Method for simultaneously detecting multiple intermediates in ceftaroline fosamil preparation process

Abstract

The invention provides a method for simultaneously detecting multiple intermediates in a ceftaroline fosamil preparation process. A high performance liquid chromatography is adopted to simultaneously detect a starting raw material-3-hydroxy cephalosporin in a synthesis process and sequentially generated intermediates: CfMs, CT-3, CT-3M, 7-APTM and 7-ATM. The method has the advantages that different analysis methods do not need to be replaced to cope with different samples, the change conditions of the six substances in the reaction process can be displayed on the same atlas, and the method is simple, convenient, accurate, rapid and clear at a glance, and is also greatly helpful for ensuring the quality of a ceftaroline fosamil product and improving the synthesis work efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and establishes a method for simultaneously detecting multiple intermediates in the preparation process of ceftaroline fosamil in order to improve the efficiency of ceftaroline fosamil synthesis and product quality. Background Art

[0002] Cefuroxime is a fifth-generation cephalosporin drug with good antibacterial activity against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus infection, multidrug-resistant Streptococcus pneumoniae infection and common Gram-negative bacteria infection. At present, Cefuroxime for Injection was approved by the US FDA on October 29, 2010 and is produced by AstraZeneca. There are no products that have been applied for or marketed in China. Today, when bacterial resistance is a global problem, Cefuroxime, as a new drug against multidrug-resistant bacteria, has broad application prospects.

[0003] A brief description of the preparation process of cefuroxime fosamil (only involving the six intermediates to be detected in the present invention):

[0004] The first step is to prepare CfMs from 3-hydroxycephalosporin

[0005]

[0006] The second step reaction, preparation of CT-3

[0007]

[0008] The third step reaction, preparation of CT-3M

[0009]

[0010] Step 4: Preparation of 7-APTM

[0011]

[0012] Step 5: Preparation of 7-ATM

[0013]

[0014] From the above findings: the synthesis of ceftaroline fosamil has many steps and the process is relatively complicated. We require monitoring of the intermediates produced in each step of the reaction. By observing the relative content of the reacted (or remaining) and newly produced substances, we can determine whether the reaction is sufficient and whether the next step of feeding can be carried out. A small amount of intermediates will remain in each reaction or some by-products will appear, all of which should be treated as impurities and controlled under this process to ensure the quality of the final product.

[0015] 3-Hydroxycephalosporin, CfMs, CT-3, CT-3M, 7-APTM and 7-ATM involved in the reaction are all identified as impurities in intermediates and products and are controlled.

[0016] In the existing preparation process, only "rough" intermediate control is often considered. For the sake of convenience, an attempt was made to use isocratic elution, which resulted in very unsatisfactory monitoring results and very poor accuracy. Using different analytical methods will greatly reduce production efficiency. Summary of the invention

[0017] The present invention aims to overcome the above-mentioned defects and provide a method for detecting various intermediates in the preparation process of ceftaroline fosamil, which does not require the replacement of different analytical methods to cope with different samples, can display the changes of six substances in the reaction process on the same graph, has the advantages of being simple, accurate, fast and clear at a glance, and is also very helpful for ensuring the quality of ceftaroline fosamil products and improving the efficiency of synthesis work.

[0018] The present invention provides a method for simultaneously detecting multiple intermediates in the preparation process of cefuroxime fosamil, using high performance liquid chromatography, characterized in that:

[0019] The mobile phase A and mobile phase B of the above-mentioned high performance liquid chromatography method are acetate buffer-acetonitrile with different compositions;

[0020] The mobile phase A is 0.04-0.12 mol / L acetate buffer-acetonitrile.

[0021] Wherein, the volume ratio of acetate buffer to acetonitrile is 99-85:1-15;

[0022] The mobile phase B is 0.04-0.12 mol / L acetate buffer-acetonitrile.

[0023] Wherein, the volume ratio of acetate buffer to acetonitrile is 10-25:90-75;

[0024] Elution mode: gradient elution.

[0025] Furthermore, the present invention provides a method for simultaneously detecting multiple intermediates in the preparation process of ceftaroline fosamil, which is also characterized in that:

[0026] The mobile phase A is 0.08 mol / L acetate buffer-acetonitrile.

[0027] The volume ratio of acetate buffer to acetonitrile was 95:5;

[0028] The mobile phase B is 0.08 mol / L acetate buffer-acetonitrile.

[0029] The volume ratio of acetate buffer to acetonitrile was 18:82.

