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Analytica Chimica Acta (v.538, #1-2)

Outside front cover - Editorial Board (pp. co1).

Calendar of forthcoming meetings (pp. n1-n3).

Contents (pp. v-vii).

Filler Ad.: ACAEES (pp. ii).

Screen-printed electrode microarray for electrochemiluminescent measurements by Benjamin P. Corgier; Christophe A. Marquette; Lo�c J. Blum (pp. 1-7).

A microarray of nine screen-printed (SP) graphite electrodes was used to develop multi-parametric electrochemiluminescent (ECL) biochip. The whole biochip is based on the ECL detection of enzymatically generated H₂O₂. The intrinsic performances of this electrode array were evaluated through the cyclic-voltammetric experiments in 0.1M ferricyanide. The variations of the specific behaviours (oxidation potential and current) of such a redox mediator were then used to reach the reproducibility of the SP arrays, i.e. 2% for electrode-to-electrode and 4% for biochip-to-biochip variations. In a similar way, electrochemiluminescent cyclic-voltammetric experiments were performed to characterize the ECL reaction triggered at the surface of a SP carbon electrode array. An optimum luminol oxidation potential of +650mV versus platinum pseudo-reference was found with a reproducibility of 4.4%.The different sensing layers were obtained through the entrapment of glucose oxidase and lactate oxidase in PVA–SbQ (poly(vinyl alcohol) bearing styrylpyridinium groups) photopolymer deposited at the surface of two from the nine electrode array. An automatic nano-spotter was employed to precisely drop spots of entrapped enzyme at an electrode surface.Therefore, ECL measurements were performed to establish calibration curves by using a charged coupled device (CCD) camera. Linear ranges, detection limits and reproducibility of these biochips were investigated as a demonstrative example of oxidase-based ECL detection/biosensor. In the present paper, the achieved biosensor arrays allowed the simultaneous detection and quantification ofl-lactate andd-glucose with detection limits of 3 and 10μM, respectively.

Keywords: Carbon electrode arrays; Biochip; Electrochemiluminescence; Glucose; Lactate; Screen-printing

Non-covalent imprinting of phosphorous esters by Andrew J. Hall; Francesca Lanza-Sellergren; Panagiotis Manesiotis; B�rje Sellergren (pp. 9-14).

The creation of molecularly imprinted polymers (MIPs) for the recognition of phosphate and phosphonate esters is reported. The single, weak hydrogen-bond acceptor site in these molecules has been targeted using a 1,3-diarylurea functional monomer. Polymers were prepared using either stoichiometric ratios of functional monomer to template or a large excess of the template during imprinting. The recognition properties of the polymers were assessed in the chromatographic mode for their ability to retain the templates and analogous analytes. The results are discussed with regards to the choice and amount of template, polymerisation conditions and the composition of the chromatographic mobile phase.

Keywords: Imprinting; Polymerisable urea; Phosphorous esters; Molecular recognition

Enantiomeric separation of cyclopropane derivatives on a polysaccharide-based chiral stationary phase by Ashraf Ghanem; Hubert Hoenen; Paul M�ller; Hassan Y. Aboul-Enein (pp. 15-24).

The enantioseparation of a set of new cyclopropanes derived from Meldrum's acid, dimethyl malonate, (silanoxyvinyl)-diazoacetates and 3,3,3-trifluoro-2-diazopropionate has been achieved on cellulose tris(3,5-dimethylphenylcarbamate) Chiralcel OD coated on 10μm silica-gel as chiral stationary phase in high-performance liquid chromatography (HPLC). The HPLC was equipped with a sensitive optical rotation detector for monitoring the optical activity of the racemic and enantioenriched cyclopropanes prepared via the Rh(II)-catalyzed asymmetric intermolecular cyclopropanations. The mobile phase was a mixture of n-hexane-2-propanol (90:10, or 99:1, v/v) with a flow-rate of 0.3ml/min. The UV detector was set at 254nm. The advantages of the liquid over the gas chromatographic analysis of the cyclopropanes are demonstrated by a comparative study of both methods. The methods provide an easy and direct determination of the enantiomeric excesses of the cyclopropanes.

Keywords: Cellulose tris(3,5-dimethylphenylcarbamate); Cyclopropanes; Enantiomeric excess; Enantioseparation; HPLC; Polysaccharide chiral stationary phase; Rhodium complexes

Stereoselective determination of the major ibuprofen metabolites in human urine by off-line coupling solid-phase microextraction and high-performance liquid chromatography by Anderson Rodrigo Moraes de Oliveira; Fernando Jos� Malague�o de Santana; Pierina Sueli Bonato (pp. 25-34).

A simple, rapid and sensitive off-line solid-phase microextraction method coupled to high-performance liquid chromatography was developed for the stereoselective analysis of the major ibuprofen metabolites in human urine samples. For this purpose, a carbowax/templated/resin coated fiber was used. Several parameters influencing the efficiency of the analyte extraction were explored. The chromatographic separation was achieved on a Chiralpak AS column using hexane:isopropanol (95:5v/v) plus 0.05% trifluoroacetic acid as the mobile phase, at a flow-rate of 1.2mL/min. Detection was carried out at 230nm. The mean recoveries were between 1.4 and 1.6%, with R.S.D. values lower than 9.5 for both 2-OHIbu enantiomers, and between 3.7 and 5.7%, with R.S.D. values lower than 13.4 for all four COOHIbu stereoisomers. The method was linear over the 5–50μg/mL concentration range and the quantification limit was 5μg/mL for all metabolite stereoisomers. Within-day and between-day assay precision and accuracy for both metabolites were studied at three concentration levels of each stereoisomer (15, 30 and 45μg/mL) and were lower than 15%.

Keywords: Off-line solid-phase microextraction; Ibuprofen metabolites; 2-Hydroxyibuprofen; Carboxyibuprofen; Enantiomeric separation; Urine

On-line solid-phase extraction and HPLC determination of polycyclic aromatic hydrocarbons in water using fluorocarbon polymer sorbents by Lubov Oliferova; Mikhail Statkus; Grigoriy Tsysin; Oleg Shpigun; Yury Zolotov (pp. 35-40).

Polyvinylidenefluoride (fluorocarbon polymer sorbent) with surface area 8.4m²g⁻¹ and particle size 15–30μm was used for on-line SPE–HPLC determination of naphthalene, biphenyl, acenaphthene, anthracene and pyrene in natural water. Desorption eluent flow rates were varied from 0.50 to 1.75mlmin⁻¹ and the volume of analyte-containing eluent stream zone was 0.10–0.11ml, while empty preconcentration column volume is 0.10ml. Detection limits of SPE–HPLC method were established: 5ngl⁻¹ (biphenyl), 7ngl⁻¹ (anthracene), 8ngl⁻¹ (acenaphthene), 30ngl⁻¹ (pyrene), 40ngl⁻¹ (naphthalene), using UV spectrophotometric detection. Several samples of natural and potable water were spiked with mixture of PAHs, thus validating the proposed technique.

Keywords: On-line; SPE; HPLC; PAH; Fluorocarbon polymers; Sorption

Development of a headspace solid-phase microextraction method combined with gas chromatography mass spectrometry for the determination of phthalate esters in cow milk by Yong-Lai Feng; Jiping Zhu; Robert Sensenstein (pp. 41-48).

Phthalate esters released from plasticized polyvinyl chloride (PVC) tubing into raw cow milk during milking at dairy farms could be a potential source of contamination by phthalate esters in dairy products. A method was developed for the determination of these phthalate esters in raw cow milk samples using a headspace solid-phase microextraction (HS-SPME) technique. The milk samples were mixed with sodium chloride and extracted for 60min at 90°C. The phthalates collected on the SPME fibre were then desorbed in the GC injection port followed by GC-MS analysis in single ion monitoring (SIM) mode. The extraction efficiency of the SPME fibre was dependent on the fat content in the milk sample. Since the fat content in each cow milk sample was different, a combined standard addition and internal standard method was used for the quantification of the phthalate esters in milk samples. The recoveries at two spiking levels were over 90% except for dimethyl phthalate. The method detection limit for di-(2-ethylhexyl) phthalate (DEHP) was from 0.31 to 3.3ngg⁻¹ for samples containing up to 10.8% fat. This method was sufficiently sensitive to detect di-(2-ethylhexyl) phthalate, dibutyl phthalate (DBP) and diethyl phthalate (DEP) in two groups of raw cow milk samples (cow milk samples collected using and without using PVC tubing). While similar levels were found in both types of samples for DBP and DEP, the level of DEHP was much higher in samples collected using PVC tubing (215.36ngg⁻¹) than once without (16.04ngg⁻¹), indicating potential leaching of DEHP from PVC tubing into raw cow milk.

