CN114166963B - Two-dimensional detection method for impurities in cefuroxime sodium - Google Patents
Two-dimensional detection method for impurities in cefuroxime sodium Download PDFInfo
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Abstract
The application relates to a two-dimensional qualitative and quantitative detection method of a polymer in cefuroxime sodium. According to the method, cefuroxime sodium bulk drug is taken to prepare a sample solution, HPLC-UV-MS detection is carried out, three impurities of cefuroxime sodium are separated through one-dimensional chromatography, and polymers in cefuroxime sodium are subjected to secondary separation under the two-dimensional chromatography condition, so that whether small molecules are wrapped in the polymers or not is determined, and the quantitative determination is more accurate.
Description
Technical Field
The invention belongs to the technical field of drug detection, and particularly relates to a two-dimensional qualitative and quantitative detection method of cefuroxime sodium polymer.
Background
Cefuroxime Xin Shuyu a second generation broad-spectrum cephalosporin, the original manufacturer was the gram-like company, which was marketed in succession in 1978 in the uk, irish, germany and italy, respectively, and subsequently marketed in a number of countries and regions worldwide. With the good performance of cefuroxime in the clinical market of cephalosporins in the world, the high-standard high-quality and good-curative imitated pharmaceutical product of cefuroxime produced by Italy ESSETI FARMACEUTICI S.R.L is marketed in Italy for the first time in 1986 and starts to enter the Chinese market in 1997.
Cefuroxime sodium is chemically named (6R, 7R) -7- [ 2-furyl (methoxyimino) acetamido ] 3-carbamoyloxymethyl-8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid sodium salt, has a molecular weight of 446.367, and has a structural formula:
cefuroxime sodium is suitable for treating infections caused by specific microbiologically sensitive strains. Has wide clinical application, such as respiratory tract infection, ear-nose-throat infection, urinary system infection, etc. In terms of safety, cefuroxime sodium has sensitization.
The sensitization strength and the impurity content of the polymer have a certain relation, domestic and foreign documents and patents are searched, the main control modes are two modes of G10 chromatographic column method and TSK chromatographic column method, but the two modes have limitations: g10 chromatography, poor separation of polymer from main component, poor reproducibility of method; some small molecule impurities in TSK chromatography front the main peak, resulting in polymer measurements that are greater than their actual content.
Patent CN101482544B discloses a method for detecting cefuroxime sodium polymer impurity, wherein the chromatographic conditions are adopted: detecting machine: a Dyan Summit high performance liquid chromatography system; chromatographic column: MKF-GPC-10 (100 mm. Times.7.8 mm,5 μm); mobile phase: phosphate buffer (pH 7.0) -acetonitrile (70:30) at 0.01 mol/L; flow rate: 1mL/min; detection wavelength: 254nm; sample injection amount: 20. Mu.L; the mass spectrum conditions are as follows: ESI ionization source, negative ion mode, polarization voltage-4200V, sample injection speed 5 μL/min, sampling frequency 1s, mass range 500-1500 Da; although the method can meet the requirements of qualitative and quantitative simultaneously, secondary separation cannot be performed, and the specificity of the method is at risk.
The HPLC method for measuring the polymer of cefuroxime sodium recorded in the journal of Chinese clinical medicine adopts a sephadex G10 chromatographic column and a mobile phase A:0.1mol/L phosphate buffer (ph=7.0); mobile phase B:0.01% sodium dodecyl sulfate solution; flow rate: 1.0mL/min; detection wavelength: 254nm; sample injection amount: 50. Mu.L; column temperature: room temperature. The method is a classical pharmacopoeia general method, but in this method the degree of separation of the polymer from the main peak is poorly tolerated, leading to poor reproducibility of the method.
With the improvement of the quality standard of medicines, higher requirements are put forward on the accuracy of medicine detection, and more strict detection and limit standards are put forward on the detection of cephalosporin polymers in particular.
Therefore, the field lacks a detection separation method capable of detecting cefuroxime sodium polymer impurities qualitatively, quantitatively and accurately with good reproducibility.
