A method for analyzing and detecting cefcapene hydrochloride intermediate cefcapene precursor acid

Abstract

The present invention belongs to the technical field of drug intermediate analysis and detection, and provides an analysis and detection method for a hydrochloric acid cefuroxime intermediate cefuroxime precursor acid. The mobile phase of the analysis and detection method of the present invention is mainly composed of methanol and water, and tetrapentyl ammonium bromide and sodium dihydrogen phosphate are added, which can not only improve the solution stability of cefuroxime precursor acid, but also ensure the accuracy of the analysis and detection method results; and, the peak type, tailing factor, separation degree and theoretical plate number of the main component are significantly improved; and, impurity A, impurity B, impurity C and impurity D all have higher response values ​​and separation effects, and more unknown impurity peaks can be separated and detected. At the same time, the operation process of the analysis and detection method of the present invention is simple and convenient, the running time is short (within 30min), and it is suitable for rapid detection of industrial production; and the equipment requirements are not high, and the brand of the chromatographic column is not limited.

Description

Technical Field

[0001] The invention relates to the technical field of drug intermediate analysis and detection, and in particular to an analysis and detection method for a cefcapone hydrochloride intermediate cefcapone precursor acid. Background Art

[0002] Cefcapone hydrochloride is 2,2-dimethylpropionyloxymethyl (6R,7R)-7-[(2Z)-2-(2-aminothiazol-4-yl)pent-2-enoylamino]-3-carbamoyloxymethyl-8-oxo-5-thio-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate monohydrochloride monohydrate, which belongs to the third generation of cephalosporin antibiotics. Cefcapone hydrochloride has a broad antibacterial spectrum in vitro (from aerobic and anaerobic Gram-positive bacteria to Gram-negative bacteria), and also exhibits antibacterial activity against penicillin-resistant Streptococcus pneumoniae and ampicillin-resistant Haemophilus influenzae. In addition, cefcapone hydrochloride has certain therapeutic effects on superficial skin infections, deep skin infections, lymphadenitis / lymphadenitis, chronic pyoderma, secondary infections (such as trauma / burns and surgical wounds, mastitis, perianal abscesses, secondary infections of pharyngitis / laryngitis), tonsillitis (including peritonsillar abscesses, peritonsillar abscesses), acute bronchitis, pneumonia, chronic respiratory tract diseases, cystitis, pyelonephritis, urethritis, cervicitis, cholecystitis, cholangitis, prostatitis, intrauterine infection, adnexitis, dacryocystitis, hordeolum, tardadenitis, otitis externa, otitis media, sinusitis, periodontitis, pericoronitis, mandibular inflammation, etc., and has certain clinical applications.

[0003] The process route for the development of cefoperazone hydrochloride API is as follows:

[0004]

[0005] It can be seen that cefcapene hydrochloride is synthesized from hydroxymethyl-7-aminocephalosporanic acid or 7-amino-3-(hydroxymethyl)-3-cephalocyclo-4-carboxylic acid (D-7-ACA) and (Z)-2-(2-tert-butyloxycarbonylaminothiazol-4-yl)-2-pentenoic acid (BAPA) through multi-step reactions, and the cost of the starting material BAPA is relatively high. Therefore, effective detection and control of the cefcapene precursor acid (7-((Z)-2-(2-((tert-butyloxycarbonyl)amino)thiazol-4-yl)pent-2-enoylamino)-3-((carbamoyloxy)methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid diisopropylamine salt) generated by the reaction of D-7-ACA and BAPA can effectively improve the yield and quality of cefcapene hydrochloride, thereby effectively controlling the cost of cefcapene hydrochloride. Therefore, an effective detection method for cefuroxime proacid is of great significance for cefuroxime hydrochloride raw materials.

