CN117330659A - High performance liquid chromatography analysis method for detecting tetrapeptide non-activated ester isomer - Google Patents
High performance liquid chromatography analysis method for detecting tetrapeptide non-activated ester isomer Download PDFInfo
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- 150000002148 esters Chemical class 0.000 title claims abstract description 66
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 21
- 238000004458 analytical method Methods 0.000 title abstract description 25
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 39
- 238000000034 method Methods 0.000 claims abstract description 39
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 26
- 238000001514 detection method Methods 0.000 claims abstract description 17
- 239000012488 sample solution Substances 0.000 claims abstract description 14
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 13
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- 239000000945 filler Substances 0.000 claims abstract description 4
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- LOUPRKONTZGTKE-WZBLMQSHSA-N Quinine Chemical compound C([C@H]([C@H](C1)C=C)C2)C[N@@]1[C@@H]2[C@H](O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-WZBLMQSHSA-N 0.000 claims description 4
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- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- DLSWIYLPEUIQAV-UHFFFAOYSA-N Semaglutide Chemical compound CCC(C)C(NC(=O)C(Cc1ccccc1)NC(=O)C(CCC(O)=O)NC(=O)C(CCCCNC(=O)COCCOCCNC(=O)COCCOCCNC(=O)CCC(NC(=O)CCCCCCCCCCCCCCCCC(O)=O)C(O)=O)NC(=O)C(C)NC(=O)C(C)NC(=O)C(CCC(N)=O)NC(=O)CNC(=O)C(CCC(O)=O)NC(=O)C(CC(C)C)NC(=O)C(Cc1ccc(O)cc1)NC(=O)C(CO)NC(=O)C(CO)NC(=O)C(NC(=O)C(CC(O)=O)NC(=O)C(CO)NC(=O)C(NC(=O)C(Cc1ccccc1)NC(=O)C(NC(=O)CNC(=O)C(CCC(O)=O)NC(=O)C(C)(C)NC(=O)C(N)Cc1cnc[nH]1)C(C)O)C(C)O)C(C)C)C(=O)NC(C)C(=O)NC(Cc1c[nH]c2ccccc12)C(=O)NC(CC(C)C)C(=O)NC(C(C)C)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CCCNC(N)=N)C(=O)NCC(O)=O DLSWIYLPEUIQAV-UHFFFAOYSA-N 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
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- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- General Physics & Mathematics (AREA)
- Immunology (AREA)
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Abstract
The invention relates to a high performance liquid chromatography analysis method for detecting a tetrapeptide non-activated ester isomer, which relates to the technical field of biological medicines and comprises the following steps: mixing the tetrapeptide non-activated ester with a diluent to prepare a sample solution; detecting the sample solution by using high performance liquid chromatography to determine the isomer content in the tetrapeptide non-activated ester, wherein the filler is silica gel surface covalent bond-quinine (8S, 9R) - (1S, 2S) -cyclohexyl sulfamic acid derivative, the mobile phase A is phosphoric acid aqueous solution, and the mobile phase B is acetonitrile; the content of the isomer of the tetrapeptide non-activated ester was measured and the content of the isomer in the tetrapeptide non-activated ester was calculated based on the peak area. The method for preparing the mobile phase is simple, time-saving and labor-saving, has good durability, is suitable for mass detection of subsequent products, and saves cost; the isomer has fast peak time, thus saving analysis time; the content of the tetrapeptide non-activated ester isomer can be detected at a quantitative limit of 0.5026 mug/mL, and at a detection limit of 0.25 mug/mL.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a high performance liquid chromatography analysis method for detecting a tetrapeptide non-activated ester isomer.
Background
Semaglutide is a long-acting GLP-1 analog developed by Nove Nordisk, inc., and is produced by gene recombination technology using yeast, and the drug is administered only once a week by subcutaneous injection. Semiglutide is used as one of representative drugs of GLP-1 analogues, and a plurality of clinical trial researches prove that the combination of different oral hypoglycemic drugs can effectively control blood sugar and can lead patients to reduce weight, reduce systolic blood pressure and improve islet beta cell functions.
