Sanford Ethan J, Chen Jianzhong, Tran Julia, Korboukh Ilia, Zhang Guangnong
Dicerna Pharmaceuticals, A Novo Nordisk Company, Lexington, MA, USA.
Bioanalysis. 2025 Jul;17(14):901-911. doi: 10.1080/17576180.2025.2535953. Epub 2025 Jul 24.
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.
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).
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.
寡核苷酸疗法（ONTs）是一类迅速发展的药物，市场上有20多种已批准的药物，还有更多药物正在针对各种适应症进行临床前和临床研究。该领域的许多研究小组正在添加化学修饰以调节组织特异性。将长链脂肪酸与siRNA分子偶联会增加分析物的疏水性，并给提取和液相色谱-质谱分析带来挑战。
我们报告了一种用于脂质偶联siRNA的固相萃取（SPE）提取方法的开发和优化。为了通过液相色谱-质谱提高测定定量，评估了一种稳定同位素标记内标（SILIS），其能够以高精度和精密度（大多数情况下为±5%）进行可靠定量。
我们证明了该测定法在小鼠血浆和组织匀浆中的性能，并将该测定法应用于一种新型脂质偶联siRNA分子的组织暴露测定和血浆药代动力学（PK）曲线分析，并建议在siRNA生物分析中，SILIS定量方法应成为标准做法。
