Quantitation of estrogens in ground water and swine lagoon samples using solid-phase extraction, pentafluorobenzyl/trimethylsilyl derivatizations and gas chromatography-negative ion chemical ionization tandem mass spectrometry

A method was developed for the confirmed identification and quantitation of 17beta-estradiol, estrone, 17alpha-ethynylestradiol and 16alpha-hydroxy-17beta-estradiol (estriol) in ground water and swine lagoon samples. Centrifuged and filtered samples were extracted using solid-phase extraction (SPE), and extracts were derivatized using pentafluorobenzy] bromide (PFBBR) and N-trimethylsilylimidazole (TMSI). Analysis was done using negative ion chemical ionization (NICI) gas chromatography-mass spectrometry-mass spectrometry (GC-MS-MS). Deuterated analogs of each of the estrogens were used as isotope dilution standards (IDS) and were added to the samples before extraction. A limit of quantitation of 1 ng/l in ground water was obtained using 500 ml of ground water sample, 1.0 ml of extract volume and the lowest calibration standard of 0.5 pg/microl. For a 25 ml swine lagoon sample, the limit of quantitation was 40 ng/l. The average recovery of the four estrogens spiked into 500 ml of distilled water and ground water samples (n = 16) at 2 ng/l was 103% (S.D. 14%). For 25 ml of swine lagoon samples spiked at 500, 1000 and 10,000 ng/l, the average recovery for the four estrogens was 103% (S.D. 15%). The method detection limits (MDLs) of the four estrogens spiked at 2 ng/l in a 500 ml of ground water sample ranged from 0.2 to 0.6 ng/l. In swine lagoon samples from three different types of swine operations, estrone was found at levels up to 25,000 ng/l, followed by estriol and estradiol up to levels at 10,000 and 3000 ng/l, respectively. It was found that pretreatment of swine lagoon samples with formaldehyde was necessary to prevent conversion of estradiol to estrone.     

表面活性剂存在下雌激素的直接电化学分析

雌二醇（E2）、雌三醇（E3）和雌酮（E1）是人体内3种重要的雌激素,它们在妇女的生殖生育方面发挥重要的作用。人体雌激素缺乏或过乘会导致诸多疾病。雌激素的检测方法已有不少报道,如气相色谱－质谱（GC－MS）、液相色谱（LC）以及化学发光免疫法等。E2,E1和E3的结构相似,且均具有疏水性及非电活性,用电化学直接测定十分困难,一般只能间接测定。本文报道了一种新的电化学分析方法。该方法对三种雌激素有十分灵敏的伏安响应,可用于微量测定。     

Bulk derivatization and direct injection of human cerebrospinal fluid for trace‐level quantification of endogenous estrogens using trap‐and‐elute liquid chromatography with tandem mass spectrometry

Although there are existing methods for determining estrogen in human bodily fluids including blood plasma and serum, very little information is available regarding estrogen levels in human cerebrospinal fluid (CSF), which is critical to assess in studies of neuroprotective functions and diffusion of neuroprotective estrogens across the blood–brain barrier. To address this problem, a liquid chromatography with tandem mass spectrometry method for the simultaneous quantification of four endogenous estrogens (estrone, 17α‐estradiol, 17β‐estradiol, and estriol) in human CSF was developed. An aliquot (300 μL) of human CSF was bulk derivatized using dansyl chloride in the sample and 10 μL was directly injected onto a restricted‐access media trap column for protein removal. No off‐line sample extraction or cleanup was needed. The limits of detection of estrone, 17α‐estradiol, 17β‐estradiol, and estriol were 17, 28, 13, and 30 pg/mL, respectively, which is in the parts‐per‐trillion regime. The method was then applied to human CSF collected from ischemic trauma patients. Endogenous estrogens were detected and quantified, demonstrating the effectiveness of this method.     

Determination of trace amounts of estriol and estradiol by adsorptive cathodic stripping voltammetry.

