Method for determining impurity content in 3, 4-diethoxy phenylethylamine by utilizing HPLC (High Performance Liquid Chromatography)

Abstract

The invention discloses a method for determining the content of impurities in 3, 4-diethoxyphenylethylamine by utilizing HPLC (High Performance Liquid Chromatography), and belongs to the technical field of pharmaceutical analysis. According to the method, a chromatographic column taking octadecyl bonded silica gel as a filling agent is adopted, a phosphoric acid aqueous solution is taken as a mobile phase A, methanol is taken as a mobile phase B, gradient elution is carried out, and impurities in a 3, 4-diethoxy phenylethylamine sample are detected by high performance liquid chromatography; the impurities are catechol and 3, 4-diethoxybenzyl cyanide. According to the method disclosed by the invention, the 3, 4-diethoxy phenylethylamine and the impurities can be better separated, the spectral purity of a destroyed sample is greater than 990 through peak purity detection under various conditions, and the specificity is good; meanwhile, when chromatographic parameters are slightly changed, the separation degrees of main components and impurities meet the specification, and the durability is good; the method can be used for rapidly, accurately and reliably detecting and separating the content of each impurity in the 3, 4-diethoxy phenylethylamine so as to meet the requirements of medicine quality control.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for determining the impurity content of 3,4-diethoxyphenethylamine by using HPLC. BACKGROUND

[0002] 3,4-diethoxyphenethylamine has a molecular formula of C 12 H 19 NO2, and a chemical structural formula is as follows:

[0003]

[0004] 3,4-diethoxyphenethylamine is a phenethylamine derivative with an ethoxy substituent, and its chemical structure endows it with certain reactivity and pharmacological potential. It can be used as a starting material or an intermediate for synthesizing various organic compounds and has wide application in the fields of medicine, pesticide, material science and the like. For example, drotaverine hydrochloride is prepared from 3,4-diethoxyphenethylamine and 3,4-diethoxyphenylacetic acid as starting materials, and drotaverine hydrochloride, as a papaverine drug, has obvious effects of relieving spasm, relieving pain and relieving symptoms caused by various reasons. Therefore, strictly controlling the quality of the starting material 3,4-diethoxyphenethylamine is crucial for the production of drotaverine hydrochloride and the like.

[0005] A common preparation process of 3,4-diethoxyphenethylamine is to use catechol as a starting material, and the starting material is prepared through ethylation, chloromethylation, cyanation and hydrogenation reduction reactions and the like. Process impurities and degradation impurities possibly generated in the production process need to be strictly controlled, such as the generation of catechol and 3,4-diethoxyphenylacetonitrile impurities. Since the detection method of 3,4-diethoxyphenethylamine related substances has not been collected in the pharmacopoeias of various countries and academic websites at home and abroad, therefore, providing a method for detecting the impurities catechol and 3,4-diethoxyphenylacetonitrile in 3,4-diethoxyphenethylamine has important significance for the quality control of 3,4-diethoxyphenethylamine. SUMMARY

[0006] In view of the deficiencies of the prior art, the technical problem to be solved by the application is to provide a high-efficiency liquid chromatography analysis method for determining the impurity content of 3,4-diethoxyphenethylamine, which has high feasibility, simple and convenient operation process, good applicability and can be widely applied to effectively control the product quality.

[0007] To solve the above technical problems, the technical scheme adopted by the application is as follows:

[0008] A method for determining the impurity content in 3,4-diethoxyphenethylamine by HPLC, using a chromatographic column with octadecyl-bonded silica gel as the filler, using aqueous phosphoric acid as the mobile phase A, and methanol as the mobile phase B, performing gradient elution, and detecting the impurities in the 3,4-diethoxyphenethylamine sample by high performance liquid chromatography; the impurities are catechol and 3,4-diethoxyphenylacetonitrile.

[0009] As a preference, the aqueous phosphoric acid has a volume percentage concentration of 0.1% and a pH of 5.5.

[0010] As a preference, the chromatographic column is a GL Sciences Inertsil ODS-3 chromatographic column with a specification of 4.6mm x 250mm and a filler particle size of 5μm, and the column temperature is 40℃.

