Analytical and Bioanalytical Chemistry (v.390, #1)

has been Professor in the Department of Physical and Analytical Chemistry of Oviedo’s University (Spain) since 1982. He is author or co-author of more than 450 scientific publications in international journals, several patents and books. Dr. Sanz-Medel’s present research interests include the following main lines: 1. New atomic detectors and ion sources for ultratrace analysis using plasmas. 2. New molecular optical sensors particularly those based on the use of quantum dots. 3. Hybrid techniques, coupling a separation unit and an atomic detector, for ultratrace and trace metal speciation to solve biological and environmental problems. 4. Speciation for proteomics, integrating MS “molecular” [MALDI- and electrospray-(MS)n] and “atomic” (ICP-MS) techniques and introducing the extensive use of ICP-MS to carry out “heteroatom-tagged proteomics”, both for qualitative and quantitative purposes. Alfredo Sanz-Medel is Editor of Analytical and Bioanalytical Chemistry since January 2002. At the Euroanalyisis XIV in Antwerp, he received the 2007 Robert Kellner Award.

Elemental mass spectrometry for quantitative proteomics by Alfredo Sanz-Medel; María Montes-Bayón; María del Rosario Fernández de la Campa; Jorge Ruiz Encinar; Jörg Bettmer (3-16).
In the last decade mass-spectrometry-based proteomics has become an indispensable analytical tool for molecular biology, cellular biology and, lately, for the emerging systems biology. This review summarises the evolution and great potential of analytical methods based on elemental mass-spectrometric detection for quantitative proteomic analysis.
Keywords: Bioanalytical methods; Genomics/Proteomics; Mass spectrometry/Inductively coupled plasma mass spectrometry; Speciation

Metal-binding molecules in the organs of Mus musculus by size-exclusion chromatography coupled with UV spectroscopy and ICP-MS by M. González-Fernández; T. García-Barrera; A. Arias-Borrego; D. Bonilla-Valverde; J. López-Barea; C. Pueyo; J. L. Gómez-Ariza (17-28).
Mus musculus mice have been investigated for the total elements content in different organs (lung, liver, spleen, kidney, brain, testicle, heart and muscle) and molecular mass distribution patterns of Mn, Ni, Cu, Zn, As, Pb, Cr, Fe, Co, Se and Cd. Some differences have been found in the organs studied, with especially relevant being the Cu-containing fraction present only in the brain and the As-containing one in the liver. Other differences related to the abundance of the metallospecies have also been found. The present paper is the first step in the study of the “metallome” of this inbred laboratory species from which the genome is completely known. This organism could be used as a model in future studies focused on wild mice and the analytical approach developed could be applied to wild mice to find markers of environmental pollution. Figure The present paper is the first step in the study of the “metallome” of the inbred laboratory specie Mus musculus from which the genome is completely known. Some interesting differences have been found in the extracts from the organs that are discussed along the text.
Keywords: Mus musculus ; Inductively coupled plasma mass spectrometry; Metallomics; Metals; Size-exclusion chromatography

Study of tungstate–protein interaction in human serum by LC–ICP-MS and MALDI-TOF by Nuria Rodríguez-Fariñas; M. Milagros Gomez-Gomez; Carmen Camara-Rica (29-35).
Oral administration of sodium tungstate is an effective treatment for type 1 and 2 diabetes in animal models; it does not incur significant side effects, and it may constitute an alternative to insulin. However, the mechanism by which tungstate exerts its observed metabolic effects in vivo is still not completely understood. In this work, serum-containing proteins which bind tungstate have been characterized. Size exclusion chromatography (SEC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) with a Phenomenex Bio-Sep-S 2000 column and 20 mM HEPES and 150 mM NaCl at pH 7.4 as the mobile phase was chosen as the most appropriate methodology to screen for tungsten–protein complexes. When human serum was incubated with tungstate, three analytical peaks were observed, one related to tungstate–albumin binding, one to free tungstate, and one to an unknown protein binding (MW higher than 300 kDa). Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of the tungsten-containing fractions collected from SEC–ICP-MS chromatograms, after desalting and preconcentration processes, confirmed the association of tungstate with albumin and the other unknown protein. Figure SEC-ICP-MS // MALDI-TOF
Keywords: Tungstate; Diabetes; LC–ICP-MS; MALDI-TOF; Human serum proteins

