Method for detecting in-vitro skin absorption amount of cocamidopropyl betaine in medical skin cleaning and repairing liquid

Abstract

The invention discloses a method for detecting the in-vitro skin absorption amount of cocamidopropyl betaine in a medical skin cleaning and repairing liquid. According to the invention, an in-vitro skin absorption test model is adopted, so that the absorption condition of cocamidopropyl betaine in human skin can be simulated, and a reliable experimental basis is provided for safety evaluation of the medical skin cleaning and repairing liquid. The method comprises the following steps: adding a reyd's salt solution to treat a precipitate, dissolving with acetone, detecting by using an ultraviolet spectrophotometer, and calculating the in-vitro skin absorption amount of cocamidopropyl betaine in the medical skin cleaning and repairing liquid according to a linear regression equation. The detection method disclosed by the invention is good in repeatability, high in sensitivity, simple and rapid, accurate in result and suitable for detecting the in-vitro skin absorption amount of cocamidopropyl betaine in the medical skin cleaning and repairing liquid.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical instrument detection, and particularly relates to a method for detecting the in-vitro skin absorption amount of cocamidopropyl betaine in medical skin cleaning and repairing liquid. BACKGROUND

[0002] Cocamidopropyl betaine (CAS No 61789-40-0) is an amphoteric surfactant with excellent dirt removal, foaming and conditioning properties, and little skin irritation, and is widely used in medical skin cleaning and repairing liquid and other products. Since the ingredient directly contacts human skin, its transdermal absorption is closely related to product safety. If the amount of cocamidopropyl betaine penetrating into the body through the skin is too high, it may cause skin allergy, inflammation and other adverse reactions, so it is of great significance to establish a method for accurately and efficiently detecting the in-vitro skin absorption amount of cocamidopropyl betaine in medical skin cleaning and repairing liquid.

[0003] At present, the detection methods for cocamidopropyl betaine mainly include high performance liquid chromatography and the like, but these methods have problems such as complex pretreatment, long detection period and high requirements for equipment when used for in-vitro skin absorption detection. At the same time, there is no unified national standard or industry standard for the detection of the in-vitro skin absorption amount of cocamidopropyl betaine in medical skin cleaning and repairing liquid, resulting in a lack of comparability between different detection results.

[0004] Therefore, it is urgent to develop a detection method with simple operation, high sensitivity, good repeatability and accurate results to meet the quality control and safety evaluation needs of medical skin cleaning and repairing liquid. SUMMARY

[0005] The purpose of the present application is to solve the above-mentioned problems, and to provide a method for detecting the in-vitro skin absorption amount of cocamidopropyl betaine in medical skin cleaning and repairing liquid.

[0006] The technical solution adopted by the present application is as follows: a method for detecting the in-vitro skin absorption amount of cocamidopropyl betaine in medical skin cleaning and repairing liquid, the method comprising the following steps:

[0007] S1. Sample pretreatment: weigh 1 g of sample into a 10 mL volumetric flask, dissolve with first-grade water, and then dilute to the mark. Mix well, take 1 mL of the solution into a test tube, and place it in an ice water bath for 30 min. Add 10 mL of Riesle salt solution (weigh 1.5 g of lithium sodium chloride, add 100 mL of first-grade water, stir for 45 min, filter, and adjust the pH value to about 1.00 with hydrochloric acid) and mix well. Place at 2-8°C for 6 h. Filter the cooled sample on a suction filtration device with a 0.22 μm organic phase filter membrane. Wash the precipitate with 20 mL of 2-8°C first-grade water for 5 times. Take out the filter membrane with the precipitate, dissolve the precipitate with 70% acetone, and dilute to 10 mL. Prepare a blank solution in the same way without adding sample;

[0008] S2. Solution preparation: take the cocoyl amido propyl betaine standard solution (concentration: 9.762 mg / mL), and remove 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL into 6 test tubes respectively to prepare a series of standard working solutions with concentrations of 0.0000 mg / mL, 0.1952 mg / mL, 0.3905 mg / mL, 0.5857 mg / mL, 0.7810 mg / mL, and 0.9762 mg / mL. Take PBS solution to obtain the detection limit and the quantification limit according to the sample pretreatment steps. Weigh 1 g of medical skin cleaning and repairing liquid into 6 10 mL volumetric flasks, dissolve with first-grade water, and then dilute to the mark. Take 1 mL of the solution, and treat it according to the sample pretreatment steps to obtain a repeatability solution. Use the sample spiking method to obtain nine accuracy solutions with three concentration levels of low, medium, and high;

