Method for determining related substances and components of polymyxin B sulfate by adopting HPLC (High Performance Liquid Chromatography)

Abstract

The invention discloses a method for determining related substances and components of polymyxin B sulfate by adopting HPLC (High Performance Liquid Chromatography), and belongs to the field of medicine detection. The components are polymyxin sulfate B1, B2, B3 and B1-I, the related substances are impurities E, F, G and M. The method is a high performance liquid chromatography method, Thermo Hypersil GOLDTM is used as a chromatographic column, a sodium sulfate solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, the detection wavelength is 215 nm, the flow velocity of the chromatographic column is 0.8 ml per minute, the column temperature of the chromatographic column is 20 DEG C, the sample injection volume is 20 microliters, the detection time is 30 minutes, and the detection time is 30 minutes. According to the present invention, the components of the polymyxin B sulfate are separated from the related substances by using the gradient elution, such that the effective separation of the four main component peaks and the adjacent peak impurities of the polymyxin B sulfate on the chromatographic column is achieved, and the contents of the related substances and the components of the polymyxin B sulfate can be rapidly, conveniently and accurately determined.

Description

Technical Field

[0001] The invention belongs to the field of drug detection, and in particular relates to a method for determining polymyxin B sulfate-related substances and components by using HPLC. Background Art

[0002] Polymyxin B Sulfate is the sulfate of polymyxin B1 and B2, produced by Bacillus polymyxa. Its English name is Polymyxin B Sulfate, and its chemical structure is shown in Formula 1.

[0003]

[0004] Polymyxin B sulfate is obtained by fermentation, so this variety has more related substances, and this variety has four components, namely polymyxin B1, B2, B3 and B1-Ⅰ, all of which are active ingredients. The chromatographic conditions of the pharmacopoeia cannot separate the polymyxin B sulfate components from the adjacent impurity peaks. The main factor affecting the safety of drug use is the control and research of impurities. In order to ensure the safety of drugs, impurities must be fully studied.

[0005] Therefore, it is necessary to establish a fast, convenient and accurate high performance liquid chromatography (HPLC) analysis method for effectively separating the four main component peaks of polymyxin B sulfate from the adjacent peak impurities and related substances of polymyxin B sulfate. Summary of the invention

[0006] The present invention aims to provide a method for determining polymyxin B sulfate-related substances and components by using HPLC.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] A method for determining polymyxin B sulfate related substances and components using HPLC, as follows:

[0009] 1. Chromatographic conditions

[0010] 1. The chromatographic column is filled with octadecylsilane bonded silica gel (Thermo Hypersil GOLDTM 4.6mm*250, 5μm or a chromatographic column with equivalent performance);

[0011] 2. Mobile phase: Sodium sulfate solution (dissolve 4.46 g of anhydrous sodium sulfate in 900 ml of water, adjust the pH value to 2.3±0.1 with dilute phosphoric acid, and then dilute to 1000 ml with water) as mobile phase A; acetonitrile as mobile phase B;

[0012] 3. The flow rate is 0.8 ml per minute;

[0013] 4. The detection wavelength is 215nm;

[0014] 5. Column temperature is 20°C;

[0015] 6. Injection volume 20μl;

[0016] 7. Gradient elution procedure is as follows

[0017]

[0018] 2. Solution Preparation

[0019] 1. Blank solution: water-acetonitrile (80:20).

[0020] 2. Test solution: Take 50 mg of the product, weigh accurately, place in a 100 ml volumetric flask, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, shake well. Or prepare a solution of equal concentration (about 0.5 mg / ml). The term "test solution" refers to the solution of the sample to be tested in the high performance liquid chromatography detection and analysis method.

[0021] 3. Reference solution: Take 50 mg of the reference substance, weigh it accurately, put it in a 100 ml volumetric flask, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, shake well. Or prepare a solution of equal concentration (about 0.5 mg / ml). The term "reference solution" refers to the solution of the standard sample in the high performance liquid chromatography detection and analysis method.

