A high performance liquid chromatography method for detecting free chiral amino acid fingerprints of protein products and its application

Abstract

The present invention discloses a high performance liquid chromatography method for detecting free chiral amino acid fingerprints of protein products and its application. First, free amino acids are extracted from protein products, and pre-column derivatization treatment is performed using Nα-(2,4-dinitro-5-fluorophenyl)-L-aminopropionamide, and separation and detection are performed using a high performance liquid chromatography-ultraviolet detector equipped with an Acclaim C18 chromatographic column, and finally the content of each amino acid is calculated using a standard curve. In this method, the derivative product has good stability within 15 hours; the analysis time for a single sample is 75 minutes; it makes up for the defects of the current high equipment cost of such methods, low stability of derivatized products, and long detection time for a single sample; it realizes the qualitative and quantitative characterization of the free chiral amino acid fingerprints of protein products, and provides key information for research on the nutritional metabolism and physiological functions of food-borne D-amino acids.

Description

Technical Field

[0001] This invention belongs to the field of chiral amino acid detection technology, specifically relating to a high-performance liquid chromatography method for detecting the fingerprint spectrum of free chiral amino acids in protein products and its application. Background Technology

[0002] The α-carbon, serving as the stereocenter of amino acids, connects the amino group, carboxyl group, hydrogen atom, and side chain group. Among the amino acids that make up proteins, all except glycine possess a chiral α-carbon atom, giving each of them two mirror-image stereoisomers (D-amino acids and L-amino acids). For a long time, research on protein amino acids has primarily focused on L-amino acids, but in recent years, more and more studies have paid attention to D-amino acids. Studies have shown that chiral amino acids are widely present in nature, especially in protein products where their abundance is relatively high. For example, yogurt can contain up to 53.8% (D-alanine), 40.3% (D-aspartic acid), and 24.2% (D-glutamic acid) of D-amino acids. Specific D-amino acids also play functions such as regulating nerve signal transduction, hormone secretion, bacterial biofilm formation, and immune system activity. Therefore, the detection of D-amino acids can provide crucial information for controlling the quality of processed protein products and studying the nutritional metabolism and physiological functions of dietary D-amino acids.

[0003] The existing methods for detecting chiral amino acids are mainly high performance liquid chromatography (HPLC) and high performance liquid chromatography-mass spectrometry (HPLC-MS / MS), in addition to gas chromatography-mass spectrometry (GC-MS / MS), capillary electrophoresis, and sensor methods.

[0004] KONYA et al. reported a method for detecting D-amino acids in yogurt based on high-performance liquid chromatography combined with chiral columns and time-of-flight mass spectrometry (Konya Y, Taniguchi M, Fukusaki E. Novel high-throughput and widely-targeted liquid chromatography-time of flight mass spectrometry method for d-amino acids in foods[J]. Journal of bioscience and bioengineering, 2017, 123(1):126-133.). The drawbacks of this method are: the chiral column used is more expensive than a conventional C18 column, and it requires a mass spectrometer for detection, resulting in a relatively high overall detection cost.

[0005] A Chinese patent application discloses a method for detecting free chiral amino acids in emulsion base materials, using a pre-column derivatization method and a high-performance liquid chromatograph (HPLC) equipped with a fluorescence detector to detect 35 free chiral amino acids in emulsion base materials. The limitations of this method are: 1) Poor stability: the analytes derived from N-isobutyryl-L-cysteine ​​and phthalaldehyde are stable for only 1.5-18 minutes at room temperature, requiring continuous and cumbersome derivatization processes for batch testing, potentially leading to inconsistent data between batches. 2) High equipment cost: the method based on N-isobutyryl-L-cysteine ​​and phthalaldehyde derivatization requires an HPLC system equipped with a fluorescence detector. 3) Long detection time for a single sample: the HPLC program for determining one sample requires 90 minutes.

[0006] Therefore, existing methods for determining free chiral amino acids in protein products using high performance liquid chromatography have drawbacks such as high equipment costs, low stability of derivatized products, and long detection time for single samples. Summary of the Invention

[0007] The technical problem to be solved by this invention is to overcome the limitations of existing methods for determining chiral amino acids, such as high equipment cost, low stability of derivatized products, and long detection time, which make it difficult to meet the requirements of rapid and efficient qualitative and quantitative analysis of chiral amino acids. This invention provides a high-performance liquid chromatography method for detecting the fingerprint spectrum of free chiral amino acids in protein products and its application.

[0008] This invention uses FDAA as a chiral derivatization reagent, and detection can be completed using a high-performance liquid chromatograph equipped only with a low-cost ultraviolet detector, overcoming the drawback of most derivatization reagents (such as phthalaldehyde, phenyl isothiocyanate, etc.) requiring the use of relatively high-cost fluorescence detectors. Furthermore, the products derived by FDAA exhibit good stability (RSD < 1.5%) within 15 hours; the analysis time for a single sample is 75 minutes, realizing the application of high-performance liquid chromatography-ultraviolet detection for the detection of free chiral amino acids in protein products.

[0009] This invention is achieved through the following technical solution:

[0010] This invention provides a high-performance liquid chromatography method for detecting the fingerprint of free chiral amino acids in protein products and its application, comprising the following steps:

[0011] (1) Preparation of standard solutions: 39 amino acid standards (D / L-aspartic acid, D / L-glutamic acid, D / L-alanine, D / L-threonine, D / L-methionine, D / L-valine, D / L-lysine, D / L-leucine, D / L-isoleucine, D / L-phenylalanine, D / L-proline, D / L-cysteine, D / L-tyrosine, D / L-tryptophan, D / L-arginine, D / L-histidine, D / L-serine, D / L-asparagine, D / L-glutamine and glycine) were dissolved in HCl solution.

