Identification and Content Determination Method for Impurity Polymer of Penem Antibiotic Drugs

Abstract

The present invention relates to a method for detecting polymer impurities in penem antibiotics, including the identification of impurity polymers and a content determination method based on the identification results. The chromatographic peaks of polymers in penem antibiotics are identified by the chromatographic peaks obtained after the sample undergoes a degradation reaction. The content of drug polymers in penem antibiotics is accurately calculated by using the external standard method with a polymer impurity reference substance.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical analysis, and particularly to a method for determining the content of impurity polymers in penem antibiotics. Background Art

[0002] Antibiotic allergens are not the drugs themselves, but the high molecular polymers formed by their degradation. They irreversibly bind to macromolecular carriers in the body, causing antigen-antibody reactions and thus a series of allergic reaction symptoms. The content of high molecular polymers in drugs directly affects the incidence of allergic reactions. Therefore, more and more antibiotic drugs require strict control of the content of high molecular polymers in penem antibiotics.

[0003] For the detection method of high molecular polymers in antibiotic drug raw materials and their preparations, the traditional method is to use size exclusion chromatography, but the determination of the content of polymers in antibiotic drugs by size exclusion chromatography is inaccurate. Currently, most of the detections of antibiotic polymers use reversed-phase high performance liquid chromatography. Summary of the Invention

[0004] Based on the above problems, the present invention aims to provide a method that can more accurately identify and determine the content of impurity polymers in antibiotic drugs.

[0005] To achieve the above object, the present invention provides a method for identifying impurity polymers in penem antibiotics. By subjecting the sample to a degradation reaction under specific conditions to generate a polymer chromatographic peak, the chromatographic peak with a relative retention time of about 2.0 - 2.6 after the main peak is generated is determined as the polymer in the penem antibiotic drug.

[0006] Further, the degradation reaction process is as follows: an appropriate amount of penem antibiotic drug is added to an acid solution with a volume 10 times that of the penem antibiotic drug. After standing for 30 minutes, it is neutralized with an alkali solution and then diluted with water to 100 volumes to obtain a degradation solution.

[0007] Preferably, the acid solution in the degradation reaction is at least one of hydrochloric acid, sulfuric acid, nitric acid, and acetic acid, and the concentration is 0.05 mol / L - 10 mol / L.

[0008] A method for identifying polymers in penem antibiotics of the present invention determines the impurity polymers in the test solution by the retention time of the chromatographic peak through high performance liquid chromatography, and specifically includes the following steps:

[0009] (1) Conduct a degradation reaction. Add an appropriate amount of penem antibiotic drug sample, about 30 - 100 mg, to 10 volumes of an acid with a concentration of 0.05 mol / L - 10 mol / L. After standing for 30 min, neutralize it with an alkali solution and dilute it with water to 100 volumes;

[0010] (2) Detect the impurities with relative retention times of approximately 2.0 - 2.6 in the solution of the degradation reaction by high performance liquid chromatography. Among them, the chromatographic peak near a relative retention time of 2.0 is Polymer 1, and the chromatographic peak near a relative retention time of 2.6 is Polymer 2.

[0011] (3) Detect the impurities with relative retention times of approximately 2.0 - 2.6 in the test sample by high performance liquid chromatography. If there is a chromatographic peak near a relative retention time of 2.0, it is identified as Impurity Polymer 1, and the impurity near a relative retention time of 2.6 is identified as Impurity Polymer 2.

[0012] Preferably, the maximum absorption wavelength of Polymer 1 is near 280 nm, and the maximum absorption wavelength of Polymer 2 is near 370 nm.

[0013] The present invention also provides a method for detecting polymers in penem antibiotics based on the above identification method, including a method for determining the content of polymers. It is characterized in that the identified polymers in the test sample solution are used, and the content of Impurity Polymer 1 and Polymer 2 in the test sample of penem antibiotics is detected by the external standard method using the reference substance of Polymer 1.

