A method for preparing and identifying unknown impurities in amikacin sulfate injection
Unknown impurities in Amikacin sulfate injection were separated by recrystallization, silica gel column separation and preparation liquid chromatography, and their structure was identified by LC-MS, which solved the problem of insufficient control of unknown impurities in the prior art and ensured product safety and quality.
Patent Information
- Application Number
- CN202210112807.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-01-29
- Publication Date
- 2025-08-15
- Estimated Expiration
- 2042-01-29
AI Technical Summary
The prior art has failed to effectively control unknown impurities in Amikacin sulfate injection, affecting product safety and use effect.
Recrystallization, silica gel column separation and preparative liquid chromatography were used to separate unknown impurities, and their structure was identified in combination with LC-MS, and their chemical formula was determined to be C16H32N4O9, and their content was controlled below 0.30% by optimizing the process.
The efficient separation and identification of unknown impurities in Amikacin Sulfate injection was achieved, ensuring product quality and drug safety, and the impurity content was controlled below the reference preparation level.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of pharmaceutical technical analysis, and particularly relates to a method for preparing and structurally identifying unknown impurities in amikacin sulfate injection. Background Art
[0002] Amikacin sulfate, chemically known as O-3-amino-3-deoxy-α-D-glucopyranosyl-(1→4)-O-[6-amino-6-deoxy-α-D-glucopyranosyl-(1→6)]-N3-(4-amino-2-hydroxy-1-oxobutyl)-2-deoxy-L-streptamine sulfate, is synthesized from the fermentation product kanamycin. Its structural characteristic is the aminohydroxybutyryl chain bound to the streptamine portion of the kanamycin A molecule. Its physicochemical, pharmacological, and pharmacokinetic properties are similar to those of other aminoglycoside antibiotics. Its active site is the 30S subunit of the bacterial ribosome, inhibiting bacterial protein synthesis to produce a bactericidal effect. Amikacin sulfate is characterized by its stability against the aminoglycoside-inactivating enzymes produced by many enteric Gram-negative bacteria, inactivating these enzymes and subsequently losing their antibacterial activity. The emergence of resistant strains is rare, and strains resistant to other aminoglycoside antibiotics remain susceptible to this drug. Amikacin can accumulate in renal cortical cells and inner ear fluid, thus causing certain nephrotoxicity and ototoxicity.
[0003] Serious adverse reactions to the domestic formulation include lumps and abscesses at the intramuscular injection site, which can be intolerable to patients. This is not the case with the original formulation. Amikacin sulfate is a commonly used medication for infectious diseases, and the quality of its formulation significantly impacts its safety and efficacy.
[0004] Based on the resistance mechanism of aminoglycoside antibiotics, amikacin was obtained by rational chemical modification of kanamycin. It is a broad-spectrum semi-synthetic aminoglycoside antibiotic. The derivatization reaction is carried out through silylation, esterification, acylation, hydrolysis, hydrazinolysis and purification reactions, so that the C-1 amino group on the deoxystreptamine in the kanamycin molecule is acylated by L-(-)-4-amino-2-hydroxybutyric acid (L-AHBH). In addition to the target C-1 amino group, the molecular structure of kanamycin also contains three amino groups that can be acylated by L-AHBH, which is not the target site. Therefore, it is possible to produce four isomers ( Figure 1 ), but the other three isomeric impurities [A(K-29), C(K-11), K-6] have almost no antibacterial activity and should be controlled during the derivatization process. The synthetic route is as follows:
[0005]
[0006] The related substances in Amikacin Sulfate Injection come from the following sources: 1. Process impurities; 2. Degradation products generated by the degradation reaction of Amikacin Sulfate. Currently, the USP, JP, ChP and other statutory standards mainly include Amikacin Sulfate Injection. Based on these statutory standards, the known impurity information of Amikacin Sulfate is shown in Table 1 below:
[0007] Table 1 Related substances in amikacin sulfate injection
[0008]
[0009]
[0010] According to the "Guidelines for the Development of Quality Standards for Relevant Impurities in Antibiotics" issued by EMA, the identification limit of unknown single impurities is 0.2%. Among them, there is an unknown single impurity exceeding 0.2% in both the reference preparation and the self-developed product. The impurities detected by the applicant are shown in Table 2 below.
[0011] While existing methods and literature have classified some impurities, this larger impurity remains an unknown impurity. Existing technology fails to control it. Amikacin sulfate contains unknown impurities, which can directly impact product safety and lead to uncontrolled adverse reactions. To further ensure drug quality, thorough research on impurities in samples, analysis of their causes, and strict control of impurity content are pressing technical challenges.
