A method for detecting related substances of piperidine-4-carboxamide

Abstract

The invention provides a method for detecting related substances of piperidine-4-carboxamide. The method adopts high performance liquid chromatography to directly detect positional isomer impurities, degradation impurities and the like in piperidine-4-carboxamide without the need for derivatization treatment and is easy to operate.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a high performance liquid chromatography method for detecting related substances in piperidine-4-carboxamide. Background Art

[0002] Piperidine-4-formamide, also known as 4-piperidinecarboxamide and isopiperidincarboxamide, is an important fine organic chemical intermediate, widely used in the fields of medicine, pesticides, and chemical industry. Piperidine-4-formamide is a commonly used material in the production of revenacin and donepezil hydrochloride raw materials. Its main use is as a molecular skeleton to participate in the synthesis of drug molecules. CAS number: 39546-32-2, molecular formula: C6H 12 N2O, molecular weight is 128.2, structural formula is as follows:

[0003]

[0004] Revefenacin is a long-acting muscarinic antagonist that is mainly used clinically for the maintenance treatment of chronic obstructive pulmonary disease, including the maintenance treatment of chronic bronchitis and emphysema accompanied by dyspnea and the prevention of acute attacks.

[0005] Donepezil Hydrochloride is an acetylcholinesterase inhibitor, which is mainly used clinically to treat mild or moderate Alzheimer's dementia symptoms.

[0006] In the production process of revenacin and donepezil hydrochloride, positional isomer impurities in piperidine-4-carboxamide, such as piperidine-2-carboxamide and piperidine-3-carboxamide, and the starting raw material piperidine-4-carboxylic acid, all contain reactive sites, namely piperidine nitrogen atoms, which can be derived and transferred to the API, posing risks to the safety and efficacy of the drug. Other impurities contained in piperidine-4-carboxamide may also remain in the API of the final product, posing risks to the safety of the finished drug.

[0007] Chinese patent application CN118169269A discloses a method for detecting multiple impurities in piperidine-4-carboxamide material, comprising the following steps: 1) sample pretreatment: taking piperidine-4-carboxamide and adding acetonitrile solution to prepare a test solution; 2) gas chromatography detection: using a gas chromatograph to measure the test solution, and the chromatographic parameters are set as follows: injection volume 0.8-1.2 μl; injection port temperature 240-260°C; detector temperature 280- 320℃; flow rate 1.5~2.5ml / min; split ratio 8~12:1; temperature program: the starting temperature of the chromatographic column is 45~60℃, and the temperature is increased to 190~210℃ at a rate of 15~25℃ per minute, maintained for 3~7 minutes, then increased to 230~250℃ at a rate of 20~35℃ per minute, maintained for 6~10 minutes, and then increased to 255~265℃ at a rate of 15~25℃ per minute, maintained for 8~12 minutes. The impurities involved in the study are piperidine-4-carboxylic acid methyl ester, N-methylpiperidine-4-carboxamide, N-methyl-3-piperidinecarboxylic acid methyl ester, and N-methyl-4-piperidinecarboxylic acid methyl ester, and do not contain the piperidine-4-carboxamide positional isomer with the highest risk of affecting drug quality.

[0008] Chinese patent application CN 117630247 A discloses a method for simultaneously determining multiple impurities in piperidine-4-carboxamide, wherein the impurities are selected from at least one of piperidine-2-carboxamide, piperidine-3-carboxamide and piperidine-4-carboxylic acid; the method is based on high performance liquid phase pre-column derivatization technology, comprising reacting a blank solution, a reference solution and a test solution with a derivatization reagent at 60°C±2°C for 50±2 minutes respectively to obtain a derivatized blank solution, a derivatized reference solution and a derivatized test solution, which are respectively injected into a high performance liquid chromatograph, the detection wavelength is 360-370nm, the chromatogram is recorded, and the determination is performed; wherein the derivatization reagent is a combination of 2,4-dinitrofluorobenzene and tris(hydroxymethylaminomethane), and the mass ratio of 2,4-dinitrofluorobenzene to tris(hydroxymethylaminomethane) is 10:3. However, this method has the following defects: (1) The derivatization reagent used is a combination of 2,4-dinitrofluorobenzene and tris(hydroxymethyl)aminomethane, of which 2,4-dinitrofluorobenzene is genotoxic and harmful to the health of test personnel and the environment; (2) The impurities controlled by this method are piperidine-2-carboxamide, piperidine-3-carboxamide and piperidine-4-carboxylic acid. The principle is that the piperidine group in the structure reacts with the derivatization reagent. Other impurities that do not react with the derivatization reagent cannot be detected, and the impurity control range is narrow; (3) It is necessary to prepare a derivatization reagent solution and a blank solution, a reference solution and a test solution. A total of four solvents, including ethanol, water, 20% acetonitrile water and acetonitrile, are used in the preparation of the solution. The blank solution, the reference solution and the test solution are reacted with the derivatization reagent at 60°C±2°C for 50±20 min, respectively. The measurement is carried out 2 minutes later. The detection process requires the preparation of multiple solutions and then derivatization. The derivatization process requires heating and precise control of temperature and reaction time. The pretreatment is cumbersome and the conditions are relatively harsh. (4) The derivatization blank solution contains multiple chromatographic peaks, and the peak positions are close to the target substances, which are easily interfered with. There is also a risk of misattribution when using relative retention times to attribute impurities.

