CN111257478B - Method for analyzing fosfomycin trometamol genotoxic impurities - Google Patents
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Abstract
The invention provides an analysis method of fosfomycin trometamol genotoxic impurities, which comprises the following steps: diluting fosfomycin trometamol by a diluent, then analyzing impurities by adopting a high performance liquid chromatography-mass spectrometry combined method, taking a formic acid solution as a mobile phase A, taking an acetonitrile solution as a mobile phase B, and carrying out gradient elution in an elution mode. In the formic acid solution, the concentration of formic acid is 0.1-10%. The diluent is an aqueous solution containing 85-95% of methanol. The method for analyzing the fosfomycin trometamol genotoxic impurities can separate and detect impurities such as diethyl allephosphonate and the like more efficiently, and has the advantages of strong specificity, high sensitivity, simplicity, rapidness and low cost.
Description
Technical Field
The invention belongs to the field of drug analysis, and particularly relates to an analysis method of fosfomycin trometamol genotoxic impurities.
Background
Fosfomycin trometamol is a white-like crystalline powder, which is fosfomycin and 2-amino-2-hydroxymethyl-1, 3-propanediol salt in a ratio of 1: 1, can directly prevent the action of pyruvate transferase necessary for the synthesis of bacterial cell walls, has an antibacterial spectrum comprising microorganisms such as escherichia coli, dysentery bacillus, spicy bacillus citrate, proteus, serratia, staphylococcus aureus and pseudomonas aeruginosa and can be used for treating diseases such as urinary tract infection and the like caused by the corresponding microorganisms.
The compounds diethyl propylene diene phosphate (impurity a), diethyl 1-propylene phosphate (impurity B), diethyl 3-phosphate-3-methyl-propylene oxide (impurity C), dimethyl 2-phosphate-3-methyl-propylene oxide (impurity D) and the like are genotoxic impurities remaining in fosfomycin trometamol, and the residue thereof must be strictly controlled. However, the quality standard of the impurities is not recorded in the domestic and foreign pharmacopoeias. According to the rules of the "limits of genotoxic impurities directive" issued by the european pharmacopoeia authority (EMEA), the maximum limit of the intake of genotoxic impurities is 1.5 ug/day, according to the Threshold of Toxicological Concerns (TTC) as the threshold for evaluating the majority of genotoxic impurities. The control limit of these impurities is not more than 0.5ppm, based on a maximum fosfomycin trometamol dose of 3 g/day. According to the detection requirement, the detection limit is about 0.1 of the control limit generally, so the detection limit is below 0.05ppm, and the conventional gas phase and liquid phase detection methods cannot meet the detection limit requirement.
Therefore, there is a need to provide an improved technical solution to overcome the technical problems in the prior art.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an analysis method of fosfomycin trometamol genotoxic impurities, which can separate and detect impurities such as diethyl propadienephosphate and the like more efficiently and has the advantages of strong specificity, high sensitivity, simplicity, rapidness and low cost.
The invention provides a method for analyzing fosfomycin trometamol genotoxic impurities, which comprises the following steps: diluting fosfomycin trometamol with diluent, performing high performance liquid chromatography-mass spectrometry to analyze impurities, and eluting with formic acid solution and acetonitrile solution as mobile phase. Through this technical scheme, the technical effect that can reach includes: can separate and detect one or more impurities of diethyl allenoate phosphate, diethyl 1-propylene phosphate, diethyl 3-phosphate-3-methyl-propylene oxide or dimethyl 2-phosphate-3-methyl-propylene oxide more efficiently, and can also detect the impurities simultaneously.
Preferably, in the aforementioned analysis method, the volume percentage of formic acid in the formic acid solution is 0.1-10%. Through the technical scheme, the achievable technical effects comprise: can further efficiently separate and detect one or more impurities of diethyl allephosphate, diethyl 1-propylene phosphate, diethyl 3-phosphate-3-methyl-propylene oxide or dimethyl 2-phosphate-3-methyl-propylene oxide, and can also simultaneously detect the impurities. The fosfomycin trometamol and genotoxic impurities can be effectively separated, the blank diluent does not interfere with the detection of the genotoxic impurities, and the method has strong specificity and is rapid.
