BUNSEKI KAGAKU Abstracts

Vol. 54 No. 9

September, 2005

Research Papers

Ultraweak Chemiluminescence from Oxygen Species Adsorbed on the Surface of Saccharides

Takayuki Tamefusa¹, Jun-ichi Kimura¹, Rie Ito¹, Yoshihiro Yoshimura², Koichi Saito¹ and Hiroyuki Nakazawa¹

(Received 12 April 2005, Accepted 14 June 2005)

Cellulose is used widely as an additive of foods and drugs. Saccharides and cereal products have been reported to emit ultraweak chemiluminescence (CL), attributed to autoxidation and hydration. We investigated the relationship between the chemical structure and the chemiluminescent mechanism. The samples used were glucose, fructose, galactose, ribose, maltose, lactose, sucrose, cellobiose and cellulose. The temperature dependence of the CL intensity emitted from each saccharide was measured in both nitrogen and oxygen atmospheres. The activation energies for all of the saccharides varied from 40 kJ/mol to 80 kJ/mol. The photoluminescence of the saccharides with UV irradiation was also examined. All of the saccharides exhibited green luminescence, which could be attributed to the phosphorescence of adsorbed oxygen. The energies of adsorption of oxygen onto saccharides were examined by using MOPAC. It was found that oxygen was adsorbed by all of the saccharides. The enthalpy change of all the saccharides became negative in the process of oxygen adsorption. Therefore, it was suggested that the adsorbed oxygen on the surface of succharides participates in the ultraweak chemiluminescence of saccharides. The chemiluminescence from the active oxygen species adsorbed on succharides was demonstrated to be a useful technique for evaluating not only the stability of saccharides in relation to heating and UV irradiation, but also the change in the surface physical properties due to oxygen adsorption.

Keywords : chemiluminescence; saccharide; cellulose; singlet oxygen.

Luminol Chemiluminescence Study on Highly Sensitive Analysis of Glycated Protein in Human Hair

Tomonari Yajima¹, Kimie Ito¹, Wataru Kitamura¹, Rie Ito¹, Koichi Saito¹, Hiroaki Kubo² and Hiroyuki Nakazawa¹

(Received 15 April 2005, Accepted 16 June 2005)

Human hair might be useful for clinical diagnosis with non-invasive sampling, storage for a long time and including the past medical history. Therefore, it can be expected to serve as an alternative sample of blood or urine. In the present study, a highly sensitive, easy and simple method was developed for diabetes by screening glycated protein in hair with luminol chemiluminescence using a microtiterplate. The optimal chemiluminescence conditions were 600 mM sodium hydroxide, 0.005 mM luminol, 0.1 mM hexacyanoferrate(III) and 40 mM hexacyanoferrate(II), respectively. The developed analytical method was able to measure the glycated protein with 1 cm length of hair, and obtain the past advanced stage of blood glucose status and medical treatment progress. Actually, this index of diabetes patients gave significantly higher values than those of healthy subjects (P<0.003). These indices remarkably correlated with the levels of glycohemoglobin (HbA1c) in diabetes patients and healthy subjects (r=0.980). Based on this study, the screening of diabetic patients for early checkups by the present method might be applicable to the clinical base with a mitigation of the burden to subjects.

Keywords : luminol chemiluminescence; glycated protein; hair; diabetes.

Study of the Sampling Method of Blood Samples for Total-Reflection X-ray Fluorescence

Yoshiko Matsuoka¹, Yoshinori Hosokawa², Masayuki Hino³ and Kouichi Tsuji^1,4

(Received 14 March 2005, Accepted 21 June 2005)

We investigated the sampling method of blood samples for total-reflection x-ray fluorescence (TXRF) analysis. Three types of samples, such as whole blood, serum and blood plasma, were prepared. After these samples were diluted by Milli-Q water, they were allowed to fall in drops onto an acrylic flat substrate, which was used as a sample carrier for TXRF analysis. In the case of a whole blood sample, the detection limits of Fe and Cl were improved by adding Milli-Q water to the sample. We consider that the improvement of the detection limits resulted from destruction of the red blood cells. This suggests that whole blood diluted by Milli-Q water is a suitable sample-preparation method for TXRF analysis without a time-consuming procedure.

Keywords : TXRF; sampling methods; whole blood; destruction of the red blood cells.

Semi-Micro Column HPLC of Three Oxicam Non-Steroidal Anti-Inflammatory Drugs in Human Blood

Akiko Nakamura¹, M. N. Nakashima¹, Mitsuhiro Wada¹ and Kenichiro Nakashima¹

(Received 11 April 2005, Accepted 22 June 2005)

A simple and rapid semi-micro column high-performance liquid chromatographic (HPLC) method with UV detection was developed for the simultaneous detrmination of oxicam non-steroidal anti-inflammatory drugs in human blood samples. Oxicams including isoxicam as an internal standard were extracted from buffered plasma samples (pH 3) with dichloromethane, and the resulting extracts were subjected to HPLC analysis. The separation was performed with a C₁₈ reversed-phase semi-micro column (250×1.5 mm i.d., 5 mm) at 35°C. The mobile phase used was a mixture of acetonitrile-0.1 M acetate buffer (pH 5.0)-methanol, and the detection wavelength was set at 365 nm. Four oxicams were well-separated within 30 min without interference by the blood components. The detection limits of tenoxicam, piroxicam, and lornoxicam were 2.3, 4.2, 6.4 ng/ml in serum, and 2.7, 4.7, 9.3 ng/ml in plasma at a signal-to-noise ratio of 3, respectively. The method was applied to the determination of lornoxicam in the sera of patients.

