Method for measuring content of single impurity in atracurium besylate injection

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and discloses a method for determining the content of a single impurity in an atracurium besylate injection. The preparation method comprises the following steps: preparing an atracurium besylate injection into a test solution by using a solvent; and respectively and precisely measuring the blank solution and the test solution, carrying out high performance liquid chromatography detection, recording a chromatogram, and calculating according to an area normalization method to obtain the content of unknown single impurities with the relative retention time of 0.95 in the atracurium besylate injection. The conditions of high performance liquid chromatography detection are as follows: the column temperature of a chromatographic column is 20-30 DEG C; the sample size of the blank solution and the sample size of the test solution are respectively 20 microliters; a mobile phase is an ammonium formate buffer solution-acetonitrile-methanol mixed system; the flow velocity is 1.0 mL / min; the detection wavelength is 270 to 290 nm. According to the method, accurate detection of unknown single impurities with relative retention time of 0.95 can be realized, so that the product quality of the atracurium besylate injection can be better controlled.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for determining the content of a single impurity in atracurium besylate injection. Background Art

[0002] Atracurium besylate injection is a skeletal muscle relaxant that has important applications in surgery and intensive care treatment. It is used as an adjuvant in general anesthesia or as a muscle relaxant in the intensive care unit (ICU), facilitating endotracheal intubation and mechanical ventilation. Atracurium besylate injection contains unknown single impurities with a relative retention time of 0.95, and the presence of these unknown single impurities may affect the safety and efficacy of the drug. Its content must be strictly controlled during the production process to ensure the quality of the drug. Summary of the invention

[0003] In view of the above problems existing in the prior art, the present application provides a method for determining the content of single impurities in atracurium besylate injection, which can achieve accurate detection of unknown single impurities with a relative retention time of 0.95.

[0004] To solve the above problems, the present invention provides the following technical solutions: The present application provides a method for determining the content of a single impurity in atracurium besylate injection, comprising the following steps: Take atracurium besylate injection and prepare the test solution with solvent; Precisely measure the blank solution and the test solution for high performance liquid chromatography detection, record the chromatogram, and calculate the content of the single impurity in atracurium besylate injection by the area normalization method; The single impurity is an unknown single impurity with a relative retention time of 0.95; The conditions for HPLC detection were: The column temperature is 20-30°C; The injection volume of blank solution and test solution was 20 μL respectively; The mobile phase was a mixture of formate buffer, acetonitrile and methanol; Flow rate 0.8-1.2 mL / min; The detection wavelength is 270-290nm.

[0005] In one embodiment of the present application, the solvent is a mixed system of ammonium formate buffer-acetonitrile-methanol.

[0006] In one embodiment of the present application, the volume ratio of formate buffer, acetonitrile and methanol is 63:20:17.

[0007] In one embodiment of the present application, the preparation method of ammonium formate buffer is: take 32.8g of ammonium formate, add 984mL of water to dissolve it, and then add 16mL of anhydrous formic acid to prepare an ammonium formate buffer with a concentration of 32.8mg / mL.

[0008] In one embodiment of the present application, the concentration of the test solution is 1.0 mg / mL.

[0009] In one embodiment of the present application, the column temperature of the chromatographic column is 25°C.

[0010] In one embodiment of the present application, the model of the chromatographic column is Boston Prime C18; the specification of the chromatographic column is 4.6×250 mm×3 μm.

[0011] In one embodiment of the present application, the flow rate is 1.0 mL / min.

[0012] In one embodiment of the present application, the detection wavelength is 280 nm.

[0013] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention can accurately detect an unknown single impurity with a relative retention time of 0.95 in atracurium besylate injection, separate other substances in atracurium besylate injection, and thus better control the product quality of atracurium besylate injection.

[0014] (2) The present invention uses the mobile phase as the solvent, the operation steps are simple, the detection data is accurate, the stability is good, the safety is high, and the detection cost is low.

[0015] (3) The present invention has good stability in the determination of unknown single impurities with a relative retention time of 0.95. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the chromatogram of the blank solution; Figure 2 is the chromatogram of the test solution; Figure 3 is the chromatogram of stability solution 1 (0 hours); Figure 4 is the chromatogram of stability solution 2 (4.5 hours); Figure 5 This is the chromatogram of stability solution 3 (10.5 hours). DETAILED DESCRIPTION

[0017] The following will provide a clear and complete description of the technical solutions in the embodiments of the present application in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments.

[0018] The "range" disclosed in this application is defined in the form of a lower limit and an upper limit, and a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of the particular range. The range defined in this way can be inclusive or exclusive of the end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a range.