[0011] As a preference, the gradient elution program is as follows: 0-20min, the volume percentage of mobile phase A decreases from 80% to 20% and the volume percentage of mobile phase B increases from 20% to 80%; 20min, the volume percentage of mobile phase A is 20% and the volume percentage of mobile phase B is 80%; 20-40min, the volume percentage of mobile phase A is 20% and the volume percentage of mobile phase B is 80%; 40min, the volume percentage of mobile phase A is 20% and the volume percentage of mobile phase B is 80%.

[0012] As a preference, the flow rate of the gradient elution is 1.0mL / min.

[0013] As a preference, in the high performance liquid chromatography, the injection volume is 20μL.

[0014] As a preference, in the high performance liquid chromatography, the detection wavelength is 280nm.

[0015] As a preference, the detection of the impurities in the 3,4-diethoxyphenethylamine sample includes the following steps:

[0016] 1) Preparation of the test sample solution and the control solution

[0017] The preparation process of the test sample solution is as follows: weigh the 3,4-diethoxyphenethylamine test sample, dissolve and dilute it with a solvent to obtain a solution containing 1mg of 3,4-diethoxyphenethylamine per 1mL, which is used as the test sample solution;

[0018] The preparation process of the control solution is as follows: accurately measure the test sample solution, dilute it with a solvent to obtain a solution containing 10μg of 3,4-diethoxyphenethylamine per 1mL, which is used as the control solution;

[0019] 2) Accurately measure the test solution and the control solution separately, inject them into the liquid chromatograph, detect them by high performance liquid chromatography, record the chromatograms, draw the standard curve, obtain the standard curve equation, and calculate the content of impurities in 3,4-diethoxyphenethylamine.

[0020] Preferably, in step 1), the solvent is methanol.

[0021] Preferably, in step 2), based on the chromatogram, a linear regression equation is plotted with peak area A as the ordinate and concentration C as the abscissa. When the detection wavelength is 280 nm, the linear equation for catechol is A = 0.3814C - 0.0787, the linear equation for 3,4-diethoxyphenylacetonitrile is A = 0.2718C - 0.0250, and the linear equation for 3,4-diethoxyphenylethylamine is A = 0.2593C + 0.0172.

[0022] Preferably, the linear concentration range of catechol is 0.5287–21.1464 μg / mL, the limit of quantitation is 0.5287 μg / mL, and the limit of detection is 0.2115 μg / mL; the linear concentration range of 3,4-diethoxyphenylacetonitrile is 0.5150–20.5996 μg / mL, the limit of quantitation is 0.5150 μg / mL, and the limit of detection is 0.2060 μg / mL; and the linear concentration range of 3,4-diethoxyphenylethylamine is 0.5084–20.3346 μg / mL, the limit of quantitation is 0.5084 μg / mL, and the limit of detection is 0.2033 μg / mL.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0024] 1) The method of the present invention can effectively separate 3,4-diethoxyphenethylamine from impurities at a detection wavelength of 280 nm. The solvent peak does not interfere with the detection of the main component and impurities. Furthermore, the peak purity of the samples destroyed under each condition is greater than 990 after peak purity detection. The impurities generated under each condition do not interfere with the detection of impurities and the main component, demonstrating good specificity.

[0025] 2) The method of the present invention ensures that the separation degree of the main component and impurities meets the requirements when the chromatographic parameters change slightly, and the detection results show no significant change, demonstrating good robustness.

[0026] 3) This invention can quickly, accurately and reliably separate and detect the content of various impurities in 3,4-diethoxyphenethylamine to meet the requirements of drug quality control. Attached Figure Description

[0027] Figure 1 This is a chromatogram of the system suitability solution under the chromatographic conditions of this invention;

[0028] Figure 2The test chromatogram under the chromatographic conditions of the present application in the destructive test; wherein, 2a is the acid destruction spectrum, 2b is the base destruction spectrum, 2c is the light destruction spectrum, 2d is the pyrolysis destruction spectrum, and 2e is the high temperature destruction spectrum, Figure 2 f is the oxidation destruction spectrum;

[0029] Figure 3 The linear standard curve under the chromatographic conditions of the present application; wherein, 3a is the catechol standard curve, 3b is the 3,4-diethoxyphenethylamine standard curve, and 3c is the 3,4-diethoxyphenylacetonitrile standard curve.

