Method for separating and preparing beta-lactam antibiotic polymer impurities

Abstract

The invention discloses a method for separating and preparing beta-lactam antibiotic polymer impurities. According to the preparation method of the polymer in the beta-lactam antibiotic, the polymer in the beta-lactam antibiotic can be efficiently and stably prepared and enriched, and a foundation is laid for passive skin allergy tests and in-vitro allergenicity evaluation research.

Description

Technical Field

[0001] This application relates to the field of analytical testing, and in particular to a method for separating and preparing β-lactam antibiotic polymer impurities. Background Technology

[0002] β-lactam antibiotics are semi-synthetic antibiotics that exert their antibacterial effect by inhibiting the production of bacterial cell wall proteins. Ampicillin is one of the commonly used drugs in clinical practice, and its common adverse reaction is immediate hypersensitivity reaction. Studies have shown that the allergens of immediate hypersensitivity reactions are usually not the drug itself, but are related to polymer impurities present in it. Therefore, the analysis and evaluation of polymers in injectable ampicillin sodium is the key to drug quality control. The current detection methods for polymer impurities in ampicillin are mainly chromatographic methods, including: 1) gel filtration chromatography, in which the separation of ampicillin polymers and the drug itself is achieved in the Sephadex G-10 dextran gel chromatography system. 2) In "Study on Ampicillin Polymers - I. Separation and Analysis of Ampicillin Polymers" (Antibiotics 1987, (04):241-245), DEAE-dextran gel A-25 ion exchange column chromatography was used to separate various polymers of ampicillin sodium from aqueous solution, and it was confirmed that they were dimers, trimers, tetramers and pentamers of ampicillin sodium. However, ion exchange chromatography is expensive and not suitable for large-scale separation of polymer impurities. 3) In the study "LC-MS / MS determination of impurity profiles of ampicillin raw materials and preparations" (Chinese Journal of Antibiotics, 2023, 48(10):1151-1162), LC-MS / MS can effectively elute and separate impurities in ampicillin and comprehensively analyze the impurity profile. However, liquid chromatography has a low desalting throughput and a long separation time. In addition to chromatography, capillary methods can also be used to separate and detect polymer impurities in ampicillin, but they also have the disadvantages of low desalting efficiency and low throughput for preparing polymer impurities.

[0003] Because ampicillin contains low levels of polymeric impurities and is unstable, traditional methods, which involve conducting animal allergy tests with the whole drug, cannot distinguish the effective sensitizing components. Therefore, there is an urgent need in this field to develop a novel method for the separation and preparation of polymeric impurities. Summary of the Invention

[0004] The purpose of this application is to provide a method for separating polymeric impurities in the preparation of β-lactam antibiotics.

[0005] Another objective of this application is to provide a method for detecting polymeric impurities in β-lactam antibiotics.

[0006] To address the aforementioned technical problems, a first aspect of this application provides a method for separating polymeric impurities in the preparation of β-lactam antibiotics, the method comprising the steps of:

[0007] Step 1: Elute the β-lactam antibiotic by gel chromatography and then collect the fraction;

[0008] Step 2: The distillate is subjected to desalting and freeze-drying processes in sequence to obtain freeze-dried powder;

[0009] The step of eluting the β-lactam antibiotic by gel chromatography includes:

[0010] At approximately 20-40°C, allow the aqueous solution of β-lactam antibiotics to stand for approximately 72-240 hours, make up to a final volume, shake well, and then elute by gel chromatography.

[0011] In some preferred embodiments, step 1, eluting the β-lactam antibiotic by gel chromatography, includes the following steps:

[0012] The aqueous solution of β-lactam antibiotic was allowed to stand for about 120 hours at about 25°C, then diluted to volume and shaken well before elution by gel chromatography.

[0013] In some preferred embodiments, step 1, eluting the β-lactam antibiotic by gel chromatography, includes the following steps:

[0014] The aqueous solution of β-lactam antibiotics was allowed to stand for about 24 hours at about 35°C, then diluted to volume and shaken well before elution by gel chromatography.

[0015] In some preferred embodiments, in step 1, the gel chromatography is Sephadex G-10 gel chromatography.

[0016] In some preferred embodiments, in step 1, the chromatographic conditions for gel chromatography include:

[0017] Mobile phase A: 0.1 mol / L phosphate buffer at pH 7.0; Mobile phase B: water;

[0018] The detection wavelength is 254nm.

[0019] In some preferred embodiments, in step 1, the chromatographic conditions for gel chromatography include:

[0020] Filler: Sephadex G-10 dextran gel (40μm);

[0021] The glass column has an inner diameter of 1.4 cm and a length of 35 cm.

