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Analytica Chimica Acta (v.573, #N1-N2,1-48)

Editorial Board (pp. co1).

Contents (pp. vii-x).

Calendar of forthcoming meetings (pp. n1).

Editorial Board (pp. co1).

Calendar of forthcoming meetings (pp. n1).

Contents (pp. vii-x).

The Fourth International Conference on Instrumental Methods of Analysis—Modern Trends and Applications, Iraklion, Crete, Greece, 2–6 October, 2005 by Nikos Chaniotakis (pp. 1-2).

pH-sensitive diamond field-effect transistors (FETs) with directly aminated channel surface by Kwang-Soup Song; Yusuke Nakamura; Yuichi Sasaki; Munenori Degawa; Jung-Hoon Yang; Hiroshi Kawarada (pp. 3-8).

We have introduced pH sensors fabricated on diamond thin films through modification of the surface-terminated atom. We directly modified the diamond surface from hydrogen to amine or oxygen with ultraviolet (UV) irradiation under ammonia gas. The quantified amine site based on the spectra obtained by X-ray photoelectron spectroscopy (XPS) is 26% (2.6×10¹⁴cm⁻²) with UV irradiation for 8h and its coverage is dependent on the UV irradiation time. This directly aminated diamond surface is stable with long-term exposure in air and electrolyte solution. We fabricated diamond solution-gate field-effect transistors (SGFETs) without insulating layers on the channel surface. These diamond SGFETs with amine modified by direct amination are sensitive to pH (45mV/pH) over a wide range from pH 2 to 12 and their sensitivity is dependent on the density of binding sites corresponding to UV irradiation time on the channel surface.

Keywords: Diamond; Solution-gate field-effect transistors (SGFETs); Directly aminated channel surface; X-ray photoelectron spectroscopy (XPS); pH sensors

Metal oxide thin films as sensing layers for ozone detection by M. Suchea; N. Katsarakis; S. Christoulakis; M. Katharakis; T. Kitsopoulos; G. Kiriakidis (pp. 9-13).

In₂O3−_x thin films with a thickness of 100–990nm were grown by dc magnetron sputtering. Their structural, electrical and ozone sensing properties were analyzed. Structural investigations carried out by electron probe micro analysis, secondary ion mass spectrometry and atomic force microscopy showed a strong correlation between stoichiometry, surface topology and gas sensitivity. Moreover, the electrical conductivity of In₂O3−_x thin films exhibited a change of over six orders of magnitude during photoreduction with ultraviolet light and subsequent oxidation in ozone atmosphere at room temperature.

Keywords: In; ₂; O; 3−; _x; thin films; Stoichiometry; Ozone; Sensing

Metal oxide thin films as sensing layers for ozone detection by M. Suchea; N. Katsarakis; S. Christoulakis; M. Katharakis; T. Kitsopoulos; G. Kiriakidis (pp. 9-13).

Keywords: In; ₂; O; 3−; _x; thin films; Stoichiometry; Ozone; Sensing

Determination of labile trace metals with screen-printed electrode modified by a crown-ether based membrane by Corinne Parat; Stéphanie Betelu; Laurent Authier; Martine Potin-Gautier (pp. 14-19).

In this work, we have undertaken the construction of a screen-printed electrode modified by a specific membrane to protect the working surface from interferences during the analysis of trace metals by anodic stripping voltammetry. Different crown-ethers selected for their metals affinity have been incorporated into a membrane then deposed on the working surface of the electrode. Each modified electrode has been first tested in an acidified KNO₃ 10⁻¹molL⁻¹ solution (pH 2) doped by free Cd²⁺ and Pb²⁺ ions. The response and selectivity of the modified electrodes have been investigated according to different parameters: (i) the substrates (commercial ink or carbon based homemade ink), (ii) the electrode support (polystyrene or transparency film) and (iii) crown-ethers nature (dibenzo-24-crown-8 and tetrathiacyclododecane 12-crown-4). The influence of the substrate on the response of the electrode is clearly demonstrated. Homemade ink appears as the most appropriate substrate to modify the working surface of the screen-printed electrode by a crown-ether based membrane. The effect of the composition of the membrane has been shown too. The best membrane developed showed a detection limit of 0.6×10⁻⁸molL⁻¹ for Cd and 0.8×10⁻⁸molL⁻¹ for Pb and a quantification limit of 10⁻⁸molL⁻¹ for Cd and 2×10⁻⁸molL⁻¹ for Pb. This method, which integrates the extraction, preconcentration and measurement, was successfully applied to environmental samples without pretreatment.

Keywords: Screen-printed electrode; Mercury film; Crown-ether; Anodic stripping voltammetry

Determination of labile trace metals with screen-printed electrode modified by a crown-ether based membrane by Corinne Parat; Stéphanie Betelu; Laurent Authier; Martine Potin-Gautier (pp. 14-19).

Keywords: Screen-printed electrode; Mercury film; Crown-ether; Anodic stripping voltammetry

Electropolymerization and characterization of polyaniline films using a spectroelectrochemical flow cell by Emma Muñoz; �?lvaro Colina; Aránzazu Heras; Virginia Ruiz; Susana Palmero; Jesús López-Palacios (pp. 20-25).

A new spectroelectrochemical flow cell is presented and its capability to provide a better understanding of reaction mechanisms is illustrated with the study of the electrosynthesis and characterization of a conducting polymer, polyaniline (PANI). A spectroelectrochemical study of electropolymerization of aniline under flow conditions has been performed for the first time. The significant influence that the flow rate of feeding monomer solution has on the electropolymerization process and, consequently, on the electrochromic properties of the resulting polymer has been demonstrated.

Keywords: Conducting polymers; Polyaniline; Spectroelectrochemistry; Flow cell; Electrochemistry

Electropolymerization and characterization of polyaniline films using a spectroelectrochemical flow cell by Emma Muñoz; Álvaro Colina; Aránzazu Heras; Virginia Ruiz; Susana Palmero; Jesús López-Palacios (pp. 20-25).

Keywords: Conducting polymers; Polyaniline; Spectroelectrochemistry; Flow cell; Electrochemistry

Preparation and characterization of diethylene glycol bis(2-aminophenyl) ether-modified glassy carbon electrode by Aybüke A. İsbir; Ali Osman Solak; Zafer Üstündağ; Selen Bilge; Zeynel Kılıç (pp. 26-33).

Diethylene glycol bis(2-aminophenyl) ether (DGAE) diazonium salt was covalently electrografted on a glassy carbon (GC) surface and behavior of this novel surface was investigated. Synthesis of DGAE diazonium salt (DGAE-DAS) and in situ modification of GC electrode were performed in aqueous media containing NaNO₂, keeping the temperature below +4°C. For the characterization of the modified electrode surface by cyclic voltammetry, dopamine (DA) was used to prove the attachment of the DGAE-DAS on the GC surface. Raman spectroscopy and electrochemical impedance spectroscopy (EIS) were used to observe the molecular bound properties of the adsorbates at the DGAE-modified GC surface (GC-DGAE). The EIS results were analyzed using the Randles equivalent circuit. The charge transfer resistance on bare GC and the modified surface were calculated using the model equivalent circuit for the ferrocene redox system. Surface coverage was found as 0.4 showing the presence of high pinhole and defects in the modified electrode. The rate constant of electron transfer through the monolayer was calculated for ferrocene. Working potential range and the stability of the DGAE-modified GC electrode was also determined.

Keywords: Cyclic voltammetry; Diethylene glycol bis(2-aminophenyl) ether-modified glassy carbon electrode; Electrografting; Raman spectroscopy; Electrochemical impedance spectroscopy

New real-time analytical applications of electrochemical quartz crystal microbalance by Milka T. Neshkova; V. Nikolova; V. Petrov (pp. 34-40).

The electrochemical quartz crystal microbalance (EQCM) has recently gained increasing popularity as a powerful tool for electrochemical interface examinations. This paper reports on its great potential for developing new real-time analytical protocols. Potentiostatic coulometry is used in conjunction with quartz crystal microgravimetry (QCMg) with controlled hydrodynamics to develop two new analytical procedures: real-time stoichiometry monitoring of electrodeposited binary chalcogenide films and phase composition quantification of electrodeposited ternary chalcogenide compounds. The newly developed EQCM methods are illustrated on the examples of Ag2+_δSe-electrodeposited films and the electroformation of ternary CuAgSe-films. Both compounds have been successfully used as ion-selective membranes for flow-injection detectors. The experimental set-up including hydrodynamic control, the main strategy of the approach used and its scope and limitations are broadly discussed. The obtained data allow for real time profile monitoring of either stoichiometry (Ag2+_δSe-case) or phase composition (CuAgSe-case).

Keywords: Electrochemical quartz crystal microbalance, real time analysis; Stoichiometry monitoring; Phase composition analysis; Ag; _y; Se-electrodeposition; CuAgSe-electrodeposition

New real-time analytical applications of electrochemical quartz crystal microbalance by Milka T. Neshkova; V. Nikolova; V. Petrov (pp. 34-40).

Keywords: Electrochemical quartz crystal microbalance, real time analysis; Stoichiometry monitoring; Phase composition analysis; Ag; _y; Se-electrodeposition; CuAgSe-electrodeposition

Application of electrochemical optical waveguide lightmode spectroscopy for studying the effect of different stress factors on lactic acid bacteria by Nóra Adányi; Edina Németh; Anna Halász; István Szendrő; Mária Váradi (pp. 41-47).

Electrochemical optical waveguide lightmode spectroscopy (EC-OWLS) has been developed to combine evanescent-field optical sensing with electrochemical control of surface adsorption processes. For bioanalytical sensing, a layer of indium tin oxide (ITO) served as both a high-refractive index waveguide and a conductive electrode. In addition, an electrochemical flow-through fluid cuvette was applied, which incorporated working, reference, and counter electrodes, and was compatible with the constraints of optical sensing.The subject of our study was to monitor how the different stress factors (lactic acid, acetic acid and hydrogen peroxide) influence the survival of lactic acid bacteria. The advantage of EC-OWLS technique is that we could carry out kinetic studies on the behaviour of bacteria under stress conditions, and after exposure of lactobacilli to acid and oxidative stress we get faster results about the status of bacteria compared to the traditional quantitative methods.After optimization of the polarization potential used, calibration curve was determined and the sensor response of different rate of living and damaged cells was studied. The bacterial cells were adsorbed in native form on the surface of the sensor by ensuring polarizing potential (1V) and were exposed to different concentration of acetic acid and hydrogen peroxide solution to 1h, respectively and the behaviour of bacteria was monitored. Results were compared to traditional micro-assay method.

Keywords: Electrochemical optical waveguide lightmode spectroscopy (EC-OWLS); Lactic acid bacteria; Indium tin oxide (ITO) covered sensor; Real-time measurement; Stress factors

Continuous oxygen monitoring in subcutaneous adipose tissue using microdialysis by Alessandro Bizzarri; Hans Koehler; Merima Cajlakovic; Alen Pasic; Lukas Schaupp; Ingo Klimant; Volker Ribitsch (pp. 48-56).

A measurement system, consisting of an optochemical glass capillary oxygen sensor, an optoelectronic measuring unit and a microdialysis catheter (CMA 60) for the extraction of the biological fluid from the subcutaneous adipose tissue of critically ill patients is reported. The capillary sensor is based on the oxygen sensitive dye platinum (II) meso-tetra(pentafluorophenyl) porphyrin (Pt-TFPP) incorporated in a polystyrene matrix. The measuring system has been tested in vitro and in vivo. In particular in vitro long-term stability of the sensor has been investigated in different measurement media (elomel, 5% mannitol, Ringer, dialysed blood). The influence of different flow rates from 0.1 up to 7.0μlmin⁻¹ on the sensor response as well as the oxygen recovery rate are discussed. The presented measurement system allows the measurement of oxygen in biological fluid in the range from 0 to 300mmHg, with a resolution better than 1mmHg and high accuracy (better than ±1mmHg absolute). Finally, the suitability of the described measurement system for the continuous oxygen monitoring in subcutaneous adipose tissue has been proved in in vivo investigations performed on test animals.

Keywords: Oxygen sensor; Optochemical sensor; Microdialysis

Luminescence lifetime-based carbon dioxide optical sensor for clinical applications by Merima Čajlaković; Alessandro Bizzarri; Volker Ribitsch (pp. 57-64).

The development of both an optical planar and capillary based carbon dioxide sensor, which final aim is pCO₂ monitoring in adipose tissue of critically ill patients, is reported. The sensor is based on the measuring principle of phase fluorometry using a dual luminophore referencing scheme (DLR) to convert the CO₂ dependent intensity signal into phase domain. The CO₂ sensors have been prepared by incorporating two appropriate luminophores and a phase transfer agent in a same hydrophobic polymer as matrix. The short-lifetime luminophore used as pH indicator is 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS). The second inert luminophore is the long-lifetime dye Ruthenium(II) tris(4,7-diphenyl-1,10-phenanthroline) (Ru(dpp)₃²⁺), which has been made insensitive to oxygen by immobilising in a suitable oxygen impermeable polymer. As phase transfer agent, tetraoctylammonium hydroxide (TOA-OH) has been chosen.Both sensor types have been characterised with respect to optimise sensitivity and mechanical stability. For this purpose, several polymers, such as ethylcellulose, eudragit RL100 (EG), copolymer eudragit/poly(ethylene glycol) (PEG) and silicone have been examined as appropriate matrix for incorporation of two indicators. The largest phase shift up to 13° and 15° has been observed in the case of silicone and copolymer EG/PEG, respectively, and they have been in detail examined in terms of sensitivity and stability. The presented sensors enable the measurement of pCO₂ in the range from 0 to 150mmHg, with a resolution of 0.5mmHg and an accuracy of ±1mmHg absolute or less than 7% of the read-out value. All measurements have been carried out only in aqueous solutions before clinical measurements.

