title: The impact of ion-pairing reagents on the selectivity and sensitivity in the analysis of modified oligonucleotides in serum samples by liquid chromatography coupled with tandem mass spectrometry.
abstract: Present study highlights the usage of various ion-pairing agents and their impact on the process of separation and ionization of oligonucleotides in the fortified human serum samples. What is more, retention studies involved different stationary phases, including: octadecyl, phenyl, pentafluorophenyl groups and ligands with embedded polar groups. It was proved that retention of oligonucleotides strongly depends on the alkyl chain branching in the structure of ion pairing reagent. Furthermore ion-pairing agents build of straight alkyl chain are more easily adsorbed on the stationary phase modified with octadecyl groups, while branching of alkyl chain caused more effective adsorption of studied compounds at phenyl groups compared to octadecyl ones. The lowest limit of quantification values were obtained for 5mMN,N-dimethylbutylamine, while the highest values are characteristic for hexylamine. Moreover it was shown that a 2-fold increase of ion-pairing agent concentration results in higher LOQ. The greatest sensitivity was obtained for 2.5mMN,N-dimethylbutylamine/150mM hexafluoroisopropanol. On the other hand Hypersil GOLD aQ column was the most appropriate in terms of time and separation effectiveness. The developed method was successfully used for the determination of studied oligonucleotides and their metabolites in human serum samples. The compounds were separated in just 3.5min with high sensitivity (0.09-0.16ng).
authors.affiliation: Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin Str., PL-87-100 Toruń, Poland. Electronic address: kowalska@chem.umk.pl.
authors.forename: Sylwia
authors.initials: S
authors.lastname: Studzińska
authors.affiliation: Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin Str., PL-87-100 Toruń, Poland.
authors.forename: Rafał
authors.initials: R
authors.lastname: Rola
authors.affiliation: Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin Str., PL-87-100 Toruń, Poland.
authors.forename: Bogusław
authors.initials: B
authors.lastname: Buszewski
mesh_terms: D000588:Amines / Q000737:chemistry; D002219:Carbamates / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry*; D009841:Oligonucleotides / Q000097:blood* / Q000737:chemistry*; D020005:Propanols / Q000737:chemistry; D012680:Sensitivity and Specificity; D044967:Serum / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods; D006845:Hydrocarbons, Fluorinated
chemical_list: D000588:Amines; D002219:Carbamates; D007202:Indicators and Reagents; D009841:Oligonucleotides; D020005:Propanols; C000614257:N,N-dimethylcarbamate; C001337:hexafluoroisopropanol; D006845:Hydrocarbons, Fluorinated; C007940:hexylamine
keywords: Amine ion-pairing reagent; Ion pair reversed phase chromatography; Phosphorothioate oligonucleotides; Separation; Serum; Tandem mass spectrometry