Method for Determining Amino Acid Content in Rice Peptides by Pre-column Derivatization - High Performance Liquid Chromatography

Abstract

The present invention discloses a method for determining the amino acid content in rice peptides by pre-column derivation-high performance liquid chromatography, which is characterized by comprising the following steps: Step S1, preparation of a standard solution; Step S2, preparation of hydrolyzed amino acids and free amino acids; Step S3, derivation of a sample; Step S4, pre-column derivation-high performance liquid chromatography determination. The present invention uses PITC as a pre-column derivatizing agent and combines an ultraviolet detector to quantitatively detect the content of 16 amino acids. This method is simple, rapid, has little interference, the derivatives are stable, and has wide applications, providing a good way for the rapid analysis of various amino acids.

Description

Technical Field

[0001] The present invention relates to the field of methods for determining the amino acid content in rice peptides. More specifically, the present invention relates to a method for determining the amino acid content in rice peptides by pre-column derivation - high performance liquid chromatography. Background Art

[0002] Currently, the detection methods for amino acids include direct determination methods and indirect determination methods. The direct determination methods include liquid chromatography - tandem mass spectrometry and ion chromatography - integrated pulsed amperometry; the indirect determination methods include pre-column derivation and post-column derivation methods. The most widely used is the post-column derivation method (such as amino acid analyzers), but it requires dedicated equipment, the instrument has a single use, and it is impossible to detect and analyze multiple amino acids simultaneously. Summary of the Invention

[0003] To achieve these and other advantages in accordance with the present invention, a preferred embodiment of the present invention provides a method for determining the amino acid content in rice peptides by pre-column derivation - high performance liquid chromatography, comprising the following steps:

[0004] Step S1, Preparation of Standard Solution

[0005] Respectively measure the mixed standard solution of 16 amino acids, and dilute it with hydrochloric acid solution into standard solutions with different concentrations to prepare a standard curve;

[0006] Step S2, Preparation of Hydrolyzed Amino Acids and Free Amino Acids

[0007] Prepare hydrolyzed amino acids and free amino acids;

[0008] Step S3, Derivation of Sample

[0009] Derive the sample using phenyl isothiocyanate acetonitrile solution and triethylamine acetonitrile solution;

[0010] Step S4, Pre-column Derivation - High Performance Liquid Chromatography Determination

[0011] Chromatographic column: Athena AAA 4.6×250mm, 5μm;

[0012] Column temperature: 30 - 40°C;

[0013] Flow rate: 0.8 - 1.2 mL / min;

[0014] Detection wavelength: 254 nm;

[0015] Injection volume: 5 μL;

[0016] Mobile phase A: methanol:acetonitrile:water = 20:60:20; Mobile phase B: 50 mmol / L sodium acetate solution.

[0017] Preferably, for step S1, the preparation of the standard solution specifically includes the following operations:

[0018] Measure 2.5 μmol / mL of the mixed standard solution of 16 amino acids respectively, with the cystine concentration being 1.25 μmol / mL. Then, gradually dilute it with 0.1 mol / L hydrochloric acid solution to prepare four concentrations of the mixed amino acid standard solutions, namely the mixed amino acid standard solution with a concentration of 1.25 μmol / mL, the mixed amino acid standard solution with a concentration of 0.5 μmol / mL, the mixed amino acid standard solution with a concentration of 0.25 μmol / mL, and the mixed amino acid standard solution with a concentration of 0.125 μmol / mL, and prepare a standard curve.

[0019] Preferably, for step S2, the preparation of hydrolyzed amino acids and free amino acids specifically includes the following operations:

[0020] Step S21, the preparation of hydrolyzed amino acids

[0021] Weigh 80.00 mg of the rice peptide sample and put it into a hydrolysis tube. Add 10 mL of 6 mol / L hydrochloric acid containing 0.1% phenol. Flush nitrogen into the hydrolysis tube for 2 - 3 minutes, and then seal the hydrolysis tube.

