Identification of Optimal Urinary Biomarkers of Synthetic Cannabinoids BZO-HEXOXIZID, BZO-POXIZID, 5F-BZO-POXIZID, and BZO-CHMOXIZID for Illicit Abuse Monitoring

Abstract

Background The continuous introduction of new synthetic cannabinoid (SC) subtypes and analogues remains a major problem worldwide. Recently, a new “OXIZID” generation of SCs surfaced in seized materials across various countries. Hence, there is an impetus to identify urinary biomarkers of the OXIZIDs to detect their abuse. Methods We adapted our previously reported two-pronged approach to investigate the metabolite profiles and disposition kinetics of 4 OXIZID analogues, namely, BZO-HEXOXIZID (MDA-19), BZO-POXIZID (5C-MDA-19), 5F-BZO-POXIZID (5F-MDA-19), and BZO-CHMOXIZID (CHM-MDA-19). First, bottom-up in vitro incubation experiments comprising metabolite identification, metabolic stability, and reaction phenotyping were performed using human liver microsomes and recombinant human cytochrome P450 enzymes. Second, top-down analysis of authentic urine samples from drug abusers was performed to corroborate the in vitro findings and establish a panel of urinary biomarkers. Results A total of 42 to 51 metabolites were detected for each OXIZID, and their major metabolic pathways included N-alkyl and phenyl hydroxylation, oxidative defluorination (for 5F-BZO-POXIZID), oxidation to ketone and carboxylate, amide hydrolysis, and N-dealkylation. The OXIZIDs were metabolically unstable, mainly metabolized by cytochromes P3A4, P3A5, and P2C9, and demonstrated mechanism-based inactivation of cytochrome P3A4. Integrating with the results of 4 authentic urine samples, the parent drug and both N-alkyl and phenyl mono-hydroxylated metabolites of each OXIZID were determined as suitable urinary biomarkers. Conclusions Drug enforcement agencies worldwide may apply these biomarkers in routine monitoring procedures to identify abusers and counter the escalation of OXIZID abuse.

Study of the Metabolic Profiles of “Indazole-3-Carboxamide” and “Isatin Acyl Hydrazone” (OXIZID) Synthetic Cannabinoids in a Human Liver Microsome Model Using UHPLC-QE Orbitrap MS

“…Our study indicated that hydroxylation occurred more frequently at the N-alkyl moiety than at the benzene ring, while the F7 (terminal fluorine atom was oxidized to a hydroxyl group) of 5F-BZO-POXIZID was the most frequently modified group, consistent with some other research results for 5F-AB-PINACA, 5F-MN-18, and MAM-2201 [34,39,54]. We considered that C5-7 and D5-6 represented oxidative dehydrogenation combined with hydroxylation (346.1554 and 332.1392 resulting from hydroxyl dehydration), but H11, H13, P8, and P10 were identified as ketone formations by Lee et al [25], although they only provided one fragment ion. In addition, we did not detect H8 (hydroxylation + ketone) reported by Lee et al [25], although we did identify the metabolites (C24-26) of dihydroxylation combined with dehydrogenation after hydrolysis of BZO-HEXOXIZID.…”

“…Overall, 12-16 metabolites were previously identified for three OXIZIDs (BZO-HEXOXIZID, BZO-POXIZID, and 5F-BZO-POXIZID) by Lee et al [25]. The major metabolic pathways were consistent with our findings, and the detected compounds included hydroxylated, ketonic, carboxylated, amide-hydroxylated, and N-dealkylated metabolites.…”

“…Several studies h comparable metabolic profiles between in vitro human liver microsome (HLM hepatocyte assays and human in vivo urine samples [16][17][18][19]. Previous studies h ined the metabolic profiles of ADB-BUTINACA and MDMB-4en-PINACA af or in vivo incubation, and some differences have been reported between the The metabolic profiles of BZO-HEXOXIZID, BZO-POXIZID, and 5-fluoro BZO have been explored, with 12-16 metabolites reported for each; however, none reported for BZO-4en-POXIZID [25].…”

“…Previous studies have examined the metabolic profiles of ADB-BUTINACA and MDMB-4en-PINACA after in vitro or in vivo incubation, and some differences have been reported between them [20][21][22][23][24]. The metabolic profiles of BZO-HEXOXIZID, BZO-POXIZID, and 5-fluoro BZO-POXIZID have been explored, with 12-16 metabolites reported for each; however, none have been reported for BZO-4en-POXIZID [25].…”

The metabolic profile of the synthetic cannabinoid receptor agonist ADB-HEXINACA using human hepatocytes, LC–QTOF-MS and synthesized reference standards

“…Similarly, a combined ester hydrolysis + ketone metabolite was the major urinary metabolite of EDMB-PINACA ( 31 ), and hydroxy + ketone products were some of the most abundant urinary metabolites for adamantyl SCRAs APINACA (AKB-48) and 5F-APINACA (5F-AKB-48) ( 32 ), showing that ketone formation is a major in vivo metabolic pathway for some compounds. Ketone metabolites have also been detected in human liver microsome (HLM) incubations of oxindole hydrazide (OXIZID) SCRAs ( 33 , 34 ), which are significantly different in structure to earlier generations of SCRAs.…”