Biophysical chemistry behind sickle cell anemia and the mechanism of voxelotor action.

Sickle cell anemia disease has been a great challenge to the world in the present situation. It occurs only due to the polymerization of sickle hemoglobin (HbS) having Pro-Val-Glu typed mutation, while the polymerization does not occur in normal hemoglobin (HbA) having Pro-Glu-Glu peptides. It is also well confirmed that the oxygenated HbS (OHbS) does not participate in the polymerization, while the deoxygenated HbS (dHbS) does, which causes the shape of red blood cells sickled. After polymerization, the blood has a low oxygen affinity. Keeping this fact into consideration, only those drugs are being synthesized that stabilize the OHbS structure so that the polymerization of HbS can be stopped. The literature data showed no systematic description of the changes occurring during the OHbS conversion to dHbS before polymerization. Hence, an innovative reasonable study between HbA and HbS, when they convert into their deoxygenated forms, was done computationally. In this evaluation, physiochemical parameters in HbA/HbS before and after deoxygenation were studied and compared deeply. The computationally collected data was used to understand the abnormal behaviour of dHbS arising due to the replacement of Glu6 with Val6. Consequently, during the presented computational study, the changes occurring in HbS were found opposite/abnormal as compared to HbA after the deoxygenation of both. The mechanism of Voxelotor (GBT-440) action to stop the HbS polymerization was also explained with the help of computationally collected data. Besides, a comparative study between GBT-440 and another suggested drug was also done to know their antisickling strength. Additionally, the effect of pH, CO, CO2, and 2,3-diphosphoglycerate (2,3-DPG) on HbS structure was also studied computationally.

Antibacterial and antibiofilm efficacy of repurposing drug hexestrol against methicillin-resistant Staphylococcus aureus.

There has been an explosion in the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) because of the indiscriminate use of antibiotics. In this study, we repurposed hexestrol (HXS) as an antibacterial agent to fight planktonic and biofilm-related MRSA infections. HXS is a nonsteroidal synthetic estrogen that targets estrogen receptors (ERα and ERß) and has been used as a hormonal antineoplastic agent. In our work, the minimum inhibitory concentrations (MICs) were determined using the antimicrobial susceptibility of MSSA and MRSA strains. Anti-biofilm activity was evaluated using biofilm inhibition and eradication assays. Biofilm-related genes were analyzed with or without HXS treatment using RTqPCR analysis of S. aureus. HXS was tested using the checkerboard dilution assay to identify antibiotics that may have synergistic effects. Measurement of ATP and detection of ATPase allowed the determination of bacterial energy metabolism. As shown in the results, HXS showed effective antimicrobial activity against S. aureus, including both type strains and clinical isolations, with MICs of 16 µg/mL. Sub-HXS strongly inhibited the adhesion of S. aureus. The content of extracellular polymeric substances (EPS) and the relative transcription levels of eno, sacC, clfA, pls and fnbpB were reduced after HXS treatment. HXS showed antibacterial effects against S. aureus and synergistic activity with aminoglycosides by directly interfering with cellular energy metabolism. HXS inhibits adhesion and biofilm formation and eradicates biofilms formed by MRSA by reducing the expression of related genes. Furthermore, HXS increases the susceptibility of aminoglycosides against MRSA. In conclusion, HXS is a repurposed drug that may be a promising therapeutic option for MRSA infection.

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%.

A yeast-based screening assay identifies repurposed drugs that suppress mitochondrial fusion and mtDNA maintenance defects.

