Method for detecting Tween-80 content in nasal spray by colorimetric method and application thereof

Abstract

The invention relates to a method for detecting the content of Tween 80 in a nasal spray by using a colorimetric method and application. The method comprises the following steps: pretreating a to-be-detected sample, adding a cobalt ammonium thiocyanate solution, carrying out a chromogenic reaction, adding an extracting agent, and carrying out determination. According to the method, the nasal spray is pretreated, so that the influence of other substances on Tween 80 detection is reduced, and the detection accuracy is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of drug quality control analysis, and in particular to a method for detecting the content of Tween 80 in a nasal spray by using a colorimetric method and an application thereof. Background Art

[0002] Tween 80, also known as polysorbate 80, is chemically known as polyoxyethylene sorbitan monooleate. It is a nonionic surfactant commonly used in pharmaceutical formulations as a cosolvent and stabilizer. Recent discoveries linking Tween 80 with some clinical adverse drug reactions necessitate the development of an accurate and effective method for measuring Tween 80 content.

[0003] Currently, the quantitative detection methods reported domestically and internationally for Tween 80 mainly fall into two categories: chromatography and spectrometry. Chromatographic methods include chromatographic analysis combined with hydrolysis pretreatment and high-performance liquid chromatography (HPLC) using an evaporative light scattering detector (ELSD). The former, primarily influenced by pretreatment conditions and the stability of the hydrolysis product, suffer from cumbersome procedures, multiple influencing factors, time-consuming processes, poor precision, and unsuitability for large-scale batch sample analysis. The latter requires regular column cleaning to ensure accurate results. Using a gradient elution system to generate a single chromatographic peak for Tween 80 is also challenging. Furthermore, the ELSD system is susceptible to sample matrix contamination, which can affect results. Spectroscopic methods commonly employ colorimetric methods based on the characteristic absorption of a blue complex soluble in organic solvents formed by the reaction of the polyethoxy groups in Tween 80 with ammonium cobalt thiocyanate. These methods are widely used because they meet the specificity and sensitivity requirements for quantitative analysis and require minimal equipment. The sample size and amount of dichloromethane used for the polysorbate 80 residue determination method in the appendix of the Chinese Pharmacopoeia were too small, and the color development conditions were unstable, resulting in poor reproducibility of the results. This affected the precision, accuracy, and reproducibility of the results.

[0004] Nasal spray prescriptions generally contain microcrystalline cellulose-sodium carboxymethyl cellulose, an excipient that is stable and difficult to remove. The residue of microcrystalline cellulose-sodium carboxymethyl cellulose will affect the detection of Tween 80. Therefore, how to effectively determine Tween 80 in nasal sprays has become an urgent problem to be solved. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a method for detecting the content of Tween 80 in nasal spray using a colorimetric method and its application. The present invention pretreats the nasal spray, reduces the influence of other substances on Tween 80 detection, and significantly improves the accuracy of detection.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for detecting the content of Tween 80 in a nasal spray using a colorimetric method, the method comprising pretreating a sample to be tested, adding an ammonium cobalt thiocyanate solution, performing a color development reaction, and then adding an extractant for determination.

[0008] In this invention, the nasal spray sample is pretreated to reduce the impact of other reagents in the nasal spray, such as microcrystalline cellulose and sodium carboxymethyl cellulose, on the test results, significantly improving the accuracy of the test. Furthermore, a color reaction is performed using an ammonium cobalt thiocyanate solution and Tween 80, followed by extraction with an extractant. The color development conditions are stable, making this method more suitable for colorimetry.

[0009] Preferably, the pretreatment step comprises shaking the sample to be tested horizontally, then fixing the volume, and centrifuging to obtain the upper layer liquid after shaking.

