Investigation of free and conjugated estrogen fate and emission coefficients in three duck farms.

Concentration animal feeding operation (CAFO) is an important source of environmental estrogen. However, to the best of our knowledge, the data on estrogen discharge during duck breeding and growth is insufficient. This study used liquid chromatography with tandem mass spectrometry (LC/MS/MS) to analyze the free and conjugated estrogen concentrations in the surface water, outlet water, groundwater, and duck manure/soil mixture at three duck farms in Taiwan. Natural estrogen species included estrone (E1), 17ß-estradiol (E2), estriol (E3), estrone-3-sulfate (E1-3S), 17ß-estradiol-3-sulfate (E2-3S), estrone-3-glucuronide (E1-3G), and 17ß-estradiol-3-glucuronide (E2-3G), whereas synthetic estrogen included 17α-ethynylestradiol (EE2) and diethylstilbestrol (DES). This study showed that the total estrogen concentrations in the surface water and groundwater were 15.4 and 4.5 ng/L, respectively, which constituted 56% and 58%, respectively, conjugated estrogen. From the pond to the outlet water, the total estrogen concentration decreased by 3.9 ng/L (23% loss) in the duck farms. However, the estrogenic potency was slightly reduced from 0.91 to 0.88 E2 equivalent/L, showing a negligible decrease. From the pond to the outlet water, the field results showed that converting the conjugated estrogen into free estrogen in the duck farm-released water increased their environmental hazard. Primarily E1, with an average concentration of 0.9 ± 1.6 ng/g, was present in the duck manure. The estrogen excreted by the ducks in the pond (from surface water to outlet water) was estimated to be 0.18 kg/million head-year. Although the estrogen concentration in the duck farms was low, the environmental impact of CAFO should not be neglected.

Free and conjugated estrogens detections in drainage tiles and wells beneath fields receiving swine manure slurry.

Although livestock manure, such as from swine (Sus scrofa domestica), have high capacity to introduce endocrine-disrupting free estrogens into the environment, the frequency of estrogen detections from reconnaissance studies suggest that these compounds are ubiquitous in the environment, perhaps resulting from historic manure inputs (e.g. cattle grazing residues, undocumented historic manure applications) or uncontrolled natural sources. Compared to free estrogens, conjugates of estrogens are innocuous but have greater mobility in the environment. Estrogen conjugates can also hydrolyze to re-form the potent free estrogens. The objective of this study was to identify the transport of free and conjugated estrogens to subsurface tile drains and groundwater beneath fields treated with swine manure slurry. Three field treatments were established, two receiving swine lagoon manure slurry and one with none. Manure slurry was injected into soils at a shallow depth (∼8 cm) and water samples from tile drains and shallow wells were sampled periodically for three years. Glucuronide and sulfate conjugates of 17ß-estradiol (E2) and estrone (E1) were the only estrogen compounds detected in the tile drains (total detects = 31; 5% detection frequency; conc. range = 3.9-23.1 ng L-1), indicating the important role conjugates played in the mobility of estrogens. Free estrogens and estrogen conjugates were more frequently detected in the wells compared to the tile drains (total detects = 70; 11% detection frequency; conc. range = 4.0-1.6 × 103 ng L-1). No correlations were found between estrogen compound detections and dissolved or colloidal organic carbon (OC) fractions or other water quality parameters. Estrogenic compounds were detected beneath both manure treated and non-treated plots; furthermore, the total potential estrogenic equivalents (i.e. estrogenicity of hydrolyzed conjugates + free estrogens) were similar between treated and non-treated plots.

Hepatic steroid sulfatase critically determines estrogenic activities of conjugated equine estrogens in human cells in vitro and in mice.

