(T1130-09-52) Development of a Robust and Sensitive Microflow LC-MS/MS Workflow for the Analysis of Oligonucleotides

Purpose: Oligonucleotides are becoming an important group of biologics resulting from their high target specificity in disease treatment. Because of their significance, robust and sensitive bioanalytical methods are in demand to enable safety and effectiveness of the biopharmaceutical products. Microflow LC-MS/MS using a trap-and-elute set-up has been shown to deliver excellent sensitivity and selectivity. While this platform enables the capability to inject large sample volumes, and the use of a trap column improves analytical column lifetime, challenges with carryover and overall ease of use can be present. In this study, a direct injection (analytical column only) workflow was compared with the trap and elute workflow for the quantitative analysis of oligonucleotides in matrix.

Methods: Extraction of rat plasma was performed using Clarity OTX SPE cartridges (Phenomenex). A mixture of oligonucleotides with the structures of the drugs fomivirsen, nusinersen, and eluforsen, plus a fully phosphorothioated 2’O-methylated 20-mer model oligonucleotide was used for this study. A 23 mer DNA oligonucleotide was used as the internal standard. LC separation was performed using a microflow LC system with a reversed-phase column and an ion pairing mobile phase system (RP-IP) with 15 mM diisopropylethylamine (DIEA) and 35 mM 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP. For the direct injection method, a flow rate of 10 µL/min was used, while for the trap-and-elute setup an analytical flow rate of 5 µL/min and a loading flow rate of 35 µL/min was used. Sample analysis was performed on a triple quadrupole mass spectrometer (SCIEX 7500 system).

Results: Initial findings using the trap-and elute method indicated an LLOQ of 0.01 to 0.03 ng/mL and a linear dynamic range (LDR) of 3-4 orders of magnitude for all the analytes examined. Typically, several hundred of analysis could be performed before the trap column started showing a significant increase in back-pressure and had to be replaced. The direct inject method, operating at a higher flow rate and allowing for lower injection volumes, is expected to be less sensitive. Carryover, which was significant (0.5%) with the trap-elute method, in initial data appears to be lower with the direct inject method, increasing the usable linear range. Ease of use of the direct injection method was improved, and its robustness will be demonstrated by analyzing a large set of samples spiked into processed plasma.

Conclusion: Demonstration of a robust and sensitive bioanalytical LC-MS/MS workflow for the quantitative analysis of oligonucleotides.