Amino acid analysis method and amino acid analyzer

Abstract

To provide a new amino acid analysis method and amino acid analysis device having a better effect.SOLUTION: A method for analyzing an amino acid included in a sample is provided, the amino acid analysis method includes: a measurement step (Step A) of measuring the Raman spectrum (SA) of the sample by using Raman spectroscopy; and an amino acid qualitative analysis step (Step B) of identifying the amino acid based on the wave number (WA) of a peak (PA) appearing according to the component of the amino acid included in the Raman spectrum (SA) of the sample. The amino acid is two or more of cystine, methionine, leucine, isoleucine, valine, tryptophan, histidine, phenylalanine, threonine, tyrosine, serine, lysine, glycine, glutamic acid, arginine, and glutamine.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for analyzing amino acids and an amino acid analyzer. [Background technology]

[0002] Conventionally, high performance liquid chromatography (for example, Patent Document 1) and ion chromatography (for example, Patent Document 2) have been used for qualitative and quantitative analysis of amino acids.

[0003] Patent Document 1 proposes the derivatization of amino acids in body fluids, the separation of the derivatized amino acids by liquid chromatography, the identification of the derivatized amino acids by mass spectrometry, and the quantification of the derivatized amino acids by comparison with structurally similar amino acids in a group of amino acid standards. Because structurally similar amino acids share important structural features (e.g., major functional groups), it is disclosed that by separating one amino acid by high-performance liquid chromatography and then identifying it by mass spectrometry, other structurally similar amino acids can be identified by the same method.

[0004] Patent Document 2 proposes a method for separating and analyzing ions in a highly concentrated sample, characterized by using a weakly acidic cation exchange separation column and a weakly acidic eluent, when measuring anions and / or cations in an aqueous solution containing one or more species selected from the group consisting of weak acids with an acid dissociation constant of 2 or more, sugars, alcohols, and amino acids by ion chromatography. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2020-187130 [Patent Document 2] Japanese Patent Application Laid-Open No. 2005-127739 DISCLOSURE OF THE INVENTION [Problem to be solved by the invention]

[0006] In an amino acid analysis method using high performance liquid chromatography (hereinafter sometimes abbreviated as HPLC), it is often difficult to separate or detect amino acids as they are, so it is necessary to use a derivatization reagent to react the amino acids with highly selective (easily detectable) amino acid derivatives to facilitate separation and detection. For example, Patent Document 1 discloses that a measurement sample is derivatized with a derivatization reagent containing phenylisothiocyanate before separation on an HPLC column.

[0007] Generally, derivatization methods include a pre-column derivatization method, in which a measurement sample is derivatized before being introduced into an HPLC column, as disclosed in Patent Document 1, and a post-column derivatization method, in which a measurement sample is derivatized after being separated in an HPLC column.

[0008] In the pre-column derivatization method, the measurement sample and derivatization reagent are directly mixed, so the yield of the target amino acid contained in the measurement sample is easily affected by the measurement sample matrix (coexisting components, solvent, etc.). This can lead to a risk of low reliability of the analytical results. Furthermore, to perform highly sensitive analysis, the combination of the measurement sample and derivatization reagent is limited to those that are less affected by the measurement sample matrix (coexisting components, solvent, etc.). On the other hand, the post-column derivatization method separates the target amino acid from the measurement sample before the derivatization reaction, so it is not affected by the measurement sample matrix and has excellent quantitative and reproducible analytical results. Therefore, there are few limitations on the combination of derivatization reagent and measurement sample, and it has the advantage of being easy to automate the analytical device by performing derivatization in-line. However, there is a risk that the cost of the derivatization reagent (analysis cost) will be high because the derivatization reagent must be continuously added from the start to the end of the analysis.

[0009] In general amino acid analysis methods using high performance liquid chromatography, an ultraviolet detector is used as the amino acid detector. In HPLC analysis using a UV detector as the detector, for example, to detect amino acids that have a carboxyl group and a benzene ring, it is necessary to change the wavelength of the UV detector during the analysis, making automation difficult.

[0010] On the other hand, in ion chromatography (hereafter sometimes abbreviated as IC), derivatization reagents are also used for the separation and detection of amino acids. Because derivatization reagents react with amino groups, if amines are present in the measurement sample, the amines will also be derivatized. As a result, there is a risk of erroneously detecting amines as amino acids. Furthermore, most amines do not have anionic functional groups such as carboxyl groups and are often more basic than amino acids. Therefore, in cation exchange, the retention time of amines is longer than that of amino acids. Therefore, cation exchange facilitates the separation of amines and amino acids, enabling analysis without interfering with the quantification of amino acids. However, this separation can take a long time; for example, it can take as long as 60 minutes to separate eight amino acids.

[0011] As described above, amino acid analysis using HPLC or IC requires high reagent costs, takes time to separate amino acids, and has the potential for false positives. Furthermore, because the HPLC and IC analytical equipment themselves are expensive, a cheaper analytical method was needed.

[0012] Therefore, an object of the present disclosure is to provide a new amino acid analysis method and amino acid analyzer that are more effective in overcoming these problems. [Means for solving the problem]

[0013] The present inventors have conducted extensive research and discovered that it is possible to identify amino acids by using Raman spectroscopy on a sample containing multiple specific amino acids, and have completed the disclosed technology.

[0014] According to one aspect of the present disclosure, A method for analyzing amino acids contained in a sample, comprising: The method for analyzing amino acids comprises: a measurement step (Step A) of measuring a Raman spectrum (SA) of the sample using Raman spectroscopy; An amino acid qualitative analysis step (Step B) for identifying the amino acid based on the wave number (WA) of a peak (PA) appearing according to the amino acid component contained in the Raman spectrum (SA) of the sample, The method for analyzing amino acids can be provided, wherein the amino acids are a plurality of amino acids selected from the group consisting of cystine, methionine, leucine, isoleucine, valine, tryptophan, histidine, phenylalanine, threonine, tyrosine, serine, lysine, glycine, glutamic acid, arginine, and glutamine. According to another aspect of the present disclosure, a light source that irradiates a sample containing amino acids with excitation light of a single wavelength; a spectrum acquisition unit that acquires a Raman spectrum (SA), which is an intensity distribution for each wave number of Raman scattered light generated in the sample; an amino acid identification unit that identifies an amino acid contained in the sample based on a wave number (WA) of a peak (PA) contained in the Raman spectrum (SA) and the Raman spectrum acquired by the spectrum acquisition unit, according to the amino acid contained in the sample, The amino acid analyzer may be characterized in that the amino acids are a plurality of amino acids selected from the group consisting of cystine, methionine, leucine, isoleucine, valine, tryptophan, histidine, phenylalanine, threonine, tyrosine, serine, lysine, glycine, glutamic acid, arginine, and glutamine. [Effects of the Invention]

