Method for separating and determining timolol intermediate and isomer impurities thereof

Abstract

The invention discloses a method for separating and determining a timolol intermediate and isomer impurities thereof, and belongs to the technical field of analytical chemistry. According to the method, normal phase chromatography is adopted for sample separation, and chromatographic conditions are as follows: a chromatographic column is a polysaccharide derivative normal phase coating type chiral chromatographic column, a mobile phase is a mixed solution of normal hexane and isopropanol, the flow velocity of the mobile phase is 0.5-1.5 mL / min, and the detection wavelength is 260-330 nm. According to the method, the timolol intermediate and enantiomer impurities thereof can be effectively separated by adopting a chiral chromatographic column taking a polysaccharide derivative as a filler and taking a mixed solution of normal hexane and isopropanol as a mobile phase. According to the method disclosed by the invention, the timolol intermediate and the isomer impurities thereof can be rapidly, effectively and accurately separated and detected, and the impurity control of the timolol enantiomer is advanced to the intermediate step, so that the risk of generating the timolol enantiomer impurities in the synthesis process is reduced, and the quality of timolol can be comprehensively and effectively controlled.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry, and particularly relates to a method for separating and determining timolol intermediates and their isomeric impurities. Background Art

[0002] S-timolol is a non-selective and potent β-blocker. It has no intrinsic sympathomimetic activity, no local anesthetic effect, and has a weak membrane-stabilizing effect. Its β-blocking effect is 5-10 times that of propranolol, and its inhibitory effect on the myocardium is milder than that of propranolol. After administration, it can slow down the heart rate, weaken myocardial contractility, and reduce myocardial oxygen consumption. Clinically, it is used to treat hypertension, angina pectoris, tachycardia, and glaucoma. Existing studies have found that its topical application also has a certain therapeutic effect on various types of hemangiomas.

[0003] Chiral isomers of drugs have different pharmacological and toxicological effects. Therefore, for chiral chemical drugs, unless there are sufficient pharmacological and toxicological studies to prove that the pharmacological and toxicological effects of compounds that are chiral isomers of each other are the same, the control of chiral isomer impurities is very important for the quality research of drugs. Based on the quality control concept of quality by design, the risk of chiral isomer control is advanced, and the chiral isomers of chiral drugs are controlled at the step where chirality is introduced. This requires the detection of the content of enantiomers in the intermediate generated in the step of introducing chirality.

[0004] In the synthesis process of S-timolol, the step of introducing chirality is as follows: generating the key intermediate (R)-4-[4-(oxiran-2-ylmethoxy)-1,2,5-thiadiazol-3-yl]morpholine.

[0005]

[0006] It can be seen from the structural formula of the S-timolol intermediate that this intermediate has an enantiomeric impurity, which participates in the synthesis reaction of the timolol compound and may produce the enantiomer of the timolol compound, thus affecting the quality of the timolol raw material and its preparation products. In order to ensure the safety of drug production, strict quality control is required during the production process.

[0007] The detection methods in the prior art are all for the separation and detection of timolol and its enantiomers. For example, the detection methods related to the enantiomeric purity of timolol in the current European Pharmacopoeia EP11.0 and the United States Pharmacopoeia USP2023; another example is that Chinese Patent CN111272944A discloses a method for analyzing and separating timolol and its optical isomers, using a reversed-phase chromatography method to separate the samples; among them, the separation column used for separating the samples is an amphoteric ion-exchange chiral column, and the mobile phase used is a reversed-phase mixed solvent. This method can simply, quickly and accurately analyze and separate timolol and its isomeric impurities. Another example is that Chinese Patent CN109991319A discloses a separation method and application of timolol compounds and / or related impurities of timolol maleate. A chromatographic column with silica gel surface-bonded with quinine derivatives as the filler is applied to high-performance liquid chromatography conditions to separate timolol compounds, timolol maleate compounds and related impurities of timolol maleate. This method uses a reversed-phase high-performance liquid system, which can effectively separate timolol compounds and related impurities of timolol maleate, with high accuracy and high sensitivity, and can be used for the quantitative detection of timolol compounds. However, the above methods are all for the separation and detection of timolol and its enantiomers. At this time, the synthesis reaction has ended, and the timolol products with unqualified quality have also been synthesized, which greatly wastes manpower and material resources. If the control of timolol enantiomeric impurities can be advanced to the intermediate step, and the chiral intermediate introduced in the production process is controlled, and qualified intermediates are screened for subsequent synthesis reactions, the risk of unqualified timolol enantiomers can be greatly reduced, and the content of enantiomeric impurities in the product can be more accurately controlled within the qualified range to ensure the quality control of timolol raw materials and their preparation products. And the existing reversed-phase chromatography method for separating timolol and its isomers cannot separate the timolol intermediate isomers.

