CN114236018A - Caspofungin acetate and detection method of isomer thereof - Google Patents
Caspofungin acetate and detection method of isomer thereof Download PDFInfo
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Abstract
The application belongs to the technical field of chromatographic analysis, and particularly relates to a caspofungin acetate and a detection method of an isomer thereof. According to the detection method, high performance liquid chromatography is adopted to detect caspofungin acetate and isomers thereof; the mobile phase in the detection process of the high performance liquid chromatography is mobile phase A and mobile phase B; the mobile phase A is a first mixed solution, and the mobile phase B is acetonitrile or/and a second mixed solution; the first mixed solution is perchloric acid and sodium chloride aqueous solution, and the second mixed solution is acetonitrile and tetrahydrofuran; passing the mobile phase through a chromatographic column by isocratic elution; the chromatographic column is a hydrophilic HILIC chromatographic column; in the high performance liquid chromatography detection process, a test sample is diluted by a diluent and then is detected. The application provides a high performance liquid analysis method of caspofungin acetate and isomers thereof, which can effectively and simultaneously detect the caspofungin acetate and the net isomers thereof.
Description
Technical Field
The application belongs to the technical field of chromatographic analysis, and particularly relates to a caspofungin acetate and a detection method of an isomer thereof.
Background
Caspofungin acetate belongs to broad-spectrum antifungal medicines and has good antibacterial effect on fungi including aspergillus and candida. Caspofungin acetate is a semisynthetic derivative of pneumocandin B0, and a caspofungin acetate isomer is gradually transferred from nemoconazin C0 in a starting material to a final caspofungin acetate bulk drug through a synthetic route, so that a non-pharmacodynamic ingredient class caspofungin (caspofungin acetate isomer, impurity C for short) is generated, and the product quality is influenced. The control of caspofungin acetate isomer is a key project for product development and quality control. At present, no detection method for caspofungin acetate isomer is recorded in pharmacopoeia.
Disclosure of Invention
In order to fill the blank of the prior art, the application provides a high performance liquid analysis method for caspofungin acetate and isomers thereof, which can effectively and simultaneously detect the caspofungin acetate and the isomers thereof.
The application provides a detection method of caspofungin acetate and isomers thereof in a first aspect,
detecting caspofungin acetate and isomers thereof by adopting high performance liquid chromatography;
the mobile phase in the high performance liquid chromatography detection process is a mobile phase A and a mobile phase B; the mobile phase A is a first mixed solution, and the mobile phase B is acetonitrile or/and a second mixed solution; the first mixed solution is perchloric acid and sodium chloride aqueous solution, and the second mixed solution is acetonitrile and tetrahydrofuran;
the volume fraction of the mobile phase A in the mobile phase is 18% -28%, the volume fraction of the mobile phase B in the mobile phase is 72% -82%, and the isocratic elution time is 0-40 min;
in the high performance liquid chromatography detection process, a isocratic elution mode is adopted to enable the mobile phase to pass through a chromatographic column; the chromatographic column in the high performance liquid chromatography detection process is a hydrophilic HILIC chromatographic column;
in the high performance liquid chromatography detection process, a test sample is diluted by a diluent and then detected;
the caspofungin acetate has a structural formula shown in a formula 1, and the caspofungin acetate isomer has a structural formula shown in a formula 2;
Wherein the sample is a sample containing caspofungin acetate and caspofungin acetate isomers.
In another embodiment, in the isocratic elution, the volume fraction of the mobile phase A in the mobile phase is 18% -23%, and the volume fraction of the mobile phase B in the mobile phase is 77% -82%.
In another embodiment, in the isocratic elution, the volume fraction of the mobile phase A in the mobile phase is 24% to 28%, and the volume fraction of the mobile phase B in the mobile phase is 72% to 76%.
In another embodiment, in the first mixed solution, the mass fraction of the sodium chloride aqueous solution is 0.075% to 0.15%, and the volume ratio of the perchloric acid to the sodium chloride aqueous solution is 1: 1000.
Specifically, the preparation method of the first mixed solution comprises the steps of taking 1.0mL of perchloric acid and 0.75-1.5 g of sodium chloride, adding water to dissolve and diluting to 1000 mL; preferably: 1.0mL of perchloric acid and 0.75g of sodium chloride were taken, dissolved in water and diluted to 1000 mL.
