Method for detecting related substances of 2-amino-3-(1, 2-dihydro-2-quinoline-4-yl) propionic acid hydrochloride

Abstract

The invention relates to the technical field of pharmaceutical analysis, and particularly discloses a method for detecting related substances of 2-amino-3-(1, 2-dihydro-2-oxoquinoline-4-yl) propionic acid hydrochloride. The detection method provided by the invention can be used for rapidly detecting related substances of the 2-amino-3-(1, 2-dihydro-2-quinoline-4-yl) propionic acid hydrochloride, is high in precision, accuracy and sensitivity and good in reproducibility and stability, can be used for quality control and comprehensive evaluation of the 2-amino-3-(1, 2-dihydro-2-quinoline-4-yl) propionic acid hydrochloride, and has a wide application prospect. A reliable guarantee is provided for improving and controlling the quality of the 2-amino-3-(1, 2-dihydro-2-oxoquinoline-4-yl) propionic acid hydrochloride.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for detecting 2-amino-3-(1,2-dihydro-2-oxoquinoline-4-yl)propionic acid hydrochloride-related substances. Background Art

[0002] Rebamipide is a gastric mucosal protective agent that protects and repairs the gastric mucosa, increases the amount of gastric mucus, and promotes the regeneration of gastric mucosal cells.

[0003] In the process of synthesizing the intermediate 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl) propionic acid hydrochloride of rebamipide, various impurities such as reaction materials, process impurities, by-products, such as impurity INT1, impurity INT2-B, impurity INT2-E, impurity INT2-F, etc., are inevitably present, which directly affect the subsequent reactions and thus affect the purity of the finished product. Among them, the specific structural formulas of 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl) propionic acid hydrochloride, impurity INT1, impurity INT2-B, impurity INT2-E and impurity INT2-F are as follows. Therefore, the purity of the intermediate and the content of each impurity need to be strictly monitored. At present, there is no report on the detection method of the related substances of the intermediate of rebamipide.

[0004] Summary of the invention

[0005] Aiming at the problem that the prior art cannot effectively detect 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride-related substances, the present invention provides a method for detecting 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride-related substances.

[0006] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0007] A method for detecting 2-amino-3-(1,2-dihydro-2-oxyquinolin-4-yl)propionic acid hydrochloride-related substances, wherein the 2-amino-3-(1,2-dihydro-2-oxyquinolin-4-yl)propionic acid hydrochloride-related substances include INT1, INT2-B, INT2-E and INT2-F, and high performance liquid chromatography is used for detection, comprising the following steps:

[0008] (1) Preparation of test solution and mixed reference solution:

[0009] Take 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride raw material and prepare the test solution with an aqueous solution containing acetonitrile and trifluoroacetic acid;

[0010] Mix the INT1 reference substance, the INT2-B reference substance, the INT2-E reference substance and the INT2-F reference substance, and prepare a mixed reference substance solution with an aqueous solution containing acetonitrile and trifluoroacetic acid;

[0011] (2) Performing high performance liquid chromatography on the mixed reference solution and the test solution, wherein the conditions of the high performance liquid chromatography are:

[0012] Chromatographic column: C18 column;

[0013] Mobile phase A: phosphoric acid aqueous solution, mobile phase B: acetonitrile;

[0014] Gradient elution, the elution program of the linear gradient elution is as follows:

[0015] 0min-5min, 88%-92% mobile phase A, 12%-8% mobile phase B;

[0016] 5min-15min, 90%→10% mobile phase A, 10%→90% mobile phase B;

[0017] 15min-20min, 8%-12% mobile phase A, 92%-88% mobile phase B;

[0018] 20min-25min, 10%→90% mobile phase A, 90%→10% mobile phase B;

[0019] 25min-30min, 88%-92% mobile phase A, 12%-8% mobile phase B.

[0020] The detection method of 2-amino-3-(1,2-dihydro-2-oxyquinolin-4-yl) propionic acid hydrochloride-related substances provided by the present invention can quickly and accurately separate and detect related substances in 2-amino-3-(1,2-dihydro-2-oxyquinolin-4-yl) propionic acid hydrochloride. After dissolving the sample 2-amino-3-(1,2-dihydro-2-oxyquinolin-4-yl) propionic acid hydrochloride in an aqueous solution containing acetonitrile and trifluoroacetic acid, high performance liquid chromatography detection is performed in combination with specific chromatographic conditions, so that 2-amino-3-(1,2-dihydro-2-oxyquinolin-4-yl) propionic acid hydrochloride and impurities can be separated, each impurity peak has no interference with the main peak, and each chromatographic peak can be completely separated. In addition, the dissolving reagent used is simple and low in cost, and the mobile phase used is simple and easy to prepare, saving time.

