Enzymatic assay of opioid analgesic, tramadol, using horseradish peroxidase

Three novel and sensitive enzymatic methods have been developed for the quantification of tramadol in pharmaceutical dosage forms. The proposed methods are based on the reaction of tramadol with 3-methylbenzothiazoline-2-one hydrazone (method A), aniline (method B) and aminoantipyrine in the presence of hydrogen peroxide and horseradish peroxidase to give colored complexes. The colored complexes obtained with 3-methylbenzothiazoline-2-one hydrazone, aniline and aminoantipyrine exhibit absorption maxima at 480 nm, 550 nm and 530 nm, respectively. Regression analysis of Beer’s plots showed good correlation for tramadol in the concentration range (μg/mL) 2-12 for the methods A & B and 4-24 for the method C. The experimental parameters were studied and optimized. The precision and accuracy of the methods were satisfactory. The proposed methods were successfully applied for the quantification of tramadol in 3 brands of commercially available tablet dosage forms. The results were compared satisfactorily with the official method.