Detection method of sodium carboxymethyl cellulose gel pharmaceutical preparation

Abstract

The invention relates to a method for detecting the content of medicine protein in a gel medicine taking sodium carboxymethyl cellulose as a gel matrix. The method comprises the following steps: firstly, adding a 1mol / L copper sulfate solution into gel as a releasing agent, so that the gel is rapidly precipitated, and meanwhile, drug protein is completely released from the gel; secondly, protein is precipitated through a sodium deoxycholate-trichloroacetic acid method, so that the influence of methylparaben and propylparaben in gel on detection is removed, trace protein in a sample is enriched, a to-be-detected sample solution with relatively high concentration is obtained, and finally, the content of the protein is measured. The method has the advantages of simplicity, accuracy, strong repeatability, good precision and accuracy, good stability, high extraction and recovery rate, no obvious matrix effect, non-toxic, harmless and pollution-free detection reagent, low detection cost and the like, and can be used for detecting the protein content of biomacromolecular active substances in the gel preparation.

Description

Technical Field

[0001] The present invention discloses a detection method for a carboxymethylcellulose sodium gel pharmaceutical preparation, belonging to the technical field of biochemical analysis. Background Art

[0002] The carboxymethylcellulose sodium gel pharmaceutical preparation is a common preparation form in the pharmaceutical field. The preparation uses carboxymethylcellulose sodium as its gel matrix to carry the drug active ingredient. Recombinant platelet-derived growth factor (PDGF) B carboxymethylcellulose sodium gel is such a preparation, in which the active ingredient platelet-derived growth factor B is a mitogenic active protein and belongs to a member of the PDGF family. It plays an important role in physiological processes such as embryonic development, organ formation, and wound healing, and has broad application prospects. Currently, there is no reported method for detecting the protein content in the gel preparations with protein activity included in the pharmacopoeia, while the protein content is crucial for the efficacy of the preparation and is a key quality control factor.

[0003] The PDGF gel preparation contains various excipients such as preservatives, thickeners, stabilizers, and inorganic salts. When measuring the protein content of the PDGF gel by the conventional method, due to the high viscosity of the sample and the strong interference of the excipients, the protein content cannot be accurately measured, thus resulting in a lack of strict control over the quality standard of the sample. Currently, the methods for measuring protein content include the Kjeldahl method, the Folin-Ciocalteu method (Lowry method), the biuret method, the BCA method, the Coomassie brilliant blue method, and the ultraviolet-visible spectrophotometry method. The Kjeldahl method has low sensitivity, cumbersome operation, and uses controlled reagents such as strong alkalis and strong acids, presenting safety problems and environmental pollution problems. The Lowry method uses sodium deoxycholate and trichloroacetic acid to remove interfering substances, but due to the high viscosity of the product, it will affect the precipitation reaction and cannot accurately analyze the protein content. The remaining methods either have low sensitivity or have many interfering substances that are difficult to remove, and are not suitable for the determination of the protein content of gel preparations. Summary of the Invention

[0004] The purpose of the present invention is to provide an analytical method for determining the protein content of a carboxymethylcellulose sodium gel pharmaceutical preparation, so as to further improve the quality control of the carboxymethylcellulose sodium gel pharmaceutical preparation.

[0005] Based on the above invention purpose, the present invention first provides a detection method for a carboxymethylcellulose sodium gel pharmaceutical preparation, wherein the carboxymethylcellulose sodium gel pharmaceutical preparation is a gel pharmaceutical using carboxymethylcellulose sodium as its gel matrix, and the method includes the following steps: (1) Add 1 mol / L copper ion solution to the gel pharmaceutical to be detected according to the ratio of 0.3 - 0.4 ml per gram of the gel pharmaceutical, mix and precipitate, stir and mash into a flocculent shape to release the protein, and take the supernatant after centrifugation; (2) Add sodium deoxycholate test solution with a volume 0.2 times that of the supernatant and a concentration of 1.5 mg / ml to the solution obtained in step (1), and then add trichloroacetic acid solution with a volume 0.2 times that of the supernatant and a concentration of 72%. Mix well, centrifuge to obtain a precipitate, and redissolve the precipitate with alkaline copper solution to obtain a redissolved solution; (3) Mix the redissolved solution obtained in step (2) with alkaline copper solution; (4) Add Folin-Ciocalteu reagent to the solution obtained in step (3), and then determine the protein content by ultraviolet-visible spectrophotometric colorimetry. Steps (3) and (4) in the method provided by the present invention are the lowry protein content determination method in the prior art.

