Method for determining related substances in starting material 2-cyanoethyl-3-oxobutyrate

Abstract

The invention discloses a method for determining the impurity content of 2-cyanoethyl-3-oxobutyrate, and belongs to the technical field of medicines. The content of impurities in 2-cyanoethyl-3-oxobutyrate is measured by adopting a high performance liquid chromatography, and the method comprises the following steps: firstly, preparing a reference solution and a test solution for later use; then setting high performance liquid detection conditions: adopting a chromatographic column taking octadecyl bonded silica gel as a filler, taking a monopotassium phosphate buffer solution as a mobile phase A, taking acetonitrile as a mobile phase B, and carrying out gradient elution; respectively sucking the test solution and the contrast solution, injecting the test solution and the contrast solution into a liquid chromatograph, and recording chromatograms. According to the present invention, with the high performance liquid chromatography method, the impurities of 2-cyanoethyl-3-oxobutyrate can be rapidly, effectively, accurately and reliably separated and detected, and the quality of the 2-cyanoethyl-3-oxobutyrate is controlled so as to improve the product quality of the finished bulk drug prepared from the material, such that the medication safety of the patient is further improved.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and specifically provides a method for determining related substances in a starting material, 2-cyanoethyl-3-oxobutyrate. Background Art

[0002] 2-Cyanoethyl-3-oxobutyrate is an important raw material for scientific research and production. Through its acetyl and cyano groups, it can undergo substitution, condensation, cyclization and other reactions to generate compounds with complex structures. Therefore, it is widely used in the fields of synthetic drugs, dyes and natural products.

[0003] 2-Cyanoethyl-3-oxobutyrate is a key starting material for the synthesis of numerous pharmaceuticals. It may produce two impurities: impurity 1 (2,2,6-trimethyl-4H-1,3-dioxin-4-one) and impurity 2 (3-hydroxypropionitrile). Currently, detection methods for related substances in 2-cyanoethyl-3-oxobutyrate suffer from low separation efficiency, insufficient accuracy in impurity identification and quantification, and poor robustness, making them difficult to meet stringent quality control requirements.

[0004] Therefore, there is an urgent need for an efficient, accurate and reliable method to achieve precise detection of relevant substances in the starting material and provide strong support for its quality control. Summary of the Invention

[0005] In view of this, the present invention proposes a method for determining related substances in the starting material 2-cyanoethyl-3-oxobutyrate. The high-performance liquid chromatography method of the present invention can quickly, effectively, accurately and reliably separate and detect the content of 2-cyanoethyl-3-oxobutyrate impurities, which is beneficial to improving the product quality of the finished raw material drug prepared from this material and improving the safety of patient use.

[0006] The technical solution of the present invention is achieved as follows: The present invention provides a method for determining related substances in a starting material, 2-cyanoethyl-3-oxobutyrate. The high-performance liquid chromatography method of the present invention can quickly, effectively, accurately and reliably separate and detect the content of 2-cyanoethyl-3-oxobutyrate impurities, which is beneficial to improving the product quality of the finished raw material prepared from this material and improving the safety of patient use. The method comprises the following steps: (1) Prepare reference solution and test solution and set aside; (2) Setting the HPLC detection conditions: using a chromatographic column filled with octadecyl bonded silica gel, 1.0-1.5 g / L potassium dihydrogen phosphate buffer adjusted to pH 2.5-3.5 with phosphoric acid as mobile phase A, and acetonitrile as mobile phase B, for gradient elution; (3) Separately inject the reference solution and the test solution into the liquid chromatograph, record the chromatogram, and calculate the content of the relevant substances using the external standard method.

[0007] In some embodiments, in the HPLC detection conditions, the ratio of mobile phase A to mobile phase B in gradient elution is: 0-5 min, 100% mobile phase A; 5-27 min, 100% mobile phase A gradually transitions to 85% mobile phase A and 15% mobile phase B; 27-35 min, 85% mobile phase A and 15% mobile phase B gradually transitions to 78% mobile phase A and 22% mobile phase B; 35-40 min, 78% mobile phase A and 22% mobile phase B gradually transitions to 65% mobile phase A and 35% mobile phase B; 40-50 min, 65% mobile phase A and 35% mobile phase B gradually transitions to 30% mobile phase A and 70% mobile phase B; 50-60 min, maintain 30% mobile phase A and 70% mobile phase B.