In vivo detection of DNA adducts induced by cisplatin using capillary HPLC–ICP-MS and their correlation with genotoxic damage in Drosophila melanogaster by Daniel García Sar; Maria Montes-Bayón; Leticia Aguado Ortiz; Elisa Blanco-González; L. María Sierra; Alfredo Sanz-Medel (37-44).
The antitumoral effect of cisplatin [cis-diamminodichloroplatinum(II)] in mammals is related to its binding to DNA components. However, there is a lack of specific chemical methods to selectively detect those adducts formed in vivo at low concentrations. In this work, a new sensitive and selective method of determining cisplatin–DNA adducts based on the use of element-selective mass spectrometry is proposed, and the method is then applied to detect cisplatin adducts induced in vivo in somatic cells of Drosophila melanogaster. The bioanalytical strategy proposed here allows the determination of the most important DNA adduct formed between adjacent guanine units of the same DNA strand with cisplatin, and it is based on the coupling of capillary liquid chromatography (cap-LC) to inductively coupled plasma mass spectrometry (ICP-MS). This set-up allows the simultaneous monitoring of the Pt (from the drug) and P (from the DNA components) present in these adducts, once they have been cleaved by enzymatic hydrolysis of the DNA samples. Using this instrumental set-up, the adducts of cisplatin formed in vivo when D. melanogaster flies are exposed to different cisplatin concentrations can be detected and their concentration determined. The results obtained show a direct correlation between the concentration of cisplatin adducts, the induced genotoxic damage (measured as DNA strand breaks using the Comet assay) and the cisplatin concentration. Figure The work illustrates the complementary use of bioanalytical and biological information to study cisplatin interactions with DNA is vivo at biologically relevant concentrations of the drug
Keywords: Cisplatin; ICP-MS; Genotoxicity; Adducts; Comet

In vitro translation with [34S]-labeled methionine, selenomethionine, and telluromethionine by Yasumitsu Ogra; Takashi Kitaguchi; Noriyuki Suzuki; Kazuo T. Suzuki (45-51).
Heteroisotope and heteroatom tagging with [34S]-enriched methionine (Met), selenomethionine (SeMet), and telluromethionine (TeMet) was applied to in vitro translation. Green fluorescent protein (GFP) and JNK stimulatory phosphatase-1 (JSP-1) genes were translated with wheat germ extract (WGE) in the presence of Met derivatives. GFPs containing Met derivatives were subjected to HPLC coupled with treble detection, i.e., a photodiode array detector, a fluorescence detector, and an inductively coupled plasma mass spectrometer (ICP-MS). The activities of JSP-1-containing Met derivatives were also measured. GFP and JSP-1 containing [34S]-Met and SeMet showed comparable fluorescence intensities and enzyme activities to those containing naturally occurring Met. TeMet was unstable and decomposed in WGE, whereas SeMet was stable throughout the experimental period. Thus, although Te was the most sensitive to ICP-MS detection among S, Se, and Te, TeMet was less incorporated into the proteins than Met and SeMet. Finally, the potential of heteroisotope and heteroatom tagging of desired proteins in in vitro translation followed by ICP-MS detection was discussed. Figure TeMet was less incorporated into GFP than Met and SeMet due to its instability in WGE
Keywords: ICP-MS; Stable isotope; Methionine; Selenomethionine; Telluromethionine; GFP; In vitro translation

Ferritin is the major iron storage protein in the biosphere. Iron stores of an organism are commonly assessed by measuring the concentration of the protein shell of the molecule in fluids and tissues. The amount of ferritin-bound iron, the more desirable information, still remains inaccessible owing to the lack of suitable techniques. Iron saturation of ferritin is highly variable, with a maximum capacity of 4,500 iron atoms per molecule. This study describes the direct isotopic labeling of a complex metalloprotein in vivo by biosynthesis, in order to measure ferritin-bound iron by isotope dilution mass spectrometry. [57Fe]ferritin was produced by cloning and overexpressing the Phaseolus vulgaris ferritin gene pfe in Escherichia coli in the presence of 57FeCl2. Recombinant ferritin was purified in a fully assembled form and contained approximately 1,000 iron atoms per molecule at an isotopic enrichment of more than 95% 57Fe. We did not find any evidence of species conversion of the isotopic label for at least 5 months of storage at −20 °C. Transfer efficiency of enriched iron into [57Fe]ferritin of 20% was sufficient to be economically feasible. Negligible amounts of non-ferritin-bound iron in the purified [57Fe]ferritin solution allows for use of this spike for quantification of ferritin-bound iron by isotope dilution mass spectrometry.
Keywords: Phaseolus vulgaris ; Ferritin; Molecular cloning; Iron; Isotope

Isotopic labelling of peptides and isotope ratio analysis using LC–ICP–MS: a preliminary study by Pritesh Patel; Phil Jones; Richard Handy; Chris Harrington; Peter Marshall; E. Hywel Evans (61-65).
Bradykinin and substance P have been derivatised with cyclic diethylenetriaminepentaacetic anhydride (cDTPA) and subsequently labelled with natural and isotopically enriched Eu3+. This enabled the detection and relative quantitation of the peptides using element-selective detection by high-performance liquid chromatography inductively coupled plasma mass spectrometry (LC–ICP–MS). Relative quantitation was achieved by differentially labelling two peptide sources, after derivatisation with cDTPA, using natural and enriched 151Eu respectively. The 151Eu:153Eu isotope ratio was measured and used to calculate the original peptide ratio. The measured ratios came within 5.2% of the known ratio. Derivatisation and chelation reactions were additionally confirmed using LC–ESI–MS.
Keywords: Relative peptide quantitation; Relative quantitation; Differential isotopic labelling; Isotope ratio; Eu tagging; Element-selective detection; Inductively coupled plasma mass spectrometry

