Method for determining content of clenbuterol hydrochloride in ambroxol oral solution by two-dimensional liquid chromatography

Abstract

The invention provides a method for determining the content of clenbuterol hydrochloride in an ambroxol oral solution, which comprises the following steps: respectively injecting a diluent, a reference solution and a test solution into a two-dimensional high performance liquid chromatograph, recording chromatograms, and calculating the content of clenbuterol hydrochloride by peak area according to an external standard method. The first-dimensional liquid chromatography conditions are as follows: a phosphoric acid buffer solution-methanol-acetonitrile is taken as a mobile phase A, methanol is taken as a mobile phase B, and gradient elution is carried out; the second-dimensional liquid chromatography conditions are as follows: phosphoric acid buffer solution-methanol-acetonitrile is taken as a mobile phase A, and switching is performed according to a specific program. The detection method disclosed by the invention is simple and convenient, the interference of a matrix in a sample can be well solved, the peak area response of a main component can be increased by about 5 times compared with that of a common high-performance liquid phase, and the detection limit is as low as 0.02 mu g / ml; the method is good in system applicability, good in specificity, good in repeatability, high in accuracy and good in solution stability, and can be used for quantitatively detecting the content of clenbuterol hydrochloride in the medicine.

Description

Technical Field

[0001] This invention belongs to the field of compound detection technology, specifically, it relates to a method for determining the content of clenbuterol hydrochloride in ambroxol oral solution using two-dimensional liquid chromatography. Background Technology

[0002] Ambroxol hydrochloride and clenbuterol hydrochloride are compound preparations used to treat symptoms such as cough, thick sputum, difficulty expectorating, and wheezing caused by acute and chronic respiratory diseases such as acute / chronic bronchitis, bronchial asthma, and emphysema. Clenbuterol hydrochloride is a selective β-receptor agonist that relaxes bronchial smooth muscle, enhances ciliary movement, dissolves mucus, and promotes sputum expectoration.

[0003] As one of the active ingredients in ambroxol oral solution, the accurate determination of clenbuterol hydrochloride content is crucial. Each milliliter of ambroxol oral solution contains 1 μg of clenbuterol hydrochloride, and this low content necessitates high requirements for the specificity, stability, sensitivity, and detection limit of the detection method. Existing technologies disclose several methods for determining the clenbuterol hydrochloride content in ambroxol oral solution, but all have some limitations.

[0004] "A Method for Simultaneous Determination of Ambroxol Hydrochloride and Clenbuterol in Ambroxol Oral Solution by Reverse High Performance Liquid Chromatography," Strait Pharmaceutical Journal, 2016, 28: 53-55, Hu Donghai and Sun Yinhua, disclose a method for determining the content of ambroxol hydrochloride and clenbuterol in ambroxol oral solution using reverse high performance liquid chromatography (RP-HPLC), employing Agilent Technologies. A C18 (4.6 mm × 250 mm, 5 μm) column was used; the mobile phase was acetonitrile: 0.01 mol / L potassium dihydrogen phosphate solution (with 0.4% triethylamine added, pH adjusted to 3.5 with phosphoric acid) (23:77) for elution; ambroxol hydrochloride and clenbuterol hydrochloride were well separated, with ambroxol hydrochloride showing good linearity in the range of 0.28475 mg / mL-2.278 mg / mL and clenbuterol hydrochloride in the range of 0.494 μg / mL-247 μg / mL; this method mainly solves the problem of simultaneous detection of ambroxol hydrochloride and clenbuterol hydrochloride, but it is no longer applicable to the detection of low concentrations of clenbuterol hydrochloride below 0.5 μg / mL; and some literature reports that the negative control peak interferes with the clenbuterol hydrochloride peak during reproducibility, indicating insufficient specificity and inaccurate determination of clenbuterol hydrochloride content.

