Method for detecting impurities of ceftriaxone sodium polymer
阅读说明：本技术 一种头孢曲松钠聚合物杂质的检测方法 (Method for detecting impurities of ceftriaxone sodium polymer ) 是由 宋更申 安百锋 朱琳琳 于 2020-12-28 设计创作，主要内容包括：本发明属于药物检测技术领域,具体涉及一种头孢曲松钠聚合物杂质的检测方法。该方法取头孢曲松钠原料药配制供试品溶液,进行HPLC检测,检测条件如下：固定相采用球状蛋白亲水改性硅胶为填充剂的色谱柱；流动相采用体积比为93～97:7～3的流动相A和流动相B构成的混合溶液,所述流动相A为0.1～0.2％的三乙胺溶液,用磷酸溶液调节pH值至5.5～6.5,所述流动相B为乙腈。利用本发明提供的方法测定头孢曲松钠中的聚合物杂质,专属性强,可操作性好且具有良好的准确度及精密度。(The invention belongs to the technical field of drug detection, and particularly relates to a method for detecting impurities of ceftriaxone sodium polymer. The method prepares ceftriaxone sodium raw material medicine into a test solution for HPLC detection, and the detection conditions are as follows: the stationary phase adopts a chromatographic column taking globular protein hydrophilic modified silica gel as a filler; the mobile phase is a mixed solution composed of a mobile phase A and a mobile phase B in a volume ratio of 93-97: 7-3, the mobile phase A is a 0.1-0.2% triethylamine solution, the pH value is adjusted to 5.5-6.5 by using a phosphoric acid solution, and the mobile phase B is acetonitrile. The method for determining the polymer impurities in the ceftriaxone sodium has the advantages of strong specificity, good operability and good accuracy and precision.)
1. A method for detecting impurities of a ceftriaxone sodium polymer is characterized by comprising the following steps: preparing a test solution from a ceftriaxone sodium raw material medicine, and carrying out HPLC detection under the following detection conditions:
the stationary phase adopts a chromatographic column taking globular protein hydrophilic modified silica gel as a filler;
the mobile phase is a mixed solution composed of a mobile phase A and a mobile phase B in a volume ratio of 93-97: 7-3, the mobile phase A is a 0.1-0.2% triethylamine solution, the pH value is adjusted to 5.5-6.5 by using a phosphoric acid solution, and the mobile phase B is acetonitrile.
2. The method for detecting ceftriaxone sodium polymer impurities as claimed in claim 1, wherein the detection wavelength of HPLC detection is 249 nm-259 nm.
3. The method for detecting ceftriaxone sodium polymer impurities as claimed in claim 1 or 2, wherein the HPLC detection column temperature is 25-35 ℃.
4. The method for detecting ceftriaxone sodium polymer impurities as claimed in any one of claims 1-3, wherein the flow rate for HPLC detection is 0.6 mL-1.0 mL/min.
5. The method for detecting ceftriaxone sodium polymer impurities as claimed in any one of claims 1-4, wherein the concentration of the sample solution is 0.3-0.7 mg/mL.
6. The method for detecting ceftriaxone sodium polymer impurities as claimed in claim 5, wherein the concentration of the sample solution is 0.5 mg/mL.
7. The method for detecting ceftriaxone sodium polymer impurities as claimed in any one of claims 1-6, wherein the sample solution comprises ceftriaxone sodium crude drug and diluent.
8. The method for detecting ceftriaxone sodium polymer impurities as claimed in claim 7, wherein the diluent is water.
9. The method for detecting ceftriaxone sodium polymer impurities as claimed in claim 1, wherein the HPLC detection adopts the following conditions:
a chromatographic column: spherical protein hydrophilic modified silica gel is used as a filling agent;
mobile phase A: 0.14% triethylamine solution, and adjusting the pH value to 6.0 by using phosphoric acid solution;
mobile phase B: acetonitrile;
column temperature: 30 ℃;
detection wavelength: 254 nm;
flow rate: 0.8 mL/min;
sample introduction volume: 20 μ L.
10. The method for detecting ceftriaxone sodium polymer impurities as claimed in claim 1, wherein the polymer impurities comprise one or more of 7-ACA dimer, 7-ACA trimer, deacetyl cefotaxime lactone dimer, 7-ACT dimer and 7-ACT trimer.
Technical Field
The invention belongs to the technical field of drug detection, and particularly relates to a method for detecting impurities of ceftriaxone sodium polymer.
Background
Ceftriaxone sodium is a long-acting, broad-spectrum, semi-synthetic cephalosporin drug synthesized by swiss Roche (Roche) in 1978, first marketed in swiss in 1982, and was first approved by the U.S. FDA in 1984 for bulk use. The existing preparation form of ceftriaxone sodium is sterile powder for injection, and British, Switzerland and Japan all have the varieties on the market.
