Ruthenium oxide nanoparticles immobilized over Citrus limetta waste derived carbon material for electrochemical detection of hexestrol.

Hexestrol is a non-steroidal estrogen which causes carcinogenic effects in animals. It is therefore important to develop sensitive and selective test methods for its early detection. Herein, we report the development of an electrochemical sensor to detect hexestrol in ultralow concentrations. In order to devise a simple and cost-effective hexestrol sensing electrode, attention is paid to the development of biomass-derived porous carbon (PCB) with large surface area and suitable porosity to immobilize ruthenium oxide nanoparticles (RuO2 NPs, 3-4 nm). The leftover Citrus limetta pulp is chosen as waste biomass since it has N and O based chemical species. Structural, morphological and compositional analysis of PCB and RuO2@PCB revealed well-dispersed RuO2 NPs over the PCB surface. High loading (5.27 at%) of Ru content is achieved due to the large surface area of PCB. Cyclic voltammetry, chronoamperometry and differential pulse voltammetry results suggest that the RuO2@PCB/ITO electrode is capable of detecting hexestrol concentration (in the range of 1 × 10-7-2 × 10-5 M). The practical application of hexestrol detection in milk samples demonstrates the recovery from 96.28 to 101%.

[Determination of four phenolic estrogens in water samples by pressure-assisted electrokinetic injection coupled with capillary electrophoresis].

A capillary electrophoresis (CE) method was developed for the simultaneous separation and determination of four phenolic estrogens (PEs), in which pressure-assisted electrokinetic injection (PAEKI) was adopted as the on-line concentration method to enrich the PEs. Several parameters affecting PAEKI conditions such as injection voltage and injection time, were systematically investigated and compared with the usual parameters. Under the optimized PAEKI conditions (-9 kV, 0.3 psi (ca. 2.1 kPa), 0.4 min), the four PEs separated completely within 7 min. Good linearities were attained in the range of 0.05-5 mg/L for hexestrol and dienestrol, and 0.1-10 mg/L for bisphenol A and diethylstilbestrol, with correlation coefficients (r) over 0.9936. The limits of detection (S/N=3) were 0.0071-0.017 mg/L, and enrichment factors ranged from 11 to 15 compared to normal hydrodynamic injection. The combined micellar electrokinetic chromatographic-PAEKI method developed was used to determine the PEs in tap water and lake water samples; limits of quantification (S/N=10) of 0.029-0.064 mg/L and 0.033-0.079 mg/L were attained, respectively, by the two sample types. Furthermore, recoveries ranged from 75.6% to 110.1% with relative standard deviations (n=5) within 4.6%-11.8%. To use this PAEKI method, researchers would only need to adjust the parameters of the CE apparatus to perform on-line enrichment of analytes, without using other reagents; this demonstrates the simplicity, rapidity, and highly automated nature of this method.

Hollow fiber membrane-coated functionalized polymeric ionic liquid capsules for direct analysis of estrogens in milk samples.

Protein removal process is always time-consuming for the analysis of milk samples. In this work, hollow fiber membrane-coated functionalized polymeric ionic liquid (HF-PIL) capsules were synthesized and used as solid-phase microextraction (SPME) sorbent for direct analysis of estrogens in milk samples. The functionalized PIL monolith sorbent was obtained by copolymerization between 1-(3-aminopropyl)-3-(4-vinylbenzyl)imidazolium 4-styrenesulfonate IL monomer and 1,6-di(3-vinylimidazolium) hexane bishexafluorophosphate IL-crosslinking agent. A group of four capsules were installed as SPME device, to determine four kinds of estrogens (estrone, diethylstilbestrol, hexestrol, and 17α-ethynylestradiol) in milk samples, coupled to high performance liquid chromatography. Extraction and desorption conditions were optimized to get satisfactory extraction efficiency. Good linearity was obtained in the range of 5-200 µg L(-1). The limits of detection were 1 µg L(-1) for diethylstilbestrol and 2 µg L(-1) for 17α-ethynylestradiol, estrone, and hexestrol. The present method was applied to analyze the model analytes in different milk samples. Relative recoveries were in the range of 85.5-112%. The HF-PIL SPME capsules showed satisfactory extraction efficiency and high resistance to sample matrix interference.