[0006] Since cefuroxime precursor acid is obtained by condensation, carbamylation and esterification of D-7-ACA and BAPA, the synthetic route of cefuroxime precursor acid was analyzed, and the possible impurities mainly include: cefuroxime acid (named as impurity A, 7-[2-(2-amino-thiazol-4-yl)-pent-2-enoylamino]-3-carbamoyloxymethyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid), endoprosthesis (named as impurity B, tert-butyl (4-((Z)-1-((1,7-dioxo-1,4,5a,6-tetrahydro-3H,7H- The impurities of the present invention are as follows: 1,1,3,3-tetramethylguanidine (Z)-7-(2-(2-((tert-butyloxycarbonyl)amino)thiazol-4-yl)pent-2-enamide)-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate), 1,1,3,3-tetramethylguanidine (Z)-7-(2-(2-((tert-butyloxycarbonyl)amino)thiazol-4-yl)pent-2-enamide)-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate), and other unknown impurities. However, there is no report on the analytical detection method of the intermediate cefuroxime hydrochloride cefuroxime precursor acid. Summary of the invention

[0007] In view of this, the object of the present invention is to provide an analytical detection method for cefcapone protoacid, an intermediate of cefcapone hydrochloride. The analytical detection method provided by the present invention can detect impurities and main components of the cefcapone protoacid to be detected, and the analytical detection method has high accuracy.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for analyzing and detecting a cefcapene hydrochloride intermediate cefcapene protease acid, comprising the following steps:

[0010] Dissolving and diluting the cefuroxime precursor acid to be tested in sequence to obtain a sample for use in an apparatus;

[0011] The sample is subjected to liquid chromatography detection to obtain chromatographic information of the cefuroxime precursor acid to be detected;

[0012] Based on a predetermined standard curve, the chromatographic information of the cefuroxime precursor acid to be tested is analyzed and calculated to obtain the material composition and content of the cefuroxime precursor acid to be tested;

[0013] The cefuroxime precursor acid to be tested includes cefuroxime precursor acid and impurities;

[0014] The impurities include 7-[2-(2-amino-thiazol-4-yl)-pent-2-enoylamino]-3-carbamoyloxymethyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, tert-butyl (4-((Z)-1-((1,7-dioxo-1,4,5a,6-tetrahydro-3H,7H-azetidinyl[2,1-b]furanyl[3,4-d][1,3]thiazin-6-yl)amino)-1- one or more of ((tert-butyloxycarbonyl)amino)thiazol-2-yl)carbamate, (Z)-2-(2-tert-butyloxycarbonylaminothiazol-4-yl)-2-pentenoic acid, 1,1,3,3-tetramethylguanidine (Z)-7-(2-(2-((tert-butyloxycarbonyl)amino)thiazol-4-yl)pent-2-enamido)-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate, and other impurities;

[0015] The parameters of the liquid chromatography detection include:

[0016] The chromatographic column is a chromatographic column with octadecylsilane bonded silica gel as a filler;

[0017] The column temperature of the chromatographic column is 20-40°C;

[0018] The mobile phase is a methanol-water solution; the volume ratio of methanol to water in the methanol-water solution is 70:30 to 60:40; the methanol-water solution also includes tetrapentylammonium bromide and sodium dihydrogen phosphate; the mass percentage of tetrapentylammonium bromide in the methanol-water solution is 1 to 10%; the mass percentage of sodium dihydrogen phosphate in the methanol-water solution is 0.1 to 3%;

[0019] Flow rate: 0.6-1.2 mL / min;

[0020] The detection wavelength is 245~275nm.

[0021] Preferably, the dissolved reagent includes methanol; the dosage ratio of the cefuroxime precursor acid to be detected and the dissolved reagent is 20 mg: 2-4 mL.

[0022] Preferably, the diluted reagent is the mobile phase.

[0023] Preferably, the concentration of the cefuroxime precursor acid to be detected in the sample is 0.3-0.6 mg / mL.

[0024] Preferably, the parameters of the liquid chromatography detection also include: the injection volume is 20 μL.

[0025] Preferably, during the liquid chromatography detection, the chromatogram is recorded to 1.5 to 5 times the retention time of the main peak.

[0026] Preferably, the predetermined standard curve comprises a predetermined standard curve of cefcaprine precursor acid, a predetermined standard curve of 7-[2-(2-amino-thiazol-4-yl)-pent-2-enoylamino]-3-carbamoyloxymethyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, a predetermined standard curve of tert-butyl (4-((Z)-1-((1,7-dioxo-1,4,5a,6-tetrahydro-3H,7H-azetidinyl[2,1-b]furanyl[3,4-d][1,3] The standard curve of (Z)-2-(2-tert-butyloxycarbonylaminothiazol-4-yl)-2-pentenoic acid and the standard curve of 1,1,3,3-tetramethylguanidine (Z)-7-(2-(2-((tert-butyloxycarbonyl)amino)thiazol-4-yl)pent-2-enamido)-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate were prepared.

