Analytical Methods (v.3, #11)

Front cover (2427-2428).

Contents (2429-2438).

A comprehensive review of CUPRAC methodology by Mustafa Özyürek; Kubilay Güçlü; Esma Tütem; Kevser Sözgen Başkan; Erol Erçağ; S. Esin Çelik; Sefa Baki; Leyla Yıldız; Şeyda Karaman; Reşat Apak (2439-2453).
Measuring the antioxidant activity/capacity levels of food and biological fluids is carried out for the meaningful comparison of the antioxidant content of foodstuffs and for the diagnosis and treatment of oxidative stress-associated diseases in clinical biochemistry. Current literature clearly states that there is no widely adopted/accepted “total antioxidant parameter” as a nutritional index available for the labeling of food and biological fluids due to the lack of standardized quantitation methods. The “parent” CUPRAC (CUPric Reducing Antioxidant Capacity) method of antioxidant measurement, introduced by our research group to world literature, is based on the absorbance measurement of Cu(i)-neocuproine (Nc) chelate formed as a result of the redox reaction of chain-breaking antioxidants with the CUPRAC reagent, Cu(ii)-Nc, where absorbance is recorded at the maximal light absorption wavelength of 450 nm; thus this is an electron-transfer (ET)-based method. From the parent CUPRAC method initially applied to food (apricot, herbal teas, wild edible plants, herby cheese etc.) and biological fluids (as hydrophilic and lipophilic antioxidants together or in separate fractions), a number of “daughter” methods have evolved, such as the simultaneous assay of both lipophilic and hydrophilic antioxidants in acetone-water as methyl-β-cyclodextrin inclusion complexes, determination of ascorbic acid alone in the presence of flavonoids (with preliminary extraction of flavonoids as their La(iii)-complexes), determination of hydroxyl radical scavenging activity of both water-soluble antioxidants (using benzoate derivatives and salicylate as hydroxylation probes) and of polyphenols using catalase to stop the Fenton reaction so as to prevent redox cycling of antioxidants, measurement of Cu(ii)-catalyzed hydrogen peroxide scavenging activity and of xanthine oxidase inhibition activity of polyphenols, TAC measurement of protein thiols in urea buffer, development of a CUPRAC-based antioxidant sensor on a Nafion cation-exchanger membrane, the off-line HPLC-CUPRAC assay and finally the on-line HPLC-CUPRAC assay of antioxidants with post-column detection. The current direction of CUPRAC methodology can be best described as a self-sufficient and integrated train of measurements providing a useful “antioxidant and antiradical assay package”. This review attempts to unify and summarize various methodologies of main and modified CUPRAC procedures that can normally be extracted from quite different literature sources.

Current separative strategies used for resveratrol determination from natural sources by Enguo Fan; Shen Lin; Daolin Du; Yunjing Jia; Lei Kang; Kai Zhang (2454-2462).
Resveratrol, occurring as trans- and cis- isomeric forms, is a phytoalexin existing in grape vines, as well as the product, wine, and a large number of plants. Owing to its diverse pharmacological activities, including anticancer and anti-cardiovascular disease activity as well as life span increasing properties, quantification methods of resveratrol are evolving rapidly, including high-performance liquid chromatography (HPLC) coupled with various detectors, gas chromatography-mass spectrometry (GC-MS) and capillary electrophoresis (CE) related techniques. In the present review, our interests are focused on analytical methods of resveratrol determination based on separative techniques, and the advantages and disadvantages of each method are summarized and compared.

Chemical amplification for in-gel DNA detection by Peng He; Eric Z. Tucker; Christopher B. Gorman; Lin He (2463-2468).
We report the use of reversible addition–fragmentation chain transfer (RAFT) polymerization as a highly efficient chemical amplification means to direct visualization of DNA in porous polyacrylamide gel. It is the first time that a dynamic polymer growth on the surface of soft medium is used in signal amplification for DNA detection. In the proof-of-concept experiment, a thin acrylamide gel on a glass microscope slide formed a thin layer of uniformly crosslinked network with porous structures. Oligonucleotides of different sequences were entrapped within the gel at separate spots. Hybridization of complementary DNA detection probes introduced chain transfer agents (CTAs) into the gel via preconjugation to the probes. Surface-initiated polymer growth was prompted on the gel surface and the growth of polymer brushes at the spot where DNA hybridization occurred was monitored using infrared spectroscopy and atomic force microscopy. Visible change in the texture of the porous gel occurred after polymer growth, which offered an attractive detection alternative for in-gel DNA analysis. Compared to the results from traditional ethidium bromide staining, better detection sensitivity and specificity were achieved.

Selective detection of Cr(vi) in aqueous media by carbazole-based fluorescent organic microcrystals by Karasinghe A. N. Upamali; Leandro A. Estrada; Douglas C. Neckers (2469-2471).
Taking advantage of its high luminescence as an aggregate, 1-cyano-trans-1,2-bis-(4-carbazolyl)-phenylethylene (CN-CPE) was evaluated as an electron-transfer probe for sensing of Cr(vi) in aqueous media. A remarkable Cr(vi)-sensitivity and selectivity over common aqueous interferents was found.

