A method for analyzing the chiral purity of amino acids and their derivatives

Abstract

The present invention relates to the field of pharmaceutical and chemical analysis and detection technology, and mainly to an analytical method for the chiral purity of an amino acid and its derivatives, which adopts pre-column derivatization-reverse-phase high performance liquid chromatography for analysis and determination, which mainly includes using 2,4-dinitrofluorobenzene for pre-column derivatization. The technical solution of the present invention is simple and easy to operate, easier to standardize operation, and more suitable for industrial application. In addition, the derivatization reaction conditions of the technical solution of the present invention are mild and rapid, and the analytical method has a stable baseline, high sensitivity, good repeatability, and a separation degree of up to 4.2 during system operation, and the test results are accurate and effective, providing reliable information for product research and development, production and purification, and laying a solid foundation for establishing quality control standards for amino acids and their derivatives.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical and chemical analysis and detection. Specifically, the present invention provides a method for analyzing the chiral purity of amino acids and their derivatives. The method adopts a pre-column derivatization-reverse-phase high-performance liquid chromatography method for analysis and determination, which includes using 2,4-dinitrofluorobenzene for pre-column derivatization. Background Art

[0002] Protein or peptide modification is a natural phenomenon in living organisms. It primarily involves the modification of peptide or amino acid residues with certain groups, which in turn regulates a range of biological activities. Amino acid modification is a common form of protein modification.

[0003] With the increasing demand for preparation and production technology of water-soluble substances such as amino acids and their derivatives, the demand for separation and purification technology is increasing, and the research and development of their analytical methods are also receiving increasing attention.

[0004] Numerous methods exist for amino acid analysis. For example, CN1053128A and CN1749748A disclose high-performance liquid chromatography (HPLC) analysis of complex solutions containing multiple amino acids, and CN111505160A discloses a method for analyzing the purity of Fmoc-protected amino acids and related substances. However, no prior art exists for analyzing the chiral purity of amino acids and their derivatives.

[0005] However, it is well known in the art that amino acids and derivatives thereof are often used as chiral drug intermediates or can be directly used as chiral drugs, so there are very high requirements for the chiral purity of amino acids and derivatives thereof. In the synthesis reaction process of bulk drugs, when used as intermediates, their chiral isomer impurities will also participate in the reaction, generating the corresponding isomer impurities of the final bulk drug, so it is difficult to reach the quality control standard, and production costs are increased. Therefore, if an analytical method that can effectively detect the chiral isomer impurities of amino acids and derivatives thereof can be developed, it will have important guiding significance for the preparation, production and separation and purification of amino acids and derivatives thereof, and strict quality control standards will be established, which will have positive significance for improving product quality.

[0006] Therefore, it is urgent to develop an analytical method for the chiral purity of amino acids and their derivatives. Summary of the Invention

[0007] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for analyzing the chiral purity of amino acids and their derivatives, which can effectively separate amino acids and their derivatives from their chiral isomers and achieve accurate qualitative and quantitative analysis of the target products.

[0008] According to the above invention purpose, the present inventors have begun to carry out systematic research on the analytical method of the chiral purity of amino acids and derivatives thereof. It is well known to those skilled in the art that for the analytical method of chiral isomers, normal phase high performance liquid chromatography is generally selected for development. However, normal phase chromatography all selects organic solvents as mobile phases, and the solubility of amino acids and derivatives thereof in organic solvents is not good, and organic solvents are very volatile, so as to cause the occurrence of situations such as poor reproducibility or substandard separation. In addition, normal phase chromatography must be thoroughly cleaned before use to remove residual substances, especially substances with large polarity, which undoubtedly increases the complexity of the analytical process. Therefore, these factors result in a lot of drawbacks if conventional normal phase chromatography is used to detect and analyze the chiral isomers of amino acids and derivatives thereof.

[0009] Therefore, the inventors attempted to develop a method for analyzing the chiral purity of amino acids and their derivatives using reversed-phase high-performance liquid chromatography. The inventors surprisingly discovered that when 2,4-dinitrofluorobenzene was used for pre-column derivatization followed by reversed-phase chromatography, a method for analyzing the chiral purity of amino acids and their derivatives with high detection sensitivity, good repeatability, high resolution, and accurate and effective detection results could be obtained.

