Determination method for related substances in carbocisteine aerosol inhalation solution

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for determining related substances of a carbocisteine aerosol inhalation solution. The method adopts a high performance liquid chromatography method, and the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler; carrying out gradient elution by taking phosphate and an ion pair buffer solution as a mobile phase A and methanol as a mobile phase B; the flow rate is 0.6 ml / min to 1.0 ml / min; the column temperature is 30-40 DEG C; the detection wavelength is 200 nm to 230 nm; the sample injection volume is 10-30 [mu] l. The method can be used for detecting the related substances of the carbocisteine aerosol inhalation solution, can detect the related substances including an impurity A, an impurity C, an impurity D, an impurity E, an impurity G and the like, and also has relatively strong detection capability on the related substances of the raw materials. The determination method provided by the invention has the advantages of stable system, durability, high efficiency, simplicity and convenience in operation and easiness in reproduction.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a high-performance liquid chromatography (HPLC) method for the determination of related substances in carbocysteine ​​nebulized inhalation solution. This method is applicable to the accurate detection of related substances (including degradation products, process impurities, etc.) in carbocysteine ​​nebulized inhalation solution during the production process, stability studies, and quality control, ensuring the safety and efficacy of the drug. Background Technology

[0002] Carbocysteine, a classic mucus modifier, plays a vital role in the treatment of respiratory diseases. It is widely used in the clinical treatment of chronic obstructive pulmonary disease (COPD), bronchial asthma, and chronic bronchitis by regulating bronchial secretions and improving sputum viscoelasticity. Nebulized inhalation solutions, as an important dosage form of carbocysteine, can act directly on the respiratory target site, with rapid onset of action and low systemic side effects, making it particularly suitable for elderly and pediatric patients. However, during the production and storage of this formulation, related substances (such as degradation products) may be generated due to the degradation of the active pharmaceutical ingredient or interactions with excipients. These impurities may affect the safety and efficacy of the drug, and even cause adverse reactions. Therefore, establishing an efficient and specific method for the determination of related substances is crucial to ensuring the quality controllability of carbocysteine ​​nebulized inhalation solutions.

[0003] Currently, neither domestic nor international pharmacopoeias (such as ChP, USP, EP, etc.) have included specific detection methods for related substances in carbocysteine ​​nebulized inhalation solutions, nor have they included detection methods for carbocysteine ​​and other related substances in formulations. The only HPLC methods reported in the literature are mostly for active pharmaceutical ingredients or oral formulations, such as patent CN 112782327 B.

[0004] Patent CN 112782327 B discloses a method for separating and determining carboxymethylcysteine ​​and its impurities using liquid chromatography. The chromatographic conditions are as follows: octadecylsilane-bonded silica gel as the packing material (ZORBAX SB-AQ, 4.6 mm × 250 mm, 5 μm or equivalent column); a mobile phase of 20 mmol / L potassium dihydrogen phosphate solution containing 9.2 mmol / L sodium octanesulfonate (pH adjusted to 1.7 with phosphoric acid); a flow rate of 1.0 mL / min; a column temperature of 25 °C; a detection wavelength of 215 nm; and an injection volume of 25 μL. The names and structural formulas of the impurities described in this patent are shown in the table below.

[0005]

[0006] The patent claims that its method can accurately and quickly separate and determine carboxymethylcysteine ​​from the seven impurities, and that the mobile phase does not contain organic solvents, thus requiring less sophisticated analytical instruments. This allows for the control of carboxymethylcysteine ​​quality in daily production, improved drug efficacy, and reduced toxic side effects.

[0007] However, the inventors of this application discovered that although the aforementioned patent can effectively separate carboxymethylcysteine ​​from seven impurities, for the carboxymethylcysteine ​​nebulization solution, there is interference from excipient peaks, mainly degrading impurity D. Furthermore, because an appropriate amount of sodium hydroxide is added to the nebulization solution formulation to increase the solubility of carboxymethylcysteine, impurities B and F will be converted into impurities C and D respectively after alkalization. Analysis of the structural formulas of impurities B and F shows that impurity B contains a thiol group (-SH), exhibiting high reactivity and easily oxidized to impurity C; impurity F contains an ester bond, is unstable in aqueous solution, and will hydrolyze to impurity D. Additionally, impurities B and F were not detected in the accelerated testing samples, therefore impurities B and F are not present in the product. This patent uses isocratic elution with pure salt, which has limited elution capacity and cannot effectively separate and detect some related impurities in the carboxymethylcysteine ​​nebulization solution. The mobile phase has a high salt concentration and low pH value, which is unfriendly to the chromatographic column. This patented method is insufficient to meet the requirements for precise control of impurities in the nebulization solution. Summary of the Invention