Herein using classic DNA binding Ru polypyridine complex and graphene oxide, we developed a label-free and simple method for fluorescence recognition of complementary double-stranded DNA and aptamer-based potassium ions detection processes.

Dual functional electrochemical sensor based on Au–polydopamine–Fe3O4 nanocomposites by Guangfeng Wang; Hao Huang; Ge Zhang; Xiaojun Zhang; Lun Wang (2475-2477).
Au–polydopamine (PDA)–Fe3O4 nanocomposites were synthesized by a seed-assisting growth method. In the synthesis, PDA was a secondary substrate for the growth of Au nanoparticles. In this paper the prepared Au–PDA–Fe3O4 nanocomposites were applied in the fabrication of an electrochemical sensor for direct electron transfer (DET) of hemoglobin (Hb) and detecting interleukin-6 (IL-6).

A laboratory comparison of analytical methods used for isocyanates by Diana M. Ceballos; Stephen G. Whittaker; Michael G. Yost; Russell L. Dills; Dhimiter Bello; Jennifer M. Thomasen; Leena A. Nylander-French; Carolyn K. Reeb-Whitaker; Phillip M. Peters; Elisa C. Weiland; Wallace W. Suydam (2478-2487).
Monomeric and oligomeric 1,6-hexamethylene diisocyanate (HDI) and isophorone diisocyanate (IPDI) are components of catalyst hardeners in two-part polyurethane coating systems. Exposure to these isocyanates in the collision repair industry has been associated with increased risk for work-related asthma; however their quantitation remains a challenging task. Four analytical methods were compared: modified version of the National Institute for Occupational Safety and Health Method 5525 (NIOSH); liquid chromatography and mass spectrometry (LC/MS) using 1-(2-methoxyphenyl)piperazine (MPP) as the derivatizing reagent; modified version of the Occupational Health and Safety Administration Method 42/PV2034 (OSHA); and modified version of the Omega ISO-CHEK® Method (WA-DOSH). Five levels of a widely used clear coat hardener were spiked onto sampling media. A reference value was calculated by using a hardener NCO titration and manufacturer composition data. Intra- and inter-method variability was determined. All methods measuring NCO-HDI monomer, NCO-IPDI monomer, NCO-IPDI oligomers, and total NCO mass (except OSHA method) compared well against the reference values (slopes ≥ 0.816, R2 > 0.90). The NCO-HDI oligomer results for the NIOSH method compared well with the reference values (slope = 1.161, standard error = 0.046 and R2 = 0.98, p < 0.001) while WA-DOSH were above (slope = 2.293, standard error = 0.055 and R2 = 0.99, p < 0.001) and LC/MS were below (slope = 0.264, standard error = 0.011 and R2 = 0.98, p < 0.001) the reference values. The principal challenges associated with comparing methods were: 1) the reporting metrics were not always directly comparable and 2) not all methods reported all isocyanate species of interest. Although this present study provided valuable information, a more extensive investigation is required in order to critically evaluate these methodological differences.

Fast and sensitive determination of aluminium with RP-HPLC using an ultra-short monolithic column by Tomas A. Remenyi; Pavel N. Nesterenko; Andrew R. Bowie; Edward C. V. Butler; Paul R. Haddad (2488-2494).
A fast and sensitive reversed-phase high-performance liquid-chromatographic method for determination of aluminium in aqueous samples has been developed. The fluorescent aluminium-lumogallion complex (λex 505 nm, λem 574 nm), was formed with a pre-column reagent and then separated on a Chromolith® RP-18e Guard column using a two-tiered, stepped gradient program, with matrix elimination (5/95 (v/v) methanol/water), followed by elution (90/10 (v/v) methanol/water). This method achieved a run time of 2.5 min without compromising sensitivity (limit of detection = 5.6 × 10−10 M, limit of quantification = 7.2 × 10−7 M), precision (3.5% at 3.71 × 10−6 M) or accuracy (recovery = 97.5% ± 3.2%, n = 5, P = 0.95). This represents an improvement in run time by >50% compared to the fastest previously published method [Lee et al., Clin. Chem., 1996, 42, 1405–1411]. This method uses MES buffer (2-(N-morpholino)ethanesulfonic acid), which compared to other buffers is more easily purified, does not complex with aluminium, and can be used in lower concentrations. This method was applied to analysis of: deionised water for column comparison; seawater for matrix interference effects; and tea-infusion for calibration and recovery studies.

