An electrochemical DNA-sensor developed with the use of methylene blue as a redox indicator for the detection of DNA damage induced by endocrine-disrupting compounds

An electrochemical biosensor capable of indirect detection of DNA damage induced by any one of the three endocrine-disrupting compounds (EDCs) – bisphenol A (BPA), 4-nonylphenol (NP) and 4-t-octylphenol (OP), has been researched and developed. The methylene blue (MB) dye was used as the redox indicator. The glassy carbon electrode (GCE) was modified by the assembled dsDNA/graphene oxide-chitosan/gold nano-particles to produce a dsDNA/GO-CS/AuNPs/GCE sensor. It was characterized with the use of electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and scanning electron microscopy (SEM) techniques. The loading/release of the MB dye by the dsDNA/GO-CS/AuNPs film was investigated, and the results showed that the process was reversible. Based on this, the sensor was used to measure the difference between the loading capabilities of intact and damaged dsDNA in the films. The sensor was then successfully applied to detect DNA damage electrochemically. The differential pulse voltammetry (DPV) peak current ratio for MB, observed before and after DNA damage, increased linearly in the presence the BPA, NP or OP compounds; the treatment range was 10–60 min, and the respective damage rates were 0.0069, 0.0044 and 0.0031 min⁻¹, respectively. These results were confirmed by the binding constants: 2.09 × 10⁶ M⁻¹ (BPA-DNA), 1.28 × 10⁶ M⁻¹ (NP-DNA) and 9.33 × 10⁵ M⁻¹ (OP-DNA), all of which were obtained with the use of differential pulse stripping voltammetry (DPSV).     

Analytical method for biomonitoring of endocrine-disrupting compounds (bisphenol A,parabens, perfluoroalkyl compounds and a brominated flame retardant) in human hair by liquid chromatography-tandem mass spectrometry

In this paper, a method for the determination of four groups of endocrine-disrupting compounds in human hair is proposed. Target compounds were a plastic monomer (bisphenol A), three parabens commonly used as preservatives (methylparaben, ethylparaben and propylparaben), six perfluoroalkyl compounds commonly used as water, oil and dirt repellents (perfluorooctane sulfonic acid and five perfluoroalkyl carboxylic acids, with alkyl chains from four to eight carbon atoms) and a brominated flame retardant (hexabromocyclododecane). All of them are of especial concern to human health because they are utilized in many everyday products. The method is based on hair incubation with methanol/acetic acid solution (85:15, v/v), extraction with acetone for 15 min in an ultrasonic bath and analysis by liquid chromatography-electrospray-tandem mass spectrometry in negative ionization mode. Limits of quantification in hair samples ranged from 0.6 ng g⁻¹ to 6.1 ng g⁻¹, except for hexabromocyclododecane (36 ng g⁻¹). Recoveries were higher than 69%. Intra-day and inter-day precision, expressed as relative standard deviation, were lower than 15% and 10%, respectively. The applicability of the method was proven by analyzing the target compounds in hair samples from six volunteers. High frequencies of detection and concentrations were obtained for bisphenol A (83% of samples; concentrations up to 158 ng g⁻¹) and parabens (100% of samples; concentrations up to 624 ng g⁻¹). Lower concentrations were detected for the perfluoroalkyl compounds (up to 13 ng g⁻¹). Hexabromocyclododecane was not detected.     

Degradation of liposome cluster caused by the interaction with endocrine disrupting chemicals (EDCs)

The interaction between endocrine disrupting chemicals (EDCs) and liposome clusters was investigated using UV-vis spectroscopy and observed by microscope. Since liposome clusters are composed of small unilamellar liposomes and membrane proteins, they are regarded as a model of plasma membranes. The size of each cluster was 10microm in diameter. UV-vis spectroscopy of liposome clusters showed an apparent absorption at 300nm which was affected by the interaction with EDCs. Four EDCs caused an alteration in absorption in a concentration-dependence manner between 0.1 and 10ppm (i.e. 0.1-10mg/l). Microscopic observation showed that the effect was caused by the degradation of liposome clusters while degradation was induced by the interaction; liposome was broken down by EDCs. This report suggests that the liposome cluster can be applied for universal detection of EDCs based on the interaction between a plasma membrane model and EDCs.     

