A method for detecting related substances in cefazolin sodium

Abstract

The invention discloses a method for detecting related substances in cefazolin sodium. The method for detecting related substances in cefazolin sodium provided by the invention comprises the following steps: using high performance liquid chromatography to detect the cefazolin sodium to be tested, wherein the elution procedure of the high performance liquid chromatography is gradient elution. The detection method of the invention realizes the detection and analysis of multiple impurities of the invention in the cefazolin sodium raw material or preparation, has strong specificity for multiple impurities, good or high sensitivity, linear relationship, accuracy and reproducibility, and can be widely used in the quality control of cefazolin sodium products.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for detecting related substances in cefazolin sodium. Background Art

[0002] The chemical name of Cefazolin Sodium is (6R,7R)-3-[[(5-methyl-1,3,4-thiadiazol-2-yl)thio]methyl]-7-[(1H-tetrazol-1-yl)acetylamino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid sodium salt. It is a semi-synthetic first-generation cephalosporin with an antibacterial spectrum similar to that of cefalexin. It has antibacterial effects on Staphylococci (including enzyme-producing strains), Streptococci (except enterococci), Streptococcus pneumoniae, Escherichia coli, Proteus mirabilis, Klebsiella, Haemophilus influenzae and Enterobacter aerogenes. The characteristic of cefazolin sodium is its strong effect on Gram-positive bacteria. Cefazolin sodium for injection is commonly used in clinical practice to treat respiratory tract infections such as otitis media, bronchitis, pneumonia, urinary tract infections, skin and soft tissue infections, bone and joint infections, sepsis, infective endocarditis, hepatobiliary system infections, and infections of the eyes, ears, nose, and throat caused by sensitive bacteria.

[0003] The detection of related substances of cefazolin sodium involves 13 or more related substances. When the 2020 edition of the Chinese Pharmacopoeia detects the related substances of cefazolin sodium raw materials or preparations, it only stipulates that impurity CL-A ≤ 0.5%, impurity CL-E ≤ 1.0%, other single impurities ≤ 1.0%, total impurities ≤ 3.5%, and reporting limit: 0.05%. However, in actual analysis and detection, when the method of the 2020 edition of the Chinese Pharmacopoeia is used to detect the related substances of cefazolin sodium raw materials or preparations, the peaks of some related substances (such as the impurity CL-K peak) will overlap with the retention time of the main component cefazolin peak, resulting in poor specificity and quantitative accuracy of this method; this method is not very suitable for the determination of related substances or content of cefazolin sodium. Therefore, there is an urgent need for a more accurate and effective new method for detecting related substances in cefazolin sodium. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the defects of the existing methods for detecting related substances in cefazolin sodium, namely, poor impurity specificity and quantitative accuracy, and to provide a method for detecting related substances in cefazolin sodium, which has strong impurity specificity for cefazolin sodium and excellent sensitivity, accuracy and reproducibility.

[0005] The present invention solves the above technical problems through the following technical solutions.

[0006] The present invention provides a method for detecting related substances in cefazolin sodium, which comprises the following steps: using high performance liquid chromatography to detect the cefazolin sodium to be tested, and the chromatographic conditions include:

[0007] Chromatographic column: C 18 Chromatographic columns;

[0008] In the present invention, the chromatographic column can be selected from various types of C 18 Chromatographic columns, such as octadecylsilane bonded silica gel as filler (YMC-Pack Pro C 18 (4.6mm*250mm,5μm) chromatographic column, or Wondasil C 18 Superb (250 mm × 4.6 mm, 5 μm) chromatographic column, or a chromatographic column of equivalent performance, preferably YMC-PackPro C 18 4.6mm*250mm, 5μm chromatographic column.

[0009] Mobile phase: The volume ratio of mobile phase A to mobile phase B is 95:5.

[0010] Wherein mobile phase A is phosphate buffer (6.8 g / L potassium dihydrogen phosphate solution, pH adjusted to 6.6-6.9 with 10% sodium hydroxide solution);

[0011] Mobile phase B was phosphate solution (6.8 g / L potassium dihydrogen phosphate solution)-acetonitrile (1:1).

[0012] Perform gradient elution as shown in the table below.

[0013]

[0014]

[0015] In the present invention, the related substances refer to any substances or impurities that affect the purity of the drug.

[0016] In the present invention, the cefazolin sodium test product can be various cefazolin sodium products, such as cefazolin sodium bulk drug or cefazolin sodium preparation.

[0017] In the present invention, before the detection, the cefazolin sodium test product is pretreated, specifically: the cefazolin sodium test product is dissolved in a diluent and left standing.

[0018] According to a specific embodiment of the present invention, the pH value of the mobile phase is preferably 6.6, 6.8 or 6.9, and the most preferred pH value of the mobile phase is 6.8.

[0019] In the present invention, the flow rate of the mobile phase can be conventional in the art, which can be 1.3 ml / min to 1.7 ml / min, preferably 1.3 ml / min, 1.5 ml / min or 1.7 ml / min, and most preferably 1.5 ml / min.

[0020] In the present invention, in the high performance liquid chromatography, the detection wavelength is: UV210nm, 254nm. The detector can be conventional equipment in the art, such as DAD or UV detector.

[0021] In the present invention, the column temperature of the chromatographic column can be conventional in the art, and can be 28°C to 35°C, preferably the column temperature is 28°C, 30°C or 35°C, and most preferably the column temperature is 30°C.

[0022] In the present invention, in the high performance liquid chromatography method, the injection volume can be conventional in the art, and can be 10-20 μL, preferably 20 μL.

[0023] Solution preparation:

[0024] Solvent: mobile phase A;

[0025] Test solution: Take about 25 mg of the product, weigh accurately, put it in a 10 ml volumetric flask, add an appropriate amount of solvent, shake to dissolve, dilute to the scale with solvent, shake well, and use it as the test solution;

[0026] Reference substance solution: Take an appropriate amount of cefazolin reference substance, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing about 25 μg per 1 ml as the reference substance solution.

[0027] System suitability solution: Take about 20 mg of this product, place it in a 10 ml volumetric flask, shake with 0.05 mol / L sodium hydroxide solution to dissolve and dilute to the scale, let it stand at room temperature for 5 minutes, take 1 ml, place it in a 25 ml volumetric flask, dilute to the scale with solvent, shake well, and use it as the system suitability solution.

[0028] Determination method: Accurately measure 20 μl of system suitability solution, test solution and reference solution, respectively, inject them into liquid chromatograph and record the chromatogram.

[0029] The standard stipulates:

[0030] System suitability: Accurately measure 20 μl of the system suitability solution, inject it into the liquid chromatograph, and record the chromatogram; the retention time of the cefazolin peak is about 32 minutes, and the separation degree between the cefazolin peak and the cefazolin-related substances (CL-L) should be no less than 8.0 (254 nm).

[0031] If there are impurity peaks in the chromatogram of the test solution, determine the detection wavelength and attribution of each peak according to the table below and calculate by the principal component external standard method with correction factor. Impurities CL-A and CL-M shall not exceed 0.5%, impurity CL-K shall not exceed 0.1%, impurities CL-B, CL-C, CL-D, CL-E, CL-G, CL-H, CL-I, CL-L, CL-N, and CL-Q shall not exceed 1.0%, other single unknown impurities shall not exceed 0.5%, and the total amount of impurities shall not exceed 3.5%. Peaks in the chromatogram of the test solution that are less than 0.05 times the main peak area of ​​the reference solution shall be ignored.