[0012] (2) Extraction of free amino acids: The protein product sample was thoroughly mixed with methanol and vortexed. After centrifugation, the supernatant was collected, dried, and the free amino acid solid was obtained. The solid was then reconstituted with water to obtain the free amino acid sample for the next step of derivatization.

[0013] (3) Derivatization conditions for standard and free amino acid samples: The derivatizing agent is a solution of Nα-(2,4-dinitro-5-fluorophenyl)-L-aminopropionamide (FDAA)-acetone containing 5-10 mg / mL. Under light-protected conditions, the standard solution in step (1) or the free amino acid sample in step (2) is mixed with the derivatizing agent, and then sodium bicarbonate solution is added for derivatization. After derivatization, the mixture is cooled, and then HCl solution and acetonitrile solution are added in sequence. The filtrate obtained after filtration with an organic phase filter membrane is the sample to be tested.

[0014] (4) High performance liquid chromatography conditions: Reversed-phase high performance liquid chromatography was used for ultraviolet detection of chiral amino acids. In the ultraviolet detection method, the ultraviolet (UV) detection wavelength was 340 nm.

[0015] The conditions for reversed-phase high-performance liquid chromatography (RP-HPLC) are as follows: Mobile phase A is a solution composed of 5.00% acetonitrile, 1.00% methanol, and 0.05%-0.08% trifluoroacetic acid (the remainder being water) by volume fraction; Mobile phase B is a solution composed of 55.00%-65.00% acetonitrile, 1.00% methanol, and 0.05%-0.08% trifluoroacetic acid (the remainder being water) by volume fraction; after elution, a chromatogram is obtained, and the content of each free chiral amino acid in the sample is calculated by calculating the standard curve of each amino acid.

[0016] Furthermore, the protein products mentioned in step (2) generally refer to animal protein products such as cow's milk and sheep's milk commonly used in this field.

[0017] Further, in step (2), the ratio of the protein product to methanol is 1g:(2-10)mL, and the vortexing time is 10-120s.

[0018] Further, in step (2), the centrifugation conditions are (1500-5000)×g for 10-30 min at 4-25℃, and the drying conditions are to dry with nitrogen at 40-50℃. The mass ratio of the free amino acid solid to the volume ratio of water is 1 mg: (100-1000) μL.

[0019] Further, in step (2), the extraction of free amino acids is carried out according to the following steps: take 0.1-1.0g of protein product, mix it thoroughly with methanol at a ratio of 1g:(2-10)mL, vortex for 10-120s, centrifuge at 1600×g for 10min at 4℃, take the supernatant, dry it under nitrogen at 40℃, add 100-1000μL of distilled water to redissolve it, and wait for the next step of derivatization.

[0020] Further, in step (3), the volume ratio of the standard solution or free amino acid sample to the volume of the derivatizing agent is 1:2, and the concentration of the derivatizing agent is 5-10 mg / mL.

[0021] Further, in step (3), the volume ratio of the sodium bicarbonate solution to the standard solution or free amino acid sample is 2:5, and the concentration of the sodium bicarbonate solution is 1.0-1.2 mol / L.

[0022] Further, in step (3), the derivatization temperature is 40-60℃, and the derivatization time is 60-120 min; the volume ratio of the HCl solution to the standard solution or free amino acid sample is 1:5; the concentration of the HCl solution is 2.0-2.4 mol / L; the volume ratio of the acetonitrile solution to the standard solution or free amino acid sample is (30-52):5, and the volume percentage concentration of the acetonitrile solution is 40%.

[0023] Further, in step (3), the derivatization of the standard or free amino acid sample is carried out according to the following steps: the derivatizing agent is a solution of Nα-(2,4-dinitro-5-fluorophenyl)-L-aminopropionamide (FDAA)-acetone containing 5 mg / mL. In a brown light-proof centrifuge tube, 50 μL of the standard solution in step (1) or the free amino acid sample in step (2) is mixed with 100 μL of the derivatizing agent, and then 20 μL of sodium bicarbonate solution with a concentration of 1.0 mol / L is added. The derivatization is carried out at 40-60℃ for 80-120 min. After the derivatization is completed, the temperature is cooled to 25℃, and then 10 μL of HCl aqueous solution with a concentration of 2.0 mol / L and 400-900 μL of acetonitrile solution with a concentration of 40% (v / v) are added in sequence. The filtrate obtained by filtering with an organic phase filter membrane with a pore size of 0.22 μm is the sample to be tested.

[0024] Furthermore, in step (3), the derivation time is 80-100 minutes.

[0025] Furthermore, in step (3), the derivation time is 90-100 minutes.

[0026] Further, in step (4), the elution procedure is as follows: 0 min - 22.5 min, the volume percentage of mobile phase A decreases from 100% to 50%; 22.5 min - 65.0 min, the volume percentage of mobile phase A decreases from 50% to 18.5%; 65.0 min - 75.0 min, the volume percentage of mobile phase A is 100%, and the flow rate is 0.9-1.1 mL / min.

[0027] Furthermore, in step (4), the elution flow rate is 1.0 mL / min.

