Method for detecting genotoxic impurities in amoxicillin bulk drug or pharmaceutical preparation

Abstract

The invention discloses a method for detecting genotoxic impurities in an amoxicillin bulk drug or a pharmaceutical preparation. Relates to the technical field of pharmaceutical analysis. The method comprises the following steps: pre-treating a sample and the like, and detecting by using a high performance liquid chromatography. A result shows that the linear range of the established method is 0.0155 to 0.2175 mu g / ml (r is equal to 0.9999), the quantitation limit is 0.0155 mu g / ml (0.04 ppm), and the detection limit is 0.0037 mu g / ml (0.01 ppm); the method has good precision (RSDlt; 2%), the accuracy is reliable (the recovery rate is 102.65%), and the solution stability within 24 hours is good. The method has the advantages of being high in sensitivity, good in specificity, high in accuracy, simple in method and rapid and accurate in detection, and is suitable for determining the genotoxic impurity methyl p-toluenesulfonate in an amoxicillin crude drug or a pharmaceutical preparation, so that the controllable drug quality is ensured, and the safety of clinical medication is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and more particularly to a method for detecting genotoxic impurities in amoxicillin raw materials or drug preparations. Background Art

[0002] Amoxicillin, also known as amoxicillin, is the main product of the second generation of penicillin. It is a white or off-white crystalline powder. It is a broad-spectrum semi-synthetic antibiotic with antibacterial effects. It is mainly used clinically for respiratory infections and urinary tract infections caused by Gram-positive and Gram-negative bacteria. It is also used to treat gastritis, gastric ulcers and combined treatment of Helicobacter pylori infection and other diseases. As a β-lactam antibiotic, it is widely used in clinical practice due to its high efficiency, low toxicity and low price.

[0003] Genotoxic impurities (GTI) are substances that can directly or indirectly react with DNA, induce DNA damage, and have the risk of causing cancer, teratogenesis, and mutagenesis. In recent years, as the research on genotoxic impurities has become more and more in-depth, people have come to realize that although genotoxic impurities exist at trace levels in drugs, their harm is self-evident.

[0004] Methyl p-toluenesulfonate is introduced into the synthesis process of amoxicillin. Methyl p-toluenesulfonate belongs to the sulfonate compound and contains a genotoxic warning structure. It is considered as a potential genotoxic impurity. It can undergo alkylation reaction with DNA, thereby inducing cancer. It has serious safety risks even at low concentrations. Therefore, it is very important to use appropriate analytical methods to control genotoxic impurities.

[0005] The maximum daily dose of amoxicillin capsules in clinical practice is about 4 g / d -1 Due to the lack of relevant toxicological data on methyl p-toluenesulfonate, the threshold of toxicological concern (TTC) method was adopted to set its safety limit at 1.5 μg·d -1 , combined with the maximum daily dose of this product, the limit of methyl p-toluenesulfonate is set at 0.375ppm.

[0006] At present, the detection methods of p-toluenesulfonate esters mainly include high performance liquid chromatography, liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. Since the impurity limit of methyl p-toluenesulfonate is extremely low, HPLC-MS / MS method is mostly used, but it is expensive. In addition, there is no research on the analysis of methyl p-toluenesulfonate genotoxic impurities in amoxicillin raw materials and preparations.

[0007] Therefore, it is an urgent problem for those skilled in the art to develop a simple, rapid, economical and practical analytical method with strong specificity and high sensitivity for detecting genotoxic impurities of methyl p-toluenesulfonate in amoxicillin raw materials and preparations. Summary of the invention

[0008] In view of this, the present invention provides a method for detecting genotoxic impurities in amoxicillin bulk drug or pharmaceutical preparation. It has the advantages of strong specificity, high sensitivity, good accuracy, and can perform sample detection simply and quickly. It includes: by repeatedly screening and studying the sample pretreatment method, mobile phase selection, chromatographic column and chromatographic conditions, suitable analysis conditions are finally explored. In order to take into account the separation requirements of the main component of amoxicillin and the genotoxic impurity methyl p-toluenesulfonate, after screening, the key detection conditions such as the appropriate solvent and mobile phase ratio, column temperature, etc. are finally selected; in order to reduce the interference of the components such as excipients in the test sample on the determination of genotoxic impurities, the sample pretreatment conditions, ultrasonic extraction time and solvent selection are screened, and the detection method finally determined achieves the purpose of the above invention.