[0019] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between liquids and solids, the percentage is volume / weight percentage; when the present invention relates to the percentage between solids and liquids, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0020] Example 1 Method for determining the unknown single impurity content with a relative retention time of 0.95 in atracurium besylate injection The high performance liquid chromatography method was used for determination, and the operation steps were as follows: (1) Establishment of chromatographic conditions and system suitability: A Boston Prime C18 4.6×250mm×3μm column was used, with ammonium formate buffer (32.8g of ammonium formate was dissolved in 984mL of water, and then 16mL of anhydrous formic acid)-acetonitrile-methanol (63:20:17) as the mobile phase, the detection wavelength was 280nm, the flow rate was 1.0mL per minute, and the column temperature was 25°C; (2) Preparation of test solution: Accurately measure 5 mL of atracurium besylate injection and place it in a 10 mL volumetric flask. Add solvent to dilute to the mark and shake well to prepare a test solution with a concentration of 1.0 mg / mL. (3) Determination: Accurately measure 20 μL of blank solution and test solution respectively and inject them into liquid chromatograph, record the chromatogram and calculate it by area normalization.

[0021] Example 2 Method for determining the unknown single impurity content at atracurium besylate injection with a relative retention time of 0.95 The high performance liquid chromatography method was used for determination, and the operation steps were as follows: (1) Establishment of chromatographic conditions and system suitability: A Boston Prime C18 4.6×250mm×3μm column was used, with ammonium formate buffer (32.8g of ammonium formate was dissolved in 984mL of water, and then 16mL of anhydrous formic acid)-acetonitrile-methanol (63:20:17) as the mobile phase, the detection wavelength was 280nm, the flow rate was 0.8mL / min, and the column temperature was 25°C; (2) Preparation of test solution: Accurately measure 5 mL of atracurium besylate injection and place it in a 10 mL volumetric flask. Add solvent to dilute to the mark and shake well to prepare a test solution with a concentration of 1.0 mg / mL. (3) Determination: Accurately measure 20 μL of blank solution and test solution respectively and inject them into liquid chromatograph, record the chromatogram and calculate it by area normalization.

[0022] Example 3 Method for determining the unknown single impurity content with a relative retention time of 0.95 in atracurium besylate injection The high performance liquid chromatography method was used for determination, and the operation steps were as follows: (1) Establishment of chromatographic conditions and system suitability: A Boston Prime C18 4.6×250mm×3μm column was used, with ammonium formate buffer (32.8g of ammonium formate was dissolved in 984mL of water, and then 16mL of anhydrous formic acid)-acetonitrile-methanol (63:20:17) as the mobile phase, the detection wavelength was 280nm, the flow rate was 1.2mL / min, and the column temperature was 25°C; (2) Preparation of test solution: Accurately measure 5 mL of atracurium besylate injection and place it in a 10 mL volumetric flask. Add solvent to dilute to the mark and shake well to prepare a test solution with a concentration of 1.0 mg / mL. (3) Determination: Accurately measure 20 μL of blank solution and test solution respectively and inject them into liquid chromatograph, record the chromatogram and calculate it by area normalization.

[0023] Example 4 Method for determining the unknown single impurity content with a relative retention time of 0.95 in atracurium besylate injection The high performance liquid chromatography method was used for determination, and the operation steps were as follows: (1) Establishment of chromatographic conditions and system suitability: A Boston Prime C18 4.6×250mm×3μm column was used, with ammonium formate buffer (32.8g of ammonium formate was dissolved in 984mL of water, and then 16mL of anhydrous formic acid)-acetonitrile-methanol (63:20:17) as the mobile phase, the detection wavelength was 280nm, the flow rate was 1.0mL per minute, and the column temperature was 20℃; (2) Preparation of test solution: Accurately measure 5 mL of atracurium besylate injection and place it in a 10 mL volumetric flask. Add solvent to dilute to the mark and shake well to prepare a test solution with a concentration of 1.0 mg / mL. (3) Determination: Accurately measure 20 μL of blank solution and test solution respectively and inject them into liquid chromatograph, record the chromatogram and calculate it by area normalization.

