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Analytical and Bioanalytical Chemistry (v.386, #7-8)

Help wanted: sage and wise chemist by John Fetzer (pp. 1939-1940).

is the author or co-author of over 130 research articles, reviews, and book chapters. He is a member of the International Advisory Board of Analytical and Bioanalytical Chemistry. Dr. Fetzer worked for over 20 years as an analytical chemist for the Chevron Corporation and now runs his own consulting company, Fetzpahs Consulting, in Hercules, CA, USA. His book Career Management for Chemists—A Guide to Success in a Chemistry Career, was published by Springer.

R.D. McDowall: Validation of chromatography data systems: Meeting business and regulatory requirements by Kazuo Iwaki; Yuzuru Hayashi (pp. 1941-1942).

Thanks to reviewers in 2006 (pp. 1943-1949).

Reduction of nonspecific protein binding on surface plasmon resonance biosensors by Jean-Francois Masson; Tina M. Battaglia; Jeff Cramer; Stephen Beaudoin; Michael Sierks; Karl S. Booksh (pp. 1951-1959).

Reduction of the nonspecific serum protein adsorption on a gold surface to levels low enough to allow the detection of biomarkers in complex media has been achieved using the N-hydroxysuccinimide (NHS) ester of 16-mercaptohexadecanoic acid. Carboxymethylated dextran (CM dextran), which is widely used, nonspecifically adsorbs enough proteins to mask the signal from target biomarkers in complex solutions such as serum or blood. The use of short-chain thiols greatly reduces the amount of nonspecific protein adsorption. Mixed layers of 11-mercaptoundecanoic acid or the NHS ester of 11-mercaptoundecanoic acid mixed layers with either 11-mercaptoundecanol or undecanethiol, and 16-mercaptohexadecanoic acid or the NHS ester of 16-mercaptohexadecanoic acid with hexadecanethiol, were also investigated for nonspecific protein binding properties as well as for biomarker signal response. The NHS ester of 16-mercaptohexadecanoic acid exhibits the largest signal for the biomarker myoglobin (including CM dextran) while offering a significantly diminished amount of nonspecific binding. The sensor has also been shown to detect interleukin-6 in cell culture media containing protein concentrations of at least 4 mg/mL.

Keywords: Short-chain thiol; CM dextran; Serum adsorption; Gold surfaces; Biomarker detection; 11-Mercaptoundecanoic acid; 16-Mercaptohexadecanoic acid

Analysis of type I and IV collagens by FT-IR spectroscopy and imaging for a molecular investigation of skeletal muscle connective tissue by Cyril Petibois; Gilles Gouspillou; Katia Wehbe; Jean-Paul Delage; Gérard Déléris (pp. 1961-1966).

Many muscular diseases result from abnormal organization of connective tissue and/or collagen network formation. Only a few molecular imaging techniques are able to analyze this collagen network by differentiating collagen types. In this study, FT-IR spectroscopy was used to analyze type I and IV collagens, the most important compounds of which are perimysium and endomysium, respectively. Secondary structure of collagen types was determined by curve-fitting the 1,700–1,480 cm⁻¹ spectral interval. Type I collagen could be differentiated from type IV by its higher amounts of triple helix and α-helix, but lower amounts of β-sheets (P < 0.01). FT-IR imaging was then used to determine structural features of perimysium and endomysium collagen network in bovine Flexor carpi radialis muscle. Secondary structure of proteins contained in perimysium and endomysium was found to be very close to type I and IV collagens, respectively. FT-IR spectroscopy and imaging are thus analytical tools that might be used for investigating biodistribution and assembly of collagen types in connective tissues. Figure Visible (left) and full spectral FT-IR (right) images of skeletal muscle tissue section (16 μm) exhibiting a vertical arrangement of fibers. + and × in FT-IR image show selected positions to obtain FT-IR spectra of perimysium and endomysium, respectively

Keywords: FT-IR imaging; Skeletal muscle; Collagen; Perimysium; Endomysium

A study on photolinkers used for biomolecule attachment to polymer surfaces by Daniela M. Dankbar; Günter Gauglitz (pp. 1967-1974).

The use of photolinkers (photoactivatable heterobifunctional crosslinkers) is a popular method to attach biomolecules to polymer surfaces. This study addresses the selection of photolinker and the adjustment of reaction conditions, such as the concentration of biomolecule applied, and irradiation time. The influence of these variables are investigated for four prominent photolinkers: ketyl-reactive benzophenone (BP) and anthraquinone (AQ), nitrene-reactive nitrophenyl azide (NPA), and carbene-reactive phenyl-(trifluoromethyl)diazirine (PTD). The influence of substrate material is discussed, and three different polymers served as representative substrates: poly(methyl methacrylate) (PMMA), polystyrene (PS), and a cycloolefin copolymer (COC). We compared the overall photolinking efficiency of all photolinkers with respect to the polymer substrate they are applied to, and we found considerable differences for certain photolinker/substrate combinations. Of all photolinkers and substrates tested, PTD as photolinker and COC as substrate showed the highest photolinking efficiencies and fastest reaction times. For this study DNA oligonucleotides were chosen as a model system of biomolecular probes, and fluorescence detection of DNA microarrays served as method of detection.

Keywords: Photo-immobilization; Photolinker; DNA microarray; Biomolecule attachment; Polymer surface

Detection and identification of IHN and ISA viruses by isothermal DNA amplification in microcapillary tubes by Erik L. McCarthy; Teressa J. Egeler; Lee E. Bickerstaff; Mauricio Pereira da Cunha; Paul J. Millard (pp. 1975-1984).

Unique base sequences derived from RNA of both infectious hematopoietic necrosis virus (IHNV) and infectious salmon anemia virus (ISAV) were detected and identified using a combination of surface-associated molecular padlock DNA probes (MPPs) and rolling circle amplification (RCA) in microcapillary tubes. DNA oligonucleotides with base sequences identical to RNA obtained from IHNV or ISAV were recognized by MPPs. Circularized MPPs were then captured on the inner surfaces of glass microcapillary tubes by immobilized DNA oligonucleotide primers. Extension of the immobilized primers by isothermal RCA produced DNA concatamers, which were labeled with fluorescent SYBR Green II nucleic acid stain, and measured by microfluorimetry. Molecular padlock probes, combined with this method of surface-associated isothermal RCA, exhibited high selectivity without the need for thermal cycling. This method is applicable to the design of low-power field sensors capable of multiplex detection of viral, bacterial, and protozoan pathogens within localized regions of microcapillary tubes.

Keywords: Bioanalytical methods; Biosensors; Fluorescence/luminescence; Virus; Molecular padlock

Study of the metabolism on tobacco-specific N-nitrosamines in the rabbit by solid-phase extraction and liquid chromatography–tandem mass spectrometry by Chenchen Li; Dawei Wen; Jianxun Zhang; Zheng Chen; Weihong Cong; Zhihua Rao; Huwei Liu (pp. 1985-1993).