Estriol and estradiol are electroinactive in the potential range from -200 to -1000 mV versus a silver-silver chloride electrode at a mercury electrode. The conversion of these estrogens into electroactive nitro derivatives of estrogens, which are used for voltammetric determination, was studied. Such nitro derivatives give a well defined cathodic stripping wave at -600 mV in pH 10.5 borate buffer. Estriol and estradiol are determined in the ranges 1 x 10(-9)-1.5 x 10(-6) and 5 x 10(-9)-2 x 10(-6) mol dm-3, respectively, by differential-pulse adsorptive stripping voltammetry at a hanging mercury drop electrode. Some steroids, such as estrone, interfere because the three estrogens have almost the same molecular structure and have similar nitro derivatives, but progesterone does not interfere and is reduced at significantly more negative potentials than the nitrated estrogens. It can be determined simultaneously with estriol or estradiol. A method was developed for the assay of estriol in pharmaceutical preparations.     

Fully automated analysis of estrogens in environmental waters by in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry

A simple, rapid and sensitive method for the determination of five estrogens, estrone, 17beta-estradiol, estriol, ethynyl estradiol, and diethylstilbestrol, was developed using a fully automated method consisting of in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS). These estrogens were separated within 8 min by HPLC using an XDB-C8 column and 0.01% ammonia/acetonitrile (60/40, v/v) at a flow rate of 0.2 mL/min. Electrospray ionization conditions in the negative ion mode were optimized for MS/MS detection of the estrogens. The optimum in-tube SPME conditions were 20 draw/eject cycles of 40 microL of sample using a Supel-Q PLOT capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME LC/MS/MS method, good linearity of the calibration curve (r > or = 0.9996) was obtained in the concentration range from 10 to 200 pg/mL for all compounds examined. The limits of detection (S/N= 3) of the five estrogens examined ranged from 2.7 to 11.7 pg/mL. The in-tube SPME method showed 34-90-fold higher sensitivity than the direct injection method (5 microL injection). This method was applied successfully to the analysis of environmental water samples without any other pretreatment and interference peaks. Several surface water and wastewater samples were collected from the area around Asahi River, and estriol was detected at 35.7 pg/mL in the effluent of a sewage treatment plant. The recoveries of estrogens spiked into river waters were above 86%, except for estriol, and the relative standard deviations were below 0.9-8.8%.     

Rapid simultaneous assay of serum estrone, estradiol, and estriol in pregnant women using methyl ether acetate derivatives by capillary gas chromatography and electron-impact mass spectrometry

A simple gas chromatographic--mass spectrometric method capable of measuring estrone, estradiol, and estriol simultaneously with a sensitivity close to that of radioimmunoassay has been developed. The estrogens in serum were extracted with diethyl ether, and internal standards (3-O-C2H3-estrone, 3-O-C2H3-estradiol, and 3-O-C2H3-estriol) were added, followed by converting to methyl ether compounds with an extractive alkylation procedure. The methyl ethers were then acetylated. Analyses were performed using a SP-2250 capillary column gas chromatograph coupled with an electron-impact mass spectrometer. The estrogen methyl ether acetate derivatives were more stable chemically and gave less fragmentation upon electron impact than the conventional trimethylsilyl derivatives. The use of selected ion monitoring of molecular ions and that of the corresponding internal standards (M + 3) provides a sensitivity down to 10 pg for estrone and estradiol and to 200 pg for estriol. The time required for the preparation of multiple samples is within 4 hours.     

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.).     

Quantitative analysis of estrogens in human urine using gas chromatography/negative chemical ionisation mass spectrometry

A sensitive procedure for the determination of estrogens in urine has been developed, using enzymatic hydrolysis and ether extraction followed by capillary gas chromatography with negative chemical ionisation mass spectrometry (GC/NCI-MS) of the pentafluorobenzoyl derivatives. These derivatives were superior for GC/NCI-MS to trifluoroacetyl, heptafluorobutyl, pentadecafluorooctanoyl and perfluorotolyl derivatives, giving characteristic negative molecular ions as the base peak for each of the naturally occurring estrogens estrone, estradiol and estriol, and for the synthetic estrogen ethynylestradiol used in contraceptive pills. The method was also suitable for determination of some metabolites of estrone and estradiol. The specificity and sensitivity of the GC/NCI-MS method simplifies sample preparation. Recovery of spiked samples was in the range 84-101% for the natural estrogens based on additions to increase the concentration by 5 and 1 microg L(-1). The repeatability of the method was 1-8% for all estrogens.     

