Method for improving quality control of cefditoren pivoxil granules

Abstract

The invention relates to a method for improving the quality control of cefditoren pivoxil granules. The method comprises injecting a cefditoren pivoxil granule sample solution into a high performance liquid chromatograph for analysis to detect impurities in the cefditoren pivoxil granules, wherein the high performance liquid chromatograph adopts a mixed solution of ammonium formate solution, acetonitrile and methanol (43-47): (23-27): (23-27) as mobile phase A, adopts a mixed solution of ammonium formate solution, acetonitrile and methanol (43-47): (53-57): (53-57) as mobile phase B, and adopts a gradient for elution, and wherein the cefditoren pivoxil granule sample solution adopts mobile phase A as a diluent for preparation. The method of the invention improves the front extension phenomenon of the cefditoren pivoxil open-ring impurity peak and the separation degree of the unknown impurity peak in the cefditoren pivoxil granules, has good reproducibility and high accuracy, thereby improving the quality control level of the cefditoren pivoxil granules.

Description

Technical Field

[0001] The present invention belongs to the field of drug analysis. Specifically, the present invention relates to a method for improving the quality control of cefditoren pivoxil granules, which comprises injecting a sample solution of cefditoren pivoxil granules into a high performance liquid chromatograph for analysis to detect impurities in the cefditoren pivoxil granules. Background Art

[0002] The chemical name of cefditoren pivoxil is 2,2-dimethylpropionyloxymethyl (6R,7R)-7-[(Z)-2-(2-amino-4-thiazolyl)-2-methoxyiminoacetylamino]-3-[(Z)-2-(4-methyl-1,3-thiazol-5-yl)vinyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate, and has the following structural formula:

[0003] .

[0004] After being absorbed by the human body, cefditoren pivoxil is metabolized into cefditoren in the intestinal wall and exerts antibacterial activity. It has strong antibacterial activity against Staphylococcus, Streptococcus, Peptostreptococcus, Branhamella catarrhalis, Propionibacterium acnes, Escherichia coli, Citrobacter, Klebsiella, Enterobacter, Serratia, Proteus (Proteus mirabilis, Proteus vulgaris), Morganella, Providencia, Haemophilus influenzae, Bacteroides, etc., and has a good therapeutic effect on infections caused by these bacteria. However, cefditoren pivoxil granules also have many adverse reactions. For example, allergic reactions: rash, itching, urticaria and fever, etc.; digestive system: nausea, vomiting, diarrhea, and like other broad-spectrum antibiotics, rare pseudomembranous colitis may occur; blood system: eosinophilia, leukopenia, etc.; there have been reports of positive Coombs' reaction when cephalosporins were used for treatment; renal function: occasional increases in blood urea nitrogen (BUN) and serum creatinine; liver function: sometimes increases in aspartate aminotransferase (GOT), alanine aminotransferase (GPT), alanine aminotransferase (ALT), alkaline phosphatase Al-P, etc.

[0005] Adverse drug reactions are sometimes not only caused by the drug itself, but also by impurities in the drug. Therefore, it is very important to control impurities in the drug, which has a great impact on the quality of the drug.

[0006] Obtaining high-quality drugs has always been the pursuit of mankind. Excellent quality means that the purity of active substances is high, and the types and contents of impurities are as low as possible to ensure the safety and effectiveness of the drugs while avoiding unexpected effects. However, the existing methods cannot completely identify all unknown impurities or cannot detect the related substances contained in cefditoren pivoxil granules with high separation, which is not conducive to controlling the quality of drugs. Therefore, it is now necessary to further improve the analysis method of related substances of cefditoren pivoxil granules to improve the quality control level of cefditoren pivoxil granules. Summary of the invention

[0007] To solve the above problems, the present invention provides a method for improving the quality control of cefditoren pivoxil granules, the method comprising:

[0008] The sample solution of cefditoren pivoxil granules is injected into a high performance liquid chromatograph for analysis to detect impurities in the cefditoren pivoxil granules, wherein mobile phase A and mobile phase B are used as eluents for gradient elution, wherein mobile phase A is a mixture of ammonium formate solution, acetonitrile, and methanol in a volume ratio of (43-47):(23-27):(23-27), and mobile phase B is a mixture of ammonium formate solution, acetonitrile, and methanol in a volume ratio of (43-47):(53-57):(53-57),

[0009] Gradient elution was used. In the gradient elution program, within 0-5 min, the volume of mobile phase A was 100% and that of mobile phase B was 0%; within 5 to 25 min, the volume of mobile phase A changed from 100% to 0%, and at the same time, the volume of mobile phase B changed from 0% to 100%; then the volume of mobile phase A was kept at 0% and the volume of mobile phase B was kept at 100% for 17-19 min.

[0010] The cefditoren pivoxil granules are dissolved using mobile phase A as a diluent to prepare a solution, which is then injected into a high performance liquid chromatograph for analysis.

[0011] Preferably, in the method of injecting the cefditoren pivoxil granules sample solution into a high performance liquid chromatograph for analysis to detect impurities in the cefditoren pivoxil granules, the gradient shown in the following Table 1 is used for elution.

[0012] Table 1

[0013] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 5 100 0 25 0 100 42-44 0 100 47-49 99-100 0-1 64-66 99-100 0-1 .

[0014] Unexpectedly, it was found that by changing the diluent used to dilute and prepare the cefditoren pivoxil granule sample and by improving the elution gradient in the high performance liquid chromatography method for detecting the related substances of the cefditoren pivoxil granule sample, a method for detecting related substances that is superior to the prior art can be provided, thereby improving the quality control of the cefditoren pivoxil granules. The advantages of the method include improving the front extension phenomenon of the cefditoren pivoxil open-ring impurity peak in the cefditoren pivoxil granules, identifying and accurately detecting unknown impurity peaks, good reproducibility, high accuracy, and especially improving the cefditoren pivoxil open-ring impurity peak, which can allow more accurate control of impurities in the cefditoren pivoxil granules, and improve the quality level of the cefditoren pivoxil granules, as shown in the examples of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a chromatogram obtained by using 75% acetonitrile-water mixture as a diluent to prepare the impurity mixed solution of cefditoren pivoxil granules in Example 1.

