Multi-mycotoxin determination in baby foods andin vitro combined cytotoxic effects of aflatoxin M1 and ochratoxin A

The co-occurrence of mycotoxins in baby foods, including aflatoxin M 1 (AFM 1 ) and ochratoxin A (OTA), has been reported in previous studies, but data on their potential combined toxic effects are still missing. The present work aimed at (1) validating an in-house multi-mycotoxin high performance liquid chromatography with fluorescence detection (HPLC-FLD) method for AFM 1 , total aflatoxins (aflatoxin B 1 (AFB 1 ), aflatoxin B 2 (AFB 2 ), aflatoxin G 1 (AFG 1 ), aflatoxin G 2 (AFG 2 )) and OTA in infant formulae (milk powders) and cereal baby foods (flours), and (2) assessing the combined cytotoxic effects of AFM 1 and OTA in an intestine-derived cell line. The HPLC-FLD method, which included a chloroform extraction, liquid-liquid extraction, immunoaffinity column clean-up and fluorescence detection after post-column derivatisation with electrochemically generated bromine, was adequate for the analysis of baby foods and met the requirements of validation and quality control for the studied working ranges. The limits of quantification for AFM 1 , AFB 1 , AFB 2 , AFG 1 , AFG 2 and OTA were 0.069, 0.032, 0.020, 0.047, 0.020 and 0.244 μg/kg, respectively. The mean recovery values were 96, 114, 112, 107, 101 and 87%, respectively. A dose-dependent cytotoxicity was observed for individual and combined AFM 1 and OTA using the Caco-2 cell line, which represents a site of contact of both mycotoxins in the body, after oral exposure. Interactions between both mycotoxins were disclosed by application of the concentration addition (CA) and independent action (IA) models, revealing the predominance of an antagonistic pattern. In conclusion, this study proposes a HPLC-FLD method for multi-mycotoxin monitoring in baby foods and provides evidence for the interaction between AFM 1 and OTA, and for the applicability of CA/IA models to predict the effect of mycotoxins mixtures, further contributing to the prevention of mycotoxins-associated adverse health effects.