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Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays
Published on: January 30, 2019
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Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay
Jean-Paul Pais de Barros1, Thomas Gautier1, Wahib Sali1
1INSERM, LNC UMR866, F-21000 Dijon, France LNC UMR866, University Bourgogne Franche-Comté, F-21000 Dijon, France LipSTIC LabEx, Fondation de Coopération Scientifique Bourgogne-Franche Comté, F-21000 Dijon, France.
Journal of Lipid Research
|May 30, 2015
Summary
Quantifying plasma lipopolysaccharides (LPS) using HPLC/MS/MS aids in diagnosing Gram-negative infections. This method, combined with the LAL bioassay, reveals how plasma components neutralize LPS activity and identifies severe illness in patients.
Area of Science:
- Biochemistry
- Microbiology
- Clinical Diagnostics
Background:
- Plasma lipopolysaccharides (LPS) are key indicators of Gram-negative bacterial infections.
- Accurate quantitation of LPS is crucial for assessing infection severity and treatment efficacy.
- Existing bioassays may not fully capture LPS presence or activity in all clinical scenarios.
Purpose of the Study:
- To develop and validate a sensitive HPLC/MS/MS method for quantifying 3-hydroxymyristate, an LPS derivative, in human plasma.
- To integrate this mass spectrometry assay with the limulus amebocyte lysate (LAL) bioassay for comprehensive LPS activity monitoring.
- To investigate the role of plasma components in LPS neutralization and assess LPS levels in patients with systemic inflammatory response syndrome (SIRS).
Main Methods:
- Extraction of LPS-derived 3-hydroxymyristate from plasma using organic solvents.
- Separation of the analyte via reversed-phase High-Performance Liquid Chromatography (HPLC).
- Quantitation using tandem Mass Spectrometry (MS/MS) and comparison with LAL bioassay results.
Main Results:
- The HPLC/MS/MS method demonstrated reliability, practicality, accuracy, and sensitivity for LPS quantitation.
- Combined LAL and HPLC/MS/MS analyses indicated that plasma lipoproteins and phospholipid transfer protein possess intrinsic LPS-neutralizing capacity.
- In SIRS patients with documented infections but negative LAL tests, significant LPS levels were detected by HPLC/MS/MS, correlating with illness severity.
Conclusions:
- HPLC/MS/MS is a valuable tool for accurate endotoxin quantitation in biological samples.
- The study provides evidence for endogenous plasma factors that neutralize LPS activity.
- This method enhances the assessment of LPS neutralization efficacy and the evaluation of LPS's in vivo proinflammatory potential, particularly in complex clinical cases.