Stacking and simultaneous determination of estrogens in water samples by CE with electrochemical detection

A rapid and sensitive method based on transient ITP and field enhancement in CE with electrochemical detection at copper disk electrode was developed for the simultaneous separation and determination of three estrogens: estrone, 17β‐estradiol, and estriol. The effects of several important factors that influence the separation and detection were investigated. Under the optimum conditions, the estrogens could be separated in 0.06 mol/L sodium hydroxide solution within 14 min. With transient ITP by addition of 0.5% NaCl, a good linear response was obtained for three estrogens from 0.2 to 10 μmol/L, with correlation coefficients higher than 0.9993. The detection limits were 8.9 × 10^?8, 6.7 × 10^?8, and 1.1 × 10^?7 mol/L (S/N = 3) for estriol, 17β‐estradiol, and estrone, respectively. This method was successfully employed to analyze different water samples from waterworks, tap water, fishpond, and river samples with recoveries in the range of 90.8–108.9%, and RSDs < 4.69%. The satisfied results demonstrated that this method was of convenient preparation, high sensitivity, and good repeatability, which could be applied to the rapid determination of environmental water samples.     

Liquid chromatography-tandem mass spectrometry analysis of estrogenic compounds in coastal surface water of the Baltic Sea

An analytical method has been developed for the determination of five naturally occurring estrogens (estradiol, estriol, estrone, genistein, daidzein), one synthetic hormone (ethynylestradiol) and three xenoestrogens (4-nonylphenol (NP), 4-tert-octylphenol (4-tert-OP), bisphenol A (BPA)) in coastal marine waters. The procedure includes a solid-phase extraction of approx. fifty litres of water samples on the solid-phase copolymer Oasis HLB followed by a clean-up on silica. Twenty-five percent aliquots were used for the analytical determination of the analytes using high performance liquid chromatography coupled with electrospray-ionisation tandem mass spectrometry (HPLC-ESI-MS/MS). Calculated extraction recoveries between 52 (4-tert-octylphenol) and 91% (nonylphenol) were obtained for the method developed. Matrix interferences occurring during electrospray ionisation were quantified by spiking the extracts prior to the measurements. Method detection limits ranged from 0.02 (estrone) to 1 ng L(-1) (estriol). The method was applied to determine environmental estrogens in coastal waters of the Baltic Sea. The analyses showed the presence of five compounds at levels between 0.10 (estrone) and 17 ng L(-1) (ethynylestradiol).     

Simultaneous determination of four trace estrogens in feces,leachate, tap and groundwater using solid–liquid extraction/auto solid‐phase extraction and high‐performance liquid chromatography with fluorescence detection

A simple and selective high‐performance liquid chromatography method coupled with fluorescence detection was developed for the simultaneous measurement of trace levels of four estrogens (estrone, estradiol, estriol and 17α‐ethynyl estradiol) in environmental matrices. For feces samples, solid–liquid extraction was applied with a 1:1 v/v mixture of acetonitrile and ethyl acetate as the extraction solvent. For liquid samples (e.g., leachate and groundwater), hydrophobic/lipophilic balanced automated solid‐phase extraction disks were selected due to their high recoveries compared to conventional C₁₈ disks. Chromatographic separations were performed on a reversed‐phase C₁₈ column gradient‐eluted with a 45:55 v/v mixture of acetonitrile and water. The detection limits were down to 1.1 × 10^?2 (estrone), 4.11 × 10^?4 (estradiol), 5.2 × 10^?3 (estriol) and 7.18 × 10^?3 μg/L (17α‐ethynyl estradiol) at excitation/emission wavelengths of 288/310 nm, with recoveries in the range of 96.9 ± 3.2–105.4 ± 3.2% (n = 3). The method was successfully applied to determine estrogens in feces and water samples collected at livestock farms and a major river in Northeast China. We observed relatively high abundance and widespread distribution of all four estrogens in our sample collections, implying the urgency for a comprehensive and intricate investigation of estrogenic fate and contamination in our researched area.     

