Insights into the metabolism of CH-PIATA-A novel synthetic cannabinoid featuring an acetamide linker.

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.