Miniaturisation of analytical steps: necessity and snobbism by M. D. Luque de Castro; F. Priego Capote (67-69).
is Professor of Analytical Chemistry at the University of Córdoba, where she began her professional activity more than 30 years ago. Her main research field has been automation —particularly concerning sample preparation and solid samples. Presently, her main research areas are metabolomics —particularly lipidomics— and the exploitation of byproducts from the Mediterranean agriculture and derived industries to obtain high added value products. F. Priego Capote is one of the most active members of the research team. is one of the most active members in Maria D. Luque de Castro’s research team. His main research areas are metabolomics and assistance of sample preparation with auxiliary energies. He is currently carrying out a postdoctoral formation in proteomics.

is Professor of Analytical Chemistry at the University of California Riverside. She has an active research program involving bioanalytical and environmental analytical applications of NMR Spectroscopy (for more information see http://www.chem.ucr.edu/faculty/larive/larive.html ). Dr. Larive is also active in curricular reform and the promotion of undergraduate research. She is editor-in-chief and principal investigator of the Analytical Sciences Digital Library ( http://www.asdlib.org ), an Internet-based resource for instructors, students, and practitioners of analytical chemistry. This digital library is a collection of peer-reviewed websites on topics including pedagogical approaches, analytical techniques, applications, and classroom resources.

Birthday chromatography challenge by Juris Meija (77-78).

Solution to “Doctrine of Signatures” challenge by Stephen G. Saupe; Katherine E. Sharpless (79-83).

was head of the Institute of Analytical Chemistry at the Department of Chemistry at Dresden University of Technology until his retirement in 2007. His research is focused on spectral imaging for fast monitoring, on polymers with biologically active functions, and on electronic media in university education. Dr. Salzer was vice president and president of the Analytical Chemistry Division of the German Chemical Society. He has been German delegate to the Division Analytical Chemistry of the European Association for Chemical and Molecular Sciences (DAC/EuCheMS) since 1997. In 2003 he was appointed head of the Study Group Education of DAC/EuCheMS. Dr. Salzer was elected reviewer of the German Research Council in 2000. He has been a member of the label committee for the Chemistry Eurobachelor® since 2006. Reiner Salzer is also a member of the Advisory Board of Analytical and Bioanalytical Chemistry

Polymer microfabrication technologies for microfluidic systems by Holger Becker; Claudia Gärtner (89-111).
Polymers have assumed the leading role as substrate materials for microfluidic devices in recent years. They offer a broad range of material parameters as well as material and surface chemical properties which enable microscopic design features that cannot be realised by any other class of materials. A similar range of fabrication technologies exist to generate microfluidic devices from these materials. This review will introduce the currently relevant microfabrication technologies such as replication methods like hot embossing, injection molding, microthermoforming and casting as well as photodefining methods like lithography and laser ablation for microfluidic systems and discuss academic and industrial considerations for their use. A section on back-end processing completes the overview.
Keywords: Microfluidics; Microfabrication; Polymers; Biochips; High throughput screening; Miniaturization

SERS: a versatile tool in chemical and biochemical diagnostics by Katharina Hering; Dana Cialla; Katrin Ackermann; Thomas Dörfer; Robert Möller; Henrik Schneidewind; Roland Mattheis; Wolfgang Fritzsche; Petra Rösch; Jürgen Popp (113-124).
Raman spectroscopy is a valuable tool in various research fields. The technique yields structural information from all kind of samples often without the need for extensive sample preparation. Since the Raman signals are inherently weak and therefore do not allow one to investigate substances in low concentrations, one possible approach is surface-enhanced (resonance) Raman spectroscopy. Here, rough coin metal surfaces enhance the Raman signal by a factor of 104–1015, depending on the applied method. In this review we discuss recent developments in SERS spectroscopy and their impact on different research fields.
Keywords: Surface-enhanced Raman spectroscopy; SERS substrates; Single-molecule detection; Tip-enhanced Raman spectroscopy

