Integrated LC-MS and SPME-GC-MS Reveal the Transformation Network of Ligustilide under Multi-Stress Conditions

Ligustilide, a major bioactive phthalide in Angelica sinensis and Ligusticum chuanxiong, is pharmacologically potent but chemically labile. Its transformation behavior under stress conditions remains incompletely characterized. Herein, we employed an integrated high resolution liquid chromatography-mass spectrometry (LC-MS) and solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) approach to profile degradation products of ligustilide after thermal-oxidative stress (80 °C, air). Thirty transformation products were tentatively identified by LC-MS, including 11 monomers, 18 dimers, and 1 trimer, with 17 previously unreported degradation products that could not be matched in the SciFinder database, and volatile degradation fragments (e.g., butanal and phthalic anhydride) were tentatively identified by SPME-GC-MS. Based on the identification results, a comprehensive transformation network of ligustilide was constructed. Subsequently, the degradation of ligustilide under different stress conditions was studied by ultra-high performance liquid chromatography (UPLC), suggesting oxidative transformation as the predominant pathway and tentatively identifying distinct routes induced by acidic conditions and Fe³⁺. Degradation kinetics indicated rapid initial loss consistent with bimolecular reactions such as oxidation and dimerization. This work provides a comprehensive transformation network of ligustilide and advances the mechanistic understanding of its degradation behavior under multiple stress conditions.