阅读说明：本技术 一种头孢孟多酯钠的含量检测方法 (Method for detecting content of cefamandole nafate ) 是由 庞海河 刘伟强 彭朝晖 王吉 袁仲 谈进 周中娥 金燕 廖孝曙 刘思川 吴中华 于 2020-11-09 设计创作，主要内容包括：本发明涉及质量控制技术领域,特别涉及一种头孢孟多酯钠的含量检测方法。该检测方法包括：将头孢孟多酯对照品和头孢孟多酯钠供试品分别溶于有机溶剂中,得到对照品溶液和供试品溶液；有机溶剂为二甲亚砜、酰胺类溶剂或醇类溶剂中的一种或几种；将对照品溶液和供试品溶液分别采用高效液相色谱法进行检测,得到头孢孟多酯钠的含量。本发明使用纯有机相溶解样品,在极大程度上抑制样品水解,样品溶液稳定、含量结果准确且重复性好；溶解样品的有机试剂可直接购买,简化了溶液配制步骤,提高了检测工作效率。(The invention relates to the technical field of quality control, in particular to a method for detecting the content of cefamandole nafate. The detection method comprises the following steps: respectively dissolving a cefamandole nafate reference substance and a cefamandole nafate test substance in an organic solvent to obtain a reference substance solution and a test substance solution; the organic solvent is one or more of dimethyl sulfoxide, amide solvents or alcohol solvents; and (3) respectively detecting the reference substance solution and the test sample solution by adopting a high performance liquid chromatography to obtain the content of the cefamandole nafate. The invention uses pure organic phase to dissolve the sample, so as to inhibit the hydrolysis of the sample to a great extent, and the sample solution is stable, the content result is accurate and the repeatability is good; the organic reagent for dissolving the sample can be directly purchased, so that the solution preparation steps are simplified, and the detection working efficiency is improved.)

1. A method for detecting the content of cefamandole nafate is characterized by comprising the following steps:

respectively dissolving a cefamandole nafate reference substance and a cefamandole nafate test substance in an organic solvent to obtain a reference substance solution and a test substance solution; the organic solvent is one or more of dimethyl sulfoxide, an amide solvent or an alcohol solvent;

detecting the reference solution and the test solution by high performance liquid chromatography respectively to obtain the content of cefamandole nafate; the conditions of the high performance liquid chromatography are as follows:

a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent;

mobile phase: a mixture of aqueous triethylamine and acetonitrile; the volume percentage concentration of the triethylamine aqueous solution is 9-11%, and the volume ratio of the triethylamine aqueous solution to the acetonitrile is (69-79): (21-31);

detection wavelength: 275-285 nm.

2. The method for detecting the content of cefamandole nafate in claim 1, wherein the concentration of cefamandole nafate in the control solution is 0.1-1.0 mg/mL, and the concentration of cefamandole nafate in the test solution is 0.1-1.0 mg/mL.

3. The content detection method according to claim 1, wherein the amide solvent is one or two of N, N-dimethylformamide and N, N-dimethylacetamide.

4. The content detection method according to claim 1, wherein the alcohol solvent is one or more of methanol, ethanol, and isopropanol.

5. The content detection method according to claim 1, wherein the specification of the chromatographic column is: 4.6X 250mm, 5 μm.

6. The content detection method according to claim 1, wherein the pH value of the triethylamine aqueous solution is adjusted to 2.3-2.7 by using phosphoric acid.

7. The content detection method according to claim 1, wherein the volume percentage concentration of the triethylamine aqueous solution in the mobile phase is 10%, and the volume ratio of the triethylamine aqueous solution to the acetonitrile is 74: 26.

8. the content detection method according to claim 1, wherein the column temperature of the high performance liquid chromatography is 30 to 40 ℃.

9. The content detection method according to claim 1, wherein the flow rate of the high performance liquid chromatography is 0.8 to 1.2 mL/min.

10. The content detection method according to claim 1, wherein the amount of sample introduction by high performance liquid chromatography is 1 to 100. mu.L.

Technical Field

The invention relates to the technical field of quality control, in particular to a method for detecting the content of cefamandole nafate.

