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Analytical and Bioanalytical Chemistry (v.386, #3)

Quo vadis? Leading the way with the younger generation of scientists by Sapna Deo; Sylvia Daunert (pp. 401-402).

Meet the Guest Editors by Sylvia Daunert; Sapna Deo; Kenji Hamase; Martin Vogel (pp. 403-404).

Comprehensive two-dimensional liquid chromatography by Robert A. Shellie; Paul R. Haddad (pp. 405-415).

Having nearly exhausted the possibilities for generating peak capacity through improvements in column technology, chromatographers are increasingly looking to alternative ways of maximising chromatographic separation. In recent years there has been increasing activity in the field of comprehensive multidimensional separations to meet analysis demands. Comprehensive two-dimensional liquid chromatography (LC×LC) approaches offer high peak capacity which leads to significantly improved analytical performance over single-column liquid chromatography. There are several closely related avenues available for achieving an LC×LC separation and this review pays special attention to the different valve-based interfaces that have been used to comprehensively couple the first and second dimension columns in LC×LC systems. A brief discussion of column choices for selected applications and the conditions employed is also presented.

Keywords: Liquid chromatography; HPLC; Comprehensive two-dimensional liquid chromatography; LC×LC; Multidimensional chromatography; Peak capacity

Recent developments in microarray-based enzyme assays: from functional annotation to substrate/inhibitor fingerprinting by Hongyan Sun; Souvik Chattopadhaya; Jun Wang; Shao Q. Yao (pp. 416-426).

Recent advances in proteomics have provided impetus towards the development of robust technologies for high-throughput studies of enzymes. The term “catalomics” defines an emerging ‘-omics’ field in which high-throughput studies of enzymes are carried out by using advanced chemical proteomics approaches. Of the various available methods, microarrays have emerged as a powerful and versatile platform to accelerate not only the functional annotation but also the substrate and inhibitor specificity (e.g. substrate and inhibitor fingerprinting, respectively) of enzymes. Herein, we review recent developments in the fabrication of various types of microarray technologies (protein-, peptide- and small-molecule-based microarrays) and their applications in high-throughput characterizations of enzymes.

Keywords: Enzyme assay; Microarray; Catalomics; Activity-based probe; Activity-based fingerprinting; Inhibitor fingerprinting; Drug discovery

Miniaturized platforms for the detection of single-nucleotide polymorphisms by Johnson Kian-Kok Ng; Wen-Tso Liu (pp. 427-434).

Conventional methods for detecting single-nucleotide polymorphisms (SNPs), the most common form of genetic variation in human beings, are mostly limited by their analysis time and throughputs. In contrast, advances in microfabrication technology have led to the development of miniaturized platforms that can potentially provide rapid high-throughput analysis at small sample volumes. This review highlights some of the recent developments in the miniaturization of SNP detection platforms, including microarray-based, bead-based microfluidic and microelectrophoresis-based platforms. Particular attention is paid to their ease of fabrication, analysis time, and level of throughput.

Keywords: Single-nucleotide polymorphisms (SNPs); Miniaturization; Microarrays; Microfluidics; Hybridization

Imaging molecular events in single living cells by Moritoshi Sato (pp. 435-443).

is a researcher at PRESTO, Japanese Science and Technology Agency. In 2005 he was promoted to lecturer at the University of Tokyo. His research interests involve building fluorescent indicators to visualize molecular events in single living cells. Fluorescence imaging could be the most powerful technique available for observing spatial and temporal dynamics of biomolecules in living cells, if fluorescent indicators for the relevant biomolecules become available. We have recently developed fluorescent indicators for a variety of second messengers or protein phosphorylations. Using the indicators, we have visualized spatial and temporal dynamics of these molecular events in single living cells. These fluorescent indicators are becoming an indispensable tool for understanding the complex mechanism of signal transduction in living cells.

Keywords: Fluorescent indicator; Fluorescence resonance energy transfer; Second messenger; Protein phosphorylation; Nuclear receptor ligand

Optical molecular imaging for systems biology: from molecule to organism by Wei Du; Ying Wang; Qingming Luo; Bi-Feng Liu (pp. 444-457).

The development of highly efficient analytical methods capable of probing biological systems at system level is an important task that is required in order to meet the requirements of the emerging field of systems biology. Optical molecular imaging (OMI) is a very powerful tool for studying the temporal and spatial dynamics of specific biomolecules and their interactions in real time in vivo. In this article, recent advances in OMI are reviewed extensively, such as the development of molecular probes that make imaging brighter, more stable and more informative (e.g., FPs and semiconductor nanocrystals, also referred to as quantum dots), the development of imaging approaches that provide higher resolution and greater tissue penetration, and applications for measuring biological events from molecule to organism level, including gene expression, protein and subcellular compartment localization, protein activation and interaction, and low-mass molecule dynamics. These advances are of great significance in the field of biological science and could also be applied to disease diagnosis and pharmaceutical screening. Further developments in OMI for systems biology are also proposed.

Keywords: Optical molecular imaging; Fluorescent protein; Quantum dots; Systems biology; Review

Biological applications of scanning electrochemical microscopy: chemical imaging of single living cells and beyond by Shigeru Amemiya; Jidong Guo; Hui Xiong; Darrick A. Gross (pp. 458-471).

Recent applications of scanning electrochemical microscopy (SECM) to studies of single biological cells are reviewed. This scanning probe microscopic technique allows the imaging of an individual cell on the basis of not only its surface topography but also such cellular activities as photosynthesis, respiration, electron transfer, single vesicular exocytosis and membrane transport. The operational principles of SECM are also introduced in the context of these biological applications. Recent progress in techniques for high-resolution SECM imaging are also reviewed. Future directions, such as single-channel detection by SECM, high-resolution imaging with nanometer-sized probes, and combined SECM techniques for multidimensional imaging are also discussed.

Keywords: Electroanalytical methods; Scanning electrochemical microscopy; Ultramicroelectrode; Single-cell analysis; Chemical imaging; Feedback mode

Investigation of structure, dynamics and function of metalloproteins with electrospray ionization mass spectrometry by Igor A. Kaltashov; Mingxuan Zhang; Stephen J. Eyles; Rinat R. Abzalimov (pp. 472-481).

is an Associate Professor at the Chemistry Department of the University of Massachusetts–Amherst. He has received the American Society for Mass Spectrometry Research Award (2000) and the Research Corporation Research Innovation Award (2001). His current research interests include biological mass spectrometry and the development of novel methods for the analysis of biomolecular architecture, dynamics and interactions. Electrospray ionization mass spectrometry (ESI MS) has emerged recently as a powerful tool for analyzing many structural and behavioral aspects of metalloproteins in great detail. In this review we discuss recent developments in the field, placing particular emphasis on the unique features of ESI MS that lend themselves to metalloprotein characterization at a variety of levels. Direct mass measurement enables the determination of protein–metal ion binding stoichiometry in solution and metalloprotein higher order structure in the case of multi-subunit proteins. MS techniques have been developed for determining the locations of metal-binding centers, metal oxidation states and reaction intermediates of metal-containing enzymes. Other ESI MS techniques are also discussed, such as protein ion charge state distributions and hydrogen/deuterium exchange studies, which can be used to measure metal binding affinities and to shed light on vital dynamic aspects of the functional properties of metalloproteins endowed by metal binding.

