Separation and purification method for degradation impurities in urea [13C] granules

Abstract

The invention provides a separation and purification method for degradation impurities in urea [13C] granules, and relates to the technical field of pharmaceutical analysis. The method comprises the following steps: carrying out an acid thermal degradation reaction on a urea [13C] granule sample by using a hydrochloric acid solution to obtain a degradation reaction solution; and carrying out liquid chromatography separation and purification on the degradation reaction liquid, and collecting a fraction rich in steviol-19-O-glucoside. And the fraction rich in steviol-19-O-glucoside can be efficiently and accurately obtained. According to the method, time-consuming and labor-consuming structural speculation and directional synthesis steps in traditional impurity analysis are avoided, the analysis cost is effectively reduced, the analysis period is effectively shortened, and meanwhile, the degradation impurity separation accuracy is improved.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical analysis technology, and in particular to a method for separating and purifying degradation impurities in urea [13C] granules. Background Technology

[0002] Helicobacter pylori (Hp) is a common pathogenic microorganism that colonizes the gastric mucosa. Its infection is closely related to gastrointestinal diseases such as chronic gastritis, dyspepsia, and peptic ulcers, and is widely recognized as an important risk factor for gastric cancer. Urea [13C] breath test kits, as a core tool for the clinical diagnosis of Hp infection, are widely used globally, and their dosage forms include capsules, powders, granules, and oral solutions. During the production, storage, and use of urea [13C] granules, environmental factors may cause degradation of the active ingredients, generating various impurities, including steviol-19-O-glucoside. If these impurities are not accurately identified and effectively controlled, they will directly interfere with the reliability of breath test results, leading to clinical diagnostic errors.

[0003] Currently, the conventional analytical process for degradation impurities in pharmaceutical formulations mainly relies on liquid chromatography-mass spectrometry (LC-MS) for preliminary sample screening, inferring the structure of potential impurities based on molecular weight information, and designing synthetic schemes based on literature and theoretical degradation pathways. This method is not only time-consuming and involves multiple chemical synthesis and verification steps, but also prone to misjudgment due to the strong subjectivity of structure inference. For example, although the synthesized impurity may have similar spectral characteristics to the target mass, its actual chemical structure may differ significantly, resulting in a significant waste of human and material resources. Especially when dealing with stereoisomers or co-eluting components with similar retention times, traditional chromatographic separation methods are difficult to distinguish accurately, further exacerbating the complexity of impurity structure analysis and hindering the study of degradation mechanisms. Therefore, existing technologies urgently need improvement to address these issues. Summary of the Invention

[0004] The purpose of this application is to provide a method for separating and purifying degradation impurities in urea [13C] granules to solve the above-mentioned problems.

[0005] To achieve the above objectives, this application adopts the following technical solution: This application provides a method for separating and purifying degradation impurities in urea [13C] granules, comprising: The urea [13C] granule sample was subjected to acid-thermal degradation reaction using hydrochloric acid solution to obtain the degradation reaction solution; The degradation reaction solution was separated and purified by liquid chromatography, and the fraction rich in stevioside-19-O-glucoside was collected.

[0006] Furthermore, the concentration of the hydrochloric acid solution is 0.05~1.0 mol / L.

[0007] Furthermore, during the acid-thermal degradation reaction, the substrate mass of the urea [13C] granules is 5-20 g.

[0008] Furthermore, during the acid-thermal degradation reaction, the volume of the hydrochloric acid solution is 3-10 mL.

[0009] Furthermore, the heating temperature for the acid thermal degradation reaction is 60-80℃.

[0010] Furthermore, sample preparation is included before performing the liquid chromatography separation and purification: The degradation reaction solution was adjusted to neutral using an alkaline solution, and then dried under reduced pressure to obtain a concentrated sample containing impurities.

[0011] Furthermore, the liquid chromatography separation and purification uses an aminopropyl bonded phase column, with a mixed solvent of acetonitrile, methanol and water as the mobile phase for isocratic elution. After collecting the target fraction, it is dried under reduced pressure to obtain purified steviol-19-O-glucoside.

[0012] Furthermore, the volume ratio of acetonitrile, methanol and water in the mobile phase is 88~90: 9~11:1.

[0013] Furthermore, the temperature for vacuum drying is 50~75℃.

