Analytica Chimica Acta (v.569, #1-2)

CONTENTS (v-vii).

Sensitivity, dynamic range and resolution have been calculated and compared from a range of analytes sensed in the literature using the techniques of interferometry, surface plasmon resonance (SPR) and luminescence. A detailed explanation of the physical and chemical/biological properties required of optical sensors is included along with the principle of operation of the sensors. Theoretical sensitivities of interferometry and SPR are also detailed along with parameters affecting these sensitivities.In the literature discussed in this review paper, the technique of luminescence, which relies intrinsically on ‘labelling’, offers the best resolutions for sensing of biomolecules (protein and DNA). Interference techniques offer the best resolutions for low molecular weight chemical liquids/vapours.Techniques which are ‘label-free’ are often desirable and it is demonstrated here that by combining the techniques of SPR with interferometry, it is possible to sense proteins with a resolution similar to that of luminescence.The future of chemo- and bio-sensing is discussed in terms of potential for multi-channel analysis, their continuous miniaturisation and their impending nanotechnology revolution.
Keywords: Optical sensors; Interferometry; Surface plasmon resonance; Luminescence; Nanoprobes; Chemosensors; Biosensors;

Sensitivity enhancement of wavelength modulation surface plasmon resonance biosensor by improving the baseline solution by Ying Sun; Xia Liu; Daqian Song; Yuan Tian; Shuyun Bi; Hanqi Zhang (21-26).
The sensitivity enhancement of wavelength modulation surface plasmon resonance (SPR) biosensor was described by improving the baseline solution that made the resonant wavelength move to longer wavelength. The baseline solutions here used are ethanol solutions of 1.0%, 2.0%, 4.0% and 6.0%. The higher is the concentration of the ethanol solution, the longer is the resonant wavelength, and the lower is the concentration of the analyte that could be detected. The heat shock protein (Hsp) 70 are studied and when the baseline solutions used are 1.0%, 2.0%, 4.0% and 6.0% ethanol solutions, the lowest concentrations of Hsp 70 that can be detected are 1.00, 0.50, 0.20 and 0.10 μg/ml, respectively.
Keywords: Wavelength modulation; Surface plasmon resonance; Baseline solution; Heat shock protein 70;

A poly(p-phenyleneethynylene) (PPE) containing pyridine and acetal groups (polymer A) was prepared by a palladium-catalyzed Sonogashira coupling reaction. Osmium complex-grafted PPE (polymer B) was prepared by post-polymerization functionalization of the polymer A via a chelate reaction of the pyridine groups with Os(bpy)2Cl2 (bpy: 2,2′-bipyridine). A cross-linkable PPE functionalized with osmium complex and aldehyde groups (polymer C) was obtained by deprotecting the acetal groups of polymer B in a hydrochloric acid solution. Both polymers B and C are highly soluble not only in common solvents such as THF, ethanol, methanol, acetone, ethyl acetate, DMF and DMSO, but also in water. The water-soluble polymer C was used to cross-link and assemble glucose oxidase (GO x ) onto amine-terminated monolayers at a gold electrode via a coupling reaction of its aldehyde groups with amine groups of the enzyme, an added polyamine cross-linking agent (triethylenetetraamine), and monolayers via formation of initial Schiff bases, which were reduced to secondary amine groups by using cyanoborohydride, a mild reducing agent. The polymer provided very efficient wiring for the enzyme, resulting in an efficient electron transfer of GO x to the electrode. Such enhancement of electron transfer is due to the hydrophilic, long and flexible tethers of osmium complex to the polymer backbone and a close contact between the polycationic polymer and polyanionic GO x via electrostatic interactions. The biosensor displayed highly sensitive responses to glucose with the apparent Michaelis constant of 8.2 mM, and good stability without substantial change of current response to glucose for 1-month storage in phosphate buffer solution (pH 7.0).
Keywords: Water-soluble conjugated polymers; Cross-linkable polymer; Glucose; Osmium complex; Electrochemistry; Biosensor;

Highly selective and sensitive chromium(III) membrane sensors based on a new tridentate Schiff's base by Mohammad Reza Ganjali; P. Norouzi; F. Faridbod; M. Ghorbani; M. Adib (35-41).
In order to get a view about the tendency of N-(1-thien-2-ylethylidene)benzene-1,2-diamine (SNS), towards chromium and some other metal ions, theoretical calculations and conductance studies were carried out. Then, a highly selective and sensitive plasticized membrane sensor for chromium(III) ions, based on SNS as membrane carrier, was prepared. The sensor shows a linear dynamic range of 1.0 × 10−6 to 1.0 × 10−1  M with a Nernstian slope of 19.9 ± 0.3 mV decade−1 and a detection limit of 7.0 × 10−7  M (∼40 ppb). It has a fast response time of <12 s and can be used for at least eight weeks without any considerable divergences in its potentials. The proposed sensor revealed very good selectivities with respect to most of the common metal ions including Li, Na, K, Rb, Cs, Be, Mg, Ca, Cu, Co, Ni, Zn, Pb, Hg, Fe, La, Ce and Eu ions. The proposed sensor could be used in a pH range of 3.0–6.6. It was also used in the determination of Cr(III) in wastewater of chromium electroplating and leather industries, showing satisfactory results. The sensor was also applied for monitoring the chromium ion level in wastewater of chromate industries.
Keywords: S-N Schiff's base; Potentiometry; Chromium(III) sensor; PVC membrane;

