NDLI: Phosphorothioate Oligonucleotide Quantification by μ-Liquid Chromatography-Mass Spectrometry

Content Provider	PubMed Central
Author	Erb, Robert Leithner, Katharina Andreas, Bernkop-schnürch Oberacher, Herbert
Abstract	Phosporothioate oligonucleotides represent an important class of therapeutic oligonucleotides, in which none-bridging oxygen atoms of the phosphate groups are replaced by sulfur. These oligonucleotides are designed to treat disease by modulating gene expression of an affected individual. As the development and application of these therapeutical oligonucleotides require analytical support, the development, validation, and application of an assay for the quantitative analysis of a phosporothioate oligonucleotide in rat plasma is described. The method employs ion-pair reversed-phase chromatography on a monolithic capillary column with acetonitrile gradients in cyclohexyldimethylammonium acetate for separation and high-resolution tandem mass spectrometry for detection of nucleic acids. Chromatographic parameters (i.e. column temperature, mobile phase composition) as well as mass spectrometric parameters (i.e. spray voltage, gas flow, and capillary position, scan mode) have been optimized for sensitive oligonucleotide quantification. Furthermore, a solid-phase extraction method was developed which enabled processing of 10 μl of plasma. The five-point calibration curve showed linearity over the range of concentrations from 100 to 1,000 nM of the oligonucleotide. The limit of detection was 50 nM. The intra- and inter-day precision and accuracies were always better than 10.2 %. Using this assay, we performed a pharmacokinetic study of the phosporothioate oligonucleotide in rat treated with a single intravenous dose of 0.39 μmol/kg. The assay sensitivity was sufficient to study the early phase elimination of the oligonucleotide. Small amounts of the oligonucleotide were detectable up to 3 h after dosing.
Related Links	http://dx.doi.org/10.1208/s12248-012-9381-2
Ending Page	737
Page Count	10
Starting Page	728
File Format	PDF
ISSN	15507416
e-ISSN	15507416
Journal	The AAPS Journal
Issue Number	4
Volume Number	14
Language	English
Publisher	Springer US
Publisher Date	2012-12-01
Access Restriction	Open
Rights Holder	Springer US
Subject Keyword	Pharmaceutical Science Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Pharmaceutical Science

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