Background

Cefamandole nafate is a prodrug of cefamandole, belongs to the second-generation semi-synthetic cephalosporin, has strong bactericidal activity and wide antibacterial spectrum, has strong antibacterial action on most gram-positive cocci, and is suitable for treating respiratory tract infection, biliary tract infection, urinary tract infection, bone and joint infection, skin soft tissue infection, abdominal infection, septicemia and the like caused by sensitive bacteria. The action mechanism of cefamandole nafate is that cefamandole nafate is combined with penicillin binding protein on a bacterial cell membrane, so that transpeptidase is acylated, the synthesis of bacterial septum and cell wall is inhibited, the cross connection of cell wall mucopeptide components is influenced, the cell division and growth are inhibited, the bacterial form is lengthened, and finally the bacterial form is dissolved and killed.

At present, two different prescriptions exist in domestic cefamandole nafate for injection: firstly, only containing cefamandole nafate raw material; ② the mixed powder of cefamandole nafate and 1 percent or 5 percent of sodium carbonate. Cefamandole nafate for injection, which is collected in foreign pharmacopoeia, is a mixed powder of cefamandole nafate and sodium carbonate in a certain proportion.

The Chinese pharmacopoeia (ChP2015) and the United states pharmacopoeia (USP41) both contain cefamandole nafate raw material medicines for injection and a content detection method of the preparation, and the content detection method of the preparation is the same as that of the raw material medicines. The Chinese pharmacopoeia method comprises the following steps: the detection method comprises the following steps: high performance liquid chromatography; a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; mobile phase: 1% triethylamine solution (pH adjusted to 2.5 with phosphoric acid) -acetonitrile (70: 30); detection wavelength: 254 nm; sample introduction amount: 20 mu L of the solution; preparing a test solution and a reference solution: taking a test sample or a reference sample, precisely weighing, adding a mobile phase for dissolution, and quantitatively diluting to prepare a solution containing about 0.1mg of cefamandole in each 1 mL. The specific method of the United states pharmacopoeia is as follows: the detection method comprises the following steps: LC; a chromatographic column: octadecyl bonding porous silica gel or inorganic oxide particle stationary phase, 250mm multiplied by 4.6mm, 5 μm; mobile phase: 10% triethylamine solution (pH adjusted to 2.5 with phosphoric acid) -acetonitrile (75: 25); detection wavelength: 254 nm; sample introduction amount: 20 mu L of the solution; preparing a test solution and a reference solution: taking a proper amount of a test sample or a reference sample, adding a mobile phase to prepare a solution containing about 0.5mg/mL of cefamandole nafate, and preparing the solution for new use.

But the methods have the technical problems of poor repeatability, low accuracy and the like of the content results of the cefamandole nafate and the cefamandole,

disclosure of Invention

In view of the above, the invention provides a method for detecting the content of cefamandole nafate. The method has the outstanding characteristics of accurate detection result, good repeatability, simple sample preparation, stable sample solution and the like.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for detecting the content of cefamandole nafate, which comprises the following steps:

respectively dissolving a cefamandole nafate reference substance and a cefamandole nafate test substance in an organic solvent to obtain a reference substance solution and a test substance solution; the organic solvent is one or more of dimethyl sulfoxide, amide solvents or alcohol solvents;

detecting the reference solution and the test solution by high performance liquid chromatography respectively to obtain the content of cefamandole nafate; the conditions of the high performance liquid chromatography are as follows:

a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent;

mobile phase: a mixture of aqueous triethylamine and acetonitrile; the volume percentage concentration of the triethylamine aqueous solution is 9-11%, and the volume ratio of the triethylamine aqueous solution to the acetonitrile is (69-79): (21-31);

detection wavelength: 275-285 nm.

The applicant finds that cefamandole nafate contains methyl ester groups in a chemical structure, is very easy to hydrolyze in an aqueous solution to generate cefamandole and formic acid, and the addition of a certain proportion of sodium carbonate in a prescription of cefamandole nafate for injection can ensure the safety and the effectiveness of the product, but the sodium carbonate can neutralize the formic acid generated in the hydrolysis process to promote the hydrolysis reaction to continuously proceed in the positive reaction direction, and the instability can easily introduce errors in the determination process of the contents of cefamandole nafate. In the cefamandole nafate content for injection in the Chinese pharmacopoeia and the United states pharmacopoeia and the cefamandole detection method, a solution (namely a mobile phase) for dissolving a sample respectively contains a 70% water phase and a 75% water phase, the sample is easy to hydrolyze, cefamandole is rapidly hydrolyzed and converted into cefamandole in the solution preparation process and the test process, the contents of cefamandole nafate and cefamandole are changed, so that the content results are inaccurate and the repeatability is poor, and a test solution and a reference solution in the United states pharmacopoeia method need to be newly used and have strict operation requirements. In addition, the product is very easy to be wetted, while the concentrations of the test solution and the reference solution in the Chinese pharmacopoeia method are low, errors are easy to generate in the actual operation process, and the content result is not parallel and accurate.