Metabolism of four tobacco-specific N-nitrosamines (TSNAs), N′-nitrosonornicotine (NNN), N′-nitrosoanatabine (NAT), N′-nitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) has been studied by solid-phase extraction (SPE) and liquid chromatography–tandem mass spectrometry (LC–MS–MS). 4-(Methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) was used as internal standard. SPE and LC–MS–MS was found to be a rapid, simple, sensitive, and selective method for analysis of TSNAs in rabbit serum. The relative standard deviation (R.S.D., n = 6) for analysis of 5 ng mL⁻¹ and 0.5 ng mL⁻¹ standards and of serum sample spiked with 5 ng mL⁻¹ standards of five TSNAs was 2.1–11% and recovery of 5 ng mL⁻¹ standards from serum was 100.2–112.9%. A good linear relationship was obtained between peak area ratio and concentration in the range of 0.2–100 ng mL⁻¹ for NNAL and 0.5–100 ng mL⁻¹ for other four TSNAs, with correlation coefficients (R ²) >0.99 (both linear and log–log regression). Detection limits for standards in solvent were between 0.04 and 0.10 ng mL⁻¹. Doses of TSNAs administered to rabbits via the auricular vein were 4.67 μg kg⁻¹ and 11.67 μg kg⁻¹, in accordance with the different levels in cigarettes. Metabolic curves were obtained for the four TSNAs and for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a metabolite of NNK; on the basis of these curves we modeled metabolic kinetic equations for these TSNAs by nonlinear curve fitting.

Keywords: Tobacco-specific N-nitrosamines; Metabolism; Solid-phase extraction; Liquid chromatography–tandem mass spectrometry

Quantitative real-time RT-PCR for determination of vitellogenin mRNA in so-iuy mullet (Mugil soiuy) by Lihui An; Jianying Hu; Zhaobin Zhang; Min Yang (pp. 1995-2001).

A quantitative real-time reverse transcription polymerase chain reaction (Q- RT-PCR) assay was developed for quantification of vitellogenin (Vtg) mRNA normalized to β-actin in so-iuy mullet. Vtg mRNA in liver samples of so-iuy mullet was induced after a single injection of E2 (0.01, 0.1, 1.0 μg/g body) and a dose–response relationship was obtained. This method was applied to determine Vtg mRNA in so-iuy mullet collected from Liaodong Bay, Bohai Bay, NanDaiHe, and a control site in north China. Compared to the control, a high level of Vtg mRNA expression was detected in so-iuy mullets collected from NanDaiHe, whereas no obvious difference between Vtg mRNA expression from Liaodong Bay and Bohai Bay was found. Thus, this method is expected to be useful for further studying the potential of Vtg mRNA as a biomarker for assessing estrogenic activity in marine environments using the so-iuy mullet as a bioindicator species.

Keywords: Biomarker; Endocrine-disrupting chemicals; Quantitative real-time reverse transcription polymerase chain reaction; Vitellogenin mRNA; So-iuy mullet

Comparison of alpha1-adrenergic receptor cell-membrane stationary phases prepared from expressed cell line and from rabbit hepatocytes by Yu Wang; Bingxiang Yuan; Xiuling Deng; Langchong He; Sicen Wang; Youyi Zhang; Qide Han (pp. 2003-2011).

A G-protein-coupled receptor-cell-membrane stationary phase (CMSP) has been prepared by immobilizing cell membranes on the surface of silica, as carrier. The resulting HEK293 α 1A adrenoceptor cell-membrane stationary phase can be used for rapid on-line chromatographic determination of potential subtype-selective α 1-adrenoceptor ligand-binding affinities for α 1-adrenoceptor subtypes. The objective of the research was to study whether cell lines stably overexpressing subtype receptors could improve the sensitivity and specificity of cell-membrane chromatography (CMC) compared with use of homogenized tissue and cells in primary culture. Effects of mobile-phase ionic strength, sample concentration, and the presence of competitive agents on ligand-receptor interaction in CMSP were also evaluated. We found that cell lines stably overexpressing subtype receptors led to improved sensitivity and specificity in CMC. The technique leads to useful procedures-cell-membrane stationary phases may, for example, facilitate exploration of ligand-receptor interaction and determination of ligand-receptor binding affinity in initial screening and separation of lead compounds or active components in Chinese traditional natural medicine and herbs. This might eventually be an important contribution and an addition to our collection of techniques.

Keywords: G-protein-coupled receptor; Cell-membrane chromatography; Cell-membrane stationary phase; α_1A adrenergic receptors; Immobilization

Determination of homocitrulline in urine of patients with HHH syndrome by liquid chromatography tandem mass spectrometry by Osama Y. Al-Dirbashi; Zuhair N. Al-Hassnan; Mohamed S. Rashed (pp. 2013-2017).

A liquid chromatography tandem mass spectrometric method is described for the analysis of homocitrulline in human urine, a key metabolite in the differential diagnosis of hyperammonemia, hyperornithinemia, homocitrullinuria (HHH) syndrome. Urine samples were prepared by mere five-fold dilution with a mixture of internal standards (²H₂-citrulline and ²H₃-creatinine) used for the simultaneous quantification of creatinine. Analytes were separated on a cyano column and eluted isocratically within seven min. Detection was achieved by monitoring transitions of 190 > 84 and 190 > 127 for homocitrulline, 178 > 115 for ²H₂-citrulline, 114 > 44 for creatinine and 117 > 47 for ²H₃-creatinine. Calibration curves were linear up to 100 micromol/L. Intraday (n = 7) and interday (n = 6) variations were less than 10%. In urine samples from three siblings confirmed to have HHH syndrome, homocitrulline levels were at 13.3 (74), 21.1 (50) and 108.2 (103) mmol/mol creatinine (micromol/L). Control values were 0–9 mmol/mol creatinine (n = 120). The current method solves specificity issues in homocitrulline determination often encountered with some ninhydrin-based systems (coelution with methionine) and some o-phthalaldehyde-based ones (coelution with taurine), and presents an attractive alternative with a relatively high throughput.

Keywords: Homocitrulline; Homocitrullinuria; HHH syndrome; LC–MS/MS

A convenient HPLC assay for the determination of fructosamine-3-kinase activity in erythrocytes by René Krause; Anett Oehme; Katja Wolf; Thomas Henle (pp. 2019-2025).

Fructosamine-3-kinase (FN3K) mediates the regeneration of lysine from fructosamines formed on proteins as a result of the ‘early’ Maillard reaction. As fructosamines and advanced glycation endproducts derived therefrom are supposed to play an adverse role in the development of diabetic complications, FN3K is discussed as a protein-repairing enzyme. In this study, a method for the determination of FN3K activity in erythrocyte lysate is described which overcomes the complexity of currently known assays. The assay is based on the FN3K-dependent conversion of the synthetic UV-active fructosamine N ^α-hippuryl-N ^ε-(1-deoxy-D-fructosyl)lysine (BzGFruK) to N ^α-hippuryl-N ^ε-(phosphofructosyl)lysine (BzGpFruK). The FN3K activity was quantified by measuring the formation of BzGpFruK using RP-HPLC with UV detection. Identification of the metabolite BzGpFruK was achieved by means of UV and mass spectroscopy. The results are related to the content of haemoglobin for standardisation. First activity measurements with a chosen number of normoglycaemic subjects confirmed the convenient applicability of the method and showed distinctly different individual activities, as already discovered recently. The new established assay needs only the equipment of a routine laboratory with HPLC instrumentation. This should facilitate further studies about a possible relationship between the FN3K activity and the development of diabetic complications.

Keywords: Fructosamine-3-kinase; Maillard reaction; Glycation; Diabetic complications

Purification and characterization of a biosurfactant produced by Pseudomonas sp. G11 by asymmetrical flow field-flow fractionation (AsFlFFF) by Sung Kwang Cho; So Hee Shim; Kyeong Ryang Park; Seong-Ho Choi; Seungho Lee (pp. 2027-2033).

