阅读说明：本技术 头孢拉定中乙酰乙酸甲酯的检测方法 (Method for detecting methyl acetoacetate in cefradine ) 是由 王付荣 吴辉 李丹 于 2020-06-24 设计创作，主要内容包括：本发明提供一种头孢拉定中杂质乙酰乙酸甲酯的检测方法,采用GC外标法对头孢拉定中的乙酰乙酸甲酯进行定性和定量分析,实验操作简捷,分析检测过程中用水做溶剂,无污染,色谱峰无干扰,峰型对称,方法学验证结果均符合要求,说明本检测方法专属性好,灵敏度高,定量准确；本发明提供的检测方法适用于头孢拉定中的乙酰乙酸甲酯的检测,便于规模化生产和质控过程中产品质量的控制。(The invention provides a method for detecting impurity methyl acetoacetate in cefradine, which adopts a GC external standard method to carry out qualitative and quantitative analysis on methyl acetoacetate in cefradine, has simple and convenient experimental operation, uses water as a solvent in the analysis and detection process, has no pollution, has no interference on chromatographic peaks, has symmetrical peak patterns and meets the requirements of methodological verification results, and shows that the detection method has good specificity, high sensitivity and accurate quantification; the detection method provided by the invention is suitable for detecting the methyl acetoacetate in the cefradine, and is convenient for large-scale production and product quality control in the quality control process.)

1. The detection method of methyl acetoacetate in cefradine is characterized in that GC is adopted to carry out quantitative and/or qualitative analysis on impurities in cefradine, and the analysis conditions are as follows:

the chromatographic column adopts a capillary column with polyethylene glycol as stationary liquid or a chromatographic column with similar polarity, and the specification of the chromatographic column is 20-60 m multiplied by 0.180-0.530 mm;

the initial temperature is 35-50 ℃, the temperature is maintained for 5-10 minutes, the temperature is raised to 140-180 ℃ at the rate of 15-25 ℃ per minute, and the temperature is maintained for 10-20 minutes; the temperature of a sample inlet is 180-250 ℃; the carrier gas is nitrogen; the detector is a flame ionization detector FID, and the temperature of the detector is 220-250 ℃.

2. The method of claim 1, wherein the impurity methyl acetoacetate has the following formula:

3. the method for detecting methyl acetoacetate in cefradine according to claim 1, wherein the sample solution and the reference solution are directly injected into the sample chamber at a temperature of 200 ℃ and the detector at a temperature of 250 ℃.

4. The method of claim 1, wherein the column size of the column is 30m x 0.530mm, 1.00 μm.

5. The method of claim 1, wherein the initial temperature of the column box is 40 ℃ for 6 minutes, and the temperature is raised to 150 ℃ at a rate of 20 ℃ per minute for 12 minutes.

6. The method of claim 1, wherein the solvent used to prepare the test sample and the control is water.

7. The method of claim 1, wherein the sample solution is prepared by dissolving the sample in a suitable amount of water, and then quantitatively diluting the solution with water to a desired concentration, wherein the concentration of the sample solution is 1-12 mg/ml.

8. The method of claim 1, wherein the concentration of the test solution is 10 mg/ml.

Technical Field

The invention belongs to the field of detection of pharmaceutical impurities, and particularly relates to a method for detecting a related substance methyl acetoacetate in cefradine.

Background

Chemical name of cefradine: (6R,7R) -7- [ (R) -2-amino-2- (1, 4-cyclohexadien-1-yl) acetamido]-3-methyl-8-oxo-5-thia-1-azabicyclo [4.2.0]Oct-2-ene-2-carboxylic acid, molecular formula: c₁₆H₁₉N₃O₄S, molecular weight: 349.40. the structural formula is as follows:

cefradine is a beta-lactam antibiotic and is used for treating respiratory tract infection, genitourinary tract infection, skin and soft tissue infection and the like caused by sensitive bacteria, such as acute pharyngitis, tonsillitis, otitis media, bronchitis, pneumonia and the like. In order to ensure the quality and the efficacy of the cefradine, the detection and the control of related substances are necessary.

At present, detection methods for cefradine related substances are loaded in pharmacopoeias of various countries such as ChP, USP, BP and the like, and the detection methods for the related substances all adopt a reversed phase high performance liquid chromatography.

