High-resolution structure of phosphoketolase from Bifidobacterium longum determined by cryo-EM single-particle analysis.

In bifidobacteria, phosphoketolase (PKT) plays a key role in the central hexose fermentation pathway called "bifid shunt." The three-dimensional structure of PKT from Bifidobacterium longum with co-enzyme thiamine diphosphate (ThDpp) was determined at 2.1 Å resolution by cryo-EM single-particle analysis using 196,147 particles to build up the structural model of a PKT octamer related by D4 symmetry. Although the cryo-EM structure of PKT was almost identical to the X-ray crystal structure previously determined at 2.2 Å resolution, several interesting structural features were observed in the cryo-EM structure. Because this structure was solved at relatively high resolution, it was observed that several amino acid residues adopt multiple conformations. Among them, Q546-D547-H548-N549 (the QN-loop) demonstrate the largest structural change, which seems to be related to the enzymatic function of PKT. The QN-loop is at the entrance to the substrate binding pocket. The minor conformer of the QN-loop is similar to the conformation of the QN-loop in the crystal structure. The major conformer is located further from ThDpp than the minor conformer. Interestingly, the major conformer in the cryo-EM structure of PKT resembles the corresponding loop structure of substrate-bound Escherichia coli transketolase. That is, the minor and major conformers may correspond to "closed" and "open" states for substrate access, respectively. Moreover, because of the high-resolution analysis, many water molecules were observed in the cryo-EM structure of PKT. Structural features of the water molecules in the cryo-EM structure are discussed and compared with water molecules observed in the crystal structure.

Development of a novel l-histidine assay method using histamine dehydrogenase and a stable mutant of histidine decarboxylase.

l-Histidine analysis is essential in physiological research and clinical applications because l-histidine concentrations in biofluids are associated with various diseases. However, an enzymatic method for l-histidine quantitation has not yet been established. Here, we describe a novel l-histidine quantitation assay using a combination of histidine decarboxylase (HDC) and histamine dehydrogenase (HDH) enzymes. Wild-type HDC is unstable and completely lost its activity within 50 days of storage at 4 °C in solution. We rationally designed a HDC C57S mutant with markedly improved stability (storage at 4 °C for over 200 days) without altering the enzyme's substrate specificity. Together with HDH, the HDC C57S mutant was applied to quantify l-histidine concentrations in human plasma. The assay showed high precision (<2.0% inter-assay variation) and high accuracy (<5.8% deviation from the results of LC/MS).

Development of a rapid and simple glycine analysis method using a stable glycine oxidase mutant.

Glycine analysis is important in research fields such as physiology and healthcare because the concentration of glycine in human plasma has been reported to change with various disorders. Glycine oxidase from Bacillus subtilis (GlyOX) is useful for quantitative analysis of glycine. However, GlyOX is not sufficiently stable for use in physiology-based research or clinical settings. In this report, site-directed mutagenesis was used to engineer a GlyOX mutant suitable for glycine analysis. The GlyOX triple-mutant (T42 A/C245 S/L301V) retained most of its enzymatic activity during storage for over a year at 4 °C. A colorimetric enzyme analysis protocol was established using the GlyOX triple-mutant to determine glycine concentrations in human plasma. The analysis showed high accuracy (-5.4 to 3.5% relative errors when compared with the results from an amino acid analyzer, and 96.0-98.7% recoveries) and high precision (<4% between-run variation). Sample pretreatments of deproteinization and derivatization were not required. Therefore, this novel enzymatic analysis offers an effective and useful method for determining glycine concentrations in physiology related research and the healthcare field.

Validity of a Self-Administered Food-Frequency Questionnaire for Assessing Amino Acid Intake in Japan: Comparison With Intake From 4-Day Weighed Dietary Records and Plasma Levels.