Three hundred and thirty two bacterial colonies were isolated from soil contaminated by an oil spill. All the bacteria were cultured in a liquid medium individually, and the surface tensions of the media were compared. The bacterium whose culture medium had the lowest surface tension was identified as Pseudomonas sp. G11. A biosurfactant was produced by cultivation of the Pseudomonas sp. G11 in the LB media. For extraction of the biosurfactant, two solvent systems were used (n-hexane and a 2:1 (v/v) mixture of chloroform/MeOH), and the results were compared. Various experimental conditions (solvent composition, flow rate, etc.) were tested to optimize the analysis of the biosurfactant by asymmetrical flow field-flow fractionation (AsFlFFF). The biosurfactant was successfully separated from the culture medium by AsFlFFF when pure water was used as the carrier. From the retention data, the hydrodynamic diameter (d _H) and molecular weight (M) of the biosurfactant were determined by AsFlFFF. The molecular weight was determined by using pullulans as the calibration standards. The d _H and M were 49 nm and 2.3 × 10⁵ Da when extracted with n-hexane, and 39 nm and 1.13 × 10⁵ Da when extracted with the 2:1 mixture of chloroform/MeOH, respectively. Figure Separation of biosurfactant from its culture medium by flow FFF

Keywords: Purification; Characterization; Biosurfactant; Pseudomonas sp. G11; Asymmetrical flow field-flow fractionation (AsFlFFF); Hydrodynamic diameter; Molecular weight

Determination of aspartate and glutamate in rabbit retina using polymer monolith microextraction coupled to high-performance liquid chromatography with fluorescence detection by Hui-Juan Zhang; Jin-Shu Li; Hong Wang; Yu-Qi Feng (pp. 2035-2042).

A simple, sensitive and low-cost method was developed for the determination of aspartate (Asp) and glutamate (Glu) in rabbit retina. Polymer monolith microextraction (PMME) using a poly(acrylamide–vinylpyridine–N,N′-methylene bisacrylamide) (AA–VP–Bis) monolithic column was combined with derivatization of Asp and Glu using 8-phenyl-(4-oxy-acetic acid N-hydroxysuccinimide ester)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (TMPAB-OSu), and this was used to analyze the derivatives of Asp and Glu by high-performance liquid chromatography (HPLC) with fluorescence detection. The conditions for the derivatization and the subsequent extraction of Asp and Glu derivatives were optimized. The enrichment factors for the derivatives of Asp and Glu were found to be 14.1 and 14.7, respectively, by PMME. The limits of detection of Asp and Glu were 0.14 and 0.53 nmol/L, respectively. The precision and recovery were evaluated with spiked retina. The inter- and intraday relative standard deviations were less than 10%. The proposed method was successfully applied to the determination of Asp and Glu levels in rabbit retina samples with different stages of intraocular hypertension.

Keywords: Polymer monolith microextraction; Derivatization; Excitatory amino acids; 8-phenyl-(4-oxy-acetic acid N-hydroxysuccinimide ester)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (TMPAB-OSu); HPLC

Quantitative determination of ginsenoside Rh₂ in rat biosamples by liquid chromatography electrospray ionization mass spectrometry by Yi Gu; Guang-Ji Wang; Jian-Guo Sun; Yuan-Wei Jia; Hai-Tang Xie; Wei Wang (pp. 2043-2053).

Ginsenoside Rh₂ is a “hot” natural compound with great potential as a new anti-cancer drug based on abundant pharmacological experiments. However, no systemic pharmacokinetic study of Rh₂ was reported because current analysis methods could not fully meet the requirements. Thus, we developed a simple LC/MS method with highly improved sensitivities for the determination of Rh₂ in rat plasma, bile, urine, feces and most tissues. The tissues and feces were firstly homogenized mechanically using buffer and methanol as the media, respectively. Plasma, bile, urine and tissue homogenates were extracted with diethyl ether for sample preparation. Feces homogenates were directly deproteinized with acetonitrile. The subsequent analysis procedures were performed on a Shimadzu LCMS2010A system (electrospray ionization single quadrupole mass analyzer), with an ODS column (150 mm × 2.0-mm i.d., 5 μm) plus a C18 guard column for separation and ammonium chloride (500 μmol) as mobile phase additive. The proportions of mobile phase were changed timely according to gradient programs. Chlorinated adducts of molecular ions [M + Cl]⁻ of Rh₂ at m/z 657.35 and internal standard digitoxin at m/z 799.55 were monitored in selective ion monitoring mode of negative ions. The method was validated to be accurate, precise and rugged with good linearity in all matrices, according to the FDA guidelines. The lower limits of quantitation in rat plasma, urine and feces were 0.2, 0.2 and 20 ng/mL respectively. Stability studies were also performed, indicating that there were no stability-related problems in the analytical procedure of Rh₂. The proposed method was successfully applied to the preclinical pharmacokinetic research of Rh₂ in rats, including plasma kinetics, tissue distribution and excretion studies.

Keywords: Ginsenoside Rh₂ ; LC/ESI/MS; Bioanalysis; Pharmacokinetic application; Rat biosamples

Nanostructured electrochemical DNA biosensors for detection of the effect of berberine on DNA from cancer cells by Renáta Ovádeková; Soňa Jantová; Silvia Letašiová; Ivan Štepánek; Ján Labuda (pp. 2055-2062).

Multi walled carbon nanotubes (MWNT) in dimethylformamide (DMF) or aqueous sodium dodecyl sulfate (SDS) solution, colloidal gold nanoparticles (GNP) in phosphate buffer solution (PBS), and a GNP–MWNT mixture in aqueous SDS solution have been investigated for chemical modification of a screen-printed carbon electrode used as the signal transducer of a dsDNA-based biosensor. Differential pulse voltammetry of the DNA redox marker $$ Co{left[ {{left( {phen} ight)}_{3} } ight]}^{{3 + }} $$ and the guanine moiety anodic oxidation and cyclic voltammetry with K₃[Fe(CN)₆] as indicator revealed substantial enhancement of the response of the biosensor, particularly when MWNT in SDS solution was used. The biosensor was used in testing of berberine, an isoquinoline plant alkaloid with significant antimicrobial and anticancer activity. Berberine had a very strong, concentration-dependent, effect on the structural stability of DNA from the human cancer cells (U937 cells) whereas non-cancer cells were changed only when berberine concentrations were relatively high 75 and 50 μg mL⁻¹. Figure Schematic illustration of preparation of the nanostructured films: (a) layer-to-layer coverage (DNA/nanomaterial/SPE); (b) mixed coverage (DNA-nanomaterial/SPE)

Keywords: DNA-biosensors; Multi walled carbon nanotubes; Gold nanoparticles; DNA from cancer cells; Berberine

SPR-based immunocapture approach to creating an interfacial sensing architecture: mapping of the MRS18-2 binding site on retinoblastoma protein by Boris Snopok; Mariya Yurchenko; Laszlo Szekely; George Klein; Elena Kashuba (pp. 2063-2073).

