Optical biosensor for pharmaceuticals, antibiotics, hormones, endocrine disrupting chemicals and pesticides in water: Assay optimization process for estrone as example

Certain contaminants at trace concentrations in surface waters can have dramatic effects on the hormonal system of organisms in the aquatic environment. Therefore, immunoanalytical methods at a very low limit of detection (LOD) and a low limit of quantification (LOQ) are becoming more and more important for environmental analysis and especially for monitoring drinking water quality. Environmental monitoring of antibiotics, hormones, endocrine disrupting chemicals, and pesticides in real water samples (e.g. surface, ground or drinking water) with difficult matrices places high demands on chemical analysis. Biosensors have suitable characteristics such as efficiency in allowing very fast, sensitive, and cost-effective detection. Here we describe an assay optimization process with a fully automated immunoassay for estrone which resulted in a LOD below 0.20 ng L⁻¹ and a LOQ below 1.40 ng L⁻¹. In contrast to common analytical methods such as GC-MS or HPLC-MS, the biosensor used requires no sample pre-treatment and pre-concentration. The very low validation parameters for estrone are the result of the continuous optimization of the immunoassay. The basis of our sensitive assay is the antibody with a high affinity constant towards estrone. During the optimization process, we reduced the amount of antibody per sample and improved the chip surface modification. Finally, this proceeding led to a calibration routine with an amount of antibody of only 3.0 ng per sample (sample volume: 1.0 mL). The reduction of the amount of antibody per sample results in better validation parameters (LOD, LOQ, and IC₅₀), but this reduction leads to the current device-related limitation of the River Analyser (RIANA).For some endocrine disrupting compounds, no effect levels (NOELs) in the lower nanogram per liter range are reported. This defines the challenge, which analytical methods have to compete with and our RIANA instrument with its improved sensitivity for the detection of a single hormone in the lower nanogram per liter range is a powerful tool in aquatic analytics in addition to the common analytical methods.     

<Emphasis Type="Italic">L</Emphasis>-Carnitine Supplementation: A New Paradigm for its Role in Exercise

Summary. Early research investigating the effects of L-carnitine supplementation has examined its role in substrate metabolism and in acute exercise performance. These studies have yielded equivocal findings, partially due to difficulties in increasing muscle carnitine concentrations. However, recent studies have proposed that L-carnitine may play a different role in exercise physiology, and preliminary results have been encouraging. Current investigations have theorized that L-carnitine supplementation facilitates exercise recovery. Proposed mechanism is as follows: 1) increased serum carnitine concentration enhances capillary endothelial function; 2) increased blood flow and reduced hypoxia mitigate the cascade of ensuing, destructive chemical events following exercise; 3) thus allowing reduced structural damage of skeletal muscle mediated by more intact receptors in muscle needed for improved protein signaling. This paradigm explains decreased markers of purine catabolism, free radical formation, and muscle tissue disruption after resistance exercise and the increased repair of muscle proteins following long-term L-carnitine supplementation.     

Quantitation of anabolic hormones and their metabolites in bovine serum and urine by liquid chromatography-tandem mass spectrometry

A specific and sensitive method based on tandem mass spectrometry with on-line high-performance liquid chromatography using atmospheric pressure chemical ionisation (LC–APCI-MS–MS) for the quantitation of anabolic hormone residues (17β-19-nortestosterone, 17β-testosterone and progesterone) and their major metabolites (17-19-nortestosterone and 17-testosterone) in bovine serum and urine is reported. [²H₂]17β-Testosterone was used as internal standard. The analytes were extracted from urine (following enzymatic hydrolysis) and serum samples by liquid–liquid extraction and purified by C₁₈ solid-phase extraction. Ionisation was performed in a heated nebulizer interface operating in the positive ion mode, where only the protonated molecule, [M+H]⁺, was generated for each analyte. This served as precursor ion for collision-induced dissociation and two diagnostic product ions for each analyte were identified for the unambiguous hormone confirmation by selected reaction monitoring LC–MS–MS. The overall inter-day precision (relative standard deviation) ranged from 6.37 to 2.10% and from 6.25 to 2.01%, for the bovine serum and urine samples, respectively, while the inter-day accuracy (relative error) ranged from −5.90 to −3.18% and from −6.40 to −2.97%, for the bovine serum and urine samples, respectively. The limit of quantitation of the method was 0.1 ng/ml for all the hormones in bovine serum and urine. On account of its high sensitivity and specificity the method has been successfully used to confirm illegal hormone administration for regulatory purposes.     

Preparation of 17β‐estradiol surface molecularly imprinted polymers and their application to the analysis of biological samples

17β‐Estradiol (E2) surface molecularly imprinted polymers have been prepared using functionalized monodispersed poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) beads as a support. The resulting polymers were found to be uniform in size (5 μm), and the surfaces of the microspheres possessed large pore‐like structures. A chromatographic experiment demonstrated that the resulting microspheres exhibited high levels of recognition and selectivity toward the target molecule. The particles were employed as a novel sorbent in a molecularly imprinted SPE protocol. A method was then developed involving the combination of the pretreatment with HPLC to determine the levels of estrogen secreted from Michigan Cancer Foundation‐7 cells. The obtained results revealed that the extraction recoveries of E2 from real samples were in the range of 73.0–97.5% with RSDs of < 7.5% (n = 3). Calibration curves were established with R values > 0.9996 for concentrations in the range of 0.50–100.00 ng/mL. The LOD of this new method was 0.14 ng/mL. Compared with traditional C₁₈ SPE agents, the particles showed high selectivity and extraction efficiency for E2 in the pretreatment process. The particles could therefore be used to determine trace estrogen in biological samples with a UV detector only.     

