Detection method for efficiently separating and determining multiple related substances of landiolol hydrochloride

Abstract

The invention discloses a detection method for efficiently separating and determining a plurality of related substances of landiolol hydrochloride, which adopts high performance liquid chromatography and takes octadecylsilane chemically bonded silica as a filling agent of a chromatographic column; carrying out gradient elution by taking a phosphate buffer solution as a mobile phase A and acetonitrile as a mobile phase B; an ultraviolet detector is adopted for detection, and the detection wavelength is 210-230 nm. The detection method provided by the invention can be used for efficiently separating and determining a plurality of related substances of landiolol hydrochloride.

Description

Technical Field

[0001] This invention relates to the field of compound analysis and detection technology, specifically to a method for efficiently separating and determining multiple related substances in brandylol hydrochloride. Background Technology

[0002] Onoact hydrochloride, a brand name, is an ultra-short-acting, highly selective β1 receptor blocker that primarily antagonizes β1 receptors present in the heart. It improves tachycardia-related arrhythmias by inhibiting the increase in heart rate induced by catecholamines. Developed by Ono Pharmaceutical Co., Ltd. of Japan, it was first approved on July 5, 2002, for "emergency treatment of intraoperative tachyarrhythmias," and was first marketed in Japan in September 2002. Clinically, it is mainly used for the emergency treatment of tachycardia-related arrhythmias (including atrial fibrillation, atrial flutter, and sinus tachycardia) during surgery. In addition, landiolol hydrochloride for injection is a novel, ultra-short-acting adrenaline beta-blocker with rapid onset of action, rapid metabolism, and rapid disappearance of beta-blocking effect after discontinuation. On March 26, 2019, it was added as an indication for "life-threatening refractory ventricular fibrillation / ventricular tachycardia", filling the treatment gap when Class III antiarrhythmic drugs are ineffective. It plays an important and irreplaceable role in the treatment of tachyarrhythmias and has become a benchmark product in the field of beta1 selective blockers.

[0003] The English name of this product is Landiolol Hydrochloride, CAS number is 144481-98-1, chemical name is [(S)-2,2-dimethyl-1,3-dioxolane-4-yl]methyl 3-[4-[(S)-2-hydroxy-3-(2-morpholinocarbonylamino)ethylamino]propoxy]phenylpropionate monohydrochloride, chemical structural formula is as follows, molecular formula is C25H39N3O8・HCl, molecular weight is 546.05.

[0004]

[0005] In this pharmaceutical research field, impurities in any active ingredient of landiolol hydrochloride may originate from the degradation and synthesis processes of the active pharmaceutical ingredient itself. Process impurities include unreacted raw materials, impurities contained in the raw materials and their chemical derivatives, intermediates, synthetic byproducts, and degradation products. Currently, Table 1 summarizes the impurities affecting the quality of landiolol hydrochloride.

[0006] Table 1. Impurities in brandylol hydrochloride

[0007]

[0008] Chinese invention CN101271085 discloses a method for detecting landiolol hydrochloride and its intermediates. This method uses isocratic elution and employs ion-pairing reagents such as sodium alkyl sulfate and sodium alkyl sulfonate in the mobile phase. These ion-pairing reagents can modify the stationary phase of the chromatographic column, causing irreversible damage. Sodium alkyl sulfonate has certain UV absorption in the ultraviolet region (especially short wavelengths <254 nm), which can lead to increased baseline noise, baseline drift, and reduced detection sensitivity, making it unsuitable for the analysis of low-concentration samples. Chinese invention CN119355180 provides a method for detecting related substances in injectable landiolol hydrochloride, which only controls individual impurities. The method uses isocratic elution and adds 0.8%-1.2% pentafluoropropionic acid to the mobile phase. Although pentafluoropropionic acid is a commonly used volatile pair reagent in HPLC, it is a strong acid (pKa≈0.3). Its strong acidity and fluorinated structure may cause slight hydrolysis of the bonded phase of the chromatographic column stationary phase. High concentrations (>0.5%) may corrode the stainless steel tubing, injection valve rotor, and other metal parts of the chromatograph.