[0030] Figure 4 The stability chromatogram of the control solution under the chromatographic conditions of the present application; wherein, 4a is the impurity control solution stability spectrum at 0h, and 4b is the impurity control solution stability spectrum at 30h.

[0031] Figure 5 The stability chromatogram of the test solution under the chromatographic conditions of the present application; wherein, 5a is the test solution stability spectrum at 0h, and 5b is the test solution stability spectrum at 30h.

[0032] Figure 6 The durability chromatogram under the chromatographic conditions of the present application; wherein, 6a is the normal condition chromatogram (flow rate 1.0mL / min, column temperature 40℃, pH of mobile phase A 5.5), 6b is the flow rate 0.95mL / min chromatogram, 6c is the flow rate 1.05mL / min chromatogram, 6d is the column temperature 38℃ chromatogram, 6e is the column temperature 42℃ chromatogram, 6f is the pH of mobile phase A 5.4 chromatogram, 6g is the pH of mobile phase A 5.6 chromatogram, and 6h is the chromatogram using different batch chromatographic columns. DETAILED DESCRIPTION

[0033] The present application will be further illustrated below in conjunction with specific examples. The examples are implemented on the premise of the technical solutions of the present application, and it should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0034] The catechol (control) used in the following examples was purchased from Aladdin Reagent (Shanghai) Co., Ltd.; 3,4-diethoxyphenylacetonitrile (control) was purchased from Leyue Reagent; 3,4-diethoxyphenethylamine (control) and 3,4-diethoxyphenethylamine (test) were both purchased from Nanjing Kexi Pharmaceutical Technology Co., Ltd.

[0035] Example 1

[0036] A method for determining the impurity content in 3,4-diethoxyphenethylamine by HPLC, comprising the following steps:

[0037] (1) Prepare a control solution and a test solution:

[0038] Preparation of the test solution: Take an appropriate amount of 3,4-diethoxyphenethylamine, dissolve it in methanol and dilute it to a solution containing 1 mg of 3,4-diethoxyphenethylamine per 1 mL, as the test solution;

[0039] Preparation of the control solution: accurately take the test solution, dilute it with methanol to a solution containing 10 μg of 3,4-diethoxyphenethylamine per 1 mL, as the control solution;

[0040] (2) Set the liquid chromatography detection conditions:

[0041] Instrument: high performance liquid chromatograph UltiMate 3000;

[0042] Detection wavelength: 280 nm;

[0043] Chromatographic column: GL Sciences Inertsil ODS-3, specification 4.6 mm x 250 mm, filler particle size 5 μm;

[0044] Mobile phase A: 0.1% phosphoric acid aqueous solution (pH adjusted to 5.5 with triethylamine);

[0045] Mobile phase B: methanol;

[0046] Gradient elution program: 0-20 min, the volume percentage of mobile phase A decreases from 80% to 20%, the volume percentage of mobile phase B increases from 20% to 80%; 20 min, the volume percentage of mobile phase A is 20%, the volume percentage of mobile phase B is 80%; 20-40 min, the volume percentage of mobile phase A is 20%, the volume percentage of mobile phase B is 80%; 40 min, the volume percentage of mobile phase A is 20%, the volume percentage of mobile phase B is 80%.

[0047] Flow rate: 1 mL / min;

[0048] Column temperature: 40℃;

[0049] Injection volume: 20 μL;

[0050] (3) precisely pipet 20 μL of the control solution and the sample solution respectively into the liquid chromatograph, the detection wavelength is 280 nm, and record the chromatogram; according to the chromatogram, take the peak area A as the vertical coordinate and the concentration C as the horizontal coordinate to draw the linear regression equation, the linear equation of the impurity o-diphenol is A=0.3814C-0.0787, the linear equation of the impurity 3,4-diethoxyphenylacetonitrile is A=0.2718C-0.0250, and the linear equation of 3,4-diethoxyphenyl- ethylamine is A=0.2593C+0.0172.

