CN110398548B - High performance liquid chromatography separation and determination method for related impurities in fosfomycin trometamol raw material medicine and preparation thereof and application - Google Patents
High performance liquid chromatography separation and determination method for related impurities in fosfomycin trometamol raw material medicine and preparation thereof and application Download PDFInfo
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Abstract
The invention discloses a high performance liquid chromatography separation determination method and application of impurities in a fosfomycin trometamol raw material medicine and a preparation, which are used for separating impurities; detecting with chromatographic column with aminopropyl silane bonded silica gel as filler in a differential detector; mixing phosphate buffer solution and methanol-acetonitrile as a mobile phase; 1. system applicability solution preparation: wetting the raw material medicine or preparation with water, heating, adding a mobile phase for dissolving and diluting to obtain a solution A; dissolving the raw material medicine or the preparation with A and diluting to obtain a system applicability solution; 2. preparing a test solution: dissolving the raw material medicine or preparation with mobile phase and diluting into fosfomycin trometamol with set content as test solution; 3. preparation of control solution: taking a test solution diluted by a mobile phase until the fosfomycin-containing tromethamine is equivalent to a solution with the mass concentration of 0.3-0.5% of the test solution as a control solution; 4. the determination method comprises the following steps: and (3) injecting the three solutions into a liquid chromatograph respectively, and calculating the content of each impurity in the raw material medicine by using a main component self-contrast method.
Description
Technical Field
The invention belongs to the technical field of medicines, and relates to a separation and determination method and application of related substances of a medicine, in particular to a high performance liquid chromatography separation and determination method and application of related impurities in a fosfomycin trometamol raw material medicine and a preparation thereof.
Background
Fosfomycin Trometamol, the English name of which is Fosfomycin Tromotal, the chemical name of which is (1R,2S) - (1, 2-epoxypropyl) phosphonic acid 2-amino-2- (hydroxymethyl) -1, 3-propanediol salt (1:1), and the chemical formula of which is C7H18NO7P, molecular weight 259.20, CAS number 78964-85-9, structural formula as follows:
the indications of fosfomycin trometamol are to treat acute simple lower urinary tract infection caused by sensitive bacteria (such as acute cystitis, acute attack of chronic cystitis, acute urethrovesical syndrome, nonspecific urethritis, asymptomatic bacteriuria during pregnancy and urinary tract infection after operation) and prevent urinary tract infection in surgery and infection caused by urinary tract diagnosis manipulation.
At present, the quality standards of fosfomycin trometamol raw material medicines are collected in European Pharmacopoeia (EP), British Pharmacopoeia (BP), Chinese Pharmacopoeia (CP) and the like, the quality standards of fosfomycin trometamol powder are also collected in Chinese pharmacopoeia, but related impurities are not controlled. The detection of related impurities in the quality standard of fosfomycin trometamol in pharmacopoeia of various countries adopts high performance liquid chromatography, and the chromatographic conditions are the same: the chromatographic column is an aminopropyl bonded silica gel column, the mobile phase is 10.89g/L potassium dihydrogen phosphate solution, and the detector is a differential detector. According to research, the chromatographic condition can effectively detect the impurity A, the impurity B and the impurity C, but because the fosfomycin chromatographic peak is seriously trailing under the chromatographic condition, the impurity D which is immediately followed to the chromatographic condition can not be effectively separated and can not be detected, and the aminopropyl bonded silica gel column is extremely easy to hydrolyze in a pure water phase mobile phase, so that the column efficiency of the amino column is rapidly reduced, and the service life of the chromatographic column is extremely short.
Wherein, the related impurity A, impurity B, impurity C and impurity D in pharmacopoeia of various countries have the following structural formulas:
impurity A:
impurity B:
impurity C:
impurity D:
in addition to the above four impurities of known structure, other impurities may also be present in fosfomycin trometamol bulk drug and its formulation. Fosfomycin trometamol is prepared by chemical synthesis, and different kinds of process impurities are generated due to the difference of starting materials and different synthesis processes. Meanwhile, due to the difference of the preparation prescription, different auxiliary materials can also influence the impurity composition of the fosfomycin trometamol preparation, particularly influence the types of degradation impurities, but the impurities have no research report of structure confirmation and detection.
As can be seen from a large number of documents and patent searches, in the prior art, although the reports of detecting related substances of fosfomycin trometamol by using capillary electrophoresis and electrospray ion trap mass spectrometry are available, the two methods have poor universality and high detection cost due to the accessibility problem of detection instruments, so that the two methods are not widely adopted.
Since the impurities are important quality control indexes affecting the safety of the medicine, the development of a high-sensitivity fosfomycin trometamol and a method for detecting substances related to the preparation thereof is of great significance to the quality control.
Disclosure of Invention
The invention aims to solve the technical problem of providing a high performance liquid chromatography separation and determination method for impurity separation, which can effectively separate fosfomycin trometamol from related impurities and prolong the service life of a chromatographic column, thereby effectively controlling the product quality, reducing the detection cost and improving the economic benefit.
The technical problem to be solved for the separation and detection of the fosfomycin trometamol impurity can be implemented by the following technical scheme.
