Recent progress of the XANES (X-ray absorption near-edge structure) spectral analysis methods for the structure characterization of metal complexes (MC) in solution is reviewed. Typical examples showing the scheme of the apparatus for XAFS (X-ray absorption fine structure) data collection, preparation and handling of solution samples, data treatment and analysis for XANES spectra, and future of XANES spectroscopy are presented. Detailed examples for the apparatus developed for laboratory scale spectrometers for XANES together with a combined spectral analyses using multi scattering method (MS) and molecular orbital (MO) calculation are discussed.

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An oxalate reagent-hydrogen peroxide-fluorescent compound chemiluminescence reaction, i.e., peroxyoxalate chemiluminescence, was introduced into micro-channel chemiluminescence analysis (MCCLA) to establish the concept of MCCLA through the direct observation of fluorescence and chemiluminescence using a fluorescence microscope-CCD camera and a microscope-CCD camera. It was confirmed from visual data that chemiluminescence in the MCCLA generated through the liquid-liquid interface collapsed during the course of molecular diffusion in the micro-channel. In addition, the visual data of chemiluminescence were transformed to line drawings on a computer to obtain chemiluminescence profiles. The chemiluminescence profiles were examined in detail at various flow rates and detection points; the relationship between the residence times and the chemiluminescence peak heights, or areas in the profiles, was easily represented as one smoothly changing reaction curve. Furthermore, the fluorescent compounds were detected with high sensitivity and good reproducibility in MCCLA by turning the syringe pumps on and off to produce determinable chemiluminescence signals; a photomultiplier tube was used as a detector. The chemiluminescence intensities in the signals of erythrosine, rhodamine B, Rose Bengal, fluorescein isothiocyanate, and eosin Y were examined; their intensities increased in this order, and eosin Y responded over the range of 1 × 10^-9 - 1 × 10^-6 M with a detection limit of 1 × 10^-9 M (S/N = 3). Introducing of the peroxyoxalate chemiluminescence reaction into MCCLA can extend the analysis system to the analysis of various types of sample and applications incorporating fluorescence labeling techniques.

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Biphasic electrospray ionization (BESI) mass spectrometry is achieved by using a dual-channel microsprayer, where channels filled with different immiscible phases meet at the Taylor cone. Two types of interfacial complexation reactions have been studied: the interfacial complexation of aqueous lead ions by thioether crown molecules, and the interfacial complexation of an aqueous dipeptide by dibenzo-18-crown-6 as ionophore. The mass spectra also give valuable information on the stoichiometry of the complexation reaction.

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Genetically encoded bioluminescent indicators for intracellular Ca²⁺ are described here with CaM-M13 interaction-induced complementation of split Renilla luciferase. The Ca²⁺-induced interaction between CaM and M13 leads to complementation of the N- and C-terminal halves of split Renilla luciferase in living cells. This intramolecular interaction results in the spontaneous and simultaneous emission of bioluminescence split Renilla luciferase. This is how intracellular Ca²⁺ is illuminated with the intramolecular complementation of split Renilla luciferase. The Ca²⁺-dependent spontaneous and simultaneous emission of bioluminescence promises to reveal Ca²⁺ dynamics in living cells, and also in vivo using the present indicators.

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The construction and electrochemical response characteristics of poly(vinyl chloride) matrix ion-selective electrodes (ISEs) for dextromethorphan (DXM) hydrobromide are described. The membranes incorporate ion-association complexes of DXM with reineckate salt {[Cr(NH₃)₂(SCN)₄]NH₄} or phosphomolybdic acid [H₃(PMo₁₂O₄₀)], as electroactive materials and dioctylphthalate or dibutylsebacate as a plasticizing solvent mediator. The sensors display a fast, stable and linear response with slopes of 54.4 to 59.5 mV/decade at pH 2.5 - 6.5 and a detection limit of 1.0 × 10^-6 M. Moreover, the sensors exhibit very good selectivity for DXM over opiate alkaloids, as well as organic and inorganic cations. The sensors proved to be useful for the determination of 5.0 × 10^-5 - 1.0 × 10^-3 M DXM hydrobromide in pure as well as in dosage forms by direct potentiometry and standard addition methods. Determination of 5.0 × 10^-4 M DXM using the standard addition method and a sensor based on phosphomolybdate and dioctylphthalate shows an average recovery of 99.8% and a relative standard deviation (RSD) of 0.4%.

