CN-121978253-A - Separation and purification method for degrading impurities in urea 13C granules

Abstract

The application provides a separation and purification method for degrading impurities in urea [13C ] granules, and relates to the technical field of medicine analysis. The method comprises the steps of carrying out acid thermal degradation reaction on urea [13C ] granule samples by using hydrochloric acid solution to obtain degradation reaction liquid, carrying out liquid chromatography separation and purification on the degradation reaction liquid, and collecting fractions rich in steviol-19-O-glucoside. Can obtain the fraction rich in steviol-19-O-glucoside efficiently and accurately. The method avoids time-consuming and labor-consuming structure estimation and directional synthesis steps in the traditional impurity analysis, effectively reduces analysis cost and period, and improves the accuracy of separation of degraded impurities.

Inventors

• Zhan Chuanhong
• WEI CUIWEN
• HE DELONG
• CHEN KAI

Assignees

• 深圳市中核海得威生物科技有限公司

Dates

Publication Date

20260505

Application Date

20260324

Claims (10)

1. 1. A separation and purification method for degrading impurities in urea [13C ] granules is characterized by comprising the following steps: Carrying out acid thermal degradation reaction on a urea [13C ] granule sample by using a hydrochloric acid solution to obtain degradation reaction liquid; And (3) carrying out liquid chromatography separation and purification on the degradation reaction liquid, and collecting fractions rich in steviol-19-O-glucoside.
2. 2. The method for separating and purifying degradation impurities in urea [13C ] granules according to claim 1, wherein the concentration of the hydrochloric acid solution is 0.05-1.0 mol/L.
3. 3. The method for separating and purifying degradation impurities in urea [13C ] granules according to claim 1, wherein the mass of the substrate of urea [13C ] granules is 5-20g when the acid thermal degradation reaction is carried out.
4. 4. The method for separating and purifying degradation impurities in urea [13C ] granules according to claim 1, wherein the hydrochloric acid solution has a volume of 3-10mL when the acid thermal degradation reaction is performed.
5. 5. The method for separating and purifying degradation impurities in urea [13C ] granules according to claim 1, wherein the heating temperature of the acid thermal degradation reaction is 60-80 ℃.
6. 6. The method for separating and purifying degradation impurities in urea [13C ] granules according to claim 1, further comprising, before the liquid chromatography separation and purification, sample preparation: The degradation reaction liquid is adjusted to be neutral by an alkaline solution, and then reduced pressure drying is carried out, so that a concentrated sample containing impurities is obtained.
7. 7. The method for separating and purifying the degraded impurities in the urea [13C ] granules according to claim 1, wherein the liquid chromatography separation and purification adopts an aminopropyl-bonded phase chromatographic column, a mixed solvent of acetonitrile, methanol and water is used as a mobile phase for isocratic elution, and the purified steviol-19-O-glucoside is obtained by drying under reduced pressure after collecting a target fraction.
8. 8. The method for separating and purifying degraded impurities in urea [13C ] granules according to claim 7, wherein the volume ratio of acetonitrile, methanol and water in the mobile phase is 88-90:9-11:1.
9. 9. The method for separating and purifying the degradation impurities in the urea [13C ] granules according to claim 7, wherein the temperature of the reduced pressure drying is 50-75 ℃.
10. 10. The method for separating and purifying the degraded impurities in the urea [13C ] granule according to any one of claims 1 to 9, further comprising performing purity measurement and/or structure confirmation on the collected steviol-19-O-glucoside.

Description

Separation and purification method for degrading impurities in urea 13C granules Technical Field The application relates to the technical field of medicine analysis, in particular to a separation and purification method for degrading impurities in urea [13C ] granules. Background Helicobacter pylori (Hp) is a common pathogenic microorganism colonizing the gastric mucosa, and its infection is closely related to gastrointestinal diseases such as chronic gastritis, dyspepsia, peptic ulcer, etc., and is recognized as an important risk factor for gastric cancer occurrence. The urea 13C expiration detecting reagent is used as the core tool for clinical diagnosis of helicobacter pylori infection and is used widely in the world, and its dosage form includes capsule, powder, granule, oral solution and other forms. During the production, storage and use of the urea 13C granules, the active ingredients may undergo degradation reactions due to environmental factors, generating various impurities including steviol-19-O-glucoside. These impurities, if not accurately identified and effectively controlled, will directly interfere with the reliability of the breath test results, resulting in clinical diagnostic bias. Currently, conventional analysis flow for degrading impurities in pharmaceutical preparations mainly relies on liquid chromatography-mass spectrometry to primarily screen samples, the structure of potential impurities is presumed through molecular weight information, and a synthetic scheme is designed by combining literature data and theoretical degradation paths. The method is time-consuming and tedious, involves multi-step chemical synthesis and verification, is extremely easy to misjudge due to strong subjectivity of structure estimation, for example, the synthesized impurities have obvious differences in actual chemical structures although similar to the spectrum characteristics of the target substances, and a large amount of manpower and material resources are wasted. Particularly, when facing stereoisomers or co-flowing out components with similar retention time, the traditional chromatographic separation means are difficult to accurately distinguish, further aggravate the complexity of impurity structure analysis, and lead the research of degradation mechanism to be in trouble. In view of the above, there is a need in the art for improvements. Disclosure of Invention The application aims to provide a separation and purification method for degrading impurities in urea [13C ] granules, so as to solve the problems. In order to achieve the above purpose, the application adopts the following technical scheme: The application provides a separation and purification method for degrading impurities in urea [13C ] granules, which comprises the following steps: Carrying out acid thermal degradation reaction on a urea [13C ] granule sample by using a hydrochloric acid solution to obtain degradation reaction liquid; And (3) carrying out liquid chromatography separation and purification on the degradation reaction liquid, and collecting fractions rich in steviol-19-O-glucoside. Further, the concentration of the hydrochloric acid solution is 0.05-1.0 mol/L. Further, the substrate mass of the urea [13C ] granule is 5-20g when the acid thermal degradation reaction is carried out. Further, when the acid thermal degradation reaction is carried out, the volume of the hydrochloric acid solution is 3-10mL. Further, the heating temperature of the acid thermal degradation reaction is 60-80 ℃. Further, before the liquid chromatography separation and purification, the method further comprises sample preparation: The degradation reaction liquid is adjusted to be neutral by an alkaline solution, and then reduced pressure drying is carried out, so that a concentrated sample containing impurities is obtained. Further, the liquid chromatography separation and purification adopts an aminopropyl-bonded phase chromatographic column, a mixed solvent of acetonitrile, methanol and water is used as a mobile phase for isocratic elution, and the target fraction is collected and then dried under reduced pressure to obtain the purified steviol-19-O-glucoside. Further, the volume ratio of acetonitrile, methanol and water in the mobile phase is 88-90:9-11:1. Further, the temperature of the reduced pressure drying is 50-75 ℃. Further, the method also comprises the step of carrying out purity measurement and/or structure confirmation on the collected steviol-19-O-glucoside. Compared with the prior art, the application has the beneficial effects that: The method provided by the application can be used for efficiently and accurately obtaining the fraction rich in steviol-19-O-glucoside by directly carrying out acid thermal degradation on the urea [13C ] granule sample and combining with liquid chromatography separation and purification. The method avoids the time-consuming and labor-consuming structure estimation and directional synthesis steps in the