ONLINE HPLC ELECTROSPRAY MASS-SPECTROMETRY OF PHOSPHOROTHIOATE OLIGONUCLEOTIDE METABOLITES

Metabolism of 2'-deoxyphosphorothioate oligonucleotides ISIS 11061 and ISIS 11637 was examined with capillary gel electrophoresis (CGE) and on-line HPLC electrospray mass spectrometry (HPLC/ES-MS). Oligonucleot ides were isolated from plasma, liver, and kidneys of rats injected wi th ISIS 11061 and ISIS 11637. Metabolites found in plasma were consist ent with 3'-exonuclease activity, Metabolites isolated from liver and kidney were consistent with 3'- and/or 5'-exonuclease activity. HPLC/E S-MS analysis of ISIS 11061 isolated from kidney indicated extensive d egradation from the 3' terminus, but metabolites consistent with 5' de gradation and combinations of 3' and 5' truncations also were observed . ISIS 11061 isolated from liver showed less extensive degradation, Th e 5' truncated metabolites represented the predominant species in cont rast to the kidney sample. Metabolites with masses consistent with com binations of 3' and 5' truncations were also observed in Liver. The me tabolic profiles generated by CGE analysis of these samples agreed qua litatively with mass spectrometric results. HPLC/ES-MS enabled the sim ultaneous determination of degradation products that are the same leng th but differ in composition, CGE could discriminate species that diff ered by one nucleotide in length. HPLC/ES-MS was shown to be a useful tool to study the complex metabolism of antisense oligonucleotides in vivo.