A method for separating and detecting impurities in cefepime hydrochloride for injection

Abstract

The invention belongs to the technical field of drug detection, and specifically relates to a method for simultaneously separating and detecting multiple impurities in cefepime hydrochloride for injection. The method comprises preparing a test solution with cefepime hydrochloride for injection, and performing high performance liquid chromatography detection by gradient elution. The method can simultaneously and accurately and sensitively detect 10 known impurities in cefepime hydrochloride for injection. The method has strong specificity, low quantitative limit and detection limit, and good repeatability and separation.

Description

Field of the Invention

[0001] The invention belongs to the technical field of drug detection, and particularly relates to a method for separating and detecting multiple impurities in cefepime hydrochloride for injection. Background of the Invention

[0002] Cefepime Hydrochloride is an anti-infective drug and a fourth-generation cephalosporin. Because it is stable to β-lactamase, it has a wider antibacterial spectrum than the antibiotics currently used in clinical practice. Its chemical name is: (6R,7R)-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-carboxylic acid hydrochloride monohydrate, and its structural formula is:

[0003]

[0004] Cefepime hydrochloride is mainly used for the treatment of moderate to severe infections caused by the above-mentioned sensitive bacteria in adults and children aged 2 months to 16 years. It has a wide range of applications, good antibacterial activity, few side effects, and a highly balanced antibacterial spectrum mechanism of action, making it a distinctive variety among cephalosporin antibiotics. Cefepime hydrochloride is easily affected by environmental factors and is prone to react and produce a variety of impurities during production and storage. Therefore, it is necessary to establish a suitable impurity separation and detection method to control its impurity spectrum. The structural formulas of the 10 currently known major impurities in cefepime hydrochloride for injection are as follows:

[0005]

[0006]

[0007] By searching domestic and foreign literature and patents, we found:

[0008] Literature: "Rapid identification of isomeric impurities in cefepime hydrochloride by LC-MS / MS, Acta Pharmaceutica Sinica. 2005(04):361", wherein the liquid phase conditions are: the chromatographic column is a C18 column (4.6mm×250mm, 5.0μm, Dalian Yilite Company); the mobile phase is acetonitrile-10mmol / L ammonium acetate (5:95); the flow rate is 0.8mL / min (post-column split 50%). This condition cannot achieve simultaneous separation and detection of multiple impurities, and can only achieve quality control of isomeric impurities in cefepime hydrochloride raw materials. The impurity structure is:

[0009]

[0010] CN101226174A Chinese patent document discloses a method for detecting the content of N-methylpyrrolidine in cefepime hydrochloride raw materials and preparations thereof, using a carboxyl cation column as an analytical column and a conductivity detector for detection, but this method is only applicable to the detection of N-methylpyrrolidine. The impurity structural formula is:

[0011]

[0012] The above documents or patents introduce methods for detecting the contents of isomers and N-methylpyrrolidine in cefepime hydrochloride raw materials and preparations thereof, but do not involve methods for separating and detecting each known impurity 1 to 10 in cefepime hydrochloride. It is impossible to control the impurity spectra and accurately control the quality of cefepime hydrochloride for injection. Summary of the invention

[0013] In view of the defects and shortcomings in the prior art, the present invention provides a method for separating and detecting 10 known impurities (impurities 1 to 10) in cefepime hydrochloride for injection, which can efficiently monitor the safety and quality of cefepime hydrochloride for injection.

[0014] The present invention simultaneously determines 10 known impurities in cefepime hydrochloride for injection, including impurities 1 to 10, and has a significant separation effect; at the same time, the present invention also performs quantitative detection on all impurities, and can evaluate and calculate the content of impurities in cefepime for injection.

[0015] The method of the present invention has remarkable separation effect and strong specificity (separation degree is greater than 1.5); the sensitivity is significantly improved (the quantitative limit can be as low as 0.057 μg / mL, and the detection limit can be as low as 0.019 μg / mL); the accuracy of quantitative determination is significantly improved, and the quantitative determination range is wider (increased by 1 to 2 orders of magnitude); the recovery rate of the determination is close to 100%; the repeatability is good (the RSD of multiple measurement results is less than 5%) and the durability is good (the RSD of the determination results under different conditions is less than 15%).

[0016] Specifically, the present invention provides the following technical solutions:

[0017] A method for separating and detecting impurities in cefepime hydrochloride for injection, the method comprising the following steps: using a diluent to prepare a test solution of cefepime hydrochloride for injection, and then using a high performance liquid chromatography method for detection;

[0018] Chromatographic conditions included:

[0019] The chromatographic column uses octadecylsilane bonded silica gel as a filler;

[0020] Mobile Phase:

[0021] Mobile phase A: phosphate buffer;

[0022] Mobile phase B: a mixture of phosphate buffer and acetonitrile, the volume ratio of the two is (45-55): (55-45); preferably, the volume ratio of phosphate buffer and acetonitrile is 50:50;

[0023] Elution mode: gradient elution.

[0024] In some preferred embodiments, the type of chromatographic column is selected from Waters T3 C18.

[0025] In some preferred embodiments, the specifications of the chromatographic column are 4.6 mm×250 mm, 5.0 μm.

[0026] In some embodiments, the pH range of the mobile phase A is 4.8-5.2; in some preferred embodiments, the pH of the mobile phase A is 5.0.

[0027] In some preferred embodiments, the elution gradient of the mobile phase is:

[0028]

[0029]

[0030] In some preferred embodiments, the detection wavelength of the chromatographic conditions is 252-256 nm; in some preferred embodiments, the detection wavelength of the chromatographic conditions is 254 nm.

[0031] In some preferred embodiments, the phosphate buffer is a potassium dihydrogen phosphate solution.

[0032] In some preferred embodiments, the column temperature of the chromatographic column is 20°C to 30°C; in some preferred embodiments, the column temperature of the chromatographic column is 25°C.

[0033] In some preferred embodiments, the column flow rate of the chromatographic column is 0.9-1.1 mL / min; in some preferred embodiments, the column flow rate of the chromatographic column is 1.0 mL / min.

[0034] In some preferred embodiments, the impurities in the cefepime hydrochloride for injection include one or more of the following, with the structural formula as follows:

[0035]

[0036]

[0037] In some preferred embodiments, the impurities in cefepime hydrochloride for injection include at least impurity 4, impurity 5, impurity 8 and impurity 10; or, include at least impurity 4 and impurity 5; or, include at least impurity 3, impurity 4, impurity 6 and impurity 9; or, include at least impurity 1, impurity 4, impurity 7 or impurity 9.

[0038] In some preferred embodiments, the solution preparation method is:

[0039] Test solution: Take cefepime hydrochloride for injection, add diluent to dissolve it, filter it and use it as the test solution.

[0040] Mixed impurity reference solution: Take cefepime hydrochloride and impurity 1 to 10 reference substances respectively, add diluent to dissolve, and prepare mixed impurity reference solution.

[0041] In some preferred embodiments, the diluent is a mixed solution of phosphate buffer and acetonitrile.

[0042] In some preferred embodiments, the solution preparation further comprises:

[0043] Impurity localization solution: Take appropriate amount of impurity 1-10 reference substances respectively, add diluent to make up to the scale, shake well to make a solution of 14 μg per 1 mL, which is used as impurity 1-10 localization solution.

[0044] The method for separating and detecting impurities in cefepime hydrochloride for injection provided by the present invention can better control the quality of cefepime hydrochloride for injection and better separate and determine the impurities present in cefepime hydrochloride for injection.

[0045] The beneficial effects of the present invention are as follows:

[0046] 1. The present invention proposes a method for separating and detecting 10 known impurities in cefepime hydrochloride for injection.

