Analytica Chimica Acta (v.560, #1-2)

Contents (v-vi).

Microfluidics technology for manipulation and analysis of biological cells by Changqing Yi; Cheuk-Wing Li; Shenglin Ji; Mengsu Yang (1-23).
Analysis of the profiles and dynamics of molecular components and sub-cellular structures in living cells using microfluidic devices has become a major branch of bioanalytical chemistry during the past decades. Microfluidic systems have shown unique advantages in performing analytical functions such as controlled transportation, immobilization, and manipulation of biological molecules and cells, as well as separation, mixing, and dilution of chemical reagents, which enables the analysis of intracellular parameters and detection of cell metabolites, even on a single-cell level. This article provides an in-depth review on the applications of microfluidic devices for cell-based assays in recent years (2002–2005). Various cell manipulation methods for microfluidic applications, based on magnetic, optical, mechanical, and electrical principles, are described with selected examples of microfluidic devices for cell-based analysis. Microfluidic devices for cell treatment, including cell lysis, cell culture, and cell electroporation, are surveyed and their unique features are introduced. Special attention is devoted to a number of microfluidic devices for cell-based assays, including micro cytometer, microfluidic chemical cytometry, biochemical sensing chip, and whole cell sensing chip.
Keywords: Microfluidic devices; Lab-on-a-chip; Miniaturized total analysis system; Biochip; Cell analysis; Cell manipulation;

Microchip-based genotoxic bioassay using sensing Escherichia coli strains has been performed. In this method, the assay was conducted in three-dimensional microfluidic network constructed by a silicon perforated microwell array chip and two poly(dimethylsiloxane) (PDMS) multi-microchannel chips. The sensing strains having firefly luciferase reporter gene under transcriptional control of umuD as an SOS promoter were put into the channels on one of the PDMS chips and immobilized in the silicon microwells. Samples containing genotoxic substances and substrates for luciferase were into the channels on the other PDMS chip. The optimum conditions of the assay in the on-chip format have been investigated using mitomycin C (MMC) as a genotoxic substance. As a result, the dose-dependence of bioluminescence intensity was obtained at once on the chip. Additionally, the response ratios of the bioluminescence between mutagen- and non-induced strains were successfully enhanced by improving the on-chip assay methods and conditions. Several well-known genotoxic substances were subjected to the on-chip assay, and were detected with the detection limits comparable to those in the conventional method with reduced time.
Keywords: On-chip bioassay; SOS response; Genotoxicity; Bioluminescence; Microfluidic network;

Automated flow enzyme-linked immunosorbent assay (ELISA) system for analysis of methyl parathion by M.A. Kumar; R.S. Chouhan; M.S. Thakur; B.E. Amita Rani; Bo Mattiasson; N.G. Karanth (30-34).
Sensitive detection of pesticides is of utmost importance in environment and food analysis. Immunological methods are widely used to detect pesticides in agricultural and environmental samples wherein antibodies are employed against the target molecules. Accurate diagnosis depends on the affinity and specificity of the antibody preparation used, and high affinity antibodies are essential for the detection of very small amounts of pesticides. Enzyme linked immuno sorbent assay (ELISA) coupled with flow injection analysis (FIA) technique provides a very high sensitivity with high throughput of analyses. Automation of this analysis scheme ensures precise detection with high accuracy. The present development aims at providing a user-friendly system for achieving this objective. It employs a 8952 microcontroller for precise flow of reagents, samples, substrate and conjugates used for analysis to be passed through an immobilized antibody column at predetermined time. With the sequence and flow control of buffers used, it also provides the option for reuse of the immobilized antibody column. The system is flexible to accommodate multiple sequences up to a maximum of 99 steps. It is customizable for different flow ELISA applications. It can control up to eight solenoid valves (dc 24 V) and two peristaltic pumps and has one 12 bit analog channel for data acquisition. With the serial interface port, the system provides convenient means for data acquisition into the computer. The system has been successfully tested for immuno analysis of organophosphorous pesticide methyl parathion.
Keywords: ELISA; Flow ELISA; Immunosensor; Methyl parathion; Microcontroller;

Determination of telmisartan in human blood plasma by Christel Hempen; Liane Gläsle-Schwarz; Ulrich Kunz; Uwe Karst (35-40).
Telmisartan is an angiotensin II receptor antagonist and a known drug against high blood pressure. In this report, the development of a new and rapid analytical technique, an enzyme-linked immunosorbent assay (ELISA) for the determination of telmisartan in human blood plasma is described. The immunoassay is based on a conversion of 4-(N-methylhydrazino)-7-nitro-2,1,3-benzooxadiazole (MNBDH) to 4-(N-methylamino)-7-nitro-2,1,3-benzooxadiazole (MNBDA), which is detected by fluorescence spectroscopy. The limit of detection was 0.1 ng/mL, the limit of quantification was 0.3 ng/mL and the working range extended from 0.3 ng/mL to 300 ng/mL.
Keywords: Telmisartan; ELISA; MNBDH; Glucose oxidase; MNBDA;