Keywords : oxicam; tenoxicam; piroxicam; lornoxicam; semi-micro column HPLC; human blood.

Determination of Adenine and Its Related Compounds Using Eosine and Silver(I)

Michiko Nishimura¹, Katsura Nakashima¹, Tsuyoshi Fujimoto¹, Takako Yamaguchi¹ and Yoshikazu Fujita¹

(Received 18 April 2005, Accepted 24 June 2005)

A spectrophotometric method for the determination of adenine and its related compounds based on an association complex between [silver-adenine] and Eosine was established. The standard procedure is as follows: The following components were mixed in a 10 ml volumetric flask: a solution containing of adenine, 0.8 ml of a 5.0×10^-4 mol dm^-3 silver(I) solution, 2.0 ml of the buffer solution, 1.0 ml of a 0.5% MC(1000~1800 cps) solution and 0.8 ml of a 5.0×10^-4 mol dm^-3 Eosine solution. The mixture was diluted to 10 ml with water, transferred into a test tube, mixed well and kept at 60°C for 20 min. After cooling for 5 min in water, the absorbance of the resultant solution was measured at 560 nm against a reagent blank prepared under the same conditions. This method can be used to determine 0.1~1.5 mg/ml adenine; the molar absorptivity is 1.1×10⁵ dm³ mol^-1 cm^-1 at 560 nm. This procedure is much more sensitive than the method using the Bratton-Marshall reagent. The relative standard deviation for 8 runs of 0.68 µg/ml adenine was 0.87%. No metal ion interfered in an equimolor ratio of adenine among the examined metal ions. A sensitive preconcentration method for the spectrophotometry of trace adenine that involves the filtration of an association complex on a membrane filter followed by its redissolution or by tristimulus colorimetry, is described. The thermodynamic parameters suggest that the colored complex formed in this reaction system is an association complex between [silver-adenine] and Eosine.

Keywords : adenine and its related compounds determination; spectrophotometry; association complex; eosine and silver(I); preconcentration using a membrane filter.

Effect of Sodium Sulfite on the Dissolution of Amorphous Silica

Shuqin Bai¹, Shinji Urabe¹, Yoshihiro Okaue¹ and Takushi Yokoyama¹

(Received 15 April 2005, Accepted 28 June 2005)

The dissolution rate and solubility of amorphous silica were determined in pure water, a sodium chloride solution and a sodium sulfite solution in the concentration range of 10^-3~10^-1 M. The initial dissolution rate (0~12 hours) was enhanced in the presence of Na⁺, while it was not affected by Cl^- and SO₃^2-. Although the solubility in pure water was the same as that in a NaCl solution, the solubility in a Na₂SO₃ solution increased depending on its concentration, suggesting the formation of complexes of silicic acid with sulfite ion. The conditional formation constants of the hypothetical 1 : 1 complex were estimated to be 105~1951 M^-1 based on difference between the solubility of amorphous silica in the sodium sulfite solution and that in sodium chloride solution.

Keywords : solubility of amorphous silica; dissolution rate of silica; silicic acid- sulfite ion complex; hydrogen bond.

Analysis of Benzimidazole Anthelmintics in Livestock Foods by HPLC/MS/MS

Shigemi Kai¹, Takeshi Akaboshi¹, Michiko Kishi¹, Hideko Kanazawa² and Shizuko Kobayashi²

(Received 15 April 2005, Accepted 28 June 2005)

A simple, selective and sensitive liquid chromato-graphic ion-trap mass spectrometry (LC-MS-MS) method was developed for the determination of benzimidazole anthelmintics, albendazole, 5-propyl-sulfonyl-¹H-benzimidazole-2-amine, flubendazole, mebendazole, oxibendazole, thiabendazole, 5-hydroxy-thiabendazole, and triclabendazole, 5-chloro-6-(2,3-dichloro-phenoxy)-benzimidazole-2-one in livestock foods. LC separation was carried out on a Discovery HS F5 column (150×2.1 mm) by using 0.2% formic acid- acetonitrile (50 : 50) as the mobile phase at a flow rate of 0.2 ml/min. Detection was carried out on an ion-trap mass spectrometer by a multiple reaction monitoring (MRM) mode and an enhanced product ion (EPI) mode via electrospray ionization (ESI). The calibration graphs for benzimidazole anthelmintics were rectilinear from 0.01 to 10 ng with the MRM mode. The precursor ion and product ions obtained under the EPI mode were key criteria to confirm the identity of benzimidazole anthelmintics in samples. The EPI mode detection limits of benzimidazole anthelmintics were 0.005 to 0.05 ng.

Keywords : benzimidazole; anthelmintics; livestock foods; liquid chromatographic ion trap mass spectrometry (LC-MS-MS).

Colorimetric and Visual Methods for Urinary Protein Determination with Tetrabromophenol Blue

Masakazu Hashimoto¹, Norio Teshima¹, Tadao Sakai¹ and Shuji Kato²

(Received 14 April 2005, Accepted 29 June 2005)

Tetrabromophenol Blue (HTBPB^-) reacted with bovine serum albumin (BSA) to form a blue ion associate at pH 3.2 in the presence of Triton X-100 (TX-100). BSA could be determined in the range of 0~20 ppm by measuring the absorbance at 625 nm. The LOD (3 σ) was 0.16 ppm BSA. The relative standard deviations (n=4) were 0.91% and 0.11% for 5 ppm and 10 ppm BSA, respectively. Moreover, a visual method based on the color changes was developed after the collection of TBPB-binding BSA on the membrane filter (MF) in the absence of TX-100. In this visual method, the standard color calibration chart on a series of MF could be made in the range of 0~9 ppm BSA for each 1 ppm. The proposed colorimetric and visual methods using TBPB could be applied to the determination of urinary protein from kidney disease patients.