[0016] Figure 2 This is a chromatogram obtained by using mobile phase A as a diluent to prepare an impurity mixed solution of cefditoren pivoxil granules in Example 2.

[0017] Figure 3 This is a chromatogram obtained by analyzing the impurity mixed solution of Cefditoren Pivoxil Granules using the method of the present invention (chromatographic condition 1) in Example 3.

[0018] Figure 4 This is a chromatogram obtained by analyzing the impurity mixed solution of Cefditoren Pivoxil Granules using the standard method (chromatographic condition 2) in Example 4. DETAILED DESCRIPTION

[0019] The specific implementation of the present invention is described in detail below. The following description is for better illustrating the present invention, but not for limiting the present invention.

[0020] In this application, all numerical values ​​used to express an amount, percentage or ratio are understood to be modified by the term "about" in all cases. The term "about" as used herein may include a range of ±5%, ±4%, ±3%, ±2%, ±1% or ±0.5% of the numerical value.

[0021] The quality of drugs not only determines the effect of disease prevention and treatment but also directly affects the safety of patients. Therefore, a clinical drug should have qualified quality to ensure safe and effective use. Quality control of quality control products should include safety, effectiveness, and controllability. Impurities are the key quality attributes of drugs and can affect the safety and effectiveness of products. Impurities in drug quality standards refer to impurities introduced by the production process or raw materials of drugs produced by processes and raw materials approved in accordance with the law, or impurities generated during storage. They do not include new impurities generated by changes in production processes or raw materials, nor do they include foreign substances that are added or contaminated. Drug impurities are usually divided into: organic impurities, inorganic impurities, and residual solvents. Organic impurities can be introduced during the production or storage of drugs, or they can be generated by the interaction between drugs and excipients or packaging structures. These impurities may be identified or unidentified, volatile or non-volatile. Among them, organic impurities with chemical structures similar to or related to active ingredients are usually called related substances. The impurities in cefditoren pivoxil granules involved in this article are organic impurities with chemical structures similar to or related to active ingredients, that is, related substances.

[0022] According to the "Imported Drug Registration Standard" standard number JX20090123, the quality standards of cefditoren pivoxil granules should include the following:

[0023] A. Properties;

[0024] B. Identification: Determined by UV-visible spectrophotometry (Appendix IVA of the 2010 edition of the Chinese Pharmacopoeia), the maximum absorption is at a wavelength of 231±2nm;

[0025] C. Inspection: (1) Loss on drying: loss of weight shall not exceed 3.0%; (2) Solubility; (3) Related substances; (4) Microbial limit; (5) Particle size;

[0026] D. Content determination.

[0027] In the quality control of Cefditoren Pivoxil Granules, the limit and control of related substances are very important contents under the inspection items. Improving the detection method of related substances makes the detected content of related substances more accurate, which helps to better control the individual content and overall content of related substances in Cefditoren Pivoxil Granules, thereby improving the quality control of Cefditoren Pivoxil Granules.

[0028] As described above, the present invention provides a method for improving the quality control of cefditoren pivoxil granules. The various method parameters involved in the method are described in more detail below.

[0029] In some preferred embodiments, mobile phase A may be a mixture of ammonium formate solution, acetonitrile, and methanol in a volume ratio of (44-46): (24-26): (24-26), and mobile phase B may be a mixture of ammonium formate solution, acetonitrile, and methanol in a volume ratio of (44-46): (54-56): (54-56). More commonly, mobile phase A is a mixture of ammonium formate solution, acetonitrile, and methanol in a volume ratio of 45:25:25, and mobile phase B is a mixture of ammonium formate solution, acetonitrile, and methanol in a volume ratio of 45:55:55.

[0030] The ammonium formate solution is an aqueous solution having an ammonium formate concentration of 1.56-1.60 g / l, and the aqueous solution is adjusted to pH 4.3-4.7, preferably to pH 4.4-4.6, preferably to pH 4.5, with diluted formic acid. The diluted formic acid solution can be an aqueous solution obtained by diluting formic acid 80-120 times, preferably 100 times, with water. Specifically, the ammonium formate solution can be prepared as follows: 1.58 g of ammonium formate is dissolved in 900 ml of water, the pH is adjusted to 4.5 with diluted formic acid (1→100), and then diluted to 1000 ml with water.

[0031] In some embodiments, elution can be performed using an elution gradient as shown in Table 2 below:

[0032] Table 2

[0033] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 5 100 0 25 0 100 43 0 100 48 100 0 65 100 0 .

[0034] In the process of researching and developing the method for detecting impurities in cefditoren pivoxil granules, it is now found that the cefditoren pivoxil open-ring impurity peak has a forward extension phenomenon. After various tests and various attempts, it is finally found that this problem can be solved by changing the diluent used to dilute the cefditoren pivoxil granule sample. Therefore, the method provided by the present invention solves the forward extension phenomenon of the cefditoren pivoxil open-ring impurity peak, improves the peak shape of the impurity peak, and makes the impurity content determination more accurate. In addition, the method provided by the present invention is also very reproducible. In addition, by improving the elution gradient, the method of the present invention can more accurately detect more unknown impurities. Since these unknown impurities are also calculated when processing the relevant impurity peaks in the chromatographic software, therefore, the improvement of the elution gradient can eventually make the content result of the relevant impurities obtained finally more accurate, thereby improving the quality control level of cefditoren pivoxil granules.

[0035] In the method described in the present application, octadecylsilane bonded silica gel is used as the stationary phase.

[0036] In the method described in the present application, an ultraviolet detector is used for detection, and the detection wavelength is 254 nm.

[0037] In the method described in the present application, the injection volume can be 8-15 μl, preferably 9-12 μl, more preferably 10 μl, and the measurement sample of cefditoren pivoxil particles can be a solution diluted with mobile phase A with a concentration of 0.5-1.1 mg / ml, preferably 0.7-0.9 mg / ml, more preferably 0.8 mg / ml; the mobile phase flow rate can be 0.8-1.2 ml / min, preferably 0.9-1.1 ml / min, most preferably 1 ml / min; the column temperature can be 20-50°C, preferably 25-45°C, more preferably 35-42°C; the elution time exceeds 44 min, preferably does not exceed 70 min, and is more commonly 50-65 min.