[0009] However, there is currently no method to comprehensively and effectively study the aforementioned known impurities. Therefore, it is of great significance to develop a simple, economical, and efficient detection method for separating and determining multiple related substances in brandylolol hydrochloride. Summary of the Invention

[0010] Purpose of the invention: The technical problem to be solved by the present invention is to provide a highly efficient method for separating and determining multiple related substances in brandylol hydrochloride, addressing the shortcomings of the existing technology.

[0011] To address the aforementioned technical problems, this invention discloses a method for detecting related substances in brandylol hydrochloride.

[0012] In some embodiments, the detection method employs high-performance liquid chromatography (HPLC), using octadecylsilane-bonded silica gel as the column packing material; gradient elution is performed using phosphate buffer as mobile phase A and acetonitrile as mobile phase B; and detection is performed using an ultraviolet detector with a detection wavelength of 210-230 nm.

[0013] In some embodiments, the related substances of brandylolol hydrochloride include one or more of impurities B, C, D, E, F, G, H, I, J, K, SM1, intermediate II, and intermediate III, and the molecular structural formulas of each impurity are shown below.

[0014]

[0015]

[0016]

[0017] .

[0018] In some embodiments, the related substances of brandylol hydrochloride include impurities B, C, D, E, F, H, I, K, SM1, and intermediate III (impurity 1); in some embodiments, the related substances of brandylol hydrochloride include impurities B, C, D, E, F, G, H, I, J, K, and intermediate II.

[0019] The detection method provided by this invention can effectively separate the aforementioned impurities and brandylol hydrochloride.

[0020] In some embodiments, the diameter of the chromatographic column is 4.0-5.2 mm, in some embodiments it is 4.3-4.9 mm, and in some embodiments it is 4.6 mm; in some embodiments, the column length is 230-270 mm, in some embodiments it is 240-260 mm, and in some embodiments it is 250 mm; in some embodiments, the particle size of the packing material is 4-6 μm, in some embodiments it is 4.5-5.5 μm, and in some embodiments it is 5 μm.

[0021] In some embodiments, the phosphate buffer is a buffer solution prepared by mixing any one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate hydrate, and dipotassium hydrogen phosphate hydrate; in some embodiments, the concentration of the phosphate buffer is 0.01-0.1 mol / L, and in some embodiments it is 0.02 mol / L, 0.05 mol / L, or 0.08 mol / L; in some embodiments, the phosphate buffer is a mixed solution of 0.02 mol / L potassium dihydrogen phosphate and 0.01 mol / L dipotassium hydrogen phosphate; in some embodiments, the pH of the phosphate buffer is 3-7, and in some embodiments, the pH of the phosphate buffer is 6; in some embodiments, the pH is adjusted by using an alkali such as sodium hydroxide.

[0022] In some embodiments, in the gradient elution process, the initial mobile phase is: mobile phase A 88%-97%, mobile phase B 3%-12%; the intermediate mobile phase is: mobile phase A 38%-52%, mobile phase B 48%-62%; and the final mobile phase is: mobile phase A 88%-97%, mobile phase B 3%-12%. In some embodiments, the initial mobile phase is: mobile phase A 90%-95%, mobile phase B 5%-10%; the intermediate mobile phase is: mobile phase A 40%-50%, mobile phase B 50%-60%; and the final mobile phase is: mobile phase A 90%-95%, mobile phase B 5%-10%.

[0023] In some embodiments, a third mobile phase may be provided between the initial mobile phase and the intermediate mobile phase: mobile phase A is 83%-87% and mobile phase B is 13%-17%, and in some embodiments mobile phase A is 85% and mobile phase B is 15%.

