Method for detecting related substances of promethazine hydrochloride bulk drug

Abstract

The invention discloses a high performance liquid chromatography analysis method for detecting related substances of a promethazine hydrochloride bulk drug. According to the method, a reversed-phase C18 column is adopted, and a ChromasirTMGhost-Sniper Column (50 * 4.6 mm) is installed between a mixer and a sample injector; carrying out gradient elution by taking ammonium acetate solution-acetonitrile (85: 15) as a mobile phase A and acetonitrile-methanol (85: 15) as a mobile phase B; and the diluent is a triethylamine-methanol (1: 1000) solution. According to the method, rapid, accurate and stable qualitative and quantitative analysis can be carried out on the promethazine hydrochloride related substance impurities.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analysis, and particularly relates to a high performance liquid chromatography analysis method for detecting related substances of promethazine hydrochloride raw materials. Background Art

[0002] Promethazine Hydrochloride can competitively block histamine H1 receptors to produce an antihistamine effect. It can counteract capillary dilation caused by histamine, reduce its permeability, and relieve wheezing caused by bronchial smooth muscle contraction. Its effect is stronger and more persistent than that of diphenhydramine hydrochloride. Because it is more easily absorbed into the brain tissue, it has an obvious sedative effect; it can enhance the central inhibitory effects of hypnotics, analgesics, and anesthetics; its anticholinergic effect is also stronger, and it has a better effect in preventing and treating motion sickness. It is used for skin and mucosal allergies, allergic rhinitis, asthma, food allergies, dermographism, as well as motion sickness, seasickness, and airsickness.

[0003] Its structural formula is as follows:

[0004] Quality control of raw materials has always been the focus and difficulty in drug research and development, and the study of impurities is the top priority in quality control. Starting materials, intermediates, by-products, and degradation products in the synthesis process of promethazine hydrochloride may all become impurities remaining in the final product, thereby affecting product quality.

[0005] Upon review, impurity A, impurity B, impurity C, and impurity D are included in the EP and USP pharmacopoeias. Impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, and impurity L are process impurities of this product. Combining with its own process route, quality research and control of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, and impurity L are planned. The specific information of the impurities is as follows:

[0006]

[0007] According to the requirements for raw material impurities in the "Technical Guidelines for the Study of Impurities in Chemical Drugs" (the quality control limit is 0.15%, and the identification limit is 0.10%), and combining with the pharmacopoeia standards of various countries and the impurity detection of multiple batches of samples, the impurity limits of this product are determined.

[0008] Currently, there are few literature reports on the research of other process impurities and degradation impurities in promethazine hydrochloride raw materials except for impurity A, impurity B, impurity C, and impurity D, as well as the research on quality control analysis methods. Summary of the Invention

[0009] The object of the present invention is to solve the problem in the prior art that there is a lack of research on starting materials, intermediates, and by-product impurities of promethazine hydrochloride, and a quality control analysis method, and to provide a high-performance liquid chromatography analysis method that is fast, accurate, and can simultaneously detect various related substances of promethazine hydrochloride.

[0010] To achieve the above object, the technical solution adopted by the present invention is as follows: The present invention provides a high-performance liquid chromatography method for related substances of promethazine hydrochloride. The inventors of the present application have deeply analyzed the possible impurities in the synthesis process of promethazine hydrochloride, and in combination with the quality standards of the European Pharmacopoeia and the United States Pharmacopoeia, a total of 12 impurities, namely impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, and impurity L of the above promethazine hydrochloride have been identified.

[0011] The high-performance liquid chromatography analysis method adopted by the present invention includes the following steps: (1) Chromatographic condition setting: An RP C18 column is used, and a ghost peak trap column needs to be installed between the mixer and the injector. Using 48 mmol / L ammonium acetate solution - acetonitrile, and acetonitrile - methanol as mobile phase B for gradient elution; (2) Sample solution preparation: Dissolve the promethazine hydrochloride raw material with a diluent to prepare a solution with a concentration of 0.5 - 0.7 mg / ml; (3) Detection: Inject the prepared promethazine hydrochloride solution into a high-performance liquid chromatograph and record the chromatogram; Among them, in step (1), the gradient elution preferably adopts the following method:

