Detection and localization of single-nucleotide mutations in synthetic oligonucleotides by ultra-high-performance...

Accurate detection of point mutations in mitochondrial DNA (mtDNA) is crucial for diagnosing various mitochondrial disorders. In this study, we developed an ultra-high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) method for the direct, label-free identification and localization of single-nucleotide mutations using synthetic 20- and 49-mer oligonucleotides as model fragments representing the pathogenic mtDNA point mutation (mt.3243 A>G). Three mobile phase systems, including ammonium bicarbonate (ABC), triethylamine/hexafluoroisopropanol (TEA/HFIP), and tributylamine/HFIP (TBA/HFIP), were systematically evaluated to assess their effects on oligonucleotide retention behavior and duplex stability under denaturing and non-denaturing conditions. The ABC buffer provided optimal performance for maintaining partial duplex integrity, while TEA/HFIP offered superior ionization efficiency for single-stranded analysis. Deconvoluted mass spectra revealed accurate monoisotopic mass differences between wild-type and mutant oligonucleotides, including ∼ + 16 Da for the sense strand (A>G), ∼ -15 Da for the antisense strand (T > C), and ∼ + 1 Da for the duplex, enabling confident mutation discrimination at the intact molecular level. High-resolution MS achieved excellent mass accuracy within ±3 ppm, and high-energy collision dissociation (HCD) MS/MS enabled sequence-specific fragmentation that localized the mutation site with high confidence when compared with theoretical fragments. Overall, this study establishes a reliable analytical framework for mutation detection in oligonucleotide models and highlights the potential of UHPLC-HRMS/MS as a complementary tool for targeted mtDNA fragment analysis.