WO2025009305A1 - アミノ酸の分析方法 - Google Patents

アミノ酸の分析方法 Download PDF

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WO2025009305A1
WO2025009305A1 PCT/JP2024/020334 JP2024020334W WO2025009305A1 WO 2025009305 A1 WO2025009305 A1 WO 2025009305A1 JP 2024020334 W JP2024020334 W JP 2024020334W WO 2025009305 A1 WO2025009305 A1 WO 2025009305A1
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mobile phase
sample
column
amino acids
derivatized
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奈津紀 岩田
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Shimadzu Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86

Definitions

  • the present invention relates to a method for analyzing amino acids using liquid chromatography.
  • amino acids have an asymmetric carbon atom at the ⁇ position, and exist as D- and L-enantiomers.
  • D- and L-amino acids There is a growing demand for the separation of D- and L-amino acids to advance research into the role of D-amino acids in the taste, storage stability, and aroma of foods and ingredients in the body, and to contribute to the development of pharmaceuticals and functional foods. Since the amount of D-amino acids in foods and living organisms is minute compared to the amount of L-amino acids, it is necessary to separate and quantify them from the L-amino acids that exist in high concentrations.
  • Patent Document 1 JP Patent Publication No. 2023-22595 discloses an amino acid analysis method that can easily analyze the D- and L-isomers of amino acids in a sample.
  • the method described in Patent Document 1 involves preparing two or more types of derivatized samples and analyzing them using a high performance liquid chromatograph (hereinafter also referred to as "HPLC").
  • HPLC high performance liquid chromatograph
  • the present invention aims to provide a new and simple analytical method that can effectively separate amino acids in a sample using liquid chromatography.
  • the first aspect of the present invention relates to a method for analyzing amino acids using a liquid chromatograph, which includes a sample preparation step of obtaining a derivatized sample by derivatizing a sample containing multiple types of amino acids with a derivatization reagent, an introduction step of injecting the derivatized sample into a mobile phase and introducing it into a column, a separation step of separating components in the derivatized sample while passing through the column, and a detection step of detecting the separated components, in which the temperature of the column is controlled to 15°C or higher and 25°C or lower.
  • the present invention provides a simple analytical method that allows for good separation and analysis of amino acids in a sample under a single set of analytical conditions.
  • FIG. 1 is a schematic diagram showing an example of a liquid chromatographic analysis system used in the amino acid analysis method according to the present invention.
  • FIG. 2 is a diagram showing a chromatogram obtained by analyzing a standard sample of amino acids in Test Example 1.
  • FIG. 2 shows chromatograms obtained by analyzing each sample in Test Example 2.
  • the present invention is a method for analyzing amino acids using a liquid chromatograph, which includes a sample preparation step in which a sample containing multiple types of amino acids is derivatized with a derivatization reagent to obtain a derivatized sample, an introduction step in which the derivatized sample is injected into a mobile phase and introduced into a column, a separation step in which components in the derivatized sample are separated while passing through the column, and a detection step in which the separated components are detected, and the temperature of the column is controlled to a temperature of 15°C or higher and 25°C or lower.
  • this method One embodiment of the amino acid analysis method according to the present invention (hereinafter sometimes simply referred to as "this method”) will be described below.
  • the amino acids to be analyzed by this method are preferably protein-constituting amino acids other than proline.
  • D- and L-forms exist in the amino acids except for glycine, which does not have an asymmetric carbon atom in the molecule.
  • D/L-amino acids a sample containing D- and L-forms of amino acids that are optical isomers
  • a derivatization reagent is used.
  • the derivatization reagent for amino acids compounds having a substituent that reacts with a free amino group to promote amino acid analysis have been conventionally used, such as ninhydrin, phenylisothiocyanate (PITC), o-phthalaldehyde (OPA), 2,4-dinitrofluorobenzene (DNFB), N ⁇ -(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA), 4-fluoro-7-nitrobenzofurazan (NBD-F), etc.
