Analytica Chimica Acta (v.584, #2)

Contents (iii-iv).

Continuous flow microfluidic device for cell separation, cell lysis and DNA purification by Xing Chen; Dafu Cui; Changchun Liu; Hui Li; Jian Chen (237-243).
A novel integrated microfluidic device that consisted of microfilter, micromixer, micropillar array, microweir, microchannel, microchamber, and porous matrix was developed to perform sample pre-treatment of whole blood. Cell separation, cell lysis and DNA purification were performed in this miniaturized device during a continuous flow process. Crossflow filtration was proposed to separate blood cells, which could successfully avoid clogging or jamming. After blood cells were lyzed in guanidine buffer, genomic DNA in white blood cells was released and adsorbed on porous matrix fabricated by anodizing silicon in HF/ethanol electrolyte. The flow process of solutions was simulated and optimized. The anodization process of porous matrix was also studied. Using the continuous flow procedure of cell separation, cell lysis and DNA adsorption, average 35.7 ng genomic DNA was purified on the integrated microfluidic device from 1 μL rat whole blood. Comparison with a commercial centrifuge method, the miniaturized device can extract comparable amounts of PCR-amplifiable DNA in 50 min. The greatest potential of this integrated miniaturized device was illustrated by pre-treating whole blood sample, where eventual integration of sample preparation, PCR, and separation on a single device could potentially enable complete detection in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.
Keywords: Microfluidic; Crossflow filtration; Cell lysis; DNA purification; MEMS;

A simple method to fabricate cylindrical carbon electrodes for use in capillary electrophoresis (CE) microchips is described. The electrodes were fabricated using a metallic wire coated with carbon ink. Several experimental variables were studied in order to establish the best conditions to fabricate the electrode. Finally, the electrodes were integrated in a poly(dimethylsiloxane) microchip and used for the analysis of phenolic compounds. Using the optimum conditions, the analysis of a mixture of dopamine, epinephrine, catechol, and 4-aminophenol was achieved in less than 240 s, showing good linear responses (R 2  = 0.999) in the 0.1–190 μM range, and limits of detection (without the use of stacking or a decoupler) of 140 and 105 nM for dopamine and epinephrine, respectively.
Keywords: Phenolic compounds; Carbon electrodes; Microchip; Capillary electrophoresis; Electrochemical detection;

Latex immunoagglutination assay in a microfluidic device is expected to be even easier than its large-sized, commercialized counterpart. However, such demonstration has had a limited success due to the difficulties in mixing in a microfluidic device, especially for the microparticles used in latex immunoagglutination assay. The primary goal of this work is to improve diffusional mixing towards the successful latex immunoagglutination in a microfluidic devices without any non-specific binding. To this end, SDS (sodium dodecyl sulfate, an ionic surfactant) or Tween 80 (polyethylene sorbitol ester, a non-ionic surfactant) was added to the antibody-conjugated polystyrene (PS) microparticle suspension. These surfactant-added particle suspensions were mixed with the target antigen solution at the Y-junction of a microfluidic device. The immunoagglutination and the diffusion behavior were visually identified with an inverted light microscope. Both surfactants showed some problems such as non-specific binding (with SDS) or very poor diffusion (with Tween 80). As an alternative approach, therefore, highly carboxylated PS microparticles, where the surface is saturated with carboxyl-terminated side chains, were evaluated without using any surfactants. These particles showed very low non-specific binding comparable to that with Tween 80 and good diffusional mixing equivalent to that with SDS.
Keywords: Latex immunoagglutination assay; Diffusional mixing; Microfluidic device; Non-specific binding;

Sensitive luminescent determination of DNA using the terbium(III)–difloxacin complex by Alla V. Yegorova; Yulia V. Scripinets; Axel Duerkop; Alexander A. Karasyov; Valery P. Antonovich; Otto S. Wolfbeis (260-267).
The interaction of the terbium–difloxacin complex (Tb–DFX) with DNA has been examined by using UV–vis absorption and luminescence spectroscopy. The Tb–DFX complex shows an up to 85-fold enhancement of luminescence intensity upon titration with DNA. The long decay times allow additional detection schemes like time-resolved measurements in microplate readers to enhance sensitivity by off-gating short-lived background luminescence. Optimal conditions are found at equimolar concentrations of Tb3+ and DFX (0.1 or 1 μM) at pH 7.4. Under these conditions, the luminescence intensity is linearly dependent on the concentration of ds-DNAs and ss-DNA between 1–1500 ng mL−1 and 4.5–270 ng mL−1, respectively. The detection limit is 0.5 ng mL−1 for ds-DNAs and 2 ng mL−1 for ss-DNA. The mechanism for the luminescence enhancement was also studied.
Keywords: UV–vis spectrometry; Luminescence; Terbium; Quinolones; Difloxacin; DNA;

