Method for detecting content of poloxamer 188 in human erythropoietin injection

Abstract

This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for detecting the content of poloxamer 188 in human erythropoietin injection. The method provided by this invention employs high-performance liquid chromatography (HPLC). Using a tandem size exclusion column and a cation exchange column, poloxamer 188 is separated and eluted from human erythropoietin protein and other excipients in the formulation. Detection is performed using a differential refractive index detector. Simultaneously, a poloxamer 188 standard is injected, and the content of poloxamer 188 in the formulation is calculated using the external standard method. The detection method provided by this invention has the advantages of simple operation, strong applicability, high accuracy, and no need for sample pretreatment. It can rapidly detect the poloxamer content in human erythropoietin and other protein formulations, enabling better control of drug quality.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for detecting the content of poloxamer 188 in human erythropoietin injection. Background Technology

[0002] Human erythropoietin (EPO) is a glycoprotein hormone that activates and regulates red blood cell production. Due to its proven efficacy and long product lifecycle, it is widely used for anemia caused by chronic kidney disease, chemotherapy, and other factors. Early EPO preparations widely used human serum albumin as a stabilizer, but it was gradually replaced because it could trigger allergic reactions, impair albumin's normal ligand binding function, and interfere with bilirubin metabolism. Poloxamer 188 is a nonionic triblock copolymer surfactant with good physicochemical properties and stability, and it exhibits good safety and tolerability in humans, leading to its widespread use. With the continuous improvement of drug quality control standards, regulatory authorities are increasingly stringent in controlling the content of pharmaceutical excipients in drugs. Therefore, establishing appropriate analytical methods to monitor excipient content is essential.

[0003] Currently, commonly used methods for detecting poloxamer 188 content in pharmaceutical preparations can be broadly categorized into size exclusion chromatography (SUC) and reversed-phase chromatography (RPC). These methods separate and detect poloxamer from other substances in the preparation based on molecular size and hydrophobic binding ability, respectively. For example, the analytical detection method for poloxamers in liquid protein samples disclosed in patent document CN 101262918A, and the detection method for poloxamer 407 content disclosed in patent document CN 118937548A, are both SUC methods. The limitation of this SUC method for detecting poloxamers in protein samples lies primarily in the limitation on the molecular weight of the protein in the sample to be separated; otherwise, separation from poloxamer is impossible, resulting in limited applicability.

[0004] Patent document CN 113424057 A discloses a method for detecting the content of poloxamer 188 in a composition using a reversed-phase chromatography column combined with an electrospray ionization detector. Patent document CN 120609920 A discloses a method for detecting the content of poloxamer 188 using octadecylsilane-bonded silica gel as the packing material, formic acid aqueous solution as mobile phase A, and a mixed solution of formic acid and isopropanol or a mixed solution of acetic acid and isopropanol as mobile phase B, eluting at isocratic or gradient rates, and using an evaporative light scattering detector. Both of these methods are reversed-phase chromatography, requiring pretreatment of the sample to remove proteins before detection, making the detection steps cumbersome and resulting in low accuracy. Furthermore, the electrospray ionization (CAD) detectors used are expensive, leading to high detection costs.

[0005] Therefore, it is necessary to provide a method that is simple to operate, highly applicable, and requires no sample pretreatment, and can rapidly detect the content of poloxamer in human erythropoietin and other protein preparations. Summary of the Invention

[0006] The purpose of this invention is to provide a simple, reliable, and widely applicable method for detecting the content of poloxamer 188 in human erythropoietin (EPO) injection. The method provided by this invention employs high-performance liquid chromatography (HPLC). Using a tandem size exclusion column and a cation exchange column, poloxamer 188 is separated and eluted from human erythropoietin (EPO) and other excipients in the formulation. Detection is performed using a differential refractive index detector. Simultaneously, a poloxamer 188 standard is injected, and the content of poloxamer 188 in the formulation is calculated using the external standard method. This detection method is simple to operate, widely applicable, and highly accurate. It requires no sample pretreatment and can rapidly detect the content of poloxamer 188 in human erythropoietin (EPO) and other protein formulations, enabling better control of drug quality.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: This invention provides a method for detecting the content of poloxamer 188 in human erythropoietin injection, comprising the following steps: Step S1, Sample preparation: Dilute the human erythropoietin stock solution with buffer solution to obtain the sample solution; Step S2, Preparation of Standards: Weigh poloxamer 188 and dissolve it in ultrapure water to obtain a standard stock solution. Dilute the standard stock solution with citrate buffer to obtain a standard solution. Step S3: Inject the sample solution obtained in step S1 and the standard solution obtained in step S2 into a high-performance liquid chromatograph for poloxamer 188 content detection. The chromatographic conditions are as follows: the chromatographic column is a size exclusion chromatography and cation exchange chromatography column, mobile phase A is citrate buffer, mobile phase B is citrate-sodium chloride solution, gradient elution is performed, and the content of poloxamer 188 in the preparation is calculated by external standard method.