BACKGROUND: Interest in the physiological roles of amino acids and their impact on health outcomes is substantial and growing. This interest has prompted assessment of the habitual intake of amino acids for use in epidemiologic studies and in clarifying the association between habitual intake and plasma levels of amino acids. Here, we investigated the validity of ranking individuals according to dietary amino acid intake as estimated using a food frequency questionnaire (FFQ) in comparison with intakes from dietary records (DRs) and plasma levels. METHODS: A total of 139 men and women selected from examinees of the cancer screening program at the Research Center for Cancer Prevention and Screening, National Cancer Center, Japan, provided 4-day weighed DRs, a semi-quantitative FFQ, and plasma samples. Plasma levels of amino acids were measured using the UF-Amino Station system. RESULTS: Spearman rank correlation coefficients of energy-adjusted intake of amino acids from the DR and FFQ ranged from 0.40 to 0.65 for men and from 0.35 to 0.46 for women. Correlation coefficients of energy-adjusted intake from the DR and plasma levels ranged from -0.40 to 0.25 for men and from -0.16 to 0.11 for women. Similarly, no significant positive correlation coefficients were observed between intake from the FFQ and plasma levels for either men or women. CONCLUSIONS: We confirmed that this FFQ has moderate validity in estimating amino acid intake when 4-day weighed DRs are used as a reference method, suggesting that it is suitable for ranking individuals living in urban areas in Japan by amino acid intake.

Plasma amino acid profiles in healthy East Asian subpopulations living in Japan.

OBJECTIVES: Profiles of plasma free amino acids (PFAAs) have been utilized as biomarkers to detect various diseases. However, few studies have investigated whether ethnicity or specific subpopulations within East Asia influence PFAA concentrations. METHODS: A total of 95 healthy volunteers living in Japan, including 31 Japanese individuals, 36 Korean individuals and 28 Chinese individuals, were enrolled. Participants' PFAA levels were measured by high-performance liquid chromatography mass spectrometry, and the effects of factors such as sex, age, body mass index (BMI) and subpopulation on PFAA profiles were analyzed. RESULTS: With the exception of glutamine and α-aminobutyric acid, there were no significant differences among the three examined subpopulations with respect to either the means or the distributions of PFAA concentrations. A multiple regression analysis revealed that most of the PFAA concentrations were significantly related to sex. Ornithine concentrations, glutamate concentrations, and glutamine and α-aminobutyric acid concentrations were significantly associated with age, BMI, and Chinese subpopulation, respectively. CONCLUSION: The study results indicate that the contributions of subpopulation within East Asia to PFAA profiles are small, particularly relative to the contributions provided by sex.

Microscopic imaging mass spectrometry assisted by on-tissue chemical derivatization for visualizing multiple amino acids in human colon cancer xenografts.

Imaging MS combined with CE/MS serves as a method to provide semi-quantitative and spatial information of small molecular metabolites in tissue slices. However, not all metabolites including amino acids have fully been visualized, because of low-ionization efficiency in MALDI MS. This study aimed to acquire semi-quantitative spatial information for multiple amino acids in frozen tissue slices. As a derivatization reagent, p-N,N,N-trimethylammonioanilyl N'-hydroxysuccinimidyl carbamate iodide (TAHS) was applied to increase their ionization efficiency and detection sensitivity. Semi-quantitative MALDI-imaging MS allowed us to visualize and quantify free amino acid pools in human colon cancer xenografts using a model of liver metastases in super-immunodeficient NOD/scid/γ(null) mice (NOG mice). Because the m/z values of several TAHS-derivatized amino acids overlap with those of the 2,5-dihydroxybenzoic acid background and other endogenous compounds, we imaged them with tandem MS. The results indicated that regional contents of glutamate, glutamine, glycine, leucine/isoleucine/hydroxyproline, phenylalanine, and alanine were significantly elevated in metastatic tumors versus parenchyma of tumor-bearing livers. On-tissue TAHS derivatization thus serves as a useful method to detect alterations in many amino acid levels in vivo, thereby enabling understanding of the spatial alterations of these metabolites under varied disease conditions including cancer.

A new approach for quantitative analysis of L-phenylalanine using a novel semi-sandwich immunometric assay.

