Overcoming oligo bioanalysis challenges: Automated oligo extraction across multiple sample matrices

In the rapidly evolving world of oligonucleotide therapeutics, researchers are constantly faced with challenges to achieve consistent, high sensitivity results across different sample matrices without the manual labor of traditional methods.

A recent study published in Bioanalysis (Karlsborn et al., 2026) demonstrates a breakthrough with a fully automated extraction workflow using Biotage^® Oligo SPE. By moving away from labor-intensive liquid-liquid extraction (LLE), researchers validated a "generic" method capable of handling nine different tissues—including heart, lung, and brain—across three species.

The problem with liquid-liquid extraction (LLE)

For years, LLE was the go-to for oligonucleotides extraction because to separate oligonucleotides from proteins and lipids. But the resulting extract can still contain matrix interreferences that cause signal suppression and require longer LC gradients. As drug pipelines evolve and move into more complex tissues—like the heart, lungs, and brain—the limitations of LLE are difficult to overcome: 

• Labor intensive: As noted by the authors, LLE is a manual, or only semi-automated, technique that involves multiple steps. In contrast, the authors highlight that the Biotage® Oligo SPE workflow is a “single-step” and can be “fully automated” reducing sample prep workload, simplifying and reducing analytical time.
• Matrix interference: Extraction of oligonucleotides by LLE often leaves behind matrix components that cause significant signal suppression and require longer analytical gradients to separate the analytes.
• Hazardous solvents: It requires significant volumes of organic solvents, creating safety and disposal concerns.

Why Biotage® Oligo SPE?

The complexity of tissue homogenates often leads to matrix interferences and poor recovery. Biotage^® Oligo SPE utilizes a mixed-mode weak anion exchange (WAX) chemistry designed specifically to:

• Eliminate interferences: Effectively remove proteins, lipids, and salts for cleaner LC-MS/MS extracts.
• Enhance sensitivity: Achieve lower limits of quantification (LLOQ) as low as 0.593 nM.
• Ensure reproducibility: Deliver batch-to-batch consistency that manual methods simply can't match.

By integrating this method with the Biotage^® Extrahera^™ automated system, the team streamlined the path to method validation. The clean extracts provided by the Oligo SPE plates allowed for surrogate matrix calibration, significantly reducing the need for rare blank tissues. This directly supports the reduction principle of the 3Rs (Replacement, Reduction, and Refinement), making it an ethically responsible choice for modern labs.

Whether you are working with ASOs or siRNAs, Biotage^® Oligo SPE coupled with the Extrahera automated sample prep workstation deliver a faster, cleaner, and efficient workflow for the bioanalysis of oligonucleotides from biological samples.