Analytica Chimica Acta (v.424, #1)

An automated system with microporous membrane liquid–liquid extraction (MMLLE) combined with HPLC has been designed for the determination of Vinclozolin in aqueous matrices. In MMLLE a porous hydrophobic membrane separates an aqueous phase from an organic phase. The pores are filled with the organic solvent. In this work, MMLLE was performed in a flow system and coupled on-line to normal phase HPLC. The performance of the system was evaluated with respect to enrichment when processing large sample volumes. Enrichment factors of 118 times were obtained and the limit of detection was around 1 ng/ml. This technique is an alternative to classical LLE and offers advantages such as reduced solvent consumption, possibility of automation and avoidance of emulsion formations.
Keywords: Microporous membrane liquid–liquid extraction (MMLLE); Automation; Vinclozolin; Normal phase HPLC;

This paper describes an evaluation of extraction methods of triazine herbicides atrazine, simazine, prometryn, and ametryn and of three chlorodealkylated atrazine metabolites from human urine using diethyl ether and C18 solid-phase extraction (Sep-Pak C18 cartridges). The extracts were analysed by capillary gas chromatography using nitrogen selective and electron capture detectors. At ng ml−1 concentrations the diethyl ether extraction recoveries of atrazine and simazine were almost quantitative (R.S.D. 10%), while the recoveries of other triazines ranged from 58% for deisopropylatrazine to 85% for deethylatrazine (R.S.D. 19–34%). The sensitivity of the method using a nitrogen selective detector ranged from 5 ng ml−1 for atrazine to 30 ng ml−1 for didealkylated atrazine. C18 solid-phase extraction with acetone as eluting solvent resulted in about 100% recoveries of all parent herbicides (R.S.D. 15–21%) with detection limits of 5 ng ml−1 for atrazine and 10 ng ml−1 for other herbicides. Comparably high recoveries of three dealkylated atrazine metabolites (R.S.D. 10–12%) were achieved from acidified urine samples (pH 2–3) only at concentrations lower than 100 ng ml−1. The diethyl ether extraction procedure served to identify occupational exposure to atrazine and simazine. Deethyldeisopropylatrazine was confirmed as the predominant urinary dealkylated metabolite followed by monodealkylated metabolites and unchanged atrazine.
Keywords: Atrazine; Simazine; Ametryn; Prometryn; Chlorodealkylated atrazine metabolites; Accumulation from urine; Occupational exposure;

A new method for the trace analysis of trans-resveratrol in wine was studied by developing a silylation on the fibre coating following solid-phase microextraction (SPME) combined with GC/MS. The silylation was performed directly by placing the fibre into the headspace of silylating reagent. The method is rapid and sensitive without going through laborious sample preparation. The effects of fibre selection, extraction and desorption times as well as the extraction temperature were studied. The optimum conditions of derivatization time and temperature were also studied. The derivatization was carried out at room temperature without damaging the fibre coating. A linear response of trans-resveratrol over a concentration range of 10 ng l−1–1 mg l−1 with a correlation coefficient of 0.9997 and detection limit 5 ng l−1 (3 s criteria) were obtained. The analytical recoveries in wine ranged from 84 to 106% with precision below 10% relative standard deviation (R.S.D.).
Keywords: Solid phase microextraction(SPME); Silylation; Gas chromatography/mass spectrometry (GC/MS); Trans-resveratrol; Wine;

Optical sensing schemes for Prussian Blue/Prussian White film system by Robert Koncki; Tomasz Lenarczuk; Stanisław Gł a ̨ b (27-35).
Optical chemical sensing schemes exploiting redox properties of Prussian Blue/Prusian White film system are reported. Transparent, thin film composed of Prussian Blue and N-substituted polypyrrole was chemically deposited on non-conducting polyester foil. The process of the film formation is induced and accelerated by UV radiation. The film is robust, crack-free and strongly adheres to the plastic support. The film was used as a receptor part of a flow-through detector cell. All spectrophotometric measurements were performed at the wavelength of 720 nm, i.e. at the absorption maximum of the Prussian Blue film. The Prussian Blue/Prussian White film sensor system exhibits excellent operational stability and long shelf-life. The Prussian Blue film is shown to be suitable for optical determination of selected strong reductants. Also potential application of the film for optical determination of alkali cations involved in the redox process is shown. In situ generated Prussian White film is mechanically stable, however, can be easily oxidized to Prussian Blue form. This feature of the Prussian White film can be applied for optical determination of various oxidants. It was also shown that the presented detector cell is useful for optical determination of redox species in non-transparent, turbid samples. The Prussian Blue/Prussian White film redox system offers a new approach to near-infrared optical sensing applications.
Keywords: Prussian Blue; Prussian White; Polypyrrole; Chemosensitive film;