Biosensor technologies based on optical readout are widely used in protein–protein interaction studies. Here we describe a fast and simple approach to the creation of oriented interfacial architectures for surface plasmon resonance (SPR) transducers, based on conventional biochemical procedures and custom reagents. The proposed protocol permits the oriented affinity-capture of GST fusion proteins by a specific antibody which is bound to protein A, which in turn has been immobilized on the transducer surface (after the surface has been modified by guanidine thiocyanate). The applicability of the method was demonstrated by studying the interaction between retinoblastoma tumor suppressor protein (pRb) and MRS18-2 proteins. The formation of the pRb–MRS18-2 protein complex was examined and the pRb binding site (A-box–spacer–B-box) was mapped. We have also shown that MRS18-2, which was detected as the Epstein–Barr virus-encoded EBNA-6 binding partner using the yeast two-hybrid system, binds to pRb in GST pull-down assays.

Keywords: Surface plasmon resonance; GST fusion proteins; Interfacial complex; Protein–protein interaction; Epstein–Barr virus

In situ electrochemical contact angle study of hemoglobin and hemoglobin–Fe₃O₄ nanocomposites by Renyun Zhang; Min Song; Xiaomao Li; Zhiqun Guan; Xuemei Wang (pp. 2075-2079).

The electrochemical redox-induced contact angle changes of hemoglobin droplets in the absence and presence of tetraheptylammonium-capped Fe₃O₄ nanoparticles have been explored by using in situ electrochemical contact angle measurements. The results indicate that the electrochemical redox process may lead to some structure changes of hemoglobin (Hb), which could further induce the hydrophobic-to-hydrophilic changes of the relative droplets. Our observations demonstrate that hemoglobin could self-assemble on the surface of the functionalized Fe₃O₄ nanoparticles as Hb–Fe₃O₄ nanocomposites, which may contribute to much more significant change of the electrochemical redox-induced contact angle values than that with free nano-Fe₃O₄. These results suggest that in situ electrochemical contact angle measurements could be readily applied as a new and convenient method to detect some specific biological process. Figure Schematic drawing of the possible process and contact angle changes for the self-assembly of hemoglobin on the tetraheptylammonium-capped Fe₃O₄ nanoparticles

Keywords: Biomaterials; Nanoparticles; Bioanalytical methods; Electroanalytical methods; UV/Vis

Determination of metformin based on amplification of its voltammetric response by a combination of molecular wire and carbon nanotubes by Xin-juan Tian; Jun-feng Song; Xin-jun Luan; Yao-yu Wang; Qi-zhen Shi (pp. 2081-2086).

Molecular wires containing copper(II) (CuMW), in the form of the coordination polymer (Cu(II)₄(bpp)₄(maa)₈(H₂O)₂).2H₂O (bpp=1,3-bis(4-pyridyl)propane, maa=2-methylacrylic acid), and multiwalled carbon nanotubes (CNT) have been combined to prepare a paste electrode (CuMW/CNT/PE). The voltammetric response of the CuMW/CNT/PE to metformin (MET) was significantly greater than that of electrodes prepared from other materials, because of both the surface effect of CuMW and CNT and coordination of MET with the Cu(II) ion in the CuMW. A novel voltammetric method for determination of MET is proposed. In pH 7.2 Britton–Robinson buffer, using single sweep voltammetry, the second-order derivative peak current for oxidation of MET at 0.97 V (relative to SCE) increased linearly with MET concentration in the range 9.0 × 10⁻⁷–5.0 × 10⁻⁵ mol L⁻¹ and the detection limit was 6.5 × 10⁻⁷ mol L⁻¹. Figure When a combination of molecular wires containing copper(II) (CuMW) and multiwalled carbon nanotubes (CNT) was used to prepare a paste electrode (CuMW/CNT/PE) the voltammetric response to metformin (curve c) was significantly higher than that at a carbon/PE (curve a) or a CNT/PE (curve b), because of the amplification effect of CNT and CuMW. A novel voltammetric method is proposed for determination of MET

Keywords: Metformin; Carbon nanotubes; Molecular wires; Copper; Modified paste electrode

Selective detection of dopamine in the presence of ascorbic acid by use of glassy-carbon electrodes modified with both polyaniline film and multi-walled carbon nanotubes with incorporated β-cyclodextrin by Tanji Yin; Wanzhi Wei; Jinxiang Zeng (pp. 2087-2094).

A simple, sensitive, and reliable method based on a combination of multi-walled carbon nanotubes with incorporated β-cyclodextrin (β-CD-MWNTs) and a polyaniline (PANI) film-modified glassy-carbon (GC) electrode has been successfully developed for determination of dopamine (DA) in the presence of ascorbic acid (AA). The PANI film had good anti-interference properties and long-term stability, because of the permselective and protective properties of the conducting redox polymer film. The acid-treated MWNTs with carboxylic acid functional groups promoted the electron-transfer reaction of DA and inhibited the voltammetric response of AA. Sensitive detection of DA was further improved by the preconcentration effect of formation of a supramolecular complex between β-CD and DA. The analytical response of the β-CD-MWNTs/PANI film to the electrochemical behavior of DA was, therefore, better than that of a MWNTs/PANI film, a PANI film, or a bare glassy-carbon (GC) electrode. Under the conditions chosen a linear calibration plot was obtained in the range 1.0 × 10⁻⁷–1.0 × 10⁻³ mol L⁻¹ and the detection limit was 1.2 × 10⁻⁸ mol L⁻¹. Interference from AA was effectively eliminated and the sensitivity, selectivity, stability, and reproducibility of the electrodes was excellent for determination of DA.

Keywords: Dopamine; β-Cyclodextrin; Multi-walled carbon nanotubes; Polyaniline

Development and characterisation of disposable gold electrodes, and their use for lead(II) analysis by Mohd F. Md Noh; Ibtisam E. Tothill (pp. 2095-2106).

There is an increasing need to assess the harmful effects of heavy-metal-ion pollution on the environment. The ability to detect and measure toxic contaminants on site using simple, cost effective, and field-portable sensors is an important aspect of environmental protection and facilitating rapid decision making. A screen-printed gold sensor in a three-electrode configuration has been developed for analysis of lead(II) by square-wave stripping voltammetry (SWSV). The working electrode was fabricated with gold ink deposited by use of thick-film technology. Conditions affecting the lead stripping response were characterised and optimized. Experimental data indicated that chloride ions are important in lead deposition and subsequent analysis with this type of sensor. A linear concentration range of 10–50 μg L⁻¹ and 25–300 μg L⁻¹ with detection limits of 2 μg L⁻¹ and 5.8 μg L⁻¹ were obtained for lead(II) for measurement times of four and two minutes, respectively. The electrodes can be reused up to 20 times after cleaning with 0.5 mol L⁻¹ sulfuric acid. Interference of other metals with the response to lead were also examined to optimize the sensor response for analysis of environmental samples. The analytical utility of the sensor was demonstrated by applying the system to a variety of wastewater and soil sample extracts from polluted sites. The results are sufficient evidence of the feasibility of using these screen-printed gold electrodes for the determination of lead(II) in wastewater and soil extracts. For comparison purposes a mercury-film electrode and ICP–MS were used for validation.

Keywords: Lead (II); Metal-ion analysis; Screen-printed gold electrodes; Stripping voltammetry; Soil samples; Wastewater samples

Surface modification of amine-functionalised graphite for preparation of cobalt hexacyanoferrate (CoHCF)-modified electrode: an amperometric sensor for determination of butylated hydroxyanisole (BHA) by S. J. Richard Prabakar; S. Sriman Narayanan (pp. 2107-2115).

