CN117420224A

CN117420224A - Method for detecting human serum albumin content in human erythropoietin injection by reversed-phase high performance liquid chromatography

Info

Publication number: CN117420224A
Application number: CN202311153771.XA
Authority: CN
Inventors: 陈金利; 张卫婷; 洪庆福; 贾思凝; 李岩异; 张红霞; 赵楠; 白燕; 张彩乔; 刘艳花; 石玉璇; 刘兴坤; 刘容麟; 贾军卫; 牛雅芳; 杨玉凤; 谢欢欢; 殷冠涛; 李会强
Original assignee: Ncpc Genetech Biotechnology Development Co ltd
Current assignee: Ncpc Genetech Biotechnology Development Co ltd
Priority date: 2023-09-08
Filing date: 2023-09-08
Publication date: 2024-01-19
Anticipated expiration: 2043-09-08
Also published as: CN117420224B

Abstract

The invention provides a method for detecting human serum albumin content in human erythropoietin injection by reversed phase high performance liquid chromatography, which adopts the chromatographic conditions as follows: c4 (4.6X105 mm,5 μm) Butylsilane-bonded silica gel column, phase A: 0.05-0.1% trifluoroacetic acid/water, phase B: gradient elution is carried out by taking 0.05-0.1% trifluoroacetic acid/acetonitrile as a mobile phase, an ultraviolet detector is adopted, the detection wavelength is 210-220nm, the column temperature is 20-30 ℃, the sample injection amount is 3.0-12 mug, and the flow rate is 0.8-1.0 ml/min. According to the method, the quaternary gradient pump is used for gradient elution, 0.05-0.1% of trifluoroacetic acid is added, the proportion of a column temperature and a mobile phase is adjusted, and therefore effective separation among various substances of sodium acetyltryptophan, human serum albumin, sodium caprylate and human erythropoietin in the human erythropoietin injection is achieved, and the separation degree is more than 2.0. Proved by methodology verification, the method has strong specificity, high accuracy and high precision, and can rapidly and accurately determine the human serum albumin content in the human erythropoietin injection.

Description

Method for detecting human serum albumin content in human erythropoietin injection by reversed-phase high performance liquid chromatography

Technical Field

The invention relates to the technical field of human serum albumin determination, in particular to a method for detecting the content of human serum albumin in human erythropoietin injection by using a reversed-phase high-performance liquid chromatography.

Background

Erythropoietin (EPO) is a glycoprotein produced primarily by the kidneys and regulates the production of erythrocytes. Since erythropoietin injection can increase the specific solubility of blood flow (i.e., increase the percentage of red blood cells in blood) in patients suffering from renal anemia, EPO has become one of the primary drugs for treating renal anemia. Today, pharmaceutical manufacturers typically use biotechnology pharmaceutical methods to produce EPO.

In order to ensure the stability of finished recombinant erythropoietin (rhEPO) drugs, a number of manufacturers use Human Serum Albumin (HSA) as an adjunct to the drug. In addition, HSA can also avoid adsorption of proteins to a portion of the container. The HSA content in human erythropoietin injection is usually 2-3mg/ml, which is more than 10 times higher than the active substance. The control of the HSA content is one of the key indexes of the human erythropoietin injection, but the detection of the HSA content is interfered by the multi-component existence in the human erythropoietin injection. Therefore, there is an urgent need to design a method for detecting finished products of EPO drugs, which can sufficiently distinguish auxiliary materials from proteins.

Some studies report analytical methods of chromatography and electrophoresis. Bietlot et al utilize High Performance Capillary Electrophoresis (HPCE) for qualitative analysis of human erythropoietin, which essentially allows separation of human erythropoietin from human albumin, but requires ultrafiltration salt exchange treatment of the formulation product, which inevitably results in loss of formulation components (HP Bietlot et al, 1997). Thus, this method is only used for qualitative analysis and cannot be applied to quantitative determination. Other studies report that the qualitative or quantitative determination of human erythropoietin or human serum albumin in a pharmaceutical preparation is carried out by adopting a reversed-phase high performance liquid chromatography method, but the method has the problem that the interference of other components in the pharmaceutical preparation (such as human serum albumin auxiliary materials of acetyl tryptophan sodium, sodium octoate and the like) on the determination is not fully considered, and the problem that the method specificity is not strong, so that the deviation of the quantitative result occurs (Shaligram S.Lane et al 2012; JD Wilczy ń ska et al 2005;A.Rehwald,et al,1994;M Girard et al,1999).