[0038] In the method described in the present application, it is preferred to use an impurity / ghost peak capture column. The capture column, also known as a capture column, can be installed between the gradient mixer and the injector of the high performance liquid chromatograph to adsorb and remove impurities with weaker polarity in the system, such as impurities in the mobile phase and impurities in the pipeline and mixer, thereby preventing the impurity peaks in the system from interfering with the target peak. The injected sample will not flow through the capture column. It is preferred to use a capture column with a model of Chromasir® Ghost-Sniper Column and a specification of 4.6mm×50mm purchased from Motion Analysis Scientific Instruments (Suzhou) Co., Ltd. or a capture column with equivalent performance.

[0039] When using the method of the present invention to detect the related impurities of cefditoren pivoxil granules, if the content of cefditoren acid and cefditoren acid △3 isomer is above 0.30%, the related substance II experiment is performed. The related substance II experiment can refer to the method of related substance ② in the standard number JX20090123 included in the "Imported Drug Registration Method". The details are as follows:

[0040] Chromatographic conditions and system suitability test:

[0041] Octadecylsilane bonded silica gel is used as filler; ammonium acetate solution (take 1.0g of ammonium acetate and dissolve it in water to 1000ml)-methanol (66:34) is used as mobile phase; the flow rate is 1.0ml / min; the detection wavelength is 230nm; the column temperature is 25℃. Take 10 microliters of the reference solution and inject it into the liquid chromatograph, record the chromatogram, and the peaks are emitted in the order of cefditoren and aminopyrine. The separation between the two peaks should be not less than 4.0. Repeat the injection, and the relative standard deviation (RSD) of the ratio of the cefditoren peak area to the aminopyrine peak area shall not exceed 1.5%.

[0042] Determination of related substances:

[0043] Accurately weigh about 40 mg of cefditoren pivoxil sample, place it in a 50 ml volumetric flask, shake vigorously with a small amount of 80% methanol aqueous solution, accurately add 10 ml of internal standard solution (take 0.1 ml of aminopyrine, dilute to 1000 ml with water, the concentration is about 0.01%), dilute to the scale with 80% methanol aqueous solution, shake well, filter, and take the filtrate as the sample solution; accurately weigh about 10 mg of cefditoren reference substance, place it in a 50 ml volumetric flask, dissolve it in water and dilute to the scale, shake well, accurately measure 2.5 ml, place it in a 50 ml volumetric flask, accurately add 10 ml of internal standard solution, dilute to the scale with 80% methanol aqueous solution, shake well, as the reference substance solution. Accurately measure 10 microliters of sample solution and reference substance solution respectively, inject them into liquid chromatograph, record the chromatogram, and calculate the content of P7 and cefditoren according to the following formula:

[0044] P7 content%=(Q T ×W S × sample dilution factor × 1000 × 1.1 × 100%) / (Q S ×W T × reference substance dilution multiple)

[0045] Cefditoren content%=(Q T ×W S × sample dilution factor × 1000 × 100%) / (Q S ×W T × reference substance dilution multiple)

[0046] Where Q T Q is the ratio of the peak area of ​​the impurity to be detected in the sample solution to the peak area of ​​aminopyrine. S W is the ratio of the main peak area to the aminopyrine peak area in the reference solution. T , W S are the weights of the sample and reference substance respectively, and 1.1 is the correction factor for cefditoren. The content of P7 and cefditoren shall not exceed 0.3%.

[0047] The solutions of each substance in the above test were prepared as follows:

[0048] Internal standard solution: Take an appropriate amount of aminopyrine, dissolve it in water and dilute it to make a solution containing approximately 0.5 mg of aminopyrine per 1 ml.

[0049] Reference substance stock solution: Take an appropriate amount of cefditoren acid, dissolve it in water and prepare a solution containing approximately 0.20 mg of cefditoren acid per 1 ml.

[0050] Reference solution: Accurately measure 1 ml of reference stock solution and 0.4 ml of internal standard solution into a 20 ml volumetric flask, dilute to the mark with 80% methanol water, and shake well to prepare the reference solution.

[0051] In the method for improving the quality control of impurities in cefditoren pivoxil granules according to the present invention, the related substances of cefditoren pivoxil granules that can be detected are as follows:

[0052] (1) Cefditoren acid

[0053] CAS No.: 104145-95-1

[0054] Chemical structure:

[0055] ;

[0056] (2) Cefditoren acid △3 isomer

[0057] Molecular formula: C 19 H 18 N6O5S3

[0058] Molecular weight: 506.60

[0059] Chemical name: (6R,7R)-7-[(Z)-2-(2-amino-1,3-thiazol-4-yl)-2-(methoxyimino)acetylamino]-3-[(Z)-2-(4-methyl-1,3-thiazol-5-yl)vinyl]-8-oxo-5-thiazolyl[4.2.0]oct-3-ene-2-carboxylic acid

[0060] Chemical structure:

[0061] ;

[0062] (3) Cefditoren Pivoxil Open-ring Impurities

[0063] Molecular formula: C 25 H 30 N6O8S3

[0064] Molecular weight: 638.76

[0065] Chemical name: 2-((Z)-2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido)-2-(5-((Z)-2-(4-methylthiazol-5-yl)vinyl)-4-((pentanoyloxy)methoxy)carbonyl)-3,6-dihydro-2H-1,3-thiazin-2-yl)acetic acid

[0066] Chemical structure:

[0067] ;

[0068] (4) Cefetamet Pivoxil Hydrochloride

[0069] CAS No.: 111696-23-2

[0070] Chemical structure:

[0071] ;

[0072] (5) Cefditoren pivoxil △3 isomer

[0073] CAS No.: 148774-47-4

[0074] Chemical structure:

[0075] ;

[0076] (6) Methoxymethyl cefditoren pivoxil

[0077] Molecular formula: C 27 H 32 N6O8S3

[0078] Molecular weight: 664.78

[0079] Chemical name: (Valeryloxy)methyl (6R, 7R)-7-((Z)-2-(methoxyimino)-2-(2-((methoxymethyl)amino)thiazol-4-yl)acetylamino)-3-((Z)-2-(4-methylthiazol-5-yl)vinyl)-8-oxo-5-thioxo-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

