Analytica Chimica Acta (v.443, #1)

Trienzymatic biosensor for the determination of inorganic phosphate by Christine Mousty; Serge Cosnier; Dan Shan; Shaolin Mu (1-8).
A biosensor for phosphate detection has been fabricated using a mixture of maltose phosphorylase (MP), mutarotase (MR), and glucose oxidase (GOD) entrapped in an inorganic LAPONITE® clay. The response of the biosensors to phosphate and glucose additions was measured by potentiostating the modified electrodes at 0.6 V versus Ag/AgCl in order to oxidize the enzymatically generated hydrogen peroxide. The bioelectrochemical response of the biosensor for phosphate detection is strongly affected by the ratio of each enzyme in the enzyme–clay mixture. The optimum pH and temperature of the biosensor are 6.5 and 40°C. Under these optimum conditions, the phosphate sensitivity and the linear range are 52.4 mA M−1  cm−2 and 1–50 μM, respectively. The maximum response current of the biosensor is stable at least for 2 weeks, a 30% decay of initial activity was observed after 42 days at 4°C.
Keywords: Biosensor; Phosphate detection; Multienzyme amperometric sensor; Clay-modified electrode;

A novel amperometric biosensor for the detection of hydrogen peroxide was described. The biosensor was constructed by electrodepositing HRP/PPy membrane on the surface of ferrocenecarboxylic acid mediated sol–gel derived composite carbon electrode. The biosensor gave response to hydrogen peroxide in a few seconds with detection limit of 5.0×10−5  M (based on signal:noise=3). Linear range was upto 0.2 mM. The biosensor exhibited a good stability.
Keywords: Hydrogen peroxide; Polypyrrole; Biosensor; Composite carbon electrode;

A recently introduced redox osmium complex of the type [Os(bpy)2Cl(pyX]+, where pyX corresponds to a pyridine derivative bearing an aldehyde group, and glucose oxidase [EC] were covalently immobilized on the glassy carbon electrode surface (upper cell body). This approach has been designed in a sealed cell to be used under continuous-flow/stopped-flow/continuous-flow processing. The operating characteristic of the integrated bioreactor/detector unit has been utilized in the determination of glucose in serum samples using the electroactive polymer as mediator in the re-oxidation of GOx and amperometric monitoring. The determinations are based on the rate of response under stopped-flow conditions. The trend in values of the apparent Michaelis–Menten constant confirms the validity of the approach to sensitive determination utilizing very small amounts of biocatalysts. The determination of glucose was possible with a limit of detection of 0.020 mM in the processing of as many as 30 samples per hour.
Keywords: Osmium; Bioreactor; Flow system; Biosensors;

A technique for the fabrication of solid-state ion sensors based on self-plasticising poly(n-butyl acrylate) matrix by photocuring is described. The poly(n-butyl acrylate) membrane was prepared by photocuring a mixture of the monomer n-butyl acrylate, cross-linker, 2-hexanedioldiacrylate and photoinitiator, 2,2-dimethoxy-2-phenylacetophenone under UV irradiation. The optimum composition of the membranes is discussed. The photocured films produced were elastic, tough and exhibited excellent adhesion on Ag/AgCl substrates. In the optimum recipe no inherent anionic site response was observed for the blank membrane, but AIBN produced anionic site impurities if used as initiator. By incorporating ionophores and lipophilic anions during photopolymerisation, ion-selective membranes could be formed in situ on Ag/AgCl substrates. Immobilisation of ionophore was also possible if a photopolymerisable ionophore such as 4-acryloylamidobenzo-15-crown-5 was included during the photocuring process. Solid-state potassium ion sensors (based on valinomycin or immobilised 4-acryloylamidobenzo-15-crown-5) and sodium ion sensor (based on bis[(12-crown-4)methyl]dodecylmethylmalonate) were fabricated using this technique. The potentiometric responses of all these solid-state ion sensors were comparable to the corresponding conventional plasticised poly(vinyl chloride) (PVC)-based ion-selective electrodes. The influence of the composition on the lower detection limit was discussed and preliminary studies of these sensors with artificial serum electrolytes indicated that the coextration of counter and interfering ions (rather than analyte buffering effects) predominated, raising the lower detection limit. Long term exposure in a complex media, e.g. real human blood serum resulted in a potential shift indicative of ion transport with full recovery of the sensor after 12 h. The overall manual fabrication reproducibility of the technique was satisfactory with sensors produced from the same batch demonstrated <5% variation in potentiometric performance.
Keywords: Cross-linked poly(n-butyl acrylate); Solid-state ion sensor; Photocure; Ion-selective membrane; Plasticiser; Blood serum electrolyte;