Determination of the pesticides considered as endocrine-disrupting compounds (EDCs) by solid-phase extraction followed by gas chromatography with electron capture and mass spectrometric detection

An SPE method followed by GC-electron capture detection (ECD) with confirmation by MS for the trace determination of four pesticides considered as endocrine-disrupting compounds (EDCs) in natural waters and sediments has been developed. Target analytes, fenarimol, fenvalerate, pendimethalin, and vinclozolin, belong to different chemical groups and are used mainly in agriculture. In the present study, analysis employs an offline SPE step for the extraction of the target analytes from natural waters. Sonication and subsequent SPE clean-up was used for extraction and purification of the sediment samples which were finally treated with activated copper powder. The type of SPE disk, eluents as well as solution parameters including pH value, and concentrations of salts and humic substances were examined for the efficiency of the method. The recoveries of all pesticides were in relatively high levels, ranging from 75 to 97% for waters and 71 to 84% for sediment samples. Both methods were applied to real water and sediment samples and the presence of the tested compounds was investigated.     

Determination of residual pharmaceuticals in edible animal tissues by continuous solid-phase extraction and gas chromatography-mass spectrometry

A sensitive, reliable method using continuous solid-phase extraction and gas chromatography-mass spectrometry was developed for the simultaneous determination of twenty pharmaceuticals including antibacterials, anti-epileptics, antiseptics, β-blockers, lipid regulators, hormones and non-steroidal anti-inflammatories at trace levels in edible animal tissues. The procedure involves deproteination and delipidation of samples by precipitation/centrifugation/filtration, followed by sample enrichment and cleanup by continuous solid-phase extraction. The proposed method was validated with quite good analytical results including low limits of detections (0.4-2.7 ng kg⁻¹ for 2 g of sample) and good linearity (r² > 0.995) throughout the studied concentration ranges. In addition, the method is quite accurate (recoveries ranged from 92 to 101%) and precise (within-day and between-day RSD values were less than 7%), which allows the determination of residual pharmaceuticals in tissues from agricultural farm and fish hatchery animals (pig, veal, lamb and chicken muscle, kidney and liver; and salmon, sea bass and sole flesh). The analytes most frequently found in the studied samples were the hormones estrone and 17β-estradiol, and the antibacterials florfenicol and pyrimethamine.     

Investigation of acidic pharmaceuticals in river water and sediment by microwave-assisted extraction and gas chromatography–mass spectrometry

Investigations were performed along the Danube river at Budapest (Hungary) by collecting water and sediment samples simultaneously for 1 year in order to clarify the possible hazard of selected acidic pharmaceuticals (ibuprofen, naproxen, ketoprofen, and diclofenac) on the water supply used for the production of drinking water by bank filtration. In the case of water samples, the sample preparation procedure included solid phase extraction (SPE), meanwhile, in the case of sediment samples, microwave-assisted extraction (MAE) followed by dispersive matrix extraction (DME) for pre-cleaning as well as SPE for enrichment. The quantification was carried out using gas chromatography–mass spectrometry (GC–MS). The calculated recoveries were 97–99% (± 7%) for the water and 95–103% (± 12%) for the sediment samples. In the river water, ketoprofen concentration was always below the limit of quantification (LOQ) level; ibuprofen, naproxen and diclofenac could be quantified in the range of 8–50, 2–30, 7–90 ng/L. In sediments, only naproxen and diclofenac were found in the range of 2–20 and 5–38 ng/g, respectively. According to the obtained results, the concentration ratios of the two phases linearly depended on the total organic carbon content (TOC) of the sediments at each sampling date. The linear regressions were 0.925 and 0.946 for naproxen and diclofenac, respectively.     

