Overcoming Reductant Interference in Peroxidase-Based Assays for Hydrogen Peroxide Quantification

Area of Science:

• Biochemistry
• Analytical Chemistry

Background:

• Peroxidase-based assays are common for hydrogen peroxide (H2O2) quantification.
• Endogenous reductants in biobased matrices interfere with these assays, causing underestimation of H2O2 levels.
• This interference stems from reductants reducing the reporter molecule used in the assay.

Purpose of the Study:

• To develop a method to circumvent interference from endogenous reductants in peroxidase-based H2O2 quantification.
• To enable accurate H2O2 measurement in complex biobased samples.

Main Methods:

• A pre-treatment step was introduced where the oxidized reporter molecule selectively oxidizes interfering compounds.
• The assay utilizes horseradish peroxidase and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the reporter molecule.
• The method was validated using garlic paste as a representative biobased matrix.

Main Results:

• The developed method effectively eliminates interference from endogenous reductants.
• Accurate quantification of hydrogen peroxide in a complex biobased matrix (garlic paste) was achieved.
• The pre-oxidation strategy prevents the underestimation of H2O2 content.

Conclusions:

• The proposed method provides a robust solution for accurate H2O2 quantification in biobased samples.
• This approach is broadly applicable to various peroxidase-based assays in complex biological systems.
• Selective pre-oxidation ensures reliable results by mitigating interference.