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.

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 x 2 mm column packed with 5 microm 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.