Validation of screening method for residues of diethylstilbestrol, dienestrol, hexestrol, and zeranol in bovine urine using immunoaffinity chromatography and gas chromatography/mass spectrometry

A method was developed, using commercially available immunoaffinity chromatography cleanup cartridges, followed by detection by gas chromatography/mass spectrometry, to screen for residues of the hormone growth promotants diethylstilbestrol, dienestrol, hexestrol, and zeranol in bovine urine. The single-laboratory, in-house validation included assessment of recoveries, repeatability, linearity of response, detection capability, and specificity (cross-reactivity) with a suite of antibiotics and other hormonal growth promotants. The method was validated for screening at a target concentration of 2.0 microg/L in urine. The detection capabilities for the analytes were diethylstilbestrol, 0.24; dienestrol, 0.15; hexestrol, 0.84; and zeranol, 0.28 microg/L.     

Determination of estrogens in animal tissue by GC-MS with negative ion chemical ionization

GC-MS with negative ion chemical ionization has been used to detect anabolic estrogens (diethylstilbestrol, dienestrol, hexestrol, and 17β- and 17α-estradiol) in bovine muscle tissue. After hydrolysis and extraction, purification by column chromatography, and derivatization, the detection limit for the estrogens is better than 0.1 ppb.     

Drug residue analysis using immunoaffinity chromatography.

The background and applicability of immunoaffinity chromatographic separations and clean-up to drug residue analysis of agricultural commodities is discussed. The uses of antibody specificity for separation and concentration of drug residues are presented. Examples of immunoaffinity chromatography for the determination of residues of (1) nortestosterone and methyl testosterone in swine muscle, urine and bile; (2) chloramphenicol in swine tissue, eggs and milk; (3) clenbuterol in calf urine; (4) zeranol and beta-zearalanolin in calf urine: (5) diethylstilbestrol, dienestrol and hexestrol in calf urine are presented. Further, examples of the successful coupling of immunoaffinity separations with other chromatographic techniques such as gas chromatography and high-performance liquid chromatography are presented.     

Development and characterization of a nanodendritic silver-based solid-phase extraction sorbent for selective enrichment of endocrine-disrupting chemicals in water and milk samples

In this study, 4-[4-phenylazo-phenoxy] butyl-1-thiol (AzSH) functionalized nanodendritic silver (AzS@AgNDs) materials were prepared as a solid-phase extraction (SPE) sorbent for the selective extraction of estrogens. AzS@AgNDs possess an extremely large surface-to-volume ratio and a small average particle size. The performance of the material was evaluated by selective enrichment of hexestrol, diethylstilbestrol, dienestrol and bisphenol A in water and milk samples followed by rapid ultra-performance liquid chromatography–electrospray ionization mass spectrometry (UPLC–ESI–MS) analyses. The results exhibited that AzS@AgNDs had excellent adsorption capability for the targeted estrogens. The limits of detection of the four estrogens ranged from 0.1 to 5.0 pg/mL. The recoveries of the estrogens spiked into tap water were over the range of 83.6–105.3% with relative standard deviations of 2.8–6.0%. The results indicated the capability of this method for the rapid determination of estrogens in milk and other environmental water samples. In addition, this method would be useful for the determination of human exposure and health risk assessments trace level of endocrine-disrupting compounds (EDCs) in drinking water.     

Determination of zeranol and beta-zearalanol in calf urine by immunoaffinity extraction and gas chromatography-mass spectrometry after repeated administration of zeranol

A method for the determination of zeranol and its metabolite beta-zearalanol in bovine urine is described. It has been applied to samples from calves given multiple subcutaneous doses of zeranol. Samples were extracted with immunoaffinity columns containing antibodies raised against zeranol and were analysed by gas chromatography-mass spectrometry. The immunoaffinity columns were prepared by coupling immunoglobulin G fractions obtained from rabbit antisera with a Sepharose matrix. The immunizing agent was carboxybutylzeranol coupled to bovine serum albumin. Gas chromatography-mass spectrometry was performed in the negative-ion chemical ionization mode, after derivatization of the compounds to their pentafluorobenzyl ethers, and allowed detection of analytes with a sensitivity of 0.01 ppb in spiked urine. The derivatization method and the gas chromatographic determination were also applied to the similar compounds zearalanone, zearalenone and beta-zearalenol. A synthesis of dideuterated zeranol and beta-zearalanol by isotopic exchange is described. These deuterated analogues had an isotopic purity of more than 99% and were used for quantitation of zeranol and beta-zearalanol by isotope dilution mass spectrometry. The recoveries of zeranol and beta-zearalanol, using the immunoaffinity columns, were determined after extraction from spiked urine and were 84 and 64%, respectively. The urines of treated calves were collected for several days after treatments and were analysed after hydrolysis with beta-glucuronidase and arylsulphatase. The samples showed variable but generally decreasing concentrations of zeranol and beta-zearalanol. The levels of beta-zearalanol ranged from less than 0.01 to 98 ppb and were 1.2-3.2 times higher than those of zeranol.     

