CN116087397A - Separation detection method for impurities in cefepime hydrochloride for injection - Google Patents

Abstract

The invention belongs to the technical field of drug detection, and particularly relates to a method for simultaneously separating and detecting various impurities in cefepime hydrochloride for injection.

Description

Separation detection method for impurities in cefepime hydrochloride for injection

Technical Field

The invention belongs to the technical field of drug detection, and particularly relates to a separation detection method for various impurities in cefepime hydrochloride for injection.

Background

Cefepime hydrochloride (Cefepime Hydrochloride) is an anti-infective field medicine, is a fourth-generation cephalosporin medicine, and has wider antibacterial spectrum than the current clinically applied antibiotic medicine because the medicine is stable to beta-lactamase. The chemical name is as follows: (6 r,7 r) -7- [ (Z) -2- (2-amino-4-thiazolyl) -2-methoxyimino ] acetamido-3- [1- (1-methylpyrrolidinyl) methyl ] -8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylate hydrochloride monohydrate, having the structural formula:

The cefepime hydrochloride is mainly used for treating moderate to severe infection caused by the sensitive bacteria of adults and children aged 2 months to 16 years, has wide application range, good antibacterial activity and small side effect, and is a special variety in cephalosporin antibiotics with a mechanism of action of highly balancing an antibacterial spectrum. Cefepime hydrochloride is easily influenced by environmental factors, and is easy to react to generate various impurities in the production and storage processes, so that a proper impurity separation detection method needs to be established for controlling the impurity spectrum. In cefepime hydrochloride for injection, the structural formula of the 10 currently known main impurities is as follows:

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by searching domestic and foreign documents and patents, the method comprises the following steps:

literature: the LC-MS/MS method is used for rapidly identifying the isomer impurities in cefepime hydrochloride, and the pharmaceutical report is provided. 2005 (04): 361", wherein the liquid phase conditions are that the chromatographic column is a C18 column (4.6 mm. Times.250 mm,5.0 μm, dalian Litt Co.); the mobile phase is acetonitrile-10 mmol/L ammonium acetate (5:95); the flow rate is 0.8mL/min (the flow is split after the column is 50%), the simultaneous separation and detection of various impurities can not be realized by adopting the conditions, the quality control of the isomer impurities in the cefepime hydrochloride bulk drug can only be realized, and the structural formula of the impurities is as follows:

CN101226174a in chinese patent literature discloses a method for detecting the content of N-methylpyrrolidine in cefepime hydrochloride raw material and its preparation, wherein a carboxyl type cation column is used as an analytical column, and a conductivity detector is used for detection, but the method is only suitable for detecting N-methylpyrrolidine. The impurity structural formula is as follows:

the above documents or patents describe a method for detecting the content of an isomer and N-methylpyrrolidine in cefepime hydrochloride raw material and a preparation thereof, and do not relate to a separation detection method of known impurities 1-10 in cefepime hydrochloride, so that the spectra of the impurities cannot be controlled, and the quality of cefepime hydrochloride for injection cannot be accurately controlled.

Disclosure of Invention

Aiming at the defects and shortcomings in the prior art, the invention provides a method for separating and detecting 10 known impurities (impurities 1-10) in cefepime hydrochloride for injection, which can efficiently monitor the safety and quality of cefepime hydrochloride medicines for injection.

The invention simultaneously measures 10 known impurities in cefepime hydrochloride for injection, including 1-10 impurities, and has obvious separation effect; meanwhile, the quantitative detection of all impurities is carried out, so that the content of the impurities in cefepime for injection can be estimated and calculated.

The method has obvious separation effect and strong specificity (the separation degree is more than 1.5); the sensitivity is obviously improved (the quantitative limit can be as low as 0.057 mug/mL, and the detection limit can be as low as 0.019 mug/mL); the accuracy of quantitative determination is obviously improved, and the quantitative determination range is wider (improved by 1-2 orders of magnitude); the recovery rate measured is close to 100%; good reproducibility (RSD of the multiple measurements less than 5%), good durability (RSD of the measurements under different conditions less than 15%).

Specifically, the invention provides the following technical scheme:

the method for separating and detecting impurities in cefepime hydrochloride for injection comprises the following steps: weighing cefepime hydrochloride for injection by adopting a diluent to prepare a sample solution, and detecting by adopting a high performance liquid chromatography method;

the chromatographic conditions include:

a chromatographic column adopts octadecylsilane chemically bonded silica gel as a filler;

mobile phase:

mobile phase a: phosphate buffer;

mobile phase B: phosphate buffer solution and acetonitrile, the volume ratio of the phosphate buffer solution to the acetonitrile is (45-55): (55-45); preferably, the volume ratio of phosphate buffer to acetonitrile is 50:50;

elution mode: gradient elution.

In some preferred embodiments, the type of chromatographic column is Waters

T3 C18。

In some preferred embodiments, the chromatographic column has a specification of 4.6mm by 250mm,5.0 μm.

In some embodiments, the mobile phase a has a pH in the range of 4.8 to 5.2; in some preferred embodiments, the pH of mobile phase a is 5.0.

In some preferred embodiments, the elution gradient of the mobile phase is:

in some preferred embodiments, the detection wavelength of the chromatographic conditions is 252 to 256nm; in some preferred embodiments, the detection wavelength of the chromatographic conditions is 254nm.

In some preferred embodiments, the phosphate buffer is a monobasic potassium phosphate solution.

In some preferred embodiments, the column temperature of the chromatographic column is from 20 ℃ to 30 ℃; in some preferred embodiments, the column temperature of the chromatographic column is 25 ℃.

In some preferred embodiments, the column flow rate of the chromatographic column is from 0.9 to 1.1mL/min; in some preferred embodiments, the column flow rate of the chromatographic column is 1.0mL/min.

In some preferred embodiments, the impurities in cefepime hydrochloride for injection include one or more of the following structural formulas:

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in some preferred embodiments, among the impurities of cefepime hydrochloride for injection, at least impurity 4, impurity 5, impurity 8 and impurity 10 are included; or at least impurity 4 and impurity 5; or at least impurity 3, impurity 4, impurity 6 and impurity 9; or at least impurity 1, impurity 4, impurity 7 or impurity 9.

In some preferred embodiments, the solution formulation method is:

test solution: taking cefepime hydrochloride for injection, adding a diluent for dissolution, and filtering to obtain a sample solution.

Mixing an impurity reference substance solution: taking cefepime hydrochloride and an impurity 1-10 reference substance respectively, and adding a diluent for dissolution to serve as a mixed impurity reference substance solution.

In some preferred embodiments, the diluent is a mixed solution of phosphate buffer and acetonitrile.

In some preferred embodiments, wherein the solution formulation further comprises:

impurity localization solution: and respectively taking a proper amount of the reference substances with the impurities of 1-10, adding a diluent to the scale, and shaking uniformly to prepare a solution with the concentration of 14 mug in each 1mL to be used as a positioning solution with the impurities of 1-10.

The method for separating and detecting the impurities in the cefepime hydrochloride for injection can better control the quality of the cefepime hydrochloride for injection and better separate and detect the impurities in the cefepime hydrochloride for injection.

The beneficial effects of the invention are as follows:

1. the invention provides a separation and detection method for 10 known impurities in cefepime hydrochloride for injection.