[0005] Patent CN114137102A discloses a method for determining the clenbuterol hydrochloride content in ambroxol oral solution. The method first derivatizes the sample before performing high-performance liquid chromatography (HPLC). The derivatization process involves diazotizing the aromatic primary amine group in the clenbuterol hydrochloride structure with sodium nitrite in the presence of potassium bromide, followed by coupling with naphthylethylenediamine to form an azo compound. This azo compound exhibits significant absorption at approximately 500 nm, while other excipients do not show peaks at 500 nm, thus providing good specificity for the determination method. The linearity of clenbuterol hydrochloride is good in the concentration range of 0.2 μg / mL to 0.6 μg / mL, which also improves the detection limit of clenbuterol hydrochloride. However, in order to reduce the damage to the chromatographic column caused by other high-concentration components in the sample and to avoid interference with the clenbuterol hydrochloride peak, the test solution needs to be fully diluted, so a lower detection limit is required. On the other hand, the derivatization processes of ambroxol hydrochloride and clenbuterol hydrochloride are competitive, and the amount of derivatization solvent needs to be controlled. Incomplete derivatization may occur during the process, resulting in a large error in the detection results.

[0006] Patent CN117054582A discloses a method for determining the content of clenbuterol hydrochloride in a compound preparation for relieving asthma and expectoration. The method is high-performance liquid chromatography (HPLC), with the following conditions: C18 column, 250 × 4.6 mm, 5 μm; column temperature: 35℃; mobile phase A: 0.005-0.02 mol / L potassium dihydrogen phosphate solution and 0.4% triethylamine (pH 2.8-3.2, v / v); mobile phase B: acetonitrile; mobile phase A and mobile phase B are eluted isocratically at a v / v ratio of 81:19; injection volume: 100 μL. This method has good system suitability and repeatability; the detection limit is 0.1 μg / mL. However, the peak area response of clenbuterol hydrochloride is relatively low, requiring a high baseline of the HPLC instrument. The peak integral is easily affected by baseline interference, leading to significant errors in the accuracy of the content determination.

[0007] Therefore, in the pharmaceutical field, further research is needed to develop a detection method for clenbuterol hydrochloride that features low detection limit, high response to main peak area detection, high recovery rate, high efficiency, convenience, and suitability for industrial production. Summary of the Invention

[0008] To address the problems of high detection limits and complex pretreatment in existing technologies, this invention provides a method for detecting clenbuterol hydrochloride in ambroxol oral solution by optimizing the conditions of two-dimensional high-performance liquid chromatography (HPLC). This method offers a lower detection limit and higher detection response. The specific scheme is as follows: This invention provides a method for determining the clenbuterol hydrochloride content in ambroxol oral solution using two-dimensional liquid chromatography, the method comprising: The diluent, reference solution, and test solution were injected separately into a two-dimensional high-performance liquid chromatograph, and the chromatograms were recorded. The clenbuterol hydrochloride content was calculated by peak area using the external standard method. Among them, the two-dimensional high performance liquid chromatograph includes: The conditions for the first-dimensional liquid chromatography are: Gradient elution was performed using phosphate buffer-methanol-acetonitrile as mobile phase A and methanol as mobile phase B. The conditions for the second-dimensional liquid chromatography are: The mobile phase A was phosphate buffer-methanol-acetonitrile. The switching procedure was as follows: the reference solution was injected into the first-dimensional liquid chromatograph to locate the clenbuterol hydrochloride peak. The retention time of the main peak of clenbuterol hydrochloride in the first-dimensional liquid chromatograph was 32-37 min. The valve cut-off time was set to 0.5 min to 1.0 min before and after the clenbuterol hydrochloride peak elution.

[0009] In one or more embodiments, the phosphate buffer in the mobile phase A is prepared by taking 3.0 g of sodium octane sulfonate and 5.0 g of potassium dihydrogen phosphate, dissolving them in water and diluting them to 1000 ml, and adjusting the pH to 3.0 with phosphoric acid.

[0010] In one or more embodiments, the volume ratio of phosphate buffer, methanol, and acetonitrile in the mobile phase A is 72.5:10:17.5.

[0011] In one or more embodiments, the gradient elution procedure in the first-dimensional liquid chromatography is as follows: From 0 to 40 minutes, the volume of mobile phase A was maintained at 100%, and the volume of mobile phase B was maintained at 0%. In 40-40.1 min, the volume of mobile phase B increased from 0% to 70%. For 40.1-50 minutes, the volume of mobile phase B is maintained at 70%. In 50-50.1 min, the volume of mobile phase B decreased from 70% to 0%; The volume of mobile phase B was maintained at 0% for 50.1-65 min.

