Detection of environmental chemicals by SPR assay using branched cyclodextrin as sensor ligand

We obtained the association constants Ka of estrogen (E2) and environmental chemicals by the surface plasmon resonance (SPR) assay using the immobilized mono-6-O-α-maltosyl-β-CD (G₂βCD) compared with the immobilized β-CD and the immobilized estrogen receptor (ER). The association behavior of G₂βCD was shown as a ER model compound. The calibration curve was determined by the initial rate of association depending on the various concentrations, and the minimum detectable concentrations in the order of parts per billion were calculated. The SPR assay has advantages that the pre-treatment of the sample is not necessary and the immobilized ligand is stable and useful for the repeated measurement.     

光周期敏感不育水稻中激素的二阶导数卷积伏安法研究

二阶导数卷积伏安法应用于水稻中痛量雌雄激素的检测，具有快速，灵敏，干扰少的特点。本文以光周期敏感核不育水稻农垦５８Ｓ为材料，以农垦５８作对照，了叶焉和穗中雌，雄性激素的含量，研究了不同光周期条件下两类激素的变化。     

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.     

Liquid chromatography tandem mass spectrometry determination of free and conjugated estrogens in breast cancer patients before and after exemestane treatment

We report liquid chromatographic separation with tandem mass spectrometry determination of 12 endogenous estrogens and their intact conjugates in blood and urine and its application to study effects of exemestane treatment on estrogen generation and metabolism in postmenopausal women with estrogen-dependent breast cancer. A 0.5 mL aliquot of each urine or serum sample is fractionated with solid phase extraction to a fraction of free estrogen and another fraction of their conjugates. The reversed phase LC/MS/MS determines dansylated estrogens with positive ionization and intact conjugates with negative ionization. The method provides reproducible separation and limit of detection as low as 1 pg mL⁻¹ for free estrogens and 0.03 ng mg⁻¹ creatinine for the conjugates in serum and urine samples. The method enabled us to acquire unique concentration profiles of 12 endogenous estrogens and their intact conjugates in 30 breast cancer patients before and after one month of exemestane treatment. Exemestane suppressed total serum and urinary estrogens by 11–97% (P < 0.0001) and 8.7–91% (P < 0.0001), respectively. Specifically, these data show that exemestane preferentially suppressed E1, E1-3S, E1-3G, and E2-17G more than other estrogens. Linear regression analysis of estrogen concentrations before and after treatment showed correlation coefficients of 0.8385 (n = 289, P < 0.0001) and 0.8863 (n = 360, P < 0.0001). This study provides urinary and blood estrogen concentration profiles in breast cancer patients to demonstrate the effect of exemestane on estrogen generation, supporting inhibition of aromatase activity.     

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。     

Picogram determination of estrogens in water using large volume injection gas chromatography–mass spectrometry

In this study, a simple and efficient large volume injection gas chromatography–mass spectrometry (GC-MS) method, via a programmable-temperature vaporizer (PTV) inlet, has been developed and applied in the determination of estrogens in environmental water samples without a prior derivatization process. Three commonly used estrogens estrone, 17 β-estradiol and 17 α-ethynylestradiol were selected as target compounds for this study. It has been demonstrated that the type of gas chromatograph liner and the initial inlet temperature can greatly affect the response intensity of estrogens. Three different types of PTV liners have been studied; the multibaffle liner generated the strongest response intensities towards the estrogen analytes. The results showed that the response intensities of estrogens reduced sharply while the initial inlet temperature increased. Various instrument conditions and sample preparation methods were studied in detail. The optimized method has been validated with good linearity, precision and accuracy. The method detection limit of each estrogen was found to be 0.041 ng/L for estrone, 0.046 ng/L for 17 β-estradiol and 0.031 ng/L for 17 α-ethynylestradiol. To the best of our knowledge, these results represent the best sensitivities achieved for estrogens analyzed in water samples via traditional GC-MS method without a derivatization process. This method has been successfully applied in the analyses of different water samples.     

Magnetic solid-phase extraction based on magnetic carbon nanotube for the determination of estrogens in milk

