Quantification of hydrogen peroxide and glucose using 3-methyl-2-benzothiazolinonehydrazone hydrochloride with 10,11-dihydro-5H-benz(b,f)azepine as chromogenic probe

A sensitive, selective, and rapid enzymatic method is proposed for the quantification of hydrogen peroxide (H₂O₂) using 3-methyl-2-benzothiazolinonehydrazone hydrochloride (MBTH) and 10,11-dihydro-5H-benz(b,f)azepine (DBZ) as chromogenic cosubstrates catalyzed by horseradish peroxidase (HRP) enzyme. MBTH traps free radical released during oxidation of H₂O₂ by HRP and gets oxidized to electrophilic cation, which couples with DBZ to give an intense blue-colored product with maximum absorbance at 620 nm. The linear response for H₂O₂ is found between 5 × 10⁻⁶ and 45 × 10⁻⁶ mol L⁻¹ at pH 4.0 and a temperature of 25 °C. Catalytic efficiency and catalytic power of the commercial peroxidase were found to be 0.415 × 10⁶ M⁻¹ min⁻¹ and 9.81 × 10⁻⁴ min⁻¹, respectively. The catalytic constant (k_cat) and specificity constant (k_cat/K_m) at saturated concentration of the cosubstrates were 163.2 min⁻¹ and 4.156 × 10⁶ L mol⁻¹ min⁻¹, respectively. This method can be incorporated into biochemical analysis where H₂O₂ undergoes catalytic oxidation by oxidase. Its applicability in the biological samples was tested for glucose quantification in human serum.