A Center of Excellence for Precision Medicine

Importance of the Topic

Precision medicine depends on ultrasensitive and reliable biomarkers to guide therapeutic decisions and predict clinical outcomes. Oxidative stress plays a central role in many diseases—hypertension, stroke, diabetes and cardiovascular disorders—but lacks a standardized, high-throughput clinical assay. F2-isoprostanes are recognized as the gold-standard markers of lipid peroxidation, yet existing methods either demand large sample volumes, lengthy derivatization steps or suffer from limited sensitivity. A streamlined LC-MS/MS workflow can close this gap, enabling robust analysis in small volumes and accelerating biomarker validation and translation from bench to bedside.

Objectives and Study Overview

Researchers at the National University Hospital in Singapore, led by Dr. Chester Drum, partnered with Waters Corporation to develop a rapid, sensitive assay for F2-isoprostanes. The goals were to reduce sample volume requirements, simplify sample preparation, improve the lower limit of quantitation (LLOQ) to near 50 pg/mL and validate the method on clinical and biobank specimens. This collaboration aimed to produce a clinically relevant oxidative stress marker suitable for both research biobanks and potential routine use in translational laboratories.

Used Instrumentation

• Waters Xevo TQ-XS tandem quadrupole mass spectrometer
• ACQUITY UPLC I-Class system
• Waters Oasis MAX solid-phase extraction (SPE) micro-elution sorbent

Methodology

Plasma or urine samples (20–50 µL) undergo SPE using Oasis MAX cartridges in a micro-elution format, eliminating evaporation and reconstitution steps. After washing and elution, the extracts are directly injected onto the ACQUITY UPLC I-Class system. Chromatographic separation precedes targeted MS/MS detection on the Xevo TQ-XS. Internal standards and optimized multi-reaction monitoring transitions ensure specificity and quantitation at low picogram levels.

Main Results and Discussion

Compared with a legacy high-sensitivity instrument, the Xevo TQ-XS achieved a five-fold improvement in LLOQ, reliably detecting 50 pg/mL of 8-F2-isoprostane in human plasma. Signal-to-noise ratios and chromatographic resolution were markedly enhanced, allowing accurate quantitation with just 20 µL of sample. The method demonstrated high recovery, precision and throughput, meeting criteria for large-scale biomarker studies. Early application to a bariatric surgery cohort showed clear reductions in isoprostane levels post-intervention, correlating with improved clinical endpoints.

Benefits and Practical Applications

The streamlined LC-MS/MS assay offers:

• High sensitivity (LLOQ ∼50 pg/mL) in minimal sample volume
• Elimination of derivatization and lengthy preparation
• Rapid turnaround suitable for both biobank research and clinical trials
• Potential to guide patient stratification based on oxidative stress status

Future Trends and Potential Applications

Ongoing work will expand validation to large biobank cohorts (n>1,200) with clinical outcomes, including all-cause mortality. Successful correlation of isoprostane levels with patient prognosis may enable interventional trials titrated to oxidative stress targets. The approach can be extended to other low-abundance biomarkers, leveraging high-resolution LC-MS/MS for precision diagnostics and drug development.

Conclusion

The collaboration between NUHS and Waters delivered a robust, high-sensitivity LC-MS/MS assay for F2-isoprostanes, overcoming key bottlenecks in sample volume, sensitivity and throughput. This platform accelerates biomarker discovery and validation, paving the way for oxidative stress testing in translational research and future clinical applications.