[0009] Among them, the chromatographic conditions for detecting the genotoxic impurity methyl p-toluenesulfonate in amoxicillin raw materials or pharmaceutical preparations by high performance liquid chromatography were optimized, the optimal chromatographic conditions were found, and the optimal conditions were used to verify the methodology for the determination of methyl p-toluenesulfonate content;

[0010] Due to the low limit of methyl p-toluenesulfonate, the concentration of the test solution needs to be increased. The concentration of amoxicillin is 0.4g / ml. Literature research found that at this concentration, amoxicillin is in a gel state in both water and methanol and cannot be extracted; although it can be dissolved in 5mol / L sodium hydroxide solution, methyl p-toluenesulfonate will hydrolyze in a strong alkaline solution; it can be dissolved in dimethyl sulfoxide, but such a high concentration of solution is very easy to precipitate in the chromatographic system, resulting in problems such as chromatographic column blockage; acetonitrile was finally selected as the solvent. At this concentration, amoxicillin can be well dispersed in acetonitrile, which is conducive to extraction treatments such as vortexing and ultrasound, and methyl p-toluenesulfonate has good stability in acetonitrile;

[0011] In order to improve the detection sensitivity, a small particle size chromatographic column Waters SunFire-C18 (4.6×150mm, 3μm) was selected for determination; after screening, the mobile phase was finally determined to be acetonitrile-water (volume ratio 46:54), which can well separate the methyl p-toluenesulfonate peak from the solvent peak and the raw material amoxicillin peak.

[0012] In order to achieve the above object, the present invention adopts the following technical solution:

[0013] A method for detecting genotoxic impurities in amoxicillin raw materials or pharmaceutical preparations comprises the following steps:

[0014] (1) Pre-treatment:

[0015] 1) Preparation of methyl p-toluenesulfonate reference solution: accurately weigh an appropriate amount of methyl p-toluenesulfonate, dissolve and dilute with acetonitrile to prepare the reference stock solution, then dilute to the required concentration, shake well, and prepare the reference solution;

[0016] 2) Preparation of test solution: Weigh an appropriate amount of amoxicillin, accurately add acetonitrile to disperse, seal, sonicate, take out, cool, vortex, let stand, centrifuge, and take the supernatant as the test solution;

[0017] 3) Preparation of system suitability solution: Weigh an appropriate amount of amoxicillin, accurately add the reference solution of step 1) to disperse, seal, sonicate, take out, cool, vortex, stand, centrifuge, and take the supernatant as the system suitability solution;

[0018] (2) Use high performance liquid chromatography to perform mass analysis on methyl p-toluenesulfonate: Chromatographic conditions: Waters SunFire-C18 column, detection wavelength: 226-228 nm; mobile phase: acetonitrile-water; flow rate: 0.5-1.0 ml / min, column temperature: 30-40°C; injection volume: 5-15 μl; elution time: 8-15 min.

[0019] Preferably, in step (1), 1) is specifically as follows: accurately weigh an appropriate amount of methyl p-toluenesulfonate, dissolve it in acetonitrile and dilute it to make a solution containing 15 μg per 1 ml as the reference substance stock solution, accurately measure 1 ml of the reference substance stock solution, add acetonitrile to dilute it to 100 ml, shake well, and use it as the reference substance solution.

[0020] Preferably, step (1) 2) is as follows: weigh 4 g of amoxicillin, accurately add 10 ml of acetonitrile to disperse, seal, ultrasonicate for 8 to 10 minutes, take out, cool at 20 to 30° C., vortex for 1 to 3 minutes, let stand for 1 to 3 minutes, centrifuge at 9000 to 12000 rpm for 10 minutes, and take the supernatant as the test solution.

[0021] Preferably, step (1) 2) is as follows: weigh 4 g of amoxicillin, accurately add 10 ml of acetonitrile to disperse, seal, ultrasonicate for 10 minutes, take out, cool at 25°C, vortex for 1 minute, let stand for 1 to 2 minutes, centrifuge at 9000 rpm for 10 minutes, and take the supernatant as the test solution.