[0012] According to the embodiments of the present invention, the inventors have tried chromatographic columns of different brands such as Waters, Phenomenex, and StarChrom Technology, and found that when the chromatographic column in step (1) is selected from Phenomenex Titank C18 (150 mm × 4.6 mm, 3 μm), an unknown single impurity front peak appears at about 32 min after the retention time of the main peak, and the reproducibility of this method is poor; when different brands of chromatographs are selected, the separation degree between impurity B and impurity G varies greatly, among which the Thermo Fisher chromatograph can meet the baseline separation, while the Waters chromatograph is completely overlapped; when the chromatographic column is selected from StarChrom Technology ArtChrom WP C18 (150 mm × 4.6 mm, 3.5 μm), it is found that impurity C and impurity L cannot meet the baseline separation; through a large number of studies, it is found that using a Waters XBridge® C18 chromatographic column with a specification of 250 × 4.6 mm and 5 μm can achieve a good separation effect for the impurities disclosed in the present invention, and this chromatographic column is preferably used in the method disclosed in the present invention.

[0013] According to an embodiment of the present invention, adding a ghost peak trapping column can improve the poor blank baseline. The ghost peak trapping column disclosed in the present invention is preferably selected from Chromasir TM Ghost-Sniper Column, with a specification of 50*4.6mm, 5μm.

[0014] According to an embodiment of the present invention, mobile phase A is selected from a 48 mmol / L ammonium acetate solution - acetonitrile, with a volume ratio of 85:15.

[0015] According to an embodiment of the present invention, the volume ratio of acetonitrile - methanol in mobile phase B is 85:15, and the initial volume ratio of gradient elution is preferably 12%.

[0016] According to an embodiment of the present invention, the inventors studied the influence of concentration on the detection of impurities in the test solution: when the concentration of the test solution in step (2) is 0.5 mg / ml, the sensitivities of impurity G, impurity I, and impurity L cannot meet the detection requirements; when the concentration of the test solution is 0.6 mg / ml, the sensitivity of impurity L still cannot meet the detection requirements. Further, when the concentration of the test solution is increased to 0.7 mg / ml, the sensitivities of each impurity and the main component can all meet the detection requirements (the quantitative limit concentrations of each known impurity are all below 0.05% of the limit in the related substance detection report of general bulk drugs).

[0017] According to an embodiment of the present invention, the inventors found that when the column temperature is selected from 20 - 30°C; the detection wavelength is selected from 247 - 252 nm; and the mobile phase velocity is selected from 1.1 - 1.3 ml / min, good separation can be achieved between each substance peak.

[0018] Furthermore, the column temperature is preferably 25°C, the detection wavelength is preferably 249 nm, and the mobile phase velocity is preferably 1.2 ml / min.

[0019] According to an embodiment of the present invention, the injection volume in step (3) is selected from 10 μl.

[0020] Advantages of the present invention: (1) The present invention discloses for the first time a detection technology for separating and determining promethazine from impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L, and other unknown impurities in the bulk drug. (2) Using the method of the present invention, qualitative and quantitative analysis of potential known impurities and other unknown impurities in the bulk drug promethazine hydrochloride can be carried out quickly and accurately. The disclosed method has good reproducibility and specificity, and other unknown impurities in the test solution do not interfere with the detection of each known impurity and the main peak (promethazine), ensuring the quality controllability of the bulk drug promethazine hydrochloride. Description of the Drawings

[0021] Figure 1 is the chromatogram of the system suitability solution in Example 1; Figure 2 is the chromatogram of the test solution in Example 1; Figure 3 is the chromatogram of the system suitability solution using the Thermo Fisher chromatograph system in Example 1; Figure 4 is the chromatogram of the system suitability solution in Example 2; Figure 5 is the chromatogram of the blank diluent in Example 3; Figure 6 is the chromatogram of the system suitability solution in Example 3; Figure 7 is the comparative chromatogram of the promethazine hydrochloride sample solution and the system suitability solution in Example 3 (from top to bottom are the sample solution and the system suitability solution); Figure 8 is the chromatogram of the system suitability solution at a column temperature of 20 °C in Example 4; Figure 9 is the chromatogram of the system suitability solution at a column temperature of 30 °C in Example 5; Figure 10 is the chromatogram of the system suitability solution at a wavelength of 247 nm in Example 6; Figure 11 is the chromatogram of the system suitability solution at a wavelength of 251 nm in Example 7; Figure 12 is the chromatogram of the system suitability solution at a flow rate of 1.1 ml / min in Example 8; Figure 13 is the chromatogram of the system suitability solution at a flow rate of 1.3 ml / min in Example 9; Figure 14 is the chromatogram of the system suitability solution with an initial proportion of mobile phase B of 10% in Example 10; Figure 15 is the chromatogram of the system suitability solution with an initial proportion of mobile phase B of 14% in Example 11; Figure 16 is the chromatogram of the system suitability solution with a test solution concentration of 0.5 mg / ml in Example 12; Figure 17 is the chromatogram of the system suitability solution with a test solution concentration of 0.6 mg / ml in Example 13. Embodiment

