The aim of the present investigation was the analysis and identification of antisense oligonucleotide metabolism products after incubation with human liver microsomes regarding four different oligonucleotide modifications. Separation and detection methods based on the use of liquid chromatography coupled with quadrupole time-of-flight mass spectrometry were developed for this purpose. Firstly, the optimization of mass spectrometer parameters was done to select those which ensure the highest possible sensitivity of oligonucleotide analysis. This step was conducted for two chromatographic modes—ion pair chromatography and hydrophilic interaction liquid chromatography—due to their common application in oligonucleotide analysis. Based on sensitivity results, ion pair chromatography coupled with mass spectrometry was selected for the separation of model oligonucleotide mixtures in order to verify its selectivity for N-deleted metabolite separation. Next, the developed method was applied in the examination of oligonucleotides in vitro metabolism. First, wide optimization of incubation parameters was conducted including the concentration of the reaction buffer components. Obtained results indicated that both 3′-exonucleases and 5′-exonucleases contributed to the biotransformation of oligonucleotides. Moreover, it may be concluded that the number of metabolites depends on oligonucleotide modification and consequently its resistance to enzymatic attack. Thus, the number of the oligonucleotide metabolites decreased with the decrease of the resultant polarity of oligonucleotide caused by chemical modification.

本研究的目的是在与人肝微粒体温育后，对四种不同的寡核苷酸修饰进行反义寡核苷酸代谢产物的分析和鉴定。为此，开发了基于液相色谱和四极杆飞行时间质谱联用的分离和检测方法。首先，对质谱仪参数进行了优化，以选择可确保尽可能高的寡核苷酸分析灵敏度的参数。由于它们在寡核苷酸分析中的普遍应用，因此对两种色谱模式（离子对色谱和亲水相互作用液相色谱）进行了此步骤。根据敏感度结果，选择离子对色谱和质谱联用来分离模型寡核苷酸混合物，以验证其对N缺失代谢物分离的选择性。接下来，将开发的方法应用于寡核苷酸的体外代谢检查。首先，对孵育参数进行了广泛的优化，包括反应缓冲液成分的浓度。获得的结果表明3'-核酸外切酶和5'-核酸外切酶均有助于寡核苷酸的生物转化。此外，可以得出结论，代谢物的数量取决于寡核苷酸的修饰，因此取决于其对酶攻击的抗性。从而，