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Related Experiment Video

Updated: Dec 9, 2025

Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients
06:28

Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients

Published on: June 24, 2019

9.7K

Novel RP-HPLC based assay for selective and sensitive endotoxin quantification.

Blanka Bucsella1, Anika Hoffmann2, Mathieu Zollinger2

  • 1University of Zürich, Department of Chemistry, Winterthurerstr. 190, Zürich, CH-8057, Switzerland and HES-SO Valais (University of Applied Sciences, Sion; Wallis), Institute of Life Technologies, Route du Rawyl 64, CH-1950 Sion 2, Switzerland. franka.kalman@hevs.ch.

Analytical Methods : Advancing Methods and Applications
|September 14, 2020
PubMed
Summary

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A new analytical method quantifies endotoxins (ETs) using HPLC-FLD, offering an economic alternative to LAL tests. This assay measures KDO, a sugar acid released from ETs, for accurate endotoxin detection.

Area of Science:

  • Analytical Chemistry
  • Biochemistry
  • Biotechnology

Background:

  • Endotoxins (ETs) are potent pyrogens requiring accurate quantification for pharmaceutical safety.
  • Current methods like the Limulus Amebocyte Lysate (LAL) test are animal-based and have limitations.
  • There is a need for reliable, economic, and instrumental methods for endotoxin detection.

Purpose of the Study:

  • To develop and validate a novel instrumental analytical assay for endotoxin quantification.
  • To provide an alternative to the LAL test using common laboratory equipment.
  • To demonstrate the assay's applicability in various research and development settings.

Main Methods:

  • Hydrolysis of endotoxins to release the rare sugar acid KDO.
  • Stoichiometric reaction of KDO with DMB to form a fluorescent derivative.

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Last Updated: Dec 9, 2025

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  • Quantification of the KDO-DMB derivative using RP-HPLC with a fluorescence detector.

    • Main Results:

    • The assay quantifies endotoxins from ~10⁹ EU/mL down to ~40 EU/mL with high accuracy.
    • Results are obtained in concentration units (e.g., ng ET/mL), convertible to EU/mL.
    • High correlation observed when comparing results with the LAL test for bacterial biopolymers.
    • Successful application in developing endotoxin-specific depth filters for downstream processing.

    Conclusions:

    • The developed assay is a sensitive, economic, and precise method for endotoxin quantification.
    • It demonstrates potential as a valuable complement to the LAL test for pharmaceutical product release.
    • The assay's utility in applied research and product development highlights its practical significance.