The tetrapeptide non-activated ester, abbreviated as tBuO-Ste-Glu (AEEA-AEEA-OH) -OtBu (chemical structural formula is shown as formula I), is a key starting material (used as a side chain of the semaglutin) for preparing the semaglutin bulk drug and analogues thereof, and the quality condition of the semaglutin bulk drug directly influences the quality of polypeptide drugs in the synthesis process of the semaglutin bulk drug. During the preparation of the tetrapeptide non-activated ester, chiral impurities are introduced: tetrapeptide non-activated ester isomer (hereinafter referred to as isomer or tetrapeptide isomer) is a very important item in quality control of tetrapeptide non-activated ester, and as isomer impurities in tetrapeptide non-activated ester may be accompanied by chemical reaction to undergo impurity transfer and conversion, so as to increase the purification difficulty of polypeptide medicine, necessary impurity research should be increased, and reasonable control limit should be formulated. Therefore, an analytical method capable of efficiently detecting the non-activated ester isomer of tetrapeptides should be developed. At present, a method for detecting the isomer content of the tetrapeptide non-activated ester is not disclosed.
Therefore, there is a need for a high performance liquid chromatography method for detecting non-activated ester isomers of tetrapeptides that is simple, low cost and robust.
Disclosure of Invention
The invention aims to provide a high performance liquid chromatography method for detecting a tetrapeptide non-activated ester isomer, which has the advantages of simple operation, low cost, rapid peak emergence and good durability, and solves the problem that the existing method for detecting the tetrapeptide non-activated ester isomer is not available. To achieve the purpose, the invention adopts the following technical scheme:
the invention provides a high performance liquid chromatography analysis method for detecting a tetrapeptide non-activated ester isomer, which comprises the following steps:
step 1: mixing the tetrapeptide non-activated ester with a diluent to prepare a sample solution;
step 2: detecting the sample solution by using high performance liquid chromatography to determine the isomer content in the tetrapeptide non-activated ester; the detection conditions of the high performance liquid chromatography are as follows,
the mobile phase A of the high performance liquid chromatography is phosphoric acid aqueous solution, and the mobile phase B is acetonitrile; the elution mode is gradient elution;
step 3: the content of the isomer in the tetrapeptide non-activated ester was measured, and the content of the isomer in the tetrapeptide non-activated ester was calculated according to the peak area.
As an embodiment of the present invention, the diluent in the step 1 is acetonitrile aqueous solution.
As an embodiment of the present invention, the volume concentration of acetonitrile in the acetonitrile aqueous solution is 30% or more, preferably 40%.
As an embodiment of the present invention, the volume concentration of phosphoric acid in the phosphoric acid aqueous solution in the step 2 is 0.05 to 0.5%, preferably 0.1%.
As an embodiment of the present invention, in the step 2, the gradient elution procedure is as follows: 0-15min,50% -80% mobile phase B;15-15.1min,80% -50% mobile phase B,15.1-20min,50% mobile phase B.
In the step 2, the column is a chiral column, and the filler is a silica gel surface covalently bonded quinine (8 s,9 r) - (1 s,2 s) -cyclohexylsulfamic acid derivative.
As an embodiment of the present invention, the chromatographic column is a macrocelluloid CHIRALPAK ZWIX (+), 150 x 3mm,3 μm.
An embodiment of the present invention is characterized in that: the detection wavelength of the high performance liquid chromatography is 200-230nm, the column temperature of the chromatographic column is 20-50 ℃, and the flow rate of the mobile phase is 0.5-2mL/min; the sample injection amount of the sample solution is 5-50 mu L, and the sample injection time is 10-50min.
As one embodiment of the invention, the detection wavelength of the high performance liquid chromatography is 200nm, the column temperature of the chromatographic column is 30 ℃, and the flow rate of the mobile phase is 1mL/min; the sample injection amount of the sample solution is 10 mu L, and the sample injection time is 20min.
The invention has the following beneficial effects: a new high performance liquid chromatography analysis method is developed, wherein a silica gel surface covalent bonding-quinine (8S, 9R) - (1S, 2S) -cyclohexyl sulfamic acid derivative is used as a filler, a phosphoric acid aqueous solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, and a gradient elution mode is adopted. The method for preparing the mobile phase is simple, time-saving and labor-saving, has good durability, is suitable for mass detection of subsequent products, and saves cost; the peak time of the tetrapeptide non-activated ester isomer is fast, and the analysis time is saved. The content of the tetrapeptide non-activated ester isomer can be detected at a quantitative limit of 0.5026 mug/mL, and at a detection limit of 0.25 mug/mL. The method provided by the invention is quick, simple, convenient, good in specificity, high in sensitivity, high in accuracy, wide in application range, low in cost and suitable for popularization and application.
