CN109358132B - Method for separating and measuring substances related to lomefloxacin aspartate glucose injection - Google Patents
Method for separating and measuring substances related to lomefloxacin aspartate glucose injection Download PDFInfo
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Abstract
The invention relates to the field of pharmaceutical analysis, in particular to a method for separating and measuring substances related to an aspartic acid lomefloxacin glucose injection. The method comprises the steps of detecting the lomefloxacin aspartate glucose injection serving as a test solution by adopting a high performance liquid chromatography, wherein the high performance liquid chromatography adopts a chromatographic column with octadecylsilane chemically bonded silica as a filler, and linear gradient elution is carried out by using a mobile phase A and fluidity B; the mobile phase A is phosphate solution-methanol; the mobile phase B is acetonitrile. The method is used for detecting related substances of the lomefloxacin aspartate glucose injection, has the characteristics of strong specificity, high accuracy, good repeatability and the like, can measure more impurity peaks compared with a standard, and has larger total detected impurity amount.
Description
Technical Field
The invention relates to the field of pharmaceutical analysis, in particular to a method for separating and measuring substances related to an aspartic acid lomefloxacin glucose injection.
Background
At present, the detection standard of lomefloxacin aspartate glucose injection is determined by the national food and drug administration standards including YBH30102005 (plant A), YBH15122004 (plant B) and WS1- (X-097) -2005Z of the national drug standards new drug transfer standard No. 64. The determination methods of related substances in the detection methods are all determined by liquid chromatography, wherein the determination methods of related substances in YBH30102005(A factory) and YBH15122004(B factory) are in accordance with the specification, the existing detection standards all use isocratic elution methods to determine related substances, the retention time is generally recorded to 3 times of the retention time of a main peak, the mobile phase is formed by mixing acetonitrile-phosphate buffer solution (6.7 g of potassium dihydrogen phosphate is weighed, 2.8ml of phosphoric acid is added, and water is added for dilution to 1000ml) -0.05mol/L tetrabutylammonium bromide solution according to the volume ratio of 15:85:4, the detection wavelength: 288nm, sample size: 20 μ l, test concentration 0.2mg/ml, control concentration: 2. mu.g/ml.
The detection method of related substances in the above standard has the following defects: the separation degree between impurity peaks is poor, 5-hydroxymethylfurfural (5-HMF) and a lomefloxacin main peak are not well separated from adjacent impurity peaks, and interference exists; impurity peaks do not belong to a good source (such as oxidation and photodegradation); the lomefloxacin has poor main peak type and a tail; the mobile phase contains an ion pair reagent (tetrabutylammonium bromide), and the long-term use of the mobile phase causes great damage to the chromatographic column. A typical high performance liquid chromatogram is shown in fig. 1 (measured according to the detection method of related substances in YBH 30102005), wherein peak 1 is a 5-hydroxymethylfurfural impurity peak; peak 2 is the other impurity peak; peak 3 is the lomefloxacin peak.
Disclosure of Invention
The invention aims to provide a method for separating and measuring lomefloxacin aspartate glucose injection related substances, which is used for detecting the aspartic acid lomefloxacin glucose injection related substances, can well separate lomefloxacin from impurities and impurities, has the characteristics of strong specificity, high accuracy, good repeatability and the like, can measure more impurity peaks compared with a legal standard, and has larger total detected impurity amount.
The above purpose of the invention is realized by adopting the following technical scheme:
the invention provides a method for separating and measuring related substances of lomefloxacin aspartate glucose injection, which comprises the steps of detecting the lomefloxacin aspartate glucose injection serving as a test solution by adopting a high performance liquid chromatography, wherein the high performance liquid chromatography adopts a chromatographic column with octadecylsilane chemically bonded silica as a filler, and linear gradient elution is carried out by using a mobile phase A and a fluidity B; the mobile phase A is phosphate solution-methanol; the mobile phase B is acetonitrile.
In the technical solution of the present invention, preferably, the preparation method of the mobile phase a is: mixing 0.6-0.7% by mass of potassium dihydrogen phosphate solution and methanol according to a volume ratio of 70: 30-80: 20, and adjusting the pH value to 2.5-3.0 by adopting phosphoric acid.
In the technical scheme of the invention, preferably, the mass percentage of the potassium dihydrogen phosphate solution is 0.67%, the volume ratio of the potassium dihydrogen phosphate solution to the methanol is 76:24, and the pH value is 2.7.
