An integrated multi-technique strategy for in vitro metabolite identification and profiling of gymnodimine A using...

Gymnodimine A (GYM-A) is a lipophilic cyclic imine toxin produced by marine dinoflagellates that accumulates in shellfish and poses potential risks to human health. However, regulatory limits have not been established worldwide, largely due to insufficient understanding of its metabolic pathways and toxicokinetic behavior. This study characterized the in vitro metabolism of GYM-A using human liver microsomes (HLM) and human liver S9 fractions (HS9) combined with liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS). An integrated identification strategy incorporating segmented data-dependent acquisition (DDA), targeted/untargeted screening, MS/MS fingerprint comparison, density functional theory (DFT) calculation, and μElution solid phase extraction (SPE) enabled confident structural elucidation of metabolites derived from low-concentration substrates in complex matrices. Four phase I hydroxylated metabolites were identified: three mono-hydroxylated derivatives (M1-M3) and one di-hydroxylated derivative (M4), with 21-hydroxy-GYM-A (M1) as the highest relative abundance. No phase II glucuronide conjugates were detected under the conditions tested. Metabolic stability studies revealed high intrinsic clearance following first-order kinetics, with elimination primarily mediated by CYP450-dependent phase I oxidation. Enzyme phenotyping identified CYP3A5 as the principal isoform responsible for GYM-A hydroxylation. Detection of metabolite M2 in urine from dosed rats supported the in vitro findings. This work provides the first systematic metabolic profile of GYM-A and advances knowledge of its kinetic behavior, while the analytical workflow may serve as a useful tool for risk assessment and metabolism studies of related marine toxins.