[0006] In a preferred technical solution, the gelling agent drug is PDGF gel.

[0007] In a more preferred technical solution, the copper ion solution in step (1) is copper sulfate solution.

[0008] More preferably, in step (1), add 1 mol / L copper ion solution to the gelling agent drug to be detected at a ratio of 1 / 3 ml per gram of the gelling agent drug.

[0009] In another preferred technical solution, the alkaline copper solution in steps (2) and (3) is 0.4% NaOH solution, 2% sodium carbonate solution, 0.0208% semi-aqueous solution of potassium sodium tartrate, and 0.0156% copper sulfate pentahydrate solution.

[0010] In a more preferred technical solution, after adding sodium deoxycholate test solution in step (2), mix well and leave it at room temperature for 10 minutes.

[0011] More preferably, in step (2), add trichloroacetic acid solution, mix well, and leave it at room temperature for at least 30 minutes.

[0012] In a preferred technical solution, the centrifugation in step (2) is carried out at a centrifugal force not less than 3500 g for 30 minutes.

[0013] In another preferred technical solution, the Folin-Ciocalteu reagent in step (4) is a Folin-Ciocalteu reagent obtained by mixing and diluting a 2N Folin-Ciocalteu reagent with ultrapure water at a ratio of 1:1. The added ratio is 1 / 12 times the volume of the solution obtained in step (3), and leave it standing for 30 minutes after adding.

[0014] In a more preferred technical solution, in step (4), the ultraviolet-visible spectrophotometric colorimetry measures the absorbance at a wavelength of 750 nm.

[0015] The detection method of the sodium carboxymethylcellulose gel pharmaceutical preparation provided by the present invention uses copper ions to precipitate sodium carboxymethylcellulose in the gel, takes the supernatant to obtain the target protein solution, then precipitates the protein by the sodium deoxycholate-trichloroacetic acid method, and measures the protein content of the obtained sample solution. Compared with the existing technical methods, the following improvements are made: (1) The pretreatment of the sample is increased, that is, the sample is treated with a copper sulfate solution to obtain the supernatant. This operation can effectively solve the problem of insufficient reaction caused by the viscosity of the sample. The sensitivity of the lowry method in the pharmacopoeia method (Pharmacopoeia of the People's Republic of China 2020 Edition, China Medical Science and Technology Press, published in May 2020, page 107) is 20 - 250 μg, but because the gel preparation contains substances that interfere with the experiment, the method of precipitating proteins needs to be used to remove impurities. High viscosity will affect the precipitation of proteins, and the sample needs to be diluted, so the sensitivity of this method is reduced to 200 - 2500 μg. By removing sodium carboxymethylcellulose in the gel, this method reduces the viscosity of the sample, so there is no need to dilute the sample, the sensitivity can reach 20 - 250 μg, and the recovery rate can reach between 90% - 108%. Compared with the existing method without sample pretreatment, the recovery rate of the method of directly centrifuging after dilution is only 44.67%, which greatly improves the recovery rate.

[0016] (2) The centrifugal precipitation method is optimized. The reaction concentration of the centrifugal precipitation reagent (1.5 mg / ml sodium deoxycholate solution and 72% trichloroacetic acid solution) is adjusted from 0.1 times the volume in the pharmacopoeia method (Pharmacopoeia of the People's Republic of China 2020 Edition, China Medical Science and Technology Press, published in May 2020, page 107) to 0.2 times the volume. After adjustment, the recovery rate is increased from 92.88% to 98.10%, increasing the precipitation rate of proteins. In addition, this step can effectively remove the interference of methylparaben and propylparaben in the sample to the method, and the absorbance of the blank matrix is reduced from 0.268 to less than 0.0083.