[0032]

[0033] Calculation formula:

[0034]

[0035] Where As(210) is the peak area of ​​impurities CL-Q and CL-N in the test solution at a wavelength of 210 nm;

[0036] Wr is the weight of the reference substance, mg;

[0037] Ar(254) is the peak area of ​​the main component in the reference solution at a wavelength of 254 nm;

[0038] Ws is the weight of the test sample, mg;

[0039] Sr is the dilution multiple of the reference substance;

[0040] Ss is the dilution multiple of the test product;

[0041] P is the content of the reference substance;

[0042] f is the correction factor for relative impurities.

[0043]

[0044] Total impurities (%) = ∑ impurities (%)

[0045] Where: As(254) is the peak area of ​​each impurity in the test solution at a wavelength of 254 nm;

[0046] Wr is the weight of the reference substance, mg;

[0047] Ar(254) is the peak area of ​​the main component in the reference solution at a wavelength of 254 nm;

[0048] Ws is the weight of the test sample, mg;

[0049] Sr is the dilution multiple of the reference substance;

[0050] Ss is the dilution multiple of the test product;

[0051] P is the content of the reference substance;

[0052] f is the correction factor for relative impurities.

[0053] According to an embodiment of the present invention, the impurity CL-A, impurity CL-M, impurity CL-K, impurity CL-B, impurity CL-C, impurity CL-D, impurity CL-E, impurity CL-G, impurity CL-H, impurity CL-I, impurity CL-L, impurity CL-N, and impurity CL-Q described in the present invention, the structural formula and impurity source of each impurity are:

[0054] The structural formula of impurity compound CL-A is: It is an intermediate in the preparation process of cefazolin sodium and is a process impurity that needs to be controlled in the raw material. In order to strictly control the product quality, the quality standard is set and the impurity control limit is 0.5%;

[0055] The structural formula of impurity compound CL-B is: It is generated by the reaction of compound CL-A and the reaction reagent pivaloyl chloride. It is a process impurity and needs to be controlled in the raw material. In order to strictly control the product quality, the quality standard is set, and the impurity control limit is 1.0%;

[0056] The structural formula of the impurity compound CL-H is: It is the starting material of the reaction and is a process impurity that needs to be controlled in the API. In order to strictly control the product quality, the quality standard is set and the impurity control limit is 1.0%;

[0057] The structural formula of impurity compound CL-C is: It is introduced into the starting material 7-ACA (impurity CL-H) and is a process impurity that needs to be controlled in the API. In order to strictly control the product quality, the quality standard is set and the impurity control limit is 1.0%;

[0058] The structural formula of impurity compound CL-D is: It is the residue of the starting material in the intermediate CL-A and participates in the subsequent reaction. It is a process impurity and needs to be controlled in the raw material. In order to strictly control the product quality, it is included in the quality standard and the impurity control limit is 1.0%;

[0059] The structural formula of impurity compound CL-E is: It is a process impurity and degradation impurity, which needs to be controlled in the raw material drug, and the product process must be strictly controlled to avoid contact with strong acids and strong bases. The product packaging and storage conditions must be strictly controlled. The impurity control limit is 1.0%;

[0060] The structural formula of impurity compound CL-G is: It is a degradation impurity, which requires strict control of product process, avoidance of high temperature conditions, strict control of product packaging and storage conditions, and an impurity control limit of 1.0%;

[0061] The structural formula of impurity compound CL-I is: It is a degradation impurity, which requires strict control of product process to avoid contact with strong alkaline conditions, strict control of product packaging and storage conditions, and an impurity control limit of 1.0%;

[0062] The structural formula of impurity compound CL-J is: It is a degradation impurity, which requires strict control of product process to avoid contact with strong alkaline conditions, strict control of product packaging and storage conditions, and an impurity control limit of 0.5%;

[0063] The structural formula of impurity compound CL-K is: It is a process impurity introduced from the starting material 7-ACA and needs to be controlled in the API. In order to strictly control the product quality, the quality standard is set and the impurity control limit is 0.1%;

[0064] The structural formula of impurity compound CL-L is: It is a process impurity and degradation impurity, which needs to be controlled in the raw material and the process conditions must be controlled to avoid contact with strong acid and strong alkali conditions. The product packaging and storage conditions must be strictly controlled. The impurity control limit is 1.0%;

[0065] The structural formula of impurity compound CL-M is: It is a process impurity and degradation impurity, which needs to be controlled in the raw material drug, and the product process must be strictly controlled to avoid contact with strong acids and strong bases, and the product packaging and storage conditions must be strictly controlled. The impurity control limit is 0.5%;

[0066] The structural formula of impurity compound CL-N is: It is a degradation impurity, which requires strict control of product process to avoid contact with strong acid and strong alkali, strict control of product packaging and storage conditions, and impurity control limit of 1.0%;

[0067] The structural formula of the impurity compound CL-Q is: It is a process impurity and degradation impurity, which needs to be controlled in the raw material and the process conditions must be controlled to avoid contact with strong acid and strong alkali conditions. The product packaging and storage conditions must be strictly controlled, and the impurity control limit is 1.0%.

[0068] According to a specific embodiment of the present invention, a method for detecting related substances in cefazolin sodium according to the present invention is as follows:

[0069] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (YMC-Pack ProC18 4.6mm×250mm, 5μm or chromatographic column with equivalent performance); mobile phase A is 6.8g / L potassium dihydrogen phosphate solution (adjust pH to 6.8 with 10% sodium hydroxide solution)-mobile phase B (95:5), mobile phase B is 6.8g / L potassium dihydrogen phosphate solution-acetonitrile (1:1); flow rate is 1.5ml per minute; linear gradient elution is performed according to the table below; column temperature is 30℃; detection wavelengths are 210nm and 254nm; injection volume is 20μl.

[0070] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 7 100 0 15 87 13 30 82 18 35 82 18 45 52 48 50 52 48 55 100 0 65 100 0

[0071] Test solution: Take an appropriate amount of the product, weigh accurately, add mobile phase A to dissolve and quantitatively dilute to make a solution containing about 2.5 mg per 1 ml.

[0072] Reference solution: Accurately weigh an appropriate amount of cefazolin reference substance, dissolve it in mobile phase A and quantitatively dilute it to make a solution containing approximately 25 μg per 1 ml.

[0073] System suitability solution: Take an appropriate amount of this product, dissolve and dilute it with 0.05 mol / L sodium hydroxide solution to make a solution containing about 2 mg per 1 ml, and let it stand at room temperature for 5 minutes (sodium hydroxide solution will degrade to produce cefazolin isomer impurity CL-L), accurately measure 1 ml, place it in a 25 ml volumetric flask, add mobile phase A to dilute to the scale, and shake well.

[0074] System suitability requirements: In the system suitability solution chromatogram, the separation between the cefazolin peak and the impurity CL-L peak should be no less than 8.0 (254nm).

[0075] Determination method: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0076] Limits: If there are impurity peaks in the chromatogram of the test solution, the detection wavelength, relative retention time, correction factor and limit of each impurity peak are shown in the table below. The total amount of impurities shall not exceed 3.5%, and any peak in the chromatogram of the test solution that is less than 0.05 times the main peak area of ​​the reference solution shall be ignored.

