CN115791989A - Separation and detection method for impurities in cefoperazone sodium or preparation thereof - Google Patents

Abstract

The invention relates to a method for detecting impurities in cefoperazone sodium or a preparation thereof. The detection method comprises the following steps: adopting a high performance liquid chromatography analysis method: octadecylsilane chemically bonded silica gel column or octylsilane chemically bonded silica gel column; temperature of the column: 25-35 ℃; flow rate: 0.8 ml/min-1.2 ml/min; detection wavelength: 250nm to 260nm; mobile phase: composed of a mobile phase A and a mobile phase B, and gradient elution is carried out; wherein the mobile phase A is a solution with the pH value of 2.0-3.0, and the mobile phase B is selected from one or more of acetonitrile, methanol and tetrahydrofuran; wherein, the impurities separated by the separation detection method comprise small molecule impurities and polymer impurities. The method has the advantages of multiple impurity types, high separation degree, high accuracy, high precision, high sensitivity and strong specificity, can detect the polymer impurities more accurately compared with the prior art, and is a reliable method for controlling the polymer impurities.

Description

Separation and detection method for impurities in cefoperazone sodium or preparation thereof

Technical Field

The invention relates to the technical field of medicine quality control, in particular to a separation and detection method of impurities in cefoperazone sodium or a preparation thereof.

Background

Drug impurity mass spectrometry control is a hot spot in current drug development. The impurity mass spectrometry can better reflect the source of impurities in the product, and further pertinently put forward a control strategy and better control the product quality. Beta-lactam antibiotic drugs (such as penicillin, cephalosporin and the like) are the most commonly used antibiotic drugs in clinic, and the types of organic impurities in the product are determined to be complex by the production process and the characteristics of the structure of the beta-lactam antibiotic drugs, wherein the beta-lactam antibiotic drugs not only comprise various isomers and degradation products, but also comprise polymer impurities generated by the polymerization reaction of active ingredients, so that the impurity spectrum analysis is difficult.

Taking cefoperazone sodium as an example, the cefoperazone sodium belongs to the third-generation cephalosporin antibiotics and is mainly used for treating respiratory system and urogenital system infection caused by sensitive bacteria. According to the Chinese pharmacopoeia 2020 edition, a high performance liquid chromatography (RP-HPLC) method and a gel chromatography method are adopted to respectively control small molecular impurities and polymer impurities of the compound, and the control method needs two detection means, so that the equipment investment cost is undoubtedly increased, and the efficiency is low.

Disclosure of Invention

At least one main purpose of the invention is to provide a method for detecting impurities in cefoperazone sodium or a preparation thereof, which can realize simultaneous separation and detection of small molecular impurities and polymer impurities of cefoperazone sodium, effectively improve detection efficiency, achieve more accurate quantitative effect, and be a reliable method for controlling polymer impurities in a raw medicine or a preparation.

In order to achieve the above purpose, the invention provides the following technical scheme:

one aspect of the present invention provides a method for separating and detecting impurities in cefoperazone sodium or a preparation thereof, which adopts a high performance liquid chromatography analysis method, wherein the conditions of the high performance liquid chromatography are as follows:

a chromatographic column: octadecyl silane bonded silica gel column or octyl silane bonded silica gel column;

temperature of the column: 25 to 35 ℃, preferably 27 to 33 ℃;

flow rate: 0.8 ml/min-1.2 ml/min;

detection wavelength: 250nm to 260nm;

mobile phase: composed of a mobile phase A and a mobile phase B, and gradient elution is carried out; wherein the mobile phase A is a solution with the pH value of 2.0-3.0, and the mobile phase B is selected from one or more of acetonitrile, methanol and tetrahydrofuran;

wherein, the impurities separated by the separation detection method comprise small molecule impurities and polymer impurities.

The cefoperazone sodium preparation can be a single preparation or a compound preparation, the compound preparation comprises typical preparations such as cefoperazone sodium sulbactam sodium for injection, cefoperazone sodium tazobactam sodium for injection and the like, and the dosage form of the single preparation or the compound preparation is not limited and is not limited to typical injection and the like.

In some embodiments, the mobile phase a is selected from one or more of a triethylamine acetic acid solution, an aqueous ammonia solution, an aqueous trifluoroacetic acid solution, an aqueous phosphoric acid solution, an aqueous citric acid solution, and an aqueous formic acid solution, preferably a triethylamine acetic acid solution or an aqueous ammonia solution, more preferably a triethylamine acetic acid solution; the mobile phase B is acetonitrile or methanol or tetrahydrofuran, preferably acetonitrile or methanol, more preferably acetonitrile.

