Method for detecting oleic acid in levosalbutamol tartrate inhalation aerosol

Abstract

The invention discloses a method for detecting oleic acid in levosalbutamol tartrate inhalation aerosol, and belongs to the technical field of medicine detection. The method comprises the following steps: removing a propellant from levosalbutamol tartrate inhalation aerosol, and diluting with methanol to obtain a test solution; analyzing the test solution by adopting ultra-high performance liquid chromatography, and identifying the existence of levosalbutamol tartrate and oleic acid through a chromatogram; chromatographic conditions are as follows: octadecylsilane chemically bonded silica is taken as a filling agent of a chromatographic column; an acidic aqueous solution with a pH value of 2.0 is used as a mobile phase A, acetonitrile is used as a mobile phase B, and isocratic elution is carried out. According to the method, detection can be completed within 3 minutes, and the analysis efficiency is high; the content of oleic acid is measured through an external standard method, the method has the advantages of being sensitive, exclusive, accurate, efficient, convenient, fast, low in cost and the like, and a basis is provided for quality control of the levosalbutamol tartrate inhalation aerosol.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and in particular relates to a method for detecting oleic acid in a levosalbuterol tartrate inhalation aerosol. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Levosalbutamol tartrate (R-AS), chemical name is (R)-4-(2-(tert-butylamino)-1-hydroxyethyl)-2-(hydroxymethyl)phenol (2R,3R)-2,3-dihydroxysuccinate, structural formula is:

[0004]

[0005] R-AS can activate β-2 adrenergic receptors in respiratory smooth muscle, which can activate adenylate cyclase and increase the concentration of intracellular cyclic adenosine monophosphate, and then activate protein kinase A, thereby inhibiting myosin phosphorylation and reducing calcium ion concentration, causing smooth muscle relaxation.

[0006] Levosalbutamol tartrate inhalation aerosol is used to prevent or treat bronchospasm symptoms in patients with reversible obstructive airway disease. Oleic acid, a pharmaceutical excipient in levosalbutamol tartrate inhalation aerosol, has a great influence on the quality of the drug. As a stabilizer, it has the function of stabilizing drug particles, ensuring that drug particles will not aggregate or crystallize due to changes in the external environment, thereby maintaining the stability and effectiveness of the drug. Oleic acid also has some other related applications and impacts. For example, in some cases, the injection of oleic acid may cause damage to the lungs, including damage to alveolar epithelial cells, macrophage activation, increased inflammatory response, pulmonary edema, and ventilation-perfusion imbalance. Therefore, it is necessary to strictly control the content of oleic acid in levosalbutamol tartrate inhalation aerosol.

[0007] The detection methods for oleic acid content in pharmaceutical excipients in domestic and foreign pharmacopoeias and related reports are mostly titration and gas chromatography. The titration method has human operation errors and is difficult to accurately quantify. Oleic acid, an excipient in salbutamol tartrate inhalation aerosol, is used as a surfactant in a small amount, and other components in the aerosol have a large interference, and the detection cost is high, so gas chromatography is not suitable.

[0008] At present, there is no report on the determination of oleic acid content by UPLC, especially the analysis and determination of oleic acid content in aerosols containing oleic acid excipients. Therefore, it is very necessary to establish a set of oleic acid content determination analysis methods with strong specificity, accurate quantitative analysis and good repeatability to provide a reference for the quality control of the drug. Summary of the invention

[0009] In order to solve the shortcomings of the prior art, the purpose of the present invention is to provide a method for detecting oleic acid in salbutamol tartrate inhalation aerosol. The method provided by the present invention uses ultra-high performance liquid chromatography to detect oleic acid in salbutamol tartrate inhalation aerosol, which has the advantages of sensitivity, exclusivity, accuracy, high efficiency, convenience, low cost, etc. The detection process is completed within 3 minutes with high efficiency.