Preferably, in the aforementioned analysis method, the diluent is an aqueous methanol solution, and the volume percentage of methanol in the aqueous methanol solution is 85-95%. Through this technical scheme, the technical effect that can reach includes: the fosfomycin trometamol and genotoxic impurities can be effectively separated, the blank diluent does not interfere with the detection of the genotoxic impurities, and the method has strong specificity and is rapid.
Preferably, in the aforementioned analysis method, a formic acid solution is used as the mobile phase a, an acetonitrile solution is used as the mobile phase B, and the elution mode is gradient elution. Through this technical scheme, the technical effect that can reach includes: can further efficiently separate and detect one or more impurities of diethyl allephosphate, diethyl 1-propylene phosphate, diethyl 3-phosphate-3-methyl-propylene oxide or dimethyl 2-phosphate-3-methyl-propylene oxide, and can also simultaneously detect the impurities.
Preferably, in the aforementioned assay method, the ratio of the weight (mg) of fosfomycin trometamol to the volume (mL) of diluent is 70-90: 1. through this technical scheme, the technical effect that can reach includes: can further efficiently separate and detect one or more impurities of diethyl allephosphate, diethyl 1-propylene phosphate, diethyl 3-phosphate-3-methyl-propylene oxide or dimethyl 2-phosphate-3-methyl-propylene oxide, and can also simultaneously detect the impurities. The fosfomycin trometamol and the genotoxic impurities can be effectively separated, the blank diluent does not interfere the detection of the genotoxic impurities, and the method has strong specificity and is rapid.
Preferably, in the aforementioned analysis method, the elution rate is 0.75-0.85 mL/min, and the temperature of the eluted chromatographic column is 28-32 ℃. Through this technical scheme, the technical effect that can reach includes: the fosfomycin trometamol and genotoxic impurities can be effectively separated, blank diluent does not interfere the detection of the genotoxic impurities, and the method has strong specificity and is rapid; can carry out quantification on the fosfomycin trometamol, and has high sensitivity, and the detection limit is below 0.05 ppm.
Preferably, in the aforementioned analytical method, the chromatographic column used in the HPLC-MS combined method is Agilent Zorbax SB-C18; the chromatographic column has a particle size of 3.5 μm, a length of 150mm and an inner diameter of 2.1 mm. Through this technical scheme, the technical effect that can reach includes: can further efficiently separate and detect one or more impurities of diethyl allephosphate, diethyl 1-propylene phosphate, diethyl 3-phosphate-3-methyl-propylene oxide or dimethyl 2-phosphate-3-methyl-propylene oxide, and can also simultaneously detect the impurities. The fosfomycin trometamol and genotoxic impurities can be effectively separated, the blank diluent does not interfere with the detection of the genotoxic impurities, and the method has strong specificity and is rapid; can carry out quantification on the fosfomycin trometamol, has high sensitivity, and has the detection limit below 0.05 ppm.
Preferably, in the analytical method as described above, the impurities include one or more of diethylpropadiene phosphate (impurity a), diethyl1-propenophosphate (impurity B), diethyl3-phosphate-3-methyl-propylene oxide (impurity C), or dimethyl2-phosphate-3-methyl-propylene oxide (impurity D). Through this technical scheme, the technical effect that can reach includes: the fosfomycin trometamol and the genotoxic impurities can be effectively separated, the blank diluent does not interfere the detection of the genotoxic impurities, and the method has strong specificity and is rapid.
Preferably, in the aforementioned analysis method, the hplc-ms method comprises using a triple quadrupole mass spectrometer under the following conditions: electrospray ion source (ESI), positive ion mode acquisition; the temperature of the drying gas is 200 ℃; flow rate of drying gas: 14.0L/min; atomization pressure: 20 psi; transmission voltage: 380V. Through this technical scheme, the technical effect that can reach includes: the fosfomycin trometamol and genotoxic impurities can be effectively separated, the blank diluent does not interfere with the detection of the genotoxic impurities, and the method has strong specificity and is rapid; can carry out quantification on the fosfomycin trometamol, has high sensitivity, and has the detection limit below 0.05 ppm.
