ELISA and HPLC analysis of Ochratoxin A in red wine collected in Croatia

Abstract

Ochratoxin A (OTA) is nephrotoxic and carcinogen mycotoxin that contaminates various commodities including wine all round the world. It was found that wine consumption might significantly increase human exposure to this toxin. In this study, enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC) for OTA analysis were tested on red must and wine samples collected in Croatia. Must samples were also tested for the presence of A. carbonarius. HPLC analysis was performed according to previously described method that includes solid phase extraction on the immuonoaffinity columns (OchraTest, Vicam, MA, USA). The standard curve was linear (r2=0.9987) and the detection limit was 5 ng/L. ELISA method performed according to the test kit manufacturer’ s instructions (I’ screen Ochratoxin A) gave unrealistically high values of OTA concentrations indicating the presence of impurities. Therefore, a step with bicarbonate purification was added. The intensity of the resulting yellow color was measured at 450 nm on the microplate reader (Victor 3, Perkin Elmer, Shelton, CT, USA). The OTA concentration was calculated comparing to standards provided by manufacturer, and the detection limit was 0.1 ng/ml. The results of ELISA and HPLC analysis of OTA in naturally contaminated red wines correlated well (r=0.821), and the correlation was better at higher OTA concentrations. In contrast to HPLC, ELISA could not detect very low OTA concentrations. OTA concentrations in must (range 19-50 ng/L) were higher than in the wines (range 0-21 ng/L). No must samples showed the presence of A. carbonarius, which is a common OTA-producing mould affecting grapes.