Category: Preclinical Development
Purpose: Demonstrating Quadrupole, Ion Trap, and Orbitrap Tribrid MS with advanced data-dependent acquisition methodology for efficient metabolite identification.
Methods: Selected model compounds at 10 µM were incubated with rat and human liver microsomes with glutathione trapping for 30 minutes. Control incubations of microsomes without the addition of the drugs were used for background subtraction. The LCMS analyses were performed on a Vanquish Flex UPLC system and Orbitrap ID-X Tribrid MS using high-resolution full scan and data-dependent MSn and AcquireX background exclusion workflow. Data were processed by Compound Discoverer and Mass Frontier.
Results: ID-X Tribrid MS AcquireX background subtraction workflow automatically updates acquisition method in real‐time, which effectively increases the MS2 and MSn fragments of low abundant metabolites and identification of these metabolites. The results show substantially higher efficiency in the collection of high‐resolution MSn data of drug metabolites using AcquireX workflow than the traditional data‐dependent one, see Figure 1. Furthermore, ID-X Tribrid MS can conduct parallel MS3 scans to enhance structural elucidation without sacrificing duty cycle time. The complete dataset with rich information of metabolites increased the efficiency of all data mining methodologies based on mass fragment analysis, including precursor ion filtering, neutral loss filtering, etc.
Conclusion: Orbitrap Tribrid ID‐X MS AcquireX data acquisition workflow including automatic background exclusion, sample inclusion list, parallel MS3 scans, feature-specific filters, and other features effectively improve quality and increase confidence of routine metabolite Identification.