A cobalt hexacyanoferrate (CoHCF)-modified graphite paraffin wax composite electrode was prepared by a new approach. An amine-functionalised graphite powder was used for the fabrication of the electrode. A functionalised graphite paraffin wax composite electrode was prepared and the surface of the electrode was modified with a thin film of CoHCF. Various parameters that influence the electrochemical behaviour of the modified electrode were studied by varying the background electrolytes, scan rates and pH. The modified electrode showed good electrocatalytic activity towards the oxidation of butylated hydroxyanisole (BHA) under optimal conditions and showed a linear response over the range from 7.9 × 10⁻⁷ to 1.9 × 10⁻⁴ M of BHA with a correlation coefficient of 0.9988. The limit of detection was 1.9 × 10⁻⁷ M. Electrocatalytic oxidation of BHA was effective at the modified electrode at a significantly reduced potential and at a broader pH range. The utility of the modified electrode as an amperometric sensor for the determination of BHA in flow systems was evaluated by carrying out hydrodynamic and chronoamperometric experiments. The modified electrode showed very good stability and a longer shelf life. The modified electrode was applied for the determination of BHA in spiked samples of chewing gum and edible sunflower oil. The advantage of this method is the ease of electrode fabrication, good stability, longer shelf life, low cost and its diverse application for BHA determination. Figure Cyclic Voltammogram of ( ) CoHCF modified electrode, ( ) in presence of 1.9 x 10⁻⁵ M of BHA and ( ) bare electrode, ( ) in the presence of 1.9 x 10⁻⁵ M of BHA in 1.0 M NaCl, pH 7.0

Keywords: Cobalt hexacyanoferrate; p-Phenylenediamine; Butylated hydroxyanisole

Simultaneous determination of uric acid and ascorbic acid at a ferrocenium–thioglycollate modified electrode by Bin Fang; Shoufeng Jiao; Maoguo Li; Haisheng Tao (pp. 2117-2122).

Self-assembled monolayers (SAMS) of chemisorbed thioglycollate on a gold electrode surface have been used as a base interface for the electrostatic adsorption of ferrocenium ion. Electrochemical impedance spectra (EIS) and cyclic voltammetry (CV) were used to evaluate the electrochemical properties of the supramolecular film. The bare gold electrode failed to distinguish the oxidation peaks of ascorbic acid (AA) and uric acid (UA) in phosphate buffer solution (PBS, pH 7.0), while the ferricinium–thioglycollate modified electrode could separate them efficiently. In differiential pulse voltammetric measurements, the prepared gold electrode could separate AA and UA signals, allowing the simultaneous determination of AA and UA. Under optimal conditions and within the linear range of 1.0 × 10⁻⁶ to 5.0 × 10⁻⁴ M, the detection limits of AA and UA achieved were 2.0 × 10⁻⁷ and 1.0 × 10⁻⁷ M, respectively. The applicability of the prepared electrode was demonstrated by measuring AA and UA in human urine without any pretreatment. Figure Fabrication process for the modified electrode

Keywords: Ferrocenium–thioglycollate modified electrode; Ascorbic acid; Uric acid; Electrocatalytic oxidation; Differential pulse voltammetry

Method optimization for determination of selected perfluorinated alkylated substances in water samples by Carmen González-Barreiro; Elena Martínez-Carballo; Andrea Sitka; Sigrid Scharf; Oliver Gans (pp. 2123-2132).

In recent years perfluorinated alkylated substances (PFAS) have appeared as a new class of global pollutant. Besides being an industrially important group of compounds, PFAS are regarded as highly toxic and extraordinarily persistent chemicals that pervasively contaminate human blood and wildlife throughout the world. They are therefore regarded as PBT (persistent, bioaccumulative, and toxic) chemicals. Two comprehensive methods have been developed for determination of eleven of the most environmentally relevant PFAS (seven perfluoroalkylcarboxylates, two perfluoroalkylsulfonates, and two perfluoroctanesulfonamides) in aqueous samples. The compounds were isolated by liquid–liquid extraction (LLE) and solid-phase extraction (SPE), and identification and quantification of the target analytes were achieved by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS–MS). With LLE detection limits ranged from 0.26 to 0.62 ng L⁻¹ for enrichment of 900-mL water samples; recovery of PFAS with a carbon chain longer than C₇ was excellent (80–93%). With SPE, carboxylates with carbon chains 10 could be extracted efficiently (70–98%) under acidic conditions, and PFOS and PFOSA could be extracted efficiently (81% and 96%, respectively) under basic conditions, resulting in MDLs between 0.25 and 0.64 ng L⁻¹. The LLE method was applied successfully to Austrian wastewater effluent samples.

Keywords: PFAS; PFOS; PFOA; LC–MS–MS; Wastewater

Fluorescence characterization of metal ion–humic acid interactions in soils amended with composted municipal solid wastes by César Plaza; Gennaro Brunetti; Nicola Senesi; Alfredo Polo (pp. 2133-2140).

Fluorescence spectroscopy has been used to probe the structural properties and Cu(II), Zn(II), Cd(II), and Pb(II)-binding behavior of humic acid (HA)-like fractions isolated from a municipal solid waste compost (MSWC) and HAs from unamended and MSWC-amended soils. The main feature of the fluorescence spectra, in the form of emission-excitation matrix (EEM) plots, was a broad peak with the maximum centered at an excitation/emission wavelength pair that was much shorter (340/437 nm) for MSWC-HA than for unamended and MSWC-amended soil HAs (455/513 and 455/512 nm, respectively). Fluorescence intensity for MSWC-amended soil HA was less than that for unamended soil HA. These results were indicative of more aromatic ring polycondensation and humification of soil HAs, and of partial incorporation of simple and low-humified components of MSWC-HA into native soil HA, as a result of MSWC amendment. Titrations of HAs with Cu(II), Zn(II), Cd(II), and Pb(II) ions at pH 6 and ionic strength 0.1 mol L⁻¹ resulted in a marked decrease of the fluorescence intensities of untreated HAs. By successfully fitting a single-site fluorescence-quenching model to titration data, the metal ion complexing capacities of each HA and the stability constants of metal ion-HA complexes were obtained. The binding capacities and stability constants of MSWC-HA were smaller than those of the unamended soil HA. Application of MSWC to soil slightly reduced the metal-ion-binding capacities and affinities of soil HAs.

Keywords: Composted municipal solid wastes; Soil amendment; Humic acids; Metal-complexing capacities and stability constants; Fluorescence spectroscopy

Authenticity control of essential oils containing citronellal and citral by chiral and stable-isotope gas-chromatographic analysis by Tran-Thi Nhu-Trang; Hervé Casabianca; Marie-Florence Grenier-Loustalot (pp. 2141-2152).

Enantioselective capillary GC on a Supelco β-DEX 225 column (heptakis(2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin SPB 20poly—20% diphenyl, 80% dimethylsiloxane) and isotope-ratio mass spectrometry, coupled online with capillary GC on an HP5 column have been used for origin-specific analysis and authenticity control of essential oils, for example lemon (Citrus limon), lemongrass (Cymbopogon citratus and Cymbopogon flexuosus), citronella (Cymbopogon nardus L.—Ceylon type and Cymbopogon winterianus—Java type), Litsea cubeba, Lippia citriodora, lemon myrtle (Backhousia citriodora), lemon gum (Eucalyptus citriodora), and, especially, precious lemon balm oil (Melissa officinalis L.). Isotope data (δ¹³C_PDB and δ²H_V-SMOW) for citral (neral + geranial) and citronellal from on-line GC–C/Py–IRMS and chiral data for citronellal in these essential oils are reported. The possibility of using these data to determine the origin of these essential oils and to detect adulteration is discussed. Principal-components analysis (PCA) of specific compounds in two essential oils of lemongrass and Litsea cubeba was performed as a practical statistical method for distinguishing between these two types of oil.

