A method for determining substances contained in levodropropizine raw materials, capsules and tablets

Abstract

The present invention discloses a method for determining substances contained in levodropropizine raw materials, capsules, and tablets, the method comprising: (1) a chromatographic column with octadecylsilane bonded silica gel as a filler; (2) drawing a predetermined amount of levodropropizine raw material capsules to be configured into a test solution; (3) injecting 10 μl of each test solution and reference solution into a liquid chromatograph; (4) using phosphate buffer as the first mobile phase A and methanol as the second mobile phase B, adding them to the liquid chromatograph for gradient elution; (5) using the liquid chromatograph to perform chromatographic detection on the test solution and the reference solution, respectively. The method of the present invention optimizes the experimental conditions, enhances the elution ability of small polar impurities, realizes the inspection of small polar impurities that cannot be detected by existing methods, has the best separation between the main peak and the adjacent impurities, has a good peak shape, and has the largest number of impurity peaks and the largest amount of impurities.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for analyzing impurities contained in levodropropizine capsules / tablets / raw materials. Background Art

[0002] Levodropropizine is included in Part II of the 2020 edition of the Chinese Pharmacopoeia, the European Pharmacopoeia EP10.0 and the Korean Pharmacopoeia 10th edition. Comparison shows that the three pharmacopoeias all use reversed liquid chromatography to control related substances. The system suitability requirements of the three pharmacopoeias are basically the same. The chromatographic conditions of the Chinese Pharmacopoeia and the European Pharmacopoeia are the same. The known impurities controlled by the related substances are all phenylpiperazine, and the impurity control limits are different, phenylpiperazine is 0.1% to 0.5%, other single impurities are 0.1%, and the total amount of impurities is 0.2% to 0.6%; Part II of the 2020 edition of the Chinese Pharmacopoeia includes levodropropizine tablets and levodropropizine capsules. The chromatographic conditions and system suitability requirements are consistent with the levodropropizine included therein, and the impurity control limits are 0.2% for phenylpiperazine, 0.2% for other single impurities, and 0.5% for the total amount of impurities.

[0003] When investigating the related substances of Levodropropizine Capsules according to the method of the Chinese Pharmacopoeia, it was found that there was an unknown impurity chromatographic peak at 8 times the retention time of the main component in the chromatogram of the test sample, with a content of about 0.05%. However, this detection method only requires recording up to 3 times the retention time of the main component, and there are cases of missed impurities. For details, see Figure 1 .

[0004] When investigating the thin layer identification of levodropropizine capsules according to the method of the Chinese Pharmacopoeia, it was found that in addition to the main component spot in the thin layer plate, there was an obvious impurity spot at the front of the solvent, indicating that there were high levels of impurities in the drug. Figure 2 . There are almost no reports on levodropropizine and related substances in its preparations after reviewing relevant literature. It was found that the current pharmacopoeia method cannot detect some impurities in levodropropizine capsules, and cannot effectively control the quality of levodropropizine, and the safety of medication cannot be guaranteed.

[0005] In view of the characteristics of the synthesis process of levodropropizine and the current quality status of commercially available levodropropizine capsules, in order to ensure effective quality control of the research and development and production quality of levodropropizine raw materials and preparations and to ensure the safety and effectiveness of drugs, it is urgently necessary to provide a method for the determination of related substances in levodropropizine raw materials and preparations. Summary of the invention

[0006] In view of the shortcomings of the prior art, the inventors of the present invention conducted numerous detection experiments and finally obtained a method for determining related substances in levodropropizine raw materials and preparations. The detection method has high detection accuracy and can be used for the detection of related substances in levodropropizine raw materials and preparations. It is accurate and reliable, has strong specificity, saves time and effort, is easy to operate, and can detect impurity components that cannot be detected by existing detection methods.

[0007] Specifically, the present invention provides a method for determining substances contained in levodropropizine raw materials, capsules, and tablets, characterized in that the method comprises:

[0008] (1) A chromatographic column using octadecylsilane bonded silica gel as a filler;

[0009] (2) taking a predetermined amount of levodropropizine raw material capsules to prepare a test solution;

[0010] (3) 10 μl of the test solution and the reference solution were injected into the liquid chromatograph respectively;

[0011] (4) using phosphate buffer as the first mobile phase A and methanol as the second mobile phase B, adding the two phases into a liquid chromatograph for gradient elution;

[0012] (5) Use a liquid chromatograph to perform chromatographic detection on the test solution and the reference solution respectively.