Here, we describe a novel method for L-phenylalanine analysis using a sandwich-type immunometric assay approach for use as a new method for amino acid analysis. To overcome difficulties of the preparation of high-affinity and selectivity monoclonal antibodies against L-phenylalanine and the inability to use sandwich-type immunometric assays due to their small molecular weight, three procedures were examined. First, amino groups of L-phenylalanine were modified by "N-Fmoc-L-cysteine" (FC) residues and the derivative (FC-Phe) was used as a hapten. Immunization of mice with bovine serum albumin/FC-Phe conjugate successfully yielded specific monoclonal anti-FC-Phe antibodies. Second, a new derivatization reagent, "biotin linker conjugate of FC-Phe N-succinimidyl ester" (FC(Biotin)-NHS), was synthesized to convert L-phenylalanine to FC-(Biotin)-Phe as a hapten structure. The biotin moiety linked to the thiol group of cysteine formed a second binding site for streptavidin/horseradish peroxidase (HRP) conjugates for optical detection. Third, a new semi-sandwich-type immunometric assay was established using pre-derivatized L-phenylalanine, the monoclonal anti-FC-Phe antibody, and streptavidin/HRP conjugate (without second antibody). Using the new "semi-sandwich" immunometric assay system, a detection limit of 35 nM (60 amol per analysis) and a detection range of 0.1-20 µM were attained using a standard L-phenylalanine solution. Rat plasma samples were analyzed to test reliability. Intra-day assay precision was within 6% of the coefficient of variation; inter-day variation was 0.1%. The recovery rates were from 92.4 to 123.7%. This is the first report of the quantitative determination of L-phenylalanine using a reliable semi-sandwich immunometric assay approach and will be applicable to the quantitative determination of other amino acids.

Development of a novel single-chain l-glutamate oxidase from Streptomyces sp. X-119-6 by inserting flexible linkers.

L-glutamate oxidase (LGOX, EC: 1.4.3.11) is an oxidoreductase that catalyzes L-glutamate deamination. LGOX from Streptomyces sp. X-119-6 is used widely for L-glutamate quantification in research and industrial applications. This enzyme encoded as a single precursor chain that undergoes post-translational cleavage to four fragments by an endogenous protease to become highly active. Efficient preparation of active LGOX by heterologous expression without proteolysis process should be indispensable for wide application of this enzyme. Thus, developing an LGOX that requires no protease treatment should expand the potential applications of recombinant LGOX. In this report, we succeeded in obtaining an active single-chain LGOX by connecting the four fragments of the mature form with insertion of flexible linkers. The most active single-chain mutant showed the similar activity to that of the mature form from Streptomyces sp. X-119-6. The structure of this mutant was determined at 2.9 Å resolution by X-ray crystallography. It was revealed that this single-stranded mutant had the similar conformation to that of mature form. This single-chain LGOX can be produced efficiently and should expand LGOX applications.

Ambient temperature structure of phosphoketolase from Bifidobacterium longum determined by serial femtosecond X-ray crystallography.

Phosphoketolase and transketolase are thiamine diphosphate-dependent enzymes and play a central role in the primary metabolism of bifidobacteria: the bifid shunt. The enzymes both catalyze phosphorolytic cleavage of xylulose 5-phosphate or fructose 6-phosphate in the first reaction step, but possess different substrate specificity in the second reaction step, where phosphoketolase and transketolase utilize inorganic phosphate (Pi) and D-ribose 5-phosphate, respectively, as the acceptor substrate. Structures of Bifidobacterium longum phosphoketolase holoenzyme and its complex with a putative inhibitor, phosphoenolpyruvate, were determined at 2.5 Šresolution by serial femtosecond crystallography using an X-ray free-electron laser. In the complex structure, phosphoenolpyruvate was present at the entrance to the active-site pocket and plugged the channel to thiamine diphosphate. The phosphate-group position of phosphoenolpyruvate coincided well with those of xylulose 5-phosphate and fructose 6-phosphate in the structures of their complexes with transketolase. The most striking structural change was observed in a loop consisting of Gln546-Asp547-His548-Asn549 (the QN-loop) at the entrance to the active-site pocket. Contrary to the conformation of the QN-loop that partially covers the entrance to the active-site pocket (`closed form') in the known crystal structures, including the phosphoketolase holoenzyme and its complexes with reaction intermediates, the QN-loop in the current ambient structures showed a more compact conformation with a widened entrance to the active-site pocket (`open form'). In the phosphoketolase reaction, the `open form' QN-loop may play a role in providing the binding site for xylulose 5-phosphate or fructose 6-phosphate in the first step, and the `closed form' QN-loop may help confer specificity for Pi in the second step.

Analytical Chemistry of Impurities in Amino Acids Used as Nutrients: Recommendations for Regulatory Risk Management.