[0022] Preferably, in step (1), 3) is specifically as follows: weigh 4 g of amoxicillin, accurately add 10 ml of the reference solution of step 1) to disperse, seal, sonicate for 10 minutes, take out, cool at 25°C, vortex for 1 minute, let stand for 1 minute, centrifuge at 9000 rpm for 10 minutes, and take the supernatant as the system suitability solution.

[0023] Preferably: step (2) chromatographic conditions: Waters SunFire-C18 column, 4.6×150 mm, 3 μm; detection wavelength: 227 nm; mobile phase is acetonitrile-water, volume ratio is 46:54; flow rate: 0.8 ml / min, column temperature: 35°C; injection volume: 10 μl; elution time is 10 min.

[0024] Preferably, the detector involved in the high performance liquid chromatography is a diode array detector.

[0025] The present invention also provides application of any of the above methods in pharmaceutical preparation.

[0026] Preferred: Medicine: amoxicillin tablets, sustained-release tablets, controlled-release tablets, dispersible tablets, orally disintegrating tablets and capsules.

[0027] Preferred: Testing for quality and safety control.

[0028] It can be seen from the above technical scheme that, compared with the prior art, the present invention discloses a method for detecting genotoxic impurities in amoxicillin raw materials or pharmaceutical preparations, and the technical effects achieved are:

[0029] 1. The present invention can effectively separate amoxicillin from the genotoxic impurity methyl p-toluenesulfonate with good separation degree, and the blank solvent acetonitrile does not interfere with the determination of methyl p-toluenesulfonate, and the detection method has strong specificity.

[0030] 2. The present invention can quantify the genotoxic impurity methyl p-toluenesulfonate with high sensitivity, and the detection limit of methyl p-toluenesulfonate is 0.0037 μg / ml (0.01 ppm). The concentration reference solution corresponding to the quantitative limit was sampled 6 times continuously, and the result showed that the quantitative limit of methyl p-toluenesulfonate was 0.0155 μg / ml (0.04 ppm). It is proved that the sensitivity of this detection method is much higher than that of LC-MS / MS method and other HPLC methods in the literature.

[0031] 3. The present invention adds a pretreatment step (dispersion, vortexing, centrifugation, etc.) to the sample, thereby greatly reducing the sample concentration in the injection solution and reducing the interference of high-concentration sample on impurity determination; and the injection volume is set at 10 μl, which protects the chromatographic column and makes the test results more accurate. The accuracy test results show that the average recovery rate is 102.65% and the RSD is 0.61%, which proves that the accuracy of this method is reliable.

[0032] 4. The elution mode in the chromatographic conditions for determining the genotoxic impurity methyl p-toluenesulfonate of the present invention is isocratic elution, and the mobile phase and diluent are simple and easy to obtain, low cost, economical and practical; and methyl p-toluenesulfonate peaks within 10 minutes, and the method is fast and simple.

[0033] 5. The present invention has been verified by methodology, and the results all meet the requirements, proving that the present invention can be used for the quantitative determination of methyl p-toluenesulfonate, a genotoxic impurity in amoxicillin. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] 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.

[0035] Figure 1 The accompanying drawing is a chromatogram of blank diluent acetonitrile under the optimal chromatographic conditions provided by the present invention.

[0036] Figure 2 The accompanying drawing is a chromatogram of the system suitability solution under the optimal chromatographic conditions provided by the present invention.

[0037] Figure 3 The accompanying drawing is a chromatogram of methyl p-toluenesulfonate under the optimal chromatographic conditions provided by the present invention.

[0038] Figure 4 The accompanying drawing is a chromatogram of the test solution under the optimal chromatographic conditions provided by the present invention. DETAILED DESCRIPTION

[0039] 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.

[0040] The embodiment of the invention discloses a method for detecting genotoxic impurities in amoxicillin raw material medicine or pharmaceutical preparation.