[0028] Further, in step (4), the chromatographic column of the reversed-phase high-performance liquid chromatography is octadecylsilane-bonded silica gel, and the octadecylsilane-bonded silica gel column is an octadecylsilane-bonded silica gel column with a specification of 5 μm and 250 × 4.6 mm; the column temperature of the octadecylsilane-bonded silica gel column is 37-43℃; and the injection volume is 20-50 μL.

[0029] Furthermore, in step (4), the water is distilled water.

[0030] Furthermore, the column temperature of the octadecylsilane-bonded silica gel column is 40°C; the injection volume is 50 μL.

[0031] Further, in step (4), mobile phase A is a solution composed of 5.00% acetonitrile, 1.00% methanol, 0.05% trifluoroacetic acid and 93.95% distilled water; mobile phase B is a solution composed of 60.00% acetonitrile, 1.00% methanol, 0.05% trifluoroacetic acid and 38.95% distilled water.

[0032] Further, in step (4), after obtaining the chromatogram of the standard amino acids, the peak area of ​​each amino acid is plotted with its corresponding concentration, and a standard curve is drawn by fitting. The content of each amino acid in the sample is obtained by calculating the peak area of ​​each amino acid in the chromatogram of the sample according to the standard curve.

[0033] Further, in step (4), the chiral amino acid includes one or more of the following: D / L-aspartic acid, D / L-glutamic acid, D / L-alanine, D / L-threonine, D / L-methionine, D / L-valine, D / L-lysine, D / L-leucine, D / L-isoleucine, D / L-phenylalanine, D / L-proline, D / L-cysteine, D / L-tyrosine, D / L-tryptophan, D / L-arginine, D / L-histidine, D / L-serine, D / L-asparagine, D / L-glutamine, and glycine.

[0034] This invention also provides the application of the high-performance liquid chromatography method for detecting the fingerprint spectrum of free chiral amino acids in protein products in the quality control of protein products, for the qualitative and quantitative analysis of free chiral amino acids in protein products.

[0035] Furthermore, the purity of FDAA is ≥95%, and the amino acid standards are all standard reagents with a purity of ≥98% (more preferably ≥99%); the purity of hydrochloric acid, acetone, and trichloroacetic acid can be of analytical grade commonly used in the art, and the purity of acetone and trichloroacetic acid is preferably chromatographic grade; the purity of methanol and acetonitrile is of chromatographic grade commonly used in the art; unless otherwise specified, the water used to prepare all solutions is distilled water.

[0036] The reagents and raw materials used in this invention are all commercially available.

[0037] Compared with existing technologies, the beneficial effects of this invention are reflected in:

[0038] (1) The present invention greatly reduces the cost of instruments and reagents required. The Nα-(2,4-dinitro-5-fluorophenyl)-L-aminopropionamide derivatization reagent used in the present invention can be detected by combining it with a common high performance liquid chromatograph equipped with an ultraviolet detector; while most other derivatization reagents require the use of a fluorescence detector for detection.

[0039] (2) In this invention, the derivative of Nα-(2,4-dinitro-5-fluorophenyl)-L-aminopropionamide after reaction with amino acids can exist stably for 15 hours at room temperature, which is beneficial for derivatizing a large number of samples at the same time for continuous determination, thereby avoiding measurement errors caused by low sample storage stability to the greatest extent.

[0040] (3) In this invention, the analysis time for a single sample is 75 minutes; it makes up for the shortcomings of the current methods, such as high equipment cost, low stability of derivatized products, and long detection time for a single sample; it realizes the qualitative and quantitative characterization of the fingerprint spectrum of free chiral amino acids in protein products, and further realizes the application of high performance liquid chromatography-ultraviolet detection method for detecting free chiral amino acids in protein products, while providing key information for the study of nutritional metabolism and physiological functions of food-derived D-type amino acids.

[0041] (4) The present invention utilizes a chiral amino acid fingerprint spectrum to detect protein products, which can stably, accurately, and reproducibly control the quality of protein products and inspect their quality. Attached Figure Description

[0042] Figure 1 This is a high-performance liquid chromatogram of a mixed standard of chiral amino acids.

[0043] Figure 2 This is a high-performance liquid chromatogram of a mixed standard of chiral amino acids.

[0044] Figure 3 This is a high-performance liquid chromatogram of a mixed standard of chiral amino acids.

[0045] Figure 4 This is a high-performance liquid chromatogram of free chiral amino acids in fermented milk.

[0046] Figure 5 This is a high-performance liquid chromatogram of free chiral amino acids in fresh milk.

[0047] Figure 6 This is a high-performance liquid chromatogram of free chiral amino acids in room-temperature milk.

[0048] Figure 7 This is a high-performance liquid chromatogram of free chiral amino acids in fresh sheep milk. Detailed Implementation

[0049] The specific implementation of the present invention will be further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0050] The instruments used in the embodiments of the present invention are as follows:

[0051] The system used was the LC-20AT Shimadzu high-performance liquid chromatography system, equipped with the SPD-20A Shimadzu UV-Vis detector and the Thermo Fisher Scientific Acclaim C18 (250×4.6mm, 5μm) column.