Determination of telmisartan in human blood plasma by Christel Hempen; Liane Gläsle-Schwarz; Ulrich Kunz; Uwe Karst (41-49).
A new liquid chromatography/atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) method with on-line sample clean-up for the determination of telmisartan in human blood plasma is presented. This technique is compared to a previously introduced enzyme-linked immunosorbent assay (ELISA), where fluorescence is used as detection method. For the LC/MS method applying an internal calibration via a deuterated internal standard, the limit of detection was 0.3 ng/mL, the limit of quantification was 0.9 ng/mL and the linear range extended from 0.9 to 1000 ng/mL. Forty-eight plasma samples from four healthy volunteers were analyzed in a pharmacokinetic study to obtain data for the method comparison. As a result, these two new and independent analytical methods for the determination of telmisartan in human blood plasma proved to yield comparable results for the amount of analyte.
Keywords: Telmisartan; ELISA; LC/MS/MS; Turboflow chromatography; MNBDH;

Stability of phospholipid vesicles studied by asymmetrical flow field-flow fractionation and capillary electrophoresis by Gebrenegus Yohannes; Kati-Henna Pystynen; Marja-Liisa Riekkola; Susanne K. Wiedmer (50-56).
The stability of zwitterionic phosphatidylcholine vesicles in the presence of 20 mol% phosphatidyl serine (PS), phosphatidic acid (PA), phosphatidyl inositol (PI), and diacylphosphatidyl glycerol (PG) phospholipid vesicles, and cholesterol or calcium chloride was investigated by asymmetrical flow field-flow fractionation (AsFlFFF). Large unilamellar vesicles (LUV, diameter 100 nm) prepared by extrusion at 25 °C were used. Phospholipid vesicles (liposomes) were stored at +4 and −18 °C over an extended period of time. Extruded egg yolk phosphatidylcholine (EPC) particle diameters at peak maximum and mean measured by AsFlFFF were 101 ± 3 nm and 122 ± 5 nm, respectively. No significant change in diameter was observed after storage at +4 °C for about 5 months. When the storage period was extended to about 8 months (250 days) larger destabilized aggregates were formed (172 and 215 nm at peak maximum and mean diameters, respectively). When EPC was stored at −18 °C, large particles with diameters of 700–800 nm were formed as a result of dehydration, aggregation, and fusion processes. In the presence of calcium chloride, EPC alone did not form large aggregates. Addition of 20 mol% of negatively charged phospholipids (PS, PA, PI, or PG) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) vesicles increased the electrostatic interactions between calcium ion and the vesicles and large aggregates were formed. In the presence of cholesterol, large aggregates of about 250–350 nm appeared during storage at +4 and −18 °C for more than 1 day.The effect of liposome storage temperature on phospholipid coatings applied in capillary electrophoresis (CE) was studied by measuring the electroosmotic flow (EOF). EPC coatings with and without cholesterol, PS, or calcium chloride, prepared from liposomes stored at +25, +4, and −18 °C, were studied at 25 °C. The performances of the coatings were further evaluated with three uncharged compounds. Only minor differences were observed between the same phospholipid coatings, showing that phospholipid coatings in CE are relatively insensitive to storage at +25, +4 °C or −18 °C.
Keywords: Asymmetrical flow field-flow fractionation; Capillary electrophoresis; Phospholipid vesicle; Liposome; Stability; Temperature;

Enantioseparation and quality control of biperiden in pharmaceutical formulations by capillary electrophoresis by Roberto Mandrioli; Alessandro Musenga; Stefano Savino Lasaponara; Maria Addolorata Saracino; Salvatore Fanali; Maria Augusta Raggi (57-63).
An original capillary electrophoretic method has been developed and applied for the enantioselective analysis of the antiparkinson drug biperiden in pharmaceutical formulations, using a modified cyclodextrin as the chiral selector. Baseline enantioseparation of the racemic compound was achieved in less than 7 min using an uncoated fused silica capillary (50 μm i.d. and 48.5, 40.0 cm, total and effective length, respectively), filled with a background electrolyte consisting of a 50 mM phosphate buffer at pH 3.5 supplemented with 3% (w/v) β-cyclodextrin sulphate and applying a voltage of 20 kV, reversed polarity. Samples were injected by pressure (50 mbar, 90 s) at the cathodic end of the capillary and detection wavelength was 195 nm (bandwidth: 10 nm). A simple and fast pre-treatment procedure allowed the complete extraction of the drug from commercial formulations (sustained release tablets and ampoules for injections) without any interference from the matrix. Good linearity was found in the 1–50 μg/mL concentration range; the limit of quantitation was 1 μg/mL and the limit of detection was 0.4 μg/mL. Precision and accuracy were good, with R.S.D. values always lower than 2.8% and a mean recovery value of 101.1%. The method was suitable for the quality control of biperiden in commercial formulations.
Keywords: Biperiden; Quality control; Enantioseparation; Capillary electrophoresis; Pharmaceutical formulations;