[0022] Place the sealed hydrolysis tube in an incubator at 165 °C for hydrolysis for 1 h, and take it out and cool it to room temperature.

[0023] Open the hydrolysis tube, transfer the hydrolysis solution to a 100 mL volumetric flask, rinse the hydrolysis tube with a small amount of water several times, transfer the hydrolysis solution into the same 100 mL volumetric flask, make up the volume, and filter.

[0024] Absorb 3 mL of the filtrate and transfer it into a test tube. Dry it under nitrogen blowing at 60 °C, completely dissolve it with 1 mL of ultrapure water, and then transfer it into a 1.5 mL centrifuge tube (the sample concentration factor is 3 times), and set aside.

[0025] Step S22, the preparation of free amino acids

[0026] Weigh 300.00 mg of the sample, make up the volume to 10 mL in a volumetric flask with 0.1 mol / L hydrochloric acid, filter, and set aside.

[0027] Preferably, for step S3, the derivation of the sample specifically includes the following operations:

[0028] At room temperature, take 200 μL of the hydrolyzed amino acid prepared in step S21 or the free amino acid prepared in step S22 and place it in a 1.5 mL centrifuge tube, add 100 μL of 0.2 mol / L phenyl isothiocyanate acetonitrile solution and 100 μL of 1 mol / L triethylamine acetonitrile solution, mix well, leave it at room temperature for 1 hour, then add 400 μL of n-hexane, vortex mixer oscillate for 5-10 s, let it stand for 10 min, draw 200 μL of the lower layer solution and 800 μL of ultrapure water, mix it, pass it through a 0.45 μm filter membrane, and obtain a sample solution for injection.

[0029] Preferably, the 16 amino acids are aspartic acid, glutamic acid, serine, glycine, histidine, arginine, threonine, alanine, proline, tyrosine, valine, methionine, isoleucine, leucine, phenylalanine and lysine.

[0030] The present invention has at least the following beneficial effects: the present invention uses PITC as a pre-column derivatization agent and combines with an ultraviolet detector to quantitatively detect the contents of 16 kinds of amino acids. The method is simple to operate, rapid, has little interference, stable derivatives, and is widely used, providing a good way for rapid analysis of multiple amino acids.

[0031] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A chromatogram showing an elution gradient of the present invention in one embodiment of the present invention;

[0033] Figure 2 This is a chromatogram of isocratic elution in one embodiment of the present invention. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0035] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art can think of other obvious variations. The basic principles of the present invention defined in the following description can be applied to other embodiments, variations, improvements, equivalents, and other technical solutions that do not deviate from the spirit and scope of the present invention.

[0036] Those skilled in the art should understand that in the disclosure of the present invention, the orientation or positional relationships indicated by the terms "longitudinal", "transverse", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. are based on the orientation or positional relationships shown in the drawings. These are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, the above terms should not be construed as limitations on the present invention.

[0037] It can be understood that the term "a" should be understood as "at least one" or "one or more". That is, in one embodiment, the number of one element can be one, while in other embodiments, the number of this element can be multiple. The term "a" should not be construed as a limitation on the quantity.

[0038] A preferred embodiment of the present invention provides a method for determining the amino acid content in rice peptides by pre-column derivation - high performance liquid chromatography, comprising the following steps:

[0039] Step S1, Preparation of standard solution

[0040] Respectively measure the mixed standard solution of 16 amino acids, and dilute it with hydrochloric acid solution into standard solutions of different concentrations to prepare a standard curve. The 16 amino acids are aspartic acid (Asp), glutamic acid (Glu), serine (Ser), glycine (Gly), histidine (His), arginine (Arg), threonine (Thr), alanine (Ala), proline (Pro), tyrosine (Tyr), valine (Val), methionine (Met), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), and lysine (Lys).