[0014] Furthermore, it also includes purity determination and / or structural confirmation of the collected steviol-19-O-glucoside.

[0015] Compared with the prior art, the beneficial effects of this application include: The method provided in this application directly degrades urea [13C] granule samples through acid-thermal degradation, combined with liquid chromatography separation and purification, enabling efficient and accurate acquisition of fractions rich in steviol-19-O-glucoside. This method avoids the time-consuming and labor-intensive structural deduction and directed synthesis steps in traditional impurity analysis, effectively reducing analytical costs and cycles, while improving the accuracy of degraded impurity separation, especially showing significant advantages in isomer differentiation, thus solving the problems of resource waste and misjudgment in existing technologies. On the other hand, the method provided in this application also provides a reference for impurity research and quality control of products that may contain new degraded impurities not included in the pharmacopoeia in urea [13C] granules. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation on the scope of this application.

[0017] Figure 1 The chromatogram of steviol-19-O-glucoside, an impurity detected in urea [13C] granules in Example 1; Figure 2 The chromatogram of the impurity steviol-19-O-glucoside obtained in Example 1 is shown below. Figure 3 This is a high-resolution mass spectrum of the impurity steviol-19-O-glucoside obtained in Example 1. Detailed Implementation

[0018] As used in this article: "Prepared from" is synonymous with "comprising". The terms "comprising", "including", "having", "containing", or any other variations thereof as used herein are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0019] The conjunction "composed of..." excludes any unspecified elements, steps, or components. If used in a claim, this phrase makes the claim closed, excluding materials other than those described, except for associated conventional impurities. When the phrase "composed of..." appears in a clause of the body of a claim rather than immediately following it, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0020] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1–5” is disclosed, the described range should be interpreted as including ranges “1–4”, “1–3”, “1–2”, “1–2 and 4–5”, “1–3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0021] In these embodiments, unless otherwise specified, the portions and percentages are all by weight.

[0022] "Parts by mass" refers to the basic unit of measurement that expresses the mass ratio of multiple components. One part can represent any unit mass, such as 1g or 2.689g. If we say that component A has "a" parts by mass and component B has "b" parts by mass, it means the ratio of the mass of component A to the mass of component B is a:b. Alternatively, it can mean that the mass of component A is aK and the mass of component B is bK (where K is any number representing a multiplier). It is important to understand that, unlike parts by mass, the sum of the mass parts of all components is not limited to 100 parts.

[0023] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0024] To better explain the technical solution provided in this application, the technical solution provided in this application will be described in general before the specific implementation.

[0025] This application provides a method for separating and purifying degradation impurities in urea [13C] granules, comprising: The urea [13C] granule sample was subjected to acid-thermal degradation reaction using hydrochloric acid solution to obtain the degradation reaction solution; The degradation reaction solution was separated and purified by liquid chromatography, and the fraction rich in stevioside-19-O-glucoside was collected.

[0026] Understandably, this application proposes using hydrochloric acid solution to perform an acid-thermal degradation reaction on urea [13C] granule samples to obtain a degradation reaction solution. However, if the concentration of the hydrochloric acid solution is not precisely defined, the conditions of the acid-thermal degradation reaction may be unstable, thereby affecting the degradation efficiency and the reproducibility of product distribution. This makes it difficult to achieve the best effect in subsequent liquid chromatography separation and purification, and may even introduce unnecessary side reactions or incomplete degradation, thus affecting the effective separation and purification of the target degradation impurities.

[0027] In one optional embodiment, the concentration of the hydrochloric acid solution is 0.05~1.0 mol / L.

[0028] Optionally, the concentration of the hydrochloric acid solution can be 0.05 mol / L, 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, 0.3 mol / L, 0.35 mol / L, 0.4 mol / L, 0.45 mol / L, 0.5 mol / L, 0.55 mol / L, 0.6 mol / L, 0.65 mol / L, 0.7 mol / L, 0.75 mol / L, 0.8 mol / L, 0.85 mol / L, 0.9 mol / L, 0.95 mol / L, 1 mol / L, or any value between 0.05 and 1 mol / L.