Determination of organotin compounds (OTC) at low levels in seawater by solid-phase extraction (SPE) and gas chromatography-pulsed flame photometric detection (GC-PFPD)

In this work, a simple, sensitive and affordable analytical method for the simultaneous determination of nine organotin compounds (butyltins, phenyltins and methyltins) in seawater using solid-phase extraction (SPE) and gas chromatography-pulsed flame photometric detection was developed and validated. The performance of three different SPE cartridges (Envi C₁₈, Oasis HLB and Oasis MCX) and three elution solvents of different polarity (hexane, methanol and acetonitrile) was evaluated. The extraction parameters, such as solvent volume, presence of complexing and ion-pairing reagents, sample volume and pH and breakthrough volume, were also investigated. Tributyltin, as the organotin compound of special interest, was efficiently extracted using any of the cartridges and solvents tested. However, the simultaneous extraction of all nine organotin compounds was the most efficient using reversed-phase Envi C₁₈ cartridge and 0.1% (w/v) tropolone in methanol as eluent. The optimised method resulted in good recovery, precision and linearity for all compounds, particularly for tri- and disubstituted species. Method detection limits ranged from 0.22 to 1.27 ng(Sn) L^?1 for butyltins, 0.37 to 4.91 ng(Sn) L^?1 for phenyltins and 0.45 to 1.16 ng(Sn) L^?1 for methyltins. The accuracy of butyltins determination was further verified by the comparison with purchased derivatised standards. The developed method was successfully applied to the environmental samples.     

Trace analysis of hetero aromatic compounds (NSO) in water and polluted groundwater by Solid phase micro-extraction (SPME)

By using Solid Phase Micro-Extraction (SPME) and Capillary Gas Chromatography (CGC) a method was developed to analyze hetero aromatic compounds (NSO) in water. Compounds with nitrogen, sulfur, or oxygen in the aromatic ring system are found in creosotecontaminated groundwater. NSO are highly water-soluble, toxic, and have adverse organoleptic characteristics. SPME is a fast, simple, and solvent-free sample preparation technique that involves exposing a polymer coated fiber to a sample to extract contaminants. Different fiber coatings were tested, and polyacrylate was found to extract NSO in the highest amounts and to be most versatile. Extraction was strongly enhanced by salting. The optimized SPME method combined with CGC coupled to an ion trap mass spectrometer (ITMS) has detection limits of 20–40 ng/L for the semi-volatile and 1–10 μg/L for the volatile NSO, response linear with respect to concentrations over three orders of magnitude, and precision of 3–14% relative standard deviation. The method was evaluated using real creosote-contaminated groundwater samples and feasibility was demonstrated. SPME-CGC/ITMS allows an unequivocal identification and quantification at a low NSO level in environmental samples.     

Capillary electrophoresis for the determination of norfloxacin and tinidazole in pharmaceuticals with multi-response optimization

Capillary electrophoresis (CE) with UV photo-diode array detection technique was utilized to adopt new method for the analysis of norfloxacin and tinidazole in pharmaceuticals. Many CE aspects including separation, rapidity, sensitivity, ruggedness as well as the repeatability of qualitative and quantitative analyses were considered simultaneously for the purpose of optimization. Experimental design approach including factorial design and response surface methods were applied to optimize electrolyte concentration and the pH while injection time, voltage and column temperature were optimized using the univariate method. Successful results were obtained using 32.5 mmol l^−l phosphate electrolyte at pH 2.5, injection time 8.0 s, voltage 25 kV and column temperature 25 °C with detection at wavelength 301 nm. The analytical characteristics including recovery, intermediate precision, linear dynamic ranges, linearity and selectivity as well as limits of detection and quantification were demonstrated and the applicability to pharmaceuticals was studied. The newly provided method enjoys the advantages of CE over HPLC with respect to rapidity, ruggedness, simplicity in reagents and sample preparation as well as saving in reagents and samples.     

