Analytica Chimica Acta (v.472, #1-2)

A rapid and simple liquid chromatographic method with photodiode array detection was developed for the simultaneous determination of six antiepileptic drugs (oxcarbazepine, carbamazepine, lamotrigine, phenobarbital, primidone and phenytoin) and two metabolites (10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine, the main active metabolites of carbamazepine and oxcarbazepine, respectively) in human plasma. Separation of the analytes was achieved in less than 11.5 min on a C18 column (150×4.0 mm, i.d. 4.5 μm) with a mobile phase composed of methanol, acetonitrile and a pH 3.0, 15 mM phosphate buffer containing 0.63% triethylamine (19.2:16.8:64.0, (v/v/v)), at 1 ml min−1 flow rate. Two procedures were tested for the pretreatment of human plasma samples: protein precipitation with perchloric acid, and solid-phase extraction. The protein precipitation procedure did not allow for the analysis of 10,11-dihydro-10,11-epoxycarbamazepine. On the contrary, solid-phase extraction with hydrophilic–lipophilic balance cartridges gave good results in terms of extraction efficiency and reproducibility and allowed for the determination of all analytes. The HPLC-DAD method developed, coupled to a careful SPE procedure, seems to be suitable for the plasma level determination of simultaneously administered antiepileptic drugs.
Keywords: Antiepileptic drugs; Liquid chromatography; Solid-phase extraction; Human plasma; Diode array detection;

Potentiometric detection of N,N′-diethylaminoethanol (DEAE) and a series of physiologically relevant lysosomotropic amino alcohols was demonstrated in cation exchange HPLC. Poly (vinyl chloride) (PVC) based liquid membrane electrode coatings were used which contained the lipophilic cation exchanger tetrakis(p-chlorophenyl)borate (TCPB) either alone or in combination with one of the macrocyclic ionophores hexakis-(2,3,6-tri-O-octyl)-α-cyclodextrin (CDX), dibenzo-18-crown-6 (CRW) or calix[6]arene hexaethylacetate ester (CLX). For all PVC membrane electrodes used, improved sensitivity over UV detection at 210 nm was obtained for the analyzed aliphatic amino alcohols. Especially, addition of CLX to the PVC-based membrane leads to highly enhanced detection limits for the low molecular weight lysosomotropic amino compounds, i.e. aminoethanol and methylamine. The performance of the proposed potentiometric detection method was demonstrated in cation exchange HPLC modes employing two different aqueous mobile phase systems with pH 2.30 comprising phosphoric or acetic acid with acetonitrile as organic modifier. Detection limits of 1.1×10−8 and 2.4×10−8  M (injected concentrations) were achieved, respectively, for N-methylaminoethanol and hexylamine by using a CLX based electrode. Sensitive and reliable determination of DEAE, a local anaesthetic agent, after liquid–liquid or SPE extraction from fortified bovine serum samples was obtained with the HPLC method coupled with the proposed potentiometric detection system. A linear free-energy relationship (LFER) based on a general solvation equation was used to predict detection limits of amino alcohols from their molecular structure in the developed potentiometric detectors employing CLX and CRW.
Keywords: Liquid membrane electrodes; HPLC; LFER; Diethylaminoethanol; Amino alcohols;

The fabrication and application of a electrochemical detector (ED) with the thioctic acid (TA)/iridium oxide (IrO2)–palladium (Pd) chemically modified electrode (CME) for liquid chromatography (LC) were described. The electrochemical behavior of dopamine (DA) and ascorbic acid (AA) at the TA/IrO2–Pd CME were investigated by differential pulse voltammetry (DPV). It was found that the CME could be permselective to DA cations and exile the AA anions, which could be used to determine the monoamine neurotransmitters and avoid the interference of AA in LC. In liquid chromatography with electrochemical detection (LC–ED), DA, norepinephrine (NE), 5-hydroxytryptamine (5-HT), epinephrine (E) and 3,4-dihydroxyphenylactic acid (DOPAC) were determined at the CME. The linear ranges of five analytes were over three orders magnitude and the limits of detection were 0.05 pmol for DA, 0.05 pmol for NE, 0.05 pmol for E, 0.10 pmol for 5-HT and 1.00 pmol for DOPAC. The application of this method coupled with microdialysis sampling for the in vivo determination of the monoamines in rat brain was also investigated.
Keywords: Thioctic acid/iridium oxide–palladium modified electrode; Neurotransmitter; LC–ED; Microdialysis sampling;

