Development and Application of a Comprehensive Non-targeted Screening Strategy for Fentanyl Analogues

The growing abuse of fentanyl and its analogues (FTNs) presents a substantial public health threat, prompting the introduction of regulatory controls by government authorities. Nevertheless, existing screening strategies for FTNs are primarily based on targeted or non-targeted approaches that utilize a limited set of mass spectrometry fragmentation data, which are far from meeting the needs of class scheduling. In this study, a comprehensive non-targeted screening strategy for FTNs was developed. Firstly, existing and potential unknown structures were obtained and a database was created by combining the substitution patterns and the characteristic fragmentation routes (CFRs). Following this, a preliminary screening was executed by matching entries from the CFRs database with empirical data to identify CFRs and suggest candidate structures. The likelihood of an analyte being an FTNs increased with the identification of multiple CFRs. After that, a final judgment was made based on the similarity of the isotope distribution of the candidate structures and the spectra obtained, along with the evaluation of chromatographic peak profile. This proposed strategy could achieve an extensive coverage of 1.25×10¹⁵. To facilitate the establishment and implementation of the strategy, a computer-aided program named FTNs_Finder was developed. To validate this method, false negative and false positive assessments were performed using the MS/MS data from 29 FTNs and 5,180 datasets from MassBank. In addition, it was further applied to the presence screening of FTNs in environmental water samples. Overall, this work could effectively promote the class scheduling of FTNs and provide a reference for the design and implementation of screening strategies for other hazardous substances.