[0012] Table 2 Unknown impurities detected in reference preparation and samples
[0013] sample Unknown impurity content (%) Reference preparation 0.40 sample 0.25 Accelerate January samples 0.24 Accelerate February samples 0.22 Accelerate March samples 0.26 Accelerate June samples 0.28 Summary of the Invention
[0014] In view of the shortcomings of the existing technology, the present invention provides a method for preparing and structurally identifying unknown impurities in amikacin sulfate injection and formulating a control strategy.
[0015] In order to achieve the purpose of the present invention, the technical solutions adopted are as follows:
[0016] A method for preparing unknown impurities in amikacin sulfate injection comprises the following steps:
[0017] (1) Recrystallizing amikacin sulfate injection and filtering to obtain a crystallization mother liquor;
[0018] (2) The crystallization mother liquor was passed through a silica gel column and eluted with a mixed solution of petroleum ether and ethyl acetate, and the components with an Rf of 0.45-0.55 were collected and concentrated to obtain a crude product of the unknown impurity;
[0019] (3) Dissolving the crude unknown impurity in water to prepare a solution, separating and eluting the solution using a preparative liquid chromatograph with a detection wavelength of 190-210 nm, collecting the fractions of the unknown impurity, and drying the fractions to obtain the unknown impurity; wherein the mobile phase A is a 4-6% acetonitrile aqueous solution, and the mobile phase B is a 9-12% acetonitrile aqueous solution, and the volume ratio of the two is 48-52:48-52.
[0020] Preferably, the content of unknown impurities in the crystallization mother liquor in step (1) is 0.8-1.0%.
[0021] Preferably, before passing through the silica gel column in step (2), the crystallization mother liquor is first decompressed to dryness, and then the sample is prepared by a dry method and loaded onto the column.
[0022] Preferably, the dry sample preparation is to reduce the pressure of the crystallization mother liquor to a dry solid sample, add dichloromethane to dissolve it, and then mix it with silica gel and reduce the pressure to dryness.
[0023] Preferably, the elution in step (2) is carried out using a petroleum ether-ethyl acetate mixed solution with a volume ratio of 20:1, 10:1, and 0:1 in sequence.
[0024] Preferably, the volumes of the petroleum ether-ethyl acetate mixed solutions with volume ratios of 20:1, 10:1, and 0:1 in step (2) are 480-520 mL respectively; and the content of the unknown impurity in the crude product of unknown impurities is 9-12%.
[0025] Preferably, the chromatographic conditions for separation by preparative liquid chromatography in step (3) are a flow rate of 1-1.5 ml / min; a detection wavelength of 200 nm, an injection volume of 0.5-2 ml, a column temperature of 35-45° C., and an impurity peak at a retention time of 9-10 min.
[0026] Preferably, the concentration of the solution in step (3) is 20-200 mg / ml.
[0027] Preferably, in step (3), the flow rate is 1.3 mL / min, the column temperature is 40° C., the injection volume is 1 mL, and the impurity peaks at 9.5-10 min.
[0028] Preferably, the mobile phase A and the mobile phase B in step (3) contain a buffer solution, and the mass concentration of the buffer solution in the mobile phase A or the mobile phase B is 4-6%, respectively. The buffer solution is prepared by taking 1.8 g of sodium octane sulfonate and 20.0 g of anhydrous sodium sulfate, adding 0.2 mol / L phosphate buffer solution with a pH of 3.0 and dissolving them.
[0029] Preferably, the phosphate buffer is prepared as a 0.2 mol / L potassium dihydrogen phosphate solution, and the pH value is adjusted to 3.0 with a 0.2 mol / L phosphoric acid solution.
[0030] Preferably, the content of the unknown impurity in step (3) is ≥80%.
[0031] Another object of the present invention is to provide a method for structural identification of an unknown impurity in amikacin sulfate injection, wherein the method comprises analyzing the unknown impurity by LC-MS to determine the structural formula of the impurity:
[0032]
[0033] Preferably, in the analysis conditions of the LC-MS, mobile phase A is 0.1% formic acid solution, mobile phase B is acetonitrile, the gradient elution conditions are: 0-4 min, 100% A; 4-8 min, 100% A→90% A; 8-10 min, 90% A; 10-10.010, 90% A→100% A; 10.010-14 min, 100% A, the column temperature is 28-32 ° C, the UV detector wavelength is 215-225 nm, and the column flow rate is 0.6-1 mL / min.
