Analysis method for related substances of erythromycin cyclic ester tablets

Abstract

The invention provides a method for analyzing related substances of erythromycin cyclic ester tablets, and relates to the field of drug detection. According to the method for analyzing the related substances of the erythromycin cyclic ester tablets, high performance liquid chromatography is adopted for detection, and chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler; an ammonium sulfate buffer solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, and isopropanol is used as a mobile phase C. The method for analyzing the related substances of the erythromycin cyclic ester tablets has good specificity, quantitation limit, detection limit, linearity, accuracy, precision (repeatability and intermediate precision), filter membrane adsorbability, solution stability and durability, can better meet the requirements of the method for analyzing the related substances of the erythromycin cyclic ester tablets, is better in quality control, and is beneficial to improvement of medicine safety.

Description

Technical Field

[0001] The invention relates to the field of drug detection, and in particular to a method for analyzing related substances of erythromycin cyclohexetate tablets. Background Art

[0002] Cyclic erythromycin tablets are an antibiotic widely used in clinical practice. They are mainly used to treat various diseases caused by bacterial infections, including tonsillitis, pharyngitis, bacterial pneumonia, mycoplasma pneumonia, stomatitis, Legionnaires' disease, diphtheria, whooping cough, scarlet fever, erythrasma, erysipelas, gonorrhea, early syphilis, chancroid, urethritis, Campylobacter enteritis, amoebic enteritis, etc.

[0003] However, some related substances (impurities) may be produced during the production process of the drug, which not only affect the efficacy of the drug, but also threaten the safety of the patient. Therefore, it is of great significance to open and improve the analysis method of related substances in erythromycin cyclopentyl tablets. Summary of the invention

[0004] In view of this, the present invention proposes a method for analyzing related substances of erythromycin cyclohexetate tablets to solve the above problems.

[0005] The technical solution of the present invention is achieved as follows: a method for analyzing related substances in cyclic erythromycin tablets, using high performance liquid chromatography for detection, the chromatographic conditions are: using octadecylsilane bonded silica gel as a filler; using ammonium sulfate buffer as mobile phase A, acetonitrile as mobile phase B, and isopropanol as mobile phase C; performing gradient elution according to the following table:

[0006] Time (min) Mobile phase A (%, v / v) Mobile phase B (%, v / v) Mobile phase C (%, v / v) 0 78 20 2 25 63 35 2 29 63 35 2 30 78 20 2 45 78 20 2

[0007] The related substances include erythromycin A, cyclic erythromycin impurity A, cyclic erythromycin impurity B, 10,11-anhydroerythromycin A, erythromycin B, erythromycin A enol ether and cyclic erythromycin enol ether.

[0008] Furthermore, the preparation method of the ammonium sulfate buffer comprises the following steps: taking ammonium sulfate, adding water to dissolve and dilute, adjusting the pH value to 7.2-7.4 with 5-15%wt ammonia solution, and the mass volume concentration of the ammonium sulfate buffer is 16-19g / L.

[0009] Furthermore, the column temperature was 63-65°C.

[0010] Furthermore, the specification of the chromatographic column is 150 mm×4.6 mm, 5 μm or a chromatographic column with equivalent performance.

[0011] Furthermore, the detection wavelength is 204-206 nm; the sample plate temperature is 4-6°C.

[0012] Further, the flow rate is 0.9-1.1 ml per minute.

[0013] Furthermore, the preparation method of the test solution includes the following steps: taking the test sample, accurately weighing, grinding, accurately weighing the powder, placing it in a volumetric flask, adding acetonitrile, ultrasonicating for 15-20 minutes, cooling to room temperature, diluting to the scale with mobile phase A, filtering, and taking the filtrate to obtain the test solution.

[0014] Furthermore, the preparation method of the reference solution includes the following steps: taking erythromycin A reference, accurately weighing, placing in a volumetric flask, adding acetonitrile, ultrasonically dissolving it, diluting with mobile phase A, and preparing a reference solution with a concentration of 0.04-0.06 mg / ml.

[0015] Furthermore, the preparation method of the system suitability solution includes the following steps: taking the cyclic erythromycin bulk drug, erythromycin A reference substance, cyclic erythromycin impurity A reference substance, cyclic erythromycin impurity B reference substance, 10,11-dehydrated erythromycin A reference substance, erythromycin B reference substance, erythromycin A enol ether reference substance and cyclic erythromycin enol ether reference substance, placing them in the same volumetric flask, adding acetonitrile, ultrasonicating for 1-2 minutes, adding mobile phase A, ultrasonically dissolving, diluting to the scale with mobile phase A, and shaking well.