[0052] Example 1: Establishment of an HPLC fingerprint detection method for chiral amino acid mixed standards

[0053] (1) Preparation of standard solutions:

[0054] Thirty-nine amino acid standards were dissolved in a 0.1 mol / L HCl aqueous solution to obtain standard solutions (D / L-glutamic acid 0.5 mM, D / L-alanine 0.5 mM, D-aspartic acid 0.2 mM, L-aspartic acid 4.0 mM, D-threonine 0.5 mM, L-threonine 1.0 mM, D / L-methionine 0.5 mM, D / L-valine 0.2 mM, D / L-lysine 0.2 mM, D / L-leucine 0. 2mM, D / L-Isoleucine 0.5mM, D / L-Phenylalanine 0.5mM, D / L-Proline 0.5mM, D / L-Cysteine ​​0.2mM, D / L-Tyrosine 0.5mM, D / L-Tryptophan 0.5mM, D / L-Arginine 0.5mM, D / L-Histidine 0.5mM, D / L-Serine 0.2mM, D / L-Asparagine 0.2mM, D / L-Glutamine 0.2mM, and Glycine 0.5mM).

[0055] (2) Derivatization process:

[0056] The volume ratio of 50 μL standard solution to derivatizing agent (acetone solution of Nα-(2,4-dinitro-5-fluorophenyl)-L-aminopropionamide) was 1:2, and the derivatizing agent concentration was 5 mg / mL. The volume ratio of sodium bicarbonate aqueous solution to standard solution was 2:5, and the concentration of sodium bicarbonate aqueous solution was 1.0 mol / L. Derivatization was carried out in brown centrifuge tubes at 40 °C for 90 min. The volume ratio of HCl aqueous solution to standard solution was 1:5, and the concentration of HCl aqueous solution was 2.0 mol / L. The volume ratio of acetonitrile aqueous solution to standard solution was 42:5, and the volume percentage concentration of acetonitrile aqueous solution was 40%. The above mixed solution was filtered through an organic phase filter membrane with a pore size of 0.22 μm to obtain the test sample.

[0057] (3) Detection:

[0058] Mobile phase A, by volume fraction, is a solution prepared with 5.00% acetonitrile, 1.00% methanol, 0.05% trifluoroacetic acid, and 93.95% distilled water; mobile phase B, by volume fraction, is a solution prepared with 60.00% acetonitrile, 1.00% methanol, 0.05% trifluoroacetic acid, and 38.95% distilled water.

[0059] The elution program was as follows: 0 min - 22.5 min, the volume percentage of mobile phase A decreased from 100% to 50%; 22.5 min - 65.0 min, the volume percentage of mobile phase A decreased from 50% to 18.5%; 65.0 min - 75.0 min, the volume percentage of mobile phase A was 100%.

[0060]

[0061] The flow rate was 1 mL / min, the column temperature was 40℃, the injection volume was 50 μL, and the detection wavelength was 340 nm.

[0062] The chromatogram obtained from the testing of the standard is as follows: Figure 1 As shown.

[0063] Table 1 shows the retention time and separation parameters of amino acids.

[0064]

[0065] Example 2: Establishment of a standard curve for chiral amino acids

[0066] Amino acid standards were prepared at a series of concentrations (0.008 mmol / L, 0.04 mmol / L, 0.2 mmol / L, 0.5 mmol / L, and 1.0 mmol / L), and then detected according to the method in Example 1. The chromatographic peak areas were recorded. A standard curve was calculated with peak area as the abscissa (x) and standard concentration as the ordinate (y). The results are shown in Table 2.

[0067] Table 2 shows the standard curve parameters for 39 amino acids.

[0068]

[0069] Example 3: Methodological Investigation of Chiral Amino Acid Fingerprint Detection Method

[0070] ①Precision test

[0071] Take one 50 μL standard solution and repeat the detection six times according to the derivatization and detection methods in Example 1. Record and analyze the high-performance liquid chromatograms, and calculate the retention time and peak area of ​​each amino acid peak. The results show that the RSD of the retention time and peak area of ​​each amino acid peak is less than 1.5%, as shown in Tables 3 and 4.

[0072] Table 3 Precision retention time results

[0073]

[0074] Table 4 Precision Peak Area Results

[0075]

[0076] ② Repeated examination

[0077] Five 50 μL standard solutions were taken and analyzed according to the derivatization and detection methods described in Example 1. The high-performance liquid chromatograms were recorded and analyzed, and the retention time and peak area of ​​each amino acid peak were calculated. The results showed that the RSD of the retention time of each amino acid peak was less than 0.04%, and the RSD of the peak area was less than 9%. Specific results are shown in Tables 5 and 6.

[0078] Table 5. Repeatability retention time results

[0079]

[0080] Table 6. Results of repeatability peak area

[0081]

[0082] ③Stability test

[0083] One 50 μL sample of standard solution was taken and analyzed at 0, 2.5 h, 6 h, and 15 h according to the derivatization and detection methods described in Example 1. The high-performance liquid chromatograms were recorded and analyzed, and the retention time and peak area of ​​each amino acid peak were calculated. The results showed that all amino acid peaks were detectable within 15 h, and the RSD of the retention time and peak area of ​​each amino acid peak was less than 1.5%, indicating that the sample had good stability within 15 h. Specific results are shown in Tables 7 and 8.

[0084] Table 7 Results of stability retention time

[0085]

[0086] Table 8 Stability Peak Area Results

[0087]

[0088] Example 4: HPLC fingerprint of chiral amino acid mixed standards

[0089] (1) Preparation of standard solutions:

[0090] Thirty-nine amino acid standards were dissolved in a 0.1 mol / L HCl aqueous solution to obtain standard solutions (D / L-glutamic acid 0.5 mM, D / L-alanine 0.5 mM, D-aspartic acid 0.2 mM, L-aspartic acid 4.0 mM, D-threonine 0.5 mM, L-threonine 1.0 mM, D / L-methionine 0.5 mM, D / L-valine 0.2 mM, D / L-lysine 0.2 mM, D / L-leucine 0. 2mM, D / L-Isoleucine 0.5mM, D / L-Phenylalanine 0.5mM, D / L-Proline 0.5mM, D / L-Cysteine ​​0.2mM, D / L-Tyrosine 0.5mM, D / L-Tryptophan 0.5mM, D / L-Arginine 0.5mM, D / L-Histidine 0.5mM, D / L-Serine 0.2mM, D / L-Asparagine 0.2mM, D / L-Glutamine 0.2mM, and Glycine 0.5mM).