In another embodiment, the volume ratio of the acetonitrile to the tetrahydrofuran in the second mixed solution is 95: 5.
In another embodiment, the diluent comprises a first mixed solution and acetonitrile; the volume ratio of the first mixed solution to the acetonitrile is (10-40) to (60-90).
Specifically, the mass fraction of the sodium chloride aqueous solution in the first mixed solution of the diluent is 0.075%, and the volume ratio of the perchloric acid to the sodium chloride aqueous solution is 1: 1000; the preparation method of the diluent comprises the steps of dissolving 1.0mL of perchloric acid and 0.75g of sodium chloride in water, diluting to 1000mL to obtain a first mixed solution, and mixing the first mixed solution with acetonitrile to obtain the diluent.
In another embodiment, the flow rate of the mobile phase in the high performance liquid chromatography detection process is 0.5-1.5 mL/min; the temperature of the chromatographic column is 25-40 ℃.
In another embodiment, the detector used in the high performance liquid chromatography is an ultraviolet detector, and the detection wavelength in the detection process of the high performance liquid chromatography is 200 nm-230 nm.
In another embodiment, the chromatographic column has a particle size of 3.0 to 5.0 μm.
Specifically, the HILIC chromatographic columns with the hydrophilic effect are SeQuat ZIC-HILIC 150mm multiplied by 4.6mm and 3.5 mu m.
In another embodiment, the caspofungin acetate has a structural formula shown in formula 1, and the caspofungin acetate isomer has a structural formula shown in formula 2;
the caspofungin acetate and caspofungin acetate isomers described herein have the structures shown in table 1.
TABLE 1
The application discloses a high performance liquid chromatography detection method of caspofungin acetate and isomers thereof, which comprises the following steps: (1) selecting a hydrophilic interaction chromatographic column; adopting mobile phase isocratic elution, wherein the phase A of the mobile phase is a first mixed solution of perchloric acid and sodium chloride, and the phase B of the mobile phase is a second mixed solution of acetonitrile and tetrahydrofuran; the detector adopted by the high performance liquid chromatography is an ultraviolet detector; (2) precisely weighing a proper amount of test sample, adding a blank solvent to dissolve the test sample, diluting the test sample into a sample solution with a certain concentration, and carrying out high performance liquid chromatography detection at a proper flow rate and a proper column temperature to analyze isomers in the sample solution. The method can effectively and simultaneously detect the caspofungin acetate and the isomers thereof, and has high sensitivity and good specificity.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a high performance liquid chromatogram of a system suitability solution provided in a comparative example of the present application;
FIG. 2 is a high performance liquid chromatogram of a solution suitable for use in the system provided in example 1 of the present application;
FIG. 3 is a high performance liquid chromatogram of a system suitability solution provided in example 2 of the present application;
FIG. 4 is a high performance liquid chromatogram of a system suitability solution provided in example 3 of the present application;
FIG. 5 is a high performance liquid chromatogram of a system suitability solution provided in example 4 of the present application;
FIG. 6 is a high performance liquid chromatogram of a system suitability solution provided in example 5 of the present application;
FIG. 7 is a high performance liquid chromatogram of a system suitability solution provided in example 6 of the present application;
FIG. 8 is a high performance liquid chromatogram for specificity verification comparison provided in example 7 of the present application;
FIG. 9 is a high performance liquid chromatogram of a system suitability solution provided in example 7 of the present application;
FIG. 10 is a linear plot of caspofungin acetate according to the detection method of the present application;
FIG. 11 is a residual map of caspofungin acetate according to the detection method of the present application;
FIG. 12 is a linear plot of caspofungin acetate isomer (impurity C) according to the detection method of the present application;
FIG. 13 is a residual plot of caspofungin acetate isomer (impurity C) according to the assay method of the present application.
Detailed Description
The application provides a caspofungin acetate and a detection method of its isomer, which can effectively detect the caspofungin acetate isomer and fill the blank of the prior art.
The technical solutions in the embodiments of the present application will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. 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 application.
The raw materials and reagents used in the following examples are commercially available or self-made.
The detection method of the following examples conforms to the specification of high performance liquid chromatography in appendix of the second part of the "Chinese pharmacopoeia" 2020 edition.
The caspofungin acetate isomer of the following examples is abbreviated as impurity C.
