Study of metabolomic profile alterations induced by opioids in murine models by UHPLC-HRMS

The proliferation of NPS and the challenges associated with their detection drove forensic toxicologists to identify suitable analytical strategies for their identification1. Untargeted metabolomics has demonstrated to be a promising approach for the detection of endogenous markers of NPS assumption2. In this study, this tool was exploited for the identification of biomarkers that may indicate the probable intake of opioids of different chemical classes - by exploiting an in-vivo study in mice and HPLC-HRMS analyses. Urine samples were collected from CD-1 mice, both males (n=8) and females (n=8). All 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 sex were treated with 30 mg/kg morphine while 6 mg/kg fentanyl was administered to the rest of the animals; then 24h urine was also collected. Samples were diluted prior to UHPLC-HRMS analysis. Mass spectra were acquired on an Orbitrap Q-Exactive mass spectrometer equipped with a HESI source; every sample was analyzed with both RP and HILIC chromatography in both polarities. Multivariate statistical analysis was carried out on the metabolomic data. The OPLS-DA results (the number of variables was reduced until the best Q2 value through the application of successive OPLS-DA models), led us to identify the main differences in terms of metabolites after drug administration; for example, 5-aminovaleric acid and creatine and others belonging to the main lipid oxidation and amino acids degradation pathways, were among the most altered metabolites. Subsequently, the peak areas for each compound in the vehicle group 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. The present study investigated alterations in metabolic pathways of CD-1 mice treated with opiates, highlighting the potential of metabolomics in forensic toxicology for investigations related to NPS.