[0015] According to the disclosed technique, a new amino acid analysis method and amino acid analyzer having superior effects can be provided. [Brief explanation of the drawings]

[0016] [Figure 1] 1(a) and 1(b) are examples of flow diagrams of the analytical method of the present disclosure. [Figure 2] FIG. 1 is an explanatory diagram showing the configuration of an amino acid analyzer of the present disclosure. [Figure 3] FIG. 2 is an explanatory diagram showing the configuration of a control unit according to the present disclosure. [Figure 4] 1 is the beginning of a flowchart illustrating an example of an amino acid identification method of the present disclosure. [Figure 5] 1 is the middle part of a flowchart showing an example of an amino acid identification method of the present disclosure. [Figure 6] 1 is the final part of a flowchart showing an example of an amino acid identification method of the present disclosure. [Figure 7] 1 is a Raman spectrum (SB) based on a standard solution of arginine according to the present disclosure. [Figure 8] 1 is a Raman spectrum (SB) based on a standard solution of cystine according to the present disclosure. [Figure 9] 1 is a Raman spectrum (SB) based on a standard solution of glutamine according to the present disclosure. [Figure 10] 1 is a Raman spectrum (SB) based on a standard solution of glycine according to the present disclosure. [Figure 11] 1 is a Raman spectrum (SB) based on a histidine standard solution of the present disclosure. [Figure 12] 1 is a Raman spectrum (SB) based on a standard solution of isoleucine according to the present disclosure. [Figure 13] 1 is a Raman spectrum (SB) based on a standard solution of leucine according to the present disclosure. [Figure 14] 1 is a Raman spectrum (SB) based on a standard solution of lysine according to the present disclosure. [Figure 15] 1 is a Raman spectrum (SB) based on a standard solution of methionine according to the present disclosure. [Figure 16] 1 is a Raman spectrum (SB) based on a standard solution of phenylalanine according to the present disclosure. [Figure 17] 1 is a Raman spectrum (SB) based on a standard solution of serine according to the present disclosure. [Figure 18] 1 is a Raman spectrum (SB) based on a standard solution of threonine according to the present disclosure. [Figure 19] 1 is a Raman spectrum (SB) based on a standard solution of tryptophan according to the present disclosure. [Figure 20] 1 is a Raman spectrum (SB) based on a standard solution of tyrosine according to the present disclosure. [Figure 21] 1 is a Raman spectrum (SB) based on a standard solution of valine according to the present disclosure. [Figure 22] 1 is a Raman spectrum (SB) based on a standard solution of glutamic acid according to the present disclosure. [Figure 23] 1 is a calibration curve for arginine of the present disclosure. [Figure 24] Fig. 24(a) is an example of a Raman spectrum (SB) of the present disclosure, and Fig. 24(b) is a conceptual diagram showing the relationship between peak (PA), peak (PB), peak (PC), and peak group (CB) of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0017] The amino acid analysis method and amino acid analyzer according to the present disclosure are described in detail below. Note that the technology according to the present disclosure is not limited to the embodiments described below. Furthermore, in the present disclosure, the term "to" used in describing a numerical value indicates a value equal to or greater than the lower limit and equal to or less than the upper limit.

[0018] In the present disclosure, the wave number of a peak refers to the wave number of the Raman shift. The Raman shift is calculated based on the following formula (1). (Formula 1) Raman shift (cm -1 )=(10 7 ) / wavelength of excitation light-(10 7) / measured wavelength

[0019] 1. Amino acid analysis methods The amino acid analysis method of the present disclosure is a method for analyzing amino acids contained in a sample, and includes a measurement step (Step A) of measuring the Raman spectrum (SA) of the sample using Raman spectroscopy, and an amino acid qualitative analysis step (Step B) of identifying amino acids based on the wavenumbers (WA) of peaks (PA) that appear according to the amino acid components contained in the Raman spectrum (SA) of the sample. wherein the amino acid is one or more of cystine, methionine, leucine, isoleucine, valine, tryptophan, histidine, phenylalanine, threonine, tyrosine, serine, lysine, glycine, glutamic acid, arginine, and glutamine.

[0020] In addition, the amino acid analysis method of the present disclosure may include a peak (PA) determination step (Step P) for determining the wavenumber (WA) of a peak (PA) that appears depending on the amino acid component, and the peak (PA) determination step (Step P) may include the following steps. (1) Prepare standard solutions adjusted to multiple concentrations for each amino acid for cystine, methionine, leucine, isoleucine, valine, tryptophan, histidine, phenylalanine, threonine, tyrosine, serine, lysine, glycine, glutamic acid, arginine, and glutamine. (2) For all standard solutions, Raman spectra based on the standard solutions (SB) are measured using Raman spectroscopy. (3) The Raman spectra (SB) based on the standard solutions of all amino acids are compared, and one or more peaks (PB) specific to each amino acid or groups of peaks (CB) specific to each amino acid containing multiple peaks are found. (4) One peak (PB) or one group of peaks (CB) arbitrarily selected from the peaks (PB) or group of peaks (CB) of all amino acids is defined as the peak (PA) that appears according to the amino acid component, and its wavenumber is defined as the wavenumber (WA). The peak (PA) determination step (Step P) may be performed at any timing before the amino acid qualitative analysis step (Step B). That is, a peak (PA) determined by a peak (PA) determination step (Step P) performed in the past may be used as the peak (PA) of the present disclosure, or a peak (PA) determined by a peak (PA) determination step (Step P) performed either before or after the measurement step (Step A) may be used as the peak (PA) of the present disclosure.

[0021] The amino acid analysis method of the present disclosure further includes an amino acid quantitative analysis step (Step C) for quantifying the concentrations of a plurality of amino acids selected from cystine, methionine, leucine, isoleucine, valine, tryptophan, histidine, phenylalanine, threonine, tyrosine, serine, lysine, glycine, glutamic acid, arginine, and glutamine in a sample, and the amino acid quantitative analysis step (Step C) can include the following steps: Figures 1(a) and (b) show an example of a flow diagram of the amino acid analysis method of the present disclosure.

