Hydroxybutyl chitosan sample pretreatment method and application thereof

Abstract

The invention discloses a hydroxybutyl chitosan sample pretreatment method and application thereof, and belongs to the technical field of protein detection. The sample pretreatment method comprises the following steps: dissolving a hydroxybutyl chitosan sample in water to obtain a hydroxybutyl chitosan aqueous solution; adding a salt solution into the hydroxybutyl chitosan aqueous solution, and extracting; and after the extraction is finished, centrifuging, and taking supernate as a sample solution. Based on the temperature sensitivity of the hydroxybutyl chitosan and the insolubility of the hydroxybutyl chitosan in a salt solution, a salt extraction-coomassie brilliant blue method is developed and is used for extracting and measuring the protein content in the hydroxybutyl chitosan, the interference of the hydroxybutyl chitosan on protein detection is removed, and the detection accuracy is improved. The method is reliable in accuracy, high in sensitivity, high in precision and good in reproducibility, thereby showing better application prospect and value in the technical field of protein detection.

Description

Technical Field

[0001] The invention belongs to the technical field of protein detection, and particularly relates to a hydroxybutyl chitosan sample pretreatment method and application thereof. Background Art

[0002] Residual protein may cause allergic reactions, thus affecting the safety of hydroxybutyl chitosan in many medical fields. Therefore, it is crucial to accurately determine the residual amount of protein. Common methods for detecting protein content include Kjeldahl nitrogen determination, Folin phenol method and Coomassie brilliant blue method. Kjeldahl nitrogen determination is known for its good reproducibility. However, this method requires specific instruments and equipment, the operation process is time-consuming and complicated, and the demand for samples is large. Therefore, it is not suitable for rapid analysis of large quantities of samples. In addition, since chitosan and its derivatives contain nitrogen, this method is not suitable for detection. Relatively speaking, the Folin phenol method and Coomassie brilliant blue method perform better in terms of sensitivity and ease of operation, and can achieve simultaneous determination of multiple samples. However, some studies have pointed out that in an alkaline environment, the amino groups of chitosan and its derivatives will react with the copper ions in the Folin phenol reagent to form a large amount of flocculent precipitates, which will interfere with the measurement results. In addition, hydroxybutyl chitosan easily reacts with Coomassie brilliant blue dye prepared with hydrochloric acid or phosphoric acid, resulting in an abnormal increase in absorbance, and the absorbance value does not change with changes in sample concentration. Therefore, an effective method for determining trace protein content in hydroxybutyl chitosan with reliable accuracy, high sensitivity and high precision is urgently needed. Summary of the invention

[0003] A hydroxybutyl chitosan sample pretreatment method comprises the following steps:

[0004] The hydroxybutyl chitosan sample is dissolved in water to obtain a hydroxybutyl chitosan aqueous solution; a salt solution is added to the hydroxybutyl chitosan aqueous solution for leaching; after the leaching is completed, the solution is centrifuged and the supernatant is taken as the sample solution.

[0005] In the above-mentioned hydroxybutyl chitosan sample pretreatment method, the mass concentration of the hydroxybutyl chitosan aqueous solution is selected from

[0006] 1.5-5.0%; preferably 1.5%, 2%, 3% or 4%.

[0007] In the above-mentioned hydroxybutyl chitosan sample pretreatment method, the salt is a neutral salt, which can be specifically selected from one or more of sodium chloride, potassium chloride, calcium chloride, potassium sulfate, and barium nitrate.

[0008] In the above-mentioned hydroxybutyl chitosan sample pretreatment method, the mass concentration of the salt solution is selected from 10 to 20%, preferably 10%.

[0009] In the above-mentioned hydroxybutyl chitosan sample pretreatment method, the mass ratio of the hydroxybutyl chitosan aqueous solution to the salt solution is selected from (0.5-2.5):(2.5-4.5); preferably 1:4.

[0010] In the above-mentioned hydroxybutyl chitosan sample pretreatment method, the extraction conditions are selected from: shaking and extracting at a speed of 100-300r / min in a 20-80°C water bath for 10-300min; preferably: shaking and extracting at a speed of 120r / min in a 40°C water bath for 4h.

[0011] In the above-mentioned hydroxybutyl chitosan sample pretreatment method, the centrifugal condition is selected from: 5000-10000 rpm for 2-15 min; preferably: 10000 rpm for 10 min.