A high performance liquid chromatography separation determination method for related impurities in a fosfomycin trometamol raw material medicine and a preparation thereof is used for separating one or more of impurity A, impurity B, impurity C and impurity D; the chromatographic column using aminopropyl silane bonded silica gel as a filling agent is characterized in that:
detecting by using a differential detector or an ultraviolet detector, taking 20-100 mmol/L phosphate buffer solution as a mobile phase A, taking methanol-acetonitrile as a mobile phase B, and mixing the mobile phase A and the mobile phase B according to the volume ratio of (95:5) - (50:50) to obtain a mobile phase; the flow rate of the mobile phase is 0.8-1.2 mL/min; the temperature of a chromatographic column incubator is 20-40 ℃; the sample amount is 1-500 mul; the method specifically comprises the following steps:
(1) the solution is prepared according to the following mixture ratio:
1) preparing a system applicability solution:
taking 0.3g of fosfomycin trometamol raw material medicine or a preparation thereof, wetting the raw material medicine or the preparation with 10-300 mu l of water, heating the raw material medicine or the preparation at 40-80 ℃ for 12-36 hours, adding a mobile phase for dissolving and diluting the raw material medicine or the preparation to 10-50 ml, and shaking the raw material medicine or the preparation uniformly to obtain a solution A;
dissolving 0.3g of fosfomycin trometamol raw material medicine or a preparation thereof by using the solution A and diluting to 2.5-10 ml to be used as a system applicability solution;
2) and preparing a test solution:
according to the new preparation of clinical application, taking a proper amount of fosfomycin trometamol raw material medicine or a preparation thereof, precisely weighing, adding a mobile phase for dissolving, and quantitatively diluting into a solution containing fosfomycin trometamol 60-240 mg in each lml as a test solution;
3) and preparing a control solution:
precisely measuring a proper amount of a test solution, and quantitatively diluting the test solution by using a mobile phase until the concentration of fosfomycin-containing tromethamine is equivalent to 0.3-0.5% (mass ratio) of the test solution, wherein the fosfomycin-containing tromethamine is used as a reference solution;
(2) the determination method comprises the following steps:
precisely measuring 1-100 mul (preferably 5-50 mul) of the system applicability solution, the reference solution and the sample solution respectively, injecting into a liquid chromatograph, and recording a chromatogram; the content of four related substances, namely impurity A, impurity B, impurity C and impurity D in the fosfomycin trometamol bulk drug or preparation and the content of other possible known or unknown impurities are calculated by adopting a main component self-comparison method.
The high performance liquid chromatography separation and determination method for the related impurities in the fosfomycin trometamol raw material medicine and the preparation thereof can also be used for separating and detecting one or more unknown impurities.
As a further improvement of the technical scheme, the mobile phase A is a buffer salt solution consisting of one or more of sodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium phosphate, dipotassium hydrogen phosphate and potassium dihydrogen phosphate.
As one preferable embodiment of the invention, the mobile phase A is 30-100 mmol/L potassium dihydrogen phosphate buffer solution.
As a further improvement of the technical scheme, the volume ratio of methanol to acetonitrile in the mobile phase B is (0: 100) to (100: 0). The ratio of methanol to acetonitrile in the mobile phase B is preferably (75:25) to (50: 50).
As a further improvement of the technical scheme, the volume ratio of the mobile phase A to the mobile phase B is (80:20) - (65: 35).
As a preferred embodiment of the invention, the chromatographic column using aminopropylsilane bonded silica gel as a filler is selected from Agilent Zorbax NH2(specification: 5. mu.m, 4.6X 250mm or 3. mu.m, 4.6X 150 mm;), Waters SpherisorbNH2(specification: 5 μm, 4.6X 250mm or 3 μm, 4.6X 150mm), Osaka Soda Capcell Pak NH2(specification: 5 μm, 4.6X 250mm or 3 μm, 4.6X 150mm), Phenomenex Luna-NH2(specification: 5 μm, 4.6X 250 mm; or 3 μm, 4.6X 150mm) or a column of equivalent column efficiency.
The invention also aims to provide an impurity reference substance for researching the fosfomycin trometamol related substances (impurities) by utilizing the separation and detection method.
Specifically, the fosfomycin trometamol reference substance is prepared by separating one or more of impurity A, impurity B, impurity C and impurity D to form an application of the impurity reference substance.
Further, the application comprises the steps of collecting the impurity A, the impurity B, the impurity C and the impurity D step by step, and desalting and freeze-drying.
Compared with the prior art, the invention has the advantages and beneficial effects that:
1. according to the chromatographic conditions, an organic phase and a buffer salt solution in a certain proportion are used as a mobile phase, so that four related substances including fosfomycin trometamol and A, B, C, D as well as unknown impurities (see unknown impurity 1 in the following table 1) can be effectively separated, particularly, the separation degree of the impurity D is more than 2.5, and the method is obviously superior to the analysis method recorded in pharmacopoeia of various countries.
