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

Abstract

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-H 2 O/TFA (0.08%) (5:1 v/v) followed by SPE. Culture medium was centrifuged and filtered through a 0.45 µm syringe filter. Analysis was conducted in a Nucleosil column, using a gradient of A (95:5 v/v H 2 O: ACN) and B (5:95 v/v H 2 O: ACN, containing TFA, pH 2), with a flow rate of 0.5 mL/min. Calibration curves (0.5-600 µg/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 µg/mL and 0.69 to 15.1 µg/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.

Simultaneous Quantification of Bisphenol-A and 4-Tert-Octylphenol in the Live Aquaculture Feed Artemia franciscana and in Its Culture Medium Using HPLC-DAD

“…Artemia metanauplii are a solid sample that require homogenization to achieve cell lysis, and this was performed by homogenization of the sample in methanol-H 2 O containing 0.08% ( v / v ) TFA and centrifugation. The previously described protocol for the extraction of BPA from bacterial cultures [ 62 ] was modified regarding the methanol aqueous phase ratio from 5:1 to about 1:3, since this amount of methanol produced better extraction of analytes. Since alkylphenols are characterized by low solubility in water compared to Bisphenol-A ( Table 1 ), methanol contributed to the increased solubility of t-octylphenol and BPA.…”

“…However, C18 cartridges provided the cleanest extracts of fish samples compared to polymeric sorbents, employed in the analysis of bisphenol and alkylphenols [ 55 ]. A flow rate of 1 mL/min during the elution of SPE, and a water bath temperature lower than 40 °C during the evaporation of methanol, after SPE, were employed as previously recommended for bacteria samples [ 62 ] to facilitate the effective recovery of both analytes and the internal standard. However, it should be noted that although simple centrifuging and filtering was sufficient for medium samples originating from bacterial cultures in basal salt medium [ 62 ], this was not the case with the Artemia medium samples, since it resulted in clogging of the injection loop, due to the higher salt concentration of the sample.…”

“…The HPLC system comprised of an LC20 AD pump and an SPD-20A photodiode array detector (DAD) (Shimadzu, Kyoto, Japan), a Rheodyne 7125 injection valve, with a loop of 80 μL volume (Rheodyne, Cotati, CA, USA) and a Nucleosil 100 C 18 column (250 × 4.6 mm, 5 μm), (Macherey-Nagel GmbH & Co., Duren, Germany) equipped with a 10 × 4.6 mm I.D., Nucleosil C 18 precolumn. Separation of BPA, t-OCT, and n-OCT was performed using a binary mobile phase system of mobile Phase A (95:5, v / v , 0.1 % TFA in H 2 O: Acetonitrile) and mobile Phase B (5:95, v / v , 0.08 % TFA in H 2 O: Acetonitrile) at room temperature, as previously reported [ 62 ] but with adjustments to the gradient, starting at 100% mobile Phase A and proceeding to an increasing mobile Phase B concentration to 20% over 2 min, 70% over 5 min, 90% at 7 min, and until the end of the analysis. Flow rate was 0.5 mL/min, and the analytes were detected at 220 nm.…”

“…The methods for the simultaneous determination of BPA and certain alkylphenols mentioned above, require specific equipment including ultrasonic extraction, fluorescence, MS or MS/MS detectors, which are not always available. A simple HPLC method, for the determination of BPA and its metabolites in bacterial cultures, was previously developed [ 18 , 62 ] and employed filtering of the medium samples and SPE (C18) extraction of BPA form bacteria, while HPLC analysis was conducted on a C18 Nucleosil column with gradient elution (acetonitrile: water containing TFA). However, this method did not include t-OCT and was validated only regarding the determination of BPA and its metabolites in cultures of bacteria in minimal salt media with BPA as the main carbon source.…”

Uptake and Distribution of Bisphenol A and Its Metabolites in Lettuce Grown in Sandy Loam and Loam Soil

“…Several methods were reported for the analysis of either the parent BPA or BPA and its analogues using GC-MS/MS or LC-MS/MS [4,8,9]. However, only one method was found to be suitable for the analysis of BPA and its metabolites (4-HAP and 4-HBA) using HPLC-DAD [10]. Since the LC-MS/MS based method is popular owing to the high selectivity and sensitivity, a new method was developed for the simultaneous analysis of BPA and its metabolites in lettuces using LC-MS/MS in the present study.…”