This work describes the potentiometric responses of liquid membranes incorporating a highly hydrophobic β-diketone-type chelating reagent, 1-phenyl-3-methyl-4-octadecanoyl-5-pyrazolone (1) to various metal ions. A membrane based on 1 without ionic additives (membrane 1) exhibited a selective potentiometric response to Yb3+ over the transition metal ions (Mn2+, Fe2+, Co2+, Ni2+, Zn2+), alkaline (Na+, K+) and alkaline earth metal (Mg2+, Ca2+, Sr2+, Ba2+) ions in a nearly Nernstian manner in the concentration range from ≈10−6 to 10−2  M at pH 5.5. The interferences of the other trivalent ions (Gd3+, La3+ and Al3+) and Cu2+ to the Yb3+ response were significant for membrane 1, and the addition of anionic and cationic sites to membranes based on 1 decreased the selectivity to Yb3+ over the other ions. This potentiometric selectivity correlates well to the separation selectivity in the solvent extraction of the metal ions with 4-acyl-5-pyrazolones into non-polar organic solutions. This correlation, and a decrease in the potentiometric responses to Yb3+ at lower pH suggest that the potentiometric responses to the metal ions result from a decrease in the deprotonated ligands at the membrane interface due to the complex formation between the metal ions and the deprotonated ligands.
Keywords: Ion-selective electrode; Trivalent lanthanoid ions; 4-Acyl-5-pyrazolone; Solvent extraction;

NO and NO2 can be detected amperometrically on both gold and platinum electrodes deposited on SPE membranes in contact with sulfuric acid. The two analytes are oxidized at +700 mV versus MSE in the double layer region of a gold electrode and in the oxide region in the case of a platinum electrode. Due to different sensitivity ratios for NO and NO2 at the two electrodes, the concentration of each analyte in a mixture of the gases can be determined. The detection limits (S/N=3, n=5) for NO and NO2 were found to be 1.2 ppb (v/v) and 3.8 ppb on a Au-Nafion electrode and 22 ppb (v/v) and 17 ppb on a Pt-Nafion electrode, respectively. The well-known interference by SO2 could be suppressed significantly by using a concentration of sulfuric acid of 10 M. At this high concentration of the acid, the cross sensitivity to SO2 is more than 10-times lower compared to a cell with 0.5 M sulfuric acid, while the sensitivities for NO and NO2 are almost identical for both acid concentrations.
Keywords: Amperometric detection; Au-Nafion electrode; Pt-Nafion electrode; NO; NO2;

The interaction of nogalamycin (NOM), an anthracycline antitumor drug, with calf thymus DNA has been studied by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The variations in the cyclic voltammetric behaviour of NOM on addition of DNA have been carried out. The experimental results reveal that the reduced form (H2NOM)red, of NOM is bound more strongly to DNA than the oxidised form (NOM)ox. The calibration graph for the determination of DNA was obtained by the decrease in the DPV peak current of NOM in the presence of DNA. Furthermore, the results are compared with the similar type of interaction behaviours of daunomycin (DAM), adriamycin (ADM) and 4′-deoxyadriamycin (DADM), mainly from the viewpoint of the influence of chemical structure. Binding constants were determined from voltammetric data, i.e. changes in limiting current with addition of DNA. In comparison with DAM, ADM and DADM, nogalamycin displayed high binding affinity to DNA (K=4.44×105  M−1), and the binding affinity increases in the sequence: DAM<DADM<ADM<NOM. The results show also that, a slight change in chemical structure causes significant changes in the binding affinity to DNA and consequently in clinical properties.
Keywords: Voltammetric studies; Anthracycline antitumor drugs; Nogalamycin; DNA; Interaction;