[0022] The following examples are only for further illustration of the content of the present invention and do not limit the present invention. The method of the present invention is not limited to impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K and impurity L either. Any method for separating and determining impurities in promethazine hydrochloride using the method of the present invention and 1 to Example 1

[0023] Instrument: Waters Arc HPLC high performance liquid chromatograph; Chromatographic column: Phenomenex Titank C18 (4.6 mm * 150 mm, 3 µm); Note: A ghost peak trap column (Chromasir TM Ghost-Sniper Column, 50 * 4.6 mm) is installed between the mixer and the injector.

[0024] Mobile phase A: 48 mmol / L ammonium acetate solution - acetonitrile (80:20); Mobile phase B: Acetonitrile; Perform linear gradient elution according to the following table:

[0025] Detection wavelength: 249 nm; Flow rate: 1.2 ml / min; Column temperature: 30 °C; Injection volume: 15 μl; Diluent: Triethylamine - methanol (1:1000).

[0026] System suitability solution: Take appropriate amounts of reference substances of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K and impurity L, dissolve and dilute with a solvent to prepare a solution containing about 0.5 mg of promethazine hydrochloride and 5 µg of each impurity per 1 ml.

[0027] Test solution: Take an appropriate amount of this product, weigh accurately, dissolve and dilute with a solvent to prepare a solution containing about 0.5 mg per 1 ml.

[0028] Determination: Take 15 μl of the diluent, system suitability solution and test solution respectively, inject them into the high performance liquid chromatograph, and record the chromatogram.

[0029] Result: The chromatogram of the system suitability solution is shown in Figure 1 , and the chromatogram of the test solution is shown in Figure 2, in the chromatogram of the system suitability solution, impurities K, D, H, I, L, C, promethazine, G, B, J, A, F and E elute in sequence, and good separation can be achieved between the peaks of each substance, indicating good specificity. However, the reproducibility of this method is poor. When the method was reproduced using a Thermo Fisher high-performance liquid chromatograph again, the chromatogram of the system suitability solution is shown in Figure 3 , and the peaks of impurity B and impurity G completely overlap. Example 2

[0030] Instrument: Thermo Scientific U3000 high-performance liquid chromatograph; Chromatographic column: StarSep ArtChrom WP C18 (150 mm × 4.6 mm, 3.5 μm); Note: A ghost peak trap column (Chromasir TM Ghost-SniperColumn, 50*4.6 mm) needs to be installed between the mixer and the injector.

[0031] Mobile phase A: 48 mmol / L ammonium acetate solution - acetonitrile (85∶15); Mobile phase B: acetonitrile; Perform linear gradient elution according to the following table:

[0032] Detection wavelength: 249 nm; Flow rate: 1.2 ml / min; Column temperature: 40 °C; Injection volume: 10 μl; Diluent: triethylamine - methanol (1∶1000).

[0033] System suitability solution: Take appropriate amounts of reference substances of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K and impurity L, dissolve and dilute them with a solvent to prepare a solution containing about 0.5 mg per 1 ml as the mother liquor of each impurity. Then take appropriate amounts of promethazine hydrochloride and the mother liquor of each impurity, dissolve and dilute them with a solvent to prepare a solution containing about 0.75 mg of promethazine hydrochloride, 1.5 μg of impurity L, 1.5 μg of impurity I, 3.75 μg of impurity C and 7.5 μg of other impurities per 1 ml.

[0034] Test solution: Take an appropriate amount of this product, weigh it accurately, dissolve and dilute it with a solvent to prepare a solution containing about 0.75 mg per 1 ml.

