Analytica Chimica Acta (v.543, #1-2)

Contents (v-vii).

Ordered mesoporous silica coating modified with octadecyl groups was introduced into the capillary for in-tube solid phase microextraction coupled to high performance liquid chromatography (in-tube SPME–HPLC). Improved extraction efficiency was demonstrated for the capillary when comparing with those of non-mesoporous structural coatings. The coatings produced by using different structure directing agents were compared and the highest sample capacity was achieved with the most ordered mesostructural coating.The capillary with the octadecyl modified ordered mesoporous silica coating was used to perform in-tube SPME–HPLC analysis of bisphenol A in water samples. Tap water and Donghu lake (Wuhan, China) water samples spiked with bisphenol A were tested under optimized conditions with the linearity ranged from 0.01 to 1 μg/mL and the detection limits were found to be 2.8 and 2.9 ng/mL, respectively. The precision of the method was satisfactory with the R.S.D. values smaller than 2.7%.
Keywords: Ordered mesoporous silica; Capillary coating; In-tube solid phase microextraction; Bisphenol A;

An approach to application of mesoporous hybrid as a fiber coating of solid-phase microextraction by Xin-Zhen Du; Ya-Rong Wang; Xiao-Juan Tao; Huang-Ling Deng (9-16).
MCM-41-typed mesoporous hybrid was investigated as the fiber coating of solid-phase microextraction (SPME) compared with bonded porous silica for the extraction of aromatic compounds in combination with HPLC. It was found that the mesoporous fiber coatings have higher sensitivity and better selectivity of extraction than bonded silica phase (BSP) coating because their larger surface area and smaller mesopores. Especially the extraction efficiency of modified mesoporous fiber coating was about 3–5 times higher than that of BSP. Moreover the values of distribution ratio for toluene and biphenyl is larger than those for p-xylene and anthracene. Desorption can be performed in mobile phase within 5 min. Vigorous stirring and higher temperature can improve the sensitivity of SPME with mesoporous coatings because mass transfer from bulk solution to the mesopores is diffusion controlled in extraction process. The mesoporous fiber coatings lose their extraction selectivity and show rapidly decreasing extraction efficiency by increasing content of methanol in water matrix. Different from general polymeric coatings of SPME. However, the mesoporous coatings show negative effect of ionic strength on extraction. Chemical modification also enhances the hydrothermal stability of mesoporous silica. Custom made SPME fiber with mesoporous hybrid coating still gives good reproducibility after 200 runs. The recovery is 99.86–100.32% and the relative standard deviation is 0.11–1.51% for the determination of studied compounds in spiked water. The limit of detection was in the range 2.00 × 10−8 to 6.91 × 10−9  mol L−1
Keywords: Mesoporous silica; Bonded silica phase; Solid-phase microextraction; HPLC; Aromatic compounds;

A novel poly(dimethylsiloxane)/β-cyclodextrin (PDMS/β-CD) coating was prepared for solid-phase microextraction (SPME) in the form of membrane instead of fiber. The surface of the membrane was uniform and has not gel cracking after dipped into methanol, acetonitrile and acetone. Non-polar polycyclic aromatic hydrocarbons (PAHs) and polar phenolic compounds were used to evaluate the character of the PDMS/β-CD solid-phase microextraction membrane by direct extraction and solvent desorption, followed by GC–MS analysis. Parameters that affect the membrane extraction process were investigated, which included the matrix of sample, extraction time, desorption solvent and time. For PAHs, the linearity was from 0.10 to 1000 μg 1−1. The limit of detection (LOD) was in the range of 0.01–0.2 μg 1−1. The repeatability was in the range of 5.4–9.3%. For phenolic compounds, the linearity was from 0.10 to 5000 μg 1−1 and LOD varied from 0.02 to 1.5 μg 1−1. Compared with the SPME fiber, the SPME membrane has a large extraction capacity, low cost of preparation. The sensitivity can be further increased by developing large-volume-injection or thermal desorption technique.
Keywords: Solid-phase microextraction membrane; Poly(dimethylsiloxane)/β-cyclodextrin; Sol–gel; Polycyclic aromatic hydrocarbons; Phenol compounds;

Aerosol-OT micelles in Sephadex gels for concentrating metal-dithizone chelates from water by Tohru Saitoh; Kazuki Hattori; Masataka Hiraide (25-30).
Anionic surfactants, sodium dodecyl sulfate (SDS), and di-2-ethylhexyl sodium sulfosuccinate (Aerosol-OT, AOT), were incorporated into a hydrated macroreticular anion-exchanger such as a DEAE- or QAE-Sephadex A-25 gel. The observation of fluorescence spectra of N-phenyl-1-naphthlamine indicated the formation of the surfactant micelles in gels. The hydrophobicity of the micelles corresponded to octanol, tetrahydrofuran, or ethyl acetate. A hydrophobic chelating agent, dithizone (1,5-diphenylthiocarbazone), and its metal chelates were incorporated into the Sephadex gels. The complex formation with dithizone and the subsequent adsorption on Sephadex gels required 10 min. The metals collected in gels were desorbed with 8 M nitric acid. As a result of 300-fold concentrations, traces of heavy metal ions at ng l−1 levels in river water were successfully determined by graphite-furnace atomic absorption spectrometry.
Keywords: Aerosol-OT; Sephadex gel; Micelles; Dithizone complex; Heavy metals; River water; Water analysis;

