Method for detecting related substances in landiolol hydrochloride for injection

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting related substances in landiolol hydrochloride for injection. According to the method, an HPLC (High Performance Liquid Chromatography) method is adopted, octadecylsilane chemically bonded silica is taken as a filler, and a mobile phase A (the volume ratio of a monopotassium phosphate solution containing pentafluoropropionic acid to methanol is 58: 42-62: 38) and a mobile phase B (the volume ratio of the monopotassium phosphate solution containing pentafluoropropionic acid to methanol is 28: 72-32: 68) are used for gradient elution to detect the related substances in landiolol hydrochloride for injection. The detection method is high in specificity, high in sensitivity, good in accuracy and convenient to operate, the medicine quality can be effectively controlled, the technical blank of detection of the landiolol hydrochloride related substances for injection is filled, a reference standard is provided for quality control of landiolol hydrochloride for injection, and therefore the safety and reliability of clinical medication are guaranteed.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for detecting related substances in landiolol hydrochloride for injection. Background Art

[0002] Landiolol hydrochloride for injection (structure as shown in Formula I) is a β-adrenergic receptor blocker that can effectively treat tachycardia combined with myocardial ischemia caused during general anesthesia. It inhibits the increase in heart rate caused by the release of norepinephrine and epinephrine from sympathetic nerve endings and adrenal medulla, and dose-dependently inhibits tachycardia induced by electrical stimulation of sympathetic nerves or isoproterenol, as well as arrhythmias induced by halothane or epinephrine. It is highly selective for cardiac β1 receptors and has important clinical applications.

[0003]

[0004] Formula I According to the instructions for use of landiolol hydrochloride for injection, its maximum daily dose reaches 31.5 mg. According to the ICH guidelines, the impurity identification limit is 0.2%. According to the concept of impurity spectrum control, in the drug quality standard, it is required that the analytical method used can not only separate the impurities actually present in the sample, but also the potential impurities. Therefore, it is very necessary to analyze the potential impurities in landiolol hydrochloride for injection and establish an accurate and reliable analytical method, which is conducive to effectively controlling the product quality of landiolol hydrochloride for injection.

[0005] At present, the detection of related substances of landiolol hydrochloride is mainly focused on the raw material of landiolol hydrochloride. For example, the "HPLC method for determining the content of related substances in the starting material B of landiolol hydrochloride" uses high-performance liquid chromatography and acetonitrile-water as the mobile phase to detect its impurity A. The "Study on the detection method of 3-[4-(3-nitrobenzenesulfonyloxy)phenyl]propionic acid in the raw material of landiolol hydrochloride" uses high-performance liquid chromatography-triple tandem quadrupole mass spectrometry to detect trace genotoxic impurities 3-[4-(3-nitrobenzenesulfonyloxy)phenyl]propionic acid. However, there are no relevant reports on the detection technology of impurities in landiolol hydrochloride for injection.

[0006] From the perspective of the structure of landiolol hydrochloride, raw materials and preparation process, combined with its IF file and forced degradation investigation results, the relevant impurities are as follows: Impurity A: Impurity B:

[0007] Impurity O: Impurity H:

[0008] Impurity I: .

[0009] The analysis method of related substances of landiolol hydrochloride is mentioned in "Quality Research and Stability Investigation of Landiolol Hydrochloride". The method in this literature was used to prepare a specific solution rich in impurities for analysis. The results showed that this method could not elute impurities H and impurity I, and the detection ability was poor. Combined with the results of the preparation stability sample investigation, impurities H and impurity I were degradation impurities. In order to ensure the safety of patients' medication, it is necessary to develop an analytical method to study and control degradation impurities, so as to effectively and strictly control the quality of drugs. Summary of the invention

[0010] Based on this, the present invention provides a method for detecting related substances in landiolol hydrochloride for injection. The method uses HPLC to strongly degrade the test sample, and effectively separates and determines the content of degradation impurities and other impurities, and has high sensitivity, good specificity, high precision, good accuracy, and accurate and reliable detection results, which provides strong support for the quality control of landiolol for injection.

[0011] In order to achieve the purpose of the present invention, the technical solution adopted is as follows: A method for detecting related substances in landiolol hydrochloride for injection adopts HPLC method, takes octadecylsilane bonded silica gel as filler, and performs gradient elution through mobile phase A and mobile phase B to detect related substances in landiolol hydrochloride for injection; wherein the mobile phase A is potassium dihydrogen phosphate solution-methanol containing pentafluoropropionic acid, and the volume ratio of the potassium dihydrogen phosphate solution containing pentafluoropropionic acid to methanol is 58:42-62:38; the mobile phase B is potassium dihydrogen phosphate solution-methanol containing pentafluoropropionic acid, and the volume ratio of the potassium dihydrogen phosphate solution containing pentafluoropropionic acid to methanol is 28:72-32:68.