[0022] Generally, Raman spectroscopic analyzers used in Raman spectroscopy (including the amino acid analyzer of the present disclosure) are inexpensive compared to HPLC, IC, and the like. Raman spectroscopy allows direct measurement without pretreatment such as derivatization of the sample (using derivatization reagents, etc.), and there is no need to continuously flow eluent during analysis as with HPLC or IC, so it has the advantage of low reagent costs and low analysis costs.

[0023] The amino acid analysis method of the present disclosure does not require a procedure for separating amino acids in advance, even in the case of a sample containing multiple amino acids, and is capable of simultaneously identifying multiple amino acids. Therefore, the amino acid analysis method of the present disclosure is labor-saving and allows multiple amino acids to be easily identified. The amino acid analysis method of the present disclosure allows simultaneous analysis of multiple amino acids in one sample, and allows measurements to be completed in a shorter time than other analytical methods. For example, when measuring a sample containing 16 types of amino acids according to the present disclosure, the measurement can be completed in 60 to 200 seconds, and the 16 types of amino acids can be individually identified and quantified. The amino acid analysis method of the present disclosure will be described below.

[0024] In this specification, the "wave number of a peak" refers to the wave number of the peak top unless otherwise specified.

[0025] 1-1. Sample The sample according to the present disclosure is a measurement sample containing multiple of the 16 amino acids, and may be either a solid sample or a liquid sample, as long as it can be placed in an amino acid analyzer (described later) and its Raman spectrum can be measured. In the case of a liquid sample, Raman spectrum measurement is possible using a measurement container such as a quartz cell. Furthermore, in the case of a solid sample, Raman spectrum measurement is also possible by dissolving or dispersing it in a solvent such as water or an organic solvent.

[0026] When measuring a solid sample as is, it is difficult to obtain a Raman spectrum from areas other than the periphery of the sample irradiated with the excitation light described below. On the other hand, in the case of a liquid sample or a solution or dispersion sample in which a solid is uniformly dissolved or dispersed in a solvent, it is preferable in that information about the components contained in the entire liquid sample or the solid sample in the form of a solution or dispersion can be obtained. When the sample is a liquid, information reflecting the entire sample can be obtained by acquiring a Raman spectrum when the liquid is homogenized. On the other hand, when a sample is solid, the components in the solid are not necessarily uniformly distributed. Therefore, the information obtained from the Raman spectrum of a sample in its solid state is biased toward the region irradiated with the excitation light described below, and may not reflect the entire sample. However, even if the sample is solid, information reflecting the entire sample can be obtained by obtaining a Raman spectrum of the sample in a state where the sample is uniformly dissolved or dispersed in a solvent.

[0027] The amino acids according to the present disclosure are a plurality of amino acids selected from the group consisting of cystine, methionine, leucine, isoleucine, valine, tryptophan, histidine, phenylalanine, threonine, tyrosine, serine, lysine, glycine, glutamic acid, arginine, and glutamine, and preferably contain only a plurality of these amino acids. In this case, the sample preferably contains only two or more amino acids selected from the group consisting of cystine, methionine, leucine, isoleucine, valine, tryptophan, histidine, phenylalanine, threonine, tyrosine, serine, lysine, glycine, glutamic acid, arginine, and glutamine (i.e., the sample does not contain any amino acids other than these 16 amino acids). These amino acids include nine essential amino acids (isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, and histidine), and are found in pharmaceuticals, supplements, functional (health) foods, foods for specified health uses, beverages, cosmetics, feed, fertilizer, and media commonly used in cell culture. Therefore, the disclosed technology is suitable for amino acid analysis of pharmaceuticals, supplements, functional (health) foods, foods for specified health uses, beverages, cosmetics, feed, fertilizers, and culture media, and is even more suitable for amino acid analysis of culture media.

[0028] 1-2. Measurement step (Step A) The measurement step (Step A) is a step of measuring the Raman spectrum (SA) of a sample using Raman spectroscopy. The Raman spectrum (SA) can be measured using a known Raman spectroscopic analyzer, but it is preferable to use an amino acid analyzer using the Raman spectroscopy described below. Here, the Raman spectrum (SA) of a sample is obtained by subtracting the Raman spectrum measured in a blank state without a sample (sometimes referred to as the background Raman spectrum) from the raw Raman spectrum of the sample. The Raman spectrum in the blank state includes Raman scattered light from HO and CO in the atmosphere, a quartz cell, etc.

[0029] 1-3. Amino acid qualitative analysis step (Step B) The amino acid qualitative analysis step (Step B) is a step in which amino acids are distinguished based on the wavenumbers (WA) of peaks (PA) that appear according to the amino acid components in the Raman spectrum (SA) of the sample measured in the measurement step (Step A), and individual amino acids contained in the sample are identified.

[0030] 1-4. Peak (PA) determination step (Step P) The peak (PA) determination step (Step P) is a step for determining the wavenumber (WA) of the peak (PA) that appears depending on the amino acid component. Here, the wave number (WA) of the peak (PA) that appears depending on the amino acid component can be determined according to the following procedure. (1) Prepare standard solutions of all amino acids, including cystine, methionine, leucine, isoleucine, valine, tryptophan, histidine, phenylalanine, threonine, tyrosine, serine, lysine, glycine, glutamic acid, arginine, and glutamine, at multiple concentrations. (2) For all standard solutions, Raman spectra based on the standard solutions (SB) are measured using Raman spectroscopy. (3) The Raman spectra (SB) based on the standard solutions of all amino acids are compared, and one or more peaks (PB) specific to each amino acid or groups of peaks (CB) specific to each amino acid containing multiple peaks are found. (4) One peak (PB) or one group of peaks (CB) arbitrarily selected from the peaks (PB) or group of peaks (CB) of all amino acids is defined as the peak (PA) that appears according to the amino acid component, and its wavenumber is defined as the wavenumber (WA). Here, when the peak group (CB) is selected as the peak (PA), the peak (PA) is all peaks in the peak group (CB) (peaks (PC) described later). Peak (PA), Peak (PB), and Peak (PC) may vary within ±2cm depending on measurement conditions such as sample concentration. -1 In such a case, the wave number of each peak can be set to the median value of the wave number variation.

[0031] Here, the Raman spectrum (SB) based on an amino acid standard solution can be the Raman spectrum of the amino acid standard solution, the spectrum obtained by subtracting the background spectrum from the Raman spectrum of the amino acid standard solution, or the spectrum obtained by further subtracting the Raman spectrum of the solvent used in the measurement from the Raman spectrum of the amino acid standard solution minus the background spectrum. The background spectrum refers to the Raman spectrum measured in a blank state without the above-mentioned sample, and refers to spectra such as Raman scattering from HO, Raman scattering from CO, and Raman scattering from a quartz cell present in the optical path of the measurement instrument. These spectra must be corrected in advance, and a sample containing nothing, such as pure water, is typically measured and used as the background. Furthermore, if each amino acid is dissolved in a solvent during measurement, the Raman spectrum (SB) can be obtained by further subtracting the Raman spectrum of the solvent.