[0008] In some embodiments, the chromatographic column is Luna Omega PS C18, with a size of 15 cm×0.46 cm, 3 μm; the column temperature is 25-35° C., preferably 30° C.; and the flow rate is 0.6-0.8 mL / min, preferably 0.7 mL / min.

[0009] In some embodiments, the detection wavelength of the high performance liquid chromatography detector is 225-235 nm, preferably 230 nm.

[0010] In some embodiments, the solvent in the reference solution and the test solution is a 1.0-1.5 g / L potassium dihydrogen phosphate solution adjusted to a pH of 2.5-3.5 with phosphoric acid, preferably a 1.2 g / L potassium dihydrogen phosphate solution adjusted to a pH of 3.0 with phosphoric acid.

[0011] In some embodiments, the test solution is prepared by taking 50 mg of the 2-cyanoethyl-3-oxobutyrate sample to be tested, placing it in a 10 ml volumetric flask, adding the above solvent to dissolve it, diluting it to the fixed volume, and mixing it evenly.

[0012] In some embodiments, the reference solution is prepared by accurately weighing 10 mg each of a 2-cyanoethyl-3-oxobutyrate reference substance and an impurity 1 (2,2,6-trimethyl-4H-1,3-dioxin-4-one) reference substance, placing the solution in a 100 mL volumetric flask, diluting the solution to the mark with the above-mentioned solvent, and shaking the solution well.

[0013] In some embodiments, when the above method is used for determination, if the chromatogram of the test solution contains impurity 1 (2,2,6-trimethyl-4H-1,3-dioxin-4-one) and impurity 2 (3-hydroxypropionitrile), the content of each impurity shall not be greater than 2.0%; the content of other individual impurities shall not be greater than 2.0%; and the total impurities shall not be greater than 10.0%.

[0014] The present invention has the following beneficial effects compared to the prior art: High separation efficiency: Through optimized chromatographic column selection (Luna Omega PS C18), mobile phase composition (potassium dihydrogen phosphate buffer-acetonitrile) and gradient elution procedure, 2-cyanoethyl-3-oxobutanoate and related substances (especially impurities 1 and 2) can be effectively separated with high resolution and no mutual interference.

[0015] High accuracy and sensitivity: The external standard method is used for quantification, combined with the optimal detection wavelength (230nm) and mobile phase parameters, to achieve a low limit of quantification for impurities (e.g., the limit of quantification for impurity 1 can reach 0.09673μg / mL) and a good recovery rate (92%-107.8%), ensuring accurate detection of trace impurities.

[0016] Strong durability: The test results are stable when the column temperature, flow rate, mobile phase pH and column batch number fluctuate, making it suitable for quality control in different laboratories and production scenarios.

[0017] Strict quality control: The upper limits of each impurity and total impurities are clearly defined. Through precise quality control of the starting material 2-cyanoethyl-3-oxobutyrate, the product quality of downstream APIs and preparations can be significantly improved, ensuring patient safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 This is a test chromatogram under the chromatographic conditions of Example 1 of the present invention; Figure 2 This is the undamaged spectrum in the destructive test of Example 2 of the present invention; Figure 3 This is the light damage spectrum in the destructive test of Example 2 of the present invention; Figure 4 This is the high-temperature destruction diagram in the destructive test of Example 2 of the present invention; Figure 5This is the pyrolysis destruction spectrum in the destructive test of Example 2 of the present invention; Figure 6 This is the acid destruction pattern in the destructive test of Example 2 of the present invention; Figure 7 This is the alkali destruction spectrum in the destructive test of Example 2 of the present invention; Figure 8 This is the oxygen destruction spectrum in the destructive test of Example 2 of the present invention; Figure 9 This is the standard curve of 2-cyanoethyl-3-oxobutyrate tested under the liquid chromatography conditions of Example 2 of the present invention; Figure 10 This is the standard curve of impurity 2 tested under the liquid chromatography conditions of Example 2 of the present invention; Figure 11 This is the standard curve of 2-cyanoethyl-3-oxobutyrate tested under the liquid chromatography conditions of Example 2 of the present invention; Figure 12 This is the standard curve of impurity 1 tested under the liquid chromatography conditions of Example 2 of the present invention;. DETAILED DESCRIPTION

[0020] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the present invention belong. If the definitions set forth in this section are contrary to or otherwise inconsistent with definitions set forth in the patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions listed in this section take precedence over the definitions incorporated herein by reference.

