論文 - 曽我　朋義

Evaluation of Metabolic Alteration in Transgenic Rice Overexpressing Dihydroflavonol-4-reductase

Takahashi, H., Hayashi, M., Sato, S., Soga, T., Nishioka, T., Tomita, M., Kawai-Yamada, M., Uchimiya, H.,

Ann. Bot. 98 （ 4 ） 819 - 825 2006年10月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Background and aims: Previous studies have shown that transgenic rice plants overexpressing YK1, which possesses dihydroflavonol-4-reductase (DFR) activity, showed biotic and abiotic stress tolerance. High throughput profiles of metabolites have also been shown in such transgenic plants by Fourier transform ion cyclotron mass spectrometry. In this study, capillary electrophoresis mass spectrometry analysis (CE/MS) was employed to identify precise metabolites such as organic acids, amino acids and sugars.
Methods: Using CE/MS, we analysed several metabolites of glycolysis, the tricarboxylic acid (TCA) cycle and the pentose phosphate pathway. In addition, the concentrations of sugars and ion were quantified.
Key results: In YK1 (DFR)-overexpressing plants, the concentrations of cis-aconitate, isocitrate and 2-oxoglutarate were higher in leaves, whereas those of fructose-1,6-bisphosphate and glyceraldehyde-3-phosphate were lower in roots. In seeds, the amounts of free amino acids and metals were altered, whereas sugars in seeds were kept constant. In YK1 calli, an approx. 3-fold increase in glutathione was observed, whereas the activities of glutathione peroxidase and glutathione reductase were concomitantly increased.
Conclusions: The overexpression of YK1 (DFR) was associated with slight changes in the amounts of several metabolites analysed in whole plants, whilst glutathione derivatives were substantially increased in suspension-cultured cells.


Metabolomics Approach for Enzyme Discovery

Saito, N., Robert, M., Kitamura, S., Baran, R., Soga, T., Mori, H., Nishioka, T., Tomita, M.

J. Proteome Res.  5 （ 8 ） 1979 - 1987 2006年08月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

The search for novel enzymes is an important but difficult task in functional genomics. Here, we present a systematic method based on in vitro assays in combination with metabolite profiling to discover novel enzymatic activities. A complex mixture of metabolites is incubated with purified candidate proteins and the reaction mixture is subsequently profiled by capillary electrophoresis electrospray ionization mass spectrometry (CE-MS). Specific changes in the metabolite composition can directly suggest the presence of an enzymatic activity while subsequent identification of the compounds whose level changed specifically can pinpoint the actual substrate(s) and product(s) of the reaction. We first evaluated the method using several Escherichia coli metabolic enzymes and then applied it to the functional screening of uncharacterized proteins. In this manner, YbhA and YbiV proteins were found to display both phosphotransferase and phosphatase activity toward different sugars/sugar phosphates. Our approach should be broadly applicable and useful for enzyme discovery in any system.

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Differential Metabolomics Reveals Ophthalmic Acid As An Oxidative Stress Biomarker Indicating Hepatic Glutathione Consumption

Soga, T., Baran, R., Suematsu M., Ueno, Y., Ikeda, S., Sakurakawa T., Kakazu, Y., Ishikawa, T., Robert, M., Nishioka, T., Tomita, M.

J. Biol. Chem.  281 （ 24 ） 16768 - 16776 2006年06月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Metabolomics is an emerging tool that can be used to gain insights into cellular and physiological responses. Here we present a metabolome differential display method based on capillary electrophoresis time-of-flight mass spectrometry to profile liver metabolites following acetaminophen-induced hepatotoxicity. We globally detected 1,859 peaks in mouse liver extracts and highlighted multiple changes in metabolite levels, including an activation of the ophthalmate biosynthesis pathway. We confirmed that ophthalmate was synthesized from 2-aminobutyrate through consecutive reactions with gamma-glutamylcysteine and glutathione synthetase. Changes in ophthalmate level in mouse serum and liver extracts were closely correlated and ophthalmate levels increased significantly in conjunction with glutathione consumption. Overall, our results provide a broad picture of hepatic metabolite changes following acetaminophen treatment. In addition, we specifically found that serum ophthalmate is a sensitive indicator of hepatic GSH depletion, and may be a new biomarker for oxidative stress. Our method can thus pinpoint specific metabolite changes and provide insights into the perturbation of metabolic pathways on a large scale and serve as a powerful new tool for discovering low molecular weight biomarkers.