[0024] In some embodiments, the initial mobile phase is the mobile phase from 0 to t1 min, the intermediate mobile phase is the mobile phase from t1 to t2 min, and the final mobile phase is the mobile phase from t2 to the total time. In some embodiments, the initial mobile phase is the mobile phase from 0 to t0 min, the third mobile phase refers to the mobile phase from t0 to t1 min, the intermediate mobile phase is the mobile phase from t1 to t2 min, and the final mobile phase is the mobile phase from t2 to the total time. In this invention, 0 < t0 < t1 < t2 < the total time of gradient elution.

[0025] In some embodiments, t0 is 8 to 12, such as 10; in some embodiments, t2-t1 is 1 to 8, such as any one of 2, 4, 5, and 7; in some embodiments, t2 is the total time minus 4 to 9 min, such as minus 1 to 8 min; in some embodiments, the total time is 57 to 73 min, such as 60 to 70 min; in some embodiments, without a third mobile phase, the total gradient elution time is 57 to 65 min, such as 60 or 63 min; in some embodiments, with a third mobile phase, the total gradient elution time is 65 to 73 min, such as 67 or 70 min.

[0026] In some embodiments, the gradient elution procedure is any one of the following:

[0027] Table 2 Gradient elution program

[0028]

[0029] or;

[0030] Table 3 Gradient elution program

[0031]

[0032] or;

[0033] Table 4 Gradient elution program

[0034]

[0035] or;

[0036] Table 5 Gradient elution program

[0037]

[0039] In some embodiments, the flow rate of the mobile phase is 0.8-1.2 mL / min, and in some embodiments it is 1.0 mL / min.

[0040] In some embodiments, the column temperature of the chromatographic column is 20-35°C, and in some embodiments it is 25°C.

[0041] In some embodiments, the detection wavelength is 220 nm.

[0042] In some embodiments, the ultraviolet detector includes a diode array detector.

[0043] In some embodiments, the injection volume is 8-12 μL, and in some embodiments it is 10 μL.

[0044] The detection method described in this invention also includes a diluent, a system suitability solution, and a test solution.

[0045] In some embodiments, the diluent is mobile phase A: mobile phase B = 100:0~0:100, in some embodiments it is mobile phase A: mobile phase B = 50-80:20-50, in some embodiments it is mobile phase A: mobile phase B = 50:50, and in some embodiments it is mobile phase A: mobile phase B = 80:20.

[0046] In some embodiments, the system suitability solution includes brandylol hydrochloride and one or more impurities selected from impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, SM1, intermediate II, intermediate III, and impurity 1. In some embodiments, the concentration of brandylol hydrochloride in the system suitability solution is 0.5-1.5 mg / mL, and in some embodiments it is 1 mg / mL. The concentration of each impurity is independently selected from 0.5-1.5 μg / mL, and in some embodiments it is 1 μg / mL. In some embodiments, brandylol hydrochloride is dissolved in a volumetric flask with diluent, and the impurity reference standard stock solution is diluted in the same volumetric flask with diluent to a solution containing 0.5-1.5 mg of brandylol hydrochloride and each impurity concentration of 0.5-1.5 μg / mL per 1 mL of the test solution, which is then used as the system suitability solution.

[0047] In some embodiments, the concentration of the test solution is 0.5-1.5 mg / mL, and in some embodiments it is 1 mg / mL; in some embodiments, the test solution is a landiolol hydrochloride solution, and the solvent of the test solution is the diluent; in some embodiments, landiolol hydrochloride raw material or its preparation is placed in a volumetric flask, dissolved and diluted with diluent to a solution containing 0.5-1.5 mg of landiolol hydrochloride per 1 mL of test solution, which is then used as the test solution.

[0048] In some embodiments, the test solution, blank diluent, etc., are injected into the liquid chromatograph for gradient elution.

[0049] The present invention provides an efficient analytical method for the separation and determination of multiple related substances in brandylol hydrochloride. It employs a high-performance liquid chromatography (HPLC) instrument with phosphate buffer and acetonitrile as the mobile phase. By adjusting the gradient elution program of the mobile phase, and considering the combined effects of flow rate and column temperature on separation and detection, and by selecting a suitable chromatographic column and detection wavelength, the detection results are optimized. This method achieves rapid and efficient separation of brandylol hydrochloride and its multiple related substances under the same chromatographic conditions. It has the advantages of being fast, simple, economical, practical, highly sensitive, and having good separation, ensuring efficient monitoring of related substances and providing a guarantee for the safety and quality control of brandylol hydrochloride.