[0008] Since no methods for determining related substances in carbocysteine ​​nebulized inhalation solutions are included in the pharmacopoeias of various countries, the purpose of this invention is to overcome the shortcomings of existing technologies and provide a highly specific, sensitive, and reproducible method for determining related substances in carbocysteine ​​nebulized inhalation solutions. Furthermore, this method validates the changes in impurity profiles, impurity linearity and range, and correction factors in forced degradation assays, providing a scientific basis for formulation process improvement and stability evaluation. The application of this invention will significantly improve the quality control level of carbocysteine ​​nebulized inhalation solutions, ensuring clinical medication safety, and also provide a reference for the development of analytical methods for similar mucolytic agents.

[0009] The technical solution provided by this invention is as follows:

[0010] A method for determining related substances in a carboxymethylcysteine ​​nebulized inhalation solution includes the following steps:

[0011] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase; phosphate and ion-pair buffer (pH adjusted to 2.1-2.3 with phosphoric acid) was used as mobile phase A; methanol was used as mobile phase B for gradient elution; flow rate was 0.6 ml / min to 1.0 ml / min; column temperature was 30℃ to 40℃; detection wavelength was 200 nm to 230 nm.

[0012] The mobile phase A comprises dipotassium hydrogen phosphate and sodium heptane sulfonate.

[0013] Furthermore, the concentration of dipotassium hydrogen phosphate in the mobile phase A is 5-10 mmol / L, and the concentration of sodium heptanesulfonate is 5-10 mmol / L.

[0014] Furthermore, the preparation method of the mobile phase A includes: taking 1.31g of dipotassium hydrogen phosphate and 1.65g of sodium heptanesulfonate, dissolving them in water and diluting them to 1000ml, and adjusting the pH value to 2.2 with phosphoric acid.

[0015] Furthermore, the method employs gradient elution.

[0016] Furthermore, the solvent used in the method is a sodium hydroxide solution.

[0017] Furthermore, the concentration of the sodium hydroxide solution is 0.05~0.15 mol / L.

[0018] Furthermore, the injection volume is 10 μl to 30 μl.

[0019] Furthermore, the chromatographic column is a Cyfel Megassil IPC-A C18, 4.6 × 250 mm, 5 μm;

[0020] Furthermore, the phosphate in the mobile phase A is preferably dipotassium hydrogen phosphate, and the concentration is preferably 7.5 mmol / L;

[0021] Furthermore, the ion-pairing reagent in the mobile phase A is preferably sodium heptanesulfonate, with a preferred concentration of 7.5 mmol / L;

[0022] Furthermore, the pH value of the phosphate and ion-pair buffer in the mobile phase A is preferably 2.2;

[0023] Furthermore, the flow rate is 0.6 ml / min to 1.0 ml / min, preferably 0.8 ml / min;

[0024] Furthermore, the column temperature is 30℃~40℃, preferably 35℃;

[0025] Furthermore, the detection wavelength is 200nm~230nm, preferably 210nm;

[0026] Furthermore, the injection volume is 10 μl to 30 μl, preferably 20 μl;

[0027] In the method of determining related substances in carboxymethylcysteine ​​nebulized inhalation solution by high performance liquid chromatography (HPLC) of the present invention, the chromatographic column used, the type and concentration of phosphate and ion-pairing reagents in mobile phase A, the pH value of mobile phase A, the column temperature and the elution gradient program are important factors affecting the detection effect.