[0027] The invention provides an analytical detection method for cefuroxime protozolin hydrochloride intermediate cefuroxime protozolin hydrochloride, comprising the following steps: dissolving and diluting the cefuroxime protozolin hydrochloride intermediate in sequence to obtain a sample for use on a machine; performing liquid chromatography detection on the sample for use on a machine to obtain chromatographic information of the cefuroxime protozolin hydrochloride intermediate; analyzing and calculating the chromatographic information of the cefuroxime protozolin hydrochloride intermediate based on a predetermined standard curve to obtain the material composition and content of the cefuroxime protozolin hydrochloride intermediate; the cefuroxime protozolin hydrochloride intermediate comprises cefuroxime protozolin hydrochloride intermediate, ... and impurities; the impurities include 7-[2-(2-amino-thiazol-4-yl)-pent-2-enoylamino]-3-carbamoyloxymethyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, tert-butyl (4-((Z)-1-((1,7-dioxo-1,4,5a,6-tetrahydro-3H,7H-azetidinyl[2,1-b]furanyl[3,4-d][1,3]thiazin-6-yl)amino)-1-oxopentyl-2-ene-2-yl )thiazol-2-yl)carbamate, (Z)-2-(2-tert-butyloxycarbonylaminothiazol-4-yl)-2-pentenoic acid, 1,1,3,3-tetramethylguanidine (Z)-7-(2-(2-((tert-butyloxycarbonyl)amino)thiazol-4-yl)pent-2-enoylamide)-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate and one or more of other impurities; the parameters of the liquid chromatography detection include: the chromatographic column is octadecylsilane bond A chromatographic column with silica gel as a filler; the column temperature of the chromatographic column is 20-40° C.; the mobile phase is a methanol-water solution; the volume ratio of methanol to water in the methanol-water solution is 70:30-60:40; the methanol-water solution also includes tetrapentylammonium bromide and sodium dihydrogen phosphate; the mass percentage of tetrapentylammonium bromide in the methanol-water solution is 1-10%; the mass percentage of sodium dihydrogen phosphate in the methanol-water solution is 0.1-3%; the flow rate is 0.6-1.2 mL / min; and the detection wavelength is 245-275 nm.

[0028] Beneficial effects:

[0029] The mobile phase of the analysis and detection method provided by the present invention mainly comprises methanol and water, and tetrapentylammonium bromide and sodium dihydrogen phosphate are added, which can not only improve the solution stability of cefcaprine precursor acid and ensure the accuracy of the analysis and detection method results; and the peak shape, tailing factor, separation degree and theoretical plate number of the main component are significantly improved; in addition, the impurities 7-[2-(2-amino-thiazol-4-yl)-pent-2-enoylamino]-3-carbamoyloxymethyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (impurity A), tert-butyl (4-((Z)-1-((1,7-dioxo-1,4,5a,6-tetrahydro-3H,7H-nitrogen)) are also significantly improved. Heterocyclobutyl[2,1-b]furanyl[3,4-d][1,3]thiazin-6-yl)amino)-1-oxopentyl-2-en-2-yl)thiazol-2-yl)carbamate (impurity B), (Z)-2-(2-tert-butyloxycarbonylaminothiazol-4-yl)-2-pentenoic acid (impurity C), and 1,1,3,3-tetramethylguanidine (Z)-7-(2-(2-((tert-butyloxycarbonyl)amino)thiazol-4-yl)pent-2-enamido)-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate (impurity D) all have high response values ​​and separation effects, and can separate and detect more unknown impurity peaks. In addition, the operation process of the analysis and detection method of the present invention is simple and convenient, the running time is short (within 30min), and it is suitable for rapid detection of industrial production; moreover, the analysis and detection method of the present invention has low requirements on equipment, and can use a very common liquid chromatography instrument, and does not limit the brand of the chromatographic column, and does not require additional low temperature control for the chromatographic column and the sample introduction plate. Finally, the analysis and detection method of the present invention can effectively detect the degradation impurities and process impurities in the cefuroxime precursor acid to be tested, which provides a good method for the effective control of the intermediate, and provides good data support for the optimization of the process. DETAILED DESCRIPTION

