Method for analyzing amino acids

Abstract

Provided is a new and simple analysis method capable of satisfactorily isolating amino acids from a sample using a liquid chromatograph.　This method for analyzing amino acids uses a liquid chromatograph, and comprises: a sample preparation step for subjecting a sample containing a plurality of types of amino acids to a derivatization process using a derivatizing reagent to obtain a derivatized sample; an introduction step for injecting the derivatized sample into a mobile phase, and introducing the mobile phase into a column; an isolation step for isolating components in the derivatized sample as the derivatized sample passes through the column; and a detection step for detecting the isolated components. The temperature of the column is adjusted to 15-25°C.

Description

Amino acid analysis methods

[0001] The present invention relates to a method for analyzing amino acids using liquid chromatography.

[0002] Many amino acids have an asymmetric carbon atom at the α-position, and exist as D- and L-enantiomers. While most amino acids found in nature, including the building blocks of proteins, are L-amino acids, it has become known in recent years that fermented foods and biological samples contain several D-amino acids in addition to many L-amino acids. Advances in research into the role of D-amino acids in the taste, shelf life, and aroma of foods and ingredients in vivo, as well as in the development of pharmaceuticals and functional foods, have led to a growing demand for the separation of D- and L-amino acids. Because D-amino acids are present in trace amounts in foods and living organisms compared to L-amino acids, there is a need to separate and quantify them from L-amino acids, which are present in high concentrations.

[0003] Patent Document 1 (JP 2023-22595 A) 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 derivatized samples and analyzing them using a high performance liquid chromatograph (hereinafter also referred to as "HPLC").

[0004] Japanese Patent Application Laid-Open No. 2023-22595

[0005] An object of the present invention is to provide a new and simple analytical method that can effectively separate amino acids in a sample using a liquid chromatograph.

[0006] As a result of extensive research, the present inventors have discovered that in an analytical method using liquid chromatography, by adjusting the temperature of a column to a predetermined temperature, amino acids in a sample can be well separated and analyzed under a single set of analytical conditions, and have completed the present invention.

[0007] A first aspect of the present invention relates to a method for analyzing amino acids using a liquid chromatograph, the method comprising: a sample preparation step of derivatizing a sample containing multiple amino acids with a derivatization reagent to obtain a derivatized sample; 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 the sample passes through the column; and a detection step of detecting the separated components, wherein the temperature of the column is controlled to a temperature of 15°C or higher and 25°C or lower.

[0008] According to the present invention, amino acids in a sample can be well separated and analyzed under a single set of analytical conditions, and therefore a simple analytical method can be provided.

[0009] 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. 2 is a diagram showing a chromatogram obtained by analyzing a standard sample of amino acids in Test Example 1. 3 is a diagram showing a chromatogram obtained by analyzing each sample in Test Example 2.

[0010] The present invention provides a method for analyzing amino acids using a liquid chromatograph, comprising: a sample preparation step of derivatizing a sample containing multiple amino acids with a derivatization reagent to obtain a derivatized sample; 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 it passes through the column; and a detection step of detecting the separated components, wherein the temperature of the column is controlled to a temperature of 15° C. or higher and 25° C. or lower. Hereinafter, one embodiment of the method for analyzing amino acids according to the present invention (hereinafter sometimes simply referred to as "the method") will be described.

[0011] [Sample to be Analyzed] The amino acids to be analyzed by this method are preferably protein-constituting amino acids other than proline. Specific examples include aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), histidine (His), threonine (Thr), arginine (Arg), alanine (Ala), tyrosine (Tyr), valine (Val), methionine (Met), cystine ((Cys)2), tryptophan (Trp), isoleucine (Ile), phenylalanine (Phe), leucine (Leu), lysine (Lys), and glycine (Gly). Of these, all amino acids, except for glycine, which does not have an asymmetric carbon atom in its molecule, exist in both D- and L-forms. This method allows simultaneous analysis of samples containing optical isomers of amino acids, both D- and L-forms (hereinafter also referred to as "D / L-amino acids"), under a single set of analytical conditions. This method allows simultaneous analysis of 37 amino acids under a single set of analytical conditions, making it possible to analyze amino acids simultaneously in a simple manner and in a short time.

[0012] [Derivatization Reagent] In this method, a derivatization reagent is used. As a derivatization reagent for amino acids, a compound having a substituent that reacts with a free amino group to promote amino acid analysis has been conventionally used, and examples thereof include ninhydrin, phenylisothiocyanate (PITC), o-phthalaldehyde (OPA), 2,4-dinitrofluorobenzene (DNFB), Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA), and 4-fluoro-7-nitrobenzofurazan (NBD-F).

