Method for detecting related substances in amoxicillin raw material and preparation thereof

Abstract

The invention discloses a method for detecting related substances in an amoxicillin raw material and a preparation thereof, and relates to the technical field of pharmaceutical analysis, HPLC conditions are as follows: the elution gradient is 0-5 min: 100% mobile phase A; in 5-25 min, 100%-94% of the mobile phase A and 0-6% of the mobile phase B are adopted; 25-40 min, 94%-84% of the mobile phase A and 6%-16% of the mobile phase B are adopted; 40-50 minutes: 84% of the mobile phase A and 16% of the mobile phase B; 50-51min: 84% of the mobile phase A, 100% of the mobile phase A and 16-0% of the mobile phase B; and in 51-60 min, 100% of the mobile phase A is adopted. The method for detecting the related substances of the amoxicillin related varieties, which is recorded and published by various Chinese pharmacopoeias and literatures, is confirmed, and a set of more effective high performance liquid chromatography is established and verified by screening and optimizing liquid phase conditions such as chromatographic columns and mobile phases, particularly chromatographic column brands, fillers, models and the like; the quality control can be simultaneously carried out on process impurities, degradation impurities, polymers and other organic impurities of amoxicillin raw materials and preparations thereof, so that the occurrence of adverse reactions such as anaphylaxis and the like is reduced, and the safety and effectiveness of medication are ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and more specifically to a method for detecting related substances in an amoxicillin raw material and a preparation thereof. Background Art

[0002] Amoxicillin, English name: Amoxicillin, is one of the most commonly used broad-spectrum β-lactam antibiotics. It is stable under acidic conditions and has a gastrointestinal absorption rate of 90%. It has a strong bactericidal effect and is one of the most widely used semi-synthetic penicillins. Its preparations include capsules, tablets, granules, dispersible tablets, etc. Its main adverse reactions are allergic reactions, nausea, vomiting and other symptoms, with an incidence of about 5% to 6%. Studies have found that the sensitization reaction of amoxicillin is mainly caused by its dimers and other polymers. For this reason, it is stipulated that a penicillin skin test must be performed before using amoxicillin, and those with positive reactions are prohibited from using it.

[0003] After consulting the data, it was found that the pharmacopoeias of various countries such as Ch.P, USP, and EP all have included the detection methods of related substances of amoxicillin and its preparations. Chinese patent CN202110130547.3 discloses a detection method for related substances of amoxicillin and clavulanate potassium, Chinese patent CN112986289 A discloses a consistency evaluation detection method for amoxicillin capsules, and the article by Qu Xilong et al. also discloses a method for determining 22 impurities of amoxicillin and clavulanate potassium by HPLC.

[0004] A comparison revealed that in the published detection methods, the research on known impurities in the samples was not comprehensive enough, and some impurities could not be completely separated, especially impurities D, E, K and O, which were racemic forms containing two or three isomers, which were not reflected in the chromatogram.

[0005] Therefore, how to detect various impurities in amoxicillin preparations is a problem that those skilled in the art need to solve urgently. Summary of the invention

[0006] In view of this, the present invention confirms the related substance detection methods of amoxicillin-related varieties included in the pharmacopoeias of various countries and published in the literature, and screens and optimizes the chromatographic column, mobile phase, especially the chromatographic column brand, filler, model and other liquid phase conditions, establishes and verifies a more effective high-performance liquid chromatography method, which can simultaneously control the quality of process impurities, degradation impurities, polymers and other organic impurities in amoxicillin raw materials and preparations, so as to reduce the occurrence of adverse reactions such as allergic reactions and ensure the safety and effectiveness of medication.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A method for detecting impurities in amoxicillin raw materials and preparations, detecting and separating by HPLC, the HPLC conditions are as follows:

[0009] The elution gradient is: 0-5min: 100% mobile phase A; 5-25min: 100%~94% mobile phase A, 0-6% mobile phase B; 25-40min: 94%~84% mobile phase A, 6-16% mobile phase B; 40-50min: 84% mobile phase A, 16% mobile phase B; 50-51min: 84% mobile phase A~100% mobile phase A, 16-0% mobile phase B; 51-60min: 100% mobile phase A.

[0010] Preferably, the detection wavelength is 220-240 nm, and the chromatographic column is 150 mm×4.6 mm, 5 μm Agilent SB-C8 column.

