Development of an LC-MS/MS assay to analyze a lipid-conjugated siRNA by solid phase extraction (SPE) in mouse plasma and tissue using a stable isotope labeled internal standard (SILIS).

IF 1.9 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Bioanalysis Pub Date : 2025-07-24 DOI:10.1080/17576180.2025.2535953

Ethan J Sanford, Jianzhong Chen, Julia Tran, Ilia Korboukh, Guangnong Zhang

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引用次数: 0

Abstract

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.

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建立了用稳定同位素标记内标（SILIS）固相萃取（SPE）分析小鼠血浆和组织中脂质偶联siRNA的LC-MS/MS分析方法。

背景：寡核苷酸疗法（ONTs）是一个快速增长的药物类别，市场上有20多种已批准的药物，还有更多的药物正在进行临床前和临床研究，用于各种适应症。该领域的许多小组正在添加化学修饰来调节组织特异性。长链脂肪酸与siRNA分子的偶联增加了分析物的疏水性，并对萃取和LC-MS提出了分析挑战。结果：我们报道了一种脂质偶联siRNA的SPE提取方法的发展和优化。为了改进LC-MS的定量分析，对稳定同位素标记内标（SILIS）进行了评估，该内标具有较高的准确度和精密度（大多数情况下为±5%）。结论：我们证明了该方法在小鼠血浆和组织匀浆中的性能，并将该方法应用于一种新型脂质共轭siRNA分子的组织暴露和血浆PK谱的测定，并建议SILIS定量方法应成为siRNA生物分析的标准做法。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Bioanalysis BIOCHEMICAL RESEARCH METHODS-CHEMISTRY, ANALYTICAL

CiteScore

3.30

自引率

16.70%

发文量

审稿时长

2 months

期刊介绍： Reliable data obtained from selective, sensitive and reproducible analysis of xenobiotics and biotics in biological samples is a fundamental and crucial part of every successful drug development program. The same principles can also apply to many other areas of research such as forensic science, toxicology and sports doping testing. The bioanalytical field incorporates sophisticated techniques linking sample preparation and advanced separations with MS and NMR detection systems, automation and robotics. Standards set by regulatory bodies regarding method development and validation increasingly define the boundaries between speed and quality. Bioanalysis is a progressive discipline for which the future holds many exciting opportunities to further reduce sample volumes, analysis cost and environmental impact, as well as to improve sensitivity, specificity, accuracy, efficiency, assay throughput, data quality, data handling and processing. The journal Bioanalysis focuses on the techniques and methods used for the detection or quantitative study of analytes in human or animal biological samples. Bioanalysis encourages the submission of articles describing forward-looking applications, including biosensors, microfluidics, miniaturized analytical devices, and new hyphenated and multi-dimensional techniques. Bioanalysis delivers essential information in concise, at-a-glance article formats. Key advances in the field are reported and analyzed by international experts, providing an authoritative but accessible forum for the modern bioanalyst.