[0080] Chemical structure:

[0081] ;

[0082] (7) Cefditoren pivoxil 3E isomer

[0083] CAS No.: 138514-32-6

[0084] Chemical structure:

[0085] ;

[0086] (8) Cefditoren Pivoxil Impurity P6

[0087] Molecular formula: C 28 H 33 N7O7S3

[0088] Molecular weight: 675.79

[0089] Chemical name: (Valeryloxy)methyl (6R,7R)-7-((2Z)-2-(2-((dimethylamino)methylene)amino)thiazol-4-yl)-2-(methoxyimino)acetylamino)-3-((Z)-2-(4-methylthiazol-5-yl)vinyl)-8-oxo-5-thioxo-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

[0090] Chemical structure:

[0091] ;

[0092] (9) Cefditoren Dipivoxil

[0093] Molecular formula: C 31 H 38 N6O9S3

[0094] Molecular weight: 734.86

[0095] Chemical name: (Valeryloxy)methyl (6R, 7R)-7-((Z)-2-(methoxyimino)-2-(2-((Valeryloxy)methyl)amino)thiazol-4-yl)acetylamino)-3-((Z)-2-(4-methylthiazol-5-yl)vinyl)-8-oxo-5-thioxo-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

[0096] Chemical structure:

[0097] ;

[0098] (10) Cefditoren Pivoxil-Pivamide

[0099] Molecular formula: C 30 H 36 N6O8S3

[0100] Molecular weight: 704.86

[0101] Chemical name: (Valeryloxy)methyl (6R, 7R)-7-((Z)-2-(methoxyimino)-2-(2-thiazol-4-yl)acetylamino)-3-((Z)-2-(4-methylthiazol-5-yl)vinyl)-8-oxo-5-thioxo-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

[0102] Chemical structure:

[0103] ;

[0104] (11) N-methoxybenzyl cefditoren pivoxil

[0105] Molecular formula: C33 H 36 N6O8S3

[0106] Molecular weight: 740.89

[0107] Chemical name: (Valeryloxy)methyl (6R, 7R)-7-((Z)-2-(2-((4-methoxybenzyl)amino)thiazol-4-yl)-2-(methoxyimino)acetamido)-3-((Z)-2-(4-methylthiazol-5-yl)vinyl)-8-oxo-5-thioxo-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

[0108] Chemical structure:

[0109] ;

[0110] (12) Cefditoren cyclodipirimipenem

[0111] Molecular formula: C 31 H 39 N6O 10 S3

[0112] Molecular weight: 751.89

[0113] Chemical name: (Valeryloxy)methyl 2-((Z)-4-(2-aminothiazol-4-yl)-13,13-dimethyl-5,8,12-trioxo-2,9,11-trioxa-3,6-diazatetradec-3-en-7-yl)-5-((Z)-2-(4-methylthiazol-5-yl)vinyl)-3,6-dihydro-2H-1,3-thiazine-4-carboxylate

[0114] Chemical structure:

[0115] ;

[0116] (13) Cefditoren Pivoxil Dimer

[0117] Molecular formula: C 51 H 56 N 12 O 14 S6

[0118] Molecular weight: 1253.50

[0119] Chemical name: Bis((pentanoyloxy)methyl)7,7'-(((2Z,2'Z)-2,2'-((methylenebis(azadiyl))bis(thiazole-2,4-diyl))bis(2-(methoxyimino)acetyl))bis(azadiyl))(6R,6'R,7R,7'R)-bis(3-((Z)-2-(4-methylthiazol-5-yl)vinyl)-8-oxo-5-thioxo-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate)

[0120] Chemical structure:

[0121] ;

[0122] (14) Cefditoren ring-opened dimer

[0123] Molecular formula: C 50 H 55 N 12 O 14 S6

[0124] Molecular weight: 1240.48

[0125] Chemical name: (Valeryloxy)methyl (6R, 7R)-7-((Z)-2-(2-((R)-2-((Z)-2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido)-2-((R)-5-((Z)-2-(4-methylthiazol-5-yl)vinyl)-4-((valeryloxy)methoxy)carbonyl)-3,6-dihydro-2H-1,3-thiazin-2-yl)acetamido)thiazol-4-yl)-2-(methoxyimino)acetamido 6-dihydro-2H-1,3-thiazin-2-yl)acetamido)thiazol-4-yl)-2-(methoxyimino)acetamido)-3-((Z)-2-(4-methylthiazol-5-yl)vinyl)-8-oxo-5-thioxo-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

[0126] Chemical structure:

[0127] .

[0128] The present invention is further described below in conjunction with specific examples. In the following examples, various processes and methods not described in detail are conventional methods known in the art, and the reagents used without indicating the source and specifications are all commercially available analytical or chromatographically pure.

[0129] In the examples, the method under the "Related Substances" item in the Cefditoren Pivoxil Granules method with standard number JX20090123 in the "Imported Drug Registration Standards" was used as a comparison. For the sake of simplicity, this method is referred to as the standard method herein.

[0130] Example

[0131] I. General Description

[0132] A. Material Information

[0133] The substance information used in this example is as follows:

[0134] Cefditoren pivoxil samples were purchased from Qilu Anti Pharmaceutical Co., Ltd.;

[0135] Cefditoren pivoxil reference substance was from Guangzhou Aiqixi Pharmaceutical Technology Co., Ltd.;

[0136] Propyl p-hydroxybenzoate, analytical grade, was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0137] Ammonium formate, analytical grade, was purchased from Guangzhou Chemical Reagent Factory;

[0138] Cefditoren acid: from Guangzhou Aiqixi Pharmaceutical Technology Co., Ltd.;

[0139] Cefditoren acid Δ3 isomer: from Guangzhou Aiqixi Pharmaceutical Technology Co., Ltd.;

[0140] Cefditoren pivoxil open-ring impurities: from Guangzhou Aiqixi Pharmaceutical Technology Co., Ltd.;

[0141] Cefditoren pivoxil Δ3 isomer: from Guangzhou Aiqixi Pharmaceutical Technology Co., Ltd.;