Mitochondria continually move, fuse and divide, and these dynamics are essential for the proper function of the organelles. Indeed, the dynamic balance of fusion and fission of mitochondria determines their morphology and allows their immediate adaptation to energetic needs as well as preserving their integrity. As a consequence, mitochondrial fusion and fission dynamics and the proteins that control these processes, which are conserved from yeast to human, are essential, and their disturbances are associated with severe human disorders, including neurodegenerative diseases. For example, mutations in OPA1, which encodes a conserved factor essential for mitochondrial fusion, lead to optic atrophy 1, a neurodegeneration that affects the optic nerve, eventually leading to blindness. Here, by screening a collection of ∼1600 repurposed drugs on a fission yeast model, we identified five compounds able to efficiently prevent the lethality associated with the loss of Msp1p, the fission yeast ortholog of OPA1. One compound, hexestrol, was able to rescue both the mitochondrial fragmentation and mitochondrial DNA (mtDNA) depletion induced by the loss of Msp1p, whereas the second, clomifene, only suppressed the mtDNA defect. Yeast has already been successfully used to identify candidate drugs to treat inherited mitochondrial diseases; this work may therefore provide useful leads for the treatment of optic atrophies such as optic atrophy 1 or Leber hereditary optic neuropathy.

[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.

Development and validation of a confirmatory method for the determination of stilbene estrogens in ostrich serum.

A simple and accurate ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the first time as a confirmatory method for the simultaneous determination of stilbenes - hexestrol and diethylstibestrol in serum. Extraction was based on a simple acid denaturation of protein followed by liquid-liquid extraction using methyl tert butyl ether. Extracts were directly injected into the UHPLC-MS/MS without further purification. Excellent recoveries in the range 82-99% and 91-128% were obtained for hexestrol and diethylstibestrol, respectively. Both within-day repeatability and between-day reproducibility were generally satisfactory with RSD <20%. The linearity of the internal standard based matrix-matched calibration curve measured as the coefficient of regression (r2) was generally >0.99 for both hexestrol and diethylstibestrol. Both matrix effect and uncertainties associated with sample preparation and instrumental analysis were significantly reduced with the use of a deuterated compound (hexestrol-d4) as internal standard. The LOD and LOQ were 0.09 and 0.08 ng/ml, and 0.28 and 0.25 ng/ml, respectively, for hexestrol and diethylstibestrol. The method was found to be suitable for the simultaneous determination of hexestrol and diethylstibestrol in serum.

Retention Models on Core-Shell Columns.

A thin, active shell layer on core-shell columns provides high efficiency in HPLC at moderately high pressures. We revisited three models of mobile phase effects on retention for core-shell columns in mixed aqueous-organic mobile phases: linear solvent strength and Snyder-Soczewinski two-parameter models and a three-parameter model. For some compounds, two-parameter models show minor deviations from linearity due to neglect of possible minor retention in pure weak solvent, which is compensated for in the three-parameter model, which does not explicitly assume either the adsorption or the partition retention mechanism in normal- or reversed-phase systems. The model retention equation can be formulated as a function of solute retention factors of nonionic compounds in pure organic solvent and in pure water (or aqueous buffer) and of the volume fraction of an either aqueous or organic solvent component in a two-component mobile phase. With core-shell columns, the impervious solid core does not participate in the retention process. Hence, the thermodynamic retention factors, defined as the ratio of the mass of the analyte mass contained in the stationary phase to its mass in the mobile phase in the column, should not include the particle core volume. The values of the thermodynamic factors are lower than the retention factors determined using a convention including the inert core in the stationary phase. However, both conventions produce correct results if consistently used to predict the effects of changing mobile phase composition on retention. We compared three types of core-shell columns with C18-, phenyl-hexyl-, and biphenyl-bonded phases. The core-shell columns with phenyl-hexyl- and biphenyl-bonded ligands provided lower errors in two-parameter model predictions for alkylbenzenes, phenolic acids, and flavonoid compounds in comparison with C18-bonded ligands.

Assessing placental corticotrophin-releasing hormone disruption by hexestrol in a cell model.

Studies have shown that corticotrophin-releasing hormone (CRH) and relaxin are associated with early delivery. Our lab previously has shown the mycotoxin zeranol increases placental CRH expression. The mycotoxin is used in the farming industry to promote cattle growth, and some synthetic hormones are also used for the same purposes. In order to complete the picture of these growth promoting agents, we attempted to examine the synthetic hormones on the placental gene expression in the current study. Among the tested compounds, hexestrol induced the CRH mRNA and protein expression at 100nM in JEG-3 cells. As signal transduction pathways have been described in the transcriptional control previously, the activations of several protein kinases were determined. P38, PKCß and JNK were activated upon hexestrol treatment. Since the P38-inhibitor SB203580 prevented hexestrol from inducing CRH in a subsequent experiment, P38 was likely involved in the transcriptional regulation. Electrophoretic mobility shift assay revealed an increase in the CRE binding activity in CRH promoter. This study showed that hexestrol exposure might be a concern for pregnant women.

