CN113219079A - HPLC detection method for polymer impurities in flucloxacillin and preparation thereof - Google Patents
HPLC detection method for polymer impurities in flucloxacillin and preparation thereof Download PDFInfo
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- 229960004273 floxacillin Drugs 0.000 title claims abstract description 58
- UIOFUWFRIANQPC-JKIFEVAISA-N Floxacillin Chemical compound N([C@@H]1C(N2[C@H](C(C)(C)S[C@@H]21)C(O)=O)=O)C(=O)C1=C(C)ON=C1C1=C(F)C=CC=C1Cl UIOFUWFRIANQPC-JKIFEVAISA-N 0.000 title claims abstract description 55
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- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 description 1
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Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
Abstract
The invention discloses an HPLC detection method for polymer impurities in flucloxacillin and a preparation thereof, which comprises the steps of separating and measuring the polymer impurities in the flucloxacillin and the preparation thereof by using a high performance liquid chromatograph and an isocratic elution method, wherein the detection wavelength is 254nm, a chromatographic column using spherical protein chromatography and hydrophilic silica gel as a filler is used, and a mixed solution consisting of 87.5-92.5% by volume of 0.01mol/L phosphate buffer solution and 7.5-12.5% by volume of organic phase is used as a mobile phase. The invention can control the quality of polymer impurities of flucloxacillin, and ensure the product quality and the medication safety.
Description
Technical Field
The invention relates to the technical field of medicine preparation and detection, and particularly relates to an HPLC (high performance liquid chromatography) detection method for polymer impurities in flucloxacillin and a preparation thereof.
Background
The flucloxacillin sodium is semi-synthetic penicillin-resistant penicillin. The action mechanism of the bactericidal composition is similar to that of penicillin G, and the bactericidal composition is combined with Penicillin Binding Proteins (PBPs) on cell membranes to inhibit the biosynthesis of bacterial cell walls, so that thalli are swollen, broken and dead, and the bactericidal composition plays a bactericidal role in the breeding period. It has better stability to penicillinase and strong bactericidal action to penicillin-producing resistant staphylococcus aureus, but the antibacterial action of penicillin-sensitized staphylococcus and various streptococcus is weaker than that of penicillin. The flucloxacillin sodium has good antibacterial activity on penicillinase-producing staphylococcus aureus, staphylococcus epidermidis, streptococcus pyogenes, streptococcus pneumoniae, gonococcus and meningococcus. Enterococcus faecalis, methicillin-resistant staphylococcus aureus, vaginal negative bacillus, pseudomonas aeruginosa and anaerobic fragile bacillus are resistant to the product. The chemical structure of flucloxacillin is shown in the following figure 1, and figure 2 shows the generation path of polymer impurities of flucloxacillin. As can be seen from the figure, the carboxyl group in the flucloxacillin structure can react with 6-APA, flucloxacillin beta-lactam ring hydrolysate and flucloxacillin beta-lactam ring hydrolysis decarboxylation to generate dimer impurity. In addition, the carboxyl in the dimer impurity can be further combined with 6-APA, flucloxacillin beta-lactam ring hydrolysate and flucloxacillin beta-lactam ring hydrolysis decarboxylation to produce polymer such as trimer. The polymer impurities belong to process impurities and degradation impurities, and can be generated, grown and accumulated in the production and storage processes of flucloxacillin raw materials and preparations thereof.
A large number of researches show that the allergen causing penicillin anaphylaxis is high molecular polymer impurities in the preparation, and the anaphylaxis can be controlled by controlling the polymer impurities. Therefore, the content level of polymer impurities in flucloxacillin and the preparation thereof directly determines the quality of the product and the safety of subsequent clinical medication. However, the existing quality standards of the flucloxacillin and preparations thereof in the current pharmacopoeia at home and abroad do not control the polymer impurities.
Therefore, in consideration of the concept that impurities need to be controlled from the source, it is necessary to establish a new HPLC (High Performance Liquid Chromatography/HPLC) also called High pressure Liquid Chromatography, High Performance Liquid Chromatography, High resolution Liquid Chromatography, modern column Chromatography, etc. for polymer impurities of flucloxacillin drug substances and preparations thereof, where the High Performance Liquid Chromatography is an important branch of the Chromatography, and uses Liquid as a mobile phase, and adopts a High pressure infusion system to pump single solvents with different polarities or mixed solvents, buffers, etc. in different proportions into a chromatographic column filled with a stationary phase, and after each component in the column is separated, the mobile phase enters a detector for detection, thereby realizing analysis of a sample, and the method becomes an important separation and analysis technology application in the fields of chemistry, medicine, industry, agriculture, commercial inspection, legal inspection, etc.), the impurities can be effectively controlled, and the quality of subsequent products is ensured.
