We improved the detection sensitivity of neutral and amino monosaccharides labeled with 2-aminoacridone by a factor of 19.6 − 48.7 and 44.4 − 65.9, respectively, with no deterioration in separation resolution, using a pH discontinuous solution system instead of the conventional solution system. The pH discontinuous solution system is simple, using only a borate solution at pH 6.0 as a sample solution and a Tris-borate solution at pH 9.3 as a separation solution, enabling an enhancement of the sensitivity by monosaccharide stacking. This technique is fully compatible with conventional microchip electrophoresis, thereby allowing us to develop efficient tools for highly sensitive and resolved detection of monosaccharides.

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We have developed a capillary chromatography system using an open capillary tube made of fused-silica, polyethylene, or poly(tetrafluoroethylene), and a water–hydrophilic–hydrophobic organic mixture carrier solution. This tube radial distribution chromatography (TRDC) system works under laminar-flow conditions. In this study, the following analytical conditions in the TRDC system using a fused-silica capillary tube and a water–acetonitrile–ethyl acetate mixture carrier solution were for the first time examined: tube temperature, 5 − 25°C; tube inner diameter, 50 − 250 μm; tube effective length, 100 − 200 cm; and flow rate, 0.2 − 1.5 μL min⁻¹. For example, the effects of temperature on the separation performance in the TRDC system were observed with an organic solvent-rich carrier solution; 1-naphthol and 2,6-naphthalenedisulfonic acid in a model mixture were eluted with baseline separation over the temperature range of 5 − 23°C. The resolution, theoretical plate number, and height equivalent to the theoretical plate were calculated from the experimental data obtained by examining the effects of the tube length. A mixture of 1-naphthol, Eosin Y, 1-naphthalenesulfonic acid, 2,6-naphthalenedisulfonic acid, and 1,3,6-naphthalenetrisulfonic acid was subjected to the present TRDC system, and the analytes in the mixture solution were eluted in this order with the organic solvent-rich carrier solution, providing good separation performance on the chromatogram.

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Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of a model peptide, Substance P (SubP), was carried out using 2,4,6-trihydroxyacetophenone (THAP) and 2,4-dihydroxyacetophenone (DHAP) with cyclodextrins (cyclodextrin-supported matrix). It was found that the use of a cyclodextrin-supported matrix simplified the mass spectrum in the low-molecular-weight region. The interaction between THAP/DHAP and cyclodextrin (CD) was studied by UV-vis absorption spectroscopy and the incorporation of matrix molecules into the cyclodextrin cavity was confirmed by ¹H-NMR spectroscopy. DHAP showed tight incorporation with βCD (βCD(DHAP)) rather than THAP and it was found that the matrix-related peaks could be weakened by less than one third of the peak intensity of a protonated analyte. The βCD(DHAP) matrix was applied to the measurements of two low-molecular-weight compounds; adenosine and adrenaline. It became clear that the cyclic structure of the CD and the host-guest interaction between βCD and the matrix molecule were important to reduce the matrix-related peaks of THAP and DHAP.

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An ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-ESI-MS/MS) method was developed to investigate 20(S)-protopanaxadiol (PPD) pharmacokinetics in rats. Rat plasma samples were treated using a solid-phase extraction with satisfactory recovery (> 81%). The method showed an excellent sensitivity that the limit of detection (LOD) and the lower limit of quantitation (LLOQ) of PPD were 0.5 and 2 ng/mL, respectively. The method was applied to the evaluation of pharmacokinetics from two types of PPD formulations. The PPD emulsion showed more rapid and efficient drug absorption, and higher and more persistent plateau concentration of PPD in plasma than PPD oil solution. PPD emulsion was demonstrated to be a promising dosage form. In spite of lower plateau plasma drug concentration, PPD oil solution was characterized by the easiness in preparation and the persistent, durative plateau plasma concentration of PPD, there is room to further improve its bioavailability.

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The ultra high performance liquid chromatography (UHPLC)-UV method for the simultaneous determination of furosemide, saluamine (furosemide metabolite), spironolactone, carnenone (spironolactone active metabolite), terbinafine, N-desmethylcarboxy terbinafine (terbinafine metabolite) and vancomycin in human plasma and urine is proposed. Good separation of the analytes was achieved with the gradient RP-UHPLC-UV with the mobile phase composed as acetonitrile and 0.1% formic acid. The determined substances were eluted from a Hypersil GOLD C₁₈e (50 mm × 2.1 mm, 1.7 μm particles) column in 3.3 min. Good linear relationships were observed for all of the analytes (R² higher than 0.994). The limit of detection (LOD) values varied from 0.01 to 0.07 μg ml⁻¹, with vancomycin as an exception (0.11 μg ml⁻¹). After protein precipitation and solid-phase extraction, samples of plasma and urine were analyzed. Thanks to the short analysis time and small quantities of urine or plasma needed, this method can be applied to routine clinical analysis.