Electrical detection of deoxyribonucleic acid hybridization based on carbon-nanotubes/nano zirconium dioxide/chitosan-modified electrodes by Yunhui Yang; Zhijie Wang; Minghui Yang; Jishan Li; Fang Zheng; Guoli Shen; Ruqin Yu (268-274).
A novel and sensitive electrochemical DNA biosensor based on nanoparticles ZrO2 and multi-walled carbon nanotubes (MWNTs) for DNA immobilization and enhanced hybridization detection is described. The MWNTs/nano ZrO2/chitosan-modified glassy carbon electrode (GCE) was fabricated and oligonucleotides were immobilized to the GCE. The hybridization reaction on the electrode was monitored by differential pulse voltammetry (DPV) analysis using electroactive daunomycin as an indicator. Compared with previous DNA sensors with oligonucleotides directly incorporated on carbon electrodes, this carbon nanotube-based assay with its large surface area and good charge-transport characteristics increased DNA attachment quantity and complementary DNA detection sensitivity. The response signal increases linearly with the increase of the logarithm of the target DNA concentration in the range of 1.49 × 10−10 to 9.32 × 10−8  mol L−1 with the detection limit of 7.5 × 10−11  mol L−1 (S/N  = 3). The linear regression equation is I  = 32.62 + 3.037 log  C DNA (mol L−1) with a correlation coefficient value of 0.9842. This is the first application of carbon nanotubes combined with nano ZrO2 to the fabrication of an electrochemical DNA biosensor with a favorable performance for the rapid detection of specific hybridization.
Keywords: Nanoparticles zirconium dioxide; Multi-walled carbon nanotubes; Hybridization; Electrochemical deoxyribonucleic acid biosensor; Daunomycin;

A sensitive method for the analysis of propoxur in environmental samples has been developed. It involves an automated solid-phase extraction (SPE) procedure using a Gilson Aspec XLi and flow-injection analysis (FI) with chemiluminescence (CL) detection. The FI–CL system relies on the photolysis of propoxur by irradiation using a low-pressure mercury lamp (main spectral line 254 nm). The resultant methylamine is subsequently detected by CL using tris(2,2′-bipyridyl)ruthenium(III), which is on-line generated by photo-oxidation of the ruthenium(II) complex in the presence of peroxydisulfate. The linear concentration range of application was 0.05–5 μg mL−1 of propoxur, with a detection limit of 5 ng mL−1. The repeatability was 0.82% expressed as relative standard deviation (n  = 10) and the reproducibility, studied on 5 consecutive days, was 2.1%. The sample throughput was 160 injection per hour.Propoxur residues below ng mL−1 levels could be determined in environmental water samples when an SPE preconcentration device was coupled on-line with the FI system. This SPE–FI–CL arrangement provides a detection limit as low as 5 ng L−1 using only 500 mL of sample. In the analysis of fruits and vegetables, the detection limit was about 10 μg kg−1.
Keywords: Propoxur; Tris(2,2′-bipyridyl)ruthenium(III) photogenerated; Flow-injection; Automated solid-phase extraction; Environmental samples;

Ultra-trace determination of beryllium in occupational hygiene samples by ammonium bifluoride extraction and fluorescence detection using hydroxybenzoquinoline sulfonate by Kevin Ashley; Anoop Agrawal; John Cronin; Juan Tonazzi; T. Mark McCleskey; Anthony K. Burrell; Deborah S. Ehler (281-286).
A highly sensitive molecular fluorescence method for measuring ultra-trace levels of beryllium has been previously described. The method entails extraction of beryllium workplace samples by 1% ammonium bifluoride (NH4HF2, aqueous), followed by fluorescence detection using hydroxybenzoquinoline sulfonate (HBQS). In this work, modification of the existing procedure resulted in a significant improvement in detection power, thereby enabling ultra-trace determination of beryllium in air filter and surface wipe samples. Such low detection limits may be necessary in view of expected decreases in applicable occupational exposure limits (OELs) for beryllium. Attributes of the modified NH4HF2 extraction/HBQS fluorescence method include method detection limits (MDLs) of <0.8 ng to ≈2 ng Be per sample (depending on the fluorometer used), quantitative recoveries from beryllium oxide, a dynamic range of several orders of magnitude, and freedom from interferences. Other key advantages of the technique are field portability, relatively low cost, and high sample throughput. The method performance compares favorably with that of inductively coupled plasma-mass spectrometry (ICP-MS).
Keywords: Air monitoring; Beryllium; Extraction; Fluorescence; Trace analysis; Workplace;

The present article describes the simultaneous phosphorimetric determination of pyrene and benzo[a]pyrene, two highly toxic polycyclic aromatic hydrocarbons, through excitation–emission phosphorescence matrices (EEPMs) and second-order calibration. The developed approach enabled us to determine both compounds at μg L−1 concentration levels without the necessity of applying separation steps, as well as significantly reducing the experimental time. An artificial neural network (ANN) approach was applied to optimize the chemical variables which have an influence on the room-temperature phosphorescence emission of the studied analytes. The present study was employed for the discussion of the scopes of the applied second-order chemometric tools: parallel factor analysis (PARAFAC) and partial least-squares with residual bilinearization (PLS/RBL). The superior capability of PLS/RBL to model the profiles of other potentially interferent polycyclic aromatic hydrocarbons (PAHs) was demonstrated. The quality of the proposed method was established with the determination of both pyrene and benzo[a]pyrene in artificial and real water samples.
Keywords: Room-temperature phosphorescence; Parallel factor analysis; Partial least-squares; Residual bilinearization; Pyrene; Benzo[a]pyrene;