In the technical solution of the present invention, preferably, the procedure of gradient elution is:
in the technical scheme of the invention, the model of the chromatographic column is Welch Ultimate XB-C18, 4.6X 250mm and 5 μm.
In the technical scheme of the invention, preferably, the detection wavelength of the high performance liquid chromatography is 280-290 nm; more preferably 287 nm.
In the technical scheme of the invention, preferably, the flow rate of the high performance liquid chromatography is 0.5-1.5 ml/min; more preferably 1.0 ml/min.
In the technical scheme of the invention, preferably, the sample injection amount of the high performance liquid chromatography is 5-20 mu L; more preferably 10. mu.L.
In the technical scheme of the invention, preferably, the temperature of a chromatographic column of the high performance liquid chromatography is 35-45 ℃; more preferably 40 deg.c.
The method for separating and measuring the related substances of the lomefloxacin aspartate glucose injection provided by the invention has the beneficial effects that:
(1) the separation and determination method provided by the invention is characterized in that Welch Ultimate XB-C18(4.6 x 250mm, 5 mu m) is used as a chromatographic column, mobile phase A (phosphate solution (6.7 g of potassium dihydrogen phosphate is weighed, and 1000ml of water is added to dissolve) -methanol (76:24) (pH value is adjusted to 2.7) by phosphoric acid) and mobile phase B (acetonitrile) are used as mobile phases for linear gradient elution, and the separation degree between related substances in the existing standard can be greatly improved, wherein the separation degree between 5-hydroxymethylfurfural, a lomefloxacin main peak and adjacent impurity peaks is more than 1.5, the separation degree between the other impurity peaks is also better, the lomefloxacin main peak is symmetrical in shape, and the tailing phenomenon of the original method is avoided. The method has the characteristics of strong specificity, high accuracy, good repeatability and the like, can measure more impurity peaks than a legal standard, and can detect more total impurities, so that the method can be better used for monitoring the quality of the aspartic acid lomefloxacin injection.
(2) According to the method, impurities generated by acid, alkali, light, oxidation and high-temperature damage can be better detected, the main peak and the front and rear adjacent impurity peaks can be better separated, and the separation effect among the impurity peaks is also better. The related substance impurities in the lomefloxacin aspartate glucose injection sample are subjected to damage by acid, alkali, light and oxidation, and a reference is provided for the improvement of the medicine quality of an enterprise.
(3) Only monopotassium phosphate, methanol, acetonitrile and water are contained in the mobile phase, and no ion pair reagent is contained, so that the damage to the chromatographic column is lower than that of the original method.
Drawings
FIG. 1 is a high performance liquid chromatogram obtained according to a prior art chromatographic method;
FIG. 2 is a high performance liquid chromatogram of the test solution obtained in example 1;
FIG. 3 is a high performance liquid chromatogram of a control solution obtained in example 1;
FIG. 4 is a high performance liquid chromatogram of the system suitability solution obtained in example 2;
FIG. 5 is a graph showing the linear relationship of lomefloxacin obtained in example 3;
FIG. 6 is a high performance liquid chromatogram of the specificity test obtained in example 7;
FIG. 7 is a high performance liquid chromatogram of the photo-disruption assay obtained in example 8;
FIG. 8 is a high performance liquid chromatogram of the photo-disruption assay obtained in example 8;
FIG. 9 is a high performance liquid chromatogram of the high temperature and strong acid attack test obtained in example 8;
FIG. 10 is a high performance liquid chromatogram of the high temperature and strong acid attack test obtained in example 8;
FIG. 11 is a high performance liquid chromatogram of the oxidative damage test obtained in example 8;
FIG. 12 is a high performance liquid chromatogram of the strong base attack test obtained in example 8;
FIG. 13 is a source assignment chart of the impurities obtained in example 8.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Unless otherwise specified, the reference lomefloxacin used in the following examples was obtained from the institute for food and drug assay (lot No. 130452-200902, content: 90.3%); the test sample lomefloxacin aspartate glucose injection provided by the embodiment of the invention has the specification of 100ml, and the lomefloxacin aspartate contained in the injection is lomefloxacin aspartate (C)17H19F2N3O3) 0.2g of glucose and 5g of glucose; the lomefloxacin aspartate raw material in the embodiment of the invention is a solid raw material medicine of lomefloxacin aspartate; the high performance liquid chromatograph used in the embodiment of the invention is of a model LC-20AT (with PDA detector) and is purchased from Shimadzu corporation; electronic balance model MS205DU, available from METTLER corporation.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1: establishment of chromatographic conditions
A chromatographic column: welch Ultimate XB-C18 (4.6X 250mm, 5 μm)
Mobile phase A: phosphate solution (Potassium dihydrogen phosphate 6.7g, water 1000ml dissolved) -methanol (76:24) (pH adjusted to 2.7 with phosphoric acid)
Mobile phase B: acetonitrile
Detection wavelength: 287nm
Flow rate: 1ml/min
Column temperature: 40 deg.C
Sample introduction amount: 10 μ l.
