Visual detection method for solubility of amoxicillin based on quantitative color gradation

Abstract

The invention discloses an amoxicillin solubility visual detection method based on quantitative color gradation, particularly relates to the field of drug detection, and aims to solve the problems of large standard gradient interval, unstable color development and lack of contrast correction in the existing visual comparison method. Preparing an amoxicillin saturated solution; the method comprises the following steps: establishing a linear regression equation, precisely preparing an amoxicillin standard solution with 9 concentration gradients, adding a copper sulfate solution to form a stable yellow-green turbidity gradient, and drawing the linear regression equation according to an absorbance value; when the sample is measured, turbidimetric analysis is carried out on the to-be-measured liquid and the standard gradient, the solubility is calculated in combination with the dilution multiple after the concentration is determined, visual estimation and reading are converted into accurate quantification through the high-density gradient standard and the linear regression model, the matching error is remarkably reduced, and the anti-interference capability and accuracy are improved; the method is suitable for rapid detection in drug research and development and quality control.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, and more specifically, to a visual detection method for amoxicillin solubility based on quantitative color gradation. Background Technology

[0002] In the field of amoxicillin solubility determination and quantitative analysis of similar substance concentrations, the visual comparison-based approach has long been used. Its core logic involves setting up a series of standard samples with known concentrations to form a visually distinguishable gradient. The sample to be tested is then visually compared with the standard samples, and the standard sample with the closest color or turbidity is selected. Its concentration is then used to approximate the concentration of the sample to be tested, ultimately deriving the solubility data. This method is widely used in grassroots laboratories or on-site rapid testing scenarios due to its simplicity and lack of complex instruments. The standard procedure is as follows: first, prepare standard solutions with gradient concentrations; after adding a specific colorimetric reagent, a stable and clearly differentiated visual gradient system is formed; then, the saturated amoxicillin solution to be tested undergoes the same colorimetric treatment; using a colorimetric device, the solution to be tested is compared one by one with the samples in the standard gradient to determine the concentration of the standard sample with the closest visual characteristics; and the solubility is calculated based on the dilution factor.

[0003] However, existing visual comparison methods have significant drawbacks: 1. Insufficiently reasonable standard gradient settings. Most schemes only set 3-5 concentration gradients, with excessively large gradient intervals, making it difficult to accurately match the test solution with the standard sample. This easily introduces large calculation deviations due to visual judgment errors. 2. Poor colorimetric stability. The reaction between commonly used chromogenic agents and amoxicillin is significantly affected by temperature and humidity, and the chromogenic products are prone to fading or precipitation. The standard gradient system is difficult to maintain stability over a long period, affecting the accuracy of the comparison. 3. Lack of a control correction mechanism. The absence of blank and positive control tubes makes it impossible to eliminate the interference of the solvent and chromogenic agent's own color on visual judgment, further reducing the reliability of the results. These problems make existing visual comparison methods insufficiently accurate in amoxicillin solubility determination, making it difficult to meet the data accuracy requirements of drug development, quality control, and other scenarios. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention provides a visual detection method for amoxicillin solubility based on quantitative color levels, which solves the problems mentioned in the background art through the following scheme.

[0005] To achieve this objective, the present invention provides the following technical solution: a visual detection method for amoxicillin solubility based on quantitative color levels, comprising: S1. Preparation of sample solution and reagents: (1) Preparation of 0.2M potassium dihydrogen phosphate solution: accurately weigh potassium dihydrogen phosphate powder, dissolve and dilute to volume to prepare 0.2M potassium dihydrogen phosphate solution; (2) Preparation of amoxicillin saturated solution: take excess amoxicillin sample powder to be tested, add the accurate volume of solution to dissolve, shake at 25°C for 24 hours to ensure that dissolution equilibrium is reached and a saturated solution is formed; S2. Establishment of linear regression equation: (1) Preparation of amoxicillin standard stock solution: accurately weigh amoxicillin reference standard, dissolve and dilute to volume to prepare amoxicillin standard stock solution with a concentration of 1000 μg / mL; (2) Preparation of a series of standard tubes: accurately measure different volumes of amoxicillin standard stock solution, dilute with solution to obtain a series of standard solutions with concentration gradients; (3) Color development and establishment of curve: quantitatively add 0.1% copper sulfate solution to each series of standard tubes, mix well and let stand. Each tube will then show a clear turbidity gradient from transparent clear liquid to yellow-green. This gradient is the visual linear regression equation used for quantitative comparison.