The detection method of the invention has the advantages that (1) has strong specificity, and the characteristic peak separation effect of 10 known impurities is remarkable (the separation degree is more than 1.5); (2) the known impurities in the cefepime hydrochloride for injection are quantitatively analyzed, and the quantitative limit and the detection limit are obviously reduced: the quantitative limit can be as low as 0.057 mug/mL, which is equivalent to 0.004% of the concentration of the solution of the test sample; the detection limit can be as low as 0.019 mug/mL, which is equivalent to 0.001% of the concentration of the solution of the test sample, and the sensitivity is obviously improved; (3) the accuracy of quantitative determination is obviously improved, and the determination range is wider; compared with the measurement accuracy required by the common standard, the quantitative measurement accuracy is improved by more than 2 orders of magnitude; (4) the recovery rate measured is close to 100%; (5) good repeatability (RSD of multiple measurements less than 5%); (6) durability was good (RSD of the measurement results under different conditions was much less than 15%).

2. The method of the invention is verified by methodology that the following indexes all reach the detection requirement, and meet the requirement of Chinese medicine supervision authorities on medicine detection:

(1) the separation degree of the main peak and the adjacent impurity peaks is larger than 1.5, the separation degree of the known impurities and the adjacent peaks is larger than 1.2, the separation effect is obvious, and good specificity is shown;

(2) the quantitative limit of detecting 10 known impurities by adopting the method can be as low as 0.057 mug/mL, which is equivalent to 0.004% of the concentration of the solution of the test sample; the detection limit can be as low as 0.019 mug/mL, which is equivalent to 0.001% of the concentration of the solution of the test sample; the quantitative limit and the detection limit have obvious reducing effect, and the sensitivity is obviously improved;

(3) in the linear concentration range, a good linear relation is shown between the peak area and the concentration of each characteristic absorption peak, and the linear correlation coefficient r is larger than 0.999; impurity 1, impurity 3, impurity 6, impurity 7, impurity 8 and impurity 9 are optimal, 1.0000 is reached, and the linear relation between the peak area and the concentration is good; compared with the correlation coefficient r which is required by the common standard and is more than 0.99, the accuracy of measurement is improved by more than 2 orders of magnitude; the detection method has higher accuracy and wider measurement range;

(4) The average recovery rate in impurity groups and the average recovery rate among groups are kept between 90% and 110%, and are close to 100%; and the recovery rate RSD of the repeated detection is less than 5%, compared with the standard that the recovery rate RSD required by the common standard is less than or equal to 10.0%, the RSD of the detection method is reduced to be less than one half of the standard requirement, the accuracy is improved by more than two times, and the accuracy is improved remarkably;

(5) the repeatability test of the parallel samples shows that the detection amount of the detected impurities is not changed obviously, the RSD of each known impurity is less than 5%, and good repeatability is shown;

(6) by changing chromatographic conditions and chromatographic column batches, the detection result RSD of the impurity content in cefepime hydrochloride is 9.2 percent and is less than 15.0 percent, which shows that the method can realize good and accurate detection effect in different condition ranges and has good and obvious durability.

3. The method can accurately control the quality of the cefepime hydrochloride for injection, is favorable for controlling the quality of the cefepime hydrochloride for injection, and is effectively and safely popularized and applied.

Drawings

FIG. 1 is a specific chromatogram of cefepime hydrochloride for injection in example 1 of the present invention;

FIG. 2 is a chromatogram of a mixed impurity control solution (including impurities 1, 2, 3, 7, 9) obtained by the chromatographic column in example 2 of the present invention;

FIG. 3 is a chromatogram of a mixed impurity control solution (including impurities 1, 2, 3, 7, 9) obtained from the mobile phase species in method 1 of example 3 of the present invention;

FIG. 4 is a chromatogram of a mixed impurity control solution (including impurities 1, 2, 3, 7, 9) obtained from the mobile phase species in method 2 of example 3 of the present invention;

FIG. 5 is a chromatogram of a mixed impurity control solution (including impurities 1 to 10) obtained in method 1 of example 4 of the present invention at a mobile phase pH of 5.5;

FIG. 6 is a chromatogram of a mixed impurity control solution (including impurities 1 to 10) obtained in method 2 of example 4 of the present invention at a mobile phase pH of 4.5;

FIG. 7 is a chromatogram of a mixed impurity control solution (including impurities 1 to 10) obtained by the elution gradient in method 1 of example 5 of the present invention;

FIG. 8 is a chromatogram of a mixed impurity control solution (including impurities 1 to 10) obtained by the elution gradient in method 2 of example 5 of the present invention;

FIG. 9 is a chromatogram of a mixed impurity solution (including impurities 1-10) obtained in example 6, method 3, wherein the ratio of phosphate buffer salt to acetonitrile in mobile phase B is 40:60;

FIG. 10 is a chromatogram of a mixed impurity solution (including impurities 1-10) obtained in example 6, method 4, wherein the ratio of phosphate buffer salt to acetonitrile in mobile phase B is 60:40.

Detailed Description

The method for separating and detecting various impurities in cefepime for injection provided by the invention is used for detecting and verifying 10 known impurities and other unknown impurities in cefepime hydrochloride for injection.

Firstly, 10 different impurities in cefepime for injection are separated, wherein the separation effect is obvious; meanwhile, the invention also carries out quantitative detection on all impurities, thereby realizing the evaluation and calculation of the impurity content in cefepime for injection.

EXAMPLE 1 specificity test

1-1, solution preparation:

a diluent: 900mL of phosphate buffer (pH 5.0) was added with 100mL of acetonitrile, and the mixture was homogenized for further use.

Blank diluent: and taking a proper amount of diluent to obtain the product.

Mixing an impurity reference substance solution: taking proper amounts of cefepime hydrochloride and an impurity 1-10 reference substance, and preparing a solution containing 1.4mg of cefepime hydrochloride and 14 mug of each impurity 1-10 mug of each impurity in each 1mL of diluent as a mixed impurity reference substance solution.

Impurity localization solution: and respectively taking a proper amount of the reference substances with the impurities of 1-10, adding a diluent to the scale, and shaking uniformly to prepare a solution with the concentration of 14 mug in each 1mL to be used as a positioning solution with the impurities of 1-10.

Test solution: weighing 24.5mg of cefepime hydrochloride for injection (14 mg of cefepime hydrochloride), precisely weighing, placing into a 10mL volumetric flask, adding a proper amount of diluent to dissolve and dilute to a scale, shaking uniformly, and filtering to obtain a sample solution.

Control solution: precisely measuring 1mL of the sample solution, placing the sample solution into a 100mL measuring flask, adding a diluent to dilute the sample solution to a scale, and shaking the sample solution uniformly to prepare a solution containing 14 mug of cefepime in each 1mL of the sample solution as a control solution.

1-2. Measurement conditions:

instrument: a high performance liquid chromatograph;

chromatographic column: waters

T3 C18，4.6mm×250mm，5μm；

Mobile phase a: phosphate buffer (0.68 g of potassium dihydrogen phosphate, dissolved in water and diluted to 1000mL, pH5.0 adjusted with sodium hydroxide solution); mobile phase B: phosphate buffer (pH 5.0) -acetonitrile (50:50);

the elution mode is gradient elution, and the elution gradient is as follows:

retention time (min)	Mobile phase a (%)	Mobile phase B (%)
			0	100	0
6	100	0
			65	40	60
75	40	60
			76	100	0
86	100	0

Column temperature: 25 ℃;

detection wavelength: 254nm;

flow rate: 1.0mL/min;

sample injection amount: 10 mu L.

1-3. Experimental steps and conclusions:

precisely measuring 10 mu L of each solution, respectively injecting into a high performance liquid chromatograph, and recording a chromatogram and a result. The results are shown in the following table, and the system applicability chromatogram is shown in FIG. 1.