Recent applications of near-infrared spectroscopy in cancer diagnosis and therapy by Venkata Radhakrishna Kondepati; H. Michael Heise; Juergen Backhaus (125-139).
In recent years, near-infrared spectroscopy (NIRS) has gained importance for non-invasive or minimally invasive diagnostic applications in cancer. This technology is based on differences of endogenous chromophores between cancer and normal tissues using either oxy-haemoglobin or deoxy-haemoglobin, lipid or water bands, or a combination of two or more of these as diagnostic markers. These marker bands provide a basis for the diagnosis and therapy monitoring of several cancers. Various applications also use advances in NIR fluorescence spectroscopy which is based on exogenous contrast-enhancing agents. In this review the literature published during the last seven years has been assessed. It will provide an overview on the importance of the NIRS tools in cancer pathology, and in the near future it is envisaged to play a crucial role in cancer diagnosis, treatment decisions, and defining therapeutic drug levels.
Keywords: Near-infrared spectroscopy; Cancer diagnosis; Therapy monitoring

What conductors are to their orchestras, biomarkers are to their associated technologies. Building fundamental science, supporting early diagnosis of diseases and following their progression, improving efficacy and safety of treatments, optimizing patient selection and adapting dosing of drugs, helping decide which therapy is most appropriate; these are examples of a few contexts in which biomarkers are key players. Technology development can definitely not escape being associated with these steps. In other words, today’s biomarkers are the thermometers of tomorrow’s therapies. This review provides an overview of recently established platforms as well as new and upcoming technologies for biomarker development in the context of drug development. The roles as well as the pros and cons of different disciplines such as genetics, genomics, proteomics, metabonomics, and assay development will be discussed.
Keywords: Biomarkers; Technologies; “Omics”; Immunoassay; Drug development

Electro-chemiluminescent biosensing by Christophe A. Marquette; Loïc J. Blum (155-168).
The present review draws a general picture of the bioanalytical applications of electro-chemiluminescent reactions (ECL). Only the two main ECL reactions—i.e. the luminol-based and $$ { ext{Ru}}{left( {{ ext{bpy}}} ight)}^{{2 + }}_{3} $$ -based reactions—are considered for application in the fields of enzyme biosensors, immunochemical biosensors, DNA biosensors, and biochips. The mechanism, principle, and experimental conditions of these two reactions are described. Then, for each category of analytical tools, experimental set-ups and performances are presented and discussed.
Keywords: Biochip; Biosensor; DNA; Electro-chemiluminescence; Enzyme; Immunochemical

A critical review of recent developments in the use of chemometric experimental design based optimization techniques in capillary electrophoresis applications is presented. Current advances have led to enhanced separation capabilities of a wide range of analytes in such areas as biological, environmental, food technology, pharmaceutical, and medical analysis. Significant developments in design, detection methodology and applications from the last 5 years (2002–2007) are reported. Furthermore, future perspectives in the use of chemometric methodology in capillary electrophoresis are considered.
Keywords: Capillary electrophoresis; Chemometrics; Optimization; Experimental design; Response surface methodology

Ferrocene-based derivatization in analytical chemistry by Bettina Seiwert; Uwe Karst (181-200).
Ferrocene-based derivatization has raised considerable interest in many fields of analytical chemistry. This is due to the well-established chemistry of ferrocenes, which allows rapid and easy access to a large number of reagents and derivatives. Furthermore, the electrochemical properties of ferrocenes are attractive with respect to their detection. This paper summarizes the available reagents, the reaction conditions and the different approaches for detection. While electrochemical detection is still most widely used to detect ferrocene derivatives, e.g., in the field of DNA analysis, the emerging combination of analytical separation methods with electrochemistry, mass spectrometry and atomic spectroscopy allows ferrocenes to be applied more universally and in novel applications where strongly improved selectivity and limits of detection are required.
Keywords: Ferrocene; Derivatization; Labeling; Electrochemistry; Mass spectrometry

Consistent treatment of measurement bias, including the question of whether or not to correct for bias, is essential for the comparability of measurement results. The case for correcting for bias is discussed, and it is shown that instances in which bias is known or suspected, but in which a specific correction cannot be justified, are comparatively common. The ISO Guide to the Expression of Uncertainty in Measurement does not provide well for this situation. It is concluded that there is a need for guidance on handling cases of uncorrected bias. Several different published approaches to the treatment of uncorrected bias and its uncertainty are critically reviewed with regard to coverage probability and simplicity of execution. On the basis of current studies, and taking into account testing laboratory needs for a simple and consistent approach with a symmetric uncertainty interval, we conclude that for most cases with large degrees of freedom, linear addition of a bias term adjusted for exact coverage (“Ue”) as described by Synek is to be preferred. This approach does, however, become more complex if degrees of freedom are low. For modest bias and low degrees of freedom, summation of bias, bias uncertainty and observed value uncertainty in quadrature (“RSSu”) provides a similar interval and is simpler to adapt to reduced degrees of freedom, at the cost of a more restricted range of application if accurate coverage is desired.
Keywords: Bias; Uncertainty; Recovery; Uncorrected bias

Analytical tools for the nano world by Renato Zenobi (215-221).