In the actual operation process, the following defects of the existing method are found:

(1) the ultraviolet absorption of the methyl acetoacetate is very weak, and the detection is carried out by adopting a reversed-phase high performance liquid chromatography detection method of related substances of cefradine in pharmacopoeia, so that the sensitivity is low;

(2) the methyl acetoacetate chromatographic peak has serious tailing, poor peak shape and inaccurate content measurement result, and when the method is used for the quality control of the cefradine in the large-scale production process, the content measurement result of the reversed-phase high performance liquid chromatography has larger error.

Disclosure of Invention

In the related research on cefradine by the inventor of the application, the byproduct can be methyl acetoacetate, the substance has weak ultraviolet absorption, high boiling point, difficult gasification and high detection difficulty, and the content of the impurity needs to be strictly controlled in order to ensure the quality of the bulk drug. However, no literature reports a detection method of methyl acetoacetate in cefradine at present. In order to definitely control the impurity content in the raw material medicine research and enable the impurity content to be controllable, the invention provides a method for detecting and controlling methyl acetoacetate impurities.

The invention provides a method for detecting methyl acetoacetate in cefradine, which adopts GC to carry out quantitative and/or qualitative analysis on impurities in cefradine, wherein the temperature rise procedure is as follows: the initial temperature is 35-50 ℃, the temperature is maintained for 5-10 minutes, the temperature is raised to 140-180 ℃ at the rate of 15-25 ℃ per minute, and the temperature is maintained for 10-20 minutes.

Further, the following chromatographic conditions were also included:

a chromatographic column: a capillary column with polyethylene glycol as stationary liquid or a chromatographic column with similar polarity;

a detector: flame ionization detector FID;

carrier gas: nitrogen gas;

detector temperature: 220-250 ℃;

further, the impurity is methyl acetoacetate, and the structural formula of the impurity is as follows:

furthermore, the sample solution to be tested and the reference solution are directly injected respectively, the injection port temperature is 180-250 ℃, the preferred injection port temperature is 200 ℃, and the preferred detector temperature is 250 ℃.

Further, the sample injection amount is 1.0-5.0 μ l, and the flow rate is 1.0-5.0 ml/min.

Further, the column size is 30m × 0.530mm, 1.00 μm, or equivalent column, preferably AgilentHP-INNOWAX

Further, the temperature-raising program is:

the initial temperature was 40 ℃ for 6 minutes, and the temperature was raised to 150 ℃ at a rate of 20 ℃ per minute for 12 minutes.

Furthermore, the solvent used for preparing the sample solution is water during detection, and the sample solution is prepared by dissolving the sample with a proper amount of water and then quantitatively diluting the sample with water to the required concentration.

Furthermore, the concentration of the test solution is 1-12 mg/ml, preferably 10 mg/ml.

Further, H₂The flow rate was 40ml/min and the air flow rate was 400 ml/min.

Furthermore, the sample injection mode is split sample injection, and the split ratio is 2-20, preferably 10.

Further, when quantitative detection is carried out, an external standard method is adopted for calculation.

The invention has the following beneficial effects: the method adopts a GC external standard method to carry out qualitative and quantitative analysis on the methyl acetoacetate in the cefradine, fully verifies the detection method from the aspects of system applicability and specificity, detection limit and quantification limit, linearity and range, precision, accuracy, durability and the like, and the verification result meets the requirements. The detection method has high sensitivity and accurate quantification. Is suitable for detecting the impurity methyl acetoacetate in the cefradine, and is convenient for controlling the product quality in the production and quality control processes.

Drawings

FIG. 1 is a GC chromatogram of example 1 of the present invention-blank solution, test solution, control solution (12.462 min), and standard test solution (12.463 min);

FIG. 2 is a chromatogram of HPLC chromatographic conditions shown in comparative example 1 of the present invention.

Detailed Description

The method for detecting related substances in cefradine of the present invention is further illustrated by the following specific embodiments. The examples of the present invention are only for illustrating the present invention and not for limiting the present invention, therefore, the simple modification or replacement of the present invention based on the method of the present invention is within the scope of the claimed invention.