Method for detecting residual protein in fermentation antibiotics by using SEC-high-sensitivity ultraviolet detector

Abstract

The invention belongs to the technical field of antibiotics, and particularly belongs to a method for detecting residual protein in fermentation antibiotics by an SEC-high-sensitivity ultraviolet detector. Comprising the following steps: preparing a detection solution, and detecting by using a chromatographic instrument under the condition of chromatography-mass spectrometry, wherein the conditions of chromatography-mass spectrometry are as follows: a chromatographic column is an SEC chromatographic column, 4.6 mm * 300 mm, 4 m; a mobile phase is a solution which is prepared from 20% acetonitrile and 100mM monopotassium phosphate and has a pH value of 6.8; the flow velocity is 0.3 mL min <-1 >; the column temperature is 35 DEG C; the sample size is 20L; carrying out isocratic elution for 40 minutes; and the detection wavelength of an ultraviolet detector is 280 nm. The method disclosed by the invention is accurate, reliable and high in sensitivity, and can be used for determining the residual protein in the fermentation antibiotics.

Description

Technical Field

[0001] The invention belongs to the technical field of antibiotics, and in particular to a method for detecting residual protein in fermentation antibiotics by using a SEC-high-sensitivity ultraviolet detector. Background Art

[0002] Antibiotics obtained by fermentation process are produced by microorganisms (including bacteria, fungi, actinomycetes, etc.) under certain conditions during the culture process. They are chemical substances that can interfere with the growth and reproduction of other microorganisms, such as vancomycin, teicoplanin, linezolid, daptomycin and other antibiotics. Although technologies such as fermentation process and purification preparation process are becoming more and more advanced and improved, macromolecules such as proteins may still remain in the final product during the fermentation process and cause adverse reactions in the human body. Therefore, it is very important to control the residual protein of fermentation antibiotics. Residual trace amounts of protein may also cause adverse reactions such as sensitization, so a highly sensitive method is currently needed to meet the detection of trace amounts of protein.

[0003] The currently reported methods include collecting proteins by gel filtration chromatography, enriching them, and then detecting them by Bradford method. This method has good versatility, but the workload of collecting proteins by gel filtration chromatography is huge and cannot be implemented in every laboratory. There are also reports of using ultrafiltration pre-treatment and then detecting them by Bradford method, but when we used this method for detection, we found that vancomycin, teicoplanin and other varieties interfered with the Bradford method detection, resulting in false positive results. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a method for detecting residual protein in fermentation antibiotics using a SEC-high-sensitivity ultraviolet detector.

[0005] The present invention solves the above technical problems through the following technical solutions.

[0006] In the first aspect, the method for detecting residual protein in fermentation antibiotics using SEC-high-sensitivity ultraviolet detector comprises the following steps: preparing a detection solution, and performing detection using a chromatograph under chromatography-mass spectrometry conditions; The chromatography-mass spectrometry conditions are: The chromatographic column is a SEC column, 4.6 mm × 300 mm, 4 µm; The mobile phase was 20% acetonitrile and 100 mM potassium dihydrogen phosphate to a pH of 6.8; Flow rate: 0.3 mL•min -1 ; The column temperature was 35°C; The injection volume was 20 µL; Isocratic elution for 40 min; UV detector detection wavelength: 280nm.

[0007] It should be noted that, in the prior art, residual proteins and other macromolecular substances in fermented antibiotics may cause adverse reactions in the human body, threatening the safety of human medication, and there is currently no universal and sensitive detection method. The present invention establishes a universal and highly sensitive SEC-high-sensitivity ultraviolet detector for detecting residual proteins in fermented antibiotics.

[0008] In one embodiment, the chromatograph is an ultra-high performance liquid chromatograph equipped with a high-sensitivity flow cell and a diode array detector.

[0009] In a second aspect, a method for preparing a detection solution is provided: The detection solution includes at least one of a blank solution, a diluent, a reference solution, a linearity verification solution, a quantitative limit solution, a detection limit solution, a test solution, and a test spike solution.

[0010] In one embodiment, the method for preparing a blank solution or a diluent comprises the following steps: Weigh urea and potassium dihydrogen phosphate into the same solvent bottle, add pure water to dissolve, add sodium hydroxide solution to adjust the pH, mix well, and obtain a blank solution or diluent.

