Mobile phase suitable for simultaneously detecting ambroxol hydrochloride and / or clenbuterol hydrochloride by high performance liquid chromatography and application of mobile phase

Abstract

The invention discloses a mobile phase suitable for simultaneous detection of ambroxol hydrochloride and / or clenbuterol hydrochloride by high performance liquid chromatography. The mobile phase is a mixed solution composed of sodium sulfosuccinate dioctyl ester methanol, water and acetic acid, wherein the mass fraction of the sodium sulfosuccinate dioctyl ester methanol is 0.3%-0.8%; the volume ratio of the sodium sulfosuccinate dioctyl ester methanol with the mass fraction of 0.3%-0.8% to the water to the acetic acid is (740-760): (240-260): 10; the pH value of the mixed solution is 2.8 to 3.2. According to the present invention, the mobile phase is combined with the high performance liquid chromatography to detect the ambroxol hydrochloride oral solution, such that the ambroxol hydrochloride and the clenbuterol hydrochloride can be simultaneously detected, the ambroxol hydrochloride and the clenbuterol hydrochloride can be effectively separated from other impurity peaks, the peak appearance time of the ambroxol hydrochloride can be significantly reduced, and the service life of the chromatographic column can be effectively prolonged.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, in particular to a mobile phase suitable for simultaneously detecting ambroxol hydrochloride and / or clenbuterol hydrochloride and an application thereof. Background Art

[0002] Ambroxol oral solution (ACS) is a compound preparation composed of ambroxol hydrochloride and clenbuterol hydrochloride as active ingredients, used to treat cough, thick sputum, difficulty in expectoration, wheezing and other symptoms caused by acute and chronic respiratory diseases (such as acute and chronic bronchitis, bronchial asthma, emphysema, etc.). Among them, ambroxol hydrochloride is a mucolytic agent, which can increase the secretion of serous glands in the respiratory mucosa, reduce the secretion of mucous glands, reduce the viscosity of sputum, promote the secretion of pulmonary surfactant, increase the movement of bronchial cilia, and make sputum easier to cough up; clenbuterol hydrochloride is a selective β-receptor agonist, which has the effects of relaxing bronchial smooth muscles, enhancing ciliary movement, dissolving mucus and promoting the discharge of sputum.

[0003] As active ingredients in ambroxol oral solution, the contents of ambroxol hydrochloride and clenbuterol hydrochloride need to be accurately determined. In ambroxol oral solution, the prescription content of clenbuterol hydrochloride is 1 μg / mL, and the prescription content of ambroxol hydrochloride is 1.5 mg / mL. The content of clenbuterol hydrochloride is much lower than that of ambroxol hydrochloride. The content of clenbuterol hydrochloride in ambroxol oral solution is extremely low. In addition, there are other impurity mixtures in ambroxol oral solution that affect the detection of ambroxol hydrochloride and clenbuterol hydrochloride. At the same time, the propylene glycol and glycerol with a relatively high content in ambroxol oral solution are more harmful to the chromatographic column in the high performance liquid chromatography. Therefore, the measurement method of clenbuterol hydrochloride and ambroxol hydrochloride needs to have both high specificity and high sensitivity, and the peak time needs to be shortened as much as possible.

[0004] The National Standard for Ambibutrol Oral Solution (WS 1-(X-118)-2006Z) records the method for determining the content of ambroxol hydrochloride and clenbuterol hydrochloride. The mobile phase for detecting ambroxol hydrochloride is: 1.0% sodium sulfosuccinate dioctyl methanol solution-water-acetic acid (740:260:10) (adjusting the pH to 3.0 with 85% phosphoric acid) as the mobile phase; the mobile phase for detecting clenbuterol hydrochloride is: 1.7% sodium sulfosuccinate dioctyl methanol solution-water-acetic acid (700:300:100) (adjusting the pH to 3.0 with 85% phosphoric acid) as the mobile phase. However, the high concentration of salt causes greater damage to the chromatographic column, resulting in a significant reduction in the tolerance of the chromatographic column. At the same time, the method recorded in the standard cannot well distinguish the impurities in the sample from ambroxol hydrochloride, and cannot achieve simultaneous detection of ambroxol hydrochloride and clenbuterol hydrochloride in one chromatogram.

[0005] Therefore, there is currently a lack of a method that can simultaneously detect ambroxol hydrochloride and clenbuterol hydrochloride in one chromatography and achieve better detection results, in which the determination of the mobile phase is the key. Summary of the invention

[0006] The purpose of the present invention is to overcome the above-mentioned shortcomings of the prior art and provide a mobile phase suitable for simultaneous detection of ambroxol hydrochloride and / or clenbuterol hydrochloride by high performance liquid chromatography and its application.

[0007] The first object of the present invention is to provide a mobile phase suitable for simultaneously detecting ambroxol hydrochloride and / or clenbuterol hydrochloride by high performance liquid chromatography.

[0008] The second object of the present invention is to provide a method for simultaneously detecting ambroxol hydrochloride and clenbuterol hydrochloride.

[0009] The third object of the present invention is to provide the use of the above mobile phase and / or the above method in simultaneously detecting the content of ambroxol hydrochloride and / or clenbuterol hydrochloride in ambroxol oral solution.

[0010] In order to achieve the above object, the present invention is implemented by the following scheme:

[0011] A mobile phase suitable for simultaneously detecting ambroxol hydrochloride and / or clenbuterol hydrochloride by high performance liquid chromatography, wherein the mobile phase is a mixed solution consisting of 0.3% to 0.8% sodium sulfosuccinate dioctyl methanol, water and acetic acid in a volume ratio of 740 to 760:240 to 260:10; and the pH value of the mixed solution is 2.8 to 3.2.

[0012] Preferably, the mobile phase is a mixed solution consisting of 0.3% by mass of sodium sulfosuccinate dioctyl ester methanol, water and acetic acid.