[0009] S3. In vitro skin absorption test process: cut the pig skin into appropriate size, wrap the sandpaper on the wooden stick, and gently rub the surface of the pig skin until the skin surface turns light red. Wash with normal saline, and dry with filter paper before use. Add appropriate amount of receiving liquid (PBS solution) to the receiving chamber, and put in a matching magnetic stirrer. Fix the pig skin, add appropriate amount of PBS solution to the receiving pool, set the electromagnetic stirrer to 200 rpm, adjust the water bath temperature to 32.0°C, and exhaust the bubbles to make the pig skin in close contact with the receiving liquid. Place 1 g of sample on the surface of the pig skin, set up 4 parallel groups, and take the same amount of PBS solution as a blank control group. After transdermal absorption, collect the target substance on the skin and in the skin at 0.5 h, 2 h, 4 h, 6 h, and 24 h, and analyze the skin surface after 24 h.

[0010] S4. Establish the test method: use ultraviolet spectrophotometer to analyze the repeatability solution, detection limit solution, quantification limit solution, standard curve solution, and accuracy solution.

[0011] S5. Method verification data analysis: the 6 repeated solution samples were injected to obtain the repeatability results; the 11 detection limit solution samples were injected for analysis, and the 3.3 times of the standard deviation of the response value of the blank test solution divided by the concentration corresponding to the slope was taken as the detection limit, and the 10 times of the standard deviation of the response value of the blank sample solution divided by the concentration corresponding to the slope of the standard curve was taken as the quantification limit; the standard curve solution was subjected to linear regression analysis to obtain the linear regression equation and establish the standard curve; the accuracy of the low, medium and high concentration solutions was analyzed to obtain the accuracy results;

[0012] S6. Quantitative analysis: the sample solution was determined by ultraviolet spectrophotometry to analyze the cocoyl amido propyl betaine in the medical skin cleaning and repairing liquid; the method has good repeatability, high sensitivity, high efficiency, fast speed, accurate results and convenient operation.

[0013] In a preferred embodiment, in step S1, the pH value of the Riesle salt solution is adjusted to about 1.00, under which condition the Riesle salt reacts more fully with the cocoyl amido propyl betaine.

[0014] In a preferred embodiment, in step S1, the precipitation reaction is placed at 2-8℃ for 6h to ensure complete precipitation.

[0015] In a preferred embodiment, in step S2, the linear range is set to 0.0000mg / mL-0.9762mg / mL, which covers the concentration range that may occur in the actual sample.

[0016] In a preferred embodiment, in step S3, when the pig skin is pretreated, the sandpaper polishing intensity is appropriate to make the skin surface become light red, which can remove the stratum corneum without damaging the dermis.

[0017] In a preferred embodiment, in step S3, the temperature of the transdermal absorption test is controlled at 32.0℃, which is close to the human skin temperature and can better simulate the in vivo environment.

[0018] In a preferred embodiment, in step S3, the method for treating part of the pig skin is: after the skin is cut into pieces, 5mL of PBS solution is added and ultrasonic is performed for 60min, and the supernatant is taken after centrifugation for standby; the subcutaneous part is treated: 2mL of the receiving liquid at 0.5h, 2h, 4h and 6h is taken in a test tube for standby.

[0019] In a preferred embodiment, in step S5, 6 prepared repeated solutions are injected in the repeatability test. Before injection, the UV spectrophotometer is preheated for more than 30 minutes, the detection environment temperature is controlled at 25℃±2℃, and the relative humidity is maintained at 45%~65%. The detection wavelength is set at 525nm, and the blank solution is used as the reference for zero adjustment. The relative standard deviation of the 6 measurement results needs to be controlled within 2.0%, and the actual measured RSD value of the method is 1.3%, indicating that the detection system has good stability in continuous operation.