Conjugated equine estrogens (CEEs), whose brand name is Premarin, are widely used as a hormone-replacement therapy (HRT) drug to manage postmenopausal symptoms in women. Extracted from pregnant mare urine, CEEs are composed of nearly a dozen estrogens existing in an inactive sulfated form. To determine whether the hepatic steroid sulfatase (STS) is a key contributor to the efficacy of CEEs in HRT, we performed estrogen-responsive element (ERE) reporter gene assay, real-time PCR, and UPLC-MS/MS to assess the STS-dependent and inflammation-responsive estrogenic activity of CEEs in HepG2 cells and human primary hepatocytes. Using liver-specific STS-expressing transgenic mice, we also evaluated the effect of STS on the estrogenic activity of CEEs in vivo We observed that CEEs induce activity of the ERE reporter gene in an STS-dependent manner and that genetic or pharmacological inhibition of STS attenuates CEE estrogenic activity. In hepatocytes, inflammation enhanced CEE estrogenic activity by inducing STS gene expression. The inflammation-responsive estrogenic activity of CEEs, in turn, attenuated inflammation through the anti-inflammatory activity of the active estrogens. In vivo, transgenic mice with liver-specific STS expression exhibited markedly increased sensitivity to CEE-induced estrogenic activity in the uterus resulting from increased levels of liver-derived and circulating estrogens. Our results reveal a critical role of hepatic STS in mediating the hormone-replacing activity of CEEs. We propose that caution needs to be applied when Premarin is used in patients with chronic inflammatory liver diseases because such patients may have heightened sensitivity to CEEs due to the inflammatory induction of STS activity.

Parent and conjugated estrogens and progestagens in surface water of the Santa Ana River: Determination, occurrence, and risk assessment.

The present study investigated the occurrence of 13 parent and conjugated estrogens and progestagens in surface water of the Santa Ana River. With the exception of the synthetic hormones 17α-ethynylestradiol and mestranol, other compounds were detected at least twice at 10 representative sites, with the ubiquitous estrone (E1) and 17ß-estradiol-3-sulfate as the dominant compounds quantified (0.24-6.37 ng/L and 0.49-9.25 ng/L, respectively). Sites near dairy farms exhibited high levels of conjugates, whereas those close to a sewage treatment plant (STP) effluent outlet displayed relatively high concentrations of E1. Principle component analysis coupled with multiple linear regression revealed dairy farms and the STP as the 2 significant contamination sources, accounting for 69.9% and 31.1% of the total hormone burden, respectively. Risk assessment results suggested E1 and 17ß-estradiol (E2) as the 2 hormones with the largest risks to aquatic organisms, and which combined, contributed >90% of the total estrogenicity. Most of the sites investigated showed that E1 and E2 posed a medium risk (0.1 < risk quotient < 1), whereas each induced a high risk (risk quotient >1) at sites severely impacted by the STP and dairy farms. These results suggest that river health would benefit from effective treatment of waste at the STP and dairy farms prior to discharge. Environ Toxicol Chem 2016;35:2657-2664. © 2016 SETAC.

Marketplace Analysis of Conjugated Estrogens: Determining the Consistently Present Steroidal Content with LC-MS.

Conjugated estrogens purified from pregnant mares urine has been used as estrogen hormone replacement therapy since 1942. Previously, methods were proposed to identify and quantify the components of this complex mixture but ultimately were withdrawn due to incomplete characterization of the product and difficulties in transferring the method between laboratories. The aim of the current study is to develop a LC method that can reliably detect multiple steroidal components in conjugated estrogen tablets and measure their relative amount. The method developed was optimized for UHPLC columns, and the elution profile was analyzed using high-resolution mass spectrometry. A total of 60 steroidal components were identified using their exact m/z, product ion spectra of known, and predicted conjugated estrogen structures. These components were consistently present in 23 lots of Premarin tablets spanning two production years. The ten conjugated estrogens identified in the USP monograph and other additional estrogens reported elsewhere are among the 60 steroidal components reported here. The LC-MS method was tested in different laboratories using multiple samples, and the obtained results were reproducible among laboratories.

Fate and transport of free and conjugated estrogens during soil passage.