[0041] The instruments not mentioned in the examples are conventional instruments, the experimental steps not mentioned are conventional experimental steps, and the raw materials not mentioned are conventional commercially available raw materials, for example:

[0042] 1. Instrument:

[0043] Agilent 1260 high performance liquid chromatograph, equipped with a diode array detector, an online degasser, an autosampler, a quaternary pump and a LabStation chromatography workstation; SB5200D ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.); XW-80 vortex mixer (Shanghai Medical University Instrument Factory); TGL-16gR centrifuge (Shanghai Anting Technology Instrument Factory); UV-2550 ultraviolet spectrophotometer (Shimadzu); METTLER TOLEDO MS105DU analytical balance (Mettler-Toledo, Switzerland).

[0044] 2. Reagents:

[0045] Amoxicillin raw material (Tonglian Pharmaceutical Co., Ltd., batch numbers: 220622008, 220622018, 220622019, 220622030, 220622031, 220622032, 220622040, 220622042, 220622045); methyl p-toluenesulfonate (Aladdin, content 98.0%, batch number E1528080); acetonitrile (chromatographic grade, batch number 230801, Concord Reagent Co., Ltd.); the remaining reagents were all domestically produced analytical grade, and the experimental water was ultrapure water.

[0046] In the embodiment, 10 μl of the reference solution is accurately measured and injected into the high performance liquid chromatograph, and the sample is injected six times continuously. The chromatogram is recorded, and the precision of the six injections is examined. The relative standard deviation shall not be greater than 2.0%; then 10 μl of the test solution is accurately measured and injected into the high performance liquid chromatograph, and the chromatogram is recorded.

[0047] Calculate by peak area using the external standard method. The calculation formula is:

[0049] In the formula: As is the average peak area of ​​impurities in the reference solution; Cs is the concentration of impurities in the reference solution (ng / mL); At is the average peak area of ​​impurities in the test solution; Ct is the concentration of impurities in the test solution (mg / mL).

[0050] Example 1

[0051] A method for detecting genotoxic impurities in amoxicillin raw materials or pharmaceutical preparations comprises the following steps:

[0052] (1) Pre-treatment:

[0053] 1) Preparation of methyl p-toluenesulfonate reference solution: Accurately weigh an appropriate amount of methyl p-toluenesulfonate, dissolve it in acetonitrile and dilute it to make a solution containing (approximately) 15 μg per 1 ml as the reference stock solution. Accurately measure 1 ml of the reference stock solution and place it in a 100 ml volumetric flask, add acetonitrile to dilute to the mark, shake well, and use it as the reference solution;

[0054] 2) Preparation of test solution: Weigh 4 g of amoxicillin (approximately), place in a 25 ml stoppered conical flask, accurately add 10 ml of acetonitrile to disperse, seal, sonicate for 10 minutes, take out, cool to 25°C, vortex for 1 minute, let stand for 1 minute, centrifuge at 9000 rpm for 10 minutes, and take the supernatant as the test solution;

[0055] 3) Preparation of system suitability solution: Weigh 4 g of amoxicillin (approximately), place in a 25 ml conical flask with a stopper, accurately add 10 ml of the reference solution in step 1) to disperse, seal, sonicate for 10 minutes, take out, cool to 25°C, vortex for 1 minute, let stand for 1 minute, centrifuge at 9000 rpm for 10 minutes, and take the supernatant as the system suitability solution;

[0056] (2) The genotoxic impurity methyl p-toluenesulfonate in amoxicillin raw materials or pharmaceutical preparations was analyzed by high performance liquid chromatography: the detector involved in the high performance liquid chromatography was a diode array detector; the chromatographic conditions were: Waters SunFire-C18 column (i.e., octadecyl bonded silica column, 4.6×150 mm, 3 μm); the detection wavelength was 227 nm; the mobile phase was acetonitrile-water (volume ratio of 46:54); the flow rate was 0.8 ml / min, the column temperature was 35°C; the injection volume was 10 μl; and the elution time was 10 min.

[0057] Example 2

[0058] The only difference from Example 1 is:

[0059] 2) Preparation of test solution: Weigh 4 g of amoxicillin (approximately), place in a 25 ml stoppered conical flask, accurately add 10 ml of acetonitrile to disperse, seal, sonicate for 8 minutes, take out, cool to 20°C, vortex for 2 minutes, let stand for 2 minutes, centrifuge at 10,000 rpm for 10 minutes, and take the supernatant as the test solution;

[0060] In the chromatographic conditions of step (2), the detection wavelength is 226 nm; the mobile phase is acetonitrile-water; the flow rate is 0.5 ml / min, the column temperature is 3° C.; the injection volume is 5 μl; and the elution time is 8 min.

