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

IF 1.9 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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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.