Optimisation of an in-tube solid phase microextraction method coupled with HPLC for determination of some oestrogens in environmental liquid samples using different capillary columns

A simple on-line method for simultaneous determination of some oestrogens including oestriol (E3), norethisterone (NORE), ethynylestradiol (EE2), D-norgestrel (NORG) and bisphenol A (BPA), in environmental liquid samples was developed by coupling in-tube solid phase microextraction (in-tube SPME) to high-performance liquid chromatography with diode array (DAD) and fluorescence (FLD) detectors. Two capillary chromatographic columns (Supel-Q? and Carboxen? 1006 porous layer open tubular) were selected to develop this method. To achieve optimum extraction performance, several parameters were investigated including number of draw/eject cycles and the sample volume for each of the columns. Reproducibility was satisfactory for inter- and intra-day precision, yielding % RSDs of less than 10% and 7.6%, respectively. Limits of detection (LODs) and quantification (LOQs) for the proposed method using a DAD detector were achieved in the ranges of 0.04–0.63?ng?mL^?1 and 0.12–1.9?ng?mL^?1, depending of the capillary column used. Fluorescence detection improved these parameters for E3, BPA and EE2, obtaining LODs of 0.005–0.03?ng?mL^?1 and LOQs of 0.015–0.08?ng?mL^?1 using Supel-Q and LODs of 0.01–0.015?ng?mL^?1 and LOQs of 0.025–0.04?ng?mL^?1 using Carboxen. The proposed method was successfully applied to spiked environmental waters obtaining recoveries greater than 80%.     

L-半胱胺尾式卟啉铜(II)修饰金电极检测苯酚

将制备的L-半胱胺卟啉铜(II)(CuL)配合物自组装在Au电极表面,获得电化学苯酚传感器(CuL/Au)。在pH 7.0的磷酸盐缓冲液(PBS)中于-0.4 V~0.5 V(vs.SCE)电位范围内有一对氧化还原峰,峰电位分别为Epa=0.09 V和Epc=-0.06 V。实验结果表明CuL能够催化氧化苯酚,通过产物在电位0.1 V下的电化学响应对苯酚进行测定。该电极对苯酚表现出快速的响应(响应时间<10 s)。传感器对苯酚的测定具有较宽的线性范围(5.0×10-7mol.L-1~2.5×10-4mol.L-1),检出限为2.0×10-7mol.L-1。该电极用于地表水中苯酚含量检测,并与标准方法4-氨基安替比林分光光度法作了对照,结果满意。     

Comparison of total fluorine, extractable organic fluorine and perfluorinated compounds in the blood of wild and pefluorooctanoate (PFOA)-exposed rats: Evidence for the presence of other organofluorine compounds

The widespread occurrence and environmental persistence of perfluorinated compounds (PFCs) received worldwide attention recently. Exhaustive analysis of all fluorinated compounds in an environmental sample can be daunting because of the constraints in the availability of analytical standards and extraction methods. Combustion ion chromatographic technique for trace fluorine analysis was used to assess the concentrations of known PFCs (e.g., PFOS, PFOA) and total fluorine (TF) in the blood of wild rats collected from Japan. The technique was further validated using tissues from PFOA-exposed rats. Six PFCs (PFOS, PFOSA, PFUnDA, PFDA, PFNA, and PFOA) were detected in all of the wild rat blood samples. Concentrations of extractable organic fluorine (EOF) in fraction 1 (Fr1; MTBE extraction) of wild rats ranged 60.9-134 ng F mL⁻¹, while those in fraction 2 (Fr2; hexane) were below LOQ (32 ng F mL⁻¹); TF concentrations in the blood of wild rats ranged from 59.9-192 ng F mL⁻¹. The contribution of known PFCs in EOF-Fr1 (MTBE) varied from 9% to 89% (56% on average), and known PFC concentrations in TF content were less than 25%. In contrast, TF concentrations in the blood of PFOA-exposed rats ranged from 46900 to 111000 ng F mL⁻¹, with PFOA contributing over 90% of TF. A comparison of results from the samples analyzed in this study and the literature revealed three distinct groups with PFOA/known PFC and TF levels (i.e., wild rats and general population, occupationally exposed workers, and PFOA-exposed laboratory rats). The mass balance analysis of the different forms of fluorine in blood suggested the presence of other forms of organic fluorine in addition to known PFCs.     

