CN102192960A - Analysis method for colistimethate sodium - Google Patents

Abstract

The invention belongs to the technical field of drug analysis, and in particular relates to an analysis method of polymyxin E sodium methanesulfonate. The invention provides an analysis method for each component in polymyxin E sodium methanesulfonate, which is simple to operate, good in reproducibility, high in sensitivity, high in accuracy, and easy to standardize, namely providing a polymyxin The component composition analysis method of E sodium methanesulfonate. The analysis method is characterized in that: the polymyxin E sodium methanesulfonate solution is converted into a polymyxin E solution by the method of high temperature hydrolysis after acidification, and then the polymyxin is characterized by the component composition of the polymyxin E The component composition of E sodium methanesulfonate.

Description

Analysis method of polymyxin E sodium methanesulfonate

technical field

The invention belongs to the technical field of drug analysis, and in particular relates to an analysis method for the content of each component of polymyxin E sodium methanesulfonate.

Background technique

Polymyxin E (colistin) is a polypeptide antibiotic composed of multiple components, mainly composed of E ₁ and E ₂ (or called A or B). In the 1950s, polymyxin E was used clinically, mainly for infections caused by Gram-negative bacteria, especially multidrug-resistant Pseudomonas aeruginosa (P. aeruginosa), Acinetobacter baumannii (Acinetobacter baumannii) and other infection-causing treatment. There are two types of colistin in clinical use, one is polymyxin E sulfate (also known as colistin sulfate) for oral or topical use, and the other is polymyxin E methyl sulfide for injection Sodium (ColistimethateSodium). These two drugs are multi-component drugs, and colistin sulfate contains at least 5 components including E ₁ , E ₂ , and E ₃ (see European Pharmacopoeia 5.0). Similarly, polymyxin E sodium methanesulfonate also contains at least five components such as polymyxin E ₁ sodium methanesulfonate and polymyxin E ₂ sodium methanesulfonate.

The general structural formula of polymyxin E is shown in formula I

For the component analysis of polymyxin E, both the United States Pharmacopoeia and the European Pharmacopoeia include the HPLC method for determining the components of polymyxin E. Unfortunately, although polymyxin E sodium methanesulfonate is also included in these pharmacopoeias, there is no corresponding HPLC method to analyze the composition of the components in the product, and only the silicotungstic acid color method is used to control Colistin E Sodium Methanesulfonate does not contain polymyxin E.

Li Jian et al. reported the analysis of colistin sodium mesylate using strong base anion exchange HPLC. (See Li J, Milne RW, Nation RL, et al. Stability of Colistin and Colistin Methanesulfonate in Aqueous Media and Plasma as Determined by High-Performance Liquid Chromatography. ANTIMICROBIAL AGENTS ANDCHEMOTHERAPY; 2003, 47(4): 1364-137) This method also separates 7 chromatographic peaks, but it cannot characterize what components these 7 chromatographic peaks are respectively. At the same time, they also reported that colistin sodium methanesulfonate can be converted into polymyxin E in water, phosphate solution or plasma, but it can not be completely converted, and only 79.6% of it can be converted after 72 hours .

Li Jian et al. also reported that polymyxin E methanesulfonate sodium in rat plasma was converted into polymyxin E by sulfuric acid hydrolysis, and then the composition of polymyxin E was determined by reverse-phase HPLC. (See Li J, MilneRW, Nation RL, et al. Simple Method for Assaying ColistinMethanesulfonate in Plasma and Urine Using High-Performance Liquid Chromatography. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY; 2002, 46(10): 3304-3307) This method is limited to Lower concentrations of colistin sodium methanesulfonate (0.025 mg/ml) do not require the presence of plasma for conversion, and complete conversion to colistin has not been demonstrated.

Contents of the invention

The object of the present invention is to provide a kind of simple operation, good reproducibility, high sensitivity, strong accuracy, the analysis method of each component in the colistin E sodium methanesulfonate that is easy to standardized operation, promptly provides a kind of polymyxin E sodium methanesulfonate Composition analysis method of bacterin E sodium methanesulfonate.

The component analysis method of polymyxin E sodium methanesulfonate provided by the invention is characterized in that: the polymyxin E sodium methanesulfonate solution is converted into polymyxin E solution by adopting the method of high temperature hydrolysis after acidification, Then the component composition of polymyxin E sodium methanesulfonate was characterized by the component composition of polymyxin E.

