Nusinersen metabolism in cerebrospinal fluid samples of patients with spinal muscular atrophy determined by LC-MS data

The repository presents data obtained during the study of nusinersen metabolism in the cerebrospinal fluid samples of patients with spinal muscular atrophy. The study used ion-pair reversed phase ultra high performance liquid chromatography coupled with mass spectrometry. Moreover, two step extraction was performed. A sample preparation method achieved high recovery, repeatability, removed matrix interferences, and allowed for a 50-fold concentration of samples. These factors were critical for detecting and identifying metabolites present at very low levels, even four months after administration. Accurate identification of metabolites depends on chromatographic separation, particularly for compounds that differ by only one nucleotide but have similar nominal m/z values. Therefore, selecting appropriate ion-pairing reagents and optimizing mass spectrometry conditions are essential to enhance separation and sensitivity for low-level metabolite detection. The findings indicate that both propylamine and can be used interchangeably; however, the latter offers better resolution. Precise mass measurements, along with characteristic fragment ions originating from methylated nucleobases and phosphorothioate groups, enabled metabolite identification. Examination of CSF samples from 17 pediatric patients with SMA, at different stages of treatment, showed significant in vivo metabolism, mainly through 3′-exonucleolytic degradation, producing various shorter oligonucleotide fragments (N-shortmers).