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.     

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。     

Determination of anabolic steroids by gas chromatography/negative-ion chemical ionization mass spectrometry and gas chromatography/negative-ion chemical ionization tandem mass spectrometry with heptafluorobutyric anhydride derivatization

A gas chromatography/mass spectrometry (GC/MS) method is described which uses negative ion chemical ionization (NCI) and tandem mass spectrometry (MS/MS) for the determination of eight anabolic steroids in human urine. Eight anabolic steroids were derivatized by heptafluorobutyric anhydride (HFBA), and were determined using GC/NCI-MS and GC/NCI-MS/MS. The linear correlation coefficients for calibration in NCI-MS/MS were in the range 0.9880-0.9988. This method of derivatization with HFBA for use with GC/NCI was useful in determinations of 19-norandrosterone, boldenone, 19-noretiocholanolone, 2-methylandrosterone, nandrolone, 1-methyleneandrosterone, 1-methylandrosterone, 4-dihydroboldenone and mesterolone. The detection limits of this procedure were 5-20 ppb at a signal-to-noise (S/N) ratio of 3.     

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.     

Determination of Estrogens by High Performance Liquid Chromatography with Pre-Label Derivatization with 2-(4-Carboxy)-5,6-Dimethylbenzimidazole (II): Shortening of Analysis Time and Application to Monitoring Estrogen in Serum From In-Vitro Fertilization Embryo Transfer (IVF-ET) Patients

Abstract

We report a sensitive high performance liquid chromatography (HPLC) method for determination of free and conjugated estrogens (estrone, estradiol and estriol) by a fluorescent pre-labeling regent, 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole, with modification of previous work. The modified method was also tried, in preliminary work, for diagnosis of the in-vitro fertilization embryo transfer (IVF-ET) process. The reagent volumes were changed to one-tenth, derivatization conditions were changed to mild conditions at 40 C, and a solid-phase extraction process by SEP-PAK could be omitted after restudy of reaction conditions. As a result analysis time could be shorted within 40 min. The proposed HPLC method was applied to monitoring of free and conjugated estrogens in the patients who attend in-vitro fertilization embryo transfer (IVF-ET). The subsequent increase of free and conjugated estrone, estradiol and estriol was observed with the progress of follicle growth following ovulation stage in the IVF-ET process. We tried to plot estrogens for assist of clinical diagnosis of IVF-ET. The free estrone: 200-600 pg/ml, estradiol: 200-600 pg/ml and estriol: 100-300 pg/ml, conjugate estrone: 1000-5000 pg/ml, estradiol: 3000-8000 pg/ml and estriol: 2000-7000 pg/ml) in the patients without hormone disease were observed before human chorionic gonadotropin stimulation (hCG) on IVF-ET process. It was expected that free estrogen values, especially E1 and E3 could be use as validation products for diagnosis of hormone disease in IVF-ET process.     

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.     

Analysis of estrogens in river water and effluents using solid-phase extraction and gas chromatography-negative chemical ionisation mass spectrometry of the pentafluorobenzoyl derivatives

A procedure was developed for the analysis of estrogens in environmental water and effluents. Samples were extracted by passing through polymer-impregnated solid-phase extraction discs or C18 cartridges, followed by gas chromatography-negative chemical ionisation mass spectrometry of the pentafluorobenzoyl derivatives. The derivatives were stable and gave diagnostic negative molecular ions as the base peak for each of the major estrogens studied. The absolute recovery of estrogens spiked into clean groundwater using the disc procedure was 84-116% at the 10 ng l(-1) level (calculation not based on use of internal standards). Using doubly deuterated estradiol as internal standard added prior to extraction, the % relative standard deviation of estrogen extraction and analysis in spiked groundwater at the 10 ng(-1) level was 2.6-9.8%. Detection limits were 0.2 ng l(-1) or below for the major estrogens, based on a 2.5 litre sample. The most abundant estrogen was estrone, with concentrations over the range 6.4-29 ng l(-1) in effluents, and 0.2 to 17 ng l(-1) in water from the River Thames.     

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).     