Endocrine disrupting chemicals, such as the free estrogens 17ß-estradiol (E2), estrone (E1) and the conjugated estrogen estrone-sulfate (E1-3S) are found at low concentration levels in the environment. This is somehow contradictory to the strong sorption and high degradation potentials found in laboratory experiments. In particular, the fate and transport behavior of conjugated estrogens is poorly understood, and the importance of enzymes triggering the transformation pathways has received little attention. To address these deficiencies, the present research uses packed laboratory soil columns with pulse injections of free estrogens, either E2 or E1, or E1-3S, to provide sound evidence of the transformation pathways. It is further shown that (i) transport of free estrogens is subject to strong retardation and degradation, (ii) the transport of conjugated estrogens is less retarded and only to a minor degree affected by degradation, and (iii) arylsulfotransferase is the enzyme triggering the transformation reaction.

Occurrence of free estrogens, conjugated estrogens, and bisphenol A in fresh livestock excreta and their removal by composting in North China.

An efficient pretreatment and analytical method was developed to investigate the occurrence and fate of four free estrogens (estrone (E1), 17ß-estradiol (17ß-E2), estriol (E3), and 17α-ethinylestradiol (EE2)), four conjugated estrogens (estrone-3-sulfate sodium salt (E1-3S), 17ß-estradiol-3-sulfate sodium salt (E2-3S), estrone-3-glucuronide sodium salt (E1-3G), and 17ß-estradiol-3-glucuronide sodium salt (E2-3G)), and bisphenol A (BPA) in three livestock farms raising beef cattle, cows, sheep, swine, and chickens in Qi County, which is located in North China. The results demonstrated that one cow and one beef cattle excreted 956.25-1,270.41 and 244.38-319.99 µg/day of total (free and conjugated) estrogen, respectively, primarily through feces (greater than 91%), while swine excreted 260.09-289.99 µg/day of estrogens, primarily through urine (98-99%). The total estrogen excreted in sheep and broiler chicken feces was calculated to be 21.64-28.67 and 4.62-5.40 µg/day, respectively. It was determined that conjugated estrogens contributed to 21.1-21.9% of the total estrogen excreted in cow feces and more than 98% of the total estrogen excreted in swine urine. After composting, the concentration of total estrogen decreased by 18.7-59.6%; however, increased levels of BPA were measured. In treated compost samples, estrogens were detected at concentrations up to 74.0 ng/g, which indicates a potential risk of estrogens entering the surrounding environment.

Occurrence and removal of free estrogens, conjugated estrogens, and bisphenol A in manure treatment facilities in East China.

The occurrence of four free estrogens, four conjugated estrogens, and bisphenol A (BPA) was investigated in three cow farms, four swine farms, and five chicken farms. The daily total estrogen (free and conjugated) excretions of a cow were 145.23-179.27 µg/d mainly through feces (92%), while swine excreted 42.56-219.25 µg/d of estrogens mainly through urine (98-99%). Estrogen conjugates contributed 14.6-48.8% to the total estrogen excretions in cow feces and more than 98% in swine urine. A chicken excreted 0.66-12.78 µg/d of total estrogens through feces, among which 34.2-100% was contributed by conjugated estrogens. The total estrogen removal efficiencies of manure anaerobic digesters and composters were 14.7-21.8% and less than 70.1%, respectively. Estrogens (E1, 17ß-E2, E1-3S, and E2-3S) still existed in treated manure at concentrations up to 2695 ± 181 ng/L (anaerobic digestate) and at contents up to 80.8 ± 6.0 ng/g (compost). BPA was found in feces and compost samples at similar contents (nd-25 ng/g), and approximately 60-70% of BPA was removed in wastewater treatment facilities.

Measuring free, conjugated, and halogenated estrogens in secondary treated wastewater effluent.

