Separation and synchronous detection method for erdosteine and intermediate thereof

Abstract

The invention discloses a method for separating and synchronously detecting erdosteine and intermediates thereof. According to the method, the erdosteine and the main intermediates thereof are separated and detected at the same time by utilizing the high performance liquid chromatography (HPLC), the specificity is high, the separation effect is stable, and the repeatability is good. The method can realize effective separation and simultaneous detection of the erdosteine and the intermediate thereof, is suitable for quality control and related research in the production process, and has both economical efficiency and practicability.

Description

Technical Field

[0001] This invention belongs to the field of drug analysis and detection technology, specifically relating to a method for the separation and simultaneous detection of erdosteine ​​and its intermediates. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD), bronchitis, bronchiectasis, and other respiratory diseases are prevalent worldwide. Their pathological features are often accompanied by airway mucus hypersecretion and mucus plug formation, leading to airway obstruction, progressive decline in lung function, and recurrent infections. Erdosteine ​​is a sulfur-containing mucolytic agent used to treat respiratory diseases. Its molecular structure contains a closed thiol group, requiring metabolism in vivo to reach its active form and exert its therapeutic effect. Because erdosteine ​​active pharmaceutical ingredient and formulations contain various impurities (such as synthetic intermediates and oxidative degradation products), and its metabolites need precise quantification in pharmacokinetic studies, establishing an analytical method capable of simultaneously detecting erdosteine ​​and its intermediates is of great significance for production process control, impurity research, and quality evaluation.

[0003] Erdostan has the molecular formula C8H. 11 NO4S2, with a relative molecular mass of 249.31, can be prepared by reacting two intermediates: DL-homocysteine ​​thiolactone hydrochloride and thioglycolic acid anhydride. The synthetic route is shown below:

[0004] .

[0005] Erdosteine ​​and its main intermediates share similar molecular polarities and sulfur functional groups, making them prone to peak tailing. Their similar retention times also facilitate co-elution, often hindering effective separation under conventional high-performance liquid chromatography (HPLC) conditions. While existing analytical methods commonly employ C18 columns and methanol or acetonitrile as the organic phase, in practice, insufficient resolution, poor peak shape, and low theoretical plate numbers are prevalent, failing to meet pharmacopoeia and industrial quality control requirements. Furthermore, current analytical methods for erdosteine ​​and its main intermediates often focus on single-component detection or only achieve qualitative analysis, making simultaneous effective separation and accurate quantification difficult. Due to the high similarity in molecular structure and polarity, and the presence of sulfur atoms, erdosteine ​​and its main intermediates are susceptible to similar retention times, peak tailing, and co-elution under conventional HPLC conditions, leading to unreliable quantitative results and failing to meet the practical needs of API release, production process monitoring, and quality consistency evaluation. Therefore, developing an analytical method with good separation and reproducibility for erdostane and its main intermediate systems remains a pressing technical problem in this field and has significant practical implications. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, this invention provides a method for the separation and simultaneous detection of erdosteine ​​and its intermediates. This invention utilizes high-performance liquid chromatography (HPLC) for the simultaneous separation and detection of erdosteine ​​and its main intermediates, exhibiting high specificity, stable separation performance, and good repeatability. This method enables the effective separation and simultaneous detection of erdosteine ​​and its intermediates, and is suitable for quality control in production processes and related research.

[0007] The present invention provides a method for the isolation and simultaneous detection of erdosteine ​​and its intermediates, comprising the following steps:

[0008] Weigh 25 mg of the sample to be tested, dissolve and dilute it in 60% acetonitrile aqueous solution, and make up to 25 mL in a volumetric flask. Send the sample for high performance liquid chromatography detection.

[0009] HPLC detection conditions and parameters were set as follows:

[0010] Detector: Diode array detector;

[0011] Chromatographic column: C18 column;

[0012] Column temperature: 30 ℃;

[0013] Wavelength: 220 nm;

[0014] Flow rate: 1 mL / min;

[0015] Injection volume: 5 μL;

[0016] Elution method: Gradient elution is performed using mobile phase A and mobile phase B, wherein mobile phase A is a 0.2% (w / w) aqueous solution of phosphoric acid and mobile phase B is methanol.

[0017] Furthermore, the gradient elution procedure is set as follows:

[0018]

[0019] Furthermore, the chromatographic column has a particle size of 20 μm and a column length of 250 mm.

[0020] Furthermore, the separation degree of erdostane and its intermediates is greater than 2.