[0013] More preferably, the volume ratio of the sodium dioctyl sulfosuccinate methanol with a mass fraction of 0.3%, water and acetic acid is 740:260:10.

[0014] Further preferably, the pH value of the mixed solution is 3.0.

[0015] A method for simultaneously detecting ambroxol hydrochloride and clenbuterol hydrochloride, the method specifically comprising:

[0016] High performance liquid chromatography is used to detect the sample containing ambroxol hydrochloride and / or clenbuterol hydrochloride; wherein the mobile phase of the high performance liquid chromatography is any one of the mobile phases described above.

[0017] Preferably, the sample to be tested is ambroterol oral solution.

[0018] More preferably, the ambroxol oral solution is an ambroxol oral solution having an ambroxol hydrochloride concentration of 0.03 to 1.5 mg / mL.

[0019] Further preferably, the ambroxol oral solution is an ambroxol oral solution having an ambroxol hydrochloride concentration of 1.5 mg / mL.

[0020] Preferably, the chromatographic conditions of the high performance liquid chromatography are:

[0021] Chromatographic column: octadecylsilane bonded silica gel chromatographic column; detection wavelength: 296nm; column temperature: 30-35°C; injection volume: 20-50μL; flow rate: 0.8-1.2mL / min.

[0022] More preferably, the chromatographic conditions of the HPLC are:

[0023] Chromatographic column: octadecylsilane bonded silica gel chromatographic column; detection wavelength: 296nm; column temperature: 30℃; injection volume: 50μL; flow rate: 1.0mL / min.

[0024] The above method is used to simultaneously detect ambroxol hydrochloride and clenbuterol hydrochloride. Ambroxol hydrochloride and clenbuterol hydrochloride can be detected simultaneously in one chromatography and are well separated from other impurities. The elution time of ambroxol hydrochloride can also be significantly reduced, and the damage of dioctyl sodium sulfosuccinate to the chromatographic column can be effectively reduced.

[0025] The present invention also claims to protect the use of any of the above-mentioned mobile phases and / or the above-mentioned methods in simultaneously detecting the contents of ambroxol hydrochloride and clenbuterol hydrochloride in ambroxol oral solution.

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

[0027] The present invention provides a mobile phase specially used for simultaneously detecting ambroxol hydrochloride and / or clenbuterol hydrochloride by high performance liquid chromatography, wherein the mobile phase is a mixed solution composed of sodium sulfosuccinate dioctyl ester methanol with a mass fraction of 0.3% to 0.8%, water and acetic acid; the volume ratio of sodium sulfosuccinate dioctyl ester methanol with a mass fraction of 0.3% to 0.8%, water and acetic acid is 740 to 760:240 to 260:10; the pH value of the mixed solution is 2.8 to 3.2. The mobile phase of the present invention is combined with high performance liquid chromatography to detect ambroxol hydrochloride oral solution, which can realize the simultaneous detection of ambroxol hydrochloride and clenbuterol hydrochloride, effectively separate from other impurity peaks, significantly reduce the elution time of ambroxol hydrochloride, and effectively improve the service life of the chromatographic column. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the HPLC chromatogram of the test solution 1 in Example 1 (4);

[0029] Figure 2 This is the chromatogram of the blank solvent in the specificity investigation;

[0030] Figure 3 This is the chromatogram of the blank excipient solution in the specificity study;

[0031] Figure 4 This is the chromatogram of the test solution that does not contain clenbuterol hydrochloride in the specificity investigation;

[0032] Figure 5 This is the standard curve of ambroxol hydrochloride;

[0033] Figure 6 According to the national drug standard (WS 1- (X-118)-2006Z) Chromatogram of ambroterol oral solution;

[0034] Figure 7 For the test solution in Comparative Example 1, the injection volume was 20 μL in accordance with the national drug standard (WS 1- (X-118)-2006Z) chromatogram detected by high performance liquid chromatography;

[0035] Figure 8 For the test solution in Comparative Example 1, the injection volume is 50 μL in accordance with the national drug standard (WS 1- (X-118)-2006Z) chromatogram detected by high performance liquid chromatography;

[0036] Fig. 9 for Figure 7 and Figure 8 Merged chromatograms;

[0037] Fig.10 The chromatogram corresponding to the method shown in Comparative Example 2;

[0038] Fig.11 The chromatogram corresponding to the method shown in Comparative Example 3;

[0039] Fig.12 It is the chromatogram corresponding to the method shown in Comparative Example 4.

[0040] Fig.13 It is the corresponding chromatogram in Comparative Example 5;

[0041] Fig.14 It is the corresponding chromatogram in Comparative Example 6. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0043] The chromatographic columns numbered W5, W29 and W36 are chromatographic columns of the same model, with specific information as follows: Waters XbridgeC18 4.6*250mm, 5μm; they were randomly selected during the inspection process.

[0044] The specific information of the chromatographic column numbered A10 is: Agilent ZORBAX Eclipse XDB-C18, 4.6mm*150mm, 5μm.

[0045] Clenbuterol hydrochloride reference substance stock solution: accurately weigh 5 mg of clenbuterol hydrochloride reference substance, place it in a 50 mL volumetric flask, add solvent (a mixed solution of 0.3% sodium sulfosuccinate dioctyl ester methanol, water and acetic acid (volume ratio of 740:260:10)) to dissolve and dilute to the scale, shake well to obtain the clenbuterol hydrochloride reference substance stock solution;

[0046] Clenbuterol hydrochloride positioning solution: dilute the clenbuterol hydrochloride reference substance stock solution by one half;

[0047] Clenbuterol hydrochloride reference solution: Accurately measure 2 mL of clenbuterol hydrochloride reference stock solution, place it in a 200 mL volumetric flask and dilute to the mark with water, shake well to obtain;

[0048] Ambroxol hydrochloride reference solution: Accurately weigh 150 mg of ambroxol hydrochloride reference, place it in a 100 mL volumetric flask and add solvent (a mixed solution of 0.3% sodium sulfosuccinate dioctyl ester methanol, water and acetic acid (volume ratio of 740:260:10)) to dilute to the scale and shake well.