[0077] Compound code Detection wavelength (nm) Relative retention time Correction Factor limit(%) CL-Q 210 0.07 2.5 1.0 CL-N 210 0.08 3.0 1.0 CL-M 254 0.18 1.4 0.5 CL-E 254 0.19 1.4 1.0 CL-H 254 0.35 1.0 1.0 CL-C 254 0.41 1.2 1.0 CL-G 254 0.50 1.5 1.0 CL-D 254 0.62 1.1 1.0 CL-A 254 0.68 0.8 0.5 CL-I 254 0.79 1.3 1.0 Cefazolin 254 1.00 - - CL-K 254 1.10 1.1 0.1 CL-L 254 1.25 1.0 1.0 CL-B 254 1.52 1.1 1.0 Other single impurities 254 - 1.0 0.5

[0078] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0079] The reagents and raw materials used in the present invention are commercially available.

[0080] The positive progress of the present invention is that compared with the impurity CL-I peak and the cefazolin peak overlap in the method of the 2020 edition of the Chinese Pharmacopoeia, the impurity CL-A separation degree is 1.27. The detection method of the present invention realizes the detection and analysis of various impurities such as impurities CL-A, CL-B, CL-C, CL-D, CL-E, CL-G, CL-H, CL-I, CL-J, CL-K, CL-L, CL-M, CL-N and CL-Q of the present invention in the cefazolin sodium raw material or preparation, and has strong specificity for various impurities, good or high sensitivity, linear relationship, accuracy and reproducibility, and can be widely used in the quality control of cefazolin sodium products. Specifically embodied in: (1) Specificity and system suitability blank solvent does not interfere with the determination. The separation between the cefazolin peak and the impurity CL-I, impurity CL-K peaks, and each known impurity peak meets the requirements and meets the system suitability requirements. (2) The injection precision is good. (3) In the degradation test of the homemade and reference preparations, the impurities produced by degradation did not interfere with the determination, and the main peak and the adjacent chromatographic peaks were baseline separated; the minimum peak purity threshold of the main peak was 1.000000; the material conservation was 90.69% to 106.26%, and the results showed that the method had good specificity. (4) In the linearity, range and correction factor test, cefazolin and each impurity had a good linear relationship in the range of 0.05% to 2.0% of the test sample concentration. (5) The concentrations of the quantitative limit solutions of each impurity and the main component were in the range of 0.07588 to 0.6099 μg / ml (quantitative limit: 1.5ng to 12.2ng), which was equivalent to 0.003% to 0.024% of the test sample concentration. The quantitative limits of each impurity were less than the reporting limit of 0.05%. This shows that the detection method of related substances has high sensitivity and is suitable for impurity control. (6) The method has good repeatability. (7) The intermediate precision results are good. (8) The recovery rate of this product is: 87.29%~114.81%; RSD is less than 10.0%, and this method has high accuracy. (9) The results show that the test sample and mixed reference solution are unstable within 24 hours at room temperature and 4℃ refrigerated conditions, so this product should be freshly prepared before use. (10) Regarding the chromatographic conditions of this product, the durability is mainly investigated from the aspects of flow rate, column temperature, mobile phase pH value, chromatographic column, etc. Under different flow rate conditions such as 1.3ml / min~1.7ml / min, the separation of each peak meets the requirements, and the content of each impurity and the total amount of impurities in the test solution are basically the same. This method has good durability for flow rate. Under different column temperature conditions such as 28℃~35℃, the separation of each peak meets the requirements, and the content of each impurity and the total amount of impurities in the test solution are basically the same. This method has good durability for column temperature. Under different mobile phase pH conditions such as mobile phase A pH of 6.6-6.9, the separation of each peak meets the requirements, and the impurity content and total impurity amount in the test solution are basically consistent. This method has good durability for mobile phase pH value.Under the conditions of different brands of chromatographic columns and different batches of chromatographic columns of the same brand, the separation of each peak meets the requirements, and the impurity content and total impurity amount in the test solution are basically the same. This method has good durability for chromatographic columns of the same brand but different batches and different brands. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 is a linear correlation diagram of impurity CL-M in Example 2; Figure 2 is a linear correlation diagram of impurity CL-E in Example 2; Figure 3 is a linear correlation diagram of impurity CL-H in Example 2; Figure 4 is a linear correlation diagram of impurity CL-C in Example 2; Figure 5 is a linear correlation diagram of impurity CL-G in Example 2; Figure 6 is a linear correlation diagram of impurity CL-D in Example 2; Figure 7 is a linear correlation diagram of impurity CL-A in Example 2; Figure 8 is a linear correlation diagram of impurity CL-1 of Example 2; Fig. 9 is the linear correlation diagram of cefazolin in Example 2;

[0082] Fig.10 is a linear correlation diagram of impurity CL-K in Example 2; Fig.11 is a linear correlation diagram of impurity CL-L in Example 2; Fig.12 is a linear correlation diagram of impurity CL-B in Example 2; Fig.13 is a linear correlation diagram of impurity CL-Q in Example 2; Fig.14 This is the linear correlation diagram of impurity CL-N in Example 2. DETAILED DESCRIPTION

[0083] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0084] Relevant inspection instruments and reagent information for the detection of relevant substances, including but not limited to: electronic analytical balance (brand or manufacturer: Mettler, specification or model: XS105DU (one hundred thousandth); brand or manufacturer: Sartorius, specification or model: MSA6.6S-CE (one millionth); brand or manufacturer: OHAUS, specification or model: Cp214 (one ten thousandth)), high performance liquid chromatograph (brand or manufacturer: Shimadzu, Japan, specification or model: LC-20AT; brand or manufacturer: ACCHROM, specification or model: S6000; brand or manufacturer: THERMO, specification or model: U3000), pH meter (brand or manufacturer: Mettler, specification or model: S210), expandable test chamber (illumination) (brand or manufacturer: Shanghai Boxun Industrial Co., Ltd., specification or model: BXS-400S), chromatographic column (brand or manufacturer: YMC-Pack Pro C 18 , specifications or models: 4.6×250mm, 5μm; Shimadzu Wondasil C18, specifications or models: 4.6×250mm, 5μm), acetonitrile (brand or manufacturer: MREDA, specifications or models: chromatographic grade), potassium dihydrogen phosphate (brand or manufacturer: Tianjin Guangfu Fine Chemical Research Institute, specifications or models: analytical grade), sodium hydroxide (brand or manufacturer: Tianjin Guangfu Fine Chemical Research Institute, specifications or models: analytical grade), hydrochloric acid (brand or manufacturer: Tianjin Guangfu Fine Chemical Research Institute, specifications or models: analytical grade), 30% hydrogen peroxide (brand or manufacturer: Tianjin Guangfu Fine Chemical Research Institute, specifications or models: analytical grade), water (homemade, purified water).