Keywords: Cow milk; di-(2-Ethylhexyl) phthalate; Fat content; Headspace solid-phase microextraction; Phthalate esters; Polyvinyl chloride tubing

Solid phase microextraction with liquid chromatography–electrospray ionization–tandem mass spectrometry for analysis of amphetamine and methamphetamine in serum by Chi-Chi Chou; Maw-Rong Lee (pp. 49-56).

This investigation evaluates solid phase microextraction (SPME) combined with liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) to determine trace levels of amphetamine (AM) and methamphetamine (MA) in serum. Chromatography was performed on a C₁₈ reversed-phase column using a linear gradient of acetonitrile–water as a mobile phase. Using sample extraction by SPME at optimum conditions, analytes were desorbed with static desorption in SPME–HPLC desorption chamber for 1min and then determined with LC–ESI–MS/MS. The detection limits for amphetamine and methamphetamine in serum were 0.3μg/L and 0.04μg/L, respectively. According to the analysis, the linearity range is from 0.5μg/L to 200μg/L with R.S.D. values below 8%. The feasibility of applying the methods to determine amphetamine and methamphetamine in real samples was examined by analyzing serum samples from methamphetamine abused suspects.

Keywords: Amphetamine and methamphetamine; Serum; Solid phase microextraction; LC–ESI–MS/MS

Development of a new clean-up tandem assay column for the detection of ochratoxin A in roasted coffee by M. Lobeau; S. De Saeger; L. Sibanda; I. Barna-Vetr�; C. Van Peteghem (pp. 57-61).

As the mycotoxin ochratoxin A (OTA) can occur in roasted coffee, regulations and maximum limits are necessary. The need for simple, rapid and inexpensive detection methods which require a minimum of equipment led to the development of a clean-up tandem assay column for the detection of OTA in roasted coffee. In this study the column comprised two superposed layers: a layer capable of adsorbing at least part of the interfering fraction of the sample and a detection layer containing antibodies that were able to capture the analyte. No expensive instruments were needed as results were visually evaluated. The cut-off level, i.e. the smallest mycotoxin concentration resulting in no colour development, was 6μgkg⁻¹. Assay validation was performed using samples fortified with OTA. The method gave a low percentage of false negative results and no false positive results. Assay performance was evaluated by screening naturally contaminated samples using the clean-up tandem assay column. The described method offers a rapid, simple and cost-effective screening tool, contributing to more effective quality control procedures.

Keywords: Abbreviations; HRP; horse-radish peroxidase; Ig; immunoglobulin; OTA; ochratoxin A; PBS; phosphate-buffered saline; EC; European commission; IARC; International Agency for Research on Cancer; HPLC-FLD; high performance liquid chromatography-fluorescence detection; ELISA; enzyme-linked immunosorbent assay; AOAC; Association of Official Analytical Chemists; rpm; revolutions per minuteOchratoxin A; Roasted coffee; Immunoassay; Field test; Column test

Determination of phenolic compounds in wastewater samples using a novel fiber by solid-phase microextraction coupled to gas chromatography by Furong Zhou; Xiujuan Li; Zhaorui Zeng (pp. 63-70).

Headspace solid-phase microextraction (SPME) coupled to gas chromatography-flame ionization detection (GC-FID) has been applied to the analysis of six phenolic compounds in water samples. A laboratory-made fiber with 25,27-dihydroxy-26,28-oxy(2′,7′-dioxo-3′,6′-diazaoctyl)oxy- p- tert-butylcalix[4]arene/hydroxy-terminated silicone oil (Amide bridged-C[4]/OH-TSO) coating was used to extract the analytes from the aqueous samples. The parameters affecting the sorption of analytes onto the fiber, such as extraction temperature and time, pH, salt addition, desorption temperature and time, were optimized. The extraction ability of this new fiber to phenols was compared with the laboratory-made C[4]/OH-TSO fiber and the commercially available polyacrylate (PA, 85μm) fiber. The results revealed that this new calixarene fiber shows high affinity for the phenolic compounds due to the introduction of the polar Amide bridge in calix[4]arene. The linearity was three or four orders of magnitude for the tested compounds with regression coefficients ranging from 0.9968 to 0.9996. The detection limits ranged from 0.47 to 9.01μg/l and the reproducibility of the measurements was <9.10%. The established method was then applied to determine the pollutant concentration in real wastewater samples from a coking plant, contaminated with phenols. The recoveries of phenolic compounds studied in wastewater were from 89.7 to 103.2% with the new fiber.

Keywords: Solid-phase microextraction; Calix[4]arene; Gas chromatography; Wastewater analysis; Phenols

Determination of carotene, tocopherols and tocotrienols in residue oil from palm pressed fiber using pressurized liquid extraction-normal phase liquid chromatography by M. Marsin Sanagi; H.H. See; Wan Aini Wan Ibrahim; Ahmedy Abu Naim (pp. 71-76).

Pressurized liquid extraction (PLE) along with normal phase liquid chromatography (NPLC) is evaluated as a new approach for the determination of β-carotene and Vitamin E isomers in residue oil obtained from palm pressed fiber (PPF). The relative extraction recoveries obtained by optimized PLE were found to be equivalent (∼100%) to conventional Soxhlet extraction. Optimized PLE was obtained (5g of sample, 80°C, 1500psi, 2× 10min static extractions with flush volume 50%) combined with NPLC system. Typical extractions give 3.7–4.0mgml⁻¹ of Vitamin E isomers and β-carotene of 3.3–3.5mgml⁻¹, which is similar to or greater than classical Soxhlet extraction with n-hexane or chloroform. The new developed method demonstrated an acceptable performance with good efficiency in terms of total extraction time, total solvent usage, total β-carotene and Vitamin E isomers contents as well as exceptional method repeatability.

Keywords: Pressurized liquid extraction; Liquid chromatography; Tocopherol; Tocotrienol; Palm oil

Determination of triamterene in pharmaceutical formulations and of triamterene and its main metabolite hydroxytriamterene sulfate in urine using solid-phase and aqueous solution luminescence by G.A. Iba�ez; G.M. Escandar; A. Espinosa Mansilla; A. Mu�oz de la Pe�a (pp. 77-84).

Solid-phase extraction (SPE) (reversed-phase and mixed-mode) and nylon membranes were investigated as solid matrices for obtaining fluorescence signals from adsorbed triamterene (TA). Although a significant emission was found in all investigated surfaces, solid-phase extraction systems yielded the best results. The analytical figures of merit obtained under the best experimental conditions, using nonpolar/strong cation mixed phase MP1 discs and 5ml of extracting sample, were: linear calibration range from 1.7 to 21ngml⁻¹ (the lowest value corresponds to the quantitation limit), relative standard deviation, 1.3% ( n=5) at a level of 12.7ngml⁻¹, and limit of detection, 0.6ngml⁻¹ (calculated according to Clayton's definition, taking into account false positive and false negative errors). An approach for the spectrofluorimetric determination of triamterene in pharmaceutical formulations was successfully applied. In addition, a simple and rapid method for the separation and fluorimetric determination of triamterene and its main metabolite (hydroxytriamterene sulfate) in urine was developed and rendered very satisfactory results, requiring in this case the use of reversed-phase C18 solid-phase extraction.

Keywords: Room temperature fluorimetry; Solid-phase extraction; Triamterene; Hydroxytriamterene sulfate

Highly selective determination of inorganic mercury(II) after preconcentration with Hg(II)-imprinted diazoaminobenzene–vinylpyridine copolymers by Yongwen Liu; Xijun Chang; Dong Yang; Yong Guo; Shuangming Meng (pp. 85-91).

Hg(II) imprinted and non-imprinted copolymers were prepared by metal ion imprinted polymer (MIIP) technique. The copolymers were obtained by copolymerizing mercury chloride (or without it), diazoaminobenzene (DAAB) and vinylpyridine (VP) using ethyleneglycoldimethacrylate (EGDMA) as cross-linker in the presence of 2,2′-azobisisobutryonitrile as initiator. The separation and preconcentration characteristics of the copolymers for Hg(II) were investigated by batch and column procedures. The results demonstrated that the Hg(II) imprinted copolymers had higher adsorption capacity with the value of 205μmolg⁻¹ of dry copolymer, about 3.5 times of and good selectivity for Hg(II) compare to non-imprinted copolymers (58.6μmolg⁻¹ of dry copolymer). The distribution ratio ( D) values of the Hg(II) imprinted copolymers increased for Hg(II) with respect to both D values of Zn(II), Cu(II), Cd(II), CH₃HgCl, CH₃CH₂HgCl and non-imprinted copolymer. The relatively selective factor ( α_r) values of Hg(II)/Cu(II), Hg(II)/Zn(II), Hg(II)/Cd(II), Hg(II)/CH₃HgCl and Hg(II)/CH₃CH₂HgCl are 45.5, 63.5, 55.1, 49.6 and 84.0, respectively. The tolerance limit for other electrolytes such as NaCl, KBr, KI, NaNO₃, Na₃PO₄, Na₂SO₄ and Mg(NO₃)₂ is also greatly improved with the values of 10–100-fold of the non-imprinted copolymer. The Hg(II) imprinted copolymers can be used at least 20 times with recoveries no less than 95%. Based on the packed columns with Hg(II) imprinted copolymers, a highly selective solid-phase extraction (SPE) and preconcentration method for Hg(II) from dilute aqueous solution was developed. The MIIP-SPE preconcentration procedure showed a linear calibration curve within concentration range from 0.13 to 25μgl⁻¹. The detection limit and quantification limit were 0.05 and 0.13μgl⁻¹ (3 σ) for cold vapor atomic absorption spectrometry (CVAAS). The relative standard deviation of the eleven replicate determinations was 2.4% for the determination of 1.0μg of Hg(II) in 100ml water sample. Determination of Hg(II) in certified river sediment sample (GBW 08603) demonstrated that the column was good enough for Hg(II) determination in matrixes containing components with similar chemical property such as Cu(II), Zn(II), Cd(II), CH₃HgCl and CH₃CH₂HgCl.