[0047] The detection method of the present invention has the following characteristics: 1. strong specificity, and significant separation effect of characteristic peaks of 10 known impurities (separation degree is greater than 1.5); 2. quantitative analysis of each known impurity in cefepime hydrochloride for injection, and significantly reduced quantitative limit and detection limit: the quantitative limit can be as low as 0.057 μg / mL, equivalent to 0.004% of the concentration of the test solution; the detection limit can be as low as 0.019 μg / mL, equivalent to 0.001% of the concentration of the test solution, and the sensitivity is significantly improved; 3. the accuracy of quantitative determination is significantly improved, and the determination range is wider; compared with the accuracy of determination required by general standards, the accuracy of quantitative determination of the present invention is improved by more than 2 orders of magnitude; 4. the recovery rate of determination is close to 100%; 5. good repeatability (RSD of multiple measurement results is less than 5%); 6. good durability (RSD of determination results under different conditions is much less than 15%).

[0048] 2. The method of the present invention has been verified by methodology, and the following indicators have met the testing requirements and are in line with the requirements of Chinese drug regulatory agencies for drug testing:

[0049] ① The specificity test analysis found that the separation between the main peak and the adjacent impurity peaks was greater than 1.5, and the separation between each known impurity and its adjacent peak was greater than 1.2. The separation effect was significant, showing good specificity;

[0050] ② The quantitative limit of the method of the present invention for detecting 10 known impurities can be as low as 0.057 μg / mL, equivalent to 0.004% of the concentration of the test solution; the detection limit can be as low as 0.019 μg / mL, equivalent to 0.001% of the concentration of the test solution; the quantitative limit and detection limit are significantly reduced, and the sensitivity is significantly improved;

[0051] ③ In the linear concentration range, the peak area of ​​each characteristic absorption peak showed a good linear relationship with the concentration, and the linear correlation coefficient r was greater than 0.999; impurities 1, 3, 6, 7, 8 and 9 were the best, reaching 1.0000, and their peak areas had a good linear relationship with the concentration; compared with the general standard requirement of correlation coefficient r>0.99, the accuracy of the determination was improved by more than 2 orders of magnitude; the detection method had higher accuracy and a wider determination range;

[0052] ④ The average recovery rate within the impurity group and the average recovery rate between groups were maintained between 90% and 110%, close to 100%; and the RSD of the recovery rate of multiple tests was less than 5%. Compared with the general standard requirement of recovery rate RSD ≤ 10.0%, the RSD of the detection method of the present invention was reduced to less than half of the standard requirement, and the accuracy was improved by more than two times, which was significantly improved;

[0053] ⑤ Repeatability determination of parallel samples revealed no significant changes in the amount of impurities detected, and the RSD of each known impurity was less than 5%, showing good repeatability;

[0054] ⑥ When the chromatographic conditions and chromatographic column batches were changed, the RSD of the detection results of the impurity content in cefepime hydrochloride was 9.2%, which was less than 15.0%, indicating that the method of the present invention can achieve good and accurate detection effects within different conditions and exhibits good and significant durability.

[0055] 3. The method of the present invention can accurately control the quality of cefepime hydrochloride for injection, which is beneficial to the quality control of cefepime hydrochloride for injection and to carry out effective and safe promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1is a specific chromatogram of cefepime hydrochloride for injection in Example 1 of the present invention;

[0057] Figure 2 It is a chromatogram of the mixed impurity reference solution (including impurities 1, 2, 3, 7, and 9) obtained by the chromatographic column in Example 2 of the present invention;

[0058] Figure 3 It is a chromatogram of a mixed impurity reference solution (including impurities 1, 2, 3, 7, and 9) obtained by the mobile phase type in method 1 of embodiment 3 of the present invention;

[0059] Figure 4 It is a chromatogram of a mixed impurity reference solution (including impurities 1, 2, 3, 7, and 9) obtained by the mobile phase type in method 2 of embodiment 3 of the present invention;

[0060] Figure 5 It is a chromatogram of a mixed impurity reference solution (including impurities 1 to 10) obtained when the mobile phase pH is 5.5 in method 1 of Example 4 of the present invention;

[0061] Figure 6 It is a chromatogram of a mixed impurity reference solution (including impurities 1 to 10) obtained when the mobile phase pH is 4.5 in method 2 of Example 4 of the present invention;

[0062] Figure 7 is a chromatogram of a mixed impurity reference solution (including impurities 1 to 10) obtained by the elution gradient in method 1 of Example 5 of the present invention;

[0063] Figure 8 is a chromatogram of a mixed impurity reference solution (including impurities 1 to 10) obtained by the elution gradient in method 2 of Example 5 of the present invention;

[0064] Fig. 9 It is a chromatogram of a mixed impurity solution (including impurities 1 to 10) obtained when the ratio of phosphate buffer salt to acetonitrile in mobile phase B is 40:60 in method 3 of Example 6 of the present invention;

[0065] Fig.10 It is a chromatogram of a mixed impurity solution (including impurities 1 to 10) obtained when the ratio of phosphate buffer salt to acetonitrile in mobile phase B is 60:40 in method 4 of Example 6 of the present invention. DETAILED DESCRIPTION

[0066] The invention provides a method for separating and detecting multiple impurities in cefepime for injection, and detects and verifies 10 known impurities and other unknown impurities in cefepime hydrochloride for injection.

[0067] Firstly, the present invention separates 10 different impurities in cefepime for injection, wherein the separation effect is remarkable; meanwhile, the present invention also performs quantitative detection on all impurities, thereby realizing the evaluation and calculation of the impurity content in cefepime for injection.

[0068] Example 1 Specificity Test

[0069] 1-1. Solution preparation:

[0070] Diluent: Take 900 mL of phosphate buffer (pH 5.0), add 100 mL of acetonitrile, mix well and set aside.

[0071] Blank diluent: Take appropriate amount of diluent.

[0072] Mixed impurity reference solution: Take appropriate amounts of cefepime hydrochloride and impurity 1-10 reference substances to prepare a solution containing 1.4 mg of cefepime hydrochloride and 14 μg of each impurity 1-10 in each 1 mL of diluent, as the mixed impurity reference solution.

[0073] Impurity localization solution: Take appropriate amount of impurity 1-10 reference substances respectively, add diluent to make up to the scale, shake well to make a solution of 14 μg per 1 mL, which is used as impurity 1-10 localization solution.

[0074] Test solution: Weigh 24.5 mg of cefepime hydrochloride for injection (containing 14 mg of cefepime hydrochloride), accurately weigh, place in a 10 mL volumetric flask, add an appropriate amount of diluent to dissolve and dilute to the scale, shake well, filter, and use as the test solution.

[0075] Control solution: Accurately measure 1 mL of the above test solution and place it in a 100 mL volumetric flask. Add diluent to dilute to the scale and shake well to make a solution containing 14 μg of cefepime per 1 mL as the control solution.

[0076] 1-2. Measurement conditions:

[0077] Instrument: High performance liquid chromatography;

[0078] Chromatographic column: Waters T3 C18, 4.6mm×250mm, 5μm;

[0079] Mobile phase A: phosphate buffer (take 0.68 g of potassium dihydrogen phosphate, dissolve in water and dilute to 1000 mL, adjust pH to 5.0 with sodium hydroxide solution); mobile phase B: phosphate buffer (pH 5.0)-acetonitrile (50:50);

[0080] The elution method is gradient elution, and the elution gradient is as follows:

[0081] 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

[0082] Column temperature: 25°C;

[0083] Detection wavelength: 254nm;

[0084] Flow rate: 1.0 mL / min;

[0085] Injection volume: 10 μL.

[0086] 1-3. Experimental steps and conclusions:

[0087] Accurately measure 10 μL of each of the above solutions and inject them into the HPLC instrument respectively, and record the chromatogram and results. The results are shown in the table below, and the system suitability chromatogram is attached. Figure 1 .