[0010] In order to achieve the above object, the technical solution of the present invention is:

[0011] The first aspect of the present invention provides a method for detecting oleic acid in a salbutamol tartrate inhalation aerosol, comprising the following steps:

[0012] After removing the propellant from the levosalbutamol tartrate inhalation aerosol, dilute it with methanol to obtain the test solution;

[0013] The test solution is analyzed by ultra-high performance liquid chromatography, and the presence of salbutamol tartrate and oleic acid is identified by the chromatogram;

[0014] Chromatographic conditions included:

[0015] The chromatographic column is filled with octadecylsilane bonded silica gel;

[0016] The mobile phase A was an acidic aqueous solution at pH 2.0 and the mobile phase B was acetonitrile, and the elution was performed isocratically.

[0017] In some embodiments of the present invention, the volume ratio of mobile phase A to mobile phase B is (18-22):(82-78), preferably 20:80.

[0018] In some embodiments of the present invention, the pH 2.0 acidic aqueous solution is one of a pH 2.0 formic acid aqueous solution, a pH 2.0 acetic acid aqueous solution and a pH 2.0 trifluoroacetic acid aqueous solution, preferably a pH 2.0 trifluoroacetic acid aqueous solution.

[0019] In some embodiments of the present invention, the chromatographic column is Thermo Hypersil Gold C18 (50 mm×2.1 mm, 3 μm) or Waters ACQUITY BEH C18 (50 mm×2.1 mm, 1.7 μm).

[0020] In some embodiments of the present invention, the chromatographic conditions also include: column temperature of 25-35°C, preferably 30°C; flow rate of 0.4-0.6mL / min, preferably 0.5mL / min; injection volume of 4.5-5.5μL, preferably 5μL; detection wavelength of 201-205nm, preferably 203nm.

[0021] In some embodiments of the present invention, the chromatographic conditions are:

[0022] The chromatographic column was Thermo Hypersil Gold C18 (50 mm × 2.1 mm, 3 μm);

[0023] Mobile phase A was trifluoroacetic acid aqueous solution at pH 2.0, mobile phase B was acetonitrile, and the volume ratio of mobile phase A to mobile phase B was 20:80; isocratic elution;

[0024] The detection wavelength was 203 nm, the column temperature was 30 °C, the flow rate was 0.5 mL / min, and the injection volume was 5 μL.

[0025] The second aspect of the present invention provides a method for quantitatively detecting oleic acid in a salbutamol tartrate inhalation aerosol, comprising the following steps:

[0026] After removing the propellant from the levosalbutamol tartrate inhalation aerosol, the aerosol is diluted with methanol to obtain a test solution; the test solution is subjected to ultra-high performance liquid chromatography analysis using the above method to obtain a chromatogram of the test solution;

[0027] The oleic acid methanol solution is subjected to ultra-high performance chromatography analysis using the above method to obtain a chromatogram of the oleic acid methanol solution;

[0028] The content of oleic acid was calculated by the peak area in the chromatogram.

[0029] In some embodiments of the present invention, the concentration of the test solution is 0.9-1.1%, volume percent.

[0030] The third aspect of the present invention provides a method for evaluating the quality of salbutamol tartrate inhalation aerosol, using the above method.

[0031] A fourth aspect of the present invention provides the use of detecting oleic acid in the quality control of salbutamol tartrate inhalation aerosol.

[0032] The beneficial effects of the present invention are:

[0033] The invention provides a method for detecting oleic acid in a levosalbuterol tartrate inhalation aerosol. The method adopts an ultra-high performance liquid chromatography method for detection, uses octadecylsilane bonded silica gel as a filler, uses a pH 2.0 acidic aqueous solution as a mobile phase A, uses acetonitrile as a mobile phase B, and performs isocratic elution. The detection can be completed within 3 minutes, and the analysis efficiency is high. The oleic acid content is determined by an external standard method, and the method has the advantages of sensitivity, exclusiveness, accuracy, high efficiency, convenience, rapidity, low cost, and the like, and provides a basis for quality control of the levosalbuterol tartrate inhalation aerosol.

