Trace determination of parabens in cosmetics and personal care products using fabric‐phase sorptive extraction and high‐performance liquid chromatography with UV detection

A simple, fast, and sensitive analytical protocol using fabric‐phase sorptive extraction followed by high performance liquid chromatography with ultraviolet detection has been developed and validated for the extraction of five parabens including methylparaben, ethylparaben, propylparaben, butylparaben, and benzylparaben. In the present work, sol‐gel polyethylene glycol coated fabric‐phase sorptive extraction membrane is used for the preconcentration of parabens (polar) from complex matrices. The use of fabric‐phase sorptive extraction membrane provides a high surface area which offers high sorbent loading, shortened equilibrium time, and overall decrease in the sample preparation time. Various factors affecting the performance of fabric‐phase sorptive extraction, including extraction time, eluting solvent, elution time, and pH of the sample matrix, were optimized. Separation was performed using a mobile phase consisting of water:acetonitrile (63:37; v/v) at an isocratic elution mode at a flow rate of 0.9 mL/min with wavelength at 254 nm. The calibration curves of the target analytes were prepared with good correlation coefficient values (r² > 0.9955). The limit of detection values range from 0.252 to 0.580 ng/mL. Finally, the method was successfully applied to various cosmetics and personal care product samples such as rose water, deodorant, hair serum, and cream with extraction recoveries ranged between 88 and 122% with relative standard deviation <5%.     

Development of a fabric phase sorptive extraction with high‐performance liquid chromatography and ultraviolet detection method for the analysis of alkyl phenols in environmental samples

A novel analytical method has been developed and validated for the quantification of alkyl phenols in aqueous and soil samples. Fabric phase sorptive extraction, a new sorptive microextraction technique, has been employed for the preconcentration of some endocrine‐disruptor alkylphenol molecules, namely, 4‐tert‐butylphenol, 4‐sec‐butylphenol, 4‐tert‐amylphenol, and 4‐cumylphenol, followed by high‐performance liquid chromatography with ultraviolet detection. Various parameters influencing the fabric phase sorptive extraction performance, namely, extraction time, eluting solvent, elution time and pH of the sample matrix, were optimized. The chromatographic separation was carried out with a mobile phase of acetonitrile/water (60:40 v/v) at an isocratic flow rate of 1.0 mL/min using a reversed‐phase C₁₈ column at λ_max 225 nm. The calibration curves of target analytes were prepared in the concentration range 5–500 ng/mL with good coefficient of determination values (R² > 0.992). Extraction efficiency values were 74.0, 75.6, 78.0, and 78.3 for 4‐tert‐butylphenol, 4‐sec‐butylphenol, 4‐tert‐amylphenol, and 4‐cumylphenol, respectively. The limits of detection range from 0.161 to 0.192 ng/mL. Subsequently, the new fabric phase sorptive extraction with high‐performance liquid chromatography and ultraviolet detection was successfully applied for the recovery of alkyl phenols from spiked ground water, river water, and treated water from a sewage treatment plant, and soil and sludge samples.     

Dispersive liquid–liquid microextraction of phenolic compounds from vegetable oils using a magnetic ionic liquid

A novel method was developed for the determination of two endocrine‐disrupting chemicals, bisphenol A and 4‐nonylphenol, in vegetable oil by dispersive liquid–liquid microextraction followed by ultra high performance liquid chromatography with tandem mass spectrometry. Using a magnetic liquid as the microextraction solvent, several key parameters were optimized, including the type and volume of the magnetic liquid, extraction time, amount of dispersant, and the type of reverse extractant. The detection limits for bisphenol A and 4‐nonylphenol were 0.1 and 0.06 μg/kg, respectively. The recoveries were 70.4–112.3%, and the relative standard deviations were less than 4.2%. The method is simple for the extraction of bisphenol A and 4‐nonylphenol from vegetable oil and suitable for routine analysis.     