[0012] Furthermore, the related substances include one or more of the following structures: Impurity A:

[0013] Impurity B:

[0014] Impurity O:

[0015] Impurity H:

[0016] Impurity I: .

[0017] The impurities detected by the present invention are degradation impurities of landiolol hydrochloride for injection. The test sample landiolol hydrochloride for injection is strongly degraded by HPLC, and the separation and detection of the degradation products and other impurities are achieved.

[0018] Furthermore, the volume percentage of pentafluoropropionic acid in the potassium dihydrogen phosphate solution containing pentafluoropropionic acid is 0.8-1.2%.

[0019] Preferably, the volume percentage of pentafluoropropionic acid in the potassium dihydrogen phosphate solution containing pentafluoropropionic acid is 1.0%. Furthermore, the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate solution containing pentafluoropropionic acid is 5.00-7.00 g / L.

[0020] Preferably, the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate solution containing pentafluoropropionic acid is 6.00 g / L.

[0021] Further, the gradient elution procedure is as follows: 0-6min: mobile phase A accounts for 100%, mobile phase B accounts for 0%; 6-16min: mobile phase A accounts for 100-80%, mobile phase B accounts for 0-20%; 16-35min: Mobile phase A accounts for 80-40%, mobile phase B accounts for 20-60%; 35-45min: Mobile phase A accounts for 40%, mobile phase B accounts for 60%; 45-45.01min: Mobile phase A accounts for 40-100%, mobile phase B accounts for 60-0%; 45.01-55min: Mobile phase A accounts for 100%, and mobile phase B accounts for 0%.

[0022] Preferably, the gradient elution procedure is as follows: 0-16min: A is 100%, B is 0%; 16-35min: A is 90%, B is 10%; 35-45.01min: A is 70%, B is 30%; 45.01-55min: A is 100%, B is 0% Furthermore, during the detection process, the flow rate of the mobile phase was 0.9-1.1 mL / min.

[0023] Preferably, the flow rate of the mobile phase is 1.0 mL / min.

[0024] Furthermore, during the detection process, the column temperature of the chromatographic column is 20-30°C.

[0025] Preferably, the column temperature of the chromatographic column is 20°C.

[0026] Furthermore, during the detection process, the detection wavelength is 220-230 nm.

[0027] Preferably, the detection wavelength is 220 nm.

[0028] Furthermore, the injection volume during the detection process is 10 μL; the injection plate is temperature controlled at 5°C.

[0029] The detection method of the present invention specifically comprises the following steps: (1) Prepare test sample solution, reference sample solution and system suitability solution; (2) Take the test solution, reference solution and system suitability solution and inject them into the HPLC for testing respectively, and record the chromatogram; (3) Calculate the impurity content in the test sample landiolol hydrochloride for injection by the principal component external standard method with the addition of correction factors.

[0030] Furthermore, the concentration of the test solution is 2 mg / mL.

[0031] Furthermore, the reference substance solution contains 4 μg of the reference substance per 1 mL.

[0032] Furthermore, the reference substance in the system suitability solution is impurity O.

[0033] Furthermore, the system suitability solution contains 2 mg of landiolol hydrochloride and 4 μg of impurity O per 1 mL.

[0034] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: (1) The present invention establishes a method for detecting related substances (impurity A, impurity B, impurity O, impurity H and impurity I) of landiolol hydrochloride for injection, which fills the technical gap in the detection of related substances (degradation products) of landiolol hydrochloride for injection, and provides a reference standard for the quality control of landiolol hydrochloride for injection, thereby ensuring the safety and reliability of clinical medication; (2) The HPLC method provided by the present invention was used to conduct a forced degradation test on the preparation. The main peak purity factor under each destruction condition was greater than 990; the separation degree between the main peak and the front and rear impurity peaks was greater than 1.5; the material conservation under acid, alkali, oxidation and high temperature destruction was between 90-110%. The method can effectively separate and determine the content of the target substance and other impurities in landiolol hydrochloride for injection, and has high sensitivity, strong specificity and good repeatability. The inspection results are reliable and accurate, which provides a technical means for the detection of related substances in landiolol hydrochloride for injection and improves the quality control system of landiolol hydrochloride for injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The chromatograms of the blank solution and the mixed reference solution of Example 1 are shown below: Figure 2 The blank solution and mixed reference solution chromatograms of Example 2 are shown in FIG. Figure 3The chromatogram of the test solution of Example 3; wherein (A) is the complete chromatogram and (B) is the enlarged view; Figure 4 The chromatograms of the test solution of Example 4 are shown in Figure 4; wherein (A) is the complete chromatogram and (B) is the enlarged view; Figure 5 is the blank solution chromatogram; Figure 6 is the chromatogram of blank excipient solution; Figure 7 is the system suitability solution chromatogram; Figure 8 This is the chromatogram of landiolol hydrochloride reference solution; Fig. 9 is the chromatogram of the test solution; (A) is the complete chromatogram, and (B) is the enlarged view; Fig.10 It is a specific solution chromatogram; wherein, (A) is a complete chromatogram, and (B) is an enlarged view; Fig.11 This is the linear standard curve of landiolol hydrochloride; Fig.12 It is the linear standard curve of impurity A; Fig.13 It is the linear standard curve of impurity B; Fig.14 It is the linear standard curve of impurity O; Fig.15 It is the linear standard curve of impurity I; Fig.16 This is the linear standard curve of impurity H. DETAILED DESCRIPTION

