Development and Validation of a HPLC‐DAD Method for Determination of Several Endocrine Disrupting Compounds in Estuarine Water

Abstract

This paper presents the development and validation of an analytical procedure, which allows the simultaneous quantification of nine endocrine disrupters (EDCs) in polluted surface waters. The compounds selected for this study were the natural hormones (17b-estradiol and estrone), the synthetic hormone (17a-ethynylestradiol), the bisphenol A, the alkylphenols (4-octylphenol and 4-nonylphenol), and the phytoestrogens (daidzein, genistein, and biochanin A). Briefly, this method consisted of the preconcentration of water samples (2 L) in 500 mg Oasis HLB cartridges, followed by a cleanup step in 1 g silica cartridges, and analysis of all EDCs by High Performance Liquid Chromatography with Diode Array Detection (HPLC-DAD). The chromatographic separation occurred in a RP-18 analytical column in a gradient mode of CH 3 CN:H 2 O (pH 2) at a flow rate of 1 mL/min. The validation parameters revealed that this method was highly specific for all assayed EDCs (.99%), the linearity of the calibration curves, obtained either by external patronization or by fortified matrix calibration techniques, always showed a correlation higher than 0.99 and their intermediate precision and repeatability, evaluated in terms of intra and inter assays, were optimal for all EDCs (RSD , 3%). The precision and accuracy, estimated as the recovery rates that occurred during the preconcentration and the cleanup processes showed, for all EDCs, excellent results (RSD , 3% and recoveries up to 116%), and preconcentration factors (enrichment) up to 10000. The limits of detection (LOD) and quantification (LOQ) were mathematically estimated from the calibration curves. As expected, these values were different for each of the nine assayed EDCs and were higher when calculated from the addition standard calibration curves. Here, 4-octylphenol was the compound with the lowest LOD and LOQ (3.8 ng/L and 12.0 ng/L) and 17a-ethynylestradiol the one with the highest levels for both cited parameters (18.0 ng/L and 54.5 ng/L). Finally, the performance of the method was checked with water samples from a highly polluted area in the Douro river estuary (Portugal). The field results revealed high levels of estrogens (up to 176 ng/L), bisphenol A (up to 5.1 mg/L), alkylphenols (up to 449 ng/L), and phytoestrogens (up to 74 ng/L), in accordance with the high local pollution load, and suggested that this method can be easily and successfully used in water monitoring surveys of, at least, highly polluted estuarine areas.

Distribution of endocrine disruptors in the Mondego River estuary, Portugal

“…In this system the identity of all quantified compounds were confirmed and, furthermore, it was investigated the presence of other chemicals that, due to their low environmental concentrations, were under the detection limits of the applied HPLC-DAD method. The recovery rates for the majority of the EDCs were higher than 94%, with the exception of BIO-A (91%), 4-OP (70%) and 4-NP (61%) that show, on average, lower recovery rates (Ribeiro et al 2007). …”

“…The wavelengths used for detection of the nine EDCs proposed in this study were 246 nm for DAID, 260 nm for GEN and BIO-A, 278 nm for BPA, 4-OP and 4-NP, and, 280 nm for E1, E2 and EE2. All working calibration curves used for the calculation of the levels of the latest EDCs showed correlation factors higher than 0.99, and peak areas were used for the present quantitative analysis (Ribeiro et al 2007). During the application of the last validated method several quality control parameters accordingly to the International Conference of Harmonization (ICH 1996) guidelines.…”

“…Furthermore, the maintenance of the retention times among samples and standards was always verified as, each analysed sample was injected both individually (n=2) and spiked with all standards (n=1). Finally only the areas of peaks showing a purity higher than 99% (this value was calculated automatically by the HPLC-DAD software) were considered for the quantitative analysis, which, was based in a calibration curve built for this propose (Ribeiro et al 2007).…”

“…Afterwards, the cartridges were kept under vacuum aspiration, for 30 min, to dry out residual water. The elution process, was performed with 20 mL of CH 2 Cl 2 : CH 3 OH (50:50, v/v), nonetheless, since the extracts from the estuarine water samples had a dark and sticky appearance, a cleanup step based in anterior protocols (Quintana et al 2004;Beck et al 2005), was implemented for the current assays (Ribeiro et al 2007). Here, all extracts were quantitatively transferred to the 1 g Sep-Pak silica cartridges, acquired from Waters Corporation (Milford, MA, USA).…”

Fabric Phase Sorptive Extraction Explained

“…Classical sample preparation techniques such as liquid-liquid extraction (LLE) and solid phase extraction (SPE) are still among popular choices for analytical sample preparation [2][3][4]. However, these procedures are time consuming, laborious, multi-step, generally utilize large volume of toxic and hazardous organic solvents, and often involve lengthy and error-prone post-extraction steps such as solvent evaporation and sample reconstitution in a suitable solvent.…”