Determination method of clarithromycin methylate related substances

Abstract

The invention relates to the field of pharmaceutical analysis, in particular to a determination method of clarithromycin methylate related substances. The determination method comprises the following steps: detecting the related substances of clarithromycin methide by adopting a high performance liquid chromatography, wherein chromatographic conditions are as follows: a mobile phase is a phosphate solution and acetonitrile in a volume ratio of (20: 80)-(40: 60), and a stationary phase is octadecylsilane chemically bonded silica. The method disclosed by the invention is simple, convenient, rapid and accurate to operate, and can be well used for detecting the related substances of the clarithromycin methylate.

Description

Technical Field

[0001] The present invention relates to the field of drug analysis, and in particular to a method for determining clarithromycin methyl-related substances. Background Art

[0002] Clarithromycin methide, chemically known as 9-[O-(1-methoxy-1-methylethyl) oxime; 6-O-methyl-2',4'-bis-O-(trimethylsilyl)erythromycin 9-[O-(1-ethoxy-1-methylethyl) oxime], is an intermediate in the synthesis of clarithromycin. It is obtained by etherifying erythromycin thiocyanate with the oxime followed by silylation, protecting the oxime hydroxyl group, and then methylating the 6-position. The production process involves four steps: oximation, etherification, silylation, and methylation. The long process and numerous chemical reactions inevitably produce reaction impurities or residual impurities from incomplete reactions, including erythromycin oxime, clarithromycin impurity G precursor, and protected substances. Therefore, quality control of intermediates is crucial for the quality of the subsequent clarithromycin product, especially the impact of the clarithromycin impurity G precursor. Existing methods have their pros and cons, including long detection times and suboptimal impurity separation. In light of this, the present invention was proposed. Summary of the Invention

[0003] In order to solve the above technical problems, the present invention provides a method for determining related substances of clarithromycin methyl. The method of the present invention is simple, rapid and accurate to operate, and can be well used for detecting related substances of clarithromycin methyl.

[0004] In order to solve the above technical problems, the present invention provides a method for determining related substances of clarithromycin methyl. The method of the present invention is simple, rapid and accurate to operate, and can be well used for detecting related substances of clarithromycin methyl.

[0005] Specifically, the present invention provides a method for determining related substances in clarithromycin methide, comprising detecting the related substances in clarithromycin methide using high-performance liquid chromatography (HPLC). The chromatographic conditions include a mobile phase consisting of a phosphate solution and acetonitrile in a volume ratio of (20:80) to (40:60), and an octadecylsilane-bonded silica gel as the stationary phase. The present invention optimizes the chromatographic conditions to more accurately reflect the quality of the product. The method is simple, efficient, and accurate to operate, and is capable of effectively separating and detecting multiple clarithromycin methide related substances, particularly the clarithromycin impurity G precursor.

[0006] The mobile phase used in the present invention is changed from a potassium dihydrogen phosphate methanol mixed solution to a potassium dihydrogen phosphate acetonitrile mixed solution, which has stronger elution ability. In addition, the viscosity of acetonitrile is smaller than that of methanol, so the generated system back pressure is lower under the same conditions, which is more beneficial to reducing the column pressure when using a small-particle chromatographic column. The cutoff wavelength of acetonitrile is 190 nm, and the cutoff wavelength of methanol is 210 nm. Using methanol as an organic solvent may generate a lot of baseline noise, and using acetonitrile is beneficial to reducing baseline interference.

[0007] Preferably, the mobile phase is a phosphate solution and acetonitrile in a volume ratio of (25:75) to (35:65), preferably 30:70. In the present invention, by optimizing the ratio of phosphate solution to acetonitrile, the peak elution time is made more appropriate, thereby improving the separation effect of impurities.

[0008] More preferably, the phosphate solution is a 0.005-0.02 mol / L potassium dihydrogen phosphate solution; and / or the pH of the phosphate solution is 3-4. The present invention uses a phosphate solution at an optimal concentration to better present the peak shape and improve the detection effect.

[0009] Preferably, the preparation of the phosphate solution comprises: dissolving potassium dihydrogen phosphate in ultrapure water, mixing and filtering, adding triethylamine, adjusting the pH with phosphoric acid, and ultrasonically degassing.

[0010] Further preferably, the preparation of the phosphate solution comprises: weighing 1.36 g of potassium dihydrogen phosphate, dissolving it in 1000 mL of ultrapure water, mixing and filtering it, adding 1 mL of triethylamine, adjusting the pH to 3.5 with phosphoric acid, and degassing by ultrasonication.