[0034] Preferably, in step (4), the fragmentation voltage is 68-72 Ev, the atomizer pressure is 58-62 psi, the N2 flow rate is 4.5-5.5 ml / min, the evaporator temperature is 345-355°C, the dryer temperature is 345-355°C, and the capillary transfer voltage is 3.2-3.8 kV.
[0035] Preferably, the LC-MS analysis in step (4) adopts ACPI positive ion detection, mass scan range: 100-1500 m / z, fragmentation voltage: 70 Ev, nebulizer pressure: 60 psi, N2 flow rate: 5.0 ml / min, evaporator temperature: 350°C, dryer temperature: 350°C, and capillary transfer voltage: 3.5 kV.
[0036] Compared with the prior art, the present invention has the following beneficial effects:
[0037] (1) The present invention uses a high performance liquid chromatograph to determine that the impurity is an impurity in the preparation process of amikacin sulfate based on the retention time, which can be confirmed by its liquid chromatogram.
[0038] The molecular weight of the unknown impurity was then determined by LC-MS: the accurate molecular ion peak m / z was 425.2 [M+H] obtained from the LC-MS spectrum. + ,447.2[M+Na] + The molecular weight of the unknown impurity is 424.2, and its molecular formula is inferred from the synthesis process: C 16 H 32 N4O9.
[0039] Analysis of the cause: [M+H] in the first mass spectrum of the unknown impurity+ The ion mass is 525.2, which is 161Da less than that of amikacin. It is speculated that the unknown impurity is a degradation product of amikacin after the six-membered ring is removed. Multi-stage mass spectrometry shows that the [M+H]+ ion at m / z: 425.2 further removes another six-membered ring (losing 161Da) to generate [M+H] + The fragment ion further loses the aminohydroxybutyryl chain (loss of 101 Da) to generate [M+H] + Fragment ion; [M+H] + Another fragmentation pathway of the ion is to first remove the aminohydroxybutyryl chain (loss of 101 Da), generating [M+H] with m / z: 324.2. + The fragment ion then loses another six-membered ring to generate [M+H] with m / z: 163.2 + Fragment ions, see the formula below for details.
[0040]
[0041] Establish the limit of this unknown impurity in amikacin sulfate injection: Considering that the detection amount of amikacin sulfate reference preparation is 0.40%, the limit of this unknown impurity in the self-developed product is set at less than 0.30% to ensure that the quality of the self-developed product is not lower than the reference preparation.
[0042] (2) The present invention uses a silica gel column for preliminary separation, and uses a preparative liquid chromatograph to achieve enrichment and purification of unknown impurities to obtain trace unknown impurities. A high performance liquid chromatograph is used to determine that the impurity is an unknown impurity in the preparation process of amikacin sulfate based on the retention time. By optimizing the preparation method and parameters, a component with a high purity content of the unknown impurity is obtained, and the chemical structure of the unknown impurity is further confirmed, and the cause of its generation is analyzed. The method is simple and can be used for impurity analysis and control of amikacin sulfate injection to ensure quality and drug safety. BRIEF DESCRIPTION OF THE DRAWINGS
[0043] Figure 1 This is the HPLC chromatogram of the sample amikacin;
[0044] Figure 2 for Figure 1 A partial enlarged view of the HPLC chromatogram of amikacin of the sample, wherein 1 is an unknown impurity;
[0045] Figure 3 LC-MS primary mass spectrum of the sample from Example 1;
[0046] Figure 4 LC-MS-MS secondary mass spectrum of the sample in Example 1. DETAILED DESCRIPTION
[0047] The present invention will be further described below with reference to specific embodiments.