[0017] This method also has the advantages of short analysis cycle, simple operation process, non-toxic, harmless and pollution-free detection reagents, and low detection cost, and is an economical and efficient detection method. Description of the Drawings

[0018] Figure 1 . Standard curve of protein content; Figure 2 . Standard curve for evaluating accuracy, repeatability and intermediate precision; Figure 3 . Standard curve for linear evaluation; Figure 4 . Standard curve for evaluating intermediate precision and durability; Figure 5 . Standard curve of copper sulfate - lowry method; Figure 6 . Standard curve of dilution centrifugation concentration method. Detailed implementation manners

[0019] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to the protection scope defined by the claims of the present invention.

[0020] For the technical solutions described in the present invention, unless otherwise specified, they are all conventional solutions in the art; for the reagents or materials, unless otherwise specified, they are all from commercial channels.

[0021] 1. Test sample: PDGF gel preparation produced by our company.

[0022] 2. Test instruments, equipment and reagents Table 1. List of test instruments, equipment and reagents used in the examples

[0023] 3. Solution preparation (1) 1 mol / L copper sulfate solution: Weigh 50 g of copper sulfate pentahydrate, add water to dissolve to 200 ml, and mix well.

[0024] (2) 1.5 mg / ml sodium deoxycholate solution: Take 15 mg of sodium deoxycholate, add 10 ml of water, mix well, and prepare freshly before use.

[0025] (3) 72% trichloroacetic acid solution: Weigh 72 g of trichloroacetic acid, add water to 100 g, and mix well.

[0026] (4) Solution A (2.5% NaOH solution, 12.5% sodium carbonate solution): Weigh 5.00 g of NaOH and 25.00 g of sodium carbonate in a 250 ml beaker, measure 200 ml of water with a measuring cylinder, and stir and mix well.

[0027] (5) Solution B1 (1.04% potassium sodium tartrate semi-aqueous solution): Weigh 0.52 g of potassium sodium tartrate semi-hydrate in a 50 ml beaker, measure 50 ml of water with a measuring cylinder, and stir and mix well.

[0028] (6) Solution B2 (1.3% copper sulfate pentahydrate solution): Weigh 0.65 g of copper sulfate pentahydrate in a 50 ml beaker, measure 50 ml of water with a measuring cylinder, and stir and mix well.

[0029] (7)Use of BSA standard: First, dilute the standard to a concentration of 200 μg / ml. Use the weighing method for dilution. Place a 15 ml centrifuge tube in a beaker, zero it on the balance after putting it on the balance, accurately weigh 1.5000 g of BSA solution, and add water to a total weight of 7.5000 g. The standard gradient is set as: 0 μg / ml (as the blank control), 20 μg / ml, 40 μg / ml, 80 μg / ml, 120 μg / ml, 160 μg / ml, 200 μg / ml; first add the corresponding different volumes of the standard, and then add different volumes of water to the test tube, with a final volume of 1 ml. Duplicate wells need to be made for each sample.

[0030] Table 2. BSA standard gradient table

[0031] (8)Test sample PDGF: Weigh 3 g of test sample PDGF (the biological activities of samples with different specifications are 25000 IU / g (50 μg / g), 50000 IU / g (100 μg / g), 10000 IU / g (200 μg / g) respectively).

[0032] Example 1. Construction of PDGF gel detection method 1. Removal of gel matrix and release of protein Add 1 ml of 1 mol / L copper sulfate solution to 3 g of the test sample of PDGF gel, mix well, and use a glass rod to mash the generated precipitate into a flocculent shape to ensure complete release of the protein. Centrifuge at 2000 rpm for 1 min, and take the supernatant for standby; add 0.2 ml of 1 mol / L copper sulfate solution to the standard solution, and mix well to obtain the standard solution.

[0033] 2. Protein precipitation Add 0.2 times the volume of 1.5 mg / ml sodium deoxycholate test solution to the standard solution and the supernatant of the test sample, vortex and mix well, let it stand at room temperature for 10 minutes, then add 0.2 times the volume of 72% trichloroacetic acid solution, vortex and mix well, and let it stand at room temperature for at least 30 min. Centrifuge at 4 °C and 3500 g for 30 minutes, gently pour out the supernatant, and use filter paper to absorb the residual liquid.