[0041] Specifically, respectively measure 2.5 μmol / mL of the mixed standard solution of 16 amino acids, in which the cystine concentration is 1.25 μmol / mL, and then gradually dilute it with 0.1 mol / L hydrochloric acid solution to prepare four concentrations of the amino acid mixed standard solution, namely the amino acid mixed standard solution with a concentration of 1.25 μmol / mL, the amino acid mixed standard solution with a concentration of 0.5 μmol / mL, the amino acid mixed standard solution with a concentration of 0.25 μmol / mL, and the amino acid mixed standard solution with a concentration of 0.125 μmol / mL, and prepare a standard curve as follows:

[0042] Table 1 Standard curve of 16 amino acids

[0043]

[0044]

[0045] Among them, 0.1 mol / L hydrochloric acid: Measure 8.3 mL of concentrated hydrochloric acid and make up the volume to 1000 mL in a volumetric flask with ultrapure water.

[0046] 1.25 μmol / mL amino acid mixed standard solution: Use a pipette to take 1 mL of 2.5 μmol / mL amino acid mixed standard solution

[0047] and add 1 mL of 0.1 mol / L hydrochloric acid filtered through a 0.22 μm membrane.

[0048] 0.5 μmol / mL amino acid mixed standard solution: Take 1 mL of 2.5 μmol / mL amino acid mixed standard solution and add 4 mL of 0.1 mol / L hydrochloric acid filtered through a 0.22 μm membrane.

[0049] 0.25 μmol / mL amino acid mixed standard solution: Take 500 μL of 2.5 μmol / mL amino acid mixed standard solution

[0050] and make up the volume to 4500 μL with 0.1 mol / L hydrochloric acid filtered through a 0.22 μm membrane.

[0051] 0.125 μmol / mL amino acid mixed standard solution: Take 250 μL of 2.5 μmol / mL amino acid mixed standard solution

[0052] and add 4750 μL of 0.1 mol / L hydrochloric acid filtered through a 0.22 μm membrane.

[0053] Step S2, Preparation of hydrolyzed amino acids and free amino acids

[0054] Step S21, Preparation of hydrolyzed amino acids

[0055] Weigh 80.00 mg of rice peptide sample and put it into a hydrolysis tube. Add 10 mL of 6 mol / L hydrochloric acid containing 0.1% phenol. Flush nitrogen into the hydrolysis tube for 2 - 3 minutes, and then seal the hydrolysis tube.

[0056] Place the sealed hydrolysis tube in an incubator at 165 °C for hydrolysis for 1 h, and take it out and cool to room temperature.

[0057] Open the hydrolysis tube, transfer the hydrolysis solution to a 100 mL volumetric flask, rinse the hydrolysis tube with a small amount of water several times, transfer the hydrolysis solution into the same 100 mL volumetric flask, make up the volume, and filter.

[0058] Absorb 3 mL of the filtrate and transfer it into a test tube. Dry it under nitrogen blowing at 60 °C, completely dissolve it with 1 mL of ultrapure water, and then transfer it into a 1.5 mL centrifuge tube (the sample concentration factor is 3 times), and set aside.

[0059] Step S22, Preparation of free amino acids

[0060] Weigh 300.00 mg of the sample, dilute to 10 mL with 0.1 mol / L hydrochloric acid, filter and set aside.

[0061] Step S3: Derivatization of samples

[0062] At room temperature, take 200 μL of the hydrolyzed amino acid prepared in step S21 or the free amino acid prepared in step S22 and place it in a 1.5 mL centrifuge tube, add 100 μL of 0.2 mol / L phenyl isothiocyanate acetonitrile solution and 100 μL of 1 mol / L triethylamine acetonitrile solution, mix well, leave it at room temperature for 1 hour, then add 400 μL of n-hexane, vortex mixer oscillate for 5-10 s, let it stand for 10 min, draw 200 μL of the lower layer solution and 800 μL of ultrapure water, mix it, pass it through a 0.45 μm filter membrane, and obtain a sample solution for injection.

[0063] Among them, 1 mol / L triethylamine acetonitrile solution: take 1.4 mL of triethylamine solution, add acetonitrile to dilute to 10 mL, and store in a refrigerator at 2-8°C.