[0061] Example 3

[0062] The only difference from Example 1 is:

[0063] 2) Preparation of test solution: Weigh 4 g of amoxicillin (approximately), place in a 25 ml conical flask with a stopper, accurately add 10 ml of acetonitrile to disperse, seal, sonicate for 10 minutes, take out, cool to 30°C, vortex for 3 minutes, let stand for 3 minutes, centrifuge at 12,000 rpm for 10 minutes, and take the supernatant as the test solution;

[0064] In the chromatographic conditions of step (2), the detection wavelength is 228 nm; the mobile phase is acetonitrile-water; the flow rate is 1.0 ml / min, the column temperature is 40° C.; the injection volume is 15 μl; and the elution time is 15 min.

[0065] Technical effect verification:

[0066] The technical effect was verified according to the method for detecting genotoxic impurities in amoxicillin bulk drug or pharmaceutical preparation provided in Example 1.

[0067] Specificity test

[0068] Measure 5 μl of each of blank solvent acetonitrile, system suitability solution, methyl p-toluenesulfonate reference solution (0.15 μg / mL), and amoxicillin test solution (batch number: 220622008) and inject them into the HPLC instrument. Record the chromatogram. The results are shown in the table. Figures 1 to 4 The results show that the retention times of the methyl p-toluenesulfonate reference substance and the main component amoxicillin are 2.910 min and 0.787 min, respectively. The separation degree of the amoxicillin peak and the methyl p-toluenesulfonate peak in the system suitability solution meets the requirements and can be well separated. The blank diluent acetonitrile does not interfere with the determination of methyl p-toluenesulfonate, indicating that the method of the present invention has good specificity.

[0069] Limit of Quantitation and Limit of Detection Tests

[0070] Take the methyl p-toluenesulfonate reference solution, add acetonitrile to dilute it step by step, accurately measure 5μl and inject it into the liquid chromatograph, record the chromatogram, and use the solution concentrations with signal-to-noise ratios of approximately 3:1 and 10:1 as the detection limit and quantification limit of p-toluenesulfonate, respectively. The result shows that the detection limit of methyl p-toluenesulfonate is 0.0037μg / ml (0.01ppm). Take the reference solution corresponding to the quantification limit and inject it continuously for 6 times, and the result shows that the quantification limit of methyl p-toluenesulfonate is 0.0155μg / ml (0.04ppm). At the same time, the precision of the retention time and peak area of ​​the quantification limit is investigated. The results of the quantification limit test of methyl p-toluenesulfonate are shown in Table 1.

[0071] Table 1

[0072]

[0073] The results show that this method has high sensitivity and the injection precision meets the requirements, which can meet the requirements of detection.

[0074] Linearity and range

[0075] Accurately measure 1 ml of the reference substance stock solution in Example 1 (step 1), place it in a 10 ml volumetric bottle, dilute it to the mark with acetonitrile, shake it up, accurately measure 0.6 ml, 0.8 ml, 1.0 ml, 1.2 ml, and 1.4 ml respectively, place it in a 10 ml volumetric bottle, dilute it to the mark with acetonitrile, shake it up, and use them as linear-2, linear-3, linear-4, linear-5, and linear-6 solutions. Take the quantitative limit solution in the above quantitative limit and detection limit test as linear-1 solution, accurately measure 10 μl respectively, and inject it into the liquid chromatograph to record the chromatogram. With peak area A as the ordinate and concentration (C) as the abscissa, perform linear regression, and the specific results are shown in Table 2.

[0076] Table 2

[0077]

[0078] The linear regression equation is A=36.906C+0.0391, and the correlation coefficient r is 0.9999 (n=6). The results show that within the concentration range of 0.0155-0.2175 μg / ml, the concentration of methyl p-toluenesulfonate has a good linear relationship with the peak area.

[0079] Injection precision test

[0080] The reference solution in Example 1 (step 1) was measured and injected six times continuously, and the chromatogram was recorded to examine the precision of the six injections. The precision of the six injections was examined by the peak area of ​​methyl p-toluenesulfonate, and the specific results are shown in Table 3.