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Conductivity detection in capillary zone electrophoresis: Inspection by PeakMaster

Jaros, M., Soga, T., Van de Goor, T., *Gas, B.

Electrophoresis 26 （ 10 ） 1948 - 1953 2005年05月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  01730835

　概要を見る

A simple rule stating that the signal in conductivity detection in capillary zone electrophoresis is proportional to the difference between the analyte mobility and mobility of the background electrolyte (BGE) co‐ion is valid only for systems with fully ionized electrolytes. In zone electrophoresis systems with weak electrolytes both conductivity signal and electromigration dispersion of analyte peaks depend on the conductivity and pH effects. This allows optimization of the composition of BGEs to give a good conductivity signal of analytes while still keeping electromigration dispersion near zero, regardless of the injected amount of sample. The demands to achieve minimum electromigration dispersion and high sensitivity in conductivity detection can be accomplished at the same time. PeakMaster software is used for inspection of BGEs commonly used for separation of sugars (carbohydrates, saccharides) at highly alkaline pH. It is shown that the terms direct and indirect conductivity detection are misleading and should not be used.

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Metabolome analysis and metabolic simulation

Ishii, N., Soga, T., Nishioka, T., Tomita, M.

Metabolomics 1 （ 1 ） 29 - 37 2005年03月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

For many decades microorganisms have been used for industrial purposes; traditional fermentations such as brewing and production of food additives, aroma molecules, organic acids and pharmaceutical-like antibiotics or recombinant proteins are instances of the industrial microorganism utilization. Therefore, microorganism modeling and simulation have been required for engineering purposes, because of demands for design, optimization and quality control of large-scale fermentation plants. Modeling has recently become more highly developed, aided by the deciphering of microorganism genomes, the completion of metabolic databases, the development of analytical methodologies and improvements in the performance of computers. This paper reviews past and recent metabolic simulation of microorganisms, and also discusses the metabolome analytical techniques and the construction of large-scale microorganism models which are now being developed in our group.

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Large-Scale Prediction of Cationic Metabolite Identity and Migration Time in Capillary Electrophoresis Mass Spectrometry Using Artificial Neural Networks

Sugimoto, M., Kikuchi, S., Arita, M., Soga, T., Nishioka, T., Tomita, M.

Anal. Chem.  77 （ 1 ） 78 - 84 2005年01月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

We developed a computational technique to assist in the large-scale identification of charged metabolites. The electrophoretic mobility of metabolites in capillary electrophoresis-mass spectrometry (CE-MS) was predicted from their structure, using an ensemble of artificial neural networks (ANNs). Comparison between relative migration times of 241 various cations measured by CE-MS and predicted by a trained ANN ensemble produced a correlation coefficient of 0.931. When we used our technique to characterize all metabolites listed in the KEGG ligand database, the correct compounds among the top three candidates were predicted in 78.0% of cases. We suggest that this approach can be used for the prediction of the migration time of any cation and that it represents a powerful method for the identification of uncharacterized CE-MS peaks in metabolome analysis.

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Simultaneous Determination of Main Metabolites in Rice Leaves using Capillary Electrophoresis Mass Spectrometry and Capillary Electrophoresis Diode Array Detection

Sato, S., Soga, T., Nishioka, T., Tomita, M.

Plant J. 40 （ 1 ） 151 - 163 2004年10月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

The study of the metabolomics of primary metabolites using conventional chemical analyses requires a high‐throughput method. Chemical derivatizations are a prerequisite for gas‐chromatographic separation, and a large sample quantity is needed for liquid‐chromatographic separation and nuclear magnetic resonance detection systems. Recently, we have developed a capillary electrophoresis–mass spectrometry (CE–MS) technology that can simultaneously quantify a large number of primary metabolites, using only a small quantity of samples, and without any chemical derivatizations. Parallel use of a capillary electrophoresis–diode array detector (CE–DAD) system further enables almost all water‐soluble intracellular metabolites to be analyzed. We demonstrate, with rice leaves, a simple and rapid method of sample preparation for CE analysis; using this method, we have successfully measured the levels of 88 main metabolites involved in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, photorespiration, and amino acid biosynthesis.

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Application of capillary electrophoresis‐mass spectrometry to synthetic in vitro glycolysis studies

Itoh, A., Ohashi, Y., Soga, T., Mori, H., Nishioka, T., Tomita, M.