[0085] Sample information for related substance testing, including but not limited to: Cefazolin reference substance (source: China Food and Drug Inspection Institute, batch number: 130421-201204), CL-A (source: China Food and Drug Inspection Institute, batch number: 130612-201102), CL-B (source: Guangzhou Oumingkai Biotechnology Co., Ltd., batch number: 53150419), CL-C (source: Guangzhou Oumingkai Biotechnology Co., Ltd., batch number: 1832609), CL-D (source: Guangzhou Oumingkai Biotechnology Co., Ltd., batch number: 1832609), Kai Biotechnology Co., Ltd., batch number: 1732609), CL-E (Source: China Food and Drug Inspection Institute, batch number: 130613-201102), CL-G (Source: Guangzhou Oumingkai Biotechnology Co., Ltd., batch number: 61010420), CL-H (Source: TRC, batch number: 2-YAS-12-1), CL-I (Source: Guangzhou Oumingkai Biotechnology Co., Ltd., batch number: 18041015), CL-J (Source: Shenzhen Excellence Biotechnology Co., Ltd., batch number: 2 0190520), CL-K (Source: Guangzhou Oumingkai Biotechnology Co., Ltd., batch number: 50130398), CL-L (Source: Guangzhou Oumingkai Biotechnology Co., Ltd., batch number: 38020424), CL-M (Source: Guangzhou Paipai Biotechnology Co., Ltd., batch number: PITBZL-E-USP-20180507-01), CL-N (Source: Guangzhou Paipai Biotechnology Co., Ltd., batch number: PITBZL-B-USP-20181020-02), CL- Q (source: Shenzhen Excellence Biotechnology Co., Ltd., batch number: 20171008), reference preparation (cefazolin sodium for injection) (source: ACS (SAGENT), batch number: LY2D, FK4L; source: haspira, batch number: JK6642), laboratory batch samples (source: Beijing Kanglisheng Pharmaceutical Technology Development Co., Ltd., batch number: 190815), pilot batch samples (source: Tongfang Pharmaceutical Group Co., Ltd., batch numbers: 200502, 200503, 200504).

[0086] Example 1. Method establishment

[0087] (1) Screening of chromatographic conditions:

[0088] The relevant substances were tested using the chromatographic conditions of the 2020 edition of the Chinese Pharmacopoeia.

[0089] Chinese Pharmacopoeia 2020 Edition: Chromatographic column: C18 column; Mobile phase: Mobile phase A: phosphate buffer (take 2.91g disodium hydrogen phosphate dodecahydrate and 0.71g potassium dihydrogen phosphate, add water to 1000ml), mobile phase B: acetonitrile. Wavelength: 254nm; Column temperature: 45℃; Flow rate: 1.2ml / min. Gradient program:

[0090] Time (min) Mobile phase A (%) Mobile phase B (%) 0 98 2 2 98 2 4 85 15 10 60 40 11.5 35 65 12 35 65 15 98 2 21 98 2

[0091] Solution preparation:

[0092] Solvent: Mobile phase A.

[0093] Each positioning solution: Weigh appropriate amount of impurity CL-A, CL-B, CL-C, CL-D, CL-E, CL-G, CL-H, CL-I, CL-J, CL-K, CL-L, CL-M, CL-N and CL-Q reference substances, add solvent to dissolve respectively, shake well to obtain.

[0094] Impurity mixed solution: Take appropriate amount of each impurity location solution and mix them evenly to obtain the solution. The test results are shown in Table 1.

[0095] Table 1. Results of the detection method for relevant substances according to the chromatographic conditions of the 2020 edition of the Chinese Pharmacopoeia

[0096]

[0097]

[0098] The results showed that in the method of the 2020 edition of the Chinese Pharmacopoeia, the impurity CL-I peak and the cefazolin peak overlapped, and the impurity CL-A separation degree was 1.27. At the same time, considering that mobile phase A is a neutral salt solution, which is very easy to grow bacteria, mobile phase A was adjusted to 6.8 g / L potassium dihydrogen phosphate solution (adjust the pH value to 6.8 with 10% sodium hydroxide solution)-mobile phase B (95:5), and mobile phase B was phosphate solution (6.8 g / L potassium dihydrogen phosphate solution)-acetonitrile (1:1), and the gradient program was fine-tuned for subsequent experiments.

[0099] (2) Screening of filter membrane:

[0100] Test solution: Take an appropriate amount of the product, weigh it accurately, add an appropriate amount of solvent, shake to dissolve and dilute to make a solution containing about 2.5 mg per 1 ml, shake well, take an appropriate amount, centrifuge, filter through polytetrafluoroethylene (PTFE) and nylon microporous membranes, discard 1 ml, 3 ml, and 5 ml of the initial filtrate, and take the subsequent filtrate as the respective test solution.

[0101] Reference substance solution: Accurately weigh an appropriate amount of cefazolin reference substance, dissolve it in a solvent and dilute it to make a solution containing about 25 μg per 1 ml as the reference substance solution.

[0102] Accurately measure 20 μl of the test solution and reference solution, inject them into the liquid chromatograph, and record the chromatogram. Calculate the content of each impurity. The results show that after the test solution is filtered through the PTFE and nylon membranes, 1 ml, 3 ml, and 5 ml are discarded, and the filtrate is compared with the centrifuged sample. There is no significant difference in the detection results, and the filter membrane has no adsorption effect on the impurities of this product. The filtration method (filter membrane: PTFE, nylon) can be used to prepare the test solution of this product.

[0103] (3) Screening of extraction methods:

[0104] Test solution: Take an appropriate amount of the product, weigh it accurately, add an appropriate amount of solvent and shake for different times (1min, 3min, 5min) to dissolve and dilute it to make a solution containing about 2.5mg per 1ml, shake well, and use it as the test solution.

[0105] Reference substance solution: Accurately weigh an appropriate amount of cefazolin reference substance, dissolve it in a solvent and dilute it to make a solution containing about 25 μg per 1 ml as the reference substance solution.

[0106] Accurately measure 20 μl of the test solution and reference solution, respectively, and inject them into the liquid chromatograph to record the chromatogram. Calculate the content of each impurity. The results show that there is no significant difference in the detection results when the test solution is shaken for different time periods (1min, 3min, 5min). The test solution of this product can be prepared by shaking and dissolving.

[0107] (4) Confirmation of detection method:

[0108] Chromatographic column: Octadecylsilane bonded silica gel as filler [YMC-PackPro C 18 (250mm×4.6mm, 5μm)]

[0109] Mobile phase A: buffered saline solution (6.8 g / L potassium dihydrogen phosphate solution, pH adjusted to 6.8 with 10% sodium hydroxide solution)-mobile phase B (95:5);

[0110] Mobile phase B: buffer solution (6.8 g / L potassium dihydrogen phosphate solution)-acetonitrile (1:1)

[0111] Gradient Program:

[0112] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 7 100 0 15 87 13 30 82 18 35 82 18 45 52 48 50 52 48 55 100 0 65 100 0

[0113] Detection wavelength: UV210nm; 254nm;

[0114] Flow rate: 1.5 ml / min;

[0115] Column temperature: 30°C;

[0116] Injection volume: 20 μl;

[0117] Solvent: Mobile phase A.

[0118] Example 2: Method validation of related substances

[0119] (1) Specificity and system applicability

[0120] Blank solvent: buffer solution (6.8 g / L potassium dihydrogen phosphate solution, pH adjusted to 6.8 with 10% sodium hydroxide solution)-mobile phase B (95:5).

[0121] Impurity localization solution: Take about 1 mg of each impurity reference substance, place it in a sampling vial, add 1 ml of solvent to dissolve it, shake well, and use it as the impurity localization solution.

[0122] System suitability solution: Take about 20 mg of this product, place it in a 10 ml volumetric flask, add 0.05 mol / L sodium hydroxide solution to dissolve and dilute to the scale, leave it at room temperature for 5 minutes (sodium hydroxide solution will destroy it to produce CL-L, i.e., cefazolin isomer), accurately measure 1 ml, place it in a 25 ml volumetric flask, dilute to the scale with solvent, shake well, and use it as the system suitability solution.