[0034] 3. Determination of protein content Add Cu 2+ to the protein solution under alkaline conditions, and the peptide bond chelates with Cu 2+ in the alkaline solution to form a protein - copper complex. This complex reduces the phosphomolybdic acid in the phenol reagent to produce a blue compound. Within a certain range, the color depth is proportional to the protein concentration, and the protein content can be calculated by measuring the absorbance. The specific steps are as follows: (1) Mix Solution A, Solution B 1, Solution B 2, and water in a volume ratio of 20:2.5:1.5:1, and dilute 5 times with water in a ratio of 1:4 to prepare an alkaline copper solution (0.4% NaOH solution, 2% sodium carbonate solution, 0.0208% potassium tartrate semi-aqueous solution, 0.0156% copper sulfate pentahydrate solution. The total volume is slightly larger than the number of samples). Add 1 ml of the alkaline copper solution to the standard and test sample precipitates to re-dissolve, then add 5 ml (equivalent to 5 times the volume of the test sample solution) of the alkaline copper solution and vortex to mix. Let it stand for 10 minutes, and start timing from the time when the first sample is vortexed.

[0035] (2) Mix ultrapure water and Folin phenol reagent (2N) in a 1:1 ratio to dilute the Folin phenol reagent. Add 0.5 ml (equivalent to 1 / 12 volume of the test solution) of the diluted Folin phenol reagent to each of the standard and test tubes to which alkaline copper has been added, vortex to mix, and let stand for 30 minutes, starting from the time when the first sample is vortexed.

[0036] (3) Pour the prepared samples into the cuvette (the cuvette is made of disposable polypropylene, standard 3.5ml, light path 1cm, can be used to measure protein concentration) in order for testing. The volume of the liquid poured in slightly exceeds two-thirds of the cuvette. Open the computer software to automatically connect, use the UV-visible spectrophotometer (single wavelength), and measure the absorbance value at a wavelength of 750nm. The blank control uses a standard with a concentration of 0μg / ml. Save the test data and organize the results according to the data.

[0037] (4) Results Perform a linear regression on the corresponding absorbance of the standard protein series concentration, substitute the absorbance of the test sample into the linear regression equation, and multiply the calculated protein content by the dilution factor to obtain the protein content of the test sample.

[0038] Table 3. Standard curve parameters

[0039] The standard curve is as follows Figure 1 shown.

[0040] Table 4. 100μg / g PDGF standard batch test parameters

[0041] Table 5. 200μg / g PDGF standard batch test parameters

[0042] Table 6. 50μg / g PDGF standard batch test parameters

[0043] Example 2. Evaluation of the PDGF Gel Detection Method 1. Specificity (1) Definition Specificity refers to the ability of an analytical method to correctly determine the analyte in the presence of other components (such as impurities, degradation products, excipients, etc.).

[0044] (2) Verification method An experimenter took 1 ml of PBS buffer, added 200 μl of copper sulfate solution, weighed about 3 g of blank gel, added 1 ml of copper sulfate solution, conducted a test, and analyzed the results once.

[0045] (3) Acceptance criteria The absorbance value of the sample at 750 nm is less than 0.01, indicating that the excipient has no effect on the experimental results.

[0046] (4) Result analysis Table 7. Specificity detection parameters

[0047] The absorbance values of PBS buffer are -0.0002 and 0.0034; the absorbance values of the blank gel are 0.0065 and 0.0083, and the results are less than 0.01. It meets the acceptance criteria.

[0048] 2. Accuracy (1) Definition Accuracy refers to the degree of closeness between the result determined by the established method and the true value or reference value, generally expressed as recovery rate (%).

[0049] (2) Verification method Samples: 1) Weigh a batch of PDGF gel at 50 μg / g; 2) A batch of PDGF gel at 100 μg / g; 3) A batch of PDGF gel at 200 μg / g; 4) Spiked sample: Sample ① + 1 ml of standard protein solution with a concentration of 80 μg / ml; 5) Spiked sample: Sample ① + 1 ml of standard protein solution with a concentration of 120 μg / ml; 6) Spiked sample: Sample ① + 1 ml of standard protein solution with a concentration of 160 μg / ml; 7) Spiked sample: Sample ② + 1 ml of standard protein solution with a concentration of 80 μg / ml; 8) Spiked sample: Sample ② + 1 ml of standard protein solution with a concentration of 120 μg / ml; 9) Spiked sample: Sample ② + 1 ml of standard protein solution with a concentration of 160 μg / ml; 10) Spiked sample: Sample ③ + 1 ml of standard protein solution with a concentration of 80 μg / ml; 11) Spiked sample: Sample ③ + 1 ml of standard protein solution with a concentration of 120 μg / ml; 12) Spiked sample: Sample ③ + 1 ml of standard protein solution with a concentration of 160 μg / ml; An experimenter prepared 1 portion of the sample and measured it once for the accuracy experiment.