[0016] Further, each milliliter of system suitability solution contains 4-6 mg of erythromycin cyclic ester, 0.02-0.03 mg of erythromycin A reference substance, 0.02-0.03 mg of erythromycin cyclic ester impurity A reference substance, 0.02-0.03 mg of erythromycin cyclic ester impurity B reference substance, 0.02-0.03 mg of erythromycin 10,11-anhydro erythromycin A reference substance, 0.04-0.06 mg of erythromycin B reference substance, 0.04-0.06 mg of erythromycin A enol ether reference substance, and 0.04-0.06 mg of erythromycin cyclic ester enol ether reference substance.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The specificity, quantitative limit, detection limit, linearity, accuracy, precision (repeatability, intermediate precision), filter membrane adsorption, solution stability and durability of the related substance analysis method of erythromycin cycloester tablets of the present invention are all good, which can better meet the related substance analysis method of erythromycin cycloester tablets, better perform quality control on erythromycin cycloester tablets, and is conducive to improving drug safety. DETAILED DESCRIPTION

[0019] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0020] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0021] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0022] The cyclic erythromycin API, erythromycin A reference substance, cyclic erythromycin impurity A reference substance, cyclic erythromycin impurity B reference substance, 10,11-anhydroerythromycin A reference substance, erythromycin B reference substance, erythromycin A enol ether reference substance and cyclic erythromycin enol ether reference substance are all commercially available products.

[0023] Table 1 Impurity information

[0024]

[0025]

[0026] Example 1

[0027] 1. Analysis method of related substances in erythromycin cyclohexetate tablets: determination by high performance liquid chromatography

[0028] Test solution: Take 10 tablets of this product, accurately weigh, grind, accurately weigh an appropriate amount of powder (equivalent to 500 mg of erythromycin cyclic), put it in a 100 ml volumetric flask, add 20 ml of acetonitrile, ultrasonicate for 15 min, cool to room temperature, dilute to the scale with mobile phase A, filter, and take the filtrate.

[0029] Reference solution: Take about 5 mg of erythromycin A reference, accurately weigh it, place it in a 10 ml volumetric flask, add 4 ml of acetonitrile, dissolve it by ultrasonication, dilute it to the scale with mobile phase A, shake well, accurately measure 2 ml, place it in a 20 ml volumetric flask, dilute it to the scale with mobile phase A, and shake well.

[0030] System suitability solution: Take about 500 mg of erythromycin cyclic ester API, about 2.5 mg of erythromycin A reference substance, about 2.5 mg of erythromycin cyclic ester impurity A reference substance, about 2.5 mg of erythromycin cyclic ester impurity B reference substance, about 5 mg of 10,11-anhydroerythromycin A reference substance, about 5 mg of erythromycin B reference substance, about 5 mg of erythromycin A enol ether reference substance and about 5 mg of erythromycin cyclic ester enol ether reference substance, put them in the same 100 ml volumetric flask, add 20 ml of acetonitrile, sonicate for 1 min, add about 20 ml of mobile phase A, sonicate to dissolve, dilute to the scale with mobile phase A, and shake well.

[0031] Chromatographic conditions: octadecylsilane bonded silica gel is used as filler (Supelcosil SuplexpKb-100, 4.6 mm×150 mm, 5 μm or a chromatographic column with equivalent performance); ammonium sulfate buffer (pH 7.3) (take 18 g of ammonium sulfate, dissolve it in water and dilute it to 1000 ml, and adjust the pH value to 7.3 with 10% wt ammonia solution) is used as mobile phase A, acetonitrile is used as mobile phase B, and isopropanol is used as mobile phase C; gradient elution is performed according to the table below; the flow rate is 1.0 ml per minute; the column temperature is 65°C; the detection wavelength is 205 nm; and the sample plate temperature is 5°C.

[0032] Table 2 Gradient elution table

[0033] Time (min) Mobile phase A (%, v / v) Mobile phase B (%, v / v) Mobile phase C (%, v / v) 0 78 20 2 25 63 35 2 29 63 35 2 30 78 20 2 45 78 20 2

[0034] 2. Validation report of the analytical method for related substances of erythromycin cyclohexetate tablets

[0035] 1. Validation Study

[0036] 1.1. Exclusivity

[0037] 1.1.1. Interference test

[0038] Test results: The blank solution and blank excipient solution had no interfering peaks at the elution points of erythromycin cyclohexetine and known impurities; the separation between adjacent peaks was 2.17-8.07, which was significantly greater than 1.5, and the separation was good.