Determination of the sodium monofluorophosphate in a hardened cement paste by ion chromatography by Fabienne Farcas; Thierry Chaussadent; Christian Fiaud; Isabelle Mabille (37-43).
In order to prevent the corrosion of steel reinforcements in concrete, sodium monofluorophosphate (MFP) is proposed as a corrosion inhibitor. When directly applied onto the concrete surface, the inhibiting action of MFP depends on the amount of product that reaches the steel. Until now, the only approach considered for determining MFP concentration has been based on water extraction; this method allows identifying the active inhibiting aspect of MFP. Nevertheless, the results obtained using this method have yielded values close to zero, hence no information is available either on the total amount of MFP applied onto the concrete surface or on the nature of MFP-insoluble compounds.This paper proposes a rapid method for quantifying the total amount of MFP in cementitious materials; this method is based on acid extraction followed by ion chromatographic analysis.During a first step, a study is carried out on pure MFP in order to determine the total hydrolysis condition of this compound. Ion chromatograms display the total MFP hydrolysis in HPO4 2− and F ions for a pH close to 1. Acid extraction with 4% HNO3, as is typically used in cement related analysis, enables meeting this condition.In a second step, a 30 wt.% MFP solution is applied to a hardened cement paste (HCP). Quantification of the HPO4 2− chromatographic peak after acid extraction reflects the total MFP amount, with a detection limit of 5 mg of MFP per gram of HCP. Moreover, it appears that the other anions resulting from cement dissociation do not interfere with HPO4 2− quantification as long as the proportion of HPO4 2− potentially present in the cement has been taken into account. Chromatograms performed following water extraction lead neither to HPO4 2− nor MFP peaks, as previously noted, which indicates that the total MFP amount is precipitated as an insoluble compound. A complementary study shows that MFP reacts with the Ca(OH)2 present in HCP; the resulting compound is an apatite identified by X-ray diffraction.
Keywords: Sodium monofluorophosphate; Ion chromatography; Apatite; Hydrolysis; Phosphates; Cement paste;

Monoclonal antibody (MAb) against streptomycin was prepared by using a streptomycin–keyhole limpet hemocyanin conjugate for the immunization of mice. Splenocytes from BALB/c immunized mice were fused with P3X63Ag8U.1 myeloma cells. This resulted in one hybridoma cell line. Using this MAb, we developed quantitative assays for dihydrostreptomycin by means of an enzyme-linked immunosorbent assay (ELISA). Fifty percent inhibition concentration (IC50) for the MAb was 2.0 ng ml−1. The detection limit was 0.1 ng ml−1 and the standard deviations were 0.5–4.7% for intra-assay and 0.9–5.9% for inter-assay, respectively. The detection limit using peroxidase was 10 ng ml−1 in milk. Using the MAb produced, a rapid test kit based on the immunochromatographic method was developed. The detection limit using the kit was 100 ng ml−1 in milk.
Keywords: Dihydrostreptomycin; Monoclonal antibody; ELISA; Immunochromatographic rapid assay; Milk;

The high sensitive dynamic reaction cell inductively coupled plasma mass spectrometry (DRC ICP-MS)-based immunoassay using ferrocene (Fc) tethered hydroxysuccinimide ester as label for the determination of 2,4-dichlorophenoxyacetic acid (2,4-D) was presented. Ferrocene tethered hydroxysuccinimide ester was directly or via horse radish peroxidase (HRP) as a bridge coupled to monoclonal antibody (mAb) of 2,4-D. Competitive immunoreactions were completed in microtiter plate and the signal of 56 Fe in the bound ferrocene labeled conjugate was detected by DRC ICP-MS. The potentially interfering 40 Ar 16 O + at the iron mass m/z 56 was reduced in intensity significantly by using NH3 as the reaction gas. The optimization process of the immunoassay was greatly simplified in the aid of enzyme linked immunosorbent assay (ELISA) procedure. The 2,4-D was determined in the dynamic range of 0.1–1000 ng/ml and the detection limits of the assays using the two ferrocene labeled conjugates were found to be 0.044 and 0.055 ng/ml, respectively. The relative standard deviation for six measurements were within 5.5–15.3%.
Keywords: Immunoassay; Inductively coupled plasma mass spectrometry; Ferrocene labeled conjugate; 2,4-Dichlorophenoxyacetic acid;

