Determination of antisense phosphorothioate oligonucleotides and catabolites in biological fluids and tissue extracts using anion-exchange high-performance liquid chromatography and capillary gel electrophoresis

J Chromatogr B Biomed Sci Appl. 1997 Apr 25;692(1):43-51. doi: 10.1016/s0378-4347(96)00499-9.


Chemically modified phosphorothioate oligodeoxynucleotides (ODNs) have become critical tools for research in the fields of gene expression and experimental therapeutics. Bioanalytical assays were developed that utilized fast anion-exchange high-performance liquid chromatography (HPLC) and capillary gel electrophoresis (CGE) for the determination of 20-mer ODNs in biological fluids (plasma and urine) and tissues. A 20 mer ODN in the antisense orientation directed against DNA methyltransferase (denoted as MT-AS) was studied as the model ODN. The anion-exchange HPLC method employed a short column packed with non-porous polymer support and a ternary gradient elution with 2 M lithium bromide containing 30% formamide. Analysis of the MT-AS is accomplished within 5 min with a detection limit of approximately 3 ng on-column at 267 nm. For plasma and urine, samples were diluted with Nonidet P-40 in 0.9% NaCl and directly injected onto the column, resulting in 100% recovery. For tissue homogenates, a protein kinase K digestion and phenol-chloroform extraction were used, with an average recovery of about 50%. Since the HPLC assay cannot provide one-base separation, biological samples were also processed by an anion-exchange solid-phase extraction and a CGE method to characterize MT-AS and its catabolites of 15-20-mer, species most relevant to biological activity. One base separation, under an electric field of 400 V/cm at room temperature, was achieved for a mixture of 15-20-mer with about 50 pg injected. Assay validation studies revealed that the combined HPLC-CGE methods are accurate, reproducible and specific for the determination of MT-AS and its catabolites in biological fluids and tissue homogenates, and can be used for the pharmacokinetic characterization of MT-AS.

MeSH terms

  • Animals
  • Carcinoma, Small Cell / chemistry
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange
  • DNA-Cytosine Methylases / genetics
  • Electrophoresis, Capillary
  • Lung Neoplasms / chemistry
  • Mice
  • Mice, Nude
  • Oligonucleotides, Antisense / analysis*
  • Reproducibility of Results
  • Thionucleotides / analysis*
  • Tissue Distribution


  • Oligonucleotides, Antisense
  • Thionucleotides
  • DNA-Cytosine Methylases