Determination of cadmium in rice by electrothermal atomic absorption spectrometry using aluminum as permanent modifier by Douglas G. da Silva; Mario M. S. Junior; Laiana O. B. Silva; Lindomar A. Portugal; Geraldo D. Matos; Sergio L. C. Ferreira (2495-2500).
In this paper, the use of aluminum as a permanent modifier for the determination of cadmium in rice by electrothermal atomic absorption spectrometry (ETAAS) was investigated. The optimized experimental conditions, which employed a full two-level factorial design, pyrolysis and atomization curves, were as follows: aluminum mass of 400 μg, pyrolysis temperature of 400 °C, pyrolysis time of 20 s and 1,800 °C for atomization temperature. Under these conditions, cadmium concentrations could be determined in rice with limits of detection and quantification of 2 and 6 ng g−1, respectively, as well as a characteristic mass of 1.3 pg. The precision, expressed as relative standard deviation (RSD%), was 1.67% for a rice sample with a cadmium concentration of 41.3 ng g−1. The accuracy was confirmed by analysis of a certified reference material of rice flour that was provided by the National Institute for Environmental Studies, Japan. Experiments involving the analyte addition technique demonstrating that the external calibration technique using aqueous standards could be used for quantification of cadmium in rice. The proposed method was used to determine the cadmium content of thirty-four rice samples acquired in supermarkets from Bahia State, Brazil. The rice samples were digested using nitric acid and hydrogen peroxide in a closed system, using a digester block and cold finger. The cadmium concentrations varied from 11.6 to 44.9 ng g−1, with an average content of 30.1 ng g−1. A preliminary study demonstrated that the cadmium contamination in rice decreased after cooking using a microwave oven.

A complete methodology for the reliable collection, sample preparation, separation and determination of organic compounds in ultrafine 30 nm, 40 nm and 50 nm atmospheric aerosol particles by José Ruiz-Jiménez; Jevgeni Parshintsev; Totti Laitinen; Kari Hartonen; Marja–Liisa Riekkola; Tuukka Petäjä; Aki Virkkula; Markku Kulmala (2501-2509).
A complete reliable methodology including several extraction and chromatographic techniques has been developed for the determination of selected organic compounds in atmospheric aerosol particles. Size separated ultrafine particles (Dp ≤ 50nm) and total suspended particles (TSP) were collected in a urban and forest environment. One third of the samples contained TS particles and the rest of the samples were size-separated (30, 40 or 50 nm particles) with a differential mobility analyzer (DMA). Gas chromatography-mass spectrometric and liquid chromatography-mass spectrometric methods were developed for the analysis of the target compounds and the sample pretreatment was shortened by exploiting ultrasonic energy. The investigated compounds included seven amines (average concentrations ranged between 0.1 and 10.9 ng m−3), eight aldehydes (0.1–11.9 ng m−3), two polyols (0.1–81.5 ng m−3) and sixteen acids (0.1–47.2 ng m−3). The whole methodology including sample preparation and analysis methods was carefully validated by comparison of the results obtained with those provided by a conventional extraction method, non-assisted by ultrasound and by standard addition methodology, respectively. There were clear differences, up to two orders of magnitude, in the concentrations of the target compounds in aerosol samples with different sizes collected from an urban and forest environment.

Molecularly imprinted-multiwall carbon nanotube paste electrode as a biosensor for voltammetric detection of rutin by Behzad Rezaei; Najmeh Majidi; Ali A. Ensafi; Hassan Karimi-Maleh (2510-2516).
A new molecularly imprinted polymer–multiwall carbon nanotube paste electrode (MIP–MWNPE) was prepared as a voltammetric sensor for rutin. The electrochemical behavior of rutin at the surface of the modified electrode was studied using cyclic voltammetry (CV) and square wave voltammetry (SWV). Under the optimized conditions, the oxidation peak current of rutin showed two linear dynamic ranges (0.08–1.4 μM and 2.0–160.0 μM) with a detection limit of 0.05 μM, using square wave voltammetric method. The RSD% for 5.0 and 10.0 μM determination of rutin were 2.7% and 2.9%, respectively. The kinetic parameters such as electron transfer coefficient (α) and charge transfer rate constant (ks/s−1) were also determined using the electrochemical approach. The modified electrode showed good sensitivity, special selectivity, and stability for rutin analysis. It was successfully applied for the determination of rutin in real samples such as urine and pharmaceutical formulation with satisfactory results.

A pyrosequencing-based method for genotyping pathogenic serotypes of S. suis by Huiyong Yang; Huan Huang; Haiping Wu; Bingjie Zou; Guohua Zhou; Tomoharu Kajiyama; Hideki Kambara (2517-2523).
Streptococcus suis (S. suis for short) can cause a variety of infections in pigs, and the infections have brought about great losses in the swine industry and some cases of deaths in human beings. In order to rapidly diagnose and control the infections of S. suis, we designed a pyrosequencing-based assay to identify the serotypes of S. suis. In the assay, pyrosequencing is used to genotype most of the pathogenic serotypes of S. suis by detecting five informative regions on the Chaperonin 60 (cpn60) gene and one species-specific region on the 16S rRNA gene, and further a few undistinguished serotypes by pyrosequencing were finely discriminated by multiplex PCR of serotype-specific fragments on the cps gene as well as species-specific fragments on the 16S rRNA gene. Through carefully designing the dispensing order of dNTP for each pyrosequencing reaction, the serotypes of S. suis could be discriminated by four pyrosequencing reactions within three hours. Five reference serotypes and three clinical strains were successfully detected and genotyped by our assay. The results indicated that our assay is a reliable, information-rich diagnostic method for the accurate detection of S. suis serotypes.