Keywords: Carbon dioxide; Optical sensor; Lifetime sensing; Dual luminophore referencing (DLR)

Luminescence lifetime-based carbon dioxide optical sensor for clinical applications by Merima Čajlaković; Alessandro Bizzarri; Volker Ribitsch (pp. 57-64).

Keywords: Carbon dioxide; Optical sensor; Lifetime sensing; Dual luminophore referencing (DLR)

Comparison of antibody and aptamer receptors for the specific detection of thrombin with a nanometer gap-sized impedance biosensor by U. Schlecht; A. Malavé; T. Gronewold; M. Tewes; M. Löhndorf (pp. 65-68).

Nanogap-impedance biosensors with electrode separations of 75nm have been fabricated by means of standard optical lithography and a sacrificial layer technique. Due to a large surface-to-volume ratio and high sensitivity, these sensors are superior compared to open interdigitated electrode structures. As a model, the blood coagulation factor thrombin was detected. As specific receptors, either an antibody or a RNA-aptamer have been used. The microwave frequency impedance measurements showed that both ligands were equally suitable for the specific detection of thrombin.

Keywords: Impedance; Biosensor; Nanogap; Thrombin; Protein-interaction

Bioelectronic sniffer for nicotine using enzyme inhibition by Kohji Mitsubayashi; Kazumi Nakayama; Midori Taniguchi; Hirokazu Saito; Kimio Otsuka; Hiroyuki Kudo (pp. 69-74).

A novel bioelectronic sniffer for nicotine in the gas phase was developed with enzyme inhibition principle to butyrylcholinesterase activity. The bioelectronic devices for nicotine in the gas and liquid phases were constructed using a Clark-type dissolved oxygen electrode and a membrane immobilized butyrylcholinesterase and choline oxidase. After the assessment of the sensor performances to choline and butyrylcholine as pre-examinations, the characteristics of the biosensor and bio-sniffer for nicotine were evaluated in the liquid and gas phases, respectively. The sensor signal of the bio-devices with 300μmoll⁻¹ of butyrylcholine decreased quickly following application of nicotine and reached to the steady-state current, thus relating the concentration of nicotine in the liquid and gas phases. The biosensor was used to measure nicotine solution from 10 to 300μmoll⁻¹. In the gas-phase experiment, the current signal of the bio-sniffer was also found to be linearly to the nicotine concentration over the range of 10.0–1000ppb including 75.0ppb as threshold limit value (TLV) by American Conference of Governmental Industrial Hygienists (ACGIH).

Keywords: Bioelectronic sniffer; Nicotine sensor; Enzyme inhibition; Butyrylcholinesterase

Bioelectronic sniffer for nicotine using enzyme inhibition by Kohji Mitsubayashi; Kazumi Nakayama; Midori Taniguchi; Hirokazu Saito; Kimio Otsuka; Hiroyuki Kudo (pp. 69-74).

Keywords: Bioelectronic sniffer; Nicotine sensor; Enzyme inhibition; Butyrylcholinesterase

Optical bio-sniffer for methyl mercaptan in halitosis by Kohji Mitsubayashi; Takeshi Minamide; Kimio Otsuka; Hiroyuki Kudo; Hirokazu Saito (pp. 75-80).

An optical bio-sniffer for methyl mercaptan (MM) one of major odorous chemicals in halitosis (bad breath) was constructed by immobilizing monoamine oxidase type A (MAO-A) onto a tip of a fiber optic oxygen sensor (od: 1.59mm) with an oxygen sensitive ruthenium organic complex (excitation: 470nm, fluorescent: 600nm). A flow cell for circulating buffer solution was applied to rinse and clean the tip of the device like nasal mucosa. In order to amplify the bio-sniffer output, a substrate regeneration cycle caused by coupling MAO-A withl-ascorbic acid (AsA) as reducing reaction with reagent system was applied to the sensor system. After evaluating the sensor characteristics using a gas flow measurement system with a gas generator, the optical bio-sniffer was applied to expired gases from healthy male volunteers for halitosis analysis as a physiological application.The optical bio-sniffer was applied to detect the oxygen consumption induced by MAO-A enzymatic reaction (and AsA chemical reduction) with gaseous MM application. The bio-sniffer was calibrated against MM vapor from 8.7 to 11500ppb with correlation coefficient of 0.977, including a MM threshold (200ppb) of pathologic halitosis and the human sense of smell level 3.5 (10.0ppb), with good gas-selectivity based on the MAO-A substrate specificity. As the result of the physiological application, the optical bio-sniffer could successfully monitor the MM level change in breath samples during daytime, which is consistent with the previously reported results.

Keywords: Optical bio-sniffer; Halitosis; Methyl mercaptan; Monoamine oxidase; Oxygen sensitive optical fiber

Optical bio-sniffer for methyl mercaptan in halitosis by Kohji Mitsubayashi; Takeshi Minamide; Kimio Otsuka; Hiroyuki Kudo; Hirokazu Saito (pp. 75-80).

Keywords: Optical bio-sniffer; Halitosis; Methyl mercaptan; Monoamine oxidase; Oxygen sensitive optical fiber

Deoxyribonucleic acid (DNA) biosensors for environmental risk assessment and drug studies by Graziana Bagni; Domenico Osella; Elena Sturchio; Marco Mascini (pp. 81-89).

In the present work, electrochemical DNA biosensors are proposed as a screening device for the rapid bio-analysis of environmental pollution and DNA–drug interaction studies. The binding of small molecules to DNA immobilised on disposable screen-printed electrodes has been measured through the variation of the electrochemical signal of guanine by square wave voltammetric scans. These kinds of biosensors were used to evaluate the soil contamination level in an Italian polluted area and the results were compared with several methods for the DNA damage detection, as Comet genotoxicity effects, aberrant anatelophases and micronucleated cells frequency on plant roots, and with fixed wavelength fluorescence (FF) by using 2-aminoanthracene as standard compound. The results showed the ability of the biosensors to distinguish in 11min low, medium and high contaminated soils with good correlation with well established techniques as well as FF, Comet and genotoxicity tests. The same kind of biosensors was also used to evaluate the interaction of DNA with some anti-proliferative metallo drugs, and the electrochemical responses reflected the kind of interaction. The reproducibility of the electrochemical measurements of DNA guanine peak was estimated as less than 10% of relative standard deviation (R.S.D.%).

Keywords: DNA-based biosensors; Environmental risk assessment; DNA damage; Anti-proliferative drugs

Deoxyribonucleic acid (DNA) biosensors for environmental risk assessment and drug studies by Graziana Bagni; Domenico Osella; Elena Sturchio; Marco Mascini (pp. 81-89).

Keywords: DNA-based biosensors; Environmental risk assessment; DNA damage; Anti-proliferative drugs

Application of “membrane-engineering�? to bioelectric recognition cell sensors for the ultra-sensitive detection of superoxide radical: A novel biosensor principle by Georgia Moschopoulou; Spiridon Kintzios (pp. 90-96).

A new, hybrid type of ultra-sensitive electrophysiological superoxide anion (O₂⁻) sensor is described, which is based on “membrane-engineered�? mammalian cells immobilized in an alginate matrix. The membrane-engineering process involved the electroinsertion of superoxide dismutase (SOD) molecules in the membranes of Vero fibroblast cells, which acted as catalytic units able to convert O₂⁻ to H₂O₂. Superoxide dismutation triggered changes to the cell membrane potential that were measured by appropriate microelectrodes, according to the principle of the bioelectric recognition assay (BERA). The sensor instantly responded to O₂⁻ with a detection limit (S/N=3) of 100pM. Combined with a 4-month storage capacity at room temperature, the novel biosensor principle offers new perspectives for monitoring ultra-low concentrations of free radical species and oxidative agents in biological systems.

Keywords: Abbreviations; BERA; bioelectric recognition assay; Cyt.; c; cytochrome; c; ESR; electron spin resonance; FCS; foetal calf serum; FITC; fluorescein isothiocyanate; MDA; malondialdehyde; PBS; phosphate buffer saline; SOD; superoxide dismutase; TBA; thiobarbituric acid; TCA; trichloroacetic acid; XOD; xanthine oxidaseBioelectric recognition assay; Cell biosensor; Hydrogen peroxide; Membrane-engineering; Superoxide; Superoxide dismutase

Application of “membrane-engineering” to bioelectric recognition cell sensors for the ultra-sensitive detection of superoxide radical: A novel biosensor principle by Georgia Moschopoulou; Spiridon Kintzios (pp. 90-96).

A new, hybrid type of ultra-sensitive electrophysiological superoxide anion (O₂⁻) sensor is described, which is based on “membrane-engineered” mammalian cells immobilized in an alginate matrix. The membrane-engineering process involved the electroinsertion of superoxide dismutase (SOD) molecules in the membranes of Vero fibroblast cells, which acted as catalytic units able to convert O₂⁻ to H₂O₂. Superoxide dismutation triggered changes to the cell membrane potential that were measured by appropriate microelectrodes, according to the principle of the bioelectric recognition assay (BERA). The sensor instantly responded to O₂⁻ with a detection limit (S/N=3) of 100pM. Combined with a 4-month storage capacity at room temperature, the novel biosensor principle offers new perspectives for monitoring ultra-low concentrations of free radical species and oxidative agents in biological systems.

A Mach-Zehnder interferometer based on silicon oxides for biosensor applications by Jongin Hong; Jung Sung Choi; Gayoung Han; Jae Kwang Kang; Chang-Min Kim; Tae Song Kim; Dae Sung Yoon (pp. 97-103).

Integrated optical devices have been increasingly interested in biosensor applications including environmental pollution, biological process and medical diagnostics. Integrated optics allows high-detection sensitivity to be achieved using optical transduction techniques in a microfluidic format. Among different transduction techniques, a Mach-Zehnder interferometer (MZI) has advantage of its inherent high sensitivity and accuracy. The evanescent wave of an optical waveguide interacts with an adjacent layer, and this can be the basis of the recognition of biomolecules. In recent years, silicon dielectrics as potential materials have been attracted in an integrated optics. The refractive index of these silicon-based materials can be easily adjusted continuously over a wide range between 1.45 (SiO₂) and 1.97 (SiO). This comes to be very attractive in terms of design and fabrication of single-mode waveguides. In this article, we tried to realize the Mach-Zehnder interferometer sensor based on silicon oxides, and the refractive index of the oxides was controlled by the oxygen concentration to achieve the single-mode behavior of a total internal reflection (TIR) waveguide. We have performed to verify the feasibility of the MZI sensor for the direct detection of immunoreactions.

Keywords: Waveguide; Biosensor; Mach-Zehnder interferometer; Single-mode; Immunoreaction

Structural development of a minimally invasive sensor chip for blood glucose monitoring by Shingo Kaimori; Takahiko Kitamura; Moriyasu Ichino; Toshifumi Hosoya; Fumiyo Kurusu; Tomoko Ishikawa; Hideaki Nakamura; Masao Gotoh; Isao Karube (pp. 104-109).

Our newly developed glucose sensor chip has the world smallest blood sample volume, as small as 200nL, which makes lancing with less pain possible. Forming the inside walls of the cavity, where the blood is collected, by screen printing of the adhesive ink instead of the conventional adhesive sheet placing drastically reduced the cavity thickness, to less than 50μm, and the blood sample volume. On this thin-cavity type sensor, the cavity thickness was proven to have the strong influence on the sensor response. This means that thinner cavity requires more accurate printing, in terms of thickness control. The printing conditions were adjusted to minimize the dispersion in the cavity geometry. One of the challenges was how to print patterns without saddles, which was achieved by selecting proper ink. After the adjustment, the contribution of the dispersion in the cavity thickness and the electrode area to the dispersion in sensor responses was estimated, respectively, which indicated the contribution of the cavity thickness still existed. Finally, the sensor was evaluated using whole sheep blood containing glucose of from 60 to 493mgdL⁻¹. Irrespective of the influence relating to the cavity thickness, the coefficients of variation of the sensor responses were from 4.4 to 7.6. In addition, the correlation curve showed linearity in this blood glucose range, and the coefficient of determination r² was 0.98. That is, the sensor was verified to have sufficient performance for practical use.

Keywords: Sensor chip; Blood glucose; Sample volume; Screen printing; Adhesive ink

Highly selective microbiosensors for in vivo measurement of glucose, lactate and glutamate by O.M. Schuvailo; O.O. Soldatkin; A. Lefebvre; R. Cespuglio; A.P. Soldatkin (pp. 110-116).

An alternative approach to production of amperometric microbiosensors, which combines electrochemical electrometallization and electropolymerisation of phenylene diamine film with covalent binding enzymes, is presented. In this respect, for a sensitive detection of hydrogen peroxide (HP) at +0.4V versus Ag/AgCl (detection limit of 0.5μM, s/n=3), carbon fiber microelectrodes (30μm in diameter and 500μm long) were covered with ruthenium. To obtain a highly selective detection of HP, in the presence of different interfering compounds (ascorbic acid, uric acid, etc.), an additive semi-permeable polymer film was formed on the top of the ruthenium layer by electropolymerisation of m-phenylene diamine ( m-PD). The enzymatic selective layers were formed by covalent cross-linking the enzymes (glucose oxidase, lactate oxidase or glutamate oxidase) with BSA by glutaraldehyde in the presence of ascorbate oxidase. An additional polymeric layer based on polyurethane and Nafion was deposited on the top of the enzymatic membrane (glucose oxidase, lactate oxidase, or glutamate oxidase) in order to extend the dynamic range of biosensors up to 4mM for glucose ( R=0.997; Y[nA]=−0.22+9.68 x[glucose, mM]), 1.75mM for lactate ( R=0.991; Y[nA]=0.43+15.36 x[lactate, mM]) and 0.25mM for glutamate ( R=0.999; Y[nA]=0.02+29.14 x[glutamate, mM]). The developed microbiosensors exhibited also negligible influences from interfering compounds at their physiological concentrations. Microbiosensors remained stable during 10h in a flow injection system at 36°C and pH 7.4. The microbiosensors developed are now used in vivo and, as an example, we report here the data obtained with the glucose biosensor.