1 preferably, in the analytical method described above, the detection wavelength is 234 nm; the injection volume was 5. mu.l. Through this technical scheme, the technical effect that can reach includes: the fosfomycin trometamol and genotoxic impurities can be effectively separated, the blank diluent does not interfere with the detection of the genotoxic impurities, and the method has strong specificity and is rapid; can carry out quantification on the fosfomycin trometamol, has high sensitivity, and has the detection limit below 0.05 ppm.
The beneficial effects of the invention are as follows:
1. the invention provides an analysis method of fosfomycin trometamol genotoxic impurities, which can separate and detect impurities such as diethyl propadienephosphate and the like more efficiently and has the advantages of strong specificity, high sensitivity, simplicity, rapidness and low cost. Can detect out the genotoxic impurity such as diethyldiallyl phosphate and the like under the condition that the detection limit is less than 0.05 ppm.
2. The fosfomycin trometamol and genotoxic impurities can be effectively separated, the blank diluent does not interfere with the detection of the genotoxic impurities, and the method has strong specificity and is rapid;
3. can carry out quantification on the fosfomycin trometamol, and has high sensitivity, and the detection limit is below 0.05 ppm.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to these drawings without inventive effort.
FIG. 1 is a typical chromatographic mass spectrum of a solvent;
FIG. 2 is a typical chromatogram mass spectrum of a control solution;
FIG. 3 is a typical chromatogram mass spectrum of a test solution.
Detailed Description
The experimental methods without specifying specific conditions in the following examples were generally determined according to national standards. If there is no corresponding national standard, it is carried out according to the universal international standard, the conventional conditions, or the conditions recommended by the manufacturer.
The features mentioned with reference to the invention or the features mentioned with reference to the embodiments can be combined. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.
In the present invention, all the technical features mentioned herein and preferred features may be combined with each other to form a new technical solution, if not specifically stated.
In order to make the technical means, the original characteristics, the achieved purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments, but the invention includes but is not limited to the embodiments.
Fosfomycin trometamol (available from northeast pharmaceutical group, inc.);
diethylallenphosphate (impurity a, available from shanghai nuoxing pharmaceutical science and technology ltd);
diethyl 1-propenoate (impurity B, available from shanghai norfloxacin medical science and technology, ltd);
3-diethyl phosphate-3-methyl-propylene oxide (impurity C, available from shanghai nuoxing pharmaceutical science and technology, ltd.);
2-dimethyl phosphate-3-methyl-propylene oxide (impurity D, available from shanghai nuoxing pharmaceutical science and technology, ltd.);
triple quadrupole mass spectrometer detector (Agilent 6490, available from Agilent technologies, china, ltd.).
Example 1 method for analyzing genotoxic impurities in fosfomycin trometamol
This example describes a method for analyzing genotoxic fosfomycin trometamol impurities, which includes: diethylallenphosphate (impurity a), diethyl1-propenophosphate (impurity B), diethyl3-phosphate-3-methyl-propylene oxide (impurity C), or dimethyl2-phosphate-3-methyl-propylene oxide (impurity D).
1. Reference substance
Diethylallenphosphate (impurity a): batch number: 20190408, content: 94.78 percent.
Diethyl 1-propenophosphate (impurity B): batch number: 20190409, content: 98.79 percent.
Diethyl 3-phosphate-3 methyl-propylene oxide (impurity C): batch number: 20190916, content: 94.34 percent.
2-dimethyl phosphate-3-methyl-propylene oxide (impurity D): batch number: 20190918, content: 94.61 percent.
2. Experimental methods
2.1 method
Taking 80mg of fosfomycin trometamol raw material medicine, precisely weighing, precisely adding 1ml of methanol-water (9: 1), swirling to dissolve, and shaking uniformly to obtain a test solution.