[0012] The invention provides application of the hydroxybutyl chitosan sample pretreatment method in detecting the protein content in hydroxybutyl chitosan.

[0013] The present invention provides a method for detecting protein content in hydroxybutyl chitosan, comprising the following steps:

[0014] The protein content in the hydroxybutyl chitosan sample was detected by the Coomassie Brilliant Blue method using the hydroxybutyl chitosan sample solution prepared in the hydroxybutyl chitosan sample pretreatment method.

[0015] In the present invention, the Coomassie Brilliant Blue method refers to the "Fifth Method - Coomassie Brilliant Blue Method" in the Chinese Pharmacopoeia Part 4 (2020 Edition) 0731 Protein Content Determination Method.

[0016] The beneficial effects of the present invention are:

[0017] The invention develops a salt extraction-Coomassie brilliant blue method based on the temperature sensitivity of hydroxybutyl chitosan and its poor solubility in salt solution, which is used for extracting and determining the protein content in hydroxybutyl chitosan. The method removes the interference of hydroxybutyl chitosan itself on protein detection, has reliable accuracy, high sensitivity, high precision and good reproducibility, and is an effective method suitable for determining the trace protein content in hydroxybutyl chitosan, thereby showing good application prospects and value in the field of protein detection technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figures are the extraction results of different salt solutions; among them, (a) is 20% sodium chloride, (b) is 20% potassium dihydrogen phosphate, and (c) is 20% sodium bicarbonate. DETAILED DESCRIPTION

[0019] The hydroxybutyl chitosan samples used in the following embodiments of the present invention are all prepared by Qingdao Chenlan Marine Bioengineering Co., Ltd. using tissue engineering grade chitosan according to the method described in patent CN107029282A. Only chemical reagents such as sodium hydroxide, isopropanol, 1,2-butylene oxide, and hydrochloric acid are added during the preparation process, and the above chemical reagents do not contain protein.

[0020] In the present invention, the protein content determination method in the standard YY / T 1699-2020 "Chitosan for Tissue Engineering Medical Devices" has the following specific steps:

[0021] (1) Solution preparation

[0022] Coomassie Brilliant Blue G-250 test solution: Weigh 100 mg of Coomassie Brilliant Blue G-250 and dissolve it in 50 mL of 95% ethanol. Add 50 mL of concentrated hydrochloric acid and dilute to 1000 mL with water. Mix well. Filter and take the filtrate, place it in a brown bottle and store at room temperature. If there is precipitation, filter it before use.

[0023] Protein standard solution (30 μg / mL): Accurately weigh the bovine serum albumin reference substance, dilute it with water to about 300 μg / mL as a stock solution, and store it at 4°C. When used, dilute it with 1% acetic acid to about 30 μg / mL.

[0024] (2) Sample preparation

[0025] Accurately weigh about 50 mg of the test sample, dissolve and dilute with 1% acetic acid to make a 5 mg / mL solution. Accurately measure 1 mL into a sample tube for inspection.

[0026] Note: Due to the flocculation effect of high molecular weight chitosan, it will produce precipitation with Coomassie Brilliant Blue, affecting the test results. After the sample is dissolved, it should be placed at 80℃ for 4 hours or other treatment methods that can degrade chitosan but do not affect the protein results should be used for sample treatment.

[0027] (3) Measurement steps

[0028] Prepare protein standard solution according to Table 1 below:

[0029] Table 1 Concentration of protein standard solution series

[0030] Test tube number 0 1 2 3 4 5 Protein standard solution / mL 0 0.1 0.2 0.4 0.8 1.0 1% acetic acid solution / mL 1.0 0.9 0.8 0.6 0.2 0 Protein concentration / (μg / mL) 0 3 6 12 24 30

[0031] Add 5 mL of Coomassie Brilliant Blue G-250 solution to each test tube of the standard solution series and the sample test tube. Use a vortex mixer to fully mix the solution in the test tube and place it at room temperature 20℃±10℃ for 15 minutes. Use tube 0 as a control and measure the absorbance of each standard tube and sample tube at 595nm according to the UV-visible spectrophotometry method in Part IV, General Rule 0401 of the Pharmacopoeia of the People's Republic of China (2015 edition).

[0032] According to the absorbance of the standard tube and the protein concentration, draw an absorbance-concentration standard curve, and calculate the protein concentration of the sample tube according to the absorbance of the sample.