A solid paraffin-based carbon paste electrode modified with 2-aminothiazole organofunctionalized silica (SiAt-SPCPE) was applied to Ni2+ determination in commercial ethanol fuel samples. The proposed method comprised four steps: (1) Ni2+ preconcentration at open circuit potential directly in the ethanol fuel sample, (2) transference of the electrode to an electrochemical cell containing DMG, (3) differential pulse voltammogram registering and (4) surface regeneration by polishing the electrode. The proposed method combines the high Ni2+ adsorption capacity presented by 2-aminothiazole organofunctionalized silica with the electrochemical properties of the Ni(DMG)2 complex, whose electrochemical reduction provides the analytical signal.All experimental parameters involved in the proposed method were optimized. Using a preconcentration time of 20 min, it was obtained a linear range from 7.5 × 10−9 to 1.0 × 10−6  mol L−1 with detection limit of 2.0 × 10−9  mol L−1. Recovery values between 96.5 and 102.4% were obtained for commercial samples spiked with 1.0 μmol L−1 Ni2+ and the developed electrode was totally stable in ethanolic solutions. The contents of Ni2+ found in the commercial samples using the proposed method were compared to those obtained by graphite furnace atomic absorption spectroscopy by using the F- and t-test. Neither the F- nor t-values exceeded the critical values at 95% confidence level, confirming that there are not statistical differences between the results obtained by both methods. These results indicate that the developed electrode can be successfully employed to reliable Ni2+ determination in commercial ethanol fuel samples without any sample pretreatment or dilution step.
Keywords: Nickel; Ethanol fuel; Solid paraffin-based carbon paste electrodes; Organofunctionalized silica; 2-Aminothiazole; Differential pulse stripping voltammetry;

A novel potentiometric zirconium – PVC matrix membrane sensor incorporating bis(diphenylphosphino) ferrocene as an electroactive material and tris(2-ethylhexyl)phosphate as solvent mediator is described. In mixed acetate buffer solution of pH 4.8, the sensor displays a rapid and linear response for zirconium ion over the concentration range 1.0 × 10−1 to 1.0 × 10−7  mol L−1 with a good slope of 59.7 ± 0.3 mV per decade and detection limit 1.8 × 10−8  mol L−1. The best performance was obtained with membrane composition 33% PVC, 65% TEHP, 1% NaTPB and 1% ionophore. The proposed electrode revealed excellent selectivity for zirconium ion over a wide variety of alkali, alkaline earth, transition and heavy metal ions and could be used in a pH range of 4.15–7.8. The electrode was applied for at least 1 month without any considerable divergence in the potential responses. The practical utility of the electrode has been demonstrated by its use as an indicator electrode in the potentiometric titration of zirconium ions with sodium fluoride and in determination of zirconium ion in some alloy, tape and waste water samples.
Keywords: Ion selective electrode; Zirconium; Potentiometry; Bis(diphenylphosphino) ferrocene;

In this work photoluminescent properties of highly Cu2+ selective organic fluoroionophore, semicarbazone derivative; bis(naphtho[2,1-b]furan-2-yl)methanone semicarbazone (BNF) was investigated in different solvents (dichloromethane, tetrahydrofuran, toluene and ethanol) and in polymer matrices of polyvinylchloride (PVC) and ethyl cellulose (EC) by absorption and emission spectrometry. The BNF derivative displayed enhanced fluorescence emission quantum yield, Q f  = 6.1 × 10−2 and molar extinction coefficient, ɛ  = 29,000 ± 65 cm−1  M−1 in immobilized PVC matrix, compared to 2.6 × 10−3 and 24,573 ± 115 in ethanol solution.The offered sensor exhibited remarkable fluorescence intensity quenching upon exposure to Cu2+ ions at pH 4.0 in the concentration range of 1.0 × 10−9 to 3.0 × 10−4  M [Cu2+] while the effects of the responding ions (Ca2+, Hg+, Pb2+, Al3+, Cr3+, Mn2+, Mg2+, Sn2+, Cd2+, Co2+ and Ni2+) were less pronounced.
Keywords: Semicarbazone derivative; Copper sensor; Fluorescence spectroscopy;