[0028] Specifically, since carboxymethylcysteine ​​is essentially not retained on a C18 column, and its structure contains two carboxyl groups and one amino group, existing in an anionic state, mobile phase A consists of phosphate and ion-pairing buffer. The phosphate is used to control the dissociation of these groups, while the ion-pairing reagent increases the retention time of carboxymethylcysteine ​​on the column. Preferred phosphates include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate, with dipotassium hydrogen phosphate being the most preferred. Preferred ion-pairing reagents include sodium pentanesulfonate, sodium hexanesulfonate, sodium heptanesulfonate, and sodium octanesulfonate, with sodium heptanesulfonate being the most preferred. Furthermore, the concentrations of phosphate and ion-pairing reagent affect the detection results; excessively high or low concentrations will affect impurity separation and the peak shape. The preferred phosphate concentration is 5 mmol / L to 10 mmol / L, with a maximum of 7.5 mmol / L, and the preferred ion-pairing reagent concentration is 5 mmol / L to 10 mmol / L, with a maximum of 7.5 mmol / L.

[0029] Regarding the pH value of mobile phase A, when the concentrations of phosphate and ion-pair reagents in the buffer solution remain constant, a low pH mobile phase can easily damage the chromatographic column, shorten its lifespan, and affect the separation effect of impurity A from unknown impurities. On the other hand, a higher pH mobile phase affects the separation degree of impurity E from impurity G and causes the excipient peak to interfere with impurity E, thereby affecting the detection effect. The preferred pH value is 2.1~2.3, and the most preferred is 2.2, which can achieve the best separation of impurities in each test sample solution.

[0030] Column temperature is also a crucial factor affecting separation efficiency. Specifically, for each test solution, as the column temperature decreases, the retention time of the main peak increases, the peak shape deteriorates, and the separation between impurity A and adjacent peaks gradually decreases until they overlap. Therefore, a suitable column temperature is particularly important for impurity separation. The inventors have concluded that the preferred column temperature is 30℃~40℃, with 35℃ being the most preferred.

[0031] The octadecylsilane-bonded silica column used in the high-performance liquid chromatography (HPLC) method can vary in performance, even when different brands use the same packing material, affecting the detection results. Specifically, the octadecylsilane-bonded silica column used in this invention can be Megassil IPC-A C18, 4.6×250mm, 5μm, Shim-pack GISTC18-AQ, 4.6×250mm, 5μm, or Caprisil C18-AQ 100A, 4.6×250mm, 5μm, with Megassil IPC-A C18, 4.6×250mm, 5μm being the most preferred.

[0032] Gradient elution programs are also crucial for detection results. Specifically, organic phases can increase the elution capacity of impurities. In a gradient program, a low proportion of organic phase may not achieve complete elution, while a high proportion of organic phase may prevent baseline separation between impurities. To improve detection efficiency and obtain better separation results, the following gradient elution program is recommended:

[0033]

[0034] The method for determining related substances in carboxymethylcysteine ​​nebulized inhalation solution by high performance liquid chromatography according to the present invention includes impurities A, C, D, E, and G. The structural and chemical formulas of these impurities are shown in the table below.

[0035]

[0036] Beneficial effects

[0037] This invention establishes a high-performance liquid chromatography (HPLC) method for determining related substances in carbocysteine ​​nebulized inhalation solutions. By optimizing chromatographic conditions (such as the selection of salt and ion pair reagents, column selection, mobile phase gradient, mobile phase pH, and column temperature), the separation efficiency and detection sensitivity of carbocysteine ​​and its degradation products (such as impurities D and E) are significantly improved. This method combines high efficiency, repeatability, and stability, allowing for precise control of impurities while effectively avoiding excipient interference, providing reliable technical support for improving product quality standards and optimizing production processes. The implementation of this invention will fill a gap in existing technologies and has significant implications for promoting the safe clinical application of carbocysteine ​​nebulized inhalation formulations. Attached Figure Description

[0038] Figure 1 The blank solvent chromatogram was obtained under the conditions specified in Example 1.

[0039] Figure 2 The chromatogram of the solution was obtained under the conditions of Example 1.

[0040] Figure 3 Chromatogram of the system suitability solution tested according to the conditions of Example 1;

[0041] Figure 4 The chromatogram of the blank excipient solution was detected according to the conditions of Example 1;

[0042] Figure 5 The chromatogram of the test solution was obtained according to the conditions of Example 1;

[0043] Figure 6 The chromatogram of the test sample solution subjected to acid degradation according to Example 2;

[0044] Figure 7The chromatogram of the test sample solution subjected to alkali degradation according to Example 2;

[0045] Figure 8 The chromatogram of the test sample solution subjected to oxidation destruction according to Example 2;

[0046] Figure 9 The chromatogram of the test sample solution subjected to high-temperature destruction is shown in Example 2.