Another 10mg of impurity A, B, C, D reference substance is precisely weighed, is placed in a 10ml measuring flask, is added with methanol for dissolution and is diluted to a scale, is shaken up, is precisely weighed for 1ml, is placed in a 100ml measuring flask, is diluted to a scale by methanol-water (9: 1), is shaken up, is precisely weighed for 1ml again, is placed in a 100ml measuring flask, is diluted to a scale by methanol-water (9: 1), is shaken up, is precisely weighed for 5ml again, is placed in a 25ml measuring flask, is diluted to a scale by methanol-water (9: 1), is shaken up to a reference substance solution.
Refer to the test method of high performance liquid chromatography (china pharmacopoeia 2015 edition four parts general rule 0431). Octadecylsilane chemically bonded silica was used as a filler (Agilent Zorbax SB-C18, 2.1 mm. times.150 mm, 3.5 μm), 0.1% formic acid solution was used as mobile phase A, and acetonitrile was used as mobile phase B; gradient elution was performed as in table 2; the flow rate was 0.8ml per minute; the column temperature is 30 ℃; a triple quadrupole mass spectrometer is adopted, in an electrospray ionization (ESI) positive ion mode and a multi-reaction monitoring mode (MRM), monitored ion pairs and collision energy parameters of the impurity A, B, C, D are shown in a table 1, the monitoring time is 1.6-4.9 minutes, and the effluent does not enter a mass spectrometer in the rest time. Precisely measuring 5 mu l of quantitative limiting solution, repeatedly injecting for 6 times, wherein the relative standard deviation of the obtained peak area is not more than 10%, and the signal-to-noise ratio of the main peak is not less than 10%. Precisely measuring the sample solution and the reference solution by 5 μ l each, injecting into a liquid chromatograph, and recording chromatogram as shown in fig. 1-3. Wherein, the impurity A, B, C, D contained in the fosfomycin trometamol bulk drug applied to the test sample is not more than 0.000025% (250 ng/g).
TABLE 1 impurity A, B, C, D monitoring ion Pair parameters
| Compound (I) | Quantitative ion pair (Collision energy) | Qualitative ion pair (Collision energy) |
| Impurity A | 177.1-120.8（10） | 177.1-103（20） |
| Impurity B | 179.1-122.8（10） | 179.1-104.9（20） |
| Impurity C | 195.1-138.9（5） | 195.1-120.9（15） |
| Impurity D | 167.1-108.9（15） | 167.1-134.7（10） |
2.2 chromatographic conditions
And (3) chromatographic column: agilent Zorbax SB-C18 (2.1 mm. times.150 mm, 3.5 μm); mobile phase: a: 0.1% formic acid solution, B: acetonitrile, gradient elution, shown in table 2; column temperature: 30 ℃; flow rate: 0.8 ml/min; the amount of sample was 5. mu.l. With the parameter settings of table 2, the degree of separation between impurities A, B, C, D was 1.5, allowing accurate quantification.
TABLE 2 gradient elution Table
| t（min） | A（%） | B（%） |
| 0 | 95 | 5 |
| 10 | 10 | 90 |
| 15 | 10 | 90 |
| 16 | 95 | 5 |
| 21 | 95 | 5 |
2.3 Mass Spectrometry conditions
Electrospray ion source (ESI), positive ion mode acquisition; the temperature of the drying gas is 200 ℃; flow rate of drying gas: 14.0L/min; atomization pressure: 20 psi; transmission voltage: 380V. The impurity A, B, C, D multiple reaction monitoring mode parameters are shown in table 2.
Methodology validation
3.1 System suitability test
The impurity D, C, A, B appears successively, and the separation degree between the peaks is respectively 13.3, 2.5 and 2.3.
