4-Hydroxy-N,N-methylpropyltryptamine (4-OH-MPT) in vitro human metabolism

Abstract

Tryptamines are indolealkylamines sharing their core structure with serotonin. Most tryptamines are psychoactive hallucinogens. Every year, new molecules are synthesized to circumvent the laws. To date, 53 tryptamine analogues are monitored by the EU Early Warning System, and, although the total number of intoxications and fatalities are still low, they are increasing. Tryptamines are generally potent and their concentrations in biological matrices are low, making detection challenging in analytical toxicology. Urinary metabolite biomarkers can be targeted to improve detection. However, like other novel psychoactive substances (NPS), there are no data available on the metabolism of the most recently encountered molecules (Malaca. International Journal of Molecular Sciences 2020;21:9279). 4-hydroxy-N,N-methylpropyltryptamine (4-OH-MPT), also known as meprocin, is a psychedelic tryptamine first identified in seizures in Europe in 2018. 4-OH-MPT intake was never reported in the literature or early warning systems in authentic cases, possibly due to lack of specific metabolite biomarkers. The aim of this research was to characterize 4-OH-MPT metabolism in human hepatocytes and identify optimal metabolite markers to identify 4-OH-MPT intake. Analyses were conducted following our in-house protocol (Di Trana. Talanta 2021;235:122740). To assist in metabolite identification, 4-OH-MPT metabolic fate was predicted using GLORYx freeware, generating a list of potential first- and second-generation metabolites with a probability score. 4-OH-MPT was incubated with cryopreserved ten-donor-pooled human hepatocytes for 3-h to simulate liver metabolism. Incubates were analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) in full-scan and data-dependent MS/MS acquisition modes to capture the signal of all metabolites and their fragmentation pattern in two injections, i.e., in positive- and negative-ionization modes. LC-HRMS/MS data were mined with Compound Discoverer (Thermo Scientific) using a targeted/non-targeted approach to identify expected and unexpected metabolites. After excluding duplicates and putative metabolites with a molecular mass lower than 100 Da, 12 first-generation and 28 subsequent second-generation metabolites were predicted in silico; major reactions were O-sulfation, O-glucuronidation, N-dealkylation, and hydroxylation, N-oxidation, and carboxylation at the N-alkyl chain. 4-OH-MPT signal intensity decreased to 47% after 3-h incubation with hepatocytes. Seven metabolites were produced through N-demethylation and N-oxidation at the alkyl side chain, and O-glucuronidation and O-sulfation at the indole ring; 4-OH-MPT-glucuronide was the metabolite with the most intense signal. These results were consistent with the metabolic fate of structural analogues. The LC gradient was purposefully long and gradual to separate potential isomers. For this reason, O-glucuronides presented a broad shoulder peak, maybe indicating the resolution of two conformers of the same metabolites. 4-OH-MPT-N-oxide and 4-hydroxy-propyltryptamine (4-OH-PT) are proposed as metabolite biomarkers of 4-OH-MPT consumption after glucuronide and sulfate hydrolysis to increase 4-OH-MPT detection capabilities. Alternatively, 4-OH-MPT-glucuronide is suggested as an additional biomarker of 4-OH-MPT intake when hydrolysis is not performed. Unfortunately, these metabolites may not be specific to 4-OH-MPT. There are currently no data on the metabolism of structural analogues 4-hydroxy-N,N-dipropyltryptamine (4-OH-DPT), 4-acetoxy-N,N-dipropyltryptamine (4-AcO-DPT), and 4-acetoxy-N,N-methylpropyltryptamine (4-AcO-MPT) that potentially could produce similar metabolites. However, 4-OH-PT might be produced by 4-OH-DPT N-depropylation and 4-AcO-DPT ester hydrolysis and N-depropylation, making 4-OH-MPT-N-oxide crucial to document 4-OH-MPT consumption. More importantly, 4-AcO-MPT and 4-OH-MPT might produce the same major metabolites, considering the reactivity of the ester bond in 4-AcO-MPT. Therefore, the distinction between 4-OH-MPT and 4-AcO-MPT consumption may not be possible without detecting 4-AcO-MPT. The detection of tryptamine metabolites in biological matrices is crucial to document consumption in clinical and forensic settings. 4-OH-MPT-glucuronide, 4-OH-MPT-N-oxide, and 4-OH-PT are metabolite biomarkers of 4-OH-MPT consumption; however, research on the metabolism of other tryptamine structural analogues is necessary to evaluate the specificity of these metabolites to identify 4-OH-MPT only.