[0050] The advantages of the technical solution of this invention are:

[0051] The method for separating and determining related substances in brandylovolone hydrochloride using HPLC described in this invention optimizes the detection results by considering the combined effects of mobile phase, gradient elution program, flow rate, and column temperature on separation and detection. It can rapidly and efficiently separate impurities B, C, D, E, F, G, H, I, J, K, SM1, intermediate II, and intermediate III impurity 1 in brandylovolone hydrochloride under the same chromatographic conditions, ensuring efficient monitoring of related substances and guaranteeing the safety and quality controllability of brandylovolone hydrochloride. Attached Figure Description

[0052] Figure 1 This is a solution spectrum for the system suitability in Comparative Example 1 of the present invention.

[0053] Figure 2 This is a solution spectrum for the system suitability in Comparative Example 2 of the present invention.

[0054] Figure 3 This is a solution spectrum for system suitability in Example 1 of the present invention.

[0055] Figure 4 This is a solution spectrum for system suitability in Example 2 of the present invention.

[0056] Figure 5 This is a solution spectrum for system suitability in Example 3 of the present invention.

[0057] Figure 6 This is the spectrum of the blank solution in Example 4 of the present invention.

[0058] Figure 7 This is a solution spectrum for system suitability in Example 4 of the present invention.

[0059] Figure 8 This is the spectrum of the test solution-1 in Example 4 of the present invention.

[0060] Figure 9 The spectrum of the test solution-2 in Example 4 of this invention. Detailed Implementation

[0061] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0062] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0063] The specific embodiments used in this invention are used to illustrate the principles and implementation methods of the invention, and are only for the purpose of helping to understand the method and core ideas of the invention. The impurities in the hydrochloric acid brandylol included in this invention are not limited to the simultaneous detection of 13 impurities, but also include the detection of any number of impurity combinations included in this invention.

[0064] For reagents and instruments used in this invention whose manufacturers are not specified, conventional products can be obtained through commercial purchase.

[0065] The test sample of landiolol hydrochloride and intermediate II used in this invention were prepared by the inventors according to the prior art literature [Chem.Pharm.Bul1:40(6)1462-1469(1992)]. SM1 was purchased from Beijing Langruibang Technology Co., Ltd., and all impurity reference standards used were purchased from Shenzhen Moke Biochemical Technology Co., Ltd.

[0066] Unless otherwise specified, all mobile phases and diluents mentioned in the following examples are volume ratios.

[0067] Comparative Example 1

[0068] Chromatographic conditions:

[0069] Octadecylsilane-bonded silica gel (Gemini C18 4.6mm × 250mm, 5μm) was used as the packing material; 0.02mol / L potassium dihydrogen phosphate (pH approximately 4.6) was used as mobile phase A, and methanol was used as mobile phase B, with gradient elution performed according to the table below; the flow rate was 1.0mL per minute; the detection wavelength was 220nm; the column temperature was 25℃; and the injection volume was 10μL.

[0070] Table 6 Gradient elution program

[0071]

[0072] Solution preparation:

[0073] Diluent: Mobile phase A: Mobile phase B = 95:5.

[0074] Stock solution of impurity reference standards: Weigh approximately 10 mg each of impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, SM1, intermediate III, impurity 1 reference standard, and intermediate II. Place them in separate 100 mL volumetric flasks, dissolve and dilute to the mark with acetonitrile, and shake well.

[0075] System suitability solution: Take about 20 mg of brandylol hydrochloride, place it in a 20 mL volumetric flask, add diluent to dissolve it, measure 0.2 mL of each impurity reference stock solution into the same volumetric flask, add diluent to dilute to the mark, and shake well to obtain the solution.