Keywords : tetrabromophenol blue; bovine serum albumin; Triton X-100; colorimetry; visual method.

Isotopic, Chemical Characteristics and Water Quality of Lagoon and River-Water Samples in Niigata Prefecture, Japan

Masaru Tazawa¹, Naoki Kano² and Hiroshi Imaizumi²

(Received 15 April 2005, Accepted 30 June 2005)

The monitoring and determination of pollutants in environmental water is very significant from the viewpoint of environmental protection. In this study, the objective was to reveal the quality and characteristics of environmental water samples from Niigata Prefecture in recent years, and to establish a new evaluation method for the behavior of environmental water. For the above-mentioned purpose, the total organic carbon (TOC), chemical oxygen demand (COD), the concentration of major ions and oxygen stable isotopic ratios (i.e., δ¹⁸O) in some river water, lagoon water and groundwater samples in Niigata Prefecture were measured. Moreover, the water temperature, pH, electric conductivity (EC) and dissolved oxygen (DO) were measured in the field. River water, circumjacent groundwater (located near the river), and lagoon water samples were taken monthly at fixed sampling points from 4 rivers (i.e. Shinano River and its two branch streams, Agano River) and 2 lagoons (i.e. Sakata and Toyanogata) in Niigata Prefecture. Consequently, the following matters have been mainly clarified: (1) The relationship between TOC and COD serve as a useful tool for estimating the effect of mixing from other kinds of river-water systems. (2) The concentration of NO₃^- in water samples from Sakata was larger than those in other environmental water samples. Particularly, those of spring water in Sakata were 30 to 60 ppm throughout the year. (3) The relationship between the δ¹⁸O values and the TOC was relatively good in the water samples from Sakata. In a lagoon, such as Sakata (not suffering from the inflow of river waters), investigating δ¹⁸O values are of interest as one of the indices of organic pollutants (originated from plankton), as well as for characterizing the origin of local water. (4) The pH values of lagoon water samples were 8 to 9, and those of groundwater samples were 5 to 6. It is considered that lagoon water is supersaturated with oxygen, and groundwater is unsaturated.

Keywords : river water; lagoon water; total organic carbon (TOC); chemical oxygen demand (COD); oxygen stable isotopic ratio (δ¹⁸O).

Aggregation behavior of Dabsylated Amino Acids in Aqueous Solution

Hiroshige Hayashi¹, Chizuru Sasaki¹, Ko-Ki Kunimoto¹, Shiro Maeda², Shinzo Hosoi³, Akio Kuwae⁴ and Kazuhiko Hanai⁴

(Received 12 April 2005, Accepted 1 July 2005)

A series of dabsylated amino acids (Dabs-AA) were prepared and their aggregation behavior in aqueous solution was studied by UV/Vis absorption and CD spectroscopies. Dabs-AAs show pH-dependent absorption in the visible region, characteristic of the dimethylamio azobenzene chromophore in a dilute aqueous solution. Upon increasing the concentration of Dabs-AAs, the visible absorption around 500 nm of the Dabs-AA monomer decreased its intensity, and a new band due to the Dabs-AA dimer appeared in the 360~400 nm region. Dimer formation depended on the DMSO co-solvent concentration, the solution temperature and the R-groups of amino acids. A split-type induced CD spectrum was observed for the dimer absorption band of Dabs-l-Phe at 363 nm: a negative first Cotton effect at 379 nm and a positive second Cotton effect at 359 nm. These results indicate that a pair of Dabs-l-Phe molecules were stacked with negative exciton chirality.

Keywords : dabsylated amino acids; dimer; induced CD; exciton chirality.

Electrospray Ionization Mass Spectrometry of Regioisomeric 1,2-Diacylglycerols

Tsuyoshi Hashidate¹ and Yutaka Itabashi¹

(Received 19 April 2005, Accepted 4 July 2005)

We investigated the identification of regioisomers (reverse isomers) of 1,2-diacylglycerols by electrospray ionization mass spectrometry (ESI-MS). The reverse isomers of diacylglycerols with various pairs of saturated and unsaturated acyl groups, which were prepared by partial Grignard degradation of the corresponding triacylglycerols, were derivatized into 3,5-dinitrophenylurethanes (DNPU), 3,5-dinitrobenzoates, nicotinates, 1-anthrylurethanes and 2-anthrylurethanes. They were then subjected to positive and negative ESI-MS using an ion-trap mass spectrometer. All of the derivatives employed gave prominent molecular related ions, [M-H]^- and [M+Na]⁺, in negative and positive ion modes, respectively, but no product ions related to the constituent fatty acids. In the ESI-MS/MS of the [M-H]^- molecules, only the DNPU derivatives gave carboxylate [RCOO]^- ions. The [R₂COO]^- ions derived from the sn-2 position of pairs of the reverse isomers of saturate/monoenoate (or dienoate) were more abundant than those from the sn-1 position, showing possible discrimination between the reverse isomers, while pairs of the reverse isomers containing polyunsaturated fatty acids, such as arachidonic and docosahexaenoic acids, were not discriminated on MS/MS. The mechanism leading to the formation of the carboxylate ions is discussed. The present study demonstrates that negative ESI-MS/MS would be useful for assigning diacylglycerol reverse isomers as DUPU derivatives.