[0047] Figure 10 The chromatogram of the test sample solution subjected to light destruction according to Example 2;

[0048] Figure 11 The chromatogram of the system suitability solution for the conditional detection in Comparative Example 1 is shown.

[0049] Figure 12 The chromatogram of the blank excipient solution under the conditions detected in Comparative Example 1 is shown.

[0050] Figure 13 The chromatogram of the test solution under the conditions of Comparative Example 1 is shown.

[0051] Figure 14 The chromatogram of the system suitability solution for the conditional detection in Comparative Example 2 is shown.

[0052] Figure 15 The chromatogram is shown for the test solution under the conditions of Comparative Example 2. Detailed Implementation

[0053] The following examples and accompanying drawings are provided to further illustrate the present invention, but are not limited to the scope of these embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0054] Example 1

[0055] Detection instruments and chromatographic conditions:

[0056] Instrument: Agilent 1260 Infinity II; VWD detector / DAD detector;

[0057] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (Cayfeld Megassil IPC-A C18, 4.6 × 250 mm, 5 μm or equivalent column); mobile phase A was a buffer solution containing 7.5 mmol / L dipotassium hydrogen phosphate and 7.5 mmol / L sodium heptanesulfonate (1.31 g of dipotassium hydrogen phosphate and 1.65 g of sodium heptanesulfonate were dissolved in water and diluted to 1000 ml, and the pH was adjusted to 2.2 with phosphoric acid); methanol was used as mobile phase B; gradient elution was performed according to the table below; the flow rate was 0.8 ml / min; the column temperature was 35 °C; the detection wavelength was 210 nm; and the injection volume was 20 μl.

[0058]

[0059] Solvent: 0.1 mol / L sodium hydroxide solution (weigh 4 g of sodium hydroxide and dissolve it in 1000 ml of water).

[0060] Test solution: Accurately measure an appropriate amount of this product and dilute it quantitatively with water to prepare a solution containing approximately 2 mg of carboxycysteine ​​per 1 ml.

[0061] Control solution: Accurately measure an appropriate amount of the test solution and dilute it quantitatively with water to prepare a solution containing approximately 4 μg of carboxymethylcysteine ​​per 1 ml.

[0062] Sensitivity solution: Accurately measure an appropriate amount of the control solution and dilute it quantitatively with water to prepare a solution containing approximately 0.4 μg per 1 ml.

[0063] Blank excipient solution: Accurately measure 1 ml of the blank preparation and place it in a 20 ml volumetric flask. Dilute with water to the mark and shake well. The blank preparation is the excipient disodium edetate, edetate, or calcium sodium edetate. After dissolving it, adjust the pH value with sodium bicarbonate, sodium hydroxide, or sodium citrate to make it consistent with the pH value of the test sample.

[0064] System suitability solution: Weigh appropriate amounts of carbocysteine ​​reference standard, carbocysteine ​​impurity 5 reference standard, carbocysteine ​​impurity E reference standard, carbocysteine ​​impurity 3 reference standard, cystine reference standard, and carboxymethylcysteine ​​lactam reference standard, add appropriate amount of solvent to dissolve, and dilute with water to prepare a mixed solution containing approximately 2 mg of carbocysteine, 4 μg each of carbocysteine ​​impurity 5, carbocysteine ​​impurity E, carbocysteine ​​impurity 3, cystine, and carboxymethylcysteine ​​lactam per 1 ml.

[0065] Injection procedure: Take 20 μl of each of the above test solutions, inject them into the liquid chromatograph, and record the chromatograms respectively.

[0066] Blank solvent and blank excipient solutions do not interfere with the detection of the carboxymethylcysteine ​​main peak and various impurities. In the system suitability solution chromatogram, the peak elution order is as follows: carboxymethylcysteine ​​impurity 5, carboxymethylcysteine ​​impurity E, carboxymethylcysteine ​​impurity 3, carboxymethylcysteine ​​lactam, carboxymethylcysteine, and cystine. The resolution between carboxymethylcysteine ​​impurity 5 and carboxymethylcysteine ​​impurity E should be no less than 1.5. In the sensitivity solution chromatogram, the signal-to-noise ratio of the main component peak height should be no less than 10. See the chromatogram below. Figures 1-5 .