On the basis of the content of cefamandole nafate for injection in Chinese pharmacopoeia (ChP2015) and a cefamandole detection method, the invention dissolves the raw material drug or preparation in dimethyl sulfoxide, an amide solvent or an alcohol solvent, optimizes chromatographic conditions, solves the problems of poor repeatability, low accuracy and the like of the cefamandole nafate and cefamandole content results, and provides experimental basis for quality standard research. The invention has the outstanding characteristics of accurate detection result, good repeatability, simple sample preparation, stable sample solution and the like.

Preferably, the concentration of the cefamandole nafate in the control solution is 0.1-1.0 mg/mL, and the concentration of the cefamandole nafate in the test solution is 0.1-1.0 mg/mL.

In the specific embodiment provided by the present invention, the concentration of cefamandole nafate in the control solution is 0.4mg/mL, and the concentration of cefamandole nafate in the test solution is 0.4 mg/mL.

Preferably, the amide solvent is one or two of N, N-dimethylformamide and N, N-dimethylacetamide.

Preferably, the alcohol solvent is one or more of methanol, ethanol and isopropanol.

Preferably, the specification of the chromatographic column is: 4.6X 250mm, 5 μm.

In the specific embodiment provided by the invention, the type of the chromatographic column is CAPCELL PAK C18 MG II or Thermo ODS Hypersil.

Preferably, the pH value of the triethylamine aqueous solution is adjusted to 2.3-2.7 by using phosphoric acid.

Preferably, the pH value of the triethylamine aqueous solution is adjusted to 2.5-2.7 by using phosphoric acid.

In the specific embodiment provided by the invention, the pH value of the triethylamine aqueous solution is adjusted to 2.5 by using phosphoric acid.

Preferably, the volume percentage concentration of the triethylamine aqueous solution in the mobile phase is 10%, and the volume ratio of the triethylamine aqueous solution to the acetonitrile is 74: 26.

preferably, the column temperature of the high performance liquid chromatography is 30 to 40 ℃.

Preferably, the column temperature of the high performance liquid chromatography is 33-37 ℃.

In the specific embodiment provided by the invention, the column temperature of the high performance liquid chromatography is 35 ℃.

Preferably, the flow rate of the high performance liquid chromatography is 0.8 to 1.2 mL/min.

In the specific embodiment provided by the present invention, the flow rate of the high performance liquid chromatography is 1.0 mL/min.

Preferably, the sample volume of the high performance liquid chromatography is 1 to 100. mu.L.

In the specific embodiment provided by the invention, the sample volume of the high performance liquid chromatography is 5 muL.

Preferably, the detection wavelength is 278-282 nm.

In the specific embodiment provided by the present invention, the detection wavelength is 280 nm.

The invention provides a method for detecting the content of cefamandole nafate. The detection method comprises the following steps: respectively dissolving a cefamandole nafate reference substance and a cefamandole nafate test substance in an organic solvent to obtain a reference substance solution and a test substance solution; the organic solvent is one or more of dimethyl sulfoxide, amide solvents or alcohol solvents; and (3) respectively detecting the reference substance solution and the test sample solution by adopting a high performance liquid chromatography to obtain the content of the cefamandole nafate. The invention has the following technical effects:

1. the pure organic phase is used for dissolving the sample, so that the hydrolysis of the sample is inhibited to the greatest extent, the sample solution is stable, the content result is accurate, and the repeatability is good;

2. the organic reagent for dissolving the sample can be directly purchased, so that the solution preparation steps are simplified, and the detection working efficiency is improved.