[0049] Example 1 Investigation of a method for simultaneously detecting ambroxol hydrochloride and clenbuterol hydrochloride

[0050] The present invention has found the optimal mobile phase suitable for the simultaneous detection of ambroxol hydrochloride and clenbuterol hydrochloride after long-term research and summary, and based on this, has designed the method conditions for the simultaneous detection of ambroxol hydrochloride and clenbuterol hydrochloride. The effect of the technical solution of the present invention is verified by the following examples.

[0051] First, the mobile phase, column temperature and concentration of the test solution in the HPLC process were changed respectively to verify and examine the chromatographic conditions.

[0052] 1. Experimental methods

[0053] (1) Validation of HPLC detection with different concentrations of test solution

[0054] The liquid obtained by filtering the ambroxol hydrochloride oral solution with a concentration of 1.5 mg / mL was used as the test solution 1 (1.5 mg / mL test solution);

[0055] Accurately measure 2 mL of the test solution 1, place it in a 10 mL volumetric flask, add water and dilute to the scale to obtain the test solution 2 (i.e., 0.3 mg / mL test solution, the content of impurity B in ambroxol hydrochloride is 0.26%);

[0056] Accurately measure 2 mL of test solution 1, place it in a 100 mL volumetric flask, add water to dilute to the scale, and obtain test solution 3 (i.e., 0.03 mg / mL test solution).

[0057] According to the chromatographic conditions shown in Table 1, the test solution 2 and the test solution 3 were subjected to high performance liquid chromatography in sequence, and the corresponding chromatograms were recorded to obtain the peak time and separation degree of each substance.

[0058] Table 1 Chromatographic conditions for test solution

[0059]

[0060] (2) Validation of mobile phase HPLC detection using different concentrations of ion-pair reagents

[0061] Four different mobile phases are set respectively, mobile phase 1 is the mobile phase shown in Table 1 of step (1); the difference between mobile phase 2 and mobile phase 1 is that the mass fraction of sodium sulfosuccinate dioctyl methanol is 0.5%; the difference between mobile phase 3 and mobile phase 1 is that the mass fraction of sodium sulfosuccinate dioctyl methanol is 0.3%; the difference between mobile phase 4 and mobile phase 1 is that the mass fraction of sodium sulfosuccinate dioctyl methanol is replaced by ethanol.

[0062] The test solution 2 obtained in step (1) is used as a test sample, and high performance liquid chromatography is performed under mobile phase 1, mobile phase 2, mobile phase 3 and mobile phase 4, respectively. Except for the mobile phase, the other chromatographic conditions are consistent with Table 1, and the corresponding chromatograms are recorded to obtain the separation of clenbuterol hydrochloride, ambroxol hydrochloride and ambroxol hydrochloride impurity B and the elution time of ambroxol hydrochloride.

[0063] (3) Validation of HPLC detection using different column temperatures

[0064] The test solution 2 obtained in step (1) is used as a test sample, and high performance liquid chromatography is performed and a chromatogram is recorded to obtain the separation degree of ambroxol hydrochloride and the impurity B of ambroxol hydrochloride and the peak time of ambroxol hydrochloride; the difference between the chromatographic conditions and the chromatographic conditions shown in Table 1 is that the mass fraction of sodium sulfosuccinate dioctyl methanol in the mobile phase is 0.3%.

[0065] The mass fraction of sodium dioctyl sulfosuccinate methanol in the mobile phase under the chromatographic conditions shown in Table 1 was adjusted to 0.3%, the column temperature was changed to 35° C., and the test solution 2 obtained in step (1) was subjected to HPLC detection, and the corresponding results were recorded.

[0066] (4) Verification of the localization of clenbuterol hydrochloride in HPLC test results using test solutions of different concentrations

[0067] The clenbuterol hydrochloride fixed solution was subjected to high performance liquid chromatography according to the chromatographic conditions shown in Table 2, and the peak time of clenbuterol hydrochloride was recorded.

[0068] Table 2 Chromatographic conditions

[0069]

[0070] The test solution 1 (1.5 mg / mL test solution) obtained in step (1) was used as a test sample and HPLC detection was performed according to the chromatographic conditions shown in Table 3 to record the elution time and peak area of ​​clenbuterol hydrochloride, the elution time of ambroxol hydrochloride impurity B, the elution time of ambroxol hydrochloride and the separation degree of each substance.

[0071] The test solution 2 obtained in step (1) is subjected to the same high performance liquid chromatography test and the test results are obtained.

[0072] 2. Experimental results

[0073] (1) In the validation study of HPLC detection of test solutions with different concentrations, the peak time and separation degree of each substance are shown in Table 3.

[0074] Table 3 Peak elution time and separation of test solution 2 and test solution 3

[0075]

[0076]

[0077] The clenbuterol hydrochloride content in test solution 2 and test solution 3 was relatively low, and the response in the chromatogram was too small, so they were not investigated.

[0078] The results showed that in the HPLC results of test solution 2 and test solution 3, the separation degrees of impurity B in ambroxol hydrochloride and ambroxol hydrochloride met the requirements, but the elution time of ambroxol hydrochloride was about 34 minutes, which was too late.

[0079] (2) In the validation study of HPLC detection using different mobile phases, the peak elution time and separation degree of each substance are shown in Table 4.

[0080] Table 4 Peak elution time and separation degree of each substance in different mobile phases

[0081]

[0082] Note: In the HPLC detection corresponding to mobile phase 4, the peak time of ambroxol hydrochloride in the test solution was too early and was not applicable, so clenbuterol hydrochloride was not examined.

