Zhang Xiao, Sha Chunjie, Zhang Wei, Zhao Fengjuan, Zhu Mingli, Leng Guangyi, Liu Wanhui
School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Yantai University, Yantai, 264005, China.
Department of Traditional Chinese Medicine, Shandong College of Traditional Chinese Medicine, Yantai, China.
Heliyon. 2024 May 14;10(10):e31213. doi: 10.1016/j.heliyon.2024.e31213. eCollection 2024 May 30.
A hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC/MS/MS) method was developed and validated for the quantitative analysis of the fully phosphorothioate modified oligonucleotide nusinersen. HILIC/MS/MS method is more robust and compatible with mass spectrometry than ion pair reversed-phase liquid chromatography-tandem mass spectrometry (IP-RP-LC/MS/MS). Various types and concentrations of additives and different pH of mobile phase affected the mass spectrometry response, chromatographic peak shape and retention of nusinersen. The optimized extraction method of nusinersen employs hydrophilic-lipophilic balance solid phase extraction, with a recovery of up to 80 %. Chromatographic quantification was performed using a gradient system on an amide column and the mobile phase consisted of ammonium acetate, acetonitrile and water in a certain proportion. The fully phosphorothioate modified nusinersen can obtain a high mass spectrometry response by providing greater peak symmetry and high ionization efficiency in a high-pH mobile phase. Moreover, the significant carry over interference was observed at the pH 6.3 of the mobile phase. Adjusting the pH value up to 10, and the carry over interference disappeared. The lower limit of quantitation of this developed HILIC/MS/MS assay was 30.0 ng/mL and the method was systematic methodology validated. This HILIC/MS/MS method provides an attractive and robust alternative for the quantitative analysis of nusinersen and was applied in the pharmacokinetic study of nusinersen in rabbits.
建立了一种亲水作用液相色谱串联质谱法（HILIC/MS/MS），用于对全硫代磷酸酯修饰的寡核苷酸诺西那生进行定量分析，并对该方法进行了验证。与离子对反相液相色谱-串联质谱法（IP-RP-LC/MS/MS）相比，HILIC/MS/MS方法更稳健且与质谱兼容。不同类型和浓度的添加剂以及不同pH值的流动相会影响诺西那生的质谱响应、色谱峰形和保留时间。优化后的诺西那生提取方法采用亲水亲脂平衡固相萃取，回收率高达80%。采用梯度洗脱系统在酰胺柱上进行色谱定量，流动相由一定比例的醋酸铵、乙腈和水组成。全硫代磷酸酯修饰的诺西那生在高pH值流动相中能提供更好的峰对称性和高电离效率，从而获得较高的质谱响应。此外，在流动相pH值为6.3时观察到明显的残留干扰。将pH值调至10，残留干扰消失。所建立的HILIC/MS/MS分析方法的定量下限为30.0 ng/mL，且该方法经过了系统方法学验证。这种HILIC/MS/MS方法为诺西那生的定量分析提供了一种有吸引力且稳健的替代方法，并应用于诺西那生在兔体内的药代动力学研究。