The elution was performed with a linear gradient according to the elution procedure in table 1 below:
TABLE 1 elution procedure
Solution preparation: taking lomefloxacin aspartate glucose injection as a test solution (containing 2.0mg/ml of lomefloxacin), precisely measuring 1ml of the test solution, adding a mobile phase A to dilute to 500ml, and taking the diluted mobile phase A as a control solution (containing 40 mu g/ml of lomefloxacin).
The determination method comprises the following steps: injecting 10 μ l of each of the test solution and the control solution into a liquid chromatograph, measuring according to the above chromatographic conditions, and recording the chromatogram, wherein the high performance liquid chromatogram of the test solution is shown in FIG. 2, and the high performance liquid chromatogram of the control solution is shown in FIG. 3.
As can be seen from fig. 2 to 3, the separation degree between related substances in the existing standard can be greatly improved by performing gradient elution with the mobile phase, wherein the separation degree between the 5-hydroxymethylfurfural (retention time of 4.915min), the main lomefloxacin peak and adjacent impurity peaks is greater than 1.5, the separation degree between the other impurity peaks is also better, the main lomefloxacin peak is symmetrical in shape, and the tailing phenomenon of the original method is avoided.
Example 2: system suitability test
About 22mg of lomefloxacin reference substance was taken, 1ml of water was added to dissolve the reference substance, 1ml of 30% hydrogen peroxide solution was added thereto, the mixture was heated in a water bath for 2 hours (oxidative destruction), cooled, diluted to 10ml with the mobile phase a, shaken up, 10. mu.l of this solution was measured precisely and injected into a liquid chromatograph, measurement was performed under the chromatographic conditions in example 1, and a chromatogram was recorded, and the results are shown in fig. 4.
As can be seen from fig. 4, the lomefloxacin peak retention time is about 22.0min, and the separation degree of the lomefloxacin peak from the adjacent impurity peak is greater than 1.5(5.7 and 2.1).
Example 3: linear relation
Precisely weighing 21.84mg of lomefloxacin reference substance, placing the reference substance into a 100ml measuring flask, adding the mobile phase A provided in the example 1 to dissolve and dilute the reference substance to a scale, and shaking up to prepare a reference substance solution with the concentration of the lomefloxacin being about 0.2 mg/ml. Then precisely measuring appropriate amount of the above control solution, and diluting with mobile phase A to obtain solutions with concentration of about 40 μ g/ml, 20 μ g/ml and 2 μ g/ml respectively. 20. mu.l, 10. mu.l and 5. mu.l of 0.2mg/ml control solutions, 10. mu.l of 40. mu.g/ml control solutions, 10. mu.l and 5. mu.l of 20. mu.g/ml control solutions, and 10. mu.l of 2. mu.g/ml control solutions were each precisely measured and injected into a liquid chromatograph, the peak area was measured under the chromatographic conditions as in example 1, and a standard curve was drawn with the peak area integrated value as ordinate and the control mass as abscissa, with the regression equation y being 5791.4x +69480, R2The standard curve obtained is shown in fig. 5, 0.9999.
As can be seen from FIG. 5, the lomefloxacin has good linearity between masses of 19.72 ng and 3944 ng.
Example 4: detection limit, quantification limit
The method comprises the steps of precisely measuring 660 mu l and 220 mu l of lomefloxacin reference solution with the concentration of about 2 mu g/ml, putting the solutions into a 10ml measuring flask, adding a mobile phase A to dilute the solutions to a scale, shaking the solutions evenly, precisely measuring 10 mu l of the solutions respectively, injecting the solutions into a liquid chromatograph to serve as detection limit and quantification limit, and displaying the signal-to-noise ratio of the solutions as 5.09 and 17.72 through a chromatogram, wherein the detection limit and the quantification limit of the lomefloxacin are respectively 0.43ng and 1.30 ng.