The component analysis method provided by the present invention specifically includes the following steps: (a) dissolving polymyxin E sodium methanesulfonate in water to make a solution with a certain concentration; Mycolistin E sodium methanesulfonate solution was transformed into polymyxin E solution; (c) the composition of polymyxin E was analyzed by reversed-phase high performance liquid chromatography; (d) the components of polymyxin E were determined Characterization of the composition of polymyxin E sodium methanesulfonate.

In the above-mentioned component analysis method: the polymyxin E sodium methanesulfonate concentration that makes in the step (a), is advisable to be more suitable for subsequent reverse phase high performance liquid chromatography analysis analysis concentration, is preferably 0.005-5mg/ ml, more preferably 0.05-3 mg/ml, most preferably 0.1-2 mg/ml.

The acidification and high-temperature hydrolysis methods used in the step (b) refer to the use of an acid solution to adjust the polymyxin E sodium methanesulfonate solution to acidity, so that the pH of the solution is 1.0-3.5, preferably 1.5-3.2, The most preferred pH is 2.0-3.0, and then the acidified solution is placed in a high temperature environment. Described acid solution can be inorganic acid or organic acid, preferably inorganic acid, more preferably hydrochloric acid, sulfuric acid or phosphoric acid, more preferably sulfuric acid, most preferably 0.1-1mol/L sulfuric acid solution, such as 0.5mol/L sulfuric acid solution. The high temperature is preferably 50-150°C, more preferably 65-120°C, most preferably 75-115°C. The time for placing in a high temperature environment is within 2 hours, preferably 0.5-1 hour.

In step (c), the composition of polymyxin E is analyzed by reverse-phase high-performance liquid chromatography. The reverse-phase high-performance liquid chromatography analysis is a method known to those skilled in the art. For example, reference can be made to the European Pharmacopoeia (version 5.0) Or the HPLC analysis method for polymyxin E related substances announced by the United States Pharmacopoeia (29 edition), preferably using the method published by the European Pharmacopoeia.

The specific analysis method can also be carried out as follows:

Component determination chromatographic conditions:

Instrument: Waters company high performance liquid chromatography (Waters 2489, ultraviolet detector, alliancee2695)

Chromatographic column: Waters XBridge C18 (5μm, 4.6×250mm);

Mobile phase: sodium sulfate solution (take 4.46g of anhydrous sodium sulfate, add 900ml of water, adjust the pH value to 2.5 with phosphoric acid, add water to 1000ml, and mix): acetonitrile (volume ratio is 78:22);

Detection wavelength selection: 215nm;

Flow rate: 1.0ml/min; column temperature: 30°C; injection volume: 20μl.

Described in the step (d) with the component composition of polymyxin E to characterize the component composition of polymyxin E sodium mesylate is based on the polymyxin E sodium mesylate can be completely converted into polymyxin Polymyxin E and polymyxa E are stable under the above acidification and high temperature conditions, and the components of polymyxin E sodium methanesulfonate will not be transformed into each other.

The invention provides a method for analyzing the content of each component in polymyxin E sodium methanesulfonate, the method skillfully transforms polymyxin E sodium methanesulfonate completely into polymyxin E, by analyzing The content analysis of each component of polymyxin E obtained the content of each component of polymyxin E sodium methanesulfonate. The method has the following advantages: (1) polymyxin E sodium methanesulfonate provided by the invention is converted into polymyxin E scheme is simple and easy, and is convenient for standardization operation, and under this transformation condition, polymyxin Sodium E methanesulfonate can be completely transformed into polymyxin E and each component will not transform into each other, while polymyxin E can remain stable under this condition. (2) The present invention adopts the existing proven high-efficiency liquid phase analysis method to measure the content of each component, which can ensure that the test results are true and effective.

