UPLC (Ultra Performance Liquid Chromatography) analysis method for content of suspension for inhalation of fluticasone propionate

Abstract

The invention belongs to the technical field of drug detection, and relates to a UPLC analysis method for the content of a suspension for fluticasone propionate inhalation. In the UPLC detection, isocratic elution is carried out in a chromatographic column by adopting a mobile phase; the volume ratio of the mobile phase A to the mobile phase B is 35: 65-45: 55, the mobile phase B is acetonitrile, the mobile phase A is an aqueous solution of methanol, and the mobile phase A contains 0.045-0.055% by mass of phosphoric acid; octadecylsilane chemically bonded silica is used as a filling agent of the chromatographic column. Through the method, the content of the suspension for inhalation of fluticasone propionate can be rapidly and accurately detected. The method disclosed by the invention is simple to operate, good in specificity, short in detection time and high in accuracy, avoids interference of auxiliary materials in the suspension for inhalation on main components, increases the solubility of the main components, shortens the analysis time, reduces the solvent dosage and reduces the analysis cost.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection and relates to a UPLC (ultra-high performance liquid chromatography) analysis method for the content of a fluticasone propionate suspension for inhalation. 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 necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Fluticasone propionate suspension for nebulized inhalation is a potent inhaled glucocorticoid, mainly used for the preventive treatment of severe asthma in adults and adolescents over 16 years old (patients who require high-dose inhaled or oral glucocorticoid treatment). Fluticasone propionate suspension for nebulized inhalation is administered by nebulized inhalation, which requires low respiratory coordination in children with asthma. Especially during acute asthma attacks, it can better enable children to receive the expected therapeutic dose and achieve the maximum therapeutic effect. It can also avoid the systemic adverse reactions of oral corticosteroids on children's growth and development.

[0004] According to the inventors' research, there is currently no UPLC analysis method for detecting the content of fluticasone propionate suspension for nebulized inhalation. Therefore, it is necessary to establish a set of content detection and analysis methods with strong specificity, good repeatability, high accuracy, simplicity and rapidity, so as to control the quality of the drug. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a UPLC analysis method for the content of fluticasone propionate suspension for inhalation. The analysis method provided by the present invention can accurately detect the content of fluticasone propionate suspension for inhalation and has a shorter analysis time.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] In a first aspect, a detection kit is provided, which is used for quantitative detection of fluticasone propionate in a fluticasone propionate suspension for inhalation by UPLC; the kit comprises a solvent for making a mobile phase, the solvent comprising methanol, water, and acetonitrile; wherein acetonitrile is used to make mobile phase B, and methanol and water are used to make mobile phase A; the volume fraction of methanol in mobile phase A is 2.5-3.5%, and mobile phase A contains phosphoric acid with a mass fraction of 0.045-0.055%, and when UPLC detection is adopted, the volume ratio of mobile phase A to mobile phase B is 35:65-45:55.

[0008] Research has found that when UPLC is used to detect the main component (fluticasone propionate) in the fluticasone propionate inhalation suspension, the difficulty in effectively separating the main component from the auxiliary materials is the main factor affecting its detection. The present invention has shown through research that adding 2.5-3.5% methanol can effectively separate the main component fluticasone propionate and the auxiliary materials sorbitan monolaurate and polysorbate 20, avoiding the interference of the auxiliary materials on the main component and causing inaccurate content detection. At the same time, adding methanol can help increase the solubility of the main component of the test sample.

[0009] In some embodiments, the detection kit further comprises a fluticasone propionate standard, which is used to prepare a fluticasone propionate reference solution for UPLC detection.

[0010] In some embodiments, the detection kit further comprises a blank excipient of fluticasone propionate suspension for inhalation, and a blank excipient solution is prepared for UPLC detection.

[0011] In a second aspect, a UPLC analysis method for the content of a fluticasone propionate inhalation suspension is provided, wherein the fluticasone propionate inhalation suspension to be tested is prepared into a test solution using a mobile phase, and the test solution is subjected to UPLC detection; in the UPLC detection, the mobile phase is used to perform isocratic elution in a chromatographic column; the mobile phases are mobile phase A and mobile phase B, the volume ratio of mobile phase A to mobile phase B is 35:65 to 45:55, mobile phase B is acetonitrile, mobile phase A is an aqueous solution of methanol, and the volume fraction of methanol in mobile phase A is 2.5 to 3.5%; the chromatographic column uses octadecylsilane bonded silica gel as a filler.