[0010] In the present invention, the nasal spray is first shaken horizontally to make the solution more uniform. Then, a quantitative amount of the shaken sample is weighed and placed in a suitable volumetric flask. Purified water is added to the volume. The solution is shaken again and then centrifuged. The shaking allows the Tween 80 in the solution to be fully and evenly dissolved in the purified water. The microcrystalline cellulose-sodium carboxymethyl cellulose in the nasal spray is removed by centrifugation.

[0011] Preferably, the horizontal shaking time is 10-20 s, for example, it can be 10 s, 11 s, 12 s, 13 s, 14 s, 15 s, 16 s, 17 s, 18 s, 19 s or 20 s.

[0012] Preferably, the horizontal shaking is performed at 1-5 cycles per second, for example, 1, 2, 3, 4 or 5 cycles per second.

[0013] Preferably, the single shaking distance of the horizontal shaking is 20-50 cm, for example, 20 cm, 25 cm, 30 cm, 35 cm, 40 cm, 45 cm or 50 cm.

[0014] Preferably, the concentration of the sample after volume fixation is 0.1-0.5 g / mL, for example, it can be 0.1 g / mL, 0.2 g / mL, 0.3 g / mL, 0.4 g / mL or 0.5 g / mL.

[0015] Preferably, the constant volume reagent is purified water.

[0016] The method of the present invention is applicable to a variety of nasal sprays and can significantly improve the accuracy of detection.

[0017] Preferably, the shaking speed is 100-250 rpm, and the shaking time is 10-20 min. The 100-250 rpm may be, for example, 100 rpm, 120 rpm, 140 rpm, 160 rpm, 180 rpm, 200 rpm, 220 rpm, 240 rpm, or 250 rpm. The 10-20 min may be, for example, 10 min, 12 min, 14 min, 16 min, 18 min, or 20 min.

[0018] Preferably, the centrifugation speed is 8000-12000 rpm, and the time is 5-15 min. The 8000-12000 rpm can be, for example, 8000 rpm, 8500 rpm, 9000 rpm, 9500 rpm, 10000 rpm, 10500 rpm, 11000 rpm, 11500 rpm, or 12000 rpm. The 5-15 min can be, for example, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, or 15 min.

[0019] Preferably, the preparation method of the ammonium cobalt thiocyanate solution comprises adding purified water to cobalt nitrate hexahydrate and ammonium thiocyanate, and then mixing them to a fixed volume.

[0020] Preferably, the added amount of the cobalt nitrate hexahydrate is 1-5 g, for example, 1 g, 2 g, 3 g, 4 g or 5 g.

[0021] Preferably, the amount of ammonium thiocyanate added is 15-25 g, for example, 15 g, 16 g, 17 g, 18 g, 19 g, 20 g, 21 g, 22 g, 23 g, 24 g or 25 g.

[0022] Preferably, the volume of the fixed volume is 50-150 mL, for example, it can be 50 mL, 60 mL, 80 mL, 100 mL, 120 mL, 140 mL or 150 mL.

[0023] Preferably, the color development reaction step comprises mixing the upper layer of the sample to be tested with the ammonium cobalt thiocyanate solution, reacting in a water bath, and then cooling.

[0024] Preferably, the volume ratio of the upper layer liquid of the sample to be tested to the ammonium cobalt thiocyanate solution is (0.5-3):1, and the (0.5-3) can be, for example, 0.5, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8 or 3.0.

[0025] Preferably, the temperature of the water bath is 90-100° C., and the time is 0.5-3 h. The 90-100° C. may be, for example, 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98° C., 99° C., or 100° C. The 0.5-3 h may be, for example, 0.5 h, 0.6 h, 0.8 h, 1.0 h, 1.2 h, 1.4 h, 1.6 h, 1.8 h, 2.0 h, 2.2 h, 2.4 h, 2.6 h, 2.8 h, or 3.0 h.

[0026] Preferably, the temperature after cooling is 20-30° C. For example, it can be 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C. or 30° C.

[0027] Preferably, the extractant comprises dichloromethane and / or chloroform.

