Analyte and probe melting temperature guided method development strategy for hybridization LC-MS/MS quantification of siRNAs

Small interfering RNA (siRNA) is a novel class of double-stranded oligonucleotide therapeutics rapidly growing in drug research and development. Accurate, sensitive, and reliable quantification of siRNA analytes in biological samples is required to study their pharmacokinetics, toxicokinetics, and biodistribution. Hybridization LC-MS/MS can achieve highly sensitive and specific bioanalysis of single-stranded oligonucleotides, e.g., antisense oligonucleotides (ASOs); however, its application for bioanalysis of siRNA or other double-stranded oligonucleotides is limited. The detailed rationale and principles for assay development are still not well understood. In this work, we systematically evaluated key steps and parameters of hybridization LC-MS/MS assays, including probes (five different types compared), hybridization procedure and temperature, elution temperature, and column temperature using patisiran, an approved siRNA drug, as the test siRNA. Based on the evaluation, a practical and efficient melting temperature (Tm) guided strategy was developed for fast and reliable method development of hybridization LC-MS/MS assays for siRNA bioanalysis. The strategy was successfully applied to siRNA-A, a test siRNA, in mouse plasma over the range of 1.00–1000 ng/mL and the resulting method has been used to support multiple mouse studies. This method-development strategy showed great value as a general approach for other siRNAs or double-stranded oligonucleotides. •First comprehensive evaluation of strategy for hybridization LC-MS bioanalysis of siRNA.•Developed a novel hybridization procedure allows the use of high hybridization temperatures.•A practical melting temperature guided strategy for fast and reliable method development of siRNA.•The strategy was successfully applied to a test siRNA in mouse plasma over the range of 1–1000 ng/mL.•The strategy can be used as a general approach for other siRNA or double-stranded oligonucleotides.