Development and Application of a Comprehensive Nontargeted Screening Strategy for Aristolochic Acid Analogues

Aristolochic acid analogs (AAAs) are naturally occurring carcinogenic and toxic compounds that pose a safety threat to pharmaceuticals and the environment. It is challenging to screen AAAs due to their lack of characteristic mass spectral fragmentation and their presence of structural diversity. A comprehensive nontargeted screening strategy was proposed by taking into account diverse factors and incorporating various self-developed techniques, and a Python3-based toolkit called AAAs_finder was developed for its implementation. The main procedures consist of virtual structure and ultraviolet and visible (UV) spectra database creation, exact mass and UV spectra-based suspect data extraction, tandem mass spectra (MS/MS) anthropomorphic interpretation, and multicondition retention time (RT) prediction-based candidate structures ranking. To initially assess screening feasibility, eight hypothetical unknown samples were subjected to nontargeted screening using the AAAs_finder toolkit and two other advanced tools. The results showed that the former successfully identified all, while the latter two only managed to identify two and three, respectively, indicating that our strategy was more feasible. After that, the strategy was carefully evaluated for false positives and false negatives, instrument dependence, reproducibility, and sensitivity. After the evaluation, the strategy was successfully applied to the screening of AAAs in real samples, such as herbal medicine, spiked soil, and water. Overall, this study proposed a nontargeted screening strategy and toolkit independent of characteristic mass spectral fragmentation and able to overcome challenges posed by structural diversity for the AAAs screening, which is also valuable for other classes of compounds.