Highly sensitive determination of endocrine disrupting chemicals in foodstuffs through magnetic solid-phase extraction followed by high-performance liquid chromatography-tandem mass spectrometry.

BACKGROUND: Endocrine disrupting chemicals (EDCs), proved to be potential carcinogenic threats to human health, have received great concerns in food field. It was essential to develop effective methods to detect EDCs in food samples. The present study proposed an efficient method to determine trace EDCs including estrone (E1), 17ß-estradiol (E2), estriol (E3) and bisphenol A (BPA) based on magnetic solid-phase extraction (MSPE) coupled high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in meat samples. RESULTS: Fe3 O4 @COF(TpBD)/TiO2 nanocomposites were synthesized via functionalization of magnetic covalent organic frameworks (COFs) with titanium dioxide (TiO2 ) nanoparticles, and used as absorbents of MSPE to enrich EDCs. The efficient EDCs enrichment relies on π-π stacking interaction, hydrogen bonding, and the interaction between titanium ions (IV, Ti4+ ) and hydroxyl groups in EDCs, which improves the selectivity and sensitivity. Under the optimized conditions, target EDCs were rapidly extracted through MSPE with 5 min. Combining Fe3 O4 @COF(TpBD)/TiO2 based MSPE and HPLC-MS/MS to determine EDCs, good linearities were observed with correlation coefficient (R2 ) ≥ 0.9989. The limits of detection (LODs) and limits of quantification (LOQs) were 0.13-0.41 µg kg-1 and 0.66-1.49 µg kg-1 , respectively. Moreover, the proposed method was successfully applied to real samples analysis. CONCLUSIONS: The established MSPE-HPLC-MS/MS method was successfully applied to determine EDCs in meat samples with rapidness, improved selectivity and sensitivity. It shows great prospects for EDCs detection in other complicated matrices. © 2020 Society of Chemical Industry.

Efficient extraction of estrogen receptor-active compounds from environmental surface water via a receptor-mimic adsorbent, a hydrophilic PEG-based molecularly imprinted polymer.

We report an efficient screening procedure for the selective detection of compounds that are actively bound to estrogen receptor (ER) from environmental water samples using a receptor-mimic adsorbent prepared by a molecularly imprinted polymer (MIP). To mimic the recognition ability of ER, we improved the typical MIP preparation procedure using a hydrophilic matrix with a polyethylene glycol (PEG)-based crosslinker and a hydrophobic monomer to imitate the hydrophobic pocket of ER. An optimized MIP prepared with methacrylic acid as an additional functional monomer and estriol (E3), an analogue of 17ß-estradiol (E2), exhibited highly selective adsorption for ER-active compounds such as E2 and E3, with significant suppression of non-specific hydrophobic adsorption. The prepared MIP was then applied to the screening of ER-active compounds in sewage samples. The fraction concentrated by the MIP was evaluated by in vitro bioassay using the yeast two-hybrid (Y2H) method and liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOFMS). Compared to an authentic adsorbent, styrene-divinylbenzene (SDB)-based resin, the fraction concentrated by the MIP had 120% ER activity in the Y2H assay, and only 25% peak volume was detected in LC-Q-TOFMS. Furthermore, a few ER-active compounds were identified only from the fraction concentrated by the MIP, although they could not be determined in the fraction concentrated by the SDB-based resin due to ion suppression along with high levels of hydrophobic compounds. These results indicated that the newly developed MIP effectively captured ER-active compounds and while allowing most non-ER-active compounds to pass through.

Countercurrent separation assisted identification of two mammalian steroid hormones in Vitex negundo.

Countercurrent separation (CCS) has been widely used for the separation of high abundance compounds. However, the identification of low abundance compounds, such as mammalian steroid hormones, from natural sources is still a challenging task. A mixture of 14 human steroid hormone reference compounds was prepared for the development of a CCS enrichment strategy. The TLC-based GUESS (Generally Useful Estimate of Solvent Systems) method along with partitioning experiments were implemented to develop a process for the enrichment of these low abundance compounds with CCS. The application of CCS to the steroid hormone enrichment of Vitex negundo extracts was demonstrated by the identification of progesterone and estriol. This method provides a CCS-driven strategy to mine plant sources for low abundance compounds.

