HPLC (High Performance Liquid Chromatography) detection method for propranolol hydrochloride genotoxic impurity 1-naphthylamine and application thereof
By using octadecylsilane-bonded silica gel chromatography column and specific mobile phase gradient elution program, the problem of insufficient sensitivity of the genotoxic impurity 1-naphthylamine detection in propranolol hydrochloride is solved, and the detection effect of high sensitivity and high accuracy is achieved to ensure the quality of the drug.
Patent Information
- Application Number
- CN202510359565.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-25
- Publication Date
- 2025-07-18
AI Technical Summary
The prior art lacks detection methods with high sensitivity to detect the content of 1-naphthylamine in propranolol hydrochloride, which makes it difficult to achieve drug quality control.
A chromatographic column with octadecylsilane bonded silica gel as filler was used, and a specific mobile phase gradient elution program was designed. A mixed solution of sodium dodecyl sulfate and tetrabutylammonium phosphate and acetonitrile were used as the mobile phase, and the concentration range was controlled within a specific value, and liquid chromatography was performed.
The rapid and accurate detection of the genotoxic impurity 1-naphthylamine in propranolol hydrochloride was achieved, with a detection limit of 0.0017μg/mL and a quantitative limit of 0.0056μg/mL. The accuracy and repetition of the test results are good, and the quality of the drug can be effectively controlled.
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Abstract
Description
Technical Field
[0001] This application relates to the technical field of analytical chemistry, and particularly relates to an HPLC detection method for 1-naphthylamine, a genotoxic impurity of propranolol hydrochloride, and its application. Background Art
[0002] Propranolol hydrochloride is a β-blocker that can antagonize sympathetic excitation and catecholamine effects. By blocking the relevant receptors on the heart, it can reduce the heart's contraction rate and speed, relieve hypertension and exertional angina; it can also be used as secondary prevention to reduce the mortality rate of myocardial infarction. 1-Naphthylamine is the starting material of propranolol hydrochloride and is an impurity with potential carcinogenic risks. Therefore, in order to ensure the safety and quality control of drugs, it is necessary to conduct effective quality control on it.
[0003] According to the maximum daily intake of propranolol hydrochloride and calculated based on rodent carcinogenicity data, the intake control limit of 1-naphthylamine should be 280 ppm. Therefore, the content detection method for the genotoxic impurity 1-naphthylamine in propranolol hydrochloride is required to have extremely high detection sensitivity; however, there is currently no relevant literature reporting or documenting the detection method for the genotoxic impurity 1-naphthylamine.
[0004] Therefore, providing a detection method with high sensitivity and strong specificity has great research significance for effectively controlling the genotoxic impurities in propranolol hydrochloride tablets and ensuring its medication safety. Summary of the Invention
[0005] In order to accurately detect the low-content toxic impurity 1-naphthylamine in propranolol hydrochloride raw material or propranolol hydrochloride injection, this application provides an HPLC detection method for 1-naphthylamine, a genotoxic impurity of propranolol hydrochloride, and its application.
[0006] In the first aspect, this application provides an HPLC detection method for 1-naphthylamine, a genotoxic impurity of propranolol hydrochloride, adopting the following technical scheme: An HPLC detection method for 1-naphthylamine, a genotoxic impurity of propranolol hydrochloride, includes the following steps: preparing a reference solution, preparing a test solution, and performing liquid chromatography detection; Liquid chromatography detection: Inject the reference solution and the test solution into a liquid chromatograph, and use the mobile phase gradient elution method for separation and detection; the chromatographic column used is a chromatographic column packed with octadecylsilane-bonded silica gel; The mobile phase gradient elution process is as follows: at 0 min, mobile phase A is 100% and mobile phase B is 0%; from 0 to 15 min, mobile phase A linearly decreases to 75% and mobile phase B linearly increases to 25%; from 15 to 30 min, mobile phase A linearly decreases to 0% and mobile phase B linearly increases to 100%; from 30 to 40 min, mobile phase A is 0% and mobile phase B is 100%; from 40 to 41 min, mobile phase A linearly increases to 100% and mobile phase B linearly decreases to 0%; from 41 to 70 min, mobile phase A is 100% and mobile phase B is 0%; both mobile phase A and mobile phase B are in volume percentages; The mobile phase A is a mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate and acetonitrile; the volume ratio of the mixed solution to acetonitrile is 70:30; The mobile phase B is a mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate and acetonitrile; the volume ratio of the mixed solution to acetonitrile is 30:70.