[0035] Determination: Take 10 μl of the diluent, the system suitability solution and the test solution respectively, inject them into the high-performance liquid chromatograph, and record the chromatogram.

[0036] Results: The chromatogram of the system suitability solution is shown in Figure 4 , in the chromatogram of the system suitability solution, impurities K, D, H, I, L, C, promethazine hydrochloride, impurity G, impurity B, impurity J, impurity A, impurity F, and impurity E elute successively. Impurities L and C cannot meet the baseline separation, and the peak shape of the main peak is poor with severe tailing. Example 3

[0037] Instrument: Thermo Scientific U3000 high performance liquid chromatograph; Chromatographic column: Waters XBridge ® C18 (250×4.6 mm, 5 μm); Note: A ghost peak trap column (Chromasir TM Ghost-SniperColumn, 50*4.6 mm) needs to be installed between the mixer and the injector.

[0038] Mobile phase A: 48 mmol / L ammonium acetate solution - acetonitrile (85:15); Mobile phase B: acetonitrile - methanol (85:15); Perform linear gradient elution according to the following table:

[0039] Detection wavelength: 249 nm; Flow rate: 1.2 ml / min; Column temperature: 25 °C; Injection volume: 10 μl; Diluent: triethylamine - methanol (1:1000).

[0040] System suitability solution: Take appropriate amounts of reference substances of impurities A, B, C, D, E, F, G, H, I, J, K, and L, dissolve and dilute them with a solvent to prepare a solution containing about 0.5 mg per 1 ml as the mother liquor of each impurity; then take appropriate amounts of promethazine hydrochloride and the mother liquor of each impurity, dissolve and dilute them with a solvent to prepare a solution containing about 0.7 mg of promethazine hydrochloride, 5 μg of impurity B, and 1.5 μg of other impurities per 1 ml.

[0041] Test solution: Take an appropriate amount of this product, weigh it accurately, dissolve and dilute it with a solvent to prepare a solution containing about 0.5 mg per 1 ml.

[0042] Determination: Take 10 μl each of the diluent, the system suitability solution, and the test solution, inject them into the high performance liquid chromatograph respectively, and record the chromatogram.

[0043] Results: The blank diluent had no interference on the determination; see the chromatogram in Figure 5 .

[0044] See the chromatogram of the system suitability solution in Figure 6 . In the chromatogram of the system suitability solution, impurity K, impurity D, impurity H, impurity I, impurity L, impurity C, promethazine, impurity G, impurity B, impurity J, impurity A, impurity F and impurity E eluted in sequence. The retention time results of the above 12 specific impurities and promethazine are shown in the following table. Good separation was achieved between the peaks of each substance, indicating good specificity. In addition, see the comparison chromatogram of the test solution and the system suitability solution in Figure 7 . By comparison, it was found that impurities K, H, I, G, J, A, F, E, and impurity P were not detected in promethazine hydrochloride, and only impurities D, L, C, B, and impurity N were detected. Other unknown impurities in the test solution did not interfere with the detection of the known impurities and the main peak (promethazine).

[0045] Example 4

[0046] Instrument: Thermo Scientific U3000 high performance liquid chromatograph; Chromatographic column: Waters XBridge ® C18 (250×4.6 mm, 5 μm); Note: A ghost peak trap column (Chromasir TM Ghost-Sniper Column, 50*4.6 mm) was installed between the mixer and the injector.

[0047] Mobile phase A: 48 mmol / L ammonium acetate solution - acetonitrile (85∶15); Mobile phase B: acetonitrile - methanol (85∶15); Perform linear gradient elution according to the following table:

[0048] Detection wavelength: 249 nm; Flow rate: 1.2 ml / min; Column temperature: 20 °C; Injection volume: 10 μl; Diluent: triethylamine - methanol (1∶1000); System suitability solution: Weigh appropriate amounts of reference substances of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K and impurity L, dissolve and dilute them with solvent to prepare solutions containing approximately 0.5 mg per 1 ml as the mother liquors of each impurity; then weigh appropriate amounts of promethazine hydrochloride and each mother liquor of impurity, dissolve and dilute them with solvent to prepare a solution containing approximately 0.5 mg of promethazine hydrochloride, 5 μg of impurity B and 1.5 μg of other impurities per 1 ml.

[0049] Determination: Inject 10 μl of the system suitability solution into the high performance liquid chromatograph respectively, and record the chromatogram.