[0083] The results showed that reducing the concentration of sodium dioctyl sulfosuccinate methanol in the mobile phase of HPLC can reduce the elution time of ambroxol hydrochloride, but if the ion pair reagent is removed, the elution time of ambroxol hydrochloride will be too early and the peak shape will deteriorate; when the mass fraction of sodium dioctyl sulfosuccinate methanol in the mobile phase is 0.3% and 0.5%, a better detection effect can be achieved; when the mass fraction of sodium dioctyl sulfosuccinate methanol is 0.3%, the HPLC detection result is the best.

[0084] (3) In the validation study of the effect of different column temperatures on HPLC detection, the peak elution time and separation degree of each substance at different column temperatures are shown in Table 5.

[0085] Table 5 Peak elution time and separation degree of each substance at different column temperatures

[0086]

[0087] The results showed that increasing the column temperature to 35°C during HPLC can shorten the elution time of ambroxol hydrochloride and reduce the chromatographic time, but it will reduce the separation degree between impurity B in ambroxol hydrochloride and ambroxol hydrochloride, and the separation condition will become worse.

[0088] (4) The peak time, peak area and separation degree of each substance in the validation study of clenbuterol hydrochloride in the HPLC test results of the test solution with different concentrations are shown in Table 6; the HPLC chromatogram of the test solution 1 is shown in Figure 1 shown.

[0089] Table 6 Peak time, peak area and separation of each substance

[0090]

[0091] The results show that in the HPLC test results of test solution 1 and test solution 2, the separation of impurity B of ambroxol hydrochloride and clenbuterol hydrochloride meets the requirements and has no significant difference (separation is greater than 1.5), but the peak area (5720) of clenbuterol hydrochloride in test solution 2 is small, which will lead to a large injection error. Therefore, using test solution 1, that is, the test solution with an ambroxol hydrochloride concentration of 1.5 mg / mL, for HPLC detection can achieve better results.

[0092] Example 2 A method for simultaneously detecting ambroxol hydrochloride and clenbuterol hydrochloride

[0093] 1. Experimental methods

[0094] High performance liquid chromatography was used to detect ambroxol hydrochloride and clenbuterol hydrochloride in ambroxol oral solution (ambroxol hydrochloride concentration was 1.5 mg / mL), and the chromatographic conditions were shown in Table 7.

[0095] Table 7 Chromatographic conditions

[0096]

[0097] After the ambroxol oral solution (ambroxol hydrochloride concentration is 1.5 mg / mL) was filtered using (Welch nylon 25 mm, 0.45 μm), 2 mL of the initial filtrate was discarded and the resulting solution was collected as the test solution. The test was performed according to the chromatographic conditions shown in Table 7, and the contents of ambroxol hydrochloride and clenbuterol hydrochloride in the ambroxol oral solution were obtained by combining the external standard method, and the content determination results were calculated.

[0098] Content determination result = (detected content ÷ labeled content) × 100%.

[0099] 2. Experimental results

[0100] The content of clenbuterol hydrochloride was 97.8%, and the content of ambroxol hydrochloride was 99.2%. The results show that the method shown in this embodiment can well detect clenbuterol hydrochloride and ambroxol hydrochloride in ambroxol oral solution.

[0101] Example 3 Methodological Study of a Method for Simultaneous Detection of Ambroxol Hydrochloride and Clenbuterol Hydrochloride

[0102] 1. Experimental methods

[0103] (1) Specificity inspection

[0104] Take blank solvent (water), blank excipient solution (excipient + water, the excipient is the other substances in the ambroxol oral solution except ambroxol hydrochloride) and the test solution (ambroxol oral solution without clenbuterol hydrochloride) and perform HPLC detection according to the chromatographic conditions shown in Table 8, and record the corresponding chromatograms and test results.

[0105] (2) Linear relationship test of ambroxol hydrochloride

[0106] Accurately measure 20 μL, 30 μL, 40 μL, 50 μL and 60 μL of the ambroxol hydrochloride reference solution, respectively, record them as reference solution 1 to reference solution 5, and perform HPLC detection according to the chromatographic conditions shown in Table 7 of Example 2.

[0107] After the chromatography is completed, the peak area corresponding to each reference solution is recorded, and the concentration of reference solution 1 to reference solution 5 is used as the abscissa and the corresponding peak area is used as the ordinate to draw a standard curve of ambroxol hydrochloride, and the corresponding standard curve formula is obtained.

[0108] (3) Injection precision study

[0109] Take ambroterol oral solution as the test sample stock solution 1; accurately measure 10 mL of the test sample stock solution 1, put it into a 20 mL volumetric flask, add water to dilute to the scale, and mix well to obtain the test sample stock solution 2.

[0110] Take the test sample solution 1 as an example: the test sample solution 1 was centrifuged at 8000 rpm for 5 min, and then the HPLC detection was performed according to the chromatographic conditions shown in Table 8, with 2 injections in total, and the corresponding peak areas of ambroxol hydrochloride and clenbuterol hydrochloride were recorded.

[0111] The difference between the HPLC detection of the sample solution 2 and the detection of the sample solution 1 is that the injection volume in the chromatographic conditions is 100 μL, 2 injections are made in total, and the corresponding peak areas of ambroxol hydrochloride and clenbuterol hydrochloride are recorded.

[0112] (4) Filter membrane adsorption investigation

[0113] The membrane adsorption test was carried out using the sample solution 1 and the sample solution 2 in the injection precision test (3).