Keywords: Solid-phase extraction (SPE); Metal ion imprinted polymer (MIIP); Preconcentration; Hg(II); Cold vapor atomic absorption spectrometry (CVAAS); Water

Fractionation of arsenic in soil and sludge samples: continuous-flow extraction using rotating coiled columns versus batch sequential extraction by P.S. Fedotov; W.J. Fitz; R. Wennrich; P. Morgenstern; W.W. Wenzel (pp. 93-98).

Fractionation of arsenic and other trace elements (TE) in environmental solid samples is usually achieved by application of batch sequential extraction procedures (SEP). In the present work a rotating coiled column (RCC) earlier used mainly in countercurrent chromatography has been applied to study continuous-flow fractionation (CFF) of As compared to conventional batch SEP. A particulate solid sample (soil or sludge) was retained in the rotating column as the stationary phase under the action of centrifugal forces while different eluents were continuously pumped through. The eluents were applied in correspondence with an optimized SEP for As addressing non-specifically sorbed, specifically sorbed, bound to amorphous and crystalline Fe oxides fractions extractable by (NH₄)₂SO₄, NH₄H₂PO₄, NH₄-oxalate buffer, NH₄-oxalate buffer+ascorbic acid at 96�C, respectively. For all samples the recoveries of As in the non-specifically sorbed fraction obtained by CFF were considerably higher than in the corresponding fraction of the batch SEP. This finding is consistent with a quickly obtained equilibrium between the solid phase and the ammonium sulphate solution in batch where no further extraction takes place. The repeated extraction occurring due to the continuous removal of the eluate in the CFF procedure enables a more complete extraction of the non-specifically adsorbed pool. For the two consecutive steps, both methods delivered virtually the same results. In contrast, since no heating was used in RCC batch SEP more efficiently extracted As bound to crystalline Fe oxides at step 4. As a result, the residual fraction was overestimated after CFF. The CFF procedure can be completely automated. In addition, investigating the elution curves allows one to study the kinetics of As (and related elements of interest) release, to test efficacy of extractants and to assess the maximum pool size of target fractions.

Keywords: Continuous-flow fractionation; Sequential extraction; Arsenic; Soil; Sludge

Qualitative and quantitative speciation analysis of water soluble selenium in three edible wild mushrooms species by liquid chromatography using post-column isotope dilution ICP–MS by Vanesa D�az Huerta; Mar�a Luisa Fern�ndez S�nchez; Alfredo Sanz-Medel (pp. 99-105).

Speciation analysis of Se in hot water extracts of three edible wild mushroom species ( Macrolepiota procera, Lepista luscina and Boletus luridus) has been performed to investigate Se species naturally occurring in wild mushrooms. Mushroom extracts have been analysed using size exclusion, reversed-phase and anion exchange chromatographies. Those chromatographic separation systems were coupled on-line to an ICP–MS, which was equipped with an octopole reaction system using H₂ as reaction gas to eliminate interfering argon dimers in Se detection. Post-column isotope dilution analysis has been used on-line with the separations to quantify Se in the eluting peaks by converting conventional intensity chromatograms into mass flow chromatograms (previous adequate mathematical corrections).⁷⁸Se/⁷⁷Se ratio was used, after correction of intensity signals for SeH⁺ formation, to quantify both total Se and individual selenocompounds. Extraction efficiencies in hot water between 47 and 91% were obtained for Se, depending on the mushroom considered. Selenomethionine was found to be the major compound of Se in the aqueous extracts of the three types of mushroom investigated. A number of unknown selenocompounds, all of them corresponding to low molecular weight species, were also detected and their Se content was quantified.

Keywords: Wild mushroom; Se speciation; Isotope dilution analysis

Determination of vanadium species in sediment, mussel and fish muscle tissue samples by liquid chromatography–inductively coupled plasma-mass spectrometry by Marinela Colina; P.H.E. Gardiner; Zulay Rivas; Federico Troncone (pp. 107-115).

Vanadium is introduced into the environment during the extraction of petrochemical products and in the production of steels and insecticides. In this study, a liquid chromatographic method for the separation of V(IV) and V(V) as ethylenediaminetetra acetic acid (EDTA) complexes was developed using reversed-phase ion-pair liquid chromatography with inductively coupled plasma-mass spectrometry detection. A C-8 reversed-phase column, 15cm long, was used to separate the species. A solution containing ammonium acetate 0.06M, tetrabutylammonium hydroxide 10mM, ammonium di-phosphate 10mM and EDTA 2.5mM at pH 6 was used as the mobile phase in order to avoid the use of organic solvents that reduce the sensitivity of the determination. To prevent changes in distribution of the vanadium species, samples should be prepared freshly. The method developed was applied to the study the vanadium speciation in sediment, mussel and fish muscle samples collected from Lake Maracaibo, Venezuela. The concentration ranges of V(IV) and V(V) in sediment samples were 0.7–61 and 1.4–2.3μgg⁻¹, respectively. The method is simple and has adequate sensitivity for these practical applications.

Keywords: Vanadium speciation; Inductively coupled plasma-mass spectrometry; Lake Maracaibo; Sediments; Fish; Mussel

Monitoring of pesticides in agricultural water and soil samples from Andalusia by liquid chromatography coupled to mass spectrometry by A. Belmonte Vega; A. Garrido Frenich; J.L. Mart�nez Vidal (pp. 117-127).

This work describes the development of an analytical method for determining 32 pesticides, widely used in agricultural areas from the south of Spain, by liquid chromatography–mass spectrometry (LC–MS) with electrospray ionization (ESI) in positive mode. The chromatographic and the MS parameters of the studied compounds were optimized in order to improve selectivity and sensitivity. The separation was carried out in a C₁₈ column using as mobile phase methanol (MeOH) with 0.01% acetic acid (A) and 0.01% acetic acid (B) with a flow of 0.2mL/min in gradient mode. The molecular ion was selected for the quantification in selective ion monitoring (SIM) mode. A solid-phase extraction (SPE) method with HLB Oasis cartridges was applied to allow preconcentration of the target analytes from water samples. However, extraction of the compounds from soil samples was carried out by sonication using liquid–solid extraction with methanol/water. In addition, a clean-up step by SPE using also HLB Oasis cartridges was necessary for soils. The proposed analytical methodology was validated in the target environmental matrices by the analysis of spiked blank matrix samples. Detection limits (LOD) varied between 10 and 50ng/L for water and 0.5–2.5μg/kg for soils, while quantification limits (LOQ) ranged from 25 to 50ng/L for water and 1.5 to 5.0μg/kg for soils. Good recoveries (60–110%) and precision values (<15%) were obtained for all compounds in the target matrices. This methodology has been successfully applied to the analysis of incurred water and soil samples from agricultural areas of Andalusia (south Spain). The results revealed the presence of some pesticides at several concentration levels both in the soil and water samples. Carbendazim, simazine and diuron were the pesticides most frequently detected in water samples, while only the herbicides simazine, diuron and terbutylazine were found in soil samples.

Keywords: Pesticides; Liquid chromatography; Mass spectrometry; Water; Soils analysis

Simultaneous analysis of N-nornuciferine, O-nornuciferine, nuciferine, and roemerine in leaves of Nelumbo nucifera Gaertn by high-performance liquid chromatography–photodiode array detection–electrospray mass spectrometry by Xubiao Luo; Bo Chen; Jingjing Liu; Shouzhuo Yao (pp. 129-133).

A novel method has been developed for analysis of N-nornuciferine, O-nornuciferine, nuciferine, and roemerine in leaves of Nelumbo nucifera Gaertn by using high-performance liquid chromatography (HPLC)–photodiode array detection (DAD)–electrospray mass spectrometry (ESI-MS). The method was carried out by using a Shimadzu VP-ODS column with a gradient solvent system of 0.1% triethylamine aqueous solution–acetonitrile. N-Nornuciferine, O-nornuciferine, nuciferine, and roemerine were identified with authentic standard compounds and with MS-spectra. The contents of these compounds were measured by employing DAD. Linearity of around three orders in the magnitude of concentration was generally obtained and limits of detection for these compounds were in the range of 30–90pg.