[0050] Result: When the column temperature is 20 °C, the chromatogram of the system suitability solution is shown in Figure 8 , at this time, the peaks of each substance can be well separated, and the separation meets the requirements (>1.5). Example 5

[0051] Apparatus: Thermo Scientific U3000 high performance liquid chromatograph; Chromatographic column: Waters XBridge ® C18 (250×4.6 mm, 5 μm); Note: A ghost peak trap column (Chromasir TM Ghost-SniperColumn, 50*4.6 mm) needs to be installed between the mixer and the injector.

[0052] Mobile phase A: 48 mmol / L ammonium acetate solution - acetonitrile (85:15); Mobile phase B: acetonitrile - methanol (85:15); Perform linear gradient elution according to the following table:

[0053] Detection wavelength: 249 nm; Flow rate: 1.2 ml / min; Column temperature: 30 °C; Injection volume: 10 μl; Diluent: triethylamine - methanol (1:1000); System suitability solution: Weigh appropriate amounts of reference substances of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K and impurity L, dissolve and dilute them with solvent to prepare solutions containing approximately 0.5 mg per 1 ml as the mother liquors of each impurity; then weigh appropriate amounts of promethazine hydrochloride and each mother liquor of impurity, dissolve and dilute them with solvent to prepare a solution containing approximately 0.5 mg of promethazine hydrochloride, 5 μg of impurity B and 1.5 μg of other impurities per 1 ml.

[0054] Determination: Inject 10 μl of the system suitability solution into the high performance liquid chromatograph respectively, and record the chromatogram.

[0055] Result: See the chromatogram of the system suitability solution in Figure 9 . When the column temperature is 30 °C, the peaks of each substance can be well separated at this time, and the separation meets the requirements (> 1.5). Example 6

[0056] Instrument: Thermo Scientific U3000 high performance liquid chromatograph; Chromatographic column: Waters XBridge ® C18 (250×4.6 mm, 5 μm); Note: A ghost peak trap column (Chromasir TM Ghost-SniperColumn, 50*4.6 mm) needs to be installed between the mixer and the injector.

[0057] Mobile phase A: 48 mmol / L ammonium acetate solution - acetonitrile (85∶15); Mobile phase B: acetonitrile - methanol (85∶15); Perform linear gradient elution according to the following table:

[0058] Detection wavelength: 247 nm; Flow rate: 1.2 ml / min; Column temperature: 25 °C; Injection volume: 10 μl; Diluent: triethylamine - methanol (1∶1000); System suitability solution: Take appropriate amounts of reference substances of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, and impurity L, dissolve and dilute them with solvent respectively to make a solution containing about 0.7 mg per 1 ml as the mother liquor of each impurity; then take appropriate amounts of promethazine hydrochloride and the mother liquor of each impurity, dissolve and dilute them with solvent to make a solution containing about 0.5 mg of promethazine hydrochloride, 5 μg of impurity B, and 1.5 μg of other impurities per 1 ml.

[0059] Determination: Inject 10 μl of the system suitability solution into the high performance liquid chromatograph respectively, and record the chromatogram.

[0060] Result: See the chromatogram of the system suitability solution in Figure 10 . When the wavelength is 247 nm, the peaks of each substance can be well separated, and the separation meets the requirements (> 1.5). Example 7

[0061] Instrument: Thermo Scientific U3000 high performance liquid chromatograph; Chromatographic column: Waters XBridge ® C18 (250×4.6 mm, 5 μm); Note: A ghost peak trap column (Chromasir TM Ghost-SniperColumn, 50*4.6 mm) needs to be installed between the mixer and the injector.

[0062] Mobile phase A: 48 mmol / L ammonium acetate solution - acetonitrile (85∶15); Mobile phase B: acetonitrile - methanol (85∶15); Perform linear gradient elution according to the following table:

[0063] Detection wavelength: 251 nm; Flow rate: 1.2 ml / min; Column temperature: 25 °C; Injection volume: 10 μl; Diluent: triethylamine - methanol (1∶1000); System suitability solution: Take appropriate amounts of reference substances of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K and impurity L, dissolve and dilute them with solvent respectively to make a solution containing about 0.7 mg per 1 ml as the mother solution of each impurity; then take appropriate amounts of promethazine hydrochloride and the mother solution of each impurity, dissolve and dilute them with solvent to make a solution containing about 0.5 mg of promethazine hydrochloride, 5 μg of impurity B and 1.5 μg of other impurities per 1 ml.