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Cyclic voltammograms of the aqueous solution containing polyammonium cations and the [Fe(CN)₆]^4-. anion at a plastic-formed carbon or platinum electrode were recorded. Polyammonium salts comprising quaternary ammonium in the main chain or sub chain and those comprising primary ammonium were tested. Under certain conditions, well-developed anodic and cathodic peak currents with the midpoint potentials different from that when the polyammonium cation is absent, that is, the midpoint potential of [Fe(CN)₆]^3- + e^- = [Fe(CN)₆]^4- were exhibited, indicating that the [Fe(CN)₆]^4-/3- anionic species associate with the polyammonium cations tested to form the polycation-[Fe(CN)₆]^4-/3- complex species. The polycation complex species can be easily localized around the electrode surface with a dialysis membrane. An application of the polycation complex species as an electron-transfer mediator in the catalytic electrode with a redox enzyme was examined.

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Bilirubin oxidase was stored in the solutions containing polyammonium salts for a given time at 30°C, and the activity was assayed. The enzyme catalyzes the reaction: 4[Fe(CN)₆]^4- + 4H⁺ + O₂ → 4[Fe(CN)₆]^3- + 2H₂O, and the activity can be measured by the absorbance at the wavelength for the absorption maxima of [Fe(CN)₆]^3-. The results show that polyammonium cations comprising quaternary ammonium in the main chain and hydrophilic groups, such as hydroxyl and amide groups, stabilize the enzyme in solution. These polyammonium cations may act like a protective colloid. The membrane-covered electrode containing the polyammonium cations, the enzyme, and [Fe(CN)₆]^4-/3- in the internal solution phase was constructed. The electrode gave a well-defined current-potential curve with a steady state limiting current due to the polycation-[Fe(CN)₆]^4-/3- complex-mediated bioelectrocatalytic current for the reduction of O₂. The time-dependent decrease of the limiting current indicates again the stabilizing effect of the polyammonium cations on the enzyme.

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An improved amperometric glucose biosensor was constructed by immobilizing glucose oxidase (GOD) in a titania sol-gel film, which was prepared by a vapor deposition method, on a Prussian Blue (PB)-modified electrode. The method combined the merits of immobilizing biomolecules in the titania sol-gel film by vapor deposition method and the synergic catalysis effects of PB and GOD molecules. Results showed that the fabricated titania sol-gel/PB membrane possessed high surface area, good mechanical stability, and good hydrophilicity, which provided a biocompatible microenvironment for maintaining the bioactivity of the immobilized enzyme and prevented the enzyme from leaking out of the film. Therefore, the present biosensor exhibited fast response time (10 s), high sensitivity (12.74 μA cm^-2 mM^-1), long-term operational stability, good suppression of interference, and a wide linear range from 0.02 to 15 mM with a low detection limit of 5 μM for the detection of glucose. In addition, this simple and controllable method could fabricate biosensors in batches with a very small amount of enzyme.

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The present work describes the dectection of cytochrome c (cyt c) at biocompatible aurum (Au) nanoparticle-structured supported bilayer lipid membrane (sBLM) modified with anionic sites. Au nanoparticles were directly deposited through sBLM modified with lauric acid (LA) to build a hybrid device of nanoscale electrode array via potential cycling in 10 mM HAuCl₄ solution containing 0.1 M KCl. The properties of Au nanoparticle-doped sBLM composite were then characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). Results indicated that Au nanoparticles grew in voids of the sBLM with size around 20 - 30 nm. With SWV, after optimization, the results of the experiments indicate that the currents of cyt c were linear functions of its concentrations over the range from 1.0 × 10^-7 to 3.2 × 10^-6 M and the limit of detection (LOD, S/N = 3) was 5 × 10^-8 M. The influences of several common base pairs, amino acids and metal ions on determination of cyt c via this Au nanoparticle-doped sBLM composite were relatively low in experiments, suggesting the excellent biocompatibility of this detection method.

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The interaction of protonated ferrocene (PF) with chicken blood DNA (CB-DNA) has been investigated in vitro by cyclic voltammetry (CV) and UV-Vis spectroscopy as well as viscosity measurements under stomach pH and body temperature. The peak potentials shift in CV, hyperchromism in UV absorption titration, an increase in the viscosity of DNA and the results of the effect of ionic strength on the binding constant strongly support the intercalation of PF into the DNA double helix. The diffusion coefficients of PF in the presence and absence of DNA were 9.54 × 10^-11 and 1.34 × 10^-10 m²/s, respectively. The binding constant of the PF-DNA complex and the number of binding sites on a DNA molecule were calculated as being 3.07 × 10² M^-1 and 2.96, with the help of the Scatchard equation. An expression by Carter et al. was used for determining the binding site size (0.17 bp). The binding constant was also determined by UV absorption titration.