  • PITC phenylisothiocyanate
  • OPA o-phthalaldehyde
  • DNFB 2,4-dinitrofluorobenzene
  • FDAA 2,4-dinitro-5-fluorophenyl)-L-alaninamide
  • NBD-F 4-fluoro-7-nitrobenzofurazan
  • the derivatization reagent derivatizes D/L-amino acids as diastereomers, and the derivatized D-amino acids and L-amino acids can be analyzed by fluorescence detection.
  • a derivatization reagent a mixture of OPA and N-acetyl-L-cysteine (NAC) (hereinafter sometimes referred to as "OPA/NAC”) or a mixture of OPA and N-isobutyryl-L-cysteine (NIBC) (hereinafter sometimes referred to as "OPA/NIBC”) is preferably used, and "OPA/NIBC" is more preferably used.
  • NIBC has an isobutyryl group and is relatively highly hydrophobic, and by utilizing this difference in hydrophobicity, it is possible to simultaneously analyze multiple amino acids, specifically, protein-constituting D/L amino acids, including glycine.
  • OPA/NIBC the derivatization reagent, D/L-amino acids can be better separated and analyzed even under one type of analysis condition.
  • Both NAC and NIBC are chiral thiols that have optically active sites, and by reacting OPA in the presence of these chiral thiols, D/L-amino acids are converted into diastereomeric fluorescent derivatives, making them analyzable by fluorescence detection.
  • the derivatization process of the sample to be analyzed may be performed manually in advance, and the derivatized sample may be prepared and analyzed.
  • an automatic sample introduction device hereinafter also referred to as an autosampler
  • a vial containing the derivatization reagent and the sample to be analyzed is set in the autosampler, which is programmed with an operation for automatically mixing the derivatization reagent and the sample for derivatization, and the derivatization process is automatically performed by executing the above settings, and the derivatized sample can be directly subjected to analysis.
  • the derivatization process in the autosampler may be performed in a vial, or if the autosampler has a function for mixing in a needle, the derivatization process may be performed in the needle.
  • a sample containing multiple types of amino acids can be derivatized with a derivatization reagent, and the resulting derivatized sample can be passed through a column together with the mobile phase for analysis by HPLC.
  • the average particle size of the packing material of the column is preferably 1-6 ⁇ m, and may have micropores and may be particles, such as silica particles, with a specific surface area of 50-600 m 2 /g. Such particles may have a binding surface that interacts with the derivatized amino acids to facilitate the separation of the amino acids.
  • Suitable binding surfaces include hydrophobic binding surfaces, such as alkyl binding surfaces that may include C4, C8 or C18 alkyl binding groups.
  • C18 columns columns using silica particles as the packing material (stationary phase) whose surfaces are modified with octadecylsilyl (ODS) groups
  • C8 columns columns using silica particles as the packing material (stationary phase) whose surfaces are modified with C 8 H 17 groups.
  • C18 columns with large hydrophobic interactions are preferred.
  • the length of the column is preferably in the range of 15 to 300 mm, and the inner diameter is preferably in the range of 0.5 to 5 mm.
  • the column is more preferably a semi-micro column.
  • a semi-micro column means a column with an inner diameter of 1 mm or more and less than 3 mm. By using a semi-micro column, it is possible to reduce the amount of solvent consumed.
  • the column can be a commercially available product, and an example of a suitable column is L-column3 C18 (manufactured by Chemicals Evaluation and Research Institute, Japan).
  • the column temperature is adjusted to 15°C or higher and 25°C or lower.
  • the column temperature is preferably adjusted to 18°C or higher and 22°C or lower, more preferably 20°C.
  • the mobile phase is composed of a plurality of mobile phases.
  • the mobile phase includes mobile phase A and mobile phase B, and mobile phase A and mobile phase B are mixed at a ratio that changes with time (hereinafter also referred to as gradient conditions).
  • Such a mobile phase is circulated through the column to perform the analysis.