The detailed study on the extraction capacity of N,N,N′,N′-tetraalkylderivatives of diglycolamide (DGA) was performed by using the novel analytical method, which determines the limit of metal concentration in the organic phase (LOC). The analytical results suggest that LOC of Nd(III) by N,N,N′,N′-tetraoctyl-diglycolamide (TODGA) depends on HNO3 concentration and temperature. In order to increase LOC and suppress the third liquid phase, the modifier, N,N-dihexyl-octanamide (DHOA) of the aliphatic diluent was employed and its effect was investigated. The modifier can raise the LOC value, and it is noteworthy that LOC corresponds to the stoichiometric value, which is obtained by the extraction reaction, by using the extraction solvent of 0.1 M TODGA—(0.5–1 M) DHOA in n-dodecane. This condition suppresses the third liquid phase. In the absence of modifier, the relation of several DGA based compounds with LOC was investigated. It is confirmed that LOC using N,N,N′,N′-tetradodecyl-diglycolamide (TDdDGA), whose extractant has the longest alkyl chain in this work and forms hardly the third liquid phase, reaches to the stoichiometric value.
Keywords: Diglycolamide; N,N′,N′-tetraoctyl-diglycolamide; Extraction; Capacity; Nd; Am;

An automated on-line method has been developed for the continuous in vivo monitoring of arsenic species, including arsenite (AsIII), arsenate (AsV), monomethvlarsonic acid (MMA) and dimethvlarsinic acid (DMA), in the blood of living rabbits. The method is based on microdialysis sampling, high performance liquid chromatography (HPLC) separation and hydride generation atomic absorption spectrometry (HGAAS) detection. Dialysates perfused through an implanted microdialysis probe were collected in the sample loop of an on-line injector for direct and automated injection into an HPLC system hyphenated with HGAAS. Operating under optimal conditions, the detection limit was found to be 2.0, 2.9, 2.4 and 4.2 ng/ml for AsIII, MMA, DMA and AsV, respectively. Typical precision values of 7.0% (AsIII), 7.0% (MMA), 5.1% (DMA) and 8.7% (AsV) were obtained, respectively at a concentration level of 10 ng/ml. Spike recoveries close to 100%, relative to an aqueous standard, were obtained for each species. The average in vivo recoveries of AsIII, MMA, DMA and AsV in the blood of living rabbits were 5.7, 12.4, 8.9, and 11.0%, respectively. The use of an on-line microdialysis–HPLC–HGAAS system permitted the continuous monitoring, with a temporal resolution of 20 min sampling, of four arsenic species in the blood of living rabbits treated with arsenic trioxide. Based on our results, we present here the first finding, to the best of our knowledge of the distribution of arsenite in the blood of a living organism after administration of arsenic trioxide.
Keywords: Microdialysis; Arsenic speciation; Blood; High performance liquid chromatography; Hydride generation atomic absorption spectrometry;

A rapid and effective analytical procedure for the determination of robenidine in animal feeds was developed. Robenidine was extracted from samples with acidified methanol using accelerated solvent extraction (ASE). Extracts were dried and subjected to clean-up with aluminium oxide. Robenidine was eluted with methanol and determined by HPLC using UV diode-array (DAD) and MS detectors. Separation was carried out on a Purosphere ODS column in isocratic mode, with methanol–water mixture (70 + 30%) acidified with 0.1% trifluoroacetic acid as an eluent. DAD UV detection was done at 317 nm. MS analysis was performed with electrospray ionisation (ES) in positive mode. The procedure has been successfully applied for a variety of animal food products. The recovery of robenidine ranged from 85 to 95%, with R.S.D. of 2%. Limit of quantitation was estimated at 0.1 and 0.02 mg kg−1 for DAD UV and MS detection, respectively.
Keywords: Robenidine; Feedstuff; ASE extraction; HPLC–DAD–MS; Coccidiostats;

2-(9-Carbazole)-ethyl-chloroformate (CEOC), a novel pre-column fluorescence labeling reagent, has been synthesized and applied for the derivatization of phenols. Taken phenol, p-chlorophenol, 2,5-dimethylphenol, 2,4-dichlorophenol and 1,4-dihydroxybenzene as testing standards, the effects of derivatization conditions, such as pH of borate buffer, reaction time and fluorescent tagging reagent concentration, have been systematically studied. Under the optimized conditions, CEOC reacts readily with the phenols to form stable derivatives with excitation and emission wavelengths, respectively, at 293 and 360 nm. The single step derivatization reaction could be finished within 20 min even at room temperature. Such a method has been successfully applied to the analysis of phenols in printing ink by high-performance liquid chromatography.
Keywords: 2-(9-Carbazole)-ethyl-chloroformate; Fluorescence derivatization; High-performance liquid chromatography; Phenols;

Liquid chromatographic determination of Vitamin D3 in infant formulas and fortified milk by S. Perales; M.M. Delgado; A. Alegría; R. Barberá; R. Farré (58-63).
An isocratic reverse-phase liquid chromatographic (LC) method with electrochemical (EC) detection has been developed and validated for determining Vitamin D3 in fortified milk and infant formulas. LC–EC determination provides linear responses in the range from 0.03 to 0.7 μg Vitamin D3/ml, with inter- and intra-day variations (R.S.D.%) of 4.1 and 4.4, respectively, and detection and quantification limits of 0.012 ng in assay (48 ng/100 g of sample) and 0.039 ng in assay (156 ng/100 g of sample), respectively. Application of the method to a set of infant formulas and fortified milk confirmed its usefulness.
Keywords: Infant formulas; Milk; Vitamin D3; Liquid chromatography; Electrochemical detection;

Patulin is a mold metabolite commonly encountered in decaying apples. The most of analytical methods consist of multiple liquid–liquid partition steps for the extraction of patulin in apple juice. In this study, two solid-phase extraction (SPE) techniques using a tandem polyvinylpolypyrrolidone-octadecyl (PVPP-C18) cartridge system and a hydrophilic lipophilic balanced (HLB) macroporous copolymer sorbent cartridge were developed for the analysis of patulin in apple juice. A portion of apple juice was applied to either tandem PVPP-C18 cartridge system or the HLB cartridge. Patulin was eluted with diethyl ether through the cartridge excluding much of the interfering phenolic impurities. The volume of apple juice applied to the cartridges and the volume of diethyl ether to elute patulin from the cartridges were optimized to exclude much of the interfering impurities during reversed-phase (C18) liquid chromatographic separation with UV detection at 276 nm, keeping the recovery higher than 90% for spiking levels of 25–100 μg/1.
Keywords: Patulin; Apple juice; Solid-phase extraction; Liquid chromatography;