[0011] Preferably, the octadecylsilane bonded silica gel used as the stationary phase is Poroshell EC-C18 100×4.6mm, 2.7μm. In the present invention, the stationary phase used, especially the preferred Poroshell EC-C18 100×4.6mm, 2.7μm, is matched with the mobile phase to better perform rapid determination, while optimizing detection time efficiency and chromatographic peak shape response, thereby improving separation effect.

[0012] More preferably, the chromatographic conditions further include: a flow rate of 1.0-1.4 mL / min, a column temperature of 20-40°C, and an injection volume of 8-12 μL. The preferred chromatographic conditions employed in the present invention can significantly improve separation and allow for more accurate determination of impurities.

[0013] Preferably, the flow rate is 1.2 mL / min, the column temperature is 30°C, and the injection volume is 10 μL.

[0014] Preferably, the detection wavelength is 200-220 nm, preferably 210 nm.

[0015] Preferably, the peak time of clarithromycin methide is 11-14 min, preferably 13.0-13.8 min, and preferably 13.305±0.05 min; preferably, the related substances include clarithromycin impurity E precursor, clarithromycin impurity G precursor and clarithromycin methide protected substance, etc. The preferred time of the clarithromycin impurity E precursor is 22.599±0.05 min, the preferred time of the clarithromycin impurity G precursor is 9.253±0.05 min, and the preferred time of the clarithromycin methide protected substance is 7.247±0.05 min, and other impurities, such as impurity number 1-25, impurity number 27, impurity number 28, impurity number 30, impurity number 36, impurity number 38, impurity number 39, impurity number 41, and impurity number 42, have peak times of 1.496±0.05 min and 1.579±0.05 min, respectively, as shown in Table 2 (±0.05 min). In the present invention, the peak elution time of clarithromycin methide offers advantages in impurity separation and time efficiency. Furthermore, the assay method provided by the present invention can detect over 40 impurities, including three known impurities: clarithromycin methide protected compound, clarithromycin impurity G precursor, and clarithromycin impurity E precursor, as well as other impurities such as impurity number 1, and achieves complete separation between each peak.

[0016] Further preferably, the method for determining clarithromycin methide-related substances further comprises: dissolving and diluting clarithromycin methide with methanol to obtain a test solution, wherein the concentration of the test solution is 0.8-1.2 mg / mL, preferably 1 mg / mL.

[0017] Further preferably, the method for determining clarithromycin methide-related substances further comprises: dissolving and diluting a clarithromycin methide reference substance with methanol to obtain a reference substance solution, wherein the concentration of the reference substance solution is 0.8-1.2 mg / mL, preferably 1 mg / mL.

[0018] The method for determining clarithromycin methide-related substances provided by the present invention can more accurately reflect the quality of the product. It is simple to operate, efficient, and accurate, and can effectively separate and detect multiple clarithromycin methide-related substances, especially clarithromycin impurity G precursor, clarithromycin impurity E precursor, and clarithromycin methide protected substances. The method can detect a large number of impurities and significantly improve separation efficiency. More than 40 impurities can be detected in the test solution, and each peak can be completely separated. The present invention can achieve rapid detection of clarithromycin methide-related substances, has better separation efficiency, and can better protect the system. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 The chromatogram of the test solution provided in Example 1 of the present invention.

[0021] Figure 2 The blank baseline chromatogram provided for Example 1 of the present invention.

[0022] Figure 3 This is a pressure line diagram provided in Example 1 of the present invention.

[0023] Figure 4 The chromatogram of the test solution provided for Comparative Example 1 of the present invention.

[0024] Figure 5 The blank baseline chromatogram provided for Comparative Example 1 of the present invention.

[0025] Figure 6 This is a pressure line diagram provided for Comparative Example 1 of the present invention.

[0026] Figure 7 The quantitative limit chromatogram provided for Example 1 of the present invention.

[0027] Figure 8 The detection limit chromatogram provided for Example 1 of the present invention.

[0028] Figure 9 The linear chromatogram provided for Example 1 of the present invention.

[0029] Figure 10 The precision chromatogram provided for Example 1 of the present invention.

[0030] Figure 11 The stability 0h chromatogram provided by Example 1 of the present invention.

[0031] Figure 12 The stability 24h chromatogram provided by Example 1 of the present invention.

[0032] Figure 13 The chromatogram of the test solution provided for Comparative Example 1 of the present invention.

[0033] Figure 14 This is a chromatogram of impurity numbers of the test solution provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0034] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0035] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to form one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed in the present invention.

