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PubMed2003

Applicability of a rapid duplex real-time PCR assay for speciation of Campylobacter jejuni and Campylobacter coli directly from culture plates.

A rapid duplex real-time polymerase chain reaction (PCR) assay for speciation of Campylobacter jejuni and Campylobacter coli using the ABI Prism 7700 sequence detection system (Applied Biosystems) was developed based on two of the genes used in a conventional multiplex PCR. A rapid turnaround time of 3 h was achieved with the use of boiled cell lysates. Applicability of the assay was tested with 6015 random campylobacter strains referred to the Campylobacter Reference Unit, with 97.6% being identified as either C. jejuni or C. coli by this technique. Rapidity, combined with specificity and sensitivity, makes this method for routine campylobacter speciation attractive to any laboratory with a Taqman system.

Campylobacter coli↗
PubMed1995

Hybrid speciation. Evolution under the microscope.

Detailed genetic maps of three related sunflower species support the view that one species originated relatively recently from the other two by hybrid speciation, providing the first good evidence for this theory of speciation.

Biological Evolution↗
PubMed2003

Climatic oscillations triggered post-Messinian speciation of Western Palearctic brown frogs (Amphibia, Ranidae).

Oscillating glacial cycles over the past 2.4 million years are proposed to have had a major impact on the diversity of contemporary species communities. We used mitochondrial and nuclear DNA sequence data to infer phylogenetic relationships within Western Palearctic brown frogs and to test the influence of Pliocene and Pleistocene climatic changes on their evolution. We sequenced 1976bp of the mitochondrial genes 16S rRNA and cytochrome b and of the nuclear rhodopsin gene for all current species and subspecies. Based on an established allozyme clock for Western Palearctic water frogs and substitution rate constancy among water frogs and brown frogs, we calibrated a molecular clock for 1425bp of the 16S and rhodopsin genes. We applied this clock to date speciation events among brown frogs. Western Palearctic brown frogs underwent a basal post-Messinian radiation about 4 million years ago (mya) into five major clades: three monotypic lineages (Rana dalmatina, Rana latastei, Rana graeca), an Anatolian lineage, and a lineage comprising Rana italica, Rana arvalis, and all Iberian taxa. Polytypic lineages radiated further in concordance with the onset of climatic oscillations ca. 3.2, 2.0, and 1.0-0.6 mya, respectively. The dated fossil record corroborates our paleobiogeographic scenario. We conclude that drastic climatic changes followed by successive temperature oscillations "trapped" most brown frog species in their southern European glacial refugia with enough time to speciate. Substantial dispersal was only possible during extensive interglacial periods of a constant subtropical climate.

Animals↗
PubMed2002

Evolution in Hawaiian cave-adapted isopods (Oniscidea: Philosciidae): vicariant speciation or adaptive shifts?

We assessed evolutionary relationships among Hawaiian cave-adapted isopods using a maximum-likelihood criterion to analyze cytochrome oxidase I nucleotide sequences. Results support morphological data that two genera of philosciid isopods have invaded caves independently in the islands. In the genus Littorophiloscia, a sister relationship between a surface-dwelling species, L. hawaiiensis, and an undescribed cave species was corroborated. This evidence, along with the known parapatric distributions between species, supports a speciation event by an adaptive shift on the island of Hawaii from a marine littoral to a terrestrial subterranean habitat. The monophyletic genus Hawaiioscia contains four known obligate cave-dwelling species, each of which occurs on a separate island. However, despite present-day allopatric distributions between Hawaiioscia species, the geographic and phylogenetic patterns are not sufficient to support a vicariant mode of speciation. Instead, we believe that the known species of Hawaiioscia evolved from a widespread ancestral surface species or a group of closely related species through multiple, independent adaptive shifts on each of the islands of Kauai, Oahu, Molokai, and Maui. This is the first molecular investigation of evolutionary relationships between surface-dwelling and cavernicolous arthropods in Hawaii and it suggests that simple vicariance is insufficient to explain the evolution of troglobites in tropical zones.

