Investigation of In‐Trap High‐Energy Collision Dissociation Methodology and Dissociation Conditions Using Dual‐Pressure Linear Ion Trap Mass Spectrometry

RATIONALE: Dissociation is a critical analytical method in mass spectrometry research. Among various dissociation techniques, higher energy collision dissociation (HCD) stands out as a pivotal tool due to its ability to generate more low-mass fragment ions and directly perform dissociation during mass spectrometric detection. However, existing HCD implementations rely on specialized instrument configurations, such as collision cell structures or triple quadrupole mass spectrometers, which inevitably increase instrument development costs and technical complexity. To address this limitation, this study proposes an HCD method based on dual-pressure linear ion trap mass spectrometry that requires no additional hardware modifications. METHODS: Instead, it achieves HCD solely by optimizing the instrument's gas flow system and control parameters. By employing a nitrogen-helium gas mixture as the collision gas and testing with a reserpine standard sample, optimal dissociation conditions were obtained through adjustments to the gas mixture ratio and ion trap DC offset voltage settings. RESULTS: This approach successfully realizes in-trap HCD directly within the dual-pressure linear ion trap, eliminating the need for a dedicated collision cell. Furthermore, it enables HCD-based MS3 functionality, a capability unavailable in traditional triple quadrupole systems. CONCLUSIONS: Validation experiments using veterinary drug residues fenoterol and ractopamine demonstrated the method's universality by comparing its dissociation results with those from conventional ion trap collision-induced dissociation (CID) and triple quadrupole method. The findings confirm that this strategy can be extended to other ion trap mass spectrometry platforms, offering broader applicability.