TABLE 1 System applicability of cefepime hydrochloride for injection-separation results of mixed impurity control solution

Conclusion:

the system suitability test was performed by high performance liquid chromatography pair, with the following results,

detecting the blank diluent, and finding that the baseline of the blank diluent is stable, and no characteristic peak appears, so that the blank diluent does not interfere the separation of cefepime hydrochloride and various impurities;

The retention time of various impurity characteristic peaks can be positioned according to the separation results of the impurity 1-10 positioning solution, the sample solution and the control solution (table 1), wherein the retention time of cefepime hydrochloride is 27.877min, and the retention time of the impurity 1-10 is respectively: 4.142min, 5.545min, 17.920min, 22.037min, 24.623min, 29.909min, 36.547min, 40.299min, 50.660min and 70.788min;

the mixed impurity reference substance solution is adopted for measurement, and the blank diluent is found to not interfere with the separation of impurities in the sample solution, and the separation degree between adjacent chromatographic peaks of cefepime hydrochloride and 10 known impurities in the mixed impurity reference substance solution is more than 1.5; the separation degree between known impurities and adjacent characteristic peak impurities is greater than 1.2 (table 1 and attached drawing 1), and the highest separation effect is remarkable as 45.96;

according to the experimental separation results, the determination method provided by the invention can simultaneously separate 10 known impurities in cefepime hydrochloride for injection, and chromatographic peaks of the cefepime hydrochloride and the 10 impurities show remarkable separation effects, so that the requirement of completely separating the 10 impurities in the cefepime hydrochloride is met, and the specificity is good.

Example 2 selection of chromatographic column

2-1, solution preparation:

a diluent: 900mL of phosphate buffer (pH 5.0) was taken, 100mL of acetonitrile was added thereto, and the mixture was homogenized for use.

Mixing an impurity reference substance solution: taking cefepime hydrochloride and 1-10 reference substances of impurities, and preparing a solution containing 1.4mg of cefepime hydrochloride and 14 mug of each impurity 1-10 in each 1mL of diluent by using the diluent to serve as a mixed impurity reference substance solution.

2-2. Test conditions:

on the basis of the example 1, the type of the chromatographic column is changed, other chromatographic conditions are kept unchanged, 10 impurities in cefepime hydrochloride for injection are separated, and the applicability of the method is examined.

Wherein the column type of example 1: waters

T3 C18，4.6mm×250mm，5.0μm；

Example 2 the column types were varied as: waters SunFire C18,4.6mm×250mm,5.0 μm; other parameters and steps were the same as in example 1.

2-3, experimental steps and conclusions:

precisely measuring 10 μl of each mixed impurity reference solution, respectively injecting into high performance liquid chromatograph, recording chromatogram and result, and the results are shown in the following table and figures 1-2.

Conclusion:

column Waters using the inventive example 1

When T3C 18 (4.6mm×250mm,5.0 μm) is used for separating the mixed impurity reference substance solution (shown in figure 1), the separation degree between a main peak (cefepime hydrochloride) and an adjacent peak is more than 2.0, the retention time of the impurity 1 is 4.1min, the impurity can be effectively retained, the separation degree of 10 impurity characteristic peaks is more than 1.5, the separation degree is good, and the known impurities 1-10 can be simultaneously separated;

Under the same conditions, when the mixed impurity control solution was separated using the column Waters SunFire C18 (4.6 mm×250mm,5.0 μm) of example 2 (see fig. 2), only the chromatographic peaks of impurity 1, impurity 2, impurity 3, impurity 7 and impurity 9 were visible, but the retention time of impurity 1 was 2.3min, the peak time was early, and the retention could not be effectively performed. Further, the degree of separation of the impurities is less than 1.0, and the known impurities 1 to 10 cannot be separated at the same time.

As can be seen from the comparison of the above-mentioned separation degrees, the type of the chromatographic column is critical to the separation effect of the method of the present invention, and only octadecylsilane chemically bonded silica of the present invention example 1 is used as a filler, for example, waters

T3C 18 (4.6mm.times.250 mm,5.0 μm) column, to separate 10 known impurities simultaneously, the separation effect is best.

In contrast, 10 known impurities in cefepime hydrochloride for injection cannot be separated simultaneously by using other chromatographic columns, for example, the Waters SunFire C18 (4.6 mm. Times.250 mm,5.0 μm) chromatographic column of example 2.

EXAMPLE 3 selection of mobile phase

3-1, preparing a solution:

a diluent: 900mL of phosphate buffer (pH 5.0) was taken, 100mL of acetonitrile was added thereto, and the mixture was homogenized for use.

Mixing an impurity reference substance solution: taking cefepime hydrochloride for injection and each impurity reference substance, and preparing a solution containing 1.4mg of cefepime hydrochloride and 14 mug of each impurity in each 1mL of diluent by using a diluent to serve as a mixed impurity reference substance solution.

3-2. Test conditions:

on the basis of example 1, the type of mobile phase was changed, and other chromatographic conditions were kept unchanged, and 10 known impurities in cefepime hydrochloride for injection were separated.

Wherein the mobile phase of example 1 is: mobile phase a: phosphate buffer (0.68 g of potassium dihydrogen phosphate, dissolved in water and diluted to 1000mL, pH5.0 adjusted with sodium hydroxide solution); mobile phase B: phosphate buffer (pH 5.0) -acetonitrile (50:50);

3-3, experimental steps and conclusions:

precisely measuring 10 mu L of each mixed impurity reference substance solution, respectively injecting into a high performance liquid chromatograph, recording the chromatograms and the results, and obtaining the results shown in the following table, figure 1 and figures 3-4.

Conclusion:

when the flow in the embodiment 1 of the invention is adopted to separate the relatively mixed impurity reference substance solution (shown in the attached figure 1), the retention time of the impurity 1 is 4.1min, the impurity 1 can be effectively retained, the separation degree between unknown impurities is more than 1.0, the separation degree is good, and 10 known impurities can be separated simultaneously;

when the mixed impurity control solution of the mobile phase in the method 1 of example 3 was used for separation (see fig. 3), only the chromatographic peaks of impurity 1, impurity 2, impurity 3, impurity 7 and impurity 9 were visible, and impurity 7 and the main peak (characteristic peak of cefepime hydrochloride) could not be separated;

When the mixed impurity control solution of the mobile phase in the method 2 of example 3 was used for separation (see fig. 4), only the chromatographic peaks of impurity 1, impurity 2, impurity 3, impurity 7 and impurity 9 were visible, and the retention time of impurity 1 was 1.717min, and the characteristic peak of impurity 1 could not be effectively retained due to too short retention time, so that 10 known impurities could not be separated at the same time;

the mobile phase type (mobile phase A phosphate buffer (taking 0.68g of monopotassium phosphate, adding water for dissolving and diluting to 1000mL, adjusting the pH to 5.0 by using sodium hydroxide solution) and mobile phase B phosphate buffer (pH to 5.0) -acetonitrile (50:50)) of the method can simultaneously separate 10 known impurities in cefepime hydrochloride for injection; the mobile phase species used in both method 1 and method 2 of example 3 cannot simultaneously separate 10 known impurities.

Thus, phosphate buffer is used as mobile phase a; phosphate buffer: acetonitrile (50:50) is a mobile phase B, and can simultaneously separate 10 impurities in cefepime hydrochloride for injection; wherein, when the mobile phase A phosphate buffer (taking 0.68g of monopotassium phosphate, adding water for dissolution and diluting to 1000mL, adjusting the pH to 5.0 by using sodium hydroxide solution); when the mobile phase B is phosphate buffer (pH 5.0) -acetonitrile (50:50), the separation effect is optimal.