[0022] Mobile phase A: 0.1 mol / L phosphate buffer at pH 7.0; Mobile phase B: water;

[0023] Flow rate: 1.5 mL / min;

[0024] Detection wavelength: 254nm.

[0025] In some preferred embodiments, step 1, collecting the fraction includes collecting the fraction corresponding to the main peak in the chromatogram.

[0026] In some preferred embodiments, in step 1, the fraction with the largest peak height or peak area is collected based on the chromatographic peak signal intensity.

[0027] In some preferred embodiments, in step 1, the chromatographic conditions for gel chromatography include:

[0028] Filler: Sephadex G-10 dextran gel (40μm);

[0029] The glass column has an inner diameter of 1.4 cm and a length of 35 cm.

[0030] Mobile phase A: 0.1 mol / L phosphate buffer at pH 7.0; Mobile phase B: water;

[0031] Flow rate: 1.5 mL / min;

[0032] Detection wavelength: 254nm

[0033] Collect the fractions that have been retained for 12-19 minutes.

[0034] In some preferred embodiments, step 2, the desalting process includes the steps of: passing the fraction through an activated desalting column in which β-lactam antibiotics are enriched, then rinsing with 95% water-5% methanol until the phosphate is substantially completely removed, and then eluting with 95% methanol-5% water as the mobile phase and collecting polymer impurities.

[0035] In some preferred embodiments, in step 2, a desalination device is used for desalination, the desalination device including a pump for controlling the flow rate of the mobile phase, a desalination column, a detector, and an eluent collector; the desalination process includes the following steps:

[0036] First, the desalting column activation procedure is performed, which includes sequentially equilibrating the desalting column with 95% methanol-5% water and 95% water-5% methanol.

[0037] The fraction is then introduced into the inlet of the desalting instrument through a pipeline, and the fraction passes through the desalting column to enrich the polymer impurities in the β-lactam antibiotic in the desalting column;

[0038] The desalting column was then rinsed with 95% water and 5% methanol to completely remove the phosphate.

[0039] Finally, the polymer impurities were eluted using a desalting column of 95% methanol and 5% water, and the polymer impurities were collected.

[0040] In some preferred embodiments, the collection of the polymer impurities includes the steps of: collecting the eluent corresponding to the main peak in the chromatogram, removing methanol from the resulting eluent by rotary evaporation, and collecting the rotary evaporation product.

[0041] In some preferred embodiments, the freeze-drying process includes: placing the rotary evaporation product in a freeze dryer for freeze-drying, wherein the freeze-drying temperature is approximately -80°C, the vacuum pressure is approximately 0 bar, and the freeze-drying time is approximately 30 hours.

[0042] In some preferred embodiments, the β-lactam antibiotic is ampicillin.

[0043] A second aspect of the present invention provides a method for detecting polymeric impurities in β-lactam antibiotics, comprising the steps of:

[0044] Step a, using the method described in the first aspect of the present invention to separate and prepare polymeric impurities in β-lactam antibiotics;

[0045] Step b, then use liquid chromatography to detect the polymer impurities, and determine the type of polymer impurities in the β-lactam antibiotic by comparing retention times.

[0046] Preferably, the method further includes step c, which involves extracting target ions by mass spectrometry to confirm the polymer impurities.

[0047] In some preferred embodiments, in step b, the chromatographic conditions for liquid chromatography include:

[0048] Using C 18 Chromatographic columns (e.g., Inertsil ODS-3 column (4.6 mm × 250 mm, 5 μm));

[0049] Mobile phase A is: 12% acetic acid solution: 0.2 mol / L potassium dihydrogen phosphate solution: acetonitrile: water = 0.5:50:50:900; mobile phase B is 12% acetic acid solution: 0.2 mol / L potassium dihydrogen phosphate solution: acetonitrile: water = 0.5:50:400:550.

[0050] Elution procedure: First, perform isocratic elution with mobile phase A:mobile phase B = 84:16. After the ampicillin peak has been eluted, perform gradient elution as shown in the table below; column temperature: 30℃; flow rate: 0.6mL / min; detection wavelength: 254nm.

[0051] Time / minute Mobile phase A% Mobile phase B% 0 84 16 30 0 100 45 0 100 50 84 16 60 84 16

[0052] In some preferred embodiments, the step of determining the type of polymeric impurities in the β-lactam antibiotic by comparing retention times includes the step of: if a peak with a retention time of approximately 32.5 minutes is present in the chromatogram, then it is determined that an open-ring dimer is present in the β-lactam antibiotic;

[0053] If a peak with a retention time of approximately 35.1 minutes is present in the chromatogram, it is determined that a closed-ring dimer exists in the β-lactam antibiotic.