[0114] Taking the sample stock solution 1 as an example, the membrane adsorption investigation was carried out as follows:

[0115] The test sample stock solution 1 was centrifuged at 8000 rpm for 5 min to obtain a centrifuged sample 1;

[0116] The test sample stock solution 1 is filtered using (Welch nylon 25mm, 0.45μm), and the primary filtrate is not discarded after filtration to obtain a filtered liquid 1;

[0117] The test sample stock solution 1 was filtered using (Welch nylon 25mm, 0.45μm), and 2mL of the initial filtrate was discarded after filtration to obtain the filtered liquid 2;

[0118] The sample stock solution 1 was filtered using (Welch nylon 25 mm, 0.45 μm), and 4 mL of the initial filtrate was discarded after filtration to obtain a filtered liquid 3.

[0119] The centrifuged sample, filtered liquid 1, filtered liquid 2 and filtered liquid 3 were respectively subjected to HPLC detection according to the chromatographic conditions shown in Table 8, wherein the injection volume was 100 μL, and the peak areas of clenbuterol hydrochloride and ambroxol hydrochloride were recorded, and the peak area change rate between filtered liquid 1 to filtered liquid 3 and the centrifuged sample was calculated.

[0120] The test sample stock solution 2 was treated in the same manner to obtain the centrifuged sample 2 and the filtered liquids 4 to 6, and the peak areas of clenbuterol hydrochloride and ambroxol hydrochloride were recorded and the corresponding peak area change rates were calculated.

[0121] (5) Solution stability study

[0122] Take the test sample stock solution 1, filter it through (Welch nylon 25mm, 0.45μm), discard 2mL of the initial filtrate, collect the filtered solution, and prepare two filtered solutions in parallel, which are recorded as test sample solution 4 and test sample solution 5.

[0123] The test solution 4 was subjected to HPLC detection according to the chromatographic conditions shown in Table 8, and the peak areas of clenbuterol hydrochloride and ambroxol hydrochloride were recorded.

[0124] After the test solution 5 was allowed to stand for 12 h, HPLC detection was performed according to the chromatographic conditions shown in Table 8, the peak areas of clenbuterol hydrochloride and ambroxol hydrochloride were recorded, and the change rate between them and the peak area of ​​the test solution 4 was calculated.

[0125] (6) Accuracy test

[0126] Accuracy test stock solution: Mix 1.5g of ambroxol hydrochloride and 10mL of clenbuterol hydrochloride reference stock solution in a 50mL volumetric flask, add water to dilute to the scale, and shake well.

[0127] 120% accuracy test sample solution: Place 3 mL of the accuracy test stock solution in a 50 mL volumetric flask, add the blank excipient solution shown in (1) to dilute to the mark, shake well, filter using (Welch nylon 25 mm, 0.45 μm), and discard 2 mL of the primary filtrate to obtain the sample solution;

[0128] 100% accuracy test solution: Place 5 mL of the accuracy test stock solution in a 100 mL volumetric flask, add the blank excipient solution shown in (1) to dilute to the mark, shake well, filter using (Welch nylon 25 mm, 0.45 μm), and discard 2 mL of the primary filtrate to obtain the solution;

[0129] 80% accuracy test sample solution: Place 2 mL of the accuracy test stock solution in a 50 mL volumetric flask, add the blank excipient solution shown in (1) to dilute to the mark, shake well, filter using (Welch nylon 25 mm, 0.45 μm), and discard 2 mL of the primary filtrate to obtain the sample solution.

[0130] Each accuracy test solution was prepared in parallel in one portion, for a total of two portions, which were respectively recorded as accuracy test solution 1 and accuracy test solution 2.

[0131] Taking the 120% accuracy test of the test solution as an example, the accuracy test is carried out as follows:

[0132] The 120% accuracy test solution was tested according to the method described in Example 2, and the concentrations of clenbuterol hydrochloride and ambroxol hydrochloride were calculated. The corresponding recovery rates were calculated after comparison with the theoretical values.

[0133] The parallel prepared accuracy test solution, 100% accuracy test solution and 80% accuracy test solution were treated identically and the results were recorded.

[0134] 2. Experimental results

[0135] (1) Specificity survey results

[0136] The chromatogram of blank solvent is as follows Figure 2 The chromatogram of the blank excipient solution is shown in Figure 3 As shown, the chromatogram of the test solution without clenbuterol hydrochloride is as follows Figure 4 The test results of the test solution without clenbuterol hydrochloride are shown in Table 8.

[0137] Table 8 Test results of the test solution without clenbuterol hydrochloride

[0138]

[0139] The results showed that the blank solvent and blank auxiliary material solution would not interfere with the test results, and the separation of impurity B of ambroxol hydrochloride and ambroxol hydrochloride was greater than 1.5, and the separation degree of the method met the requirements.

[0140] (2) Results of the linear relationship study of ambroxol

[0141] The peak areas of reference solution 1 to reference solution 5 are shown in Table 9, and the standard curve of ambroxol hydrochloride is shown in Figure 5 shown.

[0142] Table 9 Peak areas of reference solution 1 to reference solution 5

[0143]

[0144] The standard curve formula of ambroxol hydrochloride is: y = 13495782.6761x-297358.1441, R 2 =1.0000.

[0145] The results of linear investigation showed that ambroxol hydrochloride showed good linear inertia in the range of 0.6050-1.815 mg / mL, r=1.0000, and the linear equation was: y=13495782.6761x-297358.1441, R 2 =1.0000.

[0146] (3) Results of injection precision investigation

[0147] The test results of the test sample solution 1 and the test sample solution 2 are shown in Table 10.

[0148] Table 10 Test results of sample solution 1 and sample solution 2

[0149]

[0150]

[0151] The results showed that whether it was the test solution 1 or the test solution 2, the S1 / S2 of clenbuterol hydrochloride and ambroxol hydrochloride were both in the range of 0.98-1.02, and the RSD was less than 2%, which met the requirements; and compared with the test results of the test solution 2 and the test solution 1, there was no significant difference in the peak area for either the detection of ambroxol hydrochloride or the detection of clenbuterol hydrochloride, indicating that the injection precision of this method was good.