[0033] (4) Result presentation

[0034] The protein content in chitosan or chitosan salt was calculated as follows (ρ 3 , %):

[0035]

[0036] Where:

[0037] ρ 1 ——Concentration of chitosan or chitosan salt in the sample tube, μg / mL;

[0038] ρ 2 ——Protein concentration in the sample tube, μg / mL.

[0039] In the present invention, the protein content is detected by referring to the requirements of the "Fifth Method - Coomassie Brilliant Blue Method" in the 0731 Protein Content Determination Method of Part IV of the Chinese Pharmacopoeia (2020 Edition), and the specific steps are as follows:

[0040] (1) Solution preparation

[0041] Coomassie brilliant blue dye solution: weigh 0.1g of Coomassie brilliant blue, add 50mL of ethanol to dissolve, then add 100mL of phosphoric acid, dilute with water to 1000mL, and mix well. Filter and take the filtrate. Store in a brown bottle. If precipitation occurs, filter it before use.

[0042] Protein standard solution: Weigh 0.005 g (precisely weighed to 0.1 mg) of bovine serum albumin into a beaker and add sodium chloride solution to a total weight of 50 g.

[0043] (2) Measurement steps

[0044] Prepare the protein standard solution series according to Table 2 below

[0045] Table 2 Protein standard tube solution series concentration

[0046] Test tube number 0 1 2 3 4 5 6 Protein standard solution / g 0 0.02 0.05 0.1 0.15 0.2 0.3 25% sodium chloride / g 1.0 0.98 0.95 0.9 0.85 0.8 0.7 Protein concentration / (μg / g) 0 2 5 10 15 20 30

[0047] 5 mL of Coomassie Brilliant Blue G-250 solution was added to each test tube and sample test tube of the standard solution series (prepared according to the requirements of this patent), and the solution in the test tube was fully mixed with a vortex mixer. Tube No. 0 was used as a control, and the absorbance of each standard tube and sample tube was measured at 595 nm according to the UV-visible spectrophotometry method, Part IV, General Rule 0401 of the Pharmacopoeia of the People's Republic of China (2015 edition).

[0048] According to the absorbance of the standard tube and the protein concentration, draw an absorbance-concentration standard curve, and calculate the protein concentration of the sample tube according to the absorbance of the sample.

[0049] (4) Calculation of results

[0050] The protein residue in the sample was calculated as follows: 3 (%):

[0051]

[0052] Where:

[0053] ρ 1 ——Measured protein residue, μg / g;

[0054] ρ 2 ——Weighing amount of hydroxybutyl chitosan, g.

[0055] In the present invention, the recovery rate of the detection method is determined with reference to the "accuracy" requirement in the 9101 Analytical Method Validation Guidelines of the Chinese Pharmacopoeia Part IV (2020 Edition), and the specific steps are as follows:

[0056] The pharmacopoeia stipulates that accuracy refers to the degree of closeness between the result determined by the established method and the true value or reference value, and is generally expressed as a recovery rate (%). A reference substance of known purity can be used for sample addition recovery determination, that is, a certain amount of a reference substance of the component to be measured of known purity is accurately added to the test sample and measured according to the law. The recovery rate is calculated by dividing the difference between the measured value and the amount contained in the test sample by the amount of the reference substance added. Within the specified range, take the test sample of the same concentration (equivalent to a 100% concentration level) and evaluate it with the measurement results of at least 6 samples. The relationship between the content of the component to be measured in the sample and the recovery limit can be seen in Table 3.

[0057] Table 3 Relationship between the content of the components to be determined in the samples and the recovery limit

[0058]

[0059] Other materials used in the present invention, if not otherwise stated, can be obtained through commercial channels. Other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way.

[0060] Example 1

[0061] A method for detecting protein content in hydroxybutyl chitosan, the steps are as follows:

[0062] (1) Preparation of Hydroxybutyl Chitosan Aqueous Solution

[0063] 0.15 g of dry hydroxybutyl chitosan was placed in a 50 mL centrifuge tube, and purified water was added to dissolve it to prepare a hydroxybutyl chitosan aqueous solution with a mass concentration of 1.5%.

[0064] (2) Preparation of sample solution

[0065] Weigh 1g of hydroxybutyl chitosan aqueous solution into a 10mL centrifuge tube, add 4g of 10% calcium chloride solution. Cover the centrifuge tube with a sealing film, seal the tube mouth, place it in a 40℃ water bath and shake at 120r / min for 4h. Transfer it to a centrifuge, centrifuge at 10000rpm for 10min, aspirate the supernatant and accurately weigh 1g in a test tube as the sample solution. Prepare 3 sets of parallel samples in total.

