A composition of bevanolol hydrochloride and a detection method for its isomeric impurities

Abstract

The present invention belongs to the field of pharmaceutical technology, and specifically relates to a composition of bevanolol hydrochloride and a detection method for isomeric impurities thereof. The present invention provides a composition of bevanolol hydrochloride, wherein the content of ortho-isomer impurities is not higher than 0.5%, the content of para-isomer impurities is not higher than 0.5%, and the content of ortho-isomer impurities or para-isomer impurities is not 0 at the same time. The detection method of isomeric impurities in the above-mentioned composition of the present invention utilizes high performance liquid chromatography, and can separate bevanolol hydrochloride and its isomeric impurities with a low-cost C18 column as a chromatographic column. And it has strong specificity, high sensitivity, good repeatability and good accuracy, can be used to effectively control the quality of bevanolol hydrochloride and its preparations, is conducive to ensuring the safety and effectiveness of medication, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and particularly relates to a bevantolol hydrochloride composition and a method for detecting isomer impurities thereof. Background Art

[0002] Bevantolol hydrochloride is a fat-soluble selective β1 receptor blocker that can block β receptors to slow down heart rate, reduce myocardial contractility and blood pressure, and has no intrinsic sympathomimetic activity. The drug has a weak membrane stabilizing effect and can dilate blood vessels, and is used to treat hypertension and angina pectoris.

[0003] The chemical name of Bevanolol Hydrochloride is: 1-[[2-(3,4-dimethoxyphenyl)ethyl]amino]-3-(3-methylphenoxy)-2-propanol hydrochloride, and its structure is shown below:

[0004]

[0005] Lu Tao, Tang Weifang, etc. reported the synthesis process of bevanolol hydrochloride in China Pharmaceutical Industry Magazine (Volume 25, No. 1, 1994), using meta-cresol as starting material, through etherification, aminolysis, salification, to obtain bevanolol hydrochloride. Meta-cresol is separated from mixed cresols. Since mixed cresols contain o-cresol, m-cresol and p-cresol, p-cresol and m-cresol boiling points are very close, it is difficult to separate by conventional distillation methods. Therefore, a small amount of o-cresol and p-cresol are inevitably contained in m-cresol. These two impurities will participate in the reaction, be transferred to bevanolol hydrochloride, and form ortho-isomer impurities and para-isomer impurities. Its structure is as follows:

[0006]

[0007] The isomeric impurities in bevanolol hydrochloride will seriously affect the purity and quality of the drug, and are one of the key factors in determining whether the drug is qualified. Therefore, achieving effective separation and detection of isomeric impurities in bevanolol hydrochloride is of great significance for controlling product quality.

[0008] Chinese patent application CN104678013A reports a method for analyzing and detecting isomeric impurities of bevanolol hydrochloride, using reversed phase liquid chromatography, and the chromatographic conditions are as follows: chromatographic column: pentafluorophenyl column; mobile phase composition: A (aqueous phase): B (methanol); mobile phase ratio: A:B = 55:45-45:55; mobile phase flow rate: 0.5-1.5ml / min; detection wavelength: 210-280nm; column temperature: 20-40℃; detector: ultraviolet detector or diode array detector. This method can quickly and accurately detect the isomeric impurity content of bevanolol hydrochloride. However, conventional chromatographic columns such as Kromasil C18 column (4.6×250mm, 5μm) used in the patent application cannot achieve a good separation effect between bevanolol and ortho-isomers and para-isomers. Therefore, it uses an expensive and short-lived pentafluorophenyl column, which increases the cost of detecting isomeric impurities of bevanolol hydrochloride. Summary of the invention

[0009] In view of the defects in the prior art, the present invention provides a composition of bevantolol hydrochloride and a method for detecting isomeric impurities thereof, the purpose of which is to detect the isomeric impurity content in bevantolol hydrochloride, so as to replace the high-cost pentafluorophenyl column with a low-cost C18 column in the method for detecting the isomeric impurities of bevantolol hydrochloride by high performance liquid chromatography.

[0010] A composition of bevanolol hydrochloride, wherein the content of ortho-isomer impurities is not higher than 0.5%, the content of para-isomer impurities is not higher than 0.5%, and the content of ortho-isomer impurities or para-isomer impurities is not 0 at the same time;

[0011] The ortho isomer impurity structure is:

[0012] The structure of the para-isomer impurity is:

[0013] Preferably, the content of ortho-isomer impurities is not higher than 0.1%, and the content of para-isomer impurities is not higher than 0.1%.

[0014] Preferably, its HPLC chromatogram is as follows Figures 1 to 6 Any one of the pictures shown;

[0015] The chromatographic conditions are as follows: the chromatographic column uses Inertsil ODS-3, the mobile phase consists of mobile phase A and mobile phase B, the mobile phase A is an aqueous solution of cyclodextrin and / or its derivatives, and the mobile phase B is methanol.

[0016] The present invention also provides a pharmaceutical composition, which consists of the above composition and pharmaceutically acceptable adjuvants or auxiliary components.