Keywords: Amperometric microbiosensors; Carbon fiber electrode; Glucose; Lactate; Glutamate; In vitro analysis; In vivo detection

Highly selective microbiosensors for in vivo measurement of glucose, lactate and glutamate by O.M. Schuvailo; O.O. Soldatkin; A. Lefebvre; R. Cespuglio; A.P. Soldatkin (pp. 110-116).

Keywords: Amperometric microbiosensors; Carbon fiber electrode; Glucose; Lactate; Glutamate; In vitro analysis; In vivo detection

A needle-type optical enzyme sensor system for determining glucose levels in fish blood by Hideaki Endo; Yuki Yonemori; Kazuya Musiya; Masashi Maita; Toru Shibuya; Huifeng Ren; Tetsuhito Hayashi; Kohji Mitsubayashi (pp. 117-124).

A needle-type biosensor system was developed for rapid and simple determination of glucose levels in fish blood. The sensor comprises a needle-type hollow container (18-gauge needle), immobilized enzyme membrane and optic fiber probe with ruthenium complex. The enzyme membrane was prepared from glucose oxidase, azide-unit pendant water-soluble photopolymer and an ultra-thin dialysis membrane. The optic fiber probe was inserted into the rolled enzyme membrane placed in the needle-type hollow container. The calibration curve was linear for glucose levels in fish plasma. One assay was completed within 3min. A good reproducibility was observed for 60 times without exchange of the enzyme membrane. The sensor was inserted into the caudal vein of fish to measure blood glucose levels. The sensor responded immediately after insertion and glucose levels could be monitored. Good correlations were observed between values determined using the sensor and conventional methods in the range of 48–157mgdl^−l (correlation coefficient, 0.9474).

Keywords: Biosensor; Enzyme sensor; Needle; Glucose; Blood; Plasma; Fish

Enzymatic methods in food analysis: determination of ascorbic acid by Tatyana N. Shekhovtsova; Svetlana V. Muginova; Julia A. Luchinina; Anna Z. Galimova (pp. 125-132).

The feasibility and expediency of enzymatic methods application in food analysis is demonstrated by the example of ascorbic acid (AsA) determination in foods. Enzymatic determination of ascorbic acid is based on its action as a second substrate of horseradish (HRP) and peanut (PNP) peroxidases in the reactions of o-dianisidine (OD) and 3,3’,5,5’-tetramethylbenzidine (TMB) oxidation with hydrogen peroxide. The rates of the reactions are monitored spectrophotometrically by measuring the duration of the induction period on kinetic curves plotted in coordinates absorption-time. The proposed procedures are sensitive ( c_L=0.1μM), simple, and rapid. The procedure using horseradish peroxidase and the reaction of TMB oxidation was used to determine ascorbic acid in fruit juices, milk and sour-milk products for babies’ nutrition.

Keywords: Horseradish and peanut peroxidases; Ascorbic acid; Food analysis

Enzymatic methods in food analysis: determination of ascorbic acid by Tatyana N. Shekhovtsova; Svetlana V. Muginova; Julia A. Luchinina; Anna Z. Galimova (pp. 125-132).

Keywords: Horseradish and peanut peroxidases; Ascorbic acid; Food analysis

Encapsulation of biomolecules for bioanalytical purposes: Preparation of diclofenac antibody-doped nanometer-sized silica particles by reverse micelle and sol–gel processing by Fotios Tsagkogeorgas; Maria Ochsenkühn-Petropoulou; Reinhard Niessner; Dietmar Knopp (pp. 133-137).

In recent years, the sol–gel technique has attracted increasing interest as a unique approach to immobilize biomolecules for bioanalytical applications as well as biochemical and biophysical studies. For this purpose, crushed biomolecule-doped sol–gel glass monoliths have been widely used. In the present work, for the first time, the encapsulation of anti-diclofenac antibodies in silica nanoparticles was carried out by a combination of reverse micelle and sol–gel technique. Cyclohexane was used for the preparation of the microemulsion as organic solvent, while surfactant Igepal CO-520 was found to be the optimal stabilizer. The antibody source was a purified IgG fraction originating from a polyclonal rabbit antiserum. Tetramethyl orthosilicate (TMOS) was used as precursor. Rather uniform, monodispersed and spherical silica particles of about 70nm diameter size were fabricated, as was demonstrated by transmission electron microscopy (TEM) and scanning electron microscopy/energy dispersive X-ray fluorescence analysis (SEM/EDX). The biological activity of the encapsulated antibodies was evaluated by incubation of the nanoparticles with a diclofenac standard solution and analysis of the filtrate and followed washing solutions by a highly sensitive enzyme-linked immunosorbent assay (ELISA), using non-doped particles as blanks. While only about 6% of the added diclofenac was nonspecifically retained by the blank, the corresponding amount of about 66% was much higher with the antibody-doped particles. An obvious advantage of this approach is the general applicability of the developed technique for a mild immobilization of different antibody species.

Keywords: Sol–gel; Reverse micelle; Silica nanoparticles; Diclofenac; Antibodies; Encapsulation

Brownian motion of aggregating nanoparticles studied by photon correlation spectroscopy and measurements of dynamic magnetic properties by Karolina Petersson; Dag Ilver; Christer Johansson; Anatol Krozer (pp. 138-146).

We have investigated colloidal stability of magnetic nanoparticle suspensions in different buffer systems and NaCl concentrations commonly used for biological applications. We have also investigated how conjugation of proteins to magnetic nanoparticles affects colloidal stability. Two different techniques, giving complementary information on the state of the particle system studied, have been used and compared. We have monitored the rotational Brownian motion of particles using measurements of dynamic magnetic susceptibility in the frequency domain. The results were processed using an algorithm that enables us to quantify changes of particle size distribution for particle suspensions subjected to various buffer conditions. The measurements were compared to results obtained for the translational Brownian motion of the same nanoparticles using photon correlation spectroscopy (PCS). We demonstrate that the complementarity of the two techniques enables more precise characterization of particles in suspension, particularly for suspensions of particles with a wide distribution in size and shape, or systems that are close to the onset of agglomeration.

Keywords: Magnetic nanoparticles; Colloidal stability; Dynamic magnetic susceptibility; Photon correlation spectroscopy; Protein functionalisation; Brownian motion; Translational Brownian motion; Rotational Brownian motion

Rapid detection of toxicity in wastewater: Recent developments with manometric respirometry by A. Tzoris; E.A.H. Hall (pp. 147-157).

Manometric respirometry can be used for the direct toxicity assessment (DTA) of wastewater. A first prototype portable system for field use as well as the principle of operation has been reported previously [A. Tzoris, D. Cane, P. Maynard, E.A.H. Hall, Anal. Chim. Acta 460 (2002) 257; A. Tzoris, D. Fearnside, M. Lovell, E.A.H. Hall, Environ. Toxicol. 17 (2002) 284; A. Tzoris, V. Fernandez-Perez, E.A.H. Hall, Sens. Actuators B 105 (2005) 39]. In this report we examine the simple mathematical derivation of the principle and identify some design parameters that could be altered or improved. Hence by changing the geometry of the respiration chamber aiming at a larger surface area and smaller air headspace volume combined with sample stirring for improved aeration, the response of the instrument was improved by at least 50%. Data obtained from real samples as well as data from a comparative study with a lab-based respirometer routinely used in toxicity assessment is presented. Correlation was excellent with the advantage that a test could be completed in 6min or with five repeat measurements the data could be collected within a 10min measurement period. Using a 25% inhibition threshold, all samples found toxic by the standard method were also found toxic by the Baroxymeter method and dilution factors could be estimated for pretreatment of the toxic waste prior to release to a processing plant.

Keywords: Direct toxicity assessment; Respirometer; Wastewater; Biosensor; Bacterial respiration

Rapid detection of toxicity in wastewater: Recent developments with manometric respirometry by A. Tzoris; E.A.H. Hall (pp. 147-157).

Keywords: Direct toxicity assessment; Respirometer; Wastewater; Biosensor; Bacterial respiration

Combination of immunomagnetic separation with flow cytometry for detection of Listeria monocytogenes by Kyoko Hibi; Akihisa Abe; Eiji Ohashi; Kohji Mitsubayashi; Hideki Ushio; Tetsuhito Hayashi; Huifeng Ren; Hideaki Endo (pp. 158-163).

Listeria monocytogenes can grow at the low temperature commonly used in the storage and transportation of food, and the number of cases of food poisoning caused by L. monocytogenes has increased recently in the US and Europe. Several methods of detecting L. monocytogenes cells have been proposed; however, all existing methods require approximately 48h incubation. In this study, we attempted rapid detection of L. monocytogenes using flow cytometry (FCM). The method is based on measuring the number of L. monocytogenes cells by using a combination of FCM and immunomagnetic separation (IMS). First, polyclonal antibodies (anti- L. monocytogenes rabbit IgG-FITC) conjugated with fluorescein isothiocyanate (FITC) were reacted with L. monocytogenes cells, and then FCM was applied. The cell numbers were determined by FCM using a traditional colony-counting method in the range of 10⁴–10⁸cellsml⁻¹. Tetrameric antibody complexes (TAC) were used because they can recognize both magnetic and FITC molecules on the FITC-conjugated antibodies. FITC-labeled L. monocytogenes cells were reacted with a secondary antibody (TAC) bound to magnetic beads. Then, IMS was used. The method is suitable for detection in the range of 10²–10⁸cellsml⁻¹. The FCM assay enumerated the cells within 1min and the total assay time, including sample preparation, was less than 2h.

Keywords: Listeria monocytogene; Flow cytometry; Antibody; Magnetic beads; Bacteria; Detection

Miniaturized fluorescence detection chip for capillary electrophoresis immunoassay of agricultural herbicide atrazine by Kyeong-Sik Shin; Young-Hwan Kim; Jung-Ah Min; Seoung-Min Kwak; Sang Kyung Kim; Eun Gyeong Yang; Jung-Ho Park; Byeong-Kwon Ju; Tae-Song Kim; Ji Yoon Kang (pp. 164-171).

This paper reports a miniaturized fluorescence detection chip for capillary electrophoresis immunoassay of atrazine, which effectively reduces the size of fluorescence detection system. The photodiode with fluorescence filter was embedded in PDMS (polydimethylsiloxane) microfluidic chip and was placed just below the microfluidic channel. This detection chip is only few mm thick without loss of fluorescence due to the proximity of photodiode and channel. To investigate the feasibility of in situ detection of agricultural herbicide, atrazine was detected using capillary electrophoresis immunoassay in microfluidic chip. Mixture of 570nM fluorescence-labeled atrazine (Ag*) and 700nM anti-atrazine antibody (Ab) was injected and separated in 25mm long microfluidic channel. The separated peaks of Ab-Ag* immunocomplex and Ag* were detected by the miniaturized detector and the change of peak magnitude was also observed with the variation of Ab concentration. The result was verified with those of external PMT (photomultiplier tube) and commercial capillary electrophoresis system. Hence, we have demonstrated the feasibility of portable CE immunoassay of atrazine with on-chip fluorescence detector.

Keywords: Capillary electrophoresis (CE); Fluorescence detection chip; Mircofluidic channel; Polydimethylsiloxane (PDMS); Photodiode; Atrazine

Analysis of size characterized manganese species from liver extracts using capillary zone electrophoresis coupled to inductively coupled plasma mass spectrometry (CZE-ICP-MS) by Mercedes Quintana; Alkisti D. Klouda; Andrew Gondikas; Maria Ochsenkühn-Petropoulou; Bernhard Michalke (pp. 172-180).

Mn is of toxicological concern because overexposure can lead to progressive, permanent neurodegenerative damage. Monomethyl-Mn-pentadienyl-tricarbonyl (MMT) is used as an anti-knock agent in fuel. Exhausted Mn compounds are absorbed in the lung and transported to the liver. Extended exposure causes an overflow of the liver with Mn species moving e.g. to the brain, causing irreversible central nervous system (CNS) disorders like Manganism.This paper focuses on experiments for getting more information on Mn species in liver extracts. The investigations are performed with respect to (1) a size characterization and (2) a subsequent identification of the Mn species in liver extracts using preparative size exclusion chromatography (SEC) followed by capillary zone electrophoresis coupled to inductively coupled plasma mass spectrometry (CZE-ICP-MS). First, extracts were analyzed using a mass calibrated SEC column coupled to ICP-MS detection. The chromatogram showed the⁵⁵Mn-trace and proved main Mn elution between ca. 60–150kDa. Second, liver extracts were fractionated on the same SEC column, however, now the effluent was directed to a fraction collector. This resulted in fractions containing pre-purified, size characterized Mn species per fraction. It turned out that the Mn concentrations per fraction reflected roughly the previous on-line Mn trace. Third, the fractions were subject to CZE-ICP-MS, where the MS was operated additionally with dynamic reaction cell (DRC) technique. From size characterization (with SEC coupled on-line to ICP-MS or connected to a fraction collector and subsequent Mn determination in fractions) it was shown that most Mn species from liver extract were of high molecular mass (HMM) nature as they eluted mostly between 50 and 80min, corresponding to ca. 60–150kDa. With the two-dimensional speciation approach employing first SEC and then CZE-ICP-DRC-MS together with standard addition method, a series of Mn species was identified. Mn species predominantly were Mn-enzymes e.g. arginase, isocitric dehydrogenase, galactosyltransferase, prolidase, pyruvate carboxylase and oxalate oxidase. A typical Mn-transporter – Mn-albumin – was also seen, whilst Mn-transferrin obviously was degraded during SEC separation. This Mn-compound (independent whether as a standard or from liver extract) was not stable during SEC even at the finally chosen physiological conditions.