Crown bridged thiacalix[4]arenes as cesium-selective ionophores in solvent polymeric membrane electrodes by Róbert Bereczki; Viktor Csokai; Alajos Grün; István Bitter; Klára Tóth (42-49).
Novel 1,3-alternate thiacalix[4]mono- and biscrown-6 ethers were studied as ionophores in poly(vinyl chloride) membrane electrodes. Their selectivity behavior was characterized with respect to large number of cations, including potential interferents in environmental samples, and the membrane composition was optimized for cesium ion response. Among the ionophores, 1,3-alternate thiacalix[4]mono(crown-6) ether showed, especially high selectivity for cesium over other alkali-metal ions. Transition and heavy metal ions did not interfere seriously with the electrode response, which indicates that the bridging sulfur atoms do not take part in the ion recognition process. The potentiometric cesium responses of all electrodes involved in this study were found close to Nernstian and the detection limits were lower than 10−7  M. The Cs+/Na+ selectivity of the different ionophore-based sensors and the solvent extraction ability of the ligands were interpreted based on the respective constants of complex formation.
Keywords: Potentiometric sensor; Cesium ion-selective electrode; Thiacalix[4]crown-6 ethers;

A simple method for the detection of individual groups of photosynthesis-inhibiting herbicides was proposed as a practical approach for environmental monitoring. The detection method is based on fluorescent measurement of the inhibition of spinach thylakoidal activity by herbicides. The sensor selectivity was improved by combining biosensor with a sample preconcentration based on a SPE cartridge packed with a polymer, imprinted with cyanazine. The combination of biosensor with molecularly imprinted polymer (MIP) allows selective measurement of triazines herbicides (cyanazine, atrazine) and their discrimination from phenylurea (diuron) or triazinone (metribuzin) herbicides. This combination also helps improving the sensitivity of the measurement allowing the detection of photosynthesis-inhibiting herbicides in water at the level required by European Union regulations.
Keywords: Molecularly imprinted polymer; Extraction; Herbicides; Photosynthetic biosensor; Fluorescence;

9-Vinyladenine was synthesized as a novel functional monomer for molecular imprinting techniques and its structure was established with elemental analysis and 1H NMR spectroscopy. The binding mechanism between this functional monomer 9-vinyladenine and the plant hormone 1H-indole-3-acetic acid in acetonitrile was studied with UV–vis spectrophotometry. Based on this study, using 1H-indole-3-acetic acid as a template molecule, a specific 9-vinyladenine-based molecularly imprinted polymeric membrane was prepared. Then, the resultant polymeric membrane morphologies were visualized with scanning electron microscopy, and the membrane permselectivity for 1H-indole-3-acetic acid, 1H-indole-3-butyric acid and kinetin was tested with separate experiments and competitive diffusion experiments. These results showed that the imprinted polymeric membrane prepared with 9-vinyladenine exhibited higher transport selectivity for the template molecule 1H-indole-3-acetic acid than 1H-indole-3-butyric acid or kinetin. The membrane prepared with 9-vinyladenine also took on higher permselectivity for 1H-indole-3-acetic acid in comparison with the imprinted membrane made with methacrylic acid. It is predicted that the 9-vinyladenine-based molecularly imprinted membrane may be applicable to the assay of 1H-indole-3-acetic acid or for the preparation of a molecularly imprinted polymer sensor for the analysis of 1H-indole-3-acetic acid in plant samples.
Keywords: Molecular imprinting; Transport selectivity; 1H-indole-3-acetic acid; 9-Vinyladenine;

A conductometric sensor for on-line testing of haloacetic acids has been developed based on lab-on-chip device incorporated with an integrated miniaturised liquid-handling system. The sensor utilises a molecularly imprinted polymer (MIP) synthesized by the interaction between trichloroacetic acid (TCAA) template and a functional monomer, 4-vinylpyridine (VPD), together with cross-linking polymerisation of ethylene glycol dimethacrylate (EDMA). The ability of this MIP to change its conductivity in the presence of the target molecule into the imprint cavity has been used to develop the sensor, which responds well to TCAA in a continuous flow system with relatively good linearity, although this depends on the applied frequency. Thermal influences on the resistance of the sensor were in the order of 1.45% resistance signal variation per Kelvin at 3 kHz. The sensor showed high specific sensitivity to the target analyte and a stable and reasonable signal response in a solution containing inorganic anions. The sensitivity (range 0.5–5 μg l−1) and selectivity achieved with standard TCAA and five other haloacetic acids (HAAs) (dichloro-, monochloro-, tribromo-, dibromo-, and monobromoacetic acid) in water was good. Minimum sample volume required is 2.5 ml and the assay time is 2 min. The sensor has successfully been applied to haloacetic acid determination in domestic and commercial drinking water samples.
Keywords: Molecularly imprinted polymers; Disinfection by-products; Charge transfer complex; Haloacetic acids; Flow-through electrochemical system;