[0013] Preferably, when gradient elution is performed, the gradient of the mobile phase and the elution time varies as follows: 0-10 minutes, isocratic elution of mobile phase A-mobile phase B (80:20); 10-35 minutes, the proportion of mobile phase A gradually decreases from 80% to 40%, and the proportion of mobile phase B gradually increases from 20% to 60%; 35-36 minutes, the proportion of mobile phase A gradually increases from 40% to 80%, and the proportion of mobile phase B gradually decreases from 60% to 20%; 36-50 minutes, isocratic elution of mobile phase A-mobile phase B (80:20).

[0014] Preferably, when gradient elution is performed, the concentration of potassium dihydrogen phosphate in the phosphate buffer is 0.034%.

[0015] Preferably, when gradient elution is performed, the pH value of the phosphate buffer is adjusted to 2.5 with phosphoric acid.

[0016] Preferably, the detection wavelength is selected to be 241 nm.

[0017] Preferably, during liquid chromatography detection, the flow rate is 0.7 ml / min and the column temperature is 40°C.

[0018] Beneficial Effects

[0019] The method of the present invention adopts high performance liquid reverse phase chromatography technology to determine the substances related to the raw materials and preparations of levodropropizine, which makes up for the deficiencies of the current pharmacopoeia, detects impurities in levodropropizine capsules that are not detected according to the current pharmacopoeia and have a high content, thereby ensuring the safety of medication. The method of the present invention has the advantages of accuracy and reliability, short cycle, simple operation, etc. in detecting the impurities of levodropropizine. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The following is a typical liquid chromatogram of the test solution of the related substances when the levodropropizine capsules were tested using the Chinese Pharmacopoeia method;

[0021] Figure 2 This is the identification thin layer chromatogram of Levodropropizine Capsules when tested using the Chinese Pharmacopoeia method;

[0022] Figure 3 The following is a typical liquid chromatogram of a test solution of a related substance when the method of the present invention is used to detect Levodropropizine Capsules;

[0023] Figure 4 This is: High-resolution mass spectrum of impurity 2 in levodropropizine capsules;

[0024] Figure 5 The structural formula and basic information of impurity 2 in levodropropizine capsules;

[0025] Figure 6 This is: High-resolution mass spectrum of impurity 3 in levodropropizine capsules;

[0026] Figure 7 The following are the HNMR and CNMR images of impurity 3 in levodropropizine capsules;

[0027] Figure 8 DEPT90 and CNMR (DEPT135) images of impurity 3 in levodropropizine capsules;

[0028] Fig. 9 : HH-COSY, CH-HSQC, CH-HMBC diagrams of impurity 3 in levodropropizine capsules;

[0029] Fig.10 : Infrared spectrum of impurity 3 in levodropropizine capsules;

[0030] Fig.11 The following are: the inferred structure and basic information of impurity 3 in levodropropizine capsules;

[0031] Fig.12 This is: High-resolution mass spectrum of impurity 4 in levodropropizine capsules;

[0032] Fig.13The structure and basic information of impurity 4 in levodropropizine capsules. DETAILED DESCRIPTION

[0033] The present invention will be described in detail below in conjunction with the accompanying drawings and embodiments thereof, but the protection scope of the present invention is not limited to the scope of the embodiments described herein.

[0034] 1. Raw material preparation

[0035] 1.1. Preparation of test solution

[0036] Take an appropriate amount of the contents of this product, grind it finely, accurately weigh an appropriate amount of fine powder (approximately equivalent to 25 mg of levodropropizine) and place it in a 50 ml volumetric flask, add a mixed solvent (methanol and water volume ratio of 20:80), ultrasonically dissolve for 15 minutes and dilute to the scale to dissolve the levodropropizine and quantitatively dilute it to make a solution containing approximately 0.5 mg of levodropropizine per 1 ml, shake well, filter, and take the filtrate.

[0037] 1.2. Preparation of reference solution

[0038] Accurately weigh 25 mg of phenylpiperazine reference substance, place in a 50 ml volumetric flask, add methanol to dissolve and dilute to the scale, accurately measure 5 ml, place in a 50 ml volumetric flask, dilute to the scale with methanol, accurately measure 1 ml, place in a 100 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0039] 1.3. Preparation of system suitability solution

[0040] Take appropriate amounts of phenylpiperazine and levohydroxypropylpiperazine, add appropriate amounts of methanol to dissolve them, and then dilute with solvent to prepare solutions containing approximately 10 μg and 100 μg, respectively, per 1 ml.

