Analytical and Bioanalytical Chemistry (v.390, #8)
Effect-oriented environmental analysis by Marc J.-F. Suter (1957-1958).
is currently deputy department head of Environmental Toxicology at Eawag—the Swiss Federal Institute of Aquatic Science and Technology in Dübendorf, Switzerland. His main scientific interest is linking physical stress and chemical exposure to effects in aquatic organisms, currently focusing on the response at the protein level.
How to confirm identified toxicants in effect-directed analysis by Werner Brack; Mechthild Schmitt-Jansen; Miroslav Machala; Rikke Brix; Damià Barceló; Emma Schymanski; Georg Streck; Tobias Schulze (1959-1973).
Due to the production and use of a multitude of chemicals in modern society, waters, sediments, soils and biota may be contaminated with numerous known and unknown chemicals that may cause adverse effects on ecosystems and human health. Effect-directed analysis (EDA), combining biotesting, fractionation and chemical analysis, helps to identify hazardous compounds in complex environmental mixtures. Confirmation of tentatively identified toxicants will help to avoid artefacts and to establish reliable cause–effect relationships. A tiered approach to confirmation is suggested in the present paper. The first tier focuses on the analytical confirmation of tentatively identified structures. If straightforward confirmation with neat standards for GC–MS or LC–MS is not available, it is suggested that a lines-of-evidence approach is used that combines spectral library information with computer-based structure generation and prediction of retention behaviour in different chromatographic systems using quantitative structure–retention relationships (QSRR). In the second tier, the identified toxicants need to be confirmed as being the cause of the measured effects. Candidate components of toxic fractions may be selected based, for example, on structural alerts. Quantitative effect confirmation is based on joint effect models. Joint effect prediction on the basis of full concentration–response plots and careful selection of the appropriate model are suggested as a means to improve confirmation quality. Confirmation according to the Toxicity Identification Evaluation (TIE) concept of the US EPA and novel tools of hazard identification help to confirm the relevance of identified compounds to populations and communities under realistic exposure conditions. Promising tools include bioavailability-directed extraction and dosing techniques, biomarker approaches and the concept of pollution-induced community tolerance (PICT). Figure Toxicity confirmation in EDA as a tiered approach
Keywords: Effect-directed analysis; Toxicity identification evaluation; Toxicity confirmation; Structural analysis; Mixture toxicity; Hazard
The risk of altering soil and sediment samples upon extract preparation for analytical and bio-analytical investigations—a review by Thomas-Benjamin Seiler; Tobias Schulze; Henner Hollert (1975-1985).
Organic total extracts play an important role in soil and sediment risk assessment. Beside a routine application in analytical chemistry, they are used in bio-analytical investigations as a “worst-case scenario” or, e.g., in order to simulate chronic intoxication, and as samples for effect-directed analysis. While theoretically providing highly reliable data and good reproducibility, the whole process of sample handling and extract preparation can lead to extracts that might fail to accurately represent a toxic potential of their corresponding sampling site. This review identifies and discusses the most important possible alterations that have the potential to lead to over and, more often, underestimation of the effectiveness of extracts. Since incorrect data will compromise soil and sediment risk assessment as a whole, results from analytical and bio-analytical investigations of extracts demand cautious interpretation. Reliability of extract testing grows with reproducibility; experiments should therefore be repeated with independent extraction replicates. New or optimized extraction procedures should circumvent the issues mentioned here while being suitable for routine application.
Keywords: Soil; Sediment; Extraction; Risk of alteration; Bioassays; Sample treatment
Reversed-phase liquid chromatography coupled on-line to estrogen receptor bioaffinity detection based on fluorescence polarization by Jelle Reinen; Jeroen Kool; Nico P. E. Vermeulen (1987-1998).
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.
Keywords: Fluorescence polarization; High-resolution screening (HRS); Estrogen receptor α; Phytoestrogens; On-line bioaffinity assay; Receptor affinity detection (RAD)
Assessment of the acute toxicity of triclosan and methyl triclosan in wastewater based on the bioluminescence inhibition of Vibrio fischeri by Marinella Farré; Daniela Asperger; Lina Kantiani; Susana González; Mira Petrovic; Damià Barceló (1999-2007).
