title: Development of a method based on ultra high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry for studying the in vitro metabolism of phosphorothioate oligonucleotides.
abstract: Ultra high performance liquid chromatography hyphenated with quadrupole time-of-flight mass spectrometry was used to determine the products of the in vitro metabolism of phosphorothioate oligonucleotides. These compounds may be used during antisense therapy as synthetic fragments of genes. For this reason, both a sample preparation method and a qualification method were developed during this study. Liquid-liquid extraction, protein or oligonucleotide precipitation, and solid-phase extraction were tested and compared in order to select the method that yielded the highest recoveries. Ion pair chromatography was used for separation while mass spectrometry was applied for metabolite identification. The influence of the type of ion pair reagent used on the resolution and sensitivity was investigated. Results indicated that a mixture of 1,1,1,3,3,3-hexafluoro-2-propanol, N,N-dimethylbutylamine, and methanol was the best mobile phase for maximizing both of these parameters. The developed method was applied to investigate the compounds that form during the incubation of phosphorothioate oligonucleotides with human liver microsomes. Metabolites with short sequences were created after 8 hours, while oligonucleotides constructed from a large number of nucleotide units were obtained after 12 hours of incubation. Moreover, regardless of the length of the polynucleotide chain, metabolites were produced by the same mechanism: enzymatic cleavage at the 3' end of the sequence.
authors.affiliation: Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin St., PL-87 100, Torun, Poland. skowalska@o2.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 St., PL-87 100, Torun, 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 St., PL-87 100, Torun, Poland.
authors.forename: Bogusław
authors.initials: B
authors.lastname: Buszewski
mesh_terms: D000588:Amines / Q000737:chemistry; D002082:Butylamines / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D059625:Liquid-Liquid Extraction; D013058:Mass Spectrometry / Q000379:methods*; D008862:Microsomes, Liver / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000032:analysis / Q000378:metabolism / Q000493:pharmacokinetics; D054735:Phosphorothioate Oligonucleotides / Q000032:analysis / Q000378:metabolism* / Q000493:pharmacokinetics; D020005:Propanols / Q000737:chemistry; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction; D006845:Hydrocarbons, Fluorinated
chemical_list: D000588:Amines; D002082:Butylamines; D007202:Indicators and Reagents; D016376:Oligonucleotides, Antisense; D054735:Phosphorothioate Oligonucleotides; D020005:Propanols; C001337:hexafluoroisopropanol; D006845:Hydrocarbons, Fluorinated; C000601638:1,3-dimethylbutylamine
keywords: In vitro metabolism; Ion pair chromatography; Phosphorothioate oligonucleotides; Sample preparation; Separation