[0048] Example 1
[0049] The unknown impurities in amikacin sulfate injection were separated and purified by the following steps:
[0050] 80 ml of amikacin sulfate injection was heated and stirred until saturated, cooled and crystallized, and filtered. The filtrate obtained was the crystallization mother liquor, which was concentrated to dryness under reduced pressure to obtain a solid sample containing about 2% of the unknown impurity. 10 ml of dichloromethane was added to dissolve it, 8 g of 200 mesh silica gel was added, and it was concentrated to dryness under reduced pressure. Then, dry sampling was adopted, and the silica gel column was applied. 500 ml of petroleum ether-ethyl acetate mixed solution with a volume ratio of 20:1, 10:1, and 0:1 was used to elute the column, and the component with Rf of 0.5 was collected. After concentration to dryness under reduced pressure, 200 mg of 10% of the unknown impurity crude product was obtained. 10 ml of purified water was added to dissolve it and a solution was prepared. Gradient elution was performed by preparative liquid chromatography to achieve complete separation, and the sample was injected 20 times. The eluents containing different chromatographic peaks were collected separately and analyzed by HPLC to ensure that the collected chromatographic peaks were the unknown impurities. The complete spectrum of the sample HPLC amikacin chromatogram is shown in the figure. Figure 1 , where 1 is amikacin. See the partial enlarged picture of the HPLC amikacin chromatogram of the sample. Figure 2 , where 1 is an unknown impurity;
[0051] The preparative liquid chromatography separation conditions include: the chromatographic column is Dima Spursil, 4.6 mm*250 mm, 5 μm C18 column, and the injection volume is 1 ml;
[0052] Mobile phase: Dissolve 1.8 g of sodium octane sulfonate and 20.0 g of anhydrous sodium sulfate in 50 ml of 0.2 mol / L phosphate buffer (pH 3.0) (0.2 mol / L potassium dihydrogen phosphate solution, adjust the pH to 3.0 with 0.2 mol / L phosphoric acid solution) and 900 ml of water. Add 50 ml of acetonitrile and mix well. This is mobile phase A.
[0053] Dissolve 1.8 g of sodium octane sulfonate and 20.0 g of anhydrous sodium sulfate in 50 ml of 0.2 mol / L phosphate buffer (pH 3.0) and 850 ml of water, then add 100 ml of acetonitrile and mix thoroughly. This serves as mobile phase B.
[0054] Gradient elution was performed, and the elution table is shown in Table 3; the flow rate was 1.3 ml / min; the column temperature was 40° C.; under the preparative separation conditions with a detection wavelength of 200 nm, the chromatographic peak fraction of 9.984 min was collected and concentrated to dryness under reduced pressure to obtain 20 mg of an impurity sample with a content of 96%.
[0055] Table 3 Mobile phase gradient elution
[0056]
[0057]
[0058] Example 2
[0059] The unknown impurity in amikacin sulfate injection was separated and purified by the following steps: 40 ml of amikacin sulfate injection was measured and heated with stirring until saturated, cooled and crystallized, and then filtered to perform solid-liquid separation. The obtained filtrate was the crystallization mother liquor, which was concentrated to dryness under reduced pressure to obtain a solid sample containing about 2% of the unknown impurity, and then 10 ml of dichloromethane was added to dissolve it, 8 g of 200 mesh silica gel was added, and the solution was concentrated to dryness under reduced pressure. Then, a dry method was used for sample preparation, and the sample was loaded onto a silica gel column. The column was eluted with 250 ml of a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 20:1, 10:1, and 0:1, respectively, and the component with an Rf of 0.5 was collected. After concentrating to dryness under reduced pressure, 100 mg of a crude product of 10% of the unknown impurity was obtained, 5 ml of purified water was added to dissolve it, and a 20 mg / ml solution was prepared. The solution was separated again by preparative liquid chromatography and injected 5 times. The eluates containing different chromatographic peaks were collected separately and analyzed by HPLC to ensure that the collected chromatographic peaks were the unknown impurities. After combining and concentrating to dryness under reduced pressure, 10 mg of a sample with a content of 90% of the unknown impurity was obtained.
[0060] The preparative liquid chromatography separation conditions were the same as in Example 1.
[0061] Example 3
[0062] The structure of the obtained unknown impurity was elucidated by the following steps:
[0063] Molecular weight determination: Weigh approximately 2.00 mg of the unknown impurity sample into a 5 mL volumetric flask, dissolve it in purified water, and dilute to volume to prepare the unknown impurity mother solution. Remove 1 mL of the unknown impurity mother solution and place it in a 10 mL volumetric flask. Dilute to volume with purified water to prepare the unknown impurity solution. LC-MS analysis conditions preferably use an Agilent 1200-Themo-LTQ ORBITRAPXL, column: Unisil 10-120 C18 Aq 10 μm 4.6*250mm, mobile phase: phase A 0.1% formic acid solution, phase B acetonitrile solution, gradient elution (0-4min, 100% A; 4-8min, 100% A→90% A; 8-10min, 90% A; 10-10.010, 90% A→100% A; 10.010-14min, 100% A), column temperature: 30℃, UV detector wavelength: 220nm, column flow rate: 0.8ml / min, injection volume: 100μl, run time 14min, ion source: ACPI, positive ion mode, fragmentor voltage: 70Ev, nebulizer pressure: 60psi, drying gas (N2) flow rate: 5.0ml / min, evaporator temperature: 350℃, dryer temperature: 350℃, capillary transfer voltage: 3.5kV, mass scan range: 100-1500m / z. The accurate molecular ion peak m / z was obtained from the LC-MS spectrum: 425.2[M+H] + , then the molecular weight of the unknown impurity is: 424.2, and its structural formula is inferred from the synthesis process: C 16 H 32 N4O9. The structural formula is:
[0064]
[0065] The LC-MS primary mass spectrum of Example 1 is shown in Figure 3 , LC-MS-MS secondary mass spectrum is shown in Figure 4 .