An immunosensor based on the ion-selective field effect transistor (ISFET) was developed for the express determination of herbicide simazine. Polyclonal antibodies against simazine were attached to the ISFET gate via staphylococcal protein A. Two methods of simazine detection were used — (i) competitive immune assay when native (detected) and peroxidase-labelled simazine molecules competed for binding with antibodies on the ISFET surface, and (ii) sequential saturation of antibodies, left unbound after their exposure to native simazine in investigated sample, with labelled simazine. The catalytic activity of bound peroxidase was measured in the presence of ascorbic acid and H2O2. The limit of simazine detection by competitive immune assay was 1.25 ng ml−1, the linearity was observed in the range of 5–175 ng ml−1. Sequential saturation of the antibodies led to the growth of the assay sensitivity up to 0.65 ng ml−1. Acidic treatment of the ISFETs allowed using them several times without loss of the signal amplitude. The analysis had rapid kinetics, its overall time including duration of all preparation stages was around 50 min.
Keywords: Immunosensor; ISFET; Herbicide; Simazine; Competitive immunoassay;

Amperometric detection of d-sorbitol with NAD+-d-sorbitol dehydrogenase modified carbon paste electrode by S.B Saidman; M.J Lobo-Castañón; A.J Miranda-Ordieres; P Tuñón-Blanco (45-50).
A biosensor based on modified carbon paste electrodes for the amperometric detection of d-sorbitol is described. Carbon paste electrodes were modified by d-sorbitol dehydrogenase (SDH) and nicotinamide adenine dinucleotide (NAD+) and coated with a non-conducting poly(o-phenylenediamine) (PPD) film. After partial oxidation of the immobilised NAD+ by applying a potential step from 0.0 to 1.2 V (SCE), the modified electrode allowed the amperometric detection of the NADH enzymatically obtained at an applied potential of 0.0 V. The resulting biosensor responded rapidly to sorbitol up to 8×10−4  M with a detection limit of 4×10−5  M.
Keywords: Sorbitol biosensor; Amperometry; NAD+ oxidation;

Novel ceramic-carbon electrodes (CCEs) containing 1:12-phosphomolybdic acid (PMo12) were constructed by homogeneously dispersing PMo12 and graphite powder into methyltrimethoxysilane-derived gel. Peak currents for the PMo12-doped CCE were surface-controlled at lower scan rates but diffusion-controlled at higher scan rates and peak potentials shifted to the negative potential direction with increasing pH. In addition, the electrode exhibited electrocatalytic activity toward the oxidation of ascorbic acid. The PMo12-modified CCE presented good chemical and mechanical stability and good surface renewability (ten successive polishing resulted in less than 5% relative standard deviation).
Keywords: 1:12-phosphomolybdic acid; Chemically modified electrode; Sol–gel; Electrocatalytic oxidation; Ascorbic acid;

Highly sensitive electrochemical detection of alkaline phosphatase by Shuichiro Ito; Shin-ichi Yamazaki; Kenji Kano; Tokuji Ikeda (57-63).
Alkaline phosphatase (ALP), a labeling enzyme frequently used for enzyme immunoassay, was determined at the attomole level using a mushroom tyrosinase (TN)-embedded carbon paste electrode. The detection scheme consists of two successive amplifications. The first reaction is the ALP-catalyzed hydrolysis of phenyl phosphate to accumulate phenol as a product. The second is the bioelectrocatalytic detection of phenol with the TN-embedded electrode at 0 V versus Ag/AgCl, which allowed sensitive detection of phenol down to 35 nM. The two-enzymatic reactions of ALP and TN were made to proceed successively in one electrochemical cell by adjusting pH to their optimum ones. This method provided a linear relation between the ALP concentration and the steady-state current and allowed the ALP detection as low as 7 amol/10 μl sample. The ALP detection system was applied to a streptavidin-biotin binding assay. The lowest detection limit of biotin was 4 fmol/10 μl sample with an ALP reaction time of 50 min.
Keywords: Bioelectrocatalysis; Alkaline phosphatase; Enzyme immunoassay; Tyrosinase; Phenol;