The use of liquid membranes in the multi-residue extraction of stilbenes in a variety of biological matrices and their detection with LC-ES-MS

Supported liquid membrane (SLM) has been used as a sample preparation method in the simultaneous extraction of a mixture of three stilbene compounds in cow's milk, urine, bovine kidney and liver tissues matrices. The stilbene compounds analysed included, dienestrol, diethylstilbestrol and hexestrol. The liquid membrane used for trapping these compounds consisted of 5% tri-n-octylphosphine oxide (TOPO) dissolved in di-n-hexylether/n-undecane (1:1). The extraction efficiencies obtained after enrichment of 1 ng/L stilbenes in variety of biological matrices of milk, urine, liver, kidney and water, ranged from 60 - 70%, 71 - 86%, 69 - 80%, 63 - 7A% and 72 - 93% respectively. The detection limits obtained from urine extraction were 2.1 ng/L, 1.3 ng/L and 3.0 ng/L; from liver and kidney tissues were 2.9 ng/L, 1.6 ng/L and 3.8 ng/L and from milk was 3.2 ng/L, 2.5 ng/L and 4.3 ng/L for hexestrol, dienestrol and diethylstilbestrol respectively.     

Stable derivatives for the gas chromatographic determination of synthetic anabolic stilbene residues (diethylstilbestrol, dienestrol and hexestrol) in meat and organs of treated cattle in the sub-parts per billion (10(9)) level

A method for the determination of diethylstilbestrol and the related compounds dienestrol and hexestrol residues in meat and organs of treated cattle is described. After extraction and clean-up, these synthetic estrogens are subjected to reaction with pentafluorobenzoyl chloride, which gives very stable perfluoro esters that are suitable for gas chromatographic determination using an electron-capture detector. With the careful clean-up and the very sensitive response of these derivatives, it is possible to reach a limit of detection in the sub-parts per billion (10(9)) range starting with only 5 g of sample.     

Trace analysis of diethylstilbestrol, dienestrol and hexestrol in environmental water by Nylon 6 nanofibers mat-based solid-phase extraction coupled with liquid chromatography-mass spectrometry

A simple, rapid and sensitive method for the determination of diethylstilbestrol (DES), dienestrol (DE) and hexestrol (HEX) was developed by using the Nylon 6 nanofibers mat-based solid-phase extraction (SPE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS). These estrogens were separated within 8 min by LC using an ODS column and methanol/water (80/20, v/v) at a flow rate of 1.0 mL min(-1). Electrospray ionization conditions in the negative ion mode were optimized for MS detection of the estrogens. Under the optimum SPE conditions, all target analytes in 50 mL environmental water samples can be completely extracted by 1.5 mg Nylon 6 nanofibers mat at flow rate of 3.0 mL min(-1) and easily eluted by passage of 500 μL mobile phase. By using the novel SPE-LC/MS method, good linearity of the calibration curve (r(2) ≥ 0.9992) was obtained in the concentration range from 0.10 ng L(-1) to 1.0 mg L(-1) (except for DE which was 0.20 ng L(-1) to 1.0 mg L(-1)) for all analytes examined. The limits of detection (S/N = 3) of the three estrogens ranged from 0.05 ng L(-1) to 0.10 ng L(-1). This method was applied successfully to the analysis of environmental water samples without any other pretreatment and interference peaks. Several water samples were collected from Jinchuan River and Xuanwu Lake, and in Jinchuan River water DES was detected at 0.13 ng L(-1). The recoveries of estrogens spiked into tap water were above 98.2%, and the relative standard deviations were below 4.78%.     