[0017] The present invention also provides a detection method for detecting isomeric impurities in the above composition, comprising the following steps: taking the composition and detecting it by high performance liquid chromatography;

[0018] The chromatographic conditions are as follows: the chromatographic column uses Inertsil ODS-3, the mobile phase consists of mobile phase A and mobile phase B, the mobile phase A is an aqueous solution of cyclodextrin and / or its derivatives, and the mobile phase B is methanol.

[0019] Preferably, the mass volume ratio of the cyclodextrin and / or its derivatives to water is 10-30:1 mg / ml.

[0020] Preferably, the cyclodextrin is β-cyclodextrin; and the cyclodextrin derivative is selected from at least one of hydroxypropyl-β-cyclodextrin, sulfonated-β-cyclodextrin or carboxymethyl-β-cyclodextrin.

[0021] Preferably, the mobile phase A is adjusted to a pH of 2.0 to 4.0, preferably 3.0, with phosphoric acid;

[0022] And / or, the mobile phase A contains a buffer, the buffer is selected from phosphate, and the concentration of the phosphate is 30-100 mM, preferably 50 mM;

[0023] And / or, the mobile phase A contains triethylamine, and the volume ratio of triethylamine to water is 1 to 3:100 ml / ml, preferably 2:100 ml / ml.

[0024] Preferably, the phosphate is at least one of diammonium hydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate and sodium dihydrogen phosphate, preferably ammonium dihydrogen phosphate.

[0025] Preferably, the chromatographic conditions also include: the volume ratio of the mobile phase A to the mobile phase B is 58-65:35-45; and / or the detection wavelength is 210-240 nm; and / or the flow rate is 0.8-1.2 ml / min; and / or the column temperature is 20-40° C.; and / or the injection volume is 10-20 μl.

[0026] The present invention proposes to detect the isomeric impurity content in bevanolol hydrochloride, thereby controlling the quality of bevanolol hydrochloride medicine. In the chromatographic detection of the present invention, the Inertsil ODS-3 used is a C18 column, that is, a chromatographic column with octadecylsilane bonded silica gel as filler. The C18 column is the most widely used in analytical work because of its low price, simple reverse phase system, good repeatability, and long-term stable operation of the column. In order to solve the problem that the C18 column cannot separate bevanolol hydrochloride and its isomeric impurities, the present invention optimizes the chromatographic conditions such as the composition of the mobile phase, and realizes the purpose of separating bevanolol hydrochloride and its isomeric impurities using Inertsil ODS-3. The present invention has strong specificity, high sensitivity, good repeatability, good accuracy, and avoids the use of an expensive and short-lived pentafluorophenyl column, effectively reducing the detection cost of the isomeric impurities of bevanolol hydrochloride.

[0027] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0028] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 HPLC chromatogram of the test sample in Example 1;

[0030] Figure 2 HPLC chromatogram of the test sample in Example 2;

[0031] Figure 3 HPLC chromatogram of the test sample in Example 3;

[0032] Figure 4 HPLC chromatogram of the test sample in Example 4;

[0033] Figure 5 HPLC chromatogram of the test sample in Example 5;

[0034] Figure 6 HPLC chromatogram of the test sample in Example 6;

[0035] Figure 7 High performance liquid chromatogram of the test sample in Comparative Example 1;

[0036] Figure 8 High performance liquid chromatogram of the test sample in Comparative Example 2;

[0037] Fig. 9 High performance liquid chromatogram of the test sample in Comparative Example 3;

[0038] Fig.10 High performance liquid chromatogram of the test sample in Comparative Example 4;

[0039] Fig.11 High performance liquid chromatogram of the test sample in Comparative Example 5;

[0040] Fig.12 High performance liquid chromatogram of the test sample in Comparative Example 6. DETAILED DESCRIPTION

[0041] The reagents and materials used in the following examples and comparative examples are all commercially available.

[0042] The basis for judging the separation degree in the present invention is as follows: "Pharmacopoeia of the People's Republic of China" (hereinafter referred to as Chinese Pharmacopoeia) 2020 edition of the four general rules 0512 "High Performance Liquid Chromatography" stipulates that the separation degree is used to evaluate the degree of separation between the substance to be tested and the separated substance, and is a key indicator for measuring the separation efficiency of the chromatographic system. The separation degree between the substance to be tested and the known impurities can be determined, or the separation degree between the substance to be tested and a certain indicator component (internal standard substance or other difficult-to-separate substance) can be determined, or the test sample or reference substance is degraded by an appropriate method, and the separation efficiency of the chromatographic system is evaluated and adjusted by determining the separation degree between the substance to be tested and a certain degradation product. Whether it is qualitative identification or quantitative determination, it is required that there is a good separation between the chromatographic peak of the substance to be tested and the chromatographic peak of the internal standard substance or the specific impurity control chromatographic peak and other chromatographic peaks. Unless otherwise specified, the separation between the chromatographic peak of the substance to be tested and the adjacent chromatographic peak should not be less than 1.5.

[0043] Example 1

[0044] Chromatographic conditions:

[0045] Instrument: Agilent 1260 High Performance Liquid Chromatograph.

[0046] Chromatographic column: Inertsil ODS-3, 4.6 mm × 250 mm × 5 μm.

[0047] Mobile phase: mobile phase A (1.6% β-cyclodextrin + 50 mM diammonium phosphate, pH adjusted to 3.0 with phosphoric acid): mobile phase B (methanol) = 58:42.