[0051] Example 2

[0052] In combination with the production process of 3,4-diethoxyphenyl- ethylamine, the potential impurities (o-diphenol and 3,4-diethoxyphenylacetonitrile) are studied, and the detailed methodology verification is carried out. Through systematic and comprehensive study on the related substances of multiple batches of purchased 3,4-diethoxyphenyl- ethylamine, according to the research results, the quality standard of the related substances of 3,4-diethoxyphenyl- ethylamine is as follows: the standard limit of o-diphenol is ≤0.5%, the standard limit of 3,4-diethoxyphenylacetonitrile is ≤0.5%, the standard limit of the maximum unknown single impurity is ≤0.5%, and the standard limit of the total impurities is ≤2.0%.

[0053] Example 3

[0054] The detection method of Example 1 is verified from the aspects of system applicability, destructive test, quantitative limit, detection limit, linear relationship, precision, accuracy, and solution stability. The solvent used in this example is methanol.

[0055] 1. System applicability

[0056] An appropriate amount of o-diphenol, 3,4-diethoxyphenylacetonitrile, and 3,4-diethoxyphenyl- ethylamine control samples are weighed, dissolved with solvent, and diluted to prepare a control sample stock solution containing 100 μg of o-diphenol, 100 μg of 3,4-diethoxyphenylacetonitrile, and 100 μg of 3,4-diethoxyphenyl- ethylamine per 1 mL.

[0057] An appropriate amount of each control sample stock solution is taken, diluted with solvent to prepare a single positioning solution containing 10 μg of o-diphenol, 10 μg of 3,4-diethoxyphenylacetonitrile, and 10 μg of 3,4-diethoxyphenyl- ethylamine per 1 mL.

[0058] An appropriate amount of 3,4-diethoxyphenyl- ethylamine control sample is accurately weighed, and an appropriate amount of control sample stock solution is added to the same weighing bottle. The mixture is dissolved and diluted with solvent to prepare a mixed solution containing 1 mg of 3,4-diethoxyphenyl- ethylamine, 5 μg of o-diphenol, and 5 μg of 3,4-diethoxyphenylacetonitrile per 1 mL, which is used as the system applicability solution.

[0059] 20 μL each of blank solvent, single-target solutions for each impurity, and system suitability solution were injected into the high-performance liquid chromatograph (HPLC), and chromatograms were recorded to examine the separation of each impurity from the main component and adjacent impurities. Results are as follows: Figure 1 As shown in Table 1.

[0060] Table 1 Impurity Location and Separation Degree

[0061]

[0062] Depend on Figure 1 As shown in Table 1, at a detection wavelength of 280 nm, the elution order of the mixed solution is catechol, 3,4-diethoxyphenylethylamine, and 3,4-diethoxyphenylacetonitrile. There is sufficient separation between 3,4-diethoxyphenylethylamine and the impurities, and the blank solvents do not interfere with the determination of impurities in 3,4-diethoxyphenylethylamine.

[0063] 2. Destructive testing

[0064] This product: 3,4-diethoxyphenylethylamine (test sample).

[0065] Undamaged solution: Take 10 mg of this product and place it in a 10 mL volumetric flask. Dissolve and dilute to the mark with solvent, and shake well to obtain the undamaged solution.

[0066] Acid destruction solution: Take 10 mg of this product and place it in a 10 mL volumetric flask. Add 1 mL of 1 mol / L hydrochloric acid solution and destroy at room temperature for 3 h. Add 1 mL of 1 mol / L sodium hydroxide solution to neutralize. Dissolve and dilute to the mark with solvent and shake well to obtain the acid destruction solution.

[0067] Alkali destruction solution: Take 10 mg of this product and place it in a 10 mL volumetric flask. Add 1 mL of 1 mol / L sodium hydroxide solution and destroy at room temperature for 3 h. Add 1 mL of 1 mol / L hydrochloric acid solution to neutralize. Dissolve and dilute to the mark with solvent and shake well to obtain the alkali destruction solution.

[0068] Oxidative destruction solution: Take 10 mg of this product and place it in a 10 mL volumetric flask. Add 1 mL of 30% hydrogen peroxide and incubate at room temperature for 2 h. Dissolve and dilute to the mark with a solvent and shake well to obtain the oxidative destruction solution.