Comparative example
The comparative example provides a test for detecting caspofungin acetate and isomers thereof by adopting an ultra-high performance liquid chromatography method in the prior art, and the specific method comprises the following steps:
1. the EP pharmacopoeia Forum standard (Pharmeuropa 34.1 draft) uses an ultra-high performance liquid chromatograph ultraviolet visible light detector, the wavelength of the ultraviolet detector is 205nm, and an amino chromatographic column (Waters acquisition UPLC BEH Amide 150mm multiplied by 2.1mm,1.7 um) with the particle size of 1.7 μm is selected. Mobile phase a was buffered saline solution [ 2.88g ammonium dihydrogen phosphate dissolved in water and diluted to 500ml, pH adjusted to 3.2 with phosphoric acid ], mobile phase B was acetonitrile. Taking the mobile phase A: the volume ratio of the mobile phase B is 215: 785 isocratic elution was performed for 40 minutes.
2. Preparation of System suitability solutions
Taking impurity C according to the following table 2, placing the impurity C into a corresponding volumetric flask, adding a blank solvent (ultrapure water) to dissolve and dilute the impurity C to a scale mark, and shaking the mixture to prepare a mother liquor of the impurity C. And weighing 20mg of caspofungin acetate reference substance, placing the caspofungin acetate reference substance into a 100ml volumetric flask, precisely weighing 1.0ml of impurity C mother liquor, placing the impurity C mother liquor into the volumetric flask, diluting the impurity C mother liquor to the scale of the volumetric flask by using a blank solvent, and shaking up.
Table 2 table of mother liquor of each impurity of comparative example
3. The system suitability solution was tested with a sample size of 5 μ L, a flow rate of 0.35ml/min, and a column temperature of 40 ℃. The detection pattern of the solution for system suitability is shown in FIG. 1. As can be seen from FIG. 1, the present comparative example can detect caspofungin acetate and caspofungin acetate isomers.
Example 1
The embodiment of the application provides detection of caspofungin acetate and isomers thereof, and the specific method comprises the following steps:
1. an ultraviolet detector of high performance liquid chromatograph with wavelength of 220nm is used, and a hydrophilic chromatographic column (Kromasil-HILIC-D250 mm × 4.6mm, 5 μm) with particle size of 3.5 μm is selected. Mobile phase a is perchloric acid: a first mixed solution of 0.075% sodium chloride (1: 1000) (first mixed solution prepared by dissolving 1.0mL of perchloric acid and 0.75g of sodium chloride in water and diluting to 1000 mL) was prepared, and mobile phase B was acetonitrile. Taking the mobile phase A: the volume ratio of the mobile phase B is 18%: 82% was isocratic eluted and run for 30 min.
2. Preparation of System suitability solutions
Impurity C was taken according to table 3 below and placed in the corresponding volumetric flask, and the blank solvent [ mobile phase a of step 1: acetonitrile volume ratio is 18%: 82% mixed solution ] was dissolved and diluted to the scale line, and shaken to prepare a mother liquor of impurity C. And weighing 40mg of caspofungin acetate reference substance, placing the caspofungin acetate reference substance into a 20ml volumetric flask, precisely weighing 1.0ml of impurity C mother liquor, placing the impurity C mother liquor into the 20ml volumetric flask, diluting the impurity C mother liquor to the scale of the volumetric flask by using a blank solvent, and shaking up.
Table 3 table for preparation of mother liquor of each impurity of example 1
3. The system suitability solution was tested with a sample size of 20 μ L, a flow rate of 1.0ml/min, and a column temperature of 35 ℃. The detection pattern of the solution for system suitability is shown in FIG. 2. As can be seen from FIG. 2, caspofungin acetate and caspofungin acetate isomers can be detected in this example.
Example 2
The embodiment of the application provides detection of caspofungin acetate and isomers thereof, and the specific method comprises the following steps:
1. a high performance liquid chromatograph ultraviolet visible detector is used, the wavelength of the ultraviolet detector is 220nm, and hydrophilic interaction chromatographic columns (SeQuat ZIC-HILIC 150mm X4.6 mm, 3.5 μm) with the particle size of 3.5 μm are selected. Mobile phase a is perchloric acid: a first mixed solution of 0.075% sodium chloride (1: 1000) (first mixed solution prepared by dissolving 1.0mL of perchloric acid and 0.75g of sodium chloride in water and diluting to 1000 mL) was prepared, and mobile phase B was acetonitrile. Taking the mobile phase A: the volume ratio of the mobile phase B is 18%: 82% was run for 40 minutes with isocratic elution.