Disclosure of Invention
Aiming at the defects and shortcomings in the prior art, the application provides a two-dimensional detection method for cefuroxime sodium polymer, which can simultaneously realize qualitative and quantitative research on cefuroxime sodium polymer impurities. The method has high qualitative and quantitative accuracy and good reproducibility.
Cefuroxime sodium polymer and impurity 3 were detected for the first time, and the cefuroxime sodium polymer comprises impurity 1 and impurity 2. Impurity 1, retention time 11.08min, molecular weight 708.5402; impurity 2, retention time 11.78min, molecular weight 565.1060; impurity 3 retention time 12.93min, molecular weight 382.0702. Impurity 1 and impurity 2 are presumed to be polymers, and impurity 3 is a small molecule.
Specifically, the invention provides the following technical scheme:
the application relates to a detection method of cefuroxime sodium polymer, which comprises the following steps:
1) Preparing a test sample solution: weighing cefuroxime sodium to prepare a sample solution;
2) Taking the sample solution, injecting the sample solution into a liquid chromatograph, and performing one-dimensional chromatographic detection by adopting the following one-dimensional chromatographic conditions;
wherein the one-dimensional chromatographic detection conditions are as follows:
chromatographic column: TSK-gel G20SWXL gel column (7.8 mI.DX30 cm,5 μm);
mobile phase: mobile phase a: phosphate buffer, prepared from 0.5moL/L disodium hydrogen phosphate and 0.5moL/L sodium dihydrogen phosphate, in a ratio of 59:41-63:37, preferably 61:39, mobile phase aph=6.5-7.5, preferably ph=7.0; mobile phase B: acetonitrile or methanol, preferably acetonitrile;
detection wavelength: 274nm;
column temperature: 25 ℃ to 30 ℃, preferably 30 ℃;
flow rate: 0.4 to 0.8mL/min, preferably 0.6mL/min;
3) Completely separating the polymer in the sample solution from cefuroxime serving as a main component under the one-dimensional chromatographic condition, and collecting the polymer in a collecting ring;
4) And switching the collection material into a liquid chromatograph and mass spectrometer by the collection ring to perform two-dimensional chromatographic detection, wherein the chromatographic conditions are as follows:
chromatographic column: dionex Aclaim Polar Advantge I (3.0mX75m, 3 μm)
Mobile phase: mobile phase a:5mM ammonium acetate (pH adjusted = 4.5-6.5, preferably pH = 5.0, preferably pH adjusted with acetic acid) in water; mobile phase B:5 to 15% acetonitrile, preferably 10% acetonitrile;
flow rate: 0.2 to 0.5mL/min, preferably 0.3mL/min;
column temperature: 25-35 ℃, preferably 30 ℃;
detection wavelength: 274nm; and is also provided with
The mass spectrum conditions are as follows:
ion source: ESI positive ion mode
In step 3), the collection conditions of the polymer are:
the term "collection ring" refers to a ring structure, such as a metal ring tube, such as used in CN201410734233, CN201410428841, that is capable of collecting polymer from a liquid chromatograph (employing one-dimensional chromatographic conditions) and switching the collection into a liquid chromatograph (employing two-dimensional chromatographic conditions) and mass spectrometer.
The term "value" refers to a switch from 6 to 1 and 1 to 2, for example, in the case of a six-way valve.
In one embodiment, in the method of detecting cefuroxime sodium polymer of the present application, the number of collecting rings is at least 3.
In one embodiment, in the method of detecting cefuroxime sodium polymer of the present application, the mass spectrometry conditions further comprise the following:
sheath air pressure: 30-50 arb, preferably 40arb;
scanning mode: fulMS-ddMS2. FulMS: scan range (m/z 100-4000), resolution (70000). MS2: resolution (17500). Step NCE:20,40, 60.TopN: 3.
preferably, the mass spectrometry conditions further comprise the following:
spray voltage: 3500V (+);
auxiliary air pressure: 10arb;
evaporating temperature: 350 ℃;
ion transport tube temperature: 350 ℃.