[0029] Hydrochloric acid solution serves as both a catalyst and reaction medium in the acid-thermal degradation reaction, and its concentration is a key factor affecting the reaction rate, selectivity, and product stability. The concentration of hydrochloric acid solution was precisely set to 0.1 mol / L to provide a moderate and stable acidic environment. At this concentration, the hydrolysis and dissociation of degradation impurities (such as precursors or bound forms of steviol-19-O-glucoside) in urea [13C] granules can be effectively promoted, transforming them into forms that can be separated and purified by subsequent liquid chromatography. Simultaneously, this concentration avoids the problems of excessive degradation, target product structural damage, or the generation of more complex byproducts that may result from excessively high acidity, and also avoids the problems of incomplete degradation, excessively long reaction times, or low efficiency caused by excessively low acidity.

[0030] In one alternative embodiment, the substrate mass of the urea [13C] granules is 5-20 g when the acid-thermal degradation reaction is carried out.

[0031] Optionally, the substrate mass of the urea [13C] granules can be 5g, 6g, 7g, 8g, 9g, 10g, 11g, 12g, 13g, 14g, 15g, 16g, 17g, 18g, 19g, 20g, or any value between 5g and 20g, for the acid-thermal degradation reaction.

[0032] In one optional embodiment, the volume of the hydrochloric acid solution is 3-10 mL during the acid thermal degradation reaction.

[0033] Optionally, the volume of the hydrochloric acid solution can be 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, or any value between 3 and 10 mL.

[0034] In one optional embodiment, the heating temperature of the acid thermal degradation reaction is 60-80°C.

[0035] Optionally, the heating temperature for the acid thermal degradation reaction can be 60℃, 62℃, 64℃, 66℃, 68℃, 70℃, 72℃, 74℃, 76℃, 79℃, 80℃, or any value between 60℃ and 80℃.

[0036] In an optional implementation, sample preparation is further included prior to the liquid chromatography separation and purification: The degradation reaction solution was adjusted to neutral using an alkaline solution, and then dried under reduced pressure to obtain a concentrated sample containing impurities.

[0037] In an optional embodiment, the liquid chromatography separation and purification uses an aminopropyl bonded phase column, with a mixed solvent of acetonitrile, methanol and water as the mobile phase for isocratic elution. After collecting the target fraction, it is dried under reduced pressure to obtain purified steviol-19-O-glucoside.

[0038] In one optional embodiment, the volume ratio of acetonitrile, methanol and water in the mobile phase is 88:11:1 to 90:9:1.

[0039] Optionally, the volume ratio of acetonitrile, methanol and water in the mobile phase can be 88:11:1, 89:10:1, 90:9:1, or any value between 88 and 90:9 and 11:1.

[0040] In one optional embodiment, the temperature of the vacuum drying is 50~75°C.

[0041] Optionally, the temperature for vacuum drying can be 50℃, 60℃, 65℃, 70℃, 75℃, or any value between 50℃ and 70℃.

[0042] In an optional embodiment, the purity of the collected steviol-19-O-glucoside and / or structural confirmation are further included.

[0043] By employing the above technical solution, after the separation and purification process, the purity of the collected steviol-19-O-glucoside is determined and / or its structure is confirmed, effectively eliminating potential doubts about the product's quality and identity. This quality control step ensures that the obtained steviol-19-O-glucoside has a clear purity and accurate chemical structure, greatly improving the reliability of the entire separation and purification method and the credibility of the product. This is crucial for applying the purified product to subsequent fields such as analytical standard preparation, drug development, food additive production, or bioactivity research, avoiding experimental errors or product quality problems caused by the use of impure or structurally uncertain substances, thereby ensuring the effectiveness and safety of the final application.

[0044] The implementation schemes of this application will be described in detail below with reference to specific embodiments. However, those skilled in the art will understand that the following embodiments are only for illustrating this application and should not be regarded as limiting the scope of this application. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used without specified manufacturers are all conventional products that can be purchased commercially.

[0045] Example 1 This embodiment provides a method for separating and purifying degradation impurities in urea [13C] granules, the specific steps of which are as follows: Acid-thermal degradation: Accurately weigh 5g of urea [13C] granule sample and place it in a reaction flask. Add 3mL of 0.1mol / L hydrochloric acid solution. Heat the mixture in a 65℃ water bath for 3h to obtain the degradation reaction solution.