A simple and highly reliable method for the determination of mercury on a rotating gold disk electrode is reported. The signal is linear with concentration over a wide concentration range (0.2–400 nM). The stability of the electrode is excellent. No mechanical polishing between runs is required and a simple electrochemical pretreatment is applied about once in 100 runs. The detection limit in synthetic solutions, applying the subtractive mode of anodic stripping voltammetry (SASV) is 50 pM for a 120 s deposition time at 5000 rpm and 4 nM in urine sample for 180 s deposition time. The reproducibility of the analytical signal is better than 2% in solutions containing 1 nM Hg(II). The applicability of the method in urine analysis was demonstrated with the use of certified samples. No interference by lead, copper, cadmium, chromium or selenium was found at concentrations corresponding to their toxic occurrence in urine.
Keywords: Anodic stripping voltammetry; Rotating gold electrode; Mercury; Urine;

A study of the complexing properties of the α-metallothionein domain with cadmium and/or zinc was performed at pH 8, using differential pulse polarography. The applied method allows one to distinguish between the chemical form of the compound, free metal ions and complexes with the α-domain as well as providing evidence that different forms of complexation exist. This technique was used to monitor changes of different species as a function of different parameters. Polarographic results have been compared with those obtained by electrospray ionisation mass spectrometry providing the molecular mass of compounds at different proportions of metal ions to the α-domain, under similar experimental conditions. The α-domain is capable of complexing cadmium and/or zinc, separately or after simultaneous additions of metal ions. In the case of binary mixtures, Cd+α or Zn+α pure complexes are formed, whereas in the case of tertiary mixtures α+[Cd+Zn] mixed cadmium and zinc complexes prevail. When one of the two ions is added to a solution containing the α-domain complexed with the other cation, it is incorporated into the molecule resulting in a reorganisation, which is illustrated by changes in the polarographic responses. Two different conditions can be clearly distinguished: an excess of ligand and when the metal ions are in excess. For these two conditions two different and characteristic features of polarograms were obtained. So, several stable forms of cadmium and zinc complexation with the α-domain exist, depending on the composition of the solution.The combination of results obtained using two different techniques was very useful for the assignment of different cadmium and zinc electrochemical responses.
Keywords: Electrochemistry; α-MT cluster; Complexing properties;

Tetramethylammonium hydroxide (TMAH) treatment followed up with pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) was applied to characterise the structural composition of lake aquatic humic matter (HM). The HM samples were isolated with two different chromatographic methods: (1) non-ionic sorbing solid (standard XAD technique) with preacidification of a water sample to pH 2; and (2) weakly basic anion exchanger (DEAE, diethylaminoethyl cellulose) without any preacidification. A detailed structural analysis of degradation products was performed and compositional differences between different HM samples were evaluated. The main degradation products, in addition to varying proportions of different nitrogen and sulphur compounds, were methyl derivatives of different phenols, alkylphenols, phenolic acids and aliphatic mono- and dicarboxylic acids, which speak for the importance of ester linkages between the different structural subunits of aquatic HM. Despite all HM samples yielded qualitatively quite similar organic compounds, some fundamental quantitative differences existed. As a general rule, the DEAE isolate and two main XAD fractions: conventional fulvic (FA) and humic (HA) acids, seem to consist of almost similar organic matter dominating by different phenols and aliphatic acids. The other two special XAD fractions appear to belong to different structural categories yielding more alkylbenzenes/styrenes, alkanes and aliphatic monocarboxylic acids. The structural analyses and statistical calculations on the different HM samples support the postulation that the structural composition of the DEAE isolate corresponds to a certain average of the four different acidic XAD fractions.
Keywords: Pyrolysis; TMAH; Methylation; Thermochemolysis; XAD resin; DEAE cellulose; Aquatic humic substances;