Electrophoresis 25 （ 13 ） 1996 - 2002 2004年07月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

We propose an approach designed to reconstitute a metabolic pathway composed of multistep biochemical reactions, rather than to dissect the individual reactions that make up the pathway. A synthetic in vitro glycolysis was reconstructed from ten purified Escherichia coli (E. coli) enzymes to obtain a better understanding of the regulation of sequential enzymatic reactions. The key to the success of this approach is the ability to perform direct and simultaneous determination of the diverse metabolic intermediates in the pathway by capillary electrophoresis-mass spectrometry. We observed that the pathway is regulated by a delicate balance between the changing metabolite concentrations and behaves like a natural biological oscillating network that has hitherto not been reported for E. coli glycolysis. The end-product, pyruvate, was periodically synthesized from glucose at an overall efficiency of 30%, corresponding to an average of 90% conversion efficiency for each of the ten steps involved. This approach is likely useful for the synthesis of natural products requiring complex sequential biocatalytic reactions.

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Qualitative and quantitative analysis of amino acids by capillary electrophoresis‐electrospray ionization‐tandem mass spectrometry

Soga, T., Kakazu, Y., Robert.M., Tomita, M., Nishioka, T.

Electrophoresis 25 （ 13 ） 1964 - 1972 2004年07月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

We describe a method to identify and quantify amino acids using capillary electrophoresis-electrospray ionization-triple-quadrupole tandem mass spectrometry (CE-ESI-MS/MS). Amino acids, including physiological amino acids, were first separated by CE under acidic pH conditions and then detected by MS/MS. To efficiently introduce the whole sample into the capillary, no electrical potential was applied to the electrospray probe until running electrophoresis. The position of the electrosprayer with respect to the MS capillary entrance drastically affected sensitivity and generation of cluster ions. MS/MS with multiple reaction monitoring (MRM) detection was performed to obtain sufficient selectivity and sensitivity. Under optimized CE-MS/MS conditions, the minimum detectable levels for 32 free amino acids normally found in proteins and other physiological amino acids were between 0.1 and 14 micromol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. For most amino acids, this constitutes a severalfold increase in sensitivity compared to CE-MS. The relative standard deviations (% RSD) for all amino acids were better than 0.4% for migration times and between 1.4% and 8.6% for peak areas (n = 10). Since amino acids exhibited characteristic MS/MS spectra, this approach is useful for the simultaneous, selective, quantitative, and reproducible analysis of amino acids in physiological and biological samples that contain various kinds of matrices. The power of the method was demonstrated by analyzing amino acids in human urine.

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Quantitative Metabolome Analysis Using Capillary ElectrophoresisMass Spectrometry

Soga T, Ohashi Y, Ueno Y, Naraoka H, Tomita M, Nishioka T

J Proteome Res. 2 （ 5 ） 488 - 494 2003年10月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

A new approach for the comprehensive and quantitative analysis of charged metabolites by capillary electrophoresis mass spectrometry (CE-MS) is proposed. Metabolites are first separated by CE based on charge and size and then selectively detected using MS by monitoring over a large range of m/z values. This method enabled the determination of 352 metabolic standards and its utility was demonstrated in the analysis of 1692 metabolites from Bacillus subtilis extracts, revealing significant changes in metabolites during B. subtilis sporulation.

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Pressure-Assisted Capillary Electrophoresis Electrospray Ionization Mass Spectrometry for Analysis of Multivalent Anions

Soga, T., Ueno, Y., Naraoka, H., Matsuda, K., Tomita, M., and Nishioka, T.

Anal. Chem. 74 （ 24 ） 6224 - 6229 2002年12月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

We describe a method, based on pressure-assisted capillary electrophoresis coupled to electrospray ionization mass spectrometry (PACE/ESI-MS), that allows the simultaneous and quantitative analysis of multivalent anions, such as citrate isomers, nucleotides, nicotinamide-adenine dinucleotides, and flavin adenine dinucleotide, and coenzyme A (CoA) compounds. Key to the analysis was using a noncharged polymer, poly(dimethylsiloxane), coated to the inner surface of the capillary to prevent anionic species from adsorbing onto the capillary wall. It was also necessary to drive a constant liquid flow toward the MS by applying air pressure to the inlet capillary during electrophoresis to maintain a conductive liquid junction between the capillary and the electrospray needle. Although theoretical plates were inferior to those obtained by CE/ESI-MS using a cationic polymer-coated capillary, the PACE/ESI-MS method improved reproducibility and sensitivity of these anions. Eighteen anions were separated by PACE and selectively detected by a quadrupole mass spectrometer with a sheath-flow electrospray ionization interface. The relative standard deviations (n = 6) of the method were better than 0.6% for migration times and between 1.4% and 6.2% for peak areas. The detection limits for these species were between 0.4 and 3.7 μmol/L with pressure injection of 50 mbar for 30 s (30 nL), that is, mass detection limits calculated in the range from 12 to 110 fmol at a signal-to-noise ratio of 3. The utility of the method was demonstrated by analysis of citrate isomers, nucleotides, dinucleotides, and CoA compounds extracted from Bacillus subtilis cells.