2. The mobile phase of the invention is added with an organic phase composed of methanol and acetonitrile in a certain proportion, which greatly improves the stability of the aminopropyl bonded silica gel column, overcomes the serious defects of rapid loss of the fixed phase of the aminopropyl bonded silica gel column, rapid column efficiency reduction, short service life of the chromatographic column, large change of retention time and the like caused by adopting a pure water phase as the mobile phase in a pharmacopoeia method, obviously prolongs the service life of the chromatographic column and reduces the production cost.
3. The separation detection method is simple and easy to implement, can be used for preparing the fosfomycin trometamol related substance A, B, C, D, and provides a related substance reference substance and an effective quality control method for research, development and production of fosfomycin trometamol and preparations thereof, thereby further ensuring the product quality and reducing the detection cost.
Drawings
The following detailed description of embodiments of the invention refers to the accompanying drawings.
FIG. 1 is an HPLC chromatogram of a solution suitable for use in the system of example 1;
FIG. 2 is an HPLC chromatogram of a sample solution of a fosfomycin trometamol formulation in example 1;
FIG. 3 is an HPLC chromatogram of a system suitability solution in comparative example 1;
FIG. 4 is an HPLC chromatogram of a sample solution of a fosfomycin trometamol preparation in comparative example 1;
wherein, 1,2, 3, 4, 5, X1, X2 and X3 on the chromatogram map in the figure represent impurity B, impurity C, impurity A, fosfomycin, impurity D, unknown impurity 1, unknown impurity 2 and unknown impurity 3 respectively.
Detailed Description
The optimization and methodology research results of the detection method of fosfomycin trometamol raw material medicine and related substances of the preparation thereof are as follows:
(1) analytical method optimization
In view of the fact that known impurities D are controlled under the relevant substance detection items of European Pharmacopoeia (EP), British Pharmacopoeia (BP) and Chinese Pharmacopoeia (CP), but practically all pharmacopoeia methods cannot effectively detect the impurities D at present, chromatographic condition optimization is tried on the basis of the pharmacopoeia methods, the composition of a mobile phase is optimized by investigating measures such as different buffer salt concentrations, types of the organic phase, the proportion of the organic phase and a water phase, and finally the buffer salt and the organic phase are selected as the mobile phase, so that the impurities D can be detected. The system applicability and sensitivity comparison results of the optimized method (method two) and the pharmacopoeia method (method one) are shown in the following table 1:
TABLE 1 System suitability and sensitivity comparison results
Remarking: "- -" indicates that the impurities were not separated out.
The result shows that the optimized method can effectively detect the impurity D, simultaneously detect the unknown impurity 1, the unknown impurity 2 and the unknown impurity 3 which can not be detected by the pharmacopoeia method, and has higher sensitivity than the pharmacopoeia method.
(2) Results of the methodological study of the analytical methods
1) Specificity test
Forced degradation test: under the conditions of high temperature, acid, alkali and oxidation, the separation of the main peak of fosfomycin, the peaks of known impurities (A, B, C, D) and degradation products can reach baseline separation, the separation degree is more than 1.5, and the specificity of the method is good.
2) Detection limit, quantification limit
The detection limits of the impurity B, the impurity C, the impurity A and the fosfomycin are 25.2ng, 14.2ng, 49.4ng and 42.6ng respectively, which are equivalent to 0.0079%, 0.0044%, 0.015% and 0.013% of the concentration (calculated by fosfomycin) of the test solution. The quantitative limits of the impurity B, the impurity C, the impurity A and the fosfomycin are 50.3ng, 47.2ng, 164.7ng and 142.1ng respectively, which are equivalent to 0.016%, 0.015%, 0.052% and 0.044% of the concentration (calculated by fosfomycin) of the test solution, and the 6-needle sample injection peak areas RSD are 17.3%, 11.7%, 11.9% and 10.1% respectively.
3) Linear, linear
The linear relation is good (note: y is peak area corresponding to concentration response, "x" is concentration of the substance, and "r" is linear correlation coefficient) when the impurity A is 25.57 mu g/ml to 383.50 mu g/ml, the linear equation is y 29.8507x +35.3597(r is 0.9991), the impurity B is 15.98 mu g/ml to 239.69 mu g/ml, the linear equation is y 29.8507x +35.3597(r is 0.9991), the impurity C is 3.20 mu g/ml to 47.94 mu g/ml, the linear equation is y 60.0038 x-42.3982 (r is 0.9968), the fosfomycin is 15.98 mu g/ml to 239.69 mu g/ml, and the linear equation is y 29.6817 x-327.0023 (r is 0.9992).
4) Precision degree
6 parts of control solution are continuously injected into 6 needles, the RSD of the ratio of the concentration of the fosfomycin to the peak area is 1.6 percent, and the system precision is good. Three batches of the self-developing reagent are respectively detected in 2 laboratories, the maximum absolute deviation of the impurity A and the impurity B is 0.035, the impurity C, the impurity D and the unknown impurity 1 are not detected, the maximum absolute deviation of the total impurities is 0.03, and the method has good reproducibility.
5) Accuracy of the measured value
The average recovery rates of the impurity B, C, A at the three high and high concentration levels in the lower limit range are respectively 103.4%, 98.0% and 104.5%, and the recovery rates are all between 90% and 110%; the RSD of the recovery rate of the impurity B, C, A at the three concentration levels of the impurity B, C, A in the lower limit range and the higher limit range is 3.2 percent, 3.9 percent and 4.4 percent respectively, the RSD is less than 5.0 percent, and the method has good accuracy.