[0064] Determination: Inject 10 μl of the system suitability solution into the high performance liquid chromatograph respectively, and record the chromatogram.

[0065] Result: The chromatogram of the system suitability solution is shown in Figure 11 , when the wavelength is 251 nm, good separation can be achieved between the peaks of each substance, and the separation meets the requirements (>1.5). Example 8

[0066] Instrument: Thermo Scientific U3000 high performance liquid chromatograph; Chromatographic column: Waters XBridge ® C18 (250×4.6 mm, 5 μm); Note: A ghost peak trap column (ChromasirTM Ghost-SniperColumn, 50*4.6mm).

[0067] Mobile phase A: 48 mmol / L ammonium acetate solution - acetonitrile (85:15); Mobile phase B: acetonitrile - methanol (85:15); Perform linear gradient elution according to the following table:

[0068] Detection wavelength: 249 nm; Flow rate: 1.1 ml / min; Column temperature: 25 °C; Injection volume: 10 μl; Diluent: triethylamine - methanol (1:1000).

[0069] System suitability solution: Take appropriate amounts of reference substances of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K and impurity L, dissolve and dilute them with solvent respectively to prepare solutions containing about 0.5 mg per 1 ml as the mother solutions of each impurity; then take appropriate amounts of promethazine hydrochloride and each mother solution of impurities, dissolve and dilute them with solvent to prepare a solution containing about 0.5 mg of promethazine hydrochloride, 5 μg of impurity B and 1.5 μg of other impurities per 1 ml.

[0070] Determination: Inject 10 μl of the system suitability solution into the high performance liquid chromatograph respectively, and record the chromatogram.

[0071] Result: See the chromatogram of the system suitability solution in Figure 12 , when the flow rate is 1.1 ml / min, good separation can be achieved between the peaks of each substance, and the separation meets the requirements (>1.5). Example 9

[0072] Instrument: Thermo Scientific U3000 high performance liquid chromatograph; Chromatographic column: Waters XBridge ® C18 (250×4.6 mm, 5 μm); Note: A ghost peak trap column (Chromasir TM Ghost-SniperColumn, 50*4.6mm) needs to be installed between the mixer and the injector.

[0073] Mobile phase A: 48 mmol / L ammonium acetate solution - acetonitrile (85:15); Mobile phase B: acetonitrile - methanol (85:15); Perform linear gradient elution according to the following table:

[0074] Detection wavelength: 249 nm; Flow rate: 1.3 ml / min; Column temperature: 25 °C; Injection volume: 10 μl; Diluent: Triethylamine - methanol (1:1000).

[0075] System suitability solution: Take appropriate amounts of reference substances of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K and impurity L, dissolve and dilute them with a solvent respectively to prepare a solution containing about 0.5 mg per 1 ml as the mother solution of each impurity; then take appropriate amounts of promethazine hydrochloride and the mother solution of each impurity, dissolve and dilute them with a solvent to prepare a solution containing about 0.5 mg of promethazine hydrochloride, 5 μg of impurity B and 1.5 μg of other impurities per 1 ml.

[0076] Determination: Inject 10 μl of the system suitability solution into the high performance liquid chromatograph respectively, and record the chromatogram.

[0077] Result: See the chromatogram of the system suitability solution in Figure 13 , when the flow rate is 1.3 ml / min, good separation can be achieved between the peaks of each substance, and the separation meets the requirements (>1.5). Example 10

[0078] Instrument: Thermo Scientific U3000 high performance liquid chromatograph; Chromatographic column: Waters XBridge ® C18 (250×4.6 mm, 5 μm); Note: A ghost peak trap column (Chromasir TM Ghost-SniperColumn, 50*4.6 mm) needs to be installed between the mixer and the injector.

[0079] Mobile phase A: 48 mmol / L ammonium acetate solution - acetonitrile (85:15); Mobile phase B: Acetonitrile - methanol (85:15); Perform linear gradient elution according to the following table:

[0080] Detection wavelength: 249 nm; Flow rate: 1.2 ml / min; Column temperature: 25 °C; Injection volume: 10 μl; Diluent: Triethylamine - methanol (1∶1000).