[0054] If a peak with a retention time of approximately 39.1 minutes is present in the chromatogram, it is determined that the β-lactam antibiotic contains an open-ring trimer.

[0055] In some preferred embodiments, the mass spectrometry conditions for confirming the polymer impurities by extracting target ions by mass spectrometry include: mobile phase: acetonitrile: water = 1:1 (V / V);

[0056] Flow rate: 0.05 mL / min;

[0057] Electrospray ionization (ESI) source, positive ion mode;

[0058] Scan range: m / z 200-1200.

[0059] Preferably, the positive ion mode is set to 3.0 kV; the cone voltage is 25 V; the ion source temperature is 120 °C; the desolvation gas temperature is 150 °C; the cone gas flow rate is 150 L / h; and the desolvation gas flow rate is 500 L / h.

[0060] In some preferred embodiments, the confirmation of the polymer impurities by extracting target ions by mass spectrometry includes the following steps:

[0061] Target ions with m / z values ​​of approximately the following were extracted: 717, 699, 1066, 1048, 1397;

[0062] If a molecular ion peak with an m / z of approximately 717 is present in the extracted ion spectrum, it is determined that the β-lactam antibiotic contains an open-ring dimer.

[0063] If a molecular ion peak with an m / z of approximately 699 is present in the extracted ion spectrum, it is determined that a closed-ring dimer exists in the β-lactam antibiotic.

[0064] If a molecular ion peak with an m / z of approximately 1066 is present in the extracted ion spectrum, it is determined that the β-lactam antibiotic contains an open-ring trimer.

[0065] If a molecular ion peak with an m / z of approximately 1048 is present in the extracted ion spectrum, it is determined that a closed-ring trimer exists in the β-lactam antibiotic.

[0066] If a molecular ion peak with an m / z of approximately 1397 is present in the extracted ion spectrum, it is determined that a closed-ring tetramer is present in the β-lactam antibiotic.

[0067] In some preferred embodiments, steps b and c can be performed together using a liquid chromatography-mass spectrometry (LC-MS) system, wherein the chromatographic conditions of the LC-MS system include:

[0068] Waters 2.1mm×100mm, 1.7μm, using 10mmol / L ammonium acetate aqueous solution (pH adjusted to 5.2 with formic acid)-acetonitrile (98:2) as mobile phase A, with 10mmol / L -1 The mobile phase B is an aqueous solution of ammonium acetate (pH adjusted to 5.2 with formic acid) and acetonitrile (50.50).

[0069] In some preferred embodiments, the β-lactam antibiotic is ampicillin.

[0070] Based on the prior art, the present invention has at least the following advantages:

[0071] 1) This invention establishes a method for preparing polymers in β-lactam antibiotics. This method can efficiently and stably prepare and enrich polymers in β-lactam antibiotics, providing a foundation for passive skin allergy testing and in vitro sensitization evaluation studies.

[0072] 2) In a preferred embodiment, the polymer preparation method for β-lactam antibiotics established by the present invention can achieve high-throughput preparation by using an online desalting method, connecting the desalting instrument to an ultraviolet detector to facilitate real-time observation of the desalting process, resulting in high preparation efficiency.

[0073] 3) This invention provides a comprehensive confirmatory method based on liquid chromatography and mass spectrometry. Liquid chromatography compares the retention times of various polymers of β-lactam antibiotics, while mass spectrometry compares their relative molecular masses to verify that the lyophilized powder contains polymer impurities.

[0074] It should be understood that, within the scope of this application, the above-described technical features of this application and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0075] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

[0076] Figure 1 The images shown are gel chromatograms (0h, 72h, 120h) of ampicillin sodium for injection according to embodiments of the present invention.

[0077] Figure 2 The following are time-varying graphs of ampicillin polymers added to water according to embodiments of the present invention: (a) closed-ring dimer; (b) open-ring dimer; (c) open-ring trimer;

[0078] Figure 3 This is a diagram of the separation of ampicillin sodium polymers by Sephadex G-10 gel chromatography according to an embodiment of the present invention;

[0079] Figure 4 This is an ultraviolet detection image of ampicillin polymer desalination according to an embodiment of the present invention;

[0080] Figure 5 It is an ampicillin polymer powder according to an embodiment of the present invention;

[0081] Figure 6 This is a high-performance liquid chromatogram of ampicillin polymer according to an embodiment of the present invention;

[0082] Figure 7 This is the mass spectrum of the ampicillin polymer according to an embodiment of the present invention;

[0083] Figure 8 This is the LC-ESI-MS chromatogram of the ampicillin polymer according to an embodiment of the present invention;

[0084] Figure 9 This is a possible chemical structural diagram of the polymer in ampicillin sodium according to an embodiment of the present invention;

[0085] Figure 10 This refers to the stability of the ampicillin polymer according to embodiments of the present invention.

