A method for determining free amino acids in peptide products based on two-dimensional chromatography and its application

Abstract

The present invention discloses a method for determining free amino acids in peptide products based on two-dimensional chromatography separation and its application. The method of the present invention mainly comprises the following steps: pretreatment of peptide samples, high-efficiency gel filtration chromatography separation, center cutting, i.e. cutting, collecting and concentrating the free amino acid fractions in the chromatographic separation fractions, second-dimensional chromatography separation and derivatization reaction, and ultraviolet visible light detection to obtain the amino acid content in the peptide product to be tested. The present invention removes the serious interference of the main component, i.e., peptides, present in the protein peptide food ingredient sample on the detection of a small amount of free amino acids through off-line two-dimensional chromatography separation and center cutting, thereby overcoming the problem of overlapping or partially overlapping chromatographic peaks of peptides and amino acids, and achieving the effect of accurately determining the free amino acid content in peptide products.

Description

Technical Field

[0001] The invention relates to the field of free amino acid determination, and in particular to a method for determining free amino acids in peptide products based on two-dimensional chromatographic separation and application thereof. Background Art

[0002] The existing national standards or industry standards (such as GB / T22729-2008) for protein peptide products used as food ingredients all stipulate that the determination method of peptide content is: peptide content = acid-soluble protein content - free amino acid content. Since the main component of this type of product is the enzymatic hydrolysis product of protein, that is, a peptide mixture with a relative molecular mass of less than 10,000, it also contains a small amount of free amino acids. According to the requirements of the existing national standards or industry standards for peptide products, the peptide content in this type of product is generally at least 70%, and the free amino acid content is generally around 5%, and at most 10% to 15%. Among them, the method for determining the content of free amino acids is in accordance with the national standard or industry standard of relevant peptide products. The sample is treated with 5% TCA or 3.5% sulfosalicylic acid and precipitated to remove possible large molecular protein components, and then one of the following two chromatographic methods is used to separate and detect the free amino acids, namely, method 1: using the amino acid analyzer method specified in GB5009.124-2016, using cation exchange chromatography for separation, and then post-column ninhydrin derivatization and ultraviolet detection; method 2: using pre-column OPA and FMOC-CL derivatization followed by reverse phase chromatography separation, and then ultraviolet detection or fluorescence detection.

[0003] Since the peptide content in peptide samples is much greater than free amino acids, when the above-mentioned national standard or industry standard method for peptides is used to determine the peptide content, the free amino acid content is detected by amino acid analyzer or pre-column derivatization HPLC method. Since peptides also undergo derivatization reactions, the chromatographic peaks of free amino acids will be seriously interfered by peptides, that is, the chromatographic peaks of the two types of substances will mostly overlap to varying degrees, resulting in large errors in the chromatographic peak area integral of free amino acids and their content calculation results. In addition, different laboratory testers have different ways of cutting overlapping peaks, which makes the reproducibility of results in different laboratories poor. Therefore, a new liquid chromatography determination method that can more accurately determine the free amino acid content in peptide products is proposed, which has important application value for quality inspection agencies that do not yet have the conditions for liquid chromatography-tandem mass spectrometry analysis of amino acids to detect peptide products. Summary of the invention

[0004] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for determining free amino acids in peptide products based on two-dimensional chromatographic separation.

[0005] Another object of the present invention is to provide an application of the method for determining free amino acids in peptide products based on two-dimensional chromatography separation.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A method for determining free amino acids in peptide products based on two-dimensional chromatography separation comprises the following steps:

[0008] (1) Sample pretreatment

[0009] Dissolve the peptide product to be tested with trifluoroacetic acid solution or sulfosalicylic acid solution, centrifuge or filter, and take the supernatant to obtain a pretreated sample solution;

[0010] (2) Gel chromatography separation

[0011] ① Silica gel chromatography separation: A silica gel column (gel particles) is used to perform chromatographic separation on the pretreated sample solution obtained in step (1); wherein the chromatographic separation conditions are as follows: chromatographic column: silica gel column; mobile phase: 30% to 45% by volume of acetonitrile and 0.15% to 0.25% by volume of trifluoroacetic acid; detection wavelength of the ultraviolet detector: 220 nm; flow rate: 0.4 to 0.6 mL / min; column temperature: 20 to 35° C.;

