Evaluating the use of locked nucleicacid capture probes in hybrid LC–MS/MSanalysis of siRNA analytes: supplementary materials

Background: Hybrid LC–MS assays for oligonucleotides rely on capture probes to develop assays with high sensitivity and specificity. Locked nucleic acid (LNA) probes are thermodynamically superior to existing capture probes, but are not currently used for hybrid LC–MS assays. Materials & methods: Using two lipid-conjugated double-stranded siRNA compounds as model analytes, hybrid LC–MS/MS assays using LNA probes were developed. Results: The workflows demonstrated the superiority of the LNA probes, optimized sample preparation conditions to maximize analyte recovery, evaluated the need for analytespecific internal standards, and demonstrated that advanced mass spectrometric technology can increase assay sensitivity by up to 20-fold. Conclusion: The workflow can be used in future bioanalytical studies to develop effective hybrid LC–MS/MS methods for siRNA analytes.

研究背景：针对寡核苷酸的杂交型液相色谱-质谱联用（Liquid Chromatography-Mass Spectrometry, LC-MS）检测方法，依赖捕获探针以实现兼具高灵敏度与高特异性的检测效果。锁核酸（Locked Nucleic Acid, LNA）探针在热力学性能上优于现有捕获探针，但目前尚未应用于此类杂交型LC-MS检测方法中。
材料与方法：以两种脂质缀合双链小干扰核糖核酸（small interfering RNA, siRNA）化合物作为模型分析物，开发了采用LNA探针的杂交型液相色谱-串联质谱（Liquid Chromatography-Mass Spectrometry/Mass Spectrometry, LC-MS/MS）检测方法。
研究结果：本研究的实验流程验证了LNA探针的性能优势，优化了样品前处理条件以实现分析物回收率最大化，评估了针对特定分析物配置内标的必要性，并证实先进质谱技术可将检测灵敏度提升最高达20倍。
研究结论：该实验流程可应用于未来的生物分析研究，以开发针对siRNA分析物的高效杂交型LC-MS/MS检测方法。