Keywords: Citral; Citronellal; Chiral analysis; Stable isotopes; Adulteration

New analytical procedure based on a cellulose bag and ionic exchanger with p-aminobenzoic acid groups for differentiation of labile and inert metal species in aquatic systems by André Henrique Rosa; Danielle Goveia; Iramaia C. Bellin; Suzan da Silva Lessa; Newton L. Dias Filho; Pedro de Magalhães Padilha (pp. 2153-2160).

A new procedure was developed for the in situ characterization of the lability of metal species in aquatic systems by using a system equipped with a diffusion membrane and cellulose organomodified with p-aminobenzoic acid groups (DM-Cell-PAB). To this end, the DM-Cell-PAB system was prepared by adding cellulose organomodified with p-aminobenzoic acid groups (Cell-PAB) to pre-purified cellulose bags. After the DM-Cell-PAB system was sealed, it was examined in the laboratory to evaluate the influence of complexation time, mass of exchanger, pH, metal ions (Cu, Cd, Fe, Mn, and Ni), and concentration of organic matter on the relative lability of metal species. It was found that the pH and kinetics strongly influence the process of metal complexation by the DM-Cell-PAB system. At all pH levels, Cd, Mn, and Ni showed lower complexation with Cell-PAB resin than Cu and Fe metals. Note that relative lability of metals complexed to aquatic humic substances (AHS) in the presence of Cell-PAB resin showed the following order: Cu≅Fe≫Ni>Mn=Cd. The results presented here also indicate that increasing the AHS concentration decreases the lability of metal species by shifting the equilibrium to AHS–metal complexes. Our results indicate that the system under study offers an interesting alternative that can be applied to in situ experiments for differentiation of labile and inert metal species in aquatic systems.

Keywords: Aquatic humic substances; Metal species; Lability; Cellulose modified with p-aminobenzoic acid groups

Advantages of LC–MS–MS compared to LC–MS for the determination of nitrofuran residues in honey by Laure Tribalat; Olivier Paisse; Guy Dessalces; Marie-Florence Grenier-Loustalot (pp. 2161-2168).

In the framework of developing analyses for exogenous contaminants in food matrices such as honey, we have compared data obtained by high-performance liquid chromatography coupled with mass spectrometry (LC–MS) to those provided by high-performance liquid chromatography and tandem mass spectrometry (LC–MS–MS). Initial results obtained with LC–MS showed that the technique lacked selectivity, which is why the method was validated by LC–MS–MS. This method involves a solid-phase extraction (SPE) of nitrofuran metabolites and nitrofuran parent drugs, a derivatization by 2-nitrobenzaldehyde for 17 h, and finally a clean-up by SPE. The data obtained show that the limits of detection varied between 0.2 and 0.6 μg kg⁻¹ for the metabolites and between 1 and 2 μg kg⁻¹ for nitrofuran parent drugs. The method was applied to different flower honeys. The results showed that nitrofurans (used as antibiotics) are consistently present in this matrix, the predominant compound being furazolidone. Figure Working bees

Keywords: Nitrofuran; Honey; High-performance liquid chromatography; Mass spectrometry; Tandem mass spectrometry

Development of an efficient method for the preparative isolation and purification of chlorophyll a from a marine dinoflagellate Amphidinium carterae by high-speed counter-current chromatography coupled with reversed-phase high-performance liquid chromatography by Lijuan Long; Yang Song; Jun Wu; Li Lei; Kai Huang; Benwen Long (pp. 2169-2174).

In our research into chlorophylls of marine dinoflagellates, chlorophyll a was separated rapidly from the hexane extract of Amphidinium carterae in three steps. The first step was silica gel column chromatography, where elution was performed with 0–50% ethyl acetate in n-hexane. The second was high-speed counter-current chromatography using a two-phase solvent system consisting of n-hexane–ethyl acetate–methanol–water (5:5:5:1, v/v), and the third step was preparative reversed-phase high-performance liquid chromatography using a solvent system of acetone–water (89:11, v/v). HPLC analysis showed that the purity of chlorophyll a from the second step was over 83%, and after the third it was over 99%. Thirty milligrams of chlorophyll a was isolated from a crude sample of 250 mg of chlorophylls, and its structure was identified by analyzing its MS, ¹H NMR and ¹³C NMR spectra.

Keywords: Amphidinium carterae ; High-speed counter-current chromatography; Reversed-phase high-performance liquid chromatography; Preparative chromatography; Chlorophyll a

Flow-injection analysis for the determination of total inorganic carbon and total organic carbon in water using the H₂O₂–luminol–uranine chemiluminescent reaction by Shun-Li Fan; Fang Qu; Lixia Zhao; Jin-Ming Lin (pp. 2175-2182).

In the presence of carbonate and uranine, the chemiluminescent intensity from the reaction of luminol with hydrogen peroxide was dramatically enhanced in a basic medium. Based on this fact and coupled with the technique of flow-injection analysis, a highly sensitive method was developed for the determination of carbonate with a wide linear range. The method provided the determination of carbonate with a wide linear range of 1.0 × 10⁻¹⁰–5.0 × 10⁻⁶ mol L⁻¹ and a low detection limit (S/N = 3) of carbonate of 1.2 × 10⁻¹¹ mol L⁻¹. The average relative standard deviation for 1.0 × 10⁻⁹–9.0 × 10⁻⁷ mol L⁻¹ of carbonate was 3.7% (n = 11). Combined with the wet oxidation of potassium persulfate, the method was applied to the simultaneous determination of total inorganic carbon (TIC) and total organic carbon (TOC) in water. The linear ranges for TIC and TOC were 1.2 × 10⁻⁶–6.0 × 10⁻² mg L⁻¹ and 0.08–30 mg L⁻¹ carbon, respectively. Recoveries of 97.4–106.4% for TIC and 96.0–98.5% for TOC were obtained by adding 5 or 50 mg L⁻¹ of carbon to the water samples. The relative standard deviations (RSDs) were 2.6–4.8% for TIC and 4.6–6.6% for TOC (n = 5). The mechanism of the chemiluminescent reaction was also explored and a reasonable explanation about chemical energy transfer from luminol to uranine was proposed. Figure Chemiluminescence profiles in batch system. 1, Injection of 100 μL of K₂CO₃ into 1.0 mL luminol-1.0 mL H₂O₂ solution; 2-3 and 4-5, Injection in sequence of 100 μL of K₂CO₃ and 100 μL of uranine into 1.0 ml luminol-1.0 mL H₂O₂ solution; C_luminol = 1.0 × 10⁻⁷ mol/L, C_H2O2 = 1.0 × 10⁻⁵ mol/L, C_uranine = 1.0 × 10⁻⁵ mol/L, C_K2CO3 = 1.0 × 10⁻⁷ mol/L except for 4-5 where C_K2CO3 = 1.0 × 10⁻⁴ mol/L

Keywords: Chemiluminescence; Luminol; Uranine; Total organic carbon; Total inorganic carbon

Diffuse reflection FTIR spectral database of dyes and pigments by Carlos Eduardo Silva; Luciana P. Silva; Howell G. M. Edwards; Luiz Fernando C. de Oliveira (pp. 2183-2191).

