PNA‐HPLC and Hybrid LC–MS/MS as Complementary Platforms for Bioanalysis of Next‐Generation Nucleic Acid Therapeutics

Nucleic acid therapeutics (NATs), including antisense oligonucleotides and small interfering RNAs, represent an expanding class of therapeutic modalities with distinctive physicochemical, pharmacokinetic, pharmacodynamic, and biodistribution properties. Naturally, their bioanalysis requires platforms that can accurately quantify intact analytes of interest and metabolites across diverse biological matrices. Modifications ranging from 2'-modifications, alterations of the phosphate backbones, and varied ligands conjugated for targeted delivery, influence extraction recovery, matrix effects, and assay selectivity and sensitivity. Historically, ligand-binding assays and PCR-based methods were adopted due to exceptional sensitivity. However, these approaches often lacked structural resolution and overestimated intact analyte when "sequence-similar" metabolites prevailed. Conversely, two complementary methods emerged, providing higher structural resolution, i.e., peptide-nucleic acid (PNA)-based hybridization in conjunction with anion exchange high-performance liquid chromatography (PNA-HPLC assay), and liquid chromatography-tandem mass spectrometry (LC-MS/MS), enabling separation of "sequence-similar" metabolites from the parent, and additionally, metabolite identification by LC-MS and LC-MS/MS. Recent methodological advances in LC-MS/MS workflows combining sequence-specific enrichment have substantially bridged the previously observed sensitivity gap. The introduction of high-affinity capture probes has improved assay robustness and recovery for challenging analytes and enhanced signal response while minimizing matrix-effect and ion suppression. Comparative evaluation demonstrates that both the PNA-HPLC and the hybrid LC-MS/MS assays are comparably superior for metabolite profiling and tissue distribution studies. This article integrates the analytical principles, strengths, and limitations of those two assays with exemplary case studies for NATs. Practical guidance is provided for method selection, probe selection, sample preparation, assay validation, and cross-platform harmonization. Emerging trends include PNA probe engineering and high-resolution MS for structural elucidation. The integration of capture probe-based hybridization enrichment with modern LC-MS/MS detection now enables combined sensitivity and specificity. Together, these developments support increasingly robust, convergent, regulatory-compliant bioanalytical strategies for next-generation oligonucleotide therapeutics. © 2026 Wiley Periodicals LLC. Basic Protocol 1: PNA hybridization-based HPLC assay for the detection and quantification of therapeutic oligonucleotide in biological tissue samples Basic Protocol 2: Hybrid LC-MS/MS quantitative assay for identification and evaluation of NAT in biological tissue samples.