[0012] or

[0013] ② Polysaccharide gel chromatography separation: The pretreated sample solution obtained in step (1) is subjected to chromatographic separation using a polysaccharide gel chromatography column; wherein the chromatographic separation conditions are as follows: chromatographic column: polysaccharide gel chromatography column; mobile phase: 50mmol / L phosphate buffer solution and 0.15-0.25mol / L NaCl solution, pH 6.5-7.2; detection wavelength of the ultraviolet detector: 220nm; flow rate: 0.4-0.6mL / min; column temperature: 20-35°C;

[0014] (3) Heart cutting

[0015] According to the gel chromatography separation results in step ① or ②, the fractions are cut and collected, the relative molecular mass of the collection starting point is 220, and the end collection time is determined according to the time when the last chromatographic peak is completed, the number of collections is 3 to 5 times, the fractions are combined and then freeze-dried, and then dissolved and fixed with pH 2.2 sodium citrate buffer, and membrane filtered to obtain a free amino acid solution to be tested;

[0016] (4) Second dimension chromatographic separation and analysis and UV-visible light detection:

[0017] The free amino acid solution obtained in step (3) is separated by an automatic amino acid analyzer or reversed-phase HPLC chromatography, and then detected by ultraviolet visible light to obtain the amino acid content in the peptide product to be tested.

[0018] The peptide products described in step (1) are all protein peptide products (protein peptide food ingredients) in food ingredients; including protein peptide food ingredients of animal origin and plant origin; preferably oyster peptides and soybean peptides.

[0019] The concentration of the trifluoroacetic acid solution described in step (1) is 5% by volume.

[0020] The concentration of the sulfosalicylic acid solution in step (1) is 3.5% by mass volume.

[0021] The concentration of the pretreated sample solution in step (1) is 20 to 25 mg / mL, preferably 20 to 23 mg / mL.

[0022] The chromatographic column described in step (2)① is preferably a silica gel column TSK gel G2000 SWXL 300mm×7.8mm.

[0023] The mobile phase described in step (2)① is preferably: 40% by volume of acetonitrile and 0.1% by volume of trifluoroacetic acid.

[0024] The flow rate described in step (2)① is preferably 0.5 mL / min.

[0025] The column temperature described in step (2)① is preferably 25°C.

[0026] The chromatographic column described in step (2)① is preferably a polysaccharide gel chromatographic column Superdex peptide 10 / 300.

[0027] The mobile phase described in step (2) ② is preferably: 50mmol / L phosphate buffer solution and 0.15mol / L NaCl solution, pH 7.0.

[0028] The flow rate described in step (2) ② is preferably 0.4 mL / min.

[0029] The column temperature described in step (2) ② is preferably 30°C.

[0030] The time for collecting fractions by gel chromatography separation using silica gel matrix in step (3) is 24.5 min to 30 min.

[0031] The time for collecting fractions by polysaccharide gel chromatography separation in step (3) is 43 minutes to 54 minutes.

[0032] The amount of sodium citrate buffer described in step (3) is 0.5 to 1.0 mL.

[0033] The membrane filtration described in step (3) is preferably through a 0.45 μm aqueous phase filter membrane (i.e., a 0.45 μm aqueous phase microporous filter membrane).

[0034] The second-dimensional chromatographic separation analysis described in step (4) is intended to use the collected free amino acid fractions for subsequent further chromatographic separation and determination.

[0035] The second-dimensional chromatographic separation analysis and ultraviolet-visible light detection described in step (4) are preferably achieved by any of the following methods:

[0036] (I) separating the free amino acid solution obtained in step (3) using an amino acid automatic analyzer in a cation exchange chromatography separation mode, and performing post-column ninhydrin derivatization, and then performing ultraviolet visible light detection to obtain the amino acid content in the peptide product to be tested; wherein the conditions for ultraviolet visible light detection are: the detection wavelength for proline and hydroxyproline is 440 nm, and the detection wavelength for amino acids other than proline and hydroxyproline is 570 nm;

[0037] (II) The free amino acid solution obtained in step (3) is subjected to a pre-column derivatization reaction and automatic injection using an HPLC autosampler, and then separated by a reverse phase chromatographic column and then subjected to UV-visible light detection to obtain the content of amino acids in the peptide product to be tested; wherein the conditions for UV-visible light detection are: the detection wavelength for proline and hydroxyproline is 262 nm, and the detection wavelength for amino acids other than proline and hydroxyproline is 338 nm.