Keywords: N; -Nornuciferine; O; -Nornuciferine; Nuciferine; Roemerine; Nelumbo nucifera; Gaertn; HPLC–DAD–ESI-MS

Liquid chromatographic determination with fluorescence detection of B₆ vitamers and riboflavin in milk and pharmaceuticals by R. Gatti; M.G. Gioia (pp. 135-141).

A simple, reliable and selective high performance liquid chromatographic method with fluorescence detection at different programmed wavelengths has been developed for the simultaneous analysis of B₆ vitamers (pyridoxal 5′-phosphate, 4-pyridoxic acid, pyridoxal, pyridoxine and pyridoxamine) and Vitamin B₂ in commercial vitaminized milk and in woman milk. The chromatographic separations were performed on a reversed phase octyl column by using a mobile phase consisting of sodium pentanesulfonate in 1% acetic acid–methanol–tetrahydrofuran under gradient elution conditions. The fluorescence intensity of pyridoxal 5′-phosphate was enhanced by post-column photochemical conversion, giving significantly different fluorescence spectra by a on-line photoreactor switched OFF and ON under irradiation at 254nm. In addition, a simple and rapid method in isocratic conditions without the need of photochemical conversion was proposed for the analysis of Vitamin B₆ and Vitamin B₂ in pharmaceuticals. Linearity, precision, recovery, selectivity and sensitivity were found satisfactory for each analysed compound. Quantitation limits ranged from 26 to 240fmol.

Keywords: B; ₆; vitamers; Vitamin B; ₂; (Riboflavin); LC; Photochemical conversion

Determination of glycine, glutamine, glutamate, and γ-aminobutyric acid in cerebrospinal fluids by capillary electrophoresis with light-emitting diode-induced fluorescence detection by Miao-Jen Lu; Tai-Chia Chiu; Po-Ling Chang; Hsin-Tsung Ho; Huan-Tsung Chang (pp. 143-150).

In this article, we present a simple and cost-effective method for the determination of amino acids in cerebrospinal fluids (CSF) by using capillary electrophoresis-light-emitting diode-induced fluorescence detection. When using a deactivated capillary filled with 0.6% poly(ethylene oxide) (PEO, M_r 6.0×10⁵) prepared in 10mM tetraborate (pH 9.3), we have achieved the limits of detection (S/N=3) in the range of 10–30nM for naphthalene-2,3-dicarboxaldehyde (NDA) derivatized amino acids (detected in the anodic side) in the absence of electroosmotic flow (EOF). In order to further improve sensitivity, stacking and separation of CSF samples in the presence of EOF has been applied. When high voltage is applied, the analytes migrating against EOF slow down and are stacked at the boundary between 2.0% PEO ( M_r 8.0×10⁶) prepared in 10mM tetraborate (pH 9.3) and sample zone. The stacking approach provides the LODs at the nM level for the analytes (detected in the cathodic side) when injecting at 30cm height for 150s. The two proposed methods provide comparable results for the determinations of glycine (Gly), glutamine (Gln), and glutamate (Glu) in CSF samples from patients suffered from inflammation, epilepsy, and jaundice without sample preparation, but the stacking method is more sensitive and allows for the determination of γ-aminobutyric acid (GABA).

Keywords: Amino acids; Capillary electrophoresis; Cerebrospinal fluid; Light-emitting diode-induced fluorescence detection; Stacking

A surfactant enhanced stopped-flow kinetic fluorimetric method for the determination of trace DNA by Rui-Yong Wang; Xiang Gao; Ying-Tang Lu (pp. 151-158).

A novel and high-sensitive stopped-flow kinetic fluorimetric method for determining the concentration of DNA (calf thymus DNA), in the presence of SDS, based on monitoring the variation of fluorescence within a very short period has been developed. The optimum conditions for various parameters, on which the binding of YOYO-1 to DNA depends, were investigated. It was found that the initial reaction rate increased linearly with increasing DNA concentration in the range from 8.0×10⁻¹⁰ to 9.0×10⁻⁸M. The detection limit was calculated to be 3.0×10⁻¹⁰M. This method was used for determining the concentration of DNA in synthetic samples and real samples extracted from leaves of Arabidopsis thaliana, tobacco and rice with satisfactory results. The kinetics of the interaction under pseudo first-order conditions was established by the aid of single-mixing stopped-flow techniques.

Keywords: Kinetic fluoremetry; Stopped-flow; YOYO-1; DNA; Surfactants

Femtomolar immunoassay based on coupling gold nanoparticle enlargement with square wave stripping voltammetry by Kuo-Tang Liao; Hsuan-Jung Huang (pp. 159-164).

The enhancement in sensitivity for an electrochemical immunoassay by the autocatalytic deposition of Au³⁺ onto gold nanoparticles has been studied. By coupling the autocatalytic deposition with square-wave stripping voltammetry, enlarged gold nanoparticles labeled on goat anti-rabbit immunoglobulin G (GaRIgG-Au) and, thus, the rabbit immunoglobulin G (RIgG) analyte could be determined quantitatively. A variety of variables, such as concentration of AuCl₄⁻, the reducing agent used, the duration of autocatalytic deposition, and parameters for the stripping analysis were optimized. From a calibration graph over a broad dynamic range of concentrations (1–500pgmL⁻¹; R²=0.9975) a very low detection limit, 0.25pgmL⁻¹ (1.6fM), which is three orders of magnitude lower than that obtained by a conventional immunoassay using the same gold nanoparticle labels was obtained; this finding confirms applicability and effectiveness of our method of enhancing the sensitivity of gold nanoparticle label-based sandwich immunoassays. The reliability of this method was confirmed by the rather low values of RSD (2.82%, n=11; 2.44%, n=9) obtained for assays of a blank solution and for 0.02ngmL⁻¹ RIgG solution, respectively.

Keywords: Gold nanoparticle labels; Autocatalytic deposition; SWSV; Sandwich immunoassay

Analysis of the longitudinal, “sloping plateau�? effect of a planar sample in a single TE₁₀₂ rectangular electron paramagnetic resonance cavity by Milan Mazur; Jan Moncol; Marian Valko; Harry Morris (pp. 165-174).

The response of the cavity to the movement of a concentrically positioned “over full-length cavity�? planar sample of variable length, L=30, 40, and 50mm, and width,w=4, 6, and 8mm, along the vertical, common sample–cavity x-axis in a Bruker single TE₁₀₂ rectangular cavity (longitudinal effect) has been analysed. The observed dependence of the EPR signal intensity, Ipp, on the sample position, x, showed the following: (i) A plateau, over which the Ipp value remained constant within experimental error of 0.62–2.48%, for sample length of 30 and 40mm. (ii) An “sloping plateau�? region, which could be approximated by a linear function (correlation, R=0.83–0.93), for sample length of 50mm. (iii) An additional oscillating component superimposed on the “sloping plateau�? region. The trends of these dependencies with the plateau and “sloping plateau�? region were independent of the planar sample width,w, and its angular position, ϕ, in the rectangular cavity. But, the EPR signal amplitude on the plateau and “sloping plateau�? region obtained for the sample positioned at angles, ϕ=0° and ϕ=180° (which corresponded to the experimental configuration, in which the planar sample area is parallel to the waveguide in the microwave cavity) was ca 1.8 times higher compared to those obtained for the sample positioned at angles ϕ=90° and ϕ=270° (which corresponded to the experimental configuration, in which the planar sample area is perpendicular to the waveguide). Theoretical predictions of the experimental dependencies of the EPR signal intensity versus sample position along the common sample–cavity x-axis can be calculated using the “sine-squared�? function (given by longitudinal integral) modified for an “over full-length cavity�? planar sample, and the correlation between observed and theoretically computed dependencies is very good. Each of the above-mentioned phenomenon could be a serious source of significant errors in quantitative EPR spectroscopy. For maximum accuracy in quantitative EPR an “over full-length cavity�? planar sample should be compared to a reference sample of identical length which must be identically positioned in the microwave cavity.

Keywords: Planar sample; Flat cell; Longitudinal effect; Sloping plateau; EPR signal intensity

Analytical Raman spectroscopic study of cacao seeds and their chemical extracts by Howell G. M. Edwards; Susana E. Jorge Villar; Luiz Fernando C. de Oliveira; Mireille Le Hyaric (pp. 175-180).