[0008] Based on this, it is particularly important to provide a method for quickly separating and determining timolol intermediates and their enantiomeric impurities. Summary of the Invention

[0009] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a method for separating and determining timolol intermediates and their isomeric impurities. For the timolol intermediate, namely (R)-4-[4-(oxiran-2-ylmethoxy)-1,2,5-thiadiazol-3-yl]morpholine, an analytical method suitable for the determination of the content of this intermediate and its isomers is provided. The method of the present invention is simple and fast, can accurately separate and detect its content, and the resolution between the timolol intermediate and its enantiomer can reach 2.39 - 2.83, so as to realize the separation and determination of the timolol intermediate and its isomeric impurities.

[0010] The purpose of the present invention is achieved through the following technical solutions.

[0011] A method for separating and determining timolol intermediate and its isomeric impurities, which uses normal-phase chromatography for sample separation, and the chromatographic conditions are as follows: the chromatographic column is a polysaccharide derivative normal-phase coated chiral chromatographic column, the mobile phase is a mixed solution of n-hexane and isopropanol, the flow rate of the mobile phase is 0.5 - 1.5 mL / min, and the detection wavelength is 260 - 330 nm.

[0012] The present invention uses a chiral chromatographic column filled with a polysaccharide derivative as the stationary phase and a normal-phase mixed solvent as the mobile phase. Under normal-phase separation chromatographic conditions, the timolol intermediate can be effectively separated from its enantiomeric impurities, and the content of its isomers in the timolol intermediate can be accurately controlled, thereby accurately and effectively controlling the quality of timolol. The method of the present invention can quickly, effectively, and accurately separate and detect the timolol intermediate and its isomeric impurities, control the impurities of the timolol enantiomer in advance to the intermediate step, reduce the risk of generating timolol enantiomeric impurities in the synthesis process, and can comprehensively and effectively control the quality of timolol.

[0013] In the present invention, the polysaccharide derivative normal-phase coated chiral chromatographic column can be Y2 or OJ.

[0014] In the present invention, the column temperature of the polysaccharide derivative normal-phase coated chiral chromatographic column can be 20 - 30 °C, such as 20 °C, 25 °C, 30 °C, etc.

[0015] In the present invention, the volume ratio of n-hexane to isopropanol can be (50 - 70):(30 - 50).

[0016] In the present invention, the flow rate of the mobile phase can be 0.7 - 1.0 mL / min; for example, 0.8 mL / min.

[0017] In the present invention, the method specifically includes the following steps:

[0018] S1. Take an appropriate amount of timolol intermediate isomeric impurities, dissolve it in a solvent and dilute it to the mark, shake well to obtain a reference solution;

[0019] S2. Take an appropriate amount of timolol intermediate, dissolve it in a solvent and dilute it to the mark, shake well to obtain a test solution;

[0020] S3. Inject the reference solution and the test solution into a high-performance liquid chromatograph respectively, and complete the separation and determination of the timolol intermediate and its isomeric impurities according to the above chromatographic conditions.

[0021] In the present invention, the order of steps S1 and S2 can be reversed.

[0022] In the present invention, in steps S1 and S2, the solvent may be a mixed solution of n-hexane and isopropanol, or a mixed solution of ethanol and acetonitrile.

[0023] In the present invention, in step S2, the mass of timolol intermediate in each 1 mL of the test solution is 0.5 - 5 mg.

[0024] In the present invention, in step S3, the injection volume may be 2 - 20 μL, such as 10 μL, 20 μL.

[0025] In the present invention, the volume ratio of n-hexane to isopropanol is 60:40, and the volume ratio of ethanol to acetonitrile is 40:60.

[0026] Compared with the prior art, the advantages of the present invention are as follows:

[0027] (1) The present invention uses a chiral chromatographic column filled with a polysaccharide derivative and a mixed solution of n-hexane and isopropanol as the mobile phase, providing a method for separating and determining timolol intermediate and its isomeric impurities, and successfully realizing the separation and determination of timolol intermediate and its isomeric impurities.

[0028] (2) The present invention advances the control of the impurities of timolol enantiomers to the intermediate step, reducing the risk of generating timolol enantiomeric impurities in the synthesis process, and can comprehensively and effectively control the quality of timolol.