[0088] Table 1 System suitability of Cefepime Hydrochloride for Injection - Separation results of mixed impurity reference solution

[0089]

[0090] in conclusion:

[0091] The system suitability test was carried out by high performance liquid chromatography, and the results are as follows:

[0092] The blank diluent was tested and found to have a stable baseline without any characteristic peaks, indicating that the blank diluent did not interfere with the separation of cefepime hydrochloride and various impurities;

[0093] The retention times of the characteristic peaks of various impurities can be located from the separation results of the impurity 1-10 positioning solution, the test solution and the control solution (Table 1). The retention time of cefepime hydrochloride is 27.877 min, and the retention times of impurities 1-10 are 4.142 min, 5.545 min, 17.920 min, 22.037 min, 24.623 min, 29.909 min, 36.547 min, 40.299 min, 50.660 min and 70.788 min, respectively.

[0094] The mixed impurity reference solution was used for determination, and it was found that the blank diluent did not interfere with the separation of impurities in the test solution. The separation between adjacent chromatographic peaks of cefepime hydrochloride and 10 known impurities in the mixed impurity reference solution was greater than 1.5; the separation between known impurities and adjacent characteristic peak impurities was greater than 1.2 (Table 1 and Appendix Figure 1 ), the highest is 45.96, and the separation effect is significant;

[0095] It can be seen from the above experimental separation results that the determination method provided by the present invention can simultaneously separate 10 known impurities in cefepime hydrochloride for injection, and the chromatographic peaks of cefepime hydrochloride and the 10 impurities show a significant separation effect, meeting the requirement of complete separation of the 10 impurities in cefepime hydrochloride, and has good specificity.

[0096] Example 2 Selection of chromatographic column

[0097] 2-1. Solution preparation:

[0098] Diluent: Take 900 mL of phosphate buffer (pH 5.0), add 100 mL of acetonitrile, mix well and set aside.

[0099] Mixed impurity reference solution: Take cefepime hydrochloride and each impurity 1 to 10 reference substance, and use diluent to prepare a solution containing 1.4 mg of cefepime hydrochloride and 14 μg of each impurity 1 to 10 in each 1 mL of diluent, as the mixed impurity reference solution.

[0100] 2-2. Test conditions:

[0101] On the basis of Example 1, the type of chromatographic column was changed, and other chromatographic conditions remained unchanged, 10 impurities in cefepime hydrochloride for injection were separated to investigate the applicability of this method.

[0102] Among them, the chromatographic column type of Example 1: Waters T3 C18, 4.6mm×250mm, 5.0μm;

[0103] Example 2: The chromatographic column type is changed to: Waters SunFire C18, 4.6 mm×250 mm, 5.0 μm; other parameters and steps are the same as Example 1.

[0104] 2-3. Experimental steps and conclusions:

[0105] Accurately measure 10 μL of each of the above mixed impurity reference solutions and inject them into the HPLC instrument respectively. Record the chromatogram and results. The results are shown in the following table and attached. Figures 1-2 .

[0106]

[0107]

[0108] in conclusion:

[0109] Using the chromatographic column Waters of Example 1 of the present invention T3 C18 (4.6mm×250mm, 5.0μm) is used to separate the mixed impurity reference solution (see Appendix Figure 1), the resolution between the main peak (cefepime hydrochloride) and the adjacent peaks was greater than 2.0, the retention time of impurity 1 was 4.1min, which was effectively retained, and the resolution of the 10 impurity characteristic peaks was greater than 1.5, with good resolution, and each known impurity 1 to 10 could be separated simultaneously;

[0110] Under the same conditions, the mixed impurity reference solution was separated using the chromatographic column Waters SunFire C18 (4.6 mm × 250 mm, 5.0 μm) of Example 2 (see Appendix Figure 2 ), only the chromatographic peaks of impurities 1, 2, 3, 7 and 9 are visible, but the retention time of impurity 1 is 2.3 min, and the peak elution time is too early to be effectively retained. In addition, the separation degree of impurities is less than 1.0, and it is impossible to separate all known impurities 1 to 10 at the same time.

[0111] From the above separation comparison, it can be seen that the type of chromatographic column is crucial to the separation effect of the method of the present invention. Only when the octadecylsilane bonded silica gel of Example 1 of the present invention is used as a filler, for example, Waters The T3 C18 (4.6mm×250mm, 5.0μm) chromatographic column can separate 10 known impurities at the same time, with the best separation effect.

[0112] On the contrary, other chromatographic columns, such as the Waters SunFire C18 (4.6 mm×250 mm, 5.0 μm) chromatographic column in Example 2, cannot simultaneously separate the 10 known impurities in cefepime hydrochloride for injection.

[0113] Example 3 Selection of mobile phase

[0114] 3-1. Solution preparation:

[0115] Diluent: Take 900 mL of phosphate buffer (pH 5.0), add 100 mL of acetonitrile, mix well and set aside.

[0116] Mixed impurity reference solution: Take cefepime hydrochloride for injection and each impurity reference substance, and use diluent to prepare a solution containing 1.4 mg of cefepime hydrochloride and 14 μg of each impurity in each 1 mL of diluent, as the mixed impurity reference solution.

[0117] 3-2. Test conditions:

[0118] On the basis of Example 1, the type of mobile phase was changed and other chromatographic conditions remained unchanged, and 10 known impurities in cefepime hydrochloride for injection were separated.

[0119] The mobile phase of Example 1 is as follows: mobile phase A: phosphate buffer (0.68 g of potassium dihydrogen phosphate is dissolved in water and diluted to 1000 mL, and the pH is adjusted to 5.0 with sodium hydroxide solution); mobile phase B: phosphate buffer (pH 5.0)-acetonitrile (50:50);

[0120]

[0121] 3-3. Experimental steps and conclusions:

[0122] Accurately measure 10 μL of each of the above mixed impurity reference solutions and inject them into the HPLC instrument respectively. Record the chromatogram and results. The results are shown in the following table and attached. Figure 1 and attached Figures 3-4 .

[0123]

[0124]

[0125] in conclusion:

[0126] When the mobile phase in Example 1 of the present invention is used to separate the mixed impurity reference solution (see attached Figure 1 ), the retention time of impurity 1 is 4.1min, which can be effectively retained, and the separation between unknown impurities is greater than 1.0, with good separation, and 10 known impurities can be separated at the same time;

[0127] When the mixed impurity reference substance solution of the mobile phase in method 1 of Example 3 is used for separation (see attached Figure 3 ), only the chromatographic peaks of impurity 1, impurity 2, impurity 3, impurity 7 and impurity 9 are visible, and impurity 7 and the main peak (characteristic peak of cefepime hydrochloride) cannot be separated;

[0128] When the mixed impurity reference substance solution of the mobile phase in method 2 of Example 3 is used for separation (see attached Figure 4 ), only the chromatographic peaks of impurity 1, impurity 2, impurity 3, impurity 7 and impurity 9 are visible, and the retention time of impurity 1 is 1.717min. Since the retention time is too short, the characteristic peak of impurity 1 cannot be effectively retained, so it is impossible to separate 10 known impurities at the same time;

[0129] By using the mobile phase type of the method of the present invention (mobile phase A phosphate buffer (take 0.68 g of potassium dihydrogen phosphate, add water to dissolve and dilute to 1000 mL, and adjust the pH to 5.0 with sodium hydroxide solution); mobile phase B is phosphate buffer (pH 5.0)-acetonitrile (50:50)), 10 known impurities in cefepime hydrochloride for injection can be separated simultaneously; the mobile phase types of method 1 and method 2 in embodiment 3 cannot separate 10 known impurities simultaneously.