[0048] Injection volume: 10 μl.

[0049] Detection wavelength: 220nm.

[0050] Flow rate: 1.0ml / min.

[0051] Column temperature: 35°C.

[0052] Solution preparation and determination method:

[0053] Take an appropriate amount of bevanolol hydrochloride and its isomers, dissolve and dilute with 20% acetonitrile to make a solution containing 1 mg of bevanolol hydrochloride and 0.1 mg of ortho- and para-isomer impurities per 1 mL, shake well, and use it as the test solution. Inject and measure under the above conditions and record the chromatogram. The chromatogram of the test solution is shown in Figure 1 , the experimental data of the test products are shown in Table 1.

[0054] Table 1

[0055] Element Retention time [min] Peak height Peak area Peak area% Separation Para-isomer impurity 20.680 7.68861 220.20649 1.0823 / Bevanolol Hydrochloride 22.212 553.76998 19938.07854 97.9913 1.7 Ortho isomer impurity 24.654 5.31778 188.50250 0.9264 2.5 sum 20346.78753

[0056] Depend on Figure 1 As shown in Table 1, under the chromatographic conditions, the separation degree between bevanolol hydrochloride and the para-isomer impurity and the ortho-isomer impurity meets the requirements of the Chinese Pharmacopoeia.

[0057] Example 2

[0058] Chromatographic conditions:

[0059] Instrument: Agilent 1260 High Performance Liquid Chromatograph.

[0060] Chromatographic column: Inertsil ODS-3, 4.6 mm × 250 mm × 5 μm.

[0061] Mobile phase: mobile phase A (1.6% β-cyclodextrin + 2% triethylamine, pH adjusted to 3.0 with phosphoric acid): mobile phase B (methanol) = 60:40.

[0062] Injection volume: 10 μl.

[0063] Detection wavelength: 220nm.

[0064] Flow rate: 1.0ml / min.

[0065] Column temperature: 35°C.

[0066] Solution preparation and determination method:

[0067] Take an appropriate amount of bevanolol hydrochloride and its isomers, dissolve and dilute with 20% acetonitrile to make a solution containing 1 mg of bevanolol hydrochloride and 0.1 mg of ortho- and para-isomer impurities per 1 mL, shake well, and use it as the test solution. Inject and measure under the above conditions and record the chromatogram. The chromatogram of the test solution is shown in Figure 2 , the experimental data of the test products are shown in Table 2.

[0068] Table 2

[0069] Element Retention time [min] Peak height Peak area Peak area% Separation Para-isomer impurity 23.433 6.67514 227.66654 1.1259 Bevanolol Hydrochloride 25.627 517.92360 19805.39949 97.9460 2.3 Ortho isomer impurity 28.499 4.68190 187.65833 0.9280 2.8 sum 20220.72436

[0070] Depend on Figure 2 As shown in Table 2, under the chromatographic conditions, the separation degree of bevanolol hydrochloride from the para-isomer impurity and the ortho-isomer impurity meets the requirements of the Chinese Pharmacopoeia.

[0071] Example 3

[0072] Chromatographic conditions:

[0073] Instrument: Agilent 1260 High Performance Liquid Chromatograph.

[0074] Chromatographic column: Inertsil ODS-3, 4.6 mm × 250 mm × 5 μm.

[0075] Mobile phase: mobile phase A (1.6% β-cyclodextrin + 2% triethylamine, pH adjusted to 3.0 with phosphoric acid): mobile phase B (methanol) = 65:35.

[0076] Injection volume: 10 μl.

[0077] Detection wavelength: 220nm.

[0078] Flow rate: 1.0ml / min.

[0079] Column temperature: 35°C.

[0080] Solution preparation and determination method:

[0081] Take an appropriate amount of bevanolol hydrochloride and its isomers, dissolve and dilute with 20% acetonitrile to make a solution containing 1 mg of bevanolol hydrochloride and 0.1 mg of ortho- and para-isomer impurities per 1 mL, shake well, and use it as the test solution. Inject and measure under the above conditions and record the chromatogram. The chromatogram of the test solution is shown in Figure 3 , the experimental data of the test products are shown in Table 3.

[0082] Table 3

[0083] Element Retention time [min] Peak height Peak area Peak area% Separation Para-isomer impurity 39.533 4.00095 218.81390 1.0821 Bevanolol Hydrochloride 44.528 306.83698 19794.87835 97.8907 3.1 Ortho isomer impurity 50.598 2.93750 207.71514 1.0272 3.4 sum 20221.40739

[0084] Depend on Figure 3 As shown in Table 3, under the chromatographic conditions, the separation degree of bevanolol hydrochloride from the para-isomer impurity and the ortho-isomer impurity meets the requirements of the Chinese Pharmacopoeia.

[0085] Example 4

[0086] Chromatographic conditions:

[0087] Instrument: Agilent 1260 High Performance Liquid Chromatograph.

[0088] Chromatographic column: Inertsil ODS-3, 4.6 mm × 250 mm × 5 μm.

[0089] Mobile phase: mobile phase A (3.0% hydroxypropyl β-cyclodextrin + 2% triethylamine, pH adjusted to 3.0 with phosphoric acid): mobile phase B (methanol) = 64:36.