[0020] When detecting the detection limit and the quantitative limit, 11 detection limit solutions are measured respectively, and the injection interval is 2 minutes to ensure the stability of the instrument light path system. The blank test solution needs to be pretreated synchronously with the sample solution, including ice water bath cooling, Riesle salt precipitation and acetone dissolution steps, to eliminate matrix interference. By calculating the standard deviation of the response value of 11 blank solutions and combining the slope of the standard curve, the detection limit is calculated as 1.71mg, and the quantitative limit is calculated as 5.19mg. The sensitivity is sufficient to meet the detection requirements of low concentration target substances in medical skin cleaning and repairing liquid

[0021] In a preferred embodiment, in step S5, during linear regression analysis, the series of standard working solutions are injected in order from low to high concentration, and linear fitting is performed with concentration as the horizontal coordinate and absorbance as the vertical coordinate. The concentration range of the standard solution covers 0.0000mg / ml to 0.9762mg / ml, the linear regression equation is Abs=0.07068×Conc+0.00127, the correlation coefficient R reaches 0.995, indicating that the absorbance and concentration show a good linear relationship in this concentration range. In the accuracy test, 3 parallel samples were prepared for each of the low, medium and high concentration levels, and the average recoveries were 96.6%, 100.0% and 101.9% respectively after the same pretreatment steps were performed and measured. All are within the acceptable range of 92%~105%, verifying the accuracy of the method.

[0022] In a preferred embodiment, in step S6, when using UV spectrophotometry for determination, the instrument needs to be calibrated in advance for wavelength accuracy to ensure the stability of the maximum absorption peak at 525nm. The sample solution needs to be filtered through a 0.22μm organic phase filter before measurement to remove possible particulate matter interference. During measurement, 70% acetone solution is used as the reference, and the absorbance is substituted into the linear regression equation to calculate the concentration, with the results rounded to three significant digits.

[0023] The influence of temperature on the determination needs to be strictly controlled during the detection process, and the sample cell constant temperature device is set to 25 DEG C. The sample solution is balanced to the temperature before determination. For samples of different substrates such as skin, skin and receiving liquid, blank correction needs to be carried out respectively, and the substrate background absorption is deducted. After the determination is completed, the instrument automatically generates a data report, including sample concentration, relative standard deviation and recovery rate and other parameters, realizing the rapid and accurate analysis of the content of cocamide propyl betaine in the medical skin cleaning and repairing liquid.

[0024] In summary, due to the adoption of the technical scheme, the present application has the beneficial effects that:

[0025] 1. In the present application, pigskin is selected as an in-vitro skin model, and the stratum corneum state of human skin is simulated by moderate sandpaper rubbing, which not only retains the barrier function of the skin but also avoids excessive damage. During the experiment, PBS solution is injected into the receiving chamber and a water bath environment close to the temperature of human skin is maintained, and magnetic stirring is used to ensure uniform distribution of the receiving liquid, so that the skin is in close contact with the liquid. Multiple parallel tests and blank controls are set up to reduce experimental errors, and samples from different parts of the skin are collected to comprehensively reflect the absorption of the target substance in each layer of the skin. This model can truly simulate the penetration process of cocamide propyl betaine in human skin, providing intuitive and reliable experimental data for evaluating product safety.

[0026] 2. In the present application, the sample state is stabilized by ice water bath cooling, and the solution is adjusted to an appropriate acidic environment after the addition of resorcinol salt solution, which promotes the reaction of the target substance with the reagent to form a precipitate. The precipitate is allowed to stand for a sufficient period of time in a low-temperature environment to ensure complete reaction, and then filtered through a filter membrane and dissolved in acetone to a constant volume. The operation process is simple and does not require complex equipment. In the detection stage, ultraviolet spectrophotometry is used to achieve rapid quantitative analysis through a standard curve, eliminating the need for large and precise instruments and reducing detection costs. The entire process from sample processing to result output is time-consuming, suitable for batch detection requirements of conventional laboratories, while ensuring the practicality and operability of the method.

[0027] 3. In the present application, method verification covers key indicators such as linear range, detection limit, quantification limit, repeatability and accuracy. The linear range covers the concentration interval that may occur in actual samples, ensuring that target substances of different concentrations can be accurately determined; the detection limit and quantification limit are set reasonably, which can capture trace components in low-concentration samples; repeatability tests prove that the method has good stability in continuous operation through multiple parallel determinations; the high, medium and low concentration levels of the standard addition recovery rate are within the acceptable range, verifying the accuracy of the method. These systematic verification steps cooperate with each other to ensure that the detection results remain stable and reliable under different experimental conditions, providing a scientific basis for product quality control.