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In this contribution, the amounts of Ni (nickel) and Al (aluminum) in tilapias (Oreochromis niloticus) were determined using square wave voltammetry (SWV) with glassy carbon working microelectrode with a mercury thin film, platinum counter electrode, and Ag/AgCl reference electrode. Ni was studied through the formation of the dimethylglyoxime-Ni (Ni-DMG) complex, while Al was studied through the formation of the Alizarin R-Al complex. The detection limit found for Ni-DMG and Alizarin R-Al complexes were 1.70 × 10^-7 and 1.0 × 10^-8 mol L^-1, respectively. The voltammetric anodic curves for the Alizarin R-Al complex were recorded over the potential range from -0.8 to -0.05 V while the voltammetric cathodic curve for the Ni-DMG complex was recorded over the potential range from -0.7 to -1.2 V. These methods detected low concentrations of Ni and Al in biological samples efficiently.

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A square-wave voltammetric procedure for the electroanalytical determination of losartan and triamterene in Britton-Robinson buffer (pH 3.0, 0.1 mol L^-1) as a supporting electrolyte containing 30 ng mL^-1 of copper ions was developed. Opposite to the case of triamterene, losartan can not be reduced at a mercury electrode alone, but a new peak appears at -0.25 V in the presence of copper due to the formation of a complex between copper(II) and losartan. An accumulation potential of -0.30 V during 80 s for the prior adsorption of losartan-copper(II) and triamterene on the electrode surface was used. The response of the system was found to be linear in the range of 30.0 - 270.0 nmol L^-1 for losartan and two linear dynamic ranges containing 0.5 - 200.0 and 200.0 - 400.0 nmol L^-1 of triamterene. The limits of detections were 9.7 and 0.3 nmol L^-1 for losartan and triamterene, respectively. The relative standard deviations for five replicate analyses of 100.0 and 10.0 nmol L^-1 losartan and triamterene were 5.5%. Applicability to assay the drugs in urine and pharmaceutical formulations was illustrated with satisfactory results. The direct-current polarography of triamterene indicates that the reduction of a related drug is strongly dependent on the pH of the solution. A linear segment was found with slope value of -63.6 mV pH^-1 in the pH range of 2.0 - 6.0. The stoichiometry and complex formation constant (β) for losartan-Cu(II), number of transfer electrons (n), transfer coefficients (α) and number of proton transfers were also estimated.

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A simple and sensitive spectrophotometry for formaldehyde in water by membrane solubilization technique was proposed. Formaldehyde was converted into a blue cationic dye with 3-methyl-2-benzothiazolinone hydrazone, and the dye was retained on a membrane filter as an ion-associate with tetraphenylborate anion. The filter retaining the blue dye was dissolved in 2-methoxyethanol containing sulfuric acid, and the absorbance of the solution was measured at 670 nm against the reagent blank. The formaldehyde from 0.007 to 0.2 mg L^-1 was determined with an RSD of less than 5%, and the detection limit was 0.002 mg L^-1. The proposed method was very simple and rapid. Twenty minutes was sufficient for the entire analytical procedure. When the method was applied to rainwater, the analytical results were in good agreement with those obtained by GC/MS.

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Three kinds of phytoplankton were cultivated, and the contribution of dissolved organic matter (DOM) released from the phytoplankton was examined to clarify the cause of organic pollution of Lake Biwa. Microcystis aeruginosa, Staurastrum dorcidentiferum, and Cryptomonas ovata were evaluated with regard to cultivation. A significant peak (M_w: <3000 Da) was mainly detected in the algal DOM released from plankton during cultivation by gel chromatography with a fluorescence detector (E_x = 340 nm, E_m = 435 nm). Since this peak corresponds to a peak with lower molecular weight in three peaks detected in the surface water of Lake Biwa, it can be concluded that the algal DOM released from the plankton during cultivation makes a considerable contribution to the refractory organic matter in Lake Biwa. Three fluorescence maxima were observed in the cultivation of three kinds of phytoplankton: two fulvic-like fluorescence peaks (A and B) and a protein-like fluorescence peak (C). These peaks became larger as their cell counts of plankton increased. As for the fractionations of algal DOM using DAX-8, the ratio of hydrophilic DOM is fairly high in DOM produced by three kinds of phytoplankton. The order of the amount of algal DOM per cell volume during cultivation was Cryptomonas ovata > Microcystis aeruginosa > Staurastrum dorcidentiferum. These results suggest that the increase of the refractory organic matter in Lake Biwa may be attributed to a change of the predominant phytoplankton.