[0012] In one or more embodiments, the two-dimensional liquid chromatography uses a column packed with octadecyl bonded silica gel, preferably a Venusi XBP C18(L) column with dimensions of 4.6 mm × 150 mm and 5 μm.

[0013] In one or more embodiments, the detection conditions of the two-dimensional high-performance liquid chromatograph are as follows: Detection wavelength: 215nm; column temperature: 40℃; flow rate: 1.0mL / min for the first dimension and 1.5mL / min for the second dimension; collection tube: polyetheretherketone tube; preferably, the polyetheretherketone tube has an inner diameter of 0.0762cm, a length of 20m, and a volume of 9mL.

[0014] In one or more embodiments, the detector in the two-dimensional high-performance liquid chromatograph includes: a VWD detector (10 mm optical path) in the first dimension; and a DAD detector (60 mm optical path) in the second dimension.

[0015] In one or more embodiments, the diluent is mobile phase A.

[0016] In one or more embodiments, the reference solution is prepared by taking an appropriate amount of clenbuterol hydrochloride reference standard, dissolving and diluting it with mobile phase A to prepare a solution containing 0.2 μg of clenbuterol hydrochloride per 1 ml.

[0017] In one or more embodiments, the test solution is prepared by taking an appropriate amount of ambroxol oral solution and diluting it with mobile phase A to prepare a solution containing 0.2 μg clenbuterol hydrochloride per 1 ml.

[0018] Compared with the prior art, the present invention has the following advantages: (1) The detection method of this application has the characteristics of good system applicability, strong specificity, good linear range, high sensitivity, high accuracy, good repeatability and good solution stability. Under the conditions of selecting a suitable chromatographic column, optimizing the mobile phase ratio and buffer pH, it has good linearity for the detection of clenbuterol hydrochloride in the concentration range of 0.102-0.307 μg / mL, good accuracy in the concentration range of 0.102-0.307 μg / mL, and the test solution is stable for 62 hours when stored at room temperature in the dark. The detection limit is 0.02 μg / mL, which is much lower than the detection limit of existing methods. It is particularly suitable for the detection of low concentrations of clenbuterol hydrochloride in compound preparations. Based on the low detection limit of this detection method, the preparation can be fully diluted during the test, thereby reducing the damage of other high-content components to the chromatographic column and extending the service life of the chromatographic column.

[0019] (2) The selection of mobile phase ratio and pH in the detection method of this application can greatly improve the separation degree between the main peak of clenbuterol hydrochloride and the excipient peaks before and after it (both are between 2.0 and 4.4), and address the issue that most commercially available ambroxol oral solution samples will have excipient peak degradation interference at the main peak position of clenbuterol hydrochloride under extreme conditions (long-term 24 stability and influencing factor tests, etc.), which affects the detection accuracy. The method has excellent specificity, and the problem of excipient peak degradation interference can be perfectly solved by the second-dimensional liquid phase separation system. By optimizing the injection volume and using a second-dimensional high-throughput detector (DAD detector: 60mm optical path) to improve the spectral signal-to-noise ratio, the detection sensitivity and accuracy are improved.

[0020] (3) Because the compound preparation tested in this invention has high viscosity, there is a phenomenon of sticking to the wall when using conventional pipetting, the peak area will be smaller and the measured content will be smaller; while the content calculation method of this invention adopts the weighing method, which can avoid this problem and the measured content is more accurate.

[0021] (4) The method of the present invention has good system applicability, strong specificity, high accuracy, good repeatability and good solution stability; the two-dimensional liquid chromatography method is used to determine clenbuterol hydrochloride in ambroxol oral solution, which does not require special treatment of the reference standard and the test sample during the test, and is easy to realize industrial detection. Attached Figure Description

[0022] Figure 1 This is the standard chromatogram of the reference solution in Example 1.

[0023] Figure 2 This is the standard spectrum of the diluent in Example 1.

[0024] Figure 3 This is the standard chromatogram of the blank excipient solution (without clenbuterol hydrochloride) in Example 1.