[0123] Test solution: Take about 25 mg of the test sample, weigh accurately, place in a 10 ml volumetric flask, dissolve and dilute to the scale with solvent, shake well, and use as the test solution.

[0124] Mixed reference substance solution: Accurately weigh appropriate amounts of cefazolin and each impurity reference substance, dissolve in solvent and quantitatively dilute to make a mixed solution containing approximately CL-M 12.5μg, CL-A 12.5μg, CL-Q 25μg, CL-N 25μg, CL-E 25μg, CL-H 25μg, CL-C 25μg, CL-G 25μg, CL-D 25μg, CL-I 25μg, CL-K2.5μg, CL-L 25μg, CL-B 25μg, CL-J 25μg and cefazolin 25μg per 1ml. Shake well to prepare the mixed reference substance solution.

[0125] Accurately measure 20 μl of each of the above solutions and inject them into the liquid chromatograph respectively. Record the chromatogram. The results of the system suitability test are shown in Table 2.

[0126] Table 2. Method validation for related substances - system suitability

[0127]

[0128] The results showed that the blank solvent did not interfere with the determination. The separation between the cefazolin peak and the impurity CL-I, impurity CL-K, and known impurity peaks all met the requirements, satisfying the system suitability requirements.

[0129] (2) Injection precision

[0130] Accurately measure 20 μl of the mixed reference solution under the item "Specificity and system suitability", inject it 6 times continuously, record the chromatogram, and examine the RSD% value of the retention time and peak area of ​​each peak.

[0131] The results showed that after the solution was injected six times continuously, the RSD% values ​​of the retention time of each peak were all less than 1.0%, and the RSD% values ​​of the peak area were all less than 10%, indicating that the injection precision was good.

[0132] (3) Forced degradation test

[0133] Blank solvent: buffer solution (6.8 g / L potassium dihydrogen phosphate solution, pH adjusted to 6.8 with 10% sodium hydroxide solution)-mobile phase B (95:5).

[0134] Undamaged sample solution: Take an appropriate amount of this product, dissolve it in a solvent and quantitatively dilute it to make a solution containing about 2.5 mg per 1 ml, which is used as the undamaged sample solution.

[0135] Sample solution after destruction: Take an appropriate amount of this product and use acid, alkali, oxidation, high temperature and light to accelerate the destruction, and examine the specificity of the detection method of related substances of this product. The detailed test process is shown in Table 4.

[0136] Table 4. Method validation of related substances - forced degradation test method

[0137]

[0138]

[0139] Check the chromatographic conditions according to the relevant substances, accurately measure 20 μl of blank solvent and the above-mentioned destruction solution, inject them into the liquid chromatograph respectively, and detect them with the DAD detector. The destruction test results are shown in Tables 5 and 6.

[0140] Table 5. Method validation of related substances - forced degradation test results (self-made samples)

[0141]

[0142]

[0143] Table 6. Method validation of related substances - forced degradation test results (reference preparation)

[0144]

[0145]

[0146] Note: ND: Not Detected.

[0147] The material balance result is between 90% and 110%, which is the material balance. The calculation formula is as follows:

[0148]

[0149] Among them A r : total peak area after destruction; C r : Sample concentration after destruction;

[0150] A s : Total peak area is not destroyed; C s : No damage to sample concentration

[0151] Conclusion: (a) Blank solvent does not interfere with the determination; (b) Impurities produced by degradation of homemade and reference preparations do not interfere with the determination in the degradation test, and baseline separation is achieved between the main peak and adjacent chromatographic peaks; (c) The minimum peak purity threshold of the main peak is 1.000000; Material conservation is 90.69% to 106.26%. The results show that this method has good specificity.

[0152] (4) Linearity, range and correction factor

[0153] Accurately weigh appropriate amounts of cefazolin and each impurity reference substance, dissolve in solvent and quantitatively dilute to make a mixed solution containing approximately 250 μg of CL-M, CL-A, CL-Q, CL-N, CL-E, CL-H, CL-C, CL-G, CL-D, CL-I, CL-K, CL-L, CL-B and cefazolin per 1 ml, shake well and use as the linear stock solution.

[0154] ① Accurately measure 1 ml of linear solution No. ⑤ and place it in a 20 ml volumetric flask, add solvent to dilute to the scale, as the 0.05% linear solution;

[0155] ② Accurately measure 1 ml of the linear stock solution and place it in a 100 ml volumetric flask, add solvent to dilute to the mark, and use it as the 0.1% linear solution;

[0156] ③ Accurately measure 1 ml of the linear stock solution and place it in a 50 ml volumetric flask, add solvent to dilute to the scale, and use it as a 0.2% linear solution;

[0157] ④ Accurately measure 1 ml of the linear stock solution and place it in a 20 ml volumetric flask, add solvent to dilute to the scale, as a 0.5% linear solution;

[0158] ⑤ Accurately measure 1 ml of the linear stock solution and place it in a 10 ml volumetric flask, add solvent to dilute to the scale, and use it as a 1.0% linear solution;

[0159] ⑥ Accurately measure 3 ml of the linear stock solution and place it in a 20 ml volumetric flask, add solvent to dilute to the scale, as a 1.5% linear solution;

[0160] ⑦ Accurately measure 1 ml of the linear stock solution and place it in a 5 ml volumetric flask. Add solvent to dilute to the scale to obtain a 2.0% linear solution.

[0161] Accurately measure 20 μl of each of the above linear solutions and inject them into the liquid chromatograph respectively. Record the chromatogram and draw a linear curve with the peak area as the ordinate and the concentration (μg / ml) as the abscissa to examine the linearity of the main component and each impurity.

[0162] Linear data results Table 7.

[0163] Table 7. Method validation of related substances - linearity and range test results

[0164]

[0165]

[0166]

[0167]

[0168] Note: Ratio = intercept / (1.0% peak area) × 100%

[0169] The results show that the linear relationship between cefazolin and its impurities is good within the range of 0.05% to 2.0% of the test sample concentration. The calculation results of the correction factor are shown in Table 8.

[0170] Table 8. Method validation of related substances - linear correction factors

[0171]

[0172]

[0173] Note: f factor calculation formula:

[0174]

[0175] Where: K 主药 : Linear slope of the main component at 254nm wavelength;

[0176] K 杂质 : Linear slope of each impurity.

[0177] (5) Limit of detection and limit of quantification

[0178] Take appropriate amount of cefazolin reference solution and each known impurity stock solution, dilute them step by step with solvent to appropriate concentrations. When the signal-to-noise ratio is 10:1, the solution is used as the quantitative limit solution; when the signal-to-noise ratio is 3:1, the solution is used as the detection limit solution.

[0179] The results showed that the proposed inspection concentration of cefazolin sodium for injection was 2.5 mg / ml, and the concentrations of the detection limit solutions of each impurity and main component were all in the range of 0.03414-0.03499 μg / ml (detection limit: 0.68 ng-6.1 ng), equivalent to 0.001%-0.012% of the test sample concentration.

[0180] The concentrations of the quantitative limit solutions of each impurity and main component are all within the range of 0.07588-0.6099μg / ml (quantitative limit: 1.5ng-12.2ng), which is equivalent to 0.003%-0.024% of the test sample concentration, and the quantitative limit of each impurity is less than the reporting limit of 0.05%, indicating that the detection method of related substances is highly sensitive and suitable for impurity control.