In some embodiments, the pH of mobile phase a may be any value in the range of 2.0 to 3.0, such as 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, etc., more preferably pH2.4 to 2.6.

In some embodiments, the mobile phase a is a triethylamine acetic acid solution with a pH value of 2.0 to 3.0 or an aqueous ammonia solution, preferably a triethylamine acetic acid solution with a pH value of 2.4 to 2.6.

In some embodiments, the mobile phase is a combination of triethylamine acetic acid solution at a pH of 2.0 to 3.0 and acetonitrile, or a combination of triethylamine acetic acid solution at a pH of 2.0 to 3.0 and methanol, or a combination of triethylamine acetic acid solution at a pH of 2.0 to 3.0 and tetrahydrofuran.

In some embodiments, the mobile phase is a combination of triethylamine acetic acid solution at a pH of 2.4 to 2.6 and acetonitrile, or a combination of triethylamine acetic acid solution at a pH of 2.4 to 2.6 and methanol, or a combination of triethylamine acetic acid solution at a pH of 2.4 to 2.6 and tetrahydrofuran.

In some embodiments, the conditions for gradient elution are: in terms of volume ratio, 0min, A accounts for 90-92 percent, B accounts for 10-8 percent; 2min, 90 to 92 percent of A and 10 to 8 percent of B; 4min, 83-93 percent of A and 17-7 percent of B; 25min, wherein A is 83-93 percent, and B is 17-7 percent; 45min, A accounts for 60-80%, B40-20%; 55min, A is 60-80%, B is 40-20%; 56min, A is 90-92%, B is 10-8%; 60min, 90-92 percent of A and 10-8 percent of B.

In some embodiments, the conditions for gradient elution are: calculated by volume ratio, the mobile phase A is 91 percent for 0 to 2 min; reducing the mobile phase A from 91% to 88% in 2-4 min; 4-25 min, the mobile phase A is 88%; reducing the mobile phase A from 88% to 70% in 25-45 min; 45-55 min, and 70% of mobile phase A; the time is 55-56 min, and the mobile phase A rises from 70% to 91%; 56-60 min, and the mobile phase A is 91%.

In some embodiments, HPLC detection is performed at a sample size of 10-100. Mu.L, a flow rate of 1.0-1.2 mL/min, a column temperature of 25-35 deg.C, preferably 27-33 deg.C, which allows for a balance between resolution, analysis time, reproducibility, etc., or the detection wavelength can be further maintained at 254nm.

In some embodiments, the method of preparing a test solution is: dissolving cefoperazone sodium or its preparation in phosphate buffer solution, and diluting with diluent. Wherein the pH value of the phosphate buffer solution and the diluent is preferably close to the pH value of the mobile phase so as to facilitate elution. To this end, in some embodiments, the diluent consists of mobile phase a and mobile phase B, the mobile phase having a volume fraction of 90 to 92%. In some embodiments, the phosphate buffer has a pH of 2.0 to 3.0.

In some embodiments, the concentration of cefoperazone sodium in the test solution is from 2mg/mL to 5mg/mL.

In some embodiments, a separation detection method comprises: setting the high performance liquid chromatography conditions; preparing a test solution of cefoperazone sodium or a preparation thereof, and a control solution and/or a control solution; carrying out high performance liquid chromatography detection on the prepared solution, and recording a chromatogram; and calculating the content of the impurities by adopting a principal component self-comparison method and/or an external standard method. The reference solution is a solution obtained by diluting the test solution, and the reference solution is a solution to be prepared when the impurity is quantitatively calculated by adopting an external standard method.

In some embodiments, the contaminant control solution comprises an contaminant a control solution and an contaminant C control solution, the content of contaminant a and contaminant C being quantitatively calculated by the external standard method; and the contents of the impurities B, D, E and F are quantitatively calculated by a main component self-comparison method.