Adaptation, Physiological↗
PubMed2003

Clinical evaluation of resilient denture liners. Part 2: Candida count and speciation.

PURPOSE: The purpose of this study was to count and to speciate Candida isolated from 2 resilient denture liners, Molloplast-B and MPDS-SL. MATERIALS AND METHODS: A group of 20 patients each had 1 maxillary denture and 2 mandibular dentures fabricated. One mandibular denture was lined with Molloplast-B, and 1 was lined with MPDS-SL. Each denture was used for 3 months. At the end of the 3-month period, the mandibular denture was surrendered, and a 5 x 5-mm circular resilient liner sample was obtained from the tissue surface of the lingual flange. Samples were processed, and Candida was isolated and counted. Speciation of Candida was performed using CHROMagar Candida and API 20C AUX strips. RESULTS: Molloplast-B had, on average, 5 times as many CFU/sample as MPDSL-SL, but this difference was not significant (p = 0.26). A sign test gave a similar nonsignificant trend (p = 0.057). CHROMagar identified several Candida species, and confirmation was made using API 20C AUX strips. One patient was lost to follow-up. Of 19 Molloplast-B samples, 7 had no growth, 4 grew C. albicans, 3 grew C. parapsilosis, 2 grew C. glabrata, 1 grew C. tropicalis, 2 grew a Trichosporon spp., and 2 grew a nonidentifiable colony. The analogous counts for 19 MPDS-SL samples were 10, 4, 1, 3, 0, 1, and 1 (p = 0.45 for culture positively, exact McNemar test). CONCLUSIONS: Candida growth on Molloplast-B was not significantly different from growth on MPDS-SL. Several yeast species were cultured from each material. The rates of culture-positive testing did not differ between the 2 resilient denture liners.

Adult↗
PubMed1995

The speciation and subtyping of campylobacter isolates from sewage plants and waste water from a connected poultry abattoir using molecular techniques.

In this study the distribution of phenotypes of campylobacter strains in sewage and surface waters was investigated by subtyping and by speciation of isolates from various aquatic environments. These environments included two municipal sewage plants (SPA and SPB) and waste water from a poultry abattoir (WWA). Both the sewage plants SPA and SPB collected domestic and industrial waste, and SPA received drain water from WWA. SPB received no waste water from any meat-processing plant. The isolates were speciated by PCR and subtyped by PCR/RFLP based on the flagellin PCR products. From all three reservoirs, no Campylobacter lari was isolated, and approximately 80% of the isolates could be identified as C. jejuni and the rest belonged to the C. coli species. The PCR/RFLP typing technique has a high discrimination level and was reproducible between two separate laboratories. The 182 isolates tested yielded 22 distinct Dde I profiles. The results indicate that strains with profiles found in poultry are also detectable in waste water presumed to be solely from domestic and human sources. In addition some strains were unique to the known poultry-related sources, suggesting that avian-specific strains, non-pathogenic to man, may exist in the environment. In contrast some strains were unique to human waste indicating the potential importance of non-poultry sources of infection. No seasonality was observed in the profile distribution. So, at least in the Netherlands, it is unlikely that infections caused by contaminated surface waters contribute to the seasonality of human campylobacteriosis.

Abattoirs↗
PubMed2000

The capillary cold trap as a suitable instrument for mercury speciation by volatilization, cryogenic trapping, and gas chromatography coupled with atomic absorption spectrometry.

An innovative accessory for speciation analysis has been developed. The system is based on the combination of cryogenic trapping and gas chromatographic separation, carried out within the same capillary. The instrument, hyphenating derivatization, gas-phase extraction, preconcentration, and analyte separation, is semiautomated, and all operational parameters are adjustable via an in-house-developed control unit, which regulates the selected parameters throughout the analysis process. Species detection was carried out by atomic absorption spectrometry. The detection limits achieved were 33, 39, and 71 ng L(-1) for dimethylmercury, methylmercury, and inorganic mercury, respectively. A complete chromatogram could be obtained within three minutes, resulting in the duration of one whole analysis cycle of about 15 min. The proposed method was applied to mercury speciation in freeze-dried tuna fish powder after microwave-assisted extraction, finding that mercury is present at 80% as methylmercury and about 20% as inorganic mercury, in this kind of biological material.

