Quantitative Determination of A Sirna (Ad00370) in Rat Plasma Using Peptide Nucleic Acid Probe and HPLC with Fluorescence Detection

Toxicokinetic and pharmacokinetic studies of therapeutic oligonucleotides require validated bioanalytical methods for sensitive and specific quantification of oligonucleotide drug candidates in biological samples.A peptide nucleic acid (PNA) hybridization-based HPLC-fluorescence assay was developed and validated for quantification of Arrowhead Pharmaceuticals' proprietary siRNA in rat plasma samples via hybridization and anion-exchange-HPLC (AEX-HPLC) with fluorescence detection.The validated method provided a sensitive and selective approach for quantification of siRNA in biological samples at a linear quantitation range of 1-1000 ng/ml. The assay requires only 25 μl of plasma sample and shows excellent accuracy and precision even without using an internal standard, providing a useful quantification method for siRNA determination in biological matrix with limited sample volume.