[0024] Example 5 Method for determining the unknown single impurity content with a relative retention time of 0.95 in atracurium besylate injection The high performance liquid chromatography method was used for determination, and the operation steps were as follows: (1) Establishment of chromatographic conditions and system suitability: A Boston Prime C18 4.6×250mm×3μm column was used, with ammonium formate buffer (32.8g of ammonium formate was dissolved in 984mL of water, and then 16mL of anhydrous formic acid)-acetonitrile-methanol (63:20:17) as the mobile phase, the detection wavelength was 280nm, the flow rate was 1.0mL per minute, and the column temperature was 30°C; (2) Preparation of test solution: Accurately measure 5 mL of atracurium besylate injection, place it in a 10 mL volumetric flask, add solvent to dilute to the mark, and shake well; (3) Determination: Accurately measure the blank solution and the test solution respectively and inject them into the liquid chromatograph. Record the chromatogram and calculate it by area normalization.

[0025] In order to further verify the feasibility of the present invention, a series of tests were carried out, as follows: 1. Separation of unknown impurity with relative retention time 0.95 from other substances Weigh atracurium besylate and dilute it with a solvent to make a 1.0 mg / mL solution.

[0026] Weigh 54 mg of sodium citrate and 3.2 mg of anhydrous citric acid, add 100 mL of water to dissolve, measure 4 mL, place in a 200 mL volumetric flask, add solvent to dilute to the scale, shake well, and use as the blank excipient solution.

[0027] Test solution: Accurately measure 5.0 mL of the product, place it in a 10 mL volumetric flask, add solvent to dilute to the scale, and shake well.

[0028] Take atracurium besylate solution, blank excipient solution, and test solution, and determine them using the above method. The HPLC chart is shown in Figure 1~Figure 2 , the test results are shown in Table 1.

[0029] Table 1 Separation of unknown impurities with a relative retention time of 0.95 and other substances

[0030] The results showed that the unknown single impurity with a relative retention time of 0.95 was well separated from other substances, and other substances did not interfere with the detection of the unknown single impurity with a relative retention time of 0.95.

[0031] 2. Stability of unknown single impurity with relative retention time of 0.95 Take the test sample solution and examine its solution stability at different time points.

[0032] Take stability solutions 1, 2, and 3 (i.e., test solutions at different time points) and test them using the method of Example 1. The HPLC spectra are shown in Figure 3~Figure 5 The test results are shown in Table 2.

[0033] Table 2 Solution stability of unknown single impurity with relative retention time of 0.95

[0034] The results showed that the unknown single impurity with a relative retention time of 0.95 had good stability.

[0035] The present application is described in detail above in conjunction with specific implementation methods and exemplary embodiments, but these descriptions cannot be understood as limiting the present application. Those skilled in the art understand that, without departing from the spirit and scope of the present application, the technical solution of the present application and its implementation methods may be subjected to a variety of equivalent substitutions, modifications or improvements, all of which fall within the scope of the present application.

Claims

1. A method for determining the content of single impurities in atracurium besylate injection, characterized in that: The following steps are involved: Take atracurium besylate injection and prepare the test solution using ammonium formate buffer-acetonitrile-methanol mixed system; Precisely measure the blank solution and the test solution for high performance liquid chromatography detection, record the chromatogram, and calculate the content of the single impurity in atracurium besylate injection by the area normalization method; The single impurity is an unknown single impurity with a relative retention time of 0.95; The conditions for the HPLC detection are: The column temperature is 20-30°C; The injection volume of blank solution and test solution was 20 μL respectively; The mobile phase was a mixture of formate buffer, acetonitrile and methanol; Flow rate 0.8-1.2 mL / min; The detection wavelength is 270-290nm.

2. The method for determining the single impurity content in atracurium besylate injection according to claim 1, characterized in that: The volume ratio of formate buffer, acetonitrile and methanol was 63:20:

17.

3. The method for determining the single impurity content in atracurium besylate injection according to claim 2, characterized in that: The preparation method of ammonium formate buffer is as follows: take 32.8 g of ammonium formate, add 984 mL of water to dissolve it, and then add 16 mL of anhydrous formic acid to prepare an ammonium formate buffer with a concentration of 32.8 mg / mL.

4. The method for determining the single impurity content in atracurium besylate injection according to claim 1, characterized in that: The concentration of the test solution is 1.0 mg / mL.

5. The method for determining the single impurity content in atracurium besylate injection according to claim 1, characterized in that: The column temperature of the chromatographic column is 25°C.

6. The method for determining the single impurity content in atracurium besylate injection according to claim 1, characterized in that: The model of the chromatographic column is Boston Prime C18; the specification of the chromatographic column is 4.6×250 mm×3 μm.

7. The method for determining the single impurity content in atracurium besylate injection according to claim 1, characterized in that: The flow rate was 1.0 mL / min.

8. The method for determining the single impurity content in atracurium besylate injection according to claim 1, characterized in that: The detection wavelength is 280 nm.

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