[0090] Injection volume: 10 μl.

[0091] Detection wavelength: 220nm.

[0092] Flow rate: 1.0ml / min.

[0093] Column temperature: 35°C.

[0094] Solution preparation and determination method:

[0095] Take an appropriate amount of bevanolol hydrochloride and its isomers, dissolve and dilute with 20% acetonitrile to make a solution containing 1 mg of bevanolol hydrochloride and 0.1 mg of ortho- and para-isomer impurities per 1 mL, shake well, and use it as the test solution. Inject and measure under the above conditions and record the chromatogram. The chromatogram of the test solution is shown in Figure 4 , the experimental data of the test products are shown in Table 4.

[0096] Table 4

[0097] Element Retention time [min] Peak height Peak area Peak area% Separation Para-isomer impurity 24.471 10.29405 390.39764 1.1855 / Bevanolol Hydrochloride 26.260 657.44317 32223.63628 97.8532 1.5 Ortho isomer impurity 29.253 6.78937 316.54583 0.9613 2.3 sum 32930.57976

[0098] Depend on Figure 4 As shown in Table 4, under the chromatographic conditions, the separation degree of bevanolol hydrochloride from the para-isomer impurity and the ortho-isomer impurity meets the requirements of the Chinese Pharmacopoeia.

[0099] Example 5

[0100] Chromatographic conditions:

[0101] Instrument: Agilent 1260 High Performance Liquid Chromatograph.

[0102] Chromatographic column: Inertsil ODS-3, 4.6 mm × 250 mm × 5 μm.

[0103] Mobile phase: mobile phase A (1% β-cyclodextrin + 1% hydroxypropyl β-cyclodextrin + 2% triethylamine, pH adjusted to 3.0 with phosphoric acid): mobile phase B (methanol) = 60:40.

[0104] Injection volume: 20μl.

[0105] Detection wavelength: 220nm.

[0106] Flow rate: 1.0ml / min.

[0107] Column temperature: 35°C.

[0108] Solution preparation and determination method:

[0109] Take an appropriate amount of bevanolol hydrochloride and its isomers, dissolve and dilute with 20% acetonitrile to make a solution containing 1 mg of bevanolol hydrochloride and 0.1 mg of ortho- and para-isomer impurities per 1 mL, shake well, and use it as the test solution. Inject and measure under the above conditions and record the chromatogram. The chromatogram of the test solution is shown in Figure 5 , the experimental data of the test products are shown in Table 5.

[0110] Table 5

[0111] Element Retention time [min] Peak height Peak area Peak area% Separation Para-isomer impurity 20.270 13.61703 384.54113 1.1747 / Bevanolol Hydrochloride 21.618 864.87064 32032.63512 97.8526 1.5 Ortho-isomer impurities 24.010 8.93279 318.42384 0.9727 2.4 sum 32735.60009

[0112] Depend on Figure 5 As shown in Table 5, under the chromatographic conditions, the separation degree of bevanolol hydrochloride from the para-isomer impurity and the ortho-isomer impurity meets the requirements of the Chinese Pharmacopoeia.

[0113] Example 6

[0114] Chromatographic conditions:

[0115] Instrument: Agilent 1260 High Performance Liquid Chromatograph.

[0116] Chromatographic column: Inertsil ODS-3, 4.6 mm × 250 mm × 5 μm.

[0117] Mobile phase: mobile phase A (1% β-cyclodextrin + 1% hydroxypropyl β-cyclodextrin + 2% triethylamine, pH adjusted to 3.0 with phosphoric acid): mobile phase B (methanol) = 61:39.

[0118] Injection volume: 10 μl.

[0119] Detection wavelength: 230nm.

[0120] Flow rate: 1.0ml / min.

[0121] Column temperature: 35°C.

[0122] Diluent: acetonitrile-water (20:80, v / v)

[0123] Solution preparation and determination method:

[0124] Take an appropriate amount of bevanolol hydrochloride and its isomers, dissolve and dilute with 20% acetonitrile to make a solution containing 1 mg of bevanolol hydrochloride and 1 μg of ortho- and para-isomer impurities per 1 mL, shake well, and use it as the test solution. Inject and measure under the above conditions and record the chromatogram. The chromatogram of the test solution is shown in Figure 6 , the experimental data of the test products are shown in Table 6.

[0125] Table 6

[0126] Element Retention time [min] Peak height Peak area Peak area% Separation Para-isomer impurity 22.977 0.81662 28.09563 0.1725 / Bevanolol Hydrochloride 24.861 418.08394 16245.85959 99.7288 1.9 Ortho isomer impurity 27.466 0.41116 16.07599 0.0987 2.5 sum 16290.03121

[0127] Depend on Figure 6 As shown in Table 6, under the chromatographic conditions, the separation degree between bevanolol hydrochloride and the para-isomer impurity and the ortho-isomer impurity meets the requirements of the Chinese Pharmacopoeia.

[0128] Comparative Example 1

[0129] Chromatographic conditions:

[0130] Instrument: Agilent 1260 High Performance Liquid Chromatograph.

[0131] Chromatographic column: Sepax GP-Phenyl, 4.6 mm × 250 mm × 5 μm.

