(T0930-01-01) Evaluation of a Novel Solid Phase Extraction for the Determination of Oligonucleotides from Biological Matrices and Analysis Using UPLC-MS/MS

Purpose: Oligonucleotide therapeutics have gained popularity as drug candidates due to high specificity for the treatment of various genetic diseases and disorders. As a result, routine methods are required for analysis and quantitation. However, anti-sense oligonucleotides present various analytical challenges particularly when using LC/MS analysis. Sample preparation is generally required to remove unwanted endogenous matrix components that interact with targets, thereby limiting availability for detection or interfering with the analytical response. This poster investigates the use of a novel polymer-based mixed-mode weak anion exchange solid phase extraction sorbent for improved oligonucleotide detection from various biological matrices. Method performance was evaluated using plasma, urine and liver matrices and assessed for ASO extraction efficiency and overall extract cleanliness.

Methods: UPLC-MS/MS analysis was performed using a Waters Premier UPLC interfaced to a TQ Absolute triple quadrupole MS via electrospray ionization. Multiply charged precursor ions were acquired using negative ion polarity mode. HILIC chromatography was performed using a Waters Premier BEH Amide column with aq/acetonitrile ammonium bicarbonate mobile phases, ultimately resulting in the elimination of ion pair reagents from the method. Multiple anti-sense oligonucleotides, based on phosphodiester (PO) or phosphorothioate (PS) backbones were spiked into various biological matrices: plasma, serum, urine and liver. Polymer-based weak anion exchange solid phase extraction was investigated for optimum extraction efficiency. Extract cleanliness was evaluated for phospholipids using UPLC-MS/MS analysis while protein content was monitored via the use of PAGE gel electrophoresis experiments.

Results: The analyte panel consisted of a range of ASOs: 14-33 nucleotides; deoxyribose or modified ribose (2’-OMe or 2’-MOE) sugars; phosphodiester (PO) and/or phosphorothioate (PS) backbones; naked, lipid or GalNAc conjugated. The use of HILIC chromatography eliminated reliance on ion pair reagents for retention and separation within the LC/MS. Reduced charge distribution was noted when using high acetonitrile/salt concentrations under HILIC conditions. Comparison to traditional reversed phase ion pair chromatographic approaches was not undertaken in this study. Evaporative effects were investigated comparing air vs nitrogen, various antioxidants and common elution solvent combinations. Nitrogen evaporation in the presence of TCEP demonstrated reduced evaporative losses ( < 15%) when used with ammonium bicarbonate/ACN solvent combinations. Liver tissue homogenization was performed in parallel, using the Biotage^® Lysera bead mill homogenizer. Comparisons included bead size/type, matrix to solvent volume ratios and number of cycles. Finalized protocol homogenized 100mg of tissue with 500 µL of H₂O with ceramic beads. Analyte recoveries varied depending on modification and analyte properties. Subtle modification of the generic protocol resulted in improved extract cleanliness and reduced suppression effects. Typical analyte recoveries on each end of the polarity scale (lipid > GalNAc) were above 70% with corresponding RSDs below 10%. Gel electrophoresis was performed allowing evaluation of protein molecular weights from 10-220 kDa. Various “lysis” buffer options were evaluated for protein digestion in plasma. Proteinase K allowed excellent digestion of proteins whereas other “lysis” buffer options contained typical bands across the molecular weight range. However, the optimised SPE sorbent removed all trace of protein from the extracts when using each “lysis” buffer option. Protein precipitated plasma samples were used as a benchmark to evaluate the degree of phospholipid removal. Excellent phospholipid removal was observed for each matrix.

Conclusion: This study demonstrates the applicability of a novel SPE sorbent with targeted pore size distribution to the extraction and analysis of various Oligonucleotides from multiple biomatrices. High reproducible recoveries for a range of OGN targets were observed. Optimized extraction protocols demonstrated excellent removal of matrix components, specifically proteins, phospholipids and exogenous stabilizers/surfactants.

Acknowledgements: The authors declare no competing financial interest. However, salaries of all authors are paid by Biotage.

Effects of lysis buffer with respect to plasma on OGN recovery (left) and matrix factors (right).

Effects of wash combinations on OGN recovery (left) and matrix factors (right).

OGN recovery in various matrices: plasma, liver and urine. Lipid and GalNAC conjugations can be improved further with subtle method modification of wash and elution steps.