Electro-deposited poly-luminol molecularly imprinted polymer coating on carboxyl graphene for stir bar sorptive extraction of estrogens in milk.

Electrochemical polymerization of luminol molecularly imprinted polymer on carboxyl graphene (MIP/CG) was developed as stir bar sorptive extraction (SBSE) coating for selective pre-concentration and specific recognition of bisphenol A (BPA), hexoestrol and diethylstilbestrol in milk samples. Luminol was employed as monomer and BPA as the template to prepare MIP under 0-0.6V electro-polymerization. Carboxyl graphene was modified on pencil lead as the substrate to increase extraction capacity. The preparation and extraction conditions affecting the extraction efficiency were optimized. Under the optimized conditions, a good linearity of three estrogens was obtained in the range of 4-1000ngmL(-1). The average recoveries at the three spiked levels of the three estrogens ranged from 83.4% to 96.3% with the relative standard deviations (RSD)≤7.1%. The limits of detection were in the range of 0.36-1.09ngmL(-1). The developed method with low cost, high selectivity and good reproducibility can be potentially applied for determining trace estrogens in complex food samples.

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.

Picogram-level quantification of some growth hormones in bovine urine using mixed-solvent bubble-in-drop single drop micro-extraction.

Growth hormones are important biologically active compounds. However, they can cause deleterious effects if not used with care and their use in farmed animals is banned in the European Union. This study presents the development and application of a mixed-solvent "bubble-in-drop single drop micro-extraction" (BID-SDME) method for enrichment of stilbene hormones in bovine urine samples. The hormones are quantified using GC-MS showing good linearity with the coefficient of determination (R(2)) of 0.999 and 0.999 for hexestrol and diethylstilbestrol, respectively, in the concentration range 0.05-10 ng mL(-1). Excellent precision (RSD<10%) and accuracy (using a bovine urine certified reference material) were obtained. The observed detection capability (CCα, or LOD) values were 0.01 ng mL(-1) (hexestrol), 0.03 ng mL(-1) (cis-diethylstilbestrol) and 0.02 ng mL(-1) (trans-diethylstilbestrol), respectively, while the decision limit (CCß, or LOQ) values were 0.03 ng mL(-1), 0.08 ng mL(-1) and 0.07 ng mL(-1), which are comparable to or better than those reported in literature. Importantly, sample handling is significantly simplified by our method and enrichment values are greatly enhanced. The results show that a 3:1 chloroform/toluene mixture gave the highest extraction efficiency with a drop-bubble ratio of 2:1. We highlight the importance of solvent density on the success of the BID-SDME method.

Electrochemical fabrication of polymerized imidazole-based ionic liquid bearing pyrrole moiety for sensitive determination of hexestrol in chicken meat.

1-[3-(tert-Butoxycarbonylamino)propyl]-3-[3-(N-pyrrole)propyl]imidazolium tetrafluoroborate [(t-Boc-APPPI)BF4], which is a novel pyrrolyl-functionalized ionic liquid, was synthesized and characterized. Subsequently, it was electrochemically deposited onto a glassy carbon electrode surface to fabricate a polymerized ionic liquid film electrode. X-ray photoelectron spectroscopy, scanning electron microscope and electrochemical impedance spectroscopy were used to confirm the successful polymerization of ionic liquid. Voltammetric behaviors of hexestrol at the film electrode were investigated. The oxidation peak slightly shifted towards positive potential, however, dramatically increased in peak current. Experimental conditions for hexestrol determination were optimized. The oxidation peak current is linear with hexestrol concentration in the range of 1.0 × 10(-8)-1.0 × 10(-5) mol L(-1). The detection limit is estimated to be 2.1 × 10(-9) mol L(-1) (S/N=3). Hexestrol in chicken meat was determined using the film electrode with good accuracy.