[0041] 2. Testing conditions

[0042] Filler: octadecylsilane bonded silica gel (CAPCELL PAK C18 MGII 4.6mm×150mm, 3μm or chromatographic column with equivalent performance);

[0043] Mobile phase B: methanol;

[0044] Selection of phosphate concentration in mobile phase A: When using the Chinese Pharmacopoeia method to detect related substances in levodropropizine capsules / tablets, it was found that impurities were missed. By increasing the proportion of organic phase (methanol) to increase the elution capacity of impurities with low polarity, it was found that as the proportion of methanol increased, phosphate precipitated in the mobile phase and blocked the pipeline. It was not possible to achieve satisfactory results by adjusting the ratio of aqueous phase to organic phase in the original method. After multiple experiments, the concentration of potassium dihydrogen phosphate in the mobile phase phosphate buffer was adjusted from 0.681% to 0.034%, which can meet the detection requirements of related substances of this product.

[0045] Selection of pH value of mobile phase A: Phosphate buffer (weigh 0.34g of potassium dihydrogen phosphate, add 1000ml of water to dissolve, and adjust the pH value to 2.5 with phosphoric acid) was used as mobile phase A. After repeated exploration and optimization, the elution of phosphate buffer (weigh 0.34g of potassium dihydrogen phosphate, add 1000ml of water to dissolve, and adjust the pH value to the corresponding value with phosphoric acid or potassium hydroxide solution) with pH values ​​of 2.5, 3.0, 4.0, and 7.5 as mobile phase A was investigated. The results showed that the main peak of pH 2.5 was best separated from the adjacent impurities, the peak shape was good, and the number and amount of impurity peaks detected were the largest, so the pH value of mobile phase A was determined to be 2.5.

[0046] Selection of mobile phase gradient: The impurity spots detected by thin layer identification of levohydroxypropylpiperazine capsules were extracted by a suitable method to obtain a thin layer impurity spot solution, and eluted with different ratios of phosphate buffer (weigh 0.34 g of potassium dihydrogen phosphate, add 1000 ml of water to dissolve, and adjust the pH value to 2.5 with phosphoric acid)-methanol as the mobile phase. However, if the main peak is separated from its adjacent impurities, the thin layer impurities cannot be detected. If the thin layer impurities can be detected, there are unseparated impurities after the main peak. Therefore, the gradient elution method was adopted. After many experiments, the gradient elution method was determined as follows: 0-10 minutes, mobile phase A-mobile phase B (80:20) isocratic elution; 10-35 minutes, the proportion of mobile phase A gradually decreased from 80% to 40%, and the proportion of mobile phase B gradually increased from 20% to 60%; 35-36 minutes, the proportion of mobile phase A gradually increased from 40% to 80%, and the proportion of mobile phase B gradually decreased from 60% to 20%; 36-50 minutes, mobile phase A-mobile phase B (80:20) isocratic elution. By eluting in the above manner, levodropropizine and adjacent impurities and impurities can be well separated, and thin layer impurities have suitable retention time and good peak shape. Gradient elution is shown in the table below;

[0047]

[0048]

[0049] Table 1

[0050] Selection of detection wavelength: Considering that the main detection purpose of the present invention is to investigate the impurity content in the drug, since the present invention subsequently confirmed that the detected impurities 1, impurity 2, impurity 3 and impurity 4 have maximum ultraviolet absorption wavelengths of 239nm, 240nm, 241nm and 242nm, respectively, and the maximum absorption wavelength of phenylpiperazine is 239nm, the maximum absorption wavelength of the largest impurity (impurity 3), 241nm, is selected as the detection wavelength.

[0051] Flow rate: 0.7ml / min;

[0052] Column temperature: 40°C;

[0053] System suitability requirements In the system suitability solution chromatogram, the theoretical plate number calculated based on the levodropropizine peak shall not be less than 2000, and the separation degree between levodropropizine and phenylpiperazine shall comply with the regulations.