3.2 linearity
Respectively taking 10mg of each impurity A, B, C, D reference substance, precisely weighing, placing in a 10ml measuring flask, adding methanol to dissolve and dilute to scale, shaking up, and using as reference substance stock solution. A proper amount of the control stock solution was precisely measured and diluted to 50ng/ml with methanol-water (9: 1) to obtain a control solution (5). Precisely measuring 1ml, 2ml and 10ml of the reference solution (5), placing in 50ml, 10ml and 25ml measuring bottles, respectively, diluting to scale with methanol-water (9: 1), and shaking to obtain reference solutions (1), (2), (3) and (4). Precisely measuring 5 μ l of the reference solution, respectively injecting sample, and calculating linear regression equation with concentration as abscissa and peak area as ordinate. The peak area and the concentration of the impurity A, B, C, D have a good linear relationship, the regression equation and the correlation coefficient are shown in table 3, and typical chromatogram mass spectrograms are shown in fig. 1-3, wherein fig. 1 is a typical chromatogram mass spectrogram of a solvent, fig. 2 is a typical chromatogram mass spectrogram of a reference solution, and fig. 3 is a typical chromatogram mass spectrogram of a test solution. The detection result shows that the peak of the impurity A, B, C, D is completely different from the peaks of the solvent and the reference substance, so the technical parameters described in the embodiment can be used for effectively detecting the impurity A, B, C, D.
TABLE 3 Linear regression equation and correlation coefficient for impurity A, B, C, D
| Linear regression equation | Correlation coefficient | Linear range | |
| Impurity A | y=6284.46x+526.02 | 0.9998 | 1.059～52.93ng/ml |
| Impurity B | y=7763.36+714.44 | 0.9998 | 1.115～55.77ng/ml |
| Impurity C | y=190.02x-26.19 | 0.9999 | 0.9132～45.66ng/ml |
| Impurity D | y=525.84x-5.07 | 0.9997 | 0.9773～48.87ng/ml |
Example 2. method for analyzing fosfomycin trometamol genotoxic impurity
This example mainly describes a method for analyzing fosfomycin trometamol genotoxic impurities, which is different from example 1 in the following technical parameters:
(1) the mobile phase A is an aqueous solution containing 10% formic acid;
(2) the test solution is prepared by the following method: 80mg of fosfomycin trometamol raw material medicine is taken and added into water solution containing 95 percent methanol, and the mixture is swirled to be dissolved and shaken evenly to be used as test solution.
(3) The ratio of the weight (mg) of the fosfomycin trometamol raw material drug to the volume (mL) of the test solution is 90: 1;
(4) the gradient elution rate is 0.85 mL/min, and the temperature of the eluted chromatographic column is 32 ℃;
(5) the high performance liquid chromatography-mass spectrometry combined method comprises the following steps of adopting an Agilent 6490 triple quadrupole mass spectrometer, and increasing mass spectrum conditions: electrospray ion source (ESI), positive ion mode acquisition; the temperature of the drying gas is 200 ℃; flow rate of drying gas: 14.0L/min; atomization pressure: 20 psi; transmission voltage: 380V.
After setting the above technical parameters, the inventors obtained experimental results similar to those of fig. 1 to 3 of example 1 by performing detection by a high performance liquid chromatography-mass spectrometry combination method.
Example 3. method for analyzing fosfomycin trometamol genotoxic impurity
This example mainly describes a method for analyzing fosfomycin trometamol genotoxic impurities, which is different from example 2 in the following technical parameters:
(1) the mobile phase A is an aqueous solution containing 1% formic acid;
(2) the test solution is prepared by the following method: 80mg of fosfomycin trometamol raw material medicine is taken and added into water solution containing 85% methanol, and the mixture is swirled to be dissolved and shaken evenly to be used as test solution.
(3) The ratio of the weight (mg) of the fosfomycin trometamol raw material drug to the volume (mL) of the test solution is 70: 1;
(4) the gradient elution rate was 0.75 mL/min and the temperature of the eluted column was 28 ℃.
After setting the above technical parameters, the inventors obtained experimental results similar to those of fig. 1 to 3 of example 1 by performing detection by a high performance liquid chromatography-mass spectrometry combination method.
Example 4. method for analyzing fosfomycin trometamol genotoxic impurity
This example mainly describes a method for analyzing fosfomycin trometamol genotoxic impurities, which is different from example 3 in the following technical parameters:
(1) the mobile phase A is an aqueous solution containing 2% formic acid;
(2) the test solution is prepared by the following method: 80mg of fosfomycin trometamol raw material medicine is taken and added into aqueous solution containing 90% methanol, and the mixture is swirled to be dissolved and shaken evenly to be used as test solution.