Performance of gas chromatography/tandem mass spectrometry in the analysis of pyrethroid insecticides in environmental and food samples

The performance of gas chromatography coupled with tandem mass spectrometry (GC/MS/MS) was tested for the simultaneous determination of twelve pyrethroid insecticides. First, a comparison of two different ionization modes, electron ionization (EI) and negative chemical ionization (NCI), was carried out using MS and MS/MS. NCI-MS/MS provided the best results in terms of selectivity and sensitivity giving very low detection limits of 0.11 to 450 fg injected. The reliability of the method was confirmed through the evaluation of quality parameters such as accuracy (70-100%), and repeatability and reproducibility, with coefficients of variation below 15% and 10%, respectively. The applicability of the GC/MS/MS method to real samples and influence of matrix effects were evaluated through the analysis of spiked water, sediment and milk at 0.25 ng L(-1) , 5 ng g(-1) dry weight (dw) and 25 ng g(-1) (dw), respectively, of each pyrethroid insecticide considered. Using GC/NCI-MS/MS, matrix spectral interferences were minimized providing method limits of detection (MLODs) of 0.05-2.59 ng L(-1) , 0.10-87.7 pg g(-1) dw, 2.29-1071 pg g(-1) lipid weight (lw) for water, sediment and milk, respectively. To the best of our knowledge, the MLOD values found in our study were better than those reported in previous studies; in particular for sediment and food samples, they were one order of magnitude lower.     

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.     

高效液相色谱-串联质谱法检测生物样品中6种雌激素

建立了雌酮、雌二醇、雌三醇、己烯雌酚、己烷雌酚和炔雌醇6种雌激素在生物体中的HPLC-MS/MS分析方法.采用加速溶剂萃取、固相萃取技术进行提取、富集及净化,有效降低了基质的干扰.以甲醇-0.1％氨水溶液为流动相,以C18色谱柱进行分离,质谱采用电喷雾负离子扫描模式,6种雌激素的回收率为88％～104％,相对标准偏差在1.3％～8.3％之间.雌酮、雌二醇、雌三醇在生物体中的方法检出限0.35ng/g;己烯雌酚、己烷雌酚、17α-乙炔基雌二醇在生物体中的方法检出限为0.13ng/g.方法适用于生物体内雌激素的分析和检测.     

Residue determination of captan and folpet in vegetable samples by gas chromatography/negative chemical ionization-mass spectrometry

A gas chromatography/negative chemical ionization-mass spectrometry (GC/NCI-MS) method has been developed for the simultaneous determination of the fungicides captan and folpet in khaki (persimmon; flesh and peel) and cauliflower. Samples were extracted with acetone in the presence of 0.1 M zinc acetate solution in order to avoid degradation of fungicides and were purified using solid-phase extraction with divinylbenzene polymeric cartridges. Purified extracts were evaporated and dissolved in hexane prior to injection into the GC/NCI-MS system. Isotope-labeled captan and folpet were used as surrogate/internal standards, and quantification was performed using matrix-matched calibration. The method showed linear response in the concentration range tested (50-2500 ng/mL). The method was fully validated with untreated blank samples of khaki (flesh and peel) and cauliflower spiked at 0.05 and 0.5 mg/kg. Satisfactory recoveries between 82 and 106% and relative standard deviations lower than 11% in all cases (n = 5) were obtained. The limit of detection for both compounds were estimated to be 0.01 mg/kg. The developed method has been applied to treated and untreated samples collected from residue trials.     

高效液相色谱-串联质谱法测定饲料和卧床土中的环境雌激素

建立了饲料和卧床土中雌三醇、雌二醇、雌酮、双酚A和己烯雌酚5种环境雌激素的固相萃取结合高效液相色谱-串联质谱(HPLC-MS/MS)测定方法。对色谱流动相、质谱条件、固相萃取柱等影响因素进行了优化,得到的最优化条件为:样品经乙腈提取后,用固相萃取柱(NH₂-SPE)进行富集,采用Acquity UPLC^TM HSS T₃色谱柱分离,以乙腈-甲醇(4:1, v/v)与0.01%氨水为流动相,采用梯度洗脱,在负离子模式下进行MS/MS测定。在该优化条件下,5种环境雌激素的检出限(以信噪比为3计)为0.06~0.22 μg/kg,回收率为81.70%~102.20%,相对标准偏差小于10.00%。该方法用于饲料和卧床土中的5种环境雌激素残留量的测定具有简便、快速、灵敏的特点。     

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。     

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%.     