Drawings
FIGS. 1 to 2 are color charts obtained under the condition of example 1 of the present invention;
FIG. 3 is a chromatogram obtained under the conditions of example 2 of the present invention;
FIG. 4 is a chromatogram obtained under the conditions of example 3 of the present invention;
FIG. 5 is a linear plot of the resulting tetrapeptides non-activated esters under the conditions of example 4 of the present invention;
FIG. 6 is a linear diagram of the isomer obtained under the conditions of example 4 of the present invention.
Detailed Description
The present invention is further described below with reference to examples, but embodiments of the present invention are not limited thereto.
In the embodiment, the sample is tetrapeptide non-activated ester (purity is 95.6%, and the sample contains tetrapeptide non-activated ester isomer 0.03%) provided by Nanjing Hanxin medical science and technology Co., ltd; tetrapeptide non-activated ester isomer (i.e., isomer) having a purity of 98.1% and is available from Xiaomenobang Biotechnology Co., ltd.
The equipment or other reagents/materials used in the examples are commercially available.
Example 1
The method provided in this example was subjected to column chromatography screening, and this test shows that the sample was tetrapeptide non-activated ester (purity: 95.6%, and tetrapeptide non-activated ester isomer: 0.03%).
Experimental conditions:
the device comprises: high Performance Liquid Chromatography (HPLC).
Chromatographic column 1: CHIRALCEL OZ-RH,150 x 4.6mm,5 μm; PN:42724
Chromatographic column 2: CHIRALPAK ZWIX (+), 150 x 3mm,3 μm; PN:51584
Detection conditions: detection wavelength is 200nm; column temperature is 30 ℃; sampling time is 20min; the sample injection amount is 10 mu L; the flow rate was 1.0mL/min. Gradient elution procedure:
| time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 50 | 50 |
| 7 | 25 | 75 |
| 15 | 25 | 75 |
| 15.1 | 50 | 50 |
| 20 | 50 | 50 |
Preparation of mobile phase A: 1mL of phosphoric acid is removed in a 1L measuring flask, diluted with deionized water to a fixed volume to a scale, and shaken uniformly to obtain a phosphoric acid aqueous solution with the phosphoric acid volume concentration of 0.1 percent.
Mobile phase B: acetonitrile.
Preparing a diluent: and transferring 50mL of acetonitrile into a 100mL measuring flask, diluting with deionized water to a constant volume to a scale, and shaking uniformly to obtain an acetonitrile aqueous solution with the acetonitrile volume concentration of 50%.
Preparing a blank solution: as well as a diluent.
Test solution: the sample was diluted with a diluent to a solution of about 1mg of the non-activated tetrapeptide ester per 1 mL.
Preparing an isomer solution: weighing a proper amount of tetrapeptide isomer, and dissolving with a diluent to prepare a solution of 0.2 mg/ml.
Preparing a system applicability solution: and taking a proper amount of the tetrapeptide non-activated ester and isomer impurities, and diluting the mixture to a solution containing 1mg of the tetrapeptide non-activated ester and 5 mug of the tetrapeptide isomer per 1mL by using a diluent.
Assay: respectively measuring the isomer solution and the sample solution, injecting into a liquid chromatograph using a chromatographic column 1, and recording the chromatograms, wherein the chromatograms are shown in figure 1;
respectively measuring the isomer solution and the sample solution, injecting into a liquid chromatograph using a chromatographic column 2, and recording the chromatograms, see figure 2;
the related data of the tetrapeptide non-activated ester and the isomer obtained by measuring by using the chromatographic column 1 and the chromatographic column 2 are shown in the table 1, statistics of results of the chromatographic column 1 and the chromatographic column 2
Table 1 statistics of column 1 and column 2 results
Conclusion: the theoretical plate number and half-width of the main peak and isomer peak of the tetrapeptide non-activated ester under the condition of the chromatographic column 2 are superior to those of the chromatographic column 1, so that the chromatographic column 2 is selected.