[0086] Figure 11 This is a schematic diagram of the desalination process according to an embodiment of the present invention. Detailed Implementation

[0087] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the present application is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0088] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0089] Example 1

[0090] This embodiment establishes a method for preparing polymeric impurities in β-lactam antibiotics. Specifically, ampicillin sodium polymeric impurities are separated by gel permeation chromatography to remove phosphate, followed by freeze-drying to obtain ampicillin polymer lyophilized powder. Liquid chromatography and mass spectrometry confirm that the lyophilized solid powder contains ampicillin polymeric impurities.

[0091] 1.1. Separation of polymeric impurities from ampicillin sodium for injection

[0092] Polymer impurities in ampicillin sodium for injection were separated by gel electrophoresis using a dextran gel Sephadex G-10 (40 μm) as the packing material; the glass column had an inner diameter of 4 cm and a length of 35 cm; mobile phase A was 0.1 mol / L phosphate buffer at pH 7.0 [0.1 mol / L disodium hydrogen phosphate solution - 0.1 mol / L sodium dihydrogen phosphate solution (61:39)], and mobile phase B was water; the flow rate was 1.5 mL per minute; the detection wavelength was 254 nm; and the injection volume was 200 μL.

[0093] Accurately weigh approximately 0.2 g of ampicillin sodium for injection, place it in a 10 mL volumetric flask, add 0.2 mL of ultrapure water, let it stand at 25°C for 120 hours, then dilute to the mark with water, shake well, separate by gel chromatography, and collect the fraction. Collect approximately 8 mL of the fraction each time.

[0094] 1.2. Desalting and freeze-drying of ampicillin polymer solution

[0095] 1.2.1 Desalination

[0096] The collected solution was desalted online by connecting a multi-channel constant-speed pump to a UV detector to observe the desalting process. See the schematic diagram of the desalting process. Figure 11 .

[0097] First, equilibrate C with approximately 50 mL of methanol-water (95:5). 18 Desalting column. The separated ampicillin sodium polymer impurity collection solution is then passed through a C10 column at a flow rate of 1 mL / min. 18 The desalting column was washed three times with water-methanol (95:5), with each wash volume being 8 mL. Finally, 15 mL of methanol-water (95:5) was used as the eluent at a flow rate of 1 mL / min, and the eluent was collected.

[0098] 1.2.2 Rotary Evaporation

[0099] The eluent collected in 1.2.1 was rotary evaporated at a temperature of 35°C and a rotation speed of 50 rpm to remove methanol from the solvent.

[0100] 1.2.3 Freeze-vacuum drying

[0101] The solution, which was evaporated to near dryness, was placed in a freeze dryer. The freeze-drying temperature was -80°C, the vacuum pressure was set to 0 bar, and the freeze-drying time was 30 hours, resulting in a white, loose powder.

[0102] 1.3. Confirmation of the polymer in ampicillin in lyophilized powder

[0103] 1.3.1 Confirmation of polymer retention time by liquid chromatography

[0104] According to the liquid chromatography method described in "Quality Evaluation of Ampicillin Sodium for Injection" (Chinese Journal of Antibiotics, 2024, 49(03):304-309): an Inertsil ODS-3 column (4.6 mm × 250 mm, 5 μm) was used; mobile phase A was 12% acetic acid solution - 0.2 mol / L potassium dihydrogen phosphate solution - acetonitrile - water (0.5:50:50:900), and mobile phase B was 12% acetic acid solution - 0.2 mol / L potassium dihydrogen phosphate solution - acetonitrile - water (0.5:50:400:550). Initially, isocratic elution was performed with mobile phase A - mobile phase B (84:16). After the ampicillin peak was completely eluted, gradient elution was immediately performed according to Table 1. The column temperature was 30℃; the flow rate was 0.6 mL / min; and the detection wavelength was 254 nm.

[0105] Table 1 Washout Schedule

[0106] Time / minute Mobile phase A% Mobile phase B% 0 84 16 30 0 100 45 0 100 50 84 16 60 84 16

[0107] 1.3.2 Mass Spectrometry Confirmation To further confirm the structure of the ampicillin polymer impurity in the ampicillin polymer impurity powder, direct injection analysis and target ion extraction were used for confirmation.