Keywords: Manganese; Speciation; Capillary zone electrophoresis; Inductively coupled plasma mass spectrometry; Liver extract

An automated extraction approach for isolation of 24 polyaromatic hydrocarbons (PAHs) from various marine matrixes by Lucia Liguori; Karstein Heggstad; Helge T. Hove; Kåre Julshamn (pp. 181-188).

An efficient and selective analytical method for determination and quantification of 24 various polyaromatic hydrocarbons (PAHs) in blue mussel ( Mytilus edulis), salmon fillet ( Salmo salar), fish oil and fish feed has been developed. The samples were extracted by means of accelerated solvent extraction (ASE) technique followed by a purification step with gel permeation chromatography (GPC). Identification and quantification were performed by using GC/MS. The novel combination of silica and alumina in the extraction step furnishes highly purified analytes for the most of the 24 PAHs investigated, and thus a fast and selective analytical method is developed. A small limitation with the method concerns the quantification of acenaphthene (Ace), fluorene (Fl), pyrene (Py) and benzo[ a]anthracene (BaA), as the found values for these compounds do not match the certified values (SRM 2977, mussel tissue). Chrysene (Chr) and triphenylene (Tph) give unresolved peaks. The limits of detection (LOD) and limits of quantification (LOQ) found for benzo[ a]pyrene (BaP) were 1.7 and 0.44pg/g (LOD), and 5.8 and 1.5pg/g (LOQ) for salmon fillet and blue mussel, respectively. This is in a very good accordance with respect to the European Community legislation for official control of BaP levels in foodstuff. The method may be used for qualitative identification of petroleum compounds in marine matrixes.

Keywords: Polyaromatic hydrocarbons (PAH); Accelerated solvent extraction (ASE); Gel permeation chromatography (GPC); Fish; Fish oil; Fish feed; Mussels

An automated extraction approach for isolation of 24 polyaromatic hydrocarbons (PAHs) from various marine matrixes by Lucia Liguori; Karstein Heggstad; Helge T. Hove; Kåre Julshamn (pp. 181-188).

Keywords: Polyaromatic hydrocarbons (PAH); Accelerated solvent extraction (ASE); Gel permeation chromatography (GPC); Fish; Fish oil; Fish feed; Mussels

Continuous determination of volatile products in anaerobic fermenters by on-line capillary gas chromatography by V. Diamantis; P. Melidis; A. Aivasidis (pp. 189-194).

Bio-ethanol and biogas produced during the anaerobic conversion of organic compounds has been a subject of great interest since the oil crisis of the 1970s. In ethanol fermentation and anaerobic treatment of wastewaters, end-product (ethanol) and intermediate-products (short-chain fatty acids, SCFA) cause inhibition that results in reduced process efficiency. Control of these constituents is of utmost importance for bioreactor optimization and process stability. Ethanol and SCFA can be detected with precision by capillary gas chromatography usually conducted in off-line measurements. In this work, an on-line monitoring and controlling system was developed and connected to the fermenter via an auto-sampling equipment, which could perform the feeding, filtration and dilution of the sample and final injection into the gas chromatograph through an automation-based programmed procedure. The sample was continuously pumped from the recycle stream of the bioreactor and treated using a microfiltration unit. The concentrate was returned to the reactor while the permeate was quantitatively mixed with an internal standard solution. The system comprised of a gas chromatograph with the flow cell and one-shot sampler and a PC with the appropriate software. The on-line measurement of ethanol and SCFA, directly from the liquid phase of an ethanol fermenter and a high-rate continuous mode anaerobic digester, was accomplished by gas chromatography. Also, this monitoring and controlling system was proved to be effective in the continuous fermentation of alcohol-free beer.

Keywords: Bioprocess monitoring; On-line capillary gas chromatography; Short-chain volatile fatty acids; Continuous ethanol fermentation with; Zymomonas mobilis; Biogas reactors; Organic overload

Continuous determination of volatile products in anaerobic fermenters by on-line capillary gas chromatography by V. Diamantis; P. Melidis; A. Aivasidis (pp. 189-194).

Determination of organochlorine pesticides and polychlorinated biphenyls in chicken eggs by matrix solid phase dispersion by V.I. Valsamaki; V.I. Boti; V.A. Sakkas; Triantafyllos A. Albanis (pp. 195-201).

A multiresidue method for the determination of 20 organochlorine pesticides (aldrin, endrin, dieldrin, α-BHC, β-BHC, γ-BHC, δ-BHC, α-chlordane, γ-chlordane, 4,4′-DDE, 4,4′-DDT, 4,4′-DDD, endosulfan I, endosulfan II, endosulfan sulfate, endrin aldehyde, heptachlor, heptachlor epoxide, endrin ketone and methoxychlor) and eight PCB congeners (PCB 20, 28, 52, 101, 118, 138, 153, 180) in chicken eggs has been developed and validated. The samples were extracted by a simple and fast matrix solid-phase dispersion (MSPD) method using Florisil as the sorbent material and dichloromethane/hexane (1:1) as the eluting system. Further purification of the extracts was conducted using a conventional clean-up procedure with concentrated sulphuric acid. Determination and quantitation of PCBs and OCs residues was carried out using a gas chromatograph equipped with an electron capture detector (GC-ECD). A mass spectrometric detector (GC–MS) in the selected ion monitoring (SIM) mode was used for confirmation purposes. The method detection limits were <0.7ngg⁻¹ for all PCBs and OCs and the relative standard deviations for analyses of samples fortified over the range of 10–200ngg⁻¹ were <8%. All compounds provided average recoveries (spiked at five concentration levels) ranging from 82% to 110%. The proposed method was used to analyze 30 commercial products taken from local markets in the course of a 3-month sampling campaign.

Keywords: Matrix solid-phase dispersion (MSPD); Organochlorine pesticides (OCs); Polychlorinated biphenyls (PCBs); Chicken eggs

Uncertainty associated to the analysis of organochlorine pesticides in water by solid-phase microextraction/gas chromatography–electron capture detection—Evaluation using two different approaches by Nuno Ratola; Lúcia Santos; Paulo Herbert; Arminda Alves (pp. 202-208).

As the performances of analytical instrumentation are being gradually enhanced, the limits of detection become increasingly lower, allowing more environmental contaminants to be determined in different matrices. Problems emerge when dealing with data from different origins, once the uncertainty of the results is difficult to be compared, especially if its calculation procedure is rather different, as very often happens.Samples of two organochlorine pesticides of somewhat opposite characteristics (lindane and heptachlor, classified as persistent organic pollutants—POPs) were analyzed in aqueous media using headspace solid-phase microextraction (SPME) prior to gas chromatography–electron capture detection (GC–ECD) and this methodology was in-house validated. Detection limits of 0.097 and 0.050μgl⁻¹, average intermediate precision of 11.6% and 27.5%, and average recovery of 95.6% and 103.0%, for lindane and heptachlor, respectively, were found. In the absence of available interlaboratory studies to assess the reliability of the results, the expanded uncertainty was calculated following two different approaches ( bottom-up/Eurachem and modified top-down) and the results were compared.Globally it was clearly shown that, for both pesticides, the lower the concentration range, the higher the uncertainty associated to the results. Expanded uncertainties estimated by bottom-up/Eurachem approach varied from 51% to 14% for a lindane concentration range of 0.1–1μgl⁻¹, and from 48% to 24% for a heptachlor range of 0.1–2μgl⁻¹. Modified top-down approach pointed to 44% (lindane and heptachlor) in the same ranges, meaning that a uniform procedure for uncertainty estimation should be adopted.

Keywords: Lindane; Heptachlor; Method validation; Uncertainty; Solid-phase microextraction (SPME); Gas chromatography–electron capture detection (GC–ECD)

Evaluation of solid sorbents for the determination of fenhexamid, metalaxyl-M, pyrimethanil, malathion and myclobutanil residues in air samples by Nikolaos G. Tsiropoulos; Evangelos B. Bakeas; Vasilios Raptis; Stavroula S. Batistatou (pp. 209-215).

A methodology is described for greenhouse air analysis by sampling fenhexamid, pyrimethanil, malathion, metalaxyl-M and myclobutanil in solid sorbents. Pesticides were determined by gas chromatography with NP Detector. The trapping efficiency of XAD-2, XAD-4, Supelpak-2, Florisil and C-18 at different sampling conditions (rate, time and air humidity) and pesticides concentration levels has been evaluated. No breakthrough was observed in the range of concentration studied (0.10–75μg of each pesticide). In almost all the cases good stability results were obtained. Personal pumps have been used with selected sorbents (Supelpak-2 and C-18) in order to sample malathion and fenhexamid in air of experimental greenhouse after their application in a tomato crop. The dissipation process of the analytes in various time periods after application has been studied. Malathion concentrations varied between 20.1μgm⁻³ just after application and 1.06μgm⁻³ 3 days later. Fenhexamid concentrations, determined by high performance liquid chromatography with UV detection, fall rapidly; after 12h post-application being below 0.50μgm⁻³.

Keywords: Sampling; Greenhouse air; Solid sorbents; Residue analysis; Pesticides; Insecticides; Fungicides

Screening method for organophosphorus insecticides and their metabolites in olive oil samples based on headspace solid-phase microextraction coupled with gas chromatography by C. Tsoutsi; I. Konstantinou; D. Hela; T. Albanis (pp. 216-222).

This work is focused on the effectiveness of headspace solid-phase microextraction (HS-SPME) for the analysis of nine organophosphorus (OPs) insecticides (dimethoate, diazinon, fenitrothion, malathion, fenthion, parathion ethyl, methyl bromophos, methidathion, ethion) and four metabolites (omethoate, malaoxon, fenthion sulfoxide and fenthion sulfone) residues in olive oil samples. The efficiency of six fibre types with different film thickness was compared. PDMS (100μm) was found to be the most suitable fibre for the analysis of OPs in olive oils. Optimization of SPME conditions (stirring rate, extraction time, temperature, salt addition) was based on previous developed method in the laboratory that was enriched with additional analytes including major metabolites. In addition, the effect of the oil matrix on the pesticide recoveries was evaluated using spiked oil samples of different composition (acidity, fatty acids, triglycerides, sterols). It was found that only acidity and total amount of sterols are the main factors influencing the SPME efficiency. Matrix effects were compensated for, by using the internal standard method for the quantification of pesticides. The recoveries at three spiking levels were between 80% and 106% with R.S.D. (%) values below 10% in most cases. Good linearity ( R²>0.985) was observed in the 0.025–0.50mgkg⁻¹ concentration range with satisfactory R.S.D. (%) values of 4.5–10.4%. The method allowed detection of the tested compounds at concentrations below 0.010mgkg⁻¹ with GC–FTD detection. In addition, intra- and inter-day precision was satisfactory (R.S.D. (%) <10%). The performance results confirm the usefulness of the proposed methodology for the analysis of OPs in olive oils. Moreover, the maximum residue limits required by European and international regulations can be attained without difficulty. Finally, the method was applied to 30 virgin olive oil samples from major olive production areas of Greece in the framework of an extended monitoring survey of OPs residues in olive oil. The most commonly found pesticides were fenthion, dimethoate and ethion in levels that did not exceed the MRLs.

Keywords: Olive oil; Organophosphorus insecticides; Headspace solid-phase microextraction; Gas chromatography

Coupling of headspace solid phase microextraction with ultrasonic extraction for the determination of chlorinated pesticides in bird livers using gas chromatography by Dimitra A. Lambropoulou; Ioannis K. Konstantinou; Triantafyllos A. Albanis (pp. 223-230).

In the present study a combined analytical method involving ultrasonic extraction (USE), sulfuric acid clean-up and headspace solid-phase microextraction (HS-SPME) was developed for the determination of chlorinated pesticides (CPs) in bird livers. Extraction of CPs from 1g of liver was performed by ultrasonication for 30min using 20mL of solvent mixture (n-hexane:acetone (4:1, v/v)). The extract was subsequently subjected to a clean-up step for lipid removal. A comparative study on several clean-up procedures prior to the HS-SPME enrichment step was performed in order to achieve maximum recovery and optimal clean-up efficiency, which would provide suitable limits of detection in the gas chromatographic analysis. For this purpose, destructive (sulfuric acid or sodium hydroxide treatment) and non-destructive (alumina column) clean-up procedures has been assayed. The treatment of the extract with 40% (v/v) H₂SO₄ prior to HS-SPME process showed the best performance since lower detection limits and higher extraction efficiencies were obtained. The method detection limit ranged from 0.5 to 1.0ngg⁻¹ wet weight and peak areas were proportional to analyte concentrations ( r²>0.990) in the range of 5–500ngg⁻¹ wet wt. The method was found to be reproducible (R.S.D.<10%) and effective under the operational conditions proposed and was applied successfully to the analysis of CPs in liver tissues of various bird species from Greece.

Keywords: Chlorinated pesticides; Headspace solid phase microextraction; Ultrasonic extraction; Livers; Gas chromatography

Analysis of phenolic acids as chloroformate derivatives using solid phase microextraction–gas chromatography by Ivana Citová; Radek Sladkovský; Petr Solich (pp. 231-241).