2. Preparation of system applicability solution: the same as in example 1.
3. The system suitability solution was tested with a sample size of 20 μ L, a flow rate of 1.0ml/min, and a column temperature of 35 ℃. The detection pattern of the system suitability solution is shown in FIG. 3. As can be seen from FIG. 3, caspofungin acetate and caspofungin acetate isomers can be detected in this example.
Example 3
The embodiment of the application provides detection of caspofungin acetate and isomers thereof, and the specific method comprises the following steps:
1. a high performance liquid chromatograph ultraviolet visible detector is used, the wavelength of the ultraviolet detector is 220nm, and hydrophilic interaction chromatographic columns (SeQuat ZIC-HILIC 150mm X4.6 mm, 3.5 μm) with the particle size of 3.5 μm are selected. Mobile phase a is perchloric acid: a first mixed solution of 0.075% sodium chloride (1: 1000) (a first mixed solution prepared by dissolving 1.0mL of perchloric acid and 0.75g of sodium chloride in water and diluting to 1000 mL), and a mobile phase B is acetonitrile: and a second mixed solution of tetrahydrofuran (95: 5 by volume). Taking the mobile phase A: the volume ratio of the mobile phase B is 18%: 82% was run for 40 minutes with isocratic elution.
2. Preparation of system applicability solution: the same as in example 1.
3. The system suitability solution was tested with a sample size of 20 μ L, a flow rate of 1.0ml/min, and a column temperature of 35 ℃. The detection pattern of the system suitability solution is shown in FIG. 4. As can be seen from FIG. 4, caspofungin acetate and caspofungin acetate isomers can be detected in this example.
Example 4
The embodiment of the application provides detection of caspofungin acetate and isomers thereof, and the specific method comprises the following steps:
1. a high performance liquid chromatograph ultraviolet visible detector is used, the wavelength of the ultraviolet detector is set to be 220nm, and hydrophilic interaction chromatographic columns (SeQuat ZIC-HILIC 150mm x 4.6mm, 3.5 μm) with the particle size are selected. Mobile phase a is perchloric acid: a first mixed solution of 0.075% sodium chloride (1: 1000) (a first mixed solution prepared by dissolving 1.0mL of perchloric acid and 0.75g of sodium chloride in water and diluting to 1000 mL), and a mobile phase B is acetonitrile: and a second mixed solution of tetrahydrofuran (95: 5 by volume). Taking the mobile phase A: the volume ratio of the mobile phase B is 20%: 80% was run for 40 min with isocratic elution.
2. Preparation of system applicability solution: the same as in example 1.
3. Taking a system suitability solution for testing. The sample introduction was 20. mu.L, the flow rate was 0.8ml/min, and the column temperature was 30 ℃. The detection pattern of the solution for system suitability is shown in FIG. 5. As can be seen from FIG. 5, caspofungin acetate and caspofungin acetate isomers can be detected in this example.
Example 5
The embodiment of the application provides detection of caspofungin acetate and isomers thereof, and the specific method comprises the following steps:
1. a high performance liquid chromatograph ultraviolet visible detector is used, the wavelength of the ultraviolet detector is set to be 220nm, and hydrophilic interaction chromatographic columns (SeQuat ZIC-HILIC 150mm x 4.6mm, 3.5 μm) with the particle size are selected. Mobile phase a is perchloric acid: a first mixed solution of 0.15% sodium chloride (1: 1000) (a first mixed solution prepared by dissolving 1.0mL of perchloric acid and 1.5g of sodium chloride in water and diluting to 1000 mL), and a mobile phase B is acetonitrile: and a second mixed solution of tetrahydrofuran (95: 5 by volume). Taking the mobile phase A: the volume ratio of the mobile phase B is 24%: 76% was run for 40 minutes with isocratic elution.
2. Preparation of system applicability solution: the same as in example 1.
3. Taking a system suitability solution for testing. The sample introduction was 20. mu.L, the flow rate was 1.0ml/min, and the column temperature was 30 ℃. The detection profile of the system suitability solution is shown in FIG. 6. As can be seen from FIG. 6, caspofungin acetate and caspofungin acetate isomers can be detected in this example.
