Untargeted Metabolomics in Forensic Toxicology: A New Approach for the Detection of Fentanyl Intake in Urine Samples

The misuse of fentanyl, and novel synthetic opioids (NSO) in general, has become a public health emergency, especially in the United States. The detection of NSO is often challenged by the limited diagnostic time frame allowed by urine sampling and the wide range of chemically modified analogues, continuously introduced to the recreational drug market. In this study, an untargeted metabolomics approach was developed to obtain a comprehensive "fingerprint" of any anomalous and specific metabolic pattern potentially related to fentanyl exposure. In recent years, in vitro models of drug metabolism have emerged as important tools to overcome the limited access to positive urine samples and uncertainties related to the substances actually taken, the possible combined drug intake, and the ingested dose. In this study, an in vivo experiment was designed by incubating HepG2 cell lines with either fentanyl or common drugs of abuse, creating a cohort of 96 samples. These samples, together with 81 urine samples including negative controls and positive samples obtained from recent users of either fentanyl or "traditional" drugs, were subjected to untargeted analysis using both UHPLC reverse phase and HILIC chromatography combined with QTOF mass spectrometry. Data independent acquisition was performed by SWATH in order to obtain a comprehensive profile of the urinary metabolome. After extensive processing, the resulting datasets were initially subjected to unsupervised exploration by principal component analysis (PCA), yielding clear separation of the fentanyl positive samples with respect to both controls and samples positive to other drugs. The urine datasets were then systematically investigated by supervised classification models based on soft independent modeling by class analogy (SIMCA) algorithms, with the end goal of identifying fentanyl users. A final single-class SIMCA model based on an RP dataset and five PCs yielded 96% sensitivity and 74% specificity. The distinguishable metabolic patterns produced by fentanyl in comparison to other opioids opens up new perspectives in the interpretation of the biological activity of fentanyl.