Keywords: Mass spectrometry (MS); Electrospray ionization (ESI); Collision-activated dissociation (CAD); Metalloprotein; Noncovalent interactions; Hydrogen/deuterium exchange (HDX)

Advances in the analysis of dynamic protein complexes by proteomics and data processing by Peter Schubert; Michael D. Hoffman; Matthew J. Sniatynski; Juergen Kast (pp. 482-493).

Signal transduction governs virtually every cellular function of multicellular organisms, and its deregulation leads to a variety of diseases. This intricate network of molecular interactions is mediated by proteins that are assembled into complexes within individual signaling pathways, and their composition and function is often regulated by different post-translational modifications. Proteomic approaches are commonly used to analyze biological complexes and networks, but often lack the specificity to address the dynamic and hence transient nature of the interactions and the influence of the multiple post-translational modifications that govern these processes. Here we review recent developments in proteomic research to address these limitations, and discuss several technologies that have been developed for this purpose. The synergy between these proteomic and computational tools, when applied together with global methods to the analysis of individual proteins, complexes and pathways, may allow researchers to unravel the underlying mechanisms of signaling networks in greater detail than previously possible.

Keywords: Cell signaling; Computational analysis; Mass spectrometry; Post-translational modification; Protein complexes

Voltammetric study on ion transport across a bilayer lipid membrane in the presence of a hydrophobic ion or an ionophore by Osamu Shirai; Yumi Yoshida; Sorin Kihara (pp. 494-505).

This review describes voltammetric studies on ion transport from one aqueous phase (W1) to another (W2) across a bilayer lipid membrane (BLM) containing a hydrophobic ion, valinomycin (Val) or gramicidin A (GA). In particular, the ion transport mechanisms are discussed in terms of the distribution of a pair of ions between aqueous and BLM phases. By addition of a small amount of hydrophobic ion into W1 and/or W2 containing a hydrophilic salt as a supporting electrolyte, the hydrophobic ion was distributed into the BLM with the counter ion to maintain electroneutrality within the BLM phase. It was found that the counter ion was transferred between W1 and W2 across the BLM by applying a membrane potential. Facilitated transport of alkali ions across a BLM containing Val as an ion carrier compound, could be interpreted by considering not only the formation of the alkali metal ion–Val complex but also the distribution of both the objective cation and the counter ion. In the case of addition of GA as a channel-forming compound into the BLM, the facilitated transport of alkali ions across the BLM depended on the ionic species of the counter ions. It was discovered that the influence of the counter ion on the facilitated transport of alkali ions across the BLM could be explained in terms of the hydrophobicity and the ionic radius of the counter ion.

Keywords: Electroanalytical methods; Thin films; Ion transport; Ion pair; Hydrophobicity

Recent advances in methods for the analysis of catecholamines and their metabolites by Makoto Tsunoda (pp. 506-514).

is Assistant Professor at the University of Tokyo and was recently awarded the Encouragement Award from the Society for Chromatographic Sciences. His current research interests include developing sensitive methods for determination of trace amounts of biological compounds, especially catecholamines, investigating the mechanism of regulation of blood pressure, and analysis of biological compounds in animal models. Catecholamines, for example epinephrine, norepinephrine, and dopamine, are widely distributed and are important neurotransmitters and hormones in mammalian species. Several methods have been developed for analysis of catecholamines and related compounds. Determination of catecholamines in biological fluids has enabled us to clarify the physiological role played by these amines. Catecholamine levels in plasma and/or urine are also useful for diagnosis of several diseases, for example hypertension, pheochromocytoma, and neuroblastoma. This review covers reports from 2000 to the present of methods for the analysis of catecholamines and their metabolites.

Keywords: High-performance liquid chromatography (HPLC); Electrochemical detection; Fluorescence; Chemiluminescence

Anthozoa red fluorescent protein in biosensing by Suresh Shrestha; Sapna K. Deo (pp. 515-524).

is an Assistant Professor of Bioanalytical Chemistry in the Department of Chemistry and Chemical Biology at the Indiana University-Purdue University Indianapolis. Dr Deo’s research interest is at the interface of analytical and biological chemistry with the objective of developing novel bioanalytical techniques employing fluorescent and bioluminescent proteins. Dr. Deo’s laboratory is currently developing biosensing systems for microRNA detection, diagnostics, and environmental analysis. The identification and cloning of a red fluorescent protein (DsRed) obtained from Anthozoa corals has provided an alternative to commonly used green fluorescent proteins (GFPs) in bioanalytical and biomedical research. DsRed in tandem with GFPs has enhanced the feasibility of multicolor labeling studies. Properties of DsRed, for example high photostability, red-shifted fluorescence emission, and stability to pH changes have proven valuable in its use as a fluorescent tag in cell-biology applications. DsRed has some limitations, however. Its slow folding and tendency to form tetramers have been a hurdle. Several different mutational studies have been performed on DsRed to overcome these problems. In this paper, applications of DsRed in biosensing, specifically in FRET/BRET assays, whole-cell assays, and in biosensors, is discussed. In the future, construction of DsRed mutants with unique characteristics will further expand its applications in bioanalysis.

Keywords: Red flourescent protein; Bioanalytical methods; Biosensing; FRET/BRET

Radical polymerization in biosensing by Xinhui Lou; Peng He; Geoffrey O. Okelo; Lin He (pp. 525-531).

This review briefly summarizes recently published work on radical polymerization in biosensor-related applications. Advancements in surface modification aimed at improving sensor biocompatibility and reducing nonspecific background noises are discussed. Direct applications of polymers as one of the key sensing elements in which they are used either as detection probes for the biomolecular binding events or as signal transducers to amplify sensing signals are detailed. Initial applications of radical polymerization reactions in biosensing are evident and appear promising.

Keywords: Free radical polymerization; Controlled/living radical polymerization; Biosensors; Biosensing

Recent advances in fluorescent probes for the detection of reactive oxygen species by Nobuaki Soh (pp. 532-543).

Reactive oxygen species (ROS) have captured the interest of many researchers in the chemical, biological, and medical fields since they are thought to be associated with various pathological conditions. Fluorescent probes for the detection of ROS are promising tools with which to enhance our understanding of the physiological roles of ROS, because they provide spatial and temporal information about target biomolecules in in vivo cellular systems. ROS probes, designed to detect specific ROS with a high selectivity, would be desirable, since it is now becoming clear that each ROS has its own unique physiological activity. However, dihydro-compounds such as 2′,7′-dichlorodihydrofluorescein (DCFH), which have traditionally been used for detecting ROS, tend to react with a wide variety of ROS and are not completely photostable. Some attractive fluorescent probes that exhibit a high degree of selectivity toward specific ROS have recently been reported, and these selective probes are expected to have great potential for elucidating unknown physiological mechanisms associated with their target ROS. This review focuses on the design, detection mechanism, and performance of fluorescent probes for the detection of singlet oxygen (¹O₂), hydrogen peroxide (H₂O₂), hydroxyl radicals (^.OH), or superoxide anion (O₂ ^−.), a field in which remarkable progress has been achieved in the last few years.