[0021] The detection method provided by the invention can meet the requirements of rapid detection of related substances of 2-amino-3-(1,2-dihydro-2-oxyquinolin-4-yl)propionic hydrochloride, and has high precision, accuracy and sensitivity, good reproducibility and stability, and can be used for quality control and comprehensive evaluation of 2-amino-3-(1,2-dihydro-2-oxyquinolin-4-yl)propionic hydrochloride, thus providing a reliable guarantee for improving and controlling the quality of 2-amino-3-(1,2-dihydro-2-oxyquinolin-4-yl)propionic hydrochloride.

[0022] Among them, the structural formulas of 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride, INT1, INT2-B, INT2-E and INT2-F are as follows:

[0023]

[0024] Preferably, the volume ratio of acetonitrile, water and trifluoroacetic acid in the aqueous solution containing acetonitrile and trifluoroacetic acid is (490-510):(490-510):1.

[0025] Preferably, the volume concentration of the phosphoric acid aqueous solution is 0.1%-0.2%.

[0026] Preferably, the detection wavelength of the HPLC is 215nm-225nm.

[0027] Further preferably, the detection wavelength of the HPLC is 220 nm.

[0028] Preferably, the column temperature of the HPLC is 20°C-30°C.

[0029] More preferably, the column temperature of the HPLC is 25°C.

[0030] Preferably, the flow rate of the HPLC is 0.9 mL / min-1.1 mL / min.

[0031] Further preferably, the flow rate of the HPLC is 1 mL / min.

[0032] Preferably, the specification of the chromatographic column is 4.6 mm*250 mm, and the filler diameter is 3.5 μm.

[0033] Further preferably, the chromatographic column is Waters HSS T3 column.

[0034] Preferably, the injection volume of the HPLC is 20 μL.

[0035] The preferred liquid chromatography detection conditions are conducive to the effective detection of 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride-related substances and improve the detection sensitivity.

[0036] The present invention adopts a high performance liquid chromatography detection method, can realize accurate detection of 2-amino-3-(1,2-dihydro-2-oxyquinolin-4-yl) propionic acid hydrochloride related substances, is suitable for the detection of INT1, INT2-B, INT2-E and INT2-F in 2-amino-3-(1,2-dihydro-2-oxyquinolin-4-yl) propionic acid hydrochloride, and solves the problem that INT1, INT2-B, INT2-E and INT2-F in 2-amino-3-(1,2-dihydro-2-oxyquinolin-4-yl) propionic acid hydrochloride cannot be effectively controlled. In addition, the detection method is simple to operate, has high precision, and has promotion and practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is the chromatogram of the test solution in Example 2;

[0038] Figure 2 is the chromatogram of the mixed reference solution in Example 2;

[0039] Figure 3 is a linear equation diagram of 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride in Example 4;

[0040] Figure 4 is the linear equation diagram of INT1 in Example 4;

[0041] Figure 5 is a linear equation diagram of INT2-B in Example 4;

[0042] Figure 6 is a linear equation diagram of INT2-E in Example 4;

[0043] Figure 7 is a linear equation diagram of INT2-F in Example 4;

[0044] Among them, 1 is 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride. DETAILED DESCRIPTION

[0045] The technical solution of the present invention will be described clearly and completely below. It should be understood that the specific embodiments described herein can enable those skilled in the art to have a more comprehensive understanding of the present invention, but the present invention is not limited to the scope of the embodiments.

[0046] Example 1

[0047] High performance liquid chromatography conditions of the detection method of the present invention:

[0048] The HPLC conditions were:

[0049] Chromatography: Waters high performance liquid chromatograph;

[0050] Chromatographic column: Waters HSS T3, 4.6mm*250mm, 3.5μm;

[0051] Mobile phase A: 0.1% phosphoric acid aqueous solution;

[0052] Mobile phase B: acetonitrile;

[0053] Flow rate: 1 mL / min;

[0054] Injection volume: 20 μL;

[0055] Column temperature: 25°C;

[0056] Detection wavelength: 220nm;

[0057] Gradient elution, the elution program is as follows:

[0058] min A phase% Phase B % 0 90 10 5 90 10 15 10 90 20 10 90 25 90 10 30 90 10

[0059] Solvent: Add acetonitrile and trifluoroacetic acid to water to obtain a solvent, wherein the volume ratio of acetonitrile, water and trifluoroacetic acid is 500:500:1;