[0091] (2) Derivatization process:

[0092] The volume ratio of 50 μL standard solution to derivatizing agent (acetone solution of Nα-(2,4-dinitro-5-fluorophenyl)-L-aminopropionamide) was 1:2, and the concentration of the derivatizing agent was 5 mg / mL. The volume ratio of sodium bicarbonate aqueous solution to standard solution was 2:5, and the concentration of sodium bicarbonate aqueous solution was 1.2 mol / L. Derivatization was carried out in brown centrifuge tubes at 60 °C for 60 min. The volume ratio of the HCl aqueous solution to standard solution was 1:5, and the concentration of the HCl aqueous solution was 2.4 mol / L. The volume ratio of the acetonitrile aqueous solution to standard solution was 30:5, and the volume percentage concentration of the acetonitrile aqueous solution was 40%. The above mixed solution was filtered through an organic phase filter membrane with a pore size of 0.22 μm to obtain the test sample.

[0093] (3) Detection:

[0094] Mobile phase A, by volume fraction, is a solution prepared from 5.00% acetonitrile, 1.00% methanol, 0.05% trifluoroacetic acid, and 93.95% distilled water; mobile phase B, by volume fraction, is a solution prepared from 55.00% acetonitrile, 1.00% methanol, 0.05% trifluoroacetic acid, and 43.95% distilled water.

[0095] The elution program was as follows: 0 min - 22.5 min, the volume percentage of mobile phase A decreased from 100% to 50%; 22.5 min - 65.0 min, the volume percentage of mobile phase A decreased from 50% to 18.5%; 65.0 min - 75.0 min, the volume percentage of mobile phase A was 100%.

[0096]

[0097] The flow rate was 0.9 mL / min, the column temperature was 40 °C, the injection volume was 50 μL, and the detection wavelength was 340 nm.

[0098] The chromatogram obtained from the testing of the standard is as follows: Figure 2 As shown.

[0099] Table 9 shows the retention time and resolution parameters of amino acids.

[0100]

[0101] Example 5: HPLC fingerprint of chiral amino acid mixed standards

[0102] (1) Preparation of standard solutions:

[0103] Thirty-nine amino acid standards were dissolved in a 0.1 mol / L HCl aqueous solution to obtain standard solutions (D / L-glutamic acid 0.5 mM, D / L-alanine 0.5 mM, D-aspartic acid 0.2 mM, L-aspartic acid 4.0 mM, D-threonine 0.5 mM, L-threonine 1.0 mM, D / L-methionine 0.5 mM, D / L-valine 0.2 mM, D / L-lysine 0.2 mM, D / L-leucine 0. 2mM, D / L-Isoleucine 0.5mM, D / L-Phenylalanine 0.5mM, D / L-Proline 0.5mM, D / L-Cysteine ​​0.2mM, D / L-Tyrosine 0.5mM, D / L-Tryptophan 0.5mM, D / L-Arginine 0.5mM, D / L-Histidine 0.5mM, D / L-Serine 0.2mM, D / L-Asparagine 0.2mM, D / L-Glutamine 0.2mM, and Glycine 0.5mM).

[0104] (2) Derivatization process:

[0105] The volume ratio of 50 μL standard solution to derivatizing agent (acetone solution of Nα-(2,4-dinitro-5-fluorophenyl)-L-aminopropionamide) was 1:2, and the concentration of the derivatizing agent was 5 mg / mL. The volume ratio of sodium bicarbonate aqueous solution to standard solution was 2:5, and the concentration of sodium bicarbonate aqueous solution was 1.1 mol / L. Derivatization was carried out in brown centrifuge tubes at 50 °C for 120 min. The volume ratio of the HCl aqueous solution to standard solution was 1:5, and the concentration of the HCl aqueous solution was 2.2 mol / L. The volume ratio of the acetonitrile aqueous solution to standard solution was 36:5, and the volume percentage concentration of the acetonitrile aqueous solution was 40%. The above mixed solution was filtered through an organic phase filter membrane with a pore size of 0.22 μm to obtain the test sample.

[0106] (3) Detection:

[0107] Mobile phase A, by volume fraction, is a solution prepared with 5.00% acetonitrile, 1.00% methanol, 0.08% trifluoroacetic acid, and 93.92% distilled water; mobile phase B, by volume fraction, is a solution prepared with 65.00% acetonitrile, 1.00% methanol, 0.08% trifluoroacetic acid, and 33.92% distilled water.

[0108] The elution program was as follows: 0 min - 22.5 min, the volume percentage of mobile phase A decreased from 100% to 50%; 22.5 min - 65.0 min, the volume percentage of mobile phase A decreased from 50% to 18.5%; 65.0 min - 75.0 min, the volume percentage of mobile phase A was 100%.

[0109]

[0110] The flow rate was 1.1 mL / min, the column temperature was 40℃, the injection volume was 50 μL, and the detection wavelength was 340 nm.