Example 2
The present example performed screening of elution procedures for the provided method. Column selection: CHIRALPAK ZWIX (+), 150 x 3mm,3 μm. Other experimental conditions were consistent with example 1.
Assay: the gradient elution procedure of example 1 was selected and a precision measurement system applicability solution was injected into a liquid chromatograph, and the resulting chromatogram is shown in fig. 3:
example 3
The present example performed screening of elution procedures for the provided method. Column selection: CHIRALPAK ZWIX (+), 150 x 3mm,3 μm. Other experimental conditions were consistent with example 1, except that the gradient elution procedure was changed.
The gradient elution procedure was adjusted to table 2, gradient elution procedure two, and the resulting chromatogram is shown in fig. 4:
TABLE 2 gradient elution procedure two
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 50 | 50 |
| 15 | 20 | 80 |
| 15.1 | 50 | 50 |
| 20 | 50 | 50 |
Conclusion: the gradient elution procedure of example 3 was selected by screening the gradient elution procedures of examples 2-3, and the separation degree of the main peak from the front peak in examples 2 and 3 was 3.09 and 3.31, respectively, and the elution procedure of example 3 was more separated, as can be obtained from the respective chromatograms obtained.
Example 4
In this example, systematic applicability, precision, specificity, linearity, correction factors, quantitative limits, detection limits, accuracy and range of the method provided in example 3 were validated using tetrapeptide non-activated ester (purity 95.6%, including tetrapeptide non-activated ester isomer 0.03%) as a test sample. See for details the details in the methodological verification, if any.
(1) System applicability
The system applicability test parameters and acceptable criteria are based on the concept that the device, electronics, analysis operations and the sample to be analyzed form an integrated system. The system applicability test ensures that the system works properly during analysis.
The system applicability of the present application was obtained by analyzing a 6-needle system applicability solution. RSD of tetrapeptide non-activated ester peak area in 6-needle system applicability solution should not exceed 2%; the degree of separation between the tetrapeptide inactive ester and the isomer peak should be not less than 1.5. The system applicability results are shown in Table 3.
Table 3 results of system applicability
Conclusion: in the system applicability, the RSD of the peak area of the tetrapeptide non-activated ester in the 6-needle system applicability solution meets the acceptable standard and cannot exceed 2%; the degree of separation between the tetrapeptide inactive ester and the isomer peak satisfies the acceptable standard of not less than 1.5. Therefore, the system applicability results are in compliance.
(2) Precision of
The precision of the analytical method refers to the multiple sampling of a uniform sample, and the degree of agreement of each result is determined by the analytical method. The precision of the analytical method is demonstrated by the determination of the reproducibility.
The reproducibility of the present application was assessed by testing 6 portions of the system-applicability solution formulated in parallel. The RSD of the tetrapeptide isomer content in 6 parts of the system applicability solution should be not more than 15%. The repeatability results are shown in Table 4.
TABLE 4 repeatability results
| Name of the name | Isomer content |
| Repeatability 1 | 0.25％ |
| Repeatability 2 | 0.24％ |
| Repeatability 3 | 0.24％ |
| Repeatability 4 | 0.25％ |
| Repeatability 5 | 0.24％ |
| Repeatability 6 | 0.25％ |
| Relative standard deviation RSD | 2.2％ |
| Acceptable standard | Not more than 15% |
| Pass/fail | Qualified product |
Conclusion: RSD of tetrapeptide isomer content in 6 independently measured system applicability solutions meets an acceptable standard of no more than 15%. Thus, the method meets the precision related requirements.
(3) Specialization of
Specificity refers to the ability of an analytical method to accurately determine the presence of a substance under test in the presence of potentially present substances such as impurities, degradation products, and adjuvants. Specificity can be expressed in terms of peak purity and degree of separation.
The specificity of the present application is demonstrated by analysis of test solutions labeled with isomers and 100% concentrations of tetrapeptide non-activated esters and isomers. In the sample solution marked with the isomer, the separation degree of the tetrapeptide non-activated ester and the isomer peak is not less than 1.5; the peak-to-peak purity of the tetrapeptide inactive ester and isomer should be greater than 990 in 100% concentration of the tetrapeptide inactive ester and tetrapeptide isomer. The specific results are shown in Table 5.
