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

Area of Science:

• Biochemistry and Molecular Biology
• Analytical Chemistry
• Pharmacology

Background:

• Nucleic acid therapeutics (NATs) are a growing class of drugs with unique properties.
• Traditional bioanalytical methods lack structural resolution, potentially overestimating intact analytes.
• Modified NATs present challenges in extraction, matrix effects, and assay sensitivity.

Purpose of the Study:

• To compare the strengths and limitations of PNA-HPLC and LC-MS/MS assays for NAT bioanalysis.
• To provide guidance on method selection, validation, and harmonization for NATs.
• To highlight recent advances enabling sensitive and specific quantification of NATs and their metabolites.

Main Methods:

• Peptide-nucleic acid (PNA)-based hybridization coupled with anion-exchange high-performance liquid chromatography (PNA-HPLC).
• Liquid chromatography-tandem mass spectrometry (LC-MS/MS) with sequence-specific enrichment.
• Comparative evaluation of PNA-HPLC and hybrid LC-MS/MS assays for metabolite profiling and tissue distribution.

Main Results:

• Both PNA-HPLC and hybrid LC-MS/MS assays provide high structural resolution, separating sequence-similar metabolites.
• Recent LC-MS/MS advances with capture probes enhance sensitivity, robustness, and minimize matrix effects.
• Comparative studies show PNA-HPLC and hybrid LC-MS/MS assays are comparably superior for metabolite profiling and tissue distribution.

Conclusions:

• Advanced bioanalytical strategies are crucial for next-generation oligonucleotide therapeutics.
• Hybrid LC-MS/MS and PNA-HPLC assays offer robust, regulatory-compliant quantification of NATs.
• Emerging trends like PNA probe engineering and high-resolution MS will further advance NAT bioanalysis.