[0032] The standard solutions of each amino acid may be prepared at multiple concentrations, or may be commercially available. Here, the standard solutions of each amino acid may be prepared at multiple concentrations, preferably at three or more concentrations, and more preferably at four or more concentrations. If there are three or more standard solutions, the difference in the linearity of the graph (GR) becomes clear when determining the linearity of the graph (GR) created for each amino acid peak, as described below, and the accuracy of the quantitative results of the concentration of multiple amino acids contained in a sample can be improved. Furthermore, while standard solutions of each amino acid may be available in multiple concentrations, it is preferable to select them so that the concentration range is wide (so that the concentrations of the standard solutions of each amino acid are not too close). For example, the concentrations of the standard solutions can be 10 mg / dL, 30 mg / dL, 50 mg / dL, 100 mg / dL, etc. By using such a concentration range, the accuracy of the quantification results of the amino acid concentrations contained in the sample, as described below, can be improved.

[0033] The solvent used to prepare the standard solutions of each amino acid is not particularly limited; any known solvent capable of dissolving the amino acid can be used, such as pure water (purified by removing impurities using an ion exchange resin). When measuring the culture medium, the phosphate-buffered saline-based (phenol red + HEPES) simulated solution (cell culture medium simulated solution) used to prepare the culture medium is preferably used as the solvent. The phosphate-buffered saline-based (phenol red + HEPES) simulated solution is based on a solution of phosphate-buffered saline to which phenol red and HEPES have been added, and to which potassium chloride (KCl), sodium chloride (NaCl), potassium dihydrogen phosphate (KH2PO4), disodium hydrogen phosphate (Na2HPO4), and other components present in the culture medium are added. The amounts of each additive are not particularly limited and can be freely selected to match the components of the culture medium used as the analytical sample.

[0034] The Raman spectrum (SB) based on the standard solution can be measured using a known Raman spectroscopic analyzer, but is preferably measured using an amino acid analyzer employing the Raman spectroscopic analysis method described below. Furthermore, it is preferable to measure the Raman spectrum (SB) based on the standard solution using the same device and under the same conditions as the Raman spectrum (SA) of the sample, from the viewpoint of the accuracy of amino acid identification and quantification. The Raman spectrum (SB) based on the standard solution may be measured in advance, but from the viewpoint of the accuracy of amino acid identification and quantification, it is preferable to measure it close to the time of measurement of the Raman spectrum (SA) of the sample, and more preferably immediately before measurement of the Raman spectrum (SA) of the sample.

[0035] The peak (PB) is a peak specific to each amino acid contained in the Raman spectrum (SB) based on the standard solution of each amino acid, and does not overlap with the peaks of other amino acids (for example, a peak whose peak top wavenumber is 2 cm or less). -1 (peaks that are very far apart). Furthermore, the amino acid-specific peak group (CB), which includes multiple peaks, is formed from multiple peaks included in the Raman spectrum (SB), and the peaks included in the peak group (CB) are referred to as peaks (PC). While the peaks (PC) included in the peak group (CB) are not specific to each amino acid individually, the peak group (CB) is specific to each amino acid as a combination of multiple peaks (PC). In other words, the peak group (CB) in the Raman spectrum (SB) based on a standard solution of one amino acid is an amino acid-specific combination that does not match the peak group (CB) in the Raman spectrum (SB) based on the standard solutions of all other amino acids. It has been confirmed that for the amino acids (the 16 types of amino acids described above) in the present disclosure, there is one or more peaks (PB) or peak groups (CB) for each amino acid.

[0036] Here, when peak (PB) is selected as peak (PA), a single peak becomes peak (PA), and when peak group (CB) is selected, all peaks (PC) included in peak group (CB) become peak (PA). Also, the wavenumber of the peak top of peak (PA) is defined as wavenumber (WA).

[0037] A more specific explanation will be given with reference to FIG. 24. FIG. 24(a) is an example of a Raman spectrum (SB) according to the present disclosure. FIG. 24(b) is a conceptual diagram showing the relationship between peak (PA), peak (PB), peak (PC), and peak group (CB). FIG. 24(a) shows a Raman spectrum (SB) based on a standard solution of one of the 16 amino acids according to the present disclosure. In FIG. 24(a), P1 to P5 are shown in the Raman spectrum (SB). Of these, peaks (PB) are P1 (peak (PB)1 in FIG. 24(b)) and P3 (peak (PB)2), and P2, P4, and P5 will be described as being included in peak group (CB) (referred to as peak group (CB)1). Peak (PA) can be selected from any combination of three peaks, P1 and P3 as peak (PB) or P2, P4 and P5 as peak group (CB). Figure 24(b) shows an example in which peak group (CB) 1 is selected and peaks (PA) are P2, P4 and P5.

[0038] 1-5. Amino acid quantitative analysis step (Step C) The amino acid quantitative analysis step (Step C) is a step for quantifying the concentrations of multiple amino acids in the sample, including cystine, methionine, leucine, isoleucine, valine, tryptophan, histidine, phenylalanine, threonine, tyrosine, serine, lysine, glycine, glutamic acid, arginine, and glutamine.

[0039] The concentration of amino acids contained in the sample is quantified based on the peak intensity of the peaks (PD) that appear according to the amino acid components contained in the Raman spectrum (SA) of the sample measured in the measurement step (Step A) and the graph (GD) described below. Specifically, the peak intensity of the peak (PD) of each amino acid contained in the Raman spectrum is read, and the concentration of the amino acid contained in the sample is quantified from the relationship between the peak intensity of the graph (GD) of the peaks (PD) and the concentration of the amino acid.

[0040] The peaks (PD) are determined by plotting the amino acid concentrations of each standard solution and the peak intensities of the corresponding peaks (PB) or peaks (PC) for cystine, methionine, leucine, isoleucine, valine, tryptophan, histidine, phenylalanine, threonine, tyrosine, serine, lysine, glycine, glutamic acid, arginine, and glutamine measured in the peak (PA) determination step (Step P) against the peaks (PB) or peaks (PC) contained in a combination of peaks (CB). Graphs (GR) are created for each peak (PB) or peak (PC), and one peak (PB) or peak (PC) with the highest linearity is arbitrarily selected and designated as the peak (PD). The wavenumber of the peak (PD) is designated as the wavenumber (WD), and the graph (GR) is designated as the graph (GD). The peak (PD) may vary within ±2cm depending on measurement conditions such as sample concentration. -1 In such a case, the wave number of each peak can be set to the median value of the wave number variation.