[0028] Preferably, the volume ratio of the extractant to the upper layer of the sample to be tested is 1:(1-5). The (1-5) can be, for example, 1, 2, 3, 4 or 5.

[0029] In the present invention, the amount of the extractant added is increased, thereby greatly improving the extraction rate of the color-developed product, effectively reducing the error of the extraction operation, and further improving the precision and accuracy of the detection determination.

[0030] Preferably, the sample determination step includes shaking and mixing the extracted sample, and then measuring the absorbance value after standing.

[0031] Preferably, the shaking mixing time is 1-2 hours and the speed is 130-150 rpm. The 1-2 hours can be, for example, 1 hour, 1.2 hours, 1.4 hours, 1.6 hours, 1.8 hours or 2 hours. The 130-150 rpm can be, for example, 130 rpm, 135 rpm, 140 rpm, 145 rpm or 150 rpm.

[0032] Preferably, the standing time is 20-60 min, for example, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min or 60 min.

[0033] Preferably, the method further comprises the preparation of a standard.

[0034] Preferably, the standard comprises Tween 80.

[0035] Preferably, the concentration of the standard is 0-140 μg / mL, for example, 0, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL or 140 μg / mL.

[0036] Preferably, the absorbance value is determined by measuring the absorbance value of the lower layer solution at a wavelength of 622 nm after standing.

[0037] In the present invention, the absorbance of the lower layer solution of the test sample and the blank control at a wavelength of 622 nm is measured respectively, and the final test result is obtained by subtracting the blank control from the test sample result.

[0038] The blank control group includes a cobalt ammonium thiocyanate solution and an extractant having the same concentration as that in the sample to be tested.

[0039] In a second aspect, the present invention provides an application of the method for detecting the content of Tween 80 in nasal spray using colorimetry according to the first aspect in detecting Tween 80.

[0040] Compared with the prior art, the present invention has at least the following beneficial effects:

[0041] 1. The present invention pre-treats the nasal spray sample before testing, reducing the impact of other reagents on the test results and further enriching Tween 80 in the nasal spray, significantly improving the accuracy and precision of the test.

[0042] 2. Based on the research results of the kinetic process of the color development reaction between Tween 80 and ammonium cobalt thiocyanate in an aqueous medium, the present invention systematically screens and optimizes key determination conditions such as the pretreatment process of nasal spray samples, the color development reaction temperature and time. On this basis, the research establishes a colorimetric determination method suitable for the rapid and accurate quantification of Tween 80, a medicinal excipient in pharmaceutical preparations, especially nasal sprays. This provides a convenient and reliable detection method for the quantitative control of Tween 80 in related products. DETAILED DESCRIPTION

[0043] For the convenience of understanding the present invention, the present invention is given below with examples. It should be understood by those skilled in the art that the examples are only for the purpose of helping to understand the present invention and should not be regarded as specific limitations of the present invention.

[0044] The sources of the reagents used in the following examples are:

[0045] Nasal spray: Shanghai Johnson & Johnson Pharmaceutical Co., Ltd., budesonide nasal spray.

[0046] Example 1

[0047] This embodiment provides a method for detecting the content of Tween 80 in nasal spray using colorimetry

[0048] (1) Pretreatment

[0049] Take one bottle of nasal spray, remove the cap, hold the nozzle tip between your index and middle fingers, and support the bottom of the bottle with your thumb, keeping the nozzle facing upward. Shake horizontally for 15 seconds, two cycles per second, shaking at a distance of 30 cm, until the solution becomes fluid. Open the bottle. Accurately weigh 7 g of sample into a 20 mL volumetric flask, add purified water to the mark, shake well, and place on an oscillator at 180 rpm for 15 minutes. After the oscillation is complete, pour the sample from the flask into a suitable centrifuge tube. Place the centrifuge tube in a centrifuge at 10,000 rpm for 10 minutes. After centrifugation, remove the supernatant.

