Track: Bioanalytics - Biomolecular - Drug Quantification - Mass Spectrometry (LC-MS) Methods
Category: Late Breaking Poster Abstract
Improve Sensitivity for Quantification of Antisense Oligonucleotides in Plasma Using MicroLC-MRM Methodology
Purpose: As oligonucleotide therapeutics are rapidly growing as a class of therapeutic compounds, there is a need for improvements in the approaches for bioanalytical studies. Traditionally approaches include hybridization enzyme lined immunosorbent assays (ELISA), liquid chromatographic fluorescence (LC-FLR), or polymerase chain reaction (PCR) approaches. These approaches demonstrate many advantages for quantitation, including high throughput and high sensitivity, but struggle with providing desired selectivity. As the orthogonal technology, mass spectrometry has been routinely used for bioanalytical studies of oligonucleotides, but the extent of its use to date has been limited due to the ultra-low analyte levels in matrices. In this project, the implementation of microflow chromatography is reported for improved sensitivity in LC-MS assays for oligonucleotide quantitation. Methods: A curve from 0.1 ng/mL to 1000 ng/mL of fomivirsen was prepared by spiking fomivirsen into rat plasma. rat plasma samples were extracted using the OTX Clarity SPE plate using the recommended protocol. On-column MRM method development was performed because the MRM parameter optimizations are highly dependent on the LC-MS conditions. In order to compare the sensitivity difference between analytical flow LC-MS and microflow LC-MS, each sample was injected and analyzed in triplicates on a triple-quadrupole system coupled with either analytical flow or micro-flow HPLC. The acquisition methods share the same MS dependent parameters and conditions, but different source parameters with corresponding flow rates. Results: Analysis of the processed plasma samples using high flow chromatography provides high quality fomivirsen quantitation, with an LOD of 0.5 ng/ml and an LLOQ of 1 ng/ml. The blank sample shows no interference from matrix species which enables low limits of detection. A calibration curve is obtained for quantification of fomivirsen from 1 ng/ml to 1000 ng/ml. Each sample is analyzed in triplicate to demonstrate the statistical relevance. All data show CVs below 8% and accuracies from 85% to 110%. The assay LLOQ using microLC is improved. The linear is obtained over the range of 0.1 ng/ml to 1000 ng/ml, with similar accuracy and reproducibility as high flow LC assay. When directly comparing the data from the high flow and microflow experiments, the microLC data show significant improvement of sensitivity, at an average of 2.5 fold at S/N. Conclusion: A novel microLC-MS/MS workflow is reported for quantifying antisense oligonucleotides in matrix with high robustness, a wide dynamic range and great sensitivity.