[0030] The present invention provides a method for analyzing and detecting a cefcapene hydrochloride intermediate cefcapene protease acid, comprising the following steps:

[0031] Dissolving and diluting the cefuroxime precursor acid to be tested in sequence to obtain a sample for use in an apparatus;

[0032] The sample is subjected to liquid chromatography detection to obtain chromatographic information of the cefuroxime precursor acid to be detected;

[0033] Based on a predetermined standard curve, the chromatographic information of the cefuroxime precursor acid to be detected is analyzed and calculated to obtain the material composition and content of the cefuroxime precursor acid to be detected.

[0034] In the present invention, unless otherwise specified, the raw materials used in the present invention are preferably commercially available products.

[0035] The invention dissolves and dilutes the cefuroxime precursor acid to be tested to obtain a sample for testing.

[0036] In the present invention, the cefuroxime precursor acid to be tested includes cefuroxime precursor acid (7-((Z)-2-(2-((tert-butyloxycarbonyl)amino)thiazol-4-yl)pent-2-enoylamino)-3-((carbamoyloxy)methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid diisopropylamine salt) and impurities. In the present invention, the impurities include 7-[2-(2-amino-thiazol-4-yl)-pent-2-enoylamino]-3-carbamoyloxymethyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (impurity A), tert-butyl (4-((Z)-1-((1,7-dioxo-1,4,5a,6-tetrahydro-3H,7H-azetidinyl[2,1-b]furanyl[3,4-d][1,3]thiazin-6-yl)amino)-1-oxopentyl-2 -en-2-yl)thiazol-2-yl)carbamate (impurity B), (Z)-2-(2-tert-butyloxycarbonylaminothiazol-4-yl)-2-pentenoic acid (BAPA, impurity C), 1,1,3,3-tetramethylguanidine (Z)-7-(2-(2-((tert-butyloxycarbonyl)amino)thiazol-4-yl)pent-2-enoamido)-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate (impurity D) and one or more of other impurities.

[0037] In the present invention, the dissolved reagent preferably includes methanol. In the present invention, the dosage ratio of the cefcaprine precursor acid to be detected and the dissolved reagent is preferably 20 mg: 2-4 mL, and more preferably 20 mg: 3 mL.

[0038] In the present invention, the diluted reagent is preferably the mobile phase of the liquid chromatography detection; the composition of the mobile phase is preferably described in detail in the liquid chromatography detection part and is not described here. The present invention does not specifically limit the amount of the diluted reagent, as long as the concentration of the cefuroxime precursor acid to be detected in the sample on the machine meets the requirements.

[0039] In the present invention, the concentration of the cefcaprine precursor acid to be detected in the sample is preferably 0.3 to 0.6 mg / mL, and more preferably 0.4 to 0.5 mg / mL.

[0040] After obtaining the sample on the machine, the present invention performs liquid chromatography detection on the sample on the machine to obtain chromatographic information of the cefuroxime precursor acid to be detected.

[0041] In the present invention, the parameters of the liquid chromatography detection include:

[0042] The chromatographic column is a chromatographic column with octadecylsilane bonded silica gel as a filler; the model of the chromatographic column is preferably Phenomenex or YMC; the size of the chromatographic column is preferably 150 mm×4.6 mm, 5 μm.

[0043] The column temperature of the chromatographic column is 20-40°C, preferably 30°C;

[0044] The mobile phase is a methanol aqueous solution; the volume ratio of methanol to water in the methanol aqueous solution is 70:30 to 60:40, preferably 67:33 to 65:35; the methanol aqueous solution also includes tetrapentylammonium bromide and sodium dihydrogen phosphate; the mass percentage of tetrapentylammonium bromide in the methanol aqueous solution is 1 to 10%, preferably 1.5 to 8%, and more preferably 2 to 5%; the mass percentage of sodium dihydrogen phosphate in the methanol aqueous solution is 0.1 to 3%, preferably 0.5 to 2%, and more preferably 0.8 to 1%.