Chemical cytometry: the chemical analysis of single cells by Emily H. Turner; Daniella Cohen; Haley R. Pugsley; David Gonzalez Gómez; Colin D. Whitmore; Cuiru Zhu; Norman J. Dovichi (223-226).

Microfluidic enzymatic reactors for proteome research by Yun Liu; Baohong Liu; Pengyuan Yang; Hubert H. Girault (227-229).

Miniaturized mid-infrared sensor technologies by Seong-Soo Kim; Christina Young; Boris Mizaikoff (231-237).

Scanning force microscopy based amperometric biosensors by Christine Kranz; Justyna Wiedemair (239-243).

ICP-MS as a new tool for the determination of gold nanoparticles in bioanalytical applications by Andy Scheffer; Carsten Engelhard; Michael Sperling; Wolfgang Buscher (249-252).

Real-time mass spectrometry in enzymology by Thomas Letzel (257-261).

Three-dimensional elemental imaging by means of synchrotron radiation micro-XRF: developments and applications in environmental chemistry by B. De Samber; G. Silversmit; R. Evens; K. De Schamphelaere; C. Janssen; B. Masschaele; L. Van Hoorebeke; L. Balcaen; F. Vanhaecke; G. Falkenberg; L. Vincze (267-271).

Novel chromatography techniques for high-throughput analysis of proteomes by Huiming Yuan; Lihua Zhang; Zhen Liang; Yukui Zhang (273-276).

Chemical analysis of atmospheric aerosols by Urs Baltensperger; André S. H. Prévôt (277-280).

Solving fundamental problems in chromatographic analysis by Thomas Skov; Rasmus Bro (281-285).

Carbon nanotubes (CNTs)-based electroanalysis by M. Teresa Fernández-Abedul; Agustín Costa-García (293-298).

Advances in HPLC detection—towards universal detection by Bin Zhang; Xiaofeng Li; Bing Yan (299-301).

Fluorescent sensor array in a microfluidic chip by Lourdes Basabe-Desmonts; Fernando Benito-López; Han J. G. E. Gardeniers; Rob Duwel; Albert van den Berg; David N. Reinhoudt; Mercedes Crego-Calama (307-315).
Miniaturization and automation are highly important issues for the development of high-throughput processes. The area of micro total analysis systems (μTAS) is growing rapidly and the design of new schemes which are suitable for miniaturized analytical devices is of great importance. In this paper we report the immobilization of self-assembled monolayers (SAMs) with metal ion sensing properties, on the walls of glass microchannels. The parallel combinatorial synthesis of sensing SAMs in individually addressable microchannels towards the generation of optical sensor arrays and sensing chips has been developed. Figure The advantages of microfluidic devices, surface chemistry, parallel synthesis, and combinatorial approaches have been merged to integrate a fluorescent chemical sensor array in a microfluidic chip. Specifically, five different fluorescent self-assembled monolayers have been created on the internal walls of glass microchannels confined in a microfluidic chip
Keywords: Microchannels; Fluorescent sensors; Array; SAMs; Combinatorial

Detection of molecular processes in the intact retina by ATR-FTIR spectromicroscopy by Sebastiano Massaro; Theodora Zlateva; Vincent Torre; Luca Quaroni (317-322).
We used Fourier transform infrared spectromicroscopy in the attenuated total reflection configuration to study biochemical events associated with the response to light of an intact retina. We show that the technique is suitable for the detection in real time of molecular processes occurring in rod outer segments induced by light absorption. Two-dimensional correlation analysis was applied to the identification and interpretation of specific spectral changes associated to the evolution of the system. The technique allows us to observe an extensive protein translocation, which we interpret as arising from the release of transducin from the disk membrane and its redistribution from the outer segment towards the inner segment of rod cells. These results are in full agreement with our current understanding of retinal physiology and validate the technique as a useful tool for the study of complex molecular processes in intact tissue. Figure Spectral changes in the mid infrared region following exposure of an intact retina to light
Keywords: Phototransduction; ATR-FTIR; Retina; Transducin; Rod cell

Comprehensive two-dimensional gas chromatography using a high-temperature phosphonium ionic liquid column by John V. Seeley; Stacy K. Seeley; Elise K. Libby; Zachary S. Breitbach; Daniel W. Armstrong (323-332).
A high-temperature ionic liquid, trihexyl(tetradecyl)phosphonium bis(trifluoromethane)sulfonamide, was used as the primary column stationary phase for comprehensive two-dimensional gas chromatography (GC × GC). The ionic liquid (IL) column was coupled to a 5% diphenyl/95% dimethyl polysiloxane (HP-5) secondary column. The retention characteristics of the IL column were compared to polyethylene glycol (DB-Wax) and 50% phenyl/50% methyl polysiloxane (HP-50+). A series of homologous compounds that included hydrocarbons, oxygenated organics, and halogenated alkanes were analyzed with each column combination. This comparison showed that the ionic liquid is less polar than DB-Wax but more polar than HP-50+. The most unique feature of the IL × HP-5 column combination is that alkanes, cyclic alkanes, and alkenes eluted in a narrow band in the GC × GC chromatogram; whereas, these compounds occupied a much larger portion of the DB-Wax × HP-5 and the HP-50+ × HP-5 chromatograms. Each column combination was used to analyze diesel fuel. The IL × HP-5 chromatogram displayed narrow bands for three major compound classes in diesel fuel: saturates, monoaromatics, and diaromatics. The IL column was used at temperatures as high as 290 °C for several months without any noticeable changes in column performance.
Keywords: Comprehensive two-dimensional gas chromatography; GC × GC; Ionic liquid; Stationary phase; Petroleum analysis