At present, the method for measuring the content of human serum albumin in human erythropoietin injection is recorded in the general rule of Chinese pharmacopoeia, but the method is used for measuring the total protein content in products, namely human erythropoietin comprising human serum albumin as an auxiliary material and an active ingredient, and the component sodium acetyltryptophan also has interference on a detection result and also has the problem of weak specificity. Therefore, it is highly desirable to establish a method for accurately determining the human serum albumin content in human erythropoietin injection.

Disclosure of Invention

The invention aims to establish a method for detecting the human serum albumin content in human erythropoietin injection by using a reversed-phase high-performance liquid chromatography, which can effectively eliminate the interference of sodium acetyltryptophan, sodium octoate and the like in the human erythropoietin injection and has the advantages of good specificity, high precision and accuracy and good stability.

In order to achieve the above purpose, the invention provides a method for detecting human serum albumin in human erythropoietin injection by reversed phase high performance liquid chromatography, which adopts the following chromatographic conditions:

stationary phase of chromatographic column: using butane-based silane bonded silica gel or octyl silane bonded silica gel as filler;

mobile phase: phase A is 0.05-0.10% trifluoroacetic acid/water, and phase B is 0.05-0.10% trifluoroacetic acid/acetonitrile;

flow rate: 0.6-1.0 ml/min;

detection wavelength: 210-234 nm;

column temperature: 20-35 ℃;

sample injection amount: 1.5-24.0 mug;

the detector is an ultraviolet detector.

The reversed-phase high-performance liquid chromatography performs gradient elution by a quaternary gradient pump, and the adopted elution program is as follows:

time/minute	A％	B％
			0	80	20
5	35	45
			8	50	50
11	20	80
			14	20	80
16	80	20
			20	80	20

Preferably, the specification of the chromatographic column is 30nm in pore diameter, the particle size of the filler is 5 mu m, the diameter is 4.6mm, and the length is 150mm.

Preferably, the flow rate is 0.8-1.0 ml/min.

Preferably, the detection wavelength is 210-220nm.

Preferably, the column temperature is 20-30 ℃.

Preferably, the sample injection amount is 3.0-12.0 mug.

The method for detecting human serum albumin in the human erythropoietin injection by using the reversed-phase high-performance liquid chromatography comprises the following steps:

(1) Preparation of standard substance solution

Dissolving a national standard of human serum albumin in water to obtain a human serum albumin standard solution;

(2) Preparation of test sample solution

Taking a proper amount of human erythropoietin injection, and diluting with water by a proper factor to prepare a sample solution;

(3) Taking human serum albumin standard substance solution and sample solution, injecting into high performance liquid chromatograph, and performing high performance liquid chromatography analysis according to the chromatographic conditions.

The invention also provides a method for separating sodium acetyltryptophan, human serum albumin, sodium octoate and human erythropoietin by using the reversed-phase high performance liquid chromatography, which adopts the following chromatographic conditions:

flow rate: 0.6-1.0 ml/min;

detection wavelength: 210-234 nm;

column temperature: 20-35 ℃;

sample injection amount: 1.5-24.0 mug.

The elution procedure used was as follows:

The method can effectively separate sodium acetyltryptophan, human serum albumin, sodium caprylate and human erythropoietin in the human erythropoietin injection, and the separation degree between the substances is more than 2.0. Therefore, the method can be also applied to detecting the content of the sodium acetyltryptophan or the human erythropoietin in the human erythropoietin injection or to detecting the content of the human albumin, the sodium acetyltryptophan or the sodium caprylate in the human albumin injection.

The invention has the beneficial effects that:

the method realizes the detection of the human serum albumin content in the human erythropoietin injection by optimizing several key factors affecting the high performance liquid chromatography selectivity and the chromatography performance, such as column type, column temperature, mobile phase, detection wavelength, sample injection amount, flow rate and the like and setting a specific elution program, and can effectively separate acetyl tryptophan sodium, human serum albumin, sodium octoate and human erythropoietin in the human erythropoietin injection, wherein the separation degree between substances is more than 2.0. By adopting the aqueous solution of trifluoroacetic acid and the acetonitrile solution of trifluoroacetic acid as mobile phases and adjusting the proportion of trifluoroacetic acid, the serious tailing phenomenon is effectively inhibited. Through methodological verification, the method provided by the invention has the advantages of strong specificity, high accuracy and high precision.