[0060] Test solution: Take an appropriate amount of 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride, weigh accurately, dissolve with solvent and dilute to make a solution containing about 1 mg per 1 mL, accurately measure an appropriate amount of the above solution, dilute with solvent to make a solution containing about 0.1 mg of the test sample per 1 mL;

[0061] Mixed reference solution: Take appropriate amount of INT1, INT2-B, INT2-E, and INT2-F, dissolve them in solvent and dilute them to make solutions containing 0.5 mg of INT1, 0.5 mg of INT2-B, 0.5 mg of INT2-E, and 0.8 mg of INT2-F per 1 mL, respectively, as each impurity reference solution; take appropriate amount of each impurity reference solution, dilute it with solvent to make solutions containing approximately 5 μg of INT1, 5 μg of INT2-B, 5 μg of INT2-E, and 8 μg of INT2-F per 1 mL, as the mixed reference solution; take appropriate amount of 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride, accurately weigh it, dissolve it in solvent and dilute it to make solutions containing approximately 1 mg per 1 mL; take appropriate amount of the above solutions and mixed reference solution, dilute it with solvent to make solutions containing approximately INT1 per 1 mL; 0.5μg, INT2-B 0.5μg, INT2-E 0.5μg, INT2-F 0.8μg, and a mixed reference solution containing 0.1mg of 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride.

[0062] Detection method: Accurately weigh 20 μL of the test solution and the mixed reference solution and inject them into the liquid chromatograph to calculate the contents of 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride, INT1, INT2-B, INT2-E and INT2-F.

[0063] Example 2

[0064] Specificity experiment:

[0065] According to the above chromatographic conditions, accurately measure the test solution and the mixed reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram. Figure 1 and Figure 2 The experimental results are shown in Table 1.

[0066] Conclusion: The blank solvent does not interfere with the detection of related substances. The separation degrees of the main component and adjacent impurities are greater than 1.5, and the separation degrees of each impurity and adjacent impurities are greater than 1.2, all of which meet the requirements and the method has good specificity.

[0067] Table 1

[0068] Retention time Separation 2-Amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propanoic acid hydrochloride 5.146 - INT2-F 11.239 32.07 INT2-E 11.539 2.11 INT2-B 12.730 8.30 INT1 14.043 8.30

[0069] Example 3

[0070] Limit of detection and limit of quantitation tests:

[0071] 2-Amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride reference substance stock solution: Take an appropriate amount of 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride reference substance, accurately weigh it, dilute it with solvent to make a solution containing about 5μg per 1mL, and shake well.

[0072] Stock solutions of impurity reference substances of 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride: Take appropriate amount of impurity INT1, INT2-B, INT2-E, and INT2-F reference substances, accurately weigh them, quantitatively dilute them with solvent to make a solution containing about 5 μg per 1 mL, and shake well.

[0073] Quantitation limit solution: Accurately measure 1 mL of the above-mentioned 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride reference stock solution and each impurity reference stock solution, place them in the same 100 mL volumetric flask, dilute to the scale with solvent, shake well, and obtain.

[0074] The chromatographic conditions were the same as those in Example 1. The quantification limit was determined when the peak height was 10 times the baseline noise, and the detection limit was determined when the peak height was 3 times the baseline noise. The detection limit and quantification limit results are shown in Table 2-8.

[0075] Experimental conclusion: The sensitivity of the main component and each impurity meets the detection requirements; the quantitative limit solution was prepared repeatedly for 6 times, the signal-to-noise ratio was greater than 10, and the RSD of the peak area was less than 10.0%, indicating that the quantitative limit of the method has good repeatability.

[0076] Table 2

[0077] Limit of Quantitation Retention time Peak area Concentration μg / mL s / n 2-Amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propanoic acid hydrochloride 5.164 4105 0.0504 59.06 INT2-F 11.28 3646 0.0512 41.59 INT2-E 11.566 5191 0.0504 53.86 INT2-B 12.73 5146 0.0530 50.97 INT1 14.03 3771 0.0506 34.71

[0078] Table 3

[0079] Detection limit Retention time Peak area Concentration μg / mL s / n 2-Amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propanoic acid hydrochloride 5.144 1982 0.0151 17.63 INT2-F 11.278 1008 0.0154 6.43 INT2-E 11.565 1518 0.0151 7.29 INT2-B 12.727 1526 0.0159 6.9 INT1 14.028 1186 0.0152 4.87

