Application of hydrophilic interaction liquid chromatography coupled with mass spectrometry in the analysis of phosphorothioate oligonucleotides in serum

•Phosphorothioate oligonucleotides and metabolites were separated in HILIC mode.•Carbamoyl stationary phase allows for best separation of oligonucleotides mixtures.•Determination of modified oligonucleosides in serum extracts was done by HILIC MS/MS.•The advantage of HILIC MS is lower LOQ values in comparison with IPC MS.

Most of synthetic, modified oligonucleotides are candidates for therapeutics. Consequently, their quick, reliable and sensitive analysis has become a critical challenge for scientists. The main aim of the present study was an investigation of the influence of stationary phase type, mobile phase salt and its concentration on the separation and determination of the selected compounds by hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Three different columns, together with ammonium acetate and formate, were applied for this purpose. The separation of mixtures of phosphorothioate oligonucleotides and their synthetic metabolites was successfully performed. Moreover, an attempt to isolate these compounds from human serum samples was also made together with their separation, qualification and quantification by hydrophilic interaction liquid chromatography and tandem mass spectrometry. The method developed during the study appeared to be effective and sensitive, due to the limit of quantification which equaled 142-165 ppb.