Determination of kynurenine levels in rat plasma by high-performance liquid chromatography with pre-column fluorescence derivatization by Shogo Mitsuhashi; Takeshi Fukushima; Masayuki Tomiya; Tomofumi Santa; Kazuhiro Imai; Toshimasa Toyo’oka (315-321).
Kynurenine (KYN), a tryptophan metabolite, is a crucial compound for modulating neurotransmission because it can be metabolized in vivo into both quinolinic acid and kynurenic acid, which are the agonist and antagonist, respectively, of N-methyl-d-aspartate receptor. For the highly sensitive detection of KYN by high-performance liquid chromatography (HPLC), a fluorescence derivatization of KYN with a benzofurazan-type fluorogenic reagent, 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was investigated in the present study. KYN was derivatized with DBD-F (DBD-KYN) at 60 °C for 30 min, and separated on an octadecylsilica column with a gradient elution of the mobile phase, which consists of 0.1% formic acid in acetonitrile/methanol/water. DBD-KYN was detected fluorimetrically at 553 nm with an excitation wavelength of 431 nm. The limits of detection and quantification were approximately 0.30 pmol [signal-to-noise ratio (S/N) 3] and 1.0 pmol (S/N, 10) on column, respectively. Plasma KYN levels were successfully determined using 10 μL of rat plasma with satisfactory precision and accuracy. Intra- and inter-day precisions and accuracies were 1.7–6.8%, and −10 to 9.6%, respectively. KYN levels in plasma of male Sprague–Dawley rats (7 weeks old) were approximately 2.4 ± 0.32 μmol L−1 (n  = 4). The proposed HPLC method was applied to determine KYN levels in the plasma of ketamine-treated rats—the animal model of schizophrenia.
Keywords: Kynurenine; 4-N,N-Dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F); Rat plasma; High-performance liquid chromatography; Fluorescence;

The present study developed two analytical methods for quantification of acrylamide in complex food matrixes, such as Chinese traditional carbohydrate-rich foods. One is based on derivatization with potassium bromate and potassium bromide without clean-up prior to gas chromatography with micro-electron capture detector (GC-MECD). Alternatively, the underivatized acrylamide was detected by high-performance liquid chromatography coupled to quadrupole tandem mass spectrometry (HPLC-MS/MS) in the positive electrospray ionization mode. For both methods, the Chinese carbohydrate-rich samples were homogenized, defatted with petroleum ether and extracted with aqueous solution of sodium chloride. Recovery rates for acrylamide from spiked Chinese style foods with the spiking level of 50, 500 and 1000 μg kg−1 were in the range of 79–93% for the GC-MECD including derivatization and 84–97% for the HPLC-MS/MS method. Typical quantification limits of the HPLC-MSMS method were 4 μg kg−1 for acrylamide. The GC-MECD method achieved quantification limits of 10 μg kg−1 in Chinese style foods. Thirty-eight Chinese traditional foods purchased from different manufacturers were analyzed and compared with four Western style foods. Acrylamide contaminant was found in all of samples at the concentration up to 771.1 and 734.5 μg kg−1 detected by the GC and HPLC method, respectively. The concentrations determined with the two different quantitative methods corresponded well with each other. A convenient and fast pretreatment procedure will be optimized in order to satisfy further investigation of hundreds of samples.
Keywords: Acrylamide; Chinese traditional carbohydrate-rich foods; Micro-electron capture detection; Liquid chromatography tandem mass spectrometry;

Trace determination of the flame retardant tetrabromobisphenol A in the atmosphere by gas chromatography–mass spectrometry by Zhiyong Xie; Ralf Ebinghaus; Rainer Lohmann; Olaf Heemken; Armando Caba; Wilhelm Püttmann (333-342).
A simple and effective method has been developed for analysis of the flame retardant tetrabromobisphenol A (TBBPA) in environmental samples by using modified soxhlet extraction in combination with silica gel clean-up, derivatization with silylation reagent and gas chromatography–mass spectrometry (GC–MS) in selected ion monitoring mode (SIM). Satisfactory recoveries were achieved for the large volume sampling, soxhlet extraction and silica gel clean-up. The overall recovery is 79 ± 1%. The derivatization procedure is simple and fast, and produces stable TBBPA derivative. GC–MS with electronic impact (EI) ionization mode shows better detection power than using negative chemical ionization (NCI) mode. EI gives a method detection limit of 0.04 pg m−3 and enables to determine trace TBBPA in ambient air in remote area. The method was successfully applied to the determination of TBBPA in atmospheric samples collected over land and coastal regions. The concentrations of TBBPA ranged from below the method detection limit (0.04 pg m−3) to 0.85 pg m−3. A declining trend with increasing latitude was present from the Wadden Sea to the Arctic. The atmospheric occurrence of TBBPA in the Arctic is significant and might imply that TBBPA has long-range transport potential.
Keywords: Gas chromatography–mass spectrometry; Derivatization; Tetrabromobisphenol A; Atmosphere;

Simultaneous enzyme immunoassay for the screening of aflatoxin B1 and ochratoxin A in chili samples by Debjani Saha; Debopam Acharya; Dipika Roy; Dilip Shrestha; Tarun K. Dhar (343-349).
Membrane-based immunoassay has been developed for simultaneous estimation of aflatoxin B1 (AFB1) and ochratoxin A (OA) in chili samples. The combined estimation of both the mycotoxins is more economical in respect of time, work and materials than two separate assays. The method uses a low cost test device consisting of a membrane with immobilized anti-AFB1 and anti-OA antibodies and a filter paper attached to a polyethylene card below the membrane. It allows direct analysis of sample extracts containing substantial amount (40%) of methanol. This permits the use of two-fold diluted sample extracts resulting in minimum dilution error. The limit of quantitation obtained was 2 and 10 μg kg−1 for AFB1 and OA, respectively. The tolerance of 40% methanol was found to be due to the application of small size (0.8 mm diameter) spots on membranes, as the tolerance decreases to 20% with gradual increase in spot size. The combined method is capable of producing acceptable results to analyze AFB1 and OA in chili with accuracy and precision. The AFB1 and OA values obtained for spiked and naturally contaminated chili samples by the simultaneous method were in good correlation with those measured by individual ELISA. The method offers a simple, rapid and cost-effective screening tool to meet the requirements of the rapidly evolving EU legislation.
Keywords: Aflatoxin B1; Ochratoxin A; Simultaneous method;