[0108] 1.3.2.1 Direct Injection Analysis

[0109] The mass spectrometry method used the following mobile phases: 50% acetonitrile-50% water; flow rate: 0.05 mL / min; electrospray ionization (ESI) source, positive ion mode; scan range: m / z 200-1200; positive ion voltage mode set to 3.0 kV; cone voltage: 25 V; ion source voltage: 80 V; ion source temperature: 120 °C; desolvation gas temperature: 150 °C; cone gas flow rate: 150 L / hr; desolvation gas flow rate: 500 L / hr.

[0110] The prepared polymer impurity solid powder was reconstituted with ultrapure water to a concentration of 10 μg / mL, and the injection volume was 10 μL. Direct injection analysis was performed using a peristaltic pump.

[0111] 1.3.2.2 Liquid chromatography-mass spectrometry (LC-MS) for target ion extraction

[0112] To further investigate the relative proportions of different types of polymers in the lyophilized powder, we conducted a preliminary investigation using liquid chromatography-mass spectrometry (LC-MS / MS). The method was based on the LC-MS / MS method described in "Determination of Impurity Profiles of Ampicillin Raw Materials and Preparations by LC-MS / MS" (Chinese Journal of Antibiotics, 2023, 48(10):1151-1162).

[0113] Liquid chromatography column: ACE 2.1mm × 100mm, 1.7μm; mobile phase: 10 mmol / L. -1 Ammonium acetate aqueous solution (adjusted to pH 5.2 with formic acid) - acetonitrile (98:2) was used as mobile phase A, with 10 mmol / L... -1 Ammonium acetate aqueous solution (pH adjusted to 5.2 with formic acid): acetonitrile (50:50) as mobile phase B. Flow rate 0.3 mL / min; positive ion mode, spray voltage 3.0 kV; orifice voltage 25 V; ion source temperature 120 °C, desolvation gas temperature 450 °C, orifice gas flow rate 150 L / hr, desolvation gas flow rate 500 L / hr.

[0114] The lyophilized solid from step 1.2.3 was reconstituted with mobile phase A and eluted linearly according to Table 2; the column temperature was 30℃; the flow rate was 0.3 mL / min; and the detection wavelength was 230 nm.

[0115] Table 2 Washout Schedule

[0116] Time / minute Mobile phase A% Mobile phase B% 0 100 0 15 75 25 50 60 40 60 50 50 61 100 0 66 100 0

[0117] Example 2

[0118] In this embodiment, the correlation between the standing time and temperature of ampicillin sodium for injection after adding water and the polymer content was investigated.

[0119] 2.1 Instruments, reagents and test kits

[0120] Instruments: Antibiotic polymer analysis system (KGF-1, Shanghai Jinda Biochemical Instrument Co., Ltd.); electronic balance (Sartorius); ultrapure water system (Milli-Q, Merck).

[0121] Drugs and reagents: Glacial acetic acid and sodium hydroxide were of chromatographic grade; sodium dihydrogen phosphate and disodium hydrogen phosphate were of analytical grade and were purchased from Sinopharm Group; water was ultrapure water.

[0122] 2.2 Polymer Separation Conditions

[0123] Chromatographic conditions: Sephadex G-10 dextran gel (40 μm) was used as the packing material; the glass column had an inner diameter of 1.4 cm and a length of 35 cm; mobile phase A was 0.1 mol / L phosphate buffer at pH 7.0 [0.1 mol / L disodium hydrogen phosphate solution - 0.1 mol / L sodium dihydrogen phosphate solution (61:39)], and mobile phase B was water; the flow rate was 1.5 mL per minute; the detection wavelength was 254 nm; and the injection volume was 200 μL.

[0124] Test solution: Weigh 1g of ampicillin for injection into a 10mL volumetric flask, add 0.2mL of ultrapure water, and let stand at room temperature (25℃) for 0 hours, 72 hours, and 120 hours. Dilute to the mark with water and mix well. Investigate polymer growth.

[0125] Furthermore, we investigated the changes in polymer impurity mass (represented by closed-ring dimers, open-ring dimers, and open-ring trimers) at different time points (0, 12, 24, 36, 72, and 120) of ampicillin for injection after adding water at different temperatures (25°C and 35°C).

[0126] The results show that: Figure 1 , 2 As shown, the polymer peak area of ​​ampicillin increased significantly after standing for 120 hours with water at room temperature (25℃). Investigations at different temperatures (25℃ and 35℃) revealed that the content of ampicillin closed-ring dimer decreased after standing for 24 hours at 35℃, but continued to increase at 25℃. Both ampicillin open-ring dimer and ampicillin open-ring trimer showed an increasing trend with increasing standing time at both temperatures. Considering these factors, 25℃ was selected as the standing temperature for ampicillin for injection after adding water.