A fluorescence-based method for the characterization of amino loading density on the flat surface of functionalized glass tubes by Long Ye; Guorong Ma; Conghao Zhong; Yuanwei Yan; Jinting Pan; Guijun Ma; Rongxiu Li (2524-2528).
In this study, a fluorescence-based method was used to characterize the amino loading density on the flat surface of glass tubes functionalized by silylation with (3-aminopropyl)trimethoxysilane and then fluorescence-labeled with fluorescein isothiocyanate. The immobilized fluorescent molecules on the surface of the glass tubes were released by treatment with hydrofluoric acid. The fluorescence of the released solution was detected by a multifunction micro-plate reader, and the concentration of the fluorescent molecules was calculated from a calibration curve based on the reference compound 2a. A density calculation formula D = 27.95Y/S was developed, where Y represents the fluorescence intensity of the solutions and S represents the surface area of the glass tubes.

Homogeneity testing of materials used in analytical proficiency tests is usually carried out by selecting at random a number of the distribution units (commonly 10) and analysing them in duplicate. The results are assessed by one-way analysis of variance. Because of the limited amount of information available in these data, this analysis provides an imprecise estimate of the analytical variance σ2an, an even more imprecise estimate of the sampling variance σ2sam, and a test of low statistical power of the hypothesis σ2sam = 0. When a succession of similar materials are prepared for successive rounds, under conditions where statistical control of the whole process is a reasonable assumption, the greatly increased number of degrees of freedom obtained by pooling the data allows both variance components to be well estimated, even when the sampling variance is small. A new approach to homogeneity testing is proposed, in which, after an initial phase of data collection and variance estimation, a control chart is set up and the experimental design is reduced to 10 singleton measurements. This gives a considerable reduction in expenditure, with little or no loss in statistical power.

In this study, electrochemical modification of a glassy carbon (GC) electrode with apigenin was carried out and the modified electrode was used for determination of copper(ii) (Cu(ii)) in soil samples. The GC was modified through the electrochemical polymerization of apigenin (PolyApi/GC) on the electrode surface in aqueous media. The electrode surface was modified with apigenin in phosphate buffer solution (PBS), pH 7, from 0 mV to +1400 mV potential ranges, using 100 mV s−1 sweep rate and 30 cycles by cyclic voltammetry (CV). The surface characterizations of this sensor electrode were performed by CV, electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). Britton-Robinson (BR) buffer solution at pH 5 was used for determination of Cu(ii) by differential pulse voltammetry (DPV). The detection limit was obtained as lower as 1.0 × 10−11 M. By using this calibration curve, the amount of Cu(ii) was determined as 7.34 × 10−7 M in soil samples. The results showed that pH, incubation time and interferences of some cations and anions were significant.

Bi-Film on a carbon paste electrode modified with nafion film embedded with multiwall carbon nano tubes for the determination of heavy metals by M. K. Dey; A. K. Satpati; S. Sahoo; R. Kameswaran; A. V. R. Reddy; T. Mukherjee (2540-2546).
Bi-film was deposited on the carbon paste electrode and used as the modified electrode in stripping voltammetry. Bi deposition was carried out ex-situ in acetate buffer solution of pH 4.5 under mild stirring conditions. The deposition potential, deposition time and the Bi-concentration in the deposition solution were optimized and the corresponding values are −0.8 V (SCE), 300 s and 0.3 mM Bi-nitrate solution respectively. A substantial enhancement of an order of magnitude in the sensitivity has been observed when carbon paste electrode was modified with nafion coated carbon nano tube on which Bi-film was deposited. This enhanced sensitivity was attributed to the increased active surface area and also the enhanced charge transfer processes which increased the deposition processes and so the stripping current. Effect of different surfactants on the stripping peak of the heavy metal ions is investigated with and without modification by nafion. Detection limits using Bi-film electrode was obtained for Zn, Cd and Pb as 17.3, 16.9, 11.9 μg L−1, respectively, using Bi-film electrode. Analysis result of two water samples and two ayurvedic medicines are reported.

Experience with the application of the draft European Standard prEN 15768 to the identification of leachable organic substances from materials in contact with drinking water by GC-MS by Dorit Löschner; Thomas Rapp; Frank-Ullrich Schlosser; Ramona Schuster; Ernst Stottmeister; Sven Zander (2547-2556).
In this work, the draft European Standard prEN 15768 “The GC-MS identification of water leachable organic substances from materials in contact with water intended for human consumption” was applied to test 17 different commonly used plastic pipes for domestic plumbing systems for the migration of unexpected substances into drinking water. The tested pipes were made from 8 different plastic materials. Substances detected in the migration waters from the different pipe materials at concentrations exceeding 2 μg l−1 are reported. More than 60% of them could be identified by library search with the NIST mass spectral database. The migrated substances found and identified are discussed concerning their concentration trends with increasing time of testing. A significant number of substances identified were degradation products of antioxidants that are used as additives for the plastic materials. Possible degradation pathways for important antioxidants leading to frequently identified substances like 2,4-di-tert-butylphenol, 3-(3,5-di-tert-butyl-4-hydroxy-phenyl) propanoic acid, 7,9-di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione, 3,5-di-tert-butyl-4-hydroxybenzaldehyde, and 2,6-di-tert-butyl-p-benzoquinone are proposed and discussed. As a conclusion, this study showed a good performance of the preliminary standard in practice.