In the presented study, a simple and original procedure of phenolic acids derivatization treated by ethyl and methyl chloroformate performed in an aqueous media consisting of acetonitrile, water, methanol/ethanol and pyridine has been modified and optimized. Seven phenolic acid standards—caffeic, ferulic, gallic, p-coumaric, protocatechuic, syringic and vanillic were derivatized into corresponding methyl/ethyl esters and subsequently determined by the means of gas chromatography connected to the flame-ionisation detector (FID). Some selected validation parameters as linearity, detection and quantitation limits and peak area repeatability were valued. The total time of gas chromatography (GC) analysis was 24min for methyl chloroformate and 30min for ethyl chloroformate derivatization. The more suitable methyl chloroformate derivatization was used for further experiments on the possibility of multiple pre-concentration by the direct solid phase microextraction technique (SPME). For this purpose, polyacrylate (PA), polydimethylsiloxane (PDMS), carboxen/polydimethylsiloxane (CAR/PDMS) and polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibres were tested and the extraction conditions concerning time of extraction, temperature and time of desorption were optimized. The most polar PA fibre gave the best results under optimal extraction conditions (50min extraction time, 25°C extraction temperature and 10min desorption time). As a result, the total time of SPME–GC analysis was 74min and an increase in method sensitivity was reached. The limits of quantitation (LOQ) of p-coumaric, ferulic, syringic and vanillic acid esters after SPME pre-concentration were 0.02, 0.17, 0.2 and 0.2μgmL⁻¹, respectively, showing approximately 10 times higher sensitivity in comparison with the original GC method.

Keywords: Phenolic acids; Derivatization; Gas chromatography; Solid phase microextraction

Analysis of phenolic acids as chloroformate derivatives using solid phase microextraction–gas chromatography by Ivana Citová; Radek Sladkovský; Petr Solich (pp. 231-241).

Keywords: Phenolic acids; Derivatization; Gas chromatography; Solid phase microextraction

Doping control analysis in human urine by liquid chromatography–electrospray ionization ion trap mass spectrometry for the Olympic Games Athens 2004: Determination of corticosteroids and quantification of ephedrines, salbutamol and morphine by M.-H. Spyridaki; P. Kiousi; A. Vonaparti; P. Valavani; V. Zonaras; M. Zahariou; E. Sianos; G. Tsoupras; C. Georgakopoulos (pp. 242-249).

A new liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI–MS) ( n) ion trap method for the determination of corticosteroids in urine has been developed and validated. Some anabolic agents, such as epitrenbolone, trenbolone, 2-hydroxymethylformebolone, tetrahydrogestrinone, gestrinone and formoterol were included in the LC–ESI–MS method. Matrix interference, specificity, identification capability, carry over and robustness were estimated as validation parameters. Recoveries ranged from 74 to 113% at the minimum required performance limit (MRPL), which is 30ngmL⁻¹ for corticosteroids and 10ngmL⁻¹ for anabolic agents. Methods for the confirmation and quantification of norpseudoephedrine, ephedrine, methylephedrine, salbutamol, morphine and morphine glucuronide were also developed and validated and in order to minimize analysis time, direct urine injection was used. These methods proved to be specific, accurate and precise across a calibration range for each substance since matrix interference, specificity, carry over, within and between run precision, limit of detection, limit of quantification, intermediate precision and uncertainty were estimated.

Keywords: Liquid chromatography–electrospray ionization ion trap mass spectrometry; Antidoping; Corticosteroids; Ephedrines; Salbutamol; Morphine

Development of a novel liquid chromatography — Evaporative light scattering detection method for bacitracins and applications to quality control of pharmaceuticals by Artemis K. Sarri; Nikolaos C. Megoulas; Michael A. Koupparis (pp. 250-257).

A novel liquid chromatography method for the direct determination of bacitracin main components (Bc-A, -B₁, -B₂ and -B₃), a basic, cyclic polypeptide antibiotic, was developed and validated, based on ion pairs formation with trifluoroacetic acid (TFA) and evaporative light scattering detection (ELSD).The selected analytical column was the Waters Nova-pak C₈ (3.9×150mm), for which the optimum (using modified Simplex algorithm) mobile phase was H₂O–ACN (73:27, v/v) containing 0.80μLmL⁻¹ of TFA, at a flow rate of 1.0mLmin⁻¹. Optimized ELSD parameters were: nebulizing gas (nitrogen) pressure=3.5bar, evaporation temperature=50°C, detector gain=12.Retention time of Bc-B₁, -B₂, -B₃, -A and -F (oxidative degradation product of Bc-A) was 5.3, 5.8, 7.7, 8.7, 15.9min, respectively, while zinc ions and related peptides were eluted at 1.3–1.9min. A logarithmic calibration curve was obtained for each component ( r>0.998), while the concentration range of total bacitracin was 30–235μgmL⁻¹. Detection limits for the individual components were in the range 1.0–1.6μgmL⁻¹.The proposed method was applied for the direct determination of Bc components and related peptides in raw materials and pharmaceutical formulations (tablets, powder and aerosol) without tedious pretreatment (for tablets, a liquid–liquid extraction of magnesium with oxine was required). In the case of matrix interference, synthetic standards containing the same amounts of excipients or the standard addition technique were used. Recovery from spiked commercial formulations was ranged from 96.7% to 101.5% (in respect of total Bc).

Keywords: Evaporative light scattering; Liquid chromatography; Bacitracins; Polypeptide

Development of a liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI MS/MS) method for the quantification of bioactive substances present in olive oil mill wastewaters by Fotini N. Bazoti; Evangelos Gikas; Alexios Leandros Skaltsounis; Anthony Tsarbopoulos (pp. 258-266).

A novel liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI MS/MS) method was developed and validated for the simultaneous determination of the bioactive substances hydroxytyrosol, tyrosol and 2-(5-ethylidene-2-oxo-tetrahydro-2 H-pyran-4-yl)acetic acid in olive oil mill wastewater samples (OMW). The chromatographic separation was performed on a RP-C₈ column using a water–acetonitrile gradient program and the detection was achieved by tandem MS in the negative ion mode. Calibration curves were linear for all bioactive compounds over the range of 1–100ng injected, while the method exhibited good accuracy, intra- and inter-day precision. The limit of detection and the limit of quantification were in the low to mid pg range and the method was simple and rapid. Because the disposal of OMW is an environmental problem and on the other hand OMW are rich in biologically active compounds that could be recovered and exploited in various applications, the developed method was applied to the monitoring of OMW samples and the quantitative determination of the aforementioned substances. In this way, the original content in bioactive compounds could be assigned in the raw matrix, and the enrichment of the samples by various pretreatment methods could be assessed. Also, full-scan ESI MS was applied to OMW samples for the identification of several compounds known to be present in OMW.

Keywords: Olive oil; Wastewater; Hydroxytyrosol; Tyrosol; Electrospray ionization tandem mass spectrometry

Optimization and validation of a high performance liquid chromatography method for the simultaneous determination of vitamins A and E in human serum using monolithic column and diode-array detection by Lubor Urbánek; Dagmar Solichová; Bohuslav Melichar; Josef Dvořák; Iveta Svobodová; Petr Solich (pp. 267-272).

In this study a novel, simple and rapid reversed-phase high performance liquid chromatography (HPLC) procedure for simultaneous determination of vitamins A and E (retinol and alpha-tocopherol) in blood serum has been developed and validated using monolithic column and diode-array detection (DAD).The monolithic column Chromolith Performance RP-18e (100mm×4.6mm) was operated at ambient temperature. One hundred percent methanol at flow rate 2.5mlmin⁻¹ was used as a mobile phase. Detection of both compounds was performed with diode-array detector, retinol was monitored at 325nm and alpha-tocopherol at 295nm.The linear dependence between peak area and concentration ranged from 0.25 to 10.00μmoll⁻¹ for retinol and 0.5–50.0μmoll⁻¹ for alpha-tocopherol. The limit of detection (LOD) for retinol was 0.02μmoll⁻¹ and limit of quantification (LOQ) was 0.07μmoll⁻¹. The limit of detection (LOD) for alpha-tocopherol was 0.1μmoll⁻¹ and limit of quantification (LOQ) was 0.3μmoll⁻¹. Retinol was eluted in 0.8min and alpha-tocopherol in 1.4min. The simultaneous analysis of vitamin A and E can be achieved in less than 2min.The implementation of monolithic column Chromolith Performance shortens the time of analysis of both vitamins four times in comparison with using traditional particulate column Pecosphere C18 (150mm×4.6mm), 5μm. This fact may play an important role for routine clinical analysis of biological samples.

Keywords: Retinol; Alpha-tocopherol; Monolithic column; Liquid chromatography

Liquid chromatography–tandem mass spectrometry in chiral study of amlodipine biotransformation in rat hepatocytes by Bohumila Suchanova; Ludek Sispera; Vladimir Wsol (pp. 273-283).

A high proportion of drugs are chiral compounds used as racemic mixtures in a clinical practice. Very often only one of two enantiomers exhibits a desired pharmacological effect. Amlodipine, 2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl-1,4-dihydropyridine, is a chiral calcium channel blocker, currently used as a racemate in clinical practice. Racemic mixture is used even though it is known that R- and S-amlodipine do not have the same biological activity and only S-amlodipine possesses vasodilating properties.In this work a novel reversed phase liquid chromatography (RP-LC) separation method for amlodipine and its metabolites was developed. Based on this separation chiral aspects of amlodipine biotransformation were studied by incubation of amlodipine and its two individual enantiomers with primary culture of rat hepatocytes. Structure of the metabolites was elucidated using a liquid chromatography (LC) separation with ultraviolet (UV) and mass spectrometry (MS) detection. An LC–tandem MS (MS/MS) method was used to establish fragmentation pattern of amlodipine and its metabolites. Eight metabolites presented in the highest amount were identified and semiquantified by employing an LC separation. Basically two types of metabolites were detected, reduced type – dihydropyridine metabolites and oxidized type – pyridine metabolites. Other metabolic modification included changes of functional groups, e.g., methylester hydrolysis or acetylation of amino group. The results exhibited that R-amlodipine was stereoselectively metabolized by the respective biotransformation enzymes in rat liver hepatocytes and it is also demonstrated by greater extent of R-amlodipine conversion into metabolites where the values for R-amlodipine are for the most metabolites higher than those for metabolites of S-amlodipine.

Keywords: Liquid chromatography–tandem mass spectrometry; Data dependent acquisition; Amlodipine; Stereoselective metabolism; In vitro; Chiral switching

Liquid chromatography–tandem mass spectrometry in chiral study of amlodipine biotransformation in rat hepatocytes by Bohumila Suchanova; Ludek Sispera; Vladimir Wsol (pp. 273-283).

Keywords: Liquid chromatography–tandem mass spectrometry; Data dependent acquisition; Amlodipine; Stereoselective metabolism; In vitro; Chiral switching

Development and validation of a reversed-phase ion-pair liquid chromatography method for the determination of magnesium ascorbyl phosphate and melatonin in cosmetic creams by Athanasia Varvaresou; Efthimios Tsirivas; Kritonas Iakovou; Evagelos Gikas; Zaxarias Papathomas; Ariadni Vonaparti; Irene Panderi (pp. 284-290).

A reversed-phase ion-pair high-performance liquid chromatographic method was developed and validated for the determination of magnesium ascorbyl phosphate and melatonin in cosmetic creams. The determination was performed on a BDS C 18 analytical column (250×4.6mm i.d., 5μm particle size); the mobile phase consisted of 0.020M tetrabutylammonium hydroxide and 0.025M potassium dihydrogen phosphate (pH 6.8) mixed with acetonitrile in a ratio (77:23, v/v) and pumped at a flow rate 1.00mlmin⁻¹. The UV detector was operated at 260nm. The retention times of the magnesium ascorbyl phosphate, melatonin and chlorthalidone that was used as internal standard, were 6.55, 9.18 and 11.07min, respectively. Calibration graphs are linear ( r better than 0.9990, n=6), in concentration range 1.00–10.00μgml⁻¹ for magnesium ascorbyl phosphate and 0.63–6.25μgml⁻¹ for melatonin. The intra- and inter-day R.S.D. values were less than 6.0%, while the relative percentage error E_r was less than 3.5% ( n=5). The quantitation limits were 0.69 and 0.47μgml⁻¹, for magnesium ascorbyl phosphate and melatonin, respectively. The method was applied to the analysis of a cosmetic cream and proved to be suitable for rapid and reliable quality control.

Keywords: Magnesium ascorbyl phosphate; Melatonin; Ion-pair liquid chromatography; Cosmetics

Validation and global uncertainty of a liquid chromatographic with diode array detection method for the screening of azoxystrobin, kresoxim-methyl, trifloxystrobin, famoxadone, pyraclostrobin and fenamidone in grapes and wine by Susana de Melo Abreu; Pierluigi Caboni; Paolo Cabras; Vincenzo Luigi Garau; Arminda Alves (pp. 291-297).

Azoxystrobin, kresoxim-methyl, trifloxystrobin, pyraclostrobin, famoxadone and fenamidone are permitted Q_o Inhibitor (Q_oI) fungicides applied to vine in some European countries for the treatment of downy and powdery mildews. In this work, a method is validated for the analysis of these fungicides in grapes and wine. This screening method consists in a simple one step liquid–liquid extraction followed by liquid chromatography (LC) fitted with a diode array detector (DAD). Limits of detection for grapes and wine were below 0.2mgkg⁻¹ or mgl⁻¹, precision was not above 13%, and recoveries were, on average, 95±5% for grapes and 104±6% for wine. Global uncertainties evaluated in the concentration range from 0.25 to 2.50mgl⁻¹ were below 20%. A confirmatory method by gas chromatography (GC) with mass spectrometry (MS) detection was used.

Keywords: Fungicide residues; Grapes; Wine; Method validation; Liquid chromatography; Diode array detection

Sensitive and simple liquid chromatographic method with ultraviolet detection for the determination of nifedipine in canine plasma by M.V. Vertzoni; C. Reppas; H.A. Archontaki (pp. 298-304).