In this work, the contributions of triclosan and its metabolite methyl triclosan to the overall acute toxicity of wastewater were studied using Vibrio fischeri. The protocol used in this paper involved various steps. First, the aquatic toxicities of triclosan and methyl triclosan were determined for standard substances, and the 50% effective concentrations (EC50) were determined for these compounds. Second, the toxic responses to different mixtures of triclosan, methyl triclosan, and surfactants were studied in different water matrices, i.e., Milli-Q water, groundwater and wastewater, in order to evaluate (i) the antagonistic or synergistic effects, and (ii) the influence of the water matrices. Finally, chemical analysis was used in conjunction with the toxicity results in order to assess the aquatic toxicities of triclosan and its derivative in wastewaters. In this study, the toxicities of 45 real samples corresponding to the influents and effluents from eight wastewater treatment works (WWTW) were analyzed. Thirty-one samples were from a wastewater treatment plant (WWTP) equipped with two pilot-scale membrane bioreactors (MBR), and the influent and the effluent samples after various treatments were characterized via different chromatographic approaches, including solid-phase extraction (SPE), liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS), and SPE coupled to gas chromatography–mass spectrometry (GC–MS). The toxicity was determined by measuring the bioluminescence inhibition of Vibrio fischeri. In order to complete the study and to extrapolate the results to different WWTPs, the toxicity to V. fischeri of samples from seven more plants was analyzed, as were their triclosan and methyl triclosan concentrations. Good agreement was established between the overall toxicity values and concentrations of the biocides, indicating that triclosan is one of the major toxic organic pollutants currently found in domestic wastewaters.
Keywords: Triclosan; Methyl triclosan; Surfactants; Toxicity; Vibrio fischeri ; Bioluminescence; Wastewater
Activities and identification of aryl hydrocarbon receptor agonists in sediments from the Danube river by Steffen Keiter; Stefanie Grund; Bert van Bavel; Jessika Hagberg; Magnus Engwall; Ulrike Kammann; Martin Klempt; Werner Manz; Helena Olsman; Thomas Braunbeck; Henner Hollert (2009-2019).
This study is a consequence of a distinct fish decline in the Danube river since the beginning of the 1990s. In contrast to the decline of fish population, former studies have repeatedly documented that the water quality along the Danube river is improving. However, the conclusion of a pilot study in 2002 was that a high hazard potential is associated with local sediments. The present study documents that sediment samples from the Danube river showed comparatively high aryl hydrocarbon receptor mediated activity in biotests, using the cell lines GPC.2D.Luc, H4IIE (DR-CALUX®) and RTL-W1. The combination of chemical analysis, fractionation techniques and different in vitro tests revealed that priority pollutants could not explain the main induction, even though the concentrations of priority polycyclic aromatic hydrocarbons (PAHs) were very high (maximum in the tributary Schwarzach, sum of 16 EPA PAHs 26 μg/g). In conclusion, this investigation shows that nonpriority pollutants mainly mediate the high induction rates. Nevertheless, owing to the effects of PAHs towards fish and the connection between dioxin-like activity and carcinogenicity, the link between contamination and the fish population decline cannot be ruled out.
Keywords: Danube; Fish decline; Dioxin-like activity; Aryl hydrocarbon receptor agonists; Weight of evidence
Catalytic diesel particulate filters reduce the in vitro estrogenic activity of diesel exhaust by Daniela Wenger; Andreas C. Gerecke; Norbert V. Heeb; Hanspeter Naegeli; Renato Zenobi (2021-2029).
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 17β-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 m3 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.
Keywords: Diesel exhaust; Diesel particles; Diesel particulate filter; In vitro reporter gene assay; Estrogen receptor; Estrogenic activity
Development, standardization and refinement of procedures for evaluating effects of endocrine active compounds on development and sexual differentiation of Xenopus laevis by Ilka Lutz; Werner Kloas; Timothy A. Springer; Larry R. Holden; Jeff C. Wolf; Henry O. Krueger; Alan J. Hosmer (2031-2048).
Xenopus laevis has been introduced as a model to study effects of endocrine-active compounds (EAC) on development and sexual differentiation. However, variable and inconsistent data have raised questions about the reliability of the test methods applied. The current study was conducted in two laboratories to develop, refine, and standardize procedures and protocols. Larvae were exposed in flow-through systems to 17β-estradiol (E2), at concentrations from 0.2 to 6.0 μg E2 L−1 in Experiment 1A, and 0.015 to 2.0 μg E2 L−1 in Experiment 1B. In both studies survival (92%, 99%) and percentage of animals that completed metamorphosis (97%, 99%) indicated reproducible biological performance. Furthermore, minor variations in husbandry led to significant differences in snout-to-vent length, weight, and gonad size. In Experiment 1A, almost complete feminization occurred in all E2 treatment groups whereas a concentration response was observed in Experiment 1B resulting in an EC50 of 0.12 μg E2 L−1. The final verified protocol is suitable for determining effects of EAC on development and sexual differentiation in X. laevis.