[0013] In this method, the derivatization reagent derivatizes D / L-amino acids as diastereomers, enabling the derivatized D-amino acids and L-amino acids to be analyzed by fluorescence detection. As such 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 a relatively high hydrophobicity due to the isobutyryl group, 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. Using "OPA / NIBC" as the derivatization reagent allows for better separation and analysis of D / L-amino acids, even under a single set of analytical conditions.

[0014] Both NAC and NIBC are chiral thiols having an optically active site, and by reacting OPA in the presence of these chiral thiols, D / L-amino acids are converted into diastereomeric fluorescent derivatives, which can be analyzed by fluorescence detection.

[0015] In this method, the sample to be analyzed may be manually derivatized in advance to prepare the derivatized sample for analysis. However, from the viewpoint of carrying out this method more efficiently, it is preferable to carry out the derivatization using an automatic sample introduction device (hereinafter also referred to as an autosampler) that has a pretreatment function.

[0016] When an autosampler with a pretreatment function is used, a vial containing a derivatization reagent and a sample to be analyzed is set in the autosampler, which is programmed with an operation of automatically mixing the derivatization reagent and the sample to perform the derivatization treatment, and the derivatized sample can be directly subjected to analysis by executing the program. Note that the derivatization treatment in the autosampler may be performed in the vial, or if the autosampler has a function of mixing in a needle, the derivatization treatment may be performed in the needle.

[0017] A sample containing multiple amino acids can be derivatized with a derivatization reagent, and the resulting derivatized sample can be passed through a column together with a mobile phase for analysis by HPLC.

[0018] [Column] The average particle size of the column packing material is preferably 1 to 6 μm, and the column may have micropores. The specific surface area is preferably 50 to 600 m. ２ The particles may be particles, such as silica particles, having a binding surface that interacts with 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. More specifically, for example, columns using silica particles surface-modified with octadecylsilyl (ODS) groups as the packing material (stationary phase) (hereinafter also referred to as "C18 column"), C ８ H １７ Examples of suitable columns include columns using silica particles surface-modified with hydroxyl groups as a packing material (stationary phase) (hereinafter also referred to as "C8 column"). In order to distinguish between differences in hydrophobicity, a C18 column with a large hydrophobic interaction is preferred.

[0019] The column length 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 refers to a column with an inner diameter of 1 mm or more and less than 3 mm. The use of a semi-micro column can reduce the amount of solvent consumed. A commercially available column can be used, and an example of a suitable column is L-column3 C18 (manufactured by the Chemicals Evaluation and Research Institute, Japan).

[0020] In this method, the column temperature is controlled to a temperature of 15°C or higher and 25°C or lower. The column temperature is preferably controlled to a temperature of 18°C ​​or higher and 22°C or lower, more preferably 20°C. By controlling the column temperature within the above range, even D / L-amino acids can be analyzed simultaneously under a single set of analytical conditions, and it is also possible to analyze 37 amino acids simultaneously under a single set of analytical conditions. In other words, multiple types of amino acids can be analyzed simultaneously by introducing one type of derivatized sample into the column once. Therefore, amino acids can be analyzed simultaneously using a simple method in a short period of time.

[0021] [Mobile Phase] In this method, the mobile phase preferably comprises multiple mobile phases. In this method, the mobile phase preferably includes mobile phase A and mobile phase B, and mobile phase A and mobile phase B are mixed at a ratio that varies over time (hereinafter also referred to as gradient conditions). Analysis is performed by flowing such mobile phase through the column. Specifically, analysis is initiated when the mobile phase flowing through the column is highly hydrophilic, and gradient conditions are set so that the hydrophilicity of the mobile phase gradually decreases over time (by increasing the amount of hydrophobic solvent in the mobile phase). By varying the hydrophilicity of the mobile phase in this way, hydrophilic amino acids pass through the column and are eluted before hydrophobic amino acids. This shortens the analysis time compared to isocratic conditions, in which the mobile phase composition is not changed, and enables simultaneous analysis of multiple amino acids with high reproducibility.

[0022] Mobile phase A is, for example, a buffer solution. Examples of 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. Furthermore, the buffer solution is preferably one that can maintain the pH of the mobile phase after mixing mobile phase A and mobile phase B within 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 preferred. 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. By keeping the pH of the mobile phase within the above range, amino acids can be separated satisfactorily while suppressing column deterioration.