The various technical schemes of the invention can mainly separate out common small molecular impurities (including one or more of impurities A, B, C, D, E and F in the structural formula shown below) in the cefoperazone sodium product, and can also separate out polymer impurities (including cefoperazone polymer and polymer impurities 1 and 2). For the molecules with various types and large structural differences, the quantitative analysis methods which can be applicable are different, and are mainly selected from an internal standard method, an external standard method, a main component self-control method, an area normalization method and the like, and for different analysis methods, solutions which are matched with the analysis methods, such as a reference solution, a control solution and the like, need to be prepared in addition to a sample solution. Through multiple tests, the impurities A and C are more suitable for an external standard method, and the impurities B, D, E and F are more suitable for a main component self-contrast method.

In some embodiments, when formulating the test sample solution, a control solution is also formulated, as well as an impurity a control solution and an impurity C control solution.

In some embodiments, the test solution contains about 0.2mg to about 5mg of cefoperazone sodium per 1 mL; the control solution was prepared by a method comprising: taking part of the test solution, diluting the test solution into 1mL of a control solution containing 1-25 mu g of cefoperazone sodium by using a diluent; the impurity A and the impurity C are mixed in the reference solution, wherein about 3 mu g to 75 mu g of the impurity A is contained in each 1mL of the reference solution, and about 1 mu g to 25 mu g of the impurity C is contained in each 1mL of the reference solution.

The main component self-contrast method adopted by the invention is a main component self-contrast method added with a correction factor or a main component self-contrast method not added with the correction factor, compared with gel chromatography, the high performance liquid chromatography (RP-HPLC) method has strong specificity, and the main component self-contrast method is adopted for impurity quantification, so that the polymer impurity content determination is more accurate.

In addition, in order to confirm whether the polymer impurity is the index impurity in the cefoperazone sodium or the preparation thereof, the invention also carries out forced degradation treatment on the medicine, and if the content of the corresponding polymer impurity is increased, the polymer impurity can be basically identified as the index impurity.

In some embodiments, the forced degradation treatment comprises subjecting an aqueous solution containing about 200mg of cefoperazone sodium per 1mL to a forced oxidative degradation at room temperature for 10 to 60 days. By carrying out forced degradation treatment, a test solution rich in polymer impurities can be prepared, so that the polymer impurities 1 and the polymer impurities 2 can be better separated and detected. The polymer impurities can be used as the indicative impurities of the cefoperazone sodium polymer, so that the quality controllability of the cefoperazone sodium or the preparation thereof is further improved.

Based on the specific chromatographic conditions, the invention can at least achieve the following technical effects:

(1) The high performance liquid chromatography method capable of simultaneously controlling the micromolecular impurities and the polymer impurities of the cefoperazone sodium is provided, and the detection efficiency can be effectively improved. Specifically, the method comprises the following steps:

the method can separate common small molecular impurities (including impurity A, impurity B, impurity C, impurity D, impurity E and impurity F in the following structural formula) in the cefoperazone sodium product, and can also separate polymer impurities (including cefoperazone polymer and polymer impurities 1 and 2). For cefoperazone sodium, the existing separation and detection method which can simply separate small molecular impurities or polymer impurities is known, but the method can simultaneously separate small molecular impurities and polymer impurities at one time, greatly improves the detection efficiency, and has important significance for controlling the quality of medicines in actual industrial production.

(2) By improving the detection method, new polymer impurities are effectively separated, and the new polymer impurities can be used as index impurities of cefoperazone sodium or a preparation thereof so as to evaluate the stability of a raw drug/preparation. The separated polymer impurities at least comprise polymer impurities 1 and polymer 2, wherein the polymer 1 is a first chromatographic peak which shows a peak after the impurity B and has a retention time of 1.5-2.5 relative to the cefoperazone peak, the polymer 2 is a second chromatographic peak which shows a peak after the impurity B and has a retention time of 1.5-2.5 relative to the cefoperazone peak, the structure of the polymer impurities 1 is shown as the following formula (1), and the structure of the polymer impurities 1 is selected from one or more of the following formulas (2-a) and (2-B). Experiments show that after the cefoperazone sodium and the preparation thereof are subjected to forced degradation treatment, the content of polymer impurities 1 and 2 is obviously increased, and the cefoperazone sodium and the preparation thereof are proved to be capable of being used as index impurities of the cefoperazone polymer, so that compared with the existing detection method, only one detection means is needed, and the cost is lower.