[0181] (6) Repeatability

[0182] Test solution: Take about 25 mg of the test sample (batch 190815), weigh accurately, place in a 10 ml volumetric flask, dissolve with solvent and dilute to scale, shake well, and use as the test solution. Repeat 6 times.

[0183] Reference solution: Accurately weigh about 5 mg of cefazolin reference substance, place it in a 20 ml volumetric flask, dissolve it with solvent and dilute it to the scale, shake well, then accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute it to the scale with solvent, shake well, and use it as the reference solution.

[0184] According to the chromatographic conditions under the relevant substance inspection items, accurately measure 20 μl of the test solution and the reference solution, respectively, and inject them into the liquid chromatograph. Record the chromatograms and calculate the repeatability data using the principal component external standard method with the correction factor. The results are shown in Table 9.

[0185] Table 9. Method validation of related substances - repeatability test results

[0186]

[0187]

[0188] Note: ND: Not Detected.

[0189] The results showed that the method had good repeatability.

[0190] (7) Intermediate precision

[0191] On different dates, different experimenters used different instruments to conduct intermediate precision tests according to the relevant substance inspection methods in the draft quality standard of cefazolin sodium for injection, and calculated the results using the principal component external standard method with added correction factors.

[0192] The results show that the intermediate precision of this method is good. (8) Accuracy

[0193] Accuracy stock solution: Accurately weigh appropriate amounts of each impurity, dissolve in solvent and quantitatively dilute to make a mixed solution containing approximately 25 μg of CL-K, 125 μg of CL-M and CL-A, and 250 μg of CL-Q, CL-N, CL-E, CL-H, CL-C, CL-G, CL-D, CL-I, CL-L and CL-B per 1 ml. Shake well to prepare the accuracy stock solution.

[0194] Low concentration recovery sample solution (50%): Accurately weigh about 25 mg of the test sample (batch 190815), place in a 10 ml volumetric flask, accurately add 0.5 ml of the accuracy stock solution, dissolve with solvent and dilute to the scale, shake well, and use as the low concentration spiked test sample solution. Prepare 3 copies in parallel.

[0195] Medium concentration recovery sample solution (100%): Accurately weigh about 25 mg of the test sample (batch 190815), place in a 10 ml volumetric flask, accurately add 1 ml of the accuracy stock solution, dissolve with solvent and dilute to the scale, shake well, and use as the medium concentration spiked test sample solution. Prepare 3 copies in parallel.

[0196] High concentration recovery sample solution (150%): Accurately weigh about 25 mg of the test sample (batch 190815), place in a 10 ml volumetric flask, accurately add 1.5 ml of the accuracy stock solution, dissolve with solvent and dilute to the scale, shake well, and use as the high concentration spiked test solution. Prepare 3 copies in parallel.

[0197] Mixed reference solution: weigh about 5 mg of cefazolin reference substance, place in a 20 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, accurately measure 1 ml, place in a 10 ml volumetric flask, accurately add 1 ml of accurate stock solution, dilute to the scale with solvent, shake well, and use as the mixed reference solution.

[0198] According to the chromatographic conditions under the inspection item of related substance I, accurately measure 20 μl of the above-mentioned mixed reference solution and test solution, respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate the recovery test results by peak area according to the impurity external standard method.

[0199] The results showed that the recovery rate of this product was 87.29% to 114.81%; the RSD was less than 10.0%, and this method had high accuracy.

[0200] (9) Solution stability

[0201] Test sample solution: Accurately weigh about 25 mg of the test sample (batch 190815), place it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and use it as the test sample solution.

[0202] Mixed reference substance solution: Accurately weigh appropriate amounts of cefazolin and each impurity reference substance, dissolve in solvent and quantitatively dilute to make a mixed solution containing approximately 25 μg of cefazolin, 2.5 μg of impurity CL-K, 12.5 μg of CL-M and CL-A, and 25 μg of CL-Q, CL-N, CL-E, CL-H, CL-C, CL-G, CL-D, CL-I 2, CL-L and CL-B per 1 ml. Shake well and use as the mixed reference substance solution.

[0203] The above test solution and mixed reference solution were injected at room temperature and low temperature (4°C) to investigate the stability of the solution. 20 μl was accurately measured at each sampling point and injected into the liquid chromatograph, and the chromatogram was recorded to investigate the changes in peak area and impurity content (calculated by area normalization method).

[0204] The results showed that the test sample and mixed reference solution were unstable at room temperature or refrigerated at 4°C within 24 hours, so this product should be prepared fresh before use.

[0205] (10) Durability

[0206] According to the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Rules 9101, Validation of Analytical Methods for Pharmaceutical Quality Standards, the durability test was conducted. For the chromatographic conditions of this product, the durability was mainly investigated from the aspects of flow rate, column temperature, mobile phase pH value, chromatographic column, etc.

[0207] Test sample solution: Take about 25 mg of the test sample (batch 190815), accurately weigh it, put it in a 10 ml volumetric flask, dissolve it with solvent and dilute it to the scale, shake it well, and use it as the test sample solution.

[0208] Mixed reference substance solution: Accurately weigh appropriate amounts of cefazolin and each impurity reference substance, add appropriate amount of solvent to dissolve and quantitatively dilute to make a mixed solution containing approximately CL-M 12.5μg, CL-A 12.5μg, CL-Q 25μg, CL-N 25μg, CL-E25μg, CL-H 25μg, CL-C 25μg, CL-G 25μg, CL-D 25μg, CL-I 25μg, CL-K 2.5μg, CL-L 25μg, CL-B 25μg and cefazolin 25μg per 1ml. Shake well to prepare the mixed reference substance solution.

[0209] 1) Investigation of different flow rates

[0210] The detection conditions for related substances of this product stipulate that the flow rate is 1.5ml / min. The flow rate was adjusted to 1.3ml / min and 1.7ml / min, and the other conditions remained unchanged.

[0211] Accurately measure 20 μl of blank solvent, test solution, and mixed reference solution, respectively, and inject them into the liquid chromatograph. Record the chromatogram. The durability results at different flow rates are shown in Table 12.

[0212] Table 12. Method validation of related substances - Durability - Test results of test solution at different flow rates

[0213]

[0214]

[0215] Note: ND: Not Detected.

[0216] Conclusion: Under different flow rate conditions, the separation of each peak meets the requirements, and the impurity content and total amount of impurities in the test solution are basically consistent. This method has good flow rate durability.

[0217] 2) Investigation of different column temperatures

[0218] The column temperature is specified to be 30°C in the detection conditions of related substances of this product. The column temperature was adjusted to 28°C and 35°C, and the other conditions remained unchanged.

[0219] Accurately measure 20 μl of blank solvent, test solution, and mixed reference solution, respectively, and inject them into the liquid chromatograph. Record the chromatogram. The test results at different column temperatures are shown in Table 13.

[0220] Table 13. Method validation of related substances - Durability - Determination results of test solution at different column temperatures

[0221] 28℃ 30℃ 35℃ Minimum resolution (mixed reference solution) 2.01 2.05 2.92 CL-Q(%) <LOQ <LOQ <LOQ CL-N(%) 0.19 0.16 0.16 CL-M(%) ND ND ND CL-E(%) 0.41 0.32 0.30 CL-H(%) ND ND ND CL-C(%) ND ND ND CL-G(%) 0.13 0.07 0.07 CL-A(%) ND ND ND CL-I (%) ND ND ND CL-L(%) ND ND ND CL-B(%) ND ND ND CL-D(%) ND ND ND CL-K(%) ND ND ND Other individual impurities (%) 0.10 0.08 0.06 Total impurities (%) 0.95 0.74 0.70 Number of impurities 7 7 7

[0222] Note: ND: Not Detected.

