Analytica Chimica Acta (v.424, #2)

Development of an enzyme immunoassay for the detection of plant growth regulator inabenfide in rice by Eiki Watanabe; Yuriko Tsuda; Shigeyuki Watanabe; Shigekazu Ito; Masaro Hayashi; Takaho Watanabe; Yojiro Yuasa; Hiroyuki Nakazawa (149-160).
A competitive enzyme-linked immunosorbent assay (ELISA) for the plant growth regulator inabenfide was developed using a specific monoclonal antibody (MAB). The effects of rice matrices on the sensibility of ELISA can be removed by adding 0.1% Tween 20. A good reproducibility and accuracy of the proposed ELISA was obtained for rice samples spiked with different amounts of inabenfide. The average recovery for rice samples spiked at 5–500 ppb was 91.6%. Good correlations were obtained between inabenfide measured in rice samples by using the proposed ELISA and that by using a conventional HPLC or GC analysis. This assay can be used in monitoring to determine inabenfide in rice.
Keywords: Inabenfide; ELISA; Rice;

Validation of a competitive ELISA for the determination of serum keratan sulphate by Thomas Block Nikolajsen; Åse Marie Hansen; Jesper Kristiansen; Anne Helene Garde (161-167).
A competitive ELISA with an inhibition step for the determination of serum keratan sulphate (KS), was validated using bovine corneal KS as standard. The method was shown to be in statistical control. The recovery was 85% (S.D.=2.2%), hence the obtained results had to be corrected. The limit of detection (LOD) was 1.4 μg m l−1. Intra-assay CV was 3.0% (n=15) for samples used for method evaluation and 6.0% (n=18) for serum samples. Inter-assay CV was 7.0% for high KS level samples (C KS=31.2 μg m l−1) and 16.0% for low KS level samples (C KS=5.9 μg m l−1) based on the S.D. y/x from the method evaluation function and 10% (high KS level) and 16% (low KS level) based on serum samples. Validation of the method was performed using the serum samples from 102 healthy individuals. There was a statistical difference in serum KS levels between men and women, but no age related differences. Tentative biological reference intervals for men and women were established. It was also shown that due to the large between-subject variability in KS levels, the preferable monitoring strategy is to use individuals as their own controls where possible.
Keywords: Keratan sulphate; ELISA; Biological reference interval; Biomarker;

The effects of surfactant having single and two alkyl chains as a component of liposomes on the intensity of bioluminescence (BL) from firefly luciferin–luciferase reaction with adenosine 5′-triphosphate (ATP) and on the stability of liposomes were investigated by use of vesicles formed by extrusion technique. Stearyltrimethylammonium chloride (STAC) and distearyldimethylammonium chloride (DSDAC) were used as a surfactant having single and two alkyl chains, respectively. The BL intensity in the presence of liposomes containing DSDAC increased twice compared with those containing STAC. The detection limit for ATP upon using liposomes containing DSDAC was 0.5 pM in aqueous ATP standard solution, and was improved by a factor of 10 compared to that in water alone. Liposomes containing DSDAC were more stable than those containing STAC, and were superior to those containing STAC as an enhancer for the firefly BL assay of ATP.
Keywords: Firefly bioluminescence; Adenosine 5′-triphosphate; Liposomes; Surfactant; Membrane surface potential;

In this paper, the non-protected room temperature phosphorescence (NP-RTP) properties of α-naphthyloxyacetic (α-NOA) and β-naphthyloxyacetic acid (β-NOA) and effects of organic solvents have been studied to clarify the effects of position of substituent on the luminescence. Both α-NOA and β-NOA aqueous solutions can emit strong and stable RTP signal in the absence of a protecting medium by addition of KI or TlNO3 as heavy atom perturber (HAP) and Na2SO3 as deoxygenator. The fluid RTP system is completely transparent and stable. A good linearity between the RTP intensity and the concentration was obtained with detection limits of 1.0×10−8 and 1.2×10−8  mol/l, respectively, for α-NOA and β-NOA. The kind and amount of organic solvent added to the luminescent system not only affect the RTP intensity and the pre-irradiation time required to attain a stable RTP signal, but also the selection of HAP. Under the same conditions, the RTP intensity of the system using TlNO3 as HAP is much stronger than that of the system using KI as HAP, but the detection limits of both systems for analytical determination are comparable. The RTP intensity of β-NOA is lower than that of α-NOA, but the influence of organic solvent on its RTP emission is also less. Determination of α-NOA in spiked pond water using standard additions method was performed, the recoveries are 97–102%.
Keywords: α-naphthyloxyacetic acid; β-naphthyloxyacetic acid; Non-protected room temperature phosphorescence; Solvent effect; Heavy atom perturber;