An isocratic high-performance liquid chromatographic method with detection at 240nm was developed, optimized and validated for the determination of nifedipine in canine plasma. Liquid–liquid extraction was used as the sample preparation technique. Carbamazepine was used as internal standard. A Hypersil BDS RP-C₁₈ column (250mm×4.6mm, 5μm) was equilibrated with a mobile phase composed of water and methanol, 45:55 (v/v). Its flow rate was 1mlmin⁻¹. The elution time for nifedipine and carbamazepine was approximately 12 and 8min, respectively. Calibration curves of nifedipine in plasma were linear in the concentration range of 1–200ngml⁻¹. Limits of detection and quantification in plasma were 0.5 and 1.5ngml⁻¹, respectively. Recovery was greater than 98%. Intra- and inter-day relative standard deviation for nifedipine in plasma was less than 8.5 and 10%, respectively. This method was applied to the determination of nifedipine plasma levels after administration of commercially available soft gelatine capsules to dogs.

Keywords: Nifedipine; Liquid chromatography with ultraviolet detection; Canine plasma; Pharmacokinetic profile

Sensitive and simple liquid chromatographic method with ultraviolet detection for the determination of nifedipine in canine plasma by M.V. Vertzoni; C. Reppas; H.A. Archontaki (pp. 298-304).

Keywords: Nifedipine; Liquid chromatography with ultraviolet detection; Canine plasma; Pharmacokinetic profile

Modelling flow rate gradient elution in reversed-phase liquid chromatography by A. Pappa Louisi; P. Nikitas; A. Zitrou (pp. 305-310).

The fundamental equation of flow programming elution was tested in several different types of flow-rate gradients (step, linear, multilinear, parabolic and more combined gradients) implemented in the separation of two multicomponent mixtures of solutes. The retention prediction obtained for all solutes under all flow-programmed conditions was excellent. In addition, although flow programming appears quite limited in its ability to provide improved performance in liquid chromatography, there are specific flow-rate profiles which provide significant improvement in the rearrangement of the peaks within a chromatogram.

Keywords: Liquid chromatography; Flow rate gradient

Modelling flow rate gradient elution in reversed-phase liquid chromatography by A. Pappa Louisi; P. Nikitas; A. Zitrou (pp. 305-310).

Keywords: Liquid chromatography; Flow rate gradient

Contribution to the standardization of the chromatographic conditions for the lipophilicity assessment of neutral and basic drugs by Costas Giaginis; Stamatios Theocharis; Anna Tsantili-Kakoulidou (pp. 311-318).

The chromatographic conditions aiming to a better simulation of n-octanol–water partitioning using a base deactivated silica (BDS) column as stationary phase were investigated for structurally diverse basic and neutral drugs. Extrapolated retention factors log k_w, determined using different methanol fractions as organic modifier, were considered as lipophilicity indices. The effect of n-decylamine and n-octanol as mobile phase additives was examined and the appropriateness of the final retention outcome to reproduce lipophilicity data was evaluated. Moreover, the influence of n-octanol on the linearity of the log k/methanol fraction relationship and on the uniformity of the retention mechanism was investigated. 1:1 correlation between log k_w values and the logarithm of the distribution coefficient (log D) was established for basic drugs in presence of both n-decylamine and n-octanol as mobile phase additives. However, for neutral drugs n-decylamine proved to be a sufficient and more important factor than n-octanol.

Keywords: Lipophilicity; Extrapolated retention factors log; k; _w; Base deactivated silica (BDS) column; Mobile phase additives

Determination of²²⁶Ra in aqueous solutions via sorption on thin films and α-spectrometry by D. Karamanis; K.G. Ioannides; K.C. Stamoulis (pp. 319-327).

An improved spectrometric method to determine the²²⁶Ra activity in aqueous solutions is described. The method involves two stages, a preconcentration stage of²²⁶Ra sorption onto a thin manganese layer and a measurement stage using α-spectrometry. Manganese oxide thin films were prepared and characterized with X-ray diffraction (XRD) and X-ray fluorescence (XRF) analyses. The thin films were found to follow the XRD patterns and chemical formula of the K-birnessite layered exchanger. The preconcentration of radium was studied relative to the initial radium concentration, pH and salt concentrations. The preconcentration kinetics was studied as a function of manganese surface, solution volume and salt concentration. Extensive Monte Carlo calculations were performed to optimise the detection of α-particles. In this way, the thin film preparation procedure as well as the radium sorption and the measurement conditions were optimised and detection limits lower than 0.5mBqL⁻¹ were obtained for 2d of procedure completion. The method was validated with IAEA standards and it was applied for the determination of²²⁶Ra in bottled waters and also wastewaters from the major thermoelectric plant in Greece. Moreover, the²²⁶Ra distribution coefficients ( K_d) of two differently prepared powder manganese oxides, a crystalline silicotitanate and an aluminium-pillared montmorillonite were determined by γ-spectrometry.²²⁶Ra sorption experiments on silicotitanate thin films were performed and improvements in resolution and reduction of exposure time were observed.

Keywords: Radium; Manganese dioxide thin films; α-Spectrometry; Water; Sorption

Determination of²²⁶Ra in aqueous solutions via sorption on thin films and α-spectrometry by D. Karamanis; K.G. Ioannides; K.C. Stamoulis (pp. 319-327).

Keywords: Radium; Manganese dioxide thin films; α-Spectrometry; Water; Sorption

Characterization of oil spills in the environment using parallel factor multiway analysis by Vassilis Gaganis; Nikos Pasadakis (pp. 328-332).

The aim of this study was to characterize samples of petroleum spills derived from the oily free-phase zone located in the subsurface of a petroleum refinery and to reveal the contained distinct petroleum fractions, thus enabling the identification of the spill origin. The samples were collected from different monitoring wells and were analyzed using gel permeation chromatography (GPC) combined with a UV-diode array detector. The PARAFAC algorithm was employed for the analysis of the 3-D experimental data matrix, which contained the areas under the chromatographic trace, measured for distinct time slices over the 270–440nm UV range for the whole sample population. The application of the PARAFAC method revealed two significant elution profiles possessing characteristic UV signals, which were attributed to the gasoline and diesel fractions, respectively. A third elution profile was also identified which corresponded to biodegraded heavy fractions. The relative contribution of these compositional features to the oil spill samples was also identified. The presented method can be employed as a rapid and reliable fingerprinting tool in environmental studies, where petroleum pollutants of unknown composition are expected.

Keywords: Oil spills; Parallel factor analysis; Environment

Characterization of oil spills in the environment using parallel factor multiway analysis by Vassilis Gaganis; Nikos Pasadakis (pp. 328-332).

Keywords: Oil spills; Parallel factor analysis; Environment

Surface plasmon resonance imaging as a multidimensional surface characterization instrument—Application to biochip genotyping by Pierre Lecaruyer; Ilaria Mannelli; Virginie Courtois; Michel Goossens; Michael Canva (pp. 333-340).

Surface plasmon resonance imaging (SPRI) sensors allow the characterization of a metal/dielectric interface. Providing proper biochemical functionalization and spatial structuration of the functionalized surface, an optical biochip system – label free and real time – can be achieved. We study the impact of the different physical parameters on the quality of the measurements. Such a SPRI system has a great sensitivity to small variations of the physical parameters (layer optical index, thickness, etc.) occuring at the sensor surface. Precision and reliability of the measurements are provided by a multidimensional approach (4D i.e. spatial coordinates x– y, time t, angle of incidence θ) allowing multiple self-calibration procedures. Such apparatus has already been successfully applied in genomics and proteomics, studying DNA:DNA and oligosaccharide:protein interactions. In this article, we illustrate the advantages of the SPRI setup applied to the detection of gene mutations, using as a model the genetic disease Cystic Fibrosis. The results demonstrate that the system is able to monitor and analyse the interaction under investigation, allowing the diagnosis of genetic single nucleotide polymorphisms by exploiting only a part of the multidimensional potential ( x, y, t, θ).

Keywords: Surface plasmon resonance imaging; Biochip; Biomolecular interaction monitoring; Multidimensional characterization; Instrument of genetic diagnosis

Analysis of protein-based binding media found in paintings using laser induced fluorescence spectroscopy by Austin Nevin; Sharon Cather; Demetrios Anglos; Costas Fotakis (pp. 341-346).

Laser induced fluorescence (LIF) spectroscopy of intrinsic fluorophores from organic media found in paintings (casein, animal glue and egg proteins) provides novel non-invasive means of characterisation of general classes of media on the basis of fluorescence emission arising from the presence of certain amino acids and their degradation byproducts. Proteins from traditionally employed binding media include collagen, casein, albumin and other egg proteins, of animal sources (skins, milk and egg respectively). Wavelength dependence of the spectra is presented for analyses of thin films of protein-based binding media.

Keywords: Laser induced fluorescence; Protein; Binding media; Painting; Autofluorescence

Analysis of protein-based binding media found in paintings using laser induced fluorescence spectroscopy by Austin Nevin; Sharon Cather; Demetrios Anglos; Costas Fotakis (pp. 341-346).

Keywords: Laser induced fluorescence; Protein; Binding media; Painting; Autofluorescence

A study of ancient pottery by means of X-ray fluorescence spectroscopy, multivariate statistics and mineralogical analysis by Christina Papachristodoulou; Artemios Oikonomou; Kostas Ioannides; Konstantina Gravani (pp. 347-353).

Energy-dispersive X-ray fluorescence spectroscopy was used to determine the composition of 64 potsherds from the Hellenistic settlement of Orraon, in northwestern Greece. Data classification by principal components analysis revealed four distinct groups of pottery, pointing to different local production practices rather than different provenance. The interpretation of statistical grouping was corroborated by a complementary X-ray diffraction analysis. Compositional and mineralogical data, combined with archaeological and materials’ science criteria, allowed addressing various aspects of pottery making, such as selection of raw clays, tempers and firing conditions.

Keywords: Ancient pottery; Compositional data; X-ray fluorescence (XRF) spectroscopy; Principal component analysis (PCA); Mineralogical analysis

Determination of the pesticide carbaryl and its photodegradation kinetics in natural waters by flow injection–direct chemiluminescence detection by George Z. Tsogas; Dimosthenis L. Giokas; Petros G. Nikolakopoulos; Athanasios G. Vlessidis; Nicholaos P. Evmiridis (pp. 354-359).

A novel flow injection–chemiluminescence method for the quantitative assay of the pesticide carbaryl in environmental samples is presented. The determination is based on the CL-emission generated by the oxidation of the pesticide with potassium permanganate. The linear response of CL-emission versus concentration is valid in the range from 0.01 to 1.0μgmL⁻¹, yielding detection limits ( S/ N=3) as low as 14.8ngmL⁻¹. The method shows high reproducibility (R.S.D.=2.29%, n=10) and is subject to minor interferences from various organic and inorganic species likely to be found in natural waters. The suggested method is rapid and capable to be fully automated, thus resulting to a method of satisfactory sampling throughput, with low detection limits and efficient precision for routine analysis. The use of this technique to a new application of direct chemiluminescence involving the determination of carbaryl photodegradation in natural waters was successfully accomplished.

Keywords: Pesticides; Carbaryl; Chemiluminescence detection; Flow injection analysis; Potassium permanganate; Photodegradation

Kinetic separation in flow injection spectrophotometry: Simultaneous determination of copper and zinc in a single run by L.K. Shpigun; Ya.V. Shushenachev; P.M. Kamilova (pp. 360-365).

A flow injection spectrophotometric method for the simultaneous determination of copper (II) and zinc (II) in mixtures was developed, utilizing metal–zincon complex formation and kinetic differences in the ligand substitution reaction of these complexes with aminopolycarboxylic acids at pH 9.18. The linear range for the determination of zinc is 0.2–9.7μgml⁻¹ and for copper is 0.2–3.5μgml⁻¹. About 90 samples can be determined in 1h. The proposed method has been applied to the determination of zinc and copper in various vitamin formulations with no need for previous separation.

Keywords: Flow injection spectrophotometry; Kinetic separation; Ligand exchange reaction; Zinc; Copper

Kinetic separation in flow injection spectrophotometry: Simultaneous determination of copper and zinc in a single run by L.K. Shpigun; Ya.V. Shushenachev; P.M. Kamilova (pp. 360-365).

Keywords: Flow injection spectrophotometry; Kinetic separation; Ligand exchange reaction; Zinc; Copper

Automated sequential injection fluorimetric set-up for multiple release testing of topical formulation by Jana Klimundová; Kateřina Mervartová; Hana Sklenářová; Petr Solich; Miroslav Polášek (pp. 366-370).

A fully automated sequential injection analysis (SIA) device for simultaneous release testing of multiple ointment samples was devised and applied to the release testing of an ointment containing salicylic acid as an active substance. The SIA system consisted of a bi-directional 5ml syringe pump, two 8-position selection valves, auxiliary peristaltic pump, fluorescence detector and three Franz cells maintained at 32°C (water bath). The ointment sample was placed on top of a synthetic Millipore GTTP poly(carbonate) membrane (thickness 10μm, pore size 0.2μm) of the Franz cell containing 15ml of aqueous phosphate buffer of pH 7.4 as the acceptor liquid. The samples of the acceptor liquid (50μl) were aspirated in 15min intervals for the period of 6h from each of the 3 cells and dispensed to a fluorescence detector to determine the concentration of salicylic acid (excitation at 297nm and emission at 405nm). The volume of the acceptor medium taken for analysis was automatically replenished after each measurement. The calibration curve was rectilinear for 1–100μgml⁻¹ of salicylic acid. The device allowed simultaneous monitoring of the release tests for up to six cells including automated computer-aided evaluation of the release profile parameters.

Keywords: Automation; Release; Sequential injection; Salicylic acid; Fluorescence

Determination and antioxidant activity evaluation of etodolac, an anti-inflammatory drug, by sequential injection analysis by Julia B. Garcia; M. Lúcia M.F.S. Saraiva; José L.F.C. Lima (pp. 371-375).