[0045] The flow rate is 0.6 to 1.2 mL / min, preferably 0.8 to 1 mL / min;

[0046] The detection wavelength is 245 to 275 nm, preferably 255 nm.

[0047] In the present invention, the parameters of the liquid chromatography detection also include: the injection volume is preferably 20 μL.

[0048] In the present invention, during the liquid chromatography detection, the chromatogram is preferably recorded to 1.5 to 5 times, more preferably 3 to 4 times, the retention time of the main peak.

[0049] In the present invention, the detector for liquid chromatography detection is preferably an ultraviolet detector.

[0050] After obtaining the chromatographic information of the cefuroxime precursor acid to be detected, the present invention analyzes and calculates the chromatographic information of the cefuroxime precursor acid to be detected based on a predetermined standard curve to obtain the material composition and content of the cefuroxime precursor acid to be detected.

[0051] In the present invention, the predetermined standard curve includes a predetermined standard curve of cefcaprine precursor acid, a predetermined standard curve of 7-[2-(2-amino-thiazol-4-yl)-pent-2-enoylamino]-3-carbamoyloxymethyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, a predetermined standard curve of tert-butyl (4-((Z)-1-((1,7-dioxo-1,4,5a,6-tetrahydro-3H,7H-azetidinyl[2,1-b]furoxyl ... The standard curves for (1,1,3,3-tetramethylguanidine (Z)-7-(2-(2-((tert-butyloxycarbonyl)amino)thiazol-4-yl)pent-2-enamido)-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate were prepared using a standard curve for (1,1,3,3-tetramethylguanidine (Z)-7-(2-(2-((tert-butyloxycarbonyl)amino)thiazol-4-yl)pent-2-enamido)-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate).

[0052] The present invention does not specifically limit the predetermined standard curve, and a method for establishing a standard curve well known to those skilled in the art may be used.

[0053] The present invention does not specifically limit the analysis and calculation method, and any analysis and calculation method well known to those skilled in the art may be used.

[0054] The following is a detailed description of the analytical detection method of the cefcapone pivoxil hydrochloride intermediate cefcapone precursor acid provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0055] Preparation Example 1

[0056] Add 100mL of dichloromethane, 1.0g of D-7-ACA, and 1mL of tetramethylguanidine to 25mL round-bottom flask A, stir to dissolve; cool for use; add 120mL of dichloromethane and 1.5g of BAPA to 50mL round-bottom flask B, cool, add 3mL of methanesulfonyl chloride, and stir to react. After the reaction is completed, add the reaction solution in round-bottom flask A to round-bottom flask B, react for 4h, cool to below 0℃, add 1.5mL of chlorosulfonic acid isocyanate, react for 0.5h, add 10mL of purified water, let stand for stratification, add 3.4g of water to the organic layer, combine the water layers and add 20mL of acetone, react for about 0.5h; after the reaction is completed, add 10mL of acetone, let stand for stratification, wash the organic phase with water, combine the water phases and use dichloromethane to extract and combine the organic phases. 1 g of diisopropylamine was added to the organic phase to form a salt, and a solid was precipitated; the solid was collected by suction filtration and vacuum dried for 5 h to obtain cefuroxime precursor acid.

[0057] Verification Example 1 Methodological Verification of Analytical Detection Method

[0058] 1.1 Linear range

[0059] 1.1.1 Liquid chromatography conditions

[0060] Injection volume: 20 μL, detection wavelength: 255 nm, chromatographic column: octadecylsilane bonded silica gel as filler column (Phenomenex, C 18 , 150mm×4.6mm, 5μm), flow rate: 1mL / min, column temperature: 30℃; the mobile phase is a methanol-water solution with a volume ratio of 70:30, in which the mass percentage of tetrapentylammonium bromide is 3% and the mass percentage of sodium dihydrogen phosphate is 1%.

[0061] 1.1.2 Solution preparation

[0062] Preparation of reference solution: Accurately weigh a certain amount of impurity A, impurity B, impurity C, impurity D and cefuroxime precursor acid reference substance, dissolve them in appropriate amount of methanol, and dilute them with mobile phase to prepare mixed standard stock solutions with concentrations of 24μg / mL, 20μg / mL, 12μg / mL, 20μg / mL and 24μg / mL, respectively.