[0054] If there is a chromatographic peak in the test solution that is consistent with the retention time of phenylpiperazine, the peak area calculated by the external standard method shall not exceed 0.2%; other individual impurities, with the main peak area of ​​the reference solution as the reference, calculated by the peak area by the external standard method, shall not exceed 0.2%. The total amount of other impurities shall not exceed 0.5%. Peaks less than 0.1 times the main peak area of ​​the reference solution shall be ignored.

[0055] 3. Measurement process

[0056] (1) Select a chromatographic column with octadecylsilane bonded silica gel as the filler;

[0057] (2) taking a predetermined amount of levodropropizine raw material capsules to prepare a test solution;

[0058] (3) 10 μl of the test solution and the reference solution were injected into the liquid chromatograph respectively;

[0059] (4) using phosphate buffer as the first mobile phase A and methanol as the second mobile phase B, adding the two phases into a liquid chromatograph for gradient elution;

[0060] (5) Use a liquid chromatograph to perform chromatographic detection on the test solution and the reference solution respectively.

[0061] 4. Method Validation

[0062] Since no high-level impurities have been detected in the levodropropizine capsules according to the current pharmacopoeia, as described in the background art, the safety of the drug cannot be guaranteed.

[0063] During the research process, the research team of the present invention repeatedly explored and optimized the experimental conditions to find the most suitable chromatographic method: ① mobile phase A concentration; ② mobile phase gradient; ③ mobile phase A pH value; ④ detection wavelength. By optimizing these factors, the elution capacity of impurities with low polarity is increased, levodropropizine and adjacent impurities, and impurities can be better separated, the main peak and adjacent impurities are best separated, the peak shape is good, the number of detected impurity peaks and the amount of impurities are the largest, and the thin layer impurities have a suitable retention time.

[0064] During the research process, the research team of the present invention repeatedly explored and optimized the experimental conditions and results to find the most suitable experimental method.

[0065] 4.1 Linearity and correction factor

[0066] Take appropriate amounts of phenylpiperazine, impurity 3, and levohydroxypropylpiperazine reference substances, accurately weigh them, dissolve them in methanol, and dilute them to prepare reference substance stock solutions; accurately transfer 5 ml from each reference substance stock solution to the same 100 ml volumetric flask, dilute to scale with solvent (water-methanol (80:20)), shake well, and prepare linear stock solutions. Accurately measure 0.05 ml, 0.1 ml, 0.05 ml, 1 ml, 1.5 ml, 2 ml, 2.5 ml, 3 ml, and 5 ml of the linear stock solution and place them in a 10 ml volumetric flask, dilute to scale with solvent, and use them as gradient concentration solutions. Accurately measure 10 μl in sequence, inject them into the liquid chromatograph, record the chromatogram, use each peak area A as the ordinate and the corresponding concentration C as the abscissa, draw the standard curve of each component, calculate the regression equation, and the results are shown in Table 1. Calculate the correction factor F of impurity 3 according to the formula provided below, and the results are shown in Table 2

[0067]

[0068] Table 2 Linear regression of levodropropizine and known impurities

[0069]

[0070] The calculation formula for impurity 3 content is:

[0071]

[0072] Among them C R : Phenylpiperazine reference substance concentration; A R : Phenylpiperazine reference peak area; A X : peak area of ​​the test sample; F: correction factor for impurity 3.

[0073] 4.2 Confirmation of structures of impurities 2, 3 and 4

[0074] The content of impurity 1 is low, therefore, the present invention mainly conducts structural confirmation research on impurities 2, 3, and 4.

[0075] The impurity 2 was analyzed by two-dimensional liquid chromatography-high resolution mass spectrometry. Figure 4 The molecular ion peak of the impurity is m / z = 265.1912. The structural formula and basic information of impurity 2 can be found in Figure 5 .

[0076] The content of impurity 3 in some batches of samples is relatively high, up to 1.4%, so preparative liquid phase purification is used to separate and obtain the monomer of impurity 3. Two-dimensional liquid phase-high resolution mass spectrometry (such as Figure 6 impurity 3 high resolution mass spectrum), nuclear magnetic resonance (see Figure 7 Impurities in 3HNMR, CNMR, Figure 8 Impurities in 3DEPT90, CNMR (DEPT135) images, Fig. 9 Impurities in 3HH-COSY, CH-HSQC, CH-HMBC diagrams) and infrared spectrophotometry ( Fig.10 The impurity 3 infrared spectrum in the figure is analyzed to obtain the structural formula and basic information of impurity 3. Fig.11 Impurity 3 structure and basic information. Figure 2 The impurity spot before the main spot in the chromatographic separation was eluted and extracted to obtain a spot impurity. The impurity was confirmed to be impurity 3 by liquid chromatography and two-dimensional liquid chromatography-high resolution mass spectrometry, indicating that the method of the present invention can effectively detect the impurity spots detected in the chromatographic separation.