[0007] This application provides an HPLC detection method for the genotoxic impurity 1-naphthylamine in propranolol hydrochloride. By using a chromatographic column packed with octadecylsilyl-bonded silica gel and designing the elution solvent and gradient elution conditions of the mobile phase as above, the rapid detection of the content of genotoxic impurity 1-naphthylamine in propranolol hydrochloride is achieved. Moreover, the above detection method has good accuracy, high sensitivity, and excellent reliability. When the above detection method is used in the production of propranolol hydrochloride raw materials or injections, the quality of propranolol hydrochloride raw materials or injections can be effectively controlled. Specifically, this application uses a mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate and acetonitrile with a volume ratio of 70:30 as mobile phase A, and a mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate and acetonitrile with a volume ratio of 30:70 as mobile phase B, and controls the mobile phase elution program under the above conditions, which can enable the substances in propranolol hydrochloride to be fully eluted in turn, and the separation degree between the peaks of each substance is good and the peak shape symmetry is excellent, and the detection result of 1-naphthylamine obtained has high accuracy.
[0008] Optionally, in the mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate, the concentration of sodium dodecyl sulfate is 3 - 3.5 g / L and the concentration of tetrabutylammonium dihydrogen phosphate is 0.25 - 0.35 g / L.
[0009] In this application, the concentrations of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate in the mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate will affect the separation effect of the genotoxic impurity 1-naphthylamine in the chromatogram and the symmetry of its peak shape, thereby affecting the accuracy and repeatability of the detection results. Through experimental exploration, it is found in this application that by further controlling the concentrations of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate in mobile phase A and mobile phase B within the above ranges, the peak shape symmetry of the target peak in the obtained chromatogram is good, without problems such as tailing and difficult baseline separation, and the stability and repeatability of the detection results of the content of the genotoxic impurity 1-naphthylamine in the test solution are better, and the RSD of the content detection results can be as low as below 2.0%.
[0010] Optionally, the preparation method of the mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate is as follows: Take a certain amount of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate respectively, and dilute with water until the concentration of sodium dodecyl sulfate is 3 - 3.5 g / L and the concentration of tetrabutylammonium dihydrogen phosphate is 0.25 - 0.35 g / L; then add sulfuric acid with a volume content of 0.15 - 0.25%, and adjust the pH value to 3 - 3.5 with 1.5 - 2.5 mol / L sodium hydroxide solution to obtain the solution.
[0011] Optionally, in the liquid chromatography detection, the flow rate of the mobile phase is 1.4 - 1.6 mL / min.
[0012] Optionally, in the liquid chromatography detection, the column temperature is 25 - 35 °C.
[0013] Optionally, in the liquid chromatography detection, the detection wavelength is 230 nm.
[0014] Optionally, in the liquid chromatography detection, the injection volume is 80 μL.
[0015] Optionally, in the liquid chromatography detection, the chromatographic column is CERI L-column3 C18, 250 mm × 4.6 mm, 5 μm or a chromatographic column with equivalent efficiency.
[0016] Optionally, the detection limit of the genotoxic impurity 1-naphthylamine is 0.0017 μg / mL, and the quantitation limit is 0.0056 μg / mL.
[0017] In the second aspect, the present application provides the application of the HPLC detection method for the genotoxic impurity 1-naphthylamine in propranolol hydrochloride in the quality control of propranolol hydrochloride raw materials or propranolol hydrochloride injection.
[0018] To sum up, the present application has the following beneficial effects: 1. The present application provides an HPLC detection method for the genotoxic impurity 1-naphthylamine in propranolol hydrochloride, which uses a chromatographic column packed with octadecylsilyl-bonded silica gel and a specific mobile phase and gradient elution program, so as to be able to quickly and accurately detect the content of the genotoxic impurity 1-naphthylamine in propranolol hydrochloride bulk drugs and injections, and the detection has good accuracy, high sensitivity and excellent reliability, and can realize the quality control of propranolol hydrochloride bulk drugs and injections.
