-MS. Two different in vitro models, a pHLM assay and HepG2-cells, as well as an in silico prediction by GLORYx freeware assisted in metabolite formation/identification. CH-PIATA was extensively metabolized, leading to metabolites formed primarily by mono- and dihydroxylation. For urine and serum specimens, monohydroxylation at the indole core or the methylene spacer of the acetamide linker (M1.8), carboxylic acid formation at the N-pentyl side chain (M3.1) and degradation of the latter leading to a tentatively identified N-propionic acid metabolite (M5.1) are suggested as reliable markers for substance intake. The N-propionic acid metabolite could not be confirmed in the in vitro assays as it includes multiple consecutive metabolic reactions. Furthermore, CH-PIATA could be detected as parent substance in blood samples, but not in urine. Both in vitro assays and the in silico tool proved suitable for predicting metabolites of CH-PIATA. Considering effort and costs, pHLM incubations seem to be more effective for metabolite prediction in forensic toxicology than HepG2 cells. The highlighted Phase I metabolites serve as reliable urinary targets for confirming CH-PIATA use. The in silico approach is advantageous when reference material is unavailable.

Purpose: A powder found at a fatality scene, labeled as the synthetic cathinone 3’,4’-methylenedioxy-α-pyrrolidinohexiophenone (MDPHP) and most likely smoked using a crack pipe, was analyzed. The powder was identified as very potent synthetic cannabinoid ADB-BUTINACA/ADB-BINACA with a high purity (> 98%). This case highlights the risks associated with mislabeled novel psychoactive substances (NPS), particularly those purchased online. Methods: The powder was analyzed using liquid chromatography-high resolution mass spectrometry (LC-HRMS), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and nuclear magnetic resonance (NMR) spectroscopy. Comprehensive toxicological screening, including NPS, was performed in urine and blood. ADB-BUTINACA was quantified using a standard addition method on various post-mortem matrices: femoral and heart blood, urine, stomach content, bile fluid and liver tissue. Scalp hair was analyzed via external calibration to assess potential long-term exposure. Results: ADB-BUTINACA concentrations were 34.5 ng/mL in femoral blood, 101 ng/mL in heart blood and 3.1 ng/mL in urine. Traces of MDMB-BUTINACA were found in the powder and hair, but not in other biological matrices. ADB-BUTINACA metabolites were detected in all biological matrices. MDPHP was found at low concentrations (< 2 ng/mL) in blood and urine but not in the powder, indicating prior cathinone use. A toxicological significance score (TSS) of 3 was assigned for this monointoxication with ADB-BUTINACA. Conclusions: This case demonstrates fatal poisoning due to extremely high ADB-BUTINACA concentrations in post-mortem blood samples, emphasizing the severe risks associated with mislabeled substances. It underscores the importance of drug checking services to prevent poisonings and overdoses caused by highly potent NPS.

The rapid emergence of synthetic cannabinoid receptor agonists (SCRAs) poses challenges for drug testing, particularly when analyzing urine samples due to the rapid metabolization of the parent compounds. In early 2023, two novel SCRAs were reported to the European Union Drugs Agency (EUDA): ADMB-3TMS-PrINACA and Cumyl-3TMS-PrINACA, which are both indazole SCRAs featuring a trimethylsilyl propyl moiety connected to the tertiary indazole nitrogen. Peaks corresponding to metabolites of ADMB-BINACA (also known as ADB-BUTINACA) and Cumyl-4CN-BINACA observed with retention time shifts in a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detecting SCRAs were later identified as metabolites of ADMB- and Cumyl-3TMS-PrINACA. Pooled human liver microsome (pHLMs, 25 µmol/L) and pooled human hepatocyte (PHH, 20 µmol/L) assays were performed to generate metabolites. Additionally, human urine samples were analyzed by reversed phase liquid chromatography-quadrupole-time-of-flight-mass spectrometry (LC-QToF-MS), assisted by GLORYx and BioTransformer 3.0 for in silico metabolite prediction. Gas chromatography-mass spectrometry (GC-MS) was used to identify substances in seized materials. In total, 34 metabolites for ADMB-3TMS-PrINACA and 38 for Cumyl-3TMS-PrINACA were tentatively identified. Major biotransformations included side chain monohydroxylation (specific markers) and TMS-group cleavage, likely initiated by oxidative Si-demethylation followed by further hydroxylation resulting in an N -3-OH-propyl metabolite and further oxidation to the respective N -propionic acid. Most of these biomarkers were detected in the blood, urine, and stomach content of a deceased poly-drug user exposed to ADMB-3TMS-PrINACA. Overall, Cumyl-3TMS-PrINACA was more prevalent than ADMB-3TMS-PrINACA in Germany according to routine urine testing. This work provides the first investigation of the metabolic fate and suggests biomarkers for these new SCRAs.

QuEChERS is a dispersive solid phase extraction commonly applied in food analysis for residues, such as pesticides or mycotoxins for more than 20 years. Due to the quick and easy sample preparation procedure, a QuEChERS method based on ammonium acetate combined with formic acid in acetonitrile was tested for the preparation of urine samples for doping control purposes. Testing urine samples with different pH and specific gravity, using the combination of 10 M ammonium acetate with 3% formic acid in acetonitrile, 312 out of 342 tested compounds could be extracted at their respective minimum required performance levels according to the World Anti‐Doping Agency (WADA) technical documents. For nine selected analytes representing six categories of WADA's Prohibited List, we validated the QuEChERS extraction method fulfilling WADA's requirements for a confirmation procedure of the nonthreshold substances investigated. Especially for the intact stanozolol‐glucuronides analyzed by high‐resolution mass spectrometry, the described extraction method might be an alternative for confirmation procedures as it is time‐ and cost‐saving compared with the commonly applied solid phase extraction.