[0050] Recovery rate = (Measured value of spiked sample × 2 - Measured value of sample) / Concentration of added standard protein solution × 100%.

[0051] (3) Acceptable criteria For 50 μg / g and 100 μg / g PDGF gels: 85% ≤ Recovery rate ≤ 110%.

[0052] For 200 μg / g PDGF gel: 90% ≤ Recovery rate ≤ 108%.

[0053] (4) Result analysis Table 8. Statistical results table of the determination batches of 50 μg / g PDGF standard

[0054] Table 9. Statistical results table of the determination batches of 100 μg / g PDGF standard

[0055] Table 10. Statistical results table of the determination batches of 200 μg / g PDGF standard

[0056]

[0057] The recovery rates of 50 μg / g PDGF gel were 98.80%, 98.30%, and 97.90% respectively; the recovery rates of 100 μg / g PDGF gel were 102.20%, 92.90%, and 97.40% respectively; the recovery rates of 200 μg / g PDGF gel were 95.60%, 99.70%, and 99.50% respectively, meeting the acceptable criteria. The standard curve for protein determination is shown in Figure 2 .

[0058] 3. Linearity (1) Definition Linearity refers to the ability of the linear test results to be directly proportional to the concentration of the analyte in the sample within the designed range.

[0059] (2) Verification method An experimenter prepared standard solutions with concentrations of 20 μg / ml, 40 μg / ml, 80 μg / ml, 120 μg / ml, 160 μg / ml, and 200 μg / ml, namely S1, S2, S3, S4, S5, and S6. Six replicates were prepared for each and tested once. The linear correlation regression coefficient was calculated using the average absorbance value at each concentration and the corresponding standard solution concentration.

[0060] (3)Acceptance criteria The relative standard deviation RSD of the absorbance value at each concentration ≤ 10%, and the linear correlation regression coefficient r of the standard curve ≥ 0.990.

[0061] (4)Result analysis Table 11. Linear evaluation parameter table

[0062] The RSD results among parallel samples at each concentration were 1.67%, 2.47%, 0.49%, 0.76%, 0.78%, and 1.77% respectively; the linear regression coefficient R 2 = 0.9962; which meets the acceptance criteria. The curve for protein content determination is shown in Figure 3 。

[0063] 4. Precision Precision refers to the degree of closeness among the results obtained from multiple samplings and determinations of the same homogeneous test sample under the specified determination conditions.

[0064] (1)Repeatability 1) Definition The precision of the results obtained by the same analyst under the same conditions is called repeatability. 2) Verification method An experimenter took one batch of test samples of different specifications, prepared 3 samples for each batch, tested once, and conducted a repeatability experiment. Calculate the RSD among the 9 groups of data.

[0065] 3) Acceptance criteria: RSD ≤ 10%.

[0066] 4) Result analysis Table 12. Repeatability evaluation parameter table

[0067] An experimenter took one batch of test samples of different specifications, prepared 3 samples for each batch. The RSD among the 9 groups of data was 4.77%, which meets the acceptance criteria. The standard curve for protein content determination is shown in Figure 2 。

[0068] (2)Intermediate precision 1) Definition The precision between the determination results under the changed conditions in the same laboratory, such as at different times, by different analysts, using different equipment, etc., is called intermediate precision.

[0069] 2) Verification method Two experimenters each took a batch of test articles of different specifications to prepare 1 sample respectively. The two experimenters conducted the experiment at different times and measured once, which is the intermediate precision test. Calculate the RSD between the data.

[0070] 3) Acceptable standard: RSD ≤ 10%.

[0071] 4) Result analysis Table 13. Intermediate precision evaluation parameter table

[0072] Two experimenters each took a batch of test articles of different specifications at different times to prepare 1 sample respectively. The RSD between the 6 groups of data was 5.99%, meeting the acceptable standard. The standard curve for the determination of protein content is shown in Figure 2 and Figure 4 .