Animals↗
PubMed1989

Automated two-column ion exchange system for determination of the speciation of trace metals in natural waters.

A rapid method for determination of metal speciation based on an automated two-column ion exchange system is described. Two fractions of dissolved trace metal species are directly determined by on-line flame atomic absorption spectrophotometry after preconcentration by sequential columns of Chelex-100 chelating resin and AG MP-1 macroporous anion resin. A third fraction is determined by standard addition. Variables that affect the results obtained by the two-column system are studied by the use of model complexing agents. With a 10-mL sample loop, the sample throughput is 6 samples per hour and detection limits are 0.1 micrograms/L for Cu(II), 0.08 micrograms/L for Cd(II), and 0.2 micrograms/L for Zn(II). The method is used to determine the speciation of Cu(II), Cd(II), and Zn(II) in natural water samples.

Chromatography, Ion Exchange↗
PubMed1989

Speciation of aluminum in aqueous solutions using ion chromatography.

An ion chromatographic method in which aluminum (AI) is quantitatively determined via postcolumn derivatization with Tiron (4,5-dihydroxy-m-benzenedisulfonic acid) was evaluated for its utility as a method for speciating AI in aqueous solutions. Fluro-, oxalato-, and citratoaluminum complexes were identified by distinct peaks within chromatograms of AI solutions when the appropriate ligand was added. Excellent quantitative agreement between predicted species concentrations (via the thermodynamic speciation model GEOCHEM) and those determined by ion chromatography was obtained for samples prepared in the eluent matrix. The predominantly outer sphere sulfatoaluminum complexes were not observed to elute as singly charged species, but rather exhibited a retention time indistinguishable from the AI(H2O)6(3+) species. It is concluded that inner sphere AI complexes (generally possessing relatively high association constants) possess adequate kinetic stability to withstand degradation during the ion exchange process, whereas outer sphere complexes apparently readily dissociate in the presence of the sulfonate exchange sites. Deviations in sample ionic strength (mu) and pH from that of the eluent resulted in some redistribution among species, the degree of which was ligand specific.

Aluminum↗
PubMed2003

Theoretical and analytical characterization of a flow-through permeation liquid membrane with controlled flux for metal speciation measurements.

Speciation measurements with the permeation liquid membrane (PLM) technology require necessarily a good control of the flux of the analyte. In this perspective, a PLM-based multichannel flow-through cell has been designed. The first objective of this study has been to adapt the classical Levich model commonly used for electrochemical flow devices to the characteristic geometry of the PLM cell. In the latter case and contrary to the Levich model, the effects of the channel lateral walls on the flux of active species through the membrane have to be taken into account. The problem was solved by considering the existence of two parabolic Poiseuille profiles perpendicular to each other and developing along the fluid motion. The theoretical results obtained match satisfactorily with experimental data. The analytical study of this PLM system has been performed with copper(II) ions as test species and has shown that the preconcentration factor is (1) linear at least for preconcentration times of < or = 120 min, (2) reproducible on the same membrane as well as on different membranes, and (3) independent of the initial test metal ion concentration in the sample solution. The capabilities of this cell to determine metal speciation by considering lability of complexes and the flux of metal at variable flow rates of the test solution is also discussed by means of Cu(II)/sulfosalicylic complexes.

Journal Article↗
PubMed2002

On-line coupling of capillary electrophoresis to hydride generation atomic fluorescence spectrometry for arsenic speciation analysis.