Novel Extraction for Endocrine Disruptors in Atmospheric Particulate Matter

A rapid and efficient sample preparation method, which is called microwave-assisted microsolid phase extraction, was developed for the determination of endocrine disrupting chemicals in atmospheric particulate matter. The endocrine disrupting chemicals included bisphenol A, diethyl phthalate, dibutyl phthalate, and di(2-ethylhexyl) phthalate. The endocrine disrupting chemicals were isolated by microwave-assisted extraction following adsorption by copper(II) isonicotinate using microsolid phase extraction. The endocrine disrupting chemicals were subsequently determined by high performance liquid chromatography with an ultraviolet detector. The extraction was optimized for temperature, time, desorption time, and desorption solvent. Limits of detection (in the range of 2.0–8.5 nanograms per liter), limits of quantification (in the range of 6.6–28.0 nanograms per liter), and repeatability of the procedure (less than 10 percent) were established. Diethyl phthalate, diethyl phthalate, and di(2-ethylhexyl) phthalate were determined at values from 0.57 to 68.8 nanograms per cubic meter in atmospheric particulate matter collected from an urban area, a business center, and an industrial site in Dongguan, China. The concentration of bisphenol A was below the detection limit in these samples.     

Polypyrrole‐coated alginate/magnetite nanoparticles composite sorbent for the extraction of endocrine‐disrupting compounds

Magnetite nanoparticles incorporated into alginate beads and coated with a polypyrrole adsorbent were prepared (polypyrrole/Fe₃O₄/alginate bead) and used as an effective magnetic solid‐phase extraction sorbent for the extraction and enrichment of endocrine‐disrupting compounds (estriol, β‐estradiol and bisphenol A) in water samples. The determination of the extracted endocrine‐disrupting compounds was performed using high‐performance liquid chromatography with a fluorescence detector. The effect of various parameters on the extraction efficiency of endocrine disrupting compounds were investigated and optimized including the type and amount of sorbent, sample pH, extraction time, stirring speed, and desorption conditions. Under optimum conditions, the calibration curves were linear in the concentration range of 0.5–100.0 μg/L, and the limit of detection was 0.5 μg/L. The developed method showed a high extraction efficiency, the recoveries were in the range of 90.5 ± 4.1 to 98.2 ± 5.5%. The developed sorbent was easy to prepare, was cost‐effective, robust, and provided a good reproducibility (RSDs < 5%), and could be reused 16 times. The developed method was successfully applied for the determination of endocrine‐disrupting compounds in water samples.     

Fabric phase sorptive extraction/GC‐MS method for rapid determination of broad polarity spectrum multi‐class emerging pollutants in various aqueous samples

A rapid extraction and cleanup method using selective fabric phase sorptive extraction combined with gas chromatography and mass spectrometry has been developed and validated for the determination of broad polarity spectrum emerging pollutants, ethyl paraben, butyl paraben, diethyl phthalate, dibutyl phthalate, lidocaine, prilocaine, triclosan, and bisphenol A in various aqueous samples. Some important parameters of fabric phase sorptive extraction such as extraction time, matrix pH, stirring speed, type and volume of desorption solvent were investigated and optimized. Calibration curves were obtained in the concentration range 0.05–500 ng/mL. Under the optimum conditions, the limits of detection were in the range 0.009 –0.021 ng/mL. This method was validated by analyzing the compounds in spiked aqueous samples at different levels with recoveries of 93 to 99% and relative standard deviations of <6%. The developed method was applied for the determination of the emerging contaminants in tap water, municipal water, ground water, sewage water, and sludge water samples. The results demonstrate that fabric phase sorptive extraction has great potential in the preconcentration of trace analytes in complex matrix.     

Multi-residue analysis of free and conjugated hormones and endocrine disruptors in rat testis by QuEChERS-based extraction and LC-MS/MS

Endocrine disrupting compounds (EDCs) are suspected to be responsible for many disorders of the human reproductive system. To establish a causality relationship between exposure to endocrine disruptors and disease, experiments on animals must be performed with improved or new analytical tools. Therefore, a simple, rapid, and effective multi-residue method was developed for the determination of four steroid hormones (i.e., testosterone, androstenedione, estrone, and estradiol), glucuronide and sulfate conjugates of estrone and estradiol and four endocrine disruptors in rat testis (i.e., bisphenol A, atrazine, and active metabolites of methoxychlor and vinclozolin). The sample preparation procedure was based on the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) approach. An analytical method was then developed to quantify these compounds at ultra-trace levels by liquid chromatography coupled to tandem mass spectrometry. The QuEChERS extraction was optimized with regard to the acetonitrile/water ratio used in the extraction step, the choice of the cleanup method and the acetonitrile/hexane ratio used in the cleanup step. The optimized extraction method exhibited recoveries between 89% and 108% for all tested compounds except the conjugates (31% to 58%). The detection limits of all compounds were below 20 ng g⁻¹ of wet weight of testis. The method was subsequently applied to determine the levels of hormones and EDCs in seven rat testis samples.     