[0067] Experimental conclusions: Using octadecylsilane-bonded silica gel as the packing material; and a buffer solution containing 7.5 mmol / L dipotassium hydrogen phosphate and 7.5 mmol / L sodium heptanesulfonate (1.31 g of dipotassium hydrogen phosphate and 1.65 g of sodium heptanesulfonate were dissolved in water and diluted to 1000 ml, and the pH was adjusted to 2.2 with phosphoric acid) as mobile phase A, and methanol as mobile phase B, the carboxymethylcysteine ​​main peak and adjacent impurities were separated to the required degree using gradient elution, and all known impurities were effectively separated, indicating good method specificity.

[0068] Example 2

[0069] Specificity-Strong Degradation Test

[0070] To verify the feasibility of the chromatographic conditions, acid, alkali, oxidation, high temperature, and light damage tests were performed on the carboxycysteine ​​nebulized inhalation solution. To ensure the accuracy and reliability of the results, the same procedures were performed on the blank formulation to eliminate the influence of excipients on the results.

[0071] Acid destruction: Accurately measure 1 mL of this product into a 20 mL volumetric flask, add 2 mL of 1 mol / L hydrochloric acid solution, shake well, and let stand at room temperature for 24 h. Then add 2 mL of 1 mol / L sodium hydroxide solution, mix well, dilute with purified water to the mark, and shake well.

[0072] Alkali destruction: Accurately measure 1 mL of this product into a 20 mL volumetric flask, add 2 mL of 1 mol / L sodium hydroxide solution, shake well, and let stand at room temperature for 24 h. Then add 2 mL of 1 mol / L hydrochloric acid solution, mix well, dilute with purified water to the mark, and shake well.

[0073] Oxidative damage: Accurately measure 1 mL of this product into a 20 mL volumetric flask, add 1 mL of 0.1% hydrogen peroxide solution, shake well, and let stand at room temperature for 1 hour. Then, dilute to the mark with purified water and shake well.

[0074] High temperature damage: Accurately measure 1 mL of this product into a 20 mL volumetric flask, heat in an 80℃ water bath for 16 hours, cool to room temperature, dilute with purified water to the mark, and shake well.

[0075] Light damage: Place 10 ml of this product in a headspace vial and place it in a light chamber (4500±500 lx) for 12 days. After 12 days, remove the vial. Accurately measure 1 ml of the light-damaged sample into a 20 ml volumetric flask, dilute to the mark with purified water, and shake well.

[0076] The test solutions obtained from the above destructive tests were all analyzed according to the chromatographic conditions in Example 1. The results showed that:

[0077] It is sensitive to acidic, oxidizing, and high-temperature conditions, relatively sensitive to alkaline conditions, and not sensitive to light conditions.

[0078] (1) Under acid and high temperature conditions, the known impurities showed a significant increase in carboxymethylcysteine ​​lactam, complete degradation of cystine, no significant changes in other known impurities, and a large number of other unknown impurities were degraded.

[0079] (2) Under oxidizing conditions, the known impurity E of carboxymethylcysteine ​​increased significantly, while other known impurities did not change significantly. At the same time, a large number of other unknown impurities were degraded.

[0080] (3) Under alkaline conditions, the known impurities showed a slight increase in carboxymethylcysteine ​​lactam, complete degradation of cystine, no significant changes in other known impurities, and the degradation of a large number of other unknown impurities.

[0081] (4) Under illumination, known impurities show no obvious changes and no obvious unknown impurities are produced.

[0082] Under acid, alkali, oxidative, high-temperature, and photodegradation conditions, the purity of the nitrogen-based nitric acid peak was >950 under all degradation test conditions, and the material balance was between 90.0% and 110.0% under all degradation conditions. Chromatograms of the test solutions under each degradation condition are shown below. Figures 6-10 .

[0083] Experimental conclusion: Based on the results of the strong degradation test, the method for determining related substances in carboxymethylcysteine ​​nebulized inhalation solution provided by this invention has good specificity.