Development of a cellular biosensor for the detection of 2,4,6-trichloroanisole (TCA)

2,4,6-trichloroanisole (TCA) is a microbial metabolite formed from chlorophenols through the activity of several natural fungal strains present on the cork oak bark. TCA is the primary compound responsible for the mousty/mould off-odour known as “cork taint” present in cork stoppers, wine, water and alcoholic beverages. Chromatographic and electrochemical methods are currently used for the determination of TCA, however its detection at low concentrations remains a technical challenge. The aim of this study was the development of a rapid novel biosensor system based on the Bioelectric Recognition Assay (BERA). The sensor measured the electric response of cultured membrane-engineered fibroblast cells suspended in an alginate gel matrix due to the change of their membrane potential in the presence of the analyte. Membrane-engineered cells were prepared by osmotic insertion of 0.5 μg/l of specific TCA antibodies into the membrane of the cells. The BERA-based sensor was able to detect TCA in a few minutes (3-5 min) at extremely low concentrations (10⁻¹ ppt), thus demonstrating higher sensitivity than the human sensory threshold. In addition, the assay was quite selective against other haloanisoles and halophenols structurally related to or co-occurring with TCA. Finally the sensor was tested against real white wine samples from cork soaks. At this real test, the BERA sensor was able to detect TCA from cork soaks rapidly (3-5 min) at very low concentrations (1.02-12 ng/l), covering the whole range for the detection threshold for wines (1.4-10 ng/l). Therefore, this novel biosensor offers new perspectives for ultra-rapid, ultra-sensitive and low-cost monitoring of TCA presence in cork and wine and possibly also other food commodities.     

Simultaneous extraction and determination of pharmaceuticals and personal care products (PPCPs) in river water and sewage by solid-phase extraction and liquid chromatography-tandem mass spectrometry

This study features the simultaneous extraction and quantification of 18 pharmaceuticals and personal care products (PPCPs). This is a pioneering method for the quantification of acetaminophen, sulfamethoxazole, diclofenac, atenolol, metoprolol, diethyltoluamide and oxybenzone in atmospheric pressure chemical ionisation mode. The method was validated for high repeatability and reproducibility with relative standard deviations less than 10%. Instrument quantification limits for PPCPs were within the range of 0.05–1.0 µg L^?1, and the method quantification limits (MQLs) for ultrapure water were within the range of 0.3–15 ng L^?1. All samples were extracted using Oasis© hydrophilic–lipophilic balanced cartridges with optimised sample size and extraction conditions. Good accuracy was demonstrated, with solid-phase extraction recoveries above 80% for most PPCPs. In environmental matrices, the MQLs for river water, sewage treatment plant (STP) effluent and STP influent were 4–25, 10–153 and 38–386.5, respectively. The method was successfully applied to investigate occurrences of persistent PPCPs in Malaysian river and sewage samples.     

Stacked films immobilization of MBTH in nafion/sol-gel silicate and horseradish peroxidase in chitosan for the determination of phenolic compounds

The stacked-film immobilization of 3-methyl-2-benzothiazolinone hydrazone (MBTH) in hybrid nafion/sol-gel silicate film and horseradish peroxidase (HRP) in chitosan, performed in order to allow the determination of phenolic compounds, was investigated via an optical method. The stacked films were deposited onto a microscope glass slide by a spin-coating technique. The quinone or free radical product formed by the enzymatic reactions of phenolic compounds interacts with MBTH to form azo-dye products, which can be measured spectrophotometrically at a wavelength of 500 nm. The color intensity of the product was found to increase in proportion to the phenolic concentration after 5 min of exposure. The response of the biosensor was linear over concentration ranges of 0.025–0.500, 0.010–0.070 and 0.050–0.300 mM for guaiacol, resorcinol and o-cresol, respectively, and gave detection limits of 0.010, 0.005 and 0.012 mM. The sensor exhibited good sensitivity and stability for at least two months.     