[0152] (4) Results of membrane adsorption investigation

[0153] The test results of the centrifuged samples and filtered liquid corresponding to the test sample stock solution 1 are shown in Table 11; the test results of the centrifuged samples and filtered liquid corresponding to the test sample stock solution 2 are shown in Table 12.

[0154] Table 11 Test results of the centrifuged samples and filtered liquid corresponding to the test solution 1

[0155]

[0156] Table 12 Test results of the centrifuged samples and filtered liquid corresponding to the test solution 2

[0157]

[0158] The results showed that there was no significant difference in the membrane adsorption test results between the test sample solution 1 and the test sample solution 2; for clenbuterol hydrochloride, compared with the peak area of ​​the sample after centrifugation, the peak area change rates of the samples with 2 mL of the initial filtrate discarded (filtered liquid 2 and filtered liquid 5) and the samples with 4 mL of the initial filtrate discarded (filtered liquid 3 and filtered liquid 6) were all within the range of ±3% (the peak area of ​​clenbuterol hydrochloride was smaller, and the limit was relaxed to ±3%); for ambroxol hydrochloride, compared with the peak area of ​​the sample after centrifugation, compared with the peak area of ​​the sample, the peak area change rates of the samples with 2 mL of the initial filtrate discarded (filtered liquid 2 and filtered liquid 5) and the samples with 4 mL of the initial filtrate discarded (filtered liquid 3 and filtered liquid 6) were all within the range of ±2%.

[0159] This indicates that after filtering the test sample stock solution (ambroterol oral solution) and discarding 2 mL of the initial filtrate, there is no filter membrane adsorption.

[0160] (5) Solution stability test results

[0161] The test results of the test solution 4 (directly tested) and the test solution 5 (tested after standing for 12 hours) are shown in Table 13.

[0162] Table 13 Test results of test solution 4 and test solution 5

[0163]

[0164] The results showed that there was no significant difference between the test results of the samples tested after being placed for 12 hours and the samples tested directly. The peak area change rates of clenbuterol hydrochloride and ambroxol hydrochloride were both less than 2%, indicating that the test solution was stable within 12 hours.

[0165] (6) Accuracy test results

[0166] The accuracy test results are shown in Table 14 and Table 15, wherein Table 14 is the accuracy test results of clenbuterol hydrochloride, and Table 15 is the accuracy test results of ambroxol hydrochloride.

[0167] Table 14 Clenbuterol hydrochloride accuracy test results

[0168]

[0169] Table 15 Ambroxol hydrochloride accuracy test results

[0170]

[0171] The results showed that for clenbuterol hydrochloride, the recoveries of the six samples were between 102.5% and 105.8%, with an average of 104% and an RSD of 1.1%. The recovery of the test solution 1 in the 80% accuracy test was 105.8%, which was out of the range. For ambroxol hydrochloride, the recoveries of the six samples were between 99.9% and 101.2%, with an average of 101% and an RSD of 0.6%. The accuracy of ambroxol hydrochloride of all samples met the requirements.

[0172] Comparative Example 1: (WS 1 -(X-118)-2006Z) Detection of Ambroxol Hydrochloride and Clenbuterol Hydrochloride

[0173] 1. Experimental methods

[0174] According to the national drug standards (WS 1- (X-118)-2006Z) method for detecting the content of clenbuterol hydrochloride and ambroxol hydrochloride in ambroxol oral solution.

[0175] National Drug Standards (WS 1- (X-118)-2006Z) shows that the chromatographic conditions of ambroxol hydrochloride are: octadecylsilane bonded silica gel is used as the filler; 1.0% by mass fraction of sodium sulfosuccinate dioctyl ester methanol solution-water-acetic acid (740:260:10) (the pH value is adjusted to 3.0 with 85% phosphoric acid) is used as the mobile phase; and the detection wavelength is 244nm.

[0176] National Drug Standards (WS 1- (X-118)-2006Z) shows that the chromatographic conditions of clenbuterol hydrochloride are: using octadecylsilane bonded silica gel as the filler, using 1.7% sodium sulfosuccinate dioctyl ester methanol solution-water-acetic acid (700:300:100) as the mobile phase, and the detection wavelength is 296nm.

[0177] (1) Confirmation of the method for determining the content of clenbuterol hydrochloride

[0178] Clenbuterol hydrochloride reference solution 1: Accurately weigh 5 mg of clenbuterol hydrochloride reference solution, place in a 50 mL volumetric flask, add solvent (1.7% sodium sulfosuccinate dioctyl ester methanol solution-water-acetic acid (volume ratio 700:300:100)) and dilute to scale, shake well to obtain;

[0179] The test solution was ambroterol oral solution.

[0180] The clenbuterol hydrochloride reference solution 1 and the test solution were prepared according to the national drug standards (WS 1- (X-118)-2006Z) was used for high performance liquid chromatography detection, and the retention time of clenbuterol hydrochloride and the corresponding peak area were recorded.

[0181] The test solution was injected 6 times continuously and the corresponding experimental results were recorded.

[0182] (2) Confirmation of the determination method of ambroxol hydrochloride content

[0183] Ambroxol hydrochloride reference solution 1 (impurity B content is 0.04%, v / v): accurately weigh 150 mg of ambroxol hydrochloride reference, place it in a 100 mL volumetric flask, add solvent (mass fraction 1.0% sodium sulfosuccinate dioctyl ester methanol solution - water - acetic acid (volume ratio 740:260:10)) and dilute to the scale, shake well.

[0184] The test solution was ambroxol oral solution (with an impurity B content of 0.14%, v / v).