[0080] Table 4

[0081] 2-Amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propanoic acid hydrochloride Retention time ≤ 1.0% Peak area ≤10% Repeatability of quantitation limit 1 4.683 1968 Limit of Quantitation Repeatability2 4.688 1944 Limit of Quantitation Repeatability 3 4.689 1917 Limit of Quantitation Repeatability 4 4.692 1937 Limit of quantitation Repeatability 5 4.693 1912 Limit of Quantitation Repeatability 6 4.690 1891 average value 4.69 1928.17 RSD% 0.08 1.41

[0082] Table 5

[0083] INT2-1 Retention time ≤ 1.0% Peak area ≤10% Repeatability of quantitation limit 1 14.030 3771 Limit of Quantitation Repeatability2 14.028 3779 Limit of Quantitation Repeatability 3 14.031 3785 Limit of Quantitation Repeatability 4 14.020 3773 Limit of Quantitation Repeatability 5 14.021 3710 Limit of Quantitation Repeatability 6 14.028 3787 average value 14.030 3767.50 RSD% 0.03 0.77

[0084] Table 6

[0085] INT2-B Retention time ≤ 1.0% Peak area ≤10% Repeatability of quantitation limit 1 12.730 5146 Limit of Quantitation Repeatability2 12.728 5002 Limit of Quantitation Repeatability 3 12.732 5012 Limit of Quantitation Repeatability 4 12.721 5102 Limit of quantitation Repeatability 5 12.723 5002 Limit of Quantitation Repeatability 6 12.729 5217 average value 12.730 5080.17 RSD% 0.03 1.77

[0086] Table 7

[0087] INT2-E Retention time ≤ 1.0% Peak area ≤10% Repeatability of quantitation limit 1 11.566 5191 Limit of Quantitation Repeatability2 11.565 5020 Limit of Quantitation Repeatability 3 11.566 5025 Limit of Quantitation Repeatability 4 11.557 5036 Limit of quantitation Repeatability 5 11.564 4983 Limit of Quantitation Repeatability 6 11.565 5337 average value 11.564 5098.67 RSD% 0.03 2.69

[0088] Table 8

[0089] INT2-F Retention time ≤ 1.0% Peak area ≤10% Repeatability limit of quantitation 1 11.484 1853 Limit of Quantitation Repeatability2 11.483 1849 Limit of Quantitation Repeatability 3 11.492 1851 Limit of Quantitation Repeatability 4 11.491 1860 Limit of quantitation Repeatability 5 11.486 1832 Limit of Quantitation Repeatability 6 11.491 1861 average value 11.49 1851.00 RSD% 0.03 0.57

[0090] Example 4

[0091] Linear relationship:

[0092] 2-Amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride reference substance stock solution: Take an appropriate amount of 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride reference substance, accurately weigh it, dissolve and dilute it with a solvent to make a solution containing about 500μg per 1mL, and shake well.

[0093] Stock solutions of impurity reference substances of 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride: Take appropriate amount of impurity INT1, INT2-B, INT2-E, and INT2-F reference substances, accurately weigh them, dissolve them with solvents and dilute them to make solutions containing about 500 μg per 1 mL, and shake well.

[0094] A series of reference substance solutions: Take 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride and its impurity reference substance stock solutions, dilute them graded with solvent, and prepare a series of reference substance mixed solutions with the main component and impurity concentrations of approximately 20%, 50%, 80%, 100%, 150%, and 300% of the test sample concentration (0.1 mg / mL).

[0095] The test was performed according to the chromatographic conditions of Example 1: 20 μL of each reference solution was accurately measured and injected into the liquid chromatograph to record the chromatogram. The linear results are shown in Tables 9-13. Figure 3-7 .

[0096] Experimental conclusion: The correlation coefficients r of the linear equations between the main component and each impurity are all greater than 0.990, indicating that the method provided by this scheme has a good linear relationship between the concentration of each component and the peak area.

[0097] Table 9

[0098]

[0099] Table 10

[0100]

[0101] Table 11

[0102]

[0103] Table 12

[0104]

[0105] Table 13

[0106]

[0107] Example 5

[0108] Accuracy:

[0109] 2-Amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride impurity reference substance stock solution:

[0110] Take appropriate amount of impurity INT1, INT2-B, INT2-E, INT2-F reference substances, weigh accurately, dissolve with solvent, and prepare reference substance solution containing about 500μg per 1mL. Dilute with solvent to prepare reference substance mixed solution containing about 5μg per 1mL, as reference substance stock solution①.