A novel hyphenated technique, on-line coupling of capillary electrophoresis (CE) to atomic fluorescence spectrometry (AFS), was developed for speciation analysis of four environmentally significant and toxic forms of arsenic: arsenite, arsenate, monomethylarsenic acid, and dimethylarsenic acid. Baseline separation of the four arsenic species was achieved by capillary electrophoresis in a 50 cm x 100 microm i.d. fused-silica capillary at 20 kV and using a 20 mmol L(-1) phosphate buffer (pH 6.5). A hydride generation (HG) technique was employed to convert the arsenic species from the CE effluent into their respective hydrides. The CE-AFS interface was constructed on the basis of a cross design for introducing a sheath flow around the CE capillary and a Pt electrode, which provided an electrical connection for stable electrophoretic separations and allowed on-line volatile hydride formation. A laboratory-made gas-liquid separator was used to isolate the generated volatile species from the reaction mixture solution, and an argon flow was used to transport the volatile hydrides into the atomizer of AFS for on-line detection. The precision (RSD, n = 7) ranged from 2.1 to 3.1% for migration time, from 2.8 to 4.2% for peak area response, and from 2.0 to 4.1% for peak height response for the arsenic species at the 1 mg L(-1) (as As) level. The detection limits were in the range of 9-18 microg L(-1) (as As). The recoveries of the four arsenic species in locally collected water samples and urine sample ranged from 91 to 115%. The developed technique was successfully applied to the speciation of the water-methanol extractable arsenic in a sediment sample.

Arsenic↗
PubMed2003

Speciation of mercury by hydrostatically modified electroosmotic flow capillary electrophoresis coupled with volatile species generation atomic fluorescence spectrometry.

A novel method for speciation analysis of mercury was developed by on-line hyphenating capillary electrophoresis (CE) with atomic fluorescence spectrometry (AFS). The four mercury species of inorganic mercury Hg(II), methymercury MeHg(I), ethylmercury EtHg(I), and phenylmercury PhHg(I) were separated as mercury-cysteine complexes by CE in a 50-cm x 100-microm-i.d. fused-silica capillary at 15 kV and using a mixture of 100 mmol L(-1) of boric acid and 12% v/v methanol (pH 9.1) as electrolyte. A novel technique, hydrostatically modified electroosmotic flow (HSMEOF) in which the electroosmotic flow (EOF) was modified by applying hydrostatical pressure opposite to the direction of EOF was used to improve resolution. A volatile species generation technique was used to convert the mercury species into their respective volatile species. A newly developed CE-AFS interface was employed to provide an electrical connection for stable electrophoretic separations and to allow on-line volatile species formation. The generated volatile species were on-line detected with AFS. The precisions (RSD, n = 5) were in the range of 1.9-2.5% for migration time, 1.8-6.3% for peak area response, and 2.3-6.1% for peak height response for the four mercury species. The detection limits ranged from 6.8 to 16.5 microg L(-1) (as Hg). The recoveries of the four mercury species in the water samples were in the range of 86.6-111%. The developed technique was successfully applied to speciation analysis of mercury in a certified reference material (DORM-2, dogfish muscle).

Animals↗
PubMed2003

Using speciated isotope dilution with GC-inductively coupled plasma MS to determine and unravel the artificial formation of monomethylmercury in certified reference sediments.