Keywords: Xenopus laevis ; Flow-through exposure system; Standardized protocol; Development; Sexual differentiation; 17β-Estradiol
Fluorescence detection techniques for protein kinase assay by YongJin Li; WeiHong Xie; GuiJie Fang (2049-2057).
Traditional methods for protein kinase (PK) assay are mainly based on use of 32P-labeled adenosine triphosphate (ATP); applications of such methods are, however, hampered by radioactive waste and short half-life of 32P-labeled ATP. Therefore non-radioactive methods, such as fluorescence detection techniques are good alternative. In this review, we describe the principles of four fluorescence techniques (fluorescence intensity endpoint measurement, fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), and fluorescence lifetime imaging) and provide an overview of applications of these fluorescence detection techniques in protein kinase assay, underlining their relative advantages and limitations. Research trends in this field are also highlighted. Figure Schematic representation of kinase assay based on direct fluorescence polarization measurements. The fluorescent peptide, on phosphorylation by kinase, binds to a phosphospecific antibody, which leads to a high FP value
Keywords: Protein kinase; Phosphorylation; Fluorescence; Biosensor; FRET
Multivariate curve resolution–alternating least squares (MCR-ALS) applied to spectroscopic data from monitoring chemical reactions processes by M. Garrido; F. X. Rius; M. S. Larrechi (2059-2066).
This paper overviews the application of multivariate curve resolution (optimized by alternating least squares) to spectroscopic data acquired by monitoring chemical reactions and other processes. The goals of the resolution methods and the principles for understanding their applications are described. Some of the problems arising from these evolving systems and the limitations of the multivariate curve resolution methods are also discussed. This article reviews most of the applications of multivariate curve resolution applied to reacting systems published between January 2000 and June 2007. Some basic papers dated before 2000 have also been included.
Keywords: Chemometrics; MCR-ALS; Multivariate curve resolution; Reaction monitoring; Process monitoring; Rate constant determination; Kinetic data analysis
The interference of HEPES buffer during amperometric detection of ATP in clinical applications by Jean-Francois Masson; Estelle Gauda; Boris Mizaikoff; Christine Kranz (2067-2071).
HEPES-based biological buffer is subject to photooxidation upon exposure to fluorescent illumination. Thereby hydrogen peroxide is generated, which interferes with amperometric oxidoreductase-based biosensors for glucose or adenosine triphosphate (ATP). These biosensors operate at an oxidation potential above 500 mV vs. the standard calomel electrode (SCE) and involve hydrogen peroxide as the electroactive molecule detected at the electrode surface. False-positive detection of ATP was observed in HEPES buffer utilizing an amperometric microbiosensor based on the co-immobilization of glucose oxidase and hexokinase for detection of ATP in biological specimens. Electrochemical, mass spectrometric, 31P NMR, and 1H NMR studies indicate that complexation of ATP and HEPES induced by the presence of Ca2+ in HEPES buffer decreases the photooxidation of HEPES. Consequently, the hydrogen peroxide background concentration is reduced, thereby leading to erroneous ATP detection at the dual-enzyme microbiosensor, which determines an increase in ATP via a reduced hydrogen peroxide signal.
Keywords: 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); Phototoxicity; ATP microbiosensor; Hexokinase; Glucose oxidase; Ringer’s solution
A whole-cell assay for the high throughput screening of calmodulin antagonists by Emre Dikici; Sapna K. Deo; Sylvia Daunert (2073-2079).