[0009] Chinese patent application CN117929600A discloses a method for detecting piperidine-3-carboxamide, which is used to detect piperidine-3-carboxamide in (R)-3-aminopiperidine dihydrochloride. However, liquid mass spectrometry instruments are expensive, with high usage and maintenance costs, and most companies do not have the conditions for detection. In addition, this method is used for the quantitative detection of piperidine-3-carboxamide, and the separation of other substances with similar structures has not been studied. The mass spectrometer is also not suitable for the study of related substances, and the method is limited in its applicability.

[0010] Therefore, regarding the detection of piperidine-4-carboxamide related substances, the existing literature has many defects in practice and is insufficient to meet the quality control needs of piperidine-4-carboxamide. It is urgent to establish a detection method for piperidine-4-carboxamide related substances, which can effectively detect the impurities contained in piperidine-4-carboxamide, especially isomeric impurities and degradation impurities, so as to achieve quality control of revenacin and donepezil hydrochloride. Summary of the invention

[0011] The invention solves the deficiencies in the prior art and provides a method for detecting piperidine-4-carboxamide-related substances. The method adopts high performance liquid chromatography to directly detect positional isomer impurities, degradation impurities and the like in piperidine-4-carboxamide without the need for derivatization treatment and is simple to operate.

[0012] The invention provides a method for detecting related substances of piperidine-4-carboxamide, wherein the chromatographic conditions are analyzed by using a reversed-phase high performance liquid chromatograph, and the chromatographic conditions include: a chromatographic column: a chromatographic column using sulfonic acid group cation exchange bonded silica gel as a filler; a mobile phase: mobile phase A: phosphate solution-acetonitrile; mobile phase B: acetonitrile; and gradient elution.

[0013] The related substances of piperidine-4-carboxamide described in the present invention include:

[0014] .

[0015] The phosphate in the mobile phase A of the present invention is selected from ammonium dihydrogen phosphate, potassium dihydrogen phosphate, and sodium dihydrogen phosphate; preferably, the phosphate is ammonium dihydrogen phosphate. The concentration of the phosphate is 5 mmol / L to 20 mmol / L; preferably, the concentration of the phosphate is 10 mmol / L. The pH value of the phosphate solution is 3.0 to 5.0; preferably, the pH value of the phosphate solution is 4.0.

[0016] The ratio of the phosphate solution to acetonitrile in the mobile phase A of the present invention is 100:0 to 95:5. Preferably, the ratio of the phosphate solution to acetonitrile in the mobile phase A is 99:1.

[0017] The phosphate solution in the mobile phase A of the present invention further comprises a chaotropic agent, and the chaotropic agent is selected from potassium hexafluorophosphate and sodium perchlorate; preferably, the chaotropic agent is potassium hexafluorophosphate. The concentration of the chaotropic agent is 20 mmol / L to 70 mmol / L; preferably, the concentration of the chaotropic agent is 50 mmol / L.

[0018] The gradient elution conditions in the chromatographic conditions of the present invention are as follows:

[0019] ;

[0020] Preferably, the gradient elution conditions in the chromatographic conditions of the present invention are as follows:

[0021] .

[0022] The chromatographic conditions of the present invention also include:

[0023] Detector: UV detector;

[0024] Flow rate: 0.5 ml / min to 1.2 ml / min; preferably, the flow rate is 0.8 ml / min;

[0025] Column temperature: 20°C to 45°C; preferably, the column temperature is 35°C;

[0026] Detection wavelength: 190nm to 200nm; preferably, the detection wavelength is 194nm;

[0027] Injection volume: 5µl.