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A flow-injection biosensor system with an on-line microdialysis sampling system is proposed for the simultaneous detection of neurotransmitters (L-glutamate, acetylcholine and dopamine) released from rat brain cells. The dialysate collected in the sample loop from the microdialysis probe was automatically injected into the flow-injection line with a triple electrode arranged perpendicular to the flow direction. The triple electrode was constructed by hybridizing a poly(1,2-diaminobenzene) film to two enzyme sensing-parts which respond to L-glutamate and acetylcholine, and by coating a Nafion film on a remaining sensing part which responds to dopamine, respectively, without any cross-reactivity. The three sensing parts of the triple electrode responded linearly to the concentrations of L-glutamate and acetylcholine in the range of 0.002 - 5 mM and to that of dopamine in the range of 0.002 - 20 mM, respectively, without any interference from oxidizable species present in the dialysate. The proposed flow-injection analytical method could be applied to an in vivo assay of these neurotransmitters released from rat-brain cells by the continuous KCl stimulation.

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Reactive metabolic-modified proteins have been proposed to play an important role in the mechanism(s) of the hepatotoxicity and colon cancer of lithocholic acid (LCA). To identify cellular proteins chemically modified with LCA, we have generated a monoclonal antibody that recognizes the 3α-hydroxy-5β-steroid moiety of LCA. The spleen cells from a BALB/c mouse, which was immunized with an immunogen in which the side chain of LCA was coupled to bovine serum albumin (BSA) via a succinic acid spacer, was fused with SP2/0 myeloma cells to generate antibody-secreting hybridoma clones. The resulting monoclonal antibody (γ2b, κ) was specific to LCA-N^α-BOC-lysine as well as the amidated and nonamidated forms of LCA. The immunoblot enabled the detection of LCA residues anchored on BSA and lysozyme. The antibody will be useful for monitoring the generation, localization, and capture of proteins tagged with LCA, which may be the cause of LCA-induced toxicity.

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The diffusion phenomena, dispersion and mixing processes of the sample solute (Basic Blue 3 dye and KMnO₄ aqueous solutions) were directly observed in laminar flow in glass microchannels. Quasi steady-state UV-visible absorption spectrometry was carried out using CCD camera images of the colored sample dispersion and mixing processes, and the absorbance change (ΔAbs) was discussed based on the dimensionless parameter, τ which represents the flow time renormalized to the diffusion coefficient and the channel cross section. It was found that ΔAbs showed almost the same τ dependence, even though the solutions and the microchannel sizes differed in laminar flow, if the microchannel fabrication method was the same. On the basis of this fundamental result, the total microchannel length required for the reaction of 2,3-diaminonaphthalene (DAN) and NO₂^- at a flow rate of 2 µL min^-1 was calculated, and the obtained value (∼100 mm) showed very good agreement with our previous microchip research. It was concluded that both results were useful for designing the microchannel width, depth and length to control the chemical reaction time in recent microfluidic systems.

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Fe(III) and Fe(II) ions were separated and determined via in-column and post-column reactions with salicylic acid and 1,10-phenanthroline by microcolumn ion chromatography with UV-Vis detection. The separation could be achieved on a silica microcolumn using an aqueous ethanol solution of salicylic acid as eluent. The effluent from the column was then mixed with aqueous ethanol solution of 1,10-phenanthroline and detected at 518 nm. Fe(III) ion was detected as the complex with salicylic acid, whereas Fe(II) ion was detected as the complex with 1,10-phenanthroline. The detection limits with 0.25 µL injection volume at S/N = 3 were 0.21 and 0.10 mg L^-1 for Fe(III) and Fe(II), respectively. Increasing the injection volume of sample, e.g., 5.1 µL, improved the sensitivity; the detection limits at S/N = 3 were 12 and 9.2 µg L^-1 for Fe(III) and Fe(II), respectively. The present system was applied to the determination of iron in a local well water sample.

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Ethyl chloroformate was examined as a precolumn derivatizing reagent for the gas chromatographic (GC) determination of isoniazid (INH) and hydrazine (HZ). Phenylhydrazine (PHZ) was used as an internal standard. GC separation was carried out on an HP-5 column (30 m × 0.32 mm i.d.) with flame ionization detection. The elution was carried out at an initial column temperature of 150°C for 1 min at a heating rate of 10°C/min up to 250°C, nitrogen flow rate of 4 ml/min and a split ratio of 10:1, v/v. The linear calibration ranges for INH and HZ were observed between 3.5 - 37.5 and 3.5 - 35 µg/ml with corresponding detection limits of 0.18 and 0.17 ng reaching the detector. The method was subsequently applied to the determination of INH and HZ in pharmaceutical preparations, achieving a relative standard deviation (RSD) of 3.8 - 5.8%. The recovery percentage of INH from isoniazid syrup was 98% with an RSD of 5.2%.