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Combined Metabolome and Transcriptome Analysis of Bacillus subtilis Cells Cultured on Different Carbon Sources

Matsuda, K., Yamaguchi, H., Ueno, Y., Soga, T., Fujita, Y., and Nishioka, T.

Genome Informatics  13   593 - 594 2002年12月

研究論文（学術雑誌）, 共著, 査読有り

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Simultaneous Determination of Anionic Intermediates for Bacillus subtilis Metabolic Pathways by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

Soga, T., Ueno, Y., Naraoka, H., Ohashi, Y., Tomita, M., and Nishioka, T.

Anal. Chem. 74 （ 10 ） 2233 - 2239 2002年05月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

A method for simultaneous determination of anionic metabolites based on capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry is described. To prevent current drop by the system, electroosmotic flow (EOF) reversal by using a cationic polymer-coated capillary was indispensable. A mixture containing 32 standards including carboxylic acids, phosphorylated carboxylic acids, phosphorylated saccharides, nucleotides, and nicotinamide and flavin adenine coenzymes of glycolysis and the tricarboxylic acid cycle pathways were separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath-flow electrospray ionization interface. Key to the analysis was EOF reversal using a cationic polymer-coated capillary and an electrolyte system consisting of 50 mM ammonium acetate, pH 9.0. The relative standard deviations of the method were better than 0.4% for migration times and between 0.9% and 5.4% for peak areas. The concentration detection limits for these metabolites were between 0.3 and 6.7 μmol/L with pressure injection of 50 mbar for 30 s (30 nL); i.e., mass detection limits ranged from 9 to 200 fmol, at a signal-to-noise ratio of 3. This method was applied to the comprehensive analysis of metabolic intermediates extracted from Bacillus subtilis, and 27 anionic metabolites could be directly detected and quantified.

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Capillary electrophoresis method for the analysis of inorganic anions, organic acids, amino acids, nucleotides, carbohydrates and other anionic compounds

Soga, T., Imaizumi, M.

Electrophoresis 22 （ 16 ） 3418 - 3425 2001年10月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

A previously developed capillary zone electrophoresis (CZE) method with indirect UV detection for the simultaneous determination of inorganic and organic anions, amino acids and carbohydrates using 20 mM 2,6‐pyridinedicarboxylic acid (PDC) as the background electrolyte was extended to allow determination of 206 anions including those above – mentioned and physiological amino acids, nucleotides, aromatic acids, haloacetic acids, alcohols, phosphorylated saccharides, oxyhalides, metal oxoacids, metal‐ethylenediaminetetraacetic acid (EDTA) complexes, forensic anions, Good's buffers and herbicides. Every compound could be analyzed and their electrophoretic mobility determined simply by selecting detection wavelength. This method is simple and universal for anion analysis, and could be readily applied to the simultaneous determination of anionic compounds. In this work, it was used to identify and quantify important anions in sea urchin and sake.

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Direct chiral resolution of malic acid in apple juice by ligand‐exchange capillary electrophoresis using copper(II)‐L‐tartaric acid as a chiral selector

Kodama, S., Yamamoto, A., Matsunaga, A., Soga, T., Hayakawa, K.

Electrophoresis  22 （ 15 ） 3286 - 3290 2001年09月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Chiral resolution of native DL-malic acid was achieved by ligand-exchange capillary electrophoresis using copper(II)-L-tartrate as a chiral selector. Factors affecting chiral resolution, migration time, and peak area of malic acid were studied. The running conditions for optimum separation of malic acid were found to be 1 mM copper(II) sulfate-1 mM L-tartrate (pH 5.1) with an effective voltage of -20 kV at 30 degrees C, using direct detection at 280 nm, and resolution (Rs) of racemic malic acid was approximately 4. With this system, D- and L-malic acids in apple juice were analyzed successfully.

