Quantification of oligonucleotides by LC-MS/MS: the challenges of quantifying a phosphorothioate oligonucleotide and multiple metabolites

Background: LC-MS/MS allows quantification of therapeutic oligonucleotides in biological fluids at low ng/ml concentrations. Achieving selectivity between metabolites and parent molecules in a single assay is one of the biggest challenges when developing a method. We present a strategy that allows quantification of an 18-mer antisense therapeutic, trabedersen, and six metabolites in human plasma. Results/Methodology: The method utilizes phenol-chloroform and SPE with UHPLC-MS/MS to independently quantify trabedersen and the 5 ' n-1, 5 ' n-2, 5 ' n-3, 3 ' n-1, 3 ' n-2 and 3 ' n-3 metabolites in a single assay. The qualification data indicate that if the method was validated it would meet regulatory expectations for precision, accuracy and selectivity. Conclusion: We show that quantification of an oligonucleotide and multiple metabolites, including isobaric 3 ' and 5 ' metabolites, is achievable in a single assay through good sample clean-up and careful optimization of the LC-MS/MS parameters. The strategy presented here can be applied elsewhere and may be useful for other oligonucleotides and their metabolites.