[0021] Furthermore, the theoretical plate number of Erdostane and its intermediates is ≥6000.

[0022] The structure of Erdostane is shown below:

[0023] .

[0024] The structure of the intermediate is shown below:

[0025] .

[0026] Compared with the prior art, the present invention has at least the following beneficial effects:

[0027] (1) In view of the fact that erdosteine ​​and its intermediates have similar polarity and are easy to co-elute, the present invention effectively improves the peak tailing phenomenon of sulfur-containing compounds in chromatographic analysis by specifically selecting 0.2% phosphoric acid aqueous solution and methanol as mobile phase.

[0028] (2) Compared with conventional systems that use acetonitrile as the organic phase, the present invention uses methanol as mobile phase B, which significantly increases the retention time difference between erdosteine ​​and its intermediate, thus significantly improving their separation.

[0029] (3) By designing a gradient elution program, a high theoretical plate number and a stable peak shape can be obtained while ensuring good separation.

[0030] (4) The method has good repeatability and system adaptability, with RSD less than 2%, and is suitable for routine quality control.

[0031] (5) The analytical method provided by the present invention is simple to operate and economical. It is suitable for the quality control of erdostein raw material and intermediate impurities in its production process. It does not require complicated pretreatment or expensive reagents and has high industrial applicability and economy. Attached Figure Description

[0032] Figure 1 HPLC chromatogram under the conditions measured in Example 1 of this invention.

[0033] Figure 2 The HPLC chromatogram of Comparative Example 1 (simulating a conventional acetonitrile system) of this invention is obtained under the conditions measured.

[0034] Figure 3 The HPLC chromatogram of Comparative Example 2 (simulated low buffer system) of this invention is obtained under the measured conditions.

[0035] Figure 4 The HPLC chromatogram of Comparative Example 3 (simulated unbuffered system) of this invention is obtained under the measured conditions.

[0036] Figure 5 The linearity of erdostein measured under chromatographic conditions in Example 1 of this invention.

[0037] Figure 6 Linearity experimental chromatogram of erdosteine ​​intermediate measured under chromatographic conditions in Example 1 of this invention. Detailed Implementation

[0038] In establishing the method of this invention, the inventors systematically screened different organic phase systems, different buffer systems, and gradient elution programs, taking into account the similarity in structure and polarity between erdosteine ​​and its main synthetic intermediates. The study found that using a conventional acetonitrile system or low buffer concentration conditions resulted in insufficient separation between erdosteine ​​and its intermediates, making simultaneous detection difficult. However, by using an aqueous phase with a specific phosphoric acid concentration and a methanol organic phase, combined with a reasonable gradient elution program, the separation effect could be significantly improved.

[0039] The present invention will be further illustrated below with specific embodiments. It should be understood that the embodiments of the present invention are merely illustrative and not intended to limit the invention. In the following embodiments, the reagents used include methanol and acetonitrile of HPLC grade, brand name Maclean, and purified water. The high-performance liquid chromatograph used was an Agilent HPLC 1260. All raw materials used in this invention were prepared in the laboratory.

[0040] Example 1:

[0041] Preparation of solution: Weigh approximately 25 mg of erdosteine ​​test sample and its intermediate into 25 mL volumetric flasks, add an appropriate amount of 60% acetonitrile aqueous solution to dissolve and dilute to the mark, and mix well.

[0042] Chromatographic determination conditions:

[0043] Chromatographic column: C18, 4.6×250mm, 5μm;

[0044] Mobile phase: A: 0.2% aqueous phosphoric acid solution; B: methanol;

[0045] Flow rate: 1.0 mL / min;

[0046] Column temperature: 30℃;

[0047] Injection volume: 5 μL;

[0048] Gradient elution procedure:

[0049]

[0050] Under the chromatographic conditions of Example 1, the detection chromatogram of the solution is as follows: Figure 1 As shown, erdosteine ​​has a good peak shape, a retention time of 7.242 min, a tailing factor of 1.071, and a theoretical plate number of 68994; the erdosteine ​​intermediate has a retention time of 7.677 min, a tailing factor of 1.074, a resolution of 3.91, and a theoretical plate number of 71598.

[0051] Comparative Example 1:

[0052] Preparation of solution: Weigh approximately 25 mg of erdosteine ​​test sample and its intermediate into 25 mL volumetric flasks, add an appropriate amount of 60% acetonitrile aqueous solution to dissolve and dilute to the mark, and mix well.