[0010] To achieve the purpose of the present invention, the following embodiments are provided:

[0011] A method for analyzing the chiral purity of amino acids and their derivatives, characterized by comprising the following steps:

[0012] 1) Derivatization reaction:

[0013] After dissolving the amino acid and its derivatives in a mixed solution of organic solvent I and water, a certain amount of the solution is transferred to a volumetric flask, and a weak alkaline salt solution and a derivatization reagent solution are added to carry out a derivatization reaction. After completion, organic solvent I is added to the volume and allowed to stand to obtain a solution of the derivatized amino acid and its derivatives;

[0014] The derivatization reagent solution is prepared by dissolving 2,4-dinitrofluorobenzene in an organic solvent II;

[0015] 2) Analysis and testing:

[0016] A high performance liquid chromatograph consisting of a pump, a mixer, a thermostat, an ultraviolet detector, and a workstation was used. On a chiral chromatographic column, TFA (trifluoroacetic acid) solution was used as mobile phase A and acetonitrile was used as mobile phase B. At a certain injection volume, flow rate, and column temperature, gradient elution was used to determine the derivatized amino acid and its derivative solution.

[0017] Preferably, the organic solvent I in step 1) is acetonitrile, methanol, ethanol, acetone, DMF (N,N-dimethylformamide) and a combination of two or more thereof, more preferably acetonitrile.

[0018] Preferably, in the mixed solution of organic solvent I and water in step 1), the volume ratio of organic solvent I is 30-70%, more preferably 50%.

[0019] Preferably, the organic solvent II in step 1) is ethanol, acetone or a combination thereof, more preferably ethanol.

[0020] Preferably, in step 1), the molar ratio of 2,4-dinitrofluorobenzene to amino acid and its derivatives is (1-3):1, more preferably 2:1.

[0021] Preferably, the weak alkaline salt in step 1) is sodium bicarbonate, sodium carbonate, potassium carbonate, potassium bicarbonate, disodium hydrogen phosphate solution, dipotassium hydrogen phosphate solution, or a combination of two or more thereof, more preferably sodium bicarbonate.

[0022] Preferably, the molar ratio of the weakly basic salt to the amino acid and its derivatives in step 1) is (10-25):1, more preferably 20:1.

[0023] Preferably, the temperature of the derivatization reaction in step 1) is 20-60°C, more preferably 35-45°C.

[0024] Preferably, the derivatization reaction time in step 1) is 0.5-3 h, more preferably 1-2 h.

[0025] Preferably, the concentration of the TFA solution in step 2) is 0.05%-1.1%, more preferably 0.1%.

[0026] Preferably, the detection wavelength of the ultraviolet detector in step 2) is 330-350 nm, more preferably 340 nm.

[0027] Preferably, the injection volume in step 2) is 2-12 ul, more preferably 5 ul.

[0028] Preferably, the flow rate in step 2) is 0.5-1.2 ml / min, more preferably 0.7 ml / min.

[0029] Preferably, the column temperature in step 2) is 20-30°C, more preferably 25°C.

[0030] Preferably, the gradient elution in step 2) is:

[0031] Running time / min Mobile phase A (%) Mobile phase B (%) 0 55 45 30 40 60 30.1 55 45 40 55 45

[0032] Preferably, the amino acid and its derivatives have the structural formula shown in Formula I below:

[0033]

[0034] in,

[0035] Y1 is -(CH2) n -, n is 1-3, and the hydrogen on the methylene group is replaced by at least one substituent. If two hydrogens on the same methylene group are replaced by two substituents at the same time, the two substituents are different; the substituent is a C1-C6 alkyl, a halogen, or a C1-C2 alkoxy group, and the alkyl group is a straight-chain or branched alkyl group;

[0036] R1 is selected from a hydrogen atom, a C1-C6 alkyl group, a C1-C2 alkoxy group, a C2-C4 alkenyl group, or a C1-C4 alkanoyl group, wherein the alkyl group is a straight chain or branched alkyl group, and the C1-C6 alkyl group may be optionally substituted by a C1-C3 alkyl group or a halogen atom; the marked * indicates a chiral carbon atom.