[0050] (2) Preparation of ammonium cobalt thiocyanate solution

[0051] Weigh 3.0 g of cobalt nitrate hexahydrate and 20 g of ammonium thiocyanate, place in a 100 mL volumetric flask, add purified water to dissolve and dilute to the scale, shake well to obtain ammonium thiocyanate cobalt solution.

[0052] (3) Color reaction and extraction

[0053] Take 10 mL of the upper layer solution and place it in a vial. Add 10 mL of ammonium cobalt thiocyanate solution and heat in a water bath at 95°C for 2 h. Cool to 25°C. Add 6 mL of dichloromethane and shake at 180 rpm for 2 h. Let it stand for 30 min and measure the absorbance at 622 nm.

[0054] (4) Preparation of standard curve and blank solution

[0055] Prepare the reference stock solution: accurately weigh 175 mg of polysorbate 80 into a 100 mL volumetric flask, dilute to the mark with purified water, and shake well.

[0056] Prepare a standard curve: Add 10 mL of purified water, 10 mL of ammonium thiocyanate cobaltate solution, and 6 mL of dichloromethane to a transparent vial, mix them, then add 0 μL, 200 μL, 400 μL, 600 μL, and 800 μL of the reference stock solution, respectively, and measure the absorbance at a wavelength of 622 nm.

[0057] Prepare blank solution: accurately pipette 10 mL of purified water into a 50 mL transparent vial, then accurately add 10 mL of ammonium cobalt thiocyanate solution and 6 mL of dichloromethane, tighten the bottle cap, seal with parafilm, and mix well.

[0058] Example 2

[0059] This embodiment provides a method for detecting the content of Tween 80 in nasal spray using colorimetry

[0060] (1) Pretreatment

[0061] Take one bottle of nasal spray, remove the cap, hold the nozzle tip between your index and middle fingers, and support the bottom of the bottle with your thumb, keeping the nozzle facing upward. Shake horizontally for 10 seconds, five cycles per second, at a single shaking distance of 20 cm, until the solution becomes fluid. Open the bottle. Accurately weigh 2 g of sample into a 20 mL volumetric flask, add purified water to the mark, shake well, and place on an oscillator at 250 rpm for 10 minutes. After the oscillation is complete, pour the sample from the volumetric flask into a suitable centrifuge tube. Place the centrifuge tube in a centrifuge at 8000 rpm for 5 minutes. After centrifugation, remove the supernatant.

[0062] (2) Preparation of ammonium cobalt thiocyanate solution

[0063] Weigh 5.0 g of cobalt nitrate hexahydrate and 15 g of ammonium thiocyanate, place them in a 50 mL volumetric flask, add purified water to dissolve and dilute to the scale, shake well to obtain ammonium thiocyanate cobalt solution.

[0064] (3) Color reaction and extraction

[0065] Take 5 mL of the upper layer solution and place it in a vial. Add 10 mL of ammonium cobalt thiocyanate solution, heat in a water bath at 100°C for 0.5 h, and cool to 20°C. Add 5 mL of dichloromethane and shake at 180 rpm for 1.5 h. After standing for 20 min, measure the absorbance at a wavelength of 622 nm.

[0066] (4) Preparation of standard curve and blank solution

[0067] Prepare the reference stock solution: accurately weigh 175 mg of polysorbate 80 into a 100 mL volumetric flask, dilute to the mark with purified water, and shake well.

[0068] Prepare a standard curve: Add 5 mL of purified water, 10 mL of ammonium thiocyanate cobaltate solution, and 5 mL of dichloromethane to a transparent vial, mix, then add 0 μL, 200 μL, 400 μL, 600 μL, and 800 μL of the reference stock solution, respectively, and measure the absorbance at a wavelength of 622 nm.

[0069] Prepare blank solution: Accurately pipette 5 mL of purified water into a 50 mL transparent vial, then accurately add 10 mL of ammonium cobalt thiocyanate solution and 5 mL of dichloromethane, tighten the bottle cap, seal with parafilm, and mix well.