[0008] Furthermore, the buffer solution in step S1 is a human erythropoietin preparation buffer solution, which consists of the following components and their concentrations: Potassium dihydrogen phosphate 2.5~3.0 mg / ml, sodium chloride 4.5~5.0 mg / ml, methionine 0.5~0.8 mg / ml, mannitol 18~22 mg / ml, poloxamer 188 0.05~0.12 mg / ml, pH 5.9±0.3.

[0009] The preferred buffer solution for the human erythropoietin preparation is: potassium dihydrogen phosphate 2.7 mg / ml, sodium chloride 4.7 mg / ml, methionine 0.75 mg / ml, mannitol 20 mg / ml, poloxamer 188 0.1 mg / ml, with a pH of 5.9±0.3.

[0010] Further, in step S1, the concentration of the sample diluted is 0.08~0.12 mg / ml; preferably, the concentration is 0.1 mg / ml.

[0011] Further, in step S2, the standard stock solution is diluted with citrate buffer to 1.0 mg / ml, 0.5 mg / ml, 0.2 mg / ml, 0.1 mg / ml, 0.05 mg / ml, 0.02 mg / ml, and 0.01 mg / ml.

[0012] Furthermore, the high-performance liquid chromatograph in step S3 is a Waters 2695 model high-performance liquid chromatograph, the chromatographic column is a size exclusion column (GE Superose 6 Increase, 10*300 mm), the cation exchange column is a TOSOH SP-5PW, 7.5*75 mm, the mobile phase A is citrate buffer, the mobile phase B is citrate-sodium chloride solution, and the detector is a differential refractive index detector.

[0013] Further, the citrate buffer is a 18-22 mmol / L citrate buffer with a pH of 2-3; preferably, the citrate buffer is a 20 mmol / L citrate buffer with a pH of 2.8.

[0014] Further, the citrate-sodium chloride solution is a mixture of 18-22 mmol / L citrate solution and 1-3 mol / L sodium chloride solution, with a pH of 2-3; preferably, the citrate-sodium chloride solution is a mixture of 20 mmol / L citrate solution and 2 mol / L sodium chloride solution, with a pH of 2.8.

[0015] Further, the chromatographic conditions are as follows: differential refractive index detector temperature: 28~32 ℃, column temperature: 28~32 ℃, sample cell temperature: 6~8 ℃, flow rate: 0.4~0.8 ml / min, injection volume: 100 µl, and recording time: 120~160 min. The preferred chromatographic conditions are: differential refractive index detector temperature: 30 ℃, column temperature: 30 ℃, sample cell temperature: 8 ℃, flow rate: 0.5 ml / min, injection volume: 100 µl, and recording time: 160 min.

[0016] Furthermore, the mobile phase gradient is as follows: 0~40 min: mobile phase A accounts for 100%, mobile phase B accounts for 0%, and the flow rate is maintained at 0.5 ml / min; 42~52 min: mobile phase A accounts for 25%, mobile phase B accounts for 75%, and the flow rate is maintained at 0.5 ml / min; 54~160 min: mobile phase A accounts for 100%, mobile phase B accounts for 0%, and the flow rate is maintained at 0.5 ml / min.

[0017] Given that commonly used methods for detecting poloxamer 188 in formulations have limitations, such as size exclusion chromatography (MSC) which restricts the molecular weight of proteins in the sample and has narrow applicability, while reversed-phase chromatography requires protein removal before detection, which is cumbersome and has lower accuracy, this invention proposes a method for the rapid detection of poloxamer in EPO and other protein formulations that requires no sample pretreatment, is simple to operate, applicable to a variety of proteins, has high accuracy, a wide linear range, and can rapidly detect the content of poloxamer.

