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.