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An effective technique for the suppression of matrix-related ions in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry have been developed. Using typical organic matrices such as THAP (2,4,6-trihydroxyacetophenone) and CHCA (α-cyano-4-hydroxycinnamic acid) in a cyclodextrin cavity, we successfully measured the mass peaks of only protonated matrix ions and significantly suppressed their intensities and fragmentation. In addition, it became possible to analyze the mass peak of the analyte molecules (substance P and adenosine) without any interference from the matrix. We believe that this technique could be a powerful tool for MALDI mass spectrometry, particularly for low-molecular-weight compounds.

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The biological processing of Ca produces significant stable isotope fractionation. The level of isotopic fractionation can provide key information about the variation in dietary consumption or Ca metabolism. To investigate this, we measured the ⁴³Ca/⁴²Ca and ⁴⁴Ca/⁴²Ca ratios for bone and blood plasma samples collected from mice of various ages using multiple collector-ICP-mass spectrometry (MC-ICP-MS). The ⁴⁴Ca/⁴²Ca ratio in bones was significantly (0.44 - 0.84‰) lower than the corresponding ratios in the diet, suggesting that Ca was isotopically fractionated during Ca metabolism for bone formation. The resulting ⁴⁴Ca/⁴²Ca ratios for blood plasma showed almost identical, or slightly higher, values (0.03 -0.2‰) than found in a corresponding diet. This indicates that a significant amount of Ca in the blood plasma was from dietary sources. Unlike that discovered for Fe, there were no significant differences in the measured ⁴⁴Ca/⁴²Ca ratios between female and male specimens (for either bone or blood plasma samples). Similarity, the ⁴⁴Ca/⁴²Ca ratios suggests that there were no significant differences in Ca dietary consumption or Ca metabolism between female and male specimens. In contrast, the ⁴⁴Ca/⁴²Ca ratios of blood plasma from mother mice during the lactation period were significantly higher than those for all other adult specimens. This suggests that Ca supplied to infants through lactation was isotopically lighter, and the preferential supply of isotopically lighter Ca resulted in isotopically heavier Ca in blood plasma of mother mice during the lactation period. The data obtained here clearly demonstrate that the Ca isotopic ratio has a potential to become a new tool for evaluating changes in dietary consumption, or Ca metabolism of animals.

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Determination of benzophenones (BPs) in human urine samples by stir bar sorptive extraction (SBSE) and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS) is described. As analytes, BP, its metabolites benzhydrol (BP-OH) and 2-hydroxybenzophenone (2OH-BP), and its derivatives 2-hydroxy-4-methoxybenzophenone (BP-3) and 2-hydroxy-4-methoxy-4′-methylbenzophenone (BP-10) were selected. After enzymatic hydrolysis, a polydimethylsiloxane (PDMS) stir bar was placed in a urine sample diluted 1:1 with water and stirred for 60 min at room temperature. The limit of quantification (LOQ) of BPs is 0.2 - 0.5 ng ml^-1 (ppb). The method showed linearity over the calibration range (0.2 - 10 or 0.5 - 10 ng ml^-1), and the correlation coefficients were equal to or higher than 0.993 for all of the analytes. The average recoveries of BPs were equal to or higher than 98.7% (RSD: 1.5 - 4.8%, n = 6).

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A tandem preconcentration method integrating chelating resin adsorption and La coprecipitation was applied to an analysis of Lake Baikal water by high-resolution ICP-MS. As a result, 56 elements were determined in Lake Baikal water: Ca, Na, Mg, K, Si, Sr, B, Li, Ba, P, Fe, Al, Mo, Zn, Mn, Rb, U, As, V, Cu, Pb, W, Y, Cs, Se, Cr, Ni, Ti, Sb, Zr, Sn, Co, Cd, Rh, Te, Hf, Nb, Ru, Ga, Sc, Th, Bi, and all-rare earth elements (all-REEs), except for Pm. The concentrations of these elements covered a range of nine orders of magnitude, from approximately 17 µg mL^-1 of Ca to less than 50 fg mL^-1 of Tm. The elemental concentrations in Lake Baikal water were compared with reference data.

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