Example 6
The embodiment of the application provides detection of caspofungin acetate and isomers thereof, and the specific method comprises the following steps:
1. a high performance liquid chromatograph ultraviolet visible detector is used, the wavelength of the ultraviolet detector is set to be 220nm, and hydrophilic interaction chromatographic columns (SeQuat ZIC-HILIC 150mm x 4.6mm, 3.5 μm) with the particle size are selected. Mobile phase a is perchloric acid: a first mixed solution of 0.075% sodium chloride (1: 1000) (a first mixed solution prepared by dissolving 1.0mL of perchloric acid and 0.75g of sodium chloride in water and diluting to 1000 mL), and a mobile phase B is acetonitrile: and a second mixed solution of tetrahydrofuran (95: 5) (volume ratio 95: 5). Taking the mobile phase A: the volume ratio of the mobile phase B is 24%: 76% was run for 40 minutes with isocratic elution.
2. Preparation of system applicability solution: the same as in example 1.
3. Taking a system suitability solution for testing. The sample introduction was 20. mu.L, the flow rate was 1.0ml/min, and the column temperature was 35 ℃. The detection profile of the system suitability solution is shown in FIG. 7. As can be seen from FIG. 7, caspofungin acetate and caspofungin acetate isomers can be detected in this example.
Example 7
The embodiment of the application inspects the methodology of the detection method of the carprofen acetate and the net isomer thereof, and the specific method comprises the following steps:
(1) detection conditions are as follows: a high performance liquid chromatograph ultraviolet visible detector pair is used, the wavelength of the ultraviolet detector is set to be 220nm, and hydrophilic interaction chromatographic columns (SeQuat ZIC-HILIC 150mm X4.6 mm, 3.5 μm) with the particle size of 3.5 μm are selected. Mobile phase a is perchloric acid: a first mixed solution of 0.075% sodium chloride (1: 1000) (a first mixed solution prepared by dissolving 1.0mL of perchloric acid and 0.75g of sodium chloride in water and diluting to 1000 mL), and a mobile phase B is acetonitrile: and a second mixed solution of tetrahydrofuran (95: 5 by volume). Mixing the mobile phase A: acetonitrile volume ratio of 24%: and preparing a test sample by using the 76% mixed solution as a diluent. Taking the mobile phase A: the volume ratio of the mobile phase B is 24%: 76% was run for 40 minutes with isocratic elution. The sample introduction was 20. mu.L, the flow rate was 1.0ml/min, and the column temperature was 35 ℃.
(2) Preparation of the solution
a. Preparing an impurity C (caspofungin acetate isomer) positioning solution:
taking about 10mg of impurity C, precisely weighing, placing in a 50mL volumetric flask, adding a diluent to dissolve and dilute to the scale of the volumetric flask, shaking up to be used as an impurity C stock solution, precisely weighing 1mL of impurity C stock solution, placing in a 20mL volumetric flask, adding the diluent to dilute to the scale, shaking up to be used as an impurity C positioning solution. (10. mu.g/ml), the diluent used in this example was prepared by mixing the mobile phase A obtained in step 1 with acetonitrile, and the ratio of the mobile phase A: the volume ratio of acetonitrile is 24%: 76 percent.
b. Preparation of system applicability solution:
weighing about 40mg of caspofungin acetate working reference substance, placing the caspofungin acetate working reference substance into a 20ml volumetric flask, adding 1ml of impurity C stock solution, adding a diluent to dissolve and dilute the impurity C stock solution to the scale of the volumetric flask, and shaking up to obtain a system applicability solution.
c. Preparing a test solution:
taking about 40mg of caspofungin acetate, precisely weighing, placing in a 20ml volumetric flask, adding a diluent to dissolve and dilute to the scale of the volumetric flask, and shaking up to obtain the caspofungin acetate.
d. Preparing a control solution:
and d, precisely transferring 1ml of the test solution obtained in the step c, putting the test solution into a 100ml volumetric flask, adding a diluent to be diluted to the scale of the volumetric flask, and shaking up to obtain the test solution.
e. Preparing an impurity C quantitative limiting solution:
precisely measuring 3.0ml of impurity C positioning solution, placing the solution in a 10ml volumetric flask, adding a diluent to dilute the solution to the scale of the volumetric flask, and shaking up to obtain the product.