Keywords : 1,2-diacylglycerol; regioisomer; reverse isomer; electrospray ionization mass spectrometry; 3,5-dinitrophenylurethane.

Rapid Determination of Immunoglobulin A in Human Saliva by Enzyme-Linked Immunosorbent Assay on a Micro Channel Chip

Hizuru Nakajima¹, Hironori Masuda¹, Satomi Ishino¹, Tatsuro Nakagama¹, Takuya Shimosaka², Kensuke Arai³, Yoshihiro Yoshimura³ and Katsumi Uchiyama¹

(Received 11 April 2005, Accepted 7 July 2005)

An enzyme-linked immunosorbent assay for the rapid determination of Immunoglobulin A (IgA) in human saliva has been developed on a microchannel chip. First, a primary antibody (anti-IgA) was adsorbed on the surface of a polydimethylsiloxane microchannel. The channel was then filled with an antigen (IgA) solution, followed by the introduction of a secondary antibody (anti-IgA HRP labeled) solution. Finally, a substrate solution (phosphate buffer containing Amplex® Red and hydrogen peroxide) was introduced into the microchannel, and the enzyme turnover was monitored by a fluorescent imaging system equipped with a CCD camera. The calibration curve of IgA standard solution at the enzyme-substrate reaction time of 5 minutes was linear over the range of 0~50 ng/ml with a correlation coefficient of 0.998. The former assay on a 96-well microtiter plate required 150 min for the determination of IgA, while the present assay on the microchannel chip enables the determination of IgA in less than 35 min. This method was successfully applied to the determination of IgA in human saliva. An on-line flow assay system with a microchannel chip has also been developed.

Keywords : microfluidics; immunoglobulin A; saliva; stress.

Separation and Preconcentration of Trace Amounts of Copper(II) from Matrix Elements with 8-Quinolinol-Impregnated Emulsion Globules

Ryohei Ohkouchi¹, Hiroaki Matsumiya¹ and Masataka Hiraide¹

(Received 11 March 2005, Accepted 8 July 2005)

A water-in-oil type emulsion containing 8-quinolinol was prepared by dissolving 15 mg of 8-quinolinol and 30 mg of Span-80 (sorbitan monooleate) in 2.0 ml of toluene and vigorously mixing with 0.50 ml of 1.0 mol l^-1 hydrochloric acid by ultrasonic irradiation. The resulting emulsion was gradually injected into 25 ml of sample solution and dispersed by stirring for 10 min as numerous tiny globules. Copper(II) in the sample solution (pH 3~8) was transported through the toluene layer into the internal aqueous phase of the emulsion. After collecting the emulsion, it was demulsified and the copper in the segregated aqueous phase was determined by graphite-furnace atomic absorption spectrometry (GFAAS). Traces of copper(II) were successfully separated from large amounts of aluminum, cobalt, or nickel at pH 3.5 or 6.5, where the matrix elements existed as colloidal hydroxide particles. The separation factor was at 10^-4~10^-3 levels, hence no matrix-interference occurred in the subsequent GFAAS determination. The proposed method was employed as a matrix-removal technique prior to the determination of copper at low µg^-1 to ng g^-1 levels in commercially available aluminum, cobalt, and nickel salts.

Keywords : emulsion liquid membrane; copper; aluminum; cobalt; nickel; graphite-furnace atomic absorption spectrometry.

Speciation Arsenic(III) and Arsenic(V) in Natural Water by Graphite Furnace AAS after Coprecipitation with a Copper-Pyrrolidinedithiocarbamate Complex

Reiko Murata¹, Tokuo Shimizu¹ and Nobuo Uehara¹

(Received 28 March 2005, Accepted 10 July 2005)

Miniaturized coprecipitation with a copper-pyrrolidinedithiocarbamate (PDC) complex was studied for the preconcentration of mg/l levels of inorganic As(III) and As(V) in natural water. Arsenic(III) in a 10 ml of water sample could be coprecipitated with Cu(PCD)₂ at pH 5.3, while under the same conditions As(V) remained in solution. The precipitate was collected on a 10 mm membrane filter using a mini-column, and then dissolved in 225 ml of nitric acid(1+2) and 225 ml of 4% hydrogen peroxide with ultrasonic irradiation. The resulting solution was transferred into a collection vial by centrifugation. After the addition of 50 ml of water, the As(III) concentration was determined by GFAAS. A 20-fold preconcentration factor was obtained by this procedure. Arsenic(V) can be collected, as As(III), after reduction with L-cysteine. The As(V) concentration was obtained by subtracting the As(III) concentration from total the arsenic concentration. The detection limit(3 σ) was found to be 0.034 mg/l of As(III) in the initial solution. The interference of various metal ions was investigated with a mixture of metal ions at concentrations approximately equal to, or exceeding, those normally found in natural water. The mixtures of metal ions tested were found not to cause severe interferences with the determination of As(III) and As(V). The proposed method was successfully applied to river water and seawater samples.

Keywords : arsenic(III); arsenic(V); miniaturized coprecipitation method; copper-pyrrolidinedithiocarbamate complex; natural water; graphite furnace AAS.

