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24 metabolites of 5F-PY-PICA identified by LC-HR-MS/MS after hepatocytes incubation.Analytical targets identified with directional efflux from 3 d cultured SCRH.Two metabolites proposed as biliary analytical targets.Results help further understand overall disposition of synthetic cannabinoids.Analytical strategies for detecting drugs in biological samples rely on information on metabolism and elimination. 5F-PY-PICA belongs to the group of synthetic cannabinoids that are known to undergo excretion into the bile.The aims of this study were the in vitro identification of metabolites of 5F-PY-PICA and to determine which analytical targets are excreted into the bile and urine.Metabolites identified after incubation of 5F-PY-PICA with pooled human liver microsomes (pHLM), pooled human hepatocytes (pHH), or suspended and sandwich-cultured rat hepatocytes (SCRH). Rat hepatocytes were harvested following a two-step perfusion protocol and the SCRH were prepared between layers of rat-tail collagen. The biliary efflux of 5F-PY-PICA and its metabolites was determined in three-day–cultured SCRH by differential efflux into either standard buffer from intact bile canaliculi or standard buffer without divalent cations, which disrupts the bile canaliculi. The metabolites were identified using liquid chromatography–high resolution mass spectrometry/mass spectrometry (LC-HR-MS/MS).The main metabolites were the COOH-ω-metabolite (M4) in pHH, the defluoro-HO-ω-metabolite (M3) in pHLM, and the COOH-pyrrolidine-metabolite (M6) in rat hepatocytes.Efflux into standard buffer without divalent cations was significantly higher (p < 0.050) for 5F-PY-PICA, M4, and the HO-indole-glucuronide-metabolite (M22). M6 did not undergo significant biliary efflux, indicating that basolateral efflux dominates for this metabolite.5F-PY-PICA, M4, and M22 are proposed as analytical targets for bile analysis in forensic screening protocols, whereas M6 should be one of the main urinary targets for 5F-PY-PICA analysis.