Evaluating the Use of Locked Nucleic Acid Capture Probes in Hybrid LC–MS/MS Analysis of siRNA Analytes
Karan Agrawal, Lijuan Kang, Shaofei Ji, Jennyfer Tena, Wenying Jian
Abstract
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 analyte-specific 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.