The applicant shall now make further details of the method and application of the present invention with reference to specific examples.
Example 1
(1) Chromatographic conditions are as follows:
a chromatographic column: agilent Zorbax NH2(5μm，4.6mm×250mm)
Column temperature: 30 deg.C
Mobile phase: 20mmol/L phosphate buffer (Potassium dihydrogen phosphate) -methanol (70:30)
Flow rate: 1.0ml/min
And (3) detection: a differential detector that detects temperature: 35 deg.C
Sample introduction amount: 10 μ l
(2) Preparing a sample solution:
system applicability solution: taking 0.3g of fosfomycin trometamol (batch number: Y18011607), wetting with 10 μ l of water, heating at 80 deg.C for 12 hours, adding mobile phase for dissolving and diluting to 10ml, shaking up to obtain solution A; 0.3g of the product is taken out and dissolved by the solution A and diluted to 2.5ml to be used as a system applicability solution.
Sample solution: preparing for clinical application, precisely weighing appropriate amount of fosfomycin trometamol particles (batch number: 342414), dissolving with mobile phase, and quantitatively diluting to obtain a solution containing fosfomycin trometamol 120mg per lml as a test solution; precisely measuring a proper amount, and quantitatively diluting with a mobile phase until the solution containing fosfomycin trometamol is equivalent to 0.5% (mass ratio) of the test solution as a reference solution.
(3) And (3) detection: and (3) measuring the system applicability solution, the reference solution and the test solution, respectively injecting into a liquid chromatograph, recording a chromatogram, calculating the separation degree of the fosfomycin and each related substance in the system applicability solution chromatogram, and calculating the content of each related substance in the test solution according to a main component self-comparison method, wherein the result is shown in the following table 2. The results of the content determination of the relevant substances in the samples are shown in Table 3 below.
Table 2: degree of separation of impurities in different examples
Remarking: "- -" means not separated from the main peak
Table 3: the content of the related substances in the sample is determined
Remarking: "" indicates that in comparative example 1, the peak and fosfomycin gave a peak; "/" indicates less than the detection limit.
Example 2:
(1) chromatographic conditions are as follows:
a chromatographic column: osaka Soda Capcell Pak NH2(5μm，4.6×250mm)
Column temperature: 40 deg.C
Mobile phase: 80mmol/L phosphate buffer (dipotassium hydrogen phosphate) -methanol (75:25)
Flow rate: 0.8ml/min
And (3) detection: a differential detector that detects temperature: 50 deg.C
Sample introduction amount: 1 μ l
(2) Preparing a sample solution:
system applicability solution: taking 0.3g of fosfomycin trometamol (batch number: Y18011607), wetting with 60 μ l of water, heating at 60 deg.C for 12 hr, adding mobile phase for dissolving and diluting to 20ml, shaking up to obtain solution A; 0.3g of the product is taken out, dissolved by the solution A and diluted to 5ml to be used as a system applicability solution.
Sample solution: temporarily preparing, precisely weighing a proper amount of fosfomycin trometamol particles (batch number: 342414), adding a mobile phase for dissolving, and quantitatively diluting to obtain a solution containing about 240mg of fosfomycin trometamol in each lml as a test solution; precisely measuring a proper amount, and quantitatively diluting with a mobile phase until the solution containing fosfomycin trometamol is equivalent to 0.5% (mass ratio) of the test solution as a reference solution.
(3) And (3) detection: and (3) measuring the system applicability solution, the reference solution and the test solution, respectively injecting into a liquid chromatograph, recording a chromatogram, calculating the separation degree of the fosfomycin and each related substance in the system applicability solution chromatogram, and calculating the content of each related substance in the test solution according to a main component self-comparison method, wherein the result is shown in table 2. The results of the content measurement of the relevant substances of the samples are shown in Table 3.
Example 3:
(1) chromatographic conditions are as follows:
a chromatographic column: phenomenex Luna-NH2(5μm，4.6×250mm)
Column temperature: 20 deg.C
Mobile phase: 50mmol/L phosphate buffer (potassium phosphate) -methanol (50:50)
Flow rate: 1.2ml/min
And (3) detection: a differential detector that detects temperature: 15 deg.C
Sample introduction amount: 100 μ l
(2) Preparing a sample solution:
system applicability solution: taking 0.3g of fosfomycin trometamol powder (batch number: 20150501), wetting with 300 μ l of water, heating at 40 deg.C for 12 hours, adding mobile phase for dissolving and diluting to 50ml, and shaking up to obtain solution A; 0.3g of the product is taken out, dissolved by the solution A and diluted to 10ml to be used as a system applicability solution.
Sample solution: preparing for clinical application, precisely weighing appropriate amount of fosfomycin trometamol powder (batch number: 20150501), dissolving with mobile phase, and quantitatively diluting to obtain fosfomycin trometamol solution of 60mg per lml as test solution; precisely measuring a proper amount, and quantitatively diluting with a mobile phase until the solution containing fosfomycin trometamol is equivalent to 0.5% (mass ratio) of the test solution as a reference solution.