An HPLC method for the determination of vanadium using on-line preconcentration and sample clean-up has been developed employing a C18 column. 4-(2-Pyridylazo) resorcinol (PAR) and hydrogen peroxide were used as pre-column complexing agents for the determination of vanadium. Aliquots of either standard or sample solutions (100 μL and 2 mL) containing the vanadium complex were loaded onto a guard column (C18, 0.4 mm × 4.6 mm) with a carrier mobile phase comprising 20% (v/v) methanol, 5 mM acetic acid, 5 mM citric acid, l0 mM tetrabutylammonium bromide (TBABr) at pH 7 (eluent 1). A flow rate of 2 mL/min for 0.40 min was used for a 100 μL injection loop, and 1.65 min for the 2 mL injection loop, with the effluent being directed to waste. The vanadium complex was flushed onto the analytical column (C18, 150 mm × 3.9 mm, 4 μm), via an automatic switching valve, using a mobile phase comprising 32% (v/v) methanol, 5 mM acetic acid, 5 mM citric acid, and 5 mM tetrabutylammonium bromide (TBABr) at pH 7, with a flow rate of 1 mL/min for 1.60 min. The switching valve was returned to its original position, and any unwanted species remaining on the guard column were eluted with methanol for 5 min. The guard column was then re-equilibrated with eluent 1 for 5 min before the next injection. The analytes were detected at 540 nm. The detection limits (determined by a signal-to-noise ratio of 3) of vanadium with on-line preconcentration using 2 mL and 100 μL injection loops were 3.1 and 67 ng/kg, respectively.The developed method was used for the determination of vanadium in plant tissue. Chinese green mustard and tomato plant samples analysed in this study were cultured hydroponically. Vanadium concentrations measured using the HPLC technique were compared with those obtained using magnetic sector ICP-MS. Vanadium levels in plant tissue samples (root, stem and leaf of Chinese green mustard) found by HPLC agreed well (>85%) with those found using magnetic sector ICP-MS.
Keywords: Vanadium; Plant tissue; Chinese green mustard plants; Tomato plants; HPLC; On-line preconcentration;

Enantiomeric separation of organophosphorus pesticides by capillary electrophoresis by Carmen García-Ruiz; Gloria Álvarez-Llamas; Ángel Puerta; Elisa Blanco; Alfredo Sanz-Medel; Ma Luisa Marina (77-83).
The separation of the enantiomers of a group of organophosphorus pesticides (OPs) has been investigated by electrokinetic chromatography (EKC) using different anionic cyclodextrins as chiral selectors. The use of a 25 mM Tris buffer (pH 7.0), 20 mM in CM-β-CD together with an applied voltage of 24 kV and a temperature of 25 °C enabled the individual enantiomeric separation of malathion and phenthoate each one into its two enantiomers, the partial separation of the enantiomers of phenamiphos and the separation of three of the four enantiomers of isomalathion. Since naled is very reactive in aqueous solutions, its enantiomeric separation achieved in about 8 min in 25 mM borate buffer at pH 9 with 10 mM CM-β-CD resulted in the observation of a broad peak due to degradation products. A preconcentration step by disk solid-phase extraction was studied and used together with the chiral EKC method developed for the enantiomeric separation of malathion in order to determine this pesticide in tap water samples spiked at the μg mL−1 level. Finally, the precision and recovery of the method was established.
Keywords: Enantiomeric separation; Organophosphorus pesticides; Capillary electrophoresis; Malathion; Solid-phase extraction;

Carbon nanotubes paste electrodes as new detectors for capillary electrophoresis by Manuel Chicharro; Alberto Sánchez; Esperanza Bermejo; Antonio Zapardiel; María D. Rubianes; Gustavo A. Rivas (84-91).
Carbon nanotubes paste electrodes (CNTPE) prepared with short (1–5 μm length) and long carbon nanotubes (5–20 μm length) of 20–50 nm diameter have demonstrated to be highly useful as detectors in flow injection analysis and capillary electrophoresis. Compared to the classical graphite paste electrode, CNTPE improved the detection limits of dopac, ascorbic acid, dopamine, norepinephrine and epinephrine. The content of agglutinant has shown to be an important variable in the preparation of these carbon nanotubes composites. Even when no substantial differences were observed between the electrodes, those prepared with long carbon nanotubes (55.0%, w/w) and mineral oil (45.0%, w/w) have allowed us to obtain less noisy and more reproducible signals. In this article we also report the successful use of a new electrochemical cell for the detection in capillary electrophoresis that allows an easier handling and more reproducible responses. Therefore, the combination of the carbon nanotubes electrocatalytic activity with the known advantages of composite materials, the efficiency of the new electrochemical cell and the excellent separative properties of capillary electrophoresis represents a very important alternative for new electroanalytical challenges.
Keywords: Carbon nanotubes paste electrodes; Capillary electrophoresis; Nuerotransmitters; Electrochemical detection in capillary electrophoresis;

Microfluidic supported liquid membrane extraction by Xiaoyan Wang; Chutarat Saridara; Somenath Mitra (92-98).
Miniature, microfluidic devices have been designed and fabricated for the enrichment of haloacetic acids in water. The analytical approach is based on supported liquid membrane (SLM) extraction followed by direct HPLC-UV detection without any derivatization. Channel dimensions and the flow rates affected enrichment factors and extraction efficiencies. Enrichment factors as high as 54 were obtained on a 2 cm × 2 cm extraction module. Large linearity ranges with good linearity, high precisions and detection limit as low as 2 ng/mL were obtained.
Keywords: Microfludic; Supported liquid membrane extraction; Lab-on-a-chip;