[0084] Example 3

[0085] Linearity, Range, and Correction Factor

[0086] A series of standard solutions of different concentrations were prepared, including carbocysteine ​​reference standards, carbocysteine ​​impurity 5 reference standards, carbocysteine ​​impurity E reference standards, carbocysteine ​​impurity 3 reference standards, carboxymethylcysteine ​​lactam reference standards, and cystine reference standards, within their respective limit concentration ranges from approximately 1 / 2 to 150% of the limit of quantitation (using carbocysteine ​​to replace unknown impurities). Six linear solutions were selected at the limit concentrations of approximately 1 / 2, 20%, 50%, 100%, 120%, and 150% of the limit of quantitation. Linear regression analysis was performed on the measured peak areas and concentrations, regression curves were plotted, and the correlation coefficient r was calculated. Based on the linear equations obtained from the linear experiments, the correction factors for each impurity were calculated. To ensure the accuracy of the correction factors, different personnel used different instruments to conduct the tests, and the average values ​​were taken.

[0087] Table 1. Linearity and Range Results of Carbocysteine

[0088]

[0089] Table 2. Linearity and range results for carboxymethylcysteine ​​impurity 5

[0090]

[0091] Table 3. Linearity and range results for carboxymethylcysteine ​​impurity E

[0092]

[0093] Table 4. Linearity and range results for carboxymethylcysteine ​​impurity 3

[0094]

[0095] Table 5. Linearity and Range Results of Carboxymethylcysteine ​​Lactam

[0096]

[0097] Table 6. Linearity and Range Results for Cystine

[0098]

[0099] Table 7 Linear regression equations for each impurity

[0100]

[0101] Table 8. Determination of Correction Factors for Each Impurity

[0102]

[0103]

[0104] Experimental conclusion:

[0105] (1) Within the range of approximately 150% of the limit of quantitation, the peak area and concentration of carboxymethylcysteine, carboxymethylcysteine ​​impurity 5, carboxymethylcysteine ​​impurity E, carboxymethylcysteine ​​impurity 3, carboxymethylcysteine ​​lactam and cystine showed good linearity.

[0106] (2) The relative deviations of the correction factors for each impurity determined by two people were all within ±15%. The correction factor for carboxymethylcysteine ​​impurity 5 was 0.19, the correction factor for carboxymethylcysteine ​​impurity E was 0.45, the correction factor for carboxymethylcysteine ​​impurity 3 was 1.25, the correction factor for carboxymethylcysteine ​​lactam was 0.18, and the correction factor for cystine was 0.64.

[0107] Example 3 further illustrates that, over a wide range, the linearity of each impurity is good (correlation coefficients r > 0.9990), demonstrating that the method can accurately detect not only high-content impurities but also low-content impurities or trace substances, indicating that the method is stable, reliable, and has strong anti-interference capabilities. The determination of correction factors for each impurity ensures accurate impurity quantification. In summary, the method described in this invention is scientific, accurate, and reliable, providing solid data support for drug purity assessment and quality control, ultimately ensuring the safety and efficacy of drugs.

[0108] Example 4

[0109] Solution stability

[0110] The stability of the test solution after 48 hours at room temperature and the stability of the system suitability solution after 31 days at 2–8°C were investigated.

[0111] The preparation methods for each solution are described in Example 1.

[0112] The stability results of the test solution after 48 hours at room temperature are shown in Table 9, and the stability results of the system suitability solution after 31 days at 2-8℃ are shown in Table 10.

[0113] Table 9. Stability results of the test solution

[0114]

[0115] Table 10 System Suitability and Solution Stability Results

[0116]

[0117] Experimental conclusion:

[0118] (1) The absolute difference between the contents of known impurities and unknown single impurities in the test solution and 0 hours is 0.01% and does not exceed 0.05%, indicating that the test solution has good stability within 48 hours at room temperature.

[0119] (2) The relative deviation of the known impurities RRT and peak area in the system suitability solution compared with 0 hours is 14.3% at most and does not exceed 15.0%, indicating that the system suitability solution has good stability within 31 days when placed at 2~8℃.

[0120] Example 4 further illustrates that, using the solution preparation methods in Example 1, the solution stability meets the requirements under the verified conditions. The selection of solvents and solution preparation methods in Example 1 are suitable for the accurate quantification and quality control of related substances in carboxymethylcysteine ​​nebulized inhalation solutions.