A Raman spectroscopic study of cacao seeds and their extracts has been successful in the identification of biomarker bands for theobromine, a member of the caffeine group of alkaloids. Unlike caffeine detected in guarana, the spectral signatures of theobromine suggest that it occurs as the free base in cacao. No theophylline or caffeine alkaloids were detected in cacao. Raman spectroscopic monitoring of the heptane and ethyl acetate organic extracts of pulverised cacao seeds demonstrates the removal of the fatty and waxy components whereas the methanol extract is found to remove and concentrate the highly coloured aromatic polyphenolic compounds. The theobromine alkaloid is still detectable in the solid residues from the extractions. This study suggests that Raman spectroscopic monitoring of cacao seed processing should be investigated further for the purposes of industrial process analytical control.

Keywords: Cacao; Raman spectroscopy; Theobromine; Seed extractions

Determination of the energetic value of fruit and milk-based beverages through partial-least-squares attenuated total reflectance-Fourier transform infrared spectrometry by Javier Moros; Fernando A. I��n; Salvador Garrigues; Miguel de la Guardia (pp. 181-193).

The estimation of important nutritional parameters, such as carbohydrates content and energetic value (calories) in commercially available fruit juice and flavour milk shakes has been made by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) using a partial-least-square (PLS) calibration approach. A highly heterogeneous population of 65 samples obtained from the Spanish market, covering fruit juices, flavour milk shakes and milk-added fruit juices was used. The spectral range and the size of the calibration set for building the PLS model have been evaluated.Considering a calibration set comprised of 27 samples, selected via hierarchical cluster analysis, and a validation data set of 38 samples, the absolute mean difference ( d x–_y) and standard deviation of mean differences ( s x–_y) of the total carbohydrate content were 0.06 and 0.66g/100mL, respectively. The reproducibility of this determination established as the mean standard deviation of each triplicate analysis was 0.05g/100mL. In the case of energetic value, the d x–_y and s x–_y were 2.8 and 18kJ/100mL, respectively. The reproducibility of this determination corresponded to a standard deviation of 2.4kJ/100mL, for three replicate analyses. The root-mean-square error of prediction (RMSEP) was 18.4kJ/100mL and 0.72g/100mL for energetic value and total carbohydrates, respectively.The developed methodology favourably compares with that reported in previous works in much restricted sample composition and provided figures of merit which agree with the US-FDA statuary tolerance values.

Keywords: Carbohydrates determination; Fruit juice; Milk shake; Milk; Hierarchical cluster analysis; Partial-least-squares; Attenuated total reflectance; Infrared

Analysis of caffeine, theobromine and theophylline in coffee by near infrared spectroscopy (NIRS) compared to high-performance liquid chromatography (HPLC) coupled to mass spectrometry by C.W. Huck; W. Guggenbichler; G.K. Bonn (pp. 195-203).

Coffee is one of the most important raw materials within the international trade, for which highest quality is demanded. Due to the high number of samples to be analysed, new analytical techniques providing fast and reliable data about the quality are essential. Therefore, we established a new analytical method based on near infrared spectroscopy (NIRS) for the quantitation of the three main alkaloids caffeine (Caf), theobromine (Tbr) and theophylline (Tph) in roasted coffee after discrimination of the rough green beans into Arabic and Robusta. This validated method was compared to the most commonly used liquid chromatography (LC) connected to UV and mass spectrometric (MS) detection. In this course non-porous silica-C18 showed to enable the analysis of the three alkaloids with high robustness within ∼2.5min, compared to 20min employing porous silica-C18 as a stationary phase. As analysis time plays an important role in choosing a reference method for the calibration of the NIR-spectrometer, the non-porous silica-C18 phase offers a very fast method. Coupling of the optimised LC method to a mass spectrometer (MS) via an electrospray ionisation (ESI) interface not only allowed to identify Caf, Tbr and Tph by their characteristic fragmentation pattern using collisionally induced dissociation (CID), but also to quantitate the content of the three analytes, which was found to be 6% higher compared to UV-detection. The validated LC–UV method was chosen as a reference method for the calibration of the NIRS system. Analysis of 83 liquid coffee extracts in random order resulted for Caf and Tbr in values for S.E.E. (standard error of estimation) of 0.34, 0.40g/100g, S.E.P. (standard error of prediction) of 0.07 and 0.10g/100g with correlation coefficients of 0.86 and 0.85 in a concentration range between 0.10 and 4.13g/100g. Compared to LC the lower limit of detection (LOD) of the NIRS-method is found at 0.05g/100g compared to 0.244–0.60ng/100g in LC, which makes it impossible to analyse Tph by NIRS. Therefore, main focus is put on the NIRS analysis of caffeine and theobromine. Nevertheless, NIRS offers a serious alternative to LC for the coffee producing industry because of the short analysis time of a few seconds and a guaranteed high sample throughput.

Keywords: Coffee; Caffeine; Theobromine; Theophylline; Liquid chromatography; Electrospray ionisation mass spectrometry; Near infrared spectroscopy

A disposable immunomagnetic electrochemical sensor based on functionalised magnetic beads and carbon-based screen-printed electrodes (SPCEs) for the detection of polychlorinated biphenyls (PCBs) by S. Centi; S. Laschi; M. Fr�nek; M. Mascini (pp. 205-212).

A disposable immunomagnetic electrochemical sensor involving magnetic particles and carbon-based screen-printed electrodes (SPCEs) was developed and applied for the detection of polychlorinated biphenyls (PCBs). The sensor was based on a direct competitive immunoassay scheme in which antibody-coated magnetic beads were used as solid phase; then SPCEs were employed as transducers for the evaluation of the extent of the immunochemical reaction using an alkaline phosphatase (AP) labelled tracer. The α-naphthyl phosphate was used as enzymatic substrate and the α-naphthol produced during the enzymatic reaction was detected using differential pulse voltammetry (DPV).A detection limit of 0.4ng/mL for Aroclor 1248 PCB mixture was obtained. The performance of the sensor was successfully evaluated on marine sediment extracts and soil samples spiked with different Aroclor mixtures indicating that this new and sensitive technique offers great promise for decentralized environmental applications.

Keywords: Screen-printed electrodes; Immunoassay; Polychlorinated biphenyls; Electrochemical detection; Magnetic beads

Anion recognition through modified calixarenes: a highly selective sensor for monohydrogen phosphate by Vinod K. Gupta; Rainer Ludwig; Shiva Agarwal (pp. 213-218).

PVC-based membranes of 5,11,17,23,29,35-hexa- tert-butyl-37,38,39,40,41,42-hexakis(carbamoylmethoxy)calix[6]arene (I) as electroactive material with dioctyl phthalate (DOP), 1-chloronaphthalene (CN), dibutyl phthalate (DBP), o-nitrophenyl octyl ether (NPOE) and tris(2-ethylhexyl) phosphate (TEP) as plasticising solvent mediators have been prepared and used for phosphate determination. The membrane having composition PVC:I (150:5.7) exhibited the best result with linear potential response in the concentration range of 1.77×10⁻⁵ to 1.0×10⁻¹M of HPO₄²⁻ with a slope of 33.0mV/decade. The membrane worked satisfactorily in non-aqueous medium up to 5% (v/v) non-aqueous content. The selectivity coefficient values for mono- and divalent anions indicate good selectivity for phosphate over a large number of anions. The proposed sensor has been successfully used in the real sample analysis.

Keywords: PVC; Phosphate; Potentiometric sensor; Calix[6]arene

Electrodes modified with an electrosynthesised Ni/Al hydrotalcite as amperometric sensors in flow systems by B. Ballarin; M. Berrettoni; I. Carpani; E. Scavetta; D. Tonelli (pp. 219-224).

Pt electrodes, modified with a directly electrosynthesised film of Ni/Al-NO₃ hydrotalcite were evaluated as amperometric sensors for oxidizable substrates in flow systems. The optimization of the operative parameters has been carried out using EtOH as test analyte, NaOH as carrier electrolyte and applying a constant potential. Analytical results in terms of linear range, limit of detection and precision were much satisfactory. The sensors are simple and rapid to prepare, and the coatings obtained by electrosynthesis are reproducible and stable from a mechanical point of view. The detector response was stable: a very good repeatability was observed during 12h of flow experiments (R.S.D.<2.6%).

Keywords: Chemically modified electrodes; Ni/Al hydrotalcites; Electrocatalysis; Flow analysis; Amperometric detection

Characterization of an optical copper sensor based on N, N′-bis(salycilidene)-1,2-phenylenediamine by Mohammad Bagher Gholivand; Parisa Niroomandi; Abdullah Yari; Mohammad Joshagani (pp. 225-231).

We prepared a copper selective optical chemical sensor based on N, N′-bis (salycilidene)-1,2-phenylenediamine (salophen) as ionophore. This proposed membrane works on the basis of a cation-exchange mechanism and shows a significant absorbance signal change on exposure to 0.05moll⁻¹ acetate buffer solution of pH 5.5 containing copper ion. This optode membrane shows enhanced selectivity for Cu²⁺ over other transition metal ions. The sensing membrane reversibly responses to copper ion over the wide range of 5.01×10⁻⁸ to 6.32×10⁻⁴moll⁻¹ with a detection limit of 4.7×10⁻⁸moll⁻¹ (2.98ppb) and response time of less than 3min. This sensor has been used for determination of copper in some real samples containing potential interferents with satisfactory recovery.