In one embodiment, in the method of detecting cefuroxime sodium polymer of the present application, in the one-dimensional chromatography, the mobile phase a: mobile phase B is 95:5, run time is 20-40 min, preferably 30min.
In one embodiment, in the method for detecting cefuroxime sodium polymer of the present application, in step 4), the two-dimensional chromatography detection conditions are:
chromatographic column: dionex Aclaim Polar Advantge I (3.0mX75 m,3 μm);
mobile phase: a:5mM ammonium acetate (acetic acid ph=5.0) in water; b:10% acetonitrile;
flow rate: 0.3mL/min;
column temperature: 30 ℃;
detection wavelength: 274nm.
Mass spectrometry conditions:
ion source: the positive ion mode of the ESI,
spray voltage: 3500V (+);
sheath air pressure: 40arb;
auxiliary air pressure: 10arb;
evaporating temperature: 350 ℃;
ion transport tube temperature: 350 ℃;
scanning mode: fulMS-ddMS2. FulMS: scan range (m/z 200-3000), resolution (70000). MS2: resolution (17500). Step NCE:20,40, 60.TopN: 3.
in one embodiment, in the method for detecting cefuroxime sodium polymer of the present application, in step 4), the scanning pattern of the two-dimensional chromatographic detection conditions is: fulMS-ddMS2. FulMS: scan range (m/z 200-3000), resolution (70000). MS2: resolution (17500). Step NCE:20,40, 60.TopN:3, the molecular weight of the impurity 1 is 708.5402, and the fragment information is 279.15894-2739.63721; the molecular weight of the impurity 2 is 565.1060, and the fragment information is 364.05969-2752.29614; impurity 3 has a molecular weight of 382.0702 and fragmentation information of 338.08038 to 2976.70801.
The beneficial effects obtained by the invention are as follows:
the application provides a two-dimensional detection method for cefuroxime sodium polymer, which can simultaneously realize qualitative and quantitative research on cefuroxime sodium polymer impurities. The method has high qualitative and quantitative accuracy and good reproducibility.
Further, the application provides a two-dimensional qualitative and quantitative detection method for the polymer in the cefuroxime sodium, which comprises the steps of preparing a sample solution from cefuroxime sodium bulk drug, performing HPLC-UV-MS detection, separating three impurities of the cefuroxime sodium by one-dimensional chromatography, performing secondary separation on the polymer in the cefuroxime sodium under the two-dimensional chromatography condition, and determining whether the polymer is wrapped with small molecules or not, so that the quantification is more accurate.
Cefuroxime sodium polymer and impurity 3 were detected for the first time, and the cefuroxime sodium polymer comprises impurity 1 and impurity 2. Impurity 1, retention time 11.08min, molecular weight 708.5402; impurity 2, retention time 11.78min, molecular weight 565.1060; impurity 3 retention time 12.93min, molecular weight 382.0702. Impurity 1 and impurity 2 are presumed to be polymers, and impurity 3 is a small molecule.
Drawings
FIG. 1A representative spectrum of the poor peak profile of the impurity profile of the column selection of example 1;
FIG. 2. Example 2 one-dimensional mobile phase A ratio determination-principal peak forward delay, spectral representation of interference determination;
FIG. 3. One-dimensional mobile phase A ratio determination-separation good spectrum representation of example 2;
FIG. 4 shows a pH select-separation differential spectrum of mobile phase A of example 3 representing 1;
FIG. 5 shows a spectrum of poor separation at column temperature in example 4, representing 2;
FIG. 6. Example 5 flow rate determination-polymer peak broadening, poor sensitivity;
FIG. 7. Example 6-two-dimensional chromatographic peak shape good representative spectrum;
FIG. 8A two-dimensional chromatogram of the process specificity-impurity 1 of example 8;
FIG. 9. Method specificity-impurity 2 two-dimensional chromatography qualitative spectrum of example 8;
FIG. 10. Method specificity-impurity 3 two-dimensional chromatography qualitative spectrum of example 8;
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The liquid chromatograph used in the invention is mainly equipped with an ultraviolet and MS/MS detector.