(3) The method has the characteristics of high detection accuracy, high detection precision, high sensitivity, strong specificity and the like, and is a reliable method for controlling polymer impurities in the cefoperazone sodium original drug or preparation.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:

FIGS. 1 to 3 are chromatograms of a blank solution, a test solution and a system suitability solution in example 1, respectively;

FIG. 4 is an excimer peak of impurity of Polymer impurity 1;

FIG. 5 is an excimer peak of impurity for Polymer impurity 2;

FIG. 6 is a high performance liquid chromatogram of example 6;

FIG. 7 is a high performance liquid chromatogram of example 7;

FIG. 8 is a high performance liquid chromatogram of comparative example 1;

FIG. 9 is a high performance liquid chromatogram of comparative example 2;

FIG. 10 is a high performance liquid chromatogram of comparative example 3.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The raw materials, reagents or instruments used are not indicated by manufacturers, and all the raw materials, the reagents or the instruments are conventional products which can be obtained by commercial purchase or can be prepared according to the prior art.

The method is established and verified by taking a cefoperazone sodium raw material drug as an example.

Wherein, the cefoperazone reference substance and each impurity reference substance used in the following examples are: cefoperazone reference substance (content: 93.8%, china institute for food and drug testing, batch number: 130420-201105); impurity A (content: 98.1%, china institute for food and drug testing, lot number: 130428-201605); impurity B (content: 95.8%, bigdu, lot number: PN 5254-IMP-B-180719); impurity C (content: 99.8%, bigdu, lot number: PN 5254-IMP-C-170922); impurity D (content: 95.0%, TLC, lot No. 1530-014A 4); impurity E (content: 98.6%, molcan corporation, lot number 151203); impurity F (content: 100.0%, china institute for testing food and drug, lot number: 130412-200902). Impurities a to F have the structural formulae as shown above.

Example 1

Diluent agent: triethylamine acetic acid solution (pH 2.5) -acetonitrile (91: 9).

Test solution: taking a proper amount of cefoperazone sodium, precisely weighing, adding a small amount of phosphate buffer solution (weighing 1.0g of potassium dihydrogen phosphate and 1.8g of disodium hydrogen phosphate, adding 1000ml of water for dissolving to obtain the cefoperazone sodium), and quantitatively diluting with diluent [ triethylamine acetic acid solution (pH value 2.5) -acetonitrile (91: 9) ] to prepare a solution containing about 2mg of cefoperazone sodium in each 1ml of cefoperazone sodium.

Control solution: precisely measuring 1ml of the test solution, placing in a 200ml measuring flask, diluting to scale with diluent, and shaking.

Mixed impurity control solution of impurity a and impurity C: precisely weighing appropriate amount of cefoperazone impurity A reference substance and cefoperazone impurity C reference substance, dissolving with small amount of acetonitrile, performing ultrasonic treatment for 3min, and quantitatively diluting with diluent to obtain 30 μ g and 10 μ g solutions containing cefoperazone impurity A and cefoperazone impurity C respectively per 1 ml.

System applicability solution: respectively taking appropriate amount of cefoperazone reference substance and each impurity reference substance, dissolving with diluent, and diluting to obtain solution containing impurity E, impurity D, impurity C, impurity A, cefoperazone, impurity F, and impurity B about 10 μ g, 30 μ g, 2mg, 10 μ g, and 10 μ g, respectively, per 1 ml.

And (3) respectively injecting a blank solution (diluent), a test sample solution and a system applicability solution into a chromatograph for detection.

Chromatographic conditions are as follows:

agilent Eclipse XDB-C18, 150X 4.6mm,5 μm, mobile phase A: triethylamine acetic acid solution (taking 14ml of triethylamine and 5.7ml of glacial acetic acid, adding water to dilute to 100ml, shaking up, taking 1.2ml and 880ml of water to mix evenly, and adjusting the pH value to 2.5 with glacial acetic acid), mobile phase B: acetonitrile, flow rate: 1.0 mL/min ^-1 Ultraviolet detection wavelength: 254nm; column temperature: 30 ℃; sample introduction amount: 30 μ L. The elution gradient is shown in Table 1.

TABLE 1 gradient elution procedure

As a result:

as shown in fig. 1 to 3, the chromatograms of the blank solution, the test solution and the system suitability solution respectively show that: (1) not only can detect all the known impurities (impurities A, B, C, D, E and F) loaded in pharmacopoeia of various countries, but also can find two new unknown polymer impurities (as shown in figure 3) within the range of the retention time of the impurity B relative to the cefoperazone peak to be 1.5-2.5; (2) the blank solution (diluent) does not interfere with the measurement, and the separation degree of each chromatographic peak and adjacent chromatographic peaks of the solution with the applicability of the system is more than 1.5.