The sensitization of ceftriaxone sodium has a certain relation with the impurity content of the ceftriaxone sodium polymer, and the ceftriaxone sodium polymer comprises 7-ACA dimer, 7-ACA trimer, deacetylated cefotaxime lactone dimer, 7-ACT trimer and the like. The ceftriaxone sodium molecules are easy to degrade under a high-temperature condition to generate polymers, and the control of the content of the polymers is very important for the embodiment of the quality condition of the product. At present, only Chinese pharmacopoeia describes the method of the polymer. The method for determining the polymer by Chinese pharmacopoeia adopts the sephadex G-10 as a filling agent, and has poor specificity, lower theoretical plate number and poor peak pattern.
Disclosure of Invention
Aiming at the defects and shortcomings in the prior art, the invention provides the method for accurately and quickly detecting the impurities of the ceftriaxone sodium polymer, which has the advantages of good stability, good repeatability, simple operation, high theoretical plate number, high specificity, strong operability and higher accuracy and precision.
Specifically, the present invention provides the following technical solutions.
A method for detecting impurities of ceftriaxone sodium polymer comprises the following steps: preparing a test solution from a ceftriaxone sodium raw material medicine, and carrying out HPLC detection under the following detection conditions:
the stationary phase adopts a chromatographic column taking globular protein hydrophilic modified silica gel as a filler;
the mobile phase is a mixed solution composed of a mobile phase A and a mobile phase B in a volume ratio of 93-97: 7-3, the mobile phase A is a 0.1-0.2% triethylamine solution, the pH value is adjusted to 5.5-6.5 by using a phosphoric acid solution, and the mobile phase B is acetonitrile.
Preferably, in the above detection method, the detection wavelength of the HPLC detection is 249nm to 259 nm.
Preferably, in the detection method, the column temperature for the HPLC detection is 25 ℃ to 35 ℃.
Preferably, in the above detection method, the flow rate of HPLC detection is 0.6 mL-1.0 mL/min.
Preferably, in the detection method, the concentration of the sample solution is 0.3-0.7 mg/mL.
Preferably, in the detection method, the concentration of the sample solution is 0.5 mg/mL.
Preferably, in the above detection method, the test solution is composed of ceftriaxone sodium crude drug and diluent.
Preferably, in the detection method, the diluent is water.
Preferably, in the detection method, the molecular weight of the globular protein hydrophilic modified silica gel is in the range of 1000 to 10000.
Preferably, in the above detection method, the HPLC detection is performed under the following conditions:
a chromatographic column: spherical protein hydrophilic modified silica gel is used as a filling agent;
mobile phase A: 0.14% triethylamine solution, and adjusting the pH value to 6.0 by using phosphoric acid solution;
mobile phase B: acetonitrile;
column temperature: 30 ℃;
detection wavelength: 254 nm;
flow rate: 0.8 mL/min;
sample introduction volume: 20 μ L.
Preferably, in the detection method, the polymer impurities include one or more of 7-ACA dimer, 7-ACA trimer, deacetylated cefotaxime lactone dimer, 7-ACT dimer and 7-ACT trimer.
The invention has the following beneficial effects:
the detection method can realize rapid quantitative determination of the content of the polymer in the ceftriaxone sodium, and has strong operability and good accuracy and precision. The content determination method has good specificity, linearity, precision and durability through research.
Drawings
FIG. 1 is a blank solvent chromatogram provided in test example 1 of the present invention;
FIG. 2 is a chromatogram of a system suitability solution provided in test example 1 of the present invention;
FIG. 3 is a chromatogram of a control solution provided in test example 1 of the present invention;
FIG. 4 is a chromatogram of a test solution provided in test example 1 of the present invention;
FIG. 5 is a linear regression graph provided in test example 2 of the present invention.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials added in the examples are commercially conventional raw materials unless otherwise specified.
The term "product" in the invention refers to the ceftriaxone sodium crude drug unless specified otherwise, and the liquid chromatograph used in the invention is mainly equipped with an ultraviolet detector, and the chromatographic column selects spherical hydrophilic silica gel as the filler.
Example 1
1. Solution preparation
Test solution: taking a ceftriaxone sodium raw material medicine, precisely weighing, adding water for dissolving, and quantitatively diluting to prepare a solution containing about 0.5mg of ceftriaxone in every 1mL, wherein the solution is used as a test solution.
Control solution: taking a proper amount of ceftriaxone sodium reference substance, precisely weighing, adding water to dissolve, and quantitatively diluting to obtain a solution containing about 5 μ g of ceftriaxone sodium per 1mL as reference substance solution.