Example 1: selection of chromatographic columns
1. Solution preparation
Test solution: taking cefuroxime sodium, precisely weighing, adding water for dissolving, and quantitatively diluting to prepare a solution containing about 0.5mg of the main component in each 1mL serving as a test solution.
2. One-dimensional chromatographic conditions
Method 1:
mobile phase a: phosphate buffer (ph=7.0) [0.5mol/L disodium hydrogen phosphate: 0.5 moL/sodium dihydrogen phosphate (61:39) ];
mobile phase B: acetonitrile;
flow rate: 0.6mL/min;
sample injection volume: 20. Mu.L;
column temperature: 30 ℃;
detection wavelength: 274nm;
isocratic elution: mobile phase a: mobile phase B is 95:5;
run time: 30min.
Method 2:
mobile phase a: phosphate buffer (ph=7.0) [0.5mol/L disodium hydrogen phosphate: 0.5 moL/sodium dihydrogen phosphate (61:39) ];
mobile phase B: acetonitrile;
flow rate: 0.6mL/min;
sample injection volume: 10. Mu.L;
column temperature: 25 ℃;
detection wavelength: 254nm;
isocratic elution: mobile phase a: mobile phase B is 95:5.
3. experimental procedure and conclusion
Conclusion: the TSKgelG20SWXL (7.8 mm. Times.30 cm,5 μm) gel column had good separation under method 1 conditions. The inferior peak pattern represents the spectrum shown in FIG. 1.
Example 2: determination of one-dimensional chromatographic condition mobile phase A proportion
1. Solution preparation
Test solution: taking cefuroxime sodium, precisely weighing, adding water for dissolving, and quantitatively diluting to prepare a solution containing about 0.5mg of the main component in each 1mL serving as a test solution.
2. One-dimensional chromatographic conditions
Chromatographic column: TSKgel G20SWXL gel column (7.8 mI.DX30 cm,5 μm);
mobile phase a: phosphate buffer (ph=7.0) [ different ratio of 0.5moL/L disodium hydrogen phosphate to 0.5moL/L sodium dihydrogen phosphate ];
mobile phase B: acetonitrile;
flow rate: 0.6mL/min;
sample injection volume: 20. Mu.L;
column temperature: 30 ℃;
detection wavelength: 274nm;
isocratic elution: mobile phase a: mobile phase B is 95:5;
run time: 30min.
3. Experimental procedure and conclusion
Conclusion: the ratio of mobile phase A ranges from 59:41 to 63:37, the peak type of polymer impurities is good, and the optimal ratio of mobile phase A is 61:39. The results are shown in fig. 2 and 3.
Example 3: pH determination of mobile phase A under one-dimensional chromatographic conditions
1. Solution preparation
Test solution: taking cephalosporin bulk drugs, precisely weighing, adding water for dissolving and quantitatively diluting to prepare a solution containing about 0.5mg of main ingredient in 1mL serving as a test sample solution.
2. One-dimensional chromatographic conditions
Chromatographic column: TSKgel G20SWXL gel column (7.8 m I.D.times.30 cm,5 μm)
Mobile phase a: phosphate buffer
[0.5mol/L disodium hydrogen phosphate: 0.5mol/L sodium dihydrogen phosphate (61:39) ];
mobile phase B: acetonitrile;
flow rate: 0.6mL/min;
sample injection volume: 20. Mu.L;
column temperature: 30 ℃;
detection wavelength: 274nm;
isocratic elution: mobile phase a: the mobile phase B is 95:5;
run time: 30min.
3. Experimental procedure and conclusion
Conclusion: the pH range of the mobile phase A is 6.5-7.5, the impurity separation degree of each polymer meets the requirement, and the pH of the optimal mobile phase A is 7.0. The spectrum of the separation degree difference is shown in figure 4.
Example 4: determination of one-dimensional chromatographic condition column temperature range
1. Solution preparation
Test solution: taking cephalosporin bulk drugs, precisely weighing, adding water for dissolving and quantitatively diluting to prepare a solution containing about 0.5mg of main ingredient in 1mL serving as a test sample solution.