[0077] The impurity 4 was analyzed by two-dimensional liquid chromatography-high resolution mass spectrometry. Fig.12 High-resolution mass spectrum of impurity 4. The molecular ion peak of the impurity is m / z=425.255506. The structure and basic information of impurity 4 can be found in Fig.13 .

[0078] 4.3 Repeatability study

[0079] Take an appropriate amount of this product and determine the impurity content of this product according to the test method for related substances. Repeat the determination 6 times to examine the precision of the method. The results are shown in Table 3 below.

[0080] Table 3 Repeatability results of levodropropizine test solution

[0081] 1 2 3 4 5 6 average RSD Phenylpiperazine(%) 0.0038 0.0044 0.0044 0.0044 0.0036 0.0038 0.004 9.4 Impurity 1(%) 0.017 0.0161 0.0181 0.0147 0.0169 0.0188 0.0169 8.6 Impurity 2(%) 0.0727 0.0731 0.0747 0.072 0.0732 0.0712 0.0728 1.7 Impurity 3(%) 1.4065 1.455 1.4414 1.4069 1.4414 1.4555 1.4344 1.6 Impurity 4(%) 0.0132 0.014 0.0142 0.0156 0.016 0.0162 0.0148 8.4 Other impurities (%) 0 0 0 0 0 0 0 - Total impurities % (%) 1.5132 1.5626 1.5528 1.5136 1.5511 1.5655 1.5431 1.6

[0082] The experimental results show that after repeated determination for 6 times, the determination deviation of each impurity content and the total impurity content is less than 10%, and the method has good precision. Specific embodiment 1

[0084] A total of 55 batches of drugs from three commercially available manufacturers of levodropropizine capsules (Hunan Jiudian, Heilongjiang Wusulijiang and Jiangsu Zhengda Fenghai) and 2 batches of drugs from a manufacturer of levodropropizine tablets (Kunming Yuanrui) were investigated using the method of the present invention, and the results are shown in Table 4; 19 batches of levodropropizine raw materials from one manufacturer were investigated, and the results are shown in Table 5.

[0085] Related substances were determined by high performance liquid chromatography (General Rule 0512).

[0086] Solvent methanol-water (20:80)

[0087] Take an appropriate amount of the content of the product for test solution, grind it finely, accurately weigh an appropriate amount of the fine powder, add solvent, sonicate to dissolve the levodropropizine, and quantitatively dilute it to make a solution containing about 0.5 mg of levodropropizine per 1 ml, filter it, and take the filtrate.

[0088] Reference solution Take an appropriate amount of phenylpiperazine reference substance, weigh accurately, dissolve in methanol and quantitatively dilute to make a solution containing about 50μg per 1ml. Accurately measure 1ml, place in a 100ml volumetric flask, dilute to the mark with solvent, and shake well.

[0089] System suitability solution: Take appropriate amounts of phenylpiperazine and levohydroxypropylpiperazine, add appropriate amounts of methanol to dissolve them, and then dilute with solvent to prepare solutions containing approximately 10 μg and 100 μg, respectively, per 1 ml.

[0090] Chromatographic conditions: octadecylsilane bonded silica gel was used as filler; phosphate buffer (0.34 g of potassium dihydrogen phosphate was weighed, dissolved in 1000 ml of water, and the pH value was adjusted to 2.5 with phosphoric acid) was used as mobile phase A, methanol was used as mobile phase B, and gradient elution was performed according to the table below; the column temperature was 40°C, the flow rate was 0.7 ml / min, and the detection wavelength was 241 nm.

[0091] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 80 20 10 80 20 35 40 60 36 80 20 50 80 20

[0092] System suitability requirements In the system suitability solution chromatogram, the theoretical plate number calculated based on the levodropropizine peak shall not be less than 2000, and the separation degree between levodropropizine and phenylpiperazine shall comply with the regulations.