(2) And (3) detection: and (3) measuring the system applicability solution, the reference solution and the test solution, respectively injecting into a liquid chromatograph, recording a chromatogram, calculating the separation degree of the fosfomycin and each related substance in the system applicability solution chromatogram, and calculating the content of each related substance in the test solution according to a main component self-comparison method, wherein the result is shown in table 2. The results of the content measurement of the relevant substances of the samples are shown in Table 3.
Example 4:
(1) chromatographic conditions are as follows:
a chromatographic column: agilent Zorbax NH2(5μm，4.6mm×250mm)
Column temperature: 30 deg.C
Mobile phase: 30mmol/L phosphate buffer (sodium dihydrogen phosphate) -methanol (65:35)
Flow rate: 1.0ml/min
And (3) detection: a differential detector that detects temperature: 35 deg.C
Sample introduction amount: 10 μ l
(2) Preparing a sample solution:
system applicability solution: taking 0.3g of fosfomycin trometamol (batch number: Y18011607), wetting with 10 μ l of water, heating at 80 deg.C for 24 hr, adding mobile phase for dissolving and diluting to 10ml, shaking up to obtain solution A; 0.3g of the product is taken out and dissolved by the solution A and diluted to 2.5ml to be used as a system applicability solution.
Sample solution: preparing for clinical application, precisely weighing appropriate amount of fosfomycin trometamol (batch number: Y18011607), dissolving with mobile phase, and quantitatively diluting to obtain a solution containing fosfomycin trometamol 120mg per lml as a test solution; precisely measuring a proper amount, and quantitatively diluting with a mobile phase until the solution containing fosfomycin trometamol is equivalent to 0.5% (mass ratio) of the test solution as a reference solution.
(3) And (3) detection: and (3) measuring the system applicability solution, the reference solution and the test solution, respectively injecting into a liquid chromatograph, recording a chromatogram, calculating the separation degree of the fosfomycin and each related substance in the system applicability solution chromatogram, and calculating the content of each related substance in the test solution according to a main component self-comparison method, wherein the result is shown in table 2. The results of the content measurement of the relevant substances of the samples are shown in Table 3.
Example 5:
(1) chromatographic conditions are as follows:
a chromatographic column: osaka Soda Capcell Pak NH2(5μm，4.6×250mm)
Column temperature: 40 deg.C
Mobile phase: 50mmol/L phosphate buffer (sodium dihydrogen phosphate) -methanol (95:5)
Flow rate: 0.8ml/min
And (3) detection: a differential detector that detects temperature: 50 deg.C
Sample introduction amount: 1 μ l
(2) Preparing a sample solution:
system applicability solution: taking 0.3g of fosfomycin trometamol powder (batch number: 20150501), wetting with 60 μ l of water, heating at 60 ℃ for 24 hours, adding mobile phase for dissolving and diluting to 20ml, and shaking up to obtain solution A; 0.3g of the product is taken out, dissolved by the solution A and diluted to 5ml to be used as a system applicability solution.
Sample solution: preparing for clinical application, precisely weighing appropriate amount of fosfomycin trometamol powder (batch number: 20150501), dissolving with mobile phase, and quantitatively diluting to obtain about 240mg fosfomycin trometamol solution per lml as test solution; precisely measuring a proper amount, and quantitatively diluting with a mobile phase until the solution containing fosfomycin trometamol is equivalent to 0.5% (mass ratio) of the test solution as a reference solution.
(3) And (3) detection: and (3) measuring the system applicability solution, the reference solution and the test solution, respectively injecting into a liquid chromatograph, recording a chromatogram, calculating the separation degree of the fosfomycin and each related substance in the system applicability solution chromatogram, and calculating the content of each related substance in the test solution according to a main component self-comparison method, wherein the result is shown in table 2. The results of the content measurement of the relevant substances of the samples are shown in Table 3.
Example 6:
(1) chromatographic conditions are as follows:
a chromatographic column: phenomenex Luna-NH2(5μm，4.6×250mm)
Column temperature: 20 deg.C
Mobile phase: 20mmol/L phosphate buffer (potassium phosphate) -methanol (80:20)
Flow rate: 1.2ml/min
And (3) detection: a differential detector that detects temperature: 15 deg.C
Sample introduction amount: 100 μ l
(2) Preparing a sample solution:
system applicability solution: taking 0.3g of fosfomycin trometamol powder (batch number: 20150501), wetting with 300 μ l of water, heating at 40 deg.C for 24 hr, adding mobile phase for dissolving and diluting to 50ml, and shaking up to obtain solution A; 0.3g of the product is taken out, dissolved by the solution A and diluted to 10ml to be used as a system applicability solution.
Sample solution: preparing for clinical application, precisely weighing appropriate amount of fosfomycin trometamol powder (batch number: 20150501), dissolving with mobile phase, and quantitatively diluting to obtain fosfomycin trometamol solution of 60mg per lml as test solution; precisely measuring a proper amount, and quantitatively diluting with a mobile phase until the solution containing fosfomycin trometamol is equivalent to 0.5% (mass ratio) of the test solution as a reference solution.