Bioaugmentation Mitigates the Impact of Estrogen on Coliform-Grazing Protozoa in Slow Sand Filters.

Exposure to endocrine-disrupting chemicals (EDCs), such as estrogens, is a growing issue for human and animal health as they have been shown to cause reproductive and developmental abnormalities in wildlife and plants and have been linked to male infertility disorders in humans. Intensive farming and weather events, such as storms, flash flooding, and landslides, contribute estrogen to waterways used to supply drinking water. This paper explores the impact of estrogen exposure on the performance of slow sand filters (SSFs) used for water treatment. The feasibility and efficacy of SSF bioaugmentation with estrogen-degrading bacteria was also investigated, to determine whether removal of natural estrogens (estrone, estradiol, and estriol) and overall SSF performance for drinking water treatment could be improved. Strains for SSF augmentation were isolated from full-scale, municipal SSFs so as to optimize survival in the laboratory-scale SSFs used. Concentrations of the natural estrogens, determined by gas chromatography coupled with mass spectrometry (GC-MS), revealed augmented SSFs reduced the overall estrogenic potency of the supplied water by 25% on average and removed significantly more estrone and estradiol than nonaugmented filters. A negative correlation was found between coliform removal and estrogen concentration in nonaugmented filters. This was due to the toxic inhibition of protozoa, indicating that high estrogen concentrations can have functional implications for SSFs (such as impairing coliform removal). Consequently, we suggest that high estrogen concentrations could impact significantly on water quality production and, in particular, on pathogen removal in biological water filters.

Study and comparison of polydopamine and its derived carbon decorated nanoparticles in the magnetic solid-phase extraction of estrogens.

Surface functionalization enabled by bioinspired polydopamine (PDA) is recognized as a convenient route for fabrication of multifunctional nanoparticles. In the present work, magnetic nanoparticles with polymer (Fe3O4@PDA) and carbon shell (Fe3O4@C) were prepared by self-oxidation of dopamine, and carbonization of the PDA coating. The performance of the two magnetic sorbents in the extraction and determination of four estrogens, estrone (E1), estradiol (E2), estriol (E3) and diethylstilbestrol (DES) from water samples in the form of magnetic solid-phase extraction was investigated. Orthogonal array design was utilized to facilitate the optimization of the proposed sample preparation approach. The highest extraction capabilities of the two sorbents were achieved under different experimental conditions. Fe3O4@PDA was shown to be superior to Fe3O4@C in the enrichment of estrogens, suggesting stronger interactions were established between the PDA coating and the target compounds. The extraction and desorption operations were enabled more conveniently by magnetic separation and the extracts were analyzed by high-performance liquid chromatography coupled with ultraviolet and fluorescence detection. The limits of detection achieved in the proposed method were in the range of 0.072-0.15ng/mL for E1 and DES, and 0.0017-0.0062ng/mL for E2 and E3. Good precision (>0.9995) was obtained with the linearity ranging from 0.2 to 100ng/mL, and from 0.01 to 5ng/mL. The method developed was assessed by analysis of the estrogens in tap water, drain water and bottled mineral water samples.

Development of a novel magnetic molecularly imprinted polymer coating using porous zeolite imidazolate framework-8 coated magnetic iron oxide as carrier for automated solid phase microextraction of estrogens in fish and pork samples.

