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Analytical and Bioanalytical Chemistry (v.378, #3)

Cultures and languages: the worldview of science by John Fetzer (pp. 545-546).

Endocrine disruptors by Damià Barceló; Antonius Kettrup (pp. 547-548).

Endocrine disrupting compounds and other emerging contaminants in the environment: A survey on new monitoring strategies and occurrence data by M. Petrovic; E. Eljarrat; M. J. Lopez de Alda; D. Barceló (pp. 549-562).

An overview of Toxicity Identification and Evaluation (TIE) procedures, used for the effect-based analysis of endocrine disrupting compounds (EDCs) in environmental samples, is presented. Future trends in advanced chemical analysis of EDCs and some emerging contaminants are outlined. The review also gives an overview of concentration levels found in environmental samples and discusses the correlation of calculated estrogenicity (based on chemical analysis of target EDCs) with that measured by various bioassays.

Keywords: Toxicity Identification and Evaluation (TIE) procedures; Endocrine disrupting compounds; Emerging contaminants; Environmental analysis

Immunochemical determination of xenobiotics with endocrine disrupting effects by M.-C. Estévez-Alberola; M.-P. Marco (pp. 563-575).

This paper is a review with more than 100 references discussing the immunochemical methods reported in the literature for the most important man-made chemicals with suspected endocrine disrupting activity. Details regarding immunizing hapten design, antibody production, and the features (limit of detection, dynamic range, specificity) of the most important immunochemical methods developed (ELISA, FIIA, immunosorbents, immunosensors, etc.) are presented for important environmental pollutants such as bisphenol A, phthalates, alkylphenol polyethoxylates, alkylphenols, polychlorinated biphenyl compounds, and dioxins. Availability of commercial reagents and methods is reported.

Keywords: Immunochemical techniques; Endocrine disruptors; Alkylphenols; Phthalates; Bisphenol A; Dioxins

In-vitro assays for determination of oestrogenic activity by Mark D. Scrimshaw; John N. Lester (pp. 576-581).

At present, in-vitro bioassays are predominantly being seen as tools to identify, through screening programs, whether or not individual chemical compounds have an effect on the endocrine system. However, as the techniques mature, they are likely to find use in the future in monitoring of discharges to the environment for any biological effect and will compliment the range of chemical and biological techniques also available for monitoring environmental quality. Such an approach has already been utilised by a number of workers to fractionate mixtures (e.g. final effluents from STW), to isolate the oestrogenically active components and subsequently identify the compounds which are active. This paper reviews the present state of in-vitro techniques for determination of oestrogenic activity and discusses present approaches to their use in environmental monitoring in conjunction with chemical analyses in toxicity identification and evaluation.

Keywords: Environmental effects; Screening; Binding; Contaminants

Xenoestrogens: mechanisms of action and detection methods by Stefan O. Mueller (pp. 582-587).

Estrogenic compounds exert pleiotropic effects in wildlife and humans, and endogenous estrogens, like 17β-estradiol, regulate growth and development of their target tissues. Environmental, industrial, or naturally occurring chemicals that possess estrogenic and/or antiestrogenic activities are termed xenoestrogens and may interfere with endocrine systems. These xenoestrogens are therefore defined as endocrine-active or endocrine-disrupting compounds. The estrogen receptor (ER) is the major regulatory unit within the estrogen-signaling pathway and the molecular mechanisms of estrogen and ER actions are described briefly. Based on the mechanism of ER action, in vitro test systems are described that can be employed for screening but also for the elucidation of mechanisms of action of (anti)estrogenic compounds. How screening assays and mechanistic studies can aid in human risk assessment for potential endocrine-active compounds is discussed also.

Keywords: Estrogen receptor; ERα; ERβ; Endocrine-active compound; Endocrine disruptor

Biosensors for environmental monitoring of endocrine disruptors: a review article by Sara Rodriguez-Mozaz; Maria-Pilar Marco; Maria J. Lopez de Alda; Damià Barceló (pp. 588-598).

This article provides an overview of the applications of biosensors in analysis and monitoring of endocrine-disrupting compounds (EDCs) in the environment. Special attention is devoted to the various types of physical-chemical signal transduction elements, biological mechanisms employed as sensing elements and techniques used for immobilisation of the bioreceptor molecules on the transducer surface. Two different classes of biosensors for EDCs are considered: biosensors that measure endocrine-disrupting effects, and biosensors that respond to the presence of a specific substance (or group of substances) based on the specific recognition of a biomolecule. Several examples of them are presented to illustrate the power of the biosensor technology for environmental applications. Future trends in the development of new, more advanced devices are also outlined.

Keywords: Environmental monitoring; Endocrine-disrupting compounds; Biosensors

Simultaneous determination of selected endocrine disrupters (pesticides, phenols and phthalates) in water by in-field solid-phase extraction (SPE) using the prototype PROFEXS followed by on-line SPE (PROSPEKT) and analysis by liquid chromatography-atmospheric pressure chemical ionisation-mass spectrometry by P. López-Roldán; M. J. López de Alda; D. Barceló (pp. 599-609).

In this study, a new procedure, based on on-line solid-phase extraction (SPE) and analysis by liquid-chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS), has been developed for the simultaneous, multianalyte determination of 21 selected pesticides, phenols and phthalates in water. SPE was carried out on polymeric PLRP-s cartridges by percolating 20 mL-samples. For sample preconcentration, the performance of a prototype programmable field extraction system (PROFEXS) was evaluated against the commercial laboratory bench Prospekt system used for method development. The Profexs is designed for the automated on-site sampling, SPE preconcentration, and storage of up to 16 samples in SPE cartridges. These cartridges are further eluted and on-line analyzed with the Prospekt coupled to the chromatographic system. In the optimized method, where completely on-line SPE-LC-MS analysis of the samples is carried out with the Prospekt in the laboratory, detection limits lower than 100 ng/L, and satisfactory precision (relative standard deviations <25%) and accuracies (recovery percentages >75%) were obtained for most investigated compounds from the analysis of spiked Milli-Q water. The extraction efficiency achieved with the Profexs was comparable to that of the Prospekt for most compounds and somewhat lower for the most apolar analytes, probably due to adsorption on the pump filters. The completely on-line optimized method was applied to the analysis of surface water, ground water and drinking water from a waterworks in Barcelona. Some pesticides and phenols were found in both surface water and groundwater at ng/L or µg/L levels, but not in the final drinking water. Di(2-ethylhexyl)phthalate (DEHP) was present in all samples investigated, including blanks. To the author's knowledge, this is the first work describing the application of a fully automated on-line SPE-LC-MS method for the simultaneous analysis of pesticides, phenols, and phthalates in water, and the second one that examines the possibilities of the prototype Profexs for automated on-site SPE preconcentration of organic pollutants from water samples.