[0014] Further, the chromatographic conditions in the method for identifying and determining the content of polymers in penem antibiotics are the same. The chromatographic conditions are as follows: using octadecylsilane bonded phase as the stationary phase; potassium dihydrogen phosphate solution - acetonitrile as Mobile Phase A, and acetonitrile as Mobile Phase B; detection wavelength 300 nm; Mobile Phase A is maintained at 90% - 100% within the range of 0 - 10 minutes and 100% - 50% within the range of 10 - 20 minutes; flow rate is 0.8 mL / min - 1.2 mL / min, injection volume 20 μL;

[0015] Among them, the preparation method of Mobile Phase A: The preparation method of potassium dihydrogen phosphate solution - acetonitrile: Take 1.36 g of potassium dihydrogen phosphate, add 1000 ml of water, adjust the pH value to 7.00 ± 0.05 with phosphoric acid, take 990 ml and add 10 ml of acetonitrile and mix well; the stationary phase octadecylsilane bonded phase is ODS - Hypersil Thermo Scientific 4.6×150 mm 5 μm or an equivalent column.

[0016] A preferred elution method is: Mobile Phase A is maintained at 100% within the range of 0 - 5 minutes, and Mobile Phase A is 100% - 8% within the range of 5 - 20 minutes; perform gradient elution according to the following table, flow rate is 1.0 ml / min; column temperature is 25°C.

[0017] Time (min) A％ B％ 0 100 0 5 100 0 20 80 20

[0018] The method for determining the content of impurity polymers in penem antibiotics of the present invention is most suitable for the detection of imipenem raw materials and meropenem and their preparation products. Description of the Drawings

[0019] Figure 1 It is the HPLC chromatogram of the polymer in the acid degradation sample solution in Example 1;

[0020] Figure 2 It is the UV diagram of polymer 1 in the acid degradation solution in Example 1;

[0021] Figure 3 It is the MS diagram of polymer 1 in the acid degradation solution in Example 1;

[0022] Figure 4 It is the UV diagram of polymer 2 in the acid degradation solution in Example 1;

[0023] Figure 5 It is the MS diagram of polymer 2 in the acid degradation solution in Example 1;

[0024] Figure 6 It is the HPLC chromatogram of the test solution in Example 3. Detailed Description of the Invention

[0025] Example 1

[0026] The preparation and structure confirmation of impurity polymers in penem antibiotics include the following steps:

[0027] Take about 30 mg of the sample, weigh it accurately, place it in a 100 ml brown volumetric flask, add about 10 ml of 0.1 mol / l hydrochloric acid solution to dissolve it, let it stand for 30 min, add 10 ml of 0.1 mol / l sodium hydroxide solution, and then dilute it with water to the scale and shake well to obtain the degradation solution.

[0028] The chromatographic conditions are as follows: using octadecylsilane-bonded phase (ODS-Hypersil Thermo Scientific 4.6×150 mm 5 μm or equivalent column) as the stationary phase;

[0029] Potassium dihydrogen phosphate solution (dissolve 1.36 g of potassium dihydrogen phosphate in 1 L of water, adjust the pH to 7.00±0.05 with phosphoric acid)-acetonitrile (99:1) is used as mobile phase A, and acetonitrile is used as mobile phase B;

[0030] Perform gradient elution according to the following table, with a flow rate of 1.0 ml / min; the detection wavelength is 300 nm; the column temperature is 25°C; the injection volume is 20 μl.

[0031]

[0032]

[0033] The relative retention time of Polymer 1 is 2.02, and the relative retention time of Polymer 2 is 2.60. As Figure 1 shown are the HPLC chromatograms of Polymer 1 and Polymer 2 after acid degradation.

[0034] The polymer with a relative retention time of 2.02 has maximum UV absorption at 206 nm and 286 nm, which is Polymer 1 of imipenem.

[0035] For the polymer with a relative retention time of 2.02, the M+2H peak in the MS scan of this peak is 600.2046, and the molecular weight is 598.69. As Figure 3 shown, it is Polymer 1 of imipenem. Since the UV spectrum shows that this substance has an absorption peak at around 286 nm, and the sample forms an amide dimer after ring-opening degradation under acidic conditions, as Figure 2 shown.

[0036] The polymer with a relative retention time of 2.60 has maximum UV absorption at 214 nm and 372 nm, which is Polymer 2 of imipenem. As Figure 4 shown.

[0037] For the polymer with a relative retention time of 2.60, the M+2H peak in the MS scan of this peak is 600.2052, and the molecular weight is 598.69. It is Polymer 2 of imipenem. As Figure 5 shown, since the UV spectrum shows that this substance has an absorption peak at around 370 nm, and it is a polymer with a more conjugated system formed after the sample degrades under acidic conditions, as Figure 4 shown.

[0038] Example 2

[0039] Take about 60 mg of the sample, accurately weigh it, place it in a 100 mL brown volumetric flask, add 10 mL of 0.01 mol / L hydrochloric acid solution to dissolve it, let it stand for 30 min, add 10 mL of 0.01 mol / L sodium hydroxide solution, and then dilute it to the mark with water and shake well to obtain the degradation solution.