Analysis of Mineral Oils Burnt in a Fire by Solvent-Extraction and Thermal-Desorption GC/MS

Akiko Nakahara¹, Kaoru Kawagoe² and Keiko Nakamuta¹

(Received 15 April 2005, Accepted 10 July 2005)

A thermal desorption GC/MS method is able to identify traces of mineral oils from burnt residue by a fire. In that method, mineral oil is volatilized by heating, the ingredients are adsorbed to carbon resin and analyzed by GC/MS. We examined the thermal desorption GC/MS method, and compared it with the solvent extraction GC/MS method. The finger print pattern was used to identify the kinds of mineral oil on mass chromatograms (m/z=71, 85, 92, 106, 142). Throughout combustion, the peak ratio of 2-methylnaphtalene to C₁₃ (tridecane) was different between gasoline and kerosene. No influence on the identification was shown through the combustion of plastics by both methods. By using these two methods properly, mineral oils in burnt residues were detected and identified with high sensitivity. In 96 fire cases from 2001 to 2004, 34 cases were identified as involving kerosene, 5 cases involved gasoline, 13 cases involved gasoline or kerosene. No mineral oils were detected in the other 49 cases.

Keywords : thermal desorption; solvent extraction; mineral oil; GC/MS; fire cause investigation.

Observation of Annual Changes in Concentrations of Atmospheric VOCs by the Collection with Passive Samplers (2001~2004)

Kazumasa Matsushita¹, Mitsuaki Nakamura¹, Yasuro Fuse² and Etsu Yamada^1,2

(Received 15 April 2005, Accepted 20 July 2005)

A long-term monitoring method of volatile organic compounds (VOCs), such as benzene, toluene, and xylene in the atmosphere by using passive samplers was applied to analyzing the annual changes in the concentrations of atmospheric VOCs in Kyoto City from March 2001 to December 2004. The average concentrations of ambient benzene, which were higher than the environmental criterion (3.0 mg/m3), except for those on Mt. Daimonji in 2001, decreased to below 3.0 mg/m3 at all sampling stations in 2004. The average concentrations of ambient toluene and xylene in 2004 were lower than those in 2001 by 1/4~1/2 times. The decrease of atmospheric VOCs concentrations in Kyoto City was evidently observed from 2001~2004. While, the average concentrations of ambient NO2 were almost constant, or slightly increased. The decrease in ambient benzene may have been due to a decrease in the benzene content in gasoline by the end of 1999, and also by implementation of the Pollutant Release and Transfer Register (PRTR) Act in 2001. The implementation of the PRTR Act may have also affected the decrease in the atmospheric concentrations of other VOCs. Furthermore, the decrease of ambient VOCs in our institute may have been attributed to the application of the Labor, Safety and Health Law to the universities in April 2004.

Keywords : volatile organic compounds (VOCs); annual change; atmosphere; passive sampler; PRTR act.

Simple Method of PCB Determination in Transformer Oil by Improving PCB Decomposition Kit and Ion Chromatography of Chloride

Keiko Kurita¹, Makoto Nonomura¹, Yasusi Sakaguchi¹, Aiko Saiki² and Ryoko Takeda³

(Received 14 April 2005, Accepted 21 July 2005)

A PCB decomposition kit that is now available for measuring the amount of PCB in transformer oil by using a chloride ion selective electrode (ISE) has been improved to quantify 0.5 mg/kg or less of PCB by using ion chromatography (IC). The minimum limit of determination by the current ISE with the original kit is 5 mg/kg, whereas the new disposition standard of PCB in transformer oil is 0.5 mg/kg in Japan. The basic procedure of this kit is as follows; a sample of oil containing PCB is decomposed to chlorides (Cl^-) by stabilized Na and the solvent in a kit. The Cl^- are extracted in water and measured with IC. By this method, the quality of the chromatograms is not good enough for reliable high-resolution analysis. This is caused by the large amount of NaOH which is caused by Na. The removal of this Na⁺ in samples has been studied using the H⁺-form cation exchange resin, and it is found that 1 g of cation exchange resin is sufficient to remove the Na⁺ in the improved PCB decomposition kit. The amounts of stabilized Na and the solvent have also been decreased to half the amount of the original kit, which results in no change in the recovery of Cl^- and a better chromatogram. The analyzed data of real transformer oil samples containing PCB by IC and by ISE are in good agreement, but there are some differences in the data of Cl^- lower than 5 mg/kg. This new method was proved to be very useful for screening tests of 0.5 mg/kg of PCB in transformer oil.

Keywords : PCB in transformer oil; decomposition kit; ion chromatography; screening test; chloride ion; cation exchange resin.

Photochemical Behavior of Fe(II) in Coastal Seawater around Okinawa Island by HPLC with a Ferrozine Reagent

Kouichirou Okada¹, Hitomi Nakajima², Hiroyuki Fujimura¹, Takemitsu Arakaki³, Akira Tanahara⁴ and Tamotsu Oomori³

(Received 15 April 2005, Accepted 22 July 2005)

We studied the total dissolved iron concentrations and photochemical formation of Fe(II) in coastal seawater samples collected around Okinawa Island by using a Ferrozine (FZ) reagent. The total dissolved iron concentrations were the highest at sites receiving large river water. Red-soil polluted sites showed a higher total dissolved iron concentration than clean seawater sites, indicating the impacts of red soil. Fe(II) was studied by high-performance liquid chromatography using the FZ reagent (FZ-HPLC). We improved the reported FZ-HPLC method by adopting Poly Ether Ether Ketone (PEEK) materials to the instruments and joint tubing to minimize contamination from the analytical instrument. We also used a suction needle adopter so as to prevent the stainless-steel needle from contacting the injected solutions. The photochemical behavior of Fe(II) was studied by using a solar simulator, and the Fe(II) kinetics was roughly divided into three types. Type A was that the Fe(II) concentrations were almost constant during the irradiation experiments. Type B was that the Fe(II) concentrations increased for the 0 to 5 minutes of irradiation, and gradually decreased after that. Type C was that the Fe(II) concentrations increased with the irradiation time until it reached a plateau. It was inferred that the photoformation of Fe(II) was strongly affected by the dissolved Fe(III) concentrations, dissolved organic compounds, and salinity. It should be noted that since the Fe(II) concentrations were not zero at the beginning of the photochemical experiments, it became evident that oxidation-resistant Fe(II) species existed in the coastal seawater samples collected around Okinawa Island.