Capture and release of viruses using amino-functionalized silica particles by Zilin Chen; Fu-Chih Hsu; David Battigelli; Hsueh-Chia Chang (76-82).
A new virus capture/release strategy for the concentration of viral particles in water is reported. The method is an improvement upon traditional approaches that rely exclusively upon electrostatic attractions between a charged substrate and charged viral particles, which can only be reversed under extremely acidic or alkaline conditions to effect surface charge reversal and subsequent release of captured viruses. This method utilizes negatively charged silica beads functionalized with amino groups using defined length spacer molecules to yield particles with a surface density optimized for efficient virus capture. Following capture, viruses can be released using soluble proteins or amino acid-based alkaline eluents. Virus recoveries are a function of the composition of elution solution used. The zeta potentials of amino-functionalized silica particles were analyzed and used to optimize the density of functionalized groups and the charge behavior of the functionalized silica surfaces. Raman spectrometry was used for the characterization of functionalized silica beads. This method is expected to apply in the analysis of viruses.
Keywords: Functionalized silica; Virus concentration; Water sample; Zeta potential; Raman spectrometry;

A new methodology to rapidly screen for pathogenic bacteria in various liquids (e.g., potable water and juice) is described. It combines the selectivity of dye-labeled antibodies, the sample concentration capability of solid phase membrane filtration, and the facile readout of the concentrated, dye-labeled microorganisms by diffuse reflectance spectroscopy (DRS). Details about the selection of the most effective membrane filter, detection of target bacteria in different types of liquids, and evaluation of assay specificity in screening for E. coli O157:H7 are discussed. For this pathogen, the technique has a working range of 5 × 105 to 5 × 108  cells/mL and an overall work up time of ∼45 min. The amount of captured bacteria is directly determined in only 2 s by using a hand-held DRS instrument via comparisons to a calibration curve based on the Kubelka–Munk function. Overall, this assay system offers high speed, simplicity, and low cost, making it a potential alternative for screening of several types of bacterial contaminated samples in almost any location.
Keywords: Rapid detection; Pathogenic bacteria; E. coli O157:H7; Solid phase extraction; Diffuse reflectance spectroscopy;

Acetone in human blood has been regarded as the diabetic biomarker. Acetone analysis is used as the accessorial diagnosis tool for diabetes. In this work, a simple, solvent-free and low-cost technique was developed for fast determination of acetone in blood samples, which was based on headspace single-drop microextraction and simultaneous derivatization followed by gas chromatography–mass spectrometry (GC–MS). Acetone in blood was headspace extracted by using the microdrop solvent containing a derivatization agent of O-(2,3,4,5-pentafluorobenzyl)hydroxylamine (PFBHA). The extracted acetone reacted with PFBHA in the microdrop, and rapidly formed acetone oxime. Finally, the derivative in the microdrop was detected by GC–MS. The parameters of headspace single-drop microextraction and simultaneous derivatization were studied, and the method validations were also studied. The proposed method was tested by the application to the determination of acetone in blood samples from controls and diabetic patients. The results show that headspace single-drop microextraction and simultaneous derivatization followed GC–MS is a simple, rapid, solvent-free and sensitive method for the determination of acetone in blood, and also a potential tool for diagnosis of diabetes.
Keywords: Gas chromatography–mass spectrometry; Acetone; Derivatization; Headspace single-drop microextraction; Diabetic biomarker;

An HPLC method with UV detection has been established for the simultaneous quantitative determination of five quinoxaline-1,4-dioxides (carbadox, olaquindox, cyadox, mequindox, quinocetone) in porcine, chicken and fish feeds. Feed samples were extracted with methanol/acetonitrile/water (35:35:30, v/v/v) in an ultrasonic bath, and purified by solid phase extraction (SPE) on Alumina N cartridges. The samples were analyzed on an Eclipse XDB C18 liquid chromatography column using a gradient program with methanol and water. Except for cyadox (>75%), recoveries of the drugs from feed samples spiked at 5, 50 and 200 mg kg−1 ranged from 92 to 104%. Coefficients of variation were 2–13%. The decision limits (CCα) for the five compounds were <0.45 mg kg−1, and the detection capability (CCβ) were <0.75 mg kg−1. Eight commercial feed samples have been analyzed using this method, and olaquindox was detected in three out of the eight samples. The rapid and simple method could be used for the determination of multi-residue of quinoxaline-1,4-dioxides in feed and other cereal samples at wide contamination levels (1–200 mg kg−1).
Keywords: Quinoxaline-1,4-dioxides; Feeds; Ultrasonic solvent extraction; HPLC; Alumina N cartridges;

A fast method for the extraction of polycyclic aromatic hydrocarbons, PAHs, in lichens used as biomonitors of air pollution is optimized and further applied to real lichen samples. The method consists of using the dynamic sonication-assisted solvent extraction. The lichen sample is introduced in a cell through which the extracting solvent pass at 0.2 mL/min. Sixteen PAHs were extracted from 0.2 g of dried lichens, Xanthoria parietina, in 10 min with a total extraction volume of 2 mL of hexane. The extracted fraction, previously concentrated to 500 μL, was analyzed by GC–MS without any clean-up step following extraction. Both spiked and non-spiked native samples were used for the evaluation. The procedure compared with the static ultrasonic and Soxhlet extraction, showed high efficiency with respect to both recoveries, time and solvent consumption and for all the 16 PAHs recoveries higher than 70% were obtained.The limit of detection of the investigated PAHs was in the range of 0.021–0.032 μg/g. The linear range of the entire method was 0.115–1.805 μg/g. for all the investigated PAHs. An application of the method was demonstrated with the extraction of lichens samples collected from an urban area. Twelve out of 16 investigated PAHs were found.
Keywords: Extraction; Dynamic sonication extraction; Analysis; PAHs; GC–MS; Lichens;

