Development of a new micro-hptlc protocol for total antioxidant potential determination of redox-active drugs

A new micro-HPTLC method assisted by image analysis technique was proposed for rapid evaluation of the total antioxidant potential (TAP) of redox-active drugs using 2,2-diphenyl-1-picrylhydrazyl (DPPH center dot) and 2,2'-azinobis-3 ethylbenzothiazoline-6-sulfonic acid (ABTS(*+)) radicals. Colored compounds that can significantly influence the spectrophotometric and chromatographic measurements were revealed in case of selected drugs after their reaction with DPPH center dot and ABTS(*+) radical respectively. The best chromatographic conditions were selected for accurate quantification of DPPH center dot and ABTS(*+) radicals after their separation from the interfering compounds. The validation of the proposed method was performed in terms of linearity, limit of detection, limit of quantification, precision, and accuracy. Green and red color channel used for image processing were found to allow accurate quantification of DPPH center dot. For accurate quantification of ABTS(*+) the red and gray channels were selected. A linear dependence of spot area/radical concentration was obtained in the range 0.78-2.76 mu g center dot spot(-1) and 0.21-2.49 mu g/spot for DPPH center dot and ABTS(*+) radicals respectively. The developed method was found to be accurate and precise according to values higher than 92% for recovery and less than 3% for relative standard deviation parameters (RSD%). A statistical significant correlation was obtained comparing the spectrophotometric results with the chromatographic ones obtained for selected adrenergic drugs.