[0066] (3) Detection

[0067] The protein content was tested according to the requirements of the "Fifth Method - Coomassie Brilliant Blue Method" in the Chinese Pharmacopoeia Part IV (2020 Edition) 0731 Protein Content Determination Method. After testing, the protein content in hydroxybutyl chitosan was Y1 = 0.16%, Y2 = 0.13%, and Y3 = 0.15%.

[0068] (4) Recovery test

[0069] According to the "accuracy" requirements in the 9101 Validation Guidelines for Analytical Methods of the Chinese Pharmacopoeia, Part 4 (2020 Edition), 6 samples were prepared for evaluation, and the recoveries were measured to be Y1=98.4%, Y2=101.1%, Y3=99.3%, Y4=96.5%, Y5=97.1%, and Y6=98.5%, which met the requirements, indicating that the detection results of this method are more accurate.

[0070] Example 2

[0071] A method for detecting protein content in hydroxybutyl chitosan, the steps are as follows:

[0072] (1) Preparation of Hydroxybutyl Chitosan Aqueous Solution

[0073] 0.20 g of dry hydroxybutyl chitosan was placed in a 50 mL centrifuge tube, and purified water was added to dissolve it to prepare a hydroxybutyl chitosan aqueous solution with a mass concentration of 4.0%.

[0074] (2) Preparation of sample solution

[0075] Weigh 1g of hydroxybutyl chitosan aqueous solution into a 10mL centrifuge tube, add 4g of 20% sodium chloride solution. Cover the centrifuge tube with a sealing film, and place it in a 60℃ water bath at a speed of 120r / min for 2h (the sample after extraction appears as a compact white flake, such as Figure 1 (a)). Transfer to a centrifuge and centrifuge at 10000 rpm for 10 min. Take out the supernatant and accurately weigh 1 g into a test tube as the sample solution. Prepare 3 sets of parallel samples in total.

[0076] (3) Detection

[0077] The protein content was tested according to the requirements of the "Fifth Method - Coomassie Brilliant Blue Method" in the Chinese Pharmacopoeia Part IV (2020 Edition) 0731 Protein Content Determination Method. After testing, the protein content in hydroxybutyl chitosan was Y1 = 0.10%, Y2 = 0.12%, and Y3 = 0.13%.

[0078] (4) Recovery test

[0079] According to the "accuracy" requirements in the 9101 Validation Guidelines for Analytical Methods of the Chinese Pharmacopoeia, Part 4 (2020 Edition), 6 samples were prepared for evaluation, and the recoveries were measured to be Y1=97.7%, Y2=98.8%, Y3=100.6%, Y4=99.5%, Y5=101.1%, and Y6=98.6%, which met the requirements, indicating that the detection results of this method are more accurate.

[0080] Example 3

[0081] A method for detecting protein content in hydroxybutyl chitosan, the steps are as follows:

[0082] (1) Preparation of Hydroxybutyl Chitosan Aqueous Solution

[0083] 0.15 g of dry hydroxybutyl chitosan was placed in a 50 mL centrifuge tube, and purified water was added to dissolve it to prepare a hydroxybutyl chitosan aqueous solution with a mass concentration of 3.0%.

[0084] (2) Preparation of sample solution

[0085] Weigh 1g of hydroxybutyl chitosan aqueous solution into a 10mL centrifuge tube, add 4g of 15% potassium sulfate solution. Cover the centrifuge tube with a sealing film, seal the tube mouth, place it in an 80℃ water bath and shake at 120r / min for 1h. Transfer it to a centrifuge, centrifuge at 10000rpm for 10min, aspirate the supernatant and accurately weigh 1g in a test tube as the sample solution. Prepare 3 sets of parallel samples in total.

[0086] (3) Detection

[0087] The protein content was tested according to the requirements of the "Fifth Method - Coomassie Brilliant Blue Method" in the Chinese Pharmacopoeia Part IV (2020 Edition) 0731 Protein Content Determination Method. After testing, the protein content in hydroxybutyl chitosan was Y1 = 0.18%, Y2 = 0.16%, and Y3 = 0.17%.