Steroidal estrogens are potent endocrine-disrupting chemicals that enter natural waters through the discharge of treated and raw sewage. Because estrogens are detrimental to aquatic organisms at sub-nanogram per liter concentrations, many studies have measured so-called "free" estrogen concentrations in wastewater effluents, rivers, and lakes. Other forms of estrogens are also of potential concern because conjugated estrogens can be easily converted to potent free estrogens by bacteria in wastewater treatment plants and receiving waters and halogenated estrogens are likely produced during wastewater disinfection. However, to the best of our knowledge, no studies have concurrently characterized free, conjugated, and halogenated estrogens. We have developed a method that is capable of simultaneously quantifying free, conjugated, and halogenated estrogens in treated wastewater effluent, in which detection limits were 0.13-1.3 ng L(-1) (free), 0.11-1.0 ng L(-1) (conjugated), and 0.18-18 ng L(-1) (halogenated). An aqueous phase additive, ammonium fluoride, was used to increase the electrospray (negative mode) ionization efficiency of free and halogenated estrogens by factors of 20 and 2.6, respectively. The method was validated using treated effluent from the greater Boston metropolitan area, where conjugated and halogenated estrogens made up 60-70% of the steroidal estrogen load on a molar basis.

Detection, fate and transport of estrogen family hormones in soil.

Estrone (E1), 17ß-estradiol (E2), and estrone-sulfate (E1-3S) are released into the environment in significant amounts. They are known to adversely affect the endocrine systems of aquatic organisms. Although previous studies clearly demonstrate that free hormones sorb strongly to soil and degrade quickly, significant amounts of free and the more persistent conjugated estrogens can be still detected in various environmental media. To date, E1-3S has been considered as a metabolite that forms either during the animal hormone cycle or as a degradation product of precursor hormones like E2-3S. We performed small-scale laboratory column tests to investigate two major features: transport and degradation of E2, and formation of E1-3S and E1. To evaluate the influence of soil microbial activity, one portion of soil was autoclaved and the background solution treated with sodium azide. The results demonstrate that (i) E2 is degraded to E1 and E1-3S in non-autoclaved soil, and to E1 in autoclaved soil, (ii) the formation of E1-3S is biologically driven, and (iii) the transformation of E2 to E1 does not require biological interaction. An inverse modeling approach was used to quantify the transport parameters and degradation rate coefficients.

Occurrence and removal of free and conjugated estrogens in wastewater and sludge in five sewage treatment plants.

The occurrence and fate of free and conjugated estrogens were investigated in wastewater and sludge from five sewage treatment plants (STPs) in Guangdong Province, China. Estrone (E1) and 17ß-estradiol (E2) were found in all influent samples at concentrations of 69.3-280 ng L(-1) and 1.3-30 ng L(-1), respectively. The concentrations of conjugated estrogens were from ND (not detected) to 7.6 ng L(-1). High concentrations (27.6-235 ng g(-1)) of E1 were found in sludge of some STPs indicating that sorption was an important estrogen removal mechanism. According to the mass flux analyses for estrogens in STP-A, E2 was mainly removed in the anaerobic process and E1 removal was the combined efforts of biodegradation and sorption. Abnormally high concentrations of EE2 (42.6-246 ng L(-1)), detected with gas chromatography-mass spectrometry, were found in all influent samples of the STPs, therefore interlaboratory analysis with liquid chromatography-tandem mass spectrometry was conducted for confirmation, which detected no EE2 at all. In consideration of the rather lower estimated EE2 concentration than the measured value, it was speculated that the presence of interfering substances like tetracosanic acid in the matrix could lead to overestimation of EE2 concentration. Overall, the effluents still pose potential estrogenic effect to the downstream aquatic organisms.

Differentiating isobaric steroid hormone metabolites using multi-stage tandem mass spectrometry.