[0132] Mobile phase: mobile phase A (0.8% β-cyclodextrin + 2% triethylamine, pH adjusted to 3.0 with phosphoric acid): mobile phase B (methanol) = 53:47.

[0133] Injection volume: 10 μl.

[0134] Detection wavelength: 220nm.

[0135] Flow rate: 1.0ml / min.

[0136] Column temperature: 35°C.

[0137] Solution preparation and determination method:

[0138] Take an appropriate amount of bevanolol hydrochloride and ortho- and para-isomer impurities, dissolve and dilute with 20% acetonitrile to make a solution containing 1 mg of bevanolol hydrochloride and 0.1 mg of ortho- and para-isomer impurities per 1 mL, shake well, and use it as the test solution. Inject and measure under the above conditions and record the chromatogram. The chromatogram of the test solution is shown in Figure 7 , the experimental data of the test products are shown in Table 7.

[0139] Table 7

[0140]

[0141] Depend on Figure 7 As shown in Table 7, under the chromatographic conditions, the chromatographic peaks of bevanolol hydrochloride, ortho-isomer impurities and para-isomer impurities completely overlap, and have severe tailing and cannot be separated.

[0142] Comparative Example 2

[0143] Chromatographic conditions:

[0144] Instrument: Agilent 1260 High Performance Liquid Chromatograph.

[0145] Chromatographic column: Hypersil silica, 4.6 mm × 250 mm × 5 μm.

[0146] Mobile phase: mobile phase A (1.6% β-cyclodextrin + 50 mM diammonium phosphate, pH adjusted to 6.5 with phosphoric acid): mobile phase B (methanol) = 52:48.

[0147] Injection volume: 10 μl.

[0148] Detection wavelength: 220nm.

[0149] Flow rate: 1.0ml / min.

[0150] Column temperature: 35°C.

[0151] Solution preparation and determination method:

[0152] Take an appropriate amount of bevanolol hydrochloride and its isomers, dissolve and dilute with 20% acetonitrile to make a solution containing 1 mg of bevanolol hydrochloride and 0.1 mg of ortho- and para-isomer impurities per 1 mL, shake well, and use it as the test solution. Inject and measure under the above conditions and record the chromatogram. The chromatogram of the test solution is shown in Figure 8 , the experimental data of the test products are shown in Table 8.

[0153] Table 8

[0154]

[0155] Depend on Figure 8 As shown in Table 8, under the chromatographic conditions, the chromatographic peaks of bevanolol hydrochloride, ortho-isomer impurity and para-isomer impurity completely overlap and cannot be separated.

[0156] Comparative Example 3

[0157] Chromatographic conditions:

[0158] Instrument: Agilent 1260 High Performance Liquid Chromatograph.

[0159] Chromatographic column: Inertsil ODS-3, 4.6 mm × 250 mm × 5 μm.

[0160] Mobile phase: mobile phase A (1.6% β-cyclodextrin + 50 mM diammonium phosphate, pH adjusted to 6.5 with phosphoric acid): mobile phase B (methanol) = 55:45.

[0161] Injection volume: 10 μl.

[0162] Detection wavelength: 220nm.

[0163] Flow rate: 1.0ml / min.

[0164] Column temperature: 35°C.

[0165] Solution preparation and determination method:

[0166] Take an appropriate amount of bevanolol hydrochloride and its isomers, dissolve and dilute with 20% acetonitrile to make a solution containing 1 mg of bevanolol hydrochloride and 0.1 mg of ortho- and para-isomer impurities per 1 mL, shake well, and use it as the test solution. Inject and measure under the above conditions and record the chromatogram. The chromatogram of the test solution is shown in Fig. 9 , the experimental data of the test products are shown in Table 9.

[0167] Table 9

[0168] Element Retention time [min] Peak height Peak area Peak area% Separation Para-isomer impurity 38.896 18.94292 959.85273 8.3102 / Bevanolol Hydrochloride 40.530 157.34099 9735.47238 84.2881 1.1 Ortho isomer impurity 45.635 12.75790 854.90957 7.4017 3.1 sum 11550.23469

[0169] Depend on Fig. 9 As can be seen from Table 9, under the chromatographic conditions, the para-isomer impurity, bevanolol hydrochloride and the ortho-isomer impurity peak in sequence, among which the separation degree between bevanolol hydrochloride and the ortho-isomer impurity is 3.1, which meets the requirements of the Chinese Pharmacopoeia; the separation degree between bevanolol hydrochloride and the para-isomer impurity is 1.1, which does not meet the requirements of the Chinese Pharmacopoeia.

[0170] Comparative Example 4

[0171] Chromatographic conditions:

[0172] Instrument: Agilent 1260 High Performance Liquid Chromatograph.

[0173] Chromatographic column: Inertsil ODS-3, 4.6 mm × 250 mm × 5 μm.

[0174] Mobile phase: mobile phase A (1.4% β-cyclodextrin + 150 mM ammonium acetate, pH adjusted to 4.0 with acetic acid): mobile phase B (methanol) = 60:40.

[0175] Injection volume: 10 μl.

[0176] Detection wavelength: 220nm.

[0177] Flow rate: 1.0ml / min.

[0178] Column temperature: 35°C.