[0069] Pyrolysis destruction solution: Take 10 mg of this product and place it in a 10 mL volumetric flask. Add an appropriate amount of solvent to dissolve it. Heat it in an 80 °C oven for 3 h to destroy the product. Cool it to room temperature and then dissolve and dilute it to the mark with solvent. Shake well to obtain the pyrolysis destruction solution.

[0070] High-temperature destruction solution: Take 10 mg of this product and place it in a 10 mL volumetric flask. Heat it in a 100 °C oven for 2 h to destroy the product. Cool it to room temperature, then dissolve and dilute it to the mark with a solvent. Shake well to obtain the high-temperature destruction solution.

[0071] Irradiation destruction solution: take 10 mg of the product in a 10 mL volumetric flask, add an appropriate amount of solvent to dissolve, then dilute to the mark with solvent, and irradiate under UV light for 3 h as the irradiation destruction solution.

[0072] Acid-base blank solution: take 1 mL of 1 mol / L hydrochloric acid in a 10 mL volumetric flask, add 1 mL of 1 mol / L sodium hydroxide to neutralize, add solvent to dilute to the mark, and shake well as the acid-base blank solution.

[0073] Oxidation blank solution: take 1 mL of 30% hydrogen peroxide in a 10 mL volumetric flask, add solvent to dilute to the mark, and shake well as the oxidation blank solution.

[0074] Take 20 μL of the above test solution respectively into the chromatograph, and record the chromatogram. The amount of each impurity peak is calculated by normalization method. The results are shown in Table Figure 2

[0075] 1) Peak purity

[0076] The peak purity of the test solution under various destruction conditions was determined by diode array measurement. The peak purity results are shown in Tables 2-4.

[0077] Table 2 Peak purity results

[0078] Destruction conditions Unbroken Acid destruction Base destruction Oxidative destruction Pyrolytic destruction High temperature destruction Photolytic destruction Peak purity 999 999 999 999 999 999 999

[0079] As shown in Table 2, the peak purity of the destroyed sample under each condition was greater than 990.

[0080] 2) Material balance investigation

[0081] Table 3 Material balance investigation results

[0082]

[0083] As shown in Table 3, through analysis of sample material balance data, the destruction reduction amount of the main peak of 3,4-diethoxyphenethylamine was basically consistent with the decrease of its content, and the total peak area detected after destruction of samples of the same concentration under different conditions was basically similar, indicating that the chromatographic conditions could effectively detect all degradation impurities.

[0084] 3) Destruction and degradation impurity research

[0085] Table 4 3,4-diethoxyphenethylamine destruction test impurity spectrum (statistical by normalization method)

[0086]

[0087]

[0088] ​

[0089]

[0090] Note: " / " represents not detected.

[0091] From Table 4, it can be seen that 3,4-diethoxyphenethylamine did not show obvious changes in acid destruction, base destruction, pyrolysis destruction, and light destruction; a series of unknown impurities were degraded by oxidation destruction, among which the larger ones were unknown impurity 15, unknown impurity 21, unknown impurity 23, and unknown impurity 26, and unknown impurity 17 increased significantly; a series of unknown impurities were also degraded by high-temperature destruction, among which the larger ones were unknown impurity 10, unknown impurity 19, unknown impurity 20, unknown impurity 23, unknown impurity 33, unknown impurity 34, unknown impurity 39, unknown impurity 40, and unknown impurity 43, and unknown impurity 17 and unknown impurity 24 increased significantly. The peak purity of the destruction samples under each condition was not less than 990, and the materials were basically balanced before and after destruction.

[0092] 3. Quantification limit and detection limit

[0093] An appropriate amount of catechol, 3,4-diethoxyphenylacetonitrile, and 3,4-diethoxyphenethylamine reference substances were precisely weighed, dissolved and diluted with a solvent to prepare a reference substance stock solution. An appropriate amount of the above stock solution was removed, diluted with a solvent to prepare a mixed solution containing 50 μg of catechol, 50 μg of 3,4-diethoxyphenethylamine, and 50 μg of 3,4-diethoxyphenylacetonitrile per 1 mL. The quantification limit (S / N≥10) and the detection limit (S / N≥3) were determined by dilution method. The results are shown in Table 5 below.