Description of drawings

Figure 1: HPLC analysis results of polymyxin sulfate E ₁ ;

Figure ₂ : HPLC analysis results of colistin sulfate;

Figure 3: HPLC analysis results of colistin sulfate at room temperature;

Figure 4: HPLC analysis results under polymyxin E sulfate acidification and high temperature conditions;

Figure 5: HPLC analysis results of polymyxin E sodium methanesulfonate after acidification and high temperature hydrolysis;

Figure 6: HPLC analysis results of polymyxin E ₁ sodium methanesulfonate after acidification and high temperature hydrolysis;

Figure 7: HPLC analysis results of polymyxin E ₂ sodium methanesulfonate after acidification and high temperature hydrolysis;

Detailed ways

The following specific examples are used to further illustrate the content of the present invention, but it is not meant to limit the present invention in any way.

Embodiment 1: the preparation of polymyxin E sulfate

Weigh an appropriate amount of industrial-grade polymyxin E sulfate, add water to dissolve, and make a solution containing 100 mg per 1 ml, and separate it on a chromatographic column. The chromatographic conditions are as follows:

Chromatography packing: polystyrene/divinylbenzene as the skeleton and bonded phenyl, model NM-100, produced by Suzhou Nano Microbe Technology Co., Ltd.

Column specification: 45×400mm,

Column volume (CV): 635.9ml

Flow rate: 30.0ml/min

Sample volume: 500ml

Elution condition:

Wash the mobile phase: first wash 10CV with a dilute sulfuric acid solution containing 8% ethanol (adjust the pH of the solution to 2.4 with 0.05mol/L sulfuric acid).

Eluent: then use 30% ethanol dilute sulfuric acid solution (use 0.05mol/L sulfuric acid to adjust the pH value of the solution to 2.4.) to elute 10CV. The eluate was collected, after the ethanol was distilled off under reduced pressure, and vacuum-dried to obtain 33 g of polymyxin E sulfate.

Embodiment 2: the preparation of polymyxin sulfate E ₁ and E ₂

Weigh an appropriate amount of industrial-grade polymyxin E sulfate, add water to dissolve, and make a solution containing 100 mg per 1 ml, and separate it on a chromatographic column. The chromatographic conditions are as follows:

Chromatographic column: C ₁₈ SunFire Prep OBD (10μm); 19×150mm, purchased from WATERS, USA

Column volume (CV): 42.5ml

Flow rate: 10.0ml/min

Elution conditions: 30CV isocratic elution with dilute sulfuric acid solution containing 30% ethanol (use 0.05mol/L sulfuric acid to adjust the pH value of the solution to 2.3.).

Sample volume: 50ml

Collection: 10ml/tube

Analysis and processing of collected samples: Analytical HPLC analysis (for chromatographic conditions, refer to the determination of the purity of polymyxin E in European Pharmacopoeia 5.0), and the components of polymyxin E ₁ and E ₂ with a purity of more than 95% were combined , and then carry out vacuum distillation to remove ethanol, and freeze-dry at low temperature.

As a result, the single components of 1.5 g of polymyxin E ₁ sulfate and 0.5 g of polymyxin E ₂ sulfate were obtained, and their HPLC analysis diagrams are shown in Fig. 1 and Fig. 2 .

Embodiment 3: the preparation of polymyxin E sodium methanesulfonate

Weigh 10.0g of polymyxin E sulfate prepared by the method of Example 1, add water to dissolve and settle to 50ml, then add 6.2ml of formaldehyde solution, stir and adjust the pH to 7.0 with 10mol/L NaOH solution, after maintaining the reaction for 30min, 28 ml of 40% sodium bisulfite solution was added thereto, and then the pH was adjusted to 7 with NaOH solution, and the reaction was stopped after stirring for 10 h. Get this reaction solution, ultrafiltration to about 20ml with a molecular weight cut-off of 3000 ultrafiltration membrane (the product of U.S. MILLIPORE company, is a cellulose acetate membrane), then add water to about 100ml, then ultrafiltration and concentration to 20ml, so repeated for 5 times, about 15ml of ultrafiltration retentate was obtained. The temperature was controlled at 20-25°C throughout the reaction and ultrafiltration period. The ultrafiltration retentate was taken, and the sample was dried by a low-temperature freeze-drying method to obtain 7.5 g of a polymyxin E sodium methanesulfonate sample.