A robust, accurate and sensitive automated procedure for the determination of etodolac, an anti-inflammatory drug, in pharmaceutical formulas by sequential injection analysis, is reported. The same system was also applied to evaluate the antioxidant activity of the drug expressed as Trolox equivalent antioxidant capacity (TEAC). The methodology is based on measuring at 734nm the decay of absorbance of a solution with the radical 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⁺) after its reduction by etodolac. Optimum ABTS⁺-etodolac reaction was achieved with 0.329mL of sample and 0.205mL of ABTS⁺ solution. Etodolac was determined at concentrations up to 4.5×10⁻⁵molL⁻¹. A solution detection limit of 6.6×10⁻⁶molL⁻¹ was obtained under the optimised experimental conditions. A relative standard deviation ( n=10) lower than 4.7% with a sample throughput of more than 21 samples/h was obtained. No interference from excipients was observed. The developed methodology was applied in the analysis of pharmaceutical preparations and the obtained results were in good agreement with those furnished by the reference procedure with relative deviations lower than 1.4%.

Keywords: Etodolac; 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS); Antioxidant activity; Sequential injection analysis

Using on-line solid phase extraction for determination of amiloride in human urine by sequential injection technique by Jitka Huclová; Dalibor Šatínský; Ondřej Pavlí�?ek; Lucie Vedralová; Rolf Karlí�?ek (pp. 376-382).

This presented paper deals with a new methodology for the direct determination of amiloride in human urine. The methodology described is based on the sequential injection analysis technique (SIA) coupled with solid phase extraction (SPE) microcolumn. SPE microcolumn was used for selective retention of amiloride, while the urine matrix components were eluted with water carrier flow to the waste. Due to the acid-basic and polarity properties of amiloride molecule and principles of ion-exchange chromatography, it was possible to retain amiloride on the ion-exchange sorbent (SPE BAKER WCX—carboxy group). Eluting solution was 0.01M HNO₃+0.1M KCl, flow rate 20μls⁻¹. The fluorescence detection of amiloride was performed at λ_em 385nm (secondary filter). Recovery was found in the range 96.8–99.4% for 10 times diluted urine, linearity of determination in the range 0.5–100μgml⁻¹ ( r=0.998), and 3 σ limit of detection (LOD) was 0.05μgml⁻¹. The whole procedure comprising raw sample pre-treatment, analyte detection and column reconditioning took 8min. The proposed SIA–SPE method has been applied for direct determination of amiloride in human urine.

Keywords: Sequential injection analysis (SIA); Solid phase extraction (SPE); Amiloride; Urine; Sample preparation

Using on-line solid phase extraction for determination of amiloride in human urine by sequential injection technique by Jitka Huclová; Dalibor Šatínský; Ondřej Pavlíček; Lucie Vedralová; Rolf Karlíček (pp. 376-382).

Keywords: Sequential injection analysis (SIA); Solid phase extraction (SPE); Amiloride; Urine; Sample preparation

Modified tubular electrode in a multi-commutated flow system by M. Luísa S. Silva; M. Beatriz Q. Garcia; José L.F.C. Lima; E. Barrado (pp. 383-390).

This work describes the construction of a Nafion coated glassy carbon tubular electrode and its use, coupled to a multi-commutated flow system, for the voltammetric determination of acetaminophen in serum and pharmaceutical formulations. The modification of the electrode enhanced the analytical signal intensity and, simultaneously, prevented the electrode surface fouling. The multi-commutated system conferred high versatility to the manifold, allowing it to be easily adjusted to each determination without the need to introduce any physical reconfiguration. The on line enzymatic hydrolysis of acetaminophen, giving rise to 4-aminophenol, allowed the problem of interferences resulting from the oxidation of the matrix serum constituents to be overcome.The method presented a linear range up to 5.0×10⁻⁴moll⁻¹ with a detection limit of 1.7×10⁻⁵moll⁻¹, a sampling rate of 24 determinations per hour and a repeatability (expressed in relative standard deviation) always lower than 3%. The method was applied to serum samples and pharmaceutical formulations and no statistically significant difference between the results obtained by the proposed method and by the reference methods was found, for a confidence level of 95%.

Keywords: Acetaminophen; 4-Aminophenol; Modified tubular electrode; Nafion; Voltammetry; Multi-commutation; Flow analysis

Modified tubular electrode in a multi-commutated flow system by M. Luísa S. Silva; M. Beatriz Q. Garcia; José L.F.C. Lima; E. Barrado (pp. 383-390).

Keywords: Acetaminophen; 4-Aminophenol; Modified tubular electrode; Nafion; Voltammetry; Multi-commutation; Flow analysis

A smart multisyringe flow injection system for analysis of sample batches with high variability in sulfide concentration by Laura Ferrer; José Manuel Estela; Víctor Cerdà (pp. 391-398).

A fully automated smart multisyringe flow injection analysis (MSFIA) system for the monitoring of sulfide in a wide concentration range is proposed. It allows the determination of sulfide in samples containing suspended solids without requiring any preliminary batch sample treatment. The smart system is able to choose by itself the best approach to quantify the analyte, selecting either a spectrophotometric or a reflectometric detection.The method, carried out in a multi-commuted system, is based on the analyte release as hydrogen sulfide from the donor channel of the gas-diffusion module into an alkaline acceptor channel solution, which is merged with N, N-dimethyl- p-phenylenediamine (DMPD) and Fe(III). The in-line generated methylene blue (MB) dye can be delivered to an optical fiber diffuse reflectance sensor or to a flow-cell spectrophotometer according to the analyte concentration.The detection limit (3 s_b/ S) was 4.6μgl⁻¹. Two linear calibration graphs between 50–1000 and 500–10000μgl⁻¹ sulfide for reflectometry and spectrophotometry, respectively, were obtained. The potentialities of this method were assessed via the determination of sulfide at a wide range of concentrations (4.6μgl⁻¹ to 100mgl⁻¹). The high selectivity and sensitivity, the low reagent consumption and the miniaturization of the proposed automated method should be highlighted.

Keywords: Multisyringe flow injection analysis; Smart system; Gas diffusion; Optrode; Pre-concentration; Sulfide

A smart multisyringe flow injection system for analysis of sample batches with high variability in sulfide concentration by Laura Ferrer; José Manuel Estela; Víctor Cerdà (pp. 391-398).

Keywords: Multisyringe flow injection analysis; Smart system; Gas diffusion; Optrode; Pre-concentration; Sulfide

Determination of mercury by multisyringe flow injection system with cold-vapor atomic absorption spectrometry by L.O. Leal; O. Elsholz; R. Forteza; V. Cerdà (pp. 399-405).

A new software-controlled time-based multisyringe flow injection system for mercury determination by cold-vapor atomic absorption spectrometry is proposed. Precise known volumes of sample, reducing agent (1.1% SnCl₂ in 3% HCl) and carrier (3% HCl) are dispensed into a gas–liquid separation cell with a multisyringe burette coupled with one three-way solenoid valve. An argon flow delivers the reduced mercury to the spectrometer.The optimization of the system was carried out testing reaction coils and gas–liquid separators of different design as well as changing parameters, such as sample and reagents volumes, reagent concentrations and carrier gas flow rate, among others.The analytical curves were obtained within the range 50–5000ngL⁻¹. The detection limit (3 σ_b/ S) achieved is 5ngL⁻¹. The relative standard deviation (R.S.D.) was 1.4%, evaluated from 16 successive injections of 250ngL⁻¹ Hg standard solution. The injection and sample throughput per hour were 44 and 11, respectively.This technique was validated by means of solid and water reference materials with good agreement with the certified values and was successfully applied to fish samples.

Keywords: Multisyringe flow injection analysis (MSFIA); Mercury; Cold-vapor; Atomic absorption

Determination of mercury by multisyringe flow injection system with cold-vapor atomic absorption spectrometry by L.O. Leal; O. Elsholz; R. Forteza; V. Cerdà (pp. 399-405).

Keywords: Multisyringe flow injection analysis (MSFIA); Mercury; Cold-vapor; Atomic absorption

Optical fiber reflectance sensor coupled to a multisyringe flow injection system for preconcentration and determination of 1-naphthylamine in water samples by J.L. Guzmán Mar; L. López Martínez; P.L. López de Alba; J.E. Castrejón Durán; V. Cerdà Martín (pp. 406-412).

A novel optical fiber reflectance sensor is coupled to a multisyringe flow injection system (MSFIA) for the preconcentration and determination of 1-naphthylamine (NPA) in water samples using C18 disks (octadecyl groups). NPA, being a first-class carcinogen, is important from a toxicological point of view and, therefore, its quantification is of considerable interest. In this study, the Griess reaction is used for sensitive and selective spectrophotometric determination of NPA. The reaction involves conversion of nitrite into nitrous acid in acidic medium followed by diazotization of sulphanilic acid and formation of a diazonium salt. The diazonium salt is then combined with NPA to form 4-(sulphophenylazo)-1-naphthylamine, an azo dye. This compound is subsequently retained onto a C18 disk followed by spectrophotometric detection at 540nm, and it is then eluted with methanol in water (80%, v/v), so that the C18 disk is regenerated for subsequent experiments. Under the established optimum conditions, a calibration graph for NPA was constructed. Good linearity was observed within a concentration range from 10 to 160μgl⁻¹. The lineal regression equation is A=(0.0027±0.0001) [NPA]+(0.0296±0.0047), r=0.9991; relative standard deviation values obtained from the analysis of 10 samples of 10, 80 and 160μgl⁻¹ are 4.7, 1.2 and 0.6%, respectively. The mean value relative errors for concentrations of 10, 80, 160μgl⁻¹ are 3.4, 0.9 and 0.4%, respectively. The detection and quantification limits were 1.1 and 3.7μgl⁻¹. A sampling throughput of 14 injections per hour is achieved. The repeatability calculated for five different C18 disks was E_rel=2.8%. The proposed technique has been validated by replicate analysis ( n=6) of several water samples with spiked NPA, giving satisfactory results.

Keywords: Multisyringe flow injection analysis (MSFIA); Reflectance sensor; Optosensor; 1-Naphthylamine (NPA); Griess reaction; C18 disks; Water samples

Determination of arsenic(III) by flow injection solid phase extraction coupled with on-line hydride generation atomic absorption spectrometry using a PTFE turnings-packed micro-column by Aristidis N. Anthemidis; Evdoxia K. Martavaltzoglou (pp. 413-418).

A novel flow injection (FI) solid phase extraction method for the determination of arsenic(III) at trace levels was developed, using on-line hydride generation atomic absorption spectrometry (HG-AAS). Selective determination of As(III) was achieved by on-line formation and retention of the pyrrolidine dithiocarbamate arsenic complex As(III)–PDC on the PTFE turnings which are packed in the preconcentration micro-column. The retained complex was eluted by 2ml 2moll⁻¹ HCl and subsequently introduced on-line into the integrated reaction chamber/gas–liquid separator (RC–GLS). A 1.5% (m/v) NaBH₄ solution was used for arsine generation, while a gas stream of N₂ was employed for flash release and transportation towards the atomic absorption flow through cell (AAC) for atomization and measurement. The excellent performance of PTFE turnings as sorbent material and the compact design of the RC–GLS result to high sensitivity, selectivity and sampling frequency. For 60s preconcentration time and sample consumption 10.4ml a sampling frequency of 25h⁻¹ and a detection limit of c_L=0.02μgl⁻¹ were obtained. The repeatability, expressed as relative standard deviation (R.S.D.), at 1.0μgl⁻¹ As(III), was s_r=2.8%. The proposed method was successfully applied to the selective determination of As(III) in natural waters and total arsenic determination in certified reference material.

Keywords: Arsenic; Solid phase extraction; Flow injection; Hydride generation atomic absorption spectrometry; Polytetrafluoroethylene; Pyrrolidine dithiocarbamate

Flow injection potentiometric determination of total antioxidant activity of plant extracts by Liliya K. Shpigun; Marina A. Arharova; Khena Z. Brainina; Alla V. Ivanova (pp. 419-426).

A new flow injection potentiometric (FIP) method, rapid, reproducible and simple to apply, has been developed for the in vitro evaluation of antioxidative capacity of aqueous plant extracts. This method is based on the transient negative signal measurements with a flow-type platinum electrode detector due to the composition change of a [Fe(CN)₆]³⁻/[Fe(CN)₆]⁴⁻ redox-reagent solution. The variables affecting the signal height such as composition and concentration of redox-reagent, injected sample volume, flow rates of carrier and redox-reagent solution streams were studied in details and the conditions were optimized. For the compounds under study, a linear relationship was stated between the potentiometric signal height and the logarithm of antioxidant concentration. It was stated that a wide antioxidant activity range from 1μM to 10mM could be determined by the changing concentrations of the hexacyanoferrate(III) from 5 to 0.01×10⁻⁴. The present FIP method was applied to quantify relative antioxidant activity (RAA index) of the representative water-soluble antioxidants (ascorbic acid, pyrocatechol, pyrogallol, caffeic acid, chlorogenic acid, gallic acid, tannic acid, uric acid,l-cysteine, trolox). The high sampling rate (100h⁻¹) and a satisfactory reproducibility (R.S.D.=0.7–1.8%, n=5, 0.1mM each compound) were obtained.The method was also applied to estimate total antioxidant activity (TAA) of real samples (green and black tea infusions, herbal infusions and fresh fruit extracts) and the results were compared with those achieved using well-known in vitro testing methods.

Keywords: Flow injection; Potentiometry; Antioxidant; Total antioxidant activity; Plant extract

Flow injection potentiometric determination of total antioxidant activity of plant extracts by Liliya K. Shpigun; Marina A. Arharova; Khena Z. Brainina; Alla V. Ivanova (pp. 419-426).