[0028] 4、In the present application, a sampling scheme of multiple time points is designed, the receiving liquid is collected regularly during the transdermal absorption process, and the samples on and in the skin are treated after the end of the test, so as to realize the dynamic tracking of the absorption process of the target. By analyzing the samples in different time periods, the penetration trend and accumulation of cocamide propyl betaine in the skin can be clearly reflected, which provides data support for the study of its absorption rule. This dynamic monitoring mode is not only suitable for single experiment analysis, but also can be used for long-term toxicity evaluation and dose-effect relationship research, and provides comprehensive technical support for product formula optimization and safety risk assessment. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is a flow principle schematic diagram of the present application;

[0030] Figure 2 It is a diffusion cell device schematic diagram provided in the embodiment of the present application;

[0031] Figure 3 It is a drug transdermal diffusion tester schematic diagram in the embodiment of the present application;

[0032] Figure 4 It is a different time cumulative penetration amount result schematic diagram in the embodiment of the present application;

[0033] Figure 5 It is a different time diffusion percentage result schematic diagram in the embodiment of the present application. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the present application more clear and obvious, the present application is further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0035] Embodiment

[0036] Reference Figures 1-5 ,

[0037] A method for detecting the in vitro skin absorption amount of cocamide propyl betaine in medical skin cleaning and repairing liquid, the method comprising the following steps:

[0038] S1. Sample pretreatment: weigh 1 g of sample into a 10 mL volumetric flask, dissolve with first-grade water, and then dilute to the mark. Mix well, take 1 mL of the solution into a test tube, and place it in an ice water bath for 30 min. Add 10 mL of Riesle salt solution (weigh 1.5 g of lithium sodium chloride, add 100 mL of first-grade water, stir for 45 min, and then filter. Adjust the pH value to about 1.00 with hydrochloric acid to obtain the solution) and mix well. Place the solution at 2-8°C for 6 h. Filter the cooled sample on a suction filtration device equipped with a 0.22 μm organic phase filter membrane. Wash the precipitate with 20 mL of 2-8°C first-grade water for 5 times. Take out the filter membrane with the precipitate, dissolve the precipitate with 70% acetone, and dilute to 10 mL. Prepare a blank solution in the same way without adding sample.

[0039] S2. Solution preparation: take the cocoyl amido propyl betaine standard solution (concentration: 9.762 mg / mL), and respectively take 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL into 6 test tubes to prepare a series of standard working solutions with concentrations of 0.0000 mg / mL, 0.1952 mg / mL, 0.3905 mg / mL, 0.5857 mg / mL, 0.7810 mg / mL, and 0.9762 mg / mL. Take PBS solution to obtain the detection limit and the quantification limit according to the sample pretreatment steps. Weigh 1 g of medical skin cleaning and repairing liquid into 6 10 mL volumetric flasks, dissolve with first-grade water, and then dilute to the mark. Take 1 mL of the solution, and treat it according to the sample pretreatment steps to obtain a repeatability solution. Use the sample spiking method to obtain nine accuracy solutions with three concentration levels of low, medium, and high.

[0040] S3. In vitro skin absorption test process: cut the pig skin into appropriate size, wrap the sandpaper on a wooden stick, and gently rub the surface of the pig skin until the skin surface becomes light red. Wash with normal saline, and dry with filter paper before use. Add an appropriate amount of receiving liquid (PBS solution) to the receiving chamber, and put in a matching magnetic stirrer. Fix the pig skin, and then add an appropriate amount of PBS solution to the receiving pool. Set the electromagnetic stirrer to stir at a speed of 200 rpm, adjust the water bath temperature to 32.0°C, and exhaust the bubbles to make the pig skin in close contact with the receiving liquid. Place 1 g of sample on the surface of the pig skin, and set 4 parallel groups. Take the same amount of PBS solution as a blank control group. After transdermal absorption, collect the target substance on the skin and in the skin at time points of 0.5 h, 2 h, 4 h, 6 h, and 24 h, and the skin after 24 h. The skin surface is repeatedly washed with an appropriate amount of PBS solution, and the washing liquid is collected in a 10 mL volumetric flask, dissolved with first-grade water, and then diluted to the mark. Mix well, and reserve for use.

[0041] S4. Establish the test method: use the ultraviolet spectrophotometer to analyze the repeatability solution, the detection limit solution, the quantification limit solution, the standard curve solution, and the accuracy solution.

