Quantification of oligonucleotides by LC–MS/MS: the challenges of quantifying a phosphorothioate oligonucleotide and multiple metabolites. (February 2014)

• Record Type: Journal Article
• Authors: Ewles, Matthew; Goodwin, Lee; Schneider, Anneliese; Rothhammer-Hampl, Tanja
• Is Part Of: Bioanalysis. Volume 6:Number 4(2014)
• Page Start: 447
• Page End: 464
• Publication Date: 2014-02
• Subjects: Drugs -- Analysis -- Periodicals; 615.19005
• DOI: 10.4155/bio.13.319
• Languages: English
• ISSNs: 1757-6180

Abstract

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.