Creation of concentration gradients is important in the study of biological and chemical processes that are sensitive to concentration variations. This paper presents a simple method to generate a near-constant concentration gradient in electroosmotic flow in a converging–diverging microchannel. The mechanism is based on the enhanced diffusive mixing inside the converging–diverging microchannel section. By varying the converging–diverging geometry, the degree of diffusive mixing can be controlled. Different concentration gradients can be obtained by varying the applied electric field and microchannel's geometry. Concentration profiles with only 7% deviation over a channel length of 3 mm and 3% deviation over a channel length of 1 mm can be obtained for a 400 μm wide microchannel. In comparison with a simple T-shaped micromixer, the converging–diverging microchannel can produce desired concentration profiles with a much shorter mixing length (shorter by a factor of 2–3.5). A serially connected concentration gradient generator is also realized with the ability to generate two concentration gradient ranges in the same microchannel. Numerical simulations and experiments were carried out to investigate the factors that contribute to the generation of concentration gradients. The experiments were conducted in polydimethylsiloxane (PDMS)-glass chips produced by soft-lithography rapid prototyping.
Keywords: Microfluidics; Concentration gradient;

Development of a novel electrokinetically driven microfluidic immunoassay for the detection of Helicobacter pylori by Yali Gao; Frank Y.H. Lin; Guoqing Hu; Philip M. Sherman; Dongqing Li (109-116).
A novel microchip-based electrokinetically driven immunoassay was developed in this study. The microchip was made of poly(dimethylsiloxane) (PDMS)/glass using photolithography and replica molding. The immunoreaction was between the anti-Helicobacter pylori antibody and the bacterial protein antigen immobilized on the wall of the microchannel. The conditions for antigen immobilization were optimized by adjusting the plasma treatment time. By varying the concentration of blocking buffer, a buffer containing 5% (w/v) bovine serum albumin (BSA) was shown to effectively minimize non-specific binding. Rhodamine-labeled secondary antibody was employed for signal generation. The dependence of the fluorescence signal on the concentrations of both the coating antigen and the primary antibody was obtained. The detection limits for both the coating antigen and the primary antibody are 1 ng/μL. The immunoassay only took 30 min when used to detect antibody. The microchips could be prepared and stored at 4 °C for at least 10 days before the immunoassay was performed. With further development on automatic control and detection strategy, this microfluidic immunoassay technique has a potential for point-of-care analysis.
Keywords: Immunoassay; Electrokinetics; Microfluidics; Poly(dimethylsiloxane); Helicobacter pylori;

Microwave acid digestion and aqua regia extraction techniques were investigated to obtain a simple, rapid and safe method for the determination of eight trace elements in soil (Cd, Co, Cr, Cu, Mn, Ni, Pb, and Zn). Soil samples were collected between 6 October and 3 November 2004, air dried, grinded, sieved (<63 μm) and subjected to digestion. The digests were subsequently analyzed for metal content by inductively coupled plasma-mass spectrometry (ICP-MS). Various combinations and volumes of hydrofluoric, nitric and hydrochloric acids were evaluated for the microwave acid digestion efficiency. Two certified reference materials (CRM 142, light sandy soil, and CRM 143, over fertilized soil) were used in the comparison of these digestion protocols and good agreement between the two procedures and the certified values was found. The aqua regia microwave acid digestion with a mixture of 2 ml of HNO3 and 6 ml of HCl produced the fastest, safest and accurate analytical results with a recovery of 91–110% and a precision better than 5% in most cases. Very low limits of detection were obtained, below 0.05 μg g−1, for all elements, except for Zn, 2.3 μg g−1. A number of agricultural soil samples irrigated by the Tinishu Akaki River (TAR) and its tributaries, Ethiopia, were analyzed.
Keywords: Microwave digestion; Aqua regia extraction; Agricultural soil; Heavy metal; Inductively coupled plasma-mass spectrometry; Ethiopia;

Near infrared determination of Diuron in pesticide formulations by Javier Moros; Sergio Armenta; Salvador Garrigues; Miguel de la Guardia (124-129).
A simple, fast and environmentally friendly near infrared (NIR)-based methodology was developed for Diuron determination in pesticide formulations. This methodology was based on the pesticide extraction with acetonitrile and subsequent transmittance measurement determination by peak area measurement between 2021 and 2047 nm, corrected with a single point baseline established at 2071 nm. The repeatability, as relative standard deviation of five independent analysis of a 15.3 mg g−1 Diuron standard was 0.03% and the limit of detection 0.013 mg g−1. The reagent consumption was clearly reduced in front of a chromatographic reference procedure from 39.1 ml acetonitrile per sample, required by liquid chromatography (LC), to 1 ml of acetonitrile consumed for NIR. The sample measurement throughput obtained by the NIR methodology was 120 h−1, 10 times higher than that obtained by LC (12 h−1). It can be concluded that the proposed vibrational method is appropriate for the quality control of pesticide commercial formulations.
Keywords: Near infrared; Pesticide; Formulations; Agrochemicals;

Enhanced photoluminescence of up-converting phosphors in a solid phase bioaffinity assay by Katri Kuningas; Terhi Rantanen; Timo Lövgren; Tero Soukka (130-136).
For ultrasensitive bioaffinity assays, the label technology employed should produce intense signals upon specific binding of the label bioconjugate to analyte. Normally, signal levels are improved by optimizing the detection conditions of the label and by utilizing special signal enhancement methods. We have now discovered unexpectedly strong signal enhancement for up-converting (UPC) phosphors in a solid phase bioaffinity assay performed in white microtitration wells. Two commercial, micrometer-sized UPC-phosphors and down-converting europium phosphor were bead-milled to submicron-sized particles and conjugated to biomolecules along with europium(III) chelate-dyed polystyrene nanoparticles. Dilution series measurements and bioaffinity binding assays from white and transparent microtitration wells were performed to study the signal enhancement. In dilution series measurements from solid phase, signal enhancement was for UPC-phosphors about 50–155- and 70–175-fold, determined from anti-Stokes photoluminescence measurements at 535 nm, and for europium labels roughly three- to eight-fold. In bioaffinity assays enhancement was for UPC-phosphors around 150- and 250-fold and for europium labels about 10-fold. Even stronger enhancement was observed at red emission wavelength of UPC-phosphors at 660 nm. Due to signal enhancement, about 50-fold improvement in the detection limits of UPC-phosphors was observed in solid phase measurements in white wells.
Keywords: Up-conversion; Phosphor; Enhanced photoluminescence; Bioaffinity assay;