A recently proposed approach for the study of the intercalation equilibria of drugs and polynucleotides is applied to ethidium bromide and poly(inosinic)–poly(cytidylic) acid. The procedure consists of the extension of the continuous variation and mole-ratio methods by recording all the UV–VIS absorption, fluorescence and circular dichroism spectra for a set of solutions containing a range of polynucleotide:dye concentration ratios. The whole set of spectroscopic data matrices was simultaneously analyzed by a multivariate curve resolution method based on alternating least squares. This procedure allowed the detection of the intercalation complex, the recovery of the concentration profiles and pure spectra for each species and the calculation of the polynucleotide:dye ratio in the complex and the apparent equilibrium constant.The intercalation sites occur every three base pairs and the value for the logarithm of the apparent equilibrium constant at 37°C in 0.26 M NaCl was 4.6±0.1 M−1.
Keywords: Multivariate curve resolution; Factor analysis; Self-modeling; Intercalation; Polynucleotides; Ethidium bromide;

The bidimensional multivariate regression procedures: multiple linear regression (MLR), principal component regression (PCR), partial least squares regression (PLS) and continuum regression (CR), and several N-way methods such as N-way PLS (nPLS) and parallel factor analysis (PARAFAC) are tested as calibration methods for the kinetic-spectrophotometric determination of ternary mixtures in a pseudo first-order kinetic system. The different calibration procedures were first applied to computer simulated kinetic-spectrophotometric data where the effect of spectral overlap and the differences in the kinetic constants were evaluated at a low level of experimental noise. Later they were applied to the stopped-flow kinetic-spectrophotometric simultaneous resolution of Co(II), Ni(II) and Ga(III) using 4-(2-pyridylazo)resorcinol (PAR) as a chromogenic reagent. Accurate estimations of concentrations with relative standard errors of prediction of about 8% were obtained even though a high degree of spectral overlap and similar rate constants were present. The study of the influence of experimental noise on the 3-component system justifies the difference between the simulations and the experimental results for the different calibration procedures. PARAFAC and MLR did not allow the resolution of the proposed 3-component system. CR provided slightly better results than those obtained by PLS, PCR and nPLS.
Keywords: Simultaneous kinetic determination; Multivariate calibration; N-way methods; 4-(2-pyridylazo)resorcinol; Metal ions determination; Stopped-flow photodiode array spectrophotometry;

The iron(III)-catalyzed reaction of the oxidative coupling reaction of 3-methyl-2-benzothiazolinone hydrazone (MBTH) with N,N-dimethylaniline (DMA) was studied kinetically by an initial-rate method. All reactions were run at constant ionic strength 0.3 M and 40°C and followed by measuring the absorbance at 590 nm of the violet compound formed. According to results of a kinetic study of the iron(III)-catalyzed reaction, a mechanism is proposed which leads to the following rate equation: R0cat = [DMA][MBTH] P′[H +]+Q′[H +][DMA]+S′[DMA] + [DMA][MBTH] T′[H +]+U′[H +][MBTH]+V′[H +][MBTH][DMA]+W′[MBTH][DMA] [Fe]0 The equation describes in detail the quantitative behavior of the reaction throughout the range of conditions studied. The optimum conditions for iron determination by initial rate procedure were estimated using this rate equation and analytical implications of the mechanistic study are discussed.
Keywords: Mechanism of catalyzed reaction; Spectrophotometry; Iron; Oxidative coupling reaction;

A weighted least squares method is used to calculate the dissociation constants of polyprotic acids and bases from measurement of additive properties, and to evaluate the effect of the errors of measurement on these constants. The method is based on the theory of statistical adjustment of data, which was originally described in detail by Deming [W.E. Deming, Statistical Adjustment of Data, Wiley, London, 1943]. The advantage of the theory is that it makes the propagation of errors of measurements of all variables explicit and easy to analyze. The theory is presented in this paper in a general form by adapting the formalism of Wentworth [W.E. Wentworth, J. Chem. Educ. 42 (1965) 96], and then applied to the measurements of additive properties. Illustrative examples from spectrophotometry and reversed-phase high-performance liquid chromatography are presented.
Keywords: Least squares method; Dissociation constant; Polyprotic acids;