[0094] Table 5 Quantification limit and detection limit results

[0095]

[0096] From Table 5, it can be seen that under the present chromatographic conditions, the quantification limit and the detection limit of 3,4-diethoxyphenethylamine and impurities meet the requirements.

[0097] 4. Linearity and range

[0098] An appropriate amount of each impurity reference substance and 3,4-diethoxyphenethylamine reference substance was diluted with a solvent to prepare a mixed solution containing 50 μg of catechol, 50 μg of 3,4-diethoxyphenethylamine, and 50 μg of 3,4-diethoxyphenylacetonitrile per 1 mL, which was used as a linear stock solution. The above solution was diluted with a solvent according to Table 6 below to prepare linear solutions of various concentrations.

[0099] Table 6 Preparation of linear solutions

[0100]

[0101] The above solution was injected into high performance liquid chromatograph with precision pipetting 20 μL of the solution, recording chromatogram, measuring peak area, taking peak area A as ordinate, concentration C as abscissa to make linear regression. The results were as follows Figure 3 and Tables 7-9.

[0102] Table 7 Linear determination results of catechol Figure 3 a)

[0103]

[0104] Table 8 Linear determination results of 3,4-diethoxyphenethylamine Figure 3 b)

[0105]

[0106]

[0107] Table 9 Linear determination results of 3,4-diethoxyphenylacetonitrile Figure 3 c)

[0108]

[0109] The known impurity correction factor calculation results are shown in the following Table 11;

[0110] Calculation formula:

[0111]

[0112] In the formula: K 主成分 - slope of 3,4-diethoxyphenethylamine standard curve;

[0113] K 杂质 - slope of impurity standard curve.

[0114] Table 10: Known impurity correction factor

[0115] Impurities Relative correction factor Catechol 0.68 3,4-diethoxyphenylacetonitrile 0.95

[0116] The relative correction factors of all known impurities are in the range of 0.2-5, and the impurity content can be calculated according to the self-control method with correction factor.

[0117] From Figure 3 and Tables 7-9, it can be seen that catechol is in the range of 0.5287-21.1464 μg / mL, 3,4-diethoxyphenethylamine is in the range of 0.5084-20.3346 μg / mL, and 3,4-diethoxyphenylacetonitrile is in the range of 0.5150-20.5996 μg / mL, and the peak area and the measured concentration show good linear relationship.

[0118] 5. Injection precision

[0119] A mixed solution containing 5 μg of catechol, 5 μg of 3,4-diethoxyphenethylamine, and 5 μg of 3,4-diethoxyphenylacetonitrile per 1 mL was prepared, 20 μL of the mixed solution was injected into the liquid chromatograph, and the injection was repeated 6 times in succession, and the peak area was recorded. The results are shown in Table 11 below.

[0120] Table 11 Results of injection precision test

[0121]

[0122] As shown in Table 11, the injection precision of 3,4-diethoxyphenethylamine and each impurity was good when the injection was repeated 6 times in succession.

[0123] 6. Solution stability

[0124] 1) Stability of reference solution

[0125] A mixed solution containing 5 μg of catechol, 5 μg of 3,4-diethoxyphenethylamine, and 5 μg of 3,4-diethoxyphenylacetonitrile per 1 mL was prepared, and the injection was performed at 0, 1.5, 3.0, 5.0, 6.5, 8.0, 10, 14, 20, and 30 hours, respectively, to investigate the intra-day stability. The results are shown in Table 12. Figure 4

[0126] Table 12 Results of reference solution stability test

[0127]

[0128]

[0129] As shown in Table 12 and Table 13, catechol, 3,4-diethoxyphenethylamine, and 3,4-diethoxyphenylacetonitrile were stable in the solvent for 30 hours. Figure 4 2) Stability of test solution

[0130] An appropriate amount of 3,4-diethoxyphenethylamine was taken to prepare a test solution containing 0.5 mg of 3,4-diethoxyphenethylamine per 1 mL, and the injection was performed at 0, 1.5, 3.0, 5.0, 6.5, 8.0, 10, 14, 20, and 30 hours after preparation (calculated by area normalization method) to investigate the change in impurities. The results are shown in Table 13.