[0179] Solution preparation and determination method:

[0180] Take an appropriate amount of bevanolol hydrochloride and its isomers, dissolve and dilute with 20% acetonitrile to make a solution containing 1 mg of bevanolol hydrochloride and 0.1 mg of ortho- and para-isomer impurities per 1 mL, shake well, and use it as the test solution. Inject and measure under the above conditions and record the chromatogram. The chromatogram of the test solution is shown in Fig.10 , the experimental data of the test products are shown in Table 10.

[0181] Table 10

[0182]

[0183] Depend on Fig.10 As shown in Table 10, under the chromatographic conditions, the baseline noise is relatively large, and the chromatographic peaks of bevanolol hydrochloride, ortho-isomer impurities and para-isomer impurities completely overlap and cannot be separated.

[0184] Comparative Example 5

[0185] Chromatographic conditions:

[0186] Instrument: Agilent 1260 High Performance Liquid Chromatograph.

[0187] Chromatographic column: Inertsil ODS-3, 4.6 mm × 250 mm × 5 μm.

[0188] Mobile phase: mobile phase A (0.5% sulfobutyl β-cyclodextrin sodium salt + 2% triethylamine, pH adjusted to 3.0 with phosphoric acid): mobile phase B (methanol) = 60:40.

[0189] Injection volume: 10 μl.

[0190] Detection wavelength: 220nm.

[0191] Flow rate: 1.0ml / min.

[0192] Column temperature: 35°C.

[0193] Solution preparation and determination method:

[0194] Take an appropriate amount of bevanolol hydrochloride and its isomers, dissolve and dilute with 20% acetonitrile to make a solution containing 1 mg of bevanolol hydrochloride and 0.1 mg of ortho- and para-isomer impurities per 1 ml, shake well, and use it as the test solution. Inject and measure under the above conditions and record the chromatogram. The chromatogram of the test solution is shown in Fig.11 , the experimental data of the test products are shown in Table 11.

[0195] Table 11

[0196] Element Retention time [min] Peak height Peak area Peak area% Separation Para-isomer impurity 19.540 12.94724 414.42310 1.2207 / Bevanolol Hydrochloride 21.614 930.01868 33207.11356 97.8094 2.3 Ortho isomer impurity 22.903 10.10569 329.29541 0.9699 1.4 sum 33950.83208

[0197] Depend on Fig.11 As can be seen from Table 11, the separation degree between bevanolol hydrochloride and the para-isomer impurity is 1.4, which does not meet the requirements of the Chinese Pharmacopoeia; the separation degree between bevanolol hydrochloride and the ortho-isomer impurity is 2.3, which meets the requirements of the Chinese Pharmacopoeia.

[0198] Comparative Example 6

[0199] Chromatographic conditions:

[0200] Instrument: Agilent 1260 High Performance Liquid Chromatograph.

[0201] Chromatographic column: Inertsil ODS-3, 4.6 mm × 250 mm × 5 μm.

[0202] Mobile phase: mobile phase A (0.8% β-cyclodextrin + 0.8% hydroxypropyl β-cyclodextrin + 2% triethylamine, pH adjusted to 3.0 with phosphoric acid): mobile phase B (methanol) = 60:40.

[0203] Injection volume: 20μl.

[0204] Detection wavelength: 220nm.

[0205] Flow rate: 1.0ml / min.

[0206] Column temperature: 35°C.

[0207] Solution preparation and determination method:

[0208] Take an appropriate amount of bevanolol hydrochloride and its isomers, dissolve and dilute with 20% acetonitrile to make a solution containing 1 mg of bevanolol hydrochloride and 0.1 mg of ortho- and para-isomer impurities per 1 mL, shake well, and use it as the test solution. Inject and measure under the above conditions and record the chromatogram. The chromatogram of the test solution is shown in Fig.12 , the experimental data of the test products are shown in Table 12.

[0209] Table 12

[0210] Element Retention time [min] Peak height Peak area Peak area% Separation Para-isomer impurity 21.330 13.34273 392.55467 1.2193 / Bevanolol Hydrochloride 22.449 823.84983 31481.52619 97.7837 1.2 Ortho isomer impurity 24.776 8.61634 320.99635 0.9970 2.3 sum 32195.07721

[0211] Depend on Fig.12 As shown in Table 12, under the chromatographic conditions, the separation degree of bevanolol hydrochloride and the para-isomer impurity is 1.2, which does not meet the requirements of the Chinese Pharmacopoeia; the separation degree of bevanolol hydrochloride and the ortho-isomer impurity is 2.3, which meets the requirements of the Chinese Pharmacopoeia.

[0212] It can be seen from the above embodiments and experimental examples that when the Inertsil ODS-3 chromatographic column is used, under chromatographic conditions such as the preferred mobile phase, effective separation of bevanolol hydrochloride and isomeric impurities can be achieved.

[0213] The chromatographic conditions in Example 6 are methodologically verified below to further demonstrate the technical effect of the present invention.

[0214] Experimental Example 1 Methodological Verification

[0215] Chromatographic conditions:

[0216] Instrument: Agilent 1260 High Performance Liquid Chromatograph.

[0217] Chromatographic column: Inertsil ODS-3, 4.6 mm × 250 mm × 5 μm.