[0185] The ambroxol hydrochloride reference solution 1 and the test solution were prepared according to the national drug standards (WS 1- (X-118)-2006Z) is carried out high performance liquid chromatography detection, chromatogram is recorded, and the content of ambroxol hydrochloride is calculated to obtain the assay result;

[0186] Content determination result = (detected content ÷ actual content) × 100%.

[0187] (3) Investigation of the separation degree between the test sample ambroxol hydrochloride and impurity B by different chromatographic columns

[0188] According to the national drug standards (WS 1- (X-118)-2006Z) was used to detect the content of ambroxol hydrochloride in the ambroxol hydrochloride reference solution 1 and the test solution. The detection was performed twice. The first detection was performed using a chromatographic column numbered A10 with an injection volume of 20 μL; the second detection was performed using a chromatographic column numbered W5 with an injection volume of 50 μL. The corresponding chromatograms were recorded.

[0189] 2. Experimental results

[0190] (1) The retention times of clenbuterol hydrochloride reference solution 1 and the test solution are shown in Table 16. The test results of 6 consecutive injections of the test solution are shown in Table 17.

[0191] Table 16 Retention time of Clenbuterol hydrochloride reference solution and test solution

[0192] Attribution Retention time / min Theoretical plate number Clenbuterol hydrochloride reference solution 1 12.264 7553 Test solution 11.950 5216

[0193] Table 17 Test results of 6 consecutive injections of the test solution

[0194] Serial number Peak area Theoretical plate number 1 26421 4102 2 26724 4321 3 25460 2996 4 26385 3474 5 25910 3182 6 24594 2848 RSD / % 3.1 /

[0195] The results showed that the system applicability of the detection method for clenbuterol hydrochloride content in the national drug standard (standard number: WS1-(X-118))-2006Z) met the requirements, but the main peak response was low, the injection precision was poor (RSD greater than 2.0%), and as the number of injection needles increased, the main peak theoretical plate number decreased rapidly, and the ion pair reagent (mobile phase) dioctyl sodium sulfosuccinate caused great damage to the chromatographic column.

[0196] (2) According to the national drug standards (WS 1- (X-118)-2006Z) for the detection of ambroxol oral solution Figure 6 As shown, the results show that: Ambroxol hydrochloride cannot be separated from Ambroxol hydrochloride impurity B, and impurity B overlaps with the test solution; the content determination result of Ambroxol hydrochloride is 100.8%, compared with the method shown in Example 2, the determination result deviation is less than 2%, but the separation degree of Ambroxol hydrochloride is 1.11, which is less than 1.5, and Ambroxol hydrochloride impurity B and Ambroxol hydrochloride cannot be effectively separated.

[0197] (3) The test solution was chromatographically prepared using a column numbered A10 with an injection volume of 20 μL according to the national drug standard (WS 1- (X-118)-2006Z) chromatogram for high performance liquid chromatography detection Figure 7 As shown; using the chromatographic column numbered W5 and an injection volume of 50 μL, according to the national drug standard (WS 1- (X-118)-2006Z) chromatogram for high performance liquid chromatography detection Figure 8 As shown; Figure 7 and Figure 8 Combined on the same chromatogram Fig. 9 .

[0198] The results showed that according to the national drug standards (WS 1-(X-118)-2006Z) When testing ambroxol oral solution, ambroxol hydrochloride could not be separated from ambroxol hydrochloride impurity B (the content of ambroxol hydrochloride impurity B in the test solution was 0.14%, and the content of ambroxol hydrochloride impurity B in the reference solution was 0.04%); and replacing different types of chromatographic columns and changing the injection volume could not improve the separation of ambroxol hydrochloride and ambroxol hydrochloride impurity B.

[0199] Comparative Example 2 A method for simultaneously detecting ambroxol hydrochloride and clenbuterol hydrochloride

[0200] 1. Experimental methods

[0201] The chromatographic conditions of the method shown in Comparative Example 2 are different from the chromatographic conditions shown in Table 7 of Example 2 (Table 7) in that the mobile phase in the chromatographic conditions is: a mixed solution of 0.2% by mass of sodium sulfosuccinate dioctyl methanol, water and acetic acid in a volume ratio of 740:260:10 (the pH value is adjusted to 3.0 with 85% phosphoric acid).

[0202] The test object is ambroterol oral solution, and high performance liquid chromatography is performed according to the above-mentioned chromatographic method, and the corresponding chromatogram is recorded.

[0203] 2. Experimental results

[0204] The chromatogram corresponding to the method shown in Comparative Example 2 is as follows Fig.10 As shown, the results show that under the chromatographic conditions shown in Comparative Example 2, the impurity B of ambroxol hydrochloride and ambroxol hydrochloride cannot be effectively separated.

[0205] Comparative Example 3: A method for simultaneously detecting ambroxol hydrochloride and clenbuterol hydrochloride

[0206] The chromatographic conditions of the method shown in Comparative Example 3 are different from the chromatographic conditions shown in Table 7 of Example 2 in that the mobile phase in the chromatographic conditions is a mixed solution of 0.8% sodium sulfosuccinate dioctyl methanol, water and acetic acid in a volume ratio of 740:260:10 (the pH value is adjusted to 3.0 with 85% phosphoric acid).

[0207] The test object is ambroterol oral solution, and high performance liquid chromatography is performed according to the above-mentioned chromatographic method, and the corresponding chromatogram is recorded.

[0208] 2. Experimental results

[0209] The chromatogram corresponding to the method shown in Comparative Example 3 is as follows Fig.11 As shown, the results show that the impurity B of ambroxol hydrochloride and ambroxol hydrochloride cannot be effectively separated, and the peak time of ambroxol hydrochloride is relatively late.