[0025] Figure 4 It is the standard chromatogram of the test solution in Example 1.

[0026] Figure 5 This is the standard spectrum of the linear solution (50%) in Example 2.

[0027] Figure 6 This is the standard spectrum of the linear solution (75%) in Example 2.

[0028] Figure 7 This is the standard spectrum of the linear solution (100%) in Example 2.

[0029] Figure 8 This is the standard spectrum of the linear solution (125%) in Example 2.

[0030] Figure 9 This is the standard spectrum of the linear solution (150%) in Example 2.

[0031] Figure 10 This is the linear equation graph from Example 2.

[0032] Figure 11 This is the standard spectrum of repeatable solution 1 in Example 3.

[0033] Figure 12 This is the standard spectrum of repeatable solution 2 in Example 3.

[0034] Figure 13 This is the standard spectrum of repeatable solution 3 in Example 3.

[0035] Figure 14 This is the standard spectrum of repeatable solution 4 in Example 3.

[0036] Figure 15 This is the standard spectrum of repeatable solution 5 in Example 3.

[0037] Figure 16 This is the standard spectrum of repeatable solution 6 in Example 3.

[0038] Figure 17 It is the standard chromatogram of accuracy solution 1 (50% of the limit concentration) in Example 4.

[0039] Figure 18 It is the standard chromatogram of accuracy solution 2 (100% of the limit concentration) in Example 4.

[0040] Figure 19 It is the standard chromatogram of accuracy solution 3 (150% of the limit concentration) in Example 4.

[0041] Figure 20 It is the standard chromatogram of accuracy solution 4 (150% of the limit concentration) in Example 4. Detailed Implementation

[0042] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0043] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0044] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0045] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0046] The examples involve the addition amount, content and concentration of various substances, and unless otherwise specified, the percentage content refers to the mass percentage content.

[0047] All materials used in the following examples were purchased from commercially available sources; the two-dimensional liquid chromatograph used was the Agilent Technologies 1260 Infinity II model; the chromatographic column used was the VenusiXBP C18(L) 4.6mm×150mm, 5μm column manufactured by Tianjin Bona Ager Co., Ltd.; experimental methods not mentioned in the examples were conventional experimental methods.

[0048] Example 1: Study on the System Applicability and Specificity of the Detection Method Chromatographic conditions: Instrument: Two-dimensional high performance liquid chromatograph (first dimension with 10mm optical path detector, second dimension with 60mm optical path detector); Detection wavelength: 215nm; Column temperature: 40℃; Flow rate: 1.0 mL / min for the first dimension and 1.5 mL / min for the second dimension; Collection tube: Polyetheretherketone (PEEK) tube, inner diameter 0.0762 cm (0.03 inches), 20 m, volume approximately 9 mL.

[0049] First-dimensional chromatographic conditions: A Venusi XBP C18(L) 4.6mm×150mm, 5μm column was used; phosphate buffer (3.0g sodium octane sulfonate and 5.0g potassium dihydrogen phosphate were dissolved in water and diluted to 1000ml, and the pH was adjusted to 3.0 with phosphoric acid)-methanol-acetonitrile (72.5:10:17.5) was used as mobile phase A, and methanol was used as mobile phase B. Gradient elution was performed according to the program in Table 1.

[0050] Table 1 Gradient elution program

[0051] Second-dimensional chromatographic conditions: A Venusi XBP C18(L) 4.6mm×150mm, 5μm column was used; the mobile phase was phosphate buffer (3.0g sodium octane sulfonate and 5.0g potassium dihydrogen phosphate were dissolved in water and diluted to 1000ml, and the pH was adjusted to 3.0 with phosphoric acid)-methanol-acetonitrile (72.5:10:17.5); the reference solution was injected into the first-dimensional liquid chromatograph to locate the clenbuterol hydrochloride peak, and the valve cut-off time was set to 0.5min~1.0min before and after the clenbuterol hydrochloride peak elution.

[0052] The reference solution and the test solution were prepared using the following methods: Reference solution: Weigh approximately 20 mg of clenbuterol hydrochloride reference standard accurately, place it in a 100 ml volumetric flask, dissolve and dilute to the mark with mobile phase A, and shake well. Accurately measure 1.0 ml and place it in a 100 ml volumetric flask, dilute to the mark with mobile phase A, and shake well. Then accurately measure 2.0 ml and place it in a 20 ml volumetric flask, dilute to the mark with mobile phase A, and shake well (0.2 μg / ml).