[0006] S3. Sample determination and solubility calculation: (1) Sample color development: Dilute the amoxicillin saturated solution prepared in S1, add an equal amount of 0.1% copper sulfate solution, mix and let stand to obtain the test solution; (2) Turbidity analysis: Compare the test solution with the visual linear regression equation established in S2 to determine the concentration of the closest standard tube corresponding to its turbidity; (3) Calculate solubility: Calculate the solubility of the amoxicillin sample based on the concentration value determined by comparison and the dilution factor.

[0007] Preferably, the preparation process of 0.1% copper sulfate solution is as follows: weigh out sufficient amount of copper sulfate pentahydrate and dissolve it in an appropriate amount of deionized water. The resulting solution is unstable and should be stored away from light. It should be freshly prepared before the experiment to ensure the stability of the reaction activity.

[0008] Preferably, the preparation method of amoxicillin saturated solution is as follows: A conical flask containing the sample and solvent is tightly capped and placed in a constant temperature water bath shaker pre-set at 25.0±0.5°C. The shaker speed is set to 150 rpm, and the shaker is continuously shaken for 24 hours. After 24 hours, the conical flask is carefully removed and placed on the experimental table for a moment without shaking. In a well-lit area, the bottom of the flask is carefully observed from the side and bottom. If no undissolved solids are observed, it indicates that the initial amount of sample was insufficient and saturation was not achieved. In this case, a small amount of sample needs to be added, and shaking continues for several hours until excess solids are confirmed to be present.

[0009] Preferably, the amoxicillin saturated solution should be dissolved using 0.2M potassium dihydrogen phosphate solution at room temperature. To ensure complete and rapid dissolution and the formation of a homogeneous solution, any of the following methods can be used to assist dissolution: a) Mechanical oscillation method: Place the container on a vortex shaker and oscillate intermittently or continuously at a speed of not less than 2500 rpm until the solution is clear and transparent and the undissolved powder no longer decreases; b) Ultrasonic treatment method: Place the container in a water bath of an ultrasonic cleaner (power ≥300W, frequency 40kHz) and ultrasonically treat for 5 to 10 minutes, or until the solution system is clear and transparent and the undissolved powder no longer decreases.

[0010] Preferably, the solvent used to prepare the amoxicillin saturated solution and the solvent used to dilute the series of standard solutions are both 0.2M potassium dihydrogen phosphate solution to ensure the consistency of the system.

[0011] The technical effects and advantages of this invention are as follows: 1. High quantitative accuracy: By establishing a high-density gradient standard series and a visual linear regression model, a leap from visual estimation to accurate quantification is achieved, and the discrete gradient is transformed into a linear regression equation, which significantly reduces the matching error caused by the gradient interval. 2. Strong colorimetric stability: A standardized phosphate-copper sulfate colorimetric system is used to ensure the reproducibility and persistence of the turbidity gradient: 0.2M potassium dihydrogen phosphate is used as the solvent, and 0.1% copper sulfate is used to form a specific colorimetric system, forming a stable yellow-green turbidity gradient, ensuring reliable reproducibility of results.

[0012] 3. Excellent anti-interference performance: The innovative dual-control quality control design effectively identifies and eliminates non-specific interference. By setting blank and positive controls, the background color development of the phosphate-free system and the background turbidity of the drug-free system are respectively identified, which significantly improves the specificity of the method and the accuracy of the results. Detailed Implementation

[0013] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention. Example 1

[0014] S1. Preparation of sample solution and reagents: (1) Preparation of 0.2M potassium dihydrogen phosphate solution: accurately weigh potassium dihydrogen phosphate powder, dissolve and dilute to volume to prepare 0.2M potassium dihydrogen phosphate solution; (2) Preparation of amoxicillin saturated solution: take an excess of the amoxicillin sample powder to be tested, add the accurate volume of solution to dissolve, and shake at 25°C for 24 hours to ensure that the dissolution equilibrium is reached and a saturated solution is formed.

[0015] Potassium dihydrogen phosphate powder should be dissolved in sufficient deionized water at room temperature (25±5℃) until the solution is clear and transparent, ensuring no undissolved powder remains. Amoxicillin samples should be dissolved in 0.2M potassium dihydrogen phosphate solution at an ambient temperature ≤25℃ and relative humidity <40% using a gentle magnetic stirrer until the powder in the sample no longer decreases. The clear supernatant should then be aspirated to obtain a clear and transparent saturated amoxicillin solution.