An optimised method using stir bar sorptive extraction (SBSE) for the determination of nine bromodiphenil ether (Lake Michigan mix) from water samples was developed. Recoveries near to 100% were found by using 20 mm × 0.5 mm (length × film thickness) PDMS commercial stir bars with 100 mL spiked water samples and 20% methanol addition and an extraction period of 25 h.Method sensitivity, linearity, repeatability and reproducibility have been also studied. Detection limits between 0.4 and 9.4 ng/L were calculated for the combination of SBSE and thermal desorption-GC–MS. The procedure was applied a real sample, a surface water contaminated by the effluents, and the results were compared with a classical liquid–liquid extraction method.
Keywords: PBDE; Stir bar sorptive extraction; Water analysis; Thermal desorption; GC–MS;

Analysis of liquid–liquid distribution constants of nonionizable crown ethers and their derivatives by Waldemar Robak; Wiesław Apostoluk; Paweł Maciejewski (119-131).
The linear solvation energy relationships (LSER) analysis of the distribution constants of the nonionizable crown ethers between aqueous phase and organic solvents has been carried out applying the cohesive energy density and dimensionless solvatochromic parameters of dipolarity/polarizability, hydrogen donating and accepting abilities of solvents. The molar intrinsic volume and hydrophile–lipophile balance in McGowan scale have been proposed as a descriptors of different crown ethers. The derived correlations permit to predict and/or estimate: (i) distribution constants of nonionizable crown ethers and their benzo derivatives in organic solvent/water systems; (ii) hydration number of crown ethers in the organic phase; (iii) thermodynamic functions followed the distribution of nonionizable crown ethers and their benzo derivatives in organic solvent/water systems; (iv) effect of ionic strength of the aqueous phase and properties of organic solvents on the distribution constants of dicyclohexano-18-crown-6, its alkyl derivatives and higher analogues in the two phase liquid systems.
Keywords: Crown ethers; Hydrophile–lipophile balance; Distribution constants; Effect of diluents; Kamlet and Taft model;

Sodium dodecyl sulphate (SDS)-coated alumina was used for the extraction/preconcentration of benzimidazolic pesticides (BFs) [carbendazim (CB), thiabendazole (TB) and fuberidazole (FB)] from river and underground water. SDS admicelles were required to get quantitative retention of BFs. Adsolubilization of analytes occurred through hydrophobic and electrostatic interactions. Methanol (1 ml) provided quantitative elution of the target analytes for all the samples analyzed. The high breakthrough volumes obtained (400 ml for CB and 1 l for BF and FB) and the low eluent volume used resulted in very high preconcentration factors (between 400 and 1000). Calcium was found to decrease BFs retention due to the disruption of SDS admicelles; however, this interference was easily removed by precipitation with SDS prior to BFs adsolubilization. The accuracy of the proposed method was assessed by studying recoveries of BFs in natural waters at two spiking concentration levels (80, 40 and 4 ng l−1, and 400, 200 and 20 ng l−1 for CB, TB and FB, respectively); mean recoveries in the intervals 96–105% and 98–108% were obtained for river and underground water samples, respectively.
Keywords: Hemimicelle/admicelle-based solid-phase extraction; Basic polar pesticides; Benzimidazolic fungicides; LC/fluorimetry;

Glycerol–silica gel: A new solid sorbent for preconcentration and determination of traces of cobalt(II) ion by Afsaneh Safavi; Nasser Iranpoor; Narges Saghir; Safieh Momeni (139-144).
This paper reports on the synthesis and application of glycerol-bonded silica gel as a new, very stable, inexpensive and easily prepared solid sorbent for effective preconcentration of traces of Co(II) in aqueous solutions. The synthetic route for immobilizing glycerol on silica gel is a simple two-step process. Factors influencing the sorption and desorption of cobalt ion have been investigated. Aqueous sodium citrate solution has been used as eluent for desorbing cobalt ions. The amount of cobalt, which is then eluted is measured using flame atomic absorption spectroscopy. The effect of coexisting ions on percent recovery of cobalt showed no interference from most of the ions tested. The proposed method permitted large enrichment factors of about 500 and higher. The maximum capacity of the sorbent was found to be 250 μg of cobalt per gram of sorbent. The precision of the method was 1.58% (n  = 10). The method was applied to the determination of traces of Co(II) in NaCl salt (Merck) and in water samples.
Keywords: Silica-bonded glycerol; Solid-phase extraction; Cobalt(II); Water;

A sensitive microbore liquid chromatographic method was developed for analysis of protein-unbound levamisole in blood and brain of anesthetized rats. Microdialysis probes were simultaneously inserted into the jugular vein toward right atrium and the striatum of brain of the male anesthetized Sprague–Dawley rats for biological fluid sampling after the administration of levamisole (3 mg/kg, i.v.) through the femoral vein. Assay of unbound levamisole was carried out using a microbore Phenyl-Hexyl column (150 mm × 1 mm i.d.; particle size 5 μm). The mobile phase consisted of methanol–10 mM 1-octanesulfonic acid which was adjusted to pH 3.0 with orthophosphoric acid (52:48, v/v) and the flow-rate is 0.05 ml/min. The assay was performed by monitoring the wavelength of maximum UV absorbance at 213 nm. The retention time of levamisole in rat blood and brain dialysates were found to be 10.5 min. The present assay enhanced the detection sensitivity and enabled the determination of levamisole at 50 ng/ml. Average in vivo recoveries of blood and brain dialysate were 46 ± 4 and 12 ± 4%, respectively, at concentration of 1, 5, 10 μg/ml. The result indicates that this method was suitable to investigate the disposition of levamisole and its penetration into blood–brain barrier (BBB).
Keywords: Levamisole; Liquid chromatography; Microdialysis; Pharmacokinetics;