[0063] The mixed standard stock solution was diluted with the mobile phase to 33.3 times (3 mL → 100 mL), 20 times (5 mL → 100 mL), 12.5 times (8 mL → 100 mL), 10 times (10 mL → 100 mL) and 8.33 times (12 mL → 100 mL) of the original solution volume to obtain a series of standard curve solutions.

[0064] 1.1.3 Detection

[0065] Accurately pipette 20 μL of standard curve solution of different concentrations, inject into liquid chromatograph, perform sample injection and analysis according to the liquid chromatography conditions in 1.1.1, measure the peak area of ​​each peak, perform parallel determination 3 times, and take the average value of the valid data.

[0066] With the peak area (Y) as the ordinate and the mass concentration as the abscissa (X), a linear equation was plotted. The results are shown in Table 1.

[0067] Table 1 Linear equations

[0068]

[0069] It can be seen from Table 1 that the linearity of impurity A, impurity B, impurity C, impurity D and cefoperazone precursor acid measured under the chromatographic conditions is good.

[0070] 1.2 Limit of detection and limit of quantification

[0071] The detection limit (LOD) and quantification limit (LOQ) were calculated when the signal-to-noise ratio (S / N) was 3 and 10, respectively. The results are shown in Table 2.

[0072] Table 2 Results of detection limit and quantification limit

[0073]

[0074] It can be seen from Table 2 that impurity A, impurity B, impurity C, impurity D and cefuroxime precursor acid all have high response values ​​under the chromatographic conditions, and their detection limits are all less than 0.05% of the test sample concentration (0.05% is the negligible limit of impurity detection), and the sensitivity of this method is relatively high.

[0075] 1.3 Repeatability

[0076] Accurately weigh 20 mg of 6 portions of cefuroxime precursor acid obtained in Preparation Example 1, dissolve it in 3 mL of methanol, and dilute it with the mobile phase in 1.1.1 Liquid Chromatography Conditions to obtain a test solution with a concentration of 0.4 mg / mL.

[0077] Accurately pipette 20 μL of each test solution and inject it into the liquid chromatograph. Perform sample analysis according to the liquid chromatography conditions in 1.1.1 and calculate the RSD values ​​of the peak area and retention time of each peak. The results are shown in Table 3.

[0078] Table 3 Repeatability data

[0079]

[0080]

[0081] It can be seen from Table 3 that the contents of various impurities and main components of the 6 consecutive test samples have basically no large fluctuations and all meet the requirements, indicating that the repeatability of this method is good.

[0082] Example 1

[0083] Liquid chromatography conditions: injection volume: 20 μL, detection wavelength: 255 nm, chromatographic column: octadecylsilane bonded silica gel as filler column (Phenomenex, C 18 , 150mm×4.6mm, 5μm), flow rate: 1mL / min, column temperature: 30℃, the mobile phase is a methanol-water solution with a volume ratio of 70:30, in which the mass percentage of tetrapentylammonium bromide is 3% and the mass percentage of sodium dihydrogen phosphate is 1%.

[0084] Sample preparation: Take an appropriate amount of the cefuroxime precursor acid to be tested in Preparation Example 1, accurately weigh it, dissolve it with an appropriate amount of methanol, dilute it with the mobile phase to a concentration of 0.4 mg / mL, shake it well, accurately measure 20 μL and inject it into the liquid chromatograph, and record the chromatogram to 4 times the retention time of the main peak.

[0085] The results are shown in Table 4:

[0086] Table 4 Chromatographic information of Example 1

[0087] Serial number name Peak time (min) Peak area Resolution (USP) Theoretical plates Tailing Factor 1 Impurity A 2.338 44302 2.1 6929 1.2 2 Impurity B 4.909 47545 1.9 7253 0.9 3 Impurity C 6.857 10153 2.5 9879 1.1 4 Cefuroxime proacid 7.904 12132201 1.8 10225 1.1 5 Impurity D 9.942 24510 2.2 6692 0.8 6 Maximum unknown single impurity 16.076 48519 4.3 7252 1.1

[0088] Example 2

[0089] Liquid chromatography conditions: injection volume: 20 μL, detection wavelength: 270 nm, chromatographic column: octadecylsilane bonded silica gel as filler column (Phenomenex, C 18 , 150mm×4.6mm, 5μm), flow rate: 1.2mL / min, column temperature: 30℃, the mobile phase is a methanol-water solution with a volume ratio of 65:35, in which the mass percentage of tetrapentylammonium bromide is 2% and the mass percentage of sodium dihydrogen phosphate is 0.8%.

