Extending the Lower Limits of Quantification of a Therapeutic Oligonucleotide Through Microflow LC-MS/MS

Significance of the Topic

Oligonucleotide drugs offer precise modulation of gene expression and have gained regulatory approval, exemplified by fomivirsen, the first FDA-approved antisense oligonucleotide. Quantitative analysis of these molecules is essential for drug development and quality control, but conventional LC-MS methods face sensitivity and contamination challenges due to ion pairing reagents and high flow rates.

Study Objectives and Overview

This work aimed to extend the lower limit of quantification (LLOQ) for a phosphorothioate antisense oligonucleotide (fomivirsen) by comparing conventional and microflow LC-MS/MS methods. By reducing mobile phase flow and optimizing a dedicated microflow system, the study evaluated improvements in sensitivity, chromatographic performance, and instrument maintenance intervals.

Methodology

A reversed-phase ion-pairing method using 100 mM HFIP and 15 mM DIEA was applied to C18 columns of 2.1 mm and 0.3 mm internal diameters. Calibrators spanning 0.5–1,000 ng/mL were prepared in 100 µM EDTA, each spiked with a 500 ng/mL internal standard. Both conventional flow (300 µL/min) and microflow (6 µL/min) separations were evaluated on a QTRAP 6500+ system under identical MRM transitions.

Instrumentation Used

• SCIEX QTRAP 6500+ LC-MS/MS with OptiFlow™ Turbo V source (25 µm SteadySpray™ probe)
• SCIEX M5 MicroLC system for microflow separations
• Exion™ UHPLC with Ion Drive Turbo V™ source for conventional flow reference
• Phenomenex Clarity® Oligo-MS (50×2.1 mm) and Gemini® C18 (50×0.3 mm) columns

Main Results and Discussion

The microflow configuration achieved an LLOQ of 1 ng/mL and an LOD of 0.5 ng/mL, improving over the conventional LLOQ of 5 ng/mL and LOD of 2 ng/mL. Peak area and signal-to-noise ratios increased by more than an order of magnitude at both low and high concentration limits, while chromatographic resolution and peak shapes remained equivalent to standard flow.

Benefits and Practical Applications

• Enhanced sensitivity supports trace-level quantification in pharmacokinetic and toxicology studies
• Reduced mobile phase consumption and front-end contamination extend maintenance intervals
• Compatibility with existing MRM methods facilitates seamless transition to microflow formats

Future Trends and Potential Applications

Integration of microflow LC-MS with high-throughput automation and further miniaturization may push detection limits lower and reduce sample requirements. Advances in source design and column technology are expected to broaden the applicability of microflow approaches across diverse oligonucleotide platforms and other polar analytes.

Conclusion

Implementing a dedicated microflow LC-MS system significantly improves the quantitative performance of oligonucleotide assays by enhancing sensitivity, reducing solvent consumption, and minimizing contamination. Such developments are poised to streamline analytical workflows in drug development and quality control.