[0064] 0.2 mol / L phenyl isothiocyanate (PITC) acetonitrile solution: Take 250 μL of phenyl isothiocyanate and add acetonitrile to make up to 10 mL, and store in a refrigerator at 2-8°C.

[0065] 50mmol / L sodium acetate solution: Take 4.1g anhydrous sodium acetate / 6.8g sodium acetate trihydrate and dilute to 1000mL volumetric flask with ultrapure water, adjust pH to 6.5 with glacial acetic acid, filter through 0.45μm aqueous phase membrane and ultrasonicate for 10min to remove bubbles

[0066] Step S4: Pre-column derivatization-HPLC determination

[0067] Chromatographic column: Athena AAA 4.6×250mm, 5μm;

[0068] Column temperature: 30-40°C;

[0069] Flow rate: 0.8-1.2mL / min;

[0070] Detection wavelength: 254nm;

[0071] Injection volume: 5 μL;

[0072] Mobile phase A: methanol: acetonitrile: water = 20:60:20; mobile phase B: 50 mmol / L sodium acetate solution.

[0073] Table 2 Elution gradient

[0074]

[0075]

[0076] In this application, the above elution gradient is adopted, and its chromatogram is as shown in Figure 1 As shown, the peak resolution is relatively high. If other elution gradients are adopted, such as isocratic elution, specifically, isocratic elution is carried out with 10% mobile phase methanol, 30% acetonitrile, 10% water, and 50% 50 mmol / L sodium acetate solution, and its chromatogram is as shown in Figure 2 As shown, the resolution of some peaks is poor.

[0077] In order to compare the experimental effects, the amino acid contents in two samples (Sample 1 and Sample 2) were measured by an amino acid analyzer (ninhydrin post-column derivation) and PICT pre-column derivation-high performance liquid chromatography respectively, and the data are as follows.

[0078] Table 3 Comparison of amino acid content data measured by an amino acid analyzer (ninhydrin post-column derivation) and PICT pre-column derivation-high performance liquid chromatography for two samples (Sample 1 and Sample 2)

[0079]

[0080]

[0081] The hydrolyzed amino acid contents in rice peptide samples 1 and 2 were measured by ninhydrin post-column derivation and PITC pre-column derivation methods respectively. The contents of 16 amino acids could be measured. The relative standard deviation of the measurement results of the two groups was found to be less than 5% when compared, indicating that the error between the two detection methods was within a reasonable range, and both were applicable to the detection of hydrolyzed amino acids in rice peptides. From the values of individual hydrolyzed amino acids, the content of individual hydrolyzed amino acids measured by the PITC pre-column derivation method was slightly higher than that measured by the ninhydrin post-column derivation method, indicating that the PITC pre-column derivation method was more thorough in the hydrolysis of the sample in the early stage, enabling the hydrolyzed amino acids to be fully exposed. At the same time, the PITC column of the chromatographic column had a better resolution for 16 amino acids, avoiding peak overlap and tailing, and thus a higher measured content.

[0082] The specific method for calculating the amino acid content is as follows:

[0083] First, the sample concentration C is calculated using the following formula 1;

[0084]

[0085] C - sample concentration, unit: g / L;

[0086] m - mass of the sample, unit: g;

[0087] V - volume of constant volume, unit: L;

[0088] N - concentration multiple

[0089] Using the sample concentration C calculated by formula 1 and combining it with the following formula 2, calculate the content As of amino acid S;

[0090]

[0091] As - the content of amino acid s, where s refers to the type of amino acid, unit: %;

[0092] Xs - the concentration of amino acid s calculated after substituting the peak area of amino acid s into the corresponding curve, unit: μmol / mL;

[0093] Ms - the molecular weight of amino acid s, unit: g / mol;

[0094] 0.001 - unit conversion coefficient;

[0095] C - sample concentration, unit: g / L.

[0096] Although the embodiments of the present invention have been disclosed above, it is not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the examples shown and described herein.