2. Chromatographic conditions
A chromatographic column: TSKgel G2000SWXL 7.8X 300mm, 5 μm;
mobile phase A: 0.14% triethylamine solution, and adjusting the pH value to 6.0 by using phosphoric acid solution;
mobile phase B: acetonitrile;
mobile phase A: b-95: 5;
column temperature: 30 ℃;
detection wavelength: 254 nm;
flow rate: 0.8 mL/min.
3. Sample assay
Respectively taking 20 mul of the test solution and the reference solution to carry out HPLC detection according to the chromatographic conditions, and then calculating the content of polymer impurities according to the following formula:
in the formula: a. theSample (A): the sum of the peak areas of the polymers in the test solution;
Cto pair: control concentration (mg/ml);
p: content of reference substance;
Ato pair: area of main peak of control solution;
Csample (A): sample concentration (mg/ml).
Test example 1 study of specificity
Blank solvent: and (5) purifying the water.
Test solution: taking a ceftriaxone sodium raw material medicine, precisely weighing, adding water for dissolving, and quantitatively diluting to prepare a solution containing about 0.5mg of ceftriaxone in every 1mL, wherein the solution is used as a test solution.
System applicability solution: taking 10mL of sample solution, adding 1mL of 0.1mol/L sodium hydroxide solution, standing at room temperature for 10 minutes, adding 1mL of 0.1mol/L hydrochloric acid solution, and shaking up to obtain the test solution.
Control solution: taking a proper amount of ceftriaxone sodium reference substance, precisely weighing, adding water to dissolve, and quantitatively diluting to obtain a solution containing about 5 μ g of ceftriaxone sodium per 1mL as reference substance solution.
Measuring 20 μ L of the above solutions, respectively injecting into a liquid chromatograph under the same chromatographic conditions as in example 1, and recording chromatogram, wherein FIG. 1 is chromatogram of blank solvent; FIG. 2 is a chromatogram of a system suitability solution; FIG. 3 is a chromatogram of a control solution; FIG. 4 is a chromatogram of a test solution; the system suitability results are shown in table 1.
TABLE 1 Ceftriaxone sodium polymer determination methodology validation-results of specificity System applicability
Name (R)
Number of theoretical plates
Tailing factor
Degree of separation
Peak of polymer
16202
0.77
/
Main peak
23581
1.44
3.67
As can be seen from the data in Table 1, the determination method provided by the invention has good specificity and can effectively separate ceftriaxone sodium from polymers thereof.
Experimental example 2 Linear study
Preparation of linear stock solutions:
taking 10mg of ceftriaxone sodium as a reference substance, accurately weighing, placing in a 100mL measuring flask, adding water to dissolve, diluting to scale, and shaking up to obtain a linear stock solution.
Precisely measuring 5mL of linear stock solution, placing the linear stock solution into a 50mL measuring flask, adding water to dilute the linear stock solution to a scale, and shaking the linear stock solution uniformly to obtain a linear-200% solution.
Respectively and precisely measuring 0.05mL, 0.25mL, 0.5mL, 1mL, 2.5mL, 4mL, 5mL, 6mL and 7.5mL of linear-200% solution, respectively placing the linear-200% solution in a 10mL measuring flask, adding water to dilute the solution to a scale, and shaking the solution uniformly to obtain linear-1% solution, linear-5% solution, linear-10% solution, linear-20% solution, linear-50% solution, linear-80% solution, linear-100% solution, linear-120% solution and linear-150% solution.
Precisely measuring 0.5mL of linear-1% solution, placing in a 10mL measuring flask, adding water to dilute to scale, and shaking up to obtain a quantitative limiting solution.
Each 20. mu.L of each of the linear solutions was precisely measured and injected into a chromatograph under the same chromatographic conditions as in example 1, and a chromatogram was recorded, and FIG. 5 is a linear regression chart, and the linear results are shown in Table 2.
Table 2 verification of the methodology for determining ceftriaxone sodium polymer-linear results
As can be seen from the test result data in Table 2, the measurement method provided by the present invention has good linear results.
Experimental example 3 precision study
Control solution: taking a proper amount of ceftriaxone sodium reference substance, precisely weighing, adding water to dissolve, and quantitatively diluting to obtain a solution containing about 5 μ g of ceftriaxone sodium per 1mL as a reference solution.
2 mu L of the reference solution is precisely measured, sample introduction is carried out for 5 times continuously, the chromatographic conditions are the same as those in example 1, the chromatogram is recorded, and the result of sample introduction precision is shown in Table 3.