Keywords: pesticides; phenols; phthalates; water; in-field sampling; on-line analysis

Determination of decabromodiphenyl ether in sediments using selective pressurized liquid extraction followed by GC–NCI-MS by Ethel Eljarrat; Agustina de la Cal; Damià Barceló (pp. 610-614).

A method based on selective pressurized liquid extraction (SPLE) followed by gas chromatography–negative ion chemical ionization-mass spectrometry (GC–NCI-MS) has been evaluated for analysis of decabromodiphenyl ether (PBDE-209) in sediment samples. Instrumental operating conditions such as source temperature and system pressure were optimized in the NCI-MS system, giving an instrumental detection limit of 2 pg. The limit of determination of the entire SPLE–GC–NCI-MS procedure was around 50 pg g⁻¹ dry weight (dw), with repeatability of replicates between 4 and 21% relative standard deviation. Application of the method to 13 different river and marine sediment samples collected in Spain revealed that levels of decabromodiphenyl ether ranged between 2 and 132 ng g⁻¹ dry weight.

Keywords: Decabromodiphenyl ether; Selective pressurized liquid extraction; Sediment

A rapid two-step chromatographic method for the quantitative determination of vitellogenin in fish plasma by Jing Shao; Guoqing Shi; Jingfu Liu; Guibin Jiang (pp. 615-620).

By combining anion-exchange membrane purification with high-performance size-exclusion chromatography (HPSEC) analysis, a two-step chromatographic method was developed for the determination of vitellogenin (Vtg) in fish plasma. Most plasma protein interferences can be removed during anion-exchange membrane purification process. Vtg is eluted from the size-exclusion chromatography column with a retention time of about 9 min and is characterized based on the native molecular weight, with a limit of quantification of 20 μg Vtg mL⁻¹plasma. The spiked recovery and interassay variability were better than 80% and 4.8%. This method was successfully applied to analyze the plasma Vtg levels of loach (Misgurnus angaillicaudatus) and sea catfish (Enchelyopus elongatus). In addition to all the female fish, Vtg is detected in 75% of male loaches and 100% of male sea catfish. The result indicates that some chemicals or unknown factors with estrogenic activity have induced male fish to produce Vtg.

Keywords: Vtg; HPSEC; Membrane purification; Loach; Sea catfish

Development of quantitative vitellogenin-ELISAs for fish test species used in endocrine disruptor screening by Bente M. Nilsen; Karin Berg; Janne K. Eidem; Sven-Inge Kristiansen; François Brion; Jean-Marc Porcher; Anders Goksøyr (pp. 621-633).

The yolk protein precursor vitellogenin (Vtg) in plasma has proved to be a simple and sensitive biomarker for assessing exposure of fish to environmental estrogens. Within international bodies such as the Organization for Economic Cooperation and Development (OECD) work is ongoing to develop screening and testing programmes for endocrine disrupting effects of new chemicals, and in the focus of this development are the fish test species common carp (Cyprinus carpio), fathead minnow (Pimephales promelas), zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes). In this study we have developed quantitative enzyme linked immunosorbent assays (ELISAs) for Vtg in common carp/fathead minnow, zebrafish and Japanese medaka. The assays were developed using a combination of monoclonal and polyclonal fish Vtg antibodies in a sandwich format, using stabilized Vtg from the test species as a standard. The carp Vtg ELISA has a working range of 1–63 ng/mL, a minimal detection limit of 0.6 ng/mL, and may also be used for quantification of Vtg in fathead minnow. In fathead minnow whole-body homogenate samples, the practical detection limit is 400 ng/mL due to the matrix effect. The zebrafish Vtg ELISA has a working range of 0.5–63 ng/mL, a minimal detection limit of 0.4 ng/mL, and a practical detection limit of 200 ng/mL in whole-body homogenate samples. The medaka Vtg ELISA has a working range of 0.25–16 ng/mL, a minimal detection limit of 0.1 ng/mL, and a practical detection limit of 125 ng/mL in whole-body homogenate samples. The intra- and inter-assay variations were below 20% for all assays. The assays were evaluated with sets of representative samples spanning the wide dynamic range of Vtg-levels found in fish exposed to environmental estrogens, and all three assays are currently undergoing international inter-laboratory validation.

Express detection of nonylphenol in water samples by fluorescence polarization immunoassay by Julia N. Yakovleva; Anna Yu. Lobanova; Ekaterina A. Shutaleva; Maria A. Kourkina; Andrey A. Mart’ianov; Anatoly V. Zherdev; Boris B. Dzantiev; Sergei A. Eremin (pp. 634-641).

ω-(4-hydroxyphenyl)nonanoic or ω-(4-hydroxyphenyl)heptanoic acid NP derivatives designed to mimic the linear NP isomer;4-aminophenol, which potentially mimics various substituted phenolic compounds with different side-chain structures at position 4 of the benzene ring; anda mixture of branched NP isomers, conjugated to the carrier protein via a benzene ring by the Mannich reaction, and expected to be the closest mimic of NP structure by preserving its natural alkyl moiety. Fluorescence polarization immunoassays based on different combinations of antibody and labeled antigen for screening detection of NP were developed and structural aspects of assay sensitivity and specificity were investigated. The assays based on the antisera raised against ω-(4-hydroxyphenyl)nonanoic acid and NP conjugate via Mannich reaction are capable of express detection of NP with detection limit of 7 μg mL⁻¹and assay dynamic range of 18–300 μg mL⁻¹.

Keywords: Nonylphenol; Endocrine-disrupting chemicals; Antibody production; Fluorescence polarization immunoassay; Cross-reactivity

Application of chemometric methods to the investigation of main microcontaminant sources of endocrine disruptors in coastal and harbour waters and sediments by Emma Peré-Trepat; Mira Petrovic; Damià Barceló; Romà Tauler (pp. 642-654).