Keywords: Fluorescent probe; Reactive oxygen species (ROS); Singlet oxygen (¹O₂); Hydrogen peroxide (H₂O₂); Hydroxyl radical (^.OH); Superoxide anion (O₂ ^−.)

Environmental fate processes and biochemical transformations of chiral emerging organic pollutants by Charles S. Wong (pp. 544-558).

This review highlights the analytical chemistry, environmental occurrence, and environmental fate of individual stereoisomers of chiral emerging pollutants, which are modern current-use chemicals of growing environmental concern due to their presence in the environment and potential for deleterious effects. Comparatively little is known about individual stereoisomers of pollutants, which can have differential toxicological effects and can be tracers of biochemical weathering in the environment. Stereoisomers are resolved by gas chromatography (GC), high-performance liquid chromatography (HPLC), and capillary electrophoresis (CE). Separation techniques in environmental analysis are typically coupled to mass spectrometry (MS) and tandem mass spectrometry (MS/MS), as these provide the sensitivity and selectivity needed. The enantiomer composition of phenoxyalkanoic and acetamide herbicides, organophosphorus and pyrethroid pesticides, chiral polychlorinated biphenyl metabolites, synthetic musks, hexabromocyclododecane, and pharmaceuticals in the environment show species-dependent enantioselectivity from biotransformation and other biologically mediated processes affecting enantiomers differentially. These enantiomer compositions are useful in detecting biologically mediated environmental reactions, apportioning sources of pollutants, and gaining insight into the biochemical fate of chiral pollutants in the environment, which are needed for accurate risk assessment of such chemicals.

Keywords: Chiral pollutants; Environmental chemistry; Biotransformation; Source apportionment

Recent methods for the determination of peroxide-based explosives by Rasmus Schulte-Ladbeck; Martin Vogel; Uwe Karst (pp. 559-565).

In the last few years, the need to determine peroxide-based explosives in solid samples and air samples has resulted in the development of a series of new analytical methods for triacetonetriperoxide (TATP, acetone peroxide) and hexamethylenetriperoxidediamine (HMTD). In this review, after a short introduction describing the state of the art in the field, these new analytical methods are critically discussed. Particular emphasis is placed on spectroscopic and mass spectrometric methods as well as on chromatographic techniques with selective detection schemes. The potential of these methods to analyse unknown solid samples that might contain one or more of the explosives and to analyse peroxide-based explosives in air is evaluated.

Keywords: Peroxide-based; Explosives; Triacetonetriperoxide (TATP, acetone peroxide); Hexamethylenetriperoxidediamine (HMTD)

Preparation and characterization of methacrylate-based semi-micro monoliths for high-throughput bioanalysis by Tomonari Umemura; Yuji Ueki; Kin-ichi Tsunoda; Akio Katakai; Masao Tamada; Hiroki Haraguchi (pp. 566-571).

Hexyl methacrylate (HMA)-based monolithic semi-micro columns were prepared by in situ polymerization within the confines of 1.02-mm-i.d. silicosteel tubing for reversed-phase and/or precipitation–redissolution liquid chromatography. Practically useful monolithic columns with adequate separation efficiency, high permeability, and good mechanical strength were successfully obtained using a polymerization mixture comprising 24% hexyl methacrylate (HMA), 6% ethylene dimethacrylate (EDMA), 44.5% 1-propanol, and 25.5% 1,4-butanediol. The column performance was evaluated through the separations of a series of alkylbenzenes. At a normal flow rate of 50 μL min⁻¹, the produced HMA-based monolithic columns typically exhibited 3,000 theoretical plates for a 20-cm-long column, and the pressure drop was generally less than 1 MPa per 20 cm. The monolithic columns were resistant to at least 15 MPa, and could be properly operated at 15–20 times higher flow rate than normal, reducing the separation time to 1/15–1/20. The HMA-based monolithic columns were applied to rapid and efficient separations of proteins such as ribonuclease A, cytochrome c, transferrin, and ovalbumin in the precipitation–redissolution mode. Using a CH₃CN gradient elution at a flow rate of 1,000 μL min⁻¹, four proteins were baseline separated within 20 s.

Keywords: Monolithic column; Hexyl methacrylate; Reversed-phase liquid chromatography; Proteins

Simultaneous analysis of cationic, anionic, and neutral compounds using monolithic CEC columns by Masaru Kato; Yuko Onda; Kumiko Sakai-Kato; Toshimasa Toyo’oka (pp. 572-577).

A new capillary electrochromatography (CEC) column for the simultaneous analysis of cationic, neutral, and anionic compounds using CEC-ESI-MS is described. Three different silica monolith columns were prepared by changing the poly(ethylene glycol) (PEG) contents for comparison of the separation property of these columns. Different separation programs were used for the simultaneous separation of different charged compounds under the same conditions. The column prepared with 80 mg of PEG separated typical compounds within 15 min using 1 M formic acid as the electrolyte. The analytes migrated in the order of cationic, neutral, and anionic compounds, which means that the migration order was mainly determined by the electrophoresis. The hydrodynamic flow by pressure from the inlet side was significant for a stable analysis to be achieved. The effect of the composition of the sheath liquid was also examined. All analytes (14 amino acids, thiourea, urea, citric acid, and ATP) were detectable when 1% acetic acid in 50% (v/v) methanol was used as the sheath liquid.

Keywords: CEC-ESI-MS; Simultaneous analysis; Silica monolith; Matabolome; Charged and uncharged compounds

HILIC mode separation of polar compounds by monolithic silica capillary columns coated with polyacrylamide by Tohru Ikegami; Hiroshi Fujita; Kanta Horie; Ken Hosoya; Nobuo Tanaka (pp. 578-585).

HILIC mode columns were prepared by an on-column polymerization of acrylamide on a monolithic silica capillary column modified with N-(3-trimethoxysilylpropyl)methacrylamide as the anchor group. The products showed HILIC mode retention characteristics with three times greater permeability and slightly higher column efficiency compared to a commercially available amide-type HILIC column packed with 5-μm particles. The selectivity of the monolithic silica-based column was similar to that of the particulate column for each group of solutes towards nucleosides, nucleic bases and carbohydrate derivatives, although a considerable difference was observed in the selectivity for the solute groups. Although the retention of solutes based on the polar functionality was much smaller with the monolithic silica columns, which had a smaller phase ratio, than with the particle-packed column, the former can achieve better separation utilizing the high permeability and higher column efficiencies of a longer column.

Keywords: Monolithic silica; HILIC; Polyacrylamide; PA sugars; Nucleosides

Separation and identification of compounds in Adinandra nitida by comprehensive two-dimensional liquid chromatography coupled to atmospheric pressure chemical ionization source ion trap tandem mass spectrometry by Jie Zhang; Dingyin Tao; Jicheng Duan; Zhen Liang; Weibing Zhang; Lihua Zhang; Yushu Huo; Yukui Zhang (pp. 586-593).