Speciated isotope-dilution mass spectrometry (SID-MS) is claimed to be an absolute method; however, it has been found to be affected by artifact monomethylmercury (MMHg) formation in sediments. The determination of MMHg in sediments was carried out by SID-MS after open-focused microwave extraction. The extracted mercury species were then ethylated and separated by capillary gas chromatography (CGC). Isotope ratios (peak area ratios at different masses) were measured by on-line ICP-MS detection of the CGC-separated compounds. Reproducibility of 202Hg/201Hg isotope ratio measurements were 0.60% for MeEtHg and 0.69% for Et2Hg; for 202Hg/199Hg, 0.43 and 0.46%, respectively, were determined. The absolute detection limits for CGC-ICPMS measurements were better than 26 fg for 202Hg, 20 fg for 201Hg, and 24 fg for 199Hg. For the direct determination of MMHg in sediment reference materials (CRM 580, IAEA 356, and IAEA 405), higher values than the certified were always found. Systematic experiments were carried out to localize the sources of the unintentional abiotic methylmercury formation during analysis. Different spiking and derivatization procedures (either ethylation, propylation, or derivatization by Grignard reagents) were tested. In addition, isotopically enriched inorganic mercury was spiked. The amount of inorganic mercury initially present in the sample was found to be the critical factor that should be known and carefully controlled. A simple solvent extraction technique involving no critical cleanup steps was applied in order to reduce high Hg2+ amounts. The method was applied to the determination of MMHg in sediment reference material IAEA-405 with satisfactory results after organic solvent extraction. The limitations of applicability of the proposed method are evaluated as related to inorganic mercury, organic carbon, and sulfur contents. The results obtained confirmed that available sediment reference materials are adequate to achieve traceable mercury speciation analysis and to detect potential sources of MMHg artifact formation.

Journal Article↗
PubMed2004

An expert system for chemical speciation of individual particles using low-Z particle electron probe X-ray microanalysis data.

An electron probe X-ray microanalysis (EPMA) technique, using an energy-dispersive X-ray detector with an ultrathin window, designated a low-Z particle EPMA, has been developed. The low-Z particle EPMA allows the quantitative determination of concentrations of low-Z elements, such as C, N, and O, as well as chemical elements that can be analyzed by conventional energy-dispersive EPMA, in individual particles. Since a data set is usually composed of data for several thousands of particles in order to make environmentally meaningful observations of real atmospheric aerosol samples, the development of a method that fully extracts chemical information contained in the low-Z particle EPMA data is important. An expert system that can rapidly and reliably perform chemical speciation from the low-Z particle EPMA data is presented. This expert system tries to mimic the logic used by experts and is implemented by applying macroprogramming available in MS Excel software. Its feasibility is confirmed by applying the expert system to data for various types of standard particles and a real atmospheric aerosol sample. By applying the expert system, the time necessary for chemical speciation becomes shortened very much and detailed information on particle data can be saved and extracted later if more information is needed for further analysis.

Journal Article↗
PubMed2006

Atomization of hydride with a low-temperature, atmospheric pressure dielectric barrier discharge and its application to arsenic speciation with atomic absorption spectrometry.

This paper describes a novel hydride atomizer based on atmospheric pressure dielectric barrier discharge (DBD) plasma. The plasma was generated with a 3700-V, 20.3-kHz, and 5-W electrical power supply and easily sustained with inert gases (He or Ar) at a flow rate of 250 mL.min(-1) after optimization. However, it cannot be sustained with N2. This atomizer offers the advantages of low operation temperature and low power consumption in comparison with the currently used electrothermal quartz atomization operated at 900 degrees C with a power supply of several hundred watts. To confirm the utility of the proposed atomizer, four arsenic species (As(III), As(V), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA)) were determined by the present atomization technique. A hyphenation of HPLC coupled to hydride generation AAS with the optimized DBD atomizer has been successfully used for the speciation of arsenic in order to demonstrate the potential of this atomizer in the present study. The characteristics of the DBD atomizer and the effects of different parameters (discharge gas, gas flow rate, voltage, HCl concentration, KBH4 concentration) are discussed in the paper. Compared with other hydride atomization techniques, the proposed method shows the following features: (1) small size (70 mm x 15 mm x 5 mm), which is preferable for the miniaturization of the total analytical system; (2) low power consumption (< or =5 W), which indicates the possibility of the development of portable, fieldable analytical instrumentation for in situ detection using battery as power supply; (3) low atomizer temperature (approximately 70 degrees C), which is in favor of the compactness of the total instruments; (4) avoidance of residue moisture removal in comparison with the existed GD system, which leads to the facility of the system. The analytical figures of the present technique were evaluated. The detection limits of As(III), As(V), MMA, and DMA obtained with HG-DBD-AAS were 1.0, 11.8, 2.0, and 18.0 microg.L(-1), respectively. The accuracy of the system was verified by the determination of arsenic in reference material of orchard leaves SRM 1571. The concentration of As determined by the present method agreed well with the reference values. The speciation of arsenic in the freeze-dried urine SRM 2670 were carried out, and the results obtained were in agreement with the results of HPLC-ICPMS and the reported values by other laboratories.

