1. A detection method of impurities in latamoxef sodium, which adopts high performance liquid chromatography to detect impurities in latamoxef sodium for injection, and is characterized in that: the chromatographic conditions are as follows:

a chromatographic column: taking an Agilent ZORBAX SB-CN cyano bonded silica gel chromatographic column as a stationary phase;

mobile phase: 0.005-0.015 mol/L ammonium acetate solution-methanol (55-60: 40-45, V/V) is used as a mobile phase A; adopting 0.02-0.03 mol/L ammonium acetate solution-acetonitrile (85-90: 10-15, V/V) as a mobile phase B; and adjusting the pH value of the ammonium acetate solution to 5.8-6.5.

2. The method for detecting latamoxef sodium impurities as claimed in claim 1, wherein: the length and the inner diameter of the Agilent ZORBAX SB-CN cyano bonded silica gel chromatographic column are 250mm multiplied by 4.6mm, and the grain diameter of the filler is 5 mu m.

3. The method for detecting latamoxef sodium impurities as claimed in claim 1, wherein: adjusting the pH value of the ammonium acetate solution in the mobile phase A to 6.0 by acetic acid; in mobile phase B, the ammonium acetate solution was adjusted to pH 6.5 with acetic acid.

4. The method for detecting latamoxef sodium impurities as claimed in claim 1, wherein: the elution procedure is gradient elution, the volume ratio of the mobile phase A to the mobile phase B is gradually reduced from (73-78): 22-27) to (28-33): 67-72) along with the elution progress, and then is increased from (28-33): 67-72 to (73-78): 22-27), and the impurities are separated by gradient elution.

5. The method for detecting latamoxef sodium impurities as claimed in claim 5, wherein: the gradient elution procedure was:

0min, wherein the volume ratio of the mobile phase A to the mobile phase B is 78: 22;

for 10min, the volume ratio of the mobile phase A to the mobile phase B is 55: 45;

16min, wherein the volume ratio of the mobile phase A to the mobile phase B is 30: 70;

20min, wherein the volume ratio of the mobile phase A to the mobile phase B is 30: 70;

25min, wherein the volume ratio of the mobile phase A to the mobile phase B is 45: 55;

and 30min, wherein the volume ratio of the mobile phase A to the mobile phase B is 78: 22.

6. The method for detecting latamoxef sodium impurities as claimed in claim 1, wherein: the flow rate of the mobile phase is 0.5-0.8 mL/min.

7. The method for detecting latamoxef sodium impurities as claimed in claim 1, wherein: the detection wavelength is 250-260 nm.

8. The method for detecting latamoxef sodium impurities as claimed in claim 1, wherein: the injection temperature is 30-35 ℃.

9. The method for detecting latamoxef sodium impurities as claimed in claim 1, wherein: the sample injection amount is 10-20 uL, and the sample injection concentration is 1-2 mg/mL.

Background

Latamoxef sodium, chemical name is: (6R, 7R) -7- [ 2-carboxy-2- (4-hydroxyphenyl) -acetylamino ] -7-methoxy-3- { (1-methyl-1H-tetrazol-5-ylthio) methyl } -8-oxo-5-oxa-1-azabicyclo [4, 2, 0] oct-2-ene-2-carboxylic acid disodium salt. Latamoxef sodium is a third generation cephalosporin antibiotic of semisynthetic oxacephems, is used for septicemia, lower respiratory system infection, celiac and biliary system infection, complicated urinary tract infection, severe skin soft tissue infection and the like caused by sensitive gram-negative bacilli, and has the characteristics of wide antibacterial spectrum and strong antibacterial activity.

Certain impurities can be generated in the processes of preparation, transportation and storage of the conventional latamoxef sodium freeze-dried powder for injection, and effective monitoring of medicine impurities is one of important conditions for medicine quality control. At present, the detection method for impurities in latamoxef sodium bulk drugs and latamoxef sodium freeze-dried powder for injection is generally high performance liquid chromatography, for example, patent CN202010798209.2 discloses a detection method for latamoxef sodium polymer for injection and a detection method for on-line identification, which adopts multidimensional liquid chromatography conditions to separate and measure results of different target objects, the steps are complex, and the separation degree of various polymers is low. The Zhang wenting and the like adopt an HPLC method to determine related substances of latamoxef sodium for injection, the operation is simple, and the related substances can be determined under the lower detection limit of 0.02 ng. However, the latamoxef sodium freeze-dried powder for injection is not only easily affected by the drying conditions, auxiliary materials adopted in the freeze-dried powder, impurity types, impurity content differences and the like, and particularly obvious degradation substances are easily generated after strong acid and strong alkali conditions, so that in the process of measuring related substances of latamoxef sodium for injection by adopting an HPLC method, the shape of a main peak is poor, the main peak trailing factor is too large, the separation degree of the main peak, the impurity peak and each impurity peak is low, and the accurate and efficient detection and control of impurities in the latamoxef sodium freeze-dried powder for injection are greatly limited. Therefore, a method for detecting impurities of latamoxef sodium, which has simple operation steps and good detection effect, is sought, and is beneficial to accurately controlling the quality of latamoxef sodium freeze-dried powder for injection and ensuring the quality safety of medicines.