[0019] 2. The HPLC detection method provided by the present application is used to detect the content of the genotoxic impurity 1-naphthylamine in propranolol hydrochloride bulk drugs or injections, and the detection limit of 1-naphthylamine is 0.0017 μg / mL, and the quantification limit is 0.0056 μg / mL.
[0020] 3. The present application further controls the concentration of sodium dodecyl sulfate in the mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate within the range of 3 - 3.5 g / L, and the concentration of tetrabutylammonium dihydrogen phosphate within the range of 0.25 - 0.35 g / L. The obtained detection method has better stability and repeatability, and can make the RSD value of the detection result as low as below 2.00%. Description of the Drawings
[0021] Figure 1 It is the specific HPLC detection chart of the mixed reference solution; Figure 2 It is the linear relationship chart of the genotoxic impurity 1-naphthylamine; Figure 3 It is the HPLC detection chart obtained by detecting the spiked preparation test solution with the method provided in Example 1. Detailed Embodiments
[0022] The present application provides an HPLC detection method for the genotoxic impurity 1-naphthylamine in propranolol hydrochloride, including the following steps: (1) Preparation of the reference solution: Weigh accurately the 1-naphthylamine reference substance, dissolve it with mobile phase A and quantitatively dilute it to prepare a reference solution containing about 0.14 μg per 1 ml; The mobile phase A is a mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate and acetonitrile; the volume ratio of the mixed solution to acetonitrile is 70:30; The preparation method of the mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate solution is as follows: The preparation method of the mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate is: Take a certain amount of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate respectively, and dilute with water until the concentration of sodium dodecyl sulfate is 3 - 3.5 g / L and the concentration of tetrabutylammonium dihydrogen phosphate is 0.25 - 0.35 g / L; Then add sulfuric acid with a volume content of 0.15 - 0.25%, and adjust the pH value to 3 - 3.5 with 1.5 - 2.5 mol / L sodium hydroxide solution to obtain it.
[0023] (2) Prepare the test solution: Raw material test solution: Take about 10 mg of propranolol hydrochloride raw material, accurately weigh it, place it in a 10 ml volumetric flask, dissolve and dilute to the scale with mobile phase A, and shake well; Injection test solution: Propranolol hydrochloride injection.
[0024] (3) Liquid chromatography detection: Respectively absorb the reference solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram; Then calculate the content of 1 - naphthylamine in the test solution by peak area according to the external standard method. The liquid chromatography detection conditions are as follows: Use a CERI L - column3 C18 chromatographic column with a specification of 250 mm × 4.6 mm, 5 μm; The flow rate of the mobile phase is 1.4 - 1.6 mL / min, the column temperature is 25 - 35 °C, the detection wavelength is 230 nm, and the injection volume is 80 μL.
[0025] The mobile phase gradient elution process is as follows: At 0 min, mobile phase A is 100%, mobile phase B is 0%; From 0 to 15 min, mobile phase A linearly decreases to 75%, and mobile phase B linearly increases to 25%; From 15 to 30 min, mobile phase A linearly decreases to 0%, and mobile phase B linearly increases to 100%; From 30 to 40 min, mobile phase A is 0%, mobile phase B is 100%; From 40 to 41 min, mobile phase A linearly increases to 100%, and mobile phase B linearly decreases to 0%; From 41 to 70 min, mobile phase A is 100%, mobile phase B is 0%; Mobile phase A and mobile phase B are both in volume percentages; The mobile phase A is a mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate and acetonitrile; The volume ratio of the mixed solution to acetonitrile is 70:30; The mobile phase B is a mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate and acetonitrile; The volume ratio of the mixed solution to acetonitrile is 30:70.
[0026] In the mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate, the concentration of sodium dodecyl sulfate is 3 - 3.5 g / L and the concentration of tetrabutylammonium dihydrogen phosphate is 0.25 - 0.35 g / L.
[0027] In the embodiments of the present application, raw materials, reagents, solvents, etc. used can all be obtained through commercial purchase, and the source information of the main substances is shown in Table 1 below: Table 1 Source Information of Raw Materials Used in the Present Application The present application will be further described in detail below in conjunction with examples, performance detection tests, and the accompanying drawings of the specification.