Keywords: Optical chemical sensor; Salophen; Copper ion

Development of a radiochemical sensor. Part I: Feasibility study by A. Taranc�n; J.F. Garc�a; G. Rauret (pp. 233-239).

The evolution of nuclear activities and criteria for radiation protection have led to a continuous increase in measures to monitor and control the environment and therefore in the number of determinations required for such purposes. Classical analytical procedures are time-consuming, labor-intense and generate a large amount of waste. The alternative use of sensors for such determinations has seen very limited development. The present study focuses on the evaluation of the behavior of a prototype radiochemical sensor for liquid effluents. The sensor is based on a receptor made of a plastic scintillator and is capable of continuous, on-time and accurate remote quantification of the activity of alpha, beta and beta–gamma emitters. Low-level active solutions of⁹⁰Sr/⁹⁰Y,²³⁸Pu,¹³⁴Cs and⁶⁰Co in matrices of groundwater, seawater and drinking water were quantified with prediction errors lower than 10% in most cases. The study also yields information about light generation and transmission and transductor configuration that will be useful in the design of future versions of this sensor.

Keywords: Radiochemical sensor; Plastic scintillator; Optical fibers; Liquid effluents; Remote; Radioactive waste

Development of a radiochemical sensor, Part 2: Application to liquid effluents by A. Taranc�n; A. Padr�; J.F. Garc�a; G. Rauret (pp. 241-249).

Monitoring programmes to evaluate the impact of nuclear activities on the environment require an increasing number of analytical determinations. In such a scenario, the development of a sensor could be especially helpful to increase the limited analytical capabilities of laboratories. The present study continues an initial feasibility project and focuses on the evaluation of the capabilities of a radiochemical sensor for liquid effluents applicable to real samples. This sensor is capable of sending information about the specific activity and volume of a contamination episode to a remote position, on-line and continuously. The sensor is made of plastic scintillator beads and includes two receptors, one for alpha and beta particles and the other for gamma radiations. Contamination pulses of⁹⁰Sr/⁹⁰Y,¹³⁴Cs and²⁴⁰Pu of different volumes and activity levels included in a continuous stream were quantified. Relative errors obtained for both magnitudes are, in the majority of the cases, less than 10%. Lower limits for these quantifications are 5ml and around 1Bq/ml. These thresholds could be improved in new designs adapted to specific problems.

Keywords: Radiochemical sensor; Plastic scintillator; Optical fibres; Liquid effluents; Remote; Radioactive waste

Strip bioelectrochemical cell for potentiometric measurements fabricated by screen-printing by �?ukasz Tymecki; Elżbieta Zwierkowska; Robert Koncki (pp. 251-256).

Miniaturized, planar bioelectrochemical cell dedicated for potentiometric detection of urea manufactured in integrated strip format has been developed. The enzyme electrode and the reference electrode, both based on ruthenium dioxide were fabricated only by means of screen-printing. The application of the thick-film technology results in highly reproducible and cost-effective manufacturing procedure, easily adapted to large-scale production. The developed system of planar electrodes can be applied as a disposable device for drop-on measurements as well as a detector for a multiple use in flow injection analysis. The biosensor is useful for determination of urea in millimolar range of concentrations. The developed strip biosensor is also useful as a disposable device for potentiometric detection of heavy metal ions, which are able to inhibit urease. The utility of the developed devices for determination of urea in urine and serum samples is demonstrated.

Keywords: Potentiometry; Urea; Biosensor; Screen-printing; Thick-film

Potentiometric assay for acid and alkaline phosphatase by Robert Koncki; Dominika Ogończyk; Stanisław Głąb (pp. 257-261).

Simple potentiometric kinetic assay for evaluation of acid and alkaline phosphatase activity has been developed. Enzymatically catalyzed hydrolysis of monofluorophosphate, the simplest inorganic compound containing PF bond, has been investigated as the basis of the assays. Fluoride ions formed in the course of the hydrolysis of this specific substrate have been detected using conventional fluoride ion-selective electrode based on membrane made of lanthanum fluoride. The key analytical parameters necessary for sensitive and selective detection of both enzymes have been assessed. Maximal sensitivity of the assays was observed at monofluorophosphate concentration near 10⁻³M. Maximal sensitivity of acid phosphatase assay was found at pH 6.0, but pH of 4.8 is recommended to eliminate effects from alkaline phosphatase. Optimal pH for alkaline phosphatase assay is 9.0. The utility of the developed substrate-sensor system for determination of acid and alkaline phosphatase activity in human serum has been demonstrated.

Keywords: Enzyme assay; ACP; ALP; Phosphatase; Serum; Monofluorophosphate; Fluoride ion-selective electrode; Potentiometry

An analysis of distinguishing composite dissolved metal–ligand systems measurable by stripping voltammetry by Cédric Garnier; Ivanka Pižeta; Stéphane Mounier; Vlado Cuculić; Jean Yves Benaïm (pp. 263-271).

Voltammetry is a method able to distinguish in certain degree the speciation of dissolved metals. An analysis of its ability to discern composite and more complex dissolved metal–ligand systems has been carried out by simulating the experiments for determination of metal–ligand complexing parameters. Logarithmic equidistant addition of metal was presumed, covering 2.5 decades. The data obtained with the preset parameter values were subsequently fitted to the presumed models. Data points under the detection limit DL=10⁻¹⁰molL⁻¹ were eliminated and random noise following a realistic shape was added to the points to approach them to the real experiment. Four models were applied for simulation and up to five models for fitting.The analysis of the results shows that with the nowadays state-of-the-art measurement and data treatment techniques, in most of the cases it was possible to distinguish more complex and also more probable bi-ligand and mixed metal–ligand complexes from the simpler 1:1 metal–ligand systems. Statistical evidences to validate the right model were given. Its applicability has been confirmed by generating a similar data mining server (DMS) rule.

Keywords: Voltammetry; Metal–ligand complexing parameters; Multiligand systems; Mono- and bi-ligand complexes; Mixed-ligand(s) complexes; PROSECE; DMS

An electrochemical approach for detecting copper-chelating properties of flavonoids using disposable pencil graphite electrodes: Possible implications in copper-mediated illnesses by Mun’delanji Vestergaard; Kagan Kerman; Eiichi Tamiya (pp. 273-281).

We have studied the electrochemistry of eight flavonoids belonging to four flavonoid sub-classes: flavone, flavonol, flavanol and anthocyanidin using pencil graphite electrodes (PGEs). We present the electrochemistry of delphinidin, cyanidin and catechin gallate for the first time. The use of electrochemical methods in connection with PGE in the study of flavonoids and their interaction with copper ions has not been previously reported. Our results compare favorably with previously reported studies, which utilised glassy carbon electrodes (GCEs) for the detection of flavonoids. We calibrated all eight flavonoids ( r²>0.9620), six of them at at least two peak potentials. The relative standard deviation (R.S.D.) for peak potential was <5.0% and peak height was <10.0%; thus, this method could be used to characterise and quantify flavonoid-containing extracts (purified).An inverse relationship between oxidation potential and metal-chelation was established. Oxidation potential was influenced by the location of OH groups relative to each other, the oxidation state of the pyranose ring, the presence of a C-4-oxo group and the total number of OH groups. Further, we showed that the steric configuration of the compound influenced the reactivity. The order of flavonoid reactivity to Cu(II) ions was myricertin=catechin gallate>quercetin>delphinidin=baicalein>cyanidin>catechin.These findings may be significant in neuroscience and metal toxicological studies, in which copper ions have been reported to play a crucial role in initiating and/or promoting the progression of diseases such as Alzheimer's and Parkinson's.

Keywords: Flavonoids; Natural flavonoids; Antioxidants; Metal chelation; Pencil graphite electrodes (PGEs)

Rapid and quantitative electrolytic preparation and speciation of neptunium ions of various oxidation states using multi-step column electrodes by Hisao Aoyagi; Yoshihiro Kitatsuji; Zenko Yoshida; Sorin Kihara (pp. 283-289).

Redox behavior of Np(III)–Np(VI) ions in aqueous perchloric, nitric and sulphuric acid solutions was elucidated by using column electrodes connected in series in a flow system (multi-step column electrode system). Using a glassy carbon fiber working electrode as the column electrode was found to be very useful for the investigation of current–potential relations of not only reversible redox processes, such as Np(VI)/Np(V) or Np(IV)/Np(III) couple but also irreversible processes, such as Np(V)/Np(IV) or Np(V)/Np(III) couple, the latter not observed by conventional voltammetry which uses a glassy carbon or platinum electrode. Quantitative (coulometric) electrolysis could be executed even if the redox process was completely irreversible by the use of the column electrodes. By taking advantage of column electrode electrolysis, a novel method was developed for the rapid preparation of a neptunium species of a desired oxidation state, including unstable species. The multi-step column electrode system was demonstrated to be useful for the coulometric determination and speciation of Np(IV)–Np(VI) in aqueous HNO₃ solution as an example.

