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.

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.

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.

Separation and detection of the isomeric equine conjugated estrogens, equilin sulfate and delta8,9-dehydroestrone sulfate, by liquid chromatography--electrospray-mass spectrometry using carbon-coated zirconia and porous graphitic carbon stationary phases.

Equilin-3-sulfate and delta8,9-dehydroestrone-3-sulfate are two isomers found in equine conjugated estrogens that differ in structure only by the position of a double bond in the steroid B-ring. These geometric isomers were not resolved on a C18 column during the analysis of conjugated estrogen drug products by LC-MS using acetonitrile-ammonium acetate buffer as the mobile phase. While no separations of these two isomers were observed on C18 or other alkyl-bonded silica based phases using a variety of mobile phase conditions, partial separations were achieved on phenyl bonded silica phases with a resolution of 1.5 on a diphenyl phase, and baseline separations were readily achieved on two carbonaceous phases with resolutions routinely exceeding three on graphitic carbon-coated zirconia (Zr-CARB) and resolutions as high as 19 on porous graphitic carbon (Hypercarb). An examination of a selected few conjugated estrogens in the complex drug substance by LC-MS on Hypercarb is presented.

[Equine estrogens vs. esterified estrogens in the climacteric and menopause. The controversy arrives in Mexico]. / Estrógenos equinos vs. estrógenos esterificados en el climaterio y la menopausia. La controversia llega a México.

It exists controversies about if the effects and benefits of the esterified estrogens could be similar to those informed for equines, because its chemical composition and bioavailability are different. Esterified estrogens has not delta 8,9 dehydroestrone, and its absorption and level of maximum plasmatic concentrations are reached very fast. In United States of America and another countries, esterified estrogens has been marketed and using for treatment of climacteric syndrome and prevention of postmenopausal osteoporosis, based on the pharmacopoiea of that country, but the Food and Drug administration (FDA) has not yet authorized up today, a generic version of conjugated estrogens. In Instituto Mexicano del Seguro Social (IMSS) and another institutions of health sector in Mexico, starting in year 2000, it has been used esterified estrogens for medical treatment of climacteric and menopausal conditions. For this reason, in this paper we revised the most recent information about pharmacology, chemical composition, clinical use and costs of the conjugated estrogens with the purpose to guide the decisions to purchase this kind of drugs in Mexican heath institutions.

Establishment of a reliable extraction method of estrone sulfate from bovine plasma for detection of the peripheral level during the regular estrous cycle by radioimmunoassay.

A simple extraction and assay technique of estrone sulfate in bovine blood was developed with the object of detecting the peripheral level of estrone sulfate in a normal estrous cycle or in early pregnancy. Estrone sulfate in bovine plasma was extracted with a small reversed phase cartridge. The steroid conjugate retained in the cartridge was eluted with 40% (v/v) methanol. Estrone sulfate was separately recovered from other steroids by the stepwise increase in methanol concentration in the elution solvent. The recoveries of estrone sulfate eluted with 40% methanol were more than 90%, irrespective of the applied plasma volume. The concentration measured by radioimmunoassay with the eluent of 40% methanol was consistent for plasma extraction volumes of 0.5-2.0 ml. The change of estrone sulfate in bovine peripheral plasma during the regular estrous cycle was determined with a small reversed phase cartridge for extraction and 40% methanol for elution. The change in estrone sulfate was found to be similar to the change of estrone and estradiol-17 beta. The concentration of estrone sulfate was not higher than that of both estrogens in cattle.

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.

Separation of estrogen conjugates by high pressure liquid chromatography.

High pressure liquid chromatography using a prepacked commercial strong anion exchanger column (mu Partisil 10 SAX, 25 cm x 4.6 mm) was used to separate a mixture of eight estrogen conjugates. Chromatographic conditions using a 0.01 M potassium phosphate or 0.1 M NaCl as solvent in the isocratic mode are described for the separation of estrone glucosiduronate, 17beta-estradiol-3-glucosiduronate, 17beta-estradiol-17-glucosiduronate, estriol-3-glucosiduronate, estriol-16alpha-glucosiduronate, estriol-17-glucosiduronate, estrone sulfate and 17beta-estradiol-3-sulfate. This system gives high resolution of the estrogen conjugates in small eluent volumes in less than 30 min. The advantages of this high pressure liquid chromatographic system over other methods of separation are discussed.