A comprehensive two-dimensional liquid chromatographic (2D-LC) separation system based on the combination of a CN column and a Merck Chromolith Flash reversed-phase column was developed for the separation of components in Adinandra nitida, one type of traditional Chinese medicine (TCM). The two dimensions were connected by a ten-port, dual-position valve controlled automatically by software written in-house. The effluents were detected by both ultraviolet and atmospheric pressure chemical ionization source ion trap tandem mass spectrometry (MS). The calculated peak capacity of the 2D-LC–MS/MS system was above 1240. More than 57 components were resolved in the methanol extract from Adinandra nitida leaves, and five of these were identified based on their relative retention times, molecular weights and MS/MS spectra.

Keywords: Comprehensive two-dimensional liquid chromatography; Traditional Chinese medicine; Adinandra nitida ; Mass spectrometer; Monolithic column

Electrophoretic analysis of proteins and enantiomers using capillaries modified by a successive multiple ionic-polymer layer (SMIL) coating technique by Fumihiko Kitagawa; Masato Kamiya; Yukihiro Okamoto; Hiromi Taji; Satomi Onoue; Yoshiko Tsuda; Koji Otsuka (pp. 594-601).

The applicability of three-layer coatings consisting of three different polymers (A⁺-B⁻-C⁺ coating) prepared by a successive multiple ionic-polymer layer (SMIL) coating technique to the immobilization of polypeptides and/or proteins onto the inner surface of the capillaries was investigated to provide a high-performance separation medium for proteins and enantiomers in capillary electrophoresis (CE). To obtain a stable protein-coated capillary, high molecular mass poly(ethyleneimine) (PEI) was employed as the first layer in the A⁺-B⁻-C⁺ coating, and then a cationic protein was immobilized as the third layer. Comparisons of analytical performances between the A⁺-B⁻-C⁺ coating and the conventional SMIL-coated (A⁺-B⁻-A⁺ coating) capillary were conducted. The CE separation of cationic proteins was successfully achieved with the prepared capillaries. In addition, the polypeptide- and protein-coated capillaries were applied to the chiral separation of a binaphthyl compound. It should be noted that the chiral separation efficiency was strongly dependent on the second anionic polymer layer of the coating. Effects of the interaction between oppositely charged ionic polymer layers on the separation efficiency are discussed.

Keywords: Capillary electrophoresis; Successive multiple ionic-polymer layer coating; Protein analysis; Chiral separation; DNA

Projection of multidimensional GC data into alternative dimensions—exploiting sample dimensionality and structured retention patterns by J. Harynuk; B. Vlaeminck; P. Zaher; P. J. Marriott (pp. 602-613).

Comprehensive multidimensional gas chromatography (GC×GC) is a powerful separation technique. One of the features of this technique is that it offers separations with more apparent structure than that offered by conventional one-dimensional GC (1-D GC). While some previous studies have alluded to this structure, and used structured retention patterns for some simple classifications, the topic of structured retention in GC×GC has not been studied in any great detail. Using the separation of fatty acid methyl esters (FAME) on both nonpolar/polar and polar/nonpolar column sets, the interaction between the separation dimensions and the sample dimensions is explored here. The GC×GC separation of a series of compounds is presented as a projection of the sample from sample space, a p-dimensional space with dimensions defined by the dimensionality of the sample, into separation space: for GC×GC, a two-dimensional plane passing through the sample space in an orientation defined by the separation conditions. Using this conceptual model and some a priori knowledge of the sample, it is shown how the image of the sample in the separation space can be used to construct an image of the sample in alternate dimensions, such as second dimension retention factor (²k) vs. chain length in the case of FAME. These projections into alternate dimensions should facilitate the interpretation of the complex patterns found within the GC×GC chromatogram for the identification and classification of compounds.

Keywords: Comprehensive multidimensional gas chromatography; Structured retention; Dimensionality; Fatty acids; FAME

Development of an on-line immobilized-enzyme reversed-phase HPLC method for protein digestion and peptide separation by Lee Wah Lim; Mami Tomatsu; Toyohide Takeuchi (pp. 614-620).

This paper describes use of a novel glass bead-based immobilized-enzyme micro column for simple and swift on-line protein digestion then peptide separation by reversed-phase HPLC. The inexpensive and easily made immobilized-enzyme micro column was prepared from aminopropyl controlled-pore glass that was reacted first with glutaraldehyde then with trypsin in the presence of phosphate buffer. Tryptic digestion of bovine serum albumin (BSA) was achieved simply by passing pretreated protein solution through the laboratory made immobilized-trypsin column; the tryptic fragments were then separated by reversed-phase HPLC. The peptide separation was found to be identical to separation of a sample which had undergone conventional enzymatic protein digestion in solution. Digestion of BSA by the immobilized-trypsin column decreased with increasing flow rate of the solution through the column, and 1.0 μL min⁻¹ was found to be the optimum flow rate for on-line protein digestion with our system. It was also found that the sample required pretreatment with urea before injection, because of a change in the properties of the protein in the presence of urea, and the immobilized-trypsin column lost its function in the presence of acetonitrile. This on-line proteomics system enables simple and rapid protein digestion and was successfully applied to partially micro two-dimensional (2D) chromatographic separation of proteins.

Keywords: Trypsin; Enzyme immobilization; On-line protein digestion; Peptide separation; Reversed-phase HPLC; 2D liquid chromatography

Tunable design strategy for fluorescence probes based on 4-substituted BODIPY chromophore: improvement of highly sensitive fluorescence probe for nitric oxide by Yu Gabe; Tasuku Ueno; Yasuteru Urano; Hirotatsu Kojima; Tetsuo Nagano (pp. 621-626).

received his PhD at the Graduate School of Pharmaceutical Sciences at the University of Tokyo in March 2006 under the direction of Prof. Tetsuo Nagano. His current research interests are the development and applications of novel fluorescence probes for reactive nitrogen species. 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) is a well-known fluorophore, with a high molar extinction coefficient and high fluorescence quantum efficiency (Φ_fl). Furthermore, its structure can be modified to change its excitation and emission wavelengths. However, little work has been done on the structural modification of fluorines at the B-4 position with other functional groups. We synthesized 4-methoxy-substituted BODIPY derivatives in satisfactory yields, and found that they exhibited improved solubility in aqueous solution. Moreover, their oxidation and reduction potentials were greatly decreased without any change in their absorbance and fluorescence properties. These features of 4-substituted BODIPYs may be useful for developing novel fluorescence probes based on the intramolecular photoinduced electron transfer (PeT) mechanism, because it is possible to optimize the PeT process precisely by modulating the electrochemical properties of the fluorophore. The value of this approach is exemplified by its application to the development of a highly sensitive and pH-independent fluorescence probe for nitric oxide.

Keywords: Fluorescence probe; BODIPY; Nitric oxide; Photoinduced electron transfer

Longitudinal diffusion behavior of hemicyanine dyes across phospholipid vesicle membranes as studied by second-harmonic generation and fluorescence spectroscopies by Akira Yamaguchi; Masaki Nakano; Kimihisa Nochi; Tomohisa Yamashita; Kotaro Morita; Norio Teramae (pp. 627-632).