Multivariate calibration with Raman spectroscopic data: a case study by F. Estienne; D.L. Massart; N. Zanier-Szydlowski; Ph. Marteau (185-201).
An industrial process separating p-xylene from mainly other C8 aromatic compounds is monitored with an online remote Raman analyser. The concentrations of six constituents are currently evaluated with a classical calibration method. The aim of the study being to improve the precision of the monitoring of the process, inverse calibration linear methods were applied on a synthetic dataset, in order to evaluate the improvement in prediction such methods could yield. Several methods were tested including principal component regression with variable selection, partial least square regression or multiple linear regression with variable selection (stepwise or based on genetic algorithm). Methods based on selected wavelengths are of great interest because the obtained models can be expected to be very robust toward experimental conditions. However, because of the important noise in the spectra due to short accumulation time, variable selection methods selected a lot of irrelevant variables by chance correlation. Strategies were investigated to solve this problem and build reliable robust models. These strategies include the use of signal pre-processing (smoothing and filtering in the Fourier or wavelets domain), and the use of an improved variable selection algorithm based on the selection of spectral windows instead of single wavelengths when this leads to a better model. The best results were achieved with multiple linear regression and stepwise variable selection applied to spectra denoised in the Fourier domain.
Keywords: Chemometrics; Raman spectroscopy; Multivariate calibration; Random correlation;

Multivariate curve resolution of polarographic data applied to the study of the copper-binding ability of tannic acid by Boris H. Cruz; José Manuel Dı́az-Cruz; Cristina Ariño; Romà Tauler; Miquel Esteban (203-209).
The interactions between tannic acid (TA) and Cu(II) have been studied by differential pulse polarography (DPP) as a model for the metal ion–tannin interaction in the presence of simultaneously occurring phenomena such as electrodic adsorption, overlapping signals or stabilisation of intermediate Cu(I) species. The high complexity of the system hinders the direct application of the usual strategies of electrochemical hard modelling. As an alternative approach, it is proposed the use of a multivariate curve resolution (MCR) procedure which gives an estimation of the concentration profiles showing the evolution of the complexation–reduction process. Moreover, from such concentration profiles it is possible to estimate the number of Cu(II) ions which can be bound by TA under the conditions of the experiment.
Keywords: Heavy metals; Tannic acid; Polarography; Multivariate curve resolution; Soft modelling;

In-tube solid phase microextraction (SPME) is an on-line extraction technique for compounds in aqueous samples, in which analytes are extracted and concentrated from the sample directly into a coated capillary by repeated aspirate/dispense steps. In this paper, a polypyrrole (PPY) coated capillary and several commercially available capillaries (used for GC separation) were used to examine their extraction efficiencies to the organoarsenic compounds studied. Compared with commercial capillaries that were currently used for in-tube SPME, the PPY coated capillary has shown better extraction efficiency to most of the compounds studied, especially to the anionic species, due to the inherent multi-functionality of pyrrole polymer. For the first time, this PPY coated capillary has been used for automated in-tube SPME and speciation of organoarsenic species in aqueous samples when coupled with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). Organoarsenic compounds in water samples and arsenobetaine in a certified reference material (DORM-2) were analyzed by this method.
Keywords: Polypyrrole; Solid phase microextraction (SPME); Speciation; Organoarsenic compounds; HPLC; ESI-MS;

Coupling on-line preconcentration by ion-exchange with ETAAS by Jianhua Wang; Elo Harald Hansen (223-232).
A novel way of exploiting flow injection/sequential injection (FIA/SIA) on-line ion-exchange preconcentration with detection by electrothermal atomic absorption spectrometry (ETAAS) is described and demonstrated for the determination of trace-levels of nickel. Based on the use of a renewable microcolumn incorporated within an integrated micro FI-system, the column is loaded with a defined volume of small beads of an SP Sephadex C-25 cation-exchange resin and subsequently exposed to a metered amount of sample solution. However, instead of eluting the retained analyte from the organic ion-exchange resin, the beads are along with 30 μl of carrier (buffer) solution transported via air segmentation directly into the graphite tube, where they are ashed during the pyrolysis and atomization process. The ETAAS determination is performed in parallel with the preconcentration process of the ensuing sample. An enrichment factor of 72.1, a detection limit of 9 ng l−1, along with a sampling frequency of 12 h−1 were obtained with 150 s of sample loading time at a sample flow rate of 12 μl s−1 (corresponding to 0.72 ml min−1). The relative standard deviations were 3.4%. The procedure was validated by determination of the nickel contents in two certified reference materials and in a human urine sample.
Keywords: FIA/SIA on-line ion-exchange; Renewable microcolumn; SP Sephadex C-25 cation-exchange resin; Electrothermal atomic absorption spectrometry; City waste incineration ash; River sediment; Human urine; Nickel;