[0038] The pre-column derivatization described in method (II) can be selected from pre-column online automatic derivatization or other pre-column derivatization; if it is pre-column online automatic derivatization, the OPA-FMOCL derivatization method can be selected, and the reaction product is separated by reverse phase chromatography and then detected by ultraviolet light at wavelengths of 338nm (amino acids except proline and hydroxyproline) and 262nm (proline and hydroxyproline); if other pre-column derivatization reactions with more stable products are used, after reverse phase chromatography separation, other corresponding wavelengths are selected for ultraviolet or fluorescence detection; OPA-FMOCL derivatization is preferred (because it can be automatically derivatized online and has high detection efficiency).

[0039] The method for determining free amino acids in peptide products based on two-dimensional chromatography separation is used in determining the content of free amino acids in peptide products.

[0040] The peptide products are all protein peptide products in food ingredients, namely protein peptide food ingredients; including protein peptide food ingredients of animal origin and plant origin; preferably oyster peptide and soybean peptide.

[0041] Compared with the prior art, the present invention has the following advantages and effects:

[0042] 1. The method of the present invention comprises the following steps: sample treatment, high-efficiency gel filtration chromatography separation, heart cutting (i.e., cutting, collecting and concentrating the free amino acid fraction in the gel chromatography separation flow for subsequent second-dimensional chromatography separation and detection), second-dimensional chromatography separation and derivatization reaction, i.e., cation exchange chromatography separation coupled with post-column ninhydrin derivatization reaction (which can be performed using an amino acid automatic analyzer) or pre-column derivatization coupled with reversed-phase chromatography separation, and ultraviolet (or fluorescence) detection. The method removes the serious interference of the main component, i.e., peptides, present in the protein peptide food ingredient sample on the detection of a small amount of free amino acids through off-line two-dimensional chromatography separation and heart cutting, so as to achieve the effect of accurately determining the free amino acid content.

[0043] 2. When a small amount of free amino acids in peptide food ingredients are determined by conventional methods, many of the chromatographic peaks of peptides will overlap or partially overlap with the chromatographic peaks of amino acids after ultraviolet detection, which makes it difficult to accurately determine the free amino acid content and results in a large error. In the present invention, HPGFC (high performance gel chromatography)-HPCEC (high performance cation exchange chromatography) two-dimensional chromatography is combined to eliminate the influence of peptides on the determination of free amino acid content, and the free amino acid fraction and the peptide fraction in the sample are basically separated by high performance gel chromatography, and the free amino acid fraction is cut into the second chromatographic fraction by the "heart cutting" method. The free amino acids are separated in a cation exchange chromatography system, and post-column ninhydrin derivatization reaction and ultraviolet (or fluorescence) detection of each free amino acid component are performed simultaneously; or HPGFC (high performance gel chromatography)-RPLC (reverse phase chromatography) two-dimensional chromatography is combined to eliminate the influence of peptides on the detection of free amino acids, and the free amino acid fractions collected by "heart cutting" are subjected to pre-column derivatization reaction by an automatic sampler and then automatically injected into the second-dimensional chromatography, i.e., reverse phase chromatography system, to separate all free amino acids, and then online ultraviolet (or fluorescence) detection of each free amino acid component is performed. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is a schematic diagram of the cutting and collection of amino acid components in Example 1 of the present invention (the blue dotted box represents the area for collecting amino acid components).

[0045] Figure 2 It is a chromatogram of the amino acid collection components measured by an amino acid analyzer in Example 1 of the present invention.

[0046] Figure 3 It is the LC-MS / MS spectrum verified by liquid chromatography-tandem mass spectrometry in Example 1 of the present invention.

[0047] Figure 4 It is a schematic diagram of separating and collecting amino acid components using a polysaccharide gel column in Example 2 of the present invention (the blue dotted box indicates the area where the amino acid components are collected).

[0048] Figure 5 This is a chromatogram of amino acid components collected by separating a soybean peptide sample using an OPA / FMOC-CL pre-column derivatization reverse phase chromatography and then separated by a polysaccharide gel chromatography column in Example 2 of the present invention.

[0049] Figure 6 It is the LC-MS / MS spectrum verified by liquid chromatography-tandem mass spectrometry in Example 1 of the present invention.

[0050] Figure 7 It is a direct chromatographic analysis chart of the supernatant after the sample in Comparative Example 1 of the present invention is treated with 5% TCA.