24 pigments commonly used in art have been characterized by diffuse reflection infrared spectroscopy (DR). All of the compounds have also been characterized by means of infrared absorption spectroscopy to demonstrate the reliability of the DR technique. This is the first record of the use of this technique as an analytical tool in conservation science, and the results appear to be promising for the identification of unknown pigments used on historical and artwork artifacts. Although the DR technique used here is not nondestructive, it can still be usefully applied to the analysis of artwork since it requires only a very small quantity of sample for analysis.

Keywords: Pigments; DR spectra; Infrared spectra

Determination of n-octanol–water partition coefficients by hollow-fiber membrane solvent microextraction coupled with HPLC by Yu Gao Guo; Jie Zhang; Dan Ning Liu; Hua Feng Fu (pp. 2193-2198).

A novel direct method has been developed for determination of n-octanol–water partition coefficients by hollow-fiber membrane solvent microextraction (HFMSME) combined with high-performance liquid chromatography (HPLC). The compound of interest is dissolved in water with sonication and a hollow fiber containing octanol inside is placed in the sample solution to perform microextraction. After microextraction the concentrations in both the aqueous and n-octanol phases are analyzed by HPLC with UV detection. The method was evaluated with ten reference compounds and shown to be suitable for determination of the partition coefficients of organic compounds accurately, cheaply, simply, and quickly. Previously unknown n-octanol–water partition coefficients have been obtained for other compounds by use of the hollow-fiber membrane solvent-microextraction technique. Figure Schematic diagram of equipment used for hollow-fiber solvent microextraction. (1) hollow cone-shaped support; (2) aqueous sample; (3) porous hollow-fiber membrane tube (PHFMT); (4) n-octanol phase; (5) sealed end of PHFMT; (6) container (15 mL); (7) stir bar; (8) magnetic stirrer

Keywords: Hollow-fiber membrane solvent microextraction; HFMSME; log P ; n-Octanol–water partition coefficient; HPLC

A linear modulation-based stochastic resonance algorithm applied to the detection of weak chromatographic peaks by Haishan Deng; Bingren Xiang; Xuewei Liao; Shaofei Xie (pp. 2199-2205).

A simple stochastic resonance algorithm based on linear modulation was developed to amplify and detect weak chromatographic peaks. The output chromatographic peak is often distorted when using the traditional stochastic resonance algorithm due to the presence of high levels of noise. In the new algorithm, a linear modulated double-well potential is introduced to correct for the distortion of the output peak. Method parameter selection is convenient and intuitive for linear modulation. In order to achieve a better signal-to-noise ratio for the output signal, the performance of two-layer stochastic resonance was evaluated by comparing it with wavelet-based stochastic resonance. The proposed algorithm was applied to the quantitative analysis of dimethyl sulfide and the determination of chloramphenicol residues in milk, and the good linearity of the method demonstrated that it is an effective tool for detecting weak chromatographic peaks. Figure The linear modulation-based stochastic resonance algorithm (LSRA) improved the output chromatographic peak of chloramphenicol

Keywords: Stochastic resonance; Weak chromatographic peaks; Linear modulation

Marked individual variability in the levels of trimethylselenonium ion in human urine determined by HPLC/ICPMS and HPLC/vapor generation/ICPMS by Doris Kuehnelt; Dijana Juresa; Norbert Kienzl; Kevin A. Francesconi (pp. 2207-2212).

Selenium species were determined using HPLC/ICPMS and HPLC/vapor generation/ICPMS in the urine from seven human volunteers investigated at background selenium concentrations and at slightly elevated concentrations after ingestion of 200 μg Se as a selenite supplement. Trimethylselenonium ion (TMSe) was present, together with selenosugars, in the urine samples, a result that dispels recent doubts about its possible previous misidentification with a cationic selenosugar. Although TMSe was present as only a trace metabolite in urine from five of the seven volunteers (0.02–0.28 μg Se/L, equivalent to 1–5% of the sum of selenosugars and TMSe), it was a significant metabolite (up to 4.6 μg Se/L, 22%) in one volunteer, and it was the major identified metabolite (up to 15 μg Se/L, 53%) in another volunteer. This marked individual variability in the formation of TMSe was maintained in a duplicate investigation of urine from the same seven volunteers.

Keywords: Human urine; Selenium speciation; TMSe; Selenosugars; HPLC/vapor generation/ICPMS; HPLC/ICPMS

Drug–protein interaction with Vpu from HIV-1: proposing binding sites for amiloride and one of its derivatives by C. G. Kim; V. Lemaitre; A. Watts; W. B. Fischer (pp. 2213-2217).

Vpu is an 81-amino-acid auxiliary protein of the genome of HIV-1. It is proposed that one of its roles is to enhance particle release by self-assembling to form water-filled channels enabling the flux of ions at the site of the plasma membrane of the infected cell. Hexamethylene amiloride has been shown to block Vpu channel activity when the protein is reconstituted into lipid bilayers. In a docking approach with monomeric, pentameric and hexameric bundle models of Vpu corresponding to the transmembrane part of the protein, a putative binding site of hexamethylene amiloride is proposed and is compared with the site for the nonpotent amiloride. The binding mode for both ligands is achieved by optimizing hydrogen bond interactions with serines. Binding energies and binding constants are the lowest for protonated hexamethylene amiloride in the pentameric bundle. Figure The proposed binding site of the Vpu channel blocker hexamethylene amiloride within the lumen of the Vpu bundle. The bundle is a homo-pentamer with each monomer consisting of the first 32 amino acids of Vpu including the transmembrane part of the protein which is encoded by HIV-1. The bundle atoms are shown in their van der Waals representation and the helix backbone in a ribbon representation. Residues Trp-23 and Ser-24 are highlighted as sticks. Hexamethylene amiloride is shown in yellow (C atoms) and blue (N atoms)

Keywords: Viral ion channels; Vpu; HIV-1; Docking simulations; Drug–protein interactions

Toward hybridization assays without PCR using universal nanoamplicons by Z.-H. Mo; X.-L. Wei (pp. 2219-2223).

An innovative scheme for signal amplification using random tetramer-modified gold nanoparticles, termed “nanoamplicons,” has been developed for hybridization assay without PCR. Large numbers of nanoamplicons could be integrated onto one target, providing much greater amplification than the larger nanoparticles usually adopted. Using M13mp18 single-strand DNA as a target, this concept is shown to be a feasible approach to detecting 0.17 amol L⁻¹ DNA without target amplification, based on microgravimetric detection of the adsorption of the probe–target–nanoamplicons complex via thiol–gold binding. To our knowledge, this method has a sensitivity that is close to that of PCR and superior to those of nanoparticle-based methods reported previously. Additionally, this novel nanoamplicon could be prepared in the same way and used for all diagnostic tests; such universality would make the nanoamplicons highly advantageous for the generalization and standardization of bioassays, and when applying this new technology in clinical laboratories. Figure A novel signal amplification method for DNA detection with subattomolarsensitivity has been developed using random tetramer-modified gold nanoparticlesas nanoamplicons, which are easily prepared with high uniformity and can be universally adaptedto any sequences

Keywords: Hybridization assay; Nanoparticle; Signal amplification; Microgravimetry

Determination of glucosinolates in traditional Chinese herbs by high-performance liquid chromatography and electrospray ionization mass spectrometry by Kim-Chung Lee; Man-Wai Cheuk; Wan Chan; Albert Wai-Ming Lee; Zhong-Zhen Zhao; Zhi-Hong Jiang; Zongwei Cai (pp. 2225-2232).