A high-performance magnetic molecularly imprinted polymer (MIP) coating using zeolite imidazolate framework-8 coated magnetic iron oxide (Fe3O4@ZIF-8) as a carrier was developed for simultaneous automated solid phase microextraction of four estrogens in 24 food samples. The coating material, abbreviated as MZMIP, was synthesized through time-efficient layer-by-layer assembling of ZIF-8 and MIP film on Fe3O4 particles. It was characterized and automatically coated on the surface of SPME fibers by electromagnetic bonding. The extraction performance, reusability, repeatability, and validity of the MZMIP-SPME system was evaluated for high-throughput analysis of estrone (E1), estradiol (E2), estriol (E3), and ethinylestradiol (EE2). Various factors affecting the quality of MZMIP coating were optimized. Compared with traditional magnetic MIP coating based on Fe3O4@SiO2 carrier, the MZMIP coating exhibited high extraction capacity and quick adsorption and desorption kinetics to E1, E2, E3, and EE2 owing to the larger amount of imprinting sites in MZMIP. Under optimum conditions, the proposed system requires only 25min for pretreatment of all 24 samples (62.5s per sample). The limits of detection and quantitation of the proposed automated system for analysis were found to range from 0.4 to 1.7 and 1.1 to 6.2ngg(-1), respectively. During analysis of spiked fish and pork, the new coating showed better recovery and selectivity compared with Fe3O4@SiO2@MIP (MMIP) and commercially available SPME. The results indicated that the MZMIP coating could be effectively employed for pretreatment of ultra-trace level of estrogens in food.

A protein from Pleurotus eryngii var. tuoliensis C.J. Mou with strong removal activity against the natural steroid hormone, estriol: purification, characterization, and identification as a laccase.

A protein with strong removal activity against the natural estrogen estriol was purified from a culture supernatant of Pleurotus eryngii var. tuoliensis C.J. Mou. The protein was characterized as a laccase and had a molecular mass of 60kDa on SDS-PAGE. The enzyme was most active at pH 7.0 and 50°C. The partial N-terminal amino acid sequence of the enzyme showed homology with laccases from mushrooms, such as Pleurotus ostreatus, Coriolus versicolor (current name: Trametes versicolor), Pycnoporus cinnabarinus, and P. eryngii. A recombinant yeast assay confirmed that laccase treatment was very efficient for removing the estrogenic activity of steroid estrogens. Our results suggest that the enzyme may be applicable as a potential factor for removing natural steroid hormones.

Self-assembly molecularly imprinted polymers of 17ß-estradiol on the surface of magnetic nanoparticles for selective separation and detection of estrogenic hormones in feeds.

This paper reports a surface molecular self-assembly strategy for molecular imprinting on magnetic nanoparticles for selective separation and detection of estrogens in feeds. First, γ-methacryloxypropyltrimethoxysilane (MEMO) was successfully assembled at the surface magnetic nanoparticles through simple free radical polymerization, and subsequently, the copolymerization was further assembled between methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) in the presence of templates 17ß-estradiol (E2). The synthesized magnetic molecularly imprinted polymers for E2 (E2-MMIPs) showed quick separation, large adsorption capacity, high selectivity and fast binding kinetics for E2. Meanwhile, a dispersive solid-phase extraction (DSPE) based on E2-MMIPs has been established for efficient separation and fast enrichment of estrogens from the feeds. The assay exhibited a linear range of 0.1-4 µM for E2 and estriol (E3) with the correlation coefficient above 0.9996 and 0.9994, respectively. Recoveries of E2 from three kinds of feeds spiked at different concentration levels ranged from 92.7% to 97.0% with RSD<4.7%, and recoveries of E3 ranged from 71.9% to 83.1% with RSD<4.9%, respectively. The method is simple and sensitive, and can be used as an alternative tool to effectively separate and enrich the trace of estrogens in agricultural products by HPLC-UV.

Fate and levels of steroid oestrogens and androgens in waste stabilisation ponds: quantification by liquid chromatography-tandem mass spectrometry.

The capacity for removing wastewater-borne endocrine disrupting chemicals (EDCs) was investigated for two wastewater treatment plants (WWTPs) incorporating waste stabilisation ponds (WSPs) as the principal treatment technology. Samples were analysed for a number of steroidal oestrogens and androgens using liquid chromatography-tandem mass spectrometry (LC/MS/MS). Removal efficiency for steroid androgens was high for both WWTPs (93-100%) but WSP treatment was observed to be less effective for removing steroid oestrogens, particularly oestriol.

Modeling the retention of neutral compounds in gradient elution RP-HPLC by means of polarity parameter models.