f. Preparation solution of impurity C detection limiting solution:
precisely transferring 3.0ml of impurity C quantitative limit solution, placing the solution into a 10ml volumetric flask, adding a diluent to dilute the solution to the scale of the volumetric flask, and shaking up to obtain the product.
g. Preparation of caspofungin acetate quantitative limiting solution:
precisely weighing about 10mg of caspofungin acetate working reference substance, placing the caspofungin acetate working reference substance into a 100ml measuring flask, adding a diluent to dissolve and dilute the caspofungin acetate working reference substance to the scale of the measuring flask, shaking up the caspofungin working reference substance to serve as reference substance storage solution 1, precisely weighing 2ml of the solution (reference substance storage solution 1) again, placing the solution into a 20ml measuring flask, adding the diluent to dilute the solution to the scale of the measuring flask, taking the solution as reference substance storage solution 2, precisely weighing 3ml of the solution (reference substance storage solution 2) again, placing the solution into a 10ml measuring flask, adding the diluent to dilute the scale of the measuring flask, and shaking up the solution to obtain the caspofungin acetate working reference substance.
h. Preparation of caspofungin acetate detection limit solution:
and (3) precisely transferring 3.0ml of the caspofungin acetate quantitative limiting solution obtained in the step g, putting the solution into a 10ml volumetric flask, adding a diluent to dilute the solution to the scale of the volumetric flask, and shaking up the solution to obtain the caspofungin acetate quantitative limiting solution.
i. Preparation of caspofungin acetate linear stock solution:
taking 10mg of caspofungin acetate working reference substance, precisely weighing, placing into a 50ml volumetric flask, adding a diluent to dissolve and dilute to the scale of the volumetric flask, and shaking up to obtain the caspofungin acetate working reference substance.
j. Preparation of impurity C linear stock solution:
and taking about 10mg of the impurity C reference substance, precisely weighing, placing in a 50ml volumetric flask, adding a diluent to dissolve and dilute to the scale of the volumetric flask, and shaking up to obtain the product.
k. Preparation of LOQ-accurate solution of impurity C:
precisely transferring 3ml of the linear stock solution of the impurity C in the step j, putting the linear stock solution of the impurity C in a 10ml volumetric flask, and adding a diluent to dilute the linear stock solution to the scale of the volumetric flask to obtain a stock solution of an LOQ reference substance; precisely weighing about 40mg of caspofungin acetate sample, placing into a 20ml volumetric flask, precisely adding 1ml of LOQ reference substance stock solution, adding diluent, dissolving, diluting to scale, and shaking. (parallel preparation of 3 portions)
l, preparation of 100% accuracy solution of impurity C:
taking about 40mg of caspofungin acetate sample, precisely weighing, placing in a 20ml volumetric flask, precisely adding 1ml of impurity C stock solution, adding a diluent to dissolve, diluting to the scale of the volumetric flask, and shaking up to obtain a 100% accuracy solution. (3 parts were prepared in parallel).
m, preparation of 150% accuracy solution of impurity C:
taking about 40mg of caspofungin acetate sample, precisely weighing, placing in a 20ml volumetric flask, precisely adding 1.5ml of impurity C stock solution, adding a diluent to dissolve, diluting to the scale of the volumetric flask, and shaking up to obtain a 150% accuracy solution. (parallel preparation of 3 portions)
n, preparing a sample labeling solution:
taking about 40mg of caspofungin acetate test sample, precisely weighing, placing in a 20ml volumetric flask, precisely adding 1.0ml of impurity C stock solution, adding a diluent to dissolve, diluting to the scale of the volumetric flask, and shaking up to obtain a standard test sample solution.
o, preparing a sample and a standard control solution:
precisely transferring the sample in the step n, adding 1.0ml of the standard solution, placing the sample in a 100ml volumetric flask, adding a diluent to dilute the sample to the scale of the volumetric flask, and shaking up to obtain the product
(3) And carrying out methodology verification on the established high performance liquid chromatography analysis method of caspofungin acetate and the isomers thereof according to the chromatographic parameters. The verification items include system applicability, specificity, quantitation limit, detection limit, linearity, accuracy, precision (including repeatability and reproducibility), solution stability, and durability.
(4) Methodological validation results
The methodology was verified by the above method, and the verification results are shown below:
1. the specificity result of the detection method of the embodiment is as follows: the blank solution has no interference to the detection, and the separation degree of the caspofungin acetate peak and the impurity C peak (caspofungin acetate isomer peak) in the solution with system applicability is 3.9.