[0070] Example 3

[0071] This embodiment provides a method for detecting the content of Tween 80 in nasal spray using colorimetry

[0072] (1) Pretreatment

[0073] Take one bottle of nasal spray, remove the cap, hold the nozzle tip between your index and middle fingers, and support the bottom of the bottle with your thumb, keeping the nozzle facing upward. Shake horizontally for 20 seconds, one cycle per second, at a single shaking distance of 50 cm, until the solution becomes fluid. Open the bottle. Accurately weigh 10 g of sample into a 20 mL volumetric flask, add purified water to the mark, shake well, and place on an oscillator at 100 rpm for 20 minutes. After the oscillation is complete, pour the sample from the volumetric flask into a suitable centrifuge tube. Place the centrifuge tube in a centrifuge at 12,000 rpm for 15 minutes. After centrifugation, remove the supernatant.

[0074] (2) Preparation of ammonium cobalt thiocyanate solution

[0075] Weigh 1.0 g of cobalt nitrate hexahydrate and 25 g of ammonium thiocyanate, place in a 150 mL volumetric flask, add purified water to dissolve and dilute to the scale, shake well to obtain ammonium thiocyanate cobalt solution.

[0076] (3) Color reaction and extraction

[0077] Place 30 mL of the upper layer into a vial, add 10 mL of ammonium cobalt thiocyanate solution, heat in a 90°C water bath for 3 h, and cool to 30°C. Add 6 mL of dichloromethane and shake at 180 rpm for 1 h. After standing for 60 min, measure the absorbance at 622 nm.

[0078] (4) Preparation of standard curve and blank solution

[0079] Prepare the reference stock solution: accurately weigh 175 mg of polysorbate 80 into a 100 mL volumetric flask, dilute to the mark with purified water, and shake well.

[0080] Prepare a standard curve: Add 30 mL of purified water, 10 mL of ammonium thiocyanate cobaltate solution, and 6 mL of dichloromethane to a transparent vial, mix, then add 0 μL, 200 μL, 400 μL, 600 μL, and 800 μL of the reference stock solution, respectively, and measure the absorbance at a wavelength of 622 nm.

[0081] Prepare blank solution: Accurately pipette 30 mL of purified water into a 50 mL transparent vial, then accurately add 10 mL of ammonium cobalt thiocyanate solution and 6 mL of dichloromethane, tighten the bottle cap, seal with parafilm, and mix well.

[0082] Example 4

[0083] This example provides a method for detecting the content of Tween 80 in a nasal spray using a colorimetric method. The only difference from Example 1 is that horizontal shaking is not performed. All other aspects are the same as Example 1.

[0084] Example 5

[0085] This example provides a method for detecting the content of Tween 80 in a nasal spray using a colorimetric method. The only difference between this method and Example 1 is that the horizontal shaking is replaced by an oscillation at a speed of 250 rpm for 10 minutes. All other aspects are the same as those of Example 1.

[0086] Example 6

[0087] This example provides a method for detecting the content of Tween 80 in nasal spray using a colorimetric method. The only difference between this method and Example 1 is that the concentration of the sample after volume adjustment is 2 g / mL. Other aspects are consistent with Example 1.

[0088] Example 7

[0089] This example provides a method for detecting the content of Tween 80 in a nasal spray using a colorimetric method. The difference between this method and Example 1 is that the volume is fixed without shaking and then directly centrifuged. All other aspects are the same as Example 1.

[0090] Example 8

[0091] This example provides a method for detecting the content of Tween 80 in nasal spray using a colorimetric method. The only difference between this example and Example 1 is that the volume of the supernatant added to the extraction in the color development reaction is 0.3 mL. All other aspects are consistent with Example 1.