[0016] S2. Establishment of linear regression equation: (1) Preparation of amoxicillin standard stock solution: accurately weigh amoxicillin reference standard, dissolve and dilute to volume to prepare amoxicillin standard stock solution with a concentration of 1000 μg / mL; (2) Preparation of a series of standard tubes: accurately measure different volumes of amoxicillin standard stock solution, dilute with solution to obtain a series of standard solutions with concentration gradients; (3) Color development and establishment of curve: quantitatively add 0.1% copper sulfate solution to each series of standard tubes, mix well and let stand. Each tube will then show a clear turbidity gradient from transparent clear liquid to yellow-green turbid liquid. This gradient is the visual linear regression equation used for quantitative comparison.

[0017] The preparation method for amoxicillin standard stock solution is the same as that for amoxicillin saturated solution. The concentration of amoxicillin standard stock solution is calculated as follows: C = m / (m * P * V), where C is the concentration of amoxicillin standard stock solution (μg / mL), m is the mass of the reference standard (μg), P is the purity of the reference standard, and V is the final volume (mL). In addition to the blank and positive control tubes, nine concentration gradients of amoxicillin standard solutions are set up, with target concentrations of 100, 200, 300, 400, 500, 600, 700, 800, and 900 μg / mL. μg / mL; accurately measure a 1000 μg / mL amoxicillin standard stock solution, and use 0.2 M potassium dihydrogen phosphate solution as a diluent to perform serial dilutions to prepare a series of standard working solutions with different concentration gradients; the series of amoxicillin standard solutions with different concentration gradients includes blank control tubes and positive control tubes. The blank control tubes contain 1000 μg / mL amoxicillin solution dissolved in deionized water, and the solution in the blank control tubes does not contain potassium dihydrogen phosphate; the positive control tubes contain 0.2 M potassium dihydrogen phosphate solution dissolved in deionized water, and do not contain amoxicillin.

[0018] Amoxicillin reacts with copper sulfate to form a colored complex, producing turbidity. The absorbance of this turbidity at a wavelength of 550 nm is linear with the concentration of amoxicillin within a certain range. After adding 0.1% copper sulfate solution to the blank control tube, the solution in the tube is a clear yellow-brown liquid; after adding 0.1% copper sulfate solution to the positive control tube, the solution in the tube is a turbid yellow-brown liquid with obvious precipitation.

[0019] The visual linear regression equation was established based on the absorbance values ​​of amoxicillin standard solutions with a series of concentration gradients. Using a UV-Vis spectrophotometer, the instrument zero point was calibrated at 550 nm with a blank control tube. The absorbance values ​​of each standard tube and sample tube were measured sequentially. The absorbance value of the standard tube was plotted on the ordinate, and the corresponding amoxicillin concentration was plotted on the abscissa. The linear regression equation is y = a × C + b, where y is the absorbance value, C is the amoxicillin concentration in μg / mL, a is the slope, and b is the intercept. It was obtained by fitting the absorbance values ​​of amoxicillin standard solutions with a series of concentration gradients.

[0020] Table 1 Amoxicillin standard stock solution addition (ul) Add potassium dihydrogen phosphate solution (ul) Amoxicillin solution concentration (ug / ml) Blank control tube 1000 0 1000 Pipe 1 100 900 100 Pipe 2 200 800 200 Pipe 3 300 700 300 Pipe 4 400 600 400 Pipe 5 500 500 500 Pipe 6 600 400 600 Pipe 7 700 300 700 Pipe 8 800 200 800 Pipe 9 900 100 900 Positive control tube 0 1000 0 S3. Sample determination and solubility calculation: (1) Sample color development: Dilute the amoxicillin saturated solution prepared in S1, add an equal amount of 0.1% copper sulfate solution, mix and let stand to obtain the test solution; (2) Turbidity analysis: Compare the test solution with the visual linear regression equation established in S2 to determine the concentration of the closest standard tube corresponding to its turbidity; (3) Calculate solubility: Calculate the solubility of amoxicillin sample based on the concentration value determined by comparison and the dilution factor; The solubility of amoxicillin sample is calculated by the formula S=(C×D) / 1000, where S is the solubility of amoxicillin sample in solvent, in mg / mL; C is the original concentration of amoxicillin sample saturated solution determined by linear regression equation and calculated by linear regression equation, in μg / mL; D is the dilution factor of saturated solution before turbidity analysis. Example 2

[0021] S1. Sample solution and reagent preparation: Preparation of amoxicillin saturated solution: Take an excess of the amoxicillin sample powder to be tested, add deionized water as solvent, and continuously and gently stir with magnetic force under the conditions of ambient temperature ≤25℃ and relative humidity <40% until the powder in the sample solution no longer decreases. Take off the clear upper layer of solution to obtain a yellowish-brown clear and transparent amoxicillin saturated solution.

