Synthetic cannabinoids(SCs) such as 5F-MDMB-PICA(Methyl 2.［［1.(5.fluoropentyl) indole-3.carbonyl］amino］-3,3.dimethyl-butanoate) usually undergo extensive metabolization under the hydrolysis of specific cytochrome P450 enzymes and uridine diphosphate glucuronosyltransferase after being ingested.Due to their lipophilicity,many,but not all parent SCs could not be detected or could be detected in much lower proportions than their metabolites in the urine of authentic users within a relatively short duration,which brings great challenges to the monitoring of drug abuse of SCs.Therefore,to establish an analysis method for the metabolites of SCs is beneficial to confirm the use of SCs from body fluids in emergency,toxicology and forensic practices.The specific goal of this work is to study the in vivo metabolism of 5F-MDMB-PICA by zebrafish model.Adult zebrafishes were randomly divided into 2 groups with 3 fishes in each group,including the blank zebrafish group without 5F-MDMB-PICA and the experimental group.After exposed to 1 μg/mL 5F-MDMB-PICA in tank water for 24 h,the zebrafishes were transferred to a tank with clean water and euthanized.The euthanized zebrafishes were placed in a 2-mL tube containing 300 μL acetonitrile and several ceramic beads,followed by homogenization using an Omni Bead Ruptor 24 coupled with an Omni BR-Cryo cooling unit(OMNI International IM,GA) in the following conditions: speed: 6 m/s,time: 40 s,dwell: 20 s and 10 repetitions.Afterwards,the samples were centrifuged for 3 min at 12 500 r/min,then the supernatant was filtered through a 0.22-μm PTFE filter,and 5 μL of each prepared sample was injected into sample bottle for instrument analysis.5F-MDMB-PICA and its metabolites were identified and structurally illustrated by liquid chromatography-quadrupole/orbitrap mass spectrometry(LC-Q-Orbitrap MS) with Mass Frontier software.In total,22 metabolites of 5F-MDMB-PICA in the zebrafish samples were detected,including 15 phase-I metabolites and 7 phase-II metabolites.Results showed that the metabolites of 5F-MDMB-PICA in zebrafish were formed via N-dealkylation(A17) followed by hydroxylation on the aromatic ring(A2,A13),glucuronidation(A1) or sulfation(A3) followed by hydroxylation on the aliphatic chain(A21),glucuronidation(A6) or sulfation(A14),oxidative defluorination(A20) followed by conversion to pentanoic acid(A18) or glucuronidation(A7),then amide hydrolysis(A4) followed by hydroxylation(A5) and propionic acid(A16).The ester hydrolysis products(A19) were one of the most prevalent metabolites detected in zebrafish.Ester hydrolysis(A19) metabolites were further metabolized via hydroxylation(A12),glucuronidation(A8),oxidative defluorination(A11),oxidative defluorination to pentanoic acid(A10),or dehydrogenation(A22) followed by N-dealkylation(A9),or oxidative defluorination(A15).Therefore,A19 could be recommended to be a potentially good biomarker for screening the suspected intoxications arising from 5F-MDMB-PICA,which were recently reported to be detected in urine samples from 5F-MDMB-PICA drug abusers.The metabolic pathway and main metabolites of 5F-MDMB-PICA in zebrafish were preliminarily elucidated in this paper,and six phase-II metabolites,ie.A1,A3,A5,A7,A6 and A14 were reported for the first time.