Embodiment 4: the preparation of polymyxin E ₁ sodium methanesulfonate

Weigh 1.0 g of polymyxin sulfate E ₁ prepared by the method in Example 2, add water to dissolve it and set the volume to 5 ml, then add 0.5 ml of formaldehyde solution, stir and adjust the pH to 7.0 with 1 mol/L NaOH solution, and maintain the reaction for 30 minutes , 2 ml of 40% sodium bisulfite solution was added thereto, and then the pH was adjusted to 7 with NaOH solution, and the reaction was stopped after stirring for 10 h. Get this reaction solution, ultrafiltration to about 3ml with a molecular weight cut-off of 3000 ultrafiltration membrane (the product of U.S. MILLIPORE company, is a cellulose acetate membrane), then add water to about 20ml, then ultrafiltration and concentration to 3ml, so repeated for 5 times, about 5ml of ultrafiltration retentate was obtained. The temperature was controlled at 20-25°C throughout the reaction and ultrafiltration period. The ultrafiltration retentate was taken, and the sample was dried by a low-temperature freeze-drying method to obtain 0.8 g of a polymyxin E ₁ sodium methanesulfonate sample.

Embodiment 5: the preparation of polymyxin E ₂ sodium methanesulfonate

Take by weighing 1.0g of the polymyxin E2 sulfate prepared by the method of Example ₂ (the content of polymyxin _E2 is 82%), add water to dissolve and settle to 5ml, then add 0.5ml of formaldehyde solution, stir and use 1mol /L NaOH solution to adjust the pH to 7.0, after maintaining the reaction for 30min, add 2ml of 40% sodium bisulfite solution to it, then adjust the pH to 7 with NaOH solution, and react under stirring for 10h to stop the reaction. Get this reaction solution, ultrafiltration to about 3ml with a molecular weight cut-off of 3000 ultrafiltration membrane (the product of U.S. MILLIPORE company, is a cellulose acetate membrane), then add water to about 20ml, then ultrafiltration and concentration to 3ml, so repeated for 5 times, about 5ml of ultrafiltration retentate was obtained. The temperature was controlled at 20-25°C throughout the reaction and ultrafiltration period. The ultrafiltration retentate was taken, and the sample was dried by a low-temperature freeze-drying method to obtain 0.7 g of a polymyxin E ₂ sodium methanesulfonate sample.

Embodiment 6: Stability condition exploration of colistin sulfate

Take by weighing the appropriate amount of polymyxin E sulfate prepared by the method of Example 1, add water to dissolve, and make a solution containing 2 mg polymyxin E per 1 ml. Take 5ml of this solution, and adjust the pH value to 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 with 0.5mol/L sulfuric acid solution. These polymyxin E solutions with different pH values were placed in ovens at 50°C, 75°C, 100°C, 125°C, 150°C, and 200°C for 2 hours, and the content changes were measured by HPLC after taking them out. After the above-mentioned conditions are applied, the test condition that the content of polymyxin E sulfate is still greater than or equal to 95%, that is, the decomposition is less than or equal to 5%, is considered stable, otherwise it is considered that polymyxin sulfate is unstable under this condition.

The test results show that polymyxin sulfate is stable at pH 1.0-3.5 when placed at 50-150°C for 2 hours. See accompanying drawings 3 and 4 for typical HPLC analysis chromatograms.

Example 7: Exploration of polymyxin E sodium methanesulfonate acidification and high temperature hydrolysis conditions

Weigh an appropriate amount of polymyxin E sodium methanesulfonate prepared by the method of Example 3, add water to dissolve, and make a solution containing 1 mg per 1 ml. Take 5ml of this solution, and adjust the pH value to 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 with 0.5mol/L sulfuric acid solution. Put these solutions with different pH values in ovens at 50°C, 75°C, 100°C, 125°C, 150°C, and 200°C for 2 hours, and then use HPLC to determine polymyxin E methanesulfonic acid. Extent of conversion of sodium to polymyxin E. After the above-mentioned conditions act, the conversion rate of polymyxin E sodium methanesulfonate into polymyxin E is greater than or equal to 95%. The test conditions consider that the conversion is complete, otherwise it is considered that the conversion is incomplete under this condition.

The test results show that polymyxin sodium methanesulfonate can be completely converted into polymyxin at pH 1.0-3.5 and at 50-150°C.

The typical HPLC spectrum of polymyxin E sodium methanesulfonate after acidification and high temperature hydrolysis is shown in Figure 5.