[0130] Therefore, using phosphate buffer as mobile phase A and phosphate buffer:acetonitrile (50:50) as mobile phase B can simultaneously separate 10 impurities in cefepime hydrochloride for injection; among them, when mobile phase A is phosphate buffer (take 0.68 g of potassium dihydrogen phosphate, dissolve and dilute to 1000 mL, and adjust pH to 5.0 with sodium hydroxide solution); mobile phase B is phosphate buffer (pH 5.0)-acetonitrile (50:50), the separation effect is optimal.

[0131] Example 4 Selection of mobile phase pH

[0132] 4-1. Solution preparation:

[0133] Diluent: Take 900 mL of phosphate buffer (pH 5.0), add 100 mL of acetonitrile, mix well and set aside.

[0134] Mixed impurity reference solution: Take cefepime hydrochloride for injection and impurity 1 to 10 reference substances respectively, and use diluent to prepare a solution containing 1.4 mg of cefepime hydrochloride and 14 μg of each impurity in each 1 mL of diluent, as the mixed impurity reference solution.

[0135] 4-2. Test conditions:

[0136] On the basis of Example 1, the pH value of the mobile phase was changed, and other chromatographic conditions remained unchanged, and the impurities in the mixed impurity reference solution were separated.

[0137] Wherein, the pH of mobile phase A in Example 1 is 5.0;

[0138] The pH of the mobile phase A of Example 1 was changed to:

[0139] The pH of the phosphate buffer in method 1 was 5.5;

[0140] The pH of the phosphate buffer in method 2 was 4.5;

[0141] 4-3. Experimental steps and conclusions:

[0142] Accurately measure 10 μL of the above mixed impurity reference solution and inject it into the high performance liquid chromatograph, record the chromatogram and results, and the results are shown in the following table and attached: Figure 1 , 5 ~6.

[0143]

[0144] in conclusion:

[0145] The phosphate buffer solution pH of the mobile phase in Example 1 of the present invention is 5.0 (see attached Figure 1), the separation degree between the characteristic peaks of each impurity is greater than 1.0, the separation effect is good, and 10 known impurities can be separated at the same time;

[0146] When the phosphate buffer solution of the mobile phase in method 1 of Example 4 of the present invention is used, the pH value is 5.5 (see attached Figure 5 ), impurity 1 splits into two peaks, the impurity is destroyed, and it is impossible to separate 10 known impurities at the same time;

[0147] When the phosphate buffer solution of the mobile phase in method 2 of Example 4 of the present invention is used, the pH value is 4.5 (see attached Figure 6 ), impurity 4 overlaps with its subsequent adjacent peak (retention time: 20.534), and the separation degree is 0.85; impurity 9 overlaps with its subsequent adjacent peak (retention time: 50.290), and the separation degree is 0.93, both of which are less than the general standard of separation degree of 1.5, and it is impossible to separate 10 known impurities at the same time.

[0148] When the pH of the mobile phase A phosphate buffer in Example 1 of the method of the present invention is 5.0, 10 known impurities in cefepime for injection can be separated simultaneously, and the separation effect is significant. However, when the pH of the mobile phase A phosphate buffer is 5.5 or 4.5, the 10 known impurities cannot be separated simultaneously.

[0149] Therefore, the pH of the phosphate buffer in the mobile phase A in the chromatographic conditions is crucial to the separation effect of the method of the present invention. Only when the pH of the phosphate buffer in the mobile phase A of the present invention is between 4.8 and 5.2, can 10 known impurities be separated simultaneously, and the separation effect is the best; among them, when the pH is 5.0, the separation effect is optimal.

[0150] On the contrary, when the pH of the phosphate buffer in the mobile phase A in Method 1 and Method 2 of Example 4 is controlled to be 5.5 or 4.5, the 10 known impurities of the present invention cannot be separated simultaneously.

[0151] Example 5 Selection of elution gradient

[0152] 5-1. Solution preparation:

[0153] Diluent: Take 900 mL of phosphate buffer (pH 5.0), add 100 mL of acetonitrile, mix well and set aside.

[0154] Mixed impurity reference solution: Take cefepime hydrochloride for injection and each impurity reference substance, and use diluent to prepare a solution containing 1.4 mg of cefepime hydrochloride and 14 μg of each impurity in each 1 mL of diluent, as the mixed impurity reference solution.

[0155] 5-2. Test conditions:

[0156] On the basis of Example 1, the elution gradient in the chromatographic conditions was changed, and other chromatographic conditions remained unchanged, and the impurities in the mixed impurity reference solution were separated.

[0157] Wherein, the elution gradient of Example 1 is:

[0158] 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

[0159] Change the elution gradient of Example 1:

[0160] The elution gradient of method 1 is:

[0161] 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

[0162] The elution gradient of method 2 is:

[0163] 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

[0164] 5-3. Experimental steps and conclusions:

[0165] Accurately measure 10 μL of the above mixed impurity reference solution and inject it into the high performance liquid chromatograph, record the chromatogram and results, and the results are shown in the following table and attached: Figure 1 , 7 ~8.

[0166]

[0167] in conclusion:

[0168] Adopt the elution gradient of embodiment 1 of the present invention (see attached Figure 1 ), the separation between the impurity peaks in the mixed impurity reference solution was greater than 1.5, the separation effect was good, and 10 known impurities could be separated at the same time;

[0169] When the elution gradient of method 1 in Example 5 is used (see attached Figure 7 ), the separation results showed that impurity 4 and impurity 5 overlapped, and the separation degree of the main peak (characteristic peak of cefepime hydrochloride) and its adjacent impurity (retention time: 22.046) was 1.03, which was much smaller than the general standard of separation degree (>2.0), and it was impossible to separate 10 known impurities at the same time;

[0170] Adopt the elution gradient of method 2 in embodiment 5 (see attached Figure 8 ), the separation results showed that impurity 4 and impurity 5 overlapped; and the separation degree between impurity 1 and impurity 2 was 1.08, which also did not meet the general standard of separation degree (>1.5), and the separation effect was poor, and it was impossible to separate 10 known impurities at the same time;

[0171] Therefore, the elution gradient in the chromatographic conditions is crucial to the separation effect of the method of the present invention. Only under the gradient elution conditions of the present invention can the known impurities 1 to 10 in cefepime for injection be separated simultaneously with the best separation effect. However, the gradient elution parameters of method 1 and method 2 in Example 5 cannot be used to separate the 10 known impurities of the present invention simultaneously.

[0172] Example 6 Selection of Mobile Phase B

[0173] On the basis of Example 1, the volume ratio of phosphate buffer and acetonitrile in mobile phase B was changed, and other chromatographic conditions remained unchanged, and 10 impurities in the mixed impurity reference solution were separated.

[0174] Wherein, the volume ratio of phosphate buffer and acetonitrile in mobile phase B of Example 1 is 50:50;

[0175] Method 1: Change the volume ratio of phosphate buffer and acetonitrile in mobile phase B to 45:55;

[0176] Method 2: Change the volume ratio of phosphate buffer and acetonitrile in mobile phase B to 55:45;

[0177] Method 3: Change the volume ratio of phosphate buffer and acetonitrile in mobile phase B to 40:60;

[0178] Method 4: Change the volume ratio of phosphate buffer and acetonitrile in mobile phase B to 65:35;

[0179] Other test conditions were the same as those in Example 1. 10 μL of the mixed impurity reference solution prepared in Example 1 was accurately measured and injected into the high performance liquid chromatograph, and the test results were recorded.

[0180]

[0181] in conclusion:

[0182] The impurities in cefepime hydrochloride for injection were analyzed by HPLC using the three mobile phases B with different volume ratios, and it was found that:

[0183] In Example 1, when the volume ratio of phosphate buffer and acetonitrile in mobile phase B is controlled to be 50:50, and in Example 6, method 1 and method 2, when the volume ratio of phosphate buffer and acetonitrile in mobile phase B is controlled to be 45:55 or 55:45, the impurities 1 to 10 in the obtained spectrum all have peak responses, and the separation between the characteristic peaks is good, and each known impurity 1 to 10 can be separated at the same time.

