(T1130-01-03) Sensitive LC-MS/MS Method for Cationic Ligand-Conjugated siRNA Therapeutics

Purpose: The conjugation of siRNA with ligands can significantly enhance the therapeutic potential of siRNA by improving its stability, specificity, and efficiency in targeting and entering desired cells. This approach holds promise for the development of more effective and safer RNA-based therapeutics. These ligands include small molecules, lipids, peptides, aptamers, antibodies, proteins, carbohydrates, and non-coding RNA (ncRNA), etc^[1]. There is a growing need for efficient bioanalysis of oligonucleotide therapeutics. This broad class of molecules presents numerous challenges relative to traditional small-molecule therapeutics ^[2]Extraction method for several siRNAs uses liquid-liquid extraction (LLE) with phenol: chloroform: isoamyl alcohol (25:24:1), which also precipitates proteins, or dichloromethane. Using either of these solvents, the analytes are retained in the upper aqueous layer. Many methods use mixed-mode solid phase extraction (MM-SPE), which retains the analyte by a combination of reversed phase and ion exchange ^[2]. Multiple-amino ligand is a type of small molecule cationic ligand. Due to the presence of multiple amino cations on the ligand, conventional LLE methods face difficulties in extracting the siRNA from biological samples such as plasma. These multi-amino cation structures interact with various plasma components through mechanisms such as electrostatic and hydrophobic interactions, leading to significant challenges in extraction and resulting in very low or nearly non-existent mass spectrometry signals. The conventional LLE extraction method has an LLOQ of 2000 ng/mL, which does not meet the experimental dosing requirements. However, this study has developed an LLE method that is faster and more convenient than SPE. This method has a simple extraction step, high sensitivity, and avoids the cumbersome and time-consuming wash, load, and nitrogen drying operation steps required by SPE.

Methods: Add 100 µL of 50% NH₃·H₂O solution to 100 µL of rat plasma, and mix thoroughly. Then add 200 µL of RNA extraction solution and 300 µL of dichloromethane. Shake, centrifuge, and take 100 µL of the supernatant. Add 100 µL of 2%FA in water, mix well, centrifuge and take 100 µL of supernatant. All the samples were subjected to LC-MS/MS analysis. Using a methanol/water solution containing DBA and HFIP as mobile phase A, and a methanol/acetonitrile solution containing DBA and HFIP as mobile phase B, good peak shapes were obtained on an ACQUITY UPLC® Protein BEH C4 300Å column. Mass spectrometry was performed using a 6500+ system with an ESI source in the negative ion mode.

Results: To address the issue of low mass spectrometry response, ammonia solution was added to the plasma matrix to replace ammonium acetate buffer. The introduction of NH₃·H₂O leads to the deprotonation of amine groups on the ligand (NH₂⁺ → -NH), reducing their positive charge and weakening their interaction with the matrix, thus preventing co-precipitation with the matrix. At the same time, the increase in OH results in the deprotonation of the siRNA phosphate backbone (-PO₄H^- → -PO₄^2-), increasing its negative charge and enhancing hydrophilicity, which favors retention in the aqueous phase and improves extraction efficiency. This method optimized different concentrations of NH₃·H₂O. As the concentration of NH₃·H₂O increased, the response of the siRNA antisense strand (AS) significantly improved, with the LLOQ increasing from 2000 ng/mL to 10.0 ng/mL, a 200-fold enhancement.

Conclusion: The experimental results indicate that adding 50% NH₃·H₂O to the plasma matrix before extraction achieves an LLOQ of 10.0 ng/mL, which meets the experimental requirements. This method also offers the advantages of simplicity and low cost. It also demonstrates good linearity and accuracy (linear range of 10.0-1000 ng/mL, r = 0.9952). Finally, a simple and sensitive LC-MS/MS method was developed for cationic ligand-conjugated siRNA.

References: [1] Bo Hu, Liping Zhong. et al. Therapeutic siRNA: state of the art. Signal Transduction and Targeted Therapy (2020) 5:101.
[2] Matthew Ewles, Aaron R Ledvina, et al. Observations from a decade of oligonucleotide bioanalysis by LC-MS. Bioanalysis. 10.4155/bio-2024-0007.

Figure 1. Chromatograms of LLOQ before and after optimization of the LLE method.