Steroid hormones and their metabolites are currently undergoing clinical trials as potential therapeutics for traumatic brain injury (TBI). To support this work, it is necessary to develop improved procedures for differentiating isobaric species in this compound class. Equilin sulfate (E-S), estrone sulfate (E1-S), 17α-dihydroequilin sulfate (ADHE-S), and 17ß-dihydroequilin sulfate (BDHE-S) are primary constituents in hormone replacement therapies, such as Premarin, which are among pharmaceuticals being investigated for TBI treatment. The latter three compounds are isomers and can be difficult to differentiate in trace analytical determinations. In this work, a systematic study of the fragmentation of ADHE-S, BDHE-S, E1-S, and E-S under different stages of higher order tandem mass spectrometry (MS(n)) and variation of collision energy, allowed optimization of conditions for distinguishing the isomeric structures. For epimeric variants (e.g., ADHE-S versus BDHE-S; α- versus ß-stereoisomerization in the C-17 position), differentiation was achieved at MS(4) and fragmentation was demonstrated through MS(5). Computational analysis was performed to further explore differences in the fragmentation pathways due to changes in stereochemistry.

Fate and transformation of an estrogen conjugate and its metabolites in agricultural soils.

In the environment, conjugated estrogens are nontoxic but may hydrolyze to their potent unconjugated, 'free' forms. Compared to free estrogens, conjugated estrogens would be more mobile in the environment because of their higher water solubility. To identify the fate of a conjugated estrogen in natural agricultural soils, batch experiments were conducted with a (14)C labeled prototype conjugate, 17ß-estradiol-3-glucuronide (E2-3G). Initially, aqueous dissipation was dominated by biological hydrolysis of E2-3G and its oxidized metabolite, estrone glucuronide (E1-3G), both of which were transformed into the free estrogens, 17ß-estradiol (E2) and estrone (E1), respectively. Following hydrolysis, hydrophobic sorption interactions of E2 and E1 dominated. Depending on soil organic matter contents, dissolved E2-3G persisted from 1-14 d, which was much longer than what others reported for free estrogens (generally <24 h). Biodegradation rate constants of E2-3G were smaller in the subsoil (0.01-0.02 h(-1)) compared to topsoil (0.2-0.4 h(-1)). Field observations supported our laboratory findings where significant concentrations (425 ng L(-1)) of intact E2-3G were detected in groundwater (6.5-8.1 m deep) near a swine (Sus scrofa domesticus) farm. This study provides evidence that conjugate estrogens may be a significant source of free estrogens to surface water and groundwater.

Simultaneous analysis of free and conjugated estrogens, sulfonamides, and tetracyclines in runoff water and soils using solid-phase extraction and liquid chromatography-tandem mass spectrometry.

The ability to monitor multiple analytes from various classes of compounds in a single analysis can increase throughput and reduce cost when compared to traditional methods of analyses. This method for analyzing free (parent estrogen) and conjugated estrogens (metabolites) along with sulfonamides and tetracyclines utilizes a high pH (10.4) mobile phase with an ammonium hydroxide buffer for both positive- and negative-mode electrospray ionization. A single-step sample preparation by solid-phase extraction (SPE) was used to isolate and concentrate all analytes simultaneously. The analytical method was developed and validated for recoveries at 3 concentration levels for water and soil and produced recoveries of 42-123% and 21-105% respectively. Method detection limits ranged from 0.3 to 1.0 ng/L for water samples and 0.01 to 0.1 ng/g for soils. The method quantification limit ranged from 0.9 to 3.3 ng/L for water samples and 0.06 to 0.7 ng/g for soils. The single-point standard addition calibration procedure was validated across a linear range of MQL to 100 ng/L with ≥82% accuracy against a matrix matched standard curve. Furthermore, sorption of tetracyclines onto glassware was investigated and minimized by 10% using nitric acid-rinsed glassware, while separation parameters were further optimized based on retention time and signal responses. This method has been used for the quantification of estrogens, tetracyclines, and sulfonamides in soil and runoff waters with multiple compounds detected simultaneously in a single analysis.