Claims

1. A method for determining the amino acid content in rice peptides by pre-column derivation-high performance liquid chromatography, characterized in that, It includes the following steps: Step S1: Preparation of standard solution Measure the mixed standard solution of 16 kinds of amino acids respectively, and dilute it with hydrochloric acid solution into standard solutions with different concentrations to prepare a standard curve; Step S2: Preparation of hydrolyzed amino acids and free amino acids Prepare hydrolyzed amino acids and free amino acids; Step S3: Derivatization of samples Derivatize the samples with phenyl isothiocyanate acetonitrile solution and triethylamine acetonitrile solution; Step S4: Pre-column derivatization - high performance liquid chromatography determination Chromatographic column: Athena AAA 4.6×250mm, 5μm; Column temperature: 30 - 40°C; Flow rate: 0.8 - 1.2 mL / min; Detection wavelength: 254nm; Injection volume: 5μL; Mobile phase A: methanol:acetonitrile:water = 20:60:20; Mobile phase B: 50 mmol / L sodium acetate solution, and the elution gradient is as follows Step S2: Preparation of hydrolyzed amino acids and free amino acids, specifically including the following operations: Step S21: Preparation of hydrolyzed amino acids Weigh 80.00 mg of rice peptide sample and put it into a hydrolysis tube, add 10 mL of 6 mol / L hydrochloric acid containing 0.1% phenol, flush nitrogen into the hydrolysis tube for 2 - 3 min, and then seal the hydrolysis tube; Place the sealed hydrolysis tube in an incubator at 165°C for hydrolysis for 1 h, and take it out and cool it to room temperature; Open the hydrolysis tube, transfer the hydrolysis solution to a 100 mL volumetric flask, rinse the hydrolysis tube with a small amount of water several times, transfer the hydrolysis solution into the same 100 mL volumetric flask, make up the volume, and filter; Absorb 3 mL of the filtrate and transfer it into a test tube, dry it under nitrogen blowing at 60°C, completely dissolve it with 1 mL of ultrapure water and then transfer it into a 1.5 mL centrifuge tube for standby; Step S22: Preparation of free amino acids Weigh 300.00 mg of the sample, make up the volume to 10 mL in a volumetric flask with 0.1 mol / L hydrochloric acid, filter, and keep it for standby; The 16 kinds of amino acids are aspartic acid, glutamic acid, serine, glycine, histidine, arginine, threonine, alanine, proline, tyrosine, valine, methionine, isoleucine, leucine, phenylalanine, and lysine respectively.

2. The method for determining the amino acid content in rice peptides by pre-column derivation-high performance liquid chromatography according to claim 1, characterized in that, Step S1: Preparation of standard solution, specifically including the following operations: Measure 2.5 μmol / mL of the mixed standard solution of 16 kinds of amino acids respectively, with the cystine concentration being 1.25 μmol / mL, and then gradually dilute it with 0.1 mol / L hydrochloric acid solution to prepare four kinds of amino acid mixed standard solutions with different concentrations, namely amino acid mixed standard solution with a concentration of 1.25 μmol / mL, amino acid mixed standard solution with a concentration of 0.5 μmol / mL, amino acid mixed standard solution with a concentration of 0.25 μmol / mL, and amino acid mixed standard solution with a concentration of 0.125 μmol / mL, and prepare a standard curve.

3. The method for determining the amino acid content in rice peptides by pre-column derivation-high performance liquid chromatography according to claim 1, characterized in that, Step S3: Derivatization of samples, specifically including the following operations: At room temperature, take 200 μL of the hydrolyzed amino acid prepared in step S21 or the free amino acid prepared in step S22 and place it in a 1.5 mL centrifuge tube, add 100 μL of 0.2 mol / L phenyl isothiocyanate acetonitrile solution and 100 μL of 1 mol / L triethylamine acetonitrile solution, mix well, leave it at room temperature for 1 hour, then add 400 μL of n-hexane, vortex mixer oscillate for 5-10 seconds, let it stand for 10 minutes, draw 200 μL of the lower layer solution and 800 μL of ultrapure water, mix it, pass it through a 0.45 μm filter membrane, and obtain a sample solution for injection.

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