[0018] The method for detecting poloxamer 188 content in human erythropoietin injection provided by this invention employs high-performance liquid chromatography (HPLC). Using a tandem size exclusion column and a cation exchange column, based on the differences in charge properties and molecular size between poloxamer 188 and other components in EPO injection, proteins and other excipients in the injection can be directly separated and eluted. Detection is then performed using a differential refractive index detector. Simultaneously, a poloxamer 188 standard is injected, and the content of poloxamer 188 in the formulation is calculated using the external standard method, facilitating better monitoring of drug quality.

[0019] In summary, compared with the prior art, the method for detecting poloxamer 188 content in human erythropoietin injection provided by the present invention has the advantages of wide applicability, simple operation, high accuracy, stability and reliability, large linear range and low detection cost. It can effectively monitor the poloxamer 188 content in human erythropoietin injection and better control the quality of the drug. Attached Figure Description

[0020] Figure 1 Comparative chromatograms of different batches of human erythropoietin injection and poloxamer 188 standard.

[0021] Figure 2 This is a comparison spectrum of specific samples.

[0022] Figure 3 This is a linear standard curve for standard solution 1.

[0023] Figure 4 This is a linear standard curve for standard solution 2.

[0024] Figure 5This is a linear standard curve for standard solution 3.

[0025] Figure 6 The chromatograms are of seven concentrations of standard solution 1. Detailed Implementation

[0026] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. This invention provides a method for detecting the content of poloxamer 188 in human erythropoietin injection. The detection method employs differential refractive index detection, and the chromatographic conditions include: a size-exclusion column and a cation exchange column; mobile phase A is citrate buffer; mobile phase B is citrate-sodium chloride solution; and gradient elution is performed. The present invention will be further described in detail below with reference to relevant experiments and specific implementation examples and the accompanying drawings.

[0027] The poloxamer 188 used in the examples was from BASF. The EPO stock solution, EPO injection solution, and formulation buffer used were prepared by Shenzhen Saibaoer Biopharmaceutical Co., Ltd. All other reagents were of analytical grade.

[0028] Example 1: A method for detecting the content of poloxamer 188 in human erythropoietin injection. Step S1, Sample preparation: Dilute the human erythropoietin (EPO) stock solution to 0.1 mg / ml with buffer to obtain the sample solution; The buffer solution is a human erythropoietin (EPO) preparation buffer, formulated from the following components: Potassium dihydrogen phosphate 2.7 mg / ml, sodium chloride 4.7 mg / ml, methionine 0.75 mg / ml, mannitol 20 mg / ml, poloxamer 188 0.1 mg / ml, pH 5.9±0.3.

[0029] Step S2, Preparation of Standards: Weigh 100 mg of poloxamer 188 and dissolve it in 10 ml of ultrapure water to obtain a 10 mg / ml standard stock solution. Dilute the standard stock solution with 20 mmol / L (mM) citrate buffer (pH=2.8) to seven concentrations: 1.0 mg / ml, 0.5 mg / ml, 0.2 mg / ml, 0.1 mg / ml, 0.05 mg / ml, 0.02 mg / ml, and 0.01 mg / ml to obtain standard solutions.

[0030] Step S3: Take 100 µl each of the sample solution obtained in step S1 and the standard solution obtained in step S2, and inject them into a high-performance liquid chromatograph for poloxamer 188 content detection. The chromatographic column is a size exclusion chromatography and cation exchange chromatography column. Mobile phase A is citrate buffer and mobile phase B is citrate-sodium chloride solution. Gradient elution is performed, and the content of poloxamer 188 in the preparation is calculated by external standard method. The high-performance liquid chromatograph is a Waters 2695; the size exclusion column (column 1) is a GE Superose 6 Increase column (10*300 mm); the cation exchange column (column 2) is a Tosoh SP-5PW column (7.5*75 mm); mobile phase A: 20 mmol / L (mM) citrate buffer (pH=2.8); mobile phase B: 20 ​​mmol / L (mM) citrate-2 mol / L (M) sodium chloride solution (pH=2.8); all mobile phases are filtered through a 0.22 μm membrane; the detector is a differential refractive index detector.