Drawings

FIG. 1 is a chromatogram of sodium citrate buffer.

Fig. 2 is a chromatogram of auxiliary material sodium acetyl tryptophan.

FIG. 3 is a chromatogram of adjuvant sodium octoate.

FIG. 4 is a human erythropoietin chromatogram.

Figure 5 is a standard chromatogram of human serum albumin.

FIG. 6 is a full component sample chromatogram.

Figure 7 is a standard curve of human serum albumin standard.

FIG. 8 is a chromatogram showing the detection of human serum albumin content in human erythropoietin injection under different chromatographic column conditions.

FIG. 9 is a chromatogram showing the detection of human serum albumin in human erythropoietin injection under different trifluoroacetic acid ratio ranges.

FIG. 10 is a chromatogram showing the detection of human serum albumin content in human erythropoietin injection under different gradient ratios.

FIG. 11 is a chromatogram showing the detection of human serum albumin content in human erythropoietin injection under conditions of different flow rate ranges.

FIG. 12 is a chromatogram showing the detection of human serum albumin content in human erythropoietin injection under different temperature conditions.

FIG. 13 is a chromatogram showing the detection of human serum albumin content in human erythropoietin injection under different wavelength conditions.

FIG. 14 is a chromatogram of example 6 for detecting human serum albumin content in human erythropoietin injection and example 7 for detecting sodium acetyltryptophan content in human erythropoietin injection.

FIG. 15 is a chromatogram of example 7 for detecting human erythropoietin content in human erythropoietin injection.

FIG. 16 is a chromatogram of the detection of human serum albumin and sodium acetyltryptophan content in a human serum albumin injection in example 8.

FIG. 17 is a chromatogram of example 8 for detecting sodium octoate content in human serum albumin injection.

FIG. 18 is a chromatogram of comparative example 1 for detecting the content of human erythropoietin in human erythropoietin injection.

FIG. 19 is a chromatogram of comparative example 2 for detecting human serum albumin content in human erythropoietin injection.

FIG. 20 is a chromatogram of comparative example 3 for detecting human serum albumin content in human erythropoietin injection.

FIG. 21 is a chromatogram of comparative example 4 for detecting human serum albumin in human erythropoietin injection.

Detailed Description

EXAMPLE 1 specificity investigation

The specificity investigation refers to the ability of the adopted analysis method to accurately measure the measured object in the possible presence of other components (such as impurities, degradation products, auxiliary materials and the like).

The human erythropoietin injection has complex components and mainly contains auxiliary materials such as human serum albumin, sodium acetyltryptophan, sodium caprylate and the like. A series of specific solutions are respectively diluted by water for 8 times and then are injected into a liquid chromatograph, the sample injection volume is 25 μl, the detection is carried out by adopting the wavelength of 214nm, and the chromatogram is recorded.

The series of specific solutions are respectively: pH 6.8-20 mM sodium citrate buffer solution, 16mM sodium acetyltryptophan solution, 16mM sodium octoate solution, 1mg/ml human erythropoietin solution, 2.5mg/ml human albumin solution, and mixed solution of human erythropoietin injection and human erythropoietin stock solution; the volume ratio of the human erythropoietin injection to the human erythropoietin stock solution in the mixed solution is 2:1, the concentration of the human erythropoietin stock solution is 1mg/ml.

As shown in fig. 1-5, the sodium citrate buffer did not interfere with the human serum albumin assay (fig. 1); the retention time of the main peak of the auxiliary material sodium acetyl tryptophan is about 4 minutes (figure 2); the retention time of the main peak of the auxiliary material sodium octoate is about 8.6 minutes (figure 3); the main peak retention time of human erythropoietin was about 10.3 minutes (fig. 4); the main peak retention time of the human serum albumin standard was about 7.2 minutes (fig. 5). From the full component sample chromatogram of FIG. 6, it can be seen that the components in the full component sample are substantially identical to the main peak retention times of the components described above.

Therefore, all components in the full-component sample can achieve baseline separation in the analysis process, mutual interference does not exist, the separation degree is more than 2.0, and the standard is far greater than 1.5 specified in pharmacopoeia. The components are shown to have no interference to the measurement of the content of the human serum albumin. Therefore, the detection method also has the capability of accurately detecting the detected object under the condition that other auxiliary materials and buffer solution exist, and the specificity is verified to be qualified.

Example 2 linearity investigation

Linearity refers to the ability of a linear test result to directly scale with the concentration of the analyte in a sample within a designed range.