[0036] The present invention is described below by specific examples to make the technical solution of the present invention easier to understand and grasp, but the present invention is not limited thereto. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified; and different sources have no significant effect on product performance.

[0037] The reagent information used in the specific implementation process is as follows: The test sample, landiolol hydrochloride for injection, was purchased from Chengdu Tongde Pharmaceutical Co., Ltd. with batch number 91572312012 and specification of 50 mg; The blank excipient was purchased from Zhejiang Hemukang Pharmaceutical Technology Co., Ltd. with batch number Z-FLD2401001 and specification of 25 mg / mL; Landiolol hydrochloride reference substance was purchased from Lanxiha Sanlian Pharmaceutical Co., Ltd. and standardized by Zhejiang Hemukang Pharmaceutical Technology Co., Ltd.; batch number: WRSY230502; content: 99.6%; The reference material information of the relevant substances (Impurity A, Impurity B, Impurity O, Impurity H and Impurity I) is as follows: Impurity A reference substance: FDR; batch number: CD11-06L; content after salt reduction: 88.46%; Impurity B reference substance: FDR; batch number: CD15-05L; content after salt reduction: 88.24%; Impurity O reference substance: FDR; batch number: CD11-13L; content: 96.69%; Impurity H reference substance: QCS; batch number: 42400; content: 96.75%; Impurity H reference substance: FDR; batch number: CK08-31L; content: 95.28%; Impurity H reference substance: FDR; batch number: DA08-31L; content: 94.38%; Impurity I reference substance: QCS; batch number: 43074; content: 93.33%.

[0038] The structural formula is shown in Table 1: Table 1 Related substances of landiolol hydrochloride

[0039] Example 1 (1) Chromatographic conditions Chromatographic column: octadecyl silica gel column (GL Inertsil ODS, 250×4.6mm, 5μm); Mobile phase: phosphate buffer (weigh 5.44 g potassium dihydrogen phosphate, dissolve in 1000 mL of water, and adjust the pH to 6.5 with potassium hydroxide)-acetonitrile (70:30); Detection wavelength: 230nm; Column temperature: 35°C; Flow rate: 1.0 mL / min; Injection volume: 20 μL; Elution mode: isocratic elution.

[0040] (2) Solution preparation Mixed reference solution: Take appropriate amount of landiolol hydrochloride impurity A, B, O, H, I reference substances, accurately weigh, dissolve in water and dilute to make a solution containing approximately 4 μg of impurities A, B, O, H, I per 1 mL.

[0041] (3) Measurement results Accurately measure the blank solution (deionized water) and the mixed reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram. The results are as follows: Figure 1 As shown, the retention time and separation degree of each impurity peak in the mixed reference solution are shown in Table 2.

[0042] Table 2

[0043] The results showed that under the chromatographic conditions, the blank solution interfered with the peak of impurity A, the peak shape of impurity B was poor, and impurities H and I could not be eluted, failing to achieve the purpose of analysis, and was not suitable for the detection of related substances of landiolol hydrochloride for injection.

[0044] Example 2 (1) Chromatographic conditions Chromatographic column: octadecyl silica gel column (GL Inertsil ODS, 250×4.6mm, 5μm); Mobile phase: 0.05 mol / L sodium dihydrogen phosphate solution-methanol (60:40); Detection wavelength: 220nm; Column temperature: 25°C; Flow rate: 1.0 mL / min; Injection volume: 10 μL; Elution mode: isocratic elution.