Identification, resolution and distribution of main microcontaminant sources of endocrine disruptors in Spanish harbours, coastal waters and sediments are investigated using chemometric methods. We investigated eighteen different endocrine disruptor chemical compounds, including non-ionic surfactants, their degradation products and linear alkylbenzene sulfonates, found in a total number of 74 samples (35 water samples and 39 sediment samples) over a period of 16 months from March 1999 to July 2000, and in 32 different geographical sites along the Spanish Mediterranean Coast (e.g. Barcelona, Tarragona, Almeria Harbour, Malaga and the Bay of Cadiz). Main environmental contamination sources of these endocrine disruptor compounds were investigated and interpreted according to their chemical composition and according to their resolved geographical distribution profiles.

Keywords: Principal component analysis (PCA); Multivariate curve resolution alternating least-squares (MCR-ALS); Endocrine disruptors; Surfactants; Degradation products; Coastal waters; Marine sediments

Use of reporter cell lines for detection of endocrine-disrupter activity by Philippe Willemsen; Marie-Louise Scippo; Gudrun Kausel; Jaime Figueroa; Guy Maghuin-Rogister; Joseph A. Martial; Marc Muller (pp. 655-663).

We have studied stable transformed human mammary cell lines with highly inducible steroid receptor-mediated luciferase reporter gene expression. Cells responding specifically to glucocorticoids, progestagens, androgens, or estrogens are described and characterized. The use of this high-throughput, cell-based assay for analysis of steroid (ant)agonists is reported. Systematic characterization of endocrine-disrupting activity on human receptors and in a human-cell system is interpreted for a selection of xenobiotics. We show that the phytoestrogens apigenin and genistin have progestagenic and androgenic activity, respectively. Finally, application of cell-based assays to the analysis of environmental samples is discussed.

Keywords: Steroid hormones; Reporter gene; Endocrine disrupters; Residues

Recombinant human estrogen, androgen and progesterone receptors for detection of potential endocrine disruptors by Marie-Louise Scippo; Catherine Argiris; Cécile Van De Weerdt; Marc Muller; Philippe Willemsen; Joseph Martial; Guy Maghuin-Rogister (pp. 664-669).

This work reports the binding capacity of various chemicals (so-called endocrine disruptors) to recombinant human steroid receptors (hERα, hPR and hAR). The tested chemicals are organochlorine insecticides (DDT and its metabolites, methoxychlor, aldrin, dieldrin, chlordecone, lindane, trichlorobenzene), estrogenic insecticides (endosulfan, toxaphene, nonachlor), herbicides (alachlor and atrazine), fungicides (benomyl and vinclozolin), industrial chemicals (nonylphenol, bisphenol A, diphenylphtalate), antioxidants (butylated hydroxyanisol) and some phytoestrogens. Except for phytoestrogens, most of the tested chemicals (DDT and its metabolites, aldrin, α- and β-endosulfan, toxaphen, trans-nonachlor) show higher affinities for hPR than for hERα, indicating that the interaction with the progesterone receptor could contribute to the endocrine-disrupting effects imputed to these chemicals. We propose to use binding assays using recombinant human steroid receptors as screening tools for the detection of endocrine disruptors in various samples.

Keywords: Endocrine disruptors; Steroid receptors; Binding assay; Pesticides; Radioreceptor assay

Use of vitellogenin mRNA as a biomarker for endocrine disruption in feral and cultured fish by Natàlia García-Reyero; Demetrio Raldúa; Laia Quirós; Gisela Llaveria; Joan Cerdà; Damià Barceló; Joan O. Grimalt; Benjamin Piña (pp. 670-675).

The presence of the female-specific yolk protein precursor vitellogenin in blood and liver from male fish is widely used as an indicator of endocrine disruption. We studied the induction of vitellogenin mRNA in liver from several species of fish, both maintained in fish tanks or captured in the wild. Our procedure requires minute amounts of liver samples (down to 50 mg), and can be applied to field samples if the appropriate RNA-stabilisation agent is used. We used reverse-transcriptase PCR and quantitative real-time PCR for detection and precise quantitation of vitellogenin mRNA levels. Male mummichog (Fundulus heteroclitus) exposed to 17β-estradiol contained levels of vitellogenin mRNA up to 30 times higher than in untreated females and treatment with nonylphenol resulted in a weak but consistent induction of this transcript. We also studied levels of vitellogenin mRNA in a population of common carp (Cyprinus carpio) from the Anoia river, a river known for its high levels of estrogenic alkylphenols. The results were consistent with recorded data for fish from this sampling site. Finally, we also detected vitellogenin mRNA in barbs (Barbus graellsi), a species for which no vitellogenin sequence was available. The use of mRNA quantitation techniques for analysis of feral and cultured fish of different species opens the possibility of more precise detection and further control of the noxious effects of contaminants on the local fauna exposed to them.

Keywords: Vitellogenin mRNA; Biomarker; Endocrine disruption; Fish; Mummichog (Fundulus heteroclitus); Common carp (Cyprinus carpio); Barb (Barbus graellsi)

Retinol-binding protein as a biomarker to assess endocrine-disrupting compounds in the environment by Gregor Levy; Ilka Lutz; Angela Krüger; Wolf von Tümpling; Werner Kloas (pp. 676-683).

Endocrine-disrupting compounds (EDC) are predominantly investigated with respect to their ability to mimic or block estrogenic actions. However, it is well-known that EDC can act as agonists or antagonists of androgen- and estrogen-response systems. For that reason, there is an obvious need for bioassays providing the possibility of detecting (anti-)estrogenic and (anti-)androgenic effects. The retinol-binding protein (RBP) seems to be a useful molecular biomarker for assessing all modes of action of EDC, because it is regulated by sex steroid hormones. This study was conducted to establish RBP as a biomarker for determination of (anti-)estrogenic and (anti-)androgenic effects of EDC using a Xenopus laevis primary hepatocyte culture system. It could be shown that RBP mRNA expression in X. laevis hepatocytes was stimulated by estrogens in a dose-dependant manner whereas a combination of estrogen and androgen or estrogen and anti-estrogen treatment suppressed estrogenic stimulating effects. Androgens testosterone and dihydrotestosterone were able to reduce RBP mRNA expression and the anti-androgen vinclozolin could abolish the mRNA synthesis-suppressing activity of the androgen dihydrotestosterone. These results clearly demonstrated that RBP mRNA expression patterns in Xenopus laevis hepatocytes have different modes of (anti-)estrogenic and (anti-)androgenic action and can be used for examination of suspected EDC. Moreover, water samples from sewage-treatment plant effluents were applied to liver cells and expression levels of RBP and estrogen receptor mRNA (a known estrogenic biomarker) were detected. These samples had high estrogenicity but caused low to moderate induction of RBP mRNA synthesis, leading to the conclusion that RBP levels represent the sum of all possible effects (estrogenic and other effects) of EDC in environmental samples.