[0223] Conclusion: Under different column temperature conditions, the separation of each peak meets the requirements, and the impurity content and total impurity amount in the test solution are basically consistent. This method has good durability to column temperature.

[0224] 3) Investigation of different mobile phase pH values

[0225] In the detection conditions of the related substances of this product, the pH value of the mobile phase is 6.8. The pH value of the mobile phase was adjusted to 6.6 and 6.9, and the other conditions remained unchanged.

[0226] Accurately measure 20 μl of blank solvent, test solution, and mixed reference solution, respectively, and inject them into the liquid chromatograph. Record the chromatogram. The durability results of different mobile phase pH are shown in Table 14.

[0227] Table 14. Method validation of related substances - Durability - Test results of test solutions at different mobile phase pH values

[0228] pH 6.6 pH 6.8 pH6.9 Minimum resolution (mixed reference solution) 3.36 2.05 1.15 CL-Q(%) <LOQ <LOQ <LOQ CL-N(%) 0.18 0.16 0.16 CL-M(%) ND ND ND CL-E(%) 0.33 0.32 0.35 CL-H(%) ND ND ND CL-C(%) ND ND ND CL-G(%) 0.07 0.07 0.12 CL-A(%) ND ND ND CL-I (%) ND ND ND CL-L(%) ND ND ND CL-B(%) ND ND ND CL-D(%) ND ND ND CL-K(%) ND ND ND Other individual impurities (%) 0.08 0.08 0.08 Total impurities (%) 0.78 0.74 0.83 Number of impurities 7 7 7

[0229] Note: ND: Not Detected.

[0230] Conclusion: Under different mobile phase pH conditions, the separation of each peak meets the requirements, and the impurity content and total impurity amount in the test solution are basically consistent. This method has good durability for mobile phase pH value.

[0231] 4) Investigation of different chromatographic columns

[0232] The chromatographic column used in the detection of related substances in this product is YMC-PackPro C 18 (250 mm × 4.6 mm, 5 μm) replaced with chromatographic column 1 (different brand) Wondasil C 18 Superb (250 mm × 4.6 mm, 5 μm) and chromatographic column 2 (same brand, different batch number). Other conditions remain unchanged.

[0233] Accurately measure 20 μl of blank solvent, test solution, and mixed reference solution, respectively, and inject them into the liquid chromatograph. Record the chromatogram. The durability results of different chromatographic columns are shown in Table 15.

[0234] Table 15. Method validation of related substances - Durability - Determination results of test solutions on different chromatographic columns

[0235] Column 1 <![CDATA[YMC-Pack Pro C 18 ]]> Column 2 Minimum resolution (mixed reference solution) 1.91 2.05 3.19 CL-Q(%) <LOQ <LOQ <LOQ CL-N(%) 0.11 0.16 0.10 CL-M(%) ND ND ND CL-E(%) 0.27 0.32 0.31 CL-H(%) ND ND ND CL-C(%) ND ND ND CL-G(%) 0.07 0.07 0.07 CL-A(%) ND ND ND CL-I (%) ND ND ND CL-L(%) ND ND ND CL-B(%) ND ND ND CL-D(%) ND ND ND CL-K(%) ND ND ND Other individual impurities (%) 0.08 0.08 0.10 Total impurities (%) 0.63 0.74 0.68 Number of impurities 7 7 7

[0236] Note: Column 1 is the intermediate precision data result.

[0237] Conclusion: Under the conditions of different brands of chromatographic columns and different batches of chromatographic columns of the same brand, the separation of each peak meets the requirements, and the content of each impurity and the total amount of impurities in the test solution are basically the same. This method has good durability for chromatographic columns of the same brand but different batches and different brands.

[0238] Example 3: Detection of related substances in cefazolin samples

[0239] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (YMC-Pack ProC 18 4.6mm×250mm, 5μm or chromatographic column with equivalent performance); mobile phase A is 6.8g / L potassium dihydrogen phosphate solution (adjust pH to 6.8 with 10% sodium hydroxide solution)-mobile phase B (95:5), mobile phase B is 6.8g / L potassium dihydrogen phosphate solution-acetonitrile (1:1); flow rate is 1.5ml per minute; linear gradient elution is performed according to the table below; column temperature is 30℃; detection wavelengths are 210nm and 254nm; injection volume is 20μl.

[0240]

[0241]

[0242] Take an appropriate amount of the product for test solution, weigh it accurately, add mobile phase A to dissolve it and quantitatively dilute it to make a solution containing about 2.5 mg per 1 ml.

[0243] Reference solution: Accurately weigh an appropriate amount of cefazolin reference substance, dissolve it in mobile phase A and quantitatively dilute it to make a solution containing approximately 25 μg per 1 ml.

[0244] System suitability solution Take an appropriate amount of this product, dissolve and dilute with 0.05 mol / L sodium hydroxide solution to make a solution containing about 2 mg per 1 ml, and let it stand at room temperature for 5 minutes (sodium hydroxide solution will degrade to produce cefazolin isomer impurity CL-L), accurately measure 1 ml, place in a 25 ml volumetric flask, add mobile phase A to dilute to the scale, and shake well.

[0245] System suitability requirements: In the system suitability solution chromatogram, the separation between the cefazolin peak and the impurity CL-L peak should be no less than 8.0 (254nm).

[0246] Determination method: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0247] Limits: If there are impurity peaks in the chromatogram of the test solution, the detection wavelength, relative retention time, correction factor and limit of each impurity peak are shown in Table 16 below. The total amount of impurities shall not exceed 3.5%, and any peak in the chromatogram of the test solution that is less than 0.05 times the main peak area of ​​the reference solution shall be ignored.

[0248] Table 16

[0249] Compound code Detection wavelength (nm) Relative retention time Correction Factor limit(%) CL-Q 210 0.07 2.5 1.0 CL-N 210 0.08 3.0 1.0 CL-M 254 0.18 1.4 0.5 CL-E 254 0.19 1.4 1.0 CL-H 254 0.35 1.0 1.0 CL-C 254 0.41 1.2 1.0 CL-G 254 0.50 1.5 1.0 CL-D 254 0.62 1.1 1.0 CL-A 254 0.68 0.8 0.5 CL-I 254 0.79 1.3 1.0 Cefazolin 254 1.00 - - CL-K 254 1.10 1.1 0.1 CL-L 254 1.25 1.0 1.0 CL-B 254 1.52 1.1 1.0 Other single impurities 254 - 1.0 0.5

[0250] Two batches of reference preparations and three batches of verification samples were taken, and the above-mentioned related substances were inspected according to the above-mentioned methods. The sample test results are shown in Table 16.

[0251] Table 16. Test results of related substances in homemade samples and reference preparation samples

[0252]

[0253] Note: ND: Not Detected

[0254] Conclusion: From the test results of related substances in homemade and reference samples, it can be seen that the known impurities, other single impurities and total amount of impurities detected in the three batches of samples and reference preparation samples all meet the requirements.