Upon adsorbing on a solid-state substrate, water-soluble proteins are prone to denaturation and deterioration of their functions due to the conformation change. The surface electric field of a conductive substrate is one of the important factors that influence the character of adsorbed proteins. In this work, a 3D macroporous gold electrode has been prepared and served as the working electrode to study the influence of surface electric field on the adsorption kinetics and conformation of the adsorbed cytochrome c (cyt-c) with the help of electrochemical, in situ electrochemical IR spectroscopic, atomic force microscopic, and contact angle measurements. The external electric field creates excess surface charge which can manipulate the adsorption rate of proteins on the substrate by the enhanced electrostatic interactions between the electrode and protein patches by coupling with complementary charges. The amount of immobilized cyt-c with electrochemical activity on the 3D macroporous gold electrode showed a minimum at potential of zero charge (PZC) and it increased with increasing net excess surface charge. Higher electric field could influence the conformation and the corresponding properties such as direct electrochemistry, bioactivity, and surface character of the adsorbed cyt-c molecules. However, high external electric field leads to damage of the protein secondary structure. This study provides fundamentals for the fabrication of biomolecular devices, biosensors, and biofuel cells through electrostatic interactions. Figure Two cases are illustrated for the protein immobilized on electrode surfaces: a retention of protein structure under moderate excess surface charge, b denaturation and conformation change of proteins adsorbed at high excess surface charge, e.g., due to the higher external electric field.
Keywords: Surface electric field; 3D macroporous gold electrode; Cytochrome c; Conformation

A cell-based screening method for specifically detecting kinase activity by Mikiya Suda; Tsuyoshi Ishii; Hiroshi Sootome; Alastair J. King; Megumi Shibahara; Nobuhiro Noro; Keizo Yamashita; Takato Noumi (343-348).
No universal approach has been reported for specific monitoring of the catalytic activity of a wide range of kinases in cells. The present study describes an original platform for detecting the autonomous activity of serine/threonine kinases in cells through the introduction of expression vectors encoding modified substrate kinase fusion proteins. The surrogate substrate used consists of the p53 tumor suppressor protein fused with individual kinase domains (Chk1, DYRK3, and Cdk5) at its carboxy-terminal through four tandem Gly-Gly-Gly-Gly-Ser repeats. After transfection into cells, phosphorylation of the p53 moiety could be specifically induced by the catalytic activity of kinases contained in the fusion protein. Moreover, p53 phosphorylation was significantly blocked when a kinase-inactive mutant was used as the fusion partner instead of the active kinase. Using this system, the cell-based evaluation of several Cdk5 inhibitors was demonstrated. Thus, this approach provides a novel platform for the specific, cell-based screening of inhibitors of a wide prospective range of protein kinases and is of tremendous potential for drug discovery efforts.
Keywords: Drug monitoring; Drug screening; Bioanalytical methods; Enzymes

In this study, a simple and efficient large volume injection gas chromatography–mass spectrometry (GC-MS) method, via a programmable-temperature vaporizer (PTV) inlet, has been developed and applied in the determination of estrogens in environmental water samples without a prior derivatization process. Three commonly used estrogens estrone, 17 β-estradiol and 17 α-ethynylestradiol were selected as target compounds for this study. It has been demonstrated that the type of gas chromatograph liner and the initial inlet temperature can greatly affect the response intensity of estrogens. Three different types of PTV liners have been studied; the multibaffle liner generated the strongest response intensities towards the estrogen analytes. The results showed that the response intensities of estrogens reduced sharply while the initial inlet temperature increased. Various instrument conditions and sample preparation methods were studied in detail. The optimized method has been validated with good linearity, precision and accuracy. The method detection limit of each estrogen was found to be 0.041 ng/L for estrone, 0.046 ng/L for 17 β-estradiol and 0.031 ng/L for 17 α-ethynylestradiol. To the best of our knowledge, these results represent the best sensitivities achieved for estrogens analyzed in water samples via traditional GC-MS method without a derivatization process. This method has been successfully applied in the analyses of different water samples.
Keywords: Water; Estrogen; Gas chromatography–mass spectrometry; Programmable-temperature vaporization; Large-volume injection