Example 4 selection of mobile phase pH

4-1, solution preparation:

a diluent: 900mL of phosphate buffer (pH 5.0) was taken, 100mL of acetonitrile was added thereto, and the mixture was homogenized for use.

Mixing an impurity reference substance solution: taking cefepime hydrochloride for injection and an impurity 1-10 reference substances respectively, and preparing a solution containing 1.4mg of cefepime hydrochloride and 14 mug of each impurity in each 1mL of diluent by using a diluent to serve as a mixed impurity reference substance solution.

4-2. Test conditions:

based on example 1, the pH of the mobile phase was changed and other chromatographic conditions were maintained, and the impurities in the mixed impurity control solution were separated.

Wherein the pH of mobile phase a of example 1 was 5.0;

the pH of mobile phase a of example 1 was varied to be:

the phosphate buffer of method 1 had a pH of 5.5;

the phosphate buffer of method 2 had a pH of 4.5;

4-3, experimental steps and conclusions:

precisely measuring 10 mu L of the mixed impurity reference substance solution, respectively injecting into a high performance liquid chromatograph, recording a chromatogram and a result, and the results are shown in the following table and figures 1, 5-6.

Conclusion:

when the pH=5.0 (shown in the attached figure 1) of the phosphate buffer solution of the mobile phase in the embodiment 1 is adopted, the separation degree among characteristic peaks of all impurities is more than 1.0, the separation effect is good, and 10 known impurities can be separated simultaneously;

When the mobile phase phosphate buffer ph=5.5 (see fig. 5) in method 1 of example 4 of the present invention is used, impurity 1 splits into two peaks, the impurity is destroyed, and 10 known impurities cannot be separated at the same time;

when the mobile phase phosphate buffer ph=4.5 (see fig. 6) in method 2 of example 4 of the present invention was used, impurity 4 overlapped with the next adjacent peak (retention time: 20.534), and the degree of separation=0.85; impurity 9 overlaps with the next adjacent peak (retention time: 50.290), and the degree of separation=0.93, which is a common standard for the degree of separation of less than 1.5, and 10 known impurities cannot be separated simultaneously.

When the pH value of the mobile phase A phosphate buffer solution in the method embodiment 1 of the invention is 5.0, 10 known impurities in cefepime for injection can be separated simultaneously, and the separation effect is obvious. However, when the pH of the mobile phase a phosphate buffer is 5.5 or 4.5, 10 known impurities cannot be separated simultaneously.

Therefore, the pH value of the phosphate buffer solution in the mobile phase A in chromatographic conditions is critical to the separation effect of the method, and only when the pH value of the phosphate buffer solution in the mobile phase A is 4.8-5.2, 10 known impurities can be separated at the same time, so that the separation effect is the best; wherein the separation effect is optimal when the pH is 5.0.

In contrast, when controlling the ph=5.5 or 4.5 of the phosphate buffer in mobile phase a in method 1 and method 2 of example 4, 10 known impurities of the present invention cannot be separated simultaneously.

Example 5 selection of elution gradient

5-1, preparing a solution:

a diluent: 900mL of phosphate buffer (pH 5.0) was taken, 100mL of acetonitrile was added thereto, and the mixture was homogenized for use.

Mixing an impurity reference substance solution: taking cefepime hydrochloride for injection and each impurity reference substance, and preparing a solution containing 1.4mg of cefepime hydrochloride and 14 mug of each impurity in each 1mL of diluent by using a diluent to serve as a mixed impurity reference substance solution.

5-2. Test conditions:

on the basis of example 1, the elution gradient in the chromatographic conditions was changed, and the other chromatographic conditions were kept unchanged, and the impurities in the mixed impurity control solution were separated.

Wherein the elution gradient of example 1 is:

retention time (min)	Mobile phase a (%)	Mobile phase B (%)
			0	100	0
6	100	0
			65	40	60
75	40	60
			76	100	0
86	100	0

The elution gradient of example 1 was changed:

the elution gradient of method 1 is:

time (min)	Mobile phase a (%)	Mobile phase B (%)
			0	100	0
10	100	0
			30	50	50
35	50	50
			36	100	0
45	100	0

The elution gradient of method 2 is:

time (min)	Mobile phase a (%)	Mobile phase B (%)
			0	100	0
8	100	0
			45	70	30
50	70	30
			51	100	0
60	100	0

5-3, experimental steps and conclusions:

precisely measuring 10 mu L of the mixed impurity reference substance solution, respectively injecting into a high performance liquid chromatograph, recording a chromatogram and a result, and the results are shown in the following table and figures 1 and 7-8.

Conclusion:

when the elution gradient (shown in figure 1) of the embodiment 1 of the invention is adopted, the separation degree between the impurity peaks in the mixed impurity reference substance solution is more than 1.5, the separation effect is good, and 10 known impurities can be separated simultaneously;

with the elution gradient of method 1 in example 5 (see fig. 7), the separation result shows that impurity 4 and impurity 5 coincide, and the main peak (characteristic peak of cefepime hydrochloride) and the next adjacent impurity (retention time: 22.046) have a degree of separation of 1.03, which is far less than the general standard of the degree of separation (> 2.0), and 10 known impurities cannot be separated simultaneously;

when the elution gradient of method 2 in example 5 (see fig. 8) was used, the separation result showed that impurity 4 and impurity 5 were coincident; in addition, the separation degree of the impurity 1 and the impurity 2 is 1.08, and the common standard of the separation degree (> 1.5) is not satisfied, the separation effect is poor, and 10 known impurities cannot be separated at the same time;

therefore, the elution gradient in the chromatographic condition is critical to the separation effect of the method, and 1-10 of known impurities in cefepime for injection can be separated at the same time only under the gradient elution condition of the invention, and the separation effect is best; whereas 10 known impurities according to the invention cannot be separated simultaneously using the gradient elution parameters of method 1 and method 2 of example 5.

EXAMPLE 6 selection of Mobile phase B

On the basis of example 1, the volume ratio of the phosphate buffer to acetonitrile in mobile phase B was varied, and other chromatographic conditions were maintained unchanged, to separate 10 impurities in the mixed impurity control solution.

Wherein the volume ratio of phosphate buffer to acetonitrile in mobile phase B of example 1 is 50:50;

method 1 changes the volume ratio of phosphate buffer and acetonitrile in mobile phase B to 45:55;

method 2 changes the volume ratio of phosphate buffer and acetonitrile in mobile phase B to be 55:45;

method 3 changes the volume ratio of phosphate buffer and acetonitrile in mobile phase B to 40:60;

method 4 changes the volume ratio of phosphate buffer and acetonitrile in mobile phase B to be 65:35;

other test conditions were the same as in example 1, and 10. Mu.L of the mixed impurity control solution prepared in example 1 was precisely measured, injected into a high performance liquid chromatograph, and the test results were recorded.

Conclusion:

by carrying out high performance liquid chromatography analysis on impurities in cefepime hydrochloride for injection through the three mobile phases B with different volume ratios, the method has the following steps:

in example 1, mobile phase B, the volume ratio of phosphate buffer and acetonitrile was controlled = 50:50, and in mobile phase B of example 6, methods 1 and 2, the volume ratio of phosphate buffer to acetonitrile was controlled = 45:55 or 55:45, the obtained spectrograms have peak responses of the impurities 1-10, and the separation degree among the characteristic peaks is good, so that the known impurities 1-10 can be separated simultaneously.