[0053] Chromatographic determination conditions:

[0054] Chromatographic column: C18, 4.6×250mm, 5μm;

[0055] Mobile phase: A: 0.2% phosphoric acid aqueous solution; B: acetonitrile;

[0056] Flow rate: 1.0 mL / min;

[0057] Column temperature: 30℃;

[0058] Injection volume: 5 μL;

[0059] Erdosteine ​​gradient elution procedure:

[0060]

[0061] Under the chromatographic conditions of Comparative Example 1, the detection chromatogram of the solution is as follows: Figure 2 As shown. Erdostane's retention time was 6.493 min, with a tailing factor of 1.0 and a theoretical plate number of 76068; the intermediate's retention time was 6.699 min, with a tailing factor of 1.157 and a theoretical plate number of 73420. (From...) Figure 1 and Figure 2 As can be seen from the test data, under the conditions of this invention, erdosteine ​​and its main synthetic intermediate can achieve baseline separation with a separation degree of 3.9, while under the comparative conditions, the separation degree can only reach 1.7, which does not meet the pharmacopoeia requirement of separation degree ≥2.

[0062] Comparative Example 2:

[0063] Preparation of solution: Weigh approximately 25 mg of erdosteine ​​test sample and its intermediate into 25 mL volumetric flasks, add an appropriate amount of 60% acetonitrile aqueous solution to dissolve and dilute to the mark, and mix well.

[0064] Chromatographic determination conditions:

[0065] Chromatographic column: C18, 4.6×250mm, 5μm;

[0066] Mobile phase: A: 0.1% aqueous phosphoric acid solution; B: methanol;

[0067] Flow rate: 1.0 mL / min;

[0068] Column temperature: 30℃;

[0069] Injection volume: 5 μL;

[0070] Gradient elution procedure:

[0071]

[0072] Under the chromatographic conditions of Comparative Example 3, the detection chromatogram of the solution is as follows: Figure 3 As shown in the figure, the retention time of erdosteine ​​was 6.494 min, the tailing factor was 1.049, and the theoretical plate number was 74565; the retention time of the erdosteine ​​intermediate was 6.704 min, the tailing factor was 1.14, and the theoretical plate number was 78463. Furthermore, it can be seen from the figure that the separation between erdosteine ​​and the intermediate still did not meet the pharmacopoeia requirements, and the response to the intermediate was lower compared to Example 1 and Comparative Example 1.

[0073] Comparative Example 3:

[0074] Preparation of solution: Weigh approximately 25 mg of erdosteine ​​test sample and its intermediate into 25 mL volumetric flasks, add an appropriate amount of 60% acetonitrile aqueous solution to dissolve and dilute to the mark, and mix well.

[0075] Chromatographic determination conditions:

[0076] Chromatographic column: C18, 4.6 × 250 mm, 5 μm;

[0077] Mobile phase: A: pure water; B: methanol;

[0078] Flow rate: 1.0 mL / min;

[0079] Column temperature: 30℃;

[0080] Injection volume: 5 μL;

[0081] Gradient elution procedure:

[0082]

[0083] Under the chromatographic conditions of Comparative Example 3, the detection chromatogram of the solution is as follows: Figure 4 As shown in the figure, erdosteine ​​had a retention time of 7.649 min and a tailing factor of 0.525; the intermediate had a retention time of 7.649 min and a tailing factor of 11.06, with a theoretical plate number of 130, which is far below the basic requirements for plate number in the pharmacopoeia. Furthermore, as can be seen from the figure, erdosteine ​​and its intermediate were not separated, and the peak shapes were very poor, merging together and indistinguishable, with significant baseline drift.

[0084] Example 2: System Adaptability Test

[0085] Preparation of test solution: Weigh approximately 25 mg of erdosteine ​​test sample and its intermediate into 25 mL volumetric flasks, add an appropriate amount of 60% acetonitrile aqueous solution to dissolve and dilute to the mark, and mix well.

[0086] Chromatographic determination conditions:

[0087] Chromatographic column: C18, 4.6×250mm, 5μm;

[0088] Mobile phase: A: 0.2% aqueous phosphoric acid solution; B: methanol;

[0089] Flow rate: 1.0 mL / min;

[0090] Column temperature: 30℃;

[0091] Injection volume: 5 μL;

[0092] Gradient elution procedure:

[0093]

[0094] Take the test solution and inject it repeatedly 6 times. The test results are shown in Table 1 below.