2. One-dimensional chromatographic conditions
Chromatographic column: TSKgel G20SWXL gel column (7.8 m I.D.times.30 cm,5 μm)
Mobile phase a: phosphate buffer (ph=7.0) [0.5mol/L disodium hydrogen phosphate: 0.5 moL/sodium dihydrogen phosphate (61:39) ];
mobile phase B: acetonitrile;
flow rate: 0.6mL/min;
sample injection volume: 20. Mu.L;
detection wavelength: 274nm;
isocratic elution: mobile phase a: mobile phase B is 95:5;
run time: 30min.
3. Experimental procedure and conclusion
Conclusion: the chromatographic column temperature of one-dimensional chromatographic conditions is between 25 ℃ and 30 ℃, the impurity separation degree of each polymer meets the requirement, and the optimal chromatographic column temperature is 30 ℃. The spectrum of the separation degree difference is shown in figure 5.
Example 5: determination of one-dimensional chromatographic condition flow rate
1. Solution preparation
Test solution: taking cephalosporin bulk drugs, precisely weighing, adding water for dissolving and quantitatively diluting to prepare a solution containing about 0.5mg of main ingredient in 1mL serving as a test sample solution.
2. One-dimensional chromatographic conditions
Chromatographic column: TSKgel G20SWXL gel column (7.8 m I.D.times.30 cm,5 μm)
Mobile phase a: phosphate buffer (ph=7.0) [0.5mol/L disodium hydrogen phosphate: 0.5 moL/sodium dihydrogen phosphate (61:39) ];
mobile phase B: acetonitrile;
sample injection volume: 20. Mu.L;
column temperature: 30 ℃;
detection wavelength: 274nm;
isocratic elution: mobile phase a: mobile phase B is 95:5;
run time: 30min.
3. Experimental procedure and conclusion
Conclusion: the chromatographic flow rate of one-dimensional chromatographic conditions is between 0.4 and 0.8mL/min, the system applicability (peak type, separation degree, sensitivity and the like) of the chromatographic method meets the requirements, and the optimal flow rate is 0.6mL/min. The results are shown in FIG. 6.
Example 6: determination of pH value of mobile phase A under two-dimensional chromatographic conditions
1. Solution preparation
Test solution: taking cephalosporin bulk drugs, precisely weighing, adding water for dissolving and quantitatively diluting to prepare a solution containing about 0.5mg of main ingredient in 1mL serving as a test sample solution.
2. Two-dimensional chromatographic conditions
Chromatographic column: dionex Aclaim Polar Advantge I (3.0 m.times.75 m,3 μm), mobile phase: a:5mM ammonium acetate (pH adjusted with acetic acid) in water; b:10% acetonitrile, flow rate of 0.3mL/min, column temperature of 30℃and detection wavelength of 274nm.
Switching time:
3. experimental procedure and conclusion
Conclusion: the pH of the two-dimensional chromatographic condition is between 5.0 and 6.0, and the two-dimensional chromatographic peak shape is good. The results are shown in FIG. 7.
Example 7: determination of qualitative sheath gas voltage for two-dimensional mass spectrometry
1. Solution preparation
Test solution: taking cephalosporin bulk drugs, precisely weighing, adding water for dissolving and quantitatively diluting to prepare a solution containing about 0.5mg of main ingredient in 1mL serving as a test sample solution.
2. Two-dimensional chromatographic conditions
Chromatographic column: dionex Aclaim Polar Advantge I (3.0 m.times.75 m,3 μm), mobile phase A:5mM ammonium acetate (acetic acid ph=5.0) in water; mobile phase B:10% acetonitrile, flow rate of 0.3mL/min, column temperature of 30℃and detection wavelength of 274nm.
Switching time:
3. two-dimensional mass spectrometry conditions
Ion source: ESI + & spray voltage: 3500V (+). Assist gas pressure: 10arb evaporation temperature: ion transport tube temperature of 350 ℃:350 ℃ scan mode: fulMS-ddMS2. FulMS: scan range (m/z 200-3000), resolution (70000). MS2: resolution (17500). Step NCE:20,40, 60.TopN: 3.
