In Vitro Phase I Metabolism of the Recently Emerged Synthetic MDMB-4en-PINACA and Its Detection in Human Urine Samples.

Abstract

MDMB-4en-PINACA (methyl (S)-3,3-dimethyl-2-(1-(pent-4-en-1-yl)-1H-indazole-3-carboxamido)butanoate) is a recently emerged synthetic cannabinoid in Turkey. MDMB-4en-PINACA was detected in herbal material investigated by the Council of Forensic Medicine, Istanbul Narcotics Department in Turkey in April 2019. MDMB-4en-PINACA was added to the drug abuse list and quickly reported in biological samples after its first detection. In this study, the in vitro metabolism of MDMB-4en-PINACA was investigated by using a pooled human liver microsomes (HLMs) assay and liquid chromatography-high-resolution mass spectrometry (LC-HRMS). MDMB-4en-PINACA (5μmol/L) was incubated with HLMs for up to 1h, and the metabolites were identified using LC-HRMS and software-assisted data mining. The in vivo metabolism was investigated by the analysis of 22 authentic urine samples and compared to the data received from the in vitro metabolism study. Less than 7.5% of the MDMB-4en-PINACA parent compound remained after the 1h incubation. We identified 14 metabolites, which were formed via double bond oxidation, ester hydrolysis, N-dealkylation, hydroxylation, dehydrogenation and further oxidation to N-pentanoic acid or a combination of these reactions in vitro. In 10 urine samples (total n=22), MDMB-4en-PINACA was detected as the parent drug. Three of the identified main metabolites, double bond oxidation in combination with ester hydrolysis and hydroxylation metabolite (M3), MDMB-4en-PINACA butanoic acid (M14) and monohydroxypentyl-MDMB-4en-PINACA (M12), were suggested as suitable urinary markers. In vitro screening of 2,150 authentic urine samples for these identified MDMB-4en-PINACA metabolites resulted in 56 cases of confirmed MDMB-4en-PINACA consumption (2.6%).