Heart‑Cutting Two‑Dimensional Liquid Chromatography/High‑Resolution Mass Spectrometry and Complementary...

Dalbavancin hydrochloride (DB) is a semi‑synthetic lipoglycopeptide antibiotic whose pharmacopeial impurity analysis relies on mobile phases containing non‑volatile phosphate buffers. Although effective for separation, these buffers are incompatible with mass spectrometry (MS), impeding structural characterization of trace impurities. In this study, a heart‑cutting two‑dimensional liquid chromatography/high‑resolution MS (2D‑LC/HRMS) method was developed to overcome this limitation. The first dimension employed a dual‑column serial system with a phosphate‑based mobile phase to achieve high‑resolution separation of dalbavancin and its related substances. Target fractions were online transferred to a second dimension with MS‑compatible conditions for desalting and accurate mass analysis. Using this approach, 22 components-including five major components (DB‑A0, DB‑A1, DB‑B0, DB‑B1, and DB‑B2) and 17 impurities (e.g., DB‑R1, DB‑R2, DB‑D0, DB‑D1, DB‑C0, DB‑C1, RRT0.94, and RRT0.96)-were identified based on accurate mass and tandem MS fragmentation patterns, with confirmation by reference standards where available. To support routine quality control, three complementary high-performance LC methods were developed and fully validated according to ICH guidelines for the quantification of related substances, specific impurities DBa/RRT1.12/RRT1.15, and the highly polar impurity DB‑R6. Forced‑degradation studies under thermal, photolytic, acidic, alkaline, and oxidative conditions revealed distinct degradation behaviors: DB‑R1 increased markedly under thermal, acidic, and photolytic stress; DB‑R2 was the primary alkaline degradation product; DBa and RRT0.96 increased markedly under oxidative conditions; and DB‑R6 increased markedly under acidic conditions. This integrated analytical workflow provides a robust platform for both impurity characterization and routine quality control of DB, supporting process optimization and stability assessment.