[0092] Example 9

[0093] This example provides a method for detecting the content of Tween 80 in a nasal spray using a colorimetric method. The only difference between this method and Example 1 is that no water bath heating is performed. Other aspects are the same as those of Example 1.

[0094] Example 10

[0095] This example provides a method for detecting the content of Tween 80 in nasal spray using a colorimetric method. The only difference between this method and Example 1 is that the water bath heating temperature is 80° C., and all other aspects are the same as Example 1.

[0096] Example 11

[0097] This example provides a method for detecting the content of Tween 80 in nasal spray using a colorimetric method. The only difference between this method and Example 1 is that the water bath heating temperature is 70° C., and all other aspects are the same as Example 1.

[0098] Comparative Example 1

[0099] This comparative example provides a method for detecting the content of Tween 80 in nasal spray using a colorimetric method. The difference between this method and Example 1 is that the sample to be tested is not pretreated. Other aspects are the same as those of Example 1.

[0100] Test Example 1

[0101] This test case investigates the effect of water bath heating on the standard curve

[0102] Prepare the reference stock solution: accurately weigh 175 mg of polysorbate 80 into a 100 mL volumetric flask, dilute to the mark with purified water, and shake well.

[0103] Prepare standard solution: Add 10 mL of ammonium thiocyanate cobaltate solution and 6 mL of dichloromethane to a transparent vial, mix, then add 0 μL, 200 μL, 400 μL, 600 μL and 800 μL of reference substance stock solution, respectively, and measure the absorbance at a wavelength of 622 nm.

[0104] Prepare 10 suitable transparent vials, divided into Group A and Group B, with five in each group. Prepare standard solutions in each group according to the above procedure. For Group A, add 6 mL of dichloromethane at room temperature and shake on a shaker for 2 hours to extract the reactants. For Group B, place the vials in a 95°C water bath for 2 hours for color development. Remove the vials, cool to room temperature, then add 6 mL of dichloromethane to each vial, seal, and shake well. After standing for 30 minutes, measure the absorbance at 622 nm for each vial. The results are shown in Table 1.

[0105] Table 1

[0106]

[0107] From the above results, we can see that the R 2 It is significantly higher than Group A, proving that the detection accuracy is higher and the precision is higher after heating.

[0108] Test Example 2

[0109] This test example tests the results of the above examples and comparative examples.

[0110] The absorbance values of the samples in the above examples and comparative examples were measured at a wavelength of 622 nm, and the absorbance values of the blank group measured at a wavelength of 622 nm were subtracted. The content of Tween 80 was then obtained using a standard curve. The specific results are shown in Table 2.

[0111] Table 2

[0112]

[0113]

[0114] According to the test results:

[0115] (1) By comparing Example 1 with Examples 4-5, it can be seen that the results obtained by horizontal shaking are closest to the feeding amount, and other shaking methods will produce a large number of bubbles, thereby affecting the test results.

[0116] (2) By comparing Example 1 with Example 6, it can be seen that adding too much sample will reduce the detection result of polysorbate 80.

[0117] (3) By comparing Example 1 with Example 7, it can be seen that direct centrifugation without shaking after volume determination may cause the polysorbate 80 in the sample to be unable to be completely dissolved in water, thereby reducing the test results.

[0118] (4) By comparing Example 1 with Examples 9-11, it can be seen that the color development reaction speed in a 95°C water bath is significantly accelerated, thereby shortening the color development reaction time and improving the measurement efficiency. At the same time, performing the color development reaction in a 95°C water bath can effectively reduce the impact of ambient temperature fluctuations on the color development reaction, ensuring the precision, reproducibility, and accuracy of the measurement.

[0119] (8) By comparing Example 1 with Comparative Example 1, it can be seen that the present invention significantly reduces the influence of the microcrystalline cellulose-sodium carboxymethyl cellulose co-processing product in the prescription on the test results by treating the nasal spray.