[0037] Specifically, the C1-C6 alkyl group can be: methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, isobutyl, or tert-butyl; the halogen can be a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom; the C1-C2 alkoxy group can be -OCH3, or -OCH2CH3; the C2-C4 alkenyl group can be vinyl, 1-propenyl, or 1-butenyl; the C1-C4 alkanoyl group can be formyl, acetyl, propionyl, or butyryl; the C1-C3 alkyl group can be: methyl, ethyl, or n-propyl.

[0038] Preferably, the structural formula of the amino acid and its derivatives is further preferably represented by the following formula II:

[0039]

[0040] Wherein, the definitions of R1 and * are the same as above, and R is selected from C1-C6 alkyl and C1-C2 alkoxy; specifically, the C1-C6 alkyl can be: methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, isobutyl, or tert-butyl; the C1-C2 alkoxy can be -OCH3 or -OCH2CH3.

[0041] Preferably, the structural formula of the amino acid and its derivatives is more preferably represented by the following formula III:

[0042]

[0043] Wherein, R1 and * are defined the same as above.

[0044] Preferably, the R1 is further preferably a hydrogen atom, a methyl group, an ethyl group, a formyl group, an acetyl group, a methoxy group or an ethoxy group.

[0045] Exemplarily, the amino acid and its derivatives are selected from the following structural formula:

[0046]

[0047] The beneficial effects of the present invention are:

[0048] The present invention provides a method for analyzing the chiral purity of amino acids and their derivatives. The technical solution of the present invention uses pre-column derivatization-reverse-phase high-performance liquid chromatography to determine the chiral purity of amino acids and their derivatives. Compared with conventional normal-phase chromatography for chiral purity analysis, the technical solution of the present invention is simpler and easier to operate, easier to standardize, and more suitable for industrial application. In addition, the derivatization reaction conditions of the technical solution of the present invention are mild and rapid, and the analytical method has a stable baseline, high sensitivity, good repeatability, and a resolution of up to 4.2 during system operation. The detection results are accurate and effective, providing reliable information for product development, production, and purification, and laying a solid foundation for establishing quality control standards for amino acids and their derivatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The HPLC spectrum of N-methyl-D-alanine analyzed and detected in Example 1 is shown.

[0050] Figure 2 The HPLC spectrum of N-methyl-L-alanine analyzed and detected in Example 2 is shown.

[0051] Figure 3 The HPLC spectrum of N-methyl-D-alanine analyzed and detected in Example 22 is shown.

[0052] Figure 4 The HPLC spectrum of N-methyl-DL-alanine analyzed and detected in Example 23 is shown. DETAILED DESCRIPTION

[0053] In order to better understand the technical solution of the present invention, the technical solution of the present invention is further described below in conjunction with specific embodiments. The embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0054] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0055] N-methyl-D-alanine is an important raw material and intermediate in the pharmaceutical field. Its structure has a chiral center, as shown in Formula IV. Its chiral isomer impurity, N-methyl-L-alanine (abbreviated as the L-isomer, with the structural formula shown in Formula V), often coexists with it. Therefore, the content of the L-isomer impurity must be strictly controlled, and reliable and effective methods are required for analysis and detection. N-methyl-D-alanine is used below as a representative example.

[0056]

[0057] Example 1, HPLC analysis of N-methyl-D-alanine

[0058] Preparation of derivatization reagent solution: Weigh 200 mg of 2,4-dinitrofluorobenzene into a 10 mL volumetric flask, dissolve it in ethanol and make up to volume.

[0059] Preparation of sodium bicarbonate solution: Weigh 2.1 g of sodium bicarbonate into a 50 mL volumetric flask, dissolve it in water and make up to volume.

[0060] Derivatization: Weigh 40 mg of N-methyl-D-alanine into a 20 mL volumetric flask and dissolve it to volume with 50% acetonitrile. Then, pipette 0.5 mL of this solution into a 5 mL volumetric flask, add 0.4 mL of sodium bicarbonate solution and 0.2 mL of the derivatization reagent solution, mix thoroughly, and heat in a 35-45°C water bath for 1-2 hours. Remove and cool, then add acetonitrile to volume to obtain the derivatized sample solution.