Bovine colostrum: determination of naturally occurring steroid hormones by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

The aim of this study was to collect further data about levels of endogenous hormones in foodstuffs derived from animal production. Because of expected higher concentrations of especially estrogens in colostrum compared to other foodstuffs, our investigation focused on this matrix. For evaluation of endogenous steroid hormones in separated colostrum (fat and defatted fraction) and colostrum powder, the relevant free and conjugated estrogens (estradiol-17ß, estradiol-17α, estrone, and estriol) androgens (androstendione, 19nor-androstendione, 19nor-testosterone-17ß, 19nor-testosterone-17α, testosterone-17ß, and testosterone-17α), and progesterone were determined by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Upmost determined concentrations were found in the fat fraction, with 25.56 and 7.59 µg/L for estrone and androstendione, respectively. In defatted milk and colostrum powder, conjugated estrogens dominated, whereas total (free and conjugated) estrone (5.51 µg/L; 15.0 µg/kg) exceeded estradiol-17α (2.66 µg/L; 7.5 µg/kg) and estradiol-17ß (2.28 µg/L; 3.3 µg/kg). Neither 19nor-steroids nor estriol were detected in colostrum fractions or processed colostrum.

A systematic investigation to optimize simultaneous extraction and liquid chromatography tandem mass spectrometry analysis of estrogens and their conjugated metabolites in milk.

In this study, the simultaneous extraction of estrone (E1), 17beta-estradiol (E2), estriol (E3), ethinylestradiol (EE2), and their glucuronated and sulfated metabolites in milk was optimized using solid-phase extraction (SPE). The aim of this research was to analyze estrogens and their conjugated metabolites by liquid chromatography with tandem mass spectrometry (LC-MS/MS) in a single run, without the need to perform enzymatic cleavage and derivatization. Two SPE cartridges in tandem were used, consisting of sorbents based on the hydrophilic-lipophilic balance and amine-functionalized packing materials. To monitor analyte loss at every step of the SPE procedure (14)C-labeled E2 was spiked into the milk sample and the radioactivity was monitored at all stages of the SPE. In addition, non-radiolabeled standards of estrogens and metabolites were used to optimize solvent systems for the SPE and LC-MS/MS. The optimized method described in this paper can achieve recoveries ranging from 72% to 117% for the free estrogens (E1, E2, E3, and EE2), and 62% to 112% for seven conjugated metabolites. The three doubly conjugated, highly polar metabolites included in this study gave lower recoveries (< or = 43%) due to poor retention in SPE. Finally, commercial milk samples were analyzed for the presence of estrogens and their conjugated metabolites. Estrone (concentration range: 23-67 ng/L) was found to be the major free estrogen present in all milk samples. Estradiol was consistently observed in milk, but the concentrations were below the limit of detection (LOD of 10 ng/L), and no estriol and ethinylestradiol were detected. Several conjugated estrogen metabolites were identified, 17beta-estradiol-3-glucuronide (71-289 ng/L), estrone-3-sulfate (60-240 ng/L), 17beta-estradiol-3,17beta-sulfate (< LOD to 30 ng/L), and estrone-3-glucuronide (< LOQ of 25 ng/L). This method proved efficient in the simultaneous analysis of estrogens and their metabolites in milk.

Deconjugation characteristics of natural estrogen conjugates by acid-catalyzed solvolysis and its application for wastewater samples.