Proteinogenic amino acids are natural nutrients ingested daily from standard foods. Commercially manufactured amino acids are added to a wide range of nutritional products, including dietary supplements and regular foods. Currently, the regulatory risk management of amino acids is conducted by means of setting daily maximum limits of intake. However, there have been no reported adverse effects of amino acid overdosing, while impurities in low-quality amino acids have been identified as causative agents in several health hazard events. This paper reviews the analytical chemistry of impurities in amino acids and highlights major variations in the purity of commercial products. Furthermore, it examines the international standards and global regulatory risk assessment of amino acids utilized in dietary supplements and foods, recommending (1) further research on analytical methods that can comprehensively separate impurities in amino acids, and (2) re-focusing on the regulatory risk management of amino acids to the analytical chemistry of impurities.

Structural insights into the enhanced thermostability of cysteine substitution mutants of L-histidine decarboxylase from Photobacterium phosphoreum.

Enzymatic amino acid assays are important in physiological research and clinical diagnostics because abnormal amino acid concentrations in biofluids are associated with various diseases. L-histidine decarboxylase from Photobacterium phosphoreum (PpHDC) is a pyridoxal 5'-phosphate-dependent enzyme and a candidate for use in an L-histidine quantitation assay. Previous cysteine substitution experiments demonstrated that the PpHDC C57S mutant displayed improved long-term storage stability and thermostability when compared with those of the wild-type enzyme. In this study, combinational mutation experiments of single cysteine substitution mutants of PpHDC were performed, revealing that the PpHDC C57S/C101V/C282V mutant possessed the highest thermostability. The stabilizing mechanism of these mutations was elucidated by solving the structures of PpHDC C57S and C57S/C101V/C282V mutants by X-ray crystallography. In the crystal structures, two symmetry-related PpHDC molecules form a domain-swapped homodimer. The side chain of S57 is solvent exposed in the structure, indicating that the C57S mutation eliminates chemical oxidation or disulfide bond formation with a free thiol group, thereby providing greater stability. Residues 101 and 282 form hydrophobic interactions with neighboring hydrophobic residues. Mutations C101V and C282V enhanced thermostability of PpHDC by filling a cavity present in the hydrophobic core (C101V) and increasing hydrophobic interactions.

Lung cancer screening by single-shot dual-energy subtraction using flat-panel detector.

PURPOSE: The purpose of this study was to evaluate the usefulness of single-shot dual-energy subtraction (DES) method using a flat-panel detector for lung cancer screening MATERIALS AND METHODS: The subjects were 13,315 residents (5801 males and 7514 females) aged 50 years or older (50-97 years, with an intermediate value of 68 years) who underwent lung cancer screening for a period of 1 year and 6 months from January 2019 to June 2020. We investigated whether the number of lung cancers detected, the detection rate, and the rate of required scrutiny changed, when DES images were added to the judgment based on conventional chest radiography. RESULTS: When DES images were added, the number and percentage of cancer detection increased from 16 (0.12%) to 23 (0.17%) (P < 0.05). Five of the newly detected 7 lung cancers were in the early stages of resectable cancer. The rate of participants requiring scrutiny increased slightly from 1.1 to 1.3%. CONCLUSION: DES method improved the detection of lung cancer in screening. The increase in the percentage of participants requiring scrutiny was negligible.

Automated precolumn derivatization system for analyzing physiological amino acids by liquid chromatography/mass spectrometry.

An automated method for high-throughput amino acid analysis, using precolumn derivatization high-performance liquid chromatography/electrospray mass spectrometry (HPLC/ESI-MS), was developed and evaluated. The precolumn derivatization step was performed in the reaction port of a home-built auto-sampler system. Amino acids were derivatized with 3-aminopyridyl-N-hydroxysuccinimidyl carbamate, and a 3 microm Wakosil-II 3C8-100HG column (100 x 2.1 mm i.d.) was used for separation. To achieve a 13 min cycle for each sample, the derivatization and separation steps were performed in parallel. The results of the method evaluation, including the linearity, and the intra- and inter-precision, were sufficient to measure physiological amino acids in human plasma samples. The relative standard deviations of typical amino acids in actual human plasma samples were below 10%.

Multifunctional and highly sensitive precolumn reagents for amino acids in liquid chromatography/tandem mass spectrometry.

