A fully automated novel solid-phase extraction method for siRNA drug extraction and quantitation across multiple...

Area of Science:

• Pharmacokinetics and Drug Metabolism
• Analytical Chemistry
• Biotechnology

Background:

• Traditional oligonucleotide extraction methods like liquid-liquid extraction (LLE) are often multi-step and tissue-specific.
• Developing a universal one-step solid-phase extraction (SPE) method for diverse tissue types remains a challenge.

Purpose of the Study:

• To develop and validate a generic, one-step SPE method for oligonucleotide therapeutic extraction from multiple tissue types.
• To automate the sample preparation process for enhanced efficiency and reduced workload.

Main Methods:

• A one-step SPE method was developed using an antisense oligonucleotide (ASO) internal standard and small interfering RNA (siRNA) analyte.
• The method was fully automated using a liquid handler and an automated SPE station.
• Quantification was performed using liquid chromatography-mass spectrometry (LC-MS) with calibration in a surrogate matrix.

Main Results:

• The automated SPE method successfully quantified siRNA in nine different tissues across three species.
• Comparison with LLE using in-vivo samples demonstrated good agreement between the two extraction techniques.
• The surrogate matrix calibration approach proved effective for diverse tissue types.

Conclusions:

• The presented one-step SPE method significantly reduces blank tissue requirements and sample preparation workload.
• Full automation and surrogate matrix calibration streamline bioanalysis for tissue biodistribution studies.
• This method simplifies analysis, reduces time, and aligns with the 3R principles (Replacement, Reduction, Refinement).