4. experimental procedure and conclusion
Conclusion: the sheath gas voltage of the two-dimensional mass spectrum condition is between 35 and 45arb, and the response sensitivity of the mass spectrum is good.
Example 8: specificity test of the invention
1. One-dimensional chromatographic conditions
Instrument and conditions: thermo fisher liquid chromatograph, column TSKgel G20SWXL gel column (7.8 m i.d. x 30cm,5 μm), mobile phase a: phosphate buffer (ph=7.0) [0.5mol/L disodium hydrogen phosphate: 0.5 moL/sodium dihydrogen phosphate (61:39) ]; mobile phase B: acetonitrile, flow rate 0.6mL/min, sample injection volume 20. Mu.L, column temperature 30 ℃, detection wavelength 274nm, isocratic elution: mobile phase a: mobile phase B was 95:5 and run for 30min.
2. Two-dimensional chromatographic conditions
Chromatographic column: dionex Aclaim Polar Advantge I (3.0 m.times.75 m,3 μm), mobile phase A:5mM ammonium acetate (acetic acid ph=5.0) in water; mobile phase B:10% acetonitrile, flow rate of 0.3mL/min, column temperature of 30℃and detection wavelength of 274nm.
Switching time:
3. two-dimensional mass spectrometry conditions
Ion source: ESI + & spray voltage: 3500V (+). Sheath gas voltage: 40 arb. Assist gas pressure: 10arb evaporation temperature: ion transport tube temperature of 350 ℃:350 ℃ scan mode: fulMS-ddMS2. FulMS: scan range (m/z 200-3000), resolution (70000). MS2: resolution (17500). Step NCE:20,40, 60.TopN: 3.
blank solvent: mobile phase a: mobile phase b= (95:5, V/V)
System applicability solution: taking proper amount of cefuroxime sodium raw material (high temperature 60 ℃ for 30 days), precisely weighing, adding water for dissolving and quantitatively diluting to prepare a solution with the main component of about 0.5mg in each 1mL, and taking the solution as a system applicability solution.
Experimental results:
conclusion: the blank solvent does not interfere with the measurement of the sample, the peak component of the polymer impurity is single, the separation degree among the polymers meets the regulation, the method has good specificity, and the mass spectrum qualitative spectrograms are shown in figures 8-10.
Example 9: sensitivity test of the invention
Instrument and conditions: thermo fisher liquid chromatograph and mass spectrum, column is TSKgel G20SWXL gel column (7.8 m i.d. x 30cm,5 μm), mobile phase a: phosphate buffer (ph=7.0) [0.5mol/L disodium hydrogen phosphate: 0.5 moL/sodium dihydrogen phosphate (61:39) ]; mobile phase B: acetonitrile, flow rate 0.6mL/min, sample injection volume 20 μL, column temperature 30 ℃, detection wavelength 274nm, isocratic elution, mobile phase A: mobile phase B was 95:5 and run for 30min.
Blank solvent: mobile phase a: mobile phase b=95:5
Sensitivity test solution: taking a proper amount of cefuroxime sodium raw material, precisely weighing, and diluting to a signal/noise ratio (S/N) =10:1 and a signal/noise ratio (S/N) =3:1, namely the quantitative limit and the detection limit of the method.
| Name of the name | Limit of quantification (μg/mL) | Detection limit (mug/mL) |
| Cefuroxime sodium | 0.04 | 0.02 |
Conclusion: the sensitivity of each polymer was defined as cefuroxime Xin Naji, the limit of quantitation was 0.04. Mu.g/mL, and the limit of detection was 0.02. Mu.g/mL.