Keywords: Neptunium; Column electrode electrolysis; Coulometry; Preparation of oxidation state; Speciation

Phase quantification of two chlorothalonil polymorphs by X-ray powder diffraction by Qiu-ling Lu; Guang Yang; Hao Gu (pp. 291-296).

Quantitative analysis method of phase composition of two chlorothalonil polymorphs (alpha and beta) co-existed with each other was studied by X-ray powder diffraction. With the content of polymorph alpha between 1.6% and 98%, the integral area of the strongest peak of polymorph alpha (2 θ: 25.5±1°) was not a linear function of its content. In the polymorph alpha matrix the detection limit of polymorph beta (2 θ: 26.5±0.1°) was 0.099%, while in the polymorph beta matrix, the detection limit of polymorph alpha was 1.6%.The expressions of the calibration curves were given. The relative density of polymorph alpha and beta was estimated to be equal to 0.90. The calibration curves joined into one linear and went through the origin (95% confidence interval). In the mixtures of the two polymorphs, the limits of prediction of alpha content from the determined integral areas mentioned above were less than ±0.026 (95% confidence limits). Some phase transformation samples were determined by the means of this quantitative analysis method.

Keywords: Chlorothalonil; Polymorphs; Ratio of the density of alpha and beta polymorphs; Phase quantification; X-ray powder diffraction

Calculation of the contributions of scattering effects to the X-ray fluorescent intensities for light matrix samples by Xiaoyuan Han; Shangjun Zhuo; Peiling Wang; Guangyi Tao; Ang Ji (pp. 297-302).

The contributions of scattering effects to the X-ray fluorescent intensities were calculated by considering the intensity of scattered primary fluorescence radiation that is scattered into the direction of detector, the secondary enhancement by coherent scattering radiation and incoherent scattering radiation for light matrix samples on the basis of the theoretical calculations. Relative intensities for nine different characteristic X-ray lines for the synthesized fused disks were calculated and compared with the measured relative intensities. It was found from this study that the contributions of scattering effects to the fluorescent intensities of analytes in fused disk samples are sizeable. Moreover, the magnitude of contribution of scattering effects increases with the increase of atomic number of analyte or/and the decrease of the content of the fluorescent element in the fused disk samples, which is about several percents of that excited by primary radiation, or even more than 10% for PbLβ₁. The average relative error of the calculated relative intensities compared with the measured values is 0.93% after scattering effects was considered, while the average relative error of the values calculated by NRLXRF for the same lines is 2.75%. The calculated relative intensities after considering the scattering effects are more agreeable to the measured values. Therefore, more accurate results for fundamental parameter method are expected with the consideration of scattering effects, especially for light matrix samples.

Keywords: Scattering effects; X-ray fluorescence intensity; Fused disk sample; Contribution

Multi-element electrothermal AAS determination of 11 marker elements in fine ambient aerosol slurry samples collected with SEAS-II by Joseph Patrick Pancras; John M. Ondov; Rolf Zeisler (pp. 303-312).

Multi-element simultaneous electrothermal atomic absorption spectrometry (ETAAS) methods were developed for determining 11 elements, in three analytical groups (group-1, predominately crustal elements, Al, Cu, Fe, Mn, and Cr; group-2, tracers of coal and oil combustion as well as other anthropogenic sources, Se, As, Pb, and Ni; and group-3, tracers of municipal incinerator aerosol–Zn and Cd), in aqueous slurry of ambient fine airborne particles, collected, at 30min intervals with the University of Maryland–Semicontinuous Elements in Aerosol Sampler-II (SEAS-II). Combined effects of acid (0.2% v/v nitric acid) and ultrasonic treatment (15min) improved metals recovery and slurry stability. Linear calibration ranges were improved by using a 50mLmin⁻¹ carrier gas flow (mini-flow) during the atomization stage. However, analytical sensitivity for group-1 and 3 elements was compromised. Palladium (4μg) and hydrogen (5% in Argon) were found to be effective modifiers for group-1 and 2 elements. A fast furnace temperature program was developed for group-3. Detection limits for the eleven marker elements by the SEAS-II-ETAA approach were compared with traditional filter X-ray fluorescence, laser ablation-ICPMS, and instrumental neutron activation analysis (INAA) techniques used in air quality studies. The efficacy of the analytical method was evaluated by analyzing interim urban PM2.5 reference material, collected in Baltimore, by the National Institute of Standards and Technology (NIST) Gaithersburg, MD. Results obtained by the ETAAS methods agreed well with the results obtained by NIST using INAA.

Keywords: Electrothermal AAS; Slurry sampling; Fine airborne particles; Simultaneous multi-element; PM2.5 reference material

An analytical study of bioaccumulation and the binding forms of mercury in rat body using thermolysis coupled with atomic absorption spectrometry by Ru-Jin Huang; Zhi-Xia Zhuang; Yi-Ru Wang; Zhi-Yong Huang; Xiao-Ru Wang; Frank S. C. Lee (pp. 313-321).

This study concerns with the development and application of a mercury analyzer system capable of quantitative analysis and binding forms of trace mercury in different body tissues of rats after oral intake of cinnabar and HgCl₂. The analyzer consists of a temperature programmed thermolysis unit for sample decomposition and mercury release, coupled downstream to an atomic absorption spectrometry for mercury detection. No sample digestion was needed and this greatly simplifies the analytical procedure and minimizes potential sources of contamination. The precision of the method is in general better than 1.4%; the detection limit (3 σ) is 6.3ng/g; the recovery based on the analysis of standard reference materials ranges from 93.5 to 107%. The performance of the method has been compared with inductively coupled plasma–mass spectrometry (ICP–MS) technique and excellent agreements were observed between the two methods. The method has been applied to the investigation of the distribution and binding forms of mercury in rats, which had been fed orally with cinnabar and HgCl₂. The results obtained show that mercury is mainly accumulated in the tissues of liver and kidney, meanwhile, the mercury levels in different tissues change with the feeding time, dosage and the chemical form of mercury compounds. In addition to total mercury measurements, the mercury thermal release profiles obtained from temperature programmed thermolysis provides mercury binding forms information, and preliminary data suggest the presence of protein/polypeptide-bound Hg compounds in kidney samples of rats.

Keywords: Mercury; Cinnabar; HgCl; ₂; Thermolysis; Atomic absorption spectrometry; Binding forms; Protein

Online flow digestion of biological and environmental samples for inductively coupled plasma–optical emission spectroscopy (ICP–OES) by Q.Z. Bian; P. Jacob; H. Berndt; K. Niemax (pp. 323-329).

A novel type of interface for gas removal has been developed for coupling a high-temperature/high-pressure online flow digestion system to an ICP spectrometry instrument. It consists of a porous polypropylene tube with an inner rod made of PEEK which causes the formation of a thin film (100μm) of liquid at the inner surface of the porous tube, thereby considerably increasing the ratio of gas exchange area to liquid volume and simultaneously decreasing the dead volume. Thus very efficient removal of the gaseous reaction products, such as CO₂ and NO_x, which are known to affect the ICP stability, was achieved. Even at high flow rates and with a high carbon content in the samples more than 99% of the gases were removed. Applying ICP–OES detection excellent recoveries were obtained for most elements except for Sb, Ag and Ga. Good agreement between certified and measured values was obtained for online determinations of elements in various biological and environmental standard reference materials. Low standard deviation and detection limits in the lower μg/g range were found. This new tube interface combines low costs with robust properties, compact design and ease of handling.

Keywords: Trace elements in biological matrices; Slurry sample introduction; On-line high-temperature/high-pressure digestion; Flow injection; ICP–OES

Flow injection-spectrophotometeric determination of some catecholamine drugs in pharmaceutical preparations via oxidative coupling reaction with p-toluidine and sodium periodate by Loay K. Abdulrahman; Anas M. Al-Abachi; Mayada H. Al-Qaissy (pp. 331-335).

A new spectrophotometric flow injection method has been established for the determination of some catecholamine drugs (methyl dopa I, dopamine.HCl II and adrenaline III). The method is based on the oxidative coupling reaction of catecholamine with p-toluidine and sodium periodate to form an orange water-soluble dye product that has a maximum absorption at 480nm. Linearity was observed in the range of 1–50, 2–50 and 5–70, with a limit of detection (S/N=3) of 0.4, 0.2 and 0.7μgml⁻¹ for I–III, respectively.The method was applied successfully to the determination of I–III in pharmaceutical preparations with a good precision and accuracy.

Keywords: Spectrophotometric; Catecholamine; Sodium periodate; Flow injection analysis

Adsorption of penicillin–berberine ion associates at a water/tetrachloromethane interface and determination of penicillin based on total internal-reflected resonance light scattering measurements by Cheng Zhi Huang; Ping Feng; Yuan Fang Li; Ke Jun Tan; Hui Ying Wang (pp. 337-343).