Keywords : iron; Fe(II); ferrozine; seawater; photochemistry; Okinawa.

Compact Elemental Analysis System Equipped with an Inkjet Microchip for Pico-Liter Droplet Injection and a Finger-Sized Atomic Emission Detector

Hiroko Eguchi¹, Kaori Nakamura¹, Fumihiro Endo¹, Takahide Nishiyama¹, Tatsuro Nakagama¹, Nobuko Seino², Masaki Sinoda² and Katsumi Uchiyama¹

(Received 15 April 2005, Accepted 25 July 2005)

A compact elemental-analysis system equipped with an inkjet microchip for sample injection, and a finger-size atomic emission detector using a helium radio-frequency plasma was developed. The detection performance of the elemental analysis system was evaluated by using 1,1,1-trichloro-2-methyl-2-propanol (chloretone) dissolved in isopropanol as the sample. When 5100 droplets of isopropanol were ejected from the inkjet microchip, the ejection volume of one droplet was estimated to be 463 pl from the result that the 5100 droplets ejection volume was measured to be 2.36±0.04 µl (n=5). A linear relationship was obtained between the emission intensity at 837.6 nm from chlorine and the number of ejected droplets or the concentration of the sample solution The repeatability of the emission intensity (peak height) obtained with the one-droplet injection of a 2.5 mg/ml sample solution was calculated to be 7.1% RSD (n=6). The detection limit of chloretone resulted to be 184 pg (S/N=3). The detectable concentration of the sample solution was estimated to be 85 mg/ml by the injection of four droplets of the sample solution.

Keywords : inkjet microchip; atomic-emission detector; helium radio-frequency plasma; elemental analysis; chloretone.

Development of Online Solid-Phase Extraction-HPLC/MS/MS Method for the Determination of Perfluorochemicals in Human Plasma

Hisao Nakata¹, Ayako Nakata¹, Fumio Okada¹, Rie Ito¹, Koichi Inoue¹, Koichi Saito¹ and Hiroyuki Nakazawa¹

(Received 15 April 2005, Accepted 26 July 2005)

A method for determining perfluorochemicals (PFCs) such as perfluorooctanesulfonic acid (PFOS), paerfluorooctane slufoneamide (PFOSA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA), in human plasma samples was developed by online solid-phase extraction-HPLC/MS/MS, only after deproteination with acetonitrile. The limits of detection of PFOS, PFOSA, PFOA, PFNA and PFDA in human plasma at a signal to noise (ratio of 3) were 0.08~0.14 ng/ml, and the limits of quantitation of PFOS, PFOSA, PFOA, PFNA and PFDA in human plasma were 0.50 ng/ml. The average recoveries of PFOS, PFOSA, PFOA, PFNA and PFDA ranged from 93.3 to 105% (RSD, 3.0~8.9%; n=6). This method is more rapid and accurate, compared with the column-switching HPLC/MS method presented in previous reports^18)19). The developed method can be applied to the determination of PFOS, PFOSA, PFOA, PFNA and PFDA in human plasma samples for monitoring human exposure.

Keywords : perfluorooctanesulfonic acid; perfluorochemicals; MS/MS; human plasma; column-switching.

Technical Papers

Spectrophotometric Multi-Sample Determination for Trace Amounts of Hemoglobin Using Microtiter Plate Based on the Oxidative Decomposition of the Copper(II)-Phthalocyanine Complex

Takahide Horiguchi¹, Atsushi Manaka¹, Toshio Kubota¹ and Shukuro Igarashi¹

(Received 11 April 2005, Accepted 10 June 2005)

Spectrophotometric multi-sample determinations for small amounts of hemoglobin were performed using a 96-well microtiter plate based on an oxidative decomposition reaction of the copper(II)-phthalocyanine complex. As a result of examining the optimization of various conditions, the concentration of peroxomonosulfate as an oxidizing agent was 3.6×10^-2 M, the pH was 2.0, and the concentration of sodium sulfate as a salt, which was contained in the pH buffer solution, was about 6.5×10^-2 M. In the proposed method, the calibration range was 30~258 ng/ml. The detection limits (3 σ) were 16.99 ng/ml, and the relative standard deviation for 96 samples was 3.64% at the midpoint of the calibration range. As a result of examining foreign substances, cobalt(II), iron(III) and albumin had some influence, but other metal ions and organic compounds did not have any effect. The proposed method was successfully applied to the detection of trace amounts of hemoglobin in human serum samples. Moreover, the hemoglobin analysis for 96 samples was possible to be simultaneously performed within about ten minutes, including not only the time spent for the quantitative operation, but also for the measurement time.

Keywords : 96-well microtiter plate; catalytic analysis; hemoglobin; copper(II)-phthalocyanine; oxidative decomposition reaction.