[0120] Test Example 3

[0121] This test case performs repeatability test / stability test

[0122] The method of Example 1 was used to perform 10 repeatability tests, the absorbance at a wavelength of 622 nm was measured, and the content of Tween 80 was obtained using a standard curve. The specific results are shown in Table 3. As shown in the following results, the RSD value was 5.1%, indicating that the method has high accuracy and repeatability.

[0123] Table 3

[0124]

[0125] In summary, the present invention pre-treats the nasal spray, reduces the influence of other substances on Tween 80 detection, and significantly improves the accuracy of detection.

[0126] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A method for detecting the content of Tween 80 in nasal spray using colorimetry, characterized in that: The method comprises the steps of pre-treating a sample to be tested, adding an ammonium cobalt thiocyanate solution, performing a color development reaction, adding an extractant, and performing the test.

2. The method for detecting the content of Tween 80 in nasal spray using colorimetry according to claim 1, characterized in that: The pretreatment step includes shaking the sample to be tested horizontally, then adjusting the volume, and centrifuging after shaking to obtain the upper layer liquid; Preferably, the horizontal shaking time is 10-20s; Preferably, the horizontal shaking is performed at 1-5 cycles per second; Preferably, the single shaking distance of the horizontal shaking is 20-50 cm; Preferably, the concentration of the sample after volume adjustment is 0.1-0.5 g / mL; Preferably, the constant volume reagent is purified water.

3. The method for detecting the content of Tween 80 in nasal spray using colorimetry according to claim 2, characterized in that: The shaking speed is 100-250 rpm and the time is 10-20 min; Preferably, the centrifugal speed is 8000-12000 rpm, and the time is 5-15 min.

4. The method for detecting the content of Tween 80 in nasal spray using colorimetry according to any one of claims 1 to 3, characterized in that: The preparation method of the ammonium cobalt thiocyanate solution comprises adding purified water to cobalt nitrate hexahydrate and ammonium thiocyanate and then mixing them to a fixed volume; Preferably, the amount of cobalt nitrate hexahydrate added is 1-5g; Preferably, the amount of ammonium thiocyanate added is 15-25g; Preferably, the volume of the fixed volume is 50-150 mL.

5. The method for detecting the content of Tween 80 in nasal spray using colorimetry according to any one of claims 1 to 4, characterized in that: The color development reaction step comprises mixing the upper layer of the sample to be tested with the ammonium cobalt thiocyanate solution, reacting in a water bath, and then cooling; Preferably, the volume ratio of the upper layer liquid of the sample to be tested to the ammonium cobalt thiocyanate solution is (0.5-3):1; Preferably, the temperature of the water bath is 90-100°C and the time is 0.5-3h; Preferably, the temperature after cooling is 20-30°C.

6. The method for detecting the content of Tween 80 in nasal spray using colorimetry according to any one of claims 1 to 5, characterized in that: The extractant includes dichloromethane and / or chloroform; Preferably, the volume ratio of the extractant to the upper layer liquid of the sample to be tested is 1:(1-5).

7. The method for detecting the content of Tween 80 in nasal spray using colorimetry according to any one of claims 1 to 6, characterized in that: The sample determination step comprises shaking and mixing the extracted sample, and then measuring the absorbance value after standing; Preferably, the shaking mixing time is 1-2h and the speed is 130-150rpm; Preferably, the standing time is 20-60 minutes.

8. The method for detecting the content of Tween 80 in nasal spray using colorimetry according to any one of claims 1 to 7, characterized in that: The method further includes the preparation of a standard; Preferably, the standard comprises Tween 80; Preferably, the concentration of the standard is 0-140 μg / mL.

9. The method for detecting the content of Tween 80 in nasal spray using colorimetry according to any one of claims 1 to 8, characterized in that: The absorbance value is determined by measuring the absorbance value of the lower layer solution at a wavelength of 622 nm after standing.

10. Use of the method for detecting the content of Tween 80 in nasal spray using colorimetry according to any one of claims 1 to 9 in detecting Tween 80.

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