[0022] 4.1% control solution: Accurately measure 1 ml of the control solution, place it in a 100 ml volumetric flask, dilute to the mark with water-acetonitrile (80:20), and shake well. Or prepare a solution of equal concentration.

[0023] 5. Stock solution of each impurity: Take 3 mg of each impurity E, impurity F, impurity G, and impurity M, weigh accurately, place in a 10 ml volumetric flask, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, shake well. Or prepare a solution of equal concentration (about 300 μg / ml for each impurity).

[0024] Single standard solution of each impurity: accurately measure 1 ml of each impurity stock solution, place in a 20 ml volumetric flask, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, shake well. Or prepare a solution of equal concentration (about 15 μg / ml for each impurity).

[0025] 6. Mixed solution: Take 10 mg of this product, weigh accurately, put it in a 20 ml volumetric flask, add 1 ml of each impurity stock solution, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, shake well. Or prepare a solution of equal concentration (main component about 0.5 mg / ml; each impurity about 15 μg / ml).

[0026] 3. Determination method

[0027] Accurately measure blank solution, control solution, 1% control solution, and test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0028] IV. Calculation Method and Acceptance Limit

[0029] Calculated by the external standard method based on the corresponding peak area, the content of polymyxin B3 shall not exceed 6.0%, the content of polymyxin B1-Ⅰ shall not exceed 15.0%, and the total content of polymyxin B1, B2, B3 and B1-Ⅰ shall not be less than 80% based on the dry product;

[0030] If there are impurity peaks in the chromatogram of the test solution, calculated by the area normalization method, the single impurity shall not exceed 3.0%, the total amount of impurities shall not exceed 17.0%, and the peaks less than 0.7 times the area of ​​the main peak of the control solution shall be ignored.

[0031] The present invention has the following advantages: the present invention provides an HPLC method, the chromatographic column used in the method uses octadecylsilane bonded silica gel as a filler (Thermo Hypersil GOLDTM 4.6mm*250, 5μm or a chromatographic column with equivalent performance), sodium sulfate solution as mobile phase A, acetonitrile as mobile phase B, and gradient elution, so as to achieve effective separation of four main component peaks of polymyxin B sulfate (polymyxin B1, B2, B3 and B1-IB) and adjacent peak impurities (i.e. related substances E, F, G, M) on the chromatographic column, and can quickly, conveniently and accurately determine the content of related substances and components of polymyxin B sulfate. The system applicability, specificity, solution stability, linear range, detection limit and quantitative limit, accuracy, repeatability, intermediate precision and durability of the method of the present invention are verified, and the analytical method verification meets the requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a standard curve graph of polymyxin B1.

[0033] Figure 2 This is a standard curve graph of polymyxin B2.

[0034] Figure 3 This is a standard curve graph of polymyxin B3.

[0035] Figure 4 It is the standard curve graph of polymyxin B1-I.

[0036] Figure 5 This is the standard curve of impurity E.

[0037] Figure 6 This is the standard curve of impurity F.

[0038] Figure 7 This is the standard curve of impurity G.

[0039] Figure 8 It is the standard curve of impurity M. DETAILED DESCRIPTION

[0040] To illustrate the technical content of the present invention in detail, the present invention is further described below in conjunction with specific embodiments.

[0041] The main instruments and equipment involved in the present invention are:

[0042] Thermo HPLC with Vanquish Core TUV+CAD, Thermo Fisher Ultimate 3000

[0043] The reagents and medicines involved in the present invention are:

[0044] Anhydrous sodium sulfate (AR), acetonitrile (chromatographic grade), phosphoric acid (analytical grade), water (purified water), polymyxin B1 reference substance, polymyxin B2 reference substance, polymyxin B3 reference substance, polymyxin B1-I reference substance, polymyxin B sulfate reference substance, impurity E, impurity F, impurity G, impurity M, polymyxin B sulfate (sample) are shown in Table 1.