[0011] Preferably, mobile phase A: 6.8 g / L potassium dihydrogen phosphate solution; mobile phase B: acetonitrile; flow rate:

[0012] 1.0~2.5ml / min; injection volume: 20μL; column temperature: 30~50℃; running time: 60min.

[0013] Preferably, the impurities are shown in the following table:

[0014]

[0015]

[0016] Before testing, a standard solution should be prepared as a control and the peak areas of each impurity and amoxicillin should be calculated:

[0017] (1) Prepare the test solution: Take an appropriate amount of the product, add diluent to dissolve and dilute to the scale, shake well;

[0018] (2) Preparation of reference substance solution: Take an appropriate amount of reference substance, add diluent to dissolve and dilute to scale, and shake well;

[0019] (3) Preparation of system suitability solution: Take appropriate amount of each process impurity and degradation impurity, add 50% methanol to dissolve and dilute to scale, shake well; take 20 μl of each, inject into high performance liquid chromatograph, record the chromatogram, and calculate the peak area according to the main component external standard method.

[0020] The technical effect achieved by the present invention is that 15 process impurities and degradation impurities in amoxicillin raw materials and preparations thereof can be detected simultaneously and effectively, and the main peak and adjacent impurity peaks, the impurity peaks and the isomer peaks of the same impurity in the chromatogram are completely separated. The detection method of the present invention is sensitive and accurate, simple to operate, and has good reproducibility. It can fully meet the requirements for the determination of related substances in amoxicillin raw materials and preparations thereof in industrial production, and ensure the safety, effectiveness and quality control of the drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0022] Figure 1 The attached figure is a chromatogram of the blank solution.

[0023] Figure 2 The attached figure is a chromatogram of the system suitability solution.

[0024] Figure 3 The attached figure is a high temperature destruction chromatogram of amoxicillin raw material.

[0025] Figure 4 The attached figure is a chromatogram of the acid heating destruction of amoxicillin raw material.

[0026] Figure 5 The attached figure is a chromatogram of the alkali heating destruction of amoxicillin raw material.

[0027] Figure 6 The attached figure is a chromatogram of the oxidation destruction of amoxicillin raw material.

[0028] Figure 7 The attached figure is a chromatogram of amoxicillin reference substance solution.

[0029] Figure 8 The attached figure is a chromatogram of related substances of amoxicillin raw material (batch 200621035).

[0030] Fig. 9 The attached figure is a chromatogram of related substances of Amoxicillin Capsules (batch 20190303). DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The instruments and reagents used in the examples are: high performance liquid chromatograph (Agilent 1260HPLC); diode array detector (Agilent 1260DAD); electronic balance (Mettler XS205DU); pH meter (Mettler Seven Excellence); all reagents used are chromatographically pure; water (ultrapure water, homemade); amoxicillin raw material (United Pharmaceutical Co., Ltd.); amoxicillin capsules (homemade).

[0033] Chromatographic conditions:

[0034] Instrument: High performance liquid chromatography equipped with UV detector

[0035] Detection wavelength: 230nm

[0036] Chromatographic column: Agilent SB-C8 column (150mm×4.6mm, 5μm)

[0037] Mobile phase A: 6.8 g / L potassium dihydrogen phosphate solution (adjust pH to 5.0 with 2 mol / L potassium hydroxide solution)

[0038] Mobile phase B: acetonitrile

[0039] Flow rate: 2.0ml / min

[0040] Injection volume: 20 μl

[0041] Column temperature: 45°C

[0042] Running time: 60 minutes

[0043] The gradient table is shown in Table 1;

[0044] Table 1

[0045] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 5 100 0 25 94 6 40 84 16 50 84 16 51 100 0 60 100 0

[0046] Diluent: Mobile phase A

[0047] Ghost peak capture column: Welch Ghost-Buster (4.6×50mm)

[0048] Solution preparation

[0049] Test solution: Take an appropriate amount of the product, weigh accurately, dissolve in phosphate buffer and quantitatively dilute to make a solution containing about 1.7 mg per 1 ml, and shake well.

[0050] Reference solution: Take an appropriate amount of amoxicillin reference substance, weigh accurately, dissolve and dilute with phosphate buffer to make a solution containing about 17 μg per 1 ml, and shake well.

