Unraveling Chiral Recognition for Ofloxacin Enantiomers: An IMS-MS Study Enabled by Noncovalent Complexation and Theoretical Modeling

The precise analysis of chiral drugs is crucial for elucidating their stereoselective pharmacological effects. As a commonly used fluoroquinolone antibiotic, ofloxacin (OFLX) exhibits different pharmacodynamics between its R - and S -OFLX, highlighting the need for efficient chiral analysis methods. This study developed a strategy based on trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) and noncovalent complex formation for the rapid separation and quantification of ofloxacin enantiomers. Vancomycin and gentamicin C1a were selected as chiral selectors to form binary noncovalent complexes with the target enantiomers. TIMS analysis demonstrated that the complexes formed between R -/ S -OFLX and both chiral selectors achieved baseline separation, with peak-to-peak resolution values of 1.94 and 3.34, respectively. To clarify the structural basis of chiral recognition, density functional theory calculations and the independent gradient model were employed, revealing key stereospecific weak interactions within the complexes, thereby providing critical atomic-level insight into the differential migration behavior observed in TIMS experiments at the molecular level. Based on these findings, both relative and absolute quantification methods for the enantiomers were established, both demonstrating excellent linearity and high sensitivity. Finally, the method was successfully applied to determine ofloxacin in artificial urine samples, achieving recoveries of no less than 70.60%, confirming its applicability in complex real-world matrices. This derivatization-free approach provides a robust and efficient solution for the rapid analysis of chiral drugs.