Fluorometric Determination of Uric Acids by Flow Injection Analysis Using Immobilized Uricase and Horseradish Peroxidase Column

Masaaki Yoshiwara¹ and Akio Sakuragawa¹

(Received 15 April 2005, Accepted 3 July 2005)

The immobilized enzyme using chitosan as a carrier was applied to determine concentration of uric acids as a contributor to gout by a flow injection system incorporating reactor columns. The system consisted of hand-made reactor columns packed with uricase (UOD-IE) and horseradish peroxidase (POD-IE) immobilized onto chitosan beads using reaction of Sciff base by glutaraldehyde crosslinking method. A sodium chloride solution was flowed as a carrier, and sample solution was injected into the stream; it was induced into UOD-IE reaction column. After the stream was joined by 3-(p-hydroxyphenyl)-propionic acid (HPPA) reagent solution, it was induced POD-IE reaction column. In this study, uric acid was indirectly determined quantitatively from the concentration of hydrogen peroxide induced into UOD-IE reaction column. The fluorometric determination of hydrogen peroxide by a derivative reaction with HPPA catalyzed by POD-IE was used in the detection step, that is, the intensity of the fluorescence was measured at 305 nm as excitation wavelength, and 405 nm as absorption wavelength. The proposed system was able to be applied to the determination of uric acids in standard serum and control urine for the proofreading of urine analysis by standard addition method. These results agreed with reference values.

Keywords : uric acid; FIA; immobilized enzyme; uricase; horseradish peroxidase.

Characterization of PM_2.5 in the Indoor Environment by an Electron Probe Microanalysis Method

Motokazu Sato¹, Kenichirou Suzuki¹, Takanori Takii¹, Satoshi Nakano¹, Masashi Nojima², Bunbunoshin Tomiyasu³ and Yoshimasa Nihei^1,2

(Received 19 April 2005, Accepted 19 July 2005)

In this study, we paid attention to minute particles with diameters under 2.5 µm (PM_2.5) in suspended particulate matter (SPM) in an indoor environment. We also collected the suspended particles for a short time using a cascade impactor sampling device. This device is designed to have PM_2.5 cutoff characteristics for the size classification of SPM. The purpose of this study was for the collected particles to obtain detailed information about PM_2.5 in an indoor environment by a source apportionment based on the individual particle analysis using an electron probe microanalysis (EPMA) method. As a result, we could obtain the shape and components of particulate matter contained in environmental tobacco smoke (ETS). It revealed that ETS particles were distributed mainly in the particle size range of 0.5~1.5 µm. In addition, they were composed chiefly of the C-based particles whose shapes were spherical or roundish and nonspherical. To use these data, we evaluated the air environments in Kashiwa and Noda city halls in Chiba. The result showed that there was a difference between the two in terms of PM_2.5. Especially, there were many ETS particles in Noda city hall. This is related to a smoking place installed in the hall. In contrast, the presence of diesel exhaust particles was confirmed in both Kashiwa and Noda city halls as an influence of the outdoor environment. This is largely affected by Route 16 running in the vicinity of the hall. Consequently, it revealed that the indoor sources could have a significant influence on the air environment in the hall. Also, the outdoor sources had an effect on that environment. We could, therefore, understand the realities of PM_2.5 in the indoor environment by the individual particle analysis using an EPMA method, which could not be understood by the conventional method.

Keywords : PM_2.5; indoor environment; individual particle analysis; EPMA; ETS.

Simultaneous Determination of Copper and Antimony by Differential Pulse Anodic Stripping Voltammetry with a Carbon-Paste Electrode

Daisuke Watanabe¹, Takashi Furuike¹, Masahiro Midorikawa² and Tatsuhiko Tanaka¹

(Received 12 May 2005, Accepted 19 July 2005)

A carbon-paste electrode was applied to the simultaneous determination of copper and antimony, whose standard redox potentials lie close together, by differential pulse anodic stripping voltammetry. A new assembly was devised for easily preparing a rotating carbon-paste electrode. The sensitivity of the electrode was greatly improved. Also, the electrode had mechanical and long-term operational stability and could be used repeatedly at least 20 times with excellent reproducibility. The effects of the kinds of oil and the graphite powder/liquid paraffin ratio on the stripping peak heights were considered. In a hydrochloric acid solution, the anodic stripping peaks of copper and antimony were separated by adding potassium iodide and L(+)-ascorbic acid. Copper and antimony in a steel sample solution were electrodeposited on a carbon-paste electrode at -0.7 V vs. Ag/AgCl for 5 min without any removal of the iron matrix. The deposit was then anodically stripped at a scan rate of 100 mV s^-1 to 0.1 V vs. Ag/AgCl. The calibration graph (peak height vs. concentration) was linear over the range 10 to 50 ng ml^-1, and passed through the origin, with relative standard deviations below 1% for 30 ng ml^-1. The possible interferences were evaluated. The proposed method was successfully applied to the simultaneous determination of 30 µg g^-1 for copper and 95 to 194 µg g^-1 for antimony in iron and steel.

Keywords : simultaneous determination of copper and antimony; carbon-paste electrode; differential pulse anodic stripping voltammetry; steel analysis.

Notes

Development of Convenient Vitamin C Sensor Using Cucumber Microtissue Adsorbed on a Porous Carbon Material

Ryoichi Tomita¹, Kentaro Kokubun², Seiko Nakazato² and Shunichi Uchiyama¹

(Received 15 April 2005, Accepted 28 June 2005)

The utilization of biological tissues containing large amount of enzymes is effective for fabricating inexpensive enzyme sensors, but the lifetime of enzymes in a biological tissue is rather short due to its putrefaction compared with the enzyme sensors using an immobilized purified enzyme membrane. In this work, cucumber juice containing ascorbte oxidase (ASOD) was used to immobilize ASOD to a porous carbon material by adsorption; this modified carbon material was combined with an oxygen electrode to fabricate a L-ascorbic acid (AsA) sensor. We found that an excellent linear relationship between AsA concentration and current response was obtained, and the stability of the sensor response was longer than at least one month. The reason why the lifetime of the enzyme sensor increases is that the microdebris of tissue is confined into the micropores of the carbon fibers, and the leaking or unfolding of enzyme does not take place. The porous carbon material adsorbed by purified ASOD also showed a similar long lifetime and a chemically amplified response (detection limit was 2.0×10^-8 M) was observed by 0.05 M dithiothreithol (DTT). However, in the case of a microtissue adsorbed carbon material, amplification of the sensor response using DTT was not observed, because the access of DTT to enzyme in tissue is difficult, differing from purified enzyme.