[0029] (3) The method of the present invention is rapid and accurate, the separation and determination time is within 40 min, and the resolution is 2.39 - 2.83. Description of the Drawings

[0030] Figure 1 It is the HPLC chromatogram of Example 1;

[0031] Figure 2 It is the HPLC chromatogram of Example 1;

[0032] Figure 3 It is the HPLC chromatogram of Example 2;

[0033] Figure 4 It is the HPLC chromatogram of Example 3. Detailed Embodiments

[0034] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0035] Example 1 of the Present Invention

[0036] The method for separating and determining timolol intermediate and its isomeric impurities provided in this embodiment is as follows:

[0037] 1. Instruments: Shimadzu LC-20A, and the model of the detector is SPD-M20A; an electronic analytical balance from Sartorius Scientific Instruments Co., Ltd., with the model of SECURA 125.

[0038] 2. High-performance liquid chromatography conditions:

[0039] Mobile phase: n-hexane: isopropanol = 65:35;

[0040] The chromatographic column is Y2, the inner diameter of the chromatographic column is 4.6 mm, the length is 250 mm, and the particle size of the packing is 5 μm;

[0041] The detection wavelength is 295 nm;

[0042] The column temperature is 25 °C;

[0043] The flow rate is 0.8 mL / min;

[0044] The injection volume is 10 μL.

[0045] 3. Specificity investigation:

[0046] 3.1 Respectively take about 10 mg of timolol intermediate and timolol intermediate isomeric reference substance, place them in a 10 mL volumetric flask, add the mobile phase to dissolve and dilute to the scale, shake well, and use it as the system suitability solution;

[0047] 3.2 Weigh accurately 10 mg of timolol intermediate, place it in a 10 mL volumetric flask, add the mobile phase to dissolve and dilute to the scale, shake well, and use it as the test solution;

[0048] 3.3 Inject the blank solvent (mobile phase), system suitability solution, and test solution into the high-performance liquid chromatograph respectively, and conduct tests under the high-performance liquid chromatography conditions in Section 2. The results are as follows:

[0049]

[0050] The results show that the blank solvent does not interfere with the detection under this chromatographic condition, the resolution of timolol intermediate and its isomeric impurities in the system suitability solution is greater than 2, and they can be completely separated. There is no interference in the content detection of timolol intermediate isomeric impurities in the test solution, and it can be accurately determined.

[0051] Example 2

[0052] 1. Instruments: Shimadzu LC-20A with a detector model of SPD-M20A; an electronic analytical balance from Sartorius Stedim Biotech GmbH, model SECURA 125.

[0053] 2. High-performance liquid chromatography conditions:

[0054] Mobile phase: n-hexane: isopropanol = 55:45;

[0055] The chromatographic column is Y2 with an inner diameter of 4.6 mm, a length of 250 mm, and a packing particle size of 5 μm;

[0056] Detection wavelength: 295 nm;

[0057] Column temperature: 20 °C;

[0058] Flow rate: 0.8 mL / min;

[0059] Solvent: ethanol: acetonitrile (v / v) = 40:60;

[0060] Injection volume: 10 μL.

[0061] 3. Recovery investigation:

[0062] 3.1 Weigh accurately 12.5 mg of timolol intermediate isomer reference substance, place it in a 25 mL volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and use it as the reference substance stock solution;

[0063] 3.2 Take 1 mL of the reference substance stock solution under item 3.1, place it in a 25 mL volumetric flask, add solvent to dilute to the mark, and use it as the reference substance solution;

[0064] 3.3 Weigh accurately 625 mg of timolol intermediate, place it in a 25 mL volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and use it as the background stock solution.

[0065] 3.4 Take 1 mL of the background stock solution under item 3.3, place it in a 25 mL volumetric flask, add solvent to dissolve and dilute to the mark, shake well and use it as the background solution.

[0066] 3.5 Pipette accurately 12 portions of 1 mL of the background stock solution, place them in 25 mL volumetric flasks respectively, then add 0.5 mL (3 portions), 0.8 mL (3 portions), 1.0 mL (3 portions), 1.2 mL (3 portions) of the reference substance stock solution respectively, add solvent to dilute to the mark as the recovery 1 solution, recovery 2 solution, recovery 3 solution, and recovery 4 solution.