[0045] Table 1 Summary of Reference Material Information

[0046]

[0047]

[0048] Example 1 Selection of chromatographic conditions

[0049] Wavelength selection

[0050] Diluent: water-acetonitrile (80:20)

[0051] Preparation of polymyxin B sulfate solution: weigh about 10 mg of polymyxin B sulfate, place it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0052] Polymyxin B sulfate solution was used as the test sample, and the diluent was the blank calibration solution. The UV-Vis spectrophotometry (Chinese Pharmacopoeia 2020 Edition) was used to scan at 190nm-400nm. The results showed that the test solution had a maximum absorption peak at 215nm, so 215nm was determined as the detection wavelength.

[0053] Example 2 Specificity and system applicability

[0054] Chromatographic conditions: The column is filled with octadecylsilane bonded silica gel (Thermo Hypersil GOLDTM 4.6mm*250, 5μm or a column with equivalent performance), the flow rate is 0.8ml per minute, the detection wavelength is 215nm, the column temperature is 20℃, the injection volume is 20μl, and the gradient elution program is as follows:

[0055]

[0056] (1) Mobile phase A: Sodium sulfate solution: Dissolve 4.46 g of anhydrous sodium sulfate in 900 ml of water, adjust the pH to 2.3 with dilute phosphoric acid, and then dilute with water to 1000 ml.

[0057] (2) Mobile phase B: acetonitrile

[0058] (3) Diluent: water-acetonitrile (80:20).

[0059] (4) Blank solution: water-acetonitrile (80:20).

[0060] (5) Test solution: Take 50 mg of the product, weigh accurately, place in a 100 ml volumetric flask, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, shake well. Or prepare a solution of equal concentration (about 0.5 mg / ml).

[0061] (6) Reference solution: Take 50 mg of the reference substance, weigh it accurately, place it in a 100 ml volumetric flask, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, shake well. Or prepare a solution of equal concentration (about 0.5 mg / ml).

[0062] (7) 1% control solution: Accurately measure 1 ml of the control solution, place it in a 100 ml volumetric flask, dilute to the mark with water-acetonitrile (80:20), and shake well. Or prepare a solution of equal concentration.

[0063] (8) Polymyxin B1: Accurately weigh 2.2 mg of polymyxin B1 reference substance into a 10 ml volumetric flask, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, shake well. Or prepare a solution of equal concentration.

[0064] (9) Polymyxin B2: Accurately weigh 1.1 mg of polymyxin B2 reference substance into a 10 ml volumetric flask, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, shake well. Or prepare a solution of equal concentration.

[0065] (10) Polymyxin B3: Accurately weigh 1.2 mg of polymyxin B3 reference substance and place it in a 100 ml volumetric flask. Add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, shake well. Or prepare a solution of equal concentration.

[0066] (11) Polymyxin B1-I: Accurately weigh 1.2 mg of polymyxin B1-I reference substance and place it in a 100 ml volumetric flask. Add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, shake well. Or prepare a solution of equal concentration.

[0067] (12) Stock solutions of each impurity: Take 3 mg of each impurity E, impurity F, impurity G, and impurity M, weigh accurately, place in a 10 ml volumetric flask, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, shake well. Or prepare a solution of equal concentration (about 300 μg / ml for each impurity).

[0068] (13) Single standard solution of each impurity: Accurately measure 1 ml of each impurity stock solution, place it in a 20 ml volumetric flask, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, shake well. Or prepare a solution of equal concentration (about 15 μg / ml for each impurity).

[0069] (14) Mixed solution: Take 10 mg of this product, weigh accurately, place in a 20 ml volumetric flask, add 1 ml of each impurity stock solution, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, shake well. Or prepare a solution of equal concentration (main component about 0.5 mg / ml; each impurity about 15 μg / ml).

[0070] Accurately measure 20 μl of each of the above solutions, inject into the liquid phase, and record the chromatogram.

[0071] The results are shown in Table 2 and Table 3.

[0072] Table 2 System suitability test results

[0073]

[0074] Table 3 Specificity test results

[0075]

[0076]

[0077] The results showed that the blank solvent did not interfere with the detection of related substances of this product, and the specificity and system suitability were in compliance with regulations.

