Improvement of serum sample preparation and chromatographic analysis of nusinersen used for the treatment of spinal muscular atrophy

The present investigation showed that each of the three different liquid chromatography modes may be successfully used for the qualitative analysis of nusinersen metabolites in a patient's serum sample extract. However, the smallest number was detected by the hydrophilic interaction liquid chromatography. Furthermore, the response of the mass spectrometry is several times greater for ion pair chromatography compared to reversed-phase one. Various extraction methods were applied for the extraction of nusinersen metabolites from serum. Silica with bonded capture strand for hybridization was applied, as well as silica modified with amino and carboxyl groups for dispersive solid phase extraction. The hybridization allows selective extraction of nusinersen analogs, however, it fails in extraction of short metabolites. On the contrary, the efficiency of weak ion exchange-based extraction was high, even in the case of the direct extraction of nusinersen metabolites from diluted serum samples without a protein removal step. The new material is a great alternative to liquid-liquid extraction and hybridization for the isolation of nusinersen metabolites from the serum of patients with spinal muscular atrophy (SMA). It is a very simple method that uses a low concentration of organic salt and desorption occurs after changing its pH. Such complex studies were performed for the first time for nusinersen metabolites extracted from the serum of SMA patients treated with Spinraza.

Automated summary

The present study compared three different liquid chromatography modes for the qualitative analysis of nusinersen metabolites and found that hydrophilic interaction liquid chromatography detected the smallest number of metabolites. The response of mass spectrometry was higher for ion pair chromatography compared to reversed-phase chromatography. Various extraction methods were used, and it was found that hybridization allowed selective extraction of nusinersen analogs while weak ion exchange-based extraction was highly efficient.