[0036] Where specific techniques or conditions are not specified in the examples of the present invention, the techniques or conditions described in the literature in the field or in the product specifications were followed. All devices, instruments, reagents, etc. used, where the manufacturer is not specified, are conventional products available through regular channels. All experimental reagents and raw materials involved are commercially available, and all reagents are analytically pure.

[0037] The following examples use the following experimental equipment: a universal liquid chromatograph from Agilent, using a VWD detector. A clarithromycin methyl reference substance was purified in-house and had a content of 94.0%. Other reagents and materials include methanol and acetonitrile purchased from Ningbo Gaoteng Chemical Technology Co., Ltd., potassium dihydrogen phosphate from Sinopharm Group, triethylamine from Shanghai Zhanyun Chemical Technology Co., Ltd., and phosphoric acid from Shanghai MacLean Biochemical Technology Co., Ltd.

[0038] Example 1 This embodiment provides a method for determining clarithromycin methyl-related substances, which is as follows: Liquid chromatography was used to detect related substances of clarithromycin methyl. The HPLC detection conditions were as follows: 20 mg of clarithromycin methyl was placed in a 20 mL volumetric flask, and methanol was added to dissolve and dilute to the mark to serve as the test solution; 20 mg of clarithromycin methyl reference substance was accurately weighed in a 20 mL volumetric flask, and dissolved and diluted to the mark with methanol to serve as the reference solution; the HPLC test was performed using octadecylsilane bonded silica gel as the filler (Poroshell EC-C18 A 100×4.6mm, 2.7μm flash column was used, with a mobile phase consisting of a mixture of 0.01mol / L potassium dihydrogen phosphate solution (pH 3.5) and acetonitrile in a volume ratio of 30:70 (0.01mol / L phosphate solution, pH 3.5: 1.36g of potassium dihydrogen phosphate was dissolved in 1000mL of ultrapure water, mixed and filtered, and 1mL of triethylamine was added. The pH was adjusted to 3.5 with phosphoric acid, and ultrasonic degassing was performed). The flow rate was 1.2mL / min, the detection wavelength was 210nm, and the column temperature was 30°C.

[0039] Take 10 μL of the reference solution, inject it into the liquid chromatograph, and record the chromatogram. The retention time of clarithromycin methyl is about 13 minutes. Then accurately measure 10 μL of the test solution, inject it into the liquid chromatograph, and record the chromatogram.

[0040] According to the area normalization method, the purity of clarithromycin methide peak and clarithromycin impurity E precursor and other single impurities meet the relevant regulations.

[0041] Method validation: Limit of detection and limit of quantification: LOQ clarithromycin methyl = 1.6 μg / mL (approximately equivalent to 0.16% clarithromycin methyl); LOD clarithromycin methyl = 0.48 μg / mL (approximately equivalent to 0.048% clarithromycin methyl).

[0042] Range: LOQ to 120% of labeled amount.

[0043] Linear range: The content of clarithromycin methyl shows a good linear relationship in the range of 0.16% to 120% (LOQ to 120% limit). The linear equation is: Y = 0.0006X - 0.0034, R 2 =0.999.

[0044] Precision: The control solution was injected 6 times, with an RSD value of 0.57%. The test solution was injected 6 times, with an RSD of 0.51% for the purity of clarithromycin methyl, 1.5% for the purity of the precursor of clarithromycin impurity G, 0.80% for the purity of the precursor of clarithromycin impurity E, and 0.87% for the purity of the protected substance of clarithromycin methyl.

[0045] Stability: When stored at room temperature, the results of clarithromycin impurity G precursor and clarithromycin impurity E precursor are stable within 24 hours, while other impurities are degraded and are generally unstable, so they need to be freshly prepared before use.

[0046] The chromatographic conditions of this example are shown in Table 1. Figure 1-3 and Figure 14 They are respectively the test solution chromatogram, blank baseline chromatogram, pressure line diagram and impurity number diagram provided in Example 1 (see Table 2 for specific impurity descriptions). Figure 7-12 They are respectively the quantitative limit chromatogram, detection limit chromatogram, linearity chromatogram, precision chromatogram, stability 0h chromatogram and stability 24h chromatogram provided in Example 1.

[0047] Comparative Example 1 This comparative example provides a method for determining clarithromycin methyl-related substances, specifically using the same method as the document "HPLC method for determining the content of clarithromycin sustained-release tablets" in the 2008 issue of "China Practical Medicine". The specific chromatographic conditions are shown in Table 1. Figure 4-6 They are the test solution chromatogram, blank baseline chromatogram and pressure line graph provided in Comparative Example 1 respectively. Figure 13 The chromatogram of the test solution provided for Comparative Example 1.