Cell-based screening systems for pharmaceuticals are desired over molecular biosensing systems because of the information they provide on toxicity and bioavailability. However, the majority of sensing systems developed are molecular biosensing type screening systems and cannot be easily adapted to cell-based screening. In this study, we demonstrate that protein-based molecular sensing systems that employ a fluorescent protein as a signal transducer are amenable to cell-based sensing by expressing the protein molecular sensing system in the cell and employing these cells for screening of desired molecules. To achieve this, we expressed a molecular sensing system based on the fusion protein of calmodulin (CaM) and enhanced green fluorescent protein (EGFP) in bacterial cells, and utilized these cells for the screening of CaM antagonists. In the presence of Ca2+, CaM undergoes a conformational change exposing a hydrophobic pocket that interacts with CaM-binding proteins, peptides, and drugs. This conformational change induced in CaM leads to a change in the microenvironment of EGFP, resulting in a change in its fluorescence intensity. The observed change in fluorescence intensity of EGFP can be correlated to the concentration of the analyte present in the sample. Dose-response curves for various tricyclic antidepressants were generated using cells containing CaM-EGFP fusion protein. Additionally, we demonstrate the versatility of our system for studying protein-protein interactions by using cells to study the binding of a peptide to CaM. The study showed that the CaM-EGFP fusion protein within the intact cells responds similarly to that of the isolated fusion protein, hence eliminating the need for any isolation and purification steps. We have demonstrated that this system can be used for the rapid screening of various CaM antagonists that are potential antipsychotic drugs.
Keywords: Calmodulin; Calmodulin antagonists; Enhanced green fluorescent protein; Whole-cell assay; Protein-protein interactions; Tricyclic antidepressants; High throughput screening
Molecular recognition of endocrine disruptors by synthetic and natural 17β-estradiol receptors: a comparative study by Bernadette Tse Sum Bui; Anne-Sophie Belmont; Hilda Witters; Karsten Haupt (2081-2088).
A β-estradiol receptor binding mimic was synthesised using molecular imprinting. Bulk polymers and spherical polymer nanoparticles based on methacrylic acid and ethylene glycol dimethacrylate as the functional monomer and crosslinker, respectively, were prepared in acetonitrile. The selectivity was evaluated by radioligand binding assays. The imprinted polymers were very specific to β-estradiol since the control polymers bound virtually none of the radioligand. The bulk polymer was then employed to screen endocrine disrupting chemicals. Structurally related steroids like α-estradiol, estrone and ethynylestradiol showed, respectively, 14.0, 5.0 and 0.7% of relative binding to the β-estradiol polymer, whereas most unrelated chemicals did not bind at all. These results are compared to those obtained with a bioassay using stably transfected yeast cells in culture bearing the human estrogen receptor. The receptor was activated by several estrogen-like chemicals and to a lesser extent by some structurally related chemicals. Figure A molecularly imprinted polymer that was a synthetic receptor for beta-estradiol was used for the screening of endocrine disrupting chemicals that are structurally related or unrelated to beta-estradiol. The results were compared with the recognition of the compounds by the biological estrogen receptor expressed in yeast cells. Related steroids like alpha-estradiol, estrone and ethynylestradiol showed significant binding to the beta-estradiol imprinted polymer, whereas most unrelated chemicals did not bind. The biological receptor was activated by several estrogen-like chemicals, and to a lesser extent by some structurally related chemicals
Keywords: Molecular imprinting; Synthetic receptors; Endocrine disruptors; Bioassay; Screening; β-estradiol
Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging by Caroline Gurcel; Anne-Sophie Vercoutter-Edouart; Catherine Fonbonne; Marlène Mortuaire; Arnaud Salvador; Jean-Claude Michalski; Jérôme Lemoine (2089-2097).
The O-linked β-N-acetylglucosamine (O-GlcNAc) modification is an abundant post-translational modification in eukaryotic cells. This dynamic glycosylation plays a fundamental role in the activity of many nuclear and cytoplasmic proteins and is associated with pathologies like type II diabetes, Alzheimer’s disease or some cancers. However the exact link between O-GlcNAc-modified proteins and their function in cells is largely undefined for most cases. Here we report a strategy based on the 1,3-dipolar cycloaddition, called click chemistry, between unnatural N-acetylglucosamine (GlcNAc) analogues (substituted with an azido or alkyne group) and the corresponding biotinylated probe to specifically detect, enrich and identify O-GlcNAc-modified proteins. This bio-orthogonal conjugation confirms that only azido analogue of GlcNAc is metabolized by the cell. Thanks to the biotin probe, affinity purification on streptavidin beads allowed us to identify 32 O-GlcNAc-azido-tagged proteins by LC-MS/MS analysis in an MCF-7 cellular model, 14 of which were previously unreported. This work illustrates the use of the click-chemistry-based strategy combined with a proteomic approach to get further insight into the pattern of O-GlcNAc-modified proteins and the biological significance of this post-translational modification. Figure Detection of biotinylated O-GlcNAz proteins in MCF-7 cells
Keywords: O-GlcNAc; Click chemistry; Azido sugar; Biotin probe; Protein labelling
Isotope dilution quantification of ultratrace gamma-glutamyl-Se-methylselenocysteine species using HPLC with enhanced ICP–MS detection by ultrasonic nebulisation or carbon-loaded plasma by Heidi Goenaga Infante; María del Carmen Ovejero Bendito; Carmen Cámara; Linda Evans; Ruth Hearn; Sven Moesgaard (2099-2106).