Keywords: Retinol-binding protein; Endocrine-disrupting compounds; Xenopus laevis ; Hepatocyte culture; Environmental samples

Luminescent enzyme-linked receptor assay for estrogenic compounds by Martin Seifert (pp. 684-687).

The analytics of endocrine-disrupting compounds has become a major issue during recent years. Several test systems have been developed for endocrine-disrupting chemicals. Yeast reporter gene assays and MCF-7 cell-based proliferation assays (E-screen) are particularly popular. A correlation of an enzyme-linked receptor assay (ELRA) with a yeast reporter gene assay is shown. In addition, the development of an ultra-sensitive luminescent ELRA with a detection limit of 20 ng/L for 17β-estradiol in the sample is reported. Data for real sample analysis are shown in this paper. ELRA characteristics are compared with cell-based assays, and the issue of detection limits is addressed. In this context, the detection limits of the cell-based assays have been claimed to be below the ELRA detection limits. However, it is clarified that the given detection limits for the yeast estrogen screen and the E-screen are usually based on concentrations of 17β-estradiol in the well, not in the sample, whereas ELRA detection limits are concentrations in the sample.

Keywords: Estrogens; Receptor assay; ELRA; Yeast screen; Endocrine disruptor

Combined biological and chemical assessment of estrogenic activities in wastewater treatment plant effluents by Hans-Rudolf Aerni; Bernd Kobler; Barbara V. Rutishauser; Felix E. Wettstein; René Fischer; Walter Giger; Andreas Hungerbühler; M. Dolores Marazuela; Armin Peter; René Schönenberger; A. Christiane Vögeli; Marc J.-F. Suter; Rik I. L. Eggen (pp. 688-696).

Five wastewater treatment plant effluents were analyzed for known endocrine disrupters and estrogenicity. Estrogenicity was determined by using the yeast estrogen screen (YES) and by measuring the blood plasma vitellogenin (VTG) concentrations in exposed male rainbow trout (Oncorhynchus mykiss). While all wastewater treatment plant effluents contained measurable concentrations of estrogens and gave a positive response with the YES, only at two sites did the male fish have significantly increased VTG blood plasma concentrations after the exposure, compared to pre-exposure concentrations. Estrone (E1) concentrations ranged up to 51 ng L⁻¹, estradiol (E2) up to 6 ng L⁻¹, and ethinylestradiol (EE2) up to 2 ng L⁻¹ in the 90 samples analyzed. Alkylphenols, alkylphenolmonoethoxylates and alkylphenoldiethoxylates, even though found at µg L⁻¹ concentrations in effluents from wastewater treatment plants with a significant industrial content, did not contribute much to the overall estrogenicity of the samples taken due to their low relative potency. Expected estrogenicities were calculated from the chemical data for each sample by using the principle of concentration additivity and relative potencies of the various chemicals as determined with the yeast estrogen screen. Measured and calculated estradiol equivalents gave the same order of magnitude and correlated rather well (R ²=0.6).

Keywords: Endocrine disruption; Fish exposure; Yeast estrogen screen; Vitellogenin; Steroid hormones; Nonylphenol ethoxylates

Integrated procedure for determination of endocrine-disrupting activity in surface waters and sediments by use of the biological technique recombinant yeast assay and chemical analysis by LC–ESI-MS by Raquel Céspedes; Mira Petrovic; Demetrio Raldúa; Úrsula Saura; Benjamín Piña; Sílvia Lacorte; Paula Viana; Damià Barceló (pp. 697-708).

An integrated procedure using mass spectrometry and molecular biology for determination of estrogenicity in natural waters and sediments is reported. Solid-phase extraction (SPE) and pressurized-liquid extraction (PLE), respectively, were used for isolation of endocrine-disrupting compounds (EDC) from surface waters and sediments, followed by liquid chromatography–mass spectrometry using an electrospray interface (LC–ESI-MS). Twenty seven EDC were determined: non-ionic surfactants (nonylphenol ethoxylate), alkylphenols (e.g. nonylphenol and octylphenol), bisphenol A, phthalates, and natural and synthetic steroid sex hormones. Limits of detection varied from 0.02 to 0.22 μg L⁻¹ and from 1 to 10 μg kg⁻¹ in water and sediments, respectively. Recoveries ranged from 65 to 125% and 73 to 97% for waters and sediments, respectively. In addition to LC–ESI-MS determination, extracts obtained by SPE and PLE were analyzed by the recombinant yeast assay (RYA) to assess total estrogenic activity. This bioassay detects natural estrogens and xenoestrogens, producing a quantitative measurement of EDC irrespective of the identity of the chemical responsible for the activity. As a novelty, a relative estrogenicity factor was determined for 19 analytes with EC ₅₀ values ranging from 10⁻¹⁰ to 10⁻⁹ mol L⁻¹ for synthetic estrogens, from 10⁻⁷ to 10⁻⁵ mol L⁻¹ for alkylphenol derivatives, and from 10⁻⁵ to 10⁻⁴ mol L⁻¹ for phthalates and benzothiazoles. By use of this integrated chemical–ecotoxicological approach good correlation was usually established between chemical composition and estrogenic effects for surface water and sediment samples from Portugal. Estrogenic activity observed was mainly attributed to the presence of nonylphenolic compounds (with concentrations of NP ranging from 0.1 up to 44 μg L⁻¹ in waters and up to 1172 μg kg⁻¹ in sediments), and to the sporadic presence of estrogens, detected at ng L⁻¹ levels.

Keywords: Endocrine-disrupting compounds; Estrogenicity; Recombinant yeast assay; Liquid chromatography–mass spectrometry.

Bioassay-directed chemical analysis utilizing LC–MS: a tool for identifying estrogenic compounds in water samples? by Inga Heisterkamp; Juergen Gandrass; Wolfgang Ruck (pp. 709-715).