A new chiral derivatizing reagent, dehydroabietylisothiocyante (DHAIC), was synthesized and used for the enantiomeric separation of chiral compounds in capillary electrophoresis (CE). The synthetic route to obtain DHAIC is described. The separation conditions for the chiral separation of several chiral compounds, such as protein amino acids and chiral drug DOPA were optimized. Best results for the chiral separation of DHAIC derivatized amino acids and DOPA were obtained in a running buffer consisted of 50 mM borate (pH 9.5), 5 mM sodium dodecyl sulphate (SDS) and 20% acetonitrile for amino acids and 60 mM Na2HPO4 (pH 8.0), 17 mM SDS and 25% acetonitrile for DOPA. Under the conditions studied, chiral separation of five amino acids including Ser, Val, Ala, Thr, Cys and a chiral drug DOPA as their diastereomeric DHAIC derivatives has been achieved by micellar electrokinetic chromatography (MEKC).
Keywords: Enantiomeric separation; Dehydroabietylisothiocyante; Capillary electrophoresis; Derivatization; Amino acids;

A new approach to non-aqueous capillary electrophoresis based on the addition of anionic carboxylic surfactants to the basic amphiprotic organic solvent in which form neutral-surfactant aggregates was developed with a view to improving the electrophoretic resolution of charged substances. These aggregates acts as a new pseudostationary phase. The presence of these aggregates allows the effective separation of four tetracyclines with increased selectivity. The efficiency of sodium caprylate, sodium laurate and sodium palmitate as surfactants was examined. The latter two proved more effective than the former as they provided migration times reproducible to within 7% or better in all cases. The additional use of an alcohol allows peak shape to be controlled, which expands the potential of this electrophoretic technique even further. The proposed method was used to determine tetracyclines in water samples. The sensitivity of the determination was improved by using a flow manifold coupled at-line to the capillary electrophoresis system in order to preconcentrate the analytes. The limits of detection thus achieved ranged from 50 to 90 μg/l. Under optimal operating conditions, recoveries ranged from 97 to 104%, and precision from 5.4 to 7.0%.
Keywords: Tetracyclines; Non-aqueous capillary electrophoresis; Neutral-surfactant aggregates;

New applications of azamacrocyclic ligands in ion recognition, transport and preconcentration of palladium by Judit Masllorens; Anna Roglans; Enriqueta Anticó; Clàudia Fontàs (77-83).
This work deals with the evaluation of a synthesized 15-membered triolefinic azamacrocycle containing a NH group, (E,E,E)-1,6-bis(p-tolylsulfonyl)-1,6,11-triazacyclopentadeca-3,8,13-triene (R2NH), for the selective extraction of palladium and platinum from aqueous chloride matrices prior their analysis by ICP-AES. The optimal conditions for liquid–liquid experiments have been evaluated, with special emphasis given to the selection of the organic solvent and the optimal aqueous chloride concentration for the extraction of PdCl4 2− and PtCl6 2−. The selective transport and separation of palladium(II) from a mixture of Pd(II) and Pt(IV) was accomplished by means of a supported liquid membrane system containing the macrocycle as carrier dissolved in anethol and 0.5 M thyocianate solution as stripping solution. A C18 cartridge has been activated with the reagent R2NH in order to test the feasibility of achieving the preconcentration of palladium solutions. Enrichment factors close to the theoretical ones were obtained with the designed system and using thiourea as eluting solution.
Keywords: Azamacrocyclic ligands; Palladium; Platinum; Supported liquid membranes; Solid-phase extraction;

Isolation of lead from water samples and determination of 210Pb by Željko Grahek; Martina Rožmarić Mačefat; Stipe Lulić (84-93).
This paper describes the procedures of isolating lead and strontium from the larger volume of seawater and drinking water samples that enable the determination of 210Pb on gamma spectrometer and 89,90Sr on liquid scintillation counter. In one procedure, lead is directly isolated from water sample on the column filled with Sr resin by binding of lead on the Sr resin column from 0.2 M HCl in water sample, and successive elution with 0.2 and 8 M HCl. In others, lead and strontium are precipitated from sample with (NH4)2CO3, followed by isolation on an anion exchange column. Lead, strontium and yttrium are bound onto anion exchange column (filled with Amberlite CG-400 in nitrate form) from alcoholic solutions of nitric acid. Lead, Sr and Y are separated from Mg, Ca, K, and other elements by elution with 0.25 M HNO3 in the mixture of ethanol and methanol. After that, strontium and yttrium are separated from lead by elution with 0.25 M HNO3 in the mixture of ethanol and water.The procedure with the Sr resin (direct isolation) is simpler and faster in the phase of isolation on the column in comparison with the procedure with the anion exchanger. The procedure with the anion exchanger, however, makes possible the simultaneous isolation of lead, yttrium and strontium and rapid determination of 89,90Sr. These procedures were tested by determination of 210Pb and 89,90Sr in real sample. Obtained results showed that Pb can be efficiently isolated (with high recovery) from sample and activity of 6 mBq l−1 of 210Pb and higher can be determined.
Keywords: Seawater; Lead; 89,90Sr; 210Pb; Rapid determination; Gamma spectrometry;