[0022] S2. Establishment of the linear regression equation: (1) Preparation of amoxicillin standard stock solution: Accurately weigh amoxicillin reference standard, dissolve and dilute to a final volume to prepare an amoxicillin standard stock solution with a concentration of 1000 μg / mL; (2) Preparation of a series of standard tubes: Accurately measure different volumes of amoxicillin standard stock solution, dilute with the solution to obtain a series of standard solutions with concentration gradients; (3) Color development and establishment of curves: Quantitatively add 0.1% copper sulfate solution to each series of standard tubes, mix well and let stand. Each tube will then show a clear turbidity gradient from transparent clear liquid to yellow-green turbid liquid. This gradient is the visual linear regression equation used for quantitative comparison. Nine concentration gradients of amoxicillin standard solutions were prepared, excluding the blank control tube, with target concentrations of 100, 200, 300, 400, 500, 600, 700, 800, and 900 μg / mL. A 1000 μg / mL amoxicillin standard stock solution was precisely measured and serially diluted using 0.2 M potassium dihydrogen phosphate solution to prepare a series of working standard solutions with varying concentration gradients. The series of amoxicillin standard solutions included a blank control tube containing a 1000 μg / mL amoxicillin solution dissolved in deionized water.

[0023] S3. Sample determination and solubility calculation: (1) Sample color development: Dilute the amoxicillin saturated solution prepared in S1, add an equal amount of 0.1% copper sulfate solution, mix and let stand to obtain the test solution; (2) Turbidity analysis: Compare the test solution with the visual linear regression equation established in S2 to determine the concentration of the closest standard tube corresponding to its turbidity; (3) Calculate solubility: Calculate the solubility of the amoxicillin sample based on the concentration value determined by comparison and the dilution factor. Example 3

[0024] S1. Preparation of sample solution and reagents: (1) Preparation of 1% sodium hexametaphosphate solution: accurately weigh 1% sodium hexametaphosphate powder, dissolve and dilute to volume to prepare 1% sodium hexametaphosphate solution; (2) Preparation of amoxicillin saturated solution: take an excess of the amoxicillin sample powder to be tested, add an accurate volume of solution to dissolve, and shake at 25°C for 24 hours to ensure that the dissolution equilibrium is reached and a saturated solution is formed.

[0025] 1% sodium hexametaphosphate powder needs to be dissolved in sufficient deionized water at room temperature (25±5℃) until the solution is clear and transparent, ensuring no undissolved powder remains. Amoxicillin samples are dissolved in 1% sodium hexametaphosphate solution at an ambient temperature ≤25℃ and relative humidity <40% using continuous and gentle magnetic stirring until the powder in the solution no longer decreases. The clear upper layer of solution is then aspirated to obtain a clear and transparent saturated amoxicillin solution.

[0026] S2. Establishment of linear regression equation: (1) Preparation of amoxicillin standard stock solution: accurately weigh amoxicillin reference standard, dissolve and dilute to volume to prepare amoxicillin standard stock solution with a concentration of 1000 μg / mL; (2) Preparation of a series of standard tubes: accurately measure different volumes of amoxicillin standard stock solution, dilute with solution to obtain a series of standard solutions with concentration gradients; (3) Color development and establishment of curve: quantitatively add 0.1% copper sulfate solution to each series of standard tubes, mix well and let stand. Each tube will then show a clear turbidity gradient from transparent clear liquid to yellow-green turbid liquid. This gradient is the visual linear regression equation used for quantitative comparison.