  • the analysis is started in a state where the hydrophilicity of the mobile phase circulated through the column is high, and the gradient conditions are set so that the hydrophilicity of the mobile phase is gradually decreased (the amount of hydrophobic solvent in the mobile phase is increased) with the passage of time.
  • hydrophilic amino acids pass through the column and are eluted before hydrophobic amino acids, and the time required for analysis can be shortened compared to isocratic conditions in which the composition of the mobile phase is not changed, and multiple types of amino acids can be analyzed simultaneously with high reproducibility.
  • Mobile phase A is, for example, a buffer solution.
  • buffer solutions that can be used for mobile phase A include acetate buffer solutions, phosphate buffer solutions, and borate buffer solutions.
  • the pH of mobile phase A is preferably 5.0 to 12.0, more preferably 5.0 to 7.3, and even more preferably 6.0 to 7.0.
  • the buffer solution is preferably capable of maintaining the pH of the mobile phase after mobile phase A and mobile phase B are mixed in a range of preferably 5.0 to 12.0, more preferably 5.0 to 7.3, and even more preferably 6.0 to 7.0, and a phosphate buffer solution is more preferable.
  • the buffer solution may contain inorganic salts, bacteriostatic agents, surfactants, and the like, as long as the accuracy of the amino acid analysis in this method is not impaired.
  • Mobile phase B is, for example, a mixed solvent system.
  • solvents constituting the mixed solvent system that can be used for mobile phase B include water, acetonitrile, methanol, ethanol, 2-propanol, tetrahydrofuran, etc. These may be used in combination of two or more.
  • mobile phase B is preferably a mixed solvent system containing acetonitrile and 80 vol% or more of methanol.
  • the derivatization reagent and mobile phase are set as follows:
  • ⁇ Configuration of liquid chromatography analysis system> 1 is a schematic diagram of an example of a liquid chromatographic analysis system for carrying out the present method.
  • This liquid chromatographic analysis system 100 includes a mobile phase blending unit 10, a liquid delivery unit 20, an autosampler 30, a column oven 40 and a column 41, a detector 50, a control unit 60, and a display unit 70.
  • the liquid chromatographic analysis system 100 is not limited to these configurations, and any other configuration may be added, or any configuration that exhibits a similar function may be substituted.
  • the liquid delivery unit 20 has a liquid delivery pump that draws in and delivers multiple mobile phases, and a mixer that mixes the multiple mobile phases at a predetermined mixing ratio.
  • Figure 1 shows an example equipped with liquid delivery pumps 21A and 21B that deliver two types of mobile phases (mobile phase A and mobile phase B), in which a mobile phase container 11 storing mobile phase A is connected to the liquid delivery pump 21A, and a mobile phase blending unit 10 is connected to the liquid delivery pump 21B.
  • a mixer 22 is provided that mixes mobile phase A and mobile phase B, which are delivered from the liquid delivery pumps 21A and 21B, respectively, at a predetermined mixing ratio.
  • the mobile phase blending unit 10 has mobile phase containers in which different mobile phases are stored, and FIG. 1 shows an example in which mobile phase containers 11a, 11b, 11c, and 11d in which four mobile phases (hereinafter referred to as solvent a, solvent b, solvent c, and solvent d) are stored are provided.
  • the mobile phase blending unit 10 has a mixer 12 including multiple adjustable electromagnetic valves that draw in solvent a, solvent b, solvent c, and solvent d from the mobile phase containers 11a, 11b, 11c, and 11d and mix them at a predetermined mixing ratio to prepare mobile phase B, and the prepared mobile phase B is delivered by the delivery pump 21B.
  • the autosampler 30 is equipped with an autoinjector 33 that injects a fixed amount of sample.
  • the autosampler 30 preferably has a pretreatment function for the analytical sample, and can accommodate a derivatization reagent 31 and an analytical sample 32.
  • the autosampler 30 is fed with a mobile phase prepared by mixing mobile phase A and mobile phase B at a predetermined ratio via the mixer 22.
