Application of ion pair chromatography coupled with mass spectrometry to assess antisense oligonucleotides concentrations in living cells.

Antisense oligonucleotides (ASOs) are synthetic bioactive compounds used as therapeutic agents in clinical trials. They act by binding to complementary sequences of the targeted nucleic acids in cells. Assessing the efficiency of ASO delivery to cells or tissues and the stability of these compounds in different biological systems is important. To answer these questions, we developed a new, quick and reliable method to determine the concentrations of different types of ASOs in treated cells. Ultra-high performance liquid chromatography coupled with mass spectrometry was used for the first time for the separation and determination of the studied compounds in total RNA extracts. To develop a method with the highest possible sensitivity, a central composite design was used to comprehensively optimize the MS parameters. Moreover, the effects of the type and concentration of the ion pair reagent on sensitivity were also examined. Finally, a mobile phase containing methanol, hexafluoroisopropanol and N,N-dimethylbutylamine was selected. The optimized method allowed good linearity, accuracy, precision and sensitivity of ASO detection. Next, these compounds were delivered into cells via transfection at a concentration of 25 nM or 125 nM in 1 mL of cell culture medium. After 48 hours, total RNA was isolated from the treated cells and analyzed with the use of the newly developed method. For the cells treated with a higher concentration of ASO composed of phosphorothioate 2'-O-methyl RNA units, the concentration in solution was 0.96 ± 0.06 μM, while in the case of shorter ASO composed of locked nucleic acid units, it was 0.72 ± 0.06 μM in the total RNA extract.