[0028] The method for detecting related substances of piperidine-4-carboxamide of the present invention also includes the following steps: taking an appropriate amount of piperidine-4-carboxamide, adding a solvent to dissolve it and quantitatively diluting it to prepare a test solution.

[0029] The method for detecting related substances of piperidine-4-carboxamide of the present invention further comprises the following steps: taking an appropriate amount of piperidine-4-carboxamide, accurately weighing it, adding a solvent to dissolve it, and quantitatively diluting it to prepare a test solution containing about 3 mg of the test substance per 1 ml;

[0030] The method for detecting related substances of piperidine-4-carboxamide of the present invention also includes the following steps: taking appropriate amounts of piperidine-4-carboxamide, piperidine-2-carboxamide reference substances and piperidine-3-carboxamide reference substances, adding solvents to dissolve and dilute them to prepare system applicability solutions of piperidine-2-carboxamide, piperidine-3-carboxamide and piperidine-4-carboxamide, respectively.

[0031] The method for detecting related substances of piperidine-4-carboxamide of the present invention also includes the following steps: taking appropriate amounts of piperidine-4-carboxamide, piperidine-2-carboxamide reference substances and piperidine-3-carboxamide reference substances, dissolving them in solvents and diluting them to prepare solutions containing approximately 30 μg, 30 μg and 3 mg of piperidine-2-carboxamide, piperidine-3-carboxamide and piperidine-4-carboxamide per 1 ml, respectively.

[0032] The solvent described in the present invention is a diammonium phosphate solution. The concentration of the diammonium phosphate solution is 10 mmol / L. The diammonium phosphate solution also contains potassium hexafluorophosphate, and the pH value is adjusted to 4.0 with phosphoric acid, and the concentration of the potassium hexafluorophosphate is 50 mmol / L. The inventors found that the diammonium phosphate solution can dissolve the test sample and does not react with the test sample, and is basically consistent with the initial ratio of the mobile phase to avoid the solvent effect and the baseline fluctuation and solvent peak caused by the difference between the mobile phase and the solvent at low wavelength, and does not interfere with the main peak and impurity peaks.

[0033] The present invention further provides a method for detecting piperidine-4-carboxamide-related substances and its use in quality control of revenacin and donepezil hydrochloride.

[0034] The beneficial technical effects of the present invention include: the detection method of piperidine-4-carboxamide-related substances of the present invention adopts high performance liquid chromatography, can directly detect positional isomer impurities, degradation impurities, etc. in piperidine-4-carboxamide, does not require derivatization treatment, and is easy to operate. The present invention adopts high performance liquid chromatography and ultraviolet detector, has low requirements on equipment, strong applicability, and high practical value.

[0035] The present invention adopts a chromatographic column using sulfonic acid cation exchange bonded silica gel as a filler, and piperidine-4-carboxamide and its positional isomer impurities and degradation impurities are well retained and separated, with a separation degree greater than 2.0. Impurities are located and attributed using an impurity reference substance, and the method has strong specificity; the impurity quantitative limit can reach 0.025% of the concentration of the test solution, and the sensitivity is high; piperidine-4-carboxamide has good linearity within the range of 0.752 to 3601.952 μg / ml, a wide linear range, and high method precision, and can be used for the quality control of piperidine-4-carboxamide, meeting the quality control requirements of the raw material drug production process for piperidine-4-carboxamide. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The chromatograms of the mixed solution at different wavelengths in Example 1 are shown.

[0037] Figure 2 Chromatogram of the mixed solution selected for the chromatographic column in Example 2.

[0038] Figure 3 This is the separation solution chromatogram for the chromatographic column selected in Example 3.

[0039] Figure 4 Chromatogram of the mixed solution selected as the mobile phase in Example 4.

[0040] Figure 5 This is the blank solvent interference test chromatogram of Example 5.

[0041] Figure 6 This is the separation solution chromatogram of Example 6.

[0042] Figure 7 This is the linear diagram of piperidine-4-carboxamide of Example 7.

[0043] Figure 8 It is the chromatogram of the quantitative limit solution of pyridine-4-carboxamide, piperidine-4-carboxamide, piperidine-3-carboxamide and piperidine-2-carboxamide in Example 8.