[0028] Example 1 Example 1 provides an HPLC detection method for the genotoxic impurity 1-naphthylamine of propranolol hydrochloride, including the following steps: (1) Preparation of reference solution: Weigh accurately a reference substance of 1-naphthylamine, dissolve it with mobile phase A and quantitatively dilute to prepare a reference solution containing about 0.14 μg per 1 ml. The mobile phase A is a mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate and acetonitrile; the volume ratio of the mixed solution to acetonitrile is 70:30.
[0029] The preparation method of the mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate solution is as follows: Take a certain amount of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate respectively, dilute with water until the concentration of sodium dodecyl sulfate is 3.2 g / L and the concentration of tetrabutylammonium dihydrogen phosphate is 0.31 g / L; then add sulfuric acid with a volume content of 0.2%, and adjust the pH value to 3.3 with 2 mol / L sodium hydroxide solution to obtain it.
[0030] (2) Preparation of test solution: Raw material test solution: Take about 10 mg of propranolol hydrochloride raw material, weigh accurately, place it in a 10 ml volumetric flask, dissolve it with mobile phase A and dilute to the scale, and shake well; Injection test solution: Propranolol hydrochloride injection.
[0031] (3) Liquid chromatography detection: Respectively suck the reference solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram; then calculate the content of 1-naphthylamine in the test solution by the external standard method based on the peak area. The liquid chromatography detection conditions are as follows: Use a CERI L-column3 C18 chromatographic column with a specification of 250 mm × 4.6 mm, 5 μm; the flow rate of the mobile phase is 1.5 mL / min, the column temperature is 30 °C, the detection wavelength is 230 nm, and the injection volume is 80 μL.
[0032] The mobile phase gradient elution process is as follows: at 0 min, mobile phase A is 100% and mobile phase B is 0%; from 0 to 15 min, mobile phase A linearly decreases to 75% and mobile phase B linearly increases to 25%; from 15 to 30 min, mobile phase A linearly decreases to 0% and mobile phase B linearly increases to 100%; from 30 to 40 min, mobile phase A is 0% and mobile phase B is 100%; from 40 to 41 min, mobile phase A linearly increases to 100% and mobile phase B linearly decreases to 0%; from 41 to 70 min, mobile phase A is 100% and mobile phase B is 0%; both mobile phase A and mobile phase B are in volume percentages; The mobile phase A is a mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate and acetonitrile, and the volume ratio of the mixed solution to acetonitrile is 70:30; the mobile phase B is a mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate and acetonitrile, and the volume ratio of the mixed solution to acetonitrile is 30:70.
[0033] The preparation method of the mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate solution is as follows: take a certain amount of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate respectively, and dilute with water to a concentration of 3.2 g / L for sodium dodecyl sulfate and 0.31 g / L for tetrabutylammonium dihydrogen phosphate; then add sulfuric acid with a volume content of 0.2%, and adjust the pH value to 3.3 with 2 mol / L sodium hydroxide solution to obtain it.
[0034] (I) Specificity Prepare the following solutions respectively: Blank solution: mobile phase A.
[0035] Blank excipient solution: Use the propranolol hydrochloride injection without propranolol as the blank excipient solution.
[0036] Stock solution of impurity A: Take 2 mg of impurity A reference substance, accurately weigh it, place it in a 10 ml volumetric flask, dissolve it with acetonitrile and dilute to the mark, shake well, and obtain a stock solution of impurity A with a concentration of 200 μg / ml.
[0037] Localization solution of impurity A: Accurately measure 1 ml of the stock solution of impurity A, place it in a 10 ml volumetric flask, dilute it to the mark with mobile phase A, shake well, and obtain a localization solution of impurity A with a concentration of 20 μg / ml.
[0038] According to the preparation methods of the stock solution of impurity A and the localization solution of impurity A, prepare the stock solutions and localization solutions of impurity B, C, α-naphthol, β-isomer, and propranolol hydrochloride in the same way.
[0039] Stock solution of 1-naphthylamine: Take 1.4 mg of 1-naphthylamine reference substance, accurately weigh it, place it in a 100 ml volumetric flask, dissolve it with acetonitrile and dilute to the mark, shake well, and obtain a stock solution of 1-naphthylamine with a concentration of 14 μg / ml.