[0045] (2) Solution preparation Mixed reference solution: Take appropriate amount of landiolol hydrochloride impurity A, B, O, H, I reference substances, accurately weigh, dissolve in water and dilute to make a solution containing approximately 4 μg of impurities A, B, O, H, I per 1 mL.

[0046] (3) Measurement results Accurately measure the blank solution (deionized water) and the mixed reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram. The results are as follows: Figure 2 As shown, the retention time and separation degree of each impurity peak in the mixed reference solution are shown in Table 3.

[0047] Table 3

[0048] The results show that compared with the method in Example 1, under the chromatographic conditions, the blank solution does not interfere with the elution of impurity A, and the peak shape of impurity B is improved, but impurities H and impurity I still cannot be eluted, and the analysis purpose cannot be achieved. It is not suitable for the detection of related substances of landiolol hydrochloride for injection.

[0049] Example 3 (1) Chromatographic conditions Chromatographic column: octadecyl silica gel column (GL Inertsil ODS, 250×4.6mm, 5μm); Mobile phase A: potassium dihydrogen phosphate solution (weigh 6.00 g of anhydrous potassium dihydrogen phosphate, dissolve in water and dilute to 1000 mL); Mobile phase B: methanol; Detection wavelength: 220nm; Column temperature: 25°C; Flow rate: 1.0 mL / min; Injection volume: 10 μL; Sample plate temperature control: 5°C; Elution method: gradient elution, the elution program is shown in Table 4; Table 4

[0050] (2) Solution preparation Mixed reference solution: Take appropriate amounts of landiolol hydrochloride impurities A, B, O, H, I and landiolol hydrochloride reference substance, accurately weigh, dissolve them in water and dilute them to make a solution containing approximately 4 μg of impurities A, B, O, H, I and landiolol hydrochloride reference substance per 1 mL.

[0051] Test sample solution: Take an appropriate amount of the test sample (landiolol hydrochloride for injection) and dilute it with water to make a solution containing 2 mg of the test sample per 1 mL.

[0052] (3) Measurement results Accurately measure the test solution and the mixed reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram. The results are as follows: Figure 3 As shown, the retention time and separation degree of each impurity peak in the test solution are shown in Table 5.

[0053] Table 5

[0054] The results showed that: on the basis of Example 2, by adjusting the mobile phase composition and elution procedure, impurities H and I could both elute, but the unknown impurity 5 in the test solution had a poor separation from the main component.

[0055] Embodiment 4: (1) Chromatographic conditions Chromatographic column: octadecyl silica gel column (GL Inertsil ODS, 250×4.6mm, 5μm); Mobile phase A: potassium dihydrogen phosphate solution (weigh 6.00 g of anhydrous potassium dihydrogen phosphate, add 10 mL of pentafluoropropionic acid, dissolve in water and dilute to 1000 mL)-methanol (60:40); Mobile phase B: potassium dihydrogen phosphate solution (weigh 6.00 g of anhydrous potassium dihydrogen phosphate, add 10 mL of pentafluoropropionic acid, dissolve in water and dilute to 1000 mL)-methanol (30:70); Detection wavelength: 220nm; Column temperature: 25°C; Flow rate: 1.0 mL / min; Injection volume: 10 μL; Sample plate temperature control: 5°C; Elution method: gradient elution, the elution program is shown in Table 6; Table 6

[0056] (2) Solution preparation: Mixed reference solution: Take appropriate amounts of impurities A, B, O, H, I of landiolol hydrochloride and landiolol reference substance, accurately weigh, dissolve in water and dilute to make a solution containing approximately 4 μg of impurities A, B, O, H, I and landiolol per 1 mL.

[0057] Test sample solution: Take an appropriate amount of the test sample and dilute it with water to make a solution containing 2 mg of the test sample per 1 mL.

[0058] (3) Measurement results Accurately measure the test solution and the mixed reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram. The results are as follows: Figure 4 As shown, the retention time and separation degree of each impurity peak in the test solution are shown in Table 7.

[0059] Table 7

[0060] The results show that: on the basis of Example 3, pentafluoropropionic acid was added to the mobile phase composition, and the solvent ratio and elution procedure were adjusted. Under the chromatographic conditions, the blank solution did not interfere with the impurity detection; each known impurity could produce a peak; the main peak purity factor in the test solution was greater than 990, and the separation degree from the adjacent impurity peaks met the requirements. It can be seen that this detection method can effectively separate the impurities. At the same time, this method also reduces the possibility of poor mobile phase mixing and improves the peak shape of the chromatographic peak.