TABLE 5 specific results
Conclusion: the separation degree of the peak of the tetrapeptide non-activated ester and the peak of the isomer is not less than 1.5, and the peak purity of the peak of the tetrapeptide non-activated ester and the peak purity of the isomer is more than 990. Thus, the method meets the requirements of specificity.
(4) Linearity and correction factor
The linearity of the analytical method means that the analyte measurement in the sample is directly proportional to its concentration or is converted by suitable mathematics within a certain range.
The linearity of the present application is demonstrated by several parallel linear solutions. The linear equation is obtained with the concentrations (μg/ml) of the tetrapeptide non-activated ester and isomer in each linear solution as the abscissa and the peak area as the ordinate, and the regression line generated from the data should generate a correlation coefficient (R) of not less than 0.990. The correction factor is obtained by the ratio of the slope of the linear equation of the isomer to the linear equation of the tetrapeptide non-activated ester. The linear results are shown in tables 6 and 7, and the correction factor results are shown in Table 8.
TABLE 6 Linear results summary of tetrapeptides non-activated esters
TABLE 7 Linear results summary of isomers
Conclusion: the linear compliance of the tetrapeptide non-activated ester concentration at 0.4999 μg/ml to 29.9910 μg/ml (impurity level of 0.05% -3.0%) with an acceptable standard having a correlation coefficient (R) of not less than 0.990; the isomer concentration is in the range of 0.5026 μg/ml to 30.1556 μg/ml (impurity level of 0.05% -3.0%) and meets acceptable criteria for correlation coefficients (R) of not less than 0.990. Thus, the present application meets the linear correlation requirements.
TABLE 8 correction factor results
Conclusion: the correction factor is in the range of 0.9-1.1, correction is not needed, and the correction factor is defined as 1.0.
(5) Quantitative limit
The limit of quantification refers to the minimum amount of analyte in a test sample that can be measured under defined experimental conditions, with acceptable precision and accuracy.
The quantitation limit of the present application was demonstrated by analyzing 6 parts of a quantitation limiting solution (tetrapeptide non-activated ester concentration 0.4999. Mu.g/ml, isomer concentration 0.5026. Mu.g/ml, equivalent to 0.05% level) formulated in parallel. The signal to noise ratio of the tetrapeptide non-activated ester and isomer in the 6 parts quantitative limiting solution should not be less than 10, the RSD of the recovery rate should not be more than 15%, and the average recovery rate should be between 80 and 120%. The results of the limiting solution were shown in Table 9.
TABLE 9 quantitative limiting solution results
Conclusion: the quantitative limit of the isomer of the present application was 0.5026. Mu.g/mL. The average recovery of tetrapeptide non-activated esters and isomers in 6 parallel-formulated quantitative limiting solutions of the present application meets an acceptable standard of between 80-120%, and the RSD of the quantitative limiting solution recovery meets an acceptable standard of not more than 15%. The signal to noise ratio of the tetrapeptide non-activated ester and the isomer in 6 parts of the quantitative limiting solution meets the acceptable standard of not less than 10.
(6) Detection limit
The detection limit is the minimum amount of the analyte in the sample that can be detected under the prescribed test conditions, and is not necessarily required to be quantified.
The detection limit of the present application was demonstrated by analyzing 6 parts of detection limit solution (concentration of tetrapeptide non-activated ester and isomer 0.25. Mu.g/ml) formulated in parallel. The tetrapeptide non-activated ester and the tetrapeptide isomer in 6 parts of the detection limit solution prepared in parallel should be detected (signal to noise ratio S/N is more than or equal to 3). The results of the limiting solution are shown in Table 10.
Table 10 results of limiting solution
| Repeating | Tetrapeptide non-activated esters S/N | Isomer S/N |
| LOD-1 | 8.5 | 8.8 |
| LOD-2 | 8.6 | 9.8 |
| LOD-3 | 16.0 | 12.4 |
| LOD-4 | 18.8 | 11.0 |
| LOD-5 | 16.1 | 11.3 |
| LOD-6 | 13.5 | 11.6 |
| Acceptable standard | Not less than 3 | Not less than 3 |
| Pass/fail | Qualified product | Qualified product |
Conclusion: the tetrapeptide non-activated ester and isomer in 6 parts of the parallel prepared detection limit solution can be detected, and the specification is met (S/N is more than or equal to 3).