[0023] Mobile phase B is, for example, a mixed solvent system. Examples of solvents that can be used in mobile phase B include water, acetonitrile, methanol, ethanol, 2-propanol, and tetrahydrofuran. These solvents may be used in combination of two or more.

[0024] Among these, a mixed solvent containing acetonitrile and 80 vol % or more of methanol is preferred as mobile phase B. An example of such a mixed solvent is acetonitrile:methanol=10-20:90-80 (volume ratio).

[0025] In a particularly preferred embodiment of this method, the derivatization reagent and mobile phase are set as follows: the sample is derivatized with "OPA / NIBC", and the mixed solvent system in mobile phase B is acetonitrile / methanol = 15:85 (volume ratio).

[0026] [Liquid Chromatography Analysis System] The present method will be described in detail with reference to the drawings. <Configuration of Liquid Chromatography Analysis System> Figure 1 is a schematic diagram of an example of a liquid chromatography analysis system for carrying out the present method. This liquid chromatography 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. Note that the liquid chromatography analysis system 100 is not limited to these configurations, and any other configuration may be added, or any configuration that performs a similar function may be substituted.

[0027] The liquid delivery unit 20 includes 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 in which liquid delivery pumps 21A and 21B are provided to deliver two types of mobile phases (mobile phase A and mobile phase B). 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. The liquid delivery unit 20 also includes a mixer 22 that mixes mobile phase A and mobile phase B, delivered from the liquid delivery pumps 21A and 21B, at a predetermined mixing ratio.

[0028] 1 shows an example in which mobile phase blending unit 10 has mobile phase containers 11a, 11b, 11c, and 11d that store four mobile phases (hereinafter referred to as solvent a, solvent b, solvent c, and solvent d). Mobile phase blending unit 10 is equipped with mixer 12, which includes a plurality of electromagnetic valves with adjustable apertures, that draws in solvent a, solvent b, solvent c, and solvent d from mobile phase containers 11a, 11b, 11c, and 11d and mixes them at a predetermined mixing ratio to prepare mobile phase B. The prepared mobile phase B is delivered by liquid delivery pump 21B.

[0029] The autosampler 30 is equipped with an autoinjector 33 that injects a fixed amount of sample. The autosampler 30 preferably has a function of pretreating an analytical sample, and can be loaded with a derivatization reagent 31 and an analytical sample 32.

[0030] A mobile phase prepared by mixing mobile phase A and mobile phase B at a predetermined ratio is sent to the autosampler 30 via the mixer 22. Using the pretreatment function of the autosampler 30, 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, together with the mixed mobile phase, passes through a column 41 that separates the derivatized amino acid components in the time direction, and the separated derivatized amino acid components contained in the sample are detected by a 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 (e.g., a C18 column).

[0031] The detector 50 is a fluorescence detector that excites the derivatized amino acid components in the sample with excitation light of a specific excitation wavelength, causing them to emit fluorescence, and then detects the fluorescence of the specific fluorescence wavelength. The control unit 60 is electrically connected to the mobile phase blending unit 10, the liquid delivery unit 20, the autosampler 30, the column oven 40, and the detector 50, and has the function of controlling the operation of these units based on set analysis conditions and the function of performing predetermined calculations (such as creating a chromatogram) based on the detection signal. In this embodiment of the method, analysis is performed in the mixer 22 while changing the mixing ratio of multiple mobile phases over time.

[0032] The control unit 60 can set 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 chromatographic 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 operating programs required for controlling the mobile phase blending unit 10, the liquid delivery unit 20, etc., and a RAM that temporarily stores data, etc. during control.

[0033] The CPU included in the control unit 60 appropriately controls the various components of the mobile phase blending unit 10, the liquid delivery unit 20, and the pre-processing of the autosampler 30 in accordance with this operating program, thereby performing the analytical operation described below. The control unit 60 processes the data detected by the detector 50 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.

[0034] Next, an example of an analysis operation using the liquid chromatographic analysis system of FIG. 1 will be described.

[0035] <Sample Preparation Step> An analytical sample and derivatization reagent (OPA / NIBC) are placed in an automatic sample introduction device (autosampler 30) with a pretreatment function. The control unit 60 controls mobile phase B to solvent mixing ratio conditions, controls liquid delivery pumps 21A and 21B so that mobile phase A and mobile phase B have a predetermined initial mixing ratio under gradient conditions, and operates liquid delivery pumps 21A and 21B so that the mixed mobile phase has a predetermined flow rate.