[0142] Cefditoren pivoxil 3E isomer: from Guangzhou Aiqixi Pharmaceutical Technology Co., Ltd.;

[0143] Cefditoren pivoxil-tivalamide: from Guangzhou Aiqixi Pharmaceutical Technology Co., Ltd.;

[0144] N-methoxybenzyl cefditoren pivoxil: from Guangzhou Aiqixi Pharmaceutical Technology Co., Ltd.;

[0145] Cefditoren secapitoxifen: from Guangzhou Aiqixi Pharmaceutical Technology Co., Ltd.;

[0146] Cefditoren dimer: from Guangzhou Aiqixi Pharmaceutical Technology Co., Ltd.;

[0147] Cefditoren open-ring dimer: from Guangzhou Aiqixi Pharmaceutical Technology Co., Ltd.;

[0148] Cefetamet Pivoxil Hydrochloride: from Guangzhou Aiqixi Pharmaceutical Technology Co., Ltd.;

[0149] Cefditoren pivoxil impurity P6: from Guangzhou Aiqixi Pharmaceutical Technology Co., Ltd.;

[0150] Methoxymethyl cefditoren pivoxil: from Guangzhou Aiqixi Pharmaceutical Technology Co., Ltd.;

[0151] Cefditoren Dipivoxil: from Guangzhou Aiqixi Pharmaceutical Technology Co., Ltd.;

[0152] Formic acid, analytical grade, was purchased from Tianjin Concord Technology Co., Ltd.;

[0153] Acetonitrile, chromatographic grade, was purchased from Tianjin Kangkede Technology Co., Ltd.;

[0154] Methanol, chromatographic grade, was purchased from Tianjin Kangkede Technology Co., Ltd.

[0155] B. Solution Preparation

[0156] The solutions used in this example were prepared as follows.

[0157] Ammonium formate solution: Weigh 1.58 g of ammonium formate and dissolve it in 900 ml of water. Adjust the pH to 4.5 with diluted formic acid solution (dilute formic acid 100 times with water), and dilute to 1000 ml with water to obtain 1.58 g / L ammonium formate solution.

[0158] Mobile phase A solution: Use a measuring cylinder to measure 450 ml of ammonium formate solution, 250 ml of acetonitrile, and 250 ml of methanol, mix them evenly, and obtain a mobile phase A solution with a volume ratio of ammonium formate solution, acetonitrile, and methanol of 45:25:25.

[0159] Mobile phase B solution: Use a measuring cylinder to measure 450 ml of ammonium formate solution, 550 ml of acetonitrile, and 550 ml of methanol, mix them evenly, and obtain a mobile phase B solution with a volume ratio of ammonium formate solution, acetonitrile, and methanol of 45:55:55.

[0160] Sample solution: accurately weigh 40 mg of cefditoren pivoxil sample, place in a 50 ml brown volumetric flask, add 10 ml of mobile phase A, shake vigorously to dissolve the cefditoren pivoxil sample, dilute to scale with mobile phase A, shake well, filter, and take the filtrate to obtain a sample solution with a cefditoren pivoxil concentration of 0.8 mg / ml.

[0161] Reference solution: Accurately weigh 1 mg of cefditoren pivoxil reference substance, place it in a 100 ml brown volumetric flask, add 20 ml of mobile phase A to dissolve it, and dilute it to the scale with mobile phase A to obtain a reference solution with a cefditoren pivoxil concentration of 0.01 mg / ml (i.e. 10 μg / ml).

[0162] System suitability solution: Accurately weigh 10 mg of cefditoren pivoxil reference substance and 2.5 mg of propyl paraben, place in a 10 ml volumetric flask, dissolve and dilute to scale with acetonitrile, and shake well to obtain a system suitability solution with a cefditoren pivoxil concentration of 1 mg / ml and a propyl paraben concentration of 0.25 mg / ml.

[0163] The above solutions were all prepared freshly before use. The sample solution, reference solution, and system suitability solution were all stored and used in the dark.

[0164] C. Chromatographic conditions

[0165] The high performance liquid chromatograph used in this example is Ultimate 3000, purchased from Thermo Fisher Scientific Inc.

[0166] The chromatographic conditions 1 used in the method of the present invention are as follows:

[0167] Detector: UV detector;

[0168] Chromatographic column: octadecylsilane bonded silica gel filled chromatographic column, Titank C18 4.6×250mm 3μm, purchased from Motion Analysis Scientific Instruments (Suzhou) Co., Ltd.

[0169] Impurity / ghost peak capture column: Chromasir® Ghost-Sniper Column, 4.6 mm × 50 mm, purchased from Motion Analysis Scientific Instruments (Suzhou) Co., Ltd.

[0170] Mobile phase A: mobile phase A solution prepared above;

[0171] Mobile phase B: mobile phase B solution prepared above;

[0172] Flow rate: 1.0 ml / min;

[0173] Detection wavelength: 254 nm;

[0174] Injection volume: 10 μl;

[0175] Column temperature: 40°C;

[0176] Elution method: gradient elution, the elution program is shown in Table 3 below:

[0177] Table 3

[0178] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 5 100 0 25 0 100 43 0 100 48 100 0 65 100 0 .

[0179] The chromatographic conditions of the standard method 2 are as follows:

[0180] Detector, chromatographic column, impurity / ghost peak trapping column, mobile phase A, mobile phase B, flow rate, detection wavelength, injection volume, column temperature: the same as chromatographic condition 1;

[0181] The elution method is gradient elution, and the elution program is shown in Table 4 below:

[0182] Table 4

[0183] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 5 100 0 20 0 100 38 0 100 43 100 0 .

[0184] D. System suitability test

[0185] According to the system suitability requirements in the standard method, the separation degree between the propylparaben peak and the cefditoren pivoxil peak in the system suitability solution spectrum should be no less than 5.0.

[0186] Test method:

[0187] The prepared system suitability solution was injected into a liquid chromatograph for analysis, and the chromatographic conditions were respectively the chromatographic conditions 1 and chromatographic conditions 2 as described above.

[0188] The separation results obtained according to chromatographic condition 1 are shown in Table 5:

[0189] Table 5

[0190] name Retention time Relative retention time Separation Cefditoren Pivoxil 15.008 1.00 / Propylparaben 12.025 0.80 8.60 .