[0034] When the present invention quantitatively detects oleic acid in salbutamol tartrate inhalation aerosol, the sample solution and oleic acid methanol solution used do not interfere with oleic acid determination, and the specificity is good; the repeatability of 6 samples is verified, and the RSD of oleic acid content is less than 2.0%, and the precision is good; the linearity and accuracy of oleic acid in the concentration range of 1μg / mL to 15μg / mL (10% to 150%) are also verified, and oleic acid r>0.999 in the above range, and the recovery rate is between 98% and 102%, and the linearity and accuracy are good. In addition, the present invention can still accurately detect under different chromatographic conditions, indicating that the method provided by the present invention has good durability. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0036] Figure 1 The oleic acid reference substance solution spectrum of Example 2 of the present invention;

[0037] Figure 2 is the blank solvent spectrum of Example 2 of the present invention;

[0038] Figure 3 The chromatogram of the salbutamol tartrate inhalation aerosol sample solution of Example 2 of the present invention;

[0039] Figure 4 This is a specific overlay diagram of Example 2 of the present invention. DETAILED DESCRIPTION

[0040] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0041] Example 1: System Applicability

[0042] 1-1 Solution Preparation

[0043] Oleic acid reference solution: Accurately weigh an appropriate amount of oleic acid reference, dissolve it in methanol and quantitatively dilute it to make a solution containing approximately 10 μg per 1 mL, and prepare 2 parallel portions.

[0044] 1-2 Chromatographic conditions

[0045] Chromatographic column: Thermo Hypersil Gold C18 (50 mm × 2.1 mm, 3 μm);

[0046] Mobile phase: pH 2.0 trifluoroacetic acid aqueous solution: acetonitrile = 20:80;

[0047] Elution mode: isocratic elution;

[0048] The detection wavelength is 203nm;

[0049] Flow rate: 0.5 mL / min;

[0050] The column temperature was 30°C;

[0051] The injection volume was 5 μL.

[0052] 1-3 Experimental conclusion:

[0053] Accurately measure the above oleic acid reference solution, inject it into the ultra-high performance liquid chromatograph, record the chromatogram, and the results are shown in Table 1.

[0054] Table 1 System suitability results

[0055]

[0056]

[0057] Conclusion: In this system, 5 samples of reference solution were continuously injected, and the RSD of oleic acid peak area was 0.22%, which was less than 2.0%, meeting the requirements. The system applicability under this method was stable and reproducible, and it could provide a stable experimental environment for sample detection.

[0058] Example 2: Specificity

[0059] 2-1 Solution preparation

[0060] Diluent: Methanol (blank solvent).

[0061] Oleic acid reference solution: Accurately weigh an appropriate amount of oleic acid reference, dissolve it in methanol and quantitatively dilute it to make a solution containing approximately 10 μg per 1 mL.

[0062] Test solution: Take levosalbuterol tartrate inhalation aerosol, and after the propellant is completely evaporated from the cone hole, accurately measure 1 mL and place it in a 100 mL volumetric flask, dilute to the scale with methanol, and shake well to use as the test solution.

[0063] 2-2 Chromatographic conditions are the same as those in Example 1.

[0064] 2-3 Conclusion:

[0065] Accurately measure the above oleic acid reference solution and test solution, inject them into the ultra-high performance liquid chromatograph respectively, and record the chromatogram. The chromatogram of oleic acid reference solution is shown in Figure 1 , blank solvent chromatogram Figure 2 , the chromatogram of the test solution is shown in Figure 3 , see the dedicated overlay Figure 4 .

[0066] Conclusion: In the chromatogram of oleic acid control solution, the retention time of oleic acid peak is about 1.5min; the baseline of blank solvent chromatogram is stable; other chromatographic peaks in blank solvent and test solution do not interfere with the detection of oleic acid, indicating that the method of the present invention has good specificity.

[0067] Example 3: Linearity and range experiments

[0068] 3-1 Solution preparation

[0069] Accurately weigh an appropriate amount of oleic acid reference substance and prepare a series of linear concentration solutions with concentrations ranging from 1 μg / mL to 15 μg / mL (10% to 150%).