Monitoring of estrogens, pesticides and bisphenol A in natural waters and drinking water treatment plants by solid-phase extraction-liquid chromatography-mass spectrometry

A multi-residue analytical method has been developed for the determination of various classes of selected endocrine disruptors. This method allows the simultaneous extraction and quantification of different estrogens (estradiol, estrone, estriol, estradiol-17-glucuronide, estradiol diacetate, estrone-3-sulfate, ethynyl estradiol and diethylstilbestrol), pesticides (atrazine, simazine, desethylatrazine, isoproturon and diuron), and bisphenol A in natural waters. In the method developed, 500 ml of water are preconcentrated on LiChrolut RP-18 cartridges. Further analysis is carried out by liquid chromatography-mass spectrometry (LC-MS) using atmospheric pressure chemical ionisation (APCI) in the positive ion mode for determination of pesticides and electrospray in the negative ionisation mode for determination of estrogens and bisphenol A. Recoveries for most compounds were between 90 and 119%, except for bisphenol A (81%) and diethylstilbestrol (70%), with relative standard deviations below 20%. Limits of detection ranged between 2 and 15 ng/l. The method was used to study the occurrence of the selected pollutants in surface and groundwater used for abstraction of drinking water in a waterworks and to evaluate the removal efficiency of the different water treatments applied. Water samples from the river, the aquifer, and after each treatment stage (sand filtration, ozonation, activated carbon filtration and post-chlorination) were taken monthly from February to August of 2002. The presence in river water of atrazine, simazine, diuron and bisphenol A were relatively frequent at concentrations usually below 0.1 microg/l. Lower levels, below 0.02 microg/l, were usual for isoproturon. Estrone-3-sulfate and estrone were detected occasionally in the river. Most of the compounds were completely removed during the water treatment, especially after activated carbon filtration.     

Characterisation of antibodies and analytes by surface plasmon resonance for the optimisation of a competitive immunoassay based on energy transfer

The determination of binding constants using surface plasmon resonance (SPR) was introduced to optimise a competitive homogeneous fluorescence energy-transfer immunoassay (ETIA) before labelling. Steroids were chosen as model for the detection of three analytes estrone, estradiol and ethinylestradiol--by taking three polyclonal antibodies (anti estrone-, anti estradiol- and anti estrogen-antibodies) and the corresponding analyte derivatives used for the immunisation. The active concentration of the antibodies was determined before and after labelling. Inhibition curves were recorded using SPR for all possible combinations of analyte, antibody, and analyte derivatives. The experiments revealed that the active antibody concentration can be reduced to 30% whereas the antibody affinity is not affected by the labelling process. Limits of the use of SPR for determination of affinity constants in solution are discussed. All possible ETIA calibration for the quantification of estrone and estradiol was performed. The lower limits of detection for estrone (0.06 microg L(-1)) and estradiol (0.17 microg L(-1)) were reached with the anti-estrogen IgG and its derivative     

Voltammetric behavior and determination of estrogens at Nafion-modified glassy carbon electrode in the presence of cetyltrimethylammonium bromide

A method is described for the determination of estrogens (estradiol, estrone and estriol) by stripping voltammetry. These estrogens yield one well-defined oxidation peak at the Nafion-modified glassy carbon electrode in the presence of cetyltrimethylammonium bromide (CTAB). The peak current is proportional to the concentration of estradiol in the range from 2.5×10⁻⁸ to 1.5×10⁻⁶ mol/l, and the detection limit is 1×10⁻⁹ mol/l after accumulation of 6 min. The total amounts of estrogens in the blood serums were determined using the voltammetric method, and the average recovery was 106.04%. The mechanism of the oxidation of estradiol was investigated by electrochemical techniques and UV spectra.     

Determination of estrone and 17 beta-estradiol in human hair by gas chromatography-mass spectrometry

An efficient procedure is described for the determination of estrone and 17 beta-estradiol in hair by gas chromatography-mass spectrometry (GC-MS). The method involves alkyloxycarbonylation with isobutyl chloroformate (isoBCF) of phenolic hydroxy groups after alkaline digestion of hair samples. The resulting isobutyloxycarbonyl derivatives of estrone and 17 beta-estradiol are extracted with hexane and subjected to chlorodifluoroacetyl derivatization in order to protect the remaining alcoholic hydroxy groups. When GC-MS with selected ion monitoring (SIM) was used, the quantitative ions were at m/z 270 and 384 in the electron ionization mass spectra for estrone and 17 beta-estradiol, respectively. The detection limits for SIM of the steroids were 1 and 2 pg, respectively, and the SIM responses were linear with correlation coefficients varying from 0.991 to 0.994 in the concentration range 0.2-4.0 ng g-1 for the estrogens studied. The detection of estrone and 17 beta-estradiol in hair samples was possible in the concentration range of 0.24-1.30 ng g-1. The concentrations of the two estrogens detected were different in male and female hair samples.     

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.