[0036] In the example described below, a mobile phase container 11 containing a phosphate buffer solution as mobile phase A and two mobile phase containers 11b and 11c containing acetonitrile and methanol, respectively, are provided (mobile phase containers 11a and 11d may be unused or may contain water for washing). The controller 60 controls the mixer 12 of the mobile phase blending unit 10 to prepare mobile phase B by mixing acetonitrile and methanol under preset solvent mixing ratio conditions. Then, mobile phase A is delivered by the liquid delivery pump 21A, and mobile phase B is delivered by the liquid delivery pump 21B. The mobile phase mixed in the mixer 22 at a predetermined initial mixing ratio under gradient conditions is passed through the autosampler 30 to the column 41 at a constant flow rate. The solvent mixing ratio of acetonitrile and methanol in mobile phase B and the gradient conditions for mobile phase A and mobile phase B are all set in accordance with the analysis of a derivatized sample derivatized with a derivatization reagent.

[0037] Next, the autosampler 30 is controlled according to an operating program pre-stored in the control unit 60 to derivatize the analytical sample with the derivatization reagent, thereby preparing a derivatized sample. The derivatization process can be performed by weighing predetermined amounts of the derivatization reagent and analytical sample into another vial (not shown) placed in the autosampler 30 and mixing them. Alternatively, if the autosampler 30 has a pretreatment function that allows predetermined amounts of the derivatization reagent and analytical sample to be successively drawn into the needle of the autoinjector 33 and mixed within the needle, the derivatization process can be performed by mixing the derivatization reagent and analytical sample using this function. The analytical sample may be derivatized manually beforehand and then placed in the autosampler 30. However, the pretreatment function of the autosampler 30 can be used to automatically place the derivatization reagent and analytical sample in the autosampler 30, thereby reducing the labor and time required for pretreatment. Furthermore, the constant derivatization reaction time improves the reproducibility of peak area values.

[0038] <Introduction Step> When the control unit 60 issues an instruction to start analysis of a derivatized sample, 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. Note that the control unit 60 controls the column oven 40 so that the temperature of the column 41 reaches a predetermined temperature before the introduction step.

[0039] <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.

[0040] The control unit 60 also changes the mixing ratio of mobile phase A to mobile phase B in the mixer 22 over time according to gradient conditions from the time of injection of the derivatized sample. That is, during analysis, the control unit 60 supplies mobile phase B containing an organic solvent to the column 41 as a mobile phase while increasing the mixing ratio over time. The control unit 60 may include a gradient time program creation unit configured to execute gradient conditions for mobile phase A and mobile phase B.

[0041] The control unit 60 controls the operation of each component based on a preset analytical control program, thereby obtaining a detection signal from the detector 50. After the last component of the 37 amino acid components has been eluted, the analysis is terminated.

[0042] 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 values ​​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.

[0043] After the analysis is completed, a mixed solution of water and an organic solvent can be sent from the mobile phase blending unit 10 via the liquid sending pump 21B to wash the system, column, etc. This post-processing can prevent the sample from remaining on the probe into which the sample is injected and the precipitation of salts in the system and column.

[0044] This method allows the derivatization of a sample to be analyzed using a single derivatization reagent, and the analysis can be completed under a single set of analytical conditions. This method not only improves the separation performance of D / L-amino acids, but also enables the rapid simultaneous analysis of multiple amino acids.

[0045] An example of a system capable of the above-described control is the Nexera® 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 that can control the mixing ratio of multiple mobile phases. By using the mobile phase blending function and the automatic preprocessing function of the autosampler, analysis schedules with modified mobile phases and gradient conditions can be automatically created and switched, thereby reducing the effort required for mobile phase preparation and derivatization. The above-described embodiment is an example of the present invention, and it is clear that appropriate modifications, corrections, and additions can be made within the spirit and scope of the present invention.

[0046] This method can be applied to the analysis of alcoholic beverages and various foods, as well as amino acid content analysis in various fields such as biochemistry and medicine. Alcoholic beverages include fermented alcoholic beverages such as beer, sake, red wine, and white wine. Foods include fermented foods.

[0047] EXAMPLES The present invention will be specifically explained below by way of examples, but the present invention is not limited to these examples in any way.