(3) And (3) detection: and (3) measuring the system applicability solution, the reference solution and the test solution, respectively injecting into a liquid chromatograph, recording a chromatogram, calculating the separation degree of the fosfomycin and each related substance in the system applicability solution chromatogram, and calculating the content of each related substance in the test solution according to a main component self-comparison method, wherein the result is shown in table 2. The results of the content measurement of the relevant substances of the samples are shown in Table 3.
Example 7:
(1) chromatographic conditions are as follows:
a chromatographic column: agilent Zorbax NH2(5μm，4.6mm×250mm)
Column temperature: 30 deg.C
Mobile phase: 50mmol/L phosphate buffer (Potassium dihydrogen phosphate) -acetonitrile (80:20)
Flow rate: 1.2ml/min
And (3) detection: a differential detector that detects temperature: 15 deg.C
Sample introduction amount: 20 μ l
(2) Preparing a sample solution:
system applicability solution: taking 0.3g of fosfomycin trometamol (batch number: Y18011607), wetting with 10 μ l of water, heating at 80 deg.C for 36 hours, adding mobile phase for dissolving and diluting to 10ml, shaking up to obtain solution A; 0.3g of the product is taken out and dissolved by the solution A and diluted to 2.5ml to be used as a system applicability solution.
Sample solution: preparing for clinical application, taking a proper amount of fosfomycin trometamol (batch number: Y18011607), precisely weighing, adding a mobile phase for dissolving, and quantitatively diluting to obtain a solution containing fosfomycin trometamol 240mg in each lml as a test solution; precisely measuring a proper amount, and quantitatively diluting with a mobile phase until the solution containing fosfomycin trometamol is equivalent to 0.3% (mass ratio) of the test solution as a reference solution.
(3) And (3) detection: and (3) measuring the system applicability solution, the reference solution and the test solution, respectively injecting into a liquid chromatograph, recording a chromatogram, calculating the separation degree of the fosfomycin and each related substance in the system applicability solution chromatogram, and calculating the content of each related substance in the test solution according to a main component self-comparison method, wherein the result is shown in table 2. The results of the content measurement of the relevant substances of the samples are shown in Table 3.
Example 8:
(1) chromatographic conditions are as follows:
a chromatographic column: waters Spherisorb NH2 4.6×150mm 3μm
Column temperature: 30 deg.C
Mobile phase: 30mmol/L phosphate buffer (potassium dihydrogen phosphate-sodium dihydrogen phosphate (mass ratio 1:1)) -methanol (80:20)
Flow rate: 0.8ml/min
And (3) detection: a differential detector that detects temperature: 50 deg.C
Sample introduction amount: 10 μ l
(2) Preparing a sample solution:
system applicability solution: taking 0.3g of fosfomycin trometamol powder (batch number: 20180903), wetting with 60 μ l of water, heating at 60 ℃ for 36 hours, adding mobile phase for dissolving and diluting to 20ml, and shaking up to obtain solution A; 0.3g of the product is taken out, dissolved by the solution A and diluted to 5ml to be used as a system applicability solution.
Sample solution: preparing for clinical application, precisely weighing appropriate amount of fosfomycin trometamol powder (batch number: 20180903), dissolving with mobile phase, and quantitatively diluting to obtain a solution containing fosfomycin trometamol 120mg per lml as test solution; precisely measuring a proper amount, and quantitatively diluting with a mobile phase until the solution containing fosfomycin trometamol is equivalent to 0.3% (mass ratio) of the test solution as a reference solution.
(3) And (3) detection: and (3) measuring the system applicability solution, the reference solution and the test solution, respectively injecting into a liquid chromatograph, recording a chromatogram, calculating the separation degree of the fosfomycin and each related substance in the system applicability solution chromatogram, and calculating the content of each related substance in the test solution according to a main component self-comparison method, wherein the result is shown in table 2. The results of the content measurement of the relevant substances of the samples are shown in Table 3.
Example 9:
(1) chromatographic conditions are as follows:
a chromatographic column: waters Spherisorb NH2 4.6×150mm 3μm
Column temperature: 20 deg.C
Mobile phase: 30mmol/L phosphate buffer (monopotassium phosphate) -acetonitrile-methanol (75:6.25:18.75)
Flow rate: 1.2ml/min
And (3) detection: a differential detector that detects temperature: 35 deg.C
Sample introduction amount: 10 μ l
(2) Preparing a sample solution:
system applicability solution: taking 0.3g of fosfomycin trometamol powder (batch number: 20180903), wetting with 300 μ l of water, heating at 40 deg.C for 36 hr, adding mobile phase for dissolving and diluting to 50ml, and shaking up to obtain solution A; 0.3g of the product is taken out, dissolved by the solution A and diluted to 10ml to be used as a system applicability solution.
Sample solution: preparing for clinical application, precisely weighing appropriate amount of fosfomycin trometamol powder (batch number: 20180903), dissolving with mobile phase, and quantitatively diluting to obtain fosfomycin trometamol solution of 60mg per lml as test solution; precisely measuring a proper amount, and quantitatively diluting with a mobile phase until the solution containing fosfomycin trometamol is equivalent to 0.5% (mass ratio) of the test solution as a reference solution.