Determination of sulfur forms in wine including free and total sulfur dioxide based on molecular absorption of carbon monosulfide in the air–acetylene flame by Mao Dong Huang; Helmut Becker-Ross; Stefan Florek; Uwe Heitmann; Michael Okruss; Claus-Dieter Patz (361-367).
A new method for the determination of sulfur forms in wine, i.e., free SO2, total SO2, bound SO2, total S, and sulfate, is presented. The method is based on the measurement of the carbon monosulfide (CS) molecular absorption produced in a conventional air–acetylene flame using high-resolution continuum source absorption spectrometry. Individual sulfur forms can be distinguished because of the different sensitivities of the corresponding CS molecular absorption. The sensitivity of free SO2 is about three times higher than the value for bound SO2 and sulfate. The method makes use of procedures similar to those used in classic reference methods. Its performance is verified by analyzing six wine samples. Relative standard deviations are between 5 and 13% for free SO2 and between 1 and 3% for total SO2. For the validation of the accuracy of the new method, the results are compared with those of reference methods. The agreement of the values for total SO2 with values of the classic method is satisfactory: five out of six samples show deviations less than 16%. Due to the instability of free SO2 in wine and the known problems of the used reference method, serious deviations of the free SO2 results are found for three samples. The evaluation of the limits of detection focuses on the value for free SO2, which is the sulfur form having by far the lowest concentration in wine. Here, the achievable limit of detection is 1.8 mg L−1. Figure Detection of non-metal elements using continuum source flame absorption spectrometry
Keywords: Wine analysis; Molecular absorption spectrometry; Sulfur determination; Carbon monosulfide; Continuum source

Molecular interaction energy (MI) values calculated by molecular mechanics (MM2) using a model graphitic carbon phase were used for studying the selectivity of different types of graphitic carbon columns. The MI values well correlated with logk values measured on a graphitic carbon synthesized from 100% organic materials (r = 0.961, n = 13) but not with logk values measured on a graphitic carbon synthesized using silica matrix (r = 0.558, n = 17). The latter logk values correlated well with the hydrogen bonding energy values calculated using a model silica phase (r = 0.856, n = 17). The reason for the poor correlation of the logk values measured on the latter graphitic carbon is that the silica matrix might not be completely eliminated in the production process.
Keywords: Molecular interaction; Computational chemical analysis; Graphitic carbon; Aromatic compounds; Liquid chromatography

Vegetative insecticidal protein (Vip), a unique class of insecticidal protein, is now part of transgenic plants for conferring resistance against lepidopteron pests. In order to address the imminent regulatory need for detection and labeling of vip3A carrying genetically modified (GM) products, we have developed a standard single PCR and a multiplex PCR assay. As far as we are aware, this is the first report on PCR-based detection of a vip3A-type gene (vip-s) in transgenic cotton and tobacco. Our assay involves amplification of a 284-bp region of the vip-s gene. This assay can possibly detect as many as 20 natural wild-type isolates bearing a vip3A-like gene and two synthetic genes of vip3A in transgenic plants. The limit of detection as established by our assay for GM trait (vip-s) is 0.1%. Spiking with nontarget DNA originating from diverse plant sources had no inhibitory effect on vip-s detection. Since autoclaving of vip-s bearing GM leaf samples showed no deterioration/interference in detection efficacy, the assay seems to be suitable for processed food products as well. The vip-s amplicon identity was reconfirmed by restriction endonuclease assay. The primer set for vip-s was equally effective in a multiplex PCR assay format (duplex, triplex and quadruplex), used in conjunction with the primer sets for the npt-II selectable marker gene, Cauliflower mosaic virus 35S promoter and nopaline synthetase terminator, enabling concurrent detection of the transgene, regulatory sequences and marker gene. Further, the entire transgene construct was amplified using the forward primer of the promoter and the reverse primer of the terminator. The resultant amplicon served as a template for nested PCR to confirm the construct integrity. The method is suitable for screening any vip3A-carrying GM plant and food. The availability of a reliable PCR assay method prior to commercial release of vip3A-based transgenic crops and food would facilitate rapid and efficient regulatory compliance.
Keywords: Bacillus thuringiensis ; Genetically modified organisms; PCR; Transgenic cotton and tobacco; vip3A gene