[0111] The chromatogram obtained from the testing of the standard is as follows: Figure 3 As shown.

[0112] Table 10 shows the retention time and resolution parameters of amino acids.

[0113]

[0114] Example 6: Establishment of a chiral amino acid fingerprint of fermented milk

[0115] (1) Extraction of free amino acids

[0116] Mix 1g of commercially available yogurt with 5mL of methanol at a ratio of 1g:5mL, vortex for 90s, centrifuge at 1600×g for 15min at 4℃, collect the supernatant, and dry it with nitrogen at 40℃. Redissolve the free amino acid solids in water at a ratio of 1mg:1000μL to obtain a free amino acid sample for further derivatization.

[0117] (2) Derivatives

[0118] The volume ratio of the free amino acid sample obtained in step (1) to the derivatizing agent (acetone solution of Nα-(2,4-dinitro-5-fluorophenyl)-L-aminopropionamide) was 1:2, and the concentration of the derivatizing agent was 5 mg / mL. The volume ratio of the sodium bicarbonate aqueous solution to the free amino acid sample was 2:5, and the concentration of the sodium bicarbonate aqueous solution was 1.0 mol / L. The derivatization was carried out in a brown centrifuge tube at a temperature of 40℃ for 100 min. The volume ratio of the HCl aqueous solution to the standard solution was 1:5, and the concentration of the HCl aqueous solution was 2.0 mol / L. The volume ratio of the acetonitrile aqueous solution to the free amino acid sample was 42:5, and the volume percentage concentration of the acetonitrile aqueous solution was 40%. The above mixed solution was filtered through an organic phase filter membrane with a pore size of 0.22 μm to obtain the sample to be tested.

[0119] (3) Detection

[0120] Mobile phase A, by volume fraction, is a solution prepared with 5.00% acetonitrile, 1.00% methanol, 0.05% trifluoroacetic acid, and 93.95% distilled water; mobile phase B, by volume fraction, is a solution prepared with 60.00% acetonitrile, 1.00% methanol, 0.05% trifluoroacetic acid, and 38.95% distilled water.

[0121] The elution program was as follows: 0 min - 22.5 min, the volume percentage of mobile phase A decreased from 100% to 50%; 22.5 min - 65.0 min, the volume percentage of mobile phase A decreased from 50% to 18.5%; 65.0 min - 75.0 min, the volume percentage of mobile phase A was 100%.

[0122]

[0123] The flow rate was 1.0 mL / min, the column temperature was 40℃, the injection volume was 50 μL, and the detection wavelength was 340 nm.

[0124] The high-performance liquid chromatogram of the sample obtained after testing is as follows: Figure 4 As shown.

[0125] Depend on Figure 4 The amino acid content in the sample can be analyzed and is shown in Table 11.

[0126] Table 11 Amino acid content of fermented milk

[0127]

[0128] Example 7: Establishment of a chiral amino acid fingerprint of fresh milk

[0129] (1) Extraction of free amino acids

[0130] Mix 1g of commercially available fresh milk with 10mL of methanol at a ratio of 1g:10mL, vortex for 120s, centrifuge at 1800×g for 10min at 4℃, collect the supernatant, and dry it with nitrogen at 40℃. Redissolve the free amino acid solids in water at a ratio of 1mg:1000μL to obtain a free amino acid sample for further derivatization.

[0131] (2) Derivatives

[0132] The volume ratio of the free amino acid sample obtained in step (1) to the derivatizing agent (acetone solution of Nα-(2,4-dinitro-5-fluorophenyl)-L-aminopropionamide) was 1:2, and the concentration of the derivatizing agent was 10 mg / mL. The volume ratio of the sodium bicarbonate aqueous solution to the free amino acid sample was 2:5, and the concentration of the sodium bicarbonate aqueous solution was 1.0 mol / L. The derivatization was carried out in a brown centrifuge tube at a temperature of 40℃ for 85 min. The volume ratio of the HCl aqueous solution to the standard solution was 1:5, and the concentration of the HCl aqueous solution was 2.0 mol / L. The volume ratio of the acetonitrile aqueous solution to the free amino acid sample was 52:5, and the volume percentage concentration of the acetonitrile aqueous solution was 40%. The above mixed solution was filtered through an organic phase filter membrane with a pore size of 0.22 μm to obtain the sample to be tested.

[0133] (3) Detection

[0134] Mobile phase A, by volume fraction, is a solution prepared with 5.00% acetonitrile, 1.00% methanol, 0.05% trifluoroacetic acid, and 93.95% distilled water; mobile phase B, by volume fraction, is a solution prepared with 60.00% acetonitrile, 1.00% methanol, 0.05% trifluoroacetic acid, and 38.95% distilled water.

[0135] The elution program was as follows: 0 min - 22.5 min, the volume percentage of mobile phase A decreased from 100% to 50%; 22.5 min - 65.0 min, the volume percentage of mobile phase A decreased from 50% to 18.5%; 65.0 min - 75.0 min, the volume percentage of mobile phase A was 100%.

[0136]

[0137] The flow rate was 1.0 mL / min, the column temperature was 43℃, the injection volume was 50 μL, and the detection wavelength was 340 nm.

[0138] The high-performance liquid chromatogram of the sample obtained after testing is as follows: Figure 5 As shown.

[0139] Depend on Figure 5 The amino acid content in the sample can be analyzed and is shown in Table 12.

