Among novel psychoactive substances (NPS), synthetic cannabinoids (SCs) represent one of the largest subgroups. Around 2017, with the appearance of 5F-EDMB-PINACA, the ethyl analog of 5F-MDMB-PINACA (5F-ADB), the first synthetic cannabinoid carrying an ethyl ester moiety at the linked group started to circulate on the market. The unambiguous detection of the use of these substances remains challenging for forensic laboratories, especially because they tend to form common metabolites with structurally related SCs. In 2020 and 2021, further SCs with an ethyl ester moiety at the linked group emerged, among them 5F-EDMB-PICA and EDMB-PINACA, which were distributed on the German drug market. Here we present data on suitable urinary markers to prove the consumption of the ethyl ester compounds 5F-EDMB-PICA and EDMB-PINACA, facilitating differentiation from structurally similar compounds like ADB-PINACA, 5F-MDMB-PICA and 5F-EDMB-PINACA.

Twenty authentic urine samples (10 for each substance) collected for abstinence control were analyzed by means of liquid chromatography time-of-flight mass spectrometry (LC-qToF-MS) after enzymatic cleavage of glucuronides. Anticipated phase I metabolites detected in authentic urine samples were confirmed in vitro by applying a pooled human liver microsomes (pHLM) assay. The prevalence of the compounds among German users between 2020 and 2022 was estimated by analyzing forensic urine samples collected for abstinence control.

Ten phase I metabolites of 5F-EDMB-PICA and 18 metabolites of EDMB-PINACA were detected by LC-qToF-MS analysis of authentic human urine specimens. Not all of these metabolites could be confirmed in vitro using a pHLM assay. This was particularly seen for the products of multiple metabolic reactions. The first urine samples positive for 5F-EDMB-PICA were detected in October 2020, and for EDMB-PINACA in April 2021. From October 2020 to February 2022, a total of 1232 urine samples tested positive for SCs in the Institute of Forensic Medicine Freiburg, of which 66 (5.4%) were positive for 5F-EDMB-PICA and 125 (10.1%) for EDMB-PINACA. For both substances, the main in vivo metabolites were built by ester hydrolysis, coupled to further metabolic processes. However, these metabolites could be formed from other SCs as well and are, thus, non-specific. Investigation of phase-I biotransformation led to the identification of ester hydrolysis, defluorination and mono-hydroxylation products as the most suitable urinary biomarkers for 5F-EDMB-PICA. Suggested biomarkers for the use of EDMB-PINACA are represented by the products of ester hydrolysis coupled to ketone formation and mono-hydroxylation.

Due to several common metabolites among structurally related SCs, the unequivocal detection of their consumption remains challenging for forensic laboratories and requires sensitive methods to monitor multiple targets, sometimes including minor metabolites. 5F-EDMB-PICA and EDMB-PINACA, two SCs carrying an ethyl ester moiety, peaked in 2021 and seem to have vanished afterwards from the German market.