[0090] Sample preparation: Take an appropriate amount of the cefuroxime precursor acid to be tested in Preparation Example 1, accurately weigh it, dissolve it with an appropriate amount of methanol, dilute it with the mobile phase to a concentration of 0.6 mg / mL, shake it well, accurately measure 20 μL and inject it into the liquid chromatograph, and record the chromatogram to 3.5 times the retention time of the main peak.

[0091] The results are shown in Table 5.

[0092] Table 5 Chromatographic information of Example 2

[0093] Serial number name Peak time (min) Peak area Resolution (USP) Theoretical plates Tailing Factor 1 Impurity A 2.811 52813 1.9 7125 1.1 2 Impurity B 5.624 56322 1.7 7931 1.0 3 Impurity C 7.368 13021 2.4 10211 1.0 4 Cefuroxime proacid 8.412 1435632 1.9 12311 1.2 5 Impurity D 10.221 25689 2.2 7125 0.9 6 Maximum unknown single impurity 16.762 57369 4.6 6938 1.0

[0094] Comparative Example 1

[0095] Liquid chromatography conditions: injection volume: 20 μL, detection wavelength: 255 nm, chromatographic column: octadecylsilane bonded silica gel as filler column (YMC, C 18 , 150mm×4.6mm, 5μm), flow rate: 1.0mL / min, column temperature: 35℃, the mobile phase is a methanol-water solution with a volume ratio of 67:33, in which the mass percentage of tetrapentylammonium bromide is 2%, and it does not contain sodium dihydrogen phosphate.

[0096] Sample preparation: Take an appropriate amount of the cefuroxime precursor acid to be tested in Preparation Example 1, accurately weigh it, dissolve it with an appropriate amount of methanol, dilute it with the mobile phase to a concentration of 0.5 mg / mL of the sample, shake it well, accurately measure 20 μL and inject it into the liquid chromatograph, and record the chromatogram to 5 times the retention time of the main peak.

[0097] The results are shown in Table 6.

[0098] Table 6 Chromatographic information of comparative example 1

[0099] Serial number name Peak time (min) Peak area Resolution (USP) Theoretical plates Tailing Factor 1 Impurity A 3.896 40235 1.6 3025 1.9 2 Impurity B 6.789 45213 1.4 2036 2.1 3 Impurity C 8.456 98638 1.5 3756 1.8 4 Cefuroxime proacid 9.652 11536208 1.9 4998 1.9 5 Impurity D 11.596 23511 1.4 3721 1.7 6 Maximum unknown single impurity 18.011 47961 2.3 3698 2.0

[0100] Comparative Example 2

[0101] Liquid chromatography conditions: injection volume: 20 μL, detection wavelength: 260 nm, chromatographic column: octadecylsilane bonded silica gel as filler column (YMC, C 18 , 150mm×4.6mm, 5μm), flow rate: 0.8mL / min, column temperature: 25℃, the mobile phase is a methanol-water solution with a volume ratio of 69:31, in which the mass percentage of sodium dihydrogen phosphate is 1%, and does not contain tetrapentylammonium bromide.

[0102] Sample preparation: Take an appropriate amount of the cefuroxime precursor acid to be tested in Preparation Example 1, accurately weigh it, dissolve it with an appropriate amount of methanol, dilute it with mobile phase to a solution with a concentration of 0.3 mg / mL, shake it well, accurately measure 20 μL and inject it into the liquid chromatograph, and record the chromatogram to 3.5 times the retention time of the main peak.

[0103] The results are shown in Table 7.