Example 5: precision test
The method comprises the steps of precisely measuring 10 mu l of lomefloxacin reference solution with the concentration of 40 mu g/ml, injecting the lomefloxacin reference solution into a liquid chromatograph, continuously injecting samples for 6 times, recording chromatograms, wherein peak areas are 2366997, 2365606, 2366583, 2365493, 2365539 and 2366008 respectively, and RSD is 0.03%, and the method is good in precision.
Example 6: repeatability test
6 parts of lomefloxacin aspartate glucose injection (product B, lot number 161103, specification: 100ml, 0.2g lomefloxacin and 5g glucose) was taken, and the results were determined according to the chromatographic conditions provided in example 1 and are shown in Table 2:
TABLE 2 repeatability test data
As can be seen from Table 2, the RSD of the total impurities (0.3% on average) is less than 2%, and the RSD of the individual impurities is less than 6%, indicating that the method has good repeatability.
Example 7: specificity test
Blank test: taking a 5% glucose solution as a blank auxiliary material solution. Precisely measuring 10 μ l of each of the blank adjuvant solution and the mobile phase A solution, measuring according to the chromatographic conditions in example 1, and recording the chromatogram, wherein the result is shown in FIG. 6, and the curve 4 is the test result of the 5% glucose solution; curve 5 is the test result for mobile phase a.
As can be seen from fig. 6, the mobile phase A has no interference to the method, the 5-HMF peak in the blank auxiliary material appears about 5min, and no other impurity peak is detected in the sample at the position, so that the mobile phase A and the glucose basically have no interference to the detection of related substances. In addition, as can be seen from fig. 6, 5-HMF can be completely separated from impurities in the sample, which indicates that the method is also suitable for detecting 5-HMF in lomefloxacin aspartate glucose injection.
Example 8: destructive testing
The forced degradation test is to accelerate the damage of lomefloxacin aspartate under strong degradation conditions of strong acid, strong alkali, oxidation, illumination, high temperature and the like, and aims to evaluate the effectiveness and the applicability of the analysis method by inspecting the separation conditions of degradation products and main peaks of samples and known impurities.
In order to better investigate the specificity and stability of the method, a forced degradation test of acid, alkali, oxidation, illumination and high temperature is designed.
(1) Photo-destructive test
Sunlight damage: lomefloxacin aspartate glucose injection (manufactured by B, lot No. 161103, standard: 100ml: 0.2g lomefloxacin and 5g glucose) was placed under illumination at 4000lx for 24 hours and then measured according to the chromatography conditions in example 1.
Ultraviolet damage 1 (ultraviolet damage): a lomefloxacin aspartate glucose injection (manufactured by B, lot No. 161103, standard: 100ml: 0.2g lomefloxacin and 5g glucose) was placed in a glass evaporation dish, irradiated with ultraviolet rays for 2 hours, and then subjected to the measurement under the chromatographic conditions in example 1.
Comparison: lomefloxacin aspartate (without disruption) (supplied by D, lot 20151201) was added to water to make a solution containing about 2.0mg/ml of lomefloxacin and measured by chromatography as in control example 1.
Ultraviolet damage 2 (ultraviolet damage): lomefloxacin aspartate (supplied by D, lot 20151201) was subjected to ultraviolet irradiation for 6 hours and then measured under the chromatographic conditions described in example 1.
The chromatogram of the photo-damage test result is shown in fig. 7-8, in fig. 7, curve 6 is the test result of ultraviolet damage 1, curve 7 is the test result of sunlight damage, and curve 8 is the test result of contrast; FIG. 8 shows the test results of UV Damage 2.
(2) High temperature and strong acid failure test
High-temperature destruction 1: lomefloxacin aspartate (supplied by D, lot 20151201) was taken at about 27mg and dissolved in 10ml of water to prepare a solution containing about 2mg/ml of lomefloxacin, which was heated in a boiling water bath for 4 hours and then subjected to the chromatography conditions described in example 1.
High-temperature destruction 2: lomefloxacin aspartate (supplied by D, lot 20151201) was heated at 130 ℃ and 160 ℃ for 2 hours, and about 27mg of the material was dissolved in 10ml of water and measured by chromatography as described in example 1.