[0073] 5. Range (1) Definition: The range refers to the concentration or quantity interval between the upper and lower limits when the analytical method can achieve the requirements of precision, accuracy, and linearity.

[0074] (2) Acceptable standard: The concentration range that meets the requirements of accuracy, linearity, and precision in the determination is the range of this method.

[0075] (3) Result analysis: The range is 20 - 200 μg / ml.

[0076] 6. Robustness (1) Definition: Robustness refers to the degree of tolerance of the determination results to be unaffected when there are small changes in the determination conditions, providing a basis for the established method to be used in routine inspections.

[0077] (2) Verification method One experimenter took a batch of test articles of each specification and respectively conducted the operations of sucking the residual liquid at the bottom of the centrifuge tube with filter paper and without using filter paper, and measured once to calculate the RSD of the data.

[0078] (3) Acceptable standard: RSD ≤ 10%.

[0079] (4)Result analysis: Table 14. Robustness evaluation parameter table

[0080] An experimenter took three batches of test articles of each specification and performed the operations of sucking the residual liquid at the bottom of the centrifuge tube with filter paper and not sucking the residual liquid at the bottom of the centrifuge tube with filter paper respectively. The RSD of the 6 groups of data was 3.81%, meeting the acceptable standard. The standard curve for protein content determination is shown in Figure 4 .

[0081] In summary, this methodology is applicable to the protein content detection of PDGF gel.

[0082] Control Example 1. Detection results by direct Lowry method (compared with the existing technical solution, there is no sample treatment and the process of centrifugal precipitation with sodium deoxycholate-trichloroacetic acid) Currently, the methods for determining protein content include the Kjeldahl method, the Folin-Ciocalteu method (Lowry method), the biuret method, the BCA method, the Coomassie brilliant blue method, and the ultraviolet-visible spectrophotometry method. The Kjeldahl method has low sensitivity, cumbersome operation, and uses controlled reagents such as strong alkali and strong acid, resulting in safety problems and environmental pollution problems. In the Lowry method detection, due to the high viscosity of the sample, the sample cannot be accurately weighed, so it is necessary to dilute the sample or remove sodium carboxymethylcellulose. After dilution, the protein concentration decreases, and it is necessary to concentrate the protein by centrifugation for detection. In this example, the direct Lowry method was used to detect PDGF gel, and the specific method and results are as follows.

[0083] 1. Specific steps Weigh 1 g of the gel preparation, add 1.0 ml of alkaline copper test solution (mix solution A, solution B1, solution B2, and water in a volume ratio of 20:2.5:1.5:1 to obtain the alkaline copper test solution), shake well, and leave at room temperature for 10 minutes. Add 4.0 ml of Folin-Ciocalteu test solution (take the stock solution (2 mol / L acid concentration) in the Folin test solution 1→16), mix immediately, leave at room temperature for 30 minutes, and measure the absorbance at a wavelength of 650 nm according to the ultraviolet-visible spectrophotometry method (General Principles 0401); at the same time, use tube No. 0 as the blank. The standard curve for protein content determination is shown in Figure 5 .

[0084] Table 15. Results table for protein content determination by direct Lowry method

[0085]

[0086] 2. Result analysis: It can be seen from the results of the blank gel that methylparaben and propylparaben in the gel have a great interference on the experiment. After deducting the absorbance value of the blank gel and analyzing the results of the test articles, the recovery rate is only 70%, and the accuracy of the detection results is poor.

[0087] Control Example 2. Dilution and low-temperature centrifugal concentration (compared with the prior art solution, there is no sample treatment process, and the method of precipitating proteins is different from the present technical solution, which is the direct low-temperature centrifugation method). 1. Specific steps The gel was diluted several times with ultrapure water, mixed well, left to dissolve for half an hour, and centrifuged at 3900 g for 30 min at 4°C. The supernatant was discarded. Solution A, Solution B1, Solution B2, and water were mixed in a volume ratio of 20:2.5:1.5:1 to obtain an alkaline copper test solution. 1.0 ml of the alkaline copper test solution was added, shaken well, left at room temperature for 10 minutes, 4.0 ml of Folin-Ciocalteu reagent (taking the stock solution (2 mol / L acid concentration) in Folin reagent 1→16) was added to each, immediately mixed well, left at room temperature for 30 minutes, and the absorbance was measured at a wavelength of 650 nm according to the ultraviolet-visible spectrophotometry (General Principles 0401); at the same time, Tube No. 0 was used as the blank. The standard curve for protein content determination is shown in Figure 6 .