[0011] In one embodiment, the preparation method of the reference solution or the quantitative limit solution or the detection limit solution comprises the following steps: Mix BSA, diluent and Tween 20 solution, then put into an ultrafiltration tube, centrifuge at 10000g~14000g for 10~20min, then add diluent, centrifuge at 10000g~14000g for 10~20min, turn the ultrafiltration tube upside down, centrifuge at 10000g~14000g for 5~10min, take the liquid on the membrane, and add diluent to adjust the final volume to obtain the reference solution, quantitative limit solution or detection limit solution.

[0012] In one embodiment, the method for preparing the linearity verification solution comprises the following steps: BSA, diluent and Tween 20 solution were mixed to prepare mixed solutions with final concentrations of 5, 25, 50, 75 and 100 μg / mL, respectively. The mixed solutions of different concentrations were added to ultrafiltration tubes, centrifuged at 10000g~14000g for 10~20 min, and then the diluent was added, centrifuged at 10000g~14000g for 10~20 min. The ultrafiltration tube was turned upside down, centrifuged at 10000g~14000g for 5~10 min, the liquid on the membrane was taken, and the diluent was added to adjust the final volume to obtain linearity verification solutions of different concentrations.

[0013] In one embodiment, the method for preparing the test solution comprises the following steps: Weigh the antibiotic into a centrifuge tube, add the diluent to dissolve it, add Tween 20 solution, mix well, then put it into an ultrafiltration tube, centrifuge at 10000g~14000g for 10~20min, then add the diluent, centrifuge at 10000g~14000g for 10~20min, turn the ultrafiltration tube upside down, centrifuge at 10000g~14000g for 5~10min, take the liquid on the membrane, add the diluent to adjust the final volume to obtain the test solution.

[0014] In one embodiment, the method for preparing the test sample spiked solution comprises the following steps: Weigh the antibiotic into a centrifuge tube, add diluent to dissolve it, then add Tween 20 solution and reference solution, mix well, then put it into an ultrafiltration tube, centrifuge at 10000g~14000g for 10~20min, then add diluent, centrifuge at 10000g~14000g for 10~20min, turn the ultrafiltration tube upside down, centrifuge at 10000g~14000g for 5~10min, take the liquid on the membrane, add diluent to adjust the final volume to obtain the test sample spiked solution.

[0015] In one embodiment, the detection solution is pretreated, and the pretreatment method comprises the following steps: placing the original solution in an ultrafiltration tube, centrifuging at 10000g~14000g for 10~20min, then adding a diluent, centrifuging at 10000g~14000g for 10~20min, turning the ultrafiltration tube upside down, centrifuging at 10000g~14000g for 5~10min, taking the liquid on the membrane, and adding a diluent to adjust the final volume to obtain a detection solution.

[0016] It should be noted that, using the above technical scheme, the present invention uses a 10K ultrafiltration centrifuge tube (0.5mL, molecular weight cutoff of 10 kDa) to pre-treat different detection solutions, the centrifugal force is 10000g~14000g, the solution is purified, Tween 20 solution is added to the mobile phase to prevent protein adsorption, size exclusion chromatography (SEC) can well separate protein and fermentation antibiotics, and then detect them through a diode array detector of a high-sensitivity flow cell.

[0017] In a third aspect, the antibiotics involved in the present invention include fermentation antibiotics, and the fermentation antibiotics include vancomycin, teicoplanin, linezolid, and daptomycin.

[0018] The present invention develops a size exclusion chromatography (SEC) series high-sensitivity flow cell diode array detector method for detecting residual protein in fermentation antibiotics. The advantages of the present invention are: 1. Size exclusion chromatography can separate proteins and fermentation antibiotics, thereby eliminating the interference of fermentation antibiotics.

[0019] 2. The diode array detector with high sensitivity flow cell is a more commonly used UV detector. Its flow cell optical path is longer than 60mm and has higher sensitivity.

[0020] 3.SEC is connected in series with high-sensitivity UV, combining the advantages of both, making this method universal and highly sensitive, and can become a common method for detecting residual proteins in laboratories.