The decrease in fluorescence of 7-methylguanosine (m7Guo) accompanying its phosphorolysis is readily applicable to monitoring the level of purine nucleoside phosphorylase (PNP) in mammalian whole blood lysates, without prepurification of the latter. A 10-fold higher sensitivity is attained with the use of 8-azaguanine (8-azaGua), a non-reversible, highly fluorogenic, substrate in the reverse, synthetic pathway. Although its affinity for human PNP is low (K m=250 μM at pH 7), its high reaction rate and fluorescence quantum yield of the product, 8-azaguanosine (ϕ=0.55 at pH 9), permit direct fluorometric monitoring of nucleoside synthesis in 1000-fold diluted hemolysates. It is also quite selective for mammalian enzymes, 8-azaGua being a poor substrate for bacterial (E. coli) PNP. Specificity of the assay was confirmed by the competitive effects of the natural purines hypoxanthine and adenine, with IC50 values comparable to their K m values. PNP levels in animal blood, assayed with both substrates, gave comparable results. With the exception of mice, PNP blood levels in animals were lower than in humans. We have also characterized specific fluorescent/fluorogenic substrates for bacterial (E. coli) PNP. The best of these is 2,6-diaminopurine riboside. Kinetic parameters for all the foregoing substrates are listed, and shown to be favorable for analytical applications.
Keywords: Purine nucleoside phosphorylase; 8-Azapurines; 7-Methylguanosine; Fluorescence; Fluorogenic substrates; Blood enzyme levels;

Chemiluminescence assay for catechin based on generation of hydrogen peroxide in basic solution by Hidetoshi Arakawa; Mahina Kanemitsu; Noriko Tajima; Masako Maeda (75-82).
We have determined that the catechin group in basic solution efficiently produces hydrogen peroxide; moreover, a highly sensitive analysis methodology was developed to measure catechin employing a peroxalate chemiluminescence detection system. Identification of hydrogen peroxide generated by catechin was determined by ESR as well as peroxalate chemiluminescence using catalase and SOD. As a result, catechin-generated superoxide by electron reduction to dissolved oxygen in basic solution, followed by production of hydrogen peroxide through dismutation reaction. This method could measure several tea catechins, (+)-catechin (CC), (−)-epigallocatechin-3-gallate (EGCg), (−)-epicatechin-3-gallate (ECG) and gallic acid, with measurement range from 10−7 to 10−3  mol/l and sensitivity of 10−8  mol/l. This method was also applied to the determination of total catechin levels in green tea, black tea and roasted green tea.
Keywords: Chemiluminescence; Catechin; Hydrogen peroxide; Reactive oxygen; Tea;

Blue and red photoluminescence of bicapped [60]fullerene-γ-cyclodextrin complex in aqueous solutions by Timo Ala-Kleme; Riikka Mäki; Paula Laaksonen; Keijo Haapakka (83-87).
For the first time, a blue and red photoluminescence of [60]fullerene in its aqueous sample solution is reported. The water solubility of [60]fullerene is achieved by its water-soluble bicapped complex with γ-cyclodextrin. Upon UV excitation at 320 nm, this complex produces intense blue and weak red photoluminescence at 440 and 710 nm which are assigned to the radiative 2t 1g (LUMO+1)→4h u (HOMO) and 5t 1u (LUMO)→4h u (HOMO) transitions of [60]fullerene, respectively. Further, the contribution demonstrates that the blue photoluminescence can be used for a selective probing of [60]fullerene in its mixtures with higher fullerenes even at trace level.
Keywords: Fullerene; Luminescence; γ-Cyclodextrin;

The frequency doubling scattering (FDS) and second-order scattering (SOS) intensities of procaine hydrochloride solution were weak. When β-cyclodextrin was added to the procaine hydrochloride solution, it can form a 1:1 inclusion complex with procaine hydrochloride established by spectrophotometry, and the FDS and SOS intensities were enhanced greatly. Their maximum scattering wavelengths appeared at 390 nm for FDS and 480 nm for SOS, respectively. The intensities of FDS and SOS at their maximum scattering wavelengths increased with an increase in β-cyclodextrin concentration. Based on this fact, the FDS and SOS methods were first applied to the determination of the inclusion constant of procaine hydrochloride to β-cyclodextrin. The inclusion constant, K f, of the procaine hydrochloride–β-cyclodextrin complex were 1.11×102−1.19×102  l mol−1 for the FDS and SOS methods. To compare with these methods, the spectrophotometric and fluorescence experiments have been done with satisfactory results.
Keywords: Cyclodextrin; Procaine hydrochloride; Inclusion constant; Frequency doubling scattering; Second-order scattering;