Fluorescence and mass spectrometric detection schemes are developed and compared for the simultaneous activity determination of two enzymes in solution. As model system, the following reactions are used: The alkaline phosphatase catalyzed reaction with 5-fluorosalicyl phosphate yields the fluorescent 5-fluorosalicylic acid, whereas microperoxidase 11 reacts with 4-(N-methylhydrazino)-7-nitro-2,1,3-benzooxadiazole and H2O2 to the strongly fluorescent 4-(N-methylamino)-7-nitro-2,1,3-benzooxadiazole. As the emission spectra of the fluorescent products as well as the molecular masses of substrates and products do not interfere with each other, is it possible to determine both reactions in parallel with both detection schemes. The measurements resulted in the same limits of detection, limits of quantification and linear ranges of the single/simultaneous enzyme determination for fluorescence and MS detection. While the relative standard deviations were significantly lower in case of fluorescence detection (1.4–3.2%) than in mass spectrometry (5.7–10.1%), the latter proved to be the more versatile approach for multianalyte determination.
Keywords: Bioassays; Fluorescence; Electrospray ionization mass spectrometry; Microperoxidase; Phosphatase; Simultaneous determination;

Mercury–enzyme inhibition assays with an amperometric sucrose biosensor based on a trienzymatic-clay matrix by Hasna Mohammadi; Aziz Amine; Serge Cosnier; Christine Mousty (143-149).
The feasibility of amperometric sucrose and mercury biosensors based on the immobilization of invertase, glucose oxidase and mutarotase entrapped in a clay matrix (LAPONITE®) was investigated. The enzyme clay gel was deposited onto platinum electrodes and cross-linked with glutaraldehyde. The response of the biosensors to sucrose was measured by potentiostating the modified electrodes at +0.60 V versus Ag/AgCl in order to oxidize the enzymatically generated hydrogen peroxide. Mercury concentration was measured by inhibiting the invertase activity. The effect of different parameters, such as pH of the analyte solution, enzyme ratio, mercury incubation time, on the biosensor performance towards sucrose and mercury determinations was studied. The biosensor inhibition by inorganic and organic mercury was evaluated and the study of interference from other metal ions revealed a good selectivity towards mercury. Under optimized conditions, sucrose was determined from 3.3 × 10−7 to 5.1 × 10−3  M and mercury(II) in the concentration range of 1 × 10−8 to 1 × 10−6  M.
Keywords: Inhibition; Invertase; Mercury; Sucrose; Clay electrode;

A new differential pulse polarographic (DPP) and catalytic adsorptive stripping voltammetric (CAdSV) methods for the determination of vanadium(V) have been developed. The CAdSV method is based on the adsorptive accumulation of the vanadium–chloroanilic acid (CAA) complex on the surface of a HMDE and the utilization of the catalytic reaction with BrO3 . The optimal CAdSV vanadium(V) response was obtained in (0.04 ÷ 0.2) M acetic buffer at pH 4.5–4.8 in the presence of (2.0 ÷ 4.0) × 10−5  M CAA and (5.0 ÷ 10.0) × 10−3  M KBrO3 with an accumulation potential of (−0.10 ÷ −0.15) V and accumulation time of (80 ÷ 120) s. The sensitivity of the CAdSV was 42.7 nA nM−1 and the linearity range amounted from 2 × 10−10 up to 5 × 10−8  M for 100 s of accumulation. The R.S.D. was 4.5% at the 1.0 × 10−9  V(V) level. The detection limits were found to be 2.8 × 10−9  M for DP polarography and 9 × 10−12  M for CAdSV with 100 s of accumulation. Possible interferences by Sb(III), Pb(II), Cu(II), Cd(II), Fe(III), Bi(III), Sn(II), Sn(IV), U(VI), Mo(VI) and surface active substances were evaluated. The results of the determination of V(V) in certified reference material water sample showed good reproducibility (R.S.D. = 3.4%) and reliability of the elaborated CAdSV method.
Keywords: Vanadium(V); Chloranilic acid; Bromate; Polarography; Catalytic adsorptive stripping voltammetry;

Derivatives of phenoxazine dyes serve as chromoionophores in the field of chemical sensors. By studying the changes of the distribution of membrane components in ETH 5294 based, pH sensitive, ion-selective membranes using spectroelectrochemical microscopy (SpECM), the decay in the concentration of the protonated chromoionophore was generally much larger than expected upon previous studies. These recorded changes could not be interpreted with the leaching of the protonated or unprotonated forms of ETH 5294 or the tetraphenyl borate derivatives embedded in the membrane. Similarly, the changes could not be explained by the photochemical decomposition of the tetraphenyl borate derivatives (tetraphenyl borate, TPB or tetrakis(4-chlorophenyl) borate, TpClPB). The decrease in the absorbance value measured at 660 nm, at the absorbance maximum of the protonated chromoionophore has been linked to the photochemically initiated, singlet oxygen mediated decomposition of ETH 5294. The decomposition occurs only if the wavelength of the illuminating light is larger than ∼580 nm, and the rate of decomposition is facilitated by the presence of certain anions in the membrane (TPB, and TpClPB) and in the sample solution (Br, I, and Cl) as they were penetrating the membrane by acid coextraction. On the other hand, the protonated chromoionophore was found to be stable in membranes cast with tetrakis[3,5-bis(trifluoromethyl) phenyl] borate anion (TFPB) or in acidic solutions of NO3 , ClO4 , SO4 , TFPB and citrate anions. In comparison to ETH 5294 (chromoionophore I), ETH 2439 (chromoionophore II) was found completely stable, and the decomposition of ETH 5350 (chromoionophore III) was also hardly detectable. In our view, in the light of the experimental results summarized in this study some of the widely accepted views and practices in chromoionophore based sensors research should be changed and some of the results should be reevaluated.
Keywords: Chromoionophore decomposition; Phenoxazine dyes; pH sensitive electrode and optode membranes; Spectroelectrochemical microscopy;