[0012] In some embodiments, the fluticasone propionate inhalation suspension to be tested is diluted 9 to 11 times with a mobile phase to prepare a test solution. Studies have shown that the detection effect is better under this condition.

[0013] In some embodiments, the fluticasone propionate standard is prepared into a reference solution using a mobile phase, and the reference solution is subjected to UPLC detection. Specifically, the reference solution contains 0.018 to 0.22 mg of fluticasone propionate per milliliter of solution.

[0014] In some embodiments, the excipients in the fluticasone propionate inhalation suspension are prepared into a reference solution using a mobile phase, and the reference solution is subjected to UPLC detection.

[0015] In some embodiments, in UPLC detection, the detection wavelength is 234-244 nm. Studies have shown that under this condition, the fluticasone propionate in the fluticasone propionate suspension for inhalation can be better quantitatively detected.

[0016] In some embodiments, in UPLC detection, the detection flow rate is 0.3-0.5 mL / min. Studies have shown that the detection effect is better under this condition.

[0017] In some embodiments, in UPLC detection, the detection column temperature is 35-45° C. Studies have shown that the detection effect is better under this condition.

[0018] In some embodiments, in UPLC detection, the injection volume is 1.9-2.1 μL.

[0019] The beneficial effects of the present invention are:

[0020] 1. The present invention realizes effective separation of the main component and the auxiliary materials in the fluticasone propionate suspension for inhalation by selecting the mobile phase, avoids the interference of the auxiliary materials in the suspension for inhalation on the main component, increases the solubility of the main component, thereby realizing UPLC analysis of the content of the fluticasone propionate suspension for inhalation, and provides a basis for the quality control of the fluticasone propionate suspension for inhalation.

[0021] 2. The method of the present invention is used to detect the fluticasone propionate suspension for inhalation, which can significantly shorten the analysis time and analysis cost.

[0022] 3. The experiment shows that the method of the present invention has good specificity when detecting the fluticasone propionate inhalation suspension, and the repeatability of 6 samples is verified, the RSD of the content is less than 2%, and the precision is good. The present invention also verifies the linearity of 50% to 150% of the labeled concentration, and r>0.999 in the above range, verifies the recovery rates of 80%, 100%, and 120%, and the linearity and accuracy are good between 98% and 101%. The test solution of the present invention still has good stability after being placed at room temperature for 96 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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.

[0024] Figure 1 The chromatogram is detected in Example 1 of the present invention, the red line represents the blank excipient solution, the green line represents the reference solution, and the blue line represents the test solution;

[0025] Figure 2 This is a linear result diagram detected by Example 2 of the present invention;

[0026] Figure 3 This is a chromatogram detected in Comparative Example 1 of the present invention, the red line represents the blank auxiliary material solution, and the green line represents the test solution. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0028] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0029] Example 1: Specificity

[0030] 1-1 Solution Preparation

[0031] Diluent: Mobile phase

[0032] Blank excipient solution: Take 1 mL of blank excipient (water for injection containing polysorbate 20, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, and sorbitan monolaurate) without adding the main ingredient and place it in a 10 mL volumetric flask, and dilute to the scale with the mobile phase.

[0033] Test solution: Place 1 mL of fluticasone propionate suspension for inhalation in a 10 mL volumetric flask and dilute to the mark with mobile phase.

[0034] Reference solution: Accurately weigh 2 mg of fluticasone propionate and dilute with mobile phase to make a solution containing approximately 0.02 mg of fluticasone propionate per 1 mL of solution.

[0035] 1-2 Chromatographic conditions

[0036] Column: Hypersil GOLD, 100 mm × 2.1 mm, 1.9 μm;

[0037] Mobile phase: 3% methanol water (containing 0.05% phosphoric acid)-acetonitrile (40:60 v / v)

[0038] The detection wavelength is 239nm;

[0039] Flow rate: 0.4 mL / min;

[0040] The column temperature was 40°C;

[0041] The injection volume was 2 μL.

[0042] 1-3 Experimental conclusion:

[0043] Accurately measure the above solution, inject it into the liquid chromatograph, and record the chromatogram. The results show that the retention time of the main peak of the test sample and the reference sample is consistent, and the blank auxiliary material does not interfere with the detection of the main component. Figure 1 shown.