[0131] Figure 5

[0132] Table 13 Results of test solution stability test (normalization method)

[0133]

[0134] ​​​

[0135] By Figure 5 From Table 13 and Table 14, the test solution was stable and no obvious change was observed within 30 hours.

[0136] 7. Reproducibility

[0137] Take 3,4-diethoxyphenethylamine test sample, add solvent to dissolve and dilute to the mark, prepare a solution containing 0.5 mg per 1 mL as the test solution; take 1 mL of the test solution into a 100 mL volumetric flask, dilute to the mark with solvent as the self-control solution. Repeat the determination of 6 test samples. The results are shown in Table 14 below.

[0138] Table 14 Reproducibility test results

[0139]

[0140] From Table 14, the sample was determined 6 times, and the reproducibility was good.

[0141] 8. Sample recovery rate

[0142] Take an appropriate amount of each impurity reference substance, add solvent to dissolve and dilute to prepare a mixed solution containing 25 μg of catechol and 25 μg of 3,4-diethoxyphenylacetonitrile per 1 mL as the recovery rate stock solution. Precisely take the above mixed stock solution and dilute it according to Table 15 to prepare 50%, 100% and 200% solutions, each concentration is prepared in triplicate. Prepare the recovery rate solution according to Table 15.

[0143] Table 15 Preparation of recovery rate solution

[0144]

[0145]

[0146] Take 1 mL of each recovery rate solution into a 100 mL volumetric flask, dilute to the mark with solvent as the self-control solution. Take 20 μL of each sample into the sample, record the peak area of each known impurity, and calculate the recovery rate and RSD of each known impurity. The results are shown in Tables 16-17 below.

[0147] Table 16 Catechol sample recovery rate test results

[0148]

[0149] Table 17 3,4-diethoxyphenylacetonitrile sample recovery rate test results

[0150]

[0151]

[0152] Where: Original amount = Sample weight × Impurity content; Measured amount added = Total measured amount - Original amount; Recovery rate = (Measured amount added / Added amount) × 100%;

[0153] As shown in Tables 16 and 17, the average recovery rate of catechol was 97.66%, with an RSD of 5.72%; the average recovery rate of 3,4-diethoxyphenylacetonitrile was 99.35%, with an RSD of 4.87%; the RSD was less than 10% for all recoveries in the range of 90% to 108%.

[0154] 9. Durability

[0155] The robustness of the method for determining impurities in 3,4-diethoxyphenethylamine was validated primarily through four aspects: different flow rates, different mobile phase pH, different column temperatures, and different chromatographic columns. The results are as follows: Figure 6 As shown in Tables 18 and 19.

[0156] Table 18 Results of the durability test separation degree

[0157]

[0158] Table 19 Results of Durability Test Samples

[0159]

[0160]

[0161] Depend on Figure 6 The robustness test results in Tables 18 and 19 show that even with minor changes in flow rate, column temperature, mobile phase pH, and when using different batches of chromatographic columns, the resolution between each component met the requirements, and the detection results showed no significant changes. Therefore, this method has good robustness.

[0162] 10. Intermediate precision

[0163] Related substance tests were performed on the same batch of samples by the same operator using repeatability testing methods; related substance tests were performed on the same batch of samples by different operators using repeatability testing methods; related substance tests were performed on the same batch of samples by the same operator using different instruments; related substance tests were performed on the same batch of samples by the same operator at different times. The results are shown in Table 20 below.

[0164] Table 20 Results of intermediate precision test

[0165]

[0166] As shown in Table 20, the intermediate precision is good under these chromatographic conditions.

[0167] Example 4

[0168] The results are shown in Table 21 below.

[0169] Table 21 Test results of multiple batches of samples

[0170]

[0171] The above only is the preferred embodiment of the present application, it should be pointed out, for the ordinary skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for determining the content of impurities in 3,4-diethoxyphenethylamine by HPLC, characterized in that, Impurities in 3,4-diethoxyphenethylamine sample are detected by high performance liquid chromatography, using chromatographic column with octadecyl bonded silica as filler, using aqueous phosphoric acid as mobile phase A and methanol as mobile phase B for gradient elution; the impurities are catechol and 3,4-diethoxyphenylacetonitrile.