Pressurised microwave-assisted extraction was used to extract a complex mixture containing polycyclic aromatic hydrocarbons (PAHs), nitrated PAHs and heavy n-alkanes from a particularly refractory carbonaceous material resulting from the combustion in a diesel engine. A second-order central composite design was used to determine the optimal conditions of extraction in terms of time, temperature, volume and nature of extracting solvent from spiked diesel soots. To begin, methylene chloride, tetrahydrofuran and chloroform were tested for extracting the spiked diesel particulates; however, the nature of these solvents was not really an influential factor. Volume was the most influential factor and was kept at a medium level to enhance the extraction of heavy PAHs without introducing an important dilution factor. Temperature and time were not influential as main factors but interacted with the other factors. Finally, high temperature and duration associated with a medium volume of methylene chloride were better for the extractions. After this optimisation, five-ring and six-ring PAHs were nevertheless not satisfactorily desorbed. Other solvents were therefore tested. Only aromatic ones, and particularly heterocyclic aromatic solvents, managed to desorb the heaviest PAHs. Pyridine, with its both aromatic and its basic character, was the most successful solvent. Desorption was even complete with an addition of 17% of diethylamine into pyridine. So, using MAE, we succeeded in extracting quantitatively, from the spiked refractory diesel soot surface, two-ring to six-ring PAHs, heavy n-alkanes and short nitrated PAHs. However, heavy nitrated PAHs were better extracted with a small addition of acetic acid (1%) into pyridine instead of a basic cosolvent.
Keywords: Polycyclic aromatic hydrocarbons; Diesel particulate matter; Microwave-assisted extraction; Factorial design

An intercomparison to establish the performance of routine laboratories in the determination of polybrominated flame retardants in polymers was organised. Commercial poly(ethyleneterephthalate) was fortified with technical pentabromodiphenyl ether, octabromodiphenyl ether and decabromodiphenyl ether mixtures and with a decabromobiphenyl technical mixture at 0.4–0.8 g/kg. Homogeneity and stability of the total Br content in the material was confirmed. Thirty-seven laboratories from Europe, Asia and the Americas submitted results. Relative repeatability standard deviations for individual congeners ranged from 7 to 17%. Relative between-laboratory standard deviations ranged from 22 to 61%. No significant influence of a common standard, application of a standard method or method parameters could be identified. The quality and uncertainty of the results of this study are significantly worse than those reported in the environmental field and indicate a clear need for a learning process among the laboratories involved. Figure Mandel’s h (between labs): critical level: 1.91
Keywords: Quality assurance/control; Polybrominated diphenyl ethers; Polybrominated flame retardants; Polymers

Pressurized liquid extraction with an integrated carbon trap (PLE-C) has recently been developed for fast and efficient analysis of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in food and feed. The method has also been tested, but not verified, for use on more complex soil samples, such as soil, sediment and fly ash. Hence, the primary aim of this study was to verify that PLE-C can produce reliable data for PCDDs/PCDFs in various abiotic matrixes. A second aim was to find a replacement for the previously used AX21 active carbon that is currently not commercially available. The performance of the PLE-C was evaluated using both single congener concentrations and toxic equivalency potentials (TEQ-pot) of three (soil, sediment and fly ash) certified reference materials. The results clearly show that PLE-C can be used for abiotic samples and that a commercially available carbon (Norit SA 4PAH HF) can replace the AX-21 carbon in the carbon trap. The TEQ-pot values obtained for the soil and sediment samples were within the uncertainty limits of the corresponding certified values, as were the determinations of single congener concentrations. PLE-C therefore has great potential for determination of PCDDs/PCDFs in soil and sediment samples. The TEQ-pot result for the fly ash was slightly lower than the certified TEQ-pot value, but it is still within the uncertainty limits of the certified value. Out of the single congener concentrations all but four (out of 17) agreed well with the values. Hence, PLE-C may potentially be used also for fly ash—after slight modifications. The integrated PLE-C and cleanup procedure is less labour-intensive than traditional methods such as Soxhlet extraction followed by a multistep cleanup, and consumes smaller quantities of ultrapure solvents than the commonly used Power-Prep system. In addition, PLE-C is capable of larger sample throughputs than the conventional methods. Thus, PLE-C is a promising alternative to the currently used sample preparation procedures for dioxins in abiotic samples. Figure PLE with integraded carbon trap for rapid PCDD/Fs analysis
Keywords: Pressurized liquid extraction; Dioxins; Certified reference materials; In-cell cleanup; Solid samples

Fruit juice authentication by 1H NMR spectroscopy in combination with different chemometrics tools by M. Cuny; E. Vigneau; G. Le Gall; I. Colquhoun; M. Lees; D. N. Rutledge (419-427).
To discriminate orange juice from grapefruit juice in a context of fraud prevention, 1H NMR data were submitted to different treatments to extract informative variables which were then analysed using multivariate techniques. Averaging contiguous data points of the spectrum followed by logarithmic transformation improved the results of the data analysis. Moreover, supervised variable selection methods gave better rates of classification of the juices into the correct groups. Last, independent-component analysis gave better classification results than principal-component analysis. Hence, ICA may be an efficient chemometric tool to detect differences in the 1H NMR spectra of similar samples, and so may be useful for authentication of foods.
Keywords: Chemometrics; 1H NMR spectroscopy; Fruit juices; Independent-components analysis