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Updated: Sep 14, 2025
Shotgun Lipidomics of Rodent Tissues
Published on: November 18, 2022
Development of an LC-MS/MS assay to analyze a lipid-conjugated siRNA by solid phase extraction (SPE) in mouse plasma
Ethan J Sanford1, Jianzhong Chen1, Julia Tran1
1Dicerna Pharmaceuticals, A Novo Nordisk Company, Lexington, MA, USA.
Background:
Oligonucleotide therapeutics (ONTs) are a rapidly growing class of drug, with 20+ approved drugs on the market and more undergoing preclinical and clinical investigation for various indications. Many groups in the field are appending chemical modifications to modulate tissue specificity. Conjugation of long-chain fatty acids to siRNA molecules increases the hydrophobicity of the analyte and poses analytical challenges for extraction and LC-MS.
Results:
We report the development and optimization of an SPE extraction method for a lipid-conjugated siRNA. To improve assay quantitation by LC-MS, a stable isotope label internal standard (SILIS) was evaluated that enabled robust quantitation with high accuracy and precision (±5% in most cases).
Conclusion:
We demonstrate the performance of the assay in mouse plasma and tissue homogenates and apply the assay to the determination of tissue exposure and plasma PK profile for a novel lipid-conjugated siRNA molecule and suggest that a SILIS quantitation approach should be standard practice in siRNA bioanalysis.
Insights
This study optimized a solid-phase extraction method for lipid-conjugated siRNA, improving quantification accuracy. A stable isotope labeled internal standard (SILIS) is recommended for robust siRNA bioanalysis.
Area of Science:
- Pharmacology
- Analytical Chemistry
- Biotechnology
Background:
- Oligonucleotide therapeutics (ONTs) are an expanding drug class with chemical modifications for tissue specificity.
- Lipid conjugation to siRNA increases hydrophobicity, presenting analytical extraction and LC-MS challenges.
Purpose of the Study:
- To develop and optimize a solid-phase extraction (SPE) method for lipid-conjugated siRNA.
- To evaluate a stable isotope labeled internal standard (SILIS) for improved LC-MS quantitation.
- To apply the optimized assay for pharmacokinetic (PK) profiling of a novel lipid-conjugated siRNA.
Main Methods:
- Development and optimization of an SPE method for lipid-conjugated siRNA extraction.
- Evaluation of a stable isotope labeled internal standard (SILIS) for LC-MS quantitation.
- Application of the assay in mouse plasma and tissue homogenates for PK analysis.
Main Results:
- An optimized SPE method was developed for lipid-conjugated siRNA.
- SILIS enabled robust and accurate (±5%) quantitation of lipid-conjugated siRNA by LC-MS.
- The assay successfully determined tissue exposure and plasma PK profiles in mice.
Conclusions:
- The developed SPE method and SILIS approach provide accurate and precise quantification of lipid-conjugated siRNA.
- The assay is suitable for bioanalysis in biological matrices like plasma and tissue homogenates.
- SILIS quantitation should be standard practice for siRNA bioanalysis to ensure reliable results.
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