Integrated 1D‐LC‐HRMS and Heart‐Cutting 2D‐LC–MS for Impurity Profiling and Chiral Separation of Mitiglinide Drug Substance

ABSTRACT Rationale The comprehensive quality control of chiral pharmaceuticals like mitiglinide necessitates simultaneous assessment of chemical impurities and enantiomeric purity, yet conventional workflows address these separately, leading to inefficiency. This study develops a comprehensive analytical strategy to overcome this challenge for the anti‐diabetic drug mitiglinide. Methods For chiral analysis, an online heart‐cutting two‐dimensional liquid chromatography–high‐resolution mass spectrometry (2D‐LC‐HRMS) method was developed. First, impurity profiling of mitiglinide was accomplished using a one‐dimensional reversed‐phase LC‐HRMS (1D‐LC‐HRMS) method. Subsequently, the 2D‐LC‐HRMS system achieved enantiomer separation by online coupling of a C18 column (first dimension) with a polysaccharide‐based chiral column (second dimension), with the separated analytes detected by an Orbitrap mass spectrometer. Results 1D‐LC‐HRMS identified five major impurities, structurally characterizing four, with the main component accounting for only 49.12% of the total integrated peak area (relative abundance by EIC peak area normalization, not absolute purity). The 2D‐LC‐HRMS method achieved effective enantiomer separation. A consistent third minor chromatographic peak was observed across six replicate analyses, which is tentatively assigned as a potential diastereomeric impurity based on stereochemical interpretation of the chromatographic behavior; confirmatory evidence is required for definitive identification. Conclusions This work successfully establishes a comprehensive strategy that efficiently consolidates impurity profiling and chiral purity assessment for mitiglinide. It provides a reliable, more informative approach for the quality control of complex chiral pharmaceuticals.