The adsorption and longitudinal diffusion behaviors of a series of hemicyanine dyes to phospholipid vesicle membranes were studied by second-harmonic generation (SHG) and fluorescence spectroscopies. It was observed that the longitudinal diffusion of cationic hemicyanine dyes takes place immediately after the initial adsorption of these dyes to the outer surface of the vesicle membrane. In contrast, hardly any amount of a zwitterionic hemicyanine dye with a sulfonate group diffused across the vesicle membrane within the measurement time (<2000 s). Based on the difference in the time-course responses of SHG and fluorescence spectroscopies for all of the hemicyanine dyes tested, we propose that hydration of the sulfonate group is mainly responsible for the low diffusivity of the zwitterionic hemicyanine dye.

Keywords: Second-harmonic generation; Fluorescence; Phospholipid vesicle; Longitudinal diffusion

Interfacial behavior of sulforhodamine 101 at the polarized water/1,2-dichloroethane interface studied by spectroelectrochemical techniques by Hirohisa Nagatani; Shingo Suzuki; David J. Fermín; Hubert H. Girault; Kiyoharu Nakatani (pp. 633-638).

The transfer mechanism of an amphoteric rhodamine, sulforhodamine 101 (SR101), across the polarized water/1,2-dichloroethane (DCE) interface was investigated using cyclic voltammetry, differential voltfluorometry and potential-modulated fluorescence (PMF) spectroscopy. The voltammetric response for the ion transfer of SR101 monoanion from water to DCE was observed as the diffusion-controlled transfer process. An unusual voltammetric response was found at 0.15 V more negative than the formal transfer potential of SR101⁻ $${left( {Delta ^{{ ext{W}}}_{{ ext{O}}} phi ^circ prime } ight)}$$ in the cyclic voltammogram and voltfluorogram. The frequency dependence of the PMF responses confirmed the presence of the adsorption processes at negative potentials. In addition, a further transient adsorption step was uncovered at $$Delta ^{{ ext{W}}}_{{ ext{O}}} phi ^circ prime .$$ The interfacial mechanism of SR101 is discussed by comparing the results obtained from each technique.

Keywords: Sulforhodamine 101; Adsorption; Liquid/liquid interface; Potential modulated fluorescence

Measurement of antibody binding to protein immobilized on gold nanoparticles by localized surface plasmon spectroscopy by Kazuhiko Fujiwara; Hitoshi Watarai; Hideaki Itoh; Erika Nakahama; Nobuaki Ogawa (pp. 639-644).

Antibody binding to bovine serum albumin (BSA) and human serum albumin (HSA) immobilized onto gold nanoparticles was studied by means of localized surface plasmon resonance (LSPR) spectroscopy. Amine-modified glass was prepared by self-assembly of amine-terminated silane on substrate, and gold (Au) nanoparticles were deposited on the amine-modified glass substrate. Au nanoparticles deposited on the glass surface were functionalized by BSA and HSA. BSA immobilization was confirmed by LSPR spectroscopy in conjunction with surface-enhanced Raman scattering spectroscopy. Then, LSPR response attributable to the binding of anti-BSA and anti-HSA to BSA- and HSA-functionalized Au nanoparticles, respectively, was examined. Anti-HSA at levels larger than ∼10 nM could be detected by HSA-immobilized chips with LSPR optical response, which was saturated at concentrations greater than ∼650 nM of anti-HSA.

Keywords: Gold nanoparticle; Localized surface plasmon resonance; Biomolecular sensing; Antibody; Serum albumin; Protein immobilization

Potential-step coulometry of d-glucose using a novel FAD-dependent glucose dehydrogenase by Seiya Tsujimura; Shinki Kojima; Tokuji Ikeda; Kenji Kano (pp. 645-651).

is Assistant Professor of applied life science in Kyoto University. Currently his research interests focused on the design and quantitative characterization of enzyme electrode and catalytic mechanism of redox enzymes. This paper describes the construction and characterization of a batch-type coulometric system for the detection of d-glucose using a novel FAD-dependent glucose dehydrogenase. In order to overcome the problem of interferents, such as ascorbate and urate, a potential-step method was proposed to separate the electrolysis reactions of interferents and d-glucose by selecting a mediator possessing an appropriate formal potential. The rapid oxidative consumption of the interferents proceeded in the first step, whereas the mediator and glucose remained reduced. In the second step, the mediator was immediately oxidized, and subsequent bioelectrocatalytic oxidation of d-glucose occurred with the aid of aldose 1-epimerase. In this study, potassium octacyanomolybdate (IV) with a formal potential of 0.6 V vs. Ag|AgCl was chosen as a mediator, and the first and second electrolysis potentials were set at 0.4 V and 0.8 V, respectively, by considering the heterogeneous electron-transfer kinetics and the potential window. The background-corrected response in charge corresponded to 99±2 % efficiency in terms of the amount of d-glucose.

Keywords: Coulometry; Glucose; Glucose dehydrogenase; Biosensor; Ascorbate; Urate

Characterization of collagen fibers in Bruch’s membrane using chemical force microscopy by Shrestha Basu Mallick; Sandhya Bhagwandin; Albena Ivanisevic (pp. 652-657).

Bruch’s membrane is a layer composed of collagen fibers located just beneath the retina. This study validates a strategy used to map the morphological and adhesion characteristics of collagen fibers in Bruch’s membrane. Atomic force microscopy tips were functionalized with different chemical groups and used to map the hydrophilic and hydrophobic regions on the surface of the eye tissue. The largest adhesion forces were observed when tips functionalized with NH₂ groups were used. The trend in the adhesion forces was rationalized based on the distribution of different functional groups in the triple-helical structure of the collagen fibers. The results of this study can be used to design more effective strategies to treat eye diseases such as age-related macular degeneration.

Keywords: AFM (atomic force microscopy); Bioanalytical methods; Interface/surface analysis

Procedure for increasing the detection responses of estrogens in LC–MS based on introduction of a nitrobenzene moiety followed by electron capture atmospheric pressure chemical ionization by Tatsuya Higashi; Naoki Takayama; Tadashi Nishio; Emi Taniguchi; Kazutake Shimada (pp. 658-665).

A practical procedure for determining estrogens in biological fluids has been studied using liquid chromatography–electron capture atmospheric pressure chemical ionization–mass spectrometry combined with derivatization. Among the commercially available reagents (4-nitrobenzoyl chloride, 2,4-dinitrofluorobenzene, 4-nitrobenzenesulfonyl chloride and 4-nitrobenzyl bromide), 4-nitrobenzenesulfonyl chloride was of the most practical use; it rapidly and quantitatively reacted with estrogens and increased the detection responses by 8–23 times. The derivatization method allowed the reproducible and accurate quantification of serum and urine estrone and estradiol of a pregnant woman, which is useful for diagnosis of the fetoplacental function, with small amounts (10 μl) of sample and a simple pretreatment procedure. Tatsuya Higashiis Associate Professor of the Laboratory of Clinical Analytical Sciences (Professor Kazutake Shimada’s research group) at the Graduate School of Natural Science and Technology of Kanazawa University. He received the Japan Society for Analytical Chemistry Award for Young Scientists in 2003 and the Pharmaceutical Society of Japan Award for Young Scientists in 2006. His current research interests are the development of methods for increasing sensitivity in LC-MS to detect and characterize trace amounts of biologically active steroids, such as estrogens, androgens and neuroactive steroids.