A simple, rapid, sensitive, and low cost method of ultrasonic extraction (USE) and capillary electrophoresis (CE) for the determination of melamine in milk is developed. The most widely used extraction methods of melamine (SPE and LLE) were compared with ultrasonic-assisted extraction by evaluating simplicity, cost and sample clean-up effect, and the conditions of CE were optimized. The melamine was extracted by acetonitrile under the action of ultrasonic energy. After centrifuging, diluting and filtering, the extract was analyzed by CE directly. The determination used 20 mM NaH2PO4 as running buffer (pH 2.6), UV detection at 208 nm, and an applied voltage of 18 kV. The proposed method demonstrated good performance concerning linearity (R2 = 0.9989), precision (2.6–4.8%), limit of detection and quantification (LOD, 0.163 mg kg−1; LOQ, 0.542 mg kg−1) and accuracy (85.1–98.3%). The proposed method is rapid, convenient and low-cost for the determination of melamine in milk products.

A sensitive and selective electrochemical method for the determination of levodopa (LD) was developed using a ferrocenemonocarboxylic acid modified carbon nanotubes paste electrode (FMCNPE). The modified electrode exhibited good electrocatalytic activity for electrochemical oxidation of LD at pH 7.0 phosphate buffer solution. The diffusion coefficient (D = 1.44 × 10−6 cm2 s−1), and the kinetic parameters such as the electron transfer coefficient (α = 0.4) and catalytic rate constant (k = 1.8 × 10 3 M−1 s−1) of LD at the surface of FMCNPE were determined using electrochemical approaches. Differential pulse voltammetry (DPV) of LD at the modified electrode exhibited linear dynamic range with a detection limit (3σ) of 70 nM. DPV was used for simultaneous determination of LD and FA at the modified electrode. Finally, this modified electrode was also examined for the determination of LD and FA in urine samples.

In this paper, a simple method for the simultaneous preconcentration and sensitive determination of the estrogens hexestrol, diethylstilbestrol and dienestrol in environmental water samples has been developed, based on solid-phase extraction (SPE) and high-performance liquid chromatography-ultraviolet detection (HPLC-UV). Bamboo charcoal, an inexpensive material, was investigated and used as a SPE adsorbent for the enrichment and determination of these estrogens. Important parameters affecting extraction efficiencies, including the eluant and its volume, flow rate, sample pH and volume were investigated and optimized in detail. Under the optimum conditions, the experimental data exhibited excellent linear relationships between peak areas and concentrations over the range 1–100 μg L−1. The limits of detection and precision were in the range of 0.03–0.06 μg L−1 and 4.46–8.65%, respectively. The proposed method has been successfully applied for the trace analysis of these estrogens in real-world environmental water samples with satisfactory results.

Acoustic lysis of vegetative bacterial cells: Method and device development by Oana C. Marina; Claire K. Sanders; Gregory Kaduchak; Gregory R. Goddard; Steven W. Graves (2573-2578).
A critical problem of many pathogen detection assays is the availability of intracellular protein and deoxyribonucleic acid (DNA). Acoustic lysis of suspended vegetative bacterial cells in a microfluidic system offers several advantages over conventional lysis techniques. The intracellular proteins and DNA are released and available for detection. A novel acoustic lysing alternative technique to the existing lysing methods for sample preparation and lysis step is proposed. We report here an efficient lysis device that uses acoustic excitation for performing lysis of Gram-positive and Gram-negative vegetative cells and has a high yield in a short amount of time. We also verified the condition of released protein since one of the major uses of vegetative cells lysis is for protein expression studies. Fluorimetry and flow cytometry were used to assess the degree of damage induced on the cells by the actual lysis method. The acoustic device allows the delivery of proteins in a non-denatured form, without adding chemicals, particles or other substances (e.g. enzymes) that could complicate the process or the detection procedure. The lysis device operates at low power (50–400 mW) and short time (3 min) and has high efficiency in comparison to current lysis standards (>85% vs. 12–50%).

The interaction between antimicrobial peptide (JCpep7) and the living Staphylococcus aureus cells (LSACs) used as pseudo-stationary phase in Capillary Electrochromatography (CEC) was investigated in this paper. The binding constants at 298, 303 and 309 K was 3.85 × 1012 M−1, 5.04 × 1012 M−1 and 6.73 × 1012 M−1, which indicated the evident interaction between JCpep7 and LSACs. The positive enthalpy change (ΔH) and entropy change (ΔS) values showed that hydrophobic effect played a major role in the binding process. Furthermore, to better understand the binding process between JCpep7 and LSACs, conformational changes of JCpep7 when it bound to the living bacteria was also studied by fluorescence spectrum and the results were consistent with those obtained by CEC.