TABLE 3 verification of the determination methodology of ceftriaxone sodium Polymer-results of sample introduction precision
Name (R)
Retention time/min
Peak area
Reference 2-1
9.996
340269
Reference 2-2
9.995
338706
Control 2-3
9.993
338866
Reference substance 2-4
9.993
339892
Reference substance 2-5
9.993
338125
Reference substance 2-6
9.991
338380
RSD/％
0.02
0.25
The results in table 3 show that the present invention provides good measurement accuracy.
Test example 4 durability study
Blank solvent: and (5) purifying the water.
Test solution: taking the product, precisely weighing, adding water to dissolve, and quantitatively diluting to obtain a solution containing 0.5mg of ceftriaxone per 1mL as a test solution (fresh preparation).
System applicability solution: taking 10mL of sample solution, adding 1mL of 0.1mol/L sodium hydroxide solution, standing at room temperature for 10 minutes, adding 1mL of 0.1mol/L hydrochloric acid solution, and shaking up to obtain the test solution.
Control solution: taking a proper amount of ceftriaxone sodium reference substance, precisely weighing, adding water to dissolve, and quantitatively diluting to obtain a solution containing about 5 μ g of ceftriaxone sodium per 1mL as a reference solution. 2 parts are prepared in parallel.
Under the conditions of a flow rate variation of. + -. 0.2ml/min, a detection wavelength variation of. + -.5 nm, a column temperature of. + -.5 ℃ and a ratio variation of. + -. 2% of acetonitrile in a mobile phase, 20. mu.L of each of the above solutions was precisely measured, and injected into a liquid chromatograph, and a chromatogram was recorded, wherein the results of the durability system applicability are shown in Table 4, and the results of the durability test sample measurement are shown in Table 5, and the normal conditions were the chromatographic detection conditions described in example 1. Calculated as peak area by external standard method. The normal conditions are the chromatographic conditions of example 1.
Table 4 ceftriaxone sodium polymer assay methodology validation-durability system suitability results
Condition
Degree of separation of main peak from pre-impurity
Normal condition
3.67
Wavelength-259 nm
3.61
Wavelength-249 nm
3.52
Condition
Degree of separation of main peak from pre-impurity
Normal condition
3.67
Room temperature 25 deg.C
3.69
Column temperature 35 deg.C
3.53
Condition
Degree of separation of main peak from pre-impurity
Normal condition
3.67
Flow-0.6 mL/min
2.01
Flow-1.0 mL/min
3.34
Condition
Degree of separation of main peak from pre-impurity
Normal condition
3.67
Acetonitrile in the mobile phase 3%
1.64
Acetonitrile 7% in the mobile phase
3.30
TABLE 5 verification of ceftriaxone sodium Polymer assay methodology-durability test article assay results
The durability test result according to the test example 4 shows that the chromatographic conditions provided by the invention are that the ratio of the mobile phase is 93-97: 7-3; detection wavelength: 249-259 nm, column temperature: 25-35 ℃, flow rate: in the range of 0.6ml to 1.0ml per minute, the measurement results were not affected by the above-mentioned changes in conditions, and the durability of the chromatographic conditions was good.
Comparative example 1
On the basis of the normal conditions of this test example 4, the ratio of mobile phases a to B was changed to 91:9 and 99: 1; or adjusting the concentration of triethylamine to 0.05 percent and 0.25 percent; or adjusting the pH of mobile phase a to 5.0, 7.0, and examining the applicability of the method.
Blank solvent: and (5) purifying the water.
Test solution: taking the product, precisely weighing, adding water to dissolve, and quantitatively diluting to obtain a solution containing 0.5mg of ceftriaxone per 1mL as a test solution (fresh preparation).
System applicability solution: taking 10mL of sample solution, adding 1mL of 0.1mol/L sodium hydroxide solution, standing at room temperature for 10 minutes, adding 1mL of 0.1mol/L hydrochloric acid solution, and shaking up to obtain the test solution.
Precisely measuring 20 mu L of system applicability solution, injecting into a liquid chromatograph, recording a chromatogram, and inspecting the separation condition of ceftriaxone sodium and the polymer, wherein the results are shown in Table 6.
Table 6 polymer assay methodology validation-durability system suitability results
Condition
Degree of separation of main peak from pre-impurity
Normal condition
3.67
Acetonitrile 1% in the mobile phase
0.85
Acetonitrile 9% in the mobile phase
The polymer has widened peak and serious tailing
The concentration of triethylamine is 0.05%
0.92
The concentration of triethylamine is 0.25 percent
0.90
The pH of mobile phase A was 5.0
0.74
The pH of mobile phase A was 7.0
0.81
As can be seen from table 6, the chromatographic conditions adjusted as described above have significantly deteriorated resolution or polymer peak pattern as compared with the normal conditions, and thus the quantitative determination requirement cannot be satisfied.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.