[0078] Example 3 Solution Stability

[0079] Chromatographic conditions: Same as Example 2

[0080] Solution preparation

[0081] Prepare blank solution, control solution and test solution, inject into liquid chromatograph, and measure at 0, 4, 8, 16 and 24 hours under 2-8℃. Compare the relative change of peak area with that at t=0. The results are shown in Table 4.

[0082] Table 4 2~8℃ stability results

[0083]

[0084]

[0085] The results showed that the control solution and the test solution were stable at 2-8°C for 224 hours.

[0086] Example 4: Linearity, detection limit, and quantification limit

[0087] Chromatographic conditions: Same as Example 2

[0088] Solution preparation

[0089] Solvent: Mobile phase A

[0090] Principal component linearity 1 (LOQ ~ 200%)

[0091] Polymyxin B (B1+B2) stock solution: Accurately weigh reference substance B19mg and B24.5mg and place them in 10ml volumetric bottles, add diluent to dissolve and dilute to scale, shake well, and the solution is ready. Alternatively, the same method can be used to prepare solutions of equal concentration (containing approximately B10.9mg / ml and B20.45mg / ml).

[0092] 200% solution

[0093] Accurately measure 2.5 mL of polymyxin B1 and B2 stock solutions, place in a 5.0 mL volumetric flask, dilute to scale with diluent, and shake well. Or prepare solutions of equal concentrations. (Contains approximately 0.45 mg / ml for B1 and 0.225 mg / ml for B2)

[0094] 150% solution

[0095] Accurately measure 1.875 mL of polymyxin B1 and B2 stock solutions, place in a 5.0 mL volumetric bottle, dilute to scale with diluent, and shake well. Or prepare solutions of equal concentrations. (Contains approximately 0.33 mg / ml of B1 and 0.165 mg / ml of B2)

[0096] 100% solution

[0097] Accurately measure 1.25 mL of polymyxin B1 and B2 stock solutions, place in a 5.0 mL volumetric bottle, dilute to scale with diluent, and shake well. Or prepare solutions of equal concentrations. (Contains approximately 0.22 mg / ml for B1 and 0.11 mg / ml for B2)

[0098] 50% solution

[0099] Accurately measure 0.63 mL of polymyxin B1 and B2 stock solutions, place in a 5.0 mL volumetric flask, dilute to scale with diluent, and shake well. Or prepare solutions of equal concentrations. (Contains approximately 0.11 mg / ml for B1 and 0.05 mg / ml for B2)

[0100] 10% solution

[0101] Accurately measure 0.125 mL of polymyxin B1 and B2 stock solutions, place in a 5.0 mL volumetric flask, dilute to scale with diluent, and shake well. Or prepare solutions of equal concentrations. (Contains approximately 0.022 mg / ml for B1 and 0.011 mg / ml for B2)

[0102] 1% solution

[0103] Accurately measure 0.125 mL of polymyxin B1 and B2 stock solutions, place in a 50 mL volumetric flask, dilute to scale with diluent, and shake well. Or prepare solutions of equal concentrations. (Contains approximately 0.0022 mg / ml for B1 and 0.0011 mg / ml for B2)

[0104] LOQ solution

[0105] Note: If the 1% solution is not the LOQ solution, continue to dilute to the LOQ solution.

[0106] Principal component linearity 2 (LOQ ~ 200%)

[0107] Polymyxin B (B3+B1-I) stock solution: accurately weigh reference substance B3 2.4 mg and B1-I 2.4 mg and place them in 10 ml volumetric flasks respectively, add diluent to dissolve and dilute to scale, shake well, and the solution is ready. Alternatively, the same method can be used to prepare a solution of equal concentration (containing about B3 0.2 mg / ml and B1-I 0.2 mg / ml).