[0121] Comparative Example 1

[0122] Detection instruments and chromatographic conditions:

[0123] Instruments: Agilent 1260 Infinity II; VWD detector;

[0124] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (ZORBAX SB-AQ, 4.6 mm × 250 mm, 5 μm or equivalent column); the mobile phase was 20 mmol / L potassium dihydrogen phosphate solution containing 9.2 mmol / L sodium octanesulfonate (pH adjusted to 1.7 with phosphoric acid); the flow rate was 1.0 mL / min; the column temperature was 25 °C; the detection wavelength was 210 nm; and the injection volume was 20 μL.

[0125] Solvent: 0.1 mol / L sodium hydroxide solution (weigh 4 g of sodium hydroxide and dissolve it in 1000 ml of water).

[0126] Test solution: Accurately measure an appropriate amount of this product and dilute it quantitatively with water to prepare a solution containing approximately 2 mg of carboxycysteine ​​per 1 ml.

[0127] Blank excipient solution: Accurately measure 1 ml of blank preparation, place it in a 50 ml volumetric flask, dilute with water to the mark, and shake well.

[0128] System suitability solution: Weigh appropriate amounts of carbocysteine ​​reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard, impurity E reference standard, impurity F reference standard and impurity G reference standard, add appropriate amount of solvent to dissolve, and dilute with water to prepare a mixed solution containing approximately 2 mg of carbocysteine ​​and 4 μg each of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G per ml.

[0129] Injection procedure: Inject 20 μl of each of the above test solutions into the liquid chromatograph. Record the chromatograms respectively.

[0130] The results showed that in the chromatogram of the system suitability solution, the peak elution order was impurity G, impurity E, impurity D, impurity A, impurity B, carboxymethylcysteine, impurity F, and impurity C. The resolution between the main peak of carboxymethylcysteine ​​and each adjacent impurity met the requirements, and all known impurities were effectively separated. However, in the chromatogram of the carboxymethylcysteine ​​nebulization inhalation solution test sample solution, there was interference from the excipient peak, mainly degrading impurity D. (Chromatogram shown...) Figures 11-13 Furthermore, the comparative example used isocratic elution with pure salt, which has limited elution capacity and cannot effectively separate and detect some impurities in the carboxymethylcysteine ​​nebulized solution, especially unknown impurities after the main peak. The mobile phase also had a high salt concentration and low pH, which is unfavorable to the chromatographic column. In contrast, Example 1, through optimized chromatographic conditions (such as the selection of salt and ion pair reagents, column selection, mobile phase gradient, mobile phase pH, and column temperature) using gradient elution, significantly improved the separation effect and detection sensitivity of carboxymethylcysteine ​​and its degradation products (such as impurities D and E). This method combines high efficiency, repeatability, and stability, allows for precise control of impurities, and effectively avoids interference from excipients. This demonstrates that the analytical method of this invention can achieve better technical results.

[0131] Comparative Example 2

[0132] Detection instruments and chromatographic conditions:

[0133] Instruments: Agilent 1260 Infinity II; VWD detector;

[0134] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the packing material (AQ C18 column, 4.6 mm × 250 mm, 5 μm or equivalent column); mobile phase A was a solution containing 0.142% (g / ml) disodium hydrogen phosphate and 0.27% (g / ml) sodium heptanesulfonate (1.42 g of anhydrous disodium hydrogen phosphate and 2.7 g of sodium heptanesulfonate were dissolved in water and diluted to 1000 ml, and the pH was adjusted to 2.1 ± 0.2 with phosphoric acid after mixing); acetonitrile was used as mobile phase B; gradient elution was performed according to the table below; the flow rate was 1.0 ml per minute; the column temperature was 40 °C; the detection wavelength was 215 nm; and the injection volume was 20 μl.

[0135]

[0136] Solvent: 0.1 mol / L sodium hydroxide solution (weigh 4 g of sodium hydroxide and dissolve it in 1000 ml of water).

[0137] Test solution: Accurately measure an appropriate amount of this product and dilute it quantitatively with water to prepare a solution containing approximately 2 mg of carboxycysteine ​​per 1 ml.

[0138] System suitability solution: Weigh appropriate amounts of carboxymethylcysteine ​​reference standard, impurity A reference standard, impurity C reference standard, impurity D reference standard, impurity E reference standard and impurity G reference standard, add appropriate amount of solvent to dissolve, and dilute with water to prepare a mixed solution containing approximately 2 mg of carboxymethylcysteine ​​and 4 μg each of impurity A, impurity C, impurity D, impurity E and impurity G per ml.