[0061] Analysis and detection: The derivatized sample solution obtained above was measured using a high performance liquid chromatograph. The chromatographic conditions were as follows: a Phenomenex Chiral MD(2)-RH 4.6*250mm 5um column was used, 0.1% TFA solution was used as mobile phase A, acetonitrile was used as mobile phase B, the detection wavelength was 340nm, the injection volume was 5ul, the flow rate was 0.7ml / min, the column temperature was 25°C, and the gradient elution was performed according to the following Table 1:

[0062] Table 1

[0063] Running time / min Mobile phase A (%) Mobile phase B (%) 0 55 45 30 40 60 30.1 55 45 40 55 45

[0064] After the elution is completed, the test results of this embodiment are obtained: its HPLC spectrum is shown in the attached Figure 1 The corresponding data analysis is shown in Table 2 below.

[0065] Table 2

[0066]

[0067] Example 2, HPLC analysis of N-methyl-L-alanine

[0068] The derivatization reagent solution and sodium bicarbonate solution were prepared as described in Example 1.

[0069] Derivatization: Weigh 40 mg of the L-isomer standard into a 20 mL volumetric flask and dissolve it to volume with 50% acetonitrile. Then, pipette 0.5 mL of this solution into a 5 mL volumetric flask and add 0.4 mL of sodium bicarbonate solution and 0.2 mL of the derivatization reagent solution. Mix thoroughly and heat in a 35-45°C water bath for 1-2 hours. Remove and cool, then add acetonitrile to volume to obtain the derivatized sample solution.

[0070] Analysis and detection: The derivatized sample solution obtained above was measured using a high performance liquid chromatograph. The chromatographic conditions were as follows: a Phenomenex Chiral MD(2)-RH 4.6*250mm 5um column was used, 0.1% TFA solution was used as mobile phase A, acetonitrile was used as mobile phase B, the detection wavelength was 340nm, the injection volume was 2ul, the flow rate was 0.7ml / min, the column temperature was 25°C, and the gradient elution was performed according to the following Table 1:

[0071] Table 1

[0072] Running time / min Mobile phase A (%) Mobile phase B (%) 0 55 45 30 40 60 30.1 55 45 40 55 45

[0073] After the elution is completed, the test results of this embodiment are obtained: its HPLC spectrum is shown in the attached Figure 2 The corresponding data analysis is shown in Table 3 below.

[0074] Table 3

[0075]

[0076] Example 3-12, Investigation of Derivatization Reaction Conditions of N-Methyl-D-Alanine

[0077] Preparation of derivatization reagent solution: Weigh a certain amount of derivatization reagent into a 10 mL volumetric flask, dissolve it with organic solvent II and make up to volume.

[0078] Preparation of weak alkaline salt solution: Weigh a certain amount of weak alkaline salt into a 50mL volumetric flask, dissolve it in water and make up to volume.

[0079] Derivatization: Weigh 40 mg of N-methyl-D-alanine into a 20 mL volumetric flask and dissolve it to volume with 50% acetonitrile. Then, pipette 0.5 mL of this solution into a 5 mL volumetric flask, add 0.4 mL of a weakly alkaline salt solution and 0.2 mL of the derivatization reagent solution, mix thoroughly, and heat in a 35-45°C water bath for 1-2 hours. Remove and cool, then add acetonitrile to volume to obtain the derivatized sample solution.

[0080] Analysis and detection: The derivatized sample solution obtained above was measured using a high performance liquid chromatograph. The chromatographic conditions were as follows: a Phenomenex Chiral MD(2)-RH 4.6*250mm 5um column was used, 0.1% TFA solution was used as mobile phase A, acetonitrile was used as mobile phase B, the detection wavelength was 340nm, the injection volume was 5ul, the flow rate was 0.7ml / min, the column temperature was 25°C, and the gradient elution was performed according to the following Table 1:

[0081] Table 1

[0082] Running time / min Mobile phase A (%) Mobile phase B (%) 0 55 45 30 40 60 30.1 55 45 40 55 45

[0083] The selected derivatization reagent, organic solvent II, weak alkaline salt and test results are shown in Table 4 below:

[0084] Table 4

[0085]

[0086]

[0087] Note: “-” indicates no derivatization reagent was added.

[0088] Example 13-21, Chromatographic Conditions Investigation Test of N-Methyl-D-Alanine

[0089] Preparation of derivatization reagent solution: Weigh 200 mg of 2,4-dinitrofluorobenzene into a 10 mL volumetric flask, dissolve it in ethanol and make up to volume.

