Metabolic fate of drugs of abuse and new psychoactive substances: A pilot study on a novel workflow using a zebrafish embryo model combined with human microdosing.

A workflow using zebrafish embryos (ZEs) followed by human microdosing (HMD) can identify human urine biomarkers for drugs of abuse and new psychoactive substances. Metabolites of amphetamine, cocaine, LSD, MDMA, methamphetamine, THC, MDMB-CHMICA, and MDPPP were first identified in ZEs exposed via immersion or injection, then compared with known human metabolites and confirmed by HMD. Both methods identified main human urine metabolites, except for LSD (due to low dose) and cannabinoids (due to low oral bioavailability). ZEs produced more metabolites, including conjugates, than HMD. The approach provides quick, reliable data for urinary drug screening, though challenges remain with HMD, including different administration routes and low-dose detectability.

Study at a glance

Abstract

The aim of this study was to develop a novel workflow to identify human urine biomarkers for drugs of abuse and new psychoactive substances. Metabolites of amphetamine, cocaine, LSD, MDMA, methamphetamine, THC, MDMB-CHMICA, and MDPPP were first identified in a zebrafish embryo (ZE) metabolism study followed by comparison to most abundant human metabolites in literature. Finally, metabolites were confirmed by human microdosing (HMD). ZEs 4 days post fertilization were exposed via immersion in Danieau's medium containing the compound or by injection into the caudal vein. After euthanization and freeze-drying, the ZEs were extracted using methanol. HMD was performed by oral administration of individual compound solutions according to HMD guidelines. Urine was collected spontaneously over 24 h and extracted via solid-phase extraction with and without conjugate cleavage. Samples were then analysed using reversed-phase liquid chromatography coupled to high resolution tandem mass spectrometry. Overall, both ZE and HMD allowed identification of main human urine metabolites as described in literature. Exceptions for HMD were LSD due to the low applied dose and the cannabinoids, probably due to low oral bioavailability. Furthermore, ZE generally produced more metabolites compared to HMD, including conjugates. The proposed workflow of ZE exposure followed by HMD can provide quick and reliable data for prediction of analytical biomarkers for urinary drug screening. Following the initial identification of metabolites in ZE, human metabolites can afterwards be confirmed by HMD study. However, there are still challenges regarding HMD such as different routes of administration and detectability of applied low doses.