A method for the accurate determination of ultratrace selenium species of relevance to cancer research, such as gamma-glutamyl-Se-methylselenocysteine (γ-glutamyl-SeMC), using species-specific double isotope dilution analysis (IDA) with HPLC–ICP–MS is reported for the first time. The 77Se-enriched γ-glutamyl-SeMC spike was produced in-house by collecting the fraction at the retention time of the γ-glutamyl-SeMC peak from a chromatographed aqueous extract of 77Se-enriched yeast, pooling the collected fractions and freeze-drying the homogenate. The Se content of this spike was characterised using reverse isotope dilution mass spectrometry (IDMS) and the isotopic composition of this spike was checked prior to quantification of the natural abundance dipeptide species in garlic using speciated IDA. The extraction of the γ-glutamyl-SeMC species in water was performed in a sonication bath for 2 h after adding an appropriate quantity of 77Se-enriched γ-glutamyl-SeMC to 50 mg of garlic to give optimal 78Se/77Se and 82Se/77Se ratios of 1.5 and 0.6, respectively. The effect of ultrasonic nebulisation, in comparison with the loading of the ICP with carbon (through the addition of methane gas on-line), on the detection of Se associated with γ-glutamyl-SeMC using collision/reaction cell ICP–MS with hydrogen as collision gas was investigated. Sensitivity enhancements of approximately fourfold and twofold were achieved using USN and methane mixed plasma, respectively, in comparison with conventional nebulisation and conventional Ar ICP–MS. However, an approximately twofold improvement in the detection limit was achieved using both approaches (42 ng kg−1 for 78Se using peak height measurements). The use of species-specific IDMS enabled quantification of the dipeptide species at ng g−1 levels (603 ng g−1 Se) in the complex food matrix with a relative standard deviation (RSD, n = 4) of 4.5%, which was approximately half that obtained using standard addition as a confirmatory technique. Furthermore, good agreement was found between the γ-glutamyl-SeMC species concentrations obtained using both calibration methods.
Keywords: Dietary selenium; Garlic; Cancer chemoprevention; Isotope dilution analysis; 77Se-enriched gamma-glutamyl-Se-methylselenocysteine; 77Se-enriched yeast; HPLC–ICP–MS; Water extract; Ultrasonic nebulisation; Carbon-loaded plasma
Intake and excretion of disodium monomethylarsonate in horses: a speciation study by Roberta A. Assis; Ivo L. Kuchler; Norbert Miekeley; Marta B. Tozzi (2107-2113).
Capillary electrophoresis coupled to inductively coupled plasma mass spectrometry was used in a speciation study on disodium monomethylarsonate (DS-MMAV) and its metabolites in horses, to which the drug was administered by intramuscular injection on five consecutive days at a single arsenic dosage of 270 mg day−1. Samples of urine, whole blood, plasma, and mane hair were analyzed before, during, and after drug administration. The data show that blood clearing and urinary excretion of MMA is a fast process following first-order kinetics with biological half-lives of about 38 h and 44 h for urine and plasma, respectively. In the time period of 9 days studied, the only metabolite detected in urine was dimethylarsinic acid (DMAV), which 4 days after the last drug administration accounted for up to 75% of the total excreted arsenic species. This shows, for the first time, that biomethylation of MMAV to DMAV is the principal metabolic pathway of this drug in horses. Although DS-MMAV was administered only during a short 5-day period, an up to six fold increase of arsenic could be measured in the newly grown mane hair.
Keywords: Capillary electrophoresis; ICPMS; Arsenic speciation; Biomethylation; Horses
Validation of methodology for determination of the mercury methylation potential in sediments using radiotracers by Suzana Žižek; Sergio Ribeiro Guevara; Milena Horvat (2115-2122).