Packed-fiber solid-phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry for determination of diethylstilbestrol, hexestrol, and dienestrol residues in milk products.

A sensitive analytical method based on packed-fiber solid-phase extraction and high performance liquid chromatography-tandem mass spectrometry (PF SPE-HPLC-MS/MS) has been developed for determination of three synthetic stilbenes in milk. The stilbenes are extracted with acetonitrile, using sodium chloride, and purified with PF SPE using a cartridge containing electrospun polystyrene nanofibers. Parameters affecting the efficiency of PF SPE, such as pH and amount of salt, were optimized. Under optimal conditions, the limits of detection and quantification were 5-13pg/g and 15-37pg/g, respectively. Absolute recoveries varied between 60% and 85% at three different levels. The method was successfully applied for the determination of estrogenic stilbenes in a total of 69 milk samples. The method is sensitive and cost-effective in stilbene detection, and has potential in quality control of dairy products.

Dynamic liquid-liquid-solid microextraction based on molecularly imprinted polymer filaments on-line coupling to high performance liquid chromatography for direct analysis of estrogens in complex samples.

A novel sample preparation technique termed dynamic liquid-liquid-solid microextraction (DLLSME) was developed and on-line coupled to high performance liquid chromatography (HPLC) for direct extraction, desorption, and analysis of trace estrogens in complex samples. The DLLSME consists of the aqueous donor phase, the organic medium phase and the molecularly imprinted polymer filaments (MIPFs) as solid acceptor phase. The organic solvent with lesser density was directly added on top of the aqueous sample, and the dynamic extraction was performed by circulating the organic solvent through the MIPFs inserted into a PEEK tube which served as an extraction and desorption chamber. Afterwards, the extracted analytes on the MIPFs were on-line desorbed and then introduced into the HPLC for analysis. To evaluate the feasibility of the on-line system, a new DLLSME-HPLC method was developed for the analysis of five estrogens in aqueous samples by using 17ß-estradiol MIPFs as the solid phase. Under the optimized conditions, the enrichment factors of 51-70, limits of detection of 0.08-0.25 µg/L and precision within 4.5-6.9% were achieved. Furthermore, the proposed method was applied to the analysis of real samples including urine, milk and skin toner, satisfactory recovery (81.9-99.8%) and reproducibility (4.1-7.9%) were obtained. Especially, 0.59 µg/L of 17ß-estradiol was determined in female urine sample. The DLLSME offers an attractive alternative for direct analysis of trace analytes in aqueous samples and could potentially be extended to other adsorptive materials.

Preparation of a stir bar sorptive extraction coating based on molecularly imprinted polymer and its application in the extraction of dienestrol and hexestrol in complicated samples.

A new stir bar sorptive extraction (SBSE) coating based on molecularly imprinted polymer (MIP) with diethylstilbestrol as replaced template molecule was prepared. The influences of the contents of template molecule and monomer in the polymerization mixture on the extraction performance of MIP-SBSE were investigated thoroughly. The MIP was characterized by elemental analysis, scanning electron microscopy and infrared spectroscopy. In order to evaluate the usability of the new coating, the MIP-SBSE was combined with high performance liquid chromatography (HPLC) and diode array detector (DAD) with dienestrol (DS) and hexestrol (HS) as detected solutes. To achieve optimally selective extraction performance for DS and HS, several parameters, including extraction and desorption times, desorption solvent, ionic strength and pH value in sample matrix were investigated. The results showed that under the optimized experimental conditions, the present method has high selectivity and sensitivity. When drying-redissolving procedure was taken during sample preparation, the limits of detection for DS and HS were as low as 0.04 microg/L and 0.14 micorg/L, respectively. Good linearities were obtained for analytes with the correlation coefficients (R2) above 0.99. Finally, the proposed method was successfully applied to the determination of DS and HS in wastewater, honey and cow urine samples. The recoveries of spiked target compounds in real samples ranged from 61.3% to 120%. The developed method is simple, selective, sensitive and applicable for the analysis of trace DS and HS in complicated samples.

