Study of metabolomic profile alterations in murine models following the administration of opioids by UHPLC-HRMS

Background and aim: Proliferation of new psychoactive substances (NPS) and the challenges associated with their detection have driven forensic toxicologists to identify suitable strategies for their identification in seizures and biological samples. Untargeted metabolomics was recognized as a useful approach to identify endogenous markers of NPS consumption. In this study, untargeted metabolomic was carried out via UHPLC-HRMS analyses for the identification of natural or synthetic opioid consumption biomarkers in an in vivo study in mice. Methods: Urine samples were collected from CD-1 mice, males (n=8) and females (n=8). Animals were initially treated with 0.9 M NaCl-containing saline (vehicle) by intraperitoneal injection; urine was collected cumulatively over 24h. Subsequently, half of the animals of both sexes were treated with 30 mg/kg morphine, while 6 mg/kg fentanyl was administered to the rest of the animals and 24h urine was similarly collected. Samples were diluted prior to UHPLC-HRMS analysis. Mass spectra were acquired on an Orbitrap Q-Exactive mass spectrometer in full scan/data dependent acquisition mode; every sample was analyzed with both RP and HILIC chromatography in both polarities. Univariate and multivariate analyses were carried out on the data obtained. Results: The data obtained from the orthogonal partial least square-discriminant analysis (OPLS- DA), led us to identify the main differences, in terms of metabolites, after drug administration. Indeed, 5-aminovaleric acid, creatine and others belonging to the main lipid oxidation and amino acids degradation pathways were among the most altered metabolites. For each sample, to eliminate inter-individual variability, the peak areas for each compound at time 0 was subtracted from the peak areas for the compound in the same mouse after drug administration, using each mouse as its own control. Similarly, a multivariate analysis was performed to elucidate differences related to the sex of the animals. Conclusion: The study investigated alterations in metabolic pathways of CD-1 mice treated with opiates; it highlighted the potential of metabolomics in forensic toxicology for investigations related to NPS. In general, the literature can only offer a limited number of studies on the urinary metabolic profile of opiate-treated rats.