[0048] The method of Comparative Example 1 detects the methylated intermediate of clarithromycin, and the result shows that the number of impurities detected is less than that of the method of the present invention. The impurity separation effect is not ideal, the precursor of clarithromycin impurity G partially overlaps or is incorporated into the protective substance, and the related substance control method may cause safety hazards in the product. In the embodiment of the present invention, the mobile phase is changed from a potassium dihydrogen phosphate methanol mixed solution to a potassium dihydrogen phosphate acetonitrile mixed solution, which has a stronger elution ability. In addition, the viscosity of acetonitrile is smaller than that of methanol, so the system back pressure generated is lower under the same conditions, which is more beneficial to reducing the column pressure when using a small particle size chromatographic column; the cutoff wavelength of acetonitrile is 190nm, and the cutoff wavelength of methanol is 210nm. Using methanol as an organic solvent may generate a lot of baseline noise, and using acetonitrile is beneficial to reducing baseline interference.

[0049] Table 1

[0050] Table 2

[0051] Pharmacopoeias do not specify methods for determining related substances of clarithromycin methylate. In the study "HPLC Determination of the Content of Clarithromycin Sustained-Release Tablets," published in the 2008 issue of the literature "China Practical Medicine," the methylate of a clarithromycin intermediate was tested. The results showed fewer impurities than those detected by the present method, indicating less than ideal impurity separation. The precursor of clarithromycin impurity G partially overlapped or merged with the protected substance, and the method for controlling related substances may lead to potential safety hazards in the product. The method for determining related substances of clarithromycin methylate provided by the present invention more accurately reflects the quality of the product. It is simple to operate, efficient, and accurate, and can effectively separate and detect multiple related substances of clarithromycin methylate, especially the precursor of clarithromycin impurity G. The present method can detect a large number of impurities and significantly improve separation efficiency. More than 40 impurities can be detected in the test solution, demonstrating improved separation efficiency. The present invention enables rapid detection of related substances of clarithromycin methylate, has better separation efficiency, and can better protect the system.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for determining clarithromycin methide-related substances, characterized in that: include: The related substances of clarithromycin methide were detected by high performance liquid chromatography. The chromatographic conditions included: the mobile phase was phosphate solution and acetonitrile in a volume ratio of (20:80) to (40:60), and the stationary phase was octadecylsilane bonded silica gel.

2. The method for determining clarithromycin methide-related substances according to claim 1, wherein: The mobile phase is a phosphate solution and acetonitrile in a volume ratio of (25:75) to (35:65).

3. The method for determining clarithromycin methyl related substances according to claim 2, wherein: The phosphate solution is a 0.005-0.02 mol / L potassium dihydrogen phosphate solution; and / or the pH of the phosphate solution is 3-4.

4. The method for determining clarithromycin methyl related substances according to claim 3, wherein: The preparation of the phosphate solution comprises: dissolving potassium dihydrogen phosphate in ultrapure water, mixing and filtering, adding triethylamine, adjusting the pH with phosphoric acid, and ultrasonically degassing.

5. The method for determining clarithromycin methide-related substances according to any one of claims 1 to 4, characterized in that: The octadecylsilane bonded silica gel used in the stationary phase is Poroshell EC-C18 100×4.6 mm, 2.7 μm.

6. The method for determining clarithromycin methide-related substances according to claim 1, wherein: Chromatographic conditions also included: flow rate of 1.0-1.4 mL / min, column temperature of 20-40°C, and injection volume of 8-12 μL.

7. The method for determining clarithromycin methide-related substances according to any one of claims 1 to 6, characterized in that: Detection wavelength 200-220nm.

8. The method for determining clarithromycin methide-related substances according to any one of claims 1 to 7, characterized in that: The peak time of clarithromycin methyl is 11-14 minutes; the related substances include clarithromycin impurity E precursor, clarithromycin impurity G precursor and clarithromycin methyl protected substance.

9. The method for determining clarithromycin methide-related substances according to any one of claims 1 to 8, characterized in that: Also includes: Clarithromycin methide was dissolved and diluted with methanol to obtain a test solution, wherein the concentration of the test solution was 0.8-1.2 mg / mL.

10. The method for determining clarithromycin methide-related substances according to any one of claims 1 to 9, characterized in that: Also includes: The clarithromycin methide reference substance was dissolved and diluted with methanol to obtain a reference substance solution, wherein the concentration of the reference substance solution was 0.8-1.2 mg / mL.

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