[0040] 1-Naphthylamine positioning solution: Accurately measure 5 ml of 1-naphthylamine stock solution, transfer it to a 50-ml volumetric flask, dilute it to the mark with mobile phase A, and shake well to obtain a 1-naphthylamine positioning solution with a concentration of 1.4 μg / ml.
[0041] Reference solution: Accurately measure 1 ml of 1-naphthylamine stock solution, transfer it to a 100-ml volumetric flask, dilute it to the mark with mobile phase A, and shake well to obtain a reference solution with a concentration of 0.14 μg / ml.
[0042] Mixed impurity stock solution: Accurately measure 1 ml each of impurity A, B, C, α-naphthol, β-isomer, and 1-naphthylamine impurity stock solutions, transfer them to the same 10-ml volumetric flask, dilute it to the mark with mobile phase A, and shake well.
[0043] Mixed reference solution: Take about 10 mg of propranolol hydrochloride reference substance, accurately weigh it, transfer it to a 10-ml volumetric flask, accurately add 1 ml of the mixed impurity stock solution, dissolve and dilute it to the mark with mobile phase A, and shake well.
[0044] Test solution of raw material: Take about 10 mg of the raw material drug, accurately weigh it, transfer it to a 10-ml volumetric flask, dissolve and dilute it to the mark with mobile phase A, and shake well.
[0045] Test solution of preparation: Take this product to obtain it.
[0046] Spiked test solution of raw material: Take about 10 mg of the raw material drug, accurately weigh it, transfer it to a 10-ml volumetric flask, add 1 ml of 1-naphthylamine positioning solution, dissolve and dilute it to the mark with mobile phase A, and shake well to obtain a spiked test solution of raw material with a concentration of 0.14 μg / ml.
[0047] Spiked test solution of preparation: Accurately measure 1 ml of 1-naphthylamine positioning solution, transfer it to a 10-ml volumetric flask, dilute it to the mark with this product, and shake well to obtain a spiked test solution of preparation with a concentration of 0.14 μg / ml.
[0048] Accurately measure the blank solution, blank excipient solution, each test solution, reference solution, mixed reference solution, and each positioning solution, inject them into the liquid chromatograph respectively, and record the chromatogram. The results of the specificity test are shown in Table 2-3 below. The specificity test chromatogram of the mixed reference solution is as Figure 1 shown.
[0049] Table 2 Results of Specificity Test 1 Table 3 Results of Specificity Test 2 According to the specificity test results in Table 2-3, neither the blank solution nor the blank excipient solution interferes with the detection of the genotoxic impurity 1-naphthylamine in propranolol hydrochloride. The resolution between 1-naphthylamine and the adjacent peak in the mixed reference solution meets the requirements (minimum is 4.44). Therefore, it shows that the HPLC detection method for the genotoxic impurity 1-naphthylamine in propranolol hydrochloride provided in this application has good specificity.
[0050] (II) Instrument precision Precisely measure the reference solution, inject it into the liquid chromatograph, and continuously inject samples 5 times according to the chromatographic conditions in step (3) of Example 1, and record the chromatogram. The test results are shown in Table 4 below.
[0051] Table 4 Results of precision test Number of injection Retention time (min) Peak area 1 12.946 58554 2 12.943 59027 3 12.943 58860 4 12.947 58655 5 12.952 58671 RSD (%) 0.03 0.32 From the detection results in Table 4, it can be seen that for the reference solution injected continuously 5 times, the RSD value of the retention time is 0.03% (≤2.0%), and the RSD value of the peak area is 0.32% (≤2.0%). Therefore, it shows that the instrument precision of the HPLC detection method for the genotoxic impurity 1-naphthylamine in propranolol hydrochloride provided in this application is good.