[0022] Unless otherwise specified, the methods used in the following examples are conventional methods. The materials, reagents, and instruments used are conventional materials, reagents, and instruments in the art, unless otherwise specified, and can be obtained commercially by those skilled in the art.

[0023] When an amount, concentration or other value or parameter is expressed as a range, a preferred range or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper range limit or preferred value with any lower range limit or preferred value, regardless of whether the range is disclosed alone. For example, when a range "1 to 5" is disclosed, the described range should be interpreted as including the ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within the range. In the present specification and claims, range definitions may be combined and / or interchanged, and if not otherwise stated, such ranges include all subranges contained therein.

[0024] Example 1 A method for determining the impurity content of 2-cyanoethyl-3-oxobutyrate, which uses high performance liquid chromatography to determine the impurity content of 2-cyanoethyl-3-oxobutyrate, specifically comprising the following steps: (1) Prepare reference solution, test solution and mixed solution. Preparation of test solution: take 50 mg of 2-cyanoethyl-3-oxobutanoate sample to be tested and place it in a 10 ml volumetric flask, add 1.2 g / L potassium dihydrogen phosphate solution (adjust the pH to 2.9 with phosphoric acid) to dissolve it, dilute to volume, and mix well. Preparation of reference solution: accurately weigh 10 mg each of 2-cyanoethyl-3-oxobutanoate reference and impurity 1 reference and place them in a 100 ml volumetric flask, dilute to the mark with solvent, shake well, and use them as reference solution. Prepare two portions in parallel. Prepare mixed solution and inject it to test the test effect of the method of the present invention. Preparation of mixed solution: take appropriate amount of test sample, impurity 1 and impurity 2, dissolve and dilute with solvent to prepare a mixed solution containing 5 mg / ml of 2-cyanoethyl-3-oxobutanoate and 100 μg / ml of each known impurity. The solvent in reference solution and mixed solution is 1.0-1.5 g / L potassium dihydrogen phosphate solution (adjust the pH to 3.0 with phosphoric acid). (2) Set up HPLC detection conditions; Detection conditions: Luna Omega PS C18 column, size 15 cm × 0.46 cm, 3 μm, mobile phase A is 1.0-1.5 g / L potassium dihydrogen phosphate solution (pH adjusted to 2.9 with phosphoric acid); mobile phase B is acetonitrile, column temperature 30 °C, flow rate 0.7 mL / min; detection wavelength of HPLC detector is 230 nm; (3) Accurately pipette 10µL of the test solution and reference solution respectively, inject them into the liquid chromatograph, and record the chromatogram. The results are as follows: Figure 1 As shown. Figure 1It can be seen that under this chromatographic condition, the main component known impurities and unknown impurities can be well separated, and there is no minor peak interference in the blank solvent. This method is applicable.

[0025] Example 2 The detection method of Example 1 was validated from several aspects, including system applicability, destructive testing, limit of quantification, limit of detection, linearity, precision, accuracy, and solution stability, as described in detail below.

[0026] 1) System suitability determination: Take appropriate amounts of 2-cyanoethyl-3-oxobutyrate reference substance and impurity 1 and impurity 2 reference substances, accurately weigh them, add solvent to dissolve them to prepare 1 mg / ml stock solutions of each impurity, which serve as the location solutions of each impurity.

[0027] Accurately weigh an appropriate amount of the test sample into a suitable volumetric flask, then add an appropriate amount of each impurity stock solution into the same volumetric flask, dissolve and dilute with solvent to make a mixed solution containing approximately 5 mg of 2-cyanoethyl-3-oxobutyrate and 100 μg of each known impurity per 1 ml, which serves as the system suitability solution.

[0028] 10µl of each blank solution, each impurity location solution, and system suitability solution were injected into a high-performance liquid chromatograph at a detection wavelength of 230nm. Chromatograms were recorded to examine the separation of the components. The results are shown in Table 1. As shown in Table 1, at a detection wavelength of 230nm, the peak order of the mixed solution was impurity 2, 2-cyanoethyl-3-oxobutyrate, impurity 1, and 2-cyanoethyl-3-oxobutyrate. All known impurities and other impurities were effectively separated, and the solvent peak did not interfere with the determination of the main peak or the impurities.

[0029] Table 1 Impurity location and separation

[0030] 2) Destructive testing This product was destroyed by acid, alkali, oxidation, high temperature, thermal decomposition and light conditions respectively to examine the peak purity, changes in impurities and material balance.