A two-chip microdevice has been developed for the analysis of phenylephrine hydrochloride (PEH) in pharmaceutical formulations and biological fluids using (2,2′-bipyridyl)ruthenium(ii) (Ru(bipy)32+)-peroxydisulphate chemiluminescence (CL) system. It was observed that a pre-derivatization step is required to obtain an analytically useful CL signal. Using 0.5 mol L−1 formaldehyde solution as derivatization reagent, eighteen-time enhancement in the CL signal was observed. The derivatization reaction, photoreaction of (Ru(bipy)32+) with peroxydisulphate to produce (Ru(bipy)33+), reaction of (Ru(bipy)33+) with the derivatization product and detection of the produced CL signal were all carried out in this two-chip microdevice using minute quantity of reagents (27 μL per run). Various parameters that influence the derivatization reaction and the CL signal intensity were optimized. These include pH, flow rates and concentration of reagents used. A linear response was observed over the range 0.25 μg mL−1 to 15.0 μg mL−1 (R2 = 0.9999) with RSD values of 0.03% (n = 15) for 1 μg mL−1. The limit of detection (LOD) was found to be 0.027 μg mL−1 (S/N = 3). The developed method was successfully applied for the analysis of PEH in biological fluids and pharmaceutical formulations.

A carboxylated multi-walled carbon nanotubes modified glassy carbon (MWCNTs-GC) electrode was fabricated via the drop-casting of a carboxylated carbon nanotubes suspension onto a glassy carbon electrode. A headspace adsorptive accumulation on the surface of the multi-walled carbon nanotubes modified glassy carbon electrode coupled with cyclic voltammetry has been established for analyzing nitrobenzene and 4-nitrotoluene in water samples. Factors which affect the extraction efficiency (modifier amount, pH, extraction temperature, extraction time, salt addition) have been investigated. The calibration plot for the determination of nitrobenzene and 4-nitrotoluene are linear in the wide ranges of 10–4000 ng mL−1 and 20–6000 ng mL−1 respectively. Under optimal extraction conditions, the detection limits (at S/N = 3) for nitrobenzene and 4-nitrotoluene were 3.0 ng mL−1 and 6.0 ng mL−1 respectively. The proposed method has been applied successfully for the determination of nitrobenzene and nitrotoluene in water and wastewater samples within a recovery range of ca. 97.5–103.33%.

Silver nanoparticles as a cyanide colorimetric sensor in aqueous media by Salahaddin Hajizadeh; Khalil Farhadi; Mehrdad Forough; Reza Emamali Sabzi (2599-2603).
The interaction between aqueous colloidal silver nanoparticles (AgNPs) and cyanide ions was studied using UV-Vis absorption and scanning electron microscopy (SEM) techniques. It was found that AgNPs were oxidized by dissolved oxygen in the presence of cyanide ions, resulting in a considerable decrease in the intensity of the surface plasmon resonance (SPR) absorption band of AgNPs. So, we propose a simple, cost effective, rapid, sensitive and selective colorimetric sensor for the detection of cyanide using AgNPs in aqueous media. There is a linear relationship between the absorbance intensity of AgNPs and the concentration of cyanide ions over the range of 16.7 μmol L−1–133.3 μmol L−1 at 394 nm. The proposed method has been successfully used for the determination of cyanide in water samples.

An electrochemically active composite film which contains multi-walled carbon nanotubes (MWCNTs) incorporated with naphthol green B (NGB) was synthesized on glassy carbon, gold and indium tin oxide electrodes by potentiodynamic method. The presence of MWCNTs in the composite (MWCNTs-NGB) film modified electrodes mediates redox reaction of FeIII/II present in NGB, whereas no redox reaction of NGB occurred at the bare electrode. The electrochemical impedance spectroscopy studies revealed that NGB and MWCNTs present in MWCNTs-NGB composite film enhances electron shuttling between the reactant and electrode surface. The surface morphology of the MWCNTs and MWCNTs-NGB were studied using atomic force microscopy, which revealed that NGB incorporated and covered the MWCNTs on the modified electrode. The MWCNTs-NGB composite film exhibits promising enhanced electrocatalysis towards S2O82−. The response of S2O82− at MWCNTs and MWCNTs-NGB composite films was measured using both cyclic voltammetry and i–t curve techniques. The linear concentration range of S2O82− determination at MWCNTs-NGB composite film was from 15.9 μM to 7.1 mM. The sensitivity of MWCNTs-NGB composite film towards S2O82− was −301.9 μA mM−1 cm−2, and the limit of detection was 6.9 μM. These above values are better than the values obtained at MWCNTs film.

Copper(ii) doped nanoporous TiO2 composite based glucose biosensor by Xiaojun Zhang; Guangfeng Wang; Yan Huang; Liutao Yu; Bin Fang (2611-2615).
In this report we simply prepared a copper(ii) doped nanoporous TiO2 composite, which combines the capabilities of an immobilizing enzyme with electrocatalyzing glucose. Transmission electron microscopy and energy dispersive X-ray analysis were used for the characterization of the composite. Comparison experiments of the cyclic voltammetry and electrochemical impedance spectroscopy of Cu(ii) doped TiO2 composites with different doping ratio modified electrodes demonstrate that when the ratio of Cu(ii) and TiO2 is kept at 1 : 1, the electrochemical response to glucose is the best. We suggest that in the doping materials too little TiO2 would decrease the immobilization of GOx, which leads to a poorer response to glucose. However, too little Cu(ii), would decrease the electron transfer ability. Cu(ii) doped TiO2 with the ratio of 1 : 1 displays a good linear relation between the oxidation peak current and the concentration of glucose with the range from 0.5 μM–3 mM, correlation coefficients of 0.9989 and a fast response time of within 5 s. The experimental limit of detection, based on a signal-to-noise ratio of 3, was 0.1 μM and the sensitivity of the sensor was 0.9040 μA μM−1. The experimental results also showed that the sensor has good reproducibility, long-term stability and is interference free.