[0140] Table 12 Amino acid content of fresh milk

[0141]

[0142] Example 8: Establishment of chiral amino acid fingerprint profile of room temperature milk

[0143] (1) Extraction of free amino acids

[0144] Mix 0.2g of commercially available room-temperature milk with 0.4mL of methanol at a ratio of 1g:2mL, vortex for 10s, centrifuge at 1800×g for 30min at 25℃, collect the supernatant, and dry it with nitrogen at 45℃. Redissolve the free amino acid solids in water at a ratio of 1mg:200μL to obtain the free amino acid sample for further derivatization.

[0145] (2) Derivatives

[0146] The volume ratio of the free amino acid sample obtained in step (1) to the derivatizing agent (acetone solution of Nα-(2,4-dinitro-5-fluorophenyl)-L-aminopropionamide) was 1:2, and the concentration of the derivatizing agent was 5 mg / mL. The volume ratio of the sodium bicarbonate aqueous solution to the free amino acid sample was 2:5, and the concentration of the sodium bicarbonate aqueous solution was 1.0 mol / L. The derivatization was carried out in a brown centrifuge tube at a temperature of 40℃ for 90 min. The volume ratio of the HCl aqueous solution to the standard solution was 1:5, and the concentration of the HCl aqueous solution was 2.0 mol / L. The volume ratio of the acetonitrile aqueous solution to the free amino acid sample was 30:5, and the volume percentage concentration of the acetonitrile aqueous solution was 40%. The above mixed solution was filtered through an organic phase filter membrane with a pore size of 0.22 μm to obtain the sample to be tested.

[0147] (3) Detection

[0148] Mobile phase A, by volume fraction, is a solution prepared with 5.00% acetonitrile, 1.00% methanol, 0.05% trifluoroacetic acid, and 93.95% distilled water; mobile phase B, by volume fraction, is a solution prepared with 60.00% acetonitrile, 1.00% methanol, 0.05% trifluoroacetic acid, and 38.95% distilled water.

[0149] The elution program was as follows: 0 min - 22.5 min, the volume percentage of mobile phase A decreased from 100% to 50%; 22.5 min - 65.0 min, the volume percentage of mobile phase A decreased from 50% to 18.5%; 65.0 min - 75.0 min, the volume percentage of mobile phase A was 100%.

[0150]

[0151] The flow rate was 1.0 mL / min, the column temperature was 37 °C, the injection volume was 50 μL, and the detection wavelength was 340 nm.

[0152] The high-performance liquid chromatogram of the sample obtained after testing is as follows: Figure 6 As shown.

[0153] Depend on Figure 6 The amino acid content in the sample can be analyzed and is shown in Table 13.

[0154] Table 13 Amino acid content of room temperature milk

[0155]

[0156] Example 9: Establishment of a chiral amino acid fingerprint of fresh goat milk

[0157] (1) Extraction of free amino acids

[0158] 0.2g of fresh sheep milk (commercially available) was thoroughly mixed with 1.8mL of methanol at a ratio of 1g:9mL. The mixture was vortexed for 120s and centrifuged at 5000×g for 15min at 4℃. The supernatant was then collected and dried with nitrogen at 40℃. The mass ratio of the free amino acid solid to the volume ratio of water was 1mg:500μL for reconstitution to obtain the free amino acid sample for further derivatization.

[0159] (2) Derivatives

[0160] The volume ratio of the free amino acid sample obtained in step (1) to the derivatizing agent (acetone solution of Nα-(2,4-dinitro-5-fluorophenyl)-L-aminopropionamide) was 1:2, and the concentration of the derivatizing agent was 5 mg / mL. The volume ratio of the sodium bicarbonate aqueous solution to the free amino acid sample was 2:5, and the concentration of the sodium bicarbonate aqueous solution was 1.0 mol / L. The derivatization was carried out in a brown centrifuge tube at a temperature of 40℃ for 85 min. The volume ratio of the HCl aqueous solution to the standard solution was 1:5, and the concentration of the HCl aqueous solution was 2.0 mol / L. The volume ratio of the acetonitrile aqueous solution to the free amino acid sample solution was 42:5, and the volume percentage concentration of the acetonitrile aqueous solution was 40%. The above mixed solution was filtered through an organic phase filter membrane with a pore size of 0.22 μm to obtain the sample to be tested.

[0161] (3) Detection

[0162] Mobile phase A, by volume fraction, is a solution prepared with 5.00% acetonitrile, 1.00% methanol, 0.05% trifluoroacetic acid, and 93.95% distilled water; mobile phase B, by volume fraction, is a solution prepared with 60.00% acetonitrile, 1.00% methanol, 0.05% trifluoroacetic acid, and 38.95% distilled water.

[0163] The elution program was as follows: 0 min - 22.5 min, the volume percentage of mobile phase A decreased from 100% to 50%; 22.5 min - 65.0 min, the volume percentage of mobile phase A decreased from 50% to 18.5%; 65.0 min - 75.0 min, the volume percentage of mobile phase A was 100%.

[0164]

[0165] The flow rate was 1.0 mL / min, the column temperature was 39℃, the injection volume was 20 μL, and the detection wavelength was 340 nm.

[0166] The high-performance liquid chromatogram of the sample obtained after testing is as follows: Figure 7 As shown.

[0167] Depend on Figure 7 The amino acid content in the sample can be analyzed and is shown in Table 14.

[0168] Table 14 Amino acid content of fresh goat milk

[0169]

[0170] The above embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any changes, substitutions, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should be within the protection scope of the present invention.