[0027] The preparation method for amoxicillin standard stock solution is the same as that for amoxicillin saturated solution. In addition to blank and positive control tubes, nine concentration gradients of amoxicillin standard solutions are set up, with target concentrations of 100, 200, 300, 400, 500, 600, 700, 800, and 900 μg / mL. A 1000 μg / mL amoxicillin standard stock solution is accurately measured and serially diluted using 1% sodium hexametaphosphate solution to prepare a series of working standard solutions with varying concentration gradients. These solutions include blank and positive control tubes. The blank control tube contains 1000 μg / mL amoxicillin solution dissolved in deionized water and does not contain 1% sodium hexametaphosphate. The positive control tube contains 1% sodium hexametaphosphate solution dissolved in deionized water and does not contain amoxicillin.

[0028] The visual linear regression equation was established based on the absorbance values ​​of amoxicillin standard solutions with a series of concentration gradients. Using a UV-Vis spectrophotometer, the instrument zero point was calibrated at a wavelength of 550 nm with a blank control tube. The absorbance values ​​of each standard tube and sample tube were measured sequentially. The linear regression equation was plotted with the absorbance value of the standard tube as the ordinate and the corresponding amoxicillin concentration as the abscissa.

[0029] S3. Sample determination and solubility calculation: (1) Sample color development: Dilute the amoxicillin saturated solution prepared in S1, add an equal amount of 0.1% copper sulfate solution, mix and let stand to obtain the test solution; (2) Turbidity analysis: Compare the test solution with the visual linear regression equation established in S2 to determine the concentration of the closest standard tube corresponding to its turbidity; (3) Calculate solubility: Calculate the solubility of the amoxicillin sample based on the concentration value determined by comparison and the dilution factor. Example 4

[0030] S1. Sample solution and reagent preparation: (1) Prepare 1% sodium hydroxide solution: accurately weigh 1% sodium hydroxide powder, dissolve and dilute to volume to prepare 1% sodium hydroxide solution; (2) Prepare amoxicillin saturated solution: take an excess of the amoxicillin sample powder to be tested, add the accurate volume of solution to dissolve, and shake at 25°C for 24 hours to ensure that the dissolution equilibrium is reached and a saturated solution is formed.

[0031] 1% sodium hydroxide powder needs to be dissolved in sufficient deionized water at room temperature (25±5℃) until the solution is clear and transparent, ensuring no undissolved powder remains. Amoxicillin samples are dissolved in 1% sodium hydroxide solution at an ambient temperature ≤25℃ and relative humidity <40% using continuous and gentle magnetic stirring until the powder in the sample no longer decreases. The clear upper layer is then removed to obtain a clear and transparent saturated amoxicillin solution.

[0032] S2. Establishment of linear regression equation: (1) Preparation of amoxicillin standard stock solution: accurately weigh amoxicillin reference standard, dissolve and dilute to prepare amoxicillin standard stock solution with a concentration of 1000 μg / mL; (2) Preparation of a series of standard tubes: accurately measure different volumes of amoxicillin standard stock solution, dilute with solution to obtain a series of standard solutions with concentration gradients; (3) Color development and establishment of curve: add 1.0 mL of 1M NaOH solution to each tube in sequence, mix well, place at room temperature for 10-15 minutes to ensure complete hydrolysis, add 1.0 mL of 1M HCl solution to neutralize, then add an accurate volume of 0.005M iodine solution, mix well, stand in the dark for 10 minutes, finally add 1.0 mL of 1% starch indicator to each tube, dilute with water to the same volume, mix well; at a wavelength of 580 nm, with water as a reference, measure absorbance, at which time the absorbance value is negatively correlated with concentration.

[0033] The preparation method for amoxicillin standard stock solution is the same as that for amoxicillin saturated solution. In addition to blank and positive control tubes, nine concentration gradients of amoxicillin standard solutions are set up, with target concentrations of 100, 200, 300, 400, 500, 600, 700, 800, and 900 μg / mL. Amoxicillin standard stock solution with a concentration of 1000 μg / mL is accurately measured and diluted stepwise using 1% sodium hydroxide solution to prepare a series of working standard solutions with varying concentration gradients. The series of amoxicillin standard solutions includes blank and positive control tubes. The blank control tube contains a 1000 μg / mL amoxicillin solution dissolved in deionized water and does not contain 1% sodium hydroxide. The positive control tube contains a 1% sodium hydroxide solution dissolved in deionized water and does not contain amoxicillin.