[0104] Table 7 Chromatographic information of Comparative Example 2

[0105] Serial number name Peak time (min) Peak area Resolution (USP) Theoretical plates Tailing Factor 1 Impurity A 3.012 31210 1.6 3201 1.8 2 Impurity B 7.521 33569 1.4 2654 1.8 3 Impurity C 9.011 79165 1.5 3952 1.7 4 Cefuroxime proacid 10.215 7998569 1.5 5128 1.5 5 Impurity D 12.401 17569 1.3 4039 1.7 6 Other largest unknown impurities 19.320 35628 2.6 4425 2.1

[0106] From the chromatographic information of Examples 1 to 2 and Comparative Examples 1 to 2, it can be seen that the addition of disodium hydrogen phosphate and tetrapentylammonium bromide to the mobile phase can effectively improve the peak shape and tailing factor, so that the theoretical plate number of each component is higher and the tailing factor is smaller, and the impurity content can be more accurately determined.

[0107] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for analyzing and detecting the intermediate cefcapene protozolin hydrochloride, characterized in that: The following steps are involved: Dissolving and diluting the cefuroxime precursor acid to be tested in sequence to obtain a sample for use in an apparatus; The sample on the machine is subjected to liquid chromatography detection to obtain chromatographic information of the cefuroxime precursor acid to be detected; Based on a predetermined standard curve, the chromatographic information of the cefuroxime precursor acid to be tested is analyzed and calculated to obtain the material composition and content of the cefuroxime precursor acid to be tested; The cefuroxime precursor acid to be tested includes cefuroxime precursor acid and impurities; The impurities include 7-[2-(2-amino-thiazol-4-yl)-pent-2-enoylamino]-3-carbamoyloxymethyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, tert-butyl (4-((Z)-1-((1,7-dioxo-1,4,5a,6-tetrahydro-3H,7H-azetidinyl[2,1-b]furanyl[3,4-d][1,3]thiazin-6-yl)amino)- one or more of (Z)-7-(2-(2-((tert-butyloxycarbonyl)amino)thiazol-4-yl)pent-2-enamido)-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate; The parameters of the liquid chromatography detection include: The chromatographic column is a chromatographic column with octadecylsilane bonded silica gel as a filler; The column temperature of the chromatographic column is 20-40°C; The mobile phase is a methanol-water solution; the volume ratio of methanol to water in the methanol-water solution is 70:30 to 60:40; the methanol-water solution also includes tetrapentylammonium bromide and sodium dihydrogen phosphate; the mass percentage of tetrapentylammonium bromide in the methanol-water solution is 1 to 10%; the mass percentage of sodium dihydrogen phosphate in the methanol-water solution is 0.1 to 3%; Flow rate: 0.6-1.2 mL / min; The detection wavelength is 245-275nm; The predetermined standard curves include a predetermined standard curve of cefuroxime precursor acid, a predetermined standard curve of 7-[2-(2-amino-thiazol-4-yl)-pent-2-enoylamino]-3-carbamoyloxymethyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, a predetermined standard curve of tert-butyl (4-((Z)-1-((1,7-dioxo-1,4,5a,6-tetrahydro-3H,7H-azetidinyl[2,1-b]furanyl[3,4-d][1,3]thiazine The standard curve of (Z)-2-(2-tert-butyloxycarbonylaminothiazol-4-yl)-2-pentenoic acid and the standard curve of 1,1,3,3-tetramethylguanidine (Z)-7-(2-(2-((tert-butyloxycarbonyl)amino)thiazol-4-yl)pent-2-enamido)-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate were prepared.

2. The analysis and detection method according to claim 1, characterized in that: The dissolved reagent includes methanol; the dosage ratio of the cefuroxime precursor acid to be detected and the dissolved reagent is 20 mg: 2-4 mL.

3. The analysis and detection method according to claim 1, characterized in that: The diluted reagent is the mobile phase.

4. The analysis and detection method according to any one of claims 1 to 3, characterized in that: The concentration of the cefuroxime precursor acid to be detected in the sample on the machine is 0.3-0.6 mg / mL.

5. The analysis and detection method according to claim 1, characterized in that: The parameters of the liquid chromatography detection also include: the injection volume is 20 μL.

6. The analysis and detection method according to claim 1, characterized in that: During the liquid chromatography detection, the chromatogram is recorded to 1.5 to 5 times of the retention time of the main peak.

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