[0088] (4) Recovery test

[0089] According to the "accuracy" requirements in the 9101 Validation Guidelines for Analytical Methods in Part 4 of the Chinese Pharmacopoeia (2020 Edition), 6 samples were prepared for evaluation, and the recoveries were measured to be Y1=98.3%, Y2=95.2%, Y3=96.8%, Y4=99.9%, Y5=100.7%, and Y6=97.3%, which met the requirements, indicating that the detection results of this method are more accurate.

[0090] In summary, the protein content in hydroxybutyl chitosan obtained by the detection method described in the above embodiment is between 0.1 and 0.18%, and the recovery limit is between 95.2 and 101.1%, which is consistent with the relationship between the content of the component to be determined and the recovery limit in Table 3 above, indicating that the detection method of the present invention has good accuracy.

[0091] Comparative Example 1

[0092] A method for detecting protein content in hydroxybutyl chitosan, the steps are as follows:

[0093] (1) Preparation of Hydroxybutyl Chitosan Aqueous Solution

[0094] 0.15 g of dry hydroxybutyl chitosan was placed in a 50 mL centrifuge tube, and purified water was added to dissolve it to prepare a hydroxybutyl chitosan aqueous solution with a mass concentration of 1.5%.

[0095] (2) Preparation of sample solution

[0096] Take 1 mL of hydroxybutyl chitosan aqueous solution into a 10 mL volumetric flask, add water to make up to volume, and take 1 mL as the sample solution. Prepare 3 sets of parallel samples in total.

[0097] (3) Detection

[0098] The protein content was tested according to the requirements of the "Fifth Method - Coomassie Brilliant Blue Method" in the Chinese Pharmacopoeia Part IV (2020 Edition) 0731 Protein Content Determination Method. After testing, the protein content in hydroxybutyl chitosan was Y1 = 183.90%, Y2 = 185.17%, and Y3 = 186.14%. This value is obviously inconsistent with the actual situation.

[0099] Comparative Example 2

[0100] (1) Preparation of Hydroxybutyl Chitosan Aqueous Solution

[0101] 0.15 g of dry hydroxybutyl chitosan was placed in a 50 mL centrifuge tube, and purified water was added to dissolve it to prepare a hydroxybutyl chitosan aqueous solution with a mass concentration of 1.5%.

[0102] (2) Preparation of sample solution

[0103] Take 1 mL of hydroxybutyl chitosan aqueous solution into a 10 mL volumetric flask, add water to make up to volume, and take 1 mL as the sample solution. Prepare 3 sets of parallel samples in total.

[0104] (3) Detection

[0105] The test is carried out according to the protein content detection method recorded in the standard YY / T 1699-2020 "Chitosan for Tissue Engineering Medical Device Products".

[0106] After testing, the protein content in hydroxybutyl chitosan is Y1=11.27%, Y2=10.86%, and Y3=10.65%. This value is obviously inconsistent with the actual situation and is more than 80 times that of Example 1.

[0107] (4) Recovery test

[0108] According to the "accuracy" requirements in the 9101 Validation Guidelines for Analytical Methods in the Chinese Pharmacopoeia, Part 4 (2020 Edition), 6 samples were prepared for evaluation, and the recoveries were measured to be Y1=59.3%, Y2=34.9%, Y3=68.2%, Y4=43.2%, Y5=38.7%, and Y6=47.1%, which did not meet the requirements, indicating that the detection results of this method are inaccurate.

[0109] Comparative Example 3

[0110] On the basis of Example 2, a 20% potassium dihydrogen phosphate solution was used to replace a 20% sodium chloride solution to prepare a sample solution, and then the protein content in the hydroxybutyl chitosan sample was determined. In contrast, when potassium dihydrogen phosphate was used for extraction, the resulting solution was relatively turbid and contained a large amount of flocculent suspended matter, such as Figure 1 (b) as shown.

[0111] Three groups of parallel samples were prepared, and the protein content in hydroxybutyl chitosan was Y1 = 0.53%, Y2 = 0.89%, and Y3 = 0.73%. This value is also obviously inconsistent with the actual situation, which is caused by incomplete potassium dihydrogen phosphate extraction.

[0112] According to the "accuracy" requirements in the 9101 Validation Guidelines for Analytical Methods of the Chinese Pharmacopoeia, Part 4 (2020 Edition), 6 samples were prepared for evaluation, and the recoveries were measured to be Y1=74.9%, Y2=46.3%, Y3=67.2%, Y4=89.5%, Y5=77.2%, and Y6=90.0%, which did not meet the requirements, indicating that the detection results of this method are inaccurate.

