Automatic identification of oligonucleotide metabolites in complex biological samples using ultra high-performance liquid chromatography high-resolution mass spectrometry combined with the molecule profiler software

Oligonucleotide (ON) is one of the rapidly developing fields in biotherapeutics. Ultra high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) has been widely used for the identification of metabolites due to its high sensitivity, high resolution, and ability to provide structural information. The identification of ON metabolites in matrix has been reported by UHPLC-HRMS, however, manual data processing is time-consuming. In this study, an analytical strategy based on UHPLC-QTOF-MS/MS and molecule profiler software was established and employed for the automatic identification of metabolites of ONs. Fomivirsen (FMVS), a 21-mer antisense oligonucleotide with 20 phosphorothioate linkages, was selected as proof of concept. Firstly, the sample preparation and UHPLC-QTOF-MS/MS condition were optimized. Secondly, the feasibility of the automatic identification of ON metabolites by this strategy was verified using enzymatic digests of FMVS. Finally, in vitro and in vivo metabolites of FMVS were identified. As a result, the enzymatic digests of FMVS were successfully identified by the established strategy, and a total of 17 metabolites were identified from serum, plasma and tissues. FMVS was mainly metabolized by 3′-exonuclease in plasma, liver and kidney. This is the first study on metabolites identification of FMVS, and the proposed strategy would simplify the identification of ON metabolites, thus could be used for other ON metabolites.