Drawings

FIG. 1: linear merging graphs;

FIG. 2: a linear relationship diagram for cefamandole;

FIG. 3: a linear relationship diagram of cefamandole nafate;

FIG. 4: merging the accuracy graphs;

FIG. 5: repeatedly merging the graphs;

FIG. 6: merging graphs with intermediate precision;

FIG. 7: merging the solution stability graphs;

FIG. 8: durability (for chromatographic column) merged plot;

FIG. 9: durability (versus column temperature) merged plots;

FIG. 10: durability (versus flow rate) merged plots;

FIG. 11: durability (versus buffered salt pH) merged plot;

FIG. 12: durability (versus buffer salt concentration) merged plots;

FIG. 13: durability (ratio of buffer salt to organic phase) is combined;

FIG. 14: ruggedness (versus detection wavelength) merged plots;

FIG. 15: detecting the limit merging graph;

FIG. 16: quantitative limits are combined in the figure.

Detailed Description

The invention discloses a method for detecting the content of cefamandole nafate, which can be realized by appropriately improving process parameters by taking the contents of the cefamandole nafate as reference by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Interpretation of terms:

prodrug: the compound is a compound which is obtained by modifying a chemical structure of a drug, has no or little activity in vitro, and releases an active drug by enzymatic or non-enzymatic conversion in vivo to exert the drug effect.

The reagent or instrument used in the method for detecting the content of cefamandole nafate provided by the invention can be purchased from the market.

The invention is further illustrated by the following examples:

example 1

Chromatographic conditions are as follows: a chromatographic column: CAPCELL PAK C18 MG II (4.6X 250mm, 5 μm); mobile phase: 10% triethylamine solution (pH adjusted to 2.5 with phosphoric acid) -acetonitrile (74: 26); detection wavelength: 280 nm; column temperature: 35 ℃; flow rate: 1.0 mL/min; sample introduction amount: 5 μ L.

Preparing a reference substance solution: precisely weighing 20mg of cefamandole nafate control, adding N, N-dimethylformamide to dissolve, and quantitatively diluting to 50 mL.

Preparing a test solution: precisely weighing 20mg of each of the three batches of samples, respectively adding N, N-dimethylformamide to dissolve, and quantitatively diluting to 50 mL.

Respectively and precisely measuring 50 mu L of reference solution and test solution, injecting the reference solution and the test solution into a liquid chromatograph, measuring the main peak area according to the chromatographic conditions, and calculating the content RD of the cefamandole and the cefamandole nafate (calculated according to the cefamandole) of the test solution.

As a result: the contents RD of cefamandole in the three batches of samples are respectively 0.0%, 0.0% and 0.0%, and the contents RD of cefamandole nafate (calculated according to cefamandole) are respectively 0.20%, 0.21% and 0.095%, which shows that the detection results of cefamandole nafate (calculated according to cefamandole) and cefamandole nafate contents are parallel and reproducible.

Example 2 specificity experiments

Blank solution (high temperature and light degradation): n, N-dimethylformamide.

Test solution (undegraded): weighing 20mg of the product, adding N, N-dimethylformamide for dissolving, and quantitatively diluting to 50 mL.

Test solution (high temperature degradation): heating the product in an electric heating constant temperature air blast drying oven at 120 deg.C for 6h, precisely weighing 20mg of the heated sample, adding N, N-dimethylformamide to dissolve, and quantitatively diluting to 50 mL.

Test solution (photodegradation): a sample of the same batch is taken as a sample to be tested after being illuminated for 30 days, 20mg of the sample is precisely weighed, and N, N-dimethylformamide is added to dissolve the sample and quantitatively dilute the sample to 50 mL.

Blank solution (acid-base degradation): precisely measuring 1mL of 0.01mol/L hydrochloric acid solution, standing at room temperature for 10min, adding 1mL of 0.01mol/L sodium hydroxide solution for neutralization, and adding N, N-dimethylformamide for quantitative dilution to 50 mL.

Test solution (acid degradation): precisely weighing 20mg of the product, adding 1mL of 0.01mol/L hydrochloric acid solution, standing at room temperature for 10min, adding 1mL of 0.01mol/L sodium hydroxide solution for neutralization, and adding N, N-dimethylformamide for quantitative dilution to 50 mL.

Test solution (alkaline degradation): precisely weighing 20mg of the product, adding 1mL of 0.005mol/L sodium hydroxide solution, standing at room temperature for 1min, adding 1mL of 0.005mol/L hydrochloric acid solution for neutralization, and adding N, N-dimethylformamide for quantitative dilution to 50 mL.