[0031] The chromatographic conditions of the high-performance liquid chromatograph were as follows: differential refractive index detector temperature: 30 ℃; column temperature: 30 ℃; sample cell temperature: 8 ℃; flow rate: 0.5 ml / min (gradient); injection volume: 100 µl; recording time: 160 min; mobile phase gradient: 0~40 min: mobile phase A 100%, mobile phase B 0%, flow rate maintained at 0.5 ml / min; 42~52 min: mobile phase A 25%, mobile phase B 75%, flow rate maintained at 0.5 ml / min; 54~160 min: mobile phase A 100%, mobile phase B 0%, flow rate maintained at 0.5 ml / min.

[0032] Example 2: Detection of poloxamer 188 content in different batches of human erythropoietin injection 1. Detection method: The poloxamer 188 content in human erythropoietin (EPO) injections of sample batches 20260101, 20260102, and 20260103 was determined using the detection method of Example 1.

[0033] 2. Test Results: The results of poloxamer 188 content detection in different batches of human erythropoietin injection are shown in Table 1 and Figure 1 As shown.

[0034] 2.1 The results of poloxamer 188 content detection in different batches of human erythropoietin injection are shown in Table 1.

[0035] Table 1. Results of Poloxamer 188 Content Detection in EPO Injection Sample batch number Poloxamer 188 content (ug / ml) 20260101 101 20260102 103 20260103 101 2.2. Spectra of poloxamer 188 content in different batches of human erythropoietin injection are shown below. Figure 1 As shown. Figure 1 The graphs show the comparison between different batches of human erythropoietin injection and poloxamer 188 standard. The vertical axis represents the signal value in MV, and the horizontal axis represents the retention time in minutes.

[0036] From Table 1 and Figure 1 It can be seen that the poloxamer 188 content of the three batches of EPO injection solutions were 101, 103, and 101 ug / ml, respectively, which are very close to the theoretical value (0.1 mg, i.e., 100 ug / ml). This indicates that the detection method provided by the present invention can accurately detect the poloxamer 188 content in EPO injection solutions.

[0037] Example 3: Validation of the method for detecting poloxamer 188 content in human erythropoietin injection 1. Experimental Method: 1.1 Testing conditions: The content of poloxamer 188 was determined by high performance liquid chromatography; the chromatographic column was a size exclusion chromatography column and a cation exchange chromatography column, the mobile phase A was citrate buffer, and the mobile phase B was citrate-sodium chloride solution, and gradient elution was performed; The high-performance liquid chromatograph is a Waters 2695; the size exclusion column (column 1) is a GE Superose 6 Increase column (10*300 mm); the cation exchange column (column 2) is a Tosoh SP-5PW column (7.5*75 mm); mobile phase A: 20 mmol / L (mM) citrate buffer (pH=2.8); mobile phase B: 20 ​​mmol / L (mM) citrate-2 mol / L (M) sodium chloride solution (pH=2.8); all mobile phases are filtered through a 0.22 μm membrane; the detector is a differential refractive index detector.

[0038] The chromatographic conditions of the high-performance liquid chromatograph were as follows: differential refractive index detector temperature: 30 ℃; column temperature: 30 ℃; sample cell temperature: 8 ℃; flow rate: 0.5 ml / min (gradient); injection volume: 100 µl; recording time: 160 min; mobile phase gradient: 0~40 min: mobile phase A 100%, mobile phase B 0%, flow rate maintained at 0.5 ml / min; 42~52 min: mobile phase A 25%, mobile phase B 75%, flow rate maintained at 0.5 ml / min; 54~160 min: mobile phase A 100%, mobile phase B 0%, flow rate maintained at 0.5 ml / min.

[0039] 1.2 Specificity Detection: Human erythropoietin (EPO) preparation buffer, human erythropoietin (EPO) stock solution, 20 mM citrate buffer, and 0.01 mg / mL poloxamer 188 standard solution were injected into the high performance liquid chromatograph, and the samples were analyzed according to the detection conditions in 1.1.

[0040] 1.3 Linearity Detection: A 10 mg / ml poloxamer 188 standard stock solution was serially diluted to seven concentrations: 1 mg / ml, 0.5 mg / ml, 0.2 mg / ml, 0.1 mg / ml, 0.05 mg / ml, 0.02 mg / ml, and 0.01 mg / ml, resulting in seven concentrations of poloxamer 188 standard solutions. These solutions were serially diluted three times to prepare three batches of standard solutions, which were then analyzed according to the detection conditions in section 1.1. A standard curve was constructed by plotting the peak area of ​​poloxamer 188 against its concentration, and the linear correlation coefficient (R²) was calculated. 2 ).