[0044] Example 2: Linearity and range experiments

[0045] 2-1 Solution preparation

[0046] Accurately weigh an appropriate amount of fluticasone propionate to prepare a reference stock solution with a concentration of 0.2 mg / mL; measure 0.5 mL, 0.8 mL, 1.0 mL, 1.2 mL, and 1.5 mL of the reference stock solution respectively and place them in 10 mL volumetric flasks, and dilute to the scale with mobile phase to obtain a series of concentration linearity test solutions with a concentration range of 50% to 150%.

[0047] 2-2 Chromatographic conditions are the same as those in Example 1.

[0048] 2-3 Conclusion:

[0049] Accurately measure the above solution, inject it into the liquid chromatograph, and record the chromatogram. The results are shown in Table 1. Figure 2 .

[0050] Table 1 Linearity results

[0051] Linear 50％ 80％ 100％ 120％ 150％ Concentration μg / mL 10.44 16.70 20.87 25.04 31.31 Peak area 115317 188986 238833 278945 350231

[0052] Conclusion: Within the determination range, fluticasone propionate has a good linear relationship, and the linear coefficient r>0.999, which meets the methodological requirements.

[0053] Example 3: Accuracy Experiment

[0054] The accuracy was investigated through recovery test, and the study covered the limit concentration range of 80% to 120%.

[0055] 3-1 Solution preparation

[0056] Accurately measure 0.8mL, 1.0mL and 1.2mL of the reference substance stock solution and place them in a 10mL volumetric flask, add the blank excipient solution to dilute to the scale, shake well, and prepare 3 replicates at each spiked concentration level.

[0057] 3-2 Chromatographic conditions are the same as those in Example 1.

[0058] 3-3 Conclusion:

[0059] Accurately measure the above solution, inject it into the liquid chromatograph, record the chromatogram, and calculate the accuracy by peak area according to the external standard method. The results are shown in Table 2.

[0060] Table 2 Accuracy test results

[0061]

[0062]

[0063] Conclusion: Under 80%, 100%, and 120% accuracy solutions, the average recovery of fluticasone propionate was 99.6%, and the RSD was 0.56%. The test results showed that the accuracy of the invention was good.

[0064] Example 4: Precision Experiment

[0065] 4-1 Solution preparation

[0066] Test solution: Place 1 mL of fluticasone propionate inhalation suspension in a 10 mL volumetric flask and dilute to volume with mobile phase. Prepare 6 test solutions in parallel.

[0067] Reference solution: Accurately weigh an appropriate amount of fluticasone propionate and dilute it with mobile phase to make a solution containing approximately 0.02 mg of fluticasone propionate per 1 mL of solution.

[0068] 4-2 Chromatographic conditions are the same as those in Example 1.

[0069] 4-3 Conclusion:

[0070] Accurately measure the above solution, inject it into the liquid chromatograph, record the chromatogram, calculate the content, and the precision results are shown in Table 3.

[0071] Table 3 Precision test results

[0072]

[0073] Conclusion: The test results of 6 replicate sample solutions prepared in parallel showed that the RSD value of the content was less than 1%, which proved that the present invention had good repeatability.

[0074] Example 5: Solution stability experiment

[0075] 5-1 Solution preparation

[0076] Take the test solution and reference solution in Implementation Case 4, and inject samples for detection at different time points.

[0077] 5-2 Chromatographic conditions are the same as those in Example 1.

[0078] 5-3 Conclusion:

[0079] Accurately measure the above solution, inject it into the liquid chromatograph, record the chromatogram, and the solution stability results are shown in Table 4.

[0080] Table 4 Solution stability test results

[0081] time Test solution Reference solution 0h 170271 239070 96h 171277 237914 RD 0.29 -0.24

[0082] Conclusion: There was no significant change in the test solution and the reference solution after being placed at room temperature for 96 hours, indicating that the test solution and the reference solution were stable at room temperature within 96 hours.

[0083] Example 6: Durability Test

[0084] Durability refers to the degree to which the test results are not affected by slight changes in the chromatographic conditions. The blank auxiliary material solution, the reference solution and the test solution were prepared in the same manner as in Example 1. On the basis of the test conditions in Example 1, the durability of the test method was determined by changing the column temperature, flow rate, wavelength and acetonitrile ratio. The test results are shown in Table 5.