Evaluation of two membrane‐based microextraction techniques for the determination of endocrine disruptors in aqueous samples by HPLC with diode array detection

In this study, the viability of two membrane‐based microextraction techniques for the determination of endocrine disruptors by high‐performance liquid chromatography with diode array detection was evaluated: hollow fiber microporous membrane liquid–liquid extraction and hollow‐fiber‐supported dispersive liquid–liquid microextraction. The extraction efficiencies obtained for methylparaben, ethylparaben, bisphenol A, benzophenone, and 2‐ethylhexyl‐4‐methoxycinnamate from aqueous matrices obtained using both approaches were compared and showed that hollow fiber microporous membrane liquid–liquid extraction exhibited higher extraction efficiency for most of the compounds studied. Therefore, a detailed optimization of the extraction procedure was carried out with this technique. The optimization of the extraction conditions and liquid desorption were performed by univariate analysis. The optimal conditions for the method were supported liquid membrane with 1‐octanol for 10 s, sample pH 7, addition of 15% w/v of NaCl, extraction time of 30 min, and liquid desorption in 150 μL of acetonitrile/methanol (50:50 v/v) for 5 min. The linear correlation coefficients were higher than 0.9936. The limits of detection were 0.5–4.6 μg/L and the limits of quantification were 2–16 μg/L. The analyte relative recoveries were 67–116%, and the relative standard deviations were less than 15.5%.     

Quantification of bisphenol A and its selected analogs in serum using pre‐column derivatization with high‐performance liquid chromatography and tandem mass spectrometry

Due to regulation of the use of bisphenol A, several analogs serving as bisphenol A replacements have drawn substantial attention for their adverse health effects. To investigate their occurrence in humans and identify possible pollution sources, it is necessary to develop a sensitive method for total bisphenols detection. Thus, a method based on enzymolysis and liquid‐liquid extraction followed by molecularly imprinted polymer solid‐phase extraction and pre‐column derivatization with high‐performance liquid chromatography and tandem mass spectrometry was proposed. The developed method exhibited superior selectivity and sensitivity. The matrix effect can be eliminated to a great extent. The method detection limits for eight bisphenols were 0.05～0.19 ng/mL. Satisfactory recoveries (71～119%) were obtained by spiking bovine serum at three levels (0.8, 8 and, 20 ng/mL). The method was successfully applied to determine total bisphenols in the serum samples of children. Bisphenol A, bisphenol F, bisphenol S, bisphenol B and bisphenol F were detected with concentrations from below the method detection limit to 1.65, 0.45, 0.79, 2.04 and 0.17 ng/mL, respectively. These results indicate that bisphenol A remains the major pollutant among the studied bisphenols in children, whereas threats from bisphenol A analogs should also be monitored.     

Magnetic porous carbon derived from Co‐doped metal–organic frameworks for the magnetic solid‐phase extraction of endocrine disrupting chemicals

Metal–organic frameworks‐5 (MOF‐5) was explored as a template to prepare porous carbon due to its high surface area, large pore volume, and permanent nanoscale porosity. Magnetic porous carbon, Co@MOF‐5‐C, was fabricated by the one‐step direct carbonization of Co‐doped MOF‐5. After carbonization, the magnetic cobalt nanoparticles are well dispersed in the porous carbon matrix, and Co@MOF‐5‐C displays strong magnetism (with the saturation magnetization intensity of 70.17emu/g), high‐specific surface area, and large pore volume. To evaluate its extraction performance, the Co@MOF‐5‐C was applied as an adsorbent for the magnetic solid‐phase extraction of endocrine disrupting chemicals, followed by their analysis with high‐performance liquid chromatography. The developed method exhibits a good linear response in the range of 0.5–100 ng/mL for pond water and 1.0–100 ng/mL for juice samples. The limits of detection (S/N  = 3) for the analytes were in the range of 0.1–0.2 ng/mL.     