The method is beneficial to enhancing the internal control quality of the flucloxacillin raw material medicine and the preparation product thereof, and provides necessary theoretical and practical basis for the optimization of process conditions.
Disclosure of Invention
Aiming at the problems in the related art, the invention provides an HPLC (high performance liquid chromatography) detection method for polymer impurities in flucloxacillin and a preparation thereof, so as to control the quality of the polymer impurities of flucloxacillin and ensure the product quality and the medication safety.
Therefore, the invention adopts the following specific technical scheme:
an HPLC detection method for polymer impurities in flucloxacillin and a preparation thereof comprises the steps of separating and measuring the polymer impurities in the flucloxacillin and the preparation thereof by using a high performance liquid chromatograph and an isocratic elution method, wherein the detection wavelength is 254nm, a chromatographic column using globular protein chromatography and hydrophilic silica gel as a filler is adopted, and a mixed solution consisting of 87.5-92.5% by volume of 0.01mol/L phosphate buffer solution and 7.5-12.5% by volume of organic phase is adopted as a mobile phase.
Preferably, the flow rate of the mobile phase is 0.5-0.7 mL/min.
Preferably, the phosphate buffer is formulated in the following manner: mixing 0.01mol/L disodium hydrogen phosphate solution and 0.01mol/L sodium dihydrogen phosphate solution at a mass ratio of 61:39 to prepare the sodium dihydrogen phosphate/disodium hydrogen phosphate/sodium dihydrogen phosphate composite material.
Preferably, the organic phase is one of acetonitrile and methanol.
Preferably, the organic phase is acetonitrile.
Preferably, the mobile phase contains 90% by volume of the buffered salt solution and 10% by volume of acetonitrile.
Preferably, the column temperature of the high performance liquid chromatograph chromatographic column is 20-30 ℃.
Preferably, the column temperature of the column is 25 ℃.
Preferably, the flow rate of the mobile phase is 0.6 mL/min.
The invention has the beneficial effects that:
the HPLC detection method for polymer impurities in flucloxacillin and preparations thereof, which is established by the invention, has the advantages of good separation degree, simplicity, rapidness, strong specificity and high sensitivity, can control the quality of polymer impurity degradation impurities of flucloxacillin, and ensures the product quality and the medication safety.
Wherein, in the aspect of the elution step, an isocratic elution method is adopted, and separation is carried out according to the molecular weight of each substance.
The reason for adding the buffer salt to the mobile phase is that the polymer impurities contain amino and carboxyl groups, and the addition of the buffer salt to the mobile phase can enhance ionization of the analyte and increase solubility, thereby facilitating separation.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 shows the structural formula of flucloxacillin in the prior art;
FIG. 2 shows a schematic diagram of a prior art synthetic route for flucloxacillin and a production pathway for polymer impurities;
FIG. 3 shows a detection chromatogram in the first embodiment of the present invention;
FIG. 4 shows a detection chromatogram in example two of the present invention;
FIG. 5 shows a detection chromatogram in example III of the present invention.
Detailed Description
For further explanation of the various embodiments, the drawings which form a part of the disclosure and which are incorporated in and constitute a part of this specification, illustrate embodiments and, together with the description, serve to explain the principles of operation of the embodiments, and to enable others of ordinary skill in the art to understand the various embodiments and advantages of the invention, and, by reference to these figures, reference is made to the accompanying drawings, which are not to scale and wherein like reference numerals generally refer to like elements.
The first embodiment is as follows:
the instruments and settings used in this example were as follows:
high performance liquid chromatograph: shimadzu LC-20 AT;
a chromatographic column: TSK-GEL G2000Wxl, 7.8mm × 30cm × 5 μm;
flow rate of mobile phase: 0.6 mL/min;
detection wavelength: 254 nm;
column temperature of the chromatographic column: 25 ℃;
sample introduction amount: 20 uL;
wherein the mobile phase: phosphate buffer solution with the concentration of 0.01mol/L and acetonitrile are mixed according to the mass part ratio of 9: 1 is configured as a mobile phase, wherein a phosphate buffer solution with a concentration of 0.01mol/L is prepared by the following method: mixing 0.01mol/L disodium hydrogen phosphate solution and 0.01mol/L sodium dihydrogen phosphate solution at a mass ratio of 61:39 to prepare the sodium dihydrogen phosphate/disodium hydrogen phosphate/sodium dihydrogen phosphate composite material.