Example 2 structural analysis of polymer-based impurities

Test solution: taking about 20.0g of cefoperazone sodium, putting the cefoperazone sodium into a 100ml volumetric flask, adding water to dissolve and dilute the cefoperazone sodium to a scale, preparing a solution containing about 200mg/ml of cefoperazone sodium, and standing the solution at room temperature for 30 days to obtain a mother solution rich in polymer impurities. Taking 1ml of mother liquor, placing the mother liquor into a 100ml measuring flask, adding a diluent to quantitatively dilute the mother liquor to obtain a solution containing about 2mg of the mother liquor in each 1ml of the mother liquor, wherein the solution is used as a test solution rich in polymer impurities. And (3) performing mass spectrometry on the test solution rich in the polymer impurities by adopting an LC-MS method.

As a result:

the presence of excimer peaks at M/z 679.51130, M/z 717.46758 (FIG. 4), respectively, [ M + H ] + and [ M + K ] + peaks in the + ESI plot of polymer impurity 1 (FIG. 3) suggests that the molecular weight of this impurity is 678Da; the structure is shown in formula I.

The presence of excimer peaks at M/z 792.15286, M/z 130.86103 (FIG. 5), respectively, [ M + H ] + and [ M + K ] + in the + ESI plot of impurity 2 (FIG. 3) of the polymer, indicating a molecular weight of 791Da for this impurity; the structure is shown in formula 2-a or 2-b.

Example 3 linearity and correction factor, detection Limit (LOD), quantitation Limit (LOQ) of each impurity

Accurately weighing proper amounts of cefoperazone, impurities A, B, C, D, E and F respectively to prepare a series of mixed reference solutions with LOQ and limit concentrations of 40%, 80%, 100%, 120%, 200% and 400% (wherein 100% refers to the maximum limit of the allowable impurities in the drug, and the maximum limits of A, B, C, D, E and F are 1.5%, 0.5% and 0.5%, respectively), carrying out sample injection determination, and recording the chromatographic peak areas of cefoperazone and each impurity respectively. Wherein, the 10 times value of the signal-to-noise ratio is used as the quantitative limit of each impurity, and the 3 times value of the signal-to-noise ratio is used as the detection limit of each impurity. And (4) performing linear regression on the peak areas (A) and the respective mass concentrations (C) to calculate linear equations of the cefoperazone and the impurities. Two sets of chromatographs are adopted for detection: agilenggt 1260 hplc (i.e., instrumentation system 1), waters e2695 hplc (i.e., instrumentation system 2), chromatography conditions example 1.

The default correction factor for polymer impurity 1 and polymer impurity 2 in the subsequent sample impurity level calculations is 1 (i.e., the principal component self-control method without correction factor).

As a result:

(1) cefoperazone and each impurity have good linear relation in respective measuring range (table 2); (2) the detection limits (s/n = 3) of cefoperazone, impurity a, impurity B, impurity C, impurity D, impurity E and impurity F are 9.06, 1.86, 8.11, 0.62, 0.52, 0.54 and 10.05ng, respectively; (3) the quantitative limits (s/n is more than 10) are respectively 18.12, 18.60, 16.22, 2.05, 1.75, 18.16 and 20.10ng, and the RSD of the peak area of the continuous sample injection 6 needle is respectively 1.5%, 0.4%, 2.1%, 5.8%, 0.8%, 2.5% and 4.3%; (4) see table 2, some impurities have satisfactory correction factors (correction factors relative to cefoperazone); when the correction factor is not in the range of 0.7-1.3, the method is an ideal method for quantification by adopting an external standard method of an impurity reference substance. Referring to the 2020 edition of Chinese pharmacopoeia, and combining the data of Table 2, the invention selects the external standard method for quantifying cefoperazone impurity A (correction factor is 0.86) and impurity C (correction factor is 0.40), and adopts the main component self-contrast method for quantifying cefoperazone impurity B, impurity D, impurity E and impurity F (correction factors are 0.86,1.14,0.77 and 1.19 respectively), and proposes to introduce the correction factors to improve the detection accuracy.

