Simultaneous quantification of siRNA antisense and sense strands by hybrid liquid chromatography-mass spectrometry.

IF 1.8 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Bioanalysis Pub Date : 2025-02-01 Epub Date: 2025-01-31 DOI:10.1080/17576180.2025.2457894

Karan Agrawal, Shaofei Ji, Wenying Jian

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引用次数: 0

Abstract

Background: Most oligonucleotide bioanalytical assays currently only quantify the pharmacologically-active antisense strand, though there have been recent efforts to simultaneously quantify the sense strand using hybridization ELISA or solid phase extraction LC-MS. Hybrid LC-MS, which offers both high sensitivity and specificity unlike the currently used platforms, has not been applied to quantify both siRNA strands simultaneously.

Materials & methods: A hybrid LC-MS assay utilizing LNA capture probes was developed and applied to quantify both strands of a 21-mer lipid-conjugated siRNA (SIR-3) using tandem mass spectrometry (MS/MS). A similar approach using high-resolution mass spectrometry (HRMS) was also evaluated.

Results: The final LC-MS/MS method was capable of quantifying both strands of SIR-3 at concentrations between 0.600 and 1000 ng/mL in cynomolgus monkey tissue homogenates with acceptable accuracy and precision. The LC-HRMS assay demonstrated similar sensitivity and assay performance as the LC-MS/MS assay.

Conclusions: Overall, this manuscript presents orthogonal methods to existing siRNA bioanalytical workflows that with high sensitivity and specificity can provide greater information about the concentration and biotransformation of an siRNA analyte.

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液相色谱-质谱法同时定量siRNA反义链和义链。

背景：大多数寡核苷酸生物分析方法目前只能定量具有药理活性的反义链，尽管最近有人尝试使用杂交ELISA或固相萃取LC-MS同时定量反义链。与目前使用的平台不同，混合LC-MS具有高灵敏度和特异性，但尚未应用于同时量化两种siRNA链。材料和方法：利用LNA捕获探针开发了一种混合LC-MS分析方法，并使用串联质谱法（MS/MS）定量了21聚脂偶联siRNA （SIR-3）的两条链。还评估了采用高分辨率质谱（HRMS）的类似方法。结果：最终的LC-MS/MS方法能够定量食蟹猴组织匀浆中浓度在0.600 ~ 1000 ng/mL之间的SIR-3两条链，准确度和精密度可接受。LC-HRMS法具有与LC-MS/MS法相似的灵敏度和分析性能。结论：总体而言，本文提出了现有siRNA生物分析工作流程的正交方法，具有高灵敏度和特异性，可以提供有关siRNA分析物浓度和生物转化的更多信息。

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.