OLIGONUCLEOTIDE SAMPLE PREP & QUANTITATION: IMPROVED SPE PERFORMANCE USING A NOVEL SEMI- AUTOMATED EXTRACTION MANIFOLD

Importance of the Topic

Solid phase extraction (SPE) is a cornerstone technique in bioanalytical workflows, particularly for processing complex oligonucleotide samples. Effective SPE protocols reduce sample loss, improve quantification accuracy, and free analysts to focus on critical tasks. As therapeutic and diagnostic oligonucleotides gain prominence, scalable and reproducible extraction methods become essential for high-throughput laboratories and regulated environments.

Objectives and Overview of the Study

This study evaluates a traditional manual vacuum SPE approach versus a semi-automated positive pressure manifold (Otto SPEcialist) for extracting a range of oligonucleotides from aqueous samples. The goals are to compare recovery, precision, and accuracy, while demonstrating how semi-automation can streamline workflows and reduce operator variability.

Methodology

Sample Preparation:

• Oligonucleotide stocks (15–35 mer deoxythymidines and phosphorothioated analogs) were prepared in RNase-free water and diluted into 50 mM ammonium acetate buffer (pH 5.5).
• Internal standard (Gem 132) was added to reach 25 µg/mL in each sample.

Extraction Protocols:

• Manual SPE employed a vacuum manifold with Oasis WAX µElution 96-well plates.
• Semi-automated extractions used the Otto SPEcialist positive pressure manifold with programmable pressure profiles applied uniformly across wells.
• Both approaches processed 200 µL sample volumes following identical conditioning, load, wash, and elution steps.

Used Instrumentation

• Oasis WAX 96-well µElution SPE plate (Waters Corporation).
• Otto SPEcialist positive pressure manifold with software-controlled pressure profiling.
• ACQUITY Premier Oligonucleotide BEH C18 column (2.1×100 mm, 1.7 µm).
• ACQUITY Premier UPLC system.
• Xevo TQ-XS tandem quadrupole mass spectrometer (ESI-) for LC-MS/MS quantification.

Key Results and Discussion

Recovery and Reproducibility:

• Semi-automated SPE achieved higher average recoveries and lower coefficient of variation (CV < 10%) compared to manual vacuum extraction.
• Programmable pressure profiles ensured consistent flow rates and minimized sample loss.

Quantification Performance:

• Calibration curves for representative oligonucleotides spanned 5–1,000 ng/mL with R² ≥ 0.995.
• Standard curve accuracies ranged from 87–116%, meeting bioanalytical validation criteria (±15%).
• Inter-day QC samples (7.5, 75, 750 ng/mL) showed mean accuracies of 84–106% and CVs below 10% over two days.

Benefits and Practical Applications of the Method

By integrating the Otto SPEcialist:

• Analytical throughput increases due to reduced manual intervention and simplified workflow.
• Data quality improves via standardized pressure control, yielding more reliable recovery and precision.
• Laboratory efficiency is enhanced, enabling analysts to reallocate time to data analysis or method development.

Future Trends and Potential Applications

Oligonucleotide therapeutics and diagnostics will drive demand for robust SPE solutions. Semi-automated systems may evolve to:

• Support full sample-to-result platforms by integrating SPE with online LC-MS/MS.
• Incorporate machine learning–driven optimization of pressure profiles based on sample characteristics.
• Expand to accommodate emerging nucleic acid modalities, such as siRNA, ASOs, and mRNA fragments.

Conclusion

The semi-automated Otto SPEcialist manifold offers a reliable and user-friendly alternative to manual vacuum extraction. It delivers superior recoveries, tight precision, and high accuracy for oligonucleotide sample preparation, while minimizing human error and improving laboratory productivity.

References

1. Otto SPEcialist. Waters Corporation. 2021.
2. ACQUITY Premier Solution for Improved Recovery. Waters Corporation. 2021.
3. Oligonucleotide SPE: Comparing Manual vs Semi-Automated Otto SPEcialist Extraction. Waters Corporation. 2021.