Blank solution (oxidative degradation): 1mL of 1% hydrogen peroxide solution was precisely measured, and the solution was left at room temperature for 10min and quantitatively diluted to 50mL with N, N-dimethylformamide.

Test solution (oxidative degradation): precisely weighing 20mg of the product, adding 1mL of 1% hydrogen peroxide solution, standing at room temperature for 10min, and adding N, N-dimethylformamide to quantitatively dilute to 50 mL.

Precisely measuring 5 μ L of the blank solution and the sample solution, respectively, injecting into a liquid chromatograph, performing experiment according to the chromatographic conditions of example 1, and recording the chromatogram. The results show that the blank solution does not interfere with the detection of cefamandole and the cefamandole nafate peak; after the cefamandole nafate and cefamandole nafate are degraded under the conditions of acid, alkali, oxidation, high temperature and illumination, the separation degrees between the cefamandole nafate peak and the adjacent peaks thereof meet the requirement (more than or equal to 1.5), and the purities of the two main peaks meet the requirement, which shows that the detection method has good specificity.

TABLE 1 results of the specificity experiments

Remarking: "rear" -the degree of separation between a main peak and its subsequent adjacent peak; "front" -the degree of separation between a main peak and its immediately preceding adjacent peak; "pure" -the purity of the main peak meets the requirements.

Example 3 Linear experiment

Cefamandole linear range investigation: precisely weighing a proper amount of cefamandole nafate reference substances, adding N, N-dimethylformamide to prepare solutions containing cefamandole of 0.9 mu g/mL, 1.8 mu g/mL, 2.7 mu g/mL, 3.6 mu g/mL, 5.4 mu g/mL, 7.2 mu g/mL and 72 mu g/mL respectively, precisely measuring 5 mu L of the solution, injecting the solution into a liquid chromatograph, measuring the peak area of cefamandole according to the chromatographic condition of example 1 (figure 1), and performing linear regression on the injection concentration by using the peak area, wherein the regression equation is that y is 7509.58x-489.72(R is 7509.58 x-489.72)²1.0000). The results show that: the concentration of cefamandole showed a good linear relationship with the peak area in the range of 0.9-72 mug/mL (figure 2).

Linear range investigation of cefamandole nafate: precisely weighing appropriate amount of cefamandole nafate reference substance, adding N, N-dimethylformamide to prepare respectivelyPreparing a solution containing cefamandole nafate with the concentration of 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.6mg/mL and 0.8mg/mL, precisely measuring 5 mu L of the solution, injecting the solution into a liquid chromatograph, measuring the peak area of cefamandole nafate according to the chromatographic conditions of example 1 (figure 1), and performing linear regression on the injection concentration by the peak area, wherein the regression equation is that y is 5299827.15x-19858.63(R is 5299827.15 x-19858.63)²0.9999). The results show that: the concentration of cefamandole nafate in the range of 0.1mg/mL to 0.8mg/mL shows a good linear relationship with the peak area (figure 3).

Example 4 accuracy experiment

An appropriate amount of cefamandole nafate reference substance is precisely weighed, three solutions with different concentrations of 80%, 100% and 120% are respectively prepared by adding N, N-dimethylformamide, three solutions are prepared at each concentration, 5 mu L of solution is precisely measured and injected into a liquid chromatograph, an experiment is carried out according to the chromatographic conditions of example 1, and the recovery rates and RSD values of cefamandole nafate and cefamandole nafate are calculated. The experimental results show that: the recovery rate of cefamandole under three concentrations is between 100.2 and 101.7 percent, the average recovery rate is 101.1 percent, and the recovery rate RSD is 0.58 percent; the recovery rate of cefamandole nafate under three concentrations is between 99.6 and 100.4 percent, the average recovery rate is 100.1 percent, and the recovery rate RSD is 0.27 percent. The results show that the detection method of the invention has high accuracy (figure 4).

Example 5 precision experiments

1. Precision-repeatability

Precisely weighing 20mg of the sample, adding N, N-dimethylformamide to dissolve and quantitatively diluting to 50mL, preparing 6 parts in parallel, precisely weighing 5 mu L of the solution, injecting the solution into a liquid chromatograph, carrying out content measurement according to the chromatographic conditions of the example 1, and calculating the RSD value. The results show that the content of cefamandole RSD is 0.83%, the content of cefamandole nafate (calculated according to cefamandole) RSD is 0.50%, and the content of the test sample (calculated according to cefamandole) RSD is 0.49%, which shows that the detection method of the invention has good repeatability (figure 5).