[0081] System suitability solution: Take appropriate amounts of reference substances of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K and impurity L, dissolve and dilute them with solvent respectively to prepare solutions containing about 0.5 mg per 1 ml as the mother solutions of each impurity; then take appropriate amounts of promethazine hydrochloride and the mother solutions of each impurity, dissolve and dilute them with solvent to prepare a solution containing about 0.5 mg of promethazine hydrochloride, 5 μg of impurity B and 1.5 μg of other impurities per 1 ml.

[0082] Determination: Inject 10 μl of the system suitability solution into the high - performance liquid chromatograph respectively, and record the chromatogram.

[0083] Result: The chromatogram of the system suitability solution is shown in Figure 14 , when the initial proportion of mobile phase B is 10%, good separation can be achieved between the peaks of each substance, and the separation meets the requirements (>1.5). Example 11

[0084] Instrument: Thermo Scientific U3000 high - performance liquid chromatograph; Chromatographic column: Waters XBridge ® C18 (250×4.6 mm, 5 μm); Note: A ghost peak trap column (Chromasir TM Ghost - SniperColumn, 50*4.6 mm) needs to be installed between the mixer and the injector.

[0085] Mobile phase A: 48 mmol / L ammonium acetate solution - acetonitrile (85∶15); Mobile phase B: Acetonitrile - methanol (85∶15); Perform linear gradient elution according to the following table:

[0086] Detection wavelength: 249 nm; Flow rate: 1.2 ml / min; Column temperature: 25℃; Injection volume: 10 μl; Diluent: Triethylamine - methanol (1∶1000).

[0087] System suitability solution: Weigh appropriate amounts of reference substances of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K and impurity L, dissolve and dilute each with a solvent to prepare a solution containing approximately 0.5 mg per 1 ml as the mother solution of each impurity. Separately, weigh appropriate amounts of promethazine hydrochloride and the mother solutions of each impurity, dissolve and dilute with a solvent to prepare a solution containing approximately 0.5 mg of promethazine hydrochloride, 5 μg of impurity B and 1.5 μg of other impurities per 1 ml.

[0088] Determination: Inject 10 μl of the system suitability solution into the high performance liquid chromatograph and record the chromatogram.

[0089] Results: The chromatogram of the system suitability solution is shown in Figure 15 , when the initial proportion of mobile phase B is 14%, good separation can be achieved between the peaks of each substance, and the separation meets the requirements (>1.5). Example 12

[0090] Apparatus: Thermo Scientific U3000 high performance liquid chromatograph; Chromatographic column: Waters XBridge ® C18 (250×4.6 mm, 5 μm); Note: A ghost peak trap column (Chromasir TM Ghost-SniperColumn, 50*4.6 mm) needs to be installed between the mixer and the injector.

[0091] Mobile phase A: 48 mmol / L ammonium acetate solution - acetonitrile (85∶15); Mobile phase B: acetonitrile - methanol (85∶15); Perform linear gradient elution according to the following table:

[0092] Detection wavelength: 249 nm; Flow rate: 1.2 ml / min; Column temperature: 25 °C; Injection volume: 10 μl; Diluent: triethylamine - methanol (1∶1000); System suitability solution: Weigh appropriate amounts of reference substances of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K and impurity L, dissolve and dilute each with a solvent to prepare a solution containing approximately 0.6 mg per 1 ml as the mother liquor of each impurity. Separately, weigh appropriate amounts of promethazine hydrochloride and the mother liquor of each impurity, dissolve and dilute with a solvent to prepare a solution containing approximately 0.5 mg of promethazine hydrochloride, 5 μg of impurity B and 1.5 μg of other impurities per 1 ml. Determination: Inject 10 μl of the system suitability solution into a high performance liquid chromatograph respectively, and record the chromatogram. Determination: Inject 10 μl of the system suitability solution into a high performance liquid chromatograph respectively, and record the chromatogram.

[0093] Results: See the chromatogram of the system suitability solution in Figure 16 , when the concentration of the test solution is 0.5 mg / ml, good separation can be achieved between the peaks of each substance, and the separation meets the requirements (>1.5). However, impurities G, I and L do not meet the sensitivity requirements, and this sample concentration is not feasible. Example 13

[0094] Instrument: Thermo Scientific U3000 high performance liquid chromatograph; Chromatographic column: Waters XBridge ® C18 (250×4.6 mm, 5 μm); Note: A ghost peak trap column (Chromasir TM Ghost-SniperColumn, 50*4.6 mm) needs to be installed between the mixer and the injector.