[0040] Take 15 mg of the test substance, accurately weigh it, place it in a 25 mL volumetric flask, dissolve it with the diluent and dilute it to the mark, shake well. Take 1 mL and dilute it to 20 mL with water to obtain the sample solution.

[0041] The chromatographic conditions and elution process are the same as in Example 1 to obtain Polymer 1.

[0042] Take about 20 mg of Polymer 1 reference substance, weigh accurately, place it in a 250-ml volumetric flask, dissolve it with the diluent and dilute to the mark, shake well. Take 1 ml and dilute it to 100 ml with water to obtain the reference substance solution. Inject them into the liquid chromatograph respectively, record the peak areas of Impurity Polymer 1 and Impurity Polymer 2 in the test solution and Polymer 1 reference substance in the reference substance solution, and calculate the contents of Impurity Polymer 1 and Impurity Polymer 2 respectively by the external standard method according to the peak areas.

[0043] Example 3

[0044] Determination of the content of impurity polymers by the external standard method

[0045] Mobile phase A: Potassium dihydrogen phosphate solution (containing 1% acetonitrile)

[0046] Take 1.36 g of potassium dihydrogen phosphate, add 1000 ml of water, adjust the pH value to 6.8, take 990 ml and add 10 ml of acetonitrile and mix well.

[0047] Mobile phase B: Acetonitrile. Take an appropriate amount of acetonitrile and place it in a clean glass bottle.

[0048] Diluent: Water. Take an appropriate amount of water and place it in a clean glass bottle.

[0049] Test solution: Take about 15 mg of the test substance, weigh accurately, place it in a 25-ml volumetric flask, dissolve it with the diluent and dilute to the mark, shake well. Take 1 ml and dilute it to 20 ml with water.

[0050] Reference substance solution: Take about 2 mg of Polymer 1, weigh accurately, place it in a 25-ml volumetric flask, dissolve it with the diluent and dilute to the mark, shake well. Take 1 ml and dilute it to 100 ml with water to obtain the reference substance solution.

[0051] Calculate the polymer content by the external standard method according to the peak area. The contents of Polymer 1 and Polymer 2 are both less than 0.2%. As Figure 6 shown.

Claims

1. A method for identifying impurity polymers in a penem antibiotic drug, characterized in that, after the degradation reaction of the penem antibiotic drug sample, a polymer chromatographic peak is generated by high performance liquid chromatography, and the penem antibiotic drug is imipenem; the degradation reaction process is as follows: add the penem antibiotic drug sample to 10 parts by volume of an acid solution, let it stand for 30 minutes, then add an alkali solution to neutralize it, and then dilute it with water to 100 parts by volume to obtain a degradation solution; the acid solution in the degradation reaction is at least one of hydrochloric acid, sulfuric acid, nitric acid and acetic acid, and the concentration is 0.05 mol / L to 10 mol / L; the chromatographic conditions are ODS-Hypersil Thermo Scientific 4.6×150 mm 5 μm; potassium dihydrogen phosphate solution - acetonitrile 99:1 is mobile phase A, and acetonitrile is mobile phase B; the potassium dihydrogen phosphate solution is prepared by dissolving 1.36 g of potassium dihydrogen phosphate in 1 L of water and adjusting the pH to 7.00±0.05 with phosphoric acid; for the polymer with a relative retention time of 2.02, the M+2H peak in the MS scan is 600.2046, with the maximum ultraviolet absorption at 206 nm and 286 nm, and the molecular weight of 598.69 is polymer 1 of imipenem; for the polymer with a relative retention time of 2.60, the maximum ultraviolet absorption is at 214 nm and 372 nm, the M+2H peak in the MS scan is 600.2052, and the molecular weight of 598.69 is polymer 2 of imipenem; perform gradient elution according to the following table, the flow rate is 1.0 ml / min; the detection wavelength is 300 nm; the column temperature is 25°C; the injection volume is 20 μL: 。 2. A method for determining the content of impurity polymers in a penem antibiotic drug based on the method for identifying impurity polymers in a penem antibiotic drug according to claim 1, characterized in that, for the polymer identified in the test sample, the external standard method of the reference substance of polymer 1 is used to detect the contents of impurity polymer 1 and impurity polymer 2 in the test sample of the penem antibiotic drug.

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