[0184] By the attached Fig. 9It can be seen that in method 3 of Example 6, when the volume ratio of phosphate buffer and acetonitrile in mobile phase B is 40:60, the separation degree of impurity 3, impurity 4, main peak (characteristic peak of cefepime hydrochloride), impurity 6, impurity 9 and the subsequent adjacent impurity peaks (retention times are 17.190, 20.534, 27.634, 29.061, and 50.290, respectively) is at most 1.4, which are all less than 1.5, and it is impossible to separate 10 known impurities at the same time.

[0185] By the attached Fig.10 It can be seen that in Example 6 Method 4, when the volume ratio of phosphate buffer and acetonitrile in mobile phase B is 60:40, impurity 4 is split into two peaks, and the peak shapes of the impurities are all deteriorated, and it is also impossible to separate 10 known impurities at the same time.

[0186] The experimental results of the mobile phase B with different proportions above prove that when the volume ratio of phosphate buffer and acetonitrile in Example 1 is 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 to 10 can be separated simultaneously, and the separation effect is significant.

[0187] Therefore, the volume ratio of phosphate buffer and acetonitrile in mobile phase B is crucial to the separation effect of the method of the present invention. Only when the volume ratio of phosphate buffer and acetonitrile is controlled to be (45-55): (55-45), can each known impurity 1-10 be separated simultaneously; when the volume ratio of phosphate buffer and acetonitrile is 40:60 or 60:40, 10 known impurities cannot be separated simultaneously.

[0188] Example 7 Destructive Test

[0189] Forced degradation test is to accelerate the destruction of samples under more severe conditions, such as strong light irradiation, high temperature, high humidity, acid and alkali destruction, hydrolysis and oxidation destruction, etc. The purpose is to evaluate the effectiveness and applicability of the analysis method by examining the separation of the degradation products and main peaks and known impurities of the samples. At the same time, photodiode array detection is used to check the peak purity: in the spectrum obtained by the degradation experiment, when the impurity and main peak purity angle is less than the purity threshold, it is judged that the determination method meets the determination requirements.

[0190] 7-1. Solution preparation:

[0191] Diluent: Take 900 mL of phosphate buffer (pH 5.0), add 100 mL of acetonitrile, mix well and set aside.

[0192] Undegraded sample: Weigh 24.5 mg of cefepime hydrochloride for injection (containing 14 mg of cefepime hydrochloride), accurately weigh, place in a 10 mL volumetric flask, add an appropriate amount of diluent to dissolve and dilute to the scale, shake well, filter, and obtain the undegraded sample.

[0193] Oxidative degradation sample: Weigh 24.5 mg of cefepime hydrochloride for injection (containing 14 mg of cefepime hydrochloride), accurately weigh, place in a 10 mL volumetric flask, add 1 mL of 3% hydrogen peroxide, leave at room temperature for 10 min, add an appropriate amount of diluent to dissolve and dilute to the scale, shake well, filter, and take the filtrate to obtain the oxidative degradation sample.

[0194] Acid degradation sample: Weigh 24.5 mg of cefepime hydrochloride for injection (containing 14 mg of cefepime hydrochloride), accurately weigh, place in a 10 mL volumetric flask, add 1.0 mL of hydrochloric acid solution (0.5 mol / L), leave at room temperature for 10 min, neutralize with 1 mL (0.5 mol / L) of sodium hydroxide solution, add an appropriate amount of diluent to dissolve and dilute to the scale, shake well, filter, and take the filtrate to obtain the acid degradation sample.

[0195] Alkali degradation sample: Weigh 24.5 mg of cefepime hydrochloride for injection (containing 14 mg of cefepime hydrochloride), accurately weigh, place in a 10 mL volumetric flask, add 1 mL of sodium hydroxide solution (0.1 mol / L), leave at room temperature for 10 min, add 1 mL (0.1 mol / L) of hydrochloric acid solution to neutralize, add an appropriate amount of diluent to dissolve and dilute to the scale, shake well, filter, and take the filtrate to obtain the alkali degradation sample.

[0196] Hydrolysis sample: Weigh 24.5 mg of cefepime hydrochloride for injection (containing 14 mg of cefepime hydrochloride), accurately weigh, place in a 10 mL volumetric flask, add 1.0 mL of water, bathe at 30°C for 2 h, dilute to the scale with diluent, shake well, filter, and take the filtrate to obtain the hydrolysis sample.

[0197] Photodegradation sample: Take 24.5 mg of cefepime hydrochloride for injection (containing 14 mg of cefepime hydrochloride) that has been illuminated for 3 days (illuminance 4500 lx ± 500 lx), accurately weigh it, put it in a 10 mL volumetric flask, add an appropriate amount of diluent to dissolve and dilute to the scale, shake well, filter, and take the filtrate to obtain the photodegradation sample.

[0198] High temperature degradation sample: Weigh 24.5 mg of Cefepime Hydrochloride for Injection (containing 14 mg of Cefepime Hydrochloride), accurately weigh, place in a 10 mL volumetric flask, place in a 80°C water bath for 1 hour, add diluent to dilute to the scale, shake well, filter, and take the filtrate to obtain the high temperature degradation sample. 7-2. Experimental steps and conclusions:

[0199] Accurately measure 10 μL of the sample solution under the above non-degradation and degradation conditions, respectively, and inject them into the high performance liquid chromatograph, and record the chromatogram and results. The chromatographic conditions are the same as those in Example 1, and the results are shown in Tables 2-3.

[0200] Table 2 Methodology for detection of impurities in cefepime hydrochloride for injection - System suitability - 1

[0201]

[0202] Table 3 Methodology for detection of impurities in Cefepime Hydrochloride for Injection - System Suitability - 2

[0203]

[0204]

[0205] Note: “ND” means not detected.

[0206] in conclusion:

[0207] The undegraded samples and samples under various degradation conditions were tested by high performance liquid chromatography, and the results showed:

[0208] The material conservation ratio of Cefepime Hydrochloride for Injection under the degradation conditions of oxidation, acid, alkali, water, light, high temperature, etc. is maintained in the range of 98% to 102%, indicating that the material is basically conserved;

[0209] The main peak purity angle of Cefepime Hydrochloride for Injection under each degradation condition was significantly less than the purity threshold, indicating that there was no co-elution phenomenon and the peak purity could meet the impurity separation requirements; the degradation percentage of Cefepime Hydrochloride for Injection under each degradation condition was less than 5%, and the single impurity and total impurity contents increased significantly, indicating that this product was unstable under each degradation condition;

[0210] The above results show that this analytical method is effective and applicable under various degradation conditions.

[0211] The above content of the present invention separates 10 different impurities in cefepime for injection, wherein all impurity peaks can be separated, and the separation effect is remarkable. At the same time, in the following content, the present invention also performs quantitative detection on all impurities (taking 9 impurities as an example below), and can evaluate and calculate the content of impurities in cefepime for injection.

[0212] Example 8 Linearity Test

[0213] 8-1. Solution preparation:

[0214] Diluent: Take 900 mL of phosphate buffer (pH 5.0), add 100 mL of acetonitrile, mix well and set aside.

[0215] Impurity reference substance stock solution: Accurately weigh 3.5 mg of each of the impurity 1 to 9 reference substances, place them in 25 mL volumetric flasks, dissolve and make up to volume with a diluent, and shake well to obtain the impurity 1 to 9 reference substance stock solutions.

[0216] Cefepime hydrochloride stock solution: Weigh 16.6 mg of cefepime hydrochloride reference substance (containing 14 mg of cefepime hydrochloride) and place it in a 10 mL volumetric flask, add appropriate amount of diluent to dissolve and dilute to the scale, shake well, and obtain.