[0021] 4. The present invention has also been verified to have the following results: 1) It has good specificity; the linear relationship of the calibration curve is good within the protein concentration range of 5.0~100.0μg / mL (r=0.999); the recovery rate is>93% (n=6); the minimum detection limit of this method is 2.5μg / mL, equivalent to 5 ppm; the precision of this method is ≤2%.

[0022] The present invention has also been verified to have the following results: good specificity; good linearity with the calibration curve within the protein concentration range of 5.0~100.0μg / mL (r=0.999); recovery rate>93% (n=6); the minimum detection limit of the method is 2.5μg / mL, equivalent to 5 ppm; the precision of the method is ≤2%; the detection solution remains stable for at least 36h.

[0023] In summary, the method of the present invention is accurate, reliable, and highly sensitive, and can be used to determine residual protein in fermentation antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0025] Figure 1 This is a specific overlay diagram of Example 4 of the present invention; Figure 2 is a linear graph of Example 5 of the present invention; Figure 3 is the detection spectrum of sample 1 in Example 9 of the present invention; Figure 4 This is the detection spectrum of sample 2 in Example 9 of the present invention; Figure 5 This is the detection spectrum of sample 3 in Example 9 of the present invention. DETAILED DESCRIPTION

[0026] The following examples will be provided in conjunction with the accompanying drawings in the embodiments of the present invention. The following examples will help those of ordinary skill in the art to further understand the present invention, but are not intended to limit the present invention in any form. The experimental methods in the examples, unless otherwise specified, are based on conventional techniques in the art. All chemical reagents are analytically pure unless otherwise specified, and all reagents are commercially available conventional reagents that can be purchased or prepared according to the instructions.

[0027] The invention provides a method for detecting residual protein in fermentation antibiotics by using a SEC-high-sensitivity ultraviolet detector.

[0028] Example 1 1. Preparation and preparation of materials (1) Samples and main reagents: Fermentation antibiotics: Teicoplanin, AR grade; Bovine serum albumin: Thermo Fisher Scientific, USA; Ultrafiltration tube: 10K ultrafiltration centrifuge tube (0.5 mL, molecular weight cutoff 10 kDa); Millipore; Urea, Tween 20, sodium hydroxide, and potassium dihydrogen phosphate were purchased from Aladdin with AR purity.

[0029] (2) Instruments: The chromatograph is an ultra-high performance liquid chromatograph with a diode array detector connected in series with a high-sensitivity flow cell, and the diode array detector is equipped with a high-sensitivity flow cell.

[0030] Chromatograph: Vanquish Flex ultra-high performance liquid chromatograph, ThermoFisher, USA; Flow cell: high sensitivity flow cell, Vanquish™ flow cell, ThermoFisher, USA; Detector: Diode array detector, Vanquish™ diode array detector, ThermoFisher, USA.

[0031] The specific experimental method is given below: Example 2 Using the instrument of Example 1, the chromatography-mass spectrometry conditions were: Chromatographic conditions: The chromatographic column was a Waters SEC column (4.6 mm × 300 mm, 4 µm); The mobile phase was 20% acetonitrile-100 mM potassium dihydrogen phosphate solution (pH=6.8); specifically: the mobile phase was 20% acetonitrile and 100 mM potassium dihydrogen phosphate prepared into a solution with a pH of 6.8.

[0032] Flow rate: 0.3 mL•min -1 ; The column temperature was 35°C; The injection volume was 20 µL; Isocratic elution for 40 min; UV detector detection wavelength: 280nm.

[0033] Data processing: Chameleon software was used for data processing.

[0034] Example 3 Prepare the solution: 1. Preparation of blank solution / diluent: Weigh 48.08 g of urea and 275.9 mg of potassium dihydrogen phosphate into the same solvent bottle, add 200 mL of pure water to dissolve, add 5 M sodium hydroxide solution to adjust the pH to 6.8, mix well, and obtain a blank solution or diluent.

[0035] 2. Preparation of reference solution, quantitative limit solution and detection limit solution: (1) Reference solution: Pipette 40 µL of 250 μg / mL BSA stock solution, add 340 µL of diluent and 20 µL of 1 mg / mL Tween 20 solution, and mix well to obtain BSA solution.