Hexestrol- and nonylphenol-induced differential vitellogenin synthesis in female and male barfin plaice Liopsetta pinnifasciata.

The effects of hexestrol (HXS) and nonylphenol (NP) on plasma vitellogenin (Vtg) concentration in barfin plaice Liopsetta pinnifasciata was studied during spring and autumn experiment. In L. pinnifasciata two "complete" forms of Vtgs, namely VtgAa and VtgAb, were previously described which may be separated due to molecular mass of their largest polypeptide in SDS-PAGE. In spring, the injection of HXS led to an increase in Vtg concentrations in both females and males. SDS-PAGE analysis of plasma from HXS-exposed fish produced only one prominent band at a molecular mass of 180 kDa that corresponds to an increase in VtgAb levels. NP injected in fish in spring induced statistically significant increasing of Vtg concentration in males, and only one type of Vtg, as in case of HXS, accumulated in plasma. In autumn, the injection of HXS results to the increase of Vtg concentration in the plasma of females and males, electrophoretic analysis of plasma proteins showed that only a 98 kDa polypeptide, corresponding to the VtgAa-type showed a significant increase. The blood plasma ratios of VtgAa and VtgAb in experimental fish are discussed in relation to the season and stage of reproductive cycle.

[Determination of estrogen residues in drinking water by on-line solid phase extraction based on sol-gel technique coupled with high performance liquid chromatography].

A method for the determination of diethylstilbestrol (DES), hexestrol (HEX) and dienestrol (DS) residues in drinking water was established by on-line solid phase extraction (SPE) coupled with high performance liquid chromatography (HPLC). The material synthesized on the base of sol-gel technology was employed as adsorbent. This material was prepared using 3-aminopropyltriethoxysilane (APTES) as the functional monomer, tetraethoxysilane (TEOS) as the crosslinking agent, and acetic acid as the initiator. The synthesized adsorbent showed outstanding property for the estrogen extraction. The estrogen can be caught effectively from water samples and the extraction can be achieved rapidly. Some important parameters, such as pH of sample solution, eluent solvents, loading flow rate, which might influence extraction efficiency, were optimized. The results indicated that the limit of detection (S/N = 3) of the developed method could reach 0.07-0.13 microg/L under the conditions of pH 7.0 of sample solution, methanol and 1% (v/v) acetic acid aqueous solution as the eluent solvent and the loading flow rate of 2 mL/min. The recoveries of the three estrogens from the water samples at three spiked levels ranged from 82.31% to 99.43% with RSD of 1.61%-7.15%. The method was simple, rapid, and suitable to detect the trace residues of estrogens in drinking water.

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.

Comparison of the diagnostic accuracy of di-22:6-bis(monoacylglycerol)phosphate and other urinary phospholipids for drug-induced phospholipidosis or tissue injury in the rat.

Cationic amphiphilic drugs and aminoglycoside antibiotics can induce phospholipidosis (PLD), an abnormal accumulation of phospholipids in lysosome-derived vesicles, in preclinical studies. The incidence of PLD in patients and its clinical relevance are difficult to assess without noninvasive biomarkers. Di-docosahexaenoyl bis(monoacylglycerol)phosphate (di-22:6-BMP) is a phospholipid that is enriched in lysosomal membranes and a proposed urinary biomarker of drug-induced PLD. The specificity of di-22:6-BMP for PLD was compared to other phospholipid species that can increase in urine with nephrotoxicity. Using liquid chromatography coupled to mass spectrometry, 12 phospholipids were assayed in the urine of rats treated with drugs that induced PLD or caused renal or skeletal muscle injury. In receiver operating curve analyses, urinary di-22:6-BMP was a significantly better predictor of PLD and the least predictive of tissue injury of the phospholipids assayed. The data provide evidence supporting the use of di-22:6-BMP as a urinary biomarker of PLD in rats.

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%.