And (3) strong acid destruction: about 27mg of lomefloxacin aspartate (supplied by D, lot 20151201) was dissolved in 1ml of water to prepare a solution containing about 20mg/ml of lomefloxacin, 1ml of a 10mol/L hydrochloric acid solution was added, the solution was heated in a boiling water bath for 2 hours, then neutralized with 1ml of a 10mol/L sodium hydroxide solution, and then diluted to 10ml with mobile phase A, and the concentration was measured according to the chromatographic conditions in example 1.
The results of the high temperature destruction 1 and the strong acid destruction test are shown in fig. 9, wherein a curve 9 is a test result of the high temperature destruction 1, and a curve 10 is a test result of the strong acid destruction; the test results of high temperature failure 2 are shown in FIG. 10, where curve 11 is the test result at 160 ℃ and curve 12 is the test result at 130 ℃.
(3) Oxidative destruction test
And (3) oxidative destruction: taking about 27mg of lomefloxacin aspartate (supplied by D, lot 20151201), adding 1ml of water to dissolve, preparing a solution containing about 20mg/ml of lomefloxacin, adding 1ml of 30% hydrogen peroxide solution, boiling in a water bath for 2 hours, adding mobile phase A to dilute to 10ml, and measuring according to the chromatogram conditions.
Control 1: the measurement was carried out by the chromatography method in example 1, except that the aspartic acid lomefloxacin glucose injection (manufactured by B., lot No. 161103, standard: 100ml: 0.2g of lomefloxacin and 5g of glucose) was used without being destroyed.
Control 2: the measurement was carried out by the chromatography method in example 1, except that the aspartic acid lomefloxacin glucose injection (manufactured by A, lot No. 21612201-1, standard: 100ml: 0.2g lomefloxacin and 5g glucose) was used without being destroyed.
The test results are shown in FIG. 11, in which curve 13 is the test result of oxidative destruction, curve 14 is the measurement result of control 1, and curve 15 is the test result of control 2.
(4) Strong base destruction test
Strong alkali destruction 1: about 27mg of lomefloxacin aspartate (supplied by D, lot 20151201) was dissolved in 1ml of water to prepare a solution containing about 20mg/ml of lomefloxacin, 1ml of 10mol/L sodium hydroxide solution (which causes precipitation) was added, the mixture was heated in a boiling water bath for 2 hours, then neutralized with 1ml of 10mol/L hydrochloric acid solution, and diluted to 10ml with mobile phase A, and the concentration was measured according to the chromatography conditions in example 1.
Strong alkali destruction 2: about 27mg of lomefloxacin aspartate (supplied by D, lot 20151201) was taken and dissolved in 1ml of water to prepare a solution containing about 20mg/ml of lomefloxacin, about 0.4ml of 10mol/L sodium hydroxide solution (no precipitate was generated), the solution was heated in a boiling water bath for 2 hours, neutralized with 0.4ml of 10mol/L hydrochloric acid solution, diluted to 10ml with mobile phase A, and the concentration was measured according to the chromatographic conditions in example 1.
Comparison: lomefloxacin aspartate (without disruption) (supplied by D, lot 20151201) was added to water to make a solution containing about 2.0mg/ml of lomefloxacin and measured by chromatography as in control example 1.
The test results are shown in FIG. 12, in which curve 16 is the test result of strong alkaline attack 1 and curve 17 is the test result of strong alkaline attack 2, while curve 8 is set as a control.
(5) Conclusion of destructive testing
As can be seen from FIGS. 7-12, according to the method, impurities generated by acid, alkali, light, oxidation and high-temperature damage can be better detected, the main peak and the front and rear adjacent impurity peaks can be better separated, and the separation effect among the impurity peaks is also better. Secondly, the lomefloxacin aspartate aqueous solution is heated in a boiling water bath for 2 hours to be stable, the lomefloxacin aspartate raw material is heated for 2 hours at 130 ℃ to generate a small amount of impurities, the lomefloxacin aspartate raw material is heated for 2 hours at 160 ℃ to generate more impurities, and the amount of degraded impurities is greatly increased; less impurities are generated under the damage of acid and alkali; the degradation is accelerated under the illumination condition, more impurities are generated, and particularly, the color of the solution is changed into dark yellow after 2 hours of illumination under an ultraviolet lamp; most impurities are generated by adopting hydrogen peroxide oxidation destruction, and the degradation is the most severe. The results of attributing the source of each impurity are shown in fig. 13, wherein curve 18 is the results of testing (without damage) on the substances of lomefloxacin aspartate as a raw material (provided by a plant, lot No. 20151201); the remaining curves have the meanings given above; through destructive test results, the invention provides the following source attributions of various impurities, and the results are shown in table 3:
TABLE 3 Source attribution of impurities
Remarking: the retention time of the main peak of lomefloxacin is about 22min
Example 9: sample testing
In order to verify the accuracy of the test of the method, lomefloxacin aspartate glucose injection (the specification is 100ml: 0.2g of lomefloxacin and 5g of glucose) of a factory A and a factory B are respectively measured by adopting the chromatographic conditions provided by the embodiment 1 of the invention, and samples of the factory A and the factory B are respectively tested by adopting legal standards [ YBH30102005 (factory A) and YBH15122004 (factory B) ], and the test results are shown in table 4:
wherein, the method for calculating the content of the related impurities comprises the following steps: if an impurity peak (except 5-hydroxymethylfurfural) exists in the chromatogram of the test solution, the amount of the impurities in the test solution is calculated according to the main component self-contrast method.