[0036] Take 0.4 mL of the above solution into a 10KD ultrafiltration tube and centrifuge at 11000g for 10 min. Then add 0.2 mL of diluent and centrifuge at 11000g for 10 min. Turn the ultrafiltration tube upside down and centrifuge at 11000g for 5 min. Take the liquid on the membrane and add an appropriate amount of diluent to make the final volume about 0.2 mL to obtain the reference solution (reference concentration 50 μg / mL).

[0037] (2) Quantitation limit solution: Use a pipette to draw 4 µL of 250 μg / mL BSA stock solution, add 376 µL of diluent and 20 µL of 1 mg / mL Tween 20 solution, mix well, and obtain BSA solution.

[0038] Take 0.4 mL of the above solution into a 10KD ultrafiltration tube and centrifuge at 11000g for 10 min. Then add 0.2 mL of diluent and centrifuge at 11000g for 10 min. Turn the ultrafiltration tube upside down and centrifuge at 11000g for 5 min. Take the liquid on the membrane and add an appropriate amount of diluent to make the final volume about 0.2 mL to obtain the quantitative limit solution (quantitative limit solution concentration 5 μg / mL).

[0039] (3) Detection limit solution: Pipette 50 μL of the quantitative limit solution into a 1.5 mL centrifuge tube, add 50 μL of the diluent, mix well, and obtain the detection limit solution (detection limit solution concentration 2.5 μg / mL).

[0040] 3. Preparation of linearity verification solution: Different volumes of BSA stock solution were taken, and Tween 20 solution and diluent solution were added to prepare mixed solutions with final concentrations of 5, 25, 50, 75 and 100 μg / mL, respectively.

[0041] Then take 1 mL of the above mixed solution into a 10KD ultrafiltration tube and centrifuge at 11000g for 10 min. Then add 0.2 mL of diluent and centrifuge at 11000g for 10 min. Turn the ultrafiltration tube upside down and centrifuge at 11000g for 5 min. Take the liquid on the membrane and add an appropriate amount of diluent to make the final volume about 0.2 mL to obtain linear verification solutions of different concentrations.

[0042] 4. Preparation of accuracy verification solution and repeatability verification solution: (1) Test solution: Weigh approximately 100 mg of antibiotic into a 1.5 mL centrifuge tube, add 980 μL of diluent to dissolve, then add 20 μL of 1 mg / mL Tween 20 and mix well to obtain a solution.

[0043] Take 1 mL of the above solution into a 10KD ultrafiltration tube and centrifuge at 11000g for 10 min. Add 0.2 mL of diluent and centrifuge at 11000g for 10 min. Turn the ultrafiltration tube upside down and centrifuge at 11000g for 5 min. Take the liquid on the membrane and add an appropriate amount of diluent to make the final volume about 0.2 mL to obtain the test solution (test solution concentration 500 mg / mL).

[0044] (2) Test sample spiked solution: Weigh approximately 100 mg of antibiotic into a 1.5 mL centrifuge tube, add 940 μL of diluent to dissolve, add 20 μL of 1 mg / mL Tween 20, and then add 40 μL of 250 μg / mL reference solution, mix well to obtain a solution.

[0045] Take 200 μL of the above solution to a 10KD ultrafiltration tube and centrifuge at 11000g for 10 min. Add 0.2 mL of diluent and centrifuge at 11000g for 10 min. Turn the ultrafiltration tube upside down and centrifuge at 11000g for 5 min. Take the liquid on the membrane and add an appropriate amount of diluent to make the final volume about 0.2 mL to obtain the test sample spiked solution (the concentration of antibiotics in the test sample spiked solution is 500 mg / mL, and the concentration of the reference substance is 50 μg / mL, which is equivalent to 100 ppm, that is, the ratio of 50 μg / mL to 500 mg / mL is 100 ppm). Prepare 6 copies in parallel.

[0046] Prepare the test solution and the test spike solution to serve as the accuracy verification solution and repeatability verification solution.

[0047] Example 4 Test specificity: The specificity verification solutions are the blank solution, reference solution and test sample spiked solution prepared in Example 3, which are injected and analyzed according to the chromatographic-mass spectrometric conditions of Example 2, and the ultraviolet chromatograms of the three solutions are superimposed on the same horizontal axis, as shown in FIG. Figure 1 As shown in the figure, the blank solution has no interference at the target peak, and the other peaks in the test sample spiked solution have no obvious interference at the target peak. The protein reference substance BSA can be well separated from the antibiotics, indicating that the method has good specificity. For detailed results, see Figure 1 .