[0081] Table 3

[0082]

[0083] It can be seen from the above results that the injection precision of the present invention is good.

[0084] Repeatability test

[0085] According to the method of Example 1 (step 2), six test solutions (batch number: 220622008) were prepared, 5 μl of each solution was accurately measured and injected into the liquid chromatograph, and the chromatogram was recorded. The content of methyl p-toluenesulfonate in the test sample was calculated by the peak area according to the external standard method, and the precision of the six contents was examined. The specific results are shown in Table 4.

[0086] Table 4

[0087] serial number 1 2 3 4 5 6 content% Not detected Not detected Not detected Not detected Not detected Not detected

[0088] The results showed that no methyl p-toluenesulfonate was detected in this batch of samples, indicating that the samples did not contain this genotoxic impurity.

[0089] Accuracy test

[0090] Accurately measure 1 ml of the reference stock solution in Example 1 (step 1), place it in a 100 ml measuring bottle, add acetonitrile and dilute to the mark, shake up, and use it as a reference solution. Weigh about 4 g of amoxicillin (batch number: 220622008), a total of six portions, place them in 25 ml stoppered conical bottles, accurately measure 10 ml of the reference solution, shake up, seal, ultrasonicate for 10 minutes, take out, cool to room temperature, vortex for 1 minute, centrifuge at 9000 rpm for 10 minutes, and take the supernatant as the test solution. Measure 10 μl of the reference solution and the test solution, inject them into a liquid chromatograph, record the chromatogram, calculate the content of methyl p-toluenesulfonate in the test solution by peak area according to the external standard method, and compare it with the theoretical content to calculate the recovery. Specific results are shown in Table 5.

[0091] Table 5

[0092]

[0093] The test results show that the average recovery rate of methyl p-toluenesulfonate determination is 102.65% and the RSD is 0.61% (n=6), which proves that the accuracy of the present invention is good.

[0094] Solution stability test

[0095] Take the reference solution in the accuracy test, accurately measure 10 μl within 24 hours and inject it into the liquid chromatograph, record the chromatogram, and examine the stability of the reference solution. The specific results are shown in Table 6.

[0096] Table 6

[0097] Time (hours) Methyl p-toluenesulfonate peak area 0 5.668 3 5.636 5 5.611 8 5.615 10 5.638 12 5.651 24 5.679 average value 5.643 RSD% 0.45

[0098] From the above results, it can be seen that the methyl p-toluenesulfonate reference solution is stable within 24 hours.

[0099] Durability test

[0100] The separation degree of amoxicillin peak and methyl p-toluenesulfonate peak in the system suitability solution was investigated by slightly changing the analytical method parameters. The system suitability solution in Example 1 (step 3) was taken, and the durability verification range of methyl p-toluenesulfonate residual determination was confirmed according to the chromatographic conditions. The specific durability parameter scheme is shown in Table 7.

[0101] Table 7

[0102]

[0103] According to the durability investigation range confirmed in the above table, the system suitability solution in Example 1 (step 3) was measured to investigate the separation degree of the amoxicillin peak and the methyl p-toluenesulfonate peak in the system suitability solution. The specific results are shown in Table 8.

[0104] Table 8

[0105]

[0106]

[0107] The above results show that the proportion of acetonitrile in the mobile phase is 43% to 48%, the flow rate is 0.5 to 1.0 ml / min, the column temperature is 30 to 40°C, and when the chromatographic columns of the same model but different batches and the chromatographic columns of different models with the same filler are replaced, the separation degree of the methyl p-toluenesulfonate peak and the amoxicillin peak and adjacent peaks in the chromatogram of the spiked test solution meets the requirements, indicating that the method has good durability.

[0108] Example 4

[0109] According to the detection method of Example 1, methyl p-toluenesulfonate in nine batches of amoxicillin raw materials was detected. The specific detection results are shown in Table 9.

[0110] batch number Methyl p-toluenesulfonate content% 220622008 Not detected 220622018 Not detected 220622019 Not detected 220622030 Not detected 220622031 Not detected 220622032 Not detected 220622040 Not detected 220622042 Not detected 220622045 Not detected

[0111] The above results show that methyl p-toluenesulfonate was not detected in the amoxicillin raw materials, and there is no need to include this test item in the quality standard.

