Comparison of Analytical Method for Antioxidant Activity between an Image Processing Technique and Micoplate-DPPH assay

Abstract

To develop and validate method of paper chromatography coupled with DPPH (2, 2-diphenyl-1-picrylhydrazyl) scavenging assay (PC-DPPH, documented scanner and Adobe photoshop CS2 program were processed by means of an image processing program and compare with microplate assay for the purpose of quantitative determination and qualitative determination analytical method to test the antioxidant activity of health product. Validation methods were performed including linearity, % recovery, precision and sensitivity based on International Conference of Harmonization (ICH) guideline. Results of method validation used ascorbic acid as a standard substance. Linear is good regreassion line y =8266.6x-133.01 at range concentration 75-175 µg/ml and r² = 0.9962. Limit of detection (LOD) and Limit of quantitation (LOQ) were 0.009 and 0.03 µg/spot, respectively. The intra-day and inter-day precision values were 2.29-5.53% and 2.95-4.19%, respectively. The accuracy was 101.50-104.67 %. The IC₅₀ of the development method was 102.27 ± 2.97 µg/ml which calculated from computation of Graphpad prism 5. PC-DPPH test was useful for Consumer Protection Surveillance and products were available in the market and were claimed to have antioxidant properties. This is also for quality control of the product itself. The substances were environmentally friendly, low cost, simple and easy to test in the field. Specification of developed method to test spot and scope affect to reaction of DPPH.
Also inappropriate to pigment sample affect to means of an image processing program.