[0127] Example 3

[0128] In this embodiment, polymer impurities in ampicillin for injection were prepared.

[0129] 3.1 Separation

[0130] Chromatographic conditions: Same as Case 1.

[0131] Test solution: Weigh 1g of ampicillin for injection into a 10mL volumetric flask, add 0.2mL of ultrapure water, let stand at 25℃ for 120 hours, add water to make up to the mark, and shake well.

[0132] The results showed that typical separation chromatograms were as follows: Figure 3 As shown, polymer elution solutions with retention times of 12-19 minutes are collected.

[0133] 3.2 Desalination

[0134] The polymer eluent is desalted online using the desalting instrument ( Figure 2 It is connected to an ultraviolet detector to observe the desalination process.

[0135] (1)C 18 Desalting column activation: Equilibrate the desalting column with 95% methanol-5% water at a flow rate of 1 mL / min and a wash volume of 50 mL. Then equilibrate with 95% water-5% methanol at a flow rate of 1 mL / min and a wash volume of 50 mL.

[0136] (2) Injection: The ampicillin sodium polymer impurity collection solution eluted in the gel chromatography was injected at a flow rate of 1 mL / min to enrich the ampicillin sodium polymer in the desalting column.

[0137] (3) Desalting: Rinse three times with 95% water and 5% methanol, each time with a volume of 8 mL and a flow rate of 0.5 mL / min, to completely remove the phosphate.

[0138] (4) Polymer elution: Use 95% methanol-5% water as the mobile phase as the eluent, with a flow rate of 1 mL / min and an elution volume of 15 mL to elute polymer impurities.

[0139] The results show that the UV absorption spectrum observed during online desalination is as follows: Figure 4 As shown, after desalting the polymer impurity eluent, the eluent with a retention time between 40 and 60 minutes is collected.

[0140] 3.3 Rotary Evaporation

[0141] The rotary evaporation temperature was set to 35℃ and the rotation speed was set to 50 rpm to remove methanol from the solvent.

[0142] 3.4 Freeze-drying

[0143] The solution, which was evaporated to near dryness, was placed in a freeze dryer. The freeze-drying temperature was -80°C, the vacuum pressure was set to 0 bar, and the freeze-drying time was 30 hours, resulting in a white, loose powder.

[0144] The results showed that the final freeze-dried product was an off-white powder of ampicillin polymer. Figure 5 ).

[0145] Example 4: Confirmation of the polymer in ampicillin in lyophilized powder

[0146] 4.1 Confirmation of polymer retention time by liquid chromatography

[0147] Liquid chromatography method: An Inertsil ODS-3 column (4.6 mm × 250 mm, 5 μm) was used; mobile phase A was 12% acetic acid solution - 0.2 mol / L potassium dihydrogen phosphate solution - acetonitrile - water (0.5:50:50:900), and mobile phase B was 12% acetic acid solution - 0.2 mol / L potassium dihydrogen phosphate solution - acetonitrile - water (0.5:50:400:550). Initially, isocratic elution was performed with mobile phase A-mobile phase B (84:16). After the ampicillin peak was completely eluted, gradient elution was immediately performed according to Table 1; column temperature was 30℃; flow rate was 0.6 mL / min; detection wavelength was 254 nm.

[0148] Table 3. Ampicillin Polymer Retention Time

[0149] Ampicillin polymer Document retention time (min) Actual retention time (min) open-ring dimers 33 32.5 Closed-ring dimer 36 35.1 Open-ring trimer 40 39.1

[0150] The results show that: Figure 6 As shown in Table 3, the prepared ampicillin polymer impurity powder contained chromatographic peaks with retention times consistent with the corresponding polymers in the literature, suggesting that it may contain ampicillin ring-opening dimers, ring-closed dimers, and ring-opening trimers.

[0151] 4.2 Mass Spectrometry Confirmation To further confirm the structure of the ampicillin polymer impurity in the ampicillin polymer impurity powder, direct injection analysis and target ion extraction were used for confirmation.

[0152] 4.2.1 Direct Injection Analysis: The prepared polymer impurity solid powder was reconstituted with ultrapure water to a concentration of 10 μg / mL, with an injection volume of 10 μL. Direct injection analysis was performed using a peristaltic pump. Mobile phase: 50% acetonitrile-50% water; flow rate: 0.05 mL / min; electrospray ionization (ESI) source, positive ion mode; scan range: m / z 200-1200; capillary voltage set to 3.0 kV in positive ion mode; orifice voltage: 25 V; default ion source voltage: 80 V; ion source temperature: 120 °C; desolvation gas temperature: 150 °C; orifice gas flow rate: 150 L / h; desolvation gas flow rate: 500 L / h.