[0030] Furthermore, the present invention provides a method for simultaneously detecting multiple intermediates in the preparation process of ceftaroline fosamil, which is also characterized in that:

[0031] The above gradient elution program is as follows:

[0032] Time (min) A(％) B(％) 0 90-100 10-0 5 90-100 10-0 10 38～46 62～54 20 38～46 62～54 25 24～30 76～70 40 24～30 76～70 41 90-100 10-0 50 90-100 10-0

[0033] Furthermore, the present invention provides a method for simultaneously detecting multiple intermediates in the preparation process of ceftaroline fosamil, which is also characterized in that:

[0034] The above gradient elution program is as follows:

[0035] Time (min) A(％) B(％) 0 100 0 5 100 0 10 42 58 20 42 58 25 27 73 40 27 73 41 100 0 50 100 0

[0036] Furthermore, the present invention provides a method for simultaneously detecting multiple intermediates in the preparation process of ceftaroline fosamil, which is also characterized in that:

[0037] The chromatographic column used in the above-mentioned high performance liquid chromatography method is Waters Symmetry Shield RP185μm4.6*250mm.

[0038] Furthermore, the present invention provides a method for simultaneously detecting multiple intermediates in the preparation process of ceftaroline fosamil, which is also characterized in that:

[0039] The flow rate of the above HPLC method was 1.0 ml / min, the detection wavelength was 230 nm, the column temperature was 30° C., and the injection volume was 20 μl.

[0040] In addition, the method for simultaneously detecting multiple intermediates in the preparation process of ceftaroline fosamil provided by the present invention is applied to simultaneously observing the content changes of 3-hydroxycephalosporin, CfMs, CT-3, CT-3M, 7-APTM and 7-ATM during the reaction process.

[0041] Functions and effects of the present invention:

[0042] The analysis method, which is simple, accurate and rapid, can display the changes of six substances in the whole reaction process on the same graph and provide more reliable data to achieve the purpose of effectively optimizing the synthesis process and ensuring the quality of ceftaroline fosamil products. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Spectra of embodiments of the present invention;

[0044] Figure 2 For the spectrum of Comparative Example 1;

[0045] Figure 3 For the spectrum of Comparative Example 2;

[0046] Figure 4 For the spectrum of Comparative Example 3; DETAILED DESCRIPTION

[0047] This embodiment provides a method for simultaneously detecting multiple intermediates in the preparation process of ceftaroline fosamil. By using high performance liquid chromatography, the content changes of 3-hydroxycephalosporin, CfMs, CT-3, CT-3M, 7-APTM and 7-ATM during the reaction can be simultaneously observed. The characteristics of the method are as follows:

[0048] The embodiment operates according to the following steps.

[0049] Instrument: Shimadzu LC-10 series

[0050] Chromatographic column: Waters Symmetry Shield RP185μm 4.6*250mm;

[0051] Mobile phase composition: Mobile phase A is 0.04-0.12 mol / L acetate buffer-acetonitrile,

[0052] Wherein, the volume ratio of acetate buffer to acetonitrile is 99-85:1-15;

[0053] Mobile phase B is 0.04-0.12 mol / L acetate buffer-acetonitrile,

[0054] Wherein, the volume ratio of acetate buffer to acetonitrile is 10-25:90-75;

[0055] The most preferred composition of the mobile phase (i.e., the best solution of this embodiment): mobile phase A is 0.08 mol / L acetate buffer-acetonitrile (the volume ratio of acetate buffer to acetonitrile is 95:5);

[0056] Mobile phase B was 0.08 mol / L acetate buffer-acetonitrile (volume ratio of acetate buffer to acetonitrile was 18:82);

[0057] The gradient elution program is as follows:

[0058] Time (min) A(％) B(％) 0 90-100 10-0 5 90-100 10-0 10 38～46 62～54 20 38～46 62～54 25 24～30 76～70 40 24～30 76～70 41 90-100 10-0 50 90-100 10-0

[0059] The preferred composition of the gradient elution program (i.e., the best solution of this embodiment) is:

[0060] Time (min) A(％) B(％) 0 100 0 5 100 0 10 42 58 20 42 58 25 27 73 40 27 73 41 100 0 50 100 0

[0061] Flow rate: 1.0ml / min;

[0062] Detection wavelength: 230nm,

[0063] Column temperature: 30°C;

[0064] Injection volume: 20 μl;

[0065] Weighing and preparation: Weigh 3-hydroxycephalosporin: 0.0059g, CfMs: 0.0057g, CT-3: 0.0058g, CT-3M: 0.0048g, 7-APTM: 0.0052g and 7-ATM: 0.0047g. Add to a 10ml volumetric flask, dissolve with 0.08mol / L acetate buffer-acetonitrile (40:60), dilute to scale, and the prepared mixed solution is the test solution, inject, elute, and record the chromatogram until 40 minutes.

[0066] like Figure 1 As shown, the six substances appear in a certain order on the same graph, with sharp peaks and good separation.

[0067] The peak order of the six substances is as follows

[0068]

[0069]

[0070] Therefore, when this method is applied to the above synthesis process, six intermediates are produced in sequence. As the feed is added and the reaction progresses, the content of the former decreases and the content of the latter increases, one after another, and continuously advances. Thus, the changes in the peak area of ​​each intermediate and their final residual amount can be observed.

[0071] Comparative Example 1.