The experimental steps are as follows:
the method comprises the following steps: solution preparation
System adaptation solution: taking a proper amount of the product, precisely weighing, adding water to dissolve, quantitatively diluting to prepare a solution containing 1.0mg of flucloxacillin in each 1ml, taking 10ml of the solution, adding 1ml of 0.1mol/L sodium hydroxide solution, standing for 1 minute at room temperature, adding 1ml of 0.1mol/L hydrochloric acid solution, and shaking uniformly to obtain the system applicability solution. The separation degree of the main peak and the adjacent degraded impurity peak before the main peak is not less than 2.
Test solution: taking a proper amount of the product, adding a solvent to dissolve the product, quantitatively diluting the product to prepare a solution containing 1.0mg of flucloxacillin in each 1ml, and shaking up the solution to be used as a test solution.
Control solution: taking about 5mg of flucloxacillin reference substance, precisely weighing, placing in a 250ml volumetric flask, adding a solvent to dilute to a scale, and shaking up to obtain a reference solution.
Sensitivity solution: taking a proper amount of flucloxacillin reference substances, and quantitatively diluting the control substances by using a solvent to prepare a solution containing about 0.25 mu g of flucloxacillin per 1ml, wherein the solution is used as a sensitivity solution.
Step two: measurement of
Precisely measuring 20 μ l of each of the test solution and the control solution, respectively injecting into a liquid chromatograph, and recording chromatogram. If an impurity peak exists in the chromatogram of the test solution, the total amount of impurities with retention time less than the flucloxacillin peak is not more than 1.0 percent according to the external standard method by calculating the peak area of the flucloxacillin in the reference solution. Any peak in the chromatogram of the test solution that is smaller than the area of the main peak of the sensitivity solution is ignored. A typical system suitability map is shown in figure 3 below.
Example 2
The instruments and setting conditions used in this example are as follows
High performance liquid chromatograph: shimadzu LC-20 AT;
a chromatographic column: TSK-GEL G2000Wxl, 7.8mm × 30cm × 5 μm;
flow rate of mobile phase: 0.6 mL/min;
detection wavelength: 254 nm;
column temperature of the chromatographic column: 25 ℃;
sample introduction amount: 20 uL;
wherein the mobile phase: phosphate buffer solution with the concentration of 0.01mol/L and acetonitrile are mixed according to the mass part ratio of 87.5: 12.5 configuration as mobile phase, wherein phosphate buffer at a concentration of 0.01mol/L is formulated by: mixing 0.01mol/L disodium hydrogen phosphate solution and 0.01mol/L sodium dihydrogen phosphate solution at a mass ratio of 61:39 to prepare the sodium dihydrogen phosphate/disodium hydrogen phosphate/sodium dihydrogen phosphate composite material.
The experimental steps are as follows:
the method comprises the following steps: solution preparation
System adaptation solution: taking a proper amount of the product, precisely weighing, adding water to dissolve, quantitatively diluting to prepare a solution containing 1.0mg of flucloxacillin in each 1ml, taking 10ml of the solution, adding 1ml of 0.1mol/L sodium hydroxide solution, standing for 1 minute at room temperature, adding 1ml of 0.1mol/L hydrochloric acid solution, and shaking uniformly to obtain the system applicability solution. The separation degree of the main peak and the adjacent degraded impurity peak before the main peak is not less than 2.
Test solution: taking a proper amount of the product, adding a solvent to dissolve the product, quantitatively diluting the product to prepare a solution containing 1.0mg of flucloxacillin in each 1ml, and shaking up the solution to be used as a test solution.
Control solution: taking about 5mg of flucloxacillin reference substance, precisely weighing, placing in a 250ml volumetric flask, adding a solvent to dilute to a scale, and shaking up to obtain a reference solution.
Sensitivity solution: taking a proper amount of flucloxacillin reference substances, and quantitatively diluting the control substances by using a solvent to prepare a solution containing about 0.25 mu g of flucloxacillin per 1ml, wherein the solution is used as a sensitivity solution.
Step two: measurement of
Precisely measuring 20 μ l of each of the test solution and the control solution, respectively injecting into a liquid chromatograph, and recording chromatogram. If an impurity peak exists in the chromatogram of the test solution, the total amount of impurities with retention time less than the flucloxacillin peak is not more than 1.0 percent according to the external standard method by calculating the peak area of the flucloxacillin in the reference solution. Any peak in the chromatogram of the test solution that is smaller than the area of the main peak of the sensitivity solution is ignored. A typical system suitability map is shown in figure 4 below.