2. The quantitative limit result of the detection method of the embodiment is shown in table 4, and the result in table 4 shows that the quantitative limit concentration of caspofungin acetate is 3.1993 mug/ml, which is equivalent to 0.160% of the concentration of the test sample, and the peak area RSD of six-needle LOQ is 3.4%; ② the limit concentration of the impurity C is 2.9444 mug/ml, which is equivalent to 0.147% of the concentration of the sample, and the peak area RSD of six-needle LOQ is 5.6%.
TABLE 4
3. The detection limit results of the detection method of the embodiment are shown in table 5, table 6, table 7 and fig. 10 to fig. 13, and the results in table 5 show that the detection limit concentration of caspofungin acetate is 0.9598 mug/ml, which is equivalent to 0.0480% of the concentration of the test sample; ② the detection limit concentration of the impurity C is 0.8833 mug/ml, which is equivalent to 0.442 percent of the concentration of the sample. The linearity results of caspofungin acetate are shown in table 6, fig. 10 and fig. 11, and the linearity results of impurity C are shown in table 7, fig. 12 and fig. 13. The results in table 6, table 7 and fig. 10 to fig. 13 show that the correlation coefficient of caspofungin acetate is 0.9998; RSD of response factor 1.5%; the linear result ratio of the linear formula intercept to the limit concentration is 2 percent; the sum of the squares of the residuals is 0.05398, and the residual points in the residual map are not all on the same side; impurity C correlation coefficient is 0.9999; RSD of response factor 1.7%; the linear result ratio of the linear formula intercept to the limit concentration is 0%; the sum of squares of the residuals is 4.73742, the residual points are not all on the same side in the residual map, the correction factor of the impurity C is 0.91, the above results all meet the acceptance criterion, and the method of the embodiment has good linearity.
TABLE 5
TABLE 6
TABLE 7
4. The detection range result of the detection method of the embodiment is as follows: the detection range of caspofungin acetate is 3.1306-20.8709 mug/mL, which is equivalent to 0.16-1.04% of the concentration of a test sample; ② the detection range of the impurity C is 2.7707-18.4710 mug/mL, equivalent to 27.71-184.71% of the limit concentration and equivalent to 0.14-0.92% of the concentration of the test sample.
5. The accuracy results of the detection method of the embodiment are shown in table 8, and the results of table 8 show that the single recovery rate of the LOQ concentration is 118.3% -124.0%, the average recovery rate is 121.0%, and the recovery rate (n = 3) RSD is 2.4%; the single recovery rate of other concentrations is between 111.8% and 119.8%, the average recovery rate is 116.5%, and the recovery rate (n = 6) RSD is 3.2%. The experimental results all meet the acceptable standard, and the method has good accuracy.
TABLE 8
6. The precision results of the assay of this example included reproducibility and reproducibility, with the reproducibility results shown in Table 9 and the reproducibility results shown in tables 10 and 11. The results in table 9 show that no C is detected in the test sample solution in the repeatability experiment results, the single recovery rate of the impurity C in the added standard test sample solution is 95.8% -103.2%, the average recovery rate of the impurity C is 100.3%, and the recovery rate of the impurity C RSD is 2.8%. The experimental results all meet the acceptable standard, and the method has good repeatability.
Table 10 shows reproducibility results of different laboratories, table 11 shows comparison results of 12 parts of reproducibility and reproducibility results, and the results of table 10 and table 11 show that the recovery rate of impurity C in six parts of reproducible test sample solutions is 0.02%, the recovery rate of impurity C in 6 parts of standard test sample solutions is 106.7% -114.8%, the average recovery rate of impurity C is 110.1%, and the recovery rate of impurity C RSD is 2.7%; the single recovery rate of the impurity C in 12 parts of the standard sample solution is 95.8-114.8%, the average recovery rate of the impurity C is 105.2%, and the recovery rate RSD of the impurity C is 5.5%. The experimental results all accord with the acceptable standard, and the method has good precision.