We report use of β-cyclodextrin (CD)-hexamethylene diisocyanate (HMDI) copolymer(CDPU)-coated zirconia (CDPUZ) as a chiral stationary phase for separation of enantiomers of a set of 2,4-dinitrophenyl (DNP) amino acids in reversed-phase liquid chromatography. The effects of polymer loading on zirconia and HMDI/CD molar ratio in the preparation of CDPU on retention and selectivity in the separation of DNP-amino acids in were examined to find optimum polymer loading and HMDI/CD ratio. It was observed that 8% loading of the CDPU prepared with the HMDI/CD molar ratio of 4 gave the best separation of the amino acids investigated. Retention and chiral selectivity of CDPUZ were also compared to those for previously reported carboxymethyl-β-CD-coated zirconia (CMCDZ). CDPUZ gave better chiral separation than CMCDZ for the DNP-amino acids.
Keywords: β-Cyclodextrin-hexamethylene diisocyanate copolymer-coated zirconia; Chiral stationary phase; RPLC separation; Racemic DNP-amino acids;

Microcystins, a group of cyclic heptapeptide heaptoxins and tumor promoters, are bio-generated by cyanobacteria occurring in eutrophic lakes, rivers and reservoirs. In the present work, a novel liquid chromatography electrospray ionization (ESI-MS) tandem triple quadrupole mass spectrometry method was developed to determine the trace amounts of major microcystin variants in waters. Solid phase extraction (SPE) consuming 10 mL of water samples was used for sample cleaning-up and analyte enrichment. A reverse phase LC separation system consisting a symmetry300 C-18 column (4.6 mm × 75 mm i.d., 3.5 μm, pore size 300 Å) and binary gradient methanol-water mobile phase was applied for the separation of microcystin variants (MC-RR, -LR, -LW and -LF). Formic acid was spiked into the mobile phase to enhance the ionization efficiency. Tandem MS/MS analysis was performed in multi-reaction monitoring mode (MRM). Product-ion traces at m/z 519.9 → 135.0 for MC-RR, 498.4 → 135.0 for MC-LR, 1025.8 → 891.7 for MC-LW and 986.8 → 852.5 for MC-LF was used for quantification of the corresponding microcystin variants, and trace at m/z 556.1 → 278.0 used for internal standard enkephalin. Limits of quantification (LOQs) were 0.16, 0.04, 2.0, 1.0 and 0.10 μg/L for MC-RR, MC-LR, MC-LW, MC-LF and enkephalin, respectively. Intra- and inter-day precisions for the determination of the four variants were better than 5 and 6% in relative standard deviations (R.S.D.s), and recoveries for the four variants were in the range of 95–105%. The developed approach has been applied for the determination of the trace amounts of miccocystins in water samples of Taihu Lake of eastern China. The results showed that MC-RR and MC-LR were major variants detected in the water samples, and their concentrations significantly increased with the season changing from Spring to Summer.
Keywords: Microcystins; Liquid chromatography; Triple quadrupole mass spectrometry; Water analysis;

An HPLC/ESI-MS method for the simultaneous determination of taurine and 10 water-soluble vitamins including vitamin B1 (thiamine), B2 (riboflavin), B5 (pantothenic acid), B6 (pyridoxine, and pyridoxal), B8 (biotin), B9 (folic acid), C (ascorbic acid) and PP (nicotinamide and nicotinic acid) in multivitamin tablets was developed and validated. The separation was accomplished on a Johnson Spherigel C18 (250 mm × 4.6 mm) reversed phase column with methanol in an aqueous solution of heptafluorobutyric acid (5 mM) as mobile phase under gradient elution mode. Detection of target components was by ESI-MS switching continuously from positive ion mode (PI) to negative ion mode (NI). Hippuric acid was used as an internal standard for quantification. Sensitivity, precision and accuracy were determined.
Keywords: Water-soluble vitamins; Taurine; Hippuric acid; HPLC/ESI-MS; Multivitamin tablets;

Monitoring of proteinase activation in cell apoptosis by capillary electrophoresis with bioengineered fluorescent probe by Shixia Zhou; Juqiang Lin; Wei Du; Zhihong Zhang; Qingming Luo; Bi-Feng Liu; Yiqun Dai (176-181).
In this article, capillary electrophoresis (CE) was demonstrated as a novel means to monitor the activation of proteinase during cell apoptosis. A unique fluorescent probe that fused a specific amino acid sequence DEVD with two fluorescent proteins enhanced cyan fluorescent protein (ECFP) and red fluorescent protein (DsRed), ECFP-DEVD-DsRed, was engineered in HeLa cells as a substrate of proteinase caspase-3. Molecular imaging in vivo was conducted to evaluate the stability and reliability of this fusion protein probe expressed in HeLa cells. With treatment of a certain dose of cisplatin to HeLa cells, apoptosis was initiated, and then caspase-3 was activated that could specifically recognize the DEVD site and would subsequently cleave the constructed fluorescent probe into two pieces of protein fragments. Analyzing the cell lysates with CE in vitro at a series of time points gave a clear description of activation process of caspase-3 in cell apoptosis. Several parameters that influenced CE separation quality were optimized, such as buffer type, pH value, concentration and applied voltage. The employment of two color fluorescent proteins significantly simplified the separation and identification of the residues of the cleavage reaction.
Keywords: Capillary electrophoresis; Protein activation; Caspase-3; Cell apoptosis;

