Rapid and Selective Spectrophotometric Assay for Micromolar Level Determination of Hydrogen Peroxide Based on Biocatalysis of Horseradish Peroxidase

Abstract

Hydrogen peroxide (H₂O₂) is an essential compound in food, pharmaceutical, clinical, industrial and environmental analysis. H₂O₂ has been widely used for preservation of raw milk due to its bactericidal properties. However, excess of H₂O₂ can be deleterious on the nutritional value of milk due to degradation of folic acid. A simple and sensitive spectrophotometric assay was developed for the quantification of H₂O₂ in milk. This method is based on coupling of Iso-nicotinic acid hydrazide (INH) and Resorcinol (RC) as chromogenic co-substrates in presence of horseradish peroxidase (HRP) and H₂O₂ to form a red colored product, which has strong absorbance at λ_max = 520 nm. The effects of different parameters on the sensitivity of the proposed assay were studied and optimized. Under optimized condition a linearity response for H₂O₂ are 0.5 μM - 64 μM and 0.5 μM -128 μM for kinetic and fixed time methods, respectively. The detection limit, quantification limit and molar extension co-efficient values for H₂O₂ assay were found to be 0.87 μM, 2.63 μM and 0.328×105 L/mol/cm, respectively. The Michaelis-Menten (K_m) constant and catalytic efficiency were 36.27 μM and 2.0927 ×10⁵ M^-1 min^-1. The proposed method produced recovery in the range of 99-102.8 % for milk samples and 98.2-101.8 % for rain water samples. Some of the important advantages of the proposed method are, use of inexpensive reagents, simple operation conditions, rapid analysis, wide linear range, high sensitivity, free from interfering substances and precision comparable to the reference methods.

Key words:

• Horseradish peroxidase
• hydrogen peroxide assay
• milk sample
• rain water sample
• iso-nicotinicacidhydrazide
• resorcinol