Raman microprobe spectroscopy and μX-ray fluorescence were used to analyse the original composition and degradation products of the limestone and the sandstones from the facades of one historical building. Carbon particles, gypsum (CaSO4·2H2O) and some nitrate compounds were determined by Raman measurements, while elemental characterisation carried out by μXRF revealed the presence of lead, sulphur and copper as pollutants. On the one hand, the grey sandstone is the most deteriorated and in some cases pollutants reach 3 cm depth. On the other hand, quantification of soluble salts (chlorides, sulphates, nitrates, nitrites, fluorides and carbonates) was accomplished by ion chromatography. Chloride and sulphates are not significant soluble salts in the façades of the building, but the amount of nitrate is even 136 g kg−1 in some of the most deteriorated samples and of 147 g kg−1 in black-crusts. Finally, organic compounds (mainly PAHs) were characterised by GC–MS. Concentrations higher than 20 ppm of total PAHs were measured in the black-crusts of the rain-protected facades. The presence of certain PAHs indicated combustion of fuels as the main source of deterioration for the palace house building materials.
Keywords: Carbonaceous sandstone; Limestone; Raman spectroscopy, X-ray fluorescence; Ionic Chromatography; GC–MS; Diagnosis;

Data fusion in the wavelet domain by means of fuzzy aggregation connectives by Pablo M. Ramos; M. Pilar Callao; Itziar Ruisánchez (360-369).
Dual-domain classification analysis is proposed to identify pigments used in works of art studied by Raman spectroscopy and X-ray fluorescence spectrometry. By means of this methodology, Raman and X-ray fluorescence data are jointly processed by a high-level fusion approach. The system proposed aims to avoid the pre-processing stage and directly process raw data obtained from the instrument. The system is tested with spectra contaminated with background components of different shapes and intensities and with those with the background removed by line segment correction. The benefits of the approach were well demonstrated in a study of an ochre pigment classification.The approach is based on the main advantage of wavelet transform, which is multiresolution. Each spectrum is split into blocks, according to a specific frequency, to form a wavelet prism. Partial least squares-discriminant analysis (PLS-DA) is then applied to those blocks which contain the deterministic part of the signal and are not influenced by noise and background signal components. At the end, to obtain the final classification assignment, high-level data fusion of the classifications results (decision levels) obtained from PLS-DA analysis is done by means of fuzzy aggregation connective operators. Our study showed that fuzzy aggregation may be suitable for performing high-level data fusion on dual-domain data. This method can be automated so that classification can be rapid. It can handle classifications with different levels of difficulty and requires no prior knowledge of sample composition.
Keywords: Data fusion; Wavelet transform; Fuzzy aggregation connectives; Micro-Raman spectroscopy; Micro-X-ray spectrometry; Ancient pigments;

On-line HPLC combined with multivariate statistical process control for the monitoring of reactions by Lifeng Zhu; Richard G. Brereton; Duncan R. Thompson; Paul L. Hopkins; Richard E.A. Escott (370-378).
On-line high performance liquid chromatography is used to monitor a steady state reaction over 35.2 h, with 197 chromatograms recorded as the reaction progresses. For each chromatogram, peaks are detected, baseline corrected, aligned and integrated to provide a peak table consisting of the intensities of 19 peaks, two corresponding to the reactants, one to the product and one to the solvent, the remaining being impurities, by-products or intermediates. D-charts and Q-charts from multivariate statistical process control are applied to the data to determine which samples are out of control and also provide diagnostic insight into why these samples are problematic. The D-chart is best at looking at overall performance issues such as problems with mixing or difficulties with instrument operation, whereas the Q-charts are best at detecting impurities during the reaction.
Keywords: On-line high performance liquid chromatography (HPLC); Process monitoring; Chromatography; Multivariate statistical process control (MSPC);

Discrimination of transgenic tomatoes based on visible/near-infrared spectra by Lijuan Xie; Yibin Ying; Tiejin Ying; Haiyan Yu; Xiaping Fu (379-384).
VIS–NIR spectroscopy combined with multivariate analysis after the appropriate spectral data pre-treatment has been proved to be a very powerful tool for judgment of the relative pattern of the objects that have very similar properties. In this study, seventy transgenic tomatoes with antisense LeETR2 and 94 of their parents, non-transgenic ones were measured in VIS–NIR diffuse reflectance mode. Principal component analysis (PCA), discriminant analysis (DA) and partial least-squares discriminant analysis (PLSDA) were applied to classify tomatoes with different genes into two groups. Calibrations were developed using PLS regression with the leave-one-out cross-validation technique. The results show that differences between transgenic and non-transgenic tomatoes do exist and excellent classification can be obtained after optimizing spectral pre-treatment. The correct classifications for transgenic and non-transgenic tomatoes were both 100% using PLSDA after derivative spectral pre-treatment. The raw spectra with PLSDA model after the second derivative pre-treatment had the best satisfactory calibration and prediction abilities, with r c  = 0.97964, root mean square error of calibration (RMSEC) = 0.099, r cv  = 0.97963, root mean square error of cross-validation (RMSECV) = 0.0993 and a factor. The results in the present study show VIS–NIR spectroscopy together with chemometrics techniques could be used to differentiate transgenic tomato, which offers the benefit of avoiding time-consuming, costly and laborious chemical and sensory analysis.
Keywords: Visible/near-infrared spectroscopy; Transgenic tomato; Classification; Chemometrics;

