title: Determination of antisense phosphorothioate oligonucleotides and catabolites in biological fluids and tissue extracts using anion-exchange high-performance liquid chromatography and capillary gel electrophoresis.
abstract: 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.
authors.affiliation: Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
authors.forename: S H
authors.initials: SH
authors.lastname: Chen
authors.forename: M
authors.initials: M
authors.lastname: Qian
authors.forename: J M
authors.initials: JM
authors.lastname: Brennan
authors.forename: J M
authors.initials: JM
authors.lastname: Gallo
mesh_terms: D000818:Animals; D018288:Carcinoma, Small Cell / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D015257:DNA-Cytosine Methylases / Q000235:genetics; D019075:Electrophoresis, Capillary; D008175:Lung Neoplasms / Q000737:chemistry; D051379:Mice; D008819:Mice, Nude; D016376:Oligonucleotides, Antisense / Q000032:analysis*; D015203:Reproducibility of Results; D013873:Thionucleotides / Q000032:analysis*; D014018:Tissue Distribution
chemical_list: D016376:Oligonucleotides, Antisense; D013873:Thionucleotides; D015257:DNA-Cytosine Methylases