[0052] (III) Quantitation limit, detection limit Prepare the reference solution according to the method in Example 1, and then gradually dilute the reference solution according to the signal-to-noise ratio of the peak height of the reference solution. When the signal-to-noise ratio of the peak height S / N ≥ 10:1, this concentration is used as the quantitation limit; when the signal-to-noise ratio of the peak height S / N ≥ 3:1, this concentration is used as the detection limit. The results are shown in Table 5 below Table 5 Detection results of quantitation limit and detection limit From the detection results in Table 5, it can be seen that the detection method provided in this application has high sensitivity, and the detection limit for detecting the genotoxic impurity 1-naphthylamine in propranolol hydrochloride can be as low as 0.0017 μg / mL, and the quantitation limit can be as low as 0.0056 μg / mL.
[0053] (IV) Linearity and range Preparation of linear solution: Take the 1-naphthylamine positioning solution under "Specificity" as the linear stock solution, and prepare linear solutions with the concentrations shown in Table 6 respectively, then inject them into the liquid chromatograph for detection, record the peak areas of the chromatograms, and perform linear regression with the concentration (μg / ml) as the abscissa (X) and the peak area as the ordinate (Y) to obtain the linear curve. As Figure 2 shown.
[0054] Table 6 Concentrations of linear solutions and linear equations According to Table 6 and Figure 2From the detection results, it can be seen that for impurity 1-naphthylamine, within the concentration range of 0.0056 - 0.2807 mg / ml, the concentration has a good linear relationship with the peak area, the correlation coefficient r is greater than 0.998, and the response values of the Y-axis intercept within the 100% limit are all within 25%. Therefore, it shows that the HPLC detection method for the genotoxic impurity 1-naphthylamine in propranolol hydrochloride provided in this application has a good linear relationship.
[0055] (V) Repeatability Prepare 6 portions of spiked raw material test solutions 1 - 6 and 6 portions of spiked preparation test solutions 1 - 6 in parallel. The preparation method is as described in the "Specificity" section; Inject the blank solution, reference solution, and each spiked test solution into the liquid chromatograph, and perform detection according to the method provided in Example 1. Record the chromatogram, and the results are shown in Table 7 below.
[0056] Table 7 Results of the repeatability test From the detection results in Table 7, it can be seen that by using the detection method provided in Example 1 of this application to detect 6 groups of spiked raw material test solutions and 6 groups of spiked preparation test solutions in parallel, the RSD values of the detection results are 0.72 - 1.44 (<10%). Therefore, it shows that the HPLC detection method for the genotoxic impurity 1-naphthylamine in propranolol hydrochloride provided in this application has good repeatability.
[0057] Examples 2 - 9 Examples 2 - 9 respectively provide an HPLC detection method for the genotoxic impurity 1-naphthylamine in propranolol hydrochloride.
[0058] The differences between the above examples and Example 1 are as follows: In the mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate, the concentrations of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate are specifically shown in Table 8 below.
[0059] Table 8 Concentrations of each substance in the mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate in Examples 2 - 9 Comparative Example 1 Comparative Example 1 provides an HPLC detection method for the genotoxic impurity 1-naphthylamine in propranolol hydrochloride.
[0060] The differences between the above comparative example and Example 1 are as follows: Mobile phase A is a mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate and acetonitrile with a volume ratio of 80:20; Mobile phase B is a mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate and acetonitrile with a volume ratio of 20:80.
[0061] Comparative Example 2 Comparative Example 2 provides an HPLC detection method for the genotoxic impurity 1-naphthylamine of propranolol hydrochloride.
[0062] The difference between the above comparative example and Example 1 is that mobile phase A is a solution of tetrabutylammonium dihydrogen phosphate and acetonitrile with a volume ratio of 70:30; the mobile phase B is a solution of tetrabutylammonium dihydrogen phosphate and acetonitrile with a volume ratio of 30:70. The preparation method of the tetrabutylammonium dihydrogen phosphate solution is as follows: take a certain amount of tetrabutylammonium dihydrogen phosphate, add water to dilute it until the concentration of tetrabutylammonium dihydrogen phosphate is 0.31 g / L; then add sulfuric acid with a volume content of 0.2%, and adjust the pH value to 3.3 with 2 mol / L sodium hydroxide solution to obtain it.
[0063] Comparative Example 3 Comparative Example 3 provides an HPLC detection method for the genotoxic impurity 1-naphthylamine of propranolol hydrochloride.
[0064] The difference between the above comparative example and Example 1 is the mobile phase gradient elution process.