Multi-way partial least squares modeling of water quality data by Kunwar P. Singh; Amrita Malik; Nikita Basant; Puneet Saxena (385-396).
A 10 years surface water quality data set pertaining to a polluted river was analyzed using partial least squares (PLS) regression models. Both the unfold-PLS and N-PLS (tri-PLS and quadri-PLS) models were calibrated through leave-one out cross-validation method. These were applied to the multivariate, multi-way data array with a view to assess and compare their predictive capabilities for biochemical oxygen demand (BOD) of river water in terms of their relative mean squares error of cross-validation, prediction and variance captured. The sum of squares of residuals and leverages were computed and analyzed to identify the sites, variables, years and months which may have influence on the constructed model. Both the tri- and quadri-PLS models yielded relatively low validation error as compared to unfold-PLS and captured high variance in model. Moreover, both of these methods produced acceptable model precision and accuracy. In case of tri-PLS the root mean squares errors were 1.65 and 2.17 for calibration and prediction, respectively; whereas these were 2.58 and 1.09 for quadri-PLS. At a preliminary level it seems that BOD can be predicted but a different data arrangement is needed. Moreover, analysis of the scores and loadings plots of the N-PLS models could provide information on time evolution of the river water quality.
Keywords: Unfold-partial least squares (unfold-PLS); Multi-way partial least squares (N-PLS); Surface water quality; Biochemical oxygen demand (BOD); Cross-validation; Leverage; Residuals matrix;

This work presents a novel strategy for solving matrix effects using the second-order advantage and a new method called PARAllel profiles with LINear Dependencies (PARALIND). PARALIND is a generalization of parallel factor analysis (PARAFAC) and was developed to extend its use to problems with linearly dependent factors where normal PARAFAC analysis will fail to provide meaningful results. Such linearly dependent factors occur in standard addition with second-order data such as fluorescence excitation emission matrices (EEM). By successive standard addition of an analyte, the concentrations of the remaining components (interferences) remain constant and introduce linear dependency between interference concentrations in the samples. This theoretically leads to rank deficiency in the score matrix holding the relative concentrations when using PARAFAC for modeling. In practice, PARAFAC models of such data will mostly provide solutions where the score matrix is not rank deficient but a function of the noise in the data. This problem is shown to be solved by using PARALIND. In order to evaluate the applicability of the method a simulated as well as an experimental data set is tested. The results from experimental data relate to the direct determination of salicylic acid (SA), the main product of aspirin degradation, in undiluted human plasma by spectrofluorimetry.
Keywords: Parallel profiles with linear dependencies; Standard addition; Three-way data; Fluorescence excitation emission matrices;

Multivariate curve resolution with alternating least squares (MCR-ALS) is applied for the first time to the simultaneous analysis of electrochemical and spectroscopic data. Then, a data analysis is done with augmented matrices constituted by Differential Pulse Polarography and Circular Dichroism data submatrices. The use of proper, and different for each submatrix, constrains in the iterative ALS optimization allows to obtain chemically meaningful results constituted by a common matrix containing the concentration profiles, and two matrices with the pure electrochemical and spectroscopic signals. MCR-ALS is applied to the study of the complexation of Cd by Cys-Gly, a glutathione-fragment of great interest for understanding metal-phytochelatins complexation.
Keywords: Multivariate curve resolution; Cadmium; Glutathione-fragment; Circular dichroism spectroscopy; Voltammetry;

Development of a chemometric correlation technique to estimate acid–base descriptors for cationic acids in non-aqueous media by R. Carabias-Martínez; E. Rodríguez-Gonzalo; J. Domínguez-Álvarez; E. Miranda-Cruz (410-418).
We propose a procedure for estimating acid–base constants in organic solvents or mixture of solvents from the corresponding pK a values in aqueous medium and from certain properties of the organic solvents that characterize them. To accomplish this, we developed and validated a chemometric correlation for the calculation of the acid–base constants of different cationic acids in a broad variety of non-aqueous solvents. The parameters chosen for building the model were as follows: the acid–base constant of the compound in aqueous medium and those corresponding to the polarity–polarizability, basicity and acidity scales of the solvent. The results of the fitting were significant (p  < 0.01), with a root mean error in cross-validation of 18%, with no overfitting. The prediction of the acid–base constants for an external set of compounds had a mean absolute prediction error value of less than 0.8 pK a units.
Keywords: Acid–base constant; Cationic acids; Non-aqueous solvents; Model-based; Chemometric correlation;