Evaluation of PLE exhaustiveness for the extraction of PCBs from sediments and the influence of sediment characteristics by Sarah Josefsson; Rikard Westbom; Lennart Mathiasson; Erland Björklund (94-102).
Pressurized liquid extractions were performed on eight sediments in order to investigate if a modified US EPA method (100 °C, 100 bar, n-heptane/acetone (1:1), 2 × 5 min) provided exhaustive extractions of polychlorinated biphenyls (PCBs) from sediment, and to study if the extractability of PCBs from the different sediments was affected by characteristics of the sediment. The recovery from the eight native sediments, contaminated in nature, was between 96.4% and 98.9%, as an average of the recoveries from 10 PCB congeners. Hundred percent recovery was defined as the sum of two consecutive extractions (2 × 5 min each) at the stated conditions. The recoveries of the individual congeners were above 94%, except for one congener in one sediment, which had a recovery of 92%. When the recoveries and different characteristics of the sediments were compared, no correlation appeared between recoveries and sediment PCB concentration, total organic carbon (TOC), soot carbon (SC) or amorphous carbon (AC). The fact that carbon did not influence the extractions was somewhat surprising, since previous experiments have indicated a connection. Instead, statistically significant (p  < 0.05) correlations were observed for water content and carbon/nitrogen (C/N) ratio. The decrease in recoveries with decreased water content was attributed to less access of the solvent to the analytes due to less matrix swelling. The lowered recoveries with increased C/N ratio can indicate that a difference in structure of the organic matter exists, which influences the binding strength between the analytes and the matrix. The difference in structure can possibly be explained by different origin of the organic matter or by aging effects. Overall the method was found to be exhaustive and the excellent recoveries show that sediment characteristics do not influence the extractions markedly.
Keywords: Polychlorinated biphenyls; Pressurized liquid extraction; Exhaustiveness; Sediment characteristics;

Lower aliphatic carbonyl compounds, esters and alcohols were found in open atmosphere, as well as in volatiles of microorganisms, living plants and leaf litter. Quantitative determination of their emissions into the gas phase with the aid of solid phase microextraction (SPME) requires the knowledge of their distribution coefficients K fg between fiber coating and gas phase. This paper reports the results of experimental determination of K fg values for 17 volatiles belonging to the above-mentioned classes of organic compounds. Mixed Carboxen/polydimethylsiloxane stationary phase, which proved to be effective during SPME determination of more volatile pollutants, was chosen as the fiber coating. Values K fg were determined at temperature range 15–30 °C, which permitted the dependence between log  K fg and T −1 to be determined. Distribution coefficients for 23 other compounds from the same homologue series were calculated using two-parameter empirical equation, where physico-chemical parameters of compounds (boiling temperature, linear temperature programmed retention index) served as descriptors.
Keywords: Solid phase microextraction; Carboxen/PDMS fiber coating; Distribution coefficients; Carbonyl compounds; Esters; Alcohols;

Solid-phase microextraction (SPME) employing absorption type fibers coupled to gas chromatography was used to extract phthalic acid esters from water samples. Distribution coefficients between water and polydimethylsiloxane ( K fw PDMS ) and polyacrylate ( K fw PA ) for these compounds were determined. To estimate the values of K fw for compounds of the same class, which did not undergo experiments, it is proposed to use an empirical two-parameter equation in which various physico-chemical and structural characteristics weakly correlated with each other are used as descriptors. K fw values calculated on the basis of correlation with distribution coefficients in other heterogeneous systems correspond to the values determined experimentally. Distribution coefficients in polyacrylate/water system were used for quantitative determination by SPME–GC–FID method of phthalates in raw and purified municipal wastes which come from a wastewater purifying plant in Białystok. Concentration of the four phthalates detected in the wastewater (di-n-propyl phthalate, diisobutyl phthalate, di-n-butyl phthalate and di-2-ethylhexyl phthalate) ranged from 1.5 to 50 μg/l. During a purification process, decomposition of phthalates with higher polarity is the fastest whereas that of di-2-ethylhexyl phthalate is the slowest.
Keywords: Solid-phase microextraction; Distribution coefficients; Phthalates; Wastewater analysis;