[0048] Example 5 Detection linearity and range: The linear verification solution prepared in Example 3 was taken and tested according to the chromatography-mass spectrometry conditions of Example 2. The UV peak area was used as the ordinate and the concentration was used as the abscissa to draw a standard curve and obtain a regression equation. Figure 2 ,The results showed that the linear equation was y = 0.0492x–0.0373, the correlation coefficient r was 0.999, and the total protein had good linearity in the concentration range of 5.0~100.0μg / mL.

[0049] Example 6 Detection Limit and Quantitation Limit: The detection limit solution and quantification limit solution prepared in Example 3 were taken respectively, and the detection was carried out according to the chromatography-mass spectrometry conditions of Example 2. The results showed that when the concentration of the detection limit solution was 2.5 μg / mL (5 ppm), the signal-to-noise ratio S / N = 30; when the concentration of the quantification limit solution was 5.0 μg / mL (10 ppm), the signal-to-noise ratio S / N = 72, indicating that the method has good sensitivity.

[0050] Example 7 Test accuracy and repeatability: The accuracy verification solution and repeatability verification solution prepared in Example 3 were tested according to the chromatography-mass spectrometry conditions of Example 2 and calculated according to the single-point control method. In the accuracy results, no test solution was detected; the recoveries of 6 test sample spiked solutions were 93% to 99%, and the RSD of the recoveries of 6 test sample spiked solutions was 2%.

[0051] Referring to Table 1, it should be noted that the 6 test sample spiked solutions were named Re-100%-1, Re-100%-2, Re-100%-3, Re-100%-4, Re-100%-5, ​​and Re-100%-6, respectively.

[0052] Table 1

[0053] Example 8 Test solution stability: The reference solution and the test sample spiked solution prepared in Example 3 were respectively taken and tested according to the chromatography-mass spectrometry conditions of Example 2. At 2-8°C, the ratio of the concentration value of the test sample spiked solution at each time point within 30 hours to the concentration value at 0 hours was 98%-100%, indicating that the test sample spiked solution remained stable for at least 30 hours under this condition; at 2-8°C, the ratio of the concentration value of the reference solution at each time point within 36 hours to the concentration value at 0 hours was 90%-97%, indicating that the reference solution remained stable for at least 36 hours under this condition.

[0054] Table 2

[0055] Example 9 Test samples: Take 2 portions of each of the antibiotic samples from 3 batches (sample 1, sample 2, sample 3), prepare the test solution according to the method of Example 3, and measure it according to the chromatographic-mass spectrometric conditions of Example 2. The results are shown in Table 2. Figure 3-Figure 5 No protein residue was detected in any of the three batches of antibiotic samples.

[0056] In summary, the present invention has established a method for detecting residual protein in fermentation antibiotics using SEC-high-sensitivity ultraviolet detector. The method of the present invention has good universality and high sensitivity, and can become a common method for detecting residual protein in laboratories, providing a powerful and effective technical means for detecting trace protein residues in fermentation antibiotics.

[0057] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for detecting residual protein in fermentation antibiotics using SEC-high-sensitivity ultraviolet detector, characterized in that: The steps include: preparing a detection solution, and performing detection using a chromatograph under chromatography-mass spectrometry conditions; The chromatography-mass spectrometry conditions are: The chromatographic column is a SEC column, 4.6 mm × 300 mm, 4 µm; The mobile phase was 20% acetonitrile and 100 mM potassium dihydrogen phosphate to a pH of 6.8; Flow rate: 0.3 mL•min -1 ; The column temperature was 35°C; The injection volume was 20 µL; Isocratic elution for 40 min; UV detector detection wavelength: 280nm.

2. The method for detecting residual protein in fermentation antibiotics using SEC-high-sensitivity ultraviolet detector according to claim 1, characterized in that: The detection solution includes at least one of a blank solution, a diluent, a reference solution, a linearity verification solution, a quantitative limit solution, a detection limit solution, a test solution, and a test spike solution.