[0218] Mobile phase: mobile phase A (1% β-cyclodextrin + 1% hydroxypropyl β-cyclodextrin + 2% triethylamine, pH adjusted to 3.0 with phosphoric acid): mobile phase B (methanol) = 61:39.

[0219] Injection volume: 10 μL.

[0220] Detection wavelength: 230nm.

[0221] Flow rate: 1.0mL / min.

[0222] Column temperature: 35°C.

[0223] Diluent: acetonitrile-water (20:80, v / v)

[0224] Solution preparation:

[0225] Blank: diluent.

[0226] Reference substance stock solution: Weigh approximately 25 mg each of the ortho-isomer reference substance of bevantolol hydrochloride and the para-isomer reference substance of bevantolol hydrochloride, place them in the same 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain (1 mg / ml).

[0227] Dilution solution of reference substance: accurately pipette 2 ml of reference substance stock solution into a 20 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain (100 μg / ml).

[0228] Reference substance solution (STD): Accurately pipette 1 ml of reference substance dilution solution into a 100 ml volumetric flask, dilute to the mark with diluent, shake well, and obtain (1 μg / ml).

[0229] Quality control reference stock solution: Weigh approximately 25 mg each of the ortho-isomer of bevantolol hydrochloride and the para-isomer of bevantolol hydrochloride, place in the same 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain (1 mg / ml).

[0230] Quality control reference substance dilution solution: Accurately pipette 2 ml of quality control reference substance stock solution into a 20 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain (100 μg / ml).

[0231] Quality control reference solution (CHK): Accurately pipette 1 ml of reference diluted solution into a 100 ml volumetric flask, dilute to the mark with diluent, shake well, and obtain (1 μg / ml).

[0232] Sample solution: Weigh about 25 mg of bevanolol hydrochloride reference substance, place it in a 25 ml volumetric flask, dissolve and dilute to the scale with the reference substance solution, shake well, and obtain.

[0233] Test solution: Weigh about 25 mg of bevantolol hydrochloride sample, place it in a 25 ml volumetric flask, dissolve it with diluent and dilute it to the scale, shake well, and obtain (1 mg / ml; in duplicate).

[0234] 1. System applicability

[0235] Take the sample solution, STD and CHK solution, inject them into the chromatographic system according to the above chromatographic conditions, and record the chromatogram. The minimum separation between the ortho-isomer and the para-isomer and the adjacent peaks in the system suitability solution should be greater than 1.5; the signal-to-noise ratio of the ortho-isomer and the para-isomer peaks in the reference solution should be no less than 20 (based on the first injection), the relative standard deviation (RSD) of the peak area of ​​the ortho-isomer and the para-isomer of the STD solution for 6 consecutive injections should be less than 10.0%, and the recovery rate of the ortho-isomer and the para-isomer in the CHK solution should be between 90% and 110%.

[0236] The measurement results are shown in Table 13.

[0237] Table 13 System suitability test results

[0238]

[0239]

[0240] From the results in Table 13, it can be seen that in the system suitability determination, the minimum separation between the para-isomer and the ortho-isomer and the adjacent peaks is 1.9; the signal-to-noise ratio of the para-isomer peak in the reference solution is 50.5, and the signal-to-noise ratio of the ortho-isomer peak is 35.5; the RSD of the peak area of ​​the para-isomer in 6 consecutive injections of STD solution is 2.6%, and the RSD of the peak area of ​​the ortho-isomer is 5.8%; the recovery rate of the ortho-isomer in the CHK solution is 108.3%, and the recovery rate of the para-isomer is 96.2%, both of which meet the requirements.

[0241] 2. Durability

[0242] 1) Solution stability

[0243] The changes of the reference solution and the added sample solution over time were examined. At room temperature, the samples were injected and measured at a certain time to examine the stability of the solutions. The RSD of the peak area of ​​the positional isomers in the reference solution at different times was required to be no greater than 10.0%. If the relative error of the peak area of ​​the positional isomers in the added sample solution compared with the initial peak area does not exceed ±10.0%, the sample solution does not need to be newly prepared before use. Otherwise, the sample solution needs to be newly prepared before use.

[0244] Results: After the reference solution was placed for 83 hours, the RSD of the peak area of ​​the ortho isomer was 5.2%, and the RSD of the peak area of ​​the para isomer was 3.1%; after the added solution was placed for 83 hours, the relative error of the peak area of ​​the ortho isomer was -1.83%, and the relative error of the peak area of ​​the para isomer was -3.83%, indicating that the control solution and the added solution were stable within 83 hours at room temperature.

[0245] 2) Changes in chromatographic conditions

[0246] The tolerance of chromatographic conditions is evaluated by changing the flow rate ±0.1ml / min, column temperature ±2℃, and wavelength ±2nm. It is required that the relative error of the impurity content of the sample solution under each changed chromatographic parameter condition compared with that under normal conditions should not exceed ±20%.

[0247] result:

[0248] When the chromatographic conditions changed slightly (flow rate ±0.1 ml / min, column temperature ±2°C, wavelength ±2 nm), the maximum relative error of the ortho-isomer content in the sample solution was -18.6%, and the maximum relative error of the para-isomer content was -7.3%, both meeting the requirements.