[0076] Accurately measure the blank diluent and system suitability solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0077] See results Figure 1 The elution positions of each impurity were determined through localization. Under these chromatographic conditions, impurity C overlapped with impurity H (11.372 min), the adjacent impurity E (approximately 27.5 min) before the main peak showed peak splitting, impurity D (29.650 min) after the main peak did not reach baseline separation, and impurity J overlapped with intermediate II (retention time 42.422 min). These chromatographic conditions could not meet the separation requirements, and methanol was not suitable as mobile phase B.

[0078] Comparative Example 2

[0079] Chromatographic conditions:

[0080] Octadecylsilane-bonded silica gel (Gemini C18 4.6mm×250mm, 5μm) was used as the packing material; 0.02mol / L potassium dihydrogen phosphate aqueous solution-acetonitrile (50:50) was used as the mobile phase; the flow rate was 1.2mL per minute; the detection wavelength was 220nm; the column temperature was 25℃; and the injection volume was 10μl.

[0081] Solution preparation:

[0082] Stock solution of impurity reference standards: Weigh approximately 10 mg each of impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, SM1, intermediate III, impurity 1 reference standard, and intermediate II. Place them in separate 100 mL volumetric flasks, dissolve and dilute to the mark with mobile phase, and shake well.

[0083] System suitability solution: Take about 20 mg of brandylol hydrochloride, place it in a 20 mL volumetric flask, add mobile phase to dissolve it, measure 0.2 mL of each impurity reference standard stock solution into the same volumetric flask, add mobile phase to dilute to the mark, and shake well to obtain the solution.

[0084] Accurately measure the blank diluent (mobile phase) and system suitability solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0085] See results Figure 2 The retention time of the main peak of brandylol hydrochloride was 3.669 min, which is a poor retention behavior on the chromatographic column, and various impurities accumulated and could not be separated.

[0086] Example 1

[0087] Chromatographic conditions:

[0088] Octadecylsilane-bonded silica gel (Gemini C18 4.6mm × 250mm, 5μm) was used as the packing material; phosphate buffer (0.01mol / L potassium dihydrogen phosphate, pH adjusted to 3.0 with sodium hydroxide) was used as mobile phase A, and acetonitrile was used as mobile phase B, with gradient elution performed according to the table below; the flow rate was 1.2mL per minute; the detection wavelength was 210nm; the column temperature was 25℃; and the injection volume was 10μl.

[0089] Table 7 Gradient elution program

[0090]

[0091] Solution preparation:

[0092] Diluent: Mobile phase A: Mobile phase B = 50:50.

[0093] Stock solution of impurity reference standards: Weigh approximately 10 mg each of impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, SM1, intermediate III, impurity 1 reference standard, and intermediate II. Place them in separate 100 mL volumetric flasks, dissolve and dilute to the mark with diluent, and shake well.

[0094] System suitability solution: Take about 20 mg of brandylol hydrochloride, place it in a 20 mL volumetric flask, add diluent to dissolve it, measure 0.2 mL of each impurity reference stock solution into the same volumetric flask, add diluent to dilute to the mark, and shake well to obtain the solution.

[0095] Accurately measure the blank diluent and system suitability solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0096] See results Figure 3 The retention times of each chromatographic peak are shown in the table below. Baseline separation can be achieved for both brandylol hydrochloride and the impurities described in this invention.

[0097] Table 8 Retention time and resolution of brandylol hydrochloride and various impurities

[0098]

[0099] Example 2

[0100] Chromatographic conditions:

[0101] The column was packed with octadecylsilane-bonded silica gel (Welch Xtimate C18, 4.6 mm × 250 mm, 5 μm); phosphate buffer (0.05 mol / L potassium dihydrogen phosphate, pH adjusted to 6.0 with sodium hydroxide) was used as mobile phase A, and acetonitrile was used as mobile phase B. Gradient elution was performed according to the table below; the flow rate was 1.0 mL per minute; the detection wavelength was 230 nm; the column temperature was 25 °C; and the injection volume was 10 μl.

