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Because the effects of the modifications in ASOs on their metabolic stabilities are different from unmodified ASOs, studies that afford an understanding of these effects as well as propose proper methods to determine modified and unmodified ASO metabolites are imperative. An LC\u2013tandem mass spectrometry method offering good selectivity with a high\u2010quality separation using 30\u2009m<jats:sc>m<\/jats:sc> <jats:italic>N<\/jats:italic>,<jats:italic>N<\/jats:italic>\u2010dimethylcyclohexylamine and 100\u2009m<jats:sc>m<\/jats:sc> 1,1,1,3,3,3\u2010hexafluoro\u20102\u2010propanol was utilized to identify each oligonucleotide metabolite. Subsequently, the method was successfully applied to a variety of <jats:italic>in vitro<\/jats:italic> systems including endo\/exonuclease digestion, mouse liver homogenates, and then liver microsomes, after which the metabolic stability of unmodified versus modified ASOs was compared. Typical patterns of chain\u2010shortened metabolites generated by mainly 3\u2032\u2010exonucleases were observed in phosphodiester and phosphorothioate ASOs, and endonuclease activity was identically observed in gapmers that showed relatively more resistance to nuclease degradation. Overall, the degradation of each ASO occurred more slowly corresponding to the degree of chemical modifications, while 5\u2032\u2010exonuclease activities were only observed in gapmers incubated in mouse liver homogenates. Our findings provide further understanding of the impact of modifications on the metabolic stability of ASOs, which facilitates the development of future ASO therapeutics.<\/jats:p>","DOI":"10.1002\/bmc.4839","type":"journal-article","created":{"date-parts":[[2020,4,4]],"date-time":"2020-04-04T16:06:48Z","timestamp":1586016408000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":26,"title":["<i>In vitro<\/i> metabolism of 2\u2032\u2010ribose unmodified and modified phosphorothioate oligonucleotide therapeutics using liquid chromatography mass spectrometry"],"prefix":"10.1002","volume":"34","author":[{"given":"Jaeah","family":"Kim","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy The University of Georgia  Athens GA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Noha M.","family":"El Zahar","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy The University of Georgia  Athens GA USA"},{"name":"Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy Ain Shams University  Cairo Egypt"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0626-3234","authenticated-orcid":false,"given":"Michael G.","family":"Bartlett","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy The University of Georgia  Athens GA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2020,5,7]]},"reference":[{"key":"e_1_2_6_2_1","volume-title":"Second\u2010generation antisense oligonucleotides: Structure\u2014Activity relationships and the design of improved signal\u2010transduction inhibitors","author":"Altmann K.\u2010H.","year":"1996"},{"key":"e_1_2_6_3_1","doi-asserted-by":"publisher","DOI":"10.1089\/oli.2010.0252"},{"key":"e_1_2_6_4_1","doi-asserted-by":"crossref","unstructured":"Bartlett M. 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