[0034] The visual linear regression equation was established based on the absorbance values ​​of amoxicillin standard solutions with a series of concentration gradients. Using a UV-Vis spectrophotometer, the instrument zero point was calibrated at a wavelength of 550 nm with a blank control tube. The absorbance values ​​of each standard tube and sample tube were measured sequentially. The linear regression equation was plotted with the absorbance value of the standard tube as the ordinate and the corresponding amoxicillin concentration as the abscissa.

[0035] S3. Sample determination and solubility calculation: (1) Sample color development: Dilute the amoxicillin saturated solution prepared in S1, add an equal amount of 0.1% copper sulfate solution, mix and let stand to obtain the test solution; (2) Turbidity analysis: Compare the test solution with the visual linear regression equation established in S2 to determine the concentration of the closest standard tube corresponding to its turbidity; (3) Calculate solubility: Calculate the solubility of the amoxicillin sample based on the concentration value determined by comparison and the dilution factor.

[0036] Table 2

[0037] Example 2 uses deionized water instead of potassium dihydrogen phosphate solution as a solvent, resulting in rapid and unstable precipitation, which destroys the color gradient in a short time and makes effective comparison impossible. Example 3 uses 1% sodium hexametaphosphate solution instead of potassium dihydrogen phosphate, which produces precipitation too quickly and is unstable. The operator must complete the comparison in a very short time, otherwise the results are invalid, resulting in a very low error tolerance and increasing operational pressure and the risk of misjudgment. Example 4 involves multiple necessary and time-consuming steps such as alkaline hydrolysis, acid neutralization, iodination, and starch color development, making the process cumbersome, time-consuming, and unsuitable for rapid analysis.

[0038] Table 3

[0039] The process of establishing a linear regression equation by repeating the relative standard deviation (RSD) three times involves substituting the same sample into three different linear regression equations and calculating the dispersion between the measured values. The smaller the RSD value, the better the repeatability of the method and the more stable and reliable the results. The deviation from the standard value is the deviation of the absorbance of an amoxicillin solution with known solubility from that of an amoxicillin solution with the same solubility measured in this invention.

[0040] Example 2 uses deionized water as a solvent, which cannot provide a stable reaction environment. The resulting precipitate particles are of uneven size and aggregate and settle rapidly. The settling rate and state of the precipitate may vary with each measurement, causing drastic fluctuations in absorbance readings within a short period. Example 3 uses a 1% sodium hexametaphosphate solution, which provides a relatively stable and uniform reaction environment. However, sodium hexametaphosphate itself is a strong metal ion chelating agent and will competitively complex with the colorimetric reagent copper sulfate, reducing the amount of colorimetric product generated. This results in generally low absorbance values ​​and large deviations from the standard values. Example 4 has overly complicated operation steps, involving multiple steps such as hydrolysis, neutralization, iodination, and starch color development. Small operational errors in each step will accumulate and propagate to the final result, leading to relatively large differences between parallel samples. Furthermore, iodine solution is volatile and reacts easily under light. Small changes in its concentration will directly affect the color development. These minor lapses in control will ultimately translate into errors in concentration calculations.

[0041] Secondly: In the embodiments disclosed in this invention, only the structures involved in the embodiments disclosed in this invention are involved. Other structures can refer to the general design. In the absence of conflict, the same embodiment and different embodiments of this invention can be combined with each other. In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A visual detection method for amoxicillin solubility based on quantitative color gradations, characterized in that, include: S1. Preparation of sample solution and reagents: (1) Preparation of 0.2M potassium dihydrogen phosphate solution: accurately weigh potassium dihydrogen phosphate powder, dissolve and dilute to volume to prepare 0.2M potassium dihydrogen phosphate solution; (2) Preparation of amoxicillin saturated solution: take excess amoxicillin sample powder to be tested, add the accurate volume of solution to dissolve, shake at 25°C for 24 hours to ensure that dissolution equilibrium is reached and a saturated solution is formed; S2. Establishment of linear regression equation: (1) Preparation of amoxicillin standard stock solution: accurately weigh amoxicillin reference standard, dissolve and dilute to volume to prepare amoxicillin standard stock solution with a concentration of 1000 μg / mL; (2) Preparation of a series of standard tubes: accurately measure different volumes of amoxicillin standard stock solution, dilute with solution to obtain a series of amoxicillin standard solutions with concentration gradients; (3) Color development and establishment of curve: quantitatively add 0.1% copper sulfate solution to each series of standard tubes, mix well and let stand. Each tube will then show a clear turbidity gradient from transparent clear liquid to yellow-green. This gradient is the visual linear regression equation used for quantitative comparison. S3. Sample determination and solubility calculation: (1) Sample color development: Dilute the amoxicillin saturated solution prepared in S1, add an equal amount of 0.1% copper sulfate solution, mix and let stand to obtain the test solution; (2) Turbidity analysis: Compare the test solution with the visual linear regression equation established in S2 to determine the concentration of the closest standard tube corresponding to its turbidity; (3) Calculate solubility: Calculate the solubility of the amoxicillin sample based on the concentration value determined by comparison and the dilution factor.