[0249] 3. Linearity and range

[0250] Weigh an appropriate amount of bevantolol hydrochloride ortho-isomer reference substance and bevantolol hydrochloride para-isomer reference substance, add a diluent to dissolve and dilute to prepare a series of concentrations of linear test solutions, plot the measured response signal Y against the concentration X of the analyte, and then perform linear regression using the least squares method. The value of the correlation coefficient (r) should be no less than 0.995.

[0251] Results: The linear equation of the para-isomer is y=18.9780x-0.4227, and the correlation coefficient r is 0.9998, which meets the requirements. The linear equation of the ortho-isomer is y=15.4006x-0.1970, and the correlation coefficient r is 0.9971, which meets the requirements.

[0252] 4. Accuracy

[0253] The accuracy is obtained by preparing the limit of quantification (LOQ), 100%, and 120% three different concentrations of accuracy solutions and measuring the recovery rate. It is achieved by measuring the recovery rate between the determination results of ortho-isomers and para-isomers at different concentrations and the theoretical value. It is required that the recovery rate of ortho-isomers and para-isomers should be within the range of 70% to 130% at the LOQ concentration level, and the recovery rate should be within the range of 80% to 120% at the other concentration levels to confirm that the method has good accuracy.

[0254] Results: The recoveries of the ortho isomer and para isomer at the LOQ concentration level were 94.9% and 97.2%, respectively; the recoveries of the ortho isomer and para isomer at the 100% concentration level were 100.6% and 99.0%, respectively; the recoveries of the ortho isomer and para isomer at the 120% concentration level were 96.1% and 95.4%, respectively, all meeting the requirements.

[0255] 5. Repeatability

[0256] Repeatability is obtained by preparing 6 sample spike solutions and measuring the recoveries. The RSD of the recoveries of the ortho-isomer and the para-isomer in the 6 sample spike solutions should not be greater than 10.0% to verify that the method has good precision.

[0257] Results: The RSDs (n=6) of the recoveries of the ortho-isomer and para-isomer were 3.9% and 3.6%, respectively, which met the requirements.

[0258] From the above embodiments and experimental examples, it can be known that the detection method of the present invention utilizes high performance liquid chromatography, and can separate bevanolol hydrochloride and its isomeric impurities using a low-cost C18 column as a chromatographic column. And by verifying the system applicability, accuracy, repeatability, linearity, range, and durability of the detection method of the present invention, the results show that all items meet the requirements, and the above detection method provided by the present invention can be used for the determination of ortho-isomers and para-isomers in bevanolol hydrochloride, thereby effectively controlling the quality of bevanolol hydrochloride and its preparations, which is conducive to ensuring the safety and effectiveness of medication.

Claims

1. A method for detecting isomeric impurities in a composition of bevanolol hydrochloride, characterized in that: The method comprises the following steps: taking the composition and detecting it by high performance liquid chromatography; The chromatographic conditions are as follows: the chromatographic column uses Inertsil ODS-3, the mobile phase consists of mobile phase A and mobile phase B, mobile phase A is an aqueous solution of cyclodextrin and / or its derivatives, and mobile phase B is methanol; The mass volume ratio of the cyclodextrin and / or its derivatives to water is 16-30:1 mg / ml; The mobile phase A is adjusted to pH 2.0-4.0 with phosphoric acid; The volume ratio of the mobile phase A to the mobile phase B is 58-65:35-42; The detection wavelength is 210~240nm; the flow rate is 0.8~1.2 ml / min; the column temperature is 20~40℃; the injection volume is 10~20μl; The isomeric impurities are ortho-isomer impurities and / or para-isomer impurities, and the ortho-isomer impurity structure is: ; The para-isomer impurity structure is: .

2. The detection method according to claim 1, characterized in that: The content of ortho isomer impurities in the composition is not higher than 0.5%, the content of para isomer impurities is not higher than 0.5%, and the content of ortho isomer impurities or para isomer impurities is not 0 at the same time.

3. The detection method according to claim 2, characterized in that: The content of ortho-isomer impurity is not higher than 0.1%, and the content of para-isomer impurity is not higher than 0.1%.

4. The detection method according to claim 1, characterized in that: The cyclodextrin is β-cyclodextrin; the cyclodextrin derivative is selected from at least one of hydroxypropyl-β-cyclodextrin, sulfonated-β-cyclodextrin or carboxymethyl-β-cyclodextrin.

5. The detection method according to claim 1, characterized in that: The mobile phase A is adjusted to pH 3.0 with phosphoric acid; And / or, the mobile phase A contains a buffer, the buffer is selected from phosphate, and the concentration of the phosphate is 30-100 mM; And / or, the mobile phase A contains triethylamine, and the volume ratio of triethylamine to water is 1-3:100 ml / ml.

6. The detection method according to claim 5, characterized in that: The concentration of the phosphate is 50 mM; And / or, the volume ratio of triethylamine to water is 2:100 ml / ml.

7. The detection method according to claim 5, characterized in that: The phosphate is at least one of diammonium hydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate and sodium dihydrogen phosphate.

8. The detection method according to claim 7, characterized in that: The phosphate is diammonium phosphate.

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