[0139] Injection procedure: Inject 20 μl of each of the above test solutions into the liquid chromatograph. Record the chromatograms respectively.

[0140] The results showed that in the chromatogram of the system suitability solution, the peak elution order was impurity G, impurity E, impurity A, impurity D, carboxymethylcysteine, and impurity C. The resolution between the main peak of carboxymethylcysteine ​​and each adjacent impurity met the requirements, and all known impurities were effectively separated. However, in the chromatogram of the test sample solution, there was interference from the unknown impurity peak with the known impurity D, and the main peak eluted as a double peak. (Chromatograms are shown below.) Figures 14-15 Analysis suggests that the selection of acetonitrile as mobile phase B and the gradient elution program may be related to the fact that acetonitrile has a strong elution capacity. In the gradient elution program, the proportion of mobile phase B increases from 2% to 15% in the range of 0-18 min, resulting in a higher proportion of organic phase. This leads to faster elution of impurities, which in turn interferes with known impurities. It also causes instability in the chromatographic system, causing the main peak of the test sample solution to elute as a double peak.

[0141] Meanwhile, the present invention uses a DAD detector to perform spectral scanning of impurities and carboxymethylcysteine ​​from 190nm to 400nm. All of them have terminal absorption, and there is strong absorption at 210nm. Therefore, the detection wavelength of 210nm in Example 1 is better than 215nm in Comparative Example 2. At 210nm, each impurity has a higher response and the system sensitivity is higher.

[0142] In summary, due to the differences in organic phase selection and gradient elution between Example 1 and Comparative Example 2, the impurity elution and system stability of Example 1 are superior to those of Comparative Example 2. This is because methanol is more polar than acetonitrile, and a reasonable elution program set from 0 to 18 min ensures sufficient elution of impurities while effectively preventing the main peak from eluting into a double peak. Furthermore, the chromatographic system exhibits stability. This demonstrates that using the mobile phase and elution program in Example 1 yields better technical results.

Claims

1. A method for determining related substances in a carboxymethylcysteine ​​nebulized inhalation solution, characterized in that, The liquid chromatography conditions are as follows: Column: Octadecylsilane-bonded silica gel column; Mobile phase A: phosphate and ion-pair buffer, pH adjusted to 2.1-2.3 with phosphate; Mobile phase B: Methanol; Flow rate: 0.6 ml / min ~ 1.0 ml / min; Column temperature: 30℃~40℃; Detection wavelength: 200nm~230nm.

2. The method for inhaling carboxymethylcysteine ​​solution related substances according to claim 1, characterized in that, The mobile phase A comprises dipotassium hydrogen phosphate and sodium heptane sulfonate.

3. The method for inhaling carboxymethylcysteine ​​solution related substances according to claim 1, characterized in that, The concentration of dipotassium hydrogen phosphate in the mobile phase A is 5-10 mmol / L, and the concentration of sodium heptane sulfonate is 5-10 mmol / L.

4. The method for inhaling carboxymethylcysteine ​​nebulized solution related substances according to claim 1, characterized in that, The preparation method of the mobile phase A includes: taking 1.31g of dipotassium hydrogen phosphate and 1.65g of sodium heptanesulfonate, dissolving them in water and diluting them to 1000ml, and adjusting the pH value to 2.2 with phosphoric acid.

5. The method for inhaling carboxymethylcysteine ​​solution related substances according to claim 1, characterized in that, The method employs gradient elution.

6. The method for inhaling carboxymethylcysteine ​​solution related substances according to claim 1, characterized in that, The solvent used in this method is sodium hydroxide solution.

7. The method for inhaling carboxymethylcysteine ​​solution related substances according to claim 1, characterized in that, The concentration of the sodium hydroxide solution is 0.05~0.15 mol / L.

8. The method for inhaling carboxymethylcysteine ​​solution related substances according to claim 1, characterized in that, The injection volume is 10 μl to 30 μl.

9. The method for the inhalation of carboxymethylcysteine ​​solution related substances according to claim 1, characterized in that: The elution procedure is as follows:

10. The method for determining the related substances in a carboxymethylcysteine ​​nebulized inhalation solution according to claim 1, wherein the impurities are impurity A, impurity C, impurity D, impurity E, and impurity G, and the structural formulas and chemical formulas of the impurities are shown in the table below:

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