[0067] 3.6 Inject the solvent (mobile phase), background solution, reference solution, and recovery solution into the high-performance liquid chromatograph, and conduct tests under the high-performance liquid chromatography conditions in Section 2. The results are as follows:

[0068]

[0069]

[0070] The results show that the recovery rate of timolol intermediate isomers ranges from 100.38% to 101.96% within different concentration levels, confirming that the method of the present invention has good accuracy.

[0071] Example 3

[0072] The method for separating and determining timolol intermediate and its isomeric impurities provided in this example is as follows:

[0073] 1. Instruments: Shimadzu LC-20A, and the detector model is SPD-M20A; an electronic analytical balance from Sartorius Scientific Instruments Co., Ltd., model SECURA 125.

[0074] 2. High-performance liquid chromatography conditions:

[0075] Mobile phase: n-hexane: isopropanol = 60:40;

[0076] The chromatographic column is Y2, the inner diameter of the chromatographic column is 4.6 mm, the length is 250 mm, and the packing particle size is 5 μm;

[0077] Detection wavelength is 295 nm;

[0078] Column temperature is 25 °C;

[0079] Flow rate is 0.7 mL / min;

[0080] Solvent: ethanol: acetonitrile (v / v) = 40:60;

[0081] Injection volume is 10 μL.

[0082] 3. Investigation of repeatability:

[0083] 3.1 Weigh accurately 20 mg of timolol intermediate isomer reference substance, place it in a 50 mL volumetric flask, add solvent to dissolve and dilute to the mark, and shake well; then accurately transfer 1 mL and place it in a 25 mL volumetric flask, and make up the volume with solvent to obtain the reference solution;

[0084] 3.2 Weigh accurately 10 mg of timolol intermediate, place it in a 10 mL volumetric flask, add solvent to dissolve and dilute to the mark, and shake well to obtain the test solution; prepare 6 parallel portions;

[0085] 3.3 Inject the solvent, reference solution, and test solution into the high performance liquid chromatograph respectively, and conduct the test under the high performance liquid chromatography conditions in Section 2. The results are as follows:

[0086]

[0087] The results show that the content of timolol intermediate isomer calculated by the external standard method is 0.672%, and the RSD of 6 samples is 1.41, indicating good repeatability of the method.

[0088] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for separating and determining timolol intermediates and their isomeric impurities, characterized in that, The method uses normal-phase chromatography for sample separation, and the chromatographic conditions are as follows: The chromatographic column is a polysaccharide derivative normal-phase coated chiral chromatographic column, the mobile phase is a mixed solution of n-hexane and isopropanol, the flow rate of the mobile phase is 0.5 - 1.5 mL / min, and the detection wavelength is 260 - 330 nm.

2. A method for separating and determining timolol intermediate and its isomeric impurities according to claim 1, characterized in that, The polysaccharide derivative normal-phase coated chiral chromatographic column is or 3. A method for separating and determining timolol intermediate and its isomeric impurities according to claim 1, characterized in that, The column temperature of the polysaccharide derivative normal-phase coated chiral chromatographic column is 20 - 30 °C.

4. A method for separating and determining timolol intermediates and their isomeric impurities according to claim 1, characterized in that, The volume ratio of n-hexane to isopropanol is (50 - 70):(30 - 50).

5. A method for separating and determining timolol intermediates and their isomeric impurities according to claim 1, characterized in that, The method specifically includes the following steps: S1. Take an appropriate amount of timolol intermediate isomer impurity, dissolve it in a solvent and dilute to the mark, shake well to obtain a reference solution; S2. Take an appropriate amount of timolol intermediate, dissolve it in a solvent and dilute to the mark, shake well to obtain a test solution; S3. Inject the reference solution and the test solution into a high-performance liquid chromatograph respectively, and complete the separation and determination of timolol intermediate and its isomer impurity according to the chromatographic conditions described in claim 1.

6. A method for separating and determining timolol intermediate and its isomeric impurities according to claim 5, characterized in that, In steps S1 and S2, the solvent is a mixed solution of n-hexane and isopropanol, or a mixed solution of ethanol and acetonitrile.

7. A method for separating and determining timolol intermediate and its isomeric impurities according to claim 5, characterized in that, In step S2, the mass of timolol intermediate in each 1 mL of the test solution is 0.5 - 5 mg.

8. A method for separating and determining timolol intermediate and its isomeric impurities according to claim 5, characterized in that, In step S3, the injection volume is 2 - 20 μL.

9. A method for separating and determining timolol intermediate and its isomeric impurities according to claim 6, characterized in that, The volume ratio of n-hexane to isopropanol is 60:40, and the volume ratio of ethanol to acetonitrile is 40:60.

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