A rapid and environmentally friendly methodology was developed for the extraction of pyrethroid insecticides from semi permeable membrane devices (SPMDs), in which they were preconcentrated in gas phase. The method was based on gas chromatography mass–mass spectrometry determination after a microwave-assisted extraction, in front of the widely employed dialysis method. SPMDs were extracted twice with 30 mL hexane:acetone, irradiated with 250 W power output, until 90 °C in 10 min, this temperature being held for another 10 min. Clean-up of the extracts was performed by acetonitrile–hexane partitioning and solid-phase extraction (SPE) with a combined cartridge of 2 g basic-alumina, deactivated with 5% water, and 500 mg C18.Pyrethroids investigated were Allethrin, Prallethrin, Tetramethrin, Bifenthrin, Phenothrin, λ-Cyhalothrin, Permethrin, Cyfluthrin, Cypermethrin, Flucythrinate, Esfenvalerate, Fluvalinate and Deltamethrin. The main pyrethroid synergist compound, Pyperonyl Butoxide, was also studied. Limit of detection values ranging from 0.3 to 0.9 ng/SPMD and repeatability data, as relative standard deviation, from 2.9 to 9.4%, were achieved. Pyrethroid recoveries, for spiked SPMDs, with 100 ng of each one of the pyrethroids evaluated, were from 61 ± 8 to 103 ± 7% for microwave-assisted extraction, versus 54 ± 4 to 104 ± 3% for dialysis reference method. Substantial reduction of solvent consumed (from 400 to 60 mL) and analysis time (from 48 to 1 h) was achieved by using the developed procedure.High concentration levels of pyrethroid compounds, from 0.14 to 7.3 μg/SPMD, were found in indoor air after 2 h of a standard application.
Keywords: Semi permeable membrane device (SPMD); Pyrethroid; Residues; Microwave-assisted extraction; Indoor air; Mass–mass spectrometry;

Using ClO2 as chemiluminescence (CL) oxidant, a novel homogeneous CL system in neutral and near neutral aqueous solution is proposed in this paper. ClO2 could oxidize sulfite in pH 5.0–8.5 acetate buffer to produce CL emission, and pipemidic acid could sensitize the CL system. The ClO2–SO3 2−–pipemidic acid reaction was used as a model CL system and explored the possibility of highly active and eco-friendly ClO2 being used as CL oxidant in neutral and near neutral aqueous solution. Combined with flow-injection analysis, the proposed CL system was applied to the automated dissolution testing of pipemidic acid tablet. Compared with the common strong acidic or strong basic CL system, this proposed CL owns its advantages.
Keywords: Chemiluminescence; Flow injection; ClO2; Pipemidic acid; Neutral and near neutral aqueous solution;

New solid-matrix phosphorescence (SMP) methods for (±)-anti-DB[a,l]PDE-DNA adducts and B[e]P were developed. The methods can be used to detect and characterize (±)-anti-DB[a,l]PDE-DNA adducts and B[e]P by employing SMP spectra, intensities, and lifetimes acquired with the heavy-atom salt, TlNO3, on Whatman 1PS paper. With the SMP data, a number of photophysical parameters were calculated such as biexponential SMP decay curves, pre-exponential factors, and fractional contribution to SMP decay curves. The SMP results were compared with earlier SMP data for (±)-anti-BPDE-DNA adducts and tetrol I-1. The SMP results show that small molecular-weight compounds like B[e]P can be readily detected and characterized by SMP. For example, the limit of detection for B[e]P was 0.60 pmol. Comparison of the SMP properties of the (±)-anti-DB[a,l]PDE-DNA adducts with earlier SMP data for the (±)-anti-BPDE-DNA adducts showed major differences in the SMP spectra, intensities, and lifetimes. The methods developed are important for the comparison of the SMP properties of different diol epoxides of PAH bonded to DNA.
Keywords: Solid-matrix phosphorescence; Benzo[e]pyrene; Dibenzo[a,l]pyrene diol epoxide-DNA; Heavy-atom effect;

Systematic errors observed when using flame atomic absorption spectrometry (FAAS) and electrothermal atomic spectrometry (ETAAS) for the analysis of biological solid materials (seafood products) were evaluated. The effect of the sample pre-treatment method (microwave-assisted acid digestion, ultrasound-assisted acid leaching and slurry sampling) as well as the number of times that a certain pre-treatment process is repeated, were two factors evaluated. They give information about the effect of the sample pre-treatment on the uncertainty in the analysis. In addition, the number of measurements (i.e., number of times that an acid digest, an acid leachate or aqueous slurry are analysed) and the calibration technique used (aqueous calibration method or standard addition technique) were other two variables taken into account. This last factor gives information about the effect of the calibration on the results, while the replicate measurements showed the repeatability. A fifth variable named as sample matrix tests the influence of the matrix sample on the systematic error through the use of different reference materials. This variable allows the study of the effect of the trace element concentrations on the uncertainty because the trace elements contents are different in each reference material. Experimental design and principal component analysis approaches were used as chemometric tools. It has been found that the use of the slurry sampling technique in ETAAS and FAAS and the determination of high element concentrations by ETAAS have led to poor precision.
Keywords: Systematic error; Flame atomic absorption spectrometry; Electrothermal atomic absorption spectrometry; Experimental design; Principal component analysis;