Biosensor employing screen‐printed PEDOT:PSS for sensitive detection of phenolic compounds in water

Poly(3,4‐ethylenedioxythiophene) (PEDOT) and its derivatives are relatively new, and unique members of conducting‐polymers family. In this article, we present an approach for simple, reliable and cost‐efficient electrochemical biosensor for real‐time detection and quantification of phenolic compounds (PhCs). The PEDOT:poly(styrene sulfonate) (PSS) polymer, directly screen‐printed on the surface of the working electrode, was shown to act as an effective electrical conductor but also as an efficient redox mediator. It has also been found suitable for the reduction of quinone ions at low reducing potentials, close to 0 V versus Ag/AgCl, thus minimizing interferences due to other electroactive species present in real samples. Based on these properties, a biosensor based on tyrosinase immobilized on PEDOT:PSS‐modified electrodes was developed allowing the detection of PhCs in surface waters. The biosensor displayed very good performance in terms of sensitivity, detection limit and linear range. Assays using surface water previously spiked with bisphenol A showed that the biosensor was able to detect PhCs in real conditions with no matrix effect. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

超高效液相色谱-串联质谱法快速检测蔬菜中248种农药残留

采用超高效液相色谱-串联质谱（UHPLC-MS/MS）技术建立了快速检测蔬菜中248种农药残留的分析方法。蔬菜样品采用乙腈提取,盐析后无需净化,缩短了样品前处理的时间。采用正负离子多反应监测（MRM）模式对蔬菜中248种农药残留进行定性和定量分析。245种农药在各自的线性范围内线性关系良好（r>0.99）。除丁硫克百威、灭蝇胺、苯磺隆和二氯喹啉酸4种农药外,其余244种农药在3个添加水平下的平均回收率范围为63.0%～126.4%,相对标准偏差（RSD）范围为0.5%～26.7%,方法的定量限为0.001～0.030 mg/kg。该方法具有简单、快速、灵敏度高、准确度高等优点,适合蔬菜样品中农药多残留的快速检测分析。     

Determination of vitamins D2 and D3 in pharmaceuticals by supercritical-fluid extraction and HPLC separation with UV detection

Summary The supercritical-fluid extraction of vitamins D₂ and D₃ with carbon dioxide is reported for the first time. The extraction recovery was enhanced by direct addition of diethyl ether to sample contained in the extraction cell. Separation and detection of the analytes was performed off-line by reversed-phase liquid chromatography with UV-detection. The quantification limit of the method is 4.1 μg for both analytes, with precision, expressed as relative standard deviation, of 3.8 and 6.3% for vitamins D₂ and D₃, respectively (η=7). The proposed method has been applied to the determination of vitamin D in different pharmaceutical products; recoveries were between 85 and 105%.     

Determination of β-blockers in pharmaceutical preparations and human urine by high-performance liquid chromatography with tris(2,2′-bipyridyl)ruthenium(II) electrogenerated chemiluminescence detection

A highly selective and sensitive detection method based on tris(2,2′-bipyridyl)ruthenium(II) [Ru(bpy)₃²⁺] electrogenerated chemiluminescence (ECL) has been developed for the quantitative determination of β-blockers in both pharmaceutical preparations and human urine samples. The ECL emission is based on the reaction between electro-oxidized Ru(bpy)₃³⁺ and the secondary amino groups on the β-blockers. The ECL intensities for the β-blockers were strongly dependent on the pH at which the ECL detections were conducted, with the maximum intensities being obtained at pH 9.0. Under the optimal condition, the detection limit for atenolol and metoprolol were almost 0.5 μM (50 pmol) and 0.08 μM (8 pmol), respectively, with S/N of 3 and a linear working range that extends four orders of magnitude with relative standard deviations below 5% for 10 replicate injected samples. The concentrations of atenolol and metoprolol were determined in pharmaceutical preparations using flow injection analysis with Ru(bpy)₃²⁺ ECL detection based on standard addition method. The determined values by the present method showed acceptable agreement with the stated values by manufacturers. The determination of the five β-blockers in human urine samples was performed using HPLC-Ru(bpy)₃²⁺ ECL detection. The resulting chromatogram was much simpler than that obtained with HPLC-UV absorbance detection.