[0041] As with the case of peak (PA) described above, the explanation will be made with reference to FIG. 24. Each peak included in peak group (CB) is a peak (PC). Therefore, P2, P4, and P5 are peaks (PC), and are shown as peak (PC) 1, peak (PC) 2, and peak (PC) 3 in FIG. 24(b). All graphs (GR) created based on these peaks (PB) and peaks (PC) are compared, and the graph (GR) with the highest linearity is designated as graph (GD), and its wavenumber peak is designated as peak (PD). In FIG. 24(b), P3 is designated as peak (PD). Here, linearity refers to the coefficient of determination R when the graph (GR) is approximated to the first order. 2 Calculate R 2 indicates the degree to which R is close to 1. For example, 2 The graph (GD) is the graph (GR) with R close to 1. 2 It is preferable that R is closest to 1. 2 is calculated based on the following formula (2).

number

[0042] The concentration of the amino acid contained in the sample is quantified based on the peak intensity of the peak (PD) contained in the Raman spectrum (SA) of the sample and the graph (GD).

[0043] 2. Amino acid analyzer An amino acid analyzer suitable for the amino acid analysis method of the present disclosure will be described in detail with reference to Fig. 2. However, the present disclosure is not limited to this. The amino acid analyzer 1 includes a light source 2 , a spectrum acquisition unit 3 , and an amino acid identification unit 4 . The light source 2 irradiates a sample containing amino acids with excitation light of a single wavelength. The spectrum acquisition unit 3 acquires a Raman spectrum (SA), which is an intensity distribution for each wave number of the Raman scattered light generated in the sample. The amino acid identification unit 4 identifies the amino acids contained in the sample based on the wavenumber (WA) of the peak (PA) contained in the Raman spectrum (SA) and the Raman spectrum (SA) acquired by the spectrum acquisition unit 3, depending on the amino acids contained in the sample. The amino acid analyzer 1 may also include an amino acid quantification unit that quantifies the concentration of amino acids contained in a sample. In the present disclosure, as will be described later, the control unit 44 of the amino acid identification unit 4 includes a concentration detection unit 444, so that the amino acid identification unit 4 also serves as the amino acid quantification unit, but it is also possible to provide them separately.

[0044] Each component of the amino acid analyzer 1 will now be described in more detail. The light source 2 irradiates excitation light of a single wavelength onto the spectrum acquisition unit 3. A known single-wavelength laser light source can be used as the light source 2. The wavelength of the laser light source is not particularly limited, and for example, a laser of a single wavelength such as 1064 nm, 785 nm, 670 nm, 660 nm, 633 nm, 561 nm, 532 nm, 514 nm, or 473 nm can be used, and it is preferable to use a laser of a single wavelength of 785 nm, 670 nm, 633 nm, 532 nm, or 514 nm, and it is more preferable to use a laser of a single wavelength of 785 nm or 532 nm.

[0045] The spectrum acquisition unit 3 includes an irradiation unit 31, a Raman light propagation unit 32, and a spectrometer 33, and guides the excitation light emitted from the light source 2 to the sample, detects and quantifies the Raman scattered light generated in the sample as a Raman spectrum, and acquires Raman spectrum data.

[0046] The irradiation unit 31 is configured to irradiate the sample with the excitation light emitted from the light source 2. For example, the irradiation unit 31 can be configured with a mirror that reflects the excitation light emitted from the light source 2 toward the sample, and a condenser lens that condenses the excitation light reflected by the mirror onto the sample.

[0047] The Raman light propagation unit 32 is configured to propagate the Raman scattered light generated in the sample to the spectrometer 33. For example, the Raman light propagation unit 32 can be configured with a collimating lens 321 that collimates the Raman scattered light generated in the sample, and a condensing lens 323 that condenses the Raman scattered light collimated by the collimating lens 321 onto the spectrometer 33.

[0048] Furthermore, when excitation light is irradiated onto a sample, Raman scattered light having a wavelength different from that of the excitation light is generated in the sample due to the interaction between the excitation light and the sample, and Rayleigh scattered light having the same wavelength as that of the excitation light is also generated. The Raman light propagation unit 32 has a filter 322 that removes this Rayleigh scattered light. In the example shown in FIG. 2, the filter 322 is disposed between the collimating lens 321 and the condenser lens 323.

[0049] The spectrometer 33 includes a diffraction grating 331 and a photodetector 332. The diffraction grating 331 is an optical element that separates light supplied from the sample via the Raman light propagation unit 32 into wavelengths and guides the light to the light receiving surface of the photodetector 332.

[0050] The photodetector 332 has a plurality of light-receiving elements arranged one-dimensionally or two-dimensionally within its light-receiving surface. The photodetector 332 accumulates electric charges proportional to the intensity of light of each wavelength exposed to the light-receiving elements within its light-receiving surface. Data acquired by the photodetector 332 indicating the intensity distribution of the light for each wavelength is output to the amino acid identification unit 4. In the amino acid analyzer of the present disclosure, Rayleigh scattered light is removed by the filter 322 of the Raman light propagation unit 32, so that Raman scattered light is incident on the light-receiving surface of the photodetector 332. Therefore, the data output to the photodetector 332 indicates the intensity distribution of the Raman scattered light for each wavelength. Specific examples of the photodetector 332 include a photodiode, a charge-coupled device (CCD), and a complementary metal oxide semiconductor (CMOS).

[0051] The amino acid identification unit 4 is configured to identify amino acids contained in a sample based on data provided from the photodetector 332 of the spectrometer 33 and notify the identification results. When used also as an amino acid quantification unit, it is configured to calculate concentrations and, if necessary, notify the calculated concentrations of components.

[0052] The amino acid identification unit 4 will be described with reference to Fig. 3. Fig. 3 is a diagram showing an outline of the configuration of the amino acid identification unit 4 in the present disclosure. As shown in Fig. 3, the amino acid identification unit 4 includes an input unit 41, a memory unit 42, a display unit 43, and a control unit 44.