A new analytical method for d-alanine (d-Ala) based on capillary electrophoretic separation and optical fiber light-emitting diode (LED)-induced fluorescence detection has been developed. Fluorescein isothiocyanate (FITC) was used for precolumn derivatization of amino acids. The separation and determination of the derivatives was performed using a novel laboratory-built CE system with optical fiber LED-induced fluorescence detector. Optimal separation was performed at 15 kV using a background electrolyte solution consisted of 25 mmol L−1 sodium borate (pH 9.5), 18 mmol L−1 β-CD and 15 mmol L−1 SDS. High sensitive detection was obtained by the optical fiber LED-induced fluorescence detection using a blue LED as the excitation source. The limits of detection (S/N  = 3) for d-Ala was 8.0 × 10−9  mol L−1. A calibration curve ranging from 2.0 × 10−8  mol L−1 to 1.0 × 10−6  mol L−1 was shown to be linear. Using this method, the levels of d-Ala in human plasma from healthy donors were determined.
Keywords: Capillary electrophoresis; Light-emitting diode; d-Alanine; Human plasma;

The strong interactions between poly(dimethylsiloxane) (PDMS) surface and amino acids result in unavoidable adsorption on channel surface. This paper described a simple method to decrease adsorption of analytes by dynamic coating of the PDMS surface. When nonionic surfactant (Tween 20) was added in running buffer as dynamic modifier, electroosmotic flow (EOF) was well-suppressed. Moreover, arginine (Arg), proline (Pro), histidine (His) and threonine (Thr) were successfully separated within 80 s in a 3.7 cm long separation channel and then detected with an end-channel amperometric detection mode at a copper electrode. Under the optimal conditions, the linear ranges of Arg, Pro, His and Thr were all from 50 to 600 μM with detection limits of 13.6, 15.3, 10.6 and 12.8 μM, respectively. The relative standard deviations (R.S.D.s) of run-to-run, day-to-day and chip-to-chip on the coated PDMS/PDMS microchip were all below 2.4%. Tween 20 dynamic coating provides reproducible amino acids separations with good performances.
Keywords: Poly(dimethylsiloxane); Dynamic coating; Microchip electrophoresis; Nonionic surfactant; Electrochemical detection;

Extraction of C-reactive protein from serum on a microfluidic chip by Michael G. Roper; Megan L. Frisk; Janeen P. Oberlander; Jerome P. Ferrance; Brian J. McGrory; James P. Landers (195-202).
The first extraction of a protein, C-reactive protein (CRP), from unadulterated serum on a microfluidic chip is demonstrated. Two stationary phases were evaluated for their ability to selectively extract this protein directly from serum without the need for additional cleanup steps. The first extraction media tested was an affinity matrix containing monoclonal antibodies to CRP, however, after immobilization this media failed to bind CRP. The more efficient stationary phase, immobilized phosphocholine, extracted CRP in a Ca2+-dependent manner. Significant non-specific binding by more abundant serum proteins was observed using typical washing protocols with this phase and these protocols were modified and optimized to yield extractions that were selective to phosphocholine-binding proteins. Extraction efficiencies were 40% for standard CRP solutions and run-to-run extraction reproducibility was 15% for 8 μL of total serum loaded. Serum concentrations of CRP determined by this method in patients who had undergone hip replacement surgery compared favorably with those values obtained by the conventional, currently utilized methods. Thus, this newly developed miniaturized method for protein purification from serum will provide a foundation for future extractions of protein biomarkers.
Keywords: Microchip; Serum; Extraction; Proteins;

Gold-coated magnetic nanoparticle (GMP) was manufactured and used as a carrier for the immobilization of hexa-arginine-tagged esterase (Arg6-esterase). The formation of gold shell on the magnetic nanoparticle (Fe2O3) was performed by an iterative reduction method using hydroxylamine as a reductant. The surface of the GMP was further functionalized with mercapto-hexadecanoic acid (MHA) to tether the positively charged Arg6-esterase effectively. The enzymatic activity of the immobilized Arg6-esterase was investigated by monitoring the dissociation rate of its colorimetric substrate, p-nitrophenol butyrate (pNPB). Although the immobilized enzyme exhibited a lower activity (∼60%) compared to the free one, its original activity was found to be retained even after seven times repetitive uses by magnetic decantation.
Keywords: Gold-coated magnetic nanoparticle (GMP); Hexa-arginine-tagged esterase (Arg6-esterase); Magnetic separation; Enzyme immobilization;

