Sensitive LC-MS/MS quantitation of antisense oligonucleotides in plasma using the TSQ Altis Plus mass spectrometer

Importance of the Topic

A sensitive and reliable quantitation of antisense oligonucleotides (ASOs) in biological matrices is critical for evaluating pharmacokinetics, efficacy, and safety of emerging oligonucleotide therapeutics. As the number of ASO drugs entering clinical development continues to grow, robust bioanalytical methods that combine high sensitivity, specificity, and throughput are in demand for regulated environments.

Study Objectives and Overview

This application note demonstrates an LC-MS/MS method for quantifying two FDA-approved ASO drugs, fomivirsen and nusinersen, in human plasma. The workflow employs a simple liquid-liquid extraction (LLE) and an ion-pairing reversed-phase LC separation, combined with selected reaction monitoring (SRM) on a TSQ Altis Plus mass spectrometer. Chromeleon CDS software is used for data acquisition, processing, and regulated reporting.

Methodology and Instrumentation

Sample Preparation:

• Mix 100 µL of K2EDTA human plasma with 100 µL phenol/chloroform/isoamyl alcohol (25:24:1), vortex, and centrifuge.
• Transfer 25 µL of the aqueous layer, dry under vacuum, and reconstitute with 200 µL of internal standard solution.

Chromatographic Conditions:

• Column: C18, 2.1×50 mm, 2.6 µm
• Mobile phases: 15 mM DIPEA and 25 mM HFIP in water (A) and in 80:20 acetonitrile/water (B)
• Gradient: 5% B (0–1.0 min) to 24% B (3.5 min) to 80% B (4.0–6.0 min), return to 5% B by 8.0 min
• Flow: 0.25 mL/min; injection: 20 µL

MS Detection:

• Instrument: Thermo Scientific™ TSQ Altis™ Plus triple quadrupole
• Mode: Negative electrospray; SRM with optimized precursor/product transitions
• Source settings: Sheath gas 50 Arb; spray voltage 3000 V; vaporizer 350 °C

Data Processing:

• Chromeleon CDS 7.3.2 with 1/x weighted linear regression
• Retention time window ±0.25 min; Genesis peak integration; calibration report under CFR 21 Part 11 compliance

Main Results and Discussion

The method achieved a lower limit of quantitation (LLOQ) of 0.10 ng/mL for both ASOs with accuracy better than 80% and precision below 10%. Calibration curves from 0.10 to 100 ng/mL showed excellent linearity (R² = 0.998). Matrix interferences at the LLOQ level were under 10% of analyte signal. Chromatographic separation resolved fomivirsen, nusinersen, and internal standard with baseline resolution in under 5 minutes.

Benefits and Practical Applications

• Interference-free quantitation in a single LLE step to streamline sample processing
• High sensitivity for early-phase pharmacokinetic studies and therapeutic monitoring
• Robust performance across a wide dynamic range with minimal manual intervention
• Fit-for-purpose reporting suitable for regulated bioanalysis laboratories

Future Trends and Opportunities

With ongoing advances in UHPLC and mass spectrometry, future developments may include automated online extraction, higher-throughput configurations, and multiplex assays for broader ASO panels. Integration with advanced data analytics and tighter regulatory frameworks will further enhance reliability in clinical applications.

Conclusion

A streamlined IPRP-LC-SRM-MS method on the TSQ Altis Plus platform enables sensitive, accurate, and reproducible quantitation of ASO therapeutics in human plasma. The approach meets regulatory requirements and supports high-throughput bioanalysis in drug development.

Reference

1. Bennett CF, Baker BF, Pham N, Swayze E, Geary RS. Pharmacology of antisense drugs. Annu. Rev. Pharmacol. Toxicol. 2017;57:81–105.
2. Dhuri K, Bechtold C, Quijano E, Pham H, Gupta A, Vikram A, Bahal R. Antisense oligonucleotides: an emerging area in drug discovery and development. J. Clin. Med. 2020;9(6):2004.
3. Patrinos G, Danielson PB, Ansorge W. Molecular Diagnostics. 3rd ed. Elsevier; 2017.