(3) And (3) detection: and (3) measuring the system applicability solution, the reference solution and the test solution, respectively injecting into a liquid chromatograph, recording a chromatogram, calculating the separation degree of the fosfomycin and each related substance in the system applicability solution chromatogram, and calculating the content of each related substance in the test solution according to a main component self-comparison method, wherein the result is shown in table 2. The results of the content measurement of the relevant substances of the samples are shown in Table 3.
Example 10:
(1) chromatographic conditions are as follows:
a chromatographic column: agilent Zorbax NH2 4.6×250mm 5μm
Column temperature: 30 deg.C
Mobile phase: 100mmol/L phosphate buffer (Potassium dihydrogen phosphate) -acetonitrile-methanol (70:15:15)
Flow rate: 1.2ml/min
And (3) detection: a differential detector that detects temperature: 35 deg.C
Sample introduction amount: 10 μ l
(2) Preparing a sample solution:
system applicability solution: taking 0.3g of fosfomycin trometamol (batch number: Y18011607), wetting with 60 μ l of water, heating at 60 deg.C for 24 hr, adding mobile phase for dissolving and diluting to 50ml, and shaking up to obtain solution A; 0.3g of the product is taken out, dissolved by the solution A and diluted to 10ml to be used as a system applicability solution.
Sample solution: preparing for clinical application, precisely weighing appropriate amount of fosfomycin trometamol powder (batch number: 20180903), dissolving with mobile phase, and quantitatively diluting to obtain fosfomycin trometamol solution of 60mg per lml as test solution; precisely measuring a proper amount, and quantitatively diluting with a mobile phase until the solution containing fosfomycin trometamol is equivalent to 0.5% (mass ratio) of the test solution as a reference solution.
(3) And (3) detection: and (3) measuring the system applicability solution, the reference solution and the test solution, respectively injecting into a liquid chromatograph, recording a chromatogram, calculating the separation degree of the fosfomycin and each related substance in the system applicability solution chromatogram, and calculating the content of each related substance in the test solution according to a main component self-comparison method, wherein the result is shown in table 2. The results of the content measurement of the relevant substances of the samples are shown in Table 3.
Example 11:
a method for separating and preparing fosfomycin trometamol impurities comprises the following steps:
1) and the chromatographic conditions are as follows: the chromatographic column is Zorbax-NH2,5 μm,4.6mm × 250mm, the detector adopts a parallax detector, the detector temperature is 35 deg.C, and the mobile phase is 50mmol/L potassium dihydrogen phosphate-methanol (70: 30).
2) Preparing a sample solution: 0.3g of fosfomycin trometamol (batch number: Y18011607) is taken, 60 mu l of water is used for wetting, the mixture is heated at 60 ℃ for 24 hours, and a proper amount of mobile phase is added for dissolving and shaking up, thus obtaining the prepared sample solution.
3) And the preparation process comprises the following steps: 5 μ l of the prepared sample was taken into a chromatograph. And collecting component peaks of the impurity A, the impurity B, the impurity C and the impurity D step by step, desalting, and freeze-drying to obtain the high-purity impurity.
Comparative example 1:
(1) the chromatographic conditions are as follows:
detector temperature: 35 deg.C
Mobile phase: 10.89g/L aqueous potassium dihydrogenphosphate solution ()
A chromatographic column: agilent Zorbax NH2，5μm，4.6mm×250mm
Column temperature: 30 deg.C
Flow rate: 1.0ml/min
Sample introduction amount: 10 μ l
(2) And preparing a sample solution:
system applicability solution: taking 0.3g of fosfomycin trometamol (batch number: Y18011607), wetting with 60 μ l of water, heating at 60 deg.C for 24 hr, adding mobile phase for dissolving and diluting to 20ml, and shaking up to obtain solution A; 0.3g of the product is taken out, dissolved by the solution A and diluted to 5ml to be used as a system applicability solution.
Sample solution: preparing for clinical application, precisely weighing appropriate amount of fosfomycin trometamol particles (batch number: 342414), dissolving with mobile phase, and quantitatively diluting to obtain a solution containing fosfomycin trometamol 120mg per lml as a test solution; precisely measuring a proper amount, and quantitatively diluting with a mobile phase until the solution containing fosfomycin trometamol is equivalent to 0.3% (mass ratio) of the test solution as a reference solution.
(3) And (3) detection: and (3) measuring the system applicability solution, the reference solution and the test solution, respectively injecting into a liquid chromatograph, recording a chromatogram, calculating the separation degree of the fosfomycin and each related substance in the system applicability solution chromatogram, and calculating the content of each related substance in the test solution according to a main component self-comparison method, wherein the result is shown in table 2. The results of the content measurement of the relevant substances of the samples are shown in Table 3.