The mixed dissociation constants of four anticancer drugs –camptothecine, 7-ethyl-10-hydroxycamptothecine, 10-hydroxycamptothecine and 7-ethylcamptothecine, including diprotic and triprotic molecules at various ionic strengths I of range 0.01 and 0.4, and at temperatures of 25 and 37 °C – were determined with the use of two different multiwavelength and multivariate treatments of spectral data, SPECFIT32 and SQUAD(84) nonlinear regression analyses and INDICES factor analysis. A proposed strategy for dissociation constants determination is presented on the acid–base equilibria of camptothecine. Indices of precise modifications of the factor analysis in the program INDICES 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 nonlinear regression of {pK a, I} data at 25 and 37 °C: for camptothecine p K a , 1 T = 2.90 ( 7 ) and 3.02(8), p K a , 2 T = 10.18 ( 30 ) and 10.23(8); for 7-ethyl-10-hydroxycamptothecine, p K a , 1 T = 3.11 ( 2 ) and 2.46(6), p K a , 2 T = 8.91 ( 4 ) and 8.74(3), p K a , 3 T = 9.70 ( 3 ) and 9.47(8); for 10-hydroxycamptothecine p K a , 1 T = 2.93 ( 4 ) and 2.84(5), p K a , 2 T = 8.93 ( 2 ) and 8.92(2), p K a , 3 T = 9.45 ( 10 ) and 9.98(4); and for 7-ethylcamptothecine p K a , 1 T = 3.10 ( 4 ) and 3.30(16), p K a , 2 T = 9.94 ( 9 ) and 10.98(18). Goodness-of-fit tests for various regression diagnostics enabled the reliability of the parameter estimates found to be proven. Pallas and Marvin predict pK a being based on the structural formulae of drug compounds in agreement with the experimental value.
Keywords: Spectrophotometric titration; Dissociation constant; Protonation; Anticancer drug; Camptothecine; 7-Ethyl-10-hydroxycamptothecine; 10-Hydroxycamptothecine and 7-Ethylcamptothecine; SPECFIT; SQUAD; INDICES; PALLAS; MARVIN;

In this work, the effect of supplemental LiClO4 electrolytes in KCl solutions used in roughening silver substrates by electrochemical triangular-wave oxidation–reduction cycles (ORC) on surface-enhanced Raman scattering (SERS) was first investigated. To prepare SERS-active substrates by ORC procedures, electrolytes of KCl were generally employed. In contrast, LiClO4 ones were unsuitable for producing SERS-active substrates. Encouragingly, SERS of Rhodamine 6G (R6G) adsorbed on the roughened Ag substrate prepared in an aqueous solution containing KCl and LiClO4 electrolytes exhibits a higher intensity by one order of magnitude, as compared with that of R6G adsorbed on a roughened Ag substrate prepared in a solution only containing KCl. Further investigations indicate that the oxidation state of Cl on the roughened Ag substrate demonstrates decided effects on this improved SERS.
Keywords: Surface-enhanced Raman scattering; Silver Substrates; Electrolytes;

Simultaneous distillation–extraction of high-value volatile compounds from Cistus ladanifer L. by Salomé Teixeira; Adélio Mendes; Arminda Alves; Lúcia Santos (439-446).
The present paper describes a procedure to isolate volatiles from rock-rose (Cistus ladanifer L.) using simultaneous distillation–extraction (SDE). High-value volatile compounds (HVVC) were selected and the influence of the extraction conditions investigated. The effect of the solvent nature and extraction time on SDE efficiency was studied. The best performance was achieved with pentane in 1 h operation. The extraction efficiencies ranged from 65% to 85% and the repeatability varied between 4% and 6% (as a CV%).The C. ladanifer SDE extracts were analysed by headspace solid phase microextraction (HS-SPME) followed by gas chromatography with flame ionization detection (GC-FID). The HS-SPME sampling conditions such as fiber coating, temperature, ionic strength and exposure time were optimized. The best results were achieved with an 85 μm polyacrylate fiber for a 60 min headspace extraction at 40 °C with 20% (w/v) of NaCl. For optimized conditions the recovery was in average higher than 90% for all compounds and the intermediate precision ranged from 4 to 9% (as CV %). The volatiles α-pinene (22.2 mg g−1 of extract), 2,2,6-trimethylcyclohexanone (6.1 mg g−1 of extract), borneol (3.0 mg g−1 of extract) and bornyl acetate (3.9 mg g−1 of extract) were identified in the SDE extracts obtained from the fresh plant material.
Keywords: Simultaneous distillation–extraction; Solid phase microextraction; Cistus ladanifer L.; volatiles;