[0042] S5. Method verification data analysis: the 6 repetitive solution samples are injected to obtain the repeatability results; the 11 detection limit solution samples are analyzed, and the 3.3 times of the standard deviation of the response value of the blank test solution divided by the concentration corresponding to the slope is taken as the detection limit, and the 10 times of the standard deviation of the response value of the blank sample solution divided by the concentration corresponding to the slope of the standard curve is taken as the quantification limit; the standard curve solution is subjected to linear regression analysis to obtain the linear regression equation and establish the standard curve; the accuracy of the low, medium and high concentration solutions is analyzed to obtain the accuracy results;

[0043] S6. Quantitative analysis: the sample solution is determined by ultraviolet spectrophotometry to analyze the cocoyl amido propyl betaine in the medical skin cleaning and repairing liquid; the method has good repeatability, high sensitivity, high efficiency, fast speed, accurate results and convenient operation.

[0044] In the step S1, the pH value of the Riesle salt solution is adjusted to about 1.00, and under this condition, the Riesle salt reacts more fully with the cocoyl amido propyl betaine.

[0045] In the step S1, the precipitation reaction is placed at 2℃-8℃ for 6h to ensure complete precipitation.

[0046] In the step S2, the linear range is set to 0.0000mg / mL-0.9762mg / mL, which covers the concentration range that may occur in the actual sample.

[0047] In the step S3, when the pig skin is pretreated, the sandpaper polishing intensity is appropriate to change the skin surface to light red, which can remove the stratum corneum and not damage the dermis.

[0048] In the step S3, the temperature of the transdermal absorption test is controlled at 32.0℃, which is close to the human skin temperature, and can better simulate the in vivo environment.

[0049] In the step S3, the method for treating part of the pig skin is: after the skin is cut into pieces, 5mL PBS solution is added and ultrasonic is performed for 60min, and the supernatant is taken after centrifugation for standby; the subcutaneous part is treated: 2mL of the receiving liquid at 0.5h, 2h, 4h and 6h is taken in a test tube for standby.

[0050] The step S5, in the repeatability test, 6 prepared reproducible solutions were injected, and the UV spectrophotometer was preheated for more than 30 minutes before injection. The detection environment temperature was controlled at 25℃±2℃, and the relative humidity was maintained at 45%~65%. The detection wavelength was set at 525nm, and the blank solution was used as the reference for zero adjustment. The relative standard deviation of the 6 measurement results was controlled within 2.0%, and the actual measured RSD value of the method was 1.3%, indicating that the detection system had good stability in continuous operation.

[0051] When detecting the limit of detection and the limit of quantification, 11 limit of detection solutions were injected, and the interval between each injection was 2 minutes to ensure the stability of the instrument light path system. The blank test solution was pretreated synchronously with the sample solution, including ice water bath cooling, Riesle salt precipitation and acetone dissolution, etc. to eliminate matrix interference. By calculating the standard deviation of the response value of 11 blank solutions, combined with the slope of the standard curve, the detection limit was calculated as 1.71mg, and the quantification limit was 5.19mg. The sensitivity was sufficient to meet the detection requirements of low concentration target substances in medical skin cleaning and repairing liquid.

[0052] In the step S5, in the linear regression analysis, the series of standard working solutions were injected in order from low to high concentration, and the concentration was used as the horizontal coordinate and the absorbance was used as the vertical coordinate for linear fitting. The concentration range of the standard solution covered 0.0000mg / ml to 0.9762mg / ml, the linear regression equation was Abs=0.07068×Conc+0.00127, the correlation coefficient R reached 0.995, indicating that the absorbance and concentration showed good linear relationship in this concentration range. In the accuracy test, 3 parallel samples were prepared for each of the low, medium and high concentration levels, and the average recoveries were 96.6%, 100.0% and 101.9% respectively after the same pretreatment steps were operated and measured, which were all within the acceptable range of 92%~105%, verifying the accuracy of the method.

[0053] In the step S6, when the UV spectrophotometric method was used for determination, the instrument needed to be calibrated in advance for wavelength accuracy to ensure the stability of the maximum absorption peak at 520nm. The sample solution needed to be filtered through a 0.22 micron organic phase filter before determination to remove possible particulate matter interference. The absorbance was taken as the reference for zero adjustment, and the concentration was calculated by substituting the linear regression equation, with the results retaining three significant digits.