[0048] [Preparation Examples of Derivatization Reagents] ・0.1 mol / L borate buffer solution (pH 10.0): Prepared by adding 0.62 g of boric acid and 0.20 g of sodium hydroxide to 100 ml of pure water and completely dissolving. ・o-phthalaldehyde (OPA) reagent: Prepared by adding 0.3 ml of ethanol to 10 mg of OPA and completely dissolving it, followed by adding 0.7 ml of 0.1 mol / L borate buffer solution and 4 ml of pure water. ・N-isobutyryl-L-cysteine ​​(NIBC) solution: Prepared by adding 10 ml of 0.1 mol / L borate buffer solution to 10 mg of NIBC.

[0049] <Derivatization Reagent: OPA / NIBC> An OPA reagent and an NIBC solution were mixed in equal volumes and used for analysis.

[0050] [Example of preparation and injection of derivatized sample using autosampler] 2 μL of derivatization reagent was aspirated into the needle of the autosampler, followed by 1 μL of sample. After mixing in the needle, the mixture was injected into the mobile phase. The analysis sample, vial number, injection volume, number of mixings, mixing volume, waiting time, and air gap volume can be set using the derivatization program.

[0051] [Analytical equipment] 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 thermostatic bath: CTO-40C Communication bus module: SCL-40 Spectrofluorescence detector: RF-20AXS

[0052] [HPLC analysis conditions] Column: L-column 3 C18 (manufactured by Chemicals Evaluation and Research Institute, Japan) Stationary phase: C18, length 150 mm x inner diameter 2.1 mm, packing particle size 2 μm Pre-column filter: connected immediately before the column Mobile phase: <Mobile phase A> Phosphate buffer solution (10 mmol / L, pH 6.9) prepared by adding 0.78 g of sodium dihydrogen phosphate dihydrate and 1.79 g of disodium hydrogen phosphate to 1000 mL of purified water and completely dissolving them <Mobile phase B> Acetonitrile:methanol = 15:85 <Mobile phase gradient conditions (time program)> 8% B (0-7.27 min) → 27% B (18.18-30 min) → 36% B (32 min) → 41% B (40.91 min) → 46% B (54.55 min) → 50% B (58.55 min) → 52% B (65.28 min) → 80% B (66.19-74.36 min) → 8% B (75.27-87 min) <Flow rate> 0.22 mL / min Column temperature: 20°C Sample injection volume: 1 μL Vial: SHIMADZU LabTotal (registered trademark) for LC 1.5 mL, Glass Detector (FL): RF-20AXS, Ex: 350 nm, Em: 450 nm

[0053] Test Example 1: The D / L-amino acids in the sample were diastereomeric fluorescently derivatized by reacting the sample with a derivatization reagent (OPA / NIBC). The derivatized sample was analyzed by HPLC under the above-mentioned analytical conditions and fluorescence detection was performed. Derivatization was performed automatically using an autosampler, and mobile phase B was prepared using the mobile phase blending function of the solvent delivery pump.

[0054] <Evaluation of linearity of calibration curve> Thirty-seven amino acids were separated using a derivatization reagent (OPA / NIBC). The linearity of the calibration curve was evaluated using the contribution ratio (r ２ The linearity of the calibration curves for the 37 amino acids is shown in Table 1.

[0055]

[0056] A chromatogram obtained by analyzing a D / L-amino acid standard solution (37 components, 5 μmol / L each) under the HPLC analysis conditions described above is shown in Figure 2. In Figure 2, the horizontal axis represents time, and the vertical axis represents detector signal intensity. The numbers assigned to the peaks in the chromatogram in Figure 2 correspond to the numbers assigned to the amino acid species in Table 1.

[0057] The results shown in Figure 2 indicate that in this example, 37 components were separated and detected by introducing the derivatized sample (D / L-amino acid standard solution) into the column once under a single set of analytical conditions.

[0058] Test Example 2 The five samples to be analyzed (beer A, beer B, sake, red wine, and white wine) were all commercially available. The five samples to be analyzed (beer A, beer B, sake, red wine, and white wine) were reacted with a derivatization reagent (OPA / NIBC) to diastereomeric fluorescently derivatize the D / L-amino acids in the samples. The derivatized samples were then subjected to HPLC analysis under the above-described analytical conditions and fluorescence detection. Derivatization was performed automatically using an autosampler, and mobile phase B was prepared using the mobile phase blending function of the solvent delivery pump. Figure 3 shows the resulting chromatogram. In Figure 3, the horizontal axis represents time, and the vertical axis represents detector signal intensity. The numbers assigned to the peaks in the chromatogram in Figure 3 correspond to the numbers assigned to the amino acid species in Table 1.