The peak area is used as an index, and an external standard method is adopted to measure the human serum albumin content in the human erythropoietin injection. Serial dilution of human serum albumin standard solution with water to obtain standard solution with concentration of 20 μg/ml, 30 μg/ml, 60 μg/ml, 120 μg/ml, 240 μg/ml, 480 μg/ml, 960 μg/ml, 1920 μg/ml; then 25 μl is injected respectively, the peak area of the chromatogram is recorded, 5 concentration points of 60 μg/ml, 120 μg/ml, 240 μg/ml, 480 μg/ml and 960 μg/ml are selected as the abscissa, the peak area value is taken as the ordinate, and the peak area-concentration curve is drawn. As shown in fig. 7, the linear regression equation obtained is: y=21520x+13143, r ² =0.9995, indicating that the method is well linear.

The experiment is repeated for 6 times, meanwhile, the relative recovery rate is calculated, and the sample injection amount range and the optimal range are determined.

TABLE 1 relative recovery of human serum albumin sample size (%)

As can be seen from Table 1, in the method of the present invention, the optimal sample injection amount is 3.0-12. Mu.g, and at this time, the relative recovery rate of each sample injection amount of human serum albumin is 95% or more and 105% or less.

Example 3 accuracy investigation

Accuracy refers to the degree to which the results measured by established methods are close to the true or reference values, typically expressed as recovery (%), which is typically required to be 92% -105% according to the chinese pharmacopoeia requirements.

An accuracy test was performed by using the linear range determined in example 2 as a standard curve and using a standard addition method. Diluting the human erythropoietin injection with buffer solution for 4 times, mixing with 625 mug/ml human albumin standard substance solution in equal volume to obtain a marked solution, taking the measured concentration of the sample solution diluted by 8 times by the human erythropoietin injection as the theoretical concentration value of the sample, respectively injecting 25 mul, adopting wavelength 214nm for detection, recording a chromatogram, and calculating the peak area. The measurement was repeated 6 times, and the recovery rates were calculated. Recovery (%) = (measured value of solution after labeling-theoretical concentration value of test article)/theoretical value of labeling x 100%

The average recovery was calculated to be 99.9%. The method is high in accuracy.

Table 2 accuracy test results

EXAMPLE 4 precision investigation

(1) Repeatability of

Under the same condition, the same operator takes a batch of human erythropoietin injection to dilute 8 times, 6 parts of repeatability test sample solution is prepared, 25 μl of each solution is injected, detection is carried out by adopting the wavelength of 214nm, the peak area of the chromatogram is recorded, and the human albumin content in the human erythropoietin injection and the RSD (Table 3) of the 6 parts of results are calculated. The RSD was calculated to be 0.7%.

TABLE 3 method repeatability test results

(2) Intermediate precision

And three operators respectively take the same batch of human erythropoietin injection liquid to dilute for 8 times at different times to obtain intermediate precision test sample solutions, injecting 25 μl of each solution, detecting at a wavelength of 214nm, recording a chromatogram, calculating the peak area, and calculating the human serum albumin content and RSD in the test sample. The RSD was calculated to be 0.8% (table 4).

TABLE 4 results of intermediate precision experiments

The method is subjected to precision test evaluation, the repeatability RSD value of the method is 0.7%, the intermediate precision RSD value is 0.8%, and the method is proved to have good precision in determining the human serum albumin content.

Example 5 durability verification

1. Comparison with the detection results under the chromatographic column

Taking a proper amount of human erythropoietin injection sample, diluting the sample by 8 times with water, and keeping other conditions unchanged; the detection is carried out by adopting C4, C8 and C18 chromatographic columns respectively, and the detection results are observed, as shown in figure 8, under the condition that other conditions are unchanged, when the detection is carried out by adopting the C4 chromatographic column and the C8 chromatographic column, the separation of acetyl tryptophan sodium, human serum albumin, sodium octoate and human erythropoietin is achieved, and the peak type of each component is symmetrical, and the separation degree is more than 2; when the C18 chromatographic column is adopted for detection, the human serum albumin, the sodium acetyltryptophan and the human erythropoietin also achieve baseline separation, but the human serum albumin peak trailing is serious, the judgment of the starting and stopping of the baseline and the chromatographic peak and the accuracy of peak area integration are affected, the quantitative result deviation is large, the trailing part can possibly flow out together with sodium caprylate, and the method specificity is not strong.