Quantification of analytes affected by relevant interfering signals under quality controlled conditions by Ricardo J.N. Bettencourt da Silva; Júlia R. Santos; M. Filomena G.F.C. Camões (210-220).
The analysis of organic contaminants or residues in biological samples is frequently affected by the presence of compounds producing interfering instrumental signals. This feature is responsible for the higher complexity and cost of these analyses and/or by a significant reduction of the number of studied analytes in a multi-analyte method.This work presents a methodology to estimate the impact of the interfering compounds on the quality of the analysis of complex samples, based on separative instrumental methods of analysis, aiming at supporting the inclusion of analytes affected by interfering compounds in the list of compounds analysed in the studied samples.The proposed methodology involves the study of the magnitude of the signal produced by the interfering compounds in the analysed matrix, and is applicable to analytical systems affected by interfering compounds with varying concentration in the studied matrix. The proposed methodology is based on the comparison of the signals from a representative number of examples of the studied matrix, in order to estimate the impact of the presence of such compounds on the measurement quality. The treatment of the chromatographic signals necessary to collect these data can be easily performed considering algorithms of subtraction of chromatographic signals available in most of the analytical instrumentation software. The subtraction of the interfering compounds signal from the sample signal allows the compensation of the interfering effect irrespective of the relative magnitude of the interfering and analyte signals, supporting the applicability of the same model of the method performance for a broader concentration range. The quantification of the measurement uncertainty was performed using the differential approach, which allows the estimation of the contribution of the presence of the interfering compounds to the quality of the measurement.The proposed methodology was successfully applied to the analysis of pesticide residues in spiked oranges considering the quantification of the oranges ethyl acetate extract by gas-chromatography with electron capture detector. The application of the proposed methodology to the analysis of this fruit using the studied chromatographic system, allowed the quantification of an increased number of analytes in the samples.The magnitude of the measurement uncertainty estimated by the proposed methodology is fit for the purpose of monitoring pesticide residues in oranges since, frequently, the difference between the maximum residue level and the best estimation of the sample content is larger than the respective uncertainty.The proposed methodology can be useful in other analytical fields and/or instrumental methods of analysis.
Keywords: Interferences; Uncertainty; Separative techniques; Quality; Pesticides; Foodstuffs;

Paired emitter-detector light emitting diodes for the measurement of lead(II) and cadmium(II) by King-Tong Lau; Eimear McHugh; Susan Baldwin; Dermot Diamond (221-226).
A transmittance mode optical device based on using a reverse biased light emitting diode (LED) as light detector has been developed for colorimetric analysis. This new optical device was validated with bromocresol green dye for absorbance measurements before being employed for detecting cadmium(II) and lead(II) in water. Results show that the performance of this LED-based device is comparable to much more expensive bench top UV–vis instruments, but with the advantages of being low cost, low power and simple to operate.
Keywords: Paired light emitting diode (LED) detector; Cadmium(II); Lead(II); Colorimetric analysis; Optical sensing; Environmental monitoring;

A method for the removal of the interference caused by iron on electrochemical generation of stibine is proposed. It consists of a chelating resin Chelex 100 column integrated into a flow injection system and coupled to the electrochemical hydride generator quartz tube atomic absorption spectrometer (EcHG-QT-AAS). Iron, as Fe(II), is retained in the column with high efficiency, close to 99.9% under optimal conditions. No significant retention was observed for Sb(III) under same conditions and a 97 ± 5% signal recovery was achieved. An electrochemical hydride generator with a concentric configuration and a reticulated vitreous carbon cathode was employed. The system is able to determine antimony concentrations in the range of ng ml−1 in presence of iron concentrations up to 400 mg l−1. The procedure was validated by analyzing PACS-2 marine sediments reference material with a 4% (w/w) iron content and a [Fe]:[Sb] ratio of 4000:1, which caused total antimony signal suppression on the electrochemical hydride generation system. A compost sample with high iron content (0.7%, w/w), was also analyzed. A good agreement was found on both samples with the certified value and the antimony concentration determined by ICP-MS, respectively.
Keywords: Electrochemical hydride generation; On-line iron removal; Stibine; Chelating resins;

Direct identification of alizarin and lac dye on painting fragments using surface-enhanced Raman scattering by Kui Chen; Kim-Chi Vo-Dinh; Fei Yan; Musundi B. Wabuyele; Tuan Vo-Dinh (234-237).
A surface-enhanced Raman scattering (SERS) based procedure was investigated for potential applications in the direct identification of selected anthraquinone dyes on works of art objects. The simplicity and effectiveness of this procedure was demonstrated in a proof-of-concept experiment. A microscopic fragment containing alizarin or lac dye was removed from a painting. A layer of silver nanoparticles was thermally evaporated directly on the fragment to induce SERS effect. SERS spectra were collected directly from the Ag-nanoparticle-coated sample fragments with a Raman microscope. Based on their characteristic SERS spectra, the presence of alizarin or lac dye in the sample fragments can be detected. The applicability of this procedure for potential applications in minimally invasive analysis of color layer from artwork objects is discussed. Finally, the thickness of the Ag nanoparticle layer was optimized.
Keywords: Surface-enhanced Raman scattering; Alizarin; Painting; Art and archaeology;

