Overview: Oligonucleotides are short strands of nucleotides that are synthesized chemically and have various applications in fields such as diagnostics, therapeutics, and research. Analyzing oligonucleotides is an essential step to confirm their identity, purity, quality, and strength. Mass spectrometry (MS) is a powerful analytical technique that can confirm the intact mass, modification and the identity of the oligonucleotides.
Sample preparation: Prepare a solution of the oligonucleotide in an appropriate solvent, typically a polar solvent like water, or low concentration of organic solvent. The concentration of the solution is better within the range of 1-10 µM.
High-performance liquid chromatography – mass spectrometry (HPLC-MS) analysis: The oligonucleotides are separated using DNAPac high-performance liquid chromatography (HPLC). In the negative mode, the ionized oligonucleotides are analyzed using mass spectrometry to determine their mass-to-charge ratio and calculate their intact mass. Standard oligonucleotides samples are prepared in different known concentrations to depict a standard curve for determination of the absolute concentrations of oligonucleotides in plasma/serum at various time points.
Data analysis: The MS data are analyzed using software tools, such as Biopharma Finder, to determine the accurate mass of the oligonucleotide.