Keywords: Liquid chromatography–mass spectrometry; Electron capture atmospheric pressure chemical ionization; Estrogen; Derivatization; Serum; Urine

A practical procedure for determining estrogens in biological fluids has been studied using liquid chromatography–electron capture atmospheric pressure chemical ionization–mass spectrometry combined with derivatization. Among the commercially available reagents (4-nitrobenzoyl chloride, 2,4-dinitrofluorobenzene, 4-nitrobenzenesulfonyl chloride and 4-nitrobenzyl bromide), 4-nitrobenzenesulfonyl chloride was of the most practical use; it rapidly and quantitatively reacted with estrogens and increased the detection responses by 8–23 times. The derivatization method allowed the reproducible and accurate quantification of serum and urine estrone and estradiol of a pregnant woman, which is useful for diagnosis of the fetoplacental function, with small amounts (10 μl) of sample and a simple pretreatment procedure. Tatsuya Higashi is Associate Professor of the Laboratory of Clinical Analytical Sciences (Professor Kazutake Shimada’s research group) at the Graduate School of Natural Science and Technology of Kanazawa University. He received the Japan Society for Analytical Chemistry Award for Young Scientists in 2003 and the Pharmaceutical Society of Japan Award for Young Scientists in 2006. His current research interests are the development of methods for increasing sensitivity in LC-MS to detect and characterize trace amounts of biologically active steroids, such as estrogens, androgens and neuroactive steroids.

Keywords: Liquid chromatography–mass spectrometry; Electron capture atmospheric pressure chemical ionization; Estrogen; Derivatization; Serum; Urine

Identification of sulfation sites of metabolites and prediction of the compounds’ biological effects by Lin Yi; Joe Dratter; Chao Wang; Jon A. Tunge; Heather Desaire (pp. 666-674).

Characterizing the biological effects of metabolic transformations (or biotransformation) is one of the key steps in developing safe and effective pharmaceuticals. Sulfate conjugation, one of the major phase II biotransformations, is the focus of this study. While this biotransformation typically facilitates excretion of metabolites by making the compounds more water soluble, sulfation may also lead to bioactivation, producing carcinogenic products. The end result, excretion or bioactivation, depends on the structural features of the sulfation sites, so obtaining the structure of the sulfated metabolites is critically important. We describe herein a very simple, high-throughput procedure for using mass spectrometry to identify the structure—and thus the biological fate—of sulfated metabolites. We have chemically synthesized and analyzed libraries of compounds representing all the biologically relevant types of sulfation products, and using the mass spectral data, the structural features present in these analytes can be reliably determined, with a 97% success rate. This work represents the first example of a high-throughput analysis that can identify the structure of sulfated metabolites and predict their biological effects.

Keywords: Mass spectrometry; ICP-MS; Bioanalytical methods; Pharmaceuticals; Biological samples; Metabolites; Sulfation

Characterization of nucleic acid higher order structure by high-resolution tandem mass spectrometry by Jingjie Mo; Kristina Håkansson (pp. 675-681).

Mass spectrometry (MS) is extensively used for the identification and sequencing of nucleic acids but has so far seen limited use for characterization of their higher order structures. Here, we have applied a range of different tandem mass spectrometry techniques, including electron detachment dissociation (EDD), infrared multiphoton dissociation (IRMPD), activated ion (AI) EDD, and EDD/IRMPD MS³, in a Fourier transform ion cyclotron resonance mass spectrometer to the characterization of three isomeric 15mer DNAs with different sequences and predicted solution-phase structures. Our goal was to explore whether their structural differences could be directly probed with these techniques. We found that all three 15mers had higher order structures in the gas phase, although preferred structures were predicted for only two of them in solution. Nevertheless, EDD, AI EDD, and EDD/IRMPD MS³ experiments yielded different cleavage patterns with less backbone fragmentation for the more stable solution-phase structure than for the other two 15mers. By contrast, no major differences were observed in IRMPD, although the extent of backbone cleavage was higher with that technique for all three 15mers. Thus, experiments utilizing the radical ion chemistry of EDD can provide complementary structural information compared to traditional slow heating methods, such as IRMPD, for structured nucleic acids.

Keywords: Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS); Infrared mulitphoton dissociation (IRMPD); Electron detachment dissociation (EDD); Oligonucleotide; Nucleic acid; DNA

Analysis of the modification site in a small molecule-modified peptide by ion trap/time-of-flight hybrid mass spectrometry by Nariyasu Mano; Mio Kamota; Yusuke Inohana; Shin-ichi Yamaguchi; Junichi Goto (pp. 682-688).

is Associate Professor at the Tohoku University and received the Young Scientist Award from the Japanese Society for Chromatographic Sciences in 2002 for his studies on the highly selective molecular recognition of biologically active substances using liquid phase seperation, as well as the Young Scientist Award from the Japanese Society for Biomedical Mass Spectrometry in 2005. His major areas of research are the development of novel methods for the seperation and determination of biologically active substances including functional proteins by chromatographic methods combined with mass spectrometry. Ion trap/time-of-flight hybrid mass spectrometers are powerful tools for the detailed structural analysis of modified peptides. We have analyzed Met-Lys-bradykinin modified with deoxycholate at the amino-terminus or the ε-amino group as model peptides. These two modified peptides produced fragment ions with the same nominal but different exact masses in tandem mass spectrometry with low-energy collision-induced dissociation. Accurate high-resolution analysis coupled with MS³ allowed us to distinguish the deoxycholate modification sites in the modified peptides.

Keywords: Ion trap; Mass spectometry; Modification site; MSⁿ analysis; Small molecule-modified peptide; Time-of-flight

Mass spectrometric real-time monitoring of enzymatic glycosidic hydrolysis, enzymatic inhibition and enzyme complexes by Nicole Dennhart; Thomas Letzel (pp. 689-698).

is Assistant Professor at the Technical University of Munich (Prof. Dieter Langosch) in Bavaria/Germany. After developing mass spectrometric methods in environmental and pharmaceutical analysis in recent years, his research interests currently are focused on fundamental mass spectrometric studies in ‘functional proteomics’, ‘metabolic pathways’ and ‘noncovalent complexes’. The mass spectrometric development of an enzymatic assay resulting in enzymatic activity, its reaction pathway and its dissociation constant are described for the first time within a single experiment. The method combines the performance of a mass spectrometry-compatible enzyme assay with the direct detection of specific enzyme complexes using hen egg white lysozyme as a model. The continuous liquid-flow technique applied, when hyphenated with electrospray ionization (ESI)–time-of-flight (ToF)–mass spectrometry (MS), permitted the simultaneous detection of several substances involved in product screening as well as the direct observation of dissociation constants. Dissociation constants for the product inhibitor N, N′, N″-triacetylchitotriose were calculated using a Scatchard plot (12×10⁻⁶ M) and the law of mass action (18–24×10⁻⁶ M), and these are in good agreement with constants obtained in earlier mass spectrometric (6–18×10⁻⁶ M) or spectroscopic (6–8×10⁻⁶ M) studies. Finally, the enzymatic hydrolysis of glycosidic substrate was monitored by ESI–ToF–MS in the presence of various inhibitors, thus leading to decreased activities in terms of their enzyme affinities. The associated inhibitor–enzyme complexes could be detected for up to lower micromolar K d values.

