We performed RMSD calculations between the generated conformations and the QM optimized most stable conformation using the LS-align algorithm, a high-throughput virtual screening atom-level structural alignment method developed by Zhang et al. The conformation with the lowest RMSD and energy values was considered the global minima for that particular molecule using the specific conformation generation engine. If no conformation matched these criteria, it was deemed that the engine failed to find the global minima for that molecule. Furthermore, we calculated the Boltzmann average CCS values using the conformations generated by the conformation search engines and compared them with experimental values. The percentage error in predicting the CCS was reported and an error range within  3% was considered indicative of a good CCS prediction as the experimental uncertainty of CCS values within  3%. System setup. In our previous study, we extensively investigated various ionization models (protonation/deprotonation) and their impact on CCS prediction accuracy for metabolites. The predicted CCS values were compared to experimental results to identify the charge model that exhibited the lowest error percentage. In the current study, focusing on finding global minima based on CCS values, we selected the protonation state that yielded the best predicted CCS values (lowest error percentage) for further analysis. For instance, in the case of the carnosine molecule, five models were considered (model 1, model 2, model 3, model 4, and model 5) with corresponding CCS errors of 9.4%, 9.9%, 8.3%, 0.1%, and 31.0%, respectively. Model 4, exhibiting the lowest error percentage, was chosen as the representative carnosine model for the present investigation. Figure presents an overview of the metabolites included, with their respective ionization sites. The protonation site is distinctly highlighted in red, while the deprotonation site is accentuated in blue.