Machine learning– and multilayer molecular network–assisted screening hunts fentanyl compounds

Fentanyl and its analogs are a global concern, making their accurate identification essential for public health. Here, we introduce Fentanyl-Hunter, a screening platform that uses a machine learning classifier and multilayer molecular network to select and annotate fentanyl compounds using mass spectrometry (MS). Our classification model, based on 772 fentanyl spectra and spectral binning feature engineering, achieved an <i>F</i>₁ score of 0.868 ± 0.02. The multilayer network, based on spectral similarity and paired mass distances, covers more than 87% of known fentanyls. Fentanyl-Hunter identified fentanyl members in biological and environmental samples. During biotransformation, 35 metabolites from four widely consumed fentanyl derivatives were identified. Norfentanyl was the major fentanyl compound in wastewater. Retrospective screening of these biomarkers across more than 605,000 MS files in public datasets revealed fentanyl, sufentanil, norfentanyl, or remifentanil acid in more than 250 samples from eight major countries, indicating the potential widespread presence of fentanyl.