A Bioanalytical Liquid Chromatography Tandem Mass Spectrometry Approach for the Quantification of a Novel Antisense Oligonucleotide Designed for Parkinson’s Disease: A Rat Brain Biodistribution Study

Antisense oligonucleotides (ASOs) represent a unique category of therapeutics targeting disease-related RNAs. Since this new therapeutic category emerged, the immediate need to analyze ASOs in clinically relevant biological matrices has led to several methodologies, such as ligand binding assays and imaging techniques. To overcome issues in specificity and provide exact quantitative data for ASOs, a new LC-MS/MS method was developed to analyze in brain tissue a novel 4-10-4 gapmer ASO with the potential for treating Parkinson's disease with phosphorothioated backbone and 2'-O-(2-methoxyethyl) modifications. The sample pretreatment protocol to extract the ASO from brain tissue employed solid phase extraction (SPE) and protein digestion. The LC-MS/MS method was fully optimized, validated and applied to quantify the target ASO in brain tissue samples following an <i>in vivo</i> brain distribution study. The method has a Lower Limit Of Quantification of 1 ng/mg and was applied to the study's samples, demonstrating satisfactory sensitivity and providing valuable information about the ASO's distribution in different brain regions over 45 days.