The invention discloses a high performance liquid chromatography analysis method for fosfomycin trometamol and related substances of a preparation thereof, and also discloses application of the method in separating and preparing fosfomycin trometamol impurities. The method adopts high performance liquid chromatography, takes aminopropyl silane bonded silica gel as a filling agent, adopts a differential refraction detector, selects an organic phase and a buffer solution as mobile phases for elution, and effectively solves the problem of the separation degree of fosfomycin trometamol and related substances thereof. By using the method, impurities for researching methods of related substances can be separated and prepared. The method has strong specificity and good repeatability, can ensure the controllability of the fosfomycin trometamol and related substances in the preparation thereof, and overcomes the defects that the column efficiency of the aminopropyl bonded silica gel column is reduced quickly in a pure water phase environment and the detection of impurities is influenced.
Claims (4)
1. A high performance liquid chromatography separation and determination method for impurities in fosfomycin trometamol raw material medicine and its preparation for simultaneously separating impurities with molecular formula of
Impurity A of formula
Impurity B of the formula
Impurity C and molecular formula of
Impurity D of (1); the chromatographic column adopting aminopropyl silane bonded silica gel as a filling agent is characterized in that:
detecting by a differential detector at the temperature of 15-50 ℃; taking 20-100 mmol/L phosphate buffer solution as a mobile phase A, taking methanol-acetonitrile as a mobile phase B, mixing the mobile phase A and the mobile phase B according to the volume ratio of 80: 20-65: 35 to obtain a mobile phase, wherein the flow rate of the mobile phase is 0.8-1.2 mL/min; the temperature of the chromatographic column is 20-40 ℃; the sample amount is 1-500 mul; the method specifically comprises the following steps:
(1) the solution is prepared according to the following mixture ratio:
1) preparing a system applicability solution:
taking 0.3g of fosfomycin trometamol raw material medicine or a preparation thereof, wetting the raw material medicine or the preparation with 10-300 mu l of water, heating the raw material medicine or the preparation at 40-80 ℃ for 12-36 hours, adding a mobile phase for dissolving and diluting the raw material medicine or the preparation to 10-50 ml, and shaking the raw material medicine or the preparation uniformly to obtain a solution A;
dissolving 0.3g of fosfomycin trometamol raw material medicine or a preparation thereof by using the solution A and diluting to 2.5-10 ml to be used as a system applicability solution;
2) and preparing a test solution:
according to the new preparation for clinical use, taking a proper amount of fosfomycin trometamol raw material medicine or a preparation thereof, precisely weighing, adding a mobile phase for dissolving, and quantitatively diluting into a solution containing 60-240 mg of fosfomycin trometamol in each lml to be used as a test solution;
3) and preparing a control solution:
precisely measuring a proper amount of a test solution, and quantitatively diluting the test solution by using a mobile phase until the fosfomycin-containing tromethamine is equivalent to a solution with the mass ratio concentration of 0.3-0.5% of the test solution, wherein the solution is used as a reference solution;
(2) the determination method comprises the following steps:
precisely measuring 1-100 mul of system applicability solution, control solution and sample solution, respectively, injecting into a liquid chromatograph, and recording chromatogram; calculating the contents of four related substances, namely impurity A, impurity B, impurity C and impurity D, and other possible known or unknown impurities in the fosfomycin trometamol bulk drug by adopting a main component self-comparison method;
the volume ratio of methanol to acetonitrile in the mobile phase B is 75: 25-50: 50.
2. the HPLC separation and determination method for impurities in a fosfomycin trometamol raw material drug and a preparation thereof according to claim 1, wherein the chromatographic column with aminopropylsilane bonded silica gel as a filler is selected from:
agilent Zorbax NH with specification of 5 μm and 4.6X 250mm2，
Agilent Zorbax NH 3 μm, 4.6X 150mm in specification2，
Waters Spherisorb NH with specification of 5 μm and 4.6X 250mm2，
Waters Spherisorb NH with specification of 3 μm and 4.6X 150mm2，
Osaka Soda Capcell Pak NH with specification of 5 mu m and 4.6X 250mm2，
Osaka Soda Capcell Pak NH with specification of 3 mu m and 4.6X 150mm2，
Phenomenex Luna-NH with specification of 5 mu m and 4.6X 250mm2，
Phenomenex Luna-NH with specification of 3 μm and 4.6 × 150mm2，
Or one of the column equivalent.
3. The separation and determination method of any one of claims 1 to 2 is adopted to perform separation and determination on fosfomycin trometamol raw material medicine with the molecular formula of
Impurity A of formula
Impurity B of the formula
Impurity C and molecular formula of
The impurity D is separated and prepared to form the application of an impurity reference substance.
4. The use according to claim 3, comprising the steps of collecting the impurities A, B, C and D in steps, desalting and freeze-drying.
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| CN111257478B (en) * | 2020-03-24 | 2022-07-12 | 上海峰林生物科技有限公司 | Method for analyzing fosfomycin trometamol genotoxic impurities |
| CN112710773B (en) * | 2020-12-16 | 2021-10-08 | 中国环境科学研究院 | Method for simultaneously detecting fosfomycin and diol thereof by adopting ion chromatography |
| CN112730720B (en) * | 2020-12-28 | 2022-05-24 | 湖州中科院应用技术研究与产业化中心 | Liquid phase detection method for vitronectin in cosmetics |
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