Analytical Methodologies for the Characterization and Analysis of the Parent Compound and Phase I Metabolites of 4F-MDMB-BICA in Human Microsome, Urine and Blood Samples

4F-MDMB-BICA is one of the most dangerous new illicit synthetic cannabinoids (SCs) in 2020. Consumption of 4F-MDMB-BICA has been associated with a number of death cases and related serious adverse health effects in Hungary. Therefore, the use of reliable analytical methods to confirm the intake of 4F-MDMB-BICA is an important issue in forensic practice. Besides the detection of the parent compounds of SCs, the screening of their metabolites provides a reliable confirmation of their consumption, in particular, when the parent compound is under the limit of detection. To the best of our knowledge, this is the first report describing the identification of metabolites of 4F-MDMD-BICA after treatment with pooled human liver microsome (pHLM) and in human urine and blood samples using the combination of data obtained by comprehensive ultra-high performance liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC–HRMS) and semi-targeted UHPLC–HRMS-MS methods. Finally, our routine UHPLC coupled with triple-quadrupole tandem low-resolution mass spectrometry method for screening urine and blood SCs was improved by adding the parent compound and selected main biomarkers of 4F-MDMD-BICA. From the pHLM assay of 4F-MDMD-BICA, 30 phase I metabolites were characterized and structural information thus obtained provided the basis of further identification of in vivo urine and blood metabolites. Overall, 20 urinary and 13 blood in vivo metabolites of 4F-MDMD-BICA have been identified by the investigation of five authentic urine and two blood samples. The ester hydrolysis metabolite was selected as a reliable primary biomarker in urine and blood. As secondary targets, urinary mono-hydroxylation metabolite and ester hydrolysis + dehydrogenation metabolite in blood were recommended due to their abundance and selectivity. Overall, the main phase I metabolites of 4F-MDMD-BICA were successfully characterized, and our routine analytical method with related sample preparation procedure provided a reliable analytical tool for screening both 4F-MDMD-BICA and its selected metabolites in urine and blood samples.