[0093] Determination method: Accurately measure 10 μl of the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0094] If there is a chromatographic peak in the limit test solution that is consistent with the retention time of phenylpiperazine, the peak area shall be calculated using the external standard method; for other impurities, the main peak area of ​​the reference solution shall be used as a reference and the peak area shall be calculated using the external standard method.

[0095]

[0096]

[0097]

[0098] Table 4 Test results of related substances in Levodropropizine Capsules

[0099]

[0100]

[0101] Table 5 Results of the test for related substances of levodropropizine

[0102] The results of the determination of related substances using the Chinese Pharmacopoeia method and the method of the present invention show that the number and content of related substance impurities detected are significantly increased, as shown in Table 6. The impurity 3 detected by the method of the present invention is up to 1.178%, which is far more than the limit of 0.1% of other single impurities of levodeoxypropylpiperazine in the Chinese Pharmacopoeia and the limit of 0.2% of other single impurities of levodeoxypropylpiperazine tablets / capsules, as shown in Table 7.

[0103]

[0104] Table 6 Comparison of the results of determination of related substances in levodropropizine capsules / tablets

[0105] Relative retention time Impurity content (%) Checkout batch (batch) Detection rate (%) Phenylpiperazine 1.24 0～0.009 52 91.2 Impurity 1 1.88 0.001～0.036 57 100.0 Impurity 2 4.15 0.005～0.055 57 100.0 Impurity 3 6.02 0.003～1.178 57 100.0 Impurity 4 6.04 0～0.121 24 43.6

[0106] Table 7 Determination results of related substances in levodropropizine capsules / tablets

[0107] As can be seen from the above table, the method for determining related substances of levodropropizine raw materials and preparations provided by the present invention can be used for the detection of related substances of levodropropizine raw materials and preparations, which not only makes up for the omissions of existing detection technologies, but also is accurate and reliable, has strong specificity, saves time and effort, and is easy to operate.

[0108] As shown in Table 8, methodological studies have shown that the substance determination method of the present invention has good specificity, accuracy and precision, and is suitable for the determination of related substances of levodropropizine.

[0109]

[0110] Table 8 Summary of verification of substance determination methodology of the present invention

[0111] Although the principles of the present invention are described in detail above in conjunction with the preferred embodiments of the present invention, those skilled in the art should understand that the above embodiments are merely explanations of the exemplary implementations of the present invention, and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute limitations on the scope of the present invention, and any obvious changes such as equivalent transformations, simple replacements, etc. based on the technical solution of the present invention, without departing from the spirit and scope of the present invention, fall within the protection scope of the present invention.

Claims

1. A method for determining substances contained in levodropropizine raw materials, capsules, and tablets, characterized in that: The method comprises: (1) Column CAPCELL PAK C18 MGII 4.6 mm × 150 mm, 3 µm, filled with octadecylsilane bonded silica gel; (2) A predetermined amount of levodropropizine raw material capsules are drawn to prepare a test solution; (3) 10 µl of the test solution and reference solution were injected into the liquid chromatograph respectively; (4) Using phosphate buffer as the first mobile phase A and methanol as the second mobile phase B, add them into a liquid chromatograph for gradient elution; (5) Use liquid chromatography to perform chromatographic detection on the test solution and the reference solution respectively. Wherein, when gradient elution is performed, the gradient changes of the mobile phase and the elution time are as follows: 0-10 minutes, the ratio of mobile phase A to mobile phase B is 80:20 for isocratic elution; 10-35 minutes, the ratio of mobile phase A is gradually reduced from 80% to 40%, and the ratio of mobile phase B is gradually increased from 20% to 60%; 35-36 minutes, the ratio of mobile phase A is gradually increased from 40% to 80%, and the ratio of mobile phase B is gradually reduced from 60% to 20%; 36-50 minutes, the ratio of mobile phase A to mobile phase B is 80:20 for isocratic elution. When gradient elution is performed, the concentration of potassium dihydrogen phosphate in the phosphate buffer is 0.034%, and the pH value of the phosphate buffer is adjusted to 2.5 with phosphoric acid. Among them, the reference substances include phenylpiperazine, impurity 3, and levulropyrazine. The structure of impurity 3 is: ； The detection wavelength is 241 nm.

2. The method for determining substances contained in levodropropizine raw materials, capsules and tablets according to claim 1, characterized in that: During liquid chromatography detection, the flow rate was 0.7 ml / min and the column temperature was 40°C.

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