[0054] The influence of temperature on the determination needs to be strictly controlled during the detection process. The sample pool constant temperature device is set to 25℃, and the sample solution is equilibrated to this temperature before determination. For samples of different matrices such as skin, skin and receiving liquid, blank correction needs to be carried out respectively, and the matrix background absorption is deducted. After the determination is completed, the instrument automatically generates a data report, including sample concentration, relative standard deviation and recovery rate and other parameters, realizing the rapid and accurate analysis of the content of cocamide propyl betaine in medical skin cleaning and repairing liquid.

[0055] The method verification data analysis results are shown in Tables 1-4. According to the method verification data results, it is proved that the method has good repeatability, linearity and accuracy, and at the same time, the detection limit is relatively low.

[0056] Table 1 repeatability results

[0057]

[0058] Table 2 detection limit and quantification limit results

[0059]

[0060] Table 3 linearity results

[0061]

[0062] Table 4 accuracy results

[0063]

[0064] Quantitative analysis: The sample solution in the in vitro skin absorption test was determined by ultraviolet spectrophotometry, so as to analyze the cocamide propyl betaine in medical skin cleaning and repairing liquid. The analysis results are shown in Tables 5-8

[0065] Table 5 results of cocamide propyl betaine in medical skin cleaning and repairing liquid

[0066]

[0067] Table 6 transdermal results of cocamide propyl betaine

[0068] Sample No. Content in skin (mg) Content in skin (mg) Content in receiving pool (mg) Sample 1 31.69 0 0 Sample 2 29.97 0 0 Sample 3 32.55 0 0 Sample 4 29.97 0 0

[0069] Table 7 cumulative penetration amount of cocamide propyl betaine

[0070]

[0071]

[0072] Table 8 percentage of diffusion of cocamide propyl betaine

[0073]

[0074] From the above, it can be known that the present application can complete the detection of the in-vitro skin absorption amount of cocamide propyl betaine in medical skin cleaning and repairing liquid, and strict experimental design and verification process are adopted to ensure the accuracy and reliability of the in-vitro skin absorption test detection of cocamide propyl betaine in medical skin cleaning and repairing liquid. In the present application, the treated solution is detected by using an ultraviolet spectrophotometer after precipitation treatment by Riehl salt solution, and the absorption amount in the in-vitro skin absorption test of cocamide propyl betaine in medical skin cleaning and repairing liquid is calculated according to the linear regression equation. The detection method of the present application has good repeatability, high sensitivity, simplicity, rapidness, accurate results, and is suitable for in-vitro skin absorption test detection of cocamide propyl betaine in medical skin cleaning and repairing liquid.

[0075] In the present application, pigskin is selected as an in-vitro skin model, and the stratum corneum state of human skin is simulated by moderate friction treatment with sandpaper, so as to retain the barrier function of the skin and avoid excessive damage. During the test process, PBS solution is injected into the receiving chamber and a water bath environment close to the temperature of human skin is maintained, and magnetic stirring is used to ensure uniform distribution of the receiving liquid, so that the skin is in close contact with the liquid. Multiple parallel tests and blank controls are set to reduce experimental errors, and samples are collected from different parts of the skin, the middle skin and the subcutaneous skin to comprehensively reflect the absorption of the target substance in each layer of the skin. This model can truly simulate the penetration process of cocamide propyl betaine in human skin, and provide intuitive and reliable experimental data for evaluating product safety.

[0076] In the present application, the sample state is stabilized by ice water bath cooling, and the Riehl salt solution is added to adjust to a suitable acidic environment, so as to promote the target substance to fully react with the reagent to form a precipitate. The precipitate is placed in a low-temperature environment for a long enough time to ensure complete reaction, and after filtration through a filter membrane, it is dissolved and diluted with acetone. The operation process is simple and does not require complex equipment. In the detection stage, ultraviolet spectrophotometry is used to realize rapid quantitative analysis through a standard curve, which saves the use of large and precise instruments and reduces the detection cost. The whole process from sample treatment to result output takes a short time, is suitable for batch detection requirements of conventional laboratories, and at the same time ensures the practicability and operability of the method.

[0077] In the present application, method verification covers key indicators such as linear range, detection limit, quantification limit, repeatability and accuracy. The linear range covers the concentration interval that may occur in the actual sample, ensuring that the target of different content can be accurately determined; the detection limit and the quantification limit are set reasonably, which can capture trace components in low concentration samples; repeatability test proves that the method has good stability in continuous operation through multiple parallel determinations; the high, medium and low concentration level of the standard addition recovery rate is within the acceptable range, which verifies the accuracy of the method. These systematic verification steps cooperate with each other to ensure that the detection results can remain stable and reliable under different experimental conditions, and provide a scientific basis for product quality control.