A focused microwave digestion system was used to heat a mini-column of sample of crushed rock (hematite) during its successive leaching by repeated 250-μL injections of water, HNO3 1%, 10% and 30% (v/v). The mini-column was connected to the nebulizer of an inductively coupled plasma mass spectrometry instrument, which allowed a continuous monitoring of the progressive release of elements by a given leaching reagent. Quantitation of the accessible fraction of Mg, V, Cr, Mn, Co, Ni, Cu, Zn, Mo, Sb and Pb was done by calibration using 250-μL injections of standard solutions prepared in the leaching reagent matrices. Total digestion of the sample residue was also done to verify mass balance. With the exception of Mg, V and Co, where the same total amount was released with or without microwave heating, an increased release resulted from focused microwave heating, by up to an order of magnitude. Furthermore, mass balance was verified for more elements using microwave heating, presumably because of a lower relative proportion of spectroscopic interference as a result of an increased release of analytes. Using microwave energy in general resulted in the dissolution of additional phases, as evidenced by significantly different 208Pb/206Pb ratios as well as the increased release of elements with milder reagents. In fact, in the case of Pb, leaching with 30% HNO3 was no longer necessary as all the Pb was released in the first three leaching reagents. Microwave heating could therefore be used advantageously in on-line leaching for exploration geochemistry and environmental monitoring.
Keywords: On-line leaching; Microwave heating; Inductively coupled plasma mass spectrometry (ICPMS); Rock samples;

Di-(2-ethyl-hexyl)phthalate (DEHP), nonylphenol, nonylphenol mono- and diethoxylates (NPEs) and polychlorinated biphenyls (PCBs) are organic pollutants in sewage sludge which have to be monitored in the European Union according to a future Sludge Directive. In the present work, an analytical method for the simultaneous extraction and determination of DEHP, NPEs and PCBs is proposed for the routine analysis of these compounds in sludge from wastewater treatment plants. All the compounds were simultaneously extracted by sonication with hexane and analysed by gas chromatography–mass spectrometry (GC–MS) in electronic impact mode. Recoveries achieved were 105% for DEHP, 61.4–88.6% for NPEs and 55.8–108.3% for PCBs with relative standard deviation bellow 10%. Limits of quantification were 65 μg kg−1 for DEHP, from 630 to 2504 μg kg−1 for NPEs and from 5.4 to 10.6 μg kg−1 for PCBs in dried sludge. The applicability of the proposed method was evaluated by the determination of these compounds in sludge from wastewater treatment plants in Seville (South Spain).
Keywords: Sonication-assisted extraction; Di-(2-ethylhexyl)phtalate; Nonylphenol; Nonylphenol ethoxylates; Polychlorinated biphenyls; Sludge; Gas chromatography–mass spectrometry;

5,11,17,23-Tetrakis(1,1-dimethylethyl)-25,26-dihydroxy-27,28-crown-4-calix[4]arene in the cone conformation was synthesized. This p-tert-butylcalix[4]arene-1,2-crown-4 compound was then anchored with Merrifield chloromethylated resin beads. The modified polymeric resin was characterized by 1H NMR, FT-IR and elemental analysis and used successfully for the separation and preconcentration of Cu(II), Cd(II), Co(II), Ni(II) and Zn(II) prior to their determination by FAAS. Effective extraction conditions were optimized in both batch and column methods. The resin exhibits good separating ability with maximum between pH 6.0–7.0 for Cu(II), pH 6.0 for Cd(II), pH 5.0 for Co(II), pH 4.0–4.5 for Ni(II), and pH 4.5 for Zn(II). The elution studies were carried out with 0.5 mol L−1 HCl for Cu(II), Co(II) and Co(II), 1.0 mol L−1 HCl for Cd(II) and Zn(II). The sorption capacity, preconcentration factor and distribution coefficient of each metal ion were determined. The detection limits were 1.10, 1.25, 1.83, 1.68 and 2.01 μg L−1 for Cu(II), Cd(II), Co(II), Ni(II) and Zn(II). The influence of several ions on the resin performance was also investigated. The validity of the proposed method was checked for these metal ions in NIST standard reference material 2709 (San Joaquin Soil) and 2711 (Montana Soil).
Keywords: Merrifield resin; Transition metals; Calix[4]arene; Calix[4]crowns; Preconcentration;

The synthesis of a new stable chelating resin from the polystyrene divinylbenzene copolymer is reported. The polystyrene is first functionalized with a phenolic group and then allowed to couple with diazotized anthranilic acid through the ―N=N― bond. The resulting polymer containing azophenolcarboxylate with an ONO chelating environment has been characterized by elemental analysis, hydrogen ion capacity, and water regain value. Its stability towards thermal and different chemical environments has been evaluated. The sorption capacity of the chelating resin for Cr(III) and Cr(VI) as a function of pH has been studied. The interesting point is that chromium(III) is selectively retained at ca. pH 5.0 and chromium(VI) at ca. pH 2.0. When packed in a column, the new material is able to separate Cr(III) from Cr(VI). Five replicate determinations of 10 μg Cr(III) and 10 μg Cr(VI) present in 100 mL solution gave recoveries of 96.9 ± 2.9% (for Cr(III)) and 96.2 ± 2.1% (for Cr(VI)) at the 95% confidence level. Calibration graph was linear over the concentration range of 0–250 μg L−1 of chromium species with correlation coefficient (R) of 0.99994. The detection limits based on 3σ criterion were determined to be 0.6 μg L−1 for Cr(III) and 0.9 μg L−1 for Cr(VI). The developed method was successfully used for the speciation of chromium in wastewater.
Keywords: Solid phase extraction; Azophenolcarboxylate resin; Chromium speciation;