[0031] Undamaged solution: Take about 50 mg of this product, place it in a 10 ml volumetric flask, dissolve it with solvent and dilute to the scale, shake well, and use it as the undamaged test solution.

[0032] High temperature destruction: Take about 50 mg of this product and place it in a 10 ml volumetric flask. Place it in an oven at 105°C for 2 hours. After cooling to room temperature, dissolve it in a solvent and dilute to the scale. Shake well and use it as the high temperature destruction test solution.

[0033] Photodestruction: Take the undestroyed solution and place it under 254nm, 365nm, and visible light for 2 hours to use as the photodestructed test solution.

[0034] Acid destruction: Take about 50 mg of this product, place it in a 10 ml volumetric flask, add 1 ml of 0.1 mol / L hydrochloric acid solution, and let it stand at room temperature for 2 hours. Add 1 ml of 0.1 mol / L sodium hydroxide solution to neutralize it, dissolve it with solvent and make up to the scale, shake well, and use it as the acid destruction test solution.

[0035] Alkali destruction: Take about 50 mg of this product, place it in a 10 ml volumetric flask, add 1 ml of 0.01 mol / L sodium hydroxide solution, and let it stand at room temperature for 2 hours. Add 1 ml of 0.01 mol / L hydrochloric acid solution to neutralize it, dissolve it with solvent and make up to the scale, shake well, and use it as the alkali destruction test solution.

[0036] Oxidative damage: Take about 50 mg of this product, place it in a 10 ml volumetric flask, add 1 ml of 3% hydrogen peroxide, and place it at room temperature for 2 hours. Dissolve it with solvent and make up to the scale, shake well, and use it as the oxidative damage test solution.

[0037] Pyrolysis destruction: Take about 50 mg of this product, place it in a 10 ml volumetric flask, add 1 ml of solvent, place it in an oven at 80°C for 2 hours, cool to room temperature, add solvent to the scale, shake well, and use it as the pyrolysis destruction test solution.

[0038] Take 10µl of each of the above test solutions and inject it into the chromatograph, record the chromatogram, and analyze the changes of each impurity. For the test solution under various destruction conditions, use diode array measurement to measure peak purity or spectral similarity. The results are shown in Table 2 and Appendix Figure 2-8 , from Table 2 and Appendix Figure 2-8 It can be seen that under various destruction conditions, the degradation products produced by the test sample can be effectively separated from the main peak, the degradation products do not interfere with the determination of known impurities, the reduction in the destruction of the main peak should be basically consistent with the decrease in its content, the purity of the main peak is not less than 990, and the material is conserved.

[0039] Table 2 Material balance investigation results

[0040] 3) Limit of quantification and limit of detection tests Appropriate amounts of 2-cyanoethyl-3-oxobutyrate, Impurity 1, and Impurity 2 reference substances were accurately weighed, dissolved in a solvent, and diluted to create a mixed stock solution containing approximately 10 μg of each impurity per 1 ml. The limit of quantification (S / N ≥ 10) and limit of detection (S / N ≥ 3) were determined using the dilution method. The results are shown in Table 3. As can be seen from Table 3, under these chromatographic conditions, the limits of quantification and detection (LODs) for 2-cyanoethyl-3-oxobutyrate and its impurities met the requirements.

[0041] Table 3 Limits of quantitation and detection

[0042] 4) Linear relationship Take an appropriate amount of each reference substance, accurately weigh it, and dissolve and dilute it with solvent to make a mixed solution containing approximately 200 μg of 2-cyanoethyl-3-oxobutyrate, impurity 1, and impurity 2 per 1 ml. This will serve as the linearization stock solution. Prepare the linearization solution according to Table 4.

[0043] Table 4 Linear solution preparation

[0044] Accurately measure 10µl of the above solution and inject it into the high performance liquid chromatograph, record the chromatogram, determine the peak area, and perform linear regression with the peak area A as the ordinate and the concentration C as the abscissa. The results are shown in Table 5 and the attached Figure 9-12 .

[0045] Table 5 Linear relationship test results

[0046] The calculation results of the correction factors for known impurities are shown in Table 6: Calculation formula:

[0047] Where: K 主成分 :2-cyanoethyl-3-oxobutyrate standard curve slope K 杂质 : Slope of impurity standard curve Table 6 Correction factors for known impurities

[0048] It can be seen from Tables 5 and 6 that each known impurity showed good linearity with its peak area within its corresponding concentration range (r>0.999); the correction factor of impurity 2 was in the range of 0.2-5, and was determined by the main component external standard method; the correction factor of impurity 1 was less than 0.2, and was determined by the reference substance external standard method.