The maximum single impurity percent is the maximum single impurity peak area in the test sample graph/(the main peak area of the control solution multiplied by the dilution factor of the control solution) × 100 percent
The total impurity percent is the area of the impurity peak in the test sample map and/(the area of the main peak of the contrast solution multiplied by the dilution factor of the contrast solution) multiplied by 100 percent
TABLE 4 test results
As can be seen from Table 4, the new method can better distinguish the product quality of two enterprises, and the result shows that the product quality of the A factory is superior to that of the B factory, the related substance determination method established by the invention can detect more impurities than the standard method, the average total impurity amount (0.23%) is 0.1% greater than the standard result, and the method specificity is stronger.
The SPSS software is used for carrying out 'independent sample t test' on the total impurity data of the plant A and the plant B, and the result shows that when the legal standard method is adopted for testing, the mean value of the total impurities of the two plants has no obvious difference, the p value is 0.456(p is more than 0.05), which indicates that the quality difference between the plant A and the plant B cannot be distinguished by adopting the analysis of the legal standard method; when the related substance method provided by the invention is adopted for inspection, the mean value of the total impurities of the two plants has obvious difference, and the p value is 0.001(p is less than 0.01), which shows that the detection results of the samples of the two enterprises have extremely obvious difference, and the product quality of the plant A is obviously superior to that of the plant B. Therefore, compared with the existing standard method, the method provided by the invention is more accurate and can be better used for monitoring the quality of the lomefloxacin aspartate glucose injection.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (11)
1. A method for separating and measuring substances related to lomefloxacin aspartate glucose injection is characterized by comprising the steps of detecting the lomefloxacin aspartate glucose injection serving as a test sample solution by adopting a high performance liquid chromatography, wherein the high performance liquid chromatography adopts a chromatographic column with octadecylsilane chemically bonded silica as a filler, and linear gradient elution is carried out by using a mobile phase A and fluidity B; the mobile phase A is phosphate solution-methanol; the mobile phase B is acetonitrile;
the preparation method of the mobile phase A comprises the following steps: mixing 0.6-0.7% by mass of potassium dihydrogen phosphate solution and methanol according to a volume ratio of 70: 30-80: 20, and adjusting the pH value to 2.5-3.0 by adopting phosphoric acid;
the procedure for the linear gradient elution was:
2. the method of claim 1, wherein the potassium dihydrogen phosphate solution is 0.67% by weight, the volume ratio of the potassium dihydrogen phosphate solution to the methanol is 76:24, and the pH value is 2.7.
3. The method of claim 1, wherein the chromatography column is of the type Welch Ultimate XB-C18, 4.6 x 250mm, 5 μm.
4. The method according to claim 1, wherein the detection wavelength of the high performance liquid chromatography is 280 to 290 nm.
5. The method of claim 4, wherein the detection wavelength of the high performance liquid chromatography is 287 nm.
6. The method according to claim 1, wherein the flow rate of the high performance liquid chromatography is 0.5 to 1.5 ml/min.
7. The method of claim 6, wherein the high performance liquid chromatography has a flow rate of 1.0 ml/min.
8. The method according to claim 1, wherein the high performance liquid chromatography is carried out in an amount of 5 to 20. mu.L.
9. The method of claim 8, wherein the high performance liquid chromatography is performed in an amount of 10 μ L.
10. The method according to claim 1, wherein the column temperature of the high performance liquid chromatography is 35-45 ℃.
11. The method of claim 1, wherein the high performance liquid chromatography has a column temperature of 40 ℃.
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