2. Precision-intermediate precision

Under the conditions of the same laboratory, different time and different instruments and equipment, another experimenter precisely weighs 20mg of the same batch of test articles, adds N, N-dimethylformamide to dissolve and quantitatively dilute the test articles to 50mL, prepares 6 parts in parallel, precisely weighs 5 mu L of the test articles to inject into a liquid chromatograph, performs content determination according to the chromatographic conditions of the example 1, and calculates the RSD value of the content result for 12 times by combining repeatability data. The content of cefamandole RSD is 1.6 percent, the content of cefamandole nafate (calculated according to cefamandole) RSD is 0.55 percent, and the content of a test sample (calculated according to cefamandole) RSD is 0.44 percent, which shows that the intermediate precision of the detection method of the invention is good (figure 6).

Example 6 stability experiment

20mg of a sample to be tested is precisely weighed, N-dimethylformamide is added for dissolution, the solution is quantitatively diluted to 50mL, 5 mu L of sample is respectively injected for 0h, 2h, 4h, 6h, 8h, 22h, 25h, 29h and 32h at 4 ℃ according to the chromatographic conditions of example 1, and the peak areas of cefamandole and cefamandole nafate are determined. As a result: the peak area RSD of cefamandole is 2.3 percent; the peak area RSD of the cefamandole nafate is 1.1 percent, which shows that the test solution is stable within 32 hours at 4 ℃ by adopting the detection method of the invention (figure 7).

Example 7 durability test

1. Durable-pair chromatographic column

On the basis of the detection method of the embodiment 1, the detection method is verified by changing C18 chromatographic columns of different manufacturers and keeping other chromatographic conditions unchanged.

Preparing a reference substance solution: an appropriate amount of cefamandole nafate control is precisely weighed, and N, N-dimethylformamide is added to prepare a solution containing about 0.4mg of cefamandole nafate per 1 mL.

Preparing a test solution: a proper amount of the sample to be tested is precisely weighed, and N, N-dimethylformamide is added to prepare a solution containing about 0.4mg per 1 mL.

Without changing other chromatographic conditions of example 1, the main peak areas of the control solution and the test solution were measured by using a chromatographic column 1(CAPCELL PAK C18 MG II, 4.6X 250mm, 5 μm) and a chromatographic column 2(Thermo ODS Hypersil, 4.6X 250mm, 5 μm), respectively, and the contents were calculated. The content of cefamandole is 0.74% by using a chromatographic column 1, the content of cefamandole nafate (calculated as cefamandole) is 90.01%, the content of a test sample (calculated as cefamandole) is 90.8%, the content of cefamandole nafate (calculated as cefamandole) is 0.72%, the content of cefamandole nafate (calculated as cefamandole) is 90.42%, and the content of the test sample (calculated as cefamandole) is 91.1%. From the results, it can be seen that the cefamandole content, the cefamandole nafate content (in terms of cefamandole) and the test article content (in terms of cefamandole) of the chromatographic columns 1 and 2 are equivalent, indicating that the method of the present invention has good durability to the chromatographic columns (fig. 8).

2. Durability-temperature of chromatographic column

On the basis of the detection method in example 1, the temperatures of the chromatographic columns were set to 33 ℃, 35 ℃ and 37 ℃, respectively, and other chromatographic conditions were unchanged, and the main peak areas of the control solution and the sample solution were measured, respectively, to calculate the contents. The column temperature was 33 deg.C, 35 deg.C, 37 deg.C, the cefamandole content was 0.75%, 0.74%, 0.75%, the cefamandole nafate (calculated as cefamandole) content was 90.38%, 90.01%, 90.36%, the test article content (calculated as cefamandole) content was 91.1%, 90.8%, 91.1%. In conclusion, the contents of cefamandole, cefamandole nafate (in terms of cefamandole) and the contents of the test sample (in terms of cefamandole) at the column temperatures of 33 ℃, 35 ℃ and 37 ℃ are equivalent, indicating that the method of the present invention has good durability against the column temperatures (fig. 9).