2. Comparison of detection results under different trifluoroacetic acid proportion ranges

Taking a proper amount of human erythropoietin injection sample, diluting the sample with water to 8 times, and keeping other conditions unchanged; the ratio of trifluoroacetic acid was set to 0.05%, 0.10% and 0.15%, respectively, and the detection results were observed. As shown in figure 9, other conditions are unchanged, when the proportion of trifluoroacetic acid is 0.05% -0.10%, the sodium acetyltryptophan, the human serum albumin, the sodium caprylate and the human erythropoietin all reach baseline separation, and the components are symmetrical in peak type, and the separation degree is more than 2; when the proportion of trifluoroacetic acid is regulated to be 0.15%, the separation degree of sodium octoate peak and human serum albumin is 1.2, which interferes with the accurate quantification of human serum albumin.

3. Comparison of detection results under different elution gradients

Taking a proper amount of human erythropoietin injection sample, diluting the sample by 8 times with water, and keeping other conditions unchanged; the samples were examined under the conditions of gradient 1 (Table 5), gradient 2 (Table 6) and gradient 3 (Table 7), and the results were observed. As shown in fig. 10, other conditions are unchanged, when the elution program is performed under the condition of gradient 1, all of sodium acetyltryptophan, human serum albumin, sodium octoate and human erythropoietin achieve baseline separation, and the peak types of the components are symmetrical, and the separation degree is more than 2; when the detection is carried out under the condition of gradient 2 or gradient 3, the separation degree of the human serum albumin and the sodium octoate is respectively 1.3 and 1.1, which interferes the accurate quantification of the human serum albumin.

TABLE 5 gradient 1

Numbering device	Time/minute	A％	B％
				1	0	80	20
2	5	35	45
				3	8	50	50
4	11	20	80
				5	14	20	80
6	16	80	20
				7	20	80	20

TABLE 6 gradient 2

Numbering device	Time/minute	A％	B％
				1	0	80	20
2	8	50	50
				3	11	40	60
4	13	0	80
				5	14	0	80
6	16	80	20
				7	20	80	20

TABLE 7 gradient 3

Numbering device	Time/minute	A％	B％
				1	0	80	20
2	11	40	60
				3	13	0	80
4	14	0	80
				5	16	80	20
6	20	80	20

4. Comparison of detection results at different flow rates

Taking a proper amount of human erythropoietin injection sample, diluting the sample by 8 times with water, and keeping other conditions unchanged; the samples were examined under conditions of 0.6 ml/min, 0.8 ml/min, 1.0ml/min, 1.2 ml/min and 1.5 ml/min, respectively, and the results were observed. As shown in FIG. 11, other conditions are unchanged, and when the flow rate fluctuates within the range of 0.6-1.0 ml/min, the detection method has certain stability and does not influence the detection of the human serum albumin content; when the flow rate is greater than 1.0ml/min, the separation degree of human serum albumin and sodium caprylate is reduced, and accurate quantification of human serum albumin is interfered.

5. Comparison of detection results at different column temperatures

Taking a proper amount of human erythropoietin injection sample, diluting the sample by 8 times with water, and keeping other conditions unchanged; the samples were examined at 15℃at 20℃at 25℃at 30℃at 35℃at 40℃at 45℃and the results were observed. As shown in figure 12, other conditions are unchanged, when the column temperature fluctuates within the range of 20-35 ℃, the detection method has certain stability, and the detection of the human serum albumin content is not affected, wherein when the column temperature fluctuates within the range of 20-30 ℃, the separation degree of each component is the best, and the stability is higher; however, when the column temperature is lower than 20 ℃ or higher than 35 ℃, the separation degree of the sodium octoate and the human serum albumin is poor, and the accurate quantification of the human serum albumin is interfered.

6. Comparison of detection results at different wavelengths

Taking a proper amount of human erythropoietin injection sample, diluting the sample by 8 times with water, and keeping other conditions unchanged; the detection was performed at wavelengths of 210nm, 214nm, 220nm, 234nm, 254nm and 280nm, respectively, and the detection results were observed. As shown in FIG. 13, other conditions were unchanged, the human serum albumin chromatographic peak response value was higher when the detection wavelength was 210-234nm, and the human serum albumin response signal value was lower when the wavelength was greater than 234 nm.

In summary, the method for detecting the content of human serum albumin is sensitive to the detection wavelength, and the wavelength variation can have a great influence on the detection result, so that the detection wavelength is preferably 210-234nm, and more preferably 210-220nm in the detection process.