[0191] The separation results obtained according to chromatographic condition 2 are shown in Table 6:

[0192] Table 6

[0193] name Retention time Relative retention time Separation Cefditoren Pivoxil 14.260 1.00 / Propylparaben 11.987 0.84 8.53

[0194] The separation calculation formula is as follows:

[0195]

[0196] Where: is the retention time of the previous peak between two adjacent chromatographic peaks

[0197] is the retention time of the previous peak between two adjacent chromatographic peaks

[0198] and are the peak widths of the two adjacent chromatographic peaks

[0199] As shown in Tables 5-6 above, in the results obtained under chromatographic conditions 1 and 2, the separation degrees of the propylparaben peak and the cefditoren pivoxil peak were 8.60 and 8.53, respectively, both of which were not less than 5.0, indicating that this system has met the specified requirements.

[0200] II. Peak shape improvement test

[0201] When reproducing the standard method, it is found that the peak front extension of the ring-opening impurity of cefditoren pivoxil will be obvious, resulting in poor peak shape and affecting the accuracy of the test results. Various studies have been conducted on this issue, and it was unexpectedly found that when the diluent was changed from 75% acetonitrile water in the standard method to mobile phase A, the peak front extension problem of the ring-opening impurity peak of cefditoren pivoxil can be solved. In this experiment, chromatograms before and after the diluent change are provided. The specific test information is as follows.

[0202] The impurity mixed solution used in this test was prepared with 75% acetonitrile water mixed solution and mobile phase A, and the concentrations of each impurity in the prepared mixed solution were as follows:

[0203] Cefditoren acid is about 2.4 μg / ml; cefditoren acid Δ3 isomer is about 2.4 μg / ml; cefditoren pivoxil open ring impurity is about 8 μg / ml; cefditoren pivoxil Δ3 isomer is about 17.6 μg / ml; cefditoren pivoxil 3E isomer is about 4 μg / ml; cefditoren pivoxil pivalamide is about 3.2 μg / ml; N-methoxybenzyl cefditoren pivoxil is about 2.4 μg / ml; cefditoren open ring dimipivoxil is about 2.4μg / ml; cefditoren dimer is about 13.6μg / ml; cefditoren open ring dimer is about 12.8μg / ml; cefditoren pivoxil hydrochloride is about 1.6μg / ml; cefditoren pivoxil impurity P6 is about 1.6μg / ml.

[0204] Example 1 (Comparative Example)

[0205] The impurity mixed solution diluted with the 75% acetonitrile-water mixture specified in the standard method was injected into the high performance liquid chromatograph for analysis using chromatographic condition 2 (conditions in the standard method). Figure 1 , see attached Figure 1 .

[0206] Figure 1 This is the chromatogram of the impurity mixed solution diluted with 75% acetonitrile-water mixture in the standard method. It can be seen from the figure that the cefditoren pivoxil open-ring impurity peak with a retention time of 7.930 has an obvious front extension phenomenon.

[0207] Example 2 (invention example)

[0208] The impurity mixed solution diluted by mobile phase A was injected into the high performance liquid chromatograph for analysis, and the chromatographic condition 2 was also used to obtain the chromatographic Figure 2 , see attached Figure 2 .

[0209] from Figure 2 It can be seen that the peak shape of the cefditoren pivoxil ring-opening impurity peak with a retention time of 7.943 is very good, with no front extension phenomenon.

[0210] It should be noted that although chromatographic condition 2 was used in this experiment, the chromatographic condition had little effect on the improvement of peak shape.

[0211] In this experiment, especially Figure 1 and 2 It was demonstrated that changing the diluent could improve the peak shape of the ring-opening impurity peak of cefditoren pivoxil.

[0212] III. Separation test

[0213] Example 3 (invention example)

[0214] The impurity mixed solution was prepared using the mobile phase A used in Example 2. The prepared impurity mixed solution was injected into a high performance liquid chromatograph for analysis. The chromatographic conditions were the same as those in chromatographic condition 1. The chromatogram was recorded. Figure 3 , see attached Figure 3 .

[0215] The obtained chromatogram data were analyzed, and the obtained separation results are shown in Table 7 below.

[0216] Table 7 (Chromatographic Conditions 1)

[0217] Impurity No. name Retention time Relative retention time Relative peak area% Separation Active ingredients Cefditoren Pivoxil 15.163 1.00 88.36 4.94 1 Cefditoren acid / Cefditoren acid △3 isomer 2.657 / 2.743 / 0.22 / 0.24 1.12 / 27.97 2 Cefditoren Pivoxil Open Ring Impurities 7.010 0.46 0.97 24.26 3 Cefetamet Pivoxil Hydrochloride 13.493 0.89 0.21 4.25 4 Cefditoren pivoxil△3 isomer 16.297 1.07 3.81 9.02 5 Cefditoren pivoxil 3E isomer 18.447 1.22 0.63 3.27 6 Cefditoren Pivoxil Impurity P6 19.753 1.30 0.15 1.61 7 Cefditoren Pivoxil Pivalamide 27.707 1.83 0.41 2.36 8 N-methoxybenzyl cefditoren pivoxil 28.223 1.86 0.57 6.68 9 Cefditoren Sepipiroxil 29.827 1.97 0.25 19.14 10 Cefditoren Pivoxil Dimer 35.910 2.37 1.68 3.18 11 Cefditoren pivoxil ring-opened dimer 42.587 2.81 1.56 3.27 12 Unknown impurity 1 6.080 0.40 0.19 5.31

[0218] Neutralized by Table 7 above Figure 3 It can be seen that the peak shapes of the cefditoren pivoxil open-ring impurity peak with a retention time of 7.010 and the unknown impurity 1 peak with a retention time of 6.080 are very good, and the two are well separated, indicating that the use of mobile phase A as a diluent to prepare the cefditoren pivoxil granule sample solution and the use of the elution gradient in chromatographic condition 1 can accurately separate and identify the unknown impurity 1 and the cefditoren pivoxil open-ring impurity and can accurately detect these two impurities, thereby enabling technicians to better control the quality of the cefditoren pivoxil granules.