Deconjugation reactions of natural estrogen conjugates were studied here by three different solutions of 1 M hydrochloric acid (HCl) in methanol. Estrogen sulfates could be easily deconjugated even at low temperature, while deconjugation conditions of estrogen glucuronides required a higher temperature and longer time. For 1 M HCl in methanol with 8% water, the deconjugation efficiencies of the three studied estrogen glucuronides were below 59.4% at 80 degrees C for 360 min, while the corresponding deconjugation efficiencies were above 80.6% for anhydrous HCl methanol at 80 degrees C for 210 min, which suggested trace water in the solution of 1 M HCl methanol retarded the deconjugation rates of estrogen glucuronides. On the other hand, their corresponding deconjugation rates increased with the addition of ethyl acetate, and their corresponding deconjugation efficiencies were above 86.7% at 80 degrees C for 120 min. As water is a highly polar solution, and the polarity of ethyl acetate is lower than that of methanol, this may suggest that a low polar substance would favor the reaction, while a highly polar solution would prohibit the reaction. All reactions were in pseudo first-order, and higher temperature increased the reaction rate. Finally, a GC-MS method for simultaneous analysis of free estrogens and estrogen conjugates in wastewater with acid-catalyzed solvolysis was developed, and satisfactory recovery efficiencies were obtained by spiking the standard target chemicals into the influent and effluent of one municipal wastewater treatment plant (WWTP), which demonstrated the feasibility of the developed method. Compared with enzymatic hydrolysis, the acid-catalyzed solvolysis method developed here to deconjugate estrogen conjugates is cost effective and time-saving, giving it a greater potential for use with environmental samples.

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.

Estrogens and their conjugates: Determination in water samples by solid-phase extraction and liquid chromatography-tandem mass spectrometry.

A sensitive method for simultaneously determining eleven free and conjugated steroid estrogens has been developed using liquid chromatography-(electrospray)triple quadrupole-mass spectrometry (LC-(ESI)MS-MS) in negative mode with application to environmental aqueous matrices. Two selected reaction monitoring (SRM) transitions per compound were used, one of which was used for quantification and the second one for confirmation. The procedure includes a solid-phase extraction with an Oasis HLB solid-phase cartridge. Recoveries in 500 mL of river water spiked at 50 ng/L for sulfate estrogens and 100 ng/L for the rest of the compounds were 46-87%, except for E2, which had lower values (32%). Recoveries in wastewater were higher than 49% and 30% for all the compounds, except E2-17A and E2-17G (lower than 26% and 28%, respectively), in effluent (250 mL) and influent (100 mL), respectively. Ion suppression is a well-known phenomenon when using ESI; thus its impact on method recovery made us consider this effect when quantifying our samples. Limits of detection varied from 2 to 30 ng/L in river water and 10 to 100 ng/L in sewage water. The method was used to determine the target compounds in the Ebro river water where none of the analytes were found. In effluent and influent water samples, EE2, E1-3S and E2-3S were determined at concentration levels ranging from 35 to 160 ng/L.

Determination of natural and synthetic estrogens and their conjugates in sewage sludge by pressurized liquid extraction and liquid chromatography-tandem mass spectrometry.

In this study we present a pressurized liquid extraction/liquid chromatography-tandem mass spectrometry (PLE/LC-MS-MS) method to determine a group of estrogens and conjugated estrogens in sewage sludge. Parameters that affect the extraction step such as extraction solvent, temperature, pressure, static extraction time, number of cycles, purge time and flush volume have been optimized. In the LC-MS-MS system, electrospray ionization and a triple quadrupole analyzer have been used, and the multiple reaction monitoring mode has enabled low levels of target analytes to be detected. All recoveries were higher than 81% except for estrone 3-glucoronide and estradiol 17-glucoronide which were not extracted and consequently, they were not considered in the present study. The repeatability and reproducibility between days expressed as %RSD (n=3), were lower than 6% and 9%, respectively. The method developed allowed the target analytes to be quantified at low levels of microg/kg. The limits of detection were lower than 26 microg/kg of dry weight (d.w.) of sewage sludge, except for 17 alpha-estradiol, 17beta-estradiol, 17 alpha-ethinylestradiol and estradiol 17-acetate whose values were between 150 and 175 microg/kg (d.w.). The method was applied to determine these compounds in sewage sludge from two domestic sewage treatment plants. Estrone 3-sulfate, estradiol 3-sulfate, diethylstilbestrol, estrone and estriol were determined in some samples and estriol showed the highest value (406 microg/kg d.w.).