Example 10: repeatability test of the invention
Instrument and conditions: thermo fisher liquid chromatograph and mass spectrum, column is TSKgel G20SWXL gel column (7.8 m i.d. x 30cm,5 μm), mobile phase a: phosphate buffer (ph=7.0) [0.5mol/L disodium hydrogen phosphate: 0.5 moL/sodium dihydrogen phosphate (61:39) ], mobile phase B: acetonitrile, flow rate 0.6mL/min, sample injection volume 20 μL, column temperature 30 ℃, detection wavelength 274nm, isocratic elution, mobile phase A: mobile phase B was 95:5 and run for 30min.
Blank solvent: mobile phase a: mobile phase b=95:5
Repeatability test solution: taking proper amount of cefuroxime sodium raw material (high temperature 60 ℃ for 30 days), precisely weighing, adding water for dissolving, quantitatively diluting to prepare a solution containing about 0.5mg of main component in each 1mL, and preparing six parts in parallel as a sample solution.
Conclusion: the method has good repeatability, and the RSD is 1.2% or less than 2.0%.
Example 11: stability test of the invention
Instrument and conditions: thermo fisher liquid chromatograph and mass spectrum, column is TSKgel G20SWXL gel column (7.8 m i.d. x 30cm,5 μm), mobile phase a: phosphate buffer (ph=7.0) [0.5mol/L disodium hydrogen phosphate: 0.5 moL/sodium dihydrogen phosphate (61:39) ], mobile phase B: acetonitrile, flow rate 0.6mL/min, sample injection volume 20 μL, column temperature 30 ℃, detection wavelength 274nm, isocratic elution, mobile phase A: mobile phase B was 95:5 and run for 30min.
Blank solvent: mobile phase a: mobile phase b=95:5
Stability test solution: taking proper amount of cefuroxime sodium raw material, precisely weighing, adding water for dissolving and quantitatively diluting to prepare a solution with the main component of about 0.5mg in each 1mL, and taking the solution as a stability test solution.
| Time (hours) | Peak area | Change value (%) |
| 0 | 160403 | 0.0 |
| 3 | 166304 | 3.7 |
| 6 | 174035 | 8.5 |
| 12 | 194687 | 21.4 |
| 16 | 208287 | 29.9 |
Conclusion: the test sample stability investigation result shows that the test sample solution is unstable and needs to be prepared newly.
While the invention has been described in detail in the foregoing general description, embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (14)
1. A two-dimensional detection method of impurities in cefuroxime sodium comprises the following steps:
1) Preparing a test sample solution: weighing cefuroxime sodium to prepare a sample solution;
2) Taking the sample solution, injecting the sample solution into a liquid chromatograph, and performing one-dimensional chromatographic detection by adopting the following one-dimensional chromatographic conditions to completely separate impurities in the sample solution from cefuroxime serving as a main component, wherein the impurities comprise impurities 1, 2 and 3;
wherein the one-dimensional chromatographic detection conditions are as follows:
chromatographic column: TSK-gel G20SWXL gel column, 7.8 mI.Dx30cm, 5 μm;
mobile phase: mobile phase a: phosphate buffer solution prepared from 0.5mol/L disodium hydrogen phosphate and 0.5mol/L sodium dihydrogen phosphate in a ratio of 59:41-63:37, and mobile phase A pH=6.5-7.5; mobile phase B: acetonitrile or methanol;
detection wavelength: 274nm;
column temperature: 25-30 ℃;
flow rate: 0.4-0.8 mL/min;
3) Collecting the impurity 1, the impurity 2 and the impurity 3 separated under the one-dimensional chromatographic condition in the step 2) in a collecting ring respectively to obtain a collection object, wherein the collection condition of the collection object is as follows:
4) And switching the collected matter into a liquid chromatograph and mass spectrometer for two-dimensional chromatographic detection, wherein the chromatographic conditions of the two-dimensional chromatographic detection are as follows:
chromatographic column: dionexAclaim Polar Advantge I, 3.0mX75m, 3 μm;
mobile phase: a:5mM ammonium acetate aqueous solution, pH=4.5 to 6.5; b: 5-15% of acetonitrile;
flow rate: 0.2-0.5 mL/min;
column temperature: 25-35 ℃;
detection wavelength: 274nm; and is also provided with
The mass spectrum conditions are as follows: ion source: ESI positive ion mode;
wherein the mass spectrometry conditions further comprise the following:
sheath gas voltage: 35-45 arb.