Keywords: Electrospray ionization; Mass spectrometry; Enzymatic reaction; Online monitoring; Dissociation constants

Affinity-based mass spectrometry using magnetic iron oxide particles as the matrix and concentrating probes for SALDI MS analysis of peptides and proteins by Wei-Yu Chen; Yu-Chie Chen (pp. 699-704).

Silane-immobilized magnetic iron oxide particles were used as the assisting material in surface-assisted laser desorption/ionization (SALDI) mass spectrometric analysis. This approach can be used to analyze small proteins and peptides. The upper detectable mass range is approximately 16 kDa. The detection limit for peptides is about 20 fmol. Silanized iron oxide particles with negatively charged functionalities can also be used as the affinity probes to selectively trap oppositely charged species from sample solutions by adjusting the pH of the solution. A tryptic digest product of cytochrome C at a concentration as low as 10 nM can be enriched by the particles and directly analyzed by iron oxide SALDI MS without the need for elution steps. Affinity-based mass spectrometry using the bifunctional silanized magnetic iron oxide particles as the SALDI matrix and concentrating probe is demonstrated in this study.

Keywords: SALDI MS; Magnetic nanoparticles; Affinity-based mass spectrometry; Affinity probes; Inorganic matrix

Presence and origin of large amounts of d-proline in the urine of mutant mice lacking d-amino acid oxidase activity by Kenji Hamase; Sayaka Takagi; Akiko Morikawa; Ryuichi Konno; Akira Niwa; Kiyoshi Zaitsu (pp. 705-711).

Using a column-switching HPLC system combining a micro-ODS column and a chiral column, the amounts of d-proline (d-Pro) have been determined in 18 tissues, plasma and urine of mice. To avoid the enzymatic degradation of d-amino acids in vivo, a mutant mouse strain lacking d-amino acid oxidase activity (ddY/DAO⁻ mouse) was used. In the brain, relatively large amounts of d-Pro were observed in the anterior pituitary, posterior pituitary and pineal glands. In the peripheral tissues, the amounts of d-Pro were high in the pancreas and kidney. Above all, it is surprising that the ddY/DAO⁻ mice excreted large amounts of d-Pro in their urine (433 nmol/mL, 20 times that of l-Pro). The origin of d-Pro has also been investigated. By comparing germ-free mice and gnotobiotic mice, intestinal bacteria were shown to have no effect on the urinary d-Pro amount. Concerning the dietary origin, a notable amount of d-Pro was still excreted in the urine after starvation for 4 days, suggesting that some of the d-Pro is produced in the mice. Age-dependent changes in the urinary d-Pro amount have also been investigated from the postnatal 1st month up to 12 months, and ddY/DAO⁻ mice were found to excrete large amounts of d-Pro in the urine constantly throughout their lives. Kenji Hamase is Associate Professor in the Department of Bioanalytical Chemistry, Graduate School of Pharmaceutical Sciences at Kyushu University. His current research interests focus on the development of analytical methods for d-amino acids and the study of their physiological functions and diagnostic values. He received the Japanese Society for Analytical Chemistry Award for Young Scientists in 2003, and the PSJ Award for Young Scientists in 2006.

Keywords: Column-switching; HPLC; Fluorescence derivatization; Chiral separation; d-Proline; d-Amino acid oxidase

Simultaneous determination of urinary hydroxylated metabolites of naphthalene, fluorene, phenanthrene, fluoranthene and pyrene as multiple biomarkers of exposure to polycyclic aromatic hydrocarbons by Thaneeya Chetiyanukornkul; Akira Toriba; Takayuki Kameda; Ning Tang; Kazuichi Hayakawa (pp. 712-718).

is a lecturer and deputy director at the Chiang Mai University in Thailand. She received a 2003 IUPAC scholarship for young scientists and a Monbukagakucho Ph.D. candidate scholarship (2000–2004) at the Kanazawa University, Japan. Her current research interests include the development of analytical methods for the determination of polycyclic aromatic hydrocarbons (PAHs) in ecological samples and their metabolites in body fluids as biomarkers, for the estimation of carcinogenetic risk of and biomonitoring of human exposure to PAHs. A method is presented for determining monohydroxy polycyclic aromatic hydrocarbons (OHPAHs) having 2-, 3- and 4-rings in human urine by using high-performance liquid chromatography with fluorescence detection. A urine sample containing conjugates of OHPAHs was hydrolysed in the presence of β-glucuronidase/aryl sulfatase and the solution was cleaned up with a solid-phase extraction (C₁₈ and silica). Eight OHPAHs, namely 1- and 2-hydroxynaphthalenes, 2-hydroxyfluorene, 2-, 3- and 4-hydroxyphenanthrenes, 3-hydroxyfluoranthene and 1-hydroxypyrene, were separated and 1- and 9-hydroxyphenanthrenes co-eluted on an alkylamide-type reversed-phase column with fluorimetric detection. The urinary concentrations of OHPAHs were quantified by using deuterated 1-hydoxypyrene as an internal standard. The method showed good repeatability for inter- and intra-day precisions as well as good linearity of calibration curves (r ² ranged from 0.996 to 0.999). The limits of detection (S/N=3) were in the range from 2.3 fmol to 2.2 pmol per injection. This method was successfully applied to urine samples from non-smoking taxi drivers, traffic policemen and rural villagers of Chiang Mai, Thailand. The results showed higher urinary concentrations of OHPAHs in rural villagers, consistent with higher respiratory exposure to PAHs.

Keywords: Hydroxy polycyclic aromatic hydrocarbons; Urine; HPLC; Fluorescence detection

Determination of haloperidol and reduced haloperidol in human serum by liquid chromatography after fluorescence labeling based on the Suzuki coupling reaction by Naoya Kishikawa; Chiyuki Hamachi; Yoshihiro Imamura; Yoshihito Ohba; Kenichiro Nakashima; Yashuhiro Tagawa; Naotaka Kuroda (pp. 719-724).

is an Assistant Professor at the Graduate School of Biomedical Sciences, Nagasaki University, Japan. His research interests include development of sensitive and selective analytical methods based on luminescence techniques and their application to biomedical and environmental analyses. A simultaneous method for the determination of haloperidol (HP) and its metabolite, reduced haloperidol (RHP), in human serum was developed by means of high-performance liquid chromatography (HPLC) with fluorescence detection. Suzuki coupling reaction with a fluorescent arylboronic acid, 4-(4,5-diphenyl-1H-imidazol-2-yl)phenylboronic acid (DPA), was employed to convert HP and RHP into highly fluorescent compounds. HP and RHP were extracted from human serum by liquid–liquid extraction with a mixture of n-hexane and isoamyl alcohol (99:1, v/v) and subsequently labeled by reaction with DPA. Separation of DPA derivatives of HP and RHP was performed on a silica column with a mixture of acetonitrile and H₂O (90:10, v/v) containing triethylamine and acetic acid as a mobile phase. The proposed method allowed sensitive detection of HP and RHP in human serum with a detection limit (at a signal to noise ratio of 3) of 0.22 and 0.20 ng/mL, respectively. The applicability of the method for therapeutic drug monitoring (TDM) was demonstrated by analyzing human serum samples from schizophrenic patients receiving HP.