Example 6

A method for measuring the human serum albumin content in human erythropoietin injection by reverse phase high performance liquid chromatography comprises the following steps:

(1) Preparation of standard substance solution

Dissolving a national standard of human serum albumin in water to obtain a human serum albumin standard solution; then, a series of human serum albumin standard solutions were prepared at 60. Mu.g/ml, 120. Mu.g/ml, 240. Mu.g/ml, 480. Mu.g/ml, 960. Mu.g/ml, respectively.

(2) Preparation of test sample solution

And weighing a proper amount of human erythropoietin injection sample, diluting the sample with water for 8 times to obtain a test solution, and preparing 6 parts of test solution in parallel.

Taking the series of concentration standard substance solutions and the sample solutions, injecting into a high performance liquid chromatograph, and performing high performance liquid chromatography analysis according to the following chromatographic conditions.

Stationary phase of chromatographic column: column (C4) (4.6X150 mm,5 μm,30 nm) with butane-based silane-bonded silica gel as filler;

mobile phase: phase a is 0.10% trifluoroacetic acid/water and phase B is 0.10% trifluoroacetic acid/acetonitrile;

flow rate: 1.0ml/min;

detection wavelength: 214nm;

column temperature: 25 ℃ +/-5 ℃;

sample injection volume: 25 μl.

Elution procedure:

(3) Analysis of results

And establishing a standard curve by taking serial concentrations of human serum albumin standard solutions as abscissa and peak areas of various serial concentration standards as ordinate. Human serum albumin standard curve equation is y=20314x+89026, and the correlation coefficient R ² = 0.9991. Substituting the peak area of the sample to be measured into each curve equation to obtain the human serum albumin content in the sample of the human erythropoietin injection to be measured (Table 8).

TABLE 8 results of human serum albumin content

The chromatogram of human serum albumin in the human erythropoietin injection is shown in figure 14.

Example 7

The method can also measure the contents of components such as sodium acetyltryptophan and human erythropoietin while measuring the content of human serum albumin in the human erythropoietin injection, and comprises the following steps:

(1) Preparation of standard substance solution

Dissolving one sodium acetyltryptophan standard in water to obtain sodium acetyltryptophan standard solution; then, a series of sodium acetyltryptophan standard solutions with concentrations of 6.25. Mu.M/L, 12.5. Mu.M/L, 25. Mu.M/L, 50. Mu.M/L and 100. Mu.M/L were prepared.

Taking a physical and chemical reference substance solution of human erythropoietin, and preparing a series of human erythropoietin standard solutions with the concentration of 50 mug/ml, 100 mug/ml, 200 mug/ml, 400 mug/ml and 600 mug/ml respectively.

(2) Preparation of test sample solution

Sodium acetyltryptophan content determination: weighing a proper amount of human erythropoietin injection sample, diluting the sample with water for 8 times to obtain a test solution, and preparing 6 parts of test solution in parallel;

determination of human erythropoietin content: 6 parts of human erythropoietin injection sample containing about 240. Mu.g/ml are prepared;

and (3) taking the standard substance solution and the sample solution with the concentration of each component series, injecting the standard substance solution and the sample solution into a high performance liquid chromatograph, and performing high performance liquid chromatography analysis according to the chromatographic conditions of the example 7.

(3) Analysis of results

And respectively taking the serial concentrations of the sodium acetyltryptophan standard solution and the human erythropoietin standard solution as the abscissa, and the peak areas of the serial concentration standards as the ordinate, so as to establish a standard curve. Sodium acetyltryptophan standard curve equation is y=29, 630.258x+4,226.292, correlation coefficient R ² =1.000; the standard curve equation of human erythropoietin is y=13, 482.853x-182,711.053, and the correlation coefficient is R ² =0.999. And substituting the peak areas of the samples to be detected into the respective curve equations to obtain the contents of the components such as the sodium acetyltryptophan (table 9), the human erythropoietin (table 10) and the like in the samples of the human erythropoietin injection to be detected.

TABLE 9 sodium acetyltryptophan content results

TABLE 10 results of human erythropoietin content

The content chromatogram of sodium acetyltryptophan in the human erythropoietin injection is shown in fig. 14, and the content chromatogram of human erythropoietin in the human erythropoietin injection is shown in fig. 15.