[0070] 3-2 Chromatographic conditions are the same as those in Example 1.

[0071] 3-3 Conclusion:

[0072] The above series of concentration linear solutions were accurately measured and injected into the ultra-high performance liquid chromatograph to record the chromatogram. The results are shown in Table 2. A linear regression equation was made with concentration as the abscissa and peak area as the ordinate.

[0073] Table 2 Oleic acid and linear range detection results

[0074]

[0075] Conclusion: In the range of 1 μg / mL to 15 μg / mL, the oleic acid content and the oleic acid peak area show a significant linear relationship, and the regression equation is y=4131.6x-248.95, r=0.9999, indicating that the method of the present invention has a good linear relationship. Compared with the titration method and gas chromatography method, the detection sensitivity of the present invention is higher and the results are more accurate.

[0076] Example 4: Accuracy Experiment

[0077] The accuracy was investigated through recovery test, and the study covered the concentration range of 80% to 120% of the oleic acid concentration in the preparation prescription.

[0078] 4-1 Solution preparation

[0079] Oleic acid reference substance stock solution: Take an appropriate amount of oleic acid reference substance, weigh it accurately, place it in a 50mL volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and use it as the oleic acid reference substance stock solution.

[0080] Blank test solution: Take appropriate amount of salbutamol tartrate and anhydrous ethanol, weigh accurately, place in a 100mL volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and use as the blank test solution.

[0081] Accurately measure 0.8mL, 1.0mL and 1.2mL of oleic acid reference stock solution respectively, place in a 50mL volumetric flask, then accurately add 5mL of blank test solution respectively, dilute to the scale with methanol, shake well, and prepare 3 parallel copies for each spiked concentration level.

[0082] 4-2 Chromatographic conditions are the same as those in Example 1.

[0083] 4-3 Conclusion:

[0084] Accurately measure the above solution, inject it into ultra-high performance liquid chromatography, record the chromatogram, and calculate the accuracy by peak area according to the external standard method. The results are shown in Table 3.

[0085] Table 3 Accuracy test results

[0086]

[0087]

[0088] Conclusion: Under 80%, 100%, and 120% accuracy solutions, the average recovery of oleic acid was 99.9%, and the RSD was 0.36%, which was in line with the regulations. The test results showed that the accuracy of the present invention was good.

[0089] Example 5: Precision Experiment

[0090] 5-1 Solution preparation

[0091] Test solution: Take salbutamol tartrate inhalation aerosol, after the propellant is completely evaporated from the cone, accurately measure 1 mL into a 100 mL volumetric flask, dilute to the mark with methanol, shake well, and use this as the test solution. Prepare 6 portions in parallel.

[0092] Oleic acid reference solution: Accurately weigh an appropriate amount of oleic acid reference, dissolve it in methanol and quantitatively dilute it to make a solution containing about 10 μg per 1 mL. Prepare 2 portions in parallel.

[0093] 5-2 Chromatographic conditions are the same as those in Example 1.

[0094] 5-3 Conclusion:

[0095] Accurately measure the above test solution and oleic acid reference solution, inject them into ultra-high performance liquid chromatography, record the chromatogram, and calculate the oleic acid content by the external standard method. The precision results are shown in Table 4.

[0096] Table 4 Precision test results

[0097]

[0098]

[0099] Conclusion: The results of the determination of 6 replicate test sample solutions prepared in parallel showed that the RSD value of oleic acid content was 0.95%, which was less than 2.0%, proving that the present invention has good repeatability. Compared with the titration method and gas chromatography method, the present invention can quickly, effectively and accurately determine the content of oleic acid in the test sample.

[0100] Example 6: Durability Test

[0101] Durability refers to the degree to which the test results are not affected by slight changes in the chromatographic conditions. The preparation of the oleic acid reference solution and the test solution is the same as in Example 2. On the basis of the chromatographic conditions of Example 1, the column temperature, flow rate, acid solution, organic phase ratio, detection wavelength and different brands and models of chromatographic columns are changed. The results of the durability test of the detection method are shown in Table 5.