[0090] Preparation of sodium bicarbonate solution: Weigh 2.1 g of sodium bicarbonate into a 50 mL volumetric flask, dissolve it in water and make up to volume.

[0091] Derivatization: Weigh 40 mg of N-methyl-D-alanine into a 20 mL volumetric flask and dissolve it to volume with 50% acetonitrile. Then, pipette 0.5 mL of this solution into a 5 mL volumetric flask, add 0.4 mL of sodium bicarbonate solution and 0.2 mL of the derivatization reagent solution, mix thoroughly, and heat in a 35-45°C water bath for 1-2 hours. Remove and cool, then add acetonitrile to volume to obtain the derivatized sample solution.

[0092] Analysis and detection: The derivatized sample solution obtained above was measured using a high performance liquid chromatograph, and gradient elution was performed according to the following Table 1:

[0093] Table 1

[0094] Running time / min Mobile phase A (%) Mobile phase B (%) 0 55 45 30 40 60 30.1 55 45 40 55 45

[0095] Other chromatographic conditions and test results are shown in Table 5 below:

[0096] Table 5

[0097]

[0098] Example 22, HPLC Analysis of N-Methyl-D-Alanine

[0099] Preparation of derivatization reagent solution: Weigh 200 mg of 2,4-dinitrofluorobenzene into a 10 mL volumetric flask, dissolve it in ethanol and make up to volume.

[0100] Preparation of sodium bicarbonate solution: Weigh 2.1 g of sodium bicarbonate into a 50 mL volumetric flask, dissolve it in water and make up to volume.

[0101] Derivatization: Weigh 40 mg of N-methyl-D-alanine into a 10 mL volumetric flask and dissolve it to volume with 50% acetonitrile. Then, pipette 0.5 mL of this solution into a 5 mL volumetric flask, add 0.4 mL of sodium bicarbonate solution and 0.2 mL of the derivatization reagent solution, mix thoroughly, and heat in a 35-45°C water bath for 1-2 hours. Remove and cool, then add acetonitrile to volume to obtain the derivatized sample solution.

[0102] Analysis and detection: The derivatized sample solution obtained above was measured using a high performance liquid chromatograph. The chromatographic conditions were as follows: a Phenomenex Chiral MD(2)-RH 4.6*250mm 5um column was used, 0.1% TFA solution was used as mobile phase A, acetonitrile was used as mobile phase B, the detection wavelength was 340nm, the injection volume was 10ul, the flow rate was 0.7ml / min, the column temperature was 25°C, and the gradient elution was performed according to the following Table 1:

[0103] Table 1

[0104] Running time / min Mobile phase A (%) Mobile phase B (%) 0 55 45 30 40 60 30.1 55 45 40 55 45

[0105] After the elution is completed, the test results of this embodiment are obtained: its HPLC spectrum is shown in the attached Figure 3 The corresponding data analysis is shown in Table 6 below.

[0106] Table 6

[0107]

[0108] Example 23, HPLC Analysis of N-Methyl-DL-Alanine

[0109] Preparation of derivatization reagent solution: Weigh 200 mg of 2,4-dinitrofluorobenzene into a 10 mL volumetric flask, dissolve it in ethanol and make up to volume.

[0110] Preparation of sodium bicarbonate solution: Weigh 2.1 g of sodium bicarbonate into a 50 mL volumetric flask, dissolve it in water and make up to volume.

[0111] Derivatization: Weigh 40 mg of N-methyl-DL-alanine into a 20 mL volumetric flask and dissolve it to volume with 50% acetonitrile. Then, pipette 0.5 mL of this solution into a 5 mL volumetric flask, add 0.4 mL of sodium bicarbonate solution and 0.2 mL of the derivatization reagent solution, mix thoroughly, and heat in a 35-45°C water bath for 1-2 hours. Remove and cool, then add acetonitrile to volume to obtain the derivatized sample solution.

[0112] Analysis and detection: The derivatized sample solution obtained above was measured using a high performance liquid chromatograph. The chromatographic conditions were as follows: a Phenomenex Chiral MD(2)-RH 4.6*250mm 5um column was used, 0.1% TFA solution was used as mobile phase A, acetonitrile was used as mobile phase B, the detection wavelength was 340nm, the injection volume was 5ul, the flow rate was 0.7ml / min, the column temperature was 25°C, and the gradient elution was performed according to the following Table 1:

[0113] Table 1

[0114] Running time / min Mobile phase A (%) Mobile phase B (%) 0 55 45 30 40 60 30.1 55 45 40 55 45

[0115] After the elution is completed, the test results of this embodiment are obtained: its HPLC spectrum is shown in the attached Figure 4 The corresponding data analysis is shown in Table 7 below.