[0053] Test solution: Weigh 4.46g of ambroxol oral solution (containing approximately 4μg of clenbuterol hydrochloride) accurately, place it in a 20ml volumetric flask, dilute to the mark with mobile phase A, shake well, filter through a 0.2μm Jintao nylon filter membrane, discard 3ml, and take the filtrate to obtain (0.2μg / ml).

[0054] Diluent: Take mobile phase A to obtain diluent.

[0055] Blank excipient solution: Weigh approximately 4.46 g of blank excipient (excluding clenbuterol hydrochloride), place it in a 20 ml volumetric flask, dilute to the mark with mobile phase A, shake well, filter through a 0.2 μm Jintao nylon filter membrane, discard 3 ml, and collect the filtrate to obtain the blank excipient solution.

[0056] Injection procedure: Inject 100 μl each of diluent, blank excipient solution, reference solution, and test solution into the two-dimensional liquid chromatograph, and record the chromatograms for each. See details for chromatograms. Figures 1-4 The experimental results are detailed in Table 1.

[0057] Table 2 Results of System Suitability and Specificity Tests

[0058] according to Figures 1-4As shown in Table 2, the diluent and blank excipient solutions did not interfere with the peak position of clenbuterol hydrochloride. The peak purity of clenbuterol hydrochloride in both the test solution and the reference solution was greater than 99.8%. The RSD of the peak area of ​​clenbuterol hydrochloride in the reference solution after 5 consecutive injections was 0.5%, and the recovery rate of reference solution 2 was 99.1%. This indicates that the detection method has good system suitability and specificity.

[0059] Example 2: Linearity Study of the Detection Method Diluent: Mobile phase A.

[0060] Reference stock solution (1.0 μg / ml): Accurately weigh 20.65 mg of clenbuterol hydrochloride reference standard, place it in a 100 ml volumetric flask, dissolve and dilute to the mark with diluent, shake well, accurately measure 1.0 ml, place it in a 200 ml volumetric flask, dilute to the mark with diluent, shake well, and use this as the reference stock solution (1.0 μg / ml).

[0061] Linear solutions: Using pipettes and volumetric flasks, prepare linear solutions according to the table below using the stock solution of the reference standard (1.0 μg / ml). Dilute to the mark with diluent and mix well. The preparation of each linear solution is shown in Table 3.

[0062] Table 3 Preparation of linear solutions

[0063] The detection was performed under the chromatographic conditions of Example 1.

[0064] Injection procedure: Inject 100 μl of each of the above test solutions into the two-dimensional liquid chromatograph and record the chromatograms. See details of the chromatograms. Figures 5-9 The experimental results are shown in Table 4.

[0065] Table 4 Results of Linear Experiments

[0066] Experimental conclusion: The linear equation for clenbuterol hydrochloride is y = 2267.7312x - 6.0580 (e.g., ...). Figure 10 The correlation coefficient r was 0.9999, the y-intercept deviation was 1.3%, and the relative response factor of each linear concentration relative to 100% concentration was between 0.99 and 1.00. The linearity was good within the range of 0.102 μg / ml to 0.307 μg / ml (equivalent to 50% to 150% of the concentration of the test sample solution), indicating that the method has good linearity.

[0067] Example 3: Precision Study of the Detection Method

[0068] Repeatable solution: Accurately weigh 4.46 g of ambroxol oral solution (containing approximately 4 μg of clenbuterol hydrochloride), place it in a 20 ml volumetric flask, dilute to the mark with mobile phase A, shake well, filter through a 0.2 μm Jintao nylon filter membrane, discard 3 ml, and collect the filtrate to obtain (0.2 μg / ml). Prepare 6 parallel aliquots.

[0069] The detection was performed under the chromatographic conditions of Example 1.

[0070] Injection procedure: Inject 100 μl of each of the above test solutions into the two-dimensional liquid chromatograph and record the chromatograms. See details of the chromatograms. Figures 11-16 The test results are detailed in Table 5.

