Utilizing two-dose difference combined with stable isotope tracing for effective and comprehensive metabolite identification of pioglitazone via ultra-high-performance liquid chromatography-mass spectrometry.

Drug metabolite identification is an essential characterization process spanning multiple phases of drug discovery and development. Various data processing techniques have been employed in metabolite identification using high-resolution mass spectrometry. However, metabolite identification is not consistent among approaches. Thus, a more comprehensive approach to drug metabolite identification is required. This paper proposes two-dose difference in conjunction with stable isotope tracing (SIT) to identify pioglitazone (PIO) metabolites. The results of this study revealed thatincubating both native and isotope-labeled PIOs in the same tube led to more stable metabolite identification compared with separated incubation. Our approach offers a high accuracy rate in metabolite identification, with approximately 70 % of metabolites validated as potential PIO metabolites. We compared our developed approach with other 3 approaches, namely the dose-response technique coupled with SIT, mass defect filter coupled with SIT, and orthogonal partial least squares-discriminant analysis. The results revealed that our developed approach was able to identify not only all the potential PIO metabolites identified by the other 3 approaches but also additional metabolites. These results suggest that two-dose difference coupled with SIT is an effective and comprehensive approach for drug metabolite identification.