Background: Hybrid LC–MS assays for oligonucleotides rely on capture probes to develop assays with high sensitivity and specificity. Locked nucleic acid (LNA) probes are thermodynamically superior to existing capture probes, but are not currently used for hybrid LC–MS assays. Materials & methods: Using two lipid-conjugated double-stranded siRNA compounds as model analytes, hybrid LC–MS/MS assays using LNA probes were developed. Results: The workflows demonstrated the superiority of the LNA probes, optimized sample preparation conditions to maximize analyte recovery, evaluated the need for analyte-specific internal standards, and demonstrated that advanced mass spectrometric technology can increase assay sensitivity by up to 20-fold. Conclusion: The workflow can be used in future bioanalytical studies to develop effective hybrid LC–MS/MS methods for siRNA analytes.

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Papers of special note have been highlighted as: • of interest; •• of considerable interest

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Vol. 15, No. 18

Supplemental Materials

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History

Received 1 May 2023

Accepted 31 July 2023

Published online 28 August 2023

Published in print September 2023

Information

Keywords

Supplementary data

To view the supplementary data that accompany this paper please visit the journal website at: www.future-science.com/doi/suppl/10.4155/bio-2023-0079

Author contributions

K Agrawal and W Jian designed the study; K Agrawal generated data supporting the workflow; L Kang performed the probe design and initial method development for SIR-1; S Ji performed high-resolution mass spectrometry analysis for the SIR-2 capture probes; J Tena performed the melting temperature determination for the capture probes; and K Agrawal and W Jian performed data analysis and interpretation, and wrote the manuscript. All authors reviewed and approved the submitted manuscript.

Acknowledgments

The authors gratefully acknowledge the advice and assistance provided by Yanping Lin and Ying Wu during the method development process and the helpful feedback provided by Lieve Dillen during the preparation of this manuscript.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

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