Phosphorothioate Oligonucleotide Quantification by μ-Liquid Chromatography-Mass Spectrometry

Abstract

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 reversedphase 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.

The role of fluoroalcohols as counter anions for ion‐pairing reversed‐phase liquid chromatography/high‐resolution electrospray ionization mass spectrometry analysis of oligonucleotides

“…The main challenges in LC/MS analysis of oligonucleotides are (1) to fully separate oligonucleotides from their failure sequences chromatographically and (2) to detect oligonucleotides sensitively by MS. Apffel and co‐workers introduced a new buffering system containing hexafluoro‐2‐isopropanol (HFIP) and triethylamine (TEA) for analyzing oligonucleotides with both good assay sensitivity and chromatographic separation, a major milestone in LC/MS analysis of oligonucleotides. Since then, TEA has been substituted with other numerous ion‐pairing reagents (IPRs) for analyzing oligonucleotides with some success …”

“…Since then, TEA has been substituted with other numerous ion-pairing reagents (IPRs) for analyzing oligonucleotides with some success. 23,36,37 Several attempts have also been made to replace HFIP with other fluoroalcohols. 38,39 Until very recently, Basiri and co-workers compared the performance of HFIP, mainly ESI-MS detection, with another four fluoroalcohols, pentafluoropropanol (PFP), 1,1,1,3,3,3hexafluoro-2-methyl-2-propanol (HFTP), trifluoroethanol (TFE), and nonafluoro-tert-butyl alcohol (NFTB).…”

Comparison of mobile‐phase systems commonly applied in liquid chromatography‐mass spectrometry of nucleic acids

“…Huber and coworkers developed an alternative strategy to increase the compatibility of the IP‐RP‐LC mobile phase with mass spectrometric detection. They reduced the concentration of the IP reagent to 10–25 mM , increased the pH to 8.4 , and introduced new amphiphilic ions, including butyldimethylammonium and cyclohexyldimethyl‐ammonium, showing improved chromatographic and mass spectrometric performance . Acetate represented the preferred counterion.…”

Detection of small interfering RNA (siRNA) by mass spectrometry procedures in doping controls

“…These effects are not permanent, meaning that if the siRNA administration is interrupted, the gene silencing is aborted accordingly. [1,[13][14][15][16][17] Most of these methods use ion-pairing agents in order to enhance the chromatographic properties of the target analytes. By targeting performance influencing genes with siRNA in athletes, the legal requirements of a gene doping rule violation are fulfilled and open a new dimension in doping controls.…”

“…[12] The analysis of oligonucleotides by liquid chromatographymass spectrometry (LC-MS) is an established methodology in analytical chemistry and several procedures were described earlier. [1,[13][14][15][16][17] Most of these methods use ion-pairing agents in order to enhance the chromatographic properties of the target analytes. These ion-pairing modifiers (e.g.…”