[0210] Comparative Example 4: A method for simultaneously detecting ambroxol hydrochloride and clenbuterol hydrochloride

[0211] The chromatographic conditions of the method shown in Comparative Example 4 are different from the chromatographic conditions shown in Table 7 of Example 2 in that the mobile phase in the chromatographic conditions is: a mixed solution of 0.3% by mass of sodium sulfosuccinate dioctyl methanol, water and acetic acid in a volume ratio of 700:300:10 (the pH value is adjusted to 3.0 with 85% phosphoric acid).

[0212] The test object is ambroterol oral solution, and high performance liquid chromatography is performed according to the above-mentioned chromatographic method, and the corresponding chromatogram is recorded.

[0213] 2. Experimental results

[0214] The chromatogram corresponding to the method shown in Comparative Example 4 is as follows Fig.12 As shown, the results show that the impurity B of ambroxol hydrochloride and ambroxol hydrochloride cannot be effectively separated, and the elution time of ambroxol hydrochloride is more than doubled compared with the result corresponding to the method shown in Example 2, and the elution time is too late.

[0215] Comparative Example 5: A method for simultaneously detecting ambroxol hydrochloride and clenbuterol hydrochloride

[0216] 1. Experimental methods

[0217] Chromatographic column: octadecylsilane bonded silica gel as filler; flow rate: 1.0 mL / min; column temperature: 40°C; wavelength: 248 nm; injection volume: 20 μL; injector temperature: 6°C.

[0218] The mobile phases were: mobile phase A: 0.01 mol / L diammonium hydrogen phosphate solution (pH adjusted to 7.0 with phosphoric acid), mobile phase B: acetonitrile.

[0219] The isocratic elution mode of (mobile phase A: mobile phase B) = (60:40) was adopted to detect the ambroterol oral solution and obtain the corresponding chromatogram.

[0220] 2. Experimental results

[0221] The detection results corresponding to isocratic elution are shown in Table 18 and Fig.13 shown.

[0222] Table 18 Detection results corresponding to isocratic elution

[0223]

[0224]

[0225] The results showed that under the chromatographic conditions shown in Comparative Example 5, the clenbuterol hydrochloride in the ambroxol oral solution could not be completely separated from the adjacent impurities, and the ambroxol hydrochloride impurity B and ambroxol hydrochloride could not be separated either.

[0226] Comparative Example 6 A method for simultaneously detecting ambroxol hydrochloride and clenbuterol hydrochloride

[0227] 1. Experimental methods

[0228] Chromatographic column: octadecylsilane bonded silica gel as filler; flow rate: 1.0 mL / min; column temperature: 40°C; wavelength: 248 nm; injection volume: 20 μL; injector temperature: 6°C.

[0229] The mobile phases were: mobile phase A: 10 mmol / L diammonium hydrogen phosphate (pH adjusted to 8.2), mobile phase B: acetonitrile (0.1% tetrahydrofuran).

[0230] The ambroterol oral solution was detected by eluting with a gradient elution degree as shown in Table 19 to obtain a corresponding chromatogram.

[0231] Table 19 Gradient elution program

[0232] Time / min Mobile phase A / % Mobile phase B / % 0 70 30 5 70 30 15 60 40 20 40 60 21 70 30 30 70 30

[0233] 2. Experimental results

[0234] The detection results corresponding to the gradient elution are shown in Table 20 and Fig.14 shown.

[0235] Table 20 Detection results corresponding to gradient elution

[0236]

[0237] The results showed that in the ambroxol oral solution, clenbuterol hydrochloride could not be completely separated from adjacent impurities.

[0238] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A mobile phase suitable for simultaneous detection of ambroxol hydrochloride and / or clenbuterol hydrochloride by high performance liquid chromatography, characterized in that: The mobile phase is a mixed solution composed of 0.3% to 0.8% sodium sulfosuccinate dioctyl ester methanol, water and acetic acid in a volume ratio of 740 to 760:240 to 260:10; the pH value of the mixed solution is 2.8 to 3.

2.

2. The mobile phase according to claim 1, characterized in that The mobile phase is a mixed solution consisting of 0.3% by mass of sodium sulfosuccinate dioctyl ester methanol, water and acetic acid.

3. The mobile phase according to claim 2, characterized in that The volume ratio of the sodium sulfosuccinate dioctyl ester methanol with a mass fraction of 0.3%, water and acetic acid is 740:260:

10.

4. A method for simultaneously detecting ambroxol hydrochloride and clenbuterol hydrochloride, characterized in that: The method is specifically as follows: High performance liquid chromatography is used to detect a sample containing ambroxol hydrochloride and / or clenbuterol hydrochloride; wherein the mobile phase of the high performance liquid chromatography is the mobile phase described in any one of claims 1 to 3.

5. The method according to claim 4, characterized in that The sample to be tested is ambroterol oral solution.

6. The method according to claim 5, characterized in that The ambroxol oral solution is an ambroxol oral solution with an ambroxol hydrochloride concentration of 0.03 to 1.5 mg / mL.

7. The method according to claim 6, characterized in that The ambroxol oral solution is an ambroxol oral solution with an ambroxol hydrochloride concentration of 1.5 mg / mL.

8. The method according to claim 4, characterized in that The chromatographic conditions of the high performance liquid chromatography are: Chromatographic column: octadecylsilane bonded silica gel chromatographic column; detection wavelength: 296nm; column temperature: 30-35°C; injection volume: 20-50μL; flow rate: 0.8-1.2mL / min.

9. The method according to claim 8, characterized in that The chromatographic conditions of the high performance liquid chromatography are: Chromatographic column: octadecylsilane bonded silica gel chromatographic column; detection wavelength: 296nm; column temperature: 30℃; injection volume: 50μL; flow rate: 1.0mL / min.

10. Use of the mobile phase according to any one of claims 1 to 3 and / or the method according to any one of claims 4 to 9 in simultaneously detecting the content of ambroxol hydrochloride and / or clenbuterol hydrochloride in ambroxol oral solution.

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