[0088] Table 16. Results of protein content determination under different dilution and centrifugation conditions

[0089]

[0090] 2. Result analysis: The gel preparation contains substances that interfere with the experiment, so it is necessary to use a method of precipitating proteins to remove impurities. For the direct low-temperature centrifugation precipitation method after sample dilution, high viscosity will affect the precipitation of proteins. After dilution, the precipitation efficiency of the low-concentration protein solution is low, and the recovery rate is low. By adjusting the dilution factor and centrifugation speed, the recovery rate is still very low, and the accuracy of this method is poor.

[0091] Control Example 3. Dilution and deoxycholate-trichloroacetic acid method for centrifugal concentration (compared with the prior art solution, there is no sample treatment process). 1. Specific steps Dilute the gel with ultrapure water by several times, mix well, let it stand for half an hour to dissolve. Take an appropriate amount of the test solution, add 0.2 times the volume of 1.5 mg / ml sodium deoxycholate test solution, vortex to mix well, let it stand at room temperature for 10 minutes, add 0.2 times the volume of 72% trichloroacetic acid solution, vortex to mix well, and let it stand at room temperature for not less than 30 min. Centrifuge at 4°C and 3500 g for 30 minutes, gently pour out the supernatant, and suck out the residual liquid with filter paper. Mix solution A, solution B1, solution B2, and water in a volume ratio of 20:2.5:1.5:1, and dilute it 5 times with water in a ratio of 1:4 to prepare an alkaline copper solution (the total volume is slightly larger than the number of samples). Add 1 ml of the alkaline copper solution to each of the standard and test sample precipitates for re-dissolution, then add 5 ml of the alkaline copper solution and vortex to mix well, and let it stand for 10 minutes. Mix water and Folin-Ciocalteu reagent in a ratio of 1:1 to dilute the Folin-Ciocalteu reagent. Add 0.5 ml of the diluted Folin-Ciocalteu reagent to each of the standard and test sample tubes that have been added with alkaline copper, vortex to mix well, let it stand for 30 minutes, and measure the absorbance value at a wavelength of 750 nm according to the ultraviolet-visible spectrophotometry (General Principles 0401). Use the standard with a concentration of 0 μg / ml as the blank control. The standard curve for protein content determination is shown in Figure 6 。

[0092] Table 17. Results Table of Protein Content Determination under Different Dilution and Sample Treatment Conditions

[0093]

[0094] 2. Result Analysis: The gel preparation contains substances that interfere with the experiment. Therefore, it is necessary to use the method of precipitating proteins to remove impurities. After the sample is diluted, it is centrifuged and concentrated by the sodium deoxycholate-trichloroacetic acid method. High viscosity will affect the precipitation of proteins, and the precipitation efficiency of the low-concentration protein solution after dilution is low, and the recovery rate is low. By adjusting the dilution factor and centrifugation speed, the recovery rate is still very low, and the accuracy of this method is poor.