[0078] In the present application, a sampling scheme at multiple time points is designed, the receiving liquid is collected regularly during the transdermal absorption process, and the samples on and in the skin are processed after the end of the test, realizing the dynamic tracking of the absorption process of the target. By analyzing the samples in different time periods, the penetration trend and accumulation of cocamidopropyl betaine in the skin can be clearly reflected, providing data support for the study of its absorption rule. This dynamic monitoring mode is not only suitable for single experiment analysis, but also can be used for long-term toxicity evaluation and dose-effect relationship research, providing comprehensive technical support for product formula optimization and safety risk assessment.

[0079] It should be noted that, in this text, relational terms such as first and second are used only to distinguish one entity or operation from another, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. Moreover, the terms "include", "contain" or any other variants thereof are intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such process, method, article or equipment. Without more limitations, the element defined by the statement "including a" does not exclude the presence of other identical elements in the process, method, article or equipment including the element.

[0080] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for detecting the in vitro skin absorption of cocamidopropyl betaine in a medical skin cleansing and repairing solution, characterized in that: The method includes the following steps: S1. Sample pretreatment: Weigh 1g of sample into a 10mL volumetric flask, dissolve in primary water, and dilute to the mark. Mix well. Take 1mL of this solution into a test tube and place in an ice-water bath for 30min. Add 10mL of Reich's salt solution. Weigh 1.5g of Levin sodium salt, add 100mL of primary water, stir for 45min, and filter. Adjust the pH to approximately 1.00 with hydrochloric acid. Mix well and place at 2℃-8℃ for 6h. Filter the cooled sample using a vacuum filtration device equipped with a 0.22μm organic phase filter membrane. Wash the precipitate 5 times with 20mL of primary water at 2℃-8℃. Remove the filter membrane with the precipitate, dissolve the precipitate in 70% acetone, and dilute to 10mL. Prepare a blank solution without adding sample using the same method. S2. Solution Preparation: Take cocamidopropyl betaine standard working solution with a concentration of 9.762 mg / mL, and transfer 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL into six test tubes respectively to prepare a series of standard working solutions with concentrations of 0.0000 mg / mL, 0.1952 mg / mL, 0.3905 mg / mL, 0.5857 mg / mL, 0.7810 mg / mL, and 0.9762 mg / mL; take PBS solution and obtain the limit of detection and limit of quantitation according to the sample pretreatment procedure; also, take 1 g of medical skin cleansing and repair solution and dissolve it in six 10 mL volumetric flasks, then dilute to the mark with primary water, and take 1 mL of the solution and process it according to the sample pretreatment procedure to obtain a repeatability solution; obtain nine accuracy solutions at three concentration levels (low, medium, and high) by sample spiking. S3. In vitro skin absorption test procedure: Cut pigskin into appropriate sizes, wrap sandpaper around a wooden stick, and gently rub the pigskin surface until it turns light red. Wash with physiological saline and blot dry with filter paper before use. Inject an appropriate amount of receiving solution into the receiving chamber and place the matching magnetic stir bar. After fixing the pigskin, add an appropriate amount of PBS solution to the receiving cell, set the electromagnetic stirrer to a speed of 200 rpm, adjust the water bath temperature to 32.0℃, and remove air bubbles to ensure close contact between the pigskin and the receiving solution. Place 1g of sample on the surface of the pigskin and set up 4 parallel groups. Take the same mass of PBS solution as a blank control group. After transdermal absorption, collect the target substances in the diffusion cell at time points of 0.5h, 2h, 4h, 6h, and 24h, and analyze the target substances on and inside the skin after 24h. For the treatment of the skin part: take an appropriate amount of PBS solution and repeatedly wash the skin surface in small amounts. Collect the washing solution in a 10mL volumetric flask, dissolve it with primary water, and dilute to the mark. Mix well and set aside. S4. Establish test methods: Analyze the repeatability solution, detection limit solution, quantitation limit solution, standard curve solution, and accuracy solution using a UV spectrophotometer; S5. Method Validation Data Analysis: The 6 repeatability solutions were injected to obtain repeatability results; the 11 detection limit solutions were analyzed, with the detection limit calculated as 3.3 times the standard deviation of the blank test solution response value divided by the slope corresponding to the concentration, and the quantitation limit calculated as 10 times the standard deviation of the blank sample solution response value divided by the slope of the standard curve; linear regression analysis was performed on the standard curve solutions to obtain the linear regression equation and establish the standard curve; the accuracy results were obtained by analyzing the low, medium, and high concentration accuracy solutions. S6. Quantitative analysis: The sample solution was analyzed by ultraviolet spectrophotometry to determine the cocamidopropyl betaine in the medical skin cleansing and repairing solution.