[0095] Mobile phase A: 48 mmol / L ammonium acetate solution - acetonitrile (85∶15); Mobile phase B: acetonitrile - methanol (85∶15); Perform linear gradient elution according to the following table:

[0096] Detection wavelength: 249 nm; Flow rate: 1.2 ml / min; Column temperature: 25 °C; Injection volume: 10 μl; Diluent: triethylamine - methanol (1∶1000); System suitability solution: Weigh appropriate amounts of reference substances of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L and impurity N, dissolve and dilute each with a solvent to prepare a solution containing about 0.7 mg per 1 ml as the mother liquor of each impurity; then weigh appropriate amounts of promethazine hydrochloride and the mother liquor of each impurity, dissolve and dilute with a solvent to prepare a solution containing about 0.6 mg of promethazine hydrochloride and 6 μg of each impurity per 1 ml. Determination: Inject 10 μl of the system suitability solution into the high performance liquid chromatograph and record the chromatogram.

[0097] Results: The chromatogram of the system suitability solution is shown in Figure 17 . When the concentration of the test solution is 0.6 mg / ml, good separation can be achieved between the peaks of each substance, and the separation meets the requirements (>1.5). However, impurities G, I and L still do not meet the sensitivity requirements, so this sample concentration is not feasible.

Claims

1. An HPLC analysis method for detecting related substances of promethazine hydrochloride, characterized in that, The method described includes: (1) Chromatographic condition setting: Use a reverse-phase C18 column, install a ghost peak trapping column between the mixer and the injector, use ammonium acetate solution - acetonitrile as mobile phase A, and acetonitrile - methanol as mobile phase B. Set the column temperature, detection wavelength, mobile phase velocity of the chromatograph, control the injection volume, and perform gradient elution; (2) Sample solution preparation: Dissolve promethazine hydrochloride raw material with a diluent to prepare a solution with a concentration of 0.5 - 0.7 mg / ml; (3) Detection: Inject the prepared promethazine hydrochloride solution into a high-performance liquid chromatograph and record the chromatogram; Among them, the gradient elution is carried out in the following manner: , the related substances are impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K and impurity L, and the structures are as follows: 。 2. The HPLC analysis method for detecting related substances of promethazine hydrochloride according to claim 1, characterized in that, The reversed-phase C18 column described in step (1) is selected from Waters XBridge ® C18, with a specification of 250×4.6 mm, 5 μm.

3. The HPLC analysis method for detecting related substances of promethazine hydrochloride according to claim 1, characterized in that, The described ghost peak trapping column is selected from Chromasir TM Ghost-Sniper Column, with a specification of 50*4.6mm, 5μm.

4. The HPLC analysis method for detecting related substances of promethazine hydrochloride according to claim 1, characterized in that, The mobile phase A described in step (1) is selected from 48 mmol / L ammonium acetate solution - acetonitrile, and the volume ratio is 85∶15.

5. The HPLC analysis method for detecting related substances of promethazine hydrochloride according to claim 1, characterized in that, The volume ratio of the mobile phase B acetonitrile - methanol described in step (1) is 85:15, and the initial volume ratio of the gradient elution is preferably 12%.

6. The HPLC analysis method for detecting related substances of promethazine hydrochloride according to claim 1, characterized in that, The concentration of the sample solution described in step (2) is 0.7 mg / ml solution.

7. The HPLC analysis method for detecting related substances of promethazine hydrochloride according to claim 1, characterized in that, The column temperature described in step (1) is selected from 20 - 30 °C, preferably 25 °C.

8. The HPLC analysis method for detecting related substances of promethazine hydrochloride according to claim 1, characterized in that, The detection wavelength described in step (1) is selected from 247 - 251 nm, preferably 249 nm.

9. The HPLC analysis method for detecting related substances of promethazine hydrochloride according to claim 1, characterized in that, The mobile phase velocity described in step (1) is selected from 1.1 - 1.3 ml / min, preferably 1.2 ml / min.

10. The HPLC analysis method for detecting related substances of promethazine hydrochloride according to claim 1, characterized in that, The injection volume in the analysis method described is 10 μL.

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