[0217] 400% linear solution of impurities 1-4, 6-9 (equivalent to the limit concentration): accurately measure 5.0mL of reference stock solutions of impurities 7 and 9, 2.0mL of reference stock solutions of impurities 1, 2, 3, 4, 6 and 8, and 1.0mL of cefepime hydrochloride stock solution into a 25mL volumetric flask, dilute to the mark with diluent, and shake well to obtain 400% linear solutions of impurities 1-4 and 6-9;

[0218] 200%, 100%, 80% linear solutions of impurities 1-4, 6-9 (equivalent to the limit concentration): accurately measure 5.0mL, 2.5mL, 2.0mL of 400% linear solutions of impurities 1-4 and 6-9, respectively, put them into a 10mL volumetric flask, add diluent to dilute to the scale, shake well, and then 200%, 100%, 80% linear solutions of impurities 1-4, 6-9 are obtained;

[0219] 50% and 20% linear solutions of impurities 1-4 and 6-9 (equivalent to the limit concentration): accurately measure 5.0mL and 2.0mL of 100% linear solutions of impurities 1-4 and 6-9, respectively, place in a 10mL volumetric flask, add diluent to dilute to the scale, shake well, and then 50% and 20% linear solutions of impurities 1-4 and 6-9 are obtained;

[0220] 200% linear solution of impurity 5 (equivalent to the limit concentration): accurately measure 5.0mL of impurity 5 stock solution and 1.0mL of cefepime hydrochloride stock solution and place them in a 50mL volumetric flask, add diluent to dilute to the scale, shake well, and obtain a 200% linear solution of impurity 5;

[0221] Linear (150%, 100%, 80%) solutions of impurity 5 (equivalent to the limit concentration): accurately measure 7.5mL, 5mL, and 4mL of 200% linear solution of impurity 5 into a 10mL volumetric flask, add diluent to dilute to the scale and shake well to obtain 150%, 100%, and 80% linear solutions of impurity 5;

[0222] Linear (50%, 20%) solution of impurity 5 (equivalent to the limit concentration): Accurately measure 5mL and 2mL of 100% linear solution of impurity 5 into a 10mL volumetric flask, add diluent to dilute to the scale and shake well to obtain 50% and 20% linear solutions of impurity 5.

[0223] 8-2. Experimental steps and conclusions:

[0224] 10 μL of each of the linear solutions of impurities 1 to 9 were accurately measured and injected into a high performance liquid chromatograph to record the chromatogram. The chromatographic conditions were the same as those in Example 1, and the linear results are shown in Table 4.

[0225] Table 4 Impurity detection methodology in cefepime hydrochloride for injection - linearity results

[0226]

[0227] in conclusion:

[0228] In the concentration range of 0.1040 μg / mL to 59.4200 μg / mL (equivalent to 0.7% to 400% of the control solution concentration (14 μg / mL)), the linear equation between the peak area and the concentration is y=22121.5997x+3066.1813, and the linear correlation coefficient (r) is 1.0000, which is greater than 0.9999. The peak area of ​​cefepime hydrochloride has a good linear relationship with the concentration.

[0229] Impurity 1 has a concentration range of 0.0565 μg / mL to 11.2960 μg / mL (equivalent to the limit concentration of impurity 1 of 2% to 400%), and the linear equation between the peak area and the concentration is y=26708.0734x+402.1999, and the linear correlation coefficient (r)=1.0000, which is greater than 0.9999. The peak area and the concentration of impurity 1 have a good linear relationship;

[0230] Impurity 2 has a concentration range of 0.1399 μg / mL to 11.1920 μg / mL (equivalent to the limit concentration of impurity 2 of 5% to 400%), and the linear equation between the peak area and the concentration is y=10831.6518x-656.0747, and the linear correlation coefficient (r) is 0.9999, which is greater than 0.999. The peak area and the concentration of impurity 2 have a good linear relationship;

[0231] Impurity 3 has a concentration range of 0.5128 μg / mL to 10.2560 μg / mL (equivalent to the limit concentration of impurity 3 of 20% to 400%), and the linear equation between the peak area and the concentration is y=18716.1900x-2818.6074, and the linear correlation coefficient (r)=1.0000, which is greater than 0.9999. The peak area and the concentration of impurity 3 have a good linear relationship;

[0232] Impurity 4 has a concentration range of 0.1381 μg / mL to 11.0480 μg / mL (equivalent to the limit concentration of impurity 4 of 5% to 400%), and the linear equation between the peak area and the concentration is y=16074.2026x+859.8713, and the linear correlation coefficient (r) is 0.9997, which is greater than 0.999. The peak area and the concentration of impurity 4 have a good linear relationship;

[0233] Impurity 5 is in the concentration range of 0.2566 μg / mL to 14.5800 μg / mL (equivalent to the limit concentration of impurity 5 of 3.5% to 200%), the linear equation of peak area and concentration is y=19534.4613x+1803.5883, the correlation coefficient (r)=0.9992, which is greater than 0.999, and the peak area and concentration of impurity 5 have a good linear relationship;

[0234] Impurity 6 has a concentration range of 0.1813 μg / mL to 10.3600 μg / mL (equivalent to the limit concentration of impurity 6 of 7% to 400%), and the linear equation between the peak area and the concentration is y=14881.5332x-731.7108, and the linear correlation coefficient (r)=1.0000, which is greater than 0.9999. The peak area and the concentration of impurity 6 have a good linear relationship;

[0235] Impurity 7 has a concentration range of 0.1085 μg / mL to 21.7000 μg / mL (equivalent to the limit concentration of impurity 7 of 2% to 400%), and the linear equation between the peak area and the concentration is y=20151.1448x+431.1340, and the linear correlation coefficient (r) is 1.0000, which is greater than 0.9999. The peak area and the concentration of impurity 7 have a good linear relationship;

[0236] Impurity 8 has a concentration range of 0.2590 μg / mL to 10.3600 μg / mL (equivalent to the limit concentration of impurity 8 of 10% to 400%), and the linear equation between the peak area and the concentration is y=16510.0007x-92.3936, and the linear correlation coefficient (r)=1.0000, which is greater than 0.9999. The peak area and the concentration of impurity 8 have a good linear relationship;

[0237] Impurity 9 has a concentration range of 0.1608 μg / mL to 26.8000 μg / mL (equivalent to the limit concentration of impurity 9 of 2% to 400%), and the linear equation between the peak area and the concentration is y=15212.8553x+374.0952, and the linear correlation coefficient (r)=1.0000, which is greater than 0.9999. The peak area and the concentration of impurity 9 have a good linear relationship;

[0238] The above results show that within the linear concentration range, the peak area of ​​cefepime hydrochloride and impurities 1 to 9 have a good linear relationship with the concentration, and the linear correlation coefficient (r) is greater than 0.999. Impurity 1, impurity 3 and impurities 6 to 9 are the best, reaching 1.0000, and their peak area has a good linear relationship with the concentration; compared with the correlation coefficient r=0.99 required by the general standard, the determination accuracy is improved by more than two orders of magnitude; the detection method has higher accuracy and a wider determination range.

[0239] Example 9 Limit of Quantitation and Limit of Detection Test

[0240] 9-1. Solution preparation:

[0241] Diluent: Take 900 mL of phosphate buffer (pH 5.0), add 100 mL of acetonitrile, mix well and set aside.

[0242] Test solution: Weigh 24.5 mg of cefepime hydrochloride for injection (containing 14 mg of cefepime hydrochloride), accurately weigh, place in a 10 mL volumetric flask, add an appropriate amount of diluent to dissolve and dilute to the scale, shake well, filter, and use as the test solution.