A simple procedure was developed for the speciation of inorganic arsenic by electrochemical hydride generation atomic absorption spectrometry (EcHG–AAS), without pre-reduction of As(V). Glassy carbon was selected as cathode material in the flow cell. An optimum catholyte concentration for simultaneous generation of arsine from As(III) and As(V) was 0.06 mol l−1 H2SO4. Under the optimized conditions, adequate sensitivity and difference in ratio of slopes of the calibration curves for As(III) and As(V) can be achieved at the electrolytic currents of 0.6 and 1 A. The speciation of inorganic arsenic can be performed by controlling the electrolytic currents, and the concentration of As(III) and As(V) in the sample can be calculated according to the equations of absorbance additivity obtained at two selected electrolytic currents. The calibration curves were linear up to 50 ng ml−1 for both As(III) and As(V) at 0.6 and 1 A. The detection limits of the method were 0.2 and 0.5 ng ml−1 for As(III) and As(V) at 0.6 A, respectively. The relative standard deviations were of 2.1% for 20 ng ml−1 As(III) and 2.5% for 20 ng ml−1 As(V). The method was validated by the analysis of human hair certified reference material and successfully applied to speciation of soluble inorganic arsenic in Chinese medicine.
Keywords: Electrochemical hydride generation; Atomic absorption spectrometry; Speciation; Inorganic arsenic;

Sequential molecular vapor elution analysis for the separation and determination of LiCl and NaCl in river waters by Yasutoshi Shirota; Md. Nurul Amin; Yoshifumi Watanabe; Satoshi Kaneco; Hideyuki Katsumata; Tohru Suzuki; Kiyohisa Ohta (159-163).
The determination of alkali metal chloride in river waters by sequential molecular vapor elution analysis (SMVEA) using a molecular absorption detector (MA) is reported. An improved molybdenum column was developed for SMVEA. An optimum flow rate of carrier gas (pure argon or nitrogen) for separation of metal vapors was 12.0 ml min−1. Lithium chloride and sodium chloride peaks completely separated from potassium, rubidium, and cesium chlorides but CaCl2, FeCl2, MgCl2, and MnCl2 peaks did not appear at a column temperature of 1000 °C and vaporization temperature of 1000 °C. The appearing order of these metal chlorides was LiCl, NaCl, KCl, RbCl, and CsCl. It was not understood by considering the boiling points of these metal chlorides. The delay of appearing time is due to an interaction between the molecular vapors and inside surface of the column. Under the experimental conditions, the number of theoretical plates was 247 for LiCl and 268 for NaCl in the improved column. Under the optimal experimental conditions, river waters were analyzed for lithium and sodium chlorides. The analytical results agreed well with the recoveries were in the range of 94–105%. By SMVEA, it was found that lithium and sodium chlorides in waters were determined without interference of matrix elements, no chemical treatment for river water samples.
Keywords: Sequential molecular vapor elution analysis (SMVEA); Alkali metal chloride; Separation; River water; Molecular absorption detection;

An extensive monitoring programme of pesticides was carried out in soil samples from an intensive horticulture area in north of Portugal, putting into practice the needs for increased control of soil quality as far as organic pollution is concerned. The area under investigation was additionally defined as vulnerable to nitrates due to local soil and aquifer characteristics, which might be extended to pesticides contamination. Five sampling sites were selected and soils analysed at three depths in eight sampling campaigns, for the period of 2 years. A stepwise multivariate statistical approach was selected to uncover most relevant patterns inside a complex environmental data matrix. Cluster analysis was applied both to group pesticides and samples, giving a primary and unsupervised overlook of privileged relationships. Clusters of persistent pesticides and selected herbicides were identified, whereas sample classes were also formed and disposed geographically. Thirty eight percent of analysed soils samples fell into one class characterized by low contamination (class 1 in cluster analysis), which is entirely representative of the sampling site no. 1. Afterwards, linear discriminant analysis was applied to identify those pesticides, which had a higher impact in the definition of classes. Finally, factor analysis using a five component model was implemented in order to bring to light the constitution and data variance explained by each of the five main principal components, as well as, their relation to pest management practices. A factor was identified (PC1 – 22% variance) composed of chlorinated pesticides, which was representative of one of the investigated sites indicating its high contamination status. Qualitative main findings and class average concentration values were obtained through this multivariate statistical approach.
Keywords: Environmental monitoring; Soil; pesticides; Cluster analysis; Linear discriminant analysis; Factor analysis;