2. The visual detection method for amoxicillin solubility based on quantitative color gradation according to claim 1, characterized in that: The potassium dihydrogen phosphate powder must be dissolved in sufficient deionized water at room temperature (25±5℃) until the solution is clear and transparent, ensuring that there is no undissolved powder.

3. The visual detection method for amoxicillin solubility based on quantitative color gradation according to claim 2, characterized in that: The amoxicillin sample and amoxicillin reference standard were subjected to continuous and gentle magnetic stirring under ambient temperature ≤25℃ and relative humidity <40% until the powder no longer decreased. The clear upper layer of solution was then aspirated to obtain a clear and transparent amoxicillin saturated solution and amoxicillin standard stock solution, respectively.

4. The visual detection method for amoxicillin solubility based on quantitative color gradation according to claim 2, characterized in that: The concentration of the amoxicillin standard stock solution was calculated as follows: Where C is the concentration of amoxicillin standard stock solution in μg / mL, m is the mass of the reference standard weighed in μg, P is the purity of the reference standard, and V is the final volume in mL.

5. The visual detection method for amoxicillin solubility based on quantitative color gradation according to claim 1, characterized in that: The series of amoxicillin standard solutions, excluding the blank control tube and the positive control tube, includes nine concentration gradients with target concentrations of 100, 200, 300, 400, 500, 600, 700, 800, and 900 μg / mL. Amoxicillin standard stock solution with a concentration of 1000 μg / mL is accurately measured and diluted stepwise using 0.2 M potassium dihydrogen phosphate solution to prepare a series of standard working solutions with different concentration gradients.

6. The visual detection method for amoxicillin solubility based on quantitative color gradation according to claim 1, characterized in that: The series of amoxicillin standard solutions with varying concentration gradients includes blank control tubes and positive control tubes. The blank control tubes contain a 1000 μg / mL amoxicillin solution dissolved in deionized water, and the solution in the blank control tubes does not contain potassium dihydrogen phosphate. The positive control tubes contain a 0.2 M potassium dihydrogen phosphate solution dissolved in deionized water, and do not contain amoxicillin.

7. The visual detection method for amoxicillin solubility based on quantitative color gradation according to claim 6, characterized in that: The blank control tube, after adding 0.1% copper sulfate solution, produced a clear, yellowish-brown solution; the positive control tube, after adding 0.1% copper sulfate solution, produced a turbid, yellowish-brown solution with obvious precipitation.

8. The visual detection method for amoxicillin solubility based on quantitative color gradation according to claim 1, characterized in that: The visual linear regression equation was established based on the absorbance values ​​of amoxicillin standard solutions with a series of concentration gradients. Using a UV-Vis spectrophotometer, the instrument zero point was calibrated at a wavelength of 550 nm with a blank control tube. The absorbance values ​​of each standard tube and sample tube were measured sequentially. The linear regression equation was plotted with the absorbance value of the standard tube as the ordinate and the corresponding amoxicillin concentration as the abscissa.

9. The visual detection method for amoxicillin solubility based on quantitative color gradation according to claim 1, characterized in that: The linear regression equation is y = a × C + b, where y is the absorbance value, C is the amoxicillin concentration in μg / mL, a is the slope, and b is the intercept, which is obtained by fitting the absorbance values ​​of amoxicillin standard solutions with a series of concentration gradients.

10. The visual detection method for amoxicillin solubility based on quantitative color gradation according to claim 1, characterized in that: The solubility of amoxicillin sample is calculated using the formula S=(C×D) / 1000, where S is the solubility of amoxicillin sample in solvent, in mg / mL; C is the original concentration of the saturated amoxicillin solution determined by linear regression equation and calculated by linear regression equation, in μg / mL; and D is the dilution factor of the saturated amoxicillin solution before turbidimetric analysis.

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