[0046] Sample preparation: Cool the above reaction solution to room temperature, add 0.1 mol / L sodium hydroxide solution dropwise, and adjust the pH to 7.0. Transfer the neutral solution to a round-bottom flask and dry under reduced pressure in a vacuum drying oven at 60℃ to obtain a concentrated sample containing the target impurity.

[0047] Preparation and separation: Column: Thermo Hypersil GOLD Amino (250mm × 4.6mm, 5μm).

[0048] Mobile phase: Acetonitrile: Methanol: Water = 89:10:1, isocratic elution.

[0049] Flow rate: 1.0 mL / min; Detector: Diode array detector (DAD).

[0050] Inject the concentrated sample solution and collect the fractions with retention times between 17 and 35 minutes (corresponding to steviol-19-O-glucoside) based on the chromatogram.

[0051] Post-processing and confirmation: The target fractions were combined and dried under reduced pressure at 60°C to obtain a white powdery solid.

[0052] The chromatogram of the impurity steviol-19-O-glucoside detected in urea [13C] granules is shown below. Figure 1 As shown; the chromatogram of the obtained impurity steviol-19-O-glucoside is as follows. Figure 2 As shown; the high-resolution mass spectrum of the impurity steviol-19-O-glucoside is as follows. Figure 3 As shown.

[0053] Example 2 This embodiment provides a method for separating and purifying degradation impurities in urea [13C] granules, the specific steps of which are as follows: Acid-thermal degradation: Accurately weigh 20g of urea [13C] granule sample and place it in a reaction flask. Add 5mL of 0.1mol / L hydrochloric acid solution. Heat the mixture in a 65℃ water bath for 3h to obtain the degradation reaction solution.

[0054] Sample preparation: Cool the above reaction solution to room temperature, add 0.1 mol / L sodium hydroxide solution dropwise, and adjust the pH to 7.0. Transfer the neutral solution to a round-bottom flask and dry under reduced pressure in a vacuum drying oven at 60℃ to obtain a concentrated sample containing the target impurity.

[0055] Preparation and separation: Column: Thermo Hypersil GOLD Amino (250mm × 4.6mm, 5μm).

[0056] Mobile phase: Acetonitrile: Methanol: Water = 89:10:1, isocratic elution.

[0057] Flow rate: 1.0 mL / min; Detector: Diode array detector (DAD).

[0058] Inject the concentrated sample solution and collect the fractions with retention times between 17 and 35 minutes (corresponding to steviol-19-O-glucoside) based on the chromatogram.

[0059] Post-processing and confirmation: The target fractions were combined and dried under reduced pressure at 60°C to obtain a white powdery solid.

[0060] Example 3 This embodiment provides a method for separating and purifying degradation impurities in urea [13C] granules, the specific steps of which are as follows: Acid-thermal degradation: Accurately weigh 20g of urea [13C] granule sample and place it in a reaction flask. Add 10mL of 0.1mol / L hydrochloric acid solution. Heat the mixture in a 65℃ water bath for 3h to obtain the degradation reaction solution.

[0061] Sample preparation: Cool the above reaction solution to room temperature, add 0.1 mol / L sodium hydroxide solution dropwise, and adjust the pH to 7.0. Transfer the neutral solution to a round-bottom flask and dry under reduced pressure in a vacuum drying oven at 60℃ to obtain a concentrated sample containing the target impurity.

[0062] Preparation and separation: Column: Thermo Hypersil GOLD Amino (250mm × 4.6mm, 5μm).

[0063] Mobile phase: Acetonitrile: Methanol: Water = 89:10:1, isocratic elution.

[0064] Flow rate: 1.0 mL / min; Detector: Diode array detector (DAD).

[0065] Inject the concentrated sample solution and collect the fractions with retention times between 17 and 35 minutes (corresponding to steviol-19-O-glucoside) based on the chromatogram.

[0066] Post-processing and confirmation: The target fractions were combined and dried under reduced pressure at 60°C to obtain a white powdery solid.

[0067] Comparative Example 1 This comparative example provides a method for separating and purifying degradation impurities in urea [13C] granules. The only difference from Example 1 is that 3 mL of 2 mol / L hydrochloric acid solution is added in the acid-thermal degradation step. The remaining steps and parameter settings are the same as in Example 1.