A bioassay-directed chemical analysis (BDCA) scheme has been developed which combines a yeast screen for estrogenic activity with LC–MS detection after liquid–liquid extraction and fractionation by size exclusion chromatography. Focusing on sewage-treatment plant (STP) effluents, the approach aims at characterizing the substances responsible for estrogenic effects in aquatic systems. Initial results show a strong response of STP effluent extracts in the yeast screen. Estrone, bisphenol A, and nonylphenol have been identified as substances being partly responsible for observed estrogenic activity. However, confirmation experiments with synthetic samples revealed that the estrogenic effect potentials of the samples could not be completely assigned to specific compounds. Further improvement of the limits of detection of the analytical scheme is needed to enable identification and quantification of potent estrogenic compounds at low concentrations.

Keywords: Bioassay-directed chemical analysis; Estrogenic activity; Saccharomyces cerevisiae ; LC–MS–MS; Liquid–liquid extraction; Sewage-treatment plant effluent

Effects of 17α-ethinylestradiol on zoo- and phytoplankton in lentic microcosms by Burkhard A. Hense; Gabriele F. Severin; Gerhard Welzl; Karl-Werner Schramm (pp. 716-724).

The effects of 17α-ethinylestradiol (EE), an endocrine disruptor, on zoo- and phytoplankton were studied in outdoor 230-L still-water microcosms. Cell density and biomass, diversity, and community composition were analyzed. Five microcosms were treated by controlled release for six weeks, three by direct application of EE. To investigate recovery, sampling was continued for four weeks after treatment. Most characteristics of the zooplankton were not unambiguously affected by EE. Only the relative density of copepods, especially of their larvae, decreased significantly after EE application. For phytoplankton, no unambiguous concentration- or toxodose-correlated effects on any biotic characteristics could be found. However, most properties of the phytoplankton deviated from those of controls, i.e. tended to be smaller (number of species per microcosm, biomass, cell density) or covered a wider range (diversity, evenness). PCA indicated a shift of species structure in the treated microcosms. This was supported by the species scores calculated by the principal response curve method, although the principal response curve itself showed no clear EE-correlated shifts. High variability within the biocenosis between microcosms and over time, probably because of disturbance of the ecosystem before starting of the test, might have superimposed EE-dependent effects.

Keywords: 17α-Ethinylestradiol; Zooplankton; Phytoplankton; Endocrine disruptor

Analysis of nitrated polycyclic aromatic hydrocarbons by liquid chromatography with fluorescence and mass spectrometry detection: air particulate matter, soot, and reaction product studies by Christian Schauer; Reinhard Niessner; Ulrich Pöschl (pp. 725-736).

Polycyclic aromatic hydrocarbons (PAH) and their nitrated derivatives (nitro-PAH) are environmental pollutants which pose a threat to human health even at low concentration levels. In this study, efficient analytical methods for the analysis of nitro-PAH and PAH (extraction, clean-up, chromatographic separation, and spectrometric detection) have been developed, characterized, and applied to aerosol samples. The separation and quantification of 12 nitro-PAH was carried out by reversed-phase high performance liquid chromatography (HPLC), on-line reduction, and fluorescence detection. The detection limits were in the range of 0.03–0.5 μg L⁻¹ (6–100 pg in the investigated sample aliquots), and the recovery rates from soot samples were 70–90%. Nitro-PAH and PAH concentrations have been determined for different types of soot and for urban, rural, and alpine fine air particulate matter (PM2.5). For the first time, trace amounts of nitro-PAH have been detected in a high-alpine clean air environment. The on-line reduction and fluorescence technique has been complemented by atmospheric pressure chemical ionization time-of-flight mass spectrometry (APCI-TOF-MS). The MS detection allowed the analysis of partially nitrated and oxygenated PAH in laboratory studies of the heterogeneous reaction of PAH on soot and glass fiber substrates with gaseous nitrogen oxides and ozone. It led to the tentative identification of a previously unknown nitrated derivative of the particularly toxic PAH benzo[a]pyrene (BaP-nitroquinone), and provides the first experimental evidence that PAH-nitroquinones can be formed by reaction of PAH with atmospheric photooxidants.

Keywords: Nitro-PAH; Atmospheric aerosols; HPLC-fluorescence; APCI-TOF-MS; Nitroquinone

Colon cancer risk factors from nutrition by Jens Berlau; Michael Glei; Beatrice L. Pool-Zobel (pp. 737-743).

Nutrition and health are closely connected and malnutrition can seriously endanger health. The consequences are higher risks of developing diseases. Of these, cancers are of special importance. The most frequent nutrition-associated type of cancer is colon cancer. This review summarises the predisposing factors for the development of colon cancer, and molecular mechanisms responsible for sporadic colon cancer. Moreover, it is pointed out that individual genetic predisposition such as polymorphisms of biotransformation systems might affect susceptibility to cancer risk factors. Selected findings of epidemiological research and nutritional habits, foods, and metabolites that could increase cancer risk are discussed. Furthermore, toxicological assessment of food-related risk factors, e.g. heterocyclic amines, and potential protective effects of food ingredients, e.g. the induction of phase II enzymes or removal of already degenerated cells from tissue by apoptosis, inhibition of proliferation, and cell differentiation are discussed. Risks are not necessarily reduced by avoiding certain substances but by healthy and well-balanced nutrition. Knowledge of individual susceptibility will also make it possible to recognise risks and design more specific nutritional recommendations.

Keywords: Heterocyclic amines; Colon cancer; Nutrition; Toxicological assessment; Chemoprotection

Immunosensor for estrone with an equal limit of detection as common analytical methods by Jens Tschmelak; Guenther Proll; Guenter Gauglitz (pp. 744-745).

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. Biosensors have suitable characteristics such as efficiency in allowing very fast, sensitive, and cost-effective detection. Here we describe a fully automated immunoassay for estrone with 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 basis of our sensitive assay is the antibody with a high affinity constant towards estrone. The very low amount of antibody per sample results in low validation parameters (LOD, LOQ, and IC50), but this assay for estrone represents the current device-related limitation of the River Analyser (RIANA).

Keywords: River analyser; Optical immunosensor; Total internal reflection fluorescence; Estrogens; Environmental analysis

Size-based analysis of incinerator fly ash using gravitational SPLITT fractionation, sedimentation field-flow fractionation, and inductively coupled plasma-atomic emission spectroscopy by Won-Suk Kim; Mira Park; Dai Woon Lee; Myeong Hee Moon; Heungbin Lim; Seungho Lee (pp. 746-752).