2. The method for determining the content of impurities in 3,4-diethoxyphenethylamine by HPLC according to claim 1, characterized in that, The aqueous phosphoric acid has a volume percentage concentration of 0.1% and a pH of 5.

5.

3. The method for determining the content of impurities in 3,4-diethoxyphenethylamine by HPLC according to claim 1, characterized in that, The chromatographic column is GL Sciences Inertsil ODS-3 chromatographic column with a specification of 4.6mm×250mm and a filler particle size of 5μm, and the column temperature is 40℃.

4. The method for determining the content of impurities in 3,4-diethoxyphenethylamine by HPLC according to claim 1, characterized in that, The gradient elution program is as follows: 0-20min, the volume percentage of mobile phase A decreases from 80% to 20% and the volume percentage of mobile phase B increases from 20% to 80%; 20min, the volume percentage of mobile phase A is 20% and the volume percentage of mobile phase B is 80%; 20-40min, the volume percentage of mobile phase A is 20% and the volume percentage of mobile phase B is 80%; 40min, the volume percentage of mobile phase A is 20% and the volume percentage of mobile phase B is 80%.

5. The method for determining the content of impurities in 3,4-diethoxyphenethylamine by HPLC according to claim 1, characterized in that, The flow rate of the gradient elution is 1.0mL / min.

6. The method for determining the content of impurities in 3,4-diethoxyphenethylamine by HPLC according to claim 1, characterized in that, In the high performance liquid chromatography, the injection amount is 20μL.

7. The method for determining the content of impurities in 3,4-diethoxyphenethylamine by HPLC according to claim 1, characterized in that, In the high performance liquid chromatography, the detection wavelength is 280nm.

8. The method for determining the content of impurities in 3,4-diethoxyphenethylamine by HPLC according to claim 1, characterized in that, The detection of impurities in 3,4-diethoxyphenethylamine sample includes the following steps: 1) preparation of test sample solution and control solution The preparation process of test sample solution is as follows: 3,4-diethoxyphenethylamine test sample is weighed, dissolved and diluted with solvent to form a solution containing 1mg of 3,4-diethoxyphenethylamine per 1mL, which is used as the test sample solution; The preparation process of control solution is as follows: the test sample solution is precisely measured, diluted with solvent to form a solution containing 10μg of 3,4-diethoxyphenethylamine per 1mL, which is used as the control solution; 2) the test sample solution and the control solution are precisely measured and injected into the liquid chromatograph, and then detected by high performance liquid chromatography, and the chromatogram is recorded, the standard curve is drawn, and the standard curve equation is obtained, and the content of impurities in 3,4-diethoxyphenethylamine is calculated.

9. The method for determining the content of impurities in 3,4-diethoxyphenethylamine by HPLC according to claim 8, characterized in that, In step 2), according to the chromatogram, the peak area A is taken as the vertical coordinate and the concentration C is taken as the horizontal coordinate to draw a linear regression equation, and when the detection wavelength is 280nm, the linear equation of catechol is A=0.3814C-0.0787, the linear equation of 3,4-diethoxyphenylacetonitrile is A=0.2718C-0.0250, and the linear equation of 3,4-diethoxyphenethylamine is A=0.2593C+0.0172.

10. The method for determining the content of impurities in 3,4-diethoxyphenethylamine by HPLC according to claim 9, characterized in that, The linear concentration range of catechol was 0.5287-21.1464 μg / mL, the limit of quantification was 0.5287 μg / mL, and the limit of detection was 0.2115 μg / mL; the linear concentration range of 3,4-diethoxyphenylacetonitrile was 0.5150-20.5996 μg / mL, the limit of quantification was 0.5150 μg / mL, and the limit of detection was 0.2060 μg / mL; the linear concentration range of 3,4-diethoxyphenylacetamine was 0.5084-20.3346 μg / mL, the limit of quantification was 0.5084 μg / mL, and the limit of detection was 0.2033 μg / mL.

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