Bioanalysis Zone

Highly Sensitive and Accurate Quantification of Isoprostane Oxidative Biomarkers using a High-Resolution Workflow (SCIEX)

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SCIEX_LOGO_BioZonesmallAuthors: Marieke Teppner, Vicki Gallant, Axel Pähler

Increasing selectivity with high-resolution multiple reaction monitoring (MRMHR) improves the signal-to-noise ratio for better accuracy, dynamic range, and LLOQ when quantitating isobaric prostaglandin-D2α isoforms

Key challenges of isoprostane biomarker assays

  • Lack of sensitivity – Quantification is poorly reproducible at low picogram levels in complex biological matrices.
  • Overlapping interferences – Assay selectivity is hampered by interfering, co-eluting peaks.
  • Multicomponent analysis in single assay –Single isomer measurement is a poor indicator of oxidative stress due to rapid degradation and variable isomer formation.
  • Substandard data quality – Precision and accuracy are compromised at very low biomarker levels, giving results below accepted bioanalytical standards.

Key benefits of MRMHR for quantifying isoprostanes

  • Maximized sensitivity – LLOQ of 5 pg/mL was an ~10-fold improvement over the triple quad MRM method.
  • Increased selectivity and specificity – Reduced background noise enhances S/N ratios and reproducibility.
  • Wider dynamic range – Measurements (5–10,000 pg/mL) are linear over 4-orders of magnitude (r = 0.9994).
  • All-inclusive assay in one injection – Both known and unknown oxidative stress markers can be monitored simultaneously with a high-resolutionTOF-MS scan.

Key features of the MRMHR workflow on the TripleTOF® System

  • MRM-like quantitation – High-specificity is obtained using a narrow extraction width to mine high resolution TOF data.
  • Simultaneous, multicomponent analysis – Fast acquisition rates can collect full-scan, MS/MS spectra for multiple precursors without additional cycle time.
  • Fast cycle times maintained – Processing speed allows for sufficient peak coverage, even with fast LC separations.

Read the full application note here.

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