[0053] The input unit 41 is configured with hardware that can input various information such as commands and setting data in response to user operations. Specific examples of the input unit 41 include a mouse, a keyboard, a connector for a portable semiconductor memory, and a communication interface that sends and receives information via network communication or serial communication. Examples of semiconductor memory include a USB (Universal Serial Bus) memory and a CF (Compact Flash) memory.

[0054] The storage unit 42 is configured by hardware that stores data provided from the input unit 41 or the control unit 44 and can read out the stored data. Specific examples of the storage unit 42 include a magnetic disk such as a hard disk (HD), a semiconductor memory, or an optical disk.

[0055] The memory unit 42 includes a first storage area for storing various programs, a second storage area for storing various data such as setting data input from the input unit 41, and a storage area for storing programs and data. The first storage area of ​​the present disclosure stores analytical programs such as an identification program for executing an identification process based on the wavenumbers (WA) of peaks (PA) that appear in the Raman spectrum (SA) of a sample according to the amino acid components, and a quantification program for calculating the concentration of amino acids contained in a sample from calibration curve data based on the peak intensities of peaks (PD) that appear in the Raman spectrum (SA) of the sample according to the amino acid components and a graph (GD). The measurement data includes the Raman spectrum (SA) of the sample, and the setting data includes the wavenumbers (WA) of peaks (PA) and wavenumbers (WD) of peaks (PD) that appear in accordance with the amino acid components, as well as the intensities of each peak, and calibration curve data for quantifying amino acids.

[0056] The identification program identifies each amino acid based on whether or not a peak matching the wavenumber of the peak (PA) of each amino acid is present in the Raman spectrum (SA) of the sample. An example of the amino acid identification flow of the determination method (or identification program) is shown in Figures 4 to 6. Figures 4 to 6 show an example of identifying amino acids dissolved in a phosphate-buffered saline-based (containing phenol red + HEPES) simulated solution.

[0057] In Figures 4 to 6, after measuring the Raman spectrum of a sample in (S-1), a separately measured Raman spectrum of phenol red is subtracted from the Raman spectrum of the sample in (S-2), and a separately measured Raman spectrum of HEPES is further subtracted in (S-3). Here, the Raman spectrum of HEPES is obtained by subtracting the background Raman spectrum from the Raman spectrum of the sample and the Raman spectrum of phenol red. The presence or absence of each amino acid is identified by the presence of a wavenumber peak (WA) indicated for each judgment step. Specifically, in (S-4), 496 cm -1If the answer is YES, cystine is identified as an amino acid in the sample in (S-5). Then, the process proceeds to the next step. If the answer is NO, the process proceeds to the next decision step. Next, the following determination and classification processes are carried out in sequence. In (S-6), 700 cm -1 and 806 cm -1 If the result is YES, methionine is identified as an amino acid in the sample in (S-7). Then, the process proceeds to the next step. If the result is NO, the process proceeds to the next decision step. (S-8) 746cm -1 , 842cm -1 and 962 cm -1 If the result is YES, leucine is identified as the amino acid in the sample in (S-9). Then, the process proceeds to the next step. If the result is NO, the process proceeds to the next decision step. (S-10) 756cm -1 If the result is NO, the process proceeds to "B" in Fig. 5. On the other hand, if the result is YES, the following determination is further made. (S-11) 816cm -1 If the result is YES, isoleucine is identified as an amino acid in the sample in (S-12). Then, the process proceeds to the next step. If the result is NO, the process proceeds to the next decision step. (S-13) 826cm -1 If the result is YES, valine is identified as the amino acid in the sample in (S-14). Then, the process proceeds to the next step. If the result is NO, the process proceeds to the next decision step. (S-15) 868cm -1 and 1006cm -1If the result is YES, tryptophan is identified as an amino acid in the sample in (S-16). Then, the process proceeds to the next step. If the result is NO, the process proceeds to the next decision step. (S-17) 970cm -1 and 990cm -1 If the result is YES, histidine is identified as the amino acid in the sample. Then, proceed to "C" in Figure 5. If the result is NO, proceed to "C" in Figure 5. (S-19) 742cm -1 and 996 cm -1 If the answer is YES, phenylalanine is identified as an amino acid in the sample in (S-20). If the answer is NO, the process proceeds to the next decision step. (S-21) 764cm -1 and 1108 cm -1 If the result is YES, threonine is identified as an amino acid in the sample in (S-22). Then, the process proceeds to the next step. If the result is NO, the process proceeds to the next decision step. (S-23) 832cm -1 If the result is YES, tyrosine is identified as an amino acid in the sample in (S-24). Then, the process proceeds to the next step. If the result is NO, the process proceeds to the next decision step. (S-25) 852cm -1 If the result is NO, the process proceeds to "D" in Fig. 6. On the other hand, if the result is YES, the process proceeds to "E" in Fig. 6, where the following determinations are further made. (S-26) 1070cm -1 If the answer is YES, serine is identified as the amino acid in the sample in (S-28). Then, the process proceeds to the next step. (S-26) 1070cm-1 If it is determined that the peak of lysine is not included, lysine is identified as the amino acid in the sample in (S-27). Then, the process proceeds to the next step. (S-29) 892cm -1 If the result is YES, glycine is identified as an amino acid in the sample in (S-30). Then, the process proceeds to the next step. If the result is NO, the process proceeds to the next decision step. (S-31) 914cm -1 If the answer is YES, glutamic acid is identified as an amino acid in the sample in (S-32). Then, the process proceeds to the next step. If the answer is NO, the process proceeds to the next decision step. (S-33) at 1080cm -1 If the result is YES, arginine is identified as an amino acid in the sample in (S-34). Then, the process proceeds to the next step. If the result is NO, the process proceeds to the next decision step. (S-35) 1140cm -1 If the result is YES, glutamine is identified as an amino acid in the sample in (S-36). The order of identifying each amino acid in the amino acid identification flow is not particularly limited and can be determined arbitrarily as long as the effect of the present disclosure is not impaired.

[0058] The quantitative program is a program that calculates the concentration of an amino acid from the peak intensity of the peak (PD) of the amino acid determined to be present in the sample by the identification program and, for example, an approximate equation (calibration curve data) that is a first-order approximation of the graph (GD).

[0059] The display unit 43 is configured by hardware that displays information indicated in data provided from the control unit 44. Specific examples of the display unit 43 include a liquid crystal display, an EL (Electro Luminescence) display, and a plasma display.

[0060] The control unit 44 is configured by hardware that controls the amino acid identifying unit 4 based on the program stored in the first storage area of ​​the memory unit 42 and the data stored in the second storage area of ​​the memory unit 42. A specific example of the control unit 44 is a chip component having a CPU (Central Processing Unit), ROM (Read On Memory), and RAM (Random Access Memory).