Experiments to determine the mercury methylation potential were performed on sediments from two locations on the river Idrijca (Slovenia), differing in ambient mercury concentrations. The tracer used was the radioactive isotope 197Hg. The benefit of using this tracer is its high specific activity, which enables spikes as low as 0.02 ng Hg2+ g−1 of sample to be used. It was therefore possible to compare the efficiency of the methylation potential experiments over a range of spike concentrations from picogram to microgram levels. The first part of the work aimed to validate the experimental blanks and the second part consisted of several series of incubation experiments on two different river sediments using a range of tracer additions. The results showed high variability in the obtained methylation potentials. Increasing Hg2+ additions gave a decrease in the percentage of the tracer methylated during incubation; in absolute terms, the spikes that spanned four orders of magnitude (0.019–190 pg g−1 of sediment slurry) resulted in MeHg formation between 0.01 and 0.1 ng MeHg g−1 in Podroteja and Kozarska Grapa. Higher spikes resulted in slightly elevated MeHg production (up to a maximum of 0.27 ng g−1). The values of methylation potential were similar in both sediments. The results imply that the experimental determination of mercury methylation potential strongly depends on the experimental setup itself and the amount of tracer added to the system under study. It is therefore recommended to use different concentrations of tracer and perform the experiments in several replicates. The amount of mercury available for methylation in nature is usually very small. Therefore, adding very low amounts of tracer in the methylation potential studies probably gives results that have a higher environmental relevance. It is also suggested to express the results obtained in absolute amounts of MeHg produced and not just as the percentage of the added tracer.
Keywords: Mercury; Methylmercury; Mercury methylation; 197Hg radiotracer; Sediment
Comparison of methods with respect to efficiencies, recoveries, and quantitation of mercury species interconversions in food demonstrated using tuna fish by Laura H. Reyes; G. M. Mizanur Rahman; Timothy Fahrenholz; H. M. Skip Kingston (2123-2132).
Eight different analytical extraction procedures commonly used to extract mercury species from biological samples were evaluated by analyzing Tuna Fish Tissue Certified Reference Material (ERM-CE464) certified for the content of total mercury and methylmercury. Speciated isotope dilution mass spectrometry (SIDMS; US Environmental Protection Agency’s method 6800) was utilized to evaluate and effectively compensate for potential errors during measurement and accurately quantify mercury species using all the extraction methods. SIDMS was used to accurately evaluate species transformations during sample pretreatment, preparation and analysis protocols. The extraction methods tested in this paper were based on alkaline extraction with KOH or tetramethylammonium hydroxide; acid leaching with HCl, HNO3 or CH3COOH; extraction with l-cysteine hydrochloride; and enzymatic digestion with protease XIV. Detection of total mercury and mercury species from all extraction methods was carried out by inductively coupled plasma mass spectrometry (ICP-MS) and high-performance liquid chromatography–ICP-MS, respectively. Microwave-assisted extraction and ultrasound-assisted extraction were found to be the most efficient alkaline digestion protocols that caused the lowest levels of transformation of mercury species (6% or less). Extraction with 5 M HCl or enzymatic digestion with protease resulted in the second-highest extraction efficiency, with relatively lower transformation of methylmercury to inorganic mercury (3 and 1.4%, respectively). Despite frequent use of acid leaching for the extraction of mercury species from tuna fish samples, the lowest extraction efficiencies and the highest mercury species transformation were obtained when microwave-assisted extraction with 4 M HNO3 or CH3COOH was used. Transformations as high as 30% were found using some literature protocols; however, all the extractions tested produced accurate quantitation when corrected in accordance with the SIDMS method standardized in the US Environmental Protection Agency’s method 6800. Figure Determinative CRM Tuna Fish Tissue Methylmercury Calibration vs. Determinative Calculation.
Keywords: Mercury speciation; Speciated isotope dilution mass spectrometry; EPA method 6800; Extraction; High-performance liquid chromatography–inductively coupled plasma mass spectrometry; Tuna fish
Application of methyl parathion hydrolase (MPH) as a labeling enzyme by Wei Yang; Ya-Feng Zhou; He-Ping Dai; Li-Jun Bi; Zhi-Ping Zhang; Xiao-Hua Zhang; Yan Leng; Xian-En Zhang (2133-2140).
Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alkaline phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in solution is the monomer, with a molecular weight of 37 kDa; (3) its turnover number is 114.70 ± 13.19 s−1, which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody–enzyme fusion for enzyme-linked immunosorbent assay (ELISA). To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [–(Gly–Ser)5–] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 μL hemolymph from an infected shrimp and differentiated it from a healthy shrimp. Figure The 3-D structure of MPH. (a) monomer showing C- and N-terminals; (b) the crystal structure of the dimer
Keywords: Methyl parathion hydrolase; Labeling enzyme; Single-chain variable fragment; Fusion protein; White spot syndrome virus
Development and application of molecularly imprinted polymers as solid-phase sorbents for erythromycin extraction by Suquan Song; Aibo Wu; Xizhi Shi; Rongxiu Li; Zhixin Lin; Dabing Zhang (2141-2150).
Six molecularly imprinted polymers (MIPs) of erythromycin (ERY) were prepared by noncovalent bulk polymerization using methacrylic acid (MAA) as the functional monomer. On the basis of binding analysis, the MIPs with 1:2 optimum ratio of template to MAA were selected for subsequent scanning electron microscopy and Brunauer–Emmett–Teller analyses, which indicated that the MIPs had more convergent porous structures than the nonimprinted polymers. The equilibrium binding experiments showed that the binding sites of MIPs were heterogeneous, with two dissociation constants of 0.005 and 0.63 mg mL−1, respectively. Furthermore, the performance of the MIPs as solid-phase extraction (SPE) sorbents was evaluated, and the selectivity analysis showed that the MIPs could recognize ERY with moderate cross-reactivity for other macrolides. The overall investigation of molecularly imprinted SPE for cleanup and enrichment of the ERY in pig muscle and tap water confirmed the feasibility of utilizing the MIPs obtained as specific SPE sorbents for ERY extraction in real samples. Figure Schematic diagram of the preparation and application of the erythromycin imprinted molecularly imprinted polymers
Keywords: Molecularly imprinted polymers; Erythromycin; Solid-phase extraction; Pig muscle; Tissue sample
Fast purification of trace vitellogenin from Chinese rare minnow using protein A-immobilized antibody by Maoyong Song; Xuefei Lv; Hailin Wang; Guibin Jiang (2151-2157).
Vitellogenin (Vtg) is a highly responsive biomarker for environmental exposure to various estrogenically active compounds. Here we present a simple, fast, mild, and stable immobilization of anti-Vtg antibody, and demonstrate its powerful applications for preconcentration and purification of fish Vtg proteins, allowing for the monitoring and screening of environmental exposure to estrogenically active compounds. In this immobilization method, rabbit antiserum containing a specific polyclonal antibody against Vtg was directly immobilized on an antibody-binding Staphylococcal protein A matrix (SpA) without the need for prior purification. Under the unique elution conditions, the Vtg protein can be eluted out alone without any leaked specific antibody. The developed method was further used to purify Vtg from whole-body homogenate of Chinese rare minnow. Compared with previous purification methods, the isolated Vtg fraction by this method displays higher purity and well-preserved structure integrity. Moreover, our method is eight times faster. The simple one-step protein A-based specific antibody immobilization and its associated elution strategy may be extended to a number of antibodies for various application purposes, highlighting the paramount advantages over traditional immunoprecipitation and covalent immobilization of antibodies, and suggesting a wide range of promising applications in environmental monitoring and proteome analysis.
Keywords: Vitellogenin (Vtg); Affinity purification; Immobilized antibody
Novel application of modified multiwalled carbon nanotubes as a solid phase extraction adsorbent for the determination of polyhalogenated organic pollutants in aqueous solution by Mohamed Abdel Salam; Robert Burk (2159-2170).
This paper describes a novel application of pristine and chemically modified multiwalled carbon nanotubes (MWCNTs) as the packing materials for the determination of different polyhalogenated organic pollutants, pentachlorophenol, 2,4,5-trichlorophenol, 3,3′,4,4′-tetrachlorobiphenyl and 2,2′,5,5′-tetrabromobiphenyl, from aqueous solution based on solid phase extraction. The modified MWCNTs were characterized using different techniques and the results revealed the successful modification of the MWCNTs with octadecyl amine and poly(ethylene glycol), separately. Factors that maybe influence the preconcentration efficiency, such as sample flow rate, adsorbent mass, sample pH and sample volume, were studied. Desorption of the target analytes was studied using different solvents and the results showed that acetone was the best solvent for all the analytes compared with methanol and hexane. All the results indicated that the proposed method could be used for the simultaneous determination of different pollutants in environmental water samples at trace levels.