[0051] System suitability solution: Take appropriate amount of amoxicillin impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M, impurity N and impurity O reference substances, dissolve and dilute with 50% methanol to make a solution containing about 1.5 mg per 1 ml, as impurity stock solution; take about 17 mg of amoxicillin reference substance, accurately weigh, put into a 10 ml volumetric flask, accurately add 0.1 ml of each impurity stock solution, add mobile phase A to dissolve and dilute to the scale, shake well, as system suitability solution. The structural formula of each impurity is shown in Table 2.

[0052] Table 2

[0053]

[0054]

[0055] Example 1 Methodological verification of related substances of amoxicillin raw materials

[0056] ①Specificity test

[0057] Precisely measure 20 μl of blank solvent and system suitability solution respectively and inject them into the liquid chromatograph to record the chromatogram. The results show that the blank solvent has no interference, and the peak order of the system suitability solution is impurity I, impurity D1, impurity D2, impurity A, impurity B, amoxicillin, impurity E1, impurity G, impurity M, impurity E2, impurity C, impurity F, impurity H, impurity K1, impurity K2, impurity L, impurity J, impurity O1, impurity O2, impurity O3 and impurity N, and the separation between each chromatographic peak meets the requirements.

[0058] At the same time, the results of the forced degradation test showed that the separation degree of the main peak and the degraded impurity peak under each destruction condition met the requirements, and the purity of the main peak was high, the material was basically conserved, and the specificity of this method was high. Figure 1-Figure 6 , amoxicillin chromatogram see Figure 7 .

[0059] ②Linear test

[0060] Accurately weigh appropriate amounts of amoxicillin, impurity A, impurity B, impurity G, impurity H, impurity I, impurity L, impurity M, impurity N, impurity D, impurity E, impurity J, impurity K, and impurity O reference substances, place them in different volumetric bottles, add 10% methanol to dissolve and dilute to make solutions containing about 0.6 mg per 1 ml, accurately weigh appropriate amounts of impurity C and impurity F reference substances, place them in different volumetric bottles, add 50% methanol to dissolve and dilute to make solutions containing about 0.6 mg per 1 ml, as reference substance stock solutions; accurately measure 1 ml of each reference substance stock solution, place it in a 20 ml volumetric flask, add mobile phase A to dilute to the scale, shake well, and use it as linear solution-6 (2%); accurately measure 5 ml of linear solution-6 solution, place it in a 10 ml volumetric flask, add mobile phase A to dilute to the scale, shake well, and use it as linear solution-5 (1%); accurately 5 ml of linear solution-5 solution was precisely measured and placed in a 10 ml volumetric flask, diluted to scale with mobile phase A, and shaken to be linear solution-4 (0.5%); 1 ml of linear solution-5 solution was precisely measured and placed in a 10 ml volumetric flask, diluted to scale with mobile phase A, and shaken to be linear solution-3 (0.1%); 1 ml of linear solution-4 solution was precisely measured and placed in a 10 ml volumetric flask, diluted to scale with mobile phase A, and shaken to be linear solution-2 (0.05%); 3 ml of linear solution-3 solution was precisely measured and placed in a 20 ml volumetric flask, diluted to scale with mobile phase A, and shaken to be linear solution-1 (0.015%); 20 μl of each of the above linear solutions was precisely measured and injected into the liquid chromatograph respectively, and the chromatogram was recorded. The linear equation was obtained by linear regression with peak area (A) as the ordinate and concentration (C) as the abscissa. The results are shown in Table 3.

[0061] ④Sensitivity

[0062] The detection limit was calculated with a signal-to-noise ratio of S / N = 3, and the quantification limit was calculated with a signal-to-noise ratio of S / N = 10. The stock solution of each impurity reference substance was taken in turn, 20 μl was accurately measured and injected into the liquid chromatograph, and the chromatogram was recorded. The results are shown in Table 3.

[0063] ⑤Accuracy

[0064] Accurately weigh appropriate amount of amoxicillin raw material (approximately equivalent to the amount of amoxicillin in C 16 H 19N3O5S (15mg) 9 portions each, placed in different 10ml volumetric flasks, accurately added 1ml, 5ml, 10ml mixed impurity reference solution, three portions each, then dissolved and diluted to scale with mobile phase A, shaken, as low, medium and high concentration solutions (0.1%, 0.5%, 1.0%) for recovery rate experiment, accurately measured 20μl injected into liquid chromatograph, recorded chromatogram, calculated the content of each impurity (unit: μg) by peak area according to external standard method, deducted the impurity content in the sample and compared with the theoretical addition amount, calculated recovery rate. The results are shown in Table 3.