[0085] Table 5 Durability test results

[0086]

[0087]

[0088] Conclusion: When the column temperature (column temperature ±5°C, flow rate ±0.1 mL / min, wavelength ±5 nm, acetonitrile ratio ±5%) was changed, the RSD values ​​of the contents all met the requirements, and the durability of the present invention was good.

[0089] Comparative Example 1

[0090] 1. Solution preparation

[0091] Diluent: Mobile phase

[0092] Blank excipient solution: Place 1 mL of blank excipient without the main component in a 10 mL volumetric flask and dilute to the mark with the mobile phase.

[0093] Test solution: Place 1 mL of fluticasone propionate suspension for inhalation in a 10 mL volumetric flask and dilute to the mark with mobile phase.

[0094] 2 Chromatographic conditions

[0095] Column: Hypersil GOLD, 100 mm × 2.1 mm, 1.9 μm;

[0096] Mobile phase: water (containing 0.05% phosphoric acid)-acetonitrile (40:60 v / v)

[0097] The detection wavelength is 239nm;

[0098] Flow rate: 0.4 ml / min;

[0099] The column temperature was 40°C;

[0100] The injection volume was 10 μL.

[0101] 3 Experimental conclusions:

[0102] The above solution was accurately measured and injected into a liquid chromatograph to record the chromatogram. The results showed that this method could not effectively separate the auxiliary materials and the main components in the suspension for inhalation. Figure 3 shown.

[0103] 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 detection kit, characterized in that: The detection kit is used for quantitative detection of fluticasone propionate in a fluticasone propionate suspension for inhalation by UPLC; the kit comprises a solvent for preparing a mobile phase, wherein the solvent comprises methanol, water and acetonitrile; wherein acetonitrile is used for preparing a mobile phase B, and methanol and water are used for preparing a mobile phase A; the volume fraction of methanol in the mobile phase A is 2.5-3.5%, and the mobile phase A contains phosphoric acid with a mass fraction of 0.045-0.055%, and when UPLC detection is adopted, the volume ratio of the mobile phase A to the mobile phase B is 35:65-45:

55.

2. The detection kit according to claim 1, characterized in that: The detection kit also includes a fluticasone propionate standard.

3. The detection kit according to claim 1, characterized in that: The detection kit also includes blank auxiliary materials of fluticasone propionate suspension for inhalation.

4. A UPLC analysis method for the content of fluticasone propionate suspension for inhalation, characterized in that: A mobile phase is used to prepare a test solution from a suspension for fluticasone propionate inhalation to be tested, and UPLC detection is performed on the test solution. In the UPLC detection, the mobile phase is used to perform isocratic elution in a chromatographic column. The mobile phases are mobile phase A and mobile phase B, the volume ratio of mobile phase A to mobile phase B is 35:65-45:55, mobile phase B is acetonitrile, mobile phase A is a methanol aqueous solution, the volume fraction of methanol in mobile phase A is 2.5-3.5%, and mobile phase A contains phosphoric acid with a mass fraction of 0.045-0.055%. The chromatographic column uses octadecylsilane bonded silica gel as a filler.

5. The UPLC analysis method for the content of the fluticasone propionate suspension for inhalation as claimed in claim 4, characterized in that: The test solution was prepared by diluting the fluticasone propionate inhalation suspension to be tested by 9 to 11 times using the mobile phase.

6. The UPLC analysis method for the content of fluticasone propionate suspension for inhalation according to claim 4, characterized in that: The fluticasone propionate standard was prepared into a reference solution by using a mobile phase, and the reference solution was detected by UPLC.

7. The UPLC analysis method for the content of fluticasone propionate suspension for inhalation according to claim 4, characterized in that: The auxiliary materials in the fluticasone propionate inhalation suspension are prepared into a reference solution by using a mobile phase, and the reference solution is detected by UPLC.

8. The UPLC analysis method for the content of fluticasone propionate suspension for inhalation according to claim 4, characterized in that: In UPLC detection, the detection wavelength is 234 to 244 nm.

9. The UPLC analysis method for the content of fluticasone propionate suspension for inhalation according to claim 4, characterized in that: In UPLC detection, the detection flow rate is 0.3-0.5 ml / min.

10. The UPLC analysis method for the content of fluticasone propionate suspension for inhalation according to claim 4, characterized in that: In UPLC detection, the detection column temperature is 35-45°C.

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