Characterization of metabolites in microsomal metabolism of aconitine by high-performance liquid chromatography/quadrupole ion trap/time-of-flight mass spectrometry

Importance of the topic

Aconitine is a potent plant‐derived alkaloid used historically in traditional medicine. Despite its therapeutic potential, its narrow safety margin and severe cardiotoxicity necessitate detailed metabolic profiling. Understanding microsomal biotransformation pathways informs both efficacy and risk assessment in clinical and regulatory contexts.

Objectives and study overview

This work aimed to characterize the human liver microsomal metabolites of aconitine using high‐performance liquid chromatography coupled with quadrupole ion trap/time‐of‐flight mass spectrometry (HPLC–IT–TOF MS). The focus was on identifying structural modifications and proposing metabolic pathways.

Methodology and instrumentation

Sample Preparation:

• Aconitine (10 µmol/L) in phosphate buffer, preincubated at 37 °C for 3 min.
• Reaction initiated by adding NADPH (20 mmol/L), incubated for 60 min at 37 °C.
• Terminated with ice‐cold acetonitrile (3× volume), vortexed and centrifuged to remove proteins.

Instrumentation:

• LCMS‐IT‐TOF system (Shimadzu Corporation).
• UFLC XR chromatograph with Shim-pack XR-ODS II column (2.0 mm I.D. × 75 mm, 2.2 µm).
• Gradient elution: water (0.1% formic acid, 5 mm ammonium formate) and acetonitrile; 30% to 80% B over 11 min; flow rate 0.3 mL/min.

Key results and discussion

Sixteen metabolites (M0–M15) were detected, including the parent compound (M0). Biotransformations comprised O-demethylation, N-deethylation, deacetylation, dehydration, oxidation, dehydrogenation, and bidemethylation. Notably, ten metabolites (M2, M5, M6, M7, M8, M10, M11, M12, M14, M16) were reported here for the first time. Retention times ranged from 10.5 to 22.3 min. MS2 fragmentation pathways were elucidated, supporting structural assignments and revealing common neutral losses and ring‐cleavage patterns.

Benefits and practical applications

The comprehensive metabolite map improves understanding of aconitine’s pharmacokinetics and toxicology. These data support dose optimization, safety monitoring in drug development, and forensic analyses of poisoning cases.

Future trends and potential applications

Future work may include in vivo validation of identified metabolites, quantitative LC–MS/MS assays for clinical monitoring, and extension to other species or disease models. Integration with computational metabolism prediction could streamline safety assessments.

Conclusion

This study successfully identified sixteen human liver microsomal metabolites of aconitine, expanding the known biotransformation landscape. The results lay a foundation for improved therapeutic use and risk evaluation of this potent alkaloid.