[0219] Example 4 (Comparative Example)

[0220] The impurity mixed solution was prepared using the 75% acetonitrile-water mixture used in Example 1 as a diluent. The prepared impurity mixed solution was injected into a high performance liquid chromatograph for analysis. The chromatographic conditions were the chromatographic conditions 2 described above. The chromatogram was recorded. Figure 4 , see attached Figure 4 .

[0221] The obtained chromatogram data were analyzed, and the obtained separation results are shown in Table 8 below.

[0222] Table 8 (Chromatographic Condition 2)

[0223] Impurity No. name Retention time Relative retention time Relative peak area (%) Separation Active ingredients Cefditoren Pivoxil 14.277 1.00 90.95 4.55 1 Cefditoren acid / Cefditoren acid △3 isomer 2.700 0.19 0.84 15.93 2 Cefditoren Pivoxil Open Ring Impurities 6.813 0.48 0.69 1.38 3 Cefetamet Pivoxil Hydrochloride 12.820 0.90 0.04 3.76 4 Cefditoren pivoxil△3 isomer 15.257 1.07 2.37 8.56 5 Cefditoren pivoxil 3E isomer 17.057 1.19 0.51 3.16 6 Cefditoren Pivoxil Impurity P6 18.167 1.27 0.01 1.90 7 Cefditoren Pivoxil Pivalamide 24.563 1.72 0.37 1.94 8 N-methoxybenzyl cefditoren pivoxil 24.957 1.75 0.26 2.26 9 Cefditoren Sepipiroxil 26.517 1.86 0.20 2.19 10 Cefditoren Pivoxil Dimer 32.183 2.25 1.96 3.12 11 Cefditoren pivoxil ring-opened dimer 38.713 2.71 1.45 2.19

[0224] Note: In Table 7-8, Cefditoren acid / Cefditoren acid △3 isomer represents two impurities, Cefditoren acid and Cefditoren acid △3 isomer. Since these two impurities cannot be effectively separated in both methods, they are written together.

[0225] Neutralized by Table 8 above Figure 4 It can be seen that the peak of the cefditoren pivoxil open-ring impurity with a retention time of 6.813 has a front extension phenomenon, and the unknown impurity 1 cannot be identified. It is speculated that the unknown impurity 1 and the cefditoren pivoxil open-ring impurity peaks are combined and cannot be separated. This shows that the use of the 75% acetonitrile-water mixture specified in the standard method as a diluent to prepare the cefditoren pivoxil granule sample solution and the elution gradient in the chromatographic condition 2 cannot identify the unknown impurity 1, nor can it accurately detect the content of the cefditoren pivoxil open-ring impurity and the unknown impurity 1, so that the cefditoren pivoxil granules cannot be well controlled in terms of quality.

[0226] IV. Reproducibility test

[0227] This test was conducted by two experimenters. Each person conducted a system suitability test before each test. The test was conducted using the reference solution and sample solution prepared as above. Mobile phase A was used as the diluent for the solution preparation. Chromatographic condition 1 was used for analysis.

[0228] The reference solution was analyzed 5 times in the system suitability test and the sample solution was analyzed 6 times in the reproducibility test.

[0229] The experimental results of experimenter 1 are as follows.

[0230] The system suitability results are shown in Table 9.

[0231] Table 9

[0232]

[0233] Note: In Table 9 and Table 11 below, the product factor is the ratio of concentration to peak area.

[0234] The repeatability data are shown in Table 10.

[0235] Table 10

[0236]

[0237] The experimental results of experimenter 2 are as follows.

[0238] The system suitability results are shown in Table 11.

[0239] Table 11

[0240]

[0241] The repeatability results are shown in Table 12.

[0242] Table 12

[0243]

[0244] Acceptance criteria: Based on the compound concentration level and total impurity level, the test results of the 6 samples should meet the requirements shown in Table 13 below.

[0245] Table 13

[0246] Impurity Level %RSD Detection value <0.1% ≤25% 0.1%≤Detection value<0.2% ≤20% 0.2%≤Detection value<0.5% ≤10% 0.5%≤Detection value<5% ≤5.0% Detection value ≥5% ≤2.5%

[0247] The test results of the 12 samples should meet the requirements shown in Table 14 below.

[0248] Table 14

[0249] Impurity detection value %RSD Detection value <0.1% ≤35% 0.1%≤Detection value<0.2% ≤30% 0.2%≤Detection value<0.5% ≤15% 0.5%≤Detection value<5% ≤7.5% Detection value ≥5% ≤4.0% .

[0250] The conclusions of the repeatability test of experimenter 1 are as follows: the average detection value of cefditoren acid / cefditoren acid Δ3 isomer in 6 sample solutions is 0.20%, RSD is 3.16%, less than 20%; the average detection value of cefditoren pivoxil open-ring impurity is 0.55%, RSD is 0.00%, less than 5.0%; the average detection value of RRT=0.838 impurity is 0.14%, RSD is 0.00%, less than 20%; the average detection value of cefditoren hydrochloride is 0.02%, RSD is 0.00%, less than 25%; The average detection value of RRT=0.953 was 0.07%, RSD was 0.00%, less than 25%; the average detection value of cefditoren pivoxil Δ3 isomer was 1.33%, RSD was 0.57%, less than 5.0%; the average detection value of cefditoren pivoxil 3E isomer was 0.12%, RSD was 3.45%, less than 20%; the average detection value of RRT=1.348 was 0.07%, RSD was 0.00%, less than 25%; the average detection value of cefditoren dipivoxil was 0.002 %, RSD was 22.13%, less than 25%; the average detection value of cefditoren pivoxil pivalamide was 0.09%, RSD was 0.00%, less than 5.0%; the average detection value of cefditoren pivoxil dimer was 0.61%, RSD was 0.00%, less than 25%; the average detection value of cefditoren open ring dimer was 0.41%, RSD was 0.00%, less than 10%; the average detection value of total impurities was 2.87%, RSD was 0.29%, less than 5.0%, all of the above results met the requirements. The analytical repeatability passed.