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Determination of carbohydrates in food samples by capillary electrophoresis with indirect UV detection

Soga, T., Serwe, M

Food Chem. 69 （ 3 ） 339 - 344 2000年05月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

An easy and reproducible capillary electrophoretic method with indirect UV detection for the determination of carbohydrates in food samples was described. A highly alkaline pH condition was used in order to charge every carbohydrate negatively. Electroosmotic flow (EOF) was reversed to detect sugar alcohols and to improve the separation of neutral and amino sugars. Under optimized conditions 28 carbohydrates including mono- and disaccharides, uronic acids, sialic acids, amino sugars and sugar alcohols were separated in less than 25 min. The detection limits for fructose, glucose and sucrose were in the range from 12 to 16 mg l−1 with pressure injection of 50 mbar for 6 s (ca. 6 nl) at a signal to noise ratio of 3. The calibration curves were linear over the range from 50 to 10 000 mg l−1 with correlation coefficients better than 0.9999. This method could be readily applied to the analysis of carbohydrates in several foods and beverages. Sample preparation was minimal and consisted either of dilution or dilution with filtration.

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Direct chiral resolution of lactic acid in food products by capillary electrophoresis

Kodama, S., Yamamoto, A., Matsunaga, A., Soga, T., and Minoura, K.

J. Chromatogr. A. 875 （ 1-2 ） 371 - 377 2000年04月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Chiral resolution of native dl-lactic acid was performed by capillary electrophoresis using 2-hydroxypropyl-β-cyclodextrin as a chiral selector. Various factors affecting chiral resolution, migration time, and peak area of lactic acid were studied. The running conditions for optimum separation of lactic acid were found to be 90 mM phosphate buffer (pH 6.0) containing 240 mM 2-hydroxypropyl-β-cyclodextrin with an effective voltage of −30 kV at 16°C, using direct detection at 200 nm. In order to enhance the sensitivity, sample injection was done under a pressure of 50 mbar for 200 s. On-line sample concentration was accomplished by sample stacking. With this system, d- and l-lactic acids in food products were analyzed successfully.

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Amino Acid Analysis by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

Soga, T., Heiger, D.N.

Anal. Chem. 72 （ 6 ） 1236 - 1241 2000年03月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

A method for the determination of underivatized amino acids based on capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE−ESI-MS) is described. To analyze free amino acids simultaneously a low acidic pH condition was used to confer positive charge on whole amino acids. The choice of the electrolyte and its concentration influenced resolution and peak shape of the amino acids, and 1 M formic acid was selected as the optimal electrolyte. Meanwhile, the sheath liquid composition had a significant effect on sensitivity and the highest sensitivity was obtained when 5 mM ammonium acetate in 50% (v/v) methanol−water was used. Protonated amino acids were roughly separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath flow electrospray ionization interface. Under the optimized conditions, 19 free amino acids normally found in proteins and several physiological amino acids were well determined in less than 17 min. The detection limits for basic amino acids were between 0.3 and 1.1 μmol/L and for acidic and low molecular weight amino acids were less than 6.0 μmol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. This method is simple, rapid, and selective compared with conventional techniques and could be readily applied to the analysis of free amino acids in soy sauce.

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Capillary electrophoresis for the determination of forensic anions in adulterated foods and beverages

Soga, T., Tajima, I., Heiger, D.N.

Am. Lab. 32 （ 3 ） 124 - 128 2000年02月

研究論文（学術雑誌）, 共著, 査読有り

Determination of aspartic acid enantiomers in bio‐samples by capillary electrophoresis

Tsunoda, M., Kato, M., Fukushima, T., Santa, T., Homma, H., Yani, H., Soga, T., and Imai, K.

Biomed Chromatogr  13 （ 5 ） 335 - 339 1999年08月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Enantiomeric separation and detection of D,L-aspartic acid (Asp) derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) by capillary electrophoresis (CE) using modified cyclodextrins as chiral selectors was studied. Heptakis(2,3, 6-tri-O-methyl)-beta-cyclodextrin(TM-beta-CD) was most effective for enantiomeric separation of NBD-D,L-Asp with optimum conditions of 30 mM TM-beta-CD in 50 mM phosphate buffer (pH 4.0) and the limit of detection (LOD) attained was 100 nM for each enantiomer. The method proposed in the present study was convenient for both D- and L-Asp determination since the other amino compounds migrated differently and D-Asp in bio-samples such as rat pineal gland and foods was determined with a simple sample pretreatment and a short analysis run time.

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