In aqueous medium of pH 5.33, penicillin, such as ampicillin (AmP), benzyl penicillin (BP), oxacillin (OA) and amoxycillin (AmO), interacts with berberine (BB), forming ion associates through electrostatic attraction of the polar head groups of penicillin and BB. The formed ion associates are then adsorbed into the amphiphilic H₂O/CCl₄ interfaces, displaying greatly enhanced total internal-reflected resonance light scattering (TIR-RLS) signals with the maximum peak at 370nm. With the enhanced TIR-RLS data, thermodynamic parameters of adsorption of penicillin–BB ion associates into interfaces were measured. Mechanism studies show that the ion associates of penicillin with BB at H₂O/CCl₄ interfaces have 1:1 binding molar ratio, and the formation constants are in the range of 3.67×10⁴ to 5.48×10⁴mol⁻¹dm⁴. It was found that the enhanced TIR-RLS intensity was directly proportional to the concentration of penicillin in the range of 0.98×10⁻⁷ to 10.0×10⁻⁷moll⁻¹ for BP, 0.87×10⁻⁷ to 10.0×10⁻⁷moll⁻¹ for OA, 1.1×10⁻⁷ to 12.0×10⁻⁷moll⁻¹ for AmP and 1.2×10⁻⁷ to 12.0×10⁻⁷moll⁻¹ for AmO with the corresponding limits of determination (3 σ) being 3.48×10⁻⁸, 3.85×10⁻⁸, 4.22×10⁻⁸ and 4.86×10⁻⁸moll⁻¹, respectively.

Keywords: Water/tetrachloromethane interface; Total internal-reflected resonance light scattering; Penicillin; Berberine chloride

Estimation of antitubercular drugs combination in pharmaceutical formulations using multivariate calibration by Jitender Madan; A.K. Dwivedi; S. Singh (pp. 345-353).

The use of multivariate calibration methods, principal component regression (PCR) and partial least square (PLS) regression, applied on the raw and first derivative spectra, for the estimation of pyrazinamide (PYZ), isoniazid (INH) and rifampicin (RIF) in antitubercular combination formulation is presented. First derivative spectra were computed using Savitzky–Golay transformation. The three drugs show considerable degree of spectral overlap. The calibration set exhibiting central composite design was used to develop models able to predict the concentration of unknown samples containing three drugs. The calibration model was optimized by an appropriate selection of number of factors and wavelength regions to be used for building data matrix, based on minimization of predicted error sum of square (PRESS) using leave-one-out cross-validation strategy. The calibration model was validated by a separate set of 15 mixture solutions. The PCR and PLS models on the raw and first derivative spectra were statistically compared by ANOVA. All methodologies gave statistically similar results. The methodologies were successfully applied to the simultaneous estimation of PYZ, INH and RIF in pharmaceutical formulation.

Keywords: Principal component regression; Partial least square; Savitzky–Golay filter; Rifampicin; Pyrazinamide; Isoniazid

Water quality assessment and apportionment of pollution sources of Gomti river (India) using multivariate statistical techniques—a case study by Kunwar P. Singh; Amrita Malik; Sarita Sinha (pp. 355-374).

Multivariate statistical techniques, such as cluster analysis (CA), factor analysis (FA), principal component analysis (PCA) and discriminant analysis (DA) were applied to the data set on water quality of the Gomti river (India), generated during three years (1999–2001) monitoring at eight different sites for 34 parameters (9792 observations). This study presents usefulness of multivariate statistical techniques for evaluation and interpretation of large complex water quality data sets and apportionment of pollution sources/factors with a view to get better information about the water quality and design of monitoring network for effective management of water resources. Three significant groups, upper catchments (UC), middle catchments (MC) and lower catchments (LC) of sampling sites were obtained through CA on the basis of similarity between them. FA/PCA applied to the data sets pertaining to three catchments regions of the river resulted in seven, seven and six latent factors, respectively responsible for the data structure, explaining 74.3, 73.6 and 81.4% of the total variance of the respective data sets. These included the trace metals group (leaching from soil and industrial waste disposal sites), organic pollution group (municipal and industrial effluents), nutrients group (agricultural runoff), alkalinity, hardness, EC and solids (soil leaching and runoff process). DA showed the best results for data reduction and pattern recognition during both temporal and spatial analysis. It rendered five parameters (temperature, total alkalinity, Cl, Na and K) affording more than 94% right assignations in temporal analysis, while 10 parameters (river discharge, pH, BOD, Cl, F, PO₄, NH₄–N, NO₃–N, TKN and Zn) to afford 97% right assignations in spatial analysis of three different regions in the basin. Thus, DA allowed reduction in dimensionality of the large data set, delineating a few indicator parameters responsible for large variations in water quality. Further, receptor modeling through multi-linear regression of the absolute principal component scores (APCS-MLR) provided apportionment of various sources/factors in respective regions contributing to the river pollution. It revealed that soil weathering, leaching and runoff; municipal and industrial wastewater; waste disposal sites leaching were among the major sources/factors responsible for river quality deterioration.

Keywords: Gomti river; Water quality management; Cluster analysis; Factor analysis; Principal component analysis; Discriminant analysis; Source apportionment

An exact test for analytical bias detection by M�rio de Castro; Heleno Bolfarine; Manuel Galea-Rojas; M�rcio V. de Castilho (pp. 375-381).

In this paper a simple exact test statistic to detect analytical bias is proposed. The test requires neither specialized software nor iterative procedures. Comparisons with an asymptotic test using simulation studies show that the proposed test presents good behavior in terms of significance level and power. Applications to a real data set are also reported. Some general guidelines concerning the choice of the test to be used are discussed.

Keywords: Analytical bias; Hypothesis tests; Exact test; Wald test

Resolution of overlapping spectra by curve-fitting by Yaogai Hu; Jinyang Liu; Wei Li (pp. 383-389).

In this paper we present a novel method of curve-fitting based on Gaussian function, which is used to resolve the overlapping peaks. Correctly choosing the minimum separable peak–peak interval (MSI) is crucial for this type of problem. We propose a method that chooses appropriate MSI by analyzing the curve-fitting error changes with the MSI. The resolution of several kinds of overlapping peaks with computer-simulated noise has been performed and discussed in details. This method has been used for resolving of the UV–vis overlapping spectrum. The results are satisfactory and clearly show the effectiveness of the proposed method.

Keywords: Overlapping peaks; Curve-fitting; Resolving overlapping spectra; Gaussian function

Comparison of antioxidant activity of wine phenolic compounds and metabolites in vitro by D�bora Villa�o; M. Soledad Fern�ndez-Pach�n; Ana M. Troncoso; M. Carmen Garc�a-Parrilla (pp. 391-398).

A comparative study of the antioxidant activity of wine phenolics has been performed. Standards of phenolic compounds, including benzoic and cinnamic acids, flavanols, flavonols and resveratrol, as well as some of their metabolites have been analysed for their antioxidant activity. Antioxidant assays included the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging methods and the Oxygen Radical Absorbance Capacity method (ORAC).We compared the reactivity of standard phenolic compounds under selected tests and obtained a ranking order for each one. Both ABTS and DPPH methods were well correlated. Several new structure–activity relationships were observed. As the number of phenoxyl groups increases, the antioxidant capacity, determined by DPPH and ABTS tests, improves. In the case of the ORAC assay, the ortho-position of phenoxyl groups clearly determines the activity. The metabolites of phenolic compounds present antioxidant activity and their values are similar to those of the phenolic compounds themselves.The choice of solvent, the effect of concentration and the information provided by the three tests under study are considered.

Keywords: Phenolic compounds; Wine; Antioxidant activity; 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS); 1,1-Diphenyl-2-picrylhydrazyl (DPPH); Oxygen Radical Absorbance Capacity (ORAC); Metabolites

Distribution of the principal cannabinoids within bars of compressed cannabis resin by R. Lewis; S. Ward; R. Johnson; D. Thorburn Burns (pp. 399-405).

Determination by gas chromatography-mass spectrometry, after extraction into hexane, of the relative amounts of the three most abundant cannabinoids, cannabidiol (CBD), Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and cannabinol (CBN), and the six next most abundant compounds in cannabis resin all showed significant variations in their relative amounts with sampling position. Thus, data from a single sub-sample cannot be regarded as characteristic of a cannabis resin bar or without further evidence of coming from a particular resin bar or batch of bars. A cannabis resin bar stored under ambient conditions for 12months showed major loss of Δ⁹-THC and an increase in CBN content as well as variations in composition with sampling position.

Keywords: Cannabis; Cannabinoid distribution; Resin; Gas chromatography-mass spectrometry

Author Index (pp. 407-410).

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Analytica Chimica Acta (v.538, #1-2)