On-Line 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). Oligonucleotides were isolated from plasma, liver, and kidneys of rats injected with ISIS 11061 and IS...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 1997-02, Vol.69 (3), p.313-319
Main Authors: Gaus, Hans J, Owens, Stephen R, Winniman, Michael, Cooper, Scott, Cummins, Lendell L
Format: Article
Language:English
Subjects:
Analysis
Animals
Biochemistry
Biological and medical sciences
Chromatography, High Pressure Liquid
Drugs
General pharmacology
Mass Spectrometry
Medical sciences
Metabolism
Oligonucleotides - analysis
Oligonucleotides - metabolism
Pharmacology. Drug treatments
Rats
Scientific imaging
Thionucleotides - analysis
Thionucleotides - metabolism
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Summary: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). Oligonucleotides were isolated from plasma, liver, and kidneys of rats injected with ISIS 11061 and ISIS 11637. Metabolites found in plasma were consistent with 3‘-exonuclease activity. Metabolites isolated from liver and kidney were consistent with 3‘- and/or 5‘-exonuclease activity. HPLC/ES-MS analysis of ISIS 11061 isolated from kidney indicated extensive degradation from the 3‘ terminus, but metabolites consistent with 5‘ degradation and combinations of 3‘ and 5‘ truncations also were observed. ISIS 11061 isolated from liver showed less extensive degradation. The 5‘ truncated metabolites represented the predominant species in contrast to the kidney sample. Metabolites with masses consistent with combinations of 3‘ and 5‘ truncations were also observed in liver. The metabolic profiles generated by CGE analysis of these samples agreed qualitatively with mass spectrometric results. HPLC/ES-MS enabled the simultaneous determination of degradation products that are the same length but differ in composition. CGE could discriminate species that differed 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.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac960557q