Claims

1. A high performance liquid chromatography method for detecting free chiral amino acid fingerprints of protein products, characterized in that: The steps include: (1) Preparation of standard solution: Dissolve the amino acid standards D / L-aspartic acid, D / L-glutamic acid, D / L-alanine, D / L-threonine, D / L-methionine, D / L-valine, D / L-lysine, D / L-leucine, D / L-isoleucine, D / L-phenylalanine, D / L-proline, D / L-cysteine, D / L-tyrosine, D / L-tryptophan, D / L-arginine, D / L-histidine, D / L-serine, D / L-asparagine, D / L-glutamine and glycine in HCl solution; (2) Extraction of free amino acids: Take the protein product, mix it with methanol, vortex it, centrifuge it, take the supernatant, blow it dry to obtain the free amino acid solid, add water to re-dissolve it to obtain the free amino acid sample, and wait for the next step of derivatization; (3) Derivatization conditions for standard and free amino acid samples: the derivatization agent is an acetone solution of Nα-(2,4-dinitro-5-fluorophenyl)-L-aminopropionamide. Under light-proof conditions, the standard solution in step (1) or the free amino acid sample in step (2) is mixed with the derivatization agent, and then sodium bicarbonate solution is added to carry out the derivatization reaction. After the derivatization reaction is completed, the solution is cooled, and then HCl solution and acetonitrile solution are added in sequence. The filtrate obtained after filtering with an organic phase filter membrane is the sample to be tested; (4) HPLC conditions: Chiral amino acids were detected by reverse phase HPLC with UV detection; After elution by reverse phase high performance liquid chromatography, a chromatogram is obtained, and the content of each free chiral amino acid in the sample is calculated by calculating the standard curve of each amino acid; The specific HPLC conditions are as follows: using reverse phase HPLC to detect chiral amino acids with UV, in which the UV detection wavelength is 340 nm; The conditions of reversed-phase high performance liquid chromatography are as follows: By volume fraction, mobile phase A is a solution composed of 5.00% acetonitrile, 1.00% methanol, 0.05% - 0.08% trifluoroacetic acid, and the rest is water; by volume fraction, mobile phase B is a solution composed of 55.00% - 65.00% acetonitrile, 1.00% methanol, 0.05% -0.08% trifluoroacetic acid, and the rest is water; The chromatographic column of the reversed-phase high performance liquid chromatography is an octadecylsilane bonded silica gel chromatographic column, and the octadecylsilane bonded silica gel chromatographic column is an octadecylsilane bonded silica gel chromatographic column with a specification of 5 µm and 250 × 4.6 mm; the column temperature of the octadecylsilane bonded silica gel chromatographic column is 37-43°C; after obtaining the chromatogram of the standard amino acid, the peak area of ​​each amino acid is plotted against its corresponding concentration, and a standard curve is drawn by fitting, and the peak area corresponding to each amino acid in the chromatogram of the sample to be tested is calculated according to the standard curve, thereby obtaining the content of each amino acid in the sample to be tested.

2. A high performance liquid chromatography method for detecting free chiral amino acid fingerprints of protein products according to claim 1, characterized in that: In step (2), the ratio of the protein product to methanol is 1 g: (2 - 10) mL, and the vortex time is 10 - 120 s.

3. A high performance liquid chromatography method for detecting free chiral amino acid fingerprints of protein products according to claim 1, characterized in that: In step (2), the centrifugation condition is at 4 - 25 ° C and (1500 - 5000) g for 10 - 30 min, the drying condition is to blow dry with nitrogen at 40 - 50 ° C, and the ratio of the mass of the free amino acid solid to the volume of water is 1 mg: (200 - 1000) μL.

4. The high performance liquid chromatography method for detecting free chiral amino acid fingerprints of protein products according to claim 1, characterized in that: In step (3), the ratio of the volume of the standard solution or free amino acid sample to the volume of the derivatizing agent is 1:2, and the concentration of the derivatizing agent is 5 - 10 mg / mL.

5. The high performance liquid chromatography method for detecting free chiral amino acid fingerprints of protein products according to claim 1, characterized in that: In step (3), the ratio of the volume of the sodium bicarbonate solution to the volume of the standard solution or the free amino acid sample is 2:5, and the concentration of the sodium bicarbonate solution is 1.0 - 1.2 mol / L.

6. The high performance liquid chromatography method for detecting free chiral amino acid fingerprints of protein products according to claim 1, characterized in that: In step (3), the derivatization temperature is 40-60°C, the derivatization time is 60-120 min; the ratio of the volume of the HCl solution to the volume of the standard solution or the free amino acid sample is 1:5; the concentration of the HCl solution is 2.0-2.4 mol / L; the ratio of the volume of the acetonitrile solution to the volume of the standard solution or the free amino acid sample is (30-52):5, and the volume percentage concentration of the acetonitrile solution is 40%.

7. The high performance liquid chromatography method for detecting free chiral amino acid fingerprints of protein products according to claim 1, characterized in that: In step (4), the elution procedure is: 0 min - 22.5 min, the volume percentage of mobile phase A decreases from 100% to 50%; 22.5 min - 65.0 min, the volume percentage of mobile phase A decreases from 50% to 18.5%; 65.0min - 75.0 min, the volume percentage of mobile phase A is 100%, and the flow rate is 0.9 - 1.1 mL / min.

8. Application of the high performance liquid chromatography method for detecting free chiral amino acid fingerprints of protein products according to any one of claims 1 to 7 for qualitative and quantitative determination of free chiral amino acids in protein products in quality control of protein products.

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