Disclosure of Invention

In view of the above, the invention provides a method for detecting latamoxef sodium impurities, which has the advantages of simple operation steps, good repeatability, high column efficiency, moderate main peak tailing factor and retention time, good separation degree between the main peak and the impurity peaks and among the impurity peaks, high detection accuracy due to strong alkali damage, and effective separation of the main peak and the impurity peaks without interference.

The technical scheme of the invention is realized as follows:

the invention provides a method for detecting impurities of latamoxef sodium, which adopts high performance liquid chromatography to detect the impurities of latamoxef sodium for injection, and is characterized in that: the chromatographic conditions are as follows:

a chromatographic column: taking an Agilent ZORBAX SB-CN cyano bonded silica gel chromatographic column as a stationary phase;

mobile phase: 0.005-0.015 mol/L ammonium acetate solution-methanol (55-60: 40-45, V/V) is used as a mobile phase A; adopting 0.02-0.03 mol/L ammonium acetate solution-acetonitrile (85-90: 10-15, V/V) as a mobile phase B; and adjusting the pH value of the ammonium acetate solution to 5.8-6.5.

More preferably, the Agilent ZORBAX SB-CN cyano bonded silica gel chromatographic column has a length and an inner diameter of 250mm × 4.6mm and a filler particle size of 5 μm.

More preferably, the ammonium acetate solution in the mobile phase A is adjusted to the pH value of 6.0 by acetic acid; in mobile phase B, the ammonium acetate solution was adjusted to pH 6.5 with acetic acid.

More preferably, the elution procedure is gradient elution, the volume ratio of the mobile phase A to the mobile phase B is gradually reduced from (73-78): 22-27) to (28-33): 67-72) along with the elution progress, and then increased from (28-33): 67-72) to (73-78): 22-27), and the impurities are separated by gradient elution.

More preferably, the gradient elution procedure is:

0min, wherein the volume ratio of the mobile phase A to the mobile phase B is 78: 22;

for 10min, the volume ratio of the mobile phase A to the mobile phase B is 55: 45;

16min, wherein the volume ratio of the mobile phase A to the mobile phase B is 30: 70;

20min, wherein the volume ratio of the mobile phase A to the mobile phase B is 30: 70;

25min, wherein the volume ratio of the mobile phase A to the mobile phase B is 45: 55;

and 30min, wherein the volume ratio of the mobile phase A to the mobile phase B is 78: 22.

More preferably, the flow rate of the mobile phase is 0.5 to 0.8 mL/min.

More preferably, the detection wavelength is 250-260 nm.

More preferably, the injection temperature is 30-35 ℃.

More preferably, the sample injection amount is 10-20 uL, and the sample injection concentration is 1-2 mg/mL.

Compared with the prior art, the invention has the beneficial effects that: the invention establishes an analysis and detection method for latamoxef sodium impurities by using an Agilent ZORBAX SB-CN cyano bonded silica gel chromatographic column, uses 0.005-0.015 mol/L ammonium acetate solution-methanol mixed solution as a mobile phase A, 0.02-0.03 mol/L ammonium acetate solution-acetonitrile mixed solution as a mobile phase B, and simultaneously performs related substance detection on the latamoxef sodium solution for injection under the condition of adjusting the pH value of a certain ammonium acetate solution, and the result shows that the theoretical plate number of the main peak is more than 2000, the column efficiency is high, the tailing factor of the main peak is moderate, the separation degree between the main peak and the impurity peak and among the impurity peaks can be more than 1.65, the retention time of the main peak is moderate, the peak shape is good, the repeatability is good, and after the treatment under the strong alkali condition, the separation degree of any adjacent peak can reach more than 2.0, the effective separation of the main peak and each impurity peak can be realized without interference, and the retention time of the main peak is effectively maintained within 16min, the detection accuracy is high, and more accurate and efficient detection and analysis of impurities in the latamoxef sodium freeze-dried powder for injection are facilitated.

Detailed Description

In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.

The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.

The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.