  • the derivatization reagent and the analytical sample are premixed in the needle for derivatization, and a predetermined amount of the prepared derivatized sample is injected into the mobile phase by the autoinjector 33.
  • the derivatized sample injected by the autoinjector 33 passes through a column 41 that separates the derivatized amino acid components in the time direction together with the mixed mobile phase, and the separated derivatized amino acid components contained in the sample are detected by the detector 50.
  • the column oven 40 keeps the column 41 at a constant temperature during analysis.
  • the column 41 is, for example, a reverse-phase column (such as a C18 column).
  • Detector 50 is a fluorescence detector that excites derivatized amino acid components in a sample with excitation light of a specific excitation wavelength, causing them to fluoresce, and detects the fluorescence of the specific fluorescent wavelength.
  • Control unit 60 is electrically connected to mobile phase blending unit 10, liquid delivery unit 20, autosampler 30, column oven 40, and detector 50, and has the function of controlling the operation of these units based on set analysis conditions, and the function of performing predetermined arithmetic processing (creating a chromatogram, etc.) based on the detection signal.
  • analysis is performed in mixer 22 while changing the mixing ratio of multiple mobile phases over time.
  • the control unit 60 can set the analysis conditions as appropriate.
  • the analysis conditions include, for example, the type of sample, the type of mobile phase, the type of column, the column temperature, etc. This allows the liquid chromatography analysis system 100 to analyze samples under predetermined analysis conditions.
  • the control unit 60 has a built-in memory unit, which is composed of, for example, a CPU that performs logical operations, a ROM that stores the operating programs required for controlling the mobile phase blending unit 10, the liquid delivery unit 20, etc., and a RAM in which data, etc. are temporarily stored during control.
  • the CPU included in the control unit 60 appropriately controls each part of the mobile phase blending unit 10, the liquid delivery unit 20, and the pre-processing of the autosampler 30 according to this operating program, thereby performing the analytical operation described below.
  • the data detected by the detector 50 is processed by the control unit 60 to identify and quantify the amino acid components in the sample.
  • the display unit 70 is, for example, a liquid crystal display, and displays the analysis results.
  • the control unit 60 can be configured to use a personal computer or a more advanced workstation as a hardware resource, and to realize each function by executing dedicated control and processing software pre-installed on the computer, thereby controlling the entire liquid chromatographic analysis system 100.
  • sample preparation process The analytical sample and derivatization reagent (OPA/NIBC) are placed in an automatic sample introduction device (autosampler 30) having a pretreatment function.
  • the control unit 60 controls the mobile phase B to a solvent mixing ratio condition, controls the liquid delivery pumps 21A and 21B so that the mobile phase A and the mobile phase B have a predetermined initial mixing ratio under the gradient condition, and operates the liquid delivery pumps 21A and 21B so that the mixed mobile phase has a predetermined flow rate.
  • a mobile phase container 11 in which a phosphate buffer is stored as mobile phase A, and two mobile phase containers 11b and 11c in which acetonitrile and methanol are stored, respectively, are provided (mobile phase containers 11a and 11d may be unused, or may store water for washing).
  • the control unit 60 controls the mixer 12 of the mobile phase blending unit 10 to prepare mobile phase B by mixing acetonitrile and methanol under a preset solvent mixing ratio condition.
  • mobile phase A is sent by the liquid sending pump 21A
  • mobile phase B is sent by the liquid sending pump 21B, respectively
  • the mobile phase mixed in the mixer 22 at a predetermined initial mixing ratio under the gradient condition is flowed at a constant flow rate through the column 41 via the autosampler 30.
  • the solvent mixing ratio conditions of acetonitrile and methanol in mobile phase B, and the gradient conditions of mobile phase A and mobile phase B are all conditions set corresponding to the analysis of a derivatized sample derivatized with a derivatization reagent.
  • the autosampler 30 is controlled according to an operation program previously stored in the control unit 60 to derivatize the analytical sample with the derivatization reagent, and prepare the derivatized sample.