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We report on a comparison of silver colloid preparation methods for the in situ analysis of microorganisms based on surface-enhanced Raman scattering (SERS). Different colloid sols were tested and optimized regarding their suitability as SERS substrates. The silver sols produced by a modified procedure of Leopold and Lendl gave an enhancement factor of the Raman signal in the order 10⁸ for the test molecule, crystal violet. Furthermore, this SERS substrate was successfully applied for the in situ detection and identification of microorganisms immobilized on an immunoassay. The colloid preparation was carried out at room temperature and the colloids were stable for weeks. This silver nanoparticle preparation method for the label-free in situ detection of microorganisms successfully combines SERS with immunoassays. Hence, it has a great potential for the high-throughput detection of biomolecules.

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Liposomes prepared with biotinylated phospholipids and luminol entrapped were shown to be of 187 nm in size, 59% of which were unilamellar and with 43% luminol trapping efficiency. Liposome prepared from biotinylated phospholipids with a longer hydrophilic PEG2000 spacer, but not with the shorter hydrophobic caproyl one, bound efficiently and specifically with immobilized streptavidin in a microplate assay. The interactions of dinitrophenol and tobramycin with their respective antibodies, and the hybridization of 20-mers oligonucleotides were studied using the liposome as a signal generator. These reactions were shown to be specific with limits of detection of 0.58 μM, 0.96 μM and 18 nM, respectively.

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An indirect competitive chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA) for the determination of benzene, toluene, ethylbenzene, and ortho-, meta-, para-xylenes (BTEX) in soil and water was developed. The assay was optimized concerning the coating conjugate concentration, anti-BTEX antiserum dilution, incubation time effect on primary and secondary antibody incubation, and temperature effect on the competitive step and tolerance to different organic solvents. The IC₅₀ and lower limit of the detection (LDD₉₀) values were 4.6 and 0.5 μg mL⁻¹, respectively. While water samples could be analyzed directly, soil has to be extracted and diluted prior to immunochemical measurements. BTEX could be recovered from spiked soil with a yield higher than 60% using 5-min ultrasonication with methanol. Finally, the assay was applied to soil and water samples collected at a contaminated site in Italy, and was compared to GC-MS.

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In order to determine ammonium ion in water samples, we propose a method based on the Berthelot reaction of ammonia with 1-naphthol and dichloroisocyanurate to form an indophenol blue derivative and collection of the blue compound as an ion pair using Zephiramine on a pure polytetrafluoroethylene (PTFE)-type membrane filter. The ion pair on the filter was eluted with 5.0 mL of acetonitrile, and the absorbance of the eluate was measured at 725 nm. The detection limit of the method was 2.5 μg L⁻¹ of ammonium ion. We showed that the interference of foreign ions was removed by the addition of EDTA. We also demonstrated the success of the method in determining ammonia in river water and seawater.

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The measurement of total bilirubin in serum is one of the most frequently performed tests in clinical analysis. A new spectrofluorometric method was developed for the determination of trace amounts of total bilirubin. Using tetracycline-Eu³⁺ as a fluorescent probe, bilirubin can remarkably reduce the fluorescence intensity of the tetracycline-Eu³⁺ complex, and the reduced fluorescence intensity of Eu³⁺ ion is in proportion to the concentration of bilirubin. The optimum conditions for the determination of bilirubin were also investigated. This method is simple, practical, and relatively free of interference from coexisting substances. It can be successfully applied to assess total bilirubin in serum samples. We also compared the developed method with a modified Jendrassik-Grof method in clinical analysis.

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Analytical conditions of amplitude-modulated multiplexed flow analysis, the basic concept of which was recently proposed by our group, are investigated for higher sample throughput rate. The performance of the improved system is evaluated by applying it to the determination of chloride ions. The flow rates of two sample solutions are independently varied in accordance with sinusoidal voltage signals, each having different frequency. The solutions are merged with a reagent solution and/or a diluent, while the total flow rate is held constant. Downstream, the analytical signal V_d is monitored with a spectrophotometer. The V_d shows a complicated profile resulting from amplitude modulated and multiplexed information on the two samples. The V_d can, however, be deconvoluted to the contribution of each sample through fast Fourier transform (FFT). The amplitudes of the separated wave components are closely related to the concentrations of the analytes in the samples. By moving the window for FFT analysis with time, a temporal profile of the amplitudes can be obtained in real-time. Analytical conditions such as modulation period and system configuration have been optimized using aqueous solutions of Malachite Green (MG). Adequate amplitudes are obtained at the period of as low as 5 s. At this period, the calibration curve for the MG concentration of 0 − 30 μmol dm⁻³ has enough linearity (r² = 0.999) and the limit of detection (3.3σ) is 1.3 μmol dm⁻³; the relative standard deviation of repeated measurements (C_MG = 15 μmol dm⁻³, n = 10) is 2.4%. The developed system has been applied to the determination of chloride ions by a mercury(II) thiocyanate method. The system can adequately follow the changes in analyte concentration. The recoveries of chloride ion spiked in real water samples (river and tap water) are satisfactory, around 100%.

