For the success of drug discovery and development efforts aimed to treat the diseases of the central nervous system, identification of metabolites and metabolic soft spots as well as assessment of their brain distribution, are critical. To obtain this information, various experimental approaches, technologies (quantitative vs qualitative bioanalysis), laborious interpretation of spectra and in vivo data are required.The first step of analysis by mass spectrometry (MS) is the ionization process that converts the molecules to gas phase ions to allow a high transfer efficiency. The majority of biological sample analysis performed by MS coupled to liquid chromatography (LC), use electrospray ionization (ESI). However, ESI is not able to ionize all the compounds of a complex mixture with the same level of efficiency due to the factors related to the matrix composition, the sample preparation step, the dynamic range of concentration and the LC separation. Moreover, endogenous compounds as well constituents from sample preparation may lead to a signal suppression. Reference standards for all analytes are required to ensure the right MS response and to achieve accurate quantification.To address these aspects, we have explored the recent technological advances in sample preparation, in chromatographic separation (micro-Ultra Performance LC) and in mass/charge separation using High Resolution MS. Microflow leading to micro spray ionization allowed to normalize MS response across different matrices as well as between parent drug and metabolites to enable accurate quantification of all drug related material in a single injection. This technology is expected to increase our confidence in quantitative metabolite profiling. In addition to this, we took the opportunity to utilize a cutting-edge technology namely PCA iKey<sup>TM</sup>, a post column addition device developed to improve the MS response factor of the parent drug and its metabolites by normalizing the mobile phase composition.<br/>Such a combination should aid in supporting pharmacokinetics and metabolite identification studies as well as studies related to blood brain barrier. Our expectation from applying this approach to in vitro and in vivo samples is to identify and to semi-quantify unknown major metabolites (&gt;1%) in matrices from different non-clinical species and to increase our confidence in assessing major human circulating metabolites as early as possible during drug discovery and development programs.

State-of-the-art technologies for relative response normalization of metabolites in mass spectrometry

2019 Fall National Meeting

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