  • the derivatization process can be performed by weighing out a predetermined amount of the derivatization reagent and the analytical sample in another vial (not shown) placed in the autosampler 30 and mixing them.
  • the autosampler 30 has a pretreatment function that allows a predetermined amount of the derivatization reagent and the analytical sample to be continuously sucked into the needle of the autoinjector 33 and mixed in the needle, the derivatization process can be performed by mixing the derivatization reagent and the analytical sample using this function.
  • the derivatization process of the analytical sample may be performed manually in advance, and then the derivatized sample may be placed in the autosampler 30.
  • the pretreatment function of the autosampler 30 can be used to control the derivatization process to be performed automatically after the derivatization reagent and the analytical sample are placed in advance, thereby reducing the labor and time required for pretreatment.
  • the reproducibility of the peak area value is improved by making the reaction time of derivatization constant.
  • the autoinjector 33 provided in the autosampler 30 injects a predetermined amount of the derivatized sample into the mobile phase at a predetermined timing in response to the instruction.
  • the control unit 60 controls the column oven 40 so that the temperature of the column 41 becomes a predetermined temperature set in advance before the introduction step.
  • ⁇ Separation step, detection step> The injected derivatized sample is carried along with the flow of the mobile phase and introduced into column 41 . As the sample passes through column 41 , the derivatized amino acid components in the sample are separated in the time direction and eluted from the outlet of column 41 .
  • the control unit 60 also changes the mixing ratio of mobile phase A and mobile phase B in the mixer 22 over time in accordance with the gradient conditions from the time of injection of the derivatized sample. That is, during analysis, the control unit 60 supplies mobile phase B, which contains an organic solvent, to the column 41 as a mobile phase while increasing the mixing ratio over time.
  • the control unit 60 can have a gradient time program creation unit configured to execute the gradient conditions of mobile phase A and mobile phase B.
  • the control unit 60 controls the operation of each component based on a preset analysis control program, and a detection signal is obtained from the detector 50. After the last of the 37 amino acid components has been eluted, the analysis ends.
  • the control unit 60 creates a chromatogram using the obtained data, calculates the peak area values on the chromatogram for amino acids confirmed to be present in the sample, determines the concentration value for each amino acid from the peak area values by referring to a calibration curve created in advance, and creates an analysis result report.
  • the control unit 60 can have a data processing unit that creates the analysis result report.
  • a mixed solution of water and organic solvent can be pumped from the mobile phase blending unit 10 via the pump 21B to wash the system, columns, etc. as a post-processing step.
  • This post-processing step prevents the sample from remaining on the probe that injects the sample and prevents salt precipitation in the system and columns.
  • the sample to be analyzed is derivatized using one type of derivatization reagent, and the analysis can be completed under one set of analytical conditions.
  • This method improves the separation performance of D/L-amino acids and enables rapid simultaneous analysis of multiple amino acids.
  • An example of a system capable of the above-mentioned control is the Nexera (registered trademark) X3 system (manufactured by Shimadzu Corporation).
  • This system is equipped with a low-pressure gradient kit, and the low-pressure gradient unit in the kit corresponds to the mobile phase blending unit described above, and has a mobile phase blending function capable of controlling the mixing ratio of multiple mobile phases.
  • a mobile phase blending function and the automatic pretreatment function of the autosampler an analysis schedule in which the mobile phase and gradient conditions are changed can be automatically created and switched, so that the preparation of the mobile phase and the effort of derivatization can be reduced.
  • the above embodiment is an example of the present invention, and it is clear that appropriate variations, modifications, and additions can be made within the scope of the present invention.
  • Alcoholic beverages include brewed alcoholic beverages such as beer, sake, red wine, and white wine.
  • Foods include fermented foods.
  • OPA o-Phthalaldehyde
  • NIBC N-isobutyryl-L-cysteine
  • OPA/NIBC ⁇ Derivatization reagent: OPA/NIBC>
  • the OPA reagent and NIBC solution were prepared by mixing equal volumes and used in the analysis.
