Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University in Toruń, 7 Gagarin Str., PL-87-100 Toruń, Poland.
Department of Developmental Neurology, Medical University of Gdansk, 7 Dębinki Str., PL-80-952 Gdańsk, Poland.
Int J Mol Sci. 2022 Sep 5;23(17):10166. doi: 10.3390/ijms231710166.
The application of oligonucleotides as drugs for different genetic diseases is increasing rapidly. Since 2016 they are used during spinal muscular atrophy treatment with the use of nusinersen oligonucleotide. The purpose of this study was to improve methods for the analysis of serum samples of patients treated with nusinersen. The results showed that liquid-liquid extraction (with phenol/chloroform) is insufficient and an additional purification step using solid-phase extraction is necessary. The best results were obtained for microextraction by packed sorbents. Important parameters in the optimization of the method were mainly the type of amine in the mobile phase and the stationary phase. Both influenced the selectivity of metabolite separation and thus their correct identification; while amine type impacted also the intensity of signals. Finally, the highest resolution of separation and the highest peak areas were obtained for ,-dimethylbutylamine or ,-diisopropylthylamine with an octadecyl column with a terminal aryl group. Over a dozen of metabolites were successfully identified with the use of methods developed during the study. The 3' exonucleases and 5' exonucleases were mainly responsible for nusinersen metabolism, consequently, 3'end shortmers, and 5'end shortmers were observed, as well as metabolites with simultaneous loss of bases at both ends of the sequence. However, some depurination and depyrimidination products were also identified. To the best of our knowledge, this is the first report on nusinersen and its metabolite identification in serum samples by liquid chromatography and mass spectrometry.
寡核苷酸在治疗不同遗传疾病的药物中的应用正在迅速增加。自 2016 年以来，它们已被用于脊髓性肌萎缩症的治疗，使用的是 nusinersen 寡核苷酸。本研究的目的是改进用于分析接受 nusinersen 治疗的患者血清样本的方法。结果表明，液-液萃取（用苯酚/氯仿）不够充分，需要使用固相萃取进行额外的纯化步骤。使用填充吸附剂的微萃取可获得最佳结果。方法优化的重要参数主要是流动相和固定相中的胺类型。两者都影响代谢物分离的选择性，从而影响其正确鉴定；而胺类型也影响信号强度。最后，在使用研究过程中开发的方法时，获得了最高的分离分辨率和最高的峰面积，用于 -二甲基丁胺或 -二异丙基乙胺与末端芳基的十八烷基柱。使用开发的方法成功鉴定了十多种代谢物。3'外切核酸酶和 5'外切核酸酶主要负责 nusinersen 的代谢，因此观察到 3'端短链和 5'端短链，以及序列两端同时丢失碱基的代谢物。但是，也鉴定出了一些脱嘌呤和脱嘧啶产物。据我们所知，这是首次通过液相色谱和质谱法在血清样本中鉴定 nusinersen 及其代谢物。