[0061] Next, the method was validated for specificity, accuracy, precision, limit of quantification, limit of detection, linearity and range, and durability, as follows: (1) Exclusivity ①Blank interference: Blank solution: deionized water; Blank excipient solution: accurately measure 2 mL of blank excipient, place in a 25 mL volumetric flask, add water to dilute to the mark, shake well, and obtain; System suitability solution: Take appropriate amount of landiolol hydrochloride impurity O reference substance and test substance, weigh accurately, dissolve in water and dilute to make a solution containing about 4 μg of impurity O and 2 mg of test substance per 1 mL; Reference solution: Take an appropriate amount of landiolol hydrochloride reference substance, weigh accurately, dissolve in water and dilute to make a solution containing about 4µg per 1mL; Test solution: Take 100 mg of the test sample, dissolve it in water and transfer it to a 25 mL volumetric flask, dilute it to the mark with water, and shake well.

[0062] Accurately measure 10 μL of blank solution, blank excipient solution, system suitability solution, reference solution, and test solution, respectively, and inject them into the liquid chromatograph. Record the chromatogram according to the chromatographic conditions of Example 4. The results are shown in Figure 5-Figure 9 ,It can be seen from the figure that the blank solution does not interfere with the impurity detection.

[0063] ② Known impurity location: Impurity A reference substance stock solution: Accurately weigh 5.089 mg of impurity A reference substance, place in a 50 mL volumetric flask, add water to dissolve and dilute to the scale, shake well; Impurity A positioning solution: Accurately measure 2 mL of the impurity A reference substance stock solution, place it in a 5 mL volumetric flask, add water to dilute to the scale, and shake well; Impurity B positioning solution: Accurately weigh 1.262 mg of impurity B reference substance, place in a 25 mL volumetric flask, add water to dissolve and dilute to the scale, shake well; Impurity O positioning solution: Accurately weigh 1.216 mg of impurity O reference substance, place in a 25 mL volumetric flask, add water to dissolve and dilute to the scale, shake well; Impurity H localization solution: Accurately weigh 2.264 mg of impurity H reference substance, place in a 25 mL volumetric flask, add water to dissolve and dilute to the scale, shake well; Impurity I localization solution: Accurately weigh 1.110 mg of impurity I reference substance, place in a 25 mL volumetric flask, add water to dissolve and dilute to the scale, shake well; Linear stock solution: Accurately weigh 5.062 mg of impurity B reference substance, 5.340 mg of impurity O reference substance, 5.183 mg of impurity H reference substance, and 5.165 mg of impurity I reference substance, place them in a 50 mL volumetric flask, add water to dissolve and dilute to the scale, and shake well; Specific solution: Take 100 mg of the test sample, dissolve it in water and transfer it to a 25 mL volumetric flask, add 1 mL of linear stock solution and 2.5 mL of impurity A reference stock solution, dilute to the scale with water and shake well.

[0064] Accurately measure 10 μL of each of the above solutions and inject them into a liquid chromatograph. Record the chromatogram according to the chromatographic conditions of Example 4. The chromatogram of the specific solution is shown in Fig.10 , the specificity results are shown in Table 8.

[0065] Table 8

[0066] According to the results, the separation degree between each impurity in the specific solution and the previous and next impurities is greater than 1.5, the separation degree is good, and the separation and detection method provided by the present invention has good specificity.

[0067] ③Forced degradation The conditions in Table 9 were followed to investigate the degradation of the samples under strong alkali, strong acid, oxidation and high temperature conditions. The separation between the degradation products and the main peak of the samples was investigated. A diode array detector (DAD) was used to determine the purity of the main peak to further verify the specificity of this method. The results are detailed in Tables 10 and 11.

[0068] Table 9

[0069] Table 10

[0070] Table 11

[0071] According to the results in the above table: the purity factors of the main peak of forced degradation under various forced degradation conditions are all greater than 990; the separation between the main peak and the preceding and following impurity peaks is greater than 1.5; the conservation of materials destroyed by acid, alkali, oxidation and high temperature is between 90-110%; this method can effectively detect the degradation impurities of landiolol hydrochloride for injection, and the specificity meets the requirements.