[0051] Figure 8 It is a direct chromatographic analysis chart of the supernatant after the sample in Comparative Example 2 of the present invention is treated with 3.5% sulfosalicylic acid. DETAILED DESCRIPTION

[0052] The present invention will be described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0053] Example 1

[0054] This embodiment provides a method for determining free amino acids in oyster peptide products based on two-dimensional chromatography separation, which specifically includes the following steps:

[0055] (1) Sample pretreatment: Accurately weigh 200.0 mg of oyster peptide powder (commercially available product), dissolve in 10 mL of 5% (v / v) trifluoroacetic acid solution, filter and take the supernatant, inject and separate on a high performance gel chromatography system.

[0056] (2) Gel chromatography separation: A silica gel column TSK gel G2000 SWXL 300 mm × 7.8 mm was used, the mobile phase was: 40% (v / v) acetonitrile (ACN) / 0.1% (v / v) trifluoroacetic acid (TFA), the detection wavelength of the ultraviolet detector used for monitoring was 220 nm; the flow rate was 0.5 mL / min; the column temperature was: 25°C.

[0057] (3) Heart cutting: remove most of the peptide components through gel chromatography separation, and collect the free amino acid fraction. The relative molecular mass of the starting point of the gel chromatography separation fraction cutting collection is 220 (collect it appropriately in advance of the retention time of the amino acid with the largest relative molecular mass. The amino acid with the largest relative molecular mass in the protein composition is tryptophan, and its relative molecular mass is 204.22). In order to determine the corresponding retention time and facilitate accurate collection of fractions, firstly, peptide standards with different relative molecular masses were used to prepare a relative molecular mass-retention time calibration curve and its equation under the above-mentioned gel chromatography separation conditions (calibration curve equation: 1gMW=7.01-0.192T; wherein MW is the relative molecular mass and T is the retention time). The series of standards are: (I) cytochrome C, MW 12384; (II) aprotinin, MW 6512; (III) bacillus enzyme, MW 1423: (IV) tyrosine-tyrosine-arginine, MW 451; (V) tyrosine-tyrosine-tyrosine, MW 189 (the series of standards were all purchased from Sigma). The start time for collecting fractions was determined to be 24.5 min according to the retention time corresponding to the relative molecular mass of 220 calculated according to the calibration curve equation. The end time for collecting fractions was determined according to the time when the last chromatographic peak was discharged (the end time for collecting fractions in this experiment was 30 min). The fractions were collected four times, combined and freeze-dried, and then dissolved and fixed to volume with 0.5 mL of pH 2.2 sodium citrate buffer (refer to GB 5009.124-126). After passing through a 0.45-μm aqueous microporous filter membrane, the fractions were transferred to a sample injection bottle for amino acid analysis.

[0058] (4) Second-dimension chromatographic separation and analysis: The collected free amino acid fractions were analyzed and determined by an automatic amino acid analyzer. The separation was performed using a cation exchange column and post-column ninhydrin derivatization (i.e., performed according to the method described in GB5009.124-2016), followed by subsequent ultraviolet-visible light detection.

[0059] (5) UV-visible light detection: The detection wavelengths of visible light spectrophotometry are 570 nm (for amino acids other than proline and hydroxyproline) and 440 nm (for proline and hydroxyproline).

[0060] Figure 1 The relative molecular mass distribution chromatogram of the oyster peptide sample and the dotted box schematic diagram of cutting and collecting free amino acid components according to the above steps are shown in FIG. Figure 2 This is a chromatogram of the amino acid collection components determined by an amino acid analyzer: the free amino acid content in the above oyster peptide sample was analyzed and calculated by the amino acid analyzer to be 2.06 g / 100 g.

[0061] (6) Verification: Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used for verification (the method was based on the determination standard of free amino acids: GB / T 30987-2020). The free amino acid content in the oyster peptide sample was determined to be 2.15 g / 100 g (LC-MS / MS spectrum is shown in Figure 3 ), the results of the determination method of the present invention are close to those of the determination by liquid chromatography-tandem mass spectrometry, and the relative error is 4.27%, which is within the allowable range.

[0062] Example 2

[0063] This embodiment provides a method for determining free amino acids in soybean peptide samples based on two-dimensional chromatography separation. The specific operation method is as follows:

[0064] (1) Sample pretreatment: Accurately weigh 230.0 mg of soybean peptide sample (commercially available product), dissolve it in 3.5% (w / v) sulfosalicylic acid solution to a volume of 10 mL, i.e., a concentration of 23.0 mg / mL, centrifuge and filter, and then inject and separate on a high performance gel chromatography system.

