Achieving best in class sensitivity for antisense oligonucleotides in plasma using trap-and-elute microflow LC

Increasing sensitivity for oligonucleotide quantification using the SCIEX Triple Quad™ 7500 LC-MS/MS System – QTRAP ® Ready, powered by SCIEX OS Software

Remco van Soest, Eshani Nandita, Elliott Jones and Kerstin Pohl


This technical note describes how the lower limits of quantification (LLOQs) for antisense oligonucleotides (ASOs) in rat plasma are further improved by using a microflow trap-andelute method. Compared to previously published data acquired using analytical flow LC on a SCIEX 7500 System1 , sensitivity was increased by a factor of 20 (see Table 1 for achieved LLOQs). Compared to data with the same microflow method using a QTRAP 6500+ LC-MS/MS System, the sensitivity was improved by a factor of 3. 2

Oligonucleotide therapeutics and gene therapies are rapidly gaining attention as their potency improves and delivery challenges are addressed. Modalities such as ASOs are becoming more important due to their high specificity and ability to reach formerly undruggable targets. Sensitive and robust methods for quantitative analysis of oligonucleotides are needed to support the development of ASO therapeutics. Ligand binding assays using fluorescence detection can achieve very low detection limits. However, linear dynamic range is limited, and this type of assay often cannot differentiate oligonucleotides from their impurities and metabolites. LC-MS/MS can provide excellent selectivity, good sensitivity and the ability to analyze multiple oligonucleotides in a single assay. However, for studies where sample is limited, such as preclinical pharmacodynamic studies in rats or mice, sensitivity can still be insufficient.

Published data shows that the use of microflow LC lowered the LLOQ by an order of magnitude for several ASOs in an LCMS/MS assay using the QTRAP 6500+ System. 3 The use of a trap-and-elute microflow approach allowed injection of large volumes of extracted plasma samples, up to 30 µL, without increasing run times significantly. This technical note describes a further improvement of the LLOQs using the same trap-and-elute microflow method, with the more sensitive SCIEX 7500 System.

Table 1. Lower limits of quantification achieved for different ASOs in rat plasma using a trap-and-elute microflow method and the SCIEX 7500 System.

Key features of the microflow workflow using the SCIEX 7500 System for oligonucleotide quantification

  • Achieve low-pg/mL LLOQs for ASOs in rat plasma for pharmacokinetic studies faced with sample limitations that require improved specificity compared to traditional ligand binding assays

  • Large sample volume analysis without increasing method run time results in high sample throughput 

  • Save time by easily switching between analytical and microflow setup with no need for electrode position optimization, using the OptiFlow® Pro Ion Source with E Lens™ Technology

  • Reduced consumption of LC-MS grade ion-pairing reagents provides significant savings and increases robustness

  • Greater ion generation and ion transmission on the SCIEX 7500 System enables significant gains in sensitivity compared to the previous generation QTRAP 6500+ System, resulting in lower LLOQs

  • SCIEX OS Software increases productivity by providing a user-friendly interface and one single and compliance-ready platform for data acquisition, processing and management