[0044] Fig. 9 It is the detection limit solution chromatogram of pyridine-4-carboxamide, piperidine-4-carboxamide, piperidine-3-carboxamide and piperidine-2-carboxamide in Example 8.

[0045] Fig.10 This is the chromatogram of the precision test of Example 9.

[0046] Fig.11 This is the chromatogram of the solution stability test of Example 10. DETAILED DESCRIPTION

[0047] Specific embodiments of the present invention are described in detail below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0048] Instrument: High performance liquid chromatography, UV detector

[0049] Solution preparation:

[0050] Solvent: 10 mmol / L diammonium phosphate solution (containing 50 mmol / L potassium hexafluorophosphate, pH adjusted to 4.0 with phosphoric acid).

[0051] Mixed solution: Take appropriate amounts of piperidine-2-carboxamide, piperidine-3-carboxamide, piperidine-4-carboxamide, pyridine-4-carboxamide and piperidine-4-carboxylic acid, dissolve in solvent and dilute to prepare a mixed solution containing about 0.1 mg per 1 ml.

[0052] Separation solution: Take appropriate amount of piperidine-2-carboxamide, piperidine-3-carboxamide, piperidine-4-carboxamide, pyridine-4-carboxamide and piperidine-4-carboxylic acid, dissolve in solvent and dilute to make a solution containing about 30µg of piperidine-2-carboxamide, piperidine-3-carboxamide and pyridine-4-carboxamide, about 0.1mg of piperidine-4-carboxylic acid and about 3mg of piperidine-4-carboxamide per 1ml.

[0053] Positioning solution: Take appropriate amount of piperidine-2-carboxamide reference substance, piperidine-3-carboxamide reference substance, pyridine-4-carboxamide reference substance and piperidine-4-carboxylic acid reference substance, dissolve them in water and dilute them to make a solution containing about 1 mg per 1 ml.

[0054] Test solution: Take an appropriate amount of piperidine-4-carboxamide, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing about 3 mg per 1 ml.

[0055] System suitability solution: Take appropriate amount of piperidine-4-carboxamide, piperidine-2-carboxamide reference substance and piperidine-3-carboxamide reference substance, dissolve in solvent and dilute to make a solution containing approximately 30μg, 30μg and 3mg of piperidine-2-carboxamide, piperidine-3-carboxamide and piperidine-4-carboxamide per 1ml.

[0056] Example 1 Detection wavelength selection

[0057] Piperidine-4-carboxamide and its impurities are terminal absorbers on the UV detector. The response gradually weakens with the increase of wavelength. The sensitivity requirements can be met in the range of 190nm to 200nm. Figure 1 .

[0058] Example 2 Chromatographic column selection

[0059] Instrument: High performance liquid chromatography, UV detector;

[0060] Column: YMC Triart Phenyl, 4.6 mm × 250 mm, 5 μm;

[0061] Mobile phase A: 10 mmol / L potassium dihydrogen phosphate solution (containing 100 mmol / L potassium hexafluorophosphate, pH adjusted to 7.5 with phosphoric acid);

[0062] Mobile phase B: acetonitrile;

[0063] Flow rate: 0.5 ml / min; column temperature: 35 °C; detection wavelength: 200 nm;

[0064] Perform gradient elution according to the following table:

[0065] ;

[0066] Take 10 µl of the mixed solution, inject it into the liquid chromatograph, and record the chromatogram.

[0067] Results: The elution time of piperidine-4-carboxylic acid in the mixed solution was 5.536 minutes, which was interfered by the solvent peak. Figure 2 .

[0068] Example 3 Chromatographic column selection

[0069] Chromatographic column: CAPCELL PAK SCX, 4.6 mm × 250 mm, 5 μm;

[0070] Mobile phase A: 10mmol / L ammonium dihydrogen phosphate solution (containing 50mmol / L potassium hexafluorophosphate, pH adjusted to 4.0 with phosphoric acid)-acetonitrile (99:1);

[0071] Mobile phase B: acetonitrile;

[0072] Flow rate: 0.8 ml / min; column temperature: 35 °C; detection wavelength: 194 nm;

[0073] Perform gradient elution according to the following table:

[0074] ;

[0075] Take 5 µl of the separation solution, inject it into the liquid chromatograph, and record the chromatogram.

