Novel dose- and time-dependent toxicity biomarkers of fentanyl/carfentanyl: uncovered by urine-plasma metabolomics for forensics

Fentanyl analogs present a significant global risk to public health and safety due to their high abuse potential and related mortality. However, the absence of a structured research framework focused on their clinical pharmacology and toxicology has led to a scarcity of studies on the mechanisms underlying the toxicity of and addiction to these substances, particularly regarding the rapidly emerging new fentanyl analogs. In this study, we employed a nontargeted metabolomics strategy. By conducting multivariate statistical analysis on ultrahigh performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) data from rat urine and plasma to elucidate the metabolic disruptions induced by fentanyl and carfentanyl. Adult male SD rats were randomly assigned to four groups: low-dose fentanyl group, high-dose fentanyl group, low-dose carfentanyl group, and high-dose carfentanyl group. All groups underwent continuous tail vein injection of toxicants for 5 consecutive days. Plasma and urine samples were collected from the rats before the first administration and at different time points after the last administration, followed by detection and analysis. Specifically, we aimed to elucidate the dose- and time-dependent metabolic toxicity of fentanyl and carfentanyl through a nontargeted metabolomics strategy. Oxidative stress, immunosuppression, and energy dysregulation were identified as core toxic effects of fentanyl and carfentanyl, tightly linked to perturbations in taurine and glutathione pathways, taurine regulating immune function and neuronal homeostasis, glutathione maintaining redox homeostasis. Succinic acid and taurine were confirmed as common biomarkers in urine and plasma. Succinic acid showed decreased plasma levels and increased urine levels, directly indicating fentanyl and carfentanyl-induced energy metabolism impairment. Taurine exhibited similar dysregulation, reflecting drug-induced immunosuppression and neuronal excitability abnormalities. These findings provide critical references and experimental support for studies on fentanyl-related hazards, toxicological mechanisms, and forensic detection.