Development of a SPE-HPLC-PDA Method for the Quantification of Phthalates in Bottled Water and Their Gene Expression Modulation in a Human Intestinal Cell Model

Abstract

Phthalates are ubiquitous pollutants that are currently classified as endocrine disruptor chemicals causing serious health problems. As contaminants of food and beverages, they come into contact with the epithelium of the intestinal tract. In this work, a SPE-HPLC-PDA method for the determination of phthalates in water from plastic bottles was developed and validated according to the food and drug administration (FDA) guidelines. A chromatographic separation was achieved using a mobile phase consisting of ammonium acetate buffer 10 mM pH 5 (line A) and a mixture of methanol and iso-propanol (50:50 v/v, line B) using gradient elution. Several SPE cartridges and different pH values were investigated for this study, evaluating their performance as a function of recovery. Among these parameters, pH 5 combined with the SPE sep pack C18 cartridge showed the best performance. Finally, the proposed method was applied to the analysis of real samples, which confirmed the presence of phthalates. A colonic epithelial cell model was used to evaluate the effects of these phthalates at the concentrations found in water from plastic bottles. In cells exposed to phthalates, the increased expression of factors, which control the signaling pathways necessary for intestinal epithelium homeostasis, inflammatory response, and stress was detected. The proposed method falls fully within the limits imposed by the guidelines with precision (RSD%) below 7.1% and accuracy (BIAS%) within −4.2 and +6.1.