[0108] 200% solution

[0109] Accurately measure 0.5 ml of polymyxin B3 and B1-I stock solution and place it in a 5.0 ml volumetric bottle, dilute to scale with diluent, and shake well. Or prepare a solution of equal concentration. (Contains approximately 0.02 mg / ml of B3 and 0.02 mg / ml of B1-I)

[0110] 150% solution

[0111] Accurately measure 0.375 ml of polymyxin B3 and B1-I stock solutions and place them in a 5.0 mL volumetric bottle, dilute to scale with diluent, and shake well. Or prepare solutions of equal concentrations. (Contains approximately 0.015 mg / ml of B3 and 0.015 mg / ml of B1-I)

[0112] 100% solution

[0113] Accurately measure 0.25 ml of polymyxin B3 and B1-I stock solutions and place them in a 5.0 ml volumetric flask, dilute to scale with diluent, and shake well. Or prepare solutions of equal concentrations. (Contains approximately 0.01 mg / ml of B3 and 0.01 mg / ml of B1-I)

[0114] 50% solution

[0115] Accurately measure 0.125 ml of polymyxin B3 and B1-I stock solutions and place them in a 5.0 mL volumetric bottle, dilute to scale with diluent, and shake well. Or prepare solutions of equal concentrations. (Contains approximately 0.005 mg / ml of B3 and 0.005 mg / ml of B1-I)

[0116] 10% solution

[0117] Accurately measure 0.025 ml of polymyxin B3 and B1-I stock solutions and place them in a 5.0 mL volumetric bottle, dilute to scale with diluent, and shake well. Or prepare solutions of equal concentrations. (Contains approximately 0.001 mg / ml of B3 and 0.001 mg / ml of B1-I)

[0118] LOQ solution

[0119] Note: If the 10% solution is not the LOQ solution, continue to dilute to the LOQ solution.

[0120] Impurity linearity (LOQ to limit concentration 200%)

[0121] 200% solution

[0122] Accurately measure 1.0 ml of each impurity linear stock solution, place in a 20 ml volumetric bottle, dilute to scale with diluent, and shake well. Or prepare a solution of equal concentration. (Each impurity contains about 30 μg / ml)

[0123] 150% solution

[0124] Accurately measure 0.75 ml of each impurity linear stock solution, place in a 20 mL volumetric bottle, dilute to scale with diluent, and shake well. Or prepare a solution of equal concentration. (Each impurity contains about 22.5 μg / ml)

[0125] 100% solution

[0126] Accurately measure 0.5 ml of each impurity linear stock solution, place in a 20 mL volumetric bottle, dilute to scale with diluent, and shake well. Or prepare a solution of equal concentration. (Each impurity contains about 15 μg / ml)

[0127] 80% solution

[0128] Accurately measure 0.4 ml of each impurity linear stock solution, place in a 20 ml volumetric bottle, dilute to scale with diluent, and shake well. Or prepare a solution of equal concentration. (Each impurity contains about 12 μg / ml)

[0129] 50% solution

[0130] Accurately measure 0.25 ml of each impurity linear stock solution, place in a 20 mL volumetric bottle, dilute to scale with diluent, and shake well. Or prepare a solution of equal concentration. (Each impurity contains about 7.5 μg / ml)

[0131] 20% solution

[0132] Accurately measure 0.25 ml of each impurity linear stock solution, place in a 50 mL volumetric bottle, dilute to scale with diluent, and shake well. Or prepare a solution of equal concentration. (Each impurity contains about 3 μg / ml)

[0133] 10% solution

[0134] Accurately measure 0.25 ml of each impurity linear stock solution, place in a 100 mL volumetric flask, dilute to scale with diluent, and shake well. Or prepare a solution of equal concentration. (Each impurity contains about 1.5 μg / ml)

[0135] LOQ solution

[0136] Note: If the 10% solution is not the LOQ solution, continue to dilute to the LOQ solution.

[0137] Accurately measure 20 μl of blank solution and the above solution, respectively, inject them into the liquid chromatograph, and record the chromatogram. The results of the quantitative limit and detection limit test are shown in Table 5 and Table 6.

[0138] The results showed that the linear range of polymyxin B1 was 0.0014mg / ml~0.4498mg / ml; regression equation: y=207.48x-0.2837; R 2 =0.9998; the absolute value of the ratio of the Y-axis intercept to the limit concentration response value is 0.61%. The peak area has a good linear relationship with the concentration. The standard curve is shown in Figure 1 .