[0076] Results: The main peak in the separation solution was eluted at 21.478 minutes, the theoretical plate number was 13510, the tailing factor was 1.19, the peak shape was good, the separation between the main peak and the adjacent impurities and between each impurity was greater than 2.0, and the separation was good. Figure 3 .

[0077] Example 4 Selection of mobile phase

[0078] Instrument: High performance liquid chromatography, UV detector;

[0079] Chromatographic column: CAPCELL PAK SCX, 4.6 mm × 250 mm, 5 μm;

[0080] Mobile phase A1: 20mmol / L potassium dihydrogen phosphate solution (containing 50mmol / L sodium perchlorate, pH adjusted to 3.0 with phosphoric acid);

[0081] Mobile phase A2: 20mmol / L potassium dihydrogen phosphate solution (containing 50mmol / L sodium perchlorate, pH adjusted to 4.0 with phosphoric acid);

[0082] Flow rate: 0.8 ml / min; column temperature: 35 °C; detection wavelength: 194 nm;

[0083] Elution mode: isocratic elution;

[0084] Take 10μl of the mixed solution, inject it into the liquid chromatograph under different mobile phase conditions, and record the chromatogram.

[0085] Results: Under the conditions of mobile phase A1 and A2, the chromatographic peaks were well separated, indicating that the method is applicable when the mobile phase phosphate is potassium dihydrogen phosphate, the chaotropic agent is sodium perchlorate, and the pH value is within the range of 3.0-4.0. Figure 4 .

[0086] Example 5 Blank solvent interference test

[0087] The solvent used for the inspection of relevant substances is 10mmol / L diammonium phosphate solution (containing 50mmol / L potassium hexafluorophosphate, and the pH value is adjusted to 4.0 with phosphoric acid). Piperidine-4-carboxamide and its impurities have good solubility in this solvent.

[0088] Accurately measure 5 μl of solvent, inject into liquid chromatograph, and record the chromatogram. Results: Blank solvent does not interfere with the determination of related substances, see Figure 5 .

[0089] Example 6 Separation Test

[0090] Take 5 μl of separation solution and positioning solution, inject them into the liquid chromatograph respectively, and record the chromatogram. The results are shown in Table 1. Figure 6 As shown in the figure, in the separation solution, the minimum separation between the main peak and the adjacent impurity peaks and between the known impurity peaks is 3.36, which is good separation and does not interfere with each other in detection, indicating that the method has good specificity.

[0091] Table 1 Separation test results

[0092]

[0093] Example 7 Linearity Test

[0094] Take an appropriate amount of piperidine-4-carboxamide, weigh it accurately, add a solvent to dissolve it and quantitatively dilute it to make solutions containing approximately 3μg / ml (0.1%), 30μg / ml (1%), 300μg / ml (10%), 1500μg / ml (50%), and 3600μg / ml (120%) of piperidine-4-carboxamide. Accurately measure 5μl of each of the above solutions and the quantitative limit solution, inject them into the liquid chromatograph respectively, and record the chromatogram. Plot a curve of peak area versus concentration, and calculate the regression equation, correlation coefficient and intercept by the least squares method. The results are shown in Table 2. Figure 7 The results showed that within the concentration range of 0.752-3601.952 mg / ml, the peak area of ​​piperidine-4-carboxamide had a good linear relationship with the concentration.

[0095] Table 2 Linearity test results of piperidine-4-carboxamide related substances

[0096]

[0097] Example 8 Quantitative Limit and Detection Limit Test

[0098] Take an appropriate amount of each compound, add solvent to dissolve and dilute to make a solution or mixed solution of a certain concentration, dilute with solvent to different concentrations, accurately measure 5μl, inject into liquid chromatograph, record chromatogram, until the measured main peak response value is not less than 10 times and 3 times higher than the noise signal, which is the quantitative limit and detection limit of each compound. The results are shown in Table 3, Table 4 and Figure 8 , 9 .