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%.

[Enzyme immunoassay for the determination of hexestrol in meat].

An indirect competitive enzyme-linked immunosorbent assay (ELISA) of hexestrol (HES), an antibiotic forbidden for use in livestock farming, has been developed. Conditions of ELISA have been optimized by varying the concentrations of the coating conjugate (HES-ovalbumin), anti-HES antiserum, casein, and Tween 20. In the absence of Tween 20 in the reaction mixture, the detection limit (IC10) equaled 0.01 ng/ml, IC50 equaled 0.17 ng/ml, and the working range (IC20-IC80) equaled 0.03-0.86 ng/ml, while, in the presence of 0.05% Tween 20, these values equaled 0.05 ng/ml, 2.9 ng/ml, and 0.26-32.0 ng/ml, respectively. Standard deviation of the analysis results did not exceed 5.4%. If ELISA was performed in the absence of detergents, the recovery value upon HES determination in spiked beef samples ranged from 74 to 147%.

[Determination of hormone multi-residues in animal tissues by gas chromatography-mass spectrometry].

A method of gas chromatography-mass spectrometry (GC-MS) for the simultaneous determination of nine sex hormone residues, such as hexestrol, diethylstilbestrol, dienestrol, etiocholan-3alpha-ol-17-one, epitestosterone, estrone, estradiol, ethinylestradiol and estriol, in animal tissues was developed. The sex hormones were extracted with acetonitrile, then cleaned-up with a C18 solid-phase extraction (SPE) column. The microwave-assisted derivatization of the target components with N,O-bis( trimethylsilyl) trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS) (99:1, v/v) using pyridine as solvent was performed, and then the derivatives were analyzed by GC-MS. The limits of detection were 0.1-1 microg/kg for all hormones, and the limits of quantification were 0.2-2 microg/kg. The average recoveries of sex hormones were 68.8%-93.1%. The relative standard deviations (RSDs) of inter- and intra-assay were 4.1%-22.3% and 3.1%-17.9%, respectively. The real sample tests showed this method can be used for the sensitive and accurate determination of multi-sex hormones residues in biological samples.

Comparison of time-resolved fluoroimmunoassay for determining hexoestrol residues in chicken muscle tissues based on polyclonal antibodies with liquid chromatography and tandem mass spectrometry.

A time-resolved fluoroimmunoassay (TR-FIA) was developed for the determination of hexoestrol (HES) residues in animal tissues. The limit of detection (LOD) was determined to be 0.02 ng g(-1) and the limit of quantification (LOQ) was less than 0.12 ng g(-1). The results obtained by the TR-FIA and ELISA showed a good correlation. The established TR-FIA was validated for the determination of market chicken muscle tissues and confirmed by high-performance liquid chromatography and tandem mass spectrometry (LC-MS-MS). This proposed technique could be applied to routine residue analysis.

[Determination of residues of three stilbene drugs in animal tissues using gas chromatography-mass spectrometry].

A method has been developed to determine residual stilbenes such as diethylstilbestrol (DES), dienestrol (DIS) and hexestrol (HS) in animal tissues using solid phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS). The procedures for extraction, cleanup on an LC-Si solid phase extraction cartridge and derivatization of stilbenes were established and optimized. The analytes were detected by mass spectrometer with electron impact source in selected ion monitoring mode (EI/SIM), and quantified with an external standard calibration curve method. Linear calibration curves were obtained in the concentration ranges from 5 to 500 microg/L for HS and from 10 to 1000 microg/L for DES and DIS (the correlation coefficients were above 0.99). Recoveries of the stilbenes were 73.0%-86.5%, and the relative standard deviations were between 1.0% and 7.2%. The limits of detection were 0.30 microg/kg for cis-DES, 0.10 microg/kg for trans-DES and HS and 0.15 microg/kg for DIS in pork and swine liver.

Determination of hexoestrol residues in animal tissues based on enzyme-linked immunosorbent assay and comparison with liquid chromatography-tandem mass spectrometry.