[0111] Accurately weigh about 10 mg of 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride, place it in a 10 mL volumetric flask, dissolve it with solvent and dilute it to the scale, transfer 1 mL of the above solution, place it in a 10 mL volumetric flask, measure 9 portions in parallel, add appropriate amount of solvent respectively, and add 0.5 mL, 1.0 mL, and 1.5 mL of impurity reference stock solution ①, then dilute to the scale with solvent respectively, prepare 3 portions in parallel for each concentration as the recovery test solution.

[0112] According to the chromatographic conditions of Example 1, 20 μL of the test sample solution of each recovery rate was accurately measured and injected into the liquid chromatograph respectively, and the chromatogram was recorded. The results are shown in Table 14.

[0113] Experimental conclusion: The recovery rates of various impurities were all in the range of 80% to 120%, and the RSDs were all less than 10%, indicating that the method provided by this scheme has good accuracy.

[0114] Table 14

[0115]

[0116] Example 6

[0117] Repeatability:

[0118] The preparation of the test solution was the same as in Example 1. The test solution was tested 6 times according to the chromatographic conditions of Example 1. The repeatability results are shown in Table 15.

[0119] Test conclusion: The test solution was tested 6 times repeatedly, and the detected amounts of various impurities were basically consistent, indicating that the method has good repeatability.

[0120] Table 15

[0121]

[0122]

[0123] Example 7

[0124] Durability:

[0125] Based on the chromatographic conditions in Example 1, some chromatographic conditions were changed.

[0126] The preparation of the mixed solution was the same as in Example 1. The separation results are shown in Table 16.

[0127] Experimental conclusion: By fine-tuning the chromatographic conditions of Example 1, the separation between the rebamipide intermediate and the adjacent impurity peaks was greater than 1.5, and the separation between each impurity was greater than 1.2, all meeting the requirements. The method provided by the present invention has good durability.

[0128] Table 8

[0129]

[0130]

[0131] Note: Replace the chromatographic column with one of the same model.

[0132] The above-described embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting related substances of 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride, characterized in that: The 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride-related substances include INT1, INT2-B, INT2-E and INT2-F, and are detected by high performance liquid chromatography, comprising the following steps: (1) Preparation of test solution and mixed reference solution: Take 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride raw material and prepare the test solution with an aqueous solution containing acetonitrile and trifluoroacetic acid; Mix the INT1 reference substance, the INT2-B reference substance, the INT2-E reference substance and the INT2-F reference substance, and prepare a mixed reference substance solution with an aqueous solution containing acetonitrile and trifluoroacetic acid; (2) Performing high performance liquid chromatography on the mixed reference solution and the test solution, wherein the conditions of the high performance liquid chromatography are: Chromatographic column: C18 column; Mobile phase A: phosphoric acid aqueous solution, mobile phase B: acetonitrile; Gradient elution, the elution program of the linear gradient elution is as follows: 0min-5min, 88%-92% mobile phase A, 12%-8% mobile phase B; 5min-15min, 90%→10% mobile phase A, 10%→90% mobile phase B; 15min-20min, 8%-12% mobile phase A, 92%-88% mobile phase B; 20min-25min, 10%→90% mobile phase A, 90%→10% mobile phase B; 25min-30min, 88%-92% mobile phase A, 12%-8% mobile phase B.

2. The 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride-related substance according to claim 1, characterized in that: The volume concentration of the phosphoric acid aqueous solution is 0.1%-0.2%.

3. The 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride related substance as claimed in claim 1, wherein the volume ratio of acetonitrile, water and trifluoroacetic acid in the aqueous solution containing acetonitrile and trifluoroacetic acid is (490-510):(490-510):

1.

4. The 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride-related substance according to claim 1, characterized in that: The specification of the chromatographic column is 4.6 mm*250 mm, and the filler diameter is 3.5 μm.

5. The 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride-related substance according to claim 1 or 4, characterized in that: The chromatographic column is Waters HSS T3 column.

6. The 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride-related substance according to claim 1, characterized in that: The detection wavelength of the high performance liquid chromatography is 215nm-225nm.

7. The 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride-related substance according to claim 6, characterized in that: The detection wavelength of the high performance liquid chromatography is 220 nm.

8. The 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride-related substance according to claim 1, characterized in that: The column temperature of the high performance liquid chromatography is 20°C-30°C.

9. The 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride-related substance according to claim 1, characterized in that: The flow rate of the high performance liquid chromatography is 0.9 mL / min-1.1 mL / min.

10. The 2-amino-3-(1,2-dihydro-2-oxoquinolin-4-yl)propionic acid hydrochloride-related substance according to claim 9, characterized in that: The flow rate of the HPLC was 1 mL / min.

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