[0072] (2) Accuracy Impurity A reference substance stock solution: Accurately weigh 5.627 mg of impurity A reference substance, place in a 50 mL volumetric flask, add water to dissolve and dilute to the scale, shake well; Mixed reference substance stock solution: Accurately weigh 2.826 mg of impurity B reference substance, 2.573 mg of impurity O reference substance, 2.464 mg of impurity H reference substance and 2.717 mg of impurity I reference substance, place in a 25 mL volumetric flask, add water to dissolve and dilute to the scale, shake well; Test solution: Take 100 mg of the test sample, dissolve it in water and transfer it to a 25 mL volumetric flask, dilute it to the mark with water, and shake well; Recovery rate -20%: Take 200 mg of the test sample, dissolve it in water and transfer it to a 50 mL volumetric flask, accurately add 1 mL of impurity A reference substance stock solution and 0.4 mL of mixed reference substance stock solution, dilute to the scale with water, and shake well (prepare 3 copies in parallel); Recovery rate -50%: Take 100 mg of the test sample, dissolve it in water and transfer it to a 25 mL volumetric flask, accurately add 1.25 mL of impurity A reference substance stock solution and 0.5 mL of mixed reference substance stock solution, dilute to the scale with water, and shake well (prepare 3 copies in parallel); Recovery rate -100%: Take 100 mg of the test sample, dissolve it in water and transfer it to a 25 mL volumetric flask, accurately add 2.5 mL of impurity A reference substance stock solution and 1 mL of mixed reference substance stock solution, dilute to the scale with water, and shake well (prepare 3 copies in parallel); Recovery rate -150%: Take 100 mg of the test sample, dissolve it in water and transfer it to a 25 mL volumetric flask, accurately add 3.75 mL of impurity A reference substance stock solution and 1.5 mL of mixed reference substance stock solution, dilute to the scale with water, and shake well (prepare 3 copies in parallel); Recovery rate (%) = (measured amount - original amount) / added amount × 100%.

[0073] Accurately measure 10 μL of each of the above solutions, inject into a liquid chromatograph, and record the chromatogram according to the chromatographic conditions of Example 4. The recovery rates of impurity A, impurity B, impurity O, impurity I and impurity H are shown in Tables 12 to 16, respectively.

[0074] Table 12

[0075] Table 13

[0076] Table 14

[0077] Table 15

[0078] Table 16

[0079] According to the above results, the mean recoveries of impurities in the test sample solutions with 20%, 50%, 100% and 150% concentrations were all within the range of 90-110%, and the relative standard deviations (RSD%) of the 12 recoveries were all less than 5%, indicating that this method had good accuracy.

[0080] (3) Precision ①Repeatability The same experimenter prepared 6 test solutions in parallel. The preparation method of the test solutions was the same as the recovery rate under (2) accuracy - 100%. 10 μL of each solution was accurately measured and injected into the liquid chromatograph. According to the chromatographic conditions of Example 4, the chromatogram was recorded. The contents and relative standard deviations (RSD%) of the known impurities (impurity A, impurity B, impurity O, impurity I and impurity H) of the 6 test solutions were calculated by the impurity external standard method. The contents and relative standard deviations (RSD%) of other impurities and total impurities were calculated by the main component external standard method with correction factors. The repeatability results are shown in Table 17.

[0081] Table 17

[0082] ②Intermediate precision Different analysts (2 persons) prepared mixed reference solution and test solution with different instruments in the same method, accurately measured 10 μL of each of the above solutions, injected into the liquid chromatograph, recorded the chromatogram according to the chromatographic conditions of Example 4, calculated the content of known impurities and relative standard deviation (RSD%) of 6 test solutions by the impurity external standard method, calculated the content of other impurities and total impurities and relative standard deviation (RSD%) by the main component external standard method with correction factor, and the results are shown in Table 18; at the same time, the difference between the mean values ​​of the results of two different analysts was compared to examine the intermediate precision of this method, and the results are shown in Table 19.

[0083] Table 18

[0084] Table 19

[0085] According to the above results, the relative standard deviation (RSD%) of the impurity content in the 6 test solutions for repeatability and intermediate precision all meet the requirements; comparing the difference in the mean values ​​of the results of two different experimenters, the difference in the impurity content all meets the requirements, and the precision of this method is good.

[0086] (4) Limit of quantification Landiolol hydrochloride reference substance stock solution: accurately weigh 10.455 mg of landiolol hydrochloride reference substance, place in a 100 mL volumetric flask, add water to dissolve and dilute to the scale, shake well, and obtain; Impurity A reference substance stock solution: Accurately weigh 5.089 mg of impurity A reference substance, place in a 50 mL volumetric flask, add water to dissolve and dilute to the scale, shake well; Linear stock solution 1: Accurately weigh 5.062 mg of impurity B reference substance and 5.340 mg of impurity O reference substance, place in a 50 mL volumetric flask, add water to dissolve and dilute to the scale, shake well; Linear stock solution 2: Accurately weigh 5.183 mg of impurity H reference substance and 5.165 mg of impurity I reference substance, place in a 50 mL volumetric flask, add water to dissolve and dilute to the scale, and shake well; Quantitation limit solution - impurity A, landiolol hydrochloride, impurity B, impurity O: accurately measure 0.2 mL of impurity A reference stock solution in a 50 mL volumetric flask, add 0.2 mL of landiolol hydrochloride reference stock solution and 0.2 mL of linear stock solution 1, dilute to the scale with water, shake well, and obtain (prepare 6 portions in parallel); Quantitation limit solution - impurity I, impurity H: Accurately measure 0.4 mL of linear stock solution 2, place in a 50 mL volumetric flask, add water to dilute to the scale, shake well, and obtain (prepare 6 portions in parallel).

