(T1130-01-07) Metabolite Profiling and Identification Strategy for the First-In-Human Study

Purpose: Food and Drug Administration (FDA) agency encouraged to initiate metabolite profiling and identification in humans as early as possible. First in human (FIH) samples provide the earliest possible opportunity to look at circulating metabolite in humans after single ascending dose (SAD) and multiple ascending dose (SAD). The information obtained from the study is crucial in identifying major circulating metabolite as early as possible and addressing its coverage in toxicological species and evaluating drug-drug interaction and/or safety liability. The study highlights a systemic approach for metabolite profiling and identification using SAD and MAD samples from the FIH study using combination of optimal LC conditions, background subtraction and use of high-resolution mass spectrometry (HRMS) and electron activated dissociation EAD for better metabolite characterization and appropriate choice of UV wavelength for relative metabolite quantification for major circulated metabolites.

Methods: First step of method development was generation of metabolites using in vitro systems (Discovery Labware Incorporated, Woburn, MA). These samples were then used to optimize the LC-MS method using different mobile phase, column and gradient so to obtain the best separation of various metabolites. After LC-MS condition optimization, both dose and pre-dose (control blank) plasma samples were extracted, and their full mass scan (MS) were obtained using HRMS. Precursor ion (m/z) of all metabolites were then obtained from dosed samples after subtracted from that of predose samples. This list was added as an inclusion list in MS method to obtain MS/MS of all potential metabolites using information dependent acquisition (IDA). Modification site of selected metabolites was further characterized by EAD (Refrence1). Relative quantification of metabolites and the parent molecule was obtained by monitoring their UV absorption. In our experience, UV monitoring at higher wavelength reduces noise (contribution from blank) and a comparison of pre-dose and dose plasma sample, plus MS/MS profiles are very useful in identify metabolite peaks, and estimation of relative contribution of the major circulated metabolites by UV peak integration

Results: Application of our strategy for metabolite profiling and identification for the FIH study samples and how it is useful in obtaining the best results is demonstrated using a number of cases. Selection of appropriate mobile phase and gradient condition was found to be critical in separation metabolite M20 and M21, as well as M23 and compound A, while selection of UV wavelength at 310 nm vs 224/340 nm was crucial for appropriate relative quantification of M20 and the parent molecule. Similarly, use of predose sample for blank subtraction to obtain inclusion list and identification of metabolites was very useful. A comparison of results obtained using this systemic approach with that of samples obtained from a radioactive study for the same compound showed that the results (both metabolite identification and quantification wise) were very comparable. Finally, use of EAD/HRMS vs CID/HRMS has been demonstrated in identification site of N- and O-glucuronide of daroglutamide as an example (See figure).

Conclusion: The proposed strategy has been very useful not only in identification but quantification of relative percentage of various metabolites and the parent in FIH studies for a number of compounds. These systematic approaches have been utilized and applied for target and non-target metabolite identifications. The results obtained from radioactive studies of the same compound showed no surprise (no new metabolite in general and similar % metabolite contribution from both analyses). Successful implication of the strategy in the FIH studies also suggests that this strategy can be broadly applied for any other metabolite profiling and identification purpose and should be very useful to all drug metabolism scientists in general.

References: 1. Ming Yao, Nian Tong, Rahul Baghla, Qian Ruan, Advancing Structural Elucidation of Conjugation Drug Metabolites in Metabolite Profiling with Novel Electron-Activated Dissociation, Rapid Communications in Mass Spectrometry, Rapid Communications in Mass Spectrometry, 2024 Oct 30;38(20)

Acknowledgements: Kishore Katyayan (BMS), Qian Ruan (BMS), Nina Tong (BMS), Rahul Baghla (Sciex)

Structure Elucidation: Confirmation of N- and O-Glucuronide of Darolutamide