[0065] The mobile phase gradient elution process of Comparative Example 3 is as follows: at 0 min, mobile phase A is 100% and mobile phase B is 0%; from 0 to 15 min, mobile phase A linearly decreases to 40% and mobile phase B linearly increases to 60%; from 15 to 30 min, mobile phase A linearly decreases to 0% and mobile phase B linearly increases to 100%; from 30 to 40 min, mobile phase A is 0% and mobile phase B is 100%; from 40 to 41 min, mobile phase A linearly increases to 100% and mobile phase B linearly decreases to 0%; from 41 to 70 min, mobile phase A is 100% and mobile phase B is 0%; both mobile phase A and mobile phase B are in volume percentages. Detection results Precisely measure 1 ml of the 1-naphthylamine positioning solution, place it in a 10-ml volumetric flask, dilute it to the scale with the preparation test solution (propranolol hydrochloride injection, 1-naphthylamine not detected) to obtain a spiked preparation test solution with a concentration of 0.14 μg / mL; prepare 6 portions in parallel according to the above method; then inject the reference solution and the spiked preparation test solution into the liquid chromatograph, and perform detections according to the methods provided in Examples 1-9 and Comparative Examples 1-3 respectively. Obtain the content of 1-naphthylamine in the spiked preparation test solution according to the chromatogram, and take the average value and RSD value of the detection results obtained by each method. The results are shown in Table 9 below. The detection chromatogram obtained by the detection method of Example 1 for the spiked preparation test solution is as Figure 3 shown.
[0066] Table 9 Detection results obtained by the methods provided in Examples 1-9 and Comparative Examples 1-3 According to the detection results in Table 9, when the test preparation solutions of the preparations were detected by the methods provided in Examples 1-9, the average content of the genotoxic impurity 1-naphthylamine in the spiked preparation test solutions obtained was 0.1388-0.1411 μg / ml, and the RSD values were 1.06-2.64% (<3.0%). Therefore, it shows that the HPLC detection method for the genotoxic impurity 1-naphthylamine in propranolol hydrochloride provided in this application has high detection accuracy and good repeatability, and can be used to detect the content of the genotoxic impurity 1-naphthylamine in propranolol hydrochloride injection, realizing the effective control of the quality of propranolol hydrochloride injection.
[0067] When the spiked preparation test solutions were detected by the methods provided in Comparative Example 1 and Comparative Example 2, obvious irregularities, asymmetry and tailing of the chromatographic peak of 1-naphthylamine were observed in the obtained detection chromatograms, and the average content of 1-naphthylamine in the spiked preparation test solutions detected was only 0.0797-0.1260, and the RSD values were as high as 19.06-38.26%. Therefore, it shows that when a mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate with a volume ratio of 80:20 and acetonitrile were used as mobile phase A, and a mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate with a volume ratio of 20:80 and acetonitrile were used as mobile phase B, and elution was carried out according to the mobile phase gradient elution program provided in this application, the elution separation effect of 1-naphthylamine was poor, and the accuracy and repeatability of the detection results were not good.
[0068] When the spiked preparation test solution was eluted by the mobile phase gradient elution program provided in Comparative Example 3, obvious tailing of the chromatographic peak of 1-naphthylamine was observed in the obtained detection chromatogram, and it was difficult to separate the baseline. The average content of 1-naphthylamine in the spiked preparation test solution detected was only 0.0655%, and the RSD value was as high as 42.88%. It shows that when the impurity 1-naphthylamine in propranolol hydrochloride was separated and detected by the elution method provided in Comparative Example 1, the accuracy and repeatability of the detection results were poor.
[0069] From the detection results of Examples 1-9, it can be seen that the 1-naphthylamine content in the spiked preparation test solution obtained by the methods of Examples 1, 3-4, and 7-8 was 0.1394-0.1411%, and the RSD value was 1.06-1.79% (≤2.00%); while the 1-naphthylamine content in the spiked preparation test solution obtained by the methods of Examples 2, 5-6, and 9 was 0.1388-0.1411%, and the RSD value was 2.23-2.64% (>2.00%). Therefore, it shows that in this application, the concentration of sodium dodecyl sulfate in the mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate is further controlled within the range of 3-3.5 g / L, and the concentration of tetrabutylammonium dihydrogen phosphate is controlled within the range of 0.25-0.35 g / L. The resulting detection method has better stability and repeatability, and can make the RSD value of the detection result as low as below 2.00%.
