Differential Protein Precipitation-Based GalNAc-siRNA Sample Preparation with LC/MS Method Development Workflow in Plasma

Liquid chromatography-mass spectrometry (LC/MS) plays a crucial role in the quantification of small interfering RNAs (siRNAs) in biological matrices. However, the recovery of siRNA from complex biological matrices remains a significant challenge. Focusing on liver-targeted <i>N</i>-acetylgalactosamine (GalNAc)-siRNA conjugates, the primary extraction methods currently used are solid-phase extraction (SPE) and hybridization. While both methods have advantages, SPE recovery can vary depending on the analyte and is costly, whereas the hybridization method requires specific reagents, limiting its applicability. To address these challenges, we developed a novel extraction method for LC/MS bioanalysis of GalNAc-siRNA. Our innovative approach uses a differential protein precipitation method with an optimized organic solvent mix to remove large, high-abundance plasma proteins as precipitates while preserving the GalNAc-siRNAs in the liquid phase. The workflow was optimized to identify the most intense MS/MS transitions, and LC-MS/MS parameters were fine-tuned using high-resolution Orbitrap and QTRAP hybrid mass spectrometers for the highly sensitive detection of targeted siRNA molecules. This approach achieved a lower limit of quantification in the single-digit ng/mL range for four FDA-approved GalNAc-siRNAs (Givosiran, Lumasiran, Inclisiran, and Vutrisiran) and a major Givosiran metabolite, AS(N-1)3'. The applicability of this approach was successfully demonstrated by analyzing plasma samples from an in vivo rat study involving three molecules (Givosiran, Givosiran AS(N-1)3', and Inclisiran). This method is straightforward, robust, highly sensitive, and cost-effective and should be readily adaptable for the bioanalysis of diverse GalNAc-siRNAs and, potentially, for late-stage sample analyses.