A three-parameter expression, which was already employed before for the prediction of the retention time in gradient mode in reversed-phase high-performance liquid chromatography (RP-HPLC) with satisfactory results, has been tested here under a variety of gradient patterns, using methanol and acetonitrile as the organic modifiers. A wide variety of compounds have been employed as test solutes, including some complex ones used as drugs, such as hydrocortisone and estriol. Simplifications of this expression have been made by considering two- and one-parameter expressions based on the p polarity parameter model, which was successfully employed before in isocratic mode to perform predictions of retention time. The advantages that this model gives in isocratic conditions, namely simplicity and less previous experimental work, have been applied with profit in its application to gradient mode. Good correlations between the experimental retention times and the predicted ones have been obtained with both equations in most cases.

Rapid determination of free and conjugated estrogen in different water matrices by liquid chromatography-tandem mass spectrometry.

This article describes the development of a short pre-treatment method that allows the simultaneous analysis of free estrogens (estrone, 17beta-estradiol, estriol and 17 alpha-ethynylestradiol) and their sulphate and glucuronide conjugated forms. For a range of matrices, from sewage effluent to river water, the developed methodology based on solid-phase extraction and fractionation technique with ultra-performance liquid chromatography system showed effective separation of the targeted estrogens. The detection limits of this method ranged from 0.2 to 0.8 ng L(-1) for river water. The recoveries for river water and sewage effluent varied from 63% to 127%. The problems of matrix effects and ion suppression or enhancement were allowed quantitatively for in the analysis using standard addition. The developed method was used successfully to detect estrogens and their conjugates in both raw and treated wastewater, and river water at a location in Japan. High concentrations of the free estrogens estrone, 17beta-estradiol and estriol were found in the influent (22.6, 77.2, 64.6 ng L(-1), respectively) but only E1 was still present at a high concentration in the effluent which was reflected in the downstream river concentration. Estrone-3-sulphate was detected up to 18.0 ng L(-1) in influent water sample and 1.1 ng L(-1) in downstream water. For the sulphate conjugates, removal efficiencies varied from 35 to 88%. Glucuronide conjugates were detected only once in the sewage influent.

Ozone oxidation of pharmaceuticals, endocrine disruptors and pesticides during drinking water treatment.

This study investigates the oxidation of pharmaceuticals, endocrine disrupting compounds and pesticides during ozonation applied in drinking water treatment. In the first step, second-order rate constants for the reactions of selected compounds with molecular ozone (k(O3)) were determined in bench-scale experiments at pH 8.10: caffeine (650+/-22M(-1)s(-1)), progesterone (601+/-9M(-1)s(-1)), medroxyprogesterone (558+/-9M(-1)s(-1)), norethindrone (2215+/-76M(-1)s(-1)) and levonorgestrel (1427+/-62M(-1)s(-1)). Compared to phenolic estrogens (estrone, 17beta-estradiol, estriol and 17alpha-ethinylestradiol), the selected progestogen endocrine disruptors reacted far slower with ozone. In the second part of the study, bench-scale experiments were conducted with surface waters spiked with 16 target compounds to assess their oxidative removal using ozone and determine if bench-scale results would accurately predict full-scale removal data. Overall, the data provided evidence that ozone is effective for removing trace organic contaminants from water with ozone doses typically applied in drinking water treatment. Ozonation removed over 80% of caffeine, pharmaceuticals and endocrine disruptors within the CT value of about 2 mg min L(-1). As expected, pesticides were found to be the most recalcitrant compounds to oxidize. Caffeine can be used as an indicator compound to gauge the efficacy of ozone treatment.

Sorptive removal of endocrine-disruptive compound (estriol, E3) from aqueous phase by batch and column studies: kinetic and mechanistic evaluation.