[0059] The results shown in Figure 3 show that in this example, by introducing each of the derivatized samples (beer A, beer B, sake, red wine, and white wine) into the column once, it was possible to separate and detect the amino acids of 30 components of beer A, 31 components of beer B, 29 components of sake, 26 components of red wine, and 28 components of white wine under a single set of analytical conditions.

[0060] Thus, in a highly preferred embodiment of this method, a single derivatization reagent, OPA / NIBC, is used, and a total of 37 D / L-amino acids constituting proteins in a sample can be well separated and accurately quantified. Furthermore, analysis of the 37 amino acids can be completed in 66 minutes (analysis cycle of 87 minutes).

[0061] [Aspects] It will be understood by those skilled in the art that the above-described exemplary embodiments and examples are specific examples of the following aspects: (Item 1) An analytical method according to one aspect is a method for analyzing amino acids using a liquid chromatograph, comprising: a sample preparation step of derivatizing a sample containing multiple amino acids with a derivatization reagent to obtain a derivatized sample, 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 the sample passes through the column, and a detection step of detecting the separated components, wherein the temperature of the column is controlled to a temperature of 15°C or higher and 25°C or lower.

[0062] (Item 2) In the analytical method described in item 1, the column is temperature-controlled at 20°C.

[0063] (Item 3) In the analytical method described in Item 1 or 2, the sample contains L- and D-isomers of amino acids, which are optical isomers, and the L- and D-isomers are separated in the separation step.

[0064] (Item 4) In the analytical method described in any one of Items 1 to 3, the mobile phase is a mixture of mobile phase A and mobile phase B in a ratio that changes over time, and the mobile phase A is a buffer solution, and the mobile phase B contains acetonitrile and 80 vol% or more of methanol.

[0065] (Item 5) In the analytical method described in item 4, the mobile phase A is a buffer solution having a pH of 7.3 or less.

[0066] (Item 6) In the analytical method according to any one of items 1 to 5, the stationary phase comprises silica particles whose surfaces are modified with octadecylsilyl groups.

[0067] (Item 7) In the analytical method according to any one of items 1 to 6, the column is a semi-micro column.

[0068] Although the embodiments and examples of the present invention have been described above, it is also planned from the beginning that the configurations of the above-described embodiments and examples may be appropriately combined.

[0069] The embodiments and examples disclosed herein are illustrative in all respects and should not be considered limiting. The scope of the present invention is defined by the claims, not by the embodiments and examples described above, and is intended to include meanings equivalent to the claims and all modifications within the scope of the claims.

[0070] 10 Mobile phase blending unit, 11, 11a, 11b, 11c, 11d Mobile phase container, 12 Mixer, 20 Liquid delivery unit, 21A, 21B Liquid delivery pump, 22 Mixer, 30 Autosampler, 31 Derivatization reagent, 32 Analysis sample, 33 Autoinjector, 40 Column oven, 41 Column, 50 Detector, 60 Control unit, 70 Display unit, 100 Liquid chromatographic analysis system.

Claims

1. A sample preparation step involves derivatizing a sample containing multiple types of amino acids with one type of derivatization reagent to obtain a derivatized sample, An introduction step involves injecting the derivatized sample into the mobile phase and introducing it into the column, A separation step of separating the components in the derivatized sample while it passes through the column, A detection step for detecting the separated components, An analytical method for simultaneously analyzing amino acids using a liquid chromatograph having the following characteristics: The aforementioned column is temperature-controlled to a temperature between 15°C and 25°C in this analytical method.

2. The analytical method according to claim 1, wherein the column is temperature-controlled to 20°C.

3. The aforementioned sample contains L-form and D-form of an amino acid, which are optical isomers. The analytical method according to claim 1 or 2, wherein the L-form and the D-form are separated in the separation step.

4. The aforementioned mobile phase is a mixture of mobile phase A and mobile phase B in a ratio that changes over time. The mobile phase A is a buffer solution. The analytical method according to claim 1 or 2, wherein the mobile phase B comprises acetonitrile and 80 vol% or more of methanol.

5. The analytical method according to claim 4, wherein the mobile phase A is a buffer solution with a pH of 7.3 or less.

6. The analytical method according to claim 1 or 2, wherein the column comprises silica particles whose surface is modified with octadecylsilyl groups as the stationary phase.

7. The analytical method according to claim 1 or 2, wherein the column is a semi-micro column.