An electrochemical sensor for the detection of lead ions is described which is made by modifying a gold electrode substrate with self-assembled monolayers (SAMs) of 3-mercaptopropionic acid (MPA) or thioctic acid (TA) followed by covalent attachment of a lead binding peptide, human angiotensin I. Cyclic voltammetry of MPA–angiotensin modified gold electrodes complexed with lead displayed voltammograms with prominent lead peaks at E 0′, −0.29 V. A detection limit of 1 nM was achieved using Osteryoung square wave voltammetry. However, the electrodes were not stable over repeated electrochemical cycles due to partial electrochemical desorption of the SAM. The TA–angiotensin modified gold electrode showed greater stability and were able to be regenerated several times. Using Osteryoung square wave voltammetry for TA–angiotensin modified electrodes, lead concentrations down to 1.9 nM were detected. Although the detection limit of the TA–angiotensin modified electrode is higher than achieved with MPA–angiotensin, it is still well below Australian drinking water guidelines. Studies of interference effects on the Pb2+ current showed Hg2+ as a significant interferent but only at levels significantly greater than those found in natural waters.
Keywords: Biosensors; Electrochemistry; Lead; Angiotensin; Heavy metals;

Estimating the degree of similarity overlap for structural data in spectral databases by T.A. Kornakova; T.F. Bogdanova; B.G. Derendyaev; V.N. Piottukh-Peletsky (177-180).
A method for the chemical structure overlap of two spectral databases estimating is suggested. The technique matches all compounds from the infrared (IR) spectral database against all compounds from the mass-spectral database using their structural similarity. The structural similarity is estimated by comparing of structural fragment sets obtained for each database using preliminarily decomposing of all molecular graphs to exhaustive sets of structural fragments with 2–7 nodes. Quantitative estimates of the relationship between the threshold on the structural similarity and the overlap degree of two databases was revealed. High degree of structural similarity overlap provides new possibilities for structure elucidation using two spectral databases simultaneously.
Keywords: Infrared spectroscopy; Mass spectrometry; Database; Chemical structures; Structure similarity; Similarity overlap; Structure elucidation;

Boosting is one of the most important recent developments in classification methodology. It can significantly improve the prediction performance of any single classification algorithm and has been successfully applied to many different fields including problems in chemometrics. Boosting works by sequentially applying a classification algorithm to reweighted versions of the training data, and then taking a weighted majority vote of the sequence of classifiers thus produced. In this paper, we proposed a generalized boosting algorithm via Bayes optimal decision rule. Using Bayes optimal decision rule, we adjust the weights of the sequence of classifiers in the voting process of boosting algorithm. The two types of errors are introduced into the generalized boosting and make the voting process more sensible. Meanwhile, the weights of the training samples are also correspondingly adjusted according to some criterion. The generalized boosting is applied to the binary classification for chemical data. Experimental results show that it can improve the predict accuracy compared with AdaBoost algorithm especially when the difference between the two types of errors for classification is large.
Keywords: Bayes optimal decision rule; Binary chemical classification; Boosting; Chemometrics;

Design and optimization of a chemometrics-assisted spectrophotometric method for the simultaneous determination of levodopa and carbidopa in pharmaceutical products by Patricia C. Damiani; Andrea C. Moschetti; Adrián J. Rovetto; Fernando Benavente; Alejandro C. Olivieri (192-198).
A chemometrics-assisted spectrophotometric method was developed and validated for the simultaneous determination of levodopa and carbidopa in pharmaceutical preparations. Levodopa [3-(3,4-dihydroxyphenyl)-L-alanine] is an aminoacid precursor of the neurotransmitter dopamine, widely used in the treatment of Parkinson's disease, and usually associated with the decarboxilase inhibitor carbidopa [(S)-3-(3,4 dihydroxyphenyl)-2-hydrazino-2-methylpropionic acid]. The multivariate absorbance signals were recorded at a fixed optimum time (4 min) after reaction of the analytes with potassium periodate. The working pH (4.8) and oxidant concentration (1.6 × 10−3  M) were estimated using a two-factor surface response optimization procedure. Calibration and prediction were performed with the aid of the partial least-squares model. The recoveries were satisfactory and statistically comparable to those obtained by applying the reference Pharmacopoeia method of high performance liquid chromatography.
Keywords: Spectrophotometry; Partial least-squares; Levodopa; Carbidopa;

A battery powered submersible chemical analyzer, the Zn-DigiScan (Zn Digital Submersible Chemical Analyzer), has been developed for near real-time, in situ monitoring of zinc in aquatic systems. Microprocessor controlled solenoid pumps propel sample and carrier through an anion exchange column to separate zinc from interferences, add colorimetric reagents, and propel the reaction complex through a simple photometric detector. The Zn-DigiScan is capable of self-calibration with periodic injections of standards and blanks. The detection limit with this approach was 30 μg L−1. Precision was 5–10% relative standard deviation (R.S.D.) below 100 μg L−1, improving to 1% R.S.D. at 1000 μg L−1. The linear range extended from 30 to 3000 μg L−1. In situ field results were in agreement with samples analyzed by inductively coupled plasma mass spectrometry (ICPMS). This pump technology is quite versatile and colorimetric methods with complex online manipulations such as column reduction, preconcentration, and dilution can be performed with the DigiScan. However, long-term field deployments in shallow high altitude streams were hampered by air bubble formation in the photometric detector.
Keywords: Zinc; In situ; Environmental monitoring; Acid mine drainage; Near real-time;