We have developed novel precolumn derivatization reagent, p-N,N,N-trimethylammonioanilyl N'-hydroxysuccinimidyl carbamate iodide (TAHS), for sensitive analyses of amino acids using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). TAHS, an activated carbamate, was reacted briefly with the amino group to form a ureide bond under mild condition. The derivatives provided selective cleavage at the binding site between the reagent and the amino acid in the collision cell of the mass spectrometer and produced a characteristic fragment derived from the reagent moiety. Using the precursor ion scan mode of the tandem mass spectrometry, amino acids derivatized with the reagents were simultaneously measured on the chromatogram. Selective cleavage also enabled the straightforward isotope ratio analysis of amino acids by the selected reaction monitoring mode, which was applicable in (13)C metabolic flux analysis. TAHS, which contains a cationic quaternary amine, achieved subfemtomole to attomole levels of amino acids detection by measurement in the selected reaction monitoring mode. We also synthesized trideuteriummethyl-substituted TAHS, TAHS-d(3), and demonstrated that the combination of TAHS and TAHS-d(3) is useful in comparing amino acid concentrations between two different samples using a single LC/MS/MS measurement.

Precolumn derivatization reagents for high-speed analysis of amines and amino acids in biological fluid using liquid chromatography/electrospray ionization tandem mass spectrometry.

A rapid analytical method for amines and amino acids was developed, involving derivatization with the novel reagent 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS), followed by reversed-phase high-performance liquid chromatography and electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). More than 100 different analytes with amino groups, including amino acids in biological fluids such as mammalian plasma, could be measured within 10 min. The analytes were easily derivatized with APDS under the mild conditions. Selective reaction monitoring of ESI-MS/MS in positive mode was carried out to include the transitions of all of the protonated molecular ions of analytes derivatized with APDS to the common fragment at m/z 121, which was derived from the amino pyridyl moiety of the reagent. We evaluated the retention time precision, the quantification limits, the linearity, the intra- and inter-day precisions and the accuracy of 22 typical amino acids found in biological fluids, by analyzing a standard amino acid mixture and rat plasma. The intra-day relative standard deviations (RSDs) of the retention times of the 22 amino acids and their internal standards were within 0.9% and the inter-day RSDs were less than 1.1%, except for asparagines, with an RSD of 1.9%. The intra-day and inter-day RSDs of amino acid analyses in rat plasma were within 8.0% and 4.5%, respectively. The method, which facilitates the amino acid analysis of more than 100 samples in a day, represents an alternative to traditional amino acid analysis techniques, such as chromatography using postcolumn derivatization by ninhydrin.

Biaryl axially chiral derivatizing agent for simultaneous separation and sensitive detection of proteinogenic amino acid enantiomers using liquid chromatography-tandem mass spectrometry.

Novel sophisticated derivatizing agents for the efficient enantioselective separation and mass spectrometric detection of d- and l-amino acids have been developed. Two new axially chiral reagents derived from 6,6'-dimethyl-2,2'-biphenyldiamine were synthesized. Their chiral separation and detection abilities were evaluated by derivatizing proteinogenic amino acid standards in reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). The resulting diastereomers derived from the developed derivatizing agents and amino acids could be completely separated, because of the effective chiral environment constructed by the axially chiral biphenyl moiety. After optimizing the reactive group, (R)-4-nitrophenyl N-[2'-(diethylamino)-6,6'-dimethyl-[1,1'-biphenyl]-2-yl]carbamate hydrochloride ((R)-BiAC) was found to be the best reagent for highly sensitive simultaneous d,l-amino acid analysis. Using (R)-BiAC, the complete chiral separation of all derivatized proteinogenic amino acids was achieved within 11.5 min with Rs greater than 1.9, except for certain allo-isomers. An exceptional feature of this reagent was its control of elution order, i.e., it afforded elution of the diastereomers derived from d-amino acids before their l-amino acid counterparts for all 19 proteinogenic amino acids. Sensitive detection was also achieved by introducing a dialkyl amino group and selectively cleaving it at the binding site between the reagent and amino acid. Attomole (amol) detection limits (signal-to-noise ratio = 3) were obtained for the tested d,l-amino acids, in the range 7.0-127 amol. As an example of application, the method was applied to food sample analysis, and detected several d-amino acids. Consequently, the developed method seems likely to facilitate simultaneous determination of enantiomers, including the tiny amounts of d-amino acids found in nature.