3. Durability-convection velocity

On the basis of the detection method of example 1, the flow rates are set to be 0.8mL/min, 1.0mL/min and 1.2mL/min respectively, other chromatographic conditions are not changed, the main peak areas of the reference solution and the test solution are measured respectively, and the content is calculated. At flow rates of 0.8mL/min, 1.0mL/min and 1.2mL/min, the contents of cefamandole nafate (calculated as cefamandole) are respectively 0.74%, 0.74% and 0.75%, the contents of cefamandole nafate (calculated as cefamandole) are respectively 90.51%, 90.01% and 90.16%, and the contents of test samples (calculated as cefamandole) are respectively 91.2%, 90.8% and 90.9%. The results show that the flow rates of 0.8mL/min, 1.0mL/min, 1.2mL/min for cefamandole content, cefamandole nafate (in terms of cefamandole) content and the test substance content (in terms of cefamandole) are all comparable, indicating that the process of the invention is robust to flow rates (fig. 10).

4. Durability-to mobile phase pH

Based on the detection method of example 1, the pH values of the mobile phases were adjusted to 2.5 and 2.7, respectively, and the other chromatographic conditions were not changed, and the main peak areas of the control solution and the sample solution were measured, respectively, to calculate the contents. When the pH value of the mobile phase is 2.5 and 2.7, the content of the cefamandole is 0.74 percent and 0.73 percent respectively, the content of the cefamandole nafate (calculated according to cefamandole) is 90.01 percent and 89.80 percent respectively, and the content of the test sample (calculated according to cefamandole) is 90.8 percent and 90.5 percent respectively. The results show that the contents of cefamandole, cefamandole nafate (in terms of cefamandole) and the sample (in terms of cefamandole) are equivalent for a mobile phase pH of 2.5, 2.7, indicating that the process of the invention is robust to the mobile phase pH (fig. 11).

5. Durability-to buffer salt concentration

Based on the detection method of example 1, the concentrations of buffer salts were adjusted to 9%, 10%, and 11%, respectively, and other chromatographic conditions were unchanged, and the main peak areas of the control solution and the sample solution were measured, respectively, to calculate the contents. The buffer salt concentrations were 9%, 10%, and 11%, the contents of cefamandole are 0.74%, and 0.73%, respectively, the contents of cefamandole nafate (in terms of cefamandole) are 90.06%, 90.01%, and 91.00%, respectively, and the contents of the test samples (in terms of cefamandole) are 90.8%, and 91.7%, respectively. From the results, it can be seen that the cefamandole content, the cefamandole nafate content (in terms of cefamandole) and the test article content (in terms of cefamandole) of 9%, 10%, 11% buffer salt concentration are all equivalent, indicating that the method of the present invention has good durability to buffer salt concentration (fig. 12).

6. Durability-comparison of buffer salts to organic phases

Based on the detection method of example 1, the buffer salt and organic phase ratio are respectively adjusted to 69:31, 74:26 and 79:21, other chromatographic conditions are not changed, the main peak areas of the reference solution and the test solution are respectively measured, and the content is calculated. When the ratio of the buffer salt to the organic phase is 69:31, 74:26, 79:21, the content of cefamandole is 0.74%, 0.72% respectively, the content of cefamandole nafate (calculated as cefamandole) is 89.76%, 90.01%, 90.12% respectively, and the content of the test sample (calculated as cefamandole) is 90.5%, 90.8% respectively. Taken together, the contents of cefamandole, cefamandole nafate (in terms of cefamandole) and the contents of the test substances (in terms of cefamandole) were all comparable at a ratio of buffer salt to organic phase of 69:31, 74:26, 79:21, indicating that the process of the invention has a good durability compared to the buffer salt and organic phase (fig. 13).

7. Durability-to the detection wavelength

Based on the detection method of example 1, the detection wavelengths were set to 278nm, 280nm, and 282nm, and the other chromatographic conditions were unchanged, and the main peak areas of the control solution and the sample solution were measured, respectively, to calculate the content. When the detection wavelength is 278nm, 280nm and 282nm, the content of cefamandole is respectively 0.76%, 0.74% and 0.75%, the content of cefamandole nafate (calculated according to cefamandole) is respectively 90.66%, 90.01% and 90.72%, and the content of the test sample (calculated according to cefamandole) is respectively 91.4%, 90.8% and 91.5%. The results show that the cefamandole content, the cefamandole nafate content (calculated according to cefamandole) and the test article content (calculated according to cefamandole) with the detection wavelengths of 278nm, 280nm and 282nm are all equivalent, which shows that the method of the invention has good durability to the detection wavelength (figure 14).