2-Mercaptobenzimidazole, loaded on silica gel (MBI-SG) was used for preconcentration and speciation of iron(II) and iron(III) in water samples. The speciation is based upon selective retention of iron(III) on MBI-SG sorbent and quantitatively passing iron(II) ions. This type of sorbent was not only capable of separating theses two ions but also could concentrate them. The adsorbed iron(III) on MBI-SG was desorbed from the column by an SCN solution and transferred to the detection device. The effluent, then, was treated with hydrogen peroxide to oxidize iron(II) into iron(III) and subsequently was pumped through the MBI-SG column. A home-made flow injection system was coupled with a UV–VIS and an atomic absorption spectrometer, simultaneously. This method was applied to four different water samples and sub-ppb detection limit, high accuracy (>99% recovery), good precision (R.S.D.<5%), and good potential for automation were observed.
Keywords: Simultaneous preconcentration; 2-Mercaptobenzimidazole; Silica gel;

Combination of X-ray photoelectron and solid-state 13C nuclear magnetic resonance spectroscopy in the structural characterisation of humic acids by Fanny Monteil-Rivera; Eric B. Brouwer; Sonia Masset; Yves Deslandes; Jacques Dumonceau (243-255).
Leonardite humic acid (LHA) and soil humic acid (SHA), purchased from the IHSS, as well as purified humic acid from Aldrich (HA) are characterised by cross-polarisation magic angle spinning (CP-MAS) and Bloch decay 13C NMR spectroscopy and X-ray photoelectron spectroscopy (XPS). For the 13C CP-MAS NMR experiments, the effects of both the sample spinning sidebands and the influence of the cross-polarisation contact time are investigated. The functional groups distribution obtained by CP-MAS NMR is compared to the one resulting from chemical analyses. It is found that CP-MAS NMR overestimates the concentration of CO2H groups for certain humic compounds, whereas the concentration of phenolic groups is in reasonable agreement with the values determined by chemical methods. XPS was used as a complementary technique to understand the overestimation of carboxylic groups by NMR. The results from XPS, a surface characterisation technique, are in good agreement with those measured by elemental analyses of the bulk structure. High resolution C 1s and N 1s spectra show that the overestimation of the CO2H content by NMR is due to the presence of amide groups which give overlapping signals in the CO2H region of the NMR spectra.
Keywords: Humic acids; 13C MAS NMR; XPS; Carboxyl groups; Amides;

A simple, rapid and specific capillary electrophoresis (CE) method has been developed for the separation and determination of pseudoephedrine hydrochloride (PSED), paracetamol (PRCT) and dextromethorphan hydrochloride (DEMP) with UV detection. The CE was performed in 20 mmol/l sodium phosphate buffer solution (pH 7.0). The three ingredients were completely separated within 5 min, and quantified with an UV-detector at 200 nm. Relative standard deviations of 0.85% for PSED, 3.90% for PRCT and 0.84% for DEMP were achieved for seven injections of the sample containing about PSED of 30 μg/ml, PRCT of 325 μg/ml and DEMP of 15 μg/ml. The excellent linearity was obtained in the concentration range 300.0–2.5 μg/ml for PSED, 500.0–5.0 μg/m for PRCT and 500.0–10.0 μg/m for DEMP. The detection limits for PSED, PRCT and DEMP were 0.75, 1.20 and 2.50 μg/ml, respectively. The proposed method has been applied to the monitoring of commercial composite pseudoethedrine tablets and biological sample (urine). The analytical results were satisfactory.
Keywords: Capillary electrophoresis; Pseudoephedrine; Paracetamol; Dextromethorphan; Composite pseudoephedrine hydrochloride tablets;