Keywords: Multiwalled carbon nanotubes; Modification; Solid phase extraction; Polyhalogenated organic pollutants; Aqueous solution
Electroanalytical performance of a terbium(III)-selective sensor based on a neutral ionophore in environmental and medicinal samples by V. K. Gupta; A. K. Singh; Barkha Gupta (2171-2181).
A new highly selective terbium(III) electrode was prepared with a polymeric film doped using S-2-benzothiazolyl-2-amino-α-(methoxyimino)-4-thiazolethiol acetate as an electroactive material, benzyl acetate (BA) as a plasticizer, and potassium tetrakis(4-chlorophenyl) borate (KTpClPB) as an anionic site in the percentage ratio 3.17:1.58:63.4:31.7 (ionophore-KTpClPB-BA-PVC, w/w). The electrode exhibited a linear response with a near Nernstian slope of 19.5 mV/decade within the concentration range 1.5 × 10−7−1.0 × 10−2 M terbium ions, with a working pH range from 2.0 to 8.0, and a fast response time of 10 s and presented satisfactory reproducibility. The limit of detection was 9.3 × 10−8 M. The results show that this electrode can be used in ethanol media up to 30% (v/v) concentration without interference. It can be used for 3 months without any considerable divergence in the potentials. Selectivity coefficients for terbium(III) with respect to many cations were investigated. The electrode is highly selective for terbium(III) ions over a large number of monovalent, bivalent, and trivalent cations. This shows the valuable property of the proposed electrode. The stability constant of the ionophore towards Tb3+ ions was determined with the sandwich membrane method. It was successfully used as an indicator electrode in potentiometric determination of terbium(III) ions with EDTA and in direct determination in tap water and binary mixtures with quantitative results. The utility of the proposed electrode was also determined in the presence of ionic and nonionic surfactants and in the presence of fluoride ions in four pharmaceutical (mouthwash) preparations. Figure Structure of S-2-Benzothiazolyl-2-amino-α-(methoxyimino)-4-thiazolethiol acetate
Keywords: Terbium; Neutral ionophore; Potentiometry; Ion-selective electrodes; Stability constant
Size characterization of drug-loaded polymeric core/shell nanoparticles using asymmetrical flow field-flow fractionation by Dong Young Kang; Mi Jung Kim; Sun Tae Kim; Keun Sang Oh; Soon Hong Yuk; Seungho Lee (2183-2188).
Asymmetrical flow field-flow fractionation (AsFlFFF) was used to determine the size distribution of drug-loaded core/shell nanoparticles which have a lipid core of lecithin and a polymeric shell of a Pluronic. AsFlFFF provided separation of the drug-loaded core/shell nanoparticles from smaller coreless polymeric micelles, thus allowing accurate size analysis of the drug-loaded nanoparticles without interference by the coreless micelles. It was found from AsFlFFF that the drug-loaded nanoparticles have broad size distributions ranging from 100 to 600 nm in diameter. It was also found that, after the nanoparticles had been stored for 70 days, they disappeared as a result of self-degradation. Being a separation technique, AsFlFFF seems to be more useful than transmission electron microscopy or dynamic light scattering for size analysis of core/shell nanoparticles, which have broad and bimodal size distributions. Figure Separation by AsFlFFF
Keywords: Asymmetrical flow field-flow fractionation; Hydrodynamic diameter; Drug-loaded core/shell nanoparticles; Size distribution
PowerSlicing to determine fluorescence lifetimes of water-soluble organic matter derived from soils, plant biomass, and animal manures by Tsutomu Ohno; Zheming Wang; Rasmus Bro (2189-2194).
Time-resolved fluorescence spectroscopy was used to characterize water-soluble organic matter (WSOM) which plays an important role in soil ecosystem processes. WSOM was extracted from plant biomass, animal manures, and soils from controlled cropping systems studies with known histories of organic amendments. Lifetime constants were derived using the multi-way PowerSlicing method which provides a non-iterative, multi-exponential fitting of decay profiles. The lifetimes obtained by PowerSlicing were not significantly different from those obtained using the traditional discrete components analysis. The three components attributed to WSOM had lifetimes of 0.38 ± 0.14, 2.11 ± 0.72, and 7.08 ± 1.18 ns which are in agreement with previous lifetimes reported for humic substances. This study provides further support for the new paradigm for the structure of soil organic matter where the organic matter is composed of low-molecular-weight components held together by hydrogen bonding and hydrophobic interactions.
Keywords: Fluorescence spectroscopy; Dissolved organic matter; Lifetimes; Parallel factor analysis