[0243] Quantitative limit solution: Take the above linear solution and quantitatively dilute it until the signal-to-noise ratio of each impurity peak is 10. At this time, the solution concentration is the quantitative limit concentration;

[0244] Detection limit solution: Take the above linear solution and quantitatively dilute it until the signal-to-noise ratio of each impurity peak is 3. At this time, the solution concentration is the detection limit concentration.

[0245] 9-2. Experimental steps and conclusions:

[0246] Accurately measure 10 μL of the above quantitative limit and detection limit solutions, respectively, and inject them into the high performance liquid chromatograph, and record the chromatogram and results. The chromatographic conditions are the same as those in Example 1, and the quantitative limit and detection limit results of each impurity are shown in Table 5.

[0247] Table 5 Impurity detection methodology in cefepime hydrochloride for injection - results of limit of quantitation and limit of detection

[0248]

[0249]

[0250] in conclusion:

[0251] The results of the limit of quantification and the limit of detection showed that

[0252] The quantification limits of various impurities did not exceed 0.513 μg / mL, among which the quantification limit of impurity 1 was as low as 0.057 μg / mL, equivalent to 0.004% of the concentration of the test solution;

[0253] The detection limits of various impurities did not exceed 0.171 μg / mL, among which the detection limit of impurity 1 was as low as 0.019 μg / mL, equivalent to 0.001% of the concentration of the test solution;

[0254] The results of the method of the present invention show that all impurities can be effectively detected under the method of the present invention with high sensitivity.

[0255] Example 10 Recovery Test

[0256] 10-1. Solution preparation:

[0257] Diluent: Take 900 mL of phosphate buffer (pH 5.0), add 100 mL of acetonitrile, mix well, and set aside.

[0258] Impurity reference substance stock solution: Accurately weigh 14 mg of each impurity 1 to 9, place them in 100 mL volumetric flasks, dissolve them in a diluent and make up to volume, shake well, and the impurity 1 to 9 reference substance stock solutions are obtained.

[0259] Accuracy stock solution: Accurately measure 10 mL of impurity 5 reference stock solution, 2 mL of impurity 1-4 reference stock solution, and 4 mL of impurity 6-9 reference stock solution into a 20 mL volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0260] Accuracy reference solution: Accurately measure 1 mL of the accuracy stock solution and place it in a 10 mL volumetric flask. Dilute to the mark with diluent and shake well.

[0261] Accuracy test solution: Weigh an appropriate amount of cefepime hydrochloride for injection (containing 28 mg of cefepime hydrochloride), place in a 20 mL volumetric flask, weigh 9 portions in parallel, divide them into 3 groups, accurately add 1.0 mL, 2.0 mL and 3.0 mL of the above-mentioned accuracy reference solution respectively, add an appropriate amount of diluent to dissolve and dilute to the scale, shake well, and use as 50%, 100% and 150% accuracy solutions respectively.

[0262] 10-2. Experimental steps and conclusions:

[0263] Accurately measure 10 μL of each of the above accuracy solutions, inject them into the high performance liquid chromatograph, and record the chromatogram. The chromatographic conditions are the same as those in Example 1, and the results of the impurity recovery rates are shown in Table 6. The relative standard deviation is calculated as follows:

[0264]

[0265] Table 6 Impurity detection methodology in cefepime hydrochloride for injection - recovery results

[0266]

[0267]

[0268] in conclusion:

[0269] The recovery test results of each impurity showed that

[0270] Under the test solution with accuracy of 50%, 100%, and 150%, the average recovery rate within the impurity 1-9 group and the average recovery rate between groups were maintained between 90% and 110%, and the recovery rate distribution range was concentrated, which could accurately meet the quantitative determination requirements of each impurity.

[0271] The RSD% of the recoveries of the 9 samples measured in this example were all less than 4.5%. Compared with the general standard requirement of a recovery rate of RSD ≤ 10.0%, the RSD of the detection method of the present invention was reduced to less than half of the standard requirement, and the accuracy of the detection result was significantly improved by more than two times, further proving that the accuracy of the method for detecting the above impurities 1 to 9 is good.

[0272] Example 11 Repeatability Test

[0273] The changes in the impurity content were examined by repeatedly measuring the same batch of samples (100% limit spiked solution), recorded as repeatability 1 to 6, to verify and obtain the repeatability of the method. The results are shown in Table 7.

[0274] Table 7 Impurity detection methodology in cefepime hydrochloride for injection - precision test results

[0275]

[0276] in conclusion:

[0277] It can be seen from the repeatability test results of each impurity in the above table that after quantitative detection of the impurity content in 6 identical 100% limit spiked test solutions, the impurity content of impurity 1 remained the same after multiple repeated tests; among impurities 2, impurity 4, impurity 5, impurity 8 and impurity 9, the RSD of 6 tests was ≤1.0%; for impurities 6 and impurity 7, the RSD of 6 tests was ≤3.0%; the RSD of the 6 measurement results of the above impurities was ≤4.4%.

[0278] There was no significant change in the content of unknown impurities and total impurities, and the RSD did not exceed 2.3%, which met the requirements of the determination standard (RDS≤5.0%). The above test results show that the above separation and detection method has good repeatability and exhibits a significant precision effect.

[0279] Example 12 Durability Test

[0280] On the basis of Example 1, the detection wavelength (±2 nm), flow rate (±0.1 mL / min), column temperature (±5° C.), pH of mobile phase A (±0.2), sample weight of mobile phase A (±0.1 g) and different batches of chromatographic columns were changed respectively. 10 μL of the test solution and the control solution were taken respectively and injected into the high performance liquid chromatograph. The other chromatographic conditions were the same as those in Example 1. The chromatograms and results were recorded. The results are shown in Table 8.

[0281] Table 8 Impurity detection methodology in cefepime hydrochloride for injection - Durability test results

[0282]

[0283]

[0284] Note: NA means not applicable.

[0285] in conclusion:

[0286] The durability test results show that

[0287] On the basis of Example 1, when the chromatographic conditions such as detection wavelength change (±2nm), column temperature (+5°C), flow rate change (±0.2mL / min), pH value of mobile phase A (±0.2), sample weight of mobile phase A (±0.1g), and replacement of different batches of chromatographic columns were detected, the measurement results did not change: the number of impurities detected was consistent; except for changing the chromatographic condition of the mobile phase flow rate, the unknown impurities, other maximum single impurities and total impurity contents remained unchanged after the other chromatographic conditions were changed.

[0288] Experiments show that under different chromatographic conditions, the method has good durability in detecting impurities in cefepime hydrochloride for injection by controlling the detection wavelength at 252-256 nm, the column temperature at 20-30°C, the flow rate at 0.9-1.1 mL / min, the pH of mobile phase A at 4.8-5.2, the sample amount of mobile phase A at 0.58-0.78 g, and different batches of chromatographic columns.

[0289] Comparative Example 1

[0290] Referring to the detection conditions and methods of comparative document 1, 10 known impurities (impurities 1 to 10) in the test solution and the mixed impurity reference solution of the embodiment of the present invention were separated and detected.

[0291] Comparison document 1: Wang Tingting, Xiao Hui, Li Pei, Luo Jialin, Guo Yinghao, Wu Jianzhuo, Hong Jianwen. Quality evaluation of domestically produced cefepime hydrochloride for injection. Chinese Journal of Antibiotics, 2022, 47(02): 128-133.

[0292] Table 9 Comparison of impurities between this application and reference document 1

[0293]

[0294]

[0295]

[0296] Table 10 Comparison of chromatographic conditions between this application and the reference document

[0297]

[0298] in conclusion:

[0299] The impurities in the test solution of the present invention were separated and detected using the detection conditions of Comparative Example 1. The experimental results show that the conditions of Comparative Example 1 (Comparative Document 1) cannot achieve the simultaneous separation and detection of the 10 known impurities of the present invention, and cannot meet the requirements for the separation of multiple impurity peaks.