Determination of synthetic hormones in animal urine by high-performance liquid chromatography/mass spectrometry

A method was developed for the extraction of stanozolol, taleranol, zeranol, hexestrol, dienestrol, ethynylestradiol, diethylstilbestrol, and trenbolone from animal urine. The analytes were extracted from the matrix by enzymatic hydrolysis, solid-phase extraction, and liquid-liquid extraction. Detection and quantitation were performed on a high-performance liquid chromatography system coupled to a triple quadrupole mass spectrometer. The identification criteria met the European Union regulations. Validation of this method established a decision limit between 0.2 and 0.9 microg/L and a detection capability between 0.3 and 1.0 microg/L, depending on the analyte.     

Separation of structurally related estrogens using isocratic elution pressurized capillary electrochromatography

In this paper, the pressurized capillary electrochromatography (pCEC) with UV detection was utilized for the separation and determination of three structurally related estrogens, such as diethylstilbestrol (DES), hexestrol (HEX) and dienestrol (DE), which were difficult to be separated by capillary electrophoresis (CE) and HPLC due to their similarity in the structure and charge-to-mass ratios. Experiments were carried out in a commercially available pCEC instrument using a capillary column packed with 3 microm octadecyl silica (ODS). Surfactant sodium dodecyl sulfate (SDS) was introduced in the mobile phase to enhance the speed of analysis. The effective factors on the retention time and separation resolution, such as the applied voltage, supplementary pressure, the pH and the concentration of the buffer solution, the concentration of SDS, and the content of acetonitrile in the mobile phase, were evaluated. Based on the investigation, 31% (v/v) acetonitrile and 69% (v/v) of 10 mmol/L phosphate buffer (pH 6.5) containing 1.0 mmol/L SDS at an applied voltage of -12 kV and a supplementary pressure of 1000 psi were found to be the optimal conditions for pCEC to separate the three estrogens. The method also had been applied to the analysis of fish muscle samples spiked with estrogens.     

Dispersive liquid–liquid microextraction with solidification of floating organic drop combined with high performance liquid chromatography for the determination of phenolic oestrogens from human urine and water samples

A novel, simple and rapid method, termed dispersive liquid–liquid microextraction with solidification of floating organic drop coupled to high performance liquid chromatography, was developed for analysis of three phenolic oestrogens including diethylstilbestrol, dienestrol and hexestrol in human urine and water samples. The parameters of dispersive liquid–liquid microextraction with solidification of floating organic drop procedure including sample pH, type and volume of disperser solvent, and type and volume of extraction solvent were optimised. High performance liquid chromatography was applied for the phenolic oestrogens’ analysis. Under the optimum extraction and detection conditions, excellent analytical performances were attained. Good linear relationships (r ≥ 0.998) between peak area and concentration for diethylstilbestrol and dienestrol were optimised from 0.1 to 20 µg/mL, for hexestrol from 2 to 50 µg/mL. Method detection limits of 28.6–666.7 ng/mL were achieved. Satisfactory relative recoveries ranging from 72% to 122% were determined for urine, lake and tap water samples, with relative standard deviations (RSDs, n = 6) of 1.5–9.8%. The developed dispersive liquid–liquid microextraction with solidification of floating organic drop-high performance liquid chromatography method has a great potential in routine residual analysis of trace phenolic oestrogens in biological and water samples.     

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.     

基于溶胶凝胶技术的在线固相萃取与高效液相色谱联用测定饮用水中的雌激素残留

建立了固相萃取与高效液相色谱在线联用测定水样中3种雌激素(己烯雌酚、己烷雌酚、双烯雌酚)痕量残留的方法。以溶胶凝胶技术合成的聚合物为固相萃取材料,对水样中的雌激素进行萃取富集,考察了样品溶液不同pH、上样流速及洗脱溶剂等条件对合成材料富集效果的影响。结果表明,在优化的条件下,该方法对3种雌激素的检出限(S/N=3)为0.07~0.13 μg/L,样品中的加标回收率为82.31%~99.43%,相对标准偏差(RSD)为1.61%~7.15%。方法简便可靠,适用于饮用水中雌激素的痕量残留检测。     