[0065] (2) Gel chromatography separation: A polysaccharide gel chromatography column Superdex peptide 10 / 300 was used, the mobile phase was: 50 mmol / L phosphate buffer solution + 0.15 mol / L NaCl solution, pH 7.0, the detection wavelength of the UV detector was 220 nm: the flow rate was 0.4 mL / min; the column temperature was: 30°C.

[0066] (3) Heart cutting: The relative molecular mass of the starting point of the collection of the fractions separated by gel chromatography is about 220. In order to determine the corresponding retention time and facilitate accurate collection of fractions, a relative molecular mass-retention time calibration curve and its equation are first prepared using peptide standards of different relative molecular masses under the above gel chromatography separation conditions. The series of standards are the same as those in Example 1. The starting time point of collecting fractions is determined by the retention time corresponding to the relative molecular mass 220 calculated by the calibration curve equation, and the end time of collection is determined according to the time when the last chromatographic peak has finished (the start time of collecting fractions in this experiment is 43 minutes and the end time of collection is 54 minutes). Collect 3 times, merge the fractions and freeze-dry them, dissolve them in 1 mL of pH 2.2 sodium citrate buffer to make up the volume (refer to GB 5009.124-126), pass through a 0.45 micron aqueous microporous filter membrane, and transfer them to a sample injection bottle for subsequent amino acid analysis.

[0067] (4) Second-dimension chromatographic separation: The reverse-phase chromatography mode was selected, and a reverse-phase HPLC column (Hypersil ODS 4.0 mm × 250 mm, 5 μm) was used for separation. The HPLC autosampler was used for pre-column derivatization reaction and automatic injection (OPA / FMOC-CL derivatization reaction was used, and the reaction speed was fast and the reaction product was not stable enough, so it was necessary to inject the sample as soon as possible for detection. The specific chromatographic conditions were carried out according to the chromatographic conditions described in 6.3.2.2.2.4 of GB / T 22729-2008; if other pre-column derivatization methods were selected and the product was relatively stable, the pre-column derivatization reaction could also be performed manually).

[0068] (5) UV detection: Pre-column online automatic derivatization (OPA-FMOCL derivatization method) was used, and the UV detection wavelengths after reversed-phase chromatography separation were: 262 nm (proline and hydroxyproline) and 338 nm (other amino acids except proline and hydroxyproline).

[0069] Figure 4 The relative molecular mass distribution chromatogram of the soybean peptide sample separated and analyzed by polysaccharide gel column and the dotted box schematic diagram of the free amino acid components cut and collected according to the above steps. Figure 5 ) and calculated that the free amino acid content in the soybean peptide sample was 2.25g / 100g.

[0070] (6) Verification: Liquid chromatography-tandem mass spectrometry was used for verification (the method was based on GB / T30987-2020), and the free amino acid content in the soybean peptide sample was determined to be 2.39 g / 100 g (LC-MS / MS spectrum is shown in Figure 6 The results of the determination method of the present invention and those of the determination by liquid chromatography-tandem mass spectrometry are close, and the relative error is 6.03%, which is within the allowable range.

[0071] Comparative Example 1

[0072] According to the method in Example 1, the supernatant of the oyster peptide powder sample after 5% (v / v) TCA pretreatment was directly subjected to chromatographic separation (Step (4) of Example 1). The chromatographic analysis results are shown in Figure 7 .

[0073] Comparative Example 2

[0074] According to the method in Example 2, the supernatant of the soybean peptide sample after pretreatment with 3.5% (w / v) sulfosalicylic acid was directly subjected to chromatographic analysis (Step (4) of Example 2). The chromatographic analysis results are shown in Figure 8 .

[0075] Effect Example

[0076] The comparison results of Examples 1-2 of the present invention and Comparative Examples 1-2 are shown in Table 1 (all are the results of three repeated measurements).