Simultaneous determination of fifteen multiclass organic pollutants in human saliva and serum by liquid chromatography–tandem ultraviolet/fluorescence detection: A validated method

A quick and inexpensive validated method, based on sample treatment by liquid–liquid microextraction followed by liquid chromatography (LC) coupled with ultraviolet tandem fluorescence detection is proposed for the determination of 15 multiclass pollutants both in serum and in saliva, as a simple and easy to draw matrix. The method was set up and validated according to European guidelines. The compounds of interest include some endocrine‐disrupting chemicals (i.e. bisphenol A, bisphenol B, bisphenol E, bisphenol F, bisphenol AF, bisphenol A diglycidyl ether, bisphenol M, diethylhexyl phthalate, monoethylhexyl phthalate, triclosan and 4‐nonylphenol), as well as other pollutants belonging to the class of volatile organic compounds (2‐chlorophenol, 1,2 dichlorobenzene, 1,2,4,5‐tetrachlorobenzene). The limits of quantifications ranged from 2.28 × 10^?3 μg mL^?1 (bisphenol A diglycidyl ether) to 6.29 μg mL^?1 (diethylhexyl phthalate), while those of detection ranged from 0.068 × 10^?3 μg mL^?1 (bisphenol A diglycidyl ether) to 1.031 μg mL^?1 (diethylhexyl phthalate). To test method suitability, it was applied to real saliva and serum samples of healthy human volunteers and was found to meet the demands of the laboratories handling simple and relatively inexpensive equipment for screening oriented at rapid and reliable contamination assessment of a population.     

Determination of a broad spectrum of endocrine‐disrupting pesticides in fish samples by UHPLC–MS/MS using the pass‐through cleanup approach

We describe a new methodology for the simultaneous determination of the endocrine‐disrupting herbicides (acetochlor, alachlor, amitrole, and atrazine), fungicides (carbendazim, triadimefon, penconazole, and propiconazole), and insecticides (carbaryl and carbofuran) in fish samples followed by high‐performance liquid chromatography with tandem mass spectrometry. Samples were extracted and purified using the pass‐through cleanup approach. The recoveries of the pesticides were in the range 71.8–116.5%, with relative standard deviations lower than 15.28%. Limits of quantitation were in the range of 0.03–2.50 μg/kg. Validation results on linearity, accuracy, and precision, as well as on application to the analysis of the endocrine‐disrupting pesticides in 20 fish samples, demonstrated the applicability to screen the presence of pesticides in fish.     

Analytical methods for the assessment of endocrine disrupting chemical exposure during human fetal and lactation stages: A review

In the present work, a review of the analytical methods developed in the last 15 years for the determination of endocrine disrupting chemicals (EDCs) in human samples related with children, including placenta, cord blood, amniotic fluid, maternal blood, maternal urine and breast milk, is proposed. Children are highly vulnerable to toxic chemicals in the environment. Among these environmental contaminants to which children are at risk of exposure are EDCs —substances able to alter the normal hormone function of wildlife and humans—. The work focuses mainly on sample preparation and instrumental techniques used for the detection and quantification of the analytes. The sample preparation techniques include, not only liquid–liquid extraction (LLE) and solid-phase extraction (SPE), but also modern microextraction techniques such as extraction with molecular imprinted polymers (MIPs), stir-bar sorptive extraction (SBSE), hollow-fiber liquid-phase microextraction (HF-LPME), dispersive liquid–liquid microextraction (DLLME), matrix solid phase dispersion (MSPD) or ultrasound-assisted extraction (UAE), which are becoming alternatives in the analysis of human samples. Most studies focus on minimizing the number of steps and using the lowest solvent amounts in the sample treatment. The usual instrumental techniques employed include liquid chromatography (LC), gas chromatography (GC) mainly coupled to tandem mass spectrometry. Multiresidue methods are being developed for the determination of several families of EDCs with one extraction step and limited sample preparation.     