气相色谱-质谱法同时检测动物组织中多种激素类兽药的残留量

建立了不同动物基质(肌肉、肝脏、肾脏)中己烷雌酚、己烯雌酚、己二烯雌酚、还原尿睾酮、表睾酮、雌酮、雌二醇、炔雌醇和雌三醇激素残留量的气相色谱-质谱联用(GC-MS)检测方法。以乙腈为提取溶剂,固相萃取柱净化,微波辅助衍生化,用双(三甲基硅烷基)三氟乙酰胺(BSTFA)与甲基氯硅烷(TMCS)(体积比为99 :1)的硅烷化试剂在吡啶存在下进行衍生化反应。实验结果表明,9种激素的检出限为0.1～1 μg/kg。3种动物基质中9种激素的平均回收率为68.8%～93.1%,实验室内相对标准偏差(RSD)为4.1%～22.3%,实验室间RSD为3.1%～17.9%。方法的技术指标满足动物组织中激素类兽药残留分析的要求。     

Magnetic molecularly imprinted polymers synthesized by surface‐initiated reversible addition‐fragmentation chain transfer polymerization for the enrichment and determination of synthetic estrogens in aqueous solution

Magnetic molecularly imprinted polymers have attracted significant interest because of their multifunctionality of selective recognition of target molecules and rapid magnetic response. In this contribution, magnetic molecularly imprinted polymers were synthesized via surface‐initiated reversible addition addition‐fragmentation chain transfer polymerization using diethylstilbestrol as the template for the enrichment of synthetic estrogens. The uniform imprinted surface layer and the magnetic property of the magnetic molecularly imprinted polymers favored a fast binding kinetics and rapid analysis of target molecules. The static and selective binding experiments demonstrated a desirable adsorption capacity and good selectivity of the magnetic molecularly imprinted polymers in comparison to magnetic non‐molecularly imprinted polymers. Accordingly, a corresponding analytical method was developed in which magnetic molecularly imprinted polymers were employed as magnetic solid‐phase extraction materials for the concentration and determination of four synthetic estrogens (diethylstilbestrol, hexestrol, dienestrol, and bisphenol A) in fish pond water. The recoveries of these synthetic estrogens in spiked fish pond water samples ranged from 61.2 to 99.1% with a relative standard deviation of lower than 6.3%. This study provides a versatile approach to prepare well‐defined magnetic molecularly imprinted polymers sorbents for the analysis of synthetic estrogens in water solution.     

Determination of estrogens in wastewater using three-phase hollow fiber-mediated liquid-phase microextraction followed by HPLC

Three-phase hollow fiber-mediated liquid-phase microextraction followed by HPLC was used for the determination of three synthetic estrogens, namely diethylstilbestrol, dienestrol, and hexestrol, in wastewater. Extraction conditions including organic solvent, volume ratio between donor solution and acceptor phase, extraction time, stirring rate, donor phase and acceptor phase were optimized. The target compounds were extracted from a 10 mL aqueous sample at pH 1.5 (donor solution) through a 45 mm in length hollow polypropylene fiber that was immersed in 1-octanol in advance, and then the hollow fiber was filled with 10 microL 0.5 mol/L sodium hydroxide solution (acceptor phase). After a 40 min extraction, the acceptor phase was directly injected into an HPLC system for detection. Under the optimized extraction conditions, a large enrichment factor (more than 300-fold) was achieved for the three estrogens. The determination limit at an S/N of 3 ranged from 0.25 to 0.5 microg/L for the estrogens. The recovery ratio was more than 86% in the determination of these estrogens in wastewater.     

On-line coupling of pressurized capillary electrochromatography with end-column amperometric detection for analysis of estrogens

An improved technique, pressurized capillary electrochromatography (pCEC) coupling with end-column amperometric detection (AD), was developed and used for the separation and determination of estrogens. The effects of pH value, composition of mobile phase, concentration of the surfactant sodium dodecyl sulfate (SDS) and applied voltage on separation were investigated. The electrochemical oxidation of diethylstilbestrol (DES), dienestrol (DE), and hexestrol (HEX) could be reliably monitored with a carbon electrode at 0.9 V (vs. Ag/AgCl). The pCEC analyses were performed on a capillary separation column packed with 3 microm C18 particles with an acetonitrile/water (31%: 69%) mobile phase containing Tris buffer (5 mmol/L, pH 4.5) and 4 mmol/L SDS. High voltage up to 12 kV reduced the retention time dramatically and still provided a baseline resolution. In addition, supplementary pressure prevented bubble formation and provided reliability and reproducibility of the pCEC performance. The detection limits for the three estrogens ranged from 1.2 to 2.2x10(-7) mol/L, about 10 20-fold lower than those obtained with pCEC-UV detection. To evaluate the feasibility and reliability of this system, the proposed pCEC-AD method was further demonstrated with fish muscle samples spiked with estrogens.     