Fly ash has been regarded as hazardous because of its high adsorption of toxic organic and/or inorganic pollutants. Fly ash is also known to have broad distributions of different chemical and physical properties, such as size and density. In this study, fly ash emitted from a solid waste incinerator was pre-fractionated into six sub-populations by use of gravitational SPLITT fractionation (GSF). The GSF fractions were then analyzed by sedimentation field-flow fractionation (SdFFF) and ICP–AES. SdFFF analysis showed the fly ash has a broad size distribution ranging from a few nanometers up to about 50 µm. SdFFF results were confirmed by electron microscopy. Inductively coupled plasma–atomic emission spectroscopy (ICP–AES) analysis of the GSF fractions showed the fly-ash particles contain a variety of inorganic elements including Ca, Si, Mg, Fe, and Pb. The most abundant in fly ash was Ca, followed by Si then Mg. No correlations were found between trace element concentration and particle size.

Keywords: Incinerator fly ash; Inorganic elements; Inductively coupled plasma–atomic emission spectroscopy; Sedimentation field-flow fractionation; Size distribution; SPLITT fractionation

Analysis of neem oils by LC–MS and degradation kinetics of azadirachtin-A in a controlled environment by Sami Barrek; Olivier Paisse; Marie-Florence Grenier-Loustalot (pp. 753-763).

Since it was first isolated, the oil extracted from seeds of neem (Azadirachtin indica A juss) has been extensively studied in terms of its efficacy as an insecticide. Several industrial formulations are produced as emulsifiable solutions containing a stated titer of the active ingredient azadirachtin-A (AZ-A). The work reported here is the characterization of a formulation of this insecticide marketed under the name of Neem-azal T/S and kinetic studies of the major active ingredient of this formulation. We initially performed liquid–liquid extraction to isolate the neem oil from other ingredients in the commercial mixture. This was followed by a purification using flash chromatography and semi-preparative chromatography, leading to ¹³C NMR identification of structures such as azadirachtin-A, azadirachtin-B, and azadirachtin-H. The neem extract was also characterized by HPLC–MS using two ionization sources, APCI (atmospheric pressure chemical ionization) and ESI (electrospray ionization) in positive and negative ion modes of detection. This led to the identification of other compounds present in the extract—azadirachtin-D, azadirachtin-I, deacetylnimbin, deacetylsalannin, nimbin, and salannin. The comparative study of data gathered by use of the two ionization sources is discussed and shows that the ESI source enables the largest number of structures to be identified. In a second part, kinetic changes in the main product (AZ-A) were studied under precise conditions of pH (2, 4, 6, and 8), temperature (40 to 70 °C), and light (UV, dark room and in daylight). This enabled us to determine the degradation kinetics of the product (AZ-A) over time. The activation energy of the molecule (75±9 kJ mol⁻¹) was determined by examining thermal stability in the range 40 to 70 °C. The degradation products of this compound were identified by use of HPLC–MS and HPLC–MS–MS. The results enabled proposal of a chemical degradation reaction route for AZ-A under different conditions of pH and temperature. The data show that at room temperature and pH between 4 and 5 the product degrades into two preferential forms that are hydrolyzed to a single product over time and as a function of pH change.

Keywords: Neem; Triterpenoids; Azadirachtin-A; Degradation; Kinetics; HPLC–MS–MS

Chemical and physical factors affecting the extractability of methidathion from soil samples. by L. Sánchez; M. D. Mingorance; A. Peña (pp. 764-769).

Determination of methidathion in soil samples using Soxhlet extraction has been studied. Several factors were investigated for their effect on methidathion recovery. Some were related to the extraction procedure, for example solvent type used for the extraction (acetone or hexane/toluene), extraction time, soil humidity, and type of sterilisation system employed. Other factors tested included the addition of organic matter to the soil, for example urban sewage sludge and the cationic surfactant TDTMA. Experimental designs were used to determine the effects of the different factors. Acetone resulted in higher recoveries and was less affected by the presence of water. Autoclaving was the most appropriate sterilisation method. Thimerosal resulted in a decrease in insecticide recovery. Methidathion recovery increased as the amount of cationic surfactant was increased, but decreased as the amount of sewage sludge added to the soil was increased. In general, because the factors studied were not always independent of each other, a clear description of the methodology used is needed when analysing pollutants in environmental samples.

Keywords: Methidathion; Soil; Experimental designs; Sterilisation; Soil humidity; Treatment of soil with organic matter

Stripping chronopotentiometric measurements of lead(II) and cadmium(II) in soils extracts and wastewaters using a bismuth film screen-printed electrode assembly by Rashid O. Kadara; Ibtisam E. Tothill (pp. 770-775).

The key to remediative processes is the ability to measure toxic contaminants on-site using simple and cheap sensing devices, which are field-portable and can facilitate more rapid decision-making. A three-electrode configuration system has been fabricated using low-cost screen-printing (thick-film) technology and this coupled with a portable electrochemical instrument has provided a a relatively inexpensive on-site detector for trace levels of toxic metals. The carbon surface of the screen-printed working electrode is used as a substrate for in situ deposition of a metallic film of bismuth, which allows the electrochemical preconcentration of metal ions. Lead and cadmium were simultaneously detected using stripping chronopotentiometry at the bismuth film electrode. Detection limits of 8 and 10 ppb were obtained for cadmium(II) and lead(II), respectively, for a deposition time of 120 s. The developed method was applied to the determination of lead and cadmium in soils extracts and wastewaters obtained from polluted sites. For comparison purposes, a mercury film electrode and ICP-MS were also used for validation.

Keywords: Cadmium; Lead; Stripping chronopotentiometry; Screen-printed electrode; Bismuth film electrode; Soils extracts; Wastewaters

An optimization procedure for determination of metallothionein by square wave cathodic stripping voltammetry: application to marine worms by Mohamed El Hourch; Arnaud Dudoit; Jean-Claude Amiard (pp. 776-781).

Electrochemical determination of metallothionein (MT) is widely used for environmental studies. This article describes the development and optimization of the procedure for the quantification of metallothionein by square wave cathodic stripping voltammetry. The determination is based on the complexation of cisplatin and MT and the subsequent reduction of the complexes at the electrode. In order to achieve the highest possible sensitivity and resolution of the peak, an optimization of the experimental parameters has been carried out using experimental design methodology (response surface). Seven chemical and physical parameters, namely, pH, cisplatin concentration, buffer concentration, deposition potential, square wave frequency, amplitude of pulse, and step potential, have been optimized to give 9.0, 5.9 µM, 0.65 M, −0.2 mV, 229 Hz, 46 mV, and 2 mV, respectively. Method characterization has been performed, leading to a detection limit of 0.1 µg L⁻¹. Quantification of MT in polychaetes and comparison with the modified Brdička procedure were also carried out.

