Resolving the microcosmos of complex samples: UPLC/travelling wave ion mobility separation high resolution mass spectrometry for the analysis of in vivo drug metabolism studies

Abstract

In vivo drug metabolism studies with low concentrations of analytes and high matrix burden are challenging. Of special interest are ‘first-in-man’ studies in early stages of pharmaceutical development that do not use 14C labeled drug candidates. Beside conventional MS-fishing techniques which are biased towards known/expected metabolites and mass defect filtration procedures, this paper focuses on the untargeted/unbiased analysis of drug related compounds in complex matrices using two orthogonal separation techniques: UPLC and TWIMS. Standard sample material after oral administration of a drug compound to rats was investigated by UPLC/TWIMS in MSE acquisition mode using interlaced collision energies for the parallel detection of [M+H]+ parent ions and fragments. Due to the fragmentation after ion mobility separation in the transfer region of the Synapt G2-triwave device, [M+H]+ ion species are aligned with their related fragments by virtue of possessing the same retention time and drift time profile. Four dimensional data analysis of the continuum raw data was performed by automated peak picking and alignment within the MSE viewer software. As result, completely purified MS- and MS/MS-data of metabolites were extracted from raw mass data with high matrix burden and were used without compromise for structure elucidation. This analytical methodology is universally applicable for the unbiased/untargeted and robust analysis of any analyte of interest in complex matrices, including small molecules, peptides and proteins. The high quality data files can be used as data repositories for the purpose of retrospective analysis which is of particular interest for the long term process in drug development.