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of the hexoestrol (HES). Polyclonal rabbit antisera, raised against protein conjugate hexoestrol-mono-carboxyl-propyl-ethyl-bovine-serum-albumin (HES-MCPE-BSA), were utilized in immobilized antibody-based and competitive immunoassays. Assay conditions, including concentrations of antisera and Horseradish peroxidase (HRP)-HES were optimized. The effect of incubation time, surfactant concentration, ionic strength and pH of the medium were also investigated. The typical calibration curve gave an average IC50 value of 2.4 ng/ml, calibration range from 0.2 ng/ml to 30.5 ng/ml and a detection limit of 0.07 ng/ml. The specificity of the assay was tested against HES structurally related compounds, and the assay proved highly selective for HES. Assay performance was validated by using spiked pork and liver tissues samples. Moreover, it was compared with liquid chromatography-tandem mass spectrometry. The ion pair for quantification of HES was 269.4/134, and linear equation of HES was Y=0.2148X-0.0374 (r=0.9993). The two analytical methods can be applied to monitor HES and other steroid residues in edible foods.

Determination of synthetic estrogens in illegal veterinary formulations by HPTLC and HPLC.

To determine the actual amount of diethylstilbestrol, hexestrol, and dienestrol in formulations such as pellets and oily injections that are illegally available on the Brazilian market, a simple methanol extraction is used for the analysis of the pellets and an ether extraction with Sephadex columns (for clean-up) is used for the oily injections. High-performance thin-layer chromatography is used for identification (as a qualitative and semiquantitative method), and high-performance liquid chromatography is used for quantitation. The results of the analysis show that all the formulations are not in accordance with the information listed on their labels.

Analysis of diethylstilbestrol, dienestrol and hexestrol in biological samples by immunoaffinity extraction and gas chromatography-negative-ion chemical ionization mass spectrometry.

A method has been developed for the detection of diethylstilbestrol, together with dienestrol and hexestrol, using extraction with a single immunoaffinity column containing antibodies raised against diethylstilbestrol, followed by gas chromatography-negative-ion chemical ionization mass spectrometry. Immunoaffinity columns were prepared by coupling immunoglobulin G fractions obtained from rabbit antisera with a Sepharose matrix. The immunizing agent was synthesized by introducing a carboxyl group into the diethylstilbestrol molecule and coupling this product to bovine serum albumin. The columns were used for immunoadsorption of diethylstilbestrol and other estrogens, after dilution of samples with phosphate buffer, and were eluted with acetone-water (95:5 v/v). A derivatization method suitable for gas chromatographic-mass spectrometric analysis of diethylstilbestrol and other estrogens was developed using pentafluorobenzyl bromide and ethanolic potassium hydroxide as reagents. The derivatives obtained were detectable at the sub-picogram level using gas chromatography with negative-ion chemical ionization mass spectrometry. Recoveries of cis- and trans-diethylstilbestrol, dienestrol and hexestrol from the immunoaffinity columns, determined after extraction from urine, plasma and buffer, ranged from 28 to 96%. The sensitivity for diethylstilbestrol in urine samples was ca. 10 ppt. The method was applied to the analysis of urine from calves given a single dose of 10 mg of diethylstilbestrol. Free and glucuronic acid conjugated diethylstilbestrol decreased with time, but their ratio was variable.

Analysis of drug residues in tissue by combined supercritical-fluid extraction-supercritical-fluid chromatography-mass spectrometry-mass spectrometry.

The combination of supercritical-fluid extraction-supercritical-fluid chromatography-tandem mass spectrometry has been evaluated for the detection of residues of a small group of veterinary drugs in freeze-dried pig's kidney. During extraction with supercritical CO2 the drugs were retained by the column while non-polar endogenous material was not retained and thus passed to waste. Subsequent changes to the mobile phase composition eluted the drugs which were detected with high specificity by tandem mass spectrometry. Although the sensitivity in this preliminary study was not adequate for surveillance or enforcement, there is potential for further development of the approach.