Keywords: Fluorescence derivatization; Suzuki coupling reaction; Fluorescent arylboronic acid; Haloperidol; Reduced haloperidol; Therapeutic drug monitoring

Simultaneous derivatization/preconcentration of volatile aldehydes with a miniaturized fiber-packed sample preparation device designed for gas chromatographic analysis by Yoshihiro Saito; Ikuo Ueta; Mitsuhiro Ogawa; Kiyokatsu Jinno (pp. 725-732).

A novel in-needle sample preparation device has been developed for the determination of volatile aldehydes in gaseous samples. The needle device is designed for the gas chromatographic (GC) analysis of aldehydes and ketones commonly found in typical in-house environments. In order to prepare the extraction device, a bundle of polymer-coated filaments was longitudinally packed into a specially designed needle. Derivatization reactions were prompted by 2,4-dinitrophenylhydrazine (NDPH) included in the needle, and so the aldehydes and ketones were derivatized to the corresponding hydrazones and extracted with the extraction needle. A reproducible extraction needle preparation process was established, along with a repeatable derivatization/extraction process that ensures the successful determination of aldehydes. The storage performance of the extraction needle was also evaluated at room temperature for three days. The results demonstrate the successful application of the fiber-packed extraction device to the preparation of a gaseous sample of aldehydes, and the future possibility of applying the extraction device to the analysis of in-house environments.

Keywords: Derivatization; Extraction; Fiber; GC; Air/gases; Aldehydes

Capillary-assembled microchip as an on-line deproteinization device for capillary electrophoresis by Hideaki Hisamoto; Seigi Takeda; Shigeru Terabe (pp. 733-738).

A capillary-assembled microchip (CAs-CHIP), prepared by simply embedding square capillaries in a lattice polydimethylsiloxane (PDMS) channel plate with the same channel dimensions as the outer dimensions of the square capillaries, has been used as a diffusion-based pretreatment attachment in capillary electrophoresis (CE). Because the CAs-CHIPs employ square-section channels, diffusion-based separation of small molecules from sample solutions containing proteins is possible by using the multilayer flow formed in the square section channel. When a solution containing high-molecular-weight and low-molecular-weight species makes contact with a buffer solution, the low-molecular-weight species, which have larger diffusion coefficients than the high-molecular-weight species, can be collected in a buffer-solution phase. The collected solution containing the low-molecular-weight species is introduced into the separation capillary to be analyzed by CE. This type of system can be used for CE analysis in which pretreatment is required to remove proteins. In this work a fluorescently labeled protein and rhodamine-based molecules were chosen as model species and a feasibility study was performed.

Keywords: Capillary-assembled microchip; Capillary electrophoresis; Deproteinization; Polydimethylsiloxane; Square capillary

Highly selective micro-sequential injection lab-on-valve (μSI-LOV) method for the determination of ultra-trace concentrations of nickel in saline matrices using detection by electrothermal atomic absorption spectrometry by Xiangbao Long; Manuel Miró; Rikard Jensen; Elo Harald Hansen (pp. 739-748).

A highly selective procedure is proposed for the determination of ultra-trace level concentrations of nickel in saline aqueous matrices exploiting a micro-sequential injection Lab-On-Valve (μSI-LOV) sample pretreatment protocol comprising bead injection separation/pre-concentration and detection by electrothermal atomic absorption spectrometry (ETAAS). Based on the dimethylglyoxime (DMG) reaction used for nickel analysis, the sample, as contained in a pH 9.0 buffer, is, after on-line merging with the chelating reagent, transported to a reaction coil attached to one of the external ports of the LOV to assure sufficient reaction time for the formation of Ni(DMG)₂ chelate. The non-ionic coordination compound is then collected in a renewable micro-column packed with a reversed-phase copolymeric sorbent [namely, poly(divinylbenzene-co-N-vinylpyrrolidone)] containing a balanced ratio of hydrophilic and lipophilic monomers. Following elution by a 50-μL methanol plug in an air-segmented modality, the nickel is finally quantified by ETAAS. Under the optimized conditions and for a sample volume of 1.8 mL, a retention efficiency of 70 % and an enrichment factor of 25 were obtained. The proposed methodology showed a high tolerance to the commonly encountered alkaline earth matrix elements in environmental waters, that is, calcium and magnesium, and was successfully applied for the determination of nickel in an NIST standard reference material (NIST 1640-Trace elements in natural water), household tap water of high hardness and local seawater. Satisfying recoveries were achieved for all spiked environmental water samples with maximum deviations of 6 %. The experimental results for the standard reference material were not statistically different to the certified value at a significance level of 0.05.

Keywords: Brines; Nickel; Lipophilic/hydrophilic beads; Micro-sequential injection lab-on-valve; Preconcentration

A thermodynamic study on the complexation between riboflavin and a diaminotriazine derivative mediated by triple hydrogen bonds at water/oil interfaces by Shoji Ishizaka; Yoshiaki Nishijima; Noboru Kitamura (pp. 749-758).

The changes in Gibbs free energy (ΔG _int), enthalpy (ΔH _int) and entropy (TΔS _int) upon complexation between riboflavin (RF) and N,N-dioctadecyl-[1,3,5]triazine-2,4,6-triamine (DTT), mediated by triple hydrogen bonds at water/carbon tetrachloride, trichloroethylene and chloroform interfaces, were determined via temperature-controlled interfacial tension measurements. It was shown that hydrogen bonding interactions between RF and DTT were best characterized by large and negative ΔH _int values, unlike those predicted from either the polarity in each phase or the arithmetic average of the polarities in the two phases. Furthermore, the ΔH _int values became more positive as the dielectric constant of the oil phase was increased. These results strongly indicate that ΔH _int is governed by the dielectric properties of the oil phase. Adsorption of RF, DTT and the RF-DTT complex at the water/oil interface gave rise to restrictions on the translational and rotational motions of these species, as demonstrated by the ΔS _int values observed, which is another characteristic of interfacial complexation. The thermodynamic parameters evaluated in the present study revealed the characteristic complexation behavior that occurs at a water/oil interface, as mediated by hydrogen bonding.

Keywords: Thermodynamics; Hydrogen bond; Riboflavin; Liquid–liquid interface

Study of water properties in nanospace by Noritada Kaji; Ryo Ogawa; Akio Oki; Yasuhiro Horiike; Manabu Tokeshi; Yoshinobu Baba (pp. 759-764).

Here we report an anomalous behavior of water, especially its viscosity and hydrodynamic flow, in a nanometer-confined space. As a typical model of a nanometer-confined space, the nanopillar chip, which was developed for DNA size-based separation was used, and single-particle tracking (SPT) technique was applied to investigate water viscosity and hydrodynamic flow in the nanopillar chip. The diffusion coefficients of nanospheres were almost one-third of the theoretical value derived from the Stokes-Einstein equation. This result gave indirect proof that water viscosity in a nanometer-confined space is higher than in a bulk solution. In order to improve resolution and throughput of the nanopillar chip for DNA separation, these potential factors affecting performance should be seriously considered.

Keywords: Diffusion coefficient; Nanospace; Nano-particles; Single-particle tracking; Nanopillar chip

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Analytical and Bioanalytical Chemistry (v.386, #3)