This paper describes two different approaches that attempt to solve the problem of signal shift between measurements acquired with gas-sensor array systems of identical construction. Both methods provide standardization models that can be used to compensate such instrument related signal shifts by postprocessing of the measurement data. The first approach is a straightforward univariate direct standardization method, based on linear regression, where unique shift compensation models are created for each sensor. The other approach is a multivariate method, based on partial least squares regression, which can be used to design shift compensation models for the whole gas-sensor array. Both methods effectively removed signal shift after being applied on measurement data acquired with five commercial instruments of identical configuration with quartz micro balance (QMB) sensor arrays.
Keywords: Gas-sensors; Signal shift; Data standardization; Algorithms; Univariate; Multivariate; Direct standardization; PLS2;

The formation of a Prussian blue film under and above a polypyrrole film containing formate dehydrogenase and β-nicotinamide adenine dinucleotide has been investigated by electrochemical quartz crystal microbalance and cyclic voltammetry techniques. Two different deposition mechanisms of Prussian blue were identified and explained when the polypyrrole coated substrate electrode was used. Very stable current and frequency responses were obtained when the Prussian blue film was deposited under the polypyrrole film. This layered structure is potentially useful in solving the leakage problems of a biosensing system involves immobilised mediator. This advantage has been demonstrated using a formate biosensor fabricated with the enzyme, formate dehydrogenase, a co-factor, β-nicotinamide adenine dinucleotide and a Prussian blue mediator.
Keywords: Biosensor; Prussian blue; Mediator; Polypyrrole; Quartz crystal microbalance;

Amperometric determination of ethanol in beverages at copper electrodes in alkaline medium by Thiago R.L.C Paixão; Dennys Corbo; Mauro Bertotti (123-131).
The anodic oxidation of ethanol at copper surfaces in alkaline medium has been reported. The participation of electrogenerated Cu(III) species in the electrocatalysis of the oxidation of ethanol at copper surfaces was investigated by rotating ring-disc voltammetry and a mechanism involving the participation of radicals was proposed. The copper oxide sensor was assembled in a wall-jet electrochemical cell and a FIA method that incorporates flow extraction of ethanol through a PTFE membrane at room temperature was developed. Under optimal experimental conditions the peak current response (measured at 0.5 V versus Ag/AgCl) increases linearly with ethanol concentration over the range 2–10% (v/v). The repeatability of the electrode response in the FIA configuration was evaluated as 3% (n=20) and the limit of detection of the method was 0.4% (S/N=3). The analytical frequency was estimated to be 120 h−1. Experiments to investigate the selective of the PTFE membrane were carried out and they demonstrated that only small molecules such as methanol may interfere in the determination of ethanol. For some samples of different alcoholic beverages, the results showed excellent agreement (r=0.998) with the ones obtained by using gas chromatography.
Keywords: Ethanol; Copper electrodes; Amperometric detection; Flow injection analysis;

Determination of the ethylene oxide content of polyether polyols by low-field 1 H nuclear magnetic resonance spectrometry by Alison Nordon; Céline Meunier; Robert H Carr; Paul J Gemperline; David Littlejohn (133-140).
Methods have been developed and compared for the analysis of a glycerol-based polyether polyol using a low-field, medium-resolution NMR spectrometer, with an operating frequency of 29 MHz for 1 H . Signal areas in the time and frequency domains were used to calculate the ethylene oxide (EO) content of individual samples. The time domain signals (free induction decay) were analysed using a new version of the direct exponential curve resolution algorithm (FID-DECRA). Direct analysis of the 1 H NMR FT spectra gave percentage EO concentrations of reasonable accuracy (average percentage error of 1.3%) and precision (average RSD of 1.8%) when compared with results derived from high-field 13 C NMR spectrometry. The direct FID-DECRA method showed a negative bias (−0.8±0.12% w/w) in the estimation of percentage EO concentration, but the precision (average RSD of 0.9%) was twice as good as that of direct spectral analysis. When the 13 C NMR analysis was used as a reference method for univariate calibration of the 1 H NMR procedures, the best accuracy (average percentage error of 0.5%) and precision (average RSD of 0.6%) were obtained using FID-DECRA, for EO concentrations in the range 14.8–15.5% w/w. An additional advantage of FID-DECRA is that the analytical procedure could be automated, which is particularly desirable for process analysis.
Keywords: Low-field NMR spectrometry; Process analysis; Polyether polyols; Ethylene oxide; Direct exponential curve resolution algorithm;