[0095]

[0096] As shown in Table 1, after repeated injection of the same mixed sample solution 6 times, the RSD% of erdosteine ​​and its intermediate were 0.89% and 1.01%, respectively, both of which meet the requirements of RSD% < 2.0% in the pharmacopoeia. This indicates that the method of the present invention has good system adaptability and high experimental reliability.

[0097] Example 3: Repeatability Test

[0098] Preparation of test solutions: Weigh approximately 25 mg of each of the six groups of erdosteine ​​test solutions into 25 mL volumetric flasks, dissolve and dilute to the mark with 60% acetonitrile aqueous solution, mix well, and obtain mixed solutions of different mass concentrations, corresponding to test solutions numbered 1-6 respectively.

[0099] Chromatographic determination conditions:

[0100] Chromatographic column: C18, 4.6×250mm, 5μm;

[0101] Mobile phase: A: 0.2% aqueous phosphoric acid solution; B: methanol;

[0102] Flow rate: 1.0 mL / min;

[0103] Column temperature: 30℃;

[0104] Injection volume: 5 μL;

[0105] Gradient elution procedure:

[0106]

[0107] Samples of different mass concentrations (numbers 1-6) were injected separately, and the contents of each sample solution were measured as shown in Table 2 below.

[0108]

[0109] As shown in Table 2, the average content of erdosteine ​​in the sample was 98.67%, and the RSD% was 0.35%, which meets the pharmacopoeia requirement that the RSD% should not exceed 2.0%, indicating that the method of the present invention has good reproducibility.

[0110] Example 4: Linear Experiment

[0111] Under the chromatographic conditions described in Example 1, standard solutions of erdosteine ​​and its intermediates at different concentrations were prepared and linearity was investigated.

[0112] Weigh 10 mg each of erdosteine ​​reference standard and the main intermediate reference standard, and place them separately in 10 mL volumetric flasks. Dissolve and dilute to volume with 60% acetonitrile aqueous solution to prepare 1.0 mg / mL erdosteine ​​standard stock solution and 1.0 mg / mL intermediate standard stock solution. Weigh the mixed standard solution of erdosteine ​​and the intermediate separately, mix them, and dilute with 60% acetonitrile aqueous solution to prepare mixed standard working solution. Prepare 5 concentration points:

[0113] Erdostatin: 0.5, 0.8, 1, 1.2, 1.5 mg / mL.

[0114] Intermediates: 0.05, 0.08, 0.1, 0.12, 0.15 mg / mL.

[0115] Three injections were performed at each point, and the peak areas of erdosteine ​​and the intermediate were recorded. Linear regression was performed using "concentration-peak area" to obtain the regression equation and correlation coefficient R. 2 As shown in Table 3-4, Figures 5-6 As shown.

[0116]

[0117]

[0118] The above detection results show that within the specified concentration range, erdosteine ​​and its intermediates can produce clearly distinguishable detection peaks, and their peak areas exhibit a stable response relationship with changes in target concentration. The linear equation for erdosteine ​​shows a good linear relationship between peak area and concentration, with a correlation coefficient R0. 2≥0.995.

[0119] The above describes specific embodiments of the present invention. It should be understood that the present invention is not limited to the specific embodiments described above. Any modifications or alterations made by those skilled in the art to the present invention will fall within the protection scope of the present invention.

Claims

1. A method for the isolation and simultaneous detection of erdosteine ​​and its intermediates, characterized in that... Includes the following steps: Weigh 25 mg of the sample to be tested, dissolve and dilute it in 60% acetonitrile aqueous solution, and make up to 25 mL in a volumetric flask. Send the sample for high performance liquid chromatography detection to achieve effective separation and simultaneous detection of erdosteine ​​and its main synthetic intermediates under the same chromatographic analysis conditions. The structure of Erdostane is shown below: ； The structure of the intermediate is shown below: ； The detection conditions and parameters are set as follows: Detector: Diode array detector; Chromatographic column: C18 column; Column temperature: 30 ℃; Wavelength: 220 nm; Flow rate: 1 mL / min; Injection volume: 5 μL; Elution method: Gradient elution is performed using mobile phase A and mobile phase B.

2. The detection method according to claim 1, characterized in that: The mobile phase A is a 0.2% (w / w) aqueous solution of phosphoric acid, and the mobile phase B is methanol.

3. The detection method according to claim 2, characterized in that: The gradient elution program is set as follows: 。 4. The detection method according to claim 1, characterized in that: The separation degree between erdostane and the intermediate was greater than 2.

5. The detection method according to claim 1, characterized in that: The theoretical plate number of both Erdostane and the intermediate is ≥6000.

Images