As can be seen from fig. 9, in example 6, method 3, the volume ratio of the phosphate buffer solution of mobile phase B and acetonitrile=40: at 60, the separation degree of the impurity 3, the impurity 4, the main peak (cefepime hydrochloride characteristic peak), the impurity 6, the impurity 9 and the adjacent impurity peaks (retention time is 17.190, 20.534, 27.634, 29.061 and 50.290 respectively) after that is maximally 1.4, and all the separation degrees are smaller than 1.5, so that 10 known impurities cannot be separated simultaneously.

As can be seen from fig. 10, in example 6, method 4, the volume ratio of phosphate buffer and acetonitrile of mobile phase b=60: at 40, the impurity 4 splits into two peaks, and the impurity peak types are both deteriorated, and 10 known impurities cannot be separated at the same time.

As demonstrated by the above experimental results of mobile phase B in different proportions, the volume ratio of phosphate buffer and acetonitrile used in example 1 = 50:50, 10 known impurities in cefepime hydrochloride for injection can be separated simultaneously, and the main peak (characteristic peak of cefepime hydrochloride) and the characteristic peaks of impurities 1-10 can be separated simultaneously, so that the separation effect is obvious.

Therefore, the volume ratio of the phosphate buffer solution to the acetonitrile in the mobile phase B is critical to the separation effect of the method, and only the volume ratio of the phosphate buffer solution to the acetonitrile is controlled to be (45-55): (55-45), the known impurities 1-10 can be separated at the same time; when the volume ratio of phosphate buffer and acetonitrile = 40:60 or 60: at 40, 10 known impurities cannot be separated simultaneously.

EXAMPLE 7 destructive testing

The forced degradation test is to accelerate the damage to the sample under the severe conditions, such as strong light irradiation, high temperature, high humidity, acid-base damage, hydrolysis, oxidation damage and the like, so as to evaluate the effectiveness and applicability of the analysis method by examining the separation condition of the degradation product and main peak of the sample and known impurities. Meanwhile, the detection of the light-emitting diode array is adopted to carry out the detection of the peak purity: in the map obtained by the degradation experiment, when the purity angle of the impurities and the main peak is smaller than the purity threshold value, the determination method is judged to meet the determination requirement.

7-1, solution preparation:

a diluent: 900mL of phosphate buffer (pH 5.0) was taken, 100mL of acetonitrile was added thereto, and the mixture was homogenized for use.

Undegraded sample: weighing 24.5mg of cefepime hydrochloride for injection (14 mg of cefepime hydrochloride), precisely weighing, placing into a 10mL volumetric flask, adding a proper amount of diluent to dissolve and dilute to a scale, shaking uniformly, and filtering to obtain an undegraded sample.

Oxidative degradation of samples: weighing 24.5mg of cefepime hydrochloride for injection (14 mg of cefepime hydrochloride), precisely weighing, placing in a 10mL volumetric flask, adding 1mL of 3% hydrogen peroxide, standing at room temperature for 10min, adding a proper amount of diluent to dissolve and dilute to a scale, shaking uniformly, filtering, and taking a subsequent filtrate to obtain an oxidative degradation sample.

Acid degradation samples: weighing 24.5mg of cefepime hydrochloride for injection (14 mg of cefepime hydrochloride), precisely weighing, placing in a 10mL volumetric flask, adding 1.0mL of hydrochloric acid solution (0.5 mol/L), standing at room temperature for 10min, neutralizing with 1mL of sodium hydroxide solution (0.5 mol/L), adding a proper amount of diluent to dissolve and dilute to scale, shaking uniformly, filtering, and taking a subsequent filtrate to obtain an acid degradation sample.

Alkali degradation sample: weighing 24.5mg of cefepime hydrochloride for injection (14 mg of cefepime hydrochloride), precisely weighing, placing in a 10mL volumetric flask, adding 1mL of sodium hydroxide solution (0.1 mol/L), standing at room temperature for 10min, adding 1mL of hydrochloric acid solution (0.1 mol/L) for neutralization, adding a proper amount of diluent to dissolve and dilute to scale, shaking uniformly, filtering, and taking a subsequent filtrate to obtain an alkali degradation sample.

Hydrolysis of the sample: weighing 24.5mg of cefepime hydrochloride for injection (14 mg of cefepime hydrochloride), precisely weighing, placing in a 10mL volumetric flask, adding 1.0mL of water, carrying out water bath at 30 ℃ for 2 hours, adding a diluent for dilution to a scale, shaking uniformly, filtering, and taking a subsequent filtrate to obtain a hydrolysis sample.

Photodegradation sample: taking 24.5mg (containing 14 mg) of cefepime hydrochloride for injection with illumination for 3 days (with illumination of 4500 lx+/-500 lx), precisely weighing, placing into a 10mL volumetric flask, adding a proper amount of diluent to dissolve and dilute to scale, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the photodegradation sample.

High temperature degradation sample: weighing 24.5mg of cefepime hydrochloride for injection (14 mg of cefepime hydrochloride), precisely weighing, placing in a 10mL volumetric flask, placing in a water bath at 80 ℃ for 1 hour, adding a diluent to dilute to a scale, shaking uniformly, filtering, and taking a subsequent filtrate to obtain a high-temperature degradation sample. 7-2, experimental steps and conclusions:

precisely measuring 10 mu L of each sample solution under the undegraded and each degradation condition, respectively injecting into a high performance liquid chromatograph, and recording the chromatograms and the results. The chromatographic conditions were the same as in example 1, and the results are shown in tables 2 to 3.

TABLE 2 method for detecting impurities in cefepime hydrochloride for injection-System applicability-1

TABLE 3 method for detecting impurities in cefepime hydrochloride for injection-System applicability-2

Note that: "ND" is undetected.

Conclusion:

the undegraded sample and the sample under each degradation condition are detected by high performance liquid chromatography, and the result shows that:

the material conservation proportion of cefepime hydrochloride for injection is kept within the range of 98-102% under degradation conditions such as oxidation, acid, alkali, water, light, high temperature and the like, which indicates that the material is basically conserved;

the purity angle of the main peak of cefepime hydrochloride for injection under each degradation condition is obviously smaller than a purity threshold value, which indicates that no co-elution phenomenon exists, and the peak purity can meet the impurity separation requirement; the cefepime hydrochloride for injection has a degradation percentage of less than 5% under each degradation condition, and the single impurity content and the total impurity content are obviously increased, which indicates that the cefepime hydrochloride for injection is unstable under each degradation condition;

The results show that the analysis method has effectiveness and applicability under various degradation conditions.

According to the invention, 10 different impurities in cefepime for injection are separated, wherein all impurity peaks can be separated, and the separation effect is obvious. Meanwhile, in the following contents, the quantitative detection of all impurities is also carried out (9 impurities are taken as an example below), so that the content of the impurities in cefepime for injection can be estimated and calculated.

Example 8 Linear test

8-1, solution preparation:

a diluent: 900mL of phosphate buffer (pH 5.0) was added with 100mL of acetonitrile, and the mixture was homogenized for further use.

Impurity control stock: accurately weighing 3.5mg of each of the reference substances with the impurities of 1-9, respectively placing into 25mL measuring bottles, dissolving with a diluent, fixing the volume, and shaking uniformly to obtain the reference substance stock solution with the impurities of 1-9.

Cefepime hydrochloride stock solution: weighing cefepime hydrochloride reference substance 16.6mg (containing cefepime hydrochloride 14 mg), placing into a 10mL volumetric flask, adding a proper amount of diluent to dissolve and dilute to scale, and shaking uniformly to obtain the cefepime hydrochloride.