[0087] Accurately measure 10 μL of each quantitation limit solution and inject it into the liquid chromatograph respectively. According to the chromatographic conditions of Example 4, record the chromatogram, calculate the RSD% of the peak area of ​​each impurity peak and the peak area of ​​the main component peak. The quantitation limit results of impurity A, impurity B, impurity O, landiolol hydrochloride, impurity I and impurity H are shown in Tables 20 to 25, respectively.

[0088] Table 20

[0089] Table 21

[0090] Table 22

[0091] Table 23

[0092] Table 24

[0093] Table 25

[0094] The results showed that the signal-to-noise ratio of each peak in the above quantitative limit solution was greater than 10, and the relative standard deviation (RSD%) of the peak area of ​​each impurity peak was less than 10%. When the sample concentration was 2 mg / mL, the quantitative limits of impurity A, impurity B, impurity O, landiolol hydrochloride, impurity I, and impurity H were 0.02%, 0.02%, 0.02%, 0.02%, 0.04%, and 0.04% of the test sample concentration, respectively.

[0095] (5) Detection limit Detection limit solution - impurity A, landiolol hydrochloride, impurity B, impurity O: Accurately measure 2.5 mL of "quantification limit solution - impurity A, landiolol hydrochloride, impurity B, impurity O" under (4) quantification limit, place in a 5 mL volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0096] Detection limit solution - impurity I, impurity H: Accurately measure 2.5 mL of "quantification limit solution - impurity I, impurity H" under (4) quantification limit, place in a 5 mL volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0097] According to the above chromatographic conditions, accurately measure 10 μL of each of the above solutions and inject them into the liquid chromatograph respectively. According to the chromatographic conditions of Example 4, record the chromatogram. The detection limit results are shown in Table 26.

[0098] Table 26

[0099] The results showed that when the sample concentration was 2 mg / mL, impurity A, landiolol hydrochloride, impurity B, impurity O, impurity I, and impurity H could be detected when they exceeded 0.01%, 0.01%, 0.01%, 0.01%, 0.02%, and 0.02% of the test sample concentration; the method had good sensitivity.

[0100] (6) Linearity and range Landiolol hydrochloride reference substance stock solution: Accurately weigh 10.455 mg of landiolol hydrochloride reference substance, place it in a 100 mL volumetric flask, add water to dissolve and dilute to the scale, shake well, and obtain.

[0101] Impurity A reference substance stock solution: Accurately weigh 5.089 mg of impurity A reference substance, place it in a 50 mL volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0102] Linear stock solution: Accurately weigh 5.062 mg of impurity B reference substance, 5.340 mg of impurity O reference substance, 5.183 mg of impurity H reference substance, and 5.165 mg of impurity I reference substance, place them in a 50 mL volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0103] Linear solution 1 (quantification limit): same as “quantification limit solution - impurity A, landiolol hydrochloride, impurity B, impurity O” under (4) quantification limit; Linear solution 2 (quantification limit): Same as "quantification limit solution - impurity I, impurity H" under (4) Quantification limit Linear solution 3: Accurately measure 0.5 mL of linear stock solution, 0.5 mL of landiolol hydrochloride reference stock solution, and 1.2 mL of impurity A reference stock solution, respectively, and place them in a 25 mL volumetric flask, add water to dilute to the scale, and shake well to obtain; Linear solution 4: Accurately measure 0.8 mL of linear stock solution, 0.8 mL of landiolol hydrochloride reference stock solution, and 2 mL of impurity A reference stock solution respectively, place them in a 25 mL volumetric flask, add water to dilute to the scale, shake well, and obtain; Linear solution 5: Accurately measure 1 mL of linear stock solution, 1 mL of landiolol hydrochloride reference stock solution, and 2.5 mL of impurity A reference stock solution, respectively, place them in a 25 mL volumetric flask, add water to dilute to the scale, shake well, and obtain; Linear solution 6: Accurately measure 2 mL of linear stock solution, 2 mL of landiolol hydrochloride reference stock solution, and 5 mL of impurity A reference stock solution, respectively, place them in a 25 mL volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0104] Accurately measure 10 μL of each of the above linear solutions and inject them into a liquid chromatograph. According to the chromatographic conditions of Example 4, record the chromatogram, use concentration as the abscissa and peak area as the ordinate to make a linear regression equation. The results of landiolol hydrochloride, impurity A, impurity B, impurity I, impurity O and impurity H are shown in Table 1. Figure 11-Figure 16 ; and calculate the correction factors for impurities. The results for landiolol hydrochloride, impurity A, impurity B, impurity I, impurity O and impurity H are shown in Table 27.