[0041] 1.4 Accuracy Testing: This experiment selected prepared EPO injection solution and poloxamer 188 standard as samples: the poloxamer 188 content of EPO injection solution was calculated based on the standard curve; simultaneously, the peak area of ​​the standard was substituted into the standard curve to calculate the poloxamer content of the standard. The recovery rates of EPO and the standard were calculated separately to examine the accuracy of the method. The recovery rate calculation formula is shown below:

[0042] Repeat the above steps 3 times.

[0043] 1.5 Precision testing: 1.5.1 Repeatability: Personnel A took a batch of EPO injection solution and simultaneously prepared poloxamer 188 standard solutions at concentrations of 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, and 0.5 mg / mL. These solutions were then injected and analyzed according to the detection conditions in 1.1. A linear standard curve was plotted against the peak area of ​​the standard solutions against the concentration of poloxamer 188. Based on this standard curve, the poloxamer 188 content in the EPO injection solution samples was calculated. This process was repeated 6 times to prepare 6 sets of standard solutions.

[0044] 1.5.2 Intermediate precision: The operation performed by inspector B at a time different from that of inspector A is repeated by inspector A.

[0045] 1.6 Detection Limit and Quantification Limit: Based on the linear validation data in 1.3, the s / n values ​​of the main peak in the chromatograms of the 0.01 mg / ml and 0.02 mg / ml poloxamer 188 standards were calculated.

[0046] 2. Experimental Results: 2.1 The specificity detection results are shown in Table 2 and Figure 2 As shown: 2.1.1 The specificity detection results are shown in Table 2.

[0047] Table 2 Specificity Detection Results Sample Category Main peak retention time (min) EPO preparation buffer none EPO stock solution none 20 mM citrate buffer none 0.01 mg / mL Poloxamer 188 standard solution 39.656 2.1.2 Specificity detection map as follows Figure 2 As shown. Figure 2 This is a comparison spectrum of specific samples, where the vertical axis represents the signal value in MV and the horizontal axis represents the retention time in minutes.

[0048] From Table 2 and Figure 2 It was found that neither the EPO formulation buffer nor the EPO stock solution had characteristic peaks, while the 0.01 mg / mL poloxamer 188 standard solution showed a distinct characteristic peak at approximately 39.6 min. This indicates that EPO protein and other excipients in EPO formulations do not interfere with the detection, and this method has good specificity and can effectively detect the content of poloxamer 188 in EPO formulations.

[0049] 2.2 The linear detection results are shown in Table 3 and Figures 3-6 As shown: 2.2.1 The linear standard curve data of the three batches of standard solutions are shown in Table 3.

[0050] Table 3. Linear standard curve data for three batches of standard solutions.

[0051] 2.2.2 Linear standard curve of standard solution as shown in the figure Figures 3-6 As shown. Wherein: Figure 3 The graph shows the linearity of standard solution 1. Figure 4 The graph shows the linear standard curve for standard solution 2. Figure 5 This is a linear standard curve for standard solution 3. Figure 6 The chromatograms are of seven concentrations of standard solution 1, where the vertical axis represents the signal value in mV and the horizontal axis represents the retention time in minutes.

[0052] From Table 3 and Figures 3-6 It can be seen that the R-values ​​of the linear standard curves for the three batches of standard solutions are... 2 All values ​​were greater than or equal to 0.9999, and the CV of the peak area of ​​the standard solution at the same concentration was less than 5%. The linearity was good in the concentration range of 0.01 mg / ml to 1 mg / ml, and the linear range was significantly better than other existing experimental methods.

[0053] 2.3 The accuracy test results are shown in Tables 4 and 5.

[0054] Table 4. Calculation results of EPO injection recovery rate

[0055] Table 5 Calculation results of standard sample recovery rate

[0056] As shown in Tables 4 and 5, the recovery rates of EPO injection and standard solution are both between 96% and 103%, indicating that the detection method provided by this invention has good accuracy.

[0057] 2.4 The results of the precision test are shown in Table 6.

[0058] Table 6. Repeatability and intermediate precision test results

[0059] As shown in Table 6, the CV of the results of six consecutive tests by a single person and a total of 12 tests by two people are both <1%, indicating that the detection method provided by the present invention has a very small variability and good repeatability and intermediate precision.

[0060] 2.5 The detection limit and quantitation limit results are shown in Table 7.