[0095] Control Example 4. After treatment with copper sulfate, centrifuge and concentrate by the sodium deoxycholate-trichloroacetic acid method (compared with the existing technical solution, the reaction ratio of the centrifugal precipitation reagent is different) 1. Specific Steps Add 1 ml of 1 mol / L copper sulfate solution to the blank gel and the test sample, mix well, and use a glass rod to mash the formed precipitate into a flocculent shape to ensure complete release of the protein. Centrifuge at 2000 rpm for 1 min and take an appropriate amount of the supernatant. Add 0.2 times the volume of 1.5 mg / ml sodium deoxycholate test solution, vortex to mix well, let stand at room temperature for 10 minutes, add 0.2 times the volume of 72% trichloroacetic acid solution, vortex to mix well, and let stand at room temperature for not less than 30 min. Centrifuge at 4 °C and 3500 g for 30 minutes, gently pour out the supernatant, and absorb the residual liquid with filter paper. Mix solution A, solution B1, solution B2, and water in a volume ratio of 20:2.5:1.5:1, and dilute with water in a ratio of 1:4 by 5 times to prepare an alkaline copper solution (the total volume is slightly larger than the number of samples). Add 1 ml of the alkaline copper solution to each of the standard product and the test sample precipitate for re-dissolution, then add 5 ml of the alkaline copper solution, vortex to mix well, and let stand for 10 minutes. Mix water and Folin-Ciocalteu reagent in a ratio of 1:1 to dilute the Folin-Ciocalteu reagent. Add 0.5 ml of the diluted Folin-Ciocalteu reagent to each of the test tubes of the standard product and the test sample that have been added with alkaline copper, vortex to mix well, let stand for 30 minutes, and measure the absorbance value at a wavelength of 750 nm according to the ultraviolet-visible spectrophotometry (General Principles 0401). Use the standard product with a concentration of 0 μg / ml as the blank control. The standard curve for protein content determination is shown in Figure 6 。

[0096] Table 18. Results Table of Protein Content Determination under Different Precipitation Conditions

[0097]

[0098] Result analysis: By adjusting the reaction ratio of the precipitation reagents sodium deoxycholate and trichloroacetic acid, the recovery rate of 0.2 times the volume is 98.105% > the recovery rate of 0.3 times the volume is 97.12% > the recovery rate of 0.1 times the volume is 92.88%. Therefore, when using the reaction ratio of 0.2 times the volume of sodium deoxycholate and trichloroacetic acid, the recovery rate is higher and the accuracy is higher.

[0099] The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A method for detecting the drug protein content in a gelling agent drug with sodium carboxymethylcellulose as its gelling matrix, characterized in that, The method includes the following steps: (1) Add 1 mol / L copper ion solution to the gel medicine to be detected at a ratio of 0.3 - 0.4 ml per gram of the gel medicine, mix well to precipitate, stir and mash into flocs to release proteins, and take the supernatant after centrifugation; (2) Add a sodium deoxycholate test solution with a volume 0.2 times that of the supernatant and a concentration of 1.5 mg / ml to the solution obtained in step (1), then add a trichloroacetic acid solution with a volume 0.2 times that of the supernatant and a concentration of 72%, mix well, obtain a precipitate after centrifugation, and redissolve the precipitate with an alkaline copper solution to obtain a redissolved solution; (3) Add the alkaline copper solution to the redissolved solution obtained in step (2) and mix well; (4) Add Folin-Ciocalteu reagent solution to the solution obtained in step (3), and then use ultraviolet-visible spectrophotometric colorimetry to determine the protein content.

2. The method according to claim 1, characterized in that The gel medicine is PDGF gel.

3. The method according to claim 2, wherein The copper ion solution in step (1) is copper sulfate solution.

4. The method according to claim 3, wherein In step (1), add 1 mol / L copper ion solution to the gel medicine to be detected at a ratio of 1 / 3 ml per gram of the gel medicine.

5. The method according to claim 1, characterized in that, The alkaline copper solution in steps (2) and (3) is a solution containing 0.4% NaOH, 2% sodium carbonate, 0.0208% semi-aqueous potassium sodium tartrate solution, and 0.0156% copper sulfate pentahydrate solution.

6. The method according to claim 1, characterized in that, After adding the sodium deoxycholate test solution in step (2), mix well and leave it at room temperature for 10 minutes.

7. The method according to claim 1, characterized in that, After adding the trichloroacetic acid solution in step (2), mix well and leave it at room temperature for at least 30 minutes.

8. The method according to claim 1, wherein The centrifugation in step (2) is carried out at a condition of not less than 3500 g for 30 minutes.

9. The method according to claim 1, characterized in that, The Folin-Ciocalteu reagent solution in step (4) is a Folin-Ciocalteu reagent solution with a concentration of 2 N diluted with ultrapure water at a ratio of 1:

1. The added ratio is 1 / 12 times the volume of the solution obtained in step (3), and leave it standing for 30 minutes after adding.

10. The method according to claim 1, wherein In step (4), the ultraviolet-visible spectrophotometric colorimetry measures the absorbance at a wavelength of 750 nm.

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