[0072] Using 0.1 mol / L acetate buffer-acetonitrile (90:10) as the mobile phase, six intermediates were detected. Although 7-ATM could be detected, the following intermediates could not be eluted, such as Figure 2 As shown, even if there is one, the peak is low.

[0073] Comparative Example 2.

[0074] Using 0.1 mol / L acetate buffer-acetonitrile (15:85) as the mobile phase, six intermediates were detected. Although the peaks appeared quickly, Figure 2 As shown, the intermediates are all squeezed together and cannot be separated.

[0075] Comparative Example 3.

[0076] Using 0.1 mol / L acetate buffer-acetonitrile (45:55) as mobile phase, six intermediates were detected and some of them could be separated, but Figure 4 As shown, several substance peaks still cannot be effectively separated and detected.

[0077] Research has found that the six intermediates have different structural characteristics and polarities, which results in very different chromatographic performances. Therefore, an elution method with a fixed mobile phase ratio is only suitable for one or two intermediates. If other substances are to be detected, it is necessary to replace the mobile phase with other ratios, which results in the inability to accurately locate the intermediates produced in the previous steps or even the previous step, and the peak shape and separation are not good. Of course, it is even more unknown what their residual amount in the following reaction steps is and their relative ratio to the new substances. This not only results in low analysis efficiency, but also makes it impossible to continuously observe the changes of each intermediate during the entire reaction process, which is of little help to the synthesis work and the product quality cannot be guaranteed at all.

[0078] After a large number of experiments, the analytical conditions were repeatedly adjusted, and a gradient elution method was now adopted. The technical solution of the present invention is:

[0079] The chromatographic column used was Waters Symmetry Shield RP185μm 4.6*250mm. Mobile phase A was 0.08mol / L acetate buffer-acetonitrile (95:5); mobile phase B was 0.08mol / L acetate buffer-acetonitrile (18:82), gradient elution. The flow rate was 1.0ml / min, the detection wavelength was 230nm, the column temperature was 30℃, and the injection volume was 20μl.

[0080] Gradient elution program

[0081] Time (min) A(％) B(％) 0 100 0 5 100 0 10 42 58 20 42 58 25 27 73 40 27 73 41 100 0 50 100 0

[0082] Weigh appropriate amounts of the above six substances and dissolve them in 0.08 mol / L acetate buffer-acetonitrile (40:60). The amount of each substance is about 0.5 mg / ml. The prepared mixed solution is the test solution, which is injected and eluted.

[0083] Through the above comparison, it can be found that by adopting the method of the present invention, sampling to obtain a chromatogram, six substances have peaks in a certain order on the same graph, the peak shape is sharp, the separation is good, and it meets the monitoring requirements of synthetic intermediates. Therefore, the invention is of great help to the synthesis of ceftaroline fosamil.

Claims

1. A method for simultaneously detecting multiple intermediates in the preparation process of cefuroxime fosamil, using high performance liquid chromatography, characterized in that: The mobile phase A and mobile phase B of the high performance liquid chromatography method are acetate buffer-acetonitrile of different compositions; the mobile phase A is 0.04-0.12 mol / L acetate buffer-acetonitrile, Wherein, the volume ratio of acetate buffer to acetonitrile is 99-85:1-15; The mobile phase B is 0.04-0.12 mol / L acetate buffer-acetonitrile, Wherein, the volume ratio of acetate buffer to acetonitrile is 10-25:90-75; Elution mode: gradient elution.

2. A method for simultaneously detecting multiple intermediates in the preparation process of ceftaroline fosamil as claimed in claim 1, characterized in that: The mobile phase A is 0.08 mol / L acetate buffer-acetonitrile, The volume ratio of acetate buffer to acetonitrile was 95:5; The mobile phase B is 0.08 mol / L acetate buffer-acetonitrile, The volume ratio of acetate buffer to acetonitrile was 18:

82.

3. A method for simultaneously detecting multiple intermediates in the preparation process of ceftaroline fosamil as claimed in claim 1, characterized in that: The gradient elution procedure is as follows:

4. A method for simultaneously detecting multiple intermediates in the preparation process of cefuroxime as claimed in claim 1, characterized in that: The gradient elution procedure is as follows:

5. A method for simultaneously detecting multiple intermediates in the preparation process of ceftaroline fosamil as claimed in claim 1, characterized in that: The chromatographic column of the high performance liquid chromatography method is Waters Symmetry Shield RP185μm 4.6*250mm.

6. A method for simultaneously detecting multiple intermediates in the preparation process of ceftaroline fosamil as claimed in claim 1, characterized in that: The flow rate of the HPLC method was 1.0 ml / min, the detection wavelength was 230 nm, the column temperature was 30° C., and the injection volume was 20 μl.

7. Application of a method for simultaneously detecting multiple intermediates in the preparation process of ceftaroline fosamil as described in any one of claims 1 to 6 in simultaneously observing the content changes of 3-hydroxycephalosporin, CfMs, CT-3, CT-3M, 7-APTM and 7-ATM during the reaction process.

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