Example 8: Polymyxin E ₁ Sodium Methanesulfonate Acidification and High Temperature Hydrolysis Experiment

Weigh an appropriate amount of polymyxin E ₁ sodium methanesulfonate, add water to dissolve, and make a solution containing 5 mg per 1 ml. Take 5ml of this solution, adjust the pH value to 2.0 with 0.5mol/L sulfuric acid solution, then dilute with water, and dilute to 10ml to prepare a 2.5mg/ml solution. Take this solution in a glass ampoule, seal it and place it in an oven at 120°C for 30 minutes, then take it out and let it cool, and use HPLC to determine the conversion rate of polymyxin E ₁ sodium methanesulfonate to polymyxin E ₁ degree.

HPLC measurement showed that the concentration of polymyxin E ₁ in the solution was 1.67 mg/ml, which was consistent with the theoretical value of complete conversion into polymyxin E ₁ , which indicated that polymyxin E ₁ sodium methanesulfonate Can be completely transformed into polymyxin E ₁ .

The HPLC spectrum of polymyxin E ₁ sodium methanesulfonate after acidification and high temperature hydrolysis is shown in Figure 6.

Example 9: Polymyxin E ₂ Sodium Methanesulfonate Acidification and High Temperature Hydrolysis Experiment

Weigh an appropriate amount of polymyxin E ₂ sodium methanesulfonate, add water to dissolve, and make a solution containing 5 mg per 1 ml. Take 5ml of this solution, adjust the pH value to 1.5 with 0.5mol/L sulfuric acid solution, then dilute with water, and dilute to 10ml to prepare a 2.5mg/ml solution. Take this solution in a glass ampoule, seal it and place it in an oven at 115°C for 30 minutes, then take it out and let it cool, and use HPLC to determine the conversion rate of polymyxin E ₂ sodium methanesulfonate into polymyxin E ₂ degree.

HPLC measurement showed that the concentration of polymyxin E ₂ in the solution was 1.66 mg/ml, which was consistent with the theoretical value of complete conversion into polymyxin E ₂ , which indicated that polymyxin E ₂ sodium methanesulfonate Can be completely transformed into polymyxin E ₂ .

The HPLC spectrum of polymyxin E ₂ sodium methanesulfonate after acidification and high temperature hydrolysis is shown in Figure 7.

Claims (6)

1. a component analysis method of polymyxin E sodium methanesulfonate, is characterized in that: adopt the method for high temperature hydrolysis after acidification to change polymyxin E sodium methanesulfonate solution into polymyxin E solution, Then the component composition of polymyxin E sodium methanesulfonate was characterized by the component composition of polymyxin E. 2. the described analytical method of claim 1, specifically comprises the steps: (a) polymyxin E sodium methanesulfonate is dissolved in water, makes the solution of certain concentration; (b) adopts the method for high temperature hydrolysis after acidifying Colistin E sodium methanesulfonate solution was transformed into colistin E solution; (c) the composition of polymyxin E was analyzed by reversed-phase high performance liquid chromatography; (d) the components of polymyxin E were Composition to characterize the component composition of polymyxin E sodium methanesulfonate. 3. the described analysis method of claim 2, wherein the polymyxin E sodium methanesulfonate concentration of making in the step (a), it is advisable to be more suitable for the subsequent reverse phase high performance liquid chromatography analysis analysis concentration, preferably 0.005-5 mg/ml, more preferably 0.05-3 mg/ml, most preferably 0.1-2 mg/ml. 4. the analytical method described in claim 2,3, wherein the acidification used in the step (b), high-temperature hydrolysis method, refer to with acid solution, regulate polymyxin E sodium methanesulfonate solution to acidity, make solution The pH is 1.0-3.5, preferably 1.5-3.2, most preferably 2.0-3.0, and then the acidified solution is placed in a high temperature environment. 5. the described analysis method of claim 4, wherein acid solution is inorganic acid or organic acid, is preferably inorganic acid, is more preferably hydrochloric acid, sulfuric acid or phosphoric acid, is more preferably sulfuric acid, most preferably the sulfuric acid solution of 0.1-1mol/L . 6. The analysis method according to claims 2 and 3, wherein the time of placing in the high temperature environment is within 2 hours, preferably 0.5-1 hour.

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