Example 1

Adopting high performance liquid chromatography to detect impurities of latamoxef sodium for injection,

the chromatographic column is as follows: the Agilent ZORBAX SB-CN cyano bonded silica gel chromatographic column is a fixed phase, the length and the inner diameter of the chromatographic column are 250mm multiplied by 4.6mm, and the grain diameter of the filler is 5 mu m;

mobile phase: 0.005mol/L ammonium acetate solution-methanol (55: 45, V/V) as mobile phase A; adopting 0.02mol/L ammonium acetate solution-acetonitrile (85: 15, V/V) as a mobile phase B; and the pH of the ammonium acetate solution was adjusted to 5.8.

And (3) an elution mode: isocratically eluting at a volume ratio of mobile phase A to mobile phase B of 70:30, wherein the flow rate of the mobile phase is 0.5 mL/min.

Detection wavelength: 254 nm.

Sample introduction amount: 10 uL.

Temperature injection: at 30 ℃.

Example 2

Adopting high performance liquid chromatography to detect impurities of latamoxef sodium for injection,

the chromatographic column is as follows: taking an Agilent ZORBAX SB-CN cyano bonded silica gel chromatographic column as a stationary phase; the length and the inner diameter of the chromatographic column are 250mm multiplied by 4.6mm, and the grain diameter of the filler is 5 mu m;

mobile phase: 0.015mol/L ammonium acetate solution-methanol (60: 40, V/V) as mobile phase A; 0.03mol/L ammonium acetate solution-acetonitrile (90: 10, V/V) is adopted as a mobile phase B; and the pH of the ammonium acetate solution was adjusted to 6.5.

And (3) an elution mode: gradient elution is carried out, and the flow rate of a mobile phase is 0.8 mL/min; the elution procedure was:

detection wavelength: 254 nm.

Sample introduction amount: 10 uL.

Temperature injection: 35 ℃ is carried out.

Example 3

Adopting high performance liquid chromatography to detect impurities of latamoxef sodium for injection,

the chromatographic column is as follows: taking an Agilent ZORBAX SB-CN cyano bonded silica gel chromatographic column as a stationary phase; the length and the inner diameter of the chromatographic column are 250mm multiplied by 4.6mm, and the grain diameter of the filler is 5 mu m;

mobile phase: 0.01mol/L ammonium acetate solution-methanol (60: 40, V/V) as mobile phase A; 0.025mol/L ammonium acetate solution-acetonitrile (90: 10, V/V) is adopted as a mobile phase B; and the pH of the ammonium acetate solution was adjusted to 6.0.

And (3) an elution mode: gradient elution, mobile phase flow rate of 0.6mL/min, elution program:

detection wavelength: 254 nm.

Sample introduction amount: 10 uL.

Temperature injection: at 30 ℃.

Example 4

Adopting high performance liquid chromatography to detect impurities of latamoxef sodium for injection,

the chromatographic column is as follows: taking an Agilent ZORBAX SB-CN cyano bonded silica gel chromatographic column as a stationary phase; the length and the inner diameter of the chromatographic column are 250mm multiplied by 4.6mm, and the grain diameter of the filler is 5 mu m;

mobile phase: 0.01mol/L ammonium acetate solution-methanol (60: 40, V/V) as mobile phase A; 0.025mol/L ammonium acetate solution-acetonitrile (90: 10, V/V) is adopted as a mobile phase B; and the pH of the ammonium acetate solution was adjusted to 6.0.

And (3) an elution mode: gradient elution is carried out, and the flow rate of a mobile phase is 0.6 mL/min; the elution procedure was:

detection wavelength: 254 nm.

Sample introduction amount: 10 uL.

Temperature injection: at 30 ℃.

Example 5

Adopting high performance liquid chromatography to detect impurities of latamoxef sodium for injection,

the chromatographic column is as follows: taking an Agilent ZORBAX SB-CN cyano bonded silica gel chromatographic column as a stationary phase; the length and the inner diameter of the chromatographic column are 250mm multiplied by 4.6mm, and the grain diameter of the filler is 5 mu m;

mobile phase: 0.01mol/L ammonium acetate solution-methanol (60: 40, V/V) as mobile phase A; 0.025mol/L ammonium acetate solution-acetonitrile (90: 10, V/V) is adopted as a mobile phase B; and the pH of the ammonium acetate solution was adjusted to 6.0.

And (3) an elution mode: gradient elution is carried out, and the flow rate of a mobile phase is 0.6 mL/min; the elution procedure was:

detection wavelength: 254 nm.

Sample introduction amount: 10 uL.

Temperature injection: at 30 ℃.

Example 6

The difference between the impurity detection of latamoxef sodium for injection by the high performance liquid chromatography in the embodiment and the embodiment 5 is that: adjusting the pH value of the ammonium acetate solution in the mobile phase A to 6.0 by acetic acid; in mobile phase B, the ammonium acetate solution was adjusted to pH 6.5 with acetic acid.