Covalently bonded aptamer‐functionalised magnetic mesoporous carbon for high‐efficiency chloramphenicol detection

A novel aptamer‐modified magnetic mesoporous carbon was prepared to develop a specific and sensitive magnetic solid‐phase extraction method through combination with ultra‐high performance liquid chromatography‐tandem mass spectrometry for the analysis chloramphenicol in complex samples. More specifically, the chloramphenicol aptamer‐modified Mg/Al layered double hydroxide magnetic mesoporous carbon was employed as a novel magnetic solid‐phase extraction sorbent for analyte enrichment and sample clean‐up. The extraction solvent, extraction time, desorption solvent, and desorption time were investigated. It was found that the mesoporous structure and aptamer‐based affinity interactions resulted in acceptable selective recognition and a good chemical stability toward trace amounts of chloramphenicol. Upon combination with the ultra‐high performance liquid chromatography‐tandem mass spectrometry technique, a specific and sensitive recognition method was developed with a low limit of detection (0.94 pmol/L, S/N = 3) for chloramphenicol analysis. The developed method was successfully employed for the determination of chloramphenicol in complex serum, milk powders, fish and chicken samples, giving recoveries of 87.0‐107% with relative standard deviations of 3.1‐9.7%.     

Extraction and identification of matrine‐type alkaloids from Sophora moorcroftiana using double‐templated molecularly imprinted polymers with HPLC–MS/MS

Double‐templated molecularly imprinted polymers with specific recognition of three matrine‐type alkaloids were prepared using matrine and oxymatrine as the template molecules. An approach based on double‐templated molecularly imprinted solid‐phase extraction coupled with high‐performance liquid chromatography and tandem mass spectrometry was then developed to extract and purify matrine, oxymatrine, and sophocarpine from Sophora moorcroftiana in the Tibetan plateau herbs. The polymers were characterized by Fourier‐transform infrared spectroscopy and scanning electron microscopy. Their adsorption characteristics were evaluated using adsorption kinetics, isotherms, selectivity, and recycling experiments. This polymer exhibited excellent molecular recognition ability and good selectivity. The obtained polymers as adsorbent was further used for the determination of three matrine‐type alkaloids coupled to high‐performance liquid chromatography with tandem mass spectrometry, the recoveries of three matrines spiked at three concentration levels in samples were 73.25–98.42% (n = 5) with a relative standard deviation less than 6.82%. The limits of detection for the method were 9.23–15.42 μg/kg (S/N = 3). This proposed method was assessed to be an effective method for simultaneous extraction, isolation, and identification of matrine, oxymatrine, and sophocarpine from Sophora moorcroftiana.     

Development and validation of an LC‐MS/MS method for simultaneous quantitative analysis of free and conjugated bisphenol A in human urine

Bisphenol A (BPA) is an environmental endocrine‐disrupting chemicals that is widely used in common consumer products. There is an increasing concern regarding human exposure to BPA owing to the potential adverse effects associated with its estrogenic activity. For assessing environmental exposure to BPA, it is essential to have a sensitive, accurate and selective analytical method, especially one that can detect low BPA levels in complex sample matrices. In this study, we developed and validated an accurate, sensitive, and robust liquid chromatography–tandem mass spectrometry method for simultaneous quantification of free BPA and BPA β‐d ‐glucuronide (BPA‐gluc) concentrations in human urine with only a single injection. Calibration curves were linear over a concentration range of 1–100 ng/mL for BPA and 10–1000 ng/mL for BPA‐gluc. The levels of the analytes were determined quantitatively with HPLC/ESI‐MS/MS by using negative electrospray ionization in the select ion monitoring mode and a pentaflouraphenyl propyl column. The validated method was applied to the analysis of spot urine specimens collected from randomly selected healthy human subjects. Copyright © 2013 John Wiley & Sons, Ltd.     

Isotope dilution gas chromatography with mass spectrometry for the analysis of 4‐octyl phenol, 4‐nonylphenol,and bisphenol A in vegetable oils

By the combination of solid‐phase extraction as well as isotope dilution gas chromatography with mass spectrometry, a sensitive and reliable method for the determination of endocrine‐disrupting chemicals including bisphenol A, 4‐octylphenol, and 4‐nonylphenol in vegetable oils was established. The application of a silica/N‐(n‐propyl)ethylenediamine mixed solid‐phase extraction cartridge achieved relatively low matrix effects for bisphenol A, 4‐octylphenol, and 4‐nonylphenol in vegetable oils. Experiments were designed to evaluate the effects of derivatization, and the extraction parameters were optimized. The estimated limits of detection and quantification for bisphenol A, 4‐octylphenol, and 4‐nonylphenol were 0.83 and 2.5 μg/kg, respectively. In a spiked experiment in vegetable oils, the recovery of the added bisphenol A was 97.5–110.3%, recovery of the added 4‐octylphenol was 64.4–87.4%, and that of 4‐nonylphenol was 68.2–89.3%. This sensitive method was then applied to real vegetable oil samples from Zhejiang Province of China, and none of the target compounds were detected.     