Keywords: Flow injection; Potentiometry; Antioxidant; Total antioxidant activity; Plant extract

Dynamic mass spectrometer for studies of organic and inorganic molecules by A.N. Zavilopulo; O.B. Shpenik; V.A. Surkov (pp. 427-431).

Experimental technique is described and relative cross-sections of direct and dissociative ionization of CO₂, C₄H₉OH, C₂H₅OH, CH₃OH, C₆H₁₂O₆ and C₇H₈ molecules by electron impact in the near-threshold energy range are obtained. The experiment is performed on a setup with ion mass separation based on a monopole mass spectrometer which is proved to be successful for such a sort of experiments. For the incident electron energy range from 7 to 35eV the energy dependences of cross-section of appearance of the main molecule ions and the fragment ions formed due to its dissociation are given.

Keywords: Mass spectrometer; Mass spectrum; Ionization; Dissociative ionization

Dynamic mass spectrometer for studies of organic and inorganic molecules by A.N. Zavilopulo; O.B. Shpenik; V.A. Surkov (pp. 427-431).

Keywords: Mass spectrometer; Mass spectrum; Ionization; Dissociative ionization

Determination of trace elements in serum by dynamic reaction cell inductively coupled plasma mass spectrometry by S. D’Ilio; N. Violante; S. Caimi; M. Di Gregorio; F. Petrucci; O. Senofonte (pp. 432-438).

An inductively coupled plasma mass spectrometer (ICP-MS), equipped with a dynamic reaction cell (DRC) and coupled with a desolvating nebulizing system (Apex-ACM) to reduce the oxide formation, was used in the determination of Al, Co, Cr, Mn, Ni and Se in serum samples. The effect of the operating conditions of the DRC system was studied to get the best signal-to-background (S/B) ratio. The potentially interfering molecular ions at the masses m/ z²⁷Al,⁵⁹Co,⁵²Cr,⁵⁵Mn,⁶⁰Ni and⁷⁸Se, were significantly reduced in intensity by using NH₃ and H₂, as the reaction cell gases in the DRC, while a proper Dynamic Bandpass Tuning parameter q (RPq) value was optimized. The detection limits for²⁷Al,⁵⁹Co,⁵²Cr,⁵⁵Mn,⁶⁰Ni and⁷⁸Se, estimated with 3-σ method, resulted to be 0.14, 0.003, 0.002, 0.01, 0.01 and 1.8μgL⁻¹, respectively. This analytical method was developed on both a human serum certified reference material and a lyophilized animal serum produced and proposed in an intercomparison study. The results obtained for the reference samples agreed satisfactorily with the certified values. Precision (expressed as CV%) between sample replicates was better than 10% for elements determination, with the only exception of aluminium (14%).

Keywords: Trace elements; Dynamic reaction cell inductively coupled plasma mass spectrometry; Serum; Desolvating system

On-line monitoring of pine needles combustion emissions in the presence of fire retardant using a “thermogravimetry (TG)-bridge/mass spectrometry method�? by N. Tzamtzis; S. Karma; A. Pappa; M. Statheropoulos (pp. 439-444).

In this work a new method called TG-bridge/mass spectrometry is presented, for the on-line monitoring of the pine needles combustion emissions in a common lab furnace. The TG-bridge (thermogravimetry-bridge) system has been developed in-house as a TG–MS (thermogravimetry–mass spectrometry) interface, for TG–MS analysis. In this work, TG-bridge was used for directly sampling of the combustion emissions from the inside of the furnace and transferring them into the mass spectrometer (MS), without disturbing the sub-pressure conditions inside the MS ion source.The effect of Fire-Trol 931 (a long-term fire retardant) on the emissions, produced during the combustion of pine needles, is tested in the lab for future application in the field. It was shown that in treated samples, increased evolution of ammonia and aromatic compounds took place, compared to untreated samples. Maximum concentrations of specific compounds, such as benzene and toluene, evolved during the combustion experiments in the furnace, were determined.

Keywords: Thermogravimetry (TG)-bridge; Mass spectrometry; Fire emissions; Combustion; Pine needles; Fire retardant; On-line monitoring

On-line monitoring of pine needles combustion emissions in the presence of fire retardant using a “thermogravimetry (TG)-bridge/mass spectrometry method” by N. Tzamtzis; S. Karma; A. Pappa; M. Statheropoulos (pp. 439-444).

Keywords: Thermogravimetry (TG)-bridge; Mass spectrometry; Fire emissions; Combustion; Pine needles; Fire retardant; On-line monitoring

Instrumental analysis of bacterial cells using vibrational and emission Mössbauer spectroscopic techniques by Alexander A. Kamnev; Anna V. Tugarova; Lyudmila P. Antonyuk; Petros A. Tarantilis; Leonid A. Kulikov; Yurii D. Perfiliev; Moschos G. Polissiou; Philip H.E. Gardiner (pp. 445-452).

In biosciences and biotechnology, the expanding application of physicochemical approaches using modern instrumental techniques is an efficient strategy to obtain valuable and often unique information at the molecular level. In this work, we applied a combination of vibrational (Fourier transform infrared (FTIR), FT-Raman) spectroscopic techniques, useful in overall structural and compositional analysis of bacterial cells of the rhizobacterium Azospirillum brasilense, with⁵⁷Co emission Mössbauer spectroscopy (EMS) used for sensitive monitoring of metal binding and further transformations in live bacterial cells. The information obtained, together with ICP-MS analyses for metals taken up by the bacteria, is useful in analysing the impact of the environmental conditions (heavy metal stress) on the bacterial metabolism and some differences in the heavy metal stress-induced behaviour of non-endophytic (Sp7) and facultatively endophytic (Sp245) strains. The results show that, while both strains Sp7 and Sp245 take up noticeable and comparable amounts of heavy metals from the medium (0.12 and 0.13mg Co, 0.48 and 0.44mg Cu or 4.2 and 2.1mg Zn per gram of dry biomass, respectively, at a metal concentration of 0.2mM in the medium), their metabolic responses differ essentially. Whereas for strain Sp7 the FTIR measurements showed significant accumulation of polyhydroxyalkanoates as storage materials involved in stress endurance, strain Sp245 did not show any major changes in cellular composition. Nevertheless, EMS measurements showed rapid binding of cobalt(II) by live bacterial cells (chemically similar to metal binding by dead bacteria) and its further transformation in the live cells within an hour.

Keywords: Bacterial cells; Heavy metals; Metabolic processes; Spectroscopic analysis; Fourier transform infrared (FTIR) spectroscopy; Emission Mössbauer spectroscopy

Oxidative stability and radical scavenging activity of extra virgin olive oils: An electron paramagnetic resonance spectroscopy study by V. Papadimitriou; T.G. Sotiroudis; A. Xenakis; N. Sofikiti; V. Stavyiannoudaki; N.A. Chaniotakis (pp. 453-458).

The oxidative stability of extra virgin olive oils (EVOO) from the Greek island of Crete was evaluated by electron paramagnetic resonance (EPR) spectroscopy and the spin trapping technique. The spin trap N- t-butyl-α-phenylnitrone (PBN) was added to the olive oil samples and the production of free radicals was monitored during heating at 70°C. Induction time for the accelerated oxidation of virgin olive oils at 70°C was determined. The EPR results were compared with the oxidative stability values provided by the Rancimat method at 110°C and high linear correlations were found ( r=0.922). EPR spin trapping provides a sensitive and rapid method for evaluating the oxidative stability of EVOO. The same samples of Greek extra virgin olive oils were also examined for their radical scavenging activity (RSA) toward the stable galvinoxyl radical by EPR spectroscopy. The decrease of the intensity of the EPR signal upon incubation time was followed. Both oxidative stability and radical scavenging activity of EVOO samples were correlated to their content in polyphenols and tocopherols.

Keywords: Extra virgin olive oil; Antioxidants; Oxidative stability; Radical scavenging activity; Electron paramagnetic resonance spectroscopy

Applications of Fourier transform-infrared spectroscopy to edible oils by N. Vlachos; Y. Skopelitis; M. Psaroudaki; V. Konstantinidou; A. Chatzilazarou; E. Tegou (pp. 459-465).

Recent developments in Fourier transform infrared (FT-IR) spectroscopy instrumentation extend the application of this technique to the field of food research, facilitating particularly the studies on edible oils and fats. In this work, FT-IR spectroscopy is used as an effective analytical tool in order: (a) to determine extra virgin olive oil adulteration with lower priced vegetable oils (sunflower oil, soyabean oil, sesame oil, corn oil) and (b) to monitor the oxidation process of corn oil samples undergone during heating or/and exposure to ultraviolet radiation. A band shift observed at 3009cm⁻¹ assigned to the CH stretching vibration of the cis-double bond, allows the determination of extra virgin olive oil adulteration. Changes in the 3050–2800 and 1745cm⁻¹ spectral region appear after heating at elevated temperatures and aid the oxidation process monitoring. In addition, an analytical technique for the measurement of carbonylic compounds in oils, produced after heating, is applied. The possible antioxidant effect of oregano is also discussed.

Keywords: Fourier transform infrared (FT-IR) spectroscopy; Edible oils; Olive oil; Adulteration; Oxidation

Applications of Fourier transform-infrared spectroscopy to edible oils by N. Vlachos; Y. Skopelitis; M. Psaroudaki; V. Konstantinidou; A. Chatzilazarou; E. Tegou (pp. 459-465).

Keywords: Fourier transform infrared (FT-IR) spectroscopy; Edible oils; Olive oil; Adulteration; Oxidation

New simple spectrophotometric assay of total carotenes in margarines by Svjetlana Luterotti; Dane Bicanic; Romana Požgaj (pp. 466-473).

Direct and reliable spectrophotometric method for assaying total carotenes (TC) in margarines with the minimum of sample manipulation is proposed. For the first time saponification step used in determination of carotenes in margarines was omitted leading to a substantial cost saving and reduction of time needed to complete the analysis. The resulting analytical procedure is characterized in details in terms of the figures of merit. The method is sensitive, precise and accurate; for both, standard additions and calibration in soybean oil, recovery ranges between 98 and 102%. For the most accurate analyses the approach of standard additions is preferred but for quick routine analyses this latter can be replaced by the calibration in soybean oil. Limit of detection value (LOD=3S.D._B/ a, where S.D._B is the standard deviation of the blank, “ a�? is the slope of calibration line) as low as 12μg TC/100g was achieved in soybean oil enabling the sensitive detection. Concentration of TC in margarines declared as being coloured with β-carotene (carotene) ranges between 0.3 and 0.9mg/100g while in carrot extract-coloured margarine TC is 0.2mg/100g.

Keywords: Margarines; Total carotenes; Spectrophotometry; Quality control

New simple spectrophotometric assay of total carotenes in margarines by Svjetlana Luterotti; Dane Bicanic; Romana Požgaj (pp. 466-473).

Direct and reliable spectrophotometric method for assaying total carotenes (TC) in margarines with the minimum of sample manipulation is proposed. For the first time saponification step used in determination of carotenes in margarines was omitted leading to a substantial cost saving and reduction of time needed to complete the analysis. The resulting analytical procedure is characterized in details in terms of the figures of merit. The method is sensitive, precise and accurate; for both, standard additions and calibration in soybean oil, recovery ranges between 98 and 102%. For the most accurate analyses the approach of standard additions is preferred but for quick routine analyses this latter can be replaced by the calibration in soybean oil. Limit of detection value (LOD=3S.D._B/ a, where S.D._B is the standard deviation of the blank, “ a” is the slope of calibration line) as low as 12μg TC/100g was achieved in soybean oil enabling the sensitive detection. Concentration of TC in margarines declared as being coloured with β-carotene (carotene) ranges between 0.3 and 0.9mg/100g while in carrot extract-coloured margarine TC is 0.2mg/100g.

Keywords: Margarines; Total carotenes; Spectrophotometry; Quality control

Complexation efficiency of differently fixed 8-hydroxyquinoline and salicylic acid ligand groups for labile aluminium species determination in soils—comparison of two methods by Peter Matúš; Jana Kubová (pp. 474-481).

Two methods utilizing the complexation of labile Al species by 8-hydroxyquinoline (HQN) and salicylic acid (SA) ligand groups were developed for aluminium operationally defined fractionation in acid soils. First, the solid phase extraction (SPE) procedure by a short-term ion-exchange batch reaction with chelating resins Iontosorb Oxin and Iontosorb Salicyl containing both ligand groups was used previously. Second, the 8-hydroxyquinoline, salicylic acid and ammonium salicylate agents with different concentrations by a single extraction protocol were applied in this paper. The flame atomic absorption spectrometry (FAAS) and optical emission spectrometry with inductively coupled plasma were used for aluminium quantification.The comparison of results from both methods show the possibility to supersede the first laborious method for the second simpler one in Al environmental risk assessment. The use of 1% 8-hydroxyquinoline in 2% acetic acid and 0.2% salicylic acid by a single extraction protocol without a need of sample filtration can supersede the SPE procedure in the Al pollution soil monitoring. Finally, the new scheme usable in a laboratory and moreover, directly in a field was proposed for Al fractionation in solid and liquid environmental samples. The labile Al species in soils and sediments are separated after their single leaching by 8-hydroxyquinoline or salicylic acid without a need of sample filtration. The labile Al species in soil solutions and natural waters are separated after their ultrafiltration followed by the SPE procedure with Iontosorb Oxin or Iontosorb Salicyl.

Keywords: Aluminium complexation and fractionation; Solid phase extraction; Chelating ion-exchange; Iontosorb (Ostsorb) Oxin and Salicyl; 8-Hydroxyquinoline; Salicylic acid

Author index (pp. 482-486).

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Analytica Chimica Acta (v.573, #N1-N2,1-48)

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Analytica Chimica Acta (v.573, #N1-N2,1-48)