2. The method of claim 1, wherein the one-dimensional chromatographic detection conditions are:
mobile phase: mobile phase a: phosphate buffer solution prepared from 0.5mol/L disodium hydrogen phosphate and 0.5mol/L sodium dihydrogen phosphate in a ratio of 61:39, mobile phase ApH =7.0;
column temperature: 30 ℃;
flow rate: 0.6mL/min.
3. The method of claim 1, wherein the chromatographic conditions of the two-dimensional chromatographic detection are:
mobile phase: a:5mM aqueous ammonium acetate, acetic acid to adjust ph=5.0; b:10% acetonitrile;
flow rate: 0.3mL/min;
column temperature: 30 ℃.
4. The method of claim 1, wherein the mass spectrometry conditions further comprise:
sheath gas voltage: 40 and (3) arb.
5. The method of claim 1, the number of collection rings being at least 3.
6. The method of claim 1, wherein the mass spectrometry conditions further comprise: scanning mode: fulMS-ddMS2. FulMS: scan range, m/z 100-4000, resolution70000.MS2: resolution17500.Step NCE: topN, 20, 40: 3.
7. the method of claim 1, wherein the mass spectrometry conditions further comprise:
spray voltage: 3500v+;
auxiliary air pressure: 10 an arb;
evaporating temperature: 350 ℃;
ion transport tube temperature: 350 ℃.
8. The method according to any one of claims 1 to 7, wherein in the one-dimensional chromatography, the mobile phase a: the mobile phase B is 95:5, and the running time is 20-40 min.
9. The method of claim 8, wherein the run time is 30 minutes.
10. The method according to any one of claims 1 to 7, in step 4), the two-dimensional chromatography detection conditions are:
chromatographic column: dionexAclaim Polar Advantge I, 3.0mX75m, 3 μm;
mobile phase: a:5mM aqueous ammonium acetate, acetic acid to adjust ph=5.0; b:10% acetonitrile;
flow rate: 0.3mL/min;
column temperature: 30 ℃;
detection wavelength: 274nm;
mass spectrometry conditions:
ion source: ESI positive ion mode;
spray voltage: 3500v+;
sheath air pressure: 40 an arb;
auxiliary air pressure: 10 an arb;
evaporating temperature: 350 ℃;
ion transport tube temperature: 350 ℃;
scanning mode: fulMS-ddMS2. FulMS: scan range m/z is 200-3000, resolution70000.MS2: resolution17500.Step NCE: topN, 20, 40: 3.
11. the process according to any one of claims 1 to 7, wherein the cefuroxime sodium impurity has a molecular weight of: impurity 1, 708.5402; impurity 2, 565.1060; impurity 3, 382.0702.
12. The method according to any one of claims 1 to 7, wherein in step 2), retention times after the impurities in the sample solution are completely separated from cefuroxime as a main component are respectively:
impurity 1, retention time 11.08min; impurity 2, retention time 11.78min; impurity 3 retention time 12.93min.
13. The method according to any one of claims 1 to 7, wherein in step 2), retention time and molecular weight after the impurities in the sample solution are completely separated from cefuroxime as a main component are respectively: impurity 1, retention time 11.08min, molecular weight 708.5402; impurity 2, retention time 11.78min, molecular weight 565.1060; impurity 3 retention time 12.93min, molecular weight 382.0702.
14. The method according to any one of claims 1 to 7, wherein in step 4), the scanning pattern of the two-dimensional chromatography detection conditions is: fulMS-ddMS2. FulMS: scan range m/z is 200-3000, resolution70000.MS2: resolution17500.Step NCE:
TopN, 20,40, 60: 3, the molecular weight of the impurity 1 is 708.5402, and the fragment information is 279.15894-2739.63721; the molecular weight of the impurity 2 is 565.1060, and the fragment information is 364.05969-2752.29614; the molecular weight of the impurity 3 is 382.0702, and the debris information is 338.08038-2976.708.
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