Sol–gel‐graphene‐based fabric‐phase sorptive extraction for cow and human breast milk sample cleanup for screening bisphenol A and residual dental restorative material before analysis by HPLC with diode array detection

Fabric‐phase sorptive extraction has already been recognized as a simple and green alternative to the conventional sorbent‐based sorptive microextraction techniques, using hybrid organic–inorganic sorbent coatings chemically bonded to a flexible fabric surface. Herein, we have investigated the synergistic combination of the advanced material properties offered by sol–gel graphene sorbent and the simplicity of Fabric phase sorptive extraction approach in selectively extracting bisphenol A and residual monomers including bisphenol A glycerolatedimethacrylate, urethane dimethacrylate, and triethylene glycol dimethacrylate derived dental restorative materials from cow and human breast milk samples. Different coatings were evaluated. Final method development employed sol–gel graphene coated media. The main experimental parameters influencing extraction of the compounds, such as sorbent chemistry used, sample loading conditions, elution solvent, sorption stirring time, elution time, impact of protein precipitation, amount of sample, and matrix effect, were investigated and optimized. Absolute recovery values from standard solutions were 50% for bisphenol A, 78% for T triethylene glycol dimethacrylate, 110% for urethane dimethacrylate, and 103% for bisphenol A glycerolatedimethacrylate, while respective absolute recovery values from milk were 30, 52, 104, and 42%. Method validation was performed according to European Decision 657/2002/EC in terms of selectivity, sensitivity, linearity, accuracy, and precision.     

Salt‐assisted acetonitrile extraction and HPLC‐QTOF‐MS/MS detection for residues of multiple classes of pesticides in human serum samples

Detecting pesticide residues in human serum is a challenging process. In this study we developed and validated a method for the extraction and recovery of residues of multiple classes of pesticides from serum using one reagent. Salt‐assisted acetonitrile extraction and high‐performance liquid chromatography with quadrupole time of flight tandem mass spectrometry were used to quantitate 34 pesticides classified in nine groups of chemicals in human serum samples, which are frequently detected in food. The recoveries for 33 of analyzed pesticides ranged from 86 to 112% with relative standard deviations below 15%. The limits of quantitation and linearity of 31 of the pesticides were 1 µg/L and >0.990, respectively. The lower limit of quantitation has been reported in the literature particularly for multi‐classes pesticide mixtures in human serum. The salt–acetonitrile reagent was allowed to achieve good recoveries and detection limits, which could be attributed to salt altering the solvent polarity, preferentially collecting the organic phase in the solution, and promoting the extraction. The developed method was applied for two organophosphate pesticide metabolites, diethylphosphate and 3,5,6‐trichloro‐2‐pyridinol, in serum from rats that were fed a nonlethal quantity of chlorpyrifos. The concentrations of these two were 252.18 ± 15.47 and 0.63 ± 0.23 µg/L, respectively.     

Quantification of steroids and endocrine disrupting chemicals in rat ovaries by LC-MS/MS for reproductive toxicology assessment

Reproductive function is controlled by a finely tuned balance of androgens and estrogens. Environmental toxicants, notably endocrine disrupting chemicals (EDCs), appear to be involved in the disruption of hormonal balance in several studies. To further describe the effects of selected EDCs on steroid secretion in female rats, we aim to simultaneously investigate the EDC concentration and the sex hormone balance in the ovaries. Therefore, an effective method has been developed for the quantification of the sex steroid hormones (testosterone, androstenedione, estradiol, and estrone) and four endocrine disrupting chemicals (bisphenol A, atrazine, and the active metabolites of methoxychlor and vinclozolin) in rat ovaries. The sample preparation procedure is based on the so-called "quick, easy, cheap, effective, rugged, and safe" approach, and an analytical method was developed to quantify these compounds with low detection limits by liquid chromatography coupled with a tandem mass spectrometer. This analytical method, applied to rat ovary samples following subacute EDC exposure, revealed some new findings for toxicological evaluation. In particular, we showed that EDCs with the same described in vitro mechanisms of action have different effects on the gonadal steroid balance. These results highlight the need to develop an integrative evaluation with the simultaneous measurement of EDCs and numerous steroids for good risk assessment.