Column-switching reversed phase-hydrophilic interaction liquid chromatography/tandem mass spectrometry method for determination of free estrogens and their conjugates in river water

We report a column-switching liquid chromatography (LC) tandem mass spectrometry (MS/MS) method for highly sensitive determination of both free estrogens (estrone, estradiol, and estriol) and their conjugates (estrone-3-sulfate, estradiol-3-sulfate, estriol-3-sulfate, estrone-3-glucuronide, estradiol-3-glucuronide, estriol-16-glucuronide, and estriol-3-glucuronide) in river water. This technique combines reversed phase (RP) chromatographic separation of the dansyl chloride derivatized free estrogens and hydrophilic interaction liquid chromatographic (HILIC) separation of the estrogen conjugates with multiple reaction monitoring (MRM). Using this new method, sensitivity increases 100- to 1000-fold for free estrogens and 2- to 10-fold for estrogen conjugates over RPLC-MS/MS alone. Method detection limits (MDL) range from 0.038 to 6.9 ng L⁻¹ with accuracy of 68-105% and precision of 1.7-17%. We successfully used this method to analyze river water samples collected from the North Saskatchewan River at the same location and detected trace concentrations of estrone (0.042 ng L⁻¹) and estrone-3-sulfate (0.84 ng L⁻¹), demonstrating the application of this method for environmental analysis.     

Combined isolation and purification procedures prior to the high-performance liquid chromatographic-ion-trap tandem mass spectrometric determination of estrogens and their conjugates in river sediments

A fast and sensitive analytical procedure has been developed for the simultaneous separation and determination of alpha-estradiol, beta-estradiol, estriol, estrone and ethynylestradiol and their sulfate, glucuronide and acetate conjugates in river sediments. The procedure includes a microwave-assisted extraction (MAE) with aqueous methanol (25:75, v/v) at 100 degrees C in 10 min, a clean-up on Oasis WAX cartridge and a high-performance liquid chromatography-ion-trap tandem mass spectrometry (HPLC-IT-MS/MS) with electrospray ionization. The recovery for each compounds ranged from 83 to 107% and the repeatability represented as RSDs ranged from 4.9 to 9.6%. The limits of detection (LODs) were down to 1 ng g(-1). The analytical performance of the method was evaluated using determination of free and conjugated estrogens in river sediments.     

Occurrence and analysis of estrogens and progestogens in river sediments by liquid chromatography-electrospray-mass spectrometry

In this study, an analytical procedure for the determination in sediment of the most abundant and/or physiological active estrogens (estradiol, estriol, estrone, ethynyl estradiol, and diethylstilbestrol) and progestogens (progesterone, norethindrone. and levonorgestrel) is described. The procedure includes ultrasonic extraction of the lyophilized sediment, clean-up with octadecylsilica cartridges, and analysis by liquid chromatography-diode array detection-mass spectrometry (LC-DAD-MS). MS detection is performed with an electrospray interface in the positive ion mode for determination of the progestogens and in the negative ion mode for determination of the estrogens. The method was applied to the determination of the target compounds in river sediments from the area of Catalonia. Estrogens and progestogens were found at concentrations usually in the low ng g(-1) range. Estriol and norethindrone were the compounds most frequently found whereas maximum concentrations in all sediment samples were obtained for ethynyl estradiol (22.8 ng g(-1)) and estrone (11.9 ng g(-1)). Detection limits were in the range of 0.04-1.00 ng g(-1). Preliminary conjectures with regards to the environmental behavior and impact of estrogens and progestogens in rivers are made. To the authors' knowledge, this is the first work reporting a detailed method for the analysis of estrogens and progestogens in river sediments and data on the environmental occurrence of both groups of compounds.     

衍生-气相色谱-质谱法测定水中痕量雌激素

提出了气相色谱-质谱法测定水中雌二醇、雌酮、雌三醇、戊酸雌二醇、己烯雌酚、乙炔雌二醇、双酚A和壬基酚等8种雌激素含量的方法。样品经乙酸乙酯提取和HLB小柱净化后,所得净化液中的雌激素与三甲基硅基化剂(TMS)或七氟丁酸酐(HFBA)进行衍生化反应产物用正己烷定容。在气相色谱分离中用DB-5MS毛细管柱为固定相,在质谱分析中采用全扫描和选择离子监测模式。8种雌激素的峰面积与质量浓度在20.0～1 000μg.L-1范围内呈线性关系,检出限(3S/N)在0.1～0.5μg.L-1之间。方法用于水中雌激素的测定,回收率在71%～103%之间,相对标准偏差(n=6)在4.2%～15%之间。     

水中雌激素的毛细管气相色谱分析

水中天然(雌二醇,雌酮,雌三醇)与合成(炔诺酮,炔雌醇 )的环境雌激素经固相萃取柱浓缩净化,用二氯甲烷洗脱,经N,O 双(三甲基硅烷基)三氟乙酰胺(含 1%三甲基氯硅烷)衍生化后用气相色谱法进行测定.方法检出限在 8. 25~8. 99ng/L,相对标准偏差为 2. 1% ~8. 5%,加标回收率除炔雌醇较低外,其余均大于 80%.     