[0153] The quasi-molecular ions [M+H] of ampicillin polymers were determined in the LC-MS analysis of related substances in ampicillin and amoxicillin (Acta Pharmaceutica Sinica 2001(10):758-761). + As shown in Table 4:

[0154] Table 4. Ampicillin excimer ionomer [M+H] +

[0155] Ampicillin polymer <![CDATA[ESI+ MS of quasi-molecular ion m / z [M+H] + <!-- 8 -->]]> open-ring dimers 717 Closed-ring dimer 699 Open-ring trimer 1066 Closed-loop trimer 1048 Closed-loop tetramer 1397

[0156] The results show: Figure 7 The mass spectrum is a first-order mass spectrum, which includes molecular ion peaks with m / z values ​​of 717, 699, 1066, 1048, and 1397, respectively. These peaks are consistent with the molecular ions of ampicillin open-ring dimer, closed-ring dimer, open-ring trimer, closed-ring trimer, and closed-ring tetramer in Table 4, indicating that the polymer impurity powder prepared contains ampicillin open-ring dimer, closed-ring dimer, open-ring trimer, closed-ring trimer, and closed-ring tetramer.

[0157] 4.2.2 Liquid chromatography-mass spectrometry (LC-MS) for target ion extraction

[0158] Chromatographic conditions: Waters 2.1mm×100mm, 1.7μm, with mobile phase A of 10mmol / L ammonium acetate aqueous solution (adjusted to pH 5.2 with formic acid)-acetonitrile (98:2) and mobile phase B of 10mmol / L ammonium acetate aqueous solution (adjusted to pH 5.2 with formic acid)-acetonitrile (50.50).

[0159] Mass spectrometry conditions: flow rate 0.3 mL / min, same as 3.2.1

[0160] Solution preparation: Redissolve the collected lyophilized powder using mobile phase A. Perform gradient elution according to Table 2.

[0161] The results show: Figure 8 The extracted ion mass spectra are for ampicillin ring-opening dimers, ring-closed dimers, ring-opening trimers, ring-closed trimers, and ring-closed tetramers. The calculated content ratio of each polymer type is approximately (10:6:7:7:5), and the ring-opening dimers and trimers may have isomers.

[0162] Deducing the possible chemical structural formulas of the polymer in ampicillin sodium, as follows: Figure 9 , Figure 9 Among them, (a) ampicillin sodium ring-opening dimer, m / z 717.2463; (b) ampicillin sodium ring-closed dimer, m / z 699.2266; (c) ampicillin sodium ring-opening trimer, m / z 1066.3465; (d) ampicillin sodium ring-closed trimer, m / z 1048.3358; (e) ampicillin sodium ring-closed tetramer, m / z 1397.3783.

[0163] Example 5

[0164] In this embodiment, the retention time of polymer impurities was investigated.

[0165] Liquid phase method: Same as in Example 4, 4.1.

[0166] Table 5. Stability of Ampicillin Polymers

[0167]

[0168] The results show that: Figure 10 The degradation of ampicillin polymers after 18 days of storage at -20°C was investigated. During storage at -20°C, the degradation rate of ampicillin polymers was: closed-ring dimer > open-ring trimer > open-ring dimer. As shown in Table 5, the degradation rate of ampicillin polymers during storage at -20°C was: closed-ring dimer > open-ring trimer > open-ring dimer, all within 5%. Compared with storage at -20°C, the degradation rate of polymers was greater at 4°C, making -20°C a more suitable storage temperature. Repeated handling should be avoided; aliquots are necessary.

[0169] This invention effectively separates polymeric impurities from ampicillin sodium. It employs liquid chromatography and mass spectrometry, comparing the retention times and relative molecular masses of various polymers in ampicillin sodium to verify the presence of polymeric impurities in the lyophilized product. This method is accurate, reliable, and feasible.

[0170] Those skilled in the art will understand that the above embodiments are specific examples of implementing this application, and in practical applications, various changes can be made in form and detail without departing from the spirit and scope of this application.