Endocrine disruptive compounds (EDC) are a wide variety of chemicals which typically exert effects, either directly or indirectly, through receptor-mediated processes. They mimic endogenous hormones by influencing the activities of hormone activities even at nanogram concentrations and reported to disrupt the vital systems (e.g., the endocrine system) in aquatic organisms. The EDC are present in aquatic water bodies and sediments mainly due to the release of human and animal excreted waste. Estriol (E3) removal by adsorption process was investigated in this study to evaluate the potential of activated charcoal as adsorbent. Agitated non-flow batch sorption studies showed good E3 removal efficiency. Sorption kinetic data illustrated good fit with pseudo-first-order rate equation. Experimental data confirmed to linear Langmuir's isotherm model. Neutral pH condition showed comparatively good sorption of E3. Adsorption capacity showed a consistent increasing trend with increase in the operating temperature [DeltaH degrees , -9.189 kJ/mol); DeltaS degrees , 0.492 J/mol K) suggesting exothermic nature of E3 sorption process. Free energy (DeltaG degrees ) increased from 2.51 to 2.97 kJ/mol with increase in temperature from 0 to 50 degrees C. Further, E3 spiked distilled water, untreated domestic sewage and treated domestic sewage were studied in fixed bed column to assesses the potential of sorption process as tertiary unit operation in the ETP system. Total E3 concentration was determined quantitatively by employing direct competitive enzymatic-immuno assay (EIA) procedure.

Oxidative removal of aqueous steroid estrogens by manganese oxides.

This study investigated the oxidative removal of steroid estrogens from water by synthetic manganese oxide (MnO2) and the factors influencing the reactions. Using 1 x 10(-5)M MnO2 at pH 4, estrone (E1), 17beta-estradiol (E2), estriol (E3) and 17alpha-ethinylestradiol (EE2), all at 4 x 10(-6)M, were rapidly removed within 220 min, indicating the effectiveness of MnO2 as an oxidizing agent towards estrogens. E2 removal increased with decreasing pH over the tested range of 4-8, due most likely to increased oxidizing power of MnO2 and a cleaner reactive surface in acidic solutions. Coexisting metal ions of 0.01 M (Cu(II), Zn(II), Fe(III) and Mn(II)) and Mn(II) released from MnO2 reduction competed with E2 for reactive sites leading to reduced E2 removal. Observed differential suppression on E2 removal may be related to different speciations of metals, as suggested by the MINTEQ calculations, and hence their different adsorptivities on MnO2. By suppressing the metal effect, humic acid substantially enhanced E2 removal. This was attributed to complexation of humic acid with metal ions. With 0.01 M ZnCl2 in solutions containing 1 mg l(-1) humic acid, the binding of humic acid for Zn(II) was determined at 251 mmol g(-1). An in vitro assay using human breast carcinoma MCF-7 cells indicated a near elimination of estrogenic activities without secondary risk of estrogen solutions treated with MnO2. Synthetic MnO2 is therefore a promising chemical agent under optimized conditions for estrogen removal from water. Metal chelators recalcitrant to MnO2 oxidation may be properly used to further enhance the MnO2 performance.

Comparison of natural estrogen removal efficiency in the conventional activated sludge process and the oxidation ditch process.

The presence of natural estrogens, 17beta-estradiol (E2), estrone (E1) and estriol (E3), as well as estrogenic activity in wastewater influents and secondary effluents were investigated in 20 full-scale wastewater treatment plants in Japan. In all of the influent samples, natural estrogens were detected at concentrations above the minimum limits of detection (0.5ng/L). The concentrations of natural estrogens detected in the effluent of oxidation ditch plants were generally lower than previously reported values. On the other hand, in the conventional activated sludge plants, increments of E1 during biological treatment were frequently observed although E2 and E3 were removed effectively in the process. The removal rates of natural estrogens or estrogenic activity show no observed statistical relationship with the solids retention time (SRT) and the hydraulic retention time (HRT). However, the plants with high SRT or HRT generally showed high and stable removal of both natural estrogens and estrogenic activity.

Development and application of a multi-target immunoaffinity column for the selective extraction of natural estrogens from pregnant women's urine samples by capillary electrophoresis.