By attaching two right-angled prisms at a quartz cell of a common spectrofluorometer to change the propagation direction of the incident/emission light beams, a novel technique, reflected-light scattering (RFLS), was developed to detect the light scattering signals at the air/liquid interface. At pH 1.86 and ionic strength 0.04, the J- and H-aggregation of α,β,γ,δ-tetrakis(4-sulfophenyl)porphine that occurred at the interface in the presence of proteins, was assigned by measuring the enhanced resonance light scattering (RLS) signals characterized at 490.2 and 421.6 nm, respectively. With the enhanced RLS signals at 490.2 nm, a novel assay of proteins was established with the 3σ limit of detection being 18–70 ng ml−1. The protein concentrations in synthetic samples and in human serum were determined with satisfactory results.
Keywords: α,β,γ,δ-Tetrakis(4-sulfophenyl)porphine; Proteins; Reflected-light scattering; Air/liquid interface;

Three separation methods for the determination of Pu, Am, U, Th and Sr in marine sediment samples based on extraction chromatography are presented. The samples were attacked using an open focused microwave system and the radionuclides were then separated using commercially available resins (TRU Spec, U/TEVA Spec and Sr Spec). The separation methods were tested with synthetic sample solutions containing the five radionuclides with and without interference. To validate the methods, they were also applied to a sediment reference material (IAEA-135). Finally, two of the methods were applied to two sediment samples from the Mediterranean coast of northeast Spain.All the actinides were measured by alpha spectrometry, and 90Sr activity was measured using a liquid scintillation counter.While the recovery of U and Sr was satisfactory in all cases, with values higher than 50 and 75%, respectively, recovery of Pu and Am was more variable and depended on the sample matrix and separation method applied.
Keywords: Extraction chromatography; Actinides; Sr; Marine sediment; Microwave systems;

A method has been developed for the determination of interactions of metal ions and protein by using microdialysis sampling technique combined with pre-column derivation and reversed-phase ion-pair liquid chromatographic (HPLC) analysis. Cu(II), Zn(II) and human serum albumin (HSA) were chosen as model metal ions and protein, respectively. The mixed solutions of metal ions and HSA with different molar ratios buffered with 0.1 M Tris–HCl containing 0.1 M NaCl at pH 7.43 were sampled with a mirodialysis probe by keeping perfusion rate at 1 μl/min and the temperature at 37°C. The free concentrations of metal ions in microdialysates were assayed by precolumn derivatization with meso-tetra(4-sulfophenyl)-porphyrin (TPPS4) followed ion-pair HPLC analysis. The recovery (R) of microdialysis sampling was measured in vitro under similar conditions as 65.74% for Cu(II), 70.45% for Zn(II) with R.S.D. below 3.2%. The primary binding constants and number of binding site estimated by the Scatchard plot analysis are 5.04×106  M−1 and 0.85 for Cu(II), and 9.87×106  M−1 and 1.10 for Zn(II), respectively. The competition of Cu(II) and Zn(II) at the second binding site on HSA was investigated, and it was observed that there is a second site on HSA to bind Cu(II) and Zn(II), the affinity of Cu(II) is stronger than that of Zn(II) to this second site of HSA.
Keywords: Binding interaction; Metal ions; Human serum albumin; Microdialysis; HPLC;

A new method for simultaneous determination of niobium(V) and tantalum(V) is described. Capillary electrophoresis (CE) with chemiluminescence (CL) detection was used in the separation and detection of niobium(V) and tantalum(V) which catalyze the CL reaction of luminol with hydrogen peroxide (H2O2). The mixing mode used in this work allowed the CL reagents (luminol and H2O2) and the analytes to meet simultaneously at the detection window while in conventional ion chromatography chemiluminescence (ICCL) the CL reagents are mixed in advance. Therefore, in this work the luminol as a component of the separation carrier electrolyte avoided the loss of light signal which occurs when luminol and hydrogen peroxide are mixed in advance in ICCL. Signal enhancement was achieved by sample stacking injection in the electrophoresis process. A sample volume of 20 nl was injected. The metal ions were separated in less than 7 min with sodium dihydrogen phosphate buffer (pH 2.5, 10 mmol/l). The detection limits of this method for niobium(V) and tantalum(V) (S/N=3) are 826 zmol (4.13×10−10  mol/l) and 640 amol (3.2×10−8 mol/l), respectively. The sensitivity of the metal ions was satisfactory.
Keywords: Chemiluminescence; Electrophoresis; Niobium; Tantalum;