Keywords: Metallothionein; Square wave cathodic stripping voltammetry; Brdička; Polychaetes; Experimental design; Catalytic hydrogen evolution

Monitoring the air quality around an oil refinery through the use of diffusive sampling by F. De Santis; A. Fino; S. Menichelli; C. Vazzana; I. Allegrini (pp. 782-788).

Diffusive sampling has been used to study the spatial distribution of SO₂, NO₂, NO_x, NH₃ and BTX (benzene, toluene and xylenes) near an oil refinery located in Falconara, Italy, over the period from March to October 2001. Three different categories of sampling sites (roadside, residential and background) were studied. In total, 56 sites were monitored. The results were evaluated on the basis of the limit values found in the European Directives. The results of the defined study indicate that the measured concentrations were substantially lower than the ambient air quality standard with the maximum concentrations being generally found much closer to emission sources. The monitoring method described here can be used to assess integrated concentration levels over long periods of time and to identify pollution “hotspots” where concentrations are likely to be consistently high. Identification of these hotspots may help to assess air quality and to implement proper action plans, especially in locations where industrial and urban pollution coexist.

Keywords: Diffusive sampling; Air pollutants; Air quality assessment; Oil refinery; Ammonia

Speciation of dissolved silicates in natural waters containing alkaline and alkaline-earth ions by Miho Tanaka; Kazuya Takahashi; Yu Vin Sahoo (pp. 789-797).

The concentration of silica in water samples from the desert area of Xinjiang, N. W. China, has been measured by colorimetry with ammonium molybdate. The observed pattern of dependence of the concentration of silica on the concentration of sodium ion (Na⁺) in the water samples is consistent with the pattern obtained by experiments on in-vitro dissolution of silica gel in sodium chloride (NaCl) solution. This indicates that the dissolution of silica in the hydrologic system in this area depends on the concentration of Na⁺. Calcium ion (Ca²⁺), which is known to play an important role on the dissolution of silica on the basis of in-vitro experiments, was observed to take little part in the dissolution of silica in actual natural water samples. This implies that the Ca²⁺ is bound to the hydrogen carbonate anion or that the Ca²⁺ content of natural water containing salts is very low, owing to precipitation. In these samples silicate-Na⁺ was identified as the dissolution species of silica; it was also ascertained that Ca²⁺ did not form complexes with silicate species. These observations resulted from direct identification of dissolved chemical species by use of FAB-MS (fast atom bombardment mass spectrometry). The research indicates that in water samples in this critically arid region the concentration of “dissolved” silica is basically determined by the concentration of Na⁺, indicative of pure inorganic conditions in the desert area of Xinjiang, N.W. China.

Keywords: Arid land; Salt; Chemical speciation; FAB-MS; Dissolved silica; Precipitation

Doehlert matrix for optimisation of procedure for determination of nickel in saline oil-refinery effluents by use of flame atomic absorption spectrometry after preconcentration by cloud-point extraction by Marcos de A. Bezerra; André L. B. Conceição; Sérgio L. C. Ferreira (pp. 798-803).

This paper proposes a preconcentration procedure for determination of nickel in saline aqueous waste samples by flame atomic absorption spectrometry (FAAS). It is based on cloud-point extraction of nickel(II) ions as 2-(5-bromo-2-pyridylazo)-5-diethilaminophenol (Br-PADAP) complexes using octylphenoxypolyethoxyethanol (Triton X-114) as surfactant. The optimisation step was performed using a four-variable Doehlert design, involving the factors centrifugation time (CT) of system after addition of surfactant, solution pH, methanol volume (MV) added at micellar phase, and buffer concentration (BC). The analytical response used was absorbance, after volume correction. Using the established experimental conditions in the optimisation step the procedure enables nickel determination with a detection limit (3δ/S) of 0.2 μg L⁻¹, quantification limit (10δ/S) of 0.7 μg L⁻¹, and precision, calculated as relative standard deviation (RSD) of 4.7 (n=8) and 3.5% (n=8) for nickel concentration of 1 and 5 μg L⁻¹, respectively. The preconcentration factor, determined from the ratio of the slopes of the analytical curves with and without preconcentration, is 74. The recovery achieved for nickel determination in the presence of several cations demonstrated that this procedure could be applied for analysis of water samples. The robustness was checked by using saturated fractional factorial designs, centred on the established experimental conditions in the optimisation step. The results of these tests demonstrated that the variables centrifugation time and buffer concentration are robust for modification by 10% and that solution pH and methanol volume are robust for 5%. Accuracy was evaluated by using the certified material reference SLEW-3 estuarine water for trace metals. The procedure was used for determination of nickel in saline effluents from oil refinery samples. Recovery results (95–104%) indicate that the procedure has satisfactory accuracy for nickel determination in these samples.

Keywords: Oil-refinery waste; Saline effluents; Doehlert matrix; Flame atomic absorption spectrometry; Cloud-point extraction; Nickel

Improved determination of taurine by high-performance anion-exchange chromatography with integrated pulsed amperometric detection (HPAEC-IPAD) by Tommaso R. I. Cataldi; Giovanni Telesca; Giuliana Bianco (pp. 804-810).

The advantages of the high selectivity of high-performance anion-exchange chromatography (HPAEC) and the sensitive response of taurine at a gold electrode with integrated pulsed amperometric detection (IPAD) have been combined, in order to establish a new analytical method for its determination in real matrices. Potential-time settings of the potential waveform were optimized in order to get the highest amperometric response. The separation of taurine in milk samples was achieved using an alkaline eluent (100 mM NaOH) containing 1 mM Ba(OAc)₂ and a column temperature of 15 °C. The inherent merits of using a barium-modified eluent, in terms of taurine separation and detection, are demonstrated. The enhancement in sensitivity under these experimental conditions makes it suitable for taurine determination in milk. Indeed, this method allows high recovery of taurine and satisfies the necessary requirements with respect to accuracy, repeatability and sensitivity with a detection limit of 50 nmol/L, which corresponds to 2.5 pmol. The taurine content in milk samples of some common mammals was evaluated, including human milk. In goat’s milk, the taurine content ranged from 46 to 91 mg/L, whereas human and buffalo milk samples exhibited an average content of 18 mg/L and 23 mg/L, respectively.