[0071] Table 5 Repeatability Test Results

[0072] Experimental conclusion: According to the experimental results in Table 5, the RSD of clenbuterol hydrochloride content in the 6 repeatable samples was 0.7%, indicating that the method has good repeatability.

[0073] Example 4: Accuracy Study of the Detection Method

[0074] Reference stock solution (1.0 μg / ml): Accurately weigh 20.65 mg of clenbuterol hydrochloride reference standard, place it in a 100 ml volumetric flask, dissolve and dilute to the mark with diluent, shake well, accurately measure 1.0 ml, place it in a 200 ml volumetric flask, dilute to the mark with diluent, shake well, and use this as the reference stock solution (1.0 μg / ml).

[0075] For each accuracy solution: Measure 4.0 ml of blank excipient and place it into nine 20 ml volumetric flasks. Using a pipette, accurately measure the reference standard stock solution (1.0 μg / ml) according to the table below and add it to each flask. Dilute to the mark with diluent, shake well, and then filter through a 0.45 μm × 25 mm Jinteng nylon filter membrane. Discard 3 ml and collect the filtrate as the accuracy solution for each accuracy solution. See Table 6 for details on the preparation of each accuracy solution.

[0076] Table 6 Accuracy Solution Preparation

[0077] The detection was performed under the chromatographic conditions of Example 1.

[0078] Injection procedure: Inject 100 μl of each of the above test solutions into the two-dimensional liquid chromatograph and record the chromatograms. See details of the chromatograms. Figures 17-20 The test results are detailed in Table 7.

[0079] Table 7 Accuracy Test Results

[0080] Experimental conclusion: Based on Figures 17-20 As shown in Table 7, the detection method of this application has good accuracy within the research concentration range of 0.102 μg / ml to 0.307 μg / ml (equivalent to 50% to 150% of the concentration of the test sample solution), that is, the detection method has good accuracy.

[0081] Example 5: Solution stability study of the detection method

[0082] Test solution: Weigh 4.46g of ambroxol oral solution (containing approximately 4μg of clenbuterol hydrochloride) accurately, place it in a 20ml volumetric flask, dilute to the mark with mobile phase A, shake well, filter through a 0.2μm Jintao nylon filter membrane, discard 3ml, and take the filtrate to obtain (0.2μg / ml).

[0083] The test solution was analyzed at time points from 0 to 62 hours after being stored at room temperature, according to the chromatographic conditions of Example 1. The ratio of clenbuterol hydrochloride content at each time point to that at 0 hours was calculated. The test results are detailed in Table 8.

[0084] Table 8 Results of solution stability test

[0085] Experimental conclusion: The test solution is stable at room temperature for 62 hours.

[0086] In summary, based on the experimental results of Examples 1-5, the following factors must be considered to ensure the accuracy and repeatability of the analytical method for determining the clenbuterol hydrochloride content in ambroxol oral solution: 1. To improve the UV absorption response of clenbuterol hydrochloride, a high-throughput detector (60 mm optical path) must be used for detection; 2. To reduce interference from the sample matrix, while meeting the detection sensitivity requirements, the concentration of the sample matrix in the tested sample can be reduced, and the instrument detection sensitivity can be increased by adding instrument hardware to reduce the sample matrix concentration, thereby making the detection more accurate; 3. To eliminate the interference of solvent effects, a suitable mobile phase ratio needs to be selected for the experiment.

[0087] The method provided in this application has addressed the three considerations mentioned above: 1. By selecting two-dimensional liquid chromatography for detection, the sample is separated and processed in two dimensions, eliminating interference from the sample matrix; 2. A high-throughput detector (60mm optical path) has been specially developed, which improves the sample detection response by approximately 5 times. While meeting sensitivity requirements, it also reduces the concentration of the test sample (i.e., reduces the concentration of the sample matrix). Even when dealing with extreme samples (ambroxol oral solution nearing its expiration date and influencing factor test samples), the method still exhibits good specificity and can accurately detect the clenbuterol hydrochloride content in ambroxol oral solution; 3. Through experiments, various mobile phase ratios were investigated. It was found that when the mobile phase (A) ratio is phosphate buffer (3.0g sodium octane sulfonate, 5.0g potassium dihydrogen phosphate, dissolved in water and diluted to 1000ml, pH adjusted to 3.0 with phosphoric acid) - methanol - acetonitrile = 72.5:10:17.5, there is no solvent effect interference or blank excipient interference. This mobile phase ratio is preferred for testing.