[0070] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.
Claims
1. An HPLC detection method for the genotoxic impurity 1-naphthylamine of propranolol hydrochloride, characterized in that, It includes the following steps: Preparing a reference solution, preparing a test solution, and performing liquid chromatography detection; Liquid chromatography detection: Inject the reference solution and the test solution into a liquid chromatograph, and perform separation and detection using a mobile phase gradient elution method; the chromatographic column used is a chromatographic column packed with octadecylsilyl-bonded silica gel; The mobile phase gradient elution process is as follows: At 0 min, mobile phase A is 100% and mobile phase B is 0%; from 0 to 15 min, mobile phase A linearly decreases to 75% and mobile phase B linearly increases to 25%; from 15 to 30 min, mobile phase A linearly decreases to 0% and mobile phase B linearly increases to 100%; from 30 to 40 min, mobile phase A is 0% and mobile phase B is 100%; from 40 to 41 min, mobile phase A linearly increases to 100% and mobile phase B linearly decreases to 0%; At 41 - 70 min, mobile phase A is 100% and mobile phase B is 0%; both mobile phase A and mobile phase B are in volume percentages; The mobile phase A is a mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate and acetonitrile; the volume ratio of the mixed solution to acetonitrile is 70:30; The mobile phase B is a mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate and acetonitrile; the volume ratio of the mixed solution to acetonitrile is 30:
70.
2. The HPLC detection method for the genotoxic impurity 1-naphthylamine of propranolol hydrochloride according to claim 1, wherein In the mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate, the concentration of sodium dodecyl sulfate is 3 - 3.5 g / L and the concentration of tetrabutylammonium dihydrogen phosphate is 0.25 - 0.35 g / L.
3. The HPLC detection method for the genotoxic impurity 1-naphthylamine of propranolol hydrochloride according to claim 1, wherein, The preparation method of the mixed solution of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate is: Take a certain amount of sodium dodecyl sulfate and tetrabutylammonium dihydrogen phosphate respectively, dilute with water until the concentration of sodium dodecyl sulfate is 3 - 3.5 g / L and the concentration of tetrabutylammonium dihydrogen phosphate is 0.25 - 0.35 g / L; then add sulfuric acid with a volume content of 0.15 - 0.25%, and adjust the pH value to 3 - 3.5 with 1.5 - 2.5 mol / L sodium hydroxide solution to obtain it.
4. The HPLC detection method for 1-naphthylamine, the genotoxic impurity of propranolol hydrochloride, according to claim 1, is characterized in that In the liquid chromatography detection, the flow rate of the mobile phase is 1.4 - 1.6 mL / min.
5. The HPLC detection method of 1-naphthylamine, a genotoxic impurity of propranolol hydrochloride, according to claim 1, is characterized in that In the liquid chromatography detection, the column temperature is 25 - 35 °C.
6. The HPLC detection method for the genotoxic impurity 1-naphthylamine of propranolol hydrochloride according to claim 1, wherein In the liquid chromatography detection, the detection wavelength is 230 nm.
7. The HPLC detection method for the genotoxic impurity 1-naphthylamine of propranolol hydrochloride according to claim 1, wherein In the liquid chromatography detection, the injection volume is 80 μL.
8. The HPLC detection method for the genotoxic impurity 1-naphthylamine of propranolol hydrochloride according to claim 1, wherein, In the liquid chromatography detection, the chromatographic column is CERI L-column3 C18, 250 mm × 4.6 mm, 5 μm or a chromatographic column with equivalent performance.
9. The HPLC detection method for the genotoxic impurity 1-naphthylamine of propranolol hydrochloride according to claim 1, characterized in that, The detection limit of the genotoxic impurity 1-naphthylamine is 0.0017 μg / mL, and the quantitation limit is 0.0056 μg / mL.
10. The application of the HPLC detection method for the genotoxic impurity 1-naphthylamine in propranolol hydrochloride as described in any one of claims 1 - 9 in the quality control of propranolol hydrochloride raw material or propranolol hydrochloride injection.