[0139] The linear range of polymyxin B2 is 0.0007mg / ml~0.2202mg / ml; regression equation: y=198.81x-0.1052; R 2 =0.9998; the absolute value of the ratio of the Y-axis intercept to the limit concentration response value is 0.48%. The peak area has a good linear relationship with the concentration. The standard curve is shown in Figure 2 .

[0140] The linear range of polymyxin B3 is 0.0006mg / ml~0.0195mg / ml; regression equation: y=210.47x-0.0277; R 2=0.9976; the absolute value of the ratio of the Y-axis intercept to the limit concentration response value is 1.36%. The peak area has a good linear relationship with the concentration. The standard curve is shown in Figure 3 .

[0141] The linear range of polymyxin B1-I is 0.0006mg / ml~0.0198mg / ml; regression equation: y=222.85x-0.0052; R 2 =0.9979; the absolute value of the ratio of the Y-axis intercept to the limit concentration response value is 0.24%. The peak area has a good linear relationship with the concentration. The standard curve is shown in Figure 4 .

[0142] Impurity E linear range 0.2080μg / ml~20.9001μg / ml; regression equation: y=0.2679x-0.0731; R 2 =0.9988; the absolute value of the ratio of the Y-axis intercept to the limit concentration response value is 1.83%. The peak area has a good linear relationship with the concentration, and the standard curve is shown in Figure 5 .

[0143] Impurity F linear range 0.3135μg / ml~19.4640μg / ml; regression equation: y=0.2699x-0.1862; R 2 =0.9966; the absolute value of the ratio of the Y-axis intercept to the limit concentration response value is 4.80%. The peak area has a good linear relationship with the concentration, and the standard curve is shown in Figure 6 .

[0144] Impurity G linear range 0.3292μg / ml~21.6853μg / ml; regression equation: y=0.1888x-0.0621; R 2 =0.9977; the absolute value of the ratio of the Y-axis intercept to the limit concentration response value is 2.24%. The peak area has a good linear relationship with the concentration, and the standard curve is shown in Figure 7 .

[0145] Impurity M linear range 0.2157μg / ml~20.1936μg / ml; regression equation: y=0.3302x-0.0775; R 2 =0.9993; the absolute value of the ratio of the Y-axis intercept to the limit concentration response value is 1.59%. The peak area has a good linear relationship with the concentration, and the standard curve is shown in Figure 8 .

[0146] Table 5 Quantification limit results (polymyxin B1, polymyxin B2, polymyxin B3, polymyxin B1-I)

[0147]

[0148]

[0149] Table 6 Quantitation limit results (Impurity E, Impurity F, Impurity G, Impurity M)

[0150]

[0151] The results showed that under the proposed chromatographic conditions, the linearity was good, and the LOQ of each component and impurity was lower than 0.5% of the test sample concentration, which fully met the requirements for accurately determining the content of related substances and components of polymyxin B sulfate.

[0152] Example 5 Accuracy (component content)

[0153] Chromatographic conditions: Same as Example 2

[0154] Accuracy solution preparation principles

[0155] Accurately weigh an appropriate amount of each component reference substance to prepare known reference substances at 80%, 100% and 120% of the known standard limit concentration. The specific preparation is as follows.

[0156] Solution preparation

[0157] 80% concentration: accurately weigh 1 mg of polymyxin B3 and 1 mg of polymyxin B1-I into a 10 ml volumetric flask, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to scale; accurately weigh 1.76 mg of polymyxin B1 and 0.88 mg of polymyxin B2 as reference substances into a 10 ml volumetric flask, accurately pipette 1 ml of the above solution and add, dissolve and quantitatively dilute to scale with water-acetonitrile (80:20), shake well. Or prepare solutions of equal concentration (containing approximately B1 0.18 mg / ml, B2 0.09 mg / ml, B3 0.008 mg / ml, B1-I 0.008 mg / ml) in the same way. Prepare 3 copies in parallel.