[0113] Comparative Example 4

[0114] On the basis of Example 2, a 20% sodium bicarbonate solution was used to replace a 20% sodium chloride solution to prepare a sample solution, and then the protein content in the hydroxybutyl chitosan sample was determined. The sample treated with sodium bicarbonate showed a milky white gel-like mass suspended in the extract, such as Figure 1 (c) as shown.

[0115] Three groups of parallel samples were prepared, and the protein content in hydroxybutyl chitosan was Y1 = 0.42%, Y2 = 0.75%, and Y3 = 0.84%. This value is obviously inconsistent with the actual situation, which is caused by incomplete sodium bicarbonate extraction.

[0116] According to the "accuracy" requirements in the 9101 Validation Guidelines for Analytical Methods of the Chinese Pharmacopoeia, Part 4 (2020 Edition), 6 samples were prepared for evaluation, and the recoveries were measured to be Y1=81.2%, Y2=77.5%, Y3=64.2%, Y4=58.4%, Y5=85.1%, and Y6=66.8%, which did not meet the requirements, indicating that the detection results of this method are inaccurate.

[0117] Verification Example 1

[0118] According to the protein content detection method recorded in the standard YY / T 1699-2020 "Chitosan for Tissue Engineering Medical Device Products", the protein content in different batches of chitosan raw materials was tested.

[0119] The test results are shown in Table 4:

[0120] Table 4 Protein content in chitosan raw materials

[0121]

[0122] As can be seen from Table 4, the protein content in different batches of chitosan raw materials is less than 0.2%, which meets the requirements of the standard YY / T 1699-2020 "Chitosan for Tissue Engineering Medical Devices". In the process of preparing hydroxybutyl chitosan using chitosan raw materials, only chemical reagents such as sodium hydroxide, isopropanol, 1,2-butylene oxide, and hydrochloric acid were added. The above chemical reagents do not contain protein, so it can also be explained from the side that the actual protein content in the hydroxybutyl chitosan sample used in the above embodiment of the present invention should be less than 0.2%. This further proves that salt extraction has an important and beneficial effect on the accuracy of protein content detection in hydroxybutyl chitosan.

[0123] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A hydroxybutyl chitosan sample pretreatment method, characterized in that: The steps include: The hydroxybutyl chitosan sample is dissolved in water to obtain a hydroxybutyl chitosan aqueous solution; a salt solution is added to the hydroxybutyl chitosan aqueous solution for leaching; after the leaching is completed, the solution is centrifuged and the supernatant is taken as the sample solution.

2. The hydroxybutyl chitosan sample pretreatment method according to claim 1, characterized in that: The mass concentration of the hydroxybutyl chitosan aqueous solution is selected from 1.5 to 5.0%.

3. The hydroxybutyl chitosan sample pretreatment method according to claim 1, characterized in that: The salt is a neutral salt.

4. The hydroxybutyl chitosan sample pretreatment method according to claim 3, characterized in that: The neutral salt is selected from one or more of sodium chloride, potassium chloride, calcium chloride, potassium sulfate and barium nitrate.

5. The hydroxybutyl chitosan sample pretreatment method according to claim 1, characterized in that: The mass concentration of the salt solution is selected from 10 to 20%.

6. The hydroxybutyl chitosan sample pretreatment method according to claim 1, characterized in that: The mass ratio of the hydroxybutyl chitosan aqueous solution to the salt solution is selected from (0.5-2.5): (2.5-4.5).

7. The hydroxybutyl chitosan sample pretreatment method according to claim 1, characterized in that: The extraction conditions are selected from: shaking and extracting at a speed of 100 to 300 r / min for 10 to 300 min in a water bath at 20 to 80° C.

8. The hydroxybutyl chitosan sample pretreatment method according to claim 1, characterized in that: The centrifugal condition is selected from: 5000-10000 rpm for 2-15 min.

9. Use of the hydroxybutyl chitosan sample pretreatment method according to any one of claims 1 to 8 in the detection of protein content in hydroxybutyl chitosan.

10. A method for detecting protein content in hydroxybutyl chitosan, characterized in that: The steps include: The protein content in the hydroxybutyl chitosan sample is detected by the Coomassie Brilliant Blue method using the hydroxybutyl chitosan sample solution prepared in the hydroxybutyl chitosan sample pretreatment method according to any one of claims 1 to 8.

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