RESUMO
The study aimed to analyze nusinersen metabolites in the cerebrospinal fluid samples using ion-pair reversed-phase ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Three different sample preparation methods were tested for extraction and purification, but solid phase extraction appeared to be the most suitable, allowing a significant sample enrichment (40-fold). This step was necessary to detect and identify metabolites of nusinersen in the cerebrospinal fluid. The developed and applied analytical procedure enabled the identification of nusinersen metabolites: sequences shorter by several nucleotides from the 3' end; shorter by several nucleotides from both the 3' and 5' ends; and some depurination products. To the best of our knowledge, this is the first report on the analysis and identification of nusinersen metabolites in cerebrospinal fluid samples taken from children with spinal muscular atrophy treated with Spinraza.
Assuntos
Atrofia Muscular Espinal , Oligonucleotídeos , Humanos , Oligonucleotídeos/química , Cromatografia Líquida de Alta Pressão/métodos , Atrofia Muscular Espinal/líquido cefalorraquidiano , Atrofia Muscular Espinal/tratamento farmacológico , Criança , Espectrometria de Massas/métodos , Extração em Fase Sólida/métodos , Pré-EscolarRESUMO
With the development of therapeutic oligonucleotides for antisense and gene therapies, the demand for analytical methods also increases. For the analysis of complex samples, for example plasma samples, where the use of mass detection is essential, hydrophilic interaction liquid chromatography is a suitable choice. The aim of the present work was to develop a method for separation and identification of the oligonucleotide impurities and metabolites by hydrophilic interaction liquid chromatography. First of all, the effects of different chromatographic conditions (e.g. pH of the aqueous part of the mobile phase, buffer concentration, column temperature) on the retention and separation of phosphorothioate oligonucleotides standards on the amide stationary phase were investigated. A set of model oligonucleotides containing a fully modified 21mer and its typical impurities (shortmers and oligonucleotides with different number of thiophosphate modifications) was used. The results showed that the concentration of the salt in the mobile phase as well as its pH, are the most influential parameters with regard to peak shape and separation. The knowledge gained was applied to the analysis of an unpurified 18mer oligonucleotides, analogues of the drug nusinersen used for the treatment of spinal muscular atrophy. The successful separation and identification of twenty-six and twenty-eight impurities was performed with the developed HILIC method. The method was applied to analysis of nusinersen metabolites of serum samples of patients treated with Spinraza.