[0077] Table 1 Comparison of the present invention and comparative example method

[0078]

[0079] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for determining free amino acids in peptide products based on two-dimensional chromatography separation, characterized in that: The steps include: (1) Sample pretreatment Dissolve the peptide product to be tested with trifluoroacetic acid solution or sulfosalicylic acid solution, centrifuge or filter, and take the supernatant to obtain a pretreated sample solution; (2) Gel chromatography separation ① Silica gel chromatography separation: A silica gel column is used to perform chromatographic separation on the pretreated sample solution obtained in step (1); wherein the chromatographic separation conditions are as follows: chromatographic column: silica gel column; mobile phase: 30% to 45% by volume of acetonitrile and 0.15% to 0.25% by volume of trifluoroacetic acid; detection wavelength of ultraviolet detector: 220 nm; flow rate: 0.4 to 0.6 mL / min; column temperature: 20 to 35° C.; or ② Polysaccharide gel chromatography separation: The pretreated sample solution obtained in step (1) is subjected to chromatographic separation using a polysaccharide gel chromatography column; wherein the chromatographic separation conditions are as follows: chromatographic column: polysaccharide gel chromatography column; mobile phase: 50mmol / L phosphate buffer solution and 0.15-0.25mol / L NaCl solution, pH 6.5-7.2; detection wavelength of the ultraviolet detector: 220nm; flow rate: 0.4-0.6mL / min; column temperature: 20-35°C; (3) Heart cutting According to the gel chromatography separation results in step ① or ②, the fractions are cut and collected, the relative molecular mass of the collection starting point is 220, and the end collection time is determined according to the time when the last chromatographic peak is completed, the number of collections is 3 to 5 times, the fractions are combined and then freeze-dried, and then dissolved and fixed with pH 2.2 sodium citrate buffer, and membrane filtered to obtain a free amino acid solution to be tested; (4) Second dimension chromatographic separation and analysis and UV-visible light detection: The free amino acid solution obtained in step (3) is separated by an automatic amino acid analyzer or reversed-phase HPLC chromatography, and then detected by ultraviolet visible light to obtain the amino acid content in the peptide product to be tested; In step (3), the time for collecting the fractions by gel chromatography using a silica gel matrix is ​​24.5 min to 30 min; In step (3), the time for collecting fractions by polysaccharide gel chromatography separation is 43 min to 54 min.

2. The method according to claim 1, characterized in that: The second-dimensional chromatographic separation analysis and ultraviolet-visible light detection described in step (4) are achieved by any of the following methods: (I) separating the free amino acid solution obtained in step (3) using an amino acid automatic analyzer in a cation exchange chromatography separation mode, and performing post-column ninhydrin derivatization, and then performing ultraviolet visible light detection to obtain the amino acid content in the peptide product to be tested; wherein the conditions for ultraviolet visible light detection are: the detection wavelength for proline and hydroxyproline is 440 nm, and the detection wavelength for amino acids other than proline and hydroxyproline is 570 nm; (II) The free amino acid solution obtained in step (3) is subjected to a pre-column derivatization reaction and automatic injection using an HPLC autosampler, and then separated by a reverse phase chromatographic column and then subjected to UV-visible light detection to obtain the content of amino acids in the peptide product to be tested; wherein the conditions for UV-visible light detection are: the detection wavelength for proline and hydroxyproline is 262 nm, and the detection wavelength for amino acids other than proline and hydroxyproline is 338 nm.

3. The method according to claim 2, characterized in that: The pre-column derivatization described in method (II) is OPA-FMOCL derivatization.

4. The method according to claim 1, characterized in that: The chromatographic column described in step (2)① is a silica gel column TSK gel G2000 SWXL 300mm×7.8mm; The chromatographic column described in step (2)① is a polysaccharide gel chromatographic column Superdex peptide 10 / 300.

5. The method according to claim 1, characterized in that: The mobile phase in step (2) ① is: 40% by volume of acetonitrile and 0.1% by volume of trifluoroacetic acid; The flow rate in step (2) ① is 0.5 mL / min; The column temperature described in step (2)① is 25°C.

6. The method according to claim 1, characterized in that: The mobile phase in step (2) ② is: 50mmol / L phosphate buffer solution and 0.15mol / L NaCl solution, pH 7.0; The flow rate in step (2) ② is 0.4 mL / min; The column temperature described in step (2) ② is 30°C.

7. The method according to claim 1, characterized in that: The concentration of the trifluoroacetic acid solution in step (1) is 5% by volume; The concentration of the sulfosalicylic acid solution in step (1) is 3.5% by mass volume; The concentration of the pretreated sample solution in step (1) is 20-25 mg / mL; The membrane filtration described in step (3) is through a 0.45 μm aqueous phase microporous filter membrane.

8. Use of the method for determining free amino acids in peptide products based on two-dimensional chromatography separation as claimed in any one of claims 1 to 7 in determining the content of free amino acids in peptide products.

9. The use according to claim 8, characterized in that: The peptide product is a protein peptide food ingredient.

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