Investigation of anthropic effects connected with metal ions concentration, organic matter and grain size in Bormida river sediments by Emilio Marengo; Maria Carla Gennaro; Elisa Robotti; Piero Rossanigo; Caterina Rinaudo; Manuela Roz-Gastaldi (172-183).
An investigation on the sediment composition and grain size was carried out along the Bormida river (Piedmont, Northern Italy). The samples were taken both in the riverbed and on its banks. Multivariate statistical exploratory methods permitted to identify possible sources of primary pollution. In particular, principal component analysis (PCA) showed that there are probably at least three anthropogenic main causes of pollution, one connected with Cr, Ni and Sn, the second with cadmium and the last one with mercury. Some correlations were found between heavy metal ion concentrations and organic matter and/or the sediment grain size. Differences between the samples collected in the riverbed and on the banks were identified and the relationships between the principal components and the distance of the samples from the riverbed and from the hot spot represented by the ACNA industrial site were also analyzed.
Keywords: Heavy metal pollution; Kohonen artificial neural networks; Principal component analysis; River sediments; Multivariate analysis;

Stripping analysis has been widely recognised as a powerful tool for trace metal analysis. Its remarkable sensitivity is attributed to the combination of a preconcentration step coupled with differential measurements that generate an extremely favourable signal-to-noise ratio. Mercury electrodes have been traditionally employed for achieving high reproducibility and sensitivity of the stripping technique. However, because of the toxicity of mercury, new alternative electrode materials are highly desired, particularly for on-site environmental monitoring of trace pollutants. Bismuth is an electrode material characterized by its low toxicity and its ability to form alloys with some metals of interest like cadmium, lead or zinc, allowing their preconcentration at the electrode surface. We present here the preparation of Cu/Nafion/Bi electrodes and their application to heavy metal analysis by anodic stripping voltammetry. First, the main limitations of the basic Cu/Bi electrode for on-site monitoring in natural waters are highlighted. Then the modification of the Cu/Bi electrode by a Nafion membrane is presented. The analytical performances of this new electrode for trace cadmium and lead analysis were evaluated in non-deaerated solutions. Linear calibration curves were obtained in synthetic solutions for concentrations ranging from 2 to 12 and 2 to 18 μg L−1 for cadmium and lead, respectively, with relative standard deviations lower than 5% (n  = 15). The analytical methodology was then successfully applied to monitor the Cd2+ and Pb2+ content in real samples such as ground water and aquatic plant extracts. The results favourably compared to those obtained using a mercury drop electrode and were validated by ICP-MS.
Keywords: Bismuth film electrodes; Copper substrate; Nafion; Heavy metal analysis; Natural waters; Plant extracts;

A novel method of preparing fluorescence particle probe by emulsion polymerization with covalent immobilization of indicator dye was described. A terminal double bond was attached to 3-amino-9-ethylcarbazole (AEC) via methacryloyl chloride, and the resultant compound was copolymerized with butyl methacrylate. The obtained polymer particles were used as a fluorescence probe, which is almost free of dye leaching, and has higher photostability and lower toxicity in comparison with free AEC. This probe holds great potential for the applications in intracellular measurements. In present study the prepared probe was used for the determination of metronidazole. The results revealed that the probe showed good selectivity and had a linear response to the analyte in the range from 2.0 × 10−5 to 1.0 × 10−3  mol l−1 with a detection limit of 9.0 × 10−6  mol l−1.
Keywords: Covalent immobilization; Emulsion polymerization; Fluorescence probe; 3-Amino-9-ethylcarbazole (AEC); Metronidazole;

Supported capillary membrane sampling-gas chromatography on a valve with a pulsed discharge photoionization detector by Gary L. Emmert; Michael A. Brown; Zhaohui Liao; Gang Cao; Chris Duty (197-206).
The objective of this study was to develop and evaluate a simple miniaturized approach to gas chromatography termed supported capillary membrane sampling-gas chromatography on a valve (SCMS-GCOV). The prototype instrument uses a pulsed discharge photoionization detector (PDPID) as the GC detector. Two different analyzers were constructed. The first used a bench scale SCMS-GC. The second was a miniaturized SCMS-GCOV analyzer. The SCMS-GCOV analyzer was used to monitor the concentrations of trihalomethanes (THM4) in drinking water. Details are given for the construction of the SCMS-GC-PDPID and the SCMS-GCOV-PDPID analyzers. The results of method detection limit, accuracy and precision studies are reported for analysis of THM4 and are compared to other methods of THM4 analysis. Side-by-side comparison studies are conducted between the SCMS-GCOV and USEPA 502.2 with promising results.
Keywords: Pulsed discharge photoionization detector; Gas chromatography; Water analysis; Trihalomethanes (THM4); Disinfection by-products;