气相色谱-质谱法测定动物组织中三种1,2-二苯乙烯类药物的残留量

建立了动物组织中己烯雌酚(DES)、己二烯雌酚(DIS)和己烷雌酚(HS)残留量的气相色谱-质谱分析方法。动物组织样品在碱性条件下用乙酸乙酯提取,经碳酸钠溶液液-液分配净化,再用硅胶柱固相萃取净化,洗脱液均分成两份后经氮气吹干,分别用双三甲基硅基三氟乙酰氨(BSTFA)和七氟丁酸酐(HFBA)衍生,采用选择离子监测模式(SIM)进行测定,外标法定量。检出限为0.30 μg/kg(cis-DES),0.10 μg/kg(trans-DES和HS)和0.15 μg/kg(DIS)。在0.5~4.0 μg/kg添加水平,回收率为73.0%～86.5%,相对标准偏差(RSD)为1.0%～7.2%。衍生物的峰面积与样品浓度在10～1000 μg/L(DES和DIS) 和5～500 μg/L(HS)范围内呈良好的线性关系,线性回归系数大于0.99。     

Building robust calibration models for the analysis of estrogens by gas chromatography with mass spectrometry detection

Five hormonal growth promotants (diethylstilbestrol, hexestrol, dienestrol, 17-β-estradiol and 17-α-ethynylestradiol) have been analysed by gas chromatography with mass spectrometry detection (GC/MS, SIM mode) for four non-consecutive days. The aim is to build models with stable predictions. The strategies applied are internal standardization and global models carried out by gathering signals recorded on several days. Two models were examined: univariate models (with standardized peak area) and PARAFAC2 (the analyte scores were standardized by the scores of the internal standard). Internal standardization has been proved to be efficient for both models of dienestrol and ethynylestradiol. The mean relative error in absolute value when samples recorded on a different day to the calibration set are quantified by PARAFAC2 is 7.00% and 7.11% for dienestrol and ethynylestradiol, respectively. For diethylstilbestrol and estradiol, internal standardization was combined with global calibration models built with signals recorded under several sources of variability (different days). Thus predictions become steadier over time and in the estradiol example, errors decrease from 33.10% to 9.76%. The mean relative error in absolute value with PARAFAC2 updated models oscillates between 6.34% for ethynylestradiol and 10.74% for diethylstilbestrol. For univariate updated models errors range from 6.42% to 14.19% for ethynylestradiol and estradiol respectively. The combination of both strategies has been proved to be efficient independently of the analyte and of the signal order.     

Solid-phase extraction using molecularly imprinted polymer for selective extraction of natural and synthetic estrogens from aqueous samples

A method is proposed for the clean-up and preconcentration of natural and synthetic estrogens from aqueous samples employing molecularly imprinted polymer (MIP) as selective sorbent for solid-phase extraction (SPE). The selectivity of the MIP was checked toward several selected natural and synthetic estrogens such as estrone (E1), 17β-estradiol (β-E2), 17α-estradiol (α-E2), estriol (E3), 17α-ethinylestradiol (EE2), dienestrol (DIES) and diethylstilbestrol (DES). Ultrahigh pressure liquid chromatography (UHPLC) coupled to a TSQ triple quadrupole mass spectrometry (QqQ) was used for analysis of target analytes. The chromatographic separation of the selected compounds was performed in less than 2 min under isocratic conditions. The method was applied to the analysis of estrogens in spiked river and tap water samples. High recoveries (>82%) for estrone, 17β-estradiol, 17α-estradiol, estriol and 17α-ethinylestradiol were obtained. Lower but still satisfactory recoveries (>48%) were achieved for dienestrol and diethylstilbestrol. The method was validated and found to be linear in the range 50-500 ng L(-1) with correlation coefficients (R(2)) greater than 0.995 and repeatability relative standard deviation (RSD) below 8% in all cases. For analysis of 100-mL sample, the method detection limits (LOD) ranged from 4.5 to 9.8 ng L(-1) and the limit of quantitation (LOQ) from 14.9 to 32.6 ng L(-1). To demonstrate the potential of the MIP obtained, a comparison with commercially available C(18) SPE was performed. Molecularly imprinted SPE showed higher recoveries than commercially available C(18) SPE for most of the compounds. These results showed the suitability of the MIP-SPE method for the selective extraction of a class of structurally related compounds such as natural and synthetic estrogens.