[0102] Table 9 Gradient elution program

[0103]

[0104] Solution preparation:

[0105] Diluent: Mobile phase A: Mobile phase B = 80:20.

[0106] Stock solution of impurity reference standards: Weigh approximately 10 mg each of impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, SM1, intermediate III, impurity 1 reference standard, and intermediate II. Place them in separate 100 mL volumetric flasks, dissolve and dilute to the mark with diluent, and shake well.

[0107] System suitability solution: Take about 20 mg of brandylol hydrochloride, place it in a 20 mL volumetric flask, add diluent to dissolve it, measure 0.2 mL of each impurity reference stock solution into the same volumetric flask, add diluent to dilute to the mark, and shake well to obtain the solution.

[0108] Accurately measure the blank diluent and system suitability solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0109] See results Figure 4 Impurities C, H, B, intermediate III, 1, E, brandylol hydrochloride, D, I, G, K, II, J, SM1, and F eluted in sequence, and the retention times of each chromatographic peak are shown in the table below.

[0110] Table 10 Retention time and resolution of brandylol hydrochloride and various impurities

[0111]

[0112] Example 3

[0113] Chromatographic conditions:

[0114] Octadecylsilane-bonded silica gel (Inertsil ODS-3V 4.6mm×250mm, 5μm) was used as the packing material; phosphate buffer (0.02mol / L potassium dihydrogen phosphate, pH adjusted to 7.0 with sodium hydroxide) was used as mobile phase A, and acetonitrile was used as mobile phase B, with gradient elution performed according to the table below; the flow rate was 1.0mL per minute; the detection wavelength was 210nm; the column temperature was 35℃; and the injection volume was 10μl.

[0115] Table 11 Gradient elution program

[0116]

[0117] Solution preparation:

[0118] Diluent: Mobile phase A: Mobile phase B = 50:50.

[0119] Stock solution of impurity reference standards: Weigh approximately 10 mg each of impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, SM1, intermediate III, impurity 1 reference standard, and intermediate II. Place them in separate 100 mL volumetric flasks, dissolve and dilute to the mark with diluent, and shake well.

[0120] System suitability solution: Take about 20 mg of brandylol hydrochloride, place it in a 20 mL volumetric flask, add diluent to dissolve it, measure 0.2 mL of each impurity reference stock solution into the same volumetric flask, add diluent to dilute to the mark, and shake well to obtain the solution.

[0121] Accurately measure the blank diluent and system suitability solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0122] See results Figure 5Impurities C, H, B, intermediate III, 1, E, brandylol hydrochloride, D, I, G, K, J, II, SM1, and F eluted in sequence, and the retention times of each chromatographic peak are shown in the table below.

[0123] Table 12 Retention time and resolution of brandylol hydrochloride and various impurities

[0124]

[0125] Example 4

[0126] Chromatographic conditions:

[0127] Octadecylsilane-bonded silica gel (Inertsil ODS-3V 4.6mm×250mm, 5μm) was used as the stationary phase. Phosphate buffer (2.72g potassium dihydrogen phosphate and 2.28g dipotassium hydrogen phosphate trihydrate, dissolved in water and diluted to 1000mL, pH approximately 6.0) was used as mobile phase A, and acetonitrile was used as mobile phase B. Gradient elution was performed according to the table below. The flow rate was 1.0mL / min; the detection wavelength was 220nm; the column temperature was 25℃; and the injection volume was 10μl.

[0128] Table 13 Gradient elution program

[0129]

[0130] Solution preparation:

[0131] Diluent: Mobile phase A: Mobile phase B = 50:50.

[0132] Stock solution of impurity reference standards: Weigh approximately 10 mg each of impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, SM1, intermediate III, impurity 1 reference standard, and intermediate II. Place them in separate 100 mL volumetric flasks, dissolve and dilute to the mark with diluent, and shake well.

[0133] System suitability solution: Take about 20 mg of brandylol hydrochloride, place it in a 20 mL volumetric flask, add diluent to dissolve it, measure 0.2 mL of each impurity reference stock solution into the same volumetric flask, add diluent to dilute to the mark, and shake well to obtain the solution.