In this work, a novel method for the fabrication of magnetic carbon nanotubes based on 'aggregation wrap' was proposed. When carbon nanotubes and magnetic nanoparticles were vortically mixed in a solvent, the magnetic nanoparticles were wrapped into the carbon nanotube bundles that formed during the aggregation process, leading to the formation of magnetic carbon nanotubes. Thus, the resultant material can be separated from the solvent rapidly and conveniently by a magnet. Our investigation demonstrated that the 'aggregation wrap' mechanism for the preparation of magnetic composite is also applicable to other self-aggregated micro/nanomaterials, including graphene, graphite, C(60), etc. To testify the feasibility of the magnetic composites in sample preparation, the resultant magnetic carbon nanotubes were applied as sorbents for magnetic solid-phase extraction (MSPE) of estrogens in milk samples. Under optimized conditions, a rapid, convenient and efficient method for the determination of estrogens in milk samples was established by the combination of MSPE with high-performance liquid chromatography with fluorescence detector. The linearity range of the proposed method was 5-2000 μg/L with correlation coefficients (R) of 0.9983-0.9994. The limit of detection (LOD) for three estrogens ranged from 1.21 to 2.35 μg/L. The intra- and inter-day relative standard deviations (RSDs) were <9.3%. The reproducibility of the MSPE with different batches of magnetic carbon nanotubes was acceptable with RSD values <3.6%.     

Procedure for increasing the detection responses of estrogens in LC–MS based on introduction of a nitrobenzene moiety followed by electron capture atmospheric pressure chemical ionization

A practical procedure for determining estrogens in biological fluids has been studied using liquid chromatography–electron capture atmospheric pressure chemical ionization–mass spectrometry combined with derivatization. Among the commercially available reagents (4-nitrobenzoyl chloride, 2,4-dinitrofluorobenzene, 4-nitrobenzenesulfonyl chloride and 4-nitrobenzyl bromide), 4-nitrobenzenesulfonyl chloride was of the most practical use; it rapidly and quantitatively reacted with estrogens and increased the detection responses by 8–23 times. The derivatization method allowed the reproducible and accurate quantification of serum and urine estrone and estradiol of a pregnant woman, which is useful for diagnosis of the fetoplacental function, with small amounts (10 μl) of sample and a simple pretreatment procedure. Tatsuya Higashiis Associate Professor of the Laboratory of Clinical Analytical Sciences (Professor Kazutake Shimada’s research group) at the Graduate School of Natural Science and Technology of Kanazawa University. He received the Japan Society for Analytical Chemistry Award for Young Scientists in 2003 and the Pharmaceutical Society of Japan Award for Young Scientists in 2006. His current research interests are the development of methods for increasing sensitivity in LC-MS to detect and characterize trace amounts of biologically active steroids, such as estrogens, androgens and neuroactive steroids.     

Catalytic diesel particulate filters reduce the in vitro estrogenic activity of diesel exhaust

An in vitro reporter gene assay based on human breast cancer T47D cells (ER-CALUX) was applied to examine the ability of diesel exhaust to induce or inhibit estrogen receptor (ER)-mediated gene expression. Exhaust from a heavy-duty diesel engine was either treated by iron- or copper/iron-catalyzed diesel particulate filters (DPFs) or studied as unfiltered exhaust. Collected samples included particle-bound and semivolatile constituents of diesel exhaust. Our findings show that all of the samples contained compounds that were able to induce ER-mediated gene expression as well as compounds that suppressed the activity of the endogenous hormone 17beta-estradiol (E2). Estrogenic activity prevailed over antiestrogenic activity. We found an overall ER-mediated activity of 1.63 +/- 0.31 ng E2 CALUX equivalents (E2-CEQs) per m(3) of unfiltered exhaust. In filtered exhaust, we measured 0.74 +/- 0.07 (iron-catalyzed DPF) and 0.55 +/- 0.09 ng E2-CEQ m(-3) (copper/iron-catalyzed DPF), corresponding to reductions in estrogenic activity of 55 and 66%, respectively. Our study demonstrates that both catalytic DPFs lowered the ER-mediated endocrine-disrupting potential of diesel exhaust.     

Reversed-phase liquid chromatography coupled on-line to estrogen receptor bioaffinity detection based on fluorescence polarization

We describe the development and validation of a high-resolution screening (HRS) platform which couples gradient reversed-phase high-performance liquid chromatography (RP-HPLC) on-line to estrogen receptor α (ERα) affinity detection using fluorescence polarization (FP). FP, which allows detection at high wavelengths, limits the occurrence of interference from the autofluorescence of test compounds in the bioassay. A fluorescein-labeled estradiol derivative (E2-F) was synthesized and a binding assay was optimized in platereader format. After subsequent optimization in flow-injection analysis (FIA) mode, the optimized parameters were translated to the on-line HRS bioassay. Proof of principle was demonstrated by separating a mixture of five compounds known to be estrogenic (17β-estradiol, 17α-ethinylestradiol and the phytoestrogens coumestrol, coumarol and zearalenone), followed by post-column bioaffinity screening of the individual affinities for ERα. Using the HRS-based FP setup, we were able to screen affinities of off-line-generated metabolites of zearalenone for ERα. It is concluded that the on-line FP-based bioassay can be used to screen for the affinity of compounds without the disturbing occurrence of autofluorescence.