[0116] Table 7

[0117]

[0118]

[0119] Comparative Example 1, HPLC Analysis of N-Methyl-D-Alanine

[0120] Preparation of derivatization reagent solution: Weigh 2 g of o-phthalaldehyde (OPA) into a 250 mL volumetric flask, add 75 mL of methanol, 62.5 mL of 10 mmol / L KH2PO4 solution, and 30 mL of 1 mmol / L N-acetyl-L-cysteine ​​(NAC) solution (adjust pH to 5.8 with 10% NaOH), dissolve by ultrasonication, and add water to make up to volume.

[0121] Derivatization reaction: Weigh 200 mg of N-methyl-D-alanine into a 10 mL volumetric flask, dissolve it with water to make up the volume, then transfer 1.0 mL of the solution to a 50 mL volumetric flask, add 10 mL of the derivatization reagent solution, dilute to the mark with 10 mmol / L KH2PO4 solution, shake well, and place at room temperature for 0.5 h to obtain the derivatized sample solution.

[0122] Analysis and detection: The derivatized sample solution obtained above was measured using a high performance liquid chromatograph. The chromatographic conditions were as follows: a Phenomenex Chiral MD(2)-RH 4.6*250mm 5um column was used, 0.1% TFA solution was used as mobile phase A, acetonitrile was used as mobile phase B, the detection wavelength was 340nm, the injection volume was 10ul, the flow rate was 0.7ml / min, the column temperature was 25°C, and the gradient elution was performed according to the following Table 1:

[0123] Table 1

[0124] Running time / min Mobile phase A (%) Mobile phase B (%) 0 55 45 30 40 60 30.1 55 45 40 55 45

[0125] After the elution was completed, the detection results of this embodiment were obtained: N-methyl-D-alanine and its L-isomer were not derivatized, and no peak was detected.

[0126] Comparative Example 2, HPLC Analysis of N-Methyl-D-Alanine

[0127] Derivatization reaction: Weigh 20 mg of N-methyl-D-alanine into a 50 mL round-bottom flask, add 15 mL of 33% aqueous ethanol, and add one drop of phenolphthalein indicator solution, shaking well. Add 1 mol / L KOH solution dropwise until the purple color disappears. Add 40 mg of α-bromophenyl ethyl ketone, stir, and heat under reflux for 0.5 h. Cool to room temperature, then add 50 mL of ethyl acetate, mix, and shake well to obtain the derivatized sample solution.

[0128] Analysis and detection: The derivatized sample solution obtained above was measured using a high performance liquid chromatograph. The chromatographic conditions were as follows: a Phenomenex Chiral MD(2)-RH 4.6*250mm 5um column was used, 0.1% TFA solution was used as mobile phase A, acetonitrile was used as mobile phase B, the detection wavelength was 340nm, the injection volume was 10ul, the flow rate was 0.7ml / min, the column temperature was 25°C, and the gradient elution was performed according to the following Table 1:

[0129] Table 1

[0130] Running time / min Mobile phase A (%) Mobile phase B (%) 0 55 45 30 40 60 30.1 55 45 40 55 45

[0131] After the elution was completed, the detection result of this embodiment was obtained: the L-isomer could not be separated.

[0132] Example 24, High Performance Liquid Chromatography Analysis of N-Acetyl-D-Alanine

[0133] Preparation of derivatization reagent solution: Weigh 200 mg of 2,4-dinitrofluorobenzene into a 10 mL volumetric flask, dissolve it in ethanol and make up to volume.

[0134] Preparation of sodium bicarbonate solution: Weigh 2.1 g of sodium bicarbonate into a 50 mL volumetric flask, dissolve it in water and make up to volume.