(3) The ratio of the weight (mg) of the fosfomycin trometamol raw material drug to the volume (mL) of the test solution is 80: 1;
(4) the gradient elution rate was 0.8mL/min and the temperature of the eluted column was 30 ℃.
After setting the above technical parameters, the inventors obtained experimental results similar to those of fig. 1 to 3 of example 1 by performing detection by a high performance liquid chromatography-mass spectrometry combination method.
Example 5 method for analyzing fosfomycin trometamol genotoxic impurities
This example mainly describes an analysis method of fosfomycin trometamol genotoxic impurities, which are: diethylallenphosphate.
1. Reference substance
Diethylallephosphate (impurity a), content: 94.78 percent.
2. Experimental methods
2.1 methods
80mg of fosfomycin trometamol raw material medicine is weighed, 1ml of methanol-water (9: 1) is added, vortex is carried out to dissolve, and the mixture is shaken up to be used as a test solution.
Taking 10mg of the impurity A reference substance, precisely weighing, and placing in a 10ml measuring flask; adding methanol to dissolve and dilute to scale, and shaking up; precisely measuring 1ml, placing in a 100ml measuring flask, diluting with methanol-water (9: 1) to scale, and shaking; precisely measuring 1ml, placing in a 100ml measuring flask, diluting with methanol-water (9: 1) to scale, and shaking; precisely measure 5ml, place in a 25ml measuring flask, dilute to the mark with methanol-water (9: 1), shake up, as a control solution.
Refer to the test method of high performance liquid chromatography (china pharmacopoeia 2015 edition four parts general rule 0431). Octadecylsilane chemically bonded silica was used as a filler (Agilent Zorbax SB-C18, 2.1 mm. times.150 mm, 3.5 μm), 0.1% formic acid solution was used as a mobile phase A, and acetonitrile was used as a mobile phase B; gradient elution was performed as in table 2; the flow rate was 0.8ml per minute; the column temperature is 30 ℃; a triple quadrupole mass spectrometer detector is adopted, a multi-reaction monitoring mode (MRM) is adopted, parameters of a monitored ion pair and collision energy of the impurity A are shown in a table 1, the monitoring time is 1.6-4.9 minutes, and effluent does not enter a mass spectrometer in the rest time.
2.2 chromatographic conditions
A chromatographic column: agilent Zorbax SB-C18 (2.1 mm. times.150 mm, 3.5 μm);
mobile phase: a: 0.1% formic acid solution, B: acetonitrile, gradient elution, shown in table 2;
column temperature: 30 ℃;
flow rate: 0.8 ml/min;
the amount of sample was 5. mu.l.
2.3 Mass Spectrometry conditions
Electrospray ion source (ESI), positive ion mode acquisition;
temperature of the drying gas: 200 ℃;
flow rate of drying gas: 14.0L/min;
atomization pressure: 20 psi;
transmission voltage: 380V;
the impurity a multiple reaction monitoring mode parameters are shown in table 2.
Methodology validation
3.1 linearity
Taking 10mg of each impurity A reference substance, accurately weighing, placing in a 10ml measuring flask, adding methanol to dissolve and dilute to scale, shaking up, and using as reference substance stock solution. A proper amount of the control stock solution was precisely measured and diluted to 50ng/ml with methanol-water (9: 1) to obtain a control solution (5). Precisely measuring 1ml, 2ml and 10ml of the reference solution (5), placing in 50ml, 10ml and 25ml measuring bottles, respectively, diluting to scale with methanol-water (9: 1), and shaking to obtain reference solutions (1), (2), (3) and (4). Measuring 5 mul of the reference solution, injecting samples respectively, and calculating a linear regression equation by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate. The peak area and the concentration of the impurity A have a good linear relation, a regression equation and a correlation coefficient are shown in a table 3, and a typical chromatogram mass spectrum is similar to those in figures 1 to 3.
Measuring 5 μ l of quantitative limiting solution, repeatedly injecting for 6 times, wherein the relative standard deviation of the obtained peak area is not over 10%, and the signal-to-noise ratio of the main peak is not lower than 10%. Precisely measuring 5 μ l of each of the test solution and the reference solution, respectively injecting into a liquid chromatograph, and recording chromatogram. Wherein, the impurity A contained in the fosfomycin trometamol bulk drug applied to the test sample is not more than 0.000025% (250 ng/g).