[0251] The conclusions of the repeatability test of experimenter 2 are as follows: the average detection value of cefditoren acid / cefditoren acid Δ3 isomer in 6 sample solutions of a single person is 0.20%, RSD is 0.00%, less than 20%, and the RSD of 12 samples (including 6 samples of experimenter 1, the same below) is 2.13%; the average detection value of cefditoren pivoxil open-ring impurity is 0.51%, RSD is 0.00%, less than 5.0%, and the RSD of 12 samples is 3.94%; the average detection value of RRT=0.837 is 0.14%, RSD is 0. 00%, less than 20%, 12 RSD is 0.00%; the average detection value of ceftriaxone hydrochloride is 0.02%, RSD is 0.00%, less than 25%, 12 RSD is 0.00%; RRT=0.954 average detection value is 0.07%, RSD is 0.00%, less than 25%, 12 RSD is 0.00%; the average detection value of cefditoren pivoxil Δ3 isomer is 1.31%, RSD is 0.31%, less than 5.0%, 12 RSD is 0.90%; cefditoren The average detection value of the 3E isomer of pivoxil was 0.19%, RSD was 7.44%, less than 20%, and the RSD of 12 copies was 9.94%; the average detection value of RRT=1.341 was 0.07%, RSD was 0.00%, less than 25%, and the RSD of 12 copies was 0.00%; the average detection value of cefditoren dipivoxil was 0.005%, RSD was 33.70%, less than 25%, and the RSD of 12 copies was 24.76%; the average detection value of cefditoren pivoxil pivalamide was 0.08%, RSD was 0.0 0%, less than 25%, 12 RSD is 4.77%; the average detection value of cefditoren pivoxil dimer is 0.65%, RSD is 0.00%, less than 5.0%, 12 RSD is 1.58%; the average detection value of cefditoren open ring dimer is 0.43%, RSD is 0.95%, less than 10%, 12 RSD is 8.06%; the average detection value of total impurities is 3.68%, RSD is 0.49%, less than 5.0%, 12 RSD is 0.41%, all of the above results meet the requirements. The intermediate precision is good.

[0252] It can be seen from the above results that the method of the present invention has very good reproducibility.

[0253] In general, the peak shape improvement in the present application is Figure 1 and 2 It was proved that only changing the diluent can effectively improve the peak shape of the cefditoren pivoxil open-ring impurity peak, thereby making the content detection of cefditoren pivoxil open-ring impurity more accurate.

[0254] In the separation example, Tables 7-8 and Figure 3-4It is proved that the use of mobile phase A as a diluent to prepare the cefditoren pivoxil granule sample solution and the use of the elution gradient in the chromatographic condition 1 of the present invention can accurately separate and identify the unknown impurity 1 and the cefditoren pivoxil open-ring impurity and can accurately detect these two impurities, thereby enabling technicians to better control the quality of the cefditoren pivoxil granules. However, the use of a 75% acetonitrile-water mixture specified in the standard method as a diluent to prepare the cefditoren pivoxil granule sample solution and the use of the elution gradient in the chromatographic condition 2 cannot identify the unknown impurity 1, nor can it accurately detect the contents of the cefditoren pivoxil open-ring impurity and the unknown impurity 1, thereby failing to perform good quality control on the cefditoren pivoxil granules.

Claims

1. A method for improving the quality control of cefditoren pivoxil granules, the method comprising: The sample solution of cefditoren pivoxil granules is injected into a high performance liquid chromatograph for analysis to detect impurities in the cefditoren pivoxil granules, wherein the impurities include cefditoren acid, cefditoren acid △3 isomer, cefditoren pivoxil open ring impurities, cefditoren pivoxil hydrochloride, cefditoren pivoxil △3 isomer, cefditoren pivoxil 3E isomer, cefditoren pivoxil impurity P6, cefditoren pivoxil pivalamide, N-methoxybenzyl cefditoren pivoxil, cefditoren open ring dipivoxil, cefditoren pivoxil dimer, cefditoren pivoxil open ring dimer, Mobile phase A and mobile phase B are used as eluents for gradient elution. Mobile phase A is a mixture of ammonium formate solution, acetonitrile, and methanol in a volume ratio of (43-47): (23-27): (23-27). Mobile phase B is a mixture of ammonium formate solution, acetonitrile, and methanol in a volume ratio of (43-47): (53-57): (53-57). Gradient elution was used. In the gradient elution program, within 0-5 min, the volume of mobile phase A was 100% and that of mobile phase B was 0%; within 5 to 25 min, the volume of mobile phase A changed from 100% to 0%, and at the same time, the volume of mobile phase B changed from 0% to 100%; then the volume of mobile phase A was kept at 0% and the volume of mobile phase B was kept at 100% for 17-19 min. The elution gradient is as follows: ， Octadecylsilane bonded silica was used as the stationary phase. The cefditoren pivoxil granules are dissolved using mobile phase A as a diluent to prepare a solution, which is then injected into a high performance liquid chromatograph for analysis.

2. The method for improving the quality control of cefditoren pivoxil granules according to claim 1, wherein the mobile phase A is a mixture of ammonium formate solution, acetonitrile, and methanol in a volume ratio of (44-46): (24-26): (24-26), and the mobile phase B is a mixture of ammonium formate solution, acetonitrile, and methanol in a volume ratio of (44-46): (54-56): (54-56).

3. The method for improving the quality control of cefditoren pivoxil granules according to claim 1, wherein the ammonium formate solution is an aqueous solution having an ammonium formate concentration of 1.56-1.60 g / l, and the aqueous solution is adjusted to 4.4-4.6 with formic acid.

4. The method for improving the quality control of cefditoren pivoxil granules according to claim 1, wherein the injection volume is 8-15 μl, and wherein the sample solution of cefditoren pivoxil granules is a solution diluted with mobile phase A to a concentration of 0.5-1.1 mg / ml.

5. The method for improving the quality control of cefditoren pivoxil granules according to claim 1, wherein the flow rate of the mobile phase is 0.9-1.1 ml / min.

6. The method for improving the quality control of cefditoren pivoxil granules according to claim 1, wherein the detection is performed using an ultraviolet detector with a detection wavelength of 254 nm.

7. The method for improving the quality control of cefditoren pivoxil granules according to claim 1, wherein the column temperature is 35-42°C.

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