Aspects of the successive projections algorithm for variable selection in multivariate calibration applied to plasma emission spectrometry by Roberto Kawakami Harrop Galvão; Maria Fernanda Pimentel; Mario Cesar Ugulino Araujo; Takashi Yoneyama; Valeria Visani (107-115).
The successive projections algorithm (SPA) was recently proposed as a variable selection strategy to minimize collinearity problems in multivariate calibration. Although SPA has been successfully applied to UV–VIS spectrophotometric multicomponent analysis, no evidence of its ability to deal with variable sets with both high and low signal-to-noise ratios has been presented. This issue is addressed by the present work, which applies SPA to the simultaneous determination of Mn, Mo, Cr, Ni and Fe using a low-resolution plasma spectrometer/diode array detection system. This problem is of particular interest since strong interanalyte spectral interferences arise and regions with high and low signal intensity alternate in the spectra. Results show that multiple linear regression (MLR) on the wavelengths selected by SPA yields models with better prediction capabilities than principal component regression (PCR) and partial least squares (PLS) models. A standard genetic algorithm (GA) used for comparison yielded results similar to SPA for Mn, Cr and Fe, and better predictions for Mo and Ni. However, in all cases, the GA resulted in models less parsimonious than SPA. The average of the root mean square relative error of prediction (RMSREP) obtained for the five analytes was 1.4% for MLR–SPA, 1.0% for MLR–GA, 2.2% for PCR, and 2.1% for PLS. Since the computational time demanded by SPA grows with the square of the number of spectral variables, a pre-selection procedure based on the identification of emission peaks is proposed. This procedure decreased selection time by a factor of 20, without significantly degrading the results.
Keywords: Variable selection; Successive projections algorithm; Plasma emission spectrometry; Multivariate calibration;

Curve fitting must be considerably facilitated if reliable and accurate initial estimates of the number of peaks, individual peak positions, areas, and widths are known at the outset. The most important values for input to curve fitting route are the number of peaks and their positions. One of the main drawbacks involved is that as the bands become more overlapped, or the number of overlapped bands increases, the problem becomes progressively more ill-conditioned. As a consequence, small errors in the data, or errors in the estimates can be magnified, ultimately resulting in large errors in the final model. In addition, very high noise level of the analytical signal also has significant effect on the fitted results. In this work, curve fitting using wavelet transform for peak finding and an enhancement of signal-to-noise ratio was proposed, in which wavelet transform was performed prior to curve fitting to enhance noise level for subsequent fitting and to determine the number of peaks and corresponding parameters. Accordingly, the fitted conditions can be improved to the point that very accurate results could be acquired even for the simulated overlapped bands with higher noise level.
Keywords: Curve fitting; Wavelet transform; Resolving overlapped spectra; Simulated spectroscopic data;

The response of the cavity to the movement of the “over full-length cavity” cylindrical samples with lengths from 50 to 100 mm along the x-axis of the Bruker double TE104 and single TE102 rectangular cavity has been analysed. The experimentally observed dependencies of the electron paramagnetic resonance (EPR) signal intensity versus sample position in the cavity showed the following: (i) a “sloping plateau” region, which could be approximated by the linear function (correlation coefficient, r=0.85–0.96); (ii) an additional oscillating signal superimposed on the “sloping plateau” region; (iii) an identical amplitude of the maximal signal intensity on the “sloping plateau” region for all sample lengths, within an experimental error of 1.27%. The trends of the dependencies with the “sloping plateau” were independent of the length and internal diameter of the “over full-length cavity” samples, but the width of the “sloping plateau” regions progressively increased with the increase of the sample length from ca. 30 mm for the 50-mm sample to ca. 80 mm for the 100-mm sample. Theoretical predictions of the experimental dependencies of the signal intensity versus sample position in the cavity were calculated using the “modified” and “revised” sine-squared functions, and the correlation between the observed and theoretically computed dependencies is very good. The experimental dependence of the “over full-length cavity” sample position (x-coordinate), at which the signal intensity was a maximum, as a function of sample length, L, is nonlinear. The experimentally determined x-coordinate of these sample positions in the cavity oscillated between the upper, La/2, and lower, La, theoretically predicted limiting values, where a (23.5 mm) is the length of the active zone of the Bruker rectangular microwave cavity. Each of the above phenomena may be a source of serious errors in quantitative EPR spectroscopy. “Over full-length cavity” cylindrical samples to be compared should be of identical length and must be identically positioned in the microwave cavity.
Keywords: Quantitative EPR spectroscopy; Sample length; “Over full-length cavity” cylindrical sample; Double and single cavity;