The possibility of simultaneous quantification of dilute aqueous solutions of five polycyclic aromatic hydrocarbons (PAHs) with substantial overlap of fluorescence spectra without preseparation was examined by using total synchronous fluorescence spectroscopy (TSFS) with multivariate methods like N-way partial least square (N-PLS), unfolded-PLS and multivariate curve resolution-alternating least square (MCR-ALS) analysis. The PAHs chosen were anthracene, benzo[a]pyrene, chrysene, perylene and pyrene. Two calibration sets were made using different approaches. Even with significant spectral overlap, the N-PLS, unfolded-PLS and MCR-ALS models were found to be robust for the quantification of all five PAHs in the calibration set where the amounts of PAHs were changed with the fixed relative ratio. The second calibration set, which is relatively difficult to analyse, consisted of samples where amounts of PAHs were changed randomly. It was found that even for this set, predictions were satisfactory. The three calibration models based on N-PLS, unfolded-PLS and MCR-ALS in the second set were relatively more robust for chrysene, benzo[a]pyrene and perylene compared to other PAHs. Root mean square error of calibration (RMSEC) and root mean square error of prediction (RMSEP) and regression parameters were calculated for all the three calibration models. The various parameters show that the combination of TSFS and N-PLS, unfolded-PLS and MCR-ALS analysis can be used for the simultaneous quantification of PAHs in water without any preseparation even when there is substantial overlap of fluorescence.

A procedure using slurry sampling for the determination of manganese in corn flour by ET AAS by Douglas G. daSilva; Geraldo D. Matos; Ana M. P. dos Santos; Raigenis da Paz Fiuza; Robson M. de Jesus (2625-2629).
A fast and simple procedure is proposed for determination of manganese in corn flour samples employing slurry sampling and electrothermal atomic absorption spectrometry. To evaluate the particle size scanning electron microscopy was used. Slurries were prepared by weighing approximately 100 mg of sample, adding 0.014 mol L−1 nitric acid, and a solution containing 1% Triton X-100. An ultrasonic bath was employed for slurry homogenization. Analytical variables including temperature program of the graphite furnace, nitric acid concentration, amount of sample and sonication times were studied. The pyrolysis and atomization temperatures established during the optimization step were 1200 and 2100 °C, respectively, using 10 μg Pd + 3 μg Mg as the chemical modifier. The limits of detection and quantification obtained were 0.006 and 0.020 μg g−1, respectively. A characteristic mass of 0.97 pg was obtained. The precision was evaluated for a sample containing 2.17 μg g−1 of manganese and 2.6% relative standard deviation was found (n = 10). The accuracy was assessed by the analysis of two certified reference materials: NIST SRM 1568a—Rice Flour and CRM 189—Wholemeal Flour. Some experiments demonstrated that the calibration can be performed employing the external calibration technique using aqueous standards. For comparison and validation of the procedure proposed, the samples were also analyzed after complete decomposition and determined by ETAAS and good precision was obtained (at 95% confidence level). In this way, no statistical difference was observed between the results obtained by both the procedures performed. The proposed procedure has been applied for manganese determination in six samples acquired in supermarkets of Salvador (Brazil) and values varied from 0.623 to 2.17 μg g−1.

A novel method based on microextraction in packed syringe (MEPS), as a sample preparation technique in combination with gas chromatography-mass spectrometry (GC-MS) was developed using a polypyrrole (PPy) nanowires network as the extracting medium. The PPy nanowires network was prepared using a soft template technique and its characterization was studied by scanning electron microscopy (SEM). The use of micelles in this methodology has an important role in the shape of the growing polymer. The pyrrole monomer was introduced into cetyltrimethylammonium bromide (CTAB) micelles and this has led to the formation of nanowires with diameters ranging from 30 to 60 nm. The bulk PPy prepared without CTAB were all spherical particles with diameters of 100–400 nm. The PPy nanowires network was shown to have a 7–28 times higher extraction capability in comparision with bulk PPy. The developed method was applied to the analysis of some important triazine herbicides. Important parameters influencing the extraction and desorption processes were optimized and a 25 cycles of draw–eject of sample with a speed of 10 rpm gave a maximum peak area, when 2 mg of PPy nanowires network was packed in the syringe. After entrapment of the selected analytes, the desorption was performed using 150 μL of acetonitrile. Limits of detection (LODs) were in the range of 0.02 to 0.05 ng mL−1 using the time scheduled selected ion monitoring (SIM) mode. Intraday precision (RSD %) values for four replicates ranged from 7 to 10% and interday precision values obtained for three replicates measured on different days were in the range of 10–12% at a concentration level of 5 ng mL−1. The linearity of the method was in the range of 0.5–500 ng mL−1. The developed method was successfully applied to the analysis of water samples obtained from the Zayandeh-roud river and the matrix factor for the spiked real water samples was found to be in the range of 0.72–0.92.