[0061] When the control unit 44 reads out an analysis program stored in the first storage area of ​​the memory unit 42, it loads the read analysis program in the load area of ​​the memory unit 42. In this case, the control unit 44 functions as a spectrum data acquisition unit 441, a conversion unit 442, an identification unit 443, and a concentration detection unit 444, and executes an analysis process for identifying and quantifying amino acids contained in a sample. Incidentally, since the control unit 44 includes a concentration detection unit 444, the amino acid identification unit 4 can also be used as an amino acid quantification unit.

[0062] The spectrum data acquisition unit 441 appropriately adjusts the setting values ​​such as the intensity of the excitation light emitted from the light source 2 to drive the light source 2 and irradiate the sample with the excitation light. The spectrum data acquisition unit 441 also acquires from the spectroscope 33 Raman spectrum data that indicates the intensity distribution for each wavelength of Raman scattered light generated from the sample by irradiation with the excitation light.

[0063] The conversion unit 442 converts the wavelengths of the Raman spectrum into wavenumbers called Raman shift amounts based on the data acquired by the spectrum data acquisition unit 441. Specifically, each wavelength in the Raman spectrum is converted into a wavenumber, which is the number of waves contained in a unit length, and each converted wavenumber is converted into a Raman shift amount, which is the difference from the wavenumber of the excitation light emitted from the light source 2. As a result, a Raman spectrum can be obtained in which the vertical axis represents the intensity of the Raman scattered light (Raman intensity) and the horizontal axis represents the Raman shift amount.

[0064] The identification unit 443 loads an identification program stored in the memory unit 42 into the load area and identifies the amino acids contained in the Raman spectrum (SA) of the measured sample. The identification program determines whether the wavenumber (WA) of the peak (PA) that appears according to the components of 16 types of amino acids is contained in the Raman spectrum (SA), and if so, displays a result indicating that the amino acid having that wavenumber (WA) is contained in the sample.

[0065] The concentration detection unit 444 loads the quantification program stored in the memory unit 42 into the loading area, and calculates the concentration of the amino acids identified as being contained in the sample by the identification unit 443. In this case, the identification unit 443 detects the peak intensity of the peak (PD) of the amino acid contained in the sample, and calculates the concentration of each amino acid using the quantification program. [Example]

[0066] Next, the disclosed technology will be described in detail using examples, but the disclosed technology is not limited to these examples. Amino acid analyzer TSI Inc. Raman spectrometer (Model: ProRaman-L-785-B1s, measurement range: 100–3300 cm -1 ) Amino acids for standard solution preparation (1) Cystine (2) Methionine (3) Leucine (4) Isoleucine (5) Valine (6) Tryptophan (7) Histidine (8) Phenylalanine (9) Threonine (10) Tyrosine (11) Serine (12) Lysine (13) Glycine (14) Glutamic acid (15) Arginine (16) Glutamine

[0067] <Determining the peak (PA) of each amino acid> (Preparation of cell culture medium simulants, preparation of standard solutions of each amino acid) The cell culture medium simulant was prepared by mixing 10 ml of 3000 mg / L HEPES, 0–20 ml of 15 mg / L phenol red, 2.5 ml of 2000 mg / L KCl, 2.5 ml of 80,000 mg / L NaCl, 2.8–4.7 ml of 2000 mg / L KH2PO4, and 0.3–2.2 ml of 11,500 mg / L Na2HPO4, and adding ultrapure water (distilled water purified using ion exchange resin and reverse osmosis membrane) so that the total volume of the mixture did not exceed 100 ml. Tables 1–4 show the specific formulations of the cell culture medium simulant. Next, one of the 16 amino acids was added to the mixture in a predetermined amount and completely dissolved. Ultrapure water was then added to bring the total volume of the mixture to 100 ml to prepare an amino acid standard solution. The amino acid standard solutions were prepared with concentrations of 10 mg / dL, 30 mg / dL, 50 mg / dL, and 100 mg / dL. Following this procedure, standard solutions of 10 mg / dL, 30 mg / dL, 50 mg / dL, and 100 mg / dL were also prepared for the other 15 amino acids. Furthermore, standard solutions of each amino acid and its concentration were prepared in combination with all of the cell culture medium simulants listed in Tables 1-4, and these were used for Raman spectroscopy. The additives (excluding fixed amounts of HEPES) and specific formulations of the prepared cell culture medium simulant are shown in Tables 1 to 4.

[0068] [Table 1]

[0069] [Table 2]

[0070] [Table 3]

[0071] [Table 4]

[0072] The Raman spectrum was measured for all the standard solutions of each amino acid that were prepared. The Raman spectra obtained for all the standard solutions of each amino acid were subtracted from the spectra of the background, phenol red, and HEPES to obtain the Raman spectrum (SB) based on the standard solution of each amino acid. The Raman spectra (SB) obtained based on the standard solutions of each amino acid were compared to find peaks (PB) or peak groups (CB) that did not overlap with other amino acids for each amino acid. For all of these peaks (PB) and peaks (PC) contained in the peak groups (CB), a graph (GR) was created from the peak intensity and the concentration of the standard solution. A first-order approximation was performed for all the graphs (GR) created for each amino acid, and the coefficient of determination, R 2 Calculate R 2 One graph with a value close to 1 was arbitrarily selected and used as the graph (GD) for each amino acid. The peak that formed the graph (GD) was used as the peak (PD). 2 was calculated based on the following formula (2).

number

[0073] [Table 5] [Explanation of symbols]

[0074] 1. Amino acid analyzer 2 light source 3 Spectrum acquisition section 4. Amino acid identification section (also used as amino acid quantification section) 31 Irradiation unit 32 Raman light propagation section 33 Spectrometer 41 Input section 42 Storage section 43 Display section 44 Control Unit 321 Collimating Lens 322 filters 323 Condenser Lens 331 Diffraction Grating 332 Photodetector 441 Spectrum Acquisition Unit 442 Conversion Department 443 Identification Unit 444 Concentration detection unit