Development of a simple and low cost device for vapour phase Fourier Transform Infrared spectrometry determination of ethanol in mouthwashes by Sergio Armenta; Francesc A. Esteve-Turrillas; Guillermo Quintás; Salvador Garrigues; Agustín Pastor; Miguel de la Guardia (238-243).
A new vapour phase manifold coupled with Fourier Transform Infrared (FTIR) spectroscopy was developed for ethanol determination in mouthwashes. Two microliters of samples were injected, without any previous pre-treatment into a reactor heated at 70 °C, and the vapour phase generated transported to the FTIR spectrometer using a carrier nitrogen flow of 6 ml min−1. FTIR spectra were continuously recorded, as a function of time, by accumulating two scans and employing 8 cm−1 nominal resolution. Analytical measurements for ethanol were made in the range from 1130 to 992 cm−1 with a baseline defined between 1158 and 957 cm−1. After ethanol measurement the carrier flow was increased to 300 ml min−1 in order to put out of the manifold the water evolved from samples. Several commercial mouthwash samples were analysed by reference methods as titrimetry and headspace gas chromatography and results were comparable with those obtained by vapour phase FTIR. The proposed procedure is a simple, fast and environmentally friendly alternative to the reference methods providing accurate results, with a limit of detection of 0.15% (v/v) and a total analysis time of 2 min.
Keywords: Ethanol; Mouthwashes; Fourier Transform Infrared spectrometry; Vapour phase;

A method based on the peak obtained by adsorptive stripping voltammetry for the complex formed by organic matter in the presence of trace amounts of molybdenum(VI) has been tested for the determination of refractory organic matter, the so-called humic substances, in freshwaters. The method is rapid and particularly well-adapted to the determination of low amounts of refractory organic matter. It has a detection limit of 2.4 μg C L−1 for 3 min deposition time. The effect of the presence of other types of natural organic matter has been tested. The method has been applied to a wide variety of freshwaters at different trophic states.
Keywords: AdSV; Fulvic acids; Freshwaters; Humic acids; Molybdenum(VI); Refractory organic matter;

A new spectrofluorimetric method has been developed for the quantitative determination of the anti-inflammatory flufenamic and meclofenamic acids in urine samples. The method is based on second-order multivariate calibration applying parallel factor analysis (PARAFAC). The fluorescence excitation–emission matrices (EEMs) of these compounds are highly overlapped, precluding analyte direct determination without performing a previous separation procedure. The use of a micellar medium allowed for both fluorescence enhancement and analyte discrimination from the biological background. The calibration solutions were prepared in water, with concentrations in the ranges from 0.15 to 4.00 μg ml−1 for flufenamic acid and from 0.25 to 6.00 μg ml−1 for meclofenamic acid. The use of the second-order calibration method, in the standard addition mode, was required to avoid internal filter interference effects from the urine background. The measured EEMs of the two analytes as analytical signals allowed their simultaneous determination in human urine samples, in the therapeutic ranges from ca. 40 to 150 mg l−1. The proposed procedure was validated by comparing the obtained results with a capillary electrophoresis method, with satisfactory results for the assayed method.
Keywords: Second-order multivariate calibration; PARAFAC; Excitation–emission matrices; Flufenamic acid; Meclofenamic acid; Urine; Micellar media;

Statistical intervals to validate an autoanalyzer for monitoring the exhaustion of alkaline degreasing baths by E. Trullols; I. Ruisánchez; E. Aguilera; R. Lucena; S. Cárdenas; M. Valcárcel (260-266).
We describe how to use the statistical intervals for validating a qualitative method for determining the alkaline degreasing baths exhaustion. A homemade autoanalyzer based on flow-injection-evaporative light-scattering detector (FI-ELSD) coupling measures two instrumental responses related to the contents of surfactant and mineral oil. These two responses are necessary to decide whether the degreasing bath is exhausted. The instrumental responses r i are compared to their corresponding decision values i.e. cut-off response (r cut-off) and screening response (r screening). These decision values are calculated by defining the one-side prediction bound around the specification limit (SL) of both analytes. The prediction bound of each analyte must be defined differently according to their corresponding specification limit. Performance parameters, such as sensitivity, specificity, false response rates and the unreliability region, are established. The performance of this qualitative method of analysis is checked by analyzing a set of 10 real samples. Our results show that the method is accurate as far as mineral oil content is concerned.
Keywords: Statistical intervals; Flow-injection-evaporative light-scattering detector coupling; Degreasing baths; Validation;

Rank annihilation factor analysis (RAFA) was used to the spectrophotometric studies of acid–base equilibria in the presence of β-cyclodextrin (β-CD). When the conditional acidity constants (β-CD concentration dependent acidity constant) acts as an optimizing object, and simply combined with the pure spectrum of acidic and basic forms, the rank of original data matrix can be reduced. The residual standard deviation (R.S.D.) of the residual matrix after bilinearization of the background matrix is regarded as the valuation function. The performance of the method has been evaluated by using synthetic data. Spectrophotometric studies of neutral red, 4-nitrophenol and 4-nitrocatechol in the presence of varying amounts of β-CD are used as experimental model systems and the amounts of conditional acidity constants and formation constants of the inclusion complexes of β-CD with acidic and basic forms are calculated. The conditional acidity constants increased by increasing β-CD concentration for all three investigated acids. The calculated stability constants show that 4-nitrophenolate, 4-nitrocatecholate anions and basic form of neutral red (neutral form) form more stable inclusion complexes with β-CD than their acidic forms.
Keywords: Conditional acidity constant; Spectrophotometry; β-cyclodextrin; RAFA;

Author Index (277-279).