Development and Validation of an HPLC-DAD Method for the Simultaneous Extraction and Quantification of Bisphenol-A, 4-Hydroxybenzoic Acid, 4-Hydroxyacetophenone and Hydroquinone in Bacterial Cultures of Lactococcus lactis

Bisphenol-A, a synthetic organic compound with estrogen mimicking properties, may enter bloodstream through either dermal contact or ingestion. Probiotic bacterial uptake of bisphenol can play a major protective role against its adverse health effects. In this paper, a method for the quantification of BPA in bacterial cells of L. lactis and of BPA and its potential metabolites 4-hydroxybenzoic Acid, 4-hydroxyacetophenone and hydroquinone in the culture medium is described. Extraction of BPA from the cells was performed using methanol-H2O/TFA (0.08%) (5:1 v/v) followed by SPE. Culture medium was centrifuged and filtered through a 0.45 mu m syringe filter. Analysis was conducted in a Nucleosil column, using a gradient of A (95:5 v/v H2O:ACN) and B (5:95 v/v H2O: ACN, containing TFA, pH 2), with a flow rate of 0.5 mL/min. Calibration curves (0.5-600 mu g/mL) were constructed using 4-n-Octylphenol as internal standard (1 > R-2 > 0.994). Limit of Detection (LOD) and Limit of Quantification (LOQ) values ranged between 0.23 to 4.99 mu g/mL and 0.69 to 15.1 mu g/mL respectively. A 24 h administration experiment revealed a decline in BPA concentration in the culture media up to 90.27% while the BPA photodegradation levels were low. Our results demonstrate that uptake and possible metabolism of BPA in L. lactis cells facilitates its removal.