Comparative example 1

The difference between the impurity detection of latamoxef sodium for injection by adopting high performance liquid chromatography and the example 5 in the comparative example is that: an octadecylsilane bonded silica gel column TSKgel ODS-100Z C18 having a length and an inner diameter of 150X 4.6mm and a filler particle diameter of 5 μm was used as a stationary phase.

Comparative example 2

The difference between the impurity detection of latamoxef sodium for injection by adopting high performance liquid chromatography and the example 5 in the comparative example is that: the pH of the ammonium acetate solution of the mobile phase was adjusted to 7.5.

Comparative example 3

The difference between the impurity detection of latamoxef sodium for injection by adopting high performance liquid chromatography and the example 5 in the comparative example is that: the mobile phase was changed to: 0.01mol/L ammonium acetate solution-methanol (60: 40, V/V) as mobile phase A; 0.025mol/L ammonium acetate solution-methanol (90: 10, V/V) is adopted as a mobile phase B;

1. preparing a latamoxef sodium test solution for injection: precisely weighing latamoxef sodium freeze-dried powder samples (equivalent to 25mg of latamoxef sodium) for injection, placing the latamoxef sodium freeze-dried powder samples in a 25ml volumetric flask, adding water to dissolve the latamoxef sodium freeze-dried powder samples to a constant volume to obtain test solution, and preparing reference solution by using latamoxef sodium standard substance (with the purity of 97.6%) in the same method.

According to the impurity detection methods of latamoxef sodium for injection in the embodiments 1 to 6 and the comparative example 1, respectively, a sample solution of latamoxef sodium for injection is injected into a high performance liquid chromatograph, impurity detection is performed according to the different chromatographic conditions, a solvent peak and an auxiliary material peak are deducted, a theoretical plate of a main peak, a tailing factor of the main peak, retention time of the main peak and separation degree of any adjacent peak are inspected, and the results are as follows:

as can be seen from the above table, in embodiments 1 to 6 of the present invention, Agilent ZORBAX SB-CN cyano bonded silica gel chromatographic column is used as a stationary phase, an ammonium acetate solution-methanol mixed solution with a certain concentration is used as a mobile phase a, an ammonium acetate solution-acetonitrile mixed solution with a certain concentration is used as a mobile phase B, and under the condition of regulating the pH value of the mobile phase, related substance detection is performed on an injection latamoxef sodium solution, and under a test sample concentration of 1mg/mL, the theoretical plate number of a main peak is above 2000, the tailing factor is good, can be between 0.95 and 1.05, the peak shape is good, meanwhile, the main peak retention time is moderate, and the degrees of separation between the main peak and an impurity peak and between impurity peaks can be above 1.65. The detection effect of example 6 is the best, the retention time of the main peak in comparative example 1 is obviously prolonged, and the main peak is different from the impurity peaks and the separation degrees of the impurity peaks.

Meanwhile, according to the chromatographic conditions of the example 5, the results of precision and repeatability tests respectively show that the RSD is less than 0.8% (n is 5), and the precision and the repeatability of the detection method are good.

2. Strong base destruction test:

precisely weighing a latamoxef sodium freeze-dried powder sample for injection, placing the sample in a 50ml volumetric flask, adding 1ml NaOH solution with the concentration of 2mol/l, placing the sample at the room temperature of 27 ℃ for 1h, adding 1ml HCl solution with the concentration of 2mol/l for neutralization, adding water to fix the volume to a scale, shaking up, taking the solution as a sample solution, preparing a blank auxiliary material solution by the same method, and performing a strong base damage test.

10uL of latamoxef sodium test solution for injection is respectively sampled and injected into a high performance liquid chromatograph, the chromatographic conditions of the example 5 and the comparative examples 2 and 3 are respectively adopted for detection and analysis, the separation condition of the generated degradation product and the main peak retention time are observed, and the results are as follows:

as can be seen from the above table, the latamoxef sodium test solution for injection is easy to degrade to generate impurities after being destroyed under the strong alkali condition, but under the chromatographic condition of the embodiment 5, the separation degree of any adjacent peak can reach more than 2.0, the main peak and each impurity peak can be effectively separated without interference, the auxiliary materials are also free from interference, and the retention time of the main peak is effectively maintained within 16min, while the impurity peaks in the comparative examples 1 and 3 can not be effectively separated, the separation degree of each peak is poor, and the retention time of the main peak is prolonged, which indicates that the impurity detection method for the latamoxef sodium strong alkali destruction for injection is more accurate and efficient.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.