title: Quantification of oligonucleotides by LC-MS/MS: the challenges of quantifying a phosphorothioate oligonucleotide and multiple metabolites.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Covance Laboratories Ltd, Otley Road, Harrogate, HG3 1PY, UK.
authors.forename: Matthew
authors.initials: M
authors.lastname: Ewles
authors.forename: Lee
authors.initials: L
authors.lastname: Goodwin
authors.forename: Anneliese
authors.initials: A
authors.lastname: Schneider
authors.forename: Tanja
authors.initials: T
authors.lastname: Rothhammer-Hampl
mesh_terms: D002138:Calibration; D002725:Chloroform / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D006801:Humans; D055442:Metabolome; D009838:Oligodeoxyribonucleotides / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism; D019800:Phenol / Q000737:chemistry; D054735:Phosphorothioate Oligonucleotides / Q000097:blood* / Q000302:isolation & purification / Q000592:standards; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry / Q000592:standards; D013873:Thionucleotides / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D009838:Oligodeoxyribonucleotides; D054735:Phosphorothioate Oligonucleotides; D013873:Thionucleotides; D019800:Phenol; D002725:Chloroform; C525712:Trabedersen