After setting the above technical parameters, the inventors obtained experimental results similar to those of fig. 1 to 3 of example 1 by performing detection by a high performance liquid chromatography-mass spectrometry combination method.
Example 6 method for analyzing fosfomycin trometamol genotoxic impurity
This example mainly describes a method for analyzing genotoxic impurities of fosfomycin trometamol, which is different from example 5 in that the genotoxic impurity is diethyl 1-propenophosphate (impurity B).
After setting the above technical parameters, the inventors obtained experimental results similar to those of fig. 1 to 3 of example 1 by performing detection by a high performance liquid chromatography-mass spectrometry combination method.
Example 7 method for analyzing fosfomycin trometamol genotoxic impurity
This example mainly describes a method for analyzing genotoxic impurities in fosfomycin trometamol, which is different from example 5 in that the genotoxic impurities are diethyl-3-phosphate-3-methyl-propylene oxide (impurity C).
After setting the above technical parameters, the inventors obtained experimental results similar to those of fig. 1 to 3 of example 1 by performing detection by a high performance liquid chromatography-mass spectrometry combination method.
Example 8 method for analyzing fosfomycin trometamol genotoxic impurity
This example describes mainly the analysis of the genotoxic impurity fosfomycin trometamol, which differs from example 5 in that the genotoxic impurity is dimethyl-2-phosphate-3-methyl-propylene oxide (impurity D).
After setting the above technical parameters, the inventors obtained experimental results similar to those of fig. 1 to 3 of example 1 by performing detection by a high performance liquid chromatography-mass spectrometry combination method.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, and such changes and modifications are within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (6)
1. An analysis method of fosfomycin trometamol genotoxic impurities, which is characterized by comprising the following steps: diluting fosfomycin trometamol with diluent, performing high performance liquid chromatography-mass spectrometry to analyze impurities, and performing gradient elution with formic acid solution as mobile phase A and acetonitrile solution as mobile phase B, wherein the gradient elution procedure is as follows:
the gradient elution rate is 0.75-0.85 mL/min, and an Agilent Zorbax SB-C18 chromatographic column is adopted; the particle size of the chromatographic column is 3.5 mu m, the length is 150mm, and the inner diameter is 2.1 mm; the temperature of the chromatographic column is 28-32 ℃; the detection wavelength is 234 nm; the diluent is methanol water solution; the impurities are diethyldiallyl phosphate, 1-propylene diethyl phosphate, 3-diethyl phosphate-3-methyl-propylene oxide and 2-dimethyl phosphate-3-methyl-propylene oxide.
2. The analytical method of claim 1, wherein the formic acid solution contains 0.1 to 10% by volume of formic acid.
3. The analytical method of claim 1, wherein the volume percent of methanol in the aqueous methanol solution is from 85 to 95%.
4. The assay of claim 1, wherein the ratio of the weight of fosfomycin trometamol to the volume of diluent is from 70 to 90: 1.
5. the analytical method of claim 1, wherein the hplc-ms method uses a triple quadrupole mass spectrometer with the following conditions: electrospray ion source, positive ion mode collection; temperature of the drying gas: 200 ℃; flow rate of drying gas: 14.0L/min; atomization pressure: 20 psi; transmission voltage: 380V.
6. The assay of claim 1, wherein the sample volume is 5 μ L.
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| CN113655155A (en) * | 2021-07-28 | 2021-11-16 | 任贤金 | Method for measuring SPH3127 related substances |
| CN114720578A (en) * | 2021-09-16 | 2022-07-08 | 上海峰林生物科技有限公司 | Detection method of 1-propylene diethyl phosphate impurity |
| CN114720577A (en) * | 2021-09-16 | 2022-07-08 | 上海峰林生物科技有限公司 | Detection method of diethyldiallyl phosphate impurity |
| CN115166083B (en) * | 2022-07-04 | 2023-07-07 | 国药集团致君（深圳）制药有限公司 | Method for detecting fosfomycin trometamol related substances and application thereof |
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