[0158] 100% concentration: accurately weigh 1.2 mg of polymyxin B3 and 1.2 mg of polymyxin B1-I into a 10 ml volumetric flask, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to scale; accurately weigh 2.2 mg of polymyxin B1 and 1.1 mg of polymyxin B2 as reference substance into a 10 ml volumetric flask, accurately pipette 1 ml of the above solution and add, dissolve and quantitatively dilute to scale with water-acetonitrile (80:20), shake well. Or prepare equal concentration solutions (containing approximately B1 0.22 mg / ml, B2 0.11 mg / ml, B3 0.01 mg / ml, B1-I 0.01 mg / ml) in the same way. Prepare 3 copies in parallel.

[0159] 120% concentration: accurately weigh 1.4 mg of polymyxin B3 and 1.4 mg of polymyxin B1-I into a 10 ml volumetric flask, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale; accurately weigh 2.64 mg of polymyxin B1 and 1.32 mg of polymyxin B2 as a reference substance into a 10 ml volumetric flask, accurately pipette 1 ml of the above solution and add, dissolve and quantitatively dilute to the scale with water-acetonitrile (80:20), shake well. Or prepare solutions of equal concentration (containing approximately 2.64 mg / ml of B1, 1.32 mg / ml of B2, 0.012 mg / ml of B3, and 0.012 mg / ml of B1-I) by the same method. Prepare 3 copies in parallel. Solution

[0160] Accuracy (related substances)

[0161] Accuracy solution preparation principles

[0162] Accurately weigh the reference substance and add 50%, 100% and 150% of the known standard limit concentration of each known reference substance. The specific preparation is as follows.

[0163] Table 7 Solution configuration

[0164]

[0165] 50% concentration: Take 10 mg of this product, weigh accurately, place in a 20 ml volumetric flask, add 0.25 ml of each impurity stock solution, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, shake well. Or prepare an equal concentration solution (main component about 0.5 mg / ml; each impurity about 7.5 μg / ml). Prepare 3 copies in parallel.

[0166] 100% concentration: Take 10 mg of this product, weigh accurately, place in a 20 ml volumetric flask, add 0.5 ml of each impurity stock solution, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, shake well. Or prepare a solution of equal concentration (main component about 0.5 mg / ml; each impurity about 15 μg / ml). Prepare 3 copies in parallel.

[0167] 150% concentration: Take 10 mg of this product, weigh accurately, place in a 20 ml volumetric flask, add 0.75 ml of each impurity stock solution, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, shake well. Or prepare a solution of equal concentration (main component about 0.5 mg / ml; each impurity about 22.5 μg / ml). Prepare 3 copies in parallel.

[0168] Accurately measure 20 μl of each of the above solutions, inject them into the liquid chromatograph respectively, and record the chromatogram; the accuracy test results are shown in Table 8 (components) and Table 9 (related substances).

[0169] Table 8 Accuracy (component) results

[0170]

[0171]

[0172] Table 9 Accuracy (related substances) results

[0173]

[0174]

[0175] The results showed that under the proposed chromatographic conditions, the method had good recovery and high accuracy.

[0176] Example 6 Repeatability

[0177] Chromatographic conditions: Same as Example 2

[0178] (1) System applicability

[0179] Refer to the preparation process under the system suitability test

[0180] (2) Test solution: Take 50 mg of the product, weigh accurately, place in a 100 ml volumetric flask, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the mark, shake well. Prepare 6 copies in parallel.

[0181] (3) Mixed solution: Take 10 mg of this product, weigh accurately, place in a 20 ml volumetric flask, add 1 ml of each impurity stock solution, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, shake well. Or prepare a solution of equal concentration (main component about 0.5 mg / ml; each impurity about 15 μg / ml).

[0182] Table 10 Repeatability results of component content analysis of test solution

[0183]

[0184] Table 11 Repeatability results of related substance analysis of test solution

[0185]

[0186]

[0187] The results showed that this method had good repeatability.

[0188] Example 7 Intermediate Precision

[0189] Chromatographic conditions: Same as Example 2

[0190] Solution preparation: Prepare according to the repetitive solution preparation method. Under different dates, different analysts, and different equipment conditions, take the same batch of test samples for relevant substance inspection and record the chromatogram. The results of the intermediate precision test are shown in Table 12.