[0066] Comparative Example 1
[0067] The difference between this comparative example and Example 1 is that the silica gel column elution gradient is different, using a volume ratio of dichloromethane to ethyl acetate of 1:1, totaling 1500 mL, and the rest is the same as Example 1. The final unknown impurity content is 76%.
[0068] Comparative Example 2
[0069] Compared with Example 1, the preparative liquid chromatography conditions in this comparative example were the same as those in Example 1, with mobile phases A and B being the same, but the elution gradient program being different, as shown in Table 4. The flow rate was 1.0 ml / min, the column temperature was 50°C, and all other conditions were the same as in Example 1. The final unknown impurity content in the product was 81%.
[0070] Table 4 Mobile phase gradient elution
[0071] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 30 70 30 30 70 60 0 100 70 0 100 71 30 70 100 30 70
[0072] The above detailed description is a specific description of one feasible embodiment of the present invention. This embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not depart from the present invention should be included in the scope of the technical solution of the present invention.
Claims
1. A method for preparing unknown impurities in amikacin sulfate injection, characterized in that: The steps include: (1) Recrystallizing amikacin sulfate injection and filtering to obtain a crystallization mother liquor; (2) The crystallization mother liquor was passed through a silica gel column and eluted with a mixed solution of petroleum ether and ethyl acetate, and the components with an Rf of 0.45-0.55 were collected and concentrated to obtain a crude product of the unknown impurity; (3) Dissolving the crude unknown impurity in water to prepare a solution, separating and eluting the solution using a preparative liquid chromatograph with a detection wavelength of 190-210 nm, collecting the fractions of the unknown impurity, and drying the fractions to obtain the unknown impurity; wherein the mobile phase A is a 4-6% acetonitrile aqueous solution, and the mobile phase B is a 9-12% acetonitrile aqueous solution, and the volume ratio of the two is 48-52:48-52.
2. The preparation method according to claim 1, characterized in that The content of unknown impurities in the crystallization mother liquor in step (1) is 0.8-1.0%.
3. The preparation method according to claim 1, characterized in that Before passing through the silica gel column in step (2), the crystallization mother liquor is first decompressed to dryness, and then the sample is prepared by dry method and loaded onto the column.
4. The preparation method according to claim 3, characterized in that The dry method sample preparation is to reduce the pressure of the crystallization mother liquor to a dry solid sample, add dichloromethane to dissolve it, and then mix it with silica gel and reduce the pressure to dryness.
5. The preparation method according to claim 1, characterized in that The elution in step (2) is carried out by sequentially using a petroleum ether-ethyl acetate mixed solution with a volume ratio of 20:1, 10:1, and 0:
1.
6. The preparation method according to claim 5, characterized in that The volumes of the petroleum ether-ethyl acetate mixed solutions with volume ratios of 20:1, 10:1, and 0:1 in step (2) are 480-520 mL respectively; the content of the unknown impurity in the crude product is 9-12%.
7. The preparation method according to claim 1, characterized in that The chromatographic conditions for separation by preparative liquid chromatography in step (3) are a flow rate of 1-1.5 ml / min; a detection wavelength of 200 nm, an injection volume of 0.5-2 ml, a column temperature of 35-45° C., and an impurity peak at a retention time of 9-10 min.
8. The preparation method according to claim 1, characterized in that The concentration of the solution in step (3) is 20-200 mg / ml.
9. The preparation method according to claim 7, characterized in that In step (3), the flow rate is 1.3 mL / min, the column temperature is 40° C., the injection volume is 1 mL, and the impurity peaks at a retention time of 9.5-10 min.
10. The preparation method according to claim 1, characterized in that The mobile phase A and mobile phase B in step (3) contain a buffer solution, the mass concentration of the buffer solution in mobile phase A or mobile phase B is 4-6%, and the buffer solution is prepared by taking 1.8 g of sodium octane sulfonate and 20.0 g of anhydrous sodium sulfate, adding 0.2 mol / L phosphate buffer solution with a pH of 3.0 and dissolving them.
11. The preparation method according to claim 1, characterized in that The content of the unknown impurity in step (3) is ≥80%.