[0105] The relative correction factor of impurities = the slope of the linear regression equation of the main component / the slope of the linear regression equation of the impurity.

[0106] Table 27

[0107] According to the above results, the correlation coefficients (r) of the linear regression equations of landiolol hydrochloride, impurity A, impurity B, impurity O, impurity I, and impurity H were all greater than 0.995 within their respective concentration ranges, the Y-axis intercepts were all within 10% of the 100% response value, and the RSD% of the response factors were all within 10%, all meeting the requirements. This method has a good linear relationship.

[0108] (7) Durability On the basis of the chromatographic conditions in Example 4, the influence of the chromatographic conditions on the analysis results was investigated by making the following changes to the chromatographic conditions: changes in the chromatographic column (different batches) and the instrument (different models), changes in the column temperature (25°C ± 2°C), and the volume ratio of the organic phase (methanol) in the mobile phase A (40 ± 2%). The system suitability test results under various chromatographic conditions are shown in Table 28, and the impurity content test results are shown in Table 29.

[0109] Table 28

[0110] Table 29

[0111] The results show that the system suitability meets the requirements when the chromatographic column (different batches) and instrument change, the column temperature changes (25℃±2℃), and the volume percentage of the organic phase (methanol) in the mobile phase A (40±2%) changes; the difference between the impurity content measured under each condition and the impurity content under the original conditions meets the requirements, and the chromatographic conditions have good durability.

[0112] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for detecting related substances in landiolol hydrochloride for injection, characterized in that: The detection method adopts HPLC method, takes octadecylsilane bonded silica gel as filler, and performs gradient elution through mobile phase A and mobile phase B to detect related substances in landiolol hydrochloride for injection; wherein the mobile phase A is potassium dihydrogen phosphate solution-methanol containing pentafluoropropionic acid, and the volume ratio of the potassium dihydrogen phosphate solution containing pentafluoropropionic acid to methanol is 58:42-62:38; the mobile phase B is potassium dihydrogen phosphate solution-methanol containing pentafluoropropionic acid, and the volume ratio of the potassium dihydrogen phosphate solution containing pentafluoropropionic acid to methanol is 28:72-32:

68.

2. The detection method according to claim 1, characterized in that The related substances include one or more of the following structures: Impurity A: Impurity B: Impurity O: Impurity H: Impurity I: .

3. The detection method according to claim 1, characterized in that: The volume percentage of pentafluoropropionic acid in the potassium dihydrogen phosphate solution containing pentafluoropropionic acid is 0.8-1.2%; the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate solution containing pentafluoropropionic acid is 5.00-7.00 g / L.

4. The detection method according to claim 3, characterized in that: The volume percentage of pentafluoropropionic acid in the potassium dihydrogen phosphate solution containing pentafluoropropionic acid is 1.0%; the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate solution containing pentafluoropropionic acid is 6.00 g / L.

5. The detection method according to claim 1, characterized in that: The gradient elution procedure is as follows: 0-6min: mobile phase A accounts for 100%, mobile phase B accounts for 0%; 6-16min: mobile phase A accounts for 100-80%, mobile phase B accounts for 0-20%; 16-35min: Mobile phase A accounts for 80-40%, mobile phase B accounts for 20-60%; 35-45min: Mobile phase A accounts for 40%, mobile phase B accounts for 60%; 45-45.01min: Mobile phase A accounts for 40-100%, mobile phase B accounts for 60-0%; 45.01-55min: Mobile phase A accounts for 100%, and mobile phase B accounts for 0%.

6. The detection method according to claim 1, characterized in that: During the detection process, the flow rate of the mobile phase was 0.9-1.1 mL / min.

7. The detection method according to claim 6, characterized in that: The flow rate of the mobile phase was 1.0 mL / min.

8. The detection method according to claim 1, characterized in that: During the detection process, the column temperature of the chromatographic column is 20-30°C; the detection wavelength is 220-230nm.

9. The detection method according to claim 8, characterized in that: The column temperature of the chromatographic column is 25° C.; the detection wavelength is 220 nm.

10. The detection method according to claim 1, characterized in that: During the detection process, the injection volume was 10 μL and the injection plate temperature was controlled at 5°C.

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