Journal Article↗
PubMed2006

Identification and characterization of isomeric intermediates in a catalyst formation reaction by means of speciation analysis using HPLC-ICPMS and HPLC-ESI-MS.

Information on chemical speciation is much needed in mechanistic and kinetic studies on catalyst formation processes in pharmaceutical research. Speciation analysis was applied to the identification and quantification of various rhodium species involved in a ligand exchange process leading to formation of catalyst dirhodium(II) tetrakis[methyl 2-oxopyrrolidin-5(S)-carboxylate]. Inductively coupled plasma mass spectrometry (ICPMS) was used as an element-specific detector following species separation by reversed-phase high-performance liquid chromatography (RP-HPLC), and electrospray ionization mass spectrometry (ESI-MS) was used for species identification and confirmation. A novel interface between the HPLC and ICPMS, which consisted of an eluent splitter, a desolvation unit, and the ICPMS built-in peristaltic pump, enabled the use of RP-HPLC with gradient elution and up to 100% organic components in the LC eluent without organic loading in the plasma. A variety of reaction intermediates were identified and quantified along the pathway to formation of the desired product, including isomeric di-, tri-, and tetrasubstituted species previously believed to be absent. This has provided new insights into the mechanism and kinetics of the reaction. The combination of HPLC-ICPMS and HPLC-ESI-MS has proven to be a valuable tool for the investigation of species evolution in catalyst formation process.

Catalysis↗
PubMed1996

Speciation of arsenic oxides using laser desorption/ionization time-of-flight mass spectrometry.

Positive and negative ion mass spectra of arsenic trioxide (As2O3) and arsenic pentaoxide (As2O5) have been obtained by single-step laser desorption/ionization time-of-flight mass spectrometry. Pulsed UV radiation at 266 nm was used for the simultaneous desorption and ionization of the solid sample. High-mass cluster ions that are unique to the oxidation state of each oxide sample appear in the negative ion mass spectra. The As2O3 produces As3O5-, while the As2O5 yields As3O8-. The formation of unique negative cluster ions presents the capability for arsenic oxidation state speciation by laser desorption/ionization mass spectrometry. The ability of time-of-flight mass spectrometry to examine the relative amounts of each arsenic oxide present in a series of mixtures is discussed. Application of our speciation technique to a model incinerator sample is demonstrated.

Arsenic Trioxide↗
PubMed2000

Simultaneous determination and speciation of zinc, cadmium, lead, and copper in natural water with minimum handling and artifacts, by voltammetry on a gel-integrated microelectrode array.

The paper reports a new approach based on a gel-integrated Hg-plated-Ir-based microelectrode array (GIME), for measuring Cu, Pb, Cd, and Zn speciation in natural waters. This paper focuses on the quantitative discrimination between mobile and colloidal metal species (size limit of a few nanometers), for which most classical separation techniques present severe drawbacks. Previous papers have shown qualitatively that GIME combined with square wave anodic stripping voltammetry (SWASV) has the basic characteristics required to discriminate between these two fractions directly on the unperturbed sample. In addition, because of the large sensitivity provided by GIME, complexation parameters (equilibrium constants and site concentrations) can be determined in little-perturbed samples, particularly without metal addition or with small addition compared with natural concentrations. The advantages of this procedure are exemplified and the possible artifacts occurring when titrating the sample with metals, in particular intermetallic compound formation and other problems, are discussed in detail. The present paper shows that the characteristics of GIME make it a unique tool to get quantitative information on metal speciation at nanomolar or even subnanomolar levels, with only minor sample handling.

Cadmium↗