[0300] Therefore, the test conditions of Comparative Example 1 cannot meet the requirements of the present invention for the simultaneous separation and detection of 10 known impurities in cefepime hydrochloride for injection. The detection effect of the method of the present invention is significantly better than that of Comparative Document 1.

[0301] Comparative Example 2

[0302] To further verify the advantages of this method, based on the fact that impurity 7 and the main peak (characteristic peak of cefepime hydrochloride) could not be separated in method 1 of Example 3, we further changed the following conditions based on the experimental conditions of method 1 of Example 3:

[0303] The types of chromatographic columns were: Waters SunFire C18, 4.6 mm × 250 mm, 5.0 μm;

[0304] The other experimental methods and steps are the same as those in Method 1 of Example 3 for detection.

[0305] in conclusion:

[0306] The experimental results show that when the detection conditions of Comparative Example 2 are used to separate the impurities in the mixed impurity reference solution of the present invention, the results displayed by the spectrum are the same as those of Method 1 in Example 3, and only the chromatographic peaks of impurities 1, 2, 3, 7 and 9 are still visible, and good separation of impurity 7 and the main peak (characteristic peak of cefepime hydrochloride) cannot be achieved.

[0307] Comparative Example 3

[0308] To further verify the advantages of this method, based on the overlap of the characteristic peaks of impurities 4 and 5 in method 1 of Example 5, and the main peak (characteristic peak of cefepime hydrochloride) and the subsequent adjacent impurity peak (retention time of 22.046), we changed the following conditions based on the experimental conditions of method 1 of Example 5:

[0309] Comparative Example 3 - Method 1:

[0310] The types of chromatographic columns were: Waters SunFire C18, 4.6 mm × 250 mm, 5.0 μm;

[0311] The mobile phase is:

[0312]

[0313] Comparative Example 3 - Method 2:

[0314] The types of chromatographic columns were: Waters SunFire C18, 4.6 mm × 250 mm, 5.0 μm;

[0315] The mobile phase is:

[0316] Mobile phase A Take 0.57g of ammonium dihydrogen phosphate and dissolve it in 1000mL of water. Mobile phase B Acetonitrile

[0317] The other experimental methods and steps are the same as those in Method 1 of Example 5 for detection.

[0318] in conclusion:

[0319] The experimental results show that when the impurities in the mixed impurity reference solution of the present invention are separated using the detection conditions of Comparative Example 3-Method 1, the separation results show that impurity 4 and impurity 5 overlap, and the separation degree of the main peak (characteristic peak of cefepime hydrochloride) and its subsequent adjacent impurity (retention time: 22.046) is 0.9, which is still far less than the general standard of separation degree (>2.0), and it is impossible to separate 10 known impurities at the same time;

[0320] When the impurities in the mixed impurity reference solution of the present invention were separated using the detection conditions of Comparative Example 3-Method 2, the separation results showed that impurity 4 and impurity 5 overlapped, and the main peak (the characteristic peak of cefepime hydrochloride) and its subsequent adjacent impurities also overlapped, and it was impossible to separate 10 known impurities at the same time.

[0321] In summary, it can be seen from the above results that the separation degree of the 10 known impurity characteristic peaks obtained by the separation and detection conditions adopted is greater than 1.5, and the separation degree is good and significant;

[0322] The quantitative limit of 10 known impurities detected by the method of the present invention can be as low as 0.057 μg / mL, equivalent to 0.004% of the concentration of the test solution; the detection limit of 10 known impurities can be as low as 0.019 μg / mL, equivalent to 0.001% of the concentration of the test solution; the detection limit and quantitative limit are significantly reduced, and the sensitivity is significantly improved;

[0323] The recovery rates of the 10 known impurities were all maintained within the range of 90% to 110%, and the recovery RSDs of multiple tests were all less than 5%. Compared with the general standard requirement of recovery RSD ≤ 10.0%, it was reduced to less than half of the standard requirement, and the accuracy was increased by more than 2 times. The accuracy performance of the separation method of the present invention was significantly improved;

[0324] Within the limited concentration range, the characteristic absorption peak areas of cefepime hydrochloride and impurities 1 to 10 show a good linear relationship with their corresponding concentrations, and the correlation coefficient of the linear equation is r>0.999, and the optimal value is 1.0000; compared with the linear correlation coefficient r>0.99 required by general standards, the accuracy of the determination is improved by 2 orders of magnitude; the accuracy of the detection method is significantly increased, and the determination range is wider;

[0325] The separation and detection methods used have consistent detection results for each impurity with good precision;

[0326] By changing different chromatographic conditions and chromatographic column batches, the RSDs of the detection results of impurities in cefepime hydrochloride for injection were all less than 5%, showing good and significant durability.

[0327] The method for detecting 10 known impurities in cefepime hydrochloride for injection adopted by the present invention is the optimal separation method, which can accurately perform quantitative quality control on the detected impurities of cefepime hydrochloride for injection, thereby ultimately ensuring the safety, effectiveness and quality control of the drug. The high performance liquid chromatography method adopted has strong specificity and high sensitivity, and can accurately and quantitatively determine multiple impurities in cefepime hydrochloride for injection.

[0328] Finally, it should be noted that the above descriptions are only embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for separating and detecting impurities in cefepime hydrochloride for injection, the detection method comprising the following steps: using a diluent to weigh cefepime hydrochloride for injection to prepare a test solution, and then using a high performance liquid chromatography method for detection; Chromatographic conditions included: The chromatographic column uses octadecylsilane bonded silica gel as a filler; Mobile Phase: Mobile phase A: phosphate buffer, the pH range of the mobile phase A is 4.8-5.2; Mobile phase B: a mixture of phosphate buffer and acetonitrile, the volume ratio of the two is (45-55): (55-45); Elution mode: gradient elution, the elution gradient of the mobile phase is: The impurities in the cefepime hydrochloride for injection include the following, and the structural formula is as follows:

2. The method according to claim 1, wherein: The volume ratio of phosphate buffer and acetonitrile was 50:

50.

3. The method according to claim 1, wherein: The type of chromatographic column is Waters T3C18.

4. The method according to any one of claims 1 to 3, wherein: The specifications of the chromatographic column are 4.6 mm×250 mm, 5.0 μm.

5. The method according to any one of claims 1 to 3, wherein: The pH range of the mobile phase A is 5.

0.

6. The method according to any one of claims 1 to 3, wherein: The detection wavelength of the chromatographic conditions is 252-256 nm.

7. The method according to claim 6, wherein: The detection wavelength of the chromatographic conditions is 254 nm.

8. The method according to any one of claims 1 to 3, wherein: The phosphate buffer is potassium dihydrogen phosphate solution.

9. The method according to any one of claims 1 to 3, wherein: The column temperature of the chromatographic column is 20°C to 30°C.

10. The method according to claim 9, wherein: The column temperature of the chromatographic column is 25°C.

11. The method according to any one of claims 1 to 3, wherein: The flow rate of the chromatographic column is 0.9-1.1 mL / min.

12. The method according to claim 11, wherein: The flow rate of the chromatographic column was 1.0 mL / min.

13. The method according to any one of claims 1 to 3, wherein: The solution preparation method is: Test solution: Take cefepime hydrochloride for injection, add diluent to dissolve, filter, and use as the test solution; Mixed impurity reference solution: Take cefepime hydrochloride and impurity 1 to 10 reference substances respectively, add diluent to dissolve, and prepare mixed impurity reference solution.

14. The method according to any one of claims 1 to 3, wherein: The diluent is a mixture of phosphate buffer and acetonitrile.

15. The method according to any one of the preceding claims 1 to 3, wherein: Solution preparation also includes: Impurity localization solution: Take appropriate amount of impurity 1-10 reference substances respectively, add diluent to make up to the scale, shake well to make a solution of 14 μg per 1 mL, which is used as impurity 1-10 localization solution.

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