[0039] 1.1.2. Forced degradation test

[0040] Test results: The main degradation impurity peaks and the main peak of the preparation product can be well separated under strong acid, strong alkali, high temperature (solid, liquid), oxidation and strong light destruction, and the purity of the main peak meets the requirements. The main peak degradation of the preparation product is small after strong alkali, high temperature (solid, liquid) and strong light destruction, and the main peak degradation is large under strong acid and oxidation destruction conditions, indicating that the preparation is relatively stable under strong alkali, high temperature (solid, liquid) and strong light conditions, and is relatively sensitive to strong acid and oxidation conditions. The mass conservation recovery rate is in the range of 92.91% to 101.11%, indicating that the specificity of this method is good.

[0041] 1.2. Linear

[0042] Table 3 Linearity test results

[0043] name Linear equations Correlation coefficient r Erythromycin A y=2413.4223x+782.5766 0.9970 Erythromycin Cyclic A Impurity A y=7351.3195x-427.0995 0.9997 10,11-Anhydroerythromycin A y=8846.8143x+300.2075 1.0000 Erythromycin B y=1658.8621x+242.6076 0.9994 Erythromycin A enol ether y=16218.0617x-2733.8739 1.0000 Erythromycin Cyclic Acid Impurity B y=24176.3849x-144.0990 1.0000 Cyclic ester erythromycin enol ether y=17107.6608x+10745.7357 1.0000

[0044] Test results: Linearity test results show good linearity.

[0045] 1.3 Detection limit

[0046] Test results: The detection limit SN of each impurity of erythromycin A, cyclic erythromycin impurity A, 10,11-anhydroerythromycin A, erythromycin B, erythromycin A enol ether, cyclic erythromycin impurity B and cyclic erythromycin enol ether was between 3 and 11 (≥3), and the detection limit concentrations of erythromycin A, cyclic erythromycin impurity A, 10,11-anhydroerythromycin A, erythromycin B, erythromycin A enol ether, cyclic erythromycin impurity B and cyclic erythromycin enol ether were 1.9820μg / ml, 0.4027μg / ml, 0.3949μg / ml, 2.0097μg / ml, 0.2056μg / ml, 0.1446μg / ml and 0.4108μg / ml, respectively; the detection limit test results all met the validation requirements.

[0047] 1.4. Limit of quantitation

[0048] Test results: The quantification limits S / N of each impurity of erythromycin A, erythromycin ester impurity A, 10,11-dehydrated erythromycin A, erythromycin B, erythromycin A enol ether, erythromycin ester impurity B and erythromycin ester enol ether were 10-24

[0049] (≥10), the RSD of the 6-needle peak area was between 4.63% and 7.87% (≤10%), among which, the quantification limits of erythromycin A, cyclic erythromycin impurity A, 10,11-dehydrate erythromycin A, erythromycin B, erythromycin A enol ether, cyclic erythromycin impurity B and cyclic erythromycin enol ether were 4.9549μg / ml, 1.0068μg / ml, 0.9874μg / ml, 5.0242μg / ml, 0.5141μg / ml, 0.3615μg / ml and 1.0271μg / ml, respectively. The quantification limit test results all met the verification requirements.

[0050] 1.5. Accuracy

[0051] Test results: The limit concentration percentage was 100% to 150%, and the average recoveries of erythromycin A, cyclic erythromycin impurity A, 10,11-anhydroerythromycin A, erythromycin B, erythromycin A enol ether, cyclic erythromycin impurity B and cyclic erythromycin enol ether were 99.9 to 101.9%, with RSDs of 0.33 to 4.84%. The recoveries of erythromycin A, cyclic erythromycin impurity A, 10,11-anhydroerythromycin A, erythromycin B, erythromycin A enol ether, cyclic erythromycin impurity B and cyclic erythromycin enol ether all met the requirements, indicating that the method had good accuracy.

[0052] 1.6. Precision

[0053] Repeatability

[0054] Table 4 Repeatability test results

[0055]

[0056] Test results: The RSDs of erythromycin A, cyclic erythromycin impurity A, 10,11-dehydrated erythromycin A, erythromycin B, erythromycin A enol ether, cyclic erythromycin impurity B and cyclic erythromycin enol ether were all less than 5%. The repeatability test results met the validation requirements, demonstrating that this method has good repeatability.

[0057] 1.6.2. Intermediate precision

[0058] Table 5 Intermediate precision test results

[0059]

[0060] Table 6 Precision test results

[0061]

[0062] Test results: The precision test results meet the validation requirements, indicating that this method has good precision.

[0063] 1.7. Solution stability

[0064] Test results: The reference solution is stable within 30 hours at 5°C; the test solution is stable within 16 hours at 5°C.