[0135] Derivatization: Weigh 52 mg of N-acetyl-D-alanine into a 20 mL volumetric flask and dissolve it to volume with 50% acetonitrile. Then, pipette 0.5 mL of this solution into a 5 mL volumetric flask, add 0.4 mL of sodium bicarbonate solution and 0.2 mL of the derivatization reagent solution, mix thoroughly, and heat in a 35-45°C water bath for 1-2 hours. Remove and cool, then add acetonitrile to volume to obtain the derivatized sample solution.

[0136] Analysis and detection: The derivatized sample solution obtained above was measured using a high performance liquid chromatograph. The chromatographic conditions were as follows: a Phenomenex Chiral MD(2)-RH 4.6*250mm 5um column was used, 0.1% TFA solution was used as mobile phase A, acetonitrile was used as mobile phase B, the detection wavelength was 340nm, the injection volume was 5ul, the flow rate was 0.7ml / min, the column temperature was 25°C, and the gradient elution was performed according to the following Table 1:

[0137] Table 1

[0138] Running time / min Mobile phase A (%) Mobile phase B (%) 0 55 45 30 40 60 30.1 55 45 40 55 45

[0139] After the elution was completed, the detection result of this embodiment was obtained: the separation degree of the chiral isomers was 2.24.

Claims

1. A method for analyzing the chiral purity of amino acids and their derivatives, characterized in that: The steps include: 1) Derivatization reaction: After dissolving the amino acid and its derivatives in a mixed solution of organic solvent I and water, a certain amount of the solution is transferred to a volumetric flask, and a weak alkaline salt solution and a derivatization reagent solution are added to carry out a derivatization reaction. After completion, organic solvent I is added to the volume and allowed to stand to obtain a solution of the derivatized amino acid and its derivatives; the organic solvent I is acetonitrile; The derivatization reagent solution is prepared by dissolving 2,4-dinitrofluorobenzene in an organic solvent II; the organic solvent II is ethanol; 2) Analysis and testing: A high performance liquid chromatograph comprising a pump, a mixer, a thermostat, an ultraviolet detector and a workstation is used to determine the derivatized amino acid and its derivative solution by gradient elution on a chiral chromatographic column with TFA solution as mobile phase A and acetonitrile as mobile phase B at a certain injection volume, flow rate and column temperature; the chiral chromatographic column is a Phenomenex Chiral MD(2)-RH 4.6*250mm 5um chromatographic column; the concentration of the TFA solution is 0.05%-1.1%; The amino acids and their derivatives have the following structural formula: 、 ； In the step 2), the gradient elution is: 。 2. The analysis method according to claim 1, wherein the volume ratio of the organic solvent I in the mixed solution of the organic solvent I and water in step 1) is 50%.

3. The analysis method according to claim 1, wherein in step 1), the molar ratio of 2,4-dinitrofluorobenzene to amino acids and their derivatives is (1-3):

1.

4. The analysis method according to claim 3, wherein in step 1), the molar ratio of 2,4-dinitrofluorobenzene to amino acids and their derivatives is 2:

1.

5. The analysis method according to claim 1, wherein the weakly basic salt in step 1) is sodium bicarbonate, sodium carbonate, potassium carbonate, potassium bicarbonate, disodium hydrogen phosphate solution, dipotassium hydrogen phosphate solution, or a combination of two or more thereof; and the molar ratio of the weakly basic salt to the amino acid and its derivative in step 1) is (10-25):

1.

6. The analysis method according to claim 5, wherein the weakly alkaline salt in step 1) is sodium bicarbonate.

7. The analysis method according to claim 5, wherein in step 1), the molar ratio of the weakly basic salt to the amino acid and its derivatives is 20:

1.

8. The analytical method according to claim 1, wherein the temperature of the derivatization reaction in step 1) is 35-45°C; and the time of the derivatization reaction in step 1) is 1-2 hours.

9. The analysis method according to claim 1, wherein the concentration of the TFA solution in step 2) is 0.1%.

10. The analysis method according to claim 1, wherein the detection wavelength of the UV detector in step 2) is 330-350 nm; the injection volume in step 2) is 2-12 μl; the flow rate in step 2) is 0.5-1.2 ml / min; and the column temperature in step 2) is 20-30°C.

11. The analysis method according to claim 10, wherein the detection wavelength of the UV detector in step 2) is 340 nm; the injection volume in step 2) is 5 μl; the flow rate in step 2) is 0.7 ml / min; and the column temperature in step 2) is 25°C.

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