[0088] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above... The description should not be considered a limitation of the invention. Various modifications and substitutions to the invention will be apparent to those skilled in the art after reading the foregoing. Therefore, the scope of protection of the invention should be defined by the appended claims.

Claims

1. A method for determining the content of clenbuterol hydrochloride in ambrohexol oral solution by two-dimensional liquid chromatography, characterized in that, The method comprises: The diluent, the control solution, and the test solution are injected into the two-dimensional high-performance liquid chromatograph respectively, the chromatogram is recorded, and the content of clenbuterol hydrochloride is calculated by peak area according to the external standard method; In the two-dimensional high-performance liquid chromatograph: The first-dimensional liquid chromatograph condition is: Phosphoric acid buffer-methanol-acetonitrile is used as the mobile phase A, and methanol is used as the mobile phase B for gradient elution. The second-dimensional liquid chromatograph condition is: Phosphoric acid buffer-methanol-acetonitrile is used as the mobile phase A; the switching program is as follows: the control solution is injected into the first-dimensional liquid chromatograph, and the clenbuterol hydrochloride peak is positioned; the retention time of the clenbuterol hydrochloride main peak in the first-dimensional liquid chromatograph is 32-37 min, and the switching time is set to be 0.5 min-1.0 min before and after the clenbuterol hydrochloride peak elutes.

2. The method of claim 1, wherein, The phosphoric acid buffer in the mobile phase A is prepared by the following method: 3.0 g of sodium octanesulfonate and 5.0 g of potassium dihydrogen phosphate are dissolved and diluted to 1000 ml with water, and the pH value is adjusted to 3.0 with phosphoric acid.

3. The method of claim 1, wherein, The volume ratio of the phosphoric acid buffer, methanol, and acetonitrile in the mobile phase A is 72.5:10:17.

5.

4. The method of claim 1, wherein, The gradient elution program in the first-dimensional liquid chromatograph is as follows: 0-40 min, the volume of the mobile phase A is kept at 100%, and the volume of the mobile phase B is kept at 0%; 40-40.1 min, the volume of the mobile phase B is increased from 0% to 70%; 40.1-50 min, the volume of the mobile phase B is kept at 70%; 50-50.1 min, the volume of the mobile phase B is decreased from 70% to 0%; 50.1-65 min, the volume of the mobile phase B is kept at 0%.

5. The method of claim 1, wherein, Both the two-dimensional liquid chromatographs use a chromatographic column with octadecyl-bonded silica gel as the filler, and the chromatographic column is preferably Venusi XBP C18 (L) with the specification of 4.6 mm x 150 mm and 5 μm.

6. The method of claim 1, wherein, The detection condition of the two-dimensional high-performance liquid chromatograph is as follows: The detection wavelength is 215 nm; The column temperature is 40℃; the flow rate is 1.0 mL / min for the first dimension and 1.5 mL / min for the second dimension; the collection tube is a polyether ether ketone tube; preferably, the polyether ether ketone tube has an inner diameter of 0.0762 cm, a length of 20 m, and a volume of 9 mL.

7. The method of claim 1, wherein, The detector in the two-dimensional high-performance liquid chromatograph comprises: a first-dimensional VWD detector (10 mm optical path) and a second-dimensional DAD detector (60 mm optical path).

8. The method of claim 1, wherein, The diluent is the mobile phase A.

9. The method of claim 1, wherein, The preparation method of the control solution is as follows: an appropriate amount of clenbuterol hydrochloride control is dissolved and diluted with the mobile phase A to prepare a solution containing 0.2 μg of clenbuterol hydrochloride per 1 ml.

10. The method of claim 1, wherein, The preparation method of the test solution is as follows: an appropriate amount of ambroxol oral solution is diluted with the mobile phase A to prepare a solution containing 0.2 μg of clenbuterol hydrochloride per 1 ml.

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