In this paper, a methodology for the determination of three naturally occurring estrogens (estradiol, estrone and estriol) in pregnant women's urine has been described. The procedure included immunoaffinity column (IAC) extraction of 4 mL of urine sample and subsequent analysis of the extraction by micellar electrokinetic chromatography (MEKC). A multi-target polyclonal antibody that has high affinity to three estrogens was produced. Then the IAC was developed by coupling polyclonal antibody to CNBr-activated Sepharose 4B. The IAC showed high affinity for these estrogens. Recoveries of three estrogens from human serum matrix were greater than 92% with R.S.D. less than 4.5%. The final elute of urine sample was diluted with running buffer and then quantitated with MEKC. The experimental results demonstrated that IAC was a useful technique for extraction and concentration of estrogens from biological samples. Three estrogens levels in six pregnant women's urine were measured by both the present method and enzyme-linked immunoadsorbent assay (ELISA). The results of this method have been found to correlate well with those of ELISA.

The solids retention time-a suitable design parameter to evaluate the capacity of wastewater treatment plants to remove micropollutants.

Micropollutants as endocrine disrupting compounds (EDC) or pharmaceuticals are of increased interest in water pollution control. Wastewater treatment plant (WWTP) effluents are relevant point sources for residues of these compounds in the aquatic environment. The solids retention time (SRT) is one important parameter for the design of WWTPs, relating to growth rate of microorganisms and to effluent concentrations. If a specific substance is degraded in dependency on the SRT, a critical value for the sludge age can be determined. In WWTPs operating SRTs below this critical value, effluent concentrations in the range of influent concentrations or a distribution according to the adsorption equilibrium have to be expected, whereas in WWTPs operating at SRTs higher than the critical value degradation will occur. Critical SRTs were determined for different micropollutants, indicating that the design criteria based on the sludge age allows an estimation of emissions. Different treatment technologies as conventional activated sludge systems and a membrane bioreactor were considered and no significant differences in the treatment efficiency were detected when operated at comparable SRT. The results of the investigations lead to the conclusion that low effluent concentrations can be achieved in WWTPs operating SRTs higher than 10 days (referred to a temperature of 10 degrees C). This corresponds to the requirements for WWTPs situated in sensitive areas according to the urban wastewater directive of the European Community (91/271/EEC) in moderate climatic zones.

Effects of inorganic salts on solubilization of estriol in an aqueous solution of poly(ethylene oxide)/poly(propylene oxide)/poly(ethylene oxide) triblock copolymer.

The effects of inorganic salts on the solubilization of estriol was studied in an aqueous solution of a poly(ethylene oxide)/poly(propylene oxide)/poly(ethylene oxide) triblock copolymer (Pluronic L-64). The solubility of estriol increased with increasing Pluronic L-64 and salt concentrations. Furthermore, two clear transition points corresponding to the polymolecular micelle formation and a pronounced change in micellar structure of Pluronic L-64 were found on the solubility curves. The effectiveness of inorganic salts for increasing solubility of estriol followed approximately the lyotropic or Hofmeister series, except Li+.

The occurrence of estrone and estriol in Trichostrongylus colubriformis (Nematoda).

Mammalian sex steroids and cholesterol were isolated from the lipid extract of the zooparasitic nematode Trichostrongylus colubriformis. In addition to the previously identified sex steroids progesterone and testosterone, estrone and estriol were detected and isolated from the mixture. The steroids were analyzed by thin-layer, gas-liquid and high-performance liquid chromatography, and their structures confirmed by proton nuclear magnetic resonance and mass spectroscopy.

Studies on steroids. CCXIX. Separation and determination of 4-hydroxyoestriol monoglucuronides and monosulphates in biological fluids by high-performance liquid chromatography with electrochemical detection.

The separation and determination of 4-hydroxyoestriol monoglucuronides and monosulphates by high-performance liquid chromatography with electrochemical detection on a reversed-phase column has been carried out. The effects of the salt, composition and pH of the mobile phase on the resolution were investigated with a Develosil ODS-5 column. Each group of isomeric monoglucuronides and monosulphates of 4-hydroxyoestriol was efficiently resolved on this column when 0.5% sodium acetate-acetonitrile and 0.5% sodium acetate-tetrahydrofuran-acetonitrile were used as mobile phases, respectively. The use of the present method revealed that 4-hydroxyoestriol orally administered to the rat was excreted as 4-, 3-, 16-glucuronides and 4-sulphate in bile.