400% linear solutions (corresponding to limit concentrations) of impurities 1 to 4,6 to 9: precisely measuring 5.0mL of impurity 7 and impurity 9 reference substance stock solutions, respectively 2.0mL of impurity 1, 2, 3, 4,6 and impurity 8 reference substance stock solutions, respectively placing 1.0mL of cefepime hydrochloride stock solution into a 25mL volumetric flask, diluting to scale with a diluent, and shaking uniformly to obtain 400% linear solutions of impurities 1-4 and 6-9;

200%, 100% and 80% linear solutions (corresponding to the limiting concentrations) of impurities 1 to 4 and 6 to 9: precisely measuring 5.0mL, 2.5mL, 2.0mL of 400% linear solution of impurities 1-4 and 6-9 respectively, placing into a 10mL volumetric flask, adding a diluent to dilute to scale, and shaking uniformly to obtain 200%, 100% and 80% linear solution of impurities 1-4, 6-9;

50% and 20% linear solutions (corresponding to the limit concentrations) of impurities 1 to 4 and 6 to 9: precisely measuring 5.0mL and 2.0mL of 100% linear solutions of impurities 1-4 and 6-9 respectively, placing the linear solutions in a 10mL volumetric flask, adding a diluent to dilute the linear solutions to a scale, and shaking the linear solutions uniformly to obtain 50% and 20% linear solutions of impurities 1-4 and 6-9;

200% linear solution of impurity 5 (equivalent to limit concentration): precisely measuring 5.0mL of impurity 5 stock solution and 1.0mL of cefepime hydrochloride stock solution, placing into 50mL measuring bottles, adding a diluent to dilute to scale, and shaking uniformly to obtain 200% linear solution of impurity 5;

linear (150%, 100%, 80%) solutions (corresponding to limit concentrations) of impurity 5: precisely measuring 7.5mL, 5mL and 4mL of 200% linear solution of the impurity 5 respectively, placing the solution in a 10mL measuring flask, adding a diluent to dilute the solution until the scale is shaken uniformly, and obtaining 150%, 100% and 80% linear solution of the impurity 5;

linear (50%, 20%) solutions (corresponding to limit concentrations) of impurity 5: precisely measuring 5mL and 2mL of 100% linear solution of the impurity 5 respectively, placing the solution in a 10mL measuring flask, adding a diluent to dilute the solution until the scale is shaken uniformly, and obtaining 50% and 20% linear solution of the impurity 5.

8-2, experimental steps and conclusions:

and precisely measuring 10 mu L of each of the linear solutions of the impurities 1-9, respectively, injecting the solutions into a high performance liquid chromatograph, and recording a chromatogram. The chromatographic conditions were the same as in example 1 and the linear results are shown in Table 4.

TABLE 4 method for detecting impurities in cefepime hydrochloride for injection-linear results

Conclusion:

cefepime hydrochloride is in a concentration range of 0.1040 mu g/mL-59.4200 mu g/mL (which is equivalent to 0.7% -400% of the concentration of a control solution (14 mu g/mL)), a linear equation of peak area and concentration is y=22121.5997x+3066.1813, a linear correlation coefficient (r) =1.0000 is larger than 0.9999, and the peak area and the concentration of cefepime hydrochloride have good linear relation;

impurity 1 is in the concentration range of 0.0565-11.2960 mug/mL (corresponding to the limit concentration of impurity 1 of 2% -400%), the linear equation of peak area and concentration is y=26708.0734x+402.1999, the linear correlation coefficient (r) =1.0000, greater than 0.9999, the linear relation of peak area and concentration of impurity 1 is good;

impurity 2 is in the concentration range of 0.1399-11.1920 mug/mL (corresponding to 5% -400% of the limit concentration of impurity 2), the linear equation of peak area and concentration is y= 10831.6518x-656.0747, the linear correlation coefficient (r) =0.9999, and is larger than 0.999, and the linear relationship between peak area and concentration of impurity 2 is good;

The impurity 3 is in the concentration range of 0.5128-10.2560 mug/mL (which is equivalent to 20% -400% of the limit concentration of the impurity 3), the linear equation of the peak area and the concentration is y= 18716.1900x-2818.6074, the linear correlation coefficient (r) =1.0000, which is greater than 0.9999, and the linear relationship of the peak area and the concentration of the impurity 3 is good;

the impurity 4 is in the concentration range of 0.1381-11.0480 mug/mL (which is equivalent to 5-400% of the limit concentration of the impurity 4), the linear equation of the peak area and the concentration is y=16074.2026x+859.8713, the linear correlation coefficient (r) =0.9997 is larger than 0.999, and the linear relation between the peak area and the concentration of the impurity 4 is good;

impurity 5 is in the concentration range of 0.2566-14.5800 mug/mL (corresponding to 3.5% -200% of the limit concentration of impurity 5), the linear equation of peak area and concentration is y=19534.46313 x+1803.5883, the correlation coefficient (r) = 0.9992 is greater than 0.999, and the linear relationship between the peak area and concentration of impurity 5 is good;

the impurity 6 is in the concentration range of 0.1813-10.3600 mug/mL (corresponding to the limit concentration of 7-400% of the impurity 6), the linear equation of the peak area and the concentration is y= 14881.5332x-731.7108, the linear correlation coefficient (r) =1.0000, and the linear relation of the peak area and the concentration of the impurity 6 is more than 0.9999;

Impurity 7 is in the concentration range of 0.1085 mug/mL-21.7000 mug/mL (which is equivalent to the limit concentration of impurity 7 of 2% -400%), the linear equation of peak area and concentration is y=20151.1448x+431.1340, the linear correlation coefficient (r) =1.0000, which is greater than 0.9999, and the linear relation between peak area and concentration of impurity 7 is good;

the impurity 8 is in the concentration range of 0.2590-10.3600 mug/mL (which is equivalent to the limit concentration of 10% -400% of the impurity 8), the linear equation of the peak area and the concentration is y= 16510.0007x-92.3936, the linear correlation coefficient (r) =1.0000, which is greater than 0.9999, and the linear relationship of the peak area and the concentration of the impurity 8 is good;

impurity 9 is in the concentration range of 0.1608-26.8000 mug/mL (which is equivalent to the limit concentration of impurity 9 of 2% -400%), the linear equation of peak area and concentration is y=15212.85553x+374.0952, the linear correlation coefficient (r) =1.0000, which is greater than 0.9999, and the linear relationship between peak area and concentration of impurity 9 is good;

the results show that in the linear concentration range, the linear relation between the peak areas and the concentrations of cefepime hydrochloride and impurities 1-9 is good, the linear correlation coefficient (r) is more than 0.999, the impurities 1, 3 and 6-9 are optimal, the peak areas and the concentrations of the cefepime hydrochloride and the impurities are good; compared with the correlation coefficient r=0.99 required by the common standard, the measurement accuracy is improved by more than two orders of magnitude; the detection method has higher accuracy and wider measurement range.

Example 9 quantitative limit and detection limit test

9-1, solution preparation:

a diluent: 900mL of phosphate buffer (pH 5.0) was added with 100mL of acetonitrile, and the mixture was homogenized for further use.

Test solution: weighing 24.5mg of cefepime hydrochloride for injection (14 mg of cefepime hydrochloride), precisely weighing, placing into a 10mL volumetric flask, adding a proper amount of diluent to dissolve and dilute to a scale, shaking uniformly, and filtering to obtain a sample solution.

Quantitative limiting solution: taking the linear solution to quantitatively dilute until the signal to noise ratio of each impurity peak is 10, and at the moment, the concentration of the solution is a quantitative limit concentration;

detection limit solution: and taking the linear solution to quantitatively dilute until the signal to noise ratio of each impurity peak is 3, wherein the concentration of the solution is the detection limit concentration.