[0112] In summary, the present invention establishes a method for determining the genotoxic impurity methyl p-toluenesulfonate in amoxicillin and its preparations by high performance liquid chromatography, which has the advantages of high accuracy, good specificity, high precision, good sensitivity, simplicity and rapidity. The quantitative limit of the method is far lower than the limit, and it is an ideal method for determining the genotoxic impurity methyl p-toluenesulfonate in amoxicillin and its preparations.

[0113] 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.

[0114] 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 genotoxic impurities in amoxicillin bulk drug or pharmaceutical preparation, characterized in that: The following steps are involved: (1) Pre-treatment: 1) Preparation of methyl p-toluenesulfonate reference solution: accurately weigh an appropriate amount of methyl p-toluenesulfonate, dissolve and dilute with acetonitrile to prepare the reference stock solution, then dilute to the required concentration, shake well, and prepare the reference solution; 2) Preparation of test solution: Weigh an appropriate amount of amoxicillin, accurately add acetonitrile to disperse, seal, sonicate, take out, cool, vortex, let stand, centrifuge, and take the supernatant as the test solution; 3) Preparation of system suitability solution: Weigh an appropriate amount of amoxicillin, accurately add the reference solution of step 1) to disperse, seal, sonicate, take out, cool, vortex, stand, centrifuge, and take the supernatant as the system suitability solution; (2) Use high performance liquid chromatography to perform mass analysis on methyl p-toluenesulfonate: Chromatographic conditions: Waters SunFire-C18 column, detection wavelength: 226-228 nm; mobile phase: acetonitrile-water; flow rate: 0.5-1.0 ml / min, column temperature: 30-40°C; injection volume: 5-15 μl; elution time: 8-15 min.

2. The method according to claim 1, characterized in that The step (1) is as follows: accurately weigh an appropriate amount of methyl p-toluenesulfonate, dissolve it in acetonitrile and dilute it to prepare a solution containing 15 μg per 1 ml as a reference stock solution, accurately measure 1 ml of the reference stock solution, dilute it to 100 ml with acetonitrile, and shake well to prepare a reference solution.

3. The method according to claim 1, characterized in that The step (2) described in step (1) is specifically as follows: weigh 4 g of amoxicillin, accurately add 10 ml of acetonitrile to disperse it, seal it, sonicate it for 8 to 10 minutes, take it out, cool it at 20 to 30°C, vortex it for 1 to 3 minutes, let it stand for 1 to 3 minutes, centrifuge it at 9000 to 12000 rpm for 10 minutes, and take the supernatant as the test solution.

4. The method according to claim 3, characterized in that The step (2) described in step (1) is specifically as follows: weigh 4 g of amoxicillin, accurately add 10 ml of acetonitrile to disperse it, seal it, sonicate it for 10 minutes, take it out, cool it at 25°C, vortex it for 1 minute, let it stand for 1 to 2 minutes, centrifuge it at 9000 rpm for 10 minutes, and take the supernatant as the test solution.

5. The method according to claim 1, characterized in that The step (3) in step (1) is as follows: weigh 4 g of amoxicillin, accurately add 10 ml of the reference solution of step 1) to disperse, seal, sonicate for 10 minutes, take out, cool at 25°C, vortex for 1 minute, let stand for 1 minute, centrifuge at 9000 rpm for 10 minutes, and take the supernatant as the system suitability solution.

6. The method according to claim 1, characterized in that The chromatographic conditions of step (2) are as follows: Waters SunFire-C18 column, 4.6×150 mm, 3 μm; detection wavelength: 227 nm; mobile phase is acetonitrile-water, volume ratio is 46:54; flow rate: 0.8 ml / min, column temperature: 35°C; injection volume: 10 μl; elution time is 10 min.

7. The method according to claim 6, characterized in that The detector involved in the high performance liquid chromatography is a diode array detector.

8. Use of the method according to any one of claims 1 to 7 in pharmaceutical preparation.

9. The use according to claim 8, characterized in that The drugs include: amoxicillin tablets, sustained-release tablets, controlled-release tablets, dispersible tablets, orally disintegrating tablets and capsules.

10. The use according to claim 9, characterized in that Used for quality and safety control testing.

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