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An amplitude-modulated flow analysis coupled with lock-in detection is proposed for the determination of trace phosphate. The flow rate F_S of sample solution is varied in response to a periodic signal V_c of alternating waveform. A coloring reagent solution (ammonium molybdate + Malachite Green) is delivered at a constant flow rate F_R. Under the constant total flow rate F_T, both solutions are merged with a diluent (water). Phosphate ion in the sample reacts with molybdate and then with Malachite Green to form green ion pairs in acidic media. Downstream, the absorbance of the mixed solution is measured at 625 nm. The output voltage V_d from the detector is sent to a lock-in amplifier, where the wave component of V_d that has the same frequency as that of V_c is distinguished from background signals. Phosphate ion can be determined from the amplitude of the component thus extracted. The calibration curve is linear (r² > 0.998) and the limit of detection (3.3σ) is 0.17 μmol dm⁻³. Compared with a conventional flow-based method with no modulation, the present method is less susceptible to the baseline drift due mainly to the adsorption of the ion-pair on the optical window, because the quantification is based not on the absorbance itself but on the amplitude of the absorbance. Good recoveries around 100% are obtained for the phosphate ions spiked into real water samples.

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A simple and sensitive method is proposed for the electrochemical determination of enrofloxacin (ENRO) and its primary metabolite ciprofloxacin (CIPRO) at a multiwall carbon nanotubes/glassy carbon electrode (MWCNT/GCE) using a least-squares support vector machine (LS-SVM) and linear sweep voltammetry. Simultaneous determination of ENRO and CIPRO at bare glassy carbon is associated with certain difficulties due to voltammogram overlapping and their low sensitivity. The resolution of the mixture was carried out using LS-SVM as a multivariate calibration method. Under the optimum conditions at pH 7.0, the linear sweep currents increased linearly with ENRO and CIPRO concentrations in ranges of 2.0 − 780.0 μmol L⁻¹ (0.7 − 280.3 μg mL⁻¹) and 3.0 − 1200 μmol L⁻¹ (1.0 − 397.7 μg mL⁻¹), respectively. The detection limits for ENRO and CIPRO were 0.5 and 0.9 μmol L⁻¹, respectively. The proposed method was applied to simultaneously determine both compounds in human urine, plasma and in pharmaceutical samples.

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In order to discuss the matrix modifier effect of palladium, iron, and a mixture of palladium and iron for the determination of cadmium in graphite-furnace atomic absorption spectrometry (GF-AAS), we measured the absorption profiles of a cadmium line at various compositions of these elements. Variations in the gas temperature were also estimated with the progress of atomization, by using a two-line method under the assumption of a Boltzmann distribution. The atomic absorption of cadmium appeared on the way of heating from the charring temperature to the atomizing temperature while the gas temperature was still low; it was thus considered that cadmium was atomized through direct conductive heating from the wall of the graphite furnace. Therefore, the effectiveness of modifiers for cadmium would be determined through any reactions on the furnace wall at the programmed charring and atomizing temperatures. The addition of iron, palladium, and an iron-palladium mixture all enhanced the absorption signal of cadmium compared to a pure cadmium sample; however, their effects were different from one another. Each addition of iron or palladium to the sample solution led to an enhancement of the cadmium absorbance, indicating that iron or palladium individually became an effective matrix modifier for the determination of cadmium. However, the addition of palladium was ineffective for the matrix modification in the coexistence of large amounts of iron. Although these phenomena are very complicated, and thus cannot be understood completely, any metallurgical reaction between the constituent elements during heating of the furnace wall, such as the formation of solid solutions and intermetallic compounds, would cause the effect of a matrix modifier in GF-AAS.

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We have reported on a novel microfluidic mixer based on AC electroosmosis. To elucidate the mixer characteristics, we performed detailed measurements of mixing under various experimental conditions including applied voltage, frequency and solution viscosity. The results are discussed through comparison with results obtained from a theoretical model of AC electroosmosis. As predicted from the theoretical model, we found that a larger voltage (∼20 V_p-p) led to more rapid mixing, while the dependence of the mixing on frequency (1 − 5 kHz) was insignificant under the present experimental conditions. Furthermore, the dependence of the mixing on viscosity was successfully explained by the theoretical model, and the applicability of the mixer in viscous solution (2.83 mPa s) was confirmed experimentally. By using these results, it is possible to estimate the mixing performance under given conditions. These estimations can provide guidelines for using the mixer in microfluidic chemical analysis.

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We applied imaging mass spectrometry (IMS) to determine the spatial distribution of γ-aminobutyric acid (GABA). We found that GABA had a specific localization in seeds. We also visualized various biomolecules as well as GABA with higher spatial resolution than in the previous report. Our work suggests that IMS might be a powerful tool for exploring functional food factors, investigating the specific distribution of nutrients in unused natural resources, and evaluating the quality of functional foods.

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A novel method for the evaluating the transpiration rate (TR) has been proposed. Miniature temperature/humidity loggers were attached onto the leaf surface of a mangrove plant, Bruguiera gymnorrhiza, via a spacer. TR values were calculated using the mass-balance equation; the results showed good agreement with those measured using a conventional porometer when the plant root was surrounded by water. In a saline environment, on the other hand, the correlation became poor. The method was shown to require not only minimal invasion, but also a very short time for attaching leaves.

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