Genetic basis for plasma amino acid concentrations based on absolute quantification: a genome-wide association study in the Japanese population.

To assess the use of plasma free amino acids (PFAAs) as biomarkers for metabolic disorders, it is essential to identify genetic factors that influence PFAA concentrations. PFAA concentrations were absolutely quantified by liquid chromatography-mass spectrometry using plasma samples from 1338 Japanese individuals, and genome-wide quantitative trait locus (QTL) analysis was performed for the concentrations of 21 PFAAs. We next conducted a conditional QTL analysis using the concentration of each PFAA adjusted by the other 20 PFAAs as covariates to elucidate genetic determinants that influence PFAA concentrations. We identified eight genes that showed a significant association with PFAA concentrations, of which two, SLC7A2 and PKD1L2, were identified. SLC7A2 was associated with the plasma levels of arginine and ornithine, and PKD1L2 with the level of glycine. The significant associations of these two genes were revealed in the conditional QTL analysis, but a significant association between serine and the CPS1 gene disappeared when glycine was used as a covariate. We demonstrated that conditional QTL analysis is useful for determining the metabolic pathways predominantly used for PFAA metabolism. Our findings will help elucidate the physiological roles of genetic components that control the metabolism of amino acids.

[NMR analysis of a contaminant in heparin].

Recently, it has been reported that certain lots of heparin are associated with an acute, rapid onset of serious side effects indicative of allergic reaction, and (1)H NMR is one of the convenience but strong analytical methods to identify a contaminant in heparin. However, an NMR signal from the contaminant in some cases is overlapped with a satellite peak from heparin, leading a misunderstanding of the presence of the contaminant. Here, we show the satellite peak observed close to the NMR signal of the contaminant, and recommend the (13)C decoupling NMR to discriminate the satellite peak from the contaminant.

Determination of metabolic flux changes during fed-batch cultivation from measurements of intracellular amino acids by LC-MS/MS.

Metabolic flux analysis using (13)C-labeled substrates is a well-developed method for investigating cellular behavior in steady-state culture condition. To extend its application, in particular to typical industrial conditions, such as batch and fed-batch cultivations, a novel method of (13)C metabolic flux analysis is proposed. An isotopomer balancing model was developed to elucidate flux distributions in the central metabolism and all amino acids synthetic pathways. A lysine-producing strain of Escherichia coli was cultivated by fed-batch mode in a growth medium containing yeast extract. Mass distribution data was derived from both intracellular free amino acids and proteinogenic amino acids measured by LC-MS/MS, and a correction parameter for the protein turnover effect on the mass distributions of intracellular amino acids was introduced. Metabolic flux distributions were determined in both exponential and stationary phases. Using this new approach, a culture phase-dependent metabolic shift was detected in the fed-batch culture. The approach presented here has great potential for investigating cellular behavior in industrial processes, independent of cultivation modes, metabolic phase and growth medium.

Comprehensive analytical method for the determination of hydrophilic metabolites by high-performance liquid chromatography and mass spectrometry.

A method for the comprehensive analysis of hydrophilic metabolites, based on a combination of high-performance liquid chromatography and mass spectrometry, is described. We evaluated three types of stationary phases to achieve the separation of highly hydrophilic metabolites. Good chromatographic retention and separation of these metabolites were achieved on a pentafluorophenylpropyl-bonded silica column with gradient elution, using 0.1% aqueous formic acid and acetonitrile as the mobile phase. The optimized conditions allowed the comprehensive determination of the standard 49 kinds of amino acids, 6 kinds of amines, 45 kinds of organic acids, 18 kinds of nucleic bases, 5 kinds of nucleosides, and 14 kinds of nucleotides, and then the linearity, dynamic range, detection limit, and precision of the retention time and the peak area were validated. We applied this method for the targeted analysis of the components in soy sauce. The results from the quantitative determination of amino acids were compared to those obtained with an amino acid analyzer, and the accuracy was in the range between 85 and 119%. The accuracy of other detected components was confirmed to be 105-133% by the recovery rate after the addition of standard compounds. We also applied the method for the nontargeted metabolic profiling of the components in several kinds of soy sauces with the principal component analysis. They were classified by the manufacturing methods, and the components that corresponded to the differences were identified. This method could be useful for the targeted analysis of hydrophilic metabolites as well as their nontargeted metabolic profiling.