New paradigms for synthetic pathways inspired by bioorganometallic chemistry

Two series of particular examples of reactions used in bioorganometallic chemistry are described. One based on a decomplexation-complexation reaction, indicates how, starting from a cymantrenyl derivative, a range of organometallic complexes bearing various metals can be prepared. The second one refers to the easy synthesis in water of the very versatile Alberto's reagent, which leads to new organometallic radiopharmaceuticals of Tc and Re.     

Ultrasound‐assisted extraction method for the simultaneous determination of emerging contaminants in freshwater sediments

Sediments are the fate of several emerging organic contaminants, such as pharmaceuticals, personal care products and hormones, and therefore an important subject in environmental monitoring studies. In the present work, a simple and sensitive method was developed, validated and applied for the simultaneous extraction of atenolol, caffeine, carbamazepine, diclofenac, ibuprofen, naproxen, propranolol, triclosan, estrone, 17‐β‐estradiol and 17‐α‐ethinylestradiol using ultrasound‐assisted extraction from freshwater sediment samples followed by solid‐phase extraction clean‐up and liquid chromatography with tandem mass spectrometry detection. The solvent type and extraction pH were evaluated to obtain the highest recoveries of the compounds. The best method shows absolute recoveries between 54.0 and 94.4% at 50 ng/g concentration. The method exhibits good precision with relative standard deviation ranging from 1.0–16%. The detection and quantification limits ranged from 0.006–0.067 and 0.016–0.336 ng/g, respectively. The developed method was successfully applied to freshwater sediment samples collected from different sites in Jundiaí River basin of São Paulo State, Brazil. The compounds atenolol, caffeine, propranolol and triclosan were detected in all the sampling sites with concentrations of 13.8, 41.0, 28.5 and 176 ng/g, respectively.     

Hormone receptors.

Hormone receptors are proteins located on the cell membrane or in the cell cytoplasm. Their function is to recognize and bind the respective hormone; the biological action of the hormone originates from the hormone-receptor complex. Specific receptors have been found for all known hormones. Monitoring the equilibration between hormone, receptor, and hormone-receptor complex (“determination of receptor binding”) permits quantitative determinations of hormones, allows the characterization of endocrinal disturbances, and contributes to the elucidation of structure-affinity relationships.     

Turgorins,Hormones of the Endogeneous Daily Rhythms of Higher Organized Plants—Detection,Isolation, Structure,Synthesis, and Activity

Probably the most fascinating biological phenomenon is the movement which is so impressively observed in higher organized plants when, e.g., in a sort of “quick retreat”, the sensitive plant apparently vanishes on touching, or when all the pinnules of an acacia fold together pairwise at dusk, as though the whole tree were going to “sleep”. Such plants and trees do not possess muscles for the movement of their organs; instead of the contraction of a “still primitive” actomyosin system in this movement the hydrostatic internal pressure of the vacuoles—the turgor—in the parenchymal cells of the motile organs is often drastically and unilaterally reduced. After preception of an external stimulus there follows a gradual or sudden change in the semipermeability of the boundry plasma layers, and, mediated by a stimulus conduction, also of all the symplasts of a multicellular plant organism. The phytohormones which form the molecular basis of the physiology of movement discussed here have now been isolated and structurally elucidated. The chemical agonists of phytodynamics, referred to as turgorins, elicit leaf movement; moreover, they very probably also regulate—perhaps together with other phytohormones—the mechanism of change of aperture in stomatal transpiration. Hence, the turgorins facilitate not only a regulation of temperature, but even their own transport in the sap of the whole plant body.     

高效液相色谱法同时测定化妆品中9种激素

建立了化妆品中9种激素的HPLC测定方法。样品经甲醇超声提取后,经ZORBAXExtend-C18柱分离,以甲醇-水(pH=8.0,0.02mol/L磷酸盐缓冲体系)为流动相,采用紫外检测器检测。9种激素分离良好并排除了样品中杂质峰的干扰,各激素在1—100mg/L范围内呈良好线性关系(r0.999),平均回收率在93.3%—107.2%之间,相对标准偏差为0.79%—2.07%(n=7),检出限为0.05—0.2mg/L。该方法可简便、快捷、准确的同时测定化妆品中9种激素。     

Extraction and determination of hormones in cosmetics by homogeneous ionic liquid microextraction high-performance liquid chromatography

The homogeneous ionic liquid microextraction was applied to the extraction of hormones from cosmetics and the hormones were determined by high-performance liquid chromatography. 1-Hexyl-3-methylimidazolium tetrafluoroborate was used as extraction solvent. Ammonium hexafluorophosphate as used as ion-pairing agent. Several experimental parameters, including the volume of 1-hexyl-3-methylimidazolium tetrafluoroborate, amount of ammonium hexafluorophosphate and sodium chloride, extraction and centrifuge time, and the pH value, were optimized. The limits of detection and quantification for the analytes ranged from 0.03 to 0.24 ng/mL and from 0.10 to 0.79 ng/mL, respectively. The precision for determining the hormones was lower than 5.2%. The proposed method was successfully developed for the determination of hormones in real cosmetic samples.