A new method is described for the concomitant use of the Lu–Hf and Sm–Nd radiogenic isotope systems on the same rock digest. After total spiking with two mixed 176Lu–179Hf and 149Sm–150Nd enriched tracers, the sample is decomposed by fusion with a LiBO2 flux in an induction furnace. Hf is first separated from all matrix elements using U-TEVA Spec extraction chromatography material in 3 M HNO3 medium, and obtained in a single step ready for isotope ratio measurements using multiple collector ICP–MS [B. Le Fèvre, C. Pin, Anal. Chem. 73 (2001) 2453]. The rare earth elements (REE) are then separated as a group from the same sample solution by conventional cation-exchange in nitric acid-oxalic acid medium. After evaporation of the REE fraction, Nd, Sm, and Lu are isolated by extraction chromatography using two tandem columns filled with RE Spec and Ln Spec extraction chromatographic materials, respectively. This separation scheme provides fractions of the analytes with excellent recoveries (>90%) and purity, suitable for thermal ionization mass spectrometry, with satisfactory blanks. The potential and present limitations of this method, are exemplified by repeat analyses of several international standard reference materials.
Keywords: Alkaline fusion; Chromatography; Separation; Isotope dilution; Samarium; Neodymium; Lutetium; Hafnium; Sm–Nd; Lu–Hf;

A design of reaction-controlled chemiluminescence imaging and its application by Lirong Luo; Zhujun Zhang; Lifeng Ma (222-228).
The paper presented a design of reaction-controlled chemiluminescence (CL) imaging analysis method. The design was based on controlling pH to initiate CL reaction and obtain high sensitivity. The pH value of CL reaction was controlled by ammonia, which was produced by injecting NaOH solution to NH4Cl solution, and the amount of ammonia could be adjusted by varying concentration, pumping time and flow rate of NaOH solution or varying concentration of NH4Cl solution. The pH of CL reagents in 96-well microtiter plates increased continuously from the same initial value due to the absorbance of ammonia and the relative CL intensity increased with the increasing pH. Based on above reaction-controlled design, the CL reaction in 96 wells could be initiated at the same time and the total CL signal of each well could be monitored. As results of above operation, a high sensitivity and better reproducibility could be obtained. A luminol, H2O2, CL system for determination of hemoglobin (Hb) was selected to validate the presented design. The CL intensity was proportional with the concentration of Hb in the range of 1.0 × 10−9 to 1.0 × 10−7  mol l−1 and the detection limit was 3.0 × 10−10  mol l−1 (3σ), the relative standard deviation (R.S.D.) for 11 parallel measurements of 1.0 × 10−8  mol l−1 Hb was 2.7%.
Keywords: Chemiluminescence; Imaging; Design; Application;

A time-integrated sampling device interfaced with two toxicity-based assays is reported for monitoring volatile toxic industrial chemicals (TICs). Semipermeable membrane devices (SPMDs) using dimethylsulfoxide (DMSO) as the fill solvent accumulated each of 17 TICs from the vapor phase. Uptake kinetics experiments for one of these compounds (acrolein) indicated that it was significantly concentrated (i.e., 10% of the 24 h maximum) in as little as 10 min and was concentrated by a factor of over 200 for a 24 h exposure time as measured using both mass and toxicity assays. The effect of each of the TICs on the Microtox bacterial luminescence assay and IQ-Tox Daphnia magna fluorescence assay was determined both from a direct assay and a vapor accumulation assay using SPMDs. Microtox EC50 values (concentrations yielding 50% inhibition) were determined for each of the TICs analyzed. The rank order of the Microtox EC50 values for each of the compounds measured by direct dilution of the TICs into assay buffer was similar but not identical to the Apparent (App) EC50 values determined from the vapor accumulation assay. The ratios of the EC50 to the AppEC50 values were used to calculate apparent toxicity-derived concentration factors (i.e., the toxicity equivalents of compound that concentrate from vapor into the SPMD). EC50 values for the IQ-Tox assay as measured using a 90 min fluorescence assay were, in most cases, similar but not identical to the Microtox EC50 values for individual compounds.
Keywords: Toxic industrial chemicals; Acute toxicity; Daphnia magna; Vibrio fischeri;

The scope of the capillary flow hydrothermal reactor system for the UV–vis spectrophotometric detection (CHUS), which was recently developed in our group for the in situ UV–vis monitoring of hydrothermal reactions at the millisecond–second time range, has been investigated as a potential new probe for the analysis of conformations and interactions of polynucleotides at extremely high temperatures. It was possible to detect indirectly the difference between double-stranded and single-stranded DNA on the basis of the intercalation with ethidium bromide (EB). Measurements of absorbance of the DNA–EB complex were attempted at up to 200 °C by the CHUS system, and the results were consistent with the melting behavior observed by differential scanning calorimetry. This fact indicates that the CHUS system enables a rapid monitoring where the decomposition of both DNA and the intercalator did not interfere with the measurements. Since these results elucidate that the CHUS system can readily be applied for monitoring UV–vis absorbance at 100–300 °C, this study supports that the CHUS method is applicable as a new probe for exploring interactions and conformation within polynucleotides at high temperatures, such as in hyperthermophilus.
Keywords: UV–vis spectrophotometry; Fused-silica capillary; Hydrothermal reactions; DNA; Nucleotide; Intercalator;