TABLE 2 Standard Curve and Linear Range of cefoperazone and impurities

Example 4 specificity

The specificity of the method is further verified by a forced degradation test: taking about 100mg of cefoperazone sodium, and 6 parts respectively, subjecting to nondestructive, solid illumination (4500 + -500Lx, 7d), solid high temperature (60 deg.C, 7 d), oxidation (0.3% hydrogen peroxide 5mL, standing at room temperature for 4 h), acid (0.01 mol/L HCl, standing at room temperature for 25 h), alkali (0.01 mol/L NaOH, standing at room temperature for 10 min), dissolving with diluent, and diluting to obtain 2 mg. ML ^-1 The solution of (1); respectively and precisely measuring 30 mu L of the mixture, injecting the mixture into a chromatograph, and detecting the mixture under the same chromatographic conditions as the experiment I.

As a result:

(1) under the conditions of acid, alkali, oxidation and high-temperature forced degradation, cefoperazone sodium is degraded to different degrees (table 3), and each degraded sample is analyzed, the separation degree of a main peak and an impurity peak is greater than 1.5, the purity angle of the main peak is smaller than a purity threshold value, and the mass balance is between 90% and 110% (table 4).

(2) Polymer impurity 1 and polymer 2 were significantly increased in the forced oxidative degradation reaction (table 3), and could be detected in the actual sample, suggesting that this impurity may be a degradation product of cefoperazone dimer, and could be used as an index impurity for cefoperazone polymers.

TABLE 3 summary of the degraded impurities

TABLE 4 summary of the results of the forced degradation experiments

Example 5 accuracy

About 22mg of a test sample with known impurity content is precisely weighed, the test sample is respectively placed into 9 10mL measuring bottles, impurities A, B, C, D, E and F are respectively added to prepare solutions with about 40 percent, 100 percent (respectively referring to the maximum allowable limit of each impurity in the medicine) and 200 percent of each impurity, the content of each solution is measured, the recovery rate is calculated, and the chromatographic conditions are the same as those in the experiment (I).

As a result:

as shown in Table 5, the recovery rate of each impurity at each concentration level is between 90% and 108%, and the recovery rate RSD is less than 10.0%.

TABLE 5 cefoperazone and respective impurity recovery results

Example 6

The chromatographic conditions were varied from example 1 with the exception that the pH of mobile phase A was 2.6, and the chromatographic conditions were otherwise the same as in example 1.

The detection results are shown in fig. 6, and the results show that: the separation degree of the impurity D and the impurity E is reduced from 3.2 to 2.9, which shows that the pH value of the mobile phase A has a large influence on the separation degree of the impurity.

Example 7

The chromatographic conditions were varied from example 1 with the initial ratio being 10% of mobile phase B and the rest of the chromatographic conditions being the same as in example 1.

The detection results are shown in FIG. 7, and the results show that: the impurity A is sensitive to the change of the initial proportion, when the initial proportion is that the proportion of the mobile phase B is 10%, the separation degree of the impurity A and the adjacent unknown impurity is obviously reduced, and the separation degree is 1.26 and is less than 1.5.

Comparative example 1

The difference from example 1 is the change in gradient elution, which is performed according to Table 6 below, and the rest of the chromatographic conditions are the same as in example 1.

TABLE 6

Elution time	Mobile phase A volume fraction%	Mobile phase B volume fraction%
			0min	88	12
25min	88	12
			45min	75	25
55min	75	25
			56min	88	12
65min	88	12

The detection result is shown in fig. 8, and the result shows that when the initial proportion of the mobile phase B is greater than 10%, the cefoperazone impurity D and the cefoperazone impurity E cannot achieve baseline separation, which indicates that the initial proportion of the organic phase is too high, and is not beneficial to impurity separation.

Comparative example 2

The difference from example 1 is the use of mobile phase a: mobile phase B =88 isocratic elution of 12, the rest of the chromatographic conditions being the same as in example 1.

As a result, as shown in fig. 9, it was found that, when mobile phase a: mobile phase B =88 isocratic elution of 12, without increasing the organic phase ratio, small molecule impurities can be eluted, but polymer impurities cannot be eluted. It can be seen that the present invention achieves unexpected results using specific elution conditions.

Comparative example 3

The difference from example 1 is that the column temperature is different, in this example, the column temperature is selected to be 20 ℃, the other conditions are kept unchanged, and the influence of the column temperature on the impurity separation is examined.

The detection result is shown in fig. 10, and the result shows that when the column temperature is 20 ℃, cefoperazone impurity F and cefoperazone cannot achieve baseline separation, which indicates that the difference of the column temperature affects the impurity separation effect.

