Identification of urinary biomarkers of synthetic cannabinoids BZO-HEXOXIZID, BZO-POXIZID, 5F-BZO-POXIZID and BZO-CHMOXIZID from authentic urine samples in Singapore

Abstract

Following the implementation of a class-wide regulation on synthetic cannabinoids by China in July 2021, there was a notable decrease in the indole and indazole synthetic cannabinoids (SCs) worldwide. The same trend was observed in Singapore until recently, the new ‘OXIZID’ generation of SCs surfaced in seized materials, which included BZO-HEXOXIZID (MDA-19), BZO-POXIZID, 5F-BZO-POXIZID and BZO-CHMOXIZID. To detect the OXIZID abuse, it is crucial to identify the key urinary biomarkers of these OXIZIDs. The metabolites of the OXIZIDs were previously characterised in human liver microsomes (HLM) using an ultra-high-performance liquid chromatography coupled with Thermo Scientific Q-Exactive hybrid quadrupole-orbitrap mass spectrometer. Targeted masses of the metabolites were added to the detection list for screening on authentic urine samples collected from suspected OXIZIDs abusers. Metabolite peaks detected were then compared against the in vitro metabolite identification for their retention times and MS/MS spectra. Four authentic urine samples were found to contain parents and metabolites from one or more OXIZIDs. Metabolites of BZO-HEXOXIZID, BZO-POXIZID and 5F-BZO-POXIZID, generally the N-alkyl and phenyl mono-hydroxylated forms were consistently detected in the samples, alongside with the parent OXIZIDs, albeit at lower intensities compared to the mono-hydroxylated metabolites. N-alkyl di-hydroxylated metabolite was inconsistently present among the samples although some of which the identities were uncertain due to interference at the MS/MS spectrum. Parent and metabolites of BZO-CHMOXIZID were not detected from all the urine samples. The range of metabolites detected in the authentic urine samples is in agreement with the in vitro metabolism study via HLM. The results of the metabolites of BZO-HEXOXIZID and BZO-POXIZID, in particular of those resulting from oxidative reactions at the N-alkyl chain, concur with that for similar straight-chain SCs such as JWH-018 and the more recent ADB-BUTINACA. Both BZO-POXIZID and its fluorinated analogue, 5F-BZO-POXIZID were observed to have overlapping metabolite profiles and proper selection of discerning biomarkers is thus required for each OXIZID. With the emergence of OXIZIDs in Singapore and the rest of the world, our study serves as a reference to establish the urinary biomarkers for routine screening efforts and mitigate their abuse. Integrating with the authentic urine analyses, the parent drugs and both N-alkyl and phenyl mono-hydroxylated metabolites of BZO-HEXOXIZID, BZO-POXIZID and 5F-BZO-POXIZID were determined as suitable urinary biomarkers.