[0065] ⑥Precision test

[0066] Accurately measure 20 μl of the amoxicillin-related substance system suitability solution (including amoxicillin and impurities A to O), inject the sample six times continuously, and record the chromatogram. The results are shown in Table 3.

[0067] Table 3 Summary of the results of the validation of the methodology of amoxicillin raw materials

[0068]

[0069]

[0070] ⑤ Amoxicillin raw material sample testing

[0071] According to the above determined method, three batches of amoxicillin raw materials were tested for related substances. The chromatogram of batch 200621035 is shown in Figure 8 , the specific test results are shown in Table 4.

[0072] Table 4 Amoxicillin related substances test results

[0073] batch number 180221191 180311055 200621035 Impurity A Not detected Not detected Not detected Impurity B Not detected Not detected Not detected Impurity C Not detected Not detected Not detected Impurity D 0.05％ Not detected Not detected Impurity E 0.14％ 0.03％ Not detected Impurity F Not detected 0.05％ Not detected Impurity G Not detected Not detected Not detected Impurity H Not detected Not detected Not detected Impurity I Not detected Not detected Not detected Impurity J 0.05％ 0.04％ 0.05％ Impurity K Not detected Not detected Not detected Impurity L Not detected Not detected Not detected Impurity M Not detected Not detected Not detected Impurity N Not detected Not detected Not detected Impurity O Not detected Not detected Not detected Other single miscellaneous 0.06％ 0.05％ 0.04％ Total impurities 0.34％ 0.16％ 0.09％

[0074] Example 2 Detection of Related Substances in Amoxicillin Capsules

[0075] ① Methodological validation of amoxicillin capsules

[0076] According to the method determined in Example 1, amoxicillin capsules were methodologically verified. The results are shown in Table 5.

[0077] Table 5 Summary of the results of the methodological validation of amoxicillin capsules

[0078]

[0079]

[0080]

[0081] ②Testing of amoxicillin capsule samples

[0082] According to the above-mentioned method, the relevant substances of three batches of amoxicillin capsules were tested. The specific test results are shown in Table 6. The chromatogram results of the 20190303 batch of amoxicillin capsules are shown in Fig. 9 .

[0083] Table 6 Amoxicillin capsule related substances test results

[0084] batch number 20190303 20190304 20190401 Impurity C Not detected Not detected Not detected Impurity D 0.29 0.31 0.28 Impurity E 0.49 0.49 0.41 Impurity F Not detected Not detected Not detected Impurity J 0.40 0.41 0.34 Impurity K Not detected Not detected Not detected Impurity O 0.19 0.19 0.16 Other single miscellaneous 0.05 0.05 0.05 Total impurities 1.51 1.50 1.25

[0085] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0086] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting related substances in amoxicillin raw materials and preparations thereof, detecting and separating by HPLC, characterized in that: HPLC conditions were as follows: The elution gradient is: 0-5min: 100% mobile phase A; 5-25min: 100%~94% mobile phase A, 0-6% mobile phase B; 25-40min: 94%~84% mobile phase A, 6-16% mobile phase B; 40-50min: 84% mobile phase A, 16% mobile phase B; 50-51min: 84% mobile phase A~100% mobile phase A, 16-0% mobile phase B; 51-60min: 100% mobile phase A.

2. The method for detecting related substances in an amoxicillin raw material and a preparation thereof according to claim 1, characterized in that: The detection wavelength is 220-240 nm, and the chromatographic column is 150 mm × 4.6 mm, 5 μm Agilent SB-C8 column.

3. The method for detecting related substances in an amoxicillin raw material and a preparation thereof according to claim 1, characterized in that: Mobile phase A: 6.8 g / L potassium dihydrogen phosphate solution; mobile phase B: acetonitrile; flow rate: 1.0-2.5 ml / min; injection volume: 20 μL; column temperature: 30-50°C; running time: 60 min.

4. The method for detecting related substances in an amoxicillin raw material and a preparation thereof according to claim 1, characterized in that: The impurities are shown in the following table:

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