While the invention has been described with reference to specific preferred embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the following claims. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims (10)

1. A separation and detection method for impurities in cefoperazone sodium or a preparation thereof is characterized in that a high performance liquid chromatography analysis method is adopted, wherein the high performance liquid chromatography conditions are as follows:

and (3) chromatographic column: octadecyl silane bonded silica gel column or octyl silane bonded silica gel column;

temperature of the column: 25-35 ℃;

flow rate: 0.8 ml/min-1.2 ml/min;

detection wavelength: 250nm to 260nm;

mobile phase: composed of a mobile phase A and a mobile phase B, and gradient elution is carried out; wherein the mobile phase A is a solution with the pH value of 2.0-3.0, and the mobile phase B is selected from one or more of acetonitrile, methanol and tetrahydrofuran;

wherein, the impurities separated by the separation detection method comprise small molecule impurities and polymer impurities.

2. The separation detection method according to claim 1, wherein the conditions of the gradient elution are:

in terms of volume ratio, 0min, A accounts for 90-92 percent, B accounts for 10-8 percent; 2min, 90 to 92 percent of A and 10 to 8 percent of B; 4min, 83-93 percent of A and 17-7 percent of B; 25min, 83 to 93 percent of A and 17 to 7 percent of B; 45min, A accounts for 60-80%, B40-20%; 55min, A is 60% -80%, B is 40% -20%; 56min, A is 90-92%, B is 10-8%; 60min, 90-92 percent of A and 10-8 percent of B.

3. The separation detection method according to claim 1, wherein the conditions of the gradient elution are: calculated by volume ratio, the mobile phase A is 91 percent for 0 to 2 min; reducing the mobile phase A from 91% to 88% in 2-4 min; 4-25 min, the mobile phase A is 88%; reducing the mobile phase A from 88% to 70% in 25-45 min; 45-55 min, and 70% of mobile phase A; the time is 55-56 min, and the mobile phase A is increased from 70% to 91%; 56-60 min, and the mobile phase A is 91%.

4. The separation detection method according to any one of claims 1 to 3, wherein the mobile phase A is one or more selected from triethylamine acetic acid solution, aqueous ammonia solution, aqueous trifluoroacetic acid solution, aqueous phosphoric acid solution, aqueous citric acid solution and aqueous formic acid solution, preferably triethylamine acetic acid solution or aqueous ammonia solution, more preferably triethylamine acetic acid solution; the mobile phase B is acetonitrile or methanol, preferably acetonitrile; the mobile phase A is preferably a solution with a pH value of 2.4-2.6.

5. The separation detection method according to claim 1, wherein the small molecule impurities comprise at least one of:

the polymer impurity comprises polymer impurity 1 and/or polymer impurity 2, wherein the polymer impurity 1 is a first chromatographic peak which shows a peak after the impurity B and has a retention time relative to a cefoperazone peak of 1.5-2.5; the polymer impurity 2 is the second chromatographic peak which appears after the impurity B and has a retention time of 1.5-2.5 relative to the cefoperazone peak.

6. The separation detection method according to claim 5, wherein the structure of the polymer impurity 1 is represented by the following formula (1), and the structure of the polymer impurity 2 is selected from one or more of the following formulae (2-a), (2-b):

7. the separation detection method according to claim 5 or 6, characterized in that the separation detection method comprises:

setting the high performance liquid chromatography conditions;

preparing a test solution of cefoperazone sodium or a preparation thereof, and preparing a reference solution and/or a reference solution;

carrying out high performance liquid chromatography detection on the prepared solution, and recording a chromatogram; and

and calculating the content of the impurities by adopting a main component self-comparison method and/or an external standard method.

8. The separation detection method according to claim 7, wherein the reference solution comprises an impurity A reference solution and an impurity C reference solution, and the contents of the impurity A and the impurity C are quantitatively calculated by the external standard method;

and the number of the first and second groups,

the contents of the impurities B, D, E and F are quantitatively calculated by a main component self-contrast method.

9. The separation and detection method according to claim 1, wherein the cefoperazone sodium preparation is a single preparation or a compound preparation, and the compound preparation is one or more selected from cefoperazone sodium sulbactam sodium for injection and cefoperazone sodium tazobactam sodium for injection.

10. The assay method of claim 7, wherein the test solution is prepared by a method comprising: dissolving cefoperazone sodium or its preparation in water or phosphate buffer solution, and diluting with diluent.

Images