Analysis of the first and second generation of antisense oligonucleotides in serum samples with the use of ultra high performance liquid chromatography coupled with tandem mass spectrometry.

The aim of the present research was to study the impact of six various ion-pairing reagents in ion pair chromatography mode on both retention and ionization of seven antisense oligonucleotides which have the same sequence but different types of modifications and length. Furthermore, retention and selectivity studies were performed with the use of four different stationary phases, including octadecyl, octyl, and pentafluorophenyl groups as well as ligands with embedded polar groups. The results of the research showed that the main factors influencing the retention of the studied compounds are amine hydrophobicity and chain branching. The greatest retention was obtained for hexylamine, while the lowest for propylamine. Octadecyl and pentafluorophenyl columns were characterized by the best selectivity and the former was selected for further investigation. What is more, tandem mass spectrometry parameters were optimized and the sensitivity of oligonucleotide mass spectrometry determination was assessed. The greatest sensitivity was obtained for 5 mM N,N-dimethylbutylamine combined with 1,1,1,3,3,3-hexafluoroisopropanol and methanol. The developed method was successfully applied for the determination and separation of the mixture of seven different oligonucleotides in fortified serum, which was completed in 8 min. The LOQ values of the developed method were in the range of 0.15-0.56 µM (0.91-3.56 ng on column). Moreover, it has to be highlighted that it was the first time that such comprehensive investigations were carried out for various types of oligonucleotide modifications and for different stationary phases as well as ion pair reagents.