The homogeneous liquid–liquid extraction method based on the pH dependent phase-separation phenomenon was studied in the water–acetic acid–chloroform ternary solvent system. The optimum conditions were determined as follows, i.e. in order to obtain a water-immiscible small phase (approximately 50 μl) of chloroform, sodium hydroxide ([NaOH]T=1.29 M) was added to a homogeneous aqueous solution which consisted of water, chloroform (0.2 ml, 0.57 vol.%) and acetic acid (2 ml, 5.71 vol.%), where the final total volume was 35 ml. During the extraction of the 5,10,15,20-tetrakis(4N-pyridyl)porphine (TPyP), Cu(II)- and Pd(II)-TPyP complexes, when the volume ratio (V W/V O) between the water phase and organic phase was 700, the distribution ratio (D) and extraction percentage (E%) were TPyP (5600, 88.9%), Cu(II)-TPyP (6000, 90%) and Pd(II)-TPyP (6700, 91%), respectively. This extraction method was applied to the HPLC system. The calibration graphs were linear and pass through the origin over the concentration range of 2×10−8–4×10−7  M for Cu(II) and 5×10−8–8×10−7  M for Pd(II). The relative standard deviations for the central value of the calibration graph were 3.2% for Cu(II) and 3.7% for Pd(II) (10 determinations). Moreover, the detection limits (S/N=3) were 5×10−9  M for Cu(II) and 8×10−9  M for Pd(II).
Keywords: Homogeneous liquid–liquid extraction; Water–acetic acid–chloroform ternary solvent system; High powered preconcentration method for HPLC; Simultaneous determination of copper(II) and palladium(II); Water-soluble porphyrin;

Two cyclodextrin derivatives, permethylated-β-CD and heptakis(2,6-di-O-pentyl-3-O-trifluoroacetyl)-β-CD, were mixed as CGC stationary phase to investigate synergistic effects. The results showed that synergistic effects existed on the mixed stationary phases of two CD derivatives and the biggest synergistic effect was observed on the 2:1 mixed stationary phase of permethylated-β-CD and heptakis(2,6-di-O-pentyl-3-O-trifluoroacetyl)-β-CD. Not only the properties of CD derivatives, but also the size and properties of solutes and the interaction mode between CD derivatives and solutes contributed to the occurring synergistic effect. The steric fitness between two CDs and solute is the main cause for the occurring of synergistic effects.
Keywords: Gas chromatography; Mixed stationary phase; Synergistic effect; Cyclodextrin derivative;

Differentiation of products derived from Iberian breed swine by electronic olfactometry (electronic nose) by Inmaculada González-Martı́n; José Luis Pérez-Pavón; Claudio González-Pérez; Jesús Hernández-Méndez; Noelia Álvarez-Garcı́a (279-287).
The aim of the present work was to differentiate products derived from Iberian breed swine, using chemometric analysis of the data obtained from an array of gas sensors. A commercial Electronic Nose (FOX 2000) comprising six metal oxide semiconductor sensors was used to generate a pattern of volatile compounds present in the samples. Linear discriminant analysis (LDA) and independent class modelling, i.e. soft independent modelling of class analogy (SIMCA) were applied to the patterns generated to achieve several classification tasks. The procedure for obtaining the signals and the chemometric treatment are rapid and simple, allowing the classification of products derived from Iberian swine according to the type of feeding regimen they have been subject to (feed, feed+acorn, acorns alone) using samples of subcutaneous adipose tissue.
Keywords: Differentiation; Iberian breed swine; Electronic olfactometry;

The determination of the total content and the leachable aliquot by aqua regia dissolution of eight heavy metals (Cd, Co, Cr, Cu, Mn, Ni, Pb, and Zn) in soils and sediments was developed by microwave digestion technique combined with inductively coupled plasma atomic emission spectrometry. A complete digestion of soils and sediments was achieved by using an acid mixture of HF–HCl–HNO3 (1:3:1); the microwave-irradiated closed vessel system used for the determination of aqua regia leachable quota, proved to be a viable alternative to the traditional reflux system. The experimental study was conducted using five BCR standard reference materials (CRM 141R ‘Calcareous Loam Soil’, CRM 142 ‘Light Sandy Soil’, CRM 143 ‘Sewage Sludge Amended Soil’, CRM 277 ‘Estuarine Sediment’ and CRM 320 ‘River Sediment’) in two laboratories with different microwave ovens and ICP-OES. Calculated recovery, repeatability, and reproducibility confirm the good performance of the procedures adopted. A Nested Design statistical analysis was carried out for both procedures and showed that the major source of variability in the analysis was due to ICP-OES measurements rather than microwave-assisted dissolution.
Keywords: Soil; Sediment; Heavy metals; Microwave digestion; Inductively coupled plasma optical emission spectrometry;

Index (297-299).