Automated determination of vanadium(IV) and (V) in natural waters based on chelating resin separation and catalytic detection with Bindschedler’s green leuco base by Kei Okamura; Masahito Sugiyama; Hajime Obata; Masahiro Maruo; Eiichiro Nakayama; Hajime Karatani (143-151).
An automated method for determining V(V) and V(IV) in natural waters has been developed. The method is based on the combination of a selective column extraction using two kinds of chelating resins and an air-segmented continuous flow analysis (ASCFA) using catalytic detection with Bindschedler’s green leuco base (BGL)–KBrO3 system. Based on this method, V(V) in a sample solution at pHs from 2.2 to 3.8 is selectively collected on an acetylacetone-immobilized resin and then V(IV) in the eluent is collected on an 8-quinolinole-immobilized resin separating it from iron species, which interfere with the ASCFA detection. V(V) and V(IV) are successively eluted with diluted hydrochloric acid and the resulting eluent is carried into the ASCFA. The concentrations of vanadium species are determined from the absorbance of BGL. The detection range for vanadium species was 0.05–15.0 nM and the standard deviations at 5.0 nM were 1.1% for V(IV) and 0.9% for V(V), respectively. The method was successfully applied to study the behavior of vanadium species in natural waters.
Keywords: Vanadium; Speciation; Chelate resin; Natural water; Flow analysis;

A fast, selective, sensitive and automated method for determination of free chlorine in industrial formulations and water samples is proposed. The automation of the flow assembly is based on the “tandem flow” approach, which uses a set of solenoid valves acting as independent switches. The operating cycle for obtaining a typical analytical transient signal can easily be programmed by means of friendly software running in the Windows environment. The manifold is provided with a gas diffusion unit which permits the removal of interfering species as well as concentrating the chlorine. The determination of chlorine is performed on the basis of the oxidation of o-dianisidine as the colorimetric reagent to a colored product that can be monitored at 445 nm. The method (for a concentration time of 30 s) is linear over the range 0.05–1.30 μg ml−1 chlorine; the 3σ limit of detection is 0.05 μg ml−1; the reproducibility (as the R.S.D. of 42 peak heights for a 0.72 μg ml−1 chlorine solution) is 1.5% and the sample throughput is 38 h−1.
Keywords: Chlorine; Tandem flow; Gas diffusion; Porous membrane and o-dianisidine;

Rapid spectrophotometric method for the quantitation of acetaminophen in serum by Jalil Tavakoli Afshari; Tsan-Zon Liu (165-169).
A novel spectrophotometric method for rapid quantification of acetaminophen in serum is described. Free unconjugated acetaminophen is separated from other endogenous interferents by extracting the drug into ethyl acetate and hydrolysis to p-aminophenol by treatment with heat and acid. The latter compound is capable of undergoing an oxidative coupling reaction with p-xylenol (2,5-dimethylphenol) catalyzed by sodium periodate. The resultant indophenol derivative formed is measured spectrophotometrically at 635 nm. The proposed method has a linearity range from 25 to 600 μg ml−1. The method was precise with day-to-day coefficients of variation for two controls (101 and 204 μg ml−1) of 4.8 and 7.0%, respectively. A group of commonly used prescription drugs or compounds likely to be potential interferents on the basis of their chemical structures were tested and found not to interfere. Results obtained by the proposed method correlated excellently with those determined by either the nitration or the direct acid iron(III) redox methods with correlation coefficients of 0.97 and 0.98, respectively. The proposed method for acetaminophen determination is simple and rapid and is especially suitable for screening for drug overdose in an emergency situation.
Keywords: Acetaminophen; Paracetamol; Tylenol; Spectrophotometry;