Automated assay of the potency natural antioxidants potency using pipetting robot and spectrophotometry

In the food industry, in the process of creating new agricultural plant products, and in the testing ofanti-cancer drugs there is often a need to assay multiple samples of low molecular weight antioxidants, plantsamples and foods rich in antioxidants, with minimal additional costs and low degrees of uncertainty. Withthese demands in mind, we decided to study the fully automated assay of antioxidants using not onlyautomated sample measurements but also automated processing of samples and application of reagents. Theautomated pipetting system epMotion 5075 and the automated spectrophotometer BS 400 were chosen forthe assay purposes. Five methods were introduced for the automation: 2-diphenyl-1-picrylhydrazyl (DPPH)test, ferric reducing antioxidant power (FRAP) method, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid(ABTS) based test, N,N-dimethyl-1,4-diaminobenzene (DMPD) based test and the free radicals method.Samples containing one of the four antioxidants (standard rutin, quercitrin, ferulic and gallic acid) in a range1–1000 μg/ml were used throughout. All of the tested methods were found suitable for implementation in anautomated assay. However, some of them, such as the ABTS test failed to assay all tested antioxidants. Thecoefficients of determination were also unequal. From the analytical point of view, FRAP methods providedthe most reliable results in the automated assay; because of the capacity of the method, approximately240 samples per hour (one sample per 15 seconds) can be assayed using the automated protocol. We wereencouraged by the data received and we expect further interest in the practical performance of suchautomation. As a mean of testing the robustness of our method, in the next step of our study, oxidative statuswas assessed in model cell lines derived from prostate cancer (PC-3, PNT1A and 22RV1) that were culturedon ellipticine (0, 0.5, 1, 1.5, 2, 2.5, 5, 7.5, 10, 15 μmol/l) supplemented agar. Antioxidant activity wasassessed (DPPH, ABTS, FRAP, DMPD, FR) andcalculated on the phenolic antioxidant level (rutin,quercitrin, ferulic and gallic acid), and thus anestimation was formulated of the oxidative stress asa result of the impact of anti-cancer drugs. It can bedemonstrated that the new method has wideapplicability.