9-2, experimental steps and conclusions:

and precisely measuring 10 mu L of each of the quantitative limit solution and the detection limit solution, respectively injecting into a high performance liquid chromatograph, and recording a chromatogram and a result. The chromatographic conditions were the same as in example 1, and the quantitative limit and the detection limit of each impurity were as shown in Table 5.

TABLE 5 method for detecting impurities in cefepime hydrochloride for injection-quantitative limit and detection limit results

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Conclusion:

as found from the quantitative limit and the detection limit results,

the quantitative limit of each impurity is not more than 0.513 mug/mL, wherein the minimum quantitative limit of the impurity 1 is 0.057 mug/mL, which is equivalent to 0.004% of the concentration of the solution of the test sample;

The detection limit of each impurity is not more than 0.171 mug/mL, wherein the detection limit of the impurity 1 is the lowest 0.019 mug/mL, which is equivalent to 0.001% of the concentration of the solution of the test sample;

the method results show that each impurity can be effectively detected and the sensitivity is high.

Example 10 recovery test

10-1, solution preparation:

a diluent: 900mL of phosphate buffer (pH 5.0) was added with 100mL of acetonitrile, and the mixture was homogenized for further use.

Impurity control stock: respectively precisely weighing 14mg of each impurity 1-9, respectively placing into a 100mL measuring flask, dissolving with a diluent, fixing the volume, and shaking uniformly to obtain an impurity 1-9 reference stock solution.

Accuracy stock solution: precisely measuring 10mL of impurity 5 reference substance stock solution, 2mL of impurity 1-4 reference substance stock solutions respectively, and 4mL of impurity 6-9 reference substance stock solutions respectively, placing into 20mL volumetric flask, diluting to scale with diluent, and shaking to obtain the final product.

Accuracy control solution: precisely measuring 1mL of stock solution with accuracy, placing in a 10mL volumetric flask, diluting to scale with a diluent, and shaking uniformly to obtain the product.

Accuracy test solution: the cefepime hydrochloride for injection (containing 28mg of cefepime hydrochloride) is weighed in a 20mL measuring flask, 9 parts are weighed in parallel, the average is divided into 3 groups, 1.0mL, 2.0mL and 3.0mL of the accuracy control solution are respectively and precisely added, a proper amount of diluent is added to dissolve and dilute to scales, and the solutions are uniformly shaken to obtain 50 percent, 100 percent and 150 percent accuracy solutions respectively.

10-2, experimental steps and conclusions:

precisely measuring 10 mu L of each of the above accurate solutions, respectively injecting into high performance liquid chromatograph, and recording chromatogram. The chromatographic conditions were the same as in example 1, and the recovery rate of each impurity was as shown in Table 6. The relative standard deviation calculation formula is as follows:

TABLE 6 impurity detection methodology in cefepime hydrochloride for injection-recovery results

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Conclusion:

from the results of the recovery rate test of each impurity,

under the condition of 50%, 100% and 150% accuracy of sample solution, the average recovery rate in the impurity 1-9 groups and the average recovery rate between groups are kept between 90% -110%, the recovery rate distribution range is concentrated, and the quantitative determination requirement of each impurity can be accurately met.

Compared with the standard that the recovery rate RSD required by the common standard is less than or equal to 10.0%, the RSD of the recovery rate of 9 samples measured by the embodiment is reduced to be less than one half of the standard requirement, the accuracy of the detection result is obviously improved by more than two times, and further, the accuracy of the detection method for detecting the impurities 1-9 is proved to be good.

Example 11 repeatability test

The variation of the system impurity content was examined by repeatedly measuring the same batch of samples (100% limit addition of the standard solution), and was designated as repeatability 1 to 6 to verify and obtain the method repeatability, and the results are shown in Table 7.

TABLE 7 impurity detection methodology-precision test results in cefepime hydrochloride for injection

Conclusion:

as can be seen from the repeatability test results of the impurities in the table, the impurity contents in 6 identical 100% limit-added standard test sample solutions are quantitatively detected, wherein the impurity contents repeatedly detected for a plurality of times of impurity 1 are kept the same; among the impurities 2, 4, 5, 8 and 9, the RSD detected for 6 times is less than or equal to 1.0%; impurity 6 and impurity 7, RSD less than or equal to 3.0% of 6 times of detection; the RSD of the 6 measurement results of each impurity is less than or equal to 4.4 percent.

The unknown impurities and the total impurity content are not obviously changed, the RSD is not more than 2.3 percent, and the RDS is not more than 5.0 percent according with the requirement of the measurement standard; the test results show that the separation detection method has good repeatability and obvious precision effect.

Example 12 durability test

Based on example 1, the detection wavelength (+ -2 nm), the flow rate (+ -0.1 mL/min), the column temperature (+ -5 ℃), the pH (+ -0.2) of the mobile phase A, the sample weighing (+ -0.1 g) of the mobile phase A and the chromatographic columns of different batches were respectively changed, 10 mu L of each of the sample solution and the control solution was respectively taken, and injected into a high performance liquid chromatograph, and other chromatographic conditions were the same as in example 1, and the chromatograms and the results were recorded, and the results are shown in Table 8.

TABLE 8 impurity detection methodology in cefepime hydrochloride for injection-durability test results

Note that: NA indicates inapplicability.

Conclusion:

the results of the durability test showed that,

on the basis of example 1, the measurement results were unchanged when the chromatographic conditions such as wavelength change (+ -2 nm), column temperature (+ -5 ℃), flow rate change (+ -0.2 mL/min), pH value of mobile phase A (+ -0.2), sample weighing amount of mobile phase A (+ -0.1 g) and different batches of chromatographic columns were changed: the number of the detected impurities is consistent; other chromatographic conditions, other than changing the flow rate of the mobile phase, were unchanged after changing the other chromatographic conditions for unknown impurities, other maximum mono-impurities and total impurities.

Experiments show that the method has good durability for detecting the impurities in the cefepime hydrochloride for injection under the conditions of controlling the detection wavelength to 252-256 nm, the column temperature to 20-30 ℃ and the flow rate to 0.9-1.1 mL/min, the pH value of the mobile phase A to 4.8-5.2, the sample weighing amount of the mobile phase A to 0.58-0.78 g and different batches of chromatographic columns under different chromatographic conditions.

Comparative example 1

With reference to the detection conditions and methods of comparative document 1, 10 known impurities (impurities 1 to 10) in the sample solution and the mixed impurity control solution according to the embodiment of the present invention were separated and detected.

Comparative document 1: wang Tingting Shohui, li Pei, luo Jialin, guo Yinghao, wu Jianzhuo, hong Jianwen quality evaluation of domestic cefepime hydrochloride for injection, J.China antibiotics, 2022,47 (02): 128-133.

Table 9 comparison of impurities of the present application with comparative document 1

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Table 10 comparison of chromatographic conditions of the present application and comparative files

Conclusion:

the detection conditions of the comparison document 1 are adopted to separate and detect the impurities in the sample solution, and the experimental result shows that the conditions of the comparison document 1 (comparison document 1) cannot realize the simultaneous separation and detection of 10 known impurities of the invention, and cannot meet the requirements of the separation degree of various impurity peaks.

Therefore, the test condition of comparative example 1 cannot meet the requirement that the detection effect of the method of the invention is significantly better than that of comparative document 1 for simultaneous separation and detection of 10 known impurities in cefepime hydrochloride for injection.