[0191] Table 12 Intermediate precision results of component contents of test solution

[0192]

[0193]

[0194] Table 13 Intermediate precision results of related substances in test solution

[0195]

[0196]

[0197] The results showed that the method had good intermediate precision.

[0198] Example 8 Durability Test

[0199] In this test, the method detection system suitability, test solution, mixed solution, and durability test were performed by adjusting the column temperature, mobile phase pH value, and different brands of chromatographic columns under the proposed chromatographic conditions. The specific plan of the durability test is shown in Table 14.

[0200] Table 14 Durability test specific plan

[0201]

[0202] Solution preparation

[0203] 1. Reference solution: Take 50 mg of the reference substance, weigh it accurately, put it in a 100 ml volumetric flask, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, shake well. Or prepare a solution of equal concentration (about 0.5 mg / ml).

[0204] 2.1% control solution: Accurately measure 1 ml of the control solution, place it in a 100 ml volumetric flask, dilute to the mark with water-acetonitrile (80:20), and shake well.

[0205] 3. System suitability experiment: The blank should be free of interference. 5 needles of reference solution were injected continuously. In the obtained chromatogram, the RSD of the peak areas of polymyxin B1, polymyxin B2, polymyxin B3, and polymyxin B1-I were all ≤2.0%; the relative retention times of polymyxin B2, polymyxin B3, polymyxin B1-I and polymyxin B1 were approximately 0.5, 0.55, 0.8, and 1.0, respectively.

[0206] Accurately measure 20 μl of each of the above solutions, measure under different column temperatures (±2°C), different pH values ​​(±0.1) and by changing the chromatographic column, inject into the liquid phase, and record the chromatogram. The results are shown in Table 15.

[0207] Table 15 Durability test results - system suitability

[0208]

[0209]

[0210] The results show that the high performance liquid chromatography analysis method of the present invention has strong specificity, high sensitivity, good precision, and can accurately and quickly determine the contents of related substances and components of polymyxin B sulfate.

Claims

1. A method for determining related substances and components of polymyxin B sulfate using HPLC, characterized in that: The components are polymyxin sulfate B1, B2, B3 and B1-I, and the related substances are impurities E, F, G, and M. The structural formula of the related substances is as follows: The method described is a high performance liquid chromatography method, using Thermo Hypersil GOLDTM as a chromatographic column, octadecylsilane bonded silica gel as a filler, sodium sulfate solution as mobile phase A, acetonitrile as mobile phase B, the detection wavelength is 215nm, the flow rate of the chromatographic column is 0.8ml per minute, the column temperature of the chromatographic column is 20℃, the injection volume is 20μl, and gradient elution is used to separate the components of polymyxin B sulfate from related substances. The gradient elution procedure is as follows:

2. The method for determining related substances and components of polymyxin B sulfate by HPLC according to claim 1, characterized in that: The sodium sulfate solution used as mobile phase A was prepared as follows: 4.46 g of anhydrous sodium sulfate was dissolved in 900 ml of water, the pH value was adjusted to 2.3 ± 0.1 with dilute phosphoric acid, and then diluted to 1000 ml with water.

3. The method for determining related substances and components of polymyxin B sulfate by HPLC according to claim 1, characterized in that: The method comprises solution preparation, the solution comprises a blank solution, a test solution, a control solution, an impurity stock solution and a mixed solution, and the preparation methods are as follows: (1) Blank solution: water-acetonitrile solution with a volume ratio of 80:20; (2) Test solution: Take polymyxin B sulfate standard, accurately weigh it, place it in a volumetric flask, and dilute it to the required concentration; (3) Reference solution: Take polymyxin B sulfate reference substance, accurately weigh it, place it in a volumetric bottle, and dilute it to the required concentration; (4) Stock solutions of each impurity: Take impurity E, impurity F, impurity G, and impurity M, weigh them accurately, place them in a volumetric bottle, and dilute them to the required concentration. (5) Mixed solution: Take the polymyxin B sulfate standard, accurately weigh it, and place it in a volumetric bottle. Take the stock solution of each impurity, accurately weigh it, and place it in a volumetric bottle, and dilute it to the required concentration.

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