Calorimetric measurements of heat of sorption in polymer films: A molecular modeling and experimental study by A.V. Shevade; M.A. Ryan; M.L. Homer; A.K. Kisor; K.S. Manatt; B. Lin; J.-P. Fleurial; A.M. Manfreda; S.-P.S. Yen (242-248).
We report a combined molecular modeling and experimental effort to predict and measure the heat of sorption of target molecules in a polymer film. The primary focus of this work is to demonstrate and validate the applicability of molecular modeling techniques as a predictive tool to evaluate polymeric sensing films for micro-calorimetric chemical sensor applications. In the current investigation, molecular modeling studies were performed prior to experimental measurements to predict the heat of sorption (ΔH sorpt) of a target analyte in a polymer film. Experimental measurements for the heat of sorption were performed using a quartz crystal microbalance/heat conducting calorimeter setup. The system under investigation was isopropanol in ethyl cellulose polymer film. The average ΔH sorpt predicted by molecular modeling at 22 °C was found to be 39.7 kJ/mol (standard deviation = 2.6 kJ/mol), which compares well with the average measured experimental value of 30.2 kJ/mol (standard deviation = 7.2 kJ/mol).
Keywords: Molecular modeling; Polymer film; Heat of sorption; Micro-calorimeters; Quartz crystal microbalance/heat conducting calorimeter;

The interaction and binding abilities between the trans-activation response (TAR) element, residues 1–59 of the human immunodeficiency virus type 1 (HIV-1) mRNA, and four computer-simulated designed potentially small-molecule drugs were studied by capillary zone electrophoresis (CZE). Experimental results indicated that these computer-simulated small-molecule drugs could specifically bind to TAR RNA and effectively inhibit the complex formation of HIV-1 trans-activator of transcription (Tat) protein to TAR RNA – an essential component for HIV-1 transcription. The small molecular inhibitors and TAR RNA could be baselinely separated for each individual drug under optimized CE experimental conditions. Their binding constants were quantitatively determined and the data were used for the drug evaluation and screen in lead discovery.
Keywords: Binding constant; Capillary zone electrophoresis; TAR RNA;

The mixed dissociation constants of four drugs – haemanthamine, lisuride, metergoline and nicergoline – at various ionic strengths I of range 0.01 and 0.6 and at temperatures of 25 °C and 37 °C were determined using SPECFIT32 and SQUAD(84) regression analyses of the pH-spectrophotometric titration data. A proposed strategy for efficient experimentation in a dissociation constants determination, followed by a computational strategy for the chemical model with a dissociation constants determination, is presented on the protonation equilibria of haemanthamine. Indices of precise methods predict the correct number of components, and even the presence of minor ones when the data quality is high and the instrumental error is known. The thermodynamic dissociation constant p K a T was estimated by non-linear regression of {pK a, I} data at 25 °C and 37 °C: for haemanthamine p K a T = 7.22 ( 1 ) and 7.05 (2), for lisuride p K a T = 7.87 ( 1 ) and 7.59 (1), for metergoline p K a T = 7.62 ( 1 ) and 7.38 (1), for nicergoline p K a , 1 T = 7.94 ( 1 ) and 7.69 (1). Goodness-of-fit tests for various regression diagnostics enabled the reliability of the parameter estimates to be found.
Keywords: Spectrophotometric titration; Dissociation constant; Protonation; Haemanthamine; Lisuride; Metergoline; Nicergoline;

Electrochemical and theoretical complexation studies for Zn and Cu with individual polyphenols by I. Esparza; Í. Salinas; C. Santamaría; J.M. García-Mina; J.M. Fernández (267-274).
Zn and Cu interactions with three selected flavonoids (catechin, quercetin and rutin) have been electrochemically monitored. It has been shown that catechin takes one atom of metal per molecule; quercetin takes two atoms, and rutin is able to take up to three atoms. Not all ligands bind metals equally strong, and weakly bonded metals can be distinguished. Zn shows a sluggish kinetics and, at the same time, the highest conditional formation constants. The method could be applied to a real sample. Theoretical models are proposed for the most favourable compounds.
Keywords: Zn; Cu; Polyphenols; Flavonoids; Complexation; DPASV;

Influence of grapevine flower treatment with gibberellic acid (GA3) on polyphenol content of Vitis vinifera L. wine by Péter Teszlák; Krisztián Gaál; Martin Shahin Pour Nikfardjam (275-281).
Four Vitis vinifera L. cultivars (Riesling, Sauvignon blanc, Kadarka, and Lemberger) were treated at full bloom with 20 ppm gibberellic acid (GA3). Depending on variety, the respective wines showed different polyphenol contents and profiles. While GA3 seemed to have a small impact on polyphenols in Riesling (−5%), treatment of Sauvignon blanc led to significantly higher polyphenol content (+19%). Principal component analysis revealed that mainly caftaric acid and GRP account for the difference between treatments and varieties. In red varieties, GA3 generally led to a delay in ripening. In Kadarka, GA3 led to significantly higher polyphenol (+52%) and anthocyanin (+229%) content. The much higher anthocyanin content was referred to the lower Botrytis infection grade in GA3 treated grapes. Lemberger showed much lower polyphenol (−38%) but slightly higher anthocyanin (+11%) content. Principal component analysis showed that mainly caffeic acid and epicatechin as well as p-coumaric acid and rutin, respectively, are responsible for the differences between treatments and varieties. Since no uniform impact of GA3 treatment on wine composition was observed during the scope of this study and did not only occur in polyphenolic composition but also, e.g. in mineral content, it is concluded that the hormone-like influence of GA3 on the chemical composition of grapes is extremely complex and has to be studied in more detail in further research trials.
Keywords: Gibberellic acid; Vitis vinifera L.; Polyphenol; Anthocyanin;

In this paper, a general methodology to determine non-chromogenic compounds by UV–vis spectrophotometry given the competition of their inclusion complex in cyclodextrins with a structure-alike synthetic standard has been explored. The method was satisfactorily applied to quantify γ-lindane to levels as low as 0.05 μg mL−1 in tap water spiked with standard by resolving the UV–vis spectra of the complex of the structure-alike chromogenic standard and HP-γ-cyclodextrin by partial least squares (PLS).
Keywords: Cyclodextrins; Competition; UV–vis spectrophotometry; Inclusion;

Author Index (291-293).