Comparative example 2

To further verify the advantages of the present process, based on the inability to separate impurity 7 and the main peak (characteristic peak of cefepime hydrochloride) in method 1 of example 3, we further changed the following conditions based on the experimental conditions of method 1 of example 3:

the chromatographic column comprises the following components: waters SunFire C18,4.6mm×250mm,5.0 μm;

other experimental methods and procedures were performed in the same manner as in example 3, method 1.

Conclusion:

experimental results show that when the detection conditions of comparative example 2 are adopted to separate impurities in the mixed impurity control solution, the result shown by a spectrogram is the same as that of the method 1 in the embodiment 3, and only chromatographic peaks of the impurities 1, 2, 3, 7 and 9 are still visible, so that good separation of the impurities 7 and main peaks (characteristic peaks of cefepime hydrochloride) still cannot be realized.

Comparative example 3

To further verify the advantages of the present method, based on the characteristic peaks of impurity 4 and impurity 5 in method 1 of example 5, which coincide, and the main peak (characteristic peak of cefepime hydrochloride) is adjacent to the next impurity peak (retention time is 22.046), we changed the following conditions on the basis of the experimental conditions of method 1 of example 5:

comparative example 3-method 1:

the chromatographic column comprises the following components: waters SunFire C18,4.6mm×250mm,5.0 μm;

the mobile phase is:

comparative example 3-method 2:

the chromatographic column comprises the following components: waters SunFire C18,4.6mm×250mm,5.0 μm;

the mobile phase is:

mobile phase a	Dissolving 0.57g of monoammonium phosphate in 1000mL of water
		Mobile phase B	Acetonitrile

Other experimental methods and procedures were performed in the same manner as in example 5, method 1.

Conclusion:

experimental results show that when the detection conditions of the comparative example 3-method 1 are adopted to separate impurities in the mixed impurity control solution, the separation results show that the impurities 4 and 5 are overlapped, the separation degree of a main peak (a characteristic peak of cefepime hydrochloride) and the next adjacent impurities (retention time: 22.046) is 0.9, the separation degree is still far less than the general standard of the separation degree (> 2.0), and 10 known impurities cannot be separated simultaneously;

When the impurities in the mixed impurity control solution of the present invention are separated under the detection conditions of comparative example 3-method 2, the separation result shows that the impurity 4 and the impurity 5 coincide, and the main peak (characteristic peak of cefepime hydrochloride) and the next adjacent impurity also show a coincidence state, and 10 known impurities cannot be separated at the same time.

From the above results, it is clear that the degree of separation of the characteristic peaks of 10 known impurities obtained under the separation detection conditions is greater than 1.5, and the degree of separation is good and remarkable;

the quantitative limit of detecting 10 known impurities by adopting the method can be as low as 0.057 mug/mL, which is equivalent to 0.004% of the concentration of the solution of the test sample; the detection limit of 10 known impurities can be as low as 0.019 mug/mL, which is equivalent to 0.001% of the concentration of the solution of the test sample; the detection limit and the quantitative limit have remarkable reducing effect, and the sensitivity is remarkably improved;

the recovery rate of 10 known impurities is kept in the range of 90% -110%, the recovery rate RSD of repeated detection is smaller than 5%, compared with the standard that the recovery rate RSD required by common standard is less than or equal to 10.0%, the recovery rate RSD is reduced to less than one half of the standard requirement, the accuracy is improved by more than 2 times, and the accuracy performance of the separation method is remarkably improved;

In the limited concentration range, the areas of the characteristic absorption peaks of cefepime hydrochloride and impurities 1-10 and the corresponding concentrations thereof show good linear relation, and the correlation coefficient r of a linear equation is more than 0.999 and optimally reaches 1.0000; compared with the linear correlation coefficient r which is more than 0.99 and is required by the common standard, the accuracy of measurement is improved by 2 orders of magnitude; the accuracy of the detection method is obviously increased, and the measurement range is wider;

the adopted separation detection method has consistent detection results of various impurities and good precision;

the RSD of the detection result of the impurities in cefepime hydrochloride for injection is less than 5% by changing different chromatographic conditions and chromatographic column batches, and the cefepime hydrochloride for injection shows good and remarkable durability.

The method for detecting 10 known impurities in cefepime hydrochloride for injection is an optimal separation method, and can accurately perform quantitative quality control on the detected impurities in cefepime hydrochloride for injection, so that the safety, effectiveness and controllable quality of the medicine are finally ensured. The adopted high performance liquid chromatography method has strong specificity and high sensitivity, and can accurately and quantitatively determine various impurities of cefepime hydrochloride for injection.

Finally, it should be noted that: the foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related technical fields are included in the scope of the present invention.

Claims (14)

1. The method for separating and detecting impurities in cefepime hydrochloride for injection comprises the following steps: weighing cefepime hydrochloride for injection by adopting a diluent to prepare a sample solution, and detecting by adopting a high performance liquid chromatography method;

the chromatographic conditions include:

a chromatographic column adopts octadecylsilane chemically bonded silica gel as a filler;

mobile phase:

mobile phase a: phosphate buffer;

mobile phase B: phosphate buffer solution and acetonitrile, the volume ratio of the phosphate buffer solution to the acetonitrile is (45-55): (55-45); preferably, the volume ratio of phosphate buffer to acetonitrile is 50:50;

elution mode: gradient elution.

2. The method according to claim 1, wherein the chromatographic column is of the Waters type

T3 C18。

3. The method according to any one of claims 1 or 2, wherein the chromatographic column has a specification of 4.6mm x 250mm,5.0 μm.

4. A process according to any one of claims 1 to 3, wherein the pH of mobile phase a ranges from 4.8 to 5.2, preferably 5.0.

5. The method of any one of claims 1-4, wherein the elution gradient of the mobile phase is:

retention time (min) Mobile phase a (%) Mobile phase B (%) 0 100 0 6 100 0 65 40 60 75 40 60 76 100 0 86 100 0

。

6. The method according to any one of claims 1 to 5, wherein the detection wavelength of the chromatographic conditions is 252 to 256nm, preferably 254nm.

7. The method of any one of claims 1-6, wherein the phosphate buffer is a monobasic potassium phosphate solution.

8. The method according to any one of claims 1 to 7, wherein the column temperature of the chromatographic column is 20 ℃ to 30 ℃, preferably 25 ℃.

9. The method according to any one of claims 1 to 8, wherein the column flow rate of the chromatographic column is 0.9-1.1 mL/min, preferably 1.0mL/min.

10. A process according to any one of claims 1 to 9, wherein the impurities in cefepime hydrochloride for injection comprise one or more of the following structural formulae:

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11. a process according to claim 10, wherein the impurities of cefepime hydrochloride for injection comprise at least impurity 4, impurity 5, impurity 8 and impurity 10; or at least impurity 4 and impurity 5; or at least impurity 3, impurity 4, impurity 6 and impurity 9; or at least impurity 1, impurity 4, impurity 7 or impurity 9.

12. The method according to any one of claims 1 to 11, wherein the solution formulation method is:

Test solution: taking cefepime hydrochloride for injection, adding a diluent for dissolution, and filtering to obtain a sample solution.

Mixing an impurity reference substance solution: taking cefepime hydrochloride and an impurity 1-10 reference substance respectively, and adding a diluent for dissolution to serve as a mixed impurity reference substance solution.

13. The method of any one of claims 1-12, wherein the diluent is a mixture of phosphate buffer and acetonitrile.

14. The method of any of the preceding claims 1-13, wherein the solution formulation further comprises:

impurity localization solution: and respectively taking a proper amount of the reference substances with the impurities of 1-10, adding a diluent to the scale, and shaking uniformly to prepare a solution with the concentration of 14 mug in each 1mL to be used as a positioning solution with the impurities of 1-10.

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