[0134] Test solution: Take about 10 mg each of brandyl hydrochloride test sample-1 and test sample-2, place them in a 10 mL volumetric flask, add diluent to dissolve and dilute to the mark, and shake well to obtain the solution.

[0135] Accurately measure blank diluent, system suitability solution, test solution-1, and test solution-2, and inject them into the liquid chromatograph, respectively, and record the chromatograms.

[0136] See results Figure 6-7 Under these chromatographic conditions, the blank solution does not interfere with impurity detection; all known impurities elute, and the retention times of each peak are shown in the table below. The resolution between the main peak and adjacent impurity peaks is greater than 1.5, and baseline separation is achieved between all impurity peaks, making it suitable for the detection of related substances in brandylolol hydrochloride. This method is used to detect samples (see...). Figure 8-9 Both methods can effectively detect all known impurities in the sample, meeting the expected requirements.

[0137] Table 14 Retention time and resolution of brandylol hydrochloride and various impurities

[0138]

[0139] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for detecting related substances in brandylol hydrochloride, characterized in that, The detection method employs high-performance liquid chromatography (HPLC), using octadecylsilane-bonded silica gel as the column packing material; gradient elution is performed using phosphate buffer as mobile phase A and acetonitrile as mobile phase B; detection is performed using an ultraviolet detector with a detection wavelength of 210-230 nm. The related substances of the aforementioned brandylol hydrochloride include one or more of the following: impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, SM1, intermediate II, intermediate III, and impurity 1. The molecular structural formulas of each impurity are shown below. 。 2. The detection method according to claim 1, characterized in that, The diameter of the chromatographic column is 4.0-5.2 mm, optionally 4.3-4.9 mm, optionally 4.6 mm; optionally, the length of the chromatographic column is 230-270 mm, optionally 240-260 mm, optionally 250 mm; optionally, the particle size of the packing material is 4-6 μm, optionally 4.5-5.5 μm, optionally 5 μm.

3. The detection method according to claim 1, characterized in that, The phosphate buffer solution is a buffer solution prepared by mixing any one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate hydrate, and dipotassium hydrogen phosphate hydrate; optionally, the concentration of the phosphate buffer solution is 0.01-0.1 mol / L, optionally 0.05 mol / L or 0.08 mol / L; optionally, the pH of the phosphate buffer solution is 3-7.

4. The detection method according to claim 1, characterized in that, In the gradient elution process, the initial mobile phase is: mobile phase A is 88%-97%, mobile phase B is 3%-12%; the intermediate mobile phase is: mobile phase A is 38%-52%, mobile phase B is 48%-62%; and the final mobile phase is: mobile phase A is 88%-97%, mobile phase B is 3%-12%.

5. The detection method according to claim 1, characterized in that, In the gradient elution process, the initial mobile phase is: mobile phase A is 90%-95%, mobile phase B is 5%-10%; the intermediate mobile phase is: mobile phase A is 40%-50%, mobile phase B is 50%-60%; and the final mobile phase is: mobile phase A is 90%-95%, mobile phase B is 5%-10%.

6. The detection method according to claim 4 or 5, characterized in that, A third mobile phase can also be set between the initial mobile phase and the intermediate mobile phase: mobile phase A is 83%-87% and mobile phase B is 13%-17%; alternatively, mobile phase A is 85% and mobile phase B is 15%.

7. The detection method according to claim 1, characterized in that, The gradient elution procedure is any one of the following; or; or; or; 。 8. The detection method according to claim 1, characterized in that, The flow rate of the mobile phase is 0.8-1.2 mL / min, optionally 1.0 mL / min.

9. The detection method according to claim 1, characterized in that, The column temperature of the chromatographic column is 20-35℃, optionally 25℃.

10. The detection method according to claim 1, characterized in that, The detection wavelength is 220 nm; the injection volume is 8-12 μL, optionally 10 μL.

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