A quantitative, simultaneous LC-MS assay for parent and deamidated metabolite was developed and implemented for a single dose rat PK study to measure in vivo metabolite formation. The final assay was qualified and utilized for in-vivo sample analysis of rat serum. The results showed that exposure of the deamidated metabolite was more than 2-fold higher than Compound-A indicating that use of ligand binding reagents that did discriminate between parent and metabolite would yield an overestimation of parent level concentrations; it was believed that this was the cause of the divergent results observed from the two laboratories that were using ligand binding methods to quantitate the protein. This presentation will include challenges associated with chromatographic separation, stabilization of Compound-A, mass spectrometer sensitivity optimization, antibody capture, and digestion optimization as well as study results. The result confirmed the presence of deamidated metabolite confirming the hypothesis.
Learning Objectives:
Understand how LC-MS can be used to trouble shoot anomalous study data from ligand binding assay