[0061] Table 7. Signal-to-noise ratio calculation results

[0062] As shown in Table 7, when the concentration of poloxamer 188 is 0.01 mg / ml, the s / n is approximately between 5 and 8; when the concentration of poloxamer 188 is 0.02 mg / ml, the s / n is approximately between 12 and 16. Considering the injection volume is 100 µL, the detection limit of the detection method provided by this invention is approximately 0.5 μg, and the quantitation limit is approximately 1.5 μg. Considering that the amount of poloxamer added in the formulation is generally above 0.05 mg / ml, the sensitivity of the detection method provided by this invention is sufficient to meet the detection requirements.

[0063] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for detecting the content of poloxamer 188 in human erythropoietin injection, characterized in that, Includes the following steps: Step S1, Sample preparation: Dilute the human erythropoietin stock solution with buffer solution to obtain the sample solution; Step S2, Preparation of Standards: Weigh poloxamer 188 and dissolve it in ultrapure water to obtain a standard stock solution. Dilute the standard stock solution with citrate buffer to obtain a standard solution. Step S3: Inject the sample solution obtained in step S1 and the standard solution obtained in step S2 into a high-performance liquid chromatograph for poloxamer 188 content detection. The chromatographic conditions are as follows: the chromatographic column is a size exclusion chromatography and cation exchange chromatography column, the mobile phase A is citrate buffer, the mobile phase B is citrate-sodium chloride solution, gradient elution is performed, and the content of poloxamer 188 in the preparation is calculated by external standard method.

2. The method for detecting the content of poloxamer 188 in human erythropoietin injection as described in claim 1, characterized in that, The buffer solution in step S1 is a human erythropoietin preparation buffer solution, which consists of the following components and their concentrations: Potassium dihydrogen phosphate 2.5~3.0 mg / ml, sodium chloride 4.5~5.0 mg / ml, methionine 0.5~0.8 mg / ml, mannitol 18~22 mg / ml, poloxamer 188 0.05~0.12 mg / ml, pH 5.9±0.

3.

3. The method for detecting the content of poloxamer 188 in human erythropoietin injection as described in claim 1, characterized in that, In step S1, the concentration of the sample diluted is 0.08~0.12 mg / ml.

4. The method for detecting the content of poloxamer 188 in human erythropoietin injection as described in claim 1, characterized in that, The standard stock solution in step S2 is diluted to the following concentrations: 1.0 mg / ml, 0.5 mg / ml, 0.2 mg / ml, 0.1 mg / ml, 0.05 mg / ml, 0.02 mg / ml, and 0.01 mg / ml with citrate buffer.

5. The method for detecting the content of poloxamer 188 in human erythropoietin injection as described in claim 1, characterized in that, The high-performance liquid chromatograph (HPLC) used in step S3 is a Waters HPLC model 2695. The chromatographic column is a size exclusion column, model GE Superose 6 Increase, with a size of 10*300 mm, and a cation exchange column, model TOSOH SP-5PW, with a size of 7.5*75 mm. Mobile phase A is citrate buffer, mobile phase B is citrate-sodium chloride solution, and the detector is a differential refractive index detector.

6. The method for detecting the content of poloxamer 188 in human erythropoietin injection as described in claim 5, characterized in that, The citrate buffer solution is a 18-22 mmol / L citrate buffer solution with a pH of 2-3.

7. The method for detecting the content of poloxamer 188 in human erythropoietin injection as described in claim 5, characterized in that, The citrate-sodium chloride solution is a mixture of 18-22 mmol / L citrate solution and 1-3 mol / L sodium chloride solution, with a pH of 2-3.

8. The method for detecting the content of poloxamer 188 in human erythropoietin injection as described in claim 5, characterized in that, The chromatographic conditions were as follows: differential refractive index detector temperature: 28~32 ℃, column temperature: 28~32 ℃, sample cell temperature: 6~8 ℃, flow rate: 0.4~0.8 ml / min, injection volume: 100 µl, and recording time: 120~160 min.

9. The method for detecting the content of poloxamer 188 in human erythropoietin injection as described in claim 5, characterized in that, The gradient of the mobile phase is: 0–40 min: Mobile phase A 100%, mobile phase B 0%, flow rate 0.5 ml / min; 42–52 min: Mobile phase A 25%, mobile phase B 75%, flow rate 0.5 ml / min; 54–160 min: Mobile phase A 100%, mobile phase B 0%, flow rate 0.5 ml / min.

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