2. The method for detecting the in vitro skin absorption of cocamidopropyl betaine in a medical skin cleansing and repairing solution as described in claim 1, characterized in that: In step S1, the pH of the Reichelk salt solution is adjusted to around 1.00, under which the Reichelk salt reacts more fully with cocamidopropyl betaine.

3. The method for detecting the in vitro skin absorption of cocamidopropyl betaine in a medical skin cleansing and repairing solution as described in claim 1, characterized in that: In step S1, the precipitation reaction is carried out at 2℃-8℃ for 6 hours to ensure complete precipitation.

4. The method for detecting the in vitro skin absorption of cocamidopropyl betaine in a medical skin cleansing and repairing solution as described in claim 1, characterized in that: In step S2, the linear range is set to 0.0000 mg / mL-0.9762 mg / mL, which covers the concentration range that may occur in actual samples.

5. The method for detecting the in vitro skin absorption of cocamidopropyl betaine in a medical skin cleansing and repairing solution as described in claim 1, characterized in that: In step S3, during the pretreatment of pigskin, the sandpaper should be used to polish the skin until the surface turns light red, which can remove the influence of the stratum corneum without damaging the dermis.

6. The method for detecting the in vitro skin absorption of cocamidopropyl betaine in a medical skin cleansing and repairing solution as described in claim 1, characterized in that: In step S3, the temperature of the transdermal absorption test is controlled at 32.0℃, which is close to the temperature of human skin and can better simulate the internal environment.

7. The method for detecting the in vitro skin absorption of cocamidopropyl betaine in a medical skin cleansing and repairing solution as described in claim 1, characterized in that: In step S3, the method for partial processing of pig skin is as follows: cut the skin into small pieces, add 5 mL of PBS solution, sonicate for 60 min, centrifuge, and take the supernatant for later use; for subcutaneous processing: collect 2 mL of the solution at each time point of 0.5 h, 2 h, 4 h, and 6 h in test tubes for later use.

8. The method for detecting the in vitro skin absorption of cocamidopropyl betaine in a medical skin cleansing and repairing solution as described in claim 1, characterized in that: In step S5, during the repeatability test, six prepared repeatable solutions are injected. Before injection, the UV spectrophotometer is preheated for at least 30 minutes, the ambient temperature is controlled at 25℃±2℃, and the relative humidity is maintained at 45%~65%. The detection wavelength is set to 525nm, and the blank solution is used as a reference for zeroing.

9. The method for detecting the in vitro skin absorption of cocamidopropyl betaine in a medical skin cleansing and repairing solution as described in claim 1, characterized in that: In step S5, during linear regression analysis, a series of standard working solutions were injected sequentially from low to high concentration. Linear fitting was performed with concentration as the abscissa and absorbance as the ordinate. The standard solution concentration ranged from 0.0000 mg / mL to 0.9762 mg / mL. The linear regression equation was Abs = 0.07068 × Conc + 0.00127, with a correlation coefficient R of 0.995, indicating a good linear relationship between absorbance and concentration within this concentration range. In the accuracy test, three parallel samples were prepared for each of the low, medium, and high concentration levels of spiked samples. After following the same pretreatment steps, the average recoveries were 96.6%, 100.0%, and 101.9%, respectively, all within the acceptable range of 92%–105%, verifying the accuracy of the method.

10. The method for detecting the in vitro skin absorption of cocamidopropyl betaine in a medical skin cleansing and repairing solution as described in claim 1, characterized in that: In step S6, when using ultraviolet spectrophotometry, the instrument needs to be calibrated for wavelength accuracy in advance to ensure that the maximum absorption peak at 525 nm is stable. During the testing process, the sample cell temperature control device is set to 25℃, and the sample solution is equilibrated to this temperature before the measurement. For samples with different matrices such as skin surface, skin middle and receiving liquid, blank correction is required to deduct the matrix background absorption.

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