Example 3
The instruments and setting conditions used in this example were:
high performance liquid chromatograph: shimadzu LC-20AT
A chromatographic column: TSK-GEL G2000Wxl, 7.8mm × 30cm × 5 μm;
flow rate of mobile phase: 0.6 mL/min;
detection wavelength: 254 nm;
column temperature of the chromatographic column: 25 ℃;
sample introduction amount: 20 uL;
wherein the mobile phase: phosphate buffer solution with the concentration of 0.01mol/L and acetonitrile are mixed according to the mass part ratio of 92.5: 7.5 configuration as mobile phase, wherein phosphate buffer at a concentration of 0.01mol/L is formulated by: mixing 0.01mol/L disodium hydrogen phosphate solution and 0.01mol/L sodium dihydrogen phosphate solution at a mass ratio of 61:39 to prepare the sodium dihydrogen phosphate/disodium hydrogen phosphate/sodium dihydrogen phosphate composite material.
The experimental steps are as follows:
the method comprises the following steps: solution preparation
System adaptation solution: taking a proper amount of the product, precisely weighing, adding water to dissolve, quantitatively diluting to prepare a solution containing 1.0mg of flucloxacillin in each 1ml, taking 10ml of the solution, adding 1ml of 0.1mol/L sodium hydroxide solution, standing for 1 minute at room temperature, adding 1ml of 0.1mol/L hydrochloric acid solution, and shaking uniformly to obtain the system applicability solution. The separation degree of the main peak and the adjacent degraded impurity peak before the main peak is not less than 2.
Test solution: taking a proper amount of the product, adding a solvent to dissolve the product, quantitatively diluting the product to prepare a solution containing 1.0mg of flucloxacillin in each 1ml, and shaking up the solution to be used as a test solution.
Control solution: taking about 5mg of flucloxacillin reference substance, precisely weighing, placing in a 250ml volumetric flask, adding a solvent to dilute to a scale, and shaking up to obtain a reference solution.
Sensitivity solution: taking a proper amount of flucloxacillin reference substances, and quantitatively diluting the control substances by using a solvent to prepare a solution containing about 0.25 mu g of flucloxacillin per 1ml, wherein the solution is used as a sensitivity solution.
Step two: measurement of
Precisely measuring 20 μ l of each of the test solution and the control solution, respectively injecting into a liquid chromatograph, and recording chromatogram. If an impurity peak exists in the chromatogram of the test solution, the total amount of impurities with retention time less than the flucloxacillin peak is not more than 1.0 percent according to the external standard method by calculating the peak area of the flucloxacillin in the reference solution. Any peak in the chromatogram of the test solution that is smaller than the area of the main peak of the sensitivity solution is ignored. A typical system suitability map is shown in FIG. 5 below.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. An HPLC detection method for polymer impurities in flucloxacillin and a preparation thereof is characterized by comprising the steps of separating and measuring the polymer impurities in the flucloxacillin and the preparation thereof by using a high performance liquid chromatograph and an isocratic elution method, wherein the detection wavelength is 254 +/-5 nm, a chromatographic column using spherical protein chromatography and hydrophilic silica gel as a filler is used, and a mixed solution consisting of 87.5-92.5% by volume of 0.01mol/L phosphate buffer solution and 7.5-12.5% by volume of organic phase is used as a mobile phase.
2. The HPLC method for detecting polymer impurities in flucloxacillin and its preparation of claim 1, wherein the flow rate of the mobile phase is 0.5-0.7 mL/min.
3. The HPLC method for detecting polymer impurities in flucloxacillin and its preparation according to claim 1 or 2, wherein the phosphate buffer is prepared by the following method: mixing 0.01mol/L disodium hydrogen phosphate solution and 0.01mol/L sodium dihydrogen phosphate solution at a mass ratio of 61:39 to prepare the sodium dihydrogen phosphate/disodium hydrogen phosphate/sodium dihydrogen phosphate composite material.
4. The HPLC method for detecting polymer impurities in flucloxacillin and its preparation of claim 3, wherein the organic phase is one of acetonitrile and methanol.
5. The HPLC method for detecting polymer impurities in flucloxacillin and its preparation of claim 4, wherein said organic phase is acetonitrile.
6. The HPLC method for detecting polymer impurities in flucloxacillin and its preparation of claim 5, wherein the volume of the buffer salt solution is 90% and the volume of the acetonitrile is 10% in the mobile phase.
7. The HPLC method for detecting polymer impurities in flucloxacillin and its preparation according to any one of claims 1, 2, 4 or 5, wherein the column temperature of the HPLC column is 20-30 ℃.
8. The HPLC method for detecting polymer impurities in flucloxacillin and its preparation as claimed in claim 7, wherein the column temperature of the chromatographic column is 25 ℃.
9. The HPLC method for detecting polymer impurities in flucloxacillin and its preparation as claimed in claim 2, wherein the flow rate of said mobile phase is 0.6 mL/min.
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| US20180080858A1 (en) * | 2016-09-19 | 2018-03-22 | Agilent Technologies, Inc. | Functionalized support for analytical sample preparation |
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