Example 8

The method can also measure the contents of human serum albumin, sodium acetyltryptophan, sodium caprylate and other components in the human serum albumin injection, and comprises the following steps:

(1) Preparation of standard substance solution

Dissolving a sodium octoate standard substance in water to obtain a sodium octoate standard substance solution; sodium octoate standard solutions were then prepared at a series of concentrations of 1mM/L, 2mM/L, 4mM/L, 8mM/L, 16mM/L, respectively.

(2) Preparation of test sample solution

Determination of human serum albumin content: weighing a proper amount of human serum albumin, diluting with water 640 times to obtain a test solution, and preparing 6 parts of test solution in parallel;

sodium acetyltryptophan content determination: taking a proper amount of human serum albumin, diluting with water 640 times to obtain a test solution, and preparing 6 parts of test solution in parallel;

sodium octoate content determination: taking a proper amount of human serum albumin, performing ultrafiltration treatment by using a 10k ultrafiltration tube, taking a proper amount of ultrafiltration permeate, and diluting with water for 4 times to obtain a sample solution, thereby preparing 6 parts;

and (3) taking the standard substance solution and the sample solution with the concentration of each component series, injecting the standard substance solution and the sample solution into a high performance liquid chromatograph, and performing high performance liquid chromatography analysis according to the chromatographic conditions of the example 7. FIGS. 16-17 are chromatograms of human serum albumin and sodium acetyl tryptophan (FIG. 16) and sodium octanoate (FIG. 17) content of human serum albumin injection.

(3) Analysis of results

And respectively taking serial concentrations of a human serum albumin standard solution, an acetyl tryptophan sodium standard solution, a sodium octoate standard solution and a human erythropoietin standard solution as abscissa and peak areas of the serial concentration standards as ordinate to establish a standard curve. Human serum albumin standard curve equation is y=20314x+89026, and the correlation coefficient R ² = 0.9991; sodium acetyltryptophan standard curve equation is y=29, 630.258x+4,226.292, correlation coefficient R ² =1.000; standard curve equation of sodium octoate is y=60, 277.738x-2,815.375, correlation coefficient R ² =1.000. And substituting the peak areas of the samples to be detected into the respective curve equations to obtain the contents of the components such as human serum albumin (table 11), sodium acetyl tryptophan (table 12), sodium octoate (table 13) and the like in the human serum albumin samples to be detected.

TABLE 11 results of human serum albumin content

TABLE 12 sodium acetyltryptophan content results

TABLE 13 sodium octoate content results

Comparative example 1

According to literature "Shaligram s.lane et al, development and validation of RP-HPLC and RP-UPLCmethods for quantification of erythropoietin formulated withhuman serum album, 2012;2 (2): 160-165' the reversed phase high performance liquid chromatography is used for detecting the human serum albumin content in the human erythropoietin injection, and the specific chromatographic conditions are as follows:

chromatographic column: c8 (4.6 mm ID. Times.250 mm L,5 μm,)/>

flow rate: 1.5 ml/min

Column temperature: 45 DEG C

Detection wavelength: 215nm

Mobile phase a:0.1% trifluoroacetic acid/water

Mobile phase B:0.1% trifluoroacetic acid/acetonitrile

Gradient procedure: time (minutes)/mobile phase a percentage (%):

0/65,4/65,12/50,14/50,15/40,16/65,20/65。

the method can only be used for quantitatively measuring the Erythropoietin (EPO) under the condition that the Human Serum Albumin (HSA) is used as an Erythropoietin (EPO) stabilizer, and the problem of interference of other components in human erythropoietin injection to the measurement of the content of the human albumin is not fully considered, so that the quantitative detection of the human albumin cannot be realized. As shown in FIG. 18, if the human serum albumin content in the human erythropoietin injection is detected by the method, the peak type of the human serum albumin is abnormal, a double-headed peak appears, and the requirement of pharmacopoeia is not met. And the auxiliary material sodium octoate peak does not appear in the chromatogram, and can co-flow with human serum albumin, so that the problem of interference on the measurement of the human serum albumin auxiliary material exists, and the specificity is not strong.