(7) Accuracy of
The accuracy of the analysis method refers to the closeness of the test result obtained by the method to the true value. The accuracy of the analytical method should be determined within a certain range.
Analytical method accuracy the recovery rate and RSD of the analytical labeled test sample solutions (test samples labeled with five levels of tetrapeptide isomers 0.05%, 0.5%, 1%, 2% and 3%, respectively) demonstrate that the average recovery rate at each level should be between 80% and 120%, and the RSD at each concentration should not be greater than 15%. Isomer recovery and RSD are shown in table 11.
TABLE 11 isomer recovery and RSD
Conclusion: average recovery at each level of the present application meets acceptable criteria between 80-120%. RSD at each concentration meets acceptable criteria of no more than 15%. Thus, the analysis method is accurate.
(8) Range of
The range of the analysis method refers to the high concentration and low concentration range of the analyte with certain precision, accuracy and linear relation result obtained by the method.
The range of the analytical method was demonstrated by analysis of solutions at 0.05%, 0.5%, 1%, 2% and 3% levels. The average recovery rate of each grade should be between 80-120%, the RSD of recovery rate at each grade should not be more than 15%, and the linear correlation coefficient (R) should not be less than 0.990. The isomer range results are shown in Table 12.
TABLE 12 isomer range results
Conclusion: the average recovery for each grade of the present application met acceptable criteria between 80-120%. The RSD of the recovery at each level should not satisfy the acceptable standard more than 15%, and the correlation coefficient (R) should satisfy the acceptable standard more than 0.990.
Thus, the range of isomers in the analytical method was confirmed to be 0.5000. Mu.g/ml to 30.000. Mu.g/ml.
Claims (10)
1. A high performance liquid chromatography method for detecting a tetrapeptide non-activated ester isomer, comprising the steps of:
step 1: mixing the tetrapeptide non-activated ester with a diluent to prepare a sample solution;
step 2: detecting the sample solution by using high performance liquid chromatography to determine the isomer content in the tetrapeptide non-activated ester; the detection conditions of the high performance liquid chromatography are as follows,
the mobile phase A of the high performance liquid chromatography is phosphoric acid aqueous solution, and the mobile phase B is acetonitrile; the elution mode is gradient elution;
step 3: the content of the isomer in the tetrapeptide non-activated ester was measured, and the content of the isomer in the tetrapeptide non-activated ester was calculated according to the peak area.
2. The method according to claim 1, characterized in that: the diluent in the step 1 is acetonitrile water solution.
3. The method according to claim 2, characterized in that: the volume concentration of acetonitrile in the acetonitrile aqueous solution is 30% or more, preferably 40%.
4. The method according to claim 1, characterized in that: the volume concentration of phosphoric acid in the phosphoric acid aqueous solution in the step 2 is 0.05-0.5%.
5. The method according to claim 4, wherein: the volume concentration of phosphoric acid in the phosphoric acid aqueous solution in the step 2 is 0.1%.
6. The method according to claim 1, wherein in step 2, the gradient elution procedure is: 0-15min,50% -80% mobile phase B;15-15.1min,80% -50% mobile phase B;15.1-20min,50% mobile phase B.
7. The method according to claim 1, wherein in the step 2, the column is a chiral column, and the filler is a silica gel surface covalently bonded quinine (8 s,9 r) - (1 s,2 s) -cyclohexylsulfamic acid derivative.
8. The method according to claim 7, wherein: the chromatographic column is celluloid CHIRALPAK ZWIX (+), 150 x 3mm,3 μm.
9. The method according to any one of claims 1-8, wherein: the detection wavelength of the high performance liquid chromatography is 200-230nm, the column temperature of the chromatographic column is 20-50 ℃, and the flow rate of the mobile phase is 0.5-2mL/min; the sample injection amount of the sample solution is 5-50 mu L, and the sample injection time is 10-50min.
10. The method according to claim 9, wherein: the detection wavelength of the high performance liquid chromatography is 200nm, the column temperature of the chromatographic column is 30 ℃, and the flow rate of the mobile phase is 1mL/min; the sample injection amount of the sample solution is 10 mu L, and the sample injection time is 20min.
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