Claims

1. A method for detecting related substances of cefazolin sodium, comprising the following steps: using high performance liquid chromatography to detect the cefazolin sodium to be tested, and the chromatographic conditions include: The chromatographic column is C 18 Chromatographic columns; The mobile phase was: the volume ratio of mobile phase A to mobile phase B was 95:5; Mobile phase A was phosphate buffer (6.8 g / L potassium dihydrogen phosphate solution, pH adjusted to 6.6-6.9 with 10% sodium hydroxide solution); Mobile phase B was phosphate solution (6.8 g / L potassium dihydrogen phosphate solution)-acetonitrile (1:1). Perform gradient elution as shown in the table below.

2. The method for detecting related substances of cefazolin sodium according to claim 1, characterized in that: The cefazolin sodium test product is a cefazolin sodium bulk drug or a cefazolin sodium preparation; And / or, before the detection, the cefazolin sodium test product is pretreated, specifically: the cefazolin sodium test product is dissolved in a diluent and left to stand.

3. The method for detecting related substances of cefazolin sodium according to claim 1, characterized in that: The chromatographic column is YMC-Pack Pro C 18 (4.6mm*250mm,5μm) chromatographic column, or Wondasil C 18 Superb (250mm×4.6mm, 5μm) chromatographic column, or chromatographic column with equivalent performance; Preferably, the chromatographic column is YMC-Pack Pro C 18 4.6mm*250mm, 5μm chromatographic column.

4. The method for detecting related substances of cefazolin sodium according to claim 1, characterized in that: The pH value of the mobile phase is 6.6, 6.8 or 6.9, and the most preferred pH value of the mobile phase is 6.

8.

5. The method for detecting related substances of cefazolin sodium according to claim 1, characterized in that: The detection method meets one or more of the following conditions: (1) The flow rate of the mobile phase is 1.3 ml / min to 1.7 ml / min; (2) The detector is a DAD or UV detector; (3) The detection wavelength is 210nm and 254nm; (4) The column temperature of the chromatographic column is 28°C to 35°C; (5) The injection volume is 10-20 μL.

6. The method for detecting related substances of cefazolin sodium according to claim 5, characterized in that: The detection method meets one or more of the following conditions: (1) The flow rate of the mobile phase is 1.3 ml / min, 1.5 ml / min or 1.7 ml / min; (2) The detector is a DAD or UV detector; (3) The detection wavelength is 210nm and 254nm; (4) The column temperature is 28°C, 30°C or 35°C; (5) The injection volume is 20 μL.

7. The method for detecting related substances of cefazolin sodium as claimed in claim 5, characterized in that: The detection method meets one or more of the following conditions: (1) The flow rate of the mobile phase is 1.5 ml / min; (2) The detector is a DAD or UV detector; (3) The detection wavelength is 210nm and 254nm; (4) The column temperature is 30°C; (5) The injection volume is 20 μL.

8. The method for detecting related substances of cefazolin sodium according to claim 1, characterized in that: Solution preparation: Solvent: mobile phase A; Test solution: Take about 25 mg of the product, weigh accurately, put it in a 10 ml volumetric bottle, add an appropriate amount of solvent, shake to dissolve, dilute to the scale with solvent, shake well, and use it as the test solution; Reference substance solution: Take an appropriate amount of cefazolin reference substance, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing about 25 μg per 1 ml as the reference substance solution; System suitability solution: Take about 20 mg of this product, place it in a 10 ml volumetric flask, shake with 0.05 mol / L sodium hydroxide solution to dissolve and dilute to the scale, leave it at room temperature for 5 minutes, take 1 ml, place it in a 25 ml volumetric flask, dilute to the scale with solvent, shake well, and use it as the system suitability solution; Determination method: Accurately measure 20 μl of system suitability solution, test solution and reference solution, respectively, inject them into liquid chromatograph, and record the chromatogram.

9. The method for detecting related substances of cefazolin sodium according to claim 1, characterized in that: System suitability: Accurately measure 20 μl of system suitability solution, inject it into the liquid chromatograph, and record the chromatogram; the retention time of the cefazolin peak is about 32 minutes, and the separation degree between the cefazolin peak and the cefazolin-related substances (CL-L) should be no less than 8.0 (254 nm); If there are impurity peaks in the chromatogram of the test solution, determine the detection wavelength and attribution of each peak according to the table below and calculate by the main component external standard method with correction factor. Impurities CL-A and CL-M shall not exceed 0.5%, impurity CL-K shall not exceed 0.1%, impurities CL-B, CL-C, CL-D, CL-E, CL-G, CL-H, CL-I, CL-L, CL-N, and CL-Q shall not exceed 1.0%, other single unknown impurities shall not exceed 0.5%, and the total amount of impurities shall not exceed 3.5%. Peaks in the chromatogram of the test solution that are less than 0.05 times the main peak area of ​​the reference solution shall be ignored; Calculation formula: Where As(210) is the peak area of ​​impurities CL-Q and CL-N in the test solution at a wavelength of 210 nm; Wr is the weight of the reference substance, mg; Ar(254) is the peak area of ​​the main component in the reference solution at a wavelength of 254 nm; Ws is the weight of the test sample, mg; Sr is the dilution multiple of the reference substance; Ss is the dilution multiple of the test product; P is the content of the reference substance; f is the correction factor for relative impurities. Total impurities (%) = ∑ impurities (%) Where: As(254) is the peak area of ​​each impurity in the test solution at a wavelength of 254 nm; Wr is the weight of the reference substance, mg; Ar(254) is the peak area of ​​the main component in the reference solution at a wavelength of 254 nm; Ws is the weight of the test sample, mg; Sr is the dilution multiple of the reference substance; Ss is the dilution multiple of the test product; P is the content of the reference substance; f is the correction factor for relative impurities.

10. The detection method according to claims 1-9, characterized in that: Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (YMC-Pack ProC 18 4.6mm×250mm, 5μm or equivalent chromatographic column); mobile phase A is 6.8g / L potassium dihydrogen phosphate solution (adjust pH to 6.8 with 10% sodium hydroxide solution)-mobile phase B (95:5), mobile phase B is 6.8g / L potassium dihydrogen phosphate solution-acetonitrile (1:1); flow rate is 1.5ml per minute; linear gradient elution is performed according to the table below; column temperature is 30℃; detection wavelengths are 210nm and 254nm; injection volume is 20μl; Test solution: Take an appropriate amount of the product, weigh accurately, add mobile phase A to dissolve and quantitatively dilute to make a solution containing about 2.5 mg per 1 ml; Reference solution: Accurately weigh an appropriate amount of cefazolin reference substance, dissolve it in mobile phase A and quantitatively dilute it to make a solution containing about 25 μg per 1 ml; System suitability solution: Take an appropriate amount of this product, dissolve it with 0.05mol / L sodium hydroxide solution and dilute it to make a solution containing about 2mg per 1ml, leave it at room temperature for 5 minutes (sodium hydroxide solution will degrade to produce cefazolin isomer impurity CL-L), accurately measure 1ml, place it in a 25ml volumetric flask, add mobile phase A to dilute to the scale, and shake well; System suitability requirements: In the system suitability solution chromatogram, the separation between the cefazolin peak and the impurity CL-L peak should be no less than 8.0 (254 nm); Determination method: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram; Limits: If there are impurity peaks in the chromatogram of the test solution, the detection wavelength, relative retention time, correction factor and limit of each impurity peak are shown in the table below; the total amount of impurities shall not exceed 3.5%, and any peak in the chromatogram of the test solution that is less than 0.05 times the main peak area of ​​the reference solution shall be ignored;

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