[0102] Table 5 Test results of test solution

[0103]

[0104]

[0105] Conclusion: Under the conditions of changing the column temperature by ±5°C, the flow rate by ±0.1 mL / min, different acidic solutions, the mobile phase ratio by ±2%, the wavelength by ±2 nm, and changing the chromatographic columns of different brands and models, the RSD value of the oleic acid content in the test solution was less than 2.0%, indicating that the durability of the present invention is good.

[0106] Based on the above experimental results, the detection method of the present invention is used to detect the oleic acid content in the salbutamol tartrate inhalation aerosol. The method of the present invention is sensitive, exclusive, accurate, efficient, convenient, rapid, and low-cost, and can provide a basis for the quality control of the salbutamol tartrate inhalation aerosol, thereby ensuring its clinical efficacy.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting oleic acid in salbutamol tartrate inhalation aerosol, characterized in that: The steps include: After removing the propellant from the levosalbutamol tartrate inhalation aerosol, dilute it with methanol to obtain the test solution; The test solution is analyzed by ultra-high performance liquid chromatography, and the presence of salbutamol tartrate and oleic acid is identified by the chromatogram; Chromatographic conditions included: The chromatographic column is filled with octadecylsilane bonded silica gel; The mobile phase A was an acidic aqueous solution at pH 2.0 and the mobile phase B was acetonitrile, and the elution was performed isocratically.

2. The method according to claim 1, characterized in that The volume ratio of mobile phase A to mobile phase B is (18-22):(82-78), preferably 20:

80.

3. The method according to claim 1, characterized in that The pH 2.0 acidic aqueous solution is one of a pH 2.0 formic acid aqueous solution, a pH 2.0 acetic acid aqueous solution and a pH 2.0 trifluoroacetic acid aqueous solution, preferably a pH 2.0 trifluoroacetic acid aqueous solution.

4. The method according to claim 1, characterized in that The chromatographic column was Thermo Hypersil Gold C18 (50 mm×2.1 mm, 3 μm) or Waters ACQUITY BEH C18 (50 mm×2.1 mm, 1.7 μm).

5. The method according to claim 1, characterized in that The chromatographic conditions also include: column temperature of 25-35°C, preferably 30°C; flow rate of 0.4-0.6mL / min, preferably 0.5mL / min; injection volume of 4.5-5.5μL, preferably 5μL; detection wavelength of 201-205nm, preferably 203nm.

6. The method according to claim 1, characterized in that The chromatographic conditions are: The chromatographic column was Thermo Hypersil Gold C18 (50 mm × 2.1 mm, 3 μm); Mobile phase A was trifluoroacetic acid aqueous solution at pH 2.0, mobile phase B was acetonitrile, and the volume ratio of mobile phase A to mobile phase B was 20:80; isocratic elution; The detection wavelength was 203 nm, the column temperature was 30 °C, the flow rate was 0.5 mL / min, and the injection volume was 5 μL.

7. A method for quantitatively detecting oleic acid in salbutamol tartrate inhalation aerosol, characterized in that: The steps include: After removing the propellant from the levosalbutamol tartrate inhalation aerosol, dilute it with methanol to obtain a test solution; perform ultra-high performance liquid chromatography analysis on the test solution using the method described in any one of claims 1 to 6 to obtain a chromatogram of the test solution; The oleic acid methanol solution is subjected to ultra-high performance chromatography analysis using the method described in any one of claims 1 to 6 to obtain a chromatogram of the oleic acid methanol solution; The content of oleic acid was calculated by the peak area in the chromatogram.

8. The method according to claim 7, characterized in that The concentration of the test solution is 0.9-1.1%, volume percentage.

9. A quality assessment method for levosalbuterol tartrate inhalation aerosol, characterized in that: The method according to any one of claims 1 to 8 is adopted.

10. Application of oleic acid detection in quality control of salbutamol tartrate inhalation aerosol.

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