Keywords : cucumber maicrotissue; purified enzyme; porous carbon material; adsorption; L-ascorbic acid sensor.

Highly Sensitive Determination of 1,4-Dioxane in Tap Water and Raw Water in Hyogo Prefecture by GC/MS Coupled with an Improved Solid-Phase Extraction-GC/MS

Miho Yano¹, Tatsuhiko Kawamoto¹, Nobuko Makihata¹,

(Received 18 April 2005, Accepted 30 June 2005)

1,4-Dioxane was listed as a Standard Item in the New Water Quality Standards for Drinking Water, which was enforced as of April 1, 2004. Its regulated value was set at 0.05 mg l^-1. Studies of the analytical method that is used to determine 1,4-dioxane by solid phase extraction-GC/MS clarified the following: (1) 1ml of acetone is a sufficient volume for the elution of 1,4-dioxane. Therefore, any losses of 1,4-dioxane that may accompany the concentration process can be prevented. In consequence, the recovery rate can be improved. (2) Blank experiments using 16 kinds of water, including purified water, re-purified water and 14 types of mineral waters, showed that blank peaks were detected from all samples. It was also shown that the blank values of 6 of the treated mineral waters were high. Therefore, for highly sensitive analysis, the blank value from minerals should be considered. (3) The detection and quantitation limits of 1,4-dioxane by this method were 0.02 mg l^-1 and 0.05 mg l^-1, respectively. Therefore, a concentration of 1/1000 of the Water Quality Standard can be quantified by this method. Using this method, the concentrations of 1,4-dioxane in raw and tap water sampled at 20 sampling sites in Hyogo prefecture were determined. The results show that the range of concentrations that can be detected is between N.D. and 4.2 mg l^-1. Furthermore, tests that were conducted to compare water sources revealed that the concentration of 1,4-dioxane in ground water samples was higher than for surface waters.

Keywords : 1,4-dioxane; GC/MS; blank peak; drinking water; ground water.

Fractionation of Dissolved Organic Matter (DOM) as Precursors of Trihalomethane in Lake Biwa and Yodo Rivers

Kenichi Nagai¹, Sinichi Aoki¹, Yasuro Fuse² and Etsu Yamada^1,2

(Received 15 April 2005, Accepted 15 July 2005)

In order to clarify the precursors of trihalomethane (THM) in tap water, humic substances and other dissolved organic matter (DOM) in Lake Biwa and Yodo rivers were investigated to elucidate their behavior and contribution to the THM formation potential. The hydrophobic and hydrophilic fractions of DOM were isolated using XAD-7HP resin, and then the THM formation potential with the chlorination of these fractions was examined. Hydrophobic acids, such as humic substances, were 30~60% of DOC in Yodo rivers and about 25% in the northern basin of Lake Biwa. The contribution of hydrophobic acids, such as humic substances, to the THM formation potential was about 70% in Yodo rivers, while it was about 30% in the northern basin of Lake Biwa, which was almost same as the contribution of hydrophilic DOM to THM formation potential. Hydrophilic neutral DOM was 15~20% of DOC in Lake Biwa, and its contribution of THM formation potential was about 40%, which was larger than those of hydrophobic acids and hydrophilic DOM. The THM formation potential per organic carbon unit of hydrophilic fractions in the northern basin of Lake Biwa were two-times that in rivers. These results suggest that the kinds of hydrophilic DOM in Lake Biwa may be different from those in rivers.

Keywords : DOM; precursor; trihalomethane; fractionation; Lake Biwa; Yodo rivers.

Analytical Reports

Behavior of Heavy Metals in the Callus of Brassica Juncea, a Heavy Metal Accumulation Plant

Toru Yamada¹, Ryuji Takeda¹, Yusuke Tsuda², Sadayoshi Matsumoto², Sadao Komemushi² and Akiyoshi Sawabe²

(Received 14 April 2005, Accepted 19 July 2005)

To find a more efficient phytoremediation, the accumulation mechanism of elucidating heavy metals of a hyperaccumulator is important. We measured the behavior and glutathione quantity of the heavy metal accumulation level in calli using Brassica juncea, which is a hyperaccumulator of Pb and Cd. Brassica juncea from a seed was used to grow callus, which was for three months. Then, for two weeks, we conducted long-term exposure to a culture in heavy-metal component culture media (Cr, Cu, Zn, Cd, Pb, Co, Ni). Furthermore, we used a liquid culture medium; for Pb, we conducted a short-term exposure for five days. As for Cd and Pb, the absorbed dose was high, and the Cd, in particular, showed the absorbed dose to be high at a low concentration (10~100 mM), compared with other heavy-metal classes. There were many things that necrotized, in other heavy metals, the fastness was not found to influence the heavy metal. In the short-term exposure of Pb, the absorption level increased during the early phase. However, 96 hours later, according to the cell cycle, that absorption appeared to be promoted. In addition, in GSH, while a large difference was not recognized, a high concentration level was much contained it.

Keywords : phytoremediation; Pb; heavy metal; hyperaccumulator; Brassica juncea.

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