  • HPLC system Nexera X3 (Shimadzu Corporation) ⁇ Degasser: DGC-403, DGC-405 ⁇ Pump: LC-40D X3 (2 units), low pressure gradient kit (1 unit) ⁇ Autosampler: SIL-40C X3 ⁇ Column thermostat: CTO-40C ⁇ Communication bus module: SCL-40 ⁇ Spectrofluorescence detector: RF-20AXS
  • Test Example 1 The sample to be analyzed was reacted with a derivatization reagent (OPA/NIBC) to diastereomerically derivatize the D/L-amino acids in the sample, and the derivatized sample was analyzed by HPLC under the above analytical conditions to detect fluorescence. Derivatization was performed automatically using an autosampler, and mobile phase B was prepared using the mobile phase blending function of the liquid delivery pump.
  • OPA/NIBC derivatization reagent
  • the chromatogram obtained by analyzing a D/L-amino acid standard solution (37 components, 5 ⁇ mol/L each) under the HPLC analysis conditions described above is shown in Figure 2.
  • the horizontal axis indicates time and the vertical axis indicates detector signal intensity.
  • the numbers assigned to the peaks in the chromatogram in Figure 2 correspond to the numbers assigned to the amino acid types in Table 1.
  • the sample contains optical isomers of L- and D-amino acids, and the L- and D-isomers are separated in the separation step.
  • the mobile phase is a mixture of mobile phase A and mobile phase B in a ratio that changes over time, and mobile phase A is a buffer solution, and mobile phase B contains acetonitrile and 80 vol% or more of methanol.
  • the mobile phase A is a buffer solution having a pH of 7.3 or less.
  • the stationary phase includes silica particles whose surfaces are modified with octadecylsilyl groups.
  • the column is a semi-microcolumn.

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PCT/JP2024/020334 2023-07-05 2024-06-04 アミノ酸の分析方法 Ceased WO2025009305A1 (ja)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH075160A (ja) * 1993-06-15 1995-01-10 Mitsubishi Chem Corp 液体クロマトグラフィー
WO1998033064A1 (en) * 1997-01-24 1998-07-30 Amersham Pharmacia Biotech K.K. Method for separating pth amino acids
JP2004198136A (ja) * 2002-12-16 2004-07-15 Nomura Kagaku Kk 逆相液体クロマトグラフィー、液体クロマトグラフ装置およびカラム
WO2013115334A1 (ja) * 2012-01-31 2013-08-08 株式会社資生堂 分離剤及びその製造方法
CN103645266A (zh) * 2013-12-27 2014-03-19 光明乳业股份有限公司 乳基料中游离态手性氨基酸的检测方法
JP2018163155A (ja) * 2017-03-24 2018-10-18 味の素株式会社 アミノ酸の分析方法
JP2023022595A (ja) * 2021-08-03 2023-02-15 株式会社島津製作所 アミノ酸の分析方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH075160A (ja) * 1993-06-15 1995-01-10 Mitsubishi Chem Corp 液体クロマトグラフィー
WO1998033064A1 (en) * 1997-01-24 1998-07-30 Amersham Pharmacia Biotech K.K. Method for separating pth amino acids
JP2004198136A (ja) * 2002-12-16 2004-07-15 Nomura Kagaku Kk 逆相液体クロマトグラフィー、液体クロマトグラフ装置およびカラム
WO2013115334A1 (ja) * 2012-01-31 2013-08-08 株式会社資生堂 分離剤及びその製造方法
CN103645266A (zh) * 2013-12-27 2014-03-19 光明乳业股份有限公司 乳基料中游离态手性氨基酸的检测方法
JP2018163155A (ja) * 2017-03-24 2018-10-18 味の素株式会社 アミノ酸の分析方法
JP2023022595A (ja) * 2021-08-03 2023-02-15 株式会社島津製作所 アミノ酸の分析方法

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