TABLE 9
TABLE 11
7. The solution stability results of the detection method of this embodiment include the stability test results of the sample solution and the stability test results of the sample solution, and the results are shown in table 12 and table 13, and the results show that no impurity C is detected in the sample solution, the maximum value of the rate of change of the peak area of the impurity C in the sample solution added with the standard is 5.93% within 24 hours, the maximum value of the rate of change of the main peak area of the control solution added with the standard is 9.45% within 24 hours, and the number of impurities is 2 within 24 hours. By combining the above analysis, the sample solution is stable within 24h at 5 ℃.
TABLE 12
Watch 13
8. The results of the durability test method of this example include the results of the system suitability and the results of the recovery rate of the sample solution, and the results are shown in tables 14 and 15, which show that the results of the recovery rate of the impurity C in the experimental results meet the acceptable standards compared with the normal conditions when the chromatographic conditions are slightly changed (different flow rates ± 0.1 ml/min; column temperatures ± 2 ℃; different chromatographic columns). Indicating that the method is good in durability.
TABLE 14
The high performance liquid chromatogram of example 7 of the present application and the high performance liquid chromatogram of the EP pharmacopeia forum standard (Pharmeuropa 34.1 draft) are shown in fig. 8 and fig. 9, and the specific data of system applicability are compared in table 16, and the separation degree of the detection method of caspofungin acetate and its isomer of the present application is superior to that of the EP pharmacopeia forum standard (Pharmeuropa 34.1 draft); meanwhile, the system applicability, specificity, detection limit, quantitative limit, linearity, accuracy, repeatability, reproducibility and durability of the method can meet the experimental requirements and meet the requirements. Therefore, the detection method can be suitable for detecting the caspofungin acetate and the isomers thereof.
TABLE 16 comparison of System suitability results
The foregoing is only a preferred embodiment of the present application and it should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present application, and these improvements and modifications should also be considered as the protection scope of the present application.
Claims (9)
1. A method for detecting caspofungin acetate and isomers thereof,
detecting caspofungin acetate and isomers thereof by adopting high performance liquid chromatography;
the mobile phase in the high performance liquid chromatography detection process is a mobile phase A and a mobile phase B; the mobile phase A is a first mixed solution, and the mobile phase B is acetonitrile or/and a second mixed solution; the first mixed solution is perchloric acid and sodium chloride aqueous solution, and the second mixed solution is acetonitrile and tetrahydrofuran;
the volume fraction of the mobile phase A in the mobile phase is 18% -28%, the volume fraction of the mobile phase B in the mobile phase is 72% -82%, and the isocratic elution time is 0-40 min;
in the high performance liquid chromatography detection process, a isocratic elution mode is adopted to enable the mobile phase to pass through a chromatographic column; the chromatographic column in the high performance liquid chromatography detection process is a hydrophilic HILIC chromatographic column;
in the high performance liquid chromatography detection process, a test sample is diluted by a diluent and then detected;
the caspofungin acetate has a structural formula shown in a formula 1, and the caspofungin acetate isomer has a structural formula shown in a formula 2;
2. The detection method according to claim 1, wherein in the isocratic elution, the volume fraction of the mobile phase A in the mobile phase is 18% to 23%, and the volume fraction of the mobile phase B in the mobile phase is 77% to 82%.
3. The detection method according to claim 1, wherein in the isocratic elution, the volume fraction of the mobile phase A in the mobile phase is 24% to 28%, and the volume fraction of the mobile phase B in the mobile phase is 72% to 76%.
4. The detection method according to claim 1, wherein the mass fraction of the sodium chloride aqueous solution in the first mixed solution is 0.075% to 0.15%, and the volume ratio of the perchloric acid to the sodium chloride aqueous solution is 1: 1000.
5. The detection method according to claim 1, wherein the volume ratio of the acetonitrile to the tetrahydrofuran in the second mixed solution is 95: 5.
6. The detection method according to claim 4, wherein the diluent comprises a first mixed solution and acetonitrile; the volume ratio of the first mixed solution to the acetonitrile is (10-40) to (60-90).
7. The detection method according to claim 1, wherein the flow rate of the mobile phase in the high performance liquid chromatography detection process is 0.5-1.5 mL/min; the temperature of the chromatographic column is 25-40 ℃.
8. The detection method according to claim 1, wherein the detector used in the high performance liquid chromatography is an ultraviolet detector, and the detection wavelength in the detection process of the high performance liquid chromatography is 200 nm-230 nm.
9. The detection method according to claim 1, wherein the particle size of the chromatographic column is 3.0 to 5.0 μm.
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