3. The method for detecting residual protein in fermentation antibiotics using SEC-high-sensitivity ultraviolet detector according to claim 1, characterized in that: The chromatograph was an ultra-high performance liquid chromatograph with a diode array detector connected in series with a high-sensitivity flow cell.

4. The method for detecting residual protein in fermentation antibiotics using SEC-high-sensitivity ultraviolet detector according to claim 2, characterized in that: The method for preparing a blank solution or diluent comprises the following steps: Weigh urea and potassium dihydrogen phosphate into the same solvent bottle, add pure water to dissolve, add sodium hydroxide solution to adjust the pH, mix well, and obtain a blank solution or diluent.

5. The method for detecting residual protein in fermentation antibiotics using SEC-high-sensitivity ultraviolet detector according to claim 2, characterized in that: The preparation method of the reference solution or the quantitative limit solution or the detection limit solution comprises the following steps: Mix BSA, diluent and Tween 20 solution, then put into an ultrafiltration tube, centrifuge at 10000g~14000g for 10~20min, then add diluent, centrifuge at 10000g~14000g for 10~20min, turn the ultrafiltration tube upside down, centrifuge at 10000g~14000g for 5~10min, take the liquid on the membrane, and add diluent to adjust the final volume to obtain the reference solution, quantitative limit solution or detection limit solution.

6. The method for detecting residual protein in fermentation antibiotics using SEC-high-sensitivity ultraviolet detector according to claim 2, characterized in that: The method for preparing the linearity verification solution comprises the following steps: BSA, diluent and Tween 20 solution were mixed to prepare mixed solutions with final concentrations of 5, 25, 50, 75 and 100 μg / mL, respectively. The mixed solutions of different concentrations were added to ultrafiltration tubes, centrifuged at 10000g~14000g for 10~20 min, and then the diluent was added, centrifuged at 10000g~14000g for 10~20 min. The ultrafiltration tube was turned upside down, centrifuged at 10000g~14000g for 5~10 min, the liquid on the membrane was taken, and the diluent was added to adjust the final volume to obtain linearity verification solutions of different concentrations.

7. The method for detecting residual protein in fermentation antibiotics using SEC-high-sensitivity ultraviolet detector according to claim 2, characterized in that: The method for preparing the test solution comprises the following steps: Weigh the antibiotic into a centrifuge tube, add diluent to dissolve, add Tween 20, mix well, then put into an ultrafiltration tube, centrifuge at 10000g~14000g for 10~20min, then add diluent, centrifuge at 10000g~14000g for 10~20min, turn the ultrafiltration tube upside down, centrifuge at 10000g~14000g for 5~10min, take the liquid on the membrane, add diluent to adjust the final volume to obtain the test solution.

8. The method for detecting residual protein in fermentation antibiotics using SEC-high-sensitivity ultraviolet detector according to claim 2, characterized in that: The method for preparing the test sample spike solution comprises the following steps: Weigh the antibiotic into a centrifuge tube, add diluent to dissolve it, then add Tween 20 and reference solution, mix well, then put it into an ultrafiltration tube, centrifuge at 10000g~14000g for 10~20min, then add diluent, centrifuge at 10000g~14000g for 10~20min, turn the ultrafiltration tube upside down, centrifuge at 10000g~14000g for 5~10min, take the liquid on the membrane, add diluent to adjust the final volume to obtain the test sample spiked solution.

9. The method for detecting residual protein in fermentation antibiotics using SEC-high-sensitivity ultraviolet detector according to claim 2, characterized in that: The detection solution is pretreated, and the pretreatment method includes the following steps: placing the original solution in an ultrafiltration tube, centrifuging at 10000g~14000g for 10~20min, then adding a diluent, centrifuging at 10000g~14000g for 10~20min, turning the ultrafiltration tube upside down, centrifuging at 10000g~14000g for 5~10min, taking the liquid on the membrane, and adding a diluent to adjust the final volume to obtain a detection solution.

10. The method for detecting residual protein in fermentation antibiotics using SEC-high-sensitivity ultraviolet detector according to claim 7 or claim 8, characterized in that: Antibiotics include fermentation antibiotics, which include vancomycin, teicoplanin, linezolid, and daptomycin.

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