Example 8 detection Limit experiment

Precisely weighing a proper amount of cefamandole nafate reference substance, adding N, N-dimethylformamide to dissolve and gradually dilute, adjusting the concentration of the solution to enable the response value of a cefamandole nafate to be about 3 times of the noise level, measuring the peak area of cefamandole nafate according to the chromatographic conditions of example 1 (sample injection is repeated for 3 times), and recording the chromatogram to obtain the detection limit of the detection method of example 1 to be 0.9550 mug/mL (figure 15).

Example 9 quantitative Limit experiment

Precisely weighing a proper amount of cefamandole nafate reference substance, adding N, N-dimethylformamide to dissolve and gradually dilute, adjusting the concentration of the solution to enable the response value of cefamandole nafate to be about 10 times of the noise level, measuring the peak area of cefamandole nafate according to the chromatographic conditions of example 1 (sample injection is repeated for 5 times), and recording the chromatogram to obtain the quantitative limit of the detection method of example 1 to be 0.4775 mug/mL (figure 16).

Comparative example 1

Comparative example 1 the content RD of cefamandole nafate (calculated as cefamandole) in the test solution and the cefamandole nafate (calculated as cefamandole) is calculated by measuring the main peak areas in the test solution and the control solution by adopting the method for detecting the content of cefamandole nafate for injection in ChP 2015.

Chromatographic conditions are as follows: a chromatographic column: CAPCELL PAK C18 MG II (4.6X 250mm, 5 μm); mobile phase: 1% triethylamine solution (pH adjusted to 2.5 with phosphoric acid) -acetonitrile (70: 30); detection wavelength: 254 nm; column temperature: 25 ℃; flow rate: 1.0 mL/min; sample introduction amount: 20 μ L.

Preparing a reference substance solution: 10mg of cefamandole nafate control is precisely weighed, dissolved by adding a mobile phase and quantitatively diluted to 100 mL.

Preparing a test solution: 10mg of the sample was precisely weighed, dissolved in a mobile phase and quantitatively diluted to 100mL (two portions were prepared in parallel).

Respectively and precisely measuring 20 mu L of reference solution and test solution, injecting into a liquid chromatograph, measuring the main peak area according to the chromatographic conditions, and calculating the content RD of the cefamandole and the cefamandole nafate (calculated according to the cefamandole) in the test solution. The results show that the test solutions have a content RD of 25% and 1.8% of cefamandole nafate (based on cefamandole), respectively.

Comparative example 2

Comparative example 2 the content RD of cefamandole nafate (in terms of cefamandole) in the test solution was calculated by measuring the area of the main peak in the test solution and the control solution by the method for measuring the content of cefamandole nafate for injection in USP 41.

Chromatographic conditions are as follows: a chromatographic column: CAPCELL PAK C18 MG II (4.6X 250mm, 5 μm); mobile phase: 10% triethylamine solution (pH adjusted to 2.5 with phosphoric acid) -acetonitrile (75: 25); detection wavelength: 254 nm; column temperature: 25 ℃; flow rate: 1.0 mL/min; sample introduction amount: 20 μ L.

Preparing a reference substance solution: cefamandole nafate reference substance of 25mg is precisely weighed, dissolved by adding a mobile phase and quantitatively diluted to 50mL (two parts are prepared in parallel).

Preparing a test solution: the sample 25mg was weighed precisely, dissolved in a mobile phase and diluted quantitatively to 50mL (two portions were prepared in parallel).

Respectively and precisely measuring 20 mu L of reference solution and test solution, injecting into a liquid chromatograph, measuring the main peak area according to the chromatographic conditions, and calculating the content RD of the cefamandole and the cefamandole nafate (calculated according to the cefamandole) in the test solution. The results showed that the test solutions had 29% and 0.81% of cefamandole nafate (based on cefamandole) content RD, respectively.

Analysis of results of comparative examples 1 and 2

When the main peak areas of the control solution and the test solution were measured by the test method of example 1, the contents RD of cefamandole and cefamandole nafate (calculated as cefamandole) in the test solution were 0.0% and 0.095%, respectively. Comparing the results of comparative example 1, comparative example 2 and example 1 together, it can be seen that the accuracy of the results obtained by the detection methods of comparative example 1 and comparative example 2 is inferior to that obtained by the detection method of example 1.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.