A reversed-phase HPLC method has been developed for determination of twelve intact glucosinolates—glucoiberin, glucocheirolin, progoitrin, sinigrin, epiprogoitrin, glucoraphenin, sinalbin, gluconapin, glucosibarin, glucotropaeolin, glucoerucin, and gluconasturtiin—in ten traditional Chinese plants. The samples were extracted with methanol and the extracts were cleaned on an activated Florisil column. A mobile phase gradient prepared from methanol and 30 mmol L⁻¹ ammonium acetate at pH 5.0 enabled baseline separation of the glucosinolates. Glucosinolate detection was confirmed by quadrupole time-of-flight tandem mass spectrometric analysis in negative-ionization mode. Detection limits ranged from 0.06 to 0.36 μg g⁻¹ when 5 g of dried plant was analyzed. Recoveries of the glucosinolates were better than 85% and precision (relative standard derivation, n = 3) ranged from 5.3 to 14.6%. Analysis of the glucosinolates provided scientific evidence enabling differentiation of three pairs of easily confused plants. Figure Glucosinolates Analysis for the Differentiation of Easily-Confusing Herbs

Keywords: Glucosinolate; Traditional Chinese herb; HPLC; ESI–MS

Estimation of diffusive boundary layer thickness in studies involving diffusive gradients in thin films (DGT) by Ø. A. Garmo; K. Razi Naqvi; O. Røyset; E. Steinnes (pp. 2233-2237).

Recent laboratory experiments and field investigations involving diffusive gradients in thin films (DGT) have shown that the thickness (δ) of the diffusive boundary layer (DBL), which can affect the accuracy of the technique, is generally not negligible. Accordingly, the determination of δ has become a matter of considerable practical importance. Though the problem has been addressed in the recent literature, there is room for some improvement. An expression for estimation of δ is presented here, and a practical procedure for determining δ and the concentration of DGT-labile species from sparse experimental data is proposed and illustrated by analyzing data from four experiments with DGT samplers of different diffusive gel thicknesses.

Keywords: Diffusive gradients in thin films (DGT); Diffusive boundary layer (DBL); Sampling

PLS and first derivative of ratio spectra methods for determination of hydrochlorothiazide and propranolol hydrochloride in tablets by Silvana E. Vignaduzzo; Rubén M. Maggio; Patricia M. Castellano; Teodoro S. Kaufman (pp. 2239-2244).

Two new analytical methods have been developed as convenient and useful alternatives for simultaneous determination of hydrochlorothiazide (HCT) and propranolol hydrochloride (PRO) in pharmaceutical formulations. The methods are based on the first derivative of ratio spectra (DRS) and on partial least squares (PLS) analysis of the ultraviolet absorption spectra of the samples in the 250–350-nm region. The methods were calibrated between 8.7 and 16.0 mg L⁻¹ for HCT and between 14.0 and 51.5 mg L⁻¹ for PRO. An asymmetric full-factorial design and wavelength selection (277–294 nm for HCT and 297–319 for PRO) were used for the PLS method and signal intensities at 276 and 322 nm were used in the DRS method for HCT and PRO, respectively. Performance characteristics of the analytical methods were evaluated by use of validation samples and both methods showed to be accurate and precise, furnishing near quantitative analyte recoveries (100.4 and 99.3% for HCT and PRO by use of PLS) and relative standard deviations below 2%. For PLS the lower limits of quantification were 0.37 and 0.66 mg L⁻¹ for HCT and PRO, respectively, whereas for DRS they were 1.15 and 3.05 mg L⁻¹ for HCT and PRO, respectively. The methods were used for quantification of HCT and PRO in synthetic mixtures and in two commercial tablet preparations containing different proportions of the analytes. The results of the drug content assay and the tablet dissolution test were in statistical agreement (p < 0.05) with those furnished by the official procedures of the USP 29. Preparation of dissolution profiles of the combined tablet formulations was also performed with the aid of the proposed methods. The methods are easy to apply, use relatively simple equipment, require minimum sample pre-treatment, enable high sample throughput, and generate less solvent waste than other procedures.

Keywords: Hydrochlorothiazide; Propranolol hydrochloride; First derivative of ratio spectra; PLS; Dissolution test; Drug content assay

Evaluation of the applicability of backscattered light measurements to the determination of microbial cell densities in microtiter plates by Hartmut F. Zimmermann; Thomas Raebiger (pp. 2245-2247).

The turbidity of a microbial suspension sample is routinely determined by measuring the optical density (often referred to as the “absorbance”). This method requires a dilution step at moderate and high cell densities in order to ensure that measurements fall within the region where biomass concentration is linearly correlated to optical density. The measurement of backscattered light (often referred to as the “reflectance”), which has so far been mainly applied to large-scale stirred tanks, should also be applicable on the microscale. To evaluate the validity of this assumption, a standard fluorescence microplate reader was adapted to measure backscattered light. Backscattered light readings from undiluted microbial fermentation samples determined using this modified reader gave similar growth curves to optical density measurements from diluted samples determined in a standard cell photometer. Indeed, the fact that the dilution procedure is not necessary for backscattered light measurements gives them an important advantage over optical density measurements. Such an apparatus is not only suitable for manual operation, but also shows the potential for integration into fully automated robotic systems used for high-throughput experimentation.

Keywords: Optical density; Turbidimetry; Nephelometry; Microplate reader; Growth curves

Direct determination of nickel in petroleum by solid sampling–graphite furnace atomic absorption spectrometry by Geisamanda Pedrini Brandão; Reinaldo Calixto de Campos; Eustáquio Vinicius Ribeiro de Castro; Honério Coutinho de Jesus (pp. 2249-2253).

A procedure for the direct GFAAS determination of Ni in petroleum samples using a solid sampling strategy is proposed. Palladium was used as conventional modifier. Central composite design multivariate optimization defined the optimum temperature program and the Pd mass, allowing calibration using aqueous analytical solution. The limit of detection (LOD) at the optimized conditions was 0.23 ng of Ni, for typical sample masses between of 0.10 and 0.60 mg. Linearity at least up to 11 ng of Ni and a characteristic mass of 45 pg were observed, defining a dynamic range between 0.52 and 110 μg g⁻¹. Typical coefficients of variation (n = 10) in the analysis of oil reference materials were 7%. Method validation was performed both by the analysis of oil certified reference materials and by comparison with an independent method (ASTM 5863-B). No statistically significant difference was observed between obtained and expected values. The total determination cycle lasted 5 min, equivalent to a sample throughput of 6 h⁻¹ for duplicate determinations.

Keywords: Nickel; Petroleum; SS-GFAAS

Surface complexation studied via combined grazing-incidence EXAFS and surface diffraction: arsenate on hematite (0001) and (10–12) by G. Waychunas; T. Trainor; P. Eng; J. Catalano; G. Brown; J. Davis; J. Rogers; J. Bargar (pp. 2255-2255).

Development and characterization of an integrated silicon micro flow cytometer by R. Bernini; E. De Nuccio; F. Brescia; A. Minardo; L. Zeni; P. M. Sarro; R. Palumbo; M. R. Scarfi (pp. 2257-2257).

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Analytical and Bioanalytical Chemistry (v.386, #7-8)

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Analytical and Bioanalytical Chemistry (v.386, #7-8)