[0065] 1.8. Chromatographic Conditions Robustness

[0066] Test results: After adjusting the chromatographic parameters of column temperature to -2°C, mobile phase buffer concentration to 16.2 g / L~18.9 g / L, mobile phase buffer pH value to ±0.1 and replacing the chromatographic column, the change in impurity content compared with the target conditions was less than 10.0%, and the durability test results met the validation requirements, indicating that the chromatographic conditions of this method have good durability.

[0067] The above verification results prove that the analytical method for erythromycin-related substances has good specificity, quantitative limit, detection limit, linearity, accuracy, precision (repeatability, intermediate precision), filter membrane adsorption, solution stability and durability.

[0068] Comparative Example 1

[0069] The difference from Example 1 is that the mobile phase is methanol-water-acetic acid (70:30:1, v / v / v). The results show that the erythromycin cyclic ester peak interferes with the adjacent impurity peak.

[0070] Comparative Example 2

[0071] The difference from Example 1 is: Gradient elution table:

[0072] Table 7 Gradient elution Table 2

[0073]

[0074]

[0075] The results show that some adjacent impurity peaks interfere with each other.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for analyzing related substances in erythromycin cyclic ester tablets, characterized in that: The HPLC method was used for detection. The chromatographic conditions were as follows: octadecylsilane bonded silica gel was used as filler; ammonium sulfate buffer was used as mobile phase A, acetonitrile was used as mobile phase B, and isopropanol was used as mobile phase C; gradient elution was performed as shown in the following table: 。 2. The method for analyzing related substances of erythromycin cyclic ester tablets according to claim 1, characterized in that: The preparation method of the ammonium sulfate buffer comprises the following steps: taking ammonium sulfate, adding water to dissolve and dilute, and adjusting the pH value to 7.2-7.4 with 5-15%wt ammonia solution, wherein the mass volume concentration of the ammonium sulfate buffer is 16-19g / L.

3. The method for analyzing related substances of erythromycin cyclic ester tablets according to claim 1 or 2, characterized in that: The column temperature was 63-65°C.

4. The method for analyzing related substances of erythromycin cyclic ester tablets according to claim 1, characterized in that: The specification of the chromatographic column is 150mm×4.6mm, 5μm or a chromatographic column with equivalent performance.

5. The method for analyzing related substances of erythromycin cyclic ester tablets according to claim 1, characterized in that: The detection wavelength was 204-206 nm; the sample pan temperature was 4-6°C.

6. The method for analyzing related substances of erythromycin cyclic ester tablets according to claim 1, characterized in that: The flow rate is 0.9-1.1 ml per minute.

7. The method for analyzing related substances of erythromycin cyclic ester tablets according to claim 1, characterized in that: The preparation method of the test solution comprises the following steps: taking the test sample, accurately weighing, grinding, accurately weighing the powder, placing it in a volumetric bottle, adding acetonitrile, ultrasonicating for 15-20 minutes, cooling to room temperature, diluting to the scale with mobile phase A, filtering, and taking the filtrate to obtain the test solution.

8. The method for analyzing related substances of erythromycin cyclic ester tablets according to claim 1, characterized in that: The preparation method of the reference solution comprises the following steps: taking erythromycin A reference, accurately weighing it, placing it in a volumetric bottle, adding acetonitrile, ultrasonically dissolving it, diluting it with mobile phase A, and preparing a reference solution with a concentration of 0.04-0.06 mg / ml.

9. The method for analyzing related substances of erythromycin cyclic ester tablets according to claim 1, characterized in that: The preparation method of the system suitability solution comprises the following steps: taking erythromycin cyclic ester API, erythromycin A reference substance, erythromycin cyclic ester impurity A reference substance, erythromycin cyclic ester impurity B reference substance, 10,11-dehydrate erythromycin A reference substance, erythromycin B reference substance, erythromycin A enol ether reference substance and erythromycin cyclic ester enol ether reference substance, placing them in the same volumetric bottle, adding acetonitrile, ultrasonicating for 1-2 minutes, adding mobile phase A, ultrasonically dissolving, diluting with mobile phase A, and shaking well.

10. The method for analyzing related substances of erythromycin cyclic ester tablets according to claim 9, characterized in that: Each milliliter of system suitability solution contains 4-6 mg of erythromycin cyclic ester, 0.02-0.03 mg of erythromycin A reference substance, 0.02-0.03 mg of erythromycin cyclic ester impurity A reference substance, 0.02-0.03 mg of erythromycin cyclic ester impurity B reference substance, 0.02-0.03 mg of erythromycin 10,11-anhydroerythromycin A reference substance, 0.04-0.06 mg of erythromycin B reference substance, 0.04-0.06 mg of erythromycin A enol ether reference substance, and 0.04-0.06 mg of erythromycin cyclic ester enol ether reference substance.

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