Comparative example 2

Human serum albumin content in human erythropoietin injections was measured by reverse phase high performance liquid chromatography as described in the literature "JD Wilczy ń ska, I Roman, E Anuszewska, the separation of EPO from other proteins in medical products formulated with different stabilizers, acta Poloniae Pharmaceutica,2005,62 (3): 177", specific chromatographic conditions are as follows:

chromatographic column: c18 (4.6 mm ID. Times.250 mm L,5 μm, wide Pore)

Flow rate: 1 ml/min

Column temperature: there is no mention of

Detection wavelength: 215nm

Mobile phase a:0.1% trifluoroacetic acid/water

Mobile phase B:0.1% trifluoroacetic acid/acetonitrile (70:30 v/v)

Gradient procedure: time (minutes)/mobile phase B percentage (%):

0/30,12/50,12/60,19/60,43/75,44/30,60/30。

the analysis of human erythropoietin injection sample is carried out by adopting the method, as shown in the chromatogram of figure 19, the bifurcation of human serum albumin peak in the chromatogram is visible, and the method can not be used for quantitative analysis.

Comparative example 3

Human serum albumin content in human erythropoietin injections was measured by reverse phase high performance liquid chromatography as described in the literature "A.Rehwald, B.Meier, O.Sticher, qualitative and quantitative reversed-phase high performance liquidchromatographicanalysis of glycoprotein hormones in the presenceofa large excess of human serum albumin, J.chromatogrA, 1994,677 (1): 25-33", specific chromatographic conditions are as follows:

chromatographic column: c4 (4.6 mm ID. Times.25 cm L,5 μm,)

flow rate: 0.5 ml/min

Column temperature: 25 DEG C

Detection wavelength: 220nm

Mobile phase a: pH7.0,0.05M phosphate buffer

Mobile phase B: acetonitrile

Gradient procedure: time (minutes)/mobile phase B percentage (%): 0/12.5,40/50,60/65.

The obtained chromatogram is shown in figure 20, and the human serum albumin, sodium acetyl tryptophan and sodium caprylate in the chromatogram are not reserved and pass through a chromatographic column, so that the content measurement of the human serum albumin can not be realized.

Comparative example 4

Human serum albumin content in human erythropoietin injections was measured by reversed phase high performance liquid chromatography as described in the literature "M Girard, T Cyr, N Mousseau, JC Ethier, separation of human serum albumin components by RP-HPLC and CZE and their characterization by ESI-MS, chromatographicia, 1999,49 (1): S21-S27", specific chromatographic conditions are as follows:

chromatographic column: c8 (250X 4.6mm i.d.,7 μm,)

flow rate: N/A

Column temperature: 50 DEG C

Detection wavelength: 215nm

Mobile phase a:0.05% trifluoroacetic acid/10% acetonitrile water

Mobile phase B:0.05% trifluoroacetic acid/90% acetonitrile water

Mobile phase C:0.05% acetonitrile

Gradient procedure:

the obtained chromatogram is shown in figure 21, and the acetyl tryptophan sodium, the sodium octoate and the human erythropoietin in the chromatogram do not interfere with the main peak of the human serum albumin, but the main peak of the human serum albumin is asymmetric left and right, and has tailing phenomenon, so that the method can not be used for accurately measuring the content of the human serum albumin.

Claims

1. The method for detecting the human serum albumin content in the human erythropoietin injection by using the reversed-phase high performance liquid chromatography is characterized by adopting the following chromatographic conditions:

mobile phase: 0.05-0.10% of trifluoroacetic acid/water in the phase A, and 0.05-0.10% of trifluoroacetic acid/acetonitrile in the phase B;

flow rate: 0.6 to 1.0ml/min;

detection wavelength: 210-234 nm;

column temperature: 20-35 ℃;

sample injection amount: 1.5-24.0 mug.

2. The method according to claim 1, characterized in that the elution procedure used is as follows:

。

3. the method according to claim 1, wherein the chromatographic column has a size of 30nm pore diameter, 5 μm filler particle diameter, 4.6mm diameter and 150mm length.

4. The method of claim 1, wherein the flow rate is 0.8-1.0 ml/min.

5. The method of claim 1, wherein the detection wavelength is 210-220nm.

6. The method of claim 1, wherein the column temperature is 20-30 ℃.

7. The method of claim 1, wherein the sample loading is 3.0-12.0 μg.

8. A method for separating sodium acetyltryptophan, human serum albumin, sodium caprylate and human erythropoietin by using a reversed-phase high performance liquid chromatography is characterized in that the separation degree between substances reaches more than 2.0, and the following chromatographic conditions are adopted:

flow rate: 0.6 to 1.0ml/min;

detection wavelength: 210-234 nm;

column temperature: 20-35 ℃;

sample injection amount: 1.5-24.0 mug.

9. The method of claim 8, wherein the elution procedure used is as follows:

。