Development of an ion-pair reverse-phase liquid chromatographic/tandem mass spectrometry method for the determination of an 18-mer phosphorothioate oligonucleotide in mouse liver tissue

Eur. J. Mass Spectrom. 11, 209–215 (2005)
DOI: 10.1255/ejms.674

Development of an ion-pair reverse-phase liquid chromatographic/tandem mass spectrometry method for the determination of an 18-mer phosphorothioate oligonucleotide in mouse liver tissue

Anthony T. Murphy,^a* Patricia Brown-Augsburger,^a Rosie Z. Yu,^b Richard S. Geary,^b Stefan Thibodeaux^a and Bradley L. Ackermann^a
^aLilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA. E-mail: atm@lilly.com
^bISIS Pharmaceuticals, Carlsbad, CA 92009, USA

ABSTRACT:

A quantitative method for the determination of a partially modified, 2′-ribose alkoxy 18-mer phosphorothioate oligonucleotide, in liver tissue has been developed. A liquid:liquid extraction, ion-pair reverse phase chromatographic separation, and tandem mass spectrometry were used to achieve a quantitation range of 125 to 10,000 ng g^–1 mouse liver tissue. A total cycle time of 5 min was obtained while maintaining separation of three potential impurities. Separations were performed using a Discovery RP-Amide C16, 100 × 2 mm column packed with 5 µm particles. The separation was facilitated by the use of triethylamine (TEA) and hexafluoroisopropanol (HFIP) as ion-pair agents. The method has subsequently been used for the determination of other phosphorothioate oligonucleotides in support of discovery research.

Keywords: antisense, phosphorothioate oligonucleotides, liquid chromatography, tandem mass spectrometry, LC/MS/MS, HPLC.liquid:liquid extraction

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