[0099] Table 3 Quantitative limit results of piperidine-4-carboxamide related substances

[0100]

[0101] Table 4 Detection limit results of piperidine-4-carboxamide related substances

[0102]

[0103] Example 9 Precision Test

[0104] Take about 30 mg of piperidine-4-carboxamide, weigh it accurately, put it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and use it as the test solution. Prepare 6 copies in parallel. Accurately measure 5 μl, inject it into the liquid chromatograph, record the chromatogram, calculate and compare the content of piperidine-4-carboxylic acid, piperidine-3-carboxamide, piperidine-2-carboxamide, other largest single impurities and total impurities 6 times. The results show that the detection amount of piperidine-4-carboxylic acid is basically consistent (between 0.06% and 0.07%), with an RSD of 6.63%; piperidine-3-carboxamide and piperidine-2-carboxamide are not detected; the detection amount of other largest single impurities is basically consistent (between 0.12% and 0.13%), with an RSD of 3.19%; the detection amount of total impurities is basically consistent (between 0.28% and 0.29%), with an RSD of 1.81%. The precision test results of piperidine-4-carboxamide related substances were good. The results are shown in Table 5. Fig.10 .

[0105] Table 5 Results of precision test of piperidine-4-carboxamide related substances

[0106]

[0107] Example 10 Solution stability test

[0108] Test solution: Take an appropriate amount of piperidine-4-carboxamide, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing about 3 mg per 1 ml.

[0109] Place the test solution at room temperature under natural light, and accurately measure 5 μl at 0, 2.5, 5, 7.5, 10, and 24 hours, inject into the liquid chromatograph, and record the chromatogram. Compared with 0 hours, calculate and analyze the changes in the detection amount of piperidine-4-carboxylic acid, piperidine-3-carboxamide, piperidine-2-carboxamide, other largest single impurities, and total impurities in the chromatogram of the test solution. The results are shown in Table 6. Fig.11 As shown, the test solution was placed under natural light at room temperature for 24 hours, and the detection amount of piperidine-4-carboxylic acid (between 0.07% and 0.08%) had a change value of 0.01%; piperidine-3-carboxamide and piperidine-2-carboxamide were not detected, the other largest single impurity detection amount (between 0.12% and 0.13%) had a maximum change rate of 7.69%, the total impurity detection amount (between 0.29% and 0.31%) had a maximum change rate of 6.90%, and no new impurities were generated. The test solution was stable within 24 hours under room temperature and natural light conditions.

[0110] Table 6 Results of stability test of piperidine-4-carboxamide related substances test solution

[0111]

Claims

1. A method for detecting related substances of piperidine-4-carboxamide, characterized in that, Separation solution and test solution were sampled respectively, and the chromatographic conditions were analyzed by reverse phase high performance liquid chromatography, and the chromatographic conditions included: chromatographic column: a chromatographic column with sulfonic acid cation exchange bonded silica gel as a filler; mobile phase: mobile phase A: phosphate solution-acetonitrile, the concentration of the phosphate is 5mmol / L~20mmol / L, and the ratio of the phosphate solution to acetonitrile is 100:0~95:5; mobile phase B: acetonitrile; gradient elution was performed according to the following table: ； The relevant substances include:

2. The method for detecting related substances of piperidine-4-carboxamide according to claim 1, characterized in that: The separation solution is prepared by the following process: taking appropriate amounts of piperidine-2-carboxamide, piperidine-3-carboxamide, piperidine-4-carboxamide, pyridine-4-carboxamide and piperidine-4-carboxylic acid, adding a solvent to dissolve and dilute to prepare a separation solution; the test solution is prepared by the following process: taking an appropriate amount of piperidine-4-carboxamide, adding a solvent to dissolve and dilute to prepare a test solution.

3. The method for detecting related substances of piperidine-4-carboxamide according to claim 2, characterized in that, The solvent is ammonium dihydrogen phosphate solution.

4. The method for detecting related substances of piperidine-4-carboxamide according to claim 3, characterized in that: The diammonium phosphate solution also contains potassium hexafluorophosphate, and the pH value is adjusted to 4.0 with phosphoric acid.

5. The method for detecting related substances of piperidine-4-carboxamide according to claim 1, characterized in that: The phosphate in the mobile phase A is selected from ammonium dihydrogen phosphate, potassium dihydrogen phosphate, and sodium dihydrogen phosphate.

6. The method for detecting related substances of piperidine-4-carboxamide according to claim 1, characterized in that: The phosphate solution in the mobile phase A further comprises a chaotropic agent, and the chaotropic agent is selected from potassium hexafluorophosphate and sodium perchlorate.

7. The method for detecting related substances of piperidine-4-carboxamide according to claim 1, characterized in that: The chromatographic conditions also include: Detector: UV detector; Flow rate: 0.5ml / min~1.2ml / min; Column temperature: 20℃~45℃; Detection wavelength: 190nm~200nm; Injection volume: 5 μl.

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