Determination of estrogens in river water by gas chromatography-negative-ion chemical-ionization mass spectrometry.

A method for the determination of estrogens (17alpha-estradiol, 17beta-estradiol, estrone, ethynyl estradiol, and estriol) as pentafluorobenzyl-trimethylsilyl (PFB-TMS) derivatives by gas chromatography-mass spectrometry (GC-MS) with negative-ion chemical-ionization (NICI) is described. The NICI of all the derivatives produced an intense [M-PFB]- ion as the base peak. The reagent gas (methane) flow-rate and the ion source temperature were determined to be 2.0 ml/min and 240 degrees C, respectively, for the optimized NICI-selected ion monitoring (SIM) conditions. The sensitivities of the PFB-TMS derivatives in the NICI mode were 8.0-130 times higher than those of the PFB-TMS derivatives in electron ionization (EI) mode, and 12-25 times higher than those of all the TMS derivatives in the EI mode. This method was applied to the analysis of estrogens in river water using a solid-phase extraction as the sample preparation. The recoveries of the target chemicals from a river-water sample spiked with standards at 2 ng/l level were 85.8-126.5% (RSD, 6.2-13.0%). The methodical detection limits ranged from 0.10 to 0.28 ng/l.     

高效液相色谱-串联质谱法测定海水中雌酮、雌二醇、雌三醇

建立了测定海水中雌酮、雌二醇和雌三醇的高效液相色谱-串联质谱的分析方法。样品的提取方法为固相萃取,流动相为乙腈和0.1%氨水,梯度洗脱,流速0.2 mL/min,运行时间10 min。质谱采用负离子扫描模式,定量的碎片离子分别是:雌酮:269.02/144.99;雌二醇:271.04/182.96;雌三醇:287.03/170.94。仪器检出限均为0.001 ng,方法检出限均为0.2 ng/L。回收率分别是78.0~110.0%,82.2~103.2%,76.4~95.1%。该方法适用于海水中雌激素类物质的检测。     

Improved method for analyzing estrogens in water by liquid chromatography-electrospray mass spectrometry

An improved LC-electrospray ionization MS method was established for four estrogens (17beta-estradiol (E2), estriol (E3), estrone (E1), and ethynyl estradiol (EE)) in environmental water. Almost complete separation of all estrogens was achieved on a phenyl column with methanol/water as the mobile phase. Quantification was achieved in the negative ionization mode using selected ion monitoring. The instrumental detection limits were 20-30 ng/l for the four analytes. In Milli-Q spiked water, the recoveries of the four estrogens were 72-81%, which was similar to those found for river water spiked with the corresponding deuterated estrogens. The detection limits for the four estrogens in river water were 0.1-0.2 ng/l. The method was used to detect residual estrogens in the Tonghui River, which receives water from a municipal sewage treatment plant in Beijing; E1 (1.1 ng/l) and E2 (0.2 ng/l) were detected.     

Rapid Detection of Five Estrogens Added Illegally to Dietary Supplements by Combining TLC with Raman Imaging Microscope

Estrogens added illegally to dietary supplements are hazardous to human health. Traditional detection and analysis methods have many limitations, and we have developed an assay that combines thin-layer chromatography with Raman imaging microscopy (TLC-RIM). The five estrogens (estrone, estradiol, estriol, ethinyl estradiol, and diethylstilbestrol) were initially separated by TLC, then detected by area scanning Raman imaging with a 532 nm laser under a microscope. Raman spectra were obtained for each estrogen, which were used for detecting estrogen illegally added to botanical dietary supplements. The LOD of each estrogen was 0.4, 1.0, 0.8, 0.2, and 0.2 mg/mL, respectively. The matrix in the real sample did not interfere with the detection of estrogens. The method was fast, sensitive, stable, specific, and reliable.