A mechanised flow system is described aimed at extracting and pre-concentrating metal ions in a renewable water-insoluble solid phase containing a suitable ligand. The main difference between the proposed system and previously described ones for use with liquid–solid extraction is in the renewable characteristics of the solid phase, which allows for refreshing the extracting surface to be employed between pre-concentration operations. The renewable feature imposes no demands in terms of reversibility of the adsorption process as all the solid organic phase containing the complexed metal is dissolved by a non-aqueous solvent and carried to the detection system. Simultaneously, the system promotes a better compromise between metal extraction and pre-concentration as lower solvent volumes can be used to recover the absorbed species. The system has been evaluated for Zn(II) extraction in order to optimise its main geometrical and chemical variables. A simple spectrophotometric detection system employing a light emitting diode (LED) and a silicon photodiode was employed in these studies. Concentration gains of 42 and detection limits below 1.0 μg l−1 were obtained. Sample throughput and sample consume depend on the desired pre-concentration. For higher (>40) concentration factors, about 40 ml of aqueous sample and a total processing time of 20 min per sample are required.
Keywords: Renewable; Photodiode; Spectrophotometric;

A continuous-flow sequential extraction system developed in our laboratory for metal fractionation in solid materials has shown many advantages over the batch system such as speed of extraction, simple procedure, less risk of contamination and less dependency on extraction conditions. This report emphasizes the reduction of readsorption problem achieved from this system. Five metals (Pb, Cu, Zn, Ni and Cd) were studied because of their significance as environmental contaminants. A method of standard addition into extractants was used to evaluate readsorption phenomena in a three-stage sequential extraction procedure proposed by the Commission of the European Communities Bureau of Reference (BCR). The continuous-flow system showed less degree of readsorption compared with the batch system. The extent of readsorption was found to be dependent on elements, properties of soil especially organic matter content, extracting flow rate and metal concentration level. Metal readsorption from acid soluble fraction to oxidizable fraction was greater for soil of high organic matter than soil of low organic matter content. Lower extracting flow rate and lower concentration of metals in sample showed lower degree of readsorption. The behavior of metal readsorption during extraction was observed as two patterns. In pattern one, readsorption was observed at the earlier step and the adsorbed metal was leached in the following step. In pattern two, readsorption and desorption were observed in the same extraction step.
Keywords: Readsorption; Continuous-flow; Sequential extraction; Heavy metals;

Various models have been constructed and analysed for eight denominations of origin of wines bottled in the Canary Islands, using their content in different metals, with the Soft Independent Modelling Class Analogy (SIMCA) technique. The metals were grouped in three blocks: “rare earths”, “lead isotope ratios” and “other metals”. The model constructed with all the variables was taken as the reference model. This model has adequate sensibility and specificity. The contribution of each element to the model of each denomination of origin (DOs), as well as their capacity to discriminate between the DOs is shown. Cluster analysis, using the Ward method, of the SIMCA distances between the different DOs reveals the similarity of the different denominations of origin. Using only the rare earths or the lead isotope ratios it is not possible to construct adequate models for the different DOs given the low specificity obtained. The models constructed with the other metals, alone or combined with the lead isotope ratios, gave similar results to those obtained using all the variables. Finally, the similarity of the models was analysed by a weighted distance between the sensibilities and specificities of each of them compared with the rest. Cluster analysis using the Ward method shows the models which are similar as to their sensibility and specificity. The innovative aspect of the work lies in the use of cluster analysis to demonstrate the similarity between the SIMCA boxes of a model, and the definition of the distance between models based on the sensibility and specificity for the eight DOs with the five groups of variables considered.
Keywords: SIMCA models; Model similarity; Cluster analysis; Canarian wines; Trace and ultratrace metals; ICP–MS;

Author Index (175-176).