An improved radioimmunoassay for the anabolic agent hexoestrol, using a monoclonal antibody.

A monoclonal antibody was raised against hexoestrol coupled to bovine serum albumin. The antibody cross-reacted with the stilbenes, diethylstilboestrol (10%) and dienoestrol (4%), but had no cross-reaction (less than 0.01%) with other anabolic agents. A radioimmunoassay method using the monoclonal antibody has been validated and used to measure residues of hexoestrol in the urine of treated cattle. The limit of detection was 0.6 pg/ml urine at the 95% confidence limit. The results were compared with those obtained using polyclonal antibodies. Although there was a good correlation between the results, the use of monoclonal antibody gave more reliable results than those obtained with available polyclonal antibodies. The monoclonal antibody, because of its quality and theoretically limitless supply, is very suitable for use in large scale screening or monitoring programmes for regulating the use of hexoestrol.

Hexestrol residues and metabolites in the tissues of wethers injected with hexestrol dicaprylate or hexestrol.

Four young (23 kg body weight) and two mature wethers (52 and 92 kg body weight) were subcutaneously injected with hexestrol (HX) or HX dicaprylate (HX-D) and killed 41 d later. The HX residues, comprising free, glucuronide and KOH hydrolyzable forms plus metabolites were determined by gas chromatography after liquid-liquid extraction and silica gel chromatographic purification of tissue sample. The HX residues were observed to be at concentrations of .1 to 1.0 ppb in most of the tissues examined. Maturity of the animals and the two hormonal formulations resulted in little difference in residues. The KOH hydrolyzable fraction was hardly detected in the tissues examined. Free HX was a major residue in muscle, representing about 70% of HX residues. Glucuronide HX represented 70 to 80% of HX residues in liver and kidney. In lung, glucuronide and free HX were present in similar amounts. This study showed that the gross metabolic patterns of HX and its esters are similar to other estrogens and steroids.

Radioimmunoassay of hexoestrol residues in faeces, tissues and body fluids of bulls and steers.

A fast, reliable and sensitive radioimmunoassay method using either 3H-hexoestrol or a 125I-hexoestrol derivative as the competitive radioligand has been developed for the measurement of residues of hexoestrol in faeces. The blank value for untreated cattle was 113 +/- 98 pg/g faeces, the intra-assay coefficient of variation 10.8 per cent, interassay coefficient of variation 11.1 per cent and recovery of 3H-hexoestrol added to faeces was 58.7 per cent. The concentration of hexoestrol in faeces was measured in 18 steers and 14 bulls at regular intervals after implantation of 36, 45 and 60 mg of hexoestrol. Residues were detected in all samples up to 104 days after implantation and in nine out of 13 samples taken 111 to 153 days after implantation. Residues of hexoestrol were also measured in edible tissues and body fluids of 14 bulls and eight steers slaughtered between 90 and 153 days after implantation of 45 mg hexoestrol. The percentages of samples containing residues were 82, 73, 64, 82 and 27 per cent for bile, urine, liver, kidney and muscle, respectively.

The structure and function of oestrogens. VI. The quinone methide hypothesis: stability of the benzylic hydrogen atoms of meso-hexoestrol during oestrogenic stimulation in mice.

A new hypothesis for oestrogen action at the molecular level is that the phenolic moiety of an oestrogen is oxidized to the corresponding quinone methide which is rapidly reduced with, say, NADPH to regenerate the oestrogen; the feedback from the rapid consumption of oxidant and/or reductant in the target organs might be responsible for the local increase in RNA synthesis, and its consequent phenomena. This hypothesis requires continuous removal and replacement of a benzylic hydrogen atom (H9 alpha of oestradiol) in an oestrogen at the receptor site. When a physiological dose of a mixture meso-(2-14C)- and meso-(2,3,4,5-3H4)hexoestrol was administered intravaginally in mice, the 14C:3H ratio in the phenolic portion of the vaginal extracts was essentially the same as that of the pure test mixture. This indicates that there was no net exchange of benzylic hydrogen in meso-hexoestrol during its stimulation of the mouse vagina.