[0049] 5) Injection precision An appropriate amount of each impurity reference substance was dissolved and diluted with solvent to create a mixed solution containing approximately 100 μg of each of 2-cyanoethyl-3-oxobutyrate, Impurity 1, and Impurity 2 per 1 mL. This solution served as the injection precision solution. A 10-μl injection was made into the liquid chromatograph for six consecutive injections, and the peak areas were recorded. The results are shown in Table 7. As shown in Table 7, the peak area RSD% was ≤2% for six consecutive injections, indicating good injection precision.

[0050] Table 7 Injection precision results

[0051] 6) Solution stability test The stability tests of the reference solution and the test solution were performed separately.

[0052] Stability of reference solution Appropriate amounts of each impurity reference substance were dissolved and diluted with solvent to create a mixed solution containing approximately 100 μg of each of 2-cyanoethyl-3-oxobutyrate, Impurity 1, and Impurity 2 per 1 mL. Samples were injected at different times to assess their stability. The results are shown in Table 8, which shows that the reference substances exhibited good solution stability (RSD ≤ 2%) over a 50-hour period. In Table 8, SM2 represents the main component, SM2-1 represents Impurity 1, and SM2-2 represents Impurity 2.

[0053] Table 8 Impurity reference solution stability results

[0054] Stability of test solution An appropriate amount of 2-cyanoethyl-3-oxobutyrate test sample was weighed, dissolved in a solvent, and diluted to a solution containing approximately 5 mg per 1 ml. This solution was then injected at different times, and the impurity content was calculated using the area normalization method to investigate its impurity stability. The results are shown in Table 9. As can be seen from Table 9, the absolute deviation of each impurity in the test solution from the 0-hour value was ≥ 5% of the limit. The test solution was unstable and needed to be prepared immediately before use.

[0055] Table 9 Stability results of test solution

[0056] 7) Repeatability test Test solution: Weigh an appropriate amount of 2-cyanoethyl-3-oxobutyrate test sample and add an appropriate amount of each impurity stock solution into the same volumetric flask. Dissolve and dilute with solvent to make a mixed solution containing approximately 5 mg of 2-cyanoethyl-3-oxobutyrate and 100 μg of impurity 1 and impurity 2 per 1 ml. Prepare 6 parallel copies.

[0057] Unspiked test solution: Weigh an appropriate amount of 2-cyanoethyl-3-oxobutyrate test sample, dissolve it in solvent and dilute it to make a solution containing approximately 5 mg of 2-cyanoethyl-3-oxobutyrate per 1 ml.

[0058] Reference solution: Weigh appropriate amounts of 2-cyanoethyl-3-oxobutyrate and impurity 1 reference substance, dissolve and dilute with solvent to make a mixed solution containing approximately 100 μg of 2-cyanoethyl-3-oxobutyrate and impurity 1 per 1 ml, and prepare two parallel aliquots.

[0059] The reference solution was injected six times, and the test solution was injected six times, and the peak areas were recorded. The results are shown in Table 10. As can be seen from Table 10, the RSD of each impurity content in the six reproducible sample tests was ≤2%, indicating good reproducibility.

[0060] Table 10 Repeatability results

[0061] 8) Accuracy test (sample recovery rate) Take an appropriate amount of each impurity reference substance, accurately weigh it, dissolve it with a solvent and dilute it to make a mixed solution containing approximately 500 μg of impurity 1 and impurity 2 per 1 ml, which is used as the recovery rate stock solution.

[0062] Accurately weigh an appropriate amount of 2-cyanoethyl-3-oxobutyrate. Accurately dilute the mixed stock solution as shown in the table below to produce solutions containing approximately 5 mg of 2-cyanoethyl-3-oxobutyrate per 1 mL, with concentrations of each impurity at 10%, 50%, 100%, and 200% of the limiting concentration. Prepare three aliquots for each concentration. Inject 10 µl of each sample, record the peak area, and calculate the recovery and relative standard deviation (RSD). The results are shown in Table 11. Tables 12 and 13 show that the 10% recovery of the target impurity ranged from 90% to 108%, while the 50%, 100%, and 200% recoveries, as well as the average recovery, were all within the range of 92% to 105%. The RSD% for each recovery was ≤10%, indicating good recovery.