Claims

1. A method for analyzing amino acids contained in a sample, comprising: The method for analyzing amino acids comprises: a measurement step (Step A) of measuring a Raman spectrum (SA) of the sample using Raman spectroscopy and subtracting the Raman spectrum of phenol red and the Raman spectrum of HEPES from the Raman spectrum to obtain a Raman spectrum (SA) of the sample; an amino acid qualitative analysis step (Step B) of identifying the amino acid based on the wavenumber (WA) of a peak (PA) that appears according to the amino acid component contained in the Raman spectrum (SA) of the sample; an amino acid quantitative analysis step (Step C) of quantifying the concentrations of a plurality of amino acids selected from cystine, methionine, leucine, isoleucine, valine, tryptophan, histidine, phenylalanine, threonine, tyrosine, serine, lysine, glycine, glutamic acid, arginine, and glutamine in the sample; a peak (PA) determination step (Step P) of determining the wave number (WA) of a peak (PA) appearing in accordance with the amino acid component, the sample is a culture medium, the amino acids are a plurality of cystine, methionine, leucine, isoleucine, valine, tryptophan, histidine, phenylalanine, threonine, tyrosine, serine, lysine, glycine, glutamic acid, arginine, and glutamine; The amino acid quantitative analysis step (Step C) comprises: (1) For each of the amino acids, cystine, methionine, leucine, isoleucine, valine, tryptophan, histidine, phenylalanine, threonine, tyrosine, serine, lysine, glycine, glutamic acid, arginine, and glutamine, a graph (GR) is created showing the amino acid concentration of the standard solution and the peak intensity of the corresponding peak (PB) or peak (PC) for each of the peaks (PB) or peaks (PC) included in the peak group (CB). One of the peaks (PB) or peaks (PC) in the graph (GR) with the highest linearity is arbitrarily selected and designated as peak (PD). Furthermore, the wavenumber of peak (PD) is designated as wavenumber (WD), and a linear approximation is performed on the graph (GR) to calculate the coefficient of determination R2. The graph with R2 closest to 1 is designated as graph (GD). (2) quantifying the concentration of the amino acid contained in the sample based on the peak intensity of the peak (PD) contained in the Raman spectrum (SA) of the sample and the graph (GD); The method for analyzing amino acids, wherein the peak (PA) determination step (Step P) comprises the following steps: (1) For each of cystine, methionine, leucine, isoleucine, valine, tryptophan, histidine, phenylalanine, threonine, tyrosine, serine, lysine, glycine, glutamic acid, arginine, and glutamine, three or more standard solutions having different concentrations are prepared. (2) For all the standard solutions, Raman spectra (SB) based on the standard solutions are measured using Raman spectroscopy. (3) Raman spectra (SB) based on standard solutions of all the amino acids are compared to find one or more peaks (PB) or groups of peaks (CB) that are specific to each amino acid. (4) One peak (PB) or one group of peaks (CB) arbitrarily selected from the peaks (PB) or the group of peaks (CB) of all the amino acids is defined as a peak (PA) appearing according to the amino acid component, and its wavenumber is defined as the wavenumber (WA).

2. In the amino acid quantitative analysis step (Step C), the coefficient of determination R ２ The method for analyzing amino acids according to claim 1, wherein the amino acid sequence is calculated based on the following mathematical formula (2):

3. a light source that irradiates a sample containing amino acids with excitation light of a single wavelength; a spectrum acquisition unit that measures a Raman spectrum (SA), which is an intensity distribution for each wavenumber of Raman scattered light generated in the sample, and acquires the Raman spectrum (SA) of the sample by subtracting the Raman spectrum of phenol red and the Raman spectrum of HEPES from the Raman spectrum; an amino acid identification unit that identifies an amino acid contained in the sample based on a wave number (WA) of a peak (PA) contained in the Raman spectrum (SA) and the Raman spectrum acquired by the spectrum acquisition unit, depending on the amino acid contained in the sample; an amino acid quantification unit that quantifies the concentrations of a plurality of amino acids selected from cystine, methionine, leucine, isoleucine, valine, tryptophan, histidine, phenylalanine, threonine, tyrosine, serine, lysine, glycine, glutamic acid, arginine, and glutamine in the sample, the sample is a culture medium, the amino acids are a plurality of cystine, methionine, leucine, isoleucine, valine, tryptophan, histidine, phenylalanine, threonine, tyrosine, serine, lysine, glycine, glutamic acid, arginine, and glutamine; The amino acid quantification unit (1) For each of the amino acids, cystine, methionine, leucine, isoleucine, valine, tryptophan, histidine, phenylalanine, threonine, tyrosine, serine, lysine, glycine, glutamic acid, arginine, and glutamine, a graph (GR) is created showing the amino acid concentration of the standard solution and the peak intensity of the corresponding peak (PB) or peak (PC) for each of the peaks (PB) or peaks (PC) contained in the peak group (CB). One of the peaks (PB) or peaks (PC) in the graph (GR) with the highest linearity is arbitrarily selected and designated as peak (PD). Furthermore, the wavenumber of peak (PD) is designated as wavenumber (WD), and a linear approximation is performed on the graph (GR) to calculate the coefficient of determination R2. The graph with R2 closest to 1 is designated as graph (GD). (2) quantifying the concentration of the amino acid contained in the sample based on the peak intensity of the peak (PD) contained in the Raman spectrum (SA) of the sample and the graph (GD); The wavenumber (WA) of a peak (PA) contained in the Raman spectrum (SA) is determined according to the following procedure. (1) For each of cystine, methionine, leucine, isoleucine, valine, tryptophan, histidine, phenylalanine, threonine, tyrosine, serine, lysine, glycine, glutamic acid, arginine, and glutamine, three or more standard solutions having different concentrations are prepared. (2) For all the standard solutions, Raman spectra (SB) based on the standard solutions are measured using Raman spectroscopy. (3) Raman spectra (SB) based on standard solutions of all the amino acids are compared, and one or more peaks (PB) or peak groups (CB) containing multiple peaks specific to each amino acid are found. (4) One peak (PB) or one group of peaks (CB) arbitrarily selected from the peaks (PB) or the group of peaks (CB) of all the amino acids is defined as a peak (PA) appearing according to the amino acid component, and its wavenumber is defined as the wavenumber (WA).

4. 4. The amino acid analyzer according to claim 3, further comprising a memory unit having a first storage area for storing an identification program for executing an identification process based on the wavenumber (WA) of a peak (PA) that appears in the Raman spectrum (SA) of the sample according to the amino acid components, and a quantification program for calculating the concentration of the amino acid contained in the sample from calibration curve data based on the peak intensity of a peak (PD) that appears in the Raman spectrum (SA) of the sample according to the amino acid components and the graph (GD).

5. The amino acid quantification unit calculates the coefficient of determination R ２ 5. The amino acid analyzer according to claim 3, wherein the amino acid analyzer calculates the following mathematical formula (2): [Equation 2]

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