Development and fit-for-purpose validation of a LC-MS/MS assay for fibrinogen peptide A quantitation in human plasma

Bioanalysis. 2014;6(13):1759-66. doi: 10.4155/bio.14.148.

Abstract

Background: Fibrinopeptide A (FPA) is a plasma peptide, formed by the action of thrombin on fibrinogen during clog formation. FPA represents a direct indicator of thrombin activity and could potentially be used as a biomarker for anti-thrombotic therapy development. Results/Methodology: A LC-MS/MS assay with a high throughput solid phase extraction procedure was developed and validated to measure FPA in plasma. The lower limit-of-quantitation (LLOQ) of this assay was determined to be 0.16 nM. The inter- and intra-day%CV was <15%. Freeze-thaw stability of FPA was ±30% up to 3 cycles and linear response of FPA was observed for plasma dilution up to 16-fold.

Conclusion: The assay was validated and the biological variability of FPA in plasma was established (1-30 nM).

MeSH terms

  • Amino Acid Sequence
  • Chromatography, High Pressure Liquid* / standards
  • Fibrinopeptide A / analysis*
  • Fibrinopeptide A / isolation & purification
  • Fibrinopeptide A / standards
  • Freezing
  • Humans
  • Molecular Sequence Data
  • Protein Stability
  • Reference Standards
  • Solid Phase Extraction
  • Tandem Mass Spectrometry* / standards

Substances

  • Fibrinopeptide A