[0063] Table 11 Recovery solution preparation

[0064] Table 12 Impurity 1 Recovery Results

[0065] Table 13 Impurity 2 recovery results

[0066] 9) Intermediate precision The same batch of samples was tested for impurity content using the same operator at different times, different operators, and different instruments using the same operator using the repeatability test method. The results are shown in Table 14. As can be seen from Table 14, the intermediate precision of this method is good.

[0067] Table 14 Intermediate precision test results

[0068] 10) Durability test The durability of the analytical method for related substances of 2-cyanoethyl-3-oxobutanoate was mainly verified from four aspects: different flow rates, different column temperatures, different pH values, and different chromatographic columns.

[0069] Experiments were conducted according to the conditions in Table 15, and the results are shown in Tables 16 and 17. As shown in Tables 16 and 17, even with slight changes in the mobile phase ratio, flow rate, column temperature, and mobile phase pH, and with the replacement of the same model chromatographic column, all impurities were effectively separated, with no significant effect on the test results. This met the requirements and demonstrates the good durability of these chromatographic conditions.

[0070] Table 15 Durability conditions

[0071] Table 16 Durability results

[0072] Table 17 Durability separation results

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for determining related substances in the starting material 2-cyanoethyl-3-oxobutyrate, characterized in that: The high performance liquid chromatography method includes the following steps: (1) Prepare reference solution and test solution and set aside; (2) Setting the HPLC detection conditions: using a chromatographic column filled with octadecyl bonded silica gel, 1.0-1.5 g / L potassium dihydrogen phosphate buffer adjusted to pH 2.5-3.5 with phosphoric acid as mobile phase A, and acetonitrile as mobile phase B, for gradient elution; (3) Inject the reference solution and the test solution separately and record the chromatograms.

2. The method according to claim 1, wherein In the HPLC detection conditions, the ratio of mobile phase A to mobile phase B in gradient elution was: 0-5 min, 100% mobile phase A; 5-27 min, 100% mobile phase A-85% mobile phase A and 15% mobile phase B; 27-35 min, 85% mobile phase A and 15% mobile phase B-78% mobile phase A and 22% mobile phase B; 35-40 min, 78% mobile phase A and 22% mobile phase B-65% mobile phase A and 35% mobile phase B; 40-50 min, 65% mobile phase A and 35% mobile phase B-30% mobile phase A and 70% mobile phase B; 50-60 min, 30% mobile phase A and 70% mobile phase B.

3. The method according to claim 1, wherein In the HPLC detection conditions, the chromatographic column is Luna OmegaPS C18, with a size of 15 cm × 0.46 cm, 3 μm; the column temperature is 25-35°C, and the flow rate is 0.6-0.8 mL / min.

4. The method according to claim 3, wherein The column temperature was 30°C and the flow rate was 0.7 mL / min.

5. The method according to claim 1, wherein The detection wavelength of the HPLC detector was 225-235 nm.

6. The method according to claim 5, wherein The detection wavelength is 230 nm.

7. The method according to claim 1, wherein The solvent in the reference solution and the test solution is 1.0-1.5 g / L potassium dihydrogen phosphate solution adjusted to pH 2.5-3.5 with phosphoric acid; Preparation of test solution: Take 50 mg of the 2-cyanoethyl-3-oxobutyrate sample to be tested and place it in a 10 ml volumetric flask, add the solvent to dissolve it, dilute to volume, and mix well; Preparation of reference solution: Accurately weigh 10 mg each of 2-cyanoethyl-3-oxobutyrate reference and impurity 1 reference, 2,2,6-trimethyl-4H-1,3-dioxin-4-one, into a 100 mL volumetric flask, dilute to the mark with the solvent, and shake well.

8. The method according to claim 7, wherein Mobile phase A was a 1.2 g / L potassium dihydrogen phosphate solution adjusted to pH 3.0 with phosphoric acid.

9. The method according to claim 7, wherein When determined by the external standard method, if the chromatogram of the test solution contains impurity 1, 2,2,6-trimethyl-4H-1,3-dioxin-4-one and impurity 2, 3-hydroxypropionitrile, their contents shall not exceed 2.0%; other individual impurities shall not exceed 2.0%; and the total impurities shall not exceed 10.0%.

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