[0068] Comparative Example 2 This comparative example provides a method for separating and purifying degradation impurities in urea [13C] granules. The only difference from Example 1 is that in the acid-thermal degradation step, the mixture is heated in a 100°C water bath for 3 hours to obtain a degradation reaction solution. The remaining steps and parameter settings are the same as in Example 1.

[0069] Comparative Example 3 This comparative example provides a method for separating and purifying degradation impurities in urea [13C] granules. The only difference from Example 3 is that 15 mL of 0.1 mol / L hydrochloric acid solution is added in the acid-thermal degradation step. The remaining steps and parameter settings are the same as in Example 1.

[0070] Comparative Example 4 This comparative example provides a method for separating and purifying degradation impurities in urea [13C] granules. The only difference from Example 2 is that the 0.1 mol / L hydrochloric acid solution is replaced with 5 mL of water. The remaining steps and parameter settings are the same as in Example 1.

[0071] The conditions and results of the embodiments and comparative examples are shown in Table 1: Table 1. Conditions and target impurity content of the examples and comparative examples.

[0072] It can be seen that: in Comparative Example 1, the target impurity was not obtained due to excessive acid concentration and excessive degradation; in Comparative Example 2, the target impurity was measured at 0.16%, and there were many other degradation impurities, which caused significant interference; in Comparative Example 3, the amount of hydrochloric acid was increased to 15 ml, and the yield of the target impurity increased to 39.1%, but the amount of other degradation impurities increased accordingly, and the amount of hydrochloric acid was too large, making it difficult to remove them later; in Comparative Example 4, the target impurity was measured at 0.72% after replacing hydrochloric acid with water, and the yield was relatively low.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

[0074] Furthermore, those skilled in the art will understand that although some embodiments herein include certain features included in other embodiments but not others, combinations of features from different embodiments are intended to be within the scope of this application and form different embodiments. For example, in the foregoing claims, any of the claimed embodiments can be used in any combination. The information disclosed in this background section is intended only to enhance the understanding of the general background of this application and should not be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

Claims

1. A method for separating and purifying degradation impurities in urea [13C] granules, characterized in that, include: The urea [13C] granule sample was subjected to acid-thermal degradation reaction using hydrochloric acid solution to obtain the degradation reaction solution; The degradation reaction solution was separated and purified by liquid chromatography, and the fraction rich in stevioside-19-O-glucoside was collected.

2. The method for separating and purifying degradation impurities in urea [13C] granules according to claim 1, characterized in that, The concentration of the hydrochloric acid solution is 0.05~1.0 mol / L.

3. The method for separating and purifying degradation impurities in urea [13C] granules according to claim 1, characterized in that, When the acid-thermal degradation reaction is carried out, the substrate mass of urea [13C] granules is 5-20g.

4. The method for separating and purifying degradation impurities in urea [13C] granules according to claim 1, characterized in that, When the acid thermal degradation reaction is carried out, the volume of the hydrochloric acid solution is 3-10 mL.

5. The method for separating and purifying degradation impurities in urea [13C] granules according to claim 1, characterized in that, The heating temperature for the acid thermal degradation reaction is 60-80℃.

6. The method for separating and purifying degradation impurities in urea [13C] granules according to claim 1, characterized in that, Before performing the liquid chromatography separation and purification, sample preparation is also included: The degradation reaction solution was adjusted to neutral using an alkaline solution, and then dried under reduced pressure to obtain a concentrated sample containing impurities.

7. The method for separating and purifying degradation impurities in urea [13C] granules according to claim 1, characterized in that, The liquid chromatography separation and purification was performed using an aminopropyl bonded phase column, with isocratic elution using a mixed solvent of acetonitrile, methanol and water as the mobile phase. After collecting the target fraction, it was dried under reduced pressure to obtain purified steviol-19-O-glucoside.

8. The method for separating and purifying degradation impurities in urea [13C] granules according to claim 7, characterized in that, The volume ratio of acetonitrile, methanol and water in the mobile phase is 88~90: 9~11:

1.

9. The method for separating and purifying degradation impurities in urea [13C] granules according to claim 7, characterized in that, The temperature for vacuum drying is 50~75℃.

10. The method for separating and purifying degradation impurities in urea [13C] granules according to any one of claims 1-9, characterized in that, It also includes purity determination and / or structural confirmation of the collected steviol-19-O-glucoside.

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