Keywords: Taurine; Ion chromatography; Integrated pulsed amperometric detection; Milk

Application of l-cysteine-capped nano-ZnS as a fluorescence probe for the determination of proteins by Chang Qing Zhu; Dan Hua Zhao; Jin Long Chen; Yong Xin Li; Le Yu Wang; Lun Wang; Yun You Zhou; Shu Juan Zhuo; Yu Qin Wu (pp. 811-815).

Nanometer-sized l-cysteine-capped ZnS particles have been synthesized and used as a fluorescence probe to investigate the effect of proteins on fluorescent intensity. With Δλ=190 nm, maximum and constant synchronous fluorescence enhancement was produced at 267 nm and pH 5.12 in the presence of proteins. A highly sensitive synchronous fluorescence method for the rapid determination of proteins has been developed. Under optimum conditions, calibration graphs are linear over the range 0.03–8.0 μg mL⁻¹ for bovine serum albumin (BSA), 0.01–6.0 μg mL⁻¹ for human serum albumin (HSA), 0.05–8.0 μg mL⁻¹ for γ-globulin (γ-G), and 0.04–4.0 μg mL⁻¹ for ovalbumin, respectively. The relative standard deviations of seven replicate measurements were 1.75% for 1.0 μg mL⁻¹ BSA, 1.90% for 1.0 μg mL⁻¹ HSA, 1.65% for 1.0 μg mL⁻¹ γ-G, and 2.32% for 1.0 μg mL⁻¹ ovalbumin.

Keywords: l-Cysteine-capped nano-ZnS; Protein; Synchronous fluorescence

Simple and selective method for determination of iodide in pharmaceutical products by flow injection analysis using the iodine–starch reaction by D. Nacapricha; K. Uraisin; N. Ratanawimarnwong; K. Grudpan (pp. 816-821).

This work exploited the well-known iodine–starch reaction for development of a simple flow-injection (FI) method for determination of iodide in pharmaceutical samples. Iodide in an injected zone was oxidized to iodine. A gas diffusion unit enables selective permeation of iodine through a hydrophobic membrane. Detection was made very selective for elemental iodine by employing formation of the I₃ ⁻–starch complex. The detection limit (3S/N) of the system was 1 mg I L⁻¹. For a liquid patent medicine used for asthma treatment we suggested modification of the system. Direct injection of this sample, which contains a particularly high concentration level of iodide (ca. 9000 mg I L⁻¹), can be achieved by coupling a dialysis unit to the FI system. This has increased the working range to 6000–10,000 mg I L⁻¹ without employing complicated nanoliter injection.

Keywords: Flow injection; Gas diffusion; Dialysis; Iodide; Iodine–starch

Voltammetric study of glibenclamide at carbon paste and Sephadex-modified carbon paste electrodes by A. Radi (pp. 822-826).

The oxidative behaviour of the antidiabetic agent glibenclamide on a bare carbon paste electrode (CPE) and a Sephadex-modified carbon paste electrode (SMCPE) was explored by cyclic and differential pulse voltammetry (DPV). The analysis procedure consisted of an open circuit accumulation step in stirred sample solution of Britton-Robinson buffer (0.04 mol L⁻¹, pH 2.0). This was followed by medium exchange to a clean solution of Britton-Robinson buffer (0.04 mol L⁻¹, pH 5.0), and subsequently an anodic potential scan was effected to obtain the voltammetric peak. The glibenclamide oxidation peak current obtained by DPV was proportional to the concentration of the glibenclamide in the range of 1.0×10⁻⁹ mol L⁻¹ to 5.0×10⁻⁸ mol L⁻¹ for 180 s accumulation time, with a detection limit of 4.0×10⁻¹⁰ mol L⁻¹. A method was developed for the determination of glibenclamide in formulation and spiked human serum. Moreover, the proposed procedure was used to estimate the serum concentrations after oral administration of a 5 mg tablet of glibenclamide to three diabetic subjects.

Determination of two intact glucosinolates in vegetables and Chinese herbs by Zongwei Cai; Ching-Yan Cheung; Wai-Tang Ma; Wai-Man Au; Xiang You Zhang; Albert Lee (pp. 827-833).

A reversed-phase HPLC method was developed for analyzing sinigrin and gluconasturtiin in six vegetable and two Chinese herb samples. A gradient program and mobile phases using methanol and 0.05% trifluoroacetic acid containing 20 mM ammonium acetate allowed sufficient retention and separation of the glucosinolates in the sample extracts. Quadrupole time-of-flight tandem mass spectrometry in negative ion electrospray ionization was used to analyze the fractions collected from the HPLC elution to confirm the identification of the glucosinolates. The levels of sinigrin and gluconasturtiin in the vegetables and Chinese herbs were determined by using an external calibration method. Concentrations of gluconasturtiin in the Chinese herbs were more than 15 times higher than those of sinigrin. Detection limits were 18 nmol g⁻¹ for sinigrin and 4 nmol g⁻¹ for gluconasturtiin when 50 g of fresh vegetable was analyzed.

Keywords: Glucosinolates; HPLC; Sinigrin; Gluconasturtiin; ESI-QTOF-MS

Sensitive flow-injection chemiluminescence determination of terbutaline sulfate based on enhancement of the luminol–permanganate reaction by Zhouping Wang; Zhujun Zhang; Zhifeng Fu; Xiao Zhang (pp. 834-840).

A novel and highly sensitive chemiluminescence (CL) method for the determination of terbutaline sulfate, coupled with flow-injection analysis (FIA), is described in this paper. The method is based on enhancement by terbutaline sulfate of the chemiluminescence emission of the luminol–permanganate system under alkaline conditions. Under the conditions selected the concentration of terbutaline sulfate is proportional to CL intensity in the range 5×10⁻¹⁰–5×10⁻⁷ g mL⁻¹, with a detection limit of 1.7×10⁻¹⁰ g mL⁻¹ (3σ). The relative standard deviation is 2.8% for 1×10⁻⁸ g mL⁻¹ terbutaline sulfate (n=11). Ninety samples can be determined per hour. The proposed method has been used to determine terbutaline sulfate in pharmaceutical preparations and in plasma and urine samples with satisfactory results. The possible mechanism of the chemiluminescence reaction is discussed briefly.

Keywords: Chemiluminescence (CL); Flow-injection; Terbutaline sulfate; Luminol–permanganate; Plasma; Urine

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Analytical and Bioanalytical Chemistry (v.378, #3)

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Analytical and Bioanalytical Chemistry (v.378, #3)