Multi-wall carbon nanotubes as a sensor and ferrocene dicarboxylic acid as a mediator for voltammetric determination of glutathione in hemolysed erythrocyte by Jahan Bakhsh Raoof; Reza Ojani; Hassan Karimi-Maleh; Mohammad R. Hajmohamadi; Pouria Biparva (2637-2643).
A sensitive and selective electrochemical method was developed for determination of glutathione (GSH) in hemolysed erythrocyte using a ferrocene dicarboxylic acid modified carbon nanotubes paste electrode (FDCCNTPE). Cyclic voltammetry (CV), double potential-step chronoamperometry and differential pulse voltammetric (DPV) were used to investigate the suitability of ferrocene dicarboxylic acid at the surface of a multi-wall carbon nanotubes paste electrode as a mediator for the electrocatalytic oxidation of glutathione in aqueous solutions with various pH. The results showed that FDCCNTPE had high electrocatalytic activity for the electrooxidation of glutathione. Under optimum conditions (pH 7.00), oxidation of GSH occurs at a potential about 230 mV less positive than that at the unmodified carbon nanotubes paste electrode. The catalytic reaction rate constant, kh was calculated (1.98 × 104 M−1 s−1) using chronoamperometry. The electrocatalytic oxidation peak current of GSH showed two linear dynamic ranges with a detection limit of 0.2 μmol L−1 GSH. The linear calibration ranges were obtained between 0.5–24 μmol L−1 and 24–122 μmol L−1 GSH using the DPV method. Finally, the proposed method was also examined as a selective, simple and precise electrochemical sensor for the determination of GSH in real samples such as hemolysed erythrocyte.

Evaluation of analytical reflection scanometry as an analytical tool by A. Lapresta-Fernández; L. F. Capitán-Vallvey (2644-2650).
This paper describes three different methodologies for the easy readout of a potassium bulk optode film using a flatbed scanner working in reflection mode to obtain quantitative information. The analysis of the digital image through the analysis of the colour of the test zone requires the optimization of the scanner variables and further statistical evaluation of the method. In this study we used two different optical scanners, one colour calibrated scanner and another without calibration. Different image analysis protocols were applied: (i) with the calibrated scanner at 48 bits resolution and 300 dots per inch (dpi) colour depth; (ii) at 48 bits and 75 dpi without applying the image procedure and (iii) with a non-calibrated scanner but in its best scanning conditions, available in the scanner set up, of 24 bits and 600 dpi. After optimizing the scanner, variables such as resolution and colour depth, the obtained application ranges, in M units, for the different measurement system used were (i) from 4.3 × 10−7 to 0.1; (ii) 2.8 × 10−7 to 0.1; and (iii) 9.8 × 10−7 to 0.1 with a precision between 0.8 and 2.5%, 1.0 and 2.1%, 2.2 and 2.8%, respectively. Then, the trueness of the proposed scanometric methods was checked by means of a statistical study and finally, the proposed method was validated against a reference procedure using water samples from different sources and beverages, showing that there are no significant statistical differences at a 95% confidence level.

In this paper an ionic liquid (IL) 1-butylpyridinium hexafluorophosphate (BPPF6) modified carbon paste electrode was constructed and further used to establish a sensitive method for the detection of 5-fluoro-1H-pyrimidine-2 (5-FU). Compared with the carbon paste electrode (CPE), a single well-defined irreversible oxidation peak of 5-FU appeared on the IL-CPE with a great improvement of the electrochemical response, which was due to the presence of a high ionic conductive ionic liquid in the electrode that exhibited accumulation and electrocatalytic ability. The electrochemical parameters of 5-FU on the IL-CPE were calculated with the electron transfer coefficient (α) as 0.62 and the diffusional coefficient (D) as 7.02 × 10−5 cm2 s−1. Under the optimal conditions, the oxidation peak current was linear to 5-FU concentration in the range from 5.0 × 10−7 to 8.0 × 10−4 mol L−1 with a detection limit of 1.3 × 10−8 mol L−1 (3σ). Good selectivity to 5-FU detection was observed without the interference of coexisting substances and the method was further applied to determination in injection samples with satisfactory results.

Using GC-MS for the quick analysis of carbon dioxide for soil microbial biomass determination by Brenda Koerner; William Calvert; David J. Bailey (2657-2659).
Microbial biomass carbon is a common soil parameter used to evaluate microbial abundance in soil environments, and microbial biomass can change in response to disturbances and/or changing environmental conditions. Microbial biomass carbon was determined on 192 soil samples using the chloroform fumigation-incubation method, and after 10 days, gas samples were collected. Gas samples were analyzed for carbon dioxide (CO2) concentration. Determination of CO2 concentration is usually conducted by infrared gas analysis or chemical analysis (an alkali trap and subsequent titration, or soda lime absorption), and these methods were either unavailable or too time consuming for the number of samples analyzed. As a result, we have developed an analytical method for the determination of CO2 in gases using the gas chromatograph/mass spectrometer (GC-MS). This method requires a 5 μL injection, 5 minute analysis time, and the resulting figures of merit are comparable to most procedures present in the literature. Additionally, the microbial biomass carbon values obtained were consistent with previously published values in similar ecosystems.

Back matter (2660-2660).

Back cover (2661-2662).