Claims

1. A method for separating and preparing polymeric impurities in β-lactam antibiotics, characterized in that, The method includes the following steps: Step 1: Elute the β-lactam antibiotic by gel chromatography and then collect the fraction; Step 2: The distillate is subjected to desalting and freeze-drying processes in sequence to obtain freeze-dried powder; The step of eluting the β-lactam antibiotic by gel chromatography includes: At approximately 20-40°C, allow the aqueous solution of β-lactam antibiotics to stand for approximately 72-240 hours, make up to a final volume, shake well, and then elute by gel chromatography.

2. The method according to claim 1, characterized in that, In step 1, the elution of the β-lactam antibiotic by gel chromatography includes the following steps: At approximately 25°C, allow the aqueous solution of β-lactam antibiotics to stand for about 120 hours, make up to volume, shake well, and then elute by gel chromatography; or The aqueous solution of β-lactam antibiotics was allowed to stand for about 24 hours at about 35°C, then diluted to volume and shaken well before elution by gel chromatography.

3. The method according to claim 1, characterized in that, In step 1 and step 2, the desalting process includes the steps of: passing the fraction through an activated desalting column, enriching the β-lactam antibiotic in the desalting column, then washing with 95% water-5% methanol until the phosphate is substantially completely removed, and then eluting with 95% methanol-5% water as the mobile phase and collecting polymer impurities.

4. The method according to claim 3, characterized in that, In step 2, a desalination apparatus is used for desalination treatment. The desalination apparatus includes a pump for controlling the mobile phase flow rate, a desalination column, a detector, and an eluent collector. The desalination treatment includes the following steps: First, the desalting column activation procedure is performed, which includes sequentially equilibrating the desalting column with 95% methanol-5% water and 95% water-5% methanol. The fraction is then introduced into the inlet of the desalting instrument through a pipeline, and the fraction passes through the desalting column to enrich the polymer impurities in the β-lactam antibiotic in the desalting column; The desalting column was then rinsed with 95% water and 5% methanol to completely remove the phosphate. Finally, the polymer impurities were eluted using a desalting column of 95% methanol and 5% water, and the polymer impurities were collected.

5. The method according to claim 3 or 4, characterized in that, Step 2, after collecting the polymer impurities, includes the following steps: collecting the eluent corresponding to the main peak in the chromatogram, removing methanol from the obtained eluent by rotary evaporation, and collecting the rotary evaporation product.

6. The method according to claim 5, characterized in that, The freeze-drying process includes the following steps: placing the rotary evaporation product in a freeze dryer for freeze-drying, wherein the freeze-drying temperature is approximately -80°C, the vacuum pressure is approximately 0 bar, and the freeze-drying time is approximately 30 hours, thereby obtaining the product.

7. A method for detecting polymeric impurities in β-lactam antibiotics, characterized in that, The method includes the following steps: Step a, using the method described in the first aspect of the present invention to separate and prepare polymeric impurities in β-lactam antibiotics; Step b, then use liquid chromatography to detect the polymer impurities, and determine the type of polymer impurities in the β-lactam antibiotic by comparing retention times.

8. The method according to claim 7, characterized in that, The method further includes step c, which involves extracting target ions by mass spectrometry to confirm the polymer impurities.

9. The method according to claim 7, characterized in that, The step of determining the type of polymeric impurities in the β-lactam antibiotic by comparing retention times includes the following steps: if a peak with a retention time of approximately 32.5 minutes is present in the chromatogram, then it is determined that an open-ring dimer is present in the β-lactam antibiotic; If a peak with a retention time of approximately 35.1 minutes is present in the chromatogram, it is determined that a closed-ring dimer exists in the β-lactam antibiotic. If a peak with a retention time of approximately 39.1 minutes is present in the chromatogram, it is determined that the β-lactam antibiotic contains an open-ring trimer.

10. The method according to claim 8, characterized in that, The step of confirming the polymer impurities by extracting target ions by mass spectrometry includes the following steps: Target ions with m / z values ​​of approximately the following were extracted: 717, 699, 1066, 1048, 1397; If a molecular ion peak with an m / z of approximately 717 is present in the extracted ion spectrum, it is determined that the β-lactam antibiotic contains an open-ring dimer. If a molecular ion peak with an m / z of approximately 699 is present in the extracted ion spectrum, it is determined that a closed-ring dimer exists in the β-lactam antibiotic. If a molecular ion peak with an m / z of approximately 1066 is present in the extracted ion spectrum, it is determined that the β-lactam antibiotic contains an open-ring trimer. If a molecular ion peak with an m / z of approximately 1048 is present in the extracted ion spectrum, it is determined that a closed-ring trimer exists in the β-lactam antibiotic. If a molecular ion peak with an m / z of approximately 1397 is present in the extracted ion spectrum, it is determined that a closed-ring tetramer is present in the β-lactam antibiotic.

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