Capillary gas chromatographic properties of three new mono-ester permethylated β-cyclodextrin derivatives by S. Tisse; V. Peulon-Agasse; P. Cardinaël; J.-P. Bouillon; J.-C. Combret (207-217).
2I-O-Methoxycarbonylmethyl-2II–VII,3I–VII,6I–VII-eicosa-O-methyl-cyclodextrin (20Me/P2OCH2COOMe), 6I-O-methoxycarbonylmethyl-2I–VII,3I–VII,6II–VII-eicosa-O-methyl-cyclodextrin (20Me/P6OCH2COOMe), 6I-O-methoxycarbonyl-6I-deoxy-2I–VII,3I–VII,6II–VII-eicosa-O-methyl-cyclodextrin (20Me/P6COOMe) have been evaluated as chiral stationary phases (CSPs) for capillary gas chromatography. General chromatographic properties of the corresponding columns have been investigated in terms of efficiency, polarity and inertness. More than 60 solutes have been used for studying their enantioselectivity by comparison with permethylated β-cyclodextrin as a reference. Similar enantioseparation ability was established for the four studied CSPs, the 20Me/P2OCH2COOMe exhibiting in most cases a better enantioselectivity than the other selectors.
Keywords: Cyclodextrin; Chiral stationary phase; Enantiomers separation; Capillary gas chromatography;

Two-phase O-ethoxycarbonylation was performed to alkylphenols in acidic solution with ethyl chloroformate present in dichloromethane phase fortified with triethylamine with subsequent N-ethoxycarbonylation of amines after adjusting to alkaline pH. The resulting ethoxycarbonyl derivatives were subjected to pentafluoropropionylation, clean-up and concentration for analysis by gas chromatography and gas chromatography–mass spectrometry. The present method was linear (r  ≥ 0.9959) in the range of 0.5–10.0 μg ml−l with good precision (≤9.5%) and accuracy (−8.9 to 9.5%) for 20 phenols and 27 amines examined, allowing simultaneous screening for a total of one alkylphenol and four amines from wine and beer.
Keywords: Alkylphenols; Amines; Ethoxycarbonylation; Pentafluoropropionylation; Gas chromatography; Gas chromatography–mass spectrometry; Wine; Beer;

The aim of this work was to develop and validate a simple and sensitive analytical method for determining enrofloxacin (EFX) and ciprofloxacin (CFX) in equine plasma and endometrial tissue samples, as a precursor to conducting pharmacokinetic/pharmacodynamic studies on equine endometritis This was achieved in the form of a liquid chromatographic procedure, with fluorometric detection, which also gave good separation of other fluoroquinolones including marbofloxacin (MFX), danofloxacin (DFX) and ofloxacin (OFX). Analytes were separated on a C18 reversed phase column using an acidified mobile phase. The exact composition of the mobile phase differed for plasma (16% acetonitrile:methanol [13:1,v/v] 84% water containing 0.4% triethylamine and 0.4% phosphoric acid [35%]) and endometrial tissue (14% acetonitrile, 86% water, without methanol) samples. EFX and CFX were both detected at excitation and emission wavelengths of 294 and 500 nm, respectively. Prior to chromatography, EFX and CFX were purified by solid phase extraction from plasma, and a combination of solvent/solid phase extraction from endometrial tissue.Mean absolute recoveries for EFX and CFX from plasma were 94.1 and 78.0%, respectively, and from endometrial tissue, 78.0 and 57.8%, respectively, with a percentage residual standard deviation (%R.S.D.) <10% in each case. Mean relative recoveries for EFX and CFX from plasma were 91.3 and 119.4%, respectively, and from endometrial tissue, 80.2 and 108.0%, respectively, with a %R.S.D. <20% in each case.Standard curves constructed using blank plasma and endometrial tissue samples, spiked with authentic EFX and CFX in the ranges 0.005–10.0 μg mL−1 and 0.05–10.0 μg g−1, respectively, all showed acceptable linearity with correlation coefficients, r 2  ≥ 0.977. Mean intra- and inter-day precision (expressed as %R.S.D.) was <6 and <13%, respectively, with an associated accuracy (expressed as percentage relative error, %R.E.) of <20% for both analytes in both matrices. Acceptable precision and accuracy was also demonstrated at the pre-assigned LOQs of 0.005 μg